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==== Front J NeuroinflammationJournal of Neuroinflammation1742-2094BioMed Central London 1742-2094-1-141528580110.1186/1742-2094-1-14ReviewMicroglia and neuroinflammation: a pathological perspective Streit Wolfgang J [email protected] Robert E [email protected] W Sue T [email protected] Department of Neuroscience, University of Florida College of Medicine, P.O. Box 100244, Gainesville, Florida 32610, USA2 Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA3 Department of Geriatrics, University of Arkansas for Medical Sciences and GRECC/CAVHS, Little Rock, Arkansas 72205, USA2004 30 7 2004 1 14 14 8 7 2004 30 7 2004 Copyright © 2004 Streit et al; licensee BioMed Central Ltd.2004Streit et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Microglia make up the innate immune system of the central nervous system and are key cellular mediators of neuroinflammatory processes. Their role in central nervous system diseases, including infections, is discussed in terms of a participation in both acute and chronic neuroinflammatory responses. Specific reference is made also to their involvement in Alzheimer's disease where microglial cell activation is thought to be critically important in the neurodegenerative process. ==== Body Background A role for immune responses, involving antigen presentation and immune-response-generating cytokines, in neurodegenerative diseases such as Alzheimer's disease was recognized for a decade before the term neuroinflammation came into widespread use [1,2]. A PubMed search using "neuroinflammation" as the only key word yields some 300 papers, none before 1995 [3]. While some chronic/remitting neurological diseases, such as multiple sclerosis, have long been recognized as inflammatory, the term neuroinflammation has come to denote chronic, CNS-specific, inflammation-like glial responses that do not reproduce the classic characteristics of inflammation in the periphery but that may engender neurodegenerative events; including plaque formation, dystrophic neurite growth, and excessive tau phosphorylation. In this way, neuroinflammation has been implicated in chronic unremitting neurodegenerative diseases such as Alzheimer's disease – diseases that historically have not been thought of as inflammatory diseases. This new understanding has come from rapid advances in the field of microglial and astrocytic neurobiology over the past fifteen to twenty years. These advances have led to the recognition that glia, particularly microglia, respond to tissue insult with a complex array of inflammatory cytokines and actions, and that these actions transcend the historical vision of phagocytosis and structural support that has long been enshrined in the term "reactive gliosis." Microglia are now recognized as the prime components of an intrinsic brain immune system [4], and as such they have become a main focus in cellular neuroimmunology and therefore in neuroinflammation. This is not the inflammation of the adaptive mammalian immune response, with its array of specialized T-cells and the made-to-order antibodies produced through complex gene rearrangements. This is, instead, the innate immune system, upon which adaptive immunity is built [5]. Many researchers now consider this innate immune response in the brain to be a potentially pathogenic factor in a number of CNS diseases that lack the prominent leukocytic infiltrates of adaptive immune responses, but that do have activated microglia and astrocytes, i.e., neuroinflammation. The idea that neuroinflammation is detrimental implies that glial cell activation precedes and causes neuronal degeneration [2], a sequence of events that appears to be at odds with experimental models of neurodegeneration in which glial cell activation occurs secondary to neuronal damage. What is missing from this simple linear model is the understanding that chronic neurological diseases are just that – chronic, and that this chronicity introduces complex interactions and feedback loops between neurons and glia that render attempts to construct simple, linear cascades of cause and effect inelegant. In the following, we provide some basic definitions and discussion to more precisely define the idea of neuroinflammation as a CNS tissue response to injury, and the notion of neuroinflammation as a pathogenic factor in neurodegenerative diseases. Some basic definitions Inflammation is a reaction of living tissues to injury [6]. The discipline of pathology makes a fundamental distinction between acute and chronic inflammation. Acute inflammation comprises the immediate and early response to an injurious agent and is basically a defensive response that paves the way for repair of the damaged site. Chronic inflammation results from stimuli that are persistent. In the periphery, inflammation consists of leukocytic infiltrates characterized by polymorphonuclear cells (neutrophils) in acute inflammation and mononuclear cells (macrophages, lymphocytes, plasma cells) in chronic inflammation. In order to validate these principles of general pathology within the context of neuroinflammation, one must obviously consider both acute and chronic neuroinflammation and, therefore, these are addressed separately in the following sections. Acute neuroinflammation Before "neuroinflammation" became a commonly used term, neuroscientists spoke of "reactive gliosis" in describing endogenous CNS tissue responses to injury. Reactive gliosis specifically referred to the accumulation of enlarged glial cells, notably microglia and astrocytes, appearing immediately after CNS injury has occurred. In contrast to glial reactivity, which suggests a largely passive response to injury; glial activation implies a more aggressive role in responding to activating stimuli: activated glial cells release factors that act on and engender responses in target cells analogous to the responses of activated immune cells in the periphery. Activation of immune cells in the periphery leads to leukocyte infiltration of tissues, but this is notably absent in the brain unless there has been destruction or compromise of the blood brain barrier [7,8]. In the presence of such destruction or compromise, peripheral leukocytes do enter the brain producing a scenario similar to that seen in inflammatory responses in the periphery. In limited, acute reactions to injury, in the absence of blood-brain barrier breakdown, there is the subtler response of the brain's own immune system, composed largely of rapid activation of glial cells. These responses represent the other end of the spectrum of CNS injury, where limited neuronal insults trigger glial cell activation without breakdown of the blood brain barrier and without concomitant leukocytic infiltration. This form of "pure" glial response occurs in neuronal injury caused by either loss of afferents [9] or loss of efferents [10]. Axotomy, for instance, results in neuronal chromatolysis, the classic example of potentially reversible neuronal injury [9]. It is in these situations that microglial and astrocytic responses (like their peripheral counterparts) fulfill their evolutionarily programmed functions of a reparative response to the benefit of the organism as a whole. Although such specific responses might, in a strict sense, be included in the term "neuroinflammation," neuroinflammation as generally used and understood applies to more chronic, sustained cycles of injury and response, in which the cumulative ill effects of immunological microglial and astrocytic activation contribute to and expand the initial neurodestructive effects, thus maintaining and worsening the disease process through their actions. Chronic neuroinflammation The concept of chronic inflammation (as opposed to acute inflammation) is more relevant in the context of understanding CNS disease (as opposed to CNS injury), as the very term "disease" implies chronicity. Chronic multiple sclerosis is, of course, an unequivocal and long-recognized example of an inflammatory brain disease. Although the underlying cause(s) of multiple sclerosis have not been elucidated, it is probably safe to say that the persistent injurious stimulus that accounts for neuroinflammation in multiple sclerosis is a myelin-related protein that has escaped self-tolerance and become an autoimmunogen. Consistent with the chronic persistence of this autoimmunogen is a persistent accumulation of blood-derived mononuclear leukocytes in the CNS parenchyma, a phenomenon that is similar to what is found in other autoimmune diseases such as rheumatoid arthritis or polymyositis. Infections are another group of diseases that are classically recognized as inflammatory in nature, with meningeal, perivascular, or even parenchymal infiltrates of peripheral leukocytes. There are, however, exceptions. Rabies is a disease in which the peripheral immune response is slow and inadequate, and in which classic inflammatory changes are less striking than those found in other viral encephalidites. Babes, in 1897 [11], described microglial activation in rabies infection, although he did not recognize the nodules he found as clusters of activated microglia. Similar small collections of activated microglia were subsequently found to occur in a wide variety of viral brain infections. Today, the most important example of a chronic brain infection is human immunodeficiency virus (HIV). Chronic HIV encephalitis is characterized by the same nodules of activated microglia that Babes described in rabies. HIV enters and persists in the CNS via myelomonocytic cells: monocytes, perivascular cells, and microglia [12]. HIV infection is uniquely different from most other infectious diseases affecting the CNS in that the virus targets and disables precisely those cells that are key players in neuroinflammation; microglia in the brain and T lymphocytes in the periphery. It therefore comes as no surprise that prominent T cell infiltrates do not occur in HIV encephalopathy. Prion diseases represent another chronic infectious CNS disease that is not accompanied by leukocytic infiltrates. Microglial activation, again, appears to be the most prominent inflammatory component of prion diseases [13,14], although there are a few reports describing T cell infiltration as well [15,16]. Prion diseases share interesting parallels to rabies infection in that infected cells are unrecognized by peripheral immune responses. This may explain in part the unusual patterns of neuroinflammation in prion diseases – manifest not only in atypical cellular infiltrates but also in unusual cytokine profiles [17]. Both HIV and prion infections probably produce an altered microglial physiology that is likely to translate into cycles of neurodegeneration, which could be a contributing factor in the development of dementia that occurs in these conditions. Chronic microglial neuroinflammation in neurodegenerative diseases Neurodegenerative diseases – particularly Alzheimer's disease, but also amyotrophic lateral sclerosis, Parkinson's disease, and Huntington's disease – lack the prominent infiltrates of blood-derived mononuclear cells that characterize autoimmune diseases. On the other hand, there is abundant evidence that many substances involved in the promotion of inflammatory processes are present in the CNS of patients with such neurodegenerative diseases. By far the bulk of this body of evidence is related to studies in Alzheimer's disease [18]. What distinguishes Alzheimer's disease from other neurodegenerative diseases is the conspicuous presence of extracellular deposits of amyloid in senile plaques. Senile plaques in Alzheimer brain are present in different stages of maturity, ranging from diffuse to neuritic to dense core, but they all contain the amyloid beta protein (Aβ). Aβ is a peptide that forms insoluble and pathological extracellular aggregates that seem to attract microglial cells, as suggested by the clustering of microglia at sites of Aβ deposition (see [19] for a review). There is evidence from experimental studies in animals to support the idea that microglia can phagocytose and degrade amyloid [20,21], but such phagocytosis is apparently either ineffective or inadequate in Alzheimer's disease. A key question within the current context is: "Does the amyloid in Alzheimer brain by itself represent a persistent injurious stimulus that causes neuronal injury, or are additional factors involved in eliciting this outcome?" Direct injection of Aβ into the brain produces activation of microglia and loss of specific populations of neurons [21]. Furthermore, transgenic mice that overexpress human, mutant β-amyloid precursor protein (βAPP) do develop Aβ deposits with associated evidence of neuritic injury (although they do not develop Alzheimer-type neurofibrillary tangles unless they are also transgenic for human tau protein) [22]. These Aβ deposits, born of transgenic overexpression of mutant human amyloid precursor protein, invariably contain activated microglia [22,23]. β-Amyloid precursor protein βAPP functions as a neuronal acute-phase, injury-response protein. For instance, there is excessive expression of βAPP, accompanied by microglial activation and cytokine expression, after traumatic head injury [24]. With head injury, there is also Aβ deposition, both in experimental animals [25] and in humans – particularly in individuals genetically susceptible for AD (i.e. ApoE ε4-positive) [26]. These observations emphasize the complex interactions that underlie neurodegeneration in Alzheimer's disease. Conclusions Chronic microglial activation is an important component of neurodegenerative diseases, and this chronic neuroinflammatory component likely contributes to neuronal dysfunction, injury, and loss (and hence to disease progression) in these diseases. The recognition of microglia as the brain's intrinsic immune system, and the understanding that chronic activation of this system leads to pathologic sequelae, has led to the modern concept of neuroinflammation. This vision of microglia-driven neuroinflammatory responses, with neuropathological consequences, has extended the older vision of passive glial responses that are inherent in the concept of "reactive gliosis." Abbreviations Aβ: β-amyloid peptide βAPP: Aβ precursor protein CNS: central nervous system HIV: human immunodeficiency virus MS: multiple sclerosis Competing interests None declared Authors' contributions WJS conceived this review, wrote the initial draft, modified this with the comments of REM and WSTG, and wrote the final draft. REM and WSTG contributed particularly to the sections on infections and on Alzheimer's disease. All authors read and approved the final version. Acknowledgments Supported in part by NIH R21 NINDS 049185, NIH PO1 AG 12411, NIH P30 AG 19606, NIH RO1 AG 37989, and the McKnight Brain Research Foundation at the University of Florida. ==== Refs McGeer PL Itagaki S Tago H McGeer EG Reactive microglia in patients with senile dementia of the Alzheimer type are positive for the histocompatibility glycoprotein HLA-DR Neurosci Lett 1987 79 195 200 3670729 10.1016/0304-3940(87)90696-3 Griffin WST Stanley LC Ling C White L MacLeod V Perrot LJ White CL III Araoz C Brain interleukin 1 and S-100 immunoreactivity are elevated in Down syndrome and Alzheimer disease Proc Natl Acad Sci U S A 1989 86 7611 7615 2529544 Issazadeh S Mustafa M Ljungdahl A Hojeberg B Dagerlind A Elde R Olsson T Interferon gamma, interleukin 4 and transforming growth factor beta in experimental autoimmune encephalomyelitis in Lewis rats: dynamics of cellular mRNA expression in the central nervous system and lymphoid cells J Neurosci Res 1995 40 579 590 7602612 Streit WJ Kincaid-Colton CA The brain's immune system Sci Am 1995 273 54 61 8966536 Medzhitov R Janeway C Jr Advances in immunology: innate immunity N Engl J Med 2000 343 338 344 10922424 10.1056/NEJM200008033430506 Robbins SL Angell M Kumar V Basic Pathology 1981 3 Philadelphia: W.B. Saunders Sroga JM Jones TB Kigerl KA McGaughy VM Popovich PG Rats and mice exhibit distinct inflammatory reactions after spinal cord injury J Comp Neurol 2003 462 223 240 12794745 10.1002/cne.10736 Streit WJ Semple-Rowland SL Hurley SD Miller RC Popovich PG Stokes BT Cytokine mRNA profiles in contused spinal cord and axotomized facial nucleus suggest a beneficial role for inflammation and gliosis Exp Neurol 1998 152 74 87 9682014 10.1006/exnr.1998.6835 Kreutzberg GW Principles of neuronal regeneration Acta Neurochir Suppl (Wein) 1996 66 103 106 Ito K Ishikawa Y Skinner RD Mrak RE Morrison-Bogorad M Mukawa J Griffin WST Lesioning of the inferior olive using a ventral surgical approach: characterization of temporal and spatial responses at the lesion site and in cerebellum Mol Chem Neuropathol 1997 31 245 264 9336767 Babes V Sur certains caractères des lesions histologiques de la rage Ann Inst Pasteur Lille 1892 6 209 223 Garden GA Microglia in human immunodeficiency virus-associated neurodegeneration Glia 2002 40 240 251 12379911 10.1002/glia.10155 Eikelenboom P Bate C Van Gool WA Hoozemans JJM Rozemuller JM Veerhuis R Williams A Neuroinflammation in Alzheimer's disease and prion disease Glia 2002 40 232 239 12379910 10.1002/glia.10146 Perry VH Cunningham C Boche D Atypical inflammation in the central nervous system in prion disease Curr Opin Neurol 2002 15 349 354 12045736 10.1097/00019052-200206000-00020 Betmouni S Perry VH Gordon JL Evidence for an early inflammatory response in the central nervous system of mice with scrapie Neuroscience 1996 74 1 5 8843071 10.1016/0306-4522(96)00212-6 Lewicki H Tishon A Homann D Mazarguil H Laval F Asensio VC Campbell IL DeArmond S Coon B Teng C Gairin JE Oldstone MB T cells infiltrate the brain in murine and human transmissible spongiform encephalopathies J Virol 2003 77 3799 3808 12610154 10.1128/JVI.77.6.3799-3808.2003 Baker CA Martin D Manuelidis L Microglia from Creutzfeldt-Jakob disease-infected brains are infectious and show specific mRNA activation profiles J Virol 2002 76 10905 10913 12368333 10.1128/JVI.76.21.10905-10913.2002 Akiyama H Barger S Barnum S Bradt B Bauer J Cole GM Cooper NR Eikelenboom P Emmerling M Fiebich BL Finch CE Frautschy S Griffin WS Hampel H Hull M Landreth G Lue L Mrak R Mackenzie IR McGeer PL O'Banion MK Pachter J Pasinetti G Plata-Salaman C Rogers J Rydel R Shen Y Streit W Strohmeyer R Tooyoma I Van Muiswinkel FL Veerhuis R Walker D Webster S Wegrzyniak B Wenk G Wyss-Coray T Inflammation and Alzheimer's disease Neurobiol Aging 2000 21 383 421 10858586 10.1016/S0197-4580(00)00124-X Streit WJ Microglia and Alzheimer's disease pathogenesis J Neurosci Res 2004 77 1 8 15197750 10.1002/jnr.20093 Frautschy SA Cole GM Baird A Phagocytosis and deposition of vascular β-amyloid in rat brains injected with Alzheimer β-amyloid Am J Pathol 1992 140 1389 1399 1376558 Weldon DT Rogers SD Ghilardi JR Finke MP Cleary JP O'Hare E Esler WP Maggio JE Mantyh PW Fibrillar beta-amyloid induces microglial phagocytosis, expression of inducible nitric oxide synthase, and loss of a select population of neurons in the rat CNS in vivo J Neurosci 1998 18 2161 73 9482801 Irizarry MC McNamara M Fedorchak K Hsiao K Hyman BT APPSW transgenic mice develop age-related Aβ deposits and neuropil abnormalities, but no neuronal loss in CA1 J Neuropathol Exp Neurol 1997 56 965 973 9291938 Frautschy SA Yang F Irrizarry M Hyman B Saido TC Hsiao K Cole GM Microglial response to amyloid plaques in APPsw transgenic mice Am J Pathol 1998 152 307 17 9422548 Graham DI Gentleman SM Nicoll JA Royston MC McKenzie JE Roberts GW Griffin WS Altered beta-APP metabolism after head injury and its relationship to the aetiology of Alzheimer's disease Acta Neurochir – Suppl (Wein) 1996 66 96 102 Smith DH Uryu K Saatman KE Trojanowski JQ McIntosh TK Protein accumulation in traumatic brain injury Neuromolecular Med 2003 4 59 72 14528053 10.1385/NMM:4:1-2:59 Graham DI Gentleman SM Nicoll JA Royston MC McKenzie JE Roberts GW Mrak RE Griffin WS Is there a genetic basis for the deposition of beta-amyloid after fatal head injury? Cell Molec Neurobiol 1999 19 19 30 10079962 10.1023/A:1006937205905

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==== Front Int J Behav Nutr Phys ActThe International Journal of Behavioral Nutrition and Physical Activity1479-5868BioMed Central London 1479-5868-1-121528798410.1186/1479-5868-1-12ResearchWho will lose weight? A reexamination of predictors of weight loss in women Teixeira Pedro J [email protected] António L [email protected] Teresa L [email protected] Sandra S [email protected] Cláudia S [email protected] José T [email protected] Analiza M [email protected] Luís B [email protected] Department of Exercise and Health, Faculty of Human Movement – Technical University of Lisbon, Cruz Quebrada, PORTUGAL2004 2 8 2004 1 12 12 18 3 2004 2 8 2004 Copyright © 2004 Teixeira et al; licensee BioMed Central Ltd.2004Teixeira et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The purpose of this study was to analyze pretreatment predictors of short-term weight loss in Portuguese overweight and obese women involved in a weight management program. Behavioral and psychosocial predictors were selected a priori from previous results reported in American women who participated in a similar program. Methods Subjects were 140 healthy overweight/obese women (age, 38.3 ± 5.9 y; BMI, 30.3 ± 3.7 kg/m2) who participated in a 4-month lifestyle weight loss program consisting of group-based behavior therapy to improve diet and increase physical activity. At baseline, all women completed a comprehensive behavioral and psychosocial battery, in standardized conditions. Results Of all starting participants, 3.5% (5 subjects) did not finish the program. By treatment's end, more than half of all women had met the recomended weight loss goals, despite a large variability in individual results (range for weight loss = 19 kg). In bivariate and multivariate correlation/regression analysis fewer previous diets and weight outcome evaluations, and to a lesser extent self-motivation and body image were significant and independent predictors of weight reduction, before and after adjustment for baseline weight. A negative and slightly curvilinear relationship best described the association between outcome evaluations and weight change, revealing that persons with very accepting evaluations (that would accept or be happy with minimal weight change) lost the least amount of weight while positive but moderate evaluations of outcomes (i.e., neither low nor extremely demanding) were more predictive of success. Among those subjects who reported having initiated more than 3–4 diets in the year before the study, very few were found to be in the most successful group after treatment. Quality of life, self-esteem, and exercise variables did not predict outcomes. Conclusions Several variables were confirmed as predictors of success in short-term weight loss and can be used in future hypothesis-testing studies and as a part of more evolved prediction models. Previous dieting, and pretreatment self-motivation and body image are associated with subsequent weight loss, in agreement with earlier findings in previous samples. Weight outcome evaluations appear to display a more complex relationship with treatment results and culture-specific factors may be useful in explaining this pattern of association. ==== Body Background Predicting weight loss outcomes from information collected from subjects before they start weight management programs is a long-standing goal [1]. In effect, if individual variability in obesity treatment remains as high as it is presently, identifying variables that moderate outcomes (i.e., that explain for whom treatment works and under what conditions) will justifiably continue to deserve attention from researchers [2,3]. To date, however, evidence shows that individual weight change cannot be accurately predicted, with only a few variables showing positive results [4,5]. Nevertheless, advances in theoretical formulations regarding the process of weight control [6], improved research methodologies [7], and an increasing number of variables tested as potential predictors [8] suggest further progress is possible. Among the most valuable applications of valid weight loss prediction models is the early identification of individuals with the least estimated probability of success in a given treatment, who could (and perhaps should) be directed to alternative therapies. Research specifically aimed at studying these overweight/obese persons, who are more resistant to current forms of treatment, would be particularly relevant. Equally important are improvements in the matching between treatments and participants, which are dependent on the measurement of relevant pretreatment variables (i.e., that are found to predict success). More individualized programs have the potential for higher cost-effectiveness and improved overall success rates, by targeting specific areas of concern in selected participants or homogeneous groups [9]. Finally, the development of a valid and comprehensive weight loss readiness questionnaire and its use as a screening tool in obesity treatment are additional foreseeable outcomes of this research [10]. We have previously tested a large number of psychosocial and behavioral variables as predictors of short-term weight outcomes [8]. A number of significant pretreatment correlates of 4-month weight loss were identified, including previous dieting and recent weight changes, self-motivation, weight outcome evaluations, body size dissatisfaction, weight-related quality of life, self-esteem, and exercise self-efficacy and perceived barriers. Because this earlier study was primarily hypothesis-generating, confirmatory results are needed. The goal of the present study was to re-evaluate the predictive value of several of these variables in a different sample of women who underwent a comparable weight reduction program. While our previous work has studied women in the United States (US), the present analysis reports on a group of similarly-overweight/obese Portuguese females. Cross-cultural differences in social norms regarding ideal weights, in the role of physical activity, and in eating habits and relationship with food (e.g. [11]) could have an impact on how individuals respond to obesity therapies and also inform researchers about the role of pretreatment variables (moderators) in treatment success. It should be noted that this study was not designed to evaluate the overall effectiveness of the weight loss program but to analyze predictors of short-term results among participants who displayed highly variable levels of success. Methods Subjects Subjects were recruited from the community for a 2-year weight management program through newspaper ads, a website, email messages on listservs, and announcement flyers. Subjects were required to be older than 24 years, be premenopausal and not currently pregnant, have a BMI higher than 24.9 kg/m2, and be free from major disease to be eligible for the study. After several orientation sessions, 152 women signed up for the program. During the run-in phase, four women decided not to participate (reporting new time and scheduling conflicts), four did not comply with testing requirements and were excluded, three women found out they were pregnant or decided to attempt pregnancy and were also excluded, and one subject was found ineligible due to medical reasons (untreated hyperthyroidism), leaving a total of 140 women who started the intervention. An initial visit with the study physician ensured that subjects met all medical inclusion criteria. All participants agreed to refrain from participating in any other weight loss program and gave written informed consent prior to participation in the study. The Faculty of Human Movement's Human Subjects Institutional Review Board approved the study. Assessments Weight was measured twice, to the nearest 0.1 kg (average was used) using an electronic scale (SECA model 770, Hamburg, Germany) and height was also measured twice, to the nearest 0.1 cm (average was used). Body mass index (BMI) in kilograms per squared meter was calculated from weight (kg) and height (m). In addition to weight and other morphological and physiological variables assessed, subjects filled out a large psychosocial questionnaire battery prior to the first weekly treatment session. This was conducted in standardized conditions of comfort and silence, with a study technician attending every assessment period. To ensure optimal levels of concentration and avoid overburden caused by long periods of psychometric testing, subjects were required to attend three sessions, each lasting approximately 45 minutes. Portuguese versions of the Impact of Weight on Quality of Life – Lite (IWQOL-Lite, [12]), Self-Motivation Inventory (SMI, [13]), Rosenberg's Self-esteem/Self-concept (RSE, [14]), Exercise Perceived Barriers (EPB, [15]), and Exercise Self-efficacy (ESE, [16]) questionnaires were used. Details of the original English versions of these instruments are described elsewhere [8]. In brief, the IWQOL-Lite measures weight-specific perceived quality of life on five dimensions of daily life (physical functioning, self-esteem, sexual life, public distress, and work) and it also provides a summary score, which was used in this study. The SMI evaluates a general (i.e., context-unspecific) tendency to persevere, finish tasks initiated, maintain self-discipline, and motivate oneself. The RSE measures a person's self-respect and positive self-opinion. The EPB assesses the extent to which the elements of time, effort, and other obstacles are perceived barriers to habitual physical activity. The ESE measures an individual's belief or conviction that she can "stick with" an exercise program for at least 6 months under varying circumstances, in the dimensions of making time for exercise and resisting relapse. Summary scores for both the EPB and ESE were calculated and used in this study. For all instruments, higher scores indicate higher values for the constructs being measured. Forward and backward translations between English and Portuguese were performed for all questionnaires cited above. Two bilingual Portuguese researchers subsequently reviewed the translated Portuguese versions and minor adjustments were made to improve grammar and readability. In this study, Cronbach's alpha estimates were as follows, for the IWQOL-Lite (0.95, 31 items), SMI (0.88, 40 items), RSE (0.81, 10 items), EPB (0.71, 11 items), and ESE (0.77, 10 items), ensuring acceptable to high internal consistency. Number of previous diets and weight history variables were taken from a diet/weight history questionnaire developed specifically for this study. Weight outcome evaluations were assessed by 4 questions derived from the Goals and Relative Weights Questionnaire (GRWQ, [17]). Subjects were asked to indicate their "dream" weight, and also what would be their "happy", "acceptable", and "disappointing" weights by the end of the 4-month intervention. Each outcome evaluation was computed as the percentage of pretreatment measured weight. Body size dissatisfaction was assessed by the difference between self and ideal body figures selected from a list of 9 female silhouettes of increasing size [18]. High scores (i.e., larger disparity between self and ideal figure) indicate greater body size dissatisfaction. For multiple-item questionnaires, if a subject failed to correctly fill out at least 75% of all items in a summary/global scale or at least 50% of items in a subscale, the corresponding score was not calculated. However, this did not automatically eliminate a subject from analyses, if other (valid) scores could be used for the same participant. Intervention Subjects attended 15 treatment sessions in groups of 32 to 35 women, for approximately 4 months. Average attendance to the treatment sessions was 83%. Sessions lasted 120 minutes and included educational content and practical application classroom exercises in the areas of physical activity and exercise, diet and eating behavior, and behavior modification [19]. Physical activity topics included learning the energy cost associated with typical activities, increasing daily walking and lifestyle physical activity, planning and implementing a structured exercise plan, setting appropriate goals, using logs and the pedometer for self-monitoring, and choosing the right type of exercise, among many others. Examples of covered nutrition topics are the caloric, fat, and fiber content, and the energy density of common foods, the role of breakfast and meal frequency for weight control, reducing portion size, strategies to reduce the diet's fat content, preventing binge and emotional eating, planning for special occasions, and reducing hunger by increasing meal satiety (e.g., increasing fiber content). Cognitive and behavior skills like self-monitoring, self-efficacy enhancement, dealing with lapses and relapses, enhancing body image, using contingency management strategies, and eliciting social support were also part of the curriculum. The intervention team included two Ph.D.- and six M.S.-level exercise physiologists and dietitians, and one behavioral psychologist. Subjects were instructed and motivated to make small but enduring reductions in caloric intake and to increase energy expenditure to induce a daily energy deficit of approximately 300 kcal. Although weight was monitored weekly, subjects were advised that long-term (i.e., after 1–2 years), not necessarily rapid weight reduction was the primary target. In the first session, participants were informed that reaching a minimum of 5% weight loss at 6 months was an appropriate goal in this program and were subsequently instructed to individually calculate the number of kg that corresponded to. Statistical Analysis Measures of central tendency, distribution, and normality were examined for all psychosocial variables at baseline and for weight at baseline and 4 months. Following intention-to-treat principles and to include psychosocial data from all starting subjects in statistical analysis, the Last Observation Carried Forward (LOCF) method was used for 5 subjects who dropped from the program and could not be reached for testing at 4 months (the five subjects dropped after sessions number 10, 11, 12 [two subjects], and 14); in these cases, the last measured weight, which was assessed weekly for each woman with the same scale as used in laboratory testing, was entered as their final weight. The limitations of this method notwithstanding [20], variations of the LOCF are commonly used in obesity longitudinal trials (e.g., [21]). The very small number of subjects for whom 4-month weight data were imputed, all of which were derived from weights measured late in the program, should result in relatively unbiased results [22]. Furthermore, since a trend toward weight regain is common upon subjects leaving treatment, assuming no further weight change after dropping out works against the study's primary hypotheses, providing additional protection from type I error. One subject was removed from analyses that included weight outcome evaluation variables since her values were markedly lower than values from the rest of the group (i.e., it was considered a data outlier). Rank-order correlation (Spearman's ρ) was used to estimate the linear relationship between predictors and weight change. All but one among independent variables assessed at baseline displayed a non-normal distribution, warranting the use of this non-parametric technique. The dependent measure was expressed as the difference between baseline and 4-month weight. An alternative way to express weight results is to calculate the "residualized" value for 4-month weight, after the effect of baseline weight is removed (i.e., regressed out in linear regression). This method protects against overcorrection of the post by the pre score when using a subtraction score, and also effectively and completely adjusts this new "change" score for the pretreatment weight value [23]. This variable was also used as a dependent variable in analyses. Quadratic terms were produced for the two weight outcome evaluation variables, to assess the curvilinear relationship between these measures and actual weight results. Multiple regression analysis was performed to assess the multivariate relationships between the independent variables and weight change. In this regression model, the selected predictors (variables which were significant or approached significance in the bivariate analysis) were forced into the model and the squared semi-partial correlation coefficient was calculated to quantify the unique contribution of each predictor to the variance in the dependent measure [23]. Considering the relatively small subject-parameter ratio (24:1) and in the absence of strong theoretical support for a hierarchical entering of predictors into the model, this a priori (forced) model is preferable to a stepwise model as it minimizes instability in the selection of variables into the model (and in parameter estimation) caused by potential sampling biases [24]. A distribution-based criterion was employed to form three equally numbered groups, split by the two tertiles of weight change. Means of independent variables for the three subgroups were compared by analysis of variance (ANOVA), followed by post-hoc comparisons (Tukey's Honestly Significant Difference test). Type I error was set at 0.05 for all tests. Statistical analyses were completed using the Statistical Package for the Social Sciences (SPSS), version 12.0. Results Weight loss data reported in the present study refer to the initial 4 months of a longer trial. After the 4-month phase, subjects were randomly assigned to three distinct long-term interventions. Figure 1 shows individual weight changes for all 140 participants who started the program. Attrition was very low (3.5%) and average weight change was -2.9 ± 3.2 kg (-3.0 ± 3.2 kg, if only the 135 completers are considered). The range for weight change was about 19 kg, a (large) level of individual variability providing an optimal setting to study correlates of weight loss. About 53% of participants lost more than 3.3% of their initial weight (roughly the equivalent of a 5% weight loss after 6 months, in red in Figure 1), thus generally meeting or surpassing the recommended weight loss goals. Eighteen percent of all women (in grey in Figure 1) did not lose, or gained weight after 4 months. Figure 1 Individual Weight Change After 4 Months. Red bars indicate subjects who lost more than 3.3% of their initial weight; grey bars indicate subjects who did not lose weight or who gained weight. Table 1 shows descriptive statistics for the independent variables and their association with weight change. Fewer previous diets, weight outcome evaluations, and to a lesser degree self-motivation and body image were positively associated with weight loss. When the significance level was adjusted for the number of variables being tested (Bonferroni adjustment, new significance set at 0.005), the number of previous diets and weight outcome evaluations remained significantly correlated with weight results. An additional weight history question, asking whether subjects had lost at least 5 kg in the previous 2 years, was not associated with weight loss at 4 months (t = 0.71, p = 0.480, comparing subjects responding "yes" and "no"). Two additional variables from the GRWQ were also analyzed. "Dream" weight (mean ± SD, 98.1 ± 3.9%) was unrelated to baseline-adjusted weight loss (ρ = 0.001, p = 0.98) while "disappointing" weight outcome (77.4 ± 7.8% of initial weight) was associated with baseline-adjusted weight loss (ρ = 0.27, p = 0.002). Time at current weight, obesity-specific quality of life, self-esteem, and exercise variables were not associated with weight results, before or after adjusting for baseline weight. Significant predictors in the bivariate analysis (Table 1) were entered into a multivariate regression model to predict weight change. Since "happy" and "acceptable" outcome evaluations were highly intercorrelated and represent similar constructs, they were averaged into a single variable for this analysis. All variables entered in the model explained independent shares of the variance in weight loss, before (not shown) and after the inclusion of baseline weight (Table 2). Each predictor caused a significant increase in the model's R2 with weight outcome evaluations explaining the single largest share of the dependent variable. The model accounted for about 24% of the variance in 4-month weight change. Table 1 Correlation Between Pretreatment Variables and Weight Change at 4 Months Weight Change Weight Change1 n ρ p ρ p Mean SD Min Max Number of diets in past year 130 0.26 0.002 0.26 0.003 1.2 1.7 0 8 Months at current weight 127 -0.13 0.139 -0.13 0.157 24.1 24.1 0 120 "Acceptable" weight loss (% initial) 134 0.33 <0.001 0.26 0.002 92.7 4.0 77.1 100.6 "Happy" weight loss (% initial) 135 0.27 0.001 0.21 0.015 89.0 4.9 74.9 99.1 Impact of weight on quality of life 138 0.02 0.837 -0.05 0.594 79.5 14.1 37.9 100.0 Self-motivation 135 -0.19 0.030 -0.18 0.036 141.3 17.9 100.0 183.0 Body size dissatisfaction 134 0.09 0.280 0.18 0.038 2.29 0.88 0 5 Self-esteem 131 0.00 0.970 -0.01 0.930 32.4 3.77 24 40 Exercise perceived barriers 138 0.08 0.364 0.08 0.359 29.8 6.29 12 43 Exercise self-efficacy 138 -0.03 0.721 -0.03 0.750 38.3 4.78 25 49 Higher scores indicate higher value for characteristic tested (e.g. higher quality of life, higher self-motivation, higher body size dissatisfaction, more perceived barriers, etc.); Since weight change was coded as baseline weight subtracted to 4-month weight, weight loss is represented by a negative value (thus, a negative correlation coefficient indicates a positive correlation with weight loss). 1Four-month weight adjusted for baseline weight. Table 2 Multiple Regression Analysis for 4-month Changes in Weight B t p Squared semi-partial correlation (%) Baseline weight -.069 -2.481 0.015 4.0 Number of diets in past year .372 2.439 0.016 3.8 Weight outcome evaluations1 .235 3.673 <0.001 8.7 Self-motivation -.040 -2.714 0.008 4.7 Body size dissatisfaction .755 2.389 0.018 3.7 R2(×100) = 24.0 (adjusted R2(×100) = 20.5), SEE = 2.80 kg, F(df,123) = 7.84 (p < 0.001); 1Average of "happy" and "acceptable" weight outcome evaluations. Weight outcome evaluations were computed as a percentage of participants' initial weight. Thus, the lower this percentage, the more stringent (i.e., more demanding) was a subject's evaluation of her results, and vice-versa. We found significant and positive linear relationships between outcome evaluations and weight loss (Tables 1 and 2), indicating that the more demanding the evaluations of outcomes were at baseline (i.e., the lower the percentage of initial weight), the more weight was later lost (and vice-versa, i.e., the more accepting the evaluation of future weight loss, the less weight subjects lost). However, a visual inspection of these associations suggested that participants on the lower end of the outcome evaluation distribution might not be following the overall group trend. In fact, an additional analysis revealed that, for the whole group, a curvilinear pattern of association described the relationship slightly better than a linear pattern, for both "happy" and "acceptable" outcome evaluations and for the average of the two variables (Figure 2). Quadratic (squared) terms were tested in regression models, following procedures described by Cohen and Cohen [23], and were shown to produce small but significant increases in R2, in addition to the non-transformed, linear variables alone. Both linear and curvilinear relationships are depicted in Figure 2. To account for skewness in the weight outcomes data, regression analyses were repeated with the top and bottom 5% of observed values removed from analysis, yielding very similar results (y = 503.9 - 11.5x + 0.065 x2; R2 change for x2 = 0.05, p = 0.010). Figure 2 Relationship Between Weight Outcome Evaluations and Weight Loss. Dashed line shows curvilinear (quadratic term) and solid line shows linear relationship between weight outcomes evaluations (average of "happy" and "acceptable" values) and weight loss (% of initial). Regression equation includes both linear and quadratic terms and R2 change refers to the addition of the quadratic term into the model, after the linear term was already in the model. To further explore the association of the selected predictors with weight outcomes, subjects were divided into three groups based on tertiles of weight reduction adjusted for initial weight, and baseline psychosocial measures were compared among groups. Significant overall (ANOVA) differences emerged for the number of previous diets and self-motivation, with post-hoc comparisons showing significant mean differences only between the most and least successful groups (Figure 3). Considering the slightly curvilinear relationships observed for the GRWQ variables, it was not surprising that significant differences were not detected between success groups for "happy" (p = 0.284) and "acceptable" (p = 0.145) weight loss evaluations. Body size dissatisfaction scores were also not different among the three groups (p = 0.432). Table 3 shows the frequency of previous diets reported by each success group in more detail. Of all subjects reporting no diets initiated in the previous year, only 17% finished in the least successful groups. Conversely, of the 20 subjects reporting 3 or more recent diets, only 3 (15%) finished within the most successful group. Ten women reported having initiated 4 to 8 diets in the previous year, none of whom finished the 4-month program in the group of women losing the most weight. Figure 3 Comparison of Success Groups for Previous Dieting and Pretreatment Self-motivation. Groups based on tertiles for 4-month weight loss. F for ANOVA. Error bars show 95th confidence interval. Different letters indicate significant group differences in post-hoc analysis (p < 0.05). Table 3 Frequency of Diets Initiated in the Previous Year, by Weight Loss Success Group1 Most Successful Intermediate Least Successful Number of diets Freq. % Cum.% Freq. % Cum.% Freq. % Cum.% 0 25 58 58 24 52 52 10 25 25 1 7 16 74 10 22 74 14 34 59 2 8 19 93 6 13 87 6 15 73 3+ 3 7 100 6 13 100 11 27 100 Weight loss (kg) Mean -6.3 -2.7 0.3 SD 2.1 1.0 1.7 1Groups defined based on tertiles of 4-month weight loss adjusted for baseline weight. Discussion This study aimed at reexamining the association between several pretreatment individual characteristics and success in short-term behavioral weight reduction, in overweight and moderately obese women. Ten variables which had previously been shown to predict weight change [8] were analyzed in a separate sample, using a comparable research methodology. Previous dieting, self-motivation, and body image showed significant effects as predictors and in the expected direction of relationship. Participants' evaluations about possible weight outcomes were also significantly associated with weight loss in the present study, although in a direction opposite than what was hypothesized; more stringent evaluations of outcomes had predicted worse outcomes in US women [8] while the reverse was observed in Portuguese women for whom more accepting attitudes towards weight loss were associated with smaller weight changes. Earlier results for exercise, quality of life, self-esteem, and also for some variables related to weight history (time at current weight and large recent weight losses), were not confirmed in the present study. To date, the majority of research on the treatment of overweight and obesity has focused on assessing overall treatment efficacy (expressed as mean group weight change, number of individuals reaching some marker of success, etc.) and analyzing which programs work best, typically using randomly-assigned experimental treatment groups [25-27]. By contrast, much less research has been undertaken to investigate the mechanisms (mediating variables) by which treatments affect subjects, and for whom treatments are most effective (i.e., individual moderators). The potential benefits of studying moderators and mediators of outcomes within the behavioral and social sciences, including for physical activity, diet, and weight control are well described in the literature [28-30]. The identification of such variables open the way to a new generation of interventions, characterized by a higher level of individualization and overall efficacy, both by targeting those individuals more likely to succeed and through an increased focus on those mediators (treatment-related, environmental, and individual factors, and critical interactions among them) more clearly associated with outcomes [7]. Nevertheless, empirically-derived hypotheses for the role of moderators and mediators in the treatment of obesity remain scant, particularly for psychosocial variables. As a contrasting example, sufficient evidence was already available in the alcohol prevention field in the early 1990's for a large multi-center trial to be funded and carried out, aimed at testing the interaction between treatment modality and a considerable number of individual predictors/moderators such as cognitive impairment, conceptual level, motivation, social support, and patient typology [31]. In the present study and in other trials [32-36], previous dieting and weight loss attempts have emerged as reliable negative predictors of weight loss. One explanation is that the subset of women reporting more frequent dieting contains a disproportionally high number of individuals who are, for some reason, more resistant to weight control. Despite evidence showing that many individuals are successful even after many previous failed attempts [37,38], it is possible that some subjects in research-based obesity treatment programs see those programs as just one more among many solutions they have tried and failed at before, and thus are more prone to low self-confidence and impaired motivation. Frequent restriction of eating, implied in the question "how many diets have you started...?", could also be a marker for more extreme dieting behaviors that may not be sustainable after the initial boost of motivation [39]. This could also increase the probability for weight rebound. More studies are needed to investigate the mechanisms through which previous dieting affects weight control, a consisting finding in the literature. The present report also provides indication that a threshold may exist (3–4 number of diets in the previous year) which is associated with a marked reduction in the likelihood of success. Four earlier reports have examined the role of self-motivation as a predictor of weight loss [8,36,40,41] while one additional study used a general self-efficacy questionnaire worded similarly to the SMI [42]. The related construct of autonomy-oriented motivation (defined as a motivation style more related to a persons' own interests and values and less controlled by external events) has also been evaluated as a predictor [43]. With one exception [41], evidence has supported the notion that high pretreatment levels of self-motivation and an autonomy-oriented motivation are beneficial traits for subsequent weight loss. The SMI has also been shown to correlate with eating variables during weight loss [44] and to predict exercise behavior [13]. Contrary to earlier observations in US women [8,36], exercise-related variables did not predict weight loss in the present analysis. That is, while the more general personality attributes related to motivation and efficacy were stable predictors of outcomes in weight loss across studies, the moderating role of exercise self-efficacy and exercise perceived barriers (time, effort, etc.) did not translate well from the US to the Portuguese data set. Cross-national differences such as distinct levels of social awareness for exercise or differences in level of knowledge, past adoption levels, and/or perceived competence regarding exercise and physical activities, all of which may have influenced answers to the exercise questionnaires, are possible explanations for these differences. This study is among only a few that have analyzed associations between the Goals and Relative Weights Questionnaire and subsequent weight loss. Interestingly, marked differences emerged between the present and two previous analyses [8,36]. Portuguese women with more modest weight outcome evaluations were less likely to lose weight, while in US women the opposite was observed, that is, more stringent (demanding) evaluations of possible results were predictive of poorer results. Evidence for a significant effect of outcome expectancies on weight control is extremely relevant in the context of realistic versus unrealistic expectations for weight loss [45-47]. Excessively optimistic expectations are common in US treatment-seeking obese samples [17], for whom a great value is typically placed on reaching desired weights [48]. By contrast, Portuguese women, perhaps because their are comparatively less exposed to external pressures to be thin and/or because they belong to a culture where optimism is less valued than in the US, were less likely to produce very demanding weight-related evaluations. Accordingly, we have recently reported that Portuguese women do, on average, state overall less stringent evaluations of weight loss outcomes at baseline than their American counterparts [49]. This being the case, one hypothesis for the divergent associations for US and Portuguese samples is that, when a broad population is considered, the expectations-outcomes relationship is indeed curvilinear (with an yet-undetermined nadir or interval representing the more favorable goals/expectations) and that Portuguese women predominantly fall on the right (more conservative) side of the distribution while US subjects better represent the left side (more stringent). In the present study, it appeared that the weights participants would find acceptable/happy were associated with weight loss (i.e., more "optimistic" outcome evaluations, more weight loss) until a certain threshold was reached, somewhere around 85 to 90% of initial weight (10–15% weight loss); for women reporting outcome evaluations below that level no further benefit was apparent. One previous study has shown that women with more modest absolute weight loss goals were more likely to achieve their goals, and that those who achieved their weight goals had better weight maintenance after 2.5 years; however, desired weight loss did not directly predict actual weight loss [50]. Positive expectations expressed as a higher reported likelihood of reaching goal weight predicted larger short-term weight loss in subjects who showed lower level of fantasizing and daydreaming about beneficial consequences of large weight loss [51]. Other studies have shown larger weight loss goals to positively predict weight loss [41,52] and in one other case goals had small predictive value [53]. Collectively, previous results and those we now report suggest that positive and moderate expectations/outcome evaluations foretell the best overall results, particularly if accompanied by a high sense of self-assurance [52]. It should be noted that variables originating from the GRWQ are closely related but are not equivalent to the construct of outcome expectancies (the belief that certain actions will lead to the projected results [54]) or to weight loss goals. The GRWQ seems to partially measure an actual prediction of outcomes by the participant, similar to a general self-efficacy expectation (e.g., how much weight do you think you will lose by the end of this program?), while simultaneously tapping into a more attitudinal facet towards a person's weight and weight loss (how happy/accepting/disappointed would you feel at certain levels of weight loss?). To some extent, the latter could measure idealization of body weight and perceived importance of body weight and shape for self-esteem and well-being. Therefore, it is possible that moderate or "realistic" weight outcome evaluations (i.e. not too accepting but also not excessively stringent) are the most beneficial and indeed reflect a good balance between a sufficient and necessary sense of self-efficacy and low to moderate levels of thin-ideal internalization, a variable which has been shown to be a positive risk factor for body dissatisfaction, negative affect, and eating disorders [55,56]. Women reporting a larger discrepancy between self and ideal body figures, which indicates a higher body size dissatisfaction [18], were less likely to lose weight. In a previous report, the same self-ideal measure correlated similarly with short-term results, while two other measures of body image showed comparable, albeit non-significant trends [8]. Pretreatment scores in the body dissatisfaction scale of the Eating Disorders Inventory, a measure of psychological concern and dislike about one's body shape and size [57], has also been negatively associated with weight loss in two other behavioral weight loss programs [58,59]. These relationships may be explained by the negative association of body image with mood and psychological impairment [60], and also by the disappointment and lack of self-worth and self-confidence following previous failed attempts to change weight and body shape [6]. Although self-esteem did not predict outcomes, we observed significant correlations between body size dissatisfaction and self-esteem (ρ = -0.18, p = 0.042), the number of previous diets (ρ = 0.22, p = 0.013), and weight-related quality of life (ρ = -0.37, p < 0.001). Rapid and concurrent improvements in body image and eating behavior (e.g., reduction in binge episodes) have been observed after surgery-induced thinning [61], clearly suggesting a close link between attitudes towards one's body and weight control behaviors. Body image therapy has also been shown to reduce concern with food, in the context of a behavioral weight control trial [62]. Despite the sound theoretical rationale and supportive body of evidence, a note of caution must be made regarding the multidimensionality of the body image construct [63] and the proliferation of assessment instruments for body image. Although they are typically intercorrelated [60], different body image scales should be interpreted separately as they may result in different patterns of association with weight loss [8,58]. Strengths of this study are the a priori selection of variables to be analyzed as predictors, a unique population (Portuguese women), and the very low dropout rate. Limitations include a moderately-sized sample considering the known measurement error associated with questionnaire psychological assessments, the fact that some of the scales used still lack well-established validity, and the absence of a control or comparison group. Conclusions Several pretreatment variables were re-evaluated as predictors of short-term weight loss in women. Previous dieting, low self-motivation, and body size dissatisfaction were confirmed as negative predictors of weight outcomes, while the relationship of outcome evaluations with weight reduction suggested a negative and curvilinear pattern, with positive but not excessively demanding evaluations presaging the best results. These data regarding people's outcome evaluations prior to weight loss may have important clinical implications [64] and are the first evidence for such a pattern of association; thus, they await replication in other samples. Additionally, treatment decisions based on level of previous dieting (alone or included in comprehensive prediction models) may be possible in the near future, at least for overweight and moderately obese women. The more consistent predictors from this and previous studies (e.g., [8,42,59]) can and should be used in future hypothesis-testing studies of moderators of weight loss. Finally, this study highlights the fact that behavioral and psychological prediction models may, to some extent, be specific to a particular culture [65]. Hence, it is likely that some variables will emerge as moderators (and mediators) of obesity treatment in some, but not all cultures, while others will be proven as more universal correlates of success. Competing Interests None declared. Authors' Contributions PJT conceived the study, led the implementation team, performed most statistical analysis, and drafted the manuscript. ALP participated in the study's implementation, in statistical analysis, and was responsible for all psychometric assessments. TLB, SSM, CSM, and JTB were actively involved in the study's implementation and in data collection. AMS participated in the study's implementation and collected all body habitus data. LBS is the principal investigator in the research trial and contributed to the final version of the manuscript. All authors read and approved the final manuscript. Acknowledgments This study was funded by the Portuguese Science and Technology Foundation and by the Oeiras City Council. The investigators are grateful to Roche Pharmaceuticals Portugal, Becel Portugal, and Compal Portugal for small grants and donations, which contributed to the study's success. 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Int J Behav Nutr Phys Act

10.1186/1479-5868-1-12

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==== Front BMC NeurosciBMC Neuroscience1471-2202BioMed Central London 1471-2202-5-251528203410.1186/1471-2202-5-25Research ArticleBiochemical enrichment and biophysical characterization of a taste receptor for L-arginine from the catfish, Ictalurus puntatus Grosvenor William [email protected] Yuri [email protected] Andrew I [email protected] Douglas L [email protected] D Lynn [email protected] John H [email protected] Joseph G [email protected] Monell Chemical Senses Center, Philadelphia, PA 19104-3308, USA2 Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russia3 New York University College of Dentistry, New York, NY 10010, USA4 Institute of Neurological Sciences, University of Pennsylvania, Philadelphia, PA 19104, USA5 Veterans Affairs Medical Center, Philadelphia, PA 19104, USA6 Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA7 Current Address: Department of Pathology, Anatomy & Cell Biology; Thomas Jefferson University; Philadelphia, PA 19107-6799, USA8 Current Address: UCSD Thornton Hospital, San Diego, CA 92037, USA2004 28 7 2004 5 25 25 24 2 2004 28 7 2004 Copyright © 2004 Grosvenor et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The channel catfish, Ictalurus punctatus, is invested with a high density of cutaneous taste receptors, particularly on the barbel appendages. Many of these receptors are sensitive to selected amino acids, one of these being a receptor for L-arginine (L-Arg). Previous neurophysiological and biophysical studies suggested that this taste receptor is coupled directly to a cation channel and behaves as a ligand-gated ion channel receptor (LGICR). Earlier studies demonstrated that two lectins, Ricinus communis agglutinin I (RCA-I) and Phaseolus vulgaris Erythroagglutinin (PHA-E), inhibited the binding of L-Arg to its presumed receptor sites, and that PHA-E inhibited the L-Arg-stimulated ion conductance of barbel membranes reconstituted into lipid bilayers. Results Both PHA-E and RCA-I almost exclusively labeled an 82–84 kDa protein band of an SDS-PAGE of solubilized barbel taste epithelial membranes. Further, both rhodamine-conjugated RCA-I and polyclonal antibodies raised to the 82–84 kDa electroeluted peptides labeled the apical region of catfish taste buds. Because of the specificity shown by RCA-I, lectin affinity was chosen as the first of a three-step procedure designed to enrich the presumed LGICR for L-Arg. Purified and CHAPS-solubilized taste epithelial membrane proteins were subjected successively to (1), lectin (RCA-I) affinity; (2), gel filtration (Sephacryl S-300HR); and (3), ion exchange chromatography. All fractions from each chromatography step were evaluated for L-Arg-induced ion channel activity by reconstituting each fraction into a lipid bilayer. Active fractions demonstrated L-Arg-induced channel activity that was inhibited by D-arginine (D-Arg) with kinetics nearly identical to those reported earlier for L-Arg-stimulated ion channels of native barbel membranes reconstituted into lipid bilayers. After the final enrichment step, SDS-PAGE of the active ion channel protein fraction revealed a single band at 82–84 kDa which may be interpreted as a component of a multimeric receptor/channel complex. Conclusions The data are consistent with the supposition that the L-Arg receptor is a LGICR. This taste receptor remains active during biochemical enrichment procedures. This is the first report of enrichment of an active LGICR from the taste system of vertebrata. Chemical sensesTasteSignal transductionLectinIon channelReceptorImmunohistochemistryProtein purificationLipid bilayer ==== Body Background The initial event in taste transduction involves recognition of taste stimuli by plasma membrane-associated receptor proteins. These proteins are concentrated at the apical end of specialized neuro-epithelial cells (taste cells) found within multicellular end-organs known as taste buds [1,2]. The recognition binding sites for most taste stimuli face the exterior environment. The interaction of a taste stimulus with this recognition site triggers a chain of metabolic and ionic events in the taste cell, leading to alterations in membrane conductance, release of neurotransmitter, and a change in the firing rate of the afferent sensory nerve fibers with which taste cells synapse [2]. Receptor recognition is, therefore, largely responsible for maintaining the specificity of the taste transduction process. To date, 7-transmembrane G protein coupled receptors (7TM-GPCR's) for three taste modalities have been identified by both molecular cloning and through searches of the human and mouse genome. Sweet taste stimuli appear to be recognized by at least one heterodimer (T1R2/T1R3) of the three member family of 7TM-GPCR's, the T1R's [3-7]. The taste receptors for sweetness are coupled to changes in intracellular levels of either cyclic nucleotides or polyphosphoinositols [5,8-10]. Two GPCR receptor types have been implicated in the basic taste of umami (glutamate taste). One is the heterodimer of T1R1/T1R3, of the same 7TM-GPCR family as the sweet taste receptor dimer [11]. Another GPCR umami receptor is an N-terminal truncated metabotropic-type 4 glutamate receptor (taste/mGluR4) presumably coupled to an inhibition of adenylyl cyclase [12]. A third proposed, non-GPCR umami receptor is an NMDA-type ionotropic glutamate receptor [13]. Finally, a family (~40 members) of 7TM-GPCR's recognizes many bitter taste stimuli [14,15]. These bitter taste receptors are coupled through a gustducin-containing G protein [16] to changes in intracellular levels of cyclic nucleotides and polyphosphoinositide metabolites [17-19]. While these recent discoveries have markedly improved the understanding of taste transduction, it is apparent from neurophysiological, biophysical and biochemical studies that receptors and transduction processes other than the GPCR/second messenger systems are utilized by the sense of taste [2,20]. For example, several taste transduction processes make use of ion channels as the receptor recognition step [21]. Salty taste is likely transduced by an epithelial sodium channel (ENaC), and sour taste may also make use of channels such as acid sensing ion channels (ASICs) [22] and the hyperpolarization-activated, cyclic nucleotide-gated cation channel (HCN) (reviewed by [2]). Certain stimuli, such as quinine and perhaps denatonium co-opt potassium channels to alter membrane conductance of taste receptor cells [23-25]. Finally, in a variety of species, ligand-gated ion channels have been implicated as taste receptors for a number of stimuli, including sugars in the dog [26], glutamate in mouse [13,27], nicotinamide in crayfish [28], sugars and amino acids in fleshfly [29], bitter compounds in frog [30], and apparently for amino acids in the channel catfish, Ictalurus punctatus [31,32]. Little is known about the structure and function of these ligand-gated ion channel receptors (LGICR) in the taste system nor the extent to which they serve as taste receptors in other species. To evaluate the role of LGICRs in taste transduction, receptors of this class need to be identified and fully described. To date, a well characterized example of a likely LGICR class of taste receptors is found on the common channel catfish, I. punctatus. The catfish is an advantageous model system for studying taste transduction [33] because it possesses a large number of densely arrayed taste buds across its body surface, particularly on its barbel appendages and gill rakers [33-36], and shows high specificity and sensitivity to selected amino acids. Several taste transduction pathways for amino acids have been identified both biochemically and neurophysiologically, including those recognizing (1) L-alanine and other small neutral amino acids, (2) L-proline, and (3) L-arginine (L-Arg) [33,37-40]. Of these three receptor systems, the one tuned to the amino acid, L-Arg, appears to be of particular high specificity and affinity [38,41]. Calcium imaging studies on isolated catfish taste receptor cells suggest the presence of at least two subtypes of L-Arg-stimulated transduction pathways. In one, L-Arg induces a change in intracellular calcium that is independent of extracellular calcium activity. In the other, L-Arg induces an increase in intracellular calcium that is dependent upon extracellular calcium. This second type of L-Arg induced response is blocked by D-Arg, whereas the first type of L-Arg induced response is less sensitive to the D-isomer [42,43]. Intracellular and patch clamp studies on catfish taste cells are also consistent with there being two subtypes of responses to L-Arg [42]. The L-Arg induced increase in intracellular calcium independent of extracellular calcium and not blocked by D-Arg may utilize a mechanism such as a GPCR-polyphosphoinositol linked pathway with IP3 releasing calcium from intracellular stores. The other L-Arg-stimulated pathway, showing intracellular changes in calcium dependent upon extracellular calcium and blocked by D-Arg, is consistent with an LGICR mechanism. The LGICR mechanism was given additional credence through studies demonstrating that membranes from barbel epithelium (that contains taste buds), when reconstituted into lipid bilayers, show L-Arg-stimulated ion channel activity inhibited by D-Arg [31-33]. Using membrane homogenates from catfish barbel epithelium, biochemical binding studies revealed high affinity sites for L-Arg that were inhibited by D-Arg, L-arginine methyl ester, and to a lesser extent by L-lysine and L-α-amino-β-guanidino propionic acid [38]. Previous studies had also demonstrated that both of the lectins, Ricinus communis agglutinin I (RCA-I) and Phaseolus vulgaris Erythroagglutinin (PHA-E), inhibited the binding of L-Arg to its presumed receptor sites, and both lectins labelled identical SDS-PAGE-separated bands of a barbel membrane preparation [44]. L-Arg-stimulated ion channel activity of solubilized barbel (taste) membranes reconstituted into lipid bilayers was inhibited by both PHA-E [35] and RCA-I (Teeter & Brand, unpublished observations). In addition, PHA-E also labelled the apical membrane of selected taste bud cells and solitary chemoreceptor cells of catfish barbel [35]. When reacted against the exterior epithelium of fixed, unpermeabilized catfish barbel, polyclonal antibodies developed against these lectin reactive peptides immuno-labelled the apical membrane of a subset of barbel taste receptor cells [45]. While these prior studies are consistent with the hypothesis that one of the receptors for L-Arg is an LGICR, both the neurophysiological studies and the reconstitution experiments could not definitively test this hypothesis. Without actually isolating an LGICR for L-Arg, it remains possible that a GPCR moiety for L-Arg could conceivably be tightly coupled to a separate ion channel. The purpose of the studies reported here was to investigate the possibility that an LGICR exists for L-Arg, by: 1. biochemically enriching this putative LGICR to near homogeneity, and 2. biophysically characterizing the L-Arg-stimulated ion channel activity of the presumed LGICR at each step of enrichment to • demonstrate that the same LGICR is being enriched with each step, and • demonstrate that the ion channel properties and kinetics of the enriched LGICR are similar to those of the presumed LGICR in situ. This enrichment and characterization are necessary steps towards eventually cloning this putative LGICR. The data are consistent with the interpretation that a receptor for L-Arg can be solubilized in an active state and can be enriched to a point where, upon denaturation and SDS-PAGE, material containing receptor-like activity elutes as a single band. Results Lectin specificity: lectin blots and lectin histochemistry An SDS-PAGE of a detergent-solubilized epithelial membrane fraction from barbel of I. punctatus, called "Sp" (defined in methods), revealed numerous proteins labelled by silver stain (Fig. 1, Lane "Sp"). Yet the lectins, PHA-E and RCA-I, both clearly labelled only one major glycoprotein band, in the range of 82 – 84 kDa (Fig. 1, Lane "PHA" and Lane "RCA"). A few other protein bands were more lightly labelled by these two lectins, including ones near 88 – 90 kDa and ~120 kDa. This recognition specificity is notable because previous work had shown that both of these lectins inhibited binding of L-Arg to a membrane suspension of barbel epithelium with respective specificity confirmed by control studies using the hapten sugar [44]. Other lectins that did not inhibit L-Arg binding labelled other glycoproteins of barbel epithelium [44]. Previous studies showed that the lectin, PHA-E, labelled primarily exterior-facing (presumably) glycoprotein motifs of the taste buds of catfish barbel [35]. However, no comparable labelling studies were carried out using RCA-I. Since RCA-I was used in this current study as an affinity reagent, it was important to establish the labelling specificity of RCA-I – conjugated lectin to catfish barbel taste buds. Figure 2A shows that RCA-I labels primarily the taste buds on the surface of the barbel, with Figure 2B showing labelling of two taste buds in a transverse section at higher magnification. Figure 2C demonstrates that the lectin labelled primarily the apical region of the taste bud. Some of the spotty labelling scattered among the taste bud field (Fig. 2A) may due to the RCA-I recognizing an epitope on solitary chemoreceptor cells (SCC). The SCCs are a dispersed chemoreception system found in aquatic vertebrates, and are possibly related to the taste system [35,46,47]. Apparent labelling of the catfish SCC was reported with PHA-E as well [35]. The specificity shown by these lectins in binding inhibition, ion channel conductance inhibition, lectin blots, and lectin histochemistry, all give credence to use of lectin affinity chromatography as the first step in enrichment of L-Arg-stimulated channel activity. Enrichment of L-Arg-stimulated ion channel activity Step 1 – lectin chromatography Since the lectins, RCA-I and PHA-E, inhibited the binding of L-Arg but not of L-alanine [44], the assumption was made that an L-arginine receptor (L-ArgR), or a fragment thereof, was among the glycoproteins labelled by these two lectins, and lectin affinity chromatography was, therefore, chosen as the first step in enrichment of this L-ArgR. Verification of the presence of active putative L-ArgR ion channel in each eluted fraction was assessed by a bilayers – incorporation assay. This assay proved to be more readily performed and provided more reproducible results than soluble binding assays that yielded high non-specific binding and required much more material. A quantitative protein assessment of the eluted materials from the RCA-I column indicated that over 99% of the protein from fraction Sp passed through the column unbound. Only occasionally did this protein material contain minimal L-Arg-stimulated ion channel activity. In contrast, the remaining protein eluted from the affinity column by galactose and reconstituted into lipid bilayers (See below) consistently contained ion channels activated by L-Arg and inhibited by D-Arg. Silver staining of SDS-PAGE separated proteins before (Fig. 3A) and after (Fig. 3B) the RCA affinity enrichment step revealed numerous proteins from total Sp (Fig. 3A) with fewer and more heavily stained bands of protein present in galactose-eluted fractions (Fig. 3B). These included primarily protein of molecular weight ~82–84 kDa, with apparently lower abundance proteins near 115–120 kDa, 60–70 kDa, and 40–45 kDa (Fig. 3B). The 82–84 kDa band matched the general position of the principal protein labelled in the lectin blots (Fig. 1). The protein eluting at 115–120 kDa in Figure 3B may correspond to the lightly labeled glycoproteins at ~120 kDa seen in the lectin blots of Figure 1. Protein of molecular weight 60 – 70 kDa and 40 – 45 kDa observed in the galactose-eluted protein fraction of Figure 3B have no apparent match in the lectin blots of Figure 1. These may, possibly, be degradation products of the other protein fractions that are labelled by the lectins or they may be proteins of low abundance, visible here due to the enrichment of protein resulting from the affinity procedure. On the assumption that the 82–84 kDa protein was at least a subunit of this L-ArgR, the SDS-PAGE band at 82–84 kDa was electroeluted and used to develop polyclonal antibodies from three guinea pigs (See Methods). The affinity purified and pre-treated polyclonal antibodies from two of these guinea pigs proved most specific and these were labelled, "GP1" and "GP2," respectively. In Western blots of fraction Sp (Fig 4A), both of the GP antibodies labelled a wide band between 74 and 84 kDa (containing its antigen) and occasionally a second higher molecular weight band near 110 kDa. In Western blots of RCA lectin-galactose eluted proteins, a narrower band of 82–84 kDa was labelled (Fig. 4B). Little GP labelling was seen in Western blots from SDS-PAGE of the protein not retained by the RCA lectin column, but those bands that were labelled were near 84 kDa and 110 kDa (data not shown). The GP polyclonal antibodies were used as a confirmatory marker of the 82–84 kDa peptide during subsequent enrichment steps. Both the GP1 and GP2 antibodies labelled the 82–84 kDa protein band in Western blots of an SDS solubilized partial membrane preparation from catfish barbel and both immuno-labelled taste cells of the catfish (see ahead). Step 2 – gel filtration The CHAPS-solubilized, dialyzed, non-denatured protein eluted from the galactose wash of the lectin column was applied to a Sephacryl S-300 HR column and eluted with Tris/CHAPS. Each protein-containing peak of the elution was assayed for L-Arg-stimulated channel activity and subjected to SDS-PAGE with silver staining and Western blotting against the GP1 antibody. Only protein from the first peak, in fractions 1 and 2, (eluting at an equivalent molecular weight of > 670 kDa.) contained L-Arg-stimulated ion channel activity (see ahead). Silver staining of material in these first two fractions run on SDS-PAGE revealed a prominent band at 82–84 kDa (Fig 3C). The broad band near 110–115 kDa seen after lectin chromatography (Fig. 3B) was not seen while protein at 60–70 kDa and 40–45 kDa remained. The corresponding Western blot (Fig. 4C) demonstrated that the antigen to which GP1 was developed was still present in the active fraction. Step 3 – ion exchange chromatography As a final enrichment step eluted material from fractions 1 and 2 of the Sephacryl column were lyophilised and resuspended into Start Buffer (see methods) and loaded on a Hitrap Q anion exchange column. Both the pH 9 and pH 8 elution resulted in protein being released from the column, with the majority of protein eluting at pH9. However, only the pH 9 fraction contained L-Arg-stimulated ion channel activity when reconstituted into lipid bilayers. A silver stain of an SDS-PAGE of the pH 9 eluent showed a deeply staining band at 82–84 kDa along with weak staining above 200 kDa and in the 35 kDa range (Fig 3D). There was also an almost complete loss of other stained bands observed in SDS-PAGE of protein from the previous enrichment steps (compare Fig. 3D with Figs. 3C and 3B). The corresponding Western blot of 0.2 μg of the pH 9 eluent showed very strong reactivity at 82–84 kDa (Fig. 4D). The SDS-PAGE of the pH 8 eluent showed a faint band at 82–84 kDa with other less intense bands at lower molecular weight. This pH 8 eluent may contain an inactive or partially denatured form of the L-ArgR. Immunohistochemistry of GP1 and GP2 on catfish barbel The GP antibodies were developed against the 82–84 kDa fraction of solubilized catfish barbel membranes, since it was a band of this molecular weight that was labelled by the lectins, PHA-E and RCA-I. While it is expected that GP1 and GP2 would label an 82–84 kDa band by Western blots, the fact that the GP1 and GP2 antibodies faithfully marked each enriched fraction that exhibited L-Arg-stimulated ion channel activity (see below) suggests that they are immuno labels of the receptor. As such, their localization within the barbel may be a marker for this presumed L-ArgR. Figure 5 shows GP1 and GP2 immuno labelling of paraformaldehyde fixed, sectioned catfish barbel. Figure 5A, a low power image of the barbel sectioned length-wise, demonstrates that it is primarily the taste buds that are labelled by GP1 (1/8000 dilution). Figure 5B shows labelling by GP2 (1/12000 dilution) of the apical area of three taste buds from a surface viewpoint. Figures 5C and 5D show taste bud labelling by GP1 (1/16000 dilution) and GP2 (1/8000 dilution), respectively of taste buds in tangential sections. Figure 5E shows a negative control where the primary antibodies were omitted. These immunohistochemical studies suggest that the antigen epitopes for GP1 and GP2 are concentrated at the apical portion of taste buds. Characterization of the RCA lectin-, Sephacryl S 300 gel-, and ion exchange-protein reconstituted into lipid bilayers LGICR enrichment was followed and verified by measuring the L-Arg-stimulated conductance of lipid bilayers (equimolar mixture of DOPS:DOPE) into which the protein fractions derived from each purification step were fused. Nearly identical L-Arg-stimulated single channel activity was observed from active fractions of all three steps: the galactose-eluted protein from the RCA lectin column, the protein of the first peak, fractions 1 & 2, of the material eluted from the Sephacryl S-300 column, and the protein of the pH 9 elution from the ion exchange column. The fact that consistent and nearly identical L-Arg-stimulated activity was observed with material from each subsequent enrichment step indicates that the enrichment procedures were sufficiently benign so as to permit the retention of LGICR-type activity and, presumably, native receptor conformation. General agonist/antagonist channel properties. Figure 6 illustrates single channel activities observed during the enrichment steps. Since active material from each of the three steps yielded almost identical channel properties (See Table 1), only that activity seen with fractions 1 and 2 (combined) off of the Sephacryl S-300 column is shown here. The data of Figure 6 are from the same experiment. In lipid bilayers into which active material had been fused, but in the absence of added L-Arg, no spontaneous channel activity was observed (Fig. 6top panel). Addition of 10 μM L-Arg to the cis side buffer solution induced appearance of ion channels (Fig. 6middle panel). L-Arg-stimulated channel activity in positive fractions from all columns was readily blocked by the addition of 100 μM D-Arg to the same side of the chamber wherein L-Arg had been added (Fig. 6bottom panel). (In control experiments with bilayers into which no protein was incorporated, neither L-Arg nor D-Arg alone (in a range 10 – 1000 μM) induced channel activity.) While 10 μM L-Arg was generally used in the screening and assay procedures, we estimate that the threshold for L-Arg-induced channel activity of the solubilized putative L-ArgR is about 1 μM L-Arg. In addition to inhibition by D-Arg, the lectins, RCA-I and PHA-E (not shown here, but see [35]), also inhibited the L-Arg-induced ion channel activity. In contrast, none of the antibodies developed to the denatured 82–84 kDa proteins inhibited L-Arg stimulated ion channel activity. Neither L-alanine nor L-proline (up to 200 μM) activated the ion channels stimulated by L-Arg. Amplitude histogram Figure 7 illustrates an all points amplitude histogram of L-Arg activated channels from material contained in fractions 1 & 2 from the Sephacryl column elution step measured at a fixed transmembrane potential of -100 mV. These data imply that there is one major peak of current amplitude (with some fluctuation) giving a unitary current of -6.6 pA. Similar unitary currents were measured for material from both the lectin column and the ion exchange procedure. Cation/anion selectivity & current/voltage relationship The cation-anion selectivity of L-Arg activated channels found in material contained in fractions 1 & 2 off the Sephacryl column was determined for Na+ and Cl-. A potential of zero current (reversal potential) was measured after formation of a 4-fold transmembrane concentration gradient of electrolyte (100 mM NaCl at cis side and 25 mM NaCl at trans side) across the bilayer containing a few ion channels. The average reversal potential (n = 5 bilayers, ± S.D.) elicited by voltage ramps (see Fig. 8) was -14 ± 3 mV. This value corresponds to weak cation selectivity (PNa/PCl = 2.2). The current-voltage relationships for L-Arg activated channels formed by active protein fractions from all three enrichment steps are illustrated in Figure 9. The data are well fit by a linear regression (r = 0.99) with slopes of 58, 67, and 73 pS for channels formed by protein from the RCA-I lectin column (open circles), protein from Sephacryl S-300 HR column (solid boxes) and protein from the pH 9 fraction of the ion-exchange column (triangles), respectively. From these current-voltage relationships it follows that the conductance of the channels from protein at any stage in the enrichment process are nearly identical, and that L-Arg activated ion channels are essentially potential-independent. Comparison with biophysical properties of native channels The electrophysiological properties of ion channels formed by the protein fractions throughout the enrichment scheme described here closely resemble those measured for the native channels [31] (see Table I). Both native channels and channels formed by proteins after these enrichment steps 1. are activated by L-Arg and inhibited by D-Arg over the same concentration ranges; 2. display nearly the same unitary conductance (The membrane-associated channels show two conductance states, one at 40 – 60 pS, the other at 75 – 100 pS (Table I and [31])); 3. are cation selective; and 4. are potential independent. Discussion Ligand-gated ion channel receptors (LGICR) may be used for selected stimuli of several taste modalities. In spite of their likely role in taste, little is known about these LGICRs [21]. The best characterised apparent taste LGICRs that recognise non-ionic stimuli are those of the catfish, I. punctatus. This animal possesses apparent LGICRs of low affinity for L-proline and of high affinity for L-Arg [31,33]. The observation that L-Arg acts as a stimulus for the taste system of the channel catfish was first reported by Caprio [48]. Subsequent neurophysiological and biochemical binding studies demonstrated that, unlike most other vertebrate taste receptors, the catfish taste receptor(s) for L-Arg is of both high specificity and high sensitivity [32,38,41]. Contemporaneous neurophysiological cross-adaptation and single unit studies indicated that L-Arg stimulates unique sites independent of those for other amino acids such as L-alanine or L-proline [49,50,40]. The receptor sites for L-Arg have narrow structural requirements, with only a few structural analogs of L-Arg acting as cross-adapting stimuli [41]. The receptor binding studies found a high affinity site for L-Arg, with Kd of 20–50 nM, and demonstrated inhibition of L-Arg binding by D-Arg, L-arginine methyl ester, and to a lesser extent by L-lysine and L-α-amino-β-guanidino propionic acid [38]. Other amino acids were without effect at reasonable levels. Interestingly, L-Arg and D-Arg are non-reciprocal cross adaptors, where neural adaptation to D-Arg eliminates responses to L-Arg, while adaptation to L-Arg still leaves some response to D-Arg [40]. This non-reciprocal cross-adaptation predicts the presence of a receptor site for D-Arg and suggests that any receptor for L-Arg – be it a GPCR or an LGICR – should be sensitive to D-Arg. The fact that no responses to D-Arg were ever observed in the bilayer experiments suggests that the major receptor for D-Arg is a GPCR. Consistent with these receptor specificities is the behavioural observation that at micromolar levels, L-Arg induces oropharyngeal motor behavior in I. punctatus [33,51,52] with D-arginine acting as a partial antagonist of this behavior [33]. More recently, whole cell patch clamp and calcium imaging of isolated catfish taste receptor cells, along with earlier in situ intracellular electrophysiological recordings, indicated that the majority of L-Arg-induced depolarizations are generated by inward currents [42,43]. As predicted by neurophysiological cross-adaptation studies [40] and consistent with biochemical binding experiments [38] the increases in intracellular Ca2+ activity observed in taste cells stimulated by L-Arg could be blocked by D-Arg [43]. Studies in our laboratory demonstrated that plasma membrane vesicles from barbel epithelium incorporated into lipid bilayers displayed L-Arg (μM)-stimulated ion channel activity that was inhibited by D-Arg [31,32,35]. No channel activity was observed toward L-alanine, but another, apparently less abundant, channel was stimulated by mM levels of L-proline, with the L-proline response being inhibited by D-proline. The L-Arg-stimulated responses were not inhibited by D-proline, nor were the L-proline responses inhibited by D-Arg [31]. The L-Arg-stimulated channels were found to be 50–80 pS in size, cation selective, but of low ion specificity. In contrast to the taste system, the olfactory system of the catfish transduces the stimulus, L-Arg and some other basic amino acids apparently through a GPCR [53] as does the olfactory system of goldfish [54]. In addition, L-Arg is an appetitive stimulus for the leech, Hirudo medicinalis, where the transduction process for L-Arg can be influenced by bitter stimuli, suggesting an integration at the receptor cell level [55] Preliminary reports on the localization of an L-ArgR showed that the antibodies, GP1, and the lectin, PHA-E, when incubated with intact, unfixed barbels, labelled exterior-facing epitopes on catfish barbel taste buds [35,45] and SCCs scattered in the epithelium among taste buds [35]. Immunoelectron microscopy using GP1 revealed labelling primarily on those cells of the taste bud containing large microvilli [35]. Because these data were of surface labelling only, the labelling specificity towards other areas of the taste bud and the barbel epidermis for GP1 and the lectins was not known. This current report demonstrates primarily apical labelling of taste buds by both conjugated RCA-I, GP1 and GP2 (Figs 2 and 5). The scattered punctuate labelling seen with both RCA-I and the GP antibodies may represent epitope recognition on solitary chemoreceptor cells (particularly within the epidermis), the apical processes of which were previously shown to label with PHA-E [35]. As the secondary antibody controls suggest (Fig. 5E) very little of this punctuate labelling can be attributed to a second antibody effect. Considering collectively the neurophysiological, biochemical, behavioural, biophysical and localization data, a putative receptor for L-Arg emerges as one of high structural specificity, with D-Arg acting as an antagonist, one of high sensitivity, and one expressed in the apical membrane of a specific sub-class of taste receptor cells. Yet, because the previous biophysical studies were carried out with intact cells, epithelial homogenates or reconstituted membrane vesicles, the data are insufficient to permit a distinction between the L-ArgR as a single LGICR macromolecular complex and the receptor as two separate entities, a recognition molecule coupled to ion channel activity. To help make this distinction, solubilization and enrichment of the receptor were required. We assumed that if receptor activity survived solubilization and increasing enrichment, and if this receptor appeared to purify as a unitary entity by SDS-PAGE, then it is likely that the major receptor for L-Arg is indeed a LGICR. This enrichment procedure was also a necessary first step in cloning the receptor, since partial amino acid sequences may be obtained from the purified product. Enrichment of the putative L-ArgR The initial enrichment step of lectin affinity was predicated upon the observation that the lectins, RCA-I and PHA-E, inhibited the specific binding of L-Arg to its presumed receptor sites [44], and that PHA-E and RCA-I inhibited L-Arg-stimulated conductance activity of catfish barbel (taste) membranes reconstituted into lipid bilayers [35]. In addition, both lectins labelled only a few protein bands of an SDS-PAGE of barbel Sp, and only one major band, that near 82–84 kDa, was common between the two (Fig. 1). Both conjugated RCA-I and PHA-E labelled cells within the taste buds (Fig. 2 and [35]). Lectin affinity chromatography employing agarose bound RCA-I was therefore used to achieve an initial partial enrichment of the putative L-ArgR. RCA-I was chosen for lectin affinity because in our hands it was more stable and easier to work with than the agarose bound PHA-E. In pilot studies, lectin affinity chromatography with PHA-E led to results similar to those obtained with RCA-I. The two additional enrichment procedures using Sephacryl-S300 and Hitrap Q ion exchange were chosen to further enrich the putative L-ArgR because both could be carried out using solubilization buffers that were less likely to destroy the activity of the receptor and both are standard biochemical purification techniques. Each chromatography step that retained L-Arg-stimulated ion channel activity resulted in increasingly concentrated and increasingly purified protein of molecular weight near 82–84 kDa. This enrichment is readily seen in SDS-PAGE of Figure 3, where the protein profile changes dramatically over the course of each chromatography step. The Western blots of material from each step (Figs. 4A,4B,4C,4D) suggest a substantial enrichment of the 82–84 kDa protein. Throughout enrichment, the 82–84 kDa band was consistently recognized by the GP1 and GP2 antibodies in Western blots (Fig. 4). These blots suggest that the same entity(ies) labelled by the lectins was retained and enriched through the course of each chromatography step. The Western blots also speak to the specificity of the antibodies, GP1 and GP2, in that both antibodies labelled almost exclusively the 82–84 kDa band. The subsequent observation that GP1 and GP2 recognized epitopes at the apical region of the taste bud (Fig. 5) indicates that at least a portion of the proteins in the 82–84 kDa region are taste cell-related, membrane associated and, given the biophysical characteristics, likely receptors. While the 82–84 kDa protein(s) may be a major constituent of the active ion channel complex, the actual molecular weight of the active receptor, and therefore some estimate of its quarternary composition, was difficult to determine. Attempts at running native gels led to inconsistent findings. Of the procedures used for enrichment, the Sephacryl column was the one that could, theoretically, at least, give an estimate of the size of the complex. Using this column, all of the L-Arg-stimulated ion channel activity was located within the initial eluted peak (fractions 1 & 2). Calibration of the column suggested a molecular weight of > 640 kDa for eluted material at this initial peak. However, this high apparent molecular weight may not represent the actual weight of the unitary LGICR, since, like many other LGICRs, the L-ArgR may form clusters [31,56-58]. Theoretically, use of Sephacryl 400HR should be able to resolve such high molecular weights. However, when Sephacryl 400HR was used, ion channel activity was spread across many eluted fractions, making estimates of unitary molecular weight impossible. After these enrichment procedures, the L-Arg-stimulated ion channel activity was retained and the 82–84 kDa protein fraction was greatly purified. The correlation of these two observations suggests that protein in the 82–84 kDa range is at least part of the L-ArgR. Biophysical characteristics of the putative L-ArgR The biophysical characteristics of the L-Arg-stimulated channels reconstituted from each step in the enrichment scheme remained nearly unchanged from those described for the channel observed in native membrane fragments [31,32]: 1. both channels were activated by the same range of concentration of L-Arg and blocked by the same concentration range of D-Arg (Fig. 6); 2. neither were activated by L-alanine nor by L-proline; 3. both displayed similar amplitude histograms (Fig. 7 and [31]); 4. both had similar unitary conductance (see Table 1), with the enriched channel displaying unitary conductance of 73 +/- 7 pS (Fig. 7), and the channel in situ displaying two conductance ranges, 40 – 60 and 75 – 100 pS. This difference is likely due to the difference in buffers, where the bilayer studies were performed in NaCl/CaCl2/MOPS, while the reconstituted membrane in situ studies were performed with a complex ringer buffer (Table 1, Fig. 8); 5. both channel preparations when stimulated by L-Arg exhibit linear current voltage relationship (Fig. 9). The results of this study demonstrate that the isolated channel protein shows recognition-specificity for L-Arg and acts as a non-specific ion channel upon binding L-Arg, properties consistent with the in situ activity of the putative L-ArgR and consistent with expected taste receptor criteria. Conclusions Collectively, the data presented here suggest that one major taste receptor for L-Arg in the catfish, I. punctatus, is a ligand-gated ion channel receptor. The active receptor was biochemically enriched from taste bud-containing epithelium and biophysically validated. Immunohistochemical studies using an antibody raised against peptides labelled by lectins that inhibited the binding of L-Arg to a likely receptor revealed specific labelling at the apical region of the taste bud. Analogous with other LGICRs, taste receptor cell depolarization to L-Arg is suggested through L-Arg binding to a receptor site that is associated with and activates an ion channel of low ion selectivity. In the animal, activation of this channel by L-Arg or other select agonists will open the channel and allow influx of Na+ and Ca2+, present in the mucus covering the taste epithelium [32], into the receptor cell. Alternatively, given the relatively low cation/anion ratio, at least part of this charge could be carried by efflux of Cl-. This flow of charge will result in cellular depolarization, release of neurotransmitter to the innervating sensory nerve, and transmission of the taste signal to the central nervous system. This enrichment procedure can be used to generate sufficient material for obtaining partial peptide sequences necessary for eventual cloning of this LGICR. Methods Animals Use of the channel catfish for these studies was approved by the Institutional Animal Care and Use Committee of the Monell Chemical Senses Center. Channel catfish, Ictalurus punctatus, purchased from local suppliers were usually euthanized on the day of delivery, but if not, were held less than 4 days in 250 gallon aquaria under dim light and fed commercial catfish chow. Chemicals All electrolytes, buffers and other chemicals were reagent grade from Sigma (St. Louis, MO). Water was deionized and further purified through a Milli-Q Plus PF system (Bedford, MA). 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate (98%) (CHAPS), polyoxyethylenesorbitan monolaurate (TWEEN 20), phenylmethanesulfonyl fluoride (PMSF) and pepstatin A were purchased from Sigma. n-Octyl-β-D-gluco-pyranoside (n-octylglucoside) was purchased from Calbiochem (LaJolla, CA). The absolute enantiomer, L-arginine HCl, was purchased from Sigma, and the absolute enantiomer, D-arginine HCl, was a gift of the Ajinomoto Co., Tokyo, Japan. Sephacryl S-300 HR, High Range Gel Filtration Calibration Kits, and 1.0 ml Hitrap Q columns were purchased from Pharmacia Biotech (Piscataway, NJ). Agarose-bound lectins, Ricinus communis agglutinin I (RCA-I), Phaseolus vulgaris Erythroagglutinin (PHA-E), in their biotinylated forms, the ABC kits, and rhodamine-conjugated RCA-I were purchased from Vector Lab (Burlingame, CA). The second antibody, Cy3-conjugated goat anti-guinea pig IgG, was obtained from Jackson ImmunoResearch Labs. (West Grove, PA). The 4 CN Membrane Peroxidase Substrate System was purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Gels and ampholytes were purchased from BioRad (Hercules, CA). Protein was quantitated using a BioRad DC Protein Assay. Synthetic 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were purchased from Avanti Polar Lipids, Inc. (Pelham, AL). Tissue homogenate preparation and solubilization Maxillary and mandibular barbels from approximately 50 euthanized channel catfish, ~25–40 cm long, were removed and placed in a beaker containing 100 ml of 50 mM Tris-HCl buffer (pH = 7.8), 100 mM NaCl, 1 mM EDTA ("TRIS Wash"). After one exchange of buffer, barbels were removed to a 50 ml polypropylene screw cap tube filled with TRIS Wash buffer and stored at -80°C until used. The preparation of a solubilized, plasma membrane-enriched fraction from catfish barbels was adapted from Kalinoski [59]. A typical homogenate/solubilization run used barbel tissue from 150 fish, prepared in aliquots of 50 fish at a time. The entire procedure was carried out at 4°C. The epithelium of thawed barbels from 50 fish at a time in 75 ml of TRIS Wash was stripped from the supporting pseudo-cartilage with two 10 second bursts (with a 10 second interval) from a hand-held Toastmaster Hand Blender, Model 1738 (Boonville, MO). The suspension was allowed to settle, and the supernatant decanted into a 600 ml beaker through two layers of USP Type VII gauze (Kendall Co., Boston, MA). Fifty ml of TRIS Wash (4°C) was added to the remaining settled barbels, and the suspension subjected to a third 10 second burst from the Blender. This entire second suspension was rapidly poured over the gauze layers into the same beaker. A second and third tube of barbels from 50 fish were treated identically, and the filtrate from all three combined, and the volume brought to 470 ml. This homogenate, divided into two aliquots, was centrifuged at 4000 × g for 15 min. The supernatants were recovered and centrifuged at 21,500 × g for 45 min. The pellets were retained. To solubilize the pellets from the 21,500 × g spin each pellet was recovered by two, 1 ml rinses of 50 mM TrisHCl (pH 7.8), 50 mM NaCl ("Low Osmolar Buffer") (total, 4 ml) and transferred to a 15 ml Teflon/glass homogenizer. Twenty milligrams of CHAPS and 40 μl of protease inhibitor-mix (0.275 mM pepstatin A, 57.5 mM PMSF, in ethanol) were added to the ~4 ml suspension in the homogenizer. The suspension was homogenized by ten slow strokes of the Teflon pestle using a rotating motor drive at moderate speed. The homogenate was transferred to a 15 ml capped tube, diluted to 10 ml with Low Osmolar Buffer, and placed on a Clay Adams Nutator rocker/shaker overnight at 4°C to solubilize the proteins. After the overnight agitation, the suspension was centrifuged for 1 h at 100,000 × g. The supernatant was recovered and is referred to as "Sp." This Sp was then used in the subsequent lectin affinity chromatography step. Lectin affinity chromatography All steps in the lectin affinity procedure were performed at 4°C. Agarose-bound RCA I affinity resin was pre-washed by adding 8 ml of gel slurry to a 125 ml conical glass tube and bringing the final volume to 14 ml with TRIS Wash. The tube was inverted several times, then centrifuged at 500 × g for 1 min. The supernatant was discarded and the wash step repeated three additional times with TRIS Wash buffer, then two more times with Low Osmolar buffer. The resulting agarose-bound RCA I gel, with a bed volume of 4 ml, was used for affinity chromatography. The 4 ml agarose-RCA gel in the 15 ml tube was combined with ~7 ml of Sp, and the mixture equilibrated on the Nutator tube rocker for 30 min. The mixture was poured into a 1 cm × 10 cm column and the effluent collected as 1 ml fractions at a flow rate of 6.0 ml/h. Effluent from the column was monitored by absorbance at 230 nm and/or 280 nm. To remove unbound protein, the column was washed with a sufficient volume (~12 ml) of Low Osmolar Buffer until no detectable protein eluted from the column. Proteins bound to the RCA resin were then eluted from the column with 10 ml of 50 mM Tris-HCl (pH = 7.8), 200 mM NaCl, 1 mM EDTA, 20 mM D-galactose. Protein from both the Low Osmolar elution step and the D-galactose elution step was reconstituted into lipid bilayers and assayed for L-Arg-stimulated ion channel activity (See ahead). The galactose-eluted protein fractions showing L-Arg-stimulated ion channel activity were pooled and dialyzed over night against 2000 ml Milli-Q water containing 0.05% CHAPS. Dialyzed samples were lyophilized and stored at -80°C. Gel filtration chromatography All steps in the gel filtration procedure were performed at 4°C. Lyophilized proteins from the galactose elution of the lectin column were prepared for further enrichment using size exclusion chromatography by dissolution in 300 μl of TRIS Wash with the addition of 0.2% CHAPS and 10% sucrose. This preparation was loaded onto a Sephacryl S-300 HR column (1 cm × 40 cm). Fractions of 0.4 ml were eluted from the column with a buffer composed of TRIS Wash plus 0.2% CHAPS. The column was run at 2.4 ml/hr, and the effluent absorbance monitored at 230 nm and 280 nm. Eluted fractions containing measurable protein were evaluated for L-Arg-stimulated ion channel activity by incorporation into a lipid bilayer (See ahead). In addition, SDS-PAGE (See ahead) was performed on each eluted fraction. Those fractions exhibiting L-Arg-stimulated ion channel activity were dialyzed overnight against 2000 ml of Milli-Q water containing 0.05% CHAPS, lyophilized and stored at -80°C. The column was calibrated with a Gel Filtration Calibration Kit with protein standards from 158 to 669 kDa. Ion exchange chromatography All steps in the ion exchange procedure were performed at 4°C. Further enrichment of the fractions exhibiting L-Arg-stimulated ion activity was achieved using an anion exchange column (Hitrap Q). The column was prepared by washing with 5.0 ml of Start Buffer (25 mM Tris-HCl, pH = 9.0), followed by 2.0 ml of Regeneration Buffer (Start Buffer plus 1.0 M NaCl) and then 5.0 ml of Start Buffer. Lyophilized proteins of the active fraction from the gel filtration column were dissolved in 300 μl of Start Buffer and applied to the column. Prior to elution, the loaded column was washed with 2.0 ml Start Buffer. Elution was at 12 ml/h in 400 μl fractions. First, 1.0 ml of First Elution Buffer (25 mM Tris-HCl, pH = 9.0, 500 mM NaCl) was applied to the column followed by (second), 2.0 ml of Start Buffer (no NaCl), followed by (third) 4.0 ml of Second Elution Buffer (25 mM Tris-HCl, pH = 8.0, 500 mM NaCl). Effluent from the column was monitored with in line UV (230 and 280 nm) and conductivity detectors. Each fraction was assayed for L-Arg-stimulated activity (See ahead.). The active fractions were pooled, dialyzed overnight against 2000 ml of Milli-Q water containing 0.05% CHAPS, lyophilized, and stored at -80°C. Development of polyclonal antibodies The procedure for development of antibodies against the catfish barbel peptides labelled by the lectins, RCA-I and PHAE has been described previously [45]. Briefly, electroeluted material from that area of a gel congruent with an identical gel labelled by the lectins was injected into three female guinea pigs using a schedule and procedure previously found to raise high titer polyclonal antibodies [60]. Antisera were aliquoted into 500 μl lots in 1.5 ml Eppendorf tubes and kept frozen at -80°C until used. Antisera from animal #1 (GP1) and animal &2 (GP2) were found to be the most specific in that they reacted primarily with their antigen within the 82–84 kDa band in Western blots of SDS-PAGE of catfish barbel membranes. The IgG fraction of GP1 and GP2 antisera was purified using an E-Z-SEP antibody purification kit (Pharmacia). To reduce non-specific binding in the immunohistochemical studies and Western blots, the GP1 and GP2 antibodies were incubated with powder derived from an acetone precipitation of a catfish brain homogenate. One ml of E-Z-SEP-purified antibody was incubated with 10 mg of powder for 40 min at 4°C. The powder was removed by centrifugation and the procedure was repeated once with fresh powder. The resulting pretreated antibodies are called simply, "GP1" and "GP2." Gel electrophoresis, lectin blots and Western blots Tris-glycine gels (4–20%, Bio-Rad) were used for SDS-PAGE. Protein of fractions before any chromatography (i.e., Sp), as well as those from each chromatography step, were denatured by mixing 10 μl from each fraction, 1:1, with sample buffer containing 125 mM Tris-HCl (pH = 8.0), 20% glycerol, 4% SDS, 4% β-mercaptoethanol and 50 μg/ml bromophenol blue, and placing the mix in a boiling water bath for 5 min. Gels were run at a constant 20–25 mA for about 1 h, using prestained broad range molecular weight markers (Bio-Rad) in 1 or more lanes. Proteins were stained with Bio-Rad Silver Stain Plus kit. For lectin blots, proteins were electrophoretically transferred to nitrocellulose sheets (Bio-Rad Mini Trans-Blot, Hercules, CA). The sheets were incubated in a blocking solution (2% gelatine, phosphate buffered saline [(PBS) (150 mM NaCl, 100 mM sodium phosphate, pH = 7.4)] and 0.05% Tween 20) for 2 h at room temperature, and then incubated with biotinylated RCA-I or PHA-E (10 μg/ml) overnight at 4°C. Following exposure to biotinylated lectin, nitrocellulose sheets were washed extensively with blocking solution. Lectin-bound protein bands were visualized using a Vectastain peroxidase ABC kit with a 4 CN Membrane Peroxidase Substrate system. Development was stopped after one hour using 5% glacial acetic acid. For Western blots, proteins were transferred to nitrocellulose and incubated in blocking solution (PBS, pH = 7.4, 5% non-fat dry milk, 1% goat serum and 0.05% TWEEN 20) for 2 h at ambient temperature with constant slow rocking (Nutator). The nitrocellulose was incubated overnight at 4°C (rocking) with primary antibody (GP1, 1/500) in blocking solution. The nitrocellulose was washed with blocking solution and incubated with biotinylated secondary antibody (1:250) for 1 h with slow rocking. Bands were visualized as above. Lectin histochemistry and immunohistochemistry Rhodamine-conjugated RCA-I (Vector Labs) was used to estimate the specificity of lectin interaction with glycoproteins of catfish barbel. To assess the localization of the antigen contained in the 82–84 kDa band from the SDS-PAGE of a membrane fraction of catfish barbel (and thereby the likely localization of the putative taste receptor for L-Arg), immunohistochemistry was performed using the GP1 and GP2 antibodies pretreated as described above. Barbels were removed from euthanized albino channel catfish (I. punctatus) (the fish being 5 – 7 cm in length) and immediately placed in 4% buffered paraformaldehyde (PFA) (0.1 M sodium phosphate buffer, pH 7.2–7.4) for eight hours at 4°C. After washing out the PFA with several rinses of excess buffer, the barbels were placed successively in 10%, 20%, and 30% sucrose (in buffer) for 24 hr, all at 4°C. After the final cryoprotect sucrose step, barbels were cut into pieces of less than 1 cm and mounted with M-1 Embedding Matrix (Thermo Shandon, Pittsburgh, PA). The tissue was sectioned at 10 microns on a Microm HM500OM cryostat. For lectin histochemistry, ten micron sections of fixed barbel were incubated in the dark with rhodamine-conjugated RCA-I lectin (Vector Labs., Burlingame, CA), diluted 1/200 for 2 to 3 hr at ambient temperature. The sections were then washed quickly with Dulbecco's PBS (GIBCO/Invitrogen Corp), followed by three incubation washes of 10 min each. For immunohistochemistry, 10 micron barbel sections, pre-washed 3 times for 10 min each in Dulbecco's PBS, were first incubated at ambient temperature for 3 to 5 hr in blocking buffer consisting of 3% bovine serum albumin, 2% goat serum, 0.3% TritonX100, and 0.1% sodium azide in Dulbecco's PBS at pH 7.1. The sections were then incubated with primary antibody, GP1 or GP2, in blocking buffer for 18 hr at 4°C. The primary antibody solution was removed and the sections were then washed once quickly with Dulbecco's PBS, followed by three incubation washes with PBS of 10 min each. The sections were then incubated in the dark with second antibody, Cy3-conjugated goat anti-guinea pig IgG (Jackson ImmunoResearch Labs., West Grove, PA) at 1:1000 dilution for 60 min. at ambient temperature. The secondary antibody was removed and the sections washed once quickly with Dulbecco's PBS, followed by three incubation washes with PBS of 10 min each. Excess fluid was removed from the slides and the sections mounted under cover slips with VectaShield (Vector Labs). Sections were observed with a Nikon Microphot FXA fluorescence microscope, photographed, and images sized and enhanced using the GNU Image Manipulation Program software [61]. Immuno-specificity was verified by running negative controls where the primary antibody was omitted from the procedure. In all cases, this control step showed no taste bud labelling (Fig. 5E). The scattered, spotty background labelling seen with the primary antibodies is likely due to both an unknown factor in pre-immune serum and to labelling of solitary chemoreceptor cells in the barbel epithelium, as was previously documented [35]. Lipid bilayer reconstitution Reconstitution of protein fractions containing likely L-Arg-stimulated channel activity was carried out with material from the low osmolar and galactose-eluted fractions of the lectin affinity procedure, from the protein-containing fractions off the Sephacryl S-300 gel filtration column and from each fraction of the ion exchange column. Lipid vesicles were prepared by sonication of 5 mg of DOPE:DOPC (2:1) and 0.5 ml of 5 mM Tris-HCl (pH = 7.2), 300 mM NaCl and 500 mM sucrose, to which 10 μg n-octylglucoside was added. To prepare the liposome-detergent mixture for incorporation into a lipid bilayer, approximately 0.2 to 0.5 μg of presumed receptor protein was added to the liposome in a cassette dialysis unit, and the mixture dialyzed overnight against 2000 ml of the Tris/NaCl/sucrose buffer at 4°C. Virtually solvent-free lipid bilayer membranes were prepared as described [62]. The membrane-forming solution was an equimolar mixture of DOPS:DOPE in hexane. The bilayer chamber consisted of two symmetrical halves of a Teflon chamber, each with solution volumes of 1 ml divided by a 15 μm thick Teflon partition containing a round aperture of about 150 μm diameter. Hexadecane in n-hexane (1:10, v/v) was used for aperture pre-treatment. A pair of Ag-AgCl electrodes was connected to the solution in the chamber via 3 M KCl-4% agar bridges. "Virtual ground" was maintained at the trans side of the bilayer. The bilayer was bathed symmetrically with 5 mM MOPS (pH 7.2), 1 mM CaCl2, 100 mM NaCl (unless otherwise stated). Fifty to 100 μl of the dialyzed liposome vesicles containing presumed receptor was added to the cis-side of the membrane. Fusion of the vesicles was initiated mechanically by gently mixing the membrane bathing solution from the cis-side using a micro-pipette. L-Arg was added to the cis side approximately 20 min after addition of vesicles. Unless otherwise stated all other additions of reagents also were made from the cis side. Channel sidedness was determined by sensitivity of the bilayer to L-Arg. The orientation of the channels was such that the L-Arg sensitive side was normally in the cis compartment. All bilayer experiments were performed at room temperature. The current was amplified by a Dagan 3900 integrating patch-clamp amplifier (Dagan Corp., Minneapolis, MN) in the voltage clamp mode. Single channel data were digitized at 15 kHz (Digidata 1200, Axon Instruments, Foster City, CA) and analyzed using pClamp6 (Axon Instruments) and Origin 5.1 (Microcal Software, North Hampton, MA) software on an IBM compatible computer. The calculated success rate of incorporation of vesicular proteoliposomes into lipid bilayers was about 25%. Success rate is defined as the ratio of the number of successful incorporation attempts to the total number of incorporation attempts (an "incorporation attempt" refers here to the formation of a new bilayer and application of putative channel protein). Over the course of these studies, the number of incorporation attempts for each fraction tested as indicated above was about ten. Authors' contributions WG developed the enrichment procedures and drafted the manuscript. YK developed protein stabilization procedures and performed and interpreted the bilayer studies. DB performed the lectin- and immunohistochemistry and refined many of the enrichment procedures. AS developed the antibodies and performed pilot chromatography procedures. DLK initially designed the lectin procedure and carried out preliminary work on solubilization that made subsequent procedures possible. JT performed initial bilayer studies and advised YK on a continuing basis. JGB, the corresponding author and head of the laboratory, conceived the study, developed tissue preparations, designed and coordinated all phases of the study, and finalized the manuscript. All authors read and approved the final manuscript. Acknowledgements We gratefully acknowledge the efforts of Drs. Isabella Andreini and Alexander M. Feigin in the preliminary aspects of this work. We thank Ms. L. Wysocki for sectioning tissue and for advice in histological procedures, and Dr. J. Cao for formatting the manuscript. We also acknowledge the much appreciated critique of an early version of this manuscript by Dr. John Caprio. These studies were supported in part by NIH grants DC00356 (J.G.B.), DC01838 (J.H.T.), DE10754 (AIS), and Institutional NRSA T32 DC00014 (Monell Center) and by a grant from the Department of Veterans Affairs (J.G.B.). Figures and Tables Figure 1 Lectin labelling of solubilized barbel epithelial proteins SDS-PAGE (4–20%) of 10 μg of solubilized barbel homogenate stained with silver (Lane "Sp") or probed with PHA-E lectin (Lane "PHA") or RCA-I lectin (Lane "RCA") using lectins at 10 μg/ml with ABC detection. Both lectins label a band at 82 – 84 kDa and lightly label at least two other bands, one near 88 kDa, the other near 120 kDa. Figure 2 RCA-I lectin histochemistry on barbel of catfish, I. punctatus (albino). Barbels were fixed in 4% PFA, PBS, cryostat sectioned at 10 microns and histochemically probed with conjugated RCA-I at 1/200 dilution of the manufacturer's stock. (A). Surface labelling by RCA-I shows preferential recognition of binding sites primarily at the apical endings of taste buds. (B). Labelling by RCA-I of the apical region of two taste buds. (C). Labelling by RCA-I of horizontal section through the barbel showing reactive taste buds lining the epithelium. Figure 3 SDS-PAGE (4–20%) of solubilized barbel protein, visualized with silver stain, before (A) and after (B, C, D) each enrichment step. Each lane was loaded with 5 μg of protein. Lane A: Total protein of fraction Sp. Lane B: Galactose eluted protein from the RCA column containing L-Arg stimulated ion channel activity; Lane C: Protein of the first peak fractions of the Sephacryl gel filtration step containing L-Arg stimulated ion channel activity; and Lane D: Protein containing L-Arg stimulated ion channel activity from the ion exchange chromatography enrichment step. Figure 4 Western blots of SDS-PAGE protein samples before (A) and after (B, C, and D) each enrichment step. Western blots were performed using GP1. Although the identity of the samples applied to each lane was the same as illustrated in Figure 3, the amount of protein applied to each lane differed. (A). Fraction Sp, 5 μg, (B). Galactose eluted protein from the RCA column containing L-Arg stimulated ion channel activity, 1.6 μg, (C). Protein from the first peak fractions of the gel filtration step containing L-Arg stimulated ion channel activity, 0.2 μg, (D). Protein fraction containing L-Arg stimulated ion channel activity from the pH 9 fraction of the ion exchange procedure, 0.2 μg. Note that in spite of lowering protein amounts in lanes A – D, the intensity of the Western blot increases, indicative of an enrichment of the antigen protein(s) near 82–84 kDa. Figure 5 Immunohistochemistry of catfish barbel taste buds using antibodies GP1 and GP2. Barbels were fixed in 4% PFA, PBS, and cryostat sectioned at 10 microns. Sections were probed using indicated dilutions of antibodies GP1 and GP2. (A). Section through a barbel immuno-stained with GP1 at 1/8000 dilution. The lines from "TB" point to labelled taste buds. (B). The apical aspects of three taste buds immuno-stained with 1/12000 dilution of GP2. (C). Cross section through a taste bud immuno-stained with 1/16000 dilution of GP1. (D). A single taste bud immuno-stained with 1/8000 dilution of GP2. (E). Second antibody control showing barbel without exposure to primary antibodies. Figure 6 Single channel recording of the activity of the putative L-ArgR in planar lipid bilayers. Proteoliposomes containing protein from the first peak fractions off the Sephacryl S-300 elution were fused to planar lipid bilayers (DOPS:DOPE, 1:1) as described in Methods. (A). An initial control trace was obtained after addition proteoliposomes to the membrane bathing solution, before the addition of L-Arg. The three rows are from a continuous recording. (B). The addition of 10 μM L-Arg to the cis-side of the bilayer evoked regular periodic channel activity. A portion of this current record is shown at an expanded scale. (C). After several minutes of recording, the addition of 100 μM D-Arg to the cis-side resulted in the cessation of activity. Transmembrane potential was -100 mV. Traces shown in all panels are continuous records of that specific condition. Figure 7 Amplitude histogram of current fluctuations of the putative L-ArgR in lipid bilayers. Current fluctuations were calculated from studies similar to those presented in Figure 6, part B, using the same protein fraction, measured at -100 mV transmembrane potential. The histogram was fit by a Gaussian distribution and mean current values were obtained from the center of the distribution. Bilayer was DOPS:DOPE, 1:1. Figure 8 Cation-anion selectivity of L-ArgR channels. The figure shows current elicited by voltage ramps across a lipid bilayer (DOPS:DOPE, 1:1) containing several channels from the Sephacryl S-300 step, in the presence of a 4-fold gradient of NaCl across the membrane (cis and trans chambers contained 100 and 25 mM NaCl respectively). The reversal potential is -14 mV, corresponding to weak cation selectivity (PNa/PCl = 2.2). Figure 9 The unitary current/voltage (I-V) relationship of channels formed by protein of each enrichment step. The I-V relationships were obtained using L-Arg – active protein from the RCA-I lectin column (o) from the first peak fractions off the Sephacryl S-300 HR column (■) and from the pH 9 elution of the ion-exchange column (▲). Measurements were made under symmetrical conditions of 100 mM NaCl, 1 mM CaCl2 and 5 mM MOPS (pH = 7.2). Data points indicate the Mean ± S.D. The data sets are well fit by a linear regression (r = 0.99 solid and dotted lines) with slopes of 58, 67, and 73 pS respectively. Bilayer was DOPS:DOPE, 1:1. Table 1 Characteristics of L-Arg stimulated ion channel activity of the three enrichment steps compared with the same parameters from the native L-Arg stimulated channels as reported by Kumazawa et al.* Sample +Unitary Conductance pS Concentration of L-Arg, μM Concentration of D-Arg, μM Cation-anion selectivity RCA-I lectin column material 58 ± 5 ---- Sephacryl S300 column material 66 ± 3 1 – 50 10 – 200 Weak cation (PNa/PCl = 2.2) Ion-exchange column material 73 ± 7 -- Ion Channels from Taste Tissue 40 – 60 75–100 1 – 200 10 – 200 Cation * From Kumazawa [31] + Data on unitary conductance reported as Mean ± SD obtained from at least three experiments. ==== Refs Witt M. Reutter, K. Miller, I.J. 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Modulation by intracellular factors and taste stimuli J Gen Physiol 1992 99 591 613 1597680 10.1085/jgp.99.4.591 Seto E Hayashi Y Mori T Patch clamp recording of the responses to three bitter stimuli in mouse taste cells Cell Mol Biol (Noisy-le-grand) 1999 45 317 325 10386788 Spielman AI Mody I Brand JG Whitney G MacDonald JF Salter MW A method for isolating and patch-clamping single mammalian taste receptor cells Brain Res 1989 503 326 329 2605526 10.1016/0006-8993(89)91684-3 Mierson S DeSimone SK Heck GL DeSimone JA Sugar-activated ion transport in canine lingual epithelium. Implications for sugar taste transduction J Gen Physiol 1988 92 87 111 3171536 10.1085/jgp.92.1.87 Brand JG Teeter JH Kumazawa T Huque T Bayley DL Transduction mechanisms for the taste of amino acids Physiol Behav 1991 49 899 904 1679559 10.1016/0031-9384(91)90201-X Hatt H Franke C Taste receptors in crayfish: recording of single nicotinamide-activated channels Neurosci Lett 1987 73 137 142 2434888 10.1016/0304-3940(87)90007-3 Murakami M Kijima H Transduction ion channels directly gated by sugars on the insect taste cell J Gen Physiol 2000 115 455 466 10736312 10.1085/jgp.115.4.455 Tsunenari T Kurahashi T Kaneko A Activation by bitter substances of a cationic channel in membrane patches excised from the bullfrog taste receptor cell J Physiol 1999 519 Pt 2 397 404 10457058 Kumazawa T Brand JG Teeter JH Amino acid-activated channels in the catfish taste system Biophys J 1998 75 2757 2766 9826598 Teeter JH Brand JG Kumazawa T A stimulus-activated conductance in isolated taste epithelial membranes Biophys J 1990 58 253 259 1696511 Caprio J Brand JG Teeter JH Valentincic T Kalinoski DL Kohbara J Kumazawa T Wegert S The taste system of the channel catfish: from biophysics to behavior Trends Neurosci 1993 16 192 197 7685945 10.1016/0166-2236(93)90152-C Bardach JE Atema J Beidler LM The sense of taste in fishes Handbook of Sensory Physiology Vol IV: Chemical SensesPArt2: Taste 1971 New York, Springer-Verlag 293 336 Finger TE Bryant BP Kalinoski DL Teeter JH Bottger B Grosvenor W Cagan RH Brand JG Differential localization of putative amino acid receptors in taste buds of the channel catfish, Ictalurus punctatus J Comp Neurol 1996 373 129 138 8876468 10.1002/(SICI)1096-9861(19960909)373:1<129::AID-CNE11>3.0.CO;2-F Reutter K Witt M Simon SA and Roper SD Morphology of vertebrate taste organs and their nerve supply Mechanisms of Taste Transduction 1993 Boca Raton, CRC Press 29 82 Brand JG Bryant BP Cagan RH Kalinoski DL Biochemical studies of taste sensation. XIII. Enantiomeric specificity of alanine taste receptor sites in catfish, Ictalurus punctatus Brain Res 1987 416 119 128 3620948 10.1016/0006-8993(87)91503-4 Kalinoski DL Bryant BP Shaulsky G Brand JG Harpaz S Specific L-arginine taste receptor sites in the catfish, Ictalurus punctatus: biochemical and neurophysiological characterization Brain Res 1989 488 163 173 2545299 10.1016/0006-8993(89)90705-1 Cagan RH Biochemical studies of taste sensation--XII. Specificity of binding of taste ligands to a sedimentable fraction from catfish taste tissue Comp Biochem Physiol A 1986 85 355 358 2876841 10.1016/0300-9629(86)90262-8 Wegert S Caprio J Receptor sites for amino acids in the facial taste system of the channel catfish J Comp Physiol [A] 1991 168 201 211 1675294 Bryant BP Harpaz S J.G. Brand Structure/activity relationships in the arginine taste pathway of the channel catfish Chemical Senses 1989 14 805 815 Teeter JH Miyamoto T Restrepo D Zviman MM Brand JG Kurihara K, Suzuki N and Ogawa H L-Arginine-regulated conductances in catfish taste cells Olfaction and Taste XI 1994 Tokyo, Springer-Verlag 93 95 Zviman MM Restrepo D Teeter JH Single taste stimuli elicit either increases or decreases in intracellular calcium in isolated catfish taste cells J Membr Biol 1996 149 81 88 8834115 10.1007/s002329900009 Kalinoski DL Johnson LC Bryant BP Brand JG Selective interactions of lectins with amino acid taste receptors in channel catfish Chem Senses 1992 17 381 390 Grosvenor W Feigin AM Spielman AI Finger TE Wood MR Hansen A Kalinoski DL Teeter JH Brand JG The arginine taste receptor. Physiology, biochemistry, and immunohistochemistry Ann N Y Acad Sci 1998 855 134 142 9929594 Whitear M Hara TJ Solitary chemoreceptor cells Chemoreception in Fishes 1991 New York, Elsivier 506 513 Finger TE Evolution of taste and solitary chemoreceptor cell systems Brain Behav Evol 1997 50 234 243 9310198 Caprio J High sensitivity of catfish taste receptors to amino acids Comp Biochem Physiol A 1975 52 247 251 240558 Davenport CJ Caprio J Taste and tactile recordings from the ramus recurrens facialis innervating flank taste buds in the catfidsh Journal of Comparative Physiology 1982 147 217 229 Kohbara J Michel W Caprio J Responses of single facial taste fibers in the channel catfish, Ictalurus punctatus, to amino acids J Neurophysiol 1992 68 1012 1026 1432064 Holland KN Teeter JH Behavioral and cardiac reflex assays of the chemosensory acuity of channel catfish to amino acids Physiol Behav 1981 27 699 707 7323174 10.1016/0031-9384(81)90243-2 Valentincic T Caprio J Consummatory feeding behavior to amino acids in intact and anosmic channel catfish Ictalurus punctatus Physiol Behav 1994 55 857 863 8022905 10.1016/0031-9384(94)90071-X Restrepo D Boekhoff I Breer H Rapid kinetic measurements of second messenger formation in olfactory cilia from channel catfish Am J Physiol 1993 264 C906 11 8386453 Speca DJ Lin DM Sorensen PW Isacoff EY Ngai J Dittman AH Functional identification of a goldfish odorant receptor Neuron 1999 23 487 498 10433261 10.1016/S0896-6273(00)80802-8 Li Y Perruccio E Zhang X Kleinhaus AL Bitter substances suppress afferent responses to an appetitive mixture: evidence for peripheral integration of chemosensory stimuli J Neurobiol 2001 49 255 263 11745663 10.1002/neu.10003 Kossel AH McPheeters M Lin W Kinnamon SC Development of membrane properties in taste cells of fungiform papillae: functional evidence for early presence of amiloride-sensitive sodium channels J Neurosci 1997 17 9634 9641 9391018 O'Brien RJ Xu D Petralia RS Steward O Huganir RL Worley P Synaptic clustering of AMPA receptors by the extracellular immediate-early gene product Narp Neuron 1999 23 309 323 10399937 10.1016/S0896-6273(00)80782-5 Safferling M Tichelaar W Kummerle G Jouppila A Kuusinen A Keinanen K Madden DR First images of a glutamate receptor ion channel: oligomeric state and molecular dimensions of GluRB homomers Biochemistry 2001 40 13948 13953 11705385 10.1021/bi011143g Kalinoski DL Spielman AI and Brand JG Receptor binding and solubilization Experimental Cell Biology of Taste and Olfaction 1995 Boca Raton, CRC Press 179 185 Spielman AI Bennick A Isolation and characterization of six proteins from rabbit parotid saliva belonging to a unique family of proline-rich proteins Arch Oral Biol 1989 34 117 130 2783045 10.1016/0003-9969(89)90135-0 GNU Image Manipulation Program software Montal M Mueller P Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties Proc Natl Acad Sci U S A 1972 69 3561 3566 4509315

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==== Front BMC GenetBMC Genetics1471-2156BioMed Central London 1471-2156-5-221529197010.1186/1471-2156-5-22Research ArticleComparison of the accuracy of methods of computational haplotype inference using a large empirical dataset Adkins Ronald M [email protected] Children's Foundation Research Center and Center of Genomics and Bioinformatics, University of Tennessee, Memphis, TN, USA2004 3 8 2004 5 22 22 28 4 2004 3 8 2004 Copyright © 2004 Adkins; licensee BioMed Central Ltd.2004Adkins; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Analyses of genetic data at the level of haplotypes provide increased accuracy and power to infer genotype-phenotype correlations and evolutionary history of a locus. However, empirical determination of haplotypes is expensive and laborious. Therefore, several methods of inferring haplotypes from unphased genotypic data have been proposed, but it is unclear how accurate each of the methods is or which methods are superior. The accuracy of some of the leading methods of computational haplotype inference (PL-EM, Phase, SNPHAP, Haplotyper) are compared using a large set of 308 empirically determined haplotypes based on 15 SNPs, among which 36 haplotypes were observed to occur. This study presents several advantages over many previous comparisons of haplotype inference methods: a large number of subjects are included, the number of known haplotypes is much smaller than the number of chromosomes surveyed, a range in values of linkage disequilibrium, presence of rare SNP alleles, and considerable dispersion in the frequencies of haplotypes. Results In contrast to some previous comparisons of haplotype inference methods, there was very little difference in the accuracy of the various methods in terms of either assignment of haplotypes to individuals or estimation of haplotype frequencies. Although none of the methods inferred all of the known haplotypes, the assignment of haplotypes to subjects was about 90% correct for individuals heterozygous for up to three SNPs and was about 80% correct for up to five heterozygous sites. All of the methods identified every haplotype with a frequency above 1%, and none assigned a frequency above 1% to an incorrect haplotype. Conclusions All of the methods of haplotype inference have high accuracy and one can have confidence in inferences made by any one of the methods. The ability to identify even rare (≥ 1%) haplotypes is reassuring for efforts to identify haplotypes that contribute to disease in a significant proportion of a population. Assignment of haplotypes is relatively accurate among subjects heterozygous for up to 5 sites, and this might be the largest number of SNPs for which one should define haplotype blocks or have confidence in haplotype assignments. ==== Body Background Very rapid and inexpensive methods exist for determining the genotype of diploid organisms at single nucleotide polymorphisms (SNPs). Unfortunately, these high-throughput methods do not provide direct information on which SNP alleles at multiple sites coexist on the same chromosome. Instead, computational methods must be employed to infer the set of SNP alleles that are cosegregating on a single chromosome, referred to as haplotypes. However, the inference of haplotypes from phase-unknown data is computationally difficult, partly due to the fact that the number of possible haplotypes roughly increases as a power of 2 with each additional SNP. Interest in the accurate inference of haplotype structure from unphased genotypic data has increased tremendously in recent years for several reasons. Relative to analysis of single polymorphisms, haplotypes can greatly improve one's ability to infer the evolutionary history of a DNA region [1,2]. Additionally, haplotypes can provide significant increases in statistical power to detect associations between a phenotype and genetic variation [3-5]. Indeed, several disease associations with haplotypes have been detected that were not apparent from single-site analyses [6-9]. There are three principal computational approaches to inferring haplotypes from unphased SNP data. The most commonly used approach is implementation of the expectation-maximization (EM) algorithm [10]. This method is computationally intensive and is usually combined with various strategies to simplify the task (i.e., by considering only subsets of the sites at a time) or to minimize the number of potential haplotypes that must be considered [11,12]. A more recent alternative is application of Bayesian methods that incorporate prior expectations based upon population genetic principles [13-15]. A third method based on parsimony ("subtraction method"; [16]) has the limitation that haplotypes are assigned only in unambiguous cases [17], and the level of ambiguity generally increases with the number of sites considered or the number of sites at which an individual is heterozygous. This limitation is expected to be significant in large-scale analyses of SNP variation, and for this reason the subtraction method is not considered here. Unfortunately, it is unclear how accurate the EM and Bayesian approaches are or whether the EM or Bayesian method is superior in inferring haplotypes, particularly when applied to empirical data. Data simulation [18] can explore the effect of a wide range of parameters and population dynamics (i.e., linkage disequilibrium, selection, population substructuring) but is unlikely to achieve fully the complex combinations of these effects inherent in empirical data. On the other hand, comparisons using empirical data have been based on as few as six SNPs [17,19] or have employed data sets in which the number of SNPs or known haplotypes equals or greatly exceeds the number of individuals sampled [13,15]. Neither of these situations is likely to be an accurate reflection of the sample sizes or numbers of SNPs that will be assayed with the high-throughput methods available today. To understand the relative performance of the various methods of haplotype inference, there is a need for comparisons that include both larger numbers of polymorphic sites and biologically more complex correlations among the sites. In this study the performance of several leading methods of haplotype inference are compared for a large data set (154 individuals, 15 SNPs) undergoing a combination of mutation, recombination, and gene conversion. The accuracy of computational haplotype inference improves as the magnitude of linkage disequilibrium (LD) among sites increases [17]. Gene conversion, operating in conjunction with normal recombination, can complicate the normal decay of linkage disequilibrium with distance in a genomic region and can be expected to complicate the computational inference of haplotype structure. This issue has particular relevance to the human growth hormone locus. The five genes of the human growth hormone locus reside within about 45 kb on chromosome 17 [20]. Pituitary growth hormone (GH1) is by far the most thoroughly studied of the genes and lies at the 5' end of the cluster. The remaining four genes, placental growth hormone (GH2) and three chorionic somatomammotropins (CS1, CS2, and pseudogene CS5 or CSHP1), are expressed only from the placenta. The promoter region of GH1 is unusually polymorphic, with 16 SNPs having been identified in a span of 535 bp [21-23]. Most of these SNPs occur at the comparatively small number of sites that exhibit sequence differences among the five genes of the GH locus, and this has been interpreted as evidence of gene conversion [21,23,24]. A survey of 25 SNPs in the entire promoter and coding region of GH1 (Adkins et al. in review) indicates that this bias towards polymorphism at sites of intergenic divergence is quite extreme and supports the hypothesis that gene conversion plays a role in the pattern of variation in the GH1 gene in addition to mutation and recombination. In 154 recruits to the British army, Horan et al. [23] used cloning and sequencing to empirically determine 36 haplotypes based on 15 of the promoter SNPs previously identified (one site identified by [21] was invariant). This study takes advantage of the exhaustive work of Horan et al. [23] to compare the relative accuracy of some of the major implementations of the EM and Bayesian approaches to haplotype inference. Results and Discussion Characteristics of the data set The 15 sites studied by Horan et al. [23] span 535 nucleotides in the promoter of GH1, with minor allele frequencies ranging from 0.3–41.2%. Six of the sites can be considered "rare" variants with minor allele frequencies below 5% (0.3–3.6%). Standardized linkage disequilibrium (D'; [25]) among the remaining nine sites ranges from complete linkage disequilibrium (sites -301 and -308; Table 1) to effective linkage equilibrium (i.e., sites -1 and +59). Fallin and Schork [18] identified several characteristics of an unphased set of genotypes that improve the accuracy of haplotype inference, most of which are exhibited by this data set and discussed below. Therefore, this data set probably represents one that is favourable to the accurate inference of haplotypes. Table 1 Linkage disequilibrium (D') among loci with minor allele frequencies ≥ 5%1 Site Site -278 -75 -57 -31 -6 -1 +59 -308 1.000 0.653 0.892 0.741 0.458 0.192 0.646 -278 0.857 0.845 0.696 0.820 0.666 0.334 -75 1.000 0.358 0.708 0.561 0.172 -57 1.000 0.872 1.000 1.000 -31 0.410 1.000 0.538 -6 1.000 0.194 -1 0.002 1 Numbering relative to the start of transcription for GH1 Increasing sample size improves the accuracy of inferred haplotypes. In Horan's study [23] 308 chromosomes were surveyed to yield 36 haplotypes, a ratio of 8.6. Three haplotypes can be unambiguously inferred from the 27 fully homozygous individuals, and 11 subjects are heterozygous at only one site, from which an additional 11 haplotypes can be unambiguously inferred. This leaves 116 individuals (232 chromosomes) heterozygous at ≥ 2 sites upon which to attempt to infer the remaining 22 true haplotypes. The distribution of haplotype frequencies also influences the accuracy of haplotype inference in two ways. First, the presence of some haplotypes at comparatively high frequency increases the chances that those haplotypes can be unambiguously inferred from homozygotes, allowing the alternative haplotype to be inferred with high confidence in compound heterozygotes. Second, the presence of some haplotypes at near-zero frequency allows truly nonexistent haplotypes to be accurately estimated as having zero frequency. The empirical haplotype frequencies in this study exhibit considerable dispersion. Two haplotypes are relatively common (33% and 16%; Table 2). Thirty-one haplotypes have frequencies below 5%, and 19 have frequencies ≤ 1%. In multiple regression analysis, Fallin and Schork [18] found dispersion of haplotype frequencies to be the strongest predictor of the accuracy of haplotype inference. Table 2 Inferred frequencies of haplotypes. SNP Haplotype Frequency -476 -339 -308 -301 -278 -168 -75 -57 -31 -6 -1 3 16 25 59 Empirical Phase, no LD Phase, with LD Haplotyper PL-EM SNPHAP Empirical Haplotypes 1 G G G G G T A T G A A G A A T 0.334 0.312 0.321 0.325 0.333 0.326 2 G G G G T T A G G G A G A A T 0.162 0.166 0.162 0.166 0.181 0.171 3 G G T T G T A G G A A G A A T 0.091 0.097 0.097 0.101 0.098 0.102 4 G G T T G T A G - A A G A A T 0.052 0.055 0.055 0.049 0.047 0.050 5 G G G G T T G G G G A G A A T 0.042 0.052 0.052 0.052 0.049 0.050 6 G G T T G T A G - A A G A A G 0.029 0.032 0.032 0.032 0.030 0.030 7 G G G G T T A G G G T G A A T 0.026 0.032 0.032 0.032 0.028 0.029 8 G G T T G T A G G G A G A A T 0.019 0.016 0.016 0.013 0.016 0.018 9 G G G G T T A T G G A G A A T 0.019 0.013 0.013 0.013 0.011 0.011 10 G G T T G T A G - G A G A A T 0.019 0.023 0.026 0.023 0.025 0.025 11 G G G G T T G G G G A G G C T 0.016 0.016 0.016 0.016 0.014 0.014 12 G G G G T T A G G A A G A A T 0.016 0.010 0.006 0.006 0.008 0.008 13 G - G G T T G G G G A G A A T 0.016 0.016 0.016 0.013 0.010 0.013 14 G G G G T C A G G G T G A A T 0.016 0.016 0.016 0.016 0.016 0.016 15 G G T T G T A G G G T G A A T 0.013 0.010 0.010 0.010 0.006 0.009 16 G G G G T T G G G A A G A A T 0.013 0.013 0.013 0.016 0.008 0.008 17 G - G G T T A G G G A G A A T 0.013 0.013 0.013 0.013 0.011 0.011 18 G G G G T T A G - G A G A A T 0.010 - 0.006 0.010 0.007 0.008 19 A G G G T T A G G G A G A A T 0.010 0.013 0.013 0.010 0.005 0.010 20 G G G G G T A G - A A G A A T 0.010 - 0.003 0.010 0.006 0.005 21 G G G G T T G G G G A G A A G 0.010 0.010 0.010 0.010 0.011 0.011 22 G G T T G T A T G A A G A A T 0.010 0.013 0.010 0.013 0.007 0.007 23 G G G G G T A G G A A G A A T 0.006 0.016 0.013 0.006 0.006 0.008 24 G G T T G T G G - A A G A A T 0.006 - - - - - 25 G G T T G T A G G A A G A A G 0.003 - - - 0.004 0.004 26 G G G G T T G G G G T G A A T 0.003 0.006 0.006 0.006 0.007 0.007 27 G G G G T T A T G A A G A A T 0.003 0.003 0.003 - - - 28 G G G G T T A G - A A G A A T 0.003 - - - - - 29 A G G G T T A G G A A G A A T 0.003 - - - - - 30 G - G G T T A G G A A G A A T 0.003 0.003 0.003 0.003 0.003 0.003 31 G G G G T T G G - G A G A A T 0.003 0.010 - - - - 32 G G T T G T G G G G A G A A G 0.003 - - - 0.002 - 33 G G G G T T A G G G A G G C T 0.003 0.003 0.003 0.003 0.004 0.004 34 G - G G T C A G G G T G A A T 0.003 0.003 0.003 0.003 0.003 0.003 35 G G G G G T A G G A C C A A T 0.003 - - 0.003 0.003 0.003 36 G G G G T T A G G G T G A A G 0.003 - - - 0.003 0.003 Incorrect Haplotypes 1 G - G G T T G G G G A G G C T - - - 0.002 0.002 2 G G G G T T G G - A A G A A T - 0.006 - 0.009 0.008 3 G G G G T T G T - G A G A A T - - - - 0.002 4 G - G G T T G T G A A G A A T - - 0.003 - - 5 G - G G T T G T G G A G A A T - - - 0.003 0.002 6 G G G G T T G T G G A G A A T - 0.003 - - - 7 G G G G T T G T G A A G A A T - - - - 0.004 8 A G G G T T A T G A A G A A T - - 0.003 0.003 0.003 9 G G G G T T A G G G C C A A T 0.003 0.003 - - - 10 A G T T G T A G - A A G A A T - - - 0.002 - 11 A G T T G T A G G G T G A A T - - - 0.003 - 12 G G T T T T G G - G A G A A T - - 0.006 0.004 - 13 G G T T T T G G G A A G A A T - - - 0.003 - 14 G G G G G T A T - A A G A A T 0.010 - - - - 15 G G G G G T A T G G A G A A T 0.006 0.006 0.006 0.008 0.008 16 G G G G G T A T G A A G A A G 0.006 0.006 0.006 - - Minor Allele Frequency 0.013 0.036 0.247 0.247 0.399 0.019 0.114 0.367 0.133 0.412 0.065 0.003 0.019 0.019 0.049 Accuracy of haplotype inference The accuracy of computational inferences of haplotype frequencies and assignments to individuals were compared to empirical values for the full set of 15 SNPs in the promoter of GH1. Additionally, analyses were performed on a restricted set of eight SNPs with allele frequencies above 5% (and excluding site -301 which is in complete linkage disequilibrium with -308). The latter analyses were performed to better approximate the characteristics of data sets that are typically collected in genetic epidemiological studies. Although the presence of rare alleles and haplotypes improves the accuracy of haplotype inference, sites with a low frequency minor allele are often ignored due to their reduced usefulness in mapping disease loci and the assumption that such loci will contribute little to population-wide predisposition to disease. Very little difference was observed in the accuracy of haplotype inference between the two data sets. Assignment of haplotypes to individuals was very accurate by all methods (Table 3). Approximately 90% of individuals were assigned correct haplotypes. However, this number includes individuals whose haplotypes are unambiguous (heterozygotes at 0 or 1 site). Excluding those individuals, the error rate is closer to 13%. Table 3 Accuracy of computational inferences of haplotype structure of the GH1 gene promoter. 15 Promoter SNPs Error Rate Based on # of Heterozygous Sites (N) Error Rate Algorithm MSE IH # correct/# wrong IF Overall Ambiguous Individuals 2 (13) 3 (32) 4 (24) 5 (28) 6 (11) Phase v2, no LD 3.6 × 10-5 0.81 27/4 0.91 0.11 0.15 0.08 0.09 0.25 0.11 0.27 Phase v2, with LD 2.2 × 10-5 0.81 28/5 0.93 0.10 0.13 0 0.09 0.17 0.14 0.27 Haplotyper 1.0 2.0 × 10-5 0.81 28/5 0.93 0.09 0.13 0 0.06 0.13 0.18 0.27 PL-EM 1.0 2.5 × 10-5 0.82 31/9 0.92 0.11 0.15 0 0.09 0.13 0.21 0.36 SNPHAP 1.0 2.0 × 10-5 0.82 30/7 0.93 0.09 0.13 0 0.09 0.13 0.14 0.27 8 SNPs with Minor Allele Frequency ≥ 5% Phase v2, no LD 4.8 × 10-5 0.79 19/3 0.92 0.11 0.15 Phase v2, with LD 4.1 × 10-5 0.80 20/4 0.92 0.11 0.15 Haplotyper 1.0 3.8 × 10-5 0.81 19/2 0.93 0.10 0.14 PL-EM 1.0 2.3 × 10-5 0.85 22/4 0.94 0.08 0.11 SNPHAP 1.0 3.3 × 10-5 0.85 22/4 0.93 0.08 0.11 Estimation of haplotype frequencies was also highly accurate, and there was no meaningful difference in accuracy among the methods as measured by the similarity index, IF. As measured by the mean squared error (MSE) the implementation of the program Phase that ignored linkage disequilibrium among sites gave marginally lower accuracy for the full data set and for the data set composed of higher frequency alleles, but the magnitude of the MSE was small for all methods and spanned only about a two-fold difference between the best and worst value. PL-EM successfully identified the largest number of correct haplotypes, but this success rate was accompanied by the burden of the highest number of incorrect haplotypes inferred. Indeed, the aggregate frequency of incorrect haplotypes inferred by PL-EM was about 1% higher than for the other methods. This observation may have practical value for the analysis of unphased genotypic data. PL-EM may be slightly advantageous if the analytical goal of identify the largest number of correct haplotypes is much more important than minimization of the number of incorrect haplotypes inferred, which may be the case in studies of functional genetics. However, if minimization of the number of incorrect low-frequency haplotypes is more important, as will usually be the case in genetic epidemiological studies, PL-EM may not be the optimal method. Unfortunately, none of the methods is clearly superior in minimizing the number of incorrect haplotypes inferred. Importantly, none of the methods failed to identify haplotypes with frequencies above 1%. Conversely, no incorrect haplotype was assigned a frequency greater than 1%. Indeed, the aggregate frequency of incorrect haplotypes was ≤ 3.7% by all methods. These results are reassuring in two respects. First, it appears unlikely that any of the methods will fail to identify a haplotype that is a major contributor to disease risk within a study population. Second, it also is unlikely that an incorrect haplotype will be implicated as a significant disease risk. It has been noted previously [17,18] that computational methods tend to over-estimate slightly the frequency of the more common haplotypes. The four most common haplotypes in this data set have an aggregate frequency of 64%. The aggregate frequency inferred for these haplotypes ranged from 63% to 65.9% among the methods. The magnitude of error in the estimation of the frequency of the common haplotypes is very small and indicates that this should not be a significant source of error in studies of population genetics or genetic epidemiology if the present results can be generalized. Effect of number of heterozygous sites The number of possible haplotypes compatible with an individual's unphased genotype is 2k, where k is the number of heterozygous sites. For this reason, the difficulty of correctly assigning haplotypes to subjects increases dramatically as those subjects become heterozygous at more sites. Therefore, the error rate for assigning haplotypes was evaluated based on the number of sites at which subjects were heterozygous. Up to 3 heterozygous sites, the error rate is below 10%. For 4 heterozygous sites the error rate is about 15% and exceeds 20% only when 6 sites are heterozygous. Phase was an odd exception to this pattern due to an unusually high error rate for four heterozygous sites, despite the lowest error rates for five and six heterozygous sites. On the assumption that an error rate not much larger than 10% is desirable for a genetic study, it appears that computationally assigned haplotypes for subjects heterozygous at more than four SNPs should be viewed with extreme caution. Similarly, there is a current effort to define haplotype blocks in the human genome to facilitate genome-wide scans for disease loci with a minimum number of sites that must be genotyped. If the results for this gene can be generalized, it would appear unwise to define haplotype blocks based on more than 4–5 SNPs. Conclusions All of the implementations of the EM and Bayesian methods of haplotype inference had high accuracy. Therefore, if this data set is representative of other SNP genotyping studies one can have high confidence in the assignment of haplotypes and estimation of haplotype frequencies produced by any one of the programs. Each method identified every haplotype with a frequency greater than 1%. Therefore, it is unlikely that any of the methods would fail to identify a haplotype contributing to disease risk in a significant proportion of a population. Conversely, no incorrect haplotype was assigned a frequency greater than 1%, indicating a low probability of an incorrect haplotype being identified as a significant disease risk factor. Assignment of haplotypes was very accurate for subjects heterozygous for up to three SNPs, and was at least 80% accurate for up to five heterozygous sites. This suggests that haplotype blocks should perhaps be defined based on no more than five sites and that this might be the practical limit at which one can have confidence in the assignment of haplotypes to subjects. Methods Genetic analyses The empirically determined set of haplotypes from Horan et al. [23] were kindly provided by Drs. David Cooper and David Millar. To examine the accuracy of computational haplotype inference, five different algorithms (Table 3) were used to infer haplotypes based on the 15 SNPs scored by Horan et al. (2003), and the accuracy of these haplotypes was compared to their empirical determinations. The program HAPLOTYPER [13] takes a Bayesian approach to haplotype inference and a partition-ligation strategy for improving speed and accuracy that divides the data into small segments of consecutive loci during haplotype inference that are later combined. We used the default settings of the htyperv2 program, except that 50 iterations of prediction were requested before the results were reported. Like HAPLOTYPER, Phase 2.0.2 [15] employs a Bayesian approach and partition-ligation. Phase was run both with and without the assumption of decay of linkage disequilibrium (option M) with distance in order to evaluate the effect of this assumption. Phase was run with the default options with these exceptions: five restarting points (-x option), the triallelic site -1 was treated as multiallelic but without the stepwise mutation model (-d option), ten steps through the Markov chain per iteration ("thinning intervals"), and the length of the final run with all loci increased by tenfold (option -X). According to Stephens and Donnelly (2003) HAPLOTYPER and Phase differ primarily in the prior distribution that is used. Phase uses an approximate coalescent that will give greater weight to haplotype resolutions of multilocus genotypes that are most similar to previously resolved haplotypes, while HAPLOTYPER uses a Dirichlet prior that chooses randomly among possible haplotype resolutions if the genotypes can not be made to correspond to previously inferred haplotypes. The program PL-EM [12] combines partition-ligation with the EM algorithm to infer haplotypes. PL-EM was run with these settings: haplotypes with probability of appearance >0.1 reported, 3–4 loci per partition, 154 partial haplotypes passed on in each ligation step, 50 independent runs in each implementation of the EM algorithm. The program SNPHAP [11] by David Clayton also employs the EM algorithm to infer haplotypes, but differs from many implementations by adding one locus at a time and removing from consideration low probability haplotypes after each addition until all loci are added. The default settings for SNPHAP were used. Another popular implementation of the EM algorithm, EM-DeCODER [13], is limited to 100 genotypes and could not be applied to the full set of 154 subjects of Horan et al. (2003). The full data set of Horan et al. [23] includes six sites with a minor allele frequency below 5%. Sites with allele frequencies this low are often ignored in genetic studies. Therefore, haplotypes were also inferred based upon a restricted set of sites that excluded six sites (-476, -339, -168, +3, +16, and +25) with minor allele frequencies below 5% and excluded site -301 which is in complete linkage disequilibium with site -308. Additionally, the single individual bearing a C allele at sites +1 and +3 was excluded due to the extremely low frequency of that allele. This left eight sites upon which to perform haplotype inference. Pairwise D' [25], the linkage disequilibium statistic D standardized by its maximum value, was calculated for loci with minor allele frequencies above 5% using the program Arlequin v2.000 [26] based on the empirical haplotypes provided by Drs. Cooper and Millar. Measures of accuracy of haplotype inference The accuracy of haplotype inference was examined by several metrics. The mean squared error (MSE) [18] is defined as where pek and ptk are the inferred and empirically determined frequencies for the kth haplotype, and h is the number of haplotypes. IF and IH were proposed by Excoffier and Slatkin [10]. IF is another measure of how closely the inferred and empirical haplotype frequencies correspond and is given by where the variables are defined as above. IF ranges from 0 to a maximum value of 1 when the frequencies match perfectly. IH compares the number of haplotypes inferred to the number actually known to occur and ranges from 0 to 1 (complete correspondence between inferred and true). IH is defined as where mtrue is the number of haplotypes known to occur, mest is the number of inferred haplotypes with frequency ≥ 1/(2n), and mmissed is the number of known haplotypes that were not inferred. The error rate [13] is the proportion of subjects whose inferred haplotypes are not completely accurate. Acknowledgements This work was supported by funding from the Children's Foundation Research Center of Memphis at Le Bonheur Children's Medical Center, the Center of Genomics and Bioinformatics at the University of Tennessee – Memphis, and a University of Massachusetts-Baystate Medical Center Collaborative Biomedical Research Grant. I thank Dr. Julia Krushkal for analytical advice. ==== Refs Templeton AR Sing CF Kessling A Humphries S A cladistic analysis of phenotype associations with haplotypes inferred from restriction endonuclease mapping. II. The analysis of natural populations Genetics 1988 120 1145 1154 3147219 Tishkoff SA Goldman A Calafell F Speed WC Deinard AS Bonne-Tamir B Kidd JR Pakstis AJ Jenkins T Kidd KK A global haplotype analysis of the myotonic dystrophy locus: implications for the evolution of modern humans and for the origin of myotonic dystrophy mutations Am J Hum Genet 1998 62 1389 1402 9585589 10.1086/301861 Kruglyak L Prospects for whole-genome linkage disequilibrium mapping of common disease genes Nat Genet 1999 22 139 144 10369254 10.1038/9642 Judson R Stephens JC Windemuth A The predictive power of haplotypes in clinical response Pharmacogenomics 2000 1 15 26 11258593 Martin ER Lai EH Gilbert JR Rogala AR Afshari AJ Riley J Finch KL Stevens JF Livak KJ Slotterbeck BD Slifer SH Warren LL Conneally PM Schmechel DE Purvis I Pericak-Vance MA Roses AD Vance JM SNPing away at complex diseases: analysis of single-nucleotide polymorphisms around APOE in Alzheimer disease Am J Hum Genet 2000 67 383 394 10869235 10.1086/303003 Drysdale CM McGraw DW Stack CB Stephens JC Judson RS Nandabalan K Arnold K Ruano G Liggett SB Complex promoter and coding region beta 2-adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness Proc Natl Acad Sci U S A 2000 97 10483 10488 10984540 10.1073/pnas.97.19.10483 Joosten PH Toepoel M Mariman EC Van Zoelen EJ Promoter haplotype combinations of the platelet-derived growth factor alpha-receptor gene predispose to human neural tube defects Nat Genet 2001 27 215 217 11175793 10.1038/84867 Fallin D Cohen A Essioux L Chumakov I Blumenfeld M Cohen D Schork NJ Genetic analysis of case/control data using estimated haplotype frequencies: application to APOE locus variation and Alzheimer's disease Genome Res 2001 11 143 151 11156623 10.1101/gr.148401 Tishkoff SA Varkonyi R Cahinhinan N Abbes S Argyropoulos G Destro-Bisol G Drousiotou A Dangerfield B Lefranc G Loiselet J Piro A Stoneking M Tagarelli A Tagarelli G Touma EH Williams SM Clark AG Haplotype diversity and linkage disequilibrium at human G6PD: recent origin of alleles that confer malarial resistance Science 2001 293 455 462 11423617 10.1126/science.1061573 Excoffier L Slatkin M Maximum-likelihood estimation of molecular haplotype frequencies in a diploid population Mol Biol Evol 1995 12 921 927 7476138 Clayton D SNPHAP: a program for estimating frequencies of large haplotypes of SNPs (Version 1.0) Qin ZS Niu T Liu JS Partition-ligation-expectation-maximization algorithm for haplotype inference with single-nucleotide polymorphisms Am J Hum Genet 2002 71 1242 1247 12452179 10.1086/344207 Niu T Qin ZS Xu X Liu JS Bayesian haplotype inference for multiple linked single-nucleotide polymorphisms Am J Hum Genet 2002 70 157 169 11741196 10.1086/338446 Stephens M Smith NJ Donnelly P A new statistical method for haplotype reconstruction from population data Am J Hum Genet 2001 68 978 989 11254454 10.1086/319501 Stephens M Donnelly P A comparison of bayesian methods for haplotype reconstruction. Am J Hum Genet 2003 Clark AG Inference of haplotypes from PCR-amplified samples of diploid populations Mol Biol Evol 1990 7 111 122 2108305 Xu CF Lewis K Cantone KL Khan P Donnelly C White N Crocker N Boyd PR Zaykin DV Purvis IJ Effectiveness of computational methods in haplotype prediction Hum Genet 2002 110 148 156 11935321 10.1007/s00439-001-0656-4 Fallin D Schork NJ Accuracy of haplotype frequency estimation for biallelic loci, via the expectation-maximization algorithm for unphased diploid genotype data Am J Hum Genet 2000 67 947 959 10954684 10.1086/303069 Zhang S Pakstis AJ Kidd KK Zhao H Comparisons of two methods for haplotype reconstruction and haplotype frequency estimation from population data Am J Hum Genet 2001 69 906 914 11536083 10.1086/323622 Chen EY Liao YC Smith DH Barrera-Saldana HA Gelinas RE Seeburg PH The human growth hormone locus: nucleotide sequence, biology, and evolution Genomics 1989 4 479 497 2744760 Giordano M Marchetti C Chiorboli E Bona G Momigliano Richiardi P Evidence for gene conversion in the generation of extensive polymorphism in the promoter of the growth hormone gene Hum Genet 1997 100 249 255 9254859 10.1007/s004390050500 Wagner JK Eble A Cogan JD Prince MA Phillips J. A., 3rd Mullis PE Allelic variations in the human growth hormone-1 gene promoter of growth hormone-deficient patients and normal controls Eur J Endocrinol 1997 137 474 481 9405026 Horan M Millar DS Hedderich J Lewis G Newsway V Mo N Fryklund L Procter AM Krawczak M Cooper DN Human growth hormone 1 (GH1) gene expression: complex haplotype-dependent influence of polymorphic variation in the proximal promoter and locus control region Hum Mutat 2003 21 408 423 12655556 10.1002/humu.10167 Krawczak M Chuzhanova NA Cooper DN Evolution of the proximal promoter region of the mammalian growth hormone gene Gene 1999 237 143 151 10524245 10.1016/S0378-1119(99)00313-3 Lewontin RC The interaction of selection and linkage. I. General considerations; heterotic models. Genetics 1964 49 49 67 17248194 Schneider S Roessli D Excoffier L Arlequin ver 2.000 2000 Geneva, Switzerland

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==== Front BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-5-491526876610.1186/1471-2164-5-49Research ArticleThe FU gene and its possible protein isoforms Østerlund Torben [email protected] David B [email protected] Regina C [email protected] Monica [email protected]öthen Markus M [email protected] Charles E [email protected] Peter G [email protected]ård Rune [email protected] Department of Biosciences at Novum, Karolinska Institutet, SE-141 57 Huddinge, Sweden2 J.C. Self Research Institute, Greenwood Genetic Center, Greenwood, South Carolina, USA3 Department of Medical Genetics, University of Antwerp, Antwerp, Belgium4 Institute of Human Genetics, University of Bonn, Bonn, Germany5 Pharmacia Corp., Milan, Italy6 Life & Brain Center, University of Bonn, Bonn, Germany2004 22 7 2004 5 49 49 22 3 2004 22 7 2004 Copyright © 2004 Østerlund et al; licensee BioMed Central Ltd.2004Østerlund et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background FU is the human homologue of the Drosophila gene fused whose product fused is a positive regulator of the transcription factor Cubitus interruptus (Ci). Thus, FU may act as a regulator of the human counterparts of Ci, the GLI transcription factors. Since Ci and GLI are targets of Hedgehog signaling in development and morphogenesis, it is expected that FU plays an important role in Sonic, Desert and/or Indian Hedgehog induced cellular signaling. Results The FU gene was identified on chromosome 2q35 at 217.56 Mb and its exon-intron organization determined. The human developmental disorder Syndactyly type 1 (SD1) maps to this region on chromosome 2 and the FU coding region was sequenced using genomic DNA from an affected individual in a linked family. While no FU mutations were found, three single nucleotide polymorphisms were identified. The expression pattern of FU was thoroughly investigated and all examined tissues express FU. It is also clear that different tissues express transcripts of different sizes and some tissues express more than one transcript. By means of nested PCR of specific regions in RT/PCR generated cDNA, it was possible to verify two alternative splicing events. This also suggests the existence of at least two additional protein isoforms besides the FU protein that has previously been described. This long FU and a much shorter isoform were compared for the ability to regulate GLI1 and GLI2. None of the FU isoforms showed any effects on GLI1 induced transcription but the long form can enhance GLI2 activity. Apparently FU did not have any effect on SUFU induced inhibition of GLI. Conclusions The FU gene and its genomic structure was identified. FU is a candidate gene for SD1, but we have not identified a pathogenic mutation in the FU coding region in a family with SD1. The sequence information and expression analyses show that transcripts of different sizes are expressed and subjected to alternative splicing. Thus, mRNAs may contain different 5'UTRs and encode different protein isoforms. Furthermore, FU is able to enhance the activity of GLI2 but not of GLI1, implicating FU in some aspects of Hedgehog signaling. ==== Body Background The signaling molecule Hedgehog (Hh) and components of its intracellular signaling pathway have been the subject of intensive research in several species from fruit fly to man during recent years. Numerous developmental and morphogenic processes are controlled by the Hedgehog family of proteins. Much effort has been directed at identifying components of the signaling pathway and their respective roles and interactions [for an extensive review see [1]]. In Drosophila, Hh signaling to the transcription factor Cubitus interruptus (Ci) is mediated by a protein complex consisting of Ci and three other cytosolic proteins. These are the costal 2 (cos2), suppressor of fused (su(fu)) and fused (fu), where fu is a kinase domain containing protein with positive regulatory activities in Hh induction of Ci mediated transcriptional activation. Hh binds to its receptor patched (ptc), a 12 membrane spanning protein, leading to the activation of another membrane protein smoothened (smo) [2,3]. Smo is a 7 transmembrane protein that, by an unknown mechanism, signals to the Ci containing protein complex leading to activation of Ci. Vertebrate homologues of these Drosophila genes and proteins have been identified during the last decade. To a large extent the signaling pathway has been conserved in vertebrates. However, the picture is more complicated since some of the Drosophila genes have two or more vertebrate homologues. There are three Ci homologues in vertebrates, GLI1, GLI2 and GLI3. GLI1 has activation properties whereas GLI2 and GLI3 have both activation and repression activities [reviewed in [4]]. It is expected that the human homologue of fu (FU) is a positive regulator of GLI proteins, whereas the su(fu) homologue SUFU is a negative regulator. It has been shown by several groups that SUFU inhibits both GLI1 and GLI2 transcriptional activity and has major effects on the shuttling between cytosol and nucleus [5-7]. In a similar way it was shown in C3H/10T½ cells that FU is a positive regulator of GLI2 but with little effect on GLI1 [8]. FU is a 1315 residue protein with high similarity to fu in the N-terminal kinase domain. Interestingly, it was discovered that mutations in PTCH1, the human counterpart of ptc, underlie the Nevoid Basal Cell Carcinoma Syndrome (NBCCS) [9,10]. Patients with NBCCS (also known as Gorlin syndrome) have developmental abnormalities and eventually develop basal cell carcinoma (BCC) and other tumors like medulloblastoma and rhabdomyosarcoma [11,12]. Also SMO and SUFU mutations as well as overexpression of GLI1 or GLI2 can lead to BCC or medulloblastoma [13-16]. Thus, investigations of this signaling pathway, its genes and protein components, is not only important for understanding development and morphogenesis, but also for cancer biology. Here three FU cDNA clones have been identified and used for sequence analysis, identification and structural description of the FU gene, as well as for construction and subcloning of FU expression vectors. Using the available public databases the FU gene was found to be present in a sequenced BAC clone from chromosome 2. FU is located in the same region of chromosome 2q34-q36 to which the human limb malformation disorder Syndactyly type 1 (SD1) has recently been mapped in a large German pedigree [17] and confirmed in an Iranian family [18]. Its possible association with this condition was investigated by sequencing the coding exons of the FU gene in an affected member from the German family [17]. The tissue expression pattern of FU has been determined using an RNA array and Northern blots. FU is expressed in all 72 tested tissues. It is clear that not only a single transcript is expressed. Instead transcripts of different sizes are seen and some tissues apparently express more than one major transcript. From the genomic structure and the cDNA clones it was possible to predict several alternative splicing events and consequently the likely expression of different protein isoforms. Two of the isoforms were expressed in HEK293 cells and tested for their ability to regulate the activity of GLI1 and GLI2, showing positive effects on GLI2 but not on GLI1. Results Chromosomal localization of FU The sequence information derived from the FU cDNA clones 1HFU, 2HFU and Ngo3689 (see Methods) allowed the identification of the FU gene in a 200 kb BAC clone (AC009974) from chromosome 2. The gene is localized to 2q35 at 217.56 Mb using the Ensembl [19] annotation. The Ensembl gene prediction programs have identified most, but not all (21 of 29 exons; the published FU [8] predicts 26 exons) of the FU structure and named the gene STK36 (serine/threonine protein kinase 36). Chromosome 2q35 is the locus of several genetically based disorders. Both Syndactyly type 1 (SD1) and Brachydactyly type A1 (BDA1) have been mapped to this region [17,18,20]. Recently, the gene responsible for BDA1 has been identified as IHH (Indian Hedgehog) one of the vertebrate Hh homologues [21]. IHH is located in the vicinity of FU on chromosome 2 (217.94 Mb) less than 400 kb away. In order to determine if alterations of FU are responsible for SD1, the FU coding region (exons 3–29) and the flanking intronic regions were sequenced using genomic DNA from an affected member of an SD1 family whose trait maps to the 2q34-q36 region [17] and an unrelated control individual. No FU mutations were detected in this study, although three single nucleotide polymorphisms were identified. These included a T to C transition in intron 10, 17 bp 5' of exon 11 (IVS10-17T>C), causing gain of a BstNI site, and a G to A transition in exon 16, 17 bp 5' of the end of the exon (1748G>A), causing substitution of glutamine for arginine at amino acid 583 (R583Q) and loss of an AciI site. The altered restriction sites created by these sequence changes were tested in 44 CEPH unrelated individuals. The results showed that both changes are normal sequence variations as previously reported in the NCBI SNP database. The third change was a G to A transition in exon 27 (3008G>A), causing substitution of aspartic acid for glycine at amino acid 1003 (G1003D). By sequencing exon 27 in 8 affected and 6 unaffected members of the SD1 family [17], the disease variant could be observed in affected and unaffected members of the family, and a homozygous healthy individual was found. This variant has also been reported previously as a single nucleotide polymorphism in the NCBI SNP database. FU structure None of the obtained cDNA clones contain sequence from all FU exons, but they allow determination of the exon-intron organization of FU. Figure 1 shows the structure of the FU gene. The cDNA clones are outlined to account for the predicted structure. Only clone 1HFU and Ngo3689 contain exons from the 5'non-coding region. To the 5' side of the sequence encoded by exon 3 these clones are different, indicating that alternative 5' untranslated regions (UTR) from different exons can be used. Exons 3 to 9 encode the N-terminal kinase domain. None of the cDNA clones encodes the FU protein that has previously been described [8]. 1HFU lacks the sequence encoded by exon 8, which results in a frame shift and a premature termination of translation. However, it cannot be unambiguously excluded that this may encode a very short protein isoform having only a partial kinase domain. 1HFU also includes the sequence from exon 13, which encodes an in frame stop codon. Neither Ngo3689 nor 2HFU contain the sequence encoded by exon 13. It is suggested that inclusion of exon 13 gives rise to a shorter protein (S-FU) of 474 residues, encode by exons 3 to 13. The previously described [8] long form of FU (L-FU) having 1315 residues is encoded by exons 3 to 29 without inclusion of exon 13. An additional alternative splice variant is suggested from the Ngo3689 clone. The first 63 bp of the sequence in exon 24 are missing. This results in a protein that is 21 residues shorter than L-FU (encoded by exons 3 to 29 without exon 13 and the 63 bp). Since almost all of the sequence from exon 24 is missing (only 18 bp are left) this isoform is termed L-FUΔ24. The Ngo3689 clone also contains all the 289 bp from intron 17, but whether this represents a true alternative splice variant is doubtful. Figure 1 FU gene structure. All sequenced segments of the available FU cDNA clones (1HFU, 2HFU and Ngo3689) were identified in a BAC clone (AC009974) from chromosome 2. This allowed the identification of the exonic sequences and thereby also the introns. The FU protein sequence [8] and translational analysis provided information about the role of different exons. Exons 1 and 2 encode 5'UTRs shown in brown. Exon 3 contains the initiating ATG codon approximately in the middle (position 90–92 from 5'end). Exons 3 to 9 encode the kinase domain shown in red. As judged from the cDNA clones the sequences encoded by exons 8, 13 and part of exon 24 are subjected to alternative splicing. Both exon 13 and 29 encode in frame stop codons. The cDNA clones end with a poly A+ tail at the same position starting 706 bp from the stop codon (TGA) in exon 29. Multi tissue array and northern blot analyses A tissue array with poly A+ RNA from 72 human tissues was hybridized with a labeled probe 3' of the kinase domain. It was clear from this array that all examined tissues express FU to some extent. The highest amount of FU transcripts were detected in testis and pituitary (not shown). This is in agreement with the previous results by Northern blotting, showing highest FU expression in testis [8]. This analysis revealed that most tissues express an approximately 5 kb transcript [8]. The Northern blot analysis was here repeated with a larger number of tissues and a probe containing a 3' portion of the gene (exon 28). Figure 2 shows results of the three different Northern blots used. Here the transcripts are estimated to be a bit larger than the reported 5 kb, generally in the range of 6 to 7 kb. It should be emphasized that the identified cDNA clones are approximately 5 kb and that this seems closer to the correct sizes of transcripts, though they may appear larger on the Northern blots. Adult skeletal muscle, thymus, spleen, liver, small intestine, placenta, lung and leukocytes show a faint 6.5 kb band. However, the adult tissues brain, heart, colon and thyroid express a shorter transcript of 6 kb. Adrenal seems to preferentially express a band in the 6.5 to 7 kb range. In pancreas, fetal brain and fetal kidney it appears that at least two bands are expressed in the range from 6 to 7 kb. Besides, mRNA from fetal brain and lung also give rise to a band of much larger size around 9.5 kb. It is not clear if this constitutes a transcript that has not been fully processed, or whether it may contain sequences from as yet unidentified exons, for instance unknown 5'UTRs. Fetal lung and liver clearly preferentially express transcripts of different sizes, 7 and 6 kb respectively. It is confirmed that adult testis shows the highest expression but also pancreas, kidney, fetal brain and kidney stand out, in agreement with the previously reported Northern analysis [8]. Since one major alternative splicing event seems to involve exon 13, we attempted to evaluate the tissue specificity of this. A probe containing only the exon 13 sequence was used in hybridization of the Northern blots. This resulted in smeary bands irrespective of the hybridization conditions used (not shown). A likely explanation is that the probe is too short to achieve high specificity hybridization, or perhaps the transcripts containing exon 13 are degraded much faster, possibly due to the process of nonsense mediated decay [22]. Figure 2 Northern blot analysis of FU expression in human tissues. Three commercially available Northern blots were hybridized with a labeled FU probe and analyzed by phosphorimaging. The blots have RNA from endocrine organs, other adult and fetal tissues as indicated. Size markers are shown to the left. Nested PCR analyses To examine more specifically the expression of exons that are involved in alternative splicing events, nested PCR was performed on cDNA probes generated from whole tissue RNA by reverse transcription. Sequences containing segments including exons 8, 13 and 24 were amplified and analyzed on agarose gels (Fig. 3). It was not possible to detect transcripts that lack exon 8 (Fig. 3, panel A). In contrast, it appears that transcripts both with and without exon 13 are present (Fig. 3, panel B). Expression of transcripts without exon 13 is clearly most prevalent in all tissues. The expression of the longer form seems to be proportional to the expression of the shorter form. This indicates that this splicing event is not subjected to any significant tissue specific regulation. However, since a detectable amount of transcripts including exon 13 are present, it is likely that S-FU is also expressed in the tissues. The analysis of alternative splicing of the part encoded by exon 24 turned out to be difficult and several primer pairs were tested before reliable results could be obtained (Fig. 3, panel C). Interestingly, the examined tissues show very different expression patterns, suggesting that alternative splicing in this case is a regulated event. Most tissues express all of exon 24 but in small intestine and prostate clear expression of a transcript without the 63 bp is observed, and in testis expression of transcripts both with and without this segment is found. Sequence analysis shows that the 63 bp segment is likely to encode part of a leucine zipper domain and therefore a putative protein interaction may be lost in this isoform (L-FUΔ24). Figure 3 Analyses of alternative splicing by nested PCR. Nested PCR was performed on cDNA from 8 tissues, over regions implicated to undergo alternative splicing. These are encoded by exons 8 (panel A), 13 (panel B) and 24 (panel C). Available cDNA clones either with or without these parts served as controls as indicated. The PCR products were analyzed on agarose gels as shown. Functional analyses of FU isoforms Investigations into the ability of FU isoforms to regulate GLI transcription factors and SUFU has here been initiated by expression of both L-FU and S-FU as well as 2HFU, which does not have a full kinase domain, in HEK293 cells. The 293 cells, unlike the previously used C3H/10T½ cells [8], do not have a complete Hedgehog signaling pathway. Thus, it is possible to determine if FUs have direct effects on GLI proteins as it was done for SUFU [5]. The assay is based on the induction of a luciferase reporter construct having 12 consecutive binding elements for GLI transcription factors [5]. In all transfection assays GLI1 was able to induce the luciferase reporter 100–250 fold, whereas GLI2 induced the reporter some 15–30 fold, depending on cell density and the amount of construct used. Figure 4 shows the results of these expression analyses. As shown previously [5] SUFU has a strong inhibitory effect on GLI1. Moreover, a similar strong effect was seen on GLI2 (Fig 4., panel A). The results presented are typical for a large number of experiments and depend on the amount of GLI and SUFU that is used. In contrast to the strong effects seen with SUFU, none of the FUs revealed major changes (Fig. 4, panel B and C). It is clear that the FUs are not able to regulate GLI1 at all, though L-FU and 2HFU have a weak (2–3 fold) positive effect on GLI2. It appears that L-FU has a slightly stronger effect than 2HFU. This is qualitatively the same result as was obtained in C3H/10T½ cells, where the L-FU was also compared to a kinase-dead mutant and a 546 residue variant similar to S-FU. Thus, in both cases there is no evidence that the kinase domain is required for the activation of GLI2. Unlike the previous analyses [8], it cannot be confirmed that FUs have a direct effect on SUFU function (Fig. 4, panel D). The inhibitory effect of SUFU on GLI1 is not relieved by the addition of FU. The effect on GLI2/SUFU cannot be distinguished from the effect on GLI2 alone, implying that FU does not regulate SUFU but may only affect GLI2. Since 293 cells lack components of the Hedgehog signaling pathway, it is possible that an effect of L-FU on SUFU [8] requires the presence and activity of additional molecules. Figure 4 Regulation of GLI induced transcriptional activity in transfected HEK293 cells. HEK293 cells were transfected with a GLI inducible luciferase reporter construct together with a GLI1 or GLI2 expression construct. The cells were also transfected with a β-galactosidase construct that served to correct for transfection efficiency and cell density. The effects of FU and SUFU were tested by cotransfecting FU and SUFU expression constructs and compared to effects with empty vectors. Panel A shows typical examples of SUFU effects on GLI1 and GLI2. Panel B shows typical examples of the effects of different FU constructs on GLI1 and GLI2; experiments that were performed in parallel are shown, to illustrate the different impact on the GLI proteins. L-FU is shown with squares, 2HFU with triangles and S-FU with circles. Panel C shows the impact of FU constructs (400 ng) on GLI1 and GLI2 as summarized by the results of at least 4 different experiments. Panel D shows the impact of FU constructs (400 ng) on SUFU inhibited GLI1 and GLI2 as summarized by the results of 3 different experiments. In these experiments the GLI induced transcriptional activity was inhibited to 20–40 % of the non-inhibited level by the addition of SUFU (i.e. 2½ to 5 fold inhibition). Relative activity (panel A) is given as compared to activity in mock transfected cells and normalized activity (panel B-D) is given relative to activity in cells transfected with GLI and GLI+SUFU set to 1. Discussion The FU gene In the present paper, the FU gene was identified and its structure determined. FU consists of 29 exons of which exons 1 and 2 encode 5'UTRs, exon 3 contains the initiating ATG codon and exons 13 and 29 contain in frame stop codons (Fig. 1). Exon 1 and 2 may serve as alternative first exons, like the alternative exons 1, 1A and 1B found in the PTCH1 gene [23]. Exons 3 to 9 encode the kinase domain. This segment has strong similarity to Drosophila fu, whereas the remaining C-terminal part has a much weaker similarity [8]. Using the DIALIGN program [24] it is possible to align fu to L-FU in two regions in the C-terminal part (not shown). These are largely encoded by exons 15–16 and 22–29. This indicates that exons 10–14 and 17–21 may have been recruited to the FU gene during evolution. Investigations of Syndactyly patient material We investigated the possibility that FU underlies SD1 based upon the fact that FU lies within the localization interval for SD1 and that it is part of the Sonic Hedgehog signaling pathway, which participates in digital patterning [1]. Although three previously reported single nucleotide polymorphisms were identified, we did not detect any mutation in the FU coding region or flanking intronic regions. While these results do not implicate FU in the causation of SD1, it is possible that this disorder is caused by mutations in the noncoding regions not screened in this study. Alternatively, SD1 could be caused by a genomic rearrangement not identified by sequence analysis, although no altered bands were detected in an affected member of the SD1 family by Southern analysis using a FU cDNA clone as probe (data not shown). Expression analyses Analyses of FU expression have shown that transcripts are detected in all tissues examined. For the first time evidence is presented showing that more than one transcript can be expressed from this gene. The Northern blots clearly show that FU transcripts of different sizes indeed exist. Here the transcripts are estimated to be slightly bigger than previously reported and in some tissues more than one transcript is evident. It is clear from the available cDNAs and RT/PCR based transcript analyses that alternative splicing occurs. Additionally, it is also clear that different 5'UTRs are present in the transcripts. At least two protein isoforms, besides the previously described L-FU [8], may be produced. The S-FU isoform is the one that most dramatically differs from L-FU, consisting only of the N-terminal one third of L-FU. S-FU expression results from inclusion of exon 13 in the mature transcript. This alternative splicing event was detected in all tissues examined and at an apparently constant ratio. Also a case of regulated alternative splicing was detected by RT/PCR, but with a much less dramatic impact at the protein level, since it only results in the loss of 21 residues encoded by exon 24. However, the expression reveals a possible tissue specific regulation of this alternative splicing event. This may well reflect that L-FUΔ24 plays a biological role different from L-FU. Since it appears that the mRNA for L-FUΔ24 is not expressed in small intestine and prostate it can be speculated that FU has a different role there, if a leucine zipper is truly lost in L-FUΔ24. It is intriguing that testis appears to express transcripts both with and without the 63 bp segment and is also the tissue with strongest expression. Perhaps the expression of L-FUΔ24 and L-FU together is linked to the function of Desert Hedgehog which has been shown to have a particular role in spermatogenesis [25]. Whether interactions with GLI proteins, SUFU or other components of the signaling pathway are altered, and if this has any impact on GLI or SUFU activities, remains to be investigated. Certainly this adds another variable to the complicated picture of Hedgehog signaling and GLI regulation in vertebrates. Functional investigations and perspectives The assessment of functionality revealed that S-FU was not able to regulate GLI1 or GLI2 when expressed in 293 cells. In contrast, both L-FU and a variant lacking a full kinase domain (2HFU) were able to enhance GLI2 induced transcription. These results are qualitatively similar to those previously reported in C3H/10T½ cells [8]. L-FU and 2HFU were only able to enhance GLI2 activity 2 to 3 fold in 293 cells, whereas 5 to 8 fold inductions are seen in C3H/10T½ cells. This may reflect the fact that the latter cell line expresses additional components of the Hedgehog signaling pathway, which are required for full activity of FU. Unlike the previous investigations [8] it was not possible to see an effect of L-FU on SUFU. Again this difference may be explained by the various properties of the cell lines used. Understanding the signaling events downstream of SMO may reveal functional differences of the proteins involved, as compared to their fruit fly counterparts. Although SUFU inhibits GLI transcription factors and su(fu) inhibits Ci, there are still striking differences. As yet there have been no reports of a cos2 counterpart in vertebrates. Instead it has been observed that FU interacts with all GLI proteins and SUFU [8], even though fu does not bind to Ci [26]. It has also been observed that both L-FU and SUFU can be found in the nucleus [5-8], which has not been observed for fu or su(fu). It is likely that both FU and SUFU are shuttled in and out of the nucleus by binding to GLI proteins [5,8]. Though basic activities of both FU and SUFU in regulation of GLI have been conserved, it also appears that significant differences from their fruit fly counterparts exist. Clearly, FU is not having an effect on GLI1 similar to the one seen on GLI2. Additional investigations are needed in order to establish the role of FU in hedgehog signaling and GLI control. The role of the different isoforms also remains to be elucidated. These have to be tested individually for their regulation of all GLI proteins and proteolytic products. Fu is known to have at least two separate physiological functions in the fly, one of which is dependent upon the kinase domain [27]. Likewise, FU may well have two or more distinct functions in signaling, represented by different domains, isoforms and protein interactions. Conclusions FU is localized on chromosome 2q35 very close to IHH. Though SD1 has been mapped to this region, we have not identified a causative role for FU in this disorder. FU consists of 29 exons of which 1 and 2 encode 5'UTRs and 3 to 9 encode a kinase domain. For the first time it is shown that transcripts of different sizes are expressed and alternative splicing takes place, probably leading to the generation of different protein isoforms. FU protein is likely to be involved in the Hedgehog signaling pathway since it can enhance the activity of GLI2. In contrast, it has no effect on GLI1 and an effect on SUFU cannot be observed in 293 cells. Methods The FU cDNA clones Two almost full-length human FU clones were identified in the Incyte database. Both 1HFU and 2HFU were cloned in the vector pINCY. A third clone was available from Kazusa DNA Research Institute (Chiba, Japan) and termed Ngo3689 (Gene name KIAA1278). This clone was in the vector pBluescript II SK+. The human BAC clone AC009974 was obtained from Research Genetics (Huntsville, AL). The human GLI, human SUFU, 12GLI-RE-luciferase reporter and β-galactosidase vectors have been described previously [5]. FU cDNA subcloning Expression constructs for different isoforms of FU was obtained by direct PCR or extension overlap PCR, using end-primers having specific restriction sites and the high fidelity VentR DNA polymerase (New England Biolabs, Beverly, MA). The cDNA for the long form of FU (L-FU) was subcloned into pCDNA3.1-HisB using the NotI and XbaI sites. 2HFU and the short FU (S-FU) cDNAs were subcloned into pCDNA3.1-HisC using the KpnI and XbaI sites. DNA sequencing and analyses All PCR generated products were analyzed by DNA sequencing. The Big-Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA) was used according to instructions. Sequencing was performed at CyberGene AB (Huddinge, Sweden). Sequence alignments were done using the DIALIGN program [24] available at the BiBiServ from University of Bielefeld, Germany. Sequence information of proteins, clones and chromosomes were obtained from the Swiss-Pro [28], Entrez [29] and Ensembl [19] databases. Analyses of genomic DNA from family members with SD1 After informed consent was obtained, blood was taken from affected and unaffected family members and DNA extracted from peripheral blood leukocytes according to standard methods. Intronic primers were designed to amplify exons 3–29 of FU either as single exons with flanking intronic sequences or as products containing two exons with flanking intronic sequence and the complete intervening intron. The primer sequences can be obtained upon request. PCR was performed in a standard fashion and products were sequenced using either the Thermosequenase CyTM5.5 Dye Terminator or DYEnamic ET Dye Terminator Cycle Sequencing kits (Amersham Biosciences, Piscataway, NJ). Electrophoresis and analysis were performed on either an Automated Laser Fluorescence (ALF) DNA sequencer or MegaBACE DNA sequencer (Amersham Biosciences) after purification with Autoseq columns (Amersham Biosciences). For exon 27, the PCR product was purified using the enzymatic ExoI-SAP purification method, sequenced using the Terminator Cycle Sequencing kit (Amersham Pharmacia Biotech) and analysed on an ABI 3100 genetic analyzer (Applied Biosystems). PCR products containing exon 11 or exons 15/16 were digested with BstNI or AciI, respectively, and the bands resolved on 3–4% agarose gels to confirm sequence changes in the patient with SD1 and to determine their frequency in a panel of 44 CEPH individuals. Northern blot analysis Commercially available Human MTN 12-lane Blot 2, Human Fetal MTN Blot II and Human Endocrine System MTN Blot Northern blots (Clontech, Paolo Alto, CA) were obtained and used with PCR generated hybridization probes. DNA probes were made by direct PCR, amplifying the sequences corresponding to exon 13 and 28. The generated fragments were then labeled with 32P-ATP using the High Prime DNA labeling kit (Boehringer Mannheim, Mannheim, Germany) according to instructions. Hybridization of Northern blots was done with labeled DNA probes in ExpressHyp (Clontech) at 68°C according to instructions. The blots were then analyzed with a Fujix Bas 2000 phosphoimager (Fuji Photo Film, Tokyo, Japan). Expression analysis by nested PCR The expression of exon 8, 13 and 24 sequences in mRNA was assessed by nested PCR on RT/PCR generated cDNA samples from eight different tissues as provided in Human Multi Tissue cDNA Panel II (Clontech). Two sets of primers were made for each exon to be investigated. The outer pairs were used in a first PCR using 5 μl of the cDNA and Vent polymerase. In a second PCR 0.5 μl of the first PCR products was used together with the inner primer pairs. These pairs were also used for PCR of FU cDNA clones that served as controls. The primer pairs are listed in Table 1. The PCR reactions were performed using 95°C for 1 min denaturation, elongation at 72°C and 40 cycles. The exon 8 and 13 sequence PCRs were performed using 60°C 1 min annealing and 1 min elongation. The exon 24 sequence PCRs were performed using 59°C 1 min annealing and 1 min 30 sec elongation. Table 1 Primers for nested PCR analyses Sequence from exon Outer PCR primer pairs Inner PCR primer pairs 8 fwd 5'-AACATCCTCCTCGCCAAGGGT 5'-ATATGAACTGGCAGTAGGCAC rev 5'-TGCTCTCCTGACTGT GCCTGAGTAGACTCA 5'-TTACCCTTGGGGGCCAACCGA 13 fwd 5'-AACATCCTCCTCGCCAAGGGT 5'-AGCCTGTGCCTATTCAACTGA rev 5'-TGCTCTCCTGACTGT GCCTGAGTAGACTCA 5'-GCCTCCCGGCAGAAGGAATAC 24 fwd 5'-CGCAAGTGAGCCAGCCACTGC 5'-CAGCCAGCTCAGGCCATCCCT rev 5'-CTGGACCGCAGGAATCT GGAATCACATGCTATGGG 5'-CCAGGCCTGTGAGAAGGCTGA Reporter gene assays The cDNA clones were used in transfections of HEK293 cells in 24 well culture plates. Basically this was done as previously described [5]. In short, the 293 cells were transfected using Superfect Transfection Reagent (Clontech), with 100 ng of the luciferase reporter and β-galactosidase as well as different amounts and combinations of GLI, FU and SUFU constructs. For every assay there was a corresponding control with an equal amount of empty vector. The cells were harvested 24 hours after transfection with 50 μl of lysis buffer from the Galacto-Light kit (Applied Biosystems). Of this was 10 μl used for β-galactosidase assay and the rest for luciferase assay using the Luciferase Assay kit (BioThema, Dalarö, Sweden). Analyses were done on a Microplate Luminometer (Berthold Detection System, Pforzheim, Germany). Authors' contributions TØ contributed to the experimental design; participated in sequencing, sequence analysis and subcloning; did the gene analysis, Northern blots, nested PCR, cell experiments; and made the manuscript draft. DBE and CES designed and carried out the patient analysis; and contributed to the manuscript. MM provided clones; contributed with subcloning; made the array analysis; and contributed to the manuscript. MMN and RCB provided the SD1 patient material; performed segregation analyses in the SD1 family; and edited the manuscript. PGZ contributed to the experimental design and subcloning; and edited the manuscript. RT contributed to the experimental design; did data base analysis; and edited the manuscript. Acknowledgements We thank Kristin Bosse for helping recruiting the family and Chad T. Morgan for excellent technical assistance. This work was supported by the Swedish Cancer Society with a postdoctoral position to TØ and research grant to RT and PGZ, by a grant from Pharmacia to RT and by a grant from the South Carolina Department of Disabilities and Special Needs (SCDDSN) to CES. RCB was supported by a postdoctoral fellowship from the Fund for Scientific Research-Flanders (FWO). ==== Refs Ingham PW McMahon AP Hedgehog signaling in animal development: paradigms and principles Genes Dev 2001 15 3059 3087 11731473 10.1101/gad.938601 Stone DM Hynes M Armanini M Swanson TA Gu Q Johnson RL Scott MP Pennica D Goddard A Phillips H Noll M Hooper JE de Sauvage F Rosenthal A The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog Nature 1996 384 129 134 8906787 10.1038/384129a0 Marigo V Davey RA Zuo Y Cunningham JM Tabin CJ Biochemical evidence that patched is the Hedgehog receptor Nature 1996 384 176 179 8906794 10.1038/384176a0 Matise MP Joyner AL Gli genes in development and cancer Oncogene 1999 18 7852 7859 10630638 10.1038/sj.onc.1203243 Kogerman P Grimm T Kogerman L Krause D Unden AB Sandstedt B Toftgård R Zaphiropoulos PG Mammalian Supressor-of-Fused modulates nuclear-cytoplasmic shuttling of GLI-1 Nat Cell Biol 1999 1 312 319 10559945 10.1038/13031 Ding Q Fukami S Meng X Nishizaki Y Zhang X Sasaki H Dlugosz A Nakafuku M Hui C Mouse suppressor of fused is a negative regulator of sonic hedgehog signaling and alters the subcellular distribution of Gli1 Curr Biol 1999 9 1119 1122 10531011 10.1016/S0960-9822(99)80482-5 Stone DM Murone M Luoh S Ye W Armanini MP Gurney A Phillips H Brush J Goddard A de Sauvage FJ Rosenthal A Characterization of the human suppressor of fused, a negative regulator of the zinc-finger transcription factor Gli J Cell Sci 1999 112 4437 4448 10564661 Murone M Luoh S-M Stone D Li W Gurney A Armanini M Grey C Rosentahl A de Sauvage FJ Gli regulation by the opposing effects of Fused and Supressor of Fused Nat Cell Biol 2000 2 310 313 10806483 10.1038/35010610 Hahn H Wicking C Zaphiropoulos PG Gailani MR Shanley S Chidambaram A Vorechovsky I Holmberg E Unden AB Gillies S Negus K Smyth I Pressman C Leffell DJ Gerrard B Goldstein AM Dean M Toftgård R Chenevix-Trench G Wainwright B Bale AE Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome Cell 1996 85 841 51 8681379 10.1016/S0092-8674(00)81268-4 Johnson RL Rothman AL Xie J Goodrich LV Bare JW Bonifas JM Quinn AG Myers RM Cox DR Epstein EH JrScott MP Human homolog of patched, a candidate gene for the basal cell nevus syndrome Science 1996 272 1668 1671 8658145 Gorlin RJ Nevoid basal-cell carcinoma syndrome Medicine 1987 66 98 113 3547011 Toftgård R Hedgehog Signalling in Cancer Cell Mol Life Sci 2000 57 1720 31 11130178 Xie J Murone M Luoh SM Ryan A Gu Q Zhang C Bonifas JM Lam CW Hynes M Goddard A Rosenthal A Epstein EH Jrde Sauvage FJ Activating Smoothened mutations in sporadic basal-cell carcinoma Nature 1998 391 90 92 9422511 10.1038/34201 Taylor MD Liu L Raffel C Hui CC Mainprize TG Zhang X Agatep R Chiappa S Gao L Lowrance A Hao A Goldstein AM Stavrou T Scherer SW Dura WT Wainwright B Squire JA Rutka JT Hogg D Mutations in SUFU predispose to medulloblastoma Nat Genet 2002 31 306 310 12068298 10.1038/ng916 Nilsson M Unden AB Krause D Malmqwist U Raza K Zaphiropoulos PG Toftgård R Induction of basal cell carcinomas and trichoepitheliomas in mice overexpressing GLI-1 Proc Natl Acad Sci 2000 97 3438 3443 10725363 10.1073/pnas.050467397 Grachtchouk M Mo R Yu S Zhang X Sasaki H Hui CC Dlugosz AA Basal cell carcinomas in mice overexpressing Gli2 in skin Nat Genet 2000 24 216 217 10700170 10.1038/73417 Bosse K Betz RC Lee YA Wienker TF Reis A Kleen H Propping P Cichon S Nöthen MM Localization of a gene for syndactyly type 1 to chromosome 2q34-q36 Am J Hum Genet 2000 67 492 497 10877983 10.1086/303028 Ghadami M Majidzadeh-A K Haerian B-S Damavandi E Yamada K Pasallar P Najafi M-T Nishimura G Tomita H-A Yoshiura K-I Niikawa N Confirmation of genetic homogeneity of Syndactyly Type I in an Iranian family Am J Med Genet 2001 104 147 151 11746046 10.1002/ajmg.10061 Emsembl Genome View Yang X She C Guo J Yu AC Lu Y Shi X Feng G He L A locus for brachydactyly type A-1 maps to chromosome 2q35-q36 Am J Hum Genet 2000 66 892 903 10712204 10.1086/302806 Gao B Guo J She C Shu A Yang M Tan Z Yang X Guo S Feng G He L Mutations in IHH, encoding Indian hedgehog, cause brachydactyly type A-1 Nat Genet 2001 28 386 388 11455389 10.1038/ng577 Wilkinson MF Shyu A-B RNA surveillance by nuclear scanning? Nat Cell Biol 2002 4 E144 147 12042827 10.1038/ncb0602-e144 Kogerman P Krause D Rahnama F Kogerman L Unden AB Zaphiropoulos PG Toftgård R Alternative first exons of PTCH1 are differentially regulated in vivo and may confer different functions to the PTCH1 protein Oncogene 2002 21 6007 6016 12203113 10.1038/sj.onc.1205865 Dialign Bitgood MJ Shen L McMahon AP Sertoli cell signaling by Desert hedgehog regulates the male germline Curr Biol 1996 6 298 304 8805249 10.1016/S0960-9822(02)00480-3 Monnier V Ho KS Sanial M Scott MP Plessis A Hedgehog signal transduction proteins: contacts of the Fused kinase and Ci transcription factor with the Kinesin-related protein Costal2 BMC Dev Biol 2002 19 4 11914126 10.1186/1471-213X-2-4 Ohlmeyer JT Kalderon D Hedgehog stimulates maturation of Cubitus interruptus into a labile transcriptional activator Nature 1998 396 749 753 9874371 10.1038/25533 Swiss-Prot Protein knowledgebase Entrez, The Life Sciences Search Engine

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==== Front BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-5-501528385910.1186/1471-2164-5-50Research ArticleThe Retinome – Defining a reference transcriptome of the adult mammalian retina/retinal pigment epithelium Schulz Heidi L [email protected] Thomas [email protected] Juergen [email protected] Bernhard HF [email protected] Institute of Human Genetics, Biocenter, University of Wuerzburg, D-97074 Wuerzburg, Germany2 German Cancer Research Center, Central Spectroscopic Department, D-69120 Heidelberg, Germany2004 29 7 2004 5 50 50 7 4 2004 29 7 2004 Copyright © 2004 Schulz et al; licensee BioMed Central Ltd.2004Schulz et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The mammalian retina is a valuable model system to study neuronal biology in health and disease. To obtain insight into intrinsic processes of the retina, great efforts are directed towards the identification and characterization of transcripts with functional relevance to this tissue. Results With the goal to assemble a first genome-wide reference transcriptome of the adult mammalian retina, referred to as the retinome, we have extracted 13,037 non-redundant annotated genes from nearly 500,000 published datasets on redundant retina/retinal pigment epithelium (RPE) transcripts. The data were generated from 27 independent studies employing a wide range of molecular and biocomputational approaches. Comparison to known retina-/RPE-specific pathways and established retinal gene networks suggest that the reference retinome may represent up to 90% of the retinal transcripts. We show that the distribution of retinal genes along the chromosomes is not random but exhibits a higher order organization closely following the previously observed clustering of genes with increased expression. Conclusion The genome wide retinome map offers a rational basis for selecting suggestive candidate genes for hereditary as well as complex retinal diseases facilitating elaborate studies into normal and pathological pathways. To make this unique resource freely available we have built a database providing a query interface to the reference retinome [1]. ==== Body Background The mammalian retina is a highly structured tissue developmentally originating from neuroectodermal evagination of the diencephalon and subsequent invagination processes resulting in the formation of two cellular layers which ultimately give rise to the inner neural retina and the outer retinal pigment epithelium (RPE) monolayer [2]. In the adult, the neural retina consists of approximately 55 distinct cell types histologically structured into three layers of cells (photoreceptors, intermediate neurons and ganglion cells) and two layers of neuronal interconnections (outer and inner plexiform layers) [3]. The RPE is differentiated into polarized cells with an apical and a basal orientation separating the neural retina from the underlying choroidal blood supply. With its apical microvilli-like processes, the RPE establishes an intimate contact with the photoreceptor outer segments to sustain their metabolic support and maintain photoreceptor integrity [4]. Together, the neural retina and the RPE provide the structural and functional basis for light perception by ensuring the capture of photons, the conversion of light stimuli into complex patterns of neuronal impulses and the transmission of the initially processed signals to the higher visual centers of the brain. Recent progress in retinal research has greatly enhanced our current understanding of basic functional processes in the adult retina (e.g. [4,5]). A great deal of effort has focused on the molecular dissection of the phototransduction pathway and the retinoid cycle (e.g. ref. [6]). Besides elucidating physiological mechanisms in normal tissue, the identification of genes involved in hereditary retinal disease has provided another valuable source of insight into functional pathways of the retina and the RPE (reviewed in [7,8]). Despite these advances, a remaining challenge is to obtain a reference genome-wide expression map of the retina/RPE transcriptome, further facilitating the identification of retinal susceptibility genes, but most importantly, offering an invaluable resource for functional genomics studies. Initial analyses of human [9,10] and mouse [11] whole genome sequences and the use of more recent comparative gene prediction algorithms [12,13] suggest an overall number of mammalian gene loci in the range of 35,000 to 45,000. These estimates have largely been validated by experimental data on gene transcription [14,15] although alternative promoter usage, differential exon splicing during mRNA maturation, alternative usage of polyadenylation sites and other post-transcriptional modifications may further increase the genetic diversity required to encode the full complement of cellular transcripts [16,17]. In addition, there may be a considerable number of non-coding genes unaccounted for by current annotations [18]. In recent years, a number of approaches and technologies were adopted to identify genes expressed in the retina/RPE of human, cow, dog and mouse including data-mining and assembly of publically available expressed sequence tag (EST) information [19-23], sequencing of cDNA libraries generated via conventional methods [24-29] or via normalization techniques [30,31], hybridization to gene arrays of various formats [32,33] and serial analysis of gene expression (SAGE) [34,35]. Suppression subtractive hybridization (SSH) has been shown to be an efficient technique with which differentially expressed genes can be normalized and enriched over 1000-fold in a single round of hybridization [36]. Subsequently, applications of SSH to identify retina and RPE-enriched genes have been reported [37-39]. Based on a comprehensive survey of data available from 27 independent studies applying a wide spectrum of gene identification approaches we have now assembled a first genome-wide reference transcriptome of the adult mammalian retina/RPE. This reference transcriptome comprises 13,037 non-redundant transcripts and likely reflects up to 90% of the mammalian retinome. Results A total of 481,137 primary datasets on gene transcripts from the adult mammalian retina/RPE tissues have been generated in 27 independent studies (Table 1). Of these, 52,630 datasets (31,814 from retina, 11,632 from RPE and 9,184 from retina/RPE) were available and attributable to unique LocusLink identifiers (IDs). Correcting for gene redundancy within and between studies yielded a catalogue of 15,645 retinal/RPE genes. A survey of incidence and origin of each of these genes in the various studies analyzed demonstrated that 2,608 transcripts were found only once (see additional File 1) while the remaining 13,037 genes (see additional File 2) were confirmed in at least two and up to 16 independent gene identification approaches (Table 2). Thus, the latter compilation of genes may represent a more conservative description of the retinome minimizing a potential bias in data ascertainment. Of the 13K retinome, 1,411 genes were solely identified in retinal studies (see additional File 3) while 246 genes were exclusively found in the RPE datasets (see additional File 4). Table 1 Studies identifying adult mammalian retina / RPE transcripts and details on gene data retrieval Reference Source / method a) Species b) Original dataset No. of genes retrieved c) (non-redundant) No. of genes identified in ≥ 2 studies In-silico projects [20] Retina, TIGR (ID: version 3.3) Hs 1,047 30 30 [19] Retina, TIGR (ID: version 3.3) Hs 1,315 11 11 [21] Retina, UniGene (ID: build 118) Hs 4,974 1,485 1,480 [22] Retina, UniGene (ID: build 113) Hs 6,190 46 45 [23] Retina, EST (dbEST) Hs 40,000 117 110 UniGene (Build 162) d) Retina Hs 3,560 1,612 1,211 UniGene (Build 162) d) RPE Hs 1,760 1,506 1,116 UniGene (Build 162) d) Retina & RPE Hs 11,976 9,178 9,178 cDNA library sequencing [24] Fovea, conventional Hs 209 40 38 [25] Retina, conventional Hs 607 475 465 [37] Retina & RPE, SSH Hs 401 6 6 [30] Retina, subtracted Hs 137 49 48 [31] Retina, PC and subtracted Cf 173 66 65 [26] RPE, primary and amplified, conventional Hs 2,101 336 330 [38] RPE, SSH Bt 1,000 35 34 [27] and online e) Retina, PC Hs 2,701 2,096 2,059 [28] and online f) RPE, PC Hs 6,182 3,657 3,608 [29] Retina, PC (ID: MRA) Mm 1,793 421 412 [39] Retina, SSH Hs 1,080 310 301 [39] RPE, SSH Bt 2,350 343 329 Microarray Analysis [32] Retina, Affymetrix (ID: Mu11K subB) Mm 6,540 67 67 [33] Retina, custom array Hs 10,034 530 508 Serial Analysis of Gene Expression [34] Retina, adult Mm 54,009 4,233 3,974 [35] Retina (ID: HMAC2) Hs 102,359 7,269 6,919 [35] Retina (ID: HPR1) Hs 59,661 5,689 5,512 [35] Retina (ID: HPR2) Hs 105,312 7,268 6,955 [35] RPE (ID: HRPE1) Hs 53,666 5,755 5,211 Total (with inter-study redundancy) 52,630 50,022 Total (without redundancy) 15,645 13,037 a) TIGR: The Institute for Genomic Research; EST: expressed sequence tag; RPE: retinal pigment epithelium; SSH: suppression subtractive hybridization; PC: primary, conventional b) Hs: Homo sapiens; Mm: Mus musculus; Cf: Canis familiaris; Bt: Bos taurus c) Genes with LocusLink ID identified based on publically available information d) [67], For query terms see (see additional File 15 e) [68], March 2003 f) [69], March 2003 Table 2 Frequency of unique genes in studies No. of studies No. of unique genes 1 2,608 a) 2 4,162 3 2,368 4 2,002 5 1,736 6 1,282 7 768 8 385 9 178 10 84 11 37 12 23 13 6 14 2 16 4 Total 15,645 a) not included in assembly of 13K retinome (see additional File 1) To assess the degree of completeness of the adult mammalian retinome, we compared the LocusLink IDs of the 13,037 transcripts to partial lists of genes known i) to be specifically expressed in the retina/RPE (category I, n = 43) (see additional File 5), ii) to play a role in the phototransduction pathway/vitamin A cycle (category II, n = 57) (see additional File 6), iii) to encode retinal/RPE proteins verified by immunohistochemistry (category III, n = 260) (see additional File 7), and iv) to be associated with syndromic and non-syndromic retinal disease (category IV, n = 102) (see additional File 8). The data show that the compiled retinome covers all retina/RPE-specific transcripts (43/43) and 53/57 (93%) of the phototransduction pathway/vitamin A cycle genes. Known retinal/RPE proteins are represented by 204/260 (79%) transcripts while 87/102 (85%) genes known to be involved in retinal diseases are found in the 13K retinome collection (Table 3). To further evaluate the significance of these findings, partial transcriptomes of heart (n = 3,660; see additional File 9), liver (n = 5,780; see additional File 10) and prostate (n = 7,018; see additional File 11) were assembled and compared to the four selected categories. In category 1, none of the retina/RPE-specific genes were present in the heart, liver, or prostate partial transcriptomes while category 2 revealed four of 16 expected (25%, heart), 7 of 25 (28%, liver) and 9 of 31 (29%, prostate) genes. In category 3, 59/73 (81%, heart), 82/115 (71%, liver) and 92/140 (66%, prostate) genes were present. Retinal disease genes (category 4) were found at a rate of 18 of 29 (62%, heart), 26 of 45 (58%, liver) and 28 of 55 (51%, prostate) (Table 3). The expected values for the partial transcriptomes were calculated by adjusting the respective transcriptome sizes relative to the total number of transcripts of the retinome. Table 3 Representation of retinome and partial assemblies of heart, liver, and prostate transcriptomes in defined retina/RPE gene groups Transcriptome No. of non-redundant genes identified in ≥ 2 studies a) Retina / RPE-specific genes b) (n = 43) Vitamin A / phototrans-duction pathway c) (n = 57) Retina / RPE genes (verified by immunohisto-chemistry d)) (n = 260) Retinal disease genes e) (n = 102) Source / Reference Retinome 13,037 43 53 204 87 Present publication Heart (partial) 3,660 0 4 59 18 UniGene Build 166, SAGE library GSM1499 Liver (partial) 5,780 0 7 82 26 UniGene Build 166, SAGE library GSM785 Prostate (partial) 7,018 0 9 92 28 UniGene Build 166, SAGE libraries GSM685, GSM739, GSM764 a) See additional Files 2, 9, 10 and 11 b) See additional File 5 c) See additional File 6d) See additional File 7 e) See additional File 8 A comparison of the 13K retinome with partial transcriptomes of heart, liver, and prostate suggests a high degree of overlapping expression between retina/RPE and heart (3,496/3,660), liver (5,343/5,780) and prostate (6,471/7,018). A total of 2,330 genes are expressed in all tissues and represent putative "housekeeping" genes (see additional File 12). It should be noted that the low number of ubiquitously expressed genes is largely due to the fragmentary nature of the heart, liver, and prostate transcriptomes. With increasing transcriptome complexities this number is likely to increase. Analysis of the least complete transcriptome, the heart, reveals that 2,330/3,660 (64%) transcripts can be classified as ubiquitously expressed (see additional Files 9 and 12) while a maximum of 1,330/3,660 (36%) genes may display tissue-restricted or tissue-specific expression. A comparison of more complete transcriptomes may significantly reduce the latter estimate. So far 5,051 genes are only found in the retinome representing a collection of "retinome-enriched" transcripts, while 7,986 are also present in at least one of the partial transcriptomes of the heart, liver or prostate. Thirty-two genes were found to be expressed in heart, liver and prostate but not in the retinome (see additional File 13). The distribution of the mammalian retinome across the human genome was assessed by a paired comparison of the number of reference retinome genes (13,037) versus the number of human non-redundant syntenic gene predictions (SGPs) (43,109) [40] along 618 five-Mb windows (Fig. 1a). Correction for the total number of SGPs relative to the number of retinome genes positioned on average 21.1 (median 19.9) SGPs per window compared to 21.3 (median 15.0) retinal genes per window. Based on the Wilcoxon two-sample paired signed rank test, the null hypothesis assuming similar distribution of the SGPs and the retinome genes across the five-Mb windows can be rejected at p < 0.01. While their overall distribution greatly parallels that of the SGPs, retinome genes tend to cluster in several chromosomal regions most prominently on chromosomes 6p22.1-p21.31, 11q12.2-q13.1, 16p13.3, 19p13.3 and 19q13.32-q13.33 (Fig. 1a,1b). Figure 1 Chromosomal distribution of transcripts defining the reference retinome. (A) The distribution of 13,037 retinome genes over the human genome is shown as the difference between the number of observed and expected transcripts in window sizes of 5 Mb along the chromosomes (abscissa). The number of expected genes was based on 43,109 SGP-predicted transcripts. To correct for gene density per bin, the SGPs were adjusted by a factor of 0.30 (13,037/43,109). Positive/negative ordinate values indicate regions of enrichment/depletion of retinome-encoded transcripts. (B) Close-up of chromosomes 6 and 19 calculated for a window size of 1 Mb along the two chromosomes. To provide positional candidates for syndromic and non-syndromic hereditary retinopathies, the 13K reference retinome as well as the "retinome-enriched" transcripts (5,051 transcripts) were superimposed onto the disease intervals of 42 thus far uncloned retinal disorders (Table 4). In many instances, this results in a significant reduction of genes in the respective intervals offering a manageable number of candidates for retinal diseases (e.g. the RP29 locus contains 28 SGPs of which 5 are present in the reference retinome including GPM6A, WDR17, FLJ22649, VEGFC, AGA). The number of possible candidates is further reduced in the "retinome-enriched" transcript category to GPM6A and VEGFC. To make the information on the reference retinome available, we have created the interactive RetinaCentral database, a research portal which collects and stores information on genes and proteins functionally relevant to the retinal tissues [1]. We have implemented an interactive data retrieval system that presently contains linked information on the 13,037 genes of the 13K reference retinome. Database scripts were programmed to synchronize the data with LocusLink index files [41] which are updated daily [42]. Table 4 Number of genes mapped to retinal disease loci Disease Chr. Flanking DNA marker Size of locus (Mb) No. of SGPs (n = 43,109) No. of retinome transcripts a) (n = 13,307) No. of "retinome-enriched" transcripts (n = 5,051) AA 11 D11S1323 D11S902 11.21 213 65 13 AIED X DXS7 DXS72 37.33 479 129 49 AXPC1 1 D1S2692 D1S414 3.00 43 7 3 BBS3 3 D3S1603 D3S1271 2.28 28 10 1 BBS5 2 D2S142 D2S326 16.81 353 50 24 BCD 4 D4S3051 D4S1652 2.06 17 2 1 CACD 17 D17S1810 CHLC.GAT7B03 3.17 118 76 36 COD2 X DXS292 DXS1113 6.1 37 5 3 COD4 X DXS10042 DXS8060 32.44 436 117 45 CORD1 18 marker position not available - - - - CORD4 17 marker position not available - - - - CORD8 1 D1S442 D1S2681 19.86 485 222 74 CORD9 8 D8S1820 D8S532 12.79 179 43 16 CRV 3 D3S3564 D3S1578 11.2 271 157 72 CYMD 7 D7S435 D7S526 1.56 25 13 7 EVR3 11 GATA34E08 D11S4102 14.00 192 35 13 LCA3 14 D14S606 D14S610 5.95 178 36 15 LCA5 6 D6S1551 D6S1694 36.08 541 263 93 LCA9 1 D1S1612 D1S228 5.52 130 51 22 MCDR1 6 D6S249 D6S1671 2.44 45 2 0 MCDR2 4 D4S3023 D4S3022 21.00 253 57 20 MCDR3 5 D5S1981 D5S2031 19.94 184 36 14 MCDR4 14 D14S261 - 10.00 b) 58 11 4 MRST 15 D15S211 D15S152 4.70 106 24 11 OPA2 X DXS993 DXS991 14.31 245 69 24 OPA4 18 D18S34 D18S548 2.55 23 3 1 PRD X MAOB DXS426 3.92 67 18 6 RCD1 6 marker position not available - - - - RNANC 10 D10S1225 D10S1418 5.76 58 9 2 ROA1 8 D8S1702 D8S1794 11.87 150 30 13 RP6 X DXS28 DXS164 4.92 60 4 2 RP17 17 D17S1607 D17S948 7.18 130 44 17 RP22 16 D16S287 D16S420 6.33 92 40 14 RP23 X DXS1223 DXS7161 10.51 140 30 15 RP24 X DXS8094 DXS8043 7.75 74 6 3 RP28 2 D1S1337 D2S286 45.78 674 194 68 RP29 4 D4S3035 D4S2417 3.66 28 5 2 STGD4 4 D4S1582 D4S2397 16.56 187 33 10 USH1A 14 D14S99E D14S292 6.98 134 44 18 USH1E 21 D21S1905 D21S1913 11.48 77 19 10 USH2B 3 D3S1578 D3S3658 12.80 287 161 74 USH2C 5 D5S428 D5S433 18.54 269 34 13 VRNI 11 INT2 D11S873 22.41 311 89 37 WFS2 4 D4S2366 D4S3023 2.18 26 10 5 WGN1 5 D5S626 D5S2103 7.11 84 9 4 a) See additional File 14; b) Linkage to a single DNA marker reported. An arbitrary locus size of 10.0 Mb was assigned. Discussion Compiling the transcriptome of a cell or tissue is arguably more demanding than establishing the number of gene loci encoded by a given genome sequence [43]. This may mainly be explained by the dynamic nature of mRNA itself which frequently produces alternative transcripts from a single gene locus by usage of tissue-specific promoters, cryptic splice sites or variable polyadenylation signals [44,45]. In addition, variation in gene expression is known to occur within and between populations [46,47] and allele-specific expression, even from non-imprinted genes, appears to be common [48]. Further complicating transcriptome definition are effects of gender and age on RNA expression [49] as well as agonal and postmortem factors which greatly affect RNA integrity and thus frequently influence subsequent analyses [50]. Finally, differences in experimental technologies and data post-processing add an additional level of variability. Taken together, the complexities in mRNA metabolism and experimental data handling strongly suggest that there is not a single transcriptome for a given cell or tissue but implies an arbitrary number of individual transcriptomes which need to be defined by a series of parameters such as age, gender, ethnicity, cause and time of death of the tissue donor besides many others. It is therefore advisable to initially aim for a reference transcriptome providing a blueprint of an expression profile within a broadly defined time-frame. Following this line of reasoning, we here present a framework of a first reference transcriptome of the retina/RPE consisting of 13,037 unique transcripts which broadly characterize the mature state of expression in this tissue. The present meta-analysis has integrated information from 27 studies employing diverse technologies to identify retinal/RPE transcrips. Among these, SAGE represents a sensitive tool to detect low level transcription [51] while the PCR-based SSH method is well suited to enrich for differentially expressed genes [36]. The combined use of these approaches together with conventional cDNA library sequencing and microarray-based techniques provides a more solid assessment of gene expression than would each method alone. For example, SAGE is based on sequencing of hundreds of thousands of short (10, 14, or 21 bp) tags, ideally derived from a unique location of a single transcript. Rare tags could originate from infrequently expressed transcripts but could also reflect minor genomic contamination or minor sequencing errors. For the assembly of the reference retinome we have addressed these concerns by including only those transcripts that have independently been confirmed in a second unrelated study. This has led to a conservative assembly of the 13K retinome. It should be kept in mind however that this proceeding likely excludes a number of authentic transcripts. This is illustrated by the finding that the 15K retinome which comprises 15,645 transcripts including those which were solely found in a single study (Table 2), contains an additional five of the 102 known retinal disease genes (RHOK, MTATP6, CHM, LRAT, RIMS1) not included in the 13K retinome. Similarly, an additional three genes (RHOK, LRAT, GPRK7) involved in the vitamin A/phototransduction pathway are part of the 15K but not the 13K retinome. With additional transcription data on the retina/RPE becoming available, a second generation retinome map will need to address this issue. The estimation of transcriptome size represents one of the fundamental questions in molecular biology. Early studies using reassociation kinetics have calculated the number of distinct mRNA transcripts present in various mouse tissues to be between 11,500 and 12,500 [52]. Initial SAGE analyses have led to the conclusion that the number of different transcripts observed in normal and tumorous tissue may lie between 14,247 and 20,471 [53]. Recent data from comprehensive EST sequencing of a number of tissues including brain, breast, colon, head/neck, kidney lung, ovary, prostate, and uterus suggest expression of between 7,500 and 13,500 distinct genes for each tissue [54]. Although the size of the reference retinome is consistent with these estimates, the question of adequate transcript representation by the current compilation remains open. We have addressed this by defining a number of gene groups with known expression in retina/RPE and comparing these to the reference retinome. Genes exclusively expressed in retina/RPE are highly represented in the retinome (100%), as are mainly tissue-specific genes known to play a role in the vitamin A/phototransduction pathway (93%) (Table 3). A partial list of 260 genes whose encoded proteins were shown by immunohistochemistry to be expressed in the retina/RPE (but may also be present in other tissues), were represented in the reference retinome at a rate of approximately 79%. Similar numbers were obtained for the retinome coverage of retinal disease genes (85%). From these data we conclude that the 13K reference retinome is highly representative of retina/RPE-expressed genes and may describe as much as 90% of the transcript complement in the adult state. Another point of interest concerns the proportion of retinome transcripts which is uniquely expressed in this tissue. Brentani et al. [54] estimate that any two tissues may share between 73% and 84% of their transcriptomes. Comparing transcription in three tissues (breast, colon, head/neck) the authors found overlapping expression in 47% of transcripts. To investigate this in more detail, we have compiled three partial transcriptomes from heart (n = 3,660), liver (n = 5,780) and prostate (n = 7,018) by applying the same stringent criteria as defined for the retinome. Limited by the size of the partial heart transcriptome, we determined 2,330 transcripts (termed "housekeeping" genes) to be expressed in all four tissues (i.e. 64% of the heart transcriptome). Comparing the retinome to any of the partial transcriptomes revealed overlapping gene profiles between 92 % and 95 %. This would suggest that only a minor proportion of retinome transcripts is indeed unique to the retina/RPE. Thus far, we have identified a group of so called "retinome-enriched" genes comprising 5,051 transcripts which are not present in the partial transcriptomes of heart, liver and prostate. This group most likely contains additional "housekeeping" or tissue-restricted transcripts and needs further adjustment by more refined in-silico normalization to comprehensive reference transcriptomes of other tissues. Highly expressed genes including those with a ubiquitous or a tissue-specific transcription profile, have been shown to cluster in chromosomal regions of increased gene expression (termed RIDGEs) [55,56]. Functionally, this higher order structure has been related to transcriptional regulation [56,57]. To search for a possible correlation, we have determined the chromosomal distribution of the reference retinome independent of gene density. Our data show good agreement with the previously established regional expression map defining approximately 30 RIDGEs within the human genome. Overlaps are most evident for chromosomes 6, 9, 11, 17, and 19. From this we conclude that the majority of transcripts assembled in the reference retinome share characteristics of the RIDGEs including moderate to high level expression. This finding may be ascribed to the stringent selection criteria we have applied to assemble the reference retinome by excluding all transcripts (n = 2,608) that were reported in only a single study. Conversely, the RIDGE-like pattern of the reference retinome could be an indication that missing transcripts may have features compatible with chromosomal domains defined as anti-RIDGEs [56]. As opposed to RIDGEs, clustering of genes in anti-RIDGEs seems associated with significant decreased expression [56]. In contrast to their fractional occurrence in transcriptomes, the identification of such low abundant transcripts are likely to require significant resources in order to compile more complete transcriptomes. To provide positional candidates for retinal disease genes, we have mapped the transcripts representing the reference retinome to the minimal regions defined for 42 retinal disease loci with as yet undefined gene mutations. To further limit the number of candidate genes, in particular for loosely defined disease loci such as RP28 or VRNI, we have similarly integrated the "retinome-enriched" transcripts. This also accommodates for the fact that approximately 50% of retinal disease genes are retina/RPE-specific [58]. For 41 of 42 unknown disease genes we have now identified strong candidates although for some disease loci including AIED, COD4, CORD8, CRV, LCA5, RP28, and USH2B, the number of candidates may still exceed capacities of most laboratories for direct analysis. For other disease loci (e.g. BCD, BBS3, COD2, CYMD, MCDR4, OPA4, PRD, RNANC, RP24, RP29, RP6, WFS2 and WGN1), a restricted number of candidates are now available (see additional File 14). Conclusions We here present a first near-complete transcriptome of a defined tissue, the retinome, which may serve as a reference for further efforts to establish spatial, i.e. cell-specific, and developmental transcriptomes of the retina/RPE. A fundamental aspect of the current study was to integrate the available information on gene identification generated by a wide range of techniques. This ensures robustness and reliability of transcript data providing a stringent framework for further expression studies in systems biology. A similar approach for other tissues/cells would be advisable as this may greatly facilitate in-silico identification of tissue-specific genes to elucidate functional pathways vital for a defined cell population. In addition, the reference retinome may prove valuable for providing strong candidates for hereditary as well as genetically complex diseases and thus may help to further our understanding of retinal biology in health and disease. Methods Data retrieval and analysis To assemble a list of genes expressed in the adult mammalian retina and RPE we reviewed 27 studies reporting raw or processed transcript data derived from several mammalian species including H. sapiens, B. taurus, C. familiaris and M. musculus (Table 1). The data were generated by cDNA library sequencing, microarray studies, and SAGE. Publically available data analysing transcripts from adult mammalian retina/RPE tissues published until December 2003 were included. Excluded were studies investigating transcription in retina/RPE by using RNA sources such as fetal tissues, cell lines or non-mammalian species. Gene identifiers such as GenBank accession number, gene nomenclature symbol, gene description, UniGene cluster ID, cDNA sequences or tags were available from sources as detailed in additional File 15 and were used to retrieve the unique human LocusLink ID for each gene (as of December, 2003). Only genes with established LocusLink ID were included in the present study. For SAGE data, tag-to-gene assignment was done by querying the SAGEmap_tag_ug-rel dataset [59,60]. Tags assigned to multiple genes were excluded from further analysis. Human orthologous genes were established via the NCBI-curated homology database[61] or by BLAST sequence comparison [62]. To assemble partial transcriptomes of heart, liver and prostate, for each tissue data were mined from at least one SAGE library, in addition to expressed sequence tag (EST) sources (see additional File 16). Similar to the criteria for the assembly of the retinome, genes identified in only one study were disregarded. EST retrieval was facilitated by use of the Gene Library Summarizer [63] which retrieves the known genes represented by at least one EST and generated from a tissue sample with normal histology. Partial lists of genes known to play a role in the retina and/or the RPE were assembled from the literature (see additional Files 5, 6, 7 and 8). Additional File 5 summarizes genes known to be exclusively expressed in retina and/or RPE, while additional File 6 includes genes involved in the phototransduction cascade and the vitamin A cycle. Additional File 7 is a partial compilation of genes/proteins verified by immunohistochemistry to be present in adult mammalian retina and/or RPE. A list of 102 genes involved in retinal diseases was retrieved from the RetNet database, January 2004 [58] (see additional File 8). Assignment of genes and disease loci to the human genome A total of 43,109 human non-redundant syntenic gene predictions (SGP) were retrieved (as of December 2003) and chromosomally mapped to the reference sequence of the human genome (July 2003) utilizing the USCS Genome Table Browser [64]. Based on the position of their putative transcription start sites, the SGPs were assigned to 5 Mb bins along the human chromosomes. In addition, one-megabase bins were defined for refined analysis of chromosome 6 and 19 (Fig. 1b). Similarly, the chromosomal map positions of the retinome transcripts were determined by querying the USCS Genome Table Browser with the respective LocusLink, UniGene or RefSeq IDs. Mapped loci of retinal dystrophies with unknown genetic basis (n = 45) were taken from RetNet, January 2004 [58] and placed on the human genome sequence by querying the USCS Genome Table Browser with DNA marker sequences shown to flank the minimal candidate region. Three disease loci (CORD1, CORD4 and RCD1) are insufficiently mapped on the respective human chromosomes and were therefore not included in the analysis. Statistical analysis of gene distribution To determine if either of the two datasets, the 43,109 human non-redundant SGPs and the 13K retinome transcripts, is distributed in a non-parametric and distribution free manner over the genome, the Kolmogorov-Smirnov Goodness-of-Fit Test was used [65]. Statistical significance of the median difference in paired chromosomal distribution of retinome transcripts versus the SGPs was then evaluated by the non-parametric Wilcoxon two-sample paired signed rank test [66]. To carry out the test we calculated the difference between all genes versus retinal genes per 5-Mb bin. To correct for the total number of genes within the two groups, the SGPs per bin were adjusted by a factor of 13,037/43,109 = 0.30. Mean and median values per bin were 21.05 and 10.93 for all genes and 21.26 and 19.93 for retinal genes, respectively. Authors' contributions HLS participated in the design of the study, collected and processed the information from the published reports related to the retina and RPE genes. TG conceptually developed the RetinaCentral database and is involved in the curation of the site. JK carried out the statistical analyses and helped with the computational handling of data. BHFW was involved in all aspects of data assembly and prepared the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional File 1 Retina/RPE genes reported only in a single study List of genes expressed in the retina or RPE reported only in one study Click here for file Additional File 2 Gene list of the reference retina / RPE transcriptome List of 13,037 genes expressed in the retina/RPE Click here for file Additional File 3 List of genes identified exclusively in retina studies Click here for file Additional File 4 List of genes identified exclusively in RPE studies Click here for file Additional File 5 Partial list of genes known to be expressed specifically in the retina and/or RPE Click here for file Additional File 6 Partial list of genes known to be involved in the vitamin A cycle and phototranduction pathway Click here for file Additional File 7 Partial list of genes known to encode retina / RPE proteins Click here for file Additional File 8 List of known genes involved in retinal diseases Click here for file Additional File 9 Partial gene list of the heart transcriptome Click here for file Additional File 10 Partial gene list of the liver transcriptome Click here for file Additional File 11 Partial gene list of the prostate transcriptome Click here for file Additional File 12 List of genes present in the reference retinome and partial transcriptomes of heart, liver, prostate Click here for file Additional File 13 List of genes present in partial transcriptomes of heart, liver, and prostate but not in reference retinome Click here for file Additional File 14 List of retinome transcripts mapping to retinal disease intervals. Click here for file Additional File 15 Data sources used to compile the retina / RPE transcriptome Click here for file Additional File 16 Data sources used to compile partial transcriptomes of heart, liver, and prostate Click here for file Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (We1259/14-2 and 14-3) and the Bundesministerium für Bildung und Forschung (BMBF) (01KW9921/0). ==== Refs RetinaCentral - A portal to the Human Retina Chow RL Lang RA Early eye development in vertebrates Annu Rev Cell Dev Biol 2001 17 255 296 11687490 10.1146/annurev.cellbio.17.1.255 Masland RH The fundamental plan of the retina Nat Neurosci 2001 4 877 886 11528418 10.1038/nn0901-877 Marmor MF Wolfensberger TJ The Retinal pigment Epithelium 1998 Oxford: Oxford University Press Palczewski K Polans AS Baehr W Ames JB Ca(2+)-binding proteins in the retina: structure, function, and the etiology of human visual diseases Bioessays 2000 22 337 350 10723031 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==== Front BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-5-531529402710.1186/1471-2164-5-53Methodology ArticleImmobilized probe and glass surface chemistry as variables in microarray fabrication Hessner Martin J [email protected] Lisa [email protected] Jennifer [email protected] Sanaa [email protected] Xujing [email protected] The Max McGee National Research Center for Juvenile Diabetes, Department of Pediatrics, The Medical College of Wisconsin and Children's Hospital of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA2 The Human and Molecular Genetics Center, The Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA2004 4 8 2004 5 53 53 2 6 2004 4 8 2004 Copyright © 2004 Hessner et al; licensee BioMed Central Ltd.2004Hessner et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Global gene expression studies with microarrays can offer biological insights never before possible. However, the technology possesses many sources of technical variability that are an obstacle to obtaining high quality data sets. Since spotted microarrays offer design/content flexibility and potential cost savings over commercial systems, we have developed prehybridization quality control strategies for spotted cDNA and oligonucleotide arrays. These approaches utilize a third fluorescent dye (fluorescein) to monitor key fabrication variables, such as print/spot morphology, DNA retention, and background arising from probe redistributed during blocking. Here, our labeled cDNA array platform is used to study, 1) compression of array data using known input ratios of Arabidopsis in vitro transcripts and arrayed serial dilutions of homologous probes; 2) how curing time of in-house poly-L-lysine coated slides impacts probe retention capacity; and 3) the retention characteristics of 13 commercially available surfaces. Results When array element fluorescein intensity drops below 5,000 RFU/pixel, gene expression measurements become increasingly compressed, thereby validating this value as a prehybridization quality control threshold. We observe that the DNA retention capacity of in-house poly-L-lysine slides decreases rapidly over time (~50% reduction between 3 and 12 weeks post-coating; p < 0.0002) and that there are considerable differences in retention characteristics among commercially available poly-L-lysine and amino silane-coated slides. Conclusions High DNA retention rates are necessary for accurate gene expression measurements. Therefore, an understanding of the characteristics and optimization of protocols to an array surface are prerequisites to fabrication of high quality arrays. ==== Body Background The generation of reliable gene expression data with cDNA microarrays requires fabrication of quality arrays. This task encompasses the amplification of adequate amounts of concentrated PCR product for use as probe from the cDNA clone, followed by ordered arraying of the probes onto coated glass slides. The glass slide is a key variable in either spotted cDNA or oligonucleotide array fabrication since it must possess: 1) a uniform surface that yields spots of consistent shape and size, 2) low background fluorescence, and 3) high DNA retention capacity. Since the array is clearly a source of experimental variability, we have developed a novel three-color array approach where it is possible to directly visualize either cDNA or oligonucleotide arrays prior to hybridization [1-3]. For cDNA arrays, the probes are easily tagged with a third, Cy3/Cy5 compatible, fluorescent dye (fluorescein) during amplification. After purification of PCR products, which includes removal of unincorporated oligonucleotide primer, the detected fluorescein fluorescence represents deposited cDNA probe on the array. This three-color approach allows for assessment of slide fabrication independent of hybridization, thereby enabling 1) direct visualization of array/element morphology, 2) quantification of probe deposition and retention on the slide surface and 3) ultimately a means for array quality control prior to hybridization. By labeling the array itself with a third color, we have observed that arrays fabricated together are not equivalent in terms of a number of measurable physical parameters, including the amount of DNA probe deposited and retained and the amount of background arising from probe solublized and re-deposited during post-processing. In prior studies, we observed that these pre-hybridization array-based variables play a direct and significant relationship in replicate consistency, and that microarray data quality can be improved through prehybridization slide selection based upon these quality parameters [1,2]. As a result of these studies, we identified putative slide acceptance criteria: array fluorescein mean element intensity >5000 RFU/pixel, coefficient of variation (CV) in intensity <10%, mean signal to noise score (signal/signal + noise; S/S+N) >0.85, and CV in spot size <20%. In this report, using known input ratios of in vitro transcript we experimentally correlate the quantity of support bound probe to measured expression ratios, in order to validate our quality control threshold for array acceptance. We then utilize our three-color array platform to evaluate the characteristics of in-house prepared poly-L-lysine coated slides and 13 additional commercially available coating surfaces, in terms of background auto-fluorescence, spot morphology, and DNA retention. Results and Discussion The relationship between support bound probe and measured ratio reliability It has been assumed that the amount of cDNA probe deposited and retained on the array surface would have a nominal effect on observed differential expression ratios due to the competitive nature of two channel fluorescent hybridizations [4]; however this assumption has been shown to be false [1,5]. Yue et al., using unlabeled Saccharomyces cerevisiae probes and complementary Cy5 and Cy3 labeled cDNA targets derived from in vitro transcripts, indirectly demonstrated this by printing yeast probes at increasingly dilute concentrations (<50 ng/ul) and observed elimination of the measured dynamic range to where input transcript ratios of 30:1 or 1:30 were both detected as output ratios close to 1:1, illustrating that limiting bound probe results in an underestimation or failure to detect differential gene expression [5]. To expand upon these observations and place them in context with the quality control standards of our three-color array platform, we conducted similar experiments using Arabidopsis probes and transcripts. Total thymus RNA extracted from the DR+/+ and DRlyp/lyp [6] BioBreeding rats was directly labeled through reverse transcription reactions possessing cyanine dyes; these labeling reactions were spiked with known input ratios (30:1, 10:1, 1:1, and 1:0) of Arabidopsis gene in vitro transcript and hybridized to 18,000 probe rat cDNA arrays possessing serially diluted fluorescein-labeled Arabidopsis probes (cellulose synthase, chlorophyll a/b binding protein, and ribulose-1,5-bisphosphate and triosphosphate isomerase, 1:2 dilution series printed at 200 ng/ul to 6.25 ng/ul). This approach allowed comparison of known RNA input ratio to measured output ratio, enabling a direct and quantitative measure of the relationship between the amount of support-bound probe and ratio data compression (Figure 1A and 1B). Using Matarray [7,8], spots possessing low hybridized image quality (qcom) were filtered; these spots were either saturated or possessed high background. Spots with low hybridization intensities, which would normally be flagged by Matarray, were intentionally retained to study ratio compression due to low amounts of support-bound probe. After filtering, 896/1536 data points from 16 different arrays were available for analysis. Plotted on the y-axis of Figure 1b, is the measured Arabidopsis in vitro transcript output log ratio divided by the log ratio of transcript actually introduced into the Cy3 and Cy5 labeling reactions. In this analysis, a perfect measurement is represented by a value of "1". On the x-axis, is plotted the spot fluorescein intensity. When the spot fluorescein intensity falls below 5000 RFU/pixel, the data variability and data compression (underestimation of differential gene expression) dramatically increase. These results recapitulate our previous observations where replicate consistency was found to decrease when the array average spot fluorescein intensity dropped below 5000 RFU/pixel, whereas arrays possessing average fluorescein intensities above 5000 RFU/pixel were found to generate equally good data. These results further demonstrate that use of measurable array characteristics are effective quality markers for printed arrays (judged by their effect on the hybridization data) and serve to validate our array intensity quality control threshold of >5000 RFU/pixel. Figure 1 Evaluation of measured output ratio of spiked Arabidopsis in vitro transcript at known input ratios. A. Total thymus RNA extracted from the DR+/+ and DRlyp/lyp [6] BioBreeding rats spiked with known input ratios of Arabidopsis gene in vitro transcript and hybridized to 18,000 probe rat cDNA arrays possessing serially diluted fluorescein-labeled Arabidopsis probes. B. Evaluation of data compression as a function of support-bound probe. On the x-axis is plotted the average pixel fluorescein intensity per spot plotted against the Arabidopsis transcript measured output log ratio/actual input log ratio. As spot intensities fall below 5000 RFU/pixel, ratio measurements become increasingly compressed. Impact of poly-L-lysine cure-time on DNA retention capacity Clearly, the amount of immobilized probe on the coated glass surface is a critical array fabrication variable, therefore factors that affect the amount of retention characteristics, such as surface chemistry, probe concentration, spotting buffer, spotting conditions, cross-linking and blocking conditions are important to understand. Protocols for coating glass microscope slides with poly-L-lysine are readily available on-line and reasonably simple to perform (for example: ; ; ). Although most available protocols are quite similar, some recommend the curing of slides for two weeks prior to spotting, while others state that coated slides are not stable for extended periods of time and recommend not printing onto slides that are greater than 4 months old. To investigate slide coating age as a potential variable in retention capacity, we fabricated more than 1,000 rat cDNA arrays (18,000 element/slide) using in-house poly-L-lysine coated slides ranging in age from 3 to 12 weeks. These slides were coated in 26 independent sessions and utilized over 12 different print runs. After printing all arrays were post-processed [9] and imaged under standardized conditions as previously described [1,2]. A significant loss of DNA retention capacity is observed (Figure 2) when the average array spot fluorescein intensity is plotted against the coating age at the time of printing (R2 = 0.84; p < 0.0002). An average array fluorescein intensity of >15,000 RFU/pixel was observed when printing on slides 4 weeks old or less, however a nearly 50% reduction in retention capacity is observed when printing on poly-L-lysine coating greater than 10 weeks old. We speculate that the poly-L-lysine may become oxidized thereby losing its positive charge and ability to initially electrostatically interact with the negatively charged DNA. Irregardless of the type of degradation occurring to the surface coating over time, these results indicate that, at least for in house fabricated poly-L-lysine coated slides, shelf-life is a significant variable in the fabrication of quality arrays capable of yielding reliable gene expression measurements. Figure 2 Relationship between DNA retention and poly-L-lysine cure time. Retention capacity is lost as the poly-L-lysine cure time increases (R2 = 0.84; p < 0.0002). Analysis includes 999 arrays printed over 12 different print runs. Each print run consisted of 100 arrays, printed onto poly-L-lysine slides from 2 or more coating lots. Investigation of probe retention characteristics of commercially available coated slides Given the potential time-dependent variability of in-house prepared poly-L-lysine coated slides, we investigated the retention characteristics of commercially available coated slides. Our objective was to identify a surface with consistently higher retention characteristics than our "fresh" in-house slides without having to change the spotting buffer (1.5 M betaine/5% DMSO) or the nonaqueous post-processing protocol [1,2,9], since these methods were previously found to yield high quality results on poly-L-lysine coated slides prepared in-house. We obtained examples of 13 different vendor-supplied slides for evaluation that possessed either poly-L-lysine, aminosilane, or undisclosed surface chemistries. Prior to printing, background auto-fluorescence in the fluorescein, Cy3, and Cy5 channels was evaluated. Fluorescein auto-fluorescence was observed on all poly-L-lysine slides except for those produced in-house, as well as 6 of the aminosilane slides (Asper Biotech, Corning, Erie Scientific, Genetix, Telechem), and the proprietary surface from Full Moon Biosciences. Cy3 auto-fluorescence was observed on all 3 commercial poly-L-lysine slides but not those prepared in-house, however none was observed on any of the aminosilane slides. Insignificant background in the Cy5 channel was only observed on 2 commercial poly-L-lysine slides (Electron Microscopy Sciences, Polysciences Inc.). To study retention characteristics, a single 9600 element human cDNA array was spotted onto each slide in 1.5 M betaine/3%DMSO. The in-house poly-L-lysine slides were less than 6 weeks old and all vendor-supplied slides were unpacked from any special packaging immediately before printing. Five replicate arrays for each slide type were generated. The five replicates were evenly distributed over the arrayer deck (capacity 100 slides) by arranging the slides into 5 groups of 18 to account for any variance introduced through print rank order (ie first versus last), since we previously identified this as a variable that influences array average array fluorescein intensity [1]. Fluorescein images were again obtained, under strict standardized conditions, immediately after printing and again after post-processing to measure DNA deposited and retained (Figure 3A; Table 1) [1,2]. The average amount of DNA deposited per element varied considerably among slides of different sources (n = 5 per source) ranging from a low of 2,300 +/-300 RFU/pixel to a high of 20,700 +/-3,300 RFU/pixel. Among the slides evaluated, the poly-L-lysine coated slides yielded larger spot sizes, perhaps due to a lower hydrophobicity than the aminated surfaces. The average DNA retained per element after blocking/post-processing ranged from a low of 1,400 +/-200 RFU/pixel to a high of 11,100+/-2,700 RFU/pixel on poly-L-lysine slides prepared in-house (Figure 3B). These results, combined with our previous observations [1,2], indicate that DNA concentration, choice of printing buffer, slide position on the arrayer deck and slide surface chemistry all influence the amount of DNA deposited and ultimately retained, which can be effectively monitored by our three-color approach. We have previously shown that the amount of probe solublized and redistributed over the slide during post-processing is an important quality control parameter [2]. Eight of the surfaces tested generated fluorescein signal to noise values (signal/signal + noise; S/S+N) ≥ 0.90 after post-processing, a value that we have previously shown to sufficient to generate high replicate reproducibility [2]. However, the majority of vendor-supplied coated slides, did not meet or exceed our established average array element intensity value of 5,000 fluorescein RFU/pixel under the printing and post-processing methods optimized for our in-house coated slides. These results point towards the possibility that array fabrication should be optimized to the specific surface selected for use and that even the same surface chemistry from different sources may perform differently. Figure 3 A: Fluorescein images of 18,000 element rat cDNA arrays on in-house poly-L-lysine coated slide after printing (A1) and array after non-aqueous post-processing (A2). Fluorescein images of simultaneously 18,000 element rat cDNA arrays on Full Moon Biosystems coated slide (undisclosed chemistry) after printing (A3) and array after non-aqueous post-processing (A4). Note differences in amount of DNA deposited and retained. (White spots are saturated). B: Comparison of retention capacity of 14 different coating surfaces using human 9,600 probe cDNA arrays. Tabulated measurements are based upon 5 replicates slides (~48,000 elements) for each slide type evenly distributed over the arrayer deck (ie 5 slides of a given type did not occupy 5 adjacent positions on the arrayer deck). Slides 1–4 are poly-L-lysine: MCW in-house, Electron Microscopy Sciences, Polysciences, and Cel Associates, respectively. Slides 5–13 are aminated: Asper Biotech, Apogent, Bioslide, Erie Scientific, Genetix, Corning Ultra GAPS, Corning GAPS II, Sigma, and Telechem Super Amine, respectively. Slide 14 is an undisclosed chemistry offered by Full Moon Biosystems. The graph represents the average spot fluorescein intensity RFU/pixel (burgundy) +/- standard deviation (yellow). Table 1 Retention Studies on Commercial Coated Slide Surfaces Vendor Chemistry RFU/Pixel deposited × 103 RFU/pixel retained × 103 Percent Retention Spot Diameter Processed S/(S+N) MCW in-house Poly-L-lysine 19.0+/-7.7 11.1+/-2.7 58.4% 114+/-9 0.90+/-0.00 Electron Microscopy Sciences Poly-L-lysine 15.3+/-4.9 2.12+/-1.1 13.9% 100+/-28 0.85+/-0.04 Polysciences Poly-L-lysine 11.5+/-6.3 1.3+/-1.0 11.3% 125+/-6 0.86+/-0.06 Cel Associates Poly-L-lysine 5.7+/-3.6 2.3+/-0.5 40.4% 100+/-35 0.79+/-0.09 Asper Biotech Aminated 6.8+/-2.4 3.2+/-1.1 47.1% 98+/-22 0.93+/-0.05 Apogent Ezrays Aminated 6.0+/-2.9 2.8+/-0.3 46.7% 109+/-20 0.92+/-0.02 Bioslide Aminated 3.3+/-0.9 1.5+/-0.5 45.5% 93+/-7 0.87+/-0.02 Erie Scientific Aminated 11.5+/-4.1 3.2+/-0.9 27.8% 81+/-15 0.90+/-0.01 Genetix Aminated 4.1+/-1.2 1.2+/-0.2 29.3% 94+/-23 0.86+/-0.02 Corning Ultra GAPS Aminated 2.3+/-0.3 1.5+/-0.2 65.2% 103+/-13 0.92+/-0.01 Corning GAPS II Aminated 3.9+/-0.9 1.4+/-0.2 35.9% 117+/-18 0.92+/-0.01 Sigma Aminated 20.7+/-3.3 5.4+/-1.0 26.1% 93+/-8 0.79+/-0.01 Telechem Super Amine Aminated 6.7+/-2.1 1.7+/-0.5 25.4% 107+/-19 0.80+/-0.0 Full Moon Biosystems Proprietary 6.4+/-1.4 2.1+/-0.2 32.8% 78+/-7 0.90+/-0.1 It has been reported that the amount of UV irradiation may be an important array fabrication variable since the amount of hybridization signal from spotted 70-mer oligonucleotides has been found to be dependent on the amount of cross-linking [10]. In this previous report, different optimal cross-linking intensities for attachment of spotted 70-mer oligonucleotides were observed for different slide coating chemistries (poly-L-lysine, aldehyde, aminosilane, epoxide) [10]; furthermore, different cross-linking optima for probe attachment were also observed for slides with the same or similar slide chemistry from different vendors. This variable was not explored in our evaluation of vendor-supplied surfaces and may account for some of the performance differences observed. The report by Wang et al., [10] prompted us to revisit this parameter for our in-house slides and we have observed approximately 20% better DNA retention by increasing the UV cross-linking energy from 60 mJ/cm2 to 200 mJ/cm2 independent of coated slide lot. Fabrication of high quality spotted arrays is a daunting task possessing a high number of variables. The vendor supplied slides tested here were done so under conditions that have been optimized for our in-house prepared poly-L-lysine coated slides, although our optimized protocol is not drastically different than those used by other laboratories nor drastically different from any of the vendor provided protocols. Our observations, as well as the observations of others, suggest that optimization of ones protocol to a surface chemistry is an essential first step to generating reliable global gene expression measurements using in-house spotted microarrays. Methods A sequence-verified human library (Research Genetics, Huntsville, AL), consisting of 41,472 clones or a 36,000 clone rat cDNA library obtained from the University of Iowa was used as a source of probe DNA. Cultures were grown in 150 ul Terrific Broth (Sigma, St. Louis, MO) supplemented with 100 mg/ml ampicillin in 384 deep-well plates (Matrix Technologies, Hudson, NH) sealed with air pore tape sheets (Qiagen, Valencia, CA) and incubated with agitation for 14–16 hr. Clone inserts were amplified in duplicate in 384-well format from 0.5 ul bacterial culture or from 0.5 ul purified plasmid (controls only) using 0.26 μM of each vector primer (SK865 5'-fluorescein-GTC CGT ATG TTG TGT GGA A-3' and SK536: 5'-fluorescein-GCG AAA GGG GGA TGT GCT G-3' [5]) (Sigma-Genosys, The Woodlands, TX) in a 20 μl reaction consisting of 10 mM Tris-HCl pH8.3, 3.0 mM MgCl2, 50 mM KCl, 0.2 mM each dNTP (Amersham, Piscataway, NJ), 1 M betaine [11,12], and 0.50 U Taq polymerase (Roche, Indianapolis IN). Reactions were amplified with a touchdown thermal profile consisting of 94°C for 5 min; 20 cycles of 94°C for 1 min, 60°C for 1 min (minus 0.5° per cycle), 72°C for 1 min; and 15 cycles of 94°C for 5 min; 20 cycles 94°C for 1 min, 55°C for 1 min, 72°C for 1 min; terminated with a 7 min hold at 72° [13-15]. PCR reactions were analyzed for single products by 1% agarose gel electrophoresis. Products from replicate plates were pooled and then purified by size exclusion filtration using the Multiscreen 384 PCR filter plates (Millipore, Bedford, MA). Forty wells of each 384-well probe plate were quantified by the PicoGreen assay (Molecular Probes, Eugene, OR) according to the manufacturers instructions. After quantification, all plates were dried down, and reconstituted at 125 ng/μl in 3% DMSO/1.5 M betaine. It has been shown that betaine normalizes base pair stability differences, increases solution viscosity, reduces evaporation rates [11], and enhances probe binding to surfaces such as poly-L-lysine or aminosilane [1,9]. We have observed higher probe retention at much lower DNA concentrations (150–200 ng/ul) in the presence of betaine versus the typically required 4–500 ng/ul when using conventional printing solutions [2,3]. Arabidopsis thaliana PCR product and in vitro transcript were purchased from Stratagene (La Jolla, CA) as part of the SpotReport®-10 Array Validation System. Arabidopsis thaliana PCR product was cloned into the pCRII vector using the TA cloning kit (Invitrogen, Carlsbad CA) and fluorescein-labeled PCR products for photosystem I chlorophyll a/b-binding protein, RUBISCO activase, ribulose-1,5-biphosphate carboxylase/oxygenase, lipid transfer protein 6 lipid transfer protein 5, papain-type cysteine endopeptidase, root cap 1, and triosphophate isomerase were generated using vector-specific primers essentially as described above. Products were purified, quantified, and a 1:2 dilution series (200 ng/ul to 12.5 ng/ul) was prepared and printed in duplicate onto each array. Poly-L-lysine coated slides were prepared in-house as previously described [16] on Corning (Corning, NY) pre-cleaned 75 × 25 mm glass micro slides. Nine different commercially available aminosilane coated slides (Apogent Discoveries, Waltham, MA; Asper Biotech, Redwood City, CA; Bioslide Technologies, Walnut, CA; Corning Inc, Corning NY; Erie Scientific, Portsmouth, NH; Genetix, St. James, NY; Sigma, St. Louis, MO; Telechem International Inc, Sunnyvale, CA) and 3 different commercially available poly-L-lysine coated slides (Cel-Associates, Pearland, TX; Electron Microscopy Sciences, Fort Washington, PA; Polysciences Inc., Warrington, PA) were obtained for evaluation. Additionally, slides coated with a proprietary chemistry (Full Moon Biosystems, Sunnyvale, CA) were obtained. Microarrays possessing a density of 9,600 human probes/slide were printed onto coated slides using a GeneMachines Omni Grid printer (San Carlos, CA) with 16 Telechem International SMP3 pins (Sunnyvale, CA) at 40% humidity and 22°C. To control pin contact force and duration, the instrument was set with the following Z motion parameters, velocity: 7 cm/sec, acceleration: 100 cm/sec2, deceleration: 100 cm/sec2. All slides were post-processed using the previously described non-aqueous protocol [9] using 60 mJ/cm2 UV cross-linking energy. This protocol has yielded more favorable fluorescein post-blocking signal-to-noise values (signal/signal+noise; S/S+N) as compared to blocking in aqueous solutions[2]. Image files on all slides were collected prior to printing to establish background fluorescence (fluorescein, Cy3 and Cy5), after printing (fluorescein), and after blocking (fluorescein), with a ScanArray 5000 (GSI Lumonics, Billerica, MA). Array image files were analyzed with the Matarray software [7,8,17]. Isolation of mRNA, labeling, and hybridization were performed as described previously . Known input ratios of photosystem I chlorophyll a/b-binding protein (30:1); RUBISCO activase (10:1); ribulose-1,5-biphosphate carboxylase/oxygenase (5:1); lipid transfer protein 6 (1:1); 0.7 lipid transfer protein 5 (1:1); papain-type cysteine endopeptidase (1:5); root cap 1 (1:10); and triosphophate isomerase (1:30) were spiked into Cy3 and Cy5 RNA labeling reactions, respectively. After hybridization, arrays were scanned with a ScanArray 5000 (GSI Lumonics, Billerica, MA) and image files were obtained. Again, array image files were analyzed with the Matarray software [7,8,17]. Authors' Contributions MJH and XW conceived of the study, its design and coordination, and drafted the manuscript. LM, JT, and SM carried out the array fabrication and gene expression studies. XW performed the statistical analysis. All authors read and approved the final manuscript. Acknowledgments M.J.H is supported by a National Institute of Biomedical Imaging and Bioengineering Grant (EB001421) and by a special fund from the Children's Hospital of Wisconsin Foundation. ==== Refs Hessner MJ Wang X Khan S Meyer L Schlicht M Tackes J Datta MW Jacob HJ Ghosh S Use of a three-color cDNA microarray platform to measure and control support-bound probe for improved data quality and reproducibility Nucleic Acids Res 2003 31 e60 12771224 10.1093/nar/gng059 Hessner MJ Wang X Hulse K Meyer L Wu Y Nye S Guo SW Ghosh S Three color cDNA microarrays: quantitative assessment through the use of fluorescein-labeled probes Nucleic Acids Res 2003 31 e14 12582259 10.1093/nar/gng014 Hessner MJ Singh VK Wang X Khan S Tschannen MR Zahrt TC Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays BMC Genomics 2004 5 12 15018646 10.1186/1471-2164-5-12 Winzeler EA Schena M Davis RW Fluorescence-based expression monitoring using microarrays Methods Enzymol 1999 306 3 18 10432445 10.1016/S0076-6879(99)06003-6 Yue H Eastman PS Wang BB Minor J Doctolero MH Nuttall RL Stack R Becker JW Montgomery JR Vainer M Johnston R An evaluation of the performance of cDNA microarrays for detecting changes in global mRNA expression Nucleic Acids Res 2001 29 E41 41 11292855 10.1093/nar/29.8.e41 MacMurray AJ Moralejo DH Kwitek AE Rutledge EA Van Yserloo B Gohlke P Speros SJ Snyder B Schaefer J Bieg S Jiang J Ettinger RA Fuller J Daniels TL Pettersson A Orlebeke K Birren B Jacob HJ Lander ES Lernmark A Lymphopenia in the BB Rat Model of Type 1 Diabetes is Due to a Mutation in a Novel Immune-Associated Nucleotide (Ian)-Related Gene Genome Res 2002 12 1029 1039 12097339 10.1101/gr.412702 Wang X Ghosh S Guo S-W Quantitative quality control in microarray image processing and data acquisition Nucleic Acids Research 2001 29 E75 82 11470890 10.1093/nar/29.15.e75 Wang X Hessner MJ Wu Y Pati N Ghosh S Quantitative quality control in microarray experiments and the application in data filtering, normalization and false positive rate prediction Bioinformatics 2003 19 1341 1347 12874045 10.1093/bioinformatics/btg154 Diehl F Grahlmann S Beier M Hoheisel J Manufacturing DNA microarrays of high spot homogeneity and reduced background signal Nucleic Acids Research 2001 29 e38 11266573 10.1093/nar/29.7.e38 Wang HY Malek RL Kwitek AE Greene AS Luu TV Behbahani B Frank B Quackenbush J Lee NH Assessing unmodified 70-mer oligonucleotide probe performance on glass-slide microarrays Genome Biol 2003 4 R5 12540297 10.1186/gb-2003-4-1-r5 Rees W Yager T Korte J Von Hippel P Betaine can eliminate the base pair composition dependence of DNA melting Biochemistry 1993 32 137 144 8418834 10.1021/bi00052a019 Henke W Herdel K Jung K Schnorr D Loening S Betaine improves the PCR amplification of GC-rich sequences Nucleic Acids Research 1997 25 3957 3958 9380524 10.1093/nar/25.19.3957 Don RH Cox PT Wainwright BJ Baker K Mattick JS 'Touchdown' PCR to circumvent spurious priming during gene amplification Nucleic Acids Res 1991 19 4008 1861999 Hecker KH Roux KH High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR Biotechniques 1996 20 478 485 8679209 Roux KH Hecker KH One-step optimization using touchdown and stepdown PCR Methods Mol Biol 1997 67 39 45 9031128 Eisen M Brown P DNA arrays for analysis of gene expression Methods in Enzymology 1999 303 179 205 10349646 10.1016/S0076-6879(99)03014-1 Wang X Jiang N Feng X Xie Y Tonellato P Ghosh S Hessner MJ A novel approach for high quality microarray processing using third-dye array visualization technology IEEE Transactions on Nanoscience 2003 2 193 201 10.1109/TNB.2003.816233

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==== Front BMC Plant BiolBMC Plant Biology1471-2229BioMed Central London 1471-2229-4-121528203310.1186/1471-2229-4-12Methodology ArticleDiscovery of induced point mutations in maize genes by TILLING Till Bradley J [email protected] Steven H [email protected] Clifford [email protected] Nathan [email protected] Chris [email protected] Kim [email protected] Elisabeth [email protected] Christine A [email protected] Linda C [email protected] Anthony R [email protected] Elizabeth A [email protected] Luca [email protected] Steven [email protected] Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA2 Department of Biology, University of Washington, Seattle, Washington 98195, USA3 Department of Agronomy, Purdue University, West Lafayette, Indiana 47907, USA4 Department of Plant Biology, University of Minnesota, St. Paul, Minnesota, 55108 USA2004 28 7 2004 4 12 12 2 6 2004 28 7 2004 Copyright © 2004 Till et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Going from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING (Targeting Induced Local Lesions IN Genomes) method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a high-throughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community. Results We demonstrate that high-throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse-genetic resources currently available. We screened pools of DNA samples for mutations in 1-kb segments from 11 different genes, obtaining 17 independent induced mutations from a population of 750 pollen-mutagenized maize plants. One of the genes targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutations that are predicted to be strongly deleterious. Conclusions Our findings indicate that TILLING is a broadly applicable and efficient reverse-genetic strategy. We are establishing a public TILLING service for maize modeled on the existing Arabidopsis TILLING Project. ==== Body Background Rapid progress being made in genome sequencing projects provides raw material for the potential understanding of gene function, so effective reverse genetic strategies are increasingly in demand [1]. Sequence information alone may be sufficient to consider a gene to be of interest, because sequence comparison tools that detect protein sequence similarity to previously studied genes often allow a related function to be inferred. Hypotheses concerning gene function that are generated in this way must be confirmed empirically. Experimental determination of gene function is desirable in other situations as well, for example, when a genetic interval has been associated with a phenotype of interest. In such cases, the functions of genes in an interval can be deduced from the phenotypes of induced mutations. Furthermore, the dissection of gene interactions often requires the availability of a range of allele types. However, most available methods for inferring function rely on techniques that produce a limited range of mutations, are labor-intensive or unreliable, or are limited to species in which special genetic tools have been developed [2]. Just as the discovery of induced mutations led to forward genetics, the introduction of rapid reverse genetic methods can have great impact. Several general strategies have been used to obtain reduction-of-function or knockout mutations in model organisms, including insertional mutagenesis [3] and RNA suppression [4], which have been widely used in plants. Insertional mutagenesis is now largely an in silico procedure for Arabidopsis researchers, as searchable databases of flanking sequences from T-DNA and transposon insertions are available on-line [5]. RNA suppression currently requires considerable manual effort, but it has the potential of reducing expression of repeated genes, which are especially common in plants, including Arabidopsis. However, because these techniques rely either on Agrobacterium T-DNA vectors for transmission or on endogenous tagging systems, their usefulness as general reverse genetics methods is limited to very few plant species. In maize, the majority of reverse genetic resources have exploited the endogenous Mutator (Mu) and Activator (Ac) transposon families. Mutator transposable elements are frequently present in high copy number and tend to insert in or near genes [6] thus providing mostly gene knock-out and potential loss-of-function alleles due to insertions in regulatory elements. Resources to identify Mu insertions in target genes include the Trait Utility System for Corn [7], the Maize Targeted Mutagenesis project [8], and the Photosynthetic Mutant Hunt [9]. Additionally, a modified Mutator element has been engineered to allow plasmid rescue of the element and flanking genomic DNA into E. coli [10,11] facilitating the sequencing of tagged genes and allowing the generation of an insertional database analogous to the T-DNA database currently available for Arabidopsis. Activator transposons are low copy number elements that, like Mu, preferentially insert in or near genes [12]. Large-scale isolation and sequencing of Ac elements for a reverse genetics database is advantageous, because single to low copy number insertions can be obtained. Reverse genetic strategies based on induced mutations have the potential for general applicability. Two such methods have been described for plants. One is deletional mutagenesis using fast neutron bombardment, which appears to be an effective means of knocking out tandemly repeated genes [13]. Another is TILLING (Targeting Induced Local Lesions IN Genomes), in which treatment using traditional chemical mutagens causes point mutations that are then discovered in genes of interest using a sensitive method for single-nucleotide mutation detection [14]. TILLING can provide an allelic series that includes missense and knockout mutations. The utility of allelic series that has been demonstrated in traditional forward genetic studies makes TILLING an especially desirable reverse-genetic strategy as genomic sequences become increasingly available. We have introduced a high-throughput screening method for TILLING based on the use of a mismatch-cleavage endonuclease, followed by fluorescent display of cleaved products on polyacrylamide electrophoretic gels using the LI-COR analyzer system [15]. We systematized this method and established a public TILLING facility for the general Arabidopsis community [16]. Our Arabidopsis TILLING Project (ATP) screened on average ~3000 ethylmethanesulfonate (EMS)-mutagenized M2 Arabidopsis plants per 1-kb gene fragment, which resulted in the delivery of >4000 mutations in >400 genes. Analysis of the TILLING data revealed that EMS is a nearly ideal mutagen, producing G/C-to-A/T transitions >99% of the time with only minor local sequence biases [17]. Our analysis also indicated that the high-throughput cleavage-based detection method is highly efficient: at least 3/4 of all mutations present were detected and essentially all mutations detected were confirmed by sequencing. ATP demonstrated the practicality of high-throughput TILLING in a production setting. However, the small genome size of Arabidopsis and the ease with which it can be cultured might have made Arabidopsis easier to TILL than a field-grown crop plant with a large genome. To determine whether the procedures that were developed for ATP can be generalized, we chose to TILL maize, a crop plant with a genome that is ~20 times larger than Arabidopsis. We find that essentially the same procedures that provided efficient TILLING of Arabidopsis can be applied to maize, yielding comparable results. Despite the relatively small size of our mutagenized maize TILLING population, we obtained useful mutations. These include a promising allelic series for a chromomethylase gene that had been previously implicated in non-CpG DNA methylation, whose counterpart in Arabidopsis is responsible for epigenetic gene silencing and genome surveillance. Results TILLING consists of a series of steps, beginning with chemical mutagenesis of reference individuals and culminating in the determination of mutant base pairs by DNA sequencing [14]. High-throughput TILLING utilizes the CEL I mismatch-cleavage enzyme on heteroduplexes with detection of end-labeled cleavage products on electrophoretic gels [15,18]. The procedure for maize TILLING is identical to that for Arabidopsis, except that pollen rather than seed was treated with EMS (Figure 1). For this study, two separate mutagenized B73 maize populations, designated UI (M2 families from 384 mutagenized lines) and NS (366 individual M1 plants), were screened in 4-fold pools (4 UI families or 4 NS plants) in a 96-well format. To compensate for the larger genome size of maize relative to Arabidopsis, we increased the amount of genomic template DNA amplified 20-fold; otherwise, all protocols and default parameters were the same as used for Arabidopsis TILLING [16]. We found that 11 of the 14 primers gave high quality products, compared to ~90% success that is obtained for Arabidopsis. We proceeded to screen for mutations within these 11 gene fragments in the 750 DNA samples and discovered 21 point lesions (Table 1). All 21 were verified by sequencing. Of the 21 lesions, 17 appeared to be EMS-induced. These 17 mutations were G/C-to-A/T mutations, as expected for EMS [17]. The other four lesions were found in a single plant (Table 1) and only one was a G/C-to-A/T transition, suggesting that this plant is a non-B73 contaminant, likely due to cross-pollination. Contaminations seen as an excessively high frequency of polymorphisms in single plants have been occasionally observed by ATP [17,19]. The presumed EMS-induced mutations were detected in single plants, except for DMT102 G878A, where the exact same mutation was found in two different plants; this circumstance is expected and observed to occur 4% of the time in Arabidopsis based on random distribution of induced mutations, GC content of the genome and distribution of location of mutations discovered in fragments [17]. Finding one coincidence among 17 mutations is not significantly different from expectation. Furthermore, finding that all 17 mutations are G/C-to-A/T transitions effectively rules out the possibility that they are naturally occurring polymorphisms: with four possible single-base changes, the chance probability of observing that all 17 conform to the expectation for EMS mutagenesis is only (1/4)17 or ~1/1010. Importantly, each population yielded 8–9 confirmed new mutations, for an overall mutation density of approximately two mutations/megabase. Taking into account the fact that pollen treatment mutagenizes only one of two genomes, whereas seed treatment mutagenizes both (although by screening M2s in the latter case, 1/4 of the mutations are lost as +/+ segregants), the estimated mutation density for both maize populations is ~3/4 as high as our average for Arabidopsis per mutagenized genome. We have previously demonstrated reliable detection of mutations in 8-fold pools based on analysis of ATP-generated data [17]. For example, we obtained almost precisely the expected 2:1 heterozygote:homozygote ratio for ~1900 mutations in 8-fold pools, indicating that detection of 1/16 is no different from detection of 1/8 by TILLING. To confirm this detection efficiency in maize, we screened the primer sets for DMT101 and DMT103 with 8-fold pools from the 750 B73 DNA samples that had already been screened in pools of four. In this test, we detected only three of the five mutations that we had discovered by 4-fold pooling. Inadequate data quality does not account for missing these two base changes (UI20291 and NS3471.9) in 8-fold pools, because the gel images were typical of what is seen in our ATP operation. Therefore, we considered the possibility that failure to detect two of five mutations in this limited test was caused by variation of DNA amounts in the pools. One potential source of variability in DNA amount is that degraded DNA is difficult to measure accurately when visualized by agarose gel analysis. We noticed that some of the genomic DNA samples, including NS3471.9, were partially degraded. Inaccuracies in measuring the amount of DNA in a sample will compromise normalization in pools: any variation in the amount of DNA contributed by each plant in the pool will lead to reduced representation of one or more plants. As the amount of DNA in a pool from a particular plant decreases, mutation detection becomes limiting. Recognizing that the quality of genomic DNA could potentially hinder the throughput of mutation detection, we sought an alternative method of sample quantification and normalization. We have found that running samples on 3% Metaphor® (Cambrex) agarose gels reduced "smearing" of fragments, thus facilitating quantification. While lower DNA quality and inaccurate normalization could account for missing the base change in NS3471.9, this is not a likely explanation for missing the mutation in UI20291, which was from an apparently undegraded sample. Therefore, we considered that another source of sample-to-sample variation arose from the sampling of leaves from M2 sibling plants in the UI series rather than using M1 plants directly. Individual DNA samples from the UI population were generated by pooling approximately 10 individuals from an M2 family. M1 plants that are heterozygous for a new mutation will yield M2 families segregating 1:2:1 for the new mutation. A sample over a large M2 family should yield DNA that is equal parts wild type and mutant allele, assuming good viability and fertility of the mutant allele. However, a small M2 sample may be biased and contain too large a proportion of wild-type DNA which would then prevent a given target sequence containing a mutation from being detected among 8-fold pooled DNAs, depending on the limit for robust mutation detection. Direct evidence that sampling from M2 families was a problem came from our finding that two of the mutations found in plants sampled as leaves from multiple M2 plants were scored as homozygous by our usual criteria. Pollen mutagenesis can only produce M1 heterozygotes, and we interpret the homogeneity of the mutant as resulting from limited sampling of heterogeneous M2 plants. For example, if only one or two of the planted M2 seed germinated for these families, and the limited sample favored homozygotes, then they would sometimes appear to be purely homozygous in the sequence trace. Similarly, an equal or greater number of mutations will be underrepresented because wild type will be in excess, leading us to miss detecting the mutation in 8-fold, but not in 4-fold pools. This underscores the importance of using DNA from M1 pollen-mutagenized individuals and taking care to avoid collection procedures that could exacerbate degradation during DNA isolation. By assaying DNA concentrations on Metaphor gels and sampling only the M1 generation, we should be able to pool 8-fold without reducting detection efficiency. To test this, we screened a population of 768 M1 W22 maize DNAs (pollen mutagenesis) with similar degradation patterns as the B73 samples described above and normalized using 3% Metaphor agarose gels. We then made 8-fold and 4-fold pools from these 768 samples and screened with four of the 11 primer sets in the original screen. This screening of 6-Mb of total sequence led to the independent detection of the same 3 mutations in both the 4-fold and the 8-pools (data not shown). Therefore, we conclude that even partially degraded DNA from M1 pollen-mutagenized samples, such as might be extracted from material collected in the field, can be used for TILLING. The 17 induced mutations discovered in the screen were distributed as expected, consisting of 10 missense, 7 silent and no truncation mutations, compared with 51% missense, 44% silent and 5% truncation mutations based on ~4000 TILLed Arabidopsis mutations. Considering that about half of missense mutations are expected to be damaging to a typical protein [20], we expect that even a small allelic series will be useful for phenotypic analysis. Indeed, we discovered that all three different DMT102 missense mutations are likely to be deleterious to the protein, based on SIFT and PSSM Difference scores (Figure 2). The SIFT algorithm predicts deleterious missense mutations with ~75% overall accuracy based on analysis of experimental mutagenesis data [21,22] and comprehensive human polymorphism and disease data [23]. Therefore, the DMT102 allelic series appears to be essentially complete after screening a 1-kb region within only 750 maize plants. Discussion We have shown that TILLING is an efficient method for reverse genetics in a crop plant. The density of mutations that we discovered appears to be only slightly lower than what is obtained for Arabidopsis using the same methodology. For Arabidopsis, we currently screen ~2300 M2 plants to obtain a suitable allelic series, which averages ~12 mutations per 1.5-kb segment screened. Based on this work, we estimate that screening ~4000 maize plants will provide a comparable series ([4000] [17 mutations] [1.5-kb/1-kb]÷([11-kb] [750 plants]) = ~12 mutations). Since this study was completed, we have expanded the size of our mutagenized population to ~2500 M1 individuals in the B73 genetic background and ~2000 M1 plants in the W22 genetic background that are ready for screening, and populations of ~10,000 M1 plants are being prepared. For effective pooling, each individual in a pool must be represented at a concentration that is equivalent to the other members in a pool. Failure to accomplish this could result in a mutation represented in the pool below the level of detection. The goal is to maximize throughput by increasing pooling while still detecting all possible mutations. We have shown that in Arabidopsis, heterozygous mutations can be as efficiently discovered as homozygous mutations in 8-fold pools, thus providing a minimum estimate of robust discovery in a production setting of 1 in 16 [16]. We have described here two possible sources of error that might hinder the ability to pool samples effectively: inaccurate DNA quantitation, and sampling error in tissue collection. DNA quantitation using gel electrophoresis is difficult when samples are degraded, as the standard band of DNA appears as a smear, although the problem can be minimized using high percentage agarose gels. Sampling bias in the collection of individuals descended from a single mutagenized parent can also lead to non-equivalent representation of a mutation in a pool. The present study revealed normalization inaccuracy or sampling bias by detecting homozygous mutations in lines that should have yielded only heterozygous mutations. To minimize sampling bias, only DNA from M1 individuals will be used to create our library for a maize TILLING service. At least one of the maize genes that we screened yielded an excellent allelic series. We discovered three missense mutations in DMT102, all of which are predicted to damage the protein based on sequence conservation. This analysis used two different programs: SIFT (Sorting Intolerant From Tolerant [23], which uses PSI-BLAST alignments, and PARSESNP Project Aligned Related Sequences and Evaluate SNPs[24], which provides a PSSM (Position-Specific Scoring Matrix) Difference score based on alignment blocks (Figure 2). DMT102 is a member of the chromodomain-containing "chromomethylase" subfamily of cytosine-5-DNA methyltransferases. The Arabidopsis CMT3 chromomethylase is the first example of a gene to be TILLed [14], and a nonsense mutation was responsible for sharply reducing CpNpG methylation [25]. This had confirmed a study in which a Mutator insertional mutation into maize DMT102 was shown to reduce CpNpG methylation [26]. Plant chromomethylases have received considerable recent attention. For example, studies of other mutations affecting CpNpG methylation reveal the first links between DNA methylation, histone methylation [27] and the small interfering RNA (siRNA) machinery [28] in a higher eukaryote. A methylation profiling study has revealed that transposons are in vivo targets of CMT3-dependent methylation [29]. Together with the Mutator insertional mutation [26], our TILLed DMT102 allelic series may now be applied to understanding the relationship between DNA methylation, chromatin structure, siRNAs and transposon biology in maize. Conclusions TILLING has several advantages as a general reverse-genetic tool, especially for organisms for which other options are limited. The high density of mutations resulting from chemical mutagenesis means that, relative to insertional or deletional mutagenesis, far fewer plants are required for screening and much smaller genes can be effectively targeted. EMS is a stable and reliable mutagen, whereas the stability, penetrance and accuracy of RNAi-based silencing is uncertain [30,31], and insertional mutagenesis can cause chromosomal rearrangements that complicate subsequent phenotypic analysis [32]. TILLING provides an allelic series of mutations, and is the only method that can focus the search for missense mutations to just part of a protein, such as in a single domain of a multidomain protein. TILLING lines can be produced in a homogeneous wild-type genetic background, which avoids problems of heterogeneity often required for insertional mutagenesis, especially in maize. Finally, given the high regulatory and intellectual property costs associated with transgenics and the current concerns about genetically modified crop plants, there is likely to be agricultural interest in producing phenotypic variants without introducing foreign DNA of any type into a plant's genome. We are currently establishing the reference population necessary to provide TILLING as a service to the maize community. Methods Maize mutagenesis, culture and DNA preparation B73 pollen was mutagenized with EMS and applied to the silks of B73 ears [33]. Ears were harvested at 5–6 weeks post pollination. For the NS population, M1 seed were planted and a whole young leaf harvested from each plant and lyophilized for DNA sampling. For the UI population, the M1 were selfed to make M2 seed, then families of twenty M2 siblings were planted and a total of 60 leaf discs were punched from the youngest leaves using all members of the family, and the pooled sample for an entire family lyophilized. Samples were prepared from lyophilized leaf tissue essentially as described [34], except that dried tissue was homogenized into a powder in a FastPrep homogenizer before adding buffer, and 20 mg of this powder was used to prepare DNA. High-throughput TILLING The same procedure used for TILLING Arabidopsis [16] was adapted for maize with only minor modifications. Primers were designed to amplify ~1-kb segments using the CODDLE program [35] based on either known maize genomic sequence or from maize cDNAs that are orthologous to intronless rice sequence. Genes were chosen from the NSF Plant Chromatin Project web site [36] based on the availability of genomic sequence or on the prediction of an exonic region at least 1-kb in size. Because rice and maize typically have identical placement of introns, by aligning the predicted maize coding sequence with the rice gene model, we could choose maize-specific primers that would amplify only exonic DNA. To find such regions, cDNA sequences were searched against Arabidopsis and rice genomic sequences using a version of BLAST that was modified to identify large exonic regions in maize based on the corresponding regions being exonic in rice and/or Arabidopsis. In all, we were able to design 14 primer pairs from plant chromatin genes for screening, and primer sets were ordered. In other cases, maize genomic sequence was available from ChromDb. Amplification of pools and individual DNA samples in 96-well plates, annealing, cleavage by CEL I, electrophoresis, image analysis, rescreening and DNA sequencing were performed as described [16]. Screening of mutations using LI-COR gel analyzers was performed as previously described [34]. Sequence trace information was analyzed using the Sequencher program as described [17] Authors' contributions SHR, KY and EB prepared the samples, CB, CAC, LCE and ARO performed the high-throughput screening and data processing, CW and NS provided the mutagenized plant material, EAG performed the data analysis and BJT, LC and SH participated in the design and execution of the study and wrote the paper. Acknowledgements This work was supported by a grant from the Plant Genome Research Project of the National Science Foundation. We thank Jorja Henikoff for maize/rice sequence comparisons, Vicki Chandler for providing genomic DNA sequences for primer design, and Kelly Marshall, Jianhua Yu, Peter Hermanson and Virginia Zaunbrecher from the Weil and Springer labs for their work in mutagenesis and tissue collection. Figures and Tables Figure 1 Schematic diagram of maize TILLING. Fresh pollen is collected and mutagenized with ethylmethanesulfonate (EMS). Pollen is then applied to silks of wild-type plants from the same genetic background. Seeds from the resulting ears are grown into plants of the M1 generation. Plants of this generation are heterozygous for any induced mutation. Tissue is collected either from each M1 plant or from approx. 10 M2 siblings from the M1 self cross. M3 seed is generated by randomly intermating 10–12 M2 siblings. This M3 seed serves as the seed stock for future studies. DNA is extracted from collected tissue and samples are pooled to increase screening throughput. For mutation detection, sequence specific primers are used to amplify the target locus by PCR. Following amplification, samples are heat denatured and reannealed to generate heteroduplexes between mutant amplicons and their wild-type counterparts. Heteroduplexes are cleaved using CEL I endonuclease and are visualized using denaturing polyacrylamide gel electrophoresis. See reference [34] for further details. Figure 2 PARSESNP output for maize DMT102. At top is a map showing the positions of five independent mutations in the TILLed fragment, based on the gene model (red boxes for exons and lines for introns) and block alignments produced by running SIFT with DMT102 as query against SWISS-PROT-Trembl. The table provides information concerning the effect of the mutation and restriction site changes that can be used for genotyping progeny plants. PSSM Difference or SIFT Scores in red indicate that the missense mutation is predicted to damage the protein. PARSESNP also provides sequence maps (not shown). Table 1 Mutations detected by screening EMS-mutagenized maize populations. Gene1 Individual Sequence Stock Number DMT101 3D3 C553T UI20118 8A8 G464A UI20291 DMT102 3H3 C677T UI20122 11B3 G878A UI20424 12B3 G878A UI20459 15G3 G938A NS2809.1 19C2 G676A NS3457.3 21G2 A637W NS3471.92 DMT103 4A9 G116A UI20152 14E9 G514A NS2806.7 16G9 G519A NS2810.4 21G2 G120K NS3471.92 21G2 A148G NS3471.92 21G2 G789A NS3471.92 DMT106 15E2 C697T NS2808.6 HAC110 16B7 G471A NS2809.12 HDA105 4A3 G343A UI20152 9F2 C571T UI20333 13H2 G271A NS2804.1 22A9 G417A NS3478.7 1No mutations were detected in 1 kb fragments of five other genes screened: DMT105, SDG104, SDG105, SDG124 and SDG125. 2Multiple non-G/C-to-A/T mutations discovered in NS3471.9 indicate that it derives from contaminating pollen. See text for details. ==== Refs Nagy A Perrimon N Sandmeyer S Plasterk R Tailoring the genome: the power of genetic approaches Nature Gen 2003 33 Suppl. 276 284 10.1038/ng1115 Henikoff S Comai L Single-nucleotide mutations for plant functional genomics Annual Reviews of Plant Biology 2003 54 375 401 10.1146/annurev.arplant.54.031902.135009 Long D Coupland G Transposon tagging with Ac/Ds in Arabidopsis Methods Mol Biol 1998 82 315 328 9664435 Waterhouse PM Graham MW Wang MB Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA Proc Natl Acad Sci USA 1998 95 13959 13964 9811908 10.1073/pnas.95.23.13959 Krysan PJ Young JC Jester PJ Monson S Copenhaver G Preuss D Sussman MR Characterization of T-DNA insertion sites in Arabidopsis thaliana and the implications for saturation mutagenesis OMICS 2002 6 163 174 12143962 10.1089/153623102760092760 Lisch D Mutator transposons Trends Plant Sci 2002 7 498 504 12417150 10.1016/S1360-1385(02)02347-6 Bensen RJ Johal GS Crane VC Tossberg JT Schnable PS Meeley RB Briggs SP Cloning and characterization of the maize An1 gene Plant Cell 1995 7 75 84 7696880 10.1105/tpc.7.1.75 May BP Liu H Vollbrecht E Senior L Rabinowicz PD Roh D Pan X Stein L Freeling M Alexander D Martienssen R Maize-targeted mutagenesis: A knockout resource for maize Proc Natl Acad Sci U S A 2003 100 11541 11546 12954979 10.1073/pnas.1831119100 http://chloroplast.uoregon.edu/ Raizada MN Nan GL Walbot V Somatic and germinal mobility of the RescueMu transposon in transgenic maize Plant Cell 2001 13 1587 1608 11449053 10.1105/tpc.13.7.1587 http://gremlin3.zool.iastate.edu/zmdb/RescueMu.html Cowperthwaite M Park W Xu Z Yan X Maurais SC Dooner HK Use of the transposon Ac as a gene-searching engine in the maize genome Plant Cell 2002 14 713 726 11910016 10.1105/tpc.010468 Li X Song Y Century K Straight S Ronald P Dong X Lassner M Zhang Y A fast neutron deletion mutagenesis-based reverse genetics system for plants Plant J 1991 27 235 242 10.1046/j.1365-313x.2001.01084.x McCallum CM Comai L Greene EA Henikoff S Targeted screening for induced mutations Nat Biotechnol 2000 18 455 457 10748531 10.1038/74542 Colbert T Till BJ Tompa R Reynolds S Steine MN Yeung AT McCallum CM Comai L Henikoff S High-throughput screening for induced point mutations Plant Physiol 2001 126 480 484 11402178 10.1104/pp.126.2.480 Till BJ Reynolds SH Greene EA Codomo CA Enns LC Johnson JE Burtner C Odden AR Young K Taylor NE Henikoff JG Comai L Henikoff S Large-scale discovery of induced point mutations with high throughput TILLING Genome Res 2003 13 524 530 12618384 10.1101/gr.977903 Greene EA Codomo CA Taylor NE Henikoff JG Till BJ Reynolds SH Enns LC Burtner C Johnson JE Odden AR Comai L Henikoff S Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis Genetics 2003 164 731 740 12807792 Till BJ Burtner C Comai L Henikoff S Mismatch cleavage by single-strand specific nucleases Nucleic Acids Res 2004 32 2632 2641 15141034 10.1093/nar/gkh599 Comai L Young K Till BJ Reynolds SH Greene EA Codomo CA Enns LC Johnson JE Burtner C Odden AR Henikoff S Efficient discovery of DNA polymorphisms in natural populations by Ecotilling Plant J 2004 37 778 786 14871304 10.1111/j.0960-7412.2003.01999.x Markiewicz P Kleina LG Cruz C Ehret S Miller JH Genetic studies of the lac repressor. XIV. Analysis of 4000 altered Escherichia coli lac repressors reveals essential and non-essential residues, as well as "spacers" which do not require a specific sequence J Mol Biol 1994 240 421 433 8046748 10.1006/jmbi.1994.1458 Ng PC Henikoff S Predicting deleterious amino acid substitutions Genome Res 2001 11 863 874 11337480 10.1101/gr.176601 Saunders CT Baker D Evaluation of structural and evolutionary contributions to deleterious mutation predictions J Mol Biol 2002 27 891 901 10.1016/S0022-2836(02)00813-6 Ng PC Henikoff S SIFT: Predicting amino acid changes that affect protein function Nucleic Acids Res 2003 31 3812 3814 12824425 10.1093/nar/gkg509 Taylor N Greene EA PARSESNP: A tool for the analysis of nucleotide polymorphisms Nucleic Acids Res 2003 31 3808 3811 12824424 10.1093/nar/gkg574 Lindroth AM Cao X Jackson JP Zilberman D McCallum CM Henikoff S Jacobsen SE Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation Science 2001 292 2077 2080 11349138 10.1126/science.1059745 Papa CM Springer NM Muszynski MG Meeley R Kaeppler SM Maize Chromomethylase Zea methyltransferase2 Is Required for CpNpG Methylation Plant Cell 2001 13 1919 1928 11487702 10.1105/tpc.13.8.1919 Jackson JP Lindroth AM Cao X Jacobsen SE Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase Nature 2002 416 556 560 11898023 10.1038/nature731 Zilberman D Cao X Jacobsen SE ARGONAUTE4 controls of locus-specific siRNA accumulation and DNA and histone methylation Science 2003 299 716 719 12522258 10.1126/science.1079695 Tompa R McCallum CM Delrow J Henikoff JG vanSteensel B Henikoff S Genome-wide profiling of DNA methylation reveals transposon targets of CHROMOMETHYLASE3 Curr Biol 2002 12 65 68 11790305 10.1016/S0960-9822(01)00622-4 Chuang CF Meyerowitz EM Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana Proc Natl Acad Sci USA 2000 97 4985 4990 10781109 10.1073/pnas.060034297 Jackson AL Bartz SR Schelter J Kobayashi SV Burchard J Mao M Li B Cavet G Linsley PS Expression profiling reveals off-target gene regulation by RNAi Nat Biotechnol 2003 21 635 637 12754523 10.1038/nbt831 Tax FE Vernon DM T-DNA-associated duplication/translocations in Arabidopsis. Implications for mutant analysis and functional genomics Plant Physiol 2001 126 1527 1538 11500551 10.1104/pp.126.4.1527 Okagaki RJ Neuffer MG Wessler SR A deletion common to two independently derived waxy mutations of maize Genetics 1991 128 425 431 2071021 Till BJ Colbert T Tompa R Enns LC Codomo CA Johnson JE Reynolds SH Henikoff JG Greene EA Stein MN Comai L Henikoff S GrotewaldE High-throughput TILLING for functional genomics Plant Functional Genomics: Methods and Protocols 2003 Clifton, NJ, Humana Press 205 220 CODDLE Chrom DB

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BMC Plant Biol. 2004 Jul 28; 4:12

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==== Front BMC Cardiovasc DisordBMC Cardiovascular Disorders1471-2261BioMed Central London 1471-2261-4-141529869910.1186/1471-2261-4-14Research ArticleOutcome after acute myocardial infarction: a comparison of patients seen by cardiologists and general physicians Abubakar Ibrahim [email protected] David [email protected] Barbara [email protected] Jo [email protected] Peter [email protected] School of Medicine, Health Policy and Practice, University of East Anglia, Norwich, NR4 7TJ UK2 Health Protection Agency Regional Epidemiology Unit (East of England), Institute of Public Health, Cambridge CB2 2SR, UK3 Director of Public Health, South Cambridgeshire Primary Care Trust, Heron Court, Cambridge, UK4 CAMS, Institute of Public Health, Cambridge, UK5 Department of Cardiology, Peterborough District Hospital, Peterborough, UK6 Department of Cardiology, Addenbrooke's Hospital, Cambridge, UK2004 6 8 2004 4 14 14 24 5 2004 6 8 2004 Copyright © 2004 Abubakar et al; licensee BioMed Central Ltd.2004Abubakar et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The management of acute myocardial infarction (AMI) has improved over the last 50 years with the more frequent use of effective medicines and procedures. The clinical benefit of the speciality of the attending physician is less clear. The United Kingdom National Service Framework for coronary heart disease (CHD) suggested that patients with CHD are likely to benefit from cardiological supervision. We set out to assess the effect of access to cardiologists on survival among AMI patients admitted in two UK hospitals. Methods The study was conducted in a university hospital and a district general hospital in England. Information was obtained on age, sex, ethnicity, Carstairs socioeconomic deprivation category derived from postcode of residence, comorbidity, distance from hospital and medication from all patients admitted with acute myocardial infarction in two acute trusts between July 1999 and June 2000. Record linkage to subsequent Hospital Episode Statistics and Registrar General's death records provided follow up information on procedures and mortality up to eighteen months after admission. Cox proportional hazard models were used to investigate the main hypothesis controlling for confounding. The main outcome measure was 18-month survival after myocardial infarction. Results Access to a cardiologist was univariately associated with improved survival (hazard ratio 0.16, 95% CI 0.10 to 0.25). This effect remained after controlling for the effect of patient characteristics (hazard ratio 0.22, 95% CI 0.14 to 0.25). The effect disappeared after controlling for access to effective medication (hazard ratio 0.70, 95% CI 0.33 to 1.46). Conclusions Access to a cardiologist is associated with better survival compared to no access to a cardiologist among a cohort of patients already admitted with AMI. This effect is mainly due to the more frequent use of effective medicines by the group referred to cardiologists. Hospitals may improve survival by improving access to effective medicines and by coordinating care between cardiologists and general physicians. ==== Body Background The management of acute myocardial infarction (AMI) has improved over the last 50 years with the more frequent use of effective medicines and procedures. The clinical benefit of the speciality of the attending physician is less clear. The effect of the speciality of the attending physician on mortality in AMI has been studied mainly in the United States (US) with conflicting results arising from the studies [3-6]. The explanations considered for the difference in mortality observed in some of the studies include patient's condition (case mix and comorbidity)[3], volume of workload[5,7] and treatment given[3]. Previous studies show that knowledge and use of effective medicines is better among cardiologists compared with general physicians in the US [8-10]. Co-ordination of care between cardiologists and non-cardiologists also improves survival of patients seen by non-cardiologists [11]. The National Service Framework (NSF) for coronary heart disease (CHD) published in March 2000, suggested that patients with CHD are likely to benefit from cardiological supervision. To provide this level of care, all acute hospitals will eventually need a minimum of two cardiologists [1]. A recent survey by the Royal College of Physicians found an average of 1.7 whole time equivalent (WTE) cardiologists in the 211 hospitals receiving patients with AMI in the UK [2]. Eight hospitals did not have a cardiologist. There is a need to establish whether the involvement of a cardiologist in the management of AMI patients affects the quality and outcome of care, and if so to identify ways to improve the outcome of care for patients unable to gain access to a cardiologist. This study aims to assess the effect of access to cardiologists on survival among AMI patients accounting for access to effective investigation, medication, procedures and the underlying condition of the patient at presentation. Methods A retrospective cohort design was used. Study population and inclusion criteria All patients admitted to two hospitals in Eastern England between 1st July 1999 and 31st June 2000 with a discharge diagnosis of AMI were included in the analysis. A diagnosis of AMI was based on evidence of raised cardiac enzymes and/or other indicators of myocardial necrosis and on a physician's judgement of ECG changes indicative of AMI. Data collection and analysis Hospital Episode Statistics were used to extract records of all patients admitted with the ICD10 diagnostic codes for Acute Myocardial Infarction (I21, I22, I23) in any diagnostic field. The records were restructured to produce one record per patient using the new NHS number. For records with missing NHS number, a unique identifier was created using date of birth, sex and post code. The unique identifier was also used as a check for the records with NHS numbers. Records of all Finished Consultant Episodes with OPCS 4 codes of either Coronary Artery Bypass Grafting (K40–46) or Percutaneous Transluminal Coronary Angiography (K49–50) were extracted. The process was repeated for angiography (K63 – 65). Record linkage with subsequent Hospital Episode Statistics and Registrar General's records provided follow up information on procedures and death up to eighteen months after the index AMI. All cases were reviewed by examining the case notes. Detailed information on variables summarised below was obtained. List of variables extracted from patients' case notes • Hospital where patient was first admitted • Whether the patient was seen by a cardiologist or not, determined by evidence of a cardiology review in a patients case note • The average distance from residence to hospital first seen was determined by mapping the distance in miles using the RAC™ website distance calculator • A history of comorbidity – defined as the presence of diabetes, metastatic malignancy, cerebrovascular accident (CVA), asthma, chronic obstructive airway disease, renal impairment, endocrine disorder, chronic infection, dementia • Physical impairment with poor mobility • Procedures – exercise testing, angiography, CABG, PTCA • Appropriate medication – β blockers, aspirin, ACE inhibitors, thrombolysis (table 2) Table 1 Patient characteristics, and whether seen by cardiologist or by general physician Patient characteristic Physician Cardiologist (N = 275) Non Cardiologist (N = 201) Mean age (SD) 64.4 (SD 12.8) 74.8 (SD 11.4) Sex n (%) Male 275 (71.4) 201 (52.8) History of:  Congestive heart failure % 11.3 29.6  Angina % 94.9 86.7  Hypertension % 41.8 46.6  Diabetes mellitus % 12.1 17.1  Impaired mobility % 7.8 17.7  Smoking % 36.0 49.6 Impaired left ventricular function % 24.6 25.0 Table 2 Odds ratios for drug and procedure use after AMI by physician (cardiologist relative to non-cardiologist) Intervention Number (Percent) Odds ratio (95% CI) Cardiologist (275) Non Cardiologist (201) Thrombolytic therapy* 140 (51%) 66 (33%) 2.08 (1.35 – 3.23) β blocker on discharge* 206 (75%) 90 (45%) 3.70 (2.38 – 5.56) Aspirin* 264 (96%) 160 (80%) 6.67 (3.23 – 14.29) ACE inhibitors 124 (45%) 80 (40%) 1.27 (0.01 – 1.92) Exercise testing 192 (70%) 46 (23%) 7.70 (4.76 – 12.50) Coronary angiography 151 (55%) 12 (6%) 4.76 (3.22 – 7.14) Coronary angioplasty 74 (27%) 30 (15%) 1.92 (1.28 – 2.94) Coronary artery bypass grafting 66 (24%) 30 (15%) 1.75 (1.15 – 2.70) * See text for eligibility criteria • A history of Smoking Previous MI Hypertension • Impaired left ventricular function based on echocardiography result or angiography • Severity of AMI using the number of vessels affected (vessels were considered affected if a lesion of 50% or more was noted in the angiography report) • Demographic factors: age, sex, ethnicity, socioeconomic status and Carstairs socio-economic deprivation category derived from postcode of residence Baseline patient characteristics were tabulated. Categorical variables were tested for statistical significance using the χ2 test. Continuous variables were tested using the Student t test comparing patients seen by cardiologists and those seen by other physicians. The relative odds of drug and procedure use by speciality of physician were calculated. Specific variables included the use of thrombolytic therapy, β blockers, aspirin, ACE inhibitors among clinically appropriate groups, and the use of exercise testing, coronary angiography, angioplasty and coronary artery bypass grafting (CABG). Cox proportional hazards models were used to investigate the effect on survival of seeing a cardiologist. Multivariate models were fitted to control for the effect of patient and hospital characteristics. Controlled factors included: age, sex, comorbidity, hospital, distance from patients residence to hospital and Carstairs socio-economic deprivation category in the first instance. The effect of angiography, revascularisation and the use of effective medicines (aspirin and/or β blockers and/or thrombolysis – eligibility criteria listed below) were subsequently introduced into the model to investigate whether these affected outcome. The factors were included in the model either because they are known to confound the association between type of physician and survival or are known to be associated with survival and were not comparable between the two groups. Eligibility criteria for appropriate use of effective medicines [13]. Thrombolytic therapy • No warfarin therapy on admission • ST-segment elevation on initial ECG • Less than 12 hours since onset of chest pain • Systolic BP less than 180 mmHg and diastolic BP less than 110 mmHg at presentation • No severe CVA, gastrointestinal disease or chronic liver disease Aspirin • No haemorrhagic complication • No severe CVA, gastrointestinal disease or chronic liver disease or renal failure • No warfarin therapy on admission β blockers • No chronic lung disease • No cardiogenic shock, hypotension, complete heart block or decompensated heart failure The characteristics of the two hospitals are summarised figure 1. The results from the two hospitals were combined in order to make our findings more generalisable. The general characteristics did not show important differences in the organisation of care between the hospitals. It is unlikely there are important unmeasurable factors associated with a particular hospital that might cause confounding, but hospital of care is still included as a variable in the multivariate analysis to control for centre effect. Patients from the two hospitals undergo procedures in the same specialist cardiothoracic hospital. Figure 1 Characteristics of study hospitals. The main outcome was eighteen-month survival defined as time between date of AMI and death. Statistical analysis was carried out using Stata (Version 6.0). In general for Cox regression analysis, the number of events i.e. deaths (and similarly the number of non-events) per variable modelled should be at least 10 [12]. Ethical approval was obtained from the two local research ethics committees of the hospitals involved in the study. Permission from the director of research and Caldicott guardians of data in each hospital was also obtained. Results Medical records were obtained for 94% of eligible patients (476 of 506 patients). Four patients did not have enzyme changes of myocardial infarction or were clear cases of miscoding. The mean age of the cohort was 69.3 years (SD 13.2) and 34.5% were female. The patients with missing records did not differ from those with records in terms of age or sex using information from routine data (mean age was 70.4 years with SD 11.1; 36.2% were female). There were no significant differences in the proportion of data missing between the two hospitals including those for eligibility to effective medications and co morbidities. General characteristics Patients in our sample seen by a cardiologist were younger, with a higher proportion of males to females. They were more likely to have a history of CHD prior to myocardial infarction, but less likely to have a history of congestive heart failure or hypertension (Table 1). Patients with impaired mobility or who smoked were less likely to be seen by a cardiologist. The difference in the proportion of patients with echocardiographic evidence of impaired left ventricular function complicating their myocardial infarction by physician seen was not significant. Drug and procedure use The likelihood of receiving effective medication (thrombolytic therapy, β blockers, aspirin) was higher among patients seen by a cardiologist compared with those not seen by a cardiologist. Furthermore, patients seen by a cardiologist were more likely to have undergone exercise testing, angiography and revascularisation procedures (Table 2). The use of ACE inhibitors was not more frequent among cardiologists. Survival Univariate analysis indicated that patients seen by a cardiologist had better survival (hazard ratio 0.16, 95% CI 0.10 to 0.25). Effect of seeing a cardiologist after controlling for patient characteristics The survival of patients seen by a cardiologist was still better than that for those not seen by a cardiologist (hazard ratio 0.22, 95% CI 0.14 to 0.25) after controlling for the confounding effect of pre-existing comorbidity, age, sex, hospital first seen, deprivation and distance (Table 3). The effect of seeing a cardiologist remained after excluding patients that died within a week of MI. The hazard ratio was 0.21 (95% CI 0.12 – 0.37). Table 3 Cox regression analysis for eighteen month survival comparing cardiologists and non-cardiologist physicians and controlling for potential confounders. Variable Mortality (%) Adjusted hazard ratio (CI) * Adjusted hazard ratio (CI) # Seen by a Cardiologist Yes 30 (11%) 0.22 (0.14 – 0.38) 0.70 (0.33 – 1.46) No 88 (44%) Comorbidity Yes 15 (25%) 1.11 (0.62 – 1.99) 0.98 (0.49 – 1.98) No 107 (26%) Hospital A 58 (24%) 1.01 (0.67 – 1.51) 1.08 (0.71 – 1.73) B 65 (28%) Age (years) 30 – 40 1 (8%) 1.05 (1.03 – 1.08) 1.03 (0.99 – 1.06) 41 – 60 8 (7%) 61 – 70 18 (17%) 71 – 99 96 (40%) Sex Males 64 (21%) 0.91 (0.62 – 1.35) 0.83 (0.44 – 1.55) Females 58 (34%) Distance 1.0 (0.97 – 1.04) 0.96 (0.88 – 1.03) Angiography Yes 12 (7%) - 0.87 (0.24 – 3.17) No 112 (36%) Revascularisation Yes 14 (13%) - 0.25 (0.04 – 1.52) No 110 (30%) Effective medicinesγ Yes 48 (14%) - 0.15 (0.07 – 0.29) No 76 (56%) * Adjusted for patient characteristics, hospital # Adjusted for patient characteristics, hospital and the use of effective medicines and procedures γ Access to aspirin, and/or β blockers and/ or thrombolysis if appropriate Effect of seeing a cardiologist after controlling for patient characteristics and use of effective intervention The effect of seeing a cardiologist on survival became non-significant once access to angiography, revascularisation procedures and effective medicines (aspirin and/or β blockers and/or thrombolysis) had been adjusted for. The hazard ratio was 0.70 (95% CI 0.33 – 1.46). The fully adjusted model indicated that the most important factor affecting survival was access to effective medication. (Table 3). Discussion This study improves our understanding of the care of AMI by cardiologists and general physicians in UK hospitals. Access to a cardiologist was univariately associated with better survival. This effect remained after controlling for the effect of patient characteristics, including the presence of comorbidity, but disappeared when the confounding effect of access to effective medicines was controlled for. As noted in the analysis, patients seen by a cardiologist were more likely to have been prescribed these medicines and to have had exercise testing, angiography and revascularisation. This implies that the survival advantage associated with being seen by a cardiologist is due to the more frequent use of effective medicines and is similar to findings by Chen J et al in the United States [3]. Previous studies have shown a high level of miscoding in routine data for AMI patients (RM Norris cited in [2]). The use of a case note review improved the findings of this study by assisting in ascertaining the diagnosis of myocardial infarction and ensuring the accuracy and completeness of the data used in the analysis. The possibility remains that some cases not seen through the cardiology department and not recorded in the patient administration system were missed. This is likely to be a small number and unlikely to invalidate the findings. Identifying all patients with infarction is a major shortcoming of many studies that estimate mortality among patients seen by cardiologists [2]. This study includes all patients irrespective of where they were managed in the hospital; however patients dying from myocardial infarction before arriving at the hospital were not included. All cases diagnosed by the managing clinician as AMI with ECG changes and found to have enzyme changes indicating myocardial necrosis were included in the analysis after the case notes were reviewed. This may have involved some misclassification due to misdiagnosis, but this is likely to occur at random, affecting only a small number of cases, and can only underestimate the effect of each factor. Limitations of this study include potential for bias in the allocation of patients to treatments and physicians (ie cardiologist/non cardiologist) that were not measured. Another possible limitation is bias arising from medical case notes which were unobtainable. Records from routine data were used to examine whether those with missing records differed in any systematic way from those with available records; it was found that they did not differ in age or sex. The study did not use any criteria for judging clinical appropriateness of the procedures used. Incomplete recording of information in the case notes hampered the determination of appropriateness for effective medication. However there was no difference in the proportion of missing data between the two hospitals. Since doctors may selectively be referring younger patients with lower comorbidity to a cardiologist, we controlled for age and comorbidity in the analysis. However, residual confounding from age and comorbidity could still account for some of the observed difference in survival. All patients admitted to the Coronary Care Unit (CCU) in one of the hospitals are seen by a cardiologist and have a higher chance of accessing thrombolysis, angiography and revascularisation. A cardiologist also sees the majority of patients admitted to the CCU in the second hospital. The protocol for the management of AMI in both hospitals stipulates a cardiology review of all AMI patients. Access to a cardiologist may be a proxy measure of access to effective treatment and it may not be the trigger for effective treatment. These limitations mean that the findings should be interpreted with a degree of caution. In addition our inability to use formal appropriateness criteria limits the interpretation of findings. Despite these limitations the results indicate that, in the short term, acute trusts can improve survival of patients by increasing the use of effective medicines among general physicians. Conclusions We observed better survival among patients seen by a cardiologist compared with patients with no access to a cardiologist, among a cohort of patients already admitted with AMI. This effect was entirely explained by the more frequent use of effective medicines by cardiologists in the multivariate analysis. About eight hospitals in the UK Royal College of Physicians survey have no cardiologist and another 30% have a single cardiologist [2]. It will take time to provide the minimum of two cardiologists per hospital as recommended in the CHD NSF. There are several reasons why a hospital may benefit from appointing a cardiologist, ranging from the treatment of a specific subgroup of patients that will benefit from revascularisation to prompt management of angina patients via rapid access chest clinics. However, in the short term hospitals can improve the survival of patients admitted with AMI by improving access to effective medicines. Coordination of care between cardiologists and general physicians and targeted interventions using feedback from audit, research and peer education are likely to lead to more frequent use of effective cardiovascular medicines by general physicians. Competing interest None declared Authors' contributions All authors contributed in writing the paper, in addition, IA collected the data and conducted the analysis, DK conceived the idea, BA provided advice on statistical analysis, JP provided input in the conduct of the study, PW contributed in refining the research question and discussing the findings of paper. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgement We wish to thank David Lea for providing routine data, Dr Elizabeth Young for comments on early drafts and Cheryl Fortnum for collection of data from case notes ==== Refs Department of Health National service frameworks coronary heart disease London 2000 Royal College of Physicians Chen J Radford MJ Wang Y Krumholz HM Care and outcomes of elderly patients with acute myocardial infarction by physician specialty: the effects of comorbidity and functional limitations Am J Med 2000 108 460 469 10781778 10.1016/S0002-9343(00)00331-4 Jollis JG DeLong ER Peterson ED Muhlbaier LH Fortin DF Califf RM Mark DB Outcome of acute myocardial infarction according to the specialty of the admitting physician N Engl J Med 1996 335 1880 1887 8948564 10.1056/NEJM199612193352505 Casale PN Jones JL Wolf FE Pei Y Eby LM Patients treated by cardiologists have a lower in-hospital mortality for acute myocardial infarction J Am Coll Cardiol 1998 32 885 889 9768707 10.1016/S0735-1097(98)00325-8 Ayanian JZ Guadagnoli E McNeil BJ Cleary PD Treatment and outcomes of acute myocardial infarction among patients of cardiologists and generalist physicians Arch Intern Med 1997 157 2570 2576 9531225 10.1001/archinte.157.22.2570 Nash IS Corrato RR Dlutowski MJ O'Connor JP Nash DB Generalist versus specialist care for acute myocardial infarction Am J Cardiol 1999 83 650 654 10080413 10.1016/S0002-9149(98)00961-8 Ayanian JZ Hauptman PJ Guadagnoli E Antman EM Pashos CL McNeil BJ Knowledge and practices of generalist and specialist physicians regarding drug therapy for acute myocardial infarction N Engl J Med 1994 331 1136 1142 7935639 10.1056/NEJM199410273311707 Melniker LA Leo PJ Comparative knowledge and practice of emergency physicians, cardiologists, and primary care practitioners regarding drug therapy for acute myocardial infarction Chest 1998 113 297 305 9498942 Fehrenbach SN Budnitz DS Gazmararian JA Krumholz HM Physician characteristics and the initiation of beta-adrenergic blocking agent therapy after acute myocardial infarction in a managed care population Am J Manag Care 2001 7 717 723 11464429 Willison DJ Soumerai SB McLaughlin TJ Gurwitz JH Gao X Guadagnoli E Pearson S Hauptman P McLaughlin B Consultation between cardiologists and generalists in the management of acute myocardial infarction implications for quality of care Arch Intern Med 1998 158 1778 1783 9738607 10.1001/archinte.158.16.1778 Peduzzi P Concato J Kemper E Holford TR Feinstein AR A simulation study of the number of events per variable in logistic regression analysis J Clin Epidemiol 1996 49 1373 1379 8970487 10.1016/S0895-4356(96)00236-3 ACC/AHA Guidelines for the Management of Patients With Acute Myocardial Infarction

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BMC Cardiovasc Disord. 2004 Aug 6; 4:14

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==== Front BMC GeriatrBMC Geriatrics1471-2318BioMed Central London 1471-2318-4-61527293410.1186/1471-2318-4-6Research ArticleThe six-minute walk test in community dwelling elderly: influence of health status. Bautmans Ivan [email protected] Margareta [email protected] Tony [email protected] Gerontology, Free University of Brussels (VUB), Belgium2 Geriatrics, Academic Hospital of the Free University of Brussels (VUB), Laarbeeklaan 101, B-1090 Brussels, Belgium2004 23 7 2004 4 6 6 30 3 2004 23 7 2004 Copyright © 2004 Bautmans et al; licensee BioMed Central Ltd.2004Bautmans et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The 6 minutes walk test (6MWT) is a useful assessment instrument for the exercise capacity of elderly persons. The impact of the health status on the 6MWT-distance in elderly, however, remains unclear, reducing its value in clinical settings. The objective of this study was to investigate to what extent the 6MWT-distance in community dwelling elderly is determined by health conditions. Methods One hundred and fifty-six community dwelling elderly people (53 male, 103 female) were assessed for health status and performed the 6MWT. After clinical evaluation, electrocardiography and laboratory examination participants were categorized into a stratified six-level classification system according to their health status, going from A (completely healthy) to D (signs of active disease at the moment of examination). Results The mean 6MWT-distance was 603 m (SD = 178). The 6MWT-distance decreased significantly with increasing age (ANOVA p = 0.0001) and with worsening health status (ANCOVA, corrected for age p < 0.001). A multiple linear regression model with health status, age and gender as independent variables explained 31% of the 6MWT-distance variability. Anthropometrical measures (stature, weight and BMI) did not significantly improve the prediction model. A significant relationship between 6MWT-distance and stature was only present in category A (completely healthy). Conclusions Significant differences in 6MWT-distance are observed according to health status in community-dwelling elderly persons. The proposed health categorizing system for elderly people is able to distinguish persons with lower physical exercise capacity and can be useful when advising physical trainers for seniors. ==== Body Background Aging results in an important decrease of muscle power and exercise capacity[1]. Therefore, elderly often function at the limit of their capacity in order to fulfill the activities of daily living [2]. Determination of the remaining physical capacity can be important in clinical decision-making. From previous studies [3] we observed that one in five elderly patients (70 years and over) is unable to execute the classical treadmill based exercise test, either for fear of falling or because of physical or cognitive limitations. The six-minute walking test (6MWT) is a valid alternative, evaluating the exercise capacity at levels corresponding more to efforts commonly performed by elderly during daily activities. The 6MWT has first been introduced as a functional exercise test by Lipkin in 1986 [4]. Its results are highly correlated with those of the 12 minutes walk test [5] from which it was derived [6] and with those of cycle ergometer or treadmill based exercise tests [3]. The 6MWT is also a valuable instrument to assess progression of functional exercise capacity in different clinical intervention studies [7-11]. The reliability of the test in healthy elderly persons is high (Intra Class Correlation = 0.93) [12] and it is considered as a valid and reliable test to assess the exercise capacity of elderly patients with chronic heart failure (CHF) and chronic obstructive pulmonary disease (COPD) [3,6,13]. Several authors studied the determining factors of the 6MWT-distance in healthy adults and propose either reference equations or normative data for the 6MWT-outcome [14-16]. Troosters et al. [15] found that age, gender, height and weight explained 66% of the 6MWT-distance variability in 51 healthy adults (free from injury and without history of hospitalization or chronic disease influencing exercise capacity) aged 50–85 years. In another study, Enright et al. [14] administered the 6MWT to 290 healthy persons between 40 and 80 years old (health based upon age<80 years; BMI<35 kg/m2; ankle-arm blood pressure index <0.9; 1-second forced expiratory volume and absence of stroke history, use of diuretics and smoking). They report that gender-specific reference equations based upon age, height and weight explained 40% of the variance in 6MWT-distance, which was significantly less for men and women who were older and heavier, and for shorter men. Also, Rikli et al. [16], who measured the 6MWT-distance in respectively 1187 and 2721 community dwelling, functionally independent men and women (who were ambulatory without regular use of assistive device and without medical conditions or physical or cognitive limitations interfering with the test procedures) found a significant 5-year age-group decline in 6MWT-distance (after dividing the study population into gender-specific 5-year age categories) and significantly better scores for men compared to women. However, in a clinical setting one mainly has to deal with elderly having health problems and it can be presumed that their exercise capacity will generally be lower than that of healthy elderly. The available information concerning the impact of health status on the 6MWT-distance in elderly is limited to obesity [17], to the role of muscle strength in persons with mobility limitations [18] and to the influence of reduced aerobic capacity in patients with pulmonary or cardiovascular disease [13,19,20]. Since in current clinical practice geriatricians and physicians are mainly dealing with patients presenting a widespread and heterogeneous variety of co-morbidity, the results of the aforementioned studies offer only limited information concerning the prognostic value and the applicability of the 6MWT in clinical settings different from CHF and COPD. Therefore, we planned this study in order to investigate to what extent the 6MWT-distance in community dwelling elderly is influenced by a broader spectrum of health conditions. Methods Participants All members of a large Belgian Health Insurance Organization (BHIO) who registered for a health-conditioning week for seniors, organized by the BHIO, were invited by advertisement to participate in our study. The program of the health-conditioning week included general instructions for a healthy life-style and physical exercise classes. One hundred and fifty-six subjects (53 male, mean age 64.1 years, SD = 6.9; 103 female, mean age 65.5, SD = 7.7) volunteered to participate in the study and gave their informed consent. All participants were living in the community and belonged to the A-category according to Katz et al. [21] and thus were independent for basic activities of daily living. Health categories All participants underwent extensive health screening by medical doctors. First, blood & urine samples were collected after overnight fasting for determination of erythrocyte sedimentation rate, mean red blood cell corpuscular volume, leukocyte count (with differentiation), and concentration of haemoglobin, urea, alkaline phosphatase, glucose, Aspartate Aminotransferase (ASAT), Alanine Aminotransferase (ALAT), protein and electrophoresis for the blood samples and determination of protein, glucose and sediment for the urine samples (according to the SENIEUR protocol [22]). Second, by means of self-administered standardized questionnaires, which were completed by interview, information was obtained regarding medical history, actual diseases, medication use, tobacco and alcohol consumption. Next, all participants underwent physical examination and standard 12-lead electrocardiography (ECG). Based upon this information, participants were then classified into health categories by one of us (TM), before performing the 6MWT. All evaluations were performed on the same day. The classification system was originally developed in order to grade the participants according to the risk for dangerous complications during physical exercise and to allow physical therapists to adapt the scheduled program of the health-conditioning week (consisting in general instructions for a healthy life-style and physical exercise classes). Therefore, cardiovascular abnormalities were considered to present a higher risk than non-cardiovascular conditions. We distinguished four categories of decreasing health (see table 1). Subjects categorized as A were completely healthy and were considered as presenting no particular risk for any kind of physical exercise. An additional distinction can be made between those using no medication (A1) and those using preventive medication only (A2). This subdivision might be important in specific clinical contexts (e.g. assessment of Vitamin D levels in elderly); in the context of our study the distinction between health categories A1 and A2 is less relevant and therefore these participants will be considered together in all statistical analysis. Category B consisted of participants who were functioning normally, presented no major medical restrictions, but could be in need of special instructions for exercising due to their health status. Category B1 was accorded to participants having a disease that was non-cardiovascular and stable. Category B2 was given to participants using medication having cardiovascular effects. Subjects in category C had cardiovascular pathology or a history thereof; they were considered as having an increased risk of cardiovascular complications during exercise. Those belonging to category D were found to present signs of acute disease or exacerbation of chronic disease. If combinations of health conditions existed, subjects were classified in the worst health category. Table 1 Health categories Health Category Description * Clinical examples A A1 Completely healthy; no medication    A2 Completely healthy; using only preventive medication Hormonal replacement therapy, aspirin, ... B B1 Functioning normally; presence of stabilised, non cardiovascular disease; absence of cardiovascular abnormalities treated hypothyroidism, stable diabetes, ...    B2 Functioning normally; using medication with cardiovascular effect, no overt cardiovascular disease other than normalized arterial hypertension Arterial hypertension; β blocking agent, ... C (history of) cardio-vascular pathology or abnormal ECG. Bundle branch block; angina, CABG; ... D Presenting signs of acute or active disease at the moment of examination. bronchospasm, swollen joints, influenza, ... * Status after questioning, physical examination, ECG, and laboratory examination of blood, serum & urine according to the SENIEUR protocol [22]. CABG: coronary artery bypass graft Measurements Subjects were assessed for weight, height and body mass index [BMI = weight / (height)2]. Before starting the health conditioning week, all participants performed the 6MWT following a protocol as previously described [3]. All participants were naive to the 6MWT. Each participant was tested individually and was constantly observed by a physical therapist, who was unaware of the health category attribution. The 6MWT was performed outdoors upon a hardened and flat surface following a circular circuit of 121 m. Participants were instructed to try to cover as much distance as possible within six minutes without running. They wore comfortable shoes and clothing and were allowed to rest or stop when necessary. Statistical analysis Statistical analysis was performed using the SPSS (for Windows, release 11.5.1) software package. All data subsets were assessed for the presence of a normal distribution (Kolmogorov-Smirnov Goodness of Fit Test p > 0.05) before using parametric analysis. Correlations between data subsets with a normal distribution were performed using Pearson's Correlation Coefficient; non-normally distributed datasets were analyzed for correlation using Kendall's Correlation Coefficient. Differences between data subsets were analyzed using Analysis of Variance (ANOVA), Analysis of Co-Variance (ANCOVA) and Students t-test. Bonferroni post-hoc test was performed to detect significant differences between subgroups. A multiple linear regression model was designed in order to explain the variability of the 6MWT-distance. Significance level was set at p < 0.05 [23]. Results The mean 6MWT-distance was 603 m (SD = 178) for the whole group (N = 156). Overall, male participants were significantly heavier (p < 0.001), taller (p < 0.001) and covered a significantly longer distance in six minutes (p = 0.022) than female participants. There was no significant difference in age or BMI between male and female participants (table 2). Table 2 Participants' characteristics. Male N = 53 mean (SD) Female N = 103 mean (SD) 6MWT (m) 647.9 (201.8) 579.3 * (159.8) Age (years) 64.1 (6.9) 65.5 (7.7) Weight (kg) 80.5 (11.0) 69.4† (11.4) Height (cm) 171.3 (6.6) 158.2† (6.3) BMI (kg/m2) 27.4 (3.1) 27.8 (4.2) *p < 0.05, †p < 0.001 Since there was only one participant in health-category D, this subgroup was excluded from all statistic tests. Fifty-eight (37%) participants (44 in group A1 and 14 in group A2) could be considered as completely healthy. No significant difference was found regarding age or 6MWT-distance between participants of group A1 and A2 (mean age respectively 62.0 years SD = 6.6 and 62.7 years SD = 10.0, mean 6MWT-distance respectively 696.4 m SD = 150.7 and 686.9 m SD = 118.4 for A1 and A2). ANOVA testing (male and female considered together) revealed a significantly lower 6MWT-distance (p < 0.001) with worsening health category (A → C), even after correction for age (ANCOVA p < 0.001). Participants with poorer health status were significantly older (p = 0.0001) than those in better health (Figure 1 and Table 3). Figure 1 Impact of Health Status on 6-Minute Walk Distance in Community Dwelling Elderly. ■ Male Participants N = 53. Female Participants N = 102. Bars represent mean values ± SE. Significant decrease of 6 Minutes Walk Distance with worsening health category (ANCOVA corrected for age p < 0.01). * Significantly higher 6 MWT-distance than categories B2and C (Bonferroni post-hoc test p < 0.01) †Significantly higher 6 MWT-distance than category C (Bonferroni post-hoc test p < 0.05). Table 3 Characteristics of health groups. Category N Male/Female Age [years] mean (SD) A 58 20/38 62.2 *, †(6.7) B1 30 7/23 63.5 * (6.6) B2 35 11/24 69.0 (6.6) C 32 15/17 67.2 (6.6) D 1 0/1 73 All 156 53/103 65.1 (7.4) * Significantly younger than category. B2 (Bonferroni post-hoc test p < 0.01) †Significantly younger than category. C (Bonferroni post-hoc test p < 0.01) Post-hoc tests allowed detecting significant differences in the 6MWT-distance between health-categories A &B2(p < 0.01), A &C (p < 0.01), and B1 &C (p < 0.05). Subjects in health-category A were significantly (p < 0.01) younger than those in category B2 and C; those in health-category B1 were significantly (p < 0.01) younger than category B2. There was a negative correlation between the 6MWT-distance and the participant's age for the whole group (r = -0.42, p < 0.001), and for both males (r = -0.32, p = 0.019) and females (r = -0.47, p < 0.001). A correlation between 6MWT-distance and age was present within health-categories A (r = -0.38; p = 0.004), and C (r = -0.40; p = 0.023), but not within category B1 (r = -0.21; p = 0.269) or B2 (r = -0.27; p = 0.121) (table 4). No statistically significant correlations were found between 6MWT-distance and weight or BMI for the whole group or within the different health status categories. Height was significantly correlated with 6MWT-distance (r = 0.33, p = 0.012, Table 4) only in the group of completely healthy participants (category A). Table 4 Relationships between 6MWT-distance and participants' characteristics. Category Age Weight Height BMI All (N = 156) -0.42* -0.03 0.13 -0.14 A (N = 58) -0.38† 0.10 0.33* -0.20 B1 (N = 30) -0.21 0.07 0.02 0.05 B2 (N = 35) -0.27 -0.10 -0.01 -0.10 C (N = 32) -0.40* -0.14 0.08 -0.29 Male -0.32* -0.19 -0.24 -0.07 Female -0.47* -0.09 0.15 -0.18 Values represent correlation coefficients. *p 0.05, †p < 0.01 When the 156 participants were divided in age categories, again a significant (p < 0.001) decrease in 6MWT-distance was observed with increasing age (figure 2). Subjects aged 75 years and more covered a significantly (p < 0.05) shorter distance in the 6MWT than those less than 65 years of age, regardless of their health status. Two-way ANOVA revealed no significant interaction between gender, health status and age concerning the 6MWT-distance (table 5). Figure 2 Impact of Age on 6-Minute Walk Distance in Community Dwelling Elderly. ■ Male Participants N = 53. Female Participants N = 102. Bars represent mean values ± SE. Significant decrease of 6 Minutes Walk (6 MWT) Distance with increasing age category (ANOVA p < 0.01). * Significantly higher 6 MWT-distance than categories 65–69 and 75+ (Bonferroni post-hoc test p < 0.05 and p < 0.01 respectively). Significantly higher 6 MWT-distance than category 75+ (Bonferroni post-hoc test p < 0.01). ‡Significantly higher 6 MWT-distance than category 75+ (Bonferroni post-hoc test p < 0.05). Table 5 Two-way ANOVA for 6MWT-distance Source of Variation F-value P-value Health-category by Age-category 0.65 0.833 Health-category by Gender 0.20 0.937 Age-category by gender 0.40 0.807 In a first step a multiple linear regression model was computed with 6MWT-distance as dependent variable and with age, gender, weight, height and health status as independent variables. Analysis of this regression model (R2 = 0.33) revealed that neither weight nor height was a significant coefficient, contributing to the model (t = -1.4, p = 0.16 and t = -0.98, p = 0.33 for weight and height respectively). Therefore, a new multiple linear regression model was computed without weight and height as independent variables explaining 31% of the variability of the 6MWT-distance (R2 = 0.31). In figure 3 the measured individual 6MWT-distances are plotted against the predicted distances based upon the following regression equation: Figure 3 Predicted and Actual 6-Minute Walk Distance in Community Dwelling Elderly. The bullets represent individual values according to the attributed health category: Green bullets Health Category A, Yellow bullets Health Category B1, Red bullets Health Category B2 and Black bullets Health Category C. The lines represent the fit line and the 95% confidence interval. The predicted values are based upon the proposed multiple linear regression model 6MWT-distance predicted (m) = 1192 - (6 × age) - (57 × health category) -(69 × gender) with age expressed in years; 0 for male and 1 for female; 1 for health-category A, 2 for health-category B1, 3 for health-category B2, 4 for health-category C. The standard error of the estimate is 147 m. 6MWT-distance predicted (m) = 1192 - (6 × age) - (57 × health category) - (69 × gender) with age expressed in years; 0 for male and 1 for female; 1 for health-category A, 2 for health-category B1, 3 for health-category B2, 4 for health-category C. The standard error of the estimate was 147 m. In order to determine the impact of health status in the regression model, we have computed a model with 6MWT-distance as dependent variable and with age and gender as independent variables (R2 = 0.18) in a first step, in which we introduced in a second step the entry of health status as supplementary independent variable. Using this method we obtained the partial correlation coefficient (partial r = -0.40, p < 0.001) between health status and 6MWT-distance. Discussion The 6MWT presents several interesting advantages for the evaluation of the exercise capacity in elderly people. Different authors have described reference equations and tables to predict the 6MWT-distance in healthy elderly subjects. Gender, age, weight and height of these subjects appear to explain a large proportion of the variability in the 6MWT-distance [14-16]. Advancing age, however, is predominantly accompanied by an increasing burden of pathology [24] and "apparently healthy" elderly persons, willing to participate in physical training sessions, actually present a large diversity in health status. This means that the exercise capacity and the risk for complications during exercise are not the same for each person who consider themselves able to perform physical exercise. Ideally, an exercise program must be established for each person individually considering all facets of his/her health condition. It is conceivable that healthier elderly will present a better physical exercise capacity than those with a worse health condition. Therefore it is to be expected that the performance on exercise tests will reflect the aforementioned difference in health status. However, the contribution of the health status of elderly participants to the variability of the distance walked in the 6MWT has not been extensively described. In order to distinguish completely healthy participants from those with a worse health status we used criteria derived from the SENIEUR protocol [22]. This protocol was originally established to select participants for studies concerning the age-related changes of the immune system and allows the distinction between age-related and disease-related changes. The literature provides no clear guidelines to categorize elderly persons attending exercise programs according to their health status. Therefore, we developed a classification system, which stratifies health categories corresponding to an increasing risk for complications during physical exercise. Since the SENIEUR protocol allows for the presence of certain diseases and the use of medication that has no influence on the immune system, in our classification these criteria were further adapted in order to identify the "completely healthy" participants (group A) and to differentiate them from the "apparently healthy". The latter group was functioning normally, but could not be considered as completely healthy since participants either presented stabilized non-cardiovascular conditions (group B1) or were using cardiovascular medication, however, without any sign of active cardiovascular disease other than normalized hypertension and without significant abnormalities on ECG (group B2). When a history or signs of cardiovascular disease, other than hypertension, were noticed, participants were considered as belonging to group C. When evaluating large numbers of participants there is always the possibility of encountering individuals who are acutely ill; since they will not be able to fully participate in an exercise program, we choose to classify them separately (group D). The classification system was primarily designed to allow the establishment of recommendations concerning the exercise schedule (type, duration and intensity) of elderly persons in the absence of direct medical supervision. Therefore, the classification system is rather conservative and it is easy for an individual to be considered at risk for complications. Roughly, participants in category A will have no particular limitations for exercising; for those in category B1 the instructions will vary with the nature of the health problem; those in category B2 will only exercise at higher intensity (e.g. up to 80% of maximal heart rate or higher) when guided by an instructor qualified for training elderly persons; those in category C will only be allowed to exercise under supervision of an instructor and with medical guidance of the training program; those in category D will not exercise unless cleared by a physician. Since any exercise schedule will depend upon its objective, we do not go into further details here for these recommendations. It is clear that elderly persons often termed as 'apparently healthy' do not correspond to criteria for being completely healthy. In our study, only 28% (group A1 = 44 of 156) or 37% (Groups A1 and A2 = 58 of 156) of the participants were completely healthy, depending on whether the use of preventive medication was taken into account. However, 79% (groups A, B1 and B2 = 123 of 156) were functioning normally in the community and had no overt history of cardiovascular disease. Rikli et al. (1999) [16] studied the 6MWT-distance in 7183 community-dwelling older adults (5048 male, 2135 female) aged 60–94 years. According to the inclusion criteria they describe, we can assume that the participants of that study meet our classification criteria of categories A, B1 and B2. Our study, based upon a much smaller population sample, demonstrates that normative data based upon the population of the aforementioned authors is not representative for completely healthy elderly. Indeed, the mean 6MWT-distance of completely healthy participants (category A, mean age 62 years (SD = 7); mean 6MWT-distance 764 m (SD = 162) and 657 m (SD = 118) for male and female respectively) is much higher than the norm reported by Rikli et al. [16] for subjects in the same age category (60–64 years; mean 6MWT-distance 616 m (SD = 84) and 551 m (SD = 77) for male and female respectively). When age-matched categories with mixed health status are compared, our results (see figure 2) accord well with those of Rikli et al. [16]. Neither the studies of Enright et al. [14] or Troosters et al. [15] used the criteria of the SENIEUR protocol to select healthy individuals. This probably means that the health status of the populations they describe is heterogeneous and might also explain the considerable range in 6MWT-distance (383–820 m) encountered in the 'healthy' elderly (50–85 years old) participants of Troosters et al. [15] as well as the relatively low median 6MWT-distances (576 m and 494 m for male and female respectively) for 'healthy' adults (40–80 years old) in the study of Enright et al. [14]. Our study demonstrates that the proposed health-categorization is able to detect a significant reduction in physical capacity due to individual parameters such as medical history, medication use and current health status, other than age, gender (explaining only 18% of the variability) and anthropometrical parameters (no significant independent predictors when health status is included in the regression model). It is our opinion that the results of our study argue in favor of the validity of the proposed health categorization. At this moment, however, other data concerning the reliability of the classification system are not yet available. The results of the multiple linear regression analysis and the absence of a significant interaction (two-way ANOVA testing) between health status, age and gender confirm that each of these factors is an independent determinant of 6MWT-distance in the elderly. The proposed health categorization is able to discriminate the completely healthy elderly from the apparently healthy and to distinguish among the apparently healthy several categories. This results in a more diversified spectrum of the community dwelling elderly population than obtained with other categorizations like the New York Heart Association (NYHA)-classification for cardiovascular disease [25] and the American Heart Association (AHA) risk stratification criteria [26]. Currently, these systems are widely used for stratification purposes in physical training schedules. However, they were developed for the description of cardiac patients and do not consider other pathologies nor co-morbidities. A minor familiarization effect has been described for the 6MWT with better values obtained at a second testing [27]. Since repeated testing is less applicable in clinical settings, we chose to use the results from a single test administration when the participants were still naïve for the 6MWT. In our study the correlation of the 6MWT-distance with age, gender and stature as described by others [14-16] is confirmed in the group of the completely healthy participants (category A). This relationship is attenuated or disappears in subjects presenting chronic pathology even without apparent functional limitations. Our study demonstrates that there exists a significant decrease in 6MWT-distance with increasing health problems. Since the main objective of our classification system was to stratify for the risk for cardiovascular or metabolic (e.g. hypoglycemia) complications during exercise, we did not expect that the performance on the 6MWT would vary with the health categories. It is probable that the relationship between health status and 6MWT-distance reflects the influence of the status of the cardiovascular system on the general fitness. Inversely, however, it can also be explained by the negative prognostic value of lack of fitness for the ulterior development of cardiovascular disease. It has, indeed, repeatedly been reported for healthy middle-aged adults that this risk is greater for the least fit individuals [28,29]. Since most of our participants were older than those in the risk studies, a certain amount of cardiovascular pathology did occur. In a multiple linear regression model age, gender and health status explained 31% of the variability of the 6MWT-distance in the population of our study. All three independent variables had a highly significant contribution to the model (age and gender together accounted for 18% of the variability, partial correlation between health status and 6MWT-distance = -0.40, p < 0.001). The addition of weight, height and BMI to the prediction model did not significantly improve the R2-value. This is different from other studies that describe regression equations for the 6MWT-distance in healthy elderly [14,15], and indicates that the health condition of community dwelling elderly is more influencing the 6MWT-distance than their anthropometrical predisposition (weight and height are not significant independent predictors for 6MWT-distance after considering health status, age and gender). The fact that these authors obtained a much higher R2-value (R2 = 0.66 [15], R2 = 0.42 for male and R2 = 0.38 for female [14]) than ours (R2 = 0.31) can be due to the more heterogeneous composition in health status and the repartition of the population in several health categories in our study. The remaining unexplained variability in 6MWT-distance might be found in the differences in skeletal muscle strength, the training levels and the physical activity levels of the participants. However, these parameters were not measured in our study. We propose to incorporate the present health classification for elderly people as outcome of the medical screenings preceding the start of a physical exercise program. As shown in our study, significant differences can be found in physical exercise capacity according to the health status. We used the 6MWT-distance, which is an established exercise test for elderly persons, to document the physical exercise capacity of our participants. Especially in the elderly, which show increased prevalence of co-morbidities (most frequently (cardio)vascular, metabolic or rheumatological), physical exercise programs should be established according to the individual exercise capacity. Following the health classification of the elderly participant, the training instructor can adapt the modalities of the training program in order to obtain the best results at the most appropriate training intensity. This should finally lead to concrete training guidelines for elderly people in relation to their health status, diminishing the risk for injuries or complications during physical exercise. Competing interests None declared. Authors' contributions TM conceived and coordinated the study, participated in the evaluation of the health condition of the participants, the analysis and the redaction. IB performed the statistical analysis and participated in the design and the redaction. ML participated in the evaluation of the health condition of the participants, the analysis and the redaction. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements A preliminary report of this study was presented at the 24th Winter Meeting of the Belgian Society for Gerontology and Geriatrics (BVGG) in Ostend, Belgium (February 16–17 2001). ==== Refs Marcell Taylor J. 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Eur Heart J 2001 22 488 496 11237544 10.1053/euhj.2000.2310 Katz S Ford AB Moskowitz RW Jackson BA Jaffe MW Studies of Illness in the Aged. The Index of Adl: A Standardized Measure of Biological and Psychosocial Function Jama 1963 185 914 919 14044222 Ligthart GJ Corberand JX Fournier C Galanaud P Hijmans W Kennes B Muller-Hermelink HK Steinmann GG Admission criteria for immunogerontological studies in man: the SENIEUR protocol Mech Ageing Dev 1984 28 47 55 6513613 10.1016/0047-6374(84)90152-0 Kirkwood B Essentials of Medical Statistics 1988 1st ed Oxford, Blackwell Sciences Ltd 234 Izaks GJ Westendorp RG Ill or just old? Towards a conceptual framework of the relation between ageing and disease BMC Geriatr 2003 3 7 14683526 10.1186/1471-2318-3-7 Kossman CE Nomenclature and Criteria for the Diagnosis of Cardiovascular Diseases Circulation 1964 30 321 325 14210612 Fletcher GF Balady G Froelicher VF Hartley LH Haskell WL Pollock ML Exercise standards. A statement for healthcare professionals from the American Heart Association. Writing Group Circulation 1995 91 580 615 7805272 Stevens D Elpern E Sharma K Szidon P Ankin M Kesten S Comparison of hallway and treadmill six-minute walk tests Am J Respir Crit Care Med 1999 160 1540 1543 10556117 Ekelund LG Haskell WL Johnson JL Whaley FS Criqui MH Sheps DS Physical fitness as a predictor of cardiovascular mortality in asymptomatic North American men. The Lipid Research Clinics Mortality Follow-up Study N Engl J Med 1988 319 1379 1384 3185648 Sandvik L Erikssen J Thaulow E Erikssen G Mundal R Rodahl K Physical fitness as a predictor of mortality among healthy, middle-aged Norwegian men N Engl J Med 1993 328 533 537 8426620 10.1056/NEJM199302253280803

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==== Front BMC GeriatrBMC Geriatrics1471-2318BioMed Central London 1471-2318-4-61527293410.1186/1471-2318-4-6Research ArticleThe six-minute walk test in community dwelling elderly: influence of health status. Bautmans Ivan [email protected] Margareta [email protected] Tony [email protected] Gerontology, Free University of Brussels (VUB), Belgium2 Geriatrics, Academic Hospital of the Free University of Brussels (VUB), Laarbeeklaan 101, B-1090 Brussels, Belgium2004 23 7 2004 4 6 6 30 3 2004 23 7 2004 Copyright © 2004 Bautmans et al; licensee BioMed Central Ltd.2004Bautmans et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The 6 minutes walk test (6MWT) is a useful assessment instrument for the exercise capacity of elderly persons. The impact of the health status on the 6MWT-distance in elderly, however, remains unclear, reducing its value in clinical settings. The objective of this study was to investigate to what extent the 6MWT-distance in community dwelling elderly is determined by health conditions. Methods One hundred and fifty-six community dwelling elderly people (53 male, 103 female) were assessed for health status and performed the 6MWT. After clinical evaluation, electrocardiography and laboratory examination participants were categorized into a stratified six-level classification system according to their health status, going from A (completely healthy) to D (signs of active disease at the moment of examination). Results The mean 6MWT-distance was 603 m (SD = 178). The 6MWT-distance decreased significantly with increasing age (ANOVA p = 0.0001) and with worsening health status (ANCOVA, corrected for age p < 0.001). A multiple linear regression model with health status, age and gender as independent variables explained 31% of the 6MWT-distance variability. Anthropometrical measures (stature, weight and BMI) did not significantly improve the prediction model. A significant relationship between 6MWT-distance and stature was only present in category A (completely healthy). Conclusions Significant differences in 6MWT-distance are observed according to health status in community-dwelling elderly persons. The proposed health categorizing system for elderly people is able to distinguish persons with lower physical exercise capacity and can be useful when advising physical trainers for seniors. ==== Body Background Aging results in an important decrease of muscle power and exercise capacity[1]. Therefore, elderly often function at the limit of their capacity in order to fulfill the activities of daily living [2]. Determination of the remaining physical capacity can be important in clinical decision-making. From previous studies [3] we observed that one in five elderly patients (70 years and over) is unable to execute the classical treadmill based exercise test, either for fear of falling or because of physical or cognitive limitations. The six-minute walking test (6MWT) is a valid alternative, evaluating the exercise capacity at levels corresponding more to efforts commonly performed by elderly during daily activities. The 6MWT has first been introduced as a functional exercise test by Lipkin in 1986 [4]. Its results are highly correlated with those of the 12 minutes walk test [5] from which it was derived [6] and with those of cycle ergometer or treadmill based exercise tests [3]. The 6MWT is also a valuable instrument to assess progression of functional exercise capacity in different clinical intervention studies [7-11]. The reliability of the test in healthy elderly persons is high (Intra Class Correlation = 0.93) [12] and it is considered as a valid and reliable test to assess the exercise capacity of elderly patients with chronic heart failure (CHF) and chronic obstructive pulmonary disease (COPD) [3,6,13]. Several authors studied the determining factors of the 6MWT-distance in healthy adults and propose either reference equations or normative data for the 6MWT-outcome [14-16]. Troosters et al. [15] found that age, gender, height and weight explained 66% of the 6MWT-distance variability in 51 healthy adults (free from injury and without history of hospitalization or chronic disease influencing exercise capacity) aged 50–85 years. In another study, Enright et al. [14] administered the 6MWT to 290 healthy persons between 40 and 80 years old (health based upon age<80 years; BMI<35 kg/m2; ankle-arm blood pressure index <0.9; 1-second forced expiratory volume and absence of stroke history, use of diuretics and smoking). They report that gender-specific reference equations based upon age, height and weight explained 40% of the variance in 6MWT-distance, which was significantly less for men and women who were older and heavier, and for shorter men. Also, Rikli et al. [16], who measured the 6MWT-distance in respectively 1187 and 2721 community dwelling, functionally independent men and women (who were ambulatory without regular use of assistive device and without medical conditions or physical or cognitive limitations interfering with the test procedures) found a significant 5-year age-group decline in 6MWT-distance (after dividing the study population into gender-specific 5-year age categories) and significantly better scores for men compared to women. However, in a clinical setting one mainly has to deal with elderly having health problems and it can be presumed that their exercise capacity will generally be lower than that of healthy elderly. The available information concerning the impact of health status on the 6MWT-distance in elderly is limited to obesity [17], to the role of muscle strength in persons with mobility limitations [18] and to the influence of reduced aerobic capacity in patients with pulmonary or cardiovascular disease [13,19,20]. Since in current clinical practice geriatricians and physicians are mainly dealing with patients presenting a widespread and heterogeneous variety of co-morbidity, the results of the aforementioned studies offer only limited information concerning the prognostic value and the applicability of the 6MWT in clinical settings different from CHF and COPD. Therefore, we planned this study in order to investigate to what extent the 6MWT-distance in community dwelling elderly is influenced by a broader spectrum of health conditions. Methods Participants All members of a large Belgian Health Insurance Organization (BHIO) who registered for a health-conditioning week for seniors, organized by the BHIO, were invited by advertisement to participate in our study. The program of the health-conditioning week included general instructions for a healthy life-style and physical exercise classes. One hundred and fifty-six subjects (53 male, mean age 64.1 years, SD = 6.9; 103 female, mean age 65.5, SD = 7.7) volunteered to participate in the study and gave their informed consent. All participants were living in the community and belonged to the A-category according to Katz et al. [21] and thus were independent for basic activities of daily living. Health categories All participants underwent extensive health screening by medical doctors. First, blood & urine samples were collected after overnight fasting for determination of erythrocyte sedimentation rate, mean red blood cell corpuscular volume, leukocyte count (with differentiation), and concentration of haemoglobin, urea, alkaline phosphatase, glucose, Aspartate Aminotransferase (ASAT), Alanine Aminotransferase (ALAT), protein and electrophoresis for the blood samples and determination of protein, glucose and sediment for the urine samples (according to the SENIEUR protocol [22]). Second, by means of self-administered standardized questionnaires, which were completed by interview, information was obtained regarding medical history, actual diseases, medication use, tobacco and alcohol consumption. Next, all participants underwent physical examination and standard 12-lead electrocardiography (ECG). Based upon this information, participants were then classified into health categories by one of us (TM), before performing the 6MWT. All evaluations were performed on the same day. The classification system was originally developed in order to grade the participants according to the risk for dangerous complications during physical exercise and to allow physical therapists to adapt the scheduled program of the health-conditioning week (consisting in general instructions for a healthy life-style and physical exercise classes). Therefore, cardiovascular abnormalities were considered to present a higher risk than non-cardiovascular conditions. We distinguished four categories of decreasing health (see table 1). Subjects categorized as A were completely healthy and were considered as presenting no particular risk for any kind of physical exercise. An additional distinction can be made between those using no medication (A1) and those using preventive medication only (A2). This subdivision might be important in specific clinical contexts (e.g. assessment of Vitamin D levels in elderly); in the context of our study the distinction between health categories A1 and A2 is less relevant and therefore these participants will be considered together in all statistical analysis. Category B consisted of participants who were functioning normally, presented no major medical restrictions, but could be in need of special instructions for exercising due to their health status. Category B1 was accorded to participants having a disease that was non-cardiovascular and stable. Category B2 was given to participants using medication having cardiovascular effects. Subjects in category C had cardiovascular pathology or a history thereof; they were considered as having an increased risk of cardiovascular complications during exercise. Those belonging to category D were found to present signs of acute disease or exacerbation of chronic disease. If combinations of health conditions existed, subjects were classified in the worst health category. Table 1 Health categories Health Category Description * Clinical examples A A1 Completely healthy; no medication    A2 Completely healthy; using only preventive medication Hormonal replacement therapy, aspirin, ... B B1 Functioning normally; presence of stabilised, non cardiovascular disease; absence of cardiovascular abnormalities treated hypothyroidism, stable diabetes, ...    B2 Functioning normally; using medication with cardiovascular effect, no overt cardiovascular disease other than normalized arterial hypertension Arterial hypertension; β blocking agent, ... C (history of) cardio-vascular pathology or abnormal ECG. Bundle branch block; angina, CABG; ... D Presenting signs of acute or active disease at the moment of examination. bronchospasm, swollen joints, influenza, ... * Status after questioning, physical examination, ECG, and laboratory examination of blood, serum & urine according to the SENIEUR protocol [22]. CABG: coronary artery bypass graft Measurements Subjects were assessed for weight, height and body mass index [BMI = weight / (height)2]. Before starting the health conditioning week, all participants performed the 6MWT following a protocol as previously described [3]. All participants were naive to the 6MWT. Each participant was tested individually and was constantly observed by a physical therapist, who was unaware of the health category attribution. The 6MWT was performed outdoors upon a hardened and flat surface following a circular circuit of 121 m. Participants were instructed to try to cover as much distance as possible within six minutes without running. They wore comfortable shoes and clothing and were allowed to rest or stop when necessary. Statistical analysis Statistical analysis was performed using the SPSS (for Windows, release 11.5.1) software package. All data subsets were assessed for the presence of a normal distribution (Kolmogorov-Smirnov Goodness of Fit Test p > 0.05) before using parametric analysis. Correlations between data subsets with a normal distribution were performed using Pearson's Correlation Coefficient; non-normally distributed datasets were analyzed for correlation using Kendall's Correlation Coefficient. Differences between data subsets were analyzed using Analysis of Variance (ANOVA), Analysis of Co-Variance (ANCOVA) and Students t-test. Bonferroni post-hoc test was performed to detect significant differences between subgroups. A multiple linear regression model was designed in order to explain the variability of the 6MWT-distance. Significance level was set at p < 0.05 [23]. Results The mean 6MWT-distance was 603 m (SD = 178) for the whole group (N = 156). Overall, male participants were significantly heavier (p < 0.001), taller (p < 0.001) and covered a significantly longer distance in six minutes (p = 0.022) than female participants. There was no significant difference in age or BMI between male and female participants (table 2). Table 2 Participants' characteristics. Male N = 53 mean (SD) Female N = 103 mean (SD) 6MWT (m) 647.9 (201.8) 579.3 * (159.8) Age (years) 64.1 (6.9) 65.5 (7.7) Weight (kg) 80.5 (11.0) 69.4† (11.4) Height (cm) 171.3 (6.6) 158.2† (6.3) BMI (kg/m2) 27.4 (3.1) 27.8 (4.2) *p < 0.05, †p < 0.001 Since there was only one participant in health-category D, this subgroup was excluded from all statistic tests. Fifty-eight (37%) participants (44 in group A1 and 14 in group A2) could be considered as completely healthy. No significant difference was found regarding age or 6MWT-distance between participants of group A1 and A2 (mean age respectively 62.0 years SD = 6.6 and 62.7 years SD = 10.0, mean 6MWT-distance respectively 696.4 m SD = 150.7 and 686.9 m SD = 118.4 for A1 and A2). ANOVA testing (male and female considered together) revealed a significantly lower 6MWT-distance (p < 0.001) with worsening health category (A → C), even after correction for age (ANCOVA p < 0.001). Participants with poorer health status were significantly older (p = 0.0001) than those in better health (Figure 1 and Table 3). Figure 1 Impact of Health Status on 6-Minute Walk Distance in Community Dwelling Elderly. ■ Male Participants N = 53. Female Participants N = 102. Bars represent mean values ± SE. Significant decrease of 6 Minutes Walk Distance with worsening health category (ANCOVA corrected for age p < 0.01). * Significantly higher 6 MWT-distance than categories B2and C (Bonferroni post-hoc test p < 0.01) †Significantly higher 6 MWT-distance than category C (Bonferroni post-hoc test p < 0.05). Table 3 Characteristics of health groups. Category N Male/Female Age [years] mean (SD) A 58 20/38 62.2 *, †(6.7) B1 30 7/23 63.5 * (6.6) B2 35 11/24 69.0 (6.6) C 32 15/17 67.2 (6.6) D 1 0/1 73 All 156 53/103 65.1 (7.4) * Significantly younger than category. B2 (Bonferroni post-hoc test p < 0.01) †Significantly younger than category. C (Bonferroni post-hoc test p < 0.01) Post-hoc tests allowed detecting significant differences in the 6MWT-distance between health-categories A &B2(p < 0.01), A &C (p < 0.01), and B1 &C (p < 0.05). Subjects in health-category A were significantly (p < 0.01) younger than those in category B2 and C; those in health-category B1 were significantly (p < 0.01) younger than category B2. There was a negative correlation between the 6MWT-distance and the participant's age for the whole group (r = -0.42, p < 0.001), and for both males (r = -0.32, p = 0.019) and females (r = -0.47, p < 0.001). A correlation between 6MWT-distance and age was present within health-categories A (r = -0.38; p = 0.004), and C (r = -0.40; p = 0.023), but not within category B1 (r = -0.21; p = 0.269) or B2 (r = -0.27; p = 0.121) (table 4). No statistically significant correlations were found between 6MWT-distance and weight or BMI for the whole group or within the different health status categories. Height was significantly correlated with 6MWT-distance (r = 0.33, p = 0.012, Table 4) only in the group of completely healthy participants (category A). Table 4 Relationships between 6MWT-distance and participants' characteristics. Category Age Weight Height BMI All (N = 156) -0.42* -0.03 0.13 -0.14 A (N = 58) -0.38† 0.10 0.33* -0.20 B1 (N = 30) -0.21 0.07 0.02 0.05 B2 (N = 35) -0.27 -0.10 -0.01 -0.10 C (N = 32) -0.40* -0.14 0.08 -0.29 Male -0.32* -0.19 -0.24 -0.07 Female -0.47* -0.09 0.15 -0.18 Values represent correlation coefficients. *p 0.05, †p < 0.01 When the 156 participants were divided in age categories, again a significant (p < 0.001) decrease in 6MWT-distance was observed with increasing age (figure 2). Subjects aged 75 years and more covered a significantly (p < 0.05) shorter distance in the 6MWT than those less than 65 years of age, regardless of their health status. Two-way ANOVA revealed no significant interaction between gender, health status and age concerning the 6MWT-distance (table 5). Figure 2 Impact of Age on 6-Minute Walk Distance in Community Dwelling Elderly. ■ Male Participants N = 53. Female Participants N = 102. Bars represent mean values ± SE. Significant decrease of 6 Minutes Walk (6 MWT) Distance with increasing age category (ANOVA p < 0.01). * Significantly higher 6 MWT-distance than categories 65–69 and 75+ (Bonferroni post-hoc test p < 0.05 and p < 0.01 respectively). Significantly higher 6 MWT-distance than category 75+ (Bonferroni post-hoc test p < 0.01). ‡Significantly higher 6 MWT-distance than category 75+ (Bonferroni post-hoc test p < 0.05). Table 5 Two-way ANOVA for 6MWT-distance Source of Variation F-value P-value Health-category by Age-category 0.65 0.833 Health-category by Gender 0.20 0.937 Age-category by gender 0.40 0.807 In a first step a multiple linear regression model was computed with 6MWT-distance as dependent variable and with age, gender, weight, height and health status as independent variables. Analysis of this regression model (R2 = 0.33) revealed that neither weight nor height was a significant coefficient, contributing to the model (t = -1.4, p = 0.16 and t = -0.98, p = 0.33 for weight and height respectively). Therefore, a new multiple linear regression model was computed without weight and height as independent variables explaining 31% of the variability of the 6MWT-distance (R2 = 0.31). In figure 3 the measured individual 6MWT-distances are plotted against the predicted distances based upon the following regression equation: Figure 3 Predicted and Actual 6-Minute Walk Distance in Community Dwelling Elderly. The bullets represent individual values according to the attributed health category: Green bullets Health Category A, Yellow bullets Health Category B1, Red bullets Health Category B2 and Black bullets Health Category C. The lines represent the fit line and the 95% confidence interval. The predicted values are based upon the proposed multiple linear regression model 6MWT-distance predicted (m) = 1192 - (6 × age) - (57 × health category) -(69 × gender) with age expressed in years; 0 for male and 1 for female; 1 for health-category A, 2 for health-category B1, 3 for health-category B2, 4 for health-category C. The standard error of the estimate is 147 m. 6MWT-distance predicted (m) = 1192 - (6 × age) - (57 × health category) - (69 × gender) with age expressed in years; 0 for male and 1 for female; 1 for health-category A, 2 for health-category B1, 3 for health-category B2, 4 for health-category C. The standard error of the estimate was 147 m. In order to determine the impact of health status in the regression model, we have computed a model with 6MWT-distance as dependent variable and with age and gender as independent variables (R2 = 0.18) in a first step, in which we introduced in a second step the entry of health status as supplementary independent variable. Using this method we obtained the partial correlation coefficient (partial r = -0.40, p < 0.001) between health status and 6MWT-distance. Discussion The 6MWT presents several interesting advantages for the evaluation of the exercise capacity in elderly people. Different authors have described reference equations and tables to predict the 6MWT-distance in healthy elderly subjects. Gender, age, weight and height of these subjects appear to explain a large proportion of the variability in the 6MWT-distance [14-16]. Advancing age, however, is predominantly accompanied by an increasing burden of pathology [24] and "apparently healthy" elderly persons, willing to participate in physical training sessions, actually present a large diversity in health status. This means that the exercise capacity and the risk for complications during exercise are not the same for each person who consider themselves able to perform physical exercise. Ideally, an exercise program must be established for each person individually considering all facets of his/her health condition. It is conceivable that healthier elderly will present a better physical exercise capacity than those with a worse health condition. Therefore it is to be expected that the performance on exercise tests will reflect the aforementioned difference in health status. However, the contribution of the health status of elderly participants to the variability of the distance walked in the 6MWT has not been extensively described. In order to distinguish completely healthy participants from those with a worse health status we used criteria derived from the SENIEUR protocol [22]. This protocol was originally established to select participants for studies concerning the age-related changes of the immune system and allows the distinction between age-related and disease-related changes. The literature provides no clear guidelines to categorize elderly persons attending exercise programs according to their health status. Therefore, we developed a classification system, which stratifies health categories corresponding to an increasing risk for complications during physical exercise. Since the SENIEUR protocol allows for the presence of certain diseases and the use of medication that has no influence on the immune system, in our classification these criteria were further adapted in order to identify the "completely healthy" participants (group A) and to differentiate them from the "apparently healthy". The latter group was functioning normally, but could not be considered as completely healthy since participants either presented stabilized non-cardiovascular conditions (group B1) or were using cardiovascular medication, however, without any sign of active cardiovascular disease other than normalized hypertension and without significant abnormalities on ECG (group B2). When a history or signs of cardiovascular disease, other than hypertension, were noticed, participants were considered as belonging to group C. When evaluating large numbers of participants there is always the possibility of encountering individuals who are acutely ill; since they will not be able to fully participate in an exercise program, we choose to classify them separately (group D). The classification system was primarily designed to allow the establishment of recommendations concerning the exercise schedule (type, duration and intensity) of elderly persons in the absence of direct medical supervision. Therefore, the classification system is rather conservative and it is easy for an individual to be considered at risk for complications. Roughly, participants in category A will have no particular limitations for exercising; for those in category B1 the instructions will vary with the nature of the health problem; those in category B2 will only exercise at higher intensity (e.g. up to 80% of maximal heart rate or higher) when guided by an instructor qualified for training elderly persons; those in category C will only be allowed to exercise under supervision of an instructor and with medical guidance of the training program; those in category D will not exercise unless cleared by a physician. Since any exercise schedule will depend upon its objective, we do not go into further details here for these recommendations. It is clear that elderly persons often termed as 'apparently healthy' do not correspond to criteria for being completely healthy. In our study, only 28% (group A1 = 44 of 156) or 37% (Groups A1 and A2 = 58 of 156) of the participants were completely healthy, depending on whether the use of preventive medication was taken into account. However, 79% (groups A, B1 and B2 = 123 of 156) were functioning normally in the community and had no overt history of cardiovascular disease. Rikli et al. (1999) [16] studied the 6MWT-distance in 7183 community-dwelling older adults (5048 male, 2135 female) aged 60–94 years. According to the inclusion criteria they describe, we can assume that the participants of that study meet our classification criteria of categories A, B1 and B2. Our study, based upon a much smaller population sample, demonstrates that normative data based upon the population of the aforementioned authors is not representative for completely healthy elderly. Indeed, the mean 6MWT-distance of completely healthy participants (category A, mean age 62 years (SD = 7); mean 6MWT-distance 764 m (SD = 162) and 657 m (SD = 118) for male and female respectively) is much higher than the norm reported by Rikli et al. [16] for subjects in the same age category (60–64 years; mean 6MWT-distance 616 m (SD = 84) and 551 m (SD = 77) for male and female respectively). When age-matched categories with mixed health status are compared, our results (see figure 2) accord well with those of Rikli et al. [16]. Neither the studies of Enright et al. [14] or Troosters et al. [15] used the criteria of the SENIEUR protocol to select healthy individuals. This probably means that the health status of the populations they describe is heterogeneous and might also explain the considerable range in 6MWT-distance (383–820 m) encountered in the 'healthy' elderly (50–85 years old) participants of Troosters et al. [15] as well as the relatively low median 6MWT-distances (576 m and 494 m for male and female respectively) for 'healthy' adults (40–80 years old) in the study of Enright et al. [14]. Our study demonstrates that the proposed health-categorization is able to detect a significant reduction in physical capacity due to individual parameters such as medical history, medication use and current health status, other than age, gender (explaining only 18% of the variability) and anthropometrical parameters (no significant independent predictors when health status is included in the regression model). It is our opinion that the results of our study argue in favor of the validity of the proposed health categorization. At this moment, however, other data concerning the reliability of the classification system are not yet available. The results of the multiple linear regression analysis and the absence of a significant interaction (two-way ANOVA testing) between health status, age and gender confirm that each of these factors is an independent determinant of 6MWT-distance in the elderly. The proposed health categorization is able to discriminate the completely healthy elderly from the apparently healthy and to distinguish among the apparently healthy several categories. This results in a more diversified spectrum of the community dwelling elderly population than obtained with other categorizations like the New York Heart Association (NYHA)-classification for cardiovascular disease [25] and the American Heart Association (AHA) risk stratification criteria [26]. Currently, these systems are widely used for stratification purposes in physical training schedules. However, they were developed for the description of cardiac patients and do not consider other pathologies nor co-morbidities. A minor familiarization effect has been described for the 6MWT with better values obtained at a second testing [27]. Since repeated testing is less applicable in clinical settings, we chose to use the results from a single test administration when the participants were still naïve for the 6MWT. In our study the correlation of the 6MWT-distance with age, gender and stature as described by others [14-16] is confirmed in the group of the completely healthy participants (category A). This relationship is attenuated or disappears in subjects presenting chronic pathology even without apparent functional limitations. Our study demonstrates that there exists a significant decrease in 6MWT-distance with increasing health problems. Since the main objective of our classification system was to stratify for the risk for cardiovascular or metabolic (e.g. hypoglycemia) complications during exercise, we did not expect that the performance on the 6MWT would vary with the health categories. It is probable that the relationship between health status and 6MWT-distance reflects the influence of the status of the cardiovascular system on the general fitness. Inversely, however, it can also be explained by the negative prognostic value of lack of fitness for the ulterior development of cardiovascular disease. It has, indeed, repeatedly been reported for healthy middle-aged adults that this risk is greater for the least fit individuals [28,29]. Since most of our participants were older than those in the risk studies, a certain amount of cardiovascular pathology did occur. In a multiple linear regression model age, gender and health status explained 31% of the variability of the 6MWT-distance in the population of our study. All three independent variables had a highly significant contribution to the model (age and gender together accounted for 18% of the variability, partial correlation between health status and 6MWT-distance = -0.40, p < 0.001). The addition of weight, height and BMI to the prediction model did not significantly improve the R2-value. This is different from other studies that describe regression equations for the 6MWT-distance in healthy elderly [14,15], and indicates that the health condition of community dwelling elderly is more influencing the 6MWT-distance than their anthropometrical predisposition (weight and height are not significant independent predictors for 6MWT-distance after considering health status, age and gender). The fact that these authors obtained a much higher R2-value (R2 = 0.66 [15], R2 = 0.42 for male and R2 = 0.38 for female [14]) than ours (R2 = 0.31) can be due to the more heterogeneous composition in health status and the repartition of the population in several health categories in our study. The remaining unexplained variability in 6MWT-distance might be found in the differences in skeletal muscle strength, the training levels and the physical activity levels of the participants. However, these parameters were not measured in our study. We propose to incorporate the present health classification for elderly people as outcome of the medical screenings preceding the start of a physical exercise program. As shown in our study, significant differences can be found in physical exercise capacity according to the health status. We used the 6MWT-distance, which is an established exercise test for elderly persons, to document the physical exercise capacity of our participants. Especially in the elderly, which show increased prevalence of co-morbidities (most frequently (cardio)vascular, metabolic or rheumatological), physical exercise programs should be established according to the individual exercise capacity. Following the health classification of the elderly participant, the training instructor can adapt the modalities of the training program in order to obtain the best results at the most appropriate training intensity. This should finally lead to concrete training guidelines for elderly people in relation to their health status, diminishing the risk for injuries or complications during physical exercise. Competing interests None declared. Authors' contributions TM conceived and coordinated the study, participated in the evaluation of the health condition of the participants, the analysis and the redaction. IB performed the statistical analysis and participated in the design and the redaction. ML participated in the evaluation of the health condition of the participants, the analysis and the redaction. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements A preliminary report of this study was presented at the 24th Winter Meeting of the Belgian Society for Gerontology and Geriatrics (BVGG) in Ostend, Belgium (February 16–17 2001). ==== Refs Marcell Taylor J. Review Article: Sarcopenia: Causes, Consequences, and Preventions J Gerontol A Biol Sci Med Sci 2003 58 M911 916 14570858 Hortobagyi T Mizelle C Beam S DeVita P Old adults perform activities of daily living near their maximal capabilities J Gerontol A Biol Sci Med Sci 2003 58 M453 60 12730256 Peeters P Mets T The 6-minute walk as an appropriate exercise test in elderly patients with chronic heart failure J Gerontol A Biol Sci Med Sci 1996 51 M147 51 8680996 Lipkin DP Scriven AJ Crake T Poole-Wilson PA Six minute walking test for assessing exercise capacity in chronic heart failure Br Med J (Clin Res Ed) 1986 292 653 655 3081210 Cooper KH A means of assessing maximal oxygen intake. Correlation between field and treadmill testing Jama 1968 203 201 204 5694044 10.1001/jama.203.3.201 Butland RJ Pang J Gross ER Woodcock AA Geddes DM Two-, six-, and 12-minute walking tests in respiratory disease Br Med J (Clin Res Ed) 1982 284 1607 1608 6805625 Pu CT Johnson MT Forman DE Hausdorff JM Roubenoff R Foldvari M Fielding RA Singh MA Randomized trial of progressive resistance training to counteract the myopathy of chronic heart failure J Appl Physiol 2001 90 2341 2350 11356801 Rubenstein LZ Josephson KR Trueblood PR Loy S Harker JO Pietruszka FM Robbins AS Effects of a group exercise program on strength, mobility, and falls among fall-prone elderly men J Gerontol A Biol Sci Med Sci 2000 55 M317 21 10843351 Berry MJ Rejeski WJ Adair NE Zaccaro D Exercise rehabilitation and chronic obstructive pulmonary disease stage Am J Respir Crit Care Med 1999 160 1248 1253 10508815 Criner GJ Cordova FC Furukawa S Kuzma AM Travaline JM Leyenson V O'Brien GM Prospective randomized trial comparing bilateral lung volume reduction surgery to pulmonary rehabilitation in severe chronic obstructive pulmonary disease Am J Respir Crit Care Med 1999 160 2018 2027 10588623 Wright DJ Khan KM Gossage EM Saltissi S Assessment of a low-intensity cardiac rehabilitation programme using the six-minute walk test Clin Rehabil 2001 15 119 124 11330756 10.1191/026921501671364449 Mangan D Judge J Reliability and validation of the six minutes walk J Am Geriatr Soc 1994 42 SA73 Solway S Brooks D Lacasse Y Thomas S A qualitative systematic overview of the measurement properties of functional walk tests used in the cardiorespiratory domain Chest 2001 119 256 270 11157613 10.1378/chest.119.1.256 Enright PL Sherrill DL Reference equations for the six-minute walk in healthy adults Am J Respir Crit Care Med 1998 158 1384 1387 9817683 Troosters T Gosselink R Decramer M Six minute walking distance in healthy elderly subjects Eur Respir J 1999 14 270 274 10515400 10.1034/j.1399-3003.1999.14b06.x Rikli R Jones J Functional fitness normative scores for community-residing older adults, ages 60-94. Journal of Aging and Physical Activity 1999 7 162 181 Hulens M Vansant G Claessens AL Lysens R Muls E Predictors of 6-minute walk test results in lean, obese and morbidly obese women Scand J Med Sci Sports 2003 13 98 105 12641641 Bean JF Kiely DK Leveille SG Herman S Huynh C Fielding R Frontera W The 6-minute walk test in mobility-limited elders: what is being measured? J Gerontol A Biol Sci Med Sci 2002 57 M751 6 12403805 Miyamoto S Nagaya N Satoh T Kyotani S Sakamaki F Fujita M Nakanishi N Miyatake K Clinical correlates and prognostic significance of six-minute walk test in patients with primary pulmonary hypertension. Comparison with cardiopulmonary exercise testing Am J Respir Crit Care Med 2000 161 487 492 10673190 Opasich C Pinna GD Mazza A Febo O Riccardi R Riccardi PG Capomolla S Forni G Cobelli F Tavazzi L Six-minute walking performance in patients with moderate-to-severe heart failure; is it a useful indicator in clinical practice? Eur Heart J 2001 22 488 496 11237544 10.1053/euhj.2000.2310 Katz S Ford AB Moskowitz RW Jackson BA Jaffe MW Studies of Illness in the Aged. The Index of Adl: A Standardized Measure of Biological and Psychosocial Function Jama 1963 185 914 919 14044222 Ligthart GJ Corberand JX Fournier C Galanaud P Hijmans W Kennes B Muller-Hermelink HK Steinmann GG Admission criteria for immunogerontological studies in man: the SENIEUR protocol Mech Ageing Dev 1984 28 47 55 6513613 10.1016/0047-6374(84)90152-0 Kirkwood B Essentials of Medical Statistics 1988 1st ed Oxford, Blackwell Sciences Ltd 234 Izaks GJ Westendorp RG Ill or just old? Towards a conceptual framework of the relation between ageing and disease BMC Geriatr 2003 3 7 14683526 10.1186/1471-2318-3-7 Kossman CE Nomenclature and Criteria for the Diagnosis of Cardiovascular Diseases Circulation 1964 30 321 325 14210612 Fletcher GF Balady G Froelicher VF Hartley LH Haskell WL Pollock ML Exercise standards. A statement for healthcare professionals from the American Heart Association. Writing Group Circulation 1995 91 580 615 7805272 Stevens D Elpern E Sharma K Szidon P Ankin M Kesten S Comparison of hallway and treadmill six-minute walk tests Am J Respir Crit Care Med 1999 160 1540 1543 10556117 Ekelund LG Haskell WL Johnson JL Whaley FS Criqui MH Sheps DS Physical fitness as a predictor of cardiovascular mortality in asymptomatic North American men. The Lipid Research Clinics Mortality Follow-up Study N Engl J Med 1988 319 1379 1384 3185648 Sandvik L Erikssen J Thaulow E Erikssen G Mundal R Rodahl K Physical fitness as a predictor of mortality among healthy, middle-aged Norwegian men N Engl J Med 1993 328 533 537 8426620 10.1056/NEJM199302253280803

15296514

PMC512287

CC BY

2021-01-04 16:03:30

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BMC Infect Dis. 2004 Aug 5; 4:24

latin-1

BMC Infect Dis

10.1186/1471-2334-4-24

oa_comm

==== Front BMC Med GenetBMC Medical Genetics1471-2350BioMed Central London 1471-2350-5-201529196610.1186/1471-2350-5-20Research ArticleCandidate high myopia loci on chromosomes 18p and 12q do not play a major role in susceptibility to common myopia Ibay Grace [email protected] Betty [email protected] Lauren [email protected] Debra [email protected] Melissa [email protected] Heping [email protected] Taura [email protected]'Neill Jennifer [email protected] Robert [email protected] Elise [email protected]–Wilson Joan E [email protected] Dwight [email protected] Inherited Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, 333 Cassell Dr., Suite 2000, Baltimore, MD 21224, USA2 Dept. of Ophthalmology, University of Pennsylvania, 3535 Market St., Suite 701, Philadelphia, PA 19104, USA3 Owens Optometrics, 654 E. Main St., New Holland, PA 17557, USA4 Pennsylvania College of Optometry, 8360 Old York Rd., Elkins Park, PA 19027, USA2004 3 8 2004 5 20 20 15 3 2004 3 8 2004 Copyright © 2004 Ibay et al; licensee BioMed Central Ltd.2004Ibay et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background To determine whether previously reported loci predisposing to nonsyndromic high myopia show linkage to common myopia in pedigrees from two ethnic groups: Ashkenazi Jewish and Amish. We hypothesized that these high myopia loci might exhibit allelic heterogeneity and be responsible for moderate /mild or common myopia. Methods Cycloplegic and manifest refraction were performed on 38 Jewish and 40 Amish families. Individuals with at least -1.00 D in each meridian of both eyes were classified as myopic. Genomic DNA was genotyped with 12 markers on chromosomes 12q21-23 and 18p11.3. Parametric and nonparametric linkage analyses were conducted to determine whether susceptibility alleles at these loci are important in families with less severe, clinical forms of myopia. Results There was no strong evidence of linkage of common myopia to these candidate regions: all two-point and multipoint heterogeneity LOD scores were < 1.0 and non-parametric linkage p-values were > 0.01. However, one Amish family showed slight evidence of linkage (LOD>1.0) on 12q; another 3 Amish families each gave LOD >1.0 on 18p; and 3 Jewish families each gave LOD >1.0 on 12q. Conclusions Significant evidence of linkage (LOD> 3) of myopia was not found on chromosome 18p or 12q loci in these families. These results suggest that these loci do not play a major role in the causation of common myopia in our families studied. ==== Body Background Myopia is one of the leading causes of vision loss around the world[1]. In the United States, myopia affects approximately 25% of adult Americans[2]. Ethnic diversity appears to distinguish different groups with regard to prevalence. Caucasians have a higher prevalence than African Americans[3]. Asian populations have the highest prevalence rates with reports ranging from 50–90%[1,4,5]. Jewish Caucasians, one of the target populations of the present study, have consistently demonstrated a higher myopia prevalence than the general Caucasian population in both U.S. and European population surveys; Orthodox Jewish males in particular show increased susceptibility[6,7]. Despite many decades of research, little is known about the precise molecular defects and abnormal biochemical pathways that result in myopia. Compelling data from familial aggregation and twin studies indicate that susceptibility to myopia is inherited. Several familial aggregation studies have reported a greater prevalence of myopia in children of myopic parents compared to children of nonmyopic parents [8-12]. Several twin studies have demonstrated a very high heritability (estimates ranging from 60 to 90%) for myopia [13-15]. Other recent genetic studies of families with -6.00 D or more of myopia (termed high or pathological myopia) have reported significant linkage to regions on chromosome 18p11.31, 12q21-23, 17q21-22 and 7q36 [16-19]. The 18p candidate region has been confirmed in an independent study of high myopia [20]. Mutti et al.[21] examined the hypothesis that families with milder, juvenile onset myopia might show linkage to these same candidate regions. They found no evidence to support such a role in this more common form of myopia but their study was not highly powered in the presence of heterogeneity. Evidence also exists that myopia may be under environmental influences. The rapid increase in the prevalence of myopia over the last several decades suggests that environmental factors are important [22,23]. Furthermore, studies have shown a positive correlation of specific environmental factors, such as nearwork, with myopia [24,25]. It has been postulated that myopia develops in a person who engages in significant periods of sustained nearwork as an adaptive response to achieve better focus for near images[26]. Interestingly, Cordain et al.[27] suggest a positive correlation for myopia with increased consumption of carbohydrates, hyperinsulinemia and type II diabetes. Finally, experimental findings from animal studies show that the refractive state of young chicks will adapt to compensate for refractive errors induced by spectacle lenses[28]. The combination of genetic and environmental influences on the development of myopia suggests that myopia is a complex disorder and should not be classified as a simple Mendelian trait. Further evidence is shown by studies that have reported correlation coefficients for myopia between offspring and parents and between pairs of siblings to lie between 0.07–0.36 [29-32]. Due to the possible complexity of myopia, population isolates offer many advantages for genome-wide mapping studies[33]. First, they have reduced genetic complexity. Second, the people in most isolates share a common environment and culture. Differences in diet, exercise, sanitary conditions, and exposure to infectious diseases are minimized. A common language and religion usually promote social cohesion. Therefore, some of the environmental noise surrounding complex diseases that are determined by a combination of nature and nurture may be avoided. To avoid some of the complexity in mapping genes for myopia we have collected refractive measurements and DNA samples from Amish and orthodox Jewish families with myopia. The Old Order Amish are mostly rural farmers and craftsmen. They lead a culturally and technologically distinct lifestyle. They are a genetically well-defined founder population with large families and well-documented genealogies [34,35]. Family history records of the Amish in Lancaster County, Pennsylvania, beginning from 1727 are highly preserved[36]. Other features of this population include a relatively high standard of living, low migratory tendencies, and no practice of birth control, which facilitate the recruitment of large and extended families. The orthodox Jewish families in this study are all of Ashkenazi descent, a population with known founder effects in other common diseases[37]. This population also has somewhat larger family sizes than average in the US. In this initial report, we describe the design of our study and show that two regions (18p and 12q) previously reported to be linked to high myopia cannot explain the familial aggregation in these families with mostly moderate to milder forms of myopia. We had hypothesized that allelic heterogeneity might exist at these candidate loci such that in addition to highly penetrant alleles for extreme high myopia, there might also exist other susceptibility alleles of (possibly) lower penetrance that produce milder phenotypic forms of myopia. However, we found no strong evidence in support of this hypothesis. Methods Family screening The study protocol adhered to the tenet of the Declaration of Helsinki and was approved by the University of Pennsylvania and the National Human Genome Research Institute, National Institutes of Health institutional review boards. Informed consent was obtained from the subjects after explanation of the nature and possible consequences of the study. The collection of orthodox Jewish individuals was begun by a mass mailing of 3900 letters to all the known orthodox Jewish families living in Lakewood, New Jersey. Questionnaires were sent with letters explaining the study. If willing to participate, individuals completed and returned questionnaires that included their contact and physician information. Second and third mailings went out to individuals who did not respond – either positively or negatively – to the first mailing. The total number of questionnaires returned was 1,310. All Jewish individuals included in the study were of Ashkenazi heritage. Collection of Amish families was done by an advertisement in an Amish newspaper, referrals from local eye doctors in the Lancaster County community and word of mouth. Criteria for entry into the study included the following: 1) Negative history of systemic or ocular disease which may predispose to myopia, 2) negative history of a premature birth, 3) proband must be affected and must have a family history of myopia in either their parents or children, 4) only one parent (as opposed to both parents) of the proband can be myopic. For the Orthodox Jewish population, an individual's myopic status was obtained from the most recent (within 2 years) measurement of refractive error. If not recent, an individual was given a repeat exam by their local eye doctor or one of the study investigators (D.S.). For the Amish subjects, all participants were examined by a study investigator (D.S.) at the Amish Eye Clinic in Strasburg, PA. Amish participants were brought to the study site because they do not have phone access making it difficult to obtain a past history and records. Cycloplegic refractions were done on all individuals less than 40 years of age with one drop each of 1% cyclogyl, 1% mydriacyl and 2.5% phenylephrine. A manifest refraction was performed if an individual was older than 40 years of age. Classification as myopic required at least -1.00D in each meridian of both eyes. Individuals were classified as nonmyopes if they were over 21 years of age and did not meet the above criteria for myopia. Other individuals were classified as nonmyopic if they were 5–10 years old and had ≥ +3.00D in each meridian, 10–18 years old with ≥ +2.00D in each meridian or 18–21 years old with ≥ +0.50D in each meridian. All other individuals were classified as unknown for the trait. This ascertainment protocol resulted in the collection of 40 Amish families and 38 orthodox Jewish families. Of the 40 Amish families, phenotype data were available on 340 persons (170 individuals were affected and 170 were unaffected) but only 323 DNA samples were available to be genotyped. In the 38 Jewish families, phenotype data were available for 313 persons (177 affected, 122 unaffected and 14 of indeterminate phenotype) and DNA samples were available and genotyped for 290 of these family members. DNA extraction and genotyping Peripheral blood was collected from family members. High molecular weight genomic DNA extraction from the blood samples was performed with a kit (Puregene; Gentra Systems, Inc.; Minneapolis, MN, USA). Polymerase chain reactions were performed in a 17.05 ul volume containing 12–320 ng/ul of DNA; 880 uM each of dATP, dCTP, dGTP, and dTTP; 3 mM MgCl2; 10 mM Tris/HCl (pH8.3); 50 mM KCl; 0.6 uM of each primer; and 7.6 units/ul of Taq polymerase. Standard thermocycling was as follows: 94°C for 30 sec., 55°C for 30 sec. and 72°C extension time for 30 sec. Markers used included D12S85, D12S1706, D12S346, D12S78, D12S79, D12S86, D18S59, D18S481, D18S63, D18S452, D18S53, and D18S474 located in the 18p and 12q regions implicated in high myopia [16,17]. Power studies A simulation study was conducted on the first 44 Ashkenazi Jewish families collected in this study, using the computer program SIMLINK [38,39], to compare the projected power from alternative parametric trait models (five of these families contributed no information about linkage and so were not genotyped and the sixth family was dropped after genotyping because of sample problems that resulted in inadequate linkage information). It was assumed that the myopia trait is controlled by an autosomal dominant bi-allelic locus and the frequency of the high risk allele was varied in different simulations, using both 0.05 and 0.01. The actual observed pedigree structures, trait phenotypes and DNA sample availability were used to simulate the trait locus genotypes and linked and unlinked marker loci were also simulated. A highly polymorphic marker locus (9 equally frequent alleles) was assumed. The power available from these families to detect linkage was evaluated using different models for penetrance and sporadic rates. For each of the 12 models tested, simulations were performed assuming that the underlying proportion of families linked to the same marker locus (α) was 25%, 50%, 75% and 100%. Furthermore, for each model at each specified level of α, simulations were performed for six recombination distances (θ) between the disease and the marker loci (i.e., θ = 0.01, 0.05, 0.1, 0.2, and 0.5); for three maximum penetrances (0.6, 0.7 and 0.8) for gene carriers; and for two phenocopy rates (0.08 and 0.15). LOD scores assuming homogeneity were calculated for each of 100 replicates. The average LOD score over all replicates (ELod) and its standard deviation were calculated for each model simulated. The power of these families to detect a linkage (i.e., to obtain a LOD score ≥ 3.0) was tabulated for the linked marker and the probability of obtaining a LOD score greater than 1.0 when no linkage exists (Type I error) was tabulated for the unlinked marker in all simulations. Linkage analysis The data on 40 Amish and 38 Ashkenazi Jewish families were checked for misspecification of family structures, data entry errors and genotyping errors using the program SIBPAIR[40]. This program was also used to estimate allele frequencies at marker loci from the unrelated founder individuals in the families. Parametric two-point linkage analysis was performed with the MLINK program of the FASTLINK package [41,42] and the utility programs MAKEPED, Linkage Control Program, and Linkage Report Program from LINKAGE 5.1 [43-45]. Intermarker distances (Kosambi cM) of the microsatellite markers were obtained from the Marshfield database : D12S85-42.78-D12S1706-0.53-D12S346-7.22-D12S78-13.44-D12S79-9.23-D12S86; D18S59-6.94-D18S481-1.36-D18S63-10.4-D18S452-22.54-D18S53-30.08-D18S474. To carefully explore the possibility of linkage of common myopia to these high myopia candidate regions, we utilized 12 different parametric models (Table 1). Analyses were performed assuming all combinations of three different frequencies for the myopia susceptibility allele (0.0133, 0.5 and 0.10) and four different sets of genotypic penetrances for the gene carriers and non-gene carriers, respectively: 0.90 and 0.0; 0.80 and 0.0; 0.80 and 0.05; and 0.60 and 0.15. Models 1–4 (Table 1) assume an allele frequency for the putative myopia susceptibility allele of 0.0133, which is the same value used by Young et al. in their linkage studies of high myopia [16,17] and close to the value of 0.01 that showed good power in our power simulation (note that a more frequent allele frequency of 0.05 resulted in similar but always lower predicted power in our simulations than the power obtained when an allele frequency value of 0.01 was used; note also that this allele frequency applies only to the linked trait locus, so that if there are multiple loci and environmental factors involved in causing myopia under a heterogeneity model, any single locus might only account for a small proportion of all myopia cases). No sex difference was assumed in any of these models. All persons younger than age 5 were coded as unknown for the trait. This analysis assumed autosomal dominant inheritance of a myopia susceptibility allele. Recombination fractions were assumed to be equal in men and women. The program HOMOG[46] was used to test for evidence of heterogeneity in the presence of linkage in the two-point parametric linkage analyses. The heterogeneity testing was performed separately in the Jewish and Amish families and also in a joint analysis of LOD scores from the two datasets combined. Multipoint parametric and nonparametric linkage analyses were performed with the GENEHUNTER program[47]. Because of program memory constraints, one large Amish pedigree was split into three small ones for the GENEHUNTER analysis. The parametric analyses in GENEHUNTER used the same models described above, while allowing for locus heterogeneity. The nonparametric statistic NPLall, which estimates the statistical significance of alleles shared IBD between all affected family members, was calculated also, together with an estimated P value for the Amish and Jewish datasets separately. A nonparametric analysis combining the Amish and Jewish families was then performed by calculating the sum of NPL scores for each family (obtained in the separate Amish and Jewish analyses just described) divided by the square root of the total number of families (N = 78)[48] to obtain an overall combined NPL score. Table 1 Different parametric models utilized for the linkage analysis Model Allele Frequency Penetrance in DD:Dd susceptibility allele carriers Penetrance in dd normal homozygotes 1 0.0133 0.90 0.00 2 0.0133 0.80 0.00 3 0.0133 0.80 0.05 4 0.0133 0.60 0.15 5 0.05 0.90 0.00 6 0.05 0.80 0.00 7 0.05 0.80 0.05 8 0.05 0.60 0.15 9 0.10 0.90 0.00 10 0.10 0.80 0.00 11 0.10 0.80 0.05 12 0.10 0.60 0.15 Results Power simulation As expected, the estimated average maximum LOD score decreases with the distance between the linked marker locus and the trait locus, and with increasing heterogeneity. However, only minimal changes in projected power for our Ashkenazi families were observed as penetrance, phenocopy rate and disease allele were varied. Projected power was always higher when an allele frequency of 0.01 was used for the susceptibility allele at the trait locus than when an allele frequency of 0.05 was used; however, these differences in power were very small. Table 2 shows a representative sample of the predicted power results for detecting linkage to a marker 5 cM from the trait locus (the average maximum distance that a trait locus would be from our genotyped markers if it fell within the confines of either of these two candidate regions on 18p and 12q) assuming an autosomal dominant susceptibility allele frequency of 0.01. If all families were linked to one locus, these families were predicted to have 100% power to detect linkage to a marker 5 cM away from the trait locus with a LOD of 3 or more (the ELods were all ≥ 14). As less families were linked to the marker locus (i.e., as genetic heterogeneity increased) the power decreased but was still good (≥ 67%) if 50% or more of the families were linked. Even when only 25% of families were linked to the same locus, the expected LOD score was over 1.0 for all models. Of course, these LOD scores were calculated assuming homogeneity, and it is well known that power can be substantially increased when heterogeneity exists if LOD scores are calculated assuming heterogeneity (HLODs) as we have done in this study. So we would expect our actual power to detect linkage to be even higher than our simulations of heterogeneity predict. Observed Type I error rates were compatible with the nominal Type I error levels for all models. Between 0 and 1% of replicates produced a LOD score > 1 at any test map distance for unlinked markers. Table 2 Power and ELods from 100 replicates of simulated data, dominant susceptibility allele frequency of 0.01 PENETRANCE IN DD:Dd SUSC. ALLELE CARRIERS PENETRANCE IN dd NORMAL HOMOZYGOTE % FAMILIES LINKED 100% 75% 50% 25% Power1 ELod ± s.d.2 Power1 ELod ± s.d.2 Power1 ELod ± s.d.2 Power1 ELod ± s.d.2 0.8 0.08 100 14.0 ± 0.3 99 8.9 ± 0.3 82 4.7 ± 0.2 18 1.7 ± 0.1 0.8 0.15 100 14.7 ± 0.3 100 9.1 ± 0.3 73 4.8 ± 0.2 14 1.6 ± 0.1 0.7 0.08 100 14.8 ± 0.3 99 8.5 ± 0.3 77 4.8 ± 0.2 17 1.8 ± 0.15 0.7 0.15 100 15.2 ± 0.3 99 8.8 ± 0.3 70 4.5 ± 0.2 14 1.6 ± 0.1 0.6 0.08 100 15.0 ± 0.3 98 8.6 ± 0.25 67 4.3 ± 0.2 14 1.7 ± 0.1 0.6 0.15 100 14.2 ± 0.3 100 8.4 ± 0.3 73 4.3 ± 0.2 5 1.2 ± 0.1 1Power = 100 X Proportion of replicate samples that yielded a homogeneity LOD score ≥ 3.0 2ELod = average homogeneity LOD score over all replicates ± its standard deviation Linkage Parametric and nonparametric LOD scores were calculated for 40 Amish pedigrees and 38 Jewish pedigrees. The six markers on chromosome 12q21-q23 spanned 73 cM, and the six markers on chromosome 18p11.2-p11.32 spanned 70 cM. Markers D12S1706, D12S346, D18S59, D18S481 and D18S63 were previously reported by Young et al. [16,17] as showing evidence of linkage to autosomal dominant high myopia. Under all 12 parametric models, the evidence in favor of linkage to these candidate regions was minimal and this evidence varied only slightly as the assumptions of the trait model were changed across the models. Therefore, only the results from model 1 are presented here. Two-point linkage analyses in the Amish and Jewish populations Results of two-point parametric linkage analysis of myopia assuming linkage heterogeneity to the chromosome 12 and 18 markers in 40 Amish families are presented in Table 3. Statistically significant or suggestive linkage under locus homogeneity was not observed for either chromosome 12 or chromosome 18. Only one marker, D18S474 showed a two-point LOD ≥ 1.0 (LOD = 1.39 at θ = 0.3). Testing for linkage heterogeneity using HLODs in HOMOG did not significantly improve the evidence for linkage to any of these markers. Table 3 Two-point parametric LOD scores for myopia (Model 1) in 40 Amish Families RECOMBINATION FRACTION, θ MARKER 0.0 0.01 0.05 0.1 0.2 0.3 0.4 D12S85 -19.55 -12.97 -9.02 -6.27 -2.92 -1.15 -0.31 D12S1706 -39.91 -29.33 -17.82 -10.79 -3.85 -1.06 -0.18 D12S346 -36.18 -24.54 -13.68 -7.57 -1.99 -0.14 0.08 D12S78 -40.39 -28.61 -17.98 -11.46 -4.57 -1.5 -0.33 D12S79 -49.21 -32.42 -19.78 -12.36 -4.82 -1.5 -0.2 D12S86 -40.95 -32.39 -20.01 -12.52 -5.14 -1.85 -0.43 D18S59 -26.98 -20.07 -11.74 -6.77 -2.2 -0.48 -0.02 D18S481 -29.61 -19.93 -11.02 -5.99 -1.42 -0.04 0.16 D18S63 -32.32 -23.1 -12.11 -5.98 -0.87 0.5 0.35 D18S452 -38.37 -27.85 -16.16 -9.31 -2.76 -0.35 0.15 D18S53 -35.69 -25.42 -15.08 -9.07 -3.22 -0.77 -0.01 D18S474 -26.64 -14.04 -5.89 -2.01 0.94 1.39 0.7 The same markers on chromosome 12q21-q23 and chromosome 18p11.2-p11.32 were analyzed using 38 Ashkenazi Jewish families (Table 4). Statistically significant or suggestive linkage was not observed on either chromosome, no homogeneity LOD scores ≥ 1.0 were observed, and testing for linkage in the presence of heterogeneity (HLODs in HOMOG) did not alter this result. Table 4 Two-point parametric LOD scores for myopia (Model 1) in 38 Ashkenazi Jewish families RECOMBINATION FRACTION, θ MARKER 0 0.01 0.05 0.1 0.2 0.3 0.4 D12S85 -7.5 -6.71 -4.62 -2.56 -0.44 0.19 0.17 D12S1706 -36.21 -28.66 -17.67 -10.77 -3.85 -1.04 -0.15 D12S346 -32.76 -24.46 -13.34 -7.11 -1.68 -0.04 0.11 D12S78 -27.99 -20.59 -10.1 -4.65 -0.46 0.43 0.24 D12S79 -38.53 -30.26 -18.47 -11.21 -4.12 -1.24 -0.26 D12S86 -39.59 -31.68 -19.83 -12.58 -5.12 -1.8 -0.45 D18S59 -35.34 -27.25 -16.82 -10.69 -4.4 -1.51 -0.26 D18S481 -32.69 -24 -14.58 -9.19 -3.73 -1.26 -0.24 D18S63 -36.39 -28.55 -17.92 -11.36 -4.65 -1.63 -0.35 D18S452 -34.97 -25.21 -15.47 -9.72 -3.78 -1.22 -0.23 D18S53 -38.03 -26.36 -14.65 -8.62 -3.15 -0.94 -0.14 D18S474 -29.88 -22.79 -14.41 -9.29 -3.98 -1.45 -0.31 Heterogeneity testing using HOMOG in the combined Jewish and Amish families also did not yield any significant evidence of linkage in these two regions, with the maximum HLOD's being 0.39 and 0.95 on chromosomes 12 and 18 respectively. Furthermore, nonparametric two-point NPL scores did not show any significant evidence for linkage in either the Amish or Jewish populations. The observed combined NPL score of 1.37 for D12S1706 approached nominal significance at p = 0.09 but was not close to the significance level of at least p = 0.01 needed to provide confirmation of a prior linkage[49]. Multipoint linkage analyses in the Amish and Jewish populations Multipoint parametric linkage analyses assuming homogeneity were consistently negative in both the Amish and Jewish datasets. A maximum multipoint parametric HLOD of 0.92 was observed at D18S474 in the Amish population. However, multipoint parametric HLOD scores were essentially zero for the chromosome 12 region in the Amish and for both the chromosome 12 and 18 regions in the Jewish families. The multipoint nonparametric analyses did not show statistically significant evidence for linkage of myopia to either candidate region in the Amish (Figures 1 and 2) or the Jewish (Figures 3 and 4) families. Only very mild evidence for linkage of myopia in the Amish was observed between markers D18S59 and D18S481 (NPL= 1.54, p = 0.05) (Figure 2). Figure 1 Multipoint nonparametric linkage analysis of myopia to chromosome 12q in 40 Amish families Figure 2 Multipoint nonparametric linkage analysis of myopia to chromosome 18p in 40 Amish families Figure 3 Multipoint nonparametric linkage analysis of myopia to chromosome 12q in 38 Ashkenazi Jewish families Figure 4 Multipoint nonparametric linkage analysis of myopia to chromosome 18p in 38 Ashkenazi Jewish families Individual families showing linkage Only one Amish family (3061) showed marginal evidence for linkage (LOD > 1.0) to the region previously reported on chromosome 12 (D12S1706 and D12S346) for both two-point and multipoint parametric analyses (Table 5). Three Amish families gave LOD >1.0 at 2 markers on chromosome 18p. In both two-point and multipoint parametric analyses, family 3064 showed mild evidence of linkage (LOD = 1.30) to marker D18S63, and families 3049 and 3053 showed slight evidence of linkage to marker D18S474 (two-point LOD= 1.14 and 1.30, respectively). Simulations using SIMLINK (model 1) showed that for these individual families, the maximum two-point LOD score obtained when a linked marker was simulated at a recombination fraction of 0.0 ranged from 0.99 to 1.4, and the probability of obtaining a LOD over 1.0 for an unlinked marker ranged from <0.01 to 0.036. The nominal significance level corresponding to a LOD score of 1.0 is approximately 0.01. A total of three Jewish families showed marginal evidence for linkage to chromosome 12q markers for both two-point and multipoint analyses, with two-point and multipoint parametric LOD > 1.0. Simulations using SIMLINK (model 1) showed that for these individual families the maximum two-point LOD score obtained when a linked marker was simulated at a recombination fraction of 0.0 ranged from 0.93 to 1.06, and the probability of obtaining a LOD over 1.0 for an unlinked marker ranged from <0.001 to 0.05. Table 5 Families showing slight evidence for linkage of myopia (Model 1) to chromosome 12q or 18p AMISH FAMILIES FAMILY ID TWO-POINT LOD MULTIPOINT Marker Zmax LOD NPL P value 3061 D12S1706 1.04 1.04 * N/A D12S346 1.04 1.04 * N/A D12S78 1.04 1.04 * N/A 3049 D18S474 1.14 1.27 2.19 0.02 3053 D18S474 1.30 1.30 -* N/A 3064 D18S63 1.30 1.30 -* N/A JEWISH FAMILIES FAMILY ID TWO-POINT LOD MULTIPOINT Marker Zmax LOD NPL P value 20 D12S79 1.12 1.12 3.45 0.02 D12S86 1.12 1.12 3.45 0.02 58 D12S1706 1.16 1.16 3.01 0.06 D12S346 1.16 1.16 3.01 0.06 78 D12S85 1.07 1.07 3.38 0.01 * Only single affected parent-offspring pairs were genotyped in these families so the NPL analysis was uninformative in these families. However, the parametric LOD score analysis that uses both affected and unaffected family members was informative for linkage. Discussion The overall results of these preliminary studies do not indicate any strong evidence for linkage of myopia in these families to the candidate regions on chromosomes 12 or 18. Although some families show marginal evidence of linkage to one of these regions, the results could be due to chance. Our negative results for these candidate regions have several possible explanations. First, the diagnostic criteria used in the previous studies [9,10] that implicated these candidate regions were based on limiting the affection status to the sphere component of a plus cylinder refraction. An individual was considered affected with high myopia if the sphere was equal to or greater than -6D regardless of the astigmatic error. Our study required an individual to have -1D in each meridian to be considered affected. Therefore, the criterion for being affected was quite different between the two studies. Second, the study population in our study included moderate and low myopes in addition to a small number of high myopes. None of our families showed strong aggregation of high myopia. Therefore, there were no families in our study recruited exclusively for high myopia and no families that would have been highly powerful for the detection of linkage to a high myopia trait. We utilized this study design to search for allelic heterogeneity with regard to the 18p and 12q loci thinking that one or both loci may predispose to moderate/mild forms of myopia. Thus, the current linkage analysis was done to test the hypothesis that other alleles at the candidate high myopia loci on chromosomes 18 and 12 might contribute to the etiology of moderate/mild myopia. The mild evidence of linkage in a few families indicates that this hypothesis cannot be fully ruled out for a very small proportion of families with mild forms of myopia. However, there is no strong evidence in favor of this hypothesis and strong negative evidence against linkage in most of the families in this study. Previous studies attempting to confirm the high myopia loci on chromosomes 18 and 12 have yielded inconsistent results. Naiglin et al.[19]collected 23 French families with high myopia (spherical equivalent ≥ -6D) and performed a genome scan with 400 markers. Significant linkage was not found on 18p and 12q. Lam et al.[20] mapped 15 families with high myopia, ≥ -6.0D, using only 18p markers. Statistically significant (LOD > 3) linkage was not demonstrated although a multipoint LOD over 2.0 was observed, thus giving evidence of confirmation of the 18p candidate region. Mutti et al.[21] collected 53 families with varying degrees of myopia (affected ≥ -0.75D in each meridian) and genotyped the family members with 18p and 12q loci markers implicated in high myopia. No evidence of linkage to milder forms of myopia was found to the chromosome 18p and 12q loci previously associated with high myopia. Our study, although consistent with the results of Mutti et al.[21] was significantly different in design. First, Mutti et al. used a heterogeneous population that could decrease the chances of obtaining significant linkage for a minor gene effect from 18p or 12q if substantial ethnic heterogeneity exists. Both the Amish and Ashkenazi populations used in our study are more homogeneous and each sample was analyzed using marker allele frequencies estimated from the sample. Second, their study utilized 221 samples while we genotyped 613 individuals. Our power simulations predicted higher power in the presence of heterogeneity in our Ashkenazi families than was predicted for the Mutti et al. study. Our Amish families were of similar size and structure and so should have similar predicted power as the Ashkenazi families, and our combined analyses of the two data sets should provide much more power than that predicted by the simulations of the Ashkenazi families alone. The combination of the Mutti et al. study with the results presented here strongly suggest that these two candidate regions do not play a large role in the causation of moderate/mild myopia in several populations examined. These studies suggest that myopia is complex and probably caused by the interaction of multiple genes with the environment. Therefore, to understand myopia it is necessary to apply the equation: Genes + Environment=Outcome. The difficulty here is the uncertainty surrounding both terms in the equation; ideally, one set of genetic factors will interact with one set of environmental influences to produce identical outcomes, but it is unknown whether this is always going to be the case. Therefore, to lessen the problem of multiple gene interaction as well as gene-environment interaction confounding the results, strategies to limit this problem should be utilized in the genetic mapping of myopia. The use of isolated populations is one approach to limiting the heterogeneity across populations and is the approach we are using for a genome wide scan in these families. Furthermore, the definition of myopia needs to be standardized so comparisons across studies can be made accurately. Previous studies have utilized different requirements with regard to affection status making cross comparisons difficult. In conclusion, we find little evidence implicating previously described susceptibility loci for high myopia on chromosomes 12 and 18 as being important in the etiology of common, moderate/mild myopia in our two population samples. Competing interests None declared. Authors' contributions Dwight Stambolian, Lauren Reider, Debra Dana, Robert Owens, and Elise Ciner recruited patients for the study. Melissa Schlifka carried out the genotyping in chromosomes 18 and 12. Dwight Stambolian and Joan E. Bailey-Wilson performed the study design and wrote part of the manuscript. Joan Bailey-Wilson oversaw all statistical analyses; Grace Ibay performed statistical analyses and wrote part of the manuscript; Taura Holmes, Betty Doan and Jennifer O'Neill assisted with analyses of the data. All authors read and approved of the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgment This work was supported by the National Eye Institute Grant EY12226. ==== Refs Fredrick DR Myopia BMJ 2002 324 1195 1199 12016188 10.1136/bmj.324.7347.1195 Sperduto RD Seigel D Roberts J Rowland M Prevalence of myopia in the United States Arch Ophthalmol 1983 101 405 407 6830491 Katz J Tielsch JM Sommer A Prevalence and risk factors for refractive errors in an adult inner city population Invest Ophthalmol Vis Sci 1997 38 334 340 9040465 Wong TY Foster PJ Hee J Ng TP Tielsch JM Chew SJ Johnson GJ Seah SK Prevalence and risk factors for refractive errors in an adult Chinese population in Singapore Invest Ophthalmol Vis Sci 2000 41 S324 Saw SM Katz J Schein OD Chew SJ Chan TK Epidemiology of myopia Epidemiol Rev 1996 18 175 187 9021311 Baldwin W A review of statistical studies of relations between myopia and ethnic, behaviorial, and physiological characteristics Am J Opt Physiol Optics 1981 58 516 527 Zylbermann R Landau D Berson D The influence of study habits on myopia in Jewish teenagers J Pediatr Ophthalmol Strab 1993 30 319 322 Krause UH Rantakallio PT Koiranen MJ Mottonen JK The development of myopia up to the age of twenty and a comparison of refraction in parents and children Arctic Med Res 1993 52 161 165 8305093 Hui J Peck L Howland HC Correlations between familial refractive error and children's noncycloplegic refractions Vis Res 1995 35 1353 1358 7610597 10.1016/0042-6989(94)00224-A Sorsby A Leary GA Fraser GR Family studies on ocular refraction and its components J Med Genet 1966 3 269 273 16178083 Keller JT A comparison of the refractive status of myopic children and their parents Am J Optom Arch Am Acad Optom 1973 50 206 211 4511813 Yap M Wu M Liu ZM Lee FL Wang SH Role of heredity in the genesis of myopia Ophthalmol Physiol Opt 1993 13 316 319 Hammond CJ Snieder H Gilbert CE Spector TD Genes and environment in refractive error: the twin eye study Invest Ophthalmol Vis Sci 2001 42 1232 6 11328732 Lyhne N Sjolie AK Kyvik KO Green A The importance of genes and environment for ocular refraction and its determiners: a population based study among 20–45 year old twins Br J Ophthalmol 2001 85 1470 1476 11734523 10.1136/bjo.85.12.1470 Teikari JM Kaprio J Koskenvuo MK Vannas A Heritability estimate for refractive errors: A population based sample of adult twins Gen Epidemiol 1988 5 171 181 Young TL Ronan SM Drahozal LA Wildenberg SC Alvear AB Oetting WS Atwood LD Wilkin DJ King RA Evidence that a locus for familial high myopia maps to chromosome 18p Am J Hum Genet 1998 63 109 119 9634508 10.1086/301907 Young TL Ronan SM Alvear AB Wildenberg SC Oetting WS Atwood LD Wilkin DJ King RA A second locus for familial high myopia maps to chromosome 12q Am J Hum Genet 1998 63 1419 1424 9792869 10.1086/302111 Paluru P Ronan SM Heon E Devoto M Wildenberg SC Scavello G Holleschau A Makitie O Cole WG King RA Young TL New locus for autosomal dominant high myopia maps to the long arm of chromosome 17 Invest Ophthalmol Vis Sci 2003 44 1830 1836 12714612 10.1167/iovs.02-0697 Naiglin L Gazagne C Dallongeville F Thalamas C Idder A Rascol O Malecaze F Calvas P A genome wide scan for familial high myopia suggests a novel locus on chromosome 7q36 J Med Genet 2002 39 118 24 11836361 10.1136/jmg.39.2.118 Lam DSC Tam POS Fan DSP Baum L Leung Y-F Pang CP Familial high myopia linkage to chromosome 18p Ophthalmologica 2003 217 115 118 12592049 10.1159/000068554 Mutti DO Semina E Marazita M Cooper M Murray JC Zadnik K Genetic loci for pathological myopia are not associated with juvenile myopia Am J Med Genet 2002 112 355 60 12376937 10.1002/ajmg.10683 Cordain L Eaton SB Brand Miller J Lindeberg S Jensen C An evolutionary analysis of the aetiology and pathogenesis of juvenile-onset myopia Acta Ophthalmol Scand 2002 80 125 35 11952477 10.1034/j.1600-0420.2002.800203.x Hammond CJ Snieder H Gilbert CE Spector TD Genes and environment in refractive error: the twin eye study Invest Ophthalmol Vis Sci 2001 42 1232 1236 11328732 Kinge B Midelfart A Jacobsen G Rystad J The influence of near-work on development of myopia among university students. A three-year longitudinal study among engineering students in Norway Acta Ophthalmol Scand 2000 78 26 9 10726783 10.1034/j.1600-0420.2000.078001026.x Saw SM Zhang MZ Hong RZ Fu ZF Pand MH Tan DT Near-work activity, night-lights, and myopia in the Singapore-China study Arch Ophthalmol 2002 120 620 627 12003612 Goss DA Nearwork and Myopia Lancet 2000 356 1456 1457 11081523 10.1016/S0140-6736(00)02864-6 Cordain L Eaton SB Brand Miller J Lindeberg S Jensen C An evolutionary analysis of the aetiology and pathogenesis of juvenile-onset myopia Acta Ophthalmol Scand 2002 2 125 135 11952477 10.1034/j.1600-0420.2002.800203.x Wildsoet C Wallman J Choroidal and scleral mechanisms of compensation for spectacle lenses in chicks Vis Res 1995 35 1175 1194 7610579 10.1016/0042-6989(94)00233-C Sorsby A Leary GA Fraser GR Family studies on ocular refraction and its components J Med Genet 1966 3 269 273 16178083 Alsbirk PH Refraction in adult West Greenland Eskimos Acta Ophthalmol 1979 57 84 95 419981 Bear JC Richler A Burke G Nearwork and familial resemblances in ocular refraction:a population study in Newfoundland Clin Genet 1981 19 462 472 7296938 Ashton GC Segregation analysis of ocular refraction and myopia Hum Hered 1985 35 232 239 4029963 Arcos-Burgos A Muenke M Genetics of population isolates Clin Genet 2002 61 233 247 12030885 10.1034/j.1399-0004.2002.610401.x McKusick VA Medical Genetic Studies of the Amish 1978 Baltimore, MD, Johns Hopkins University Press Cross HE Population studies and the Old Order Amish Nature 1976 262 17 20 934323 Church Directory of the Lancaster County Amish 2002 Gordonsville, PA, Peqaea Publishers Osterer H A genetic profile of contemporary Jewish populations Nat Rev Genet 2001 2 891 898 11715044 10.1038/35098506 Ploughman LM Boehnke M Estimating the power of a proposed linkage study for a complex genetic trait Am J Hum Genet 1989 44 543 551 2929597 Boehnke M Estimating the power of a proposed linkage study: a practical computer simulation approach Am J Hum Genet 1986 39 513 527 3464203 Duffy DL Sibpair: a program for nonparametric linkage/association analysis Am J Hum Genet suppl 1997 61 A197 Cottingham RW JrIdury RM Schaffer AA Faster sequential genetic linkage computations Am J Hum Genet 1993 53 252 263 8317490 Schaffer AA Gupta SK Shriram K Cottingham RW Jr Avoiding recomputation in linkage analysis Hum Hered 1994 44 225 237 8056435 Lathrop GM Lalouel JM Easy calculations of lod scores and genetic risks on small computers Am J Hum Genet 1984 36 460 465 6585139 Lathrop GM Lalouel JM Julier C Ott J Strategies for multilocus linkage analysis in humans Proc Natl Acad Sci U S A 1984 81 3443 3446 6587361 Lathrop GM Lalouel JM White RL Construction of human linkage maps: likelihood calculations for multilocus linkage analysis Genet Epidemiol 1986 3 39 52 3957003 Ott J Linkage analysis and family classification under heterogeneity Ann Hum Genet 1983 47 311 320 6651220 Kruglyak L Daly MJ Reeve-Daly MP Lander ES Parametric and nonparametric linkage analysis: a unified multipoint approach Am J Hum Genet 1996 58 1347 1363 8651312 Xu J Combined analysis of hereditary prostate cancer linkage to 1q24-25: results from 772 hereditary prostate cancer families from the International Consortium for Prostate Cancer Genetics Am J Hum Genet 2000 66 945 957 10712209 10.1086/302807 Lander E Kruglyak L Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results Nat Genet 1995 11 241 247 7581446

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==== Front BMC Pregnancy ChildbirthBMC Pregnancy and Childbirth1471-2393BioMed Central London 1471-2393-4-151529196510.1186/1471-2393-4-15Research Article'Perinatal outcome in preterm premature rupture of membranes with Amniotic fluid index < 5 (AFI < 5) Borna Sedigheh [email protected] Hajieh [email protected] Soghra [email protected] Sedigheh [email protected] Assistant Professor of Gynecology & Obstetrics, Department of Gynecology & Obstetrics, Tehran University of Medical Sciences, Tehran, Iran2 Assistant Professor of pediatrics, Department of pediatrics, Shahed University of Medical Sciences, Tehran, Iran2004 4 8 2004 4 15 15 13 4 2004 4 8 2004 Copyright © 2004 Borna et al; licensee BioMed Central Ltd.2004Borna et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Our purpose was to determine whether AFI<5 cm after preterm premature rupture of the membranes (PPROM) is associated with an increased risk of perinatal morbidity. Methods We performed a prospective cohort study of 95 singleton pregnancies complicated by preterm premature rupture of the membranes (PPROM) with delivery between 26 and 34 weeks gestation. Patients were categorized in two groups on the basis of amniotic fluid index<5, (AFI<5 cm)(n = 26) or AFI ≥ 5 cm (n = 69). Categorical data were tested for significance with the χ2 and Fisher exact tests. Continuous data were evaluated for normal distribution and tested for significance with the student t test. All 2-sided p values < 0.05 were considered significant. Results Both groups were similar with respect to selected demographics, gestational age at rupture of the membranes, gestational age at the delivery, birth weight. Both groups were similar with respect to selected variable, latency until delivery, early onset neonatal sepsis, RDS and neonatal death. Patients with AFI<5 cm demonstrated greater frequency of C/S delivery for non reassuring fetal tests (23%vs 2.8%) (p = 0.001). Our study demonstrated that patients in group I had a significant increase in the frequency of clinical chorioamnionitis (P < 0/001). Post partum infections were not seen in 2 groups. Conclusions An AFI<5 cm after PPROM between 26 and 34 weeks gestation is associated with an increased risk of maternal infections and frequency of C/S. PROMchorioamnionitisneonatal sepsisAFI<5 ==== Body Background Preterm premature rupture of the membranes (PPROM) is one of the most common complications of the pregnancy. Preterm PROM is an important cause of perinatal morbidity and mortality, particularly because it is associated with brief latency from membrane rupture to deliver, perinatal infection, and umbilical cord compression due to oligohydramnios. PPROM is multifactor in nature. In any given patient, one or more path physiologic processes may be evident. Choriodecidual infection or inflammation appears to play an important role in etiology of preterm PROM, especially at early gestational ages. It has also been proposed that amniotic fluid posse's certain bacteriostatic properties that protect against potential infections processes and that a decrease in amniotic fluid volume may impair the gravid women ability to combat such infections, this latter hypothesis was tested by Vintziuleos, et al [2]. These investigations demonstrated that patients with olighhydramnios (AFI<5), were at greater risk of having chorioamnionitis and subsequent sepsis in the neonate [2,8]. Our purpose was to determine whether patients with PPROM and amniotic fluid index <5 cm (AFI< cm) are at an increased risk of having perinatal morbidity. Methods We performed a prospective cohort study of infants delivered between 26 weeks – 34 weeks gestation after preterm premature rupture of the membranes. This study was performed at Vali-e-Asr hospital at the medical university of Tehran between October 2000 and February 2002. All patients were at between 26 and 34 of weeks of pregnancy as best estimated by LMP, and confirmed by ultrasonography. In all patients rupture of the membrane was diagnosed by sterile speculum examination using pooled fluid, fern test and Nitrazine paper test. Patients with clinical chorioamnionitis, nonreassuring fetal status, obstetrical indication for immediate delivery, major congenital anomalies and advanced labor (cervical dilatation >3), and a growth-restricted fetus at initial admission were excluded. 95 singleton pregnancies have observed until 34 weeks of gestation. At admission, all patients had an ultrasonographic examination, which included confirmation of the estimated gestational age, and cumulative 4 guardant AFI measurements, as previously described by Phelan et al. [3] All patients received antibiotic prophylaxis at admission consisting of Ampicillin plus Erythromycin for 7 days following. In addition all patients received a single course of Betamethasone, consisting of two 12 mg Betamethasone injections during the first 24 hours after admission. Fetal surveillance incorporated daily non-stress testing. For fetuses with non-reassuring, non stress test results, biophysical profile assessments were performed. Indications for delivery included: labor, the diagnosis of clinical chorioamnionitis or non-reassuring fetal test results. Eligible patients were subsequently categorized into 2 groups on the basis of the admission AFI measurement. Patients in group 1 were those with an AFI<5 cm, whereas those in group 2 had AFI ≥ 5 cm. The 2 groups were compared for demographic characteristics, the estimated gestation age at both rupture of the membranes and delivery, latency until delivery, mode of delivery, birth weight, the development of clinical chorioamnionitis, postpartum endometritis, early onset neonatal sepsis and respiratory distress syndrome. Neonatal sepsis was diagnosed by positive blood, urine, or cerebrospinal fluid cultures. Possible neonatal sepsis was diagnosed when two or more of the following criteria were present: white blood cell count less than 5000/mm3, polymorphonuclear counts less than 1800/mm3, ratio of bands to total neutrophil counts greater than 0.2. Early onset neonatal sepsis was defined as sepsis in a neonate with positive culture results or possible sepsis within the first 48 hours of life and prior to the antibiotic administration. The clinical diagnosis of chorioamnionitis was made in presence of two or more of the following criteria: maternal fever greater than 38 C, maternal tachycardia (120 beats per minute or more), leukocytosis (greater than or equal to 20,000/mm3 white blood cell), fetal tachycardia (greater than 160 beats per minute), uterine tenderness, and foul-smelling amniotic fluid. Categorical data were tested for significance with the χ2 and Fisher exact tests. Continuous data were evaluated for normal distribution and tested for significance with the student t test. All 2-sided p values <.05 were considered significant. Results A total of 95 patients with preterm premature rupture of membranes were included in the study; 26 were included in group I (AFI<5 cm) and 69 in group II (AFI ≥ 5 cm). The 2 groups were similar with respect to maternal age, Parity, and gestational age at admission (table I). Gestational age at delivery and latency period until delivery, birth weight were not significantly different between the 2 groups. Both 2 groups had similar proportions of vaginal deliveries, however, in group I cesarean delivery was more likely to be performed because of non-reassuring fetal status (table I). Table 1 Demographic variable between two groups with PPPROM Variable AFI<5 (n = 26) AFI ≥ 5 (n = 69) Statistical significance Maternal age 25,1 ± 5.2 26.3 ± 4.9 Ns Parity 3 ± 1,5 2.4 ± 1.2 Ns Gestational age at admission 31.5 ± 2.00 33.5 ± 1.8 Ns Gestational age at delivery 32.6 ± 4.0 34.5 ± 3.7 Ns Latency (Mean ± SD) 7.6 ± 4.0 6.6 ± 5.2 Ns C/S rate for fetal distress 6(%23) 2(28%) P = 0.001, s Birth weight (mean) 2120 gr 2445 gr Ns Ns = not significant P-value < 0.05 = significant Our study demonstrated that patients in group I had a significant increase in the frequency of clinical chorioamnionitis (P < 0/001). Post partum infections were not seen in 2 groups. Early onset neonatal sepsis, respiratory distress syndrome (RDS), neonatal deaths were not significantly different between the 2 groups. (Table II) Table 2 Maternal and neonatal outcome comparison between two groups with PPROM Out come AFI<5(26) AFI ≥ 5 (69) Statistical significant Chorioamnionitis 5(19/2%) 2 (3%) p < 0/001 Early onset sepsis 7(30/4%) 19(27/9%) p = 0.819 RDS 6(26/1%) 8 (11/8%) p = 0.1 Neonatal death 4(17/4%) 5(7/4%) p = 0.163 P-value < 0.05 was considered significant Discussion Complications of preterm premature rupture of membranes count for approximately 25% to 33% of all preterm deliveries. Approximately, 75% of women will be delivered within 1 weeks of presentation [1,5]. More recent evidence suggests that membrane rupture is also related to biochemical processes, including disruption of collagen within the extracellular matrix of the amnion and the chorion and programmed death of cells in the fetal membranes. It has been proposed that the fetal membranes and the maternal uterine lining (decidua) respond to various stimuli, including membrane stretching and infection of the reproductive tract, by producing mediators, such as prostaglandins, cytokines, and protein hormones that govern the activities of matrix-degrading enzymes. When the fetal membranes rupture at term or before, the options are expectant management (with close observation for signs of labor, non reassuring fetal-heart-rate patterns, or intrauterine infection) or induction of labor [10,11]. Expectant management with antenatal antibiotics and corticosteroid administration are recommended the standard of care in the setting of PPROM at gestational ages of ≤ 34 [1,4,9,10]. Current evidence suggests adjunctive antibiotic therapy to reduced gestational age-dependent and infectious infant morbidity. Amniocentesis and amniotic fluid volume have been advocated as a useful adjunct for identifying these patients [9]Several studies have implicated oligohydramnios in patients with preterm premature rupture of the membranes as a significant risk factor for perinatal infection, and fetal distress, cesarean delivery, and neonatal death [5-8,10]. In our study the finding of an AFI<5 cm after preterm premature rapture of the membranes was associated with the development of chorioamnionits. However, patients in the group with AFI<5 did not have a shorter latency until delivery. Our study did not demonstrate an association between the development of chorioamnionitis and latency interval in patients with ruptured membranes (P = 0/783), because the latency period in our study were not significantly different between 2 groups. Other investigators have demonstrated an association between the development of chorioamnionitis and a shorter latency in patients with PPROM [5-8]. Post partum infections were not seen in our study. Perhaps, decreasing of post partum infections rates in our cases were the reason of using antibiotics after C/S. We were used intravenous Cephazolin for 48 h and then oral Cephalexin for 5 days after C/S. Our study demonstrated that the patients with oligohydramnios were more likely to undergo cesarean delivery because of non-reassuring fetal heart rate patterns and is consistent with the findings of these other studies [5,6,8]. This study didn't show an increased frequency of early onset sepsis in the group I (AFI<5 cm), because all newborn infants in the study were treated possible sepsis with clinical symptom and laboratory evidence. 7 of the included neonates had positive blood cultures or spinal fluid cultures; as a result, there was not sensitive mechanism for appropriately determining the diagnosis of early sepsis. The negative cultures in the neonates with possible sepsis may be related to inadequate culturing techniques or the inherent difficulty encountered by most laboratories in isolating anaerobic bacteria. Perhaps, Diagnosis of early onset neonatal sepsis and close observation for early signs of sepsis and more aggressive evaluation and early treatment for neonatal sepsis have decreased early onset sepsis in 2 groups. Preterm premature rupture of the membranes is associated with a significant decrease in the frequency of neonatal respiratory distress syndrome. In the Sims EJ study (2002), The frequency of respiratory distress syndrome in the neonate complicated with PPROM was (17%).11 This study evaluate the effect of AFI on the frequency of respiratory distress syndrome among two group that are complicated with PPROM. The frequency of respiratory distress syndrome in the neonate was not significantly lower in the group (II) than in the group I. (11/8% vs26/1%) (P < .01). The identification of oligohydramnios, defined as an AFI<5 cm, patients with preterm PPROM appear to indicate a significant risk of chorioamnionitis and early onset neonatal sepsis. These finding can aid in the counseling of patients with PPROM and may have several clinical application. Management of PPROM requires an accurate diagnosis as well as evaluation of costs and the risks and the benefits of continued pregnancy or expeditious delivery. It is important that the patient be well informed regarding the potential for subsequent maternal, fetal, and neonatal complications regardless of the management approach. Pre-publication history The pre-publication history for this paper can be accessed here: ==== Refs Yoon BH Kim Ya Romero R Kim JC Park KH Association of oligohydramnios in women with preterm premature rupture of membranes with an inflammatory response in fetal amniotic and maternal compartments Am J Obstet Gynecol 1999 181 784 8 10521729 Vintzileos AM Cmpbell WA Nochimson DJ degree oligohydramnios and pregnancy outcome in patients with prom Obstet Gynecol 1985 66 162 7 3895069 Phelan JP Smith CV Broussard P Smal lM Amniotic fluid volume assessment with four-quadrant technique at 36–12 week's gestation J repord Med 1987 32 540 2 Cox SM Leveno KJ International delivery versus expectant management with PROM at 30–34 weeks gestation Obestet Gynecol 1999 86 875 9 10.1016/0029-7844(95)00303-9 Moberg IJ Garete TJ Freeman RK Fetal heart rate patterns of fetal distress inpatient with prom Obestet Gynecol 1984 64 60 4 Joong Shin Yoon BH Romero R Moon JB The relationship between olighydrmnios and the onset of preterm labor in prom Am J Obestet Gynecol 2001 184 459 62 10.1067/mob.2001.109398 Macmillan WE Mann SE Shmoys SM Amniotic fluid index as predictor of latency after preterm premature rupture of the membranes Am Jperinatol 1994 11 249 52 Vermillion S Kooba A Amniotic fluid index value after prom and subsequent perinatal infection Am J obstet Gynecol 2000 183 271 6 10942458 10.1067/mob.2000.107653 Mercer BM Crocker L Boen Sibai B Induction versus expectant management in PROM with mature amniotic fluid at 32–36 weeks Am J Obestet Gynecol 1993 82 775 82 Mercer BM Preterm premature rupture of the membranes Obestet Gynecol 2003 101 178 90 10.1016/S0029-7844(02)02366-9 Sims EJ Vermillion ST Soper DE Preterm premature rupture of the membranes is associated With a reduction in neonatal respiratory distress syndrome Am J Obstet Gynecol 2002 187 268 72 12193910 10.1067/mob.2002.126203

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BMC Pregnancy Childbirth. 2004 Aug 4; 4:15

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==== Front BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-4-421529196410.1186/1471-2407-4-42Research ArticleElevated expression of MMP-13 and TIMP-1 in head and neck squamous cell carcinomas may reflect increased tumor invasiveness Culhaci Nil [email protected] Kubilay [email protected] Eray [email protected] Emel [email protected] Department of Pathology, Adnan Menderes University, Faculty of Medicine, Aydin, Turkey2 Department of Ear, Nose and Throat, Adnan Menderes University, Faculty of Medicine, Aydin, Turkey3 Department of Plastic and Reconstructive Surgery, Adnan Menderes University, Faculty of Medicine, Aydin, Turkey2004 3 8 2004 4 42 42 13 1 2004 3 8 2004 Copyright © 2004 Culhaci et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Matrix metalloproteinases [MMPs], which degrade the extracellular matrix, play an important role in the invasion and metastasis of squamous cell carcinomas. One MMP, MMP-13, is thought to play a central role in MMP activation. The purpose of this study was to investigate MMP-13 and TIMP-1 expression in squamous cell carcinomas of the head and neck and to relate these levels of expression to histologic patterns of invasion. Methods This study included T1 lesions obtained via biopsy from the larynx, tongue, and skin/mucosa of 78 patients with head and neck squamous cell carcinomas. The relationship between expression of MMP-13 and TIMP-1 and the mode of tumor invasion [MI] was evaluated immunohistochemically, using breast carcinoma tissue as a positive control. Results Increased expression was observed in highly invasive tumors, as reflected by the significant correlation between the degree of staining for MMP-13 or TIMP-1 and MI grade [p < 0.05]. There was no significant relationship between the degree of staining for MMP-13 or TIMP-1 and patient age, sex, tumor site, or tumor histologic grade. In addition, levels of staining for MMP-13 did not correlate with levels of staining for TIMP-1. Conclusion The expression of MMP-13 and TIMP-1 appears to play an important role in determining the invasive capacity of squamous cell carcinomas of the head and neck. Whereas additional studies are needed to confirm these findings, evaluating expression of these MMPs in small biopsy samples may be useful in determining the invasive capacity of these tumors at an earlier stage. ==== Body Background The invasion of surrounding tissues by neoplastic cells is one of the most important steps in tumor progression. Proteolytic enzymes such as matrix metalloproteinases [MMPs] contribute to tumor expansion by degrading components of the extracellular matrix [ECM]. MMPs are a 21-member family of zinc-dependent endopeptidases, which are capable of degrading most ECM components including collagen, elastin, fibronectin, and gelatin. Whereas this facilitates processes such as wound healing, enhanced MMP activity has been observed in a variety of pathologic conditions including osteoarthritis, rheumatoid arthritis, cardiovascular disease, and neoplasia [1-6]. MMPs can be divided into subgroups, which include collagenases, stromelysins, stromelysins-like MMPs, gelatinases, membrane-type MMPs, and others [5]. MMP-13 [collagenase 3] is a member of the collagenase family. It was first identified in human breast cancer and is active against a wide variety of ECM components [7]. MMP-13 also plays a central role in the MMP activation cascade, both activating and being activated by several MMPs [8]. Elevated MMP-13 expression has been found in a number of different malignancies, and expression has been related to tumor behavior and patient prognosis [7,9,10]. MMPs can be inactivated by specific tissue inhibitors of matrix metalloproteinases [TIMPs]. Thus far, four different TIMPs [TIMP-1, -2, -3, -4 ] have been identified [11] and implicated in connective tissue turn-over and remodeling. TIMPs inhibit MMPs by binding to them and forming non-covalent complexes [5]. Recent studies have linked increased MMP expression and decreased TIMP expression with tumor aggressiveness; however, other studies have shown overexpression of TIMPs in some patients with advanced tumors [12,13]. Squamous cell carcinoma [SCC] of the head and neck region is a major problem. Many studies have shown that MMPs are expressed in SCCs [5,14,15], with particular involvement by MMP-2 and MMP -9 [5,13,16]. MMP-13, which plays a central role in MMP activation, has also been shown to be highly expressed in head and neck SCCs [HNSCCs] [5]. As MMPs appear to be essential for tumor invasion and metastatic spread, characterizing their expression might help to determine a patient's treatment or prognosis. Therefore, we investigated the expression of MMP-13 and TIMP1 in biopsy specimens of HNSCCs to determine the relationship between the expression of these proteins and the mode of tumor invasion. Methods Patient and tumor characteristics Between 1998 and 2003, we obtained 78 incisional and excisional biopsy samples from the primary tumors of 78 patients with invasive SCC who were admitted to the Head and Neck Surgery and Plastic Surgery Departments. Fifty-four men and 24 women were included in the study, and the mean patient age was 64 years [range: 26 to 88 years]. T1 stage tumors from the larynx, tongue, and skin or mucosa of the face, cheek, lip, nose, and ear were studied. Table 1 summarizes clinical characteristics of the patients. None had undergone prior chemotherapy or radiotherapy. All patients provided informed consent to participate, and the study protocol was approved by our institution's ethics committee. Tumors were histologically graded as well [G1], moderately [G2], or poorly [G3] differentiated [Table 1]. The mode of tumor invasion [MI] was graded as follows: grade 1, well-defined border; grade 2, less-marked border; grade 3, groups of cells with no distinct border; grade 4, diffuse invasion [a, cordlike; b, widespread] [Table 1]. Immunohistochemical procedures The most representative block of tumor tissue was chosen in each case, and 5-μm sections were obtained and mounted on poly-L-lysine-coated slides for immunohistochemical staining. A standard streptavidin-biotin immunoperoxidase method was used for immunostaining with MMP-13 [7 ml, MS-825-R7, Ready-to-use, Neomarkers, USA] and TIMP-1 [7 ml, MS-608-R7, Ready-to-use, Neomarkers, USA] antibodies. The tissue sections were deparaffinized in xylene, rehydrated in an alcohol series, and immersed in distilled water. The sections were then boiled in a citrate buffer solution [10 mmol/L, pH= 6.0] in a microwave oven X3 for 10 minutes for antigen retrieval of both the MMP-13 and TIMP-1 antibodies. Endogenous peroxidase activity was blocked by exposing sections to a 0.3% solution of hydrogen peroxidase in phosphate-buffered saline [PBS] for 10 minutes at room temperature. After the sections had been rinsed with TRIS buffer, primary antibodies were applied for 60 minutes at room temperature followed by TRIS buffer. Linking antibody and streptavidin peroxidase complex were then added consecutively for 10 minutes at room temperature, and sections were washed again in TRIS buffer. After applying AEC chromogen, the sections were washed in deionized water, counterstained and mounted. Breast carcinoma tissue (which showed positive staining) was used as positive control during the evaluation of MMP-13 and TIMP-1 immunostaining. Evaluation/scoring of the staining One pathologist [NC] evaluated the stained slides associated with each case. The degree of staining for MMP-13 and TIMP-1 was scored as follows: 0, no staining of the tumor or stromal cells; 1+, weak [< 50%] positive staining of the tumor cells and/or weak staining of stromal cells; 2+, moderate [> 50%] positive staining of the tumor cells and/or moderate staining of stromal cells; 3+, extensive staining of the tumor cells and/or strong staining of stromal cells [Figs. 1,2] [12]. No normal epithelial cells were stained [Fig. 3]. Follow-up If necessary, surgery or radiotherapy was performed after the diagnosis. All patients were followed-up postoperatively for a mean interval of 20 months [range: 7 to 36 months]. During this period, 2 of 78 [2.6%] patients died from their tumors [one in larynx, one in tongue], and 1 patient died of an unrelated cause. Seventy-five of 78 [96.2%] patients were free of disease at the end of the follow-up period. Statistical analysis The chi-square test was used for statistical analysis. Data were analyzed using SPSS for Windows 10,0. A p level <0.05 was considered to be statistically significant. Results Although expression of cytoplasmic MMP-13 was primarily detected in tumor cells, it also was seen in stromal cells. MMP-13 expression was evident in tissue from 44 [56.4%] of the 78 patients. High levels of MMP-13 were noted in highly invasive tumors. There was no difference in the amount of staining between tumor centers or margins. Significant TIMP-1 expression was detected in tissue from 42 [53.8%] patients. Prominent labeling was confined to the stroma surrounding the tumor cells. Table 2 shows the relationship between MMP-13 expression and MI in the 78 biopsy specimens. Increased expression of MMP-13 was observed in highly invasive tumors [MI grades 4a and b]. The relationship between MMP-13 expression and MI grade was statistically significant [p < 0.05]. Table 3 summarizes the relationship between TIMP-1 expression and MI in the 78 biopsy specimens. Increased expression of TIMP-1 was observed in highly invasive tumors. The relationship between TIMP-1 expression and MI grade was statistically significant [p < 0.05]. There was no statistically significant relationship between the degree of staining for MMP-13 or TIMP-1 and patient age, sex, tumor site, or histologic grade. Also, the degree of staining for MMP-13 did not significantly correlate with the degree of staining for TIMP-1. Discussion As tumor invasion and metastasis affect a patient's prognosis, it is important to predict the invasive potential of tumors such as SCCs at an early stage. Several steps are involved in the invasion and metastasis of malignant cells including the attachment of cells to the ECM, the breakdown of matrix components, cell detachment, and migration of cells through the degraded matrix. This complex process requires several proteases, and the local balance between these proteases and protease inhibitors appears to be crucial. The MMPs represent one family of degrading proteases, which are expressed in various tumors. Many studies have shown that MMPs, especially MMP-2, -3, -9, are expressed in SCCs [5,12,16-18]. In the present study, we characterized the expression of one MMP–MMP-13, which plays a key role in the MMP activation cascade–and one inhibitor of MMPs, TIMP-1, in HNSCCs. Our finding that expression of both proteins was upregulated in markedly invasive tumors is consistent with prior reports and may have important therapeutic and prognostic implications. The expression of several MMPs has been investigated in SCCs including MMP-9 and MMP-13. MMP-9 expression was found to correlate the most strongly with advanced pathological stages, and patients with HNSCCs also may have elevated serum concentration of MMP-9 [17,19]. Furthermore, expression of some MMPs such as MMP-9 appears to vary not only between the primary tumor and sites of lymph node metastasis, but also between the early and late stages of lymph node metastasis [16]. MMP-13 is a member of the collagenase family, which degrades fibrillar collagens of types I, II, III, IV, X, and XIV, tenascin, fibronectin, aggrecan, versican, and fibrillin-I. It is now accepted that MMP-13 plays a key role in the MMP activation cascade, both activating and being activated by several MMPs [MT1-MMP, MMP-2, MMP-3] [8,11]. MMP-13 expression is also susceptible to stimulation by several cytokines and growth factors such as TNF-α and TGF-β [11,15]. Elevated MMP-13 expression has been documented in numerous malignancies [e.g., breast carcinoma, colorectal cancer, vulvar SCC, skin SCC, HNSC, BCC (in focal areas of keratinized cells)] and associated with tumor behavior and patient prognosis [1,7-10,20]. Both small and large tumors appear to express MMP-13, with expression being the most prevalent in undifferentiated tumors [11]. MMP-13 expression has also been observed in transformed, but not primary, human epidermal keratinocytes [11,15]. Although MMP-1, -2, -3 have been detected in actinic keratosis [12,21], premalignant and benign tumors were mostly negative for MMP-13 in one study [20]. It is thought that MMP-13, alone, can markedly enhance the invasive capacity of malignant cells [22]. In transformed keratinocytes, p 53 plays an important role in suppressing MMP-13 expression [23]. Whereas some investigators have not found a significant relationship between MMP-13 expression and HNSCC behavior [17], we found significant MMP-13 expression in highly invasive tumors; this finding suggests that MMP-13 likely plays a role in regulating tumor invasion. In regard to location, cellular events at the tumor-stromal interface are thought to be more closely related to metastatic potential than events at the tumor's center [17,24]. Others have reported that MMP-13 is not only expressed by tumor cells in the invading periphery of most SCCs but also by stromal cells in a subset of tumors [15]. In the present study, we found no difference in levels of MMP-13 expression between tumor centers or margins. TIMP-1 is another protein that has been implicated in tumor growth. TIMP-1 mRNA has been detected in well-differentiated cancer cells, proliferated keratinocytes, and endothelial cells [2,14], and TIMP-1 overexpression has been observed in almost every case of HNSCC [24]. Whereas some investigators have reported that increased expression of TIMP-1 and TIMP-2 correlates with less aggressive tumors, others have reported the opposite finding [12,13]. In the present study, TIMP-1 expression was significantly increased in markedly invasive tumors, suggesting that TIMP-1 also plays a role in regulating tumor invasiveness. In some studies, no association between MMP-13 and TIMP-1 expression and any clinicopathological variables was found, or TIMP expression did not correlate with tumor growth [13,17]. Our results are not compatible with these studies. However, we did fail to identify any statistically significant relationship between the degree of staining for MMP-13 or TIMP-1 and the patient's age, sex, tumor site, or tumor histologic grade. Also, there was no statistically significant correlation between the degree of staining for MMP-13 and TIMP-1. This suggests that the balance between MMP and TIMP expression may not be as important to tumor invasion as their overexpression; that is, MMP and TIMP may play important separate roles in tumor invasion in which they act via different mechanisms. Because of the relatively short follow-up interval, we are unable to demonstrate whether MMP-13 and TIMP-1 expression is related to long-term survival. However, 75 of our 78 patients were free of disease after a mean follow-up interval of 20 months. As poor outcomes are the result of local recurrences and distant metastasis, factors that reflect the invasive and metastatic potential of SCCs, such as MMPs, could be helpful in predicting patient prognosis. Thus, further investigation of MMPs may not only clarify their role in tumor invasion but also may facilitate the development of new therapeutic approaches. Conclusion The results of this study suggest that MMP-13, which plays a central role in MMP activation, and the MMP inhibitor, TIMP-1, help regulate the invasiveness of HNSCCs. Although other methods [e.g., Western blots] are needed to confirm these findings, examination of MMP-13 and TIMP-1 expression in small biopsy samples might be useful in determining the invasive capacity of these tumors at an earlier stage. Competing interests None declared. Authors' contributions NC drafted and wrote the manuscript. KM and EC performed the surgery and follow-up of the patients. ED participated in the design and coordination of the study. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgement The authors would like to thank Pinar Okyay from the Department of Public Health of Adnan Menderes University for her help with the statistical analysis of data. This study was supported by Adnan Menderes University Research Foundation No: TPF-01016. Figures and Tables Figure 1 Moderate MMP-13 staining of the tumor cells in MI grade 2 SCC (anti-MMP-13, original magnification, × 100) Figure 2 Moderate TIMP-1 staining of the stromal cells in MI grade 3 SCC (anti-TIMP-1, original magnification, × 200) Figure 3 Normal epithelial cells at the top were not stained with MMP-13 (anti-MMP-13, original magnification, × 200) Table 1 Clinicopathologic features Characteristic Number of cases Age  < 60 years 27  > 60 years 51 Sex  Male 54  Female 24 Tumor site  Larynx 26  Tongue 8  Skin and mucosa 44 Histologic grade  Differentiated (G1) 39  Poorly differentiated (G2, G3) 39 Mode of tumor invasion  Well defined (1 + 2) 18  No distinct border (3) 43  Diffuse invasion (4a + 4b) 17 Table 2 Relationship between level of MMP-13 expression and MI MMP-13 staining Grade of tumor invasion Negative/weak (0 + 1) Moderate/ extensive (2 + 3) Total 1 (1 + 2) 15 (83.3%) 3 (16.7%) 18 2 (3) 14 (32.6%) 29 (67.4%) 43 3 (4a + 4b) 5 (29.4%) 12 (70.6%) 17 Total 34 44 78 MI: mode of tumor invasion (expressed as grade). p values: 0.001 Table 3 Relationship between level of TIMP-1 expression and MI TIMP-1 staining Grade of tumor invasion Negative/weak (0 + 1) Moderate/ extensive (2 + 3) Total 1 (1 + 2) 13 (72.2%) 5 (27.8%) 18 2 (3) 16 (37.2%) 27 (62.8%) 43 3 (4a + 4b) 7 (41.2%) 10 (58.8%) 17 Total 36 42 78 MI: mode of tumor invasion (expressed as grade). p values: 0.03 ==== Refs Nakopoulou L Giannopoulou I Gakiopoulou H Liapis H Tzonou A Davaris PS Matrix metalloproteinase-1 and -3 in breast cancer: correlation with progesterone receptors and other clinicopathologic features Hum Pathol 1999 30 436 442 10208466 10.1016/S0046-8177(99)90120-X Vaalamo M Leivo T Saarialho-Kere U Differential expression of tissue inhibitors of metalloproteinases (TIMP-1, -2, -3, and -4) in normal and aberrant wound healing Hum Pathol 1999 30 795 802 10414498 10.1016/S0046-8177(99)90140-5 Iki K Tsutsumi M Kido A Sakitan H Takahama M Yoshimoto M Motoyoma M Tatsumi K Tsunada T Konishi Y Expression of matrix metalloproteinase-2 (MMP-2), membrane-type 1 MMP and tissue inhibitor of metalloproteinase 2 and activation of pro MMP-2 in pancreatic duct adenocarcinomas in hamsters treated with N-nitrosobis (2-oxopropyl)amine Carcinogenesis 1999 20 1323 1329 10383907 10.1093/carcin/20.7.1323 Maatta M Soini Y Liakka A Autio-Harmainen H Localization of MT1-MMP, TIMP-1, TIMP-2, TIMP-3, messenger RNA in normal, hyperplastic, and neoplastic endometrium. Enhanced expression by endometrial adenocarcinomas is associated with low differentiation Am J Clin Pathol 2000 114 402 411 10989641 Werner JA Rathcke IO Mandic R The role of matrix metalloproteinases in squamous cell carcinomas of the head and neck Clin Exp Metastas 2002 19 275 282 10.1023/A:1015531319087 Sternlicht MD Werb Z How matrix metalloproteinases regulate cell behaviour Annu Rev Cell Dev Biol 2001 17 463 516 11687497 10.1146/annurev.cellbio.17.1.463 Freije JMP Diez-Itza I Balbin M Sanchez LM Blasco R Tolivia J Lopez-Otin C Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas J Biol Chem 1994 269 16766 16773 8207000 Leeman MF McKay JA Murray GI Matrix metalloproteinase 13 activity is associated with poor prognosis in colorectal cancer J Clin Pathol 2002 55 758 762 12354802 10.1136/jcp.55.10.758 Johansson N Vaalamo M Grenman S Hietanen S Klemi P Saarialho-Kere U Kahari VM Collagenase-3 (MMP-13) is expressed by tumor cells in invasive vulvar squamous cell carcinomas Am J Pathol 1997 154 469 480 Nielsen BS Rank F Lopez JM Balbin M Vizoso F Lund LR Dano K Lopez-Otin C Collagenase-3 expression in breast myofibroblasts as a molecular marker of transition of ductal carcinoma in situ lesions to invasive ductal carcinomas Cancer Res 2001 61 7091 7100 11585740 Thomas GT Lewis MP Speight PM Matrix metalloproteinases and oral cancer Oral Oncol 1999 35 227 233 10621841 10.1016/S1368-8375(99)00004-4 Kurahara S Shinohara M Ikebe T Nakamura S Beppu M Hiraki A Takeuchi H Shirasuna K Expression of MMPs, MT-MMP, and TIMPs in squamous cell carcinoma of the oral cavity: correlations with tumor invasion and metastasis Head Neck 1999 21 627 638 10487950 10.1002/(SICI)1097-0347(199910)21:7<627::AID-HED7>3.3.CO;2-U Yoskizaki T Maruyama Y Sato H Furukawa M Expression of tissue inhibitor of matrix metalloproteinase-2 correlates with activation of matrix metalloproteinase-2 and predicts poor prognosis in tongue squamous cell carcinoma Int J Cancer 2001 95 44 50 11241310 10.1002/1097-0215(20010120)95:1<44::AID-IJC1008>3.3.CO;2-D Polette M Clavel C Birembaut P Localization by in situ hybridization of mRNAs encoding stromelysin 3 and tissue inhibitors of metallo-proteinases TIMP-1 and TIMP-2 in human head and neck carcinomas Path Res Pract 1993 189 1052 1057 8302724 Johansson N Airola K Grenman R Kariniemi AL Saarialho-Kere U Kahari VM Expression of collagenase-3 (matrix metalloproteinase-13) in squamous cell carcinomas of the head and neck Am J Pathol 1997 151 499 508 9250162 Sato H Shimada Y Watanabe G Uchida S Makino T Imamura M Expression of vascular endothelial growth factor, matrix metalloproteinase-9 and e-cadherin in the process of lymph node metastasis in oesophageal cancer Brit J Cancer 1999 80 1366 1372 10424737 10.1038/sj.bjc.6690530 O-charoenrat Rhys-Evans PH Eccles SA Expression of matrix metalloproteinases and their inhibitors correlates with invasion and metastasis in squamous cell carcinoma of the head and neck Arch Otolaryngol Head Neck Surg 2001 127 813 820 11448356 Imanishi Y Fuji M Tokumaru Y Tomita T Kanke M Kanzaki J Kameyama K Otani Y Sato H Clinical significance of expression of membrane type 1 matrix metalloproteinase and matrix metalloproteinase-2 in human head and neck squamous cell carcinoma Hum Pathol 2000 31 895 904 10987249 10.1053/hupa.2000.9756 Riedel F Götte K Schwalb J Hörmann K Serum levels of matrix metalloproteinase-2 and -9 in patients with head and neck squamous cell carcinoma Anticancer Res 2000 20 3045 3050 11062721 Airola K Johansson N Kariniemi AL Kahari VM Saarialho-Kere U Human collagenase-3 is expressed in malignant squamous epithelium of the skin J Invest Dermatol 1997 109 225 231 9242512 10.1111/1523-1747.ep12319441 Tsukifuji R Tagawa K Hatamochi A Shinkai H Expression of matrix metalloproteinase-1, -2 and -3 in squamous cell carcinoma and actinic keratosis Brit J Cancer 1999 80 1087 1091 10362121 10.1038/sj.bjc.6690468 Ala-Aho R Johansson N Baker AH Kahari VM Expression of collagenase-3 (MMP-13) enhances invasion of human fibrosarcoma HT-1080 cells Int J Cancer 2002 97 283 289 11774278 10.1002/ijc.1619 Ala-Aho R Grenman R Seth P Kahari VM Adenoviral delivery of p53 gene suppresses expression of collagenase-3 in squamous carcinoma cells Oncogene 2002 21 1187 1195 11850838 10.1038/sj.onc.1205198 Charous SJ Stricklin GP Nanney LB Netterville JL Burkey BB Expression of matrix metalloproteinases and tissue inhibitor of metalloproteinases in head and neck squamous cell carcinoma Ann Otol Rhinol Laryngol 1997 106 271 278 9109715

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==== Front BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-4-431529402410.1186/1471-2407-4-43Research ArticleHedgehog-interacting protein is highly expressed in endothelial cells but down-regulated during angiogenesis and in several human tumors Olsen Catherine L [email protected] Pin-Pin [email protected] Jens [email protected] Gabor M [email protected] Alan R [email protected] Department of Gene Therapy, Berlex Laboratories, Inc., Richmond, CA 94806, USA2 SBU DG&RP, Schering AG, Berlin, Germany3 Present location: Exelixis, Inc., South San Francisco, CA 94083, USA2004 4 8 2004 4 43 43 6 4 2004 4 8 2004 Copyright © 2004 Olsen et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The Hedgehog (Hh) signaling pathway regulates a variety of developmental processes, including vasculogenesis, and can also induce the expression of pro-angiogenic factors in fibroblasts postnatally. Misregulation of the Hh pathway has been implicated in a variety of different types of cancer, including pancreatic and small-cell lung cancer. Recently a putative antagonist of the pathway, Hedgehog-interacting protein (HIP), was identified as a Hh binding protein that is also a target of Hh signaling. We sought to clarify possible roles for HIP in angiogenesis and cancer. Methods Inhibition of Hh signaling by HIP was assayed by measuring the induction of Ptc-1 mRNA in TM3 cells treated with conditioned medium containing Sonic hedgehog (Shh). Angiogenesis was assayed in vitro by EC tube formation on Matrigel. Expression of HIP mRNA was assayed in cells and tissues by Q-RT-PCR and Western blot. HIP expression in human tumors or mouse xenograft tumors compared to normal tissues was assayed by Q-RT-PCR or hybridization of RNA probes to a cancer profiling array. Results We show that Hedgehog-interacting protein (HIP) is abundantly expressed in vascular endothelial cells (EC) but at low or undetectable levels in other cell types. Expression of HIP in mouse epithelial cells attenuated their response to Shh, demonstrating that HIP can antagonize Hh signaling when expressed in the responding cell, and supporting the hypothesis that HIP blocks Hh signaling in EC. HIP expression was significantly reduced in tissues undergoing angiogenesis, including PC3 human prostate cancer and A549 human lung cancer xenograft tumors, as well as in EC undergoing tube formation on Matrigel. HIP expression was also decreased in several human tumors of the liver, lung, stomach, colon and rectum when compared to the corresponding normal tissue. Conclusion These results suggest that reduced expression of HIP, a naturally occurring Hh pathway antagonist, in tumor neo-vasculature may contribute to increased Hh signaling within the tumor and possibly promote angiogenesis. ==== Body Background The Hedgehog family of genes encodes secreted signaling molecules that regulate cell proliferation and cell fate determination. In mammals, there are three such genes: Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). All three Hedgehog (Hh) proteins function by binding to the transmembrane receptor, Patched-1 (Ptc-1), leading to the de-repression of the membrane-bound inhibitor, Smoothened [1,2]. This results in activation of the transcription factor Gli-1, which induces expression of target genes that include Ptc-1 and Gli-1 itself. The increase in expression of Ptc-1 may limit the range of action of Hh by sequestering it at the surface of Hh-responsive cells [3]. Hedgehog-interacting protein (HIP) was discovered by screening a mouse cDNA expression library for proteins that bound to Shh [4]. HIP binds all three Hh proteins with an affinity equal to that of Ptc-1, and in mouse embryos it is expressed in cells adjacent to those expressing Shh, positioning it appropriately for in vivo interactions. Ectopic expression of Shh leads to ectopic HIP expression, indicating that HIP is a transcriptional target of Hh signaling [4]. Transgenic mice that overexpress HIP in the endochondral skeleton displayed a phenotype similar to that of Ihh knockout mice, consistent with an inhibitory role for HIP in Hh signaling [4]. Although it has been shown that overexpression of HIP in cells making Shh reduced the amount of Shh secreted into the media [5], no data has been published specifically demonstrating that expression of HIP in responding cells inhibits the activation of the Shh signaling pathway. During gastrulation in the mouse, Ihh is secreted by the endoderm and is sufficient to activate hematopoiesis and vasculogenesis [6]. In addition to its role in developmental processes, Shh was shown to induce angiogenesis in a murine corneal angiogenesis model, probably through the induction of the angiogenic factors VEGF, Ang-1, and Ang-2 [7]. Moreover, inhibition of Shh in the ischemic hindlimb of mice through the use of a neutralizing antibody inhibits endogenous angiogenesis [8]. Hh is also required for normal angiogenesis in the murine yolk sac, as Ihh-/- mice can initiate vasculogenesis and hematopoiesis but are defective in vascular remodeling to form blood vessels [9]. In the murine cornea, fibroblasts were identified as the Shh-responsive cells, while endothelial cells in the corneal neovessels, as well as human umbilical vein endothelial cells and microvascular endothelial cells cultured in vitro, were unable to respond to Shh, even though they express the receptor Ptc-1 [7]. Misregulation of the Hh signaling pathway has been implicated in several different types of cancer, including basal cell carcinomas (BCCs), medullablastomas, and gliomas (reviewed in [10]. Mutations in Ptc-1, which lead to constitutive activation of the Hh pathway, are the underlying factor in basal cell nevus syndrome, a familial condition characterized by a predisposition to BCC development [11]. In addition, Gli-1 was originally identified as a gene overexpressed in a human glioma line [12]. Recently, it was reported that Hh pathway activity is upregulated in digestive tract, pancreatic, and small-cell lung tumors and is required for the growth of these tumors [13-15]. Here we show that overexpression of HIP in cultured mouse Leydig cells inhibits the ability of these cells to respond to exogenous Shh, confirming that HIP can block Hh signaling when expressed in the same cells as the receptor Ptc-1. We have also shown that HIP is expressed predominantly in vascular endothelial cells and is downregulated in HUVEC during in vitro angiogenesis. Strikingly, HIP mRNA levels were decreased or even absent in several types of human tumors, as well as in highly vascularized human tumors grown in nude mice. Taken together, these results indicate a correlation between angiogenesis and decreased expression of HIP, lending support to the role of HIP as a naturally occurring regulator of Hh signaling and neovessel formation in the adult organism. Methods Conditioned media production Adherent cultures of 293-EBNA cells were transfected with a Shh-pCDNA3 expression plasmid kindly provided by Dr. Pao-Tien Chuang (UCSF) using LipofectAMINE 2000 and OptiMem reduced-serum medium (Invitrogen). Three to five hours after transfection, cells were re-fed with serum-containing growth medium, and three or four days later conditioned medium was collected and filtered through 0.2-μm cellulose acetate filters. Endothelial cell tube formation assays For RNA isolation, human aortic endothelial cells (HAEC) were cultured in 12-well plates with or without Matrigel (BD Biosciences) for 16 hours, then cells were lysed, digested with proteinase K, and total RNA was extracted using the RNeasy Mini Kit (Qiagen). HIP expression was assayed by real-time quantitative reverse transcriptase PCR (Q-RT-PCR) and normalized to 18s rRNA (see below). Transfection and conditioned medium assays The mouse testicular epithelial cell line, TM3, was grown in complete medium (DMEM/F12 with 2.5% FBS, 5% horse serum, 15 mM Hepes, and 4 mM glutamine) at 37°C and 5% CO2. Cells were transfected in 12-well or 24-well plates at 80–90% confluence using LipofectAMINE 2000 transfection reagent and OptiMem reduced-serum medium (Invitrogen). For conditioned medium (CM) assays, cells were transfected with the plasmid MycHIP-pCDNA3 (gift of Dr. Pao-Tien Chuang, UCSF), which encodes full-length mouse HIP, or empty vector (mock). Mock or Shh CM was added to cells 36 hours after transfection, then RNA was harvested 36–48 hours later. Collection of mouse tissues PC3 human prostate cancer cells were implanted subcutaneously into male nude mice (strain nu/nu), and on day 39 the resulting tumors were excised, weighed, placed on dry ice, then stored at -80°C prior to RNA isolation. A549 lung cancer cells were implanted subcutaneously into female nude mice and harvested as described above on day 9. Normal livers or skin from 5- to 6-week-old male or 4- to 5-week-old female nude mice were excised immediately following cervical dislocation and frozen in liquid nitrogen, then stored at -80°C. RNA isolation RNA was extracted from cultured cells using the RNeasy kit (Qiagen), including on-column DNase I digestion, according to the manufacturer's instructions. To extract RNA from frozen normal mouse tissues and tumors, hard-frozen tissue samples of 100–300 μg were pulverized over dry ice, placed in denaturing buffer, and disrupted using a Mixer Mill (Retsch) or rotor-stator homogenizer (Omni International). RNA was isolated from the homogenate either by phenol-chloroform extraction followed by precipitation with isopropanol and washing with 70% ethanol (ToTALLY RNA kit, Ambion) or by using the RNeasy kit (Qiagen). All RNA samples were treated with DNase I (Qiagen) prior to quantitative RT-PCR. Concentrations of RNAs were determined by reading the absorbance at 260 nm. Quantitative RT-PCR Quantitative RT-PCR was performed using the ABI PRISM™ 7700 Sequence Detection System and TaqMan™ chemistry. Reactions contained 0.3 μM of each primer, 0.1 μM probe, 0.25 U/ml MultiScribe reverse transcriptase, 0.4 U/ml RNase inhibitor, and 1x PCR master mix (Applied Biosystems). Primers and probes used to quantitate the mRNA level of various genes were as follows: mouse Ptc-1 (forward primer 5'GCCAATGGCCTAAACCGACT, reverse primer 5'AAACCGGACGACACTTGGAG, probe 5'6FAM-CCCACTCCTTCGCCTGAGCCG-TAMRA), mouse HIP (forward primer 5'CCACTGACCTCCGATTGCTC, reverse primer 5'TGCAGCAGCACTTGCCAG, probe 5'6FAM-CGGCTCTGTCGAAACGGCTACTACACC-TAMRA), mouse vWF (forward primer 5'CCGGAAGCGACCCTCAGA, reverse primer 5'CGGTCAATTTTGCCAAAGATCT, probe 5'6FAM-TGGCCTCTACCAGTGAGGTTTTGAAGTACACAC-TAMRA), mouse CD146 (forward primer 5'GGGCCTCAGGCAACTTCA, reverse primer 5'TTGGTGCACACGGAAAATCA, probe 5'6FAM-CTCCTTGTGAATCAAAAACCAGTCCACTTGG-TAMRA), human HIP (forward primer 5'CCCACACTTCAACAGCACCA, reverse primer 5'GCACATCTGCCTGGATCGT, probe 5'6FAM-CCCCGAAGTGTTTGCTCATGGGCT-TAMRA), human vWF (forward primer 5'TGAAGTATGCGGGCAGCC, reverse primer 5'GCGGTCGATCTTGCTGAAG, probe 5'6FAM-CCTCCACCAGCGAGGTCTTGAAATACAC-TAMRA). Mouse and human GAPDH and ribosomal RNA probe and primer sets were purchased from Applied Biosystems. RNA quantities were determined from a standard curve of serially diluted total cellular or tissue RNA run in parallel with each set of reactions. Standard curves had a slope between -3.1 and -3.3 and correlation coefficients of 0.98 or greater. Western blot Total cell lysates were made by lysing cells in 2% SDS, 10% glycerol, and 0.063 M Tris-HCl (pH 6.8) containing a cocktail of protease inhibitors (Pierce). Lysates were heat denatured at 95°C for 10 minutes, passed through a 26G needle to shear DNA, and centrifuged at 10,000 g for 30 minutes to remove insoluble material. Protein concentrations were determined using the BCA assay (Pierce). Conditioned medium samples were diluted 1:1 in Laemmli sample buffer (Sigma). 50 μg of cell lysate or 20 μl of conditioned medium/sample buffer was resolved on 4–15% polyacrylamide gels (BMA) and transferred to nitrocellulose (Invitrogen). Even loading of protein samples was verified by staining with Ponceau S. After overnight blocking with 5% nonfat dry milk, 0.05% Tween-20 in phosphate-buffered saline, blots were incubated with antibodies against Shh (sc-1194, Santa Cruz), Myc tag (Invitrogen), or HIP (provided by Dr. Pao-Tien Chuang, UCSF). Antibody-antigen complexes were visualized using horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoLabs, Santa Cruz) and SuperSignal West Pico chemiluminescent substrate (Pierce). Hybridization of RNA probes to cancer array Linearized templates containing a 716-bp fragment of human HIP cDNA (bases 521–1236 of human HIP coding sequence) or a 777-bp fragment of human vWF cDNA (bases 7301–8077 of human vWF coding sequence) were radiolabelled with 32P-dUTP by in vitro transcription using the MAXIscript kit (Ambion). The array was pre-hybridized in Clontech ExpressHyb solution with 100 μg/ml boiled, sheared salmon testis DNA (SS-DNA) for 2–12 hours at 68°C with rotation. Pre-hybridization solution was replaced with fresh hybridization solution with SS-DNA, plus a total of 1.5–1.7 × 107 cpm of 32P-labelled HIP or vWF probe. Hybridization of the probe proceeded for 16–18 hours at 68°C, followed by four washes in 2X SSC/0.5% SDS at 68°C, one wash in 0.2X SSC/0.5% SDS at 68°C, and one wash in 2X SSC at room temperature. The array was exposed to a phosphor-screen for 24–72 hours and scanned in a Storm phosphor-imager at a resolution of 50 μm. After hybridization to the HIP probe, the same array was stripped by boiling in 0.5% SDS and re-scanned to verify the absence of residual probe before hybridization to the vWF probe. Relative levels of HIP and vWF expression were determined using ImageQuant software (Molecular Dynamics). Statistical analysis All results are expressed as mean ± standard deviation unless otherwise noted. Statistical significance of differences was determined using a two-tailed Student's t-test. Results HIP is highly expressed in endothelial cells Ptc-1 and Smo mRNA are expressed in human aortic endothelial cells (HAEC) at levels comparable to that of GAPDH, as determined by comparing the threshold cycle numbers obtained by quantitative RT-PCR (Q-RT-PCR) (data not shown). Thus HAEC express components of the Hh pathway that are known to mediate a response to Hh ligands. However, when HAEC or human umbilical vein endothelial cells (HUVEC) were treated with conditioned medium containing functional Shh, the Hh pathway was not activated as determined by measuring the levels of mRNA for Ptc-1 and Gli-1, two genes known to be responsive to Hh signaling (data not shown). Shh mRNA levels in HAEC and HUVEC were near the limits of detection by the Q-RT-PCR assay and could therefore be estimated at less than 10-3 copies per cell based on the typical sensitivity of this assay. Hedgehog-interacting protein (HIP) binds Hh proteins with an affinity equal to that of Ptc-1 and can function as an antagonist of Ihh signaling in vivo [4]. Thus we hypothesized that HIP may be expressed in EC and function to block Hh signaling. Indeed, we found that HAEC and HDMEC expressed amounts of HIP mRNA similar to that of GAPDH, as measured by Q-RT-PCR. Endothelial cell-predominant expression of HIP was demonstrated by Q-RT-PCR analysis of a variety of human and mouse primary cell strains and immortalized cell lines and human cancer cell lines representing different tissues. When normalized to the level of GAPDH mRNA, the level of HIP mRNA was 100- to 10,000-fold higher in vascular endothelial cells than in the other cell types examined (Figure 1). Expression of HIP protein in HAEC, but not in ZR75-1 or HT-29, was confirmed by Western blot (Figure 1, inset). The observation that HIP mRNA is expressed at a high level in human endothelial cells suggests that HIP may inhibit Hh signaling in these cells and may explain why these cells are unresponsive to Shh. HIP inhibits Ptc induction by Shh Conditioned media from 293 cells co-transfected with Shh and full-length (membrane-anchored) HIP contained a reduced amount of Shh and had a reduced ability to induce differentiation in C3H10T1/2 cells compared to cells transfected with Shh alone, demonstrating that HIP can sequester Shh [5]. However, there is no direct evidence that cells in which HIP is overexpressed are less responsive to Shh. In order to test this directly, 293-EBNA cells were transfected with a Shh expression plasmid. Western blot analysis demonstrated the presence of Shh protein in the conditioned medium from these cells (Figure 2, inset left). Exposure of TM3 mouse Leydig cells, a cell line with no detectable endogenous HIP mRNA (Figure 1), to increasing amounts of conditioned media containing Shh resulted in induction of the Shh pathway, as measured by a dose-dependent increase in the amount of endogenous Ptc-1 mRNA (Figure 2). When TM3 cells were transfected with a plasmid expressing full-length, N-terminally Myc-tagged mouse HIP, the induction of Ptc-1 mRNA by Shh was reduced by 79 percent (p < 0.05; Fig. 2), demonstrating that HIP functions as an inhibitor of the Shh pathway when expressed in the responding cells. Expression of HIP in transfected TM3 cells was verified by Western blot using a Myc antibody (Fig. 2, inset right). HIP is downregulated in endothelial cells during tube formation in vitro Shh has been shown to induce angiogenesis [7], and HIP is able to antagonize the Shh pathway (Figure 2). Therefore, we tested whether HIP expression is downregulated in EC during tube formation, an essential step in the angiogenic process. Expression of HIP mRNA in HAEC that had formed tubes on Matrigel as measured by Q-RT-PCR was 2.9-fold lower (p < 0.05) than in the same cells cultured in standard plastic dishes (Figure 3A). To investigate whether there was a coordinated downregulation of Hh-regulated genes during tube formation on Matrigel, the mRNA level of Ptc-1, a known Hh-responsive gene, was measured. In contrast to HIP, levels of Ptc-1 mRNA increased 2.5-fold (p < 0.05) in cells on Matrigel compared to plastic (Figure 3B). HIP is downregulated in human tumors compared to normal tissues Angiogenesis is a common feature of tumor growth (reviewed in [16,17]. It is conceivable that HIP is downregulated in this process, similar to the reduction we observed in endothelial cells during tube formation. Therefore, we measured HIP mRNA levels in a variety of tumor samples. Tumor and corresponding normal tissue RNA samples (normal and tumor tissues were from different individuals) were obtained from two different commercial sources (Ambion and BioChain), and the endothelial cell marker vWF was used to normalize HIP expression to the number of endothelial cells present in the tumors. In all tissue and tumor RNA samples assayed, vWF mRNA was detectable. In both pairs of liver tissue examined, HIP mRNA was expressed in normal tissue but was undetectable in the tumor (Table 1). One of two kidney tumor-normal pairs showed no difference in normalized HIP expression, while the other showed a 5.9-fold reduction of normalized HIP in tumor relative to normal tissue. Of the two breast tissue pairs, one had undetectable levels of HIP in both normal and tumor tissues (data not shown), while the other showed a 3.7-fold decrease in HIP expression in tumor compared to normal. In one lung tissue pair, vWF levels in normal tissue were over 900-fold lower than in the tumor, such that normalization of HIP to vWF resulted in an apparent 30-fold increase in HIP in tumor compared to normal tissue. In the other lung tissue pair, normalized HIP was 8.1-fold lower in tumor than in normal tissue. To further investigate the frequency of the change in HIP expression in human tumors, a cancer profiling array (Clontech) containing cDNA from 154 tumor and corresponding normal tissues from individual patients was used to compare the expression of HIP in a larger set of samples. A 32P-labelled HIP probe corresponding to bases 521–1236 of human HIP cDNA was hybridized to the array, and relative levels of HIP expression were quantitated on a phosphoimager. HIP expression levels were normalized to the expression of the endothelial marker vWF, by re-probing the same array with a fragment of the human vWF cDNA, as different tumors and normal tissues may contain different degrees of vascularization. In liver, stomach, colon, and rectum, all or most of the paired samples showed a reduction of HIP in tumor compared to normal tissue (Table 2). In total, 28 samples had a decrease in the tumor, while only 3 samples showed a slight increase (1.3- to 1.7-fold), and 2 samples showed no change. The lung samples did not show a consistent patten of HIP mRNA expression. Five had a decrease of HIP mRNA in the tumor, while four had an increase and one showed no change. The remaining sets of paired normal and tumor tissues, including samples from breast, ovary, vulva, uterus, cervix, prostate, testis, thyroid, skin, bladder, small intestine, and pancreas, showed no marked difference in HIP expression between normal and tumor, or had undetectable HIP expression in both normal and tumor. HIP expression is very low or undetectable in xenograft tumors Mouse xenograft tumors resulting from transplantation of PC3 (human prostate cancer cell line) or A549 (human lung cancer cell line) cells into nude mice are known to have high levels of neovascularization as a result of angiogenesis, which is required for their continued growth [18,19], so we hypothesized that HIP expression would be lower in these tumors than in normal tissues that also contain normal vessels. RNA extracted from PC3 or A549 tumors weighing an average of 350 mg that were excised 39 (PC3) or 9 (A549) days after subcutaneous implantation, or from liver or skin from non-implanted nude mice, was assayed for murine HIP, vWF, CD146, and GAPDH mRNA by Q-RT-PCR. The Q-RT-PCR assay for murine HIP is species-specific, i.e. does not detect human HIP mRNA (data not shown). Therefore, any HIP mRNA detected using this assay must be from host mouse cells present in the tumor, not from the human tumor cells themselves. Murine rather than human HIP was assayed because tumors have been shown to incorporate vasculature from the surrounding vessels [20-22] and there is evidence that tumors can recruit endothelial and hematopoietic precursor cells [23] to form their own vasculature. RNA from tumors and normal liver and skin contained measurable amounts of the murine endothelial cell markers vWF and CD146, indicating the presence of endothelial cells (Figure 4). For the PC3 xenograft experiment, the levels of vWF and CD146 mRNA normalized to GAPDH were actually higher in the tumors than in the normal liver, consistent with the highly vascularized nature of these tumors. In contrast, mouse HIP mRNA was abundant in all liver and skin samples examined but was 16- to 30-fold lower in A549 tumors and undetectable in any of the PC3 tumor samples (Figure 4A). These data demonstrate that although the tumors contained endothelial cells, they expressed markedly reduced or undetectable amounts of HIP mRNA. Discussion Our demonstration that HIP functions to inhibit Ptc-1 upregulation in mouse testicular epithelial cells (TM3) exposed to Shh-containing conditioned media is the first direct evidence to date that Shh signaling is attenuated in cells that express full-length, membrane-bound HIP. The decrease in Ptc-1 induction caused by transfection with HIP is consistent with previous reports indicating that HIP can bind and reduce the availability of Shh and presumably prevent it from signaling through Ptc-1 [4,5]. In human aortic endothelial cells (HAEC), which express high levels of HIP, the addition of Shh-containing conditioned medium did not cause induction of Ptc-1, even though these cells express the receptor components Ptc-1 and Smo. In addition, others have shown that although Shh induces angiogenesis in a mouse model of hindlimb ischemia, it has no effect on endothelial cell proliferation or migration in cell culture [7]. Our finding that vascular EC express abundant amounts of HIP mRNA may explain the inability of these cells to respond to Shh. An analysis of various human cell lines and primary cells indicated that HIP is absent or expressed at low levels in other cell types, suggesting that in adults HIP is expressed primarily in EC. These results are supported by gene chip data analysis of more than 30 normal human tissues showing that HIP is most highly expressed in blood vessels or in vascular-rich tissues such as liver, lung, brain, and pancreas (data not shown). These results suggest a role for HIP in the normal function of blood vessels. Several lines of evidence support the hypothesis that HIP expression is decreased in EC participating in angiogenesis. Firstly, we describe that HIP is downregulated in HAEC forming tubes on Matrigel. In contrast, Ptc-1 mRNA levels were increased in EC forming tubes on Matrigel, suggesting that the decrease in HIP mRNA under these conditions does not reflect a general downregulation of Hh-responsive genes. Given that transcription of HIP and Ptc-1 are both activated by Hh signaling, it is likely that the decrease in HIP expression in EC on Matrigel is mediated by a pathway independent of Hh signaling. Secondly, we have observed that HIP mRNA levels are decreased in human tumors and xenograft human tumors grown in mice, situations in which angiogenesis occurs. Previous studies have demonstrated a requirement for Shh in angiogenesis in the ischemic mouse limb and embryonic yolk sac [8,9]. The pro-angiogenic activity of exogenously supplied Shh protein has been attributed to the induction of factors such as VEGF and angiopoietin in fibroblasts [7]. Our data suggest the possibility that in situations where HIP expression is downregulated, Hh may also act directly on endothelial cells. The Shh pathway has been implicated in several different types of cancer, where upregulation of signaling, associated with mutations in Patched or amplification of Gli, is often the suspected cause of malignancy [11,12,24-26]. In at least one type of human cancer, basal cell carcinoma (BCC), HIP upregulation has been demonstrated [27,28], probably as a result of dysregulation of the Shh signaling pathway. More recently it has been reported that Hh pathway activity is activated in a wide variety of digestive tract tumors by overexpression of the Hh ligand, and is essential for tumor growth [13]. Aberrant expression of Shh and activation of Hh signaling was also demonstrated to occur in pancreatic cancer [14] and small cell lung cancer [15]. Hh ligands secreted by tumor cells have the potential to induce Hh pathway activation in nearby non-tumor cells, including fibroblasts and endothelial cells. Our observation that HIP mRNA expression decreases in several human tumor types relative to normal tissues suggests that modulation of HIP expression may also promote increased Hh signaling in certain tumors and contribute to cancer progression. However, since expression of HIP is known to be induced by activation of the Hh pathway, an alternative interpretation is that a reduced level of HIP mRNA in tumors is indicative of reduced Hh signaling, as suggested by Hu et al. [29]. Our finding that, in endothelial cells in Matrigel, HIP mRNA levels decrease while Ptc-1 levels increase demonstrates that a mechanism other than Hh signaling can modulate the expression of these Hh target genes. Thus, the down-regulation of HIP in tumors may be unrelated to Hh pathway activity. The precise mechanism of HIP regulation by the Hh pathway in the context of tumors remains to be elucidated by future studies. Among the tumor types represented on the Clontech Cancer Profiling Array, liver and several areas of the digestive tract (stomach, colon, rectum) exhibited consistent downregulation of HIP in the tumor relative to normal tissue. Tumors from other tissues, including breast, testis, and kidney, did not show a significant change in HIP expression from the normal tissue, suggesting that this phenomenon may be restricted to certain tumor types. Differences in the degree of vascularization of the various tumors may also explain why a decrease in HIP was not seen in all tumor types. Xenotransplantation of PC3 or A549 cells into immunocompromised mice is a commonly used animal model for prostate or lung cancer, respectively. Extensive neovascularization occurs in the resulting tumors, and it has been demonstrated that angiogenesis inhibitors slow their growth [30,31]. The discovery that vWF-normalized mouse HIP mRNA levels were markedly decreased in A549 tumors and undetectable in all of the PC3 tumors we examined gives further weight to the hypothesis that decreased HIP expression is associated with tumor angiogenesis. Angiogenesis has been well-characterized as a prerequisite to continued tumor growth and metastasis [16,17,32], and many approaches have been developed to attempt to control the progression of cancer by inhibiting angiogenesis [32,33]. A more complete analysis of the gene expression changes in endothelial cells during developmental and pathological processes should help to identify additional signaling pathways that are altered and that could be manipulated to control angiogenesis and disease progression. The identification of Shh as an angiogenic signaling molecule [7], along with the characterization of HIP as an inhibitor of Shh that is downregulated in certain tumors, strongly suggests that inhibition of the Shh pathway may be of therapeutic use in various cancers that rely on angiogenesis for their continued growth. Conclusions We have shown that HIP is expressed predominantly in EC but at low or undetectable levels in other cell types, and that the high expression of HIP in EC may be responsible for their inability to respond to Shh. Expression of HIP in TM3 cells attenuated their response to Shh, demonstrating that HIP antagonizes Hh signaling when expressed in the responding cell. The reduction of HIP in a variety of human tumor samples as well as PC3 and A549 xenograft tumors, and in EC undergoing tube formation on Matrigel, suggests that modulation of HIP may play a role in tumor angiogenesis. Competing interests None declared. Authors' contributions CLO participated in experimental design and carried out CM production and assays, tissue and RNA processing, Q-RT-PCR, Western blot, and cancer array hybridization. PPH participated in the design of the study and performed tube formation assays, tissue and RNA processing, Q-RT-PCR, and Western blot. JG participated in initial characterization of HIP and provided supporting data. GMR was involved in the planning and organization of the study and reviewed the manuscript. ARB initiated, planned, and supervised the entire study. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements We are grateful to Dr. Pao-Tien Chuang for providing HIP expression vectors and helpful advice. We thank Ying Zhu and Wei Xia for help with protein expression, Bruno Alicke for providing mouse tissues, and Rhonda Humm, Eileen Paolo-Chrisco, and Jean MacRobbie for cell culture assistance. We also thank Rick Harkins for helpful discussions on the research and the manuscript. Figures and Tables Figure 1 Comparison of HIP expression in different cell types. Total RNA from each cell type was assayed for HIP and GAPDH mRNA levels by Q-RT-PCR. Relative HIP mRNA expression was normalized to GAPDH and plotted on a log scale. Open bars, immortalized or cancer cell lines; filled bars, normal cells. TM3, mouse testicular epithelial cell line; ZR75-1, human breast cancer cell line; HT-29, human colon cancer cell line; HepG2, human liver cancer cell line; HEK293, human kidney cancer cell line; 1321N1, human brain cancer cell line; HeLa, human cervical cancer cell line; AMM, activated mouse macrophage; HMEC, normal human mammary epithelial cells; WI-38, normal human lung fibroblasts; HuSKMC, normal human embryonic skeletal muscle cells; HDMEC, normal human dermal microvascular endothelial cells; HAEC A and HAEC B, normal human aortic endothelial cells from two different donors (obtained from Cascade Biologics and Clonetics, respectively). Figure 2 Expression of HIP in responding cells blocks Ptc-1 induction by Shh. Untransfected mouse TM3 cells (first three pairs of columns) or TM3 cells transfected with empty vector or a vector expressing mouse HIP (last two pairs of columns) were exposed to Shh conditioned media at the indicated concentrations (% v/v in normal growth media) for two days, after which Ptc and GAPDH mRNA levels were measured by Q-RT-PCR. Ptc-1 mRNA level was then normalized to that of GAPDH. Values shown for Shh conditioned media treatment (filled bars) are normalized to those obtained with mock conditioned media (open bars). Results shown are the mean of three experiments performed in duplicate. Error bars show standard deviation. The decrease in normalized Ptc mRNA level for mock- vs. Shh-treated (*) and for HIP- vs. vector-transfected (+) cells was statistically significant (p < 0.005, two-tailed Student's t-test). Left inset, Western blot showing Shh protein expression in conditioned media from Shh-transfected 293-EBNA cells. Right inset, Western blot showing HIP protein expression in conditioned media from HIP-transfected TM3 cells. Figure 3 HIP mRNA expression under in vitro angiogenic conditions. A, HIP or B, Ptc-1 expression in HAEC cultured on plastic (control, filled bar) or on Matrigel (open bar). RNA was harvested from cells 16 hours after seeding on plastic or Matrigel, when tubes had fully formed on Matrigel, and assayed for HIP and 18s rRNA, or Ptc-1 and GAPDH mRNA, by Q-RT-PCR. The mean data are plotted as relative HIP mRNA normalized to 18s rRNA, or Ptc-1 mRNA normalized to GAPDH mRNA, and are the average of three (HIP) or two (Ptc-1) independent experiments performed in triplicate. Error bars show standard deviation. Differences in normalized HIP or Ptc-1 expression were significant for control vs. Matrigel (*p < 0.05, two-tailed Student's t-test). Figure 4 HIP mRNA expression in normal mouse tissues vs. PC3 and A549 tumors. 100 ng of RNA extracted from A, normal livers or PC3 human prostate xenograft tumors from nude mice, or B, normal livers, normal skin, or A549 tumors from nude mice, were assayed for vWF, CD146, and HIP mRNA expression by Q-RT-PCR. The vWF (white bars), CD146 (gray bars), and HIP (black bars) mRNA levels were normalized to the GAPDH mRNA level in each RNA sample and are presented as the mean with standard deviation (A, N = 3 for normal liver, N = 8 for PC3 tumor; B, N = 5 for normal liver, N = 5 for normal skin, N = 3 for A549 tumor). nd = not detectable (no signal from Q-RT-PCR assay after 40 cycles). Table 1 HIP mRNA expression in human normal vs. tumor tissues. HIP/vWF* Tissue Normal Tumor Tumor/normal Liver 1 0.107 0 0 Liver 2 2.357 0 0 Kidney 1 0.306 0.051 0.17 Kidney 2 0.272 0.299 1.10 Breast 0.051 0.014 0.27 Lung 1 0.018 0.002 0.11 Lung 2 0.026 0.779 29.97 *Values are HIP mRNA expression normalized to that of vWF. Table 2 Fold change in HIP for human tumor vs. normal tissues. Tissue Sex Race Age Tumor additional information HIP tumor/normal* Liver M C 59 Primary cancer of the liver 0.41 M C 36 Primary cancer of the liver 0.06 M C 58 Primary cancer of the liver 0.03 HIP decreases in 3/3 Stomach M C 63 Adenocarcinoma 0.16 M C 57 Gastric cancer 1.61 M C 47 Gastric cancer 0.24 M C 65 Adenocarcinoma 0.21 M C 61 Adenocarcinoma 0.24 M C 50 Mucinous adenocarcinoma 0.53 F C 72 Adenocarcinoma 0.68 F C 67 Adenocarcinoma 0.37 M C 75 Adenocarcinoma 0.03 F C 68 Adenocarcinoma 0.21 HIP decreases in 9/10 Colon F C 67 Adenocarcinoma 0.25 F C 58 Tubulovillous adenoma 0.23 F C 43 Adenocarcinoma 0.85 F C 69 Adenocarcinoma 1.34 F C 35 Adenocarcinoma 0.41 M C 58 Adenocarcinoma 0.4 M C 63 Adenocarcinoma 0.24 F C 73 Adenocarcinoma 0.16 F C 65 Adenocarcinoma 1.68 F C 65 Adenocarcinoma 0.14 HIP decreases in 8/10 Rectum F A 44 Mucinous adenocarcinoma 0.25 M C 68 Adenocarcinoma 1.1 F C 49 Adenocarcinoma 0.18 F C 59 Adenocarcinoma 0.22 F C 53 Mucinous adenocarcinoma 0.08 F C 70 Adenocarcinoma 1.05 M C 42 Adenocarcinoma 0.79 F C 71 Adenocarcinoma 0.59 F C 59 Adenocarcinoma 0.15 M C 43 Adenocarcinoma 0.56 HIP decreases in 8/10 Lung F C 69 Bronchiolo-alveolar adenocarcinoma 6.45 M C 72 Central cancer of the right lung 0.36 M C 66 Central cancer of the middle lobe of rt lung 0.23 M C 51 Peripheral cancer of the upper lobe of rt lung 0.45 M C 63 Squamous cell carcinoma, keratinizing type 5.49 M C 62 Squamous cell carcinoma, keratinizing type 0.19 M C 67 Squamous cell carcinoma 0.4 M C 71 Squamous cell carcinoma, keratinizing type 0.95 M C 65 Adenocarcinoma 4.9 M A 52 Squamous cell carcinoma 7.08 HIP decreases in 5/10 *HIP tumor/normal value is the vWF-normalized quantity of HIP for the tumor, divided by the vWF-normalized quantity of HIP for the corresponding normal tissue. ==== Refs Kalderon D Transducing the hedgehog signal Cell 2000 103 371 374 11081624 10.1016/S0092-8674(00)00129-X Nybakken K Perrimon N Hedgehog signal transduction: recent findings Curr Opin 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12615704 Fernandez A Udagawa T Schwesinger C Beecken WD Achilles-Gerte E McDonnell TJ D'Amato RJ Angiogenic potential of prostate carcinoma cells overexpressing bcl-2 J Natl Cancer I 2001 93 208 212 10.1093/jnci/93.3.208 Gavish Z Pinthus JH Barak V Ramon J Nagler A Eshhar Z Pines M Growth inhibitionof prostate cancer xenografts by halofuginone The Prostate 2002 51 73 83 11948962 10.1002/pros.10059 Carmeliet P Jain RK Angiogenesis in cancer and other diseases Nature 2000 407 246 257 10.1038/35025220 Scappaticci FA The therapeutic potential of novel antiangiogenic therapies Expert Opin Inv Drugs 2003 12 923 932 10.1517/eoid.12.6.923.21795

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==== Front BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-4-441529871610.1186/1471-2407-4-44Research ArticleThe oncogenic fusion protein RUNX1-CBFA2T1 supports proliferation and inhibits senescence in t(8;21)-positive leukaemic cells Martinez Natalia [email protected] Bettina [email protected] Heidemarie [email protected] Claire [email protected] Hans-Peter [email protected] Arnold [email protected] Gerhard [email protected] Alfred [email protected] Jürgen [email protected] Olaf [email protected] Department of Molecular Biology, Institute for Cell Biology, Faculty of Biology, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany2 Department of Haematology, Haemostaseology and Oncology, Hannover Medical School, 30625 Hannover, Germany3 Alnylam Europe AG, Fritz-Hornschuch-Str. 9, 95326 Kulmbach, Germany2004 6 8 2004 4 44 44 9 6 2004 6 8 2004 Copyright © 2004 Martinez et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The fusion protein RUNX1-CBFA2T1 associated with t(8;21)-positive acute myeloid leukaemia is a potent inhibitor of haematopoetic differentiation. The role of RUNX1-CBFA2T1 in leukaemic cell proliferation is less clear. We examined the consequences of siRNA-mediated RUNX1-CBFA2T1 depletion regarding proliferation and clonogenicity of t(8;21)-positive cell lines. Methods The t(8;21)-positive cell line Kasumi-1 was electroporated with RUNX1-CBFA2T1 or control siRNAs followed by analysis of proliferation, colony formation, cell cycle distribution, apoptosis and senescence. Results Electroporation of Kasumi-1 cells with RUNX1-CBFA2T1 siRNAs, but not with control siRNAs, resulted in RUNX1-CBFA2T1 suppression which lasted for at least 5 days. A single electroporation with RUNX1-CBFA2T1 siRNA severely diminished the clonogenicity of Kasumi-1 cells. Prolonged RUNX1-CBFA2T1 depletion inhibited proliferation in suspension culture and G1-S transition during the cell cycle, diminished the number of apoptotic cells, but induced cellular senescence. The addition of haematopoetic growth factors could not rescue RUNX1-CBFA2T1-depleted cells from senescence, and could only partially restore their clonogenicity. Conclusions RUNX1-CBFA2T1 supports the proliferation and expansion of t(8;21)-positive leukaemic cells by preventing cellular senescence. These findings suggest a central role of RUNX1-CBFA2T1 in the maintenance of the leukaemia. Therefore, RUNX1-CBFA2T1 is a promising and leukaemia-specific target for molecularly defined therapeutic approaches. ==== Body Background The chromosomal translocation t(8;21) (q22;q22), which is associated with 10–15% of all cases of acute myeloid leukaemia, fuses the DNA binding domain of the transcription factor RUNX1 (also called AML1 or CBFα) to the almost complete open reading frame of CBFA2T1 (also named MTG8 or ETO) [1,2]. The resulting fusion protein RUNX1-CBFA2T1 (AML1/MTG8, AML1/ETO) interferes with haematopoetic gene expression by recruiting histone deacetylases via N-CoR and mSin3 to promoters, thereby inhibiting the transcription of the respective target gene [3-7]. Moreover, by directly binding to and sequestering transcription factors, such as SMAD3, C/EBPα or vitamin D receptor, RUNX1-CBFA2T1 interferes with signal transduction pathways controlling differentiation and proliferation [8-12]. Consequently, RUNX1-CBFA2T1 blocks myeloid differentiation and promotes self-renewal of haematopoetic progenitors [13-16]. The influence of RUNX1-CBFA2T1 on the control of proliferation and apoptosis is less clear. On the one hand, its ectopic expression in several cell types, including leukaemic cell lines such as U937, inhibits proliferation and enhances apoptosis [13]. On the other hand, RUNX1-CBFA2T1 may interfere with p53-dependent cell cycle arrest and apoptosis by suppressing the p53-stabilizing protein p14ARF [17]. RUNX1-CBFA2T1 expression supports the expansion of haematopoetic progenitor cells, which has been mainly attributed to its anti-differentiation capabilities, but which may also depend on a proliferation supporting activity of RUNX1-CBFA2T1 [18-21]. RUNX1-CBFA2T1 alone is not sufficient to cause leukaemia [22,23]. Instead, secondary mutations have to be acquired in addition to RUNX1-CBFA2T1 to induce leukaemia [24-27]. Cellular senescence limits the proliferative capacity of cells and is characterized by an irreversible G1 arrest [28]. Senescent cells cannot be stimulated with mitogens to enter the S phase of the cell cycle. Nevertheless, senescent cells are still viable and metabolically active [29]. They can be distinguished from non-senescent cells by the expression of senescence-associated β-galactosidase activity, which can be detected at slightly acidic pH [30]. In the case of replicative senescence, cells enter G1 arrest after the telomeres have shortened below a critical length [29]. After exposure to stresses, cells may also undergo stress-induced senescence instead of apoptosis or transient cell cycle arrest [28]. The molecular mechanisms of senescence are still very incompletely understood. However, several regulators of cell cycle progression such as pRb, p53 or the cdk inhibitors p16Ink4a or p27Kip1 are involved in the establishment of senescence [31]. Furthermore, overexpression of oncogenic H-Ras in murine embryonic fibroblasts (MEFs) induce premature senescence in a PML-dependent fashion [32,33]. Similarly, overexpression of RUNX1 in MEFs induces senescence likely by upregulating p19Arf [17]. However, control of senescence by an endogenously expressed oncogene such as RUNX1-CBFA2T1 in t(8;21)-positive leukaemic cells has not been shown yet. The specific inhibition of gene expression by small interfering RNAs (siRNAs) provides a new approach to investigate the functions of oncogenes in the development of cancer, thereby complementing other approaches such as ectopic (over-) expression [34-36]. We and others have used siRNAs to specifically down-modulate leukaemic fusion genes such as BCR-ABL or RUNX1-CBFA2T1 [14,37-39]. We have shown that the siRNA-mediated depletion of RUNX1-CBFA2T1 led to a sensitization towards myeloid differentiation inducing agents such as TGFβ and vitamin D3 [14]. Here, we report the consequences of RUNX1-CBFA2T1 depletion on CD34 expression (a surface marker, indicator of differentiation), for the proliferation and clonogenicity of in t(8;21)-positive leukaemic cell lines. We demonstrate that RUNX1-CBFA2T1 supports the clonogenicity and proliferation of t(8;21) positive Kasumi-1 cells by interfering with the establishment of cellular senescence. Methods siRNAs The siRNAs siAGF1 and siAM targeting the fusion site of the RUNX1-CBFA2T1 mRNA, the mismatch control siAGF6, and the unrelated controls targeting luciferase (siGL2) (for sequences see ref. 13) or MLL-AF4 (siMLL14; sense 5'-AAA CCA AAA GAA AAG CAG ACC-3', antisense 5'-GGU CUG CUU UUC UUU UGG UUU UU-3') were synthesized by either Alnylam Europe AG (Kulmbach, Germany) or MWG Biotech (Ebersberg, Germany) and hybridized as described [14]. Cell culture and siRNA transfection The t(8;21)-positive acute myeloid leukaemia (AML) cell lines Kasumi-1 (Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ) No. ACC 220) [40] and SKNO-1 [41], and the t(8;21)-negative leukaemic lines MV4-11 (DSMZ No. ACC 102), NB4 (DSMZ No. ACC 207), HL60 (DSMZ No. ACC 3), U937 (DSMZ No. ACC 5) and K562 (DSMZ No. ACC 10) were cultivated and electroporated together with siRNAs as described [14,42,43]. Briefly, cells were electroporated in 100–500 μl culture medium at a density of 5 × 106/ml in 4 mm electroporation cuvettes. SiRNAs were added immediately before electroporation. If not otherwise indicated, the siRNA concentration during electroporation was 100 nM. Electroporations were performed using a rectangle pulse EPI 2500 electroporator (Fischer, Heidelberg, Germany; ). Parameters were 330 V and 10 ms (milliseconds) for Kasumi-1 cells, and 350 V and 10 ms (milliseconds) for all other cell lines. Fifteen minutes after electroporation, cells were diluted twenty fold in culture medium and incubated at 37°C, 5% CO2 and 92% humidity. Using this protocol, we routinely observe less than 10% of dead cells after electroporation with an siRNA transfection efficiency close to 100% [14,42,43]. Immunoblotting To obtain total cell lysates, cells were washed with phosphate-buffered saline, lysed in Urea lysis buffer (9 M urea, 4% (w/w) 3-[(3-Cholamidopropyl)-dimethylammonio]-propansulfonat (CHAPS), 1% (w/w) Dithiothreitol) and treated for 30 min on ice with 2.5 u/ml Benzonase (Merck, Darmstadt, Germany). Total (10–20 μg) and nuclear lysates (10 μg) were analyzed as described [14]. Band intensities were determined by scanning the exposed films followed by quantification using the Image Gauge 3.0 software (Fujifilm, Düsseldorf, Germany). The following antibodies were used for detection: AML1/RHD (2.5 mg/l; Oncogene Research PC285, Boston, USA); p27 C-19 (200 μg/l; Santa Cruz Biotechnology sc-9737; Heidelberg, Germany); Tubulin Ab-4 (1 mg/l, NeoMarkers MS-719-P0, Fremont, USA). Real-time RT-PCR Total RNA was isolated using the RNeasy Kit (Qiagen, Hilden, Germany). Reverse transcription was performed with random hexamers using MMLV-RT, RNase H- (Promega, Heidelberg, Germany) as suggested by the manufacturer. Real time PCRs were performed using Sybr-Green Mix (Applied Biosystems, Warrington, UK), 300 nM primers and 20% v/v diluted RT reaction mix using standard conditions on a 7000 Sequence detection system (Applied Biosystems, Foster City, CA, USA). The primer sequences for STAT1 (5'-CAT CAC ATT CAC ATG GGT GGA (forward primer) and 5'-GGT TCA ACC GCA TGG AAG TC (reverse primer)), for CD34 (5'-TCC AGA AAC GGC CAT TCA G (forward primer) and 5'-CCC ACC TAG CCG AGT CAC AA (reverse primer)), for G-CSF (5'-CCC ACC TTG GAC ACA CTG C (forward primer) and 5'-AGT TCT TCC ATC TGC TGC CAG (reverse primer)) and for GAPDH (5'-GAA GGT GAA GGT CGG AGT C (forward primer) and 5'-GAA GAT GGT GAT GGG ATT TC (reverse primer)) were designed with Primer-Express software (Applied Biosystems, Foster City, CA, USA). Proliferation, single cell expansion and colony formation assays To determine proliferation rates and doubling times, cells were counted using trypan blue. For single cell expansion assays, 24 h after electroporation cells were diluted to a density of 10 cells/ml and plated in 100 μl aliquots into 96 well tissue culture plates. Colony formation assays were performed 24 h after electroporation with a density of 5,000 cells/ml in semisolid medium (RPMI1640 containing 20% fetal calf serum and 0.5625% methyl cellulose). Colonies were counted 14 days after plating. The cytokine concentrations were 20 ng/ml for G-CSF and GM-CSF in the cell cycle and senescence assays, and 10 ng/ml for all growth factors in the colony formation assays. Cell cycle analysis Cell cycle analysis was performed as described [44]. Four days after electroporation, 106 cells were suspended in 200 μl citrate buffer (250 mM sucrose, 40 mM sodium citrate pH 7.6) followed by the addition of 800 μl staining solution (phosphate-buffered saline containing 20 mg/l propidium iodide, 0.5% (w/w) NonIdet P40, 500 μM EDTA) and 10 μl boiled RNase A (10 g/l). Cells were kept for 30 minutes on ice and analyzed by flow cytometry (FACSCalibur, Becton Dickinson, Heidelberg, Germany). Data analysis was performed with FCSPress 1.3 . Analysis of apoptosis and of CD34 expression For quantification of apoptotic cells, cells were stained with FITC-labeled annexin V as suggested by the supplier (Bender, Vienna, Austria). CD34 expression was monitored by staining with anti-human CD34-FITC (clone 581, BD Pharmingen #555388, Heidelberg, Germany). Analysis was performed by flow cytometry (FACSCalibur, Becton Dickinson, Heidelberg, Germany) using FCSPress 1.3 for data analysis. Results Time course of RUNX1-CBFA2T1 protein depletion by siRNAs Electroporation of t(8;21)-positive cells with the RUNX1-CBFA2T1 siRNA siAGF1, but not with the mismatch control siRNA siAGF6, resulted in a three- to fourfold reduction of RUNX1-CBFA2T1 fusion protein in both Kasumi-1 cells (fig. 1A, lanes 1–3) and SKNO-1 cells (fig. 1A, lanes 4–6). One day after electroporation with siAGF1, RUNX1-CBFA2T1 protein levels were already reduced by 75% (fig. 1B). This finding suggests that RUNX1-CBFA2T1 protein has a half-life of less than 24 hours. The depletion of RUNX1-CBFA2T1 lasted for at least 7 days with a slow recovery of RUNX1-CBFA2T1 protein levels from day 5 on (fig. 1B), in agreement with the previously reported time-course of siRNA-mediated RUNX1-CBFA2T1 mRNA reduction [14]. Transfection with siRNAs may induce an interferon response leading to non-specific effects on cellular processes such as proliferation or differentiation [45]. Since STAT1 is an interferon-stimulated gene, and since several siRNAs have been shown to induce STAT1 expression [45], we examined a possible induction of STAT1 gene expression by RUNX1-CBFA2T1 siRNAs or by mismatch control siRNAs in Kasumi-1 cells. Neither the electroporation with RUNX1-CBFA2T1 siRNA nor with the control siAGF6 caused a substantial change in STAT1 mRNA levels (data not shown), suggesting that these siRNAs do not induce an interferon response in Kasumi-1 cells. RUNX1-CBFA2T1 siRNAs decrease the clonogenic growth of Kasumi-1 cells In comparison to mock-transfected cells, electroporation with RUNX1-CBFA2T1 siRNAs resulted in a ten- to twenty fold decreased clonogenicity in both single cell expansion (data not shown) and colony formation assays (fig. 2). Electroporation with the mismatch control siRNA siAGF6 affected the clonogenicity in neither of these assays. We did not observe major differences in colony morphology. However, the colonies derived from cells treated with RUNX1-CBFA2T1siRNAs tended to be smaller in size than those derived from control cells (data not shown). We also examined the effects of RUNX1-CBFA2T1 siRNA on the clonogenicity of AML cell lines not expressing RUNX1-CBFA2T1. Colony formation of the t(8;21)-negative myeloid leukaemia cell lines MV4-11, NB4, HL60, U937 and K562 was not affected upon electroporation with RUNX1-CBFA2T1 siRNA (data not shown), which argues against a general, unspecific effect of the siRNAs used in this study on leukaemic clonogenicity. Therefore, the application of RUNX1-CBFA2T1 siRNAs is sufficient to inhibit the clonogenic growth of Kasumi-1 cells, indicating that RUNX1-CBFA2T1 is essential for the clonogenicity of these leukaemic cells. RUNX1-CBFA2T1 depletion reduces CD34 expression To examine the effects of RUNX1-CBFA2T1 on the progenitor status of t(8;21)-positive leukaemic cells, we analyzed the CD34 expression levels in siRNA-treated Kasumi-1 cells. When compared to mock-transfected cells, depletion of RUNX1-CBFA2T1 caused a twofold decrease in CD34 surface expression 7 days after electroporation (fig. 3). Control siRNA-treated cells contained only slightly lower amounts of CD34 surface marker. RUNX1-CBFA2T1 suppression inhibits proliferation in suspension cell culture In contrast to its effects on clonogenicity, a single electroporation with RUNX1-CBFA2T1 siRNA was not sufficient to substantially inhibit Kasumi-1 proliferation in suspension culture. However, repeating the electroporation every three to four days strongly inhibited cell proliferation (fig. 4A). During a course of three to four electroporations with or without mismatch control siRNA, the cell numbers steadily increased with a doubling time of 3 days (fig. 4B). RUNX1-CBFA2T1 siRNA treatment increased the doubling time twofold. Therefore, repetitive siRNA applications causing a long-lasting RUNX1-CBFA2T1 depletion inhibited cell proliferation of Kasumi-1 cells. These results suggest that RUNX1-CBFA2T1 is essential for the proliferation of t(8;21)-positive leukaemic cells in suspension culture. RUNX1-CBFA2T1 suppression interferes with cell cycle progression Next, we examined possible consequences of RUNX1-CBFA2T1 depletion for the cell cycle distribution of Kasumi-1 cells. Three days after an electroporation with RUNX1-CBFA2T1 siRNA we observed 25% less cells being in S phase (data not shown). Two or more consecutive electroporations with RUNX1-CBFA2T1 siRNA caused a reduction of the amount of S phase cells by 50% and a 30% reduction in G2/M phase cells, with a corresponding increase of the G1/G0 fraction by 20% (fig. 4C). Treatment of Kasumi-1 cells with the mismatch control siRNA did not affect the cell cycle distribution. The changes in the cell cycle distribution are associated with changed expression levels of CDKN1B (p27Kip1), a general inhibitor of Cyclin-CDK complexes and, thereby, of cell cycle progression. Two electroporations with RUNX1-CBFA2T1 siRNA caused a twofold increase in CDKN1B protein levels in RUNX1-CBFA2T1-depleted cells, but not in control cells (fig. 4D). Therefore, siRNA-mediated inhibition of RUNX1-CBFA2T1 expression may interfere with the transition of Kasumi-1 cells from the G1 to the S phase of the cell cycle. RUNX1-CBFA2T1 controls apoptosis A possible reason for the observed inhibition of proliferation is the induction of apoptosis upon suppression of RUNX1-CBFA2T1. We addressed this possibility by examining the amount of apoptotic cells by annexin V staining and of hypodiploid (subG1) cells by flow cytometry. Electroporation of Kasumi-1 cells together with the RUNX1-CBFA2T1 siRNA siAGF1 neither increased the amount of hypodiploid cells nor the amount of annexin V positive cells when compared to control cells. Instead, after siAGF1 treatment we observed a slight but reproducible decrease in the amount of apoptotic cells in both assays. This decrease was more pronounced after two consecutive electroporations with siRNAs. Repeating the siAGF1 electroporation after 4 days resulted in a threefold decrease of the amount of annexin V stained cells (fig. 5) indicating that RUNX1-CBFA2T1 depletion has an antiapoptotic effect on Kasumi-1 cells, and that the reduced proliferation upon siRNA treatment is not related to increased apoptosis. RUNX1-CBFA2T1 siRNAs induce senescence in Kasumi-1 cells The observed G1 arrest upon RUNX1-CBFA2T1 depletion could be either reversible or irreversible. An irreversible G1 arrest is a hallmark of cellular senescence. To address the question of the nature of the G1 arrest, and to analyze a possible influence of RUNX1-CBFA2T1 on cellular senescence, we stained siRNA- and mock-treated cells for senescence-associated β-galactosidase activity. After two or more electroporations with RUNX1-CBFA2T1 siRNA, but not with control siRNA, a significant fraction of the Kasumi-1 cells stain positive for β-galactosidase (fig. 6A). Cell counting revealed up to 50% of senescent cells, whereas mock or control siRNA treatment caused only a minor increase in senescent cells compared to untreated cells (fig. 6B). Therefore, depletion of RUNX1-CBFA2T1 results in an increase in cellular senescence. This implies that the observed G1 arrest is irreversible for a major fraction of RUNX1-CBFA2T1 siRNA treated cells. Haematopoetic growth factors cannot rescue RUNX1-CBFA2T1-depleted cells in the long term A possible mechanism of how RUNX1-CBFA2T1 prevents cell cycle arrest and senescence is the autocrine production of growth factors. Kasumi-1 cells express, for instance, G-CSF (fig. 7A), but not GM-CSF (data not shown). Depletion of RUNX1-CBFA2T1 caused a twofold decrease in G-CSF transcript levels (fig. 7A). Therefore, we examined whether addition of haematopoetic growth factors rescued siRNA-treated cells from cell cycle arrest and senescence. Depletion of RUNX1-CBFA2T1 for 16 days reduced the fraction of S phase cells twofold compared to control cells. This decrease is independent of G-CSF or GM-CSF (fig. 7B). When analyzing the cell cycle distribution siRNA or mock-treated cells in dependence on the duration of siRNA treatment, the fraction of S phase cells already decreases within 4 days of leukaemic fusion protein depletion and reaches its minimum after further 4 to 8 days (fig. 7C). Addition of G-CSF, and particularly of GM-CSF delayed this decrease, but could not prevent it. Furthermore, in RUNX1-CBFA2T1 siRNA treated cells, neither G-CSF nor GM-CSF addition caused any substantial change in the generation of senescent cells compared to cells without growth factors (fig. 7D). In each setting, the fraction of senescent cells increased from 2% at day 4 to 30–50% at days 12 and 16. Control groups contained between 1–10% of senescent cells. Finally, we examined the effects of several haematopoetic growth factors on colony formation of siRNA-treated Kasumi-1 cells. Supplementing the semisolid medium with the corresponding growth factors increased colony numbers for both control cells and RUNX1-CBFA2T1-depleted cells (fig. 8). G-CSF enhanced colony formation by control cells twofold, and by RUNX1-CBFA2T1-depleted cells threefold, and GM-CSF three- and fivefold, respectively. However, in comparison to control cells, neither G-CSF nor GM-CSF could completely restore colony formation of RUNX1-CBFA2T1-depleted cells. Taken together, the addition of G-CSF or GM-CSF delays, but does not prevent the G1 arrest in RUNX1-CBFA2T1-depleted cell. Furthermore, senescence is not inhibited by these growth factors. In line with these findings, neither of these growth factors completely restored the impaired clonogenicity of such cells. These results indicate that neither G-CSF nor GM-CSF can rescue leukaemic cells suffering of siRNA-mediated RUNX1-CBFA2T1 depletion. Discussion The leukaemic fusion protein RUNX1-CBFA2T1 inhibits haematopoetic differentiation by repressing gene expression via recruitment of histone deacetylases [4-7], and by sequestering transcription factors such as C/EBPα, SMADs or vitamin D3 receptor [8-11]. In addition, RUNX1-CBFA2T1 inhibits cell proliferation by inducing apoptosis in a variety of different cell types [13,46], in spite of repressing the proapoptotic gene p14Arf [17]. The notable exception are haematopoetic stem cells (HSCs); ectopic expression of RUNX1-CBFA2T1 causes an increased clonogenicity and results in their expansion both ex vivo and in vivo [20,21]. In addition to the block of differentiation, RUNX1-CBFA2T1 may also support proliferation, as, for instance, RUNX1-CBFA2T1-expressing HSCs maintain their telomere lengths and their CD34 expression [21]. We show that RUNX1-CBFA2T1 also supports proliferation, CD34 expression and colony formation in the t(8;21)-positive leukaemic cell line Kasumi-1. Thus, RUNX1-CBFA2T1 functions are similar in HSCs and in Kasumi-1. In the latter, support of proliferation is not caused by an inhibition of cell death. Instead, as observed with many other cell types [13,46], RUNX1-CBFA2T1 expression is associated with a certain extent of apoptosis. This observation disagrees with a report employing ribozymes to interfere with RUNX1-CBFA2T1 expression [47]. In contrast to our study, no clear reduction of either fusion transcript or protein was shown. It may, thus, be possible that the ribozyme effects observed in this study are not related to a suppression of RUNX1-CBFA2T1. The reduced proliferation of Kasumi-1 cells upon RUNX1-CBFA2T1 depletion is paralleled by a cell cycle arrest in G1. Particularly from a therapeutic point of view, it is important to know, whether this G1 block will be relieved by recovering RUNX1-CBFA2T1 expression, or by growth factors, or whether this arrest is irreversible. We observe that RUNX1-CBFA2T1 depleted cells show enhanced levels of senescence-associated β-galactosidase activity, a hallmark of cellular senescence. Therefore, the inhibition of G1 to S transition is associated by the induction of senescence indicating that this G1 arrest is irreversible. Interestingly, the elevated levels of p27Kip1 (CDKN1B) during RUNX1-CBFA2T1 depletion are in line with the recently demonstrated central role of this CDK inhibitor in the establishment of senescence [49,50]. Furthermore, RUNX1 was shown to induce senescence in murine embryonic fibroblasts, probably by inducing p19Arf [17]. Since RUNX1-CBFA2T1 inhibits the expression of p19Arf and of its human homologue, p14Arf [17], an increased expression of this regulator of p53 may also account for the induction of senescence in RUNX1-CBFA2T1-depleted Kasumi-1 cells. An important point to consider is a possible relation between the effects of RUNX1-CBFA2T1 on clonogenicity, proliferation and senescence, and its anti-differentiation activity. For instance, increased β-galactosidase activity may also be associated with late monocytic differentiation [48]. Moreover, later stages of myeloid differentiation are characterized by a stop of proliferation. Therefore, the observed inhibition of proliferation upon RUNX1-CBFA2T1 depletion might be directly related to myeloid differentiation. Kasumi-1 and SKNO-1 cells express the myeloid differentiation marker CD11b when treated with RUNX1-CBFA2T1 siRNA in combination with TGFβ and vitamin D3 [14]. However, in the absence of differentiation inducers, even extended siRNA treatment only results in a small fraction of less than 5% of CD11b-positive cells [14], and data not shown) which cannot be accountable for the up to 50% β-galactosidase positive cells. Nevertheless, depletion of RUNX1-CBFA2T1 causes an onset of myeloid differentiation; therefore, a possible link between the antiproliferative effects of RUNX1-CBFA2T1 suppression and early differentiation events cannot be entirely excluded. The haematopoetic growth factors G-CSF and GM-CSF were shown to support clonogenicity and proliferation in Kasumi-1 cells [40]. However, neither of these factors prevented senescence in RUNX1-CBFA2T1-depleted cells. Furthermore, G1-S transition was only transiently restored; after extended terms of RUNX1-CBFA2T1 depletion, the fraction of S phase cells also diminished in the presence of G-CSF or GM-CSF. Finally, clonogenicity of RUNX1-CBFA2T1-depleted cells was not completely restored in the presence of G-CSF or GM-CSF. These findings show that at least some haematopoetic growth factors cannot rescue leukaemic cells treated with RUNX1-CBFA2T1 siRNAs, and suggest that targeting of leukaemic cells with such siRNAs in vivo might interfere with the development and maintenance of leukaemia. However, a remaining possibility might be that the stroma environment with its complex mixture of growth factors and cell-cell interactions may be able to rescue RUNX1-CBFA2T1-depleted cells. We are currently investigating this question both in cell culture and in vivo. A major point of concern for siRNA applications is the specificity of the corresponding siRNA [51]. Reasons for "off-target" effects may be (i) the siRNA-induced RNA cleavage of transcripts with imperfect homology to the siRNA [52,53], (ii) a possible competition with endogenously expressed micro RNAs (miRNAs), which may be involved in the control of, for instance, cell differentiation, for RISC protein components [54], and (iii) the induction of an interferon response [45,55]. However, neither RUNX1-CBFA2T1 siRNAs nor the mismatch control siRNA affected the clonogenicity of five different leukaemic cell lines not carrying the translocation t(8;21). Furthermore, expression of the interferon-inducible gene STAT1 is not changed upon electroporation with RUNX1-CBFA2T1 siRNA or mismatch control siRNA. These results do not argue for general "off-target" effects being responsible for the observed changes in cell proliferation and clonogenicity of RUNX1-CBFA2T1 siRNA-treated Kasumi-1 cells. Instead, the data suggest the siRNA-mediated depletion of this leukaemic fusion protein as the cause for the observed phenotypic changes. Conclusions In summary, we show that RUNX1-CBFA2T1 does not only inhibit the myeloid differentiation [14], but is also essential for the proliferation and clonogenicity of the t(8;21)-positive leukaemic cell line Kasumi-1 by interfering with the establishment of senescence. Since this cell line was derived from a patient suffering of leukaemia refractory to chemotherapy [40], these findings suggest a central role of RUNX1-CBFA2T1 not only in the expansion of preleukaemic progenitor cells, but also in the maintenance of the leukaemia. Moreover, they imply that this leukaemic fusion gene is a promising target for molecularly defined therapeutic approaches. Competing interests HPV is an employee of Alnylam Europe AG, which also provided some of the siRNAs used in this study. Authors' contributions NM performed the proliferation, senescence and cell cycle assays. BD analyzed the clonogenicity of growth-factor treated Kasumi-1 cells and of t(8;21) negative cell lines. HR performed the immunoblot analyses and was involved in the analysis of clonogenicity and apoptosis. CC examined the expression levels of CD34 and of STAT1. HPV, AG, GH and AN participated in the coordination of the study. JK and OH conceived and designed the experiments. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements The authors thank Dagmar Reile and Kerstin Görlich for excellent technical assistance. This study was supported by a grant from the Deutsche Krebshilfe (10-2104-He 2) to O.H., G.H., J.K., A.N. and A.G, and by two grants from the Deutsche Jose Carreras Leukämie-Stiftung to J.K. (DJCLS-R01/08) and to O.H. (DJCLS-R03/10). Figures and Tables Figure 1 Depletion of RUNX1-CBFA2T1 protein by siRNAs A. RUNX1-CBFA2T1 and RUNX1 protein levels in siRNA-treated Kasumi-1 and SKNO-1 cells. Nuclear lysates were prepared 4 days after electroporation with 100 nM siRNAs and analyzed by immunoblotting. The electroporated cells and siRNAs are indicated on top. Arrows on the right mark RUNX1-CBFA2T1 and RUNX1 proteins. Markers are shown on the left, and the relative ratios between RUNX1-CBFA2T1 and RUNX1 are indicated below the blot. B. Time course of siRNA-mediated RUNX1-CBFA2T1 depletion. Kasumi-1 nuclear lysates were isolated at the indicated days after electroporation with 100 nM siRNA and analyzed by immunoblotting. Values were normalized to the control siRNA siAGF6. Figure 2 RUNX1-CBFA2T1 depletion reduces the clonogenicity of Kasumi-1 cells The columns represent the means of four independent experiments. Error bars indicate standard deviations. ‡, p < 0.001 according to Student's t-test. Figure 3 RUNX1-CBFA2T1 depletion diminishes CD34 expression Seven days after electroporation with the indicated siRNAs, cells were analyzed for CD34 surface marker by flow cytometry as described in Material and Methods. Figure 4 RUNX1-CBFA2T1 depletion inhibits cell proliferation and G1-S transition in Kasumi-1 A. Proliferation curve of siRNA treated cells. Kasumi-1 cells were electroporated at days 0, 4, 8 and 12. Cell numbers were determined at the indicated days using trypan blue counting. Arrows indicate electroporations. B. Graphical representation of cell doubling times. The columns represent the means of three independent experiments. Error bars indicate standard deviations. *, p < 0.05 according to Student's t-test. C. Graphical representation of the cell cycle phase distribution. Kasumi-1 cells were electroporated at days 0 and 4, and were examined at day 8 using FACS analysis. The columns and error bars represent the mean values and standard deviations of three independent experiments. †, p < 0.01 according to Student's t-test. C. RUNX1-CBFA2T1 suppression is associated with increased CDKN1B (p27Kip1) levels. After electroporation at days 0 and 4, total cell lysates were prepared at day 8 and analyzed using immunoblotting. After CDKN1B detection, the membrane was stripped and reprobed with an anti-tubulin antibody. The siRNAs are indicated on top. Arrows on the left mark RUNX1-CBFA2T1, RUNX1, CDKN1B (p27Kip1) and tubulin proteins. The relative ratios between CDKN1B and tubulin are indicated below the blot. Figure 5 Effects of RUNX1-CBFA2T1 and RUNX1 suppression on extent of apoptosis in Kasumi-1 cells Kasumi-1 cells were electroporated at days 0 and 4, and were examined at day 8 by annexin V and propidium iodide staining followed by FACS analysis. One of two experiments yielding very similar results is shown. The percentage of cells is given for each quadrant of the plots. Figure 6 RUNX1-CBFA2T1 siRNAs induce senescence in Kasumi-1 cells A. Senescence-associated β-galactosidase stained cells. Kasumi-1 cells were electroporated with siRNAs at days 0 and 4, and were examined at day 8 for senescence by staining for senescence-associated β-galactosidase. B. Quantification of cellular senescence. Cells were treated as above, and the ratio of β-galactosidase positive cells was examined by counting. Error bars represent the standard deviations of single-blinded counts performed by three different experimentators. †, p < 0.01 according to Student's t-test Figure 7 Effects of growth factors on the cell cycle distribution and extent of senescence of siRNA-treated Kasumi-1 cells A. Autocrine G-CSF expression in dependence on the siRNA treatment. Kasumi-1 cells were electroporated at days 0 and 4. Total RNA was isolated on day 8 and analyzed by real-time RT-PCR. The columns and error bars represent the means and standard deviations of three independent experiments. B. Cell cycle distribution of siRNA-treated cells in the absence and presence of growth factors. Kasumi-1 cells were analyzed on day 8 by FACS analysis as described in Materials and Methods. C. Amount of S phase cells in dependence on the length of RUNX1-CBFA2T1 depletion. D. Amount of senescent cells dependence on the length of RUNX1-CBFA2T1 depletion. Growth factors are indicated below the graphs. The data shown in B, C and D were obtained from the same time course experiment. Cells were electroporated with the indicated siRNAs at days 0, 4, 8 and 12, and were analyzed on days 4, 8, 12 and 16. Figure 8 Influence of haematopoetic growth factors on the colony formation of siRNA-treated Kasumi-1 cells Kasumi-1 cells were electroporated with the indicated siRNAs, plated in semi-solid medium containing the indicated growth factors, and analyzed as described in Material and Methods. 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==== Front BMC PsychiatryBMC Psychiatry1471-244XBioMed Central London 1471-244X-4-201527122210.1186/1471-244X-4-20Research ArticleFamily-based clusters of cognitive test performance in familial schizophrenia Hoti Fabian [email protected] Annamari [email protected] Jari [email protected] Timo [email protected]öm Lasse [email protected]önnqvist Jouko [email protected] Department of Mathematics and Statistics, University of Helsinki, Finland2 Department of Mathematical Sciences, University of Oulu, Finland3 Department of Mental Health and Alcohol Research, National Public Health Institute of Finland, Helsinki, Finland2004 22 7 2004 4 20 20 2 4 2004 22 7 2004 Copyright © 2004 Hoti et al; licensee BioMed Central Ltd.2004Hoti et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Cognitive traits derived from neuropsychological test data are considered to be potential endophenotypes of schizophrenia. Previously, these traits have been found to form a valid basis for clustering samples of schizophrenia patients into homogeneous subgroups. We set out to identify such clusters, but apart from previous studies, we included both schizophrenia patients and family members into the cluster analysis. The aim of the study was to detect family clusters with similar cognitive test performance. Methods Test scores from 54 randomly selected families comprising at least two siblings with schizophrenia spectrum disorders, and at least two unaffected family members were included in a complete-linkage cluster analysis with interactive data visualization. Results A well-performing, an impaired, and an intermediate family cluster emerged from the analysis. While the neuropsychological test scores differed significantly between the clusters, only minor differences were observed in the clinical variables. Conclusions The visually aided clustering algorithm was successful in identifying family clusters comprising both schizophrenia patients and their relatives. The present classification method may serve as a basis for selecting phenotypically more homogeneous groups of families in subsequent genetic analyses. ==== Body Background Schizophrenia is a severe mental illness which tends to run in families. Moreover, schizophrenia is a complex disorder with multiple environmental as well as genetic predisposing effects. Previous studies have shown that many neuropsychological functions are impaired in schizophrenia patients, and, to a lesser degree, also in their unaffected relatives [1-3]. Consequently, the continuous traits derived from neuropsychological tests have been suggested as one type of endophenotypes of schizophrenia to be included in genetic analyses [4-9], for a review see Egan and Goldberg [10]. Identifying more homogeneous subgroups of families with a similar pattern of cognitive test performance would further refine the data to be included in these analyses. Recently, cluster analysis of verbal learning and memory tests was used to divide patients with schizophrenia into subtypes. Categorization by these cognitive traits resulted in meaningful subgroups of schizophrenia [11]. In another study, extended neuropsychological test data of patients with schizophrenia were included in a hierarchical and iterative partitioning cluster analysis [12]. Four clusters were identified, ranging from good performance to profound global dysfunction. In Sautter et al. [13] an exploratory study comparing clustering of neuropsychological test performance in schizophrenia patients with familial history to those without was performed. In their analysis, patients with family history fell into three distinct clusters, while only one homogeneous cluster was found for the non-familial group. However, only patients were included in the analyses of these studies. As schizophrenia is likely to be a multifactorial disorder with low penetrance, the inclusion of relatives in the clustering analyses would be a powerful way to reveal subgroups based on the endophenotype of interest. In the present study, we report a new visually aided clustering approach aimed at identifying clusters of multiply affected families with schizophrenia on the basis of performance in neuropsychological tests. In the clustering process, each family was represented by the test scores of its affected and unaffected members, and the closeness the families was defined by the maximum pairwise distance between the members of the families. To our knowledge, this is the first study in which the clustering has been applied to families instead of solely to affected subjects with schizophrenia. Methods Subjects and data collection From a general population cohort of people born between 1940 and 1976 inclusive in Finland, a northern European country with approximately 5 million inhabitants, we identified 33,731 individuals with a diagnosis of schizophrenia, schizoaffective disorder or schizophreniform disorder. Data on the diagnosis were derived from three nation-wide computerized health care registers covering the years 1969 to 1998: the Hospital Discharge Register, the Free Medicine Register, and the Pension Register. Linking the personal identification numbers of the affected subjects to the National Population Register database allowed us to identify their family members and to construct pedigrees. Information on families with at least two members with schizophrenia, schizoaffective disorder or schizophreniform disorder, and at least two members with no diagnosis of psychiatric disorder was received from the aforementioned registers for 895 families from the whole of Finland. A blood sample for subsequent genetic analyses was drawn from 2295 subjects of 643 families. All available case note records were collected for those with a diagnosis of schizophrenia, schizoaffective disorder or schizophreniform disorder in any of the three registers. Two psychiatrists independently assessed the lifetime diagnoses for each case, according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) [14]. One of the assessors also completed the Operational Criteria Checklist for Psychotic Illness (OPCRIT) [15]. The collection of blood samples complied with the Declaration of Helsinki and its amendments. The protocol was accepted by the Ethics Committee of the National Public Health Institute, and the study was approved by the Ministry of Social Affairs and Health. Of those multiply affected families who already had given the blood samples, a subsample was targeted for collection of more detailed phenotypic information. This sample was selected randomly based on the data from the registers and the OPCRIT process. All subjects from the families gave a written informed consent for the study protocol comprising a diagnostic research interview and neuropsychological testing. Both patients and their family members were interviewed using the Structured Clinical Interview for DSM-IV (SCID-I for axis I disorders and SCID-II for axis II disorders) [16]. All the interviewers were trained in a similar manner for the use of these instruments. The final consensus diagnoses were based on the data collected from the records, the OPCRIT process, and the SCID interview. A total of 281 subjects from 54 families fulfilled the inclusion criteria and thus included at least two siblings with schizophrenia, schizoaffective disorder or schizophreniform disorder, and at least two siblings without these disorders. Altogether 16 patients were excluded because of being too psychotic (n = 6), having a current substance use diagnosis (n = 6), or being mentally retarded (n = 4). Of the family members to whom no psychiatric diagnosis was assigned for their lifetime, 6 were excluded because of high age, or for a defect in vision or hearing. The final sample thus comprised 165 subjects with a psychiatric diagnosis and 94 unaffected family members from 54 families. Of the 165 subjects with a diagnosis, altogether 82 subjects had schizophrenia, while 13 subjects suffered from schizoaffective disorder, 10 from schizophreniform disorder and 12 from bipolar disorder. A nonpsychotic disorder was assigned to 48 individuals. The 94 unaffected subjects did not get any current or lifetime psychiatric diagnosis. In 51 families, at least one of the patients included in the analysis suffered from pure schizophrenia. In the remaining three families, at least one subject with schizoaffective or schizophreniform disorder was included. All families from which the subjects for the present study were drawn, represent familial schizophrenia, as in each of them there were at least one sibling with a diagnosis of pure schizophrenia, plus at least one other sibling with schizophrenia, schizoaffective disorder or schizophreniform disorder. Test procedures A neuropsychological test battery was administered to all subjects in fixed order by well-trained examiners either after the interview during the same day, or the following day. All examiners were psychologists or advanced psychiatric nurses extensively trained and supervised with the test battery. Experienced psychologists scored all the tests. Auditory attention was assessed with the Digit Span Forward task, and verbal working memory with the Digit Span Backward task of the Wechsler Memory Scale-Revised (WMS-R) [17]. According to Finnish normative data, the test-retest reliability coefficients of the Span subtests vary with age from 0.74 to 0.82 [18]. The Visual Span forward subtest of the WMS-R [17] was used to assess visual attention. The backward condition of the span task was used for measuring visual working memory. According to Finnish normative data, the test-retest reliability coefficients of the Visual Span subtests vary with age from 0.72 to 0.80 [18]. The Logical Memory story A, of the WMS-R [17], immediate and delayed, was used to assess recall and retention in a story format. Visual memory was measured by the Visual Reproduction subtest of the WMS-R [17], immediate and delayed. In Finnish normative data, the test-retest reliabilities of these subtests have varied with age from 0.84 to 0.91, and 0.31 to 0.34, respectively. Verbal learning and memory were assessed with the California Verbal Learning Test (CVLT) [19] which examines recall and recognition of word lists over a number of trials. The present study reports the following variables derived from the test: verbal learning (total recall over 5 trials), semantic clustering, and recognition memory (discriminability). No reliability data for Finnish subjects exist, but the split-half reliability of the CVLT is 0.77 to 0.86, according to the test manual [19]. Controlled Oral Word Association test (COWA) [20] was used to assess verbal fluency. The quantity of words the subject produces in one minute, both with words beginning with a designated letter (S,K), and within a category (animals), was assessed. No reliability data for Finnish subjects are available. Four subtests of the Wechsler Adult Intelligence Test – Revised (WAIS-R) [21] were used. Verbal abilities were measured with the Vocabulary and Similarities subtests. Vocabulary is considered the best single measure of general ability [22]. The Similarities subtest is a task of abstraction and concept formation. The Block Design and Digit Symbol subtests have a motor component as the trials are timed. The former is a measure of visuospatial reasoning and abstraction. The latter subtest measures psychomotor performance. According to Finnish normative data, the test-retest reliabilities for Vocabulary, Similarities, Block Design, and Digit Symbol are 0.89–0.95, 0.69–0.88, 0.78–0.83, and 0.82–0.86, respectively, depending on age [23]. Clustering and statistical analyses Notation and imputation of missing values The variables used in cluster analysis included 17 neuropsychological test variables together with the age and the sex of the subjects. With a total of M = 19 variables and N = 259 subjects, the data formed an M × N matrix x = (xik), where xik is the the value of the ith variable for the kth subject. However, there were 85 (1.7 %) missing values as not all test results were obtained for all subjects. The missing values were handled by the following procedure, which replaces an individual's missing value with an estimate obtained from a linear fit between the test with the missing value and the test that correlates with it most and that also has the individual's test result available. 1. Pairwise correlations were calculated between all test variables using only subjects with results available for both tests. We denote such correlation between the tests i and j by cij. 2. Given a missing value in the test i for the subject k, we found the test j = j0 which had the highest value of |cij| among the tests with the value xjk available and set where the coefficients a and b were found by computing linear regression of the test j0 on the test i using only subjects with results available for both tests. Cluster analysis The families were clustered using a complete-linkage clustering algorithm. Each variable was normalized by subtracting the mean value and dividing by the standard deviation. The normalization was done to ensure that each variable contributes equally to the clustering procedure. Denote by xk = (x1k,...,xMk) the normalized data for subject k and define the distance between two clusters Cr and Cs by drs = max{||xk - xl|| : xk ∈ Cr and xl ∈ Cs}, that is, drs is the maximum pairwise distance between members of the two clusters. Here ||·|| denotes the euclidean distance, . In the sense of this distance measure, two clusters are close when all subjects in both clusters are close. Clustering was carried out using the following algorithm. 1. Initial clusters are defined by the families. 2. The two clusters with the smallest inter-cluster distance drs are merged into one larger cluster. 3. Steps 2 and 3 are repeated until a desired number of clusters remains. In Figure 1, two steps of the above procedure are demonstrated. Three clusters are depicted by the green solid lines. The two nearest clusters are combined (the dashed green line). Their inter-cluster distance drs is shown by the solid red line. The inter-cluster distance between the two remaining clusters is shown by the dashed red line. Note that by using a different inter-cluster distance measure, such as the minimum pairwise distance, a different merging order would result (see the Discussion). Figure 1 Visualization of clustering. Two merging steps of the clustering algorithm (see the text). Visualization of clusters We introduce a visualization technique that helps in identifying candidate clusters and also gives an overall picture of the main differences between the produced clusters as measured by all variables simultaneously. The method gives information about the dynamics of the clustering process and the characteristics of the candidate clusters. The upper part of Figure 2 presents the data matrix as what is called the "color histogram" [24] or "the data image" [25]. The rows correspond to variables and the columns correspond to subjects. The values of the neuropsychological tests and other variables are visualized using color coding. The color of a variable changes from blue to red as its value increases. To better utilize the dynamic range of coloring, 5 % of the highest values and 5 % of the lowest values were set to the 95th percentile and the 5th percentile of the test results, respectively. The variables were then shifted and scaled to the interval [0,1] and color coded (0 = blue, 1 = red). On the last line of the color histogram, clusters are depicted with different colors. Figure 2 Visualization of clustering result. A visualization of the data and the cluster solution using the data image and the dendrogram. On the last line of the data image clusters 1, 2 and 3 are depicted with different colors. Both the subjects and the neuropsychological tests were ordered by a complete linkage clustering algorithm (see the text). To further improve the visual impression of the clusters, the neuropsychological test variables (the rows of the data image) were ordered using essentially the same procedure that was used in clustering the families. The initial clusters were now the individual variable vectors xi = (xi1,...,xiN) and the pairwise distance between two clusters Cr and Cs was defined as drs = max{1/|cij| : i ∈ Cr, j ∈ Cs}, where cij denotes the correlation between the variables i and j. Thus, at each step, the algorithm merged clusters with the highest correlating variables. The lower part of Figure 2 visualizes the actual clustering process using the dendrogram. The history (vertical direction) of the mergings is shown from the beginning (one family in each cluster) to the end (all families in one cluster). By simultaneously exploring the two images, a reasonable value for the number of clusters can be found and the characteristics of the cluster solution visualized in a useful manner. It is also helpful to monitor the inter-cluster distance measure for possible large jumps which indicate that two distant clusters are being merged (Figure 3). Figure 3 Merging distances. The inter-cluster distance (merging distances) as a function of the number of clusters. The vertical line indicates the suggested three cluster result, after which there is a clear jump in the merging distances. Validation of cluster result The clusters were obtained by treating families as single objects whose dissimilarity was measured by the pairwise test performance differences between the family members. One may therefore ask whether the clusters found still appear to be distinct groups when viewed simply as sets of individual subjects. We examined this question by dividing repeatedly the 54 families into three random clusters that had the same number of families as in the proposed three cluster solution and by computing, for each of the three pairs of the generated clusters, the ratio BWr,s = Br,s/(Ws + Wr), where Br,s is the mean distance between subjects from clusters r and s (in the 19-dimensional space) and Wr is the mean distance between subjects within cluster r. The statistic BWr,s takes on a large value if the distance between the subjects from the different clusters is large compared to the distance between the subjects within the clusters themselves indicating that the two clusters are separated in the 19-dimensional space defined by the variables used. If the values of BWr,s for the proposed three clusters are significantly higher than for a random partition we take this as evidence that the clusters found indeed constitute meaningful groups also at the level of individual subjects. Further, after the cluster analysis, the proposed family clusters were examined for differences on demographic and neuropsychological measures. In addition, the patients included in the clusters were examined for the differences in clinical variables as evaluated by the OPCRIT (premorbid social adaptation, response to neuroleptic treatment, chronicity, age of onset) of the disorder. In comparing the demographic and clinical variables, the Chi-square test, or t-test, both two-tailed, were applied. The differences in the quantitative neuropsychological measures were analyzed using the linear mixed effects (LME) model, which takes into account the dependence between the subjects, who, a priori, came from the same families. Thus, family was included as a random effect in all models with age and sex as the fixed effects. In addition, post hoc models were conducted with education years as an added fixed effect, a known confounder for cognitive functions. In all these analyses, the probability level < 0.05 indicated statistical significance. Analyses were performed using the S-Plus statistical software, version 3.4 [26]. Results The cluster solution Three clusters of families were successfully identified from the study sample. The first cluster comprised 94 subjects from 17 families, the second cluster 50 subjects from 12 families, and the third cluster 115 and 25. Adding more neuropsychological test variables or leaving out the sex or the age of the subjects had little effect on the solution. The data image (Figure 2) indicated that the overall performance of the subjects was higher in the first cluster than in the second, and that the performance in the third cluster was between the other two. The three clusters were therefore identified as consisting of subjects that were relatively well-performing, impaired and intermediate, respectively. A three cluster solution is supported by the homogeneity of the within-cluster test performance patterns of the proposed groups (Figure 2). As shown by the dendrogram, the two-cluster solution would combine the impaired and the intermediate clusters, and the four-cluster result would divide the well-performing cluster into two subclusters one of which is very small, consisting only of six families. Stopping the merging process even earlier does not appear to suggest any interesting alternative cluster solutions. Note also the jump in the distance function of Figure 3 after 3 clusters. In Figure 4 the three family clusters are further visualized by classic metric multidimensional scaling (MDS) [27,28]. Thus, with a total of 19 variables, the 54 families comprising the three clusters are represented as points in the 19-dimensional euclidean space so that the pairwise distances between the points match the original distances between the families (maximum pairwise euclidean distances between subjects in the families). The two-dimensional projection of the 19-dimensional space, although capturing only 27.0% of the total variation, shows the two most important directions (the directions with the highest variance) and provides evidence on the success of the clustering process itself, i.e., in the sense of the distance measured used, clustering does appear to produce three separate classes of families. Further visualization of the clusters, including an animation, is provided by the supplementary material to this article (See Additional file 1, 2, 3, 4 and 5. Figure 4 Multidimensional scaling visualization. A two-dimensional visualization using multidimensional scaling (MDS) of the families in the three clusters found. The similarity measure employed in MDS was the same one that was used in the family clustering procedure, with the natural modification that the distance between a family and itself was set to zero. The horizontal and vertical axes are the directions with the highest and the second highest variance, respectively. In each of the three pairwise comparisons of the statistic BWr,s the randomly generated clustering solution almost always had a smaller value than the proposed clustering solution (the fraction of opposite results in 10 000 trials was less than 0.01). This lends support to the visual impression that the three clusters are separate groups when viewed as subsets of individual subjects. Results were similar when the family structure was ignored and the random clusters were generated allowing subjects from the same family to be assigned to different clusters. Demographic and clinical characteristics The demographic characteristics of the clusters of families are shown in Table 1, and Table 2 shows the clinical characteristics of the subjects with schizophrenia, schizoaffective disorder or schizophreniform disorder. The three clusters did not differ by age or sex distribution. The well-performing cluster had significantly more years of education than the two others (p <0.001 in contrasts versus both other clusters). Overall, the clusters did not differ in clinical characteristics, except that the well-performing cluster showed better premorbid adaptation than the intermediate cluster (p = 0.04). The age of onset did not differ between the clusters (mean 25.9, SD 7.8, mean 24.7, SD 7.6, mean 23.7, SD 7.6 in clusters 1, 2, and 3, respectively, all p-values > 0.20). The impaired cluster did not include any patients with schizoaffective disorder, bipolar disorder or other affective psychotic disorders, while in the well-performing and intermediate clusters, these diagnoses were assigned to 14% and 11% of the subjects, respectively. About 36% of family members in all three clusters were unaffected. Table 1 Demographic characteristics Demographic characteristics of the family members in the well-performing (Cluster 1), impaired (Cluster 2), and intermediate (Cluster 3) clusters. Cluster 1 (n = 94, 17 families) Cluster 2 (n = 50, 12 families) Cluster 3 (n = 115, 25 families) Mean SD Mean SD Mean SD Sex (F/M) 48/46 24/26 50/65 Age 48.8 9.4 52.3 12.6 48.5 11.3 Education years 11.3a,b 3.0 9.3 2.4 9.9 2.3 aContrast versus cluster 1 and 2, p < 0.001, t-test, two-tailed. bContrast versus cluster 1 and 3, p < 0.001, t-test, two-tailed. Table 2 Clinical characteristics Clinical characteristics of the affected family members in the well-performing (Cluster 1), impaired (Cluster 2), and intermediate (Cluster 3) clusters Cluster 1 (n = 35) Cluster 2 (n = 27) Cluster 3 (n = 43) p Yes No Yes No Yes No 1 vs. 2 * 1 vs. 3* 2 vs. 3* Poor premorbid social adjustment 14 21 13 14 27 16 ns 0.04 ns Response to neuroleptics 30 5 21 6 34 9 ns ns ns Chronic course of the disorder 15 20 17 10 24 19 ns ns ns * Chi square, two-tailed. Neuropsychological variables The impaired cluster scored lowest in all measured neuropsychological variables, and the intermediate cluster showed consistently worse performance than the well-performing one (Table 3). The differences between the family clusters in the neuropsychological variables were tested by the within-family linear mixed effect models. In these models, the impaired cluster was found to achieve significantly lower scores than both other clusters in almost all traits (Table 4). The only variable not reaching statistical significance in differentiating any of the clusters was auditory attention. Table 3 Neuropsychological test performance Means and Standard Deviations in neuropsychological test performance (raw scores) among the family members in the well-performing (Cluster 1), impaired (Cluster 2), and intermediate (Cluster 3) clusters Cluster 1 (n = 94) Cluster 2 (n = 50) Cluster 3 (n = 115) Mean SD Mean SD Mean SD Auditory attention 6.7 2.1 5.8 1.9 6.3 1.8 Verbal working memory 6.1 2.3 4.5 2.0 4.7 1.7 Visual attention 8.6 2.1 6.8 1.9 7.6 1.8 Visual working memory 7.8 2.1 5.3 2.7 6.9 2.0 Story recall immediate 20.1 7.9 11.7 6.8 14.5 7.1 Story recall delayed 16.6 8.0 8.0 6.9 11.2 6.8 Visual reproduction imm 32.1 6.9 23.5 11.4 27.6 8.4 Visual reproduction del 27.4 9.9 16.2 12.8 21.2 10.5 Vocabulary 41.8 12.1 27.8 14.3 33.1 11.6 Similarities 24.7 4.6 18.5 6.7 21.1 5.3 Digit Symbol 39.4 16.5 26.9 13.6 34.4 13.8 Block design 28.6 11.6 17.7 12.5 22.3 10.8 Verbal learning 45.2 12.2 30.9 11.5 36.6 11.7 Semantic clustering 13.2 8.4 6.7 4.8 8.1 6.6 Recognition 93.2 6.0 81.1 16.6 87.2 10.3 Verbal fluency 29.9 11.5 22.0 10.4 25.2 9.3 Verbal fluency, animals 20.2 6.2 14.2 5.2 16.2 5.0 Table 4 Differences in neuropsychological test performance Differences in neuropsychological test performance between the well-performing (Cluster 1), impaired (Cluster 2), and intermediate (Cluster 3) clusters. Linear mixed effects models with family as a random effect, and sex and age as fixed effects Cluster 2 vs. Cluster 1 Cluster 1 vs. Cluster 3 Cluster 2 vs. Cluster 3 Coeff SD Wald p Coeff SD Wald p Coeff SD Wald p Auditory attention -0.81 0.43 -1.87 0.07 0.40 0.35 1.12 0.28 -0.41 0.41 -1.00 0.32 Verbal working m -1.52 0.46 -3.31 0.002 1.40 0.38 3.74 < 0.001 -0.11 0.44 -0.27 0.80 Visual attention -1.65 0.39 -4.23 < 0.001 1.00 0.31 3.19 0.002 -0.65 0.37 -1.74 0.08 Visual working m -2.31 0.46 -4.98 < 0.001 0.91 0.38 2.42 0.02 -1.40 0.44 -3.17 0.003 Story recall imm -8.18 1.65 -4.95 < 0.001 5.71 1.33 4.29 < 0.001 -2.47 1.56 -1.58 0.11 Story recall del -8.38 1.59 -5.27 < 0.001 5.37 1.28 4.20 < 0.001 -3.01 1.50 -2.00 0.05 Visual reprod imm -7.82 1.56 -5.02 < 0.001 4.49 1.23 3.65 < 0.001 -3.33 1.49 -2.23 0.03 Visual reprod del -10.20 2.03 -5.03 < 0.001 5.93 1.60 3.71 < 0.001 -4.26 1.93 -2.21 0.03 Vocabulary -14.39 2.39 -6.03 < 0.001 8.64 1.92 4.50 < 0.001 -5.74 2.29 -2.51 0.02 Similarities -9.95 2.43 -4.09 < 0.001 3.51 0.84 4.17 < 0.001 -2.45 1.00 -2.45 0.02 Digit Symbol -10.01 2.66 -3.91 < 0.001 4.42 2.13 2.08 0.04 -5.66 2.54 -2.23 0.03 Block design -8.48 2.76 -3.07 < 0.004 6.64 1.96 3.38 0.001 -3.31 2.31 -1.43 0.16 Verbal learning -13.74 2.11 -6.53 < 0.001 8.22 1.69 4.87 < 0.001 -5.52 2.03 -2.72 0.009 Semantic clust -6.20 1.21 -5.12 < 0.001 4.92 0.97 5.10 < 0.001 -1.28 1.17 -1.09 0.28 Recognition -11.21 1.82 -6.14 < 0.001 5.79 1.45 3.98 < 0.001 -5.41 1.77 -3.07 0.004 Verbal flu -7.50 2.21 -3.40 0.001 4.59 1.79 2.57 0.01 -2.90 2.08 -1.40 0.17 Verbal flu, anim -5.80 1.00 -5.78 < 0.001 4.01 0.80 5.03 < 0.001 -1.79 0.96 -1.87 0.07 Effect of education As the clusters differed significantly from each other in education years, we conducted post hoc linear mixed effects models with family as the fixed effect, and age, sex and education years as the random effects (data not shown). This did not eliminate the significant differences in cognitive functioning between the well-performing and the impaired cluster. In contrasts between the well-performing and intermediate cluster, all other differences remained significant, except in the scores of Visual immediate recall, Digit Symbol, and Verbal fluency, which lost their significance. Between the intermediate and the impaired cluster, scores in Vocabulary and Digit Symbol were no longer significantly different after controlling for education years. Discussion We report on the application of a visually aided clustering algorithm to data based on performance in a set of neuropsychological test measures, these being potential endophenotypic traits in schizophrenia. We were able to successfully detect three separate family clusters comprising both schizophrenia patients and their family members. In the impaired cluster, the families scored significantly worse than those in the other two. The well-performing cluster received the highest scores in each cognitive test, and the intermediate cluster scored consistently between the other two. However, the clusters of families did not differ from each other in age, sex distribution, and, regarding the affected subjects, in the age of onset, or in most of the other clinical features. The well-performing cluster was significantly more educated than the two others, but controlling for education years did not change the main results. We tested the differences in the diagnostic class distributions (including those with no diagnosis), and although the differences did not reach statistical significance, we find it interesting that none of the subjects with schizoaffective disorder, bipolar disorder, or other affective psychotic disorders ended up into the impaired cluster. We consider this as supporting the validity of particularly the poor cluster, which seems to represent a subsample of core schizophrenia with the most defected cognitive functioning. This cluster included the same proportion of unaffected subjects than the other two clusters, and based on the clustering algorithm, these family members without any psychiatric diagnoses during their lifetime performed generally poorly, too. Global verbal memory, including the story recall from the WMS-R [17] and verbal learning from the CVLT [19], were among the measures that differentiated well the clusters. This is in line with results by Heinrichs and Zakzanis [29], who found the best effect sizes in these functions in differentiating schizophrenia patients from controls. However, against a background of global dysfunction, any selective impairments such as those in verbal memory, are only relative [29]. The present study suggests that it is possible to characterize families with convergent cognitive performance using variables from several domains of cognition, such as attention, verbal memory, executive functioning, and intelligence. In efforts aiming at sample homogeneity, the best method may be using multiple endophenotypic measures. In part, our results are also comparable to those by Erlenmeyer-Kimling et al [30], who found that impairments in multiple cognitive measures best predicted future schizophrenia in high risk subjects. Our results suggest that molecular genetic analyses could benefit from prior appliance of our method, revealing meaningful family subgroups in a representative sample of familial schizophrenia. It would allow the resources to be targeted primarily for gene hunting projects among more homogeneous groups of families. Our new approach to combining data visualization and clustering appears to offer a valuable tool for identifying clusters in family-based data. Applying hierarchical clustering and the data image interactively helps to identify a reasonable value for the number of clusters in the cluster solution. By ordering the variables in the data image suitably, one gains useful insight into the test performance characteristics of the subjects in the clusters. As suggested in Palmer et al. [31], there may be a group of schizophrenia patients with no observed global impairment in cognition. One result of the present cluster analysis was the detection of a group of schizophrenia families with clearly better performance than the families in the two other clusters. This finding, together with those of previous studies, warrants further research for detecting putative factors protecting the cognitive development of these patients and their family members. Interestingly, attention, as measured by the simple auditory attention task (verbal span forwards), did not differentiate the clusters. The mean score in this task was also below the national normative mean in all clusters. This may indicate a fundamental impairment of attention in schizophrenia [32], observed also in patients and family members who otherwise perform well. When education was controlled for, it was further found that score in Digit Symbol, a test measuring information processing speed, and verbal fluency, a measure of executive function, did not any more separate the clusters. These results are in line with those in Weickert et al. [33], who found a selective impairment in executive function and attention in a group of schizophrenia patients defined as cognitively preserved. The present study is the first one in which cluster analysis of neuropsychological test variables has been conducted among a representative sample of familial schizophrenia comprising both affected and unaffected family members. The sample of the present study was randomly selected from a nationwide familial schizophrenia cohort. However, the results may not be generalizable to families with only one patient with the disorder. The similar patterns in neuropsychological performance in the clusters may be due to a variety of familial environmental effects, which are difficult to define ex post facto. Furthermore, our set of neuropsychological measures did not cover all those cognitive domains that previous studies have suggested as valid cognitive endophenotypes. However, it has been demonstrated in twin and in family studies [3,7,34], that the cognitive traits from our test selection measuring attention, working memory, verbal memory and visual memory do show genetic effects. Furthermore, in the present sample, the included test variables discriminated the affected and unaffected subjects, both in the whole sample and within clusters (data not shown). In the absence of a control sample, the present study could not test the possibility that the same clustering solution would emerge in normal families from the population. However, to our knowledge, such family clustering studies have not been conducted. In a study by Horan and Goldstein [35], a cluster analysis was conducted both in a patient and in a non-psychotic patient control group. The clustering solutions in these groups did not resemble each other, suggesting a specific pattern in the schizophrenic population. It is known that family members of schizophrenia patients tend to perform worse than subjects from control populations [1-3], and particularly those in multiply affected families [8,34]. Indeed, the aim of the present study was to explore the clustering of families in multiply affected families with schizophrenia. Thus the generalizability of the results may be limited to such samples representing about one fifth of all schizophrenia cases [36]. Clearly, the choice of the inter-cluster distance measure can greatly influence the merging process and hence the cluster solutions obtained. The maximum pairwise distance between subjects adopted in our analysis assigns a small distance between clusters only if all subjects in the clusters are close to each other in their test performance. We also experimented with the minimum pairwise distance but the results were poor. An explanation for this emerges by studying the minimum distance measure along the first few principal component directions of the normalized test results (the directions of largest variance). It turns out that, along these directions, most families have a member with nearly average performance and whose test results therefore closely match those of many members of other families. The variance of the distribution of the pairwise minimum distances is small and modest changes in the test results can lead to significantly different cluster solutions. The mean of pairwise distances between two clusters would be a compromise between the maximum and the minimum distances, but it turned out to behave much like the minimum distance and was therefore not used. Conclusions The new approach which combines clustering and data visualization was effective in identifying homogeneous subgroups of schizophrenia families with convergent cognitive test performance. Our results emerging from a sample of familial schizophrenia patients are in line with previous studies in which two extreme clusters have consistently emerged, characterized by a well-performing and a dysfunctional group of subjects, and at least one intermediate [11,12,37]. Our results agree with those in Sautter et al. [13], in which neuropsychological data of familial schizophrenia patients formed three clusters with respect to the level of performance. The fact that our findings, after including both affected and unaffected subjects agree with prior evidence, suggest further use of the cognitive traits as valid endophenotypes to be used in genetic linkage analyses. This method seems valid for partitioning the schizophrenia families by a relevant phenotypic category, resulting in more homogeneous subgroups. The method and results of the present study may be exploited in selecting whole families for subsequent analyses using the actual genetic marker data. Competing interests None declared. Authors' contributions FH and LH made the cluster analysis and wrote the corresponding sections, ATH supervised the neuropsychological test data collection, performed the differences testing analyses and drafted the manuscript, TP was the clinical and diagnostic study leader and commented on the text, JH designed the models for differences testing, JL conceived the research project. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional file 1 Comparison of Cluster 1 (well-performing) and Cluster 2 (impaired). The original variables (neuropsychological test variables + age) are visualized using the parallel coordinate plot. The subjects classified as well-performing are colored green and those classified as impaired are colored red. Yellow indicates overlap of green and red. There is overlap in all the original variables. The above figure was produced using the Crystal Vision software. The software uses a so-called grand tour technique to systematically (and in a continous manner) go through all possible rotations in the data space. The tour can be visually monitored using the parallel coordinate plot. Click here for file Additional file 2 Smaller version of Additional file 1. Comparison of Cluster 1 (well-performing) and Cluster 2 (impaired). The original variables (neuropsychological test variables + age) are visualized using the parallel coordinate plot. The subjects classified as well-performing are colored green and those classified as impaired are colored red. Yellow indicates overlap of green and red. There is overlap in all the original variables. The above figure was produced using the Crystal Vision software. The software uses a so-called grand tour technique to systematically (and in a continous manner) go through all possible rotations in the data space. The tour can be visually monitored using the parallel coordinate plot. Click here for file Additional file 3 A parallel coordinate plot of the data after a rotation transformation in the data space. The axes correspond now to linear combinations of the original variables. The 6th axis from the top shows an interesting one-dimensional projection of the data. In this direction the two clusters are well separated. The above figure was produced using the Crystal Vision software. The software uses a so-called grand tour technique to systematically (and in a continous manner) go through all possible rotations in the data space. The tour can be visually monitored using the parallel coordinate plot. Click here for file Additional file 4 Smaller version of Additional file 3. A parallel coordinate plot of the data after a rotation transformation in the data space. The axes correspond now to linear combinations of the original variables. The 6th axis from the top shows an interesting one-dimensional projection of the data. In this direction the two clusters are well separated. The above figure was produced using the Crystal Vision software. The software uses a so-called grand tour technique to systematically (and in a continous manner) go through all possible rotations in the data space. The tour can be visually monitored using the parallel coordinate plot. Click here for file Additional file 5 A movie which demonstrates the use of the software can be downloaded here. Smaller resolution CrystalVision.mpg(0.86 Mb). A higer resolution version of this movie is available at Reference for Crystal Vision software CrystalVision, copyright (c) 2000 by Crystal Data Technologies (Qiang Luo, Edward J. Wegman, and Xiaodong Fu), is a Windows 95/98/NT package for Wintel computers. A demonstration version of CrystalVision is available at Click here for file Acknowledgements The study was supported in part by Millennium Pharmaceuticals, Inc., and American Home Products Corp., Wyeth-Ayerst Research Division. Diagnosticians and field workers collecting data from the families are warmly acknowledged. The work of F. Hoti was partially supported by the Finnish Graduate School in Stochastics and by Grant 202324 from the Academy of Finland. ==== Refs Park S Holzman PS Goldman-Rakic PS Spatial working memory deficits in the relatives of schizophrenia patients Arch Gen Psych 1995 52 821 828 Faraone SV Seidman LJ Kremen WS Toomey R Pepple JR Tsuang MT Neuropsychological functioning among the nonpsychotic relatives of schizophrenic patients: a 4-year follow-up study J Abnorm Psychol 1999 108 176 181 10067004 10.1037//0021-843X.108.1.176 Cannon TD Huttunen MO Lönnqvist J Tuulio-Henriksson A Pirkola T Glahn D Finkelstein J Hietanen M Kaprio J Koskenvuo M The inheritance of neuropsychological dysfunction in twins discordant for schizophrenia Am J Hum Genet 2000 67 369 382 10880296 10.1086/303006 Gottesman II Shields J Schizophrenia and genetics: a twin study vantage point 1972 New York: New York: Academic Press Toomey R Faraone SV Seidman LJ Kremen WS Pepple JR Tsuang MT Association of neuropsychological vulnerability markers in relatives of schizophrenia patients Schizophr Res 1998 31 89 98 9689713 10.1016/S0920-9964(98)00025-5 Freedman R Adler LE Leonard S Alternative phenotypes for the complex genetics of schizophrenia Biol Psychiatry 1999 45 551 558 10088045 10.1016/S0006-3223(98)00321-7 Tuulio-Henriksson A Haukka J Partonen T Varilo T Paunio T Ekelund J Cannon TD Meyer JM Lönnqvist J Heritability of neurocognitive functions and number of quantitative trait loci contributing to them in families with schizophrenia Am J Med Genet (Neuropsychiatric Genet) 2002 114 483 490 10.1002/ajmg.10480 Tuulio-Henriksson A Arajärvi R Partonen T Haukka J Varilo T Schreck M Cannon TD Lönnqvist J Familial loading associates with impaired visual span among healthy siblings of schizophrenia patients Biol Psychiatry 2003 54 623 628 13129657 10.1016/S0006-3223(03)00232-4 Gottesman II Gould TD The Endophenotype Concept in Psychiatry: Etymology and Strategic Intentions Am J Psychiatry 2003 160 636 645 12668349 10.1176/appi.ajp.160.4.636 Egan MF Goldberg TE Intermediate cognitive phenotypes associated with schizophrenia Methods in Molecular Medicine 2003 77 163 197 12298369 Turetsky BI Moberg PJ Mozley LH Moelter ST Agrin RN Gur RC Gur RE Memory-delineated subtypes of schizophrenia: relationship to clinical, neuroanatomical, and neurophysiological measures Neuropsychology 2002 16 481 490 12382987 10.1037//0894-4105.16.4.481 Hill SK Ragland DJ Gur RC Gur RE Neuropsychological profiles delineate distinct profiles of schizophrenia, an interaction between memory and executive function, and uneven distribution of clinical subtypes J Clin Exp Neuropsych 2002 24 765 780 10.1076/jcen.24.6.765.8402 Sautter FJ McDermott BE Cornwell J Johnson J Borges A Wilson AF Vasterling JJ Foundas AL A preliminary study of the neuropsychological heterogeneity of familial schizophrenia Schizophr Res 1995 18 1 7 8929755 10.1016/0920-9964(95)00015-1 Diagnostic and Statistical Manual of Mental Disorders 1994 4 Washington, DC: American Psychiatric Press McGuffin P Farmer AE Harvey I A polydiagnostic application of operational criteria in psychotic illness: development and reliability of the OPCRIT system Arch Gen Psychiatry 1991 48 764 770 1883262 First MB Spitzer RL Gibbon M Williams JBW Structured Clinical Interview for DSM-IV Axis I Disorders – Clinician Version (SCID-CV) 1997 Washington, DC: American Psychiatric Press Wechsler D Wechsler Memory Scale – Revised (WMS-R). Manual The Psychological Corporation, San Antonio: Harcourt Brace Jovanovich, Inc 1987 Wechsler D WMS-R Käsikirja Helsinki: Psykologien Kustannus 1996 In Finnish Delis DC Kramer JH Kaplan E Ober BA California Verbal Learning Test. Manual Research, The Psychological Corporation Harcourt Brace & Company, San Antonio 1987 Benton AL Hamsher K Multilingual Aphasia Examination Manual Iowa City: University of Iowa 1989 Wechsler D Wechsler Adult Intelligence Scale – Revised (WAIS-R). Manual The Psychological Corporation, Cleveland: Harcourt Brace Jovanovich, Inc 1981 Lezak MD Neuropsychological Assessment 1995 3 New York: Oxford University Press Wechsler D WAIS-R Käsikirja Helsinki: Psykologien Kustannus 1992 In Finnish Wegman E Hyperdimensional data analysis using parallel coordinates J Am Stat Assoc 1990 85 664 675 Minnotte MC West BW The Data Image: a tool for exploring high dimensional data sets In 1998 Proceedings of the ASA Section on Statistical Graphics 1999 25 33 MathSoft, Data Analysis Products Division S-Plus Version 34 for Unix-supplement, Seattle 1996 Young G Householder AS Discussion of a set of points in terms of their mutual distances Pyschometrika 1938 3 19 22 Cox TF Cox MAA Multidimensional scaling 1994 Chapman & Hall, London Heinrichs RW Zakzanis KK Neurocognitive deficit in schizophrenia: A quantitative review of the evidence Neuropsychology 1998 12 426 445 9673998 10.1037//0894-4105.12.3.426 Erlenmeyer-Kimling L Rock D Roberts SA Janal M Kestenbaum C Cornblatt B Adamo UH Gottesman II Attention, memory, and motor skills as childhood predictors of schizophrenia-related psychoses: The New York High-Risk Project Am J Psychiatry 2000 157 1416 1422 10964857 10.1176/appi.ajp.157.9.1416 Palmer BW Heaton RK Paulsen JS Kuck J Braff D Harris MJ Zisook S Jeste DV Is it possible to be schizophrenic yet neuropsychologically normal? Neuropsychology 1997 3 437 446 9223148 Cornblatt BA Malhotra AK Impaired attention as an endophenotype for molecular genetic studies of schizophrenia Am J Med Genet 2001 105 11 15 11424979 10.1002/1096-8628(20010108)105:1<11::AID-AJMG1045>3.3.CO;2-7 Weickert TW Goldberg TE Gold JM Bigelow LB Egan MF Weinberger DR Gognitive impairments in patients with schizophrenia displaying preserved and compromized intellect Arch Gen Psychiatry 2000 57 907 913 10986554 10.1001/archpsyc.57.9.907 Faraone SW Seidman LJ Kremen WS Toomey R Pepple JR Tsuang MT Neuropsychologic functioning among the nonpsychotic relatives of schizophrenic patients: the effect of genetic loading Biol Psychiatry 2000 48 120 126 10903408 10.1016/S0006-3223(99)00263-2 Horan WP Goldstein G A retrospective study of premorbid ability and aging differences in cognitive clusters of schizophrenia Psychiatry Research 2003 118 209 221 12834815 10.1016/S0165-1781(03)00078-7 Gottesman II Shields J Schizophrenia The Epigenetic Puzzle 1982 Cambridge: Cambridge University Press Goldstein G Allen DN Seaton BE A comparison of cluster solutions for cognitive heterogeneity in schizophrenia J Int Neuropsych Soc 1998 4 353 362

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==== Front BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-4-331529651210.1186/1471-2458-4-33Research ArticleField assessments in western Kenya link malaria vectors to environmentally disturbed habitats during the dry season Carlson John C [email protected] Brian D [email protected] Francois X [email protected] Department of Tropical Medicine Tulane University, 1430 Tulane Avenue SL-17 New Orleans, LA 70112, USA2 International Centre of Insect Physiology and Ecology, PO Box 44, Kisii, Nyanza Province, Kenya2004 5 8 2004 4 33 33 7 5 2004 5 8 2004 Copyright © 2004 Carlson et al; licensee BioMed Central Ltd.2004Carlson et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Numerous malaria epidemics have occurred in western Kenya, with increasing frequency over the past 20 years. A variety of hypotheses on the etiology of these epidemics have been put forth, with different implications for surveillance and control. We investigated the ecological and socioeconomic factors promoting highland malaria vectors in the dry season after the 2002 epidemic. Methods Investigations were conducted in Kisii District during the dry season. Aquatic habitats in were surveyed for presence of malaria vectors. Brick-making pits were further investigated for co-associations of larval densities with emergent vegetation, habitat age, and predator diversity. Indoor spray catches were completed in houses near aquatic habitats. Participatory rural appraisals (PRAs) were conducted with 147 community members. Results The most abundant habitat type containing Anopheles larvae was brick-making pits. Vegetation and habitat age were positively associated with predator diversity, and negatively associated with mosquito density. Indoor spray catches found that houses close to brick-making sites had malaria vectors, whereas those next to swamps did not. PRAs revealed that brick-making has grown rapidly in highland swamps due to a variety of socioeconomic pressures in the region. Conclusion Brick-making, an important economic activity, also generates dry season habitats for malaria vectors in western Kenya. Specifically, functional brick making pits contain less that 50% as many predator taxa and greater than 50% more mosquito larvae when compared with nearby abandoned brick making pits. Further evaluations of these disturbed, man-made habitats in the wet season may provide information important for malaria surveillance and control. ==== Body Background The World Health Organization estimates that 300 to 500 million people are diagnosed with malaria annually, causing 1.1 to 2.7 million deaths. Approximately 1 million of these deaths are among children in sub-Saharan Africa, where 90% of all malaria cases occur [1]. During the 1950's and 1960's, a coordinated world-wide effort succeeded in eliminating malaria transmission within countries with temperate climates, and dramatically reduced malaria transmission in many other countries. Since the collapse of this campaign, malaria has since resurged, surpassing pre-campaign infection rates in many places, and entering previously unaffected locations [2]. The global resurgence of malaria has been attributed to a number of factors: drug-resistant parasites, insecticide-resistant vectors, population shifts, war-damaged infrastructures, altered meteorological conditions, and drastic ecological transformation [1,2]. The first recorded epidemic of malaria in the highlands of western Kenya occurred in 1918/19, with additional epidemics occurring periodically until 1950 [3]. These initial epidemics were associated with both population movements and progressive construction of roads and a railway through the highlands. With these human activities, new aquatic habitats were created, facilitating a gradual spread of parasites and vector mosquitoes into the highlands from the low-lying hyperendemic-disease areas [4]. Adoption of an extensive malaria control program [5] temporarily kept the highlands free from epidemics. Since the 1980s, however, the incidence of highland malaria and frequency of epidemics have been increasing, with severe outbreaks in 1995, 1998/99, [6-9] and most recently in May through July, 2002. Fifteen districts are now considered epidemic-prone by the Kenyan government [10]. Climate changes have poorly predicted epidemics in the East African highlands [11-14]. Hay et al. [11] suggest that intrinsic population dynamics are more likely the cause of vector fluctuations. Investigators reviewing Kericho District tea plantation records [9,15,16] found no obvious change in average temperature or rainfall over the previous 20–30 years, stable tribal/ethnic composition in the study area, and a properly maintained health care system. These studies suggest a failure of medications was the most likely cause for the recurrence of malaria epidemics in the highlands. However, due to the retrospective approach, these studies were unable to investigate environmental/ecological changes in the highlands or evaluate the role of malaria vectors, as had been done for earlier epidemics [4]. Following the 2002 epidemic, this study was conducted to identify the ecological and socioeconomic variables affecting malaria vector densities and distributions. In this paper we connect specific man-made aquatic habitats present during the dry season with malaria vector larvae in western Kenya. Methods Study area The highlands of western Kenya are an ecologically diverse and densely populated region situated east of Lake Victoria. Detailed investigations have been conducted in Mosocho Division, a sub-section of Kisii Central District with a predilection for epidemics and high rates of malaria (Ministry of Health Kisii, personal communication). Kisii District covered 648 Km2 and contained 491,786 people in 2001 (759 people/Km2) [17]. Mosocho Division covered 97 hectares and contained 105,309 people (1,086 people/Km2). Agriculture was the primary industry in the area, conducted on family plots which surrounded smaller industries, such as quarrying, and extended to the borders of wetlands. Survey of aquatic habitats in Mosocho Division All sites with standing water within Mosocho Division were evaluated by standard dipping for presence of Anopheles mosquito larvae over the course of two weeks in the dry season (September 2002). Standing water was identified by canvassing Mosocho Division by vehicle and by foot, with the assistance of local inhabitants in the areas investigated. This technique especially relied on information from inhabitants when locating the more remote habitats, which may therefore be under-represented in the survey. Once identified, habitats were evaluated for presence of mosquito larvae using standard aquatic dippers. Dipping was performed around the perimeter of the habitat, with three dips performed at approximately one meter intervals. No effort was made to determine larval densities due to the patchiness of larval distribution within each habitat. Dipping was not performed more than one meter into the habitats, which may have led to an under representation of less abundant species. Cross-sectional survey of brick-making pits in Kisii District At each of five brick-making sites in Kisii District, three functional brick-making pits and three abandoned pits were evaluated for pit size, larval densities, percent emergent vegetation and predator taxa present in the habitat. (See Figure 1.) Functional pits were chosen by asking brick makers to identify sites in which bricks were currently being produced that had been in use for at least two weeks. Abandoned pits were chosen near functional pits that had not been used for brick-making during the previous four months according to brick makers. Surface area of the rectangular pits was estimated by multiplying the measurements of the habitat at maximum and minimum lengths. Larval densities were obtained by standard dipping method, averaging the yields of five dips per pit. Percent emergent vegetation was estimated visually. An estimation of predator diversity was obtained by adding the number of Vertebrata orders, Odonata suborders, Hemiptera families, and adult Dytiscidae (Coleoptera) size-categories collected from the pits with 20 cm diameter fine-mesh sieves capable of collecting organisms down to 1 mm in size[18]. Predators were surveyed after obtaining larval dips, with one minute spent in each pit. Figure 1 Brick-making site in the Kenyan highlands. Assessments of the shallow pools dug by brick-makers is taking place in the foreground as brick makers continue to excavate soil, combine the soil with water, mould the resulting mud into bricks, and then left to dry before firing. Spatial data To assess the spatial relationship between aquatic habitats and the presence of malaria vectors inside houses, a series of indoor spray catches (described by [19]) was performed on 5 September 2002. Nine houses were chosen along a transect from a valley used for brick-making up to a maximum elevation (1920 meters), and then down into the next valley, containing swamps. Houses were chosen for possession of a thatched roof, not having had a fire inside since the previous evening, and permission of the owner. Socioeconomic survey Forty nine Chiefs and ninety eight brick makers were interviewed at four locations within Kisii and Gucha Districts to determine the social and economic factors promoting brick-making in the highlands, as well as the historic development of the industry. Standard Participatory Rural Appraisal methodology was used to obtain qualitative data [20]. Statistical analysis The means of the variables in the cross-sectional survey were compared for independence between the 15 functional and 15 abandoned brick-making pits (SPSS 11.0 t-test for independence). GPS coordinates taken for correlating presence of Anopheles in a house with distance to brick-making pits and swamp were converted into decimal degree coordinates and integrated as map layers within a Geographical Information System (ArcView 8.2). The distances from each house to the nearest brick making pit and swamp boundary were generated using the distance tool within the ArcView software. Spearman's correlation coefficient (SPSS 11.0) was calculated to correlate presence of Anopheles in a house with distance to swamp, distance to brick-making pits, and elevation. Results Survey of aquatic habitats in Mosocho Division A dry-season survey of aquatic habitats in Mosocho Division was conducted to identify potentially important larval habitats of Anopheles mosquitoes. (See Table 1.) A total of 53 standing aquatic habitats were assessed, with 16 (30.2%) containing Anopheles larvae. 37.5% of Anopheles-positive habitats were functional brick-making sites containing only An. gambiae s.l. larvae, and 18.8% were abandoned brick-making habitats containing only An. gambiae s.l. larvae (combined for 56.3%). Of the four quarries positive for Anopheles larvae, three were functional (created in the previous year) and one was abandoned (unused > two years), both with An. gambiae s.l and An. funestus. A portion of a drainage canal dug near a brick-making sites constituted an additional Anopheles-positive habitat, containing An. gambiae s.l. larvae. Of natural habitats sampled, only two contained malaria vectors. One swamp had An. funestus larvae and one tree hole in an ornamental Flamboyant tree (Delonix regia) had An. gambiae s.l. larvae; 87.5% of Anopheles-positive habitats were of direct human origin. Table 1 Dry season survey of aquatic habitats Habitat Number of assessed habitats Number of habitats with Anopheles larvae % of habitats with Anopheles larvae % of total Anopheles larval habitats BMS (F) 6 6 100 37.5 Quarry (F) 4 3 75 18.8 BMS (A) 14 3 21.4 18.8 Quarry (A) 10 1 10 6.3 Tree hole 5 1 20 6.3 Swamp 6 1 16.7 6.3 Drainage 7 1 14.3 6.3 Fish pond 1 0 0 0 Stream pool 2 0 0 0 Total 53 16 30.2 100.3 BMS (F): Functional Brick-making site BMS (A): Abandoned Brick-making site Cross-sectional survey of brick-making pits in Kisii District Mean mosquito larvae densities were higher in functional than abandoned pits for both An. gambiae s.l. (2.87/dip in functional, 0.91/dip abandoned, p = 0.002) and Culex spp. (3.77/dip in functional; 1.32/dip in abandoned, p = 0.025). No An. funestus larvae were found in this study. This corresponded with an increase in predator biodiversity found in the abandoned sites (9.07 taxa in abandoned; 5.13 taxa in functional, p= 0.001) and increased percent emergent vegetation (0.27% in functional; 94.27% in abandoned, p < 0.001). (See Table 2.) Functional brick-making pits were an average of 7.8 square meters (standard deviation= 6.38; range = 1.65 to 22.5 square meters). Table 2 Ecological survey of brick-making pits Use N Mean Std. Deviation t-statistic p Percent vegetation Functional 15 0.27 0.46 -47.116 <0.001 Abandoned 15 94.27 7.71 Habitat age (months) Functional 15 0.62 0.17 -5.734 <0.001 Abandoned 15 24.20 15.92 Predator biodiversity Functional 15 5.13 2.36 -3.586 0.001 Abandoned 15 9.07 3.53 Average number of Anopheles/ dip (5 dips per pit) Functional 15 2.87 2.04 3.402 0.002 Abandoned 15 0.91 0.90 Average number of Culex/dip Functional 15 3.77 3.60 2.435 0.025 Abandoned 15 1.32 1.51 Spatial data Spray catches in nine houses during the dry season resulted in one Anopheles mosquito in five different houses. Distance from brick-making sites was negatively correlated with presence of Anopheles in houses (p = 0.002, r = -0.868), while distance from swamp was positively correlated with presence of Anopheles mosquitoes (p = 0.018, r = 0.758). Elevation was not correlated with presence of Anopheles in a house (p = 0.829, r = -0.085). Brick-making and socioeconomic survey According to brick-makers, making bricks is predominantly a dry-season activity due to the damage caused by heavy rains to drying bricks. Although variation in technique exists, universally used stages in the brick-making process are: Excavation, Fermentation, Moulding, Drying, and Kilning. During the Moulding stage, water is brought into the excavated clay pits and mixed with soil. During this stage, which lasts from several days to one month, water is continually supplied to the pit through irrigation systems, ground water, or is brought by bucket from a nearby water source. Once abandoned during the Kilning stage and afterwards, the pit accumulates rain water and ground water, which can be subsequently used as a water source for newly excavated pits. Over years, abandoned brick-making pits degrade until they are continuous with the surrounding swamp. Interviews with Chiefs and brick makers revealed different scales of brick-making operations, from individuals working on their own land to large-scale industries (Figure 1) where wetland plots are rented to brick-makers who employ a large number of casual laborers for the mass production of bricks. Brick-making is an increasingly popular means of obtaining income, spreading to new communities when skilled brick makers are hired to work new land for short-term projects. While brick-making has been passed through multiple generations, the large-scale industries have developed steadily over the previous 20 years. A primary socioeconomic factor described by brick-makers that promotes brick-making is the desire to make use of agriculturally unfavorable wetlands as human population densities increase. Nearly all brick makers work in wetlands, leading to progressive deforestation of the highland swamps. Money from brick-making was used principally for paying school fees and malaria medication. According to brick-makers, brick houses are considered to be more prestigious than traditional houses, are associated with lower long-term costs than traditional mud-thatched houses, and are less permeable to mosquitoes. Discussion Our study shows that man-made larval habitats were the predominant (87.5%) source of malaria vectors in Mosocho Division during the dry season. In particular, the continuously disturbed, functional brick-making pits contained high densities of malaria-vectoring mosquitoes. PRAs revealed that small brick-making groups have developed into large-scale industries over the past two decades, and brick-making is now dispersed throughout the highlands in unfarmable wetlands. Because brick-making occurs predominantly in the dry season, it may aid in maintaining vector populations year-round. Additionally, houses closest to brick-making pits had malaria vectors present within them. While the number of mosquitoes captured during a single transect of spray-catches was only one per house, this represents a real, if low, possibility of malaria transmission during the dry season at these locations. Thus brick-making areas may function as refugia for malaria parasites and their vectors over the dry season, facilitating spread of malaria when habitats become more plentiful in the wet season. In this study we found emergent vegetation to be negatively associated with presence of malaria vectors in man-made habitats. This association may be a result of an association of both emergent vegetation and predator diversity with habitat age. In continually disturbed habitats (such as functional brick-making pits), the habitats are kept at a low stage of biological succession, possessing fewer species of both plants and animals. There may also be a direct effect of vegetation on the trophic dynamics in ground pools. In structurally simple habitats, intraguild predation has been shown to suppress the diversity of important predators [21-23]. Whether due to co-associated variables, direct effects, or some combination, there was a sizable 57% increase in predator taxa found in abandoned, vegetated pits and a >50% reduction of Anopheles larval densities. More research into the ecology of small aquatic pools will help clarify the interrelationships of these variables. In ground pools, predators are typically thought to regulate the density of mosquito larvae [24,25]. Predators may exert an effect by consuming larvae or through deterring oviposition into an otherwise suitable habitats[26]. During the dry season, disturbed, man-made habitats (such as functional brick-making pits and quarries) provide a developmental habitat in which Anopheles larvae escape the high degree of predation found in the natural environment. These habitats should be specifically targeted during larval control programs. With substantial socioeconomic motivation for brick-making in the highlands, traditional source reduction (eliminating standing water) is unsustainable, and larvicides should be employed. Further research is needed on the use of these habitats by mosquito larvae during the wet season. The negative correlation between predator diversity and mosquito density suggests that pesticide application may exacerbate epidemics by decreasing predation pressure if used in natural habitats. Pesticide applications can be selectively harmful to larger predators such as dragonflies and large dytiscid beetles which take months to years to develop[27]. In contrast, larvicides have a smaller impact on rapidly developing insects such as mosquitoes, which can reach maturity in a week. Rapid recolonization of predator-free habitats by mosquitoes would lead to vector resurgence [8,28]. Thus, targeting larvicide application to disturbed aquatic habitats should lead to a better mosquito control than treating all available habitats. The social and economic benefits accompanying disease reduction through vector control was demonstrated in the Zambian copperbelt [29]. As the population density of the highlands of western Kenya grows, the social and economic costs of malaria are likely to grow as well, unless vector-centered interventions (including environmental management, larvicide application, and vector surveillance systems) are used to confront the disease. Competing interests None declared List of abbreviations PRA: Participatory Rural Appraisal Km: Kilometer BMS: Brick making site NGO: Non governmental organization Authors' contributions JCC designed and conducted evaluations of larval habitats and houses, performed statistical analysis, and wrote the initial draft of the manuscript. BDB performed GIS analysis. FXO identified BMS as sources of vector mosquitoes, designed and conducted PRAs, and aided in the production of the manuscript's final draft. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements The authors wish to thank John Gimnig for an insightful review of the manuscript. Funding for this research was provided by the Government of Finland through grant 24811201 and by BioVision Switzerland. Travel expenses for JCC were provided through NIH ICIDR grant U19 AI45511 and personal financial support provided through CDC fellowship training grant CCT 622308-02. ==== Refs WHO WHO Expert Committee on Malaria World Health Organ Tech Rep Ser 2000 892 i v, 1-74 10892307 WHO Africa Malaria Report 2003 1 69 Matson AT The history of malaria in the highlands in Nandi East Afr Med J 1957 34 431 441 13473734 Graham PCC The incidence of malaria at high altitudes J Nat Malaria Soc 1948 7 275 284 Roberts JMD The control of epidemic malaria in the highlands of western Kenya. Part III. After the campaign. J Trop Hyg 1964 59 230 237 Hay SI Noor AM Simba M Busolo M Guyatt HL Ochola SA Snow RW Clinical epidemiology of malaria in the highlands of western Kenya Emerg Infect Dis 2002 8 543 548 12023907 Hay SI Cox J Rogers DJ Randolph SE Stern DI Shanks GD Myers MF Snow RW Climate change and the resurgence of malaria in the East African highlands Nature 2002 415 905 909 11859368 10.1038/415905a Oloo AJ Vulule JM Koech DK Some emerging issues on the malaria problem in Kenya East Afr Med J 1996 73 50 53 8625864 Malakooti MA Biomndo K Shanks GD Reemergence of epidemic malaria in the highlands of western Kenya Emerg Infect Dis 1998 4 671 676 9866748 MoH National Malaria Strategy 2001-2010 2001 Nairobi, Republic of Kenya Division of Malaria Control 48 Hay SI Myers MF Burke DS Vaughn DW Endy T Ananda N Shanks GD Snow RW Rogers DJ Etiology of interepidemic periods of mosquito-borne disease Proc Natl Acad Sci U S A 2000 97 9335 9339 10922081 10.1073/pnas.97.16.9335 Hay SI Rogers DJ Randolph SE Stern DI Cox J Shanks GD Snow RW Hot topic or hot air? Climate change and malaria resurgence in East African highlands Trends Parasitol 2002 18 530 534 12482536 10.1016/S1471-4922(02)02374-7 Hay SI Simba M Busolo M Noor AM Guyatt HL Ochola SA Snow RW Defining and detecting malaria epidemics in the highlands of western Kenya Emerg Infect Dis 2002 8 555 562 12023909 Lindsay SW Martens WJ Malaria in the African highlands: past, present and future Bull World Health Organ 1998 76 33 45 9615495 Shanks GD Biomndo K Hay SI Snow RW Changing patterns of clinical malaria since 1965 among a tea estate population located in the Kenyan highlands Trans R Soc Trop Med Hyg 2000 94 253 255 10974991 Shanks GD Hay SI Stern DI Biomndo K Snow RW Meteorologic influences on Plasmodium falciparum malaria in the Highland Tea Estates of Kericho, Western Kenya Emerg Infect Dis 2002 8 1404 1408 12498655 MoFP Kisii Central District Development Plan 2002-2008, Effective management for sustainable economic growth and poverty reduction 2001 Nairobi, Republic of Kenya Merritt RW Cummins KW An introduction to the aquatic insects of North America 1996 3rd Dubuque, Kendall/Hunt Publishing Company 862 WHO Manual on practical entomology in malaria. 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Methods and techniques 1975 13 Geneva, World Health Organization McCracken Grant The long interview 1998 Thousand Oaks, Sage Publications Rypstra AL, PE Carter, RA Balfour, and SD Marshal Architectural features of agricultural habitats and their impact on the spider inhabitants J Arachnol 1999 27 371 377 Clark TF Messina FJ Foraging Behavior of Lacewing Larvae (Neuroptera: Chrysopidae) on Plants with Divergent Architectures Journal of Insect Behavior 1998 11 303 317 10.1023/A:1020979112407 Finke DL Denno RF Intraguild Predation Diminished in Complex-Structured Vegetation: Implications for Prey Suppression Ecology 2002 83 643 652 Sunahara T Ishizaka K Mogi M Habitat size: a factor determining the opportunity for encounters between mosquito larvae and aquatic predators J Vector Ecol 2002 27 8 20 12125876 Washburn JO Regulatory factors affecting larval mosquito populations in container and pool habitats: implications for biological control J Am Mosq Control Assoc 1995 11 279 283 7595462 Angelon KA Petranka JW Chemicals of predatory mosquitofish (Gambusia affinis) influence selection of oviposition site by Culex mosquitoes J Chem Ecol 2002 28 797 806 12035927 10.1023/A:1015292827514 Service MW Mortalities of the immature stages of species B of the Anopheles gambiae complex in Kenya: comparison between rice fields and temporary pools, identification of predators, and effects of insecticidal spraying J Med Entomol 1977 13 535 545 845895 Roberts DR Andre RG Insecticide resistance issues in vector-borne disease control Am J Trop Med Hyg 1994 50 21 34 8024082 Utzinger J Tozan Y Doumani F Singer BH The economic payoffs of integrated malaria control in the Zambian copperbelt between 1930 and 1950 Trop Med Int Health 2002 7 657 677 12167093 10.1046/j.1365-3156.2002.00916.x

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==== Front BMC Musculoskelet DisordBMC Musculoskeletal Disorders1471-2474BioMed Central London 1471-2474-5-241529196210.1186/1471-2474-5-24Research ArticleAltered mRNA expression of genes related to nerve cell activity in the fracture callus of older rats: A randomized, controlled, microarray study Meyer Martha H [email protected] Wiguins [email protected] Ralph A [email protected] Orthopaedic Research Laboratory, Department of Orthopaedic Surgery, Carolinas Medical Center, P.O. Box 32861, Charlotte, NC 28232-2861 USA2004 3 8 2004 5 24 24 25 3 2004 3 8 2004 Copyright © 2004 Meyer et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The time required for radiographic union following femoral fracture increases with age in both humans and rats for unknown reasons. Since abnormalities in fracture innervation will slow skeletal healing, we explored whether abnormal mRNA expression of genes related to nerve cell activity in the older rats was associated with the slowing of skeletal repair. Methods Simple, transverse, mid-shaft, femoral fractures with intramedullary rod fixation were induced in anaesthetized female Sprague-Dawley rats at 6, 26, and 52 weeks of age. At 0, 0.4, 1, 2, 4, and 6 weeks after fracture, a bony segment, one-third the length of the femur, centered on the fracture site, including the external callus, cortical bone, and marrow elements, was harvested. cRNA was prepared and hybridized to 54 Affymetrix U34A microarrays (3/age/time point). Results The mRNA levels of 62 genes related to neural function were affected by fracture. Of the total, 38 genes were altered by fracture to a similar extent at the three ages. In contrast, eight neural genes showed prolonged down-regulation in the older rats compared to the more rapid return to pre-fracture levels in younger rats. Seven genes were up-regulated by fracture more in the younger rats than in the older rats, while nine genes were up-regulated more in the older rats than in the younger. Conclusions mRNA of 24 nerve-related genes responded differently to fracture in older rats compared to young rats. This differential expression may reflect altered cell function at the fracture site that may be causally related to the slowing of fracture healing with age or may be an effect of the delayed healing. ==== Body Background Bone formation to bridge the fracture gap following skeletal fracture slows with age in both humans [1-6] and rats [7-9]. While young, 6-week-old rats reach radiographic union by 4 weeks after femoral fracture, adult, 26-week-old rats require 10 weeks, and older, 52-week-old rats need in excess of 6 months [7]. Despite this increased time to radiographic union with age, there was no increase in the time of expression of Indian hedgehog or any of the bone morphogenetic proteins in the fracture callus for adult rats [10] or for older rats [11,12]. Radiographic union for adult and older rats occurred well after the time of expression of these skeletally active cytokines [10,11]. Except for markers of osteoblast activity and bone matrix formation, few genes remain up-regulated during the time period when bone forms to bridge the fracture gap [10-12]. These earlier studies done with RT-PCR revealed a paucity of data for genes differentially expressed by age. We had hypothesized that bone formation to bridge the fracture gap would be under a negative-feedback control system. Thus, the genes which stimulate bone formation should be up-regulated in adult or older rats to attempt to accelerate their slower progression of bony healing. This was not observed in adult [10] or older [11,12] rats. Either bone formation to bridge the fracture gap is not subject to negative-feedback control, or the genes up-regulated to control this bone formation are not those normally thought of as being involved in skeletal homeostasis. This suggested the need for a wider search for genes active during the fracture reparative process. In this project, mRNA gene expression was measured by DNA microarray technology at various time points after fracture for young, adult, and older rats. The goal was to identify genes whose expression following fracture was altered by age. Such genes may either show reduced expression, if the age-related slowing of healing is caused by inadequate expression levels, or they may show enhanced expression, in an attempt to stimulate some poorly responding pathway. Among the genes which were differentially expressed at the fracture site with age were genes related to nerve cell activity. In this study, we explored whether abnormal mRNA expression of genes related to nerve cell activity was associated with the slowing of skeletal repair in older rats. Abnormalities in the innervation of the fracture site will slow skeletal healing clinically [13-15] and experimentally [16-18]. Methods Rats Intact female Sprague-Dawley rats (Harlan Sprague-Dawley, Inc., Indianapolis, IN) were purchased at one or six months of age and housed in our vivarium in pairs until they were the proper age for experimentation. The rats were fed Teklad Rodent Diet [W] (#8604, Harlan Teklad, Madison, WI) and tap water ad libitum. The work was done in an AAALAC-accredited vivarium under protocols approved by our Institutional Animal Care and Use Committee. Surgery Intact female Sprague-Dawley rats at 6 (young), 26 (adult) or 52 (older) weeks of age, weighing 154 ± 11 g (mean ± SD), 281 ± 25 g, and 330 ± 30 g respectively, were anaesthetized with an intraperitoneal injection of ketamine and xylazine (30 mg and 5 mg/kg body weight respectively) as described earlier [7,11]. The left knee was shaved, scrubbed with Betadine Solution (Purdue Frederick, Stamford, CT), and draped with sterile sheets. A medial incision was made at the knee, the patella was deflected laterally and a 1.0 mm hole was drilled into the intercondylar notch. An intramedullary rod (1.0 mm diameter, stainless steel, type 304V, O-SWGX-400, Small Parts, Miami Lakes, FL) was placed retrograde into the left femur [11]. The incision was closed with wound clips. A closed simple transverse mid-diaphyseal femoral fracture was induced with a Bonnarens and Einhorn device [19]. Randomly selected rats from among those scheduled for surgery were used for 0 time no-fracture sham controls. Rats were euthanized at 0, 0.4, 1, 2, 4, and 6 weeks after fracture for a total of 6 time points at each of the 3 ages. Six rats per time point per age group were selected for microarray analysis (2 rats/array). Radiographs were made at fracture, at 1 week after fracture, and at euthanasia. The femora were rapidly harvested, and one third of the femoral length, centered on the fracture site, was collected. This contained the fracture callus with associated cortical bone and marrow and was frozen in liquid nitrogen and stored at -75 C. RNA Sample Preparation and Microarray Processing Samples were prepared as described in the Affymetrix GeneChip Expression Analysis Technical Manual (copyright 2001, Affymetrix, Inc., Santa Clara, CA, Rev. 1, Part number 701021, ). The sample preparation is described here in brief. Total RNA was extracted from the tissue by TRIzol (Invitrogen Life Technologies, Carlsbad, CA) with disruption of the tissue in a Brinkman Polytron homogenizer. RNA from two rats of the same age and time point was pooled for each microarray sample. Samples with 30 μg RNA were purified on RNeasy columns by Qiagen (Valencia, CA, P/N 74104) and then converted to double-stranded cDNA with a Superscript Double Stranded cDNA Synthesis Kit (Invitrogen Life Technologies, P/N 11917-010). The cDNA was then expressed as biotin-labeled cRNA by in vitro transcription (IVT) with the Enzo RNA Transcript Labeling Kit (Affymetrix, P/N 900182). Each sample was spiked with bioB, bioC, bioD, and cre (Affymetrix P/N 900299). The biotin-labeled cRNA was fragmented non-enzymatically. The fragmented cRNA was hybridized to 54 Rat U34A microarrays (Affymetrix P/N 900249) in the Affymetrix hybridization buffer for 16 hours at 45 C. The hybridized arrays were washed and stained in the Affymetrix Fluidics Station 400 to attach fluorescent labels to the biotin, followed by biotin-labeled antibody, and then a second staining with fluorescent labeling of the biotin. Each array was scanned twice by the Agilent GeneArray Scanner G2500A (Agilent Technologies, Palo Alto, CA). Three arrays from three independent samples (six rats) were done for each age at each time point. Data Analysis The Rat U34A GeneChip Microarray has probe sets for over 8,700 rat genes. Most probe sets have 20 different probes for the same gene on each array with 20 additional mismatch controls. The data were analyzed with Affymetrix Microarray Suite 5.0 and Affymetrix Data Mining Tool 3.0 software. Microarray Suite was used to scale the mRNA expression (signal value) of all genes to an average of 500 for each array. For each gene, the software reported a signal value and a Present/Marginal/Absent call. This latter algorithm was a statistical comparison of the variation among the several probe sets for each gene compared to the noise level and gave a call for each gene as Present, Marginal, or Absent. The program then compared the signal value of each gene in the fractured samples against the signal value of the same gene in the unfractured control sample. The difference between the two signal levels, relative to the variability between the multiple probes for each gene, yielded a probability of change due to chance alone. Genes with p less than 0.005 were judged significantly different from the same gene in the unfractured sample. This more conservative p value was employed to minimize false positive responses. The Data Mining Tool was used for cluster analysis with the Self Organizing Map (SOM) algorithm. The data were clustered on the signal values between 20 and 20,000 with the maximum/minimum ratio of at least 3.0 and the maximum – minimum difference of at least 100. One hundred clusters were specified. Nerve-related genes were identified by searches for nerve-related names in the gene descriptions of each gene on the microarray. This association was confirmed by a review of the information for that gene in the NetAffx web site and in the PubMed database . GenBank accession numbers and names are shown for each gene. Each graph shows the average ± SEM of the three microarrays that were done for each time point for each age. Significant changes in gene expression were demonstrated by t test and linear regression [20]. This report conforms to the MIAME standards of MGED . A copy of the full microarray data set has been deposited in the NCBI Gene Expression Omnibus as series GSE594. Results Radiology In all young rats, bone bridged the fracture gap by four weeks after surgery. By six weeks after fracture, remodeling was beginning to obscure the fracture site (Fig. 1). In contrast, bone bridging in the adult rats progressed more slowly. The adult rats did have a vigorous periosteal reaction at the site of the fracture and were approaching radiographic union by six weeks after surgery (Fig. 1). In the older, one-year-old rats, bridging of the fracture gap by bone progressed the slowest. They had a minimal periosteal reaction at six weeks after surgery (Fig. 1). General results On each array, on average, 5,200 genes were scored as absent, and 3,300 as present. Of these, 1,159 were significantly up-regulated and 928 were significantly down-regulated at two weeks after fracture in the adult rats of the first series (see Additional File 1). Up-regulated genes included cytokines and matrix genes for both cartilage and bone. Down-regulated genes included genes related to blood cell synthesis and mitochondrial function. SOM clusters identified genes up- or down-regulated by fracture. Most genes affected by fracture followed the same time course at all three ages. These genes showed approximately the same peak expression level and regressed to baseline at about the same time point at all three ages. Among the genes affected by fracture were a number of genes associated with nerve cells. These were selected for more intense analysis. Similar responses at all three ages Up-regulated nerve-related genes are shown in Table 1. Two examples are shown in the upper two graphs in Figure 2. Both of these genes were significantly up-regulated from the 0 time control (P < 0.001 by t test for 9 samples (3 ages × 3 replicates) of 0 time vs. 0.4 week (Fig. 2, top graph) or vs. 0 time vs. 2 week (Fig. 2, middle graph)). Other nerve-related genes were down-regulated by fracture at all three ages (Table 2). These regained near normal activity by six weeks after fracture. An example is shown in the bottom graph of Figure 2. This gene (TAG-1) had a significant down-regulation after fracture (P < 0.001 by t test for 9 samples of 0 time vs. 0.4 week), followed by a significant increase at 6 weeks after fracture compared to 0.4 week after fracture (P < 0.001 by t test for 9 samples). Defects in the older rats SOM cluster analysis identified three types of defects in the older rats. In the first type, a number of genes were down-regulated by fracture at all three ages. However, while genes in the younger rats were returning to pre-fracture expression levels by six weeks after fracture, there was less recovery in the older rats. These genes are shown in Table 3, and three examples of these genes are shown in Figure 3. All three of these genes had a significantly decreased mRNA expression levels at 1 week after fracture compared to 0 time control (P < 0.001 by t test for 9 samples (3 ages × 3 replicates)). At 4 and 6 weeks after fracture, the young rats showed faster recovery in mRNA expression than did the older rats for the three genes in Fig. 3 (P < 0.01 by t tests for 4 and 6 week young vs. 4 and 6 week old). In the second type of defect, other genes were up-regulated by fracture, but the response was weaker in the older rats. These genes are shown in Table 4. Three examples are shown in Figure 4. The broad peaks of the genes in Figure 4 permitted the t test to demonstrate a significantly higher expression level in the young rats at 1 and 2 weeks after fracture in comparison to the same time points of older rats. These comparisons for the three genes in Figure 4 were significant at P < 0.001 (top graph, AF034963), P < 0.02 (middle graph, AB005541) and P < 0.01 (bottom graph, U09357) for 6 samples per age group (2 time points pooled for 3 replicates). In the third type of defect, genes were also up-regulated by fracture. However, the response was stronger in the older rats than in the younger rats. These genes are shown in Table 5, and three examples are shown in Figure 5. The peak values for these three genes significantly increased with age by linear regression (P < 0.001 (top graph, AF030089), P = 0.01 (middle graph, M88469), and P < 0.001 (bottom graph, X89963) for 9 data points (3 ages × 3 replicates)). Present/Marginal/Absent calls For each gene for each array, the Microarray Suite software reported a statistical decision as to whether the mRNA was Present, Marginal, or Absent. We have reviewed these calls for the genes shown in Figures 2,3,4,5. For Figure 2, the Present/Marginal/Absent calls were: Fig. 2 Top: 53/1/0; Fig. 2 Middle: 39/2/13; and Fig. 2 Bottom: 0/0/54. For Figure 3, the calls were: Fig. 3 Top: 45/2/7; Fig. 3 Middle: 7/0/47; and Fig. 3 Bottom: 32/6/16. For Figure 4, the calls were: Fig. 4 Top: 0/0/54; Fig. 4 Middle: 0/0/54; and Fig. 4 Bottom: 51/0/3. For Figure 5, the calls were: Fig. 5 Top: 45/1/8; Fig. 5 Middle: 52/0/2; and Fig. 5 Bottom: 54/0/0. Discussion In this study, as in our earlier work [7,10-12], the time required to reach radiographic union after femoral fracture increased with age in the female rat. This slowing of fracture repair with age is associated with changes in the mRNA expression of specific genes within the healing fracture site [10-12]. To study this further, microarray technology was used to identify additional genes whose mRNA expression was affected by skeletal fracture. More than two-thirds of the detectable genes on the rat U34A microarray have a change in mRNA expression level following fracture [21]. Most of these genes were not known to participate in the healing process of bone before the advent of microarray technology [21,22]. This reflects changes in both the types of cells at the fracture site as well as changes in the activity of the existing cells. Among the cells affected by fracture are nerve fibers. Protein and mRNA of genes related to neuronal functioning are found in intact bone and in the fracture callus [23-29]. Since proper innervation of the fracture site is needed for fracture repair clinically [13-15] and experimentally [16-18], this led to the hypothesis that the age-related slowing of fracture repair may be related to the abnormal nerve cell activity at the fracture site. To evaluate this hypothesis, nerve-related genes were studied from among the genes present on the Affymetrix Rat U34A microarray. Genes were identified for which the mRNA response to femoral fracture was changed in the older rats compared to the young rats. Three types of change with age were found: 1. The mRNA expression levels of the genes shown in Table 3 and Figure 3 were decreased by fracture. While gene expression in the young rats was approaching pre-fracture levels by six weeks after fracture, gene expression showed minimal return to normal in older rats. Genes in this category were all related to signaling molecules or to signal receptors (references shown in Table 3). 2. Other nerve-related genes had strong up-regulation after fracture in young rats but only mild up-regulation in older rats. These are shown in Table 4 and Figure 4. This partial loss of function with age was observed in genes associated with nerve cell differentiation or cell cycle or genes related to synaptic structure (references cited in Table 4). 3. A third set of genes was increased in mRNA expression by fracture, but the increase was greater in the older rats. These are shown in Table 5 and Figure 5. Many of these genes were related to cell adhesion or to cell signal or signal transduction (references cited in Table 5). All three classes of genes showed altered expression in the older rats compared to young rats. We hypothesize that bone fracture may physically disrupt nerve fibers in bone. A sub-population of these skeletal nerve fibers may regrow into the fracture site or regain function at a slower rate in older rats. This may account for the failure to recover from low mRNA values for the first group (prolonged down-regulation) or the failure to up-regulate mRNA expression adequately after fracture in the older rats in the second group (diminished up-regulation). Other genes in the third group with increased levels of mRNA after fracture in the older rats may represent attempts to stimulate nerve regrowth or other processes that are not responding. This may represent negative-feedback-induced up-regulation caused by effector cell resistance. Taken together, these changes in nerve cell function with age may contribute to the slowing of fracture repair in older rats. It must be pointed out that the associations noted here do not necessarily reflect cause and effect. It is also possible that the delayed re-innervation of the fracture site is an effect of the delayed healing in the older rats and not a cause of the delayed healing. Experimental studies have been done to detect the role of innervation on fracture healing. Studies of sectioning the sciatic nerve in concert with tibial fracture have been reported to speed fracture healing [30-33]. However, sectioning both femoral and sciatic nerves inhibits fracture healing [18]. Aro et al. [16] have reported mechanoreceptors (Pacinian corpuscles) in the periostium of the rat fibula, which, if removed, lead to non-union [17]. Direct application of nerve growth factor to the fracture site increases healing in the rat rib [34]. In humans, abnormal bone healing is also associated with lack of nerve activity at the fracture site. Nagano et al. [13] have noted scaphoid nonunion in the wrists of patients with neuroarthropathy from a long-standing nerve palsy. Santavirta et al. [14] have found a lack of peripheral innervation at the fracture site of noninfected fractures with delayed union or nonunion of diaphyseal bones. Nordstrom et al. [15] have found a lack of stromal innervation associated with delayed union or pseudoarthrosis in spondylolysis. Humans [1-6] show a slowing of fracture healing with increasing age as do rats [7-9]. The cause of the slowing of fracture healing with age is not well understood. The femora of young rats regain normal biomechanical properties by 4 weeks after fracture, while adults take 12 weeks, and older rats require in excess of 6 months [7]. This model presents an opportunity to elucidate novel genes important to this healing process. The slowing could reflect a loss of function as some processes essential for the rapid healing of fractures in young animals are inhibited with age. Alternatively, the slowing of skeletal repair with age may be caused by partial resistance of the healing process to stimulation in adult or older individuals. Such resistance should result in enhanced stimulation by regulatory systems to attempt to evoke a healing response. Both patterns were seen among the genes studied in this report. These genes are candidates for further study. These changes with age are not limited to genes related to neuronal activity. We have also noted similar changes in genes related to mitochondrial activity [35]. It is likely that the age-related changes in fracture repair are caused by failure of several metabolic pathways. Methods, such as DNA microarrays, which sample many different biological pathways will be useful in defining these novel, multi-faceted defects. The specificity of these changes is seen in the majority of the nerve-related genes for which the expression pattern following fracture was unaffected by age. These transcripts had similar increases or decreases following fracture in the young, adult, and older rats. These uniform responses suggest that most metabolic patterns were unaffected by age. Nerve-related genes similarly up-regulated by femoral fracture at all three ages were broadly related to differentiation and growth of nerve cells, to known up-regulation following nerve injury, or to association with apoptosis (references cited in Table 1). Some of these genes were slower to return to baseline values in older rats, such as galanin and TAG-1. In contrast, nerve-related genes similarly down-regulated by femoral fracture at all three ages were broadly related to the nerve growth cone or to synaptic signaling pathways (references cited in Table 2). In this study gene expression was measured by quantification of the mRNA level for each gene with microarray technology. It must be kept in mind that there are other control systems which influence the protein synthetic rate and also protein degradation. Protein synthesis will be low in the absence of mRNA for that gene, but elevated mRNA levels are not a guarantee that protein levels will also be elevated for that gene. Changes noted at the mRNA level will need to be confirmed at the protein and structural levels. Assignment of the genes studied herein as nerve-related is made on the basis of currently available information. Other cell types in the fracture callus may also express these genes. Histological studies will permit the association of these genes with specific cell types within the fracture callus. These experiments are now in progress. We have compared mRNA gene expression by microarray to that measured by reverse transcription – polymerase chain reaction (RT-PCR)[36]. Good correlation was found between the two methods if the transcripts were judged mostly present, the signal level did not approach the upper limit of the detector, and the probe sets or PCR primers were from the same region of the gene [36]. Some other genes, even though most samples were judged absent, also gave good correlation between the two methods. These latter genes were at the upper range of the absent calls and had good precision between samples [36]. The genes reported herein have the marked variation in mRNA levels that have been reported previously in fracture samples [10,11] with large changes in expression after fracture which return to the prefracture levels as healing progresses. The finding here of moderate signal levels, good precision among the three samples for each time point at each age, and a strong response to fracture indicate the ability of this technology to report changes in mRNA levels for these genes. Conclusions In summary, most genes respond to bone fracture with altered mRNA gene expression, including genes related to neuronal functioning. However, a number of these genes responded to fracture differently in older rats than in young rats. Such differential expression with age may reflect altered cell functioning at the fracture site that may be related to the slowing of fracture healing in older rats. Competing interests None declared. Authors' contributions MM, WE, and RM participated in the surgery and the sample collection. MM and WE prepared and hybridized the samples and analyzed the data. RM prepared the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Additional File 1. Response of adult rats at two weeks after fracture. This spreadsheet is a listing of all genes altered by fracture in the first adult rat sample at two weeks after fracture compared to the first adult no-fracture sample. The data are sorted on the probability value for the comparison of each gene between the two arrays. For each array, the signal (mRNA transcript level), detection (present, absent, or marginal) and detection p-value are given. In addition, for the two-week sample, the change (increase, marginal increase, marginal decrease or decrease) and change p-value are given for the comparison of the two-week sample to the no-fracture sample. These two arrays are archived in the GEO repository as samples GSM9028 (no-fracture sample) and GSM9031 (2-week sample). Click here for file Acknowledgements This study was supported in part by grants from the North Carolina Biotechnology Center and the Orthopaedic Trauma Association. We thank Drs. Scott Porter, James Fiechtl and Daniel Heiner for their surgical assistance. We thank Ms. Virginia Mooney, Mr. Michael Steuerwald, and Ms. Jessica Heath for their technical assistance and Ms. Carolyn Ayers for her secretarial assistance. A preliminary report of this study has appeared [37]. Figures and Tables Figure 1 Radiographs. Radiographs of the fractured left femora at 6 weeks after fracture in rats 6(A), 26(B), and 52(C) weeks old at fracture. Figure 2 mRNA levels of three nerve-related genes affected by fracture in young, adult, and older rats. The first two genes were up-regulated at all three ages (0.4 week (top graph) and 2 weeks (middle graph) exceed 0 time control at P < 0.001), while the third gene was down-regulated at all three ages (0.4 week significantly less than 0 time control at P < 0.001). Rats were 6, 26 and 52 weeks of age at fracture respectively. Samples were collected at the indicated times after fracture. The 0 time samples were no-fracture controls. Each bar is the mRNA expression level for the indicated gene for the average ± SEM of three DNA microarrays in arbitrary units of fluorescence. mRNA from two rats of the same age and time after fracture were pooled for each array. Gene identifications are shown with their GenBank accession number. Axonal glycoprotein (TAG-1) is also known as contactin 2. Figure 3 mRNA levels of three nerve-related genes with more prolonged down-regulated after fracture in the older rats. The 1 week values were significantly less than 0 time controls for all three graphs (P < 0.001). Note the failure of the mRNA activity to return to pre-fracture levels in the older rats (4 and 6 week values for young exceeded older rat values at P < 0.01). The data are presented as in Figure 2. Figure 4 mRNA levels of three genes related to nerve function and up-regulated more in young rats than in older rats following femoral fracture. The decline with age was significant at P < 0.001 (top graph), P < 0.02 (middle graph) and P = 0.01 (bottom graph). The data are presented as in Figure 2. Figure 5 mRNA levels of three genes related to nerve function and up-regulated more in older rats than in younger rats following femoral fracture. The increase with age was significant at P < 0.001 (top graph), P = 0.01 (middle graph), and P < 0.001 (bottom graph). The data are presented as in Figure 2. Table 1 Nerve-related genes up-regulated by fracture at all three ages GenBank Name Reference Genes related to apoptosis S67769 Cardiac sodium/calcium exchanger (Slc8a1) [38] M82824 Leucopus neurofibromatosis protein – type 1 (NF1) [39] L11319 Signal peptidase complex 18kD [40] Genes up-regulated by injury J03624 Galanin (shown in Fig. 2) [41–43] E12625 Rat novel protein expressed with nerve injury (sterol-C4-methyl oxidase-like) [44] Genes related to nerve cell differentiation and growth Z11558 Glia maturation factor beta [45–47] A03913 Glia-derived neurite-promoting factor (serine (or cysteine) proteinase inhibitor, clade E, member 2) [48,49] E05646 Hippocampal cholinergic neurostimulating peptide (phosphatidylethanolamine binding protein) [50] AB011531 MEGF 5, slit homolog 3 [51] AA924772 Metallothionein 3 [52,53] AF097593 N-cadherin, Testicular (cadherin 2) [54,55] AF023087 Nerve growth factor induced factor A (early growth response 1) [56,57] AI102795 Pleiotrophin (heparin binding growth factor 8, Hbnf, neurite growth promoting factor 1) (Fig. 2) [58–60] L19180 Receptor-linked protein tyrosine phosphatase type D (protein tyrosine phosphatase, receptor type D) [61] AF044910 Survival motor neuron [62] U64689 Synaptotagmin interacting protein zygin II (fasciculation and elongation protein zeta 2) [63] Other genes L07281 Carboxypeptidase E [64,65] U03416 Neuronal olfactomedin-related ER localized protein [66] U67140 PSD-95/SAP 90 associated protein-4 (disks large-associated protein 4) [67] M83680 RAB14 (GTPase Rab 14) [68] AF016247 RTK40 homolog (tyro 10, discordin domain receptor family, member 2) [69] X65454 SC65 – Synaptonemal complex protein [70] AJ006855 Synaptojanin 1 [71] M96601 Taurine transporter (Slc6a6) [72] U35245 Vacuolar protein sorting homolog r-vps33b [73] Alternative names are shown in parentheses. References to physiological roles are shown to the right. Table 2 Nerve-related genes down-regulated by fracture at all three ages Genbank Name Reference Genes associated with the growth cone of nerve fibers M31725 Axonal glycoprotein TAG-1 (Contactin 2) (shown in Fig. 2) [74] S82649 Neuronal activity-regulated pentraxin (Narp) [75] X59149 Neural cell adhesion molecule L1 [76–78] Genes associated with synaptic signaling pathways L08496 GABA-A receptor delta subunit [79] S68944 Na+/Cl--dependent neurotransmitter transporter [80] S76742 Neurotransmitter transporter rB21a (X transporter protein 3) [81] U39549 Synaptogyrin (synaptogyrin 1) [82] D28512 Synaptotagmin III (synaptotagmin 3) [83] AF007758 Synuclein 1 (synuclein alpha) [84] Other genes AF081557 Glial cells missing protein homolog (Gcm1, glial cells missing homolog a) [85] L15305 Glial-derived neurotrophic factor [86] M24852 Neuron-specific protein PEP-19 (Purkinje cell protein 4) [87] D17521 Protein kinase C-regulated chloride channel [88] Data presented as in Table 1. Table 3 Nerve-related genes with prolonged down-regulation after fracture in older rats GenBank Name Reference L09119 C kinase substrate calmodulin-binding protein (neurogranin) (shown in Fig. 3) [89] S77867 G coupled protein receptor UHR-1 (G protein coupled receptor 10) [90] AF109405 GABA-B receptor 2 (G protein-coupled receptor 51) [91] U08290 Neuronatin alpha (neuronatin) [92] M15880 Neuropeptide Y (shown in Fig. 3) [93,94] S56508 Fatty acid coenzyme A ligase, long chain 6 [95] AF041083 RoBo-1 (Slc11a1) (shown in Fig. 3) [55,96,97] X86789 Sensory neuron synuclein (gamma-synuclein) [98,99] Data presented as in Table 1. All genes are related to signals and signal receptors. Table 4 Nerve-related genes with a greater up-regulation in younger rats GenBank Name Reference Genes related to nerve cell differentiation and the cell cycle X86003 Neuron-derived orphan receptor-2 [100] AB005540 PCTAIRE 2 [101] AB005541 PCTAIRE 3 (shown in Fig. 4) [102] U09357 Protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (shown in Fig. 4) [103] Genes related to the synapse and sensory perception AI179632 Proton gated cation channel DRASIC [104] AF034863 Synaptic scaffolding molecule (S-SCAM, activin receptor interacting protein 1) (shown in Fig. 4) [105,106] S56141 Sodium-dependent neurotransmitter transporter [107] Data presented as in Table 1. Table 5 Nerve-related genes with a greater up-regulation in older rats GenBank Name Reference Genes related to signals and signal transduction X74832 Acetylcholine receptor alpha subunit (cholinergic receptor, nicotinic, alpha polypeptide 1) [108] M16112 Brain type II calcium/calmodulin dependent protein kinase beta subunit [109] Genes related to cell adhesion M88469 F-spondin (shown in Fig. 5) [110] AF060879 Neurocan (chondroitin sulfate proteoglycan 3) [111,112] U16845 Neurotrimin [113] M96375 Non-processed neurexin 1-beta (neurexin 1) [114] X89963 TSP-4 (thrombospondin-4) (shown in Fig. 5) [115–117] Other genes AF030089 Activity and neurotransmitter-induced early gene 4 (ania-4) (shown in Fig. 5) [118] D88250 Serine protease (complement component 1, s subcomponent) [119] Data presented as in Table 1. ==== Refs Bronk JT Ilstrup D Melton J Bolander ME Age and gender effect fracture repair in the humerus Trans Orthop Res Soc 1997 22 258 (Abstract) Skak SV Jensen TT Femoral shaft fracture in 265 children. 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==== Front BMC EcolBMC Ecology1472-6785BioMed Central London 1472-6785-4-91528799110.1186/1472-6785-4-9Research ArticlePreliminary inventory and classification of indigenous afromontane forests on the Blyde River Canyon Nature Reserve, Mpumalanga, South Africa Lötter Mervyn C [email protected] Hans T [email protected] Terrestrial Services, Scientific Services, Mpumalanga Parks Board, Private Bag X1088, Lydenburg, 1120, South Africa2 Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115 USA2004 2 8 2004 4 9 9 31 1 2004 2 8 2004 Copyright © 2004 Lötter and Beck; licensee BioMed Central Ltd.2004Lötter and Beck; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Mixed evergreen forests form the smallest, most widely distributed and fragmented biome in southern Africa. Within South Africa, 44% of this vegetation type has been transformed. Afromontane forest only covers 0.56 % of South Africa, yet it contains 5.35% of South Africa's plant species. Prior to this investigation of the indigenous forests on the Blyde River Canyon Nature Reserve (BRCNR), very little was known about the size, floristic composition and conservation status of the forest biome conserved within the reserve. We report here an inventory of the forest size, fragmentation, species composition and the basic floristic communities along environmental gradients. Results A total of 2111 ha of forest occurs on Blyde River Canyon Nature Reserve. The forest is fragmented, with a total of 60 forest patches recorded, varying from 0.21 ha to 567 ha in size. On average, patch size was 23 ha. Two forest communities – high altitude moist afromontane forest and low altitude dry afromontane forest – are identified. Sub-communities are recognized based on canopy development and slope, respectively. An altitudinal gradient accounts for most of the variation within the forest communities. Conclusion BRCNR has a fragmented network of small forest patches that together make up 7.3% of the reserve's surface area. These forest patches host a variety of forest-dependent trees, including some species considered rare, insufficiently known, or listed under the Red Data List of South African Plants. The fragmented nature of the relatively small forest patches accentuates the need for careful fire management and stringent alien plant control. ==== Body Background Mixed evergreen forests form the smallest, most widely distributed and fragmented biome in southern Africa [1]. Originally classified as Undifferentiated Afromontane forest [2], 44% of this vegetation type within South Africa has been transformed [2]. The forest biome only covers 0.56 % of South Africa [3], yet it contains 5.35% of South Africa's plant species [4]. These forests have a relatively high species richness of 0.58 species km-2, exceeding the grassland biome with 0.25 species km-2 and lagging only the fynbos with 1.36 species km-2[5]. The forest biome occurring in Mpumalanga is now recognized as Mpumalanga Mistbelt Forest [6], and it covers only 0.51% of the province's surface area [7]. Cooper [8] conducted a study on the conservation status of indigenous forests within Transvaal, Natal and the Orange Free State, in which he estimated the Blyde River Canyon Nature Reserve's (BRCNR) forests at 352 ha. Recently, ownership of a 700 ha forest tract bordering on BRCNR at the base of the Drakensberg Escarpment was transferred to the Mpumalanga Parks Board (MPB). Therefore, it was thought that just over 1000 ha of forest occurred on BRCNR. To begin to understand the nature and distribution of forests in the BRCNR landscape, we need to have an accurate surface area inventory, a species composition list, and an identification and classification of the communities. Standard ecological fieldwork coupled with remotely sensed data and GIS allow for an efficient analysis. Prior to this investigation of the indigenous forests on BRCNR very little was known about the size and floristic composition of the forest biome conserved within the reserve. In comparison, the grassland biome has received much attention on BRCNR with a monitoring program set up to assess the status of the grasslands. We report here an inventory of forest size, fragmentation, species composition and the basic floristic communities along environmental gradients. Study area BRCNR is situated on the northern Drakensberg Escarpment, Mpumalanga Province, South Africa (24°40'S longitude, 30°51'E latitude; Figure 1). The Mpumalanga Parks Board administers this reserve, which is approximately 29 000 ha in size. The elevation on BRCNR ranges from 560 m to 1944 m above sea level. The stratigraphy of the northern Drakensberg Escarpment region is composed of sediment rock types (quartzite, shale, and dolomite) of the Transvaal Supergroup [9], dominated by Black Reef Quartzite and Wolkberg Group [10]. Rainfall varies from 541 mm to 2776 mm per annum. Variation in altitude and rainfall, associated with a landscape of geological and pedological extremes, has created a very diverse flora. The landscape is prone to lightning-induced burning [11] and is topographically complex, hosting a variety of habitats [12], including grassland plateaus, wetlands and sponge areas, grassland slopes, afromontane forest, riparian forest, moist woodlands, dry woodlands and shrublands. The vegetation type is classified as the Northeastern Drakensburg High-Mountain Sourveld ecoregion [13], and the reserve encompasses four veld types: Afromontane Forest, North-eastern Mountain Sour Grassland, Sour Lowveld Bushveld, and Mixed Lowveld Bushveld [14]. Figure 1 Reserve location. The Blyde River Canyon Nature Reserve is located in northeastern Mpumalanga Province, South Africa. Results Spatial distribution of forests We found a total area of 2111 ha of afromontane forest on BRCNR. This comprises 7.3% of the reserve's total surface area. Figure 2 shows the distribution of the forest on BRCNR. The forest is fragmented, with a total of 60 forest patches recorded, varying from 0.21 ha to 567 ha in size. The number of patches per size class illustrates this level of fragmentation (Figure 3). Unfortunately, 15 % of the larger forest patches have their patch size delimited by political boundaries. This means that a few forest patches continued over BRCNR's borders onto adjacent land and only the portions occurring on BRCNR were included in the calculations. If the average patch size is only calculated from patches with natural boundaries within the reserve, then the average size of forest patches is only 23 ha. This small patch size accentuates the fragmented nature of the afromontane forest on BRCNR. Figure 2 Forest distribution. Distribution of afromontane forest fragments in the Blyde River Canyon Nature Reserve. Figure 3 Forest patches per size class. Within the BRCNR, a total of 60 forest patches are recorded, varying from 0.21 ha to 567 ha in size. Flora and forest classification The forest flora recorded in 22 relevés is listed in Appendix A (see additional file 1). The total of 167 species includes 38 ferns and fern allies, 3 conifers and 140 flowering plants (11 monocots, 131 dicots). Previous botanical field work and logistical reconnaissance of the forest fragments within the park boundaries showed the forest flora to be qualitatively similar. However, some areas of the fragments were unreachable or technically difficult to visit and study due to the escarpment topography. For purposes of this vegetation analysis, the relevés were located (Figure 4) in two of the five largest forest patches, located in the central region of the BRCNR, where forest fragments were at least reasonably accessible. At this time, we consider these relevés to be representative. While the forest flora of BRCNR has not been exhaustively surveyed, this species list represents our best knowledge collected to date. Figure 4 Forest fragment study plots. Distribution of forest study plots (yellow dots) within the Blyde River Canyon Nature Reserve boundaries (red lines) shown on Landsat 7 satellite image. From our analysis of the forest vegetation in the relevés, two main plant communities – moist afromontane forest at high altitude and dry afromontane forest at lower altitude – are identified and described below. An eigenvalue of 0.33 was produced from the TWINSPAN algorithm at the first division into two communities. This value is strong enough to represent beta diversity and the division into two plant communities is accepted. Each community was then further broken down again into two communities, whose eigenvalues were lower and therefore taken as a measure of alpha diversity. The four proposed sub-communities are accepted as variants within the two larger communities. A complimentary analysis of forest communities using DECORANA was carried out and supports the results of the CANOCO analysis. Figure 5 displays the results from the CANOCO analysis. This joint plot data is analyzed for relationships with environmental data. These environmental variables are displayed as arrows radiating from the center of the diagram with the length of the arrows representing each environmental variable's contribution to explaining the variation in the sample scores on each axis. Altitude contributed most of the variation in samples along the bottom axis. Axis 1 is therefore based on an altitudinal gradient. Other environmental variables that contributed towards the floristic variation include drainage, slope, rockiness, shrub density, herb density, climber density and canopy density. The effects of aspect and topography were excluded, as their contribution was small. An altitudinal gradient accounts for most of the variation within the forest communities. Figure 5 Relationship between plant communities and environmental gradients. A CANOCO joint plot showing the relationship between the proposed plant communities and environmental gradients. The length and position of the arrows indicate the strength of the relationship and the direction of change. Moist high-altitude afromontane forest This was a tall forest community that was heavily logged at the turn of the 20th century. The canopy is still very uneven as the sub-canopy trees have not yet matured or replaced those trees which were originally removed. This forest occurs in the mist-belt along the escarpment at altitudes of between 1450 m and 1700 m. The slopes are steep and generally scattered with large boulders. Preferential species identified for this community include Clivia caulescens, Ochna arborea, Podocarpus latifolius, Rapanea melanophloeos, Vernonia wollastonii and Blechnum attenuatum. Epiphytes are common in these forests. Within this community, two variants are recognized based on canopy development. Degraded moist high-altitude forest A very broken and open canopy with a large shrub component characterizes this forest type. A number of species within this community are shade-intolerant and have established themselves under an open canopy. This sub-community has been so over-utilized that the canopy has never been able to close and this community is sometimes dominated by the exotic invader species Acacia mearnsii. Other preferential species include Rubus spp., Myrsine africana and Helichrysum chrysargyrum. This community is only temporary as it will eventually be replaced by climax species during the next seral stage when the canopy closes. Developing moist high-altitude forest This forest type has a closed canopy with only a few pioneer, shade-intolerant species, such as Rhus tumulicola, Maesa lanceolata and Acacia mearnsii, found in the canopy. The shrub component is not nearly as dense as the degraded sub-community community. Preferential species include Behnia reticulata, Dovyalis lucida, Olea capensis subsp. macrocarpa, Asplenium rutifolium and Jasminum abyssinicum. Dry low-altitude afromontane forest Parts of this forest type were also logged at the turn of the 20th century. This dry forest community occurs just below the escarpment mist-belt, from 1200 m to 1450 m above sea level. Slopes are steep to gentle, with scattered large boulders. Preferential species in this forest community include Brachylaena transvaalensis, Podocarpus falcatus, Adenopodia spicata, Lauridia tetragona and Pteris captoptera. Within this community, two variants are recognized based on degree of slope. Dry forest on gentle slopes or along drainage lines This forest type occurs in areas with a gentle slope or along drainage lines. Both the shrub and herb layer densities are high with the increase in soil moisture gained directly from drainage lines or as a result of poor drainage on gentle slopes. Differential species include Acacia ataxacantha, Combretum kraussii, Eugenia natalitia, Gymnosporia mossambicensis and Faurea galpinii. Dry forest along steep slopes These low altitude forests occur along steep slopes. Overall canopy density is high with a poorly developed herb layer. This sub-community eventually grades into the moist high-altitude afromontane forest at higher altitudes. Preferential species include Combretum edwardsii, Cryptocarya transvaalensis, Chionanthus peglerae, Xymalos monospora, Oxyanthus speciosus and Prosphytochloa prehensilis. Species responses Our analysis showed that altitude appears to be the primary environmental variable responsible for the distribution of forest plant species along environmental gradients. Specific species responses to environmental variables are depicted in Figure 6. With 167 species recorded in the plots, only those species with a cumulative fit of more than 0.35 are displayed. Canopy density is used as a surrogate for measuring disturbance, or forest dynamics. It is expected that forest dynamics would have an influence on the distribution of forest species. Figure 6 Species responses to environmental variables. Upper graph is a scatter diagram produced from CANOCO using the CCA procedure, displaying species responses to environmental variables. Lower graph displays strength and position of environmental variables. Asparagus virgatus, Dicliptera clinopodia, Setaria megaphylla and Desmodium repandum are a few of the herbaceous plants that prefer the lower lying forests. Some of the trees include Euclea crispa, Rhamnus prinoides, Combretum kraussii, Myrsine africana, Lauridia tetragona, Protorhus longifolia and Scolopia mundii. Species that occur in the higher lying forests include Oxyanthus speciosus, Xymalos monospora, Rothmannia capensis and Cassipourea malosana. Some of the species that appear to prefer rocky areas include Clivia caulescens and Blechnum attenuatum. On the other hand, Prosphytochloa prehensilis, Rawsonia lucida and Ptaeroxylon obliquum seem to favor areas almost devoid of rocks or boulders. Species that favor low canopy densities, or higher levels of disturbance, are Rubus sp., Blotiella natalensis, Acacia mearnsii and Schefflera umbellifera. Discussion Geldenhuys [1] states that species diversity within forest patches is determined by patch size and proximity to other forests, which together explain 82% of the species richness in South Africa's forests. The floristic variation within BRCNR's forests can be attributed predominantly to variation in species composition along an altitudinal gradient. Extremes in environmental variables and gradients, associated with a history of over-utilization, have resulted in a floristically and dynamically diverse forest type. Fire exclusion, clearing of old plantation sites, and a history of intensive and selective logging have all contributed to the variation in forest dynamics currently occurring on BRCNR. The afromontane forests occurring on BRCNR are extremely fragmented, and yet over 2100 ha of forest patches are conserved within its boundaries. A large proportion of forest-dependent tree species are offered protection within BRCNR's borders. Of special interest was the discovery of Jasminum abyssinicum, Combretum edwardsii and Olinia radiata, all of which occurred in relatively large numbers in the forest. Jasminum abyssinicum had a status of Insufficiently Known under the old Transvaal threatened plants programme [15], and Hilton-Taylor [16] currently lists this species under the Red Data List of South African Plants. Jasminum abyssinicum occurred in 64% of the sample plots and in both forest communities identified. Combretum edwardsii is similarly listed as Insufficiently Known and also occurs under the Red Data List of South African Plants. Combretum edwardsii occurred in 73% of the sample plots. Pooley [17] lists O. radiata as very rare in KwaZulu Natal and Transkei and fails to mention its occurrence north of Natal. Palmer & Pitman [18] describe O. radiata as a rare species restricted to Natal forests from Pondoland to Zululand. This species was surprisingly common in the canopy of BRCNR's forests and occurred in 82% of the sample plots. Some severely over-utilized forest patches are currently in a state of recovery; however, current and future damage from invasive trees is a threat to this recovery. Some of the forest patches on BRCNR have a forest margin largely comprised of the invasive black wattle (Acacia mearnsii). The flammable nature of this species, compared to natural forest margin species, allows grassland fires to penetrate forests, resulting in a reduction in forest patch size. With a large edge effect resulting from many small forest patches, there is a need for careful fire management and stringent alien plant control. According to Geldenhuys [5], conservation status implies the extent to which populations, species or communities have been modified by the influences of man and the degree to which they might be expected to maintain their genetic diversity and ecological processes in the medium term (10 to 100 years). We see two different aspects to the conservation of the afromontane forest biome in the BRCNR. Firstly, it is the maintenance of the components and critical processes within a forest ecosystem. The disturbed and unstable state of the forest margins are identified as an area requiring further investigation. The effects of the alien tree species (e.g., black wattle) and the destructive burning of forest margins should be of concern to management authorities, as the forest patches are being reduced in size and the impact of edge effects is being amplified on the forest interior. Secondly, it is the maintenance of gene flow between the fragmented forest patches through management of the land surrounding the forest and forest corridors. As the BRCNR forest vegetation is situated along an altitudinal gradient, it therefore seems possible to identify certain forest patches (which may have been harvested in the past, or possibly will in the future) as critical adjuncts for conserved forest patches at the same altitude. From the evidence we gathered, no "climax" forests exist on BRCNR. Although it is known that BRCNR's forests were utilized, the impact, extent and degree of the utilization are still not quantified. An investigation into the successional and dynamic state of the five largest forest patches is currently underway. Very little of the neighbouring forest on Mariepskop was harvested for its timber, and this forest seems to be the obvious control site for further comparative research. Conclusions This study shows that BRCNR has a fragmented network of small forest patches that together make up 7.3% of the reserve's surface area, almost twice the area that was generally known. Two afromontane forest communities are recognized and associated along an altitudinal gradient, one within the moist mist belt and one within drier micro-climates outside the mist belt; further, within each community, variants were recognized based on either available soil moisture or degree of past utilization. These forest patches host a variety of forest-dependent trees, including some species considered rare, insufficiently known, or listed under the Red Data List of South African Plants. The fragmented nature of the relatively small forest patches accentuates the need for careful fire management and stringent alien plant control. Methods Forest size and fragmentation All forest patches greater than 0.25 ha in size were mapped on a Geographical Information System (GIS). Forest boundaries were marked on 1:10 000 orthophotos, and the data were digitized onto the Mpumalanga Parks Board's GIS program, SPANS [19]. Total forest size and patch numbers are calculated from the digitized data. Floristic description Twenty-two plots (0.04 ha each) were subjectively distributed throughout two of the five largest forest patches occurring on BRCNR (Figure 4), including the Op-de-Berg and Hebronberg forests. Plots covered an altitudinal range from 1240 to 1660 m above sea level and were sampled along environmental gradients, including the factors of slope, surface rockiness, drainage, topography, and disturbance (from logging). Sample relevés included a list of all the species present in a sample plot as well as cover-abundance values for each species, according to the Braun Blanquet cover-abundance scale [20]. A forest flora (see additional file 1 – Appendix A) is compiled from the species composition lists recorded in the 22 relevés. Data processing and analyses Eigenvalues produced from the TWINSPAN and DECORANA programs are a measure of the degree of separation in the data. According to Jongman, ter Braak & van Tongeren [21], low eigenvalues would represent a poor separation of samples and can be regarded as a measure of alpha diversity (species turnover within a plant community). High eigenvalues would represent a strong separation of samples, which can therefore be a measure of beta diversity (species turnover between plant communities). The 22 relevés were analyzed for a circumscription of possible plant communities. Firstly, a complementary analysis was run using the hierarchical classification program TWINSPAN [22,23] and the indirect ordination program DECORANA [24,25]. Complementary analyses of the two different multivariate analysis results provide for an accurate interpretation and description of plant communities [26]. Qualitative cover-abundance values were used for data input instead of quantitative values. Appendix A lists the 167 species recorded in the forest and used in the classification of plant communities. Secondly, the relevés were analyzed for relationships between plant communities and environmental gradients. Canonical correspondence analysis is a direct ordination technique that analyzes and presents such relationships between many species and numerous environmental variables. For this study, we used the program CANOCO [27]. No formal syntaxonomical classification was done in this study. An informal classification was performed with preferential species indicating the names of different vegetation associations. Specific species responses to environmental variables are a useful way of displaying the impact a certain environmental variable may have on a species. Direct ordinations relate species presence to environmental variables on the basis of species and environmental data from the same set of sample plots [28]. A scatter diagram produced from the CANOCO program depicts the relationship between species and environmental variables. Only those species with a cumulative fit of more than 0.35 are displayed. This ensures that only those species that most positively contributed to the ordination scores in the scatter plot are displayed. Canopy density is used as a surrogate for measuring disturbance, or forest dynamics. Authors' contributions ML conducted the field work and the map digitization. Both authors performed the statistical analyses. Both authors read and approved the final manuscript. Supplementary Material Additional File 1 Appendix 1. Forest flora in 22 relevés on Blyde River Canyon Nature Reserve Click here for file ==== Refs Geldenhuys CJ Southern African Forests: their biogeography, conservation and utilization MAB/UNESCO Regional seminar on forests 1996 Sri Lanka White F The vegetation of Africa: a descriptive memoir to accompany the UNESCO/AETFAT/UNSO map of Africa Natural Resources Research 20 1983 Paris, UNESCO Lubke R McKenzie B Lowe A B and Rebelo A G Afromontane forest Vegetation of South Africa, Lesotho and Swaziland 1996 Pretoria, Department of Environmental Affairs & Tourism Geldenhuys CJ Macdevette DR Huntley BJ Conservation status of coastal and montane evergreen forest Biotic diversity in southern Africa: concepts and conservation 1989 Cape Town, Oxford University Press Geldenhuys CJ Requirements for improved and sustainable use of forest biodiversity: examples of multiple use of forests in South Africa Forum: Biodiversity--Treasures in the world's forests 1998 Germany Von Maltitz G Mucina L Geldenhuys CJ Lawes M Eeley H Adie H Vink D Fleming G Bailey C Classification system for South African indigenous forests: an objective classification for the Department of Water Affairs and Forestry. Environmentek Report, ENV-P-C 2003-17 2003 Pretoria, CSIR Lötter MC Emery AJ Williamson SD Emery AJ, Lötter MC and Williamson SD Forests Determining the conservation value of land in Mpumalanga 2002 Nelspruit, South Africa, Mpumalanga Parks Board Cooper KH The conservation status of indigenous forests in Transvaal, Natal and OFS, South Africa 1985 Durban, Wildlife Society of Southern Africa Viljoen MJ Reimold WU An Introduction to South Africa's Geological and Mining Heritage 1999 Randburg, South Africa, Mintek Geological Survey of South Africa Geology Map. 2430 Pilgrim's Rest. 1:250 000 Geological Series 1986 Pretoria, Government Printer Knight RS Manry DE Lightning density and burning frequency in South African vegetation Vegetatio 1986 66 67 76 Schmidt E Lötter M McCleland W Trees and shrubs of Mpumalanga and Kruger National Park 2002 Johannesburg, Jacana Acocks JPH Veld types of South Africa 1988 3rd Pretoria, Botanical Research Institute, Dept. of Agriculture and Water Supply Low AB Rebelo AG Vegetation of South Africa, Lesotho and Swaziland 1996 Pretoria, Department of Environmental Affairs & Tourism Fourie SP The Transvaal, South Africa, threatened plants programme Biological Conservation 1986 37 23 42 10.1016/0006-3207(86)90032-7 Hilton-Taylor C Red data list of South African plants 1996 Pretoria, National Botanical Institute Pooley Elsa The complete field guide to the trees of Natal, Zululand and Transkei 1994 Durban, Natal Flora Publications Trust Palmer E Pitman N Trees of southern Africa 1972 Cape Town, A.A. Balkema SPANS SPANS GIS 1997 Ontario, Canada, TYDAC Research Inc. Mueller-Dombois D Ellenberg H Aims and methods of vegetation ecology 1974 New York, John Wiley & Sons Jongman RH Ter Braak CJF Van Tongeren FR Data analysis in comunity and landscape ecology 1987 Wageningen, The Netherlands, Pudoc Hill MO TWINSPAN: a FORTRAN program for arranging multivariate data in an ordered two-way table by classification of the individuals and the attributes 1979 New York, Section of Ecology and Systematics, Cornell University TWINSPAN TWINSPAN: Two-way indicator species analysis 1994 7.0 Phar Lap Software Inc. Hill MO DECORANA: a FORTRAN program for detrended correspondence analysis and reciprocal averaging 1979 New York, Section of Ecology and Systematics, Cornell University DECORANA DECORANA: Detrended correspondence analysis. 1994 7.0 Phar Lap Software Inc. Kent Martin Coker Paddy Vegetation description and analysis: a practical approach 1992 Boca Raton, Florida, USA, CRC Press 363 CANOCO CANOCO: Canonical correspondence analysis. 1998 4.0 Wageneningen, The Netherlands, Centre for Biometry ter Braak Cajo J. F. The analysis of vegetation-environment relationships by canonical correspondence analysis Vegetatio 1987 69 69 77

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==== Front BMC Evol BiolBMC Evolutionary Biology1471-2148BioMed Central London 1471-2148-4-251529197110.1186/1471-2148-4-25Research ArticleOrganization of the mitochondrial genomes of whiteflies, aphids, and psyllids (Hemiptera, Sternorrhyncha) Thao MyLo L [email protected] Linda [email protected] Paul [email protected] Microbiology Section, University of California, One Shields Ave., Davis, California, USA, 95616-86652004 3 8 2004 4 25 25 10 6 2004 3 8 2004 Copyright © 2004 Thao et al; licensee BioMed Central Ltd.2004Thao et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background With some exceptions, mitochondria within the class Insecta have the same gene content, and generally, a similar gene order allowing the proposal of an ancestral gene order. The principal exceptions are several orders within the Hemipteroid assemblage including the order Thysanoptera, a sister group of the order Hemiptera. Within the Hemiptera, there are available a number of completely sequenced mitochondrial genomes that have a gene order similar to that of the proposed ancestor. None, however, are available from the suborder Sternorryncha that includes whiteflies, psyllids and aphids. Results We have determined the complete nucleotide sequence of the mitochondrial genomes of six species of whiteflies, one psyllid and one aphid. Two species of whiteflies, one psyllid and one aphid have mitochondrial genomes with a gene order very similar to that of the proposed insect ancestor. The remaining four species of whiteflies had variations in the gene order. In all cases, there was the excision of a DNA fragment encoding for cytochrome oxidase subunit III(COIII)-tRNAgly-NADH dehydrogenase subunit 3(ND3)-tRNAala-tRNAarg-tRNAasn from the ancestral position between genes for ATP synthase subunit 6 and NADH dehydrogenase subunit 5. Based on the position in which all or part of this fragment was inserted, the mitochondria could be subdivided into four different gene arrangement types. PCR amplification spanning from COIII to genes outside the inserted region and sequence determination of the resulting fragments, indicated that different whitefly species could be placed into one of these arrangement types. A phylogenetic analysis of 19 whitefly species based on genes for mitochondrial cytochrome b, NADH dehydrogenase subunit 1, and 16S ribosomal DNA as well as cospeciating endosymbiont 16S and 23S ribosomal DNA indicated a clustering of species that corresponded to the gene arrangement types. Conclusions In whiteflies, the region of the mitochondrial genome consisting of genes encoding for COIII-tRNAgly-ND3-tRNAala-tRNAarg-tRNAasn can be transposed from its ancestral position to four different locations on the mitochondrial genome. Related species within clusters established by phylogenetic analysis of host and endosymbiont genes have the same mitochondrial gene arrangement indicating a transposition in the ancestor of these clusters. ==== Body Background Whiteflies, psyllids, and aphids correspond to superfamilies within the suborder Sternorrhyncha (Hemiptera) [1]. These insects share a number of common properties that are a consequence of their utilization of plant phloem as their diet. This mode of feeding is accomplished by means of needle-like stylets that probe plant tissues between plant cells until they enter the phloem-sieve elements. Due to this mode of feeding, some species are of major agricultural importance in that they vector plant pathogens and in high numbers may cause plant debilitation due to excessive nutrient consumption [1]. Whiteflies, psyllids, and aphids have an obligatory association with prokaryotic endosymbionts localized in specialized cells called bacteriocytes that constitute a larger structure called the bacteriome [2-4]. In the past, numerous studies have been performed on the phylogeny of some of these endosymbionts and their hosts [2-5]. The results have indicated congruence between the endosymbiont and the host derived phylogeny. This observation has been interpreted as being the consequence of an infection of an insect ancestor by a prokaryote and the vertical transmission of the endosymbiont resulting in cospeciation or cocladogenesis. In a recent study of whiteflies, we compared the phylogeny based on endosymbiont 16S*-23S* rDNA to the phylogeny of the host based on several mitochondrial genes [6]. During this study, we found that in the whitefly, Bemisia tabaci, the order of some of the mitochondrial genes was quite different from the frequently found order of genes in the mitochondria of the class Insecta. This observation led us to obtain the full sequence of the mitochondrial genome of representatives of the suborder Sternorrhyncha. Due to the observed differences in the order of genes in the mitochondrial genome of whiteflies, we obtained additional mitochondrial sequences from species representative of the major phylogenetic clusters previously established on the basis of whitefly mitochondrial and endosymbiont genes [6]. Previous studies of the phylogenetic relationships of member of the Sternorrhyncha, using host 18S rDNA, indicated that it is a monophyletic group [7-9]. These studies also showed that aphids and whiteflies were more closely related to each other than to psyllids. In animals, the mitochondrial genome is generally circular (14–17 kb), is maternally transmitted and has a relatively simple genetic structure, and a rapid rate of sequence change [10-12]. Of the thirty seven genes found in animal mitochondria, thirteen encode for proteins, consisting of three subunits of cytochrome oxidase (COI, COII, COIII), two subunits of ATP synthase (atp6, atp8), seven subunits of NADH dehydrogenase (ND1, ND2, ND3, ND4, ND4L, ND5, ND6), and cytochrome b (cytB). Two genes encode for the large subunit of ribosomal RNA (16S) and the small subunit of ribosomal RNA (12S). In addition, there are 22 tRNAs, two for leucine and two for serine, and one tRNA each for the remaining eighteen amino acids. In general, there is conservation of the gene order within phyla but variation between phyla [10,13-17]; the tRNA genes are subject to more change in their position than the genes for proteins and rRNAs. The order of mitochondrial genes has been suggested to be a good phylogenetic marker for studies of relationships [14]. The animal mitochondrial genome is generally very compact with few if any intergenic spaces. Usually there is one (or rarely more) noncoding region, frequently following 12S rDNA. Such a region most often has a reduced G+C content and all or some of the following properties: a) direct repeats, b) inverted repeats, c) stretches of "T"s, "A"s, or "TA"s. By analogy with other well-studied mitochondria, such a region is considered to be a putative origin of DNA replication and a region from which transcription is initiated [10,12]. Variation in mitochondrial size is generally a consequence of variation in the length of the repeats in the noncoding region and not in the number of structural genes. Early studies within the class Insecta suggested conservation of the gene order over a wide range of different organisms indicating an ancestral gene order for this group [10,18]. However, more recent studies have shown that within the Hemipteroid assemblage, there is considerable variation in the order of genes in the orders Phthiraptera, Psocoptera, and Thysanoptera, but no variation in the order Hemiptera (that includes the suborder Sternorrhyncha) [18-21]. The complete sequence of the mitochondria of a representative of the Phthiraptera (wallaby louse) and the Thysanoptera (plague thrips) has been obtained [18,20]. The latter shows major differences from the ancestral gene order. The Hemiptera and the Thysanoptera are sister groups and it was consequently of interest to obtain sequences of the mitochondrial genomes of the former. Since the sequence of mitochondrial genomes is poorly conserved, sequence determination of a portion of the genome is useful for the study of closely related species or the population structure within a species [22,23]. The availability of completely sequenced mitochondrial genomes is also an aid to the design of primers for the PCR amplification of the regions selected for population studies. Results Evolutionary relationships within the Sternorrhyncha Table 1 gives the properties and the accession numbers of the mitochondrial DNA sequences determined in this study. An unrooted phylogenetic tree showing the relationships of whiteflies, psyllids and aphids, based on mitochondrial cytB (partial), ND1, and 16S rDNA is presented in Fig. 1. A similar tree is obtained when the amino acid sequence of CytB (partial) and ND1 is used. The sole difference is the position of Neomaskellia andropogonis which becomes part of the cluster containing Bemisia tabaci, Tetraleurodes acaciae, Aleurochiton aceris, and Trialeurodes vaporariorum. Whiteflies, psyllids and aphids have associations with different primary endosymbionts that are transmitted vertically and are essential for the survival of the insect host [2-6]. The time for the establishment of these endosymbiotic associations and the emergence of the composite organism is generally estimated to be between 100 and 200 million years ago [2]. The representative species chosen for study (Fig. 1) probably span the range of diversity within whiteflies, psyllids, and aphids. The maximum % difference in the DNA sequence of these organisms is 33.5 % for whiteflies, 29.7% for psyllids and 13.1% for aphids suggesting that the rate of mitochondrial sequence change in aphids is considerably less than that in whiteflies and psyllids. Resolution of the order of branching among these insect groups is not possible using mitochondrial sequences, since due to their rapid rate of change they are saturated. Table 1 Properties and accession numbers of mitochondrial DNA sequences determined in this study. Organism Type Mitochondrial sequence Size (bp) G+C content GenBank accession number Bemisia tabaci whitefly-A complete 15,322 25.9 AY521259 Tetraleurodes acaciae whitefly-B complete 15,080 28.0 AY521626 Neomaskellia andropogonis whitefly-C complete 14,496 18.7 AY572539 Aleurochiton aceris whitefly-D complete 15,388 22.1 AY572538 Trialeurodes vaporariorum whitefly-Y complete 18,414 27.7 AY521265 Aleurodicus dugesii whitefly-Y complete 15,723 13.8 AY521251 Pachypsylla venusta psyllid complete 14,711 26.3 AY278317 Schizaphis graminum aphid complete 15,721 16.1 AY531391 Bemisia argentifolii whitefly-A cytB-COIII 4, 796 23.2 AY521257 Bemisia sp. whitefly-A 12S-COIII 985 19.4 AY572845 cytB-12S AY521257a Aleuroplatus sp. whitefly-B cytB-COIII 4, 540 27.4 AY521256 Tetraleurodes mori whitefly-B cytB-COIII 4,416 25.3 AY521263 Vasdavidius concursus whitefly-C cytB-COIII 3,374 20.0 AY648941 Siphonius phillyreae whitefly-D cytB-12S 4,561 22.3 AY521268 Bactericera cockerelli psyllid cytB-12S 3,077 28.0 AY601890 Calophya schini psyllid cytB-12S 3,044 26.3 AY601891 Glycaspis brimblecombei psyllid cytB-12S 3,081 26.8 AY601889 Diuraphis noxia aphid cytB-12S 3,180 15.8 AY601892 Melaphis rhois aphid cytB-12S 3,184 17.0 AY601894 Schlechtendalia chinensis aphid cytB-12S 3,188 16.1 AY601893 Daktulosphaira vitifoliae phylloxera cytB-12S 3,215 22.6 AY601895 aFrom [6]. Figure 1 Unrooted phylogenetic tree showing the relationships of members of the Sternorrhyncha (whiteflies, aphids, and psyllids). The tree is based on mitochondrial cytB, ND2, and 16S rDNA sequences. Maximum likelihood analysis, values at nodes are for bootstrap percentages from 500 replicates, only nodes supported by 70% or greater are shown. * by species name designates the organisms for which the complete mitochondrial sequence has been determined. Mitochondrial genomes with a similar gene order With some notable exceptions, within the class Insecta the order of the mitochondrial genes is highly conserved and has led to the proposal of an ancestral gene order [10,18]. An identical or a similar gene order has been observed in the mitochondrion of Pachypsylla venusta (psyllid), Schizaphis graminum (aphid), as well as Aleurodicus dugesii and Trialeurodes vaporariorum (whiteflies) (Fig. 2). In pysllids and aphids, tRNA-C is followed by tRNA-Y (Fig. 2, extreme right) which corresponds to the ancestral Insecta gene order. In most whiteflies (Fig. 2, 3, 4, 6), the order of these tRNA genes is reversed and this probably constitutes the whitelfly ancestral gene order. In the mitochondria of T. vaporariorum, tRNA-G is transposed from its position between COIII and ND3 to a position between tRNA-W and tRNA-Y (Fig. 1). tRNA-S1 was not detected in the mitochondria of S. graminum; this tRNA and tRNA-Q was not detected in A. dugesii. Figure 2 Mitochondrial gene arrangements of psyllids, aphids, and selected whitefly species all of which have a highly similar gene order. Genes are transcribed from left to right except for the underlined genes which are transcribed in the opposite direction. Dot in box indicates a putative origin of replication and/or a region of direct repeats. Empty box indicates 40–60 nt that do not code for a readily identifiable tRNA. Horizontal bar between genes indicates that they are contiguous with little or no nucleotides between them. The change in the position of tRNA-G in T. vaporariorum is traced by lines and the 5X preceding the box containing tRNA-S2, indicates that this putative tRNA is present five time in a direct repeat. Figure 3 Differences of the A type gene order from the postulated whitefly ancestral gene order. The principal changes are indicated by thick lineswith complete arrowheads. Direction of the arrowheads or half arrowheads indicates direction of transcription. Figure legend same as for Fig. 2. Dashed double headed arrow indicates the sequenced genes from other listed whitefly species. Figure 4 Differences of the B type gene order from the postulated whitefly ancestral gene order. Figure legend same as in Fig. 2 and 3. Figure 6 Differences of the D type gene order from the postulated whitefly ancestral gene order. Figure legend same as in Fig. 2 and 3. Mitochondria of whiteflies with transposition of COIII-(tRNA-G)-ND3-(tRNAs-A-R-N) A number of whitefly mitochondria had transpositions of DNA fragments containing COIII-(tRNA-G)-ND3-(tRNAs-A-R-N). In most cases in which these genes are removed, there is a change in the direction of transcription of the adjacent downstream tRNA-S1 from clockwise to counter clockwise (Fig. 3, 5, 6). There is variation in the mitochondrial position into which these genes are transposed. In addition, there are differences with respect to the retention of the number and the order of the excised tRNA genes at the mitochondrial location in which the genes are inserted. The maximal insertion involves all of the genes from the excised fragment in their original order (Fig. 3) (tRNAs-A-R-N)-ND3-(tRNA-G)-COIII), the minimal insertion involves ND3-(tRNA-G)-COIII (Fig. 4, 5). In all insertions, the transcription direction is altered from that in the original position. Based on the location of the insertions and the adjacent genes, we have subdivided these transpositions into four types (A-D) (Fig. 3,4,5,6). In all cases, it would appear that the excision involved the removal of COIII-(tRNA-G)-ND3-(tRNAs-A-R-N). However, the DNA that is inserted always contains COIII-(tRNA-G)-ND3 and may contain all or only some of the tRNA genes. Figure 5 Differences of the C type gene order from the postulated whitefly ancestral gene order. Figure legend same as in Fig. 2 and 3. Transposition of the A type is shown in Fig. 3. In this case, COIII-(tRNA-G)-ND3-(tRNAs-A-R-N) is removed from the inferred ancestral position and placed between 12S rDNA and tRNA-I. Additional changes involve the position of tRNA-D, tRNA-Q and the direction of transcription of tRNA-S1 and tRNA-E. There is a total of 5 difference between the A type gene arrangement and the ancestral whitefly gene order. Sequence determination of smaller DNA fragments from two related species (cytB-COIII) were consistent with the same gene order (Fig. 3). Transposition of the B type is shown in Fig. 4. In this case, ND3-(tRNA-G)-COIII is inserted into a location downstream of 12S rDNA and is bounded by tRNAs that have also changed locations (tRNAs-Q-V and tRNAs-R-D). In addition, the position of tRNA-A is changed as compared to the ancestral position. There were 6 differences from the putative ancestral gene order. No tRNA genes for N, S1, and I were detected. Sequence determination of a smaller DNA fragment (cytB-COIII) from two related species was consistent with the same gene order. Transposition of the C type is shown in Fig. 5. In this case, ND3-(tRNA-G)-COIII is inserted downstream of 16S rDNA between tRNA-P and tRNA-C. Another major difference is the change in the direction of the transcription of tRNA-V and 12S rDNA. Other differences include the change in the order and the position of tRNA-Y and tRNA-C, the change of position of tRNA-P, and the direction of transcription of tRNA-S1. Putative tRNA-W is transcribed clockwise. A small change in the span of the DNA fragment resulted in a putative tRNA-S2, transcribed counter clockwise. The initially adjacent tRNAs-A-R-N as well as tRNA-I were not detected. There was a total of 7 differences between the whitefly ancestral gene order and the C type gene order. Sequence determination of a fragment of mitochondrial DNA from a related whitefly species was consistent with the C type gene order. The D type gene order is shown in Fig. 6. In this case,(tRNAs-R-A)-ND3-(tRNA-G)-COIII is found after tRNA-S2 and before tRNA-N. Additional differences from the ancestral gene order involve the change in position of tRNA-N and tRNA-Q and the direction of transcription of tRNA-S1. tRNA-I was not detected. The total number of differences between the ancestral gene order and the D type gene order is 4. The sequence of a mitochondrial DNA fragment from a related species indicated a gene order of the D type (Fig. 6). PCR-based screening for excision of COIII-(tRNA-G)-ND3-(tRNAs-A-R-N) and identification of transposition types We have devised a set of oligonucleotide primers complementary to COII and ND5 that allow the amplification of the DNA between these two genes. The size of the resulting fragments is a potential indication of the presence or absence of COIII-(tRNA-G)-ND3-(tRNAs-A-R-N) between COII and ND5 (Fig. 2). Fig. 7 shows the results obtained with insects containing mitochondria that have these genes in the ancestral position (lanes 6–8, bands of 3.7 kb) and those in which they have been excised from this position (lane 2–5, bands of 2.2 to 2.3 kb). Figure 7 Agarose gel electrophoresis of PCR products amplified from whole insect DNA using primers complementary to regions encoding COII and ND5. A, B, C, D, refers to different gene arrangement types; Y, ancestral arrangement. Lanes 1 and 9 molecular size markers; lane 2, Bemisia tabaci; lane 3, Tetraleurodes acaciae; lane 4, Neomaskellia andropogonis; lane 5, Aleurochiton aceris; lane 6, Aleyrodes elevatus; lane 7, Trialeurodes vaporariorum; and lane 8, Aleurodicus dugesii. In addition, we have devised a set of PCR primers that allow the distinction of the four types of transpositions. Using oligonucleotide primers complementary to COIII and cytB, the PCR fragments shown in Fig. 8 were obtained. The sizes characteristic of arrangement types A, B, C and D, were 4.9, 4.5, 3.5, and 1.5 kb, respectively. Figure 8 Agarose gel electrophoresis of PCR products amplified from whole insect DNA using primers complementary to regions encoding COIII and cytB. A, B, C, D, refers to different gene arrangement types; Y, ancestral arrangement. Lanes 1 and 8 molecular size markers; lane 2, Bemisia tabaci; lane 3, Tetraleurodes acaciae; lane 4, Neomaskellia andropogonis; lane 5, Aleurochiton aceris; lane 6, Trialeurodes vaporariorum; and lane 7, Aleurodicus dugesii. Non-coding regions Mitochondrial genomes of insects are very compact. The principal non-coding segments of the genome are a low G+C content region usually following 12S rDNA [10-12]. The low G+C region usually has stretches of "T"s or "A"s as well as multiples of the sequence "TA." Another feature of this region may be inverted and direct repeats. Fig. 9 presents a diagrammatic summary illustrating some of the properties of the non-coding regions of the mitochondria of the studied insects. Only direct repeats and their sizes are indicated in this figure. No consistent pattern of inverted repeats was found and these are not indicated in the diagrams. All of the non-coding regions in the vicinity of 12S rDNA had a G+C content lower than the G+C content of the full genome (Fig. 9). The decrease ranged from 3.2 to 10.0%. Some of these regions of lower G+C content, adjacent to 12S rDNA, contained direct repeats (Fig. 7, Adu, Tva, Aac). In Bta and Nan (Fig. 9), the direct repeats were in a non-coding region following COIII that also had a decrease in the G+C content. The noncoding regions of Tac, containing direct repeats, had a G+C content that was actually higher than that of the full Tac mitochondrial genome. However, the segment before the repeats had regions with a lower G+C content. In Sgr (Fig. 9), the direct repeats were between tRNA-E and tRNA-F and had essentially no decrease in the G+C content. In this organism and Pve (Fig. 9), the region following 12S rDNA had a decrease G+C content but did not contain substantial direct repeats. Figure 9 Summary of the properties of non-coding regions found in the mitochondrial genomes of psyllids, aphids and whiteflies. Letter abbreviations for amino acids, denote tRNAs for the designated amino acid; thin lines, length of non-coding region; thick lines, direct repeats; numerals above line, length of direct repeats, numbers in parentheses at end of fragment denote its length in bp; column of numbers at right represent the difference between the G+C content of the full mitochondrial genome and the considered segment. Pve, psyllid, Pachypsylla venusta; Sgr, aphid, Schizaphis graminum; Adu, whitefly, Aleurodicus dugesii; Tva, whitefly, Trialeurodes vaporariorum; Bta, whitefly, Bemisia tabaci;Tac, whitefly, Tetraleurodes acaciae; Nan, whitefly, Neomaskellia andropogonis; Aac, whitefly, Aleurochiton aceris. tRNA anticodons In general, the anticodons found in the tRNAs of the mitochondria of whiteflies, psyllids, and aphids were those expected of insect mitochondria [12]. Some exceptions were "TTT" (instead of CTT) for tRNA-K for B. tabaci and A. dugesii, and "TCT"(instead of GCT) for tRNA-S1 for B. tabaci, A. aceris, N. andropogonis and T. vaporariorum. The latter codon maybe the usual tRNA-S1 anticodon in whiteflies; this tRNA was not detected in T. acaciae and A. dugesii. Discussion The novel aspect of this study is the finding that whitefly mitochondria contain a region of their genome spanning COIII-(tRNA-G)-ND3-(tRNAs-A-R-N) that is prone to excision followed by insertion as a unit or as fragments in different parts of the mitochondrial genome. Based on the collection of whiteflies we have examined, this event occurred three or four different times in the ancestors of the studied species. These conclusions are summarized in Fig. 10 where the phylogeny of the whiteflies is compared to the gene arrangement types. The designation Y refers to the ancestral arrangement established in the species under this designation. Species bracketed under A and B have a similar insertion position for COIII-(tRNA-G)-ND3) (Fig. 3, 4) but differ in adjacent tRNAs, so that it is possible these differences followed the insertion of the transposed fragment in a common ancestor. The positions of the transpositions in species of clusters C and D are very different and are probably the results of independent events. For purposes of this discussion we have chosen the simplest interpretation but this does not exclude other more complex scenarios. Cluster C is of additional interest since it is related to two species of Aleyrodes that have the ancestral (Y) arrangement. From the 16S*-23S* rDNA sequence divergence of Portiera (the primary endosymbiont of whiteflies) and the estimated rate of endosymbiont sequence change [24], it is possible to estimate the time of divergence of cluster C and the two Aleyrodes species. This value corresponds to 30–60 million years ago which is the maximum time for the occurrence of the transposition in an ancestor of cluster C. Figure 10 Summary of the major transpositions occurring in the mitochondria of whiteflies and the relationship of these changes to the phylogeny of whitefly species. The phylogenetic tree was obtained on the basis of combined mitochondrial cytB-N22-16S rDNA and Portiera endosymbiont 16S* and 23S* rDNA using the maximum likelihood method. Numbers at nodes correspond to bootstrap values after 500 replicates. The combination of the host and endosymbiont sequence data is justified by their cospeciation [6]. A, B, C, D indicate transposition type; Y, indicates mitochondria with an ancestral gene arrangement. Large arrowhead in mitochondrial genome indicates the original position of the transposed genes. Small arrowheads indicate the position of the insertion of the genes. Arrows outside circle indicate the direction of the transcription of the transposed genes. Arrow by the arrowhead of the B type transposition indicates the changed direction of transcription of the 12S rDNA. tRNAs have been omitted. (*), by species names indicate that the full mitochondrial genome was sequenced. (+), by species names indicates that a DNA fragment containing all or a part of the gene encoding for COIII and adjacent genes was sequenced. (o), by species name indicates that using oligonucleotide primers to COII and ND5 a PCR product was obtained corresponding to a size that was consistent with the presence of COIII-(tRNA-G)-ND3-(tRNAs-A-R-N) in the ancestral position (Fig. 8). The excision of the same mitochondrial fragment at least four times during the evolutionary history of whiteflies suggests that this fragment is prone to transposition. In spite of the apparent similarity of the excisions, we have not been able to find any conserved sequence properties either adjacent to the region of the excised fragment or adjacent to its insertion site. The excision appears to be associated with a change in the direction of transcription of the previously adjacent tRNA-S1 (Fig. 3, 5, 6) and a change in the direction of transcription of the relocated fragments. As previously noted the order of the mitochondrial genes is conserved in most insects [10,14]. The major exceptions are within the three hemipteroid orders Phthiraptera, Psocoptera, and Thysanoptera [18,20,21]. The rearrangements are different within these three orders, being rather extreme in the Thysanoptera. In most insects, the order of the rRNA genes is 16S-(tRNA-V)-12S and the genes are transcribed in the counterclockwise direction [10,18]. A major exception is in the mitochondrion of Thrips imagines where these two genes are distant from each other and transcribed in opposite orientations [18]. In the C type gene order, there is an inversion of 12S-(tRNA-V) that is possibly associated with the insertion of ND3-(tRNA-G)-COIII between 16S and 12S rDNA (Fig. 5, 10). This situation resembles that found in Thrips imagines in that the rRNA genes are transcribed in opposite directions. In whiteflies, besides rearrangements involving COIII-(tRNA-G)-ND3-(tRNAs-A-R-N), there are also substantial rearrangements involving single tRNAs. The physiological significance (if any) of these rearrangements is not known. Genes that are highly expressed (16S, 12S rDNA) when separated and transcribed in opposite orientations would have to become part of different transcription units. In addition, we are not certain of the validity or significance of our inability to find a few of the tRNAs. In some cases, this may stem from our inability to recognize them. In other cases, such as tRNAs-A-R-N that are absent in the type C gene order, there would not appear to be any room for these genes on the mitochondrion and it might be that the tRNAs for these amino acids are provided by the host [25]. The mitochondrion of A. dugesii has a G+C content of 13.8 moles % (Table 1). All the other sequenced whitefly mitochondria have G+C contents of 18.7 to 27.7 moles % (Table 1). On the basis of morphological classification, Aleurodicus has been placed into a subfamily Aleurodicinae, while the remaining whitefly species listed in Fig. 10 have been placed into the subfamily Aleyrodinae [1]. This separation is supported by a phylogenetic analysis of mitochondrial DNA, host 18S rDNA, as well as Portiera DNA from different whitefly species [6-8]. It is possible that the common ancestor of whiteflies had a higher G+C content in its mitochondria and that in Aleurodicus there was a decrease. Alternatively, it is possible that the ancestral G+C content was low and increased in the Aleyrodinae. Our work points to the uncertainty inherent in making generalizations from one or a few organisms assumed to be representative of a group. We were fortunate that in the whiteflies the first mitochondrion we chose to study was that of B. tabaci which had an altered gene order. Had we started with our second or third choice (T. vaporariorum, A. dugesii) we would have concluded that the whiteflies have the ancestral mitochondrial gene order and not pursued further studies of mitochondria within this group of insects. Previously, evidence was found of a correlation between the rate of nucleotide sequence change and the rate of gene rearrangement [26]. If this has general applicability one would expect conservation of the mitochondrial gene order in aphids which have a low rate of sequence change (Fig. 1) and perhaps some changes in the gene order of psyllids as has been observed with whiteflies. The relatively localized different changes observed in several whitefly lineages may be of use in the study of the phylogeny and taxonomy of these organisms as is already indicated from the relatively small sample of organism studied in the present work. Conclusions Psyllids, aphids, and many whiteflies have mitochondria in which the order of the genes resembles the proposed Insecta ancestral gene order. However, in a variety of whitefly species there is a change in the gene order. In these organisms, there is an excision of a DNA segment containing COIII-(tRNA-G)-ND3-(tRNAs-A-R-N) from the ancestral position, between atp6 and tRNA-S1, and the insertion of all of these genes or fragments containing COIII-(tRNA-G)-ND and tRNAs into different locations on the mitochondrial genome. On the basis of the insertion positions, four gene arrangement types were identified. A phylogenetic analysis of 19 whitefly species involving mitochondrial and endosymbiont genes showed that each arrangement type was characteristic of a cluster of related whitefly species indicating that the transposition occurred in a common ancestor of the related species. The reason for the "restlessness" of this DNA segment in whiteflies and the physiological significance of these rearrangements are not known. Methods Amplification and sequencing of mitochondrial genomes In all cases, the starting material was whole insect DNA that was prepared and used in a previous study [6]. In our initial attempts at cloning mitochondrial DNA, we used methods previously developed for obtaining clones of insect endosymbiont DNA that have been described in detail [27]. In outline this involved obtaining a homologous probe for COI using previously described primers [28], followed by restriction enzyme and Southern blot analysis of insect DNA. Appropriate sized fragments were electroeluted from agarose gels and cloned into λ-ZAP (Stratagene, La Jolla, California). Following excision of the insert-containing plasmid, the DNA sequence was determined using a double stranded nested deletion kit (Pharmacia, Piscatawy, New Jersey) and where necessary custom-made oligonucleotides. As new sequence data was acquired for the mitochondria of several insect species our ability to design more specific oligonucleotide primers was improved. This allowed us to use pairs of primers, in combination with PCR, to obtain the full mitochondrial genome in 2–4 overlapping fragments. Conserved regions of the whitefly mitochondrial genome that are of use for the design of oligonucleotide primers, based on comparisons of six mitochondrial genomes, are given in Table 2. Usually the oligonucleotide primers had added sequences at the 5'-ends for restriction enzymes. Table 2 Oligonucleotide primers for PCR amplification of whitefly mitochondrial DNA fragments.a Primer Gene Position on AY5212656 Nucleotide sequence of conserved regions (5'->3') F-COI-1 COI 172–221 TCWCATGCWT TTATYATAAT TTTTTTYATR ACWATGCCTT TDGTWATTGG F-COI-2 COI 673–710 GAYCCHATTT TRTATCAACA YTTDTTTTGATTTTTTGG R-COI COI 1133–1069 ACATAATGAA AATGDGCAAC AACAAAATAWGTATCATGHA RACAHACATC HACHGAAGAA TTACC F-COII COII 1845–1876 CCTTCTATYC GDATTTTDTA TYTAATRGAT GA R-COII COII 2093–2067 AGGAACHGTY CAAGAATGHA AAACATC F-COIII COIII 3854–3879 TTAACWGGHT TTCAYGGNTT HCATGT R-COIII COIII 4002–3974 CARACWAHRT CDACRAAATG TCAGTATCA F-CYTB CYTB 9169–9215 GCTTTTATRG GBTATATYTT RCCTTGRGGY CARATATCTT TTTGRGG R-CYTB CYTB 9566–9544 GCTATAATAA AATTTTCTGA ATC F-ND1 ND1 10275–10314 ATTCAATRTT AAAWCCWGAA ATWARYTCTG AYTCTCCTTC R-ND1 ND1 10506–10484 CAAYTAATTT CDTATGAAAT TAA F-16S-1 16S 11053–11087 ACCTGGCTTA CGCCGGTCTG AACTCAGATC ATGTA F-16S-2 16S 11202–11220 GCTGTTATCC CTTAGGTAA R-16S-1 16S 11377–11354 AAAAGACAAR AAGACCCTTT AGAA R-16S-2 16S 11526–11483 TTAAATAGCT GCAGTAWATT DACTGTACTA AGGTAGCATA ATAA F-12S-1 12S 12273–12308 ACTTTCCAGT AADTTTACTT TGTTACGACT TATCTT F-12S-2 12S 12318–12342 AAGAGTGACG GGCRATTTGT ACATA R-12S-1 12S 12643–12619 CTTCAAACTT AAAAAATTTG GCGGT R-12S-2 12S 12861–12843 GTGCCAGCAG TWGCGGTTA aUnderlined sequences indicate primers used in this study. bMitochondrial genome of Trialeurodes vaporariorum. We will illustrate the approach by describing how the full genome of the mitochondrion of T. acaciae was obtained in three overlapping fragments. Using primers F-CYTB and R-12S-2 (Table 2) and PCR, a 3.6 kb DNA fragment was obtained. Similarly, using the pairs of primers F-COI-2 and R-CYTB and F-12S-2 and R-COI fragments of 7.3 and 5.5 kb, respectively, were obtained. For fragments of 4 kb or less, the PCR reaction mixture (10 ul) contained 10 ng insect DNA, 1 ug bovine serum albumin, 5 mM MgCl2, 0.2 mM dNTP, 10 pmoles of each primer, 0.6 U Bio-X-Act DNA polymerase, in Opti-Buffer (Bioline, London, United Kingdom). The PCR program was 94°C for 3 min, 30 cycles of, 94°C 30 sec, 55.0–65.0°C (predetermined optimal annealing temperatures) 30 sec, 70° 5 min, followed by 70°C 10 min. For the 5.5 and 7.3 kb DNA fragments, the PCR reaction mixture was modified by the increase of dNTPs to 0.3–0.4 mM and Bio-X-Act to 0.8 U. The PCR program was 94°C for 2 min, 10 cycles of 92°C 20 sec, 55.0–65.0°C (predetermined optimal annealing temperatures) 30 sec, 68° 10 min, followed by 20 cycles of 92°C 20 sec, optimal annealing temperature 30 sec, 68°C 10 min with increases of 15 sec each cycle, followed by 68°C 10 min. The DNA fragments were purified by means of the Wizard SV gel and PCR clean-up system (Promega, Madison, Wisconsin) as directed by the manufacturer. Following digestion with restriction enzymes the mitochondrial DNA fragments were cloned into pBluescript (Stratagene). In some cases where difficulty was experienced with using this vector due to possible toxicity of the inserts, the low copy number plasmid pWSK130 was used [29]. The DNA sequence was obtained as described above. Sequences were determined at the University of Arizona (Tucson) LMSE sequencing facility. In some cases, PCR fragments of 1 to 4 kb were directly sequenced after gel purification using custom made oligonucleotide primers. PCR amplification of other mitochondrial fragments CytB-12S mitochondrial DNA fragments were amplified and cloned into pBluescript as previously described [6]. CytB-COIII DNA fragments were obtained using oligo WF-CYTB-3 (BamHI, SacII; 5'-GCAGGATCCG CGGCCWTGRG GHCAAATATC WTTTTGRGGD GC-3') and WF-COIII-3 (KpnI, 5'-GTGCGGTACC TTCWATTTGR TATTGRCATT TYGTTGA-3') and cloned into pBluescript. COII-ND5 DNA fragments (indicative of the presence or absence of COIII-(tRNA-G)-ND3-(tRNAs-A-R-N) were obtained by use of oligo WF-COII (5'-TGYTCAGAAA TYTGTGGRGT TAATCAYAGR TTTATRCC-3') and WF-ND5 (5'-TCAGCMTTAG TYCAYTCWTC AACAYTAGTW ACAGCAGG-3'). CytB-COIII fragments (size diagnostic of the arrangement type) were obtained by use of WF-CYTB-1 (5'-TTTATRGGBT ATATYTTRCC TTGRGG-3') and WF-COIII-1 (5'-TATTCWRTWT GATATTGACA TTTYGT-3'). The PCR reaction mixture (10 ul) differed from those above in containing 0.1 mM dNTP, and 0.8 U Bio-X-Act DNA polymerase. The PCR program was 94°C for 5 min, 30 cycles of, 94°C 30 sec, 56.0–63.1°C (predetermined optimal annealing temperatures) 30 sec, 70° 5 min, followed by 70°C 10 min. Identification of genes and phylogenetic analyses The protein-coding and rRNA genes were identified by BLAST searches [30] of GenBank. tRNA genes were identified by tRNAscan-SE [31], DOGMA [32] and in some cases by eye from the anticodons and inferred secondary structures. The methods used for the phylogenetic analyses have been described [6]. In Fig. 1, the phylogenetic analysis of mitochondrial cytB-ND1-16S was based on 2730 characters; the analysis in Fig. 10, which besides cytB-ND1-16S also included cospeciating endosymbiont 16S*-23S* rDNA [6], was based on 6860 characters. List of abbreviations used tRNAs tRNA-one letter amino acid abbreviation (parenthesis three letter amino acid abbreviation followed by anticodons): tRNA-A (ala, TGC), tRNA-C (cys, GCA), tRNA-D (asp, GTC), tRNA-E (glu, TTC), tRNA-F (phe, GAA), tRNA-G (gly, TCC); tRNA-H (his, GTG), tRNA-I (ile, GAT); tRNA-K (lys, TTT or CTT), tRNA-L1 (leu, TAG), tRNA-L2 (leu, TAA), tRNA-M (met, CAT), tRNA-N (asn, GTT), tRNA-P (pro, TGG), tRNA-Q (gln, TTG), tRNA-R (arg, TCG), RNA-S1 (ser, TCT or GCT), RNA-S2 (ser, TGA), tRNA-T (thr, TGT), tRNA-V (val, TAC), tRNA-W (trp, TCA), and tRNA-Y (tyr, GTA). Other structural genes atp6 (ATP synthase, subunit 6), atp8 (ATP synthase, subunit 8), COI (cytochrome oxidase, subunit I), COII (cytochrome oxidase, subunit II), COIII (cytochrome oxidase, subunit III), ND1 (NADH dehydrogenase, subunit 1), ND2 (NADH dehydrogenase, subunit 2), ND3 (NADH dehydrogenase, subunit 3), ND4 (NADH dehydrogenase, subunit 4), ND4L (NADH dehydrogenase, subunit 4L), ND5 (NADH dehydrogenase, subunit 5), ND6 (NADH dehydrogenase, subunit 6), 12S (small subunit of mitochondrial ribosomal DNA [rDNA]), 16S (large subunit of mitochondrial rDNA), 16S* (small subunit of primary endosymbiont rDNA), 23S* (large subunit of primary endosymbiont rDNA). Other abbreviations %G+C (moles percent guanine+ cytosine in DNA). Authors' contributions MLT cloned and sequenced the mitochondrial genomes of whiteflies. LB cloned and sequenced the mitochondrial genome of a psyllid and an aphid as well as smaller fragments of mitochondrial DNA from psyllids and aphids. PB directed the research and in collaboration with MLT and LB performed the data analysis and wrote the paper. Acknowledgements This material is based on work supported by National Science Foundation Awards DEB-9978518 (N. A. Moran, P. Baumann) and MCB-9807145 (P. Baumann) and the University of California Experiment Station (P. Baumann 26V12N13A14F21O19-15). ==== Refs Gullan PJ Martin JH Resh VH, Cardé RT Sternorrhyncha (jumping plant-lice, whiteflies, aphids and scale insects) In Encyclopedia of Insects 2003 New York: Academic Press/Elsevier Science 1079 1089 Baumann P Moran NA Baumann L Dworkin M Bacteriocyte-associated endosymbionts of insects In The Prokaryotes 2000 New York: Springer Moran NA Telang A Bacteriocyte-associated symbionts of insects: a variety of insect groups harbor ancient prokaryotic endosymbionts BioScience 1998 48 295 304 Wernegreen JJ Genome evolution in bacterial endosymbionts of insects Nature Reviews 2002 3 850 861 12415315 10.1038/nrg931 Thao ML Moran NA Abbot P Brennan EB Burckhardt DH Baumann P Cospeciation of psyllids and their primary prokaryotic endosymbionts Appl Environ Microbiol 2000 66 2898 2905 10877784 10.1128/AEM.66.7.2898-2905.2000 Thao ML Baumann P Evolutionary relationships of primary prokaryotic endosymbionts of whiteflies and their hosts Appl Environ Microbiol 2004 70 3401 3406 15184137 10.1128/AEM.70.6.3401-3406.2004 Campbell BC Steffen-Campbell JD Gill RJ Evolutionary origin of whiteflies (Hemiptera: Sternorrhyncha: Aleyrodidae) inferred from 18S rDNA sequences Insect Mol Biol 1994 3 73 88 7987524 Campbell BC Steffen-Campbell JD Gill RJ Gerling D, Mayer RT Origin and radiation of whiteflies: an initial molecular phylogenetic assessment In Bemisia 1995: Taxonomy, Biology, Damage Control and Management 1996 Andover, United Kingdom: Intercept 29 51 von Dohlen CD Moran NA Molecular phylogeny of the Homoptera: a paraphyletic taxon J Mol Evol 1995 41 211 223 7666451 Boore JL Animal mitochondrial genomes Nucleic Acids Res 1999 27 1767 1780 10101183 10.1093/nar/27.8.1767 Scheffler IE Mitochondria 1999 New York: Wiley-Liss Wolstenholme DR Animal mitochondrial DNA: structure and evolution Int Rev Cytol 1992 141 173 216 1452431 Adams KL Palmer JD Evolution of mitochondrial gene content: gene loss and transfer to the nucleus Mol Phylogen Evol 2003 29 380 395 10.1016/S1055-7903(03)00194-5 Boore JL Brown WM Big trees from little genomes: mitochondrial gene order as a phylogenetic tool Curr Opin Genet Devel 1998 8 668 674 9914213 10.1016/S0959-437X(98)80035-X Burger G Gray MW Lang BF Mitochondrial genomes: anything goes Trends Genet 2003 19 709 716 14642752 10.1016/j.tig.2003.10.012 Lang BF Gray MW Burger G Mitochondrial genome evolution and the origin of eukaryotes Annu Rev Genet 1999 33 351 397 10690412 10.1146/annurev.genet.33.1.351 Nosek J Tomáska L Mitochondrial genome diversity: evolution of the molecular architecture and replication strategy Curr Genet 2003 44 73 84 12898180 10.1007/s00294-003-0426-z Shao R Barker SC The highly rearranged mitochondrial genome of the plague thrips, Thrips imagines (Insecta: Thysanoptera): convergence of two novel gene boundaries and an extraordinary arrangement of rRNA genes Mol Biol Evol 2003 20 362 370 12644556 10.1093/molbev/msg045 Cranston PS Gullan PJ Resh VH, Cardé RT Phylogeny of insects In Encyclopedia of Insects 2003 New York: Academic Press/Elsevier Science 882 898 Shao R Campbell NJH Barker SC Numerous gene rearrangements in the mitochondrial genome of the wallaby louse, Heterodoxus macropus (Phthiraptera) Mol Biol Evol 2001 18 858 865 11319269 Shao R Campbell NJH Schmidt ER Barker SC Increased rate of gene rearrangement in the mitochondrial genomes of three orders of Hemipteroid insects Mol Biol Evol 2001 18 1828 1832 11504862 Downie DA Locating the sources of an invasive pest, grape phylloxera, using mitochondrial DNA gene genealogy Mol Ecol 2002 11 2013 2026 12296945 10.1046/j.1365-294X.2002.01584.x Frohlich DR Torres-Jerez I Bedford ID Markham PG Brown JK A phylogeographical analysis of the Bemisia tabaci species complex based on mitochondrial DNA markers Mol Ecol 1999 8 1683 1691 10583831 10.1046/j.1365-294x.1999.00754.x Ochman H Elwyn S Moran NA Calibrating bacterial evolution Proc Natl Acad Sci USA 1999 96 12638 12643 10535975 10.1073/pnas.96.22.12638 Schneider A Maréchal-Drouard L Mitochondrial tRNA import: are there distinct mechanisms? Trends Cell Biol 2000 10 509 513 11121736 10.1016/S0962-8924(00)01854-7 Shao R Dowton M Murrell A Barker SC Rates of gene rearrangement and nucleotide substitution are correlated in the mitochondrial genomes of insects Mol Biol Evol 2003 20 1612 1619 12832626 10.1093/molbev/msg176 Baumann L Thao ML Funk CJ Falk BW Ng JCK Baumann P Sequence analysis of DNA fragements from the genome of the primary endosymbiont of the whitefly Bemisia tabaci Curr Microbiol 2004 48 77 81 15018108 10.1007/s00284-003-4132-3 Moran NA Kaplan ME Gelsey MJ Murphy TG Scholes EA Phylogenetics and evolution of the aphid genus Uroleucon based on mitochondrial and nuclear DNA sequences Syst Entomol 1999 24 85 93 10.1046/j.1365-3113.1999.00076.x Wang RF Kushner SR Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli Gene 1991 100 195 199 2055470 10.1016/0378-1119(91)90358-I Altschul SF Gish W Miller W Myers EW Lipman DJ Basic local alignment search tool J Mol Biol 1990 215 403 410 2231712 10.1006/jmbi.1990.9999 Lowe TM Eddy SR tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence Nucleic Acids Res 1997 25 955 964 9023104 10.1093/nar/25.5.955 Wyman SK Jansen RK Boore JL Automatic annotation of organellar genomes with DOGMA Bioinformatics

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020245Research ArticleGenetics/Genomics/Gene TherapyPlant SciencePlantsUnused Natural Variation Can Lift Yield Barriers in Plant Breeding Natural Variation Can Lift Yield BarriersGur Amit 1 Zamir Dani [email protected] 1 1The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Faculty of AgricultureThe Hebrew University of Jerusalem, RehovotIsrael10 2004 24 8 2004 24 8 2004 2 10 e2457 3 2004 1 6 2004 Copyright: © 2004 Gur and Zamir.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Natural Biodiversity Breaks Plant Yield Barriers Natural biodiversity is an underexploited sustainable resource that can enrich the genetic basis of cultivated plants with novel alleles that improve productivity and adaptation. We evaluated the progress in breeding for increased tomato (Solanum lycopersicum) yield using genotypes carrying a pyramid of three independent yield-promoting genomic regions introduced from the drought-tolerant green-fruited wild species Solanum pennellii. Yield of hybrids parented by the pyramided genotypes was more than 50% higher than that of a control market leader variety under both wet and dry field conditions that received 10% of the irrigation water. This demonstration of the breaking of agricultural yield barriers provides the rationale for implementing similar strategies for other agricultural organisms that are important for global food security. Dramatic improvements in crop yield (in tomatoes, in this case) can be achieved by breeding selected genetic regions from wild species into domesticated varieties ==== Body Introduction Plant evolution under domestication has led to increased productivity, but at the same time it has narrowed the genetic basis of crop species (Ladizinsky 1998). A major objective in modern breeding is to return to the wild ancestors of crop plants and employ some of the diversity that was lost during domestication for the improvement of agricultural yields under optimal as well as stress field conditions (Bessey 1906; Tanksley and McCouch 1997; Lee 1998; Zamir 2001). Most of the genetic variation present in wild species has a negative effect on the adaptation of plants to agricultural environments; hence, the challenge is to identify and utilize the advantageous traits in a breeding program. DNA markers have facilitated quantitative trait loci (QTL) mapping studies in segregating populations, showing that certain genomic regions derived from wild germplasm have the potential to improve yield, e.g. for rice (Septiningshi et al. 2003), wheat (Huang et al. 2003), barley (Pillen et al. 2003), soybean (Concibido et al. 2003), chickpea (Singh and Ocampo 1997), tomato (Bernacchi et al. 1998), and pepper (Rao et al. 2003). In the above studies, and many others that are not cited, plants in the segregating populations generally contained a number of wild-species chromosome segments which masked the magnitude of some of the favorable effects that were clearly identified for certain introgressed alleles. As a result, the yield-promoting QTL did not have a substantial contribution to the phenotype and the best lines were inferior to intensively bred varieties that are in wide commercial cultivation. A major advantage for the above populations is that they can easily lead to the development of introgression lines that are discussed below. The main question addressed in the present study is whether it is possible to incorporate favorable wild-species QTL into genetic backgrounds that will consistently out-perform the leading varieties in the market. To enhance the rate of progress of breeding based on wild-species resources, we developed a population of tomato segmental introgression lines (ILs). The ILs comprised marker-defined genomic regions taken from the drought-tolerant wild species Solanum pennellii and introduced (through genetic crosses) onto the genetic background of the elite inbred variety M82 (Eshed and Zamir 1995; see Knapp et al. 2004 for the new taxonomic classification of tomato species in the genus Solanum). The ILs constitute an “exotic library” where the entire wild-species genome was partitioned among 76 lines each carrying a single homozygous introgressed segment. Implementation of this resource for QTL mapping is based on the nearly isogenic nature of the lines such that any phenotypic difference between M82 and an IL, or the hybrid of M82 with an IL (ILH), is attributable to the S. pennellii genomic segments (Figure 1). Similar population structures were recently shown to greatly facilitate the detection of naturally occurring variation in inbred mice (Singer et al. 2004). Figure 1 Genetic Sources of BY Variation (Exotic variation) Since M82, the multiple-introgression line (IL789), and their hybrid IL789 × M82 (ILH789) differ only in three S. pennellii segments (green chromosomes), any BY difference between them is associated with the exotic allelic variation. (Cultivated variation) Yield differences between M82 (red chromosomes), the four tomato tester inbreds (pink chromosomes), and their hybrids (M82 × Testers) result from allelic variation present in the cultivated tomato gene pool. (Cultivated + Exotic) The yield of the hybrids of IL789 with the four testers (IL789 × Testers) results from both cultivated and exotic variation. Phenotyping is the rate-limiting component in the utilization of such an exotic genetic resource. This is particularly true for quantitative traits where the reproducibility of the phenotype has to be evaluated in different seasons, environments, and genetic backgrounds. Over the past ten years the ILs and their hybrids have been assayed for yield-associated traits, and the data are presented, in silico, in a search engine that displays a range of statistical and graphical outputs that describe the components of the genetic variation (http://zamir.sgn.cornell.edu/; Gur et al. 2004). A review of the tomato QTL data and results of the QTL mapping studies from other species indicate that it is unlikely that a single introgression will induce a striking improvement in a yield-associated phenotype. However, pyramiding a number of independent introgressions in a single genotype, each with a positive effect on the desired trait, could be a strategy to greatly improve performance. The nearly isogenic nature of the ILs provides a relative advantage over other segregating populations in the rapid implementation of a pyramiding approach through crosses and marker analysis. We demonstrate here that the ILs make an efficient reagent for the discovery and utilization of genes that underlie traits of agricultural value and constitute a resource to explore the interactions among independent yield-associated QTL. We show that the pyramiding of independent yield-promoting segments can lead to novel varieties that reproducibly increase productivity relative to leading commercial genotypes both under normal cultivation conditions and in the stress environment of drought. Results/Discussion Exotic Variation In “ketchup tomatoes,” which are used to produce various concentrated products, agricultural yield is made up of the total weight of the fruits harvested per unit area (yield [Y], measured in kg/m2) and their soluble-solids content (mainly the sugars glucose and fructose), which is measured in refractometer brix (B) units (expressed as a percentage). Therefore, agricultural yield of processing tomatoes is the total sugar output per unit area (brix × yield [BY], measured in g/m2); the industry is searching for varieties that excel in BY. Our experiments were conducted in wet and dry fields, where the BY of the control variety M82 in the dry conditions was only 50% of that produced in the wet treatment, which received 10-fold greater irrigation (184 g/m2 and 353 g/m2, respectively; average BY from 3 y). In this study we focused on three independent introgressions from Chromosome 7 (IL7-5-5), Chromosome 8 (IL8-3), and Chromosome 9 (IL9-2-5) that affect the components of BY. Figure 2 summarizes the data of the yield components from three growing seasons in wet and dry fields. The data from the different years were pooled, as we detected no significant year x genotype interactions. IL7-5-5 was dominant in its effect on Y, as both the homozygous IL and heterozygous ILH increased Y in the wet fields by 30% compared to M82 and by 12% to 22% (nonsignificant) in the dry fields. IL7-5-5 did not affect B, while for BY it was dominant. IL8-3 was greatly inferior to M82 for Y (−55% and −34% for the wet and dry treatments, respectively), but the ILH increased Y by 45% and 25% (wet and dry, respectively). This result indicates a strong overdominant effect for the introgression (d/[a] = 2.5; see “Statistical analyses” for definitions). The homozygous IL had double the effect on B relative to the ILH; this resulted in a strong overdominant effect on BY in both environments (70% and 40% increases relative to M82 in the wet and dry fields, respectively; d/[a] = 5). The reduced Y of IL8-3 was caused by a pleiotropic effect of a leaf necrosis gene that was observed in all lines that were homozygous for this introgression; the necrosis was particularly severe in the dry treatment. IL9-2-5 significantly increased Y only in the homozygous condition in the wet treatment. For B and BY, ILH9-2-5 was intermediate between M82 and the homozygous IL, showing an additive mode of inheritance. The nature of the genes that improve BY in the above introgressions is unknown, with the exception of IL9-2-5, which harbors at least two QTL that affect the components of BY (Fridman et al. 2002). One of the QTL is Brix9-2-5, which resides in a 484-bp interval within the apoplastic invertase (LIN5) that increases sugar content of the fruit as a result of a modification of enzyme functions (Fridman et al. 2000 and unpublished data). LIN5 and three other invertase family members reside on segmental duplications in the near-collinear genomes of tomato and potato. These chromosomal segments are syntenically duplicated in the model plant Arabidopsis and in rice, thus facilitating the research of synteny-based orthologs and their relationship to yield components (Fridman and Zamir 2003). Figure 2 Pyramiding of S. pennellii Introgressions That Increase Agricultural Yield Components Introgression lines IL7-5-5, IL8-3, IL9-2-5, and IL789, which combines all three segments, were compared to M82 (percent difference from M82) in a homozygous (IL) and heterozygous (ILH) condition in wet and dry fields (1 plant/m2). The bars represent total yield (Y), brix (B), and brix × yield (BY) means (± standard error) from three growing seasons; these data were pooled, since no season × genotype interactions were found. The base line represents M82, where the mean BY values of M82 from the three seasons were 353 g/m2 in the irrigated treatment (455 g/m2 in 2001, 285 g/m2 in 2002, and 320 g/m2 in 2003) and 184 g/m2 in the dry treatment (244 g/m2 in 2001, 186 g/m2 in 2002, and 122 g/m2 in 2003). The additive effect (a) is half of the difference between each IL and M82. The dominance deviation (d) is the difference between ILH and the mid-value of its parents. Values marked by an asterisk are significant (p < 0.05). The bars in the gray background and their corresponding a and d values represent the expected values of IL789 and ILH789 assuming complete additivity of the introgression effects. Asterisks above the expected-value bars represent significant deviations from the observed means for IL789 or ILH789 as determined by a t test at a confidence level of 95%. All experiments were transplanted in a randomized block design with the following number of replications for each genotype under each irrigation regime: 2001, 10 replications; 2002, 15 replications; 2003, 15 replications. The three S. pennellii segments were pooled, using marker-assisted selection, into a single M82 line designated IL789 (homozygous for IL7-5-5, IL8-3, and IL9-2-5). IL789 showed a strong interaction with the environment: In the wet treatment it produced a Y similar to that produced by M82, while in the dry fields Y was reduced by 36% as a result of the pleiotropic effect of the recessive leaf necrosis gene on IL8-3. In the heterozygous condition IL789 dramatically increased Y in both field environments, and combined with the increases in B, ILH789 improved BY by 109% in the wet field and 58% in the dry fields. As described in earlier studies (Eshed and Zamir 1995) and demonstrated here for IL789, the positive effects of the wild introgressed segments on yield-associated traits were often more pronounced in the heterozygous condition due to linked deleterious recessive genes originating from S. pennellii. In a previous study based on the ILs, Eshed and Zamir (1996) showed less-than-additive epistatic interactions for yield QTL. For a large number of lines carrying pairs of different introgressed segments, they detected a trend of lower BY values than were expected based on the sum of the effects of the individual ILs. This phenomenon was more pronounced for combinations of ILs that affected the same component of BY (either B or Y). The less-than-additive mode of interaction was suggested as an underlying genetic model to explain canalized characters, where the phenotype is kept within narrow boundaries despite genetic and environmental disturbances. For quantitative traits affected by a large number of QTL, the less-than-additive interaction ensures that a “loss” of an allele will have a minimal effect on the canalized phenotype. In the present study we compared the observed phenotypic values of IL789 and ILH789 with the expected value based on the assumption of additivity of the effects of the three introgressions that were pyramided into these lines. In the wet treatment all the observed values for IL789 were lower than expected; however, this trend was statistically significant only for B. In the dry fields the recessive leaf necrosis gene on IL8-3 exerted a strong epistatic effect which nullified the contribution of the other introgressions. Thus in all cases the observed values for IL789 were lower than expected and very similar to those for IL8-3 (Figure 2). From the plant breeding point of view, it is noteworthy that by pyramiding three heterozygous introgressions that affect the different components of BY, we achieved in ILH789 81% and 70% additivity (wet and dry, respectively) of the effects of the individual ILHs. Thus the heterozygous wild-species introgression pyramid improved BY in the genetic background of M82 by 109% in the wet fields and 58% in the dry fields. Cultivated Variation Our breeding program within the Solanum lycopersicum gene pool over the past several years has generated tester inbreds of different origins that give a sampling of the genetic diversity of processing tomatoes. Four testers, whose hybrids with M82 exhibited the highest BY, were selected for this experiment, which was aimed at exploring the breeding potential of the genetic variation within the processing tomato germplasm (Figure 3A). The BY values of the inbred testers in the wet and dry treatments were not significantly different from that of M82, whereas the four hybrid combinations with M82 had higher mean BY in both environments (71% in the wet treatment and 51% in the dry treatment; Figure 3B). This improvement over the parents reflects the genetic variation present in the cultivated tomato gene pool, which was expressed as hybrid vigour originating from crossing of the preselected diverse inbreds (see Figure 1). As a reference outgroup for the entire experiment we selected the commerical hybrid BOS3155, which has been a processing tomato market leader in California for the past five years (http://www.ptab.org/). BY of BOS3155 was in a range similar to that of our experimental hybrids, indicating that the experiment was conducted in elite genetic backgrounds. Figure 3 Contribution of Cultivated and Exotic Variation to BY (A) The contribution of cultivated and exotic variation to BY in five genetic backgrounds. BY phenotypes in the Akko wide-spacing wet and dry experiments that involved four independent tester inbreds and their hybrids with M82 and IL789 are shown. Included are mean values for a control background of M82 and BOS3155. The four inbreds are represented as horizontal lines with circles in the gray bars. Experiments were transplanted in a randomized block design with 20 replications for each genotype under each irrigation regime. In all cases BY of the hybrids containing the exotic introgressions (IL789 × Testers) was significantly higher than that of their nearly isogenic cultivated tomato hybrids (M82 × Testers; t test, p < 0.01). The exotic effect, which represents the BY differences between the IL789 × Testers hybrids and the M82 × Testers hybrids, was consistent for all genetic backgrounds in each of the irrigation regimes. This was determined by genetic background × exotic effect two-way ANOVA (p for the interaction is 0.75 for the wet treatment and 0.88 for the dry field). (B) The mean contribution of cultivated and exotic variation to BY in wet and dry fields. BOS3155 is a leading commercial tomato hybrid that was used as a reference. Values of tester inbreds, M82 × Testers, and IL789 × Testers (shown as Δ% from M82) represent the means of the four genotypes included in each group (see Figure 3A). Base line and the letters attached to it represent M82. Means for each irrigation treatment with different letters are significantly different using a multiple-range means comparison (Tukey-Kramer; p < 0.01). The deduced exotic effect on BY is marked as black bars, and the contribution of the cultivated variation to BY is marked in gray. The absolute BY values of M82 were 303 g/m2 in the wet treatment and 122 g/m2 in the dry treatment. Combining Exotic and Cultivated Variation A correct assessment of the potential of exotic QTL is in the context of high-yield genetic backgrounds—those close to the “yield barrier.” This was achieved by crossing IL789 with the four inbred tester lines in a manner that combined the contribution of both the cultivated and the exotic variation (see Figure 1). These IL789 × Testers hybrids were nearly isogenic to the M82 × Testers hybrids, and thus the differences between them reflect the effect of the exotic alleles on BY. The effects of the three heterozygous introgressions on BY were consistent in all the hybrid combinations, and no genetic background × exotic effect interactions were found in any of the environments (Figure 3A). The mean BY increase of the four IL789 × Testers hybrids, compared to M82, was 170% (wet) and 115% (dry), while the mean contribution of the M82 × Testers hybrids was 71% (wet) and 51% (dry) (Figure 3B). Based on the consistent effect of the introgressions in the different genetic backgrounds, we could estimate the contribution of the S. pennellii pyramid as the mean difference between the nearly isogenic hybrid groups: 100% (wet) and 65% (dry). These estimates for the exotic effects on BY are very close to those obtained in the uniform M82 background, indicating additivity of the exotic and cultivated effects (see Figure 2). The highest BY was measured for the hybrid of IL789 with inbred #76. This hybrid was compared with M82 and BOS3155 in six independent experiments that differed in location, planting density, and irrigation regime (Figure 4). The BY advantage of the IL789 × 76 hybrid relative to M82 was not accompanied by other negative traits originating from the wild species and was observed in all field environments, ranging from 60% in Mevo-Hama with wide spacing and wet treatment to 200% in Akko under similar conditions. Significantly, the mean BY improvement of this IL789 × 76 hybrid over the market leader BOS3155 was 67% in the irrigated conditions and 58% in the dry conditions. Figure 4 BY Phenotypes of M82, BOS3155, and the Best Hybrid Combination (IL789 × 76) in Six Independent Trials Plants were grown in two locations, under two planting densities and two irrigation regimes. The locations were (i) the Western Galilee Experimental Station in Akko and (ii) Kibbutz Mevo-Hama (MH) in the Golan Heights. The planting densities were (i) single plants (SP; 1 plant per m2) and (ii) plots (14 plants per 4 m2, or 3.5 plants/m2). The irrigation regimes were (i) wet (320 m3 of water per 1,000 m2 of field throughout the growing season) and (ii) dry (30 m3 of water per 1,000 m2 of field). All experiments were transplanted in a randomized block design with the following number of replications: Akko-SP-wet, 15 replications; Akko-SP-dry, 20 replications; Akko-plots-wet, 8 replications; Akko-plots-dry, 8 replications; MH-SP-wet, 15 replications; MH-SP-dry, 15 replications. Means not connected by the same letter are significantly different using a multiple-range means comparison (Tukey-Kramer; p < 0.01). The results of IL789 × 76 and the control varieties in the different environments provided the means to explore genotype × environment (G × E) interactions and the stability of BY improvement associated with the heterozygous S. pennellii introgressions (Table 1). Generally, strong G × E interactions of new varieties indicate the lack of a predictable response, which is undesirable in breeding (Dudley and Moll 1969). In the wet fields we detected significant genotypic, environmental, and G × E interaction effects for the varieties tested. However, the IL789 hybrid always had significantly higher BY than the commercial varieties, and the interaction was caused in part by differences between the commercial varieties M82 and BOS3155 (Figure 4). In the dry trials there was a highly significant genotypic effect and marginally significant effects for the environment; no interaction between the two components was detected, and IL789 × 76 had higher BY in all experiments. This analysis highlights the potential of wild germplasm to affect yield stability in diverse environments, which has long been recognized as an important objective in plant breeding. Table 1 ANOVA of Genotype by Environment Interaction for BY in IL789 × 76, BOS3155, and M82 Combined analysis of variance of genotype (G), environment (E), and G × E interaction for BY of three varieties and three environments in each of the dry and wet treatments a df, degrees of freedom bMS, mean square We have demonstrated that an IL population derived from a wild tomato species, with no yield potential, can make a wide array of previously unexplored genetic variation rapidly available to plant breeders to improve crop productivity. The effectiveness of the introgressions in diverse genetic backgrounds indicates that alleles similar to those of the wild species are not present in the cultivated tomato gene pool. The results presented here using the tomato ILs establish a genetic infrastructure to explore the molecular basis underlying yield heterosis. With the coming sequence of the tomato genome it will be easier to isolate those factors that are responsible for the strong overdominant effects, such as those observed for IL8-3 and some additional lines that are described in the IL phenotypic database Real Time QTL (Gur et al. 2004). Finally, our approach of pyramiding beneficial wild-species chromosome segments provides an alternative to the genetically modified organism strategy for crop improvement and offers a new paradigm to revitalize plant breeding (Tanksley and McCouch 1997; Zamir 2001; Morgante and Salamini 2003; Koornneef et al. 2004). Can these results be extrapolated to the breeding of other crop plants? Wild species that are distantly related to crop plants can be viewed as vast naturally mutagenized resources where every gene and regulatory element has been refined and defined by evolution. We propose that for crops that rely on a rather narrow genetic basis (rice, wheat, soybean, etc.) and have rich biodiversity resources, the construction and screening of ILs will lead to dramatic improvements in yield and other quality traits that are important for human well-being (Rosegrant and Cline 2003). As we are able to make a wider range of natural genetic diversity accessible to breeders, we will make progress in improving our global food security. Materials and Methods Field trials The results presented are from three growing seasons. In 2001 and 2002 all field trials were conducted at the Western Galilee Experimental Station in Akko, Israel, at a wide-spacing planting density of 1 plant per m2. In 2003, trials were conducted in two locations: Akko and Mevo-Hama, in the Golan Heights. In Akko, experiments were both at wide-spacing planting density and in plots of 14 plants per 4 m2 (3.5 plants/m2). In Mevo-Hama all experiments were at the wide-spacing density. The seedlings were grown in a greenhouse for 35–40 d and then transplanted in the field, at the beginning of April in Akko, and at the beginning of May in Mevo-Hama. In all seasons and locations both wet and dry trials were conducted. Both the wet and dry fields started the growing season at “field capacity,” which represents the maximum amount of water that the soil could hold. For the dry treatment only 30 m3 of water was applied per 1,000 m2 of field immediately after transplanting. In the wet treatment 320 m3 of water was applied per 1,000 m2 of field throughout the growing season according to the irrigation protocols in the area. All experiments were transplanted in a randomized block design. Genotyping and phenotyping To ensure the nearly isogenic nature of the ILs, we bred an M82 line that was heterozygous for all three introgressions and used RFLP markers to genotype 128 F2 plants segregating for the three S. pennellii genomic segments (CT252 for IL7-5-5, CT148 for IL8-3, and GP263 for IL9-2-5). Lines homozygous for each of the segments and IL789 homozygous for all three introgressions were selected and verified with RFLP markers that flanked the introgressed segments from both ends (http://www.sgn.cornell.edu/). Phenotyping of the plants for Y, B, and BY was performed according to published protocols (Fridman et al. 2002). Statistical analyses Statistical analyses were performed using the JMP V.5 software package (SAS Institute, Cary, North Carolina, United States). Mean values for the parameters measured for the tested genotypes were compared using the “Fit Y by X” function and “Compare all pairs” (Tukey-Kramer). All calculations were performed with the phenotypic values, while some of the results are presented as the percent difference from M82. The additive effect (a) was half of the difference between each IL and M82, and its significance level was determined by the comparison between the IL and M82. The dominance deviation (d) is the difference between ILH and the mid-value of its parents. Its significance level was calculated by contrasting the ILH (+1) with M82 (−0.5) and the appropriate IL (−0.5). The degree of dominance for each introgression (d/[a]) was calculated by dividing the mean dominance deviation by the mean additive effect. Deviation of the observed yield component values of the pyramided genotypes (IL789 and ILH789) from the expected values based on the assumption of additivity of the effects of the individual introgressions was tested using a t test at p < 0.05. G × E interaction was tested using a two-way ANOVA. We thank Gabi Gera and Uri Luchinsky for their assistance with the field trials and Dr. Michael Friedmann for his valuable comments. This research was supported by grant no. M21-025 from the Middle East Regional Cooperation Program of the United States Agency for International Development. Conflicts of interest. The authors have declared that no conflicts of interest exist. Author contributions. AG and DZ conceived and designed the experiments. AG performed the experiments. AG and DZ analyzed the data. AG and DZ wrote the paper. Academic Editor: Jeffrey Dangl, University of North Carolina, Chapel Hill Citation: Gur A, Zamir D (2004) Unused natural variation can lift yield barriers in plant breeding. PLoS Biol 2(10): e245. Abbreviations Bbrix BYbrix × yield G × Egenotype × environment ILintrogression line ILHIL hybrid QTLquantitative trait loci Yyield ==== Refs References Bernacchi D Beck-Bunn T Emmatty D Eshed Y Inai S Advanced backcross QTL analysis of tomato. II. Evaluation of near-isogenic lines carrying single-donor introgressions for desirable wild QTL-alleles derived from Lycopersicon hirsutum and L. pimpinellifolium Theor Appl Genet 1998 97 170 180 Bessey CE Crop improvement by utilizing wild species 1906 American Breeders' Association II 112 118 Concibido VC La Vallee B Mclaird P Pineda N Meyer J Introgression of a quantitative trait locus for yield from Glycine soja into commercial soybean cultivars Theor Appl Genet 2003 106 575 582 12595984 Dudley JW Moll RH Interpretation and use of estimates of heritability and genetic variances in plant breeding Crop Sci 1969 9 257 262 Eshed Y Zamir D An introgression line population of Lycopersicon pennellii in the cultivated tomato enables the identification and fine mapping of yield associated QTL Genetics 1995 141 1147 1162 8582620 Eshed Y Zamir D Less-than additive epistatic interactions of quantitative trait loci in tomato Genetics 1996 143 1807 1817 8844166 Fridman E Zamir D Functional divergence of a syntenic gene family in tomato, potato and Arabidopsis Plant Physiol 2003 131 603 609 12586884 Fridman E Pleban T Zamir D A recombination hotspot delimits a wild-species quantitative trait locus for tomato sugar content to 484 bp within an invertase gene Proc Natl Acad Sci U S A 2000 97 4718 4723 10781077 Fridman E Liu YS Carmel-Goren L Gur A Shoresh M Two tightly linked QTL modify tomato sugar content via different physiological pathways Mol Genet Genomics 2002 266 821 826 11810256 Gur A Semel Y Cahaner A Zamir D Real Time QTL of complex phenotypes in tomato interspecific introgression lines Trends Plant Sci 2004 9 107 109 15058274 Huang XQ Coster H Ganal MW Roder MS Advanced backcross QTL analysis for the identification of quantitative trait loci alleles from wild relatives of wheat (Triticum aestivum L) Theor Appl Genet 2003 106 1379 1389 12750781 Knapp S Bohs L Nee M Spooner DM Solanaceae – a model for linking genomics with biodiversity Comp Funct Genomics 2004 5 285 291 18629162 Koornneef M Alonso-Blanco C Vreugdenhil D Naturally occurring variation in Arabidopsis thaliana Annu Rev Plant Biol 2004 55 141 172 15377217 Ladizinsky G Plant evolution under domestication 1998 Dordrecht Kluwer Academic Press 262 Lee M Genome projects and gene pools: New germplasm for plant breeding? Proc Natl Acad Sci U S A 1998 95 2001 2004 9482824 Morgante M Salamini F From plant genomics to breeding practice Curr Opin Biotechnol 2003 14 214 219 12732323 Pillen K Zacharias A Leon J Advanced backcross QTL analysis in barley (Hordeum vulgar e L) Theor Appl Genet 2003 107 340 352 12677407 Rao GU Ben Chaim A Borovsky Y Paran I Mapping of yield-related QTLs in pepper in an interspecific cross of Capsicum annuum and C. frutescens Theor Appl Genet 2003 106 1457 1466 12750789 Rosegrant MW Cline SA Global food security: Challenges and policies Science 2003 302 1917 1919 14671289 Septiningshi EM Prasetiyono J Lubis E Tai TH Tjubaryat T Identification of quantitative trait loci for yield and yield components in an advanced backcross population derived from the Oryza sativa variety IR64 and the wild relative O. rufipogon Theor Appl Genet 2003 107 1419 1432 14513215 Singer JB Hill AE Burrage LC Olszens KR Song J Genetic dissection of complex traits with chromosome substitution strains of mice Science 2004 304 445 448 15031436 Singh KB Ocampo B Exploitation of wild Cicer species for yield improvement in chickpea Theor Appl Genet 1997 95 418 423 Tanksley SD McCouch SR Seed banks and molecular maps: Unlocking genetic potential from the wild Science 1997 277 1063 1066 9262467 Zamir D Improving plant breeding with exotic genetic libraries Nat Rev Genet 2001 2 983 989 11733751

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020248Research ArticleEcologyEvolutionZoologyInsectsConflict over Male Parentage in Social Insects Male Parentage in Social InsectsHammond Robert L [email protected] 1 Keller Laurent 1 1Department of Ecology and Evolution, Bâtiment de BiologieUniversity of Lausanne, LausanneSwitzerland9 2004 24 8 2004 24 8 2004 2 9 e24828 1 2004 3 6 2004 Copyright: © 2004 Hammond and Keller2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Policing Relative Conflicts of Interest in Social Insects Mutual policing is an important mechanism that maintains social harmony in group-living organisms by suppressing the selfish behavior of individuals. In social insects, workers police one another (worker-policing) by preventing individual workers from laying eggs that would otherwise develop into males. Within the framework of Hamilton's rule there are two explanations for worker-policing behavior. First, if worker reproduction is cost-free, worker-policing should occur only where workers are more closely related to queen- than to worker-produced male eggs (relatedness hypothesis). Second, if there are substantial costs to unchecked worker reproduction, worker-policing may occur to counteract these costs and increase colony efficiency (efficiency hypothesis). The first explanation predicts that patterns of the parentage of males (male parentage) are associated with relatedness, whereas the latter does not. We have investigated how male parentage varies with colony kin structure and colony size in 50 species of ants, bees, and wasps in a phylogenetically controlled comparative analysis. Our survey revealed that queens produced the majority of males in most of the species and that workers produced more than half of the males in less than 10% of species. Moreover, we show that male parentage does not vary with relatedness as predicted by the relatedness hypothesis. This indicates that intra- and interspecific variation in male parentage cannot be accounted for by the relatedness hypothesis alone and that increased colony efficiency is an important factor responsible for the evolution of worker-policing. Our study reveals greater harmony and more complex regulation of reproduction in social insect colonies than that expected from simple theoretical expectations based on relatedness only. Workers of social insects prevent other workers laying eggs to increase colony efficiency and not -- as traditionally thought - purely because workers are more related to the queen of the colony ==== Body Introduction Major evolutionary transitions (Maynard-Smith and Szathmáry 1995) require the evolution of mechanisms that moderate within-group conflict (Keller 1999; Queller 2000; Michod and Roze 2001). One such mechanism is mutual policing, where members of a group collectively prevent individuals from acting in their own selfish interests (Frank 1995). The best example of mutual policing behavior in nature is found in social insects, where workers police worker reproduction (worker-policing) by selectively removing worker-laid eggs that would otherwise develop into males (Ratnieks and Visscher 1989; Foster and Ratnieks 2000, 2001a; Halling et al. 2001; Oldroyd et al. 2001), or by directing aggression toward workers with developing ovaries (Monnin and Ratnieks 2001; Iwanishi et al. 2003). Selection for worker-policing depends upon two variables: the relative relatedness of workers to queen- and worker-produced males (relatedness hypothesis) and the colony-level cost of workers reproducing (efficiency hypothesis). Worker-policing theory (Starr 1984; Woyciechowski and Lomnicki 1987; Ratnieks 1988), an extension of kin selection theory (Hamilton 1964), has typically highlighted relatedness as the all-important variable that explains when workers should lay male-destined eggs and when they should police one another's reproduction. In contrast, the costs of worker reproduction (Ratnieks 1988) have been largely ignored or given low prominence in the literature, with the effect that the relatedness hypothesis has become widely accepted as the explanation for worker-policing (Whitfield 2002). Empirical investigations of worker-policing behavior initially focused on species with colony kin structures that predicted the behavior under the relatedness hypothesis, and worker-policing was first demonstrated in the multiply mated honey bee, Apis mellifera (Estoup et al. 1994; Visscher 1996). Subsequently, similar patterns have been found in other multiply mated members of the genus Apis (Halling et al. 2001; Oldroyd et al. 2001; Wattanachaiyingcharoen et al. 2002) and in the multiply mated wasp Vespula vulgaris (Foster and Ratnieks 2001a). Support for the relatedness hypothesis comes from contrasts between these species and closely related species that are singly mated (Peters et al. 1999; Foster and Ratnieks 2001c) and from an intraspecific study of the vespine wasp Dolichovespula saxonica, in which worker-policing behavior is facultative and occurs only in colonies headed by multiply mated queens (Foster and Ratnieks 2000). There are, however, problems with the conclusion that relatedness is the underlying cause of policing behavior, because phylogeny is not controlled for in the interspecific comparisons described above. This is an important problem, because these species are clustered with respect to phylogeny (e.g., four Apis species), and related wasp species, such as Vespa crabro, show patterns of worker reproduction and worker-policing behavior that are consistent with the efficiency hypothesis but not the relatedness hypothesis (Foster et al. 2002). The relatedness hypothesis explicitly predicts that the parentage of males (male parentage) is dependent upon colony kin structure. Importantly, males should be worker-produced in colonies headed by single, once-mated queens, and queen-produced in colonies headed by multiple related queens, or by multiply mated queens, because worker reproduction is prevented by worker-policing. By contrast, the efficiency hypothesis predicts no association of male parentage or worker-policing with colony kin structure. In this paper we test these predictions by analyzing, using methods that control for phylogenetic dependence, how the proportion of worker-produced males (WPM) varies with both colony kin structure and colony size. The theoretical difference in relatedness of workers to queen- and worker-produced males (r diff) was used to make predictions about male parentage based upon colony kin structure. We included colony size in our analyses because it potentially alters expected patterns of male parentage (Bourke 1999) by altering power relationships within the colony. In small colonies a single individual may have the power to dominate male production completely, but such reproductive dominance becomes less likely as colony size increases. Results We found data for 50 species: 16 ants, 20 bees, and 14 wasps (Table 1; Figure 1). WPM varied considerably (0%–85%), but in most species, queens produced the majority of males, with less than 10% of males being worker-produced in 72% of species surveyed. In only 10% of species were more than 50% of males worker-produced. There was great variation in the number of males (n m = 13–1,426) and likewise in the number of assignable males (n a = 10–677, where n a is the sample size corrected for the probability of nondetection [Foster et al. 2001]) that were used to estimate the WPM. However, in those species for which we had relevant data, there was no significant correlation of n m or n a with WPM (Spearman's rank correlation: n m versus WPM: ρ = 0.17, n = 45, p = 0.27; n a versus WPM: ρ = 0.11, n = 27, p = 0.59), suggesting that there was no systematic bias in our dataset. Figure 1 Composite Phylogeny Used in Comparative Analyses Phylogeny includes within-species variation. Duplicated species labeled 1 or 2 (e.g., Leptothorax acervorum 1 and 2) refer to taxa in which within-species variation was included in some analyses (see text for details). Dotted lines, r diff is negative; solid lines, r diff is positive. Horizontal bars indicate WPM. Table 1 The WPM, Colony Kin Structure, and Colony Size in a Sample of Queenright Colonies of Eusocial Hymenoptera a Estimates based on pedigree relatedness NA, maximum likelihood methods were used; n c, number of queenright colonies in which male parentage was analyzed; n q, average number of queens per colony; n w, colony size defined as the number of workers Comparative Analysis Tests of serial independence showed that there was significant phylogenetic dependence for all variables when within-species variation was ignored (log10WPM, p = 0.016; r diff, p < 0.001; log10 of colony size [log10 n w], p < 0.001) and when within-species variation was included (log10WPM, p = 0.002; r diff, p < 0.001). This confirmed that a comparative approach using an analysis of independent contrasts was warranted (Abouheif 1999; Freckleton et al. 2002). The WPM was not significantly correlated with colony kin structure in any of our comparative analyses. Ignoring within-species variation, the slope of the line of regression of contrast in log10WPM against contrast in r diff was not significantly different from zero (Figure 2A; slope β = −2.14, t = –1.53, df = 48, p = 0.13), and the mean contrast in log10WPM (–1.70 ± 5.4) was not significantly different from zero when r diff was coded categorically (t = 0.31, df = 2, p = 0.78). Likewise, neither analysis that included within-species variation was significant (Figure 2B; β = –1.31, t = –1.36, df = 55, p = 0.18; mean contrast in log10WPM = –0.14 ± 4.81, t = 0.03, df = 7, p = 0.98). The power was high (Figure 3; power greater than 0.75) for both analyses of regression to detect a large effect of relatedness on WPM, and there was relatively high power (see Figure 3; power greater than 0.6) to detect a medium effect in the analysis that included within-species variation. The WPM also did not show any significant relationship with colony size when all species were included (Figure 4A; β = –0.12, t = –1.04, df = 48, p = 0.30) or when relatedness was controlled for and we included only species with positive r diff values (Figure 4B; β = –0.14, t = –1.05, df = 41, p = 0.30). Figure 2 Variation in Worker Reproduction with Colony Kin Structure Axes show standardized independent contrasts in WPM (log10WPM) and in r diff. (A) is based on species values; (B) includes intraspecific variation for seven species (see text). Lines of regression are forced through the origin. Figure 3 Statistical Power As a Function of the Slope β (Effect Size) in Comparative Analyses of r diff on WPM On the graph, + data points show the power of tests in which within-species variation was ignored, and × show the power of tests in which within-species variation was included. Figure 4 Variation in Worker Reproduction with Colony Size Axes show standardized independent contrasts in the proportion of worker-produced males (log10WPM) and in colony size (log10 n w). (A) includes all species; (B) includes only species in which relatedness predicts worker-produced males (i.e., r diff is positive). Lines of regression are forced through the origin. Discussion Our survey revealed that queens produced the majority of males in most of the species, and in less than 10% of the species did workers produce more than half of the males, in line with earlier surveys based largely on behavioral data (Bourke 1988; Choe 1988). Since workers of all the species included in our survey have functional ovaries, this demonstrates that self-restraint and worker-policing are widespread and powerful mechanisms that regulate reproduction in colonies of social Hymenoptera. Our comparative study did not support the view that intra- and interspecific variation in male parentage can be accounted for by the relatedness hypothesis only. First, and most importantly, the proportion of males produced by workers was not significantly associated with colony kin structure. This was true both when within-species variation in colony kin structure was included and when it was ignored. In fact, although the relatedness hypothesis predicts a positive relationship between WPM and r diff, the analyses of relatedness revealed a tendency for a negative relationship. Importantly, our study included data from 50 species, and our power analyses showed that we had enough power to detect a relationship between male parentage and colony kin structure if it was of moderate or large effect. A second line of evidence against the relatedness hypothesis came from the finding that workers produce only very few males in a large number of species where, on purely relatedness grounds, they would benefit from producing males. Workers produce less than 10% of males in 30 of the 43 species (70%) in which workers were more related to worker-produced than to queen-produced males. A third line of evidence came from within-species comparisons. Only in Dolichovespula saxonica (Foster and Ratnieks 2000) were patterns of male parentage compatible with the relatedness hypothesis. By contrast, patterns of male parentage contradicted the relatedness hypothesis in the ants Leptothorax acervorum (Hammond et al. 2003), Lasius niger (Fjerdingstad et al. 2002), Formica exsecta (Sundström et al. 1996; Walin et al. 1998), and Myrmica tahoensis (Evans 1998). Interestingly, intraspecific variation in colony sex ratios in agreement with relatedness predictions have been shown in L. acervorum (Chan and Bourke 1994; Chan et al. 1999; Hammond et al. 2002), F. exsecta (Sundström et al. 1996), and M. tahoensis (Evans 1995, 1998). This suggests that although workers in these species can assess within-colony relatedness, they do not appear to respond to it in the context of the conflict over male parentage (Walin et al. 1998; Hammond et al. 2003). The lack of association between kin structure and the degree of male parentage by workers indicates that factors others than relatedness effectively act as a brake on worker reproduction. The finding of no significant effect of colony size on WPM suggests that the ratio of queens to workers is not an important general factor regulating reproductive division of labor in social Hymenoptera. The low instance of worker reproduction is therefore unlikely to be the consequence of queens using aggression or pheromones to suppress worker reproduction, except, perhaps, in the few species with very small numbers of workers (e.g., Strassmann et al. 2003). Most importantly, unchecked worker reproduction is likely to reduce overall colony productivity and may therefore reduce the average fitness of colony members. For example, reproductive workers have been found to spend time engaged in dominance interactions and egg-laying (Cole 1986) that otherwise would be used for foraging and brood rearing. Unchecked worker reproduction could also cause a “tragedy of the commons” (Hardin 1968; Frank 1995, 1996), because there would be more male brood than can be reared by the colony. If queens conceal the sex of their eggs (Nonacs 1993), these costs may also include workers mistakenly replacing queen-laid diploid eggs with their own male eggs. Furthermore, costs incurred by workers biasing colony sex ratios can select for worker-policing behavior (Foster and Ratnieks 2001b). Theory shows that these costs do not have to be large for worker-policing and self-restraint to be selected (Ratnieks 1988). Our data showed considerable variation across species in the origin of males, raising the question, what are the factors underlying interspecific variation in male parentage? The efficiency hypothesis predicts that the extent of worker-produced males should depend largely on the shape and slope of the function relating colony productivity and worker efficiency. This property is expected to vary across species, and it is conceivable that closely related species, which are likely to live in similar habitats and have similar life histories, also have similar functions relating colony productivity and worker efficiency. Consistent with this prediction, our analysis revealed a significant phylogenetic signal, with closely related species being more similar in terms of the origin of males than expected by chance. Importantly, this similarity was not due to a greater similarity in kin structure and colony size between closely related species, because these two factors had no significant effect on the origin of males. Previous evidence for the view that the relatedness hypothesis can account for variation in male parentage comes mostly from matched comparisons between honey bees (genus Apis) and singly mated stingless bees (tribe Meliponini) (Ratnieks 1988; Peters et al. 1999) and comparisons within vespine wasps (Foster and Ratnieks 2001c). However, a closer inspection of these matched comparisons reveals problems. In the matched comparison with honey bees, stingless bees are generally assumed to have worker-produced males. However, there is considerable variation in levels of worker reproduction, with males in the majority of species being exclusively queen-produced (Figure 1). Moreover, workers of some stingless bee species are completely sterile (Suka and Inoue 1993; Boleli et al. 2000), indicating that considering stingless bees as a taxon with generalized worker reproduction is not warranted. Similarly, the matched comparison in vespine wasps also has problems. It is true that males are queen-produced, and that workers police one another in Vespula vulgaris, a species in which queens are multiply mated (Foster and Ratnieks 2001a), whereas at least some males are worker-produced in Dolichovespula, a species in which queens are singly mated (Foster et al. 2001). However, the wasp most basal in the phylogeny (Vespa crabro) is singly mated, yet males are all queen-produced because workers police one another (Foster et al. 2000, 2002). Considering Vespa, Vespula, and Dolichovespula together, the most parsimonious explanation is that worker-policing is the ancestral state in vespines and it has been lost, or at least reduced, in Dolichovespula. In short, neither of these traditional lines of support for the relatedness hypothesis stand up to close scrutiny. In conclusion, our comparative analysis does not support relatedness as the general explanation of patterns of male parentage and occurrence of worker-policing in social Hymenoptera. The concentration of published examples of worker-policing in multiply mated bees and wasps probably reflects the influence of the relatedness hypothesis on the selection of study taxa, rather than relatedness being the ultimate explanation of worker-policing. Moreover, recent studies have revealed worker-policing in species in which the relatedness hypothesis predicts males to be produced by workers (Kikuta and Tsuji 1999; Foster et al. 2002; Hartmann et al. 2003; Iwanishi et al. 2003). We conclude that costs associated with worker reproduction are likely to be significant and variation in these costs to be the main factor underlying differences across species in the origin of males. Experimental investigations of the colony-level costs of worker reproduction have begun (Lopez-Vaamonde et al. 2003). More are needed. It will also be important to conduct behavioral assays to determine whether worker-policing, by either egg-eating or aggression toward workers with developing ovaries, is responsible for the lack of worker reproduction in the stingless bee genera Trigona and Plebeia. Finally, we would like to stress that the finding that kin structure alone cannot account for the intra- and interspecific variation in male parentage does not amount to saying that kin structure is unimportant. Rather, it may work in concert with costs as a force influencing patterns of male parentage in social insects. Thus, this study reveals greater harmony and more complex regulation of reproduction in social insect colonies than that expected from simple theoretical expectations based on relatedness alone. Materials and Methods Male parentage For all analyses, the response variable was WPM (see Table 1). For almost all studies, estimates of WPM took into account the power of the genetic markers to detect worker reproduction using either exclusion (Foster et al. 2001) or maximum likelihood approaches (Arévalo et al. 1998). Where this type of analysis was not included in the original paper we reanalyzed data using the exclusion-based approach of Foster et al. (2001). Specific details of how we treated data are given for each species in Protocol S1. With comparative analyses there is always the difficult question of deciding “quality control” criteria to ensure that data are reliable and comparable. We collated data from published, in-press, and unpublished sources where colony genetic structure and male parentage were known accurately from molecular genetic markers. We restricted our survey to those including molecular genetic data, because recent genetic studies have shown that colony kin structures inferred from behavioral observations are often incorrect (e.g., mating frequency in Leptothorax nylanderi c.f. Plateaux 1981; Foitzik et al. 1997; Foster and Ratnieks 2001c), and in some social insect taxa (e.g., stingless bees and ants), workers lay trophic eggs that mistakenly could be counted as reproductive (Bourke 1988). We also restricted our analysis to queen-containing (queenright) colonies and species in which workers have ovaries. We did this because our aim was to investigate the outcome of worker–queen and worker–worker conflict. For those studies that included data on both queenright and queenless colonies, we considered male parentage in queenright colonies only (Protocol S1; e.g., Vespula germanica [Goodisman et al. 2002]). For all but two species, Leptothorax unifaciatus and Epimyrma ravouxi (L. Keller, J. Heinze, and A. F. G. Bourke, unpublished data), data were for adult or pupal males. For these two exceptional species, we had estimates of WPM at only the egg stage. However, as we found few worker-laid male eggs in both species (see Table 1), our estimate of WPM at the egg stage most likely reflected WPM in adults. In our comparative analyses we used log10WPM. Colony genetic structure We made predictions about the parentage of males based on colony kin structure by calculating r diff, the theoretical difference in relatedness of workers to queen- (r w–qm) and to worker-produced males (r w–wm) (see Table 1). The relatedness hypothesis predicts that if r diff is positive, males are worker-produced, and if r diff is negative, males are queen-produced, because workers should police one another. For colonies headed by single queens, where variation in colony genetic structure is caused by variation in the effective mating frequency of queens (Pamilo 1993), we calculated r diff as (2r w–w – 1)/4, where r w–w is the relatedness among adult workers. For species with variation in queen number (polygyny), predictions about worker reproduction are more complicated because both queen number and queen relatedness are important (Pamilo 1991). For these species, we estimated r diff from the actual relatedness of workers to queens (r w–q) and among workers (r w–w) as r diff = (r w–w – r w–q)/2. In our comparative analyses we used r diff as a continuous explanatory variable, or we coded r diff categorically as one when r diff was greater than zero (worker-produced males predicted), or as zero when r diff was less than zero (queen-produced males predicted). Colony size We defined colony size as the number of adult workers per nest (n w; see Table 1). Where only ranges of worker number were given, we took the midpoint value, and if more than one estimate was available, we combined data by calculating unweighted means. In our comparative analysis we used log10 n w as an explanatory variable. Comparative analysis We constructed an ant, bee, and wasp phylogeny (see Figure 1) by combining published phylogenies. For ants, we based our phylogeny on Keller and Genoud's (see Figure 3 in Keller and Genoud [1997]), which we modified in light of a recent combined molecular and morphological phylogeny (Ward and Brady 2003); for bees, we based it on a combined DNA and morphological phylogeny (see Figure 5 in Cameron and Mardulyn [2001]), and for wasps, on a morphological and behavioral phylogeny (Smith et al. 2001). In addition, we added phylogenetic details for the Meliponini (stingless bees) following Velthuis (1997), and for leptothoracine ants, we used the molecular phylogeny of Baur et al. (1996). We placed bees basal to ants and wasps (see Figure 1) (Brothers and Carpenter 1993; Brothers 1999). We set all branch lengths equal, corresponding to a punctuational view of evolutionary change, and we considered ambiguous nodes to be unresolved. Using this tree, we tested the assumption of the phylogenetic independence of our three variables (log10WPM, r diff, and log10 n w) by a test for serial independence (Abouheif 1999) calculated by the program Phylogenetic Independence (Reeve and Abouheif 2003). For these analyses, we rotated nodes within our dataset 10,000 times and randomly shuffled our data 10,000 times to generate our null distribution. As all three variables showed significant phylogenetic nonindependence (see Results), we used Felsenstein's method of independent contrasts in our comparative analyses (Felsenstein 1985). Analyses using r diff coded categorically were carried out using the “Brunch” algorithm in CAIC (Purvis and Rambaut 1995), whereas analyses using r diff and log10 n w coded as continuous variables were analyzed using the program PDTREE (Garland et al. 1999; Garland and Ives 2000). We tested Brunch analyses for significance by comparing the mean independent contrast against zero using t-tests. We tested for the significance of contrasts generated by PDTREE by regression through the origin. We did not reduce the number of degrees of freedom (df), as has been suggested for phylogenies containing polytomies (Purvis and Garland 1993), because none of our analyses were significant without such adjustment. Power analyses (see below) were calculated using R (http://www.r-project.org/). All other statistical tests were performed using SPSS (version 11). We tested the hypothesis that colony kin structure determines patterns of male parentage both when within-species variation in kin structure was ignored and when it was included. In our first set of two analyses, we used estimates of WPM and r diff that were mean values for each species. We calculated independent contrasts between log10WPM and r diff, and with r diff coded as a categorical variable. In our second set of two analyses, we included within-species variation in colony genetic structure that was present in seven species because of facultative variation in queen number or queen mating frequency (see Table 1). We did this by calculating r diff per colony and grouping colonies into those where r diff was positive (worker-production of males was predicted), and those where r diff was negative (males were predicted to be queen-produced because of worker-policing). We then estimated WPM for each group. We modified the phylogeny by adding an additional bifurcation at the tips corresponding to these seven species (see Figure 1). Although it is not necessary to control for phylogeny when testing hypotheses within species, doing so enabled us to combine evidence from within- and among-species comparisons (Garland et al. 1992). Using our modified dataset, we calculated independent contrasts between log10WPM and r diff, and with r diff coded as a categorical variable. We tested the role of colony size in two ways. First, we ignored any effect of relatedness and simply compared contrasts in log10WPM with contrasts in log10 n w. Second, we controlled for relatedness by limiting our analysis to species in which workers were more related to worker- than to queen-produced males (i.e., r diff was positive), and then compared contrasts in log10WPM with contrasts in log10 n w in this subset of the data. Statistical power To investigate the power of our analysis, we first determined the expected relationship between WPM and r diff in our dataset. To do that we set WPM to 0% when r diff was less than zero, to 100% when r diff was greater than zero, and to 50% when r diff was equal to zero. An analysis of independent contrasts based on this hypothetical relationship gave a highly significant relationship between WPM and r diff both when within-species variation was ignored (β = 5.48, t = 6.57, df = 48, p < 0.0001) and included (β = 6.59, t = 9.12, df = 55, p < 0.0001). On the basis of these slopes, we conducted a power analysis by assuming two types of effects. We considered r diff to have a “large” effect on WPM when β was greater than 4.0, and a “moderate” effect when β was between 2.0 and 4.0. To test the power that our analysis had to detect a large and moderate effect, we used the model y = βx + “resampled residual of y,” where x is the observed standardized contrast in rdiff and “resampled residual of y” is the residual of y estimated by resampling the distribution of residuals from our observed regressions through the origin. From this model, we defined power as the proportion of regressions (forced through the origin) in 1,000 simulated datasets that were significant at α ≤ 0.05 for a given slope β (the effect size). We investigated how power varied with effect size by increasing β incrementally from 1 to 5 in steps of 0.1 (see Figure 3). Supporting Information Protocol S1 Details of Data Selection Methods and Sources A detailed synopsis of how data used in this paper were selected from published and unpublished sources. (86 KB DOC). Click here for additional data file. We thank the following people for help in compiling the data: Andrew Bourke, Mark Brown, Anne Dollin, Jay Evans, Michael Goodisman, Else Fjerdingstad, Kevin Foster, Juergen Heinze, Joan Herbers, Michael Henshaw, Ben Oldroyd, Robert Paxton, Matthias Sanetra, Lotta Sundtröm, Eva Tóth, Palle Villesen, and Tom Wenseleers. We thank Ted Garland and Nick Isaac for advice on the comparative analysis and Jerome Goudet for help in estimating the power of our analyses. Michel Chapuisat, Sara Helms Cahan, Kevin Foster, Max Reuter, Tom Wenseleers, three anonymous referees, and the academic editor kindly gave us comments on previous versions of the manuscript. This work was funded by the European Union “Improving Human Potential” research-training network “INSECTS” under contract HPRN-CT-2000-00052. Conflicts of interest. The authors have declared that no conflicts of interest exist. Author contributions. RH and LK conceived and designed the experiments. RH analyzed the data. RH and LK wrote the paper. Academic Editor: Ross Crozier, James Cook University Citation: Hammond RL, Keller L (2004) Conflict over male parentage in social insects. PLoS Biol 2(9): e248. 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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020261Research ArticleCell BiologyMammalsThe Rab5 Effector Rabankyrin-5 Regulates and Coordinates Different Endocytic Mechanisms Rabankyrin-5 in MacropinocytosisSchnatwinkel Carsten 1 Christoforidis Savvas 2 Lindsay Margaret R 3 Uttenweiler-Joseph Sandrine 4 ¤Wilm Matthias 4 Parton Robert G 3 Zerial Marino [email protected] 1 1Max-Planck Institute of Molecular Cell Biology and GeneticsDresdenGermany2Laboratory of Biological Chemistry, Medical SchoolUniversity of Ioannina, IoanninaGreece3Institute for Molecular Bioscience, Centre for Microscopy and MicroanalysisSchool of Biomedical Sciences, University of Queensland, Brisbane, QueenslandAustralia4European Molecular Biology LaboratoryHeidelbergGermany9 2004 24 8 2004 24 8 2004 2 9 e26119 12 2003 11 6 2004 Copyright: © Schnatwinkel 2004 et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Protein Helps Orchestrate Cells' Fluid Uptake The small GTPase Rab5 is a key regulator of clathrin-mediated endocytosis. On early endosomes, within a spatially restricted domain enriched in phosphatydilinositol-3-phosphate [PI(3)P], Rab5 coordinates a complex network of effectors that functionally cooperate in membrane tethering, fusion, and organelle motility. Here we discovered a novel PI(3)P-binding Rab5 effector, Rabankyrin-5, which localises to early endosomes and stimulates their fusion activity. In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid-phase uptake, whereas its downregulation inhibits these processes. In polarised epithelial cells, this function is primarily restricted to the apical membrane. Rabankyrin-5 localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells, and its overexpression in polarised Madin-Darby canine kidney cells stimulates apical but not basolateral, non-clathrin-mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)pinocytosis, our studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, its active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells. Rabankyrin-5, which is activated by the small GTPase Rab5, coordinates two disparate methods that cells use to take in solids and fluids, playing a key role in both endosome fusion and macropinocytosis ==== Body Introduction In mammalian cells multiple mechanisms of endocytosis operate within a single cell to perform nutrient uptake, cellular homeostasis, neurotransmission, signal transduction, antigen presentation, and defence against pathogens. Endocytosis comprises two major categories, phagocytosis and pinocytosis, depending on the uptake of particles or fluid, respectively (reviewed in Conner and Schmid 2003). Pinocytosis encompasses various membrane entry routes and mechanisms. Clathrin-mediated endocytosis, the best-studied route at the molecular level to date, primarily serves receptor-mediated uptake. Caveolae assemble on sphingolipid and cholesterol rafts and may internalise molecules partitioning in these lipid microdomains. To a certain extent, ligand-receptor complexes and raft components can follow both entry routes but can also be internalised via clathrin- and caveolae-independent endocytosis. Large volumes of fluid are engulfed by the closure of plasma membrane protrusions in a process termed macropinocytosis. Although it was the first mode of pinocytosis reported (Lewis 1931), little is known on the molecular mechanisms underlying this physiologically very important endocytic process. Constitutively active in immature dendritic cells, macropinocytosis favors antigen sampling (Steinman and Swanson 1995). However, it can be transiently induced in most cells by growth factors (Haigler et al. 1979; Shao et al. 2002), tumor-promoting chemicals such as phorbol 12-myristate 13-acetate (PMA), or oncogenes like H-Ras or v-Src (Bar-Sagi and Feramisco 1986; Veithen et al. 1996), and it has been proposed to downregulate signalling molecules from the cell surface. Since macropinosomes arise from membrane ruffles, regions of intense actin remodelling which are also a trait of motile cells, they have been implicated in directed cell locomotion (Carpentier et al. 1991). Macropinocytosis is exploited by several invasive pathogens as entry route (Francis et al. 1993; Sansonetti 1997) but differs from phagocytosis with respect to regulation (e.g., receptor mediated) and cargo (e.g., uptake and degradation of opsonised particles) (Galan and Zhou 2000). Macropinosomes are distinct from early endosomes, morphologically and biochemically. In epidermal growth factor (EGF)-stimulated A431 cells, whereas macropinosomes can fuse homotypically, they seldom fuse with early endosomes (Hewlett et al. 1994). However, this partition is not absolute, since in dendritic cells macropinosomes do fuse with endosomes (Racoosin and Swanson 1993). The intracellular trafficking properties of macropinosomes may therefore be governed by cell type-specific mechanisms and fulfil specialised functions. Finally, macropinocytosis shares mechanistic features with apical fluid-phase endocytosis in polarised cells (Gottlieb et al. 1993; Jackman et al. 1994; Holm et al. 1995; Amyere et al. 2002). In kidney, apical pinocytosis contributes an essential function to the physiology of the renal system by contributing to vectorial fluid-phase transport across the membranes (Goligorsky and Hruska 1986). Given the dependence on actin remodelling, it is no surprise that Rho GTPases (West et al. 2000), ARF6 (Radhakrishna et al. 1996), and type 1 phosphatidylinositol-3 kinases (PI3-Ks) (Hooshmand-Rad et al. 1997) are involved in macropinocytosis, presumably through their role in membrane ruffling. Much less clear is the function of two Rab GTPases, Rab5 and Rab34/Rah, both of which are implicated in the formation of macropinosomes (Li et al. 1997; Sun et al. 2003). Rab34/Rah colocalises with actin to membrane ruffles and nascent macropinosomes and its overexpression promotes macropinocytosis (Sun et al. 2003). Rab5 regulates fluid-phase and receptor-mediated uptake (Bucci et al. 1992; McLauchlan et al. 1998), and its activity has been linked to Ras-stimulated fluid-phase endocytosis (Li et al. 1997). However, Rab5 colocalises to some, but not all, macropinosomes harbouring Rab34/Rah (Sun et al. 2003). Furthermore, Rab5 together with its effector PI3-K, hVPS34, regulates the recruitment of a set of cytosolic FYVE-finger effector proteins on early endosomes. These molecules cooperate in the tethering and fusion of clathrin-coated vesicles (CCVs) with early endosomes and homotypic endosome fusion (Gorvel et al. 1991; Bucci et al. 1995; Rubino et al. 2000) as well as the motility of early endosomes along microtubules (Nielsen et al. 1999). However, in view of the function of Rab5 on the early endosomes, its role in macropinocytosis is difficult to assess. Specifically, it is unclear whether the large vacuoles induced by Rab5 activation, either via the expression of activated mutants (Stenmark et al. 1994) or via signalling molecules that stimulate the GTP loading on Rab5 (Lanzetti et al. 2000; Tall et al. 2001), correspond to either macropinosomes or enlarged endosomes originated by increased homotypic early-endosome fusion, or both (Hewlett et al. 1994). Here, the identification of a novel Rab5 FYVE-finger effector, Rabankyrin-5, led us to revisit the role of Rab5 in fluid-phase (macro)pinocytosis in both nonpolarised and polarised cells. Results Identification of a Novel 130-kDa Rab5 Effector We have previously purified a large number of Rab5 effectors by an affinity chromatography approach based on glutathione S-transferase (GST)-Rab5-GTPγS (Christoforidis et al.1999a). Among the numerous proteins purified, we focused on a prominent 130-kDa protein (Figure 1A). The protein was digested with trypsin and the amino acid sequences of the tryptic fragments were determined by nanoelectrospray tandem mass spectrometry (Wilm et al. 1996). The identified peptides matched the mouse sequence of Ankhzn (unpublished data). The corresponding human cDNA was obtained by PCR, using primers derived from the mouse DNA sequence and a random primed HeLa cDNA library as template (Zerial et al. 2001). The sequence of the human cDNA matched the recently revised hAnkhzn gene sequence (NP 057460.2) (Kuriyama et al. 2000). The predicted human p130 primary sequence consists of an N-terminal BTB/POZ domain, a C-terminal FYVE-finger, and 21 successive ankyrin (ANK) repeats in between these two domains (Figure 1B). In light of the fact that p130 is a Rab5 effector (this study), we propose to amend its name to Rabankyrin-5 (Rab5 binding and ankyrin repeats containing protein). Figure 1 A Protein of 130 kDa Is a New Rab5 Effector (A) GST-Rab5-GDP and GST-Rab-GTPγS were loaded on beads and incubated with bovine brain cytosol. Bound proteins were eluted and analysed by SDS-PAGE followed by Coommasie Blue staining. The positions of the already known Rab5 effectors (EEA1, Rabaptin-5, hVps34, p110β, and Rabenosyn-5) and of the new Rab5 effector are indicated. (B) Schematic representation of the domain organisation in Rabankyrin-5. ANK, ankyrin repeats. (C) Bovine brain cytosol or HeLa cell cytosol was incubated with GST-Rab5-GDP– or GST-Rab5-GTPγS–loaded beads. Subsequently the beads were washed, and bound proteins were eluted and analysed by Western blotting using anti–Rabankyrin-5 antibodies. (D) GST-Rab4, -5, -7, and -11 fusion proteins were preloaded with GDP or GTPγS and incubated with in vitro-translated 35S-methionine–labelled Rabankyrin-5 full-length protein. As a control, bound and unbound material was analysed by SDS-PAGE followed by phosphoimager analysis. (E) Rabankyrin-5 binds most strongly to PI(3)P. Recombinant full-length Rabankyrin-5 was incubated with liposomes containing 2% of the indicated phosphoinositide. Bound Rabankyrin-5 was detected by Western blotting. To investigate the function of human Rabankyrin-5, we raised antibodies against the recombinant full-length protein. By Western blotting, these antibodies detected a predominant band of 130 kDa in both bovine brain and HeLa cytosol, in the corresponding GST-Rab5-GTPγS but not GST-Rab5-GDP affinity column eluates (Figure 1C). Evidence for a direct interaction with Rab5 was obtained by incubating in vitro-translated Rabankyrin-5 with beads displaying GST-Rab5, -Rab4, -Rab7, and -Rab11, preloaded with either GDP or GTPγS. Figure 1D shows that human Rabankyrin-5 binds to GST-Rab5-GTPγS, but neither to beads containing GST-Rab5-GDP nor to other Rab proteins. We conclude that Rabankyrin-5 binds to Rab5 specifically, directly, and GTP dependently. Association of Rabankyrin-5 with Two Types of Rab5-Positive Vesicles After EEA1 (Stenmark et al. 1996) and Rabenosyn-5 (Nielsen et al. 2000), Rabankyrin-5 is the third Rab5 effector containing a FYVE-finger. Since the FYVE-finger is a key determinant for targeting these proteins to early endosomes via its binding to phosphatidylinositol-3-phosphate [PI(3)P] (Kutateladze et al. 1999), we tested whether this holds true also for Rabankyrin-5. First, recombinant Rabankyrin-5 interacted significantly with PI(3)P in a liposome-binding assay (Figure 1E). Second, endogenous Rabankyrin-5 colocalised significantly with EEA1 and Rab5-positive early endosomes (approximately 80%) in A431 cells by confocal immunofluorescence microscopy analysis (see Figure 3A and 3B below). Figure 3 Rabankyrin-5 Associates with Two Types of Rab5-Containing Vesicles in A431 Cells, Early Endosomes, and Macropinosomes (A) A431 cells stably transfected for GFP-Rab5wt were immunostained for endogenous Rabankyrin-5 and EEA1. While there is perinuclear overlap between Rab5, Rabankyrin-5, and EEA1 in nontransfected cells (arrowheads), some smaller peripheral structures are devoid of EEA1 (arrows). (B) Overexpression of Rabankyrin-5 in A431 by using a recombinant adenovirus construct of Rabankyrin-5 causes an accumulation of peripheral, enlarged Rab5-positive structures, costained mainly by Rabankyrin-5 (arrows) but not detectable for EEA1. (C) Rabankyrin-5 localises on EGF-induced and -enriched macropinosomes. Serum-starved A431 cells (16 h) were incubated for 7 min with 100 ng/ml rhodamine-conjugated EGF to induce macropinocytosis and 1 μg/ml Cy5-labelled transferrin. Endogenous Rabankyrin-5 localises to enlarged EGF-containing macropinosomes, indicated by the lack of transferrin labelling (arrows), but also to EGF- and transferrin-containing endosomes (arrowheads). (D) Rabankyrin-5 structures contain tyrosine-phosphorylated proteins. A431 cells, stably transfected for GFP-Rab5, were stimulated with 50 ng/ml EGF for 7 min and immediately processed for immunofluorescence. Costaining of Rabankyrin-5 and tyrosine-phosphorylated proteins (α-4G10) reveal the localisation of Rabankyrin-5 to plasma membrane ruffles. Scale bars represent 10 μm. We next investigated whether Rabankyrin-5 plays a role in homotypic early endosome fusion as well as fusion of CCVs with early endosomes, similar to EEA1 (Christoforidis et al. 1999a) and Rabenosyn-5 (Nielsen et al. 2000). Quantitative immunodepletion of the cytosolic pool of Rabankyrin-5 using anti–Rabankyrin-5 antibodies (Figure 2A) had only minor effects on early endosome fusion in vitro compared to preimmune serum (Figure 2B). Considering the possibility that the early-endosome–associated pool of Rabankyrin-5 may still be sufficient for fusion, we attempted to neutralise its function by the addition of anti–Rabankyrin-5 antibodies on top of Rabankyrin-5–depleted cytosol. This treatment strongly inhibited early-endosome fusion (Figure 2B). The fusion of CCVs with early endosomes was minimally affected under these conditions (Figure 2C), suggesting that Rabankyrin-5 is not essential in this process, at least in vitro. However, addition of increasing amounts of recombinant Rabankyrin-5 (at concentrations in the range of the endogenous protein; see Materials and Methods) on either immunodepleted cytosol or nontreated cytosol significantly enhanced both homo- and heterotypic fusion activity. We conclude from these experiments that endogenous Rabankyrin-5 colocalises with EEA1 on Rab5 endosomes in vivo, through the interaction with Rab5 (see Figure 1D) and PI(3)P (see Figure 1E). It plays a modulatory role in early-endosome fusion since it can stimulate both homotypic and heterotypic fusion of CCVs with early endosomes. However, it does not appear to be strictly required in the latter reaction in vitro. Figure 2 Rabankyrin-5 Stimulates Homotypic Fusion between Early Endosomes and Heterotypic Fusion between Early Endosomes and CCVs In Vitro (A) Rabankyrin-5 was efficiently immunodepleted from HeLa cytosol without affecting the EEA1 concentration. Twenty micrograms of HeLa cell cytosol, either treated with control IgGs and immunodepleted for Rabankyrin-5 or nontreated, was applied to SDS-PAGE and probed for the indicated antigens by Western blotting. Anti-γ tubulin was used as a loading control. (B and C) Addition of recombinant Rabankyrin-5 stimulates homotypic and heterotypic fusion. Fusion (B) between early endosomes (EE-EE fusion) and (C) of early endosomes to CCVs (CCV-EE fusion) in vitro was performed in the presence of 3 mg/ml HeLa cytosol and an ATP-regenerating system (basal) and in the absence or presence of the indicated reagents. For some conditions, cytosol was immunodepleted for Rabankyrin-5 (columns 8–13) and in addition supplemented with anti–Rabankyrin-5 antibodies to neutralise the function of membrane-associated proteins (column 10). Background fusion activity is demonstrated by ATP (−Energy) or cytosol (−Cytosol) omission. Surprisingly, we noted that some peripheral structures harbouring Rabankyrin-5 overlapped with Rab5 but either poorly or not at all with EEA1 in A431 cells (Figure 3A, arrows). Upon overexpression, Rabankyrin-5 induced the accumulation of several vesicles of variable size in the periphery of the cells, most of them lacking EEA1 (Figure 3B, arrows). The irregular size, intracellular distribution (Swanson 1989), and lack of endosomal markers (see “Rabankyrin-5 localises to macropinosomes” below) (Hewlett et al. 1994) of these structures were suggestive of (macro)pinocytic vesicles. We therefore set out to investigate the putative role of Rabankyrin-5 in macropinocytosis. Rabankyrin-5 Localises to Macropinosomes Macropinosomes have been so far defined by a combination of criteria rather than specific molecular markers. To test whether the Rabankyrin-5 vesicles fulfil the previously described requirements of macropinosomes, we determined whether they (1) are stimulated by growth factors (Haigler et al. 1979; West et al. 1989; Amyere et al. 2000), (2) do not promote receptor-mediated endocytosis of transferrin, (3) engulf large amounts of extracellular fluid (Hewlett et al. 1994; Amyere et al. 2000), (4) depend on PI3-K activity (Araki et al. 1996; Zhou et al. 1998), and (5) follow actin rearrangements at the plasma membrane. A431 and NIH3T3 cells are established experimental systems that exhibit different features in the regulation of macropinocytosis. In A431 cells, macropinocytosis can be transiently induced upon growth factor stimulation (Hewlett et al. 1994), whereas NIH3T3 cells exhibit constitutive macropinocytic activity (Dharmawardhane et al. 2000). We first tested whether EGF can induce the formation of Rabankyrin-5–positive macropinocytic structures. Upon treatment of A431 cells with rhodamine-conjugated EGF, endogenous Rabankyrin-5 colocalised to enlarged structures which, importantly, were largely devoid of simultaneously internalised transferrin (Figure 3C). Interestingly, EGF was not excluded but rather enriched in these structures. In addition, we observed that such vesicles were also enriched in tyrosine-phosphorylated proteins, presumably EGF-receptor and downstream signalling molecules (Figure 3D). In NIH3T3 fibroblasts, the staining pattern of endogenous Rabankyrin-5 was similar to that of EGF-stimulated A431 cells. Rabankyrin-5 was detected on enlarged structures, predominantly at the outermost periphery and in membrane protrusions of the cell (Figure 4A, arrows). Consistent with these observations, immunoelectron microscopy revealed that antibodies to Rabankyrin-5 labelled electron-lucent vesicular structures underlying the plasma membrane in the cell periphery and in cell protrusions (Figure 5). The labelled structures were sparse in untransfected cells, but in Rabankyrin-5– overexpressing cells fortuitous sections revealed concentrated areas of Rabankyrin-5–labelled structures. Unfortunately, we failed to distinguish these structures from conventional early endosomes by double immunolabelling experiments due to technical limitations. By light microscopy, the appearance of vacuole-shaped structures devoid of the early endosomal marker EEA1 was dramatically increased upon Rabankyrin-5 overexpression. In addition, after a 3-min pulse, these structures were intensively labelled by a fluid-phase marker (see Figure 4B, arrows) but not by transferrin (unpublished data). Given the potent induction of macropinosomes by Rabankyrin-5 in NIH3T3 cells, we used this experimental system to explore further the regulatory parameters of macropinocytosis. Figure 4 Rabankyrin-5 Localises to Macropinosomes in NIH3T3 Cells (A) NIH3T3 cells directly fixed and immunostained for endogenous Rabankyrin-5 and EEA1 reveal segregation of Rabankyrin-5 from EEA1-containing structures (arrows) in the cell periphery, while there is colocalisation in the cell centre (arrowheads). (B) Overexpression of Rabankyrin-5 increases the number of peripheral enlarged structures devoid of EEA1. NIH3T3 cells were infected with recombinant adenovirus for Rabankyrin-5 for 18 h. Dextran (2,5 mg/ml) uptake was performed for 3 min at 37 °C, fixed, and immunostained for the indicated antigens. (C and D) Formation of enlarged Rabankyrin-5 structures requires PI3-K activity. NIH3T3 cells either (C) DMSO treated or (D) pretreated for 20 min with wortmannin (WM; 100 nM) were incubated for 8 min with 0,5 μg/ml rhodamine-labelled transferrin and 2,5 mg/ml FITC-labelled dextran (MW, 10.000), fixed, processed for immunofluorescence, and analysed by confocal scanning microscopy. (E) NIH3T3 cells transiently transfected for YFP-Rabankyrin-5 and CFP-actin were imaged using time-lapse video microscopy to visualise the formation of macropinosomes by actin-driven membrane ruffles. Images were taken for the indicated time points. The arrowhead points towards Rabankyrin-5 association to an enlarged vesicle driven by actin dynamics over time. Scale bars represent 10 μm. Figure 5 Immunolocalisation of Endogenous and Overexpressed Rabankyrin-5 in NIH3T3 Cells Untransfected NIH3T3 cells or cells transfected with Rabankyrin-5 were labelled with antibodies to Rabankyrin-5 followed by 10 nm protein A gold. (A) Transfected cell showing labelling of a group of vesicular structures underlying the plasma membrane (pm). (B and C) In control (untransfected) cells, low but specific labelling for Rabankyrin-5 (arrowheads) is associated with compartments close to the pm. Scale bars represent 200 nm. In NIH3T3 cells, Rabankyrin-5–enlarged structures were labelled by an 8-min uptake of fluorescein isothiocyanate (FITC)-dextran (MW, 10.000). Consistent with their macropinocytic nature, these structures were largely depleted of transferrin (see Figure 4C, arrows) and susceptible to the PI3-K inhibitors wortmannin (see Figure 4D) and LY 294002 (unpublished data). These drugs both reduced the formation of peripheral enlarged vesicles and almost completely blocked the internalisation of high-molecular-weight dextran (MW, 70.000) (Figure S1A) that enters the cell almost exclusively via macropinocytosis (Dharmawardhane et al. 2000). Interestingly, we noted that, unlike EEA1, Rabankyrin-5 did not readily dissociate from membranes (see Figure 4D) upon inhibition of PI3-K activity, suggesting that endosome-targeting determinants other than PI(3)P contribute to its localisation. Neither markers of late endosomes (Rab7) (Chavrier et al. 1990), nor of caveosomes (caveolin) (Pelkmans et al. 2001), nor of the clathrin-independent pathway which is used by glycosyl-phosphatidylinositol (GPI)-anchored proteins (GFP-GPI) (Sabharanjak et al. 2002) overlapped significantly with the peripheral enlarged Rabankyrin-5 structures (unpublished data). To confirm that the Rabankyrin-5 structures originated from the plasma membrane through an actin-driven process, we employed time-lapse video fluorescence microscopy on NIH3T3 cells transiently coexpressing yellow fluorescent protein (YFP)-Rabankyrin-5 and cyan fluorescent protein (CFP)-β-actin. In the sequence depicted in Figure 4E (see also Video S1), membrane ruffling driven by actin resulted in the formation of a large, plasma membrane-derived vesicle acquiring Rabankyrin-5. Some newly formed vesicles shed off Rabankyrin-5 over time and seemed to regurgitate back to the plasma membrane via an actin comet-tail. Altogether, the data support the hypothesis that the enlarged structures containing Rabankyrin-5 are macropinosomes and can be molecularly distinguished from early endosomes by being mostly, albeit not exclusively, devoid of EEA1. Moreover, and contrary to earlier findings (West et al. 1989), we found that in A431 cells, EGF but not transferrin could be enriched in macropinosomes. Both Rab5 and Rabankyrin-5 Are Required for Macropinocytosis The GTP-dependent interaction with Rab5 suggests that Rabankyrin-5 may function as a downstream effector of Rab5 in the macropinocytic pathway. Accordingly, one would expect that Rab5 localises to macropinosomes and, together with Rabankyrin-5, is required for macropinocytosis. We found that Rab5 indeed colocalises with Rabankyrin-5 on macropinosomes (see Figure 3B, 3D, and 6A; unpublished data), which are operationally defined as vesicles (1) enriched in fluid-phase marker but depleted of transferrin receptor (Racoosin and Swanson 1992), (2) of large size (0,2–2 μm) (Swanson 1989; Hewlett et al. 1994), and (3) negative for EEA1 but positive for Rabankyrin-5. Consistent with a requirement for Rab5 in macropinocytosis, overexpression of RN-tre, a GTPase-activating protein (GAP) for Rab5 (Lanzetti et al. 2000), significantly reduced (by 60% ± 10%) the uptake of large fluorescent dextran (MW, 70.000) into transferrin- and EEA1-negative structures in NIH3T3 cells (Figure 6B and 6C). Similar results were also obtained when NIH3T3 cells were cotransfected for Rab5S34N and Rabankyrin-5 (unpublished data), implying that Rabankyrin-5 induces macropinocytosis Rab5-GTP dependently. Figure 6 Inhibition of Rab5 Activity Decreases Fluid-Phase Uptake NIH3T3 cells transiently transfected for either (A) GFP-Rab5wt or (B) RN-tre were subjected to a 30-min uptake of rhodamine-conjugated transferrin (1 μg/ml) or dextran (MW, 70.000; 3 mg/ml) at 37 °C and further processed for confocal imaging with indicated antibodies. (A) Rab5wt transfected cells show colocalisation of Rabankyrin-5–labelled macropinocytic structures, indicated by the lack of transferrin accumulation, with Rab5. (B) Cells transiently transfected for RN-tre (asterisk) show a significant reduction of fluid-phase uptake compared to nontransfected cells. (C) Fluid-phase dextran quantification of single cells transfected for RN-tre by measuring internalised fluorescence intensity (p > 0,001). Values shown are means ± standard deviation of at least 15 cells. Scale bars represent 10 μm. Whereas the aforementioned results demonstrate a general requirement for Rab5 and Rabankyrin-5 in pinocytosis, they do not discriminate between clathrin-dependent and-independent endocytosis. To distinguish between the two pathways, we compared the effects of Rabankyrin-5 overexpression on transferrin and horseradish peroxidase (HRP) uptake in NIH3T3 cells, biochemically. A recombinant adenovirus was generated to overexpress Rabankyrin-5 in these cells. In comparison with mock-infected cells, Rabankyrin-5 overexpression increased the uptake of HRP, particularly after longer (10 min) incubation times (Figure 7A). In contrast, the rate of transferrin uptake was unaffected, suggesting that the increase of fluid phase was not due to increased clathrin-mediated endocytosis (Figure 7C). The increased intracellular accumulation of HRP induced by Rabankyrin-5 seems to be due to increased uptake rather than inhibited recycling, as the latter process was only slightly decreased (Figure 7B). Figure 7 Rabankyrin-5 Overexpression Increases, whereas Knock-Down Decreases Fluid-Phase Uptake, Specifically (A–C) NIH3T3 cells were either mock-infected or infected with recombinant adenovirus encoding Rabankyrin-5. Simultaneous uptake of HRP (5 mg/ml) and biotinylated transferrin (2 μg/ml) was performed at 37 °C for the indicated time points. (C) NIH3T3 cells were pulsed with HRP (10 mg/ml) for 10 min. Recycled HRP into the medium was determined after the indicated time points. (D–F) A431 cells were treated with esiRNA against Rabankyrin-5 or control treated for 4 d. (D) Western blot analysis revealed a 50% reduction of Rabankyrin-5 in whole-cell lysate. (E and F) Serum-starved cells (1 h) were stimulated with 50 ng/ml EGF in complete medium for the indicated time points in the presence of 5 mg/ml HRP and 2 μg/ml biotinylated transferrin. Values shown are means ± standard deviation and were performed in duplicates. The results are representatives of at least two independent experiments. To determine whether Rabankyrin-5 is also required for macropinocytosis, we attempted to ablate the protein by RNA interference using endoribonuclease-prepared small interfering RNA (esiRNA) (Yang et al. 2002). Since we optimised this technique in human cells, we conducted these experiments in EGF-stimulated A431 cells where an approximately 55% knock-down of Rabankyrin-5 was achieved (Figure 7D). EGF stimulation of control-treated cells enhanced HRP uptake 2-fold versus control-treated, non-EGF-stimulated cells, as reported previously. Reduction of Rabankyrin-5 expression in esiRNA-transfected cells caused an inhibition of HRP of up to approximately 50% by selectively affecting the burst of pinocytosis induced by EGF (within 5 min) (Figure 7E). Under the same conditions, transferrin uptake was marginally inhibited (Figure 7F). We conclude from these experiments that inhibition of fluid-phase uptake occurs due to an inhibition of (macro)pinocytosis rather than of clathrin-mediated endocytosis. To confirm the requirement of Rabankyrin-5 for fluid-phase endocytosis we used an independent method: quantification of the uptake of fluorescent dextran in NIH3T3 cells microinjected with affinity-purified anti–Rabankyrin-5 antibodies. Following microinjection, FITC-dextran uptake was internalised for 30 min to label macropinosomes. Microinjection of anti–Rabankyrin-5 antibodies inhibited FITC-dextran uptake by 53% ± 5% when compared with cells injected with preimmune serum (Figure S2), whereas the uptake of rhodamine-transferrin was not significantly changed. Altogether, these results suggest that Rabankyrin-5 is a Rab5 effector required for the formation of macropinosomes. Rabankyrin-5 Regulates Apical Fluid-Phase Endocytosis in Polarised Epithelial Cells Apical pinocytosis in polarised epithelial cells and growth factor-induced macropinocytosis share mechanistic properties, like dependence on actin (Gottlieb et al. 1993) and PI3-K activity (Tuma et al. 1999; Sandvig et al. 2000). In view of the role of Rab5 in endocytic trafficking of polarised cells such as epithelial cells and neurons (Bucci et al. 1994; de Hoop et al. 1994), we explored the role of Rabankyrin-5 in apical endocytosis in polarised epithelial cells. First, we determined the intracellular localisation of Rabankyrin-5 on cryosections of adult mouse kidney, since the epithelial cells in this organ, especially proximal tubule cells (Christensen et al. 1998), exhibit high levels of apical pinocytosis under physiological conditions. Apical endocytic vesicles were pulse-labelled by injecting mice with HRP followed by fixation of the kidney through perfusion after 5 min. Prominin-1 was used as marker for the apical brush border of proximal tubules (Weigmann et al. 1997) in combination with affinity-purified anti–Rabankyrin-5 antibodies. Basement membrane staining was demonstrated by immunolabelling of endogenous mouse immunoglobulin G (IgG). As illustrated in Figure 8, Rabankyrin-5 immunoreactivity was mainly detected on vesicle-like structures underneath the apical, prominin-1–labelled brush border. Most of the vacuole-like Rabankyrin-5 structures contained HRP, suggesting that they correspond to apical endocytic structures (Figure 8D, arrows). By further investigating the nature of these Rabankyrin-5– and HRP-labelled structures, we observed that some vesicles were immunoreactive for LAMP-1 (Figure 8D, arrowheads), suggesting that Rabankyrin-5 (macro)pinocytic vesicles can acquire characteristics of late endocytic compartments. Figure 8 Rabankyrin-5 Localises on Vesicular Structures Underlying the Apical Brush Border in Mouse Kidney Proximal Tubules Mice were perfused either (A–C) directly with 4% PFA or (D) 5 min after injection of HRP into the femoral vein. Immunofluorescence was performed on semi-thin cryosections (500 nm). (C) Higher magnification of the inset in (B). Scale bars represent 10 μm. This was confirmed and extended by immunogold electron microscopy on ultrathin frozen sections of mouse proximal tubule. Rabankyrin-5 showed weak labelling of the apical microvilli-covered surface of proximal tubule cells but labelled large electron-lucent structures underlying the apical surface (Figure 9A and 9B). Consistent with the immunofluorescence analysis, double labelling with LAMP-1 antibodies confirmed that many of these structures were LAMP-1 negative or weakly labelled. However, a significant number of Rabankyrin-5–positive structures were also LAMP-1 positive. Rabankyrin-5 immunoreactivity was also observed in distal tubule cells and other segments of the nephron. Figure 9 Immunolocalisation of Rabankyrin-5 in the Mouse Kidney Mouse kidney cortex was processed for frozen section immunoelectron microscopy. Sections were (A and B) single labelled for Rabankyrin-5 (arrowheads, 10 nm) or (C and D) double labelled (arrows, 5 nm) for Rabankyrin-5 and LAMP-1. (A) Low-magnification view of the apical region of two proximal tubule cells demonstrates low labelling for Rabankyrin-5 on apical microvilli (M) but stronger labelling (arrowheads) of large subapical electron-lucent vesicular structures (asterisks). One of these structures is shown at higher magnification in (B). L, lateral membrane. (C) Rabankyrin-5 labels LAMP-1–negative subapical structures as well as compartments showing low LAMP-1 labelling (arrows and asterisk). (D) Rabankyrin-5 (arrowheads) associates with compartments, which show no or weak labelling for LAMP-1 (asterisks). In addition, low Rabankyrin-5 labelling is associated with more strongly labelled LAMP-1–positive compartments. Note that there is some nonspecific labelling of mitochondria (m). Scale bars represent 500 nm. To functionally address the role of Rabankyrin-5 in pinocytosis, we used filter-grown Madin-Darby canine kidney (MDCK) cells as an established system to study polarised trafficking. Alexa 488 dextran was internalised for 13 min from either the apical or the basolateral chamber. Although endogenous Rabankyrin-5 exhibited partial colocalisation with fluid-phase marker internalised from the basolateral side (unpublished data), a striking degree of overlap between the internalised tracer and Rabankyrin-5 on often ring-shaped structures underlying the apical region was observed (Figure 10A). These large vesicles were neither detectable in the nuclear area (Figure 10B) nor close to the basolateral domain (Figure 10C). We next tested whether Rabankyrin-5 overexpression could stimulate fluid-phase uptake from the apical plasma membrane domain. Rabankyrin-5 was overexpressed using the recombinant adenovirus system in MDCK cells grown on coverslips and the cells stained for Rabankyrin-5 and EEA1. As in A431 and NIH3T3 cells, and similar to endogenous Rabankyrin-5 in MDCK cells (Figure 11A), the overexpressed protein exhibited significant colocalisation with endogenous EEA1, but in addition, it induced the formation of enlarged structures devoid of EEA1 in the periphery of the cell (Figure 11B). The recombinant adenovirus was next applied to infect filter-grown MDCK cells. In these cells, it induced enlarged Rabankyrin-5 structures predominantly underlying the apical domain that contained dextran internalised from the apical side. Some of these structures were EEA1 positive, whereas others lacked this marker of early endosomes (see Figure 10D, arrows). These structures were also detectable to some extent in the nuclear region (see Figure 10E) and basolateral membrane domain (see Figure 10F). In addition, there were some enlarged Rabankyrin-5 vesicles that were labelled for neither dextran nor EEA1. These may correspond to macropinosomes that were formed prior to the labelling with the fluorescent fluid-phase marker. Figure 10 Rabankyrin-5 Colocalises to Apically Labelled Structures in Filter-Grown MDCK Cells MDCK cells were seeded on polycarbonate filters for 4 d and either (A–C) mock-infected or (D–F) infected with an adenovirus construct expressing full-length Rabankyrin-5 for another 18 h. Cells were then incubated for 13 min with 5 mg/ml Alexa 488 dextran, added to the apical medium, fixed, and stained for the indicated antigens. Images represent confocal scans just beneath (A and D) the apical plasma membrane, (B and E) the nuclear area, and (C and F) the basal plasma membrane. Endogenous Rabankyrin-5 and EEA1 localise to several dextran-labelled structures, often with ring-shaped appearance in the apical region. Overexpression of Rabankyrin-5 seems to increase the number of enlarged and dextran-labelled structures, some of which are depleted of EEA1 (arrows) (B). Scale bars represent 10 μm. Figure 11 Overexpression of Rabankyrin-5 Stimulates Fluid-Phase Uptake from the Apical Plasma Membrane (A and B) MDCK cells plated on coverslips were (A) mock-infected or (B) infected with Rabankyrin-5 and processed for immunofluorescence. Under both conditions EEA1 colocalised almost completely with Rabankyrin-5, while there were some Rabankyrin-5 structures devoid of EEA1. Upon Rabankyrin-5 overexpression these EEA1-lacking structures were enlarged macropinocytic-like vesicles localised predominantly in the periphery of the cell, while other enlarged compartments containing EEA1 were centripetally located. (C–F) Filter-grown MDCK cells were either mock-infected or infected with recombinant adenovirus for full-length Rabankyrin-5. HRP (5 mg/ml) or biotinylated Fab fragments (50 μg/ml) were internalised from the indicated chambers for various time points. Whereas Rabankyrin-5 overexpression (C) specifically increased HRP uptake from the apical domain, (D) basolateral HRP uptake and (E and F) Fab uptake at both domains were unaffected (p < 0,01). Values shown are means ± standard deviation of at least three independent experiments. Scale bars represent 10 μm. We next measured fluid-phase uptake biochemically by quantifying the internalisation of HRP either from the apical or the basolateral side for various periods of time. Filter-grown MDCK cells were either infected with adenovirus encoding Rabankyrin-5 or control virus prior to HRP internalisation. While internalisation of HRP from the basolateral plasma membrane did not significantly vary (Figure 11D), uptake from the apical membrane was increased up to 65% in cells overexpressing Rabankyrin-5 when compared with control cells (Figure 11C). Given that Rabankyrin-5 was overexpressed in only 50–60% of the cells, this value may be an underestimate. Since the kinetic profiles resembled the ones obtained in NIH3T3 cells, we also tested whether the increase in HRP uptake may have been due to an inhibition of recycling or transcytosis. Neither of these trafficking pathways was significantly affected (unpublished data). To verify that the increase in fluid-phase uptake was not due to stimulation of clathrin-mediated endocytosis, we measured the internalisation of FcLR 5–27, a chimeric receptor between the IgG Fc receptor and the LDL receptor which, when expressed in MDCK cells, is targeted to, and internalised from, both apical and basolateral plasma membrane domains (Matter et al. 1992). Rabankyrin-5 overexpression did not affect significantly the internalisation of FcLR 5–27, as revealed by the uptake of a biotinylated Fab-fragment of the 2.4G2 monoclonal anti-FcRII antibody (Figure 11E and 11F). Taken together, these findings indicate that Rabankyrin-5 is predominantly localised to apical endocytic structures and specifically stimulates apical, non-clathrin-mediated fluid-phase endocytosis upon overexpression in polarised epithelial cells. Discussion Rabankyrin-5 in Macropinocytosis Since the pioneering work of Bar-Sagi and Feramisco (1986) almost two decades ago, it has been established that certain oncogenes and signalling molecules induce actin-dependent membrane ruffling and macropinocytosis. Subsequent work has advanced our understanding of the signalling pathway that led to growth factor-dependent actin remodelling and membrane ruffling (Ridley 1994). Despite macropinocytosis being the most ancient form of pinocytosis and an endocytic process of high physiological importance (Amyere et al. 2002), progress in determining the molecular mechanisms that regulate the generation and trafficking of macropinosomes has advanced to a lesser extent. It has been demonstrated that macropinocytosis differs from clathrin-mediated endocytosis (West et al. 1989) and that the two pathways lead to distinct endocytic structures (Hewlett et al. 1994). Rab5, an established regulator of clathrin-mediated endocytosis and endosome dynamics (Gorvel et al. 1991; Bucci et al. 1992; McLauchlan et al. 1998), has also been implicated in macropinocytosis (Li et al. 1997). This hypothesis was based on the expression of mutants that induced or inhibited fluid-phase endocytosis and formation of giant endocytic vesicles. In those studies, however, an accurate assessment of whether the enlarged compartments corresponded to macropinosomes or were produced by the coalescence of multiple endosomes by homotypic fusion was not determined. Here, we provide evidence that Rabankyrin-5 is a novel Rab5 effector which, in addition to being localised to early endosomes, is associated with macropinosomes and promotes their formation according to previously established criteria. First, Rabankyrin-5 localised to enlarged vesicles that were stimulated by growth factors (e.g., EGF). Second, these vesicles differed from early endosomes by the lack of constitutively endocytosed molecules, such as transferrin, and components of the transport machinery, such as EEA1. Third, the vacuoles originated from the plasma membrane and engulfed extracellular fluid. Fourth, their formation depended on PI3-K activity, and fifth, they followed actin rearrangements at the plasma membrane. Possible Functions of Rabankyrin-5 Macropinosomes are formed by the closure of membrane protrusions generated upon actin-mediated membrane ruffling. Although membrane ruffling is required for macropinocytosis, it seems not to be sufficient for macropinosome formation (Araki et al. 1996; West et al. 2000). Our data suggest that Rabankyrin-5 is a novel regulator of this endocytic mechanism. One possibility is that it could play an active role in the generation of macropinosomes (see Figure 7). The deduced primary structure of Rabankyrin-5 predicts the existence of several protein-protein interaction motifs, suggesting a role as a multifunctional adaptor protein. The N terminus of Rabankyrin-5 contains a BTB/POZ motif, which is present in proteins involved in signalling, development, and tumorigenesis and mediates homo- and heterodimerisation (Bardwell and Treisman 1994; Kobayashi et al. 2000). It contains 21 ANK repeats which, by analogy with the function of ANKs (Bennett and Chen 2001; Denker and Barber 2002a), could interact with different proteins on the membrane, including Rab5, and mediate the assembly of a multiprotein complex. Such a scaffolding function has been postulated to bridge the membrane to the actin cytoskeleton but also to link proteins involved in endocytosis and signal transduction (Pryciak and Hartwell 1996; Lubman et al. 2004). Rabankyrin-5 could assemble a membrane-cytoskeleton scaffold committing the ruffling membrane to generate a pinocytic vesicle or directly participate in the closure of the plasma membrane sealing the vesicle. Alternatively, Rabankyrin-5 could be recruited onto macropinosomes concomitantly or following their formation to prevent their back-fusion with the plasma membrane or regulate their onward trafficking. This possibility receives support from our time-lapse video microscopy analysis, where the dissociation of Rabankyrin-5 from the membrane precedes what appears to be the regurgitation of macropinosomes back to the plasma membrane (as shown in Video S1). Interestingly, it has recently been proposed that ANK repeats may not be simply anchoring domains but may be part of a mechanical sensory system that transmits tension from the cytoskeleton to ion channels in the membrane (Corey and Sotomayor 2004; Howard and Bechstedt 2004). In the case of Rabankyrin-5, the ANK repeats may confer a sensory function to detect and adapt to rearrangements of the actin cytoskeleton during membrane ruffling and membrane closure. Interactions of the ANK repeats with ion channels may sense changes in water-salt homeostasis and pH, and respond to them via modulation of macropinocytosis and macropinosome trafficking (de Baey and Lanzavecchia 2000; Morris and Homann 2001; Denker and Barber 2002b). Functional Relation between Macropinosomes and Endosomes How does the function of Rab5 and Rabankyrin-5 in macropinocytosis relate to early endosome trafficking? We have previously proposed a model whereby Rab5, via the recruitment of its effectors, generates and maintains a spatially restricted and functionally specialised membrane domain on the early endosomes (Zerial and McBride 2001). The Rab5 effector hVps34 (Christoforidis et al. 1999b) is a PI3-K that generates PI(3)P. Together with Rab5, this phosphoinositide serves as a binding determinant for the FYVE-finger Rab5 effectors EEA1 (Simonsen et al. 1998) and Rabenosyn-5 on the early-endosome membrane (Nielsen et al. 2000). The Rab5 effectors form oligomeric complexes (McBride et al. 1999) and play distinct but cooperative roles in membrane tethering, fusion, and motility of early endosomes along microtubules (Nielsen et al. 1999). Our data extend the list of PI(3)P-binding Rab5 effectors to Rabankyrin-5. Rabankyrin-5 colocalises with Rab5 and EEA1 to early endosomes and plays a role in homotypic early-endosome fusion, further underscoring the activity of Rab5 as organiser of an endosomal Rab5 domain enriched in PI(3)P-binding effector proteins. Rabankyrin-5 played a minor modulatory role in the fusion of CCVs with early endosomes, suggesting mechanistic differences between homotypic early-endosome fusion and the heterotypic fusion of CCVs with early endosomes. Clearly, Rabankyrin-5 exerted the most striking effects on fluid-phase rather than clathrin-mediated endocytosis. Clathrin-dependent endocytosis and macropinocytosis are independent but interconnected pathways. Whereas macropinosomes and early endosomes remain segregated in EGF-stimulated A431 cells (Hewlett et al. 1994), they can fuse in other cells, such as dendritic cells and macrophages (Racoosin and Swanson 1993). Rabankyrin-5 alone may not be sufficient for macropinosomes to fuse with endosomes, and this activity may require the additional recruitment of EEA1 and Rabenosyn-5. The extent to which macropinosomes can recruit Rabankyrin-5, release or retain it, and further acquire late endocytic components or other Rab5 effectors acting in endosome tethering and fusion may depend on the cellular context. In kidney cells, for example, several Rabankyrin-5–positive structures appeared also to contain EEA1 and late endocytic markers (LAMP-1), suggesting that macropinosomes can communicate with other endocytic organelles and undergo maturation as it has been described for phagocytosis (Allen and Aderem 1996). Also intriguing is the weak interaction of Rabankyrin-5 with PI(5)P (see Figure 1E) and recent findings that BTB domain-containing proteins interact with ubiquitin ligases (Furukawa et al. 2003). Since both components have been implicated in the biogenesis of multivesicular bodies (Katzmann et al. 2001), Rabankyrin-5 may function in the trafficking to late endocytic components, as it was described previously for PIKfyve, another FYVE-finger–containing protein (Ikonomov et al. 2003). Stimulation of epithelial cells with either EGF or phorbol esters increases fluid-phase uptake while reducing internalisation via clathrin-mediated endocytosis (Sandvig and van Deurs 1990). Our results provide a possible explanation for how the two endocytic routes can be quantitatively balanced. Since Rab5 appears to be rate limiting for both receptor-mediated and fluid-phase endocytosis, its activity could be shifted between these two endocytic pathways, depending on the stimuli and the cell type. The shared activity of Rab5 and Rabankyrin-5 would ensure coordination between endosome trafficking and macropinocytosis, regulate the kinetics, and limit the extent of both endocytic processes, thus preserving plasma membrane homeostasis. Rabankyrin-5 in Apical, Actin-Dependent Endocytosis in Polarised Epithelial Cells In addition to macropinocytosis in nonpolarised cells, we have found that Rabankyrin-5 regulates apical, non-clathrin-mediated pinocytosis in polarised epithelial cells. The role of apically stimulated pinocytosis is crucial for the physiology of various organs. For example, it plays an important role in the reabsorption of proteins from the glomerular filtrate in the renal proximal tubule (Christensen et al. 1998). Rabankyrin-5 may be a regulator of this process and this possibility is supported by our findings, which show that it (1) localises to subapical compartments in kidney proximal tubules and (2) induces fluid-phase, clathrin-independent uptake from the apical but not basolateral side in polarised MDCK cells. As for macropinocytosis, apical endocytosis depends on actin remodelling mediated by Rho family GTPases (Gottlieb et al. 1993; West et al. 2000) and ARF6 (Altschuler et al. 1999). Since macropinocytosis appears to occur primarily from the apical side, the involvement of epidermal growth factor receptor as demonstrated for nonpolarised cells (Haigler et al. 1979; West et al. 1989) is unlikely since it is primarily localised to the basolateral domain of polarised epithelial cells (Gesualdo et al. 1996). However, apical pinocytosis depends on other components of receptor tyrosine kinase signalling pathways such as PI3-K (Tuma et al. 1999) and protein kinase C and can be induced by oncogenes (e.g., v-Src) that stimulate macropinocytosis in nonpolarised cells (Holm et al. 1995; Amyere et al. 2002). It is not known which PI3-K subtype functions in apical pinocytosis, but an attractive candidate is PI3-Kβ, as it has been identified as a Rab5 effector (Christoforidis et al. 1999b). Its activity could be regulated by Rab5 constitutively and/or be subjected to stimulation by apical or axonal signalling molecules (Pillion et al. 1989; Kryl et al. 1999). In neurons, Rac- and actin-dependent endocytosis of Eph receptor-ephrin complexes is required to control repulsive versus attractive cell movement during tissue patterning in embryonic development (Marston et al. 2003; Zimmer et al. 2003) and may play a role in the structural plasticity of synapses (Holt et al. 2003). It will be interesting to determine whether Rabankyrin-5 and the macropinocytic machinery play a role in this event. In conclusion, the identification of Rabankyrin-5 opens new opportunities for investigating the molecular principles underlying macropinocytosis and its regulation by signalling molecules. The dual role of Rab5 and Rabankyrin-5 in endosome and macropinosome function argues for a role in coordinating these two different endocytic mechanisms. Future work is required to identify the molecular partners of Rabankyrin-5 and to establish the mechanisms responsible for its membrane targeting and its role in endocytic membrane dynamics. Materials and Methods Reagents and cell lines. Phospholipids were purchased from Sigma (St. Louis, Missouri, United States) and Calbiochem (San Diego, California, United States). FITC- or Texas Red-conjugated dextran (lysine fixable; MW, 10.000 and 70.000), rhodamine-conjugated EGF, and rhodamine-conjugated transferrin were purchased from Molecular Probes (Eugene, Oregon, United States). Wortmannin and LY 294002 were from Calbiochem. A431 and NIH3T3 cells were grown in DMEM (high glucose) supplemented with 10% (v/v) heat-inactivated foetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine. MDCK II cells were grown in MEM plus 10% FCS. MDCK II cells stably transfected for FcLR 5–27 (Matter et al. 1992) were a gift of Professor Ira Mellman. Amino acid sequence determination and Rabankyrin-5 cloning The 130-kDa protein band was excised from gels and enzymatically digested. The tandem mass spectroscopy protein sequencing procedure was performed as described previously (Wilm et al. 1996). Bovine peptides determined from Rabankyrin-5 were from bovine brain and were used to identify corresponding expressed sequence tags using BLAST similarity searches at NCBI. A random primed HeLa cDNA library was used in a PCR reaction with primers based on the mouse cDNA to obtain the full-length clone of Rabankyrin-5. Preparation of recombinant Rabankyrin-5 Recombinant proteins of Rabankyrin-5 were expressed in SF plus insect cells according to the manufacturer's instructions using Rabankyrin-5 cDNA subcloned into pfastBAC vector or subcloned into pGEX-6P series and expressed in BL21 cells. Antibodies, plasmids, and recombinant adenovirus Human anti-EEA1 serum (1:10.000) was a gift from Ban Hok Toh (Monash Medical School, Adelaide, Australia), whereas a mouse monoclonal EEA1 antibody, a sheep polyclonal anti-HRP antibody and an antibody against phosphotyrosine (α-4G10) were purchased from BD Bioscience (Heidelberg, Germany), Abam (Cambridge, United Kingdom), and Upstate Biotechnology (Lake Placid, New York, United States), respectively. Fluorescently labelled secondary antibodies were purchased from Molecular Probes. Recombinant full-length Rabankyrin-5 was used to raise a polyclonal antibody in rabbit. Human full-length Rabankyrin-5 was cloned into pEYFP vectors containing an N-terminal tag (pEYFP-C1; Clontech, Palo Alto, California, United States). Recombinant adenovirus encoding full-length Rabankyrin-5 was generated according to the manufacturer's protocol (AdEasy). cDNAs encoding Rab5wt were fused to the amino termini of ECFP or EYFP, as previously described (Sonnichsen et al. 2000). The antibody for the FcLR 5-27 chimeric receptor was purified from 2.4G2 hybridoma supernatant (Unkeless 1979) by ammonium sulfate precipitation. For the preparation and biotinylation of Fab fragments of 2.4G2, 6 mg of purified 2.4G2 IgG was digested with insoluble papain enzyme as described by the manufacturer (Sigma). Fab fragments were biotinylated with NHS-LC-biotin or NHS-SS-biotin (Pierce, Rockford, Illinois, United States). In vitro binding assays The GST-Rab5 affinity chromatography was performed as in Christoforidis and Zerial (2000). 35S-Methionine–labelled proteins were transcribed and translated in vitro using a TnT-coupled transcription-translation kit (Promega, Madison, Wisconsin, United States). For Rab5 effector recruitment assays, in vitro-translated proteins were incubated with glutathione-sepharose beads complexed with GST-Rab5-GTPγS or GST-Rab5-GDP and eluted as described previously (Christoforidis et al. 1999a). Confocal immunofluorescence microscopy and transfection Cells were transfected with plasmids using FuGENE6 according to the manufacturer's instructions (Roche, Basel, Switzerland). After 16–24 h, transfected cells were washed twice with PBS and fixed with 3% paraformaldehyde and immunofluorescence labelling was performed according to standard procedures. Cells were mounted in moviol and examined on a confocal microscope (Leica TCS SP2; Leica, Wetzlar, Germany) with a 100×/1.40 plan-Apochromat objective. Fluorescent images were collected using the Leica IM500 Image Manager and processed using Adobe Photoshop v5.5 (Adobe Systems, San Jose, California, United States). Endocytosis of FITC-labelled dextran and rhodamine-labelled transferrin, and quantification NIH3T3 cells grown on glass coverslips and transfected with plasmids encoding RN-tre were incubated with 3 mg/ml FITC-labelled dextran (MW, 70.000) in DMEM supplemented with 1% FCS and 20 mM HEPES (pH 7,4), fixed, and stained with rhodamine-conjugated antirabbit secondary antibody. Dextran uptake was determined by quantifying grey values of thresholded, fluorescent images of at least 15 cells using MetaVue 6.1r3 and ImageJ (NIH). In vitro endosome fusion and early endosome/liposome recruitment assays In vitro fusion assays were performed using early endosomes labelled with biotinylated transferrin or antitransferrin antibody as well as CCVs labelled with biotinylated transferrin prepared from HeLa cells as described previously (Horiuchi et al. 1997). The concentration of Rabankyrin-5 in HeLa cell cytosol in the in vitro early-endosome fusion assay was estimated to be approximately 50 nM, as determined by comparison with a serial dilution of recombinant Rabankyrin-5 analysed by SDS-PAGE, followed by Western blotting analysis. Recruitment of cytosolic proteins to early endosomes was performed as described in Christoforidis et al. (1999b). Recombinant Rabankyrin-5 was recruited to phosphoinositides as described previously (Nielsen et al. 2000) using liposomes (98% phosphatidylcholine, 2% phosphoinositides; 2 mg/ml final concentration in 30 mM HEPES-NaOH [pH 7,2], 120 mM NaCl, 0,5 mM EGTA) prepared as reported previously (Otter-Nilsson et al. 1999). Time-lapse video microscopy and data processing Time-lapse epifluorescence video microscopy was performed using an Olympus IX70 (Olympus, Hamburg, Germany) inverted microscope equipped with a polychrome II monochromator (TILL Photonics, Martinsried, Germany), a custom filter block for simultaneous visualization of YFP and CFP (AHF Analysentechnik, Tübingen, Germany), a 100× oil-immersion objective (NA 1.35, Olympus) attached to a PIFOC z-SCAN (Physik Instrumente, Waldbronn, Germany), an incubation chamber (37 °C), and a 12-bit CCD digital camera IMAGO (0,134 μm pixel-1; 2 times 2 binning) (TILL Photonics), controlled by TILLvisION v3.3 software (TILL Photonics). Time-lapse video sequences of YFP and CFP images were merged as RGBs using TILLvisION v3.3. They were exported as single TIFF files and either further processed using Adobe Photoshop 5.5 and Illustrator 8.0 or converted into QuickTime movies using ImageJ (NIH). HRP staining of the apical endocytic machinery in mouse proximal tubules Anaesthetized mice were perfusion fixed with 4% PFA, 100 mM HEPES (pH 7,25), and 0,2% sucrose either directly or 5 min after injection with 5 mg/g body weight HRP (Sigma) into the femoral vein. Kidneys were excised and postfixed 6 h at 4 °C. Some tissue pieces were cryoprotected in 2,3 M sucrose overnight at 4 °C, frozen in liquid nitrogen, and cryosectioned on a Leica UCT (Tokuyasu et al. 1984). Adenovirus infection and continuous uptake measurement with HRP, biotinylated transferrin, and biotinylated Fab (2.4G2). MDCK cells were grown on 12-mm, 0,4-μm pore Transwell polycarbonate filters (Corning Costar, Cambridge, Massachusetts, United States). Ninety-six hours after seeding, cells were infected from the apical side for 4 h at 37 °C with adenoviruses in 250 μl of complete medium. After changing medium, cells were incubated for 18 h at 37 °C and then used for biochemical assays or microscopy. NIH3T3 and MDCK cells grown on coverslips in 24-well plates were infected in 500 μl of medium. HRP uptake was performed as described previously (Bomsel et al. 1989). In brief, cells were incubated in incubation medium (IM; DMEM, 1% FCS, 24 mM HEPES [pH 7,4]) at 37 °C for 1 h. For internalisation experiments coverslips or Transwell filters (Costar) were incubated with IM containing 5–10 mg/ml HRP, 2 μg/ml biotinylated transferrin, or 5 μg/ml biotinylated Fab (2.4G2). In the case of MDCK cells, HRP and Fab were applied from either the apical or the basolateral side. After internalisation, cells were briefly washed with warm IM followed by three washes with ice-cold PBS supplemented with 1 mM CaCl2 and 1 mM MgCl2 (PBS++) containing 2 mg/ml BSA. Cell surface-bound transferrin was efficiently removed (95%) by three incubations over 10 min in IM (pH 3,4). Extracellular HRP was quenched in the cold by incubating the cells for 20 min with 20 mM MESNA in 100 mM NaCl, 50 mM Tris (pH 8,7), followed by two washes with ice-cold PBS and a further incubation for 10 min with ice-cold 50 mM iodoacetamide in PBS. For recycling experiments, cells were transferred again to 37 °C in fresh medium that was collected at the indicated time points. Thereafter, cells were washed once more with PBS and extracted for 15 min at 4 °C with 300 μl of lysis buffer (1% w/v Triton X-100, 0,1% w/v SDS, 20 mM HEPES [pH 7,4], and 100 U/ml DNase). Total HRP activity present in the cells was determined in duplicates out of at least three independent experiments using the previously described enzymatic method (Steinman et al. 1976) and standardised to the protein content of each well. Fab and transferrin uptake assay was performed as described previously (Zacchi et al. 1998). Knock-down of Rabankyrin-5 in A431 cells by RNA interference A431 cells were seeded in 24-well plates the day prior to transfection. Cells were transfected using oligofectamine (Invitrogen, Carlsbad, California, United States) with either 300 ng/ml double- or single-stranded (control) esiRNA derived from the I.M.A.G.E. clone (IMAGE:258664) according to the protocol of Yang et al. (2002). After 4 d, cells were starved in MEM containing 2 mg/ml BSA and 24 mM HEPES (pH 7,3) for 1 h and stimulated with 50 ng/ml EGF in complete medium containing 24 mM HEPES (pH 7,3), 5–10 mg/ml HRP, and 2 μg/ml biotinylated transferrin for the indicated time points (see “Adenovirus infection and continuous uptake measurement with HRP, biotinylated transferrin, and biotinylated Fab (2.4G2)” above). Electron microscopy Cells or tissues were fixed with 8% PFA in PHEM buffer and then processed for frozen sectioning according to published methods (Liou et al. 1996). Thawed sections were either single or double labelled. For single labelling a polyclonal anti–Rabankyrin-5 antibody followed by protein A 10 nm gold (University of Utrecht) was used. For double labelling the polyclonal anti–Rabankyrin-5 antibody was incubated together with rat anti–LAMP-1 (1D4B; courtesy of Professor M. Desjardins, University of Montreal, Montreal, Canada). The sections were then incubated with a mixture of anti-rabbit 10 nm gold and anti-rat 5 nm gold (BBI International, Cardiff, United Kingdom). Supporting Information Figure S1 Wortmannin Treatment Inhibits Fluid-Phase Uptake into Enlarged Rabankyrin-5 Structures NIH3T3 cells were pretreated for 20 min with wortmannin (100 nM) and incubated for 25 min with 0,5 μg/ml rhodamine-labelled transferrin and 2,5 mg/ml FITC-labelled dextran (MW, 70.000), fixed, processed for immunofluorescence, and analysed by confocal scanning microscopy. Scale bar represents 10 μm. (8.4 MB EPS). Click here for additional data file. Figure S2 Microinjection of a Function-Blocking Antibody against Rabankyrin-5 Decreases Fluid-Phase Uptake NIH3T3 cells were either microinjected with (A and D) preimmune or (B and C) affinity-purified polyclonal α–Rabankyrin-5 antibodies and subjected to a 30-min incubation at 37 °C with (A and B) 3 mg/ml FITC-conjugated dextran (MW, 70.000) or (C and D) 1 μg/ml rhodamine-conjugated transferrin. Cells injected with immune but not preimmune antibodies showed a significant reduction of the fluid-phase marker, while the uptake of transferrin was not significantly perturbed. (D) Fluid-phase dextran quantification of single cells microinjected for indicated antibodies by measuring internalised fluorescence intensity (p > 0.001). Values shown are means ± standard deviation of at least 15 cells. Scale bars represent 10 μm. (44.0 MB EPS). Click here for additional data file. Video S1 Rabankyrin-5 Localizes to Macropinosomes Generated by Actin Dynamics Images were taken every 3 s over a time period of 15 min. RGB stacks were converted to QuickTime format using ImageJ (NIH). The movie is played at 15 frames per second. (11.1 MB MOV). Click here for additional data file. Accession Numbers The LocusLink (http://www.ncbi.nlm.nih.gov/LocusLink) accession numbers of proteins discussed in this paper are H-Ras (ID 3265), v-Src (ID 6714), Arf6 (ID 382), hVps34 (ID 5289), p110β (ID 5291), Rab34 (ID 83871), Rab5 (ID 5868), Ankhzn (ID 51479), Rab4 (ID 5867), Rab7 (ID 7879), Rab11 (ID 8766), Rabenosyn-5 (ID 64145), EEA1 (ID 8411), Caveolin-1 (ID 857), RN-tre (ID 9712), Transferrin (ID 7018), EGF (ID 1950), LAMP-1 (ID 16783), Prominin-1(ID 8842), PIKfyve (ID 200576), RhoA (ID 387), and PKC-α (ID 5578). We are indebted to Pierre Courtoy and Marcel Mettlen for valuable comments on the paper during the revision process. Moreover, we thank D. Corbeil for sharing antibodies against Prominin-1 and Angelika Giner for excellent technical assistance. We are also grateful to B. Hoflack, L. Pelkmans, K. Simons, M. Miaczynska, and J. Rink for comments on the manuscript. This work was supported by grants from the Human Frontier Science Program (HFSP) (RG-0260/1999-M), the European Union (HPRN-CT-2000-00081), and the Max Planck Society. Conflicts of interest. The authors have declared that no conflicts of interest exist. Author contributions. CS and SC conceived and designed the experiments. CS, SC, and MRL performed the experiments. CS, SC, MRL, SUJ, RGP, and MZ analyzed the data. CS, SC, MRL, SUJ, and MW contributed reagents/materials/analysis tools. CS and MZ wrote the paper. Academic Editor: Peter Walter, University of California at San Francisco ¤ Current address: IPBS, CNRS, Toulouse Cedex, France Citation: Schnatwinkel C, Christoforidis S, Lindsay MR, Uttenweiler-Joseph S, Wilm M, et al. (2004) The Rab5 effector rabankyrin-5 regulates and coordinates different endocytic mechanisms. PLoS Biol 2(9): e261. 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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020296Research ArticleCell BiologyGenetics/Genomics/Gene TherapyPhysiologySaccharomycesSir2-Independent Life Span Extension by Calorie Restriction in Yeast Calorie Restriction and Sir2Kaeberlein Matt 1 Kirkland Kathryn T 2 Fields Stanley 1 3 Kennedy Brian K [email protected] 2 1Departments of Genome Sciences and Medicine, University of WashingtonSeattle, Washington, United States of America2Department of Biochemistry, University of WashingtonSeattle, Washington, United States of America3Howard Hughes Medical Institute, University of WashingtonSeattle, WashingtonUnited States of America9 2004 24 8 2004 24 8 2004 2 9 e29619 5 2004 7 7 2004 Copyright: © 2004 Kaeberlein et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. New Route to Longer Life Calorie restriction slows aging and increases life span in many organisms. In yeast, a mechanistic explanation has been proposed whereby calorie restriction slows aging by activating Sir2. Here we report the identification of a Sir2-independent pathway responsible for a majority of the longevity benefit associated with calorie restriction. Deletion of FOB1 and overexpression of SIR2 have been previously found to increase life span by reducing the levels of toxic rDNA circles in aged mother cells. We find that combining calorie restriction with either of these genetic interventions dramatically enhances longevity, resulting in the longest-lived yeast strain reported thus far. Further, calorie restriction results in a greater life span extension in cells lacking both Sir2 and Fob1 than in cells where Sir2 is present. These findings indicate that Sir2 and calorie restriction act in parallel pathways to promote longevity in yeast and, perhaps, higher eukaryotes. This study indicates that calorie restriction and Sir2 promote longevity in yeast through distinct pathways. This undermines the accepted view, and has implications for aging in higher organisms ==== Body Introduction The budding yeast Saccharomyces cerevisiae has served as a useful model for aging research, leading to the identification of new longevity genes and pathways whose counterparts can be examined in higher eukaryotes (Kaeberlein et al. 2001). One measure of aging in yeast is the finite replicative life span (RLS) of mother cells, defined as the number of mitotic cycles completed prior to senescence (Mortimer and Johnston 1959). Alternatively, the survival of nondividing yeast cells over time can be monitored and has been termed chronological aging (Fabrizio and Longo 2003). It has been proposed that replicative aging in yeast may be a suitable model for the aging of dividing cells in mammals, such as germ cells; whereas, chronological aging of yeast may be related to the aging of postmitotic tissues. Replicative aging of yeast can be caused by the accumulation of extrachromosomal rDNA circles (ERCs) in the mother cell nucleus (Sinclair and Guarente 1997), and mutations that decrease ERC formation correlate with increased life span. One example of such a mutation is deletion of the gene encoding the rDNA replication fork barrier protein Fob1, which results in a dramatic decrease in ERC levels accompanied by a 30%–40% increase in mean and maximum RLS (Defossez et al. 1999). In addition to Fob1, the Sir2 protein has also been found to affect longevity by regulating the rate at which ERCs are formed (Kaeberlein et al. 1999). Sir2 is an NAD-dependent histone deacetylase (Imai et al. 2000; Landry et al. 2000; Tanner et al. 2000) necessary for transcriptional silencing near telomeres (Aparicio et al. 1991), HM loci (Ivy et al. 1986; Rine and Herskowitz 1987), and rDNA (Bryk et al. 1997; Smith and Boeke 1997). Deletion of Sir2 increases both rDNA recombination (Gottlieb and Esposito 1989) and ERC formation, while shortening life span by approximately 50% (Kaeberlein et al. 1999). Conversely, overexpression of Sir2 increases life span by 30%–40%. Overexpression of Sir2 in the context of FOB1 deletion fails to further extend life span, consistent with the idea that Sir2 and Fob1 both impact aging by regulating ERC levels (Kaeberlein et al. 1999). Calorie restriction (CR) of yeast cells can be accomplished by a reduction in the glucose concentration of growth media from 2% to 0.5% (or lower) and results in a 30%–40% increase in life span (Lin et al. 2000). Several genetic models of CR have also been described. In one model, deletion of the HXK2 gene, coding for hexokinase, reduces the availability of glucose for glycolysis; while in the others, deletion of other genes, including gpa2Δ and gpr1Δ, decreases cAMP-dependent protein kinase activity (Lin et al. 2000). Growth in low glucose and the various genetic models of CR have been treated as experimentally interchangeable. While they are clearly not identical, evidence to date suggests that they behave in a similar manner with respect to yeast aging (Lin et al. 2000, 2002, 2004; Kaeberlein et al. 2002, 2004). Several reports have suggested a link between the enhanced longevity associated with CR and increased activity of Sir2 (Koubova and Guarente 2003). In one genetic model of CR, cdc25-10 is reported to decrease both rDNA recombination and ERC levels (Lin et al. 2000). In addition, deletion of Sir2 has been shown to prevent life span extension by cdc25-10 and low glucose (Lin et al. 2000, 2002). These data have been used to support a model whereby CR activates Sir2, thus causing decreased ERC accumulation and increased life span. It was initially proposed that life span extension by CR is the consequence of a metabolic shift resulting in increased cellular NAD available as a substrate for Sir2-dependent histone deacetylation (Lin et al. 2002). More recently, this theory has been supplanted by two competing models for activation of Sir2 by CR: (1) a decrease in cellular nicotinamide (a product inhibitor of Sir2) via upregulation of PNC1 (Anderson et al. 2003a), and (2) a decrease in cellular NADH (a competitive inhibitor of Sir2) (Lin et al. 2004). We present here evidence that CR and Sir2 act in different genetic pathways to promote longevity and show that Sir2 is not required for full life span extension in response to CR. In addition, we offer data suggesting that previous experiments were misinterpreted. Finally, we propose a model that reconciles our findings with earlier reports and suggests a greater level of conservation between aging in yeast and higher eukaryotes. Results We recently carried out a large-scale study of more than 40 single-gene deletions reported to affect aging in yeast (unpublished data). This analysis was performed in the BY4742 genetic background, which has a mean life span significantly longer than most other yeast strains commonly used for aging research (Table 1). Included in this analysis were three genetic models of CR (hxk2Δ, gpa2Δ, and gpr1Δ) and fob1Δ. As previously reported for shorter-lived strain backgrounds (Defossez et al. 1999; Lin et al. 2000), each of these single-gene deletions resulted in a 30%–40% increase in life span in BY4742 (Figure 1A). Figure 1 Regulation of Longevity by CR and Fob1 (A) Life span analysis for three genetic models of CR and deletion of FOB1. Strains shown (and mean life spans) are BY4742 (26.7), fob1Δ (37.8), gpa2Δ (34.9), gpr1 (34.4), and hxk2Δ (36.7). (B) fob1Δ and hxk2Δ increase life span additively. Strains shown (and mean life spans) are BY4742 (26.6), fob1Δ (37.3), hxk2Δ (36.7), and fob1Δ hxk2Δ (48.3). (C) fob1Δ and gpa2Δ increase life span additively. Strains shown (and mean life spans) are BY4742 (27.2), fob1Δ (37.8), gpa2Δ (36.7), and fob1Δ gpa2Δ (54.5). Table 1 BY4742 Is Long Lived Relative to Other Yeast Strains Commonly Used in Aging Research Reported mean RLS (MRLS) for each strain and the percent difference relative to BY4742 ((Strain MRLS − BY4742 MRLS)/Strain MRLS) are shown Since both CR and deletion of FOB1 increased life span individually in BY4742, we examined the effect of CR combined with deletion of FOB1. It is notable that this experiment has not to our knowledge been previously reported. We constructed a fob1Δ hxk2Δ double mutant and determined the replicative aging potential of this strain. As expected, both single mutants lived longer than wild-type mother cells (p < 0.001). However, the life span of the fob1Δ hxk2Δ double mutant greatly exceeded that of either single mutant (p < 0.001), suggesting an additional effect on longevity as a result of combining deletion of FOB1 with CR (Figure 1B). In order to demonstrate that this synthetic lengthening of life span in combination with fob1Δ was not specific to the hxk2Δ model of CR, we determined the life span of a fob1Δ gpa2Δ double mutant. As observed for HXK2, deletion of GPA2 combined with deletion of FOB1 resulted in a mean life span significantly greater than was observed for either single mutant (p < 0.001), and nearly double that of wild-type cells (Figure 1C). With mean and maximum life spans of 54.5 and 94 generations, respectively, the fob1Δ gpa2Δ and fob1Δ hxk2Δ double mutants are to our knowledge the longest-lived yeast strains reported to date. The observation that CR further increases the long life span of a fob1Δ mutant is inconsistent with the model that CR increases life span solely by activation of Sir2. Since overexpression of SIR2 is sufficient to increase the life span of wild-type cells but fails to further extend the life span of a fob1Δ mutant (Kaeberlein et al. 1999), CR (acting through Sir2) should also fail to further extend the life span of a fob1Δ strain, by this model. Our data therefore suggest the existence of a Sir2-independent pathway by which CR enhances longevity. In order to test this possibility, we determined whether CR would increase life span in the absence of Sir2. As observed in other strain backgrounds, deletion of SIR2 shortens life span by approximately 50% in BY4742 (Figure 2A), likely because of an elevated level of ERCs (Kaeberlein et al. 1999). Neither deletion of HXK2 nor deletion of GPA2 conferred increased life span to the sir2Δ mutant. As expected, deletion of FOB1 was sufficient to suppress the life span defect of cells lacking Sir2 (Figure 2B), consistent with the idea that accelerated ERC accumulation is responsible for the severe life span defect of the sir2Δ strain. Surprisingly, in the sir2Δ fob1Δ double mutant, deletion of HXK2 resulted in a robust life span extension (Figure 2C; p < 0.001). Similarly, the life span of sir2Δ fob1Δ gpa2Δ triple mutant cells was significantly longer than that of sir2Δ fob1Δ double mutant cells (Figure 2D; p < 0.001). In fact, the life spans of sir2Δ fob1Δ hxk2Δ and sir2Δ fob1Δ gpa2Δ cells did not differ significantly (p ≈ 0.4) from fob1Δ hxk2Δ and fob1Δ gpa2Δ cells, respectively. Thus, CR clearly enhances longevity in the absence of both Sir2 and Fob1, but not in the absence of Sir2 alone. While seemingly contradictory (see below), these findings demonstrate that Sir2 is dispensable for life span extension by CR, at least in the context of reduced ERC levels (as a result of fob1Δ). Figure 2 Life Span Extension by CR Does Not Require Sir2 (A) CR fails to increase life span of a sir2Δ mutant. Strains shown (and mean life span) are BY4742 (26.7), sir2Δ (14.0), hxk2Δ sir2Δ (12.4), and gpa2Δ sir2Δ (11.7). (B) Deletion of FOB1 suppresses the short life span of a sir2Δ strain. Strains shown and mean life spans are: BY4742 (27.5), sir2Δ (14.0), sir2Δ fob1Δ (30.0). (C) Deletion of HXK2 increases the life span of a sir2Δ fob1Δ double mutant. Strains shown (and mean life spans) are BY4742 (26.5), sir2Δ fob1Δ (30.0), and sir2Δ fob1Δ hxk2Δ (45.3). (D) Deletion of GPA2 increases the life span of a sir2Δ fob1Δ double mutant. Strains shown (and mean life spans) are BY4742 (26.6), sir2Δ fob1Δ (30.0), and sir2Δ fob1Δ gpa2Δ (51.0). Genetic models of CR, such as hxk2Δ and gpa2Δ, have been used as convenient surrogates for CR by growth on low glucose (Lin et al. 2000, 2002); however, it is possible that these genetic models of CR may not completely recapitulate the effects of glucose deprivation. Additionally, unlike genetic models of CR, growth on low glucose provides an opportunity to control the degree of CR by manipulating the glucose concentration within a range of values (Kaeberlein et al. 2002). Taking advantage of this property, we examined the life span of wild-type and sir2Δ fob1Δ double mutant cells on 2%, 0.5%, 0.1%, and 0.05% glucose (Figure 3; Figure S1). Wild-type cells showed an increase in mean life span ranging from 15% to 25%, with maximal increases observed at 0.05% glucose (p < 0.05). The effect of growth on low glucose was even more pronounced in the sir2Δ fob1Δ double mutant, with mean life span increased by 25% on 0.5% glucose (p < 0.01) and by 60% on 0.05% glucose (p < 0.001). Figure 3 CR Is More Effective at Enhancing Longevity in a sir2Δ fob1Δ Double Mutant than in Wild-Type Cells Percent increase in mean life span relative to growth on 2% glucose was determined for 20 mother cells from each strain at 0.5%, 0.1%, and 0.05% glucose. Our data conflict with the report that CR fails to increase the life span of a sir2Δ fob1Δ double mutant (Lin et al. 2000). However, all of these prior experiments, as well as nearly all of the published life span data on CR in yeast, were carried out in the PSY316 strain background (Lin et al. 2000, 2002, 2004; Anderson et al. 2002, 2003a, 2003b; Bitterman et al. 2002). We therefore asked whether strain-specific effects might account for this apparent discrepancy. Consistent with prior reports, we observed that growth on low glucose fails to increase the life span of a sir2Δ fob1Δ double mutant derived from strain PSY316 (unpublished data). However, the previous experiments demonstrating that either deletion of FOB1 or overexpression of SIR2 increase life span were carried out in W303R (Kaeberlein et al. 1999), a genetic background apparently unrelated to PSY316. Notably, life span phenotypes for a fob1Δ mutant or SIR2-overexpressing strain in PSY316 have not been reported. Thus, we created these strains and measured their life span. Neither deletion of FOB1 (p = 0.29) nor overexpression of SIR2 (p = 0.76) was sufficient to increase life span in the PSY316 background (Figure 4A). In fact, PSY316 behaves differently from the majority of other yeast strains with respect to the roles of SIR2 and FOB1 as regulators of longevity, since overexpression of SIR2 or deletion of FOB1 has been found to increase longevity in multiple genetic backgrounds, including BY4742 (Table 2). Figure 4 CR Increases the Life Span of Cells Overexpressing SIR2 (A) Neither deletion of FOB1 nor overexpression of SIR2 impact longevity in PSY316. Strains shown (and mean life spans) are PSY316 (21.1), PSY316 fob1Δ (20.7), and PSY316 SIR2-ox (21.7). (B) Overexpression of SIR2 and CR increase life span additively in BY4742. Strains shown (and mean life spans) are BY4742 on 2% glucose (26.1), BY4742 on 0.05% glucose (31.8), BY4742 SIR2-ox on 2% glucose (34.6), and BY4742 SIR2-ox on 0.05% glucose (42.2). Table 2 FOB1 Deletion or SIR2 Overexpression Increase Life Span in Multiple Genetic Backgrounds The percent effect on mean RLS ((mutant − wild-type)/wild-type × 100) as a result of FOB1 deletion or SIR2 overexpression is shown for each strain relative to the parental wild-type (strain background). The reported mean RLS for fob1Δ and SIR2-overexpressing mutants in each strain is also shown in parentheses. To our knowledge, PSY316 is the only background in which these interventions do not increase longevity Unlike in PSY316, overexpression of SIR2 in BY4742 significantly increases life span (Figure 4B; p < 0.001). Further, growth of SIR2-overexpressing cells on low glucose results in an additional life span increase (p < 0.001), similar to that observed for sir2Δ fob1Δ double mutant cells on low glucose. The observation that CR further enhances the already long life span of cells in which SIR2 is overexpressed reinforces our model that CR and SIR2 promote longevity by influencing different pathways (Figure 5). Figure 5 Two Pathways Determine Yeast Longevity The longevity of mother cells can be modified by at least two independent interventions: altered ERC levels and CR. In cells lacking Sir2 but containing Fob1, senescence due to ERCs predominates, causing an extremely short life span that cannot be increased by CR. In cells lacking FOB1, ERCs are greatly reduced and the CR pathway predominates. The presence or absence of Sir2 does not impact the longevity benefits of CR under this condition. Discussion We present substantial genetic evidence that CR and Sir2 act in different genetic pathways to promote longevity. The combination of CR with SIR2 overexpression results in an additive life span increase, as expected for two genetic interventions acting in parallel pathways. Further, in the context of FOB1 deletion, CR results in a larger relative increase in life span in the absence of Sir2 than in cells where Sir2 is expressed. Finally, the ability of CR to promote longevity in a strain lacking Sir2 definitively demonstrates the existence of a Sir2-independent aging pathway responsive to CR. Experiments have previously suggested that life span extension by CR in yeast is partially Sir2-independent (Jiang et al. 2002). It is important to note, however, that the conditions employed for these experiments involved maintaining the cells on defined medium, which is known to slow growth rate and shorten life span by about 50% (Jiang et al. 2000). Under these conditions, CR is reported to modestly increase mean life span of sir2Δ mother cells from seven generations to nine generations. This differs from our results, which demonstrate that CR has no significant effect on life span in the sir2Δ background when cells are grown under standard conditions (see Figure 2A). We speculate that the apparently toxic effects of growth on defined medium (as evidenced by dramatically reduced life span and fitness) are partially mitigated by CR in a Sir2-independent manner. It is not clear whether this modest effect (two generations) is related in any way to the robust (20–30 generations) Sir2-independent life span extension caused by CR under standard growth conditions. The seemingly disparate findings that CR fails to extend the life span of a sir2Δstrain (see Figure 2A) but dramatically extends life span in a sir2Δ fob1Δ double mutant background (see Figure 2C–2E) can be explained by a model in which there are (at least) two pathways that regulate aging in yeast: one is ERC accumulation and the other is undefined at a molecular level, but responsive to CR (see Figure 5). In our long-lived wild-type background, both processes influence longevity. To explain why CR fails to extend life span in the sir2Δ strain, we postulate that the ERC pathway predominates in this mutant. Cells lacking Sir2 exhibit elevated rDNA recombination and increased levels of ERCs (Kaeberlein et al. 1999), resulting in the premature death of nearly all mother cells prior to an age where the CR pathway becomes limiting. Thus, CR, acting through the alternative pathway, fails to impact aging in the sir2Δ mutant. In the fob1Δ mutant or sir2Δ fob1Δ double mutant, strains in which ERCs are greatly reduced (Defossez et al. 1999; Kaeberlein et al. 1999), CR slows aging through the Sir2-independent alternative pathway. This independent pathway should be more important when ERC levels are reduced, and, consistent with this model, we find that CR has a more pronounced effect on life span under these conditions (see Figure 3; Figure S1). Our findings do not preclude the possibility that CR enhances Sir2 function through previously proposed mechanisms. However, the fact that the life spans of sir2Δ fob1Δ hxk2Δ and sir2Δ fob1Δ gpa2Δ triple mutants do not differ significantly from those of fob1Δ hxk2Δ and fob1Δ gpa2Δ double mutants, respectively, suggests that any role for Sir2 in the CR pathway is, at best, minor. Alternatively, it is possible that another protein can substitute for Sir2 as a downstream effecter of CR when Sir2 is absent. This model seems unlikely, however, given a need to postulate that the hypothetical Sir2-like protein could function as a substitute for Sir2 only in a strain lacking Fob1, since CR fails to increase life span in the sir2Δ single mutant. The most likely candidate for such a Sir2-like protein is the Sir2 homolog, Hst1. We find that deletion of HST1 has no effect on life span (unpublished data), suggesting that, at least under normal conditions, Hst1 is not an important determinant of longevity. Nearly all of the evidence supporting a role for Sir2 in CR-mediated life span extension is derived from experiments carried out in PSY316, further weakening the case for a Sir2-dependent model. The inability of SIR2 overexpression, in particular, to increase life span in the PSY316 background supports the idea that Sir2 does not play a primary role in CR-mediated life span extension, as it is not straightforward to postulate a model whereby CR would increase life span via activation of Sir2 in a strain background that is insensitive to Sir2 dosage. Further, the inability of the fob1Δ mutation to increase life span in PSY316 provides a plausible explanation for why CR is unable to enhance longevity in the PSY316 sir2Δ fob1Δ double mutant, and suggests that either deletion of FOB1 fails to impact ERCs in this background or ERCs are not limiting for life span. While we cannot rule out the possibility that the Sir2-independent nature of CR is unique to BY4742, we note that BY4742 behaves like the majority of other strains with respect to increased life span in response to deletion of FOB1 or overexpression of SIR2, while PSY316 is the only strain (to our knowledge) that is unresponsive to these interventions (Table 2). The observation that CR further increases the long life span of a fob1Δ strain (see Figure 1B and 1C) suggests that the mechanism of enhanced longevity by CR is unrelated to ERCs, as cells lacking Fob1 have dramatically reduced ERC levels. However, it is still possible that ERCs limit the life span of fob1Δ cells and that CR slows ERC accumulation by a second pathway that is insensitive to both Fob1 and Sir2. This seems unlikely, since CR is more effective at enhancing longevity in a sir2Δ fob1Δ double mutant than in wild-type cells. It has been observed that, while life span is comparable between wild-type and sir2Δ fob1Δ cells, ERCs are much reduced in the double mutant (Kaeberlein et al. 1999), suggesting that sir2Δ fob1Δ cells are not senescing as a result of ERCs. Thus, life span extension by CR in this context is likely to be unrelated to ERCs. The existence of an ERC-independent aging pathway in yeast that is modulated by CR is of particular relevance to aging in higher organisms. CR is the only intervention shown to extend life span in a wide range of eukaryotes, including mammals (Weindruch and Walford 1988). In contrast, there is no evidence that ERCs affect aging in organisms other than budding yeast. Nevertheless, in Caenorhabditis elegans, increased expression of the Sir2 ortholog, Sir-2.1, has been found to extend life span in a manner dependent on the Daf-16 transcription factor (Tissenbaum and Guarente 2001). Similarly, the mammalian Sir2 ortholog, SirT1, has recently been reported to regulate the activity of murine Foxo3A (Brunet et al. 2004; Motta et al. 2004). These experiments support a role for Sir2 proteins in eukaryotic aging, linking Sirtuin activity to insulin/IGF-1 signaling. Evidence is accumulating, however, that CR and insulin/IGF-1 act in different pathways to regulate aging in complex eukaryotes. Life span extension by CR is independent of Daf-16 in C. elegans (Lakowski and Hekimi 1998; Houthoofd et al. 2003), and CR can further extend the life span of long-lived insulin/IGF-1 pathway mutants in both C. elegans and mice (Lakowski and Hekimi 1998; Bartke et al. 2001). We present similar evidence that the effects of CR and Sir2 are genetically distinct in yeast, raising the intriguing possibility that aspects of both aging pathways have been conserved. Materials and Methods Strains and plasmids. All yeast strains used in this study are congenic derivatives of BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0), except for PSY316AR (MATα RDN1::ADE2 his3- 200 leu2-3,112 lys2 ura3-52), PSY316AR fob1Δ::kanMX, and PSY316AR SIR2-ox. All gene disruptions were verified by PCR. In addition, sir2Δ mutants were verified by the sterility phenotype associated with this mutation. Strains overexpressing Sir2 were constructed by genomic integration of an extra copy of SIR2, as described (Kaeberlein et al. 1999), and life span was determined for four independent transformants. RLS analysis. Yeast strains for RLS analysis were removed from frozen stock (25% glycerol, −80 °C) and streaked onto YPD. After 2 d of growth, single colonies were selected and patched to YPD. The next evening, cells were lightly patched to the plates used for life span analysis (4–6 strains per plate). After overnight growth, cells were arrayed onto solid medium using a micromanipulator and allowed to undergo 1–2 divisions. Virgin cells were selected and subjected to life span analysis. Cells were grown at 30 °C during the day and stored at 4 °C at night. Daughter cells were removed by gentle agitation with a dissecting needle and tabulated every 1–2 cell divisions. All life span experiments were carried out on standard YPD plates (2% glucose), except for the low glucose experiments, which were performed on YEP plates supplemented with the indicated amounts of glucose. In order to prevent introduction of bias, strains were coded such that the researcher performing the life span experiment had no knowledge of the strain genotype for any particular strain. For each experiment, each strain was randomly coded at the time of removal from frozen stock. One individual was responsible for assigning codes (K. T. K.) while a different individual (M. K. or B. K. K.) performed the micromanipulation and was unaware of the genotypes of the strains being analyzed. Statistical analysis of data For statistical analysis, life span datasets were compared using a two-tailed Wilcoxon Rank-Sum test. Mother cell life span and p-value matrices for each figure are available in Dataset S1; life span data for individual mother cells are available in Dataset S2. Wilcoxon p-values were calculated using the MATLAB ranksum function. Data shown in each figure and used to calculate p-values were derived from pair-matched, pooled experiments where each mutant was compared to wild-type cells examined within the same experiment(s). Strains are stated to have a significant difference in life span for p < 0.05. Supporting Information Dataset S1 P-Value Matrices for Figures 1–4 Each matrix contains the Wilcoxon Rank-Sum p-values for a two-tailed test in which the life span data for the strain in the corresponding row were compared against the life span data for the strain in the corresponding column. Significant p-values (p < 0.05) are colored yellow. P-values were calculated using the MATLAB ranksum function. (46 KB PDF). Click here for additional data file. Dataset S2 Raw Mother Cell Life Span Data for Figures 1–4 (16 KB TXT). Click here for additional data file. Figure S1 CR Increases Life Span in Wild-Type and sir2Δ fob1Δ Mother Cells (A) Life span extension by CR is maximized at 0.05% glucose in BY4742 mother cells. Mean life spans are shown for cells grown on 2% glucose (24.8), 0.5% glucose (28.3), 0.1% glucose (30.1), and 0.05% glucose (32.1). (B) Life span extension by CR is maximized at 0.05% glucose in sir2Δ fob1Δ mother cells. Mean life spans are shown for cells grown on 2% glucose (26.0), 0.5% glucose (32.9), 0.1% glucose (40.8), and 0.05% glucose (42.0). (88 KB PS). Click here for additional data file. Accession Numbers The Saccharomyces Genome Database (http://www.yeastgenome.org/) accession numbers for the yeast genes and gene products discussed in this paper are CDC25 (SGDID S0004301), FOB1 (SGDID S0002517), GPA2 (SGDID S0000822), GPR1 (SGDID S0002193), HST1 (SGDID S0005429), HXK2 (SGDID S0003222), PNC1 (SGDID S0003005), and SIR2 (SGDID S0002200) The LocusLink (http://www.ncbi.nlm.nih.gov/LocusLink/) accession numbers for the non-yeast genes and gene products discussed in this paper are C. elegans Daf-16 (LocusLink 172981), C. elegans Sir-2.1 (LocusLink 177924), mouse Foxo3A (LocusLink 2309), and mouse SirT1 (LocusLink 23411). We thank L. Guarente, S. Lin, G. Martin, and T. Powers for helpful discussion and G. Liszt for providing strains. MK is supported by National Institutes of Health training grant P30 AG013280. This work was funded by awards to BK from the University of Washington Nathan Shock Center of Excellence for the Basic Biology of Aging and the American Federation for Aging Research. SF is an investigator of the Howard Hughes Medical Institute. Conflicts of interest. The authors have declared that no conflicts of interest exist. Author contributions. MK, KTK, SF, and BKK conceived and designed the experiments. MK, KTK, and BKK performed the experiments and analyzed the data. MK, KTK, SF, and BKK contributed reagents/materials/analysis tools. MK and BKK wrote the paper. Academic Editor: Andy Dillin, Salk Institute Citation: Kaeberlein M, Kirkland KT, Fields S, Kennedy BK (2004) Sir2-independent life span extension by calorie restriction in yeast. PLoS Biol 2(9): e296. Abbreviations CRcalorie restriction ERCextrachromosomal rDNA circle RLSreplicative life span ==== Refs References Anderson RM Bitterman KJ Wood JG Medvedik O Cohen H Manipulation of a nuclear NAD+ salvage pathway delays aging without altering steady-state NAD+ levels J Biol Chem 2002 277 18881 18890 11884393 Anderson RM Bitterman KJ Wood JG Medvedik O Sinclair DA Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae Nature 2003a 423 181 185 12736687 Anderson RM Latorre-Esteves M Neves AR Lavu S Medvedik O Yeast life-span extension by calorie restriction is independent of NAD fluctuation Science 2003b 302 2124 2126 14605207 Aparicio OM Billington BL Gottschling DE Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae Cell 1991 66 1279 1287 1913809 Bartke A Wright JC Mattison JA Ingram DK Miller RA Extending the lifespan of long-lived mice Nature 2001 414 412 11719795 Bitterman KJ Anderson RM Cohen HY Latorre-Esteves M Sinclair DA Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of yeast sir2 and human SIRT1 J Biol Chem 2002 277 45099 45107 12297502 Borghouts C Benguria A Wawryn J Jazwinski SM Rtg2 protein links metabolism and genome stability in yeast longevity Genetics 2004 166 765 777 15020466 Brunet A Sweeney LB Sturgill JF Chua KF Greer PL Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase Science 2004 303 2011 2015 14976264 Bryk M Banerjee M Murphy M Knudsen KE Garfinkel DJ Transcriptional silencing of Ty1 elements in the RDN1 locus of yeast Genes Dev 1997 11 255 269 9009207 Defossez PA Prusty R Kaeberlein M Lin SJ Ferrigno P Elimination of replication block protein Fob1 extends the life span of yeast mother cells Mol Cell 1999 3 447 455 10230397 Fabrizio P Longo VD The chronological life span of Saccharomyces cerevisiae Aging Cell 2003 2 73 81 12882320 Gottlieb S Esposito RE A new role for a yeast transcriptional silencer gene, SIR2, in regulation of recombination in ribosomal DNA Cell 1989 56 771 776 2647300 Houthoofd K Braeckman BP Johnson TE Vanfleteren JR Life extension via dietary restriction is independent of the Ins/IGF-1 signalling pathway in Caenorhabditis elegans Exp Gerontol 2003 38 947 954 12954481 Imai S Armstrong CM Kaeberlein M Guarente L Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase Nature 2000 403 795 800 10693811 Ivy JM Klar AJ Hicks JB Cloning and characterization of four SIR genes of Saccharomyces cerevisiae Mol Cell Biol 1986 6 688 702 3023863 Jiang JC Jaruga E Repnevskaya MV Jazwinski SM An intervention resembling caloric restriction prolongs life span and retards aging in yeast FASEB J 2000 14 2135 2137 11024000 Jiang JC Wawryn J Shantha Kumara HM Jazwinski SM Distinct roles of processes modulated by histone deacetylases Rpd3p, Hda1p, and Sir2p in life extension by caloric restriction in yeast Exp Gerontol 2002 37 1023 1030 12213553 Kaeberlein M McVey M Guarente L The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms Genes Dev 1999 13 2570 2580 10521401 Kaeberlein M McVey M Guarente L Using yeast to discover the fountain of youth Sci Aging Knowledge Environ 2001 pe1 2001 14602950 Kaeberlein M Andalis AA Fink GR Guarente L High osmolarity extends life span in Saccharomyces cerevisiae by a mechanism related to calorie restriction Mol Cell Biol 2002 22 8056 8066 12391171 Kaeberlein M Andalis AA Liszt G Fink GR Guarente L Saccharomyces cerevisiae SSD1-V confers longevity by a Sir2p-independent mechanism Genetics 2004 166 1661 1672 15126388 Kennedy BK Austriaco NR Zhang J Guarente L Mutation in the silencing gene SIR4 can delay aging in S. cerevisiae Cell 1995 80 485 496 7859289 Kirchman PA Kim S Lai CY Jazwinski SM Interorganelle signaling is a determinant of longevity in Saccharomyces cerevisiae Genetics 1999 152 179 190 10224252 Koubova J Guarente L How does calorie restriction work? Genes Dev 2003 17 313 321 12569120 Lakowski B Hekimi S The genetics of caloric restriction in Caenorhabditis elegans Proc Natl Acad Sci U S A 1998 95 13091 13096 9789046 Landry J Sutton A Tafrov ST Heller RC Stebbins J The silencing protein SIR2 and its homologs are NAD-dependent protein deacetylases Proc Natl Acad Sci U S A 2000 97 5807 5811 10811920 Lin SJ Defossez PA Guarente L Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae Science 2000 289 2126 2128 11000115 Lin SJ Kaeberlein M Andalis AA Sturtz LA Defossez PA Calorie restriction extends Saccharomyces cerevisiae life span by increasing respiration Nature 2002 418 344 348 12124627 Lin SJ Ford E Haigis M Liszt G Guarente L Calorie restriction extends yeast life span by lowering the level of NADH Genes Dev 2004 18 12 16 14724176 Lin SS Manchester JK Gordon JI Sip2, an N-myristoylated beta subunit of Snf1 kinase, regulates aging in Saccharomyces cerevisiae by affecting cellular histone kinase activity, recombination at rDNA loci, and silencing J Biol Chem 2003 278 13390 13397 12562756 Mortimer RK Johnston JR Life span of individual yeast cells Nature 1959 183 1751 1752 13666896 Motta MC Divecha N Lemieux M Kamel C Chen D Mammalian SIRT1 represses forkhead transcription factors Cell 2004 116 551 563 14980222 Rine J Herskowitz I Four genes responsible for a position effect on expression from HML and HMR in Saccharomyces cerevisiae Genetics 1987 116 9 22 3297920 Sinclair DA Guarente L Extrachromosomal rDNA circles—A cause of aging in yeast Cell 1997 91 1033 1042 9428525 Smith JS Boeke JD An unusual form of transcriptional silencing in yeast ribosomal DNA Genes Dev 1997 11 241 254 9009206 Takeuchi Y Horiuchi T Kobayashi T Transcription-dependent recombination and the role of fork collision in yeast rDNA Genes Dev 2003 17 1497 1506 12783853 Tanner KG Landry J Sternglanz R Denu JM Silent information regulator 2 family of NAD-dependent histone/protein deacetylases generates a unique product, 1-O-acetyl-ADP-ribose Proc Natl Acad Sci U S A 2000 97 14178 14182 11106374 Tissenbaum HA Guarente L Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans Nature 2001 410 227 230 11242085 Weindruch RH Walford RL The retardation of aging and disease by dietary restriction 1988 Springfield (Illinois) Charles C Thomas 436

15328540

PMC514491

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2021-01-05 08:21:14

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PLoS Biol. 2004 Sep 24; 2(9):e296

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PLoS Biol

10.1371/journal.pbio.0020296

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020308SynopsisGenetics/Genomics/Gene TherapyYeast and FungiSaccharomycesNew Route to Longer Life Synopsis9 2004 24 8 2004 24 8 2004 2 9 e308Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Sir2-Independent Life Span Extension by Calorie Restriction in Yeast ==== Body Ever since the early Greeks recast humans as the center of the universe and remade God in their own image, Western philosophers and poets have grappled with the limits of human mortality. Philosophers found relief from Keats's “unwilling sleep” by dividing human existence into body and soul and asserting that the true essence of humanity lies in the immortal soul, not in the body. Ironically, as this decidedly nonscientific subject has lost favor with modern-day philosophers, it has captured the imagination of scientists. But, for now at least, the interest is in prolonging life rather than escaping mortality. Over the past twenty years, mounting evidence from a wide range of organisms indicates that a longer life awaits those who eat less. In yeast, calories can be restricted directly, by limiting yeast's glucose supply, or indirectly, by inhibiting yeast's ability to metabolize glucose. Either way, many studies have suggested that the increased longevity associated with calorie restriction is linked to increased activity of a gene called SIR2. Now, Brian Kennedy and colleagues show that calorie restriction and SIR2 promote longevity through distinct genetic pathways—and that aging in yeast and higher organisms may be more similar than previously thought. One of the causes of aging in yeast is the accumulation of coiled bits of DNA, called extrachromosomal ribosomal DNA circles (ERCs), in the nucleus of a mother cell (which divides to create two identical daughter cells). An overabundance of these rDNA circles wreaks havoc on a cell and eventually kills it. Genetic mutations that reduce their levels are linked to increased life span. Mutations that disrupt the FOB1 gene, for example, dramatically reduce ERC levels and increase the reproductive life span of cells by 30%–40%. In contrast, mutations that disrupt SIR2 increase ERC levels and cut life span in half, while increasing SIR2 activity increases life span by 30%–40%. In previous experiments, several groups have identified a link between calorie restriction, SIR2, and the accumulation of ERCs. The idea is that calorie restriction somehow activates the protein encoded by SIR2, which in turn decreases ERC accumulation. Now, Kennedy's team has found that the combination of calorie restriction and FOB1 mutation increases life span more than either approach does alone. This finding was unexpected because previous studies showed that combining increased SIR2 activity with FOB1 deletion mutations did not extend life span. If calorie restriction extends life through SIR2, then combining either caloric restriction or SIR2 overexpression with FOB1 mutations should produce the same result. This contradiction raised the possibility that calorie restriction operates through another mechanism, independent of SIR2. In support of this view, caloric restriction enhances life span to a greater extent in FOB1 mutants lacking SIR2 than in FOB1 mutants with an intact SIR2 gene. This and other genetic experiments indicate that calorie restriction does not always work through SIR2. That suggests, the authors explain, that calorie restriction functions either by regulating ERC levels or by some still unknown molecular pathway. They conclude that the enhanced longevity seen in calorie-restricted FOB1 mutants is not related to ERCs, because these yeast strains already have low ERC levels. Since calorie restriction is the only demonstrated approach to increasing life span in a diverse range of organisms, including mammals, and since there's no evidence that ERCs affect the aging of any organism besides yeast, these results bode well for understanding how calorie restriction works in higher organisms. And the finding that calorie restriction and SIR2 operate through genetically distinct pathways in yeast, the authors conclude, suggests that certain aspects of both pathways might have been conserved through evolution. Working out the details of these pathways in yeast is the first step toward understanding which, if any, of these components might enhance longevity in humans. Of course, as any student of Greek mythology knows, longevity without eternal youth comes with a price.

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PMC514492

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2021-01-05 08:21:13

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PLoS Biol. 2004 Sep 24; 2(9):e308

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PLoS Biol

10.1371/journal.pbio.0020308

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020318SynopsisCell BiologyMammalsProtein Helps Orchestrate Cells' Fluid Uptake Synopsis9 2004 24 8 2004 24 8 2004 2 9 e318Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. The Rab5 Effector Rabankyrin-5 Regulates and Coordinates Different Endocytic Mechanisms ==== Body You are what you eat and drink. Steak can sit in your stomach or orange juice wind through your intestines, but they only become part of your body once they're taken up by your cells. First, foods must be reduced to a soup of proteins, fats, sugars, and so on. But even then, getting these materials into a cell isn't as simple as sticking them in your mouth. For one, there's the membrane enclosing a cell. Simply puncturing a hole in the membrane would spill the cell's contents, harming or killing the cell. Instead, all eukaryotes—organisms whose cells have nuclei—use a carefully orchestrated process called endocytosis to bring materials into their cells. Eukaryotic cells first form cavities in their cell membrane that surround nearby particles or fluid. These pockets seal shut and bud off into the cell to form small membrane-bound sacs called vesicles. When taking in fluids, eukaryotic cells use two distinct mechanisms—to take tiny sips or huge gulps. With one process, called pinocytosis, cells continually form small pockets in the cell membrane that enclose small droplets of fluid in vesicles called pinosomes. These newly formed vesicles, called early endosomes, bud off from the membrane and fuse with other early endosomes. In one form of pinocytosis, the vesicles are encaged by a protein called clathrin that tightly constrains their size. These carriers incorporate membrane constituents (for example, growth factors) with very high selectivity. In macropinocytosis, on the other hand, large ruffles in the membrane engulf mass quantities of fluid in vesicles known as macropinosomes. Beyond taking in nutrients, these processes are essential to the function of many organs—from the brain, where nerve cells receive other cells' chemical signals by pinocytosis, to the kidney, where cells use macropinocytosis to take in waste fluids for processing. Macropinocytosis is also relevant to cancer cells; it has long been known that oncogenes dramatically induce this endocytic process, affecting the signaling status of these cells. But compared with other types of endocytosis, molecular biologists know surprisingly little of the mechanisms behind macropinocytosis. They do know that the Rab5 protein—an enzyme that coordinates a complex network of other proteins, called effectors—is crucial for both pinocytosis and macropinocytosis. Now, as reported in this issue of PLoS Biology, Marino Zerial and colleagues have found a new protein, which they named Rabankyrin-5, that forms a further link between these two mechanisms for fluid uptake. The protein is necessary for macropinocytosis, and its levels control the rate of this process. In addition, Rabankyrin-5 helps regulate endosome trafficking and coordinates this mechanism with macropinocytosis. In two commonly used human and mouse cell lines, the researchers found the protein Rabankyrin-5 along with Rab5 on both types of pinosomes, early endosomes and macropinosomes. The early endosomes usually fuse with one another inside the cell, but when the researchers blocked Rabankyrin-5 activity, this fusion fell sharply. Suppressing Rabankyrin-5 activity also stifled macropinocytosis; overexpressing the effector, on the other hand, sent macropinocytosis into overdrive. The researchers also looked at endocytosis in mouse kidney and canine kidney cell lines. Inside the kidney, fluid-carrying ducts are lined with epithelial cells that take up liquids through their exposed surface. The researchers found Rabankyrin-5 predominately on vesicles at this surface, and as in the other experiments, overexpression of the protein promoted macropinocytosis. Together, these findings suggest Rabankyrin-5 plays a role in regulating this form of fluid uptake and plays a role in kidney function. The discovery of Rabankyrin-5 involvement in macropinocytosis also has implications for other physiological and pathological mechanisms such as the immune system response, defense against pathogens, and hyperactivation of signaling pathways in cancer cells. Rabankyrin-5 contains various regions that bind other proteins and also lipids found in cell membranes, suggesting the protein plays a mechanical role in forming vesicles. The protein also has regions found on other proteins that are involved in signaling and development, so it may help direct vesicles' traffic within the cell. The protein also has regions characteristic of proteins involved in clathrin-dependent endocytosis, which fits with the researchers' finding that Rabankyrin-5 affects pinocytosis. Rabankyrin-5 (green) colocalizes with rhodamine-conjugated EGF on macropinosomes after growth factor stimulation All told, Rabankyrin-5 appears to form a bridge between two distinct mechanisms, pinocytosis and macropinocytosis, that cells use to take in fluids. While the details of how Rabankyrin-5 functions are still unclear, these findings give researchers a new handle for grasping how macropinocytosis works and how cells control when and how much they drink in their surroundings.

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PMC514493

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2021-01-05 08:21:19

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PLoS Biol. 2004 Sep 24; 2(9):e318

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PLoS Biol

10.1371/journal.pbio.0020318

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020324SynopsisEcologyEvolutionZoologyInsectsPolicing Relative Conflicts of Interest in Social Insects Synopsis9 2004 24 8 2004 24 8 2004 2 9 e324Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Conflict over Male Parentage in Social Insects Workers of social insects prevent other workers laying eggs to increase colony efficiency and not -- as traditionally thought - purely because workers are more related to the queen of the colony ==== Body The order and harmony that appears to bless the lives of many social insects, from ants to bees, has long fascinated naturalists. That individual workers seem to routinely sacrifice their own (reproductive) interests for the good of the colony has also piqued the interest of philosophers and kings, for obvious reasons. But scratch the surface and that blissful harmony reveals a complex feat of social engineering that is both exquisitely organized and potentially ruthless. One of the altruistic behaviors that social insects are famous for is that one or a few queens perform most or all of the reproduction in a colony, while workers are, for the most part, non-reproductive. The evolution of this social structure partly stems from the unusual sex determination system of social insects, in which unfertilized eggs (of either workers or queens) develop into males and fertilized eggs (produced only by queens) develop into female queens and workers. This creates unusual relationships between family members that affect how W. D. Hamilton's theory of “kin selection” operates in these species. Kin selection, as elegantly summarized by “Hamilton's Rule,” predicts that the altruistic behavior of workers—that is, investing in the reproduction of others in the colony rather than in their own reproduction—can evolve if the indirect reproductive payoff to workers (i.e., via reproduction by relatives) is higher than the cost of the missed opportunity for direct reproduction. Kin selection revolves around relatedness because relatedness determines the magnitude of indirect reproductive payoffs. However, based on a survey of 50 species of ants, wasps, and bees, Rob Hammond and Laurent Keller now demonstrate that the behavior in the colony cannot be accounted for simply based on relatedness patterns, but that it is necessary to consider how colony efficiency influences behavior. In some social insect colonies, workers do lay eggs, in a sense “cheating” on the other workers who are investing in the queen's reproduction rather than in their own. Such action can be severely penalized by other workers, who aggressively police the colony for the illicit offspring—or behavior—of their guilty colleagues. In honeybees, where this behavior was first shown, workers remove worker-laid eggs within hours by eating them, and, in some ants, more draconian methods lead to the mutilation of the culprit caught in the act of laying. Conflict between ants (Photo: Christian König, www.konig-photo.com) Why workers police worker reproduction in some colonies and not in others can also be influenced by relatedness. If a queen is monogamous and mates only once, then each worker will actually be more related to her nephew (produced by a sister worker) than to her brother (produced by the queen); in this case, workers should tolerate other workers' male offspring. But if the queen mates more than twice (as in honeybees) or if there are multiple queens heading a colony, then the relationship between workers becomes diluted (they do not all have the same father), and workers are more closely related to brothers than to nephews. In this case, workers should clamp down hard on any worker breeding and raise only the queen's sons (in addition to her daughters). But workers policing the reproduction of their fellow workers could also be advantageous if the energy invested by workers into laying eggs—which would otherwise be used in foraging and legitimate brood rearing—detracts from the overall efficiency and growth of the colony. Although there is some evidence for this “efficiency hypothesis,” it is widely accepted that the driving force behind policing is primarily explained by patterns of relatedness. By doing a detailed comparative phylogenetic analysis of different species, Hammond and Keller put the “relatedness hypothesis” to the test and—contrary to expectations—found evidence that this genetic incentive for workers to police the reproduction of other workers cannot account for its widespread prevalence among social insects. One prediction from the relatedness hypothesis is that the extent to which workers produce male offspring is determined by the relatedness of the workers. By contrast, the efficiency hypothesis predicts no such relationship. In line with this, Hammond and Keller's survey reveals that no matter how related workers are to each other, most males across this broad range of species are produced by queens. In other words, worker-policing does not depend on relatedness, so other factors—such as colony efficiency—must act as an important constraint on worker reproduction. This, Hammond and Keller emphasize, does not amount to showing that kin selection is unimportant—but it does mean that the harmony and regulation of reproduction in social insects is much more complex than expected from simple theoretical expectations based solely on relatedness.

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PMC514494

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2021-01-05 08:21:14

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PLoS Biol. 2004 Sep 24; 2(9):e324

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PLoS Biol

10.1371/journal.pbio.0020324

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020331SynopsisBiotechnologyGenetics/Genomics/Gene TherapyPlant SciencePlantsNatural Biodiversity Breaks Plant Yield Barriers Synopsis10 2004 24 8 2004 24 8 2004 2 10 e331Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Unused Natural Variation Can Lift Yield Barriers in Plant Breeding ==== Body The birth of agriculture, some 10,000 years ago in the Middle East's Fertile Crescent, revolutionized human culture and society. Refined farming techniques led to increased yields and freed humans from the demands of constant foraging. Along with that freedom came social complexity, division of labor, improved standards of living, and a measure of leisure time. Agriculture also led to overpopulation followed by starvation, conflict over fertile farming land, and environmental damage. For the Maya and other civilizations, such consequences proved fatal. Many consumer and environmental groups believe that modern industrial agricultural practices like factory farming of animals and genetic engineering of crops threaten to bring similar ruin. But with 6 billion people living on the planet—a figure that's expected to increase 50% in just 50 years—many plant scientists believe that feeding a burgeoning population will require the tools of biotechnology. Plant breeders face the daunting challenge of developing high-yielding, nutritious crops that will improve the global quality of life without harming the environment or appropriating dwindling natural habitats for agricultural production. A major roadblock to feeding the world is a continuing decline in the genetic diversity of agricultural crops, which has in turn limited their yield improvement. (Domestication often involves inbreeding, which by definition restricts the gene pool.) Now Amit Gur and Dani Zamir of Hebrew University report a way to lift these productivity barriers by tapping into the natural diversity of wild plants. Traditional plant breeders improve the quality and yield of crops by crossing plants with desired traits to create a new, hopefully improved, hybrid strain. But traditional breeding is limited by the available gene pool of a cultivated plant species and eventually hits a wall—reshuffling the same genetic variation can boost yield only so much. With the advent of biotechnology, plant scientists were buoyed by the prospect of improving plants through genetic modification. But aside from a few successes with introducing single-gene herbicide- and pest-resistant traits, most plant traits have proved too complex to repay the incorporation of a single transgene—that is, a gene taken from a different species—with the hoped-for response. Biotech-based investigations and applications in plant science have also been hampered by consumer reaction against genetically modified organisms. (For more on the techniques of modern plant breeding, see the essay “Diversifying Selection in Plant Breeding,” also in this issue.) Faced with these limitations, Gur and Zamir tried another approach—a back-to-nature approach. “Natural biodiversity is an unexploited sustainable resource that can enrich the genetic basis of cultivated plants,” they explain in the report. The distantly related wild cousins of cultivated plants can be seen as a “huge natural mutagenesis resource” with novel gene variants that can increase productivity, quality, and adaptability. Not only that, the genetic material of wild plants—every gene and regulatory element—has already been refined and tested by over a billion years of evolution and natural selection. To identify genomic regions in wild tomato species that affect yield, Gur and Zamir created a population of hybrid crosses of a wild tomato species and a cultivated tomato species; each line had a single genomic region from the wild tomato inserted into the cultivated plant. Rather than introducing a single wild tomato gene into the cultivated plants, the authors used a “pyramided” strategy that combined three independent yield-enhancing genomic regions from the wild species into the new plant line. Plants were grown over three seasons, during which they were exposed to different environments, including drought. By combining traditional phenotyping techniques—which characterize the plant's physical traits based on its genetic makeup—with genetic marker analysis, the authors identified a number of wild tomato genomic regions that increased yield. Their results demonstrate that an approach based on biodiversity—which takes advantage of the rich genetic variation inherent in wild relatives of cultivated crops—can produce varieties that outperform a commercially available hybrid tomato in both yield and drought resistance. Gur and Zamir attribute the improved performance to their unique pyramiding strategy. Their hybrid model—applying the tools of modern genomics to traditional plant breeding—offers plant breeders a powerful approach to improving the quality and yield of cultivated plants by taking advantage of the inherent biodiversity of the natural world. It's a strategy that may well apply to rice, wheat, and other vital staples of the world's food supply.

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PLoS Biol. 2004 Oct 24; 2(10):e331

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10.1371/journal.pbio.0020331

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==== Front Malar JMalaria Journal1475-2875BioMed Central London 1475-2875-3-261528203010.1186/1475-2875-3-26ResearchMalaria morbidity and immunity among residents of villages with different Plasmodium falciparum transmission intensity in North-Eastern Tanzania Lusingu John PA [email protected] Lasse S [email protected] Bruno P [email protected] Chris J [email protected] Caroline [email protected] Juma [email protected] Zacharia X [email protected] Andrew Y [email protected] Martha M [email protected] Thor G [email protected] National Institute for Medical Research, Amani Medical Research Centre, Amani & NIMR Headquarters, Dar es Salaam, Tanzania2 Centre for Medical Parasitology at Institute of Medical Microbiology and Immunology, University of Copenhagen, and Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark3 Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK2004 28 7 2004 3 26 26 16 4 2004 28 7 2004 Copyright © 2004 Lusingu et al; licensee BioMed Central Ltd.2004Lusingu et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The relationship between the burden of uncomplicated malaria and transmission intensity is unclear and a better understanding of this relationship is important for the implementation of intervention programmes. Methods A 6-month longitudinal study monitoring risk factors for anaemia and febrile malaria episodes was conducted among individuals aged below 20 years, residing in three villages of different altitude in areas of high, moderate and low malaria transmission intensity in North-Eastern Tanzania. Results The burden of anaemia and malarial fever fell mainly on the youngest children and was highest in the village with high transmission intensity. Although a considerable percentage of individuals in all villages carried intestinal worms, logistic regression models indicated that Plasmodium falciparum was the only significant parasitic determinant of anaemia. Interestingly, children who carried low-density parasitaemia at the start of the study had a lower risk of contracting a febrile malaria episode but a higher risk of anaemia during the study period, than children who were slide negative at this point in time. Conclusion Young children living in the high transmission village carried a very high anaemia burden, which could be attributed to malaria. The overall incidence of febrile malaria was also highest in the high transmission village particularly among those under five years of age. These data suggest that in rolling back malaria, available resources in prevention programmes should primarily be focussed on young children, particularly those residing in areas of high malaria transmission. ==== Body Background Plasmodium falciparum malaria remains an important public health problem in sub-Saharan Africa. To develop and assess the efficacy of control measures, it is important to obtain a better understanding of how the malaria disease burden is distributed among population groups and how this burden is affected by changes in malaria transmission intensity [1]. In areas of high malaria transmission infants and young children carry a very high disease burden [2], but protective immunity is developed in early childhood. Adults and older children are able to control parasitaemia and therefore only rarely suffer from mild malaria symptoms [3,4]. In areas of low malaria transmission, immunity develops slowly and malaria affect all age groups [5,6]. It has been suggested that the societal burden of malaria does not necessarily increase with transmission intensity, but peaks at a certain level of transmission after which it remains constant and may even decrease [7-9]. To address this issue we have compared the malaria situation in three communities situated North-Eastern Tanzania, which show differences in transmission intensity. In this area, transmission intensity is determined by altitude and large differences in transmission can be found within a limited geographical area [10-12]. This study reports the results of six months morbidity follow-up, during which the incidence of febrile malaria episodes and the prevalence of anaemia were assessed in cohorts of 0–19 year old individuals. Methods Study area The study was conducted in three villages in Tanga region, North-Eastern Tanzania. The three villages were Mgome (5°12'S, 38'51'E) at an altitude of approximately 200 meters, Ubiri (4°72'S, 38°29'E) at an altitude of approximately 1,200 meters, and Magamba (4°75'S, 38°29'E) at an altitude of approximately 1,700 meters. The climate in the area is characterized by variations in rainfall and temperature related both to season and altitude [12]. The long rainy period occurs during April-May, while short rains occur in November-December. Mean daily temperatures are highest in January and lowest in July. Generally, the malaria transmission season peaks just after the rainy seasons with most consistent transmission in lowland sites from April to July. Previous studies have reported parasite prevalence rates to be in the ranges of 79–90% in the lowlands, 27–46% at intermediate altitudes and 8–16% in the highlands [10]. Entomological surveys in the study areas have shown that Anopheles gambiae is the most prevalent vector in the lowlands, while Anopheles funestus predominates in the highlands [10]. The entomological inoculation rates (EIR) have been reported to be in the range between 91–405 in the lowlands, and between 1.8–34 at intermediate altitudes [10]. In the highlands, mosquito densities are too low to allow reliable EIR measurements, but an EIR of 0.03 has been extrapolated [10]. Villagers living at low and intermediate altitudes perceive malaria as a major problem among both children and adults, but at the highest altitudes villagers consider that malaria is not a major part of the disease burden in either adults or children. There is little difference in treatment seeking behaviour for febrile illness between the altitudes. Treatment is generally sought for symptoms rather than for the disease and first treatment is almost universally an anti-pyretic drug bought from local shops (Caroline Jones, unpublished data). For all three villages, the nearest health facility is located within a distance of 13 km. Mgome is served by Umba Dispensary (10 km), Masaika Dispensary (5 km), Mkuzi Health Centre (7 km) and Muheza Designated District Hospital (14 km). Ubiri village is served by Lushoto District Hospital at a distance of approximately 13 km. Magamba village has a government and a private missionary dispensary both within the village, and is served also by Lushoto District Hospital at a distance of about 15 km. At the time of the study, sulphadoxine-pyrimethamine (SP) was the first-line treatment for uncomplicated malaria in Tanzania. It has been documented that the level of SP resistance is high in the Mgome area [13], whereas the situation has not been monitored previously in Ubiri and Magamba. Land use in the lowland areas is characterized by subsistence farming of maize, rice, bananas, beans, cassava, coconuts, fruits and other crops, as well as large-scale production of sisal. In the highlands, there is subsistence farming, mainly of maize, beans, bananas, potatoes, cabbages, tomatoes and fruits, and also large-scale production of tea and coffee. Study population Prior to the study, census surveys were done in each village and study individuals randomly selected from a census list. Mgome village is inhabited mainly by the Bondei tribe (60%), while Ubiri and Magamba are inhabited by Sambaa at 97% and 57%, respectively. The aim was to recruit a total of 250 individuals below the age of twenty years from each village, distributed in different age groups as follows: 0–1 year: n = 25, 1 year: n = 25, 2 years: n = 25 3 years: n = 25, 4 years: n = 25, 5–6 years: n = 25, 7–9 years: n = 25, 10–14 years: n = 40 and 15–19 years: n = 40. Cross-sectional surveys Malariometric surveys were conducted in each village in April, July and September 2001. During the first survey, the purpose of the study was explained and consent to participate obtained from each study individual or their parents/guardians. Baseline demographic data were collected together with a history of migration and recent movements. The use of malaria preventive measures was also recorded. A history of recent illness was obtained, emphasizing symptoms suggestive of malaria. Physical examination on signs related to malaria such as temperature, pulse, spleen size, pallor and respiratory rate was conducted. Axillary temperature was measured using digital thermometers. Height, weight and upper-arm-circumference were recorded for estimation of nutritional status. For any individual diagnosed with mild disease, appropriate drugs were administered in the field. Individuals with symptoms of malaria were treated with SP. Participants with severe disease were referred to the nearby hospital. Five millilitres of venous blood were collected from study individuals aged three years and above into vacutainer tubes containing citrate buffer. For children below three years, 300–400 μl of capillary blood from a fingerprick were collected into eppendorf tubes containing EDTA. The haemoglobin (Hb) of each participant was measured from drops of blood using a HemoCue® photometer (Ångelholm, Sweden). Whole blood was used to prepare thick and thin blood smears for malarial microscopy. These were stained with 10% Giemsa stain for 15–20 minutes after fixing thin smears with methanol. Asexual and sexual parasites were counted against 200 and 500 white blood cells, respectively. The differentiation of malaria parasite species was confirmed by microscopy of thin smears. A blood smear was declared negative only after examination of 200 high power fields. The density of asexual parasites was calculated assuming 8000 leucocytes per μl of blood and expressed as parasites per μl. During the first cross-sectional survey, study participants were asked to collect stool and urine specimens in special containers. Direct smear-technique was used to check for the presence of hookworm ova and other intestinal parasites. A pinhead of stool was collected, put on a slide and emulsified in a drop of normal saline. A cover slip was then applied and the slide examined using low-power microscopy. Longitudinal monitoring of febrile episodes Local village helpers (two community members per village) and health workers at nearby health facilities performed passive case detection during the 6-month study period. The village helpers were provided with first-line antimalarial drugs (SP), paracetamol, slides, blood lancets, treatment charts, febrile case detection forms and storage boxes. Villagers could seek treatment at any time from these helpers. Patients with symptoms of malaria were treated with first-line antimalarial drugs or, if they had severe symptoms or did not respond adequately to the first-line treatment, they were referred to a health facility. Prior to treatment the village helpers collected clinical information and a malaria blood smear. At each nearby health facility, two permanent staff members monitored study participants seeking medical treatment at the facility. If a study participant presented at the facility with history of fever and/or an axillary temperature ≥ 37.5°C, a form was completed and a blood smear collected. Once per month active febrile case detection was undertaken by the research team. During active case detection, each study participant was seen by a trained physician and a blood smear was taken from any study participant reporting a history of fever within two days and/or those who had an axillary temperature ≥ 37.5°C Case definitions Anaemia was defined as haemoglobin < 11.0 g/dl [14,15]. To adjust for the physiological effect of altitude on haemoglobin concentration, a correction factor was calculted with haemoglobin values being normalized to sea level for direct comparison between the study villages. The correction factor assumed a linear relationship between increasing altitude and haemoglobin, although the relationship may not necessarily always be exact [16]. For Mgome (200 m), the correcting factor was a reduction of 0.1 g/dl, for Ubiri (1,200 m) the factor was 0.8 g/dl and for Magamba (1,700 m) the factor was a reduction of 1.0 g/dl. Febrile malaria episodes were defined as an axillary temperature ≥ 37.5°C and /or a history of fever within the previous 48 hours in the presence of asexual P. falciparum parasites above a defined density cut-off level. Many individuals carried low density asymptomatic parasitaemia, and fever among parasitaemic individuals may also have been caused by other illness [17]. Thus, to account for the variation in levels and point prevalence of asymptomatic parasitaemia between study villages [18–20], as well as the different age groups involved in the study [21], different P. falciparum density cut off levels were applied in each village. To balance between sensitivity and specificity in diagnosing a febrile malaria episode, we aimed at a febrile malaria case specificity >80%. In Magamba (the low transmission village), a cut-off of 40 parasites/μl was applied, while cut-offs of 1000 parasites/μl and 5000 parasites/μl were used in Ubiri (the moderate transmission village), and Mgome (the high transmission village), respectively. Age-specific incidence rates of febrile malaria episodes were calculated as the number of episodes divided by the number of days that individuals in the age group were at risk during the follow-up. After a febrile malaria episode an individual was censored for 28 days [6]. The effect of using different parasite density cut-offs in the definition of a febrile episode was evaluated by not applying a cut-off in the definition or by applying age specific cut-offs [21]. Statistical methods All data were double-entered into a database in Epi-lnfo Version 6.04d (CDC, Atlanta, USA) and statistical analyses were performed with Stata version 8 (Stata Corporation, Texas, USA). Univariate analyses and multivariate logistic regression were performed to determine risk factors for anaemia and febrile malaria episodes. For Mgome village, a logistic regression model was developed to determine whether the result of the first slide reading in April could be used to predict the subsequent risk of developing anaemia or febrile malaria during the following six months of morbidity surveillance. In this model, P. falciparum parasitaemia was categorised as no parasitaemia if no parasites were detected microscopically, low-density if parasitaemia was between 40 parasites/μl and 4999 parasites/μl, and high-density if the level was above or equal to 5000 parasites/μl. Thus, the first slide reading of individuals who did not have fever/had normal haemoglobin levels at enrolment was used to predict the risk of developing a subsequent episode of malarial fever/anaemia. Ethical considerations Ethical clearance was granted by the Medical Research Co-ordinating Committee of the National Institute for Medical Research, Tanzania. Prior to the study, meetings were held with local authorities and with the villagers in each study village, during which the aims of the study were explained. Informed consent documents for the study were prepared in English and translated into Kiswahili before administration to both village leaders and participants. Written informed consent to participate was obtained from each study individual or from his/her parents or guardians. Study individuals were free to withdraw from the study at any time without giving any reasons, or being disqualified from any medical services that were provided to all villagers throughout the study period. At the end of the study, preliminary findings were presented at village meetings. Results Prevalence and densities of Plasmodium species and other infections Three study villages were selected to represent areas of markedly different malaria transmission intensity. In each village, approximately 250 individuals under the age of 20 years were recruited. Few individuals reported using anti-malarial preventive measures (Table 1). Repeat investigations on the same individuals were undertaken at enrolment in April 2001, and during subsequent cross-sectional surveys in July and September 2001. Only about 10% of the study participants were lost to follow-up in each village. Table 1 Baseline characteristics of the study villages Baseline characteristic Mgome Ubiri Magamba Altitude (m) [range] 196 [165, 208] 1216 [1174, 1262] 1585 [1659, 1751] Enrolled (0–19 years) (Male/Female) 254 (115/139) 250 (139/111) 255 (132/123) Use of preventive measures Nets (%) 18/254 (7.1) 5/250 (2.0) 14/255 (5.5) Burning coils (%) 8/254 (3.1) 1/250 (0.4) 0/255 (0) Neem (%) 0/254 (0) 1/250 (0.4) 0/255 (0) Spray (%) 0/254 (0) 0/250 (0) 3/255(1.2) Prophylaxis (%) 0/254 (0) 1/250 (0.4) 1/255 (0.4) As expected, P. falciparum prevalence and parasite densities (Figure 1) were higher in Mgome than in Ubiri and Magamba (trend test, z = 15.64, p < 0.001). In Mgome, the carrier rate was particularly high for children aged 1–9 years and then declined in the older age groups (trend test, z = -3.2, p < 0.001). In Ubiri, carrier rates were low in infants, peaked at the age of two years, but showed little variation in the age groups between 4 and 19 (Figure 1). Although carrier rates in Ubiri were slightly higher in April than in July and September, there were no marked seasonal changes in carrier rate by age in any of the villages. The parasite densities in those carrying parasites did not differ between villages after the age of six years. Among the under fives, children from Mgome and Ubiri carried higher levels of parasitaemia in July than in April and September 2001 surveys. Interestingly, between April and July surveys, there was a marked difference in the age-specific pattern of parasite density in Mgome. In April, the peak parasite density was noted in age group of 2 years, whereas the youngest had the highest parasite density in July surveys. Based on these findings, we categorised Mgome as a high transmission (holoendemic) village, Ubiri as a moderate transmission (mesoendemic) village, and Magamba as low transmission (hypoendemic) village. P. falciparum was the most predominant malarial parasite accounting for more than 95% of all malaria infections. The other malaria species were mainly found as mixed infections. The April prevalence rates of Plasmodium malariae in Mgome and Ubiri were 8.3 % and 3.9%, respectively, while these rates for Plasmodium ovale was 1.0% and 0%. In Magamba, only P. falciparum was found. Figure 1 Age-specific P. falciparum prevalence and geometric mean densities (positives only) by village and season. Panels A, B, and C show age-specific P. falciparum densities and prevalence in Magamba (low transmission), Ubiri (moderate transmission) and Mgome (high transmission), respectively. Lines indicate the prevalence rate for each survey. Solid lines with filled circle for April 2001, dotted lines with filled triangle for July 2001, and dashed lines with filled box for September 2001. Bars indicate P. falciparum densities (positives only) for each survey. Empty bars indicate the April 2001 surveys, hatched bars indicate the July 2001 surveys, and crossed hatched bars indicate the September 2001 surveys. Error bars indicate 95% confidence interval. A total of 492 individuals from the three villages submitted stool and urine samples, which were investigated for worms. Worms were found in 35.0% of study participants living in Mgome, and in 29.2% and 7.8% of individuals from Ubiri and Magamba, respectively. In Mgome, spleen enlargement was common (about 49.21%) and associated with age (Spearman rho = 0.238, p < 0.001) while in the two other villages the prevalence of splenomegaly was low and with no distinct age-pattern (data not shown). This distribution of splenomegaly remained stable during the study period. Haemoglobin levels and anaemia Haemoglobin levels were measured in all 759 individuals during enrolment and among those who reported for the subsequent cross-sectional surveys in July and September 2001. Regardless of the season, haemoglobin levels increased with both altitude and age (Figure 2). Univariate analysis indicated that age, altitude of residence, and presence of P. falciparum parasitaemia were associated with anaemia (Table 2) and this was supported by multivariate analyses in which P. falciparum was the only parasitic infection showing a statistically significant association to anaemia (Table 2). Figure 2 Age-specific anaemia prevalence and mean haemoglobin levels by village and season. Panels A, B, and C show results of surveys conducted in April, July and September 2001, respectively. The lines and symbols show patterns of anaemia prevalence for each village (Mgome: solid lines with filled circle, Ubiri: dotted lines with filled triangle, Magamba: dashed lines with filled square). Bars indicate mean altitude adjusted haemoglobin levels (g/dl) and 95% confidence intervals in each village (Mgome: empty bars, Ubiri: hatched bars, Magamba: crossed hatched bars). Table 2 Crude and adjusted odds ratios for risk factors for anaemia Explanatory variable Crude odds ratio (95% Cl) p-value Adjusted odds ratio (95% Cl) p-value Age group (years) 0–2 21.38 (8.36–54.65) <0.001 20.41 (7.44 – 56.0) <0.001 3–4 7.38 (2.82–19.25) <0.001 5.42 (1.93–15.20) <0.001 5–9 3.43 (1.36–8.66) 0.009 2.18 (0.82–5.81) 0.118 10–14 1.86 (0.70–4.99) 0.215 1.51 (0.54–4.27) 0.435 15–19 1 1 Sex Male 1.04 (0.70 – 1.54) 0.85 1.16 (0.71 – 1.90) 0.551 Female 1 1 Village Mgome 19.99 (7.08–56.42) <0.001 15.55 (4.78–50.65) <0.001 Ubiri 8.02 (2.80–23.82) <0.001 6.44 (2.08–19.92) 0.001 Magamba 1 1 Parasites P. falciparum 3.77 (2.44 – 5.83) <0.001 2.0 (1.11–3.62) 0.021 Hookworm 1.41 (0.83–2.39) 0.201 1.43 (0.76–2.69) 0.264 Ascariasis 0.86 (0.52–1.44) 0.564 1.04 (0.54–1.92) 0.952 Amoeba 0.16 (0.02–1.20) 0.074 0.16 (0.02–1347) 0.091 Schistosoma 0.42 (0.12–1.46) 0.174 0.28 (0.07–1.20) 0.086 Malaria morbidity during follow-up Of the 759 individuals enrolled in the study, 669 (88%) adhered to the follow-up scheme and were included in the analysis of febrile episodes. Loss to follow-up was due to death (three individuals) or emigration. Using the village-specific density cut-off described above, 54 individuals had febrile malaria episodes in Mgome, 10 in Ubiri and none in Magamba during the six-month follow-up period. The mean age of febrile malaria individuals was 1.97 years (95% Cl: 1.50, 2.59) and 3.23 years (95% Cl: 1.59, 5.86) for Mgome and Ubiri, respectively. Children below five years carried the major burden of febrile malaria episodes in Mgome (Figure 3, panel A). The data was also analysed using age-specific parasite cutoffs [21] in the case definition (data not shown) and using a definition in which all fevers accompanied by a positive slide were considered a febrile malaria episode (Figure 3, panel B). The latter definition increased the incidence rates in Mgome and Ubiri slightly, but the overall conclusion that the incidence rates were by far the highest in the children under five years living in Mgome was not affected by the case definition used. Figure 3 Incidence rates of febrile malaria episodes by age group. Panel A: Incidence rates calculated using village specific parasite density cut-offs in the definition of episodes. In Mgome (solid lines with filled circle) the cut-off was 5000 parasites per μl, in Ubiri (dotted lines with filled triangle) 1000 parasites per μl, and in Magamba (dashed lines with filled square) 40 parasites per μl. Panel B: Incidence rates calculated using a definition in which all fevers accompanied by positive slide were considered a febrile malaria episode. In Mgome, host age and the presence of low-density parasitaemia at the start of the study were consistently found to be associated with decreased risk of suffering a febrile malaria episode during the morbidity follow-up. Other variables such as sex, splenomegaly and use of a mosquito net did not contribute significantly to the model. In logistic regression models correcting for age, those who carried parasites at low densities in April had a four-fold lower risk (P < 0.03) of developing a febrile malaria episode during follow-up than those who were slide negative (Table 3). Table 3 Logistic regression model showing the risk of developing a febrile malaria episode during the 6 month morbidity surveillance according to age and the result of the slide at the initiation of the study in Mgome Explanatory variable Crude odds ratio (95% Cl) p-value Adjusted odds ratio (95% Cl) p-value Low parasite density1 0.34 (0.15–0.78) 0.011 0.22 (0.06–0.89) 0.033 High parasite density2 3.0 (1.09–8.29) 0.034 1.36 (0.27–8.82) 0.706 No parasitaemia3 1 1 Age (years) 0.66 (0.58 – 0.76) <0.001 0.51 (0.332 – 0.772) 0.002 Age squared 0.98 (0.97 – 0.998) 0.007 1.03 (1.007–1.047) 0.007 1Parasitaemia in April between 40 and 4999 parasites/μl 2Parasitaemia in April >4999 parasites/μl 3Slide negative in April In Mgome, 112 of the 254 individuals were not anaemic on enrolment in April 2001. Out of these, 68 (mean age (years) and 95% Cl: 11.2 [10.2, 12.3]) had normal haemoglobin levels during the July and September cross sectional surveys, while 44 developed anaemia during the study (mean age (years) and 95% Cl: 7.9 [6.4, 9.5]). Logistic regression models correcting for age showed that the risk of developing anaemia during the study was 4.4 times (p = 0.038) higher in individuals carrying low-density parasitaemia in April than in those who were slide negative (Table 4). Table 4 Logistic regression model showing the risk of developing anaemia during the 6 month morbidity surveillance according to age and the result of the slide at the initiation of the study in Mgome Explanatory variable1 Crude odds ratio (95% Cl) p-value Adjusted odds ratio (95% Cl) p-value Low parasite density 3.98 (1.276–13.095) 0.02 4.38 (1.10–17.69) 0.038 High parasite density 3.17 (0.601–16.692) 0.17 2.43 (0.35–16.73) 0.369 No parasitaemia 1 1 Age (years) 0.86 (0.789 – 0.94) 0.001 0.51 (0.332–0.772) 0.002 Age squared 0.99 (0.99 – 0.998) 0.007 1.03 (1.007–1.047) 0.007 1Refer to table 3 Discussion This prospective longitudinal study was designed to compare the burden of uncomplicated malaria in three similar villages situated in areas of markedly different transmission intensity. Not surprisingly, the study showed that individuals living in the village with very high malaria transmission carried a markedly higher burden of both anaemia and febrile malaria episodes compared to villagers at the sites with lower transmission. This result is in agreement with results from previous studies in the area [11]. The villages in the highlands are prone to malaria epidemics [12], but such epidemics did not occur during the study period. If they had, it is conceivable that the incidence of febrile malaria episodes at these sites would have reached or even exceeded the incidence found in the high transmission village [6,9]. Never the less, the anaemia burden in the high transmission village was very high among infants and young children. The burden among these children was much greater than among individuals of the corresponding age groups in the other two villages. During the first survey, villagers were also investigated for the presence of parasites in urine and faeces, but neither of these was shown to be a significant risk factor for anaemia in the multivariate logistic regression models (Table 2). Thus, the difference in anaemia burden between the sites appears likely to have been due to differences in malaria transmission intensity. The reason that hookworm infection did not constitute a risk factor for anaemia is likely to be a consequence of the fact that the villagers receive regular deworming medication as part of health promotion programmes, and therefore, the worm burden was rather low [22]. The heavy burden of anaemia carried in populations exposed to high malaria transmission has recently been highlighted [14]. The results of this study support the findings that malaria plays a major role in the burden of anaemia and these results are further corroborated by the fact that malaria interventions such as insecticide treated nets and intermittent preventive treatment in infants (IPTi) considerably reduce the incidence of anaemia [23-25]. From a public health perspective, our results reinforce the view that malaria prevention programmes should focus their attention on high-transmission areas and concentrate particularly on children under five years of age. Our study was not designed to compare incidences of severe disease and malaria deaths. It has previously been suggested that this malaria burden is in fact higher in populations exposed to moderate transmission than in populations living in areas of very high transmission [8]. The results of a large hospital-based study recently conducted in North-Eastern Tanzania over a wide range of transmission intensities suggested, however, that there was a positive correlation between severe malaria outcomes and intensity of transmission (Reyburn et al., submitted for publication). The longitudinal design of our study allowed an exploration of whether P. falciparum carriage at the beginning of the study influenced the risk of developing febrile malaria episodes or anaemia during the following study period. Interestingly, multivariate logistic regression models indicated that children carrying low-density parasitaemia during the first cross sectional survey were at a lower risk of developing a febrile malaria episode than children without detectable parasitaemia or children with higher levels of parasitaemia. This apparent protective effect of low-grade parasitaemia was recently also reported in a study from Ghana [26], but in our study this protection came with a price since children who controlled the parasite density at low levels were at markedly higher risk of developing anaemia. Conclusions The overall burden of malaria morbidity was found to be highest in the high-transmission area, where infants and children carried a very high malaria burden in the form of febrile episodes and anaemia. Populations in the areas of moderate and low transmission suffered a significantly lower morbidity. Therefore, in order to roll back malaria, available resources in malaria control programmes should focus on underfives residing in the high-transmission areas. Authors' contributions JPAL and LSV participated in the planning of the study, carried out field surveys, analysed the data and drafted the manuscript. BPM participated in designing the study, carried out field surveys and managed the data. CJD participated in study planning, in the fieldwork and in editing of the manuscript. CJ participated in the planning of the study and conducted the socio-economic analysis of study villages. JA and ZXS participated in the field surveys and performed microscopy of all blood smears. AYK, MML and TGT participated in study planning, coordination, and analysis of data. All authors participated in the paper writing and approved the final manuscript. Acknowledgements All study participants including their parents or guardians, as well as village helpers and health management teams in Tanga region are highly acknowledged. Anne Corfitz, Magreth Hamisi, Fabio-Avit Massawe, John Hiza, William Chambo, Donald Mwanjeluka and Seth Nguhu are thanked for excellent technical assistance throughout the study. Dr. Thomas Scheicke is thanked for statistical advice. The study was conducted under the auspices of the Joint Malaria Programme, a collaborative research initiative between Centre for Medical Parasitology at the University of Copenhagen and Copenhagen University Hospital, Kilimanjaro Christian Medical College, London School of Hygiene and Tropical Medicine and the Tanzania National Institute for Medical Research. JPAL is a PhD scholar under the Gates Malaria Partnership. The field study was funded by the ENRECA programme of the Danish International Development Agency (DANIDA). ==== Refs Ellman R Maxwell C Finch R Shayo D Malaria and anaemia at different altitudes in the Muheza district of Tanzania: childhood morbidity in relation to level of exposure to infection Ann Trop Med Parasitol 1998 92 741 753 9924532 10.1080/00034989858989 Kitua AY Smith T Alonso PL Masanja H Urassa H Menendez C Kimario J Tanner M Plasmodium falciparum malaria in the first year of life in an area of intense and perennial transmission Trop Med Int Health 1996 1 475 484 8765455 10.1046/j.1365-3156.1996.d01-89.x Christophers SR The mechanism of immunity against malaria in communities living under hyper-endemic conditions Indian J Med Res 1924 12 273 294 Bruce-Chwatt LJ A longitudinal survey of natural malaria infection in a group of West African adults W Afr Med J 1963 12 141 173 14052649 MacDonald G The epidemiology and control of malaria 1957 London: Oxford University Press Giha HA Rosthoj S Dodoo D Hviid L Satti GMH Scheike T Arnot DE Theander TG The epidemiology of febrile malaria episodes in an area of unstable and seasonal transmission Trans R Soc Trop Med Hyg 2000 94 645 651 11198648 Snow RW Omumbo JA Lowe B Molyneux CS Obiero JO Palmer A Weber MW Pinder M Nahlen B Obonyo C Newbold C Gupta S Marsh K Relation between severe malaria morbidity in children and level of Plasmodium falciparum transmission in Africa Lancet 1997 349 1650 1654 9186382 10.1016/S0140-6736(97)02038-2 Snow RW Bastos de Azevedo I Lowe BS Kabiru EW Nevill CG Mwankusye S Kassiga G Marsh K Teuscher T Severe childhood malaria in two areas of markedly different falciparum transmission in East Africa Acta Trop 1994 57 289 300 7810385 10.1016/0001-706X(94)90074-4 Rogier C Commenges D Trape JF Evidence for an age-dependent pyrogenic threshold of Plasmodium falciparum parasitemia in highly endemic populations Am J Trop Med Hyg 1996 54 613 619 8686780 Bodker R Akida J Shayo D Kisinza W Msangeni HA Pedersen EM Lindsay SW Relationship between altitude and intensity of malaria transmission in the Usambara Mountains, Tanzania J Med Entomol 2003 40 706 717 14596287 Maxwell CA Chambo W Mwaimu M Magogo F Carneiro IA Curtis CF Variation of malaria transmission and morbidity with altitude in Tanzania and with introduction of alphacypermethrin treated nets Malar J 2003 2 28 14585106 10.1186/1475-2875-2-28 Balls MJ Bodker R Thomas CJ Kisinza W Msangeni HA Lindsay SW Effect of topography on the risk of malaria infection in the Usambara Mountains, Tanzania Trans R Soc Trop Med Hyg 2004 98 400 408 15138076 10.1016/j.trstmh.2003.11.005 Alifrangis M Lemnge MM Ronn AM Segeja MD Magesa SM Khalil IF Bygbjerg IC Increasing prevalence of wildtypes in the dihydrofolate reductase gene of Plasmodium falciparum in an area with high levels of sulfadoxine/pyrimethamine resistance after introduction of treated bed nets Am J Trop Med Hyg 2003 69 238 243 14628937 Schellenberg D Schellenberg JR Mushi A Savigny D Mgalula L Mbuya C Victora CG The silent burden of anaemia in Tanzanian children: a community-based study Bull World Health Organ 2003 81 581 590 14576890 Menendez C Fleming AF Alonso PL Malaria-related Anaemia Parasitology Today 2000 16 469 476 11063857 10.1016/S0169-4758(00)01774-9 Berglund B High-altitude training. Aspects of haematological adaptation Sports Med 1992 14 289 303 1439397 Smith T Hurt N Teuscher T Tanner M Is fever a good sign for clinical malaria in surveys of endemic communities Am J Trop Med Hyg 1995 52 306 310 7741165 Trape JF Peelman P Morault-Peelman B Criteria for diagnosing clinical malaria among a semi-immune population exposed to intense and perennial transmission Trans R Soc Trop Med Hyg 1985 79 435 442 3909550 10.1016/0035-9203(85)90054-9 Smith T Schellenberg JA Hayes R Attributable fraction estimates and case definitions for malaria in endemic areas Stat Med 1994 13 2345 2358 7855468 Boisier P Jambou R Raharimalala L Roux J Relationship between parasite density and fever risk in a community exposed to a low level of malaria transmission in Madagascar highlands Am J Trop Med Hyg 2002 67 137 140 12389936 Bloland PB Boriga DA Ruebush TK McCormick JB Roberts JM Oloo AJ Hawley W Lal A Nahlen B Campbell CC Longitudinal cohort study of the epidemiology of malaria infections in an area of intense malaria transmission II. Descriptive epidemiology of malaria infection and disease among children Am J Trop Med Hyg 1999 60 641 648 10348241 Beasley NM Tomkins AM Hall A Kihamia CM Lorri W Nduma B Issae W Nokes C Bundy DA The impact of population level deworming on the haemoglobin levels of schoolchildren in Tanga, Tanzania Trop Med Int Health 1999 4 744 750 10588768 10.1046/j.1365-3156.1999.00486.x Massaga JJ Kitua AY Lemnge MM Akida JA Malle LN Ronn AM Theander TG Bygbjerg IC Effect of intermittent treatment with amodiaquine on anaemia and malarial fevers in infants in Tanzania: a randomised placebo-controlled trial Lancet 2003 361 1853 1860 12788572 10.1016/S0140-6736(03)13504-0 Schellenberg D Menendez C Kahigwa E Aponte J Vidal J Tanner M Mshinda H Alonso P Intermittent treatment for malaria and anaemia control at time of routine vaccinations in Tanzanian infants: a randomised, placebo-controlled trial Lancet 2001 357 1471 1477 11377597 10.1016/S0140-6736(00)04643-2 Maxwell CA Msuya E Sudi M Njunwa KJ Carneiro IA Curtis CF Effect of community-wide use of insecticide-treated nets for 3–4 years on malarial morbidity in Tanzania Trop Med Int Health 2002 7 1003 1008 12460390 10.1046/j.1365-3156.2002.00966.x Ofori MF Dodoo D Staalsoe T Kurtzhals JAL Koram K Theander TG Akanmori BD Hviid L Malaria-induced acquisition of antibodies to Plasmodium falciparum variant surface antigens Infect Immun 2002 70 2982 2988 12010988 10.1128/IAI.70.6.2982-2988.2002

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==== Front Malar JMalaria Journal1475-2875BioMed Central London 1475-2875-3-271528202910.1186/1475-2875-3-27ResearchCare-seeking patterns for fatal malaria in Tanzania de Savigny Don [email protected] Charles [email protected] Eleuther [email protected] Honorati [email protected] Abdulatif [email protected] Yahya [email protected] Conrad [email protected] Harun [email protected] Graham [email protected] Tanzania Essential Health Interventions Project, P.O. Box 78487, Dar es Salaam, Tanzania2 International Development Research Centre, Box 8500, Ottawa, Canada3 Ifakara Health Research and Development Centre, Box 56, Ifakara, Tanzania4 Rufiji Demographic Surveillance System, Ikwiriri, Tanzania5 Ministry of Health, Box 9083, Dar es Salaam, Tanzania2004 28 7 2004 3 27 27 18 6 2004 28 7 2004 Copyright © 2004 de Savigny et al; licensee BioMed Central Ltd.2004de Savigny et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Once malaria occurs, deaths can be prevented by prompt treatment with relatively affordable and efficacious drugs. Yet this goal is elusive in Africa. The paradox of a continuing but easily preventable cause of high mortality raises important questions for policy makers concerning care-seeking and access to health systems. Although patterns of care-seeking during uncomplicated malaria episodes are well known, studies in cases of fatal malaria are rare. Care-seeking behaviours may differ between these groups. Methods This study documents care-seeking events in 320 children less than five years of age with fatal malaria seen between 1999 and 2001 during over 240,000 person-years of follow-up in a stable perennial malaria transmission setting in southern Tanzania. Accounts of care-seeking recorded in verbal autopsy histories were analysed to determine providers attended and the sequence of choices made as the patients' condition deteriorated. Results As first resort to care, 78.7% of malaria-attributable deaths used modern biomedical care in the form of antimalarial pharmaceuticals from shops or government or non-governmental heath facilities, 9.4% used initial traditional care at home or from traditional practitioners and 11.9% sought no care of any kind. There were no differences in patterns of choice by sex of the child, sex of the head of the household, socioeconomic status of the household or presence or absence of convulsions. In malaria deaths of all ages who sought care more than once, modern care was included in the first or second resort to care in 90.0% and 99.4% with and without convulsions respectively. Conclusions In this study of fatal malaria in southern Tanzania, biomedical care is the preferred choice of an overwhelming majority of suspected malaria cases, even those complicated by convulsions. Traditional care is no longer a significant delaying factor. To reduce mortality further will require greater emphasis on recognizing danger signs at home, prompter care-seeking, improved quality of care at health facilities and better adherence to treatment. ==== Body Background Malaria continues to be the largest single component of the burden of disease in sub-Saharan Africa, even though simple, effective and affordable treatments exist. Malaria's pervasive morbidity and high mortality persist because of failed transactions between those at risk of malaria transmission and available preventive and curative health systems. The consequence is not just an intolerable burden for individuals, their families and national health systems, but is also a devastating and continuing impediment to socio-economic development on the continent. Unlike HIV and TB, the other major fatal communicable diseases in Africa, malaria deaths can be prevented by prompt treatment with relatively affordable and efficacious drugs. Yet this goal continues to be elusive. The paradox of a continuing, but easily preventable, cause of high mortality raises important questions for policy makers and health systems in Africa. Malaria in Tanzania The United Republic of Tanzania has a population of 34.5 million, all of whom are at risk of malaria. However, endemicity and risk of transmission varies and have recently been mapped by the MARA collaboration [1](Figure 1). This GIS-based analysis reveals that 75% of the population is subject to stable perennial or stable seasonal malaria transmission; 8% to unstable highly seasonal transmission; and 17% to no malaria transmission in the average year, but still at risk of epidemic malaria. Tanzania has the third largest population at risk of stable malaria in Africa after Nigeria and the Democratic Republic of Congo (MARA-Lite Software 3.0.0, ). Malaria is the leading cause of out-patient and in-patient health service attendance for all the ages and the leading cause of death in both children and adults in all regions of Tanzania [2]. In Tanzania, malaria is believed to be directly or indirectly responsible for about 16 million annual malaria episodes and 100,000 to 125,000 annual deaths (70–80,000 in under-fives) [3]. Figure 1 Risk of malaria transmission. Length of malaria transmission season in Tanzania based on the MARA climate model. (Source, Ministry of Health TEHIP and MARA-Tanzania). National Responses Increasing global political commitment to malaria control in recent years stimulated by the Roll Back Malaria partnership and the Global Fund to fight AIDS, TB and Malaria, has been reflected in renewed attention to malaria in Tanzanian national level policies, and to a lesser extent, in local government practices. The National Malaria Control Program's strategic plan is built around four pillars: 1) improved malaria case management; 2) national scale use of insecticide treated nets (ITNs); 3) prevention of malaria in pregnancy; and 4) malaria epidemic prevention and control [3]. Integrated Management of Childhood Illnesses (IMCI), intermittent presumptive treatment in pregnancy (IPT) and Insecticide Treated Nets (ITNS) are all part of Tanzania's national package of essential health interventions. In late 2001 the national antimalarial drug policy ceased chloroquine as the first line drug due to high drug resistance. On average there was 52% total treatment failure in sentinel surveillance of antimalarial drug efficacy [4]. The new policy includes sulfadoxine-pyrimethamine (SP) as first line, amodiaquine as second line and quinine as third line antimalarials. In 1998 a district-scale, and later in 2000, a national-scale social marketing programme for ITNs was implemented by the Ministry of Health and its NGO and donor partners in order to develop and test processes for increasing affordable supply, demand and coverage for ITNs and to stimulate the commercial market for ITNs. As part of the health sector reforms, a sector-wide approach to financing places per capita resources under the control of local government councils at district level where they can be used to support the provision of the national package of health interventions, including malaria interventions at both public and non-governmental health facilities. Household responses Tanzanians enjoy relatively good geographic access to primary health services, with 90% of the population within one hour of a government health service [5]. Government health services for children under five years of age and for pregnant women are officially free. However, household health needs and demands are great. Prevalence of overall morbidity is high, with 28.3% of the population reporting illness or injury in the previous four weeks. Utilization of the health system is relatively good and 67.1% of these episodes were reported to attend a health provider (predominantly government). The most commonly reported complaint resulting in a health service consultation is fever or malaria – reported in 69.3% of ill children (less than 15 years of age) and 60% of ill adults (15+ years). Non-governmental health providers are also common and work in partnership with government facilities at rural level. Private-for-profit health providers are relatively new and still largely available only in urban areas and large towns. Over-the-counter drugs are increasingly available in rural settings through private pharmacies, shops and kiosks [6]. Nevertheless, the most accessible health service for the rural household, both in socio-economic as well as spatial-temporal terms, is traditional medicine and traditional healers. Economic considerations Coincident with and consequent to having one of the highest malaria burdens, Tanzania is also one of the poorest countries in the world with an annual GDP of $213 USD per capita (2000) and 36% of the population below the basic needs poverty line. Malaria is estimated to consume 3.4% of GDP or about $240 million USD dollars annually [5]. This is stifling for an already fragile economic performance [7]. Tanzania spends about USD $11.37 per person per year on health [8]. Of this, $2.14 is spent on malaria services. About 75% of malaria expenditures are borne by the household, with the government contributing 20% and partners 5% [9]. Of the household malaria expenditure, about one-third is spent on antimalarial drugs and almost half on bed nets, insecticides, coils and other preventive strategies. This burden is greatest on the poorest households and contributes to the continuing cycle of poverty. Care-seeking There have been a number of studies of care-seeking for malaria in Africa reviewed by McCombie in 1996 [10] and updated in 2002 [11] with much additional work since then [12-18]. Many of these studies involve qualitative and sometimes quantitative analyses of data from illness narratives for recalling episodes of recent illness. Common themes emerge which can be summarized as follows: almost every study identified local community or folk perceptions, terminology or explanations of illness that overlap with malaria disease in ways that distinguished fever, malaria and convulsions as distinct in aetiology and required treatment. Care-seeking patterns for simple fever or uncomplicated malaria were more likely managed initially at home while cases with convulsions or severe malaria were more likely to seek care from a health care practitioner. Multiple care-seeking events and switching between types of providers were common. Cases with simple fever or uncomplicated malaria were more likely to seek formal, modern biomedical care and antimalarial drugs, while cases with convulsions were more likely to be managed by traditional healers or traditional practices, as well as modern care. The hierarchy of such events is likely to affect timely access to effective care. One feature of much of this prior work is that, because severe and fatal malaria is relatively rare, nearly all studies based on illness recall ask what people would do if they/their child experienced a severe illness such as "degedege" (cerebral malaria with convulsions) rather than what they did do. Rationale Although malaria mortality rates are high, fatal malaria is still relatively infrequent when compared to the number of malaria illness episodes. It is possible that the care-seeking patterns of the majority who are ill, but survive, will potentially mask different patterns of those whose care-seeking choices fail and result in a fatal outcome. To understand how best to reduce malaria mortality through improved access to antimalarials, it will be important to examine the care-seeking of individuals who actually died from what they or the health system considered was malaria. No studies in Africa have specifically focused on short-term recall of care-seeking patterns for fatal malaria to see whether and how the general themes above prevail in this sub-group of greatest interest [19]. In this paper an analysis is reported of care-seeking events in a large series of malaria deaths recorded in the course of longitudinal demographic surveillance. Methods Study area The general context of malaria and malaria control in Tanzania has been outlined in the background. The specific setting of this study is in the stable perennial malaria transmission belt that runs along the coast of Tanzania and up the Rufiji and Kilombero River basins (Figure 1). This transmission risk is typical of that experienced by the majority (75%) of Tanzanians and of sub-Saharan Africa in general. There are two main rainy seasons, October-December and February-May. The specific data for the study comes from a demographic surveillance system (DSS) in the Rufiji District of Coast Region, managed by the Ministry of Health and the Tanzania Essential Health Interventions Project (TEHIP). Details of the study populations, DSS methods, life tables and results are available for the Rufiji DSS [20]. Household characteristics of the Coast Region are provided in Table 1. These are shown to be generally representative of rural mainland Tanzania. Table 1 General household-level characteristics of Coast Region in comparison to Tanzania rural mainland Basic Indicators National Mainland Coast Region* Household and Housing Average Household Size 4.9 4.9 Percentage of female-headed households 23 18 Percentage of households with a modern roof 43 24 Percentage of households with modern floor 25 10 Percentage of households with modern walls 25 1 Percentage of households with electricity 12 6 Percentage of households using a toilet 93 98 Mean distance to firewood (km)(rural households only) 3.1 1.7 Mean distance to a shop (km)(rural households only) 1.8 1.0 Mean distance to a bank (km)(rural households only) 37.5 31.3 Education, Health and Water Percentage of adult men without any education 17 24 Percentage of adult women without any education 33 52 Percentage of adults literate 71 58 Primary net enrollment ratio 59 56 Percentage of individuals ill in 4 weeks before survey 28.3 34 Percentage of ill individuals who consulted any health provider 69 83 Percentage of above who consulted a government provider 54 69 Percentage of households within 6 km of primary health facility 75 69 Mean distance to a dispensary / health centre 4.7 3.5 Mean distance to a hospital (km) 25.6 25.9 Percentage of households with a protected water source 57 23 Percentage of households within 1 km of drinking water 55 51 Mean distance to a primary school (km) 1.8 1.7 Mean distance to a secondary school (km) 12.6 13.1 Economic Activities Percentage of adults whose primary activity is agriculture 63 62 Percentage of children age 5–14 years who are working 62 57 Mean area of land owned by rural households (acres) 6 2.9 Consumption and Poverty Consumption expenditure per capita (2000/01 TZS / month) 10,120 9,922 Percentage of consumption expenditure on food 65 71 Percentage of population below the food poverty line 19 27 Percentage of population below the basic needs poverty line 36 46 * Rural result provided where available; ** Exchange rate, January 2001: TZS/USD = 803 Source: Government of Tanzania, National Bureau of Statistics, Tanzania Household Budget Survey 2000/01 Rufiji District is 178 km south of Dar es Salaam on the Indian Ocean coast and has a population of 203,000 in 2002 in an area of 14,500 km2. The district is entirely rural with 94 registered villages, no urban areas or towns, and has a large area set aside as a game reserve. The economy is predominantly subsistence farming and fishing. Rufiji district is home to several ethnic groups. The largest is the Ndengereko who, according to oral tradition, are the original inhabitants of the area. Other groups include the Matumbi, Nyagatwa (concentrated in the delta area), Ngindo, Pogoro and Makonde. The majority of the people are Moslems (98%) with a few Christians (1.3%) and followers of traditional religions. In addition to local languages, Kiswahili is widely spoken; English is not commonly used in the area. The population has access to 57 formal health facilities: two hospitals (one government and one NGO), five health centres with in-patient facilities (all government) and 50 outpatient dispensaries (46 government). Over-the-counter drugs are available from many private shops and kiosks in the villages. People also obtain services from traditional healers including traditional birth attendants. Immunization coverage ranges from 85% for BCG (tuberculosis) to 66% for measles in children 12–23 months of age. Acute febrile illness and malaria are the leading causes of attendance at health facilities, and the largest cause of mortality. For malaria, the district provides Integrated Management of Childhood Illness (IMCI), Intermittent Presumptive Treatment of malaria in pregnancy (IPT), and first, second and third line antimalarial services at all formal health services, as well as social marketing of insecticide-treated nets (ITNs). Demographic Surveillance The Rufiji District hosts a sentinel DSS area that covers 1,800 km2 north of the Rufiji River and west of the Rufiji Delta (7.470 to 8.030 south latitude and 38.620 to 39.170 east longitude). The Rufiji DSS monitors a total population of 85,000 people in 17,000 households in 32 villages. All residents are registered in the system and all births, deaths, in-migrations, out-migrations, pregnancies and other vital events are monitored and registered. Events are recorded in the Demographic Surveillance Area (DSA) by 150 village key informants and verified by DSS staff. Twenty-eight full-time enumerators update the population register every four months by household survey cycles. The field and data system is based on the Household Registration System Software [21]. The database also includes key household level information on household structure, socio-economics and assets, food-security and environmental features that are updated annually. All households and community structures have been geo-located by global positioning satellite (GPS) systems. The Rufiji DSS is part of the Ministry of Health's National Sentinel System (NSS) for monitoring health and poverty status and serves as a sentinel for rural coastal districts. Annual Burden of Disease profiles are produced by the DSS and used for district planning purposes in the NSS. Verbal Autopsy The Rufiji DSS continuously records vital events within households and among individuals over time in a systematic way. The vital events reporting system consists of key informants who notify the system of any death occurring in the DSS area. This information is passed to a DSS key informant supervisor (or DSS enumerator who informs the key informant supervisor). The key informant supervisor visits the households in which death has been reported within two weeks and contacts the DSS data centre for verification of the registry status. A verbal autopsy (VA) (post mortem interview) is then scheduled and administered to one of the deceased's relatives or the individual who is most well informed of events and details of illness of the deceased. A DSS VA supervisor, who is also a trained clinical officer or health officer, conducts the VA interview. Respondents are not aware of the health care qualifications of VA interviewers. Enumerators also ascertain death events at fixed enumeration rounds three times per year, using specific event forms that are reconciled with the mortality database. There is no population sampling. The entire population of the DSS area is in the DSS and all deaths to DSS residents are subject to VA. Population compliance in both the DSS and VA interviews was very high resulting in high completeness of death registration for registered members. Verbal autopsy was available on 97.7% of deaths, missing only those where the family out-migrated shortly after the death or declined the VA interview. The VA tool used is that of National Sentinel System [22] based on an evolution of forms developed by the Adult Morbidity and Mortality Project (AMMP) [23] and very similar to that proposed by INDEPTH . It uses individual specific standard questionnaires for: a) children under 31 days of age; b) children under five years but 31 days and older; and c) population aged five years and older. The questionnaires and responses are in Kiswahili. Information such as household ID number, name, age and sex are re-collected for confirmation. In addition, data is collected by open-ended and closed questions on history of events leading to death, together with previously diagnosed medical conditions as well as signs and symptoms before death. Questions about use of health facilities prior to death, reasons for using or not using a particular health facility and confirmatory evidence of medical care and cause of death (if available) are also asked and recorded in the questionnaires. A typical bereavement interview in the course of a VA takes 45 to 60 minutes. The tentative cause of death is established from the sequence and severity of signs and symptoms, as well as the available confirmatory evidence, by the VA supervisor and recorded on the forms. However, it is physician coding that determines the final cause of death that is subsequently entered in the database. Completed questionnaires are coded independently by two physicians according to a list of causes of death based upon the tenth revision of the International Classification of Diseases. A third physician independently codes the VA in case of discordant results from the first two physicians. Where there are three discordant codes, the cause of death is registered as undetermined (about 6% of cases). A single cause is assigned as the main cause, with contributing causes also indicated. All death coded as the following were included as suspected to be directly or indirectly due to malaria and included in the study: acute febrile illness 1–4 weeks; acute febrile illness < = 7 days; acute febrile illness including malaria; acute febrile illness with convulsions; acute febrile illness with anaemia; cerebral malaria; fever plus malnutrition; malaria; malaria confirmed; and unspecified acute febrile illness. Quantitative methods All data from the DSS and the VA were entered, cleaned and managed using FoxPro (Microsoft Corp). Databases were linked and selected data transferred to Stata 7.0 (Stata Corp) for analysis. The VA database was linked to the household registration database to obtain other indices, such as the socio-economic status. In a separate study, we determined socio-economic indices for individuals in 14,440 rural households in the Rufiji DSS area for the year 2000. The index was based on principal components analysis of the presence or absence of items from a list of 22 specific household assets and nine household characteristics dealing with household ownership, construction features, water supply, sanitation and type of fuel. Further details on the socio-economic index are provided elsewhere [24]. The household index was applied to each individual in the respective household and all deaths due to malaria were partitioned into socio-economic quintiles by this index. Univariate analyses were used to assess the affect of age, sex, socio-economic status, household headship and severity of malaria on initial choices from 13 potential categories of health care providers. Chi-square was used to identify significant factors associated with choice of care sought during the final illness. Qualitative methods The health behaviour research component of the Tanzania Essential Health Interventions Project (TEHIP) investigated the care-seeking and compliance patterns for malaria in a separate study in the Rufiji District from 1998–2001. Eight villages were purposely selected to include four villages with a local health facility and four villages far from a health facility. From these villages 80 households with children under-five years of age were selected by simple random sampling. Ethnographic approaches (semi-structured interviews, case histories and focus group discussions) were used to explore and describe households' responses to childhood illnesses including malaria. A two-step coding strategy was used. In the field, a research assistant, using a provided guide, performed initial thematic coding of the data. Field coding was supervised and consistency checked by a senior social scientist. At computer data entry level, field codes were replaced by corresponding thematic codes written in text macros by experienced data clerks. A data manager supervised the data entry and was responsible for quality and further consistency checks. All qualitative data was processed in a text editor and analysed using text analysis software, Text-Base Beta (Centre for Qualitative Research, University of Aarhus, Denmark). The codes allowed retrieval and compilation of text segments of interest for thematic analysis. Terminology The ethnographic literature on treatment seeking in Africa uses a variety of terms, none of which are wholly satisfactory in capturing the nature and complexity of available health systems. In this paper the term "modern care" is used to describe what conventionally includes biomedical, western, pharmaceutical, professional, official or formal health care and the term "traditional care" is used to describe what conventionally includes traditional medicine, traditional healers, traditional providers, lay providers, traditional practices or folk care. Ethical Considerations All household visits, surveys and questionnaires in the DSS and TEHIP surveys were administered with individual informed consent. All individual and household data are confidential. All reports are based on summary data that cannot be linked to individuals or individual households. The Ministry of Health, National Institute for Medical Research's Tanzania Medical Research Coordinating Committee has approved the research protocols of TEHIP and its Rufiji DSS. Information is fed back to the communities concerned on a semi-annual basis and provided to the local council authorities and the Ministry of Health for planning purposes on an annual basis. Results Qualitative themes: illness terminology Qualitative studies confirmed that the population refers to the signs and symptoms associated with the biomedical condition of malaria as three distinct conditions, each with its own aetiology, treatment-seeking patterns and prognosis. The three conditions are: "homa" (fever, vomiting, feeling cold, loss of appetite, limp body, red eyes, not considered life threatening); "malaria" (high fever, vomiting, loss of appetite, feeling cold, some caretakers considered life threatening) and "degedege" (high fever, loss of appetite, stiffness of body, rolling of eyes, lips twisted sideways, twitching, considered life threatening). These are well recognized by most households in the study population. " [...] you are able to recognize an episode of degedege in one day. It begins with mild fever and the next day the fever becomes more severe and results in symptoms of epilepsy. The child opens the eyes wide and the black spot cannot be seen, he begins to twist the arm and leg, and then, even if you pour cold water over the child, does not react..." (Female respondent aged 37 from Bungu – Rufiji). Although the local population distinguish between the illness "homa" and malaria the distinction is not always very clear to them. Analysis of case studies revealed that the illness term "malaria" has been obtained from modern health care. When mothers take their children to these health services with what had been diagnosed at home as "homa", they are told it is malaria. The following is illustrative of experiences reported: "I first thought it was normal homa (fever) and I could see the child had homa. Now, when I took the child to the hospital, they checked the child's blood and informed me the child had malaria. [...] the child was not playing. I touched the child and the body was like fire (mwili wake ulikuwa wa moto), the body was very hot". (Female respondent aged 29, Bungu, Rufiji). Anaemia is not often recognized, and where recognized, is not associated with malaria. Qualitative themes: aetiology Although "homa" and especially "malaria" were seen as associated with malaria and mosquitoes, in most cases the signs and symptoms of "degedege" are not attributed to malaria. Life threatening malaria with convulsions is not only perceived as a different illness from malaria through local symptom definition but is attributed to different causes than malaria. Few households mention the mosquito as a cause of the illness described as "degedege". Popular beliefs as to the cause of "degedege" were found to include: fever, evil spirits and a change in weather/wind. The following translation is typical of quotes obtained from respondents on perceived causes of "degedege": "....Evil spirits or demons cause degedege. If it happens that evil spirits or demons pass in front of the child, then the child is likely to get degedege. This may result in paralysis of the body or leg or arm or any part of the body..." (Male respondent aged 46, from Kilimani, Rufiji). Qualitative themes: Care-seeking pattern "Homa" and "malaria" are seen as conditions that can be managed at least initially at home with modern medicine available from shops and from health facilities. But "degedege" is perceived as a serious life-threatening condition for which prompt treatment-seeking is required. People reported different sources of care they used for the treatment of "degedege". These sources encompass more than the biomedical health system and fall into three broad categories: home treatment, traditional healers and biomedical. Home treatment was reported to include the use of modern medicines, such as aspirin from local shops, in the early stages the illness. If the illness reaches a severe stage (convulsions) people claim to use traditional healers in the home or outside the home. Biomedical care ranging across government hospitals, health centres, dispensaries and equivalent private facilities was used in the later stage, when convulsions had subsided. However, some respondents perceive traditional healers as not competent to deal with such illness and claim to seek care from biomedical providers at the beginning of the illness. "We use traditional remedies only to treat degedege. They (remedies) must have a very bad smell for this will chase away the evil spirit. It is just like telling you to stay in the latrine; surely you will have to find another place because of the bad smell. This is just the same case for the evil spirit attacking the child because of the bad smell it will have to find another place to stay...." (Male respondent aged 57 years, Kiomboni, Rufiji). "I had gone to the dispensary for treatment; my child was suffering from homa. The first day he was given panadol tablets and chloroquine injection and was asked to return the next day for chloroquine injection. The next day while I was there at the dispensary waiting for treatment my child started convulsing. This I believed to be a sign of degedege. Immediately I left the dispensary in search of a traditional healer. Degedege is never treated in the dispensary. Child may die after being injected." (Female respondent, 39 years old, Kiomboni, Rufiji). "When my child developed degedege I was at Kibiti. I had to look for transport to take the child to Songa Hospital (Mchukwi Missionary hospital). There you have reliable service because you find almost all kinds of investigations. I don't like going to traditional healers because they are not reliable and do not have equipment to investigate well your child. They end up telling you things related to superstition." (Female respondent aged 42, Bungu, Rufiji). Quantitative results: care-seeking pattern In the period January 1999 to December 2001 inclusive, the Rufiji DSS conducted 243,042 person years of follow-up. In this series, 3,023 deaths occurred to resident members and 2,953 (97.7%) verbal autopsies were conducted. Of these, 24.4% (722) had a cause of death suggestive of malaria as the direct or underlying cause, of which 44.3% (320) were in children less than five years of age. Among these child deaths, there was no difference in frequency between sexes, with 51.3% being male and 48.7% female. Of the child malaria-attributed deaths, 282 (88.1%) sought care at least once before death, while 38 (11.9%) did not, or could not, seek care. Convulsions (possible cerebral malaria) were recorded in 30 (9.4%) of these fatal cases. The verbal autopsies contained both an open-ended narrative account of the final illness and a specific chronological account of where and in what sequence care was sought. There were 13 possible sources of treatment that were collapsed for purposes of certain analyses into three sub-categories of care types (Modern Care; Traditional Care; and No Care) and into six sub-categories of provider types (Government; Home/Shops; Non-Government; Traditional Medicine at Home; Traditional Medicine at Practitioner; and No Care). Table 2 compares the level and detailed source of initial care in acute febrile illness (malaria) for children less than five years of age compared with older cases. The initial treatment-seeking choice for children less than five years of age was modern care (78.7%), whereas only 9.4% used traditional care initially. The remainder (11.9%) sought no care (Figure 2). Table 2 Level and source of initial care in fatal acute febrile illness / malaria by age group in the Rufiji DSS sentinel area, 1999–2001 Level of Care Provider Age <5 5+ Government VHW 0.0% 0.7% Dispensary 19.4% 11.2%** Health Centre 20.0% 14.4%* Hospital 5.3% 5.0% Home Mothers 2.5% 2.2% Family 9.4% 13.2% Drug Shops 8.1% 20.6%** Non-Government Dispensary 10.3% 5.5%* Health Centre 1.6% 2.0% Hospital 2.2% 2.5% TM at Practitioner 6.6% 6.5% TM at Home 2.8% 1.7% None None 11.9% 14.3% 100% 48% Number 320 402 Total 722 * Significant at 5% level; ** Significant at 1% level TM Traditional Medicine or Practice Figure 2 Initial care-seeking patterns. Care of first resort sought during the final illness by 320 fatal "malaria" cases in children less than five years of age in the Rufiji DSS sentinel area, 1999–2001. Within modern care, government providers were most prominent (44.7%) followed by home care with antimalarials from private shops (20%) (Table 3). Children were statistically more likely to be taken to government health centres and government and non-government dispensaries and less likely to be served by drug shops as the initial resort to care (p < 0.05). There were no significant differences between treatment-seeking patterns for male and female patients regarding the broad choices of modern, traditional or no care. Even though there was no difference in the proportion of males and females receiving traditional care, within the traditional care group, females were statistically more likely to be kept home to receive traditional medicine, and males were more likely to be taken out of the home to see a traditional healer (p < 0.05). There were no significant differences in specific or general care-seeking patterns by sex of the household head. There was no difference in treatment-seeking patterns when comparing choices made by households in the poorest quintile and households in the least poor quintile. Table 3 Type and provider of initial care in fatal acute febrile Illness / malaria by age group, sex, socio-economic status, and type of illness in the Rufiji DSS sentinel area, 1999–2001 Type of Care Provider Age Sex of Child Sex of HH Head Poverty Quintiles Convulsions <5 5+ Male Female Male Female Poorest Least Poor With Without Modern Care Government 44.7% 31.1%** 46.4% 42.9% 38.6% 33.3% 42.6% 51.0% 55.6% 43.3% Home / Shops 20.0% 36.1%** 21.3% 18.6% 28.2% 31.9% 22.2% 15.7% 19.4% 20.1% Non-Government 14.1% 10.0% 12.2% 16.0% 12.6% 9.8% 9.3% 9.8% 2.8% 15.5% Traditional Care TM at Practitioner 6.6% 6.5% 7.3% 5.8% 2.1% 2.9% 7.4% 5.9% 16.7% 5.3%* TM at Home 2.8% 1.7% 1.2% 4.5% 6.0% 7.8% 1.9% 3.9% 0.0% 3.2% No Care None 11.9% 14.4% 11.6% 12.2% 12.0% 14.3% 16.7% 13.7% 5.6% 12.7% 100% 33% 100.0% 100.0% 100% 100.0% 100% 100% 100% 95% Number 320 402 164 156 485 204 54 51 36 284 Total 722 320 689 105 320 * Significant at 5% level; ** Significant at 1% level TM Traditional Medicine or Practice HH Household. Note, 33 households had a change in headship during the study period and were excluded from the analysis in the sex of HH Head column. Cases with convulsions were as likely to receive initial modern care as cases without convulsions (77.8% and 78.9% respectively) (Table 3). However, cases with convulsions were less likely to receive no care. Therefore, although the predominant choice of care was modern, inclusion of care from traditional healers was significantly more frequent in those with convulsions than in those without convulsions (p < 0.05). All traditional care was provided by traditional healers and no case claimed to give traditional medicine at home, which is contrary to what is often described in non-fatal treatment seeking. Among children for whom care was actively sought, 82.4% of those with convulsions and 90.3% without convulsions sought modern care as the initial care (Table 3). Multiple episodes of care-seeking were common. More than half of cases had two or more treatment-seeking events for the same illness involving a different type of provider (Figure 3). There is also a difference in pattern when initial care choices and cumulative care choices are compared (Table 4). The latter indicates important switching between providers over time and this phenomenon is most apparent when comparing malaria without convulsions to malaria with convulsions. Multiple provider care-seeking was more common if convulsions were present. These synchronic choices (frequency of use of a particular resort to care) are shown in Figures 3 and 4. In the multiple-care-seeking group, switching between modern care and traditional care can be a factor in the delay of effective care. Of the multiple-care-seeking group that did not have convulsions, 88.4% and 99.4% had used modern care at least once by their first or second choice respectively. In this group, of those who started with modern care, only 0.9% switched to traditional care as the second choice. Of the few who started with traditional care as their first choice, most (94%) switched to modern care for their second choice. For the group that had convulsions, 90% chose modern care as their first choice, but by the second choice, 29.6% switched to traditional as the second provider. Switching did not seem to be based on differences in likelihood of receiving treatment. All provider categories were generally able to supply the expected treatment, the poorest being government providers who were able to give treatment for 94% of cases and the best being traditional healers at 96.8% of cases. Figure 3 Frequency of care-seeking events. Distribution of frequency of care-seeking events at differing categories of provider among those who sought care during the final illness in fatal episodes of malaria in 320 children under five years of age with (dark shading) and without convulsions (light shading). Table 4 Level and source of accumulative care in fatal acute febrile illness / malaria, all ages, in the Rufiji DSS sentinel area, 1999–2001 Level of Care Provider Cumulative Events No. % Government VHW* 5 0.8% Dispensary 92 14.5% Health Centre 104 16.4% Hospital 67 10.6% Home Mothers 19 3.0% Family 64 10.1% Drug Shops 36 5.7% Non-Government Dispensary 77 12.2% Health Centre 39 6.2% Hospital 30 4.7% TM** at Practitioner 73 11.5% TM** at Home 27 4.3% Total care seeking 633 100.0% VHW* Village Health Worker; TM** Traditional Medicine or Practice Figure 4 Loyalty to first provider. Comparison of loyalty to first provider of modern or traditional care during the final illness in fatal cases (all ages) that saw two or more providers. Discussion Limitations of verbal autopsy methods for malaria deaths have long been recognized, especially with regards to specificity and sensitivity [25,26]. This has provoked efforts to improve and validate verbal autopsy procedures in the settings in which they are used [23,27-34]. The general consensus is that, although imperfect, verbal autopsies are reasonably reliable in determining major causes of death at population level, but may not be suitable for detecting specific impacts of interventions. However, recent work applying adjustments for sensitivity and specificity at differing prevalence levels based on validation studies shows how VA data could be used to monitor progress towards malaria-specific mortality reduction [35]. It must be emphasized that not all of the cases identified as "malaria" in this series are malaria, especially those with unspecified acute febrile illness at older ages. Undoubtedly, some malaria deaths were coded as a cause other than malaria-related. For example, severe and life threatening anaemia, likely to be due to malaria, is prevalent in young children over six months of age in the study area [36] yet VA coded deaths due to anaemia with malaria are infrequent. Despite improvements in verbal autopsy methods in recent years, any study based on verbal autopsy is subject to bias. The recall abilities of respondents can be faulty, although for major events such as a death in the family, it tends to be better than recall of less significant events [33]. In the current study of care-seeking as reported in verbal autopsy, respondents might inflate the number of care-seeking events or exaggerate the choice of modern care if they perceive the DSS to be an instrument of the modern health system or if they feel guilt regarding the care-seeking decisions they took. This would tend to bias responses in favour of more modern care. Much has been learnt in recent years concerning treatment seeking for malaria in Africa, largely through ethnographic research on illness recall narratives [10-12,14,37-42]. This literature confirms that, for the majority of cases deemed as uncomplicated malarial fevers, modern care based on antimalarial drugs is favoured over traditional medicine. Usually treatment starts at home using anti-pyretics and antimalarials obtained over-the-counter from local shops or left over from previous episodes. Knowledge of appropriate treatment regimens is lacking on the part of the public as well as on the part of private providers [43,44]. Under-dosing in home-based care is common. Malaria is perceived by adult care givers as a mild disease, and if it becomes serious or life threatening, then, it is generally believed that the perceived diagnosis changes from malaria to something that is more likely to be treated with traditional medicine or practices. These beliefs are not rigid. Every case is subject to a process of continuing debate and re-evaluation such that modern pharmaceuticals are also sought, albeit with delay, when convulsions fail to resolve or reoccur after traditional medicine [16,45]. If this is the case in studies of illness recalls, where most patients recover, the question remains whether this general and widespread pattern of treatment seeking holds in those cases where effective treatment seeking clearly failed and the patient died. Since most cases of malaria death in Africa occur at home rather than in health facilities, facility-based data and studies cannot answer this question. The increasing use of demographic surveillance field sites to monitor health at population level in Africa [46] presents an opportunity to examine large series of verbal autopsy findings. Modern verbal autopsy goes beyond cause of death data to collect additional contextual data on, for example, care-seeking events prior to death. This study confirms that the general patterns seen in illness recalls for uncomplicated malaria in Africa also apply to what people actually do in episodes of fatal malaria in a holoendemic area of Tanzania. Modern care is the first choice for children in over 78% of all child malaria deaths. Government health facilities and shopkeepers were the main source of modern antimalarial drugs. Traditional care may have caused delay in modern care in only 9.4% of fatal cases. 11.9% had no care of any kind. This general pattern held over broad age, sex and socio-economic status groups. Among children with and without the complication of convulsions for whom care was actively sought, 82.4% and 90.3% respectively sought modern care as the initial care (Table 3). In the case of convulsions, although the majority of initial care-seeking was modern, the use of traditional healers increased while the no-care group decreased accordingly. Among those of all ages who sought care two or more times in the course of fatal malaria, modern care was included in the first two choices in 99.4% of cases excluding convulsions and in 90% of cases with convulsions. Clearly, the perceived severity and danger signs posed by convulsions provoke polyvalent treatment seeking. Nevertheless, modern care is now more popular than previous reports and qualitative studies suggest. One other study of care-seeking patterns in a large series of verbal autopsy reports from the mid 1980's from Tanzania analysed a similar number of all-cause child deaths from Bagamoyo District, a nearby district in the Coast Region [47]. In that study, malaria deaths were not analysed separately, but government providers were the choice in only 45% of deaths. At that time government providers were often without an adequate drug supply and a preference for traditional healers was cited by 41% of mothers as the reason for not using government providers. At the time of the present study in Rufiji, all government providers had adequate drug supplies under the health reforms and offered the integrated management of childhood illness (IMCI) strategy. This could be a factor in the current popularity of government providers. A relatively small proportion (21.3%) of malaria-attributable child deaths failed to seek modern care (9.4%) or any care (11.9%). This is considerably better than was seen in the mid-eighties, when 55% of children who died had not utilized any modern care [47]. It is also better than seen for deaths in general in the same area during the same period, when 20% of all-cause deaths had no prior care-seeking events [48]. Part of these non-care groups would include those who had sudden death following apparently mild illness, including severe anaemia. This study shows that most patients now include modern care early in their treatment seeking patterns for eventually severe and fatal malaria, including malaria with convulsions. So why is malaria still the largest single component in the burden of mortality? With belief systems for malaria treatment seeking now firmly on the side of modern care, there is obviously something still failing in 1) the transaction to obtain this care; 2) the quality of the care and referral once it is reached; and/or 3) patient adherence to treatment once it is obtained. This would suggest that policies, efforts and implementation research aimed at improving early recognition of symptoms and danger signs at home, prompt treatment or treatment seeking, the quality and efficacy of the antimalarial available and compliance with the full course of treatment, are now, more than ever, highly justified. When appropriate care-seeking is as high as it is in Tanzania, continuing malaria deaths should be considered as sentinel events deserving of close scrutiny and audit to identify the best remedial strategies for the health system. There are promising developments. IMCI has recently been introduced in the study area. It places heavy emphasis on training care-givers on early recognition of danger signs and the need for prompt treatment and on improving quality of assessment and care at primary health facilities [36,49]. Replacement of chloroquine with directly observed treatment with sulfadoxine-pyrimethamine (SP) and its simpler single dosing schedule should result in less under-dosing while the introduction of pre-packaged doses has also been shown to be effective in improving provider and client adherence [50,51]. This study was conducted over the last three years of a policy period that used chloroquine as the first line antimalarial. It will be repeated for a similar time frame over the initial three year period of a new policy that uses SP to see if the care-seeking and care-getting patterns change. A qualitative analysis is also planned for the narrative portion of the verbal autopsy questionnaire to look at categories and sub categories of health care related themes in VA reports. This would focus on reasons for delay in seeking modern care (e.g. tried to treat at home without antimalarials, transport, beliefs, poor recognition of severity, lack of confidence in modern care, no power to decide, insufficient finances); delay in receiving modern care (e.g. outside of working hours, weekends, long queues, satisfaction); ineffective modern care (poor communication, no referral, drugs not available, abusive health worker). Conclusions This preliminary study examined what families of children who died from malaria in a holoendemic setting in Africa actually did in terms of treatment-seeking choices and sequence. It confirms that modern medicine in the form of antimalarial pharmaceuticals from shops or government or non-governmental heath facilities is now the preferred choice in an overwhelming majority of cases (78.7% and 97% as their first or second choice respectively). Traditional medicine could only be implicated in a possible delay of modern care in 9.4% of cases. 11.9% sought no care of any kind. There were no differences in these broad patterns of choice by sex of the child, sex of head of household, socioeconomic status of the household or presence or absence of convulsions. Contrary to what is concluded from much of the historical and qualitative work on this subject, modern care is now the care of first choice, even for those who seek care for children with malaria with convulsions (82.4%), although traditional medicine also played an important role in later choices. But despite high rates of modern care-seeking for all forms of malaria, and despite relatively high attendance and utilization of modern care as seen in Tanzania, malaria mortality remains high. This must, therefore, be due to excessive delay in seeking modern care, and/or poor quality of modern care (providers and/or drugs) once sought, and/or poor patient adherence to treatment regimens once obtained. Certain policy and practice implications arise: 1) public messages need to focus aggressively on improving early recognition of malaria and severe malaria at home and improving promptness of treatment seeking (within 24 hours of onset of malaria symptoms or immediately in the case of severe malaria); 2) quality of modern care providers and modern care must be improved in all sectors, private, NGO and Government; and 3) patient adherence with modern care at home must be simplified and reinforced. List of abbreviations DSA Demographic Surveillance Area DSS Demographic Surveillance System GIS Geographic Information System HBS Household Budget Survey HH Household IMCI Integrated Management of Childhood Illness ITN Insecticide-treated netting MARA Mapping Malaria Risk in Africa Collaboration SP Sulfadoxine-pyrimethamine TM Traditional Medicine VA Verbal Autopsy Authors' contributions DD conceived the study, participated in the design, coordination and quantitative analysis and co-wrote the article. CM conceived, conducted and analysed the qualitative studies and co-wrote the article. HM led the analysis of quantitative data. EM managed the surveillance system and participated in design and coordination. AM managed and cleaned the quantitative data. YM managed the field work. CM, HK and GR participated in the coordination and management of the study. Acknowledgements The authors wish to thank Dr. Andrew Kitua, the Director General of the National Institute for Medical Research, Tanzania, for support and for permission to conduct this research. This study was funded in part by grants from the International Development Research Centre (IDRC, Canada). The Rufiji DSS was supported by IDRC Canada through the Tanzania Essential Health Interventions Program, by DFID UK through the Adult Morbidity and Mortality Project and by the US Centers for Disease Control through the IMPACT project. Further support from the National Academy of Sciences, Institute of Medicine for additional data analysis is gratefully acknowledged. Particular thanks are extended to Dr. Saidi Mkikima, District Medical Officer for Rufiji District and to Dr. Alex Mwita, National Malaria Control Programme Manager for the Tanzania Ministry of Health. ==== Refs Le Sueur D Binka F Lengeler C de Savigny D Snow RW Teuscher T Toure Y An atlas of malaria in Africa. 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10.1186/1475-2875-3-27

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==== Front Filaria JFilaria Journal1475-2883BioMed Central London 1475-2883-3-71529870910.1186/1475-2883-3-7ResearchMapping the distribution of Loa loa in Cameroon in support of the African Programme for Onchocerciasis Control Thomson Madeleine C [email protected] Valérie [email protected] Joseph [email protected] Jacques [email protected] Samuel [email protected] Innocent [email protected] Peter [email protected] Jan H [email protected] David H [email protected] Michel [email protected] Liverpool School of Tropical Medicine, Liverpool, UK, L3 5QA2 Laboratoire mixte Institut de Recherche pour le Développement (IRD) – Centre Pasteur du Cameroun d'Epidémiologie et de Santé publique, Centre Pasteur du Cameroun, BP 1274 Yaoundé, Cameroon3 Department of Life Sciences, Faculty of Science, University of Buea, P.O. Box 63, Buea, Cameroon4 Department of Public Health, Faculty of Medicine and Biomedical Sciences, University of Yaoundé I, P.O. Box 1364, Yaoundé, Cameroon5 Tropical Medicine Research Station, P.O. Box 55, Kumba, Cameroon6 UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), WHO, Geneva, Switzerland2004 6 8 2004 3 7 7 8 5 2004 6 8 2004 Copyright © 2004 Thomson et al; licensee BioMed Central Ltd.2004Thomson et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Loa loa has recently emerged as a filarial worm of significant public health importance as a consequence of its impact on the African Programme for Onchocerciasis Control (APOC). Severe, sometimes fatal, encephalopathic reactions to ivermectin (the drug of choice for onchocerciasis control) have occurred in some individuals with high Loa loa microfilarial counts. Since high density of Loa loa microfilariae is known to be associated with high prevalence rates, a distribution map of the latter may determine areas where severe reactions might occur. The aim of the study was to identify variables which were significantly associated with the presence of a Loa microfilaraemia in the subjects examined, and to develop a spatial model predicting the prevalence of the Loa microfilaraemia. Methods Epidemiological data were collected from 14,225 individuals living in 94 villages in Cameroon, and analysed in conjunction with environmental data. A series of logistic regression models (multivariate analysis) was developed to describe variation in the prevalence of Loa loa microfilaraemia using individual level co-variates (age, sex, μl of blood taken for examination) and village level environmental co-variates (including altitude and satellite-derived vegetation indices). Results A spatial model of Loa loa prevalence was created within a geographical information system. The model was then validated using an independent data set on Loa loa distribution. When considering both data sets as a whole, and a prevalence threshold of 20%, the sensitivity and the specificity of the model were 81.7 and 69.4%, respectively. Conclusions The model developed has proven very useful in defining the areas at risk of post-ivermectin Loa-related severe adverse events. It is now routinely used by APOC when projects of community-directed treatment with ivermectin are examined. ==== Body Background Knowledge of the spatial distribution of Loa loa is important in countries involved in the African Programme for Onchocerciasis Control (APOC) and is also now a significant issue for the Global Programme to Eliminate Lymphatic Filariasis (GPELF). Both programmes rely on the wide-scale distribution of anti-helminthic drugs to poor communities using community-directed drug distribution schemes. A problem was first observed in Cameroon where a series of reports of severe and sometimes fatal encephalopathic reactions to ivermectin (Mectizan®) in individuals with high Loa loa microfilarial counts was made [1-3]. Similar problems may occur when albendazole (used to control lymphatic filariasis) is distributed in Loa loa endemic areas although evidence for this is contradictory [4-7]. Loa loa is associated with tropical "eye worm" (migration of adult worms across the sub-conjunctiva), Calabar swelling, oedemas and prurities but is not considered as pathogenic as other filarial worms, and is consequently less well studied. It is transmitted by species of horse-flies (Chrysops spp.), most commonly Chrysops dimidiata and C. silacea which inhabit the forest areas of West and Central Africa, extending to the Ethiopian border. Severe and fatal reactions to ivermectin have been associated with individuals with high Loa loa microfilarial loads (more than 30,000 microfilariae (mfs) per ml of blood) and those with more than 50,000 mfs/ml are considered at very high risk [1,8-10]. Changes in the protocols for drug administration and post-treatment surveillance in areas considered to be at-risk of severe reactions to ivermectin have been implemented [11,12], but the detailed geographic distribution of Loa loa remains unclear [13]. Such information is essential in terms of developing safe treatment, and above all surveillance strategies across the region and, given the vast area which may be affected, it is recognized that rapid assessment methods must be developed to evaluate the risk of severe reactions in communities co-endemic for loiasis and onchocerciasis. Recent studies have shown that there is a close relationship between intensity of microfilariae infection and prevalence rates of Loa loa [14,15] suggesting that a distribution map based on prevalence of infection alone (and not intensity, which would require time-consuming counting of mfs) would provide sufficient information to delineate areas of high risk of severe reactions. The aim of our study was to develop a map indicating the areas where Loa loa infection may be high enough (i.e. with a prevalence of Loa microfilaraemia in adults exceeding 20%) that poses an operational problem for drug distribution by the Community-Directed Treatment with Ivermectin (CDTI) strategy [16]. As a preliminary step we created a risk model for Loa loa in West and Central Africa based on the relationship of crude Loa loa prevalence data (obtained from a literature search) to a wide range of environmental variables. Initial results suggested that land/forest cover derived from NOAA-AVHRR satellite data (i.e. collected by the Advanced Very High Resolution Radiometer on board the satellite series operated by the National Ocean and Atmospheric Administration of the USA), and soil type (from the FAO digitised soil map of Africa), are significant predictors and a preliminary risk map was produced [17]. However, when this first model was tested against field data from Cameroon it was found to poorly represent areas of high risk of infection in certain districts [18]. Possible reasons for this include: (a) the low spatial resolution of the satellite and environmental data used (1 km) which was unable to identify narrow gallery forests in savannah areas and/or (b) the use of prevalence data from various sources which had not been standardised by age or sex. In order to improve the quality of epidemiological data being used to develop the model, we created a new prevalence database from a series of surveys conducted by the team of the Institut de Recherche pour le Développement (IRD) at the Centre Pasteur du Cameroun (CPC). A second independent data set, collected as part of a project supported by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) to calibrate a rapid clinical diagnosis of Loa loa (RAPLOA) [15], was used to validate the results of the model. Methods Epidemiological data Epidemiological data used to create the model was obtained from a series of field studies undertaken by the IRD-CPC laboratory in Cameroon during the period 1991–2001 in which the presence of Loa loa parasites in the blood of individuals was assessed. The data come from five regions: (a) the forest-savannah mosaic area of the Mbam and Kim division (Central province); (b) the degraded forest area of the Lekie division (Central province); (c) the dense forest area of the southern part of the Central province; (d) the highland savannah area of the Western Province; and (e) the savannah areas of the districts of Banyo and Bankim, in the Adamaoua province. The Ministry of Public Health of Cameroon provided ethical approval for these surveys. The total number of subjects included in the analysis was 14,225 individuals for 94 villages. Study participants consisted of individuals over the age of 5 years who gave their consent or for whom consent was obtained from the parent or guardian. Individuals who had had filaricidal drugs, such as ivermectin or diethylcarbamazine, in the previous five years were not included in the study. The arrival of the research team in the villages was announced to the population one week before the event through the local authorities and teachers. The objective of the examinations were clearly explained, and it was stated that each individuals results would be returned. On the given day, the team settled at a central place of the village (usually at the chief's home, or in a school), and all the volunteers were examined between 10.00 and 16.00 hours. A standardized quantity of capillary blood was obtained with a non-heparinized micro-capillary tube. Prior to 1994, the amount taken was 30 μl, and thereafter, 50 μl. After Giemsa staining, the slides were examined under a microscope and the presence of Loa mfs was recorded. The latitude and longitude of all study villages were obtained from either the ordinance survey map or a global positioning system. Thus the data set included village name, longitude and latitude, alongside data on individuals examined (age, sex, standard size of blood sample taken and presence/absence of Loa loa infection). A summary of the epidemiological data used is presented in Table 1. Table 1 Epidemiological data sets used in development of the spatial model for predicting the prevalence of Loa microfilaraemia Region Sex* Blood sample (μl) Prevalence of Loa loa Age range No. subjects No. villages Banyo-Bankim M 50.00 22.21 5–98 1783 16 Banyo-Bankim F 50.00 20.00 5–90 1815 South of Central Province M 30.00 32.81 5–80 381 5 South of Central Province F 30.00 27.37 5–80 453 South of Central Province M 50.00 24.76 5–86 941 17 South of Central Province F 50.00 18.49 5–90 1206 Lekie Department M 30.00 26.33 5–91 1052 14 Lekie Department F 30.00 19.10 5–92 1340 Lekie Department M 50.00 23.96 5–80 359 3 Lekie Department F 50.00 21.41 5–90 369 Mbam et Kim M 30.00 12.37 5–99 1293 24 Mbam et Kim F 30.00 4.53 5–95 1281 Western Province M 50.00 6.22 15–99 916 15 Western Province F 50.00 3.76 15–99 1036 * M = male and F = female General principles of the analysis The aim of the study was to identify variables which were significantly associated with the presence of a Loa microfilaraemia in the subjects examined. Besides data on individuals (age, sex, size of blood sample), we investigated whether some variables describing the environment of their place of residence (see list of variables below) were significantly related to the microfilaraemia. For these environmental variables, distance operatives indicating whether or not villages were within 5 km of potential at-risk variables were created. Distance operatives were based on the normal dispersal range of Chrysops which has been shown to be within 5 km of their breeding sites [19]. In order to design a valid modelling structure, univariate analysis was first undertaken of the relationship between, on the one side, individual and environmental variables, and on the other side the variable of interest, i.e. the presence/absence of Loa loa mfs in the individual. Then we developed a series of logistic regression models (multivariate analysis) to describe variation in the prevalence of Loa loa microfilaraemia using individual level co-variates (age, sex, μl of blood taken for examination) and village level environmental co-variates. Logistic regression is useful to predict the presence or absence of a characteristic or outcome based on values of a set of predictor variables. It is similar to a linear regression model but is suited to models where the dependent variable is dichotomous. Logistic regression coefficients can be used to estimate odds ratios for each of the independent variables in the model. In our example the dichotomous variable is the presence or absence of Loa loa infection in each individual in the sample. Those variables which did not have a significant relationship with age and sex adjusted prevalence were excluded from further analysis. All variables were analysed at individual level. Environmental variables and data sets included in the analysis The distribution of the human population in sub-Saharan Africa is relatively well described from population census data obtained at the national and sub-national level. The population data for 1990 used in this analysis was obtained from the Global Resource Information Database of the United Nations Environment Programme (UNEP-GRID), Eros Data Centre [20]. These population density surfaces (with 1 km resolution) have been derived from a model including population estimation for countries, population of major urban centres and transportation network and accessibility. They have been used in numerous studies involving population distribution in Africa, including studies on estimating the burden of malaria [21]. The USGS (United States Geological Survey) hydrologic digital dataset provides detailed descriptions of the topography of the area, including elevation (Digital Elevation Model, DEM), slope, aspect (direction of maximum rate of change in elevation between each cell and its eight neighbours and representing direction of slope), flow accumulation (defining amount of upstream area draining into each cell) [22]. Satellite data indicating the 'greenness' of the environment (Normalised Difference Vegetation Index, NDVI) was obtained from the VEGETATION sensor launched in 1998 onboard the SPOT 4 satellite system. NDVI images have been specifically tailored to monitor global land surfaces' parameters on a daily basis with a medium spatial resolution of one km [23]. They permit monitoring of seasonal and inter-annual variation in vegetation status and have been shown elsewhere to be important correlates of the spatial and temporal changes in the distribution of insect vectors of disease [24]. Free access is given to 10 daily synthesized maximum value composite products 3 months after insertion in the VEGETATION archive [25]. Thirty-six decadal NDVI images were obtained from SPOT VEGETATION satellite sensor data archive for 1999. The VEGETATION data used consists of 10 daily NDVI products, compiled from daily synthesis over the previous ten days, for the entire African continent, to which both radiometric corrections and geometric corrections have been applied to enhance product quality. The true value of NDVI was calculated from the 8 bit decadal images (1–255) using ((digital number*0.004)-0.1). From the true values images, mean, minimum, maximum, median, standard deviation were calculated for the year 1999. In order to take into account the flight distance of the vector, 5 km buffers were created around each study village and the mean value of the NDVI variable was obtained and used in the development of the logistic regression model. Validation of the model Epidemiological data used to validate the model was obtained from a multicentric study supported by TDR which was designed to assess the relationship between clinical and parasitological indicators of Loa loa endemicity [15,26]. The surveys were conducted by three research teams (comprising epidemiologists, parasitologists, social scientists and clinicians) based in Buea and Yaoundé, both in Cameroon, and in Calabar, Nigeria. Only data from Cameroon are used in our validation process. The Buea study sites were in South-West and North-West Provinces of Cameroon; in these areas, a total of 4532 individuals over the age of 15 years, living in 42 villages, participated in the survey. The study sites for the Yaoundé team were located in the East Province of Cameroon, where 3181 persons of the same age, living in 32 localities, were examined. A standardized questionnaire was administered to participants from whom finger-prick blood samples were collected and examined for Loa loa mfs. Model validation was undertaken through correlating model outputs with the independent data set. Results Univariate analysis While Loa loa prevalence when regressed against population density was not found to be significantly correlated, a strong and significant linear relationship (r2 = 0.7699; P < 0.001) was found with age below 40 years (Figure 1). In our analysis we used this linear relationship for all individuals below 40 but then treated all those above 40 as though they remained at this age since any subsequent change with age was deemed non-significant. This broken stick approach was considered to be a reasonable strategy and much simpler than fitting a non-linear model. Figure 1 Relationship between prevalence of Loa loa microfilaraemia and age. Prevalence rates were significantly (P < 0.001) higher in males than in females (19.8% n = 6725 and 15.2% n = 7500 respectively). Sex was therefore entered (as a categorical variable) in the logistic regression model. As one would expect, prevalence rates were significantly higher (P < 0.001) in individuals from whom the blood smear was prepared using a volume of 50 μl as compared with those from whom 30 μl of blood were taken. Blood sample size was therefore entered (as a categorical variable) in the logistic regression model. After investigating the value of the DEM and its associated files (slope, aspect, flow accumulation) in predicting microfilaraemia prevalence using univariate and multivariate regression statistics, the DEM alone was chosen as a predictive variable for the development of a logistic regression model. Because the relationship between Loa loa prevalence and the DEM was found to be non-linear (Figure 2) the DEM data was divided up into 250 m interval classes and treated as a categorical variable. Figure 2 Relationship between prevalence of Loa loa microfilaraemia and elevation. Since univariate analysis of the SPOT VEGETATION NDVI data against prevalence indicated a non-linear relationship [Figure 3a and 3b], the satellite data were entered into the model as a series of numeric variables (the original, the square and the cube) for the mean, the minimum, the maximum and the standard deviation. Standard deviation and maximum NDVI were most strongly correlated with prevalence using univariate analysis. Figure 3 Relationship between prevalence of Loa loa microfilaraemia and standard deviation of NDVI (a); and between prevalence of Loa loa microfilaraemia and maximum annual NDVI (b). Multivariate analysis Separate and combined models were created which included all individual level co-variates and altitude for both buffered and non-buffered SPOT VEGETATION data variables. Final model choice was based on (a) the simplicity and biological validity of the model (b) the predictive capacity of the model when assessed using bootstrap methodology (where half of the data was extracted randomly from the data set and tested against model results developed from the remaining half) and (c) the ability of the model to be extended over the large geographic area involved in the APOC programme. Thus, besides the three individual-level covariates (sex, age and blood sample size), only three village level environmental co-variates were kept in our model: maximum annual NDVI, standard deviation of the NDVI, and elevation. In the final model, the probability of a female individual being infected with Loa loa is represented by: 1/(1+e-z), where (for our example a female aged ≥ 40 years, and a blood sample of 50 μl): [Z] = ([Sex]*0.221) + ([Age] * 0.049) - ([Blood sample size]*0.542) + ([Maximum NDVI2] * 74.38) - ([Maximum NDVI3] * 58.816) + ([Standard deviation NDVI] * 11.788) + (([Elevation] < 250) * 1.133) + (([Elevation] = 250-500) * 0.865) + (([Elevation] = 500-750) * 0.981) + (([Elevation] = 750-1000) * 1.171) - (([Elevation] = 1000-1250) * 0.623) - (([Elevation] = 1250-1500) * 1.516) - (([Elevation] = 1500-1750) * 1.342) - (([Elevation] = 1750-2000) * 0.669) - 22.883 (constant), where [Sex] = 0 for females, and 1 for males; and [Blood sample size] = 0 for 30 μl, and 1 for 50 μl. Thus for our example, age, sex and blood sample size variables are presented for a fixed value because of their non-spatial nature. The probability of infection increases (+) with increasing value for sex (0 versus 1), with increasing age, and with increasing standard deviation of NDVI and Maximum NDVI2; it decreases (-) with Maximum NDVI3 (indicating that the relationship to maximum NDVI is non linear), and increases for categories of elevation between 0 and 1000 metres after which it decreases. The probability of infection decreases with increasing value of blood sample size (0 versus 1) even if larger blood sample should normally allow better detection of the infection. This could be due to the fact that larger samples have been taken often in places where infection rates are low, in the highlands, and this is presumably a result of interacting with the other variables. A similar model can be made for males and individuals at any other age. The model was developed using 50% of the data (randomly selected) and then tested on the remaining 50% of the data. When the model results were plotted against the observed data an r2 of 0.6033 was obtained (Figure 4). In addition, when one takes the threshold of 20% as the Loa microfilaraemia above which there is a risk of post-ivermectin encephalopathy, the sensitivity and specificity of the model (respectively: the proportion of villages with a measured prevalence ≥ 20%, and which were correctly predicted as such by the model; and the proportion of villages with a measured prevalence <20%, and correctly identified as such by the model) were found to be 77.1 and 80.0%, respectively (Table 2). Figure 4 Relationship between observed prevalence of Loa loa microfilaraemia and predicted prevalence. Table 2 Distribution of the villages examined, according to their observed and predicted prevalence of Loa microfilaraemia Observed prevalence <20% ≥ 20% Predicted prevalence First dataset <20% 48 8 (collected by IRD-CPC) ≥ 20% 12 27 Dataset used for validation <20% 20 5 (collected by TDR) ≥ 20% 18 31 Mapping the model results: an Environmental Risk Map for Loa loa In order to standardize the variables relating to individuals (which cannot be mapped) we chose values that best represent the average adult population (above the age of 15) i.e. 50:50 male – female ratio, age 40 and a blood sample of 50 μl. Age 32 was the approximate average age of individuals in the data sets used to develop the model. Using age 40 (the average age of individuals >15 years of age) in the model was therefore likely to slightly over-represent infection in these data sets but the final model would be directly comparable with current policy to exclude individuals below the age of 15 from rapid epidemiological surveys for loiasis [14,15]. In order to create a model, which included both males and females, two separate models were created, one for each sex and the mean of the two model results taken. Validation of the model Since the chosen model was based on environmental variables, which could be mapped across the entire country, it was possible to extrapolate the model results to the whole of Cameroon (Figure 5) and compare them with the results of the independent TDR survey. Correspondence between observed prevalence from the TDR verification data set and the model results was found to be very close: the sensitivity and the specificity of the model, when using the prevalence threshold of 20%, were 86.1 and 52.6%, respectively. Five villages were observed to have high prevalence rates (>20%) despite being model predictions for prevalence below 20% (Table 2). Two villages, Nguri and Ngu, located in the North-West province, were classified as extremes (Figure 6). Inaccuracies in the georeferencing of the village location or the spatial data used to create the model may account for this, as could the possibility that the local population regularly visit the Chrysops infested area nearby. The three other villages (Ntem, Boum and Baktala) are all villages from the Eastern Province of Cameroon which were sited on very localised areas where model predictions for <20% prevalence occurred. These villages were all within 1 km of areas where model results predicted >20% prevalence. Figure 5 Predictive model of Loa loa prevalence for Cameroon overlaid with the observed prevalence data. Figure 6 Inset of verification villages in North-West province of Cameroon (note the position of Nguri and Ngu). When one considers both datasets as a whole, and the prevalence threshold of 20%, the sensitivity and the specificity of the model were 81.7 and 69.4%, respectively. Discussion Detailed information on distribution of disease and levels of endemicity is a prerequisite for effective planning of control programmes. This has been an important element in the planning of the African Programme for Onchocerciasis Control (APOC) where community-directed treatment with ivermectin (Mectizan®) has been targeted at areas of hyper- and meso-endemicity, identified by Rapid Epidemiological Mapping [27]. The expansion of the APOC programme in Cameroon has been delayed due to severe (sometimes fatal) adverse reactions in patients receiving ivermectin who were co-infected with Loa loa. This has led to caution in defining new areas for the expansion of the programme and a requirement for identifying areas of high risk of Loa loa using a potentially rapid and extensive methodology. Thomson et al. [17] developed a preliminary model using satellite mapping based on existing knowledge of distribution and prevalence, and recently a method of rapid assessment using a simple questionnaire, called RAPLOA, has been developed [15]. This paper further develops the satellite-derived risk map of Loa loa by using improved satellite data sets and detailed information on infection rates in villages in various ecological zones in Cameroon. The resulting model is able to predict prevalence risk throughout southern Cameroon with a greater accuracy than hitherto available. The final model has been chosen because of the good fit between observed and predicted prevalence and is based on elevation and 1 km SPOT VEGETATION data. In order to understand the value of the risk map it is important to note that (a) while the 1 km resolution of the environmental data cannot reveal the small and localised muddy breeding sites of the Chrysops vectors, their general habitat is well captured by the model and it is recognised that the 1 km resolution is much less than the dispersal capacities of the adult flies; and (b) the model is not dependant on the seasonal or inter-annual variability of the vector density, since this is not reflected in fluctuations in Loa microfilaraemias, because the adult worm life-span is much longer (4–17 years). Among the areas in which the prevalences of Loa loa are particularly high, according to the Environmental Risk Map (ERM), two should be pointed out, because they are also those where most of the cases of serious adverse events (SAEs) have been recorded so far. The first one is the Lekie Department (Central Province), in the Sanaga valley, where 53 of the 63 probable or possible Loa loa encephalopathy cases recorded between 1989 and 2001 in Cameroon have occurred [3]. Besides the high level of endemicity, this cluster of cases may be related to the high population density there, as well as the proximity of the area with the capital, Yaoundé, which probably led to a high reporting rate; however, other hypothesis should be considered, such as specific susceptibility of the local human populations, or special pathogenicity of the local Loa "strain" [28,29]. The latter possibility is currently investigated in Cameroon, and in the Mayumbe forest (Democratic Republic of Congo), another area where some 15 cases of fatal SAEs were reported in December 2003. The second area of interest shown by the ERM in Cameroon is the Tikar plain, near Bankim, between the Western, North-West and Adamaoua provinces, where several cases have been recently reported, though the area's vegetation is of shrub savanna type. The ERM also shows those areas where CDTI projects against onchocerciasis may be implemented in a near future, and where the risk of SAEs is probably high: the Eastern and Southern Provinces, and the north-western part of the Littoral Province. While the use of satellite-derived environmental data for evaluation of disease risk has been developed for several vector borne diseases (malaria, Rift Valley fever, visceral leishmaniasis, tick-borne encephalitis) [24], limited use of such data in public health programmes for the control of infectious diseases has been achieved. This paper defines a model which identifies areas of potential high risk of severe adverse reactions to ivermectin, and will contribute to APOC's programme development by enabling resources to be effectively targeted to areas deemed at risk. The Technical Consultative Committee of APOC, and the Mectizan Expert Committee have developed a series of recommendations aimed at "facilitating effective detection and management of SAEs following treatment with Mectizan in known and suspected Loa loa endemic areas". Three types of mass treatment strategies have been defined, according to the levels of endemicity of onchocerciasis and of loiasis, the latter being defined by the prevalence of Loa microfilariae (<20 % versus ≥ 20%) or of history of eye worm passage (<40 % versus ≥ 40%). In those areas where onchocerciasis is meso- or hyperendemic, and the prevalence of Loa mfs exceeds 20%, a number of detailed measures should be taken, regarding training of community distributors and medical personnel, availability of medical supplies, duration of distribution, surveillance of the treated populations, and management of the patients who develop a SAE. Though lower, the risk that SAE occur in areas where the prevalence of Loa microfilaraemia is lower than 20% is not nil, and thus guidelines have also been developed regarding the strategy to apply in such situations [30]. The model developed is by no means a definitive product but provides a basis for decision making in terms of where rapid epidemiological surveys for loiasis should now be targeted. Modelling in this way permits an iterative process between field epidemiologist and modeller which not only means that decisions are made on the best available data but that such data is updated rapidly as new survey results are entered in the database and the model refined. Models are valuable in so far as they reflect reality. Given the complexity of disease transmission processes where human, parasite, vector and environment interact, it is impossible to think that all relevant factors can be incorporated into a general model which can be applied on a regional scale. What is significant here is the fact that important decisions need to be made now with regard to the likely spatial extent of the distribution of Loa loa. As it stands, the model presented here uses environmental features known to be associated with the biology of the vector, is robust for the area of data collection and predicts areas within Cameroon where Loa loa has been found in the past. It provides a rapid assessment methodology of areas where adverse reactions to ivermectin may occur and will be further developed in conjunction with the RAPLOA procedure which APOC intends to apply to CDTI areas potentially endemic for Loa loa [15]. The next step in the process will be to update the model with new data (e.g. the verification data set) from all the countries where loiasis is endemic, and to explicitly represent uncertainty in the model outputs so that decision makers will be able to assess for themselves the quality of the model results for their area of interest. List of abbreviations APOC African Programme for Onchocerciasis Control CDTI Community-Directed Treatment with Ivermectin CPC Centre Pasteur du Cameroun DEM Digital Elevation Model ERM Environmental Risk Map for Loa loa GIS Geographic Information System GPELF Global Programme to Eliminate Lymphatic Filariasis IRD Institut de Recherche pour le Développement Mfs Microfilariae NDVI Normalised Difference Vegetation Index NOAA-AVHRR National Ocean and Atmospheric Administration – Advanced Very High Resolution Radiometer RAPLOA Rapid Assessment of the Prevalence and Intensity of Loa infection SAE Serious Adverse Event SPOT Satellite Pour l'Observation de la Terre TDR UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases UNEP-GRID United Nations Environment Programme-Global Resource Information Database USGS United States Geological Survey Competing interests None declared. Authors' contribution MCT designed the study, planned the analysis, supervised the modelling, interpreted the results, and wrote the paper. VO prepared the data, developed the model, prepared the maps, and wrote the paper. JK and JG collected the field data used for the development of the model. SW, IT and PE collected the field data used for the validation of the model. JHR supervised the study thanks to which the validation data were collected. DHM proposed the study, contributed to its design and interpretation of the results. MB designed the study, planned the analysis, collected the field data, interpreted the results, and wrote the paper. Acknowledgements Mark Cresswell and Tudor Davies are acknowledged for assistance in data processing. This work was undertaken with financial support from The African Programme for Onchocerciasis Control (TSA 08/181/451/AP/98 and 08/181/502/AP/99). Dr. A. Sékétéli, Director of APOC, and Dr. M. Noma, Chief of the Epidemiology and Vector eradication Unit of APOC, are thanked for their encouragement and support. The independent data that were used for validation of the model was collected with financial support from the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR). ==== Refs Gardon J Gardon-Wendel N Demanga-Ngangue Kamgno J Chippaux JP Boussinesq M Serious reactions after mass treatment of onchocerciasis with ivermectin in an area endemic for Loa loa infection Lancet 1997 350 18 22 9217715 10.1016/S0140-6736(96)11094-1 Boussinesq M Gardon J Gardon-Wendel N Kamgno J Ngoumou P Chippaux JP Three probable cases of Loa loa encephalopathy following ivermectin treatment for onchocerciasis Am J Trop Med Hyg 1998 58 461 469 9574793 Twum-Danso NA Serious adverse events following treatment with ivermectin for onchocerciasis control: a review of reported cases Filaria J 2003 2 S3 14975060 10.1186/1475-2883-2-S1-S3 Klion AD Massougbodji A Horton J Ekoué S Lanmasso T Ahouissou NL Nutman TB Albendazole in human loiasis: results of a double-blind, placebo-controlled trial J Infect Dis 1993 168 202 206 8515109 Blum J Wiestner A Fuhr P Hatz C Encephalopathy following Loa loa treatment with albendazole Acta Trop 2001 78 63 65 11164753 10.1016/S0001-706X(00)00159-5 Kamgno J Boussinesq M Effect of a single dose (600 mg) of albendazole on Loa loa microfilaraemia Parasite 2002 9 59 63 11938697 Tsague-Dongmo L Kamgno J Pion SDS Moyou-Somo R Boussinesq M Effects of a 3-day regimen of albendazole (800 mg daily) on Loa loa microfilaraemia Ann Trop Med Parasitol 2002 96 707 715 12537632 10.1179/000349802125001933 Chippaux JP Boussinesq M Gardon J Gardon-Wendel N Ernould JC Severe adverse reaction risks during mass treatment with ivermectin in loiasis-endemic areas Parasitol Today 1996 12 448 450 15275280 10.1016/0169-4758(96)40006-0 Boussinesq M Gardon J Challenges for the future: loiasis Ann Trop Med Parasitol 1998 92 S147 S151 9861281 Boussinesq M Gardon J Gardon-Wendel N Chippaux JP Clinical picture, epidemiology and outcome of Loa-associated serious adverse events related to mass ivermectin treatment of onchocerciasis in Cameroon Filaria J 2003 2 S4 14975061 10.1186/1475-2883-2-S1-S4 Anonymous Treating onchocerciasis in loiasis endemic areas Mectizan Programe Notes 2000 25 1 2 Haselow NJ Akame J Evini C Akongo S Programmatic and communication issues in relation to serious adverse events following ivermectin treatment in areas co-endemic for onchocerciasis and loiasis Filaria J 2003 2 S10 14975067 10.1186/1475-2883-2-S1-S10 Boussinesq M Gardon J Prevalences of Loa loa microfilaraemia throughout the area endemic for the infection Ann Trop Med Parasitol 1997 91 573 589 9425360 10.1080/00034989760671 Boussinesq M Gardon J Kamgno J Pion SDS Gardon-Wendel N Chippaux JP Relationships between the prevalence and intensity of Loa loa infection in the Central province of Cameroon Ann Trop Med Parasitol 2001 95 495 507 11487371 10.1080/00034980120073184 Takougang I Meremikwu M Wanji S Yenshu EV Aripko B Lamlenn SB Braide LE Enyong P Meli J Kale O Remme JH Rapid assessment method for prevalence and intensity of Loa loa infection Bull World Health Organ 2002 80 852 858 12481206 Obsomer V Boussinesq M Kamgno J Mayaux P Connor SJ Molyneux DH Thomson MC Use of geographical information systems and remote sensing technologies in the mapping of Loa loa Trans R Soc Trop Med Hyg 2002 96 364 Thomson MC Obsomer V Dunne M Connor SJ Molyneux DH Satellite mapping of Loa loa prevalence in relation to ivermectin use in west and central Africa Lancet 2000 356 1077 1078 11009145 10.1016/S0140-6736(00)02733-1 Kamgno J Boussinesq M Hyperendémicité de la loase dans la plaine Tikar, région de savane arbustive du Cameroun Bull Soc Pathol Exot 2001 94 342 346 11845532 Chippaux JP Bouchité B Demanou M Morlais I Le Goff G Density and dispersal of the loaiasis vector Chrysops dimidiata in southern Cameroon Med Vet Entomol 2000 14 339 344 11016443 10.1046/j.1365-2915.2000.00249.x UNEP/GRID – Sioux Falls Snow RW Craig M Deichmann U Marsh K Estimating mortality, morbidity and disability due to malaria among Africa's non-pregnant population Bull World Health Organ 1999 77 624 640 10516785 Hydro 1K Africa Spotimage Thomson MC Connor SJ Environmental information systems for the control of arthropod vectors of disease Med Vet Entomol 2000 14 227 244 11016429 10.1046/j.1365-2915.2000.00250.x Free vegetation products Wanji S Tendongfor N Esum M Ndindeng Atanga S Enyong P Heterogeneity in the prevalence and intensity of loiasis in five contrasting bioecological zones in Cameroon Trans R Soc Trop Med Hyg 2003 97 183 187 14584374 Ngoumou P Walsh JF Macé JM A rapid mapping technique for the prevalence and distribution of onchocerciasis: a Cameroon case study Ann Trop Med Parasitol 1994 88 463 474 7979636 Duke BOL Overview: Report of a scientific working group on serious adverse events following Mectizan treatment of onchocerciasis in Loa loa endemic areas Filaria J 2003 2 S1 14975058 10.1186/1475-2883-2-S1-S1 Higazi TB Klion AD Boussinesq M Unnasch TR Genetic heterogeneity in Loa loa parasites from southern Cameroon: a preliminary study Filaria J 2004 3 4 15225348 10.1186/1475-2883-3-4 The Mectizan® Expert Committee and the Technical Consultative Committee Recommandations for the treatment of onchocerciasis with Mectizan® in areas co-endemic for onchocerciasis and loiasis

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==== Front Cardiovasc UltrasoundCardiovascular Ultrasound1476-7120BioMed Central London 1476-7120-2-111528578010.1186/1476-7120-2-11ReviewStress echocardiography in heart failure Agricola Eustachio [email protected] Michele [email protected] Matteo [email protected] Alberto [email protected] Division of Non-Invasive Cardiology, Cardiothoracic Department, San Raffaele Hospital, IRCCS, Milano, Italy2004 30 7 2004 2 11 11 12 7 2004 30 7 2004 Copyright © 2004 Agricola et al; licensee BioMed Central Ltd.2004Agricola et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Echocardiography has the ability to noninvasively explore hemodynamic variables during pharmacologic or exercise stress test in patients with heart failure. In this review, we detail some important potential applications of stress echocardiography in patients with heart failure. In patients with coronary artery disease and chronic LV dysfunction, dobutamine stress echocardiography is able to distinguish between viable and fibrotic tissue to make adequate clinical decisions. Exercise testing, in combination with echocardiographic monitoring, is a method of obtaining accurate information in the assessment of functional capacity and prognosis. Functional mitral regurgitation is a common finding in patients with dilated and ischaemic cardiomyopathy and stress echocardiography in the form of exercise or pharmacologic protocols can be useful to evaluate the behaviour of mitral regurgitation. It is clinical useful to search the presence of contractile reserve in non ischemic dilated cardiomyopathy such as to screen or monitor the presence of latent myocardial dysfunction in patients who had exposure to cardiotoxic agents. Moreover, in patients with suspected diastolic heart failure and normal systolic function, exercise echocardiography could be able to demonstrate the existence of such dysfunction and determine that it is sufficient to limit exercise tolerance. Finally, in the aortic stenosis dobutamine echocardiography can distinguish severe from non-severe stenosis in patients with low transvalvular gradients and depressed left ventricular function. stress echocardiographyheart failurediastolic dysfunctionmitral regurgitation ==== Body Background The identification of viable hibernating myocardium in patients with coronary artery disease and chronic left ventricular (LV) dysfunction is, up to today, the most common use of stress echocardiography in patients with heart failure. However, to search viable myocardium or the presence of contractile reserve is only one of plugs of the physiopathologic puzzle in a failing heart (Figure 1 and 2). If we consider the ability of echocardiography to provide valuable haemodynamic information accurately and non-invasively, it is ideally suited for application during stress testing to objectively assess other physiopathologic components of heart failure. These include the study of exercise physiology, the presence and the behaviour of concomitant mitral regurgitation (MR), the prediction of response to resynchronization therapy etc. Figure 1 Physiopathologic components of systolic heart failure that can be potentially explored with stress echocardiography. Figure 2 Physiopathologic components of diastolic heart failure assessable with stress echo. Therefore, the present review will detail some important potential applications of stress echocardiography in patients with heart failure in the evaluation of the different clinical and physiopathologic aspects of heart failure syndrome. Systolic heart failure Searching the myocardial viability The most common cause of heart failure in the Western world is coronary artery disease, accounting for up to 60% of cases [1]. In patients with coronary artery disease and chronic LV dysfunction, it is crucial to distinguish between viable and fibrotic tissue to make adequate clinical decisions. Noncontractile but viable myocardium may correspond to different states that are important but difficult to distinguish, i.e., ischemia, stunning, nontransmural infarction, or hibernation and in individual patients these pictures may coexist [2]. After brief episodes of coronary occlusion and reflow a reversible global LV dysfunction can occur. This phenomenon was called myocardial stunning [3]. It is characterized as prolonged mechanical dysfunction after coronary reflow despite resumption of normal perfusion and lack of permanent tissue damage. Stunning seems to result from alterations in contractile proteins in response to sublethal ischemic insults. This phenomenon can occur in several settings, including after acute reperfused myocardial infarction and after CABG. In humans, the return of functional recovery may require days to weeks [4]. Hence, diagnostic methods to distinguish stunning from necrosis are particularly relevant for clinical investigation and management in patients with acute, severe LV dysfunction or cardiogenic shock after revascularization. Persistent wall motion abnormalities can be observed by echocardiography at a time when chest pain, ST segment deviation, and regional perfusion had recovered. The presence of contractile reserve during dobutamine infusion identifies the stunning but viable myocardium from myocardial necrosis. The term "Hibernating myocardium" was first termed by Rahimtoola to indicate the state of reversible dysfunctional myocardium, which was considered to be the result of a state of persistently impaired myocardial function at rest, caused by reduced coronary blood flow, and which could be partially or completely restored to normal either by improving blood flow or reducing oxygen demand [5]. Echocardiography can detect viable myocardium during infusion of drugs which have ability to elicit an enhanced contractile response by recruiting contractile proteins. At least two drugs have these proprieties: the dobutamine, a synthetic β1 agonist with additional α1- and β2-stimulating properties and the enoximone that inhibits cyclic adenosine monophosphate-specific phosphosdiesterase [6,7]. Routinely, the dobutamine is the most common stressor used, whereas the enoximone is particularly useful in patients on beta-blocker therapy [7,8]. The mechanism by which dobutamine stimulation elicits a contractile response in hypoperfused dysfunctional segments without precipitating ischemia has been demonstrated by Sun et al. [6]. By using positron emission tomography and echocardiography, they demonstrated that the improvement in contractile function during dobutamine infusion was associated with a concomitant increase in myocardial blood flow. The increase in myocardial blood flow occurs because there is persistent, albeit reduced, coronary flow reserve distal to a stenosis which dobutamine may exploit. Another mechanism whereby contractile response may be elicited during dobutamine infusion is through its peripheral vasodilator effect, which causes reduction in LV end-systolic wall stress by reducing afterload [6]. Moreover, dipyridamole echocardiography (up to 0.84 mg/kg over 10 minutes) can identify regions with myocardial viability [9]. Dipyridamole leads to transiently increased coronary flow, which leads to improved contractility in viable myocardium [9]. A small study comparing dipyridamole with dobutamine revealed 93% concordance [10]. Combined dipyridamole-dobutamine (low-dose dipyridamole followed by low-dose dobutamine) has also been proposed and found to recruit a contractile reserve in some asynergic segments that were nonresponders after dobutamine or dipyridamole alone [11]. An initial evaluation of end diastolic wall thickness of akinetic segments with resting echocardiography can be used as an initial screening technique for assessment of viability. Indeed, akinetic regions with an end diastolic wall thickness <6 mm do not contain viable myocardium and do not improve in function after revascularization [12]. However, in segments with a thickness ≥ 6 mm, additional testing is needed because approximately 40% of these regions do not contain viable myocardium and will not improve after revascularization [12]. Therefore, myocardial thinning should not be equated with the lack of myocardial viability, and in some patients, these regions can improve in contractile function after revascularization [13]. The detection of subendocardial infarcts became clinically relevant because the quantification of non-viable myocardium in addition to viable myocardium in that region of LV is important in predicting contractile improvement following revascularization. Thus, the ratio of viable to total myocardium (viable plus non-viable) in the dysfunctional region was more accurate that absolute amount of viable myocardium alone in predicting functional improvement [13]. Unfortunately, currently available techniques, such as single photon emission computed tomography, dobutamine stress echocardiography and positron emission tomography are still insufficient to provide a comprehensive assessment including the evaluation of subendocardial infraction with respect to magnetic resonance imaging [14]. During stress echocardiography is possible to observe four response patterns based on regional wall function: normal, ischemic, viable and necrotic. In the normal response, a segment is normokinetic at rest and normal or hyperkinetic during stress. In the ischemic response, a segment worsens its function during stress from normokinesis to dyssynergy. In the necrotic response, a segment akinesia remains akinetic during stress. In the viability response, a segment with resting dysfunction improves during stress. During pharmacologic stress, a viable response at low dose can be followed by ischemic response at high dose (biphasic response). This biphasic response is suggestive of viability and ischemia, with jeopardized myocardium fed by a critically stenosed coronary artery [15]. A resting akinesia which becomes dyskinesia during stress reflects a purely passive mechanical phenomenon and should not be considered a true active ischemia. The overall sensitivity and specificity of dobutamine echocardiography for predicting recovery of regional function after revascularization was 84% and 81% respectively [16]. In a study by Afridi et al., the best predictive value for recovery of function after revascularization was most often noted in patients demonstrating an ischemic response during low and high doses of dobutamine infusion [17]. On the other hand, sustained improvement of regional function during dobutamine infusion was a poor marker of recovery function. Sensitivity of dobutamine echocardiography may be affected by several factors such as the severe reduction of myocardial blood flow that can preclude the contractile response, the premature interruption of dobutamine infusion, resting tachycardia that may renders the myocardium ischemic and dobutamine can only augment ischemia [16]. On the contrary, the specificity may be affected by the tethering effect, the injured subendocardial portion of myocardium that does not respond to dobutamine when the infarction is confined to subendocardium, and also specificity may be reduced in myocardial regions that do not develop an ischemic response [16]. The main clinical issue to search the myocardial viability is that patients with evidence of hibernating myocardium who do not undergo revascularization have poor prognosis with high incidence of cardiac events [18]. In contrast, evidence of viable myocardium in patients undergoing successful revascularization is associated with longer survival and improvement of both symptoms and LV function [19]. However, the presence of myocardial viability is only relevant in patients with severely depressed LV function and has a prognostic impact only when a significant amount of viable myocardium is present. Therefore, the final end point of searching the myocardial viability is to predict the recovery of global myocardial function after revascularization. At this purpose there is a relation between improvement in left ventricular ejection fraction (LVEF) and the number of segments with contractile reserve, indicating that extent of jeopardized but viable myocardium determine the magnitude of improvement of LV function after revascularization. Usually a level of ≥ 4 viable segments, which corresponds an improvement in wall motion score index >0.25 (about 20% of left ventricle), is advised as a cutoff value to predict improvement of LVEF [20]. However, despite the presence of substantial viability, in some patients LVEF does not improve after revascularization because not only the amount of dysfunctional but viable tissue but also LV remodelling and enlargement determines the improvement in function following revascularization [21]. Thus, patients with a high end systolic volume (≥ 140 ml) due to LV remodelling have a low likelihood of improvement of global function [21] (Figure 3). Figure 3 A schematic flow chart for searching segmental and global systolic function in chronic ischemic LV dysfunction. Assessing the functional capacity In most patients with chronic heart failure, symptoms are not present at rest but become limiting with exercise. Despite this, the major measures used to characterise the symptoms, the severity, the mechanisms and the prognosis of heart failure are obtained at rest. Exercise testing, in combination with echocardiographic monitoring, may be an attractive and practical method of obtaining accurate information which can aid in the diagnosis of heart failure as well as the assessment of functional limitation and prognosis. Exercise rather than dobutamine is the stressor of choice to evaluate functional capacity due to the possibility to combine echocardiographic variables with common parameters available during physiologic exercise. Symptom limited exercise testing can be undertaken using either treadmill or bicycle exercise protocols. Available data about the safety of exercise testing in patients with significant heart failure suggest a very low incidence of serious adverse events such as arrythmias or hypothension. The echocardiographic monitoring during exercise testing may have an additional value overt the conventional parameters assessed during exercise testing such as functional capacity, symptoms and peak oxygen uptake that become part of the final interpretation. Indeed, several haemodynamic parameters can be noninvasively obtained with echocardiography such as LVEF at rest and during stress deriving the contractile reserve, the behaviour of mitral valve function, the pulmonary artery pressure, the right ventricular function, the diastolic function (Table 1). In this way, it is possible to observe the variation of the monitored variables and to correlate these with the appearance of symptoms, i.e. impairment of global contractile function followed by increase in pulmonary pressure with dyspnoea. The critical level to define the presence of contractile reserve is generally defined as an increase of at least 5% (in absolute terms) in the global LVEF [22] (Figure 4). The change in the systolic pulmonary artery pressure (sPAP) at rest and during exercise is among others, the most frequently utilized echocardiographic variable. It can reliably be estimated by adding the right atrial pressure derived from the tricuspid reguritation jet velocity [23]. The right atrial pressure can be estimated at rest by the response of inferior vena cava to deep inspiration and assumed to be constant throughout exercise. Sometimes the use of echocardiographic contrast agents such as agitated saline solution may help to enhance Doppler signals. Pulmonary hypertension determined by echocardiography has been defined as a peak of sPAP >30 mmHg at rest and >45 mmHg during exercise [24]. Right ventricular dysfunction predicts impaired exercise capacity and decreased survival in patients with both moderate and advanced heart failure [25]. There are several clinically validated methods to detect right ventricular dysfunction. Tricuspid annular plane systolic excursion (TAPSE) visualized from the apical four-chamber view is an easy measure and can be used a surrogate of right ventricular function. A TAPSE value of 14 mm or less means the presence of right ventricular dysfunction and is a significant adverse prognostic indicator [26]. More recently, the evalution of tricuspid systolic annular tissue Doppler velocity has been introduced as index of right ventricular function and a value less than 10.8 cm/s indicates patients with abnormal right ventricular function [27]. Table 1 Potential parameters obtainable during exercise echocardiography. Common variables during exercise test Additional echocardiographic variables during exercise test Duration of exercise Contractile reserve Peak VO2 Mitral valve function Anaerobic threshold Pulmonary systolic pressure Oxygen pulse Right ventricular function VO2 workload ratio Diastolic function O2 saturation Figure 4 Echocardiographic apical four-chamber images (end-systolic frames) from two patients with and without contractile reserve. Moreover, these evaluations are useful not only for the diagnosis but also for predicting the outcome of patients overt the symptoms of heart failure. Indeed, some patients with marked reduction in myocardial contractility at rest, but with good residual contractile reserve, have a favourable exercise capacity and prognosis, whereas patients with mild symptoms and similar degree of abnormal myocardial contractility at rest, but without contractile reserve, have poor outcome [28]. Although a VO2max <14 ml/kg/min is well known as a measure for deciding on eligibility for cardiac transplantation, it has been clearly shown that there is no absolute threshold for adverse prognosis and that VO2max uptake should be considered as a continuous variable. In term of discriminating survivors from non survivors, it appears that VO2max <10 ml/kg/min definitively defines high risk, while a value >18 ml/kg/min defines low risk; those values in between may represent a grey zone. Thus stress echocardiography yields the greatest incremental prognostic value in patients with intermediate values of VO2max (10–14 ml/Kg/min) and helps to further stratify the risk of patients with intermediate (Table 2) [29]. Table 2 Additive prognostic value of stress echo in patients with intermediate values of VO2max (10–14 ml/Kg/min). Risk Low (5–10% year) High (≥ 25–30% year) Exercise capacity ≥ 8–10 min <8 min Contractile reserve yes no Pulmonary hypertension <45 mmHg >45 mmHg Right ventricular dysfunction no yes Mitral regurgitation ↓ or = ↑↑ Looking at the behaviour of mitral valve MR is a common finding in heart failure patients. In patients with dilated and ischaemic cardiomyopathy, the MR is typically functional and reflects geometric distortions of LV chamber, which displaces the normal valve and subvalvar closing mechanisms. This functional MR is a consequence of adverse LV remodelling and increased sphericity of the chamber. It is typically dynamic and a marker of adverse prognosis. The 5-year survival of heart failure patients with functional MR ranges from 39.9% to 48.7% depending on the degree of MR [30]. Stress echocardiography in the form of exercise or pharmacologic protocols can be useful in the assessment of MR. Exercise echocardiography is usually preferred due to the possibility to reproduce physiological setting. Even though supine bike protocol allows to obtain good image acquisition, upright bicycle or treadmill protocols are more frequently utilized in the practical setting. Treadmill exercises can be performed using the standard protocols such as Bruce or modified Bruce, while gradual increase in the bike workload of 20–25 W every 2–3 minutes is often applied until the patients achieves either the target heart rate or develops symptoms of fatigue or shortness of breath. Sometimes, pharmacologic stress is used with dobutamine protocol at low or intermediate doses infusion. In the collection of echocardiographic data should be included: the MR jet to evaluate the MR jet area and the vena contracta width, the velocity time integrals (mitral and aortic) to calculate the regugitant volume and the effective regurgitant orifice area (EROA), the LV volumes to assess the myocardial contractility and the tricuspid regurgitant jet velocity to measure the sPAP that is an useful index of the haemodynamic burden of MR. Exercise echocardiography can play several roles in the assessment of the behaviour of mitral valve in heart failure patients. First, in symptomatic patients with LV dysfunction and a clinical picture suspicious for new or worsening MR, but not evident at resting echo examination, exercise echocardiography can demonstrate a worsening of MR which helps to correlate this scenario with the patient's symptoms (Figure 5) [31]. Second, LV contractility, in presence of MR, can impair or improve during exercise with consequent modification of MR. Patients with presence of contractile reserve show a decrease in MR [32], whereas generally a fall in stroke volume is associated with an increase in mitral regurgitant volume during isometric exercise, which increases systemic resistances and thereby afterload [33]. These observations support the concept of presence of a vicious circle between LV function and behaviour of MR. Therefore, to study these patients with exercise echocardiography may be important for assessing the response of MR to medical therapy and for the following prognostic implications. Indeed, third, in patients with ischemic MR and LV dysfunction, quantitative assessment of exercise-induced changes in the degree of MR provides independent prognostic information. Significant exercise-induced increases in MR (increase in ERO ≥ 13 mm2 ) unmask patients at high risk of poor outcome. The cardiac mortality rate of medically treated patients with dynamic MR during exercise is 39% at 20 months which represents excess mortality in patients in functional class II or III (Figure 6) [34]. Figure 5 Apical four-chamber view at rest and during exercise in patients with ischemic mitral regurgitation showing a large exercise-induced increase in mitral regurgitation. SPAP = Systolic pulmonary pressure. Figure 6 Relationship between contractile reserve, mitral regurgitation and pulmonary pressure and its contribution in defining the prognosis in patients with functional mitral regurgitation. MR = mitral regurgitation; sPAP = systolic pulmonary pressure. Finally, dobutamine protocol has a different role in the contest of ischemic MR. Generally, in this setting it is used to evaluate the behaviour of MR in relation with the presence or absence of myocardial viability (Figure 7). Dobutamine infusion has the ability to decrease MR volume due to a reduction of afterload and mitral orifice size that may occur as a result of the vasodilatory and inotropic effects of dobutamine [35,36]. Therefore, if during dobutamine protocol we find myocardial viability and a concomitant reduction of MR, these results should be interpreted with caution because we cannot assume a direct effect of the presence of myocardial viability on the MR. Thus, the complex interplay between haemodynamic effects of dobutamine, myocardial viability and behaviour of MR has to be taken in mind during clinical management of patient with LV dysfunction and ischemic MR, i.e. revascularization alone versus revascularization plus mitral valve surgery. Figure 7 Targets and effects of dobutamine stress echo in patients with mitral regurgitation and chronic ischemic left ventricular dysfunction. EROA = Effective regurgitant orifice area. Evaluating the contractile reserve beyond hibernating myocardium It is commonly believed that the assessment of contractile reserve is only confined and clinically useful to search the myocardial viability in patients with LV dysfunction and coronary artery disease. Growing published data suggest the utility in searching the presence of contractile reserve in non ischemic dilated cardiomyopathy (DCM). While in the ischemic cardiomyopathy the search of myocardial viability is focused to find the presence of reversible segmental myocardial dysfunction and its possible effect on global systolic LV recovery after revascularization, in DCM the primary end point is to evaluate the presence of residual global contractile reserve. Both dobutamine and exercise testing have been used in the study patients with DCM, but there is a clear predominance for the use of dobutamine test. The doses of dobutamine utilized vary from investigators, but safety in its use in this population has been documented in doses as high as 40 μg/kg per minute. In the interpretation of results both wall motion score index and the LV volume to derive LVEF must be calculated. LV systolic function at the time of diagnosis has been proposed to be the strongest predictor of survival in DCM, but now the presence of contractile reserve recognised by dobutamine echocardiography seems to be the best marker of good prognosis in patients with severe LV dysfunction at rest [37,38]. Patients with significant improvement in their wall motion score index and LVEF during dobutamine infusion have a better survival rate and increase in the LVEF during follow-up period [37]. The data extracted from dobutamine study can be used as an adjunct or alternative to predict VO2max and exercise capacity of patients with heart failure, especially when the patients fall into the gray zone of VO2max (10–14 ml/kg/min) or when there is limitation to ambulation [29]. Moreover, the response to dobutamine infusion predicts the improvement in LVEF with beta-blocker therapy in patients with advanced heart failure. Patients with contractile reserve experienced a greater improvement in LVEF with beta-blocker by biologically augmenting myocyte a chamber contractility [39]. Whereas, in the absence of contractile reserve (when myocytes have been replaced by fibrosis), ventricular function cannot improve by this biological mechanism because there are not enough contractile units and the sympatholytic effects of beta-blocker prevail [39]. However, the clinical use of dobutamine stress echocardiography in patients with chronic heart failure may be limited by a substantial proportion of patients already receiving beta-blocker therapy at time of evaluation. In these patients enoximone echocardiography might be a valid alternative to low-dose dobutamine for evaluating contractile reserve showing a more potent and a better safety profile than dobutamine [8]. Stress echocardiography may also help in the identification of patients in the initial phase of cardiomyopathy overt normal resting echocardiographic parameters. Both dobutamine and exercise have to be used to screen for the presence of latent myocardial dysfunction in patients who had exposure to cardiotoxic agents [40]. Diastolic heart failure The prevalence of diastolic heart failure in the community is now to be at least as high as that reported in previous studies of hospitalised patients; almost half of all patients with heart failure have diastolic heart failure [41]. The term asymptomatic diastolic dysfunction is used to refer to an asymptomatic patient with a normal LVEF and abnormal echo-Doppler pattern of LV filling; this is often seen, for example, in patients with hypertensive heart disease. If such patients exhibit symptoms of effort intolerance and dyspnoea, especially if there are evidence of venous congestion and edema, the term diastolic heart failure can be used [42]. Resting echocardiography is most useful in the assessment of LV size, LVEF and the use of Doppler-derived indices of diastolic function has impact on the identification of diastolic dysfunction. However, to determine whether an abnormality of diastolic function is the cause of the patient's symptoms, we need to demonstrate the existence of such dysfunction and determine that it is sufficient to limit exercise tolerance. Therefore, the stress echocardiography, in particular exercise echocardiography could be useful in dyspnoeic patients with apparently normal LV function to unmask the presence of diastolic dysfunction (signs of elevated LV filling pressure) during exercise as cause of symptoms. Patients with diastolic heart failure, as well as those with diastolic dysfunction and little or no congestion, exhibit exercise intolerance for several reasons. First, an elevated LV diastolic and pulmonary venous pressure during exercise causes reduction in lung compliance, which increases work of breathing and evokes the symptom of dyspnoea [42]. Second, a substantial number of patients who have LV hypertrophy, high relative wall thickness and small end diastolic volume exhibit a low stroke volume and a depressed cardiac output [43]. These hearts exhibit a limited ability to utilize the Frank-Starling mechanism during exercise. Such limited preload reserve, specially if coupled with the chronotropic incompetence limits the cardiac output during exercise [44]. Third, elevated LV diastolic and pulmonary venous pressures in patients with normal LVEF are directly related to abnormalities in the diastolic proprieties of the ventricle. This is not to say contractile function is entirely normal, but subtle and latent abnormalities of contractile function could be present in many patients, in whom, however, diastolic dysfunction is the dominant feature [42]. All these aspects can be assessed during exercise echocardiography (Table 3). In particular the assessment of diastolic function during exercise has been shown to be feasible [45]. Combining transmitral flow velocity with annular velocity obtained at level of the mitral annulus with tissue Doppler (E/E') has been proposed as a tool for assessing LV filling pressures that combines the influence of transmitral driving pressure and myocardial relaxation [46]. Patients with rest E/E' >15 can be classified as having elevated filling pressure. A rest E/E' <8 suggests normal filling pressure and a range of 8 to 15 represents a gray zone. E and E' velocities increased significantly after exercise. In normal subjects because of proportional increases of both velocities, the E/E' ratio do not change significantly with exercise; this observation can be taken as a normal diastolic response during exercise [45]. Indeed, if E/E' ratio increases up to 15 we can suppose a pathological increase of LV filling pressure during exercise. This evaluation must be associated to the assessment of cardiac output and sPAP during exercise with appearance of symptoms. Finally, with the evaluation of systolic LV function during exercise it is possible to discover the portion of patients with concomitant latent myocardial dysfunction but predominant diastolic abnormality (Figure 8). Table 3 Useful echocardiographic parameters to evaluate diastolic function during exercise test in patients with suspected diastolic heart failure. Transmitral Doppler indices E/E' ratio Cardiac output Preload reserve Contractile reserve Pulmonary systolic artery pressure Figure 8 Schematic diagnostic algorithm in patients with suspected diastolic heart failure. LV = Left ventricle; LVEF = Left ventricular ejection fraction. Aortic stenosis with left ventricular dysfunction Stress echocardiography with dobutamine infusion is particularly useful in clinical decision making in patients with aortic stenosis with LV dysfunction and low transvalvular gradients. In this group of patients, an important clinical question rises: is the low gradient a consequence of low cardiac output due to a severe aortic stenosis which has led to LV dysfunction or is the low gradient a consequence of LV dysfunction is unrelated to aortic stenosis and the aortic stenosis is an incidental finding? It is well known that the transvalvular gradients are flow-depentent parameters so that they are influenced by LV function. The aortic valve area can be accurately determined by Doppler echocardiography with continuity equation and that correlate well with Gorlin formula [47]. However, it has been shown that valve areas calculated by the Gorlin formula is flow-dependent and usually increase with flow, probably due to the flow dependence of the empirical constant C of the Gorlin formula, which represents the ratio of effective to anatomical orifice area. Burwash et al., with dobutamine stress-echocardiography, demonstrate a flow-dependent increase in actual orifice aortic valvular area calculated with continuity equation [48]. Therefore, the assessment of valve area does not solve the diagnostic dilemma in these patients, because we cannot distinguish between severe fixed from flow-dependent (relative) aortic stenosis. Thus, it is important to perform pharmacological manoeuvres to increase cardiac output so that valve area can be calculated at higher flow rate. Dobutamine stress echocardiography until 20 γ/kg/min with concomitant evaluation of cardiac output, aortic valve area and gradients, is a useful and reliable test to distinguish between severe fixed from relative aortic stenosis in presence of low gradient and LV dysfunction. On the basis on test results, it is possible to distinguish 3 groups of patients [49] (Figure 9): 1. Patients with an improvement of contractile function but no significant increase in valve area and an increase of transvalvular gradients. These patients have severe fixed aortic stenosis and are good candidate for surgery with an acceptable peri-operative surgical risk. 2. Patients with contractile reserve with an increase of aortic valve area without substantial increase in transvalvular gradients. These patients have a non critical aortic stenosis and the LV dysfunction is not related to the aortic stenosis and should be treated conservatively. 3. Finally, patients without contractile reserve and no modification of valve area and transvalvular gradients. These patients represent an ambiguous group, because can represent patients with end-stage severe aortic stenosis with severe LV dysfunction or patients with severe LV dysfunction without contractile reserve and incidental aortic stenosis. However, this group has very poor prognosis. Figure 9 Possible results during dobutamine stress echocardiography in presence of aortic stenosis, low cardiac output and low transvalvular gradients. AVA: Aortic valve area; CO: Cardiac output. When interpreting the results of a dobutamine study in these patients to rule out or confirm definitively the presence of a severe fixed aortic stenosis, it is advisable to use an absolute cut-off value of the aortic valve area at peak of dobutamine >1 cm2 rather than an increase of ≥ 0.3 cm2 from baseline alone [49,50]. Conclusions Beyond the identification of viable hibernating myocardium, stress echocardiography is particular useful in patients with systolic and diastolic heart failure to assess the different physiopathologic component of heart failure syndrome and can aid to an appropriate clinical decision making. List of abbreviations LV: left ventricular MR: mitral regurgitation LVEF: left ventricular ejection fraction DCM: dilated cardiomyopathy TAPSE: Tricuspid annular plane systolic excursion sPAP: Systolic pulmonary artery pressure Competing interests The manuscript is not under consideration elsewhere and the data presented have not been previously published. All authors have read and approved the manuscript. No financial support was received for this study. The content of this manuscript is not associated with any financial interest or other relations that could lead to a conflict of interest. Author's contribution Concerning the authorship, the listed authors have contributed as follows to the manuscript: EA and MP: 1) conception, design, analysis and interpretation of data, 2) drafting of the manuscript and 3) final approval of the manuscript MO and AM: 1) critical revision of the manuscript for important intellectual content, and 3) final approval of the manuscript. ==== Refs Teerlink JR Goldhaber SZ Pfeffer MA An overview of contemporary etiologies of congestive heart failure Am Heart J 1991 121 1852 1853 2035416 10.1016/0002-8703(91)90072-P Shulz R Heusch G Characterization of hibernating and stunned myocardium Eur Heart J 1995 16 19 25 8746933 Braunwald E Kloner RA The stunned myocardium: prolonged, postischemic ventricular dysfunction Circulation 1982 66 1146 1149 6754130 Kloner RA Bolli R Marban E Reinlib L Braunwald E Medical and cellular implications of stunning, hibernation, and preconditioning: an NHLBI workshop Circulation 1998 97 1848 1867 9603540 Rahimtoola SH The hibernating myocardium Am Heart J 1989 117 211 221 2783527 10.1016/0002-8703(89)90685-6 Sun KT Czernin J Krivokapich J Lau YK Bottcher M Maurer G Phelps ME Schelbert HR Effects of dobutamine stimulation on myocardial blood flow, glucose metabolism, and wall motion in normal and dysfunctional myocardium Circulation 1996 94 3146 3154 8989122 Lu C Carlino M Fragasso G Maisano F Margonato A Cappelletti A Chierchia SL Enoximone echocardiography for predicting recovery of left ventricular dysfunction after revascularization: a novel test for detecting myocardial viability Circulation 2000 101 1255 1260 10725284 Ghio S Constantin C Raineri C Fontana A Klersy C Campana C Tavazzi L Enoximone echocardiography: a novel test to evaluate left ventricular contractile reserve in patients with heart failure on chronic beta-blocker therapy Cardiovasc Ultrasound 2003 1 13 14596683 10.1186/1476-7120-1-13 Picano E Marzullo P Gigli G Reisenhofer B Parodi O Distante A L'Abbate Identification of viable myocardium by dipyridamole-induced improvement in regional left ventricular function assessed by echocardiography in myocardial infarction and comparison with thallium scintigraphy at rest Am J Cardiol 1992 70 703 710 1519518 10.1016/0002-9149(92)90545-A Varga A Ostojic M Djordjevic-Dikic A Sicari R Pingitore A Nedeljkovic I Picano E Infra-low dose dipyridamole test. 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Cardiovasc Ultrasound

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==== Front Microb Cell FactMicrobial Cell Factories1475-2859BioMed Central London 1475-2859-3-91528203110.1186/1475-2859-3-9ResearchCharacterisation of the Escherichia coli membrane structure and function during fedbatch cultivation Shokri Atefeh [email protected] Gen [email protected] The Swedish Centre for Bioprocess Technology, Albanova University Centre, KTH SE-106 91 Stockholm, SWEDEN2004 28 7 2004 3 9 9 30 3 2004 28 7 2004 Copyright © 2004 Shokri and Larsson; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Important parameters during recombinant protein production in Escherichia coli, such as productivity and protein activity, are affected by the growth rate. This includes the translocation of protein over the membrane to gain better folding capacity or reduced proteolysis. To vary the growth rate two techniques are available: fedbatch and continuous cultivation, both controlled by the ingoing feed rate. Results During fedbatch cultivation, E. coli contains phosphatidylethanolamine, phosphatidylglycerol, cardiolipin and saturated fatty acids in amounts which are stable with growth rate. However, the levels of cardiolipin are very high compared to continuous cultivation. The reason for fedbatch triggering of this metabolism is not known but hypothesised to result from an additional need for carbon and energy. The reason could be the dynamic and sometimes rapid changes in growth rate to which the fedbatch cell has at all times to adjust. The membrane flexibility, essential for translocation of various components, is however to some degree sustained by production of increased amounts of unsaturated fatty acids in phosphatidylglycerol. The result is a functionally stiff membrane which generally promotes low cell lysis and is constant with respect to protein leakage to the medium. At comparatively high growth rates, when the further stabilising effect of cyclic fatty acids is gone, the high level of unsaturated fatty acids results in a pronounced effect upon sonication. This is very much in contrast to the membrane function in continuous cultivation which shows very specific characteristics as a function of growth rate. Conclusions The stiff and unchanging fedbatch membrane should promote a stable behaviour during downstream processing and is less dependent on the time of harvest. However, optimisation of protein leakage can only be achieved in the continuously cultivated cell where leakage is twice as high compared to the constant leakage level in fedbatch. If leakage is undesired, continuous cultivation is also preferred since it can be designed to lead to the lowest values detected. Induction at low growth rate (<0.2 h-1) should be avoided with respect to productivity, in any system, since the specific and total protein production shows their lowest values at this point. ==== Body Background For several recombinant proteins produced in Esherichia coli there seems to be a strong dependence of the productivity and product quality on the limiting substrate feed rate at induction [1-8]. Different feed profiles in fedbatch cultivation have thus been used to affect the growth rate and thus the production of the desired product. A common strategy to increase cell productivity and product quality is furthermore to export products to the periplasm. This reduces proteolysis since the periplasm contains less proteases and the more oxidising environment is favourable for correct folding of proteins containing disulphide bridges. Furthermore, the translocation to the periplasm can result in a primary purification provided that the outer membrane can selectively be removed which requires a stable inner membrane. Of particular importance for protein translocation processes are the anionic phospholipids but also unsaturated fatty acids. It was reported a great number of times that increased amounts of phosphatidylglycerol could facilitate protein translocation [9,13-15]. In continuous cultivation data has shown that there is clear growth rate coupling of the membrane phospholipid/fatty acid structure [9,10] but also a coupling between the structure and function [9]. It is also known that the accumulated amount of protein translocators are dependent on for example the medium composition but are also influenced by metabolic events like catabolite repression [11,12]. The latter process is known to be a function of the growth rate [12]. In continuous cultivation it was observed that conditions for establishment of an optimum in phosphatidylglycerol and unsaturated fatty acids could be achieved. An optimum in the accumulation of these compounds occurred at a growth rate of 0.3 h-1 simultaneously to an optimum in β-lactamase leakage over the outer membrane [9]. It was further shown that at already at growth rates lower than 0.3 h-1 unsaturated fatty acids where replaced by cyclic fatty acids [9] which was earlier thought to take place first in stationary phase, as shown in batch cultivation experiments [26,27]. If the formation of cyclic fatty acids is allowed to take place this influences some important functions of the membrane due to the higher rigidity caused by this rapid accumulation. This is of great concern since this generally constitutes the point of induction in protein producing processes but also since the cell organelles have a particular influence on the unit operations of the cell harvest and further in the downstream processing performance. There is thus a strong incentive to select and operate processes which allow the establishment of controlled growth conditions and to understand the specific growth characterises established with respect to the membrane at particular feed rates. Two main techniques are at present available: fedbatch and continuous cultivation. We have previously established data from continuous cultivation [9] which we here will compare to the present data from fedbatch cultivation. The fedbatch technique is used when a high cell density is the primary goal. A variety of feeding strategies can be applied for different research purposes where exponential feed can be used to create conditions that resemble a steady state in all points except for the continuous cell mass accumulation. This technique can however only be used for a few number of generations since the oxygen transfer rate rapidly becomes limiting. In order to reach a high cell density, i.e. cell densities of 50 g, l-1 and above, the traditional fedbatch concept established early in the former century based on bakers yeast and later on for antibiotics production, is generally used. The feed profile, characterised by this general concept, is initially exponential until the oxygen or heat transfer capacity of the reactor is reached and thereafter constant. This implies that the substrate concentration and the specific growth rate decline with cultivation time after the constant feed profile is adopted. This constitutes the advantage: the declining growth rate allows the cell mass productivity to increase at constant oxygen consumption/heat production. A further advantage of this cultivation mode, in comparison to a standard batch process with substrate overflow and to fedbatch with high exponential feed, is the possibility to avoid inhibitory by-products such as acetate. At a preset time during the feeding process, induction is performed. The drawback is that when the cell mass is very high the substrate consumption is very low and this point might not represent the optimal condition for high product formation, achievement of high product quality or facilitated transport of the protein. In continuous cultivation the feed/dilution rate is also used to control the growth rate where there is a limit of the chemostat to cultivation at the maximum or near maximum rates when the pH-auxostat is the preferred method [16]. Continuous cultivation relies on a theoretical steady state in all variables and the production can thus take place under constant growth conditions. Fedbatch cultivation for high cell density production, on the other hand, is a dynamic operation, as described above, where the cell at all times has to adapt to a new environment. Although the processes can be designed for induction at the same growth rate, the overall cell response might still severely differ due to the difference in process conditions preceding the induction, sometimes referred to as a result of the microbial "memory" of the recent past. One reason is that the time of adaptation of important cellular processes related to production and product quality by far exceeds those of the changes in substrate uptake. Thus, after a rapid change in the feed rate it might very well take some time before seen in a product variable. In spite of all benefits and drawbacks characterising both techniques, the fedbatch technique, using the concept described above, is the most common production technique in industry for recombinant production of protein pharmaceuticals in Escherichia coli. The goal of this work is to show how growth rate controls the performance in fedbatch cultivation with respect to the structure of the membrane and to compare these changes to the function of some selected process variables and methods. These data will, in turn, be compared to earlier data derived from continuous cultivation [9]. From this, conclusions could be drawn as to which are the bottlenecks of either method at a particular event which will facilitate the choice of production method and enhance the design of the growth rate profile of the chosen method to achieve an increased cultivation but also harvest and downstream processing performance. Results Escherichia coli W3110, a K-12 derived strain, was grown in fedbatch cultivation. The fedbatch concept was characterised by an initial exponential feed, started at time zero, of the growth limiting component, glucose, leading to the establishment of a specific growth rate of 0.6 h-1. This was followed by a constant feed phase where the growth rate declined asymptotically to a value of approximately 0.05 h-1. During this phase the cell mass increases linearly to a cell density of approximately 27 g, l-1, as expected (Figure 1). The characteristic and rapid declination in the growth rate, following the shift from exponential to constant feed, is also shown in the figure where the growth rate thus decreases by half in a little more than two hours. This feed process was designed to cover the larger part of the possible growth rates achievable with this strain of E. coli to permit comparison with earlier data from continuous cultivation [9]. However, the profile follows the general industrial feed profile concept except for the levels, which are generally kept below the limit for acetic acid production [9]. During the exponential feed phase acetic acid is thus formed however only in minor amounts due to the high glucose influx which induces overflow metabolism. This amount of acetic acid is consumed concomitantly to glucose during the rapid declination of the growth rate from 0.6 h-1 to 0.4 h-1. The phospholipid content of the E.coli membranes is shown as a function of time from constant feed start in Figure 2A. The amount of phosphatidylethanolamine (PE) is always predominant and constitutes 76–77% of the phospholipids in the cell membranes at all growth rates. The content of phosphatidylglycerol (PG) and cardiolipin (CL) is kept at a level slightly below 12 % throughout the cultivation and does not vary to a great extent. At a growth rate of approximately 0.3 h-1 a minimum of CL of 10.9 % is indicated which is also the time of the maximum PG level. At lower growth rates there is less PG than CL which is directly opposite to the amounts of these compounds at high growth rates. Apart from these small changes the phospholipid values are constant throughout the cultivation. The fatty acids are shown as a function of the time from constant feed start in Figure 2B. The fatty acids predominant in the E.coli cytoplasmic and inner part of the outer membrane, are saturated (SFA: C14:0, C16:0), unsaturated (UFA: C16:1, C18:1), and cyclic (CFA: C17:cyc). From this figure is evident that the amount of SFA's is the most stable during the declination of growth. There is only a slight reduction of the amount at a growth rate of approximately 0.3 h-1, which is accompanied by a maximum in the amount of unsaturated fatty acids. The unsaturated fatty acids are markedly decreased as the growth rate goes below 0.3 h-1 to 56/70 % of their maximum values, respectively. These maximal values, which coincide with the above mentioned minimum in CL/maximum in PG, thus constitutes a switch-point in the fatty acid and phospholipid metabolism during fedbatch cultivation. At this point there is also a minimum of cyclic fatty acids. The levels of the cyclic fatty acids are then rapidly increasing the lower the growth rate and from its minimum the cyclic fatty acids are dramatically increased by almost 100 % in less than two hours. This occurs although there is a supply of nutrients at all times which is in contrast to earlier literature where it is stated that it is the lack of carbon and energy in the stationary phase which results in cyclic fatty acid formation. During the continuing declination of the growth rate, the cyclic fatty acids continues to increase to a final of three times their minimum value. It should be noted that since the fatty acids of the lipopolysaccharide (LPS) were removed during the sample extraction, the results are only reflecting the inner part of the outer membrane and the cytoplasmic membrane. The three phospholipids were furthermore examined for their individual fatty acid content. The results are shown in Figure 3. Since the dominating phospholipid is PE the pattern of fatty acids in PE consequently mirrors the overall results seen in Figure 2B. The amount of saturated fatty acids does not change to a high degree in any phospholipid, the differences lie more in the changes of unsaturated and cyclic fatty acids. In PG the same general pattern as in PE is observable but the increase in C18:1 with growth rate is more pronounced and this increase is made at the expense of C17:cyc. In CL, it is obvious that the levels of the fatty acids do not change very much at all except for the well-documented increase in cyclic fatty acids towards low growth rate. Since the metabolic pathway depicts that the formation of CL is a condensation of two molecules of PG and since the fatty acid pattern in CL is not mirrored by the one in PG it is thus evident that there is a modification also of the fatty acids during the condensation. This is in favour of considerably less unsaturated fatty acids in CL specifically with respect to C18:1 and also less cyclic fatty acids compared to PG. The difference is however less at lower growth rates. The effects of these membrane changes were investigated on basis of the function of some selected methods and process variables: the mechanical strength of the membrane to ultrasound, the effect of osmotic chock, the shredding of endotoxin to the medium, the leakage to the medium and the amount of cell lysis. The mechanical strength of the membrane during different growth rates was quantified as the release of the periplasmic marker, β-lactamase, at different time of sonication using the same amount of cell and sonication effect. Figure 4 illustrates the release compared to French press data. A full release of the amount of the specific protein is achieved in all cases by a two-minute sonication except for the samples at the lowest growth rate. There is no evident change in the pronounced effect of sonication at growth rates down to 0.3 h-1 but from this point on there is continuously increased membrane stability. A comparison to the pattern of unsaturated fatty acid accumulation in Figure 2B shows the close resemblance between structure and performance and where the high amounts of the double bond might be the reason to the effect of the ultrasound. Osmotic shock is a frequently used method to release protein in the small scale. The methodology chosen as a model system gives a step-wise permeabilisation of the membrane, which is followed by breakage of the cell wall murein saccculus and formation of sphaeroplasts when lysozyme enters the cell [17]. Several conditions must be met before lysozyme can efficiently penetrate the outer membrane which was shown for several methods in the publication by Witholt et al [17]. The employed method relies six individual steps, as further described in material and methods. Figure 5 shows the drop in absorbance caused by each addition as a function of sampling at selected growth rates. At the top is shown the initial values and each following line in the diagram refers to the effect compared to the preceding addition. Consequently a large distance between two lines is a large effect and vice versa. The figure shows that the sucrose addition and the late addition of water results in the major effect on permeabilisation and, for unknown reason, also the initial dilution by Tris-HCl. The addition of lyzosyme, in itself, has no effect which is a result of the fact that it can only penetrate the membrane by the addition of water [17]. The effect, read as a change in absorbance is due to the change in the shape of the cells which was also clearly visible in the microscope. The growth rate dependent changes were evident between comparison of the first and second measuring points at high growth rate, specifically the result of the addition of water, but the effect stays thereafter more or less constant shown by the constant distance between the lines. The release of lipopolysaccharide, LPS, to the medium was measured by means of detection of the presence of a specific inner part of this structure, lipid A, which has properties of an endotoxin. This compound has to be removed in downstream processing of pharmaceutical proteins since it gives a toxic response in patients and must always be proven absent in the final product. It was considered likely that the changes in the underlying phospholipid and fatty acid structure might have large consequences for the release of this component to the medium which was to some extent confirmed by literature studies [12]. In Figure 6A the volumetric accumulation of endotoxin in the medium is seen as a function of time compared to cell mass accumulation. In this graph is also included the measurements of the cell metabolite, cyclic adenosine monophosphate (cAMP), which is important for carbon utilisation and which was proposed to constitute the signal for changes leading to membrane reorganisation during statis and stress [18]. The figure shows that all metabolites are accumulated with time. In Figure 6B are plotted the specific accumulation of the parameters of figure 6A and it can be concluded that endotoxin and cAMP accumulation are constant and follows the cell mass accumulation. A major concern of this work was the effect of membrane structural changes on protein leakage from the periplasm to the medium. Since it is clear that a periplasmic protein can be present in the medium both by cell lysis and by true leakage from the periplasm, cell lysis was registered as a function of cultivation time. This is shown in Figure 7A. The leakage to the medium due to cell lysis was detected both by total protein analysis and by presence of DNA in the medium (the latter results not shown). Both variables showed the same pattern. At low growth rates there seems to be a marked change of the amount of total protein in the medium but it should be noted that the value is exceedingly low. The conclusion is that during the present fedbatch operation cell lysis is at all times very low and does not exceed 1 %. Earlier data from continuous cultivation [9] has shown that both total and specific protein synthesis can vary at different growth rates. To understand if this takes place also in fedbatch cultivation the total protein was measured at different growth rates. In Figure 7A is seen that total protein accumulation in each cell is constant down to a growth rate of 0.2 h-1 where after it slowly declines from 0.50 to a final value of 0.42 g protein, g cells-1. In Figure 7B is plotted the time course of the formation of the specific protein of interest i.e. periplamsic protein β-lactamase, which is constitutively produced from its own promoter. The total β-lactamase production is shown as well as the specific production values in the medium per gram of cells. It is clear that during fedbatch cultivation the total amount of this specific protein follows the total protein accumulation pattern and is constant independent if calculated per cell mass or per total cell protein except for the values at very low growth rate. The β-lactamase value per gram of cells in the medium is thus slightly higher during the first part of the cultivation down to μ = 0.3 h-1 where after the value declines. At very low growth rate there is yet another period of increased specific accumulation. It is realised that this latter period of slowly increased accumulation in the medium coincides with an increased however small lysis in the end of the cultivation. Since the leakage is not increased during this period lysis seems most likely cause for the accumulation. The level of β-lactamase in the medium amounts to approximately 10% of the total β-lactamase production throughout the cultivation as seen from the quotient of β-lactamase in the medium in relation to the total value. Discussion The conditions during fedbatch leads to a structure, which is approximately constant with respect to phospholipids and saturated fatty acids compared to growth rate. The changes in lipid components arrive from changes in unsaturated fatty acids where the increased values above the maximum at 0.3 h-1 is formed in expense of cyclic fatty acids which are accumulating at low growth rate. Compared to literature data the amount of cardiolipin is approximately 70 % higher than from earlier observations [9]. The resulting effects on the membrane function are several but are generally and overall characterised by their stability as a function of growth rate. Cell lysis is low at all times i.e. the membrane is not easily breaking-up and this is due to the high content of cardiolipin at all times which is further emphasised by high levels of cyclic fatty acids specifically at growth rates below 0.3 h-1. The high content of cardiolipin is to some extent balanced by high level of unsaturated fatty acids, which compensates the need for flexibility, permeability and movement of compounds within the membrane which growth rate. The growth dependent accumulation and the generally high level of unsaturated fatty acids influence the results of sonication. The high levels above μ = 0.3 h-1 thus supports a weaker membrane, in this respect, while the substitution of unsaturated to cyclic fatty acids reduces the effect at low growth rate. The transport of protein over the membrane is however only slightly higher at higher amounts of unsaturated fatty acids which is implicated by the larger effect of enzyme/water penetration during osmotic chock and the leakage of the model product, β-lactamase, at high growth rate. In total, β-lactamase is however always present in the medium at a level of approximately 10 % of the total production due to that the membrane flexibility caused by unsaturated fatty acid is balanced by the high and constant amount of cardiolipin. There is a very slight increase in cell lysis at the end of cultivation. The reduced capacity of this cell for total and specific protein production at low growth rate, which might be due to a reduced amount of ribosomes [1], low supply of carbon and energy and the comparatively high maintenance demand, makes it likely that also the protein content in the membranes is reduced with increased susceptibility to lysis as a result. The changes at low growth rate often results in a change in the shape of the cells which are known to become smaller and more round [19] which might influence the performance. Flow cytometric studies could however not reveal any major changes of these cells during the time of the process (data not shown). Continuous cultivation gives theoretically always the highest productivity due to the reduced shut-down periods for cleaning and refilling. However this technique is rarely used in industrial production of recombinant protein production due to problems of separation of growth and product formation. The pharmaceutical industry will furthermore need a validation protocol, which must be derived for this technique and where the concept of batch reproducibility needs some clarification. There is also a tendency to use techniques which are since long established and to which the personnel are educated. However, sometimes reasons are not well developed and a problem is the lack of data for a scientific choice of production technique. In table 1 and 2 continuous cultivation and fedbatch data are summarised as a function of growth rate in a process based on the same cell, the same vector, the same medium and the same product. Table 1 shows selected values of membrane components and data has been collected as to represent either a relatively high (0.6 h-1) or a low (0.05 h-1) growth rate. The table furthermore indicate if curves, with respect to growth rate, show a trend which reveals the existence of maximum/minimum values of the specific compound. Table 2 shows the comparative effects on a selection of functional parameters at the same growth rates. One of the most significant structural differences between the cultivation techniques is the high content of CL in fedbatch cells in expense of both PG and PE. The formation of large amounts of CL is an obvious cell strategy to save energy and carbon by the release of glycerol to further metabolisation. Since the only difference between the two cultivation techniques is the constant conditions in continuous cultivation and the dynamically changing ones in fedbatch the increased need for carbon/energy must lie in the rapidly changing growth environment. In fedbatch there are also more unsaturated fatty acids accumulated, which develop in expense of saturated fatty acids, which are higher in continuous cultivation. The fatty acid level is also more or less constant in CL, which promotes stability. The conclusion is that cells in fedbatch cultivation interpret the environmental signals in a way that leads to a membrane structure with several features that generally only develop during nutrient exhaust in stationary phase. This is not the case for cells growth in continuous cultivation which develop distinct characteristics at each stable growth rate. This leads to a fedbatch membrane that is rigid compared to cells in continuous cultivation. The overall result is fewer changes in the membrane function due to growth rate. This is represented by constant leakage, constant lysis and an almost constant effect of permeabilisation. The fedbatch membrane shreds also less endotoxin although levels are low for both cultivation techniques. However, the fedbatch membrane is sensitive to sonication, specifically at high growth rate due to the comparatively high amount of unsaturated fatty acids and thus releases more β-lactamase due to the growth dependent changes of this fatty acid. From the comparative data of table 1 and 2, from both process techniques it is evident that there is a growth dependent switch-point in the membrane regulation at approximately 0.3 h-1. However, this is not allowed to develop in fedbatch to the extent seen in continuous cultivation where a specific growth rate is kept much longer of the process time. The result is that no optimum in the leakage to the medium is clearly established in fedbatch cultivation and the fedbatch process cannot be designed for this since a constant growth rate of 0.3 h-1 cannot be kept while at the same time leading to a high cell accumulation. Protein transport is generally considered to benefit from a high content of PG and unsaturated fatty acids. This accumulation leads to high leakage which is verified by the continuous cultivation leakage optimum [9] and which leads to a comparatively high overall level in fedbatch (present data). The high fedbatch content of unsaturated fatty acids is however counteracted by signals leading to reduce levels of PG in favour of CL. The control of cellular activities at the switch-point leading to the establishment of the membrane compound pattern cannot be clarified at this point. However, it is clear that it is not the signals relating to the accumulation of cAMP. This compound is indeed a function of the growth rate, but not of the accumulation of the structural components. The overall conclusion is thus that if leakage is undesired, fedbatch should not be used but the process could well be run in continuous cultivation at low growth rate. If an increased leakage is preferred this can be optimised in the continuous operation mode principally by change of growth rate but probably also by mutants accumulating a combination of phosphatidylglycerol and unsaturated fatty acids if these are stable enough for prolonged cultivation. Conclusion The stability of the fedbatch membrane, which is not growth dependent, is the strength of this cultivation technique. The time of cell harvest is thus not critical since the membrane strength and function does not rapidly change which allows for flexibility in operation. The stability of the fedbatch derived cell membrane might thus also by the same reasoning be an advantage in further downstream processing however depending on the chosen unit operations. The merits of the cell characteristics in continuous cultivation are due to the possibility to run the cultivation at a feed rate which is optimal for membrane leakage independent if this is to be small or maximised. Total and specific protein production is lowered at low growth rate and the data points to that recombinant protein production should not be performed below a growth rate of 0.2 h-1 and induction should thus take place well above this value. This is a considerable drawback in fedbatch cultivation, which relies on high cell density accumulation leading to low growth rates before induction but easily controlled in continuous cultivation. The coupling of the productivity and even product quality to a specific growth rate further strengthens the implication of using continuous cultivation. The conclusion is that the drawbacks of industrial operation of continuous cultivation in the biopharmaceutical industry earlier mentioned should be preferably be solved to explore the benefits that are obviously present with this technique. Methods Strain and medium Escherichia coli W3110 (F-, λ-, IN (rrnD-rrnE)) [20], a K12 derivative with a maximum growth rate, in the selected medium and temperature, of 0.65 h-1, was used. The plasmid pBR322 (Pharmacia Biotech, gene Bank Accession number V0119) was transferred to the host cell. This plasmid carries the genes bla and tet where β-lactamase (TEM-1) was used as the model product. The maximum levels were approximately 1 % of the total protein. The medium for the bioreactor and the feed was a mineral salts medium composed of (g, l-1): (NH4)2SO2, 7.0; KH2PO4, 1.6; Na2HPO4 *2H2O, 6.6; (NH4)2-H-citrate, 0.5. Glucose was autoclaved separately and added to bioreactor together with a sterile filtered trace element solution and 1.0 M MgSO4 (1 ml, l-1, respectively). A further addition of magnesium, of the same amount, was done at eight hours of feeding. The glucose concentration in the feed was 300 g, l-1. The trace elements solution was composed of (per litre): CaCl2 *2H2O, 0.5g; FeCl3 *6H2O, 16.7 g; ZnSO4 *7H2O, 0.18 g; CuSO4 *5H2O, 0.16g; MnSO4 *4H2O, 0.15 g; CoCl2 *6H2O, 0.18 g; Na-EDTA, 20.0 g. Cultivation Conditions Four independent fedbatch cultivations were performed in the following manner: inoculum was prepared from a shake flask culture grown overnight at 37°C at 150 rpm. This was transferred into the bioreactor when an optical density at 600 nm (OD600) of 2–3 was reached from which point the fedbatch cultivation was performed. The experiments were carried out in a 14-l bioreactor with a working volume of 8.0 l. The bioreactor was equipped with an air sparger and the air-flow was 0.5–1.0 vvm. By varying of the stirrer speed up to 1000 rpm the dissolved oxygen concentration did never go below (30 ± 2) % air saturation. A pH of 7.0 was kept by addition of 28% (w/w) ammonia solution. Temperature was controlled at 37°C. The cultivations were started as batch cultures with an initial glucose concentration of 2 g, l-1. The feed was started when glucose was almost exhausted. The feed profile consists of two different phases where an exponential feed was followed by a constant feed phase. The exponential feed profile was calculated from the following equation: F(t) = F0 * e-μt where F is the feed rate (L/h), μ the desired specific growth rate (1/h) and t is the process time (h). The calculation of the initial feed rate, F0, is based on a mass balance at the time of the feed i.e. after the batch growth assuming: pseudo steady state growth during the feed phase, a theoretical yield coefficient (Yx/s) of 0,5 g, g-1 and a negligable amount of the limiting substrate in the reactor. F0 =(μ *V*X)/(Si*Yx/s) Where Yx/s is the cell yield on glucose (g/g), X is the cell concentration at F0 (g/L), V is the culture volume (L) and Si is the glucose concentration in the feed (g/L). The continuous cultivation conditions and data, compared to in the conclusions section, were reported elsewhere [9]. These were designed for steady states at similar growth rates to those reported from the fedbtach cultivations of this paper. Steady state was kept for approximately eight generations and the same strains, vectors and media were used. Analyses Cell mass, substrate and metabolites The cell growth was followed by optical density at 600 nm. Biomass concentration was determined as cell dry weight (CDW, g, l-1) by 10 min centrifugation (4500 rpm, 2250 g) of 3*5 ml of cell suspension in preweighed test tubes and drying of the pellet overnight at 105°C before weighing. The supernatant from the CDW samples was sterile filtered and used for acetic acid and cyclic adenosine monophosphate, cAMP, analysis. Acetic acid was measured with the enzymatic method from Boehringer Mannheim GmbH (Cat. No 148 261) and cAMP was analysed by an enzyme immunoassay system from Amersham Pharmacia Biotech AB (code RPN 225). Samples for glucose analysis were taken as described earlier [21]. The β-lactamase assay was performed as described in reference [22]. Inductively Coupled Plasma (ICP) analysis was used for magnesium determination. Total protein and DNA analysis were used for cell lysis detection. Total-protein was measured according to the method by [23] using bovine serum albumin as standard (Sigma Chemical Co, USA). DNA was measured by a fluorescence assay and quantified with Hoechst 33258 (Sigma Chemical Co, USA) according to the Dyna Quant 200 application method (Pharmacia, Sweden). The amounts of endotoxin i.e. lipid A, the inner part of the lipopolysaccharide (LPS) structure, was measured by the chromogenic Limulus method (Chromogenix, LAL, U.S. License No. 1197). Mechanical strength of the cell membrane The mechanical strength was estimated by sonication (Vibra-Cell, Sonics & Materials, USA) at constant frequency, cell dry mass, sample volume and acoustic power. Samples were placed in a water bath at +4°C during cell disruption to prevent overheating. The release of β-lactamase protein was determined at the time events of 0.5, 1.0, 1.5 and 2.0 minutes and compared to cell that was disintegrated in a high-pressure homogeniser (French press FA-073) at 55 bar. Data from French press was set as the 100% disruption level and all other values were taken as a percentage of this value. Chemical strength of the cell membrane Chemical strength was estimated by permeabilisation as described by Witholt et al [17]. The following steps were thus undertaken: (1) t = 0 min, 0.5 g, l-1 cultures were harvested and suspended in 0.3 ml, 200 mM Tris-HCL (pH 8.0), (2) t = 1 min, 0.5 μl, 100 mM EDTA (pH 7.6) was added, (3) t = 2 min, 0.5 ml, 1 M sucrose (pH 8.0) was added thus giving excess of EDTA over Mg2+ ions, (4) t = 3.5 min, egg white lysozyme (EC 3.2.1.17, C. F. Boehringer und Soehne GmbH, Mannheim, Germany) was added to a final concentration of 60 μg/ml, (5) t = 4 min, the cell suspension was exposed to a mild osmotic shock by two-fold dilution in water to trigger lysozyme penetration of the outer membrane, and finally (6) t = 8 min, the cell suspension was diluted 11-fold in 10 mM EDTA (pH 7.6). Permeabilisation was recorded by optical density at 450 nm. The samples taken at different growth rate were diluted to the same cell concentration (0.5 g, l-1) and the same volume (0.2 ml) was used at the sampling point. Phospholipids and fatty acids analysis Phospholipids and fatty acids were determined by the method of Arneborg [24] where the total and individual phospholipid content was quantified by an inorganic phosphorous assay after extraction and thin liquid chromatography. The presented value is the mol% of individual to the total phospholipids. The standard deviation was +/- 0.03 %. Fatty acids were released from extracted phospholipids by transmethylation with KOH in methanol. The methyl esters are thus representing the actual fatty acid. Chromatography was performed with C13:0 as an internal standard as described by [25]. The presented value is the mol% of individual to the total fatty acids (shortened for the number of carbon atoms according to: C14:0, C16:0, C16:1, C18:1 and 17:cyc). The methodology is further described in reference [9]. Author's contributions AS made the fermentations, analyses and data treatments and evaluation. The project was conceived by GL who further participated in the project planning, design and data evaluation. All authors read and approved the final manuscript. Acknowledgement This work was sponsored by the Swedish Centre for Bioprocess Technology, CBioPT which is gratefully acknowledged. Figures and Tables Figure 1 Time course of the fed-batch cultivation with E.coli W 3110. The process is started at time zero with an exponential feed phase corresponding to a specific growth rate of 0.6 h-1, followed by constant feed at approximately 2,5 h. Filled circles: specific growth rate (h-1), filled squares: acetic acid accumulation (mg, l-1), triangles: cell dry weight (CDW,g, l-1) and open squares: glucose accumulation (mg, l-1). Figure 2 A. Accumulation of phospholipids as a function of cultivation time. Filled squares: phosphatidylethanolamine (PE), open squares: phosphatidylglycerol (PG), circles: cardiolipin (CL). B. Accumulation of fatty acids as a function of time. Open squares: C14:0, filled squares: C16:0, filled triangles: C16:1, open triangles: C18:1, circles: C17cyc. The specific growth rate is indicated in the figure by a dashed line. Figure 3 Accumulation of fatty acids in the specific phospholipids PE, PG and CL. The five bars from left to right at each time event in the figure are: C14:0, C16:0, C16:1, C18:1, C17:cyc. The specific growth rate is indicated in the figure by a dashed line. Figure 4 Effect of sonication time on E. coli W3110 shown as β-lactamase release compared to French press data. Top filled squares: French press data. From top to bottom: 2, 1.5, 1, 0.5 minutes sonication at constant effect and cell mass. The specific growth rate is indicated in the figure by a dashed line. Figure 5 Effect of osmotic chock on E.coli W3110 cells represented by a drop in absorbation units at a specific addition. Addition from top to bottom: initial value, Tris-HCl, EDTA (100 mM), sucrose, lysozyme, water, EDTA (10 mM). The specific growth rate is indicated in the figure by a dashed line. Figure 6 Accumulation of endotoxin (lipid A of lipopolysaccharide layer) and cAMP as a function of cultivation time. The specific growth rate is indicated in the figure by a dashed line. A. Volumetric accumulation of cAMP and endotoxin compared to CDW accumulation. The arrow indicates an addition of magnesium sulphate. B. Accumulation of cAMP and endotoxin in μmol and mg per CDW, respectively. Figure 7 A. Total protein accumulation per CDW (filled squares), protein accumulation per CDW in the medium (circles) and cell lysis (protein in the medium/total protein, open squares) as a function of time. B. β-lactamase accumulation per CDW (filled squares), β-lactamase accumulation in the medium per CDW (circles) and leakage from periplasm to medium (β-lactamase in medium/total β-lactamase, open squares). The specific growth rate is indicated in the figure by a dashed line. Table 1 Membrane structural changes as a function of growth rate and cultivation technique STRUCTURE 1 Continuous cultivation Fed-batch cultivation Comment Parameter Low μ Max/Min High μ Low μ Max/Min High μ Min/Max values which correlate with μ = 0.3 h-1 Phospholipids (%) PE 70 75 78 77 n.p. 77 PG 16 18 15 12 ** 11 Max value only in CC CL 8 7 7 12 * 12 PS 7 - - - - - Compound not produced in FB Fatty acids (%) C14:0 3 1 2 3 n.p. 3 Min value only in CC C16:0 53 49 52 44 * 44 Min value only in CC C16:1 16 22 21 19 28 26 Max value only in CC C18:1 8 20 14 12 21 18 Max value only in CC C17:1 20 7 11 22 9 9 Min value only in CC 1Summary of the structural differences in continuous and fed-batch cultivation as a function of growth rate. Results compared to data from Shokri et al 2002. N.p.: max/min not present. CC: continuous cultivation, FB: fed-batch cultivation. * value is slightly decreased compared to the value at high μ. ** value is slightly increased compared to the values at high μ. A high growth rate refers to μ = 0.6 h-1 and a low growth rate to μ = 0.05 h-1. Table 2 Membrane functional changes as a function of growth rate and cultivation technique FUNCTION 2 Continuous cultivation Fed-batch cultivation Comment Parameter Low μ Min/Max High μ Low μ Min/Max High μ Total protein production (per CDW) R High High R High High Yield drops from the theoretical 0.5 to approx. 0.4 Specific protein production (per tot prod.) R High High. R High High Model substance: periplasmic β-lactamase Cell lysis (%) 8 1 1 1 <1 <1 Effect of sonication (% release of specific protein) 56 78 73 57 80 85 Result of 1 min sonication Endotoxin release (% of CDW) 1 1 1 <1 <1 <1 Periplasmic leakage 1.5 19 7 10 10 9 2Summary of the structural differences in continuous and fed-batch cultivation as a function of growth rate. Results compared to data from Shokri et al 2002. A high growth rate refers to μ = 0.6 h-1 and a low growth rate to μ = 0.05 h-1. ==== Refs Sandén AM Prytz I Tubulekas I Förberg C Le H Hektor A Neubauer P Pragai Z Harwood C Ward A Picon A Teixera de Mattos J Postma P Farewell A Nyström T Reeh S Pedersen S Larsson G Limiting factors in Escherichia coli fed-batch production of recombinant proteins Biotechnol Bioeng 2003 81 158 166 12451552 10.1002/bit.10457 Seo JH Bailey JE Effects of recombinant plasmid content on growth properties and cloned gene product formation in Escherichia coli Biotechnol Bioeng 1985 XXVII 1668 1674 Ryan W Collier P Loredo L Pope J Sachdev R Growth kinetics of Escherichia coli and expression of a recombinant protein and its isoforms under heat shock conditions Biotechnol Prog 1996 12 596 601 8879154 10.1021/bp960045x Ramirez DM Bentley WE Fed-batch feeding and induction policies that improve foreign protein synthesis and stability by avoiding stress responses Biotechnol Bioeng 1995 47 596 608 Hellmuth K Korz DJ Sanders EA Deckwer WD Effect of growth rate on stability and gene expression of recombinant plasmids during continuous and high cell density cultivation of Escherichia coli J Biotechnol 1994 32 289 298 7764721 10.1016/0168-1656(94)90215-1 Curless C Pope J Tsai L Effects of preinduction specific growth rate on recombinant alpha consensus interferon synthesis in Escherichia coli Biotechnol Prog 1990 6 149 152 1366547 Riesenberg D Menzel K Schulz V Schumann K Veith G Zuber G Knorre WA High cell density fermentation of recombinant Escherichia coli expressing human interferon alpha1 Appl Microbiol Biotechnol 1990 34 77 82 1366975 Jensen EB Carlsen S Production of recombinant human growth hormone in Escherichia coli : Expression of different precursors and physiological effects of glucose, acetate and salts Biotechnol Bioeng 1990 36 1 11 Shokri A Sandén AM Larsson G Growth rate dependent changes of Escherichia coli membrane structure and recombinant protein leakage Appl Microbiol Biotechnol 2002 58 386 392 11935192 10.1007/s00253-001-0889-0 Arneborg N Salskov-Iversen AS Mathiasen TE The effect of growth rate and other growth conditions on the lipid composition of Escherichia coli. Appl Microbiol Biotechnol 1993 39 353 357 Seoh HK Tai PC Carbon source-dependent synthesis of SecB, a cytosolic chaperone involved in protein translocation across the Escherichia coli membranes J Bact 1997 179 1077 1081 9023186 Shokri A Sandén AM Larsson G Cell and process design for targeting of recombinant protein into the culture medium of Escherichia coli. Appl Microbiol Biotechnol 2003 60 654 664 12664143 Van Klompenburg W de Kruijff B The role of anionic lipids in protein insertion and translocation in bacterial membranes J Membr Biol 1998 162 1 7 9516232 10.1007/s002329900336 Thommassen J de Vrije T de Cock H Bosch D de Kruijff B Involvement of membrane lipids in the protein export in Escherichia coli J Cell Sci Suppl 1989 11 73 83 2693462 Nesmeyanova MA On the possibility of participation of phospholipids in the translocation of secreted proteins through the bacterial cytoplasmic membrane FEBS letters 1982 142 189 193 7049732 10.1016/0014-5793(82)80131-2 Larsson G Pham H Enfors SO The PH-auxostat as a tool for studying microbial dynamics in continuous fermentation Biotech Bioeng 1990 36 224 232 Witholt B Heerikhuizen Van H de Leij L How dose lysozyme penetrate through the bacterial outer membrane? Biochim Biophys Acta 1976 443 534 544 822877 DiRusso CC Nyström T The fats of Escherichia coli during infancy and old age: regulation by global regulators, alarmones and lipid intermediates Mol Microbiol 1998 27 1 8 9466250 10.1046/j.1365-2958.1998.00645.x Prytz I Sandén AM Nyström T Farewell A Wahlström Å Förberg C Pragai Z Barer M Harwood C Larsson G Fed-batch production of recombinant β-galactosidase using the universal stress promoters uspA and uspB in high cell density cultivations Biotechnol Bioeng 2003 83 595 603 12827701 10.1002/bit.10716 Smith MW Neidhardt FC Proteins induced by anaerobiosis in Escherichia coli. J Bacteriol 1983 154 336 343 6339476 Larsson G Törnkvist M Rapid sampling, cell inactivation and evaluation of low extracellular glucose concentrations during fed-batch cultivation J Biotechnol 1996 49 69 82 8879165 10.1016/0168-1656(96)01534-9 Georgiou G Shuler ML Release of periplasmic enzymes and other physiological effects of β-lactamase overproduction in Escherichia coli Biotechnol Bioeng 1988 32 741 748 Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein dye binding Anal Biochem 1976 72 248 254 942051 10.1006/abio.1976.9999 Arneborg N Influence of ethanol on the lipid composition of Saccharomyces cerevisiae. PhD thesis, Technical University of Denmark, Copenhagen 1994 Patton GM Fasulo Jm Robins SJ Separation of phospholipids and individual molecular species of phospholipids by high-performance liquid chromatography J Lipid Res 1982 23 190 196 7057106 Nikaido H Outer membrane In Esherichia coli and Salmonella, Cell and Molecular Biology 1996 1 Ed in chief: FC Neidhardt, ASM Press Washington DC Cronan JE Phospholipid alterations during growth of Escherichia coli J Bact 1968 95 2054 2061 4876126

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==== Front BMC Med ImagingBMC Medical Imaging1471-2342BioMed Central London 1471-2342-4-21528387010.1186/1471-2342-4-2Research ArticleDiagnostic accuracy of ultrasonography compared to unenhanced CT for stone and obstruction in patients with renal failure Ather M Hammad [email protected] Aftab H [email protected] M Nasir [email protected] Section of Urology, Department of Surgery, Aga Khan University, Karachi, Pakistan2004 29 7 2004 4 2 2 10 3 2004 29 7 2004 Copyright © 2004 Hammad Ather et al; licensee BioMed Central Ltd.2004Hammad Ather et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background To determine accuracy of ultrasound (US) kidney, ureter and bladder (KUB) compared to un-enhanced helical CT (UHCT) in patients with renal failure in the diagnosis of stone and obstruction. Methods This is a case controlled study conducted in the period from June 2000 to July 2003 at a university hospital. All patients had both US and UHCT scan. Patients with serum creatinine ≥ 1.8 mg/dl were included in the study. Only direct visualization of stone was considered as confirmatory. In both the studies, UHCT and US, presence of stone and obstruction were noted. The relevant biochemicals, radiological and clinical records of all the patients were analyzed. Data was analyzed using commercially available software. Results During the period of study 864 patients had UHCT for evaluation of the urinary tract in patients presenting with flank pain. Out of these 34 patients had both UHCT and US done within a span of one day and had serum creatinine of ≥1.8 mg/dl. Mean age was 48 ±15.8 years and 59% of patients were males. UHCT identified renal stones in 21 (62%), whereas 17 of these were identified on US, with a sensitivity of 81%. Of the four patients with renal stones missed on US, three were identified on plain x-ray; the mean size of stones missed was 6.3 mm. Of the 22 (65%) patients with ureteric stone on UHCT, US could only identify 10; a further 7 were identified on x-ray KUB, giving a sensitivity of 45% (US alone) and 77% (US with x-ray KUB). Conclusions US is sensitive and specific for renal stones, 81% and 100% and for hydronephrosis, 93% and 100%, respectively. Its sensitivity to pick ureteric stone (46%) and to identify hydroureter (50%) is low. Addition of x-ray KUB abdomen increases the sensitivity for ureteric stones to 77%. ==== Body Background Intravenous urography (IVU) has been the gold standard for the radiological survey of intra renal collecting system, ureter and bladder. Choice of imaging for urinary tract in patients with raised serum creatinine is limited to non-contrast enhanced studies. These considerations have led to the use of other modalities like combination of plain abdominal radiography (KUB) and gray scale ultrasound (US) kidney, ureter and bladder. More recently use of non-contrast enhanced CT (UHCT) and magnetic resonance urography (MRU) in the evaluation of flank pain has received increasing attention [1,2]. Work in the past decade has shown UHCT to be highly sensitive and specific [1,3,4]. It is highly sensitive for both renal and ureteric stone [3]. The probability of misdiagnosis in distal ureter with multiple phleboliths is still a significant problem. Presences of tissue rim [3,5] and comet tail [5] signs along with secondary signs of obstruction are helpful in these situations. Ultrasound has many inherent advantages, which includes lack of radiation, universal availability, in expensive and non-invasive. It is useful in the diagnosis of renal and ureteric calculi. Stones on US are characteristically demonstrated as highly echogenic foci with distinct acoustic shadowing. The greatest challenge with regard to US is the identification of ureteral calculi, particularly in it's abdominal, and upper pelvic course. This limitation of US is due to its inability to scan retroperitoneum due to overlying bowel loop, and bony structures [4,6] Plain abdominal radiograph also lacks specificity, as phleboliths are not readily differentiated from ureteric calculi. Plain radiographs are also not sensitive to radiolucent calculi and non-calculus obstruction. In the present study we have compared the diagnostic accuracy of UHCT with US with x-ray KUB for the diagnosis of renal and ureteric stones in patients with raised serum creatinine precluding the use of contrast enhanced study. Methods This is a case controlled study conducted in the period from June 2000 to July 2003 at a university hospital. All patients who had both US and un-enhanced helical CT (UHCT) scans performed within a span of 24 hours and a serum creatinine ≥ 1.8 mg/dl were included in the study. Serum creatinine of 1.8 mg/dl is considered as a cut off for use of intravenous contrast by our radiology department. The radiologist's reports on CT, CT films and medical records of patients for suspected renal/ureteral colic were reviewed. The UHCT were obtained on a Cti/pro single slice helical CT scanner (General Electrical medical systems, Milwaukee, WI). The exposure factors setting were KVp 130 and mAS 200–250. All scans were obtained from the upper border of T12 vertebral body to the lower border of symphysis pubis using 5–7 mm collimation, without the use of oral or intravenous contrast material. Patients were placed in supine position with full urinary bladder at the time of the UHCT. Additional prone films were taken whenever the radiologist needed a better description of suspected distal ureteric calculi. Ultrasound KUB was done using 3.75 MHz surface probe. All ultrasounds were seen and reported after being reviewed by a senior radiologist. Secondary signs of obstruction, like hydronephrosis, hydroureter, nephromegaly, perinephric and periureteric stranding were also noted but only direct visualization of stone was considered confirmatory. The relevant biochemicals, radiological and clinical records of all the patients were analyzed. In the studies, UHCT, and US presence of stone and obstruction were noted. Data was analyzed using commercially available software (statistical package for social sciences version 8.0). Results During the 38-month period of study 864 patients had UHCT for evaluation of the urinary tract in patients presenting with flank pain. Out of these 34 patients had both UHCT and US done within a span of one day and had serum creatinine of ≥ 1.8 mg/dl. UHCT was considered as a reference point in the study as all stones identified on the CT were subsequently reconfirmed with interventional treatment or history of spontaneous passage. Mean age was 48 ± 15.8 years (range 20–76 years), 59% of patients were males. UHCT identified renal stones in 21 and ureteric stones in 22 patients. Forty-two (98%) of these stones were confirmed clinically (history of spontaneous passage), or during treatment with ureteroscopy, percutaneous nephrolithotomy and extracorporeal shock wave lithotripsy. Of the 21 renal stones, only 17 were identified on US, with a sensitivity of 81%, specificity and positive predictive value of 100% and negative predictive value of 77%. Of the four patients with renal stones missed on US, three were identified on x-ray KUB; the mean stone size of stones missed on US was 6.3 mm. In all cases US and x-ray KUB were performed prior to the UHCT. Of the 22 patients with ureteric stone, on UHCT, US could only identify 10. Twelve patients with ureteric stones identified on UHCT were missed on US. The mean size of stones missed was 6.1 mm (range 3–15 mm). The sensitivity, specificity, positive and negative predictive values were 46, 100, 100 and 50% respectively. A further 7 patients, missed on US, were identified on x-ray KUB. The overall sensitivity of US and x-ray KUB was 77%. The impact of location of stones missed on US is shown in table 1 and 2. Table 1 The impact of location on detection of stone and hydronephrosis by UHCT and US. Upper ureter Middle ureter Distal ureter n 6 14 2 Identified on CT 6 14 2 Identified on US 4 5 1 Hydrouretero-nephrosis on CT 6 14 2 Hydrouretero-nephrosis on US 4 8 1 CT un enhanced helical CT US gray scale ultrasound Table 2 Site and size of stones missed on US, the mean size of stones missed was 6.1 mm. Stone identified/total Mean size Upper ureter 2/6 (33%) 6 mm Middle ureter 9/14 (64%) 5 mm Distal ureter 1/2 (50%) 7 mm Discussion IVU has been the traditional imaging modality of choice for evaluation of patients suspected of having urolithiasis and obstruction. Choice of imaging for urinary tract in patients with renal insufficiency and renal failure is limited to non-contrast enhanced studies. Gray scale ultra sonography is the most effective way to exclude sub acute or chronic obstruction. However, regular gray scale US is not accurate in minimally dilated obstruction, such as with partially obstructing ureteric stone; in one series, 4–5% of patients with obstruction showed minimal or no upper tract dilatation [7]. Duplex Doppler is less effective in acute and incomplete obstruction since obstruction for longer than six hours is necessary to show a consistently elevated resistive index (RI) [8]. Therefore, we did not evaluate RI values or ureteric jets in our study. Others have also recently examined the role of RI with disappointing results. Cronan showed that the addition of RI did not improve the 77% sensitivity of gray scale US in that series [9]. US has high sensitivity for renal stones and presence of hydronephrosis. But its sensitivity for ureteral calculi is low. In one study, where IVU was compared with US, the sensitivity of US for ureteral calculi was only 37% (direct visualization) and when hydronephrosis was included as positive sign for ureteral calculi the sensitivity increased to 74% [10]. Recent studies have demonstrated that UHCT is an excellent method for demonstrating urolithiasis and obstruction in patients presenting with flank pain [1-3]. Smith et al [3] showed UHCT to be more effective than IVU in identifying ureteral stones. In another comparative study, Sommer et al [4] noted that reformatted (see Figure 2), UHCT images are superior to US and plain radiographs. Data from our institution showed that UHCT has a sensitivity of 99% and specificity of 98% in the diagnosis of ureteric calculi [1](US and plain radiograph Figure 1 and Figure 2). Additionally UHCT could also suggest additional, non-urinary tract abnormalities as cause of flank pain in 12% of patients [11]. Sensitivity of US is reported to be 96 % for renal stones and is 100% sensitive for stones larger than 5 mm in reported literature [1,12]. In our study US had sensitivity and negative predictive value of 81 and 77%. If x-ray KUB is added the sensitivity increased to 95%. The four patients with renal stones missed on US had a mean stone size of 6.3 mm. Lower sensitivity in our work could be due to small sample size. In the present study US alone had a sensitivity of only 46% for direct visualization of ureteric stones, in combination with x-ray KUB it increased to 77%. The 12 stones missed on US had a mean size of 6.1 mm (range 3–15 mm). Majority of stones missed on US were in the middle ureter (n = 9), 2 were in the proximal ureter and one in the distal ureter. X-ray KUB identified 7 of the 12 stones missed on US. Of these 12 patients with ureteric stones missed only 2 had hydroureter. Presence of hydroureter in patients with ureterolithiasis is valuable as it allows the ureter to be traced to the level of obstruction. Majority (9 out of 12) of stones missed on US were in the middle ureter, an area often obscured by bowel gas. Conclusions In summary, US is the first imaging study for evaluating the patients with previously undiagnosed renal failure. It helps the clinician to separate end stage renal disease from potentially reversible obstructive uropathy secondary to urolithiasis. US is highly sensitive and specific for renal stones in patients with renal failure, it lacks sensitivity for ureteric calculi particularly when they are in the middle ureter. Even addition of x-ray KUB to US misses about a quarter of ureteric stones; we therefore recommend using UHCT if ureterolithiasis is clinically suspected or US and x-ray KUB examinations are equivocal. Due to small sample size, findings of this study should be validated by other studies on a larger cohort of patients. Competing interest None declared. Authors' contribution MHA conceived the idea, analyzed data and drafted the manuscript. AHJ collected the data and analyzed the results. MNS analyzed results and drafted the manuscript. Figure 1 US of 65 years old male presented to emergency room with bilateral flank pain, nausea and vomiting for the past 1 week. He had an ultrasound in a peripheral hospital, which identified hydronephrosis on the right side, and percutaneous nephrostomy tube was placed. His left kidney showed hydronephrosis with renal stone (upper picture). This scan shows small-scarred right kidney (middle picture), pigtail catheter could be identified (arrow) and a proximal ureteric stone could also be seen (lower picture). Figure 2 Reformatted unenhanced helical CT image of the same patient showing small-scared right kidney with proximal ureteric calculus and hydroureter. Left kidney shows hydronephrosis and renal calculus. Pre-publication history The pre-publication history for this paper can be accessed here: ==== Refs Ahmed NA Ather MH Rees J Unenhanced helical computed tomography in the evaluation of acute flank pain Int J Urol 2003 10 287 292 12757595 10.1046/j.1442-2042.2003.00628.x Sudah M Vanninen RL Partannen K patients with acute flank pain: comparison of MR urography with un enhanced helical CT Radiology 2002 223 98 105 11930053 Smith RC Rosenfield AT Acute flank pain: comparison of non-contrast enhanced CT and intravenous pyelography Radiology 1995 194 789 794 7862980 Sommer FG Jeffrey RB Rubin GD Detection of ureteral calculi in patients with suspected renal colic: value of reformatted non contrast helical CT AJR Am J Roentgenol 1995 165 509 13 7645461 Guest AR Cohan RH Korobkin M Assessment of clinical utility of the rim and comet-tail signs in the differentiating ureteral stones from phleboliths AJR Am J Roentgenol 2001 177 1285 91 11717067 Dunnick RN Sandler CM Newhouse JH Amis ES Jr Nephrocalcinosis and nephrolithiasis In Text book of uroradiology 2001 3 Philadelphia, Pa: Lippincott Williams & Wilkins 178 194 Spital A Volvo JR Segal AJ Non-dilated obstructive uropathy Urology 1988 31 478 482 3287742 10.1016/0090-4295(88)90211-7 Platt JF Rubin JM Ellis JH Acute renal obstruction: evaluation with intrarenal duplex Doppler and conventional US Radiology 1993 186 685 688 8430174 Cronan JJ Tublin ME Role of the resistive index in the evaluation of acute ureteral obstruction AJR Am J Roentgenol 1995 164 377 78 7839973 Aslaksen A Gothilin JH Ultrasonic diagnosis of ureteral calculi in patients with acute flank pain Eur J Radiol 1990 11 87 90 2253643 10.1016/0720-048X(90)90153-3 Ahmed NA Ather MH Rees J Incidental diagnosis of disease on un-enhanced helical computed tomography for ureteric colic BMC Urol 2003 3 2 12675951 10.1186/1471-2490-3-2 Middleton WD Dodds WJ Lawson TL renal calculi: sensitivity for detection with US Radiology 1988 167 239 244 3279456

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BMC Med Imaging. 2004 Jul 29; 4:2

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BMC Med Imaging

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==== Front Filaria JFilaria Journal1475-2883BioMed Central London 1475-2883-3-51528386510.1186/1475-2883-3-5Short PaperFailed attempts at experimental transplantation and transmission of nocturnally-periodic simian Loa from monkey to man Duke BOL [email protected] River Blindness Foundation, 2 Hillside, Lancaster LA1 1YH, UK2004 29 7 2004 3 5 5 19 5 2004 29 7 2004 Copyright © 2004 Duke; licensee BioMed Central Ltd.2004Duke; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper describes unsuccessful attempts to induce a nocturnally-periodic infection with simian Loa in a human volunteer (the author of this paper) by means of 1. Transplanting adult simian Loa worms from a wild drill (Mandrillus leucophaeus) to man; and 2. Infecting the same volunteer by sub-cutaneous inoculation with infective larvae of simian Loa from a laboratory-bred, experimentally infected Chrysops silacea. ==== Body Findings In the mid 1950s, one of the main lines of research followed by the Helminthiasis Research Unit (HRU), at Kumba in the then British Cameroons was to establish the relationship between human infections with Loa loa and the highly similar Loa parasites which were found in several species of local monkey, especially in the drill (Mandrillus leucophaeus) but also, less frequently, in several long-tailed monkey species, Cercopithecus nictitans martini, C. mona mona and C. preussi. Observations on 109 young drills (found to be free from natural Loa infection after a quarantine period of 6–8 months in screened cages), which were experimentally infected by transplantation of live adult simian Loa worms extracted from wild drills shot in the local forests, revealed that the parasites exhibited a nocturnal microfilarial periodicity with microfilariae (mf) that were significantly longer than those of human L. loa. By contrast, drills which had been infected by inoculation of infective larvae from the day-biting Chrysops silacea or C. dimidiata, that had been experimentally infected 10 days previously by feeding on the blood of Loa-infected human volunteers, demonstrated diurnally-periodic microfilarial infections, whose parasites (both adult worms and mf) were of shorter length than the normal wild monkey parasites [1]. Further studies on the biting habits of local Chrysops species revealed that in nature the diurnally periodic human Loa loa was transmitted among humans by the day-biting species C. silacea and C. dimidiata, whereas the nocturnally periodic simian parasite was being transmitted among monkeys by two forest-canopy-dwelling species, C. langi and C. centurionis, both of which bite in the forest canopy at night and are presumed to feed mainly on sleeping monkeys [2]. No naturally-acquired diurnally periodic microfilarial infection was seen in 21 wild drills, the periodicities of whose adult worms were examined by transplantation into uninfected animals; despite the fact that male and female worms of human and simian Loa were quite capable, when transplanted together under experimental conditions into uninfected drills, of inter-breeding and producing microfilariae of intermediate dimensions and periodicity [3]. (This is not to say that under natural conditions diurnally-periodic worms of human Loa are never transmitted to monkeys, but such events, if they do occur, appear to be rare). Later, Belgian workers in the Mayumbe District in the south-western part of the Democratic Republic of the Congo (DRC), commented on the local human Loa infections in that area being particularly liable to give rise to cases of Loa-encephalopathy after treatment with diethylcarbamazine citrate (DEC). They also noted that out of 547 patients from this area, whose blood films were examined both by day and night, 197 showed Loa mf only by day, 322 showed them by day and by night; and 16 showed mf only at night. Although the latter cases, with a complete reversal of the normal periodicity, showed only light microfilarial loads (12 cases with 1–5 mf per examination, and 4 cases with 6–9 mf per examination), among those persons who showed Loa mf by day and by night, 62 showed microfilarial concentrations that were nearly as high by night as by day and 10 showed more mf by night than by day (two of them showing 500 – 1,000 mf by night as compared with 300 – 500 mf by day) [4-6]. As some of the cases of loiasis from Mayumbe were abnormal in displaying a primarily nocturnal periodicity of the microfilariae, it is possible that the local strain of Loa responsible for them may be closely related to the simian parasite. Recently in the Republic of Cameroon, cases of Loa-encephalopathy have been reported following mass treatment with ivermectin by the African Programme for Onchocerciasis Control (APOC) in areas where loiasis is co-endemic with onchocerciasis [7-11]. A remarkable clustering of many of these cases was found in the Lékié Division, a forest and forest/savannah mosaic area some 80 km from the capital, Yaoundé. So far, the reason for this clustering has not become apparent but the occurrence of these cases of Loa-encephalopathy has had a deleterious effect on the popularity of the APOC campaign in that area [12]. Furthermore, at the end of 2003 in the Mayumbe area of the DRC, some 100,000 persons were treated with a standard single dose of ivermectin distributed as part of the activities of APOC, and 41 cases of serious adverse reactions (SAEs) were reported, of which 14 were fatal despite appropriate management of the patients. This is an incidence rate even higher than that reported in Lékié Division of Cameroon and has led to the establishment of a commission to examine the matter (Dr B. Thylefors, personal communication). The patho-biological reasons for the occurrence of Loa-encephalopathy following treatment with DEC or with ivermectin, mainly seen in patients heavily infected with Loa microfilariae, are not well understood, and co-factors may exist that account for the fact that some patients do not develop SAEs despite having high Loa microfilaraemia. Experimental work by Dr Samuel Wanji using an animal model is currently trying to reproduce heavy microfilaraemic infections of human L. loa in experimentally infected monkeys (mainly Mandrillus spp) and to investigate the biochemical and pathological changes that accompany the development of any Loa-encephalopathy following ivermectin treatment. In the original work at the HRU, Kumba, where it was relatively easy to infect young drills experimentally (either by inoculation of infective larvae or by transplantation of adult worms) with either the nocturnally-periodic simian Loa parasite or with the diurnally-periodic human parasite, it was obviously far more difficult to determine whether the nocturnally-periodic simian parasite could be transferred to man. Nevertheless, at that time, before the discovery of the potentially deadly viruses such as Ebola, Marburg and HIV that are believed to originate from monkeys, attempts to infect a human (the author) experimentally with a simian strain of the Loa parasite were undertaken. Today, such experiments would not only be viewed as unethical but also as potentially life-threatening. In 1954 and 1955, the author (who at that time had no signs or symptoms of loiasis and who was not taking any medication, apart from 200 mg proguanil (Paludrine) daily as a prophylactic for malaria), took part in two such experiments, which have not been previously published but are relevant in the light of the localised occurrence of Loa-encephalopathy in some individuals following treatment with ivermectin. These attempts at experimental infection of a human being with simian Loa are as follows: In July 1954, a large male drill, which had been shot in the forest near Kumba some 3–4 hours previously, was brought into the laboratory by the Unit's hunter. It was immediately dissected and a total of seven male and fifteen female mature simian Loa worms, all alive, undamaged and motile, were collected from the subcutaneous and intermuscular tissues. The worms were placed in sterile normal saline solution, along with a small quantity of merthiolate, in the same manner that had been used previously to transplant adult simian Loa worms successfully into other monkeys. Two and a half hours later, 12 of these live, motile, adult female worms and five males were inserted, under local anaesthesia, into the upper, anterior part of the right thigh of the author by the Medical Officer-in-Charge of the Kumba Medical Field Unit. After making a 3-inch, longitudinal incision through the skin and the superficial fascia, the simian Loa worms were inserted, some into the sub-cutaneous tissue and others under the deep fascia of the rectus femoris muscle. The fascial layers were then sewn up, the skin closed with nylon sutures, and intramuscular penicillin was given to counteract infection. Over the ensuing week the transplant area became considerably swollen and painful over an area of approximately 6–8 × 5–6 inches and over the following month it itched frequently. Day and night blood films (50 cu. mm) were taken once a week over a period of six months, and thereafter fortnightly for the next six months, but none of them detected any microfilariae. No Loa worm(s) appeared under the skin or crossing the eye, nor did any Loa mf appear in the peripheral blood over the next 46 years. (It is possible that all the worms died fairly soon after being inserted, or it may be that they remained alive but failed to produce a detectable microfilarial infection). Eighteen months after the transplant, the author also injected himself subcutaneously in the left thigh with 35 live, motile, infective larvae of simian Loa, which had been dissected out in normal saline from a laboratory-bred female Chrysops silacea that had taken a blood-meal 10 days previously from a captive drill infected with the nocturnally-periodic simian strain of Loa. Previous experimental work with monkeys had shown that this method of infection was more effective than trying to induce a Chrysops containing infective larvae of Loa to feed on, and thus transmit infective Loa larvae to, an uninfected person. There was a slight reddening and itching of the skin in the area around the injection site over the following week but otherwise no papular eruption or other reaction developed. No Loa microfilariae were detected in the peripheral blood by day or by night over the ensuing eight years; nor, over the same period was any skin reaction seen that could have been attributed to the death of L3 or subsequent stages of L. loa dying in or under the skin, as were reported subsequently when infective larvae of L. loa from the bite of an experimentally-infected Chrysops silacea were subsequently killed by dosage with diethylcarbamazine citrate (DEC) used as a chemoprophylactic [13]. Both these attempts failed to infect a healthy human volunteer with the nocturnally-periodic simian strain of Loa. Obviously the failure to infect a single individual must be interpreted with caution. Nevertheless these observations are worth recording especially now that research is in progress to try and ascertain: 1. The factors leading to the localised occurrence of Loa-encephalopathy in certain areas of central Africa, where ivermectin is now being used currently for the control of onchocerciasis, or where in the past it has occurred following treatment with diethylcarbamazine; and 2. The biochemical and histological changes that may be associated with a Loa-encephalopathy if it can be induced in drills heavily infected with Loa mf when treated with ivermectin. Competing Interests None declared. Authors' contributions Brian Duke was sole author ==== Refs Duke BOL Wijers DJB Studies on loiasis in monkeys. 1. The relationship between human and simian Loa in the rain-forest zone of the British Cameroons Ann Trop Med Parasitol 1958 52 158 175 13559953 Duke BOL Studies on the biting habits of Chrysops. V. The biting cycles and infection rates of Chrysops silacea, C. dimidiata, C. langi and C. centurionis at canopy level in the rain-forest at Bombe, British Cameroons Ann Trop Med Parasitol 1958 52 24 35 13521701 Duke BOL Studies on loiasis in monkeys IV. Experimental hybridization of the human and simian strains of Loa Ann Trop Med Parasitol 1964 58 390 408 14249018 Kivits M Quatre cas d'encephalite mortelle avec invasion du cephalo-rachidien par microfilariae à Loa Ann Soc Belge Med Trop 1952 32 235 242 12976895 Fain A Elsen P Wéry M Maertens K Les filarioses humaines au Mayumbe et dans les régions limitrophes (République du Zaire) Evaluation de la densité microfilarienne Ann Soc Belg Med Trop 1974 54 5 34 Fain A Les problèmes actuels de la loase Bull World Health Organ 1978 56 155 167 96952 Gardon J Gardon-Wendel N Demanga-Ngangué Kamgno J Chippaux JP Boussinesq M Serious reactions after mass treatment of onchocerciasis with ivermectin in an area endemic for onchocerciasis and loiasis Lancet 1997 350 18 22 9217715 10.1016/S0140-6736(96)11094-1 Boussinesq M Gardon J Gardon-Wendel N Kamgno J Ngoumou P Chippaux JP Three probable cases of Loa loa encephalopathy following treatment for onchocerciasis Am J Trop Med Hyg 1998 58 461 469 9574793 Boussinesq M Gardon J Kamgno J Pion SDS Gardon-Wendel N Chippaux JP Relationship between the prevalence and intensity of Loa loa infection in the Central Province of Cameroon Ann Trop Med Parasitol 2001 95 495 507 11487371 10.1080/00034980120073184 Boussinesq M Gardon J Gardon-Wendel N Chippaux JP Clinical picture, epidemiology and outcome of Loa-associated serious adverse events related to mass ivermectin treatment of onchocerciasis in Cameroon Filaria J 2003 2 S4 14975061 10.1186/1475-2883-2-S1-S4 Twum-Danso NAY Serious adverse events following treatment of onchocerciasis in Cameroon Filaria J 2003 2 S3 14975060 10.1186/1475-2883-2-S1-S3 Haselow NJ Akamé J Evini C Akongo S Programmatic and communication issues in relation to serious adverse events following treatment in areas co-endemic for onchocerciasis and loiasis Filaria J 2003 2 S10 14975067 10.1186/1475-2883-2-S1-S10 Duke BOL Studies on the chemoprophylaxis of loiasis. II. Observations on diethylcarbamazine citrate (Banocide) as a prophylactic in man Ann Trop Med Parasitol 1963 57 82 96

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Filaria J. 2004 Jul 29; 3:5

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==== Front Mol CancerMolecular Cancer1476-4598BioMed Central London 1476-4598-3-211529870710.1186/1476-4598-3-21ResearchColorectal cancers with microsatellite instability display mRNA expression signatures characteristic of increased immunogenicity Banerjea Ayan [email protected] Shafi [email protected] Rebecca E [email protected] Fei [email protected] Xia [email protected] Peter M [email protected] Roger [email protected] Stephen A [email protected] Sina [email protected] Centre for Academic Surgery, Barts and The London Queen Mary School of Medicine and Dentistry. The Royal London Hospital, Whitechapel, London, E1 1BB, UK2 Department of Pharmacogenomics, Bristol Myers Squibb, 311 Pennington-Rocky Hill Road, Pennington, NJ 08534, USA3 Institute of Pathology, The Royal London Hospital, Whitechapel, London, E1 1BB, UK2004 6 8 2004 3 21 21 17 5 2004 6 8 2004 Copyright © 2004 Banerjea et al; licensee BioMed Central Ltd.2004Banerjea et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Colorectal cancers displaying high-degree microsatellite instability (MSI-H) have an improved prognosis compared to microsatellite stable (MSS) cancers. The observation of pronounced lymphocytic infiltrates suggests that MSI-H cancers are inherently more immunogenic. We aimed to compare the gene expression profiles of MSI-H and MSS cancers to provide evidence for an activated immune response in the former. Results We analysed tissue from 133 colorectal cancer patients with full consent and Local Ethics Committee approval. Genomic DNA was analysed for microsatellite instability in BAT-26. High-quality RNA was used for microarray analysis on the Affymetrix® HG-U133A chip. Data was analysed on GeneSpring software version 6.0. Confirmatory real-time RT-PCR was performed on 28 MSI-H and 26 MSS cancers. A comparison of 29 MSI-H and 104 MSS cancers identified 2070 genes that were differentially expressed between the two groups [P < 0.005]. Significantly, many key immunomodulatory genes were up-regulated in MSI-H cancers. These included antigen chaperone molecules (HSP-70, HSP-110, Calreticulin, gp96), pro-inflammatory cytokines (Interleukin (IL)-18, IL-15, IL-8, IL-24, IL-7) and cytotoxic mediators (Granulysin, Granzyme A). Quantitative RT-PCR confirmed up-regulation of HSP-70 [P = 0.016], HSP-110 [P = 0.002], IL-18 [P = 0.004], IL-8 [0.002] and Granulysin [P < 0.0001]. Conclusions The upregulation of a large number of genes implicated in immune response supports the theory that MSI-H cancers are immunogenic. The novel observation of Heat Shock Protein up-regulation in MSI-H cancer is highly significant in light of the recognised roles of these proteins in innate and antigen-specific immunogenicity. Increased mRNA levels of pro-inflammatory cytokines and cytotoxic mediators also indicate an activated anti-tumour immune response. ==== Body Background Colorectal cancer remains a leading cause of cancer death in the Western world despite recent advances in surgery, radiotherapy and chemotherapy [1]. Immunotherapy has attracted attention as a novel treatment modality that may exploit the host immune response against tumour cells. However, definitive evidence that colorectal cancer cells can stimulate a specific immune response has been elusive. Approximately 15–20% of sporadic colorectal cancers and nearly all large bowel malignancies in the Hereditary Non-Polyposis Colorectal Cancer (HNPCC) syndrome are characterised by widespread microsatellite instability [2,3]. Microsatellites are very short repetitive nucleotide sequences, distributed throughout the human genome, that are prone to insertion and deletion mutations during DNA replication. These mutations are normally corrected by the inherent proofreading capacity of DNA polymerase and a group of genes involved in mismatch repair (MMR). Defective mismatch repair allows the accumulation of errors in microsatellites and this is termed microsatellite instability (MSI). In HNPCC a germline mutation in a mismatch repair gene is inherited and a subsequent "second hit" leads to failure of MMR, resulting in MSI. In sporadic cancers epigenetic silencing by hypermethylation of the MMR genes has been implicated. Despite these differences in their molecular genesis the two groups share common clinicopathological features [4]. Several studies have confirmed that patients with tumours displaying a high degree of microsatellite instability (MSI-H) appear to possess a survival advantage over those with cancers that are microsatellite stable (MSS) [5-7]. This improvement in outcome appears to be an inherent feature of the unstable phenotype. It has been shown that MSI-H cancers generate abnormal peptides that can be used to excite cytotoxic T cell responses in in vitro experiments [8,9]. These peptides may act as Tumour specific antigens (TSA's) in vivo and hence, excite a host immune response. In keeping with this observation MSI-H cancer is characterised by the presence of a significant infiltrate of lymphocytes, a feature that has been previously associated with better patient prognosis [10]. Lymphocytes that infiltrate tumour epithelium (intra-epithelial lymphocytes, IEL's) are specifically associated with improved survival and may be involved in an immune response [11]. Immunohistochemical analyses have shown that the IEL's infiltrating MSI-H colorectal cancers are predominantly cytotoxic, activated and release mediators of target cell death [12]. Follow-up analyses confirm improved survival in patients with these tumours [13]. Increased apoptosis has also been demonstrated in MSI-H cancers but the link between increased lymphocyte infiltrate and apoptotic cell death has not yet been proven. Some argue that these infiltrates are secondary phenomena with no biological relevance [14] and it has been suggested that intra-epithelial lymphocyte populations in MSI-H colorectal cancers simply represent proliferation of resident lamina propria lymphocytes with no immunological activation or role. The development of high-density data analysis techniques such as microarray technology allows rapid gene expression profiling of tissue-derived RNA to give an mRNA expression signature for the tissue under study. The gene expression signature of a tumour microenvironment reflects the interactions between tumour, stroma and host response therein. We aimed to compare these signatures between groups of MSI-H and MSS colorectal cancers to identify genes that are differentially expressed between the two phenotypes. Specifically, we focus on genes involved in anti-tumour immune responses whose activity may be modified in colorectal cancers, in order to clarify the nature of any immune response in MSI-H colorectal cancer. Results We analysed 133 colorectal tumours of which 29 (22%) tumours were identified as MSI-H (Table 1). This is at the upper end of accepted figures but reflects the frequency of MSI-H in a subset of our tumour bank that yielded high-quality RNA. The overall prevalence of MSI-H colorectal cancer in our tumour bank is lower (16%) and consistent with other large series. As expected the MSI-H group showed a statistically significant association with the right side of the colon (P < 0.0001, χ2 test). During histological assessment each tumour was graded for lymphocytic infiltration on standard Haematoxylin and Eosin stained sections by a consultant pathologist (RF). Tumours with minimal or mild infiltration were scored 1, those with moderate infiltration scored 2 and those with pronounced lymphocytic infiltrates were scored 3. As expected the MSI-H cancer group had higher proportions of tumours with moderate and pronounced infiltration but this difference did not reach statistical significance (P= 0.287, χ2 test for trend). Table 1 Summary of patient demographics for microarray and RT-PCR analyses. Microsatellite Stable (MSS) Microsatellite Unstable (MSI-H) Microarray analysis Patients 104 27 Mean age (SD) (yrs) 69.6 (12.3) 65.5 (15.6) Male: Female (%) 62:42 (60:40) 13:14 (48:52) Cancers (n) 104 29 Right: Left (%) 28:76 (27:73) 19:10 (66:34) Dukes' Stage A (%) 14 (13.5) 3 (10.3) B (%) 48 (46.1) 15 (51.7) C (%) 39 (37.5) 10 (34.5) D (%) 3 (2.9) 1 (3.4) Lymphocyte score 1(%) 78(75) 19(65.5) 2(%) 17(16.3) 6(20.7) 3(%) 9(8.7) 4(13.8) RT-PCR analysis Patients 26 26 Mean age (SD) (yrs) 71.4 (13.6) 66.4 (15.1) Male: Female (%) 14:12 (54:46) 12:14 (46:54) Cancers (n) 26 28 Right: Left (%) 17:9 (65:35) 19:9 (68:32) Dukes' Stage A (%) 2 (7.7) 3 (10.7) B (%) 15 (57.7) 15 (53.6) C (%) 8 (30.8) 10 (35.7) D (%) 1 (3.8) 0 For RT-PCR analysis groups were matched for age, tumour side and Dukes' Stage. Two MSI-H patients had two tumours each. An initial comparison of the gene expression profiles of MSI-H versus MSS tumours, using a parametric unpaired t-test (with Welch's correction for unequal variances) and the Benjamini and Hochberg False Discovery Rate (multiple correction method), identified 2070 genes that were differentially expressed at a significance of p < 0.005 (Additional Table 1 ). This represents 9.3% of those included on the chip and statistically less than 0.5% of these genes would be selected by chance. 1293 genes (62.5%) had significantly increased signal intensity in MSI-H cancers and 777 genes (37.5%) had reduced signal intensity. The clear differences between the two groups can be demonstrated in a cluster map (Figure 1). This was generated using 542 of the most significant genes in our list, selected by performing a group comparison at p < 0.05 and the more stringent Bonferoni multiple testing correction (Additional Table 1 ). The expression signatures of the MSI-H group on the right of the cluster map display marked homogeneity, in contrast to the heterogeneous MSS cancers. Four tumour profiles came from patients who satisfied family history criteria for HNPCC (Amsterdam criteria). These profiles were clustered amongst the other MSI-H cancers but did not form a distinct group. Analysis after exclusion of these HNPCC tumour profiles yielded very similar gene lists (Additional Table 2 ). The small number of HNPCC MSI-H profiles (<5) precluded meaningful comparison of expression profiles with the sporadic group. Figure 1 Two-way hierarchical clustering by the 542 most significantly differentially expressed genes between MSS and MSI-H colorectal cancers. Samples arranged along the x-axis and genes along the y-axis. Each square represents the expression level of a given gene in an individual sample. Red represents increased expression and blue represents decreased expression relative to the normalised expression of the gene across all samples. Samples with similar gene expression profiles are clustered together. 1328 of our 2070 genes (64.2%) had a recognised function. We noted differences in several cancer-related genes that were consistent with our existing knowledge of the genetic profiles of MSI-H colorectal cancers (Table 2). Notably, the mismatch repair gene hMLH1 had reduced signal intensity in our MSI-H group, as did the TGFβ RII and IGFIIR genes. The mismatch repair gene PMS2 was also underexpressed in our MSI-H cancers (P = 0.003, Fold change 1.4). The mRNA of TP53 gene was more abundant in MSI-H tumours when compared to MSS tumours. Similarly, the β catenin gene also had a high signal in our MSI-H tumours. Significantly, several transcripts related to the heat shock protein family (HSP 70, 110 and 90) were up-regulated in MSI-H tumours (Table 2), as were several other genes that may be involved in a putative antigen-directed immune response. We focussed on genes relevant to immunological responses but many other interesting differences are evident in our list but are not discussed in this paper. Table 2 A highly selective list of gene specific probes that are differentially expressed (P < 0.005) between MSI-H and MSS colorectal cancers with a fold-change of at least 1.5. Genes Up-regulated in MSI-H Colorectal Cancer Gene name P value Fold change Catenin (cadherin assoc. protein) beta 1 6.5 × 10-12 2.9 Interleukin 8* 1.7 × 10-4 2.8 Granulysin* 1.3 × 10-7 2.8 Caspase 2* 6.0 × 10-8 2.4 Interleukin 24 0.004 2.3 Heat shock protein (HSP 110 family)* 2.4 × 10-5 2.2 TP53 (Li Fraumeni)* 2.5 × 10-4 2.2 Toll-like receptor 2 (TLR-2) 1.9 × 10-4 1.9 Heat shock protein 70* 3.5 × 10-6 1.8 Granzyme A 0.001 1.7 Interleukin 1β 0.004 1.7 Survivin 0.001 1.6 Calreticulin 2.9 × 10-5 1.6 Human Natural killer Cell enhancing factor 0.001 1.6 CD68 antigen 4.8 × 10-4 1.6 ICAM 1 (CD54) 0.003 1.6 Interleukin 18 (interferon-gamma inducing factor)* 0.003 1.5 Interleukin 7 4.6 × 10-4 1.5 Interleukin 15* 0.002 1.5 Genes down-regulated in MSI-H colorectal cancer Gene name P value Fold change Insulin-like growth factor 2 (somatomedin A) 1.9 × 10-5 4.3 TGFβ RII 9.3 × 10-5 3.0 hMLH 1* 6.7 × 10-5 2.4 P values denote results of Welch's t test with Benjamini and Hochberg False Discovery Rate. * denotes genes selected for RT-PCR analysis. To validate our microarray results we used real time RT-PCR to confirm the findings on nine genes of immunological interest. The RT-PCR results confirmed the significant differences between the two groups (Table 1) in seven out of the nine genes selected. The mismatch repair gene hMLH1 was significantly down-regulated in MSI-H (Figure 2a), whilst transcription of TP53 was significantly higher in the unstable group (Figure 2b). Similarly the heat shock protein (HSP) 70 (Figure 2c) and HSP-110 (Figure 2d), the Interleukins (IL) 18 (Figure 2e) and IL-8 (Figure 2f), and the protease Granulysin (Figure 2g), were all significantly up-regulated in MSI-H when compared to the MSS group. Two analyses, IL-15 (p = 0.17) and Caspase 2 (p = 0.16), had reduced sample numbers in each group and did not reach statistical significance. However, both showed trends of up-regulation in MSI-H cancers consistent with the microarray analysis. Figure 2 Boxplots showing RT-PCR data analysis of seven genes of interest:A hMLH1, B TP53, C HSP-70, D HSP-110, E IL-18, F IL-8, and G Granulysin. Data analysed using non-parametric Mann Whitney test (P values as shown). Discussion Our study examines the differences in overall gene expression profiles in the tumour micro-environments of MSI-H and MSS colorectal cancers. The observation that a large number of pro-inflammatory genes are upregulated in MSI-H colorectal cancer is a strong indicator that an immune response is indeed activated in these tumours. These results support the notion that the lymphocytic infiltrates in these cancers represent immune activation rather than simple proliferation of resident lymphocytes. The exact nature of the immune response remains unclear but our novel observation that heat shock proteins are upregulated in MSI-H colorectal cancer may be highly significant. Microarray data analysis demonstrated that several members of the HSP family are up-regulated in MSI-H cancers (Table 2) and RT-PCR analyses confirmed increased levels of both HSP-110 and HSP-70 mRNA in our MSI-H cancers. Heat shock proteins have roles in both innate and adoptive immunity and have excited much interest as natural adjuvants for immunotherapy [15,16]. Some members of the HSP family act directly to excite an innate immune response that might be more marked in MSI-H colorectal cancer. Such a response might be mediated by Natural Killer cells that characteristically release Granulysin to induce tumour cell death, a recognised feature of MSI-H colorectal cancer, which in turn releases intra-cellular TSA's. Alternatively, the concept of "effete malignancy", in which accumulation of mismatch errors overwhelms the tumour cells' viability, [17] may explain increased tumour cell death and release of TSA's. In fact, these two possible explanations of increased apoptosis in MSI-H colorectal cancers may actually be complementary rather than mutually exclusive. However, the release of TSA's into the tumour micro-environment is the pivotal step in the generation of an antigen-specific immune response. Heat shock proteins act as chaperone proteins in the processing and presentation of antigenic peptides [15,16]. They promote antigen uptake and induce expression of antigen presenting and co-stimulatory molecules on dendritic cells. By example, HSP-70 has been shown to recruit dendritic cells (and T cells) and enhances their ability to uptake antigen [18]. This is a crucial step in the cross-priming of dendritic cells necessary for an antigen-directed immune response [19]. The CD68 antigen, expressed by immature dendritic cells and macrophages that are ready to take up antigen, is included in our list of genes up-regulated in MSI-H colorectal cancer, as is the TLR-2 gene, one of a family of receptors to which HSP's bind to activate dendritic cells. Heat Shock Proteins have also been shown to induce cytokine profiles that promote antigen-specific responses. This role may underscore our observation that several immunogenic interleukins are up-regulated in MSI-H cancers. An important function of these cytokines is to promote the presentation of antigen by dendritic cells (and macrophages) to effector T cells. This represents another crucial step in the development of an antigen-specific immune response, prior to the interaction of primed cytotoxic (CD8+) and helper (CD4+) T cells with the tumour cells. This interaction subsequently results in lytic tumour cell death. Specifically, such cytokines also modulate which arm of the T helper system is activated: Th1 activity favours immune activity whilst Th2 pathways favour tolerance. In this context, our data reveals significant up-regulation of IL-18 and other pro-inflammatory cytokines in MSI-H cancers that promote Th1 activity. Our microarray data are validated by the existing knowledge of key gene expression in MSI-H colorectal cancers. The presence of hMLH1 mRNA was reduced in MSI-H cancers, as shown by both microarray and RT-PCR analyses. This observation reflects the fact that hMLH1 is frequently silenced, due to promoter region hypermethylation, in sporadic cancers, which formed the majority of our MSI-H group. Other genes known to be affected by the MSI pathway such as TGFβ RII, IGFIIR, TP53, APC, β catenin and Bcl-2 were also differentially expressed between our two groups [20,21]. The inclusion of HNPCC cancers within our MSI-H group appears not to affect the differentially expressed genes we identify and this is likely to reflect their small number. Accrual of further HNPCC gene expression profiles should allow us to sub-analyse the MSI-H group in the future. However, the immunological focus of this paper appears unaffected by any differences in the biology of these cancers. As expected a large number of pro-apoptotic genes were upregulated in our MSI-H group as these cancers are characterised by increased apoptosis (Table 3). The increased levels of Bcl-2 related transcripts are consistent with previous findings despite the anti-apoptotic functions of this gene [22,23]. Clearly, the interaction between pro-and anti-apoptotic agents in MSI-H cancers is complex and needs further elucidation. Table 3 Additional genes related to apoptosis and the Major Histocompatibility Complex shown to be up-regulated in MSI-H colorectal cancer in comparison to MSS cancers. Apoptosis related genes up-regulated in MSI-H colorectal cancers (P < 0.005, Benjamini and Hochberg False Discovery Rate) Gene name Fold change TNF-induced protein 1.9 Tumour necrosis factor receptor superfamily 10d 1.9 Tumour necrosis factor receptor superfamily 9 1.7 Tumour necrosis factor receptor superfamily 10b 1.7 Tumour necrosis factor receptor superfamily 6 1.6 Death-associated protein kinase 1 1.5 DNA fragmentation factor, beta polypeptide (caspase-activated DNase) 1.4 Bcl-2-related protein A1 2.2 Bcl-2/adenovirus E1B 19 kDa interacting protein 3-like 1.8 Bcl-2 antagonist/killer 1.4 Bcl-2 associated athanogene 3 1.4 Bcl-2/adenovirus E1B 19 kDa interacting protein 1 1.2 Baculoviral IAP repeat-containing 3 1.6 Baculoviral IAP repeat-containing 2 1.2 Chemokine (C-X-C motif) receptor 4 1.8 CD27-binding (Siva) protein 1.5 Thioredoxin-like, 32 kDa 1.4 Mitogen-activated protein kinase kinase kinase 5 1.4 Microtubule associated protein tau 1.4 Macrophage erythroblast attacher 1.3 Testis enhanced gene transcript (BAX inhibitor 1) 1.2 Major histocompatibility complex-related genes up-regulated in MSI-H colorectal cancer (P < 0.05, Benjamini and Hochberg False Discovery Rate) Gene name Fold change Major histocompatibility complex, class II, DR alpha 2.0 Major histocompatibility complex, class II, DQ beta 1 1.8 Major histocompatibility complex, class II, DP alpha 1 1.8 H. sapiens HLA-DMA gene 1.7 Major histocompatibility complex, class II, DR beta 1 1.6 Major histocompatibility complex, class II, DR beta 5 1.5 HLA-B associated transcript 1 1.2 Major histocompatibility complex, class I, C 1.2 It is of interest that reduction of the stringency of our statistical comparison to P < 0.05 (Benjamini and Hochberg False Discovery Rate) generates a list of 4788 differentially expressed genes (Additional Table 1 ). These include several more genes with key immunomodulatory functions. Transcripts specific to co-stimulatory molecules (CD80 and CD86), HSP60, MHC peptides, pro-inflammatory cytokine receptors, Perforin 1 and Caspase 9 were amongst those that were up-regulated in MSI-H colorectal cancers. These subsidiary data provide further evidence that these cancers excite an antigen-specific immune response. Closer study of HLA molecules that were up-regulated in MSI-H cancers reveals that the majority are Class II-related (Table 3). This finding is consistent with previous studies [24,25] and supports the notion that immunogenicity of these cancers relies on antigen presentation by Antigen Presenting Cells (cross-priming) rather than directly by the tumour. The HLA Class I molecule β2-microglobulin has been shown to be a target for mutation in the MSI-H pathway and this renders HLA Class I machinery ineffective in these tumours [26]. This gene does not, however, appear in our list of differentially expressed genes. This study compares gene expression profiles in 133 primary human cancers and confirms the previous finding on a smaller sample set that microarray profiling can differentiate cancers according to microsatellite stability status [27]. A recent report on differential gene expression from microarray profiling of smaller numbers of MSI-H (n = 8) and MSS (n = 14) colorectal cancer tissue samples yielded findings similar to ours in genes such as TP53, IGF2, RAN, MORF4L1, ZFP36L2 and CCNF [28]. Their observation that EIF3S2 is downregulated in MSI-H was confirmed in our study, as was the downregulation of TGFβ RII. However, they did not observe differences in MMR genes, such as hMLH1 and PMS2, or indeed the immunomodulatory genes that we report. The likely explanation is that the smaller sample numbers used in their analysis as well as the smaller size of their spotted cDNA array (8000 genes) limits the sensitivity of their microarray comparisons. Previously two groups have reported the results of cDNA microarray comparisons of MSI-H and MSS cancer cell lines but both were restricted to very small numbers [29,30]. Interestingly, of the 122 differentially expressed genes identified by these two studies 33 transcripts (27%) were also included in our list of 4788 genes (P < 0.05, Benjamini and Hochberg False Discovery Rate). However, some genes noted to be down-regulated in MSI-H cancer cell lines were up-regulated in the MSI-H cancers in our analysis, and vice versa. These disparities can be attributed to the fact that cancer cells cultured in vitro behave differently to cells from primary tumours. Indeed Bertucci et al report that colorectal cancer lines show overexpression of genes involved in cellular proliferation and underexpression of several gene clusters, including a cluster associated with immunomodulatory genes, when compared to colorectal cancer tissue samples using microarrays [28]. These biological differences and the small numbers used in the cell line experiments render any concordance between our data and cell line analyses altogether encouraging. We acknowledge that mRNA profiles cannot be presumed to reflect functional significance at a protein level. However, the upregulation of such a large number of genes, known to be involved in innate and antigen-specific immune responses, in MSI-H colorectal cancer indicates a genuine difference in host-tumour interactions. These findings are entirely novel and add considerable weight to the argument that MSI-H cancers excite an immune response. Clearly, additional work on protein expression specific to the genes we have identified will help to elucidate the exact nature of immune mechanisms in these cancers. In this study microdissection was deliberately eschewed as our focus was the interaction between tumour, stroma and inflammatory cells and the expression profiles therefore reflect contributions from each of these groups. Microdissection would have excluded the contribution of certain cell populations which may have important roles in the modulation of host-tumour interactions. We have previously extensively analysed the composition of tumour biopsies obtained by sampling exophytic areas of resected specimens. We consistently found that a random sample taken from a fresh frozen segment of tumour tissue contained at least 80% tumour cells (Unpublished data). We know from experience that necrotic tumour does not yield high quality RNA and our study included only those samples that yielded high quality RNA and thus, the sampling technique we used inherently excludes necrotic tumour. It is therefore unlikely that our results are attributable to the presence of tumour necrosis. Furthermore, a DNA microarray analysis of the gene expression profiles of naïve versus activated tumour-specific lymphocytes did not show differences of gene expression in heat shock proteins or the interleukins noted in our study [31]. This suggests that our observations do not simply reflect the more pronounced lymphocyte infiltrates of MSI-H colorectal cancer although we accept that this remains a possibility. Conclusions Certainly, our results provide new evidence to support the notion that MSI-H colorectal cancers are immunogenic and new insights into the pathways that may be involved. Further focussed study of these cancers may clarify the immunology of colorectal cancer and, specifically, may provide useful targets for directing immunotherapeutic strategies. Methods Patient population and microsatellite analysis The Local Ethics Committee approved this study and all patients gave informed consent prior to surgery. Tissue was available from a bank of 223 colorectal cancers resected at The Royal London Hospital between December 1997 and March 2003. Tissue samples of tumour and normal mucosa were taken within 20 minutes of resection and snap frozen in liquid nitrogen. Normal mucosa and tumour DNA was extracted and used in PCR reactions to amplify the mononucleotide BAT-26 marker, as described elsewhere [32]. Products were separated and visualised on micro-fabricated chips to identify tumours displaying bandshifts characteristic of a high degree of microsatellite instability [33]. Microarray profiling and analysis Total RNA was prepared from samples using an RNeasy® kit (QIAGEN, Hilden, Germany) and quality was assessed on the Agilent Bioanalyser 2100. Only 129 samples, from our tumour bank, that yielded high quality mRNA with minimal degradation and clear 18S/28S ribosomal peaks, were included in the analysis. Preparation of in vitro transcription (IVT) products, oligonucleotide array hybridization and scanning were performed according to Affymetrix® (Santa Clara, California) protocols. In brief, 5 μg of total RNA from each colon tumour and T7-linked oligo-dT primers were used for first-strand cDNA synthesis. IVT reactions were performed in batches to generate biotinylated cRNA targets, which were chemically fragmented at 95°C for 35 minutes. Fragmented biotinylated cRNA (10 μg) was hybridized at 45°C for 16 hours to Affymetrix® high density oligonucleotide array human HG-U133A chip, which contains 22,283 probe sets representing more than 14,500 well-substantiated human genes. The arrays were washed and stained with streptavidin-phycoerythrin (SAPE, final concentration of 10 μg/ml). Signal amplification was performed using a biotinylated anti-streptavidin antibody. The array was scanned according to the manufacturer's instructions (Affymetrix Genechip® Technical Manual, 2001). Scanned images were inspected for the presence of obvious defects (artefacts or scratches) on the array. Defective chips were excluded and the sample was re-analysed. To minimize discrepancies due to variables such as sample preparation, hybridization conditions, staining, or array lot, the raw expression data was scaled using Affymetrix® Microarray Suite 5.0 software. The trimmed mean signal of all probe sets on the HG-U133A chip was adjusted to a user-specified target signal value (1500) for each array for global scaling. No specific exclusion criteria were applied. Comparative analysis between expression profiles for MSI-H and MSS samples was carried out on GeneSpring™ software version 6.0 (Silicongenetics, Redwood, California). The "Cross gene error model for deviation from 1.0" was active. Gene expression data was normalised in two ways: "per chip normalisation" and "per gene normalisation". For "per chip normalisation" all expression data on a chip is normalised to the 50th percentile of all values on that chip. For "per gene normalisation" the data for a given gene is normalised to the median expression level of that gene across all samples. The data sets are then assigned to the two groups MSI-H and MSS, and the expression profiles of the two groups were compared using unpaired t-tests (with Welch's correction for unequal variances) and multiple testing corrections to identify genes that were differentially expressed between them. RT-PCR Analysis Nine genes were further analysed using quantitative real-time RT-PCR. To compare similar groups each MSI-H tumour was matched to an MSS tumour from patients of similar age, the same side of the colon and, where possible, same Dukes' Stage. Sample RNA was extracted, quantified and quality-controlled as for microarray analysis. High-quality RNA was available on 28 tumours of the 29 tumours with MSI-H. These were matched to a group of 26 MSS cancers (Table 1). Three analyses were performed on smaller groups (Table 4) because high-quality RNA was no longer available for analysis. Gene-specific primers and probe sets for each gene (Table 4) were obtained from Assays-on-Demand Gene Expression Products (Applied Biosystems, Warrington, UK). RT-PCR reactions were carried out in a one-tube system for seven genes but reactions for two genes were sub-optimal in this set-up and so a two-tube system was used instead (Table 4). Sample RNA was processed in duplicate with serial dilutions of Human Universal Reference RNA (Stratagene, LaJolla, California), in triplicate, and No Template Controls on the same 96-well plate. Standard curves were constructed from the Universal RNA wells with arbitrary units of 1 Unit equivalent to 1 picogram of Universal RNA. Duplicate wells that differed by more that one Ct count were repeated or were excluded from the analysis. The results were compared using the non-parametric Mann-Whitney test and significance was taken at P < 0.05. Table 4 Summary of RT-PCR details and the numbers of samples in each analysis. Analysis of hMLH1, IL-15 and Caspase2 restricted to smaller numbers due to limited availability of high-quality RNA. Gene name GENBANK NUMBER Chemistry (Applied Biosystems, Warrington, UK) Sample Numbers (n) MSS MSI-H hMLH1 NM_000249 High-Capacity cDNA Archive Kit + TaqMan Universal PCR Master Mix (with AmpErase UNG) 12 16 TP53 NM_000546 TaqMan® EZ-RT-PCR Kit 26 27 HSP-70 kD 1B NM_005346 TaqMan® EZ-RT-PCR Kit 26 28 HSP-110 NM_014278 TaqMan® EZ-RT-PCR Kit 26 28 IL-18 NM_001562 TaqMan® EZ-RT-PCR Kit 24 28 IL-8 NM_000584 TaqMan® EZ-RT-PCR Kit 26 28 IL-15 NM_000585 High-Capacity cDNA Archive Kit + TaqMan Universal PCR Master Mix (with AmpErase UNG) 12 16 Granulysin NM_006433 TaqMan® EZ-RT-PCR Kit 26 27 Caspase 2 NM_001224 TaqMan® EZ-RT-PCR Kit 21 18 List of abbreviations MSI = Microsatellite instability MSI-H = High degree microsatellite instability MSS = Microsatellite stable (including low degree instability) Authors' contributions AB carried out RT-PCR experiments and analysis, microarray data analysis and drafted the manuscript. SA participated in microarray experiments and carried out microarray data analysis. RH, FH, XH and PS coordinated and performed microarray experiments. RF performed histopathological analysis of cancers. SB and SD conceived the study, supervised the project and drafted the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional File 1 2070 genes differentially expressed between MSI-H and MSS colorectal cancers (P < 0.005, Benjamini and Hochberg False Discovery Rate). 4788 genes differentially expressed between MSI-H and MSS colorectal cancers (P < 0.05, Benjamini and Hochberg False Discovery Rate). 542 genes differentially expressed between MSI-H and MSS colorectal cancers (P < 0.05, Bonferoni Mutilple Testing Correction). Click here for file Additional File 2 2184 genes differentially expressed between MSI-H (HNPCC profiles excluded) and MSS colorectal cancers (P < 0.005, Benjamini and Hochberg False Discovery Rate). 1436 genes upregulated in sporadic MSI-H cancers (with fold change). 748 genes upregulated in MSS cancers (with fold change). 4874 genes differentially expressed between MSI-H and MSS colorectal cancers (P < 0.05, Benjamini and Hochberg False Discovery Rate). Click here for file Acknowledgements This work was funded by MRC Grant (G0000141) and Bowel and Cancer Research (Registered Charity No. 328667). 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Cancer Res 2003 63 4577 4582 12907634 Bertucci F Salas S Eysteries S Nasser V Finetti P Ginestier C Charafe-Jauffret E Loriod B Bachelart L Montfort J Victorero G Viret F Ollendorff V Fert V Giovaninni M Delpero JR Nguyen C Viens P Monges G Birnbaum D Houlgatte R Gene expression profiling of colon cancer by DNA microarrays and correlation with histoclinical parameters. Oncogene 2004 23 1377 1391 14973550 10.1038/sj.onc.1207262 Dunican DS McWilliam P Tighe O Parle-McDermott A Croke DT Gene expression differences between the microsatellite instability (MIN) and chromosomal instability (CIN) phenotypes in colorectal cancer revealed by high-density cDNA array hybridization Oncogene 2002 21 3253 3257 12082642 10.1038/sj.onc.1205431 Yuan Z Sotsky Kent T Weber TK Differential expression of DOC-1 in microsatellite-unstable human colorectal cancer. Oncogene 2003 22 6304 6310 13679870 10.1038/sj.onc.1206609 Zhang X Chen Z Huang H Gordon JR Xiang J DNA microarray analysis of the gene expression profiles of naive versus activated tumour-specific T cells. Life Sci 2003 71 3005 3017 12384184 10.1016/S0024-3205(02)02164-1 Banerjea A Phillips SM Dorudi S Bustin SA Colorectal cancers with mononucleotide microsatellite instability can be identified using microfabricated chip technology. Anal Biochem 2003 322 130 133 14705790 10.1016/j.ab.2003.07.006

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2021-01-04 16:36:34

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Mol Cancer. 2004 Aug 6; 3:21

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Mol Cancer

10.1186/1476-4598-3-21

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==== Front Cardiovasc UltrasoundCardiovascular Ultrasound1476-7120BioMed Central London 1476-7120-2-101526876310.1186/1476-7120-2-10ResearchEarly endothelial dysfunction in cholesterol-fed rabbits: a non-invasive in vivo ultrasound study. Drolet Marie-Claude [email protected] Éric [email protected] Bruno [email protected] Jacques [email protected] Marie [email protected] Groupe de recherche en valvulopathies, Institut de cardiologie de Québec, Centre de recherche Hôpital Laval, Université Laval, 2725 Chemin Sainte-Foy, Sainte-Foy (Québec) G1V 4G5 Canada2004 21 7 2004 2 10 10 1 7 2004 21 7 2004 Copyright © 2004 Drolet et al; licensee BioMed Central Ltd.2004Drolet et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Endothelial function in hypercholesterolemic rabbits is usually evaluated ex vivo on isolated aortic rings. In vivo evaluation requires invasive imaging procedures that cannot be repeated serially. Aim We evaluated a non-invasive ultrasound technique to assess early endothelial function in rabbits and compare data with ex vivo measurements. Methods Twenty-four rabbits (fed with a cholesterol diet (0.5%) for 2 to 8 weeks) were given progressive infusions of acetylcholine (0.05–0.5 μg/kg/min) and their endothelial function was assessed in vivo by transcutaneous vascular ultrasound of the abdominal aorta. Ex vivo endothelial function was evaluated on isolated aortic rings and compared to in vivo data. Results Significant endothelial dysfunction was demonstrated in hypercholesterolemic animals as early as 2 weeks after beginning the cholesterol diet (aortic cross-sectional area variation: -2.9% vs. +4% for controls, p < 0.05). Unexpectedly, response to acetylcholine at 8 weeks was more variable. Endothelial function improved in 5 rabbits while 2 rabbits regained a normal endothelial function. These data corroborated well with ex vivo results. Conclusion Endothelial function can be evaluated non-invasively in vivo by transcutaneous vascular ultrasound of the abdominal aorta in the rabbit and results correlate well with ex vivo data. endothelial functionatherosclerosischolesterol-fed rabbitsultrasound. ==== Body Background Endothelial dysfunction occurs early in the development of atherosclerosis. Historically, evaluation of endothelial function in small animals has been performed on isolated vessel segments, or vessels exposed by surgical procedures. Very few attempts were made to develop a method of analysis of endothelium-dependent relaxation in vivo[1-5]. In those studies, an invasive intravascular ultrasound approach was used. Correlation between results obtained in vivo and data on isolated arteries ex vivo was never assessed. Non-invasive methods to study endothelial function in humans (e.g. ultrasound of the brachial artery) have been used for many years and have yielded an important amount of data [6-12]. Unfortunately this non-invasive technique has never been transposed to animal studies. The objective of the current study was to assess the reliability of transcutaneous vascular ultrasound in order to evaluate endothelial function in vivo in rabbits and to compare this non-invasive method with results obtained ex vivo on isolated aortic rings. Material and methods Materials Acetylcholine, nitroglycerin and sodium nitroprusside were from Sigma (Markham, ON, Canada). Angiotensin II and endothelin-1 peptides were acquired from Peninsula Laboratories Inc. (San Carlos, CA) Animals and Study Design Twenty-four male New Zealand White rabbits (3–4 kg body weight) were used in this study. Animals were treated in accordance to the Guide to the care and use of experimental animals published by the Canadian Council on Animal Care and the protocol was approved by the Animal Protection Committee of the Université Laval. Sixteen rabbits were divided in two groups (n = 8) and all animals were fed with standard rabbit chow supplemented with 0.5% cholesterol (w/w) (Harlan, Indianapolis, IN) for 2 or 8 weeks respectively. The other 8 animals received normal rabbit chow for eight weeks (normal controls). After 2 weeks, 8 randomly chosen cholesterol-fed rabbits were killed; the others were kept alive for an additional 6 weeks (total of 8 weeks of hypercholesterolemic diet) as for the normal control group. When animals were sacrificed abdominal and thoracic aortas were excised and immediately rinsed in freshly prepared Krebs buffer in preparation for the ex vivo experiments. Plasma samples were drawn from the marginal ear vein every week and plasma cholesterol levels were determined using a commercially available spectrophotometric assay kit (Roche Molecular Biochemicals, Saint-Laurent, Canada). In vivo evaluation of endothelial function by trans-abdominal ultrasound Ultrasound evaluation of endothelial function of the abdominal aorta was performed at baseline, 2 weeks and 8 weeks. Rabbits were sedated using midazolam (0.5 mg/kg), butorphanol (0.5 mg/kg) and ketamine (30 mg/kg) IM. Marginal ear vein and artery were cannulated for drug infusions and arterial blood pressure monitoring, respectively (Figure 1A). Heart rate was monitored continuously throughout the procedure. The abdomen was shaved and the animal was put in dorsal decubitus (Figure 1B and 1C). The abdominal aorta was located using two-dimensional and color Doppler ultrasound. Image settings were optimized to allow the best and clearest definition of the endothelial-blood interface (Figure 1D). All studies were performed with a vascular 7.5 MHz probe coupled to a Sonos 5500 echograph (Phillips, Andover, MA). Figure 1 In vivo assessment of endothelial function in rabbits. Rabbits under sedation were serially infused for two minutes in the marginal ear (A) vein with the vehicle. An ink mark (Panel B) was made on the rabbit abdomen for probe location for each exam. Images (Panels C and D) of the abdominal aorta were recorded throughout the procedure. Once the imaging of the aorta was considered optimal, the animals received the following drug perfusions I.V sequentially for 2 minutes each: 1) saline at 1 ml/min; 2) acetylcholine (Ach) at 0.05 μg/ml/min and Ach at 0.5 μg/ml/min. Nitroglycerin (5 μg/ml/min) was used as positive control. Typical arterial blood pressure recordings are illustrated in Figure 2. At the end of a drug infusion, blood pressure was allowed to come back to baseline for at least one minute before the next infusion was started. Images of the abdominal aorta were recorded continuously through the entire procedure on standard S-VHS videotape for off-line analysis. Figure 2 Representative blood pressure recordings during vehicle and drug infusions. Video sequences from the first 15 seconds (void volume) of drug infusion at baseline and between 40 to 60 seconds of drug infusion were digitized and used for analysis. Image analysis Video sequences from the first 15 seconds (void volume) of drug infusion at baseline and between 40 to 60 seconds of drug infusion were digitized (Dazzle Video Creator, Dazzle Multimedia, Fremont CA) and stored on a computer for analysis (Figure 2). Still frames of the aorta from both the baseline and drug infusion (n = 5 each) synchronized to the beginning of the QRS and the respiratory phase were analyzed. The maximal diameter was measured (5 beats averaged) using the SigmaScan Pro software (SSSP Science, Chicago IL). Care was taken to measure the same segment for each beat using anatomic landmarks as a guide. The mean of 5 diameters for each image of the aorta was calculated. Vessel cross-sectional area was then calculated assuming the aortic section is circular using the formula: Area = π (D/2)2 where D is the diameter of the aorta. Area was expressed in percent of change from baseline. Inter and intra-observer variability was assessed on 10 randomly selected studies. Ex vivo endothelial function evaluation in isolated rabbit aortic rings At the end of the protocol rabbits were given a sub-lethal dose pentobarbital (25 mg/kg) and were sacrificed by exsanguination. The middle part of the descending thoracic aorta as well as the abdominal aorta were removed and dissected free of adhering fat and connective tissues. The aorta was placed in warm Krebs solution. Rings of 5 mm thickness were suspended in individual organ chambers filled with 5 ml of oxygenated Krebs (37C pH 7.4). The segments were connected to force transducers and any variations in force were recorded continuously (WIN-SMT software, PO-NE-MAH inc., Gould, Valley View, OH.). Contractile response Baseline contractile response was evaluated by a 30 to 60 minutes exposition to KCl (80 mM) where the rings were gradually stretched to a resting tension of 2 g until steady state was reached. Following this initial experiment, contractile capacity was further evaluated by exposing the rings to other vasoconstrictors. Briefly, when the rings had recovered their resting tension after the initial KCL exposure, they were exposed sequentially to cumulative concentrations of L-phenylephrine (PE, 10-9 to 10-5 M), angiotensin II (10-10 to 10-7 M) and endothelin-1 (10-9 to 10-6 M). Results were compared to the initial response obtained with KCL 80 mM. Relaxation response Relaxation studies were performed after a precontraction with PE (10-6 M). Cumulative concentrations of acetylcholine (10-9 to 3 × 10-6 M) or sodium nitroprusside (10-10 to 3 × 10-5 M) were used. Sodium nitroprusside was used as a non-endothelial dependant vasodilator while acetylcholine evaluated the endothelial-dependant vasodilatation. Relaxation was expressed as a percent of change from the pre-contracted tension with PE. Statistical analysis Results are expressed as mean ± standard error of the mean (SEM). Differences between the various conditions in the in vivo endothelial function experiments were evaluated with an ANOVA for repeated measures using the Tukey's post hoc test to evaluate significance. In the in vitro study, Student t-test was used when two values were compared. Differences were considered significant when p < 0.05. Results Total cholesterol circulating levels Total cholesterol levels were measured weekly in the serum of cholesterol-fed rabbits. As illustrated in figure 3, total cholesterol levels rose sharply after one week of hypercholesterolemia and stabilized around 20 mM from Week 3 to Week 8. Figure 3 Total cholesterol circulatory levels in rabbits fed with the cholesterol diet. Results are expressed as mean ± SEM in mmoles/l (mM). (n = 8 animals) In vivo experiments As expected, saline alone had no effect. Low doses of acetylcholine (ACh 0.05 and ACh 0.5 μg/kg/min) had only a minor and transient lowering effect on blood pressure. As illustrated in Figure 4, both saline and ACh 0.05 infusion had no effect on abdominal aortic cross-sectional area compared to baseline in both normal (Week 0) and hypercholesterolemic (Week 2 and 8) rabbits. As expected, ACh 0.5 infusion induced a dilatation of the aorta in animals at week 0 but had a paradoxical effect (contraction) at Week 2. Interestingly, while the response was clear and homogeneous at 2 weeks, it was more heterogeneous after eight weeks of hypercholesterolemia as endothelial function had improved in 5/8 rabbits (62%) and 2/8 (25%) regained a normal endothelial function. Although a trend towards contraction was recorded overall at week 8, it did not reach statistical significance. Figure 4 In vivo assessment of endothelial function in rabbits. Change in the area of the abdominal aorta in response to acetylcholine infusions. Rabbits (n = 8) were fed with a cholesterol diet for the indicated period of time. The endothelial function was assessed before (week 0) and after 2 (week 2) and 8 (week 8) of hypercholesterolemia. Rabbits under sedation were serially infused for two minutes (saline (vehicle); 1 ml/min) in the marginal ear vein with the vehicle (left), acetylcholine (Ach) at 0.05 and at 0.5 mg/ml/min. *: P < 0.05 vs. vehicle and ¶ P < 0.05 vs Week 0. Ex vivo endothelial function measurements In order to confirm the validity of the in vivo results, we performed sections isometric contraction-relaxation experiments on isolated aortic rings. In Figure 5 are illustrated the contraction experiments using phenylephrine (Fig. 5A and 5B), angiotensin II (Fig. 5C and 5D) and endothelin-1 (Fig. 5E and 5F). All results are expressed relative to a control contraction using potassium chloride (80 mM). Except for endothelin-1, responses to vasoconstrictor were similar between abdominal and thoracic portions of the rabbit aorta. Thoracic aorta was less responsive to endothelin-1 than the abdominal portion. The amplitude of this response to endothelin-1 was also clearly less in the thoracic aorta. Figure 5 Contraction to phenylephrine (PE), angiotensin II (ATII) and to endothelin-1 (ET-1) of abdominal (panels A, C and E) and thoracic (panels B, D and F) aortic rings from rabbits fed or not with a cholesterol diet for 2 or 8 weeks. Aortic rings were exposed to cumulative doses of the indicated agent. Values are presented as percentage of contraction relative to a KCl (80 mM) control contraction. Values are expressed as mean ± SEM (n = 16). *P < 0.05 vs Week 0. We then studied the endothelium-dependent relaxation using acetylcholine on our aortic rings after a pre-contraction with phenylephrine (1 μM). As illustrated in Figure 6A and 6B, abdominal and thoracic aortic ring of rabbits fed for two weeks with a cholesterol diet had a decreased vasodilatory response compared to normal rabbits (Week 0). Figure 6 Relaxation to acetylcholine (ACh) and to sodium nitroprusside (SNP) of abdominal (panels A and C) and thoracic (panels B and D) aortic rings from rabbits fed or not with a cholesterol diet for 2 or 8 weeks Aortic rings were exposed to cumulative doses of the indicated agent. Values are presented as percentage of relaxation relative to a phenylephrine precontraction (1 μM). Values are expressed as mean ± SEM (n = 8). *P < 0.05 vs Week 0. As seen in vivo, the endothelial function of the animals fed 8 weeks with the cholesterol diet was heterogeneous. In those animals hypercholesterolemia had no effect on the acetylcholine-induced relaxation of thoracic aortic rings while for the abdominal aortic sections; the response to acetylcholine was highly variable. As illustrated in Figure 6C and 6D the endothelium-independent response to sodium nitroprusside of aortic rings was normal and similar for all treatments and controls. Discussion Our results clearly show that endothelial function can be assessed non-invasively by transcutaneous ultrasound of the abdominal aorta in hypercholesterolemic rabbits. The method was easily feasible in all animals and yielded very reproducible results. We also show that this in vivo method correlates very well with the ex vivo evaluation of endothelial function on isolated aortic rings. To our knowledge, this is the first demonstration of such a comparison. Ultrasound imaging of the brachial artery in response to reactive hyperaemia has been used in many studies in humans [6-12]. Normal arteries dilate in response to reactive hyperaemia while arteries with an abnormal endothelial function show a decreased or absent response to this stimulus. Ultrasound evaluation of the aorta in rabbits has been performed in the past mostly to evaluate the extent of atherosclerotic plaques in response to a hypercholesterolemic diet. Intravascular ultrasound has been used to document endothelial dysfunction but has never been compared to ex vivo evaluation of endothelial function on aortic rings [1-5]. Our method is much less invasive for the animals than intravascular ultrasound. It is compatible with longitudinal studies requiring repeated measurements in the same animal and correlates very well with the ex vivo data [13,14]. The extent of endothelial dysfunction observed after 2 weeks of hypercholesterolemic diet was surprising although this parameter has not been studied very much after such a short exposure to hypercholesterolemia in rabbits [2]. The paradoxical contraction in response to acetylcholine signs the presence of endothelial dysfunction and this is confirmed in bath studies. However, the results obtained after 8 weeks were unexpected. Indeed, a significant number of animals had an improved endothelial function after 8 weeks of hypercholesterolemia compared to the two weeks data both in vivo and ex vivo. This transient improvement of endothelial function in the early phases of the atherosclerotic process has never been described before to our knowledge and the underlying mechanisms responsible for this paradoxical response need to be explored. This dysfunction may relate to an initial stress response of the aortic endothelium to hyperlipidemia then evolving with the development of atherosclerosis lesions. Conclusion Endothelial function can be evaluated non-invasively in rabbits using a standard vascular ultrasound probe by a trans-abdominal approach. Results correlate well with in vitro data. A transient improvement in endothelial function can occur after 8 weeks of hypercholesterolemia in some animals for reasons that remain unclear. Acknowledgments The authors wish to thank Mr. André Blouin for his expert technical support with the animal studies. This work was supported by grants from the Heart and Stroke Foundation of Canada (Quebec) and from the Institut de cardiologie de Québec to Jacques Couet. Jacques Couet, Marie Arsenault and Bruno Battistini are scholars from the Fonds de la recherche en santé du Québec. ==== Refs Chin HP Liu CR Liu CH Blankenhorn DH Very early aortic responses during atherosclerosis induction in rabbits: measurement by duplex ultrasound. I. Non-invasive study of aortic hyperresponsiveness to serotonin Atherosclerosis 1990 83 1 8 2202316 Mano T Masuyama T Yamamoto K Naito J Kondo H Nagano R Tanouchi J Hori M Inoue M Kamada T Endothelial dysfunction in the early stage of atherosclerosis precedes appearance of intimal lesions assessable with intravascular ultrasound Am Heart J 1996 131 231 238 8579013 Hiromoto M Toma Y Tomochika Y Umemoto S Matsuzaki M Echographical assessment of the early stage of experimental atherosclerosis of the descending aorta in rabbits Jpn Circ J 1996 60 691 698 8902587 10.1253/jcj.60.691 Schwarzacher S Weidinger F Krejcy K Raberger G Assessment of changes in vasomotor tone in vivo using intravascular ultrasound J Pharmacol Toxicol Methods 1992 28 143 147 1489985 10.1016/1056-8719(92)90076-D Ochiai R Kisanuki A Asada Y Asato M Tamura S Sumiyoshi A Intravascular ultrasound imaging in the assessment of atherosclerotic plaques in rabbit abdominal aorta: comparison with histologic findings Invest Radiol 2002 37 309 313 12021586 10.1097/00004424-200206000-00001 Ramsey M Goodfellow J Bellamy M Lewis M Henderson A Non-invasive detection of endothelial dysfunction Lancet 1996 348 128 129 8676693 Sorensen KE Kristensen IB Celermajer DS Atherosclerosis in the human brachial artery J Am Coll Cardiol 1997 29 318 322 9014983 10.1016/S0735-1097(96)00474-3 Celermajer DS Sorensen KE Gooch VM Spiegelhalter DJ Miller OI Sullivan ID Lloyd JK Deanfield JE Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis Lancet 1992 340 1111 1115 1359209 10.1016/0140-6736(92)93147-F Sorensen KE Celermajer DS Spiegelhalter DJ Georgakopoulos D Robinson J Thomas O Deanfield JE Non-invasive measurement of human endothelium dependent arterial responses: accuracy and reproducibility Br Heart J 1995 74 247 253 7547018 Lambert J Aarsen M Donker AJ Stehouwer CD Endothelium-dependent and -independent vasodilation of large arteries in normoalbuminuric insulin-dependent diabetes mellitus Arterioscler Thromb Vasc Biol 1996 16 705 711 8963729 Kobayashi H Yoshida A Kobayashi M Nakao S Non-invasive detection of endothelial dysfunction with 30 MHz transducer Lancet 1996 347 1336 1337 8622535 10.1016/S0140-6736(96)90986-1 Corretti MC Plotnick GD Vogel RA Technical aspects of evaluating brachial artery vasodilatation using high-frequency ultrasound Am J Physiol 1995 268 H1397 H1404 7733339 Ludwig M Stumpe KO Sauer A Kolloch R Goertz U Vetter H Effects of a high-cholesterol diet on arterial wall thickness and vascular reactivity in young rabbits Clin Investig 1992 70 105 112 1600336 Ludwig M Stumpe KO Sauer A Kolloch R Goertz U Vetter H Short-term high dietary cholesterol in early life induces persistent increases in arterial wall thickness and vascular reactivity to noradrenaline J Hypertens Suppl 1991 9 S136 S137 1818914

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2021-01-04 16:38:29

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Cardiovasc Ultrasound. 2004 Jul 21; 2:10

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Cardiovasc Ultrasound

10.1186/1476-7120-2-10

oa_comm

==== Front Health Qual Life OutcomesHealth and Quality of Life Outcomes1477-7525BioMed Central London 1477-7525-2-401528581210.1186/1477-7525-2-40ResearchSad, blue, or depressed days, health behaviors and health-related quality of life, Behavioral Risk Factor Surveillance System, 1995–2000 Kobau Rosemarie [email protected] Marc A [email protected] Matthew M [email protected] David G [email protected] Daniel [email protected] Health Care and Aging Studies Branch, Division of Adult and Community Health, National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention, 4770 Buford Highway NE, Mailstop MS K-51, Atlanta, GA 30341, USA2 Office of the Director, National Center for HIV, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, GA, USA3 Emerging Investigations and Analytical Methods Branch, Division of Adult and Community Health, National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention, Atlanta, GA, USA2004 30 7 2004 2 40 40 24 5 2004 30 7 2004 Copyright © 2004 Kobau et al; licensee BioMed Central Ltd.2004Kobau et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Mood disorders are a major public health problem in the United States as well as globally. Less information exists however, about the health burden resulting from subsyndromal levels of depressive symptomatology, such as feeling sad, blue or depressed, among the general U.S. population. Methods As part of an optional Quality of Life survey module added to the U.S. Behavioral Risk Factor Surveillance System, between 1995–2000 a total of 166,564 BRFSS respondents answered the question, "During the past 30 days, for about how many days have you felt sad, blue, or depressed?" Means and 95% confidence intervals for sad, blue, depressed days (SBDD) and other health-related quality of life (HRQOL) measures were calculated using SUDAAN to account for the BRFSS's complex sample survey design. Results Respondents reported a mean of 3.0 (95% CI = 2.9–3.1) SBDD in the previous 30 days. Women (M = 3.5, 95% CI = 3.4–3.6) reported a higher number of SBDD than did men (M = 2.4, 95% CI = 2.2–2.5). Young adults aged 18–24 years reported the highest number of SBDD, whereas older adults aged 60–84 reported the fewest number. The gap in mean SBDD between men and women decreased with increasing age. SBDD was associated with an increased prevalence of behaviors risky to health, extremes of body mass index, less access to health care, and worse self-rated health status. Mean SBDD increased with progressively higher levels of physically unhealthy days, mentally unhealthy days, unhealthy days, activity limitation days, anxiety days, pain days, and sleepless days. Conclusion Use of this measure of sad, blue or depressed days along with other valid mental health measures and community indicators can help to assess the burden of mental distress among the U.S. population, identify subgroups with unmet mental health needs, inform the development of targeted interventions, and monitor changes in population levels of mental distress over time. ==== Body Background Mood disorders are a major public health problem in the United States as well as globally, imposing a substantial burden of disability, impaired quality of life, and death if they remain untreated [1-3]. National estimates for 12-month prevalence of depressive disorders for adults aged 18 and over range between 6.3%-11.3% depending on the assessment tools, criteria used, and populations studied [1,4-6]. The lifetime prevalence of six selected mood disorders, including major depressive episode, dysthymia, and bipolar disorder as assessed by the Diagnostic Interview Schedule [7] among 7,667 respondents aged 17–39 years to the third National Health and Nutrition Examination Survey, was 8.6% for major depressive episode; 7.7% for severe major depressive episode; 6.2% for dysthymia; 3.4% for combined major depressive episode and dysthymia, 1.6% for any bipolar disorder, and 11.5% for any mood disorder [2]. In the Alameda County Study, 6.6% of men and 10.1% of women aged 50 years or older met DSM-III-R and DSM IV [8,9] symptom criteria for major depression within the past two weeks [10]. By 2020, depression will become the second leading cause for disease burden [11] Mental health disorders due to depression, anxiety and substance use are not only burdensome by themselves, but they can complicate existing physical disorders and also increase risk for other physical comorbidity [3,12,13]. For example, psychological distress might interfere with medication adherence for an existing disorder such as hypertension, but also increase the likelihood of adopting unhealthy behaviors such as smoking, excessive alcohol use, or overeating that can further impair physical health. Both major depressive disorder and subsyndromal levels of depression are associated with similar demographic, social, psychiatric and physical health predictors [14,15]. Results from the 1980–1985 Epidemiologic Catchment Area Study indicated that almost 30% of the population reported having experienced a period lasting at least two weeks in their lifetime when they felt sad, blue or depressed or lost interest in previously pleasurable things or activities [16]. The inclusion of lesser levels of depressive symptomatology when calculating estimates of the prevalence of diagnosable depression could inflate such estimates [17]. However, an examination of subsyndromal levels of depression to determine what proportion of the population is at risk for major depressive disorder can be useful for communities interested in preventing depression [12]. Examining subsyndromal depression and its associated risk with unhealthful behaviors, furthermore, can highlight associations between feeling sad, blue or depressed and behaviors risky to health [3], and is of public health interest [12,18-20]. For at least one year since 1995, more than one-third of state health departments have assessed the number of recent days that adults "felt sad, blue or depressed" using the Behavioral Risk Factor Surveillance System (BRFSS). This measure has good construct validity when compared with other BRFSS health-related quality-of-life (HRQOL) domains related to mental health [21], and has acceptable reliability and criterion validity when compared with the mental health scales of the Medical Outcomes Study Short-Form 36 (SF-36) [22] and with the Center for Epidemiologic Studies Depression Scale (CES-D) [23] among older, low-income African-American men [24]. Using a large multi-state sample, this study is the first to focus on the prevalence of self-reported "sad, blue or depressed" days (SBDD) overall and in sociodemographic subgroups in the United States. It also examines the construct validity of the measure. Methods The BRFSS, which is designed to monitor behavioral health risks in the United States, is an annual random-digit-dialed telephone survey of the non-institutionalized civilian population aged 18 years or older conducted in all states and the District of Columbia [25]. As part of an optional Quality of Life survey module that was added to the BRFSS and used in 38 states and the District of Columbia in one or more years from 1995 through 2000, a total of 166,564 BRFSS respondents answered the question, "During the past 30 days, for about how many days have you felt sad, blue, or depressed?" The module also contains questions on the number of recent days of pain, anxiety, sleeplessness and on other HRQOL domains. Respondents answered standard BRFSS questions about age, race/ethnicity, education, employment, income, marital status, health status, physical health, mental health, activity limitation, access to care, and the presence of certain health conditions such as hypertension and diabetes. Respondents also answered questions about how often they engaged in behaviors risky to health, such as smoking and binge drinking, and in health promoting behaviors, such as using a seat belt and exercising. Each respondent's body mass index (BMI), (weight in kilograms divided by the square of height in meters), was classified according to the National Institutes of Health criteria as either underweight (<18.5 kg/m2), normal weight (18.5< 25.0 kg/m2), overweight (25.0 to < 30.0 kg/m2), or obese (≥ 30.0 kg/m2) [26]. Individual responses were weighted to reflect the age and sex distribution of each state's population during each survey year. To account for the BRFSS's complex sample survey design, means (M) and 95% confidence intervals (CI) for SBDD and other HRQOL measures were calculated using SUDAAN (Research Triangle, release 8.0.0, Research Triangle Park, NC: 2001). Because mean SBDD varied by five-year age groups, the analyses were directly standardized to the age distribution of adults aged 18 years or older from the 2000 U.S. Census to control for confounding by age. Results Respondents reported a mean of 3.0 (95% CI = 2.9–3.1) SBDD in the previous 30 days. About 43.4% of respondents reported one or more SBDD including 7.9% who reported 14 or more SBDD. Women (M = 3.5, 95% CI = 3.4–3.6) reported a higher number of SBDD than did men (M = 2.4, 95% CI = 2.2–2.5) (Table 1). Young adults aged 18–24 years reported the highest number of SBDD, whereas older adults aged 60–84 reported the fewest, with the gap in mean SBDD between men and women decreasing with increasing age (Table 1). Table 1 Mean number of days U.S. adults felt sad, blue or depressed (Behavioral Risk Factor Surveillance System)* Males Females Males & Females N Mean 95% CI N Mean 95% CI N Mean 95% CI Demographic, behavioral risk group variable 5 year age group 18–19 yrs. 1,704 2.8 2.4–3.2 1,914 4.5 4.0–5.0 3,618 3.6 3.2–3.9 20–24 yrs. 4,899 2.8 2.5–3.0 6,697 4.0 3.7–4.2 11,596 3.4 3.2–3.6 25–29 yrs 6,439 2.4 2.2–2.6 8,881 3.4 3.2–3.6 15,320 2.9 2.7–3.1 30–34 yrs. 7,163 2.2 2.0–2.4 10,016 3.6 3.4–3.8 17,179 2.9 2.7–3.0 35–39 yrs. 7,763 2.5 2.3–2.6 11,061 3.9 3.7–4.1 18,824 3.2 3.0–3.3 40–44 yrs. 7,589 2.6 2.4–2.8 10,430 3.7 3.5–3.9 18,019 3.2 3.0–3.3 45–49 yrs. 6,729 2.7 2.5–2.9 9,297 3.8 3.5–4.0 16,026 3.2 3.0–3.4 50–54 yrs. 5,722 2.6 2.4–2.9 7,986 3.8 3.5–4.1 13,708 3.2 3.0–3.4 55–59 yrs. 4,525 2.2 1.9–2.4 6,424 3.6 3.3–3.9 10,949 3.0 2.7–3.2 60–64 yrs. 3,806 2.2 1.9–2.6 5,489 3.2 2.9–3.4 9,295 2.7 2.5–2.9 65–69 yrs. 3,733 1.8 1.6–2.1 5,905 2.6 2.4–2.9 9,638 2.3 2.1–2.5 70–74 yrs. 3,232 1.9 1.6–2.2 5,629 2.8 2.5–3.1 8,861 2.4 2.2–2.6 75–79 yrs. 2,287 2.2 1.8–2.5 4,627 2.8 2.5–3.1 6,914 2.5 2.3–2.8 80–84 yrs. 1,270 2.3 1.8–2.8 2,949 2.6 2.3–3.0 4,219 2.5 2.2–2.8 85+ yrs. 616 2.4 1.7–3.1 1,782 3.1 2.6–3.7 2,398 2.9 2.4–3.4 All categories 67,477 2.4 2.2–2.5 99,087 3.5 3.4–3.6 166,564 3.0 2.9–3.1 Race/ethnicity Asian/Pacific Islander 1,098 1.6 1.2–2.0 1,216 2.5 2.0–2.9 2,314 2.0 1.7–2.3 White 54,502 2.3 2.2–2.4 77,761 3.3 3.3–3.4 132,263 2.8 2.7–2.9 Hispanic 4,395 2.9 2.5–3.2 6,455 4.3 3.9–4.6 10,850 3.6 3.3–3.8 Black 5,852 2.9 2.7–3.2 11,463 4.5 4.2–4.7 17,315 3.8 3.6–4.0 Native American Indian/Alaska Native 679 3.0 2.3–3.7 956 5.0 4.1–5.9 1,635 4.0 3.4–4.6 Other 431 3.0 2.0–4.0 538 5.2 4.0–6.4 969 4.2 3.2–5.1 All categories 66,526 2.4 2.3–2.5 97,851 3.6 3.4–3.7 165,346 3.0 2.9–3.1 Education < High school 7,772 3.7 3.4–4.0 12,468 5.6 5.3–5.9 20,240 4.7 4.5–4.9 HS graduate 20,727 2.6 2.4–2.7 32,902 3.8 3.7–3.9 53,629 3.2 3.1–3.3 Some college 17,770 2.4 2.2–2.5 27,700 3.4 3.3–3.5 45,470 2.9 2.8–3.0 College graduate 21,068 1.7 1.6–1.8 25,826 2.4 2.3–2.5 46,894 2.0 1.9–2.1 All categories 67,337 2.4 2.3–2.5 98,896 3.6 3.5–3.7 166,233 3.0 2.9–3.1 Employment Employed (wages) 41,465 1.9 1.8–2.0 51,567 2.9 2.8–3.1 93,032 2.4 2.3–2.5 Self-employed 7,497 2.3 2.0–2.5 5,354 3.0 2.7–3.3 12,851 2.5 2.3–2.7 Retired 12,054 3.6 2.4–4.9 19,880 2.4 1.5–3.2 31,934 3.0 2.0–4.0 Student 1,997 2.0 1.6–2.4 3,019 3.8 3.1–4.5 5,016 3.3 2.7–3.9 Homemaker 168 2.7 1.6–3.9 11,549 3.5 3.3–3.8 11,717 3.5 3.3–3.7 Unemp. < 1 yr. 1,238 4.5 3.9–5.1 2,094 6.4 5.5–7.3 3,332 5.7 4.9–6.5 Unemp. ≥1 yr. 766 5.3 4.4–6.2 1,657 6.3 5.6–7.0 2,423 6.1 5.5–6.7 Unable to work 2,225 9.6 8.5–10.6 3,878 10.7 10.0–11.5 6,103 10.2 9.5–10.8 All categories 67,410 2.4 2.3–2.5 98,998 3.6 3.5–3.7 166,408 3.0 2.9–3.1 Income <$15,000 5,194 5.4 5.0–5.8 13,012 6.5 6.2–6.8 18,206 6.1 5.8–6.3 $15,000–$24,999 10,765 3.3 3.1–3.4 18,662 4.5 4.3–4.7 29,427 3.9 3.8–4.0 $25,000–$49,999 23,046 2.2 2.1–2.4 29,419 3.2 3.0–3.3 52,465 2.7 2.6–2.8 ≥$50,000 20,046 1.7 1.6–1.8 21,382 2.4 2.3–2.6 41,428 2.0 1.9–2.1 All categories 59,051 2.5 2.4–2.6 82,475 3.6 3.5–3.7 141,526 3.0 2.9–3.1 Marital status Currently married 38,756 2.1 1.8–2.3 49,671 3.0 2.8–3.1 88,427 2.5 2.4–2.6 Never married 14,429 3.0 2.8–3.2 15,847 3.6 3.3–3.8 30,276 3.3 3.1–3.4 Divorced 8,060 3.6 3.1–4.2 13,600 5.0 4.7–5.2 21,660 4.4 4.1–4.8 Unmarried couple 1,536 3.8 2.6–5.0 2,008 4.7 3.7–5.6 3,544 4.5 3.6–5.5 Widowed 2,980 4.2 3.4–5.0 14,562 6.6 5.5–7.7 17,542 6.0 5.1–6.9 Separated 1,609 5.4 4.6–6.1 3,164 6.3 5.7–6.9 4,773 6.0 5.5–6.5 All categories 67,370 2.4 2.3–2.5 98,852 3.6 3.5–3.6 166,222 3.0 2.9–3.1 Participate in any physical activity Yes 36,039 2.1 2.0–2.2 49,472 3.1 3.0–3.2 85,511 2.6 2.5–2.7 No 13,388 3.1 3.0–3.3 22,607 4.6 4.4–4.8 35,995 3.9 3.8–4.1 All categories 49,427 2.4 2.3–2.5 72,079 3.6 3.5–3.7 121,506 3.0 2.9–3.1 Body mass index Underweight 608 4.6 3.6–5.6 3,192 4.5 4.0–5.0 3,800 4.5 4.1–5.0 Normal 23,561 2.5 2.4–2.6 47,187 3.0 2.9–3.1 70,748 2.8 2.7–2.9 Overweight 30,305 2.1 2.0–2.2 25,987 3.8 3.6–3.9 56,292 2.7 2.6–2.8 Obese 12,295 2.8 2.6–2.9 17,031 5.0 4.7–5.2 29,326 3.8 3.7–4.0 All categories 66,769 2.4 2.3–2.5 93,397 3.6 3.5–3.7 160,166 3.0 3.0–3.1 Ever drank ≥ 5 drinks in past 30 days at once Yes 6,403 2.6 2.4–2.9 2,968 5.0 4.4–5.5 9,371 3.3 2.9–3.6 No 18,206 2.2 2.0–2.3 29,554 3.3 3.2–3.4 47,760 2.8 2.7–2.9 All categories 24,609 2.3 2.2–2.4 32,522 3.4 3.3–3.5 57,131 2.8 2.7.2.9 Smoking status Never smoked 31,088 1.9 1.8–2.0 57,611 3.0 2.9–3.1 88,699 2.6 2.4–2.7 Former smoker 18,973 2.2 2.1–2.4 19,639 3.5 3.4–3.7 38,612 2.9 2.8–3.0 Smokes <1 pack/day 7,112 3.1 2.8–3.3 11,357 5.0 4.7–5.2 18,469 4.1 3.9–4.3 Smokes ≥1 pack/day 8,751 4.2 3.8–4.6 8,688 6.1 5.7–6.4 17,439 5.0 4.7–5.2 All categories 65,924 2.4 2.3–2.5 97,295 3.6 3.4–3.7 163,219 3.0 2.9–3.1 Seatbelt use Always 7,960 2.2 2.0–2.4 13,769 3.1 2.9–3.2 21,729 2.7 2.6–2.8 Nearly always 2,142 2.0 1.7–2.3 2,469 3.5 3.1–3.9 4,611 2.7 2.4–2.9 Sometimes 1,287 2.8 2.4–3.3 1,291 4.4 3.8–4.9 2,578 3.5 3.1–3.8 Seldom 698 3.1 2.4–3.8 578 5.5 4.5–6.4 1,276 3.9 3.4–4.5 Never 901 4.0 3.3–4.7 639 6.1 5.0–7.1 1,540 4.7 4.1–5.3 All categories 12,988 2.4 2.3–2.5 18,746 3.4 3.2–3.5 31,734 2.9 2.8–3.0 Time when could not afford to get medical care in past year Yes 5,629 5.4 5.0–5.7 11,867 6.8 6.5–7.1 17,496 6.2 6.0–6.5 No 61,740 2.1 2.0–2.2 87,069 3.1 3.0–3.2 148,809 2.6 2.5–2.7 All categories 67,369 2.4 2.3–2.5 98,936 3.6 3.4–3.7 166,305 3.0 2.9–3.1 Have any kind of health plan? Yes 58,645 2.2 2.1–2.3 87,693 3.3 3.2–3.4 146,338 2.8 2.7–2.9 No 8,657 3.7 3.4–4.1 11,236 5.1 4.7–5.4 19,893 4.4 4.1–4.6 All categories 67,302 2.4 2.3–2.6 98,929 3.6 3.4–3.7 166,231 3.0 2.9–3.1 Self-rated health Excellent 16,485 1.4 1.3–1.5 22,518 1.9 1.8–2.0 39,003 1.6 1.5–1.7 Very good 22,980 1.7 1.6–1.8 33,099 2.7 2.6–2.8 56,079 2.2 2.1–2.3 Good 18,980 2.5 2.3–2.6 28,081 3.9 3.7–4.0 47,061 3.2 3.1–3.3 Fair 6,441 4.8 4.5–5.1 11,041 7.2 6.8–7.5 17,482 6.0 5.8–6.3 Poor 2,464 11.3 10.2–12.4 4,154 11.2 10.3–12.1 6,618 11.2 10.5–12.0 All categories 67,350 2.4 2.3–2.5 98,893 3.6 3.4–3.7 166,243 3.0 2.9–3.1 Note. *Selected U.S. states, 1995–2000; all variables except age-groups are age-adjusted. Asians/Pacific Islanders reported the fewest SBDD (M = 2.0, 95% CI = 1.7–2.3), whereas Hispanics, Blacks, American Indians and Alaska Natives, and non-whites of another race/ethnicity reported about 4–5 SBDD; in the last three of these groups, the gaps between men and women were larger. Adults with more education reported fewer SBDD, with the gap between men and women diminishing with more education. Respondents who were unemployed or unable to work reported more SBDD than the employed. The number of SBDD decreased with increasing levels of annual household income. Widowed or separated adults reported about 6 SBDD, whereas respondents who were currently married reported the fewest number of SBDD (M = 2.5); the gap between men and women was least among those who had never married. SBDD was associated with an increased prevalence of behaviors risky to health, extremes of BMI, less access to health care, and worse self-rated health status. Respondents who reported physical inactivity, binge drinking, seldom-use or nonuse of seatbelt, or any or more cigarette smoking reported substantially higher numbers of SBDD than those who did not report engaging in these risky behaviors (Table 1). Subjects who were either underweight or obese reported a higher number of SBDD than those of normal weight or overweight. Obese women reported more SBDD (M = 5.0, 95% CI = 4.7–5.2) than obese men (M = 2.8, 95% CI = 2.6–2.9). However, underweight men and women reported the same number (about 4.5) of SBDD. Respondents who could not afford to see a physician at least once during the past year or who had no health care insurance coverage reported more SBDD than those who could afford to see a physician or had such coverage. Although subjects with excellent self-rated health status reported 1.6 SBDD, those with poor health reported 11.2 SBDD, with the largest gap (2.4 days) occurring between men and women with fair health status. Additional Construct Validity SBDD were associated with other physical and mental HRQOL domains in expected ways. Mean SBDD increased with progressively higher levels (i.e., 0, 1–2, 3–13, 14–29, and 30 days) of physically unhealthy days, mentally unhealthy days, unhealthy days, activity limitation days, anxiety days, pain days, and sleepless days (Table 2). Similarly, subjects who reported more days when they felt "very healthy and full of energy" reported fewer SBDD. The gap between men and women was smaller at lower levels of these other HRQOL domains. Table 2 Mean number of days adults felt sad, blue or depressed by HRQOL* and sex** HRQOL variable Males Females Male & Females N Mean 95% CI N Mean 95% CI N Mean 95% CI Physically unhealthy days 0 days 48,188 1.7 1.6–1.8 63,192 2.6 2.5–2.7 111,380 2.2 2.0–2.3 1–2 days 6,792 2.3 2.1–2.5 10,855 3.0 2.8–3.2 17,647 2.6 2.5–2.8 3–13 days 6,371 3.6 3.4–3.9 12,631 4.6 4.4–4.8 19,002 4.2 4.1–4.4 14–29 days 1,959 5.9 5.3–6.5 4,614 7.5 7.0–8.0 6,573 6.9 6.5–7.3 30 days 3,539 8.1 7.3–8.8 6,207 9.3 8.6–9.9 9,746 8.7 8.3–9.2 all categories 66,849 2.4 2.3–2.5 97,499 3.5 3.4–3.6 164,348 3.0 2.9–3.1 Mentally unhealthy days 0 days 50,042 1.1 1.0–1.2 63,446 1.5 1.4–1.6 113,488 1.3 1.2–1.4 1–2 days 5,446 2.1 1.9–2.2 9,644 2.2 2.0–2.3 15,090 2.1 2.0–2.2 3–13 days 6,717 4.9 4.6–5.2 14,219 5.2 5.0–5.3 20,936 5.0 4.8–5.2 14–29 days 2,194 11.1 10.5–11.8 5,318 12.6 12.2–13.1 7,512 12.1 11.7–12.5 30 days 2,366 16.1 15.3–16.9 5,173 17.2 16.6–17.7 7,539 16.8 16.3–17.2 all categories 66,765 2.4 2.3–2.5 97,800 3.5 3.4–3.6 164,565 3.0 2.9–3.1 Unhealthy days+ 0 days 38,831 0.9 0.8–1.0 45,983 1.3 1.2–1.4 84,814 1.1 1.0–1.2 1–2 days 7,675 1.4 1.3–1.5 11,093 1.5 1.4–1.6 18,768 1.5 1.4–1.6 3–13 days 10,963 2.9 2.8–3.0 20,785 3.4 3.3–3.5 31,748 3.2 3.1–3.5 14–29 days 3,344 6.7 6.3–7.2 7,935 7.7 7.4–8.0 11,279 7.4 7.1–7.6 30 days 5,446 10.7 10.2–11.3 10,678 12.4 12.1–12.8 16,124 11.7 11.4–12.1 all categories 66,259 2.4 2.3–2.5 96,474 3.5 3.4–3.6 162,733 3.0 2.9–3.1 Activity limitation days 0 days 56,202 1.7 1.6–1.8 77,711 2.6 2.5–2.7 133,913 2.1 2.0–2.2 1–2 days 3,556 2.5 2.2–2.7 6,446 3.3 3.1–3.5 10,002 3.0 2.8–3.1 3–13 days 3,462 5.3 4.9–5.7 7,169 6.6 6.3–6.9 10,631 6.1 5.8–6.4 14–29 days 1,305 10.2 9.4–11.0 2,887 11.8 11.2–12.5 4,192 11.2 10.7–11.7 30 days 1,962 12.3 11.3–13.4 3,204 13.6 12.7–14.5 5,166 13.0 12.2–13.7 all categories 66,487 2.4 2.3–2.5 97,417 3.5 3.4–3.6 163,904 3.0 2.9–3.1 Anxiety days 0 days 32,664 0.6 0.5–0.7 38,600 0.8 0.7–0.9 71,264 0.7 0.6–0.8 1–2 days 11,739 1.1 1.0–1.2 17,592 1.3 1.2–1.4 29,331 1.2 1.1–1.3 3–13 days 14,076 3.1 3.0–3.3 25,130 3.7 3.6–3.8 39,206 3.4 3.3–3.5 14–29 days 3,946 9.4 8.9–9.9 7,834 10.2 9.9–10.6 11,780 9.9 9.6–10.1 30 days 4,018 13.9 13.3–14.5 8,017 15.0 14.6–15.5 12,035 14.5 14.2–14.9 all categories 66,443 2.4 2.3–2.5 97,173 3.6 3.4–3.7 163,616 3.0 2.9–3.1 Pain days 0 days 52,475 1.7 1.6–1.8 72,697 2.6 2.5–2.7 125,172 2.2 2.0–2.2 1–2 days 4,421 2.6 2.3–2.8 7,143 3.5 3.3–3.8 11,564 3.1 2.9–3.3 3–13 days 4,815 3.8 3.6–4.1 8,598 5.5 5.2–5.8 13,413 4.8 4.5–5.0 14–29 days 1,762 6.8 6.1–7.4 3,642 8.6 8.1–9.2 5,405 7.9 7.4–8.3 30 days 3,363 9.1 8.3–9.9 5,612 10.3 9.6–11.0 8,975 9.7 9.2–10.3 all categories 66,836 2.4 2.3–2.5 97,692 3.6 3.4–3.7 164,528 3.0 2.9–3.1 Sleeplessness days 0 days 24,107 1.6 1.5–1.7 31,809 2.2 2.1–2.3 55,916 1.9 1.8–2.0 1–2 days 7,750 1.1 1.0–1.2 10,410 1.6 1.5–1.8 18,160 1.4 1.3–1.5 3–13 days 20,007 2.1 2.0–2.2 29,594 2.8 2.7–2.9 49,601 2.5 2.4–2.6 14–29 days 8,407 5.0 4.6–5.3 13,633 6.0 5.8–6.3 22,040 5.5 5.3–5.7 30 days 6,347 6.4 6.0–6.8 12,087 7.9 7.6–8.3 18,434 7.3 7.0–7.6 all categories 66,618 2.4 2.3–2.5 97,533 3.6 3.4–3.7 164,151 3.0 2.9–3.1 Vitality days 0 days 6,247 7.1 6.6–7.5 11,536 9.0 8.6–9.4 17,783 8.2 7.9–8.5 1–2 days 1,450 5.9 5.2–6.6 3,118 7.4 6.8–8.0 4,568 6.8 6.4–7.3 3–13 days 8,912 4.2 4.0–4.4 14,809 5.4 5.2–5.6 23,721 4.9 4.7–5.0 14–29 days 28,280 1.7 1.6–1.8 41,496 2.5 2.3–2.6 69,776 2.1 2.0–2.2 30 days 20,990 1.1 0.9–1.2 24,687 1.3 1.2–1.4 45,677 1.2 1.1–1.3 all categories 65,879 2.4 2.3–2.5 95,646 3.6 3.4–3.7 161,525 3.0 2.9–3.1 Note. *Health-Related Quality of Life (HRQOL) questions are: Now thinking about your physical health which includes physical illness and injury, for how many days in the past 30 days was your physical health not good?; Now thinking about your mental health which includes stress, depression, and problems with emotions, for how many days in the past 30 days was your mental health not good?; During the past 30 days, for about how many days did poor physical or mental health keep you from doing your usual activities, such as self-care, work, or recreation?; During the past 30 days, for about how many days did pain make it hard for you to do your usual activities, such as self-care, work, or recreation?; During the past 30 days, for about how many days have you felt worried, tense, or anxious?; During the past 30 days, for about how many days have you felt you did not get enough rest or sleep?; During the past 30 days, for about how many days have you felt very healthy and full of energy? **Behavioral Risk Factor Surveillance System; Selected U.S. states 1995–2000; all variables are age-adjusted. +A calculated measure which results from the sum of physically unhealthy days and mentally unhealthy days with a maximum of 30 unhealthy days for an individual. Discussion In our study, U.S. adults reported an average of about 3 days during the past 30 days when they felt "sad, blue or depressed." Our results are consistent with previous studies documenting the increased prevalence of depressive symptoms among the following groups: women [27-31], in certain minority racial and ethnic groups [32], people with lower levels of education and income [32,33], people of lower employment status [27,34,35], people formerly married or living together but not married [27,36], and in those with limited or no access to health care [32,37,38]. The gap in the number of SBDD between men and women was less pronounced as socioeconomic status improved. Respondents who reported a higher number of SBDD also reported engaging in unhealthy behaviors such as cigarette smoking, binge drinking, and physical inactivity. Underweight and obese adults also reported higher numbers of SBDD than did normal or overweight adults. These findings extend previous public health studies that have documented an association between self-reported mental distress and behaviors risky to health [39-42]. Given the cross-sectional design of the BRFSS, we were unable to determine whether risky behaviors preceded or followed SBDD. Nonetheless, our findings provide additional evidence for the association of considerable public health importance between negative mood and unhealthful behaviors [43]. For example, in the prospective Stirling County Study (1952–1992), subjects who became depressed were more likely to initiate smoking, continue smoking, and refrain from quitting smoking than those who had never become depressed [44]. Negative mood adversely influences self-efficacy to adopt and maintain healthful behaviors and may thwart other self-motivating processes (e.g., attitudes, outcome expectations, and goals) associated with engaging in healthful behaviors [45]. Perceived inefficacy can foster additional despondency. This finding has implications for public health interventions. For example, psychosocial interventions that elicit positive emotions, instill confidence in adopting health-promoting behavior, and improve people's coping skills might be more effective for individuals with despondent mood than interventions designed to arouse fear regarding the consequences of engaging in risky behaviors–which can foster inefficacy and increased despondency [45]. Our findings support the construct validity of the SBDD measure in this study because SBDD were associated with other physical and mental HRQOL domains in expected ways. Groups with progressively higher numbers of physically unhealthy days, activity limitation days, and pain days reported a higher number of SBDD. Moreover, these associations were more pronounced with mentally unhealthy days and anxiety days, than with physically unhealthy days, activity limitation days, and pain days. We found an exception to the linear relationship between SBDD and HRQOL measures with our measure for sleeplessness. Adults who reported 1–2 days of sleeplessness reported fewer SBDD than those who reported no days of sleeplessness. Sleep disturbance, both insomnia and hypersomnia are symptoms of depression. Those reporting no days of sleeplessness, but more SBDD, might be those with hypersomnia. Additional studies are warranted to examine this hypothesis. Besides the cross-sectional design, this study has other limitations. Only 38 states and the District of Columbia included the HRQOL supplemental module that assessed SBDD. All states and the District of Columbia, however examined mentally unhealthy days–the number of days respondents experienced poor mental health due to stress, depression or problems with emotions. Mean mentally unhealthy days in the states that assessed SBDD with the HRQOL supplemental module did not differ significantly from that in states that did not. Given the positive correlation between mentally unhealthy days and SBDD (r = 0.6), states that did not assess SBDD would most likely report similar SBDD as states that did include this measure, suggesting similar study results had all states assessed SBDD. Second, BRFSS excludes people who do not have telephones, live in institutions, and persons younger than 18 years. Third, BRFSS may under represent the severely impaired because functional capacity is required to participate in BRFSS. Including this group however, would probably only strengthen the associations we found because the variability of SBDD would increase because the severely impaired would be more likely to report more SBDD. Finally, because our findings on SBDD are based on respondents' self-reports rather than on professionally administered psychiatric evaluations, people who experience SBDD may differ from people with clinical depression. Conclusion The 1999 Surgeon General's report states that mental health and mental illness "are not polar opposites but may be thought of as points on a continuum" [1]. Although most people who report feeling sad, blue or depressed several days each month probably do not have a diagnosable mental disorder, those above a certain threshold of SBDD might be at increased risk for mental illness and physical illness. Additional studies that examine this hypothesis are warranted. Findings from this study, moreover, highlight the relationship between feeling sad, blue or depressed and engaging in risky behaviors, thereby suggesting the need for appropriately designed interventions specifically targeted to a person's individual and social context [18]. Use of this measure of "sad, blue or depressed days" along with other valid mental health measures can help to assess the burden of population mental distress, identify subgroups with unmet mental health needs, inform the development of targeted interventions, and monitor changes in population levels of mental distress over time [12]. Future research might examine in more detail the associations among SBDD, anxiety, vitality, and sleeplessness and their ability to assess mood, anxiety, and sleep disorders. It would also be useful to examine the prevalence and demographic characteristics of those who report 14 or more SBDD, and the criterion validity of this measure with other screening instruments and clinical assessments. While SBDD does not provide a strict measure of diagnosable depression as would validated screening and diagnostic assessments, SBDD and other measures such as activity limitations, alcohol or substance abuse, physical inactivity, and employment status can be useful community indicators for addressing the prevention and treatment of depressive symptoms and associated comorbidity [12]. Abbreviations SBDD sad, blue or depressed days HRQOL health-related quality of life BRFSS Behavioral Risk Factor Surveillance System CDC Centers for Disease Control and Prevention ==== Refs U.S. Department of Health and Human Services Mental Health: A Report of the Surgeon General Rockville, MD: US Department of Health and Human Services, Substance Abuse and Mental Health Services Administration, Center for Mental Health Services, National Institutes of Health, National Institute of Mental Health 1999 Jonas BS Brody D Roper M Narrow WE Prevalence of mood disorders in a national sample of young American adults Soc Psychiatry Psychiatr Epidemiol 2003 38 618 624 14614549 10.1007/s00127-003-0682-8 World Health Organization Prevention and Promotion in Mental Health Geneva, Switzerland: World Health Organization 2002 Center for Mental Health Services Manderscheid RW, Henderson MJ Mental Health, United States, 2002 DHHS Pub No (SMA) 3938 Rockville, MD: Substance Abuse and Mental Health Services Administration 2004 92 104 Kessler RC McGonagle KA Zhao S Nelson CB Hughes M Eshleman S Wittchen HU Kendler KS Lifetime and 12-month prevalence of DSM-III-R psychiatric disorders in the United States Arch Gen Psychiatry 1994 51 8 19 8279933 National Institute of Mental Health The Numbers Count: Mental Disorders in America, 2001 Robins L Helzer JE Croghan J Williams JBW Spitzer RL NIMH Diagnostic Interview Schedule Version III Rockville, MD: National Institute of Mental Health 1981 American Psychiatric Association Diagnostic and Statistical Manual, Third edition, revised (DSM-III-R) 1987 Washington, DC: APA American Psychiatric Association Diagnostic and Statistical Manual, Fourth edition, revised (DSM-IV) 1994 Washington, DC: APA Roberts RE Kaplan GA Shema SJ Strawbridge WJ Prevalence and correlates of depression in an aging cohort: the Alameda County Study J Gerontol B Psychol Sci Soc Sci 1997 52B S252 S258 9310097 Murray JL Lopez AD Eds Summary: The Global Burden of Disease Boston: Harvard School of Public Health 1996 Institute of Medicine Improving Health in the Community: A Role for Performance Monitoring 1997 Washington, DC: National Academy Press Gaynes BN Burns BJ Tweed DL Erickson P Depression and health-related quality of life J Nerv Ment Dis 2002 190 799 806 12486367 10.1097/00005053-200212000-00001 Hybels CF Blazer DG Pieper CF Toward a threshold for subthreshold depression: an analysis of correlates of depression by severity of symptoms using data from an elderly community sample Gerontologist 2001 41 357 65 11405433 Solomon A Haaga DA Arnow BA Is clinical depression distinct from subthreshold depressive symptoms?: A review of the continuity issue in depression research J Nerv Ment Dis 2001 189 498 506 11531201 10.1097/00005053-200108000-00002 Weissman MM Bruce ML Leaf PJ Florio LP Holzer C Robins LE, Regier DA Affective Disorders In Psychiatric Disorders in America: The Epidemiologic Catchment Area Study 1991 New York: The Free Press 53 80 Mechanic D Is the prevalence of mental disorders a good measure of the need for services? Health Affairs 2003 22 8 20 14515877 10.1377/hlthaff.22.5.8 Institute of Medicine Health and Behavior: The Interplay of Biological, Behavioral, and Societal Influences 2001 Washington, DC: National Academy Press Cassano P Fava M Depression and public health: an overview J of Psychosom Res 2002 53 849 57 12377293 10.1016/S0022-3999(02)00304-5 Judd LL Schettler PJ Akiskal HS The prevalence, clinical relevance, and public health significance of subthreshold depressions Psychiatr Clin North Am 2002 25 685098 Centers for Disease Control and Prevention Measuring Healthy Days Atlanta: CDC 2000 Newschaffer CJ Validation of Behavioral Risk Factor Surveillance System (BRFSS) HRQOL measures in a statewide sample Atlanta: US Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion 1998 Radloff L The CES-D Scale: a self-report depression scale for research in the general population Appl Psychol Measurement 1977 1 385 401 Albert SM Validation of BRFSS QOL Items: Harlem Prostate Screening Project Atlanta: US Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion 1997 Centers for Disease Control and Prevention Behavioral Risk Factor Surveillance System National Heart, Lung and Blood Institute Clinical Guidelines on the identification, evaluation, and treatment of obesity in adults 1998 Bethesda, MD: National Heart Lung, and Blood Institute Turner RJ Wheaton B Lloyd DA The epidemiology of social stress Am Socid Rev 1995 60 104 125 Kuehner C Gender differences in unipolar depression: an update of epidemiological findings and possible explanations Acta Psychiatrica Scandinavica 2003 108 163 74 12890270 10.1034/j.1600-0447.2003.00204.x Blehar MC Public health context of women's mental health research Psychiatr Clin North Am 2003 26 781 799 14563109 Sloan DM Kornstein SG Gender differences in depression and response to antidepressant treatment Psychiatr Clin North Am 2003 26 581 594 14563099 Steiner M Dunn E Born L Hormones and mood: from menarche to menopause and beyond J Affect Disord 2003 74 67 83 12646300 10.1016/S0165-0327(02)00432-9 Borawski E Wu G Jia H Substance Abuse and Mental Health Services Administration, Centers for Disease Control and Prevention Self-Reported Frequent Mental Distress Among Adults–United States, 1993–1996 MMWR 1998 47 325 31 Lorant V Deliege D Eaton W Robert A Philippot P Ansseau M Socioeconomic inequalities in depression: a meta-analysis Am J Epidemiol 2003 157 98 112 10.1093/aje/kwf182 Link BG Lennon MC Dohrenwen BP Socioeconomic status and depression: The role of occupations involving direction, control, and planning AJS 1993 98 1351 1387 10.1086/230192 Brown DW Balluz LS Ford ES Giles WH Strine TW Moriarty DG Croft JB Mokdad AH Associations between Short- and Long-Term Unemployment and Frequent Mental Distress among a National Sample of Men and Women J Occup Environ Med 2003 45 1159 1166 14610397 10.1097/01.jom.0000094994.09655.0f Brown SL The effect of union type on psychological well-being: Depression among cohabitors versus marrieds J Health Soc Behav 2000 41 241 255 11011503 Dunlop DD Song J Lyons JS Manheim LM Chang RW Racial/ethnic differences in rates of depression among preretirement adults Am J Public Health 2003 93 1945 1952 14600071 Institute of Medicine Hidden Costs, Value Lost: Uninsurance in America 2003 Washington, DC: National Academy Press Strine TW Balluz LI Chapman DP Moriarty DG Owens MG Mokdad AH Risk behaviors and health care coverage among adults by frequent mental distress status, 2001 Am J Prev Med 2004 26 213 216 15026100 10.1016/j.amepre.2003.11.002 O'Koro CA Mokdad AH Brewer RD Naimi TS Moriarty DG Giles WH Binge drinking and health-related quality of life: Do popular perceptions match reality? Am J Prev Med 2004 26 230 232 15026103 10.1016/j.amepre.2003.10.022 Ford ES Moriarty DG Zack MM Mokdad AH Chapman D Self-reported body mass index and health-related quality of life: findings from the Behavioral Risk Factor Surveillance System Obes Res 2001 9 21 31 11346664 Brown DW Balluz LS Heath GW Moriarty DG Ford ES Giles WH Mokdad AH Associations between recommended levels of physical activity and health-related quality of life–Findings from the 2001 Behavioral Risk Factor Surveillance System Prev Med 2003 37 520 528 14572437 10.1016/S0091-7435(03)00179-8 Stroebe W Changing conceptions of health and illness: Social psychology and health 2000 Buckingham, Philadelphia: Open University Press 1 9 Murphy JM Horton NJ Monson RR Laird NM Sobol AM Leighton AH Cigarette smoking in relation to depression: Historical trends from the Stirling County study Am J Psychiatry 2003 160 1663 1669 12944343 10.1176/appi.ajp.160.9.1663 Bandura A Perceived self-efficacy in health-promoting behavior In Self-efficacy: The Exercise of Control 1997 4 New York: W.H. 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==== Front Health Qual Life OutcomesHealth and Quality of Life Outcomes1477-7525BioMed Central London 1477-7525-2-421529195810.1186/1477-7525-2-42ResearchThe AMC Linear Disability Score project in a population requiring residential care: psychometric properties Holman Rebecca [email protected] Robert [email protected] Marinus [email protected] Haan Rob J [email protected] Department of Clinical Epidemiology and Biostatistics, Academic Medical Center, Amsterdam, The Netherlands2 Department of Neurology, Academic Medical Center, Amsterdam, The Netherlands2004 3 8 2004 2 42 42 22 6 2004 3 8 2004 Copyright © 2004 Holman et al; licensee BioMed Central Ltd.2004Holman et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Currently there is a lot of interest in the flexible framework offered by item banks for measuring patient relevant outcomes, including functional status. However, there are few item banks, which have been developed to quantify functional status, as expressed by the ability to perform activities of daily life. Method This paper examines the psychometric properties of the AMC Linear Disability Score (ALDS) project item bank using an item response theory model and full information factor analysis. Data were collected from 555 respondents on a total of 160 items. Results Following the analysis, 79 items remained in the item bank. The remaining 81 items were excluded because of: difficulties in presentation (1 item); low levels of variation in response pattern (28 items); significant differences in measurement characteristics for males and females or for respondents under or over 85 years old (26 items); or lack of model fit to the data at item level (26 items). Conclusions It is conceivable that the item bank will have different measurement characteristics for other patient or demographic populations. However, these results indicate that the ALDS item bank has sound psychometric properties for respondents in residential care settings and could form a stable base for measuring functional status in a range of situations, including the implementation of computerised adaptive testing of functional status. ==== Body Background It is now widely accepted that examining quality of life is an important aspect in the treatment and evaluation of many conditions. Functional status is seen as an important determinant of quality of life. A wide variety of instruments have been developed to quantify functional status [1]. These instruments tend to have a fixed length and all items are administered to the whole group of patients under scrutiny. However, currently interest is moving towards the more flexible framework offered by item banks. An item bank is a collection of items, for which the measurement properties of each item are known [2,3]. When using an item bank, it is not essential for all respondents to be examined using all items. This enables the burden of testing to be considerably reduced for both patients and researchers. It is even possible to select the 'best' items for individual patients using computerised adaptive testing algorithms [4]. Furthermore, results from studies using different selections of items from an item bank can be directly compared. Item banks, measuring concepts such as quality of life [2,5], the impact of headaches [6] or functional status [7,8], have been developed. The AMC Linear Disability Score (ALDS) project item bank was developed to quantify functional status [7,9]. The ALDS item bank covers a large number of activities, which are suitable for assessing respondents with a very wide range of functional status and many types of chronic condition. The item bank is particularly suitable for use in the Netherlands. The ALDS items were obtained from a systematic review of generic and disease specific functional health instruments [1]. Five psychometric aspects of the ALDS item bank need to be considered before it can be implemented. These are: (a) there needs to be enough variation in the response categories used for each item [9]; (b) estimates of the item response theory model parameters should not depend on patient characteristics such as age or gender [10,11]; (c) estimates of the item response theory model parameters, which are stable across different subsets of items from the instrument and based on a sufficiently large sample [12] of respondents, should be available [9]; (d) an examination of the extent to which the ALDS items represent a single construct; and (e) testing whether a simpler item response theory model is suitable for the set of items. This paper examines these five aspects of the ALDS item bank using the responses given by residents of supported housing schemes, residential care and nursing homes in and around Amsterdam, the Netherlands. This, mainly elderly, population has been chosen because they generally experience some level of functional restriction and consume a large amount of health care services. Methods Data collection This paper considers 160 items, which were considered to be applicable in a residential care setting. Each item has two response categories: 'I could carry out the activity' and 'I could not carry out the activity'. If a respondent had never had the opportunity to experience an activity 'not applicable' was recorded. In the analysis, responses in the category 'not applicable' were treated as if the individual items had not been presented to the individual respondents [13]. It was felt that presenting all 160 items to each respondent would place an unnecessary and unacceptable burden on those responding to the items. Therefore, the data described in this paper were collected using an incomplete, anchored calibration design [7,9,14,15] with four sets of 80 items. Item sets A and B have half their items in common, as do item sets B and C, item sets C and D and item sets A and D. The items in common between two sets of items are known as 'anchors' and allow all items and patients to be calibrated on the same scale. The patterns of missing data in this type of design are, in statistical terms, ignorable [16]. The item sets were administered randomly to 150 respondents (item set A), 143 respondents (item set B), 138 respondents (item set C) and 124 respondents (item set D). Respondents A total of 555 residents of supported housing, residential care and nursing homes were interviewed. The median age was 84 years (range 37 to 101 years), while 444 (80%) were female. Since the respondents were interviewed 'at home', accurate data on medical conditions were not available. All respondents gave informed consent. The study was approved by the medical ethics committee in our hospital. The item response theory models In this paper the data were analysed using the two-parameter logistic item response theory model [7,9,17,18]. In this model, the probability, Pik, that patient k responds to item i in the category 'can' is modelled using where θk denotes the ability of patient k to perform activities of daily life. The discrimination parameter (αi) and difficulty parameter (βi) describe the measurement characteristics of item i. The larger the value of βi, the more difficult item i is. In addition, the larger the value of αi, the better an item is a distinguishing between abilities above and below βi. If the values of αi are constrained to be equal for all items, the model in equation 1 becomes the one-parameter logistic item response theory model [19]. The model in equation 1 can be extended to test whether the values of βi for, say males and females, are significantly different. If the values of βi for different groups of respondents are significantly different, then there is evidence of differential item functioning. Full-information factor analysis also uses an extension of the model in equation 1. These approaches are described in mathematical terms in the Appendix. In this paper, estimates of αi and βi were obtained using a marginal maximum likelihood based procedure [20]. This method assumes that the ability parameters (θk) follow a Normal distribution and can account for incomplete designs, as described in the Appendix. Expected a posteriori methods were used to estimate θk [21]. Statistical analysis To achieve the objectives of this study, there were five steps in the statistical analysis. In step (a), the amount of variation in the response categories used for each item [9] was considered and items demonstrating too little variation were removed. Items were excluded from further analysis if fewer than 10% or more than 90% of the patients responded in the category 'cannot'. In step (b), the items were examined to investigate whether the value of the item difficulty parameter (βi) was similar for male and female patients and for patients younger than 85 years and those aged 85 or older. The model is described in depth in the Appendix. Items were excluded from further analysis if the value of the item difficulty parameter was significantly different (1% level) between gender or aged based groups. In this step, the fit of the model to the data from each item was not assessed. In step (c), estimates of the item parameters (αi and βi) were obtained. The fit of the model to the data from each item was assessed using G2 statistics [22]. Items, for which the fit statistic had a p-value of less than 0.01, were excluded from the item bank. In addition, the stability of the estimates of the item parameters over different sets of items was examined using the model from step (b). Items were excluded from further analysis if the value of the item difficulty parameter was significantly different (1% level) between item sets A and B, B and C, C and D or A and D. Furthermore, a Kolmogornov-Smirnov test was carried out to examine whether the ability parameters (θk) were Normally distributed. In step (d), the dimensionality of the item bank was examined using item response theory based full information factor analysis [18,22,23]. The number of latent roots greater than 1 is regarded as an indicator of the number of factors in the data set. This method is described in more depth in the Appendix. Four exploratory factor analyses were carried out, one on each of the anchors between item sets A and B (293 respondents), B and C (281 respondents), C and D (262 respondents) or A and D (274 respondents). A fifth, confirmatory, factor analysis was carried out on the whole data set (555 respondents). In addition, Cronbach's coefficient alpha was calculated for each anchor and the whole data set [24,25]. In step (e) the one-parameter logistic item response theory model was fitted to the remaining items. The differences between the -2log likelihoods of this model and the two-parameter model fitted in step (c) was tested using a χ2 test. The analysis in steps (a), (b), (c) and (e) was carried out in Bilog, version 3.0 [22]. The analysis in step (d) was carried out using TESTFACT, version 4.0 [22]. Results Of the 160 items included in the item bank, one was removed because it was worded differently in two different item sets. Of the 159 remaining items, 77 were removed from the item bank. This process is described in Table 1. In step (a), 28 items were excluded from further analysis because fewer than 10% or more than 90% of responses were in the category 'cannot'. In step (b), 26 items were removed because they had significantly different estimates of the item difficulty parameter (βi) for for males and females and/or for younger and older respondents. Of these 26 items, 19 had different measurement characteristics for females and for males, 5 items had different measurement characteristics for those aged under 85 and for those aged 85 or over, and 2 items had different measurement characteristics for both males and females and for older and younger respondents. In step (c), 23 items had an item fit statistic p-value of less than 0.01. In addition, 3 items were excluded from further analysis because the value of the item difficulty parameter (βi) was significantly different between two item sets of items. Hence, 79 psychometrically sound items remained in the item bank. A short description of the content of the 79 items in the final version of the calibrated item bank, together with estimates of the dispersion (α) and difficulty (β) parameters and their standard errors, are given in Tables 2a and 2b. Following step (c) of the analysis, the anchors between the sets of items contained between 13 and 23 items. In addition, there was no evidence to suggest that estimates of θ do not follow a Normal distribution (Kolmogorov-Smirnov test, p-value = 0.637). In step (d), the full information factor analysis indicated that, for three of the four anchors between the item sets, there was only one latent root of the correlation matrix larger than 1. In the fourth item set, a second latent root was marginally above 1. The percentage of the variance explained by the first factor varied between 67% and 72%. The values of Cronbach's alpha coefficient for the four anchors were between 0.86 and 0.93. The confirmatory factor analysis carried out on the whole data set indicated that 70% of the variance was explained by the first factor. Cronbach's alpha coefficient for the whole data set equalled 0.98. In step (e), the one-parameter logistic item response theory model was fitted to the 79 items remaining after step (c). This model fitted the data significantly less well than the two-parameter model (p-value < 0.0001). For 3 items, the item fit statistic had p-value < 0.01. After removal of these items, the two-parameter model was still significantly better than the one-parameter model (p-value < 0.0001). Table 1 The number of items proceeding to each step of the analysis The number of and examples of items removed at each stage of the psychometric analysis. Stage of analysis Number of items removed Reason for removal Examples 1 Concerns about the way the item was presented (a) 28 < 10% or > 90% of responses in 'cannot' Reaching for a cup and taking a sip of water Combing hair at a sink Cycling on a heavily laden bicycle (b) 26 Significant difference between M and F and/or under and over 85 years Washing up (easier for older respondents) Crossing the street (easier for younger respondents Preparing a warm meal (easier for female respondents) (c) 26 Item fit p-value < 0.01 or estimates of βi not stable Taking oral medication Cycling Getting money out of the bank using an ATM In item bank 79 See Table 2 Total 160 Table 2 The 79 items remaining in the calibrated item bank. The items remaining in the calibrated item bank. The number of respondents, to whom the item was offered (Offered to), the number responding in the category 'not applicable' (NA), the number responding in the category 'can' (can) and the number responding in the category 'cannot' (cannot) are given. The discrimination (β) and difficulty (β) parameters are given along with their standard errors in parentheses. Description of item content Offered to Item response category Location parameter (β) Discrimination parameter (α) NA can cannot Walking up stairs with a bag 262 0 19 243 -3.607 (2.404) 1.122 (0.892) Mopping a flight of stairs 262 5 16 241 -2.830 (1.708) 0.447 (0.411) Cleaning the top of a high cupboard 281 2 27 252 -2.816 (1.946) 0.480 (0.393) Cleaning a bathroom 293 1 37 255 -2.621 (2.061) 0.323 (0.338) Vacuuming 274 0 33 241 -2.408 (1.844) 0.287 (0.280) Going for a walk in the woods 281 0 31 250 -2.343 (1.636) 0.362 (0.340) Fetching groceries for 3–4 days 293 0 36 257 -2.262 (1.623) 0.353 (0.343) Mopping the floor 281 2 41 238 -2.225 (1.902) 0.339 (0.374) Caring for plants on a balcony 262 1 32 229 -2.108 (1.616) 0.314 (0.325) Travelling by bus or tram 281 0 44 237 -2.093 (1.835) 0.308 (0.370) Walking up two flights of stairs 274 0 39 235 -1.921 (1.532) 0.277 (0.298) Cleaning a fridge 293 2 64 227 -1.406 (1.464) 0.171 (0.236) Going to a restaurant 293 3 60 230 -1.335 (1.238) 0.159 (0.188) Carrying a tray 281 1 75 205 -1.304 (1.774) 0.222 (0.326) Going for a long walk (15+ minutes) 281 0 72 209 -1.290 (1.629) 0.178 (0.277) Going to the dentist 293 18 75 200 -1.283 (1.881) 0.205 (0.299) Sweeping the floor 262 1 70 191 -1.239 (1.902) 0.196 (0.313) Cutting toe nails 262 0 45 217 -1.175 (0.786) 0.136 (0.144) Walking up a hill or bridge 281 3 73 205 -1.133 (1.425) 0.137 (0.198) Walking up one flight of stairs 274 2 67 205 -1.127 (1.382) 0.155 (0.222) Going to a concert 262 0 57 205 -0.996 (0.860) 0.113 (0.128) Going to the pharmacist 262 2 75 185 -0.976 (1.572) 0.131 (0.201) Hanging a load of washing out 293 10 84 199 -0.960 (1.514) 0.144 (0.240) Going to the post office or bank 274 0 88 186 -0.948 (1.881) 0.151 (0.248) Going to a party 281 1 69 211 -0.924 (0.878) 0.109 (0.129) Filling an official form in 281 1 68 212 -0.896 (0.790) 0.106 (0.119) Using a washing machine 281 6 95 180 -0.851 (1.743) 0.153 (0.244) Visiting an outpatients' clinic 293 0 95 198 -0.815 (1.317) 0.112 (0.161) Taking bottles to the bottle bank 281 5 108 168 -0.675 (1.840) 0.153 (0.312) Short walk (less than 15 minutes) 274 0 95 179 -0.645 (1.358) 0.108 (0.166) Putting a rubbish bag outside 293 5 108 180 -0.573 (1.338) 0.116 (0.198) Reaching into a high cupboard 274 0 95 179 -0.569 (1.097) 0.099 (0.172) Using a dustpan and brush 262 2 103 157 -0.537 (1.865) 0.135 (0.335) Opening and closing a high window 281 0 140 141 -0.078 (1.290) 0.096 (0.166) Fetching groceries for one day 262 0 128 134 -0.043 (1.373) 0.099 (0.184) Using a public toilet 293 5 159 129 0.139 (1.835) 0.110 (0.241) Putting flowers in a vase 293 2 162 129 0.169 (1.787) 0.107 (0.240) Frying an egg 281 3 154 124 0.178 (1.982) 0.115 (0.261) Warming up a tin of soup 293 1 164 128 0.203 (1.919) 0.113 (0.232) Cleaning a toilet 262 0 149 113 0.308 (1.528) 0.105 (0.203) Putting socks and lace up shoes on 281 1 167 113 0.314 (1.165) 0.093 (0.157) Changing the bulb in a table light 281 1 177 103 0.533 (1.541) 0.116 (0.174) Cleaning a bathroom sink 281 5 170 106 0.564 (2.089) 0.126 (0.302) Cutting finger nails 262 0 168 94 0.605 (1.337) 0.114 (0.173) Rubbing lotion into whole body 262 4 164 94 0.627 (1.469) 0.115 (0.184) Reaching into a low cupboard 274 0 184 90 0.672 (1.131) 0.106 (0.152) Picking something up off the floor 262 0 172 90 0.712 (1.466) 0.129 (0.198) Making porridge 293 2 191 100 0.714 (1.704) 0.119 (0.216) Getting in and out of a car 281 3 185 93 0.738 (1.656) 0.132 (0.209) Shaking a tablecloth out 274 2 190 82 0.906 (1.438) 0.125 (0.204) Making a bed 281 0 193 88 1.003 (2.028) 0.152 (0.292) Preparing breakfast or lunch 262 1 186 75 1.117 (1.729) 0.169 (0.279) Using the lift in a public building 262 2 199 61 1.208 (1.299) 0.158 (0.186) Putting an alarm clock right 281 4 216 61 1.319 (1.431) 0.165 (0.199) Pulling a blanket up 293 0 253 40 1.485 (0.898) 0.167 (0.149) Visiting the neighbours 293 1 231 61 1.548 (1.685) 0.221 (0.252) Travelling as a passenger in a car 274 3 230 41 1.592 (1.126) 0.222 (0.192) Shaving face or applying make up 274 1 233 40 1.593 (1.075) 0.180 (0.164) Watering a house plant 262 3 204 55 1.600 (1.681) 0.226 (0.259) Opening and closing a window 262 0 201 61 1.735 (2.137) 0.246 (0.343) Putting trousers on 293 2 224 67 1.821 (2.372) 0.295 (0.406) Making coffee or tea 293 0 235 58 1.832 (1.936) 0.237 (0.290) Peeling an apple 281 1 233 47 1.859 (1.631) 0.226 (0.219) Making a bowl of cereal 281 1 225 55 1.860 (1.921) 0.222 (0.256) Eating a meal at the table 293 0 255 38 2.081 (1.509) 0.253 (0.225) Hanging clothes up in a cupboard 262 0 203 59 2.105 (2.595) 0.344 (0.481) Opening and closing curtains 262 0 216 46 2.129 (1.958) 0.366 (0.357) Moving between two dining chairs 281 0 237 44 2.214 (1.905) 0.389 (0.375) Putting a scarf and gloves on 293 1 259 33 2.364 (1.617) 0.306 (0.246) Making a cheese sandwich 281 1 243 37 2.416 (1.856) 0.392 (0.333) Moving to sit on the edge of a bed 262 1 231 30 2.457 (1.658) 0.309 (0.244) Putting a coat on 274 0 227 47 2.463 (2.323) 0.425 (0.395) Putting a shirt or blouse on 262 0 228 34 2.495 (1.842) 0.360 (0.287) Washing upper body at a sink 274 0 243 31 2.705 (1.875) 0.420 (0.327) Answering the front door 274 1 233 40 2.792 (2.373) 0.481 (0.449) Getting out of bed into a chair 262 0 232 30 3.019 (2.132) 0.581 (0.448) Washing lower body at sink 293 1 241 51 3.037 (3.098) 0.722 (0.761) Putting a T-shirt on 293 2 257 34 3.440 (2.664) 0.718 (0.630) Locking a door 262 0 230 32 3.366 (2.512) 0.970 (0.749) NA denotes that the category 'not applicable' was chosen Discussion In this study, the psychometric properties of the item bank have been examined using a sample of 555 respondents and an incomplete calibration design. Each item was presented to between 262 and 293 respondents. These figures are above the minimum, of 200 respondents, regarded as necessary to implement the models used in this paper [12]. It could be argued that it would have been desirable for all respondents to be presented with all items, but this would have placed an unacceptable burden on the, often frail, population in this study. Incomplete calibration designs are regularly implemented in the development and maintenance of item banks used in educational testing [4,14] and have gained some recognition in health related applications [15]. Developments in psychometric theory mean that it is now possible to perform the same types of analysis on data resulting from incomplete designs, as is performed on data from complete calibration designs [22,23,25]. The number of items in the anchors following the analysis, indicate that the design was still amply linked [9]. One of the major assumptions underlying the use of the item response theory models described in this paper is that the items reflect a single latent trait (θ). This has been examined using item response theory based full-information factor analysis. Part of the full-information factor analysis was performed on sub-sets of the data, as exploratory analyses on incomplete designs may lead to instable results. However, the confirmatory factor analysis was performed on all data. The results, together with the high level of internal consistency, as measured by Cronbach's alpha, and the acceptable fit of the two-parameter logistic item response theory model to the data indicate that the items presented in this paper probably represent a unidimensional construct in a population of respondents requiring residential care. Another important assumption when using item response theory models in conjunction with marginal maximum likelihood estimation procedures is that the values of the latent trait (θ) follow a pre-specified, usually Normal, distribution. In this study, there was no evidence that these values did not follow a Normal distribution. This is in contrast to many previously published studies into health and quality of life outcomes, where a strongly skewed distribution was found. The authors feel that there are two reasons for this contrast. Firstly, in this study, the respondents all had some level of restriction in their ability to perform activities of daily life. Secondly, the item bank includes items well above and well below the level of functional status enjoyed by the respondents. This means that the item bank did not have a ceiling or floor effect with respect this this population. In this study, 81 (51%) of 160 items were removed from the item bank because they did not conform to the psychometric standards required of the item bank. This is a much higher level than would be expected in the calibration of an item bank for use in educational measurement. However, when the results are examined more carefully, 28 items were removed because they were too difficult or too easy for the population in this study. In addition, 26 items were removed because they had different item parameters for different groups of respondents. These problems would have been identified much earlier in an educational item bank. Hence, only 26 (25%) of 106 items were removed due to item misfit. The number of items retained in the item bank may have been higher if a more flexible model, based on, for example, non-parametric smoothing techniques had been used [26]. However, this type of model is less suitable as a base for implementing modern testing algorithms, such as computerised adaptive testing. In addition, it is possible that more items could be made available if the items demonstrating differential item functioning were included in the item bank with different item location parameters (βi) for males and females or for younger and older respondents. This may seem complicated, but is straightforward in the framework of a computerised item bank. This paper has concentrated on the two-parameter logistic item response theory model. However, the one-parameter logistic item response theory model was also fitted to the 79 items remaining in the item bank. This model fitted the data significantly less well than the two-parameter model, even after 3 items demonstrating misfit at the item level were removed. This confirms the choice of the two-parameter model. This model was chosen because it allows the probability of responding in the category 'can' to be modelled more flexibly than when the one-parameter logistic model is used. This enables a more realistic model for the data to be built than when the more restrictive approach associated with the one-parameter model is chosen [18]. This paper has examined the calibration of the ALDS item bank in a population requiring residential care. It has been shown that the item bank has sound psychometric properties and could form a stable base for a wide range of applications. However, it is possible that the items will have different measurement characteristics for patients requiring treatment for specific chronic conditions or in other countries. Hence, it is important that the ALDS item bank is tested carefully before it is used to assess the functional status in other groups of respondents or in other countries. Conclusions Now that the measurement properties of the ALDS item bank have been examined carefully, the item bank can be used as a foundation for quantifying functional status. If modern algorithms, such as computerised adaptive testing, are implemented, it will be possible to obtain accurate measurements, whilst keeping the burden of testing on respondents and interviewers to a minimum. Items can be selected for use in further research, for allocation individuals to appropriate care settings and for calculating institutional funding based on the actual care load. It is hoped that the ALDS item bank will play an important part in the implementation of computerised adaptive testing of functional status. Appendix Differential item functioning The model in equation 1 can be extended to test whether different groups of respondents to have different values of βi. This is known as differential item functioning. For instance, if interest is in possible differences in βi between males and females, then the probability, Pik, that patient k responds to item i in the category 'can' is written as where βiM is the item difficulty for male respondents, βiF-M is the difference between the item difficulty for males and for females and Ik is an indicator variable taking the value 0 if respondent k is male and the value 1 if respondent k is female. The hypothesis H0 : βiF-M = 0 can be tested to examine whether item i has the same measurement characteristics for males and for females. Item parameter estimation in incomplete designs In this study, the item parameters (αi and βi) were estimated using marginal maximum likelihood methods. The likelihood, L, over n items and K (K = 555) respondents can be written as where Iik is an indicator variable taking the value 1 if respondent k was offered item i and the value 0 otherwise and where Jik is an indicator variable taking the value 1 if respondent k responded to item i in the category 'can' and the value 0 otherwise. Furthermore, the probability, Pik, that respondent k responded to item i in the category 'can' is as in equation 1, or, where appropriate, as in equation 2 or 4. In the estimation process, the values of θk or θkm were assumed to follow a Normal distribution with mean equal to 0 and unknown variance, σ2, and were integrated out of the likelihood to obtain the marginal likelihood. The marginal likelihood was maximised using an EM algorithm [20]. Full information factor analysis Full information factor analysis is a technique based on multidimensional item response theory models where the ability is represented by M variables, denoted θkm where m = 1, 2,..., M [22,23]. The model, in equation 1, for the probability, Pik, that person k responds to item i in the category 'can' can be extended to where θkm denotes the value of the latent variable θm associated with person k and αim denotes the discrimination parameter for item i with respect to the latent variable θm. Furthermore, δi is a difficulty type parameter. The loading, aim of item i on factor m can be calculated using The value of the standard difficulty parameter, (βi), can be calculated using Generally, the parameters αim and δi are estimated using marginal maximum likelihood methods. Abbreviations ALDS = AMC Linear Disability Score Competing interests None declared. Funding RH and RL were supported by a grant from the Anton Meelmeijer fonds, a charity supporting innovative research in the Academic Medical Center, Amsterdam, The Netherlands. Authors contributions RL conceived the study and supervised the data collection. RH prepared the first draft and carried out the analyses. RL, MV and RJdH critically reviewed the manuscript. RH prepared the final version. ==== Refs Lindeboom R Vermeulen M Holman R de Haan RJ Activities of daily living instruments in clinical neurology. Optimizing scales for neurologic assessments Neurology 2003 60 738 742 12630419 Bode RK Lai JS Cella D Heinemann AW Issues in the development of an item bank Arch Phys Med Rehabil 2003 84 S52 60 12692772 10.1053/apmr.2003.50247 McHorney CA Ten recommendations for advancing patient-centered outcomes measurement for older persons Ann Intern Med 2003 139 403 409 12965966 van der Linden WJ Glas CAW Computerized Adaptive Testing Theory and Practice 2000 Kluwer Academic Publishers, Dordrecht, the Netherlands Accessed 8th July 2004 Accessed 8th July 2004 Holman R Lindeboom R Vermeulen M Glas CAW de Haan RJ The Amsterdam Linear Disability Score (ALDS) project. The calibration of an item bank to measure functional status using item response theory Quality of Life Newsletter 2001 27 4 5 Webster K Cella D Yost K The functional assessment of chronic illness therapy (facit) measurement system: properties, applications, and interpretation Health Qual Life Outcomes 2003 1 79 14678568 10.1186/1477-7525-1-79 Holman R Lindeboom R Glas CAW Vermeulen M de Haan RJ Constructing an item bank using item response theory: the AMC Linear Disability Score project Health Services and Outcomes Research Methodology 2003 4 19 33 10.1023/A:1025824810390 Holman R Lindeboom R Vermeulen M Glas CAW de Haan RJ The Amsterdam Linear Disability Score (ALDS) project. Differential item functioning with regard to gender Quality of Life Newsletter 2002 29 13 14 Holman R Lindeboom R de Haan RJ Gender and age based differential item functioning in the AMC Linear Disability Score project Quality of Life Newsletter 2004 32 1 4 Zimowski MF Mukari E Mislevy RJ Bock RD BILOG-MG Multiple group IRT analysis and test maintenance for binary items 1996 Scientific Software International, Inc. Chicago, IL Holman R Glas CAW Zwinderman AH de Haan RJ Practical methods for dealing with 'not applicable' item responses in the AMC Linear Disability Score project Health Qual Life Outcomes 2004 2 29 15200681 10.1186/1477-7525-2-29 Kolen MJ Brennan RL Test Equating 1995 Springer, New York van Buuren S Hopman-Rock M Revision of the ICIDH severity of disabilities scale by data linking and item response theory Stat Med 2001 20 1061 1076 11276036 10.1002/sim.723 Mislevy RJ Chang H Does addaptive testing violate local independence? Psychometrika 2000 65 149 156 Birnbaum A Some latent trait models and their use in inferring an examinee's ability. Chapter in Lord FM, Novick MR Statistical theories of mental test scores 1968 Addison-Wesley, Reading, MA Thissen D Wainer H Test Scoring 2001 LEA, Mahwah, NJ Rasch G Probabilistic models for some intelligence and attainment tests 1960 Danish Institute for Educational Research, Copenhagen, Denmark Bock RD Aitkin M Marginal maximum likelihood estimation of item parameters: An application of the EM algorithm Psychometrika 1981 46 443 459 Bock RD Mislevy RJ Adaptive EAP estimation of ability in a microcomputer environment Applied Psychological Measurement 1982 6 431 444 du Toit M editor IRT from SSI: Bilog-MG, Multilog, Parscale, Testfact 2003 Scientific Software International, Inc, Lincolnwood, IL Bock RD Gibbons RD Muraki E Full information item factor analysis Applied Psychological Measurement 1988 12 261 280 Cronbach LJ Coefficient alpha and the internal structure of tests Psychometrika 1951 16 297 334 Harvey WR Estimation of variance and covariance components in the mixed model Biometrics 1970 26 485 504 Ramsay JO Kernal smoothing approaches to non-parametric item characteristic curve estimation Psychometrika 1991 56 611 630

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==== Front Health Res Policy SystHealth Research Policy and Systems1478-4505BioMed Central London 1478-4505-2-51528202510.1186/1478-4505-2-5CommentaryPublic – private 'partnerships' in health – a global call to action Nishtar Sania [email protected] Heartfile, Islamabad, Pakistan2004 28 7 2004 2 5 5 30 3 2004 28 7 2004 Copyright © 2004 Nishtar; licensee BioMed Central Ltd.2004Nishtar; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The need for public-private partnerships arose against the backdrop of inadequacies on the part of the public sector to provide public good on their own, in an efficient and effective manner, owing to lack of resources and management issues. These considerations led to the evolution of a range of interface arrangements that brought together organizations with the mandate to offer public good on one hand, and those that could facilitate this goal though the provision of resources, technical expertise or outreach, on the other. The former category includes of governments and intergovernmental agencies and the latter, the non-profit and for-profit private sector. Though such partnerships create a powerful mechanism for addressing difficult problems by leveraging on the strengths of different partners, they also package complex ethical and process-related challenges. The complex transnational nature of some of these partnership arrangements necessitates that they be guided by a set of global principles and norms. Participation of international agencies warrants that they be set within a comprehensive policy and operational framework within the organizational mandate and involvement of countries requires legislative authorization, within the framework of which, procedural and process related guidelines need to be developed. This paper outlines key ethical and procedural issues inherent to different types of public-private arrangements and issues a Global Call to Action. private sectorpublic sectorpartnershipsconflict of interest. ==== Body Public-private partnerships in health – a global call to action Public-private partnerships are being increasingly encouraged as part of the comprehensive development framework. The need to foster such arrangements is supported by a clear understanding of the public sectors inability to provide public goods entirely on their own, in an efficient, effective and equitable manner because of lack of resources and management issues. These considerations have necessitated the development of different interface arrangements, which involve the interfacing of organizations that have the mandate to offer public good on one hand, and those that could facilitate this goal. Within the health sector, public-private partnerships are also the subject of intensely fueled debate [1]. Several examples, which fall within this framework, highlight a potential for the creation of a powerful mechanism for addressing difficult problems by leveraging on the strengths of different partners; however, these also illustrate complex issues, as such arrangements bring together a variety of players with different and sometimes conflicting interests and objectives, working within different governance structures [2]. This paper focuses on public-private partnerships that are intended to address broad questions of providing sustainable health outcomes rather than on the day-to-day interaction that occurs when the government buys a health service from a private supplier or where it leaves the entire matter of health service supply to the private sector. The public sector in this paper refers to national, provincial/state and district governments; municipal administrators, local government institutions, all other government and inter-governmental agencies with the mandate of delivering 'public goods'. The word private denotes two sets of structures; the for-profit private encompassing commercial enterprises of any size and the non-profit private referring to Non Governmental Organizations (NGOs), philanthropies and other not-for-profits. The word partnership in this paper refers to long term, task oriented, and formal relationships. There has been ample critique relating to the convention of using the word partnership to describe such arrangements; much of this debate is valid, given that there are certain requisites for coining such an association. For the same reasons it also needs to be differentiated from privatization, which involves permanent transfer of control through transfer of ownership right or an arrangement in which the pubic sector shareholder has waived its right to subscribe. A distinction also needs to be made between partnerships and contractual arrangements, particularly with regard to the relationship between the public sector and NGOs. Although such arrangements can be used for strategic purposes, they are inherently distinct from partnerships. Types of public-private interface arrangements the database of the Initiative on Public-Private Partnerships for Health of the Global Forum for Health Research lists 91 international partnership arrangements in the health sector, which can qualify to be called public-private partnerships. Of these, 76 are dedicated to infectious disease prevention and control, notably AIDS, tuberculosis and malaria; four focus on reproductive health issues, three on nutritional deficiencies whereas a minority focus on other issues (health policy and research {1}, injection and chemical safety {2}, digital divide {1}, blindness and cataract {4})[3]. This categorization takes into account large transnational public-private partnerships. There are, however, many other arrangements at a country level, which bring in their wake similar challenges as the ones posed by larger partnerships. Several classifications have been proposed to conceptualize and categorize public-private partnerships. These may be based on the terms of the constituent membership or the nature of activity [4,5]. However by virtue of the definitions and the characteristics of the public and private sectors, it can be stated that public-private arrangements are fostered either when governments and inter-governmental agencies interface with the for-profit private sector to tap into resources, or the non-profit private sector for technical expertise or outreach. Several varieties of arrangements of various sizes, forms and scope at a global, regional or country level qualify to fall within this categorization. Transnational partnerships involving a visible role of the for-profit sector are at one end of the spectrum. These usually involve larger partnerships and a complex grouping; depending upon their structure, they may bring together several governments, local and international NGOs, research institutions and UN agencies in transnational programs, often also involving the non-profit sector. Such partnerships can be housed and coordinated by different sources [6]. They can be owned by the pubic sector and have private sector participants such as in the case of Global Alliance for Vaccines and Immunization (GAVI) [7], Roll Back Malaria (RBM) [8], Stop TB partnership (Stop TB) [9], Safe Injections Global Network (SIGN) [10], Global Polio Eradication Programme (PEI) [11], the Special Programme for Research and Training in Tropical Diseases (TDR) [12], and the Special Programme for Research Development and Research Training in Human Reproduction (HRP) [13]. Partnerships can be principally orchestrated by companies such as in the case of Action TB [14], and can be legally independent such as the International Aids Vaccine Initiative (IAVI) [15], Medicines for Malaria Venture (MMV) [16], Global Alliance for TB Drug Development (GATBDD) [17], and the Concept Foundation (CF) [18]. Large partnerships can also be hosted by a civil society NGO; examples include the Malaria Vaccine Initiative (MVI) [19], the Mectizan Donation Programme (MDP) [20], and the HIV Vaccine Initiative (HVI) [21]. At the other end of the spectrum, there are examples of individual governments forming partnerships with the for-profit private sector [22]. There are also examples of situations when a government partners with an NGO with a particular technical strength, technical or outreach related. The recent evolution of a public-private partnership for the prevention and control of non-communicable diseases in Pakistan is an example of this approach, where the government leverages on the technical strength of the private sector partner for addressing an emerging health challenge [23]. Examples also exist of NGOs seeking support from corporate partners both at a national and an international level. The World Heart Federation has recently structured transparent and successful business relationships with the corporate sector for supporting global programs with initial encouraging results [24,25]. Partnerships in the health sector can be for various purposes; categories as stated by the Initiative on Public-Private Partnerships for Health have been summarized in Table 1. Such partnerships are novel arrangements and potentially present an opportunity for more than one partner(s) to contribute to the same goal. Many of these have positively contributed to health outcomes in the past; developing technologies for tropical diseases, surveillance and screening strategies, contributing to technical aspects of sustainable drug development and vector control are amongst a few examples [26,27]. Notwithstanding, partnerships involving the for-profit private sector bring in their wake many concerns as they involve a donor-recipient relationship [28]. Table 1 Categorization of public-private partnerships based on the purpose they serve Purpose Partnership 1 Product development GATBDD, IAVI, MMV and MVI. 2 Improving access to healthcare products CF, MDP, Accelerated Access Initiative (AAI) [48], Global Alliance to Eliminate Leprosy (GAEL) [49], Global Alliance to Eliminate Lymphatic Filiariasis (GAELF) [50] and the Global Polio Eradication Initiative (GPEI) [51]. 3 Global coordination mechanisms GAVI, RPS, Stop TB, Global Alliance for Improved Nutrition (GAIN) [52], and the Micronutrient Initiative (MI) [53]. 4 Strengthening health services Alliance for Health Policy and Systems Research (AHPSR) [54], Multilateral Initiative on Malaria (MIM) [55], African Comprehensive HIV/AIDS Partnerships (ACHAP) [56]. 5 Public advocacy and education Alliance for Microbicide Development (AMD) [57], African Malaria Partnership (AMP) [58], Global Business Coalition on HIV and AIDS (GBC) [59] and Corporate Council on Africa (CCA) [60]. 6 Regulation and quality assurance The International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) [61], Pharmaceutical Security Institute (PSI) [62] and the Anti-Counterfeit Drug Initiatives [63] In many countries, there are long established links of the public sector with NGOs. Theoretically, since NGOs are not driven by a profit generating motive, many of the ethical challenges that potentially exist in partnering with the for-profit are not of relevance in this case. However, it could also plausibly be argued that NGOs, who though objective and altruistic, may, in fact, have quite complex motives. In promoting public-private partnerships therefore, several issues need to be clearly flagged in an attempt to address them in tandem with efforts that aim to foster such relationships. Within that context, a set of ethical and process related challenges are summarized hereunder: Ethical challenges, which are largely generic across the range of public-private partnerships relate to the following dimensions 1. Global norms and principals: many of the large partnerships involving a variety of players are of a transnational nature. However, against this backdrop, there are no global norms and principals, to set a framework within which global public health goals can be pursued in a partnership arrangement. 2. Impartiality in health: if public-private partnerships are not carefully designed, there is a danger that they may reorient the mission of the public sector, interfere with organizational priorities, and weaken their capacity to uphold norms and regulations. Such a shift is likely to displace the focus from the marginalized and may therefore be in conflict with the fundamental concept of equity in health. 3. Social safety nets: it has been increasingly argued that engaging in a partnership mode provides the public sector an opportunity to renounce their responsibilities; this in a sense may lead to withdrawal of social safety nets. Failure to commit to maintain the role of the state in such partnerships may result in a laissez-faire attitude, prejudicial to the interest of the most vulnerable groups. 4. Conflict of interest: many partnerships are initiated on the premise that they fulfill a social obligation, and can involve good intentions on part of individuals and organizations. However the basic motive that drives the 'for-profit' sector demands that these involve a financial pay off in the long term. In such cases, the difference between corporate sponsorships and scientific philanthropic donations with long term visible public health goals needs to be clearly separated. This issue has been further complicated in recent years as many global health initiatives funded by endowments generated by foundations have partnerships with the private sector as a key feature [29]. Such donor-recipient relationships bring in their wake many concerns. These include concerns relating to such arrangements providing the 'for profit' private sector an opportunity to improve their organizational image by engaging in cause-related marketing and concerns relating to these engagements facilitating access of the commercial sector to policy makers. On the other hand, many NGOs even in the developing countries are little more than lobby groups with a particular interest, which may or may not be aligned to public good. 5. Redirecting national health polices: there are also concerns that such partnerships redirect national and international health polices and priorities and have the potential to defeat crucial local and national efforts. 6. Fragmentation of the health system: partnerships generally tend to aim for short term high profile goals and tend to pick the lowest lying fruits. Partnerships do have the mandate and cannot be held accountable to synchronize their activities with emerging processes within countries aimed at developing their health systems. Therefore if they are instituted in countries with weak health systems they have the potential to fragment the healthcare system by instituting independent vertical programs. The changing global agenda around 'vaccines' helps to highlight many of these issues. Previously polices around vaccination were grounded in the general principal that promoted equitable access to few vaccines around the world. However new initiatives and their vertical systems have less of a focus on sustainability, may not contribute to strengthening of the health system and have the potential of redirecting national health policies, which focus on equitable care [30]. 7. Contribution to common goals and objectives: it is common for partners to have different objectives while pursuing a relationship though it may be implicit that partnerships are contributing to common goals. 8. Lack of outcome orientation: many a times, partnerships exist in form and do not contribute to improvements in quality and efficiency. Operational and process-related challenges in public private partnerships relate to the following dimensions 1. Legislative frameworks, polices and operational strategies: many developed countries have legislation to interface with the private sector [31]. However in the developing world, there is a general failure, to have overarching legislation relating to public-private partnerships. As a result, such arrangements develop on an ad hoc and opportunistic basis and may have questionable credibility; as a results of this failure, polices and specific operational strategies fail to develop. 2. Participatory approach to decision making: the expression 'partnership' gives the impression of equality. However many a times, a participatory approach to the decision making process is usually not optimally accomplished. This has implications of varying degrees. Almost all the large 91 transnational partnerships referred to earlier are focused on the developing world. However, among these, 85 have their secretariats in Europe and North America; the United States and Switzerland being the commonest host countries. Cleary this lack of proximity to the intended beneficiaries has a bearing on the manner in which the beneficiaries have a role in the decision making process [32]. The decision-making process in a partnership may also be biased because of the stronger partners' influence. At a county level and in the case of governments interfacing with NGOs, the stronger partner, which his usually the Government generally tends to make the rules. On the other hand, in the case of relationships with the 'for-profit' private sector, there is the danger of the financially stronger partner influencing the public sectors decision making process on policies, regulatory and legislative matters, which have implications for their profit-making motive. 3. Governance structures: workable partnerships require a well-defined governance structure to be established to allow for distribution of responsibilities to all the players. Public-private partnerships may run into problems because of ill-defined governance mechanisms. Recent evaluation of the RBM project while acknowledging the successes of the partnership in drawing global attention to the scale of the problem posed by Malaria has outlined serious governance-related issues [33]. More recently, independent evaluation of the Global Stop TB partnership has also resulted in the issuance of detailed recommendations for improved governance [34]. 4. Power Relationships: skewed power relationships are a major impediment to the development of successful relationships. Governments in developing countries usually tend to assume core responsibility of the joint initiative and take charge of the weaker partner. In case of NGOs with outreach-related strengths, this usually takes the form of a 'contractual relationship' without much regard to the participatory processes, which should be key to a public-private partnership arrangement. In case of relationships with NGOs with technical strength, there are issues relating to power relationships of a more serious nature with regard to who assumes the leadership role. 9. Criteria for selection: the criteria for selection are an important issue both from an ethical and process-related perspective as it raises the questions of competence and appropriateness. In many instances the public sector is vague about important issues related to screening potential corporate partners and those in the non-profit sector. 10. Sustainability: the question of long-term sustainability is often ignored in public-private partnerships. An analysis of the operation of GAVI has concluded that it overemphasizes high technology vaccines, lacks sustainability, relies too heavily on the private sector and consequently, runs the risk of compounding health inequities in the poorest countries [35]. 11. Accountability: many partnerships do not ensure that all players are held accountable for the delivery of efficient, effective and equitable services in a partnership arrangement. Often in public-private relationships it is unclear as to whom are these partnerships accountable to, according to what criteria, and who sets priorities? To hold partners accountable for their actions, it is imperative to have clear governance mechanisms and clarify partner's rights and obligations. Clarity in such relationships is needed in order to avoid ambiguities that lead to break up of partnerships. A case in point is the recent breakup of GAEL with the exit of the International Association of Anti-leprosy [36]. The Call to Action In the world we live today, global agendas are being increasingly shaped by the private sector. The 'for-profit' private sectors' immense resources make it an irresistible partner for public health initiatives. These arrangements can also be mutually synergistic. Governments and international agencies can tap into additional resources to full fill their mandate whereas the commercial sector can fulfill its social responsibility, for which it is being increasingly challenged. Additionally, the recent SARS epidemic and bio-terrorist threats should help to make the private sector understand the value of investment in health for reasons beyond fulfilling their social obligations. Active involvement of the 'non-profit' sector and donor coordination in country goals is also being increasingly encouraged within comprehensive development frameworks; this approach is synchronous and in harmony with the Poverty Reduction Strategy Paper Framework [37]. The development and health actors have highlighted the need to harness the potential that exists in collaborating with the private sector to advance public health goals. This is also becoming increasingly essential as both the public and the private sector recognize their individual inabilities to address emerging public health issues that continue to be tabled on the international and within country policy agendas. Public-private partnerships therefore seem both, unavoidable and imperative. However in building such collaborations, certain measures must be taken at a global level to assist global partnerships and set a framework within which efforts at a country level can emanate. As a first step, there is a need to develop a set of global norms and ethical principles; a broad-based agreement over these must be achieved. The transnational nature and global outlook of emerging partnerships necessitate that these stem from a broad-based international dialogue. It is critical that the driving principles for such initiatives be rooted in 'benefit to the society' rather than 'mutual benefit to the partners' and should center on the concept of equity in health. Norms must stipulate that partnerships contribute to strengthening of social safety nets in disadvantaged settings and should be set within the context of 'social responsibility' as the idea is not meant for private funds to be put to public use nor to privatize public responsibilities. Global principles must specify that partnerships should be in harmony with national health priorities; they should complement and not duplicate state initiatives and should be optimally integrated with national health systems without any conflict of interest. Norms must make it mandatory for all partners in a 'partnership' arrangement to contribute to common goals as a true partnership is one in which the partners, though having different motivation and values have a shared objective. Global norms must outline that partners be committed to making contributions, sharing risks and the decision making process. Principles should emphasize an outcome orientation. Development of a public-private partnership in itself should not be seen as an outcome, but a process and an output; it is important for partnerships not to just exist in form but to contribute to improvements in health outcomes. It must be made binding for international agencies to develop transparent policy and procedural frameworks. Many international agencies have established guidelines on interacting with the private sector [38-45]. However there is a need for comprehensive polices and operational strategies, which are crucial to ensuring transparency and protecting public interest [46]. Inviting third party reviews and ensuring an open process for deliberations will help to ensure transparency and reflect that these processes are indeed being structured in public interest. Global efforts should demand, encourage and assist the development of policy and legislative frameworks shaping public-private partnerships within countries [47]. However in the setting of developing countries, there is a need for international actors to guide these and for them to emanate within the framework of global norms and standards. Assisting with capacity development through donor coordination may be a necessary prerequisite to this approach. Legislative and policy frameworks within countries will help to legitimize public-private relationships, lend credence to this approach, help to foster an enabling environment and provide a mandate for the development of ethical guidelines to further direct these initiatives. Within stipulated legislative and policy frameworks, support must be provided to developing countries to develop specific guidelines to steer such relationships. Guidelines can assist with the development of selection criteria and help specify roles of the public and the private sectors. They can also assist with the development of models that outline combined governance structures, clearly aimed at improved systems of governance. Guidelines must also articulate a clear policy on a participatory approach to the decision making process. In addition, they should assist with assigning responsibilities to various levels of Government and then hold people and institutions both within Governments and those in the private sector that partner with them accountable for their performance. Though an evidence-based approach and ethical considerations must never be compromised in such endeavors and every effort should be made to ensure that goals are mutually compatible, guidelines also need to be flexible in order to accommodate each partner's organizational requirements and integrity. Moreover they need to be pragmatic. The public sector needs to recognize the basic legitimacy of the private sector and the profit motive that drives it. It is also essential for the public sector to respect the organizational autonomy and priorities of the non-profit sector. In this context, partnerships and contractual relationships need to be carefully differentiated. Partnerships must also be the subject of noteworthy empirical research, which would enable a detailed assessment of the specific issues inherent to the various types of public-private partnership arrangements from an ethical and methodological perspective. The impetus for driving global and national efforts in creating a transparent and conducive environment for public-private partnerships needs to come from the public sector. This raises the issue of capacity within countries; the gap needs to be bridged by assistance from UN agencies, which have the mandate of harnessing and coordinating support among a variety of players for global actions. However, the results of such actions will only be as good as Governments make them; weak and poorly informed Governments cannot remedy their own deficiencies by seeking to yolk the private sector to their own uncertain cart. In conceptualizing a framework that assists with setting global norms and guidelines and within-country legislative actions, it needs to be recognized that the dynamics of public-private partnership arrangements are generic across social sector. It may therefore be useful to allow this commonality to prevail in initiating global and country-specific actions. Acknowledgements The author would like to thank Mr. Michael Francino and Mr. Mohammad Ghalib Nishtar, for their valuable suggestions and comments and Dr. Shahzad Ali Khan for help with literature search. ==== Refs Health Action International, 2002. Statement to 109th WHO Executive Board meeting; 2002 accessed Jun 22, 04 Reich MR Public-private partnerships for public health Nat Med 2000 6 617 620 10835666 10.1038/76176 Initiative on Public-Private Partnerships for Health accessed June 22, 03 Mitchell-Weaver C Manning B Public-Private partnerships in Third World Development The 20th Norma Wilkinson Memorial Lecture Geographical Paper; Reading University, Reading 1990 Leading companies to mobilize against global AIDS epidemic Press Release. Geneva, UNAIDS 23 October 1997 Widdus R Public-Private Partnerships for Health and Health Care in the Developing World Initiative on Public-private Partnerships for Health accessed Jun 23, 04 Global Alliance for Vaccines and Immunization accessed Jun 23, 04 Roll Back Malaria; World Health Organization accessed Jun 23, 04 Stop TB accessed Jun 23, 04 Injection Safety: Safe Injection Global Network; World Health Organization accessed Jun 23, 04 Global Polio Eradication Program accessed Jun 23, 04 Tropical Disease Research; World Health Organization accessed Jun 23, 04 Special Programme for Research Development and Research Training in Human Reproduction; World Health Organization accessed Jun 23, 04 Glaxo SmithKline: Global Community Partnerships; Action TB Initiative accessed Jul 13, 04 International Aids Vaccine Initiative accessed Jun 23, 04 Medicines for Malaria Venture; World Health Organization accessed Jun 23, 04 Global Alliance for TB Drug Development: Tropical Diseases Research; World Health Organization accessed Jun 23, 04 Concept Foundation accessed Jun 23, 04 Malaria Vaccine Initiative accessed Jun 23, 04 Mectizan Donation Programme accessed Jun 23, 04 HIV Vaccine Initiative; World Health Organization accessed Jun 23, 04 Slack K Savedoff WD Public purchaser-private provider contracting for health services; examples from Latin America and the Caribbean. Inter-American Development Bank Technical Paper series 2001 National Action Plan for Non-Communicable Diseases Prevention, Control and Health Promotion in Pakistan accessed May 12, 04 Bayes de Luna A Tse TF de Figueiredo MB Maranhao M Voute J Nishtar S World Heart Federation. World Heart Day: a World Heart Federation enterprise promoting the prevention of heart disease and stroke across the world Circulation 2003 108 1038 40 12952824 10.1161/01.CIR.0000089504.27796.2C World Heart Day: World Heart Federation accessed Jun 23, 04 WHO-Aventis Collaborative Working Group One cornerstone of a global alliance for controlling and eliminating African sleeping sickness TDR News no 70 2003 assessed Jan 9, 03 Blanks J Richards F Beltran F Collins R Alvarez E Zea Flores G The Onchocerciasis Elimination Program for the Americas: a history of partnership Rev Panam Salud Publica 1998 3 367 74 9734217 Brugha R Starling M Walt G GAVI, the first steps: lessons for the Global Fund Lancet 2002 359 435 8 11844535 10.1016/S0140-6736(02)07607-9 Public Private Partnerships for health – Report of the Director General. Executive Board, 105th session. provisional agenda item 2. EB1-5/8; World Health Organization; Geneva, Switzerland 1999 Yamey G Faltering Steps towards partnerships BMJ 2002 325 1236 40 12446545 10.1136/bmj.325.7374.1236 Newman CL Comparative study of NGO/Government partnerships accessed Jun 23, 04 Buse K Governing public-private infectious disease partnerships Brown Journal of World Affairs 2004 2 225 42 Feachem R Medlin C Daniels D Dunlop D Mshinda S Petko J Achieving impact: Roll Back Malaria in the next phase Final Report of the external evaluation of Roll Back Malaria August 29, 2002. accessed Jun 23, 04 Independent external evaluation of the Global Stop TB Partnership. Institute for Health Sector Development accessed July, 04 Yamey G Global vaccine initiative creates inequity, analysis concludes BMJ 2001 322 754 10.1136/bmj.322.7289.754 Skolnik B Agueh F Justice J Lechat M Independent Evaluation of the Global Alliance for the Elimination of Leprosy 2003 The World Bank: Poverty Reduction Strategy Papers accessed Jul 13, 04 Maher D Boldrini F Pathania V Alli B Gabriel P Kisting S Norval P Guidelines for Workplace TB Control accessed Jul 13, 04 Barrett D HIV/AIDS and Business in Africa and Asia: A Guide to Partnerships accessed Jul 13, 04 Drucker PF Jossey B Meeting the Collaboration Challenge Workbook accessed Jul 13, 04 Bennett S Quick JD Velasquez G Public-Private Roles In The Pharmaceutical Sector: Implications For Equitable Access And Rational Drug Use accessed Jul 13, 04 Perrot J Carrin G Sergent F The Contractual Approach: New Partnerships For Health In Developing Countries accessed Jul 13, 04 The World Health Organization Vaccine Vial Monitor: Training Guidelines accessed Jul 13, 04 The World Health Organization Vaccine Vial Monitors For All: Technical Session accessed Jul 13, 04 Smith E Brugha R Zwi A Working with Private Sector Providers for Better Health Care accessed Jul 13, 04 Buse K Waxman A Public-private health partnerships: a strategy for WHO Bull World Health Organ 2001 79 748 54 11545332 Widdus R Public private partnerships for health require thoughtful evaluation – Bull World Health Organ 2003 81 235 12764487 Accelerated Access Initiative: UNAIDS accessed Jul 13, 04 Leprosy Elimination Group World Health Organization accessed Jul 13, 04 The Global Alliance to Eliminate Lymphatic Filiariasis accessed Jul 13, 04 Polio Eradication accessed Jul 13, 04 Global Alliance for Improved Nutrition accessed Jul 13, 04 Micronutrient Initiative accessed Jul 13, 04 Alliance for Health Policy and Systems Research accessed Jul 13, 04 The Multilateral Initiative on Malaria accessed Jul 13, 04 African Comprehensive HIV/AIDS Partnerships accessed Jul 13, 04 Alliance for Micro biocide Development accessed Jul 13, 04 Glaxo Smithkline: Global Community Partnerships; African Malaria Partnership accessed Jul 13, 04 Global Business Coalition on HIV/AIDS accessed Jul 13, 04 Corporate Council on Africa accessed Jul 13, 04 The International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use accessed Jul 13, 04 Pharmaceutical Security Institute accessed Jul 13, 04 Anti-Counterfeit Drug Initiative accessed Jul 13, 04

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==== Front Health Res Policy SystHealth Research Policy and Systems1478-4505BioMed Central London 1478-4505-2-61529650910.1186/1478-4505-2-6ResearchHealth policy and systems research agendas in developing countries Gonzalez-Block Miguel A [email protected] Manager Alliance for Health Policy and Systems Research World Health Organization CH 1211 Geneva 27, Switzerland2004 5 8 2004 2 6 6 6 2 2004 5 8 2004 Copyright © 2004 Gonzalez-Block; licensee BioMed Central Ltd.2004Gonzalez-Block; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Health policy and systems research (HPSR) is an international public good with potential to orient investments and performance at national level. Identifying research trends and priorities at international level is therefore important. This paper offers a conceptual framework and defines the HPSR portfolio as a set of research projects under implementation. The research portfolio is influenced by factors external to the research system as well as internal to it. These last include the capacity of research institutions, the momentum of research programs, funding opportunities and the influence of stakeholder priorities and public opinion. These dimensions can vary in their degree of coordination, leading to a complementary or a fragmented research portfolio. Objective The main objective is to identify the themes currently being pursued in the research portfolio and agendas within developing countries and to quantify their frequency in an effort to identify current research topics and their underlying influences. Methods HPSR topics being pursued by developing country producer institutions and their perceived priorities were identified through a survey between 2000 and 2002. The response to a call for letters of intent issued by the Alliance in 2000 for a broad range of topics was also analyzed. The institutions that were the universe of this study consisted of the 176 institutional partners of the Alliance for Health Policy and Systems Research producing research in low and middle income countries outside Europe. HPSR topics as well as the beneficiaries or issues and the health problems addressed were content analyzed. Topics were classified into 19 categories and their frequency analyzed across groups of countries with similar per capita income. Agendas were identified by analyzing the source of funding and of project initiation for projects under implementation. Results The highest ranking topic at the aggregate level is "Sector analysis", followed by "Disease burden" and "Management and organization". Categories at the bottom of this ranking are "Equity", "Policy process", "Economic policy and health" and "Information systems". "Disease burden" is more often funded than other topics for which there is more demand or perceived priority. Analysis suggests few although important differences across priorities, demand for funding and actual project funding. The donors' agenda coincides most with the ranking of research topics overall. Ranking across country income groups shows important differences. Topics that gain prominence in low income countries are "Disease burden" and "Accessibility". In lower middle income countries "Insurance" gains prominence. In upper middle income countries "Decentralization/local health systems", "Equity" and "Policy process" are more prominent. "Program evaluation" is the most consistently ranked topic across income regions, showing a neutral influence by donors, governments or researchers. Conclusions The framework proposed offers a basis to identify and contrast research needs, projects and products at the international level and to identify the actor agendas and their influence. Research gaps are suggested when comparing topic ranking against the challenges to health system strengthening and scaling up of disease control programs. Differences across per capita income groups suggests the need for differentiated priority setting mechanisms guiding international support. Data suggests that stakeholders have different agendas, and that donors predominate in determining the research portfolio. High-level consensus building at the national and international levels is necessary to ensure that the diverse agendas play a complementary role in support of health system objectives. The Ministerial Summit for Health Research to be held in Mexico in November 2004 should be an opportunity to analyze further data and to commit funding for priorities identified through sharing and discussion of agendas. ==== Body Background Countries and international agencies have made a qualitative leap in the funding of the global disease challenges. The Global Fund for AIDS, TB and Malaria has received pledges totalling over US$ 2 billion. Bilateral donors are also making important funding contributions. In this context, strengthening of health systems has become a critical issue. Research can play a major role to identify the best policies to channel massive efforts, to ensure that vertical approaches do not fragment fragile health systems and to monitor and evaluate progress. How relevant is the research effort being undertaken in developing countries, and how is the agenda being driven? WHO is organizing the Ministerial Summit on Health Research, to be held in Mexico City, 23 to 26 November, 2004. The main theme will be the role of health research in meeting the Millennium Development Goals. Health policy and systems research (HPSR) will have a prominent role in the context of the scaling up of efforts against major diseases and child and maternal mortality. Looking towards the Summit, WHO established a task force to identify HPSR priorities as an effort to advocate for major funding in this area. The Alliance for Health Policy and Systems Research, an initiative of the Global Forum for Health Research in collaboration with WHO, has been promoting since its inception in 2000 the identification of research priorities among producer institutions in developing countries. This paper proposes a conceptual framework and a methodology to think about the HPSR research portfolio, the agendas that influence it and the priority setting process. The paper makes the case for the formulation of HPSR priorities at national, international and global levels. A previous article [1] looked at research capacity among Alliance partner institutions in the South and identified the need to increase funding to establish long term research projects as a basis for sustainable capacity building. Using data from the same survey, indicators are proposed to assess the priority setting process on the basis of various types of data. The scope and value of the conceptual framework and its indicators are illustrated by presenting the HPSR topic ranking on the basis of Alliance partner contributions and the influence upon it of country income and actor agendas. A forthcoming paper will present findings from a new survey now being completed and covering the universe of research producers in the South. Conceptual framework Priority setting efforts are often bogged down because of inadequate methods of categorization of the research that is undertaken and the influences shaping it. These influences, in turn, are often not adequately understood, including the nature and role of priorities. The conceptual framework proposed here strives to offer some simple definitions of the research portfolio and its various influences, as well as indicators to measure and relate these concepts. The nature of overall health research has been defined in terms of basic, applied and strategic research. These categories are useful to guide investment decisions which might maximize benefit [2,3]. However, within the field of health policy and systems research, little effort has been given to the classification of what is studied. Before priorities can be identified, it is important to be able to agree on what is studied through an analysis of the dimensions that characterize the object of research. Furthermore, it is important to establish the distinction between the object of the research and the factors shaping this choice at various levels. The HPSR portfolio To identify the object of research the concept of the research portfolio can be useful. The HPSR portfolio can be defined as the current set of research projects on health system structures, functions, processes and results at national, international and global levels. Projects, as distinct from plans or priorities, would include the commitment of resources towards a specific, time-bound aim and a set of objectives. It has been proposed that the research portfolio should be analyzed along three dimensions of strategic importance: where to make investments, that is, the identification of the object of research or thematic areas in which investments are made; the type of investment research and development (R & D) instrument used and the resources spent through each area and type of instrument [3]. While our contribution aims to develop the first dimension, it is useful to expand on the other two to understand their interrelations. The Ad Hoc Committee on Health Research proposed three types of R & D instruments: discovery oriented research to develop new health products and interventions; innovation research to adapt efficacious but unaffordable interventions to make them cost-effective, and implementation R & D to achieve greater efficiency in the use of existing interventions [2]. Harrison has argued for the need to consider a fourth instrument of equity R & D to ensure that the research portfolio responds to the poor and the underlying health problems in developing countries. With regard to funding, there is a need not only to consider investments, but also funding sources and mechanisms. Four broad types of sources can be recognized: bilateral and multilateral donors, government commissioning, private commissioning, and funding through resources available to research institutions as part of their budgets. Each will have different implications for the kind of knowledge produced and for its possible influence on the health system [1]. This subject has been explored for Alliance partners in developing countries, identifying the amounts and sources of funding for their research portfolio [4]. The object of research Previous analyses have revealed a complex heterogeneity along which researchers classify HPSR in developing countries, which is not surprising in an interdisciplinary field [5]. However, five overarching dimensions can be recognized: • concepts reflecting the health system, such as policy and financial structures, regulatory functions, processes such as technology evaluation and quality monitoring, and results such as satisfaction and health gain • the levels of the health system, such as the households and the community, first level facilities and hospitals • the issues or problems pertaining to the health system such as priorities, equity and the public private mix • the populations addressed by the system, such as children, mothers and the elderly, or rural and urban populations • the health needs addressed, whether in terms of risks or disease. While these dimensions can be useful to characterize the research portfolio, it is clear that there will be overlaps; for example, equity is both an issue and an attribute of the health system, particularly if it has been integrated in monitoring and regulation. In order to make use of these dimensions it is proposed to consider as the project topic the first dimension of concepts pertaining to the health system structures, functions, processes and results. The topic could then be classified following normative or theoretical frameworks or by using the categories researchers apply in their own research. The other four dimensions can be used to qualify the research topic as to provide a more detailed description. These four dimensions could be selectively used or aggregated to facilitate description according to the needs at hand. Analysis of portfolio characteristics Each of the five project dimensions can be analyzed in terms of the range of items considered. The ranking and emphasis of each item can also be revealed by analyzing its frequency. A research portfolio at any level can be very focused and comprise a narrow set of topics. Or it can be wide-ranging across many health system functions, structures, processes and results. It has been argued that research portfolios should be more focused on improving immediate health problems through operations research in low income countries, where funding and human resources are very limited [3]. However, the challenges of scaling up disease control programs call for research at the health systems level also. The level of the research portfolio The level at which the portfolio is analyzed is important, as it will have different characteristics and uses. At national level, the HPSR portfolio would be the set of projects addressing health and health system problems within the confines of national borders and governmental authority as well as sector-wide and inter-sectoral issues. Examples would be the impact on equity of decentralization policies, or the roles played by conflicting policy actors in scaling up of services. The international research portfolio would be topic areas which are common across a number of countries or regions. Identification of the international portfolio could serve, among other purposes, to fund research at regional or international levels, to strengthen the critical mass of research available to inform country policy making and lesson learning across countries, and to extend the range of methodological approaches through comparative research [6]. The HPSR portfolio at global level refers to research themes and projects that are, by their very nature, supra-national. This would involve, for example, international financing of immunization efforts, intellectual property rights in health research, and development of international disease control measures. The HPSR agendas The HPSR portfolio at any level is influenced by factors within and outside the research system [7]. Within the research system the following factors can be identified: research capacity; research trends and preferences expressed by researchers and research institutions; research funding and market opportunities; and research preferences voiced by policy makers, service managers and public opinion. Outside the health system the broad factors shaping the portfolio are the health conditions and health system problems as well as the cultural, economic and political context. As a whole, these factors shape actor-specific research agendas that express ethical, professional and political values and that influence the allocation of scarce resources towards alternative project portfolios. It is clear that if actor agendas have few areas in common there will be no consensus and therefore no overall priorities. Significant overlap of interest, on the other hand, can lead to the identification and formulation of shared research priorities. Research priorities are therefore defined as the explicit areas of agreement on, and ranking of, the object of research across diverse actor agendas. Priorities can then become policy instruments to coordinate diverse agendas towards a common end without forcing a single research agenda. The characteristics of agendas and of priorities can be identified through the same kinds of dimensions and indicators as the research portfolio. That is, preferences can be classified in terms of topics, issues or beneficiaries, and health problems. The characteristics of the HPSR agendas and priorities can also be studied in terms of the range of topics, issues, levels, populations and health problems. They can also be ranked and their emphasis revealed by frequency analysis. In this way, the agendas can be compared across themselves, priorities can be identified as common topics and issues and with similar ranking and emphasis, and the influence of agendas and priorities on the actual research portfolio can be assessed. Priority setting through agenda co-ordination It has been argued that co-ordination of the various influences shaping the HPSR portfolio can increase the impact of research on equity and can contribute to its strategic role for development [8]. Co-ordination would involve developing a consensus of researcher, policy maker and investor agendas. Such a consensus should ideally result in a highly coherent set of topics across the various actors' research agendas and, eventually, a high degree of correspondence between agendas and the research portfolio. HPSR portfolio change through co-ordination would involve a gradual process of adjustment to new priorities, project completions, maturation of research capacity and funding opportunities. Coordination requires interfaces and mechanisms such as Research Forums and Essential National Health Research Mechanisms [9] to develop a consensus while allowing, and even encouraging, critical differences. In sum, the HPSR portfolio can encompass a differing range of topics with diverse rankings and emphases; can be more or less coherent with respect to researcher, funding and policy maker agendas; and can be more or less co-ordinated along a set of shared priorities. The interplay of these dimensions can give rise to a number of scenarios, of which three are here illustrated. a) Co-ordination could lead to focusing the HPSR portfolio on a few, highly cost-effective topics in a situation of few resources and well identified, high priority needs. There would be, eventually, a high degree of coherence across the portfolio and agendas held by various actors. The risks here would be lack of diversity to foster innovation and healthy criticism. b) In a situation of plentiful resources the research portfolio and its driving agendas could be wide-ranging and have little overlap. Specific portfolio segments would correspond with particular agendas, thus satisfying multiple interests. However, co-ordination through overarching mechanisms could ensure integration of knowledge around high level priorities. In this manner a unified, although highly diverse, HPSR field of enquiry would be obtained. c) In a situation of lack of coordinating mechanisms, with low resources, the portfolio could focus on a reduced set of topics, each satisfying a particular agenda and thus fragmenting resources and hindering support to health system development. If resources are more plentiful, lack of coordination could lead to a rich but highly dispersed and inefficient research portfolio with little impact on development. Irrespective of the availability of resources, co-ordinating mechanisms are likely to be important to ensure an efficient use of research resources. Methodology and Indicators Two sources of information were used to illustrate the conceptual framework proposed. The first was a survey of Alliance-HPSR partner institutions in developing countries detailing research priorities and project information. Researchers reported here on the priorities they had received from policy makers in the course of diverse consultations in the past year. The second source was a database of letters of intent (LOI) submitted to the Alliance for funding, where projects were justified on the basis of priorities negotiated by researchers and policy makers or service managers. Content analysis This proceeded in several steps. A preliminary list with 24 research topics was identified through an inductive analysis using the research statements expressed in the LOI, which were the most detailed. This list was then used to classify research topics in the projects and priorities expressed in the Alliance partners' profiles. Beneficiaries/issues and diseases/health problems addressed by the LOI, projects and priorities were also categorized and classified at this stage. While projects may have contained more than one topic or beneficiary/issue, the most prominent one was selected. In a few cases where several topics were considered this was indicative of a sector-wide analysis and classified accordingly. The beneficiaries/issues of the research were classified to include any of four alternative dimensions: a) the demographic group: elderly or children/adolescents, b) level of care: community, primary or hospital, c) the geographical focus: urban or rural, and d): gender, equity/poverty, indigenous populations, and public-private mix. Whenever more than one dimension or aspect was applicable (which occurred only in a small number of statements), a decision was made to include the most prominent. The concept "equity" was classified both as a topic and as an issue. It was assigned as a topic whenever equity was the main objective of the research and it was addressed through a number of health system attributes such as financing, access, and service delivery. Equity/poverty was considered as an issue when the poor were identified as the main subjects of research or when the equity implications of research directed mainly to another topic were highlighted as a major concern. The distribution of statements was analyzed according to country income group: low income, lower middle income and upper middle income. These income groups correlate highly with geographical regions, with LI being mostly in Africa and Asia, LMI mostly in Asia and with a particular weight by China, and UMI in Latin America and the Caribbean (table 1). Table 1 Glossary of Health Policy and Systems Terms Used for Content Analysis TOPIC TERMS FOUND IN RESPONSES Accessibility Health seeking behaviour, determinants of utilization, coverage, outreach, referral, barriers to care, willingness and capacity to pay, cost-sharing, price regulation, prices, equity in access, demand for health services. Community participation Community-based strategies, community participation in governance, empowerment, school health, family health strategies, social support networks. Costing & cost effectiveness Determination & evaluation of costs, cost-benefit of services, economic evaluation, cost-effectiveness of resource allocation, alternative uses for resources. Decentralisation/local health systems Decentralization policy and process, impact of decentralization on services and health outcomes, district health system development, healthy cities, municipal health services, local government, devolution, community participation in local health services. Disease burden Prevalence and incidence of diseases, mortality and morbidity, disease profiles, health status, health needs, burden of disease studies, risk factors, determinants of health and disease other than economic or social policy. Economic policy and health Free trade agreements and health, TRIPPS and health, economic crises and health, impact of poverty reduction and adjustment policies on health, debt reduction and health, social policy and health, social assistance and health issues, intersectoral co-ordination, labour policies and health. Equity Equity of health system, impact of health reforms on equity, equity and poverty, poverty targeting of services, poverty and health, exclusion. Financing Financial mobilization, financial allocation, financing policies, national & district health accounts, financial equity, community health financing, financing of specific programmes. Human resources Personnel management, deployment, migration, motivation, knowledge, attitudes and practices of health personnel, satisfaction, quality of life, human resource policy, human resource performance, traditional healers, training and education of human resources, medical education curriculum assessment, evaluation of medical and nursing teaching programmes. Information, education and communication (IEC) Information and communication for the general public, health education strategies and impacts, knowledge attitudes and practices (KAP). Information systems Information needs, informatics, surveillance mechanisms and systems, strengthening of information systems, health monitoring systems, establishment of public domain databases, development of indicators for service management and policy. Insurance Risks and benefits covered by insurance schemes, community based health insurance, options for health insurance, insurance reform, impact of insurance on health and service outcomes. Management & organization Health service provider performance, delivery of services, administration, service management strengthening, contracting and provider payment mechanisms, impact of privatization on services, performance agreements, impact of hospital autonomy on service delivery, stakeholders in service management, community participation in management. Pharmaceutical policy & management Rational drug use, procurement, logistics, herbal medicine, dispensing practices, pharmaceutical regulation, national drug policy, essential lists. Policy process Stakeholder analysis, role and relationships of actors in the formulation and implementation of policy, role of government agencies in policy formulation, role of community and NGOs in policy formulation, factors influencing policy process, perceptions of policy, decision-making processes, policy negotiation. Programme evaluation Evaluation and assessment of impact of policies or programmes on specific diseases or services. Quality Clinical practice guidelines, evidence-based medicine, quality assurance, patient satisfaction. Research to evidence Health systems research training, health systems research training, outcomes of research, research impact, policy utilization and impact of research, research methods, creation of national HPSR database, priority setting of health research, research ethics, essential national health research, dissemination of research. Sector Analysis Health sector reforms and implications, health systems development, private health service development, intersectoral collaboration and co-ordination, public/private mix health care, health care organization, regulation, policy formulation on specific diseases, on programmes or on aspects of the health system, sector-wide and system-wide performance. Two researchers classified all statements independently and disagreements were discussed and resolved. The 24 topic categories were reduced to 19 to avoid groups with less than 2% of the total number of statements while maintaining topic coherence. Table 1 presents the glossary of terms included under each topic. The frequency of responses by country for all types of statements is generally proportional to country population, with China, India, Brazil and Bangladesh at the top of the frequency. However, countries with a strong health systems research presence are over-represented, such as Colombia, Argentina, Philippines, Thailand, South Africa, Uganda, Ghana, Cuba, Costa Rica, Benin, Jamaica and Tanzania. Identification of agendas The range and emphases of the HPSR portfolio and agendas were mapped through topic content and frequency analysis (Figure 1). Project and agenda data were also aggregated from the two sources to obtain a general mapping of topics. This was used to assess coherence across actor agendas and with the portfolio and to increase the number of observations to enable analysis by income level. Figure 1 Concepts and indicators Two methods were used to assess the coherence between the HPSR portfolio and the agendas held by researchers, policy makers and international donors and partners. The first method used the survey data to infer the agendas by observing the topic frequency of: projects proposed and funded by researchers without external assistance; projects initiated and funded exclusively by government, and projects initiated and funded exclusively by international stakeholders or research partners. The second method compared the research portfolio against the agenda expressed by policy makers. The policy maker agenda was observed through the survey as reported by researchers and through the LOI as negotiated with researchers. A negligible influence by the donor, in this case the Alliance-HPSR, would be expected in the LOI given that the call requested priorities within the generic definition of HPSR presented above. Each of the three modes of identification of agendas could have method-specific biases. In the first method, preferences are derived from the portfolio itself, that is, from research projects in implementation. Furthermore, the method isolates the preferences expressed by each actor. As such, this method could be deemed to reveal most objectively preferences behind each actor funding or initiating a project. However, projects under implementation may hide topic preferences that are not translated into projects or topics that were generated and funded through joint actor participation. The observation of negotiated priorities expressed in LOI captures the mix or balance of researcher-side influences and policy maker needs. It will therefore reflect a consensus position across each actor. However, it will exclude the influence of funding opportunities, will not reveal actor-specific preferences and will be limited by the constraints placed on the LOI (see below). Priorities based on consultations between researchers and policy makers and expressed by researchers through a survey will reveal the understanding and conceptual framework of researchers and may underplay policy-maker needs. Furthermore, these priorities will be influenced by the research projects under execution and reported in the same instrument. Assessment of coordination between portfolio and agendas The analysis of relationships or influences across the portfolio and each of the actor agendas, as well as of similarities or differences between agendas, was undertaken by correlating topic frequencies across lists and by undertaking a qualitative analysis of changes in rank order and emphasis. Analysis of the range and rank of topics across groups of countries by income was undertaken by aggregating project and priority topic data into a reference list representing the combined set of influences on the agenda-setting process, including the portfolio itself. The aggregation of data into a reference list was mainly a strategy to increase the observations and make the analysis more reliable, although it may have validity if it describes the overview of the agenda-setting factors at play. That is, the actual portfolio can be conceived as a force shaping the agendas, together with other factors. Survey and LOI database The survey of HPSR producer institutions in developing countries was described in detail elsewhere [1] and includes information for 108 of the 176 Alliance-HPSR partners (61% response rate) who produced research in low and middle income countries outside Europe between 2000 and 2001. The database contains information on the current research portfolio (294 projects were declared) as well as research priorities (402 priorities were stated, with a maximum of 5 per survey). Information on project initiation and source of funding is available for 270 projects. A total of 39 developing countries out of a total of 133 were contacted. Respondents are close to one sixth of the close to 650 institutions known to the Alliance to be producing HPSR in developing countries. Biases in the partner database could have occurred as a result of preferences by certain type of institutions in joining the Alliance HPSR and in answering the questionnaire required from partners. Over-representation at both levels could have occurred of more competitive and productive institutions with larger project portfolios and funding, and more interest in international funding. On the other hand, larger institutions may have been discouraged from responding given the larger number of projects to be reported, although they would also have more capacity to respond. Furthermore, the response rate could have been lower among institutions where producing HPSR is not a main function. The LOI database has 403 submissions for research funding in response to a call by the Alliance-HPSR in 2000. Applicants requested funding for one year projects in high priority areas identified jointly by them and national policy makers and stakeholders. A limitation of this database is the exclusion of funding requests for projects over one year as well as topics that would be formulated solely by researchers. Analysis of the partners' database indicates that 24% of projects are of longer duration and that up to 34% of projects undertaken are initiated by the research institution without stakeholder collaboration. Expansion and standardization The frequencies of statements for each income region were expanded proportional to population to make comparisons across regions possible. The frequency of statements across the three types of statements (projects, LOI and priorities) was standardized to give each equal weight when aggregating them to analyze the combined representation of the research portfolio and the agenda-setting process as a whole. Responses show a distribution across income regions proportional to population in some cases and with significant biases in others (table 2). The low income region (LI) has 50% of the population and 47% of statements, while the upper middle income (UMI) region has only 12% of the population but twice the number of statements, with 22%. The lower middle income (LMI) region is also somewhat under-represented, with 38% of the population and 31% of statements. Table 2 Distribution of Statements According to Type, Content Category and Geographical Region by Income Group LI LMI UMI TOTAL % Types of statements Total % Total % Total % Priorities 198 49 143 36 61 15 402 100 Letters of intent 193 48 101 25 109 27 403 100 Projects 124 42 97 33 73 25 294 100 TOTAL 515 47 341 31 243 22 1099 100 Content categories Topics 482 46 330 31 237 23 1049 100 Beneficiaries or Issues 217 53 113 28 80 20 410 100 Health Problems 132 58 48 21 48 21 228 100 Total statements by geographical region Africa 247 77 15 5 57 18 319 100 Asia 260 53 214 44 13 3 487 100 Latin America and the Caribbean 8 3 112 38 173 59 293 100 TOTAL 515 47 341 31 243 22 1099 100 Total population in Geographical region 50 38 12 100 The frequency of statements on priorities is as would be expected for the population in each region. However, project statements and demand for funding are biased in favour of UMI, with 25% and 27% of the statements, respectively, against 12% of population share. Ranking This was done for each topic or category within the topic by rounding percentage differences to integers and grouping in the same rank all categories falling within the same percentage. Results This article does not attempt to provide an exhaustive analysis given the fact that data is limited to Alliance partner producer institutions in the South. The purpose here is to illustrate the potential of the proposed methodology and to present the most robust findings. HPSR topics are first presented and analyzed aggregating in a single list the topics in the research portfolio as well as in the policy maker and researcher agendas. This aggregate representation is then analyzed by groups of countries according to their per capita income. The influence exerted on the HPSR portfolio by various actors is then analyzed. Characteristics of HPSR producers in developing countries HPSR producer institutions are generally small with an average of 3 projects, 8 researchers and a project portfolio worth $155,226 [1]. Only 19% of researchers have a PhD qualification, although researchers in key disciplines are well represented and better qualified. Research capacity and funding are similar across income regions, although inequalities are apparent. Overview of topics A total of 19 research topics were identified when aggregating portfolio (project) and priority (voiced preferences) data into the reference list. Topics ranged in frequency from 2% to 11% and were ranked in 8 classes (Table 3). The highest ranking topic is "Sector analysis" with 11% followed by "Disease burden" with 9% and "Management and organization" with 8%. From here three topics rank lower equally at 7%, two rank at 6%, seven rank at 4% and then two each at 3% and 2%. Categories at the bottom of this ranking are "Equity", "Policy process", "Economic policy and health" and "Information systems". The emphasis of topics at the top end is then about five times as greater as those at the bottom end of the range. Table 3 Ranking of Topic at the Aggregate Level Rank Topic % 1 Sector Analysis 11 2 Disease burden 9 3 Management & organization 8 4 Accessibility 7 Programme evaluation 7 Research to evidence 7 5 Financing 6 Human resources 6 6 Community participation 4 Costing & cost effectiveness 4 Decentralisation/local health systems 4 Information, Education and Communication 4 Insurance 4 Pharmaceutical policy & management 4 Quality 4 7 Equity 3 Policy process 3 8 Economic policy and health 2 Information systems 2 Rank Beneficiaries/Issue % 1 Community 15 2 Equity/poverty 14 3 Hospital 12 4 1st level 11 Gender/women 11 Rural areas 11 5 Children/adolescents 10 6 Public private mix 6 7 Urban areas 5 8 Elderly 4 9 Indigenous peoples/traditional medicine 3 Rank Health Problem % 1 Reproductive health 30 2 HIV-AIDS 11 Nutrition 11 TB 11 3 Chronic 7 Environmental health 7 Malaria 7 Mental health 7 4 Other infectious 6 5 Other 3 The fact that "Equity" appears so low in the aggregated ranking could be partly attributable to the fact that this topic was defined to include only projects and priorities having equity as the central topic and measuring it through multi-dimensional approaches such as health conditions, access to services and financing. A subsidiary analysis was thus undertaken to include under "Equity" those projects or priorities addressing equity or poverty as a secondary, qualifying, role of research on other topics. This broadened topic "Equity" climbs to fourth rank, at the same level as "Accessibility", "Program evaluation" and "Research to policy". Public and private institutions show no significant changes in topic ranking (corr = 0.70). "Community participation" and "Accessibility" are the only topics with major differences, ranking higher among private institutions. Topic analysis by beneficiary/issue Out of the total topics classified in the reference list, only 38% (404) were sufficiently focused or detailed to be able to attribute a beneficiary or specific issue (Table 4). This was mainly the case with priority statements, which by their very nature were generic. The beneficiary or issue statements were spread across the 11 categories identified through content analysis. The category with least statements had only 2% of the total, and that with most 13%. Table 4 Beneficiaries/Issues According to Topic Beneficiaries or Issues Topic Elderly Children Community Primary Hospital Urban Rural Equity Gender Indigenous peoples/traditional medicine Public private mix TOTAL A B A B A B A B A B A B A B A B A B A B A B n = B Accessibility 2 3 10 7 7 7 10 8 7 12 19 20 17 14 19 17 2 11 7 7 42 10 Community participation 9 20 4 3 43 19 13 7 13 12 4 3 13 7 23 6 costing & cost effectiveness 13 3 13 2 38 6 13 4 13 3 13 2 8 2 Decentralisation/local health systems 13 5 27 7 7 2 13 8 27 10 7 2 7 2 15 4 Disease burden 15 50 21 19 3 2 3 2 6 4 12 15 9 8 6 4 24 17 3 11 34 8 Economic policy and health 14 10 43 8 14 2 29 4 7 2 Equity 6 10 6 2 78 28 11 22 18 4 Financing 39 17 4 4 9 5 39 18 4 2 4 2 23 6 Human resources 29 9 35 15 6 2 6 4 18 8 6 11 17 4 Inform. Educ. & Communication 21 8 7 2 7 2 14 8 14 5 36 11 14 3 Information systems 25 4 38 7 13 2 13 3 13 2 8 2 Insurance 44 13 13 8 19 8 13 4 6 11 6 2 16 4 Management & Organization 2 3 9 7 9 10 49 48 2 4 11 13 2 2 4 4 13 14 47 12 Pharmaceutical policy & Mgmnt. 17 5 33 8 8 3 25 6 8 2 8 11 12 3 Policy process 50 6 17 4 33 4 6 1 Programme evaluation 2 10 22 24 2 2 17 17 5 4 12 19 2 3 2 2 2 2 32 30 41 10 Quality 7 2 20 7 47 15 7 3 13 4 7 2 15 4 Research to evidence 33 2 33 2 33 2 3 1 Sector Analysis 11 11 11 10 8 8 11 8 8 7 3 11 47 40 36 9 None 26 14 5 2 11 5 5 3 47 20 5 11 0 19 5 n 10 37 54 41 48 26 40 50 46 9 43 404 100 % 2 100 9 100 13 100 10 100 12 100 6 100 10 100 12 100 11 100 2 100 11 100 A = % across the row ; B = % down the column The topics with least identification of beneficiary or issue were "Costing and cost effectiveness", "Policy process" and "Research to Evidence", with 79% to 96% in this situation. By contrast, "Community participation" and "Management & Organization" were the topics least frequently unidentified with beneficiary/issue, at between 30% and 41%. The most frequent beneficiary/issues were Community, Equity/poverty Hospital, Gender Primary care and Rural areas. The three beneficiary/issues least identified were Urban areas, elderly and Indigenous peoples/traditional medicine. The analysis of the correlation between beneficiary/issue and topic is tentative at this stage given the low frequency in many of the cells of the 19 by 11 matrix. Two beneficiaries/issues account for a large part of the focus of research topics: Community and Hospital as a focus on levels of care. At the community level the topics of community participation, financing, health insurance, decentralization, policy process, information systems and human resources are all prominent. At the hospital level the topics of costing and cost effectiveness, pharmaceutical policy, quality of care and management and organization are most prominent. By contrast, the topic of program evaluation is fairly widely spread across several issues or beneficiaries. The following topics show also a fairly discreet relationship to beneficiaries/issues: Research on accessibility is mostly focused on rural areas. Research on disease burden is prominent among the elderly and children. Economic policy and health focuses on children. Gender is also an important component of these three topics. Equity is focused on indigenous populations. The topic of information, education and communication is prominent among children. Sector analysis focuses mainly on the public private mix. Topic analysis by income level The differences in ranking of the topics in the reference list across income regions are shown in the first three columns of Table 5. Larger differences occur in 9 topics, mostly in lower middle income and upper middle income countries. This suggests that the reference list reflects more closely lower income country needs. Largest differences were observed in health insurance, decentralization/local health systems and, equity and policy process, topics that are more highly ranked in upper middle income countries. Table 5 Ranking of Topics in the Reference List and Differences by Income Region and by Project Initiation RANK TOPIC INCOME REGION PROJECT INITIATION Low Lower Middle Upper Middle Donors Govnt. Research Institution 1 Sector Analysis -- - 2 Disease burden -- 3 Management & organization + -- 4 Accessibility + + -- -- Programme evaluation -- Research to evidence - - + 5 Financing - ++ ++ -- Human resources + 6 Community participation - + + ++ Costing & cost effectiveness -- + + +++ Decentralisation/local health systems - +++ + Information, Education and Communication + Insurance -- +++ - + Pharmaceutical policy & management Quality + 7 Equity - +++ + +++ Policy process - +++ 8 Economic policy and health - + + + Information systems + + Cells with a rank difference of 1, 0 or -1 are blank. Changes of 4 or more = +++; 3 = ++; 2 = +; -2 = -; -3 = --; -4 or more = --- Comparing the research portfolio and the agendas The overall ranking of topics in the reference list was compared against the ranking of topics in projects initiated by each actor. The relationship between actor preferences and the reference list was assessed through an analysis of their rank congruence. As described in a previous paper in more detail [1], the research institution is the initiator in 34% of projects, while 31% are initiated by a donor agency, international research partner or by a private contractor. Governments initiate in 24% of cases. 12% of projects are reported as a mix of the above and are not considered for this analysis. The agendas across actors differ substantially, and none can be said to be close to the other. As a result, the reference list shows marked difference with respect to each actor's agenda. Government initiation shows preference for financing and cost-effectiveness as compared to the overall ranking. Government initiation tends to give lower regard to disease burden studies and for research on accessibility. International donor initiation matches best the reference list but gives somewhat less preference to sector-wide analyses. Research institution initiation is more marked for equity and community participation and less so for management and organization, accessibility and program evaluation. International donor initiation preferences are associated to the top ranking topics in the overall listing, suggesting a predominance of their agenda on the reference list. Conclusions The analysis of the research portfolio and priorities at the international level shows a widely diversified set of topics, ranging from sector wide issues to more focused program evaluation. The emphasis on sector wide issues reflects the challenges to health systems today and suggests that countries consider as important the macro-level analysis as the micro. Micro approaches with a focused attention to beneficiaries or specific issues are well identified, particularly under the topic of program evaluation. However, the evidence also suggests a gap between the research that is actually being undertaken and the challenges for strengthening and scaling up of disease control programs. Such a gap is evident in the low emphasis given to research on human resources, policy process, equity, economic policy and health and information systems. By contrast, the analysis suggests a high degree of attention at the community level, although much attention is also given at the hospital level. Primary care thus seems to be under-emphasized. Considering the disease focus, whenever this was made evident, the data do not suggest a bias towards problems that would not be evidently important at country level. The fact that the public or private character of research institutions is insignificant for the agenda suggests the capacity of diverse institutions to work within a common agenda. There are significant differences in the research portfolio across groups of countries based on per capita income, suggesting the need for priority setting mechanisms at both national and international levels that reflect such diversity. The greater congruence between donor preferences and the international research agenda highlight the importance of consensus building between national and international actors. While it is appropriate for governments and international donors to fund different aspects of the research portfolio, this requires high-level priority setting and consensus mechanisms to ensure they complement each other rather than lead to fragmentation. More research is required to establish the relationships between actors' agendas and the research portfolio at the international level. There is also a need to discuss the most desirable balance of influences and to increase the voice of developing country actors. Evidence-based HPSR priorities emerging through such a process would then be able to support scaling up of research efforts on a par with scaling up of health system strengthening and disease control. Regional and global meetings, such as the Ministerial Summit for Health Research to be held in Mexico in November 2004, are good opportunities to present and discuss the evidence and to commit funding accordingly. Attention must be given to encouraging consensus building on research priorities within regions comprising countries with similar needs. The interests of donors, governments, health workers, the community and researchers must all be taken into consideration so that research funding leads not only to fund relevant research but to build the necessary interfaces for utilization. Competing interests None declared. ==== Refs Gonzalez-Block MA Mills A Assessing capacity for health policy and systems research in low and middle income countries Health Research Policy and Systems 2003 1 1 12646072 10.1186/1478-4505-1-1 Ad hoc committee on health research concerning future intervention options, Investing in health research and development Geneva, WHO 1996 Harrison D Neufeld V, Johnson N Health research: an essential tool for achieving development through equity In Forging links for health researchPerspectives from the Council on Health Research for Development 2001 Ottawa, IDRC Stokes D Pasteur's quadrant Basic science and technological innovation Washington DC, The Brookings Institution 1997 Alliance for Health Policy and Systems Research, Development of an HPSR Thesaurus Newsletter of the Alliance-HPSR, Geneva, No 4 2001 Gonzalez-Block MA Comparative research and analysis methods for shared learning from health system reforms Health Policy 1997 42 187 209 10176300 10.1016/S0168-8510(97)00072-9 Pang T Sadana R Health research systems A framework for the future Bulletin of the World Health Organization 2003 81 159 12764510 Commission on Health Research for Development, Health research essential link to equity in development Oxford, Oxford University Press 1990 Global Forum for Health Research The 10/90 report on health research 2001–2002 Geneva, GFHR 2003

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==== Front Genet Vaccines TherGenetic Vaccines and Therapy1479-0556BioMed Central London 1479-0556-2-61529195710.1186/1479-0556-2-6ResearchThe significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events Zhang Bing [email protected] Pat [email protected] Howard [email protected] Kay AO [email protected] Geoff [email protected] Malcolm J [email protected] Ming Q [email protected] Department of Medicine, University of Queensland, Prince Charles Hospital, Brisbane, AUSTRALIA2 Queensland Institute of Medical Research, Brisbane, AUSTRALIA3 Department of Paediatrics and Child Health, Royal Children's Hospital, Brisbane, AUSTRALIA2004 4 8 2004 2 6 6 24 10 2003 4 8 2004 Copyright © 2004 Zhang et al; licensee BioMed Central Ltd.2004Zhang et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Although lentiviral vectors have been widely used for in vitro and in vivo gene therapy researches, there have been few studies systematically examining various conditions that may affect the determination of the number of viable vector particles in a vector preparation and the use of Multiplicity of Infection (MOI) as a parameter for the prediction of gene transfer events. Methods Lentiviral vectors encoding a marker gene were packaged and supernatants concentrated. The number of viable vector particles was determined by in vitro transduction and fluorescent microscopy and FACs analyses. Various factors that may affect the transduction process, such as vector inoculum volume, target cell number and type, vector decay, variable vector – target cell contact and adsorption periods were studied. MOI between 0–32 was assessed on commonly used cell lines as well as a new cell line. Results We demonstrated that the resulting values of lentiviral vector titre varied with changes of conditions in the transduction process, including inoculum volume of the vector, the type and number of target cells, vector stability and the length of period of the vector adsorption to target cells. Vector inoculum and the number of target cells determine the frequencies of gene transfer event, although not proportionally. Vector exposure time to target cells also influenced transduction results. Varying these parameters resulted in a greater than 50-fold differences in the vector titre from the same vector stock. Commonly used cell lines in vector titration were less sensitive to lentiviral vector-mediated gene transfer than a new cell line, FRL 19. Within 0–32 of MOI used transducing four different cell lines, the higher the MOI applied, the higher the efficiency of gene transfer obtained. Conclusion Several variables in the transduction process affected in in vitro vector titration and resulted in vastly different values from the same vector stock, thus complicating the use of MOI for predicting gene transfer events. Commonly used target cell lines underestimated vector titre. However, within a certain range of MOI, it is possible that, if strictly controlled conditions are observed in the vector titration process, including the use of a sensitive cell line, such as FRL 19 for vector titration, lentivector-mediated gene transfer events could be predicted. ==== Body Background Multiplicity of infection (MOI) is a parameter that has been commonly used to predict viral infectivity in a population of target cells. With wild type viruses, an "infectious unit" refers to the smallest amount of virus capable of producing an infection in a susceptible cell. The titre of the original suspension is defined as the number of infectious units per unit volume of the preparation [1]. In the field of gene therapy where viral vectors are used for gene transfer, MOI was adopted to represent the ratio of input infectious units (titrated on the target cell line) to the number of cells available for transduction [2]. Ideally, there should be a simple linear relationship between the viral vector titre, its dilution, the volume of viral vector suspension used, and the proportion of cells infected, taking into account the probabilistic nature of the infective process when the number of viral vector particle approximates the number of cells. However, at present, the number of viable vector particles (or vector titre) in a given vector stock is determined by a vector-mediated transduction process, which is of a non-linear nature and can be influenced by various factors. If MOI is based on vector titre that is "variable", then MOI is complicated by all of the factors that influence vector titration and determination. Unfortunately, the extent of which is poorly understood. Recently, lentivirus-based gene transfer vectors have been developed and have shown considerable promise for gene therapy research. It is evident that this vector system has several distinct advantages, and rapidly emerges as the vector of choice for in vitro and in vivo gene therapy studies [3,4]. Most current lentiviral vectors in use are based on Human Immunodeficiency Virus (HIV) type 1. A transient, three or four-component, HIV-1 based vector system consisting of one or two packaging constructs, a transfer vector and a Vesicular Stomatitis Virus G glycoprotein (VSV-G) envelope has recently been described and widely used [5-10]. Several reports have demonstrated that the HIV-based vectors effectively transduced dividing and non-dividing cells in vitro and in vivo including hematopoietic stem cells [7,11], terminally differentiated cells such as neurons [9], retinal photoreceptors [8], muscle, liver cells [5] and dendritic cells [12]. Other lentivectors, such as those based on the feline immunodeficiency virus (FIV) [13], equine infectious anaemia virus (EIAV) [14], caprine arthritis/encephalitis virus (CAEV) [15], Jembrana disease virus (JDV) [7], bovine immunodeficiency virus [16] and visna virus [17], are examples of recently developed non-primate lentiviral vectors that have also demonstrated efficient gene transfer to various types of cells. Just as with Moloney murine leukaemia virus (MoMLV) based retroviral vectors, many variables could theoretically affect the measurement of infectivity of lentiviral vector particles, such as target cell type, number, cycle, other modulators of cell membrane ingredients, the time needed for vector uptake and vector viability/susceptibility, half life during the transduction process or even Brownian motion in which the vector makes way to the target cell [18]. In addition, the issue of particle variation within the population of artificially assembled vector "infectious" units could be a contributory factor to between-preparation variation in the predictability of their infectious behaviour. Arai et al (1999) found that the ratio of cells transduced with the VSV-G-pseudotyped retroviral vectors based on MoMLV correlated with the result predicted from a Poisson distribution [9]. Generally with retroviral vectors using an ecotrophic or amphotrophic envelope, MOI at 1–3 is commonly used and results in around 30% of cells being transduced. The efficiency of gene transfer reaches a plateau after this. Higher MOI may reduce the number of transduced cells [3,19]. However, with lentiviral vector-mediated gene transfer, experiments employing MOI even greater than 1000 have been explored [12]. The rational behind the usage has obviously distinguished lentiviral vector from MoMLV based retroviral vectors. Unfortunately, there are, at present, no data available as to how lentiviral vectors behave in an in vitro transduction process, and how the variables affect vector titre determination and MOI usage. In this study, we characterised factors that influenced the in vitro vector titration process, including the number of target cells being transduced, total number of viral vector particles, inoculum volumes (well beyond the depth of relevance to diffusion), vector decay and the period of vector adsorption (and thus vector decay). We also examined the use of various MOIs on several commonly used cell lines and tried to establish the relationship of MOI with the efficiency of gene transfer. Methods Cell cultures Cell lines used in this study were a fetal rat liver carcinoma cell line, FRL 19; a human embryonic kidney cell line, 293 and its derivative, 293T; and a murine embryonic fibroblast cell line, NIH 3T3. FRL 19 was maintained at 37°C in Ham and Dulbecco's modified Eagle's medium (1:1 ratio, DMEM; Life Technologies Inc) containing 2 mM glutamine, 4% Fetal Calf Serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin, 1 μg of fungizone per ml (Ham and DMEM), 10-7 M of insulin, and 10-7 M of dexamethasone in a 5% CO2 incubator. All other cells were maintained at 37°C in DMEM containing 2 mM glutamine, 10% Fetal Calf Serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin, similarly in a 5% CO2 incubator. 293, 293T and NIH3T3 were maintained in DMEM containing 10% FCS, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomcycin at 37°C similarly in a 5% CO2 incubator. Cells were seeded at 5 × 105 on 10 cm or 7.5 × 105 on 15 cm plate and were at 70 – 80% confluence at the time of transfection or transduction. Viral vector production Replication-defective retroviral particles were generated by transient co-transfection of 293T cells with the three plasmids (pHR' CMVGFP or pHIV-CSGFP, pCMVΔR8.2 pr pCMVΔR8.9 and pHCMV-G), using a CaPO4 precipitation method as we previously reported [21]. Briefly, 293T cells were grown on 10 cm plates to 70–80% confluence and co-transfected with 10 μg pHCMV-G, 10 μg pHR' CMVGFP or pHIV-CSGFP and 20 μg pCMVΔR8.2 or pCMVΔR8.9. The plasmid DNA was diluted into 250 mM CaCl2 in 1/10-TE buffer (1 mM Tris HCl, 0.1 mM EDTA, pH 7.6) in 0.5 ml before an equal volume of 2× HBS (140 mM NaCl, 1.5 mM Na2HPO4, 50 mM HEPES, pH 7.05) was added and mixed by gently bubbling air through the mixture for 1 min. The solution was then added drop-wise onto the cells (100 μl per 1 ml of culture media). The cell cultures were rinsed with PBS and given fresh media within 10–12 hr after initiating transfection. The medium was harvested 48 hr post-transfection, centrifuged at low speed to remove cell debris and filtered through a 0.45 μm filter. The supernatant was stored at 4°C no more than 24 hr before it was used for transduction. Ultracentrifugation This was performed as reported previously [20,21]. Briefly, 30 mL of vector-producing cell (VPC) supernatant was added to each polypropylene ultra-centrifugation tube (6 × 30 mL), and ultracentrifuged at 50,000 g for 2 hr at 4°C on AH629 rotors in a Beckman refrigerated centrifuge. After centrifugation, the tubes were promptly removed and supernatant decanted. The viral pellet was resuspended in 0.6 mL of DMEM and stored at -20°C. In vitro transduction and determination of lentivector titre This was performed as we previously reported [20]. Briefly, cultured 293T cells were seeded at 5 × 105 cells and transduced with serially diluted and concentrated viral vector stocks 16–18 hours after seeding when cells were about 70% confluent. For each transduction, 8 μg/mL of polybrene (Sigma) was included in the transducing inoculum. Forty-eight hours after transduction, EGFP positive fluorescent cells were counted using epifluorescent microscope (Nikon eclipse E600, Japan) with the fluorescein isothiocyanate (FITC) excitation-emission filter set at 470 nm. The viral vector titre was determined as the average number of EGFP positive cells per 20 1-mm2 fields multiplied by a factor to account for dilution of the viral stock as well as plate size and thus total cell number. Alternatively, 48 hours after transduction, cells were harvested, resuspended and sent for FACs analyse at a local FACS facility (Queensland Institute of Medical Research, QIMR, Brisbane, Australia). Transduction – studies of target cell volume and number 293T cells at 1 × 103, 3 × 104 or 1 × 105 per well were seeded in triplicate in 24-well plates. Transduction was performed with the same stock of viral supernatant using volumes of 100 μl, 300 μl and 1 ml for 2 hours in the presence of 10 μg/ml polybrene. After the incubation period the cells were washed with fresh growth medium twice and allowed to grow for 2 days before the cells were trypsinised and fixed with 2% formaldehyde + 0.2% glutaraldehyde in PBS. EGFP positive and total cell numbers were counted with a haemocytometer using epi-fluorescence microscopy. Transduction – studies of variable vector-cell contact and adsorption periods 293T cells were grown in 24-well plates to approximately 70% confluence. The cells were incubated with 500 μl of pHR' CMVLacZ supernatant for 10 min, 30 min, 1 hr, 2 hr, 4 hr and 17 hours. After the indicated incubation period the viral supernatant was removed and replaced with fresh media. Forty-eight hours post-transduction, cells were stained to check for the presence of LacZ with the following solution: 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6.3H2O, 2 mM MgSO4, and 1 mg/ml X-gal in PBS. Blue cells or colonies were counted as positive for gene transfer. Transduction – studies of vector decay Cell-free viral vector-containing supernatant was incubated at 37°C for 30 min, 2 hr, 4 hr, or 24 hr prior to being used as the transducing medium (500 μl), with experimental samples in triplicate. 293T cell at 70% confluent cultures were exposed to the transducing media for 2 hours, after which the inoculum was removed and the cultures replenished with fresh media. Forty-eight hr post-transduction, cells were stained to check for the presence of LacZ with the X-gal solution. Blue cells/colonies were counted in 3 fields and the average used as the titre at that time point. Transduction – studies of MOI and transgene expression 293T cells were plated in a 10 cm plate at 1 × 105 cells/plate. Transduction was performed with viral vector stocks at a MOI of 2, 4, 8, 16 and 32 in the presence of 10 μg/ml polybrene (Sigma). Transduced cells were passaged every three days and EGFP positive cells sorted at a local FACS facility (QIMR, Brisbane, Australia). Flow cytometry Flow cytometry analysis was performed to evaluate the expression of lentivirus vector-mediated gene transfer. Cells were washed with PBS, and then fixed with 1% paraformaldehyde before the analysis. Samples were analysed on a FACScan flow cytometry in QIMR. Results The inoculum volume of the vector and the number of target cells affect vector titre determination, but not proportionally Figure 1 shows that during the lentiviral vector titration process, the higher the inoculum volume of the vector (ie. more viral vector particles) the more numbers of positively transduced cells. This was true over a range of target cells tested from 1 × 103 to 1 × 105 cells/ml. The results suggest the higher inoculum volume of the vector the more opportunity for a viral vector to reach a given target cell. Figure 1 Higher inoculum volumes (more vector particles) and increased number of target cells resulted in higher efficiency of gene transfer. This was true over a range of target cells from 1 × 103 to 1 × 105 and volumes from 0.1 ml to 1 ml. However the increase in gene transfer was not proportional to the increase in inoculum volume. e.g. a 10 fold increase in volume resulted in only a 3.7 to 4.7 fold increase in transduction efficiency. The values represent mean ± SD (n = 4). However, the results contradicted the data from an amphotropic MoMLV viral vector-mediated gene transfer where it was found that by keeping the virus vector concentration constant while the inoculum volume varied, the infectivity remained the same [19]. This discrepancy was not accounted for by the depth of fluid as in the present experiments, in the wells (area = 2 cm2) of the cell culture, the depth of the fluid varied from 0.5 mm (with a volume of 0.1 ml) to 1.5 mm for the 0.3 ml volume, and a depth of 5.0 mm for 1 ml of the vector preparation. All of these depths were well beyond the diffusion limit of relevance to the adsorption of 95% of a retrovirus preparation. This was because the rate decreased with the square of the depth, equating to 0.16 mm for a 2.5 hours adsorption period [2]. Similarly, vector titre was also affected by the number of target cells used in the vector titration process. A very significant increase in vector titre was noticed with increasing the cell numbers, but the increase was also not proportional. For a 30-fold increase in target cell number between 1 × 103 and 3 × 104 there was only an average of 9.17-fold increase in total number of transduced cells (for all transducing volumes). For a further 3.3-fold increase in cell number exposed in the same area, there was only a further 2.3 fold increase in total number of transduced cells. Thus, overall for a 100-fold increase in cell numbers (from 1 × 103 – 1 × 105) exposed to vectors there was only a 21.3-fold increase in total number of transduced cells. Interestingly, the increase of the number of positively transduced cells was not proportional to the increase of the vector inoculum volume. The increase in the number of transduced cells was proportionally less than the increase in inoculum volume, e.g. a 10-fold increase in inoculum volume resulted in only a 3.7 to 4.7-fold increase in the number of positively transduced cells. Vector decay and the period of vector adsorption to target cells were significant factors in influencing the transduction process The length of period of vector adsorption to target cells was shown to alter the transduction efficiency significantly. As the incubation period increased so did the number of transduced cells (Figure 2a). At 4 hours less than half of the active vectors had adsorbed on to the cells. Since vector adsorption to cells was often protracted, the issue of thermostability of the vector preparation arose as a negative modulator of transduction efficiency with increasing time, thereby producing further variation in the estimated titre and thus the "MOI". Figure 2 The period of adsorption (a) and vector decay (b) were significant factors in determining transduction efficiency. The duration of the adsorption period was also shown to alter the transduction efficiency significantly. As the incubation period increased so did the number of transduced cells. At 4 h less than half of the active vectors had adsorbed to the cells. To estimate the t(1/2) of the vector system used here, we pre-incubated the inoculum for increasing periods of time before applying aliquots to the target cell monolayer. By applying the following equations VA = VAo exp (-kdt) and t(1/2) = ln(2)/kd to the data, {where VA is the concentration of active virus at time t, VAO is the initial concentration of active virus, and Kd is the virus decay rate constant}, the half-life of the vector was in the 8–9 hr range. The values represent mean ± SD (n = 4). To estimate the half time (t(1/2)) of the vector system used here, we pre-incubated the inoculum for increasing periods of time before applying aliquots to the target cell monolayer for vector titre determination. The length of time for which the viral supernatant harvest was left at 37°C (in a cell-free environment) prior to use, noticeably affected the value of the vector titre (Figure 2b). The viral vector activity decayed logarithmically with time. By applying the following equations: VA = VAo exp (-kdt) and t(1/2) = ln(2)/kd to the data, {where VA is the concentration of active virus at time t, VAO is the initial concentration of active virus, and Kd is the virus decay rate constant}, the half-life of the vector was in the 8–9 hours range. This is the first time that lentivector stability has been examined. This estimation was twice as long as that for wild-type HIV [1], suggesting that lentivector is much more stable. Variations in viral vector titration further complicated the use of MOI for predicting gene transfer events Lentiviral vector titre (transducing unit per millitre, TU/ml) was calculated using the number of TU/ml times the dilution factor of the vector stock, divided by the volume of vector used in the transduction. As shown in the above results, the number of positively transduced cells changed when the transduction conditions varied. Therefore, the vector titre was affected by inoculum volume, vector stability and target cell numbers. If vector titres were to be calculated using the existing formula that was developed based on retroviral vector-mediated gene transfer, i.e. EGFP-positive cells (TU) ÷ volume of vector inoculum (ml), the titre of the original vector suspension would result in absurdly different figures (see Table 1), with ranges from 2.2 × 102 TU/mL to 1.2 × 104 TU/mL for the same viral suspension, more than a 50 fold difference. Likewise, because MOI is based on vector titre (MOI = titre × TD volume / number of cells), the use of MOI was thus affected. Table 1 Different titres and MOI were obtained for the same vector stock when different numbers of target cells and volumes of inoculum were used. The number of positively transduced cells and thus the transduction efficiency, was also affected by the number of target cells in the transduction process, eg.: a thirty-fold increase in cell numbers resulted in a 53% decrease in efficiency. The transduction efficiency was highest with the smallest cell number and largest inoculum volume. Titre TU/mL (followed by MOI) Number of target cells 1 mL of VI Vol. 0.3 mL of VI Vol. 0.1 mL of VI Vol. 2.24 × 102 (0.224) 3.96 × 102 (0.119) 6.08 × 102 (0.061) 1 × 103 2.14 × 103 (0.071) 3.77 × 103 (0.038) 5.14 × 103 (0.017) 3 × 104 5.58 × 103 (0.056) 7.79 × 103 (0.023) 1.19 × 104 (0.012) 1 × 105 TU – Transducing Unit; VI Vol – Volume of Inoculum. Considerable differences existed in the sensitivity of lentiviral vector-mediated gene transfer in several conventional cell lines The sensitivity of lentivector-mediated EGFP gene transfer to commonly used target cell lines has never been directly compared previously. In this study, 3 commonly used cell lines plus a new cell line FRL-19 were included for comparison. All cells were seeded in 12 well plate at 5 × 104 cells/well 16–18 hours before transduction. Concentrated viral vectors with unknown titre were added to each well at 50 μl, 100 μl, 200 μl, and 400 μl. Medium was changed every day. All cells were harvested 72 hours after transduction, washed twice with PBS, and then analysed by FACS. Figure 3 showed that the percentage of EGFP positive cells was 88.1% for FRL-19 cells, 52.9% for 293T cells, 34.7% for NIH 3T3 cells, and 27.8% for 293 cells respectively when 50 μl viral vector was used for transduction. Clearly transduction efficiency of lentivector-mediated EGFP gene transfer to FRL-19 was the highest amongst the four cell lines tested. It reached 96.7% when 400 μl of viral vector was used while the transduction efficiency of lentivectors was only 87.9% for 293T cells, 77.1% for NIH 3T3 cells, and 63.9% for 293 cells for the same volume of vector (Fig. 3A). When a third generation of lentiviral vector packaging system (pMDg/p, pRSV-Rev, gifts from Professor Didier Trono, Department of Genetics and Microbiology, CMU., Switzerland) were used to package a HIVCS-CMV-EGFP vector, a very similar transduction efficiency was obtained (Zhang et al., unpublished data). These results convincingly demonstrated that conventional cell lines were less sensitive to lentiviral vector-mediated gene transfer than FRL19, thus grossly underestimating vector titre. Figure 3 Efficiency of lentivector-mediated gene transfer to commonly used target cell lines (A) under different MOI (B). Four cell lines were seeded at 5 × 104/well in 12 well plates. Several different inoculum volumes of lentivectors without known titre (A) or with known titre, ie.: different MOI (B) were added were added to each well (A) or as indicated. The media was changed daily. Cells were harvested three days after transduction, and washed three times with PBS. Transduction efficiency of lentivectors in different cell lines was obtained using flow cytometric analysis. Data represents mean value ± SD (n = 4). The sensitivity of cell lines to lentivectors was generally MOI dependent We further examined whether the sensitivity of these cell lines to lentivectors-mediated EGFP gene transfer was dependent on the MOI. All four cell lines were seeded in 12 well plate at 5 × 104 cells/well 16–18 hrs before transduction. Viral vectors with known titre were added to each well at different MOI (MOI = viral titre/cell number). Medium was changed every day, with cells harvested 72 hrs after transduction, washed twice with PBS, and then examined by FACS analysis. Figure 4a shows that transduction efficiency of lentivectors was higher on the FRL-19 cell line than the other three cell lines. Transduction efficiency was 67.4% in FRL-19 cells, 33.1% in 293T cells, 23.1% in NIH 3T3 cells, and 8.7% in 293 cells at a MOI of 32. Generally, it was the higher the MOI, the higher the transduction efficiency (Fig 3B). Discussion We showed in this study that a number of factors within the vector titration process, ie.: the volume of inoculum, the number of target cells, cell type and viability/susceptibility, vector exposure time for uptake and vector half life affected vector titre determination. We were also surprised to find that the volume of inoculum (with a constant virus concentration) played such an important role in the determination of transduction efficiency. It has been demonstrated that above the cell's surface in MoMLV based retroviral vector mediated gene transfer, a fluid layer of 0.1–1 mm thick remained stationary, and this layer is seen to be the major source of origin of the transducing elements. The large effects seen with non-agitated cultures in the present series of experiments with lentiviral vectors indicated some fundamental differences in the processes of the transduction pathways of MoMLV based retroviral vectors and lentivirally derived vectors. During the transduction process, the rate of collision between the virions and the surface of the target cells could be predicted from Brownian theory even when the viral suspension was being shaken continuously [22]. This appears to suggest that successful transduction depends on the concentration of virus and not the overall number of virions present, due to the layer effect. The fact that viral vector titre may vary from the transduction process and that the MOI was calculated based on the viral titre, suggested that different vector titres and MOIs could be generated from a single lentivector stock, making direct comparison of data difficult, especially when the difference in vector titre was as high as 50 fold. Therefore, the titre obtained this way obviously did not represent the true value of active vector concentration. Rather, it was grossly underestimated when commonly used cell lines were used as target cells for vector titration. The viral stocks of most lentiviral vectors are generally produced from a 293 or 293T cell lines and the titre calculated by determining the number of foci (effect of the marker gene expression) produced in the cell line [23]. For example, if 100 μl of the vector suspension gives rise to 1 × 105 cells positive for a given marker gene expression, then the titre of the vector stock would be 1 × 106 TU/ml. When this vector stock is further used to transduce a new cell line, MOI is then determined by simply dividing the number of viral vector units added (ml added × TU/ml) by the number of target cells added (ml added × cells/ml). The average number of viral vector particles per cell in a transduction experiment could be less than 0.1 or more than 1000 depending upon how the experiment is designed. However, recent research showed that if MOI is too low, one may not get enough gene transfer and transgene expression [24]. If MOI is too high, the efficiency of gene transfer may not be very high, but many copies of transgene may integrate into the chromosomes of the target cells instead, thus causing chromosomal instability [24]. Employing MOI from 0–32, we demonstrated that efficient transduction of four different cell lines (293, 293T, NIH3T3, FRL19) resulted in a near liner relationship of MOI to transduction efficiency, the higher the MOI, the higher the transduction efficiency. This was somewhat surprising and contradicted traditional MoMLV based vectors, which showed an obvious plateau when the MOI was increased to about 3 [3]. The reason for this is unclear, but the fact that lentiviruses are more complicated retroviruses, having more sophisticated machinery for replication and integration than MoMLV, as well as that lentiviral vectors were exploiting the pseudotyped envelope (VSV-G utilises a different receptor), may probably explain the difference in gene transfer efficiency. The VSV-G envelope, binds to its target in cell membranes which are known to be phospholipids, such as phosphatidylcholine (PC) and phosphatidylserine (PS), (the receptors for VSV-G). PC is the most abundant membrane phospholipid while PS domains are present in much smaller quantity but bind more strongly and fuse faster with the VSV-G protein [25]. This issue is probably one of the most overlooked variables in vector transduction. Membrane phospholipid movement is highly dynamic. Its biosynthesis and degradation are very much dependent on cell type and positions in the cell cycle and/or metabolic activity. Also, the rate of degradation is rapid in G1, slows drastically during S phase, and picks up the pace again as cells exit mitosis and re-enters G1[26], which suggests that the cell cycle phase may be an important variable for VSV-G protein coated lentiviral transduction, and may contribute to the time dependence of the transduction efficiency observed in the present experiments. A further contribution to the volume effect may be increased cellular phospholipid uptake from the serum in the expanded volume of medium used for the delivery of the increased total vector or possibly enhanced phospholipid synthesis in a more generous nutritional environment. Cells double their phospholipid mass while maintaining the correct relative composition prior to cytokinesis [27]. Theoretically, during the intermitotic period the target cells will double the number of target binding sites for the viral vectors as well as allowing a period with the more favourable conditions (DNA synthesis) for integration. The amount of PC in the total membrane mass varies from 40–80% of the total P-lipid, depending on the cell type [27] and this variation may explain the discrepancies in transduction efficiencies observed with different cell lines using inocula of the same volume and titre of vector. In the real world of gene transfer experiments, transduction conditions will be optimised to achieve the maximum efficiency. Generally, a high MOI is needed for satisfactory levels of gene transfer. Ideally, with a MOI of 2, every single cell might be expected to experience an average of two gene transfer events in a given transduction experiment, but probabilistic considerations of viral and vector-cell interactions ensure that this does not occur (i.e. only 67% of the cells would be "infected"). As seen in the current data, however, the efficiencies of transduction are very much less than the theoretical outcomes. Our study with lentiviral vector convincingly showed that the higher the MOI, the higher the efficiency of gene transfer and the level of gene expression. However, experiments employing MOI even greater than 1000 have still resulted in less than 100% of cells transduced [11,28,29] indicating the presence of unexplained variables in the cell dependence of the transduction process. Conclusions MOI is only a useful term for predicting transduction efficiency under very carefully defined experimental conditions. The assumption is not valid that changes in any one of the variables shown to be important in the in vitro vector titration process will cause proportional changes in the magnitude of the transduction efficiency. It is thus evident that MOI is not applicable as a simply manipulable quantity in most gene therapy uses of the lentiviral vector system. Since clinical applications are an important outcome of gene transfer manipulations, and ultimately this may be done by in vivo delivery, the awesome task of evaluating the efficiency of transduction via this route will require considerable ingenuity. If MOI for lentiviral vector transduction has to be used for rigorous comparisons of data, then the specific experimental conditions for vector titration, with using the most sensitive cell lines, such as FRL 19, must be strictly observed for infectivity outcomes to be predictable. List of Abbreviations CAEV, caprine arthritis/encephalitis virus; DMEM, Dulbecco's modified Eagle's medium; EGFP, enhanced green fluorescent protein; EIAV, equine infectious anaemia virus; FCS, Fetal Calf Serum; FITC, fluorescein isothiocyanate ; FIV, the feline immunodeficiency virus; HIV, Human Immunodeficiency Virus; JDV, Jembrana disease virus; MOI, Multiplicity of Infection; MoMLV, Moloney murine leukaemia virus; PC, phosphatidylcholine; PS, phosphatidylserine; VPC, vector-producing cell; VSV-G, Vesicular Stomatitis Virus G glycoprotein; Competing interests None declared. Authors' contributions BZ performed the use of MOI to predict gene transfer events in the four cell lines; PM performed titration of lentiviral vectors; HJ performed the statistics and Table 1. KE helped with design of the experiments in examining various conditions in in vitro transduction; GC provided some in BZ and HJ's work; M West provided advice on analysis of the data and manuscript writing; M Wei helped with the design and day to day supervision of all the experiments, assisted with analysing the data and prepared and prove read the manuscript. Acknowledgments The authors wish to thank Mrs Polla Hall for FACS analysis. BZ is a Royal Children's Hospital Foundation/Chinese Club PhD scholar. This work was partly supported by project grants to MQW from the National Heart Foundation and the Queensland Cancer Fund, Brisbane, Australia. ==== Refs Higashikawa F Chang L Kinetic analyses of stability of simple and complex retroviral vectors Virology 2001 280 124 31 11162826 10.1006/viro.2000.0743 Andreadis S Lavery T Davis HE Le Doux JM Yarmush ML Morgan JR Toward a more accurate quantitation of the activity of recombinant retroviruses: alternatives to titer and multiplicity of infection J Virol 2000 74 3431 3439 10755888 10.1128/JVI.74.7.3431-3431.2000 Amado RG Chen RS Lentiviral vectors – the promise of gene therapy within reach? 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D Verma IM Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector Proc Natl Acad Sci USA 1996 93 11382 11388 8876144 10.1073/pnas.93.21.11382 Uchida N Sutton RE Fridera AM He D Reitsma MJ Chang WC Veres G Scollay R Weissman IL HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/G1 human hematopoietic stem cells Proc Natl Acad Sci USA 1998 95 11939 11944 9751769 10.1073/pnas.95.20.11939 Sutton RE Reitsma MJ Uchida N Brown PO Transduction of human progenitor hematopoietic stem cells by human immunodeficiency virus type 1-based vectors is cell cycle dependent [in process citation] J Virol 1999 73 3649 3660 10196257 Case SS Price MA Jordan CT Yu XJ Wang L Bauer G Haas DL Xu D Stripecke R Naldini L Kohn DB Crooks GM Stable transduction of quiescent cd34(+)cd38(-) human hematopoietic cells by hiv-1-based lentiviral vectors Proc Natl Acad Sci USA 1999 96 2988 2993 10077624 10.1073/pnas.96.6.2988 Poeschla EM Wong-staal F Looney DJ Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors Nat Med 1998 4 354 357 9500613 Olsen JC Gene transfer vectors derived from equine infectious anemia virus Gene Ther 1998 5 1481 1487 9930301 10.1038/sj.gt.3300768 Mselli-lakhal L Favier C Da Silva teixeira MF Chettab K Legras C Ronfort C Verdier G Mornex JF Chebloume Y Defective RNA packaging is responsible for low transduction efficiency of caev-based vectors Arch Virol 1998 143 681 695 9638141 10.1007/s007050050323 Berkowitz R Ilves H Lin WY Eckert K Coward A Tamaki S Veres G Plavec I Construction and molecular analysis of gene transfer systems derived from bovine immunodeficiency virus J Virol 2001 75 3371 3382 11238863 10.1128/JVI.75.7.3371-3382.2001 Berkowitz R Ilves H Plavec I Veres G Gene transfer systems derived from visna virus: analysis of virus production and infectivity Virology 2001 279 116 129 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mediated by a lentiviral vector Gene Ther 2000 7 196 200 10694795 10.1038/sj.gt.3301075 Hamaguchi I Woods NB Panagopoulos I Andersson E Mikkola H Fahlman C Zufferey R Carlsson L Trono D Karlsson S Lentivirus vector gene expression during ES cell-derived hematopoietic development in vitro J Virol 2000 74 10778 10784 11044122 10.1128/JVI.74.22.10778-10784.2000

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==== Front BMC PharmacolBMC Pharmacology1471-2210BioMed Central London 1471-2210-4-151529651610.1186/1471-2210-4-15Research ArticleProteasome inhibitors: Their effects on arachidonic acid release from cells in culture and arachidonic acid metabolism in rat liver cells Levine Lawrence [email protected] Department of Biochemistry, Brandeis University Waltham, MA 02454, USA2004 5 8 2004 4 15 15 22 3 2004 5 8 2004 Copyright © 2004 Levine; licensee BioMed Central Ltd.2004Levine; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background I have postulated that arachidonic acid release from rat liver cells is associated with cancer chemoprevention. Since it has been reported that inhibition of proteasome activities may prevent cancer, the effects of proteasome inhibitors on arachidonic acid release from cells and on prostaglandin I2 production in rat liver cells were studied. Results The proteasome inhibitors, epoxomicin, lactacystin and carbobenzoxy-leucyl-leucyl-leucinal, stimulate the release of arachidonic acid from rat glial, human colon carcinoma, human breast carcinoma and the rat liver cells. They also stimulate basal and induced prostacycin production in the rat liver cells. The stimulated arachidonic acid release and basal prostaglandin I2 production in rat liver cells is inhibited by actinomycin D. Conclusions Stimulation of arachidonic acid release and arachidonic acid metabolism may be associated with some of the biologic effects observed after proteasome inhibition, e.g. prevention of tumor growth, induction of apoptosis, stimulation of bone formation. ==== Body Background The proteasome degrades many cellular proteins, several with regulatory functions. It is not surprising that proteasome inhibitors affect many biologic processes [1] including prevention of cancer [2]. The effect of proteasome inhibition on cell growth and possible cancer chemoprevention has been reviewed by Adams [3]. Epoxomicin, an α'-β'-epoxyketone, appears to be the most selective proteasome inhibitor. Based on its anti-tumor activity, this product was originally isolated from an actinomycetes strain C-996-17 [4]. It inhibits the chymotrypsin-like activity (cleavage after large hydrophobic residues), trypsin-like activity (cleavage after basic residues) and peptidyl-glutamyl peptide hydrolyzing (PGPH) activity (cleavage after acidic residues) of proteasomes. Activities of the Ca++-dependent proteases, calpain, papain, chymotrypsin, trypsin and cathepsin are not affected by epoxomicin even at a 50 μM concentration [5]. The β-lactone, lactacystin, is relatively selective but can inhibit cathepsin A [6]. Peptide aldehydes, in addition to inhibiting proteasome activity, can also inhibit lysosomal and Ca++-activated proteases [7]. The peptides containing the carboxyvinylsulfone moiety inhibit cysteine proteases [8,9]. I have shown that inhibition of proteolysis by phenylmethylsulphonyl fluoride, the peptide aldehydes carbobenzoxy-leucyl-leucyl-norvalinal and carbobenzoxy-leucyl-leucyl-leucinal (ZLLL) and lactacystin stimulate induced prostaglandin (PGI2) production in rat liver cells [10,11]. Lactacystin stimulates arachidonic acid (AA) release from these cells [11]. Others have reported that proteasome inhibition up-regulates cyclooxygenase-2 (COX-2) and stimulates PGE2 production in neuronal cells [12]. In this report, evidence is presented that proteasome inhibitors stimulate PGI2 production by rat liver cells as well as the release of AA from rat liver, rat glial, human colon carcinoma and human breast carcinoma cells in culture. The stimulation of AA release from rat liver cells is partially inhibited by preincubation of the cells with actinomycin D. Results and Discussion Of the cells examined (C-9 rat liver, C-6 rat glial, HT-29 human colon carcinoma and BT-20 human breast carcinoma) the prostanoid metabolic profile has been described only for C-9 rat liver cells (95% is PGI2 and less than 5% is PGE2 and PGF2α) [13]. At the low cell densities used in this study, only PGI2, the main product of COX-mediated synthesis, can be quantitatively estimated. The rat liver cells express COX-2 both constitutively and after induction [14]. The effect of time on basal and 12-0-tetradecanoylphorbol-13-acetate (TPA) induced PGI2 synthesis during incubation of cells with epoxomicin is shown in Fig. 1. Figure 1 Time-course of (A) basal and (B) TPA-induced 6-keto-PGF1α production during incubation with 1.2 μM epoxomicin. In (B) 16.7 nM TPA was used. Analyses were performed with duplicate and triplicate dishes. Mean values are shown. The stimulation of basal PGI2 production by epoxomicin and TPA-induced PGI2 production by epoxomicin and lactacystin as a function of dose is shown in Fig. 2. As little as 0.3 μM epoxomicin stimulates TPA-induced PGI2 production significantly (Fig. 2-B). It is 10 to 15 times more effective than lactacystin (compare Fig. 2-B and 2-C). Using purified bovine erythrocyte proteasomes, epoxomicin inhibits the chymotrypsin-like activity, about 4 to 5 times more effectively than does clasto-lactacystin β-lactone, the derivative of lactacystin [5]. They are almost equally effective on inhibiting the trypsin-like and PGPH-like activities [5]. Assuming that epoxomicin and lactacystin have equal access to the proteasome and that proteasome activity is regulating COX-2 in rat liver cells similarly to neuronal cells [12] then COX-2 may be degraded in the proteasome by cleavage after large hydrophobic residues. Figure 2 Effect of epoxomicin on (A) basal and (B) TPA-induced 6-ketoPGF1α production and (C) effect of lactacystin on TPA-induced 6-ketoPGF1α production. Cells were incubated with the reagents for 6 hours. The analyses were performed with triplicate dishes *- statistically significant vs MEM/BSA. **- Statistically significant vs TPA. The amplification of PGI2 production (Figs. 1 and 2) after inhibition by epoxomicin could reflect not only stabilization of COX-2 but also an intracellular increase in the concentration of the substrate i.e. the AA that is produced during hydrolysis of the membrane phopholipids by PLase activity [15]. Extracellular and/or intracellular release of AA will depend, in part, on the localization of the phospholipids in the membrane, e.g. in its inner or outer leaflet [16]. Release of AA in response to several agonists has been described [17-20]. The effect of a 2, 4 or 6-h incubation on AA release from rat liver and rat glial cells by 1.0 μM epoxomicin was determined. Only after the 6-h incubation were the differences significant statistically. Regulation of PLase activity by the proteasome pathway appears to be a relatively slow process. After a 6-h incubation, epoxomicin, lactacystin and ZLLL stimulate the release of extracellular AA from rat liver cells (Fig. 3) and AA release after TPA-induction (3.7% vs 13.5% in the presence of 1.0 μM epoxomicin). Epoxomicin also stimulates the release of AA from rat glial, human colon carcinoma and human breast carcinoma cells (Table 1). The stimulation of AA release from the rat liver cells after incubation with epoxomicin is partially inhibited by pre-incubation of the cells for 2-h with actinomycin (Fig. 4) suggesting that a fraction of the PLase is induced. As expected, the inhibition of TPA-induced PGI2 production by actinomycin D is complete (Fig. 5). Thus, some mechanisms leading to maximum AA release appear to be genomic. The induced PLase activity, probably PLA2, could reflect expression of either a secretory or cytosolic PLA2 or some combination of both enzymes [21]. Figure 3 Dose-response of epoxomicin, lactacystin and ZLLL on AA release from rat liver cells. After incubation for 6 hours. The analyses were performed with triplicate dishes. *- Statistically significant vs MEM/BSA. Table 1 Effect of Epoxomicin on AA Release from Rat Glial, Human Colon Carcinoma and Human Breast Carcinoma Cells. Cell Type % AA Release MEM/BSA control With epoxomicin Rat glial (C-6) 9.0 ± 0.07 11.4 ± 0.28 Human Colon Carcinoma (HT-29) 7.8 ± 0.05 9.4 ± 0.33 Human Breast Carcinoma (BT-20) 12.1 ± 0.35 14.2 ± 0.31 Cells were incubated in the presence and absence of 4.5 μM epoxomicin for 6-h, 12-h or 9-h (C-6, HT-29, BT-20 respectively). Analyses were performed with quadruplicate (C-6 and HT-29) or quintuplicate (BT-20) dishes. All values are expressed as Mean ± SE (n = 4 or n = 5). All data with epoxomicin are statistically significant vs MEM/BSA. Figure 4 Effect of Actinomycin D on AA release from rat liver cells. Cells were preincubated with and without 1.0 μM actinomycin D for 2-h, then incubated in the presence and absence of epoxomicin and actinomycin D for another 6-h. The analyses were performed with triplicate or quadruplicate dishes. *- Statistically significant vs epoxomicin in the presence of actinomycin D. Figure 5 Effects of actinomycin D on TPA-induced 6-Keto-PGF1α production. Cells were preincubated with 1.0 μM actinomycin D for 2-h and then incubated in the presence and absence of 0.6 μM epoxomicin and/or 16.7 μM TPA for another 6-h. The analyses were performed with triplicate dishes. *- Statistically significant vs TPA in the absence of actinomycin D. **- Statistically significant vs epoxomicin plus TPA in the absence of actinomycin D. The release of AA from rat liver cells, most likely resulting from PLase activation, is associated with cancer chemoprevention [14,17-19], [22-24]. In addition to its intrinsic biologic activities, AA regulates production of lipoxygenase, cytochrome P-450, and epoxygenase products as well as COX activities. Prostanoid profiles differ with cell type and individual AA metabolites have different pharmacological properties [15]. COX-2 activity, as measured by PGI2 production, is stimulated by proteasome inhibition (Fig. 1 and 2). Thus, some biologic effects of proteasome inhibition, e.g. stimulation of bone formation [25], may reflect the metabolism of the intracellular AA. Inhibition of COX-2 activity is one possible mechanism that has been proposed to prevent colon cancer [26]. However, rather than inhibiting, tamoxifen and raloxifene, statins and epoxomicin stimulate COX-2 activity and AA release from rat liver cells [14,17-19]. As shown in Table 1, epoxomicin stimulates AA release from human colon carcinoma, breast carcinoma and rat glial cells. Tamoxifen and simvastatin also stimulate AA release from the human colon carcinoma and human breast carcinoma cells (unpublished data). These drugs have been reported to prevent cancer [27,28]. At least as measured by the COX activity of rat liver cells, tamoxifen, raloxifene, statins and proteasome inhibitors could be preventing cancer by a COX independent mechanism. AA resulting from proteasome inhibition has many intrinsic biologic properties [reviewed in [29]]. Some of these activities may trigger PLase activity. The causal relationship of AA to cancer prevention (if any) is unclear. Production of AA by the tumor-suppressive type-II phospholipase A2 (PLA2G2A) may be related to the cancer prevention [22-24]. It is not surprising that control of PLase activities present an attractive area for cancer prevention studies [30]. Methods The rat liver (C-9 cell line) and human breast carcinoma (the BT-20 cell line) were purchased from the American Type Culture Collection (Manassas, VA, USA). The rat liver glial cells (C-6 cell line) was obtained from Dr. Elaine Lai of the Department of Biology, Brandeis University and the human colon carcinoma (the HT-29 cell line) was obtained from Dr. Basil Rigas, American Health Foundation, Valhalla, NY, USA. They were maintained in Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum. [3H] AA (91.8 Ci/mmol) was obtained from NEN Life Science Products, Inc. (Boston, MA, USA). Epoxomicin, lactacystin and ZLLL were purchased from Biomol (Plymouth Meeting, PA, USA). All other reagents were from Sigma Chemical Co. (St. Louis, MO, USA). Rat liver cells incubating with lactacystin (5.4 μM) or ZLLL (1.0 μM) for 6-h have been tested for viability by a tetrazolium-based assay and found not to be toxic [31]. Two days prior to experiments, the rat liver cells were treated with 0.25% trypsin-EDTA and, after addition of minimal essential media (MEM) containing 10% fetal calf serum, the floating cells were seeded onto 35 mm culture dishes. The plating densities varied from 0.1 to 0.5 × 105 cells/35 mm dish. The freshly seeded cultures were incubated for 24-h to allow for cell attachment. After decantation of MEM containing the fetal bovine serum, 1.0 ml fresh MEM containing 10% fetal bovine serum and [3H] AA (0.2 μCi/ml) were added and the cells incubated for another 24-h. The cells were washed 4 times with MEM and incubated for various periods of time with 1.0 ml of MEM containing 1.0 mg BSA/ml (MEM/BSA) and different concentrations of each compound. The culture fluids were then decanted, centrifuged at 2000 × g for 10 min, and 200 μl of the supernate counted for radioactivity. Radioactivity recovered in the washes before the incubation was compared to input radioactivity to calculate the % radioactivity incorporated into the cells [31]. For PGI2 production, 1.0 ml of MEM supplemented with 10% fetal bovine serum, void of [3H]AA, was added after the first 24-h incubation. The cells were incubated for another 24-h, washed three times with MEM, then incubated with the compounds in MEM/BSA for various periods of time. The culture fluids were decanted and analyzed for 6-keto-PGF1α, the stable hydrolytic product of PGI2, by radioimmunoassay [32]. The [3H] AA release is presented as a percentage of the radioactivity incorporated by the cells. Except for the time-course experiments which used duplicate dishes, three to five culture dishes were used for each experimental point. The data are expressed as mean values ± SEM. The data were evaluated statistically by the unpaired Student's t-test. A P value < 0.05 was considered significant. Acknowledgements My thanks to Hilda B. Gjika for preparation of the manuscript and to Dr. Armen H. Tashjian, Jr., Department of Genetic and Complex Diseases, Harvard School of Public Health, for his continuing interest in these studies. ==== Refs Kisselev AF Goldberg AL Proteasome inhibitors: from research tools to drug candidates Chem Biol 2001 8 739 758 11514224 10.1016/S1074-5521(01)00056-4 Mitchell BS The proteasome – an emerging therapeutic target in cancer N Engl J Med 2003 348 2597 2598 12826633 10.1056/NEJMp030092 Adams J Potential for proteasome inhibition in the treatment of cancer Drug Discov Today 2003 8 307 315 12654543 10.1016/S1359-6446(03)02647-3 Hanada M Sugawara K Kaneta K Toda S Nishiyama Y Tomita K Yamamoto H Konishi M Oki T Epoxomicin, a new antitumor agent of microbial origin J Antibiot (Tokyo 1992 45 1746 1752 1468981 Meng L Mohan R Kwok BH Elofsson M Sin N Crews CM Epoxomicin, a potent and selective proteasome inhibitor, exhibits in vivo antiinflammatory activity Proc Natl Acad Sci U S A 1999 96 10403 10408 10468620 10.1073/pnas.96.18.10403 Ostrowska H Wojcik C Omura S Worowski K Lactacystin, a specific inhibitor of the proteasome, inhibits human platelet lysosomal cathepsin A-like enzyme Biochem Biophys Res Commun 1997 234 729 732 9175783 10.1006/bbrc.1997.6434 Mellgren RL Specificities of cell permeant peptidyl inhibitors for the proteinase activities of mu-calpain and the 20 S proteasome J Biol Chem 1997 272 29899 29903 9368065 10.1074/jbc.272.47.29899 Bogyo M McMaster JS Gaczynska M Tortorella D Goldberg AL Ploegh H Covalent modification of the active site threonine of proteasomal beta subunits and the Escherichia coli homolog HslV by a new class of inhibitors Proc Natl Acad Sci U S A 1997 94 6629 6634 9192616 10.1073/pnas.94.13.6629 Bromme D Klaus JL Okamoto K Rasnick D Palmer JT Peptidyl vinyl sulphones: a new class of potent and selective cysteine protease inhibitors: S2P2 specificity of human cathepsin O2 in comparison with cathepsins S and L Biochem J 1996 315 85 89 8670136 Levine L Proteolysis negatively regulates agonist-stimulated arachidonic acid metabolism Cell Signal 1998 10 653 659 9794247 10.1016/S0898-6568(98)00008-4 Levine L Lactacystin stimulates arachidonic acid metabolism in rat liver cells: effects of cell density on arachidonic acid release, PGI2 production and cyclooxygenase activity Prostaglandins Leukot Essent Fatty Acids 2000 63 371 375 11133174 10.1054/plef.2000.0230 Rockwell P Yuan H Magnusson R Figueiredo-Pereira ME Proteasome inhibition in neuronal cells induces a proinflammatory response manifested by upregulation of cyclooxygenase-2, its accumulation as ubiquitin conjugates, and production of the prostaglandin PGE(2) Arch Biochem Biophys 2000 374 325 333 10666314 10.1006/abbi.1999.1646 Rigas A Levine L Arachidonic acid metabolism by rat liver cells (the C-9 cell line) J Pharmacol Exp Ther 1984 231 230 235 6436471 Levine L Tamoxifen and the raloxifene analog LY117018: their effects on arachidonic acid release from cell in culture and on prostaglandin I2 production by rat liver cells BMC Cancer 2004 Smith WL The eicosanoids and their biochemical mechanisms of action Biochem J 1989 259 315 324 2655580 Edidin M The state of lipid rafts: from model membranes to cells Annu Rev Biophys Biomol Struct 2003 32 257 279 12543707 10.1146/annurev.biophys.32.110601.142439 Levine L Does the release of arachidonic acid from cells play a role in cancer chemoprevention? FASEB J 2003 17 800 802 12724337 10.1096/fj.02-0906hyp Levine L Tamoxifen stimulates arachidonic acid release from rat liver cells by an estrogen receptor-independent, non-genomic mechanism BMC Cancer 2003 3 24 14498998 10.1186/1471-2407-3-24 Levine L Statins stimulate arachidonic acid release and prostaglandin I2 production in rat liver cells Lipids Health Dis 2003 2 1 12689340 10.1186/1476-511X-2-1 Levine L Nuclear receptor agonists stimulate release of arachidonic acid from rat liver cells Prostaglandins Leukot Essent Fatty Acids 2002 67 453 459 12468267 10.1054/plef.2002.0456 Diaz BL Arm JP Phospholipase A(2) Prostaglandins Leukot Essent Fatty Acids 2003 69 87 97 12895591 10.1016/S0952-3278(03)00069-3 Praml C Amler LC Dihlmann S Finke LH Schlag P Schwab M Secretory type II phospholipase A2 (PLA2G2A) expression status in colorectal carcinoma derived cell lines and in normal colonic mucosa Oncogene 1998 17 2009 2012 9788445 10.1038/sj.onc.1202121 Cormier RT Hong KH Halberg RB Hawkins TL Richardson P Mulherkar R Dove WF Lander ES Secretory phospholipase Pla2g2a confers resistance to intestinal tumorigenesis Nature Genetics 1997 17 88 91 9288104 10.1038/ng0997-88 MacPhee M Chepenik KP Liddell RA Nelson KK Siracusa LD Buchberg AM The secretory phospholipase A2 gene is a candidate for the Mom1 locus, a major modifier of ApcMin-induced intestinal neoplasia Cell 1995 81 957 966 7781071 10.1016/0092-8674(95)90015-2 Garrett IR Chen D Gutierrez G Zhao M Escobedo A Rossini G Harris SE Gallwitz W Kim KB Hu S Crews CM Mundy GR Selective inhibitors of the osteoblast proteasome stimulate bone formation in vivo and in vitro J Clin Invest 2003 111 1771 1782 12782679 10.1172/JCI200316198 Subbaramaiah K Dannenberg AJ Cyclooxygenase 2: a molecular target for cancer prevention and treatment Trends Pharmacol Sci 2003 24 96 102 12559775 10.1016/S0165-6147(02)00043-3 Jordan VC Morrow M Tamoxifen, raloxifene, and the prevention of breast cancer Endocr Rev 1999 20 253 278 10368771 10.1210/er.20.3.253 Brower V Of cancer and cholesterol: studies elucidate anticancer mechanisms of statins J Natl Cancer Inst 2003 95 844 846 12813163 10.1093/jnci/95.12.844 Brash AR. Arachidonic acid as a bioactive molecule J Clin Invest 2001 107 1339 1345 11390413 Laye JP Gill JH Phospholipase A2 expression in tumours: a target for therapeutic intervention? Drug Discov Today 2003 8 710 716 12927514 10.1016/S1359-6446(03)02754-5 Levine L Stimulated release of arachidonic acid from rat liver cells by celecoxib and indomethacin Prostaglandins Leukot Essent Fatty Acids 2001 65 31 35 11487305 10.1054/plef.2001.0284 Levine L Rose NR, Friedman H, Fahey JL Measurement of arachidonic acid metabolites by radioimmunoassay. Manual of Clinical Laboratory Immunology 1986 3 Washington DC: American Society for Microbiology 685 691

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020288Research ArticleBioengineeringPhysiologyMus (Mouse)Loss of Skeletal Muscle HIF-1α Results in Altered Exercise Endurance HIF-1α and EnduranceMason Steven D 1 Howlett Richard A 2 Kim Matthew J 1 Olfert I. Mark 2 Hogan Michael C 2 McNulty Wayne 1 Hickey Reed P 3 Wagner Peter D 2 Kahn C. Ronald 4 Giordano Frank J 3 Johnson Randall S [email protected] 1 1Molecular Biology Section, Division of BiologySchool of Medicine, University of California, San Diego, CaliforniaUnited States of America2Division of Physiology, School of MedicineUniversity of California, San Diego, CaliforniaUnited States of America3Cardiology Division, Yale University Medical SchoolNew Haven, Connecticut United States of America4Joslin Diabetes Foundation, Harvard Medical SchoolBoston, MassachusettsUnited States of America10 2004 24 8 2004 24 8 2004 2 10 e2889 2 2004 29 6 2004 Copyright: © 2004 Mason et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. A Skeletal Muscle Protein that Regulates Endurance Skeletal Muscle Fiber Type: Influence on Contractile and Metabolic Properties The physiological flux of oxygen is extreme in exercising skeletal muscle. Hypoxia is thus a critical parameter in muscle function, influencing production of ATP, utilization of energy-producing substrates, and manufacture of exhaustion-inducing metabolites. Glycolysis is the central source of anaerobic energy in animals, and this metabolic pathway is regulated under low-oxygen conditions by the transcription factor hypoxia-inducible factor 1α (HIF-1α). To determine the role of HIF-1α in regulating skeletal muscle function, we tissue-specifically deleted the gene encoding the factor in skeletal muscle. Significant exercise-induced changes in expression of genes are decreased or absent in the skeletal-muscle HIF-1α knockout mice (HIF-1α KOs); changes in activities of glycolytic enzymes are seen as well. There is an increase in activity of rate-limiting enzymes of the mitochondria in the muscles of HIF-1α KOs, indicating that the citric acid cycle and increased fatty acid oxidation may be compensating for decreased flow through the glycolytic pathway. This is corroborated by a finding of no significant decreases in muscle ATP, but significantly decreased amounts of lactate in the serum of exercising HIF-1α KOs. This metabolic shift away from glycolysis and toward oxidation has the consequence of increasing exercise times in the HIF-1α KOs. However, repeated exercise trials give rise to extensive muscle damage in HIF-1α KOs, ultimately resulting in greatly reduced exercise times relative to wild-type animals. The muscle damage seen is similar to that detected in humans in diseases caused by deficiencies in skeletal muscle glycogenolysis and glycolysis. Thus, these results demonstrate an important role for the HIF-1 pathway in the metabolic control of muscle function. Mice that can't express HIF-1α in skeletal muscle don't induce glycolytic genes and thus switch to aerobic metabolism allowing them to run uphill longer than the their wild-type littermates ==== Body Introduction During exercise in normoxia, the partial pressure of oxygen in muscle tissue has been shown to dip to as low as 3.1 mm Hg, whereas in the capillary, it remains at 38 mm Hg (Hoppeler et al. 2003). In order to maintain effort, skeletal muscle exertion must be able to rely on pathways designed to help the tissue cope with oxygen stress after oxygen delivery capacity is exceeded. A switch between aerobic and nonaerobic metabolism during strenuous exertion requires mechanisms to adjust metabolic function, and this need is acute in extended exertion in skeletal muscle. It is clear that the transcription factor hypoxia-inducible factor 1α (HIF-1α) is an essential factor in maintenance of ATP levels in cells (Seagroves et al. 2001). In fact, although HIF-1α is typically thought of as acting only during hypoxia, its loss has an effect on both normoxic and hypoxic ATP levels in a number of tissue types (Seagroves et al. 2001; Cramer et al. 2003), and this implicates the factor in regulation of metabolic function even during conditions of normal physiologic oxygenation. In skeletal muscle, signaling of fatigue has been studied extensively, and signaling of exhaustion involves, to some degree, elevated systemic lactic acid, a by-product of the glycolytic pathway of metabolism (Myers and Ashley 1997). Thus, the glycolytic pathway is intrinsically involved in muscle function and fatigue, and this in turn is linked to the response to hypoxia. To understand how the primary hypoxia-responsive transcription factor controls skeletal muscle function, we targeted mouse skeletal muscle for tissue-specific deletion of HIF-1α via the use of a conditionally targeted allele of the gene (Ryan et al. 2000; Schipani et al. 2001). This mouse strain was crossed into a strain transgenic for the skeletal-muscle-specific muscle creatine kinase (MCK) promoter, which drives expression of the cre recombinase gene (Bruning et al. 1998; Sauer 1998). We found that loss of the regulation of hypoxic response in muscle has a profound effect on the function of the muscle during exertion, with effects that mimic human metabolic myopathies. Results/Discussion In 4-mo–old mice with the skeletal-muscle HIF-1α gene knocked out (HIF-1α KOs), the frequency of excision was evaluated through real-time PCR techniques. We saw deletion frequencies consistent with those described previously for this cre recombinase transgene (Bruning et al. 1998) with some variation in penetration; mean frequency of deletion was 54.9%, with the highest frequency of muscle-specific deletion of HIF-1α being 72% in the gastrocnemius of 4-mo–old mice homozygous for the loxP-flanked allele (Table 1). This transgene is expressed at a lower level in cardiac tissue, and cardiac deletion was detected (Table 1); however, none of the phenotypes described below were seen in cardiac myocyte-specific deletions of HIF-1α (Figure 1A). Gross muscle sections were evaluated histologically to evaluate both vascularization and fiber type (Tables 2 and 3), and ultrastructurally to determine number of mitochondria (Figure 1B). No changes were detected in any of these features in HIF-1α KOs, except for a slight but statistically significant decrease in type IIA fibers in the soleus muscles (Table 3). Similar hematocrit and blood hemoglobin levels were seen in HIF-1α KOs and wild-type (WT) mice (Figure 2). Figure 1 Exercise Capacity of Cardiac HIF-1α KOs and HIF-1α/MCK/cre Mitochondrial Density (A) Mice lacking cardiac HIF-1α perform no differently in endurance running trials than WT mice, showing that the increase in exercise capacity seen in MCK/Cre mice is due to deletion of HIF-1α in skeletal muscle, not cardiac tissue. (B) Mice lacking skeletal muscle HIF-1α have a slight but nonsignificant increase in mitochondrial density as measured by the number of mitochondria per electron microscope field of view. Figure 2 Hematocrit and Hemoglobin Levels in HIF-1α KOs and WT Mice (A) Hematocrit levels are virtually identical in both HIF-1α KOs (n = 3) and WT (n = 4) mice, indicating that loss of HIF-1α in skeletal muscle does not affect oxygen carrying capacity of the blood. (B) In addition to similar hematocrit levels, WT mice and HIF-1α KOs have very close blood hemoglobin levels. Table 1 Excision of HIF-1α in Various Tissues Deletion levels are the average percent of HIF-1α deleted ± SE Table 2 Fiber Typing of Gastrocnemius Muscle Values are percent ± SE Table 3 Fiber Typing of Soleus Muscle Values are percent ± SE * p < 0.05, WT vs. KO As can be seen in Table 4, significant changes in HIF-1α–dependent gene expression occur in muscle during exercise, including changes in genes involved in glucose transport and metabolism. Vascular endothelial growth factor (VEGF), which increases vascular permeability, and glucose transporter 4 (GLUT4), the muscle-specific glucose transporter, show increased levels in exercise and likely increase the availability of glucose to the muscle. The muscle-specific form of phosphofructokinase (PFK-M), phosphoglycerate kinase (PGK), and lactate dehydrogenase-A (LDH-A) are also up-regulated at the mRNA level by exercise, and this up-regulation is inhibited by the loss of HIF-1α, further demonstrating that HIF-1α is important for transcriptional response during skeletal muscle activity. Table 4 Relative Gene Expression Levels Expression levels are means relative to resting WT for each gene ± SE Percent increase indicates percent increase of postexercise average gene expression over resting average *p < 0.05, rest vs. postexercise; **p < 0.01, rest vs. postexercise In Table 5, we show the changes in enzymatic activity in a number of key glycolytic enzymes affected by deletion of HIF-1α. As can be seen from the data, several of the enzymes assayed showed a decrease in activity in response to exercise. In particular, the activity of one of the key rate-limiting enzymes, PFK, was significantly lower following exercise in HIF-1α KOs compared to WT mice, indicating that HIF-1α KOs may have difficulty maintaining optimal PFK activity. The responses of other glycolytic enzymes to exercise were fairly similar between WT mice and HIF-1α KOs. These include no significant changes in phosphoglucose isomerase activity and significant, yet similar, decreases in aldolase, glyceraldehyde 3-phosphate dehydrogenase, and PGK activities. An exception to this is that WT muscles were able to significantly increase pyruvate kinase (PK) activity (see Table 4; p < 0.05). LDH activity was also increased in the WT mice, although the level did not reach statistical significance. Activities of both PK and LDH were not significantly changed in HIF-1α KO muscles following exercise. Increased activities of PK, and subsequently LDH, could be expected to lead to increased levels of lactate in the WT mice relative to HIF-1α KOs.In Figure 3A, it can be seen that the decrease in PFK activity in the HIF-1α KOs is correlated with a trend approaching significance (p = 0.10) toward an increased amount of hexose monophosphates (HMPs), which are pre-PFK glycolytic metabolites, following stimulation of the HIF-1α KO muscle. This increase was not due to differences in glucose uptake, since animals of both genotypes were able to significantly increase intramuscular glucose to a similar degree (Figure 3B). Consistent with decreased flow through the glycolytic pathway, however, the increased amount of HMPs was correlated with increased muscle glycogenolysis (Figure 3C) and increased depletion of phosphocreatine (PCr) (Figure 3D), with a resultant decrease in the PCr/ATP ratio in HIF-1α KO muscle (Figure 3E), although there was only a nonsignificant drop in overall muscle ATP concentrations (Figure 3F). Intramuscular levels of lactate did increase in both HIF-1α KOs and WT mice during stimulation, although lactate accumulation did not differ significantly between them (Figure 3G). In order to evaluate whether these changes had any effect on overall muscle force, we measured force and calcium release in isolated single fibers; as can be seen in Figure 4A and 4B, there were no significant changes in these parameters, indicating that the muscle can compensate at this level for the metabolic changes induced by loss of HIF-1α. Figure 3 Intramuscular Metabolite Levels at Rest and Following Stimulation (A) Glycolytic intermediates were measured from gastrocnemius muscles following the isolated stimulation protocol. Resting values represent levels in the unstimulated gastrocnemius from the same animals. HIF-1α KOs had a trend toward greater accumulated levels of HMPs during the stimulation protocol, although the difference did not reach statistical significance (p = 0.10). This difference could be indicative of a blockage in the glycolytic pathway at PFK. (B) No significant differences were seen between HIF-1α KOs and WT intramuscular glucose levels at rest or following stimulation. Both HIF-1α KO and WT muscles were able to significantly increase glucose uptake, leading to greater levels of intramuscular glucose in response to stimulation (WT, p < 0.001; KO, p < 0.05). (C) HIF-1α KOs have more stored glycogen than do WT mice. Glycogen levels were measured following the same stimulation protocol as in (B). The change in glycogen from rest to poststimulation was also greater in the HIF-1α KOs, indicating that they metabolized more glycogen in response to stimulation (p < 0.01; *p < 0.05, WT at rest vs. KO at rest). (D) HIF-1α KOs utilize more PCr in response to stimulation than do WT mice. Similar levels of PCr were seen at rest, but HIF-1α KOs metabolized significantly more during stimulation (p < 0.05) and had much lower levels following the protocol (**p < 0.01, WT poststimulation vs. KO poststimulation). (E) A trend toward lower PCr/ATP concentration ratios was seen in HIF-1α KOs relative to WT mice following stimulation, although the difference did not quite reach statistical significance (p < 0.10). A trend toward a greater drop from rest to poststimulation in the PCr/ATP ratio was also seen in HIF-1α KOs following stimulation (p < 0.10), indicating that they had to rely more heavily on PCr for ATP generation. (F) Slight but nonsignificant differences were seen in whole-muscle ATP levels at rest or following stimulation. Although HIF-1α KOs exhibited altered substrate utilization, they were able to meet their ATP demands during the protocol. (G) Both HIF-1α KOs and WT animals produced significant intramuscular lactate during the stimulation protocol; however, there was no significant difference in the amount produced by either genotype. Resting intramuscular lactate levels were also similar for WTs and HIF-1α KOs. Figure 4 Force Generation and Ca2+ Release in Isolated Muscle Fibers during Stimulation (A) No differences were seen in total force generation in isolated muscle fibers. Mechanically dissected fibers from the flexor brevis muscle were subjected to a fatiguing protocol. Neither initial nor final forces differed between HIF-1α KO and WT fibers. (B) Ca2+ release and reuptake in HIF-1α KO and WT fibers was not different during the stimulation protocol. Ca2+ levels were measured in individual fibers through use of fura-2 Ca2+ indicator. The altered substrate utilization did not affect the ability of the fibers to maintain proper Ca2+ flux. Table 5 Glycolytic Enzyme Activity Levels from Gastrocnemius Muscles Activities are in U/mg protein ± SE GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PGI, phosphoglucose isomerase *p < 0.05, WT vs. HIF-1α KO for given exercised or resting state; **p < 0.05, rest vs. postexercise within given genotype Given altered levels of glycolytic throughput without significant changes in intramuscular ATP levels, it is likely that there is increased activity of oxidative pathways in the HIF-1α KO muscle. Increased muscle oxidative activity is typical in patients with myopathies involving muscle glycolysis or glycogenolysis, including phosphofructokinase disease (PFKD) and McArdle's disease (Vissing et al. 1996). We analyzed the activity of citrate synthase (CS), a key allosteric enzyme of the citric acid cycle, in WT and HIF-1α KO muscle (Figure 5A), and found that it was up-regulated in HIF-1α KOs. CS is a mitochondrial enzyme that responds to decreases in ATP concentration allosterically, allowing for increased oxidative activity in the mitochondria. In addition, significant up-regulation of the mitochondrial enzyme beta-hydroxyacyl CoA dehydrogenase (B-HAD) was seen in HIF-1α KO muscle (Figure 5B). B-HAD is also affected by energy levels in the cell, and decreases in NADH/NAD+ concentration ratios cause the enzyme to increase mitochondrial oxidation of fatty acids (Nelson and Cox 2000). Increased activity of oxidative pathways in the muscle should result in more rapid lactate clearance, as in fact occurs in PFKD patients during exercise; this phenomenon gives rise to a “second wind” in these patients, and under some circumstances allows for an increase in exercise endurance (Vissing et al. 1996; Haller and Vissing 2002), although this was disputed in one recent study (Haller and Vissing 2004). This decreased lactate accumulation postexercise clearly occurs in the HIF-1α KOs, as can be seen in Figure 5C. This systemically lower level of lactate postexercise indicates that there may be a shift toward a more oxidative metabolism in skeletal muscle. Figure 5 Oxidative Metabolism and Serum Lactate Production in HIF-1α KOs and WT Mice (A) HIF-1α KOs have higher resting levels of CS activity. CS is an enzyme in the Krebs cycle that can be regulated allosterically by ATP levels. Increased CS activity is indicative of increased muscle oxidative capacity, which is common in patients with glycogenolytic or glycolytic myopathies (# p < 0.10, KO vs. WT). (B) HIF-1α KOs have higher resting levels of B-HAD activity, which is indicative of a greater ability to oxidize fatty acids (**p < 0.01, WT vs. KO). (C) Lower serum lactate levels were seen in HIF-1α KOs following a timed 25-minute run (*p < 0.05, WT vs. KO). As mentioned above, patients with muscle glycolytic deficiencies demonstrate both increased exercise-induced muscle damage and a “second wind”; the latter phenomenon allows them to exercise for extended periods of time at submaximal levels. This is thought to be due to an increase in rates of oxidative ATP production, and a decreased utilization of and need for muscle glycogen (Vissing et al. 1996; Haller and Vissing 2002). To assess whether this is also the case in the HIF-1α KOs, both WT mice and HIF-1α KOs were subjected to endurance tests to assess muscle function. To first determine whether HIF-1α KOs were capable of extended activity during exercise, the animals were given a swimming endurance test. As can be seen in Figure 6A, HIF-1α KOs were capable of significantly longer-duration swimming activity when compared to matched WT controls (p < 0.05). Figure 6 Endurance Capabilities of Untrained Mice (A) HIF-1α KOs have greater endurance in swimming tests as shown by their ability to swim on average more than 45 min longer than WT (*p < 0.05, WT vs. KO). (B) HIF-1α KOs have greater endurance than WT mice in uphill running tests. Although only a 10-min difference is seen between run times, it is to be noted that because of the protocol, this 10 min included two velocity increases (**p < 0.01, WT vs. KO). (C) HIF-1α KOs have less endurance than WT mice in downhill running tests. The same protocol was used as in Figure 4A, except the mice were run on a 10° decline (*p < 0.05, WT vs. KO). (D) RER uphill vs. downhill in WT mice. As would be expected from eccentric exercises relying more heavily on glycolytic fibers, the RER values are higher in mice running downhill than in those running uphill. (E) RER uphill vs. downhill in HIF-1α KOs. Once again, higher RER values are observed for mice running downhill than those running uphill. Further testing was done to determine the parameters of this increased endurance. HIF-1α KOs were run on an enclosed treadmill, with a 5° incline and an initial velocity of 10 m/min, with an increase in velocity every 5 min. In their first runs, HIF-1α KOs again had significantly greater endurance, as shown by their consistently longer run times compared to WT controls (p < 0.01, Figure 6B). As it has been shown that muscle groups and fibers respond differently to eccentric exercise (i.e., downhill running) than to concentric exercise (i.e., uphill running) (Nardone and Schieppati 1988), mice from both genotypes were run on a 10° decline with the same velocity and time parameters as in the uphill runs. Eccentric exercises have been shown to recruit primarily fast-twitch glycolytic fibers for contraction, as opposed to the traditional recruitment of slower, smaller, oxidative motor units in concentric contraction, where animals with an increased capacity for muscle oxidation would be at an advantage (Nardone and Schieppati 1988). Now, the trend from swimming and uphill running tests was reversed, with WT mice able to run for a significantly longer time than HIF-1α KOs (p < 0.05, Figure 6C). Within genotypes, WT mice ran for significantly longer times downhill than uphill (p < 0.01); HIF-1α KOs did the reverse, and ran for significantly shorter times downhill than uphill (p < 0.05). Substrate utilization confirms the shift toward glycolytic fibers in downhill running; both genotypes had higher average respiratory exchange ratio (RER) values when running downhill compared with running uphill (Figure 6D and 6E). PFKD and McArdle's disease demonstrate significant myopathic effects in muscle, including soreness and cramping induced by bouts of exercise. After 1 d of recovery from endurance testing, HIF-1α KOs had increased levels of the MM isoform of creatine kinase in their serum (unpublished data), indicative of skeletal muscle damage. To further investigate this finding, mice were run on a treadmill daily for 4 d. By the second day, the trend for increased endurance in the HIF-1α KOs was absent, and by the final day, HIF-1α KOs were running for significantly shorter times than they had on the first day (p < 0.01, Figure 7A). In addition, a repeated measures ANOVA performed on run times showed that the response of the HIF-1α KOs to the protocol was significantly different than that of the WT mice (p < 0.05). Histological examination of gastrocnemius tissue following 1 d of recovery revealed significantly greater amounts of muscle damage in HIF-1α KO tissue than WT tissue (Figure 7B). Staining of the tissue for proliferating cellular nuclear antigen (PCNA) and counts of positive nuclei (Olive et al. 1995) also revealed more cell division in HIF-1α KOs than in WTs, another indication that HIF-1α KOs had been subject to greater tissue damage (Figure 7C and 7D). Figure 7 Increased Muscle Damage in HIF-1α KOs Following Repeated Exercise (A) WT mice and HIF-1α KOs underwent a 4-d endurance test, in which animals were run to exhaustion on each of four successive days with a minimum of 22 h rest between trials. HIF-1α KOs demonstrated initially greater endurance under the protocol; however, by the second day, their endurance advantage was eliminated, and by the fourth day, HIF-1α KOs were running for a significantly shorter time (**p < 0.01) than on the first day, while WT animals were running for approximately similar times as on the first day. Repeated measures ANOVA revealed that the decrease in performance on each successive day was unique to HIF-1α KOs (p < 0.05). (B) Example of hematoxylin and eosin staining of gastrocnemius muscles after 1 d of recovery by mice after the 4-d endurance test. Evidence of greater damage can be seen in HIF-1α KO muscles compared to WT muscles. (C) Example of PCNA staining of gastrocnemius muscles from exercised mice, demonstrating increased levels of muscle regeneration in HIF-1α KOs. (D) Number of PCNA-positive nuclei per square millimeter in gastrocnemius muscles of WT mice (n = 5) and HIF-1α KOs (n = 7) that ran repeatedly for 4 d. Although HIF-1α KOs have almost twice as many PCNA-positive nuclei per square millimeter, the difference is not significant, because of wild variations in that population. F-test analysis of the data reveals that the variance is much greater in the HIF-1α KO population than the WT population (p < 0.05). As noted above, both PFKD and McArdle's disease are marked by increased resting intramuscular levels of glycogen, a failure of serum lactate to rise during exertion, an exercise-induced “second wind,” and signs of muscle damage following exertion, including elevated levels of creatine kinase in the serum (Tarui et al. 1965; Layzer et al. 1967). In addition, PFKD is characterized by elevated levels of HMPs (Tarui et al. 1965; Layzer et al. 1967; Argov et al. 1987; Grehl et al. 1998) and greater PCr utilization during contraction (Argov et al. 1987; Grehl et al. 1998). We see many of these hallmarks of muscle deficiencies in glycolytic processing in HIF-1α KOs. The effects are not likely due to glucose uptake, as WT and HIF-1α KO intramuscular glucose levels were not different at rest or following stimulation (see Figure 3B), and both types of mice responded similarly to a glucose tolerance test (Figure 8A). Periodic acid–Schiff (PAS) staining of tissue from mice of both genotypes gave further demonstration of increased glycogen levels in resting muscles from HIF-1α KOs (Figure 8B). Figure 8 Glucose Tolerance and Glycogen Storage (A) No significant differences were seen in resting blood glucose levels in HIF-1α KOs or WT mice. Following injection of glucose at a dosage of 2 g/kg, no differences were seen in the maximum levels of blood glucose or the rate of glucose disappearance in either genotype. (B) Representative PAS staining of gastrocnemius muscle from WT mice and HIF-1α KOs. HIF-1α KOs demonstrate darker staining, indicating more stored glycogen. Given the differences in performance observed in the HIF-1α KOs in eccentric and concentric exercise, it is clear that the HIF-1 pathway and hypoxic response have a central role in determining the capacity for work and endurance through regulation of glycolysis. It is also clear that these mice will provide an important model system to investigate the physiology of muscle response during work and oxygen depletion, and may be useful as a model for a group of very debilitating myopathic syndromes in humans. Materials and Methods Mouse strains and crosses. Mice were generated from HIF-1α loxP-flanked allele mouse stocks backcrossed into a C57Bl6/J background. These were crossed into a C57Bl6/J strain containing the MCK/cre transgene. Controls were in all cases littermates that were genotyped as containing only the loxP-flanked HIF-1α allele or only the MCK/cre transgene. No phenotypic differences were seen in the two controls, so they were considered interchangeably as WT control animals. Genotyping and real-time PCR for HIF-1α deletion Mice from the above crosses were genotyped using DNA extracted from tail sections. DNA was then extracted from the gastrocnemius, heart, liver, and uterus of eight 4-mo–old, loxP-flanked HIF-1α–positive and MCK/cre–positive mice. HIF-1α levels were measured by real-time PCR analysis using the Universal PCR Master Mix Kit (Applied Biosystems, Foster City, California, United States) and the ABI Prism 7700 Sequence Detector (Applied Biosystems). Conditions for the PCR were one 10-min incubation at 95 °C (polymerase activation), followed by 40 cycles of 15 s at 95 °C (denaturation) and 1 min at 60 °C (anneal/extend). The degree of excision was calculated by comparing HIF-1α DNA levels to c-Jun DNA levels. HIF-1α real-time PCR primers and probe were as follows: forward primer, HIFLOX501/F 5′-CTATGGAGGCCAGAAGAGGGTAT-3′; reverse primer, HIFLOX574/R 5′-CCCACATCAGGTGGCTCATAA-3′; probe, HIFLOX/P 5′-(6FAM)AGATCCCTTGAAGCTAG(MGBNFQ)-3′. Muscle histology and electron microscopy. Paraffined gastrocnemius sections were deparaffinized and stained with Gill II hematoxylin. Sections were then washed successively in water, a bluing agent, water again, and 95% ethanol, and restained with eosin. Hematoxylin and eosin staining was performed by the University of California at San Diego (UCSD) Cancer Center Histology Resource (La Jolla, California, United States). Imaging was performed on sections mounted on slides using Cytoseal 60 (VWR, West Chester, Pennsylvania, United States). Electron microscopy was performed by standard methods on gastrocnemius muscle. Briefly, fixation was by 25.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Postfix was in 1% osmium tetroxide. The section was stained in 2% uranyl acetate in sodium maleate buffer (pH 5.2), then placed in Epon resin (VWR, West Chester, Pennsylvania, United States), and cured overnight at 60 °C. Fiber typing was performed using the metachromatic dye ATPase method (Ogilvie and Feeback 1990). PAS staining was performed as has been described (Bancroft and Stevens 1996). Assessment of exhaustion. Untrained, age-matched WT mice and HIF-1α KOs (WT, n = 10; KO, n = 14) were run either on an Omnipacer treadmill (Columbus Instruments, Columbus, Ohio, United States) or on an enclosed-chamber modular treadmill (Columbus Instruments) with a 5° incline at an initial velocity of 10 m/min. Velocity was increased by 2 m/min every 5 min during the assessment. Exhaustion was determined to be the point at which the animal would not resume running when provoked through a low-voltage power grid. Gas flow (O2 and CO2) into and out of the enclosed chamber treadmill was monitored using the Paramax O2 sensor and a CO2 sensor (Columbus Instruments) and analyzed using Oxymax software (Columbus Instruments) to determine metabolic parameters. The downhill running assessment (WT, n = 8; KO, n = 6) was carried out in the enclosed-chamber modular treadmill at a 10° decline using the same protocol as above. In the swimming exhaustion assessment, a second group of WT and HIF-1α KOs (n = 8 for each class) was placed in a 30 °C water bath with mild turbulence. Exhaustion was determined to be the point at which the animal experienced three successive periods below the surface of more than 3 s. Isolated stimulation and metabolic analysis. The Achilles tendon was surgically freed from live, anesthetized mice (WT, n = 8; KO, n = 6) and attached to a force transducer to record contractile force. Muscles were electrically stimulated through excitation of the sciatic nerve. Stimulation was in the form of 8–10-V direct titanic contractions using 200-ms trains at 70 Hz with 0.2 ms duration. Initial frequency of tetanic contraction was one every 8 s and was increased every 2 minutes to one every 4 s and one every 3 s, up to the end point of 6 min. Isolated muscles were then immediately harvested and snap-frozen for ATP, lactate, phosphocreatine, and glycogen analyses. Samples were freeze-dried and analyzed by enzymatic assay as has been previously described (Bergmeyer 1974). The unstimulated gastrocnemius muscle from each mouse was used as a resting control. Real-time PCR measurement of gene expression. For basal gene expression levels, total RNA was isolated from gastrocnemius tissue from seven WT and five HIF-1α KOs using RNA-Bee (Tel-Test, Friendswood, Texas, United States). Reverse transcription was performed using the Superscript First Stand Synthesis System for RT-PCR (Invitrogen, Carlsbad, California, United States). Amplification was performed using the ABIPrism 7700 as described above. Reverse transcription real-time PCR primers and probes were as follows. For PGK-1: reverse primer, PGK/R 5′-CAGGACCATTCCAAACAATCTG-3′; forward primer, PGK/F 5′-CTGTGGTACTGAGAGCAGCAAGA-3′; probe, PGK/P 5′-(6∼FAM)TAGCTCGACCCA-CAGCCTCGGCATAT(TAMRA)-(phosphate)-3′. For VEGF-A: reverse primer, VEGF/R 5′-ATCCGCATGATCTGCATGG-3′; forward primer, VEGF/F 5′-AGTCCCATGAAGTGATCAAGTTCA-3; probe, VEGF/P (6∼FAM)TGCCCACGTCAGAGAGCAACATCAC(BHQ∼6∼FAM). For GLUT4: reverse primer, GLUT-4/R 5′-CCCATGCCGACAATGAAGTT-3′; forward primer, GLUT-4/F 5′-TGTGGCCTTCTTTGAGATTGG-3′; probe, GLUT-4/P 5′(6-FAM)TGGCCCCATTCCCTGGTTCATT(BHQ1-Q)-3′. For PFK-M: reverse primer, PFK-M/R 5′-AAGTCGTGCAGATGGTGTTCAG-3′; forward primer, PFK-M/F 5′-GCCACGGTTTCCAATAACGT-3′; probe, PFK-M/P 5′-(6-FAM)CCTGGGTCAGACTTCAGCATCGGG(BHQ1-Q)-3′. For LDH-A: reverse primer, LDH-A/R 5′-ATGCACCCGCCTAAGGTTCTT-3′; forward primer, LDH-A/F 5′-TGCCTACGAGGTGATCAAGCT-3′; probe, LDH-A/P 5′-(6- FAM)TGGCAGACTTGGCTGAGAGCAT(BHQ1-Q)-3′. For changes in gene expression due to exercise, age-matched male mice (WT, n = 5; KO, n = 6) were run on a treadmill at 25 m/min for 30 min. Following the run, mice were euthanized and RNA was isolated and analyzed as described above. Analysis of enzyme activity levels For changes in enzyme activity levels with exercise, mice (WT, n = 5; KO, n = 12) were run on a treadmill using the same protocol as for the gene expression analysis. Tissue was harvested after the run and from resting mice (WT, n = 6; KO, n = 10), and enzymes were extracted and analyzed spectrophotometrically as has been described (Reichmann et al. 1983), with the exception that fructose 1,6-bisphosphate was replaced with fructose 2,6-bisphosphate for stabilization of PFK. Units of activity were normalized to milligrams of total protein using a BCA protein quantification kit (Pierce Biotechnology, Rockford, Illinois, United States). Creatine kinase, serum lactate, hematocrit, and hemoglobin levels. Creatine kinase levels were analyzed from serum from WT mice and HIF-1α KOs 24 h after running-induced exhaustion using a kit from Sigma (St. Louis, Missouri, United States). Creatine kinase isoforms were analyzed enzymatically and then fractionated by gel electrophoresis. Serum lactate levels were analyzed by the UCSD Comparative Neuromuscular Laboratory from blood obtained by cardiac puncture from six WT mice and six HIF-1α KOs following 25 min running time on the treadmill ramp at 25 m/min. Hematocrit and hemoglobin levels were measured from resting mice (WT, n = 6; KO, n = 4) by the UCSD Animal Care Program Diagnostic Laboratory. Glucose tolerance curve. Animals were assigned into either a sham (WT, n = 5; KO, n = 4) or glucose tolerance group (WT, n = 8; KO, n = 8). Experimental animals were injected with 0.3 g/ml glucose in PBS to achieve a dosage of 2 g/kg. Sham animals were injected with an equivalent amount of PBS. Blood was drawn from the tail at time intervals of 0, 15, 30, 60, and 120 min. Samples were then centrifuged to isolate plasma. Plasma blood glucose was quantified using the Infinity Glucose Kit (Sigma). Calcium uptake measurements. Intact individual muscle fibers (WT, n = 6; KO, n = 4) were mechanically dissected from the flexor brevis muscle and loaded with fura-2. Fibers were then stimulated while force generation and Ca2+ release were monitored. Four-day endurance test Endurance was tested by running 24 animals (WT, n = 10; KO, n = 14) on the Omnipacer Treadmill or the enclosed-chamber modular treadmill using the same exhaustion protocol described above. Mice ran according to this protocol every day for 4 d with a minimum of 22 h of rest between trials. Following the fourth trial, mice were given 24 h of rest and then euthanized. Tissue was harvested and stained using hematoxylin-eosin (as described above) and α-PCNA (Pharmingen, San Diego, California, United States) combined with a DAB Kit (Vector Labs, Burlingame, California, United States). Statistical analysis Statistical analyses (unpaired Student's t-test, Mann-Whitney test, ANOVA) were carried out using StatView software (SAS Institute, Cary, North Carolina, United States) or Prism software (GraphPad Software, San Diego, California, United States).  Conflicts of interest. The authors have declared that no conflicts of interest exist. Author contributions. RSJ conceived and designed the experiments. SDM, RAH, MJK, IMO, WM, RPH, and FJG performed the experiments. SDM, RAH, MJK, IMO, MCH, PDW, FJG, and RSJ analyzed the data. CRK and RSJ contributed reagents/materials/analysis tools. SDM and RSJ wrote the paper. Academic Editor: Michael Bate, University of Cambridge Citation: Mason SD, Howlett RA, Kim MJ, Olfert IM, Hogan MC, et al. (2004) Loss of skeletal muscle HIF-1α results in altered exercise endurance. PLoS Biol 2(10): e288. Abbreviations B-HADbeta-hydroxyacyl CoA dehydrogenase CScitrate synthase GLUT4glucose transporter 4 HIF-1αhypoxia-inducible factor 1α HIF-1α KOskeletal-muscle HIF-1α knockout mouse HMPhexose monophosphate LDH-Alactate dehydrogenase-A MCKmuscle creatine kinase PASperiodic acid–Schiff PCNAproliferating cellular nuclear antigen PCrphosphocreatine PFKDphosphofructokinase disease PFK-Mmuscle-specific form of phosphofructokinase PGKphosphoglycerate kinase PKpyruvate kinase RERrespiratory exchange ratio VEGFvascular endothelial growth factor WTwild-type ==== Refs References Argov Z Bank WJ Maris J Leigh JS Chance B Muscle energy metabolism in human phosphofructokinase deficiency as recorded by 31 P nuclear magnetic resonance spectroscopy Ann Neurol 1987 22 46 51 2957951 Bancroft JD Stevens A Theory and practice of histological techniques, 4th ed 1996 New York Churchill Livingstone 766 Bergmeyer HU Methods of enzymatic analysis 1974 New York Academic Press 2302 Bruning JC Michael MD Winnay JN Hayashi T Horsch D A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance Mol Cell 1998 2 559 569 9844629 Cramer T Yamanishi Y Clausen BE Forster I Pawlinski R HIF-1α is essential for myeloid cell-mediated inflammation Cell 2003 112 645 657 12628185 Grehl T Muller K Vorgerd M Tegenthoff M Malin JP Impaired aerobic glycolysis in muscle phosphofructokinase deficiency results in biphasic postexercise phosphocreatine recovery in 31 P magnetic resonance spectroscopy Neuromuscul Disord 1998 8 480 488 9829278 Haller RG Vissing J Spontaneous “second wind” and glucose-induced second “second wind” in McArdle disease: Oxidative mechanisms Arch Neurol 2002 59 1395 1402 12223025 Haller RG Vissing J No spontaneous second wind in muscle phosphofructokinase deficiency Neurology 2004 62 82 86 14718702 Hoppeler H Vogt M Weibel ER Fluck M Response of skeletal muscle mitochondria to hypoxia Exp Physiol 2003 88 109 119 12525860 Layzer RB Rowland LP Ranney HM Muscle phosphofructokinase deficiency Arch Neurol 1967 17 512 523 4228297 Myers J Ashley E Dangerous curves: A perspective on exercise, lactate, and the anaerobic threshold Chest 1997 111 787 795 9118720 Nardone A Schieppati M Shift of activity from slow to fast muscle during voluntary lengthening contractions of the triceps surae muscles in humans J Physiol 1988 395 363 381 3411483 Nelson DL Cox MM Lehninger principles of biochemistry, 3rd ed 2000 New York Worth Publishers 1200 Ogilvie RW Feeback DL A metachromatic dye-ATPase method for the simultaneous identification of skeletal muscle fiber types I, IIA, IIB and IIC Stain Technol 1990 65 231 241 1703671 Olive M Blanco R Rivera R Cinos C Ferrer I Cell death induced by gamma irradiation of developing skeletal muscle J Anat 1995 187 127 132 7591973 Reichmann H Srihari T Pette D Ipsi- and contralateral fibre transformations by cross-reinnervation: A principle of symmetry Pflugers Arch 1983 397 202 208 6878008 Ryan HE Poloni M McNulty W Elson D Gassmann M Hypoxia-inducible factor-1α is a positive factor in solid tumor growth Cancer Res 2000 60 4010 4015 10945599 Sauer B Inducible gene targeting in mice using the Cre/lox system Methods 1998 14 381 392 9608509 Schipani E Ryan HE Didrickson S Kobayashi T Knight M Hypoxia in cartilage: HIF-1α is essential for chondrocyte growth arrest and survival Genes Dev 2001 15 2865 2876 11691837 Seagroves TN Ryan HE Lu H Wouters BG Knapp M Transcription factor HIF-1 is a necessary mediator of the Pasteur effect in mammalian cells Mol Cell Biol 2001 21 3436 3444 11313469 Tarui S Okuno G Ikura Y Tanaka T Suda M Phosphofructokinase deficiency in skeletal muscle: A new type of glycogenosis Biochem Biophys Res Commun 1965 19 517 523 14339001 Vissing J Galbo H Haller RG Paradoxically enhanced glucose production during exercise in humans with blocked glycolysis caused by muscle phosphofructokinase deficiency Neurology 1996 47 766 771 8797477

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2021-01-05 08:21:14

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PLoS Biol. 2004 Oct 24; 2(10):e288

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PLoS Biol

10.1371/journal.pbio.0020288

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020315SynopsisBioengineeringPhysiologyMus (Mouse)A Skeletal Muscle Protein That Regulates Endurance Synopsis10 2004 24 8 2004 24 8 2004 2 10 e315Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Loss of Skeletal Muscle HIF-1α Results in Altered Exercise Endurance ==== Body It's a common runner's complaint. Just when you've built up enough strength and endurance to make running fun, those niggling aches and pains won't go away. Every time your foot hits the ground, a force equal to about twice your weight shoots through your body, eventually chipping away at bones, cartilage, muscles, tendons, ligaments, and joints. For those lucky souls who can take the pounding, the main limitation to running performance stems from muscle fatigue. Now, Randall Johnson and colleagues report that a protein found in skeletal muscle profoundly influences muscle endurance. Running, like any sustained skeletal muscle activity, consumes large quantities of adenosine triphosphate (ATP), a molecule that fuels many essential cell processes. A number of metabolic pathways supply muscle tissue with the ATP needed to power muscle contraction and sustain ongoing exercise. Which pathway predominates depends on factors like speed, duration, and type of activity, as well as on the availability of oxygen, which fluctuates during activity. (For more on muscle cell type and endurance, see the synopsis titled “Gene Targeting Turns Mice Into Long-Distance Runners.”) Say you start a half-hour run with a sprint. Within a few seconds, your body uses up the oxygen in its muscles and has to switch to anaerobic pathways, which metabolize sugars and fats to regenerate ATP. Aerobic pathways operate inside mitochondria, the cell's major power generators. Anaerobic pathways like glycolysis function in the cytoplasm. Hypoxia (the physiological state that occurs when oxygen levels drop below normal levels) governs how ATP is recycled and which energy-producing substrates (for example, glucose or fatty acids) are used; it also generates metabolic by-products, like lactic acid, during strenuous exercise. (Runners know the “lactic acid burn” associated with reduced blood pH.) Glycolysis—the primary source of anaerobic energy in animals—uses glucose, stored as glycogen in muscle cells, to produce ATP. When blood oxygen levels drop, the gene transcription factor hypoxia-inducible factor 1α (HIF-1α) triggers the glycolytic pathway. To understand how HIF-1α regulates skeletal muscle function, Johnson's team generated mice that couldn't express HIF-1α in skeletal muscle. Normal and mutant mice went through exercise routines that included swimming and running on treadmills. After exercise, the normal mice had increased levels of gene transcripts and enzymes involved in glucose transport and metabolism. In the mutant mice, expression of these glycolysis-associated genes and enzymes was significantly lower. The mutants' ATP levels, however, were normal. Without the molecular machinery to engage anaerobic metabolism, their muscles switched to aerobic pathways. The presence of enzymes that respond to reduced ATP levels by increasing mitochondrial ATP production, combined with low levels of lactic acid, confirmed the switch. During endurance tests, the mutants could swim and run uphill (on treadmills tilted upward) longer than the normal mice, but when it came to running downhill, the normal mice prevailed. Downhill running, it turns out, favors glycolytic metabolism; uphill running and swimming favor oxidative pathways, which the mutants were predisposed toward. But their inappropriate use of this pathway came at a cost. By the final day of a four-day exercise routine, the mutants' run time was significantly shorter and their muscles were clearly damaged. The mutants displayed a number of the trademark muscle defects seen in human patients with glycolytic processing disorders. These patients often have reduced lactate levels and elevated levels of mitochondrial enzymes, which apparently can cause a second wind and enhance endurance. This inappropriate use of oxidative pathways—which compensates for the inability to trigger glycolysis—may account for the exercise-induced muscle damage associated with these diseases. These results demonstrate that losing the molecular wherewithal to engage hypoxia response pathways has serious consequences for muscle function during exercise; it can give increased endurance, but at a high price. The mouse model presented here will help researchers explore how muscles normally function in response to low oxygen and how metabolic deficiencies cause debilitating muscle disease.

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PLoS Biol. 2004 Oct 24; 2(10):e315

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PLoS Biol

10.1371/journal.pbio.0020315

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==== Front BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-5-1101530789410.1186/1471-2105-5-110Methodology ArticleTests for finding complex patterns of differential expression in cancers: towards individualized medicine Lyons-Weiler James [email protected] Satish [email protected] Michael J [email protected] Tony E [email protected] Department of Pathology, Center for Biomedical Informatics, and Interdisciplinary Biomedical Graduate Program, University of Pittsburgh, PA 15232 USA2 Clinical Genomics Facility, Center for Pathology Informatics, Benedum Center for Oncology Informatics, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15232 USA3 Departments of Surgery and Human Genetics, University of Pittsburgh Medical School, Pittsburgh, PA 15232 USA4 Mount Sinai School of Medicine, One Gustave Levy Place, Box 1668, East Building, Room 1070C, New York, NY 10029 USA2004 12 8 2004 5 110 110 11 2 2004 12 8 2004 Copyright © 2004 Lyons-Weiler et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Microarray studies in cancer compare expression levels between two or more sample groups on thousands of genes. Data analysis follows a population-level approach (e.g., comparison of sample means) to identify differentially expressed genes. This leads to the discovery of 'population-level' markers, i.e., genes with the expression patterns A > B and B > A. We introduce the PPST test that identifies genes where a significantly large subset of cases exhibit expression values beyond upper and lower thresholds observed in the control samples. Results Interestingly, the test identifies A > B and B < A pattern genes that are missed by population-level approaches, such as the t-test, and many genes that exhibit both significant overexpression and significant underexpression in statistically significantly large subsets of cancer patients (ABA pattern genes). These patterns tend to show distributions that are unique to individual genes, and are aptly visualized in a 'gene expression pattern grid'. The low degree of among-gene correlations in these genes suggests unique underlying genomic pathologies and high degree of unique tumor-specific differential expression. We compare the PPST and the ABA test to the parametric and non-parametric t-test by analyzing two independently published data sets from studies of progression in astrocytoma. Conclusions The PPST test resulted findings similar to the nonparametric t-test with higher self-consistency. These tests and the gene expression pattern grid may be useful for the identification of therapeutic targets and diagnostic or prognostic markers that are present only in subsets of cancer patients, and provide a more complete portrait of differential expression in cancer. ==== Body Background Studies of differential expression of individual genes often find genes that are up-regulated in some tumors, and down-regulated in others. Microarray studies typically seek to identify differentially expressed genes using use fold-change [1], t-tests [2], and models [3-6]. Studies of global gene expression patterns in cancer have focused largely on the identification of novel cancer subtypes via classification [7-13] or the identification of differentially expressed genes [14-18]. Such studies typically use fold-change [1], t-tests [2], and models [3-6]. The methods of analysis for identifying differentially expressed genes in data from microarray experiments vary widely [20-45], but all are focused on the question of whether genes are over-expressed or under-expressed in samples in group A (e.g., tumor, or treatment, or metastastic, or responder) compared to samples in group B (e.g., normal, or control, or quiescient, or nonresponder). These patterns can efficiently be referred to as AB (overexpressed in A) and BA (underexpressed in A) patterns. Typically, researchers use study designs that favor biological replication to maximize the ability to detect reproducibly genes that are differentially expressed in a patient population, at a sacrifice of the ability to detect individual-specific patterns of differential expression with technical replication. Most cancers are diseases with heterogeneous etiologies; moreover, the development of every primary tumor in different individuals is a unique biological event. Thus, the expression levels of genes in the individual patient are also important; some important gene dysregulation may be highly specific to each individual. Statistical methods that average gene expression may hide important expressotypes (expression phenotypes). Current tests that compare mean group expression intensities are not likely to find genes that are in fact significantly dysregulated in only a proportion of the individuals in the case population, unless the magnitude of differential expression is very high in the subset of individuals. Unsupervised clustering can be used to attempt to identify unknown partitions, or subgroups within patients, but clustering is not a well-defined method for finding differentially expressed genes, and, upon discovery of novel groups, researchers are restricted to comparing group means, and cannot identify genes that may be dysregulated in subsets of patients where the combined patterns of dysregulation patterns do not suggest coherent subgroups. Results A remarkable pattern emerges when the PPST test is applied to published cancer data sets, including breast cancer [7], ovarian cancer [16], colon cancer (epithelial-rich normals only [17,47]), lymphoma [18], and lung cancer [19] at the 99th percentile. We find an abundance of AB and BA pattern genes, with roughly the same number of genes called significant under the parametric t-test. We also find large numbers of genes with significant ABA test scores, and some with 'BAB' pattern genes (Table 1). There is a marked tendency in most data sets for more ABA (cancer-normal-cancer) type genes than BAB pattern genes. These patterns are also reflected in 'expression pattern grids' of gene with significant s3(ABA) statistics (Fig. 1). These patterns are reproducible at more stringent levels of α (Table 1). The capability of the PPST test to identify genes that are in fact differentially expressed in only a subset of patients is made evident by a comparison of genes that are found to be significant under the PPST test, but missed by, for example, the t-test (even without Bonferroni-type adjustments). These are listed in Table 2, for the lymphoma data18, and notably include B-cell growth factor 1 (IL4; ABA pattern). Furthermore, 'classic' oncogenes such as cyclin D1 are found by the PPST test in the lung cancer data set [19] are not reported to be significant by the t-test. Cyclin D1 ranks 1009th among significant genes in the colon cancer data set under the t-test but ranks 90th under the PPST test (AB/BA pattern results only). Discussion Our initial results are compelling in that they suggest that we can expect biomarkers of high clinical significance for subsets of patients to be important for distinct subsets of patients. This also suggests that clinical validation of the utility of biomarkers should examine panels of expression biomarkers, not individual biomarkers. Disruption of genomic function via these patterns cannot be studied in the population level biomarker framework for the simple reason that methods that compare, say, group means, will find no difference between the sample groups if the number of case samples found in the two tails are even approximately equal. This is a sensible approach even from within the framework of population-based hypothesis testing, because the PPST test can be expected to be more robust to one or two outliers that might mislead simple parametric tests. Note that a number of genes are 'nearly significant' under the t-test but are strongly significant under the PPST test for the AB/BA patterns (e.g., Table 2). Our re-analysis of two independently generated data sets on astrocytoma progression demonstrates the utility of extending analysis to include a search for genes that are differentially expressed in a subset of patients. Of the tests examined, the parametric t-test showed the least internal consistency, while the PPST exhibited the highest internal consistency in identifying progression markers. Comparison to the non-parametric t-tests demonstrates that PPST is most similar to the nonparameteric t-test, but is more self-consistent. While the ABA test showed the least internal consistency across populations, it also exhibited low overlap with any other test, so the genes reported are unique and tend not to be found by others tests, matching expectations. Our results are consistent with Knudsen's 'two-hit' hypothesis on the genomic etiologies of cancer [49] with some insight into the diversity of genomic pathologies (functional 'hits') that may be relevant in patient populations. Studies of differential gene expression – and its role in the etiology of cancer and its responses to treatment – should seek these types of genes in addition to population-wide biomarkers, because they represent a subset of the genes that are expressed differentially in a significant subset of cancer patients. We recommend a major shift in perspective on the study on gene expression dysregulation away from the study of 'tumor populations' – which do not exist – toward the study of genomic pathologies in individual patients. For example, tumor subtypes are typically characterized by morphological characters, and these classifications may conflict with important expressotype subtypes that do not follow classical morphological tumor classes. Imposition of these subtypes on a study design may interfere with identifying expressotypes that provide high diagnostic, prognostic and theranostic value to the individual – and sets of individuals with similar expressotypes. This view is also consistent with the Hanahan-Weinberg model of oncogenesis [50], which envisions multiple possible mechanistic strategies to the acquisition of characteristics and capabilities of cancers including self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion & metastasis, limitless replicative potential, sustained angiogenesis and evasion of apoptosis. We also expect that individual cancers in different patients will be found to have evolved unique sets of solutions to each of these problems. Current prevailing methods for finding differentially expressed genes such as fold-change and t-tests do not allow for such complexities. Our comparison of the methods (Table 3) highlights the uniqueness of the ABA test. It is an extension of the PPST test; it specifically focuses on genes that are differentially expressed in subsets of patients. This ability is extremely important in search of genes with expression patterns that correlate with drug response. The ABA and the two-tailed t-test are not comparable because the ABA test allows us to find genes that the t-test specifically cannot (genes that are simultaneously overexpressed in some patients while underexpressed in others). Such test will have high variance (leading to a low t-test score) and low mean difference, and will thus not be significant. The PPST and the ABA tests extend our abilities beyond the t-test. Other improvements or even superior alternatives to these tests may be possible. The performance of these tests and all tests described to date for the AB type patterns and now for ABA patterns should be compared using extensive numerical simulations and cross-validation. Developments are needed to determine how best to select a threshold to allow deliberate control of the false positive and false negative error rates. Conclusions The two major conclusions these results suggest are (1) that the most commonly applied tests for identifying differentially expressed genes will miss important genes that are dysregulated in only a fraction of patients, and (2) that important aspects of differential expression may be, to a degree, highly individualistic in most cancers. Some potentially important genes with this form of unusual differential expression (ABA; Table 2) would be missed by methods that compare group means, because the means of the two sample groups would be approximately identical, and the variance in tumors would be high, leading to a large error term. The high internal consistency of PPST compared to the non-parametric t-test and our observation that the PPST test exhibited high consistency with the nonparametric t-test suggests that the PPST test may be of interest to researchers interested in identifying both population-level biomarkers and biomarkers important to a subset of patients. An online implementation of this test, it source code (Java), and that for many other methods of analysis, are accessible online in the Cancer Gene Expression Data Analysis tool . It is hoped that the development and application of more approaches like this will lead to a more complete representation of differential expression, leading to more meaningful and specific hypotheses of dysregulation, and thus a better comprehension of how diverse genomic pathologies contribute to the etiologies of cancers, and thereby facilitate the identification of targets that may lead to individual-specific therapies. Methods We have developed a test we call the Permutation Percentile Separability Test (PPST), which attempts to refute a null hypothesis that is slightly different from A = B, but which is capable of detecting AB, BA, ABA and BAB patterns. Under this test, we are interested in the question "are there are statistically significant number of samples in group A (e.g., tumor) that exhibit expression intensities beyond a particular percentile of the observed expression intensities in group B (e.g., normal)?" and vice versa. By 'statistically significant' we mean that the number of samples that exhibit apparent overexpression (or underexpression) exceeds that expected under the null distribution. To test these hypotheses, we count the number of samples in both groups that are found beyond the nth percentile of the samples in the opposite group. This provides two scores, s1, and s2, for each gene (PPST scores). s1 is the number of samples in group A that are beyond the upper percentile (say, 95th) of group B plus the number of samples in group B that are below the lower 95th percentile of group A. This measure will tend to be large when all samples in both groups are significantly distinct from the alternate group in the same way (comparisons consistent with A > B). It can also be significant when a surprising number of samples in only one group varies from the expression levels in the alternate group. s2 is the number of samples with correspondingly opposite pattern (comparisons consistent with B > A). Sample class label permutations are used to generate an arbitrarily large number of permuted data sets. These scores s1 and s2 are calculated in each permuted data set to produce unique null distributions for each gene. For the sake of convenience of interpretation, we use -s2 when reporting s2 to denote underexpression. Genes with values of s1 beyond the specified acceptable Type 1 error risk (e.g., α = 5%) are determined to be significantly overexpressed in sample group A relative to B. Individuals in sample group A with expression intensity values over the 95th percentile of sample group B for a given gene may be considered overexpressed. Similarly, genes with values of s2 beyond the specified Type 1 risk for s2 are deemed underexpressed in sample group B relative to A. Varying the percentile threshold allows direct control over the false discovery rate. Test for ABA patterns (ABA Test) Genes that exhibit both significant s1 and s2 scores in this comparison may be considered 'ABA pattern genes' (Fig. 1); however, for stronger inference, permutation tests are also used to calculate s3, to determine, for a given gene, the number of samples from one group (A) that can expected to be distributed both in the upper and lower nth percentile tails of the intensity distribution of that gene in the other group (B); i.e., in the ABA (s3) or BAB (s4) pattern. These scores are not redundant to but rather allow for exploration of distribution-wise (upper and lower) false discovery rates. The application of the PPST test to find ABA patterns is called the 'ABA' test. Under the ABA test, differential expression of a gene may be deemed to be significant in both directions at once, i.e., simultaneously significantly over-expressed and under-expressed in a surprising number of patients in the case population. Both the PPST test and the ABA test will perform optimally when the variation in expression intensities in the normal sample population is well characterized. A collection of published microarray data sets we have placed 'on-tap' in the caGEDA (Gene Expression Data Analysis) web application [51] were subjected to the PPST test and the ABA test. To avoid idiosyncracies that can result from the study of extreme values, we ran the tests at a fairly relaxed Type 1 error risk (α = 0.05 in both tails, or α = 0.10 overall). To compare the self-consistency of the parametric t-test, the nonparametric t-test, the PPST test and the ABA test, we re-analyzed two published data sets from independent astrocytoma progression studies [52,53]. Details of these studies are available in the original papers. In brief, Khatua et al. [52] studied global gene expression profiles from 6 early stage and 7 late-stage astrocytoma patients, while van den boom et al. [53] studied global gene expression profiles from 8 early stage and 8 late-stage astrocytoma patients. We calculated the overlap in the gene lists using our online Overlap tool . Abbreviations PPST: permutation percentile separability test. ABA test: a test that can detect genes with both A > B (gene is overexpressed in sample A compared to sample group B) and B < A (gene is overexpressed in sample B compared to sample group A) patterns. s1, s2, s3, s4: measures of the number of samples that exhibit expression intensities beyond a specified percentile in an alternate group; used as scores in the determination of significance under the PPST and ABA tests. Author contributions JLW conceived of the PPST and ABA tests and executed the analyses, SP encoded the methods in caGEDA, TG provided direction, input and scientific motivation for pursuing a test with the capabilities of the PPST and ABA tests, MJB provided, direction, input and coordination of the research. All authors read and approved the final manuscript. Acknowledgments This research was funded by JLW's faculty recruitment funds, provided by MB and Dr. Ronald Herberman using funds from a grant from the Claude Worthington Benedum Foundation . Eleanor Feingold is thanked for her critical read of the manuscript and of the implementation of the PPST and ABA tests. Figures and Tables Figure 1 Conceptual representation of AB, BA, and ABA patterns of differential expression. The colored tails represent the placement of expression values of a given gene in tumors when compared to the distribution of expression values in normal samples. Standard AB and BA patterns are represented by red and black, respectively. Cases in which a surprising number of samples are distributed in both tails for a given gene are represented here as green (BA) and red (AB), respectively, and are painted similarly in Fig. 2 for specific samples. Figure 2 Gene Expression Pattern Grid of genes with significant ABA patterns from a comparison of epithelial-like normal colon tissue (blue samples) to colon cancers (red samples; Alon et al, 1999). We have previously determined that 5 samples in the Alon et al. data set were epithelial-like normal using unsupervised bootstrap cluster analysis and removed the remaining muscle-like normals from this analysis. These, and many other published cancer microarray data sets are 'on-tap' in our GEDA web application. The Gene Expression Pattern Grid, which is generated for any set of differentially expressed genes with the GEDA web application, summarizes the types of differential expression in a way that is related to the PPST test. Color signifies that an individual in one group exhibits an expression value that is significantly different from the expression pattern in the other group (red = overexpression; green = underexpression). Black signifies that an individual exhibits an expression value within the specified upper and lower percentiles in the other group. Tumor samples that fall within the upper and lower 95th %tiles of the distribution of expression values from the normal samples are labeled black, showing which genes for which a sample is are not different from normal. This representation includes information on both the population-level informativeness as well as which individuals appear to exhibit uniquely differentially expressed profiles. Samples within sample group are arranged according to their relative position in a hierarchical agglomerative clustering with pairwise distance = 1-Pearson's correlation coefficient. 'Not expressed' is a hypothesis generated in these graphs when the expression intensity value of that gene for that individual falls in the lower 95th %tile of the entire data set. Expression pattern grids were produced online with the Gene Expression Data Analysis web application . Table 1 Number of Genes with Significant Differences between Tumor and Normal Class in Various Cancer Types under the PPST and ABA tests and the parametric t-test (for comparison) α = 0.1 α = 0.05 Data Set PPST ABA test (ABA/BAB) PPST ABA test (ABA/BAB) parametric tα = 0.05 ** breast7 572 40/5 326 28/49 313 melanoma15 662 60/202 312 38/133 347 colon45 1788 55/153 1558 46/55 1378 ovarian16 3344 253/63 2060 189/22 1813 lymphoma18 2077 286/42 1114 194/30 1370 lung19 614 40/3 506 35/3 389 *A = tumor sample group, B = normal sample group **pooled variance t Table 2 Exemplar Genes Found to the Significant (p < 0.05) under the PPST test in the lymphoma data set[18], but missed under the t-test. Gene PPST Score* Pattern p-value under t-test B-cell growth factor (IL-4) 13 ABA 0.301 CCND1 Cyclin D1 (PRAD1; parathyroid adenomatosis 1) 13 AB 0.087 homeobox protein Cdx2 mRNA 12 AB 0.232 LIF Leukemia inhibitory factor (cholinergic differentiation factor) 10 AB 0.181 tumor susceptiblity protein (TSG101) mRNA -16 BA 0.526 carcinoembryonic antigen -18 BA 0.328 CCND2 Cyclin D2 -18 BA 0.076 VIM Vimentin -18 BA 0.976 *PPST Score = s1 or -s2 (for AB or BA pattern) and s3 (for ABA pattern) Table 3 Summary of the overlap study of the two astrocytoma progression marker data sets. A. Internal consistency of the methods under comparison. k = Khatua et al. data set51; vdb = van den Boom et al. data set52 B. Number of significant genes that overlap between the two data sets in the significant gene list for each method. C. Comparison (% overlap) of methods in the k data set. D. 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For 72 additional studies of gene expression patterns in cancer, see the University of Pittsburgh Cancer Gene Expression Data Link Database Knudsen AG Mutation and cancer: Statistical study of retinoblastoma Proc Natl Acad Sci USA 1971 68 820 823 5279523 Hanahan D Weinberg RA The hallmarks of cancer Cell 2000 100 57 70 10647931 10.1016/S0092-8674(00)81683-9 Patel S Lyons-Weiler J caGEDA: A web application for the integrated analysis of global gene expression patterns in cancer Applied Bioinformatics 2004 3 49 62 16323966 Khatua S Peterson KM Brown KM Lawlor C Santi MR LaFleur B Dressman D Stephan DA MacDonald TJ Overexpression of the EGFR/FKBP12/HIF-2alpha pathway identified in childhood astrocytomas by angiogenesis gene profiling Cancer Res 2003 63 1865 1870 12702575 van den Boom J Wolter M Kuick R Misek DE Youkilis AS Wechsler DS Sommer C Reifenberger G Hanash SM Characterization of gene expression profiles associated with glioma progression using oligonucleotide-based microarray analysis and real-time reverse transcription-polymerase chain reaction Am J Pathol 2003 163 1033 1043 12937144

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==== Front BMC Evol BiolBMC Evolutionary Biology1471-2148BioMed Central London 1471-2148-4-261529870410.1186/1471-2148-4-26Research ArticleSimilarities in transcription factor IIIC subunits that bind to the posterior regions of internal promoters for RNA polymerase III Matsutani Sachiko [email protected] Division of Microbiology, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan2004 9 8 2004 4 26 26 30 3 2004 9 8 2004 Copyright © 2004 Matsutani; licensee BioMed Central Ltd.2004Matsutani; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background In eukaryotes, RNA polymerase III (RNAP III) transcribes the genes for small RNAs like tRNAs, 5S rRNA, and several viral RNAs, and short interspersed repetitive elements (SINEs). The genes for these RNAs and SINEs have internal promoters that consist of two regions. These two regions are called the A and B blocks. The multisubunit transcription factor TFIIIC is required for transcription initiation of RNAP III; in transcription of tRNAs, the B-block binding subunit of TFIIIC recognizes a promoter. Although internal promoter sequences are conserved in eukaryotes, no evidence of homology between the B-block binding subunits of vertebrates and yeasts has been reported previously. Results Here, I reported the results of PSI-BLAST searches using the B-block binding subunits of human and Shizosacchromyces pombe as queries, showing that the same Arabidopsis proteins were hit with low E-values in both searches. Comparison of the convergent iterative alignments obtained by these PSI-BLAST searches revealed that the vertebrate, yeast, and Arabidopsis proteins have similarities in their N-terminal one-third regions. In these regions, there were three domains with conserved sequence similarities, one located in the N-terminal end region. The N-terminal end region of the B-block binding subunit of Saccharomyces cerevisiae is tentatively identified as a HMG box, which is the DNA binding motif. Although I compared the alignment of the N-terminal end regions of the B-block binding subunits, and their homologs, with that of the HMG boxes, it is not clear whether they are related. Conclusion Molecular phylogenetic analyses using the small subunit rRNA and ubiquitous proteins like actin and α-tubulin, show that fungi are more closely related to animals than either is to plants. Interestingly, the results obtained in this study show that, with respect to the B-block binding subunits of TFIIICs, animals appear to be evolutionarily closer to plants than to fungi. ==== Body Background Phylogenetic relationships among animals, fungi, and plants have been a controversial issue. Although fungi traditionally had been considered more closely related to plants than to animals, Whittaker and Margulis [1] classified the fungi as a separate kingdom in their five-kingdom classification: the three major multicellular groups of animals, fungi, and green plants were each given the status of kingdoms derived from different protistan lineages of uncertain affinities. With the determination of the primary structures of homologous macromolecules in various organisms, spanning several kingdoms, molecular phylogenetic techniques resulted in new hypotheses about the relationships among eukaryotes. Small subunit rRNA, and the proteins like actin and α-tubulin, exist ubiquitously and their primary structures are highly conserved. Thus, these sequences have been used to make molecular trees [for examples, [2,3]]. Most of these studies place fungi as more closely related to animals than either is to plants [3-6]. Eukaryotic RNA polymerase III (RNAP III) transcribes a variety of small RNAs like tRNAs, 5S rRNA, and several viral RNAs [7]. Short interspersed repetitive elements (SINEs) are also transcribed by RNAP III [8]. Genes for these small RNAs have internal promoters that consist of two regions called the A and B blocks [9]. These promoter sequences are well-conserved in diverse eukaryotes [9]. Transcription by RNAP III requires the multisubunit transcription factor TFIIIC, which plays an important role in transcription initiation [10]. TFIIIC contains a B-block binding subunit, which recognizes the RNAP III promoter in the transcription of tRNAs and several viral RNAs, orienting its associated subunits along the DNA [11]. TFIIIC that is oriented toward the start site, promotes TFIIIB binding and assists in directing accurate initiation by RNAP III [12,13]. In TFIIIC of Saccharomyces cerevisiae, a subunit of 138 kDa binds to a B-block, and its gene which is called TFC3, has been cloned [14]. The open reading frame for the B-block binding subunit is interrupted by one intron [14]. TFC3 is a single-copy gene and essential for cell viability [14]. In human and rat, the B-block binding subunits of TFIIICs are 243 kDa and 220 kDa respectively [14,15], and there is great similarity between them at the amino acid sequence level [15]. However, B-block binding subunit from mammals has been thought to show no homology to the S. cerevisiae 138 kDa subunit, although all of them bind to similar DNA regions, suggesting a significant degree of evolutionary divergence for RNAP III factors [15,16]. Huang et al. [17] identified several subunits of S. pombe TFIIIC from the S. pombe sequence database by homology searches using the S. cerevisiae TFIIIC subunits as queries; one of these subunits named Sfc3p, is similar to the S. cerevisiae B-block binding subunit. It has been thought that, like the B-block binding subunit from S. cerevisiae, Sfc3p does not share homology with the human B-block binding subunit. On the other hand, Sfc1p, Sfc4p, and Sfc6p, which are other subunits of S. pombe TFIIIC, show homologies not only to S. cerevisiae TFIIIC subunits but also to human TFIIIC subunits [17]. It has been shown that Sfc1p, Sfc3p, Sfc4p, and Sfc6p are associated in vivo, and the isolated Sfc3p complex is active in the in vitro RNAP III-mediated transcription of S. pombe tRNA genes [17]. The lack of clear homology between the yeast and human B-block binding subunits is strange, because transcription of tRNA genes by RNAP III is initiated by the binding of these subunits to the B-block regions, and the internal promoter sequences are highly conserved between vertebrates and yeasts (see above). Interestingly, bacterial tRNA genes also have conserved RNAP III promoters (Fig. 1) [18]. Although these genes are transcribed from the upstream promoters by the bacterial RNA polymerase, RNAP III can transcribe some of them in vitro [18]. Thus, understanding the relationship of the B-block binding subunits from human and yeast TFIIICs is important for understanding the evolution of the RNAP III transcription machinery. Here, I demonstrate that, at the amino acid sequence level, the B-block binding subunits of the vertebrate and yeast TFIIICs do have important homologies. These homologies are found by comparing the B-block binding subunits with Arabidopsis proteins, which appear to be the homologs of both the human and yeast subunits. Figure 1 Sequences of the internal promoters for RNA polymerase III. In the sequences of the Xenopus tRNAMet gene [37], the Xenopus 5S rRNA gene [38], adenovirus VAI [39], human Alu [40], and the E. coli tRNAAsp gene [18], the nucleotides identical to those in the consensus sequence [9] are shown by capital letters. Results PSI-BLAST search using the human B-block binding subunit as a query When the B-block binding subunits of human and S. cerevisiae are aligned using the early Clustal program, they show little sequence similarity [16]. To search for potentially homologous B-block binding regions, first I carried out a PSI-BLAST search where the human subunit was used as a query. PSI-BLAST is known to be useful in finding distantly related proteins [19]. After three iterations, twelve sequences were found with strong similarities to the sequence of human B-block binding subunit. Fig. 2A shows a summary of the result of a PSI-BLAST search, including proteins with alignment scores better than 200. Proteins that showed only local alignment similarities (shorter than about 300 amino acids) were omitted. The human B-block binding subunit was very similar to the rat subunit (75% identity and 84 % positive) as reported by Lagna et al. [15] (Fig. 2A). The nuclear protein of 238 kDa in the mosquito Chironomus tentans (GenBank identification number: 18073910) is structurally similar to the human B-block binding subunit [20]; in BLAST and FASTA searches of non-redundant databases using C. tentans protein as a query, the best hits were the human and rat B-block binding subunits with 28 % identities. A comparable level of similarity has been found also in the Drosophila hypothetical protein, but no other related proteins were identified in the database [20]. Immunoelectron microscopy shows that this mosquito protein is located at sites of transcription, suggesting the role of the protein in transcription initiation [20]. In this study, when a PSI-BLAST search was performed using the human sequence as a query, the Chironomus and Drosophila proteins were hit (23 % and 20 % identities respectively) (Fig. 2A). In addition to these, hypothetical proteins from the mosquito Anopheles gambiae (GI 31212899), Mus musculus (GIs 38087408, and 21595152), Caenorhabditis briggsae (GI 39594187), and Arabidopsis thaliana (GIs 25402830, 9665127, 15218016, 25404859, and 30685327), were also hit with low E-values: E-values were 0, 0, 0, 0, 0, e-169, e-167, e-148, and 5e-87, respectively (Fig. 2A). Interestingly, in Arabidopsis proteins of GIs 25402830, 9665127, and 15218016, both the N- and C-terminal end regions corresponded to those of the human subunit, suggesting that these Arabidopsis proteins are orthologs of the B-block binding subunits (Fig. 2A). Figure 2 Proteins which show homologies to the B-block binding subunits of human and S. pombe, and the Arabidopsis protein GI 25402830. A. The result of a PSI-BLAST search using the human subunit (GI 4753161) as a query. B. The result of a PSI-BLAST search using the S. pombe subunit (GI 19112919) as a query. C. The result of a PSI-BLAST search using the Arabidopsis protein (GI 25402830) as a query. In A-C, only the proteins that had bit scores more than 200 are shown. Bold horizontal lines (black, dark gray, and pale gray lines) are the regions that appeared as convergent iterative alignments. The numbers above the lines are the amino acid positions in the query sequence, and those beneath the lines are the amino acid positions in the hit sequences. When more than two regions were hit in the same sequence, the highest bit score and E-value are shown. Black lines are the alignments that had the best bit scores and E-values. Gray-colored alignments had the worse scores. Dark gray lines represent the alignments with the bit scores more than 200, and the scores of the pale grey alignments are lower than 200. The proteins that had similarities only in short regions (smaller than about 300 amino acids) were omitted, even if the bit scores were more than 200. The case of the Ustilago sequence is exceptional. This result was from the PSI-BLAST which was limited a search of the fungi database. The bold horizontal lines are the regions that appeared as convergent iterative alignments, and below them E-values are shown. PSI-BLAST using the yeast B-block binding subunits as queries The S. cerevisiae B-block binding subunit exhibits 21 % identity and 39 % similarity to the S. pombe Sfc3p protein, and these similarities extend to the overall sequences (Background; [17]). When I performed a PSI-BLAST search using the S. cerevisiae B-block binding subunit as a query, five sequences were hit with E-values better than threshold after three iterations: the Neurospora crassa hypothetical protein (GI 32412546) with 0.0, S. pombe Sfc3p with e-159, the Magnaporthe grisea hypothetical protein (GI 38108450) with e-121, the Saccharomyces bayanus hypothetical protein fragment (GI 10863079) with e-100, and the Arabidopsis hypothetical protein (GI 15218016) with 0.002 (data not shown). It is noteworthy that the Arabidopsis protein of GI 15218016 was hit although the E-value was not so good. This protein was hit also in the PSI-BLAST search using the human B-block binding subunit as a query (Fig. 2A). Next, I performed a PSI-BLAST search using the S. pombe Sfc3p protein as a query. Fig. 2B is a summary of the result. The Magnaporthe grisea and Neurospora crassa hypothetical proteins (GI 38108450 and GI 32412546), were hit with very good E-values (0 and e-168 respectively) (Fig. 2B). The Aspergillus nidulans protein GI 49107000 also was hit with a robust E-value (data not shown). Four hypothetical proteins of Arabidopsis thaliana were hit with E-values worse than those of the two fungi proteins but well above the threshold: GI 15218016 with e-113, GI 25402830 with e-88, GI 9665127 with 2e-88, and GI 25404859 with 7e-70 (Fig. 2B). The Arabidopsis protein GI 15218016 was found also in the result of the PSI-BLAST search using the S. cerevisiae subunit as a query, but in the search with S. pombe Sfc3p it had a much better E-value. Surprisingly, these four Arabidopsis proteins were identical to the proteins that were hit with low E-values in the PSI-BLAST search using the human B-block binding subunit as a query (Fig. 2A). While both the N-terminal half regions and C- terminal end regions were similar between the Arabidopsis proteins and the human subunit, their similarities to S. pombe Sfc3p were only in the N-terminal halves (Figs. 2A and 2B). The B-block binding subunits of rat and human also were hit in this search, but with E-values of 5e-5 and 0.20, respectively (data not shown); the N-terminal 350 amino acid sequences of the rat and human subunits showed similarities to the N-terminal region of S. pombe Sfc3p protein. PSI-BLAST using the B-block binding subunit homolog found in Arabidopsis as a query The four Arabidopsis proteins (GIs 25402830, 9665127, 15218016, and 25404859), were hit with low E-values in both of the PSI-BLAST searches using the human and S. pombe subunits as queries. Thus, I performed a PSI-BLAST search using one of these Arabidopsis proteins (GI 25402830) as a query. Fig. 2C shows a summary of the result. Six hypothetical Arabidopsis proteins were hit with E-values of 0, and three of them were identical to the proteins which were hit in the PSI-BLAST searches using the human and S. pombe subunits as queries. In addition to these, the human and rat subunits were hit with low E-values (e-132 and e-108 respectively). The N-terminal 700 amino acids of the human and rat subunits were most similar to the Arabidopsis proteins, but the short regions of the C-terminal ends also were similar (Fig. 2C). The hypothetical mouse protein (GI 38087408) which was hit in the PSI-BLAST search using the human subunit as a query, also was hit in the PSI-BLAST search with Arabidopsis GI 25402830 (Fig. 2C). The B-block binding subunits of S. pombe and S. cerevisiae were hit with E-values worse than threshold (0.024 and 1.1 respectively) (data not shown). However, it should be noted that these Arabidopsis proteins were hit with E-values better than threshold when a PSI-BLAST search was performed using the S. pombe B-block binding protein as a query. It is interesting that the four Arabidopsis proteins were similar to the B-block binding subunits of vertebrates and yeasts, despite the fact that vertebrate and yeast subunits share no recognizable homology [15-17]. These results seemed to imply that the Arabidopsis proteins of GIs 25402830, 9665127, 15218016, and 25404859 represent a 'missing link' between the vertebrate and yeast B-block binding subunits. Alignment of the B-block binding subunits and their homologs When the primary structures of human and rat B-block binding subunits and their homolog in Drosophila are compared, the most conserved sequences are located within the N-terminal two-thirds of the proteins, while the C-terminal one-third is much less conserved [20]. The Chironomus tentans protein, which probably binds to the B-block in the RNAP III promoter, also has similarities to the human, rat, and Drosophila proteins in the N-terminal region [20]. The results of PSI-BLAST searches here also showed that conserved sequences in the human subunit, and the three Arabidopsis homologs (GIs 25402830, 9665127, and 15218016), mainly are located in the N-terminal halves of the proteins (Fig. 2A). Similarly, conserved sequences in the S. pombe subunit and the three Arabidopsis homologs (GIs 25402830, 9665127, and 15218016), are located in the N-terminal one-third regions of the proteins (Fig. 2C). Thus, I used the Clustal W program [21] to align the N-terminal one-third regions of the proteins of human, rat, mouse, Drosophila, Chironomus, Arabidopsis, and yeasts. The Clustal W alignment of the N-terminal ends of about 60 amino acids corresponded to each of the alignments obtained by the PSI-BLAST searches between the query and hit sequences (Fig. 3A). However, the rest of the Clustal W alignment did not correspond to the alignments obtained by PSI-BLAST (data not shown). Therefore, I compared all of the PSI-BLAST alignments further by eye. Three regions including the N-terminal ends were found to be most conserved between the PSI-BLAST alignments; these three regions were aligned separately by Clustal W (Fig. 3). Previously, Rozenfeld and Thuriaux [22] performed PSI-BLAST using the S. cerevisiae B-block binding subunit as a query. They identified two domains of about 70 amino acids, which were conserved in S. cerevisiae subunit and Arabidopsis thaliana protein of 1808 amino acids [22]: amino acid (aa) positions 333–361 of the S. cerevisiae subunit correspond to positions 334–362 of the A. thaliana protein, and positions 1079–1111 in S. cerevisiae correspond to positions 1716–1748 in A. thaliana. These local homologies are detected also in the human B-block binding subunit by visual inspection (aa positions 367–397 and 1987–2019) (Fig. 2 in [22]). The Arabidopsis protein reported in Rozenfeld and Thuriaux [22] appears to be the GI 9665127 protein hit in the PSI-BLAST searches here, because the lengths of their amino acid sequences are the same and the partial sequences shown in their paper are identical to those in the GI 9665127 protein. The alignment shown in Fig. 3C contains the domain reported by Rozenfeld and Thuriaux [22], and the sequences in the alignment shown in their paper are identical to those in this study. Figure 3 Clustal W alignments of the sequences conserved in the B-block binding subunits and their homologs. The GI numbers of the proteins are shown in parentheses. The amino acid positions of the sequences are shown to the right. The cases of the Clamydomonas sequences are exceptional. These sequences were hit by tblastn of the C. reinhardtii genome sequence using the Arabidopsis protein GI 25402830 as a query (see also Fig. 5). A. Alignment of the N-terminal end sequences. B and C. Alignments of the internal sequences of the proteins. D. Alignment of the C-terminal end sequences. When a PSI-BLAST search was performed using the human B-block binding subunit as a query, it was shown that the C-terminal region also is conserved (Fig. 2A); for examples, Chironomus, Anopheres, Drosophila, and Arabidopsis (GIs, 25402830, 9665127, and 15218016) proteins are hit with E-values of e-29, 3e-7, 6e-54, 2e-20, 2e-39, and 2e-21 respectively (data not shown). These regions contain the domains shown to have sequence similarities [22] (see above). When a PSI-BLAST search was performed using the S. pombe B-block binding subunit as a query, alignments consisting of its C-terminal region and each of the Magnaporthe, Neurospora, and S. cerevisiae sequences were generated, but no homology to the C-terminal regions of the Arabidopsis proteins was suggested (Fig. 2B). However, in agreement with the result of Rozenfeld and Thuriaux [22], when the S. cerevisiae B-block binding subunit was used as a query, the C-terminal region of the Arabidopsis protein (GI 9665127) was hit after four iterations; aa positions 1032–1146 of the S. cerevisiae subunit aligned to positions 1673–1774 of the Arabidopsis protein (GI 9665127) with an E-value of 5e-21 (data not shown). Consequently, the C-terminal sequences of the human, rat, mosquitoes, Drosophila, Arabidopsis and fungi proteins, can be aligned by Clustal W (Fig. 3D). Are the HMG boxes in the B-block binding proteins? The aa positions 1–68 and 1037–1110 of the S. cerevisiae B-block binding subunit had been tentatively identified as HMG boxes [14]. The HMG box is a small eukaryotic DNA binding motif (70–80 amino acids in size) found in many proteins including transcription factors [23]. Interestingly, the regions of HMG boxes predicted for the S. cerevisiae subunit overlap the N- and C-terminal regions conserved in many B-block binding proteins. Therefore, I investigated whether these conserved regions could be homologs of HMG boxes. HMG boxes are diverse, and it is sometimes difficult to determine whether a given protein belongs to the HMG box superfamily [24]. However, in alignments of known HMG boxes a loose consensus sequence can be defined, in which many basic and aromatic residues are conserved [24-26] (see also Fig. 4B). Structures of several HMG boxes have been determined by the NMR spectroscopy and X-ray diffraction [for examples, [25,27,28]] (Fig. 4B); most of them have three α-helices arranged in L-shapes, and tertiary structures stabilized by conserved aromatic residues. It should be noted that in alignments of the N- and C-terminal end regions of B-block binding proteins, several basic and aromatic residues also are conserved (Figs. 3A and 3D). Since HMG boxes contain three helices (see above), I predicted the secondary structures of N- and C-terminal regions of B-block binding proteins using the PSIPRED method [29]. The results are shown in Figs. 4A and 4C. In the N-terminal end regions, all of the sequences (except for Drosophila) were predicted to contain three α-helices with corresponding locations (Fig. 4A). Although the Drosophila N-terminal sequence was predicted to have only two helices, their locations corresponded to the middle and posterior helices in the other sequences (Fig. 4A). In the C-terminal end regions, all of the sequences were predicted to contain one helix in the same corresponding location (Fig. 4C). Fig. 4 shows alignments of B-block binding protein sequences and HMG boxes, arranged to show relationships between them. Numerous gaps were inserted into the sequences by visual inspection in order to relate the positions of basic and aromatic residues and locations of the α-helices (Fig. 4). It appears possible that HMG boxes are present in B-block binding proteins, particularly in their N-terminal regions, however, strong evidence for this relationship is not clear from comparing their amino acid sequences. Figure 4 Alignments of the N- and C- terminal end sequences of the B-block binding proteins, which are compared with the HMG box sequences. Gaps were inserted into the sequences by visual inspection to relate the positions of the conserved basic and aromatic residues and the locations of the α-helices between the alignments. The amino acid residues common among the three alignments are shown in boldface. A. Alignment of the N-terminal end sequences. Above the amino acid sequences, are the secondary structures predicted from them, where H, E, and C represent helix, strand, and coil respectively. B. Alignment of HMG boxes. The identification codes for PDB entries are shown to the left. The regions shown by black characters form α-helices (see ). C. Alignment of the C-terminal ends, with the predicted secondary structures shown above the amino acid sequences. Evolutionary relationships of the B-block binding proteins PSI-BLAST searches presented here provide the evidence that the B-block binding subunits of vertebrates and yeasts are homologous, and that the Arabidopsis proteins can be used to link these subunits. These results suggest that, with respect to the B-block binding subunits of TFIIICs, animals are evolutionarily closer to Arabidopsis than to yeasts. These results are intriguing because phylogenetic analyses using sequences of small subunit rRNA, elongation factor 1, actin, α-tublin, β-tubulin, and heat shock protein 70, show that animals and fungi are most closely related, to the exclusion of the broad diversity of eukaryotic phyla including plants [3-6]. To confirm that the B-block binding subunits of animals and plants are closely related, I decided to carry out an extensive comparison of the sequences from additional plant taxa. However, the PSI-BLAST searches using the B-block binding subunits of human and S. pombe, and Arabidopsis homolog (GI 25402830) as queries, did not significantly hit any of the plant sequences except the Arabidopsis proteins shown in this study. Therefore, I performed a tblastn search of the Viridiplantae database using the Arabidopsis protein GI 25402830 as a query. The Oryza sativa sequence GI 37990182 was hit with the best E-value (8e-85) among Viridiplantae sequences, except the Arabidopsis genes already described (Fig. 5A). In this Oryza sequence, there are four regions that show high similarities to the query Arabidopsis protein (Fig. 5A). This result suggests that the Oryza sequence GI 37990182 encodes a B-block binding protein. Subsequently, I performed the PSI-BLAST searches using three conserved domains from the Oryza sequence of GI 37990182 (Fig. 5B). The query sequence from the 5' end of the Oryza gene had greater similarities to the B-block binding proteins in animals than those in fungi: for examples, the human and rat subunits were hit with E-values of e-26 and 7e-26 respectively, while the S. pombe subunit was hit with an E-value of 0.005, and other fungal sequences were not hit with E-values better than 10 (Fig. 5B). The results for the query sequence from 3' region of the Oryza gene also indicate that the animal and plant proteins are more closely related: for examples, the human and rat subunits were hit with E-values of 0.009 and 0.12 respectively, while the yeast subunits were not hit with E-values better than 10 (Fig. 5B). The result for the query sequence from the middle region of the Oryza gene, however, did not retrieve animal subunits, although the S. cerevisiae subunit was hit with an E-value of 0.21 (Fig. 5B). Figure 5 Protein coding regions in the nucleotide sequences of Oryza sativa and Chlamydomonas reinhardtii with predicted amino acid sequences similar to B-block binding proteins. A. Protein coding regions in the Oryza sativa nucleotide sequence GI 37990182 with amino acid sequences similar to the Arabidopsis protein GI 25402830. Coding regions are shown as boxes and the regions hit by a tblastn search are the filled boxes. E-values are shown below the filled boxes. B. The B-block binding proteins hit by PSI-BLAST searches using the Oryza three amino acid sequences as queries. The corresponding bp positions are indicated above the queries, and hit sequences are below the Oryza queries. Only proteins with E-values better than 10 are shown. C. Protein coding regions in the Chlamydomonas reinhardtii nucleotide sequence with amino acid sequences similar to the Arabidopsis protein GI 25402830. Coding regions are shown as boxes, and the regions hit by a tblastn search are shown as filled boxes. E-values are shown below the filled boxes. D. The B-block binding proteins which were hit by PSI-BLAST searches using the Chlamydomonas amino acid sequences as queries. The corresponding bp positions are indicated above the queries, and hit sequences are below the Chlamydomonas queries. Only proteins with E-values better than 10, are shown. I also searched for a B-block binding protein homolog in the genome of green alga Chlamydomonas reinhardtii. I performed a tblastn search using the Arabidopsis protein GI 25402830 as a query and the C. reinhardtii genome sequence ver2 in the Joint Genome Institute website (see Methods). The Arabidopsis sequences at aa positions of 83–144, 234–262, 698–713, and 1812–1847 showed similarities to sequences corresponding to bp positions of 539027-538842, 527862-537776, 534044-533997, and 526942-526835 in the C. reinhardtii scaffold 16, with E-values of 9.7e-5, 80.2, 80.2, and 9.7e-5 respectively (Fig. 5C). The amino acid sequences deduced from bp positions 539027-538842 and 526942-526835 corresponded to the domains conserved among the B-block binding proteins that were aligned by Clustal W, as shown in Figs. 3B and 3D. These results suggest that the Chlamydomonas B-block binding protein is encoded in these DNA regions. Subsequently, I performed PSI-BLAST searches using the two amino acid sequences of C. reinhardtii with the highest similarities to the Arabidopsis protein (Fig. 5D). The query sequence from bp positions 539231-538197 in C. reinhardtii, showed similarities to the rat B-block binding subunit and its homolog in mouse (E-values of 7.1 and 5.8 respectively) (Fig. 5D). Although these E-values are not robust, no fungal B-block binding proteins was hit with E-values better than 10. The other query sequence encoded in bp positions 527524-526466 of C. reinhardtii also had greater similarities to the B-block binding proteins in animals than to those in fungi: for examples, the human and rat subunits were hit with E-values of 4e-68 and 2e-56 respectively, while no fungal proteins were hit with E-values better than 10 (Fig. 5D). The results of these PSI-BLAST searches with Oryza and Chlamydomonas query sequences indicate that the greater similarity in TFIIIC B-block binding proteins between animals and plants, with yeast as more distant, across the broad diversity of the animal and plant kingdoms. Because yeasts may not be representative of all fungi, it is important to demonstrate that the greater similarity between the animal and plant B-block binding proteins extends beyond the yeast taxa. To this end, I searched for homologs of the B-block binding protein in the basidiomycete genomes. I performed a PSI-BLAST search using the S. pombe subunit as a query, limiting the search to the fungi database. The sequence hit with the best E-value among the basidiomycete sequences, was the Ustilago maydis protein GI 461005911 (Fig. 2B). The Cryptococcus and Coccidiodes sequences were not hit with E-values better than 10. In the Ustilago sequence GI 461005911, three regions show similarities to the S. pombe subunit, particularly the N-terminal one-third and the C-terminal regions as is true of other homologs in fungi (Fig. 2B). Subsequently, a PSI-BLAST search was performed using the human B-block binding subunit as a query of the fungi database. Although the S. cerevisiae B-block binding subunit was hit with an E-value of 5e-7, the Ustilago sequence of GI 46100591 was not hit with an E-value better than 10 (data not shown). Moreover, a PSI-BLAST search performed using the Arabidopsis homolog (GI 25402830) as a query of the fungi database also did not hit the Ustilago sequence of GI 46100591 with an E-value better than 10, although the S. cerevisiae and S. pombe B-block binding subunits were hit with E-values of 0.93 and 9.7 respectively (data not shown). These results indicate that animal and plant B-block binding subunits are more similar to the yeast subunits than to the Ustilago protein GI 46100591. The overall results in this section demonstrate that the greater similarity between the plant and animal B-block binding proteins extends to the green alga protein, and the greater differences in fungi go beyond the yeast taxa. Discussion In this study, I have demonstrated that the B-block binding subunits of TFIIICs in vertebrates are apparently homologous to those of yeasts, by identifying the homologs of each in Arabidopsis. The Arabidopsis proteins (GIs 25402830, 9665127, 15218016, and 25404859), which show strong similarity to B-block binding subunits, are the hypothetical proteins translated conceptually from the nucleotide sequences of the chromosome I [[30]; see also ]. The lengths of the inferred amino acid sequences of three of these Arabidopsis proteins (GIs 25402830, 9665127, and 15218016), are close to those of the amino acid sequences of the vertebrate subunits (Fig. 2). These Arabidopsis proteins probably function as the B-block binding subunits in vivo. B-block binding subunits act on the RNAP III promoters, the sequences of which are conserved in diverse eukaryotes (Background; Fig. 1). Thus, the domains of the subunits that bind to these promoters also should be conserved in vertebrates and yeasts. The N-terminal one-third regions of the human, yeast, and the Arabidopsis homologs found in this study probably associate with the B-block sequences. It is interesting that the HMG boxes predicted in the S. cerevisiae B-block binding subunit [14] overlap with the regions conserved in many of these putative B-block binding subunits. There is a striking degree of similarity in most of the RNAP III transcription machinery in human, S. pombe, and S. cerevisiae; RNAP III, TFIIIA, TFIIIB, and the TFIIIC subunits that interact with the transcription initiation site, are highly conserved in these three organisms [31]. On the other hand, the TFIIIC subunits, which interact with downstream promoter regions including the B-block binding subunits, are more divergent [31]. There is the possibility that substitution rates of the amino acid residues in the B-block binding subunits vary among animals, fungi, and plants, resulting in the high divergence between the human and fungi proteins, and the similarity between the human and plant proteins. Alternatively, evolutionary inferences based on the RNAP III transcription machinery may be different from those of the genes that generally have been used to examine phylogenetic relationships in animals, fungi, and plants. RNAP III transcribes genes encoding tRNA, 5S rRNA, and several viral RNAs, and SINEs (Background). It was reported that molecular phylogenies based on tRNA sequences place plants as the sister group to the animals, although the tRNA data set available at the time was small [32]. Generally, it is thought that 5S rRNA is convenient for intrakingdom phylogenies, but cannot resolve the question of the animal-plant-fungal divergence because of its short length and high divergence [32,33]. It should be noted that more recent investigations of the proteins involved in RNA metabolism, the mRNA capping apparatus, and several key components that regulate the cell cycle, also suggest a close relationship between animals and plants, with fungi as more distant [34-36]. Conclusions Previously, no evidence of homology between the B-block binding subunits of TFIIICs of vertebrates and yeasts has been reported. PSI-BLAST searches presented here provided the evidence that these subunits are homologous, and that the Arabidopsis proteins can be used to link them. These results imply that, with respect to the B-block binding subunits, animals are evolutionarily closer to Arabidopsis than to yeasts. Comparisons of the B-block binding proteins from additional plant taxa showed that the greater similarity between plants and animals extends to the green algae Chlamydomonas. It was also demonstrated that the differences in fungi go beyond the yeast texa, and occur in basidiomycetes. These are interesting because molecular phylogenetic analyses using the small subunit rRNA and ubiquitous proteins, show that fungi are more closely related to animals than either is to plants. Methods To search for similarities between the B-block binding subunits of vertebrates and yeasts, I used the PSI-BLAST program in the NCBI website [19]. PSI-BLAST searches were performed by default: matrix was BLOSUM62; gap costs were Existence 11 and Extension 1; and the E-value of threshold was 0.005. Peptide sequence databases used for the PSI-BLAST searches were all non-redundant GenBank CDS translations, RefSeq proteins, PDB, SwissProt, PIR, and PRF (total 1605642 sequences). The PSI-BLAST was limited searches of the eukaryota databases, when the amino acid sequences from the Oryza and Chlamydomonas coding regions were used as queries. The fungi database was used in a search for homologs of the S. pombe B-block binding subunit in basidiomycetes. PSI-BLAST was run three times for each of the queries. To search for homologs of the Arabidopsis protein GI25402830 in the plant sequences, the tblastn program was used [19]. To search for homologs of the Arabidopsis protein GI25402830 in the Chlamydomonas reinhardtii sequences, the tblastn program at the Joint Genome Institute website , was used. Clustal W in the EMBL-EBI website was used to align the multiple amino acid sequences [21]. Clustal W was performed by default: matrix was Gonnet 250; the penalty for opening a gap was 10; the penalty for extending a gap was 0.05; and gap separation penalty was 8. 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What messenger RNA capping tells us about eukaryotic evolution Nature Reviews Mol Cell Biol 2002 3 619 625 10.1038/nrm880 Oakenfull EA Riou-Khamlichi C Murray JAH Plant D-type cyclins and the control of G1 progression Phil Trans R Soc Lond B Biol Sci 2002 357 749 760 12079670 10.1098/rstb.2002.1085 Hofstetter H Kressman A Birnstiel ML A split promoter for a eucaryotic tRNA gene Cell 1981 24 573 585 7237560 10.1016/0092-8674(81)90348-2 Sakonju S Brown DD Engelke D Ng SY Shastry BS Roeder RG The binding of a transcription factor to deletion mutants of a 5S ribosomal RNA gene Cell 1981 23 665 669 7226225 10.1016/0092-8674(81)90429-3 Rohan RM Ketner G A comprehensive collection of point mutations in the internal promoter of the adenoviral VAI gene J Biol Chem 1987 262 8500 8507 3597384 Perez-Stable C Ayres TM Shen CKJ Distinctive sequence organization and functional programming of an Alu repeat promoter Proc Natl Acad Sci USA 1984 81 5291 5295 6089189

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==== Front BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-5-521528796210.1186/1471-2164-5-52Research ArticleRetrieving sequences of enzymes experimentally characterized but erroneously annotated : the case of the putrescine carbamoyltransferase Naumoff Daniil G [email protected] Ying [email protected] Nicolas [email protected] Bernard [email protected] Institut de Génétique et Microbiologie, CNRS UMR 8621, Université Paris Sud, Bâtiment 409, 91405 Orsay Cedex, France2 Microbiology, Free University of Brussels (VUB) and J.M. Wiame Research Institute 1, ave E. Gryzon, B-1070, Brussels, Belgium3 State Institute for Genetics and Selection of Industrial Microorganisms I-Dorozhny proezd, 1, Moscow 117545, Russia2004 2 8 2004 5 52 52 8 4 2004 2 8 2004 Copyright © 2004 Naumoff et al; licensee BioMed Central Ltd.2004Naumoff et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Annotating genomes remains an hazardous task. Mistakes or gaps in such a complex process may occur when relevant knowledge is ignored, whether lost, forgotten or overlooked. This paper exemplifies an approach which could help to ressucitate such meaningful data. Results We show that a set of closely related sequences which have been annotated as ornithine carbamoyltransferases are actually putrescine carbamoyltransferases. This demonstration is based on the following points : (i) use of enzymatic data which had been overlooked, (ii) rediscovery of a short NH2-terminal sequence allowing to reannotate a wrongly annotated ornithine carbamoyltransferase as a putrescine carbamoyltransferase, (iii) identification of conserved motifs allowing to distinguish unambiguously between the two kinds of carbamoyltransferases, and (iv) comparative study of the gene context of these different sequences. Conclusions We explain why this specific case of misannotation had not yet been described and draw attention to the fact that analogous instances must be rather frequent. We urge to be especially cautious when high sequence similarity is coupled with an apparent lack of biochemical information. Moreover, from the point of view of genome annotation, proteins which have been studied experimentally but are not correlated with sequence data in current databases qualify as "orphans", just as unassigned genomic open reading frames do. The strategy we used in this paper to bridge such gaps in knowledge could work whenever it is possible to collect a body of facts about experimental data, homology, unnoticed sequence data, and accurate informations about gene context. ==== Body Background As a consequence of the deluge of completely sequenced genomes belonging to a large array of species, one can expect to identify many homologues of enzymes which have been previously well studied at the experimental level. This seems to be the general rule and the public sequence databanks (DDBJ/EMBL/GenBank) are now inundated by putative amino acid sequences which have been annotated uniquely by the widely used two-step process : (1) detection of a homologous relationship by a pairwise sequence similarity search at the level of primary structure and (2) inference of functional similarity from this detected homology. However, the opposite might be true. For various reasons one can either miss or misinterpret the actual function of a putative protein when annotating by homology, resulting in a wrong function transfer. Several studies have already emphasized this point (see, for example, [1-3]). On the other hand, beside these now well identified errors which are often due to automatic processes, more subtle mistakes may occur when some of the numerous effects of divergent evolution are overlooked. In particular, one of the insufficiently appreciated problems of functional assignment is that homologous proteins might catalyse different biochemical reactions. Here, we discuss an instance of erroneous annotation (misannotation) in genes of nitrogen metabolism which to our knowledge has not yet been brought up. We explain why this is so and draw attention to the fact that similar cases must actually be rather frequent. Results and Discussion Annotating distant carbamoyltransferases Our group ([4,5]) is presently involved in deciphering the evolutionary relationships between two ubiquitous and essential proteins, aspartate carbamoyltransferase (ATCase, EC 2.1.3.2) which catalyses the first committed step of de novo pyrimidine biosynthesis and ornithine carbamoyltransferase (OTCase, EC 2.1.3.3) which plays a crucial role in both anabolism and catabolism of arginine. In a recent study of the phylogeny of the 245 available OTCases (paper in preparation), we confirmed the existence of two families, OTC alpha and OTC beta, previously proposed on the basis of phylogenetic studies [4]. However, the advent of many new sequences further led to a more complex topology of the distance tree schematized on Fig. 1. It appears now that a significant number of sequences which have been annotated as OTCases are distantly related to either family as outlined on Fig. 1. These sequences, which are forming several clusters emerging in different locations between the root and the two families OTC alpha and OTC beta, have been provisionally annotated as UTC (unknown carbamoyltransferases). Indeed, among these UTC we found two sequences which do not correspond to classical OTCases. Figure 1 A schematic evolutionary tree of carbamoyltransferases. A distance tree of the 245 available ornithine carbamoyltransferases has been obtained using the PhyloTree programme [33]. This tree was rooted by a few paralogous aspartate carbamoyltransferases belonging to either family ATC I or ATC II ([4], [5]). The root is indicated by a closed circle. The two families OTC alpha and OTC beta are schematized as triangles. The homologous carbamoyltransferases which are branching far from these ATC or OTC families are labelled as unknown (UTC) even if some of them have been annotated as ornithine carbamoyltransferases (see text). The few UTC sequences which have been annotated as carbamoyltransferase-like are enclosed in ovals. The clade of reannotated putrescine carbamoyltransferases is framed in red. The YgeW protein encoded by Escherichia coli and its close homologue from Clostridium botulinum are both located on a long branch emerging at the basis of this OTCases tree. YgeW is annotated as belonging to the ATCase/OTCase family (see, for example, the SwissProt knowledgebase [6]). On the branch which is next to the root we find the sequence of a protein which has been reported to be essential for arginine biosynthesis in the anaerobic bacterium Bacteroides fragilis [7]. This protein has been crystallized and characterized as a carbamoyltransferase-like protein since it does not display OTCase activity in vitro [7]. Indeed, several of its residues have been substituted in sites which are viewed as crucial for OTCase activity. Moreover, Dashuang et al. [7] indicated that a similar protein has been found in Xylella fastidiosa. Our phylogenetic data are in agreement with this observation since the protein annotated as OTCases in two strains of X. fastidiosa and its close relative present in two species of the Xanthomonas genus are found to branch close to that of B. fragilis. Therefore, the functional identification of these different UTC is certainly not straightforward and requires further investigations. Furthermore, it occurred to us that, more than thirty years ago, another carbamoyltransferase was discovered by Roon and Barker [8]. A putrescine carbamoyltransferase (PTCase, EC 2.1.3.6) was found to be synthesized by the Gram-positive bacterium Streptococcus (now Enterococcus) faecalis when it was grown on agmatine but not arginine as primary energy source. This PTCase was easily separated from the OTCase synthesized by the same organism grown on arginine [8]. This putrescine carbamoyltransferase had further been studied by V. Stalon's group ([9-12]). Two features of this study – which had apparently been overlooked in recent genome annotations – appear now to be crucial for the interpretation of the data shown on Fig. 1. (1) Wargnies et al. [9] showed that the putrescine carbamoyltransferase of E. faecalis had a weak but unambiguous OTCase activity (7.4% in terms of Vmax, with KM for putrescine and L-ornithine of 0.029 mM and 13.0 mM respectively); (2) A short NH2-terminal sequence (29 residues) was published ten years later [13]. Since the complete genome of E. faecalis has now been sequenced [14], we could identify that one of the three putative ornithine carbamoyltransferases encoded by this genome, the open reading frame EF0732 annotated as ArgF-2 [14], is actually the putrescine carbamoyltransferase previously studied by the group of Stalon. A family of putrescine carbamoyltransferases In a second step, we extended this reannotation of a wrong OTCase as a PTCase to six other sequences encoded by Lactococcus lactis, Streptococcus mutans, Pediococcus pentosaceus, Lactobacillus brevis (and a very close partial sequence in Lactobacillus sakei) , Listeria monocytogenes and Mycoplasma mycoides, respectively. Indeed, these eight sequences, which have been annotated as either ArgF or ArcB (Table 1), (i) share high identity at the level of their amino acid sequence; (ii) they form a monophyletic group (Fig. 1) and (iii) they match the previously published E. faecalis NH2-terminal 29 residues sequence [13]. Moreover, as shown on Fig. 2, these sequences share several specific motifs which are not found in the homologous OTCases. These motifs, especially the five longer ones, are well conserved, even in M. mycoides which is however more distant. When these motifs are used together to query either the UniProt knowledgebase [15] or the nr (non-redundant) database using the PHI-Blast programme, we obtain only these putative PTCase sequences (including that of M. mycoides) to the exclusion of any other carbamoyltransferase. Table 1 The seven ornithine carbamoyltransferases sequences which have been reannotated as putrescine carbamoyltransferases. Species name gene name CDS Swissprot TREMBL Web link Enterococcus faecalis argF-2 EF0732 Q837U7 Lactococcus lactis argF2 LL1700 Q9CEY4 Streptococcus mutans arcB SMU.262 Q8DW19 Pediococcus pentosaceusa - Scaffold 18 Gene 459 - Lactobacillus brevisa - Scaffold 15 Gene 476 - Lactobacillus sakeib argF - Q8RPX3 Listeria monocytogenes arcB LMO0036 Q8YAS7 Mycoplasma mycoides arcB MSC_0703 CAE77322 a : draft genome b : fragment Figure 2 Long motifs specific of putrescine carbamoyltransferases. The amino acid sequence of the open reading frame EF0732 of E. faecalis annotated as ArgF-2 [14] but now reannotated as putrescine carbamoyltransferase is shown. The NH2-terminal 29 residues sequence [13] which helped to this reannotation is indicated by a red bar above the respective residues. The amino acids which are specifically conserved in putrescine carbamoyltransferases and not in ornithine carbamoyltransferases are printed in bold. Inside the long motifs (> 15 residues) found along the whole sequence and numbered as "Motif 1" to Motif 5"are indicated the few substitutions present in the other sequences (lines above the E. faecalis sequence) including those of M. mycoides which is more distant (line in italic below the E. faecalis sequence). The residues forming the respective binding sites of carbamoylphosphate and of either putrescine or ornithine are also in bold and framed. Gene context, another tool for gene reannotation In a third step, the reannotation of this clade of OTCase sequences as PTCases was confirmed by a comparative study of the neighbourhood of their encoding genes present in the four genomes completely sequenced and published (E. faecalis, Lc. lactis, S. mutans and L. monocytogenes). As shown on Fig. 3, the same set of neighbouring genes were present in these four species. We have successively a transcriptional regulator, the reannotated PTCase, an amino acid permease (probably an antiporter), a conserved hypothetical protein and finally the carbamate kinase ArcC-3 (EC 2.7.2.2). In a next step, we found that the so-called conserved hypothetical protein is homologous to the agmatine deiminase (or agmatine iminohydrolase, EC 3.5.3.12) of Bacillus cereus [15]. Fig. 3 further shows that the gene order found in E. faecalis, is completely conserved in Lc. lactis and S. mutans and slightly modified in L. monocytogenes. Figure 3 Conservation of the gene context of putrescine carbamoyltransferases. The open reading frame (ORF) EF0732 of E. faecalis is shown with neighbouring ORFs. Under each numbered ORF (EF0731 to EF0735) is indicated the putative function proposed by the annotators of this complete genome [14]. Below is the reannotation we are proposing. This schematic representation is repeated for the similar genomic regions present in three other completely sequenced genomes, those of Lc. lactis, S. mutans and L. monocytogenes. The homologous sequences are indicated by using the same color : white for transcriptional regulator, yellow for PTCase, pink for permease, green for agmatine deiminase, blue for carbamate kinase, respectively. Thus, these four clusters of genes appear to encode the full set of enzymes which are expected to form the catabolic agmatine deiminase pathway [10]. Agmatine deiminase, PTCase and carbamate kinase were already known to become coinduced by agmatine in E. faecalis when it is used as sole energy source [10], strongly suggesting that these gene clusters are functional operons. In Pseudomonas aeruginosa PAO1, the homologous agmatine deiminase is encoded by the gene aguA belonging to an operon aguBA induced by agmatine and N-carbamoylputrescine ([16,17]) but in this species N-carbamoylputrescine is converted by a N-carbamoylputrescine amidohydrolase (EC 3.5.1.53, the aguB product) into putrescine and CO2 + ammonium rather than into putrescine and carbamoylphosphate. More recently, a similar pathway for polyamine biosynthesis has been identified by homology in higher plants [18]. In the alternative pathway corresponding to the analogous sets of genes shown on Fig. 3, it is thus a PTCase which catalyzes the second step and catabolically converts N-carbamoylputrescine to putrescine and carbamoylphosphate which can further be used to synthesize ATP via carbamate kinase [19]. Furthermore, when we compare the clusters of genes shown in Fig. 3 to those surrounding gene argF or arcB, encoding the catabolic OTCase functioning in the arginine deiminase (ADI) pathway present in many microbial genomes (see [20-22]), we observe a very similar distribution, namely a transcriptional regulator, the arginine deiminase (EC 3.5.3.6), an arginine/ornithine antiporter and a (sometimes two) carbamate kinase. There is thus a very close analogy between the set of genes encoding the enzymes catalyzing the different steps of the agmatine deiminase pathway found in these different Firmicutes and that encoding the enzymes catalyzing the different steps of the arginine deiminase pathway. Conclusions Genome annotation requires both reliable tools for identifying gene function and manual expertise. The frustration due to the high percentage of orphan genes found in all genomes is often compounded with another – more vicious – problem which may occur when a very strong sequence similarity is obscuring the actual functional identity of another kind of orphan. The analysis described in this paper illustrates the difficulty in identifying such a potential source of misannotation and delineates at least two fundamental parameters which must be considered especially when the results appear to be straightforward. First, one must keep in mind that proteins sharing a high level of identical residues may have different functions. A routine step for challenging the functional annotation of any putative coding sequence should be a phylogenetic analysis. Any CDS found to branch far from its homologues in an evolutionary tree, as observed in the case of the carbamoyltransferases (Fig. 1), should be examined with caution before assigning it a putative function. Another example of the usefulness of the phylogenetic approach to correct misannotations can be found in a comparative analysis of ureohydrolases [20]. The second parameter which must be considered is the striking lack of information in the various public databases. For example, in the case studied here (the putrescine carbamoyltransferase EC 2.1.3.6) it is reported that there is no sequence available in various first-rate databases specialized in enzymatic and/or metabolic data such as ENZYME [23], BRENDA [24], KEGG [25], BIOCYC [26], etc...as well as in the Gene Ontology (GO:0050231) Consortium [27]. A significant part of this deficit of information appears to be due to not correlating biochemical data [8-11] previously published and well recorded in BRENDA [24], for example, with the incomplete amino acid sequence which was not taken into account although it had been published by the same group [13] who studied this enzyme. The specific point we would like to stress in this paper reflects a more general gap – which is widely ignored – between the enormous quantity of information buried in the sequence data and the refined knowledge built up over several decades of studies on gene regulation and protein biochemistry (recorded in [23] to [27]). In this respect, experimentally studied proteins not correlated with sequence data also qualify as "orphans". In the present case, such a resulting gap in knowledge could be bridged only because we used the experimental approach detailed in this paper. After being alerted by the unusual topology of the phylogenetic tree (Fig. 1) and the rediscovery of the partial sequence [13], a confirmation of the reannotation as PTCases was obtained when considering their signature (Fig. 2) and the gene context (Fig. 3) which differentiate them clearly from their OTCase homologues. The strategy we used to identify such orphan sequences could work in any other case where it is possible to collect a body of facts about experimental data, homology, unnoticed sequence data, and accurate informations about gene context. Note that we incidentally used such a strategy to annotate the genes encoding a putative agmatine deiminase in the genomes listed in Fig. 3. It is highly probable that this approach can be applied to many similar cases. Therefore, our community should feel encouraged to dig in old lab books, unpublished data buried in doctoral thesis and similar documents, in order to retrieve information crucial for correct genome annotation. Moreover, it becomes urgent to design new approaches in order to efficiently explore what has been called the "bibliome" [28]. This could help to bridge important gaps in knowledge – such as exemplified in this paper- which lead to numerous errors in genome annotation. Accordingly, it would become possible to (re)annotate conserved hypothetical proteins for which there is an apparent lack of information in the various public databases. Methods Collecting sequences Near 450 carbamoyltransferases (ATCases and OTCases) sequences were collected from the public databases SwissProt, TREMBL and TREMBLNEW [15]. To facilitate the management of these data which are continuously growing up with the onset of new completely sequenced genomes, we assemble them in a relational database (available on request). Moreover, in the case of unpublished but completely sequenced genomes, it was often possible to recover bona fide sequences from specific sequencing groups sites (Joint Genome Institute [29] and Sanger Institute [30]) using either BlastP or tBlastN queries. We retained only unpublished sequences aligning along their whole length with bona fide carbamoyltransferases and sharing no less than 30% identity with it, using at least two distantly related seeds. Reconstructing phylogenetic trees Rooted phylogenetic trees were derived from multiple alignements of ATCases and OTCases using two different approaches. (1) New sequences were manually added and aligned to the previously published [4] multiple alignement using the BioEdit sequence alignment editor [31]. These additions were made effortless by introducing each new sequence near its closest partner (the first hit in a routine BlastP check). This processive approach minimized the risk of introducing any bias when adding numerous new sequences. However, the soundness of this manual alignement was routinely checked using automatic programmes (both ClustalX and DARWIN, see below) to verify that we did not miss any conserved motifs. We further ascertained this multiple alignement (especially the introduction of gaps) by using the informations available from the known 3D structures of ATCases and OTCases. Maximum parsimony and distance trees were derived from this alignment using the PROTPARS and NEIGHBOR programmes of the PHYLIP package [32], respectively. This PHYLIP package was further used to derive confidence limits for each node of either parsimony or distance trees using a bootstrap approach (programmes SEQBOOT and CONSENSE). (2) The PhyloTree programme of the DARWIN package [33] allows to build a multiple alignement and to derive a distance tree which is an approximation to maximum likelihood tree since the deduced evolutionary distances are weighted by computing their variance when reconstructing the tree. Authors' contributions NG dug up the "ancient" data on putrescine carbamoyltransferase, contributed his knowledge about carbamoyltransferases and made important additions to the manuscript. YX brought essential informations about the genetics and biochemistry of the enzymes involved in arginine metabolism and their evolution. DGN participated in the collection of new carbamoyltransferase sequences and their manual alignment and identified which sequence of E. faecalis is the putrescine carbamoyltransferase. BL carried out the phylogenetic analyses, the gene context study and drafted the manuscript which was further improved (and approved) by all authors. Acknowledgements We thank the DOE (Department of Energy, USA) and the Wellcome Trust (United Kingdom) for making available unpublished sequences from genomic projects produced by different Sequencing Groups at either the JGI [29] or the Sanger Institute [30]. This work was supported by the Flanders Foundation for Joint and Fundamental Research and by the CNRS (UMR 8621). Daniil Naumoff was supported by a postdoctoral grant from the French Ministère de la Recherche. ==== Refs Brenner SE Errors in genome annotation Trends Genet 1999 15 132 133 10203816 10.1016/S0168-9525(99)01706-0 Gerlt JA Babbitt PC Can sequence determine function? 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EMBO Reports 2002 3 200 203 11882534 10.1093/embo-reports/kvf059 Joint Genome Institute (Department of Energy, USA) Sanger Institute (Wellcome Trust, United Kingdom) Hall TA BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT Nucl Acids Symp Ser 1999 41 95 98 Felsenstein J Inferring phylogenies from protein sequences by parsimony, distance and likelihood methods Methods Enzymol 1996 266 418 27 8743697 10.1016/S0076-6879(96)66026-1 Gonnet GH Hallett MT Korostensky C Bernardin L Darwin v. 2.0: an interpreted computer language for the biosciences Bioinformatics 2000 16 101 103 10842729 10.1093/bioinformatics/16.2.101

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BMC Genomics. 2004 Aug 2; 5:52

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==== Front BMC Mol BiolBMC Molecular Biology1471-2199BioMed Central 1471-2199-5-111529870110.1186/1471-2199-5-11Research ArticleThe p68 and p72 DEAD box RNA helicases interact with HDAC1 and repress transcription in a promoter-specific manner Wilson Brian J [email protected] Gaynor J [email protected] Samantha M [email protected] David J [email protected] Neil D [email protected] Frances V [email protected] Department of Molecular and Cellular Pathology, Ninewells Medical School, University of Dundee, DD1 9SY, UK2 Division of Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dow Street, DD1 5EH, UK3 Molecular Oncology Group – McGill University Health Centre, 687 Pine Avenue West, Montreal, Quebec, H3A 1A1, Canada4 Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, Oxford, OX3 9DU, UK5 Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, H3A 2B4, Canada2004 6 8 2004 5 11 11 11 3 2004 6 8 2004 Copyright © 2004 Wilson et al; licensee BioMed Central Ltd.2004Wilson et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background p68 (Ddx5) and p72 (Ddx17) are highly related members of the DEAD box family and are established RNA helicases. They have been implicated in growth regulation and have been shown to be involved in both pre-mRNA and pre-rRNA processing. More recently, however, these proteins have been reported to act as transcriptional co-activators for estrogen-receptor alpha (ERα). Furthermore these proteins were shown to interact with co-activators p300/CBP and the RNA polymerase II holoenzyme. Taken together these reports suggest a role for p68 and p72 in transcriptional activation. Results In this report we show that p68 and p72 can, in some contexts, act as transcriptional repressors. Targeting of p68 or p72 to constitutive promoters leads to repression of transcription; this repression is promoter-specific. Moreover both p68 and p72 associate with histone deacetylase 1 (HDAC1), a well-established transcriptional repression protein. Conclusions It is therefore clear that p68 and p72 are important transcriptional regulators, functioning as co-activators and/or co-repressors depending on the context of the promoter and the transcriptional complex in which they exist. ==== Body Background The DEAD/H box family of RNA helicases has been demonstrated to be involved in virtually all processes that require manipulation of RNA including transcription, pre-mRNA and pre-rRNA processing, RNA export, ribosome assembly and translation [1]. Although, in vitro, several members of this family have been shown to unwind RNA duplexes, relatively few appear to be true processive helicases and it is clear that, in the cell, many are likely to be involved in unwinding of short base paired regions of RNA or in the modulation of RNA-protein interactions. DNA helicases belong to a superfamily of proteins that is distantly related to DEAD/H box RNA helicases and includes the Werner syndrome protein (WRN) [2] and the Xeroderma pigmentosum XPB and XPD proteins [3], which have well established roles in transcription. Although the functions of DEAD/H box RNA helicases in other cellular processes, such as pre-mRNA processing and translation have been well studied, their role in transcriptional regulation is only now emerging. Examples of DEAD/H box RNA helicases involved in transcription include RNA helicase II (RHII/Gu) and RNA helicase A (RHA/NDHII). RHII/Gu was demonstrated to be a cofactor for c-Jun-activated transcription [4] and was shown to translocate from the nucleolus to the nucleoplasm after UV or anisomycin treatment (which activates JNK signalling). Although RHII/Gu was found to associate with phosphorylated c-Jun in a non-phosphorylated state, this association was observed to increase after anisomycin treatment, implying a stronger interaction when c-Jun is phosphorylated [4]. RHA is a homologue of the Drosophila maleless (MLE) gene product [5] and is thought to be important for gene dosage compensation on the X-chromosome [6]. RHA has been shown to be required for complex formation between the transcriptional co-activator, CREB binding protein (CBP), and RNA polymerase II [7]. Furthermore, different regions of the RNA helicase protein were found to interact with both CBP and RNA polymerase II. The association of RHA with RNA polymerase II was further investigated, and narrowed down to a 50 amino acid stretch, outwith the conserved helicase motifs [7]; this study also showed that RHA could regulate CREB-dependent transcription either through recruitment of Pol II or by ATP-dependent mechanisms. A later study reported that RHA acts as a bridging molecule between the breast tumour specific transcriptional activator, BRCA1 and the RNA polymerase II holoenzyme complex [8]. These reports thus provide clear evidence of a role for RNA helicases as transcription factors. p68 is a prototypic member of the DEAD box family of proteins [9] and an established RNA helicase [10]. The subsequent discovery of p72 [11] and the finding that p68 and p72 share remarkable homology (90% over the central conserved core and 60% and 30% at the N- and C-terminal extensions respectively) suggests that these proteins may form a specific sub-group of DEAD box proteins and may have similar, but perhaps subtly different, functions in the cell, perhaps through interaction with different RNA substrates or proteins. In vitro, both proteins exhibit the RNA-dependent ATPase and RNA helicase activities characteristic of members of the DEAD box family [10-14] and have also been reported to catalyse rearrangement of RNA structure via branch migration [13]. Moreover p68 and p72 can interact with each other, as well as self-associate, and appear to preferentially form heterodimers in cells [15]. This provides the potential for a wide range of functions for p68 and p72 with the possibility of their co-operation in some contexts. More recently p68 and p72 have been shown to be involved in a range of processes in the cell, including pre-mRNA and pre-rRNA processing and alternative splicing [16-18]. p68 and p72 have also been shown to be growth- and developmentally-regulated [19-22] and, furthermore, p68 appears to be over-expressed and poly-ubiquitylated in colorectal tumours [23]. Interestingly p68 has been shown to act as a transcriptional co-activator, specific for the activation function 1 (AF-1) domain of estrogen receptor alpha (ERα) [24]. This interaction was dependent upon phosphorylation of AF-1 at serine118, a residue phosphorylated by mitogen-activated protein kinase (MAPK). Interestingly the RNA helicase function of p68 appeared to be dispensable for this activity as a mutant p68 (Lys144 to Arg) in the ATP binding site (conserved motif I) retained the ability to co-activate ERα, although in a later study RNA binding appeared to be required [25]. p72 was subsequently shown to share this property and both proteins were shown to interact with the activation domain 2 (AD2) of p160 co-activators [25]. Furthermore p68 was also found to interact with the CBP co-activator and RNA polymerase II [26]. More recently p68 has been shown to be recruited to the promoter of the ERα target gene pS2 [27], suggesting a direct involvement in transcriptional regulation. In this report we explore further potential mechanisms through which p68 and p72 may contribute to transcriptional regulation. We find that, in some contexts, p68 and p72 can also act as transcriptional repressors and that these proteins exhibit clear promoter specificity in this function. By directing GAL4-tagged p68/p72 (GAL4 DNA binding domain aa1-147) to promoters containing GAL4 binding sites, we show that both p68 and p72 can repress transcription from the herpes virus thymidine kinase (TK) promoter but not the simian virus 40 promoter/enhancer. Moreover, while p72 can repress transcription from the Adenovirus major late promoter, p68 appears to have no effect, suggesting that these proteins do not behave in an identical way in all contexts. Furthermore we show, by co-immunoprecipitation, that both p68 and p72 interact with histone deacetylase 1 (HDAC1) and that HDAC1/p68/p72 co-elute by gel filtration, indicating that they exist in the same complex in the cell and suggesting a possible mechanism by which these proteins may exert their repressive effect. Results p68 and p72 differentially repress constitutively active promoters/enhancers In order to determine whether, in addition to their reported role as co-activator proteins [25], p68 and p72 helicases have any intrinsic transcriptional activity we generated plasmids to express p68-/p72-GAL4 DNA binding domain (aa1-147) fusion proteins (p68G4 and p72G4). U2OS cells were then co-transfected with p68G4, p72G4, or GAL4-tagged pcDNA3 (pcG4) plasmid as a control, and a Herpes virus thymidine kinase promoter/chloramphenicol acetyl transferase (CAT) reporter plasmid (TK-CAT) bearing 5 copies of the GAL4 binding site, and CAT activity was measured in standard assays. The amount of TK-CAT used had previously been titrated to give a basal level of activity within the linear range of measurement in the CAT assay system used (data not shown). Interestingly, we observed a marked decrease in CAT activity for both p68G4 and p72G4 (Figure 1a). This was confirmed using other cell lines, including MCF-7 and 293 HEK (data not shown). As many transcription factors have been shown to act differentially depending upon promoter context [28], we also tested the transcriptional activity of p68 and p72 with the Adenovirus major late promoter (MLP-CAT), and the SV40 promoter/enhancer (SV40-CAT). In each case we used the same amount of p68G4/p72G4 DNA as previously and appropriate amounts of the reporter constructs that would give similar basal levels of CAT activity using the GAL4 control plasmid (Figure 1a). The amounts of reporter plasmid DNA transfected had again been titrated previously to give similar basal levels, which were within the linear range (data not shown). We also confirmed that p68 and p72 were not limiting under these conditions (see Figure 2c and data not shown). Surprisingly p68 and p72 acted differentially with the MLP promoter, with p72 acting as a repressor, while p68 had no significant effect on CAT activity. Furthermore, neither p68 nor p72 repressed transcription from the simian virus 40 promoter/enhancer (SV40-CAT-Figure 1a). Western blotting, using an antibody against GAL4 confirmed that p68 and p72 were expressed at similar levels in these cells (Figure 1b). Taken together these results reveal not only that p68 and p72 appear to have a previously unknown transcriptional repressive ability, but also that this activity is variable depending on the promoter context. Figure 1 Effect of GAL4-tagged p68 and p72 on transcriptional activity as measured by CAT assays using the TK-CAT, MLP-CAT and SV40-CAT promoter-reporter plasmids, each harbouring 5 copies of a GAL4 binding site fused to the promoter. The pcDNA3-GAL4 expression vector (pcG4) was used as a control. In each case U2OS cells were co-transfected with pcG4 or plasmids expressing GAL4-tagged p68/p72 (p68G4/p72G4) and the appropriate promoter-reporter construct. The amounts of DNA transfected were: pcG4-, p68G4-/p72G4- 1 μg; TK-CAT- 2.5 μg; MLP-CAT- 9 μg; SV40-CAT- 0.5 μg. The amount of DNA used had been previously titrated to achieve appropriate levels of baseline CAT activity. a) Transcriptional repression by p68 and p72 as measured by CAT activity, which is shown as % conversion of 14C-labelled chloramphenicol to acetylated forms. The data represent results from 5 independent assays, which were each performed in triplicate. b) Western blot, using a GAL4-specific antibody, showing expression levels of the pcG4, p68G4 and p72G4 plasmids. Figure 2 Control CAT assays to examine repression of the TK-CAT promoter-reporter in U2OS cells by p68/p72. The amounts of DNA transfected in each assay are indicated below and in all cases the % conversion of 14C-labelled chloramphenicol to acetylated forms is shown as an average of three independent experiments. a) Effect of untagged p68/p72 on TK-CAT transcription. 7.5 μg of control pcDNA3 vector, pcDNA3-p68 (p68) or pcDNA3-p72 (p72) were co-transfected with 2.5 μg of TK-CAT. b) Effect of GAL4-tagged p68 and p72 on transcriptional activity of a TK-CAT promoter-reporter which incorporated a 1.6 kb DNA 'spacer' between the GAL4 binding sites and the promoter (TK-S-CAT). 1 μg of pcDNA3-GAL4 (pcG4) or GAL4-tagged p68/p72 (p68G4/p72G4) were co-transfected with 5 μg of TK-S-CAT. The amount of TK-S-CAT had previously been titrated to achieve an appropriate baseline level of CAT activity. c) Titre of repression of TK-CAT activity by GAL4-tagged p68/p72. 2.5 μg of TK-CAT were co-transfected with different amounts of pcG4 vector, p68G4 and p72G4 as indicated. d) Effect of p300 and CBP on repression of TK-CAT transcription by GAL4-tagged p68/p72. 2.5 μg of TK-CAT were co-transfected with 1 μg of pcG4 vector, p68G4 or p72G4 together with 6.5 μg of either bluescript (as control) or p300/CBP. The repression of transcription is an active process To confirm that the repressive effect observed is not due to an artefact of the assay conditions we initially repeated the experiment with the TK-CAT reporter plasmid, using non-tagged p68 or p72 or the pcDNA3 expression plasmid vector alone (Figure 2a). In this experiment neither p68 nor p72 significantly reduce transcription of the TK-CAT reporter, suggesting that the repression observed with the GAL4-tagged p68/p72 plasmids (Figure 1) is not due to competing out of an essential factor required for TK-CAT transcription but, instead, implies an active mechanism of repression in which the p68/p72 proteins are required to be directed to the TK-CAT promoter via the GAL4 tag. The possibility still remained that p68 and p72 were repressing transcription by direct interference with the transcriptional machinery of the TK-CAT promoter (e.g. perhaps by physically blocking the promoter). To rule out this possibility, we used a similar TK-CAT promoter construct, which had, however, a 1.6 kb DNA 'spacer' between the GAL4 binding sites and the promoter (the inserted DNA is reported to have no effect on transcription [29]). Both GAL4-p68 and -p72 retained the ability to repress transcription, almost to the same degree as previously (Figure 2b). Furthermore, a titration using the GAL4- p68/p72 fusion proteins clearly shows a dose-dependent concentration curve (Figure 2c) as would be expected for active repression. In addition, p68 and p72 have been observed to interact with p300/CBP co-activators [24,26]. Therefore it remained possible that the observed transcriptional repression was due to competition for, or interference with, p300/CBP. If this were the case co-expression of p300/CBP would be expected to relieve repression by p68/p72. As shown in Figure 2d, no such relief of expression was observed. Thus our findings that transcriptional repression by p68/p72 was dose-dependent and not due to steric blocking of the promoter or competition/interference with p300/CBP, suggest that it is an active process. Deletion experiments do not identify a distinct repression domain for p68 or p72 but reveal an activation domain To determine whether specific regions or domains of p68 and p72 act as transcriptional repressors, we performed CAT assays, again using the TK-CAT reporter, but with a range of deletion derivatives of p68/p72 covering the entire coding region. Deletion derivatives encompassing domains between amino acids 1–478 and 1–474 for p68 and p72 respectively repressed transcription in this assay while the C-terminal region for both proteins (aa 477–614 for p68 and 468–650 for p72) acted as a strong transcriptional activator (Figure 3a,3b). Residues 1–478 of p68 and 1–474 of p72 include all the conserved motifs that characterise the DEAD box family of proteins (Figure 3c). Within this conserved core we have shown that there are three domains, which can independently repress transcription (Figure 3c), while the complete region (aa 1–478/474) can repress as well as the full-length respective proteins. Additionally, the finding that the C-terminal regions of p68 and p72 activate transcription is consistent with earlier reports of transcriptional activation by p68/p72 [25,26]. Interestingly, ATPase inactive mutants of p68 and p72 (in which the DEAD motif had been mutated to NEAD) as well as the more recently identified p82 (a derivative of p72 which uses an alternative non-AUG upstream translation initiation codon [30]) also repress transcription (Figure 3b). Thus ATPase and helicase activity appear to be dispensable for transcriptional repression suggesting that this function of p68/p72 may not specifically require RNA unwinding; again this is consistent with reports that helicase activity is not required for co-activation of ERα transcriptional activity [24,25] although another report suggested that p68 helicase activity is required for synergism with the transcriptional co-activators CBP/p300 [26]. Figure 3 Deletion mapping of potential repression/activation domains in a) p68 and b) p72 as observed in CAT assays using the TK-CAT promoter-reporter plasmid. The pcDNA3-GAL4 expression vector (pcG4) and full-length GAL4-tagged p68/p72 were used as controls. All p68/72 deletion derivatives were expressed as GAL4-tagged fusion proteins in pcG4 and included the amino acids indicated. Additional proteins tested in this assay included the ATPase/helicase GAL4-tagged inactive mutants of p68/p72 (p68N/p72N) and the alternative upstream initiation product of the p72 gene (p82). The amounts of DNA used in the transfections were; TK-CAT- 2.5 μg; pcG4 and all p68/p72 constructs- 1 μg. The % conversion of 14C-labelled chloramphenicol to acetylated forms is shown as an average of five independent experiments. c) Diagram correlating the deletion end-points to the position of the motifs conserved in the DEAD box family of proteins. p72 immunoprecipitates a HDAC activity Many studies have implicated histone deacetlyase (HDAC) proteins in active repression of transcription, and many transcriptional repressors have been shown to associate with HDACs [31]. To test whether the observed transcriptional repression by p68 and p72 was dependent on HDAC activity, CAT activity assays were performed as before, using the TK-CAT and MLP-CAT reporter plasmids, in the presence and absence of the HDAC inhibitor trichostatin A (TSA). The relief of repression by TSA was then determined for p68-/p72-GAL4 compared with that observed for the GAL4 vector alone since TSA will also increase basal levels of transcription. No effect was observed with the TK-CAT promoter (data not shown). For the MLP-CAT promoter repression was relieved two-fold in the case of p72 while no effect was seen with p68 (Figure 4a). This is not surprising since p68 does not repress transcription from this promoter (Figure 1). These findings therefore suggest that HDAC activity appears to be important for transcriptional repression of the MLP promoter by p72 and imply that repression of the TK-CAT may employ a different mechanism [32]. Figure 4 The involvement of HDAC activity in transcriptional repression by p68/p72. a) Relief of p68/p72 repression of MLP-CAT transcription by TSA. 1 μg of pcDNA3-GAL4 (pcG4) or GAL4-tagged p68/p72 (p68G4/p72G4) were co-transfected with 9 μg of MLP-CAT and TSA was added 16 hr after transfection, at a final concentration of 300 nM. The values for p68G4 and p72G4 are given relative to the baseline value for the pcG4 vector control, which was set at 1, and represent the average from three experiments. b) Immunoprecipitation/western blotting of myc-tagged p68 and p72 from 293 cells expressing these proteins. Myc-tagged proteins were immunoprecipitated with the anti-myc epitope antibody, 9E10, and western blotted with the same antibody to detect the presence of p68-myc and p72-myc fusion proteins. A myc-tagged pSG5 vector control is included. pSG5, p68 and p72 all refer to myc-tagged versions. H denotes cross reaction with the antibody heavy chain. Molecular weight markers (in kDa) are indicated. Equal amounts of these immunoprecipitated proteins were used in the HDAC activity assay shown in c. c) HDAC activity assay of immunoprecipitated p68 and p72 (see b). HDAC activity in the presence and absence of TSA is shown relative to that of the myc-tagged pSG5 vector control, which was set at 1, and represent the average from three experiments. To examine further the involvement of HDACs in transcriptional repression by p68/p72 we determined whether p68 and/or p72 co-immunoprecipitate a HDAC activity in cells. Plasmids expressing myc-tagged p68 and p72 were expressed in 293 cells (with myc tag plasmid vector as a negative control) and the tagged proteins were immunoprecipitated from cell lysates using the myc epitope antibody, 9E10. Equal amounts of immunoprecipitated protein bound to the antibody, as confirmed by western blotting (Figure 4b), were then used in an HDAC activity assay (Biomol) in the presence and absence of TSA. In this assay (Figure 4c) p72 was found to co-immunoprecipitate a HDAC activity, which is abolished by TSA, while p68 did not. These findings are consistent with p72 interacting with a HDAC and repressing transcription in a HDAC-dependent manner. p68 and p72 associate with HDAC1 in cells Three classes of HDACs have been described. Class I HDACs, which include HDAC1, 2 and 8, are expressed in the nucleus and have been shown to bind several transcription factors and to mediate transcriptional repression [31]. Since HDAC1 is the prototypical member (in mammalian cells) and has been well studied we decided to examine whether p68 and/or p72 associate with HDAC1 in cells. We had previously shown that a large proportion of p68 and p72 co-elute by gel filtration [15]. Therefore we examined fractions from a DNAse/RNAse-treated 293 cell lysate, which had been separated by gel filtration, by western blotting using antibodies against p68, p72 and HDAC1, and showed that a significant proportion of HDAC1 co-elutes with the majority of p72 and a substantial proportion of p68 in the cell in complexes that are of a size consistent with p68/p72 interacting with HDAC1 (Figure 5). Since co-elution does not, in itself, indicate an interaction, we went on to examine whether p68 and or p72 co-immunoprecipitate with HDAC1 from cell lysates. For p68, nuclear extracts were prepared from U2OS cells and HDAC1 was immunoprecipitated with an HDAC1-specific antibody. Immunopreciptiated proteins were then separated by SDS-PAGE and the presence of p68 was detected by western blotting with a p68-specific antibody (Figure 6a). As currently available antibodies against p72 cross react with other nuclear proteins [15] 293 cells were transfected with myc-tagged p72 and interactions between the myc-tagged p72 and HDAC1 were examined. In this case, therefore, nuclear extracts were prepared from transfected cells, HDAC1 was immunoprecipitated as before and associated myc-tagged p72 was detected by western blotting using the myc epitope-specific antibody, 9E10 (Figure 6b). In each case, the presence of HDAC1 in the immunoprecipitate was confirmed by western blotting with the HDAC1-specific antibody (Figure 6a,6b). As an additional control we carried out a reciprocal co-immunoprecipitation experiment in which nuclear extracts from 293 cells transfected with myc-tagged p68/p72 were prepared, the myc-tagged p68/p72 proteins were immunoprecipitated using the myc epitope-specific antibody and associated HDAC1 was detected by western blotting using the HDAC1-specific antibody (Figure 7a). Cells that had not been transfected were used as a control. Additionally, since deletion derivatives encompassing residues 1–478 of p68 and 1–474 of p72 can repress transcription as well as the respective full-length proteins, we examined whether these deletions could co-immunoprecipitate with HDAC1. We therefore transfected 293 cells with GAL4-tagged proteins containing residues 1–478 of p68 and 1–474 of p72, prepared nuclear extracts, immunoprecipitated HDAC1 and western blotted for associated GAL4-tagged p68/p72 with a GAL4-specific antibody. As shown in Figure 7b, these deletion derivatives co-immunoprecipitate efficiently with HDAC1, suggesting that this region of p68/72 interacts with HDAC1. These findings thus indicate that p68 and p72 associate with HDAC1 in cells and that the interaction appears to be mediated by the regions which, in our system, are responsible for the transcriptional repression activity of p68 and p72. Moreover, since the immunoprecipitations were performed with extracts that had been treated with DNase/RNase (see Materials and Methods), the interactions of p68/p72 and HDAC1 are not mediated by nucleic acid. Figure 5 Western blots showing gel filtration elution profiles of p68, p72 and HDAC1. p68, p72 and HDAC1 in the fractions were detected by western blotting using appropriate antibodies. Note that the antibody raised against p72 also recognises p82 and cross-reacts with NFAR-2 [15]. All lysates had been treated with DNase and RNase prior to gel filtration. The void volume and elution position of the Pharmacia FPLC size markers are indicated, as are molecular weight markers (in kDa). Figure 6 Co-immunoprecipitation of a) p68 and b) p72 with HDAC1. a) HDAC1 was immunoprecipitated from U2OS nuclear extracts using an HDAC1-specific antibody. Immunoprecipitated proteins were separated by SDS-PAGE and the presence of HDAC1 and associated p68 was detected by western blotting with p68- and HDAC1-specific antibodies. b) HDAC1 was immunoprecipitated from nuclear extracts of 293 cells expressing myc-tagged p72 using an HDAC1-specific antibody. Immunoprecipitated proteins were separated by SDS-PAGE and the presence of HDAC1 and associated myc-tagged p72 was detected by western blotting with HDAC1- and myc epitope- specific antibodies. In both experiments a control immunoprecipitation (IP) was performed using an irrelevant rabbit IgG. An aliquot of nuclear extract (NE) was also included in the western blots (west.) as an additional control. H denotes cross reaction with the antibody heavy chain. Molecular weight markers (in kDa) are indicated. Figure 7 a) Reciprocal co-immunoprecipitation of p68 and p72 with HDAC1. Myc-tagged p68 and p72 were immunoprecipitated from nuclear extracts of 293 cells expressing these proteins using a myc epitope-specific antibody and associated HDAC1 was detected by western blotting with an HDAC1-specific antibody. 293 cells, which had not been transfected, were used as control. b) Co-immunopreciptiation of p68/p72 deletion derivatives with HDAC1. HDAC1 was immunoprecipitated from nuclear extracts of 293 cells expressing GAL4-tagged p68 and p72 deletion derivatives, which encompass residues 1–478 and 1–474 of p68 and p72 respectively. Associated p68/p72 were detected by western blotting with a GAL4-specific antibody. 293 cells, which had not been transfected, were used as control. NE-nuclear extract, IP-immunopreciptiation. Molecular weight markers (in kDa) are indicated. Discussion We have shown that the highly related DEAD box RNA helicases p68 and p72 act as repressors of transcription in a promoter-context manner. When targeted to the TK-CAT promoter-reporter construct they both strongly repress transcription (Figure 1). Furthermore, this transcriptional repression does not appear to be due to either squelching or physical blocking of the transcription apparatus (Figure 2), implying an active transcriptional mechanism. Moreover repression of TK-CAT was observed in several cell lines (U2OS, 293, MCF-7) suggesting that it is not cell line dependent. In order to determine whether this repression activity exhibited any promoter specificity we also tested the ability of p68 and p72 to repress transcription of other constitutively active promoter-reporter constructs with high basal levels of transcription, namely MLP-CAT and SV40-CAT. Interestingly MLP-CAT revealed a difference in the ability of p68 and p72 to repress transcription, with p72 strongly repressing transcription of this promoter-reporter and p68 having no effect (Figure 1). This observation suggests that, although highly homologous (70% overall identity at the amino acid level [11]) p68 and p72 proteins may act differently in some contexts, perhaps through the association with different protein partners. Neither p68 nor p72 repressed transcription of SV40-CAT (Figure 1) suggesting that the repression by p68 and p72 is promoter context-dependent, an observation that has been reported for other transcription factors [33]. These findings thus are consistent with the observed repression activity of p68/p72 being an active process. Interestingly, in this context, another DEAD box protein DP103 (Ddx20) has been found to act as a co-repressor of the Ets repressor METS/PE1 [34]. Using a series of deletion derivatives of p68 and p72 we identified three domains, within the core conserved among the DEAD box family of proteins, which can independently repress transcription (Figure 3c). Moreover regions encompassing residues 1–478 of p68 and 1–474 of p72, which contain the complete conserved core (Figure 3c), can repress transcription as well as the respective full-length proteins (Figure 3a,3b). In contrast, the C-terminal extension of both proteins acts as a transcriptional activator in this context (Figure 3a,5) consistent with earlier reports of these proteins acting as transcriptional co-activators [24,25]. Thus, using this system, we have shown that there are separable transcriptional repression and activation domains within p68 and p72. Since HDAC proteins have been extensively implicated in the repression of transcription, it was important to examine whether these proteins are likely to play a role in transcriptional repression by p68 and/or p72. Firstly, the ability of the HDAC inhibitor, TSA, to relieve repression was examined. No effect was observed on repression of TK-CAT (data not shown) implying the involvement of a HDAC-independent mechanism in repression of the TK promoter. In contrast, repression of MLP-CAT by p72 was relieved two-fold compared with the vector control (Figure 4a) suggesting the involvement of HDACs in this process. (Since p68 did not repress MLP-CAT transcription, the lack of effect by TSA is not surprising.) Supporting these data, p72 was found to co-immunoprecipitate a HDAC activity which was abolished by the addition of TSA, while p68 did not (Figure 4b). We chose to investigate whether p68 and/or p72 associate with HDAC1, since it is a well-studied example of Class I HDACs. Both p68 and p72 co-immunoprecipitate with HDAC1 (Figures 6 and 7); furthermore HDAC1, p68 and p72 co-elute in similar sized complexes by gel-filtration, which are of an appropriate size (Figure 5) supporting the idea of interactions between p68/p72 and HDAC1 in cells. Moreover, the finding that these proteins co-immunoprecipitate and co-elute from extracts which had been treated with DNase/RNase suggest that these represent protein-protein interactions rather than merely interactions via nucleic acid. While an interaction between HDAC1 and p68 is not supported by the results of the HDAC assay, or the TSA experiment, it is possible that, in some instances, p68 does recruit HDAC1 and that this mechanism is not being triggered in the MLP-CAT assay or HDAC assay. Alternatively, it is possible that the observed co-immunoprecipitation of p68 and HDAC1 is occurring through the interaction between p68 and p72 [15] or that HDAC1 associated with p68 may have other, possibly non-transcriptional, roles [35,36]. However, the data are entirely consistent with p72 recruiting HDAC1 to achieve active repression of transcription. Future investigations should also reveal whether the differential ability of p68 and p72 to recruit active HDAC proteins is responsible for the difference observed upon the MLP promoter. Our attempts at correlating the different repressive functions of p68 and p72 to specific domains of the respective proteins, using deletion derivatives, were unsuccessful, as the equivalent regions of both either caused transcriptional repression or activation. While this might suggest that both helicases repress transcription in the same manner, it is more likely that the HDAC recruitment by p72 may be an additional mechanism of repression, used at specific promoters. We also cannot rule out the recruitment of other repression complexes at this stage. Our findings that p68 and p72 differ in their ability to repress the MLP promoter and to recruit HDAC activity suggest that, at least in some contexts, p68 and p72 repress transcription by different mechanisms. In summary, it is clear that p68 and p72 act to repress transcription in a differential manner dependent upon promoter context. It will be important to determine which endogenous promoters are subject to repression by p68/p72 in a physiological context. However, until the signal transduction pathways, which target these proteins to the appropriate promoters, are elucidated it will be necessary to use a targeting system (such as GAL4) to undertake a molecular analysis of transcriptional repression by p68 and p72. Since it is now clear that p68/p72 can act both to activate and repress transcription future work will involve dissection of the transcriptional activation/repression complexes in which p68 and p72 are involved, as well as characterisation of the molecular 'switch' which determines whether these proteins will be part of transcriptional activation or repression complexes. Conclusions We have shown that the highly related RNA helicases p68 and p72 can repress transcription in a promoter context-dependent manner. Both proteins associate with HDAC1, a well-established transcriptional repressor protein. Strikingly, however, p68 and p72 behave differently in their ability to repress transcription from different promoters and in their ability to recruit HDAC activity suggesting that they may, at least in some contexts, repress transcription by different mechanisms. Methods Cell culture U2OS human osteosarcoma cells and 293 human embryo kidney cells were maintained at 5% CO2 at 37°C in DMEM with 10% FBS, 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin (all supplied by Invitrogen). Plasmids A pcDNA3-GAL4 expression plasmid was used to express full-length and deletion derivatives of p68/p72/p82 tagged at the N-terminus with the DNA binding domain (amino acids 1–147) of GAL4 [37]. The majority of deletion derivatives were created by PCR and inserted, as BamHI/EcoRI fragments, in frame with the GAL4 tag. The CAT reporter constructs have been described previously: TK-CAT [38], TK-Spacer-CAT [29], MLP-CAT and SV40-CAT [39]. The MLP-CAT and SV40-CAT plasmids were a kind gift from Douglas Dean (Washington University, St Louis, USA), and the TK-spacer-CAT plasmid was kindly provided by Dr. Alain Nepveu (McGill University, Canada). Untagged p300, CBP, p68, p72 were expressed from pcDNA3. A myc-tagged derivative of pSG5 (Stratagene) [15] was used to express the myc-tagged p68/p72 or the myc epitope alone as negative control. Antibodies p68: The antibodies used were the mouse monoclonal antibody PAb 204 and the rabbit polyclonal antibody 2906, generated against the C-terminal 15 amino acids of p68 [19]. PAb 204 was originally generated against the SV40 large T antigen but it cross-reacts with p68 [9]. It is specific for p68 in cells that are not infected or transformed by SV40. p72: A rabbit anti-peptide polyclonal antibody generated against amino acids 624 to 638 [15] was used to detect p72/p82 in fractions from gel filtration. Myc epitope: A mouse monoclonal antibody (9E10) was used to detect proteins tagged with the myc epitope (MRQKLISEEDL). HDAC1: A rabbit polyclonal antibody (Oncogene Research Products) was used both for immunoprecipitation and western blotting. GAL4- a mouse monoclonal antibody (Santa Cruz) was used to detect GAL4-tagged proteins. A negative control rabbit IgG antibody for immunoprecipitation was obtained from R&D systems and appropriate anti-mouse and anti-rabbit secondary antibodies were obtained from DAKO. Transient transfections and chloramphenicol acetyl-transferase (CAT) assays 3×105 U2OS cells were seeded for transfections, which were performed as previously described [38]. Cells were co-transfected with the appropriate CAT reporter construct and GAL4-tagged p68/p72, and in each case the DNA was made up to a total of 10 μg with Bluescript DNA (Stratagene). CAT activity was determined 48 hr after transfection using 100 μg of total protein from cleared whole cell lysates. Typically each experiment was performed in triplicate within each assay, and each assay was repeated 3 to 5 times. Where applicable, the HDAC inhibitor Trichostatin A (TSA) (Upstate Biotechnology) was added, at a final concentration of 300 nM, 16 hr post transfection. Nuclear extract preparation and co-immunoprecipitation Nuclear extracts were prepared from U2OS and 293 cells essentially as described in [40] except that the NaCl concentration was reduced to 330 mM NaCl, then diluted to 150 mM NaCl (after nuclear lysis) and treated with RNAse/DNAse. The extract was pre-cleared in buffer D [20 mM Hepes (pH7.9), 150 mM NaCl, 0.5 mM DTT, 20% (v/v) glycerol, 10 mM NaF and protease inhibitor cocktail (Roche)] with protein G sepharose beads for 30 min. at 4°C. Immunoprecipitations were carried out in the presence of appropriate antibodies and protein G sepharose beads for one hour at 4°C. After washing in buffer D plus 0.1% Igepal (Sigma), immunoprecipitated proteins were separated by SDS-PAGE and western blotted using standard conditions and appropriate primary and secondary antibodies. Immunoreactive proteins were detected using the ECL method (Amersham). Gel filtration 293 cell lysates were prepared in RIPA buffer [50 mM Tris-HCl (pH8.0), 150 mM NaCl, 1% Igepal, 0.1% SDS, 1% Na deoxycholate, 1 mM EDTA and protease inhibitor cocktail (Roche)]. After treatment with RNase/DNase, lysates were fractionated on a Pharmacia Superose 6 HR column in 50 mM Tris-HCl (pH7.5), 150 mM NaCl, 10% glycerol and 1 mM benzamidine, using a Pharmacia AKTA FPLC system. 0.5 ml fractions were collected and alternate fractions were analysed by western blotting. Molecular weight standards from Pharmacia were used to calibrate the column. HDAC assay 293 cells were transfected with either pSG5-myc vector, pSG5-p68-myc, or pSG5-p72-myc. Cells were lysed in buffer B 48 hr after transfection. The lysate was then diluted in buffer A and myc-tagged proteins immunoprecipitated with 9E10 (myc epitope) antibody as described above. The HDAC assay employs Fleur de lys substrate, which contains an acetylated lysine side chain (Biomol) and was performed according to manufacturers instructions. Antibody-bound beads were washed in HDAC assay buffer prior to being added to the 96-well plate, to remove immunoprecipitation buffer. Reactions were incubated for 30 min at 37°C with or without the addition of 1 μM TSA. Samples were excited at 360 nm and emitted at 460 nm and were read in a fluorometer. Typically each assay was performed 3 times. Authors' contributions BJW carried out the transcriptional and HDAC activity assays, the deletion mapping and the gel filtration, GJB and SMN carried out the co-immunoprecipitation experiments, DJG carried out the original experiments showing that p68 and p72 could act as transcriptional repressors, and NDP and FFP participated in the design of this study. FFP co-ordinated the study and prepared the final draft of the manuscript. All authors read and approved the manuscript. Acknowledgements We thank David Lane and David Meek for helpful discussions. 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==== Front BMC Plant BiolBMC Plant Biology1471-2229BioMed Central London 1471-2229-4-131528578510.1186/1471-2229-4-13Research ArticleAssessment of genetic diversity in Trigonella foenum-graecum and Trigonella caerulea using ISSR and RAPD markers Dangi Rakhee S [email protected] Meena D [email protected] Lal B [email protected] Prabhakar K [email protected] Vidya S [email protected] Plant Molecular Biology Group, Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008. Maharashtra State. India2 Taxonomy and Biodiversity Division, National Botanical Research Institute, India. Lucknow 2260012004 30 7 2004 4 13 13 12 1 2004 30 7 2004 Copyright © 2004 Dangi et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Various species of genus Trigonella are important from medical and culinary aspect. Among these, Trigonella foenum-graecum is commonly grown as a vegetable. This anti-diabetic herb can lower blood glucose and cholesterol levels. Another species, Trigonella caerulea is used as food in the form of young seedlings. This herb is also used in cheese making. However, little is known about the genetic variation present in these species. In this report we describe the use of ISSR and RAPD markers to study genetic diversity in both, Trigonella foenum-graecum and Trigonella caerulea. Results Seventeen accessions of Trigonella foenum-graecum and nine accessions of Trigonella caerulea representing various countries were analyzed using ISSR and RAPD markers. Genetic diversity parameters (average number of alleles per polymorphic locus, percent polymorphism, average heterozygosity and marker index) were calculated for ISSR, RAPD and ISSR+RAPD approaches in both the species. Dendrograms were constructed using UPGMA algorithm based on the similarity index values for both Trigonella foenum-graecum and Trigonella caerulea. The UPGMA analysis showed that plants from different geographical regions were distributed in different groups in both the species. In Trigonella foenum-graecum accessions from Pakistan and Afghanistan were grouped together in one cluster but accessions from India and Nepal were grouped together in another cluster. However, in both the species accessions from Turkey did not group together and fell in different clusters. Conclusions Based on genetic similarity indices, higher diversity was observed in Trigonella caerulea as compared to Trigonella foenum-graecum. The genetic similarity matrices generated by ISSR and RAPD markers in both species were highly correlated (r = 0.78 at p = 0.001 for Trigonella foenum-graecum and r = 0.98 at p = 0.001 for Trigonella caerulea) indicating congruence between these two systems. Implications of these observations in the analysis of genetic diversity and in supporting the possible Center of Origin and/or Diversity for Trigonella are discussed. ==== Body Background The family Fabaceae includes many crops useful for food, forage, fiber, wood and ornamental purposes. In this family, a few legumes such as chickpea, soybean, fababean, fenugreek, lentil, pea etc. are consumed as grain legumes. The grain legumes are plants used as food in the form of unripe pods, mature seeds or immature dry seeds, directly or indirectly [1]. The grain legumes not only provide variety to human diet but they also supply dietary proteins for vegetarian populations that lack animal and fish protein in their diet. Considering the growing problem of malnutrition, use of legume species as high-protein food is very important. Moreover, legumes are also capable of symbiotic nitrogen fixation enriching the soil condition suitable for a crop following the legume crop [2]. The genus Trigonella is one of the largest genera of the tribe Trifoliatae in the family Fabaceae and sub-family Papilionaceae [3]. Among Trigonella species, Trigonella foenum-graecum (commonly known as fenugreek) is a flowering annual, with autogamous white flowers occasionally visited by insects. Indigenous to countries on the eastern shores of Mediterranean, fenugreek is widely cultivated in India, Egypt, Ethiopia, Morocco and occasionally in England [4]. Trigonella foenum-graecum is extensively grown in the tropical and subtropical regions of India. Different parts of the plant such as leaves and seeds are consumed in India. It is also used for medicinal purpose. According to ancient medicinal system, the Ayurveda, it is a herbal drugs that is bitter or pungent in taste. It is effective against anorexia and is a gastric stimulant [5]. Fenugreek is becoming popular around the world with its extract used to flavor cheese in Switzerland, artificial maple syrup and bitter-run in Germany, roasted seeds as coffee-substitute in Africa, seed powder mixed with flour as fortification to make flat-bread in Egypt, as an anti-diabetic herb in Israel, whole seed and dried plant used as insect and pest repellent in grain storage, and oil used in perfumery in France [6]. Research reports in recent years have indicated that fenugreek can be a remedy to diabetes by lowering blood sugar and cholesterol levels [7]. T. caerulae, from the same family and commonly known as the Blue fenugreek, on the other hand, is a less commonly grown herb. This flowering annual with autogamous blue flower is found in Alps and in the mountains of eastern and south eastern Europe. Terminal leaves are mainly used for cooking while young seedlings are eaten with oil and salt. Dried and powdered leaves as well as flowers are used for flavoring and coloring bread, cheese, etc in China and Germany. They are also used as a condiment in soups and potato dishes and a decoction of leaves is used as aromatic tea [8]. Grain legumes like T. foenum-graecum and T. caerulea although important in food and medicine are rarely grown outside their native habitat. Across the world only known and well-defined cultivars are grown in specific areas. Gene banks also harbor scanty germplasm collection of Trigonella species [9]. The neglected and the under-use status of these locally important crops indicates a risk of disappearance of important plant material developed over thousands of years of cultivation. One of the important factors restricting their large-scale production and development of better varieties is that very little information is available about their genetic diversity, inter and intraspecific variability and genetic relationship among these species. Therefore, attempts to analyze possible untapped genetic diversity become extremely essential for breeding and crop improvement. The present study was undertaken with the objective of analyzing genetic diversity in various accessions of T. foenum-graecum and T. caerulea representing various countries where they are grown using molecular marker technology. Results Assessment of genetic diversity in T. foenum-graecum and T. caerulea using ISSR and RAPD markers A set of 100 ISSR primers was used for initial screening of 7 accessions of T. foenum-graecum of which 40 gave amplification. However, only 14 ISSR primers detected intraspecific variation in I7 accessions of T. foenum-graecum generating clear reproducible patterns and revealing 100 bands in the range of 500 bp to 2 kb. Among these seventy-two bands were polymorphic amounting to 72% polymorphism [Table 3]. Furthermore, during ISSR analysis 11 unique bands were obtained, where 6 were contributed by accession TMP = 8714 from Yemen, 4 by accession TMP = 8691 from Turkey, and 1 by accession TMP = 8685 from Iran. Similarly, in T. caerulea, of the 100 ISSR primers used for initial screening, 47 gave amplification. Of these, 18 primers detected intraspecific variation in 9 accessions of T. caerulea showing 93.64% polymorphism [Table 4]. With these 18 primers, 16 unique bands were produced, where 9 and 7 bands were contributed by accessions 206901 and 206486, respectively, from Turkey alone. In case of RAPD analysis, 100 RAPD primers were used for initial screening in T. foenum-graecum of which 22 primers generated polymorphic patterns revealing 70.12% polymorphism [Table 3]. Eight unique bands were produced by these primers of which 3 were contributed by accession TMP = 8714 from Yemen, 2 by accession TMP = 8698 from Egypt, 1 by accession TMP = 8707 from Afghanistan and 1 each by accession TMP = 8691 and TMP = 8690 from Turkey. Similarly in T. caerulea of the 40 primers used for initial screening, 10 primers produced polymorphic pattern giving 95.83% polymorphism [Table 4]. Eight unique bands were produced with these primers wherein; the maximum numbers of unique bands (4 each) were again produced by the same accessions 206901 and 206486 from Turkey. Dendrogram analysis for T. foenum-graecum and T. caerulea Genetic similarity was calculated from the Nei's similarity index value for all the 17 accessions of T. foenum-graecum considering ISSR and RAPD approaches individually as well as together. Based on ISSR markers alone, the similarity index values ranged from 0.69 to 0.92. These values were used to construct a dendrogram using Unweighted Pair Group Method with Arithmetic averages (UPGMA). In the ISSR based dendrogram T. foenum-graecum genotypes formed 4 clusters [Figure not shown]. The first cluster grouped together accessions from Afghanistan (8707), Canada (1065), Pakistan (8717,8718), Iran (8675), and Turkey (8690). The second cluster contained accessions from India (8686,8689,8675). Remaining one accession from India (8687) and one from Nepal (8706) formed the third cluster. The fourth cluster contained accessions from Egypt (8698,8679) and Turkey (8692). Accessions from Ethiopia (8696), Turkey (8691) and Yemen (8714) out grouped from the main clusters. Three accessions from Turkey analyzed in the present study fell into different clusters. Based on RAPD markers alone, the similarity index values ranged from 0.71 to 0.91. In the RAPD based dendrogram, T. foenum-graecum genotypes formed 2 main clusters [Figure not shown]. The first cluster had two subgroups, the first subgroup contained accessions from Afghanistan (8707), India (8686,8687,8675), Turkey (8690) and Egypt (8698) while the second subgroup contained accessions from Pakistan (8717,8718), Turkey (8692), and Egypt (8679). The accession from Canada associated with these clusters. The second cluster contained accession from Iran (8685) and Nepal (8706) and the accession from India associated with this cluster. Accessions from Ethiopia (8696), Turkey (8691) and Yemen (8714) out grouped from these two clusters. In the RAPD based dendrogram also accessions from Turkey fell into different clusters. Based on both the marker systems together the similarity index values ranged from 0.65 to 0.89 [Fig. 1]. Here the T. foenum-graecum accessions from Egypt (8698,8679) were grouped together. Accessions from Pakistan (8717, 8718) and Afghanistan (8707) were grouped together in one cluster. Accessions from India (8686,8689,8675,8687), Nepal (8706) and Iran (8685) were grouped together. However, all the three accessions from Turkey fell in different clusters and did not group among themselves. Bootstrapping was done using the WinBoot program to estimate the relative support at clades. The robustness of the cluster was not very strong in T. foenum-graecum (50–70%). In T. caerulea, genetic similarity was calculated from the Nei's similarity index value considering ISSR and RAPD approaches individually as well as together. Based on ISSR marker system, the similarity index values ranged from 0.41 to 0.92 while that on the basis of RAPD markers ranged from 0.34 to 0.93. Similarity indices values based on both the marker systems together ranged from 0.38 to 0.92 indicating more diversity in case of T. caerulea. The dendrograms based on ISSR and RAPD markers showed similar clustering pattern when used individually [Figures not shown] as well as together [Fig. 2]. Here the dendrogram showed two main clusters. The first cluster grouped together accessions form Turkey (TMP = 8704) and Australia (PI = 186283). The second cluster contained 3 accessions of unknown geographic origin and 1 accession each from U.S.A (PI = 345743) and Spain (PI = 244288). The remaining 2 accessions i.e. accessions PI = 206486 and PI = 206901 from Turkey, out grouped from these two main clusters. Thus the accessions from Turkey did not cluster together as observed in T. foenum-graecum. The bootstrap values obtained using the WinBoot program in T. caerulea were very high (90–100%). Heterozygosity and marker index Heterozygosity was calculated using ISSR and RAPD marker systems individually as well as together as detailed in Table 3 for T. foenum-graecum and in Table 4 for T. caerulea. In T. foenum-graecum the Hav values calculated for ISSR, RAPD and ISSR+RAPD were 0.214, 0.203 and 0.211, respectively. In case of T. caerulea the Hav values for the same were 0.330, 0.346 and 0.338, respectively. For both, T. foenum-graecum and T. caerulea, the Hav values for ISSR and RAPD systems did not differ significantly. The marker index (MI) values calculated for ISSR and RAPD for T. foenum-graecum were 1.53 and 1.51, respectively while in T. caerulea they were 3.17 and 3.35, respectively. Thus MI values in both the species did not differ significantly for ISSR and RAPD systems. Correlation between measures of similarity In T. foenum-graecum, when the similarity matrices generated using ISSR and RAPD markers were compared, a value of r = 0.78, at P = 0.001 indicated a good correlation between data generated by both the systems [Fig. 3]. Similarly when the similarity matrices generated using ISSR and RAPD systems were compared in case of T. caerulea, a value of r = 0.98 indicated a very good correlation between the two marker systems [Fig. 4]. Discussion The two marker systems, ISSR and RAPD used in the present study have also been used as effective tools to evaluate genetic diversity and to throw light on the phylogenetic relationships in Brassica napi [rapeseed, [10]], Allium sect. Sacculiferum [Alliaceae, [11]] and Asimina triloba [pawpaw, [12] and [13]]. Genetic diversity analysis using ISSR and RAPD markers in legumes like Cicer [[14] and [15]] and Cajanus [[17] and [18]] has been carried out in our own laboratory. These studies have given important clues in understanding species relationship, which may further assist in developing and planning breeding strategies. However, no such reports on genetic diversity using molecular markers were available in the genus Trigonella. In the present study, an attempt has been made to examine the level of genetic variation within T. foenum-graecum and T. caerulea accessions obtained from germplasm collection centers at Saskatoon (Plant Gene Resources of Canada) and Pullman (USDA-ARS Plant Introduction Station, Washington). The T. foenum-graecum accessions were selected in order to represent most of the countries where it is grown. In case of T. caerulea all the nine accessions available at Saskatoon and Pullman were used in present study. Analysis of polymorphism detected in T. foenum-graecum and T. caerulea Polymorphism in a given population is often due to existence of genetic variants represented by the number of alleles at a locus and their frequency of distribution in a population. Heterozygosity corresponds to a probability that two alleles taken at random from a population can be distinguished using the marker in question. Thus a convenient quantitative estimate of marker utility and the polymorphism detected can be given in terms of the mean heterozygosity and the marker index [18]. In T. foenum-graecum as well as in T. caerulea the Hav and the marker index (MI) values for ISSR and RAPD markers [Table 3 and 4], respectively, did not differ significantly indicating that similar levels of polymorphism were detected by both the marker systems in the given germplasm pools. This was also confirmed by the high correlation co-efficent for ISSR and RAPD marker systems obtained for T. foenum-graecum and T. caerulea [Fig. 3 and 4]. Genetic diversity as measured by the heterozygosity was higher in T. caerulea (0.33) as compared to T. foenum-graecum (0.21). Based on allozyme diversity, the estimated mean heterozygosity values have been reported for self-pollinating species, Vigna unguiculata, Hav = 0.027 [19] and Vicia tetrasperma, Hav = 0.342 [20]. The heterozygosity value for Vigna unguiculata was lower while that for Vicia tetrasperma was higher as compared to T. foenum-graecum and T. caerulea. Based on ISSR markers, the estimates of genetic diversity, Hav = 0.358 reported in cultivated pawpaw (Asimia triloba), was higher as compared to T. foenum-graecum and T. caerulea [13]. Estimation of genetic relatedness in T. foenum-graecum and T. caerulea Data collected with ISSR and RAPD marker systems were used to compare genetic similarity between various accessions of T. foenum-graecum and T. caerulea. The accessions could be a mixture of different genotypes. Therefore, to have a complete representation of a specific accession, DNAs from fifteen plants were mixed in equal proportion. Thus within accession diversity was eliminated and a complete banding profile of the accession was used for the analysis. In T. foenum-graecum, ISSR and RAPD could detect almost similar level of polymorphism (72% with ISSR and 70.12% with RAPD). In the UPGMA analysis, T. foenum-graecum accessions from one country and the nearby region grouped together in some cases while they were placed in different clusters in certain cases. Accessions from Pakistan and Afghanistan grouped together in one cluster while accessions from India and Nepal grouped in another cluster. Moreover, the three accessions from Turkey fell in different clusters inspite of being geographically very close to each other. Thus, there was no clear clustering pattern of geographically closer accessions in the present study indicating that the association between genetic similarity and geographical distance was less significant. However, it is necessary to use more number of accessions from each geographical location to confirm the available pattern. In T. caerulea also, ISSR (93.64%) and RAPDs (94.83%) detected almost equal level of polymorphism. T. caerulea showed more polymorphism as compared to T. foenum-graecum. In case of T. caerulea also, the UPGMA analysis showed that plants from different geographical regions were distributed in different groups. Here again, the accessions from Turkey were not grouped together. Two accessions from Turkey out grouped from the main cluster while one was grouped in the first cluster with Australia. In T. caerulea we could obtain three accessions from Turkey and only one accession from other countries. Therefore, it would be premature to comment precisely about the correlation of geographic distance and genetic diversity in this case. To confirm the available pattern it is necessary to use more number of accessions from each geographical location. Center of Origin and /or Center of Diversity for Trigonella The place of origin of a species as explained by Vavilov is an area that contains a large amount of genetic variability of that species. According to him variation is a function of time, hence the region containing the greatest variation in a species would have supported and sustained that species for a longer time than the other regions suggesting that region to be the Center of Origin and/or Diversity. He set up eight geographic centers, two of which namely the Near Eastern and Mediterranean Centers extent into Turkey [21]. It has been postulated by Vavilov that the Near East region extending from Israel through Syria, Southern Turkey into Iran and Iraq and the Mediterranean Center including Spain, Morocco and Turkey is the Center of Origin of Trigonella, Trifolium and Medicago species [22]. In the present study both, T. foenum-graecum and T. caerulea accessions from Turkey exhibited more diversity. These results support Vavilov's hypothesis. The Indian accessions of T. foenum-graecum i.e. accession number 8686 (Khandwa), 8685(Mumbai), and 8687 (Patiala) separated by an aerial distance of 52 km, 104 km, and 135 km, respectively from each other, were genetically more similar (similarity index 0.893) and clustered together [Fig. 1]. However, the accessions of T. foenum-graecum from Turkey i.e. accession number 8692 (Malatya) and 8691 (Elbistan) separated by a distance of 100 km (similarity index value 0.875-0.745) were out grouped and were genetically more distant from each other although morphologically they were similar to each other as well to the accessions from other countries. Turkey is one of the significant and unique countries in the world from the point of view of plant genetic resources and plant diversity. The country has more than 3,000 endemic plants and immense diversity has been reported in many legumes such as Vicia, Medicago, Trifolium, Lathyrus, Onbrychis, Trigonella, Pisum, Cicer, Lens, etc [23]. Many genera of cultivated plants like Cicer, Lens, Pisum, Amygladus, Prunus, Triticum, etc have their Center of Origin and/or Diversity in this country [22]. Vavilov designated southeastern Turkey and the adjoining Syria as the primary Center of Origin (now the center of diversity) for chickpea [24]. Similar to chickpea (and other grain legumes also), in T. foenum-graecum the large seeded cultivars are abounded around the Mediterranean region whereas the small seeded cultivars are predominated eastwards. Thus, Turkey may also be the primary Center of Origin of T. foenum-graecum and T. caerulea. However, this hypothesis needs to be confirmed by considering more accessions distributed over a wide geographic range especially from the Near East and the Central Mediterranean region. Conclusions In conclusion, molecular markers allowed us to estimate the overall genetic diversity in T. foenum-graecum and T. caerulea and simultaneously revealed molecular based genetic relationship. In the UPGMA analysis, no significant correlation was observed between geographic distance and genetic diversity. Our data further supported the hypothesis of the Near East and the Central Mediterranean to be the Center of Origin and/or Diversity for Trigonella as put forth by Vavilov. Methods Plant material and DNA extraction Seeds of T. foenum-graecum accessions were obtained from Plant Gene Resources of Canada (PGRC), Saskatoon, Canada. These accessions along with the TMP numbers and the country from where they have been collected are outlined in Table 1. Seeds for various T. caerulea accessions were obtained from PGRC, Saskatoon, Canada and USDA-ARS Plant Introduction Station at Pullman, Washington (W-6), and are detailed in Table 2. Fifteen plants of each accession were grown in pots for DNA isolation. Two gram of young leaf tissue was harvested for each plant and frozen in liquid nitrogen for DNA extraction. Plant DNA was extracted by the Doyle and Doyle method [25] and equal amount of DNA from each of the fifteen plants was pooled together for each accession. PCR amplification ISSR A set of 100 anchored micro satellite primers was procured from University of British Columbia, Canada. PCR amplification of 20 ng of DNA was performed in 10 mM Tris-HCI pH 7.5, 50 mM KCI, 1.5 mM MgCl2, 0.5 mM spermidine, 2% formamide, 0.1 mM dNTPs, 0.3 uM primer and 0.8 U of Taq DNA polymerase (Ampli-Taq DNA polymerase, Perkin Elemer, USA) in a 25 ul reaction using Perkin Elmer 9700 thermocycler. After initial denaturation at 94°C for 5 minutes, each cycle consisted of 30 seconds denaturation at 94°C, 45 seconds of annealing at 50°C, 2 minutes extension at 72°C along with 5 minutes extension at 72°C at the end of 45 cycles. RAPD RAPD analysis was performed using arbitary decamer primers procured from University of British Columbia, Canada. The reaction mixture (25 ul) contained 10 mM Tris-HCI pH 7.5, 50 mM KCI, 1.5 mM MgCl2, 0.5 mM spermidine, 0.1 mM dNTPs, 15 pmoles of primer, 20 ng genomic DNA, and 0.8 U of Taq DNA polymerase (Ampli-Taq DNA polymerase, Perkin Elmer, USA). Amplification was carried out using Perkin Elmer 9700 thermocycle for 40 cycles, each consisting of a denaturing step of 1 minute at 94°C, followed by annealing step of 1 minute at 36°C and an extension step of 2 minutes at 72°C. The last cycle was followed by 5 minutes of extension at 72°C. Amplified products were electrophoresed on 2% agarose gel using 0.5× TAE buffer (10 mM Tris HCl and 1 mM EDTA pH. 8.0) and visualized by ethidium bromide staining. The patterns were photographed and stored as digital pictures in gel documentation system. The reproducibility of the amplification was confirmed by repeating each experiment three times. Agarose gel electrophoresis Amplified products were electrophoresed on 2% agarose gel using 0.5x TAE buffer (10mM Tris HCl and 1mM EDTA pH. 8.0) and visualized by ethidium bromide staining. The patterns were photographed and stored as digital pictures in gel documentation system. The reproducibility of the amplification was confirmed by repeating each experiment three times. Data analysis Unequivocally reproducible bands were scored and entered into a binary character matrix (1 for presence and 0 for absence). The genetic similarity among accessions was determined by Nei’s genetic distance [26]. A dendrogram was constructed based on the matrix of distance using Unweighted Pair Group Method with Arithmetic averages. Scores entered in matrix were analyzed using TAXAN version 4.0 software based on the degree of bands sharing. Similarity matrix was generated using Dice coefficient as SI = 2Nab/Na+Nb where Na = total number of bands present in lane a, Nb = total of bands in lane b, Nab number of bands common to lanes a and b [27]. The dice values were then used to perform the UPGMA analysis. To evaluate the robustness of the grouping formed the binary data matrix was subjected to bootstrapping using WinBoot program [28]. The phenogram was reconstructed 1000 times by repeating sampling with replacement and the frequency with which the groups were formed was used to indicate the strength of the group. Correlation co-efficient for the similarity matrices generated by ISSR and RAPD data in both, T.foenum-graecum and T.caerulea, were calculated by method of Smouse et al [29]. The expected heterozygosity, Hn for a genetic marker was calculated as: Hn = 1 - pi2, where pi is the allele frequency of the ith allele [26]. The arithmetic mean heterozygosity Hav for each marker class was calculated as Hav = Hn/n, where n = number of markers or loci analyzed [18]. The average heterozygosity for polymorphic marker (Hav)p was further derived as; (Hav)p = Hn/np; where np is the no. of polymorphic markers or loci [18]. Marker index (Ml) for each marker system was also calculated as, MI = E (Hav)p; where E = effective multiplex ratio [E = nβ where β is the fraction of polymorphic marker or loci,18]. Authors' contributions RD carried out the PCR and molecular genetic study, participated in the design of the study and performed statistical analysis. ML participated in procuring the seeds from different sources and monitoring experiments and result. LC a taxonomist participated in procuring, growing and analyzing the seeds obtained from different sources. PR and VG conceived of the study, participated in its design and co-ordination and monitored the experiments and the results. All authors read and approved the final manuscript. Acknowledgement The authors are thankful to the Department of Biotechnology, Government of India for financial support. We also gratefully acknowledge the Plant Gene Resources of Canada (PGRC), Saskatoon, Canada and the United States Department of Agriculture (USDA), Washington, USA for providing the seed material used in this study. Figures and Tables Figure 1 ISSR+RAPD dendrogram based on algorithm of Unweighted Pair Group Method With Arithmetic Averages in T. foenum-graecum. Figure 2 ISSR+RAPD dendrogram based on algorithm of Unweighted Pair Group Method With Arithmetic Averages in T. caerulea. Figure 3 Regression analysis of similarity matrices generated using RAPD and ISSR marker systems in T. foenum-graecum. Figure 4 Regression analysis of similarity matrices generated using RAPD and ISSR marker systems in T. caerulea. Table 1 List of T. foenum-graecum accessions used in the present study Sr. No. TMP. No. Country 1 8707 Afghanistan 2 1065 Canada 3 8698 Egypt 4 8696 Ethiopia 5 8686 India 6 8689 India 7 8687 India 8 8685 India 9 8675 Iran 10 8706 Nepal 11 8717 Pakistan 12 8718 Pakistan 13 8690 Turkey 14 8691 Turkey 15 8692 Turkey 16 8679 Egypt 17 8714 Yemen Table 2 List of T. caerulea accessions used in present study Sr. No. Source TMP\PI No. Country 1 Saskatoon TMP = 8704 Turkey 2 Saskatoon TMP = 8738 Unknown 3 USDA PI = 345743 U.S 4 USDA PI = 244288 Spain 5 Saskatoon TMP = 8750 Unknown 6 USDA PI = 206486 Turkey 7 USDA PI = 186283 Australia 8 USDA PI = 206901 Turkey 9 Saskatoon TMP = 8752 Unknown Table 3 Comparison of DNA marker systems in T. foenum-graecum Marker System No. of primers % Polymorphism Average No. Of bands/Primer Hav (Hav)p MI ISSR 14 72 7.14 0.2145 0.297 1.53 RAPD 22 70.12 7.45 0.203 0.289 1.51 ISSR + RAPD 36 70.8 7.33 0.211 0.298 - Table 4 Comparison of DNA marker systems in T. caerulea Marker System No. Of primers % Polymorphism Average No. Of bands/Primer Hav (Hav)p MI ISSR 18 93.64 9.61 0.330 0.354 3.17 RAPD 10 95.83 9.7 0.346 0.361 3.35 ISSR + RAPD 28 94.05 9.65 0.338 0.359 - ==== Refs Rachie KO Roberts LM Grain legumes of the lowland tropics Advances in Agronomy 1974 26 1 32 Bromfeild S Butler G Barran LR Temporal effect on the composition of a population of Sinorrhizobium meliloti associated with Medicago sativa and Medicago alba Can J Microbiol 2001 47 567 573 11467732 10.1139/cjm-47-6-567 Balodi B Rao RR The genus Trigonella L. (Fabaceae) in the Northwest Himalaya J Econ Tax But 1991 5 11 16 Polhil RM Raven PH Advances in legume systematic Royal Botanical Gardens, Kew, England 1981 2 Rajagopalan MS Fenugreek a savory Medicinal Supplement industry Executive 2001 5 43 44 Rajagopalan MS Fenugreek, what this herb can offer? Naturally 1998 1 1 4 Sharma RD Effect of fenugreek on blood glucose and serum lipids in type-1 diabetes European Journal of Clinical Nutrition 1990 44 301 306 2194788 Ken Fern Plants For A Future: Edible and useful Plants for a Healthier World: 1997 Permanent Publications, England Hymowitz T Janick J, Simon JE Grain Legumes In Advances in new crop 1990 Timber Press, Portland, OR 54 57 Chao-Zia MA Ting-dong FU Stine Tuevesson Bo Gertsson Genetic diversity of Chinese and Swedish Rapeseed (Brassica napi) analyzed by Inter Sequence Simple Repeat (ISSRs) Agricultural Sciences in China 2003 2 137 143 Gang H Dong-hee-lee June S NamSook L A study of taxonomical relationship among species of Allium sect. Sacculiferum (Alliaceae) and the related species using Inter Sequence Simple Repeat (ISSR) markers Bot Bull Acad Sin 2002 43 63 68 Huang H Layne DR Kubisiak TL Molecular characterization of cultivated pawpaw (Asimina triloba) using RAPD markers J Amer Soc Hort Sci 2003 128 85 93 Pomper KW Crabtree SB Brown SP Jones SC Bonney TM Assessment of genetic diversity of Pawpaw (Asimina Triloba) cultivars with Inter Simple Sequence Repeat markers J Amer Soc Hort Sci 2003 128 521 525 Sant VJ Patankar AG Gupta VS Sarode ND Mhase LB Sainani MN Deshmukh RB Ranjekar PK Potential DNA markers in detecting divergence and in analyzing heterosis in Indian elite chickpea cultivars Theor App Genet 1999 98 1217 1225 10.1007/s001220051187 Rajesh PN Tekeogul M Gupta VS Ranjekar PK Muehlbauer FJ Genetic relationships among annual and perennial wild species of Cicer using inter simple sequence repeat (ISSR) polymorphism Euphytica 2003 129 15 23 10.1023/A:1021567821141 Ratnaparkhe MB Gupta VS Ven Murthy MR Ranjekar PK Genetic fingerprinting of pigeonpea (Cajanus cajan) and its wild relatives using RAPD markers Theor App Genet 1995 91 893 898 Dabak MM Ranade SA Dhar MS Gupta VS Ranjekar PK Molecular characterization of pigeonpea genome Ind J Biochem Biophys 1988 25 230 236 Powell W Morgante M Andre C Hanafey M Voges J Tingey S Rafalski A A comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis Mol Breed 1996 2 225 230 Pasquet RS Allozyme diversity of cultivated cowpea, Vigna unguiculata Theor App Genet 2002 101 211 219 10.1007/s001220051471 Man Kyu Huh Hoong Wook Huh Genetic diversity and population structure of wild tare lentil Crop Sci 2001 41 1940 1946 Vavilov NI The origin, variation, Immunity, and Breeding of cultivated Plants Chronica Botanica 1951 13 1 366 Vavilov NI Centers of origin of cultivated plants Trends Pract Bot Gener Sel 1926 16 3 24 Oltraco Sabanci Aarora PK, Mathur PN, Riley KW Lathyrus Genetic Resources of Turkey In Lathyrus genetic resources of Asia 1996 Adham 77 87 Van Der Masen LJG Sexena MC, Singh KB Origin, History, and Taxonomy In The Chickpea 1987 11 34 Doyle JJ Doyle JL A rapid DNA isolation procedure for small amount of fresh leaf tissue Phytochem Bull 1987 19 11 15 Nei M Estimation of average heterozygosity and genetic distance from a small number of individuals Genetics 1987 83 583 590 Nei M Li WH Mathematical model for studying genetic variation in terms of restriction endonucleases Proc Natl Acad Sci 1979 76 5269 5273 291943 Yap I Nelson RJ A program for performing bootstrap analysis of binary data to determine the confidence limit of UPGMA based dendrograms IRRI Discussion Paper series 14 International Rice Research Institute 1996 Smouse PE Long JC Sokal RR Multiple regression and correlation extension of the Mantel test of matrix correspondence Sys Zool 1996 35 627 632

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==== Front BMC Cardiovasc DisordBMC Cardiovascular Disorders1471-2261BioMed Central London 1471-2261-4-131529196710.1186/1471-2261-4-13Research ArticleArrhythmia-provoking factors and symptoms at the onset of paroxysmal atrial fibrillation: A study based on interviews with 100 patients seeking hospital assistance Hansson Anders [email protected]ärdig Bjarne [email protected] Olsson S [email protected] Department of Cardiology, University Hospital, Lund, Sweden2004 3 8 2004 4 13 13 26 3 2004 3 8 2004 Copyright © 2004 Hansson et al; licensee BioMed Central Ltd.2004Hansson et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Surprisingly little information on symptoms of paroxysmal atrial fibrillation is available in scientific literature. Using questionnaires, we have analyzed the symptoms associated with arrhythmia attacks. Methods One hundred randomly-selected patients with idiopathic paroxysmal atrial fibrillation filled in a structured questionnaire. Results Psychic stress was the most common factor triggering arrhythmia (54%), followed by physical exertion (42%), tiredness (41%) coffee (25%) and infections (22%). Thirty-four patients cited alcohol, 26 in the form of red wine, 16 as white wine and 26 as spirits. Among these 34, red wine and spirits produced significantly more episodes of arrhythmia than white wine (p = 0.01 and 0.005 respectively). Symptoms during arrhythmia were palpitations while exerting (88%), reduced physical ability (87%), palpitations at rest (86%), shortage of breath during exertion (70%) and anxiety (59%). Significant differences between sexes were noted regarding swollen legs (women 21%, men 6%, p = 0.027), nausea (women 36%, men 13%, p = 0.012) and anxiety (females 79%, males 51%, p = 0.014). Conclusion Psychic stress was the commonest triggering factor in hospitalized patients with paroxysmal atrial fibrillation. Red wine and spirits were more proarrhythmic than white wine. Symptoms in women in connection with attacks of arrhythmia vary somewhat from those in men. Paroxysmal atrial arrhythmiatriggering factorssymptomsalcohol. ==== Body Background Atrial fibrillation (AF) is nowadays divided into three different forms; paroxysmal, persistent and permanent [1]. Even if the pathological, electrical and physiological phenomena leading to AF have been described in ever more detail, the mechanisms underlying these changes remain largely unknown. The relative occurrences of paroxysmal atrial fibrillation (PAF) and other forms of this arrhythmia in the population are also not well known. In a material based on hospital observations, 35% of all fibrillation was described as being of paroxysmal type [2]. Despite being common, surprisingly limited information about possible triggering factors and symptoms at the onset of arrhythmia in larger groups of patients is available [3]. Our aim in this investigation was to throw further light on the factors believed by patients to have caused their arrhythmia and on the symptoms experienced at the onset of attacks of AF. Methods A group of one hundred patients suffering ECG-verified PAF and whose symptoms prompted hospital care were asked to fill in a structured questionnaire with 58 questions covering arrhythmia-triggering factors, time at which the attack starts and symptoms during attack. All patients completed the data by personal interviews by one of the authors. The vast majority of all patients were recruited during two periods of totally 14 months. In some questions, extra information could be supplied by the patients in their own words. (The complete questionnaire could be seen in additional file 1: AHansson_enkat.pdf). All patients had earlier had attacks of AF which stopped either spontaneously or following medication. Most of them had earlier been treated in hospital for the arrhythmia. Patients who previously had myocardial infarction, pericarditis, diseases of the thyroid or diabetes were not included since these diseases may be the underlying cause of the arrhythmia [4-10]. Our group of patients thus had idiopathic PAF. Permission for the investigation was obtained from the local Ethical Committee and all patients were informed about the investigation both orally and in writing. Statistics Data were compared using Fisher's Exact Test and Mann Whitney U-test. Values of p < 0.05 were considered significant. Results Material One hundred completed questionnaires were filled in by 72 men and 28 women. Twenty-four of these had on some occasion been treated for hypertension. The median (range) age of the entire group was 59.9 (22.4–79.2) years. Patients with previous histories of hypertension were significantly older than those without 65.0 (46.6–78.6) years as against 58.6 (22.4–79.2) years respectively. The women in the group were significantly older than the men 67.9 (54.1–78.6) years and 58.2 (22.4–79.2) years respectively, p = 0.02 (Fig. 1). Since patients with hypertension differed from the others only as regards age, the ensuing analyses were made without respect to its incidence. Seventy six of all patients were given genuine antiarrhythmic drugs, sotalol being the most common. Twenty four of the patients were not taking any antiarrhythmic pharmacological treatment (Table 1). Figure 1 Age-distribution by sex. In the group investigated, the median age was 59.9 (22.4–79.2) years median (range). The women were significantly older than the men. Table 1 Current pharmacological antiarrhythmic treatment Sotalol 40 Disopyramid 13 Digoxin 13 Metoprolol 5 Flekainid 5 Propranolol 3 Atenolol 3 Verapamil 3 Bisoprolol 1 Amiodaron 1 No antiarrhythmic medication 24 Attacks of arrhythmia Seventy-two patients believed that their attacks usually started at about the same time of day, typically starting in the evenings or at night (Fig. 2). The time of onset appeared not to depend on sex. Figure 2 Time at which palpitations started. Seventy-two patients thought that arrhythmia always started at the same time of day, typically in the evening or at night. Some, however, gave more than one time-interval. For most patients (64%), attacks typically lasted less than one day, while a further 17% gave 1–7 days. No attack lasted more than one week. Thirty-five percent of the entire group woke up with episodes of fibrillation, 34% stated that attacks began with psychic stress, as defined by the patients. Thirty one percent of the episodes started during rest and 22% of the attacks followed physical exertion. Twenty-five percent could not think of any special factor initiating attacks. No differences in the onset of attacks between men and women could be established. Eighty-five percent of the group succeeded in identifying some sort of triggering-factor (Table 2). The most common of these was psychic stress followed by physical exertion, tiredness and infection. As regards foodstuffs, 25% thought that coffee was the triggering-factor. Those who cited sympathetic tone anamnesis with stress as the triggering-factor usually stated that the onset of arrhythmia was in the evening or at night. Table 2 Possible arrhythmiatriggering-factors as identified by patients with paroxysmal atrial fibrillation Triggering factor % Mental stress 54 Physical effort 42 Tiredness 41 Any alcohol 34 White wine 16 Red Wine 26 Liquor 26 Coffee 25 Infections 22 Cold drinks 8 Large meal 3 Food 18 Onions 5 Nuts 4 Chocolate 3 Ice cream 2 Spiced food 2 Cream 1 Strawberries 1 Fish 1 Sweets 1 Beans 1 Shellfish 1 Garlic 1 No triggering-factor 15 Alcohol was named as a triggering-factor by 34 patients, in 26 cases in the form of red wine, in 26 as spirits and in 16 white wine. Some patients named more than one kind of alcohol as a triggering factor. Two of those who reacted to red wine did not drink spirits and hence could not say whether spirits, too, trigger arrhythmia. In the alcohol-triggered group, red wine and spirits triggered more episodes of arrhythmia than white wine (p = 0.01 and p = 0.05 respectively). There were no significant differences in the arrhythmia-provoking effects of spirits and red wine nor between men and women (Table 2, Fig. 3). Time delay between onset of trigger and subsequent onset of the AF episode was not explored in the questionnaire. Figure 3 Any alcohol as a pro-arrhythmic factor. Thirty-four patients cited various forms of alcohol as a factor triggering arrhythmia. Some patients named more than one kind of alcohol. Twenty-six cited red wine, 26 spirits and 16 white wine. In this group, red wine and spirits caused significantly more episodes of arrhythmia than white wine. Foodstuffs such as onion, nuts, chocolate and ice-cream were cited as agents producing fibrillation by a few patients (Table 2). Seventy-four percent considered that the episodes of AF they experienced affected their lifestyles, while 26% thought this was not the case. Among answers in their own words, a common reply was that they did not dare to exercise as much as they would have liked (13 patients). Eight patients did not dare to travel. Sixty-six percent stated that their episodes of AF affected their relatives, while 32% answered this question in the negative. In their own words, they remarked that the main problem was their relatives' uneasiness. During attacks of AF, the most common symptoms were palpitations in connection with strain, reduced physical performance, palpitations when at rest, breathlessness during exertion and anxiety (Table 3). Females showed significantly higher frequencies of swollen legs (p = 0.02), indisposition (p = 0.012) and anxiety (p = 0.014) than males. Table 3 Symptoms in association with the onset of paroxysmal atrial fibrillation by gender Symptoms Total n = 100 Males n = 72 Female n = 28 p-value Pre-symptoms 32 20 (28%) 12 (43%) 0.159 Pains in the chest 25 14 (19%) 11 (39%) 0.070 Dizziness 52 33 (46%) 19 (68%) 0.074 Syncope 7 4 (6%) 3 (11%) 0.396 Breathlessness when resting 41 32 (44%) 9 (32%) 0.365 Breathlessness when working 70 53 (74%) 17 (61%) 0.230 Swollen ankles 10 4 (6%) 6 (21%) 0.027 Palpitation at rest 86 62 (86%) 24 (86%) >0.999 Palpitation at exercise 88 59 (82%) 23 (82%) >0.999 Nausea 19 9 (13%) 10 (36%) 0.012 Vomiting 2 1 (1%) 1 (4%) 0.484 Abdominal pain 5 2 (3%) 3 (11%) 0.312 Loss of appetite 31 20 (28%) 11 (39%) 0.336 Anxiety 59 37 (51%) 22 (79%) 0.014 Reduced physical capacity 87 62 (86%) 25 (89%) >0.999 Polyuria* 40/75 25/52 (48%) 15/23 (65%) 0.213 *this question was put to only 75 patients. Discussion Although PAF is one of the most common heart-disturbances causing patients to get in touch with medical care centres, surprisingly little information is available about the factors which trigger it and the symptoms associated with the onset of arrhythmia in larger groups of patients. This study has therefore been undertaken to determine which factors patients consider responsible for triggering arrhythmia and the symptoms that occur in connection with episodes of arrhythmia. What provokes arrhythmia? From observations of heart-rate in sinus rhythm shortly before its onset, a separation of PAF into sympathetically-mediated and vagal forms has been suggested [11]. Earlier studies have shown a degree of daily variation in the onset of AF. Thus, attacks are more common in the morning and at night [12], but higher frequency has also been reported during daytime [13]. A possible explanation is that arrhythmia often starts in younger patients at night and in older ones during the day [14]. A weekly variation has also been reported, with fewer attacks on Saturdays [12]. An annual variation with fewer attacks during the last months of the year has also been reported [12]. In our study, the 72 patients who thought that their attacks of arrhythmia usually occurred at about the same time of day gave this as the evening or at night. Hence a large fraction of those investigated should have vagal PAF since this often starts at night [11]. Despite this, the majority of the patients considered that the triggering-factor was some kind of situation in which increased levels of catecholamines can be discerned. Even those with positive stress-related anamnesis (triggered by physical exertion and psychic stress) had often attacks starting in the evening or at night. However, this need not imply an absolute correlation in time, but rather the probable existence of a certain latent period between stress and the onset of arrhythmia. In earlier studies, it has been proposed that attacks of PAF are often due to variations in the tonus of the autonomic nervous system. Arrhythmia is stimulated particularly when an initial adrenergic increase is followed by an abrupt change to vagal dominance [15]. Alcohol has long been considered to play an etiological role in PAF, a correlation underlined in the expression "holiday heart" [11]. Since temporary enhanced alcohol consumption is frequent, it is difficult to prove a direct correlation [8,16]. Episodes of AF have been triggered by the acute effects of alcohol on atrial refractoriness and conduction, but also by the effects of chronic misuse of alcohol leading to subclinical heart dysfunction [17]. Other mechanisms have also been discussed [18]. Every third patient in our study considered alcohol to be a triggering-factor, but white wine was blamed less than red wine and spirits. Why arrhythmia should be triggered less frequently by white wine than by red wine or spirits remains unclear. It is now generally accepted that most attacks of PAF are induced by ectopic impulses originating in the pulmonary veins [19,20]. Both automaticity and triggered automaticity in these cells have been demonstrated in experimental conditions [19,20]. The influence of the autonomic nervous system, alcohol and other factors inducing arrhythmia on this mechanism is, however, uncertain. Symptoms at the onset of fibrillation Although a number of patients with PAF are without any symptoms [21], in general patients with this form of arrhythmia show more symptoms than those with permanent AF [22]. Studies with telephone-transmitted ECG have, however, shown a sensitivity of symptomatic registrations of up to 89% with PAF [23]. There is thus good correlation between the symptoms and ECG-verified AF. The limited amount of literature on the symptomology of PAF includes Quality of Life investigations, Case Reports and quantification of a few symptoms [24]. Investigations based on "Quality of Life" forms have earlier shown that patients with PAF have lower scores for physical function, emotional function, vitality and general health [24]. The symptoms commonly reported include palpitations, giddiness, dyspnoea, tachycardia, perspiring, chest pains, coldness, anxiety [23-25], tiredness, weakness, indisposition, vomiting and epigastrical discomfort [26]. The most frequent symptoms in periods of AF reported by our group of patients included palpitations, reduced physical performance, palpitations when at rest, breathlessness when exerting oneself and anxiety. In an earlier report, the most pronounced symptoms were palpitations and anxiety as well as giddiness [24]. That females showed significantly higher frequencies of swollen legs, indisposition and anxiety than males has not previously been reported. These differences can possibly be accounted for since earlier studies have reported that attacks of AF in women last longer and cause higher heart-rates [27]. We could not, however, establish any significant differences in the lengths of attacks between men and women in our material. Swollen legs can also be accounted for due to right-sided cardiac failure in some patients. That most patients experience definite symptoms following acute but transient attacks of AF can possibly depend on the increased activity of the sympathetic nervous system triggered by an attack of AF [28]. It is plausible to assume that the autonomic nervous system plays a considerable part in both the genesis of and the symptoms observed during a period of AF [24]. Limitations This material was taken at a hospital and is thus not representative of all patients with PAF. The symptoms of our patients are so far advanced that hospitalization or a visit to a hospital was required. Ongoing treatment can have modified patients' recollections of anamnestic factors. The material was not taken consecutively, but randomness was favoured by lack of a systemic inclusion mechanism. Patients with hypertension are not excluded, even if subtle diastolic changes in the left ventricle and hence the left atrial performance could be caused by hypertension [29]. Although symptoms associated with the onset of PAF may be age related, the present material is too limited to allow exploration of this relation. Conclusions Most of the patients in a group being treated at a hospital for PAF consider psychic stress to be the factor triggering their arrhythmia. Red wine and spirits seems more prone to trigger attacks of AF than white wine. The symptoms of PAF are many and occur frequently. In women, PAF leads to significantly higher frequencies of swollen legs, indisposition and anxiety than in men. Competing interests None. Authors' contributions Author AH designed the investigation, collected all patient data, performed the statistical analysis and interpretation of the results, as well as the preparation of the manuscript. Author BMH assisted with the statistical analysis and the preparation of the manuscript. Author SBO supervised and designed the investigation as well as participated in the preparation of the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 The complete questionnaire. The structured questionnaire with 58 questions covering arrhythmia-triggering factors, time at which the attack starts and symptoms during attack. Click here for file Acknowledgements The Swedish Heart and Lung Foundation and the Franke & Margareta Bergqvist Foundation supported this study. ==== Refs Task Force Rapport ACC/AHA/ESC Guidelines for the management of patients with atrial fribrillation Eur Heart J 2001 22 1852 1923 11601835 10.1053/euhj.2001.2983 Alpert JS Petersen P Godtfredsen J Atrial fibrillation; Natural history, complications and management Ann Rev Med 1988 39 41 52 3285783 10.1146/annurev.me.39.020188.000353 Parkinson J Campbell M Paroxysmal auricular fibrillation. 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Mechanisms and Management 1992 New York: Raven Press, Ltd 93 108 Freedberg AS Papp JG Williams EMV The effects of altered thyroid state on atrial intracellular potentials J Physiol 1970 207 357 369 5499024 Levy S Epidemiology and classification of atrial fibrillation J Cardiovasc Electrophysiol 1998 9 S78 82 9727680 Coumel P Falk RH, Podrid PJ Neural aspects of paroxysmal atrial fibrillation Atrial Fibrillation. Mechanisms and Management 1992 New York: Raven Press, Ltd 109 125 Viskin S Golovner M Malov N Fish R Alroy I Vila J Laniado S Kaplinsky E Roth A Circadian variation of symptomatic paroxysmal atrial fibrillation. Data from almost 10 000 episodes Eur Heart J 1999 20 1429 1434 10487804 10.1053/euhj.1999.1632 Rawles JM Metcalfe MJ Jennings K Time of occurrence, duration, and ventricular rate of paroxysmal atrial fibrillation: the effect of digoxin Br Heart J 1990 63 225 227 2337493 Yamashita T Murakawa Y Hayami N Sezahi K Inone M Futuri E Ornata M Relation between aging and circadian variation of paroxysmal atrial fibrillation Am J Cardiol 1998 82 1364 1367 9856920 10.1016/S0002-9149(98)00642-0 Bettoni M Zimmermann M Autonomic tone variations before the onset of paroxysmal atrial fibrillation Circulation 2002 105 2753 2759 12057990 10.1161/01.CIR.0000018443.44005.D8 Rich EC Siebold C Campion B Alcoholrelated acute atrial fibrillation: a case control study and review of 40 patients Arch Intern Med 1985 145 830 833 3158290 10.1001/archinte.145.5.830 Ettinger PO Wu CF De La Cruzc Weisse AB Ahmed SS Regan TJ Arrhythmias and the holiday heart: Alcohol associated cardiac rhythm disorders Am Heart J 1978 95 555 562 636996 10.1016/0002-8703(78)90296-X Koskinen P Kupari M Olsson SB, Allessie M, Campbell R The role of alcohol in atrial fibrillation Atrial Fibrillation Mechanisms and Therapeutic Strategies 1994 Armonk, NY: Futura Publishing Company, Inc 225 236 Chen Y-J Chen S-A Chen Y-C Yeh HI Chan P Chang MS Lin CI Effects of rapid atrial pacing on the arrhytmogenic activity of single cardiomyocytes from pulmonary veins. Implication in initiation of atrial fibrillation Circulation 2001 04 2849 2854 11733406 Chen Y-J Chen Y-C Yeh H-I Lin C-I Chen S-A Electrophysiology and arrhythmogenic activity of single cardiomyocytes from canine superior vena cava Circulation 2002 105 2679 2685 12045176 10.1161/01.CIR.0000016822.96362.26 Neutel J Smith D Jankelow D Pretorius J Myburgh P The aeromedical implications of atrial fibrillation Aviation, Space, and Environmental Medicine 1990 1036 1038 2256879 Jung W Herwig S Camm J Dorian P Newman D Akhtar M Wood K Ayers G Luderitz B Are there differences in quality of life measures depending on clinical type of atrial fibrillation? PACE 1998 21 890 Bhandari A Anderson J Gilbert E Alpert B Henthorn R Waldo A Cullen M Hawkinson R Pritchett E Correlation of symptoms with occurrence of paroxysmal supraventricular tachycardia or atrial fibrillation: a transtelephonic monitoring study. The Flecainide Supraventricular Tachycardia Study Group Am Heart J 1992 124 381 386 1636582 10.1016/0002-8703(92)90601-Q Van den Berg M Hassink R Tuinenburg A van Sonderen E Lefrandt J de Kam P van Gelder I Smit A Sanderman R Crijns H Quality of life in patients with paroxysmal atrial fibrillation and its predictors: importance of the autonomic nervous system Eur Heart J 2001 22 247 253 11161936 10.1053/euhj.2001.2180 Lau CP Leung WH Wong CK Cheng CH Haemodynamics of induced atrial fibrillation; A comparative assessment with sinus rhythm, atrial and ventricular pacing Eur Heart J 1990 11 219 224 2318225 Schlepper M Weppner HG Merie H Haemodynamic effects of supraventricular tachycardias and their alterations by electrically and verapamil induced termination Cardiovasc Res 1978 12 28 33 639077 Hnatkova K Waktare J Murgatroyd F Xiahoua G Camm J Malik M Age and gender influences on rate and duration of paroxysmal atrial fibrillation PACE 1998 21 2455 2458 9825366 Anderson JL Gilbert EM Alpert BL Henthorn R Waldo A Bhandari A Hawkinson R Pritchett E Prevention of symptomatic recurrences of paroxysmal atrial fibrillation in patients initially tolerating antiarrhythmic therapy Circulation 1989 1557 1570 2513143 Allessie MA Boyden PA Camm AJ Pathophysiology and prevention of atrial fibrillation Circulation 2001 103 769 777 11156892

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==== Front BMC Med Res MethodolBMC Medical Research Methodology1471-2288BioMed Central London 1471-2288-4-201529651510.1186/1471-2288-4-20DebatePublication bias in situ Phillips Carl V [email protected] Center for Philosophy, Health, and Policy Sciences, Inc., Houston, USA2 Management, Policy and Community Health Division, University of Texas School of Public Health, RAS E-311, 1200 Pressler, Houston, TX 77225, USA3 Center for Clinical Research and Evidence-Based Medicine, University of Texas Medical School, Houston, USA2004 5 8 2004 4 20 20 1 12 2003 5 8 2004 Copyright © 2004 Phillips; licensee BioMed Central Ltd.2004Phillips; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Publication bias, as typically defined, refers to the decreased likelihood of studies' results being published when they are near the null, not statistically significant, or otherwise "less interesting." But choices about how to analyze the data and which results to report create a publication bias within the published results, a bias I label "publication bias in situ" (PBIS). Discussion PBIS may create much greater bias in the literature than traditionally defined publication bias (the failure to publish any result from a study). The causes of PBIS are well known, consisting of various decisions about reporting that are influenced by the data. But its impact is not generally appreciated, and very little attention is devoted to it. What attention there is consists largely of rules for statistical analysis that are impractical and do not actually reduce the bias in reported estimates. PBIS cannot be reduced by statistical tools because it is not fundamentally a problem of statistics, but rather of non-statistical choices and plain language interpretations. PBIS should be recognized as a phenomenon worthy of study – it is extremely common and probably has a huge impact on results reported in the literature – and there should be greater systematic efforts to identify and reduce it. The paper presents examples, including results of a recent HIV vaccine trial, that show how easily PBIS can have a large impact on reported results, as well as how there can be no simple answer to it. Summary PBIS is a major problem, worthy of substantially more attention than it receives. There are ways to reduce the bias, but they are very seldom employed because they are largely unrecognized. ==== Body Background A study is more likely to appear in the literature, and thus be indexed and publicly available, if it shows a strong, statistically significant, or otherwise "better" result. A study that does not show such results has a greater chance of remaining hidden in a file drawer, either because the author (or funder) does not think the result is worth mentioning, or because journals are less interested in publishing studies that find "nothing". This form of publication bias, wherein readers have access to only a biased sample of the full distribution of results, is well studied, particularly in the literature on meta-analysis and other systematic reviews where the problem is most apparent (a primer on the topic can be found at the Cochrane Collaboration webpage [1]). Even well-read lay people are, at the time of this publication, aware of the problem due to the controversy about pharmaceutical companies selectively releasing research about their products. Far less attention is paid to the bias that occurs when some results from a study are published, but the choice of which results to publish produces bias. As with traditional publication bias, the tendency is to analyze data and choose to present results which are statistically significant, further from the null, or closer to what the researchers believe is the true value. The implications for the literature as a whole, though, are much the same as the file drawer bias. I label this "publication bias in situ" (PBIS) because the biased reporting of study findings exists within each individual research report (with any metaphoric references to cancer – the usual context of the phrase "in situ" in health science – left as an exercise for the reader). Despite the similarities between the file drawer bias and PBIS, there are fundamental differences. In particular, the bias from some studies having no published findings exists only at the level of the whole literature (no particular study can be said to be biased), while PBIS exists within the results reported from a single study (and thus exists in the literature as a whole by aggregation). More practically, PBIS is substantially more difficult to even identify, let alone correct. This paper defines and describes PBIS and identifies some of the choices that create it. The purpose of the paper is not to make novel technical or statistical claims. Indeed, there is probably no single statistical observation here that will not be clear to a skilled data analyst (or, indeed, that could not be explained to anyone competent in middle-school-level math). The literature includes many observations about issues relating to PBIS. Yet the challenge to the validity of the entire health science literature posed by PBIS – arguably a greater issue than conflict of interest, traditional publication bias, or any other commonly discussed threat to the integrity of the literature – has not received the attention it deserves. In the literature about meta-analysis and systematic reviews there is substantial attention to file-drawer publication bias (though most of this considers only bias from reporting statistically significant results, ignoring preferences for reporting more dramatic results or those that agree with authors' or journals' previous reports). But there is considerably less attention to possible picking and choosing which study results to report or statistical methods to use. To the extent that this is considered, it is often bundled with questions about whether ex ante protocols were stated and followed, and is thus put on par with many protocol violations that could be considered mere technicalities. For example, a few years ago, a highly-publicized meta-analysis [2] called into question the evidence on the benefits of screening mammography by citing apparent problems in the methods of most of the relevant randomized trials. But little was done to distinguish a few of these problems that appeared to be PBIS and others that were minor technical points. The literature and pedagogy related to observational inference, where the potential for PBIS is considerably greater than for well-designed experiments, seems to pay even less attention. Epidemiology textbooks typically discuss some of the methods that can reduce PBIS (e.g., well-defined protocols), but say little or nothing about the possible bias. Indeed, many study and data-analysis methodologies (some of which are noted below) that are typically taught in classes or by apprenticeship seem designed to create PBIS. Despite this, highly-trained experts summarize claims reported in the literature without mention of the likely bias, indicating an unawareness of the major implications of PBIS. In a notable exception, Hahn and colleagues [3,4] discussed some sources of PBIS and argued that they receive too little attention compared to bias from selective publication. Their analyses addressed selective reporting of subgroup results in the context of randomized trials, a topic further discussed below, and reporting a selected subset of multiple measured endpoints. They do not mention the other sources of PBIS discussed below. Unfortunately, their findings do not appear to have had the impact they deserved. It is not clear how common PBIS is or how large the resulting bias, but a few efforts to find it suggest that the potential is great enough that it deserves much more attention. (Labeling might matter: Hahn et al. label the problem "within-study selective reporting." The more dramatic name suggested here, with its emphasis on the bias that results, might catch more attention.) Only by systematically addressing the problem are we likely to substantially affect it. Moreover, as will be discussed, statistical and research methods that ostensibly address some of the sources of PBIS are unsatisfactory, and a systematic attempt should be made to find better solutions. Discussion Many degrees of freedom Reviewers (systematic or otherwise) of the literature can only see what researchers choose to report and highlight in their publications, and that choice can be biased in a number of ways. Researchers have great freedom in deciding exactly what to analyze, how to analyze it, and what to report. All research results are derived from data that can be used to measure many associations. Even the most narrowly focused clinical trial can be analyzed with the endpoint defined in different ways, stratified by age, etc. As with traditionally defined publication bias, the analyses that are deemed unworthy of publication are largely invisible to the scientific and policy community. Among the dimensions of freedom researchers have in deciding what to analyze and report are three choices illustrated by examples in this paper: (1) Which exposures and outcomes to consider in datasets with many variables. (2) Which functional forms to use to represent variables (e.g., how to divide continuous variables into categories). (3) Whether to conduct separate analyses by subgroup, and which subgroup results to emphasize. Making such choices is a legitimate part of research. Indeed, the choices must be made. But when those choices are primarily driven by what produces stronger (or otherwise "better") results, bias is created. This creates a difficult challenge: It is easy to recognize traditional publication bias (paper in journal = no contribution to bias; paper in file cabinet = contribution to bias). But since there is no clearly correct option for any of the above choices (indeed, any particular analysis gives the right answer to some question), there is no clearly wrong choice, and thus no clear way of concluding that a particular choice was biased. Fortunately, as should become apparent from the following analysis, PBIS results less from the choices made and more from what the choices are based on (which can often be inferred) and, to an even greater extent, the generally overlooked issue of how the results are presented (which can be easily observed). Publication bias, either PBIS or the file-drawer effect, can be seen most clearly as an interaction between random errors and researcher choices (e.g., when random sampling error leads to a weaker result, the result is less likely to be reported), creating a bias from what would be unbiased random variation. Systematic errors (confounding, measurement error, etc.) and methods for correcting for them create many additional opportunities for PBIS; however, for simplicity, systematic errors are ignored in this paper. Multiple analyses from the same data Choice (1) in the above list has probably received the most attention in previous literature (for example, debates over whether to correct for multiple hypothesis testing and the appropriateness of data dredging). Despite the disproportionate attention, this choice is probably not the major source of PBIS, but it provides a familiar starting point. Many epidemiologic datasets are characterized by thousands or even millions of possible combinations of exposures, endpoints, and covariates. It is frequently assumed that statistical science tells us the "right" way to deal with this challenge, but current practice (not to mention the confusion of students coming out of epidemiology and biostatistics classes) makes clear that there is substantial disagreement among viewpoints about how to apply statistical methods when dealing with multiple hypotheses or measurements. Further consideration makes it clear that statistical rules cannot actually provide clear answers. At one extreme are viewpoints such as, "We must correct measures of statistical certainty (significance levels for p-values (α-levels) or confidence interval widths) whenever multiple comparisons are made using the same dataset" and even, "statistical analysis can only be legitimate for a short list of pre-specified hypotheses." At the other extreme are viewpoints such as, "regardless of how many comparisons are examined, each can be considered and statistically tested as if it were the only one," and, "it does not matter at all if a hypothesis was proposed after looking at the data." The impasse in this debate seems to stem from both sides attacking straw men, without recognizing that each side has a stronger case some of the time. This can be illustrated with examples. Example: unrelated results from the same dataset A cohort dataset originally used to report the relationship of drinking water source and the occurrence of Helicobactor pylori infection contains data that is later used to look at the relationship of household crowding and performance in school. It is difficult to understand why we would make an adjustment when doing the second analysis because we have already done the first (or, worse, disallow the analysis because it was not pre-specified in the study or because we have already "used up" our .05 worth of α with the H. pylori analysis, and so cannot analyze anything more with this data at all). A logical extension of that argument would be to consider the dataset that contains all quantitative human knowledge (which is logically an epistemologically legitimate definition), and declare that we have to adjust for every statistical analysis ever done, effectively precluding further statistical analysis. Example: multiple comparisons that will support claims of the "same" relationship Researchers investigate the hypothesis that poor nutrition increases the risk of H. pylori infection. The dataset contains dozens of different measures of food and nutrient intake, as is usually the case for nutrition data. This, plus multiple diagnostic tests for H. pylori which are sometimes discordant, creates a large number of statistical comparisons, any of which could be described as supporting the plain language claim, "poor nutrition affects H. pylori status." A typical approach is to find individual comparisons that support the hypothesis, presenting only these comparisons with statistical tests as if each were the only analysis conducted. The claim about the relationship between the particular measure of nutritional status and the particular measure of H. pylori status is accurate, as are the test statistics reported for that association. But the plain language conclusion (which would probably be drawn) was very likely to be supported by some relationship in the data by chance alone, even in the absence of any true underlying association. This fact is obscured by the reported unadjusted tests statistics or confidence intervals. As the second example illustrates, unrestricted picking and choosing of comparisons leads to publication bias. A lot of associations that were not deemed worthy of reporting never appeared in the literature, while the few that were "interesting" did. This problem is well known (though few probably realize that it can lead to hundreds of instances of publication bias, in situ within a single published article, making it a bias of much greater magnitude than the file-drawer effect). The solutions offered by statistical rules – corrections for multiple hypothesis testing or restricting analysis to ex ante hypotheses – is inadequate. Such rules produce absurd implications, noted in the first example. Trying to eliminate the absurdity by exempting from statistical adjustment analyses with disjoint exposures and outcomes, as in the first example, does not work; the second example offers options for disjoint analyses also. Most important (and widely overlooked), correcting for multiple comparisons does not affect the reported biased estimates of effect size; changing test statistics and confidence interval widths does nothing to reduce the bias. This alone shows that the standard statistical corrections for multiple tests do nothing to solve the problem. The other standard method for trying to reduce PBIS, rules that limit analyses to pre-specified hypotheses and protocols, will throw away a lot of potentially valuable findings and is nearly impossible to operationalize because detailed protocols require advanced knowledge that may not exist and can never be specified unambiguously. Frequentist statistical theory cannot offer a solution to this problem because PBIS, like the file-drawer problem, is not a matter of statistics. The second example illustrates where the problem primarily lies: in the plain-language reporting of results. The statistics that describe the relationship between a particular exposure and outcome measure could be exactly right, but the claim about good nutrition (as a generic concept) and infection status (as if we had a gold standard measure), which will likely be emphasized in the paper and its title (and press releases) and will likely stick readers' minds, might be misleading. Consider how the result would be interpreted if there were a table reporting every analyzed comparison, most of which showed little or no association. Most scientifically literate readers would realize the result was not so convincing, even though those same readers seldom think to object when – as is typical – only one or a few results are reported. By contrast, if the researchers in the first example reported the result of the previous study, it would be unlikely to change most readers' assessments. This suggests the simplest partial solution to the problem. By reporting a table of results from other comparisons considered, researchers could report their interesting result (rather than not informing the world due to the lack of a specific ex ante hypothesis or having "used up" the α), but without creating the PBIS that would result otherwise. Indeed, this appears to summarize the most obvious generic rule to reduce PBIS (and publication bias in general): publish everything. An immediate implication of this is that online publications, like this journal, allow researchers to publish less biased articles. Online articles can usually be whatever length is appropriate to report the results (which is of particular value in the health sciences, where paper journals have extremely restrictive length limits), and can include dozens or thousands of alternative analyses in appendices or links to data or software that allow the reader to review still more results. Of course, this opportunity is beneficial only if authors choose to take advantage of it (or editors and reviewers demand that they do). Bias from the choice of functional form The implications of choice (2), the functional form for variables, can be clearly illustrated with a simple example. Consider a study with an exposure variable measured as 10 ordered categories (i.e., values 1,2,...,10, with larger numbers representing greater exposure). Assume researchers wish to analyze the association of a disease endpoint and a dichotomous definition of exposure. If there is no clear cutpoint for defining exposure, there are many options. There are 9 cutpoints that divide the observations into two categories, defining those above the cutpoint to be exposed and those below unexposed. Other options include comparing a group of highest categories to a group of lowest categories, leaving out the middle, such as 8–10 versus 1–3, yielding an additional 36 possibilities. How will the researchers choose a definition of exposure? A typical procedure is to let the data inform the choice: The cutpoint that provides the clearest contrast between the exposed and unexposed is judged to be the right one, the "most sensitive" to the presumed effect. It should be immediately obvious that this procedure will bias the result away from the null. To illustrate this, consider a case-control study with 200 subjects (throughout the examples, subjects are half cases and half non-cases). Calculations for this example and others are based on Monte Carlo simulation of different realizations of the data based on the assumed underlying relationship. All simulations were performed using Crystal Ball (Decisioneering Inc., Denver, Colorado, USA). Assume that each of the 10 exposure categories is equally likely for cases and non-cases. If the researchers consider only the 9 cutpoints that dichotomize the data and choose the cutpoint for "exposed" to get the largest odds ratio (OR), the median result will be 1.5. Since the exposure and disease are not associated, this is clearly an upward bias. For those inclined to focus on statistical significance, the chance of observing a significant positive association at a one-tailed significance level of .025 is 13%. (Of course, for any single definition of exposure, the median OR is 1.0 and the chance of seeing a significant relationship is about 2.5%.) Even if researchers do not analyze their data in every possible way and report the strongest association, any decision to report results that is based on associations in the data (e.g., choosing between a cutpoint at 5 or at 6 based on which produces a stronger association) will create bias. Although this observation should be obvious to anyone with an understanding of statistics, letting the data have some influence on the choice is probably more the rule than the exception among researchers. It is often defended on the grounds that there was no way to know what the "right" cutpoint was before doing the study, and the study data is the only existing answer to the question. This is a legitimate point, but it does not reduce the resulting bias. Less scrupulous researchers – who are trying to support a preferred answer to further a policy agenda or advance their careers – need make no such explanation and can intentionally choose the extreme results. As with the file-drawer effect, results in the literature will tend to show effects greater than the true value. Most important, whichever analysis is reported, the plain-language result will be "we found an association between the exposure and the disease," and so collections of studies that each report an exposure-disease comparison with a greater-than-average association, and will seem to be confirming the same result, even though the comparisons are not the same. Extending this example to illustrate how PBIS can compare to traditional publication bias, assume now that there is a positive association between the exposure and disease. In particular, non-cases are still equally likely to be in each of the ten categories, while cases have respective probabilities for each category of (.069, .072, .077, .084, .093, .102, .112, .121, .131, and .139). These values were chosen so that the true OR is similar, whichever of the 9 cutpoints is chosen (for those interested, the numbers follow a logistic curve). True ORs round to 1.5 for all cutpoints. Consider a collection of studies of varying sizes, with fewer larger studies, as we would typically see in the literature, specifically 100 randomly generated studies (more than would likely exist, but better to illustrate) of random size (drawn from a triangular distribution with modal probability at a minimum value of 100 subjects, diminishing linearly to a maximum of 1600 subjects). Note that to avoid committing the very type of transgression discussed in this paper – repeating an analysis until "good" results are found – the reported results are from the first and only run of the simulation. For a single definition of "exposed" (values >5), a typical result appears in Figure 1 in the form of a funnel plot of study results vs. study size [1,5,6]. The results that are statistically significantly different from 1.0 at the two-tailed .05 level are represented by solid dots. The other results (represented by open circles) might never be reported – the simplest form of publication bias – though they could be inferred from the asymmetry of the distribution that would occur if only the significant results were published. Notice that the distribution for all the studies is unbiased and would lead to an estimate very close to 1.5, while a naive summary estimate based only on the statistically significant studies (possibly the only ones published) could almost double the estimated effect size. Figure 1 Traditional publication bias from simulated studies. Simulated results from case control studies with varying populations (half cases, half non-cases) for true odds ratio of 1.5. Solid circles represent statistically significant results at the 2-tailed, .05 level. (Note: x-axis scale chosen for compatibility with other figures.) Compare this to the results for the same 100 studies where the cutpoint is chosen based on the largest OR (Figure 2). The distribution is also substantially biased, with PBIS leading to results above the single-definition results of 1.5. A summary estimate of effect size would turn out to have substantially greater bias than would reporting only significant results as in Figure 1. Notice that though there is a skew, it is much harder to discern a pattern like the asymmetry in Figure 1 that would show a systematic reviewer that the literature is biased. Figure 2 Publication bias in situ from simulated studies. Simulated results from case control studies with varying populations (half cases, half non-cases) for true odds ratio of 1.5. For each simulated study, the largest odds ratio (choosing among 9 different cutpoints for exposure definition) is reported. (Note: one outlier odds ratio estimate not shown.) Unbiased random errors, when combined with picking and choosing functional forms, lead to biases in reported results. A solution to this problem is much less obvious than its existence. The commonly proposed solution of only reporting results for pre-specified functional forms is not satisfactory, because it is difficult to enforce (most every pre-specification has some room for interpretation in retrospect; intentional cheating is difficult to detect; there may be little basis for selecting any particular pre-specified functional form) and it forces us to ignore real unpredicted findings. Sticking to pre-specified analyses is especially unrealistic in research studies that collect data on many different risk factors and outcomes. Simply labeling all results that were not pre-specified with the caveat, "hypothesis generating," accomplishes nothing. If such results were actually treated as not yet "real", the problems of determining exactly which results were pre-specified and the loss of important serendipitous findings are reintroduced. Of course, results with the caveat are very seldom treated as less real than any others in the literature. Moreover, the "generated" hypotheses will never be retested in exactly the form reported, so the label is simply disingenuous. In sum, the proposed solutions to this type of PBIS are no more realistic or satisfactory as solutions than trying to eliminate traditional publication bias by requiring that all studies be adequately powered. A better family of solutions would be to establish a standard of reporting results for alternative variable definitions (perhaps in online appendices). Not only does this directly reduce PBIS by publishing more results, but it provides readers with a choice of results if they prefer different definitions (information that would be discarded by a pre-specified hypothesis rule). If results for every cutpoint from the 100 trials in the example were reported, the results, as pictured in Figure 3, would be unbiased. Naturally, researchers could emphasize the variable definitions they think best, but by acknowledging other possibilities they would be forced to justify their choice. Figure 3 Publication bias in situ eliminated by reporting all results. Results from Figure 2, but with results for all possible cutpoints reported. In many cases there will not be an obvious short list of variable definitions, but some alternative definitions should be obvious and others could be found in previous literature. A simple, but very useful, improvement would be a standard practice of reporting the closest possible replication of previous published analyses using data from the new study. This would directly address the problem of PBIS resulting from data-driven picking and choosing of functional forms (though it might require the cooperation of previous authors to provide details about what analysis they reported, given the typically abbreviated reporting of methods in published papers – another problem with paltry word limits). Repeating whatever analyses that previous researchers happened to choose is somewhat arbitrary, but each round of new research can also add a new preferred functional form. The key is that results based on previously published functional forms cannot be data driven and, unlike the standard practice of new studies that make different comparisons but describe them with the same plain language, would actually replicate (or fail to replicate) previous results. Bias from the analysis of subgroups In 2003, VaxGen Corporation (Brisbane, California, USA) released results of a large HIV vaccine trial in the United States, one of the highest-profile clinical trials of the year. The disappointing result showed a trivial reduction in incidence among the treatment group compared to the placebo group. But the three non-white racial groups (black, Asian, and "other") each showed a substantial reduction in incidence (Table 1). VaxGen reported the overall failure of the trial to the popular and business press [7,8]. A technical report describing the drug and the trial results, written by a third party, appeared later in an indexed journal [9], though the New York Times articles actually contained more complete study results. VaxGen tried to salvage some hope for the drug by pointing out the results for non-whites, suggesting that maybe it held promise for some populations [8]. VaxGen's search for a silver lining resulted in rash of criticism from the research community (focused on the reporting of a result that was not a pre-specified hypothesis and the failure to correct for multiple hypothesis testing) and a shareholder lawsuit, alleging that statistically illicit reporting was used to inflate stock prices [9-12]. Table 1 Results of VaxGen HIV vaccine trial total vaccine placebo RR subjects subjects incidence subjects incidence white 4511 3003 179 1508 81 1.11 nonwhite 498 327 12 171 17 0.37  black 314 203 4 111 9 0.24  Asian 73 53 2 20 2 0.38  other 111 71 6 40 6 0.56 total 5009 3330 191 1679 98 0.98 Source: The New York Times [7] and author's calculations. Given the failure to publish the results in a scientific journal, some might argue that VaxGen was guilty of traditional publication bias – not publishing unfavorable results about its products – a charge that is currently being leveled at many drug companies. However, the company actively released fairly complete study results and accompanying analysis to the press, and the results appeared in forums that are more widely read than all scientific journals combined, so the results were clearly not buried in a file drawer. But despite reporting their results, VaxGen was biasing what they published. Singling out of the result for non-whites is a clear case of PBIS. VaxGen did not give equal emphasis to the apparently harmful effect of the vaccine for whites. The company's subsequent report that the different results for whites and non-whites could not be due to chance [9] does not diminish the apparent bias, since it basically repeats the same information contained in the original (data-driven, biased) reporting of the result for the non-white subgroup. Setting aside some controversy that erupted about systematic errors in the data, what can we say about the results from the perspective of PBIS and chance alone? To look at the result for non-whites, with its one-tailed p-value of .002, ignoring the fact that the subgroup definition was clearly data-driven, would overstate the finding, as suggested in the preceding examples. But a naive correction for multiple hypothesis testing would make the opposite error. Setting aside the possibility that other covariates would have been used to stratify the data had they produced subgroups with positive findings, the combination of the 4 racial groups implies a test of 24-1 = 15 different hypotheses. To adjust for 15 implicit hypotheses makes it very unlikely that any will pass the statistical test, including the population as a whole, even for substantial associations. An alternative is to ask the question, "if the vaccine has no effect, what are the chances of seeing, in any racial group or combination thereof, a result at least as strong as the observed 63% reduction?". Phrased that broadly, simulation shows the answer is about 20%. However, most of the 20% comes from the unstable results for the two smallest groups, Asians and "other". Restricting the analysis to combinations of racial groups that contain black, with or without Asians or other, the probability is only 2.1% (the probability of seeing a 63% reduction by chance alone for any group that includes the whites is vanishingly small). So, what is the right answer? We must return to the observation that there is never a single Right Answer from a study; the quality of an answer always depends on what question was being asked. Did VaxGen find a successful vaccine? Clearly not, as the relative risk for the whole population shows. Should the result for non-whites be considered unlikely to be due to chance (i.e., statistically significant)? It depends on whether you consider it the answer to the question "does the vaccine show a result for non-whites?", in which case the answer is 'yes' (though the effective study size is small), or "does the vaccine show a result for any racial group?" in which case the answer is 'it is fairly likely we would see such a result due to chance alone.' It is worth noting how this illustrates a popular fallacy in data analysis: Frequentist hypothesis testing is not the objective exercise that some think it to be; it depends on subjective decisions about what to test. We might decide to infer that VaxGen would have emphasized the results from any racial subgroup that showed a positive result (and the company did claim the original protocol called for analysis by racial and other subgroups [9]), and thus that they were answering the latter question. Notice that none of the options that are typically practiced or recommended are satisfying. To just report the subgroup analysis as if it were the only analyzed result obviously leads to bias. (It is worth noting that in a less high-profile research project, that might well have happened, without anyone questioning the result.) But it is not satisfying to suppress the tantalizing findings about non-whites, either because there was not really an ex ante hypothesis that the vaccine would work only in non-whites or because the multiple-hypothesis correction for hundreds of possible racial and other subgroups makes it non-significant. A general rule requiring us to ignore interesting but surprising findings is a huge waste of information. Requiring a data-driven subgroup analysis to be biologically plausible before reporting it offers no solution, since we can usually construct a story to explain whatever associations appear in the data (it has been speculated that some genotypes get a benefit from the vaccine, and the frequency of those genotypes is strongly correlated with race). To offer the "hypothesis generating" caveat would make little difference, scientifically or in the securities market. It is unrealistic to suggest that this "generated" hypothesis will be re-examined given the overall disappointing result. Two studies in Thailand (one completed later [13], which also found the vaccine ineffective, and another by the U.S. National Institute of Allergy and Infectious Disease that may continue to use the vaccine anyway [14]) are likely the closest anyone will come to re-examining the hypothesis, but a population of Thais is hardly the same as non-white Americans. Furthermore, this example shows how epistemologically absurd the hypothesis generating caveat is: The result could originally have been considered hypothesis generating. But a few days after the results were released the company claimed that they had an ex ante plan to analyze racial and other subgroups [9], which would presumably promote the result "hypothesis confirming". However, that claim by the company, accurate or not, was completely uninformative about the effect of the vaccine, telling us nothing about the certainty of the findings, and so cannot legitimately change our conclusions. It does not matter whether the hypothesis was pre-specified. Debating whether the company really proposed the subgroup analysis ex ante, as if that should change our interpretation of the result, seems particularly absurd. When Hahn et al. [3] observed apparent selective reporting of subgroup analyses, they suggested identifying subgroups in the protocol, keeping that list as short as possible, and implicitly called for reporting results for all pre-specified subgroups. But since every measured covariate creates two, several, or a continuum of possible subgroups, this approach would require ignoring a lot of the results of a study, no matter how interesting they are (as well as severely taxing the imagination of the researchers about which subgroups are the right ones). Since it is unrealistic to expect researchers to not report interesting results (let alone to not even do the analysis that would produce those results) after spending months or years gathering data, we need methods that allow the reporting of results but with less bias. The obvious general solution is to report all subgroup analyses with equal prominence. Any reporting (be that a research paper, abstract, paper title, or press release) that suggests there is a beneficial effect for some people should equally emphasize any apparent harmful effects for other people (and vice versa). The fact that one result is statistically significant and the other is not should be of no consequence. Indeed, selecting which results to report based on statistical significance guarantees there will be publication bias (and, more generally, the inappropriate emphasis on statistical significance may be the source of a large amount of PBIS, but this point must be left for future analyses). The reporting of the multiple subgroups results should be accompanied by statistics similar to those calculated here, instead of standard test statistics, so that readers know the probability that an estimated effect (or test statistic) at least as great as the one found would result from chance alone for any of the subgroup analyses. Such information will allow readers (researchers working on related projects, policy makers, investors) to focus on what they consider to be the answer to their own questions. Summary The opportunities for PBIS, along with the almost universal failure to report research results in ways that avoid it, create the possibility that biased study results are very prevalent in the health science literature. Some of the causes of PBIS are well understood, but the enormity of its implications is largely ignored. PBIS can produce very misleading results, leading to widespread misperceptions and misguided policies. The examples presented here show just a few of the many ways that PBIS can result from random error and researchers' (usually innocent, almost always invisible, possibly quite reasonable) choices. Neither the problem nor the solution lies in the mathematics of data analysis, so answers will not be found by appealing to statistical theory. The critical issue is the completeness of reporting and the plain-language interpretation of results. The simple solutions offered by statisticians are not satisfying or even realistic. All they really let us do is observe that in almost every research report, "the rules" have been violated. This is not helpful. Rather than a right-vs.-wrong view of proper use of statistics that would condemn most of the literature as invalid, we need a realistic way of addressing this problem. The solutions, like the solutions for traditional publication bias, will generally consist of doing a more complete job of reporting what can be reported. List of abbreviations HIV = human immunodeficiency virus OR = odds ratio PBIS = publication bias in situ Competing interests None declared Authors' contributions Single author Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgments The author thanks Brian Guenzel for research assistance and Karen J. Goodman and the journal editor for helpful suggestions. ==== Refs Alderson P Green S Eds Cochrane Collaboration open learning material for reviewers. The Cochrane Collaboration Accessed July 26, 2004 Olsen O Gotzsche PC Cochrane review on screening for breast cancer with mammography Lancet 2001 358 1340 1342 11684218 10.1016/S0140-6736(01)06449-2 Hahn S Williamson PR Hutton JL Investigation of within-study selective reporting in clinical research: follow-up of applications submitted to a local research ethics committee J Eval Clin Pract 2002 8 353 359 12164983 10.1046/j.1365-2753.2002.00314.x Hahn S Williamson PR Hutton JL Garner P Flynn EV Assessing the potential for bias in meta-analysis due to selective reporting of subgroup analyses within studies Stat Med 2000 19 3325 3326 11122498 10.1002/1097-0258(20001230)19:24<3325::AID-SIM827>3.0.CO;2-D Light RJ Pillemer DB Summing up The science of reviewing research 1984 Cambridge, Harvard University Press Egger M Davey Smith G Schneider M Minder C Bias in meta-analysis detected by a simple, graphical test BMJ 1997 315 629 634 9310563 Pollack A Altman LK Large trial finds AIDS vaccine fails to stop infection New York Times A1 February 24, 2003 Altman LK Official Hopes to explain AIDS vaccine disparities New York Times A24 February 25, 2003 Adis International Ltd. HIV gp120 vaccine – VaxGen: AIDSVAX, AIDSVAX B/B, AIDSVAX B/E, HIV gp120 vaccine – Genentech, HIV gp120 vaccine AIDSVAX – VaxGen, HIV vaccine AIDSVAX – VaxGen Drugs RD 2003 4 249 253 Cohen J AIDS vaccine results draw investor lawsuits Science 2003 299 1965 12663890 10.1126/science.299.5615.1965b Watanabe M Skeptical scientists skewer VaxGen statistics Nature 2003 9 376 10.1038/nm0403-376b Johnson & Perkinson announces class action lawsuit against Vaxgen, inc Business Wire 2003 The Associated Press Test AIDS vaccine fails in Thai study New York Times November 13, 2003 Cohen J Public health. AIDS vaccine still alive as booster after second failure in Thailand Science 2003 302 1309 1310 14631004 10.1126/science.302.5649.1309a

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BMC Med Res Methodol. 2004 Aug 5; 4:20

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BMC Med Res Methodol

10.1186/1471-2288-4-20

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==== Front BMC GastroenterolBMC Gastroenterology1471-230XBioMed Central London 1471-230X-4-161529872010.1186/1471-230X-4-16Research ArticleNitric oxide-an endogenous inhibitor of gastric acid secretion in isolated human gastric glands Berg Anna [email protected] Stefan [email protected] Ann-Charlott [email protected]östrand Sven Erik [email protected] Department of Biomedicine and Surgery, Division of Cell Biology, Linköping University, Linköping, Sweden2 Surgery Department, University Hospital, Linköping, Sweden2004 6 8 2004 4 16 16 25 1 2004 6 8 2004 Copyright © 2004 Berg et al; licensee BioMed Central Ltd.2004Berg et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Endothelial nitric oxide synthase (eNOS) has previously been detected in the glandular part of the human gastric mucosa. Furthermore, nitric oxide (NO) has been shown to influence gastric secretion in various animal models. The present study was conducted to investigate the influence of exogenously and endogenously derived NO on histamine- and cAMP-stimulated gastric acid secretion in isolated human oxyntic glands. Methods Oxyntic glands were isolated from human gastric biopsies and were subsequently pre-treated with NO donors and nitric oxide synthase inhibitors and then exposed to histamine or dibutyryl-cAMP (db-cAMP). The secretory response of the glands was determined as accumulation of [14C]aminopyrine. Results The histamine- or db-cAMP-induced acid secretion was attenuated by L-arginine, a known source of endogenous NO, and also by the NO-donors sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP). Pre-treatment with either of the NOS inhibitors NG-nitro-L-arginine methyl ester (L-NAME) or NG-nitro-L-arginine (L-NNA) enhanced the secretory response. Conclusion Our results show that NO inhibits gastric acid secretion in isolated human gastric glands, and that there is endogenous formation of NO within the glandular epithelium in the vicinity of the parietal cells. ==== Body Background Nitric oxide (NO) is produced from L-arginine in a reaction catalyzed by the enzyme nitric oxide synthase (NOS)[1,2]. NO is an important biological signalling molecule that influences circulation by regulating vascular smooth muscle tone and modulating systemic blood pressure. Furthermore, NO is involved in neurotransmission; it is a critical factor in the inflammatory response and immunity [3-5]; and it has been shown to exert positive effects on mucosal defence in the gastrointestinal system. In several studies (for review, see [6]), chemically induced mucosal damage seemed to be reduced by simultaneous addition of NO and impaired by removal of NO from the gastric mucosa. An explanation for those findings might be that NO increases mucosal blood flow[7], and it has been suggested that NO augments the release of mucus[8]. It is likely that NO is also involved in the regulation of other secretory processes in the gastrointestinal system. Takeuchi and co-workers [9] have reported that NO inhibits the secretion of duodenal bicarbonate, whereas other investigators have proposed that bicarbonate secretion is stimulated by NO [10,11]. In addition, several studies have indicated that NO affects the secretion of gastric acid [12-16]. Animal experiments have provided conflicting information about the interaction between NO and gastric acid secretion. For instance, studies in vitro have shown that NO stimulates secretion of gastric acid in the mouse[17,18] and bullfrog[19]. In addition, similar results have been obtained in dogs [12]. However, other investigations have shown that NO inhibits gastric acid secretion in the rat [13,14], in gastric glands isolated from rabbits [15], and in mucosa from toads [16]. Studies of humans have provided data indicating that NO can both inhibit and augment intragastric pH [20,21], but it is not yet known how this compound participates in gastric acid secretion in humans. In an earlier study, we found morphological support that endogenous NO plays a role in regulation of parietal cell function [22]. Also, the immunohistochemical data from that investigation revealed the presence of endogenous NOS in epithelial cells of the normal human oxyntic mucosa, more precisely, in both surface mucous cells and endocrine cells. In addition, we observed that there were close contacts between eNOS-positive cells and parietal cells either because the eNOS-positive cells contacted parietal cells via cytoplasmic processes or were invaginated by a parietal cell. Based on these findings, together with the chemical properties of NO, we concluded that NO derived from the endocrine-like cells might be a paracrine regulator of gastric acid secretion. In the present study, our aim was to verify the effect of exogenous NO on histamine- and cAMP-stimulated gastric acid secretion in humans, and also to determine whether endogenously derived NO has a functional effect on human parietal cells. Methods Subjects and ethical approval Twenty-four healthy men ranging in age from 22 to 31 years were recruited as paid volunteers. The selection criteria stipulated that the subjects had to be free from disease and should not have taken any medications or imbibed alcohol for at least one week prior to examination. The men fasted for at least six hours before examination. Pharyngeal anaesthesia was induced with lidocaine spray (Xylocain®, AstraZeneca, Södertälje, Sweden), after which routine gastroscopy was performed using an Olympus GIF-100 endoscope. Pinch biopsy forceps (Olympus FB 24K-1) were used to take tissue specimens from the greater curvature, immediately distal to the fundus. In all subjects, the gastric mucosa appeared to be normal, both macroscopically and histologically. All subjects tested negative for Helicobacter pylori infection in the urease breath test (Diabact® UBT 50 mg 13C-urea, Diabact AB, Uppsala, Sweden). The experimental procedures were approved by the Regional Ethics Committee for Human Research at University Hospital, Linköping, Sweden (File no. 02-039), and all subjects gave informed consent. Secretory study Isolation and incubation of gastric glands The current experiments were based on a technique that was first described in 1976 for use in rabbits in vitro [23] and is now well established for indirect determination of gastric acid secretion induced by various stimuli. The method of isolating gastric glands was initially developed for animal tissue, but it was later refined so that it could also be applied to small amounts of human tissue [24]. The human oxyntic mucosal biopsies used in our study were washed and stored no longer than 15 minutes in ice-cold oxygenated phosphate-buffered saline (PBS). The tissue specimens were cut into smaller pieces with a pair of scissors and transferred to oxygenated (100% O2) collagenase enzyme solution (130.0 mM NaCl, 12.0 mM NaHCO3, 3.0 mM Na2HPO4, 3.0 mM K2HPO4, 2.0 mM MgSO4, 1.0 mM CaCl2, 0.1 mM N(alfa)-tosyl-L-lysine chloromethyl ketone [TLCK], 10 μM indomethacin, 10 mM glucose, 2 mg/ml human serum albumin [HSA; Sigma], and 1 mg/ml collagenase type IA [Sigma]). The mixture was placed in a 37°C water bath and was gently stirred for 120 minutes, after which most of the treated specimen had disintegrated, leaving mainly isolated gastric glands. The mixture was subsequently filtered through a 200 μm mesh. The isolated glands were washed and re-suspended in pre-warmed (37°C) respiratory medium (132.4 mM NaCl, 1.0 mM NaH2PO4, 1.2 mM MgSO4, 5.4 mM KCl, 5.0 mM Na2HPO4, 1.0 mM CaCl2, 10 μM indomethacin, 10 mM glucose, and 2 mg/ml HSA). The glands were then transferred to vials containing fresh respiratory medium to which we added one of the following: the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 1 mmol/L) or equivalent amounts of its biologically inactive enantiomer NG-nitro-D-arginine methyl ester (D-NAME); the NOS inhibitor NG-nitro-L-arginine (L-NNA; 0.1 mmol/L); either of the two NO donors sodium nitroprusside (SNP; 1 mmol/l) and S-nitroso-N-acetyl-penicillamine (SNAP; 0.1 mmol/L); the substrate for endogenous NO production, L-arginine (0.1 mmol/L) [15]. All gland suspensions, including those that were not stimulated, were incubated in a shaking water bath at 37°C for 30 minutes, after which we added histamine to a final concentration of 50 μmol/L or dibutyryl-cAMP (db-cAMP) to a final concentration of 1 mmol/L. To prevent degradation of cyclic nucleotides, we added 0.1 mmol/L 3-isobutyl-1-methylxantine (IBMX) to all stimulations. Determination of the [14C]aminopyrine accumulation ratio A well-established method used to indirectly measure acid secretion by isolated gastric glands is to determine accumulation of 14C-labeled aminopyrine (AP) in the glands themselves and in the supernatant after centrifugation and then calculate the ratio between those two values (called the AP ratio) [23]. In short, acid secretion was stimulated at 37°C for 40 minutes, and after that 0.5 μCi [14C]labeled aminopyrine was added to the vials, which were then further incubated at 37°C for 90 minutes. Thereafter, the gland suspension was transferred to previously dried and weighed tubes, which were centrifuged at 4,000 rpm for two minutes. The supernatant was removed and transferred to scintillation vials. The pellets (glands) were dried at 100°C, and the dry weight was determined, and the glands were subsequently re-suspended in 0.5 mol/L NaOH at 60°C and transferred to scintillation vials. The radioactivity of the glands and the supernatant was determined in a liquid scintillation counter (1214 Rackbeta, LKB), and the AP ratio was calculated using the following formula[24]: where IGW is intraglandular water volume (= 2 × the dry weight of glands in mg). Background accumulation of AP is included in the values representing the secretory response. The AP ratios for background and histamine-and db-cAMP-stimulated conditions differed between the individuals. Therefore, for each subject, we determined the AP ratio for stimulated glands and considered that value to be 100% and used it as an individual reference value. All values are based on single analyses. Statistics Data were analysed by one-sample sign tests comparing median values using MINITAB™ Statistical Software. P values less than 0.05 were considered significant. Immunohistochemistry Isolated gastric glands from five test subjects were placed on charged Super Frost*/Plus glass slides (Menzel-Gläser, Germany) and then washed with PBS and permeabilized with 100% ethanol at -70°C for 5 min. Thereafter, the slides were incubated at room temperature overnight with rabbit anti-NOS3 antibody (1:1000; Santa Cruz Biochemicals) and then washed thoroughly in PBS and incubated for 1 h with biotinylated goat anti-rabbit secondary antibody. Slides where primary antibody had been left out served as negative controls. The slides were subsequently washed again, and biotinylated antibody was detected by exposure to 20 μg/ml Texas Red® Avidin (Vector Laboratories) for 1 h. Following that treatment, the slides were washed and coverslipped using Vectashield® mounting medium. A Nikon Eclipse® E800 fluorescence microscope with a VFM EPI-fluorescence attachment was used to examine and evaluate the slides. A band-pass filter with a wavelength range of 520–560 nm and a long-pass filter with cut-on wavelength at 590 nm (for emitted light) were employed to visualize the Texas Red® molecules. Hematoxylin and eosin staining For morphological evaluation, glands were fixed on glass slides and stained with Harris hematoxylin for five minutes and 0.5% eosin Y for two minutes. Each step was followed by a rinse in tap water. Results We studied the effects of NO on acid secretion induced by various stimulants in gastric glands isolated from stomach biopsies from human. Morphological examination of the hematoxylin-eosin-stained slides revealed that the isolation procedure had successfully yielded whole-gland preparations and that parietal cells were present in the gastric glands. Immunohistochemical analysis showed that the isolated glands contained eNOS-immunoreactive cells (Fig. 1), which agrees with results obtained using other types of mucosal preparations [22]. Control experiments were primary antibody was excluded showed no immunoreactivity. Figure 1 Immunofluorescence of an isolated gastric gland. Immunolocalization of eNOS (arrowheads) in a gastric gland isolated from human oxyntic mucosa was achieved using a rabbit anti-eNOS polyclonal antibody. The results were visualized with Texas Red®-conjugated goat anti-rabbit IgG. Bar = 30 μm. Background AP-accumulation Background AP accumulation was observed in the isolated glands, with a median AP ratio of 8.6 (range 2.5–22.1; n = 19). After administration of 50 μmol/L of histamine or 1 mmol/L of db-cAMP, the median AP ratios were 24.7 (5.8–64.5; n = 16) and 38.2 (range 7.6–47.8; n = 11) respectively. The response to both histamine and db-cAMP exceeded the background by a factor of about 2–4 in all preparations (Figs. 2 and 3). Figure 2 Accumulation of 14C-labeled aminopyrine in histamine-stimulated gastric glands. All values are expressed as percent of the gastric acid secretion induced by histamine (considered to be 100%), which was calculated separately for gastric glands isolated from each of the healthy volunteers. Each symbol represents the results for one individual. a) Accumulation of 14C-aminopyrine in glands pretreated with the NO donor sodium nitroprusside (SNP, 1 mmol/L) or with L-arginine (0.1 mmol/L), the substrate for endogenous NO production. It can be seen that SNP markedly reduced AP accumulation (median = 48%; p < 0.05), which indicates that NO inhibits acid secretion from the isolated glands. Background 14C-aminopyrine accumulation (bg) is also shown. b) Accumulation of 14C-aminopyrine in gastric glands pretreated with the NOS inhibitors L-NNA (0.1 mmol/L) and L-NAME (1 mmol/L), respectively. L-NAME caused increased accumulation (median = 147%; p < 0.05), which suggests that acid secretion is elevated when endogenous NO production is prevented, indicating an inhibitory role for endogenous NO in human gastric glands. D-NAME, which is the biologically inactive stereo isomer of L-NAME, did not have an effect on acid secretion, and it was therefore used as a control substance. Figure 3 Accumulation of 14C-labeled aminopyrine in db-cAMP-stimulated gastric glands. All values are expressed as percent of a value representing the gastric acid secretion induced by db-cAMP (considered to be 100%), which was calculated separately for each of the studied subjects. Each symbol represents the data for one individual. a) Pretreatment with SNP (median = 63%; p < 0.05) or SNAP (median = 81%; p < 0.05) to release exogenous NO before adding db-cAMP to stimulate acid secretion reduced the accumulation of 14C-aminopyrine in gastric glands, as compared to levels of secretion seen in untreated glands. This indicates that NO can inhibit acid secretion in gastric glands isolated from humans. b) Treatment with L-NAME to inhibit NOS in the gastric glands increased the accumulation of 14C-aminopyrine after stimulation with db-cAMP (median = 152%; p < 0.05). Those results indicate that NO inhibits db-cAMP-induced acid secretion. The NO substrate L-arginine reduced the accumulation of 14C-aminopyrine in db-cAMP-stimulated glands (median = 77%; p < 0.05). Background accumulation (bg) is also shown. Effect of NO donors and NOS inhibitors on histamine-stimulated gastric acid secretion Pre-treatment of isolated glands with the exogenous NO donor SNP (1 mmol/L) reduced the histamine response to a median of 48% (n = 7) of the response seen in non-pre-treated glands (100%). In four out of five gland preparations, the substrate for endogenous NO formation, L-arginine (0.1 mmol/L), decreased the AP ratio. This effect however was not statistically significant (Fig. 2a). When isolated glands were pre-treated with NOS inhibitor L-NAME (1 mmol/L), and then exposed to histamine, the AP ratio was markedly elevated to a median value of 147% (n = 13). Although a small number of individuals were tested, L-NNA (0.1 mmol/L) yielded a similar result; 172% (n = 2). By comparison, in control experiments, the L-NAME analogue D-NAME (1 mmol/L) had no effect at all on histamine-stimulated acid secretion (Fig. 2b). Effect of NO donors and NOS inhibitors on db-cAMP-stimulated gastric acid secretion Exposing the isolated glands to db-cAMP increased the secretion of gastric acid compared to the background level. Compared to untreated glands, those that were pre-treated with SNP (1 mmol/L) or SNAP (0.1 mmol/L) accumulated less AP after stimulation with db-cAMP; 63%(n = 9) and 81%(n = 8) respectively (Fig. 3a). Moreover, similar to histamine-stimulated secretion, the db-cAMP-induced secretion was increased to a median of 152% (n = 9) in glands that had been pre-treated with L-NAME (1 mmol/L). Although no effect on L-arginine on histamine-stimulated acid secretion could be seen, there was a significant effect of L-arginine on db-cAMP-induced secretion. Acid output was inhibited to a median of 77% (n = 10) in those pre-treated with L-arginine (0.1 mmol/L)(Fig. 3b). Discussion The method of using isolated gastric glands in vitro is well suited for studying the interaction between gastric acid secretion and various endocrine signals. This technique has been thoroughly evaluated, chiefly in experiments on animals, and it has been reported to offer good reproducibility [23]. Furthermore, in a study of gastric glands isolated from stomach biopsies taken from humans [24], it was found that both histamine and db-cAMP induced secretory responses that were reproducible when repeated using gland preparations from the same subject, although there was considerable interindividual variation. Fellenius et al. [24] and Haglund et al. [25] have reported that both histamine and db-cAMP stimulated gastric acid secretion from isolated human gastric glands, and this was observed as a two- to threefold increase in AP accumulation compared to the background level. Those results agree with our data obtained using histamine and db-cAMP. It is known that SNP releases NO [26], and in our experiments SNP reduced secretion of gastric acid from isolated glands. Hence, NO inhibits acid secretion in isolated human gastric glands. Some of the effects of SNP may be due to cytotoxic interactions, although no such impact was found in a study of isolated rabbit gastric glands [15]. To rule out the possibility of a cytotoxic influence, we performed complementary experiments using the NO donor SNAP, which has chemical properties that differ from those of SNP. In those experiments, we observed the same reduction in AP accumulation after stimulation with db-cAMP, which further favours the conclusion that NO is in fact responsible for observed results. In db-cAMP-stimulated glands, the induced secretory response was reduced by L-arginine, a compound that depends on an endogenous factor (i.e., eNOS) to generate NO, although that reduction was not as pronounced as the decrease induced by SNP and SNAP. There are a number of possible explanations for that observation. If there was already enough L-arginine in the glands to sustain NO production at the time of the experiment, addition of L-arginine was therefore without effect. Furthermore, L-arginine may have had a weak impact because the effect of NO occurred through up- or down regulation of the enzyme NOS and was not influenced by access to substrate. Notwithstanding, L-arginine did decrease the accumulation of AP, albeit not as much as the exogenous NO donors did at the present doses. This indicates that some specific process is responsible for generating NO from L-arginine in isolated gastric glands. These results are consistent with studies showing that SNP, SNAP, and L-arginine inhibited histamine-stimulated acid secretion from gastric glands isolated from animals [13,15]. L-arginine has also been observed to reduce carbachol-stimulated acid secretion in toads [16]. In a previous study conducted by our research group [22], examination of the glandular epithelium of specimens of human oxyntic mucosa revealed that one particular type of cells contained NOS. These eNOS-immunoreactive cells, defined as endocrine cells, were in close contact with parietal cells. These two characteristics suggest that cells of this type release NO, which thus might be a paracrine regulator that directly affects the function of parietal cells. That assumption may be supported by a number of other conditions. For example, NO can easily penetrate cell membranes, which may indicate an intracellular site of action. Also, NO has a rather short life span, which implies that sources needed to generate this oxide must be available close to the NO target cell. In this study, the occurrence of eNOS in the glands is shown, but earlier extensive investigations using antibodies against both nNOS and iNOS have not revealed presence of any of the two isoforms in the glandular epithelium of normal human subjects (unpublished observation). Similar to results obtained in a study of isolated rabbit gastric glands[15], we found that the NOS inhibitors L-NAME and L-NNA, but not D-NAME, amplified the secretion-stimulating effect of histamine, which further indicates that the isolated human glands we used contained the enzyme NOS. Both the increase in the AP ratio that we observed following inhibition of NOS and the decrease in secretory responses that we noted in glands treated with L-arginine strengthen the hypothesis that NO is produced by cells in the glandular epithelium and, when released, it interferes with stimulated acid secretion. Interestingly, the mentioned observations suggest that the release of NO is sustained, regardless of whether acid secretion is stimulated, which implies that NO functions as an endogenous inhibitor of gastric acid secretion. The intensity of this inhibition probably depends on the number of eNOS-containing endocrine-like cells that are present in the vicinity of the parietal cells. The exact mechanisms behind this paracrine regulation of gastric acid secretion is yet to be elucidated. There are several different pathways within the parietal cell that might be affected by NO. It can induce ADP ribosylation of G-actin [27], thereby influencing the cytoskeleton. This could be essential for the morphological changes that parietal cells exhibit during acid secretion. Accordingly, if NO does have a persistent impact on an element such as the cytoskeleton, it might play a role in the membrane recycling hypothesis proposed by Forte et al. [28]. Briefly, that theory suggests that parietal cells undergo the following morphological alterations: they have a large active secretory surface during the stimulatory phase, and they display a minimal active secretory surface during the resting phase. Since it is known that guanylate cyclase is a general target of NO in many cell systems, some investigators have suggested that NO exerts its effects via cyclic guanosine 3',5'-monophosphate (cGMP) in both rat and rabbit parietal cells [13,15]. An ongoing study in our laboratory will show whether this is the case in human parietal cells. Downstream effects of cGMP may include activation of a number of effectors, such as ion channels, protein kinases, and phosphodiesterases [29]. It is also plausible that NO can exert its effect alone, without acting through other signalling molecules. Under experimental conditions, NO can induce nitrosylation and nitration of cellular proteins, although that is probably not the case in vivo, since those two processes often result permanent damage to vital functions [30]. There is a possibility that the suppression of acid secretion occurs not only at parietal cell level, but via other cell types. ECL-cells are probably present in the glandular preparation and in the rat, these cells have the ability to release histamine in response to increased intracellular levels of cAMP [31]. NO can inhibit this histamine-release [14] and thereby further contribute to the inhibition of acid secretion. Although there is little known about human ECL-cells and the effects of NO on histamine-release there are studies that indicate species differences in other histamine-secreting cells. For example, rat mast cells have been shown to produce an "NO-like factor" which inhibits histamine-release [32] while there are investigations that indicate that NO does not affect histamine-secretion in human basophils [33]. At present we can only establish differences in inhibitory response to l-arginine for histamine and db-cAMP stimulation. Further investigations are needed to clarify the role of NO in parietal cell function. Conclusions The findings of the present study suggest that NO produced endogenously in the human oxyntic mucosa can reduce the stimulatory effects of histamine or db-cAMP on gastric acid secretion. We obtained uniform results for gastric glands isolated from different healthy human subjects, which implies that NO released from specific cells within the secretory mucosa plays an important physiological role in the regulation of gastric acid secretion. Competing interests None declared. Authors' contributions AB participated in the design and coordination of the study, performed the secretory studies, carried out the immunohistochemical procedures, and drafted the manuscript. SR was the surgeon in charge and carried out all gastroscopic procedures. ACE and SES were involved in the design of the study and in drafting of the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements We thank Ulf Hannestad for carrying out the urease breath tests, Inga-Lill Andersson and Gunilla Strand for help with the gastroscopic procedures, and Marja Tjädermo for technical assistance. This work was supported by AstraZeneca R & D, Sweden. ==== Refs Bredt DS Snyder SH Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme Proc Natl Acad Sci U S A 1990 87 682 5 1689048 Palacios M Knowles RG Palmer RM Moncada S Nitric oxide from L-arginine stimulates the soluble guanylate cyclase in adrenal glands Biochemical & Biophysical Research Communications 1989 165 802 9 2480784 Furchgott RF Zawadzki JV The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine Nature 1980 288 373 6 6253831 Ignarro LJ Buga GM Wood KS Byrns RE Chaudhuri G Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide Proceedings of the National Academy of Sciences of the United States of America 1987 84 9265 9 2827174 Moncada S Palmer RM Higgs EA Nitric oxide: physiology, pathophysiology, and pharmacology Pharmacological Reviews 1991 43 109 42 1852778 Calatayud S Sanz MJ Canet A Bello R de Rojas FD Esplugues JV Mechanisms of gastroprotection by transdermal nitroglycerin in the rat British Journal of Pharmacology 1999 127 1111 8 10455256 Wallace JL Granger DN The cellular and molecular basis of gastric mucosal defense FASEB Journal 1996 10 731 40 8635690 Brown JF Hanson PJ Whittle BJ Nitric oxide donors increase mucus gel thickness in rat stomach European Journal of Pharmacology 1992 223 103 4 1335874 10.1016/0014-2999(92)90824-N Takeuchi K Ohuchi T Miyake H Niki S Okabe S Effects of nitric oxide synthase inhibitors on duodenal alkaline secretion in anesthetized rats European Journal of Pharmacology 1993 231 135 8 7680316 10.1016/0014-2999(93)90694-D Sababi M Nilsson E Holm L Mucus and alkali secretion in the rat duodenum: effects of indomethacin, N omega-nitro-L-arginine, and luminal acid Gastroenterology 1995 109 1526 34 7557135 Holm M Johansson B Pettersson A Fandriks L Acid-induced duodenal mucosal nitric oxide output parallels bicarbonate secretion in the anaesthetized pig Acta Physiologica Scandinavica 1998 162 461 8 9597112 10.1046/j.1365-201X.1998.0307f.x Bilski J Konturek PC Konturek SJ Cieszkowski M Czarnobilski K Role of endogenous nitric oxide in the control of gastric acid secretion, blood flow and gastrin release in conscious dogs Regulatory Peptides 1994 53 175 84 7846293 10.1016/0167-0115(94)90166-X Brown JF Hanson PJ Whittle BJ The nitric oxide donor, S-nitroso-N-acetyl-penicillamine, inhibits secretory activity in rat isolated parietal cells Biochemical & Biophysical Research Communications 1993 195 1354 9 8216268 10.1006/bbrc.1993.2192 Kato S Kitamura M Korolkiewicz RP Takeuchi K Role of nitric oxide in regulation of gastric acid secretion in rats: effects of NO donors and NO synthase inhibitor British Journal of Pharmacology 1998 123 839 46 9535011 Kim H Kim KH Effects of a nitric oxide donor and nitric oxide synthase inhibitors on acid secretion of isolated rabbit gastric glands Pharmacology 1996 53 331 9 9032797 Molero M Hernandez IM Lobo P Cardenas P Romero R Chacin J Modulation by nitric oxide of gastric acid secretion in toads Acta Physiologica Scandinavica 1998 164 229 36 9805110 10.1046/j.1365-201X.1998.00413.x Hasebe K Horie S Yano S Watanabe K Inhibitory effect of N(omega)-nitro-L-arginine on gastric secretion induced by secretagogues and vagal stimulation in the isolated stomach European Journal of Pharmacology 1998 350 229 36 9696412 10.1016/S0014-2999(98)00248-9 Hasebe K Horie S Komasaka M Yano S Watanabe K Stimulatory effects of nitric oxide donors on gastric acid secretion in isolated mouse stomach Eur J Pharmacol 2001 420 159 64 11408038 10.1016/S0014-2999(01)00995-5 Kawauchi S Sugamoto S Furukawa O Mimaki H Takeuchi K Stimulation by nitric oxide of gastric acid secretion in bullfrog fundic mucosa in vitro J Physiol Pharmacol 2001 52 93 105 11321516 Hirsch DP Tiel-Van Buul MM Tytgat GN Boeckxstaens GE Effect of L-NMMA on postprandial transient lower esophageal sphincter relaxations in healthy volunteers Dig Dis Sci 2000 45 2069 75 11117586 10.1023/A:1005611221617 Konturek JW Fischer H Gromotka PM Konturek SJ Domschke W Endogenous nitric oxide in the regulation of gastric secretory and motor activity in humans Alimentary Pharmacology & Therapeutics 1999 13 1683 91 10594405 10.1046/j.1365-2036.1999.00598.x Berg A Kechagias S Sjostrand SE Ericson AC Morphological support for paracrine inhibition of gastric acid secretion by nitric oxide in humans Scand J Gastroenterol 2001 36 1016 21 11589372 10.1080/003655201750422594 Berglindh T Obrink KJ A method for preparing isolated glands from the rabbit gastric mucosa Acta Physiol Scand 1976 96 150 9 1258668 Fellenius E Elander B Wallmark B Haglund U Helander HF Olbe L A micro-method for the study of acid secretory function in isolated human oxyntic glands from gastroscopic biopsies Clin Sci (Lond) 1983 64 423 31 6297844 Haglund U Elander B Fellenius E Leth R Rehnberg O Olbe L The effects of secretagogues of isolated human gastric glands Scand J Gastroenterol 1982 17 455 60 6182608 Butler AR Glidewell C McGinnis J Bisset WI Further investigations regarding the toxicity of sodium nitroprusside Clin Chem 1987 33 490 2 3829378 Clancy R Leszczynska J Amin A Levartovsky D Abramson SB Nitric oxide stimulates ADP ribosylation of actin in association with the inhibition of actin polymerization in human neutrophils Journal of Leukocyte Biology 1995 58 196 202 7643014 Forte TM Machen TE Forte JG Ultrastructural changes in oxyntic cells associated with secretory function: a membrane-recycling hypothesis Gastroenterology 1977 73 941 55 902958 McDonald LJ Murad F Nitric oxide and cGMP signaling Advances in Pharmacology (New York) 1995 34 263 75 Hanafy KA Krumenacker JS Murad F NO, nitrotyrosine, and cyclic GMP in signal transduction Med Sci Monit 2001 7 801 19 11433215 Chen D Marvik R Ronning K Andersson K Waldum HL Hakanson R Gastrin-evoked secretion of pancreastatin and histamine from ECL cells and of acid from parietal cells in isolated, vascularly perfused rat stomach. Effects of isobutyl methylxanthin and alpha-fluoromethylhistidine Regulatory Peptides 1996 65 133 138 8884980 Salvemini D Masini E Pistelli A Mannaioni PF Vane J Nitric oxide: a regulatory mediator of mast cell reactivity Journal of Cardiovascular Pharmacology 1991 17 S258 S264 Peh KH Moulson A Wan BY Assem EK Pearce FL Role of nitric oxide in histamine release from human basophils and rat peritoneal mast cells European Journal of Pharmacology 2001 425 229 238 11513842 10.1016/S0014-2999(01)01205-5

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BMC Gastroenterol. 2004 Aug 6; 4:16

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==== Front BMC GastroenterolBMC Gastroenterology1471-230XBioMed Central London 1471-230X-4-171530789310.1186/1471-230X-4-17Research ArticleSynergic effect of chronic hepatitis C infection and beta thalassemia major with marked hepatic iron overload on liver fibrosis: a retrospective cross-sectional study Ardalan Farid Azmoudeh [email protected] Mohammad RF [email protected] Mohsen N [email protected] Guiti [email protected] Department of Pathology, Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, Iran2 Department of Gastroenterology, Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, Iran2004 12 8 2004 4 17 17 24 4 2004 12 8 2004 Copyright © 2004 Ardalan et al; licensee BioMed Central Ltd.2004Ardalan et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Increased hepatic iron is assumed to potentiate progression towards liver fibrosis in chronic hepatitis C virus (HCV) infection. In this study we have evaluated the potentiating effect of marked hepatic iron overload and chronic HCV infection on hepatic fibrosis in thalassemic patients. Methods Liver biopsies of one group of patients with beta thalassemia major and chronic HCV infection (group 1) was compared with two groups of patients (groups 2&3) with either chronic HCV infection or thalassemia major, respectively (20 patients in each group). Necroinflammation, fibrosis, and iron overload were graded and compared. Results Stage of fibrosis in group 1 patients was significantly higher than the other two groups (p < 0.05). Necroinflammatory grade was significantly lower, but iron score was significantly higher in thalassemic patients (group 3) in comparison to groups 1 and 2 (p < 0.05). Conclusion Our results indicate that marked liver iron overload and HCV infection in thalassemic patients have potentiating effect on hepatic fibrogenesis. ==== Body Background Thalassemia major is an inherited disorder particularly common in people of Mediterranean, African, and South-east Asian ancestry. It is characterized by decreased production of beta chain of hemoglobin. Clinical features result from anemia, markedly expanded marrow space, and transfusional and absorptive iron overload. In these patients iron overload is often inevitable, especially when iron chelating agents are not used properly [1]. Hepatic iron overload leads to different degrees of liver fibrosis, the severity of which is closely correlated with the severity of liver iron overload [2]. The pattern of iron deposition seen in the initial stages of thalassemia major is preferentially sinusoidal with a more or less diffuse distribution within the acinus. With significant loading, hepatocytes, bile duct epithelia, and fibrous tissue of portal tracts or septa will also show iron deposition [3]. HCV is responsible for 80–90% of post-transfusional cases of hepatitis in patients who have received blood transfusion(s) prior to the introduction of routine blood products screening in 1990 [4]. More than 75% of HCV infections become chronic and up to 20–30% progress to cirrhosis [3-5]. Increased hepatic iron may potentiate progression towards liver fibrosis in chronic HCV infection, and may contribute to poor response to interferon therapy [3,6,7]. Acceleration of hepatic fibrosis in patients with combined hereditary hemochromatosis (HH) and chronic hepatitis C infection has also been shown [7]. Nevertheless, the pattern of iron deposition in HH is initially hepatocellular and different from that of thalassemia major. In this study we have evaluated the potentiating effect of marked hepatic iron overload and chronic HCV infection on hepatic fibrosis in thalassemic patients. To the best of our knowledge this synergic effect has been studied just in another study on bone marrow transplanted thalassemic patients [8]. Methods A retrospective cross-sectional study was performed on sixty patients in three different groups as outlined below: Group 1: Twenty patients (10 males, 10 females) with the diagnosis of thalassemia major and chronic HCV infection (BTM/CHI). The only risk factor of HCV infection in these patients was blood transfusion before 1990. Group 2: Twenty patients (13 males, 7 females) with chronic HCV infection (CHI). The route of infection and the duration of disease were not known in most of the patients. Group 3: Twenty patients (10 males, 10 females) with thalassemia major. Thalassemia major was diagnosed by appropriate clinical and laboratory findings, and confirmed by hemoglobin electrophoresis. All the thalassemic patients had received multiple blood transfusions since childhood, and only those with at least moderate hepatic iron overload (2/4 based on Marx and Sindram hepatic iron scoring [9]) were included. CHI was confirmed by positive anti-HCV (enzyme-linked immunosorbant assay), a positive HCV RNA by polymerase chain reaction and appropriate findings on liver biopsy. Patients with history of alcohol intake, smoking, positive HIV serology, positive HBs antigen or HBc antibody, and other liver diseases (e.g., autoimmune hepatitis, drug hepatotoxicity, Wilson's disease, alpha-1 antitrypsin deficiency, hereditary hemochromatosis) were excluded from all the three groups. All of the patients whose liver biopsies were submitted to the Central Pathology Department of Imam Khomeini Hospital (a Terhan University of Medical Sciences affiliated hospital) between April 2001 and January 2004 were retrieved. During this period, there were only 20 thalassemic patients with moderate to marked hepatic iron overload, so all of them were included in our study. Since the sex and age distribution of patients with thalassemia major and BTM/CHI were relatively the same, 20 patients with BTM/CHI and at least moderate hepatic iron overload were randomly selected from all the patients with this diagnosis. On the other hand, patients with CHI had a considerably higher age, so to match the age of the patients, we selected 20 of the youngest patients. For all the sixty patients, we had hematoxylin and eosin, Masson's trichrome and Perls' Prussian blue stains. All the slides were reviewed by a single pathologist (FAA) who was blind to the diagnoses. The necroinflammation, fibrosis, and iron deposition were scored using modified Hepatitis Activity Index (HAI) grading, modified HAI staging, and Marx/Sindram hepatic iron scoring systems, respectively [9,10]. Presence or absence of macrovesicular steatosis was also assessed. Statistical analysis The results are presented as mean value +/- standard deviation (for age), median and range values (for modified HAI grade, stage, and iron score) and percentages (for steatosis). Group comparisons were made using the two-tailed independent Student's t-test for age and two-sided Fisher's exact or Chi2 tests for other variables with p < 0.05 considered to be significant. For each patient with BTM/CHI (doubly exposed cases), there were two different unexposed cases, i.e. thalassemic cases (unexposed to HCV) and CHI cases (unexposed to iron overload). Considering "disease" as higher stages of fibrosis (modified HAI stage ≥ 3), our guesstimate of the expected frequency of disease in unexposed and exposed patients were about 25% and 75%, respectively. So the sample size was calculated as about 20 patients for each group with confidence level of 95% and power of 80%. Results and Discussion The results of the study are summarized in table 1. This study supports the synergic effects of CHI and marked hepatic iron deposition in thalassemic patients on liver fibrosis. The patients with BTM/CHI had higher stages of liver fibrosis in comparison to patients with either CHI or thalassemia major alone. The patients with BTM/CHI had higher stages of fibrosis despite lower scores for iron overload in comparison to thalassemic patients. The potentiating effect of hepatic iron overload and CHI has been shown in other studies [7,8,11,12]. The cause of iron overload in the majority of studies has been hereditary hemochromatosis or HFE mutations. Table 1 Demographic and histopathologic findings in the three groups Group 1 Group 2 Group 3 P value Group 1 vs. 2 Group 1 vs. 3 Age(years)* 21.55+/-4.74 29.7+/-9.16 18.7+/-4.45 0.001 0.057 Sex (M/F) 10/10 13/7 10/10 0.33 1 Modified Stage‡ 3 (1–6) 2 (0–6) 2 (1–4) 0.02 0.01 Modified HAI grade‡ 5 (1–8) 6 (2–11) 2 (0–6) 0.36 0.001  Periportal inflammation‡ 1.5 (0–3) 2 (0–4) 0 (0–2) 0.77 0.004  Confluent Necrosis‡ 0 (0–1) 0 (0–2) 0 (0–1) 0.13 0.091  Focal Necrosis‡ 1 (0–2) 2 (1–2) 1 (0–2) 0.10 0.45  Portal Inflammation‡ 2 (0–3) 2 (1–4) 1 (0–2) 0.87 0.002 Iron Score‡ 3 (2–4) 0 (0–1) 4 (2–4) 0.000 0.024 Steatosis (%) 10 20 0 0.66 0.48 * mean+/-standard deviation ‡Median (minimum-maximum) Group 1: Patients with beta thalassemia major and chronic HCV infection Group 2: Patients with chronic HCV infection Group 3: Patients with beta thalassemia major Angelucci et al showed for the first time the role of iron overload and HCV positivity as independent risk factors for hepatic fibrosis progression in thalassemic patients following successful bone marrow transplantation [8]. The Angelucci's study has a few advantages over our study: firstly, serial liver biopsies have been studied and the rate of liver fibrosis progression assessed. Secondly, hepatic iron concentration was used instead of scoring of stainable iron. Thirdly, two pathologists reviewed the slides independently. And finally, the sample size was much larger, but that study does not show whether the rate of hepatic fibrosis progression in BTM/CHI patients is greater than patients with only CHI. The potentiating effect of iron overload and HCV infection can be explained by the fact that both these agents produce oxidative stress in the liver [7,13]. In our study the grade of necroinflammation in BTM/CHI patients was not significantly different from CHI patients. Other studies have shown the same results [3,7]. Limitations of our study were that it was a retrospective study with limited number of patients in each group. The duration of HCV infection and genotype of virus were not known in our patients. The routes of infection in most of the CHI patients were not known and where probably different from our thalassemic patients. The age of our CHI group was significantly higher than the other two groups. Since older age at the time of infection is considered to be a risk factor for progression of chronic hepatitis C, the stage of fibrosis should have been higher in our CHI patients than BTM/CHI patients if iron had not had any potentiating effect on liver fibrosis. Our study was performed on thalassemic patients with moderate to severe liver iron deposition. At these stages of iron overload, the pattern of hepatic iron deposition in HH and thalassemic patients are nearly the same. So this study does not reveal the effect of only sinusoidal iron deposition on progression of fibrosis in HCV infected thalassemic patients. Conclusions Our results show that moderate to severe liver iron overload and chronic HCV infection in thalassemic patients have potentiating effect on hepatic fibrogenesis. So the proper use of chelating agents in HCV-infected thalassemic patients seems to be of great importance in delaying progression of the liver disease. The presence of liver siderosis has been shown to be related to poor response to interferon alpha (IFN) in non-thalassemic patients [3,6,7,12,14,15]; hence one can expect a poor response to IFN therapy because of transfusion related siderosis in thalassemic patients. However studies have shown that in thalassemic subjects, there is a promising response to IFN therapy (as high as 50% sustained response in some series) [16,17]. Perhaps in these patients (BTM/CHI), the therapeutic protocol for chronic HCV infection should differ from those without significant iron overload, but further studies are needed to confirm or refute these suggestions. Competing interests None declared. Authors' contributions FAA reviewed all of the slides, participated in study design and statistical analysis of results, and prepared the manuscript. MRFO assisted in collection and entering of the data, cooperated in reviewing slides, statistical analysis of results, and preparing the manuscript. MN was the clinical consultant. GI reviewed some of the problematic slides. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements This study was Dr. Mohammad RF Osquei's postgraduate thesis. The authors wish to thank Tehran Thalassemia Clinical Center for providing the clinical information of some of the thalassemic patients. ==== Refs Forget BG Hoffman R, Benz E J, Shathl S J, Furie B, Cohen H J, Silberstein L E and McGlare P Thalassemia syndromes Hematology basic principles and practice 2000 3rd Philadelphia, Churchill Livingstone 485 510 Risdon RA Barry M Flynn DM Transfusional iron overload: the relationship between tissue iron concentration and hepatic fibrosis in thalassaemia J Pathol 1975 116 83 95 1151528 Ferrel LD Theise ND Scheuer PJ MacSween R N M, Burt A D, Portmann B C, Ishak K G, Scheuer P J and Anthony P P Acute and chronic viral hepatitis Pathology of liver 2002 4th London, Churchill Livingstone 344 349 Costello M Yungbluth M Henry J B Viral infections Clinical diagnosis and management by laboratory methods 2001 20th Philadelphia, W. B. Saunders 1064 1066 Dore GJ Freeman AJ Law M Kaldor JM Is severe liver disease a common outcome for people with chronic hepatitis C? J Gastroenterol Hepatol 2002 17 423 430 11982723 10.1046/j.1440-1746.2002.02730.x Fargion S Fracanzani AL Rossini A Borzio M Riggio O Belloni G Bissoli F Ceriani R Ballare M Massari M Trischitta C Fiore P Orlandi A Morini L Mattioli M Oldani S Cesana B Fiorelli G Iron reduction and sustained response to interferon-alpha therapy in patients with chronic hepatitis C: results of an Italian multicenter randomized study Am J Gastroenterol 2002 97 1204 1210 12014729 Diwakaran HH Befeler AS Britton RS Brunt EM Bacon BR Accelerated hepatic fibrosis in patients with combined hereditary hemochromatosis and chronic hepatitis C infection J Hepatol 2002 36 687 691 11983453 10.1016/S0168-8278(02)00018-1 Angelucci E Muretto P Nicolucci A Baronciani D Erer B Gaziev J Ripalti M Sodani P Tomassoni S Visani G Lucarelli G Effects of iron overload and hepatitis C virus positivity in determining progression of liver fibrosis in thalassemia following bone marrow transplantation Blood 2002 100 17 21 12070002 10.1182/blood.V100.1.17 Lee GR Lee G R Storage and metabolic disorders Diagnostic liver pathology 1994 1st St. Louis, Mosby 237 280 Ishak K Baptista A Bianchi L Callea F De Groote J Gudat F Denk H Desmet V Korb G MacSween RN et al. Histological grading and staging of chronic hepatitis J Hepatol 1995 22 696 699 7560864 10.1016/0168-8278(95)80226-6 Tung BY Emond MJ Bronner MP Raaka SD Cotler SJ Kowdley KV Hepatitis C, iron status, and disease severity: relationship with HFE mutations Gastroenterology 2003 124 318 326 12557137 10.1053/gast.2003.50046 Fong TL Han SH Tsai NC Morgan TR Mizokami M Qian D Phan C Goad K Redeker AG A pilot randomized, controlled trial of the effect of iron depletion on long-term response to alpha-interferon in patients with chronic hepatitis C J Hepatol 1998 28 369 374 9551672 10.1016/S0168-8278(98)80308-5 Britton RS Metal-induced hepatotoxicity Semin Liver Dis 1996 16 3 12 8723319 Olynyk JK Reddy KR Di Bisceglie AM Jeffers LJ Parker TI Radick JL Schiff ER Bacon BR Hepatic iron concentration as a predictor of response to interferon alfa therapy in chronic hepatitis C Gastroenterology 1995 108 1104 1109 7698578 Van Thiel DH Friedlander L Fagiuoli S Wright HI Irish W Gavaler JS Response to interferon alpha therapy is influenced by the iron content of the liver J Hepatol 1994 20 410 415 8014455 Di Marco V Lo Iacono O Almasio P Ciaccio C Capra M Rizzo M Malizia R Maggio A Fabiano C Barbaria F Craxi A Long-term efficacy of alpha-interferon in beta-thalassemics with chronic hepatitis C Blood 1997 90 2207 2212 9310471 Mirmomen S Ghofrani H Pishbijary HF Daryani NE Farahvash J Sharifian RA Azmodeh F Malekzadeh R Safety and efficacy of interferon alfa for the treatment of chronic hepatitis C infected subjects with transfusion dependent thalassemia in Iran Medical Journal of the Islamic Republic of Iran 2003 17 87 95

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BMC Gastroenterol. 2004 Aug 12; 4:17

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==== Front BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-4-271531040110.1186/1471-2334-4-27Research ArticlePatterns of geohelminth infection, impact of albendazole treatment and re-infection after treatment in schoolchildren from rural KwaZulu-Natal/South-Africa Saathoff Elmar [email protected] Annette [email protected] Jane D [email protected] Chris C [email protected] Department of Nutrition, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115 USA2 Danish Bilharziasis Laboratory, Jaegersborg Allé 1D, DK-2920 Charlottenlund, Denmark3 Child, Youth and Family Development, Human Sciences Research Council, Private Bag X07, Dalbridge, 4014, South Africa4 School of Life and Environmental Sciences, University of KwaZulu-Natal, Durban 4041, South Africa2004 13 8 2004 4 27 27 23 6 2004 13 8 2004 Copyright © 2004 Saathoff et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Geohelminth infection is a major health problem of children from rural areas of developing countries. In an attempt to reduce this burden, the Department of Health of the province of KwaZulu-Natal (KZN) established in 1998 a programme for helminth control that aimed at regularly treating primary school children for schistosomiasis and intestinal helminths. This article describes the baseline situation and the effect of treatment on geohelminth infection in a rural part of the province. Methods Grade 3 schoolchildren from Maputaland in northern KZN were examined for infections with hookworm, Ascaris lumbricoides, and Trichuris trichiura, treated twice with 400 mg albendazole and re-examined several times over one year after the first treatment in order to assess the impact of treatment and patterns of infection and re-infection. Results The hookworm prevalence in the study population (83.2%) was considerably higher than in other parts of the province whereas T. trichiura and especially A. lumbricoides prevalences (57.2 and 19.4%, respectively) were much lower than elsewhere on the KZN coastal plain. Single dose treatment with albendazole was very effective against hookworm and A. lumbricoides with cure rates (CR) of 78.8 and 96.4% and egg reduction rates (ERR) of 93.2 and 97.7%, respectively. It was exceptionally ineffective against T. trichiura (CR = 12.7%, ERR = 24.8%). Re-infection with hookworm and A. lumbricoides over 29 weeks after treatment was considerable but still well below pre-treatment levels. Conclusion High geohelminth prevalences and re-infection rates in the study population confirm the need for regular treatment of primary school children in the area. The low effectiveness of single course albendazole treatment against T. trichiura infection however demands consideration of alternative treatment approaches. ==== Body Background Geohelminth infections are among the most prevalent diseases in developing countries. Children are the group with the highest prevalences and infection intensities and are also very vulnerable to the effects of worm infection. These include nutritional deficiencies [1,2] and impaired physical and mental development [3-5], resulting in additional insults to an already disadvantaged group. Thus, even though the proportion of cases that develops clinical disease is relatively low, the enormous number of infections warrants attention to this public health problem [6]. Consequently in 1998 the KwaZulu-Natal (KZN) Department of Health initiated a pilot helminth control programme which aimed to regularly apply treatment for intestinal helminths and schistosomiasis to primary school children [7]. This group was treated without prior screening of infection status. The rationale behind this and similar programmes elsewhere is not to eliminate infection, since this could most likely only be achieved by a combination of health education, improvements in sanitation, population chemotherapy and general development which would presently overburden most developing countries. Instead the aim is to keep infection intensities in this vulnerable age group low in order to prevent serious morbidity [8,9]. Our objectives were to describe the initial pattern of geohelminth infection, assess the impact of treatment with 400 mg albendazole and to monitor re-infection after treatment in order to develop recommendations for further local and regional control efforts. Methods Study area and population, treatment and ethics The study was conducted in central Ingwavuma district in northern KwaZulu-Natal (Figure 1). This area was selected because of its high geohelminth prevalences [10]. It is situated on both sides of the perennial Pongola river (Figure 2), covering approximately 28 × 16 km. The climate in the area is tropical to subtropical with a hot and wet summer (November – February) and a cooler and dry winter (June – August) (Figure 3). The study population was limited to one grade in order to keep disturbance of the school routine low. Grade 3 was selected because it should represent the infection situation in a primary school relatively well [11]. All pupils attending grade 3 at the start of the study in all ten primary schools in the area were eligible for participation with one exception: during the baseline survey two out of five and one out of four grade 3 classes in two large schools had to be excluded due to time constraints. These classes were however included during treatments and successive surveys. Otherwise only children who refused to participate or who were absent or unable to produce a specimen during each of our repeated visits were not included in the analysis of the respective surveys due to lack of data. They were however included for those parts of the study where they participated in order not to increase bias due to the likely difference in infection status between absentees and pupils who attended school [12]. High rates of absenteeism during treatment and stool collections thus result in variable sample sizes which are therefore reported together with the results. Characteristics of the pupils who took part in the first survey are given in Table 1. Treatment in all primary schools in the entire district was carried out by school nursing teams from the two local hospitals as part of the helminth control programme. Consenting children from all grades were treated for intestinal helminths with 400 mg albendazole (Zentel®, SmithKline Beecham). Treatment in the studied schools was administered in April and in October 1998. The study team assisted the nurses with treatment and also recorded those of the study population who were treated and those who were not. During the first round of treatment the pupils were also treated for schistosomiasis with 40 mg/kg praziquantel (Biltricide®, Bayer). However, results regarding schistosomiasis will be reported elsewhere. After the end of the study the participants were included in the normal treatment routine of the control programme. Ethical clearance was obtained from the Ethics Committee of the Faculty of Medicine of the University of Natal/Durban and the study was also approved by the Central Medical Ethics Committee in Denmark. Before the onset of the study, information meetings were held with the staff and parents of the schools in the study. At these meetings informed consent was obtained from the parents and the staff were asked for their unpaid co-operation. The children were asked for their consent directly before the first specimen collection. Specimen collection and processing After an initial survey to monitor the infection situation at baseline, two separate treatments were each followed by a survey to monitor treatment success 3 weeks later. Re-infection was assessed 16 weeks after the first and 18 and 29 weeks after the second treatment. The timing of treatments and assessments was done so as to accommodate the necessities of the treatment programme and was also restricted by the school terms. On our visits to the schools pupils were provided with sampling equipment and asked to provide a stool specimen. Each school was visited three times during each survey in order to include children who were absent or unable to deliver a specimen on the first occasion. Apart from the pre-treatment baseline survey, where only one specimen was collected due to the above mentioned time constraints, an effort was made to obtain two stool specimens per pupil. Pupils who provided only one stool specimen (between 7 and 14% in each post-treatment survey) were nevertheless included in the analysis in order not to increase bias. Specimens were kept cool until preparation of the slides. Duplicate 50 mg Kato-Katz cellophane thick smears were prepared from each faecal sample. They were examined by trained microscopists twice: first for hookworm eggs within one hour of preparation and later for A. lumbricoides and T. trichiura eggs within one day after preparation [13]. Accurate egg counts for both thick smears were recorded. Repeat counts by different microscopists were done on a sub sample of about 5% of the slides for quality control purposes. These counts revealed no bigger discrepancies. Diarrhoeal specimens and slides that were too dark were not examined. Instead the respective pupils were asked to provide another specimen. Infection intensities are expressed as eggs per gram of faeces (EPG) calculated as the arithmetic mean number of eggs per thick smear multiplied by 20. Statistics Data were double entered into Microsoft Excel 97 and corrected for data entry errors. Data analysis was carried out in SPSS 10.0.5 for Windows. Prevalence ratios (PR) and their 95% confidence intervals were calculated using the SPSS "Tables" procedure [14]. Cure rates (CR) and egg reduction rates (ERR) were calculated for the examinations three weeks after treatment using the formulae below [11]: Only data from the first of the two samples we received per pupil in each post-treatment survey were used for the above calculations in order to make them comparable to the baseline survey. Otherwise differences in sensitivity between surveys would have caused misleading results. Re-infection was however calculated for both obtained samples because the main purpose was not to compare with the baseline situation but to estimate trends in re-infection reliably. Results Infection patterns at baseline The cumulative prevalence of all three geohelminths combined was as follows: At baseline 90.0% of the pupils were infected with one or more species, 55.9% were infected with two or more species and 30.9% of the children were infected with all three geohelminths. Figure 4 shows the intensity distribution of the three helminths in the study population and Table 1 demonstrates significant differences between the sexes for A. lumbricoides infection. With a PR of 0.71, males were about 30% less likely to be Ascaris infected than females and they also had lower infection intensities. The prevalence and intensity of hookworm infection were slightly (but nevertheless statistically significantly) higher in male pupils and the prevalence of T. trichiura was nearly identical in the two groups. Treatment With more than 96% of the infected children cured three weeks after the first treatment and the total egg output reduced by more than 97%, albendazole was highly effective against A. lumbricoides infection (Table 2). It was slightly less effective against hookworm infection, but nevertheless more than 75% of the pupils were cured after the first, and more than 90% three weeks after the second treatment, and the ERR already exceeded 90% after one treatment. Albendazole treatment was considerably less effective for T. trichiura infection: even after two treatments only one third of the participants were cured, and the ERR was still below 50%. Re-infection The prevalences of hookworm and A. lumbricoides infection at 29 weeks after the second treatment were about 40% of those measured at baseline. Mean infection intensities were still considerably below this level (Table 3). Hookworm re-infection was low during the 16 weeks after the first treatment with only 10% of those pupils who were found uninfected after treatment acquiring new infections. It was markedly higher 18 weeks after the second treatment, with 21% acquiring new infections. In contrast, A. lumbricoides re-infection rates during both periods were fairly similar to each other. Due to the low treatment efficacy it is difficult to make detailed conclusions about re-infection with T. trichiura. It is however remarkable that prevalence and intensity of this helminth apparently decreased during the re-infection period following the first treatment and that the prevalence during the 29 weeks after the second treatment appears to have been nearly stable. Discussion Infection patterns at baseline The hookworm prevalence in the study population was higher than in comparable populations from other parts of KZN [15,16]. The predominant type of hookworm in the area seems to be Necator americanus although Ancylostoma duodenale has also been found close to the study location[16]. Prevalences of T. trichiura and A. lumbricoides were lower than in other areas on the KZN coastal plain where they usually exceed 70% [16]. A possible explanation for the latter difference is the greater distance of our study area from the coast and the resulting decrease in rainfall [17] when compared to the narrower southern parts of the coastal plain where all schools assessed [16] were relatively close to the sea. The patterns reported here are in agreement with those reported by SCHUTTE et al. [10] who surveyed 45 schools in Maputaland about twenty years earlier. They also found considerably lower A. lumbricoides and T. trichiura prevalences in four schools that were situated in the area of our study than in schools closer to the coast. The high hookworm prevalence is probably attributable to high temperature and humidity and the sandy soils in the study area which all seem to favour hookworm transmission [18,19]. Higher A. lumbricoides prevalences and intensities in females when compared to males are a common occurrence in many parts of the world [20] and are often attributed to different patterns of soil contact. In this study population the higher incidence of soil eating among girls might be one reason for this difference as shown in an earlier paper [21]. However, that same article found that even when geophagy preference was adjusted for, girls were still at a higher risk for A. lumbricoides infection at baseline. Interestingly this was not true for re-infection after treatment where boys were more often infected than girls. Treatment A review that summarises numerous studies reports a median CR of 95% for A. lumbricoides infection and a median ERR of over 99% after a single dose of 400 mg albendazole. The corresponding numbers for hookworm infection are just above 80% and just below 90% [22]. Our results are well in agreement with these rates for both helminths. In contrast, the CR and ERR for T. trichiura infection found in our study were among the lowest documented in the literature. The above cited review reports a median CR of 38% and a median ERR of 80%. Our respective rates are only about 1/3 of these. The lowest CR found in any of the 28 studies that were analysed in the review was 4.9%, the lowest ERR (23 studies) was about 27% which is still about 2% higher then the ERR that we found in this study. A study that was conducted in Durban only about 350 km south of the study area found that albendazole reduced the median EPG of children infected before treatment by 44.1% [23]. The same outcome measure when calculated for our data is only 31.0%. But a study that was conducted in three schools about 50 km east of the area of our study also found that single dose albendazole treatment had little effect on T. trichiura: 6 months after treatment the treated pupils had the same prevalence (61%) as a placebo treated control group [24]. Although the CR and ERR as measured in most field studies including ours are notoriously unreliable and can only approximately indicate the true treatment success [25], the very low success rates that we found for the treatment of T. trichiura infection warrant attention. It is unlikely that the low CR and ERR in our study are due to low compliance: Tablet intake was monitored by the school nurses and the study team and only pupils recorded as treated were included in the analysis. Moreover, if a considerable number of children had only pretended to take the tablets, this would also have affected the impact of treatment on hookworm and A. lumbricoides infection. It is also unlikely that the low efficacy is due to drug resistance because of prior treatment. There were no previous control programmes in the area, to our knowledge hospitals and clinics had not been using albendazole or any other benzimidazole until recently and benzimidazoles were also not available in local shops. High infection intensities, as they are frequently found in schoolchildren, are often mentioned in the literature as a reason for low cure rates [22,26] and this may partly explain our results. As expected the CR (but not the ERR) in our population was a lot lower in children with high pre-treatment intensities of infection than in children with lower intensities (data not shown), and in a school-based study on Pemba Island, where intensity and prevalence rank among the highest in the world, the CR for T. trichiura was only 10.5% [27]. But even in this population the geometric mean ERR including uninfected children was 73.3% which is in good agreement with the above cited median ERR of 80% [22] but about twice as high as the same measure for our study (36.1%). Furthermore, although it is obvious that high infection intensities can result in lower CRs, it is not clear why they should also negatively influence the ERR. Currently there are no drugs available that are highly effective against T. trichiura infection as single dose treatments, but other studies show that two or three repeated doses of albendazole on consecutive days are more effective than a single dose [24,26,28,29]. It may be worthwhile to test whether this is also the case in the study area. If applied intermittently with single dose treatments this would increase drug costs but not necessarily increase the workload for the school nursing teams, as the additional doses could be administered by teachers, which is common practice in other control programmes [6,30]. The proportion of children treated in our study can not be regarded as representative for the control programme in general, because study participants were more informed about it than their schoolmates. Therefore it was not analysed statistically. However, it should be noted that although only seven pupils openly refused to be treated, absenteeism was unusually high during the first round of treatment. This improved greatly in the second treatment, when – according to the opinion of school staff – pupils and their parents had realised that treatment was beneficial. Re-infection In accordance with the pre-treatment infection levels, hookworm re-infection over 29 weeks after the second treatment was relatively high and thus confirms the need for regular treatment. The moderate re-infection with A. lumbricoides is also in accordance with the pre-treatment situation. The markedly higher re-infection with hookworm during the 18 weeks after the second treatment when compared to the 16 weeks after the first treatment is most likely only partly due to the slightly longer re-infection period. The difference in season could also be important [19,31]: The 16 weeks' period (April to August) fell into the relatively cool and very dry winter, whereas the 18 weeks after the second treatment (October to February) fell into the hot and humid summer. Contrastingly the absence of a clear difference between these two periods regarding re-infection with A. lumbricoides is not too surprising. As opposed to hookworm that is restricted to the humid tropics and subtropics, this helminth also occurs in temperate climates [20]. Its eggs are relatively robust and are thus less dependent on suitable weather conditions than the fragile free-living hookworm larvae. Although the low efficacy of treatment for T. trichiura complicates conclusions, the data seem to indicate that re-infection was low. Therefore, if a more effective treatment approach could be utilised, the resulting decrease in prevalence and intensity of T. trichiura infections might be relatively long-lasting. Conclusion This study has shown that the high prevalence and intensity of geohelminth infection necessitate regular treatment of primary schoolchildren in the study area according to WHO criteria [6]. This is underlined by the fact that our baseline data and those from Schutte et al. are little different despite the passage of 20 years which indicates that transmission and epidemiology appear not to have changed much. This means that without intervention, geohelminths will likely remain a problem in the area. Because high prevalences in primary schoolchildren may also indicate relatively high community prevalences [32] it should be investigated whether other high risk groups e.g. pre-schoolers and pregnant women also need treatment for geohelminths [33,34]. Our study has also demonstrated that a single course of treatment with 400 mg of albendazole is a powerful tool to control hookworm and A. lumbricoides infection, but that it is much less effective for the treatment of T. trichiura infection in the study area. It would be important to find out whether albendazole treatment is similarly ineffective against T. trichiura in other parts of the province and to test the effectiveness of treatment alternatives. To answer the question whether hookworm transmission is really seasonal as this study seems to suggest would necessitate a cohort study over at least one year. Nevertheless it might be worthwhile to investigate when in the year transmission is highest in order to schedule treatment accordingly. Competing interests None declared. Authors' contributions ES conceived of the study and designed it with input from all authors. He conducted the field work with contributions from the other authors, did the statistical analysis and drafted the manuscript. All authors contributed to the final version of the manuscript and read and approved it. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements We would like to thank the children, staff and parents of the participating schools and the school nursing teams of Manguzi and Mosvold hospitals. The study was funded by the Danish Bilharziasis Laboratory. The KwaZulu-Natal Department of Health provided treatment, laboratory space and logistic support. The MRC National Malaria Research Programme in Durban provided logistic support and Prof. Wilhelm Becker from Hamburg University provided valuable input during the planning and field work of the study. ES was supported by a PhD scholarship from Evangelisches Studienwerk Villigst/Germany. Figures and Tables Figure 1 Location of the study area in northern KwaZulu-Natal Figure 2 Map of the study area Figure 3 Long-term monthly averages of rainfall and temperature in the area. Data from 1966 to 1990 for Makatini research station, about 30 km south of the study area [35]. Figure 4 Cumulative prevalence of geohelminth infection at baseline (n = 1017). The prevalence of infection > = any intensity threshold of interest can be read from the percentage scale (x-axis). 10 EPG correspond to one egg on one of two slides, the minimum for a positive reading. Thus the intersection of each graph with the x-axis corresponds to the total prevalence of infection of the respective helminth. Table 1 Prevalence and intensity (EPG) of geohelminth infection at baseline Males Females M/F Ratio* (95%CI or P) n = 445 572 Median Age (Inter quartile range) 11.2 (10.1–12.3) 10.7 (9.7–11.8) Prevalence (%) Hookworm 86.5 80.6 1.07 (1.02 to 1.13) A. lumbricoides 15.7 22.2 0.71 (0.54 to 0.92) T. trichiura 57.3 57.2 1.00 (0.90 to 1.12) Mean Intensity (EPG)† Hookworm 984 792 1.24 (P < 0.001) A. lumbricoides 876 2948 0.30 (P = 0.008) T. trichiura 240 332 0.72 (P = 0.671) * Male to Female ratios for prevalence and mean intensity of infection and 95% confidence interval (for prevalence) or P-value of 2-sided Mann-Whitney-U-test (for differences in mean EPG). † Arithmetic mean EPG including uninfected pupils. Table 2 Cure rates and egg reduction rates at three weeks after the first and after the second treatment with 400 mg albendazole (n = 592)* Hookworm A. lumbricoides T. trichiura Prevalence (%)  Baseline Survey 82.9 22.0 59.8  After Treatment 1 17.6 0.8 52.2  After Treatment 2 5.7 0.5 39.9 Cure rate (%)  1 Treatment 78.8 96.4 12.7  2 Treatments 93.2 97.7 33.3 Arithmetic Mean EPG†  Baseline Survey 881 2213 319  After Treatment 1 60 52 240  After Treatment 2 11 3 166 ERR (%)  1 Treatment 93.2 97.7 24.8  2 Treatments 98.8 99.8 47.8 * Including only pupils who participated in both treatments, the baseline survey and in the surveys directly after the first and second treatment and calculated only using the first of two samples that were obtained in the post-treatment surveys. † Calculated including uninfected children. Table 3 Development of prevalence and intensity after one and after two rounds of treatment with 400 mg albendazole* Post-Treatment 1‡ Re-infection 1 Post-Treatment 2‡ Re-infection 2a Re-infection 2b n = 801 790 803 739 731 Time of survey May/Jun 98 Aug 98 Oct/Nov 98 Feb 99 Apr/May 99 Weeks since last treatment 3 16 3 18 29 Prevalence (%)  Hookworm 21.2 24.8 10.1 24.6 36.7  A. lumbricoides 1.0 4.3 0.7 3.0 8.6  T. trichiura 54.6 50.9 44.0 44.8 43.4 Mean intensity (EPG)†  Hookworm 77 184 14 65 139  A. lumbricoides 55 115 3 59 351  T. trichiura 227 215 116 174 175 * Included are all pupils who participated in both treatments (n = 864) and in the respective survey (n given in table). Calculated using both samples that were obtained in each survey. † Arithmetic mean EPG for all pupils including uninfected. ‡ The two treatments were administered in April and October 1998 respectively. ==== Refs Stephenson LS Impact of helminth infections on human nutrition 1987 London: Taylor & Francis Olsen A Magnussen P Ouma JH Andreassen J Friis H The contribution of hookworm and other parasitic infections to haemoglobin and iron status among children and adults in western Kenya Trans R Soc Trop Med Hyg 1998 92 643 9 10326110 10.1016/S0035-9203(98)90795-7 Watkins WE Pollitt E "Stupidity or worms": do intestinal worms impair mental performance? Psychol Bull 1997 121 171 91 9100486 10.1037//0033-2909.121.2.171 Nokes C Grantham McGregor SM Sawyer AW Cooper ES Robinson BA Bundy DAP Moderate to heavy infections of Trichuris trichiura affect cognitive function in Jamaican school children Parasitology 1992 104 539 547 1641252 Kvalsvig JD Cooppan RM Connolly KJ The effects of parasite infections on cognitive processes in children Ann Trop Med Parasitol 1991 85 551 568 1809249 WHO Prevention and Control of Schistosomiasis and Soil-transmitted Helminthiasis- Report of a WHO Expert Committee Geneva: World Health Organization 2002 Kvalsvig JD Appleton CC Archer CE Mthethwa P Memela C Mpanza JT Mweni SL Ngcoya M Nkomazi J Qotyana P The KwaZulu-Natal parasite control programme, 1998-Report to the Department of Health, KwaZulu-Natal 2001 42 Warren KS Bundy DAP Anderson RM Davis AR Henderson DA Jamison DT Prescott N Senft A Jamison DT, Mosley WH, Measham AR, Bobadilla JL Helminth infection Disease Control Priorities in Developing Countries 1993 Oxford: The World Bank/Oxford University Press 131 160 Albonico M Crompton DW Savioli L Control strategies for human intestinal nematode infections Adv Parasitol 1999 42 277 341 10050275 Schutte CHJ Eriksson IM Anderson CB Lamprecht T Intestinal parasitic infections in black scholars in northern KwaZulu S Afr Med J 1981 60 137 141 7256455 Montresor A Crompton DW Bundy DAP Hall A Savioli L Guidelines for the evaluation of soil-transmitted helminthiasis and schistosomiasis at community level Geneva: World Health Organization 1998 Nokes C Bundy DAP Compliance and absenteeism in school children: implications for helminth control Trans R Soc Trop Med Hyg 1993 87 148 152 8337713 10.1016/0035-9203(93)90464-2 WHO Basic Laboratory Methods in Medical Parasitology Geneva: World Health Organization 1991 Thompson ML Myers JE Kriebel D Prevalence odds ratio or prevalence ratio in the analysis of cross sectional data: what is to be done? Occup Environ Med 1998 55 272 7 9624282 Appleton CC Gouws E The distribution of common intestinal nematodes along an altitudinal transect in KwaZulu-Natal, South Africa Ann Trop Med Parasitol 1996 90 181 188 8762408 Appleton CC Maurihungirire M Gouws E The distribution of helminth infections along the coastal plain of KwaZulu-Natal province, South Africa Ann Trop Med Parasitol 1999 93 859 68 10715680 10.1080/00034989957862 Maud RR Bruton MN, Cooper KH The climate and geology of Maputaland Studies on the ecology of Maputaland 1980 Grahamstown & Durban: Rhodes University & The Natal Branch of the Wildlife Society of Southern Africa 1 7 Smith G Schad GA, Warren KS The ecology of the free-living stages: a reappraisal Hookworm disease: current status and new directions 1990 London: Taylor & Francis 89 104 Mabaso ML Appleton CC Hughes JC Gouws E The effect of soil type and climate on hookworm (Necator americanus) distribution in KwaZulu-Natal, South Africa Trop Med Int Health 2003 8 722 7 12869093 10.1046/j.1365-3156.2003.01086.x Crompton DW Crompton DW, Nesheim MC, Pawlowski ZS Prevalence of Ascariasis Ascariasis and its prevention and control 1989 London: Taylor & Francis 45 69 Saathoff E Olsen A Kvalsvig JD Geissler PW Geophagy and its association with geohelminth infection in rural schoolchildren from northern KwaZulu-Natal, South Africa Trans R Soc Trop Med Hyg 2002 96 485 90 12474473 10.1016/S0035-9203(02)90413-X Bennett A Guyatt H Reducing intestinal nematode infection: efficacy of albendazole and mebendazole Parasitol Today 2000 16 71 4 10652492 10.1016/S0169-4758(99)01544-6 Jackson TF Epstein SR Gouws E Cheetham RF A comparison of mebendazole and albendazole in treating children with Trichuris trichiura infection in Durban, South Africa S Afr Med J 1998 88 880 3 9698717 Taylor M Jinabhai CC Couper I Kleinschmidt I Jogessar VB The effect of different anthelmintic treatment regimens combined with iron supplementation on the nutritional status of schoolchildren in KwaZulu-Natal, South Africa: a randomized controlled trial Trans R Soc Trop Med Hyg 2001 95 211 6 11355564 10.1016/S0035-9203(01)90171-3 Davis A Clinical trials in parasitic diseases Trans R Soc Trop Med Hyg 2004 98 139 41 15024922 10.1016/S0035-9203(03)00036-1 Horton J Albendazole: a review of anthelmintic efficacy and safety in humans Parasitology 2000 121 S113 32 11386684 10.1017/S0031182000007290 Albonico M Smith PG Hall A Chwaya HM Alawi KS Savioli L A randomized controlled trial comparing mebendazole and albendazole against Ascaris, Trichuris and hookworm infections Trans R Soc Trop Med Hyg 1994 88 585 589 7992348 10.1016/0035-9203(94)90174-0 Olsen A Nawiri J Friis H The impact of iron supplementation on reinfection with intestinal helminths and Schistosoma mansoni in western Kenya Trans R Soc Trop Med Hyg 2000 94 493 9 11132373 Hall A Nahar Q Albendazole and infections with Ascaris lumbricoides and Trichuris trichiura in children in Bangladesh Trans R Soc Trop Med Hyg 1994 88 110 2 8153985 10.1016/0035-9203(94)90525-8 Montresor A Gyorkos TW Crompton DWT Bundy DAP Savioli L Monitoring helminth control programmes Geneva: World Health Organization 1999 Chandiwana SK Schad GA, Warren KS Hookworm population ecology in Zimbabwe Hookworm disease : current status and new directions 1990 London: Taylor & Francis 165 176 Brooker S Donnelly CA Guyatt HL Estimating the number of helminthic infections in the Republic of Cameroon from data on infection prevalence in schoolchildren Bull World Health Organ 2000 78 1456 65 11196498 Montresor A Stoltzfus RJ Albonico M Tielsch JM Rice AL Chwaya HM Savioli L Is the exclusion of children under 24 months from anthelmintic treatment justifiable? Trans R Soc Trop Med Hyg 2002 96 197 9 12055814 10.1016/S0035-9203(02)90303-2 Allen HE Crompton DW de Silva N LoVerde PT Olds GR New policies for using anthelmintics in high risk groups Trends Parasitol 2002 18 381 2 12377247 10.1016/S1471-4922(02)02386-3 Climatological Information Section Long term climatological data for Makathini weather station Pretoria: South African Weather Service 2000

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==== Front BMC Pregnancy ChildbirthBMC Pregnancy and Childbirth1471-2393BioMed Central London 1471-2393-4-161529871810.1186/1471-2393-4-16Research ArticleMisoprostol for treating postpartum haemorrhage: a randomized controlled trial [ISRCTN72263357] Hofmeyr G Justus [email protected] Sandra [email protected] V Cheryl [email protected] Lindeka [email protected] Mandisa [email protected] Zukiswa [email protected] Babalwa [email protected] Zonke [email protected] Gijs [email protected]ülmezoglu A Metin [email protected] Effective Care Research Unit, University of Witwatersrand and Fort Hare, and East London Hospital Complex, East London, South Africa2 Department of Nursing, University of the Western Cape, Cape Town, South Africa3 Tembisa Hospital Effective Care Research Unit, Tembisa, South Africa4 Aga Khan Health Services, Aiglemont, France5 HRP/RHR, World Health Organisation, Geneva, Switzerland2004 6 8 2004 4 16 16 26 2 2004 6 8 2004 Copyright © 2004 Justus Hofmeyr et al; licensee BioMed Central Ltd.2004Justus Hofmeyr et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Postpartum haemorrhage remains an important cause of maternal death despite treatment with conventional therapy. Uncontrolled studies and one randomised comparison with conventional oxytocics have reported dramatic effects with high-dose misoprostol, usually given rectally, for treatment of postpartum haemorrhage, but this has not been evaluated in a placebo-controlled trial. Methods The study was conducted at East London Hospital Complex, Tembisa and Chris Hani Baragwanath Hospitals, South Africa. Routine active management of the third stage of labour was practised. Women with more than usual postpartum bleeding thought to be related to inadequate uterine contraction were invited to participate, and to sign informed consent. All routine treatment was given from a special 'Postpartum Haemorrhage Trolley'. In addition, participants who consented were enrolled by drawing the next in a series of randomised treatment packs containing either misoprostol 5 × 200 μg or similar placebo, which were given 1 orally, 2 sublingually and 2 rectally. Results With misoprostol there was a trend to reduced blood loss ≥500 ml in 1 hour after enrolment measured in a flat plastic 'fracture bedpan', the primary outcome (6/117 vs 11/120, relative risk 0.56; 95% confidence interval 0.21 to 1.46). There was no difference in mean blood loss or haemoglobin level on day 1 after birth < 6 g/dl or blood transfusion. Side-effects were increased, namely shivering (63/116 vs 30/118; 2.14, 1.50 to 3.04) and pyrexia > 38.5°C (11/114 vs 2/118; 5.69, 1.29 to 25). In the misoprostol group 3 women underwent hysterectomy of whom 1 died, and there were 2 further maternal deaths. Conclusions Because of a lower than expected incidence of the primary outcome in the placebo group, the study was underpowered. We could not confirm the dramatic effect of misoprostol reported in several unblinded studies, but the results do not exclude a clinically important effect. Larger studies are needed to assess substantive outcomes and risks before misoprostol enters routine use. ==== Body Background Excessive bleeding from the genital tract after birth, or postpartum haemorrhage (PPH) is the major cause of maternal deaths in many low-income countries. The global estimate is 125,000 deaths per year [1]. In South Africa, 240 of 2,445 maternal deaths reported between 1999 and 2001 were due to postpartum haemorrhage, the third most common cause [2]. In a community-based study in Senegal, estimates of maternal mortality ratio in three regions ranged from 436 to 852 per 100,000 live births. Two-thirds were due to direct obstetric causes, the commonest being haemorrhage [3]. In the United Kingdom, the risk of maternal death from haemorrhage is about 1 in 100 000 births [4]. The potential to save mothers' lives with medical interventions for haemorrhage is thus considerable. There is good evidence that a policy of active management of the third stage of labour [5], and one component of active management, the routine administration of uterotonics (drugs to contract the uerus) such as oxytocin [6] or oxytocin and ergometrine [7], are effective in reducing the risk of postpartum haemorrhage. When postpartum haemorrhage occurs, a number of medical and surgical interventions are used to control the bleeding [8]. A crucial aspect of both prevention and treatment of postpartum haemorrhage is uterotonic therapy. The most commonly used agents are injectable oxytocin and/or ergometrine. Misoprostol is a methyl ester (a synthetic analogue) of natural prostaglandin E1. It is marketed and registered for use in the prevention and treatment of peptic ulcer disease caused by prostanglandin synthetase inhibitors. Administered orally or sublingually, peak plasma concentrations of misoprostolic acid are achieved in less than 30 minutes [9]. It is a thermostable drug [9] and is relatively inexpensive. Administered orally or vaginally, it is an effective agent for the induction of abortion [10] and of labour [11]. Misoprostol for routine prevention of postpartum haemorrhage Oral and rectal misoprostol have been used for routine management of the third stage of labour (after the birth). Several small trials have given conflicting results. The main side-effects have been shivering and pyrexia, which are dose-dependent [12]. In the large WHO Collaborative Trial of Misoprostol in the Management of the Third Stage of Labour [13] and a systematic review of the topic [14], blood loss >1,000 ml and use of additional oxytocics (the primary outcomes in the WHO trial) were more frequent with misoprostol than with injectable oxytocics, indicating that injectable oxytocics should remain the drug of choice for routine prophylaxis. On the other hand, blood transfusion was used less frequently in the misoprostol group. This may have been a chance occurrence (it was not a primary outcome for the trial). Secondly, there could be a synergistic pharmacological effect between misoprostol and conventional uterotonics, which were given to most women with increased blood loss. Many women in the misoprostol group thus received misoprostol as well as conventional uterotonics, whereas those in the oxytocin group received only conventional oxytocics. Thirdly, we have suggested in the report of a pharmacokinetic study linked to the WHO trial that the longer time to peak levels of misoprostol (20–30 minutes) than syntocinon (3 minutes) may account for more early blood loss with misoprostol [15]. This does not exclude the possibility that misoprostol may have an effect on more persistent bleeding. Physiological studies have also shown a more rapid onset of uterine contractions following syntometrine than misoprostol after delivery [16]. Misoprostol has been widely recommended for the prevention of postpartum haemorrhage when other methods are not available [17]. Misoprostol for treatment of postpartum haemorrhage Apart from the prophylactic use of misoprostol in the third stage of labour, the therapeutic use of misoprostol for the treatment of postpartum haemorrhage has been promoted on the basis of unblinded studies. In a systematic review [18] we located 6 uncontrolled reports (41 women) [19-24] and one small, unblinded randomised trial [25]. This method has entered clinical use, particularly in developing countries, without systematic research to document the optimal route and dosage, effectiveness or risks of this treatment. The urgent need for randomised trials of this new intervention has been emphasised [26]. If effective it could have a major impact on maternal mortality, particulary in under-resourced countries. If not, its use should be discouraged because of the dangers of side-effects, and the risks associated with widespread introduction of misoprostol into health systems in which it might be used for labour induction in inappropriate doses, with the risk of fetal compromise and uterine rupture [27,28]. Route and dosage of misoprostol The most common regimens reported for the treatment of postpartum haemorrhage are 800 [23] or 1,000 μg [19,21] rectally. We reviewed the literature on the pharmacokinetics of misoprostol administered by various routes [18]. The oral route has the most rapid uptake, but the shortest duration. The rectal route has slow uptake but prolonged duration. The buccal or sublingual route has rapid uptake, prolonged duration and greatest total bioavailability. In order to improve the rapidity of onset of action and the overall bioavailability, we modified the reported practice of using 1,000 μg rectally, by administering 200 μg orally, 400 μg sublingually and 400 μg rectally. Objective The objective of this study was to determine the side-effects and effectiveness of high-dose misoprostol for the treatment of postpartum haemorrhage by means of a double-blind, placebo-controlled, randomised clinical trial. The hypothesis was that measured blood loss of 500 ml or more in the hour after enrolment would be significantly less frequent in the misoprostol than in the placebo group. Methods The study was conducted at the East London Hospital Complex (Frere Maternity and Cecilia Makiwane Hospitals, Eastern Cape) and Tembisa and Chris Hani Baragwanath Hospitals, Gauteng, South Africa, between Jan 2002 and Dec 2003. All are busy referral hospitals. Treatment packs were prepared independently, ordered in computer-generated random sequence and numbered consecutively. The packs contained either misoprostol 5 × 200 μg or inactive placebo. The treatment sequence was kept sealed, and the code broken only after complete entry and checking of all trial data. A confidential interim analysis, blind to which group was which, was performed by AMG after the first 100 enrolments. Evidence of clear benefit or harm for either group would have prompted stopping the trial, which was not the case. Women in labour were given basic information about the trial in the form of notices in the labour rooms (Table 2 [see additional file 1 ]). Management of labour 3rd stage was routine active management with oxytocin 10 units or syntometrine one ampoule soon after the birth. Women aged 18 or more with bleeding judged by the clinician to be more than expected at least 10 minutes after delivery, and thought to be due to uterine atony and requiring additional uterotonic therapy, were given all the routine treatment for PPH (intravenous infusion, uterotonics, etc) from a special 'PPH Trolley' kept in the labour ward. The uterotonics used were oxytocin by intravenous infusion, and/or oxytocin/ergometrine, at the discretion of the attending clinician. Prostaglandin preparations such as sulprostone were not routinely available in the labour wards, and were not part of routine management of postpartum haemorrhage in the participating units. Once all emergency treatment was instituted, and if the women were in a position to give fully informed consent, they were given detailed information about the trial and asked whether they wished to participate. Information about the trial was given verbally and in writing in English or Xhosa (Table 3 [see additional file 2 ]). Those who gave written consent were enrolled in the trial. Baseline data were documented. The next treatment pack containing either misoprostol 5 × 200 μg or inactive placebo was drawn and the number recorded. In addition to routine management, misoprostol or placebo (an inactive base) were given, 1 orally, 2 buccally/sublingually and 2 rectally, and the time recorded. A low-profile plastic 'fracture bedpan' was placed under the woman's buttocks. In previous studies we have shown this to be an efficient method of collecting ongoing blood loss with very little spillage [29]. Any small swabs soaked with blood were dropped into the bedpan. After 1 hour, the blood collected in the bedpan was measured in a graduated measuring jug, the temperature was measured sublingually with a mercury thermometer, and any shivering was subjectively recorded as 'mild' (not causing any distress), 'moderate' or 'severe'. All other management was by hospital staff according to the hospital routine for the management of postpartum haemorrhage. After 24 hours, blood was collected for haemoglobin estimation and the hospital records checked for use of additional uterotonics and any other complications such as blood transfusion. The primary outcome measures were specified prior to commencing the study: (1) measured blood loss ≥500 ml in 1 hour after enrolment; (2) mean measured blood loss in 1 hour after enrolment; (3) haemoglobin level day 1 after birth <6 g/dl or blood transfusion; (4) Side-effects (pyrexia 38.5°C or more, moderate or severe shivering 1 hour after enrolment). Secondary outcome measures were: (1) blood loss ≥1,000 ml in 1 hour after enrolment; (2) blood transfusion; (3) haemoglobin level 1 day after birth <8 g/dl or blood transfusion; (4) additional uterotonic given after enrolment; (5) manual removal of the placenta; (6) evacuation of retained products of conception; (7) hysterectomy; (8) maternal death. Sample size calculation In the WHO misoprostol trial control group (routine syntocinon), significant postpartum haemorrhage (blood loss >1000 ml) occurred in 2.8% of women. Of these 25% lost >1,500 ml. To reduce blood loss >500 ml after enrolment from 25% to 10%, would require 112 per group (α = 95%, β = 80%). Data were collected on a data form, entered onto an excel spreadsheet, checked for accuracy, then analysed using Epi-info 2002 (United States Department of Health and Human Services, Centres for Diseases Control and Prevention, Epidemiology Program Office) and Review Manager (RevMan) [Computer program] version 4.2 for Windows. Oxford, England: The Cochrane Collaboration, 2003. Results were expressed as relative risks or mean difference with 95% confidence intervals. Analysis was by intention to treat. Ethical considerations All women enrolled in the trial received all the conventional management available for postpartum haemorrhage, in addition to the trial medication (misoprostol or placebo). Rapid conventional therapy was facilitated by the ready availability of the 'PPH trolley' with all the necessary materials. This trolley was also available for women not enrolled in the trial. Participation was limited to women who were in a position to give fully informed consent. Ethical approval was given by the Committee for Research on Human Subjects, University of the Witwatersrand, and the Ethics Committee, East London Hospital Complex. The trial complied with the Helsinki Declaration for research on human subjects. Results Altogether 244 women were enrolled in the trial. The pack numbers for 6 women were incompletely filled in on the data sheets. The group allocation of these women was therefore unknown, and they could not be included in the analysis. Of these 6 women, the highest measured blood loss after enrolment was 220 ml; none had pyrexia >37.5°C, one had moderate shivering, one had a blood transfusion and none had day 1 haemoglobin level below 8 g/dl or other complications. There was no reason to expect that these exclusions occurred in any selective way which would introduce bias into the final samples, nor could their results have materially altered the conclusions of the study. All the remaining 238 women were included in the analyses. The baseline characteristics are shown in table 1. As would be expected in a randomised trial, the groups were well matched. Parity was not recorded at all hospitals, but was similar between groups where recorded (misoprostol mean 1.61, SD 1.14; placebo 1.75, SD 1.26). Table 1 Comparison of outcomes between women who received misoprostol or placebo, expressed as proportions (percent) or mean values [standard deviation]. Differences are expressed as relative risks or mean differences, with 95% confidence intervals. Misoprostol (117) Placebo (121) Baseline characteristics n n Age (years) 116 27.1 [6.0] 119 26.2 [6.2] Ergometrine before enrolment 106 36 (34%) 104 34 (33%) Oxytocin ≥20 U before enrolment 116 82 (71%) 117 78 (67%) Primary outcomes: RR/ MD 95% Conf. interval Blood loss ≥500 ml* 117 6 (5.1%) 120 11 (9.2%) 0.56 0.21 to 1.46 Mean blood loss* [SD] (ml) 117 168 [163] 120 176 [173] -8 -51 to 35 24 hour Haemoglobin <6 g/dL or blood transfusion 110 20 (18.2%) 116 17 (14.7%) 1.24 0.69 to 2.24 Pyrexia at 1 hour ≥38, 5°C 114 11 (9.6%) 118 2 (1.7%) 5.69 1.29 to 25 Shivering at 1 hour ≥ moderate 116 63 (54%) 118 30 (25%) 2.14 1.50 to 3.04 Secondary outcomes: Blood loss ≥ 1,000 ml* 117 1 (0.85%) 120 0 (0%) Blood transfusion 115 19 (17%) 119 15 (13%) 1.31 0.70 to 2.45 24 hour haemoglobin <8 g/dL or blood transfusion 110 43 (39%) 116 37 (32%) 1.23 0.86 to 1.75 Additional uterotonic after enrolment 111 63 (57%) 112 63 (56%) 1.01 0.80 to 1.27 Manual removal of the placenta 117 1 (0.85%) 121 4 (3.3%) 0.26 0.03 to 2.28 Evacuation of retained products of conception 117 2 (1.7%) 121 0 (0%) Hysterectomy** 117 3 (2.6%) 121 0 (0%) Maternal death** 117 3 (2.6%) 121 0 (0%) RR = relative risk; MD = mean difference; Conf = confidence; SD = standard deviation *Measured, within 1 hour after enrolment ** One woman died after hysterectomy and is counted in both outcomes The outcomes are shown in table 1. In the placebo group, the primary outcome (measured blood loss within 1 hour after enrolment of 500 ml or more) was less frequent (9.2%) than the 25% prediction on which the sample size calculation was based. This may have been because the criteria for enrolment were loosely defined as 'more than expected blood loss', resulting in the enrolment of women with less severe blood loss than anticipated. The fact that enrolment was limited to women who were able to received detailed information about the study, may have caused selection of less severe cases. Although the intention was to enrol women who had not responded to conventional therapy, the time taken for counselling of the women and obtaining informed consent may have resulted in some women responding to the primary treatment, and therefore the low rate of ongoing blood loss in the placebo group. The study was thus underpowered to detect a statistically significant reduction in the primary outcome. With misoprostol there was a trend to reduced blood loss ≥500 ml in 1 hour after enrolment (6/117 vs 11/120, relative risk 0.56; 95% confidence interval 0.21 to 1.46). Other proxy estimations of blood loss showed no significant differences between the groups. The well-known side-effects of misoprostol, pyrexia and shivering, were significantly more frequent in the misoprostol than the placebo group. Serious morbidity or mortality occurred in 5 women, all from the misoprostol group. Two women who continued to bleed despite conservative therapy underwent abdominal hysterectomy and recovered well. There were 3 maternal deaths: Case 1 A 22-year old woman with one previous pregnancy developed postpartum haemorrhage after vaginal delivery, managed with a 40-unit oxytocin infusion. She was enrolled and received the trial medication 85 minutes after delivery. Thereafter she received a second 40-unit oxytocin drip, oxytocin/ergometrine (5 units/0.5 mg) and intravenous cyclokapron. In the hour after randomisation, measured blood loss was 125 ml. Subsequently bleeding continued and a sub-total hysterectomy was performed. Coagulopathy developed, bleeding continued through an abdominal drain, and she died 2 days after delivery despite re-laparotomy and multiple blood product transfusions. Case 2 A 32-year-old woman, para 2, gravida 4, delivered normally after labour was induced with misoprostol, 25 μg vaginally and 4 doses of 50 μg orally. Before enrolment she received oxytocin10 units, ergometrine 0.5 mg and a 20-unit oxytocin infusion. She was enrolled and received the trial medication 140 minutes after delivery. After enrolment she received a further 40-unit oxytocin infusion. Measured blood loss in the hour after enrolment was 380 ml. Bleeding continued and a blood transfusion was commenced. Six and a half hours after delivery she suffered a cardiac arrest and was resuscitated. Examination in theatre found the uterus to be empty and intact. The main source of bleeding appeared to be a torn cervix, which was sutured. At 7.5 hours after delivery she suffered a second cardiac arrest and could not be resuscitated. The estimated total blood loss was 3,000 ml. Case 3 A woman with one previous delivery by caesarean section, developed postpartum haemorrhage of about 500 ml after a vaginal delivery. She was enrolled 85 minutes after delivery. Measured blood loss over the next hour was 425 ml, after which she developed hypotension and cardiorespiratory arrest. As no post-mortem examination was performed, the possibility of internal bleeding from a dehisced caesarean section scar could neither be confirmed nor excluded. Unresponsive uterine atony was therefore not confirmed as the main cause of any of the deaths. Discussion Pyrexia and shivering were common side-effects of misoprostol, as found in previous studies. Three women, all in the misoprostol group, had severe pyrexia >40°C. Previous studies of high-dose misoprostol for the treatment of postpartum haemorrhage have mostly used the rectal route, and none has had sufficient numbers to be likely to detect rare adverse outcomes such as severe pyrexia. These side-effects may be related to the rapid absorption of misoprostol given orally, and the rapid absorption and high bioavailability when given sublingually. Because of these serious side-effects, we would recommend that for future trials the dose be reduced. Several previous unblinded studies have reported dramatic effects of misoprostol when used to treat postpartum haemorrhage. In 6 observational studies [19-24], 41 women with postpartum haemorrhage were treated with misoprostol 1,000 μg rectally (32 women), 200 μg rectally (5 women), 200 μg orally 2-hourly (2 women) or 800 μg intrauterine (2 women). In all but 2 of the women (who received 1,000 μg rectally), a prompt response to the treatment was reported. In a single blind randomised trial [25] subjective prompt cessation of bleeding was reported in 30/32 women who received misoprostol 800 μg rectally compared with only 21/32 who received oxytocin 5 units plus ergometrine 0.5 mg intramuscularly. Conclusions In our double blind trial we have been unable to confirm the dramatic effects of misoprostol reported previously in unblinded studies. Our results, however, are consistent with anything from a large beneficial effect to a smaller adverse effect (relative risk of additional blood loss of 500 ml or more anywhere between a reduction of 79% and an increase of 46% with misoprostol). It is important that this possible benefivial effect be assessed by sufficiently powered randomised trials before the treatment enters routine clinical use. List of abbreviations PPH: postpartum haemorrhage; SD: standard deviation Competing interests None known. Authors' contributions GJH prepared the first draft of the protocol and the final manuscript, and oversaw the clinical work CN assisted with the design of the protocol and data collection sheet, initiated enrolment at two sites, supervised some of the data collection and contributed to the manuscript SF, ZM, LM, MS and ZJ commented on the protocol, conducted the clinical procedures and collected data. SF entered and collated the data BM undertook the literature search and commented on the protocol. GW contributed to the protocol development AMG contributed to the protocol development and undertook the blinded interim analysis All authors read and approved the final manuscript Additional files Additional file 1 - Table 2. Notice informing women about the trial, MS Word file: HofmeyrFile1.doc Additional file 2- Table 3: Information and consent form. MS Word file: HofmeyrFile2.doc Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Table 2. Notice informing women about the trial Click here for file Additional File 2 Table 3: Information and consent form Click here for file Acknowledgements We acknowledge the women who participated in the trial. Jose Villar, WHO HRP, for contributions to the development of our research programme in this field. Norma Pirani for conducting clinical procedures at Chris Hani Baragwanath Hospital, Soweto, South Africa. ==== Refs Selo-Ojeme DO Primary postpartum haemorrhage J Obstet Gynaecol 2002 22 463 469 12521409 10.1080/0144361021000003555 Moodley J for Committee on Confidential Enquiries into Maternal Deaths in South Africa Saving mothers. Second report on Confidential Enquiries into Maternal Deaths in South Africa 1999–2001 Department of Health, South Africa Kodio B de Bernis L Ba M Ronsmans C Pison G Etard JF Levels and causes of maternal mortality in Senegal Trop Med Int Health 2002 7 499 505 12031071 10.1046/j.1365-3156.2002.00892.x Department of Health, Welsh Office, Scottish Home and Health Department, Department of Health and Social Security Report of confidential enquiries into maternal deaths in the United Kingdom 1991–1993 HMSO, London 1996 Prendiville WJ Elbourne D McDonald S Active versus expectant management in the third stage of labour (Cochrane Review) The Cochrane Library 2003 Chichester, UK: John Wiley & Sons, Ltd Elbourne DR Prendiville WJ Carroli G Wood J McDonald S Prophylactic use of oxytocin in the third stage of labour (Cochrane Review) The Cochrane Library 2003 Chichester, UK: John Wiley & Sons, Ltd McDonald S Prendiville WJ Elbourne D Prophylactic syntometrine versus oxytocin for delivery of the placenta (Cochrane Review) The Cochrane Library 2003 Chichester, UK: John Wiley & Sons, Ltd Mousa HA Walkinshaw S Major postpartum haemorrhage Curr Opin Obstet Gynecol 2001 13 595 603 11707663 10.1097/00001703-200112000-00008 Tang OS Schweer H Seyberth HW Lee SWH Ho PC Pharmacokinetics of different routes of administration of misoprostol Human Reproduction 2002 17 332 336 11821273 10.1093/humrep/17.2.332 Norman JE Thong KJ Baird DT Uterine contractility and induction of abortion in early pregnancy by misoprostol and mifepristone Lancet 1991 338 1233 1236 1682644 10.1016/0140-6736(91)92102-8 Hofmeyr GJ Gulmezoglu AM Alfirevic Z Misoprostol for induction of labour: a systematic review Br J Obstet Gynaecol 1999 106 798 803 10453829 Lumbiganon P Hofmeyr J Gulmezoglu AM Pinol A Villar J Misoprostol dose-related shivering and pyrexia in the third stage of labour Br J Obstet Gynaecol 1999 106 304 308 10426235 Gülmezoglu AM Villar J Ngoc NN Piaggio G Carroli G Adetoro L Abdel-Aleem H Cheng L Hofmeyr GJ Lumbiganon P Unger C Prendiville W Pinol A Elbourne D El-Refaey H Schulz KF for the WHO Collaborative Group To Evaluate Misoprostol in the Management of the Third Stage of Labour The WHO multicentre double-blind randomized controlled trial to evaluate the use of misoprostol in the management of the third stage of labour Lancet 2001 358 689 695 11551574 10.1016/S0140-6736(01)05835-4 Gülmezoglu AM Forna F Villar J Hofmeyr GJ Prostaglandins for prevention of postpartum haemorrhage (Cochrane Review) The Cochrane Library 2003 Chichester, UK: John Wiley & Sons, Ltd Abdel-Aleem H Villar J Gulmezoglu AM Mostafa SA Youssef AA Shokry M Watzer B The pharmacokinetics of the prostaglandin E1 analogue misoprostol in plasma and colostrums after postpartum oral administration Eur J Obstet Gynecol Reprod Biol 2002 4419 1 4 Chong YS Chua S El-Refaey H Choo WL Chanrachakul B Tai BC Rodeck C Arulkumaran S Postpartum intrauterine pressure studies of the uterotonic effect of oral misoprostol and intramuscular syntometrine Br J Obstet Gynaecol 2001 108 41 47 10.1016/S0306-5456(00)00014-0 McCormick ML Sanghvi HC Kinzie B McIntosh N Preventing postpartum hemorrhage in low-resource settings Int J Gynaecol Obstet 2002 77 267 275 12065142 10.1016/S0020-7292(02)00020-6 Hofmeyr GJ Walraven G Maholwana B Gülmezoglu AM Villar J Misoprostol to treat postpartum haemorrhage: a systematic review of evidence relevant to efficacy, dosage and route of administration Br J Obstet Gynaecol 2004 Adekanmi OA Purmessur S Edwards G Barrington JW Intrauterine misoprostol for the treatment of severe recurrent atonic secondary postpartum haemorrhage Br J Obstet Gynaecol 2001 108 541 542 10.1016/S0306-5456(00)00110-8 Oboro VO Tabowei TO Bosah JO Intrauterine misoprostol for refractory postpartum hemorrhage Int J Gynecol Obstet 2003 80 67 68 10.1016/S0020-7292(02)00301-6 O'Brien P El-Refaey H Gordon A Geary M Rodeck CH Rectally administered misoprostol for the treatment of postpartum hemorrhage unresponsive to oxytocin and ergometrine: a descriptive study Obstet Gynecol 1998 92 212 214 9699753 10.1016/S0029-7844(98)00161-6 Ozan H Bilgin T Ozsarac N Ozerkan RK Cengiz C Misoprostol in uterine atony: a report of 2 cases Clin Exp Obstet Gynecol 2000 27 221 222 11214957 Abdel-aleem H El-Nashar I Abdel-Aleem A Management of severe postpartum hemorrhage with misoprostol Int J Gynecol Obstet 2001 72 75 76 10.1016/S0020-7292(00)00321-0 Shojai R Piechon L d'Ercole C Boubli L Ponties JE [Rectal administration of misoprostol for delivery induced hemorrhage. Preliminary study] (French) J Gynecol Obstet Reprod Biol 2001 30 572 575 Lokugamage AU Sullivan KR Niculescu I Tigere P Onyangunga F El Refaey H Moodley J Rodeck CH A randomized study comparing rectally administered misoprostol versus Syntometrine combined with an oxytocin infusion for the cessation of primary post partum hemorrhage Acta Obstet Gynecol Scand 2001 80 835 839 11531635 10.1034/j.1600-0412.2001.080009835.x Darney PD Misoprostol: a boon to safe motherhood...or not? Lancet 2001 358 682 683 11551569 10.1016/S0140-6736(01)05846-9 Hofmeyr GJ Gülmezoglu AM Vaginal misoprostol for cervical ripening and induction of labour (Cochrane Review) The Cochrane Library 2003 Chichester, UK: John Wiley & Sons, Ltd Hofmeyr GJ Misoprostol in obstetrics and gynaecology – unregistered, dangerous and essential S Afr Med J 1998 88 535 536 9638117 Hofmeyr GJ Nikodem VC de Jager M Gelbart BR A randomised controlled trial of oral misoprostol in the third stage of labour Br J Obstet Gynaecol 1998 105 971 975 9763047

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==== Front BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-4-451529871510.1186/1471-2407-4-45Research ArticleGenome-wide array comparative genomic hybridization analysis reveals distinct amplifications in osteosarcoma Man Tsz-Kwong [email protected] Xin-Yan [email protected] Kim [email protected] Laszlo [email protected] Charles P [email protected] Shishir [email protected] Marc [email protected] Richard [email protected] Ching C [email protected] Pulivarthi H [email protected] Texas Children's Cancer Center, Baylor College of Medicine, Houston, TX, USA2 Spectral Genomics, Houston, TX, USA3 Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York, USA4 Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, New York, USA2004 7 8 2004 4 45 45 4 5 2004 7 8 2004 Copyright © 2004 Man et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Osteosarcoma is a highly malignant bone neoplasm of children and young adults. It is characterized by extremely complex karyotypes and high frequency of chromosomal amplifications. Currently, only the histological response (degree of necrosis) to therapy represent gold standard for predicting the outcome in a patient with non-metastatic osteosarcoma at the time of definitive surgery. Patients with lower degree of necrosis have a higher risk of relapse and poor outcome even after chemotherapy and complete resection of the primary tumor. Therefore, a better understanding of the underlying molecular genetic events leading to tumor initiation and progression could result in the identification of potential diagnostic and therapeutic targets. Methods We used a genome-wide screening method – array based comparative genomic hybridization (array-CGH) to identify DNA copy number changes in 48 patients with osteosarcoma. We applied fluorescence in situ hybridization (FISH) to validate some of amplified clones in this study. Results Clones showing gains (79%) were more frequent than losses (66%). High-level amplifications and homozygous deletions constitute 28.6% and 3.8% of tumor genome respectively. High-level amplifications were present in 238 clones, of which about 37% of them showed recurrent amplification. Most frequently amplified clones were mapped to 1p36.32 (PRDM16), 6p21.1 (CDC5L, HSPCB, NFKBIE), 8q24, 12q14.3 (IFNG), 16p13 (MGRN1), and 17p11.2 (PMP22 MYCD, SOX1,ELAC27). We validated some of the amplified clones by FISH from 6p12-p21, 8q23-q24, and 17p11.2 amplicons. Homozygous deletions were noted for 32 clones and only 7 clones showed in more than one case. These 7 clones were mapped to 1q25.1 (4 cases), 3p14.1 (4 cases), 13q12.2 (2 cases), 4p15.1 (2 cases), 6q12 (2 cases), 6q12 (2 cases) and 6q16.3 (2 cases). Conclusions This study clearly demonstrates the utility of array CGH in defining high-resolution DNA copy number changes and refining amplifications. The resolution of array CGH technology combined with human genome database suggested the possible target genes present in the gained or lost clones. ==== Body Background Osteosarcoma (OS) is a primary malignant tumor of bone arising from primitive bone-forming mesenchymal cells and it accounts for approximately 60% of malignant bone tumors in the first two decades of life [1]. These tumors typically arise in the metaphyseal regions of long bones, with the distal femur, proximal tibia and proximal humerus. A significant number of osteosarcomas are of conventional type which can be subdivided into three major categories based on their predominant differentiation of tumor cells: osteoblastic, chondroblastic, and fibroblastic. Currently, only the histological response (degree of necrosis) to therapy represent gold standard for predicting the outcome in a patient with non-metastatic osteosarcoma at the time of definitive surgery [2]. Patients with lower degree of necrosis have a higher risk of relapse and poor outcome even after chemotherapy and complete resection of the primary tumor. Therefore, a better understanding of the underlying molecular genetic events leading to tumor initiation and progression could result in the identification of potential diagnostic and therapeutic targets. Chromosomal aberrations in osteosarcoma are highly complex and characterized by high frequency of amplifications. These amplifications may result in the overexpression of genes and contribute to the genomic instability in osteosarcoma. The identification of genes within the amplified sites is crucial for understanding the biology and clinical behavior of osteosarcoma. Until, recently gene amplification has been detected by PCR, southern blot analysis or FISH-based approach using gene specific probes. These techniques are inherently restricted to the previously known amplified genes in the genome. In contrast, genome-wide screening of amplified chromosomal regions with CGH has become an important tool for the detection of amplified regions in the tumor genome. So far published chromosomal CGH studies in osteosarcoma have identified several high-level chromosomal amplifications at 1p22, 1p31, 1q21, 1q23, 2q24, 3p25, 3q26, 6q24.3, 4q12, 5p14-p15, 5q33, 6p12-p21, 6q24.3, 7p21-p22, 8q12-q23, 10p21, 10q11.1, 10q22, 11q13, 11q23, 12p13, 12q12-q15, 17p11.2, 17q21, 18q22, 19p13.1 and 20p11.2 [3-7]. However, conventional CGH has limited sensitivity and resolution (~10–15 megabases) because of its dependence on the morphology of metaphase chromosomes. In addition, extensive follow-up work is required to identify candidate genes after regions of gain or loss have been identified. Recently, novel method termed as array-based comparative genomic hybridization (array CGH) has been described, which enables high throughput quantitative measurement of high-resolution DNA copy number changes throughout the genome [8]. This method is based on hybridization of differentially labeled test and reference DNAs to an array of mapped human genomic DNA fragments (~100–200 kb) and has been recently applied to human and mouse tumors [9-14]. To identify high-resolution copy number, we used array CGH to the panel of 48 tumors. The resolution of array CGH technology combined with human genome database not only allowed a precise identification of amplicons but also suggested the possible target genes within the amplicons. Methods Patient samples A total of 48 tumors from 42 patients (20 males and 22 females) were collected from the Texas Children's Cancer Center, Houston, TX (tumors 193, 196, 204, 209, 221, 226, 248, 274, 295, 311, 326, 341, 345, 360, 400, 464, 481, 501, 527, 591 and 606) and Memorial Sloan Kettering Cancer Center, New York (tumors 06, 15, 24, 25, 27, 29, 32, 34, 40, 48, 68, 76, 78, 79, 80, 82, 83, 85, 88, 95, 98, 99, 102, 123, 423, 425, and 474). All tissues in this study were obtained after IRB approved informed consents were signed. The age at diagnosis ranged from 5 years to 71 years at diagnosis. The histological information of 42 patients is presented in Table 1. Array CGH The array used in this study consists of 967 human BACs, which were spaced approximately 3 megabase across the whole genome. These arrays were obtained from Spectral Genomics, Houston, TX. The experiments were performed according to the manufacturer's protocol. Arrays were pre-hybridized with human Cot-I DNA (GIBCO Invitrogen, Carlsbad, CA) and salmon testes DNA to block the repetitive sequences on BACs. One microgram of normal DNA (reference) and tumor DNA (test) was labeled with cy5-dUTP and cy3-dUTP respectively, by random priming. To avoid dye bias, we performed dye swap experiments for each sample. The probe mixture is dissolved in hybridization mixture, denatured, cooled, and mounted with 22 × 60 mm coverslip. Hybridizations were performed in sealed chambers for 16–20 hours at 60°C. After post hybridization washes, arrays were rinsed, dried with compressed air, and scanned into two 16-bit TIFF image files using Gene Pix 4000A two-color fluorescent scanner (Axon Instruments, Inc., Union City, CA) and quantitated using GenePix software (Axon Instruments, Union City, CA). Data processing and analysis After scanning of the slide, the fluorescent intensities of the green and red channels were background subtracted. The resulting values were normalized by intensity based local weighted regression method (Lowess) to correct for systematic bias in dye labeling and fluorescent intensity [15]. Then the ratio of the red/green channel of each clone was calculated and log base 2 transformed (log ratios). Each experiment was repeated once with dye reversal to addressing the confounding effect of the dye and experiment. The average of the dye-reversal experiment pair was calculated by reversing the sign of one experiment so that the log ratio reflects the tumor versus normal ratio. We developed a new analytical method called invariant analysis to define the significant copy number changes. This method is designed to: i) increase the power of the analysis by combining all the cases in our dataset to define an invariant population (unchanged population); and, ii) to address the signal to noise differences among individual cases due to sample and hybridization variability. Our goal is to define a set of unchanged clones that can be used to calculate the upper and lower bound thresholds of the log ratios for the unchanged population in each experiment. First, we calculated the variance of each clone from all the experiments. We computed the p-values of the each clone by comparing to the clone with median variance using chi-square distribution . The clones that have p-value greater than preset cutoff 0.9 were considered as invariant clone set, i.e. clones that do not vary significantly in all experiments. Then the mean and standard deviation of the log ratios of these invariant clones in each experiment were calculated. The clones with log ratios that exceed mean +/- 2 × SD of the invariant set were considered gains and losses, respectively. For amplification and homozygous deletions, clones were defined to have at least 2 fold of the upper bound threshold and 4-fold of lower bound threshold, respectively. The gene(s) present in the clones were identified using UCSC browser by downloading gene table (refFlat) from human gene assembly, July 2003. We search the candidate genes based on linear mapping position, which include 100 kb up and downstream from the clone center position. The supplemental data for this article is available at: Statistical analysis Significant clones in 6p, 8q, 12q and 17p amplicons were calculated using 2-sample t-test with randomized variance model . The experiments in each of the two groups, amplification and normal, used for comparison were defined based on the invariant analysis (see above). The clones that have p <0.001 were considered as significant. We chose a stringent cutoff to minimize the multiple testing problem. FISH FISH was performed to validate and quantify chromosomal amplicons using clones from 6p12-p21 (RP11-91E11, AL391415, RP11-81F7, RP11-79I2, RP11-90H17 and RP11-79F13), 8q24.3 (RP11-89K10), and 17p11.2-p12 (RP11-64B12, RP11-89K6 and RP11-189D22 on tumors metaphase/interphase cells from cases 274, 364, 425, 426, 527 and 628. We confirmed the map positions of all clones on normal human metaphase cells by FISH. The bacterial artificial chromosome (BAC) clones, and centromeric clone from 6 (pEDZ6) were labeled with Spectrum Red or Spectrum Green (Vysis, Downers Grove, IL) by nick translation. Hybridization and FISH analysis was performed as described previously [16]. Results To define the gains and losses in our experiments, we used invariant analysis for the first time to describe genomic changes by array CGH. In this method, we defined an invariant clone set that has low variance of log ratios among all the array experiments. After the mean and standard deviation of the log ratios in the invariant set of each experiment were calculated, clones that have higher or lower log ratios than the mean +/- 2SD of the invariant set (upper bound and lower bound) were used to define gains and losses. We chose to use this method because it addresses some of the shortcomings of the modeling method, such as using all information provided in a set of experiment to determine the unchanged population instead of using one experiment at a time. However, the variation of each experiment is accounted for because the thresholds are calculated using the invariant set from each experiment. It also does not require a separate reference set for comparison. Finally, it provides an adjustable cutoff to optimize the thresholds to the training data, if provided. The amplified and homozygously deleted clones were defined to have at least 2 fold of the upper bound and 4-fold of lower bound, respectively. Figure 1 summarizes the high-resolution DNA copy number changes identified by array CGH in 48 osteosarcomas derived from 42 patients. Copy number changes were detected involving small genomic regions, whole chromosomes, and chromosomal arms showing homozygous deletions and high-level amplifications. Overview of genomic profiles Copy number changes excluding clones from sex chromosomes were involved in a significant fraction of most tumor genome. The estimated average genomic distance between clones was ~3–4 Mb. The frequency of clones showing gains (79%) was greater than losses (66%). High-level amplifications and homozygous deletions constitute 28.6% and 3.8% of tumor genome respectively. The most frequently deleted clones were identified from the chromosomal regions 2q31.1, 3p14.1, 4p16.2, 6q12, 6q21, 6q27, 7q35, 10p15.1, 10q22-q23, 10q25-q26, 11q25, 13q12.2, 13q14.3, 13q22.1, 17p13.3 and 17q12 (Table 2). Most frequently gained clones were mapped to chromosome 1p36, 4p16, 6p12-p21, 8q21, 8q23-q24, 12q14.3, 16p13.3, 16q24.3, 17p11-p12, 19p13.3 and 21q22.3 (Table 3). We explored the possible statistical relationship between copy number alterations and histological and clinical parameters. We found no significant relationship between copy number changes and primary/metastatic disease, or histological type or histological response. This may be due to the involvement of large number of genomic loci and insufficient sample size. Homozygous deletions were noted for 32 clones (3.8%). Recurrent homozygous deletions were noted for 7 clones that are were mapped to 1q25.1 (4 cases), 3p14.1 (4 cases), 13q12.2 (2 cases), 4p15.1 (2 cases), 6q12 (2 cases), 6q12 (2 cases) and 6q16.3 (2 cases). Figure 2A is showing a homozygous deletion at 3p14.1 in tumor 06. Loss of 6q12 region was noted in 35% of the osteosarcomas. This region was covered with four clones spanning ~4.2 Mb. Two tumors (tumor 27 and 345) showed low intensity ratios indicting homozygous deletions in this region, one tumor (tumor 345) showed all 4 deleted clones spanning ~4.2 Mb with RP1-129L7 having the lowest ratio intensity decrease. In another case (tumor 27), two clones (RP1-46B1 and RP1-129L7) showed decreased intensity ratios indicating homozygous deletions. Both these clones spanning approximately 2.6 Mb of 6q12 region. Amplification is a frequent phenomenon in osteosarcoma Previous studies using CGH have identified several chromosomal amplification sites in osteosarcoma. Because of the limitation of the method, it fails to pinpoint the precise site of amplicon. However, the present study by array CGH has identified 238 clones (28.6%) with high-level amplifications. Recurrent amplifications were noted in ~37% of the total amplified clones (Figure 3). These amplified clones were mapped to 1p22, 1p31.1 (ROR1), 1p36.1 (PRDM16), 1q21, 1q23 (TNFF6), 2q24, 3p25, 3q26.1, 4p16.3, 5p14, 5q33, 6p11.2-p21, 7p21, 8q12.1, 8q24.13, 10p21, 10q11.1, 10q22 (KCNMA1), 11q13, 11q23 (GRIK4), 12q12, 12q13-q15, 12q21-q21.33, 17p11.2-p12, 17q21 (NGFR), 18q22, and 19p13.1 (NFAT). Of these amplified sites, 6p11.2-p21, 8q12.1, 8q24.13, 12q12, 12q13-q15, 12q21-q21.33, 16p13 and 17p11.2-p12 were frequent. Gain of clones from 6p12-p21 regions was noted in 33/48 (~65%) cases analyzed. High-level amplification of the clones from same region was noted in 25% of the cases by array CGH. We found that most of the cases with amplification of 6p12-p21 displayed either increased or slightly varying degree of copy number increase across the 6p12-p21 region. The combined log ratios from all the cases defined the boundaries of amplification between RP3-329A5 and RP11-79F13. The amplicon spans approximately 9.4 Mb with amplification peak for clone RP11-81F7. Further, we used FISH to validate 6p amplicon on tumor metaphase and interphase cells from cases 274, 364, 426 and 527. Increased copy numbers for clones RP11-91E11, AL391415, RP11-81F7, RP11-79I2, RP11-90H17 and RP11-79F13 were noted in interphase cells with maximum copy number increase for clone RP11-81F7 (Figure 4A). This was consistent with amplification peak for clone RP11-81F7 in the tumors profiled by array CGH (Figure 2B). In addition, we used 2-sample t-test with randomized variance model to define significant clones from 6p12-p21 amplicon. By this method, we identified RP11-79F13 (p = 0.00000007), RP11-79I2 (p = 0.00000007) and RP11-81F7 (p = 0.00000007) as statistically significant clones. Most cases with 8q gain, displayed varying degree of copy number increase predominantly from 8q12.1 (16.9%), 8q21.13 (29%), and 8q24.3 (35%). High-level amplifications were also noted from 8q12.1 (RP11-550I15 – 6.3%; Figure 2C), 8q21.13 (RP11-89H1 – 6.3%), 8q24.3 (RP11-89K10 – 6.3%) and RP11-637F16 (12.5%). FISH using clone RP11-89K10 (p = 0.00049) on interphase cells from case 527 confirmed the amplification (10–12 copies) (Figure 4B). Amplification of 12q was noted in 14/51 (~27%) tumors analyzed by array CGH. Three distinct amplicons – AMP1 (12q12), AMP2 (12q14.1) and AMP3 (12q21.33) were noted across the entire long arm of chromosome 12 (Figure 2D). Of these 14 cases, four of them (80, 123, 248, 341) displayed all three amplicons. The AMP1 was noted in 10 cases covering 1.8 Mb region between RP11-91K15 and RP11-90I21 with peak amplification for clone RP11-91K15 (p = 0.00000004). Another amplicon (AMP2) was noted 24.48 Mb distal to AMP1 between RP11-91K23 and RP11-89P15. The AMP3, which was 23.3 Mb distal to AMP2 containing RP11-89F6. Amplification of 17p11.2 was noted in 27% of the cases analyzed by array CGH. The amplicon was composed of three clones RP11-64B12 (p = 0.0000014), RP11-89K6 (p = 0.00000005) and RP11-189D22 (p = 0.0000001) and covering 3.7 Mb region on the short arm of chromosome 17 (Figure 2E). We used these three clones as FISH probes to validate 17p amplicon in tumors 274, 364, 425 and 628 on interphase/ metaphase cells. The distribution of copy number for this amplicon in all the cases ranged from 4–14 copies with peak amplification for clone RP11-189D22 (10–14 copies), followed by and RP11-89K6 (8–10 copies) RP11-64B12 (6–8 copies) (Figure 4C). Discussion This study represents the first application of genome-wide copy number changes by array CGH in osteosarcoma. Recent studies in breast, renal and bladder cancer showed the potential assessment of this technology in detecting high-resolution copy number changes [9,11,14]. This approach will augment the identification of cancer causing genes by relating the clone information directly with sequence information from human genome database. In this study, we used array CGH to screen for high-resolution DNA copy number changes and precise identification of amplifications in a panel of 48 osteosarcomas. Gene amplification is an important genetic mechanism in human cancers, as it clearly associated with tumor progression and has a prognostic significance and has even provided a target for therapeutics [17,18]. These amplifications are often seen at the cytogenetic level as homozygously staining regions (hsrs) or double minute chromosomes (dms). However, cytogenetic recognition of amplifications doesn't contribute to the mapping and identification of amplified DNA sequences. The advent of CGH points an ever-increasing number of chromosomal amplifications in various tumors. These amplifications contribute to the genomic instability in tumors. We have recently shown that the mutation of p53 significantly correlates with genome-wide DNA instability and seems to represent a major genetic factor contributing to the extremely high levels of genomic instability found in high-grade osteosarcomas [19]. Our analysis have identified frequently amplified clones from 6p11.2-p21, 8q12.1, 8q24.13, 12q12, 12q13-q15, 12q21-q21.33, 16p13 and 17p11.2-p12. Amplification of clones from 6p12-p21 region was noted in 25% of the cases analyzed. This was consistent with the previously published results by CGH. By array CGH, we refined the 6p amplicon to 9.4 Mb with amplification peak for clone RP11-81F7. We recently demonstrated the origin of 6p amplicon as consequence of tandem duplication of clones RP11-81F7 and RP11-79F13 [7]. Based on combined array CGH and FISH analysis suggest CDC5L, HSPCB, and NFKBIE, and HGNC and MRPL14 are the target genes from 6p12-p21 amplicon. Of these genes, CDC5L may be an important gene in cancer because of its role as a positive cell cycle regulator for G2/M transition[20]. Consistent with our analysis, overexpression of HSPCB was shown recently by cDNA microarray studies on osteosarcoma [21]. This protein was shown to play an important role in assemble/disassembly of tubulin by inhibiting tubulin polymerization. High-level amplifications were also noted from 8q12.1 (RP11-550I15 – 6.3%), 8q21.13 (RP11-89H1 – 6.3%), 8q24.3 (RP11-89K10 – 6.3%) and RP11-637F16 (12.5%). There were no candidate genes present in clones RP11-550I15, RP11-89H1 and RP11-637F16, but clone RP11-89K10 contained NSE2 (breast cancer membrane protein 101 kDa) gene. High-level amplification of clones on 12q revealed three distinct sites of amplifications – AMP1 (12q12), AMP2 (12q14.1) and AMP3 (12q21.33). Pervious studies have shown the amplification GLI, CHOP, SAS, HMGI-C, CDK4, HDM2, and PRIM1 from 12q13-q15 region in osteosarcoma [22,23]. The present array CGH analysis identified a possible target gene IFNG from AMP2 (RP11-298M11; p = 0.0000001), which is physically mapped close to the HDM2 oncogene locus[24]. Previous studies demonstrated that T-cell production of IFNG strongly suppresses osteoclastogenesis by interfering with the RANKL-RANK signaling pathway. IFNG induces rapid degradation of the RANK adaptor protein, TRAF6, resulting in strong inhibition of the RANKL-induced activation of the transcription factor NFKB and JNK [25]. The AMP3, which was 23.3 Mb distal to AMP2 containing RP11-89F6. Our analysis from AMP3 revealed two interesting candidate genes: transcription factor ELK3 and PCTAIRE protein kinase 2 (PCTK2). ELK3 is a member of the ETS-domain transcription factor family and the protein is activated by signal-induced phosphorylation [26]. The protein encoded by PCTK2 belongs to the cdc2/cdkx subfamily of the ser/thr family of protein kinases and play an important role in the regulation of the mammalian cell cycle [27]. High-level amplification of three clones from 12p13 was noted in case 27 and the amplicon span 4.6 Mb with peak amplification for clone RP11-89D16. No candidate genes contained with in this BAC. Amplification 12p has been reported previously in 9/19 high-grade osteosarcomas by CGH. Recent FISH analysis has identified the amplification of CCND2, ETV6, and KRAS2 from 12p region [28]. Amplification of 17p11.2 was noted in 27% of the cases analyzed by array CGH. Our array CGH analysis has identified three clones with high-level amplifications that spans ~3.7 Mb region on 17p11.2. Several candidate genes were identified within these clones (TPP3A, SMCR5, DRG2, FL11, MYCD, SOX 17, ELAC2, and PMP22). Recent studies have shown the amplification of some of the genes identified in the present study (PMP22, and TOP3A) from 17p11.2-p12 in high-grade OS by semi-quantitative PCR and cDNA microarrays [29,30]. The present array CGH analysis has identified seven recurrent clones exhibiting homozygous deletions from 1q25.1, 3p14.1, 13q12.2, 4p15.1, 6q12, 6q12 and 6q16.3. These chromosomal regions were consistent with previously reported studies by loss of heterozygosity (LOH) and CGH [3-7,31]. The clone, RP11-90M15 (13q12.2) contain possible candidate gene MTMR6, a protein-tyrosine phosphatase gene and shown to be present within a cloned region that encompasses a translocation breakpoint t(8;13) in an atypical myoproliferative disorder [32]. Homozygous deletions of two clones spanning approximately 2.6 Mb of 6q12 region containing candidate genes – nuclear fragile X mental retardation protein interacting protein 1 pseudogene (NUFIP1P) and BAI3 gene (brain-specific angiogenesis inhibitor gene), which is to homologous to BAI1 and shown to suppress glioblastoma [33]. Conclusions In summary, high resolution array-based CGH revealed large number of chromosomal aberrations previously identified in osteosarcoma by chromosomal CGH and conventional cytogenetic methods. The present study allowed precise identification of smaller DNA copy number alterations, which suggest the presence of specific target genes in osteosarcoma. Although this study suggested several possible target genes from amplified regions from 6p, 8q, 12q and 17p, but these genes should be validated by other molecular and immunohistochemical approaches on well-defined large patient samples. Further, interaction or association studies between small genomic losses and gains will facilitate the identification of new genetic pathways in the pathogenesis of osteosarcoma. Competing interests None declared. Authors contributions TKM and KJ have contributed towards the data analysis. LP, ML, RG, and CL were assisted in sample collection and clinical information of the patients. X-YL has involved in array CGH experiments and data collection. CPH has involved in extracting the gene information from BAC clones. SS has provided the arrays used in this study. PHR was involved in the planning, and organization of the project. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements We thank Prof Rocchi, University of Bari, Italy for providing chromosome 6 centromeric clone and Dr. Richard Simon and Amy Peng for BRB ArrayTools software. This work was supported in part by NIH grant CA88126 and by grants from the Dunn Foundation and the Kleberg Foundation to C.C.L. Figures and Tables Figure 1 Frequency of DNA copy number changes identified by array CGH in 48 osteosarcomas. The gains and losses are depicted as green and red color bars respectively. Clones are arranged from chromosome 1 to 22 and within each chromosome on the basis of UCSF mapping position. Figure 2 A representative chromosome profiles showing homozygous deletions (A, E) and high-level amplifications (B- E). The clones showing homozygous deletions from 3p14.1 (RP11-89A12) and 17q12 (RP11-79O9) in tumor 06 (A, E) and high-level amplifications from 6p12-p21 in tumor 248(B), 8q12 in tumors 06 and 341 and 8q23-q24 in tumor 06, (C), 12q13-q15 in tumor 48, (D) and 17p11.2 in tumor 06(E), are shown as log ratios (Y-axis). The clones on each chromosome are arranged (pter to qter) on the basis of UCSC mapping positions. Figure 3 Ideogram showing recurrent homozygous deletions (left) and high-level amplifications (right) identified by array CGH in 48 cases. The gene(s) contained within the BAC clone are shown in parentheses of the respective clones. Gene(s) present in the BAC clones were identified using UCSC browser by downloading gene table (refFlat) from human gene assembly, July 2003. The ideograms for chromosomes 6, 8, 12 and 17 are shown separately. Figure 4 FISH validation of some of the high-level amplifications (6p12.1, 8q24.3 and 17p11.2) identified by array CGH. Interphase cells hybridized with centromere 6 (red)/RP11-81F7 (green) in case 274 (A), RP11-89K10 (red) in case 527 (B) and RP11-189D22 (red) in case 364 (C). The ploidy of these cases was determined based on the modal chromosome number of the respective cases, e.g. diploid (case 426) triploid (cases 274 and 364), and tetraploid (case 527). Table 1 Histological information on 48 osteosarcoma samples Tumor No. Sex Age-Dx Site Histological Subtype Type Metastatic Disease Huvos Grade Response 06 Female 7 Distal Femur NA Biopsy L II PR 15 Male 35 Distal Femur Osteoblastic+MFH-Like Biopsy L 24 Female 16 Clavicle Chondroblastic Biopsy L Minimal 25 Male 8 Skull Osteoblastic Mets L 27 Female 34 Ischium Mixed Chondroblastic+Fibroblastic-Like Mets L 29 Male 19 Ilium NA Biopsy M II PR 32 Female 25 Ilium Chondroblastic Biopsy L Minimal 34 15 Femur Chndroblastic And Osteoblastic Biopsy L II PR 40 Female 11 Left Distal Femur Giant Cell Definitive None IV GR 48 Female 32 Proximal Tibia Osteo/Fibro/Chondroblastic Definitive None I PR 68 Male 18 Femur Telangectactic Mets 76 Female 20 Thigh N/A Biopsy L 78 Male 5 Humerus N/A Biopsy L III GR 79 Male 9 Tibia Telangectactic Mets L III GR 80 Male 46 Tibia N/A Biopsy L I PR 82 Male 23 Humerus Osteoblastic Mets L I PR 83 Female 12 Femur Telangiectatic Biopsy L IV GR 85 Male 34 Femur Fibroblastic Biopsy L I PR 88 Male 17 Humerus Chndorblastic Biopsy L IV GR 95 Male 71 Femur Giant Cel Rich Biopsy L 98 Female 31 Ilium Epithelioid Biopsy L 99 Female 22 Humerus N/A Biopsy L 102 Male 70 Humerus Fibrohistiocytic Mets L 123 Female 16 Femur N/A Biopsy L I PR 209 Female 17 Distal Femur Osteoblastic Biopsy No II PR 221 Female 17 Femur Osteoblastic Biopsy No IV GR 248 Female 13 Tibia Pleiomorphic Biopsy No ? 311 Female 13 Distal Femur Osteoblastic Definitive No III GR 326 Female 20 Femur Osteoblastic Pul Met Yes IV GR 341 Male 12 Lemur Fibroblastic Biopsy N II PR 345 Male 10 Distal Femur Osteosarcoma Biopsy No IV GR 360 Female 18 Distal Femur Osteoblastic Biopsy Yes IV GR 400 Female 15 Distal Femur Chondroblastic Yes 423 Male 30 Proximal Humerus Giant Cell Definitive None I PR 425 Male 24 Tibia N/A Biopsy Proximal Femur I PR 474 Female 17 Pelvis Chondroblastic Biopsy None II PR 591 Male 15 Proximal L Tibia Telangectactic Definitive No II PR 193a Female 17 Distal Femur Osteoblastic Biopsy No 196a Female 15 Distal Femur Osteoblastic Pul Met Yes 274b Male 13 Distal Femur Osteoblastic//Chondroblastic Biopsy No II PR 295b Male 13 Femur Osteoblastic//Chondroblastic Yes II PR 464c Female 15 Distal Femoral Head Osteoblastic/Spindle Cell/Chondroblastic Biopsy No II PR 501c Female 15 Femur Osteoblastic Definitive No II PR 606c Female 14 Distal Femur Osteoblastic Biopsy Yes II PR 481d Male 10 Distal Femur Osteoblastic Biopsy No II PR 527d Male 10 Proximal Tibia Osteoblastic Definitive Yes III GR 204e Female 18 Distal Femur Osteoblastic Biopsy No I PR 226e Female 18 Distal Femur Telangectactic Definitive No I PR a, b, c, d, e-Tumor samples obtained from the same patient. Table 2 Most frequently lost clones. Clone Map Position (Mb) Cyto Position Frequency (%) Genes RP11-79K15 31.9 17q12 27 RP1-140C12 170.4 6q27 22.9 PSMB1 RP11-90M15 24.8 13q12.2 20.8 RP11-79I4 73.4 13q22.1 20.8 KLF12 RP11-79K22 101.7 6q16.3 20.8 MTMR6, NUPL1 AC004889 148.401 7q35 20.8 OR2A4 RP11-89H7 116.5 10q25.3 18.8 AL359836 128.22 10q26.11 18.8 RP11-80L16 67 6q12 18.8 RP11-80D10 2.8 10p15.1 16.7 AC021027 84.029 10q22.3 16.7 RP11-79E24 88 10q23.2 16.7 RP11-90B19 131.5 10q26.3 16.7 RP11-835G21 143.17 11q25 16.7 RP11-80H2 50 13q14.3 16.7 RP11-81D9 72.8 13q22.1 16.7 RP5-1029F21 1.21 17p13.3 16.7 AC020681 175.95 2q31.1 16.7 PDK1 RP11-89A12 68 3p14.1 16.7 RP11-9A1 71.28 3p14.1 16.7 RP11-492I23 3.64 4p16.2 16.7 RP1-46B1 69.4 6q12 16.7 BAI3 RP3-454N4 106 6q21 16.7 Table 3 Most frequently gained clones. Clone Map Position (Mb) Cyto Position Frequency (%) Genes RP11-81F7 43.8 6p21.1 45 HGNC, MRLP14 RP1-163G9 2.6 1p36.32 43 PRDM16 RP11-79F13 44.6 6p21.1 37.5 CDC5L, HSPCB, NFKBIE RP11-90H17 46.5 6p12.3 37.5 UCP4 RP11-64L12 0.68 16p13.3 37.5 MSLN, SOX8 RP11-637F16 144.61 8q24.3 35.4 RP3-447E21 46 6p21.1 35.4 CLIC5 RP11-79I2 43.4 6p21.1 31.2 EGFL RP11-80F24 78.3 8q21.13 29 RP4-753D5 50.9 6p12.3 29 TFAP2B AC005263 0.95 19p13.3 29 AMH, GNRPX, DIT1L RP11-189D22 19.56 17p11.2 29 TPP3A, SMCR5, DRG2, FLI RP11-89P19 119.5 8q23.3 27 RP11-89H1 77.4 8q21.13 25 RP11-88N2 43.7 21q22.3 25 SNF1LK RP1-283E3 1.5 1p36.33 25 CDC2L2, GNB1 RP11-89P9 125.4 8q24.13 22.9 MTSS1 AL391415 43.06 6p21.2 22.9 GLO1, DNAH8 RP11-492I23 3.64 4p16.2 22.9 RP1-163M9 16.2 1p36.13 22.9 RP11-383B15 2.89 19p13.3 22.9 RP11-89K6 13.1 17p12 22.9 RP11-89K10 127.3 8q24.13 20.8 NSE2 RP11-90D11 98.8 8q22.1 20.8 RP11-91E11 37.4 6p21.2 20.8 PIM1 RP3-417I1 63.04 6p11.2 20.8 BAG2, RAB23 RP11-79O4 19.9 17p11.2 20.8 ULK2, AKAP10 RP11-46C24 101.27 16q24.3 20.8 RP11-91K23 67 12q14.3 20.8 ==== Refs Huvos A Huvos A Bone Tumors: Diagnosis, Treatment and Prognosis, 1991 Philadelphia, WB Saunders Provisor AJ Ettinger LJ Nachman JB Krailo MD Makley JT Yunis EJ Huvos AG Betcher DL Baum ES Kisker CT Miser JS Treatment of nonmetastatic osteosarcoma of the extremity with preoperative and postoperative chemotherapy: a report from the children's cancer group J Clin Oncol 1997 15 76 84 8996127 Tarkkanen M Karhu R Kallioniemi A Elomaa I Kivioja AH Nevalainen J Bohling T Karaharju E Hyytinen E Knuutila S Gains and losses of DNA sequences in osteosarcomas by comparative genome hybridization Cancer Res 1995 55 1334 1338 7882332 Tarkkanen M Elomaa I Blomqvist C Kivioja AH Kellokumpu-Lehtinen P Bohling T Valle J Knuutila S DNA sequence copy number increase at 8q: a potential new prognostic marker in high-grade osteosarcoma Int J Cancer 1999 84 114 121 10096241 10.1002/(SICI)1097-0215(19990420)84:2<114::AID-IJC4>3.0.CO;2-Q Stock C Kager L Fink F-M Gadner H Ambros PF Chromosomal regions involved in the pathogenesis of osteosarcoma Genes Chromosomes Cancer 2000 28 329 336 10862039 10.1002/1098-2264(200007)28:3<329::AID-GCC11>3.0.CO;2-F Zielenska M Bayani J Pandira A Toledo S Marrano P Andrade J Petrilli A Thorner P Sorensen P Squire JA Comparative genomic hybridization analysis identifies gains of 1p35-36 and chromosome 19 in osteosarcoma Cancer Genet Cytogenet 2001 130 14 21 11672768 10.1016/S0165-4608(01)00461-7 Lau CC Harris CP Lu XY Perlaky L Gogineni S Chintagumpala M Hicks J Johnson ME Davino NA Huvos AG Meyers PA Healy JH Gorlick R Rao PH Frequent amplification and rearrangement of chromosomal bands 6p12-p21 and 17p11.2 in osteosarcoma Genes Chromosomes Cancer 2004 39 11 21 14603437 10.1002/gcc.10291 Pinkel D Segraves R Sudar D Clark S Poole I Koebel D Collins C Kuo WL Chen Zhai Y Dairkee SH Ljung BM Gray JW Albertson D High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays Nature Genet 1998 20 207 211 9771718 10.1038/2524 Albertson DG Ylstra B Segraves R Collins C Dairkee SH Kowbel D Kuo WL Gray JW Pinkel D Quantitative mapping of amplicon structure by array CGH identifies CYP24 as a candidate oncogene Nat Genet 2000 25 144 146 10835626 10.1038/75985 Cai WW Mao JH Chow CW Damani S Balmain A Bradley A Genome-wide detection of chromosomal imbalances in tumors using BAC microarrays Nat Biotechnol 2002 20 393 6 11923847 10.1038/nbt0402-393 Wilhelm M Veltman JA Olshen AB Jain AN Moore DH Presti JC JrKovacs G Waldman FM Array-based comparative genomic hybridization for the differential diagnosis of renal cell cancer Cancer Res 2002 62 957 60 11861363 Hackett CS Hodgson JG Law ME Fridlyand J Osoegawa K de Jong PJ Nowak NJ Pinkel D Albertson DG Jain A Jenkins R Gray JW Weiss WA Genome-wide array CGH analysis of murine neuroblastoma reveals distinct genomic aberrations, which parallel those in human tumors Cancer Res 2003 63 5266 73 14500357 O'Hagan RC Brennan CW Strahs A Zhang X Kannan K Donovan M Cauwels C Sharpless NE Wong WH Chin L Array comparative genome hybridization for tumor classification and gene discovery in mouse models of malignant melanoma Cancer Res 2003 63 5352 5356 14500367 Veltman JA Fridlyand J Pejavar S Olshen AB Korkola JE DeVries S Carroll P Kuo WL Pinkel D Albertson D Cordon-Cardo C Jain AN Waldman FM Array-based comparative genomic hybridization for genome-wide screening of DNA copy number in bladder tumors Cancer Res 2003 63 2872 80 12782593 Yang YH Dudoit S Luu P Lin DM Peng V Ngai J Speed TP Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation Nucleic Acids Res 2002 30 e15 11842121 10.1093/nar/30.4.e15 Singh B Gogineni SK Sacks PG Shaha AR Shah JP Stoffel A Rao PH Molecular cytogenetic characterization of head and neck squamous cell carcinoma and refinement of 3q amplification Cancer Res 2001 61 4506 4513 11389082 Lengauer C Kinzler KW Vogelstein B Genetic instabilities in human cancers Nature 1998 396 643 649 9872311 10.1038/25292 Schwab M Oncogene amplification in solid tumors Review Semin Cancer Biol 1999 9 319 325 10.1006/scbi.1999.0126 Overholtzer M Rao PH Favis R Lu XY Elowitz MB Barany F Ladanyi M Gorlick R Levine AJ The presence of p53 mutations in human osteosarcomas correlates with high levels of genomic instability Proc Natl Acad Sci U S A 2003 100 11547 52 12972634 10.1073/pnas.1934852100 Bernstein HS Coughlin SR A mammalian homolog of fission yeast Cdc5 regulates G2 progression and mitotic entry J Biol Chem 1998 273 4666 4671 9468527 10.1074/jbc.273.8.4666 Wolf M El-Rifai W Tarkkanen M Kononen J Serra M Eriksen EF Elomaa I Kallioniemi A Kallioniemi OP Knuutila S Novel findings in gene expression detected in human osteosarcoma by cDNA microarray Cancer Genet Cytogenet 2000 123 128 32 11156738 10.1016/S0165-4608(00)00319-8 Wei G Lonardo F Ueda T Kim T Huvos AG Healey JH Ladanyi M CDK4 gene amplification in osteosarcoma: reciprocal relationship with INK4A gene alterations and mapping of 12q13 amplicons Int J Cancer 1999 80 199 204 9935200 10.1002/(SICI)1097-0215(19990118)80:2<199::AID-IJC7>3.0.CO;2-4 Yotov WV Hamel H Rivard GE Champange MA Russo PA Leclecrc JM Bernstein ML Levy E Amplification of DNA Primase 1 (PRIM1) in human osteosarcoma Genes Chromosomes Cancer 1999 26 62 69 10441007 10.1002/(SICI)1098-2264(199909)26:1<62::AID-GCC9>3.0.CO;2-F Bureau JF Bihl F Brahic M Le Paslier D The gene coding for interferon-gamma is linked to the D12S335 and D12S313 microsatellites and to the MDM2 gene Genomics 1995 28 109 112 7590734 10.1006/geno.1995.1114 Takayanagi H Ogasawara K Hida S Chiba T Murata S Sato K Takaoka A Yokochi T Oda H Tanaka K Nakamura K Taniguchi T T-cell-mediated regulation of osteoclastogenesis by signaling cross-talk between RANKL and IFN-gamma Nature 2000 408 600 605 11117749 10.1038/35046102 Lopez M Oettgen P Akbarali Y Dendorfer U Libermann TA ERP, a new member of the ets transcription factor/oncoprotein family. cloning, characterization, and differential expression during B-lymphocyte development Molec Cell Biol 1994 14 3292 3309 7909357 Meyerson M Enders GH Wu C-L Su L-K Gorka C Nelson C Harlow E Tsai L-H A family of human cdc2-related protein kinases EMBO J 1992 11 2909 2917 1639063 Gisselsson D Palsson E Hoglund M Domanski H Mertens F Pandis N Sciot R Dal Cin P Bridge JA Mandahl N Differentially amplified chromosome 12 sequences in low-and high-grade osteosarcoma Genes Chromosomes Cancer 2002 33 133 40 11793439 10.1002/gcc.1219 van Dartel M Cornelissen PW Redeker S Tarkkanen M Knuutila S Hogendoorn PC Westerveld A Gomes I Bras J Hulsebos TJ Amplification of 17p11.2 approximately p12, including PMP22, TOP3A, and MAPK7, in high-grade osteosarcoma Cancer Genet Cytogenet 2002 139 91 96 12550767 10.1016/S0165-4608(02)00627-1 Squire JA Pei J Marrano P Beheshti B Bayani J Lim G Moldovan L Zielenska M High-resolution mapping of amplifications and deletions in pediatric osteosarcoma by use of CGH analysis of cDNA microarrays Genes Chromosomes Cancer 2003 38 215 25 14506695 10.1002/gcc.10273 Yamaguchi T Toguchida J Yamamuro T Kotoura Y Takada N Kawaguchi N Kaneko Y Nakamura Y Sasaki MS Ishizaki K Allelotype analysis in osteosarcoma: frequent allele loss on 3q, 13q, 17q, and 18q Cancer Res 1992 52 2419 2423 1568211 Laporte J Blondeau F Buj-Bello A Tentler D Kretz C Dahl N Mandel J-L Characterization of the myotubularin dual specificity phosphatase gene family from yeast to human Hum Molec Genet 1998 7 1703 1712 9736772 10.1093/hmg/7.11.1703 Shiratsuchi T Nishimori H Ichise H Nakamura Y Tokino T Cloning and characterization of BAI2 and BAI3, novel genes homologous to brain-specific angiogenesis inhibitor 1 (BAI1) Cytogenet Cell Gene 1997 79 103 108

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==== Front BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-4-461530169110.1186/1471-2407-4-46Research ArticleDMBT1 expression is down-regulated in breast cancer Braidotti P [email protected] PG [email protected] J [email protected] A [email protected] C [email protected] A [email protected] G [email protected] G [email protected] S [email protected] GG [email protected] University of Milano, Department of Medicine, Surgery and Dentistry, S.Paolo Hospital and IRCCS Ospedale Maggiore, Milan, Italy2 Molecular Pathology Unit, FIRC Institute of Molecular Oncology, Milan, Italy3 Department of Molecular Genome Analysis, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany4 Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical School, Philadelphia, PA 19104, USA2004 9 8 2004 4 46 46 19 3 2004 9 8 2004 Copyright © 2004 Braidotti et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background We studied the expression of DMBT1 (deleted in malignant brain tumor 1), a putative tumor suppressor gene, in normal, proliferative, and malignant breast epithelium and its possible relation to cell cycle. Methods Sections from 17 benign lesions and 55 carcinomas were immunostained with anti DMBT1 antibody (DMBTh12) and sections from 36 samples, were double-stained also with anti MCM5, one of the 6 pre-replicative complex proteins with cell proliferation-licensing functions. DMBT1 gene expression at mRNA level was assessed by RT-PCR in frozen tissues samples from 39 patients. Results Normal glands and hyperplastic epithelium in benign lesions displayed a luminal polarized DMBTh12 immunoreactivity. Normal and hyperplastic epithelium adjacent to carcinomas showed a loss of polarization, with immunostaining present in basal and perinuclear cytoplasmic compartments. DMBT1 protein expression was down-regulated in the cancerous lesions compared to the normal and/or hyperplastic epithelium adjacent to carcinomas (3/55 positive carcinomas versus 33/42 positive normal/hyperplastic epithelia; p = 0.0001). In 72% of cases RT-PCR confirmed immunohistochemical results. Most of normal and hyperplastic mammary cells positive with DMBTh12 were also MCM5-positive. Conclusions The redistribution and up-regulation of DMBT1 in normal and hyperplastic tissues flanking malignant tumours and its down-regulation in carcinomas suggests a potential role in breast cancer. Moreover, the concomitant expression of DMTB1 and MCM5 suggests its possible association with the cell-cycle regulation. ==== Body Background DMBT1 (Deleted in Malignant Brain Tumor) at chromosome 10q25.3–26.1, was considered a tumour suppressor gene because frequent deletions and/or lack of expression had been found in malignant tumours of the brain [1-3], the gastrointestinal tract [4,5], and the lung [6,7]. However, recent evidence has shown that DMBT1 is not a typical tumour suppressor gene because it is up-regulated in normal or inflamed epithelium adjacent to lung carcinomas as well as in some primary lung cancers and glioblastomas [8]. It was also shown that mutations in combination with loss of heterozygosity do not play a role in DMBT1 inactivation in many tumours including breast cancer [9,10] DMBT1 is a multi-functional protein related to the Mac-2 binding protein and mucins that have a protective function and mediate cell-extracellular matrix interactions [8,11]. DMBT1GP340(glycoprotein-340) and DMBT1SAG (Salivary Agglutinin) are DMBT1 variants, present in the respiratory tract and the oral cavity, respectively, that have been linked to innate host defense and epithelial regeneration [3,12-16]. In the respiratory tract, DMBT1GP340is involved in innate host defence by its interaction with the lectins surfactant protein D and A (Sp-D, Sp-A) and by its ability to stimulate alveolar macrophage migration [12,13,17]. Another lectin, mannan binding protein (MBP), has been shown to have an anti tumour effect against gliomas and colorectal carcinomas in vitro and in vivo [18,19]. While this anti tumor property remains to be demonstrated for Sp-A and Sp-D, we have previously reported the presence of surfactant protein A (Sp-A) in the ductal epithelium of the normal breast and its variable expression in mammary carcinomas. We found that in the normal ductal epithelium Sp-A immunoreactivity is present on the luminal side, whereas in carcinomas it is present both on the luminal side as well as on the baso-lateral surfaces. In carcinomas, the Sp-A immunoreactivity is not only redistributed but also decreased as the histological tumour grade increases [20]. Remarkably, this resembles the changes of DMBT1 expression and localisation observed in other tumor types originating from mono-layered epithelia such as primary esophageal adeno-carcinomas and lung carcinomas [5,8]. Hensin, the rabbit homologue of DMBT1, triggers epithelial terminal differentiation by mediating cell-extracellular matrix (ECM) interactions, and this may apply also to DMBT1 [3,21-24]. CRP-ductin, DMBT1 homologue in the mouse, and a novel homologue in the rat (DMBT1 4,7 kb), have been linked to initiation of cell proliferation, differentiation and repair [25,26]. It has been proposed that translocation of DMBT1 to the ECM triggers cellular differentiation, whereas a loss of expression of DMBT1 favours tumor cell growth [8]. These findings led us to explore whether similar principles apply to breast epithelium. Here we examined the expression of DMBT1 in the morphologically normal, hyperplastic, and neoplastic breast epithelium. We also examined in morphologically normal epithelium a possible association of DMBT1 expression with that of an early proliferative marker such as MCM5 (Minichromosome Maintenance protein 5). MCM5 is one of the 6 pre-replicative complex proteins (MCM 2–7) [27] and its expression was compared with that of Ki 67 proliferation index. Methods Tissue and cell-lines From the files of the Surgical Pathology at S.Paolo University Hospital, we retrieved paraffin blocks of 72 consecutive breast lesions surgically resected. This material included 16 cases of fibrocystic disease (FD), 1 case of gynecomastia (GM), 10 samples of ductal carcinoma in situ (DCIS) and 45 cases of infiltrating carcinoma (IC) (36 ductal, 2 lobular, 2 mixed lobular and ductal, 2 mucinous, 1 apocrine, 1 cribriform, 1 papillary). RT-PCR (Reverse-Transcriptase Polymerase Chain Reaction) was performed on 39 samples: i.e. 37 samples out of the 55 carcinomas used for the immunohistochemical studies, 1 sample of normal breast tissue that had been removed with a neoplastic lesion and 1 sample of fibrocystic disease. All 39 samples had been quickly frozen in liquid nitrogen after surgical resection and stored at -80°C. As confirmed by histopathologic examination, in all tumor specimens the amount of tumor cells equalled or exceeded 80% of the overall sample. As controls for each test a frozen sample of the same case positive with immunohistochemical method was used. We also analysed by RT-PCR two breast cancer cell lines (Hs578T and T-47D) obtained from the American Type Culture Collection and maintained as recommended. Immunohistochemistry Sections from blocks containing the predominant lesion were incubated with anti DMBT1h12 monoclonal antibody [3] as well as anti Ki67 Mib1 (Dako Cytomation DK). Double labelling with anti-DMBT1 and anti MCM5 monoclonal antibodies (Novocastra Laboratories Ltd, Newcastle upon Tyne, U.K.) was performed on 17 benign lesions and a subset of 19 in situ and invasive carcinomas where residual normal breast tissue was well represented. On these selected samples a single incubation with anti MCM5 and anti Ki 67 antibody was performed in order to compare the two respective immunoreactivities. Antibodies were diluted as follows: anti-DMBT1h12, 1:500; anti MCM5, 1:40; anti Ki 67 Mib1, 1:100. For anti-DMBT1 and Ki 67 Abs, sections were pre-treated for antigen retrieval in boiling 0.01 M sodium citrate buffer pH 6, in microwave (MW) oven for 12 minutes. DMBT1 and MCM5 double labelling was performed in two steps following a modification of the method of Lan et al. [28], briefly: 1) first antigen retrieval 6 minutes in 0.5 M EDTA pH8 in MW oven three times, followed by 1 h incubation with anti DMBT1h12. The reaction was developed with alkaline phosphatase using Fucsin + (Dako) as chromogen. 2) same antigen retrieval followed by over-night incubation with anti MCM5 Ab and development with peroxydase and diaminobenzidine (DAB) as chromogen. All slides were incubated in an automated slides stainer (Biogenex, S.Ramon, CA, U.S.A.); reaction products were visualized by En Vision product (Dako). Negative and positive controls were added in each experiment. Three investigators (AM, PB, PGN) evaluated separately DMBTh12 immunopositivity of morphologically normal, hyperplastic, and malignant cells. Immunopositivity was scored in 4 distinct categories according to the percentage of positive cells: 0 = negative, 1 = < 10%, 2 = 10%–50% and 3 = > 50%. MCM5 and Ki67 positivity in morphologically normal epithelium was determined with a semi-quantitative method: percentage of positive glandular structure and number of positive cells observed in each positive gland divided number of glands. RNA extraction Total RNA was extracted from breast specimens using Trizol reagent, (Invitrogen s.r.l., S. Giuliano Milanese, Italy) according to the manufacturer's instructions. Total RNA was quantified and checked for purity by UV spectrophotometry. cDNA synthesis Reverse transcription of RNA was done in a final volume of 20 μl containing 1x buffer (50 mM KCl, 15 mM Tris-HCl, pH 8.3), 500 μM each deoxynucleotide triphosphate, 5 mM MgCl2, 20 units of RNase inhibitor, 50 units of MuLV Reverse Transcriptase, 2.5 μM Random hexamers and 1 μg of total RNA. All reagents were purchased by the supplayer (Applied Biosystems, Foster City, CA, USA). The samples were incubated at 25°C for 10 min, 42°C for 60 min and at 95°C for 5 min. PCR amplification Polymerase chain reaction (PCR) of the cDNA was performed in a final volume of 50 μl containing all four dNTPs (each at 200 μM), 1X buffer, 1.5 mM MgCl2, 1.25 units of Taq Gold DNA polymerase and each primer at 0.3 μM. The PCR amplification of DMBT1 was carried out in two steps, following a nested procedure. In the first step we used 2 μl of cDNA from the reverse transcription and in the second 2 μl of the first PCR product as template. Primers and thermal condition were used according to Mollenhauer et al [3]. The specificity of the PCR product was confirmed by sequencing with BigDyeTM terminator chemistry using the genetic analyser ABI Prism 310 (Applied Biosystems), β-2-microglobulin (as control for sample integrity) was amplified in the amplification mixture described above and using the following primers: 5'-TCTGGGTTTCATCCATCCGA-3' and 5'-CCCCAAATTCTAAGCAGAGTATGTAA-3'. The thermal cycling parameters were: initial step of 10 min at 94°C, 39 cycles at 94°C for 30 sec, 58°C for 30 sec, 72 °C for 30 sec, and a final step for 5 min at 72°C. All the PCR products were separated by electrophoresis on a 1.5% agarose gel containing ethidium bromide and visualized under UV transillumination. Amplification product of 602 bp (β-2-microglobulin) and 435 bp (DMBT1) were obtained. Data analysis Statistical differences between immunopositive cases/total number of cases were calculated by Fisher's exact test. p value less than 0.05 were considered statistically significant. T-test for independent values was used to evaluate statistically significant differences between immunoreactivity of normal and hyperplastic epithelium in benign, pre-invasive and invasive lesions. Results Immunohistochemistry Different patterns of immunopositivity were observed in the histologically normal and hyperplastic breast tissue, present in the sections of benign and malignant lesions: a) single supra-nuclear, intra-cytoplasmic dot polarized toward luminal surface [Figure 1a]; b) a strong surface decoration of luminal cells [Figure 1b], c) intra-luminal immuno-reactive material [Figure 1c], d) multiple, randomly distributed peri-nuclear, irregular dots [Figure 1d], e) marked supra-nuclear and/or baso-lateral accumulation of reaction product [Figure 1e] and f) strong diffuse granular intra-cytoplasmic decoration [Figure 1f]. Morphologically normal ducts showed the pattern of immunopositivity described in section a). In addition to pattern a), the epithelium in benign lesions showed immunoreactivity patterns b) and c). Hyperplastic tissues flanking carcinoma frequently showed depolarization of immuno-reaction of patterns d), e), and f). Normal and hyperplastic mammary structures displayed a variable DMBTh12 immunoreactivity: some ducts were decorated while others were negative. Moreover a variable percentage of cells was immunopositive. This heterogeneity suggests that there is no constitutive expression of DMBT1 at protein level in the mammary gland epithelium. In tumour areas, only 3 out of 55 carcinomas showed variable degrees of immunopositivity with anti-DMBTh12 Ab: in one case there was a widespread and strong decoration of numerous tumour cells (immunopositivity score = 3) with irregularly distributed dots [Figure 1g]; in the second case only few cells had cytoplasmic positivity (immunopositivity score = 1) [Figure 1h] and in the third case only weak granular decoration in a small clusters of cells was present (immunopositivity score = 1). All other carcinomas were completely negative (immunopositivity score = 0) [Figure 1i]. Down-regulation of DMBT1 expression in the cancerous lesions compared to the normal and/or hyperplastic epithelium was statistically significant for both, ductal carcinoma in situ (DCIS) (1/10 versus 33/42 normal and/or hyperplastic epithelia; p = 0.0001) and invasive carcinoma (IC) (2/45 versus 33/42 normal and/or hyperplastic epithelia; p < 0.0001). The results of DMBT1 expression at protein and mRNA level are summarized in table 1. According to the 4 category scoring system, there was no statistically significant difference of DMBT1 immunoreactive cells between the normal and the hyperplastic epithelium that flanked benign lesions, in situ and invasive carcinomas (Table 2). Double immunostaining with DMBTh12 Ab and anti MCM5 Ab revealed a co-expression of the two antigens, respectively, in the cytoplasm and the nucleus of the same cells in morphologically normal breast epithelium (Figure 2a,2b). Conversely, glands that were negative with anti DMBT1 were negative also with anti MCM5 antibody (Figure 2b). DMBT1 and MCM5 positive or negative glands were morphologically undistinguishable. In comparison to MCM5, very few morphologically normal epithelial cells labelled by Anti DMBT1 Ab, expressed Ki67 (Figure 3a,3b,3c). 30–50% of glands were positive with anti MCM5 Ab with a mean number of 4,5 positive cells per positive glandular structure versus less than 10% of positive glands with a mean number of 1,46 positive cell per positive glandular structure with anti Ki 67 Ab. As expected for a pre-replicative marker, anti MCM5 Ab decorated diffusely all benign proliferative and malignant lesions. The percentage of Ki 67 positive cells of carcinomas varied and was assessed as part of the protocol for the surgical pathology diagnosis of breast neoplasia together with other prognostic parameters i.e. hormonal receptors status and Her2 Neu positivity. These data did not correlate with DMBT1 expression. Expression analysis by RT-PCR In 28/39 cases (72%) RT-PCR confirmed immunohistochemical results, whereas 11 tumours that were negative with anti-DMBTh12 revealed PCR-products after RT-PCR amplification (Table 1). In two of these discordant cases we could document the presence in the sample of residual normal epithelial cells. DMBT1 mRNA was detected in both the breast carcinoma cell lines analysed: Hs 578T (Figure 4) and T47D (not shown). The RT-PCR positivity of samples did not correlate with histological parameters or with IHC expression of hormonal receptors, proliferation index Ki 67 and Her 2/Neu. Discussion In the present study we investigated DMBT1 expression by immunohistochemistry and RT-PCR in benign and malignant breast epithelial lesions. By immunohistochemistry we found a variable DMBTh12 positivity in the morphologically normal and hyperplastic epithelium adjacent to benign (88%) and malignant (78%) lesions. Among 55 carcinomas we could detect immunopositivity only in 3 tumours (5%); in one case of infiltrating carcinoma the immunopositivity was widespread with a pattern similar to that seen in hyperplastic lesions near carcinoma; in the other two cases (one carcinoma in situ and one infiltrating ductal carcinoma), it was limited to few tumour cells. DMBT1 expression was detected by RT-PCR in 13 of 35 (37%) infiltrating carcinomas and in 2 breast cancer cell lines. Eleven tumours, that were negative with anti-DMBTh12, revealed PCR-products after RT-PCR amplification. This discordance between IHC and RT-PCR results may be due to residual normal epithelial cells present in the sample. Furthermore other possible reasons of this discrepancy could be: 1) the greater sensitivity of molecular technique like RT-PCR in detecting very low expression levels of DMBT1 that were below detection by immunohistochemistry, 2) sampling problems i.e. the presence of few positive tumour cells only in the frozen samples or 3) few immunoreactive cells have been missed by microscopic examination and finally 4) DMBT1 protein could have an altered epitope not recognized by its paratope in the antibody. By either immunohistochemistry or molecular technique we found no correlation between DMBT1 expression and tumor histotype, grade, hormonal receptors status, proliferation index, Her2 Neu expression or lymph-nodal metastases. In a recent paper on breast carcinogenesis, qualitatively similar data were reported for DMBT1 in human breast cancer, but an indirect weak correlation between the degree of differentiation of the breast carcinomas and the extent of the immunoreactivity was found [10]. The discrepancy in the results between our and this study may be due to sampling problems, the size of the sample and heterogeneous expression of DMBT1 in breast cancer or to other poorly-defined factors such as race, age or hormonal status of the population under study. Mollenhauer et al. point out that DMBT1 expression may be up-regulated in pathophysiological conditions such as inflammation, liver injury or after carcinogen administration and it is down-regulated in tumors to achieve a growth advantage in a dynamic and complex model. Also according to these authors, the possibility that DMBT1 loss may be a consequence rather than a primary event in carcinogenesis is unlikely (10). Although the normal mammary gland has been reported to have low DMBT1 expression by RT-PCR [12], we found variable DMBTh12 immunopositivity in breast epithelium flanking benign and malignant lesions. Immunopositive epithelial cells in our material appeared either morphologically normal and indistinguishable from immunonegative cells or hypertrophic with abundant cytoplasm and large elongated nuclei oriented perpendicularly to the basement membrane [Figure 1i]. Moreover, DMBTh12 immunoreactivity was also maintained in hyperplastic epithelium flanking carcinomas. This resembles the DMBT1 expression patterns observed in the human respiratory tract and in associated inflammation and carcinomas [8] and suggest that the variable DMBT1 expression in the normal mammary gland epithelium may result from the necessity of DMBT1 induction. Indeed, in parallel in vivo studies, it was found that DMBT1 is strongly up-regulated in the mammary gland tissue of the mouse shortly after carcinogen-administration and prior to the onset of histo-pathologically visible symptoms [10]. It has been reported that Sp-D and Sp-A are ligands of DMBT1 products [29]. We recently demonstrated that Sp-A, in breast carcinoma tissues, has a pattern of expression resembling that of its interaction partner [20]; thus, we hypothesize that co-ordinated induction of both SP-A and DMBT1 in breast tissue may be part of a protective response as suggested by Kang et al [30] for DMBT1 and Sp-D in pulmonary epithelium. While this interaction may be attributed to lumenally secreted DMBT1, the depolarisation of the staining patterns indicates that DMBT1 may also be secreted to other destinations, i.e. the ECM, by the mammary gland epithelial cells. In this regard, the second putative function of DMBT1, i.e. triggering of epithelial differentiation, is noteworthy. It may prevent hyperproliferative epithelial cells from out-of control proliferation and progression to the tumor state. This view is supported by two present findings. The correlation between MCM5 and DMBT1 expression may point to a coupling to cell-cycle processes: basolateral relocation and hyper-expression of DMBT1 at the ECM may provide cells that are licensed to proliferate with protection from unregulated proliferation. On the other hand loss of DMBT1 expression in a significant numbers of DCISs and ICs is consistent with the notion that DMBT1 inactivation may offer growth advantages to neoplastic cells [10]. The loss of DMBT1 expression appears to take place early, i.e. already at the time of transition from hyperplastic lesions to carcinoma, as it has been reported in oesophageal squamous cell carcinomas [5]. The exact mechanisms for DMBT1 inactivation remain unclear. An inactivation by mutation appears not to be a major mechanism for DMBT1 inactivation in tumors [10,31] The studies of Munoz and co-workers suggest that epigenetic silencing of either DMBT1 or genes that regulate DMBT1 expression takes place in some tumors [32]. Accordingly, except for direct epigenetic silencing of the DMBT1 promotor, loss of expression or mutation of DMBT1-regulatory genes might represent alternative candidate mechanisms. Conclusions We have shown that DMBT1 expression and cellular location in breast epithelium varies in normal, hyperplastic and neoplastic cells. In normal or hyperplastic tissue flanking carcinomas it is up-regulated and polarized toward glandular lumen or the basolateral membrane while it is down-regulated in carcinomas. These findings closely resemble the patterns observed for its interaction partner SP-A in an earlier study. Additionally we have shown that DMBT1 is co-expressed with MCM5 in normal and hyperplastic tissue. We hypothesize that hyper-expression and basolateral relocation of DMBT1 in these epithelia prevents the out of control growth that characterizes neoplastic epithelium. List of abbreviations used DMBT1: Deleted in Malignant Brain Tumor 1, MCM5: Minichromosome Maintenance protein, 5 RT-PCR: Reverse Transcriptase Polymerase Chain Reaction GP 340: Glycoprotein 340 SAG: salivary Agglutinin Sp-A, Sp-D: Surfactant Protein A, D MBP: Mannan Binding Protein ECM: Extracellular Matrix FD: Fibrocystic Disease GM: gynecomastia DCIS: Ductal Carcinoma In Situ IC: Invasive Carcinoma MW: Micro Wave EDTA Ethylenediaminetetraacetic Acid DAB: Diaminobenzidine Competing interests None declared. Autors Contributions PB designed the study, participated in the evaluation of immunohistochemical results and drafted the manuscript. PGN participated in the evaluation of immunohistochemical results and performed statistical analysis. JM participated in sequence alignment, criticism and produced DMBTh12 antibody. AP participated in sequence alignment and data evaluation. CP Carried out RT-PCR. AM participated in the evaluation of histologic samples and immunohistochemical results. GB participated in data evaluation and sequence alignment GC participated in study design and data evaluation. SB participated in study design, sequence alignment, criticism and coordinated molecular study. GGP participated in study design, criticism and general revision of paper. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements We thank Mrs. Patrizia Doi and Mr. Robert Rey for technical assistance. The study has been supported in part by grants from Università degli Studi di Milano (FIRST), Associazione Italiana per la Ricerca sul Cancro (AIRC), and the Deutsche Krebshilfe grant 1835-Mo1 and by the Wilhelm Sander-Stiftung grant no. 1999.018.2. Figures and Tables Figure 1 Immunoreactivity of mammary epithelium with anti DMBTh12 Ab: a, supranuclear dot positivity in morphologically normal breast sample; b, strong, abundant surface immunoreactivity in a case of fibrocystic disease; c, intraluminal immunoreactive material in a case of gynecomastia; d, numerous supranuclear and randomly distributed positive dots in intraductal papillomatosis near an infiltrating ductal carcinoma; e, strong supranuclear and baso-lateral cytoplasmic immunoreactivity of hyperplastic ductal cells in a case of mucinous infiltrating carcinoma; f, granular diffuse intracytoplasmic positivity of hyperplastic intraductal cells in a case of carcinoma in situ; g, numerous positive malignant tumour cells of an infiltrating ductal carcinoma; h, few positive cells in an infiltrating ductal carcinoma; i, completely negative neoplastic cells of an invasive carcinoma; the arrow points to positive cells with large elongate nuclei in a non neoplastic duct facing normal negative cells. Figure 2 a, double staining of morphologically normal mammary epithelium: with anti DMBTh12 Ab (red stain in the cytoplasm) and with anti MCM5 Ab (Brown stain in the nucleus); b, lower magnification of double labelled mammary tissue; arrow points to a structure negative for both antigens. Figure 3 Immuno-reactivity of normal mammary epithelium in three serial sections incubated with: a, anti DMBTh12 Ab; b, anti MCM5 Ab and c, anti Ki 67 antibody where only few cells are labelled. Inset: higher magnification of the same ductal structure. Figure 4 DMBT1 expression assessed by RT-PCR. Both DMBT1 (435 bp) and β-2microglobulin (602 bp) products were loaded in the same well. Lane M: size marker (50 bp ladder), lane 1: cell line Hs 578T, lanes 2, 3, 4, 5: breast carcinomas, lane 6: negative control without template. Table 1 Immunohistochemical and RT-PCR expression of DMBT1 in benign and malignant mammary epithelium as number of positive cases/total number of cases. We considered positive the cases in which we found any percentage of positive cells. FD/GM Normal/Hyperplastic DCIS IC IHC 15/17 (88%) 33/42 (78%) 1/10 (10%) 2/45 (4%) RT-PCR 1/1 1/1 0/2 13/35 (37%) IHC= immunohistochemestry FD/GM = fibrocystic disease and gynecomastia Normal/Hyperplastic = morphologically normal and/or hyperplastic tissues flanking carcinoma DCIS = ductal carcinoma in situ IC = invasive carcinoma Table 2 DMBTh12 staining scores in normal (N) and hyperplastic epithelium (H) flanking fibrocystic disease and gynecomastia (FD/GM), ductal carcinoma in situ (DCIS) and invasive carcinoma (IC) Immunohistochemical postivity score Epithelium type FD/GM cases/total number* DCIS cases/total number* IC cases/total number* 0 N 4/14 0/8 11/37 H 3/14 0/4 16/22 1 N 0/14 6/8 16/37 H 1/14 1/4 10/22 2 N 8/14 1/8 9/37 H 8/14 2/4 4/22 3 N 2/14 1/8 1/37 H 2/14 1/4 2/22 * total number of cases in which was present normal or hyperplastic epithelium. ==== Refs Mollenhauer J Wiemann S Scheurlen W Korn B Hayashi Y Wilgenbus KK Von Deimling A Poustka A DMBT1, a new member of the 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Deleted in Malignant Brain Tumors 1 Is a Versatile Mucin-like Molecule Likely to Play a Differential Role in Digestive Tract Cancer Cancer Res 2001 61 8880 8886 11751412 Takeshita H Sato M Shiwaku HO Semba S Sakurada A Hoshi M Hayashi Y Tagawa Y Ayabe H Horii A Expression of the DMBT1 gene is frequently suppressed in human lung cancer Jpn J Cancer Res 1999 90 903 908 10551316 Wu W Kemp BL Proctor ML Gazdar AF Minna JD Hong WK Mao L Expression of DMBT1, a Candidate Tumor Suppressor Gene, Is frequently Lost in Lung Cancer Cancer Res 1999 59 1846 1851 10213490 Mollenhauer J Helmke B Müller H Kollender G Lyer S Diedrichs L Holmoskov U Ligtenberg T Herbetz S Krebs I Wiemann S Madsen J Bikker F Schmitt L Otto HF Poustka A Sequential changes of the DMBT1 expression and location in normal lung tissue and lung carcinomas Genes Chromosomes Cancer 2002 35 164 169 12203780 10.1002/gcc.10096 Mollenhauer J Müller H Kollender G Lyer S Diedrichs L Helmke B Holmoskov U Ligtenberg T Herbetz S Krebs I Madsen J Bikker F Schmitt L Wiemann S Scheurlen W Otto HF von Deimling A Poustka A The SRCR/SID region of DMBT1 defines a complex multi-allele system representing the major basis for its variability in cancer Genes Chromosomes Cancer 2002 35 242 255 12353266 10.1002/gcc.10115 Mollenhauer J Helmke B Medina D Bergmann G Gassler N Müller H Lyer S Diedrichs L Renner M Wittig R Blaich S Hamann U Madsen J Holmskov U Bikker F Ligtenberg A Carlén A Olsson J Otto HF O'Malley B Poustka A Carcinogen Inducibility in Vivo and Down-Regulation of DMBT1 During Breast Carcinogenesis Genes Chromosomes Cancer 2004 39 185 194 14732920 10.1002/gcc.10309 Mollenhauer J Deichmann M Helmke B Muller H Kollender G Holmskov U Ligstemberg T Krebs I Wiemann S Bantel-Shaal U Madsen J Bikker F Klauk SM Otto HF Moldenhauer G Pouskta A Frequent downregulation of DMBT1 and galectin-3 in epithelial skin cancer Int J Cancer 2003 105 149 157 12673672 10.1002/ijc.11072 Holmskov U Mollenhauer J Madsen J Vitved L Grønlund J Tornøe I Kliem A Reid KBM Poustka A Skjødt K Cloning of gp-340, a putative opsonin receptor for lung surfactant protein D Proc Natl Acad Sci USA 1999 96 10794 1799 10485905 10.1073/pnas.96.19.10794 Tino MJ Wright JR Glycoprotein-340 Binds Surfactant Protein-A (SP-A) and Stimulates Alveolar Macrophage Migration in an SP-A-Independent Manner Am J Resp Cell Mol 1999 20 759 768 Prakobphol A Xu F Hoang VM Larsson T Bergstrom J Johansson I Frängsmyr L Holmskov U Leffler H Nilsson C Borén T Wright JR Strömberg N Fisher SJ Salivary Agglutinin, Which Binds Streptococcus mutans and Helicobacter pylori, Is the Lung Scavenger Receptor Cysteine-rich Protein gp-340 J Biol Chem 2000 275 39860 39866 11007786 10.1074/jbc.M006928200 Ligtenberg TJ Bikker FJ Groenink J Tornoe I Leth-Larsen R Veerman EC Nieuw Amerongen AV Holmskov U Human salivary agglutinin binds to lung surfactant protein-D and is identical with scavenger receptor protein gp-340 Biochem J 2001 359 243 248 11563989 10.1042/0264-6021:3590243 Kang W Reid KBM DMBT1, a regulator of mucosal homeostasis through the linking of defense and regeneration? FEBS Letters 2003 540 21 25 12681477 10.1016/S0014-5793(03)00217-5 Holmskov U Lawson P Teisner B Tornøe I Willis AC Morgan C Koch C Reid KBM Isolation and Characterization of a New Member of the Scavenger Receptor Superfamily, Glycoprotein-340 (gp-340), as a Lung Surfactant Protein-D Binding Molecule J Biol Chem 1997 272 13743 13749 9153228 10.1074/jbc.272.21.13743 Fujita T Taira S Kodama N Matsushita M Fujita T Mannose-binding protein recognizes glioma cells: in vitro analysis of complement activation on glioma cells via the lectin pathway Jpn J Cancer Res 1995 86 187 192 7730143 Ma Y Uemura K Oka S Kozutsumi Y Kawasaki N Kawasaki T Antitumor activity of mannan-binding protein in vivo as revealed by a virus expression system: Mannan-binding protein dependent cell-mediated cytotoxicity Proc Natl Acad Sci USA 1999 96 371 375 9892640 10.1073/pnas.96.2.371 Braidotti P Cigala C Graziani D Del Curto B Dessy E Coggi G Bosari S Pietra GG Surfactant Protein A Expression in Human Normal and Neoplastic Breast Epithelium Am J Clin Pathol 2001 116 721 728 11710690 10.1309/DU7F-XYP8-1DF6-WM9E Takito J Hikita C Al-Awqati Q Hensin, a New Collecting Duct Protein Involved in the In Vitro Plasticity of Intercalated Cell Polarity J Clin Invest 1996 98 2324 2331 8941650 Al-Awqati Q Vijayakumar S Hikita C Chen J Takito J Phenotipic plasticity in the intercalated cell: the hensin pathway Am J Physiol 1998 275 F183 F190 9691006 Vijayakumar S Takito J Hikita C Al-Awqati Q Hensin Remodels the Apical Cytoskeleton and Induces Columnarization of Intercalated Epithelial Cells: Processes that Resemble Terminal Differentiation J Cell Biol 1999 144 1057 1067 10085301 10.1083/jcb.144.5.1057 Hikita C Vijayakumar S Takito J Erdjument-Bromage H Tempst P Al-Awqati Q Induction of Terminal Differentiation in Epithelial Cells Requires Polymerization of Hensin by Galectin 3 J Cell Biol 2000 151 1235 1246 11121438 10.1083/jcb.151.6.1235 Cheng H Bjerknes M Chen H CRP-ductin: A Gene Expressed in Intestinal Crypts and in Pancreatic and Hepatic Ducts Anat Rec 1996 244 327 343 8742698 10.1002/(SICI)1097-0185(199603)244:3<327::AID-AR5>3.3.CO;2-A Bisgaard HC Holmskov U Santoni-Rugiu E Nagy P Nielsen O Ott P Age E Dalhoff K Rasmussen LJ Tygstrup N Heterogeneity of Ductular Reactions in Adult Rat and Human Liver Revealed by Novel Expression of Deleted in Malignant Brain Tumor 1 Am J Pathol 2002 161 1187 1198 12368192 Stoeber K Tlsty TD Happerfield L Thomas GA Romanov S Bobrow L Williams ED Williams GH DNA replication licensing and human cell proliferation J Cell Sci 2001 114 2027 2041 11493639 Lan HY Mu W Nikolic-Paterson DJ Atkins RC A novel, simple, reliable, and sensitive Method for multiple immunoanzyme staining: use of microwave oven heating to block antibody crossreactivity and retrieve antigens J Histochem Cytochem 1995 43 97 102 7822770 Crouch E Wright JR Surfactant proteins A and D and pulmonary host defense Annu Rev Physiol 2001 63 521 554 11181966 10.1146/annurev.physiol.63.1.521 Kang W Nielsen O Fenger C Madsen J Hansen S Tornoe I Eggleton P Reid KBM Holmskov U The scavenger receptor, cysteine-rich domain-containing molecule gp-340 is differentially regulated in epithelial cell lines by phorbol ester Clin Exp Immunol 2002 130 440 458 10.1046/j.1365-2249.2002.01992.x Mueller W Mollenhauer J Stockhammer F Poustka A von Deimling A Rare mutations of the DMBT1 gene in human astrocytic gliomas Oncogene 2002 21 5956 5959 12185598 10.1038/sj.onc.1205733 Munoz J Lazcoz P del Mar Inda M Nistal M Pestana A Encio IJ Castresana JS Homozygous deletion and expression of PTEN and DMBT1 in human primary neuroblastoma and cell lines Int J Cancer 2004 109 673 679 14999773 10.1002/ijc.20055

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==== Front BMC PsychiatryBMC Psychiatry1471-244XBioMed Central London 1471-244X-4-221530603110.1186/1471-244X-4-22Research ArticleDrop-out and mood improvement: a randomised controlled trial with light exposure and physical exercise [ISRCTN36478292] Leppämäki Sami [email protected] Jari [email protected]önnqvist Jouko [email protected] Timo [email protected] Department of Mental Health and Alcohol Research, Unit for Epidemiology and Genetics of Mental Health, National Public Health Institute, Helsinki, Finland2 Department of Psychiatry, Jorvi Hospital, Helsinki University Central Hospital, Espoo, Finland2004 11 8 2004 4 22 22 18 2 2004 11 8 2004 Copyright © 2004 Leppämäki et al; licensee BioMed Central Ltd.2004Leppämäki et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Combining bright light exposure and physical exercise may be an effective way of relieving depressive symptoms. However, relatively little is known about individual factors predicting either a good response or treatment failure. We explored background variables possibly explaining the individual variation in treatment response or failure in a randomised trial. Methods Participants were volunteers of working-age, free from prior mental disorders and recruited via occupational health centres. The intervention was a randomised 8-week trial with three groups: aerobics in bright light, aerobics in normal room lighting, and relaxation/stretching in bright light. Good response was defined as a 50% decrease in the symptom score on either the Hamilton Depression Rating Scale (HDRS) or 8-item scale of atypical symptoms. Background variables for the analysis included sex, age, body-mass index, general health habits, seasonal pattern, and sleep disturbances. Results Complete data were received from 98 subjects (11 men, 87 women). Of them, 42 (5 men, 37 women) were classified as responders on the HDRS. Overall, light had a significant effect on the number of responders, as assessed with the HDRS (X2 = .02). The number needed to treat (NNT) for light was 3.8. Conclusions We investigated the effect of bright light and exercise on depressive symptoms. Problems with sleep, especially initial insomnia, may predict a good response to treatment using combined light and exercise. Bright light exposure and physical exercise, even in combination, seem to be well tolerated and effective on depressive symptoms. phototherapybright lightseasonaldepressive symptomsexercise ==== Body Background At northern latitudes reduced vitality, increased appetite and sleep complaints are common symptoms during wintertime, even among people considered healthy [1]. Atypical depressive symptoms, which are often seen in seasonal depression, appear to correlate with decreased illumination [2]. Exposure to natural light also appears to have a substantial effect on well-being in twins with bipolar disorder [3]. Disorganised circadian clockwork, related to the shortening photoperiod and changes in this most important external time-giver, is thought to play a role in the pathophysiology of seasonal mood changes. Bright (>2500 lx) light therapy has proven effective for season-related major depressive episodes, and also for milder, subsyndromal symptoms [4]. An interesting alternative to bright light is dawn simulation (short, timed pulses of light during the natural awakening), which may be helpful in seasonal affective disorder [5] and sleeping problems, even in the general population [6]. Exercise would seem to be the ideal treatment for depression: available, affordable, with minimal side effects. Unfortunately, many exercise treatment studies suffer from methodological weaknesses and lack of adequate follow-up to determine long-term efficacy. In their systematic review, Lawlor and Hopker even concluded that the effectiveness of exercise in reducing depressive symptoms cannot be determined because of a lack of good quality research, though exercise was found to be more effective than no treatment, and as effective as cognitive therapy [7]. Fortunately, absence of evidence does not necessarily indicate evidence of absence. A recent 16-week study with rigorous methodology indicates that exercise is as effective as standard medication (sertraline) for treatment of depression [8]. A 6-month follow-up of the study subjects showed that remitted subjects in the exercise group had significantly lower relapse rates than remitted subjects in the medication group, and that continued exercise was associated with lower rates of depression [9]. Other studies on the efficacy of exercise treatment for depression are also conducted, to address e.g. the question of a dose-response relationship [10]. Several mechanisms may explain the mood-lifting effects of exercise: psychological (increased sense of self worth, positive feedback), social (an increase in social contacts), and physiological (changes in central endorphin and monoamine concentrations). Exercise may induce phase-shifts in the human circadian rhythms [11], so it is possible that exercise also may exert part of its action on mood by influencing the circadian clock. Adding light exposure to exercise for the treatment of depression and depressive symptoms seems a promising intervention. Combinations of bright light exposure and physical exercise have achieved beneficial effects on mood in trials on healthy adult populations [12,13]. Natural light exposure (one hour walk outdoors) may also be effective in treating seasonal affective disorder [14]. It remains to be determined, however, whether it is possible to define a subgroup of people who are especially likely to benefit from this kind of intervention, and what, if any, are the individual factors predicting a good response to light or exercise, or their combination? And on the contrary, another important subgroup to identify, in determining the efficacy of any intervention, is the subjects who drop out of the study prematurely. Several predictors of response to light treatment in winter depressives have been identified: the ratio of atypical to classical symptoms of depression [15] and hypersomnia, increased eating and younger age [16]. Meesters et al. found that a large diurnal variation had a negative predictive value on response to light treatment [17]. Temperament dimensions have also been investigated. Although higher harm avoidance scores have been linked to non-response to light therapy in one study [18], another study found no predictive value of avoidance scores [19]. With regard to exercise treatment of depression, baseline levels of self-reported anxiety and life satisfaction were found to be best predictors of both dropout and treatment success, when exercise alone was compared to antidepressive medication or exercise with medication [20]. In the present study we tried to address this question: who will benefit from light, exercise or their combination? A variety of background variables were investigated: age, seasonality, depressive and atypical symptoms, treatment adherence, fitness, body mass index, self-perceived quality of sleep, and alcohol consumption. We did not measure diurnal variation or personality dimensions. Methods Adult volunteers of working age were invited to participate in a study of light and exercise via occupational health centres. The enrolled subjects were randomly allocated to three intervention groups: aerobics training in bright light (>2500 lx, measured at eye-level), similar training in the normal lighting of the gym (400–600 lx), and relaxation and stretching sessions in bright light. The training or relaxation sessions (45 minutes each, starting at 7:30 a.m. or 8:30 a.m. Monday through Friday, and at 10:00 a.m. or 11:00 a.m. on Saturdays) were scheduled two times a week over eight weeks. Study was conducted between November 25, 1997, and January 25, 1998. The length of daylight on these dates was 6 h 48 min and 7 h 23 min, respectively. Intervention Both the aerobics training and relaxation groups were led by 3 physiotherapists, each of whom supervised one third of each group's training or relaxation sessions. The training and relaxation sessions were structured to maintain treatment consistency. In the aerobics groups, the intensity of the training was checked with a heart rate monitor, the target rate being 120 to 150 beats per minute. Stretching/relaxation training was designed to avoid raising the pulse. All training sessions were in the same gym, the ceiling of which was equipped with 30 extra light fixtures with cool-white (6000 K) fluorescent lamps (F58W/186, Sylvania, Germany), which were turned on for the bright light groups. Assessment Mood during the study period was recorded using the Structured Interview Guide for the Hamilton Depression Rating Scale – Seasonal Affective Disorders Version Self-Rating Format (SIGH-SAD-SR) [21], which includes the 21-item Hamilton Depression Rating Scale (HDRS) plus an eight-item addendum for atypical symptoms (ATYP). The SIGH-SAD-SR was filled in at baseline, after week 4 and at the end of the 8-week study period. At the start of the study and at weeks 4 and 8, all subjects were weighed to assess body-mass index (BMI). Before and after the study period all subjects in the Aerobics training groups participated in a 2-km walking test, which predicts maximal oxygen uptake using a model with age, sex, walking time, BMI, and heart rate at the end of the test as variables [22]. At baseline, sleep quality was assessed with the Basic Nordic Sleep Questionnaire (BNSQ) [23], and the subjects also completed an abbreviated, 26-item FINRISK questionnaire [24] concerning smoking, alcohol consumption, dietary fat intake, and habitual exercise. The Seasonal Pattern Assessment Questionnaire (SPAQ) [25] measures seasonal changes in mood and behaviour. The SPAQ includes a 6-item scale yielding the Global Seasonality Score (GSS). Based on the GSS the subjects were thereby divided into seasonals and non-seasonals, according to the criteria for subsyndromal seasonal affective disorder presented by Bartko and Kasper [26]. Baseline demographic data on all participants included sex, age, and educational level. Ethics All subjects returned a written informed consent prior to participation. The ethics committee of the National Public Health Institution approved the study protocol. Statistics All analyses were done on SPSS for Windows (Release 11.5.1)-statistical package (SPSS Inc., Chicago, Illinois). Dropouts Participants were classified as dropouts if they, for any reason, did not finish the eight-week study protocol. Analysis of variance (ANOVA)-models were used to compare the baseline characteristics of dropouts with those who completed the study. In the models, the baseline characteristic was the dependent variable, and the outcome (dropouts vs. completers) and treatment group (Light & Exercise, Exercise, Light) were factors. The Outcome × Treatment group interaction was also tested in each model. Background characteristics examined were age, GSS, BMI, fitness, and alcohol consumption, all as continuous variables. Baseline HDRS and ATYP were included, as well as percentage of sessions attended. From the BNSQ, following variables were chosen: initial, middle, and late insomnia, and quality of sleep. Treatment benefit was defined separately as response and, with stricter criteria, as remission. Treatment response The main outcome measures were changes in the HDRS, ATYP and the SIGH-SAD-SR over the 8-week study period. A 50 % decrease on the HDRS, ATYP or the SIGH-SAD-SR total score was used to divide subjects into responders and non-responders. To assess the effect of baseline characteristics, logistic regression models were formulated, with the defined clinical response as the dependent variable. Light therapy, physical exercise, and sex were constant in the models. Other independent baseline variables in the analysis were age (over/under 40 years), seasonality (from the SPAQ), initial, middle and late insomnia, quality of sleep, feeling tired after waking (from the BNSQ), serum total cholesterol levels, current smoking, physical training, and consumption of alcoholic beverages (from the FINRISK questionnaire). All categorical variables were dichotomised for the analysis. The best-predicting co-variates were found by backward step-wise selection. Analysis of variance (ANOVA) was applied to compare means between groups, and associations were analysed by calculating partial correlation coefficients, after controlling for age and sex. Pearson chi-square (two-sided) was used when applicable. Remission Stricter criteria were applied for the definition of remission. To increase clinical meaningfulness and to avoid 'flooring effect', subjects with low symptom scores were excluded from these analyses. HDRS Only subjects with a baseline HDRS of eight or higher were included. Remission was defined as at least a 50% reduction on the HDRS during the trial, and a score of less than eight at the end of the study period. SIGH-SAD Subjects with a baseline SIGH-SAD-total score of fourteen or more were included. Remission was defined as at least a 50% reduction on the score during the eight-week study, and a final SIGH-SAD-score of eight or less. All analyses were done 'intention-to-treat', i.e. dropouts were classified as treatment failures. With remitted subjects, we desisted from using logistic regression because of lower number of subjects, which would limit the number of variables. Instead, the background variables were examined one-by-one with ANOVA-models (see Dropouts for description). Results Complete data were received from 98 subjects (11 men, 87 women, see Figure 1) with a mean (s.d.) age of 43.4 (9.5), ranging from 26 to 63 years. Sixty-nine subjects were assigned to the light therapy groups, and 61 subjects to the aerobic exercise treatment groups. There were 37 of these subjects in the combined group, and their mean (s.d.) score on the HDRS was 10.5 (6.3), on the ATYP 5.9 (4.2), and on the SIGH-SAD-SR 16.4 (9.4). On average (s.d.), the GSS was 10.5 (4.9), and the BMI 24.2 (3.7). At baseline, the GSS was negatively associated with habitual training (r = -.26, p = .01), and correlated with initial insomnia (r = .20, p = .05), low quality of sleep (r = .30, p = .003), and feeling tired after waking (r = .35, p = .001). Subjects reporting initial insomnia on the BNSQ (n = 28) had, as expected, a higher score on the HDRS at baseline than other subjects (F = 11.2, p = 0.001), and they also had a higher ATYP score (F = 6.70, p = 0.01) and a trend towards higher GSS (F = 3.92, p = 0.05). Twenty-three of these subjects received light therapy, and 13 (57%) of them were classified as responders based on the changes in HDRS scores (X2 = .04). Figure 1 Study protocol There was a negative correlation between response on the HDRS and alcohol consumption (more than 7 drinks a week) (r = -.23, p = .03), and high levels of serum total cholesterol (r = -.21, p = .04). Treatment response Based on the HDRS, 42 subjects (5 men, 37 women, X2 = .9) were classified as responders. Their mean age (s.d.) was 41.3 (9.5) years, ranging from 26 to 58. Thirty-five (83%) had had light therapy, 24 (57%) had been in the aerobic exercise groups, and 17 subjects (40%) in the combined group. Overall, light had a significant effect on the number of responders, as assessed with the HDRS (X2 = .02). The number needed to treat (NNT) for light was 3.8. On the basis of the ATYP scores, 51 subjects (8 men, 43 women) were classified as responders. Their mean age (s.d.) was 41.9 (9.8) years, ranging from 26 to 63. Thirty-seven (73%) had received light therapy, 30 (59%) had been in the exercise groups, and 16 (31%) in the combined group. Response on the SIGH-SAD-SR was negatively associated with baseline self-reported alcohol consumption (r = -.26, p = .01). There were 45 responders (5 men, 40 women) on the SIGH-SAD-SR, with a mean age (s.d.) of 41.5 (9.5) years, ranging from 26 to 58. Thirty-seven (82%) had received light therapy, 26 (58%) had done aerobic exercise, and 18 (40%) had been in the combined group. The effect of light therapy was significant (X2 = .03). Based on these figures, the NNT for light was 3.8. The logistic regression models for the HDRS, ATYP, and SIGH-SAD-SR total scores are presented in Table 1. Table 1 Key results from logistic regression models. Variable coefficient s.e. p value HDRS Age group* -.8 .50 .09 Total cholesterol** -1.6 .66 .01 Alcohol consumption† -1.6 .62 .008 ATYP score Age group* -1.0 .52 .05 Initial insomnia‡ 1.8 .72 .01 Quality of sleep ± -2.1 .69 .002 Alcohol consumption† -1.2 .60 .04 SIGH-SAD-SR total score Age group* -1.1 .53 .04 Initial insomnia‡ 1.5 .70 .03 Quality of sleep ± -1.6 .66 .02 Alcohol consumption† -1.8 .62 .005 *Age group: over 40 years vs. under 40 years **Total cholesterol: previous high total serum cholesterol, yes vs. no †Alcohol consumption: over 7 drinks per week vs. 7 drinks or less per week ‡Initial insomnia: trouble falling asleep once a week to daily vs. less than once week ± Quality of sleep: bad or rather bad vs. normal to good Dropouts There were 26 (21%) subjects classified as dropouts (4 male, 22 female, X2 = .6), 8 (20%) in the light & exercise group, 13 (31%) in the exercise group, and 5 (12%) in the light group (X2 = .1). Thirteen subjects (50%) had received light therapy (X2 = .05). Table 2 presents the background variables investigated from all subjects and by treatment group. Dropout-status was significantly influenced by the GSS (F = 5.40, p = .02) and treatment sessions attended (F = 143.0, p = .000). There was a trend towards baseline HDRS having an effect on dropout-status (F = 4.00, p = .05), but baseline HDRS was also influenced by treatment group (F = 4.38, p = .02), despite random assignment to groups. The pre-intervention fitness test result (in the exercise groups) was predictive of dropout status (F = 11.1, p = .001), and there was also a significant Treatment group × Dropout interaction (F = 7.82, p = .007). Initial insomnia, derived from the BNSQ, was also a significant factor (F = 6.00, p = .02, Treatment group × Dropout interaction F = 5.43, p = .006). Table 2 Comparison of baseline variables (S.D) of drop-outs vs. completers Analysis of variance Variable Treatment group Dropout group Interaction All Exercise&Light Exercise Light F p F p F p Age .50 .61 2.33 .13 2.20 .12 Drop-outs 39.5 (8.2) 41.3 (7.2) 35.4 (7.2) 43.4 (9.6) Completers 43.4 (9.6) 41.8 (9.2) 45.5 (10.2) 43.2 (9.3) GSS 1.35 .26 5.40 .02 .98 .38 Drop-outs 13.1 (4.5) 13.0 (5.0) 11.1 (3.3) 15.8 (4.5) Completers 10.5 (4.9) 10.1 (5.2) 10.5 (4.6) 10.9 (5.1) HDRS 4.38 .02 4.00 .05 1.55 .22 Drop-outs 13.6 (8.4) 16.3 (10.9) 8.29 (3.9) 16.6 (5.6) Completers 10.5 (6.4) 10.8 (7.0) 9.00 (5.2) 11.4 (6.6) ATYP* 1.18 .31 2.42 .12 .23 .79 Drop-outs 7.45 (5.3) 7.50 (5.4) 6.14 (5.0) 9.20 (6.0) Completers 5.92 (4.3) 6.22 (4.9) 5.07 (3.7) 6.32 (4.2) BMI** .30 .74 1.88 .17 1.33 .27 Drop-outs 25.5 (4.9) 27.0 (6.2) 24.9 (4.5) 24.4 (3.8) Completers 24.2 (3.7) 23.6 (3.1) 24.1 (3.6) 24.7 (4.3) Sessions attended (%) 2.70 .07 143 .000 2.29 .11 Drop-outs 32.5 (24.4) 25.8 (20.6) 30.6 (24.9) 48.0(27.2) Completers 82.1 (14.4) 82.7 (14.4) 80.5 (15.2) 82.9(14.1) Fitness 2.61 .11 11.1 .001 7.82 .007 Drop-outs 93.3 (14.3) 83.7 (11.3) 99.1 (13.0) ---- Completers 103.2 (12.1) 105.1 (13.6) 101.0 (10.2) --- BNSQ Initial insomnia 3.00 .06 6.00 .02 5.43 .006 Drop-outs 2.61 (1.2) 3.50 (.84) 2.14 (1.1) 2.20 (1.3) Completers 2.06 (.93) 1.84 (.81) 1.83 (.93) 2.43 (.93) Middle insomnia 1.91 .15 .23 .64 1.34 .27 Drop-outs 3.21 (1.4) 3.57 (1.4) 2.43 (1.1) 3.80 (1.6) Completers 3.11 (1.3) 3.10 (1.3) 3.03 (1.3) 3.19 (1.4) Late insomnia 2.97 .06 .55 .46 1.15 .32 Drop-outs 1.68 (.95) 1.86 (1.1) 1.14 (.38) 2.20 (1.1) Completers 1.92 (.92) 1.78 (.83) 1.79 (.90) 2.14 (.98) Quality of sleep 1.615 .20 .10 .753 .70 .50 Drop-outs 2.47 (1.1) 2.86 (1.2) 2.00 (0.0) 2.60 (1.3) Completers 2.42 (1.1) 2.34 (1.0) 2.21 (1.0) 2.65 (1.2) Alcohol consumption† .079 .92 .31 .58 .098 .91 Drop-outs 6.05 (5.6) 5.63 (6.3) 6.57 (6.7) 6.00 (3.4) Completers 5.34 (5.5) 5.00 (4.4) 4.97 (5.9) 5.92 (6.0) *ATYP – atypical symptom score ** BMI – Body Mass Index †Alcohol consumption – drinks per week Remitted subjects HDRS (see Table 3) Table 3 Comparison of baseline variables (S.D) of subjects in remission (HDRS) with those who did not remit during the study Analysis of variance Variable Treatment group Remitted group Interaction All Exercise&Light Exercise Light F p F p F p Age .11 .90 1.56 .22 .11 .89 Remitted 40.5 (10.5) 39.4 (11.6) 39.6 (11.2) 41.7(10.2) Not remitted 43.4 (9.0) 43.8 (8.0) 42.9 (9.6) 43.4 (9.7) GSS 1.59 .21 1.86 .18 .24 .79 Remitted 11.2 (4.7) 10.2 (5.2) 9.60 (5.0) 12.8 (3.9) Not remitted 12.6 (5.0) 12.8 (5.5) 11.4 (4.3) 13.5 (5.1) HDRS 3.09 .05 .76 .38 .11 .90 Remitted 14.2 (4.9) 16.3 (5.7) 11.0 (3.5) 14.0 (4.3) Not remitted 15.1 (6.4) 16.7 (8.1) 12.6 (3.7) 15.9 (6.2) ATYP 1.51 .23 .26 .61 .17 .84 Remitted 7.56 (5.0) 8.22 (4.7) 5.00 (3.5) 8.18 (5.6) Not remitted 7.76 (4.5) 8.27 (5.7) 6.69 (4.1) 8.28 (3.9) BMI .63 .54 .088 .77 .51 .60 Remitted 24.5 (3.9) 24.8 (2.6) 22.4 (1.8) 25.3 (5.2) Not remitted 24.5 (4.4) 24.3 (5.4) 24.4 (3.8) 24.7 (4.4) Sessions attended (%) .58 .56 8.66 .004 1.35 .27 Remitted 80.3 (15.8) 82.2 (10.0) 81.3 (27.2) 78.2(14.6) Not remitted 61.6 (30.1) 52.0 (33.6) 59.2 (31.1) 71.9(24.2) Fitness .34 .57 .003 .96 .30 .59 Remitted 100.1 (13.7) 98.3 (16.5) 103.4 (6.8) --- Not remitted 100.6 (12.8) 100.5 (16.0) 100.7 (10.2) --- BNSQ Initial insomnia 3.83 .03 1.19 .28 1.87 .16 Remitted 2.32 (1.0) 2.22 (.97) 1.40 (.55) 2.82 (.98) Not remitted 2.42 (1.1) 2.71 (.99) 2.13 (1.2) 2.44 (.98) Middle insomnia .43 .65 .00 .99 .47 .63 Remitted 3.36 (1.3) 3.22 (1.3) 3.20 (1.5) 3.55 (1.2) Not remitted 3.31 (1.2) 3.64 (.93) 3.00 (1.2) 3.33 (1.4) Late insomnia 1.37 .26 .69 .41 .90 .41 Remitted 2.04 (.94) 2.00 (1.0) 2.00 (1.0) 2.09 (.94) Not remitted 1.85 (.90) 1.93 (.83) 1.38 (.50) 2.22 (1.1) Quality of sleep 2.72 .07 .00 .99 .52 .60 Remitted 2.84 (1.2) 2.78 (1.1) 2.20 (1.1) 3.18 (1.3) Not remitted 2.71 (.99) 3.00 (.96) 2.31 (.70) 2.83 (1.2) Alcohol consumption .17 .85 1.08 .30 1.29 .28 Remitted 5.08 (3.9) 6.78 (5.4) 3.80 (2.2) 4.27 (2.5) Not remitted 6.63 (6.8) 5.20 (5.4) 6.38 (7.5) 8.06 (7.2) Abbreviations as in Table 2 A total of 74 subjects (10 male, 64 female) were included in the analyses. Twenty-five subjects (3 male, 22 female, X2 = .8) were considered remitted on the HDRS after the study period. Nine had been in the light & exercise group, 5 in the exercise group, and 11 in the light group (X2 = .5). Thus, 20 of the subjects had received bright light therapy (X2 = .3) and 14 had been in the exercise groups (X2 = .5). Proportion of sessions attended had a significant impact on remission status (F = 8.66, p = .004). The BNSQ initial insomnia-variable was influenced by treatment group assignment (F = 3.83, p = .03). SIGH-SAD (see Table 4) Table 4 Comparison of baseline variables (S.D) of subjects in remission (SIGH-SAD total score) with those who did not remit during the study Analysis of variance Variable Treatment group Remitted group Interaction All Exercise&Light Exercise Light F p F p F p Age .26 .77 .068 .80 .54 .58 Remitted 42.0 (10.5) 43.6 (11.4) 37.5 (6.4) 41.8 (11.2) Not remitted 42.0 (9.5) 40.1 (8.6) 41.7 (10.2) 43.6 (9.7) GSS 1.60 .21 5.23 .03 .17 .85 Remitted 10.62 (4.9) 10.3 (4.8) 7.00 (1.4) 11.8 (5.4) Not remitted 13.2 (4.7) 13.3 (5.7) 11.9 (4.3) 14.4 (4.1) HDRS 1.71 .19 1.07 .31 .093 .91 Remitted 14.5 (5.3) 15.3 (6.7) 11.5 (2.1) 14.5 (4.6) Not remitted 15.6 (6.2) 18.3 (7.9) 12.7 (3.8) 16.2 (5.7) ATYP 1.89 .16 2.68 .11 .52 .60 Remitted 7.71 (4.7) 8.29 (4.2) 3.50 (.71) 8.25 (5.4) Not remitted 8.88 (4.2) 10.3 (4.8) 7.76 (3.8) 8.84 (4.1) BMI .63 .54 .57 .45 1.79 .18 Remitted 24.3 (2.9) 25.7 (2.5) 21.0 (1.4) 23.9 (2.8) Not remitted 24.7 (4.5) 23.4 (4.2) 24.6 (3.8) 25.6 (5.3) Sessions attended (%) .64 .53 2.66 .11 .60 .55 Remitted 82.4 (14.9) 84.8 (6.34) 66.7 (47.1) 84.2 (8.68) Not remitted 65.3 (28.3) 59.0 (31.6) 64.3 (30.5) 70.9 (24.0) Fitness 1.06 .31 .12 .73 2.01 .17 Remitted 99.7 (16.5) 96.6 (17.7) 110.5 (2.1) --- Not remitted 101.4 (11.6) 102.7 (13.8) 100.5 (10.1) --- BNSQ Initial insomnia 2.46 .09 .69 .41 1.26 .29 Remitted 2.35 (.93) 2.00 (1.0) 1.50 (.71) 2.88 (.64) Not remitted 2.39 (1.0) 2.54 (.78) 2.12 (1.2) 2.53 (1.1) Middle insomnia .19 .83 .88 .35 .707 .50 Remitted 3.18 (1.4) 3.00 (1.4) 3.00 (.00) 3.37 (1.6) Not remitted 3.47 (1.2) 3.92 (.95) 3.24 (1.2) 3.37 (1.3) Late insomnia 1.48 .24 .003 .96 .068 .94 Remitted 2.06 (.90) 2.00 (1.0) 1.50 (.71) 2.25 (.89) Not remitted 1.92 (.95) 1.85 (.99) 1.65 (.70) 2.21 (1.1) Quality of sleep 1.87 .16 .21 .65 1.35 .27 Remitted 2.82 (1.1) 2.43 (.98) 2.00 (.00) 3.38 (1.1) Not remitted 2.76 (1.1) 2.92 (1.2) 2.53 (.87) 2.84 (1.3) Alcohol consumption .059 .94 1.49 .23 .26 .77 Remitted 4.06 (2.8) 4.43 (3.3) 4.50 (3.5) 3.63 (2.4) Not remitted 6.50 (6.1) 5.36 (5.7) 6.76 (7.0) 7.11 (5.8) Abbreviations as in Table 2 Sixty-seven subjects (8 male, 59 female) were included in the analyses. After the trial, sixteen of them (1 male, 15 female, X2 = .4) were considered to be in remission on the SIGH-SAD-scale. Seven subjects had been in the light & exercise group, 1 in the exercise group, and 8 in the light group (X2 = .08). Fifteen subjects (94%) had been in groups with bright light exposure (X2 = .03), and eight (50%) in the exercise groups (X2 = .4). In the ANOVA-models, remission was significantly influenced only by the GSS (F = 5.23, p = .03). Discussion Treatment response The results of this study confirm earlier findings from light and exercise trials that these interventions are well tolerated and effective. Considering the study population was not a clinical one, but consisted of volunteers not suffering from any major mental or physical disorder, the NNT for light therapy was high: 4 subjects need to be treated for one subject to respond, as assessed with both the HDRS and the SIGH-SAD-SR. The overall response rate, on all the scales, was about 50 %. Initial insomnia at the start of the study was an independent variable predicting good response, both on the ATYP and the SIGH-SAD-SR. Disorders of sleep are common in depressive states, and also in the general population [27]. The mean GSS of our study subjects was relatively high, suggesting that they had a marked degree of seasonal variation in mood and behaviour and might be predisposed to desynchrony between the sleep-wake cycle and circadian rhythms in winter. It is probable that entrainment of internal clocks by environmental stimuli is impaired in depression. Light and exercise are both capable of entraining the circadian rhythms, which could be one, but not the only mechanism of action reflected externally as improved sleep and mood [28]. However, a limitation of our study was that we did not measure circadian rhythms. Ageing changes sleep-wake habits. This may be due to a deteriorating impact of light with age on the flexibility of the internal clock [29]. No studies comparing the young and the old on the benefits of bright light therapy have been published to our knowledge. Age has not been a significant factor in trials of bright light therapy. Baehr et al. have examined the circadian phase-shifting effects of exercise in two age groups (20–32 and 55–73 years), and no significant differences were found [30]. However, our hypothesis that the older age group would benefit from this kind of intervention was not supported; in fact, the opposite occurred. One explanation for this is the frequency of the intervention: two times a week may be a signal powerful enough to entrain circadian rhythms for younger subjects, but not for older subjects, even when light and exercise interventions are combined. We found that even moderate consumption of alcohol (>7 drinks per week) predicted a poorer response on all the assessed scales. One explanation to this might be the negative effect of alcohol on circadian rhythms and especially on sleep [31,32]. Dropout from the study The dropout rate was relatively low (combined 21%), and did not differ significantly between the treatment conditions. There seemed to be a trend towards subjects in groups receiving bright light therapy adhering to the study more closely than subjects in the exercise group. Subjects might have felt bright light therapy is a novel, more attractive treatment option than plain, 'old-fashioned' exercise. We tried to minimise this problem by emphasising 'exercise trial' in leaflets provided to possible volunteers. It was not possible to avoid this problem altogether, as reflected by those four subjects who dropped out of the study after hearing they had been randomised into the exercise group. When these four subjects, now counted as dropouts, are excluded, the dropout rates of the light & exercise and exercise groups are virtually identical, and similar to the rates reported previously [20]. The dropout rate in the light group was considerably lower than in the exercise groups, but the difference failed to reach statistical significance. Subjects possibly experienced bright light exposure without the strenuous exercise very comfortable, which is understandable. An interesting finding is that completers were in a better physical condition in the pre-intervention fitness test than were dropouts. Regular exercising may be a representation of self-motivation trait, which would increase adherence to a therapeutic exercise program [33]. A major limitation is of course that the subjects in the bright light group did not perform the fitness test. This had to be omitted from the study protocol for economical reasons. Remitted subjects Using the strict remission criteria yielded few results. Attendance to treatment sessions did predict remission on the Hamilton scale (but not the SIGH-SAD total score), but post-hoc analyses showed that this was caused by dropouts, which were automatically labeled as treatment failures. A lower GSS was predictive of remission on the SIGH-SAD total score. Again, post-hoc analyses showed this effect was caused by dropouts with higher than average scores. In previous research on the use of light therapy, atypical depressive symptoms have been predictive of treatment response and remission. This effect was not seen in the present study. We did separate analyses for hypersomnia, hyperphagia, increased appetite / carbohydrate craving, and reduced vitality, but none of these individual atypical symptoms predicted remission either. Assessment The study subjects were not patients with clinically diagnosed depression, but volunteers with varying degrees of depressive symptoms. This poses a major question of definition of treatment response and remission, and also measurement of depressive symptoms, e.g. using scales originally designed for the follow-up of depressed in-patients [34]. Our primary focus in planning this study was practical: to find an intervention that would benefit the public at large. Study subjects were not a random sample of population, but volunteers invited through occupational health centres, and free of pre-existing, diagnosed or medicated mental illness. However, we decided to use established methods for the assessment of depressive symptoms. In their systematic review on exercise studies, Lawlor and Hopker demand the use of dichotomous outcomes, arguing them to be more understandable and more important outcomes in clinical terms [7]. We agree, and use the concepts of response and remission in this study. The cut-offs selected were based on previous studies [35], done on depressed patients. The application of these criteria to a trial with healthy subjects may seem artificial, but we feel this increases the clinical meaningfulness of the results. Future research More studies, with expanded methodology, are clearly needed to shed light on the individual factors separating responders and non-responders in exercise trials, with or without bright light. Interesting research subjects would be circadian phenotyping (with Morning-Eveningness Questionnaire; [36]), and motor activity measurement. Pre-treatment expectations should be assessed, to estimate the possible placebo-effect. Conclusions We investigated the effect of bright light and exercise on depressive symptoms in working-age men and women, free from mental disorder and/or psychotropic medication. We found that problems with sleep, especially initial insomnia, may predict a good response to treatment using combined light and exercise. Regular intake of alcoholic beverages (over 7 drinks per week) seems to have an opposite effect. Bright light exposure and physical exercise, even in combination, seem to be well tolerated and effective on depressive symptoms, but more research is needed to confirm these findings. Competing interests None declared. Authors' contributions SL, TP, and JL planned the study protocol and supervised the study. JH planned the statistical analyses. SL, TP, and JH analysed the data. SL and TP wrote the manuscript, which was commented by JH and JL. All authors have read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements This study was supported by grants from The Signe and Ane Gyllenberg Foundation and The Finnish Foundation for Psychiatric Research. ==== Refs Saarijärvi S Lauerma H Helenius H Saarilehto S Seasonal affective disorders among rural Finns and Lapps. 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Psychiatry Res 2003 119 89 97 12860363 10.1016/S0165-1781(03)00106-9 Herman S Blumenthal JA Babyak M Khatri P Craighead WE Krishnan KR Doraiswamy PM Exercise therapy for depression in middle-aged and older adults: predictors of early dropout and treatment failure. Health Psychol 2002 21 553 563 12433007 10.1037//0278-6133.21.6.553 Williams JBW Link MJ Rosenthal NE Terman M Structured Interview Guide for the Hamilton Depression Rating Scale – Seasonal Affective Disorders Version (Self-Rating Format), Revised. 1991 New York, NY: New York Psychiatric Institute Oja P Laukkanen R Pasanen M Tyry T Vuori I A 2-km walking test for assessing the cardiorespiratory fitness of healthy adults. Int J Sports Med 1991 12 356 362 1917218 Partinen M Gislason T Basic Nordic Sleep Questionnaire (BNSQ): a quantitated measure of subjective sleep complaints. J Sleep Res 1995 4 150 155 10607192 Puska P Tuomilehto J Salonen J Nissinen A Virtamo J Björkqvist S Koskela K Neittaanmäki L Takalo T Kottke TE Mäki J Sipilä P Varvikko P Community control of cardiovascular diseases. The North Karelia project. 1981 Copenhagen: WHO Regional Office for Europe Rosenthal NE Sack DA Gillin JC Lewy AJ Goodwin FK Davenport Y Mueller PS Newsome DA Wehr TA Seasonal affective disorder. A description of the syndrome and preliminary findings with light therapy. Arch Gen Psychiatry 1984 41 72 80 6581756 Bartko JJ Kasper S Seasonal changes in mood and behavior: a cluster analytic approach. Psychiatry Res 1989 28 227 239 2748773 10.1016/0165-1781(89)90049-8 Ancoli-Israel S Roth T Characteristics of insomnia in the United States: results of the 1991 National Sleep Foundation Survey. I. 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Alcohol Res Health 2001 25 101 109 11584549 Dishman RK Ickes W Self-motivation and adherence to therapeutic exercise. J Behav Med 1981 4 421 438 7338896 Hamilton M A rating scale for depression. J Neurol Neurosurg Psychiatry 1960 23 56 62 14399272 Mulder RT Joyce PR Frampton C Relationships among measures of treatment outcome in depressed patients. J Affect Disord 2003 76 127 135 12943942 10.1016/S0165-0327(02)00080-0 Horne JA Östberg O A self-assessment questionnaire to determine morningness-eveningness in human circadian rhythms. Int J Chronobiol 1976 4 97 110 1027738

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==== Front BMC PsychiatryBMC Psychiatry1471-244XBioMed Central London 1471-244X-4-231531039510.1186/1471-244X-4-23Research ArticleMental health first aid training in a workplace setting: A randomized controlled trial [ISRCTN13249129] Kitchener Betty A [email protected] Anthony F [email protected] Depression & Anxiety Consumer Research Unit, Centre for Mental Health Research, Australian National University, Canberra, Australia2 Centre for Mental Health Research, Australian National University, Canberra, Australia2004 15 8 2004 4 23 23 9 3 2004 15 8 2004 Copyright © 2004 Kitchener and Jorm; licensee BioMed Central Ltd.2004Kitchener and Jorm; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The Mental Health First Aid training course was favorably evaluated in an uncontrolled trial in 2002 showing improvements in participants' mental health literacy, including knowledge, stigmatizing attitudes, confidence and help provided to others. This article reports the first randomized controlled trial of this course. Methods Data are reported on 301 participants randomized to either participate immediately in a course or to be wait-listed for 5 months before undertaking the training. The participants were employees in two large government departments in Canberra, Australia, where the courses were conducted during participants' work time. Data were analyzed according to an intention-to-treat approach. Results The trial found a number of benefits from this training course, including greater confidence in providing help to others, greater likelihood of advising people to seek professional help, improved concordance with health professionals about treatments, and decreased stigmatizing attitudes. An additional unexpected but exciting finding was an improvement in the mental health of the participants themselves. Conclusions The Mental Health First Aid training has shown itself to be not only an effective way to improve participants' mental health literacy but also to improve their own mental health. It is a course that has high applicability across the community. ==== Body Background In 2000 we developed a Mental Health First Aid course in response to the findings of two large national mental health surveys in Australia [1,2]. These findings included a high prevalence rate of mental health problems (approximately 20% of adults in any one year), the poor mental health literacy of members of the Australian public (poor recognition and knowledge of symptoms and causes of mental health problems, where to seek help and what are the most effective treatments) and the widespread stigma towards people with mental health problems. Regular first aid courses are recognised as improving the public's giving of initial and appropriate help at medical emergencies but, unfortunately, most of these courses do not include mental health problems. The Mental Health First Aid course consists of three weekly sessions of three hours each. The content covers helping people in mental health crises and/or in the early stages of mental health problems. The crisis situations covered included suicidal thoughts and behavior, acute stress reaction, panic attacks and acute psychotic behavior. The mental health problems discussed included depressive, anxiety and psychotic disorders. The co-morbidity with substance use disorders is also covered. Participants learn the symptoms of these disorders, possible risk factors, where and how to get help and evidence-based effective help. The initial evaluation trial of the Mental Health First Aid course was an uncontrolled one with 210 members of the public with pre, post and 6-month follow-up. This trial showed that participants improved: their recognition of mental disorders, their beliefs about what treatments were helpful, attitudes towards people with mental illness, the amount of help provided to people with mental health problems, and their confidence in providing help to these people [3]. The next step in our evaluation of this course was to conduct a randomised trial involving a wait-list control group. The present article reports this study, which was carried out in a workplace setting. Methods Participants Eligible participants (approximately 4800) were all Canberra-based employees of two Australian government departments: Health and Ageing, and Family and Community Services. The trial was advertised to staff by email. Participants had to agree to be randomly assigned to receive the training in either Month 1 or Month 6. Training was delivered and data collected at the worksite during office hours. Interventions The course content has been described in the Background and previously [3] and further details can be found at the Mental Health First Aid website [4]. The training followed set lesson plans and all participants were given a Mental Health First Aid Manual to keep [5]. Training was administered at the worksite in classes of 6–18 participants. Participants did not necessarily stay in the same class, but moved between classes to complete the course as necessitated by their work schedule. One instructor carried out all the training. She is the developer of the Mental Health First Aid course and had trained over 1000 people before the start of the trial. Participants received training either immediately (June) or after a five-month delay (November). Those who received training immediately constituted the intervention group and the wait-listed group was the control. To monitor whether the intervention was actually received, an attendance roll was kept for each class. Objectives The main objective was to assess whether Mental Health First Aid training improved mental health literacy and helping skills relative to a wait-list control. A secondary objective was to assess any benefits to the participants' own mental health. Outcomes Outcomes were measured in the month before intervention (the pre-test assessment) and in the fifth month after intervention (the follow-up assessment). The intervention group received training in Month 1 (immediately after pre-test) and the wait-list control group received training in Month 6 (immediately after the follow-up). All outcomes were measured by self-completed questionnaires based on the ones used in the uncontrolled trial of Mental Health First Aid [3]. The pre-test questionnaire (see Additional File 1) covered the following: socio-demographic characteristics of the participant, why they were interested in doing the course, history of mental health problems in participant or family, confidence in providing help, contact with people who have mental health problems in previous 6 months and help offered, recognition of a disorder in vignettes describing a person with depression and one with schizophrenia, belief about the helpfulness of various interventions for the persons described, a social distance scale to assess stigmatizing attitudes [7], and whether the participant or a family member or friend had ever had a problem like the one in the vignette. To score the items on beliefs about treatment, a scale was created showing the extent to which participants agreed with health professionals about which interventions would be useful. For depression, there is a professional consensus that GPs, psychiatrists, clinical psychologists, antidepressants, counseling and cognitive-behavior therapy are helpful [6]. Thus, participants received a score from 0 to 6 according to the number of these interventions endorsed as helpful and this was converted into a percentage. For schizophrenia, there is a professional consensus that GPs, psychiatrists, clinical psychologists, antipsychotics and admission to a ward are helpful for schizophrenia [6]. "Helpful" ratings were summed to give a score from 0 to 5 and converted to a percentage. The questionnaire ended with the SF-12, which provided scales assessing the participant's mental and physical health [8]. These scales were scored using Andrews' [9] integer scorer. The follow-up questionnaire was the same as the pre-test questionnaire except that it omitted the sociodemographic questions and asked about contact with anyone with a mental health problem over the 5 months since the last questionnaire (rather than 6 months). The questionnaires were sent out via internal departmental mail by a human resources staff member in each place of employment. The questionnaires were completed anonymously with only an ID number and posted back to the researchers at the Centre for Mental Health Research. The IDs of any non-responders were sent back to the human resources staff member who sent out a reminder. The researchers were never told the names of individual respondents and the human resources staff member in the place of employment never saw any completed questionnaires or individually identifiable data. Sample size The study was planned to have a sample of 300. The sample size was determined by practical constraints: when it was convenient to run classes that fitted the employees' work schedule and the workload on the instructor. It was determined that this sample size had excellent power to detect medium effect sizes for both continuous and dichotomous outcomes [10]. The trial was originally planned to involve only one workplace, but was extended to a second one because the number of participants recruited was smaller than expected. The lower recruitment appeared to be due to the requirement that participants agree to random assignment to training at either of two periods. Randomization and blinding A staff member in the human resources section of the place of employment kept a list of participants' names and ID numbers. The researchers only had access to the IDs. One of the researchers (Jorm) randomly assigned participants to training or control groups by ID number using the Random Integers option at the website [11]. After recruitment, participants were assigned an ID by the staff member in human resources. These staff assigned participants to groups based on the randomized IDs provided to them. Random allocation occurred only after all participants within a place of employment were recruited and assigned ID numbers. The instructor (Kitchener) provided the human resources staff member with the names of attendees to check that participation was as allocated. Blinding was not possible with the Mental Health First Aid intervention. Ethics Ethical approval for the study was given by the Australian National University Human Research Ethics Committee. Statistical methods Repeated measures analysis of variance was used to analyze continuous measures, with two groups (intervention and control) and two time points (pre-test and follow-up). The principal interest was in the group × time interaction effect. Logistic regression was used to analyze change in dichotomous measures, with group and pre-test score as the predictors and follow-up score as the outcome. Place of employment was also investigated to see if there was a difference in the effects of training. However, no interaction effects involving place of employment were found, so this variable was dropped from all analyses reported below. The analysis was carried out according to intention-to-treat principles, so that all persons who completed a pre-test questionnaire were included, even if they subsequently dropped out. In such cases, the pre-test score was substituted for the missing value, so that no improvement was assumed. Results Recruitment An email inviting participation was sent to all staff of the relevant departments based in Canberra. The email was sent out in May 2002 for the Department of Health and Ageing and March 2003 for the Department of Family and Community Services. In order to participate, staff had to send back a consent form and fill out a pre-test questionnaire before the start of classes. Participant flow Figure 1 shows the flow of participants at each stage of the trial. There were two deviations from plan. Firstly, 18 of the 146 participants (12.3%) assigned to receive Mental Health First Aid training did not complete the whole course. Secondly, 39 out of 146 participants (26.7%) in the intervention group did not complete follow-up questionnaires, compared to only 22 out of 155 (14.2%) in the control group. Figure 1 Flow diagram showing progress through the phases of the trial. Participants' characteristics In terms of sociodemographic characteristics, 78.1% of the participants were female, 49.2% were aged 18–39 years, 50.2% were aged 40–59 and 0.7% aged 60+ years. There were 60.6% with a university degree, 1.3% were aboriginal and 8.6% did not have English as their first language. 13.0% described themselves as mental health consumers, 9.6% as carers for a person with a mental health problem, and 6.3% as health service providers. When asked their reason for doing the course, 27.2% cited reasons relating to their workplace, 11.7% reasons relating to family or close friends, 4.9% reasons relating to their own mental health status, 20.5% cited duty as a citizen, 29% said they were just interested, and 6.7% wanted more accurate or updated information on mental health. 165 (54.8%) of the participants worked at the Department of Health and Ageing and 136 (45.2%) at the Department of Family and Community Services. Numbers analyzed The data were analyzed according to intention-to-treat principles, so that all persons who completed a pre-test questionnaire were included, even if they subsequently dropped out. For every analysis, there were 146 participants analyzed in the intervention group and 155 in the control group. Perception of mental health problem in self or family Participants were asked about whether they themselves had ever experienced a mental health problem or whether anyone in their family had. Table 1 shows that around half reported having personally experienced a mental health problem and around three-quarters reported that a family member had a mental health problem. However, participating in the Mental Health First Aid course did not affect these variables. Table 1 Percent reporting history of mental health problem in self or family. Mental health problems in: MHFA group Control group P-value for group × time interaction Self .577  Pre-test 60.0% 49.7%  Follow-up 65.5% 55.6%  Change (95% CI) 5.5% (0.5 to 10.6) 5.9% (0.6 to 11.1) Family .849  Pre-test 74.5% 73.0%  Follow-up 77.2% 75.7%  Change (95% CI) 2.8% (-3.9 to 9.4) 2.6% (-3.5 to 8.7) Recognition of disorder in vignette Table 2 shows the percentage who correctly recognized the disorders in the vignettes. For the schizophrenia vignette, mention of either "schizophrenia" or "psychosis" was considered correct. The table also shows the percentage who got both vignettes correct. Although there tended to be greater improvement in recognition in the group receiving Mental Health First Aid, there were no significant differences from the control group. Table 2 Percent correctly recognizing the disorder in a vignette. Type of vignette MHFA group Control group P-value for group × time interaction Depression .091  Pre-test 90.2% 87.7%  Follow-up 95.8% 90.3%  Change (95% CI) 5.6% (0.5 to 10.7) 2.6% (-2.8 to 8.0) Schizophrenia .083  Pre-test 74.6% 83.9%  Follow-up 82.6% 81.9%  Change (95% CI) 8.0% (1.5 to 14.4) -2.0% (-6.8 to 2.8) Both vignettes .189  Pre-test 70.6% 76.5%  Follow-up 80.2% 77.8%  Change (95% CI) 9.6% (2.8 to 16.4) 1.3% (-5.2 to 7.9) Beliefs about treatments Table 3 shows the data on whether beliefs about treatments became more concordant with those of health professionals. There was significantly greater improvement in concordance in the Mental Health First Aid group when both depression and schizophrenia were considered together. However, the trends failed to reach significance at the .05 level when the disorders were considered separately. Table 3 Changes in beliefs about treatment and in social distance. Scale MHFA group Control group P-value for group × time interaction Beliefs about treatment for depression .062  Pre-test mean (SD) 82.10 (17.27) 83.00 (18.95)  Follow-up mean (SD) 86.29 (18.30) 83.42 (18.48)  Change (95% CI) 4.19 (1.18 to 7.20) 0.42 (-2.20 to 3.04) Beliefs about treatment for schizophrenia .096  Pre-test mean (SD) 84.28 (19.33) 88.21 (16.76)  Follow-up mean (SD) 87.41 (18.26) 88.41 (16.11)  Change (95% CI) 3.13 (0.30 to 5.96) 0.20 (-1.87 to 2.27) Beliefs about treatment for both disorders .036  Pre-test mean (SD) 83.28 (16.65) 85.51 (15.05)  Follow-up mean (SD) 86.98 (16.78) 85.89 (14.42)  Change (95% CI) 3.70 (1.16 to 6.24) 0.38 (-1.46 to 2.23) Social distance from person with depression .005  Pre-test mean (SD) 8.74 (2.80) 8.63 (2.63)  Follow-up mean (SD) 7.86 (2.50) 8.46 (2.54)  Change (95% CI) -0.88 (-1.23 to -0.53) -0.18 (-0.51 to 0.16) Social distance from person with schizophrenia .211  Pre-test mean (SD) 12.12 (3.53) 12.13 (3.50)  Follow-up mean (SD) 11.27 (3.50) 11.62 (3.35)  Change (95% CI) -0.84 (-1.23 to -0.46) -0.51 (-0.87 to -0.15) Social distance from both .020  Pre-test mean (SD) 20.88 (5.79) 20.79 (5.53)  Follow-up mean (SD) 19.14 (5.43) 20.07 (5.30)  Change (95% CI) -1.73 (-2.37 to -1.10) -0.72 (-1.29 to -0.14) Social distance Table 3 shows data on social distance from the person in each vignette. There was greater improvement in social distance in the Mental Health First Aid group overall, but when the two vignettes were examined separately, this improvement was confined to the depression vignette. Help provided to others Table 4 shows data on confidence in providing help and actual help provided to others in the period before completing the questionnaire. Confidence improved more in the Mental Health First Aid group. There was no change in the percentage who reported contact with anyone with a mental health problem or in the percentage reporting giving "some" or "a lot" of help. However, while the control group showed a decline in the percentage advising professional help, the Mental Health First Aid group did not, leading to a significant difference between groups. Table 4 Changes in confidence and help provided to others. Outcome MHFA group Control group P-value for group × time interaction % Feeling confident in helping someone ("moderately", "quite a lot" or "extremely") .001  Pre-test 54.5% 49.7%  Follow-up 74.5% 57.4%  Change (95% CI) 20.0% (12.6 to 27.4) 7.7% (1.3 to 14.1) % Had contact with anyone with mental health problem .157  Pre-test 71.5% 70.8%  Follow-up 72.9% 65.6%  Change (95% CI) 1.4% (-6.9 to 9.6) -5.2% (-13.5 to 3.1) % Provided help ("some" or "a lot") .525  Pre-test 37.0% 37.5%  Follow-up 39.0% 36.2%  Change (95% CI) 2.0% (-5.5 to 9.6) -1.3% (-9.6 to 6.9) % Advised professional help .007  Pre-test 28.1% 27.1%  Follow-up 29.4% 16.8%  Change (95% CI) 1.4% (-6.8 to 9.5) -10.3% (-18.0 to -2.6) Participants' mental health Table 5 shows changes in the mental and physical health of participants. The Mental Health First Aid group showed significantly greater improvement in mental health. No difference between groups was found in physical health, but none was expected. The physical health scale is included in the table only to show the specificity of the effect on mental health. Table 5 Changes in mental and physical health. Scale MHFA group Control group P-value for group × time interaction Mental health .035  Pre-test mean (SD) 45.43 (11.40) 45.40 (10.17)  Follow-up mean (SD) 47.48 (11.11) 45.11 (11.25)  Change (95% CI) 2.06 (0.39 to 3.72) -0.29 (-1.72 to 1.14) Physical health .506  Pre-test mean (SD) 51.38 (7.97) 51.97 (8.11)  Follow-up mean (SD) 50.74 (8.14) 51.90 (8.68)  Change (95% CI) -0.64 (-1.80 to 0.53) -0.07 (-1.29 to 1.16) Adverse events Given that an educational intervention was evaluated with a non-clinical sample, there was no justification for a systematic inquiry into adverse events. Informally, no adverse events were reported. Discussion This trial has found a number of benefits from Mental Health First Aid training. Relative to the control group, the intervention group showed greater confidence in providing help to others, greater likelihood of advising people to seek professional help, improved concordance with health professionals in beliefs about treatment, decreased social distance from people suffering from depression, and improved mental health of the participants themselves. Recognition of disorders in vignettes did not improve, but there was a very high recognition at pre-test, limiting the scope for improvement. A potential criticism of Mental Health First Aid training is that it will lead to excessive labeling of life problems as mental disorders by members of the public. To check this possibility we asked participants about mental health problems in themselves and family members. Although a high prevalence rate was reported, we found that the course had no effect on these rates. A surprising effect was that the course improved the participants' scores on the SF-12 mental health scale. We included this scale to explore whether there was any impact on mental health, but did not have any strong expectation that it would. The course is not aimed at the participants' own mental health and does not include any therapy. Furthermore, only 5% of participants cited their own mental health as a reason for doing the course. Nevertheless, the participants' mean score on the mental health scale was around half a standard deviation below Australian population norms [9], showing that some were having on-going problems. The cause of the improvement in mental health is not clear. It is unlikely to be a placebo effect because the course gave no expectation of personal change in mental health and only a small percentage did the course for their own benefit. Furthermore, there was no corresponding change on the SF-12 physical health scale. We speculate that the evidence-based information given in the course allowed participants to take action to benefit their own mental health. A similar therapeutic effect has recently been reported from a trial of a web site giving evidence-based information on depression [12]. The data analysis involved a conservative intention-to-treat strategy in which participants who failed to complete the whole course were included and those who failed to respond to the follow-up questionnaire were assumed to show no change. A particular limitation in the present study is that participants in the intervention group showed a poorer response to the follow-up questionnaire than controls. The reason for this poorer response is unknown, but we believe it occurred because the intervention group had already received the course and had nothing to gain by filling out a further questionnaire. By contrast, the controls were still waiting to receive their training and may have believed that filling out the questionnaire would assist this. Whatever the reason, the poorer response in the intervention group meant that more of them were assumed to show no change, thus minimizing any benefits of the training. It is likely that the true effects of Mental Health First Aid training are greater than the present data indicate. The present trial evaluates efficacy rather than effectiveness. The trial was carried out in a workplace setting with well-educated employees who were allowed to do the course during working hours. There was only one instructor, who was the developer of the Mental Health First Aid course, limiting the generalizability of the findings to other instructors. Further research is needed to evaluate the course as taught by other instructors in more typical settings. We are currently engaged in an effectiveness trial with members of the public in a large rural area, with local health service staff trained to run the courses. The Mental Health First Aid training evaluated in this trial was 9 hours long. Based on feedback from participants that the course needed to be longer, we now routinely run the course over 12 hours. This longer course expands on each of the topics covered, especially substance use disorders. Whether this longer course has additional benefits remains to be evaluated. However, our expectation is that it would produce greater effects on beliefs about treatment, confidence in providing help and actual help to provided to others. Conclusions Mental Health First Aid training appears to be effective in improving some aspects of mental health literacy, confidence in providing help to others, and the type of help provided. The training also benefits the mental health of participants. The course is highly acceptable in a workplace setting and could be widely applied. Over 100 Mental Health First Aid instructors have now been trained and the course is available throughout much of Australia and in Scotland, Hong Kong and New York State, USA. Dissemination in other localities is planned in the near future. Competing interests The authors were the developers of the Mental Health First Aid course. Authors' contributions BAK co-designed the study and the evaluation questionnaire, taught the Mental Health First Aid courses, and co-wrote the manuscript. AFJ co-designed the study and the evaluation questionnaire, analyzed the data, and co-wrote the manuscript. Both authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Pre-test questionnaire. questions used in pre-test questionnaire Click here for file Acknowledgements Thanks to Kelly Blewitt for assistance with the organization of the trial and Claire Kelly for data entry. We also wish to thank the staff of the Department of Health and Ageing and the Department of Family and Community Services for their assistance with recruitment, data collection and class organization, in particular Christine Scicluna, Jaime Castles, Deborah Sydenham, and Hannah Gillespie. ==== Refs Andrews G Hall W Teeson M Henderson S The Mental Health of Australians Canberra: Mental Health Branch, Commonwealth Department of Health and Aged Care 1999 Jorm AF Korten AE Jacomb PA Christensen H Rodgers B Pollitt P "Mental health literacy": a survey of the public's ability to recognise mental disorders and their beliefs about the effectiveness of treatment Med J Aust 1997 166 182 186 9066546 Kitchener BA Jorm AF Mental health first aid training for the public: evaluation of effects on knowledge, attitudes and helping behavior BMC Psychiatry 2002 2 10 12359045 10.1186/1471-244X-2-10 Mental Health First Aid Website Kitchener BA Jorm AF Mental Health First Aid Manual 2002 Canberra: Centre for Mental Health Research Jorm AF Korten AE Jacomb PA Rodgers R Pollitt P Christensen H Henderson S Helpfulness of interventions for mental disorders: beliefs of health professionals compared with the general public Br J Psychiatry 1997 171 233 237 9337975 Link BG Phelan JC Bresnahan M Stueve A Pescosolido BA Public conceptions of mental illness: labels, causes, dangerousness, and social distance Am J Public Health 1999 89 1328 1333 10474548 Ware JE Kosinsk M Keller SD A 12-item short-form health survey: construction of scales and preliminary tests of reliability and validity Med Care 1996 34 220 233 8628042 10.1097/00005650-199603000-00003 Andrews G A brief integer scorer for the SF-12: validity of the brief scorer in Australian community and clinic settings Aust N Z J Public Health 2002 26 508 510 12530792 Cohen J A power primer Psychol Bull 1992 112 155 159 10.1037//0033-2909.112.1.155 Random.org Website Christensen H Griffiths KM Jorm AF Delivering depression interventions using the internet: positive results from a large randomized controlled trial BMJ 2004 328 265 14742346 10.1136/bmj.37945.566632.EE

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==== Front BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-4-321529402310.1186/1471-2458-4-32Research ArticleEstimating the beginning of the waterpipe epidemic in Syria Rastam Samer [email protected] Kenneth D [email protected] Thomas [email protected] Wasim [email protected] Syrian Center for Tobacco Studies, Aleppo, Syria2 Center for Community Health, University of Memphis, Memphis, USA3 Department of Psychology, Virginia Commonwealth University, Richmond, USA4 Institute of Epidemiology and Social Medicine, University of Muenster, Muenster, Germany2004 4 8 2004 4 32 32 30 3 2004 4 8 2004 Copyright © 2004 Rastam et al; licensee BioMed Central Ltd.2004Rastam et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Waterpipe smoking is becoming a global public health problem, especially in the Eastern Mediterranean region (EMR). Methods We try in this study, which is a cross sectional survey among a representative sample of waterpipe smokers in cafes/restaurants in Aleppo-Syria, to assess the time period for the beginning of this new smoking hype. We recruited 268 waterpipe smokers (161 men, 107 women; mean age ± standard deviation (SD) 30.1 ± 10.2, response rate 95.3%). Participants were divided into 4 birth cohorts (≤ 1960, 1961–1970, 1971–1980, >1980) and year of initiation of waterpipe smoking and daily cigarette smoking were plotted according to these birth cohorts. Results Data indicate that unlike initiation of cigarette smoking, which shows a clear age-related pattern, the nineties was the starting point for most of waterpipe smoking implicating this time period for the beginning of the waterpipe epidemic in Syria. Conclusion The introduction of new flavored and aromatic waterpipe tobacco (Maassel), and the proliferation of satellite and electronic media during the nineties may have helped spread the new hype all over the Arab World. ==== Body Background Waterpipe smoking is becoming increasingly a worldwide phenomenon, with populations in the Eastern Mediterranean region (EMR) being especially affected [1]. This centuries-old tobacco use method comes under many different names (e.g., shisha, hookah, narghile, arghile), shapes, and sizes, depending on the region, with the term waterpipe implying a unifying feature of all these forms; the passage of smoke through water before inhalation by the smoker [2], Recent evidence shows that a quarter of some populations in the EMR currently smoke the waterpipe [3]. This trend is worrisome because of tobacco's known harmful effects to human health, and because prevailing norms the EMR may put certain slices of the society at increased risk of acquiring the habit, particularly women and children [6,7]. Although research on the health effects of waterpipe is still scarce, preliminary evidence links waterpipe use to respiratory, cardiovascular, and cancer diseases [8-11]. Developing effective intervention strategies to curb this emerging public health problem requires clear understanding of factors influencing the take up of this habit, as well its time course [1]. According to waterpipe smokers, the recent resurgence in waterpipe popularity is due to the introduction of Maassel (a specially prepared tobacco with sweetened fruit flavors and mild aromatic smoke), the media, and social trends [6]. Understanding the context in which these factors operate as well as being able to follow the secular course of the waterpipe epidemic requires estimation of the time frame for the beginning of the waterpipe hype. In this study we try to identify this time frame as well as provide evidence for the increase in waterpipe smoking. Methods The current analysis is drawn from a survey conducted in 2003 among a representative sample of waterpipe smokers visiting cafes/restaurants in Aleppo, Syria. The survey details can be found elsewhere [12], but briefly a cross sectional interviewer-administered survey was conducted in 17 randomly selected (out of total 112) café/restaurants in Aleppo, Syria. Overall, 268 waterpipe smokers were recruited (161 men and 107 women; mean age ± SD 30.1 ± 10.2, age range 18–68 years; response rate 95.3%). Participants were asked about their waterpipe use frequency, cigarette smoking status, current age, age of initiation of waterpipe smoking, and age of initiation of daily cigarette smoking. The protocols and informed consent documents for this study were approved by the SCTS' IRB and the University of Memphis' IRB. The questionnaire was anonymous and informed consent was obtained prior to all interviews. Analysis First, year of birth was calculated by subtracting current age of participant from the year of survey (2003), while year of initiation of waterpipe smoking and daily cigarette smoking were calculated by adding the age of initiation of smoking to the year of birth. Year of birth was then divided into four decade-long categories (people born in/before 1960; during 1961–1970; during 1971–1980; in/after 1981), and year of smoking initiation into three decade-long categories (initiation in/before 1990, initiation during 1991–2000, and initiation in/after 2001). The Chi-Square test was used to assess differences between the three smoking initiation time-groups for each birth cohort, with p level <0.05 considered significant. Results and conclusions Figure 1a,1b compares between year of initiation of waterpipe and cigarettes among study participants, respectively, according to their birth cohort. It shows that while cigarette initiation displays an age-related pattern with peak initiation of participants occurring during in their twenties, most of waterpipe initiation and for all birth cohorts is commencing in the 1990s. Figure 2, depicts number of waterpipe smokers according to their year of initiation, and suggests indirectly the rapid increasing trend of this smoking method. Taking into consideration possible limitations of this study, particularly the use of cross-sectional design to examine time trends and the relative youngness of the study sample, these results suggest that the current hype of waterpipe smoking is a recent one, commencing mostly in the nineties of the 20th century, and showing an increasing trend. Based on these results and our previous data regarding factors related to the spread of waterpipe [6], we present the following scenario for the current surge of popularity of waterpipe smoking. In the nineties, Maassel was introduced [13] simplifying waterpipe preparation and attracting more people to its mild aromatic smoke. Out of curiosity about this new tobacco or of modeling of others, people started experimenting with the waterpipe. The increasing number of waterpipe users together with the spread of satellite channels and electronic media, occurring during the same time period, may have contributed further to the creation and spread of this new smoking trend. Since waterpipe smoke contains considerable amounts of the addictive substance nicotine [14,15], nicotine dependence can sustain the habit among experimenters creating a stable base of waterpipe smokers within the society and contributing further to its spread. It remains to be seen, the possible role of resurgence of local identities in contrast to western culture in the adoption of this "oriental" method of smoking. The dramatic increase of this addictive smoking method within a short period of time and its potential health risks mandate that active surveillance and in depth research into its risk profile should become a priority for health systems in the EMR. Policy makers should also be alerted to this eminent public health threat, which is so far escaping current regulations and restrictions imposed on cigarettes, such as the ban on advertisement and the inclusion of health warnings on waterpipe tobacco products. Figure 1 a: Shows the proportion of current waterpipe smokers of different birth cohorts according to their year of initiation categorized into three decade-long categories. b. The same parameters are shown for cigarette smoking. Figure 2 Shows number of study participants according to their year of initiation of waterpipe smoking. Competing interests None declared. Authors' contributions SR designed the study, conducted the analysis and wrote the first draft of the manuscript. KW and TE contributed to the design of survey and revision of the manuscript. WM contributed to the design of survey and wrote the final draft of the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgement This work is supported by USPHS grant R01 TW05962 ==== Refs Maziak W Eissenberg T Klesges RC Keil U Ward KD Adapting Smoking Cessation Interventions for Developing Countries: A Model for the Middle East Int J Tuberc Lung Dis 2004 8 403 413 15141730 Maziak W Ward KD Afifi Soweid RA Eissenberg T Tobacco smoking using a waterpipe: a re-emerging strain in a global epidemic Tobacco Control 2004 Maziak W Fouad MF Hammal F Asfar T Bachir EM Rastam S Eissenberg TE Ward KD Prevalence and characteristics of narghile smoking among university students in Aleppo, Syria Int J Tuberc Lung Dis 2004 8 882 889 15260281 Chaaya M Awwad J Campbell OM Sibai A Kaddour A Demographic and psychosocial profile of smoking among pregnant women in Lebanon: public health implications Matern Child Health J 2003 7 179 186 14509413 10.1023/A:1025136421230 Tamim H Terro A Kassem H Ghazi A Khamis TA Hay MM Musharrafieh U Tobacco use by university students, Lebanon, 2001 Addiction 2003 98 933 939 12814499 10.1046/j.1360-0443.2003.00413.x Maziak W Eissenberg T Asfar T Rastam S Hammal F Bachir ME Fouad MF Ward KD Beliefs and attitudes related to narghile smoking among university students in Syria Ann Epidemiol 2004 The global youth tobacco survey collaborative group Tobacco use among youth: a cross-country comparison Tob Cont 2002 11 252 70 10.1136/tc.11.3.252 Bedwani R el-Khwsky F Renganathan E Braga C Abu Seif HH Abul Azm T Zaki A Franceschi S Boffetta P La Vecchia C Epidemiology of bladder cancer in Alexandria, Egypt: tobacco smoking Int J Cancer 1997 73 64 7 9334811 10.1002/(SICI)1097-0215(19970926)73:1<64::AID-IJC11>3.0.CO;2-5 Gupta PC Murti PR Bhonsle RB Epidemiology of cancer by tobacco products and the significance of TSNA Crit Rev Toxicol 1996 26 183 98 8688160 Jabbour S El-Roueiheb Z Sibai AM Narghile (water-pipe) smoking and incident coronary heart disease: a case-control study [abstract] Ann Epidemiol 2003 13 570 10.1016/S1047-2797(03)00165-0 Kiter G Ucan ES Ceylan E Kilinc O Water-pipe smoking and pulmonary functions Respir Med 2000 94 891 94 11001082 10.1053/rmed.2000.0859 Maziak W Eissenberg TE Ward KD Factors related to level of narghile use: the first insights on tobacco dependence in narghile users Drug Alcohol Depend 2004 Kandela P Nargile smoking keeps Arabs in wonderland Lancet 2000 356 1175 11030308 Shafagoj YA Mohammed FI Levels of maximum end-expiratory carbon monoxide and certain cardiovascular parameters following hubble-bubble smoking Saud Med J 2002 23 953 58 Shihadeh A Investigation of mainstream smoke aerosol of the argileh water pipe Food Chem Toxicol 2003 41 143 152 12453738 10.1016/S0278-6915(02)00220-X

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==== Front BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-4-351529871210.1186/1471-2458-4-35Research ArticleThe National Women's Health Study: assembly and description of a population-based reproductive cohort Maconochie Noreen [email protected] Pat [email protected] Susan [email protected] Department of Epidemiology and Population Health, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK2004 7 8 2004 4 35 35 7 2 2004 7 8 2004 Copyright © 2004 Maconochie et al; licensee BioMed Central Ltd.2004Maconochie et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Miscarriage is a common event but is remarkably difficult to measure in epidemiological studies. Few large-scale population-based studies have been conducted in the UK. Methods This was a population-based two-stage postal survey of reproductive histories of adult women living in the United Kingdom in 2001, sampled from the electronic electoral roll. In Stage 1 a short "screening" questionnaire was sent to over 60,000 randomly selected women in order to identify those aged 55 and under who had ever been pregnant or ever attempted to achieve a pregnancy, from whom a brief reproductive history was requested. Stage 2 involved a more lengthy questionnaire requesting detailed information on every pregnancy (and fertility problems), and questions relating to socio-demographic, behavioural and other factors for the most recent pregnancy in order to examine risk factors for miscarriage. Data on stillbirth, multiple birth and maternal age are compared to national data in order to assess response bias. Results The response rate was 49% for Stage 1 and 73% for the more targeted Stage 2. A total of 26,050 questionnaires were returned in Stage 1. Of the 17,748 women who were eligible on the grounds of age, 27% reported that they had never been pregnant and had never attempted to conceive a child. The remaining 13,035 women reported a total of 30,661 pregnancies. Comparison of key reproductive indicators (stillbirth and multiple birth rates and maternal age at first birth) with national statistics showed that the data look remarkably similar to the general population. Conclusions This study has enabled the assembly of a large population-based dataset of women's reproductive histories which appears unbiased compared to the general UK population and which will enable investigation of hard-to-measure outcomes such as miscarriage and infertility. ==== Body Background Despite improvements in obstetric care in the UK over the past fifty years, it is estimated that around one in five pregnancies will end in miscarriage (fetal death before 24 weeks) [1,2]. The personal and public health impact of pregnancy loss is a neglected area in medical research and strategies of prevention remain outside mainstream medical services. Although many large-scale population-based studies of miscarriage risk have been conducted elsewhere [3-10], relatively few such studies have been conducted in the UK, and most of these have been occupational [11-14]. There are no registers of miscarriage or routine data collection systems which would allow linkage of miscarriages to individual women in the UK . There are thus no national prevalence estimates which can be used as reference for UK-based clinical or epidemiological studies. In addition, although there is now greater knowledge of how the risk of miscarriage changes with maternal age and previous history of miscarriage [6], the influence and interaction of biological, behavioural and social risk factors are less well-understood. The lack of reliable information on risk factors, and the confusion surrounding ad hoc reports of spurious associations, makes research in this area of great importance. Studies of miscarriage have tended to be clinical-based, and are thus subject to selection bias. For example, gestations are later among miscarriages reaching hospital-based clinics. Many miscarriages are managed at home, and some are not reported to a clinician. Not only is miscarriage hard to measure, and different clinical sources rarely see the full range of cases, but reported risks of miscarriage tend to be pregnancy-rather than woman-based: estimates of risk tend to relate to the proportion of pregnancies ending in miscarriage, and there are very few studies examining the risk of experiencing one, two or more miscarriages, or the chances of conceiving following a miscarriage [15]. Large prospective cohort studies are theoretically the ideal design, but take time and are prohibitively expensive [2]. An alternative and practical approach is a survey asking the women themselves for their full reproductive history, including all fetal losses at all gestations. An increasing number of couples are also seeking help for problems achieving a pregnancy. Although it is estimated that up to 15% couples experience such problems [16], few population-based prevalence studies have been conducted in the UK, particularly where fertility problems have been treated solely by the general practitioner using ovarian stimulation. We now report on a large UK population-based survey of reproductive health, the National Women's Health Study. The study design was developed from several other large epidemiological surveys of reproductive outcome which showed that a postal method could be used to obtain full reproductive histories from large study populations [13,14,17,18]. The aim of the study was to obtain population-based prevalence estimates relating to miscarriage and infertility, and to obtain good quality data on potential risk factors for miscarriage to be used when advising and counselling women who have suffered miscarriage and those who wish to reduce their risk of future pregnancy loss. The design of the study, together with response rates and description of the study population, is presented in this report. Further reports on risk factors for miscarriage, plus population-based estimates of miscarriage and of pregnancies conceived using assisted reproduction techniques will follow. Methods Sample selection This was a population-based cross-sectional postal survey of reproductive histories of adult women living in the United Kingdom in 2001, designed to enable the construction of a retrospective population-based reproductive cohort and a case-control study of risk factors for miscarriage. A sample of women was randomly selected from electronic electoral registers for England, Wales, Scotland and Northern Ireland held by the company Eurodirect [19]. All UK citizens aged 18 and over are eligible to vote; registration is voluntary, but in 2001 around 98% of the entire resident population were on the electoral register [20], the remainder being largely non-UK citizens and iterant population. At the time of survey there was no opt-out clause for those who did not wish to be on an electronic version of the electoral register, so the sampling frame contained all UK residents eligible (and registered) to vote. In order to reduce possible biases associated with memory, we aimed for a sample aged 55 years and under at survey. Date of birth is not, however, routinely recorded on the electoral register. To avoid unnecessary mailing and expense, we therefore made use of a probabilistic process offered by Eurodirect based on forename, whereby the sampling frame was restricted to women thought likely to be aged 55 and under on the basis of their name. This process was based on empirical data relating to birth certificates going back to the beginning of the 20th century, from which it could be calculated that, for example, those named "Elsie" are likely to be aged over 55, and those named "Kylie" under 55 years. Predictions are further refined by examination of combinations of names within a household (a "Jane" married to or living with an Alfred likely to be older than a "Jane" married to or living with a "Darren") and length of residency (e.g. someone registered to vote at the same address for 12 years has to be over 30). We requested a random sample of 61,000 women likely to be aged 55 and under (sample size calculations based on achieving at least 80% power for key risk factors in the case-control analysis, and cost). After removing those known to be under age 18 at study (those turning 18 in the year of registration are allowed to register early, giving date of birth), the final sample consisted of 60,814 women. The study received approval from the Trent Multi-Centre Research Ethics Committee and the Ethics Committee of the London School of Hygiene & Tropical Medicine. Postal survey The postal survey had two stages. Stage one consisted of a single-page "screening" questionnaire which asked for details of all pregnancies experienced by study participants, as well as periods of infertility and infertility treatment. This form was sent to the whole sample and included "opt-out" boxes to be ticked if the recipient had never been pregnant and had never attempted to have children, and/or was over age 55, and/or did not wish to take part. The second stage of the study consisted of a longer postal questionnaire which was sent to all those responding to Stage 1 who had ever been pregnant or who reported ever attempting to conceive and who agreed to be re-contacted. Excluded from this second stage were women who had had one or more termination for non-medical reasons (i.e. for reasons other than that a defect had been identified in the fetus or that continuing the pregnancy would put the mother at risk) and no other pregnancies. The Stage 2 questionnaire requested more general detail about the women (including height, age at menarche, educational level, marital status and details of infertility problems, treatment and diagnosis, if appropriate); detailed information on all pregnancies (including whether the pregnancy was the planned, the result of infertility treatment, father's date of birth and whether father had remained the same); plus socio-demographic and behavioural details relating to the most recent pregnancy. These details included questions relating to weight at start of pregnancy, nausea, smoking, coffee and alcohol consumption, diet, vitamin intake, ill health, air travel, sexual intercourse, occupation and stress levels. The most recent pregnancy was selected to minimise biases related to recall, and since it could be at the start, middle or end of the reproductive careers of these women whose ages at survey ranged from 18 to 55 years potential biases relating to ending reproductive careers on a "success" were not expected to be large. For those whose most recent pregnancy had ended in miscarriage (defined as fetal death at <24 weeks gestation), brief information relating to clinical management of miscarriage and the advice given was also requested. Permission to access clinical notes relating to outcomes reported in the questionnaire, and to contact the women for further study if needed, was also requested. In order to increase the number of cases for the case-control analysis of risk factors for miscarriage, women who had had a miscarriage recently (since 1995) but whose last pregnancy was not a miscarriage were sent a third questionnaire. This was a shortened version of the Stage 2 questionnaire, containing only those questions relating to biological, socio-demographic and behavioural details of the most recent pregnancy, but now requesting these details in relation to the most recent miscarriage. Such women then had two pregnancies in case-control analyses and standard errors were computed using a robust method based on the "sandwich estimate" to account for this statistically. A free telephone helpline was run throughout the study, to answer queries and refer on to other organizations for professional help, if appropriate, and this was well used. Statistical methods All analyses in this paper were performed using Stata statistical software [21]. To investigate possible selection bias we compared stillbirth and multiple delivery rates with rates in the general population. For this we obtained annual registered stillbirth risks and registered multiple delivery rates by maternal age for England and Wales, 1980–2001 [22] (data for 2002 was estimated from that for 2001). Standardised registered stillbirth ratios (SRSR) and standardised multiple delivery rates (SMDR) were then calculated using logistic regression analysis (offsetting the log odds of the population risk) [23]. The unit of analysis for stillbirths was a registered birth. A registered livebirth is defined as a baby born alive at any gestation, registered stillbirth being defined as a fetal death at 28 weeks or more gestation until the end of 1992, and at 24 weeks or more gestation from 1993 onwards. Where gestational age was not available from Stage 2 data, a pregnancy was considered to be a stillbirth if it was so described. Forty-one (40%) of the total 102 stillbirths in the analysis fell into this category. For multiple delivery, the unit of analysis was a pregnancy containing at least one livebirth or registered stillbirth (as described above). For the purposes of the analyses presented in this paper (comparisons with the general population), a pregnancy was only considered multiple if it contained two or more babies who were liveborn or (registered) stillborn in order to be consistent with the definitions used in the national data. Thus, for example, a twin pregnancy occurring before 1993 and resulting in a livebirth and a fetal death at less than 28 weeks was considered to be a singleton pregnancy in this analysis. Average maternal age at first birth, if live, was also compared with that in the general population. Annual average maternal age at first (registered) birth, if live, was obtained with denominators for England and Wales, 1980–2001 [22] and re-calculated for 5-year periods. This national data was available for births within marriage only. Marital status of mother at time of birth was known only for the most recent pregnancy (or most recent miscarriage since 1995) in this dataset. For the NWHS average maternal age was therefore calculated for all first registered births, if live. No formal statistical comparisons of maternal age were made, partly because the numbers were so large that slight, non-meaningful, nuances in the data would give a statististically significant result, and render the comparison meaningless, and partly because the average ages in the general population, though comparable, were expected to be similar but slightly older in the general population data owing to the fact that the data related to births within marriage only. Births where the date of birth or maternal age were not known were excluded from all comparisons with population data. Results Stage 1 The response to the first stage of the study is summarised in Table 1. 29,721 (49%) of all the questionnaires were returned to us, though for 3,591 (6%) this was to say that the addressee had moved, and for 70 (0.1%) that the woman had died. A total of 26,050 questionnaires were returned by the addressee, a response rate of 46% assuming that all questionnaires not returned undelivered had reached the correct recipient. Of these, 11% (5% overall) did not wish to participate in the study, and a further 21% were aged over 55 (n = 5,499) or were otherwise ineligible (n = 65). 27% of the 17,748 women who were eligible on the grounds of age, reported that they had never been pregnant and had never attempted to conceive a child, the remaining 13,035 women reporting their full reproductive history. Table 1 The National Women's Health Survey – response rates STAGE 1 No. Crude % Adjusted1 % TOTAL QUESTIONNAIRES POSTED 60,814 100% - Returned undelivered2 3,661 6% - Responded 26,050 43% 46%  Did not wish to participate 2,738 5% 5%  Aged >55 years or otherwise ineligible3 5,564 9% 10%  Aged < = 55 years but never attempted to have children 4,713 8% 8%  Aged < = 55, ever attempted to have children 13,035 21% 23%  Among whom,   - Never pregnant 340 3% -   - Ever pregnant 12,695 97% - STAGE 2 TOTAL QUESTIONNAIRES POSTED 10,828 100% - Returned undelivered 16 0.2% - Responded 7,882 73% 73%  No longer wished to participate 180 2% 2%  Completed questionnaire 7,702 71% 71%  Among whom,   - Attempted pregnancy, never pregnant 194 3%   - Ever pregnant4 7,508 97% 1 Adjusted for undelivered mail 2 Includes 70 women who died before the study start 3 Under 18 at study start (6th November 2001); male; foreign national; or too ill to participate 4 344 women who had had a miscarriage since 1995, but whose last pregnancy was not a miscarriage, were sent a second stage 2 questionnaire and were asked to supply details in relation to their most recent miscarriage. 285 (83%) of the women responded to this third questionnaire. 12,695 women aged under 55 at survey had been pregnant at least once. These 12,695 women, whose average age at survey was 40.5 years, had started their reproductive careers from 1963 to 2002, 75% having their first pregnancy in 1980 or later (Table 2). 486 women had conceived their first pregnancy less than 40 weeks before the study commenced, 126 of whom were pregnant when they filled in the questionnaire. Overall these 12,695 women reported a total of 30,661 pregnancies, 80% of which occurred in 1980 or later. Outcome of these pregnancies is described in Table 2. Table 2 NWHS Stages 1 and 2 – description of women reporting one or more pregnancy, and of the pregnancies they reported STAGE 1 STAGE 2 n (%) n (%) TOTAL NO. WOMEN IN ANALYSIS 12,695 (100) 7508 (100) Age at survey (years)  <30 1247 (9.8) 685 (10.6)  30–34 2007 (15.8) 1284 (20.6)  35–39 2618 (20.6) 1629 (28.6)  > = 40 6678 (52.6) 3910 (39.3)  Not known 145 (1.1) -  Mean age (SD)1 40.5 (8.45) 40.4 (8.24) Year of first pregnancy  <1980 3201 (25.2) 1798 (24.0)  1980–84 1902 (15.0) 1131 (15.1)  1985–89 2091 (16.5) 1259 (16.8)  1990–94 2158 (17.0) 1356 (18.1)  1995–99 2079 (16.4) 1406 (18.7)  2000–02 7882 (6.2) 5583 (7.4)  Not known 476 (3.8) - Total number of pregnancies reported per woman  1 2607 (20.5) 1403 (18.7)  2 5077 (40.0) 3162 (42.1)  3 2962 (23.3) 1749 (23.3)  4 1573 (12.4) 818 (10.9)  5 285 (2.2) 229 (3.1)  > = 6 191 (1.5) 147 (1.9)  Median (range) 2 (1 – 18) 2 (1 – 18) Pregnancy history No dates given for any pregnancies 436 (3.4) - All pregnancies occurred before 1980 1495 (11.8) 853 (11.4) Pregnancies before and after 1980 1707 (13.5) 945 (12.6) Pregnancy history commenced 1980 onwards 9057 (71.3) 5710 (76.1)  All pregnancies conceived after 31/03/2000 486 (3.8) 329 (4.4) TOTAL REPORTED PREGNANCIES 30661 (100) 18391 (100) Outcome of pregnancy Livebirth, surviving >7 days 24081 (78.9) 14782 (80.4) Livebirth, early neonatal death 95 (0.3) 56 (0.3) Stillbirth 188 (0.6) 110 (0.6) Miscarriage4 3512 (11.5) 2326 (12.7) Ectopic 226 (0.7) 102 (0.6) Termination for medical reasons5 312 (1.0) 89 (0.5) Termination for non-medical reasons6 1424 (4.6) 562 (3.1) Molar pregnancy 47 (0.2) 26 (0.1) Ongoing (current) pregnancy 482 (1.6) 338 (1.8) Not known 294 (1.0) - Year of pregnancy end <1980 6093 (19.9) 3486 (18.0) 1980–84 4503 (14.7) 2623 (14.3) 1985–89 5028 (16.4) 3000 (16.3) 1990–94 5549 (18.1) 3434 (18.7) 1995–99 5808 (18.9) 3865 (21.0) 2000–02 27217 (8.9) 19838 (10.8) Not known 959 (3.1) - 1 Where date of birth given 2 Includes 486 women whose first pregnancy was conceived after 31st March 2000, 126 of whom were currently pregnant for the first time at time of survey 3 Includes 329 women whose first pregnancy was conceived after 31st March 2000, 73 of whom were currently pregnant for the first time at time of survey 4 Fetal death at <24 weeks gestation. Includes missed miscarriages (fetal death at <24 weeks without spontaneous expulsion of fetus) and blighted ova (anembryonic pregnancy) 5 Termination of pregnancy because of a defect identified in the baby, or because continuing the pregnancy would put the mother's health at risk 6 Termination of pregnancy for reasons other than a defect identified in the baby or risk to mother's health 7 1,718 of these pregnancies were conceived after 31st March 2000 8 1,232 of these pregnancies were conceived after 31st March 2000 Stage 2 11,424 (88%) women ever attempting to have children (successfully or unsuccessfully) agreed to participate in the second stage of the study. Of these 596 (5%) were not sent a Stage 2 questionnaire, 212 because they had only ever had one or more termination of pregnancy for non-medical reasons, and 384 because their Stage 1 form arrived back after mailing had ended. A total of 10,828 women were thus sent a second stage questionnaire. The response to this second stage was high (73%), though 2% of women had decided that they no longer wished to participate (Table 1). The 7,702 women completing a Stage 2 questionnaire, and the 18,391 pregnancies they reported, are described in Table 2. Their characteristics are almost identical to those of Stage 1, indicating that Stage 2 responders were an apparently unbiased subset of those responding to Stage 1. 5,777 (75%) women responding to Stage 2 gave signed consent for us to access their medical notes, with 6,963 (90%) agreeing to be contacted again in the future, if required. Comparison with national data Comparisons of Stage 1 data, and the subset Stage 2 data, with national rates are presented in Table 3. There was no evidence to suggest that stillbirth differed from expectation in either Stage 1 (SRSR 115 (95% CI 94 – 139), P = 0.17), or Stage 2 data (SRSR 102 (95% 79 – 132), P = 0.86). Multiple delivery was also in line with expectation from national rates for both stages (Stage 1 SMDR 111 (95% CI 99 – 126), P = 0.08), Stage 2 SMDR 108(95% CI 93–126, P = 32)). Although the inference from this is unambiguous for both stages of the study, the point estimates were noted to be closer to unity for Stage 2 data where almost all pregnancies had known gestational age. This reflects the fact that there might be some slight misclassification of registered stillbirth prior to 1993 in the Stage 1 data where gestational age was only known for 61% of reported stillbirths, some of which might legally be classified as miscarriages. Table 3 Comparison with population birth data of reported births in Stages 1 and 21 of the National Women's Health Study occurring since 19802 REGISTERED STILLBIRTH3 No. stillbirths3 Total livebirths & stillbirths3 SRSR4 (95% CI) Stage 1 1980–2002 102 18,740 115 (94 – 139) Stage 2 1980–2002 59 12,061 102 (79 – 132) MULTIPLE (REGISTERED) DELIVERY5 No. multiple deliveries5 Total deliveries5 SMDR4 (95% CI) Stage 1 1980–2002 264 18,391 111 (99 – 126) Stage 2 1980–2002 169 11,887 108 (93 – 126) AVERAGE MATERNAL AGE AT FIRST6(LIVE)BIRTH (years) No. first6 livebirths Mean (SD) age7 England & Wales8 Mean age Stage 1 Year of delivery 1980–84 1,724 25.2 (4.12) 25.5 1985–89 1,916 25.9 (4.56) 26.4 1990–94 2,058 27.1 (4.85) 27.8 1995–99 2,026 28.6 (5.01) 29.0 2000–02 699 29.4 (5.06) 29.6 Stage 2 1980–84 1,032 25.5 (4.02) 25.5 1985–89 1,182 26.0 (4.45) 26.4 1990–94 1,325 27.3 (4.78) 27.8 1995–99 1,432 28.8 (4.81) 29.0 2000–02 540 29.7 (4.89) 29.6 1 Stage 2 data are a subset of Stage 1 data (see methods). 2 Pregnancies with missing maternal age have been excluded from this analysis. 3 Registered stillbirths 1980–2002, defined as fetal death at ≥ 28 weeks prior to 1992, or at ≥24 weeks thereafter. 41 (40%) of stillbirths had no gestational age, but were described as stillbirths by the mother. Unit of analysis is a baby; multiple births counted as many times as there are babies. Denominator contains all reported livebirths and registered stillbirths 1980–2002. 4 Standardised Registered Stillbirth Ratio (SRSR) and Standardised registered Multiple Delivery Ratio (SMDR). Standardised for maternal age (5-year intervals) and single year of birth using data for England and Wales 1980–2002. 5 Unit of analysis is a delivery (pregnancy) containing one or more registered live or stillbirth; multiple pregnancies counted once only. Multiple pregnancies containing only one registered birth (with another non-registrable outcome, such as miscarriage) considered as singleton in this analysis. 6 First registered birth, if live. 7 NWHS data relates to livebirths both within and outside marriage 8 Livebirths within marriage only Age at first (live) birth was remarkably similar to national data for both Stage 1 and Stage 2 data (Table 3). Exactly as expected, though showing no evidence to suggest any biases with respect to maternal age, average age at first birth was very slightly higher for the national data, since it related to births within marriage only, whereas the NWHS data related to all births (marital status at delivery was unknown). Discussion Using a novel method, the National Women's Health Study has enabled a large UK population-based dataset to be assembled, comprising full reproductive histories, including any history of infertility, for 13,035 women, 12,695 of whom had conceived 30,661 pregnancies. We have obtained further detailed information for 7,702 of these women (18,391 pregnancies), including fertility diagnoses for both male and female partner (if appropriate), and lifestyle and behavioural risk factors for the most recent pregnancy. Seventy-five percent of these women consented to their medical notes being accessed in relation to information reported in the questionnaire, and 90% agreed to be contacted again, thus providing the means to carry out a population-based cohort study of these women at some time in the future. UK population-based data, collected at government level by England & Wales, Scotland and Northern Ireland, relate to registered births (live and still) and terminations of pregnancy, with Scotland also routinely collecting maternity data on hospital deliveries at any gestation. The National Women's Health Study goes one step further than this, providing the whole reproductive picture. Rather than being a pregnancy-based, cross-sectional survey, the data collected for each woman covers the complete spectrum of reproductive outcomes from infertility problems through miscarriage, ectopic pregnancies and terminations (for both medical and non-medical reasons), to live and stillbirths, and does not rely on legal definitions for inclusion in the dataset. Furthermore, unlike most epidemiological studies of adverse reproductive outcome such as miscarriage, the data source is not clinical (which, for miscarriage, leads to inevitable biases relating to gestational age), but relates to women selected randomly from the UK electoral register. And for outcomes such as infertility no other data currently exist to enable estimation of how many pregnancies in the population as a whole result from fertility treatment. The study does rely on maternal recall and this could be a source of bias. Studies of self-reported reproductive history and exposures relating to reproductive events have, however, found maternal recall to have acceptably high reliability, and to be little affected by time from event [24-26]. In terms of the key reproductive indicators of stillbirth, multiple delivery rates and maternal age at first birth, the data look remarkably similar to the general population. We therefore feel confident that response was unlikely to be related to adverse reproductive outcome. Indeed, the average age at survey of around 40 years, coupled with average ages at first birth which are exactly as would be expected from general population data, could be seen to indicate that non-responders to the survey tended to concentrate among younger women who had not yet tested their fertility. In addition, we feel confident that those responding to the more detailed Stage 2 questionnaire are an unbiased sample of those responding to Stage 1. Both Stage 1 and Stage 2 data can thus can be considered unbiased with respect to reproduction, and representative of patterns among all women in the UK population who have ever tried to have children, hence prevalence estimates might be taken as unbiased estimates of hard-to-measure outcomes such as miscarriage and pregnancies conceived through assisted reproduction techniques. Such data will be invaluable as population-based reference data for epidemiological studies of reproduction. In addition to both pregnancy-and woman-based population prevalence estimates, further papers to follow include reports of case-control analyses of behavioural and lifestyle risk factors for miscarriage. Conclusions In summary, we have assembled a large population-based dataset of women's reproductive histories which appears representative of the general UK population and which will enable investigation of hard-to-measure outcomes such as miscarriage and infertility. Competing interests None declared Authors' contributions NM and PD initiated the research and participated in protocol design, data collection, analysis and writing the paper. SP participated in data collection and analysis. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements We thank Ruth Bender Atik and Barbara Hepworth-Jones from The Miscarriage Association who instigated the study. The project was funded by the National Lottery Community Fund (through the Miscarriage Association), and by the Miscarriage Association. ==== Refs Garcia-Enguidanos A Calle ME Valero J Luna S Dominguez-Rojas V Risk factors in miscarriage: a review Eur J Obstet Gynecol Reprod Biol 2002 102 111 9 11950476 10.1016/S0301-2115(01)00613-3 Savitz DA Hertz-Picciotto I Poole C Olshan AF Epidemiologic measures of the course and outcome of pregnancy Epidemiol Rev 2002 24 91 101 12762085 10.1093/epirev/mxf006 Naylor AF Warburton D Sequential analysis of spontaneous abortion. II. Collaborative study data show that gravidity determines a very substantial rise in risk Fertil Steril 1979 31 282 6 437160 Risch HA Weiss NS Clarke EA Miller AB Risk factors for spontaneous abortion and its recurrence Am J Epidemiol 1988 128 420 30 3273482 Rowland AS Baird DD Shore DL Weinberg CR Savitz DA Wilcox AJ Nitrous oxide and spontaneous abortion in female dental assistants Am J Epidemiol 1995 141 531 8 7900720 Nybo Andersen A-M Wohlfahrt J Christens P Olsen J Melbye M Maternal age and fetal loss: population based register linkage study BMJ 2000 320 1708 12 10864550 10.1136/bmj.320.7251.1708 Li DK Odouli R Wi S Janevic T Golditch I Bracken TD Senior R Rankin R Iriye R A population-based prospective cohort study of personal exposure to magnetic fields during pregnancy and the risk of miscarriage Epidemiology 2002 13 9 20 11805581 10.1097/00001648-200201000-00004 Slama R Werwatz A Boutou O Ducot B Spira A Hardle W Does male age affect the risk of spontaneous abortion? An approach using semiparametric regression Am J Epidemiol 2003 157 815 24 12727675 10.1093/aje/kwg056 Xiong X Fraser WD Demianczuk NN History of abortion, preterm, term birth, and risk of preeclampsia: a population-based study Hum Reprod 2002 17 1649 56 12042293 10.1093/humrep/17.6.1649 Osborn JF Cattaruzza MS Spinelli A Risk of spontaneous abortion in Italy, 1978-and the effect of maternal age, gravidity, marital status, and education Am J Epidemiol 2000 151 98 105 10625179 Pandya PP Snijders RJ Psara N Hilbert L Nicolaides KH The prevalence of non-viable pregnancy at 10–13 weeks of gestation Ultrasound Obstet Gynecol 1996 7 170 3 8705407 10.1046/j.1469-0705.1996.07030170.x Elliott RC Jones JR McElvenny DM Pennington MJ Northage C Clegg TA Clarke SD Hodgson JT Osman J Spontaneous abortion in the British semiconductor industry: An HSE investigation. Health and Safety Executive Am J Ind Med 1999 36 557 72 10506738 10.1002/(SICI)1097-0274(199911)36:5<557::AID-AJIM8>3.0.CO;2-Q Doyle P Maconochie N Roman E Davies G Smith P Beral V Fetal deaths and congenital malformation in the offspring of nuclear industry employees: a report from the Nuclear Industry Family Study Lancet 2000 356 1293 1299 11073016 10.1016/S0140-6736(00)02812-9 Maconochie N Doyle P Davies G Lewis S Prior S Sampson P Pelerin M The study of reproductive outcome and the health of offspring of UK veterans of the Gulf war: methods and description of the study population BMC Public Health 2003 3 4 12523940 10.1186/1471-2458-3-4 Regan L Braude PR Trembath PL Influence of past reproductive performance on risk of spontaneous abortion BMJ 1989 299 541 5 2507063 Templeton A Infertility-epidemiology, aetiology and effective management Health Bull (Edinb) 1995 53 294 8 7490200 Roman E Doyle P Ansell P Bull D Beral V The health of children of radiographers in the UK Occup Environ Med 1996 53 73 79 8777454 Doyle P Roman E Beral V Brookes M Spontaneous abortion in dry cleaning workers potentially exposed to perchloroethylene Occup Environ Med 1997 54 848 53 9470891 Eurodirect Office for National Statistics Parliamentary electors on the 2000 and 2001 electoral registers – revised figures. United Kingdom; England; Wales; Scotland; Northern Ireland London 2001 Statacorp Stata Statistical Software: Release 8.0. College Station, TX: Stata Corporation 2003 Office for National Statistics Birth Statistics London: HMSO, 1970–2001 Series FM1 nos 13,18 27 30 Breslow NE Day NE Statistical methods in cancer research The analysis of cohort studies Lyons: International Agency for Research on Cancer 1980 II Olson JE Shu XO Ross JA Pendergrass T Robison LL Medical record validation of maternally reported birth characteristics and pregnancy-related events: a report from the Children's Cancer Group Am J Epidemiol 1997 145 58 67 8982023 Farrow A Farrow SC Little R Golding J The repeatability of self-reported exposure after miscarriage. ALSPAC Study Team. Avon Longitudinal Study of Pregnancy and Childhood Int J Epidemiol 1996 25 797 806 8921459 Yawn BP Suman VJ Jacobsen SJ Maternal recall of distant pregnancy events J Clin Epidemiol 1998 51 399 405 9619967 10.1016/S0895-4356(97)00304-1

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BMC Public Health. 2004 Aug 7; 4:35

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==== Front BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-4-371530602910.1186/1471-2458-4-37Research ArticleAssessing computer skills in Tanzanian medical students: an elective experience Samuel Miriam [email protected] John C [email protected] J Jaime [email protected] Rob [email protected] Eoin JW [email protected] Pejman [email protected] PRHO The Ipswich Hospital, Ipswich, Suffolk IP4 5PD, UK2 PRHO, Ealing Hospital, Middlesex UB1 3H, UK3 International Health Electives Co-ordinator, International Health and Medical Education Centre, University College London, London N19 5LW, UK4 PRHO, University College Hospital, London WC1E 6DB, UK5 Research Fellow, Centre for Health Informatics and Multiprofessional Education, University College London, London N19 5LW, UK2004 12 8 2004 4 37 37 26 4 2004 12 8 2004 Copyright © 2004 Samuel et al; licensee BioMed Central Ltd.2004Samuel et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background One estimate suggests that by 2010 more than 30% of a physician's time will be spent using information technology tools. The aim of this study is to assess the information and communication technologies (ICT) skills of medical students in Tanzania. We also report a pilot intervention of peer mentoring training in ICT by medical students from the UK tutoring students in Tanzania. Methods Design: Cross sectional study and pilot intervention study. Participants: Fourth year medical students (n = 92) attending Muhimbili University College of Health Sciences, Dar es Salaam, Tanzania. Main outcome measures: Self-reported assessment of competence on ICT-related topics and ability to perform specific ICT tasks. Further information related to frequency of computer use (hours per week), years of computer use, reasons for use and access to computers. Skills at specific tasks were reassessed for 12 students following 4 to 6 hours of peer mentoring training. Results The highest levels of competence in generic ICT areas were for email, Internet and file management. For other skills such as word processing most respondents reported low levels of competence. The abilities to perform specific ICT skills were low – less than 60% of the participants were able to perform the core specific skills assessed. A period of approximately 5 hours of peer mentoring training produced an approximate doubling of competence scores for these skills. Conclusion Our study has found a low level of ability to use ICT facilities among medical students in a leading university in sub-Saharan Africa. A pilot scheme utilising UK elective students to tutor basic skills showed potential. Attention is required to develop interventions that can improve ICT skills, as well as computer access, in order to bridge the digital divide. ==== Body Background Developments in information and communication technology occur at an astonishing rate. The World Wide Web (WWW) doubled in size during the first 6 months of 2000 and by 2005 the number of Internet users is likely to pass the one billion mark [1]. This has had huge implications for medical practice throughout the world. One estimate suggests that by 2010 more than 30% of a physician's time will be spent using information technology tools [2]. But these developments are occurring in a world that many of our colleagues cannot access. The International Labour Organization's World Employment Report for 2001 noted that barely 6% of people in the world had ever logged onto the Internet, and 85–90% of these are in the industrialized countries [3]. In September 2000, the digital divide was highlighted by the World Health Organization as 'more dramatic than any other inequity in health or income' [4]. In a world afflicted by poverty, debt and HIV, why has the digital divide continued to trouble so many academics and development policymakers? The basic concern is that the spread of information and communication technologies in developed countries is leaving the rest of the world behind. The development of online databases allows medical professionals throughout the developed world immediate access to hundreds of e-journals at the touch of a button, a striking contrast to the plight of many of their colleagues in developing countries who are forced to trawl empty libraries. Highlighting one of the greatest tragedies of the digital divide, it threatens the very communities that could benefit the most from the developments in ICT. Many programmes have concentrated on increasing the number and spread of telephones and computers [5]. Other schemes have minimised cost barriers to accessing Internet resources [6]. Beyond this classic access gap, several other factors have been identified as contributing to the divide. These include a gap in ability to use ICT, measured as the skills base; a gap in the actual use, measured as amount of time spent utilising ICT facilities [5]; and a gap in the impact of use, measured by financial, economic and clinical returns. In other words, equipment alone is useless unless people are able to use it effectively and informed of the potential benefits of its use. During January and February 2003 we studied ICT skills of medical students at Muhimbili University College of Health Sciences (MUCHS), University of Dar es Salaam, in Dar es Salaam, Tanzania. We also report a pilot intervention of peer mentoring training in ICT by medical students during the elective period. Methods Setting MUCHS is the largest medical school in east Africa and the only public medical school in Tanzania. A lack of resources has resulted in the university library being filled with out-dated textbooks. The majority of its graduates go on to serve rural communities throughout Tanzania and its neighbouring countries, often as the only qualified doctor serving populations of over 100,000. Access to computer facilities is a key problem. The University of Dar es Salaam is aiming for a ratio of one computer for every ten students. One-hundred-and-twenty computers would therefore be required for the 1200 MUCHS students. Help from international donors has allowed MUCHS to secure the presence of 40 computers, but only 25 are fully functional at any one time. Of these, less than half were connected to the Internet or loaded with basic software, bringing the real ratio of students to adequately equipped computers to around 100:1. Most MUCHS students found commercial Internet cafes too expensive to use on a regular basis. The cost of one hour at an Internet café can often be as high as $1, an important limiting factor considering that over 50% of an estimated 36 million people live in extreme poverty, surviving on less than US $1 per day [7]. Methods The abilities and attitudes of the fourth year MUCHS medical students (MD4s) towards ICT was assessed using Questionnaire 1 [see Additional File 1], an adapted version of a questionnaire developed by Jeannette Murphy [email protected] at the Centre for Health Informatics and Multiprofessional Education (CHIME, ) in London, UK, to assess ICT skills amongst first year medical students (MD1s) attending University College London (UCL). The questionnaires were distributed to all MD4 students by Tanzanian student representatives, to be filled in independently, and were then collected by the representatives. The questionnaire addressed different ICT-related variables associated with generic skills (Figure 1) and specific skills (Figure 2). A self-reporting assessment of competence (none, very basic, average or advanced, the equivalent of 0, 1, 2 or 3 points respectively) on several topics was evaluated as part of a generic ICT score. A specific ICT score aimed to address similar abilities (presence or not, 0 or 1 point) but asking about their abilities to perform such tasks. The generic ICT score has a range between 0 and 33 (11 items studied), and the specific one between 0 and 16 (16 items in total). Figure 1 Generic ICT Skills of 92 Medical Students at Muhimbili University College of Health Sciences. The data are shown as percentages of students reporting average and advanced competences. Figure 2 Specific ICT Skills of 92 Medical Students at Muhimbili University College of Health Sciences. The data are shown as percentages of students reporting to have the abilities to perform these tasks. Information related to frequency of computer use (hours per week) and years of computer use was also gathered. Access to computers at home or at educational facilities, last time of computer use, reasons for use and resources for reference in students' medical studies was also evaluated. The data were analysed using SPSS v.11 to calculate frequencies and percentages. Pearson Rho coefficient was used to look at the correlation between variables, with logarithmic transformation where necessary. Comparisons of frequencies before and after tutoring were performed using Wilcoxon's test. Data are shown as mean (± SD) for normally distributed data and median (interquartile [IQ] range) for skewed data. Results Ninety two (72.7%) of 120 Tanzanian MD4s completed the questionnaires. 76% of them did not have a computer at home and 74% never use a computer as part of any course either at school or university. Only 48 students (52%) felt that they understood the basic terminology and concepts of computing. The mean (± SD) of the generic and specific ICT scores were 11.1 (± 7.6) (out of a maximum score of 33) and 7.7(± 4.1) (out of a maximum score of 16) respectively. The two scores were significantly correlated, r = 0.81, p < 0.001. The highest levels of competence, assessed using the generic ICT score, were for email, Internet and file management (see Figure 1). For the remaining items most respondents reported low levels of competence. Over 60% of the Tanzanian students in each of the generic areas indicated that they had taught themselves these skills. Of the remainder most students had learnt the skills at school, with a small number learning them at work. The results for the tasks evaluated in the specific ICT score are shown in Figure 2. Of 16 skills evaluated, only 2 (12.5%) were present in 90% of the participants. The majority of skills were found in between 40 and 60% of participants. The majority of the students claimed to use the available computers very regularly, 25% of students using them daily and nearly two-thirds at least once a week. The median hours per week of computer use was 3.8 (2–10). The median years of computer use was 3 (2–5) years. The main reasons for using a computer during the last year was to communicate by email (75%), Internet navigation (33%), learning purposes (27%), and to prepare reports (22%). Only 21 students (23%) had ever consulted an electronic journal, and nearly 70% did not use any electronic resource. The participants unanimously agreed that medical students should receive training in the use of computers. Piloting a peer mentoring training scheme During our time in Tanzania we piloted an ICT peer mentoring training scheme aimed at the fourth year Tanzanian medical students whose skills had been assessed. We utilized tutorials that had been developed by CHIME for underskilled UCL medical students [8]. Topics covered included file management, Internet, email, word processing, Excel and PowerPoint. The willingness of the Tanzanian students to participate was identified as vital to the success of the scheme, since one of our main concerns was that we would we be seen as a neo-colonial force trying to impose our values. This was not the case: the response from the students was overwhelmingly positive. The majority of those who returned the questionnaires wished to receive peer mentoring training. Four tutorials (1.0–1.5 hours) were conducted utilising MUCHS computers. The course covered computing skills such as file management, word processing, spreadsheet use, Internet access and email. Competence levels were compared for 12 students before and after 4 to 6 hours of peer mentoring training using Questionnaire 2 [see Additional File 2]. The generic ICT score in these students increased from 8.8(+5.2) to 18.4 (+4.8) out of a maximum score of 33 (p = 0.007). The specific ICT score increased from 6.8 (+3.6) to 12.1(+2.2) out of a maximum score of 16 (p = 0.003). There were significant improvements (p < 0.05) in 7 of the 11 components of the generic ICT score and 5 of the 16 items of the specific score. Discussion We have assessed ICT competence of a representative sample of 4th year medical students in a Tanzanian medical school and have found substantial limitations in computing skills. A mean generic score of 11.1 would be equivalent to approximately 1 point (very basic) in each of the 11 skills studied. The corresponding score among first year UK medical students in 2002 was 18.5. Initially at UCL, students with an overall score of less than 10 are considered to have low skills and are offered peer mentoring training. Using this criterion, around 50% of the Tanzanian students would fall into the low skills category compared with 9% of first year UCL medical students in 2002. Scores amongst UCL medical students have been rising in recent years (J Murphy, personal communication). The mean of the specific ICT score was 7.7, in comparison with 11.9 for first year UCL medical students. (J Murphy, personal communication). The strong correlation between generic and specific scores suggests that the students were good at judging their own ability, thus decreasing a potential bias from self-assessment in our results. Two studies from Nigeria show that there is poor knowledge of computer use. Ajuwon [9] reports that only 42.6% of the sample studied could use the computer. In Lagos [10], 79% of students had little or no computer skills. Although we did not evaluate self-reporting ability to use a computer, we can assume that this figure is similar considering the low averages of the scores reported in our study. The Internet was the most common application utilised in our sample in Tanzania, especially for email communications (75%). This is concordant with the reports from Nigeria where 76.4% of first year clinical and nursing students in Ibadan [9] and 58% of final year medical and dental students in Lagos [10] have used email. This high rate of Internet and email use amongst medical students is also similar in other countries, such as Denmark [11], Finland [12], India [13], Malaysia [14] and the United Kingdom [15]. Around half of the Tanzanian students were able to print a document, cut and paste information from one application to another and word process an essay, letter or their CV. These results, clearly addressed through items in the specific ICT score, show us that even though the students are able to communicate by email and use the Internet, they cannot perform some basic skills necessary for working with files. The lack of confidence in use of packages such as PowerPoint and Excel was also noticeable in the reported score and in the reported reasons for using the computer. The median duration of computer use was 3 years, meaning that most doctors in training had their first contact with a computer at university. Access is one of the main issues when one considers the fact that the current student/computer ratio at MUCHS is 100:1, compared to 35:1 in Portugal, 9:1 in the UK and 5:1 in Norway [16]. Seventy-six percent of students in our sample did not have a computer at home. This figure is in stark contrast with the 71.7% of first year medical students that do have access to a computer at home in Aarhus, Denmark [11] and 86% in California, USA [17]. These findings have substantial implications for addressing the digital divide in this population. These range from a marked limitation of knowledge of basic packages to the possibility of making incorrect assumptions that students who are able to communicate electronically are automatically able to perform basic tasks such as word processing. Graduates recruited by foreign institutions to pursue postgraduate training would face additional difficulties in their learning experience due to their problems with ICT. Also important is the fact that several academic institutions are now looking to expand their teaching programmes using online courses. Again, course organisers could erroneously enrol participants with a lack of basic ICT skills, based solely on the ability to communicate by email. The peer mentoring training piloted in the present study shows promising results, achieving a doubling of competence scores. This programme [18,19], which uses foreign medical students visiting MUCHS to teach local ones, is a cheap intervention and feasible to replicate in the same context, and perhaps in other countries, in a similar way to the buddy system that has been suggested by Ajuwon in Nigeria [9]. Conclusions Our study has found a low level of ability to use ICT facilities among medical students in one university in sub-Saharan Africa. These findings reinforce the idea that more is needed to bridge the digital divide than simply increasing the number of computers. A pilot scheme utilizing visitors from the developed world to tutor basic skills showed potential, but it would be naïve to think that volunteers alone can bridge the gap. Some would argue that increasing the number and distribution of computers will eventually result in an improved skills base. Our experiences would support this. Tanzanian medical students are keen to learn new skills and are aware of the implications of being left behind in the technological revolution. The main concern of such a scenario is that without direct intervention the time required to attain these skills increases, to the further detriment of some of the world's most vulnerable societies. Competing interests None declared. Authors' contributions JCC, RM, MS, EJWY and JJM took part on the initial planning of the project. JCC, RM, MS and EJWY did the field work and the peer mentoring training to Tanzanian medical students. PA and JJM did the data input and analysis. MS, JJM and JCC wrote the initial version of the paper. All the authors contributed to subsequent versions of the paper. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Questionnaire 1 Instrument used to assess baseline ICT skills amongst MUCHS medical students. ©Jeannette Murphy Click here for file Additional File 2 Questionnaire 2 Instrument used to assess skills after the ICT peer mentoring training amongst MUCHS medical students. © Jeannette Murphy Click here for file Acknowledgements Our sincere appreciation to Professor John S Yudkin, IHMEC for his support in the preparation of this manuscript; to Jeannette Murphy, CHIME for discussions on the methodology and for providing us with the questionnaires and materials for the peer mentoring training; and to Nick Wright for his input on early stages of this process. We would like to express also our gratitude to the MUCHS community. The full version of the questionnaires used is available from Jeannette Murphy [email protected] and the course materials for the peer mentoring training are available at . Ms. Jeannette Murphy is the Copyright holder for the questionnaires used in this work. ==== Refs Larkin M Non-US web use rising fast Lancet 2000 356 1451 Skinner H Biscope S Poland B Quality of internet access: barrier behind internet use statistics Soc Sci Med 2003 57 875 80 12850112 10.1016/S0277-9536(02)00455-0 International Labour Organization The World Employment Report 2001: Life at work in the information economy 2001 Geneva: ILO Accessed July 4, 2004 Edejer TT Disseminating health information in developing countries: the role of the internet BMJ 2000 321 797 800 11009519 10.1136/bmj.321.7264.797 Fink C Kenny CJ W(h)ither the digital divide? Unpublished paper 2001 Accessed July 4, 2004 Kmietowicz Z Deal allows developing countries free access to journals BMJ 2001 323 65 11451770 10.1136/bmj.323.7304.65 USAID Africa: Tanzania Accessed July 4, 2004 Centre for Health Informatics and Multiprofessional Education, University College London IT skills for medical students: Course Materials Accessed July 4, 2004 Ajuwon GA Computer and internet use by first year clinical and nursing students in a Nigerian teaching hospital BMC Med Inform Decis Mak 2003 3 10 14498997 10.1186/1472-6947-3-10 Odusanya OO Bamgbala OA Computing and information technology skills of final year medical and dental students at the College of Medicine University of Lagos Niger Postgrad Med J 2002 9 189 93 12690677 Dorup J Experience and attitudes towards information technology among first-year medical students in Denmark: longitudinal questionnaire survey J Med Internet Res 2004 6 e10 15111276 Virtanen JI Nieminen P Information and communication technology among undergraduate dental students in Finland Eur J Dent Educ 2002 6 147 52 12410665 10.1034/j.1600-0579.2002.00251.x Inamdar SC Rotti SB Computer use among medical students in an institution in southern India Natl Med J India 2004 17 8 10 15115224 Nurjahan MI Lim TA Yeong SW Foong AL Ware J Utilization of information technology in medical education: a questionnaire survey of students in a Malaysian institution Med J Malaysia 2002 57 58 66 12733195 Asgari-Jirhandeh N Haywood J Computer awareness among medical students: a survey Med Educ 1997 31 225 9 9231143 James E Learning to bridge the digital divide OECD Observer 2001, January 14 Jerant AF Lloyd AJ Applied medical informatics and computing skills of students, residents, and faculty Fam Med 2000 32 267 72 10782374 Young E Melvin R Coombes J Samuel M Elect to teach Student BMJ 2004 12 36 Young E Melvin R Samuel M Coombes J Peer mentoring in ICT: UK medical students sharing skills in developing countries Health Informatics Europe March 10, 2004. Accessed July 4, 2004

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BMC Public Health. 2004 Aug 12; 4:37

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10.1186/1471-2458-4-37

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==== Front BMC Musculoskelet DisordBMC Musculoskeletal Disorders1471-2474BioMed Central London 1471-2474-5-251529196110.1186/1471-2474-5-25Research ArticleDoes preoperative abduction value affect functional outcome of combined muscle transfer and release procedures in obstetrical palsy patients with shoulder involvement? Aydin Atakan [email protected] Turker [email protected] Defne [email protected] Department of Plastic and Reconstructive Surgery, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey2004 3 8 2004 5 25 25 19 1 2004 3 8 2004 Copyright © 2004 Aydin et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Obstetric palsy is the injury of the brachial plexus during delivery. Although many infants with plexopathy recover with minor or no residual functional deficits, some children don't regain sufficient limb function because of functional limitations, bony deformities and joint contractures. Shoulder is the most frequently affected joint with internal rotation contracture causing limitation of abduction, external rotation. The treatment comprises muscle release procedures such as posterior subscapularis sliding or anterior subscapularis tendon lengtening and muscle transfers to restore the missing external rotation and abduction function. Methods We evaluated whether the preoperative abduction degree affects functional outcome. Between 1998 and 2002, 46 children were operated on to restore shoulder abduction and external rotation. The average age at surgery was 7.6 years and average follow up was 40.8 months. We compared the postoperative results of the patients who had preoperative abduction less than 90° (Group I: n = 37) with the patients who had preoperative abduction greater than 90° (Group II: n = 9), in terms of abduction and external rotation function with angle measurements and Mallet classification. We inquired whether patients in Group I needed another muscle transfer along with latissimus dorsi and teres major transfers. Results In Group I the average abduction improved from 62.5° to 131.4° (a 68.9° ± 22.9°gain) and the average external rotation improved from 21.4° to 82.6° (a 61.1° ± 23°gain). In Group II the average abduction improved from 99.4°to 140°(a40.5° ± 16°gain) and the average external rotation improved from 33.2°to 82.7° (a 49.5° ± 23.9° gain). Although there was a significant difference between Group I and II for preoperative abduction (p = 0.000) and abduction gain in degrees (p = 0.001), the difference between postoperative values of both groups was not significant (p = 0.268). There was also no significant difference between the two groups in the preoperative external rotation, the external rotation gain and the postoperative external rotation (p = 0.163, p = 0.181 and p = 0.803, respectively). Conclusions Obstetric palsy patients with shoulder sequela who had a preoperative abduction less than 90°hadas good functional results using latissimus dorsi, teres major muscle transfer and subscapularis muscle release as the patients who hada preoperative abduction greater than 90°. ==== Body Background Although the extent and severity of the deformity in obstetrical palsy may vary from patient to patient, shoulder is the most frequently affected joint. Many clinical and radiological classification systems had been described to address the problem and tailor the solution at the shoulder [1-5]. Among deformities affecting this joint, internal rotation contracture causing limitation of abduction and external rotation and dislocation of the shoulder is commonly observed on the follow up [4]. The shoulder instability was not caused directly by obstetrical trauma but related to a dynamic phenomenon of muscle imbalance. It was discovered that subscapularis muscle usually recovers more quickly than the external rotators and abductors [6,7]. Despite initial conservative therapy, patients with partial recovery may develop internal rotation and adduction contractures at the shoulder. If this persists long enough, flattening of the humeral head and joint incongruence will ensue. Under these conditions the main goal of the treatment is to reestablish the muscle equilibrium. The first step is to treat the muscle release such as the posterior subscapularis slide or the modified anterior subscapularis tendon lengtening procedures. Afterwards, muscle transfer is performed to restore the missing external rotation and abduction function in patients with congruent glenohumeral joint and limited glenohumeral deformity. However external rotational humeral osteotomy is preferred in patients with similar external rotation weakness, internal rotation contracture but with severe glenohumeral deformity [1]. Usually release and transfer procedures are seperated depending on the age of the patient and condition of the shoulder but in older age group (>4 years), it is preferred to combine the shoulder release and muscle transfer simultaneously [6-8]. Some authors perform other muscle transfers like levator scapulae and trapezius along with latissimus dorsi and teres major only for total flail shoulders [9], others claim that if the preoperative abduction degree of the shoulder is less than 90° indicating a week deltoid and external rotator muscles, a trapezius muscle transfer must be added to latissimus dorsi and teres major transfers to achieve significant improvement of both abduction and external rotation [10]. In this study, we compared the results of combined release and tendon transfer operations performed at the same stage because of the late presentation of the cases, for shoulder abduction and external rotation in two groups of patients who did not have any surgical treatment before. In one group preoperative abduction degree was less than 90°, while in the other group it was equal or more than 90°. We compared the gains of abduction and external rotation in two groups and evaluated whether the preoperative abduction degree affects the functional outcome and in patients with preoperative abduction degree <90° and also another muscle transfer is needed along with latissimus dorsi and teres major transfers. Methods General data about the patients Between 1998 and 2002, 46 children with obstetrical brachial plexus palsy who had no surgical treatment before (30 male and 16 female) were operated to restore shoulder abduction and external rotation in our clinic. The average age at surgery was 7.6 years (3–16). The patients had an average follow up of 40.8 months (range 24 to 60 months). Involvement of right side was seen in 30 patients and the left side in 16 patients. No patient had bilateral involvement. All of the patients were vaginally delivered with vertex presentations. Obstetrical history revealed that most mothers were multiparous and 8 of the patients were delivered with the help of forceps or vacuum. The mean birth weight of the patients was 4.5 kg (3–6.6 kg). The pool of the patients consisted 12 of patients with C5–C6 spinal root involvement, while 11 of the patients had additional C7 involvement. Finally 23 of the patients had total brachial plexus roots involvement. Accompanying birth complications were fracture of clavicle in one case, injury of sternocleidomastoideus muscle in one case and Horner's Syndrome in 2 cases. Table 1 [see Additional file 1] summarizes the specific qualifications, preoperative and postoperative evaluation values of all patients. We compared the postoperative results of the patients who had preoperative abduction less than 90° [Group I: n = 37, mean abduction 62.5° (20°–85°) and mean external rotation 21.4° (0–80°)] with the patients who had preoperative abduction equal or more than 90° [Group II: n = 9, mean abduction 99.4° (90–110) and mean external rotation 33.2° (0–65)], in terms of abduction and external rotation function with angle measurements and Mallet classification. The statistical analysis was performed with analysis of variance (ANOVA) and Student's t-test. Statistical significance was presumed at p < 0.05. Preoperative and postoperative evaluation Preoperative and postoperative active and passive range of motion degrees of abduction and external rotation were measured, videos were recorded during shoulder abduction and external rotation and Mallet scores (Figure 1) were noted. Abduction degree is measured at standing position and external rotation is measured in prone position with 90° shoulder abduction and at 90° elbow flexion. Radiography of the shoulder in adduction and 90° abduction and axial magnetic resonance imaging of the shoulder was performed. Shoulder deformity was classified according to Waters-Peljovich grading system, Table 2, and the patients with type I and type II deformities were included in the series [1]. Most of the times, preoperative physical examination revealed weakness of the deltoid muscle and external rotators as well as co-contraction of pectoralis major, latissimus dorsi and teres major muscles at the anterior and posterior margin of the axillary fossa during shoulder elevation specially in Group I patients. Operative technique In our series we used a technique similar to the Hoffer technique [8]. We observed tightness and/or hypertrophy of latissimus dorsi, teres major, subscapularis and sometimes pectoralis major muscles intraoperatively. We performed subscapularis muscle release and the latissimus dorsi and teres major muscle transfer at the same session since the mean age at surgery was 7.6 years. Patients were placed in the lateral decubitus position and conjoined tendon of latissimus dorsi and teres major was explored with a posterior zigzag incision parallel to the lateral border of scapula to prevent scar contracture (Figure 2). Extensive dissection of the conjoined tendon and the related muscles from the surrounding structures was needed so that the conjoined tendon could reach to its new insertion easily. While preparing the muscles for transfer care must be taken not to injure the pedicles, which were located at medial side of these muscles (Figure 3). The next step was detachment of the conjoined tendon from its insertion on the inner side of the humerus by retracting the neurovascular structures of the arm superiorly. Afterwards a tunnel was prepared between long head of triceps and deltoid muscle with an extensive care to prevent injury to the axillary nerve. Rotator Cuff Quick Anchor® Plus (Johnson & Johnson) which had size 2 green ethibond polyster sutures on it, was applied at the insertion point of infraspinatus muscle on the rotator cuff (Figure 4). The suture material was transferred to the posterior incision from the tunnel and the conjoint tendon was interwoven with this suture material. Then conjoined tendon was transferred to the posterior deltoid incision. Finally, reinsertion of the conjoined tendon to humerus was achieved while the arm was at 90° abduction and full external rotation (Figure 5). Fractional tenotomy or Z plasty procedure was applied to the pectoralis major tendon, if necessary, through an anterior axillary incision or by retracting posterior incision superiorly. Subscapularis muscle was released from the anterior surface of the scapula subperiostally from a small incision at the lateral side of the scapula carefuly, in order to prevent injury to the pedicle of lattisimus dorsi and teres major muscles (Figure 6). A cast, stabilizing the shoulder at 90° abduction and 90° external rotation and elbow at 90° flexion was applied for five weeks and physiotherapy was started after the removal of the cast under the control of custom-made splint. Results The average abduction degree improved from 62.5° (20°–85°) to 131.4° (90°–165°) and the average external rotation degree improved from 21.4° (0°–80°) to 82.6° (30°–95°) in Group I. We obtained 68.9° ± 22.9° (109%) gain for abduction and 61.1° ± 23° (285%) gain for external rotation. The difference between preoperative and postoperative values of abduction and external rotation was significant (ANOVA, F = 265 p = 0.000, F = 201 p = 0.000, respectively). The average abduction degree improved from 99.4° (90°–110°) to 140° (110°–170°) and the average external rotation degree improved from 33.2° (0°–65°) to 82.7° (45°–90°) in Group II. We obtained 40.5° ± 16° (40%) gain for abduction and 49.5° ± 23.9° (149%) gain for external rotation. The difference between preoperative and postoperative values of abduction and external rotation was significant (ANOVA, F = 25 p = 0.000 and F = 32.3 p = 0.000, respectively). The results were summarized in Table 3. Although difference between preoperative abduction and abduction gain values in terms of degrees of Group I and Group II were significant (ANOVA, F= 43.1 p = 0.000 and F = 12 p = 0.001, respectively), the difference between postoperative values of both groups was insignificant (ANOVA, F = 1.257 p = 0.268). There was also no significant difference between the preoperative external rotation, the external rotation gain and the postoperative external rotation for both groups (ANOVA, F = 2.017 p = 0.163, F = 1.848 p = 0.181 and F = 0.063 p = 0.803, respectively). The results of both groups were shown in graphics in Figure 7. Mean Mallet scores increased from 2.8 to 3.9 for global abduction, from 2.5 to 3.9 for global external rotation, from 2.1 to 3.6 for hand to head and from 2.5 to 3.5 for hand to mouth for Group I. Hand to back Mallet score decreased from 2.5 to 2.2. All the differences between preoperative and postoperative values were found significant according to the Student's t-test (t = -17.14 p = 0.000 for abduction score, t = -12.04 p = 0.000 for external rotation score, t = -13.06 p = 0.000 for hand to head score, t = 2.372 p = 0.023 for hand to back score and t = -7.361 p = 0.000 for hand to mouth score). Mean Mallet scores increased from 3.5 to 4 for global abduction, from 2.8 to 4 for global external rotation, from 2.7 to 4 for hand to head and from 3.2 to 3.5 for hand to mouth for Group II. Hand to back Mallet score decreased from 2.8 to 2.1. All the differences, apart from hand to mouth score, between preoperative and postoperative values were found significant according to the Student's t-test (t = -2.53 p = 0.035 for abduction score, t = -3.592 p = 0.007 for external rotation score, t = -4.4 p = 0.002 for hand to head score and t = 2.8 p = 0.023 for hand to back score). The difference between preoperative and postoperative hand to mouth scores was found insignificant (t = -2, p = 0.081). While the difference between preoperative Mallet scores of Group I and Group II concerning abduction, hand to head and hand to mouth was significant (ANOVA, F = 23.211 p = 0.000, F = 11.407 p = 0.002 and F = 7.692 p = 0.008, respectively), the difference was insignificant for external rotation and hand to back scores (ANOVA, F = 1.393 p = 0.244 and F = 2.475 p = 0.123, respectively). The difference between postoperative values of both groups was insignificant (ANOVA, F = 0.239 p = 0.627 for abduction, F = 0.76 p = 0.388 for external rotation, F = 2.764 p = 0.106 for hand to head, F = 0.354 p = 0.555 for hand to back and F = 0.363 p = 0.555 for hand to mouth). The results were summarized in Table 4. Example cases from each group can be seen in Figure 8 and 9. Discussion Internal rotation contracture is the most frequent and important secondary deformity of the shoulder in birth palsy. The problem is sometimes addressed by muscle release procedures such as the posterior subscapular slide or an anterior subscapularis tendon lenghtening operations. Once passive external rotation is improved, the child is later assessed for muscle transfers to reconstruct active external rotation if necessary [4]. According to Chang et al [5] there are two types of residual muscle impairment after recovery in the late obstetric brachial plexus palsy: motor recovery with cross-innervation and paralysis or paresis. Contractures of the pectoralis major, teres major, brachialis and biceps muscles, which are most frequently observed, cause the deformity of the shoulder and elbow. The reconstructive strategy include releasing of the antagonistic muscles (elongation of the pectoralis major and latissimus dorsi muscles) and augmentation of the paretic muscles (teres major transfer to the infraspinatus muscle for augmentation of shoulder external rotation and abduction and reinsertions of both ends of the clavicular part of the pectoralis major laterally for deltoid augmentation). However, there are still many controversies concerning donor muscle choice for transfer, timing and operative tecniques of palliative surgical theraphy for the shoulder deformity. The infraspinatus muscle works to center the humeral head in the glenoid throughout elevation. External rotation of the shoulder allows greater arm elevation by clearing the greater tuberosity from impingement by the coracoacromial arch. External rotation of the humerus also positions the long head biceps centrally to aid in its function as humeral stabilizer and loosens the inferior glenohumeral ligaments, thereby allowing greater arm elevation. Hence the infraspinatus muscle plays a key role in shoulder elevation as a humeral head stabilizer, an active abductor, and an external rotator of the shoulder [6]. The importance of transferring the teres major and latissimus dorsi as one conjoined tendon and anchoring into the posterior aspect of the greater tuberosity at the insertion of the infraspinatus similar to Hoffer method is augmentation of the weakened infraspinatus. Transfer with this technique instead of rerouting around humeral neck enables a stronger external rotator power because of the increased mechanical advantage at its insertion in the humeral head as opposed to the humeral shaft. The reason for the dramatic improvement of shoulder abduction after latissimus muscle transfer is probably because the transfer enhances the stabilizing effect of the rotator cuff which enables the deltoid to act more effectively, this phenomenon was called "force couple" effect by Phipps and Hoffer [11]. In many centers, muscle release procedures are performed before the age of two years, however for older children tendon transfer to restore abduction and external rotation is added [12]. It is accepted that the corrective procedures to rebuild the muscle equilibrium are best undertaken before permanent bony deformity occurs at 3 to 4 years of age [13]. Gilbert [14] suggested that release of the subscapularis is indicated if the external rotation does not improve more than 20°. Based on his 5 years of follow up, he reported excellent results after subscapularis release especially in patients before the age of 2 years. Raimondi also waits for the active external rotation due to the reinforcement of weak external rotator muscles after subscapular muscle release procedure in early ages but since recovery of the external rotators cannot be expected, he preferres the tendon transfer and muscle release operations at the same time in children older than 4 years of age [9]. Muhlig et al [12] described a common policy accepted by most of the centers. According to this; if passive external rotation of the shoulder stays < 30°, surgical treatment is indicated. If there is no posterior displacement of the humeral head than a subscapular slide will be used. However, if there is posterior displacement of the humeral head than subscapular lengthening by an anterior approach will be preferred. Indications for tendon transfer for improving external rotation and abduction are determined as well. If infraspinatus muscle does not show signs of reinnervation by the age of 2 years, a muscle transfer should be added to the subscapularis lengthening to avoid recurrence. If there is a fixed medial rotation contracture and posterior luxation of the humeral head with deformities of the glenoid than derotational osteotomy of the humerus should be added to the subscapularis lengthening. As all of our patients were older than 2 years of age, we performed latissimus dorsi and teres major transfer at the same session with subscapularis and pectoralis major release. In total flail shoulders, despite a certain degree of innervation, the functional results of shoulder corresponds to zero with the absence of a strong latissimus dorsi. In that condition, the levator scapulae muscle is utilised as an intrinsic stabilizer of glenohumeral joint and trapezius muscle is used as a prime mover for shoulder abduction with or without latissimus dorsi and teres major transfers [9]. Gilbert in his series of 44 patients with transfer of latissimus dorsi, the improvement of abduction was satisfactory in the shoulders which preoperatively coded as Grade III (Shoulder abduction is between 90°–120°, external rotation is between 0°–30°) or more, but not in those coded as Grade II (Shoulder abduction is between 45°–90°, external rotation is to neutral) or less. Hence he thought it may be necessary to add a concomitant transfer of the trapezius to the patients whose abduction of the shoulder was weak or absent [15]. Chen et al [10] asserted the need for an additional trapezius muscle transfer for shoulder of the patients who had less than 90° abduction to increase the success of the classic latissimus dorsi + teres major transfer. They transferred latissimus dorsi by fixing its tendon to the insertion of the infraspinatus and tenotomized the teres major and then attached to the belly of the latissimus dorsi and found out in their early stage of treatment that, 10 of 18 cases with abduction less than 90°, with transfer of the latissimus dorsi and the teres major, patients gained no improvement of abduction but some recovery of external rotation, while five of seven patients with abduction equal or more than 90°, made significant progress in both abduction and external rotation. Al-Qattan [16] performed latissimus dorsi transfer on 12 children with variable preoperative shoulder abduction (range 60–150°, mean 100°) and postoperatively ten children achieved a modified Mallet score of 4 and were able to reach the occiput easily and they had mean 140° active shoulder abduction (range 90–170°). Hence the author also did not find any difference in patients with weak or strong preoperative abduction. It is our opinion that in our Group I patients, the co-contraction between shoulder abductors (supraspinatus, infraspinatus and deltoid) and adductors (mainly, pectoralis major, teres major and latissimus dorsi) and also subscapularis muscle tightness cause limitation of shoulder elevation. If antagonistic muscles (teres major and latissimus dorsi) are transferred for the paretic muscles (infraspinatus) and the pectoralis major and subscapularis muscles are released with preserving their shoulder stability function, these children can have as succesfull postoperative shoulder abduction and external rotation as the children in Group II, who has less cross innervation hence better preoperative abduction value. Group I and II patient had almost similar postoperative mean abduction (131.4° & 140°, respectively) and external rotation values (82.6° & 82.7°, respectively). Extensive dissection of latissimus dorsi and teres major muscles from the surrounding structures gave us the opportunity to utilize both muscles for transfer, without any difficulty during the passage of the conjoined tendon through the tunnel which was prepared between long head of triceps and deltoid muscle, and also during reinsertion to the humerus. Several authors reported recurrences of the deformity in terms of reduction of external rotation and abduction gain. Two of the 12 children in Al-Qattan series [16] and three of 35 cases in Phipps and Hoffer series [11] had recurrence of the deformity. Al-Qattan [16] classified the possible cause of this late complication as recurrence of the internal rotation contracture (mainly in the subscapularis), gradual contracture of the teres major as part of the inferior glenohumeral angle contracture and co-contraction of the muscles. We did not have any recurrence of the deformity during the follow-up period which may be related to the use of rigid fixation with bone anchors for reinsertion of the conjoint tendon. We believe that in cases who has congruent glenohumeral joint (Type I-III Waters-Peljovich grading system), and deltoid muscle strength of M3–M4 (British Medical Research Council evaluation) but weak or absent external rotation, if the latissimus dorsi and teres major muscles have sufficient strength (M3 or more), the ideal procedure is transfer of latissimus dorsi and /or teres major onto the posterior aspect of the greater tuberosity of humerus, at the insertion of the infraspinatus. So we are not totally convinced about adding trapezius muscle transfer concomitantly with the latissimus dorsi + teres major transfer session. We rather preserve this muscle for the patients that could not achieve enough shoulder abduction after the first operation. Conclusions Since our study was not randomised to treatment, the groups did not comprise equal number of patients, the assessments were not performed by independent observers but by a physiotherapy group of our team at postoperative 24 – 60 months (not at the same time for everyone), we would like to interpret our conclusions cautiously. Almost near normal shoulder function can still be reached in children who could not receive primary early neural reconstruction, by combined muscle release and muscle transfer operations which are performed before the severe glenohumeral deformities occur. We found out that the patients with obstetric palsy shoulder sequela who had a preoperative abduction value less than 90° could have good functional results as the patients who had preoperative abduction values equal or more than 90°, with latissimus dorsi, teres major muscle transfer and subscapularis muscle release. Competing interests None declared. Authors' contributions All authors participated in the design of the study, operations and drafting the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Preoperative and postoperative range motion degrees and Mallet scores of the patients. Patients with bold numbers are in Group II with preoperative abduction values ≥ 90° and the others are in Group I with preoperative abduction values < 90° (Abd Deg: abduction degree, Ex. Rot. Deg: external rotation degree). Passive range of motion degrees are in parenthesis. Click here for file Figures and Tables Figure 1 Mallet classification. Grade 0 indicates no movement in the desired plane, Grade V is full movement. Figure 2 Operative incision. Posterior zigzag incision parallel to the lateral border of scapula is planned for preparing the conjoined tendon of latissimus dorsi and teres major. Figure 3 Conjoined tendon preperation. The conjoined tendon is ready for the transfer and its pedicle is pointed medial side of the muscles. Arrow indicates muscle pedicle. Figure 4 Rigid fixation. Rotator Cuff Quick Anchor which had size 2 with green ethibond polyster sutures on it, was applied at the insertion point of infraspinatus muscle. Figure 5 Tendon reinsertion. The conjoined tendon was transferred to the posterior deltoid incision and reinsertion of the conjoined tendon to humerus was achieved while the arm was at 90° abduction and full external rotation. Figure 6 Subscapular muscle release. Subscapularis muscle is released from the anterior surface of the scapula subperiostally. Figure 7 Preoperative and postoperative abduction (abd) and external rotation (ext rot) range of motion degrees of Group I and Group II patients. Figure 8 An example case from Group I, Case 6 in Table 1: 7 years old girl with left brachial plexus palsy. (a), (b) Anterior preoperative views of the patient while performing abduction and external rotation of her left arm reveals co-contraction of pectoralis major, latissimus dorsi and teres major muscles at the anterior and posterior margin of the axillary fossa. Preoperative abduction degree was 60° and external rotation degree was 45°. (c) Anterior postoperative view of the patient while performing abduction. (d) Lateral postoperative view of the patient while placing her hand at the nape of her neck. Postoperative abduction degree is 135° and external rotation degree is 87°. Figure 9 An example case from Group II, Case 38 in Table 1: 5 years old boy with left brachial plexus palsy. (a), (b) Anterior views of the patient while performing abduction of his both arms. Preoperative abduction degree is 110° and external rotation degree is 24°. (c) Anterior postoperative view of the patient while performing abduction. (d) Lateral postoperative view of the patient while placing his hand at the nape of his neck. Postoperative abduction degree is 170° and external rotation degree is 90°. Table 2 Radiographic classification of glenohumeral deformity (Waters and Peljovich). Type I Normal glenoid (<5° retroversion difference versus contralateral normal) Type II Minimal deformity (>5° retroversion difference) Type III Moderate deformity (posterior humeral head subluxation) Type IV Severe deformity (posterior humeral head subluxation with false glenoid) Type V Severe flattening of humeral head and glenoid +/- complete dislocation Type VI Infantile glenohumeral dislocation Type VII Proximal humeral growth arrest Table 3 Abduction and external rotation evaluation of patients in Group I and Group II. Preoperative Abduction Postoperative Abduction Abduction Gain Preoperative External Rotation Postoperative External Rotation External Rotation Gain Group I (n = 37) 62.5°# (20°–85°) 131.4° * (90°–165°) 68.9° ± 22.9 # (109%) 21.4° (0–80°) 82.6° * (30°–95°) 61.1° ± 23° (285%) Group II (n = 9) 99.4°# (90°–110°) 140° * (110°–170°) 40.5° ± 16°# (49.5%) 33.2° (0–65°) 82.7° * (45°–90°) 49.5° ± 23.9° (149%) "*" sign represents significant difference between pre and postoperative values in the groups and "#" sign represents significant difference between Group I and Group II. Table 4 Preoperative and postoperative Mallet scoring. Mallet Classification Abduction External Rotation Hand to Head Hand to Mouth Hand to Back Preop Postop Preop Postop Preop Postop Preop Postop Preop Postop Group I (n = 37) 2.8# 3.9* 2.5 3.9* 2.1# 3.6* 2.5# 3.5* 2.5 2.2* Group II (n = 9) 3.5# 4* 2.8 4* 2.7# 4* 3.2# 3.5 2.8 2.1* "*" sign represents significant difference between pre and postoperative values in the groups and "#" sign represents significant difference between Group I and Group II. ==== Refs Waters PM Allan E Peljovich E Shoulder reconstruction in patients with chronic brachial plexus birth palsy Clin Orthop 1999 364 144 152 10416403 10.1097/00003086-199907000-00019 Zancolli EA Classification and management of the shoulder in birth palsy Orthop Clin North Am 1981 12 433 457 7243246 Birch R Gilbert A Medial rotation contracture, posterior dislocation of the shoulder (Ed) Brachial plexus injuries 2001 Hampshire (UK), Taylor & Francis 249 259 Al-Qattan MM Classification of secondary shoulder deformities in obstetric brachial plexus palsy J Hand Surg [Br] 2003 28 483 6 12954262 10.1016/S0266-7681(02)00399-6 Chuang DC Ma HS Wei FC A new strategy of muscle transposition for treatment of shoulder deformity caused by obstetric brachial plexus palsy Plast Reconstr Surg 1998 101 686 94 9500384 Price AE Grossman AI A management approach for secondary shoulder and forearm deformities following obstetrical brachial plexus injury Hand Clin 1995 11 607 617 8567742 Egloff DV Raffoul W Bonnard C Stalder J Palliative surgical procedures to restore shoulder function in obstetric brachial palsy Hand Clin 1995 11 597 606 8567741 Hoffer M Wickenden R Roper B Brachial plexus birth palsies. Results of tendon transfers to the rotator cuff J Bone Joint Surg 1978 60(A) 691 695 681392 Raimondi PL Muse A Saporiti E Gilbert A Palliative surgery: shoulder paralysis Brachial plexus injuries 2001 London, Martin Dunitz Ltd 225 238 Chen L Gu Y Hu S Applying transfer of trapezius and/or latissimus dorsi with teres major for recontruction of abduction and external rotation of the shoulder in obstetrical brachial plexus palsy J Recons Microsurg 2002 18 275 80 10.1055/s-2002-30183 Phipps GJ Hoffer MM Latissimus dorsi and teres major transfer to rotator cuff for Erb's palsy J Shoulder Elbow Surg 1995 4 124 9 7600163 Muhling RS Blaauw G Slooff ACJ Kortleve JW Tonino AJ Gilbert A Conservative treatment of obsterical brachial plexus palsy (OBPP) and rehabilitation Brachial plexus injuries 2001 London, Martin Dunitz Ltd 173 187 Covey DC Riordian DC Milstead ME Albright JA Modification of the L'Episcopo procedure for brachial plexus birth palsies J Bone Joint Surg [Br] 1992 74 897 901 1447254 Gilbert A Brockman R Carlioz H Surgical treatment of brachial plexus birth palsy Clin Orthop 1991 264 39 47 1847671 Gilbert A Tubiana R Obstetric brachial plexus palsy The hand 1993 Philadelphia, WB Saunders 576 601 Al-Qattan MM Latissimus dorsi transfer for external rotation weakness of the shoulder in obstetric brachial plexus palsy J Hand Surg [Br] 2003 28 487 490 12954263 10.1016/S0266-7681(02)00393-5

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==== Front BMC DermatolBMC Dermatology1471-5945BioMed Central London 1471-5945-4-81529402210.1186/1471-5945-4-8Research ArticleDLQI scores in vitiligo: reliability and validity of the Persian version Aghaei Shahin [email protected] Manouchehr [email protected] Peyman [email protected] Nazila [email protected] Andrew Y [email protected] Department of Dermatology, Shiraz University of Medical Sciences, Shiraz, Iran2 Department of Biostatistics, Shiraz University of Medical Sciences, Shiraz, Iran3 Burn Hospital, Shiraz University of Medical Sciences, Shiraz, Iran4 Department of Dermatology, University of Wales College of Medicine, Cardiff CF4 4XN, UK2004 4 8 2004 4 8 8 15 5 2004 4 8 2004 Copyright © 2004 Aghaei et al; licensee BioMed Central Ltd.2004Aghaei et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The objective of this study was to translate and to test the reliability and validity of the 10-item Dermatology Life Quality Index (DLQI) questionnaire in Iranian patients with vitiligo. Methods Using a standard "forward-backward" translation procedure, the English language version of the questionnaire was translated into Persian (the Iranian official language) by two bilinguals. Seventy patients with vitiligo attending the Department of Dermatology, Saadi Hospital, Shiraz, Iran, were enrolled in this study. The reliability and internal consistency of the questionnaire were assessed by Cronbach's alpha coefficient and Spearman's correlation, respectively. Validity was performed using convergent validity. Results In all, seventy people entered into the study. The mean age of respondents was 28.3 (SD = 11.09) years. Scores on the DLQI ranged from 0 to 24 (mean ± SD, 7.05 ± 5.13). Reliability analysis showed satisfactory result (Cronbach's α coefficient = 0.77). There were no statistically significant differences between daily activity (DA) and personal relationship (PR) scale mean scores in generalized versus focal-segmental involvement in sufferers (P = 0.056, P = 0.053, respectively). There were also strong differences between the mean scores of the PR (personal relationship) scale with the involvement of covered only and covered/uncovered areas (P= 0.016) that was statistically significant in the second group. Conclusions The study findings showed that the Persian version of the DLQI questionnaire has a good structural characteristic and is a reliable and valid instrument that can be used for measuring the effects of vitiligo on quality of life. ==== Body Background Vitiligo is an important skin disease having major impact on the quality of life of patients suffering from vitiligo. Appearance of skin can condition an individual self-image, and any pathological alteration can have psychological consequences [1,2]. Many vitiligo patients feel distressed and stigmatized by their condition. These patients often develop negative feeling about it, which are reinforced by their experiences over a number of years. Most patients of vitiligo report feelings of embarrassment, which can lead to a low self-esteem and social isolation [3]. The Dermatology Life Quality Index questionnaire is designed for use in adults, i.e. patients over the 16. It is self-explanatory and can be simply handed to the patient who is asked to fill it in without the need for detailed explanation. It is usually completed in one to two minutes. The questions were classified to 6 headings items: symptoms and feelings (questions 1 and 2), daily activities (questions 3 and 4), leisure (questions 5 and 6), and personal relationships (questions 8 and 9) each item with maximum score 6; work and school (question 7), and treatment (question10) each item with maximum score 3 [4]. The DLQI is calculated by summing the score of each question resulting in a maximum of 30 and a minimum of 0. The higher the score, the more quality of life is impaired. The DLQI can also be expressed as a percentage of the maximum possible score of 30. The scores for each of these sections can also be expressed as a percentage of either 6 or 3. Since the DLQI is a brief, simple, easy to complete, and its application in research settings as a screening tool is well documented, it was decided to translate the DLQI into Persian (the Iranian official language) and to examine reliability and validity of this questionnaire in an Iranian population with vitiligo. Methods The standard "forward-backward" procedure was applied to translate the questionnaire from English into Persian. Two independent bilinguals translated the items and two others translated the response categories and after cultural adaptation, a provisional version was provided. Subsequently, it was back translated into English and then the final version was provided. The cultural adaptation was done by the translation of the "partner" to the "spouse" in Persian language and adding of it in question 8 and 9, respectively. The final draft of the Persian version was administered to a sample of patients with vitiligo that referred to Department of Dermatology, Saadi Hospital, Shiraz, Iran. There were no restrictions on patient selection with regard to extension of lesions. The patients were introduced to the subject of this study and informed about the personal nature of the questionnaire, and all those who gave consent were given the DLQI questionnaire to complete. The questionnaires were completed by the patients whom were referred to our clinic for psoralen and UVA (PUVA) therapy. The patients were categorized by extension of lesions into covered only (vitiligo patches are covered by cloths) and covered/uncovered involvement and by severity of disease to focal-segmental (focal is defined as a single or a few depigmented macules that are located in a discrete area, segmental is the unilateral localization of one or more macules to one area of the body) [5], which in this study were settled in one category, and generalized involvement groups (widespread distribution of numerous macules over the integument in a random pattern) [6]. Age of patients, marital status and the number of treatment sessions were recorded. Responses on the DLQI were scored according to the guidelines of Finlay and Khan [4]. All statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS 11.0 for Windows). To test the reliability, the internal consistency of the questionnaire was assessed by Cronbach's alpha coefficient and alpha equal to or greater than 0.70 was considered satisfactory [7]. Validity was performed using convergent validity to demonstrate the extent to which the DLQI correlated with global quality of life. Construct validity was checked by factor analysis. Results Seventy patients aged 18 to 68 (mean ± SD, 28.3 ± 11.09) years completed the questionnaire. We did not have any incomplete questionnaires; therefore we included all questionnaires in our study. Scores on the DLQI ranged from 0 to 24(mean ± SD, 7.05 ± 5.13). The reliability of the questionnaire was obtained by Cronbach's alpha coefficient (α = 0.77). There were no statistically significant differences between the sex, item scores and mean DLQI score. Table 1 shows the results of item convergent validity tests. The scaling success rates were 100% for convergent validity of each scale. Table 1 Item scaling tests: convergent validity for DLQI scales Scale No. of items per scale Convergent validity (range of correlation) Scaling Success1 Scaling Success Rate2 Internal consistency (Cronbach's α) SF 2 0.69–0.78 2/2 100 0.70 DA 2 0.71–0.78 2/2 100 0.76 L 2 0.67–0.88 2/2 100 0.71 WS 1 1.00 1/1 100 1.00 PR 2 0.50–0.99 2/2 100 0.79 T 1 1.00 1/1 100 1.00 SF (Symptoms and feelings), DA (Daily activities), L (Leisure), WS (Work and School),PR (Personal relationships), T (Treatment) 1- Number of correlation between items and hypothesized scale corrected for overlap ≥ 0.4/ total number of convergent validity tests. 2- Scaling success rate is the previous column as a percentage. Cronbach's α coefficient by gender, marital status, severity, and extension of disease are shown in Table 2. Table 2 Cronbach's coefficient by gender, marital status, severity, and extension of disease Variable Cronbach's coefficient (n1) Gender   Male 0.60 (27)   Female 0.80 (43) Marital status   Single 0.79 (42)   Married 0.75 (28) Severity   Focal/Segmental 0.58 (18)   Generalized 0.79 (52) Extension   Covered/Uncovered 0.78 (54)   Covered 0.67 (16) 1 The number of patients in each category. There were no statistically significant differences between item and mean DLQI scores of males versus females and married versus single cases. Cronbach's α reliability coefficients ranged from 0.69 to 0.78 for symptoms and feelings (SF) scale, 0.71 to 0.78 for daily activities (DA) scale, 0.67 to 0.88 for leisure (L) scale, and 0.50 to 0.99 for personal relationships (PR) scale. The reliability coefficient for work and school (WS) scale was equal to treatment (T) scale that was 0.100. Table 2 shows comparison of Cronbach's α in each scale separately. The DA scale was found to have a strong association with gender (female scores were greater than male ones). The L scale was found to have significant relationship with the severity of the disease (generalized versus focal/segmental) (P value = 0.018). The DA and PR scales, also had no statistical association with severity factor (P = 0.056 and P = 0.053, respectively). The PR scale had strong correlation with the type of the extension of lesions (covered only versus covered/uncovered type) (P value = 0.016). There was no association between the numbers of treatment sessions with the type of disease (generalized versus focal/segmental). The number of treatment sessions and mean DLQI score was found to have a positive correlation coefficient (P value = 0.02, r = 0.28) but this correlation was statistically significant in the generalized type only (P value = 0.008, r = 0.37). Spearman's correlation coefficient of severity of the disease with questions 5, 6, and 8 were 0.25, 0.24, and 0.26 respectively. For question 8 and the extension of disease and also for question 1 and the stage of disease it was equal to 0.26. The result in question 4 and gender status was statistically significant (0.008) there was no statistically significance correlation between the age and each of questions. The result of question 9 and marital status was statistically significant (P = 0.002) and it was higher in married patients. The range of the paired correlations between the items was 0.17–0.68. Factor analysis is performed to determine the Persian version is a two-dimensional measure including social and psychological parameters (Table 3). Table 3 Factor loadings (rotated) 1 of two-factor solution DLQI items Social factor Psychological factor Q 1 .086 .485 Q 2 .545 .305 Q 3 .535 .374 Q 4 .470 .285 Q 5 .564 .473 Q 6 .190 .468 Q 7 .088 .619 Q 8 .813 .186 Q 9 .681 -.195 Q 10 .099 .500 1 Varimax Discussion The DLQI questionnaire is a well-known instrument for measuring dermatological distress and has been translated into a variety of languages [8,9] and [10]. The translation process set by the international quality of life assessment (IQOLA) project was built on lessons from cross-cultural psychology and other health survey projects to develop protocols for translating, validating, and norming health status questionnaires, entails forward translation by at least two translators who were native speakers of the target language, rating of translation equality by two other bilinguals, and back translation by two translators who were native speakers of American-English or British-English [11]. Because native English speakers were unavailable, we did not fully adhere to this strategy. Two independent Iranian health professionals translated the items and subsequently, it was back translated into English by two others and then the final version was provided. Vitiligo is an acquired depigmentation disorder of great cosmetic importance affecting 1–4% of the world's population. The disease has a major impact on quality of life of patients, many of whom feel stigmatized by their condition [12]. Porter et al. studied the effect of vitiligo on sexual relationships and found that embarrassment during sexual relationships was especially frequent for men with vitiligo [13]. Salzer and Schallreuter reported that 75% found their disfigurement moderately or severely intolerable [14]. Weiss et al compared the difficulties faced by patients with vitiligo with those with leprosy in India [15]. There may be a relationship between stress and the development of vitiligo. Al-Abadie et al. indicated that psychological stress increases levels of neuroendocrine hormones, affects the immune system and alters the level of neuropeptides, which may be the initial steps in pathogenesis of vitiligo [16]. In general, the finding of this study indicated that mental health in vitiligo patients is poor and it is strongly associated with their quality of life. Since the patients with higher DLQI scores responded less favorably to a given therapeutic modality [12], improving quality of life in this group becomes very important task.Severity (generalized versus focal/segmental) and extension of lesions on covered only or covered/uncovered areas has an effect on quality of life of patients. This study reports data from a validation study of the 10-item DLQI questionnaire in Iran. In general the findings showed promising results and were comparable with other research finding throughout the world [12]. The two-dimensional Persian version of DLQI questionnaire assessed the social and psychological difficulties as other studies [12]. There was no relationship of DLQI score with gender, which is consistent with the study of Parsad et al. [12]. The mean DLQI score in this study was 7.05 that is lower than that obtained by Finlay and Khan (mean 7.3) [4] and Parsad et al. (mean 10.67) [12], and it is higher than Kent and Al-Abadie's study (mean 4.82) [17]. Reliability was associated by internal consistency of the questionnaire reporting Cronbach's alpha coefficient and validity was examined by convergent validity showed satisfactory results (α = 0.77). Cronbach's α was < 0.7 for males, focal/segmental, and covered vitiligo that may be related to small sample size and cultural differences. The Persian version of the DLQI questionnaire proved to be acceptable to patients and it is worth nothing that occasionally the questionnaire was administered by a trained nurse in face-to-face interviews. However this was done in illiterate patients and some ones indicated that some questions were difficult to answer, especially question 8. Perhaps this was the reason why a weaker correlation was found for this item with its corresponding subscale. Conclusions The study finding showed that the Persian version of the DLQI questionnaire has a good structured characteristic and is a reliable and valid instrument that can be used for measuring the effects of the vitiligo on quality of life. Especially, the reliability of this questionnaire was high in females and patients with generalized involvement, because of the great cosmetic importance in these groups. Competing interests None declared. Abbreviations DLQI: Dermatology Life Quality Index SD: standard deviation IQOLA: international quality of life assessment Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgement The Persian version of DLQI (dlqifarsi) can be reached via the internet ==== Refs Mattoo SK Handa S Kaur I Gupta N Malhotra R Psychiatric morbidity in vitiligo: prevalence and correlates in India J Eur Acad Dermatol Venereol 2002 16 573 578 12482039 10.1046/j.1468-3083.2002.00590.x Parsad D Dogra S Kanwar AJ Quality of life in patients with vitiligo Health and Quality of Life Outcomes 2003 1 58 14613564 10.1186/1477-7525-1-58 Savin J The hidden face of dermatology Clin Exp Dermatol 1993 18 393 395 8252755 Finlay AY Khan G Dermatology Life Quality Index (DLQI): a simple practical measure for routine clinical use Clin Exp Dermatol 1994 19 210 16 8033378 Ortonne JP Mosher DB Fitzpatrick TB eds Vitiligo and Other Hypomelanosis of Hair and Skin New York: Plenum Press 1983 Koga M Vitiligo: A new classification and therapy Br J Dermatol 1977 97 255 261 921895 Nunnally JC Bernstein IH Psychometric Theory New York: McGraw-Hill 1994 3 De Tiedra AG Mercadal J Badia X Mascaró JM Herdmann M Lozano R Adaptación transcultural al Español del cuestionario Dermatology Life Quality Index (DLQI): el Índice de Calidad de Vida en Dermatologia Actas Dermo-Sifiliograficas 1998 89 692 700 Schafer T Staudt A Ring J German instrument for the assessment of quality of life in skin diseases (DIELH). Internal consistency, reliability, convergent and discriminant validity and responsiveness Hautarzt 2001 52 624 8 11475643 10.1007/s001050170102 Etemesi BA Quality of life in Tanzanian adults with chronic skin disease Ann Dermatol Venereol 2002 129 1S253 Bullinger M Alonso J Apolone G Translation health status questionnaire and evaluating their quality: The IQOLA Project Approach J Clin Epidemiol 1998 51 913 23 9817108 10.1016/S0895-4356(98)00082-1 Parsad D Pandhi R Dogra S Kanwar AJ Kumar B Dermatology life quality index score in vitiligo and its impact on the treatment outcome Br J Dermatol 2003 148 373 4 12588405 10.1046/j.1365-2133.2003.05097_9.x Porter J Beuf A Lerner A The effect of vitiligo on sexual relationship J Am Acad Dermatol 1990 22 221 2 2312803 Salzer BA Schallreuter KU Investigations of the personality structure in patients with vitiligo and a possible association with catecholamine metabolism Dermatology 1995 190 109 15 7727831 Weiss M Doongaji D Siddartha S The explanatory model interview catalogue (EMIC) Br J Psychiatry 1992 160 819 30 1617366 Al-Abadie MSK Kent G Gawkrodger DJ The relationship between stress and the onset and exacerbation of psoriasis and other skin conditions Br J Dermatol 1994 130 199 203 8123572 Kent G Al-Abadie MSK Factors affecting responses on Dermatology Life Quality Index among vitiligo sufferers Clin Exp Dermatol 1996 21 330 3 9136149 10.1046/j.1365-2230.1996.d01-219.x

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==== Front BMC EcolBMC Ecology1472-6785BioMed Central London 1472-6785-4-101529871110.1186/1472-6785-4-10Research ArticleRecovery of frog and lizard communities following primary habitat alteration in Mizoram, Northeast India Pawar Samraat S [email protected] Gopal S [email protected] Binod C [email protected] Wildlife Institute of India, Chandrabani, Dehradun 248 001, India2 Section of Integrative Biology, University of Texas, Austin, TX 78712, USA2004 6 8 2004 4 10 10 28 1 2004 6 8 2004 Copyright © 2004 Pawar et al; licensee BioMed Central Ltd.2004Pawar et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Community recovery following primary habitat alteration can provide tests for various hypotheses in ecology and conservation biology. Prominent among these are questions related to the manner and rate of community assembly after habitat perturbation. Here we use space-for-time substitution to analyse frog and lizard community assembly along two gradients of habitat recovery following slash and burn agriculture (jhum) in Mizoram, Northeast India. One recovery gradient undergoes natural succession to mature tropical rainforest, while the other involves plantation of jhum fallows with teak Tectona grandis monoculture. Results Frog and lizard communities accumulated species steadily during natural succession, attaining characteristics similar to those from mature forest after 30 years of regeneration. Lizards showed higher turnover and lower augmentation of species relative to frogs. Niche based classification identified a number of guilds, some of which contained both frogs and lizards. Successional change in species richness was due to increase in the number of guilds as well as the number of species per guild. Phylogenetic structure increased with succession for some guilds. Communities along the teak plantation gradient on the other hand, did not show any sign of change with chronosere age. Factor analysis revealed sets of habitat variables that independently determined changes in community and guild composition during habitat recovery. Conclusions The timescale of frog and lizard community recovery was comparable with that reported by previous studies on different faunal groups in other tropical regions. Both communities converged on primary habitat attributes during natural vegetation succession, the recovery being driven by deterministic, nonlinear changes in habitat characteristics. On the other hand, very little faunal recovery was seen even in relatively old teak plantation. In general, tree monocultures are unlikely to support recovery of natural forest communities and the combined effect of shortened jhum cultivation cycles and plantation forestry could result in landscapes without mature forest. Lack of source pools of genetic diversity will then lead to altered vegetation succession and faunal community reassembly. It is therefore important that the value of habitat mosaics containing even patches of primary forest and successional secondary habitats be taken into account. ==== Body Background Evaluation of the importance of various processes determining community structure and function is an important topic in ecology. Unlike just a decade or so ago, few studies today question whether or not community assembly is strictly random, recognizing the role of both stochastic and deterministic processes [1]. This change can be attributed to accumulating data on organisation in experimental and natural communities, and new perspectives gained from the fields of evolutionary population ecology and phylogenetics ([2] e.g., [3,4]). It is worth noting that empirical tests for much of this theory have been attempted relatively recently in experimental microcosms research in particular, yielding valuable insights into the role of processes in driving long-term community dynamics [5]. This newfound view of community ecology is an excitingly realistic one, and has the potential to make valuable contributions to conservation biology as well [6,7]. However, though studies on long term dynamics of communities are obviously important, they are extremely difficult to implement. A vast majority of field studies are restricted to exploring correlates and predictors of species diversity and other emergent community properties. This is in part due to the problems associated with studying complex natural communities. But to a great extent, difficulties also arise because community ecology studies have traditionally been skewed towards relatively long-lived vertebrate groups, or are restricted to short study periods due to logistical constraints, especially in the tropics [8,9]. Although these studies yield valuable information, they are carried out at timescales that at best, give insight into short-term processes, providing limited information about community organization, persistence, or assembly. Circumventing this problem is obviously very difficult. One possible approach is to study communities along gradients of habitat succession using space-for-time substitution (SFT) to obtain chronosequential communities [10]. Thus, instead of studying changes in a single community over time, successional habitats of known ages that can be arranged on a temporal gradient are compared. This method can reveal changes in community structure, environmental predictors of these changes, and provide estimates of the rate of community change [11-14]. Although many studies have examined recovery of faunal communities with tropical forest regeneration, a vast majority have been restricted to one or two vertebrate (birds, small mammals) and invertebrate (ants, beetles) groups [10]. For example, 19 of the 33 studies reviewed by Dunn [10], were on vertebrates, out of which only two were on amphibians and/or reptiles. Considering that amphibians and reptiles are ectothermic and have life history traits different from mammals and birds [9], more data on these taxonomic groups is important to test the generality of conclusions about effects of tropical habitat alteration on fauna. This study takes an SFT approach to compare changes in frog and lizard community structure in two contrasting habitat succession gradients: (a) 1-yr jhum fallows giving way to mature forest, and (b) 1-yr jhum fallows planted over with teak, leading to monoculture stands. Slash-and-burn or shifting cultivation (jhum) agriculture involves clearing and burning of forest patches, so the original rainforest communities are effectively obliterated, and succession involves recovery of communities from scratch. The following questions were addressed in this study: 1. How much does frog and lizard community succession differ between the two gradients of habitat recovery? 2. Does composition of the entire community change in synchrony, or does the recovery pattern differ between subcommunities such as frogs vs. lizards and guilds? 3. What aspects of habitat change influence frog and lizard community recovery, and if habitat parameters are linked to niche axes, do they predict changes in guild composition? 4. Do successional changes in guilds also show trends in phylogenetic structure? This last question is expected to yield interesting insights into possible evolutionary mechanisms underlying changes in community composition [15], but has not explicitly been addressed in previous work on faunal recovery during tropical forest regeneration (cf. [10], and references therein). In this paper, a chronosere is defined as a habitat that has recovered from perturbation for a known length of time, and can be assigned a place in the SFT. An assemblage is the set of all species of a taxonomic group in a landscape of interest. Ecological groups (EGs) are species' subsets of the assemblage with similar niche characteristics. Communities comprise species of the assemblage which share a habitat stratum (i.e., chronosere) in the landscape. Guilds are members of the EGs that actually coexist in the same chronosere i.e., belong to the same community, and are thus likely to have ecological and evolutionary interactions (cf. [16]). To draw inferences about what aspects of habitat change determine sequential communities, habitat and frog-lizard community data were analysed hierarchically. As a first step, species richness and turnover of frog and lizard communities along habitat recovery gradients was summarised, and the entire assemblage classified into ecological groups (EGs) based on niche similarities. Guilds identified from this classification were then examined for phylogenetic structure. Using factor analysis, orthogonal combinations of variables that described biotic and abiotic aspects of habitat transition were extracted. We then tested for correspondence between these composite variables and composition of frogs and lizard communities and guilds. Based upon the relationships between different habitat factors and frog and lizard communities, variables were interpreted as composite adaptive zones, and we tested whether they predicted successional changes at different levels of community organization. Results and Discussion Gradients of vegetation recovery Table 1 shows details of the chronosere sampling plots (see Additional file 2 for photographs of chronoseres). Both the 1-yr post-jhum fallows (plots jh1A and B) were dominated by herbaceous plants, tall grass, shrubs and wild bananas, along with saplings and surviving crop plants. The 4 to 5-yr post-jhum plot (jh5) was dominated by almost homogeneous stands of the bamboo Melocanna baccifera, interspersed with a few shrubs and trees. Herbs were rare, and the understorey sparse. The 7 to 10-yr post-jhum plot (jh10) was very similar to jh5. However, here the bamboo culms were more sparsely distributed, and along with M. baccifera, two other bamboos- Dendrocalamus longispathus and Bambusa tulda were in greater abundance, and woody plants were relatively more common. Compared to the other plots, a larger area was included in the 30 to 35-yr jhum plot (jh35) because it contained a greater range of ages and hence perhaps more variability. Also, this was the only accessible site in the study area that represented a chronosere aged between 30–50 years. Although M. baccifera was common, this site had a greater abundance of other bamboos and trees than any of the previous stages. Though most trees were small, woody vegetation formed a significant part of the canopy. Herbs and shrubs were rare, and the understorey generally sparse. Table 1 Details of sampling plots. Plot ages were determined by consultation with local people. The labels in the first column are used to identify plots throughout the rest of the paper. Plot Details Size (ha.) jh1A 1 year jhum fallows, cultivated and abandoned in 1998 3–4 jh1B 1 year jhum fallows, cultivated and abandoned in 1998 3–4 jh5 Two adjoining, indistinguishable 4–5 year jhum fields cultivated and abandoned in 1994 & 1996 respectively 4–6 jh10 Three adjoining, indistinguishable 7–10 year jhum fields cultivated and abandoned between 1988 & 1991 4–6 jh35 Five adjoining, indistinguishable 30–35 year post-jhum fields cultivated and abandoned between 1963 & 1969 8–10 tk4 4 year old teak plantation, planted in 1994 3–4 tk 22 Subset of a 22 year old teak plantation, planted in 1977 4–6 matA Subset of slightly disturbed contiguous mature forest 4–6 matB Subset of undisturbed contiguous mature forest 4–6 matC Subset of undisturbed contiguous mature forest 4–6 The three mature forest plots (matA, B, and C) were of untraceable age (probably >100 years old; [12]). They were characterized by high tree density, canopy cover, and a sparse understorey with few herbs or true shrubs (not tree saplings). One of the sites (matA) was slightly disturbed by dead wood and palm leaf extraction, and had a relatively dense understorey in places. Bamboos were mainly restricted to moist gullies, and occasionally in the understorey. The 4-yr teak plot (tk4) was a young plantation characterized by a monodominant stand of teak trees. The understorey was sparse, with some tall grass, shrubs (mainly Lantana camara) and occasional herbs. The 22-yr teak plantation site (tk22) had a monotonous, uniform structure characteristic of a mature, managed teak monoculture. Undergrowth was sparse, consisting mostly of tall grass and Lantana sp. Table 2 summarises differences in four habitat parameters that show broad contrasts between chronoseres. These results are very similar to those of a another study in the same region [12]. For the purposes of these comparisons, data for the two undisturbed mature forest plots matB and matC are presented together because they are were very similar in macrohabitat characteristics. Table 2 Differences in four macro-habitat parameters across plots. All variables were normally distributed, but not homoscedastic (Levene's test, p < 0.001), so Tamhane's T2 (a conservative pair wise comparisons test based on a t test) was used as a post-hoc multiple range test (F ⇒ F-ratio of one-way parametric ANOVA; ** ⇒ p < .005; * ⇒ p < .05; – ⇒ Not significant;). (a) Tree density (F = 79.232) Plot Mean / 250 m2 ± S.E. jh1A&B jh5 jh10 tk4 tk22 jh35 matA jh1A&B 00.41 ± 0.26 jh5 01.52 ± 0.96 - jh10 03.38 ± 0.95 - - tk4 34.05 ± 3.04 ** ** ** tk22 24.45 ± 0.87 ** ** ** ** jh35 15.32 ± 1.30 ** ** ** ** ** matA 27.67 ± 2.05 ** ** ** - - ** matB+C 20.33 ± 1.76 ** ** ** ** - - - (c) Bamboo culm density (F = 194.30) Plot Mean / 25 m2 ± S.E. jh1A&B jh5 jh10 tk4 tk22 jh35 matA jh1A&B 00.00 jh5 96.36 ± 5.05 ** jh10 62.86 ± 4.85 ** ** tk4 00.00 - ** ** tk22 00.00 - ** ** - jh35 30.26 ± 3.85 ** ** ** ** ** matA 00.01 ± 0.21 - ** ** - - ** matB+C 01.14 ± 0.15 - ** ** - - ** - (b) Canopy cover (F = 139.38) Plot Mean (%) ± S.E. jh1A&B jh5 jh10 tk4 tk22 jh35 matA jh1A&B 12.07 ± 1.67 jh5 67.91 ± 2.79 ** jh10 69.79 ± 1.71 ** - tk4 37.25 ± 2.58 ** ** ** tk22 51.33 ± 4.61 ** ** ** ** jh35 76.70 ± 1.62 ** - - ** ** matA 72.64 ± 2.03 ** - - ** ** - matB+C 80.68 ± 1.86 ** ** * ** ** - - (d) Shrub density (F = 12.21) Plot Mean / 25 m2 ± S.E. jh1A&B jh5 jh10 tk4 tk22 jh35 matA jh1A&B 25.85 ± 2.86 jh5 25.59 ± 3.31 - jh10 19.58 ± 2.29 - - tk4 11.28 ± 1.19 ** - - tk22 13.99 ± 2.08 ** - - - jh35 27.58 ± 3.21 - - - * - matA 45.24 ± 4.26 ** ** ** ** ** * matB+C 39.37 ± 4.04 * - ** ** ** - - Eight factors were extracted after Principal Component Analysis (PCA), and by Varimax rotation of the factor structure, which explained a cumulative 85.8% of the variation (see methods). Eigenvalues, factor loadings and factor scores are given in Additional file 3. The ordination of the sampling plots based on scores of the first two PCA factors is shown in Figure 1. This ordination was very similar to one obtained by non-metric multidimensional scaling (NMDS) [17]. The two gradients of vegetation recovery have very different trajectories of change in habitat attributes. The predominant macro-habitat characteristic along the factor 1 axis is high bamboo abundance, while high positive loading on the factor 2 axis indicates tree-forest dominated habitat. The gradient towards mature forest succession includes intermediate stages dominated by bamboo, which are succeeded by a tree dominated forest. Figure 1 Plot of scores of first two PCA factors. Vectors are drawn to show the trajectories of the two gradients of habitat recovery. The chronosere codes are as follows- jh1A, 1B, 5, 10 &35: jhum fallows ranging from 1 to 35 years; tk4 &22: teak plantation 4 and 22 years old; matA, B, &C: mature forest plots. See methods and Table 1 for more details. In general, although there is a change towards a tree dominated habitat in both recovery gradients, the end result is very different because the 22 year teak plantation is a monoculture, whereas the mature forest consists of a diverse tree community. Successional changes in frog and lizard communities The three sampling techniques used in conjunction during the study (see methods) yielded sixteen frog and seventeen lizard species. Figure 2 shows changes in species richness, and Figure 3 differences in community composition for frogs and lizards along the two recovery gradients. Clearly, there are distinct similarities in overall community composition between the early jhum fallows and teak plantation communities on one hand, and the mature forest and the 35 year jhum fallows on the other. Figure 2 Change in species richness of frogs and lizards with chronosere age along teak plantation and mature forest recovery gradients. Both gradients have 1 yr jhum fallows (jh1A&B) as the starting point. The number of species increases logarithmically with succession towards mature forest for both taxonomic groups, but the change is much more striking in the case of frogs. Species richness does not seem to change much with recovery time on the teak gradient. The age of mature forest, known to be >100 years old, was assigned an arbitrary value of 150 years. Figure 3 Dendrogram of similarities in frog and lizard (combined) communities across plots. See text for explanation. Community overlap was calculated with the Bray-Curtis measure, and sites clustered using the UPGMA algorithm. The pattern of recovery is very different for the two gradients. For the mature forest succession gradient, the rate of frog and lizard community recovery is similar to that found for birds by Raman [12] in the same region in Northeast India, with the community approaching mature forest characteristics after about 30 years. Even more remarkably, this timescale is also comparable with those reported by similar studies on other fauna elsewhere in the tropics [10]. The gradient from jhum to mature teak plantations on the other hand, seems to show little change in species richness or composition even after 22 years of plantation growth. It is worth noting that there are dissimilarities in the manner of species accumulation for frogs vs. lizards. There is much less augmentation of species number in the case of the latter, the main reason for this being that younger chronoseres support more lizard than frog species richness. Species accumulation curves (see Additional file 4) show that these differences are not sampling artefacts. Moreover, across chronosere species turnover (see methods) for lizards is significantly higher than that for frogs (Student's t-test, two tail p < 0.05), indicating that lizard community succession was characterized by relatively high replacement and low accumulation of species. Guilds Ecological groups defined by non-metric multidimensional scaling (NMDS) of the niche based dissimilarity matrix for the entire assemblage are shown in Figure 4. The NMDS configuration was derived in 2 dimensions with low stress and high RSQ values, indicating a very good representation of actual niche dissimilarities [18]. On dimension 1, the dominant niche characteristic determining high negative loadings is arboreality, and high positive values indicate that the species is predominantly terrestrial. On dimension 2, higher positive values indicate predominantly diurnal diel activity pattern, and negative values indicate crepuscular and/or nocturnal activity pattern. Identities of species in each group are in Additional file 4. Of the five EGs, two consist of both frogs and lizards: the nocturnal arboreal (NA) group with eight species of frogs (mostly tree frogs) and four species of lizards (all gekkonid lizards), and the crepuscular-nocturnal terrestrial (CT) group, which consists of seven frogs and one crepuscular-nocturnal lizard. The diurnal arboreal group (DA) consists of five agamid lizards, some of whom are occasionally terrestrial. The diurnal terrestrial (DT) group consists of six skinks and one lacertid lizard. More detailed natural history descriptions of these species can be found in Pawar [17]. Figure 4 NMDS configuration showing ecological groups (EGs) of frogs and lizards in the assemblage. Each point represents a species. For this configuration, stress = 0.14712 and RSQ =.90076 [17]. Broad characteristics of EGs are as follows: DA= Diurnal, arboreal; NA(L)= Nocturnal, arboreal, all lizards; NA(F)= Nocturnal, arboreal, all frogs; CT= Crepuscular-nocturnal, terrestrial; DT= Diurnal, terrestrial. See Additional file 4 for EG species' identities. Figure 5 shows how EGs are represented along the two recovery gradients. In this paper, representatives of each EG in a sampling plot are considered guilds of that chronosere. Clearly, the number of guilds as well as number of species per guild increases with succession along the gradient leading to mature forest, but not along the one leading to teak monoculture. The species accumulation during natural forest succession is mainly due to augmentation of crepuscular and nocturnal guilds. It is also worth noting that the DT and DA groups, which are consistently present along both gradients of recovery, also have the maximum niche overlap (distance between pairs of species is the smallest for these groups in the NMDS niche space). The implication of this fact is discussed below. It is due to these two guilds that successional lizard communities show the high species turnover and low accumulation noted above. Figure 5 Representation of ecological groups along gradients of habitat recovery. Each EG for a particular habitat is effectively a guild. Note that the number of guilds increases with succession along the jhum to mature forest gradient, but not along the teak gradient. See Figure 4 for guild identities, and Additional file 4 for species' that make up each EG. Phylogenetic structure Figure 6 shows the ratio of species to genera (S/G ratio) of guilds in different chronoseres. The S/G ratio increased with succession towards mature forest in the NA guild, and to a lesser extent, in the CT guild. The S/G ratio of the NA, DT and DA guilds was variable, and did not change directionally with habitat recovery. In general, across all chronoseres irrespective of which recovery gradient they belonged to, the number of guilds represented in each chronosere was positively correlated with phylogenetic structure (S/G ratio averaged across guilds; Spearman R = 0.92, p < 0.0002) and the species richness of the chronosere (R = 0.88, p < 0.01). This suggests that the ability of chronoseres to support a larger number of guilds predicts species number as well as phylogenetic structure. Figure 6 Trends in phylogenetic structure (species/genus ratio) in five guilds across chronoseres. An "x" indicates that a guild is absent. The S/G ratio increases with time of habitat recovery in the nocturnal guilds, but not for the diurnal guilds. See text for discussion. These results on successional changes in guild structure and representation indicate a distinctly non-random sequence of community assembly, as certain guilds appear in later stages, followed by increase in their species richness and in many cases, phylogenetic structure. Habitat attributes that determine these changes are explored in the next section (see below). Correspondence between habitat factors and frog-lizard community succession Table 3 shows the results of correspondence tests between Euclidean dissimilarity matrices calculated from the eight PCA factors and frog-lizard species compositional dissimilarity matrices for different levels of community structure. As the factor structure of the PCA analysis was rotated to maximize the orthogonality of factor loadings, these matrix correspondence tests are statistically similar to performing partial mantel tests (partial correlation) with multiple variables [19]. The hierarchical nature of the correlations in Table 3 and the fact that guilds are correlated with different, orthogonal composite variables is interesting, and offers answers to the third question addressed this paper: what aspects of habitat change influence frog and lizard community recovery at different levels of community organization? Table 3 Correlation between dissimilarity matrices based on eight PCA factors (unsquared Euclidean distances), and different levels of community organisation (Jaccard's index). The correlation coefficients are followed by significance (p) values in parentheses. The p-values were estimated by 1000 Monte Carlo randomizations of each pair of matrices. Correlations with p values >0.05 not reported. See the text and Additional file 3 to see loadings of habitat variables for the PCA factors. Community/guild Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Factor 6 Factor 7 Factor 8 Frogs + Lizards 0.762 (0.003) Frogs 0.5031 (0.003) 0.467 (0.019) Lizards 0.672 (0.005) 0.375 (0.033) CT 0.331 (0.035) 0.417 (0.016) 0.402 (0.016) DA 0.630 (0.008) DT 0.608 (0.003) NA(F) 0.347 (0.025) 0.482 (0.037) NA(L) 0.362 (0.004) 0.417 (0.030) Higher-order community structure The strongest association is between factor 2 and overall species composition (frogs and lizards combined) across chronoseres. Factor 2 was strongly and non-linearly correlated with age along both teak and mature forest succession gradients (logarithmic fit, R2 = 0.85, and 0.97, respectively), and represents deterministic, linear aspects of vegetation succession. It has high positive loadings for tree species richness, and macro-habitat variables such as tree density, canopy cover, and canopy height, most of which increase deterministically along both gradients of habitat change. Among the measured variables, these are primary and independent, which over time drive changes in secondary (microhabitat) variables such as bamboo density, shrub abundance, and various measures of habitat heterogeneity (see methods). This factor clearly influences species composition at all levels of frog and lizard community structure. Frog vs. Lizards Along with factor 2, the frog subcommunity was also associated with factor 7, which shows no significant age determinacy along either gradient of habitat recovery. This factor has high positive loading for soil moisture content, which is an important limiting factor for frogs. The lizard subcommunity was associated with factor 8 along with factor 2. Factor 8 has high loading for soil moisture variability, which is highest in chronoseres with spatial variation in insolation. This factor is crucial for diurnal lizards, many of which are heliotherms. Factors 7 and 8 are probably also surrogates of unmeasured or unclassified variables which influence successional changes in these two communities. Guilds Three out of five guilds are secondarily correlated with factors orthogonal to factor 2. The two that were not, i.e., the diurnal-arboreal (DA) and diurnal-terrestrial (DT) groups, were correlated with factor 2. This suggests that in contrast to other guilds, these two, which are both made up only of lizards, are directly influenced by a hierarchically higher order of habitat attributes. These were also the two groups that showed non directional trends in species richness as well as phylogenetic trends along habitat recovery gradients (Figures 5 and 6). Along with factor 2, the crepuscular-nocturnal terrestrial (CT) group was correlated with factor 1 and 4. Factor 1 scores increase and then decrease with plot age along both habitat recovery gradients (2nd order polynomial fit, R2= 0.99, and 0.82, respectively). This factor had high loading (defined as ≥ ± 0.65; see Additional file 3) positive for both macro- and micro-habitat variables such as bamboo density canopy cover, leaf litter cover and depth and negative loadings for many habitat heterogeneity variables such as CVs of canopy cover and litter cover. These variables are interpretable as ones that are associated with, or influence ground microhabitat conditions. Factor 4 decreased logarithmically with age along both teak and mature forest gradients (R2 = 0.62, and 0.34, respectively). This factor had no strong loadings, but is associated with shrub density, canopy height variability and tree density, all of which also affect ground cover, and can be considered macrohabitat variables. The nocturnal arboreal frog group (NA(F)), was correlated with factor 7, which is non-deterministic with respect to chronosere age. This factor as a high loading for soil moisture, which by itself is difficult to interpret as a variable directly affecting this ecomorphological group. It is likely that this factor is a surrogate for an unmeasured or unclassified variable. Lastly, the nocturnal arboreal lizard group (NA(L)) is correlated with factor 8 along with factor 2. Factor 8 shows a weak negative linear relationship with recovery age along both gradients gradient (R2 = 0.209 and 0.18 for teak and mature forest gradients, respectively). It has high positive loading for CV of soil moisture, which as mentioned above, is highest in chronoseres with spatial and/or temporal variation in insolation. Among the measured variables, factor 2 probably subsumes most habitat parameters that affect both NA groups directly (see next section). Adaptive zones? Can biologically meaningful adaptive zones be interpreted from these associations? As each factor is orthogonal with respect to the others, factors subsume different habitat variables, or their variability in the same variable. Note that the composite variable represented by each factor consists of negative as well as positive loadings of variables. This means that if a guild was associated with a factor, both positive and negative trends in different variables affected it simultaneously, together representing a composite adaptive zone. However, an important fact to consider here is that these "adaptive zones" thus identified may actually be surrogates for actual sets of unmeasured variables. Raman [12] inferred that floristics (tree species composition) and physiognomy (vertical stratification) were the dominant habitat attributes that independently predicted changes in bird species composition at the level of communities, but not at much at the level of guilds. In the case of frogs and lizards, factor 2, which includes a measure of floristic attributes (tree species diversity), is a strong predictor of frog and lizard community composition at all levels. But factor 2 also includes numerous structural attributes that are correlated with tree species diversity, from canopy height to understorey and ground habitat structure, all of which have equal or higher positive loadings. Also, Figure 7 shows that understorey habitat complexity increases with post-jhum succession towards mature forest. Figure 7 The relationship between transect sampling time and habitat complexity. The NAW time varies across habitat, and increases with habitat complexity, whereas DAW is constant. The reason for this is that more complex habitats needed more searching time. The index of complexity was calculated by summing the coefficients of variance for various understorey habitat structure variables. Sample sizes of transects were: jh1A = 15, jh1B = 13, jh5 = 14, jh10 = 16, tk4 = 15, tk22 = 15, jh35 = 38, matA = 17, matB = 28, matC = 21. Thus, it is difficult to infer the extent to which tree species diversity per se influences frog and lizard community structure. Previous work has shown that unlike endothermic vertebrates, amphibian and reptile distributions are likely to be influenced more strongly by abiotic rather than biotic features [20]. The effect of physiognomy on the other hand, is definitely important, though at a different scale than for birds. The idea that a habitat with higher structural complexity will support more species [21-25], and have a strong influence on re-colonisation success [26], has been shown for amphibians and reptiles (but see [27]). It can therefore be inferred that factor 2 subsumes nested subsets of biotic and abiotic variables that directly affect the (mean) fitnesses of species' populations in different guilds. Also, it is clear that guilds are also associated with other, independent variables sets that can be considered to comprise additional aspects of each member species' adaptive zone. Factors 3, 5, and 6 showed no significant association with any level of community composition. The obvious reason for this appears to be that unlike other factors, these are completely non-deterministic with respect to age of succession, thus representing temporally and/or spatially stochastic attributes that were unlikely to show any influence on the conspicuously deterministic nature of frog and lizard community and guild (except for the DT and DA groups) succession (See Figures 2,3,5 and 6). At this resolution, it is impossible to say whether these variables are adaptively significant for certain subgroups/species of frogs and lizards or not. Nevertheless, this complex, nested pattern of these alleged adaptive zones, is ecologically realistic (see [28]). Although difficult to interpret at this level of resolution, this hierarchical partitioning of variables is an indicator of which attributes of habitat change influence community assembly and turnover with such gradients of vegetation succession. Composite variables and successional changes in community characteristics Table 4 shows the predictive ability of the different habitat factors for species richness, guild abundance, and phylogenetic structure in communities and guilds. As expected, factor 2 predicts increase in overall species richness, number of guilds represented, and number of species per guild. However, it does not predict overall phylogenetic structure (measured as the average of S/G ratios across guilds represented in each chronosere). Instead, it predicts the phylogenetic structure of all guilds except DT. Factor 1 predicts species richness as well as S/G ratio in the DA group, and factor 4 predicts the S/G ratio of NA(L). No community characteristics were correlated with factors 3,5,6,7 or 8. Table 4 Correlation between availability of composite variables (factor scores) and various measures of community change. Coefficients are Spearman's R, with p-levels in parentheses. Correlations with p > 0.05 not reported. No community characteristics were correlated with Factors 3,5,6,7 or 8. Factor 1 Factor 2 Factor 4 Species Richness  Overall 0.77 (0.009)  DA 0.81 (0.004)  DT  CT 0.91 (0.000)  NA(F) 0.82 (0.003)  NA(L) Number of Egs 0.69 (0.028) Species per EG  Overall 0.72 (0.019)  CT  DA  DT  NA(F)  NA(L) S/G ratio  Averaged across EGs  CT 0.66 (0.036)  DA 0.85 (0.002)  DT  NA(F) 0.71 (0.020)  NA(L) 0.80 (0.006) The DT group does not show correlation with any of the factors. Interestingly, this ecological group along with the DA group, also occupies the smallest niche space (having the maximum niche overlap in the NMDS space; see Figure 4), has the most consistent presence across chronoseres (but with species turnover) and a phylogenetic structure that varies non-directionally along successional gradients (Figure 6). Similar patterns have been observed for diurnal terrestrial herpetofauna (which are largely lizards) elsewhere [29]. Members of this group also have the highest population densities, and most have wide geographic distributions (Pawar, unpublished data). All these data strongly suggest that this guild is not resource constrained in chronoseres along the habitat recovery gradients, and is more randomly assembled during recovery than any of the other groups. In general, these results help explain the trends seen in Figures 2,3,5 and 6 by indicating attributes of habitat change that drive changes in community structure in successional frog and lizard communities. The succession of jhum fallow towards mature forest involves a deterministic, directional change in attributes that allow coexistence of successively more speciose and phylogenetically structured communities. In terms of change in species richness, these results are qualitatively similar to those of similar work on bird, butterfly, and reptile communities [11-13]. Previous work has not however attempted to look at phylogenetic structure for such successional communities. The jhum to teak monoculture gradient also has many aspects of deterministic habitat change, but apparently not for the variables that are essential for a diverse community. The trajectory of habitat change also indicates that this pattern is unlikely to change with transition towards older plantations either. No previous data exists on herpetofaunal community changes in post-jhum monoculture plantations. Conclusions By comparing disparate trajectories of habitat change and recovery of different taxonomic groups, this study provides useful insights into faunal community change in response to habitat recovery. To summarise, the results show that (1) The two gradients of habitat recovery are very different and accordingly affect frog and lizard community assembly differently, (2) Although both groups increased in species richness with habitat recovery, lizards had higher species turnover, combined with lower species augmentation within each recovery gradient (3) Looking at a finer scale of community organization, assembly appears to be driven by changes in guild representation and composition, where some guilds change directionally with age of habitat recovery by species augmentation, while others change by species turnover (4) Guilds that showed directional increase in species richness also increased in phylogenetic structure (5) Hierarchies of community organisation were affected by composite, nested habitat attributes that correspond to particular niche axes, and (6) the increase in species richness along the mature forest gradient in contrast to lack of change along the teak gradient was due to availability (or lack thereof) of variables that comprise these complex adaptive zones. Also, the results show that a niche-based guild classification reveals patterns that would have been hidden in the gross response pattern of the entire community. Some indication of the qualitative nature of potential evolutionary and ecological processes in community turnover comes from the fact that changes in phylogenetic structure are tied to guild structure in the communities. Using phylogenetic techniques, recent work has demonstrated the importance of evolutionary adaptation in assembling ecological communities [4]. It is clear that specialisation on different subsets of resources, in a habitat drive the origin and as well as persistence of diversity [30]. Frogs and lizards have incongruent patterns of community succession, mainly because they generally differ in fundamental niche dimensions axes such as diel activity [31]. However, although most lizards are diurnal and most frogs nocturnal, there are many sub-lineages that are an exception, and species do share niche space transcending taxonomic boundaries (ecological groups in this paper). Such subgroups probably have congruent ecological and evolutionary dynamics. It is an open question as to what extent vegetation succession leads to changes in the number of adaptive peaks and corresponding changes in mean fitnesses of species' populations such that multiple species can persist in the same habitat. In more ecological terms this is same as asking how habitat succession leads to changes in niche availability, occupancy, and overlap (due to character displacement, for example). Another related question, that was partly explored using the S/G ratios in this paper, is whether similar adaptive zones (or niches or adaptive peaks) tend to be occupied by more closely related taxa. The results here do indicate that this may be true for such gradients of community change, as phylogenetic and guild structure increase directionally and in tandem with succession towards mature forest. Whether this change is driven by immigration from the regional gene pool or due to local divergent adaptation is an interesting question [15]. Reptiles, and to a greater extent amphibians, have limited dispersal ability compared to most vertebrates. This distinction in itself may drive differences in local adaptation and community assembly from other biotic groups. Conservation issues The time scale of recovery on the jhum-rainforest succession gradient, which is about 30 years for both frogs and lizards, and suggests that recovery of diverse communities can be relatively fast, as has been reported for other fauna [10]. However, this pattern of community recovery (or re-assembly) is tightly coupled to changes in certain sets of habitat attributes, which in turn are dependent upon vegetation succession wherein post-jhum chronoseres are gradually replaced by trees. This vegetation succession is obviously reliant on seed rain/dispersal from nearby mature forests. In this region and many other areas Southeast Asia, apart from continued pressure from shifting cultivation and shortening cultivation cycles, it has also become popular practice to plant and maintain monocultures of timber species. As the results of this study indicate, such plantations are unlikely to support natural recovery of faunal communities, and will harbour lower biological diversity compared to primary forest. It is possible that the combined effects of short jhum cycles, plantation forestry and invasion by non-native species such as Lantana and Eupatorium will lead to the local extirpation of even remnant forest patches. This loss of recolonisation pools for flora and fauna, will alter natural trajectories of succession, and strongly impact the biological diversity supported by the landscape. It is therefore important that conservation and prioritisation agencies in these areas consider the value of habitat mosaics containing even small patches of primary forest vegetation. Methods The study was carried out from November 1998 to April 1999 in and around Ngengpui Wildlife Sanctuary (WLS; 21°56'N – 24°31'N and 92°16'E – 93°26'E) in south Mizoram, Northeast India. The study area covers about 200 sq. km. (see Additional file 1 for maps). A combination of high annual precipitation and temperature, and low elevation supports a predominantly tropical evergreen [32] climax vegetation in the area. Shifting cultivation is the primary mode of agriculture here. While most of the area within Ngengpui WLS is mature or primary forest, the surrounding areas are a mosaic of bamboo-dominated sites, mature forest fragments, teak Tectona grandis plantations and abandoned shifting cultivation (jhum) fallows of varying ages. All primary forest is referred to as "mature forest" throughout the paper because it is often difficult to determine the age of ostensibly primary tropical forest, especially in areas with poorly known history of land use and recovery [12,33]. Further details and supporting literature about the geology, vegetation, and land use patterns in the study area can be found in Pawar [17]. Sampling plots Ten sampling plots representing mature and successional vegetation stages of known ages were established [17] (Table 1). To control for recolonisation potential, all secondary plots were selected such that at least 50% of the perimeter abutted mature forest, and the edge was within 100 m from contiguous mature forest. All plots had a slope of 0–20° and were within an altitudinal range of ca. 150–350 m above sea level. As the study was focused on terrestrial frogs and lizards, all plots were at least 100 m away from large perennial water bodies. To minimize spatial autocorrelation, all plots were at least 2 km (straight distance) from each other, with the replicates (e.g., the two 1 yr fallows) being the furthest apart (ca. 10 km). Vegetation sampling, habitat variables and gradients of habitat recovery Vegetation composition and habitat structure variables were sampled on randomly located 10 × 25 m belt transects [13,17]. Transects were cut short whenever an edge of the site was reached. The number of transects sampled were, six each in Jh1A, Jh1B, Jh5, Jh10, and Jh35, and five each in tk4, tk22, matA, matB and matC. Tree density and tree species richness was sampled on the whole area of each transect. All trees >20 cm GBH were enumerated, while the rest were classified as 'shrubs'. Density of bamboo culms, shrubs, palms, bananas, and tall grass clumps was estimated in each of six 2 m radius circular plots laid at 5 m intervals on the transect, beginning from the starting point of the transect. Percentage cover of herbaceous forms and leaf litter, dead woody matter abundance and liana abundance in each circular plot were estimated visually. Percentage canopy cover from ground level was estimated with a hand-held canopy densiometer from the centre of each circular plot. Litter depth was gauged by pressing a blunt rod of 0.5-cm diameter at 5 random points in each circular plot, and counting the number of leaves pinned under it. Principal Components Analysis (PCA) was used to identify different aspects of habitat change with vegetation succession and collapse the list of raw variables into composite factors that could potentially predict frog and lizard community structure. The factor structure was rotated using the Varimax method to obtain clear loading patterns [18]. As additional variables, within-habitat coefficient of variation (CV) of variables was also used along with the raw data as measures of habitat heterogeneity. Within habitat variation was considered potentially informative as it is an important feature of the adaptive landscape [30]. Habitat data were collected at comparable times of each month for all plots, and these CVs are unlikely to be due to temporal fluctuations. Data were square root transformed if they deviated from normality. For a list of variables used in the analyses, see Additional file 3. As the objective of the analysis was to combine variables into composite, orthogonal factors that could potentially account for community and guild structure, all factors with eigenvalues = 0.8 were extracted, irrespective of the number of factors thus extracted. Although somewhat arbitrary, in essence this eigenvalue threshold ensured that a factor was included only if it extracted approximately as much as one raw variable [18]. Although all extracted factors were used as predictors of community structure (see below), in order to obtain a graphic, low dimensional representation of the two gradients of habitat recovery, scores of only the first two factors were plotted. Deterministic sets of variables that changed directionally with chronosere age were identified by regressing scores of each factor against chronosere ages. Frog and lizard sampling The low abundance of amphibians and reptiles and unstandardised sampling methodology in tropical Asia reduces the reliability of species diversity estimates and hence community structure analyses [8]. Taking this problem into consideration, three techniques were used in conjunction to maximize inventorying effort – (i) belt transects, (ii) pitfall trapping and (iii) systematic searching. All these techniques are oriented towards sampling terrestrial, and non-canopy arboreal species, and to further increase the sampling efficiency, the study was restricted to terrestrial, non-fossorial, and non-canopy frogs (Amphibia: Anura) and lizards (Reptilia: Sauria, excluding family Varanidae). Although this excluded a few amphibian and reptile groups, it ensured that taxonomic groups unsuited for the chosen sampling techniques were not unnecessarily included, thus augmenting the reliability of the data. To distribute sampling effort effectively among the ten plots, sampling was carried out in sampling 'sessions' of ten days each. Eleven such sessions (= 110 days) were completed, starting from 15th December 1998, to the end of April 1999. Sufficient time was allocated to all three sampling techniques during each session. Belt transects To improve detection and gather information for delineation of EGs (see below), the traditional transect method was modified by eliminating pseudoreplication and sampling both nocturnal and diurnal species on the same transect [34,35]. The former was achieved by establishing fresh 50(length) × 3(width) × 3(height) m transects during each session, which were sampled only once. To detect both nocturnal and diurnal taxa, each transect was walked in both directions (to and fro) by two observers. The diurnal animal walk (DAW) was first, and was conducted at a steady pace fixed for all plots (ca. 20 min/50 m). Any animal seen leaving the transect area was recorded as being present on it. Care was taken to cause minimal disturbance to the habitat, and no active searches were done. The nocturnal animal walk (NAW), conducted in the direction opposite to the DAW, was focused on intensive microhabitat searching within the same 50 × 3 × 3 m area. All nocturnal animals found on the DAW were included in the analyses, but to reduce the possibility of re-recording the same animal, diurnal animals found on the NAW were not. Behavioural and microhabitat data were recorded for every animal detected (see below). All transects were sampled immediately after they were established, between 1000–1400 hrs during winter (mid-December to February) and 0900–1300 during summer (March and April). There was no noticeable species turnover with season, so winter and summer data were not analyzed separately [17]. The belt transects also yielded abundance data, which are not used in this paper [17]. Although time taken for the NAW was more or less constant within a chronosere, it varied considerably across plots. The DAW time on the other hand, was more or less consistent. This strategy was used because just as sampling effort needs to be proportional to habitat heterogeneity, higher microhabitat complexity calls for proportionally greater searching effort. Figure 7 shows how well this sampling strategy was implemented. There is a strong positive correlation between an index of microhabitat complexity (calculated as the sum of the coefficients of variance for various understorey habitat structure variables listed in Additional file 3) and time spent on the NAW, but not the DAW. Thus, though no extra time was needed to sight active (diurnal) animals in more complex habitats, the time needed for microhabitat searching (and hence NAW time) increased along a gradient of increasing habitat complexity from the 1-yr fallows and teak plots to mature forest. A total of 192 belt transects were completed, from a minimum of thirteen in jh1B to thirty-eight in jh35 (See Figure 7 for sample sizes). Pitfall trapping This technique was used to supplement species inventorying from the belt transects, and for an unbiased measure of the effects of weather on herpetofaunal activity and hence sampling efficiency. Comparisons of trapping frequency across plots over the study period are not used in this paper. Each pitfall array was, 'Y'-shaped, with three terminal (30 cm diam. × 60 cm depth) and one central (50 cm diam. × 70 cm depth) cylindrical aluminium funnel pitfall traps buried in the ground. The traps were connected with three opaque plastic-sheet fences (the arms of the 'Y') 0.4 m high and 5 m long, held up by bamboo stakes. In all, 22 arrays were placed, with two in each plot except for the large Jh35, which had four. Arrays were at comparable distance from plot edges, and on similar slope. Systematic trapping was initiated ten days after trap were established. Traps were opened for 5–10 consecutive days, and checked according to habitat characteristics, taking into consideration the level of exposure trapped animals were likely to be subjected to; plots with open habitat, such as jh1A were checked most (every alternate day) and those with relatively closed habitat such as matA were checked least frequently (every third day). Most specimens (95.2 %) obtained from pitfall trapping were released a minimum of 100 m away from the array, either in the same site, or in similar habitat elsewhere. A few were retained as voucher specimens. Systematic searching At the end of a sampling session in a plot, far ranging searches were carried out. This augmented species inventorying, and provided information crucial for EG classification (see below). Periodically, nocturnal searches were also made to collect information about the refuge of diurnally active animals, and also to confirm the presence or absence of species in different chronoseres. Identification of taxa Irrespective of the sampling technique, animals detected were caught whenever possible, and identified in hand. All those that escaped were identified to a justifiable level or excluded from the analyses. A few individuals of taxonomically problematic species or taxa were preserved for later identification. Sampling efficiency The effectiveness of sampling was evaluated by species accumulation curves (see Additional file 4), and the effort adjusted after a mid-fieldwork examination of species richness data across chronoseres. While all the early succession stages and teak plantations reach an asymptote very soon, the 30–35 year fallow stopped yielding new species only by the eighth sampling session, while mature forest continued to yield new species till the final sampling session. Characterization of frog and lizard community succession Overlap between recovering frog and lizard communities was measured with the Bray-Curtis measure between all possible pairs of chronoseres using presence absence data of all species (see Additional file 4). The resultant dissimilarity matrix was then used to generate a dendrogram using the UPGMA clustering algorithm [18]. Species turnover in sequential frog vs. lizard communities was compared using the mean Jaccard's dissimilarity value between all chronosere pairs calculated from separate presence absence data for the two groups [36]. Ecological group classification and phylogenetic structure Life history and behavioural traits were used to group species. These are often called guilds (e.g., [37]), but are referred to as ecological groups (EGs) here because the classification covers species from all chronoseres, including those that belonged to separate, non-interacting communities. The representatives of each EG in a particular community or chronosere on the other hand, can be considered a guild of that habitat's community. The life history and behavioural traits used for the EG classification were: diel activity period, habitat use when active, habitat use when resting, substrate temperature when active, air temperature when active, relative humidity when active, substrate moisture when active, resting refuge, resting refuge temperature, resting refuge substrate moisture and foraging tactic. To validate this data, information from literature and consultations with regional herpetologists was also used. These data, collected at different measurement scales, were rescaled to discrete categories to which species were allocated as absent or present. From this binary data, a dissimilarity matrix was calculated between all species using the Bray-Curtis measure [19]. The dissimilarity matrix was then scaled using non-metric multidimensional scaling (NMDS). NMDS geometrically represents dissimilarities in a graphical, low-dimensional space, and is a robust method to represent ecological distance [18,19]. See Additional file 4 for the list of species included in the EG classification. Phylogenetic structure was measured as the ratio of number of species to the number of genera (S/G ratio) in each EG. A similar approach has been used in studies addressing questions about phylogenetic structure in ecological communities [15]. Community – Habitat interrelationships To test which habitat attributes influenced community structure, Mantel tests of correspondence between dissimilarity (distance) matrices [18,19,38,39] were used. Dissimilarity matrices between sites were generated based on differences in set of composite variables (factors) extracted by the PCA analysis (unsquared Euclidean distances), and for different levels of frog and lizard community composition (from entire community to guilds defined by the EG classification using Jaccard's index) [18,19]. Significance of correlation coefficients was tested by 1000 row-column Monte Carlo randomizations for each pair of matrices. To test whether the availability of composite variables (PCA factors) that predicted community and guild structure identified by the matrix correspondence tests did indeed influence community succession and phylogenetic structure along gradients of habitat recovery, correlations between sums of factor scores and species richness, ratio of species number/guild number and S/G ratios across chronoseres were tested. Authors' contributions SSP conceived the study, carried out the fieldwork, performed the data analyses, and drafted the manuscript. BCC and GSR participated in design and coordination of the study. GSR also supervised the vegetation identification and habitat classification. All authors read and approved the final manuscript. Supplementary Material Additional File 2 Photographs of habitat types. Representative photographs of habitat types Click here for file Additional File 3 Results of PCA along with list of habitat variables used. PCA Results Click here for file Additional File 4 Species accumulation curves and frog and lizard species' lists. Species accumulation curves and lists Click here for file Additional File 1 Maps of study area. Location map of study area sampling plots with respect to vegetation types Click here for file Acknowledgements This study was funded by a fellowship from the Ministry of Environments and Forests, Government of India and a grant from the Bombay Natural History Society, to SP. The Mizoram forest department kindly gave research permits and logistical support to work in Ngengpui WLS. We are particularly grateful to Pu Lal Fala, Mr. N.R. Pradhan, Pu Lathlamuana Pachuau, Rijkzova (Zokima) and the people of Kawrthindeng village for their wonderful enthusiasm and kind support. We also thank C. 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Hofer U Bersier LF Borcard D Spatial organization of a herpetofauna on an elevational gradient revealed by null model tests Ecology 1999 80 976 988 Pianka ER Ecology and Natural History of Desert Lizards: Analyses of the Ecological Niche and Community Structure, 1986 New Jersey: Princeton University Press Schoener TW Gans C Competition and the niche In Biology Of The Reptilia 1977 London: Academic Press,Inc 35 136 Schoener TW Resource partitioning in ecological communities Science 1974 185 27 39 Sexton OJ Heatwole H Knight D Correlation of microdistribution of some Panamanian reptiles and amphibians with structural organization of the habitat Caribbean J Sci 1964 4 261 295 Scott NJ The abundance and diversity of herpetofauna of tropical forest litter Biotropica 1976 8 41 58 Petren K Case TJ Habitat structure determines competition intensity and invasion success in gecko lizards PNAS 1998 95 11739 11744 9751735 10.1073/pnas.95.20.11739 Tews J Brose U Grimm V Tielborger K Wichmann MC 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first 100 years of succession Trends Ecol Evol 1996 11 119 124 10.1016/0169-5347(96)81090-1 Pearman PB Velasco AM L¢pez A Tropical amphibian monitoring: A comparision of methods for detecting inter-site variation in species' composition Herpetologica 1995 51 325 337 Heyer WR Donnelly MA McDiarmid RW Hayek LC Foster MS Measuring and Monitoring Biologial Diversity: Standard Methods for Amphibians, 1994 Washington: Smithsonian Institution Press Legendre L Legendre P Numerical Ecology, 1998 Amsterdam, Netherlands: Elsevier Scientific Publications Williams SE Hero JM Rain forest frogs of the Australian wet tropics: guild classification and the ecological similarity of declining species Proc R Soc Lond B 1998 265 597 602 9881468 10.1098/rspb.1998.0336 Manly BJF Randomization and Monte Carlo Methods in Biology, 1991 London: Chapman and Hall Dutilleul P Stockwell JD Frigon D Legendre P The Mantel test versus Pearson's correlation analysis: Assessment of the differences for biological and 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==== Front BMC EcolBMC Ecology1472-6785BioMed Central London 1472-6785-4-111530419810.1186/1472-6785-4-11Research ArticleFitness benefits of trypsin proteinase inhibitor expression in Nicotiana attenuata are greater than their costs when plants are attacked. Zavala Jorge A [email protected] Ian T [email protected] Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, Beutenberg Campus, Hans-Knöll Strasse 8, Jena 07745, Germany2004 10 8 2004 4 11 11 25 3 2004 10 8 2004 Copyright © 2004 Zavala and Baldwin; licensee BioMed Central Ltd.2004Zavala and Baldwin; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The commonly invoked cost-benefit paradigm, central to most of functional biology, explains why one phenotype cannot be optimally fit in all environments; yet it is rarely tested. Trypsin proteinase inhibitors (TPIs) expression in Nicotiana attenuata is known to decrease plant fitness when plants compete with unattacked conspecifics that do not produce TPIs and also to decrease the performance of attacking herbivores. Results In order to determine whether the putative benefits of TPI production outweigh its cost, we transformed N. attenuata to silence endogenous TPI production or restore it in a natural mutant that was unable to produce TPIs. We compared the lifetime seed production of N. attenuata genotypes of the same genetic background with low or no TPI to that of genotypes with high TPI levels on which M. sexta larvae were allowed to feed freely. Unattacked low TPI-producing genotypes produced more seed capsules than did plants with high TPI levels. Caterpillar attack reduced seed capsule production in all genotypes and reversed the pattern of seed capsule production among genotypes. M. sexta larvae attacking genotypes with high TPI activity consumed more TPI, less protein, and move later to the young leaves. Larval masses were negatively correlated (R2 = 0.56) with seed capsule production per plant. Conclusions Our results demonstrate that the fitness benefits of TPI production outweigh their costs in greenhouse conditions, when plants are attacked and that despite the ongoing evolutionary interactions between plant and herbivore, TPI-mediated decreases in M. sexta performance translates into a fitness benefit for the plant. ==== Body Background The cost-benefit paradigm is central to functional biology and to ecological and evolutionary theory because fitness costs and benefits associated with a trait determine its equilibrium value in a population. If the trait offers fitness benefits to the population rather than costs, then selection should lead to beneficial allele(s) being fixed, which reduces variability [1]. Alternatively, when the fitness benefit of the trait also has a cost, intermediate frequencies of the trait may be favored because the benefit varies [1-3]. For example, resistance against natural enemies has costs as well as obvious benefits for fitness, as has been shown in insect-parasite, insect-parasitoid, plant-pathogen and plant-insect systems [4-7]. Herbivores can reduce seed production and other correlates of plant fitness, and this reduction can result in natural selection for either constitutively expressed or inducible plant defenses [8-10]. Current theory predicts that one benefit of induced defenses is to allow a plant to optimize its allocation of limiting resources to defense, growth, and reproduction [9]. Although defenses might benefit plants in the presence of herbivores, plant resistance to herbivores can be costly in the absence of enemies, and inducible expression of resistance traits allow plants to forgo or, to pay the potential fitness cost of resistance traits when they are needed [3,5,11-14]. Evidence for the existence of resistance costs and benefits from studies using plant species with constitutive and inducible defenses is increasing [3,14-16]. Experiments on natural populations of plants as diverse as Arabidopsis, Ipomea, Pastinaca and Trifolium have provided evidence for costs [2,17-20]. These experiments typically use quantitative genetic approaches to determine whether, in the absence of enemies, fitness and resistance are inversely correlated. However, attribution of fitness consequences to the expression of a particular defense trait in an environment either with or without herbivory is difficult, because genes that control the expression of defensive traits may have pleiotropic effects [21]. Ideally, one should assess the costs and benefits of inducible defenses in plants that differ only in the expression of genes that control (induced) resistance but are otherwise genetically identical [15]. Transformation technology provides a means of manipulating traits with unparalleled precision. Although the benefits of plant traits that provide resistance against herbivores are expected to equal or exceed their cost when the system is at evolutionary equilibrium [22-25], very few direct tests have been done. While costs and putative benefits of defense traits have been studied in separate experiments, their currencies are usually not comparable (i.e., plant fitness for the cost; herbivore performance for the benefits). Tests of the cost-benefit model using the same currency are few [5] and these studies do not consider the heterogeneity of the plant. Ecological interactions can be viewed as the net outcome of a series of cost-benefit optimizations in which both players respond to the variability in each others' defense traits. For example, there is enormous within-plant heterogeneity of defensive secondary metabolites. This heterogeneity could motivate within-plant movement of herbivores, so that they eat leaves of low fitness value rather than leaves of high fitness value, or it could motivate herbivores to move off plants and onto neighboring competitors [26,27]. Herbivores, in turn, can both readjust their metabolism to cope with the secondary metabolites as well as adjust their feeding positions to maximize their performance [27-29]. We present here a cost-benefit analysis of a plant-insect interaction in which the costs and benefits of a defensive protein are evaluated in the currency of plant fitness. Nicotiana attenuata [Torr. Ex Wats. (synonymous with Nicotiana torreyana Nelson and Macbr.)], a postfire annual native tobacco inhabiting the Great Basin Desert, has a number of well-described herbivore-induced direct and indirect defenses [30], which increase the fitness of plants under attack in natural populations [5,31]. Trypsin proteinase inhibitors (TPI) play an important defensive role in addition to nicotine [30]. We isolated cDNA from N. attenuata that coded for a TPI precursor belonging to the potato PI-II family with a 7-repeat TPI domain. The normal constitutive expression of this gene increases 4-fold after herbivore attack [32,33]. The elicitation of TPI expression in N. attenuata varies with ontogeny and leaf age [34], as is true for nicotine [35] and volatile emissions [36]. The within-plant pattern of systemic TPI induction at the rosette stage of growth suggests that the signal(s) triggering remote TPI induction follows a source-sink relationship; regardless of ontogenetic stage, if young sink leaves are damaged, TPI levels increase only in the attacked leaf, while older leaves are less sensitive to leaf damage and produce a less intense response in the attacked leaf, the systemic responses in young leaves is dramatic [34]. The spatial and temporal variability in N. attenuata's ability to deploy certain defenses against herbivores can be correlated with the relative fitness values of leaves growing at particular nodes. Removal of young and mature leaves at the elongation stage in N. sylvestris had a greater negative effect on fitness than did the removal of old leaves, but not at either the rosette or flowering stages, demonstrating the different fitness values of leaves growing at different nodes on a plant. Damage to younger leaves increases nicotine contents more than damage to older leaves does, suggesting that defense allocation is proportional to the fitness value of the tissue, as predicted by Optimal Defense (OD) theory [10,23,35,37]. Manduca sexta, a specialized lepidopteran herbivore, prefers elongating N. attenuata plants to rosette-stage plants for oviposition and places eggs on leaves in the middle section of the stem (from S1 to S3; Figure 1; [38]). TPIs of N. attenuata leaves reduce the growth of M. sexta larvae [32,33]. However, insects may adapt to high TPI levels, replacing the inhibited trypsin with the secretion of trypsins that are insensitive to the particular TPIs of the diet [29,39]. Intra-plant movement of the first instar larvae is very rare but common in the second-to fourth-larval instars [38]. Larger instars are heavier and more difficult to handle by insect predators, and also better able to defend themselves against an attack in their natural environment by vigorous movement and regurgitation (A. Kessler, personal communication). Larvae are particularly sensitive to jasmonate-induced defenses during the third instar (approx. 11 days after hatching), and can be motivated to move between adjacently growing plants [26] by the plant's induced defense. When M. sexta larvae were placed on MeJA-induced plants, larvae left the induced plants 1–3 days earlier than did larvae placed on uninduced plants, which dramatically reduced the leaf area consumed and larval weight gain [40]. Figure 1 Sketch of Nicotiana attenuata plant showing different leaf positions on either the rosette or the stem [38] and larval location. Larva depicts the leaf growing at node S1 on which a single M. sexta neonate was placed. TPI expression in N. attenuata is known to decrease lifetime seed production in unattacked but competing plants [32] and to decrease M. sexta performance in attacked plants [32]. Whether the TPI-mediated decrease in herbivore performance translates into a fitness benefit for the plant is unknown. In other systems, plants expressing high PI levels caused herbivores to grow more slowly, but they compensated by eating more tissue, a potential fitness detriment for the plant [41]. Here we provide a critical test of whether the fitness benefits of TPI expression outweigh their costs. We compare lifetime seed production of N. attenuata genotypes with either low or no TPI production to that of TPI-producing genotypes on which M. sexta larvae were allowed to feed freely for 11 days. TPI and protein content were measured in all genotypes at all leaf positions. M. sexta larval mass and movement were recorded, and we calculated and simulated the amount of TPI and protein consumed by the larvae from the larval movement and the TPI and protein concentration at each leaf position from each genotype. We used two independently transformed N. attenuata lines in which the expression of the pi gene was down-regulated by antisense expression of a 175 bp fragment of the N. attenuata pi gene (AS –, AS-), and untransformed wildtype plants (WT) of the same genetic background (an inbred line collected from Utah). In addition, we used a natural N. attenuata genotype collected from Arizona, which has a mutation in the endogenous 7-domain pi gene and does not produce pi transcripts or TPI activity (A). We transformed this genotype with the full-length cDNA of the 7-domain pi gene in a sense orientation under control of a constitutive promotor (S++), so that after 11 days of caterpillar attack it produced TPIs at 74 % of the level found in the stem leaves of the wildtype Utah genotype. Our analysis demonstrates that the fitness benefits of TPIs production outweigh their cost when plants are attacked. Results Spatial and temporal distribution of plant TPI/protein contents In order to determine the effect of caterpillar attack on TPI activity, measurements were made from all rosette and stem leaves before, and 4 and 11 d after larvae started to feed on S1 leaves (Fig. 1) from transformed (AS –, AS-, and S++) and untransformed (WT and A) genotypes (Fig. 2 and Fig. 1-4/Appendix 1 [see Additional file 1 ]). All genotypes had high within-plant heterogeneity of TPI activity and protein contents. Constitutive TPI levels in all genotypes on day 0 (before larvae started to feed) were higher in rosette leaves than in stem leaves (F1,70-AS – = 217.13; P <0.0001; F1,70-AS- = 357.76; P <0.0001; F1,70-WT = 209.8; P <0.0001; F1,70-S++ = 4.27; P = 0.04), while protein content showed the opposite pattern, with higher levels in stem leaves than in rosette leaves (F1,70-AS – = 331.8; P <0.0001; F1,70-AS- = 256.6; P <0.0001; F1,70-WT = 289.6; P <0.0001; F1,70-S++ = 1.87.1; P <0.0001; Fig. 1-4/Appendix 1) which persisted through the samplings performed on day 4 and 11 (data not shown). A-genotype plants had a similar pattern in protein content (data not shown; F1,70-A = 245.5; P <0.0001). Caterpillar attack increased levels and within-plant heterogeneity of TPI activity. Larval damage to WT plants increased TPI activity 2.5-fold in S1 leaves (F1,14 = 197.0; P <0.0001) and 1.7-fold in unattacked (F1,110 = 17.3; P <0.0001) stem leaves, and did not alter TPI activity in older rosette leaves 4 d after neonates started to feed (F1,62 = 0.04; P = 0.8; Fig. 2 and Fig. 1/Appendix 1). By day 11, TPI activity had increased in WT S1 leaves 4-fold (F1,22 = 183.3; P <0.0001), 2.5-fold in the stem leaves (S avg; F1,334 = 337.0; P <0.0001; Fig. 2), and also marginally (1.1-fold) on the rosette leaves (F1,94 = 8.6; P <0.004; Fig. 1/Appendix 1). Figure 2 TPI activity (mean ± SEM) from stem leaves and the leaf growing at node S1 of untransformed wild type Nicotiana attenuata plants of the Utah genotype (WT); two homozygous T3 independently transformed lines of the Utah genotype that had been transformed with a construct containing a 175 bp pi gene fragment in an antisense orientation (AS –, AS-); plants of a homozygous T3 transformed line of the Arizona genotype transformed with a construct containing the full-length pi gene in a sense (S++) orientation before attack (day 0); and either unattacked or attacked by M. sexta larvae 4 and 11 d after neonates started to feed on the leaf at S1 position. Thin bars indicate ± SEM. Levels and within-plant heterogeneity of TPI activity were either intermediate or low in AS compared to WT plants after larval damage. After 4 days of caterpillar attack, TPI levels in AS – and AS-genotypes were 60 % and 40 % lower than those of unattacked WT (F1,190-AS–total = 62.4; P < 0.0001; F1,190-AS-total = 23.4; P < 0.0001; Figs 2 and 3/Appendix 1). Caterpillar attack increased TPI activity 2.4-fold in S1 leaves in AS plants, attaining values that were 19% and 48% in AS – and AS-plants, respectively of that in attacked WT plants (F1,14-AS–S1 = 630.3; P < 0.0001; F1,14-AS-S1 = 193.2; P < 0.0001; Fig. 1); TPI levels in the stem leaves were 37% and 55% of that found in induced WT plants (F1,190-AS– = 89.8; P < 0.0001; F1,190-AS- = 42.1; P < 0.0001; Fig. 2). By day 11, TPI levels in stem leaves were 22% in AS – and 65% in AS-of the WT levels (F2,501= 225.5; P < 0.0001; Fig. 2). As expected, caterpillar attack did not affect either levels or within-plant heterogeneity of TPI activity of S++ plants. Compared to the constitutive levels of TPI activity in the WT, levels in S++ plants on day 4 were 30% higher (F1,190-total = 23.8; P < 0.0001; Fig. 4/Appendix 1). Caterpillar attack did not alter TPI activity in S++ plants (F1,430-total = 0.1; P = 0.06; Fig. 4/Appendix 1) which remained at approximately 90% of the induced WT plants in the S1 leaf and 1.1-fold at the plant level (averaged across all measured leaf positions; F1,14-S++-S1 = 3.8; P = 0.06; F1,190-total = 0.8; P = 0.3; Fig. 2). By day 11 d, TPI activity in S++ plants were 56% in the S1 leaf (F1,22 = 48.8; P < 0.0001) and 74% in stem leaves of the induced WT levels (F1,430 = 72.3; P < 0.0001; Fig. 2). As expected, the untransformed A genotype showed no TPI activity even after caterpillars had fed on the plant for 4 or 11 d. Protein levels did not differ significantly among genotypes. In summary, TPI levels in AS – and AS-genotypes in S1 and stem leaves were lower than in WT plants without differences in protein contents. Absolute TPI levels were substantially lower in the AS genotypes after caterpillar attack and S++ genotype produced TPI levels that were 74% of the activity found in induced WT plants. Within-plant movement of M. sexta larvae To determine the effect of TPI on within-plant movement of M. sexta larvae, we measured the position of each larva on each plant daily. Caterpillars on low TPI genotypes left the S1 leaf and moved to the BOTTOM of the plant earlier than those feeding on high TPI genotypes (Fig. 3). While larvae on WT plants started to move from the S1 leaf to the BOTTOM of the plant after 5 d, larvae on AS – plants started to move 2 d earlier (day 3), and those that fed on AS-plants started to move 1 d earlier (day 4; Fig. 3). This early larval movement resulted in more larvae on the TOP and MIDDLE parts of plants from the AS – genotype (51 %, 77 %, and 80%) than on WT plants (11 %, 37 %, and 49 %) during subsequent days (days 6–8; Mann-Whitney U-test; P <0.0001; Fig. 3). If caterpillars prefer to feed on leaves with low TPI levels, then we would expect to have higher defoliation levels of plants with either no or low TPI compared to those with high TPI levels, increasing the number of caterpillars on the BOTTOM after some days. By day 11, 65 % of the larvae on WT plants were on the top and 19 % were on the BOTTOM, while on AS – plants, 30 % were on the TOP and 48 % on the BOTTOM, and on AS-plants 44 % were on the TOP and 36 % on the BOTTOM (Mann-Whitney U-test; PWT-AS–TOP = 0.001; PWT-AS–BOTTOM = 0.03; PWT-AS-TOP = 0.01; PWT-AS-BOTTOM = 0.3; Fig. 3). Figure 3 Relative number of M. sexta larvae on different plant locations on WT, AS –, AS-, S++, and Arizona (A) genotypes during 11 days on leaves growing at node S1, or the bottom, middle or top part of the plant (Figure 1). A single M. sexta neonate was placed on the leaf growing at node S1 and larval movement was monitored. Similar movement patterns were found in larvae on S++ and A genotypes. Larvae on A plants moved earlier (day 4) from the leaf at node S1 and toward the MIDDLE and TOP of the plant compared to larvae on S++ plants (Fig. 3). This earlier movement was reflected in the number of larvae on the MIDDLE and TOP of the plant from day 6 to 9 with a greater percentage in A (23 %, 65 %, 69 %, and 84 %) than in S++ (8 %, 16 %, 28 %, and 56 %) genotypes (Mann-Whitney U-test; P <0.0001; Fig. 3). On day 11, there were no differences in the number of caterpillars between A and S++ plants at the BOTTOM and at TOP of the plant (Mann-Whitney U-test; PA-S++TOP = 0.35; PA-S++BOTTOM = 0.1; Fig. 3). In summary, lighter caterpillars moved later than heavier caterpillars upward within the plant during the first days, and on day 11 they moved later to the BOTTOM of the plant. Calculated and simulated TPI and protein consumed by M. sexta larvae We calculated the amount of TPI and protein consumed by M. sexta larvae during the first, second, and third instars from each larvae's instar-specific feeding site, the concentration of leaf protein and TPI at the feeding site, and the instar-specific consumption from literature values (Tables 1a and b/Appendix 1). Plant genotype strongly influenced the calculated amount of TPI and protein consumed. Calculated total TPI and TPI consumed during the first, second and third instars were the highest for larvae on WT (16.7 g total) and the lowest for larvae on AS – (2.9 g total) plants (F2,76-Total = 888.6; P < 0.0001; F2,76-First = 28419.3; P < 0.0001; F2,76-Second = 442.8; P < 0.0001; F2,76-Third = 671.2; P < 0.0001; Fig. 5/Appendix 1). During the second instar, larvae on AS – plants consumed the highest calculated amount of protein (1.5 g), larvae on WT plants, the lowest (0.8 g), but no differences were found between genotypes during the first and second instars (F2,76-First = 1.8; P = 0.1; F2,76-Second = 87.14; P < 0.0001; F2,76-Third = 2.1; P = 0.1; Fig. 5/Appendix 1). As expected, the calculated total amount of protein consumed was higher on larvae fed on AS – (7.0 g) than those fed on either AS-(6.6 g) or WT (6.3 g) genotypes (F2,76-Total = 11.6; P < 0.0001; Fig. 5/Appendix 1). Larval mass of caterpillars fed on WT, AS –, and AS-genotypes was affected by the amount of TPI (F2,76-11d = 10.2; P = 0.0001) but not by protein consumed. Similar results were found when larvae fed on S++ and A genotypes. Second instar larvae on A consumed more protein (1.3 g) than those on S++ (0.8 g) plants, but no differences were found during the first and third instars (F1,49-Second = 152.9; P < 0.0001; F1,49-Third = 1.0; P = 0.3; Fig. 5/Appendix 1). The calculated total amount of protein consumed was higher for larvae on A (7.0 g) than on S++ (6.3 g) genotypes (F1,49-Total = 7.8; P = 0.007; Fig. 5/Appendix 1). Larval mass of caterpillars on A and S++ genotypes was affected by the amount of TPI and protein consumed (F1,49-11d = 49.8; P < 0.0001). In summary, caterpillar fed on high TPI-genotypes consumed more TPI and less protein than those larvae fed on low TPI-genotypes. We estimated the effect of the differences in larval movement by simulating TPI and protein consumption by transposing movement and consumption patterns from untransformed (WT and A) to transformed (AS –, AS-, and S++) plants as explained in the supplemental section (Fig. 6 and Tables 1a and b/Appendix 1). Patterns of larval movement on WT plants (SWT) did not alter TPI consumed on the AS (AS – and AS-) genotypes when WT movement data were transposed to larvae on AS genotypes (PAS – = 0.9; PAS- = 0.09); the highest values were found in the calculated WT genotype (F4,126 = 545.5; P < 0.0001; Fig. 6/Appendix 1). WT daily movement patterns decreased SWT protein consumed from AS – genotype plants (F4,126 = 11.7; P < 0.0001; Fig. 6/Appendix 1). Larval movement on AS – plants increased TPI and protein consumed on WT plants (F4,127-TPI = 473.8; P < 0.0001; F4,127-Protein = 8.1; P < 0.0001; Fig. 6/Appendix 1). Figure 6 Seed capsule production per plant of N. attenuata genotypes (WT, AS –, AS-, S++ and A), regressed against M. sexta larvae mass (g) 11 d after neonates started to feed on the leaf at S1 position (elongation stage). Line represents a regression fitted to the points (Y = -12.773 (g) + 42.229; R2 = 0.5642). Larval movement on A plants did not change the amount of TPI consumed on S++ genotype plants (F1,49 = 1.5; P = 0.2) but did increase the amount of protein consumed (F2,74 = 7.4; P = 0.001; Fig. 6/Appendix 1); larval movement on S++ plants did not change protein consumed on A genotype (F2,73 = 3.8; P = 0.2; Fig. 6/Appendix 1). In summary, when larval movement patterns on low TPI plants were transposed to high TPI genotypes, protein and TPI consumption increased. Transposing WT movement patterns to AS – genotype decreased the amount of protein consumed. Fitness consequences of TPI expression for plants attacked by M. sexta larvae To determine whether expression of TPIs increases N. attenuata's fitness when plants are attacked by M. sexta larvae, we measured caterpillar mass on and capsule number per plant from transformed and untransformed genotypes with either low or no TPI activity (A, AS –, and AS-) and high TPI activity (WT and S++). Larval mass of caterpillars fed on low TPI genotypes were higher (45-21 %) than those fed on genotypes with high TPI activity (F4,35-4d = 20.0; P < 0.0001; F4,195-11d = 8.6; P < 0.0001; Fig. 4), those that fed on either WT or S++ (F1,14-4d = 0.02; P = 0.9; F1,78-11d = 0.1; P = 0.6) or AS – or A (F1,14-4d = 0.01; P = 0.9; F1,78-11d = 1.6; P = 0.2) did not differ (Fig. 4). Figure 4 M. sexta mass (mean ± SEM) at 4 and 11d after neonates started to feed on leaves at S1 position (elongation stage) from WT, AS –, AS-, S++, and Arizona (A) genotypes. Bars with the same letter are not significantly different at P < 0.05 determined by one-way ANOVA. Thin bars indicate ± SEM. We measured lifetime seed capsule number per plant on unattacked and attacked plants and calculated the mean differences and the percentage mean differences between treatments in order to estimate fitness consequences of constitutive and inducible TPI production. As expected, mean capsule number in unattacked plants was higher on genotypes with either low or no TPI activity (A and AS –) than on genotypes with intermediate and high TPI activity (WT, S++, and AS-; Fig. 5), which reflects the fitness cost of TPI production. Eleven days of caterpillar attack reduced seed capsule production per plant in all genotypes and reversed the pattern of seed capsule production among high and low TPI-containing genotypes. Within the group of transformed (AS – and AS-) and untransformed (WT) unattacked plants from the Utah genotype, mean capsule number was higher (22–25 %) on the genotype with low TPI activity (AS –) than on genotypes with intermediate and high TPI activity (AS-and WT; F2,81 = 8.6; P = 0.004; Fig. 5); however after 11 d of caterpillar attack, mean capsule number, absolute and relative mean difference in capsule number were the highest on WT (15 capsules) and the lowest on AS – (4 capsules) genotypes (F2,81 = 25.3; P < 0.0001; Fig. 5 and Table 1). Within the Arizona genotypes, mean capsule number of unattacked plants was higher on the genotype with no TPI activity (A; 49 capsules) than on the genotype with high TPI activity (S++; 35 capsules; F1,54 = 16.4; P = 0.0002; Fig. 5). However, when plants were attacked, mean capsule number as well as absolute and relative mean difference in capsule number were higher on S++ (23 capsules) than on A genotypes (17 capsules; F1,54 = 7.9; P = 0.006; Fig. 5 and Table 1). Figure 5 Mean capsule number from WT, AS –, AS-, S++, and A genotypes that were either unattacked or attacked by Manduca sexta larvae for 11 days. Bars with the same letter within a group are not significantly different at P < 0.01 determined by one-way ANOVA. Thin bars indicate ± SEM. Table 1 Absolute and relative mean differences between treatments in seed capsule production and TPI levels from either untransformed wildtype (WT) or homozygous T3 independently transformed lines of a WT genotype of Nicotiana attenuata which had been transformed with constructs containing the pi gene in an anti-sense orientation (AS –, AS-); absolute and relative mean differences between untransformed plants of the Arizona (A) genotype and plants of the Arizona genotype transformed with constructs containing the full-length pi gene in a sense (S++) orientation, that were either unattacked or attacked by Manduca sexta larvae for 11 days. Genotypes Mean diff. in capsule number % Mean diff. in capsule number P TPI levels WT 18.04 54.18 <0.0001 High AS-- 40.36 91.35 <0.0001 Low AS- 23.14 68.14 <0.0001 Intermediate S++ 12.64 35.47 <0.0001 High A 32.25 65.58 <0.0001 No TPI P-values are from one-way ANOVAs between treatments. In order to determine the effect of caterpillar attack on seed capsule production per plant, we regressed caterpillar mass against seed capsule production per plant from transformed and untransformed genotypes and found that a linear equation (Y = -12.7 (g) + 42.2; R2 = 0.5; Fig. 6; P < 0.0001) represented the best fit. The relationship suggests that the higher the M. sexta larvae mass, the lower the seed capsule number production per plant. Discussion Our experiments demonstrate that the benefits of TPI expression in N. attenuata grown in greenhouse conditions outweigh their costs when plants are attacked by M. sexta larvae. Unattacked plants with low constitutive TPI levels produced more seed capsules (AS–: 44, AS-: 34 and A: 49 capsules) than did plants with high TPI levels (WT: 33 and S++: 35 capsules), and 11 days of M. sexta attack reduced seed capsule production per plant in all genotypes and reversed the pattern of seed capsule production with higher reductions in AS (AS–: 91 % and AS-: 68 %) and A (65 %) than in WT (54 %) and S++ (35 %) plants (Fig. 5 and Table 1). This differential reduction in seed capsule production amongst genotypes correlated negatively with larval mass. Across all genotypes, the larger the larval mass, the lower the number of capsules per plant (Fig. 6). This result is consistent with previous demonstrations that endogenous TPIs decrease the performance of M. sexta [33] and with the central prediction of the Optimal Defense theory, namely that defense is costly [23,35,37]. Moreover, the results highlight the heuristic value of the cost-benefit paradigm for functional studies. However, conclusive evidence that TPI expression in N. attenuata outweigh their costs when plants are attacked will require field experiments in which both ecological and allocation costs of defense can arise. For example, constitutive and inducible TPI production incurs large fitness costs in N. attenuata when plants where grown with competitors [32,42], one of the dominant selective factors for this species [30]. In addition, other factors such as temperature and M. sexta predators can affect feeding damage [38]. Despite the central role of the cost-benefit model of inducible defenses, the vast majority of research in this area examines how inducible defenses influence either herbivore performance or plant fitness in separate experiments and their currencies are usually not comparable (i.e., plant fitness for the cost; herbivore performance for the benefits). Few studies have tested the cost-benefit model by measuring both costs and benefits in the same currency (plant fitness for both the costs and benefits) and have elicted plant defenses by either applying methyl jasmonate or damaging leaves [5,11]. However, because of the pleiotorpic effects of the elicitors, the observed fitness differences do not arise solely from the expression of the resistant trait [30,43], and therefore these studies are likely to overestimate the fitness cost of resistance. Direct genetic manipulation of TPI expression allowed us measure the costs and benefits of a defensive protein in a plant-insect interaction in the common currency of plant fitness. Transformation technology gave us the means to manipulate TPI expression with high precision. Antisense expression of the pi gene reduced constitutive and caterpillar induced TPI levels in AS – and AS-genotypes (by 35–80% of the activity of WT) in S1 and stem leaves without influencing protein contents. Caterpillar attack increased TPI levels 2–2.5-fold in either WT or AS genotypes (Fig. 2; Figs 1-3/Appendix 1) but the absolute levels were substantially lower in the AS genotypes. Transformation of the A genotype with a functional TPI gene under the control of a constitutive promoter (S++ genotype) produced TPI levels that were 74% of the activity found in caterpillar attacked WT plants (Fig. 2; Fig. 4/Appendix 1). Because these transformed lines did not differ in any other measured defense traits [42], they allowed us to examine the defensive function of TPIs by constraining plant responses to herbivore attack and observe unconstrained herbivore behavior in response to these constrained plant responses. In this way, the dynamics of the plant responses, or the lack thereof, are reflected in the herbivore behavior. Low constitutive TPI expression in the host plant may increase proteolytic enzyme activity in the guts of neonates, digestion efficiency and the growth rates [44] (Fig. 4). This early increase in larval growth rate translates into increases in pupal mass, which is an accurate proxy for fecundity in Lepidoptera [45,46], but may also profoundly influence larval movement. Given the large within-plant heterogeneity in food quality, it is reasonable to expect a complex resource-oriented larval behavior that changes with instars [27,47]. Moving has been shown to be costly during the first 3 instars [26,38], but these costs are thought to decrease with size [48]. Larvae with larger mass (on either low or no TPI-producing genotypes) left the S1 leaf 1–2 days earlier than did those with lower mass (on high TPI-producing genotypes; Fig. 3). The heavier larvae moved earlier than lighter larvae to young leaves which typically have higher levels of protein and water contents [27,48,49]. Based on the calculations, larvae fed on high TPI genotypes consumed 3–4 fold more TPI and 10 % less protein than did larvae feeding low TPI genotypes over the 11d of the experiment (Fig. 3 and 4; Fig. 5/Appendix 1). Since we did not measure the amount of leaf consumed by larvae and the values used for the calculation of protein consumption are from plants with natural TPI levels, the calculations likely underestimate the amount of protein consumed. These results suggest that a high TPI content keeps caterpillars from feeding on the high-protein younger leaves at the TOP of the plants possibly by decreasing larval mass and thereby their ability to move. Larval movement influences the caterpillar's ability to compensate for variation in diet quality. By moving, caterpillars can exploit the high within-plant heterogeneity in food quality to compensate for nutritional imbalances. For example, Helicoverpa zea larvae feed on multiple plant structures to balance their amino acid requirements [50]. M. sexta larvae fed low protein and nutritionally unbalanced diets compensated not only for the decreased protein intake [51] but also for unbalanced nutrition by selecting diets high in the missing nutrients which increased larval growth rates [52,53]. Growth depends on nutrient ratios, and insects may use behavioral and post-ingestive mechanisms to compensate for nutrient imbalances [54,55]. To estimate the consequence of higher caterpillar mass on movement, we transposed the larval location data of caterpillars from those observed on low-to high-TPI genotypes, and found increased larval protein (10 %) and TPI (12 %) consumption (Fig. 6/Appendix 1). Transposing daily larval location data in the opposite direction decreased (by 10 %) protein consumed but did not influence TPI consumption (Fig. 6/Appendix 1). These calcuations suggest that M. sexta caterpillars may adjust their feeding positions to minimize TPI consumption and maximize protein intake. Hence the naturally occurring high TPI levels delay larval growth and prevent caterpillars from feeding on high-quality younger leaves, which may have a high fitness value for the plant [35,50,56]. The interaction between N. attenuata and M. sexta starts with moths ovipositing on leaves at the bottom of the plant; oviposition is influenced by temperature, food quality and quantity, and predation risk [38]. Plants respond by increasing TPI levels, which decreases larval mass and survivorship [33], and by increasing the emission of volatile organic compounds, which alters oviposition choices and attracts the generalist predator Geocoris pallens to feeding larvae [31]. Geocoris is size selective and preferentially attacks eggs and larvae in the first three instars. The up-regulation of TPIs by herbivore attack slows larval growth and keeps larvae in stages that are more vulnerable to the predator, thus increasing larval mortality [57]. Interestingly, the volatile signals that function as indirect defenses by attracting Geocoris to feeding larvae are elicited by the same signals that elicit TPI production [34,36,58], providing the mechanism of coordination among these defense system. Once larvae reach a mass that can compensate for the cost of movement, they leave the leaf with high TPI levels and move upward within the host plant and feed preferentially on young leaves with high levels of protein and nicotine, which increases larval mass and decreases plant fitness [35,38,51]. A starvation period during the firsts instars was found to reduce M. sexta larval development more than feeding on fully JA-induced (high TPIs) N. attenuata leaves [40]. Thus for these larval instars, the costs of movement, which include increases in starvation and predation risks are likely greater than the costs of coping with a plant's induced defenses. Other generalist herbivores on N. attenuata, namely noctuid larvae and weevil beetles, usually attack older leaves that are lower in nutrients as well as nicotine [30,35,38]. Nicotine is not an efficient defense against M. sexta, because this insect is adapted to feed on N. attenuata and larvae can detoxify nicotine [59-61]. Moreover, its attack down-regulates nicotine production which could be sequestered by the herbivore and maybe co-opted and used as a defense against parasitoids [30,62,63]. Hence the plant relies on other defenses when attacked by M. sexta larvae: TPIs, for example, decrease larval mass and prevent caterpillars from feeding on leaves with high fitness value for the plant. This delayed in caterpillar movement upward within the plant, maybe a result of larvae adaptation to high leaf-TPI levels by increasing the production of insensitive gut proteases to TPIs [29]. Eliciting only those defenses that confer resistance to the attacking herbivore (targeting), rather than the entire defensive repertoire, may minimize the cost of resistance [14]. Conclusions We conclude that despite the ongoing evolutionary interaction between N. attenuata and M. sexta, TPI-mediated decreases in herbivore performance translates into a fitness benefit for the plant. Methods Plant material and transformation N. attenuata used in this study were grown from seeds collected from either Utah [5] or Arizona [32] and inbred 10 and 4 generations, respectively. In order to silence the expression of N. attenuata's pi gene in the genotype collected in Utah (WT), WT was transformed by an Agrobacterium-mediated transformation procedure with pNATPI1, which contains 175 bp of N. attenuata's 7-repeat domain pi gene in an anti-sense orientation (AS), as described in [32]. Southern gel blot analysis confirmed that all T3 lines were single-copy independent transformants [42]. In this study, we used a genotype of N. attenuata collected from Arizona (A), with methyl jamonate (MeJA)-inducible nicotine levels identical to that found in WT plants, but completely lacking the ability to produce TPIs or accumulate TPI mRNA [32]. More recently, the mutation in the 7-domain repeat pi of A plants has been characterized and found to be located in the 5'signal peptide, resulting in a premature stop codon (J. Wu and I.T. Baldwin, unpublished data). Because we never detected TPI activity with radial diffusion assay in A genotype [34], nor have we detected TPI mRNA transcript with either northern blot analysis or reverse transcriptase-PCR, we suggest that this transcript is rapidly silenced [33]. Plants of the A genotype were transformed with a binary transformation vector pRESC2PIA2 containing the full-length 7-domain N. attenuata pi gene from the WT genotype in the sense orientation under control of the constitutive CaMV 35S promotor [42]. Several T3 lines harboring a single copy of the transgene [42] were screened for TPI activity, and all had TPI activity comparable to that of elicited WT plants. One of these A lines (S++) with 60% of the activity of MeJA-elicited WT plants was selected for study. Arizona non-transformed plants (A) had no detectable TPI activity. All of these transformed and untransformed genotypes were used in the experiments and the quality of the seeds that these genotypes produce do not differ from the seed quality of the WT. Bioassay experiments and plant fitness determination In order to determine the effect of M. sexta herbivory on the fitness of N. attenuata's genotypes using either down-regulation or restored expression of the pi gene, a single M. sexta neonate was placed on the leaf growing at node S1 (Fig. 1) of 48 soil-grown plants in elongation stage of AS lines (AS – and AS-), on A line transformed to express the functional pi (S++), and on untransformed genotypes (WT and A). Larvae were allowed to move and feed freely on plants for 11 days. Their mass was determined 4 and 11 days after hatching. Larval movement on the plant during this time was monitored, and larval location on the plant classified as follows: S1 (leaf where larvae started to feed), BOTTOM (from 0 to S3 leaf position), MIDDLE (from S4 to S5 leaf position), and TOP (from S6 to S9 leaf position; Fig. 1). Eggs of Manduca sexta L. (Lepidoptera: Sphingidae) were obtained from Carolina Biological Supply Company (Burlington, North Carolina, USA) and placed in plastic containers (200 mL) on a moist tissue. The containers were kept in climate chambers at 28°C and 65 % relative humidity under a 16:8 h light:dark photoperiod until the eggs hatched. Seeds were germinated in diluted liquid smoke solutions as described in [64]. Seedlings were transplanted in 1-L pots in a glasshouse under the conditions described in [42] with 1000 – 1300 μmol m-2 s-1 PPFD supplied by 450 W Na-vapor HID bulbs. To compare the lifetime reproductive performance among genotypes after being either unattacked or attacked by M. sexta larvae, we recorded the number of seed capsules per plant from 28 plants (8 + 12 plants were used to TPI determination) of each genotype and treatment combination two weeks after last watering day. Daily watering stopped 21 d after neonates started to feed on the leaf, in order to mimic the drying and termination of growth in the plant's natural habitat, the Great Basin Desert. The number of capsules per plant reflects the lifetime reproductive output (seeds) in N. attenuata under natural or glasshouse conditions [5,65]. Constitutive and TPI activity induced by caterpillar damage were determined from stem and rosette leaves before the larvae were placed on the leaf at node S1 (8 plants; 4 rosette leaves; 5 stem leaves; Fig. 1), and 4 (8 plants; 4 rosette leaves; 8 stem leaves) and 11 (12 plants; 4 rosette leaves; 14 stem leaves) days after the larvae started to feed. During the last harvest TPI activity was also determined on axillary leaves from S1 to S4 nodes. Protein concentrations and TPI activity were measured and expressed as nmol mg-1 as described in [34]. Larvae TPI and protein consumption were calculated and simulated as explained in the supplemental section. Statistical analysis Data were analyzed with Stat View, Version 5.0 (SAS, 1998). The TPI, protein and larval mass, and calculated and simulated values were analyzed by ANOVAs followed by Fisher's protected LSD post-hoc comparisons in all experiments. Differences in larval number on plants were analyzed with the Mann-Whitney U-test. Author's contributions JAZ carried out the experiments and analyzed the data, while planning of the experiment and writing of the manuscript was a joint effort by JAZ and ITB. Supplementary Material Additional File 1 Calculation of TPI and protein consumed by M. sexta larvae. Table 1. Combination of TPI and protein from different N. attenuata genotypes and larval location of different genotypes that have either calculated (C) or simulated (S) values. Fig. 1: TPI activity (mean ± SEM) and protein content from different leaf positions of WT plants at the elongation stage. Fig. 2: TPI activity (mean ± SEM) and protein content from different leaf positions of AS – plants at the elongation stage. Fig. 3: TPI activity (mean ± SEM) and protein content from different leaf positions of AS-plants at the elongation stage. Fig. 4: TPI activity (mean ± SEM) and protein content from different leaf positions of S++ plants at the elongation stage. Fig. 5: Calculated TPI and protein consumed by M. sexta larvae fed on WT, AS–, AS-, S++, and A genotypes during the first, second and third instars. Fig. 6: Calculated and simulated TPI and protein consumed by M. sexta larvae. Click here for file Acknowledgments We thank the Max Planck Gesellschaft for financial support, M. Lim for plant transformations, B. Berger for technical assistance in TPI determinations and E. Wheeler for editorial assistance. ==== Refs Simms EL Rausher MD Costs and benefits of plant resistance to herbivory. Am Nat 1987 13 570 581 10.1086/284731 Mauricio R Costs of resistance to natural enemies in field populations of the annual plant Arabidopsis thaliana. Am Nat 1998 151 20 28 10.1086/286099 Cipollini DF Purrington CB Bergelson J Costs of induced responses in plants Basic Appl Ecol 2003 4 79 85 Kraaijeveld AR Ferrari J Godfray HCJ Costs of resistance in insect-parasite and insect-parasitoid interactions. Parasitology 2002 125 S71 S82 12622330 10.1017/S0031182002001762 Baldwin IT Jasmonate-induced responses are costly but benefit plants under attack in native populations Proc Natl Acad Sci USA 1998 95 8113 8118 9653149 10.1073/pnas.95.14.8113 Milks ML Myers JH Leptich MK Cost and stability of cabbage looper resistance to nucleopolyhedrovirus. 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Science 1985 230 895 899 Agrawal AA Phenotypic plasticity in the interactions and evolution of species Science 2001 294 321 326 11598291 10.1126/science.1060701 van Dam NM Hermenau U Baldwin IT Instar-specific sensitivity of specialist Manduca sexta larvae to induced defenses in their host plant Nicotiana attenuata Ecol Entom 2001 26 578 586 10.1046/j.1365-2311.2001.00366.x Denno RF McClure MS Variable plants and herbivores in natural managed systems. Academic Press. 717 p. 1983 Cloutier C Jean C Fournier M Yelle S Michaud D Adult colorado potato beetles, Leptinotarsa decemlineata compensate for nutritional stress on Oryzacystatin I- Transgenic potato plants by hypertrophic behavior and over-production of insensitive proteases. Arch Insect Biochm Physiol 2000 44 69 81 10.1002/1520-6327(200006)44:2<69::AID-ARCH2>3.0.CO;2-6 Jongsma MA Bakker PL Peters J Bosch D Stiekema WJ Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition. Proc Natl Acad Sci USA 1995 92 8041 8045 7644535 Baldwin IT An ecologically motivated analysis of plant-herbivore interactions in native tobacco Plant Physiol 2001 127 1449 1458 11743088 10.1104/pp.127.4.1449 Kessler A Baldwin IT Defensive function of herbivore-induced plant volatile emissions in nature. 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Proc Natl Acad Sci USA 2004 101 1607 1612 14757829 10.1073/pnas.0305096101 Hermsmeier D, Schittko U Baldwin IT Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata: III. Fatty acid-amino acids conjugates in herbivore oral secretion are necessary and sufficient for hebivore-specific plant responses. Plant Physiol 2001 125 683 700 11161026 10.1104/pp.125.2.683 Zalucki MP Clarke AR Malcom SB Ecology and behavior of first instar larval lepidoptera. Annu Rev Entomol 2002 47 361 393 11729079 10.1146/annurev.ento.47.091201.145220 Awmack SC Leather SR Host plant quality and fecundity in herbivorous insects. 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Arch Insect Biochm Physiol 1996 32 107 130 10.1002/(SICI)1520-6327(1996)32:1<107::AID-ARCH7>3.3.CO;2-7 Woods HA Patterns and mechanism of growth of fifth-instar Manduca sexta caterpillars following exposure to low- or high-protein food during early instars. Physiological and Biochemical Zoology 1999 72 445 454 10438682 10.1086/316678 Simpson SJ Simmons MSJ Blaney WM A comparison of dietary selection behavior in larval Locusta migratoria and Spodoptera littoralis. Physiol Entomol 1988 13 225 238 Thompson SN Redak RA Wang LW Altered dietary nutrient intake maintains metabolic homeostasis in parasitized larvae of the insect Manduca sexta L. The Journal of Experimental Biology 2001 204 4065 4080 11809781 Raubenheimer D Simpson SJ Integrating nutrition: a geometrical approach. Ent Exp App 1999 91 67 82 Raubenheimer D Simpson SJ The geometry of compensatory feeding in the locust. Anim Behav 1993 45 953 964 10.1006/anbe.1993.1114 Mattson WJ Herbivory in relation to plant nitrogen content. Ann Rev Ecol Syst 1980 11 119 161 10.1146/annurev.es.11.110180.001003 Williams IS Slow-growth, high-mortality - a general hypothesis, or is it? Ecol Entom 1999 24 490 495 10.1046/j.1365-2311.1999.00217.x Halitschke R, U. Schittko G. Pohnert W. Boland Baldwin IT Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. III. Fatty acid-amino acid conjugates in herbivore oral secretions are necessary and sufficient for herbivore-specific plant responses. Plant Physiol 2001 125 711 717 11161028 10.1104/pp.125.2.711 Snyder MJ Err-Lieh H Feyereisen R Induction of cytochrome P-450 enzyme activities by nicotine in the tobacco hornworm, Mannduca sexta. J Chem Ecol 1993 19 2903 2916 Appel HM Martin MM Significance of metabolic load in the evolution of host specificity of Manduca sexta Ecology 1992 73 216 228 Campo del ML Renwick JA Dependence on host constituents controlling food acceptance by Manduca sexta larvae Ent Exp App 1999 93 209 215 10.1046/j.1570-7458.1999.00580.x Barbosa P Gross P Kemper J Influence of plant allelochemicals on the tobacco hornworm and its parasitoid, Cotesia congregata Ecology 1991 72 1567 1575 Winz RA Baldwin IT Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. IV. Insect-induced ethylene suppresses jasmonate-induced accumulation of nicotine biosynthesis transcripts. Plant Physiol 2001 125 2189 2202 11299398 10.1104/pp.125.4.2189 Baldwin IT, Staszakkozinki L Davidson R Up in smoke: I. Smoke-derived germination cues for the post-fire annual Nicotiana attenuata Torr. Ex. Watson J Chem Ecol 1994 20 2345 2371 Baldwin IT Gorham D, Schmelz EA, Lewandowski CA Lynds GY Allocation of nitrogen to an inducible defense and seed production in Nicotiana attenuata Oecologia 1998 115 541 552 10.1007/s004420050552

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==== Front BMC Complement Altern MedBMC Complementary and Alternative Medicine1472-6882BioMed Central London 1472-6882-4-101529402110.1186/1472-6882-4-10Case ReportThe effect of Fucus vesiculosus, an edible brown seaweed, upon menstrual cycle length and hormonal status in three pre-menopausal women: a case report Skibola Christine F [email protected] School of Public Health, Molecular Epidemiology and Toxicology Laboratory, University of California, Berkeley, CA 94720, USA2004 4 8 2004 4 10 10 14 1 2004 4 8 2004 Copyright © 2004 Skibola; licensee BioMed Central Ltd.2004Skibola; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Rates of estrogen-dependent cancers are among the highest in Western countries and lower in the East. These variations may be attributable to differences in dietary exposures such as higher seaweed consumption among Asian populations. The edible brown kelp, Fucus vesiculosus (bladderwrack), as well as other brown kelp species, lower plasma cholesterol levels. Since cholesterol is a precursor to sex hormone biosynthesis, kelp consumption may alter circulating sex hormone levels and menstrual cycling patterns. In particular, dietary kelp may be beneficial to women with or at high risk for estrogen-dependent diseases. To test this, bladderwrack was administered to three pre-menopausal women with abnormal menstrual cycling patterns and/or menstrual-related disease histories. Case Presentation Intake of bladderwrack was associated with significant increases in menstrual cycle lengths, ranging from an increase of 5.5 to 14 days. In addition, hormone measurements ascertained for one woman revealed significant anti-estrogenic and progestagenic effects following kelp administration. Mean baseline 17β-estradiol levels were reduced from 626 ± 91 to 164 ± 30 pg/ml (P = 0.04) following 700 mg/d, which decreased further to 92.5.0 ± 3.5pg/ml (P = 0.03) with the1.4 g/d dose. Mean baseline progesterone levels rose from 0.58 ± 0.14 to 8.4 ± 2.6 ng/ml with the 700 mg/d dose (P = 0.1), which increased further to 16.8 ± 0.7 ng/ml with the 1.4 g/d dose (P = 0.002). Conclusions These pilot data suggest that dietary bladderwrack may prolong the length of the menstrual cycle and exert anti-estrogenic effects in pre-menopausal women. Further, these studies also suggest that seaweed may be another important dietary component apart from soy that is responsible for the reduced risk of estrogen-related cancers observed in Japanese populations. However, these studies will need to be performed in well-controlled clinical trials to confirm these preliminary findings. ==== Body Background Epidemiological studies show that incidence rates of estrogen-dependent diseases such as cancers of the breast, endometrium and ovary are among the highest in Western, industrialized countries, while rates are much lower in China and Japan [1,2]. These disparities may be attributable, in part, to differences in dietary and environmental exposures associated with affluent and modern lifestyles that promote estrogenic stimulation and hormone imbalances [3-5]. Although the mechanisms are not fully understood, epidemiological and experimental data suggest that exposure to estrogens, through endogenous production and exogenous exposures resulting in an imbalance in the estrogen/progesterone ratio, may be the most critical determinants in disease risk [6-8]. In estrogen-sensitive tissues, estrogen triggers cell proliferation, and through prolonged stimulation, hyperplasia [9] and possibly neoplasia can occur. Reproductive factors associated with increased exposure to menstruation resulting in persistent and sustained estrogenic stimulation, such as shorter menstrual cycles, reduced parity, early menarche, and late menopause, are known to increase risk of endometriosis and estrogen-dependent cancers [10,11], while post-menopausal obesity, hormone replacement therapy and alcohol consumption may be associated with increased breast cancer risk [12-14]. Therefore, limiting exposure to estrogens and reducing the overall number of menstrual cycles in one's lifetime through dietary and lifestyle changes may be the simplest means to reduce disease risk. In particular, the identification of dietary compounds that have estrogen- reducing effects holds great promise in developing chemopreventive strategies to abrogate risk of these diseases. Studies show that Japanese women have longer menstrual cycle lengths (greater than the 28 day average) and lower circulating estrogen levels compared to Western populations [15-17], which until now has been at least partly attributed to the increased intake of soy protein among Asian populations [18-20]. Another less explored component but main staple of the Japanese diet is seaweed, which accounts for approximately 10–25% of their food intake [21,22]. Other reported estimated daily intakes are as high as 3–13 g/day [23]. A major source of dietary seaweed among Japanese populations is the edible brown kelp, wakame (Undaria pinnatifida) and kombu (Laminaria japonica). These species and the Atlantic brown kelp, bladderwrack (Fucus vesiculosus), have been shown to exert powerful anti-hypertensive activity related to angiotensin-I-converting enzyme inhibition [24], to possess antibacterial and antioxidant properties related to their high polyphenolic content [25], and to prevent dioxin absorption and accelerate dioxin excretion in rats [26]. Other chemopreventive properties such as antiviral activity [27,28], immunostimulatory effects [29], anti-proliferative effects on 7,12-dimethylbenz(a)-anthracene-induced rat mammary tumors [30,31], and anti-tumor and anti-metastatic activities in xenograft mouse models [32], have been associated with the high level of sulfated polysaccharides, also known as fucoidans, found in brown seaweed. Intake of bladderwrack, as well as other brown kelp species, also has been shown to alter cholesterol metabolism and to significantly lower plasma cholesterol levels [33,34]. A possible mechanism of action involves competitive inhibition by fucosterols found in kelp. Since cholesterol is the precursor involved in steroid hormone biosynthesis, a reduction in cholesterol bioavailability could lower circulating plasma 17β-estradiol levels that may lead to alterations in menstrual cycling patterns in pre-menopausal women. Until now, no studies have been performed in humans to determine the effects of brown kelp on menstrual cycling patterns and sex hormone status in pre-menopausal women, particularly in women with or at risk for estrogen-dependent diseases. To explore the hypothesis that kelp consumption could reduce circulating17β-estradiol levels and attenuate menstrual cycle irregularities, bladderwrack was administered to three pre-menopausal women with abnormal menstrual cycling patterns and/or menstrual-related disease histories. Case presentation Three pre-menopausal women with abnormal menstrual cycling histories volunteered for the present study. An abnormal menstrual cycle was defined as one or more of the following: menstrual cycles of <26 or >32 days in length; menstrual cycles consisting of >8 menstruating days; or anovulatory menstrual cycling. Study subject characteristics are outlined in Table 1. Subject 1 had a history of hypermenorrhea (excessive blood loss during menstruation), polymenorrhea (shorter than average menstrual cycle length of 28 days), anovulatory menstrual cycles, and was diagnosed with luteal phase deficiency and endometriosis (through laparoscopy). Subject 2 suffered from hypermenorrhea and polymenorrhea. Subject 3 suffered from hypermenorrhea and was diagnosed with endometriosis. All three women reported a history of dysmenorrhea (painful menses). Otherwise, all women were in general good health and free of any chronic diseases. All women were active and exercised approximately three times per week. No hormones or other medications were taken for >3 months prior to the inception of the study. No soy protein products were consumed during the study period. Table 1 Study Subject Characteristics Subject Age Body weight (lb) Menstrual cycle history Medical conditions/health status Medications 1 43 142 hypermenorrhea, polymenorrhea, dysmenorrhea, luteal phase deficiency endometriosis; otherwise healthy none 2 42 138 hypermenorrhea, polymenorrhea, dysmenorrhea general good health none 3 21 126 hypermenorrhea, dysmenorrhea endometriosis; otherwise healthy none The protocol was approved by the Committee for the Protection of Human Subjects of the University of California at Berkeley. The nature of the study was explained, and written informed consent was obtained from all study subjects. Source and dose of bladderwrack (Fucus vesiculosus) Dried, powdered bladderwrack was obtained from Maine Coast Sea Vegetables (Franklin, ME) and encapsulated in 350 mg capsules. Two capsules were administered daily for the low dose treatment (700 mg) and four capsules were administered daily for the high dose treatment (1.4 g). Bladderwrack dosage levels were chosen to fall within the range of reported dietary seaweed intakes (10–25%) of the total diet reported for Japanese populations [21,22]. This was calculated by assuming a total 500 g/d total dietary intake and a range between 50–125 g/d (wet weight) or 0.5–1.25 g/d (dry weight) seaweed intake. This calculation falls below the estimated 3–13 g/d intake reported by Teas et al. [23]. Experimental protocols Details of the study protocol are outlined in Table 2. All women provided self-reported menstrual cycling histories for the three months prior to the treatment period. In addition, 17β-estradiol and progesterone serum measurements were taken for Subject 1 throughout the course of the study as outlined in Table 2. Ovulation was monitored through body basal temperature. Since the average length of her cycle was 16 days prior to treatment and she was not ovulating at the inception of the study, baseline hormone levels were ascertained on a set day (menstrual cycle day 12) for two consecutive cycles prior to the administration of 700 mg/d bladderwrack for two additional cycles. During the treatment period, serum hormone levels were measured on days 12 and 21 for the first cycle (which was another anovulatory cycle) and on day 21 thereafter during the treatment period. Subjects 2 and 3 were administered 700 mg/d of bladderwrack beginning on day 21 of their menstrual cycles and followed for two consecutive cycles. Subsequently, Subjects 1 and 3 agreed to continue the experiment for two additional cycles at which time they received a daily dose of 1.4 g/d kelp. Menstrual cycling logs were maintained on all subjects during the entire course of the experiment. Subjects were monitored at least weekly to insure compliance to the supplement regimen. Table 2 Treatment protocol and timeline Subject Pretreatment menstrual cycling history obtained Pretreatment serum 17β-estradiol and progesterone levels ascertained (Cycle and day) Treatment period/dose Treatment serum 17β-estradiol and progesterone levels ascertained (Cycle and day) Treatment period/dose Treatment serum 17β-estradiol and progesterone levels ascertained (Cycle and day) 1 Cycles 1–3 Cycle 2, day 12; Cycle 3, day 12 Cycle 4–5/ 700 mg/d Cycle 4, day 12; Cycle 4, day 21; Cycle 5, day 21 Cycle 6–7/ 1.4 g/d Cycle 6, day 21; Cycle 7, day 21 2 Cycles 1–3 NA Cycle 4–5/ 700 mg/d NA NA NA 3 Cycles 1–3 NA Cycle 4–5/ 700 mg/d NA Cycle 6–7/ 1.4 g/d NA Hormone assays All hormone assays were performed by Quest Laboratories (San Diego, CA), an outside-certified clinical laboratory. Serum 17β-estradiol and progesterone levels were measured in duplicate by radioimmunoassays. Blank and control sera were run with each assay. The coefficient of variation (a measure of laboratory error) was consistently low (<15%) for 17β-estradiol and progesterone. Statistical methods Statistical analyses were performed by unpaired t-tests (2-sided) with a commercially available statistical software package (Stata, College Station, Texas). All statistical tests were considered significant for p ≤ 0.05. Results are referred to as borderline significant for 0.05 < p ≤ 0.10. Clinical findings There were no adverse side effects reported and bladderwrack was well tolerated by all three women. Effects of treatment on length of menstrual cycle and total days of menstruation Following treatment, all women exhibited a significant increase in menstrual cycle lengths (Figure 1). Specifically, in Subject 1, who had a 30-year history of irregular menses, the menstrual cycle length increased from an average of 16.3 ± 0.6 days to 26.0 ± 1.4 days with the low dose treatment (P < 0.002), which increased by approximately 5 additional days to 31.2 ± 1.1 days following administration of the higher dose (P < 0.001). In Subject 2, the average cycle length increased 5.5 days, from 23.0 ± 1.7 to 28.5 ± 0.7 days (P = 0.03). Subject 3 exhibited a 4-day increase in menstrual cycle length from 27.3 ± 0.6 to 31.5 ± 0.7 days with the 700 mg dose (P = 0.005) that increased by approximately 6 more days to 36.0 ± 2.8 days with the 1.4 g dose (P = 0.01). Figure 1 Average menstrual cycle length in days for Subjects 1–3 at baseline (black bars) and following bladderwrack administration of 700 mg/d (diagonal striped bars) and 1.4 g/d (white bars). Black bars indicate the averages of 3 menstrual cycles; diagonal striped and white bars indicate the averages of 2 menstrual cycles; and whiskers indicate standard deviations. * P value <0.05. Along with increased menstrual cycle lengths, all women reported marked reductions in blood flow and average number of days of menstruation following bladderwrack treatment (Figure 2). Subject 1 reported the most significant reduction in total days of menstruation, changing from an average 9.3 ± 0.6 to 6.3 ± 1.8 days (P = 0.06) with the low dose and to 4.5 ± 0.7 average days with the high dose (P < 0.003). Subject 2, who only took the low dose, also experienced a marked reduction in number of days of menstruation, from 8.0 ± 1.0 to 5.3 ± 2.5 days (P = 0.06). Subject 3 exhibited a decrease in total menstruating days averaging from 6.3 ± 1.5 to 5.8 ± 0.4 days (P = 0.65) with the low dose, and to 3.5 ± 0.7 days (P = 0.10) with the 1.4 g/d dose. Subjects 1 and 3, who both suffered from endometriosis, reported substantial alleviation from pain during menstruation and throughout the menstrual cycle following bladderwrack treatment. Figure 2 Average number of days of menstruation per cycle for Subjects 1–3 at baseline (black bars) and following bladderwrack administration of 700 mg/d (diagonal striped bars) and 1.4 g/d (white bars). Each bar indicates averages from two menstrual cycles; whiskers indicate standard deviations. * P value <0.05. Subject 1 also reported an ovulatory cycle following 2 months on the 700 mg/d kelp intervention, and continued to have ovulatory cycles while on the 1.4 g/d dose. Effects of treatment on serum estradiol and progesterone levels A significant anti-estrogenic and progestagenic dose response was observed in plasma estradiol and progesterone levels in Subject 1 (Table 3). Specifically, the mean baseline 17β-estradiol levels were reduced from 626 ± 91 to 164 ± 30 pg/ml (P = 0.04) with the low dose (700 mg/d), which decreased further to 92.5 ± 3.5 pg/ml (P = 0.03) with the higher dose (1.4 g/d). Furthermore, mean baseline progesterone level rose from 0.58 ± 0.14 to 8.4 ± 2.6 ng/ml with the lower 700 mg/d dose (P = 0.1), which increased further to 16.8 ± 0.7 ng/ml with the 1.4 g/d dose (P = 0.002). Table 3 Average circulating plasma 17β-estradiol and progesterone levels prior to and during kelp intervention for Subject 1 Pre-treatment baseline levels 700 mg/d dose P-value 1.4 g/d dose P-value 17β-estradiol (pg/ml) 626 ± 91 164 ± 30 0.04 92.5 ± 3.5 0.03 Progesterone (ng/ml) 0.58 ± 0.14 8.4 ± 2.6 0.10 16.8 ± 0.7 0.002 All measures are from 2 month averages ± S.D. Discussion of clinical findings The results of this preliminary pilot study suggest that bladderwrack consumption can effectively increase the length of the menstrual cycle and reduce the total number of days of menstruation in pre-menopausal women. These effects were most marked in the two women that had shorter than average cycles (16 and 23 days) versus the normal range of 26 to 32 days seen in women in Western populations. Menstrual cycles were further lengthened with increasing dose, which may suggest a linear dose-response effect. However, since there was not a sufficient washout period between the 700 and 1400 mg/d doses, an effect of length of time of dosing rather than a dose response effect cannot be ruled out. Nonetheless, these marked increases in menstrual cycle length may have beneficial health effects in lowering risk of estrogen-dependent diseases such as endometriosis and ovarian, endometrial, and breast cancers as reported in a number of studies [16,35-38]. Menstrual characteristics are surrogate markers that may reflect a woman's overall exposure to and production of endogenous hormones. Shorter menstrual cycle lengths and prolonged menstruation confer longer follicular and luteal phases where estrogen and progesterone levels and endometrial and breast cell proliferation rates are at their highest. A nearly fourfold increase in mitotic activity in the breast lobules occurs during the luteal phase of the menstrual cycle [39], while the highest proliferation rates (nearly 100-fold) in the endometrium occur during the follicular phase [40]. Therefore, fewer menstrual cycles over a woman's lifetime would decrease the amount of time during which the breast and endometrial epithelia would be exposed to high levels of proliferation, which may decrease overall disease risk. Bladderwrack consumption also led to a marked reduction in circulating 17β-estradiol levels and an increase in progesterone levels in a subject who exhibited high serum estrogen levels and progesterone deficiency prior to the intervention. While estrogen's proliferative effects on mammary gland development and endometrial and breast tumorigenesis are well documented, progesterone's role in these processes is not as well defined. Studies show that progesterone deficiency is associated with increased endometrial cancer incidence [41], and that progesterone inhibits estrogen-induced luminal epithelial proliferation in the uterus [42]. However, progesterone has been shown to both stimulate and inhibit the growth of experimental mammary tumors [43], and the use of synthetic progestins in hormone replacement therapy has been associated with an increased risk of breast cancer [43]. Experimental rat models have elucidated progesterone's vital role in pregnancy-induced morphological changes in the breast, which confer protection against breast cancer [44]. Further, epidemiological studies suggest that it is not pregnancy alone but early first parity and increasing number of pregnancies that are associated with reduced breast cancer risk [45,46]. These studies suggest that the effects of progesterone in breast cancer risk may be dependent on timing and the type and level of progesterone/progestin exposure. Thus, the anti-estrogenic/progestagenic activity of kelp observed in this study warrants further investigation in its role in breast cancer and other hormone-dependent diseases. Study limitations Due to the small number of subjects and the lack of a control group, this study will need to be repeated in a larger, randomized population of women with placebo controls. Other weaknesses of the present study are the lack of data on luteinizing hormone and follicular stimulating hormone levels which would provide pertinent information regarding the effects of dietary bladderwrack on ovulation and the luteal and follicular phases of the menstrual cycle. The potential beneficial impact that dietary bladderwrack may have on abrogating symptoms of endometriosis warrants a closer look at a larger population of women suffering from this disease. However, studies should also be performed in women with normal menstrual cycles who have sex hormone levels within clinically normal ranges to determine the impact of dietary kelp on menstrual cycling patterns and hormone levels in the general population. Conclusions The observed responses to bladderwrack consumption in this study suggest that dietary modification may lead to significant changes in the regulation of the menstrual cycle by increasing the length of the cycle, stimulating ovulation, and lowering the estrogen/progesterone ratio in pre-menopausal women. Such changes may be beneficial particularly with regard to women at high risk of estrogen-dependent diseases or who are experiencing fertility problems. Results from these preliminary experiments also suggest that bladderwrack administration may alleviate hypermenorrhea and dysmenorrhea, which may provide some relief in the treatment of endometriosis. Although these reported effects are generally in a beneficial direction, their clinical significance is yet to be determined in a well-controlled study. Future Directions The critical role of hormones in breast, endometrial, and ovarian cancers in women and prostate cancer in men has long been recognized. Given the vast rise of these cancers in the U.S. and our limited success with prevention and treatment, there is clearly a need for the identification of novel, non-cytotoxic chemopreventive agents. Future investigations should clarify the role of bladderwrack and other seaweed species on estrogen and progesterone metabolism, to evaluate its potential binding affinity to estrogen and progesterone receptors, and to determine its effects on proliferation in hormone-sensitive tissues. These investigations should also be expanded to include effects of bladderwrack on other sex hormones including the androgens and gonadotropins. In this regard, animal and in vitro studies are currently underway in our laboratory to elucidate the potential mechanisms and clinical relevance of bladderwrack bioactivity, and to identify and isolate the active components involved. Competing Interests None declared. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements Written consent was obtained from the patients for publication of study. This work was supported by the National Foundation for Cancer Research and NIH grant P30 ES01896. Thanks to Dr. Martyn T. Smith for his guidance and support. ==== Refs Parkin DM Pisani P Ferlay J Estimates of the worldwide incidence of 25 major cancers in 1990 Int J Cancer 1999 80 827 841 10074914 10.1002/(SICI)1097-0215(19990315)80:6<827::AID-IJC6>3.0.CO;2-P Parkin DM Pisani P Ferlay J Global cancer statistics CA Cancer J Clin 1999 49 33 64, 1 10200776 Madigan MP Troisi R Potischman N Dorgan JF Brinton LA Hoover RN Serum hormone levels in relation to reproductive and lifestyle factors in postmenopausal women (United States) Cancer Causes Control 1998 9 199 207 9578297 10.1023/A:1008838412423 McTiernan A Behavioral risk factors in breast cancer: can risk be modified? 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==== Front BMC Clin PharmacolBMC Clinical Pharmacology1472-6904BioMed Central London 1472-6904-4-61530169010.1186/1472-6904-4-6Case ReportIntrathecal baclofen withdrawal syndrome- a life-threatening complication of baclofen pump: A case report Mohammed Imran [email protected] Asif [email protected] Department of Internal Medicine, Mercy Hospital of Pittsburgh, 1400 Locust Street, Pittsburgh, Pennsylvania 15219, United States of America2 Department of Pharmaceutical Chemistry, Jamia Hamdard University, Hamdard Nagar, New Delhi 110062, India2004 9 8 2004 4 6 6 29 3 2004 9 8 2004 Copyright © 2004 Mohammed and Hussain; licensee BioMed Central Ltd.2004Mohammed and Hussain; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Intrathecal baclofen pump has been used effectively with increasing frequency in patients with severe spasticity, particularly for those patients who are unresponsive to conservative pharmacotherapy or develop intolerable side effects at therapeutic doses of oral baclofen. Drowsiness, nausea, headache, muscle weakness, light-headedness and return of pretreatment spasticity can be caused by intrathecal pump delivering an incorrect dose of baclofen. Intrathecal baclofen withdrawal syndrome is a very rare, potentially life-threatening complication of baclofen pump caused by an abrupt cessation of intrathecal baclofen. Case presentation A 24-year-old man with a past medical history of cerebral palsy and spastic quadriparesis developed hyperthermia, disseminated intravascular coagulation, rhabdomyolysis, acute renal failure and multisystem organ failure leading to a full-blown intrathecal baclofen withdrawal syndrome. Intrathecal baclofen pump analysis revealed that it was stopped due to some programming error. He was treated effectively with supportive care, high-dose benzodiazepines and reinstitution of baclofen pump. Conclusion The episodes of intrathecal baclofen withdrawal syndrome are mostly caused by preventable human errors or pump malfunction. Educating patients and their caregivers about the syndrome, and regular check-up of baclofen pump may decrease the incidence of intrathecal baclofen withdrawal syndrome. Oral baclofen replacement may not be an effective method to treat or prevent intrathecal baclofen withdrawal syndrome. Management includes an early recognition of syndrome, proper intensive care management, high-dose benzodiazepines and prompt analysis of intrathecal pump with reinstitution of baclofen. ==== Body Background Baclofen is a gamma-aminobutyric acid (GABA) analog that has inhibitory effects on spinal cord reflexes and brain. Intrathecal baclofen (ITB) therapy consists of long-term delivery of baclofen to the intrathecal space. Intrathecal baclofen has been used to treat spasticity due to cerebral palsy, brain or spinal cord injury, multiple sclerosis, dystonia, stroke and stiff-man syndrome, particularly for those patients who are unresponsive to conservative pharmacotherapy or develop intolerable side effects at therapeutic doses of oral baclofen [1]. Side effects such as drowsiness, nausea, headache, muscle weakness and light-headedness can occur as a result of the pump delivering an incorrect dose of baclofen. Sudden cessation of ITB administration can cause mild symptoms like reappearance of baseline level of spasticity associated with pruritis, anxiety and disorientation [2]. These mild symptoms represent "loss of drug effect". All patients experience "loss of drug effect" when ITB is discontinued, only a small (but unknown) proportion of patients develop a full-blown potentially life-threatening withdrawal syndrome. We report a case of ITB withdrawal syndrome developing hyperthermia, severe spasticity, disseminated intravascular coagulation, rhabdomyolysis, acute renal failure and multisystem organ failure. Case presentation A 24-year-old man was admitted to our intensive care unit (ICU) with a possible diagnosis of seizure disorder and sepsis. He had a past medical history of cerebral palsy and spastic quadriparesis. Three years ago, he had an ITB pump implanted for spasticity refractory to the high doses of oral baclofen. He had a significant improvement in spasticity, social and functional capacity in the past three years. Later, he developed some disorientation and increased spasticity. He was taken to a local physician who prescribed oral baclofen (120 mg daily in four divided doses) for his increased spasticity. He also advised him to have his ITB pump checked immediately. The following day, his spasticity increased even after taking oral baclofen. He developed multiple seizures and respiratory distress in the next 24-hour period. Subsequently, he was admitted in a local hospital where he was orally intubated and transferred to our ICU for aggressive management. On presentation, his temperature was 104.6°F (40.3°C), heart rate 127 beats per minute, and the blood pressure was 85/45 mm/Hg. His ventilator settings were: assist-control ventilation mode; respiratory rate, 15 breaths per minute; tidal volume, 650 mL; positive end expiratory pressure (PEEP), 5 cm H2O; and FiO2, 60%. His spontaneous respiratory rate was 18 breaths per minute and an oxygen saturation of 100% was noted on pulse oximetry. In the local hospital, he was documented to have a high fever of 107°F (41.6°C) and he had received intravenous lorazepam, phenytoin, pantoprazole, piperacillin/tazobactem and dopamine. On physical examination, neurologically he was unconscious with decerebrate posturing and his Glasgow coma scale was 6. He had an absent corneal and gag reflexes. He was moving all four limbs in response to noxious stimuli. He was also noted to have an extreme spasticity in all four limbs. Lung examination revealed decreased breath sounds in the left lower base. Cardiac examination was unremarkable. He had a palpable baclofen pump on abdominal wall and bowel sounds were heard. The differential diagnoses were septic shock, meningitis, neuroleptic malignant syndrome and malignant hyperthermia. The initial laboratory results showed serum creatinine phosphokinase (CPK) 5250 U/L (Normal, 25–235 U/L) and CPK-MB fraction 12.1 ng/ml (Normal, 0.5–6.3 U/L). Serum chemistry revealed sodium 142 mmol/L, potassium 5.1 mmol/L, chloride 120 mmol/L, bicarbonate 13 mmol/L, and creatinine 2.1 mg/dl. Hemogram showed white blood cell count 12.2 K/UL, hemoglobin 16.5 g/dl and platelet count 9 K/UL (Normal, 130–400 K/UL). Liver function test showed aspartate aminotransferase (ALT) 1128 U/L, alanine aminotransferase (AST) 1140 U/L, alkaline phophatase 90 U/L, total bilirubin 1.2 mg/dl, conjugated bilirubin 0.7 mg/dl, prothrombin time 20.2 seconds (Normal, 10–12.5 seconds), and INR 2.0 (Normal, 0.9–1.1). Blood and urine cultures were obtained. Chest radiograph was normal. A computed tomography (CT) scan of the chest revealed atelectasis of the left lung base. His CT scan of head did not show any acute infarct or bleeding. His initial management included intravenous fluids, norepinephrine, platelet transfusion, phenytoin, propofol and broad-spectrum antibiotics (vancomycin, ceftriaxone) for suspected meningitis and septic shock. He received intravenous lorazepam (4–8 mg every four hours) for his spasticity. Next day, his spasticity improved and an ITB specialist investigated his baclofen pump. His baclofen pump analysis revealed that it was stopped due to some programming error, which was restarted at a previously prescribed baclofen rate (260 μg/day). On third hospital day, his serum CPK was 15,878 U/L, AST was 2566 U/L, ALT was 2993 U/L, while CPK-MB fraction came down to 3.4 ng/ml. His urine output decreased (<400 ml/ day) and serum creatinine increased in the range of 5–6 mg/dl. Later, he was hemodialyzed few times during the course of hospitalization due to acute renal failure. His echocardiogram showed left ventricular ejection fraction of 20–25% and severe global hypokinesis. His electroencephalogram did not reveal any epileptogenic activity. He developed full-blown multisystem organ failure with an evidence of shock liver, renal failure, respiratory failure, disseminated intravascular coagulation and myocardial depression. His nutrition was started on nasogastric tube feedings, and proper ventilator care was taken through a tracheostomy tube. His serum baclofen obtained at the time of admission was less than 0.02 μg/ml (Expected values, 0.08–0.4 μg/ml). After a three-week course of aggressive management in ICU, he was weaned off from the ventilator and his multiple organ shock resolved. At a six-month follow-up, he was observed in a nursing home with his baseline functional, social, and family activities. Discussion Intrathecal baclofen provides an effective improvement in the spasticity of patients whose spasticity is not sufficiently managed by oral baclofen or other oral anti-spastic medications [1]. Regardless of the cause of spasticity (cerebral or spinal), anti-spastic effects of baclofen occur at spinal level. Poor response of oral baclofen in many patients can be explained by the fact that the spinal cord represents only about 2% mass of the brain, and receives proportionately a lower blood flow as a fraction of cardiac output. Therefore, cerebral side effects often occur before therapeutic anti-spastic effects of oral baclofen are observed. A programmable ITB pump provides a direct, pattern-controlled delivery of baclofen to the spinal cord. Precise delivery of the ITB pump yields better spasticity reduction at 1000 times lower than the doses of oral baclofen. In addition, the adverse effects are minimized as compared with oral baclofen. ITB provides reduced tone, spasms and pain, improves speech, mobility, sleep quality and bladder control, with a response rate up to 97% in adults and children [3,4]. The ITB pump is approximately 3 inches wide and 1 inch thick. It is surgically implanted in the subcutaneous tissue of anterior abdominal wall. Baclofen is delivered via a silicone rubber catheter into the lumbar subarachanoid space. The ITB pump delivers approximately 100–900 μg/day of baclofen, and it is titrated for the desired clinical response. It is also equipped with an alarm that signals low volume, low battery, or malfunction. Nine years ago, catheter or pump-related malfunction that leads to an overdose or withdrawal has been reported in 40 % of the patients with ITB pump [5]. Catheter system, operative techniques and programming system of the ITB pump have now been improved significantly to reduce the incidence of an overdose or withdrawal. The precise mechanism of action of baclofen as a muscle relaxant and anti-spasticity agent is not fully understood. Baclofen inhibits both monosynaptic and polysynaptic reflexes at the spinal cord level [6], possibly by decreasing excitatory neurotransmitter release from primary afferent terminals, although actions at supraspinal sites may also contribute to its clinical effects. Baclofen also causes enhancement of vagal tone and inhibition of mesolimbic and nigrostriatal dopamine neurons (directly or via inhibiting substance P) [7]. Baclofen is a structural analog of the inhibitory neurotransmitter GABA, and may exert its effects by stimulation of the GABAB receptor to cause muscle relaxation. Baclofen reduces increased muscle tone, Babinski sign, tendon reflexes, ankle clonus and sometimes decreases muscle force. Long-term ITB infusion causes down-regulation of GABAB receptors in the CNS and spinal cord. Down-regulation of GABAB receptors accounts for the decreased sensitivity to the baclofen over time. Although GABAB receptors are down-regulated, it is the baclofen itself that causes increased inhibitory tone in the CNS and spinal cord [8]. Therefore, abrupt ITB withdrawal results in a predominance of excitatory effects and simulates other conditions that are associated with CNS hyperexcitability and severe spasticity. Sudden cessation of ITB administration can cause mild symptoms like reappearance of baseline level of spasticity associated with pruritis, anxiety and disorientation. These mild symptoms represent "loss of drug effect". All patients experience "loss of drug effect" when ITB is discontinued. However, more severe symptoms like hyperthermia (109.4°F), myoclonus, seizures [9-12], rhabdomyolysis, disseminated intravascular coagulation, multisystem organ failure [10], cardiac arrest, coma and death [9,12,13] have been well reported, and represents a full-blown life-threatening ITB withdrawal syndrome. Food and drug administration (FDA) of USA has included a drug label warning for baclofen withdrawal syndrome in April 2002 [1,13]. Differential diagnoses include malignant hyperthermia, neuroleptic-malignant syndrome, autonomic dysreflexia, sepsis and meningitis. ITB withdrawal syndrome has been fatal in some cases. Six patients have died out of 27 cases reported to FDA [13]. Most reported episodes of ITB withdrawal were caused by preventable human errors or oversights. However, catheter dislodgement, catheter migration and kinks, and other catheter-related issues might be more common than pump-related malfunctions [1,14]. Close attention to pump refilling and programming procedures may reduce the incidence of ITB withdrawal syndrome. Benzodiazepines are helpful in controlling spasticity and seizures during ITB withdrawal syndrome [1,10]. Benzodiazepines activate central receptors and GABAA receptors of spinal cord by different mechanisms [1]. Therefore, ITB induced down-regulation of GABAB receptors do not interfere with benzodiazepine's mechanism of action. During a planned removal of ITB pump due to infection or other causes, premedication with high doses of benzodiazepines and augmented oral baclofen is usually administered in the hospitals to prevent spasticity. Similarly, high doses of oral baclofen is also tried in some cases of ITB withdrawal syndrome [15,16]. But failure of high doses of oral baclofen (80 mg three times daily) have been reported recently [17]. High doses of oral baclofen may not be adequate to treat or prevent ITB withdrawal because of down-regulation of central GABAB receptors due to chronic ITB administration. Moreover, it has been suggested that it may take many hundreds of grams of oral baclofen to achieve a therapeutic baclofen level in the cerebrospinal fluid, compared to the patients who had effective spasticity control with an ITB pump [17]. Although, our patient received oral baclofen (120 mg daily in four divided doses) initially but these doses may be low enough to prevent ITB withdrawal syndrome. Failure of high doses of oral baclofen suggests that resumption of GABAB receptor agonist by prompt restoration of ITB pump and proper supportive care might be the best treatment. Similarly, high-dose benzodiazepines may be effective because of similar mechanism of action on widespread CNS GABAA receptors. High-dose benzodiazepines could be an initial life saving strategy even before analysis and restoration of ITB pump is achieved, or in cases, where resumption of ITB administration is not as simple as correcting a programming error. Intrathecal baclofen bolus is appropriate, but due to the risk of inadvertent overdose, an experienced physician should immediately perform reinstitution of ITB pump. We had restarted ITB in our patient at a previously prescribed dose. However, a much higher dose of baclofen could have safely been given to overcome the spasticity since the patient was in an ICU. High-dose dantrolene (a direct muscle relaxant that acts on sarcoplasmic reticulum of skeletal muscle) has been tried to reduce spasticity and fever in ITB withdrawal syndrome [18]. Reduction in fever may be due to cessation of repetitive and thermogenic contractions of muscle fibers. It is unlikely that dantrolene has any GABA agonistic effects, and administration of dantrolene may not be accompanied by any correction in anomalies of CNS functions. Therefore, seizures, autonomic instability and death may occur in ITB withdrawal syndrome even after controlling spasticity with dantrolene. Cyproheptadine (a non-selective serotonin antagonist) has also been used postulating that ITB withdrawal may be a form of serotonergic syndrome that occurs from the loss of GABAB receptor-mediated presynaptic inhibition of serotonin [19]. We did not consider dantrolene or cyproheptadine in our patient due to lack of sufficient clinical support in the treatment of ITB withdrawal syndrome. There was a three-day period of delay in diagnosing the ITB withdrawal syndrome leading to deterioration and multisystem organ failure. However, our patient had a successful recovery in response to restoration of baclofen pump and adequate intensive care management. Conclusions Baclofen withdrawal syndrome is a potentially life-threatening complication of intrathecal baclofen pump. Empty pump reservoir, catheter leaks or displacement, pump malfunction, programming error and refill of pump with improper drug concentration are the possible mechanisms which could lead to an ITB withdrawal syndrome. Regular check-up of the ITB pump by a specialist, educating patients and their caregivers may decrease the incidence of ITB withdrawal syndrome. Oral baclofen replacement may not be an effective method to treat or prevent ITB withdrawal syndrome. Early recognition of syndrome, high-dose benzodiazepines, prompt analysis of the ITB pump with reinstitution of baclofen, and proper intensive care management are mainstays for the management of ITB withdrawal syndrome. List of abbreviations GABA -gamma amino butyric acid, ITB – intrathecal baclofen, CPK – creatinine phosphokinase, ALT – aspartate aminotransferase, AST – alanine aminotransferase, CT – computed tomography. Competing interests None declared. Authors' contributions IM: Direct patient care, article conception, critical and extensive revision of article for important intellectual content, review and drafting of the original article. AH: Literature search, case review and summary, drafting of the original article. Both authors read and approved the final manuscript and contributed equally to the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements Written consent was obtained from the patient's mother for publication of this case report. ==== Refs Coffey RJ Edgar TS Francisco GE Graziani V Meythaler JM Ridgely PM Sadiq SA Turner MS Abrupt withdrawal from intrathecal baclofen: recognition and management of a potentially life-threatening syndrome Arch Phys Med Rehabil 2002 83 735 741 12048649 10.1053/apmr.2002.32820 Coffey JR Cahill D Steers W Park TS Ordia J Meythaler J Herman R Shetter AG Levy R Gill B Smith R Wilberger J Loeser JD Chabal C Feler C Robertson JT Renn RD Clarke A Burchiel KJ Leibrock LG Intrathecal baclofen for intractable spasticity of spinal origin: results of a long-term multicenter study J Neurosurg 1993 78 226 232 8421205 Penn RD Intrathecal baclofen for spasticity of spinal origin: seven years of experience J Neurosurg 1992 77 236 240 1625011 Van SP Nuttin B Lagae L Schrijvers E Borghgraef C Feys P Intrathecal baclofen for intractable cerebral spasticity: a prospective placebo-controlled, double-blind study Neurosurgery 2000 46 603 609 10719857 Penn RD York MM Paice JA Catheter systems for intrathecal drug delivery J Neurosurg 1995 83 215 217 7616263 Allerton CA Boden PR Hill RG Actions of the GABAB agonist, (-)-baclofen, on neurones in deep dorsal horn of the rat spinal cord in vitro Br J Pharmacol 1989 96 29 38 2924075 Peng CT Ger J Yang CC Tsai WJ Deng JF Bullard MJ Prolonged severe withdrawal symptoms after acute-on-chronic baclofen overdose J Toxicol Clin Toxicol 1998 36 359 363 9711203 Kroin JS Bianchi GD Penn RD Intrathecal baclofen down-regulates GABAB receptors in the rat substantia gelatinosa J Neurosurg 1993 79 544 549 8410224 Green LB Nelson VS Death after acute withdrawal of intrathecal baclofen: case report and literature review Arch Phys Med Rehabil 1999 80 1600 1604 10597813 10.1016/S0003-9993(99)90337-4 Sampathkumar P Scanlon PD Plevak DJ Baclofen withdrawal presenting as multiorgan system failure Anesth Analg 1998 87 562 563 9728828 10.1097/00000539-199809000-00011 Reeves RK Stolp-Smith KA Christopherson MW Hyperthermia, rhabdomyolysis, and disseminated intravascular coagulation associated with baclofen pump catheter failure Arch Phys Med Rehabil 1998 79 353 356 9523793 10.1016/S0003-9993(98)90021-1 Meinck HM Tronnier V Rieke K Wirtz CR Flugel D Schwab S Intrathecal baclofen treatment for stiff-man syndrome: pump failure may be fatal Neurology 1994 44 2209 2210 7969992 MedWatch Safety Alert – Lioresal (baclofen injection) 2002 Kao LW Amin Y Kirk MA Turner MS Intrathecal baclofen withdrawal mimicking sepsis J Emerg Med 2003 24 423 427 12745045 10.1016/S0736-4679(03)00039-8 Siegfried RN Jacobson L Chabal C Development of an acute withdrawal syndrome following the cessation of intrathecal baclofen in a patient with spasticity Anesthesiology 1992 77 1048 1050 1443727 Al-Khodairy AT Vuagnat H Uebelhart D Symptoms of recurrent intrathecal baclofen withdrawal resulting from drug delivery failure: a case report Am J Phys Med Rehabil 1999 78 272 277 10340425 10.1097/00002060-199905000-00018 Greenberg MI Hendrickson RG Baclofen withdrawal following removal of an intrathecal baclofen pump despite oral baclofen replacement J Toxicol Clin Toxicol 2003 41 83 85 12645974 10.1081/CLT-120018277 Khorasani A Peruzzi WT Dantrolene treatment for abrupt intrathecal baclofen withdrawal Anesth Analg 1995 80 1054 1056 7726408 10.1097/00000539-199505000-00038 Meythaler JM Roper JF Brunner RC Cyproheptadine for intrathecal baclofen withdrawal Arch Phys Med Rehabil 2003 84 638 642 12736874 10.1016/S0003-9993(03)00105-9

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==== Front BMC Med EducBMC Medical Education1472-6920BioMed Central London 1472-6920-4-121529871010.1186/1472-6920-4-12Research ArticleMedical Students' and Residents' preferred site characteristics and preceptor behaviours for learning in the ambulatory setting: a cross-sectional survey Schultz Karen W [email protected] John [email protected] Dianne [email protected] Marshall [email protected] Sarita [email protected] Richard [email protected] Chris [email protected] Rachelle [email protected] Department of Family Medicine, Queen's University, 220 Bagot St., PO Bag 8888, Kingston, ON, Canada, K7L 5E92 Faculty of Education, Queen's University, 511 Union St., Kingston, ON, Canada, K7M 5R43 Department of Psychology, Queen's University Kingston, ON, Canada, K7L 3N62004 6 8 2004 4 12 12 8 12 2003 6 8 2004 Copyright © 2004 Schultz et al; licensee BioMed Central Ltd.2004Schultz et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Medical training is increasingly occurring in the ambulatory setting for final year medical students and residents. This study looks to identify if gender, school, level of training, or speciality affects learner's (final year medical students and residents) preferred site characteristics and preceptor behaviours for learning in the ambulatory setting. Methods All final year medical students and residents at the five medical schools in Ontario (N = 3471) were surveyed about the site characteristics and preceptor behaviours most enhancing their learning in the ambulatory setting. Preferred site characteristics and preceptor behaviours were rank ordered. Factor analysis grouped the site characteristics and preceptor behaviours into themes which were then correlated with gender, school, level of training, and speciality. Results Having an adequate number and variety of patients while being supervised by enthusiastic preceptors who give feedback and are willing to discuss their reasoning processes and delegate responsibility are site characteristics and preceptor behaviours valued by almost all learners. Some teaching strategies recently suggested to improve efficiency in the ambulatory teaching setting, such as structuring the interview for the student and teaching and reviewing the case in front of the patient, were found not to be valued by learners. There was a striking degree of similarity in what was valued by all learners but there were also some educationally significant differences, particularly between learners at different levels and in different specialities. Key findings between the different levels include preceptor interaction being most important for medical students as opposed to residents who most value issues pertaining to patient logistics. Learning resources are less valued early and late in training. Teaching and having the case reviewed in front of the patient becomes increasingly less valued as learners advance in their training. As one approaches the end of ones' training office management instruction becomes increasingly valued. Differences between specialities pertain most to the type of practice residents will ultimately end up in (ie: office based specialties particularly valuing instruction in office management and health care system interaction). Conclusions Preceptors need to be aware of, and make efforts to provide, teaching strategies such as feedback and discussing clinical reasoning, that learners have identified as being helpful for learning. If strategies identified as not being valued for learning, such as teaching in front of the patient, must continue it will be important to explore the barriers they present to learning. Although what all learners want from their preceptors and clinic settings to enhance their learning is remarkably similar, being aware of the educationally significant differences, particularly for learners at different levels and in different specialities, will enhance teaching in the ambulatory setting. ==== Body Background "The ideal preceptor should be like Captain Picard from Star Trek, who has a good grasp of situations but lets his subordinates push themselves to their limits without interfering/imposing his views and methods"! (survey comment) Medical care is being delivered primarily in the ambulatory setting in an increasing number of specialties. Since learning is best done contextually [1,2] it is appropriate and necessary that medical training also increasingly occur in the ambulatory setting. Theory suggests that trainees at different levels [3-8] and in different specialties [3,6,9-11] may have different learning needs. Students early in their training may be looking to be taught certainties about facts and concepts, corresponding to Perry's concept of simple dualism, ie; right versus wrong, and to not find it helpful to be engaged in discussions about "softer" emotional and social issues [5]. Stritter found first year residents preferred being told what to do, whereas higher-level residents preferred more autonomy and more explanations from their preceptors [7]. Work looking at learning styles in different specialities has been mainly based on Kolb's work, who outlined four different learning styles, and suggests those in different specialities learn differently (ie: surgeons learn best by hands-on practical application of ideas [11], while pathologists learn best using abstract theoretical models [10]). An article by Kernan [12] outlined site accommodations and preceptor behaviours that third year medical students felt facilitated their learning during a one-month ambulatory internal medicine rotation. A pilot project at our institution asking first year family medicine residents to rank Kernan's study items found differences between the two groups. What was not clear was if these differences were due to school attended, level of training or specialty. Given that trainees at all levels and in all specialties are increasingly being trained in the ambulatory setting, it seemed important to understand if there truly are differences between different types of students in what is perceived as being most helpful for learning. If differences are identified it will then be important to study whether adjusting to these differences actually improves learning. We surveyed all final year medical students and residents in Ontario about the site characteristics and preceptor behaviours that they find most enhance learning in the ambulatory setting and determined if these were related to demographic factors, level of training or residency program. Implications for teaching in the ambulatory setting are explored based on these results. Methods All medical students (n = 532) and residents (n = 2939) at the five medical schools in Ontario were surveyed using a four part questionnaire which collected information on demographics, preferred site characteristics, preferred preceptor behaviours, and approaches to learning and perceptions of learning climate. Questions for the site characteristics and preceptor behaviours included previously validated questions [12-15] and questions believed to be important by study group consensus. The approaches to learning and perceptions of learning climate questionnaire was validated by Kirby et al [16] and is not reported here. Students rated 24 site characteristics and 38 preceptor behaviours on a Likert scale from 1 (very important for learning) to 5 (not at all important for learning) or D (detrimental for learning). Within each section they indicated the five most important and 5 most unimportant or detrimental items for learning. A section for general comments was included at the end of the survey. The survey was piloted with a group of Queen's University residents and final year medical students checking for ambiguity and content. Ethical approval was granted by the Queen's University General Research Ethics Board. To ensure privacy for their students schools requested that the questionnaires be addressed by their own undergraduate and postgraduate offices. Coded questionnaires were thus sent with student's names and bulk mailed to the undergraduate and postgraduate medical schools who then addressed and forwarded the questionnaires to their final year medical students and residents. Entry into a draw for a Personal Digital Assistant or equivalent monetary prize was offered for completed surveys. Non-responders were identified by a lack of a returned coded questionnaire. Two subsequent mailings were sent to the non-respondents through their schools' undergraduate or postgraduate office. In addition an email reminder was sent to everyone between the second and third mailing. Data were analyzed using SPSS for Windows, version 11.0 [17]. A systematic effort to look for out-of-range data was conducted by doing frequency distributions for each of the variables, identifying out-of-range entries and correcting the errors by going back to the original data sheets. Double entry data assessment was not done. Frequency distributions for demographic factors, valued site characteristics, and preceptor behaviours were compiled. Counts were derived for each site characteristic or preceptor behaviour by calculating percentages of respondents giving the item a score of 1 or 2 on the Likert scale. Detrimental items were tallied from the frequency data. Factor analysis of the site characteristics and preceptor behaviours was carried out. Cronbach alpha coefficients were calculated for the identified factors. (Factor analysis is a means of reducing a large number of items to a smaller, more manageable number of dimensions, based on the ways in which the items correlate with each other. Cronbach alpha coefficients can then be calculated for internal consistency of the scales based on the identified factors. The resulting factors/scales need to be interpreted, but may provide a view of underlying constructs that are responsible for the observed variables and their correlations. Both the choice of the number of factors to extract and the interpretation of the factors/scales are matters of interpretation [18].) Counts (derived by calculating percentages for each item ranked 1 or 2 on the Likert scale) were generated for gender, school, level of training and residency for each factor. The Post-Graduate Year 2 (PGY2) group had an additional, possibly confounding feature, containing a large number of family medicine residents, who would be at the end of their training, instead of half way through their training like the remainder of the group. A subanalysis was done on the level of training data removing family medicine residents from the PGY2 data to analyse the impact of this on preferred site characteristics and preceptor behaviours. Initial data interpretation for residency used 11 residencies. Residencies were then collapsed into five groups (medicine; family medicine, paediatrics, psychiatry; lab/path, radiology; surgery, emergency, ob/gyn; and intensivists,anaesthetists) based on similarity of practice patterns. Logistic regression analysis[17] was used to compare gender, school, level of training and residency with respect to the site characteristic and preceptor behaviour factors. Each independent variable (ie: gender, school, level of training and residency grouping) was entered individually into a regression procedure as a categorical variable and the proportion of positive responses (1 or 2 on the Likert scale) for each level of the variable was compared to the proportion of positive responses in the full sample. Results Survey response was 48% (1642/3430). Of these 44 had not worked in an ambulatory setting and so were eliminated from further analysis (N = 1598). The demographics of the five medical schools are listed in Table 1. Demographic characteristics of responders are shown in Table 2. Comparisons to all Ontario and Canadian clerks and residents revealed more women, junior residents, McMaster and Family Medicine residents and fewer PGY6-fellows and Toronto trainees responded. Table 1 Demographics of the five medical schools School City Size # final year medical students (2001–02)* # residents (2001–02)* Queen's University 113,000 71 248 University of Toronto 4,700,000 167 1268 University of Western Ontario 432,000 98 353 University of Ottawa 823,000 85 398 McMaster University 662,000 103 396 * Data from Association of Canadian Medical Colleges Table 2 Demographics of Study Group Demographic Study Numbers N (%) Gender N = 1642 Male 805 (49) Female 837 (51) Level of Training N = 1641 Clerks 279 (17.0) First year residents 377 (23.0) Second year residents 366 (22.3) Third year residents 231 (14.1) Fourth year residents 165 (10.1) Fifth year residents 185 (11.3) Sixth and above year residents including Fellows 38 (2.3) University N = 1642 Queen's U. 172 (10.5) U. of Toronto 611 (37.3) U. of Western Ontario 243 (14.8) Ottawa U. 296 (18.0) McMaster U. 317 (19.3) Mean Age Residents 29.9 Training Program N = 1356*,** Medicine 298 (22.0) Family Medicine 351 (25.9) Paediatrics 100 (7.4) Surgery 226 (16.7) Psychiatry 104 (7.7) Radiology 56 (4.1) Intensivists 7 (0.5) Anaesthesia 102 (7.5) Laboratory 24 (1.8) Obstetrics/Gynaecology 65 (4.8) Emergency 23 (1.7) *N = total number-clerks **7 not specified The rank ordering of site characteristics and preceptor behaviours, including missing data and number judging an item to not only be unhelpful but detrimental for learning are shown in Tables 3 and 4. The five most and five least important items for learning essentially matched the rank ordering and thus are not separately reported. Table 3 Ranking of Site Characteristics Rank Question Number saying important to learning (%) Number not answering question Number saying detrimental for learning (%) 1 Effective teachers 1569 (98.4) 4 3 (0.2) 2 Opportunity to see patients independently 1592 (97.3) 6 0 3 Opportunity to see a large variety of patients 1511 (94.8) 4 1 (0.1) 4 Opportunity to see an adequate number of patients 1496 (93.9) 5 2 (0.1) 5 Preceptors readily available 1490 (93.5) 4 2 (0.1) 6 Opportunity to do procedures 1357 (85.3) 8 3 (0.2) 7 Readily available examination room 1348 (84.8) 9 1 (0.1) 8 Opportunity to see patients in follow-up visits 1275 (80.1) 6 1 (0.1) 9 Opportunity to observe preceptor if desired 1239 (77.8) 6 0 10 Opportunity to interact with consultants and/or referring doctors 1227 (77.1) 7 0 11 Block rotation 1094 (68.8) 7 3 (0.2) 12 Efforts to meet objectives made by preceptor 1059 (66.5) 5 1 (0.1) 13 Teaching of medical record keeping skills 956 (60.0) 4 0 14 Computer learning resources available in the clinic 947 (59.4) 3 0 15 Orientation to the practice 937 (59.0) 11 1 (0.1) 16 Teaching of time management skills 904 (56.7) 3 3 (0.2) 17 Teaching of office management skills 872 (54.7) 4 2 (0.1) 18 Clearly defined site objectives for the rotation 843 (52.9) 5 4 (0.2) 19 Library resources available in the clinic 778 (48.8) 4 0 20 Existence of a site-coordinator 762 (48.4) 22 2 (0.1) 21 Longitudinal/horizontal rotation 603 (38.4) 29 42 (2.7) 22 Limited number of preceptors 443 (27.9) 13 233 (14.7) 23 Presence of other trainees in the clinic 432 (27.1) 4 74 (4.6) 24 Close proximity of clinic to campus 366 (23.0) 8 5 (0.3) Table 4 Preceptor Behaviours Ranking Rank Question Number saying important for learning (%) Number not answering question Number saying detrimental for learning (%) 1 Is open to questions 1540 (96.7) 5 1 (0.1) 2 Gives constructive feedback 1522 (95.6) 6 1 (0.1) 3 Demonstrates enthusiasm for teaching 1515 (95.1) 5 1 (0.1) 4 Reviews differential diagnoses 1507 (94.6) 5 0 5 Delegates appropriate responsibility for patient care 1491 (93.7) 7 1 (0.1) 6 Gives timely feedback 1445 (90.7) 5 0 7 Has a strong command of his or her specialty 1433 (90.1) 7 2 (0.1) 8 Discusses clinical topics in an organized way 1416 (88.9) 5 0 9 Makes student feel like a valued member of the practice 1407 (88.3) 5 1 (0.1) 10 Identifies and responds to student's specific learning needs 1398(87.9) 8 1 (0.1) 11 Discusses own clinical reasoning processes 1396 (87.3) 8 1 (0.1) 12 Asks for students' ideas before giving own 1372 (86.1) 5 0 13 Discusses clinical topics concisely 1361 (85.5) 6 1 (0.1) 14 Demonstrates a caring attitude towards students 1347 (84.6) 5 1 (0.1) 15 Sets time aside to discuss topics unable to be discussed during busy clinics 1340 (84.3) 10 4 (0.3) 16 Provides a role model of professional behaviour 1327 (83.5) 8 0 17 Asks students differing complexities of questions 1302 (81.8) 6 3 (0.2) 18 Welcomes differing points of view 1294 (81.3) 7 2 (0.1) 19 Demonstrates a caring attitude towards patients 1279 (80.3) 5 0 20 Facilitates student's participation in follow-up care 1263 (79.4) 7 0 21 Teaches physical examination 1218 (76.8) 13 3 (0.2) 22 Monitors quality of the rotation 1216 (76.4) 6 1 (0.1) 23 Seeks to understand student's ideas 1186 (74.5) 6 0 24 Suggests relevant reading 1172 (73.6) 5 0 25 Connects new ideas to existing knowledge 1149 (72.4) 10 0 26 Defines student's role 1087 (68.4) 8 3 (0.2) 27 Provides a role model of a balance between personal and professional life 1079 (67.9) 9 0 28 Teaches appropriate use of health care resources 1072 (67.4) 8 0 29 Teaches use of community resources 1005 (63.2) 7 0 29 Demonstrates effective interactions with support staff 1005 (63.2) 9 0 30 Observes clinical interactions directly 966 (60.8) 8 7 (0.4) 31 Teaches communication skills 940 (59.2) 10 2 (0.1) 32 Discusses limitations of his or her own knowledge 899 (56.5) 7 1 (0.1) 33 Provides background on patients before students sees patient 602 (37.8) 5 36 (2.3) 34 Outlines specific task(s) to be done during a clinical encounter 595 (37.5) 12 30 (1.9) 35 Teaches in the patient's presence 429 (27.1) 14 116 (7.3) 36 Focuses on one teaching theme per clinic 348 (21.9) 9 71 (4.4) 37 Reviews case in the patient's presence 281 (17.7) 10 242 (15.1) Six factors, accounting for 55 % of the variance for the 24 site characteristics, and 7 factors, accounting for 54% of the variance for the 38 preceptor behaviours, were identified (Tables 5,6). Labels describing the factors were decided by group consensus among the researchers. 7 items that failed to load on any factor were eliminated from the analysis. The site characteristic factors were office management, patient logistics, objectives, learning resources, clinic set-up and preceptor interaction. The preceptor behaviour factors were professional role modeling, teaching, learning climate, feedback, direction, patient presence and health care system interaction. Cronbach alpha coefficients for the factors identified in the factor analysis ranged from 0.52 to 0.83. Table 5 Factor Analysis makeup for Site Characteristics Factor Items making up factor Factor Loading Alpha Analysis Office Management Teaching of time management skills .832 .62 Teaching of medical record keeping skills .760 Teaching of office management skills .746 Patient Logistics Opportunity to see an adequate number of patients .766 .69 Opportunity to see a large variety of patients .542 Opportunity to see patients independently .538 Readily available examination room .473 Opportunity to see patients in follow-up visits .442 Objectives Clearly defined site objectives for the rotation .806 .53 Efforts to meet objectives made by preceptor .776 Learning Resources Library resources available in the clinic .794 .60 Computer learning resources available in the clinic .756 Clinic Set-up Close proximity of clinic to campus .442 .55 Presence of other trainees in the clinic .418 Existence of a site co-coordinator .386 Longitudinal/horizontal rotation .364 Orientation to the practice .342 Preceptor Interaction Effective teachers .514 .55 Preceptors readily available .506 Opportunity to observe preceptor if desired .491 Table 6 Factor analysis for Preceptor Behaviours Factor Items Making Up Factor Factor Loading Alpha Analysis Professional Role Modeling Provides a role model of professional behaviour .681 .79 Demonstrates effective interactions with support staff .565 Provides a role model of a balance between personal and professional life .557 Teaches communication skills .526 Discusses limitations of his or her own knowledge .500 Discusses own clinical reasoning processes .426 Teaching Discusses clinical topics in an organized way .739 .82 Discusses clinical topics concisely .650 Suggests relevant reading .462 Identifies and responds to student's specific learning needs .390 Is open to questions .365 Asks students differing complexities of questions .362 Has a strong command of his or her area of specialty .340 Asks for students' ideas before giving own .334 Sets time aside to discuss topics unable to be discussed during busy clinics .323 Monitors quality of the rotation .301 Learning Climate Makes student feel like a valued member of the practice .613 .83 Demonstrates a caring attitude towards students .591 Seeks to understand student's ideas .563 Demonstrates a caring attitude towards patients .512 Demonstrates enthusiasm for teaching .365 Welcomes differing points of view .328 Facilitates student's participation in follow-up care .301 Feedback Gives constructive feedback .730 .73 Gives timely feedback .709 Reviews differential diagnosis .473 Direction Outlines specific task(s) to be done during a clinical encounter .588 .69 Focuses on one teaching theme per clinic .507 Provides background on patients before student sees patient .447 Teaches physical examination .432 Defines student's role .404 Patient Presence Teaches in the patient's presence .759 .77 Reviews case in the patient's presence .720 Health Care System Interaction Teaches use of community resources .531 .82 Teaches appropriate use of health care resources .516 Logistic regression analysis of the independent variables revealed striking similarities, but some significant differences, in valued site characteristics and preceptor behaviours for male and female students and those in different schools, at different levels of training and in different residencies (Figures 1,2,3,4,5,6,7,8). Similarities are evident by mainly flat, nonintersecting lines on the graphs indicating similar percentages of subgroups of learners valuing a factor and similar relative valuing of factors respectively. Differences are apparent where lines intersect within a graph and/or percentages are statistically higher (indicated by *, **, ***) or lower (#, ##, ###) than the means. Figure 1 Gender and site characteristics Figure 2 Gender and preceptor behaviours Figure 3 School and site characteristics Figure 4 School and preceptor behaviours Figure 5 Training level and site characteristics Figure 6 Training level and preceptor behaviours Figure 7 Residency and site characteristics Figure 8 Residency and preceptor behaviours Male and female residents rank ordered all site characteristics and preceptor behaviours identically. Women ranked all factors, with the exception of teaching in the patient's presence, higher than men, usually significantly so (Figures 1,2). Across schools the rank ordering of factors was identical with the exception of Toronto and Ottawa ranking office management instruction higher than the other schools. Toronto valued six factors significantly more than the other schools, Queen's and Western ranked three and two items respectively significantly less than the other schools (Figures 3,4). Across levels the only difference in rank ordering was clerks ranking preceptor interaction as the most important site characteristic whereas all other groups ranked patient logistics as most important. Those at the beginning and end of their training valued having learning resources available less than all other levels. Clerks were most different from all the other levels in what they valued or did not value (indicated by the number of * and # for this group) (Figures 5,6). Subanalysis of the PGY2 data removing family medicine residents significantly decreased the importance of office management and health care system interaction instruction (52.2% of all PGY2's rated office management instruction important versus 44.8% removing family medicine residents, and 60.1% of all PGY2's rated health care system interaction instruction important versus 50.5% removing family medicine residents). Residency groups again showed mainly similarities in rank ordering, the exceptions being the family medicine/paediatrics/psychiatry group ranking office management and learning climate higher, the lab/path/radiology group patient logistics and learning climate lower and the surgery/emergency/ob/gyn group health care system interaction lower than the rest. There were a large number of responses significantly different from the group averages throughout all the residency groups (Figures 7,8). Combining residencies into five groups lost only two pieces of information, that of ob/gyn residents being similar to the family medicine, paediatrics, psychiatry group in relatively highly valuing office management instruction and that of anaesthesia residents being similar to lab/path, radiology residents in relatively less valuing feedback, teaching, and learning climate than other groups. Discussion The ambulatory teaching site characteristics most valued by clerks and residents are having an adequate number and variety of patients while being supervised by enthusiastic and available preceptors. These characteristics have been identified before and are well summarized by Bowen and Irby [19]. Little value is placed on having other trainees in the clinic despite social learning theory that suggests this enhances learning. Bowen [20] and Lesky [21] suggest that students learn by teaching and may feel less threatened asking questions that reveal a lack of knowledge of a fellow student than of a preceptor. Although what students value may not translate into effective learning, it is still important to understand why something is valued or not valued. Without reliable learning outcome measures perceived learning value is a proxy measure of learning effectiveness. Further studies should assess what learners do not like about having other trainees present. Computer resources were more valued than books, likely reflecting a generation of learners who are comfortable accessing electronic information. Proximity of the clinic to university campus was unimportant. In contrast to other studies [22-24] we found block rotations were valued more than longitudinal rotations. Some programs, particularly Canadian Family Medicine programs, encourage longitudinal rotations to enhance the continuity of care experience. Merenstein et al [25] however recently reported there to be no difference in continuity of care provided by residents in longitudinal rotations. Exploration of the value of block versus longitudinal rotations is an area for further research. Valued preceptor behaviours identified in this study are feedback by enthusiastic, open preceptors who are willing to discuss their reasoning processes and delegate responsibility. Recent studies report 3rd year medical students to also value these preceptor behaviours[26,27]. Lesky and Borkan [21] suggest that pathogenesis and natural histories of disease can be learned from a variety of resources, including books and computers but problem solving, decision making and dealing with uncertainty are learned mainly from preceptors and practice. This study supports students' perceived value of these aspects and suggests them as priorities for teachers in ambulatory settings. We have confirmed the value of feedback found in most studies [26,28-31] (a study by O'Malley [32] being the exception). As one respondent commented "constructive and honest feedback in a timely manner is by far the most important (item)". Feedback leads to positive learning outcomes. Cope [33] demonstrated that giving feedback to residents improved their patient satisfaction scores, which in turn has been correlated with improved patient outcomes[34]. Unfortunately this teaching behaviour is underutilized. Irby [1], in a review of studies, reports that feedback is given only 3–6% of the time (range 0–16%). This is an effective teaching behaviour that is valued by students and deserves high priority. Meaningful feedback about many aspects of students' patient care is best based on direct observation[35]. Only 61% of our respondents actually value direct observation by their preceptors. Some of the reasons for more not valuing this may be similar to why students do not want to be taught in front of the patient (see next section). Since direct observation is a necessary component of good teaching it will be important to explore further why more students do not value this important preceptor behaviour. A number of strategies have been suggested to improve efficiency in the ambulatory teaching setting including teaching in the patient's presence and preceptors directing tasks to be covered in the interview [36-38]. A significant proportion of our respondents rated reviewing the case and teaching in the patient's presence, structuring the interview by providing patient information background, outlining tasks to be done during the interview and focusing on one teaching theme per clinic not only to be unimportant for learning but detrimental. Kernan similarly found 3rd year medical students to not value being taught in front of the patient[26]. Comments from students in this study give some indication why teaching in front of the patient is disliked ("it would undermine a therapeutic alliance with the student", "it gives a tense atmosphere more often than not", "...impairs free thinking of student because student feels inhibition in front of patients", "makes it difficult for students to ask questions, not wanting to scare/worry the patient"). Teaching however occurs within a larger context where providing background information on patients may be necessary for ongoing patient care and safety and to model continuity of care. Teaching in the patient's presence may be necessary for efficiency and maintaining a relationship between the preceptor and the patient.Further studies are needed to determine if explanation or teaching methods can overcome this aversion. Analysis of the impact of gender, school, level of training or residency on valued site characteristics and preceptor behaviours revealed striking uniformity between the groups. There were some statistically significant differences between the groups, many of which do not appear to be educationally relevant, others which likely are important. Male and female students rank ordered site and preceptor behaviour factors identically. It is of interest that female students ranked all factors, with the exception of teaching in the patient's presence, as being more important for learning than male students. The literature [39-41] suggests that women predominantly emphasize relationship issues, which may partially explain this finding. It would appear however with respect to the items surveyed that there are no gender-based educationally important differences in valued site characteristics and preceptor behaviours. The five schools also essentially rank ordered the factors identically. One school did stand out from the others in frequently ranking factors significantly higher than the rest. This school is the largest of the five schools with the most trainees and teaching sites. It would be valuable to know the ratio of students to preceptors at the different schools. If this were high at the larger school, perhaps resulting in residents feeling relatively anonymous, it may partially explain why these students there particularly value factors like learning climate, professional role modeling and clinic set up. Within level of training preceptor interaction is most important for clerks. This is the only group to rank this item more important than patient logistics. This may reflect the clerks' developmental stage of being eager to go beyond textbook lists and start to put clinical decisions into patient context–skills best learned by preceptor interaction. Learning resources are significantly less valued by those at either end of their training–clerks for perhaps the above reason and PGY6's/fellows presumably because they are confident in their theoretical knowledge. Beyond clerkship patient logistic factors usurp preceptor interaction as the highest ranked site characteristic. Becoming an expert clinician involves, in part, connecting disparate units of knowledge into networks [3,5,42]. This encapsulating of knowledge occurs when students learn with patients. The residents in this study recognize this, ranking seeing an adequate and large variety of patients independently as the most important site characteristic for their learning. Having objectives defined with efforts made to meet them was third in importance for most levels, superceding available learning resources, office management skills instruction, and clinic setup items. Office management instruction is relatively more important for PGY2's and those at the end of their training. Subanalysis of the PGY2 data removing family medicine residents who would be at the end of their training and leaving those in the middle of their training significantly decreased the importance of office management and health care system interaction instruction. Teaching these aspects thus seems most important for those at the end of their training. Directing the clinical encounter and teaching in the patient's presence is valued less as residents gain seniority and presumably identify themselves more as the patients' physicians. Increasing desire for autonomy and decreasing potential for undermining their relationship with the patient may be reasons for these trends. Within almost all residencies patient logistics and preceptor interaction are the most valued site characteristics; feedback, teaching and learning climate the most important preceptor behaviours. Lab/path, radiology and anaesthesia residents value all these preceptor behaviours less than other residents. Arguably these are areas of medicine where decision making is more clear-cut without as much patient input, which may explain these results. Other significant differences between the specialties seem best explained by considering future practice ie: office-based specialties (paediatrics, psychiatry, family medicine) most valuing office management and health care system interaction instruction. Strengths of this study are the large multi-institutional sample size (n = 1642) encompassing students at multiple levels in all specialties. The response rate (48%) limits the external validity of the result. A confounding factor within the level of training data set may be the variability in residency lengths as suggested by the subanalysis of the PGY2 data. Rather than years from graduation from medical school what seems to influence valued site characteristics and preceptor behaviours more are years from independent practice. Conclusions "Software" (patient encounters and enthusiastic preceptors who delegate, give feedback and explain clinical reasoning) is valued more than "hardware" (clinic set-up, learning resources). All learners value the above preceptor behaviours; most do not value, and a significant number consider detrimental, having the structure of the patient encounter dictated to them and having the patient present during review and teaching. Future work is needed to explain why learners do not value these practices. Learners at all levels and in all specialties are strikingly similar in what they value from their preceptors and clinic sites for their learning. There are some differences between levels and residencies however that require consideration when teaching these different groups. Educationally significant differences within levels include preceptor interaction being paramount for medical students; patient logistics (adequate number and variety of patients seen independently and in follow-up) being second. The reverse is true for residents. Proportioning time accordingly deserves attention. The more senior the learner the more being taught or having the case reviewed in the patients' presence is not valued. Sensitivity to the patient-learner relationship is required if these practices are utilized but particularly so for more senior learners. Finally relevance not surprisingly dictates importance. Office management instruction is valued by those at the end of their training and those primarily in office-based specialties. Similarly office-based specialties appreciate instruction in health care system interaction. This study identifies preceptor behaviours and site characteristics valued by medical students and residents for their learning in the ambulatory setting. Further studies are needed to determine the effect of providing these valued site characteristics and preceptor behaviours on learning outcomes. Competing interests None declared. Authors' contributions KS conceived of the study, prepared the manuscript and participated in the conceptual planning and design of the study and data interpretation. JK, DD, and MG participated in the conceptual planning and design of the study, statistical analysis and data interpretation and manuscript revision. SV and RB contributed to the design of the study and manuscript revision. CK contributed to the design of the study. All authors read and approved the final manuscript. RS participated in the statistical analysis. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements I am grateful to CIHR-ACMC for funding this project. I would like to thank Jason Schmelzle for his diligent data entry and Debbie Jones for assisting with finalizing the manuscript. ==== Refs Irby DM Teaching and learning in ambulatory care settings. 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==== Front BMC Med EducBMC Medical Education1472-6920BioMed Central London 1472-6920-4-131530168910.1186/1472-6920-4-13Research ArticleExplaining computation of predictive values: 2 × 2 table versus frequency tree. A randomized controlled trial [ISRCTN74278823] Steckelberg Anke [email protected] Andrea [email protected] Jürgen [email protected]ühlhauser Ingrid [email protected] Unit of Health Sciences and Education, University of Hamburg, Martin-Luther-King Platz 6, 20146 Hamburg, Germany2 Institute of Mathematics and Computer Science in Medicine, University Hospital Hamburg, Martinistr. 52, 20246 Hamburg, Germany2004 10 8 2004 4 13 13 28 2 2004 10 8 2004 Copyright © 2004 Steckelberg et al; licensee BioMed Central Ltd.2004Steckelberg et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Involving patients in decision making on diagnostic procedures requires a basic level of statistical thinking. However, innumeracy is prevalent even among physicians. In medical teaching the 2 × 2 table is widely used as a visual help for computations whereas in psychology the frequency tree is favoured. We assumed that the 2 × 2 table is more suitable to support computations of predictive values. Methods 184 students without prior statistical training were randomised either to a step-by-step self-learning tutorial using the 2 × 2 table (n = 94) or the frequency tree (n = 90). During the training session students were instructed by two sample tasks and a total of five positive predictive values had to be computed. During a follow-up session 4 weeks later participants had to compute 5 different tasks of comparable degree of difficulty without having the tutorial instructions at their disposal. The primary outcome was the correct solution of the tasks. Results There were no statistically significant differences between the two groups. About 58% achieved correct solutions in 4–5 tasks following the training session and 26% in the follow-up examination. Conclusions These findings do not support the hypothesis that the 2 × 2 table is more valuable to facilitate the calculation of positive predictive values than the frequency tree. ==== Body Background Diagnostic procedures are increasingly expected by consumers to ensure their health; "certainty" has become a product [1]. Assuming that test results are certain, only a minority is aware about false positive and false negative alarms. Previous research has shown that even physicians have great difficulties in estimating the positive predictive values of diagnostic tests [2-4]. One study reported that 95 out of 100 physicians estimated the positive predictive value of screening mammography to be between 70–80% rather than 7.8% [2]. Similar results were reported for AIDS counselors for low-risk clients. The majority of counselors assured that false positives would never occur and half of the counselors incorrectly assured that if a low-risk person tests positive, it is absolutely certain (100%) that he or she is infected with the virus [5]. An incorrect probability judgment may result in unnecessary tests or pseudo certainty. Therefore, the understanding, presentation and communication of test quality are a challenge for both: lay people and professionals. Involving lay people in decision making on diagnostic procedures requires a basic level of statistical thinking. Help for computing Bayesian inference is needed. Statistical thinking can be enhanced by representing statistical information in terms of natural frequencies rather than probabilities [6,7]. This is explained by the evolution of the human reasoning system. Gigerenzer proposed that human reasoning is algorithms designed for information that comes in a format that was present in the "environment of evolutionary adaptiveness" [8]. Human reasoning processes are adapted to natural frequencies. Also Bayesian computations are easier when the information is communicated this way. In cognitive psychology the frequency tree is used as visual help for the representation of frequencies, a variant of a tree structure often used in decision analysis to teach computing the positive predictive value the simple way (Figure 1) [4]. This format allows a multistage presentation of the numerical information and demonstrates the reasoning process. Figure 1 Frequency tree. In contrast, in medical science the 2 × 2 table is the standard method to teach computing predictive values (Figure 2) [9,10]. In addition, the 2 × 2 table is used for other calculations, e.g. odds ratios or relative risks [9]. Figure 2 2 × 2 table. In the present study, we compare the two visual helps in non-medical students. We hypothesized that the 2 × 2 table is more eligible than the frequency tree to facilitate correct answers in tasks of calculations of positive predictive values 4 weeks after an initial training-session. We also describe students' ability to calculate positive predictive values, analyzing the transfer of the numerical information into the visual help and the correct computation. Methods Participants We approached 238 students without prior statistical training to recruit the necessary 184 students who agreed to participate. (See power calculation below) Students attending the University of Hamburg (health sciences, biology and sports), a vocational college (health and nursing) or taking part in an in-service training (nursing and public health) were informed about the timing and content procedure of the study during their courses. Procedure The study was carried out between October 2000 and July 2001 and consisted of two supervised sessions lasting about 1 h each. The recruited 184 students were randomly assigned either to the frequency tree group (n = 94) or to the 2 × 2 table group (n = 90) using blocked randomization in blocks of 10. Concealed allocation based on computer-generated random numbers was done by an external person. In addition, the external person prepared sealed envelopes for both sessions including the tutorial with the tasks and a questionnaire for survey of age, gender, years of school, mark in mathematics and social state. The training consisted of a written step-by-step self-learning tutorial (Additional file 1, 2, 3). The participants had to compute 5 positive predictive values in each session. The tutorial and tasks followed the recommendations for the presentation of numerical information [4]. Participants were asked to reveal how they achieved their solutions. Participants were allowed to use a pocket calculator. Correct results were presented and discussed after each session. In the follow-up examination participants were again asked to solve 5 different diagnostic problems of similar level of difficulty but without having the tutorial instructions at their disposal (Additional file 4,5,6). Participants who missed the date were repeatedly contacted by letter, phone or e-mail. Efforts were discontinued after 4 weeks. Assessing performance Correct solution of the tasks A solution was classified correct, when the documented positive predictive value was equivalent to the correct solution rounding up or down to the next full percentage point. If a participant used the correct computation (correct positives divided by all positives) but made a calculation error either in the transfer of the numerical information into the visual help or within the division, we ignored calculation errors. Whenever a different computation such as rule of three – a mechanical method for solving proportions – was used or the calculation protocol was missing the rounded solutions were classified likewise as correct by congruence. If the protocol indeed showed that a correct rounded solution resulted from an incorrect computation such as positive predictive value = correct positives / false positives the answer was classified as incorrect. Tasks that had not been worked on were also classified incorrect. Correct transfer To evaluate the usefulness of the different visual helps, we evaluated the ability of correct transfer of the numerical information into the charts. A transfer was classified as correct, when the numerical information of the problems was inserted into the gaps provided. It was sufficient to insert the relevant values for the computation, calculation errors were ignored. Correct computation The computation was classified as correct Bayesian approach when the following computation was used: positive predictive value = correct positives / (correct positives + false positives) or positive predictive value = correct positives / all positives. The computation was classified as Non-Bayesian approach when the computation was used with false values. Other computations were classified as other strategies. Statistical power and analyses Table 1 shows the hypothesized distribution of correct answers within the different categories as primary outcome measure between the two study groups (Table 1). By using the Wilcoxon (Mann-Whitney) rank-sum Test in a sample of 92 persons in each group (84 + 10% drop-out) the hypothesized differences are detected with a power of 80% at a 2- tailed α of 0.05. For our one-sided hypothesis that the 2 × 2 table is superior to the frequency tree the power is 88% at sample size of n1 = n2 = 80. Table 1 Hypothesized distribution of correct answers after 4 weeks between the two study groups Categories* (numbers of correct answers) Frequency tree 2 × 2 table 0 0.40 0.30 1 0.15 0.05 2 0.15 0.05 3 0.10 0.20 4 0.10 0.20 5 0.10 0.20 * category 0 = 0 answers correct category 1–5 = 1–5 answers correct Analysis is based on the intention-to-participate principle that includes all randomised participants as randomised. Drop outs were considered as having solved none of the positive predictive values correctly. Results Figure 3 shows the flow of participants through the trial (Figure 3). There were 18% drop outs in the frequency tree group and 20% in the 2 × 2 table group resulting in a power of 78% for the two-sided and 86% for the one-sided hypothesis. For grouping into three categories as used for analyses the power is 81% for the two-sided and 89% for the one-sided hypothesis. Figure 3 Flow of participants. The groups were similar regarding demographic variables (Table 2). Table 2 Baseline characteristics* Frequency tree (n = 94) 2 × 2 table (n = 90) Age Median (range) 29 (20–54) 26 (19–51) Missing values 3 (3) 2 (2) Gender Male 15 (16) 20 (22) Female 77 (82) 67 (75) Missing values 2 (3) 3 (3) Years of school < 10 years 1 (1) 1 (1) 10–12 years 22 (23) 19 (21) > 12 years 68 (72) 67 (75) Missing values 3 (3) 3 (3) Mark in mathematics 1 (highest level) 6 (6) 6 (7) 2 20 (21) 18 (20) 3 35 (37) 32 (36) 4 14 (15) 17 (19) 5 (lowest level) 5 (5) 9 (10) Missing values 14 (15) 8 (9) Group University of Hamburg 59 (63) 55 (61) Vocational College 14 (15) 15 (17) Non-academic students 21 (22) 20 (22) *Values are numbers (percentages) of participants unless stated otherwise Correct solutions of the tasks Table 3 shows the solutions of both sessions with regard to the primary outcome. Within the training session 20% of participants in both groups calculated only 0–1 answers correctly; 58% (95% CI, 47%–68%) (2 × 2 table) and 59% (95% CI, 48%–69%) (frequency tree), respectively, solved 4 or 5 tasks correctly. In the follow-up examination most participants could not solve more than 0–1 tasks correctly (72% frequency tree and 67% 2 × 2 table). Table 3 Numbers of correct solutions of positive predictive values* Category Training session Follow-up examination Frequency tree (n = 94) 2 × 2 table (n = 90) Frequency tree (n = 74) 2 × 2 table (n = 75) 0–1 (0–1 answer correct) 19(20) 18(20) 53 (72) 50 (67) 2–3 (2–3 answers correct) 20 (21) 20 (22) 2 (3) 5 (7) 4–5 (4–5 answers correct) 55 (59) 52(58) 19 (26) 20(27) * Values are numbers (percentages) of participants Within the category 4–5 correct answers 27% of participants (95% CI, 17%–38%) (2 × 2 table) and 26% (95% CI, 16%–37%) (frequency tree) had correct solutions. The differences between the two study groups were not statistically significant neither in the training session (p = 0.95 {0.49 one-sided}) nor in the follow-up examination (p = 0.48 {0.24} for the analysis on intention-to-participate and p = 0.61 {0.31} for the analysis on-participation (Table 3). In addition, we analyzed every single task in terms of correct solution. In the training session 66% of all questions [(n = 309/470 (frequency tree); n = 297/450 (2 × 2 table)] were solved correctly in both groups. The amount of correct solutions decreased to 26% (n = 98/370) and 31% (n = 115/375), respectively, in the follow-up examination. Differences between groups were not statistically significant (Table 4). Table 4 Analysis of each task regarding correct solutions, transfer of numerical information and Bayesian computations* Correct solution Training session Follow-up examination frequency tree (n = 94) 2 × 2 table (n = 90) frequency tree (n = 74) 2 × 2 table (n = 75) Task A 67 (71) 66 (73) 18 (24) 25 (33) Task B 63 (67) 64 (71) 22 (30) 24 (32) Task C 69 (73) 63 (70) 19 (26) 23 (31) Task D 67 (71) 54 (60) 21 (28) 23 (31) Task E 43 (46) 50 (56) 18 (24) 20 (27) Correct transfer Training session Follow-up examination frequency tree (n = 94) 2 × 2 table (n = 90) frequency tree (n = 74) 2 × 2 table (n = 75) Task A 84 (89) 79 (88) 53 (72) 57 (76) Task B 83 (88) 78 (87) 52 (70) 57 (76) Task C 71 (76) 65 (72) 45 (61) 53 (71) Task D 73 (78) 67 (74) 42 (57) 49 (65) Task E 54 (57) 53 (59) 42 (57) 48 (64) Correct Bayesian Computation Training session Follow-up examination frequency tree (n = 94) 2 × 2 table (n = 90) frequency tree (n = 74) 2 × 2 table (n = 75) Task A 62 (66) 59 (66) 13 (18) 18 (24) Task B 60 (64) 58 (64) 17 (23) 18 (24) Task C 70 (75) 60 (67) 15 (20) 15 (20) Task D 69 (73) 52 (58) 16 (22) 17 (23) Task E 46 (49) 44 (49) 15 (20) 15 (20) * Values are numbers (percentages) of tasks Correct transfer Transfer of the numerical information into the visual help in the training session could be managed in 78% (n = 365/470 frequency tree) and 76% (n = 342/450 2 × 2 table) of the tasks. In the follow-up examination in 63% (n = 234/370) and 70% (n = 264/375), respectively, the information was correctly transferred into the visual helps (Table 4). Correct computation The application of the Bayesian computation in the training session was correctly used in 65% (n = 307/470 frequency tree) and in 61% (n = 273/450 2 × 2 table). In the follow-up examination 21% (n = 76/370) and 22% (n = 83/375), respectively, used correct Bayesian computation (Table 4). Incorrect Bayesian approaches Table 5 shows the commonly used incorrect Bayesian approaches which lead to incorrect solutions of the tasks (Table 5). Table 5 The commonly used incorrect Bayesian approaches* Training session Follow-up examination total Frequency tree 2 × 2 table total Frequency tree 2 × 2 table correct positive rate/ false positive rate 41 26 (63) 15 (37) 16 11 (69) 5 (31) disease yes / all positives 14 7 (50) 7 (50) 37 20 (54) 17 (46) correct positives / disease yes 11 6 (55) 5 (45) 22 11 (50) 11 (50) all positives / total 4 4 (100) 0 (0) 14 6 (43) 8 (57) all positives / 100 0 0 (0) 0 (0) 6 6 (100) 0 (0) disease yes / correct positives 4 1 (25) 3 (75) 1 1 (100) 0 (0) all positives/ correct positives 4 0 (0) 4 (100) 5 5 (100) 0 (0) not identified 23 13 (57) 10 (43) 29 14 (48) 15 (52) total 101 57 (56) 44 (44) 130 74 (57) 56 (43) * Values are numbers (percentages) of incorrect Bayesian approaches. Discussion Differences between the 2 × 2 table and the frequency tree groups were neither meaningful nor statistically significant with regard to the primary outcome measure of correct calculation of the positive predicted values. In the training session the majority of participants were able to calculate the positive predictive value of all tasks correctly. In the reexamination after 4 weeks the proportion of participants with solutions of all tasks decreased to 26% in both groups. The transfer of the numerical information into the visual helps was comparable between the two sessions. However, participants had major difficulties in applying the correct computation as a precondition of a correct solution. In all our tasks we have used frequency formats following the recommendation of Gigerenzer & Hoffrage [4]. In those earlier studies the frequency tree without caption has been used and we adopted this format of the frequency tree in our study. However, in more recent studies a captioned frequency tree has been used [11]. Therefore, we cannot exclude that when comparing the 2 × 2 table with a captioned frequency tree the results might be different. Our study is the first that has compared the two visual helps 2 × 2 table and frequency tree. Previous studies have concentrated on teaching methods using either one of the visual helps or both in combination [4,12]. These previous studies addressed different target groups, mainly medical students and physicians and focused different questions. In contrast, we addressed non-medical students without prior statistical knowledge as a first approach to lay people. Therefore, the overall results of our study are difficult to compare to previous publications. The primary aim of our study was not to investigate different teaching methods for computing predictive values. We have tried to apply the most appropriate method according to actual research at the initiation of the study. However, overall performance of our students was poor. In the training session 58% of participants were able to calculate the positive predictive value of 4 or 5 tasks correctly. In the follow-up examination after 4 weeks the proportion of correct solutions in 4 or 5 tasks decreased to 26%. In addition, after 4 weeks participants had major difficulties in applying the correct computation as a precondition of a correct solution whereas there was only a minor deterioration with respect to the transfer of the numerical information into the visual helps. A recent study used a computerized tutorial programme to teach Bayesian inference [11]. Within the study carried out in a rather small sample of mostly medical students, the role of the graphical aids captioned frequency tree presenting data as natural frequencies versus probability tree presenting data as probabilities in teaching Bayesian inference was explored. After 3 month participants who used the frequency tree reached 100% Bayesian solutions compared with 57% of participants using the probability tree. The authors hypothesized that it is much more important whether the proper representation is used than which graphical aid is applied [11]. Kurzenhauser & Hoffrage studied the effects of a classroom tutorial using both visual helps to teach Bayesian reasoning [12]. They achieved 47% correct answers after 2 months. Participants of the study were medical students in their second and third semester. Generalisability of the results with respect to the overall correct solutions of our study may be limited by the prevalent innumeracy that has lately been ascertained for Germany within the OECD Programme for international student assessment (PISA). Mathematics literacy was stated to be poor in Germany especially in girls [13]. A high percentage of participants in our study were women which corresponds to the distribution of students. Transferring the self-learning tutorial to people without general qualification for university entrance would probably result in an even lower amount of correct solutions. Conclusions In conclusion, our findings do not support the hypothesis that the 2 × 2 table is more valuable to facilitate the calculation of positive predictive values than the frequency tree. Regardless which visual help is used there is a need for improvement of teaching methods to approach lay people who want to participate in medical decision making. Competing interests None declared. Authors' contributions AS as the principal investigator planned and performed the study analysed the data and wrote the paper. AB contributed to planning and performance of the study. JB calculated the power of the study and carried out the statistical analysis of data. IM contributed to all parts of the study. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Original questionnaire used in the 2 × 2 table group in the 1. session in German language. Click here for file Additional File 2 Original questionnaire used in the frequency tree group in the 1. session in German language. Click here for file Additional File 3 Tasks used in the questionnaires of the training session in English language. Click here for file Additional File 4 Original questionnaire used in the 2 × 2 table group in the 2. session in German language. Click here for file Additional File 5 Original questionnaire used in the frequency tree group in the 2. session in German language. Click here for file Additional File 6 Tasks used in the questionnaires of the follow-up examination in English language. Click here for file ==== Refs Gigerenzer G Reckoning with risk: Learning to live with uncertainty 2002 London: Penguin Books Eddy DM Kahnemann D, Slovic P, Tversky A Probabilistic reasoning in clinical medicine: problems and opportunities In Judgment under uncertainty: heuristics and biases 1982 Cambridge: Cambridge University press 249 267 Steurer J Fischer JE Bachmann LM Koller M Ter Riet G Communicating accuracy of tests to general practitioners: a controlled study BMJ 2002 324 824 826 11934776 10.1136/bmj.324.7341.824 Gigerenzer G Hoffrage U How to improve Bayesian reasoning without instruction: frequency formats Psychol Rev 1995 102 684 704 10.1037//0033-295X.102.4.684 Gigerenzer G Hoffrage U Ebert A AIDS counselling for low-risk clients AIDS Care 1998 10 197 211 9625903 10.1080/09540129850124451 Hoffrage U Lindsey S Hertwig R Gigerenzer G Communicating statistical information Science 2000 290 2261 2262 11188724 10.1126/science.290.5500.2261 Hoffrage U Gigerenzer G Using natural frequencies to improve diagnostic inferences Acad Med 1998 73 538 540 9609869 Gigerenzer G Cummins DE, Allen C Ecological intelligence: an adaptation for frequencies In The evolution of the mind 1998 New York: Oxford University Press 9 29 Sackett DL Straus SE Richardson WS Rosenberg W Haynes RB Evidence-based medicine: How to practice and teach EBM 2000 2 Edinburgh, London, New York, Philadelphia, St Louis, Sydney, Toronto: Churchill Livingstone Johnson KV The two by two diagram: a graphical truth table J Clin Epidemiol 1999 52 1073 1082 10527001 10.1016/S0895-4356(99)00087-6 Sedlmeier P Gigerenzer G Teaching Bayesian reasoning in less than two hours J Exp Psychol 2001 130 380 400 10.1037//0096-3445.130.3.380 Kurzenhauser S Hoffrage U Teaching Bayesian reasoning: an evaluation of a classroom tutorial for medical students Med Teach 2002 24 516 521 12450472 10.1080/0142159021000012540 PISA, the OECD programme for international student assessment

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==== Front Malar JMalaria Journal1475-2875BioMed Central London 1475-2875-3-291528578110.1186/1475-2875-3-29ResearchA simulation model of African Anopheles ecology and population dynamics for the analysis of malaria transmission Depinay Jean-Marc O [email protected] Charles M [email protected] Gerry [email protected] Bart [email protected] John [email protected] John [email protected] Jonathan [email protected] Peter [email protected] Henry [email protected] John [email protected] Abdoulaye M [email protected] McKenzie F [email protected] Fogarty International Center, National Institutes of Health, 16 Center Drive, Bethesda MD 20892, USA2 Kenya Medical Research Institute, Centre for Geographic Medicine Research – Coast, P.O. Box 428, Kilifi, Kenya3 International Centre of Insect Physiology and Ecology, P.O. Box 30772, Nairobi, Kenya4 Ifakara Health Research and Development Centre, PO Box 53 Ifakara, Kilombero District, Tanzania5 Entomology Unit, FAO/IAEA Agriculture and Biotechnology Laboratory, A-2444 Seibersdorf, Austria6 Global Public Health Program, University of Miami, South Campus, 12500 SW 152nd Street, Building B, Miami, FL 33177, USA7 Tulane University, New Orleans, LA 70118, USA8 University of Aberdeen, Zoology Building, University of Aberdeen, Aberdeen AB24 2TZ, UK9 School of Mathematics, Statistics and IT, University of Natal, Private Bag X01 Scottsville, 3209 Pietermaritzburg, South Africa10 Faculty of Medicine, Pharmacy, and Dentistry, Malaria Research and Training Center; B.P. 1805 Bamako, Mali2004 30 7 2004 3 29 29 15 12 2003 30 7 2004 Copyright © 2004 Depinay et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Malaria is one of the oldest and deadliest infectious diseases in humans. Many mathematical models of malaria have been developed during the past century, and applied to potential interventions. However, malaria remains uncontrolled and is increasing in many areas, as are vector and parasite resistance to insecticides and drugs. Methods This study presents a simulation model of African malaria vectors. This individual-based model incorporates current knowledge of the mechanisms underlying Anopheles population dynamics and their relations to the environment. One of its main strengths is that it is based on both biological and environmental variables. Results The model made it possible to structure existing knowledge, assembled in a comprehensive review of the literature, and also pointed out important aspects of basic Anopheles biology about which knowledge is lacking. One simulation showed several patterns similar to those seen in the field, and made it possible to examine different analyses and hypotheses for these patterns; sensitivity analyses on temperature, moisture, predation and preliminary investigations of nutrient competition were also conducted. Conclusions Although based on some mathematical formulae and parameters, this new tool has been developed in order to be as explicit as possible, transparent in use, close to reality and amenable to direct use by field workers. It allows a better understanding of the mechanisms underlying Anopheles population dynamics in general and also a better understanding of the dynamics in specific local geographic environments. It points out many important areas for new investigations that will be critical to effective, efficient, sustainable interventions. ==== Body Background Not so long ago, in 1998, Sherman declared: "Of all the human afflictions, the greatest toll has been exacted by malaria. Even today, malaria, which is caused by protozoan parasites of the genus Plasmodium, disables and kills more people than any other infectious disease." [1] In line with the pioneering models of Ross (1911) and Macdonald (1957), malaria interventions such as breeding-site reduction and insecticide use have been considered the most effective and practical ones for reducing malaria transmission. Bednets and house screening serve as personal protection, and bednet-associated effects on malaria prevalence appear to be greater than can be accounted for by personal protection [2]. These interventions have produced good results, but in much of the world malaria remains uncontrolled. Furthermore, malaria vectors are increasingly developing insecticide resistance. At every level of research, policy and practice, malaria control can be helped by models that are both more comprehensive and closer to the day-to-day realities of malaria (K. Dietz in [3]). As Bradley (1982) has pointed out, "for real progress, the mathematical modeller, as well as the epidemiologist, must have mud on his boots." The aim of this study is to provide a framework and a tool for modelers to work closely with field workers in malariology, particularly entomologists. The study also aims to achieve a broader analysis and deeper understanding of the complex mechanisms involved in malaria transmission, in order to aid intervention programs. The idea of controlling malaria through the introduction of genetically modified mosquitoes is gaining increasing attention, for instance, but will first need to be tested critically, in trials that will necessarily involve models. Thus the work presented below represents only a beginning, and it has two major aims. First, it introduces an approach to help researchers account for ecological variables that are key determinants of malaria vector population dynamics. When fully calibrated, this approach will provide an integrated platform for hypothesis testing with complex temporal and spatial data; ultimately, it should help by providing forecasting capabilities. Of perhaps even greater importance, this first model provides a vehicle for assembling and structuring existing knowledge, thereby pointing out critical areas in which knowledge is lacking and very much needed. Thus it is a means of identifying and organizing important research priorities and indicating their epidemiological implications. One of the most important strengths of this model is to combine biological and environmental variables. As stated by [4], the combination of intrinsic and extrinsic determinants of mosquito-borne disease incidence should be the focus of future research. This is critical both in controlling these diseases and reducing the severity of epidemics by predicting them. Approximately 70 species of Anopheles have been implicated in malaria transmission worldwide. In Africa the major vectors are Anopheles gambiae sensu lato, which is considered the most important in most regions, Anopheles arabiensis, which is part of the preceding complex but with distinct characteristics, and Anopheles funestus, which is often reported as the second most important species in terms of malaria transmission and, more particularly, is considered the end-of-rainy-season vector that sustains the parasite. This work focuses on the major vector in sub-Saharan Africa An. gambiae, but much of what follows may be applicable to An. arabiensis, and even to An. funestus separately and all together, with inter-as well as intra-species competition. This paper describes the first model of malaria vector population dynamics integrating both biological and environmental factors. Methods The model incorporates basic biological requirements for Anopheles development on an individual basis and, using local environmental data as input, allows the simulation of the aggregate dynamics of Anopheles populations. The life cycle of each individual proceeds through four stages: three immature stages, which occur in a water body – egg, larva, pupa – and then the mature stage, a flying adult. An adult female disperses from the natal water body and begins a cycle which is maintained throughout the rest of life-alternating between obtaining a bloodmeal and ovipositing in a water body. Five major factors are considered here as characterizing Anopheles population dynamics, by means of mechanisms detailed below (see figure 1 for a schematic): Temperature is a critical regulator of growth and development within each stage, in determining the end of one stage and the beginning of the next and in regulating the length of the gonotrophic cyle. Moisture, in the form of precipitation and relative humidity, is a second key abiotic factor, with effects that in part interact with those of temperature. Nutrient competition is a major potential regulator which is considered to induce mortality in the larval stage. In addition, there is a minimum weight requirement for the transition from larva to pupa, and, through its influence on adult weight, the relation of larval weight to fecundity. Predation and Disease, in which pathogens are included, is a second important mortality-inducing factor, which is considered in local terms relative to the water body. Dispersal, or the adult female's movement in space, is a critical factor in the cycle of seeking blood meals and oviposition sites. The model explicitly represents spatial locations of individual adults, though it does not fully engage this capacity in the analyses presented here. The model is implemented as a software package in the C++ object-oriented programming language, in the Microsoft Windows 98 operating system, and is available from the corresponding author upon request. It was developed and run on a personal computer with a Pentium 3 processor 933 MHz and a relatively small memory of 256 Mb. Temperature Because malaria vectors are poikilothermic, temperature is a critical variable in malaria epidemiology. For instance, in the range of 18°C to 26°C, a change of only 1°C in temperature can change a mosquito's life span by more than a week [5]. Here, in line with the work of Focks et al. [6] on Aedes aegypti, the enzyme kinetics model derived by Sharpe and DeMichele [7] is used, based on absolute reaction rate kinetics of enzymes for the temperature-dependent developmental rates of eggs, larvae and pupae and the duration of the gonotrophic cycle, in the simplified form derived by Schoofield et al. [8]. This equation is derived on the basic assumption that poikilotherm development is regulated by a single control enzyme whose reaction rate determines the development rate of the organism [7,8]. This is of special interest because each parameter of the equation has a biological significance that may have an epidemiologic impact. At time step tn of t0, t1, ..., tn, the development within each of the four stages, during the time step Δtk = tk - tk-1, is defined by: dk = r(Ttk)·Δtk.     (1) is the mean temperature (°K) over the time interval k and r() the developmental rate per hour at temperature T(°K), given by the following equation: where ρ25°C is the development rate per hour at 25°C, under the assumption that there is no temperature inactivation of the critical enzyme; is the enthalpy of activation of the reaction catalyzed by the enzyme (cal·mol-1); ΔHL is the enthalpy change associated with low temperature inactivation of the enzyme (cal·mol-1); is the temperature (°K) where 50% of the enzyme is inactivated by low temperature; ΔHH is the enthalpy change associated with high temperature inactivation of the enzyme (cal·mol-1); is the temperature (°K) where 50% of the enzyme is inactivated by high temperature; and R is the universal gas constant (1.987cal·mol-1). The cumulative development, depending only on temperature at each time step tn, of each of the three stages (egg, larvae, pupae) and the length of the adult gonotrophic cycle is defined as: with dk defined above in equation 1. As detailed below, other factors are also considered, including a particular case for the larval stage that takes food requirements into account. Variability is allowed for in the cumulative development time, CD(tn), with a default value of 10% and a stage is considered completed, such that the next stage begins when: CD(t) >CDf = 1 + G(0,0.l)     (4) where G is a normal random variable. A survey of the literature reveals how very little developmental-rate data is available for Anopheles, even for the most important African malaria vectors. The deficit is striking for all of the three major malaria vector species in Africa. We have fit the curve defined by equation 3 to all of the relevant published data. Those data are compiled in tables 1 and 2, for An. gambiae sensus lato. One reference provided only the total An. gambiae development time from egg to adult [5], we have then estimated the development time for each of the three constituent stages in according with the other data, and also assumed longer development times at low temperatures. The only gonotrophic cycle data available in relation to temperature was for An. arabiensis, part of the An. gambiae complex. All three curves shown in figure 2, for different parameters of equation 2, provide similar fits to the An. gambiae data in tables 1 and 2. These different curves have important implications for vector population dynamics and reinforce the need for more data for these species, particularly at the temperature extremes (low and high), in order to fit an optimal curve. Until there is data for the extreme temperatures, any number of curves might fit the data. Three such curves are illustrated in figure 2. For the purposes of this paper the middle of these three curves has been chosen, with parameters shown in table 3. The curves for all four stages are shown in figures 3 and 4, with parameters in table 3. An. gambiae females are one-day old when they take their first blood meal, according to [9]. This greater length of the first gonotrophic cycle has been taken into account [9][10] by defining a coefficient UFirstGon which represents the time lag before the first blood meal expressed as a percentage of the gonotrophic cycle length. Therefore, the first gonotrophic cycle is considered completed if: CD(t) >CDf = 1 + UFirstGon + G(0,0.1)     (5) UFirstGon has been set to 0.5 for An. gambiae. All subsequent gonotrophic cycles follow equation 4. Thermal mortality Although the range of variation of water temperature is very wide, it is rarely taken into account in the literature. Some authors have recorded temperatures close to 40°C in small pools [5,11,12]. Such temperatures exceed the thermal death point of many species, including An. funestus [5,12]; this may help to explain why these species are rarely found in small pools. Based on these observations [5,12], a daily mortality in the larval stage of 10%, 50% and 100% for a maximum water temperature of 1, 2 and 3°C above the thermal death point, respectively, has been considered. According to [5] the thermal death point for An. gambiae is set to 40°C. Moisture Anopheles usually develop in natural water bodies, such as puddles, pools or streams [11-14]. The model must take into account two critical parameters in a water body, the temperature and the volume of water. In this stage of the project it was not possible to develop a full water-balance model to estimate those parameters but it should be possible in the future. Cloud coverage is likely to be relatively important because of its impact on the water temperature, but this variable is rarely available in climate data. However, it is known that a relative humidity of 100% is usually associated with complete cloud coverage and rain and a relative humidity less than 50% with dryness and almost no clouds. Hence an estimate of cloud coverage as a function of relative humidity RH was made. A clear sky, without clouds (0), for relative humidity below 50%, linearly increases to completely cloudy (1) for relative humidity above 95%, as follows: The maximum water temperature of a water body depends on the cloud coverage and a user-defined coefficient USunExpo that describes the water body's sun exposure. This user-defined coefficient represents the coverage or shaded percentage of the particular water body, ranging from 0 for complete shade to 1 for complete sunlight exposure. By default it is set to 1. If the maximum air temperature in degrees Celsius is TM, it is estimated that the maximum water temperature in accord with the water volume x (in liters) is , where: with CSE = USunExpo·CloudCover(RH). The minimum water temperature is taken as the minimum air temperature. The following formula estimates the daily dynamics of water height WH in a water body: where UIF is the fixed daily water intake in mm·day-1 (e.g. from a stream, pipeline, human activity, etc.); its default value is 0. UIV, the variable daily water intake in mm·day-1is set in accord with the precipitation and the surrounding area's topology. Its default value is 1, which would apply to a water body in a flat area, such that only direct rainfall fills the water body. The user can set a particular value: for a water body on a slope, this coefficient should reflect the volume of water intake given 1 ml of precipitation in the area. P is the precipitation in mm per day, and RH is the relative humidity. UO, in mm·day-1, is the daily loss of water due to soil infiltration and evapotranspiration. By default, this parameter is set to a mean value of 3 mm·day-1. The water bodies are approximated by means of simple geometric objects, such as cubes and cylinders. The default geometric object is a box; its dimensions (length, width, depth) can be entered by the user. Therefore, the volume of water available in the water body is calculated from the particular shape of the water body and the water height calculated above (equation 6). Aestivation and diapause Unlike the eggs of Aedes aegypti, which, it has been shown, can survive in dry soil for more than two months [6], recent work [15] indicates that Anopheles eggs cannot survive more than 15 days on dry soil. Thus, since some African regions with endemic malaria experience drought periods longer than two months, the only plausible alternative seems to be adult aestivation. This is another aspect of Anopheles biology in which much more data is needed. The different survival probability during aestivation has been arbitrarily set as shown in table 4. Aestivation or diapause is triggered by the non-availability of water (when water bodies are completely dry) for all stages. For the adult stage, aestivation is also triggered by a relative humidity arbitrarily chosen here at less than 40%, though even this may prove to be high in some area. Nutrient competition Some combination of regulatory mechanisms limits the size of any population of any species. The most important, for many species, can be described as density-dependent regulation, or competition for space and/or food, which is assumed to summarize or integrate complex, difficult-to-measure mechanisms, such as food mass conversion. For the sake of simplicity and practicality, the basic ecological concept of carrying capacity [16] has been used here. This concept has been applied primarily to the larval stage since it is the longest immature stage and is the only immature stage in which the mosquitoes feed and is, therefore, likely to be the most sensitive to competition. For each water body i a carrying capacity K(i) (in mg) has been defined as: K(i) = LMax·S(i)·UCarrying     (7) where LMax is the maximum larval biomass density, defined for all species j by: where Nj is the larval population size per surface unit (m2) for species j, and Wj is the approximate mean weight of species , with and being the maximum and minimum possible weight in species j, respectively), divided by 2 in equation 8 to correct for the greater size of the low-weight larval population. LMax = 300 mg·m-2 has been arbitrarily set for larvae. S(i) is the available water surface in water body i, and UCarrying is a positive user-defined coefficient for each water body, to correct for particular water-body characteristics; by default it is set to 1. Thus, for each water body at peak season periods, the maximum larval biomass density LMax is estimated by measuring the larval population size at its maximum. Density-dependent mortality Resource competition is considered as a cause of mosquito mortality only for the larval stage. For species j [16] the natural increase of the total larval population size, N, (without mortality) can be defined by: where p is the proportion of larvae that is newly-hatched eggs, estimated by: where ΔNe(t) is the number of individual eggs entering the larval stage. The carrying capacity K(i) of a particular water body i is defined above (Equation 7). In general, the larval population increase is given by: where W(t) is the current larval biomass overall (in contrast to Wj, the approximate mean weight of species j; see equation 8). The larval per capita density-dependent mortality rate m for all species can be approximated by: Weight As noted above, the larval stage is the only immature stage with food intake and, therefore, with weight changes. Thus, this stage is the key determinant of the final adult weight. where and is a coefficient that describes food availability for an individual i of species j, is the maximum possible weight for species j, W(t) is the current larval biomass, K is the carrying capacity of the water body, and Wi, j(t) the weight of individual i of species j at time t. For each time step k, for species j, the weight of individual i increases linearly as , where dk is the thermal development in time period k (equation 2). The weight in the larval stage is then calculated as: This formula allows the individual larva to have a maximum weight in accord with its species when the larval biomass W << K. At the other extreme the weight increase will be almost zero if W ≈ K. Note that this formula allows both intra-and inter-species competition for food. From [5,17-19] the weight parameters for each species have been set as shown in table 5. For the purpose of stochastic simulation variability has been allowed, again with a default value of 10%, as follows: Wi, j = Wi, j + G(0, 0.1)     (16) where G is a normal random variable. The larval stage is regarded as completed, such that the pupa stage begins, when the thermal development CD is completed (Eq. 4) and Weight >WeightMin. The relative weight of an individual within its species is used as an important factor in subsequent subsections on fecundity and number of blood meals, in which the following coefficient is used: Predation and Disease Predators and pathogens are an important regulating factor and are sometimes reported to be the major cause of mortality [20]. Egg Little has been reported about An. gambiae egg mortality, from predation or any other cause, beyond an observation (Beier, personal observation) that up to 83% of eggs hatch after one day of drying on sandy loam soil. Without more information, the total egg mortality for each species was arbitrarily set at 5% as a fixed pre-development mortality for the overall batch and a daily survivorship of 0.99. Larvae and Pupae Service [20] points out that An. gambiae population sizes rise to a peak just after a drought period and then decrease to a roughly stationary level. Life cycles of predators on immature An. gambiae are generally longer than those of their prey, and during the latter phases predators are found in non-predatory stages (i.e. not preying on immature An. gambiae) [20]. Intensity of predation appears to be highly related to the early peak in prey, but there is still a regulatory effect even in the absence of predators. Hence, it is likely that predation is not the only major cause of mosquito mortality [20]. Service [20] evaluated immature An. gambiae sensu lato mortality from predation in two experiments, one in which predator density was high and another in which spraying had reduced predator density. His results are summarized in table 6. With respect to pathogens and parasites, he found that 2.1% to 15.9% of An. gambiae were infected. Active predation exhibits a lag time around the mean life-cycle length of the prey [20]. During the lag period l, if t = 0 is the start of this period, a curve should show a gradual increase in predation. The conditions leading to a new predator lag period could occur, for instance, when a dry water body gains water or after a control intervention killing the predators. If (fig. 5): with and p = 0.001, then the total larval and pupal mortality due to predators and pathogens for species j, can be expressed as: Note that Δmj(t) differs from m(t) in equation 12, which represents density-dependent mortality. For all species j the following were arbitrarily set: = 25% for larvae and = 10% for pupae. = 25% is converted to a daily mortality rate as: where T is the individual's developmental time. Thus at t = 0, the beginning of the lag period, Δmj(t) ≈ 0, and at t ≥ l, for species j. On adding to the density-dependent mortality mj the mortality due to predation and pathogens Δmj(t), for each species j, we obtain a new equilibrium Kp <K, given K in equation 11, where where Nj is the larval population size for species j (N(t) = . Adult There are several published studies of adult mortality rates [9,21] for An. gambiae and An. funestus. The causal mechanisms are not clear, but some authors report adult predators preying on adult mosquitoes at oviposition sites [20]. It is assumed that predation-related adult mortality is focused at the water body and that survivorship is greater with fewer predators present. Oviposition typically occurs every two to three days (see above). Accounting for the low predation during the previously-defined predator lag time, the daily adult survival probability is taken to be 0.911 for a non-ovipositing day and 0.911 - 0.1·CLag(t) for An. gambiae sensus lato. Dispersal The mechanisms governing mosquito dispersal in general remain unknown. Wind strength and direction are likely to be important factors, for instance, but relevant data are rarely reported. Very little is known about the relative attractiveness of individual humans and individual water bodies to Anopheles, but these cues, along with distance, must be key factors in dispersal. In most tropical regions, bloodmeals are taken at night, between 6:00 pm and 6:00 am. As the mosquitoes are active during the night, for simplicity bites were modelled only in houses. Bloodmeal source selection is modelled by a two-step process, first a choice of house and second a choice of individual human within the house. Anthropophily, the proportion of bites taken on humans, can be set for each Anopheles species overall; the default value of this parameter is 1. Exophily is expressed as the proportion of fed mosquitoes that leave the house during the first half of the gonotrophic cycle. For An. gambiae the default value of this parameter is 75%. The model explicitly, dynamically represents individual locations in space, but at this stage the adult female alternately chooses at random among some number of water bodies for an oviposition site, and at random among some number of houses and individuals within the chosen house, for a bloodmeal. That is, the choices do not reflect relative distance, attractiveness, wind or other features the model is designed to address in future phases of development. Multiple bloodmeals and multiple bites In addition to the greater length of the first gonotrophic cycle (Equation 5), Brengues [9] has shown that, to complete their first gonotrophic cycle, 42% of female An. gambiae and 63% of female An. funestus require a second bloodmeal one day after the first one. Here the probability of having a second bloodmeal within the first gonotrophic cycle is related to the weight of the individuals: there is a second bloodmeal when the coefficient Cweight is less than 0.4 for An. gambiae. For multiparous females, there is a second bloodmeal when Cweight is less than 0.1. According to [22], 14% of female An. funestus and 19% of female An. gambiae that had just fed had taken only a partial bloodmeal. These figures are used to represent the proportion of females that take a subsequent bite within what is considered the same bloodmeal. Fecundity The number of eggs oviposited by individuals shows a wide range of variation, both within and between experiments [17,18,23,24]. The mean number of eggs oviposited is defined by m = 100, with a standard deviation s = 50. In the absence of more precise information these values are assumed. The number of eggs oviposited is simulated as: N = G(m, s)·UEgg     (21) where UEgg is a positive user-defined coefficient set to fit local observations, by default set to 1, and G is a normal random variable. Because fecundity is closely tied to body size, a variability of 50% of the number of eggs is allowed as a function of the individual's weight, as follows (see [18][23]): N' = N·(0.5 + 0.5·Cweight)     (22) The male-female ratio at emergence from the pupa stage is assumed to be 1:1. Results A simple example is used to show how the model can help to achieve a better understanding of vector population dynamics and determine key underlying factors. In particular, the influence of temperature, moisture, predation and nutrient competition on adult abundance is investigated. The example is taken as a small cluster of six houses, each with five residents, and a total of three oviposition sites (figure 6 and table 7. An attempt has been made to reproduce some important characteristics of a local environment by considering two types of pools: a semi-permanent pool, P1, and two temporary pools, P2 and P3 (see figure 7 and table 7. As noted above, at this stage each mosquito in the model chooses at random among oviposition sites and among houses and residents at the appropriate points in her gonotrophic cycle. Temperature and moisture inputs were obtained based on data from Kilifi, on the coast of Kenya. Figures 8 and 9 show daily precipitation, minimum and maximum temperature and relative humidity reported there over the 20 months from May 1, 2000 to December 31, 2001. In this region there are two primary rainy seasons: April-June and October-November. Except where noted, the default values were used for parameters, as given above. Effects of temperature In the first set of simulations there are 300 eggs and 10 adults, with all six houses but only pool P1 present. Figure 10 shows the variability and mean of twenty replicates realizations of the simulation model, an effect of the stochasticity allowed in the cumulative development time (equation 4), length of initial gonotrophic cycle (equation 5) and number of eggs oviposited (equation 21). The abundance curve is predicted from the preceding environmental data, with each run started on May 1, 2000. This An. gambiae adult mean curve shows similarities to several published curves, at much wider scales [25], in that there are relatively low levels of mosquitoes throughout the year, with fluctuations in abundance that may correspond to the limitations of competition and/or predation and several very high peaks in short time intervals. To analyse the effects of temperature, two additional temperature curves were used, one in which the actual temperatures are increased by two degrees and one in which they are lowered by two degrees Celsius, the results are shown in figure 11. Table 8 shows the impact of temperature on adult abundance. For An. gambiae (figure 11), with increasing temperature there is a general increase in the level and number of peaks. As detailed above in the section on Temperature (table 1 et seq.), the egg-to-adult development time is shortened with higher temperature, thus producing more mosquitoes. The two-degree temperature rise increases An. gambiae adult abundance over the full 19 months by 15%; the two-degree temperature drop decreases it by 17% overall. Recall that multiple factors interact to determine the adult abundance at each point; however, predation is probably not a critical biotic regulating factor by the time of the initial peak, for instance, but nutrient competition/carrying capacity probably can have a strong impact at late stages of this initial peak. In general, although the drought period from March 12, 2001 to March 31, 2001 has the effect of allowing a first big peak in adult abundance for An. gambiae, it also synchronizes the first peak, and might be important for control intervention purposes. The overall pattern of adult abundance appears well-conserved, and the variability relatively minor. However, as noted above, the aim here is simply to suggest the potential of the model. Figure 10 shows the standard deviation (variability) of the twenty replicate for each date. Effects of temporary pools Here An. gambiae is considered and examined for the effect on adult abundance of adding pools P2 and P3 to the semi-permanent pool P1, beginning with 10 adults and 300 eggs in each pool. Pools P2 and P3 may be classified as temporary, since they dry two or three times during the year (see figure 7). Beside the expected increase in the total number, there is a much more dramatic fluctuation in the mosquito abundance curve, with six added major peaks (figure 12). Effects of interventions Here An. gambiae is considered, with pool P1 only, and show how the model might be of help in reducing peaks in adult abundance by helping to optimize the control of larval and adult populations. Recall that the goal here is not to allege or prove a particular finding, which can depend on a specific environmental situation, but to show how the model could help address a given question in a specific environmental situation, and help in understanding the mechanisms involved. The aim is to show examples, with graphical representation, of how such a model can be a powerful tool in research on malaria vector dynamics. For the purpose of the first analysis the predator population is excluded from any effects of the larval control intervention. Therefore, the impact of the predator as described above (in the Predation section) will remain constant. Although the focus is the first major peak in adult abundance, the analysis could be transposed to any period. Interventions that take effect in two periods are compared, the first beginning on May 6, 2000, at the beginning of the first major peak, and the second beginning 15 days later, on May 21, 2000. A successful one-time larval control intervention is simulated by imposing 80% mortality on all larvae present during 10 consecutive days. An adult control intervention that consists of spraying surfaces inside houses with residual insecticide is simulated by imposing 75% mortality on blood feeding adults during a 25-day period. Figure 13 indicates that the later larval-control intervention (5/21/00), though done at the highest adult abundance rates, would have almost no effect on overall adult abundance, since it happens at a period of lower larval abundance. Still worse, it could lead to the production of bigger mosquitoes by diminishing the nutrient competition. On the other hand, a larval-control intervention that began only 15 days earlier would nearly eliminate the entire first peak in adult abundance. This emphasizes the need of good forecasting tools. Similarly, for an adult-control effort (figure 14), the later control intervention would have very little impact, but the first peak in adult abundance could be decreased consequently by an effort that began only 15 days earlier. At this stage the model does not take into account such important factors as insecticide resistance and mosquito avoidance behavior, which would tend to diminish the impact of spray programs. A combined control intervention (figure 15) shows similar patterns and suggests that the single most effective intervention approach would be an early focus on larval control. Effects of interventions on predators In this analysis the same conditions are considered as the preceding section but the potential impact of the control interventions on predators is also taken into account. In the case of the larval control intervention, 80% mortality in the predator population is assumed, as was observed by [20]. The predator pressure returns to its normal level after a time lag of 21 days (see Predator section). To the best of our knowledge, no study has focused on predators on adult Anopheles within houses, but spiders in particular are thought to be very efficient in preying on mosquitoes. Here the impact of the destruction of these predators is investigated under an assumption that they represent an adult mosquito mortality of 5%. It is also assumed that the predator-pressure returns to its normal level after a time lag of 21 days. Figures 16, 17 and 18 show the impact of predators on the vector population. Figure 16 shows that the removal of predators has a big impact on the effect of a larval control intervention: the first peak is much less flattened, as it was in the previous section, and is displaced by about seven days. The lack of predator pressure allows a much quicker reconstruction of the larval population. For the adult control intervention, the curves in figure 17 show almost no differences. However, the half-life of the adult mosquito population increases by one day (from 4.6 to 5.7 days), which is of great epidemiological interest since this would increase the vectorial capacity by allowing more mosquitoes to become infectious. Figure 18 considers the effects of a combined larval and adult control intervention for 10 and 25 days respectively and makes several points. First, the combined control intervention seems to have a stronger impact in terms of reducing the adult population. However, it was noted that the peak in adult abundance (with the predator simulation) is higher than the one without the predator simulation and also that there is a dramatic three-day increase in adult half-life (from 4.6 to 7.5 days). Furthermore, if the larval control intervention is delayed by 20 days, the consequences include not only the persistence of a fairly high first peak but also a higher second one. Therefore, such a model could be very important in helping to assess the optimal timing for vector control interventions. Discussion This model integrates important mechanisms underlying Anopheles population dynamics in an explicit, transparent way. It focuses on five basic factors, two of them abiotic – temperature and moisture – and three biotic – nutrient competition, predation or death by disease, and dispersal. Little of the published literature takes into account the effects of temperature on vector populations. It may be that temperature shows little fluctuation compared to countries with marked seasonality, but most African regions like Kenya exhibit temperature fluctuations ranging from 16°C to 35°C, which can be critical. Futhermore, temperature range is a key determinant for species dispersal and is, therefore, of high epidemiological importance: the species have different vectorial capacities and require different control programs. Each parameter in equation 2 is individually related to the slopes of the curves for each stage of insect development (see Schoofield et al. [8]), and therefore may reflect a species' adaptation to different climates. Particularly, , ΔHH and ΔHL, should reflect the sensitivity of each species to temperature changes in temperate, high and low temperature areas respectively, and thus could be highly informative. Many studies focus on vector breeding site characteristics, which the model addresses simply in terms of moisture. As yet no particular variables have been found to be crucial determinants of breeding site selection or success, but when these are determined, the model can implement them relatively easily. The transient patterns of breeding sites are taken into account as key determinants of predator and vector disease dynamics, however. Nutrient competition is considered one of the major regulators of vector populations. Here the carrying capacity concept is used to allow both intra-and inter-species competition. Very few studies of vector predators and pathogens have been undertaken to date, but some literature suggests that this may also be an important determinant, so it has been incorporated accordingly. Little is known about Anopheles dispersal, though this is clearly a critical factor. Here simple random dispersal has been used, but it may be possible to implement a more sophisticated dispersal algorithm soon. Thus, a basic tool has been developed for use by field workers and will be vastly improved by their efforts. First, more complete and precise data on Anopheles biology is needed: if nothing else, the model provides an organized view of the huge gaps in the existing information. A framework has been developed by exploiting what is available, but, at this point, far too many parameters and mechanisms involve arbitrary values or estimates. Nonetheless, as an example, a vector population was simulated for a 20-month period, from May 1, 2000 to December 31, 2001, with meteorological data from Kilifi in Kenya and it was possible to roughly assess the sensitivity of vector population dynamics to four of the five basic factors – temperature, moisture, competition, and predation. The focus was on adult abundance curves. Temperature is very important to the adult abundance curve and, particularly, to the occurrence of the initial peak after a drought period; this may be critical for control purposes. Moisture is a key determinant of particular high peaks that occur not only after a drought period but throughout the year for temporary breeding sites. These peaks were attributed to the lower larval mortality proceeding from lower predation and disease pressure. These peaks may be of great epidemiological importance, in that they could bring malaria prevalence in humans above a threshold at which relatively high transmission could occur despite a low vector density. One concern with such large fluctuations is that the proportion of people susceptible may be very high at the beginning of the peak period. Furthermore, the earliest emergent adult mosquitoes may have a higher vectorial capacity; with almost no food competition, their weight is greater, which implies a longer life [26]. With different initial conditions, when high density competition induces longer development time, the occurrence of the first peak can be delayed by more than a week. Preliminary results on species competition suggest the existence of competitive exclusion, i.e. the survival of only one species in a given habitat, which highlights the necessity of niche differentiation for species coexistence. The example also suggests that if insecticides impact populations of predators on Anopheles, the resulting de-regulation may backfire, producing a vicious cycle that leads to ever-increasing insecticide use. This further supports the argument that great improvements in our understanding of Anopheles ecology and population dynamics are needed. The model is based on the data and knowledge currently available, and it can reproduce some broad, diverse patterns found in the field; its mechanisms and rules are explicit, and they allow us to provide detailed analyses and explanations of vector population dynamics. However, it requires considerable, continued application in the field to improve the data and our understanding of the underlying mechanisms. This is exactly the plan for subsequent research, to contribute to improved control of the scourge of malaria. Table 9 shows the parameters in the most immediate need of field testing and measurement. However, with the default parameter setting, the model can currently be run by users with only: 1. A description of the geographical area with the pools and houses. 2. Climate information (temperature, precipitation, relative humidity) for the period considered. Conclusions This model made it possible to structure existing knowledge of Anopheles vector population dynamics, and highlight crucial elements that are missing. The data and other information currently available made it possible to build a model that can reproduce diverse patterns found in the field. It incorporates explicit mechanisms and rules that can provide detailed analyses and explanations, and thus is a tool to help the malaria research and intervention community gain a better understanding of vector dynamics. The model should be greatly improved as more precise data and hypotheses become available and as it is applied in the field. Authors contributions • JMD contributed conceptualisation and design of the model, main literature review and authorship of the paper. • CM contributed conceptual and data input, review and comments. • GK, BK, JB and JC contributed conceptual input, review and comments. • JD, PB, HM, JG and AT contributed review and comments. • FEM contributed the initial concept and general supervision. All authors read and approved the manuscript. Acknowledgements We wish to thank Lizette Koekemoer of the South African Institute of Medical Research for a advice and provision of unpublished data. Figures and Tables Figure 1 Model description. Figure 2 Three possible curves fit to An. gambiae larvae development rate data. Figure 3 Egg and adult development rates. Figure 4 Larvae and pupae development rates. Figure 5 Predation percentage function of time (lag time). Figure 6 Example schematic. Figure 7 Water height in pools. Figure 8 Rainfall and Temperature Figure 9 Relative humidity Figure 10 Simulated An. gambiae adult abundance at actual temperatures for 20 simulations Figure 11 An. gambiae adult abundance, mean of 20 simulations for each temperature level Figure 12 An. gambiae adult abundance with all three pools (P1, P2 and P3) mean of 20 simulations Figure 13 An. gambiae adult abundance with larval control intervention Figure 14 An. gambiae adult abundance with adult control intervention Figure 15 An. gambiae adult abundance with larval and adult control intervention Figure 16 An. gambiae adult abundance with larval control intervention and predators Figure 17 An. gambiae adult abundance with adult control intervention and predators Figure 18 An. gambiae adult abundance with larval and adult control intervention and predators Table 1 Published or estimated (*) An. gambiae sensu lato immature stage developmental times (in days). The last point (**) is derived from the Jepson catenary curves. Temp. °C Egg Larvae Pupae Egg-Adult Species Reference 15.1 2* 25.8* 2* 29.8 SL [27] 16.3 2* 27.2* 2* 31.2 SL [27] 18 1,5* 19.1* 1.5* 22.1 SL [27] 18 1 21.3 1 23.3 ss [28] 20 1 18.4 1 20.4 ss [28] 21.9 1 12 1 14 SL [29] 22 1 15.5 1 17.5 SS [28] 22.1 1 14 1 16 SL [29] 23 1 19 1 21 SL [5] 23.5 1 10 1 12 SL [29] 24 12.9 SS [30] 24 1 11.5 1 12.5 ss [28] 24.6 1 9 1 11 SL [29] 25 1* 13.1* 1* 15.1 SL [27] 25.3 1 10 1 12 SL [5] 25.4 1 8 1 10 SL [29] 25.5 1 13 2 16 SL [5] 25.5 1 8 1 10 SL [29] 26 1 11 1 13 SL [5] 26 1 9.5 1 11.5 SS [28] 26.8 1 8 1 10 SL [29] 27 10.2 SS [30] 27.2 1 9 1 11 SL [29] 27.5 1 6 1 8 SL [29] Table 2 Published or estimated (*) An. gambiae sensu lato immature stage developmental times (in days). The last point (**) is derived from the Jepson catenary curves (continuing). Temp. °C Egg Larvae Pupae Egg-Adult Species Reference 28 10.88 SS [31] 28 1 7.8 1 9.8 SS [28] 28.1 1 11 2 14 SL [5] 28.2 1 7 1 9 SL [29] 28.4 1 7 1 9 SL [29] 28.4 1 7 1 9 SL [29] 28.9 1 6 1 8 SL [5] 28.9 1 6 1 8 SL [29] 29.6 1 72 10 SL [5] 30 8.3 SS [30] 30 1 8 1 10 SS [28] 30.7 1 5 1 7 SL [29] 30.8 1 6 2 9 SL [5] 30.8 1* 6.1* 2* 9.1 SL [27] 31.2 7.9 SL [32] 31.3 1 4 1 6 SL [29] 31.4 1 8 1 10 SL [5] 31.7 1 72 10 SL [5] 32 1 8.2 1 10.2 SS [28] 32.7 1 5 1 7 SL [29] 32.8 1 6 1 8 SL [5] 33.7 1 6 1 8 SL [29] 37 1* 5.5* 1* 7.5** SL [5] Table 3 An. gambiae developmental rate parameters. ρ25°C ΔH L ΔH H Egg 0.0413 1 -170644 288.8 1000000 313.3 Larvae 0.037 15684 -229902 286.4 822285 310.3 Pupae 0.034 1 -154394 313.8 554707 313.8 Adult 0.02 1000 -75371 293.1 388691 313.4 Table 4 Aestivation daily survival. Daily survival Egg 0.8 Larvae 0.1 Pupae 0.3 Adult 0.96 Table 5 Vector weight parameters. Vector Weight Min (mg) Weight Max (mg) An. gambiae 0.236 0.383 An. arabiensis 0.33 0.45 An. funestus 0.2 0.33 Table 6 Proportion of death attributable to predation in An. gambiae larvae and pupae. Stage duration (days) With predators Without predators Attributable to Predators Larvae 9.98 90.9 79.58 11.34 Pupae 1.79 73.49 35.63 37.86 Total 11.77 97.6 86.85 0.11 Table 7 Water body characteristics sun exposure coef water fill water fix intake (mm) water fix lost (mm) max biomass density (mg·m-2) Pool 1 1 4 0 0.01 30 Pool 2 1 7 0 0.02 60 Pool 3 0.7 4 0 0.028 70 Table 8 Impact of temperature on adult abundance. Temperature Mosquitoes +2oC 47550.3 Normal 41449.9 -2oC 36199 Table 9 Parameters to define. 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(Diptera: Culicidae): the disadvantage of being small J Med Entomol 1998 35 639 645 9775585 Lyimo EO Takken W Effect of body size on fecundity and pre gravid rate of Anopheles gambiae females in Tanzania Med Vet Entomol 1993 7 328 332 8268486 Koella JC Lyimo EO Variability in the relationship between weight and wing length of Anopheles gambiae (Diptera: Culicidae) J Med Entomol 1996 33 261 264 8742532 Service MW Mortalities of the immature stages of species B of the Anopheles gambiae complex in Kenya: Comparison between rice fields and temporary pools, identification of predators, and effects of insecticidal spraying J Med Entomd 1977 13 535 545 Clements A Paterson GD The analysis of mortality and survival rates in wild populations of mosquitoes J Appl Ecol 1981 18 373 399 Charlwood J Smith T Kihonda J Heiz B Billingsley P Takken W Density independent feeding success of malaria vectors (Diptera: Culicidae) in Tanzania Bull Entomol Res 1995 85 29 35 Hogg JC Thompson MC Hurd H Comparative fecundity and associated factors for two sibling species of the Anopheles gambiae complex occuring sympatrically in the gambia Med Vet Entomol 1996 10 385 391 8994142 Maharaj R Effects of temperature and humidity on adults of the Anopheles Gambiae complex (Diptera: Culicidae) in South Africa – implications for malaria transmission and control PhD thesis University of Natal 1995 Patz JA Strzepek K Lele S Hedden M Greene S Noden B Hay SI Kalkstein L Beier J Predicting key malaria transmission factors, biting and entomological inoculation rates, using modelled soil moisture in Kenya Trop Med Int Health 1998 3 818 827 9809915 10.1046/j.1365-3156.1998.00309.x Ameneshewa B Service MW The relationship between female body size and survival rate of the malaria vector Anopheles arabiensis in Ethiopia Med Vet Entomol 1996 10 170 172 8744710 Le Sueur D The ecology, over-wintering and population dynamics of the pre-imaginal stages of the Anopheles Gambiae Giles complex (Diptera culicidae) in northern natal, South Africa PhD thesis, University of Natal, South Africa 1991 MN B SW L Effect of temperature on the development of the aquatic stages of Anopheles gambiae sensu stricto (Diptera: Culicidae) Bull Entomol Res 2003 93 375 381 14641976 10.1079/BER2003259 Holstein MH Biology of Anopheles gambiae : research in French West Africa Tech rep, World Health Organization, Palais des Nations, Geneva 1954 Monograph Series No9 Lyimo EO Takken W Koella J Effect of rearing temperature and larval density on larval survival, age at pupation and adult size of Anopheles gambiae. Entomol Exp Appl 1992 63 265 271 Schneider P Takken W McCall PJ Interspecific competition between sibling species larvae of Anopheles Arabiensis and An. Gambiae. Med Vet Entomol 2000 14 165 170 10872860 10.1046/j.1365-2915.2000.00204.x Gimnig JE Ombok M Otieno S Kaufman MG Vulule JM Walker ED Density-dependent development of Anopheles gambiae (Diptera: Culicidae) larvae in artificial habitats J Med Entomol 2002 39 162 172 11931252

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==== Front Malar JMalaria Journal1475-2875BioMed Central London 1475-2875-3-301529651110.1186/1475-2875-3-30MethodologyIn vivo transcriptional profiling of Plasmodium falciparum Daily Johanna P [email protected] Roch Karine G [email protected] Ousmane [email protected] Xuemin [email protected] Yingyao [email protected] Omar [email protected] Soulyemane [email protected] Ali [email protected] Elizabeth A [email protected] Dyann F [email protected] Department of Immunology and Infectious Disease, Harvard School of Public Health, Boston, Massachusetts 02115, USA2 Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA3 Faculty of Medicine and Pharmacy, Cheikh Anta Diop University, Dakar, Senegal4 Genomics Institute of the Novartis Research Foundation, San Diego California, 92121, USA5 Department of Department of Biostatistics, Harvard School of Public Health, Boston, Massachusetts 02115, USA2004 5 8 2004 3 30 30 1 6 2004 5 8 2004 Copyright © 2004 Daily et al; licensee BioMed Central Ltd.2004Daily et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Both host and pathogen factors contribute to disease outcome in Plasmodium falciparum infection. The feasibility of studying the P. falciparum in vivo transcriptome to understand parasite transcriptional response while it resides in the human host is presented. Methods A custom made oligonucleotide array with probes based on the P. falciparum 3D7 laboratory strain chromosome 2 sequence was used to detect in vivo P. falciparum transcripts. This study analyzed transcripts from total RNA derived from small blood samples of P. falciparum infected patients and compared the in vivo expression profile to the in vitro cultivated 3D7 strain transcriptome. Results The data demonstrated that in vivo transcription can be studied from a small blood sample, despite the abundance of human RNA. The in vivo transcriptome is similar to the 3D7 ring stage transcriptome, but there are significant differences in genes encoding a sexual stage antigen and surface proteins. Conclusions Whole genome transcription analysis of P. falciparum can be carried out successfully and further studies in selected patient cohorts may provide insight into parasite in vivo biology and defense against host immunity. ==== Body Background Plasmodium falciparum infection remains a major health problem worldwide. Its complex life cycle has hampered standard methods for the study of pathogenesis. New approaches to elucidate parasite biology using whole genomic methods have provided insight into gene function, transcriptional regulation and stage specific biology [1-4]. Characterization of the in vivo biology of this pathogen, through adaptation of a whole genome approach, would provide insight into the host-parasite relationship, parasite virulence factors and inform new strategies for intervention. Genomic scale transcriptional profiling of P. falciparum during a natural infection is presented. Small amounts of parasite RNA, isolated from a few milliliters of a blood sample are found to be sufficient for whole genome transcriptional analysis. This data show that several genes are differentially expressed in vivo, indicating differences between the transcriptional program of 3D7 laboratory strain parasites growing in culture and naturally occurring infections in the human host. Whole genome expression has been used in studies of bacterial pathogenesis to identify genes that are specifically transcribed under in vivo conditions [5-7]. For example, genes involved in amino acid transport and metabolism are upregulated in Pasteurella multocida in vivo as compared to in vitro conditions [8]. Similarly, analysis of P. falciparum gene expression patterns, particularly the subset of genes that are specifically expressed in the in vivo state may identify unique parasite biology when it resides in the host environment. Processes involving parasite metabolism, immune evasion and transmission may be altered in the highly specialized environment of the human host as compared to in vitro conditions. In addition, approximately 12% of P. falciparum's predicted genes have not been found to be expressed in any of the life cycle stages previously studied [9]. Whole genomic analysis of the parasite in vivo may reveal the unique expression of such genes in vivo, providing additional targets for intervention. Methods Parasite isolates This study was conducted as part of an ongoing P. falciparum chloroquine resistance study in Senegal [10]. Patients with mild P. falciparum malaria gave consent for the study and were enrolled at an outpatient health clinic. Patients underwent venipuncture using K3 EDTA coated Vacutainers (Beckton Dickinson) and from this sample, 1.6–2.5 ml of whole blood was collected and passed through a white cell depletion filter using a 20 ml syringe. The filtered sample was centrifuged for 5 minutes at 3,200 rpm in a clinical centrifuge and placed in Tri-Reagent BD (Molecular Research Center). The samples were vortexed and stored at minus 70°C. Samples were thawed in a room temperature bath one month later and isolation of RNA was completed per Molecular Research Centers protocol. Three samples obtained in Senegal that had the highest parasitemia and largest blood volume are presented. A 14 ml blood sample from a P. falciparum infected traveler from Nigeria was similarly processed in Boston. Oligonucleotide array analysis Labeling and hybridization of total RNA was performed as described [2]. Expression levels were calculated using the Match Only Integral Distribution Algorithm (MOID) [11]. The presence or absence of gene expression was determined using methods previously described [9]. The design of the probes to human ESTs was based on UniGene version 116. Real time PCR To confirm array data, a subset of genes (PFB0120, PFB0100, PFB0270 PFB0355, and PFB0065) that vary from high to low level abundance by array were quantified using real time PCR from cDNA. PFB0120: forward primer 5'-CAG CCC TCT TAG CTC TCA ACT TC-3', reverse primer 5'-AGC AAC AGC AGA GGC TAT AGA ACT-3', PFB0100: forward primer 5'-CAC CAA ATG GCT ATG CTT ATG GA-3', reverse primer 5'-TTC CAG GAG CAC CAT TAA ATC CT-3', PFB0270: forward primer 5'-ACA CTT ACT GGT ATT TCG GAA TTT-3', reverse primer 5'-TAA TTG TCC ATA TTC TTC AAT ATA T-3', PFB0355: forward primer 5'-ATT GTA AGA AAT AGT TGG GGT-3', reverse primer 5'-TAT ATC ATG CTC CTT CTT ATC A-3', PFB0065: forward primer 5'-CGT TGG TAG TGC GTT CCT TAC AA-3', reverse primer 5'-GTT CCT GCT ATA TCA GGA GCA CCA-3'. Sequence analysis confirmed the identity of the amplification products. 3D7 strain parasites were cultivated under standard conditions and synchronized with 5% sorbitol to obtain ring stage parasites for extraction of total RNA [12,13]. cDNA was synthesized from total RNA from the Nigerian in vivo sample and 3D7 ring stage total RNA using Super Script 1st Strand synthesis system (Invitrogen). Duplicate reactions using real time PCR were performed with 1 μl cDNA with gene specific primers in 50 μl reaction volume using fluorescent dye SYBR Green (SYBR Green PCR Master Mix, Applied Biosystems). The reactions were carried out on an ABI PRISM model 7700-sequence detector and all PCR reactions amplified a single product as determined by dissociation curve analysis (Dissociation Curve Software, Applied Biosystems). Statistical tests Variation between samples was assessed using Kruskal-Wallis method (non-parametric ANOVA) to test the null hypothesis. To normalize samples, the mean gene expression level was calculated for all Plasmodium genes between the 10th and 90th percentile with at least six probes. Analysis based on rank was a second method used; in each experiment the probe intensity was ranked and this resulted in equivalent quantile distribution for all probes between two experiments. This method is more conservative and will define relative rank changes between experiments and is independent of potential normalization artifacts. Human subjects Patient blood samples were collected after informed consent was obtained. The study was approved by the institutional review boards at Harvard School of Public Health, Brigham and Women's Hospital and Cheikh Anta Diop University. Results To evaluate the integrity of the RNA transcripts from the in vivo isolated samples a denaturing RNA gel was carried out (Figure 1). The ribosomal bands are sharp with minimal RNA degradation. Despite buffy coat depletion there are human ribosomal bands present in addition to P. falciparum ribosomal bands. Human ribosomal bands are not seen on a denaturing RNA gel from in vitro cultivated 3D7 (data not shown). The most abundant transcript of human origin in the in vivo was haemoglobin RNA (Table 1). Human transcripts are also detected in 3D7 in vitro samples, but at a lower level of abundance. Figure 1 Denaturing gel of total RNA isolated from P. falciparum infected patient blood reveals both human and parasite ribosomal RNA. Total RNA was electrophoresed on a 1.3% formaldehyde agarose gel and stained with ethidium bromide. Marker (M) marks RNA size ladder. Three patient samples were run in lane 1–3. Closed arrows represent human ribosomal RNA 28s: 4700 bp, 18s:1900 bp; Open arrows mark P. falciparum ribosomal RNA 28s:4104 bp, 18s:1384 bp. Table 1 Human transcripts detected in P. falciparum infected patient blood samples and in vitro cultivated 3D7 samples Comparison of expression level of the most abundant human transcripts detected in in vivo blood and in 3D7 in vitro cultivated samples. Average expression level of in vivo isolated RNA derived from the average expression level of three samples from Senegal and one from Nigeria compared with the average expression level from three 3D7 ring stage in vitro isolated RNA samples. Transcripts are listed in order of highest abundance in vivo as detected by array. Expression levels are reported as expression units (EU), using the MOID algorithm. Expression Units Gene Description in vivo in vitro 3D7 Hs.155376_at haemoglobin, beta 757266 101307 Hs.36977_at haemoglobin, delta EST, similar to tctp human translationally controlled tumor protein H. sapiens 395849 12265 Hs.203820_at 241506 6932 Hs.21295_at EST, Weakly similar to KIAA0902 protein H. sapiens 190454 9321 Hs.247921_at haemoglobin, theta 1 188961 4528 Hs.87246_at Bcl-2 binding component 3 187575 5688 Hs.256047_at ESTs, similar to B24264 proline-rich protein MP3 -M. musculus 160290 5681 Hs.14587_at ESTs, similar to AF1 51 859_1 CGI-101 protein H. sapiens 155768 6389 Hs.117848_at haemoglobin, epsilon 1 150921 5903 Hs.168073_at DKFZP727M231 protein 147492 6615 Hs.24545_at hypothetical protein FLJ11137 146324 6292 Hs.6318_at peroxisomal short-chain alcohol dehydrogenase 145846 5136 Hs.155833_at ESTs, similar to spliceosomal protein SAP 155 H. sapiens Homo sapiens mRNA; cDNA DKFZp434H0820 (from clone DKFZp434H0820); partial cds 140563 5508 Hs.109857_at 138856 7397 Hs.248677_at ESTs, similar to A48018 mucin 7 precursor, salivary – H. sapiens 136390 5253 Hs.4205_at hypothetical protein FLJ20124 ATPase, H+ transporting, lysosomal (vacuolar proton pump), member D 132244 4050 Hs.106876_at 127582 3930 Hs.172914_at retinol dehydrogenase 5 (11-cisand 9-cis) 125310 4560 Hs.23898 at paraneoplastic antigen 124648 5631 The corresponding peripheral blood smears for the four in vivo samples contained only ring forms. Notably, only ring stages are present in the peripheral blood of P. falciparum infected patients; later stages are sequestered in the microvasculature. For this reason, the in vivo whole genome transcription data was compared to the in vitro chromosome 2 ring stage transcriptome. Three samples with parasitemias that were less than 0.3% and total volumes of up to 2.5 ml from Senegal were studied: this resulted in the detection of fewer transcripts than the sample obtained from a Nigerian patient who had parasitemia of 0.4% and underwent a larger blood draw. However, 50% of the top twenty five expressed transcripts in all four samples were shared (data not shown). Further analysis was performed on the Nigerian sample. Only one parasite line was detected in this sample through DNA genotyping of the K1, MAD20, RO33 alleles of msp1 and FC27 and IC1 alleles of msp2 using primers and methods previously reported [14]. After total RNA was isolated, aliquots of 8 μg of total RNA were labeled using a modified Eberwine procedure [2]. To maximize parasite transcript detection, 15 μg to 120 μg of cRNA were hybridized to the array and a quantitative expression level was calculated using the MOID algorithm for the Plasmodium genes on the array [2]. Correlation coefficients comparing P. falciparum chromosome 2 expression levels utilizing 15 μg to 30 μg or 15 μg to 60 μg cRNA were 0.95 and 0.92, respectively. However, as cRNA concentration was increased to 120 μg, the background to noise ratio increased significantly, resulting in a decreased correlation coefficient (R = 0.72) (Figure 2a,2b and 2c). Figure 2 Scatter plot of expression level variance between samples to define the highest signal to background ratio of calculated expression units for P. falciparum probes. Increasing concentrations of cRNA from the Nigerian sample were hybridized to the array and expression levels (Expression Units) for each transcript were derived using the MOID algorithm. 15 μg cRNA data is presented on the Y axis (a) 15 μg v. 30 μg of starting cRNA. (b) 15 μg v. 60 μg starting cRNA. (c) and 15 μg v. 120 μg starting cRNA. (R = correlation coefficient). The most abundant transcripts detected from the Nigerian in vivo sample are listed in Table 2. Genes in bold are uniquely expressed in the in vivo sample compared to 3D7 ring stage previously reported using the Kruskal-Wallis method [2]. Notably, a number of genes encoding surface proteins such as rifins and SERA antigens appear overexpressed in vivo. Table 2 P. falciparum genes expressed in vivo encoded by chromosome 2. In vivo transcripts from the Nigerian sample were defined as present as compared to uninfected blood control hybridization. Asterisk (*) denotes transcripts that were also detected in a Senegal derived blood sample. Genes in bold are uniquely expressed in vivo and were not found to be expressed in the previously reported 3D7 ring stage transcriptome. Gene locus is from PlasmoDB 4.1 . gene locus description gene locus description membrane proteins cellular function PFB0120w* etramps 2 PFB0175c* prt of the MAK16 family PFB0405w* transmission blocking target antigen PFB0205c prt with 5'-3' exonucl. domain PFB0015c rifin PFB0210c monosaccharide transporter PFB0025c rifin PFB0265c* RAD2 endonucl. PFB0035c rifin PFB0295w* adenylosuccinate lyase (OO) PFB1010w* rifin PFB0380c phosphatase (acid phosphatase family) PFB1020w* rifin PFB0390w* ribosome releasing factor (OO, TP) PFB1035w* rifin PFB0410c* phospholipase A2-like a/b fold hydrolase PFB1050w* rifin PFB0445c elF-4A-like DEAD family RNA helicase PFB0330c SERA antigen/papain-like protease PFB0510w GAF domain prt PFB0340c SERA antigen/papain-like protease PFB0525w asparaginyl-tRNA synthetase (OO, TP) PFB0345c* SERA antigen/papain-like protease PFB0585w Leu/Phe-tRNA prt transferase PFB0355c* SERA antigen/papain-like protease PFB0595w* prt with DnaJ domain, DNJ1/SIS1 family PFB0360c SERA antigen/papain-like protease PFB0760w Mtn3/RAG1IP-like prt PFB0095c membrane protein, PfEMP3 PFB0795w ATP synthase alpha chain PFB0975c PfEMP1 fragment PFB0815w* calcium-dept. prt kinase PFB1045w Pf EMP1 fragment PFB0875c chromatin-binding prt (SKI/SNW family) PFB1055c PfEMP1 (var gene) hypothetical proteins PFB0085c* prt with DnaJ domain (RESA-like) PFB0135c hypothetical protein PFB0100c* knob-associated His-rich prt PFB0490c* hypothetical protein PFB0300c merozoite surface antigen MSP-2 PFB0575c hypothetical protein PFB0475c predicted multiple-TM membrane prt PFB0580w hypothetical protein PFB0770c* predicted multiple-TM membrane prt PFB0630c hypothetical protein PFB0125c predicted membrane associated prt PFB0705w hypothetical protein PFB0735c predicted integral membrane protein PFB0745w hypothetical protein PFB0275w* membrane transporter PFB0870w hypothetical protein PFB0465c membrane transporter other PFB0675w predicted secreted protein PFB0990c predicted secreted protein To confirm the accuracy of the results from the oligonucleotide array and to confirm the in vivo overexpression of a SERA antigen (PFB0355), the relative expression of five genes that had varying transcript abundance by array was carried out using real time PCR of cDNA generated from total RNA isolated from the Nigerian in vivo sample and a 3D7 in vitro ring stage sample. There is good correlation between the array results and those obtained by real time PCR (Figure 3). To compare abundance of PFB0355 cDNA between the in vivo and in vitro samples, the data is normalized to cDNA of PFB0120 to account for differences in starting parasite cDNA, secondary to human cDNA. The in vivo sample contained 0.15 ng cDNA of PFB0355c and 3D7 ring stage cDNA had 0.09 ng by real time PCR. When PFB0355c is normalized to PFB0120c, it was found to be ten fold overexpressed in vivo as compared to in vitro, consistent with the array results. Figure 3 Correlation of expression levels derived from the oligonucleotide array with ng cDNA determined by real time PCR. Log ng of cDNA from the Nigerian sample and the in vitro 3D7 ring stage sample was determined for five genes that vary from high to low level abundance by array: PFB0120, PFB0100, PFB0270, PFB0355, and PFB0065. cDNA concentration was determined with real time PCR using a standard curve based on 3D7 genomic DNA. These results were correlated to the expression units (EU) found by the array. (R2 = correlation coefficient) Discussion This data confirms that in vivo whole genomic expression can be performed despite the potential technical challenges of scarce RNA contained in a small blood volume sample and presence of abundant human RNA. Tri-Reagent BD was used to stabilize RNA and the samples were stored at -80°C before transport to the US. The denaturing gel suggests that the RNA remains intact using this reagent. Notably Kyes et al. reported that minus 80°C rather than 4°C or minus 20°C is the optimal temperature to store field sample RNA for detection of long transcripts such as var [15]. Surprisingly, abundant human RNA was detected in the denaturing gel despite buffy coat depletion of the samples. In addition, the degree of hybridization to human probes on the oligonucleotide array used here was not seen in the previously studied in vitro sample [2]. Haemoglobin is found to be the most abundantly expressed human transcript (Table 1) with considerably higher levels noted in the in vivo samples as compared to the 3D7 in vitro sample. Although human red cells are used for culturing in the in vitro system, reticulocytes which contribute to the haemoglobin detected are not abundant in in vitro samples. This is most likely due to the observation that reticulocyte levels display a 75% loss at 48 hours, when placed at 37°C, which is the condition of in vitro culture [16]. In addition, other human RNA such as ribosomal RNA is more abundant in the in vivo samples and may be secondary to white cell contamination. Cross hybridization of human RNA to parasite probes may occur. However, Zhou et al have shown that human genomic DNA does not highly cross hybridize to the parasite probes on this custom array [17]. The high specificity of this array is due to the nature of the highly AT rich parasite genome as compared to the human genome and the careful selection of parasite unique 25 mers probes [2,18,19]. Due to this high specificity, it is likely that buffy coat depletion is not necessary for analysis of in vivo parasite transcripts when using these probes. The amount of blood volume necessary for comprehensive detection of transcription depends on level of parasitemia and method of microarray analysis. This analysis utilized the Affymetrix system which requires very little starting RNA as compared to other methods [20]. Due to the presence of human RNA in the in vivo samples it was not possible to determine how much parasite RNA is required for whole genome analysis. The samples from Senegal were of low parasitemia and small volumes, whereas the 14 ml blood sample with 0.4% parasitemia was sufficient to detect a greater number of chromosome 2 transcripts. Four to five mls of packed blood in a patient with a greater than 2% parasitemia should provide sufficient material for whole genome analysis using these methods. The data demonstrates the reproducibility of the method by independent hybridizations and that maximal senstivity can be achieved with up to 60 μg of cRNA, using this array. PFB0120w is the most abundant in vivo transcript in all samples encoded on chromosome 2. This gene is a member of a recently described gene family, etramps, expressed at early ring stage encoding a protein thought to be involved in erythrocyte remodeling; this was also the most highly expressed transcript in in vitro ring stage cultures [2,21,22]. The in vivo expressed genes from the Nigerian sample was compared to that of the in vitro 3D7 ring stage chromosome 2 transcriptome [2]. As expected, there was a good correlation between the in vivo ring stage transcriptome and the 3D7 ring stage transcriptome. Most of the genes that are expressed in vivo are also expressed in vitro particularly those involved in cellular function and genes encoding hypothetical proteins (Table 2) [2]. A number of differentially expressed genes involved in transmission and antigenic variation were identified. There is high transcription level of transmission blocking target antigen (PFB0405w) in the in vivo samples. Previously this gene has been demonstrated to be expressed and transcribed only in the sexual transmission stage [3,9]. Since no transmissible forms were identified by microscopy this suggests that the in vivo samples may have more parasites that are undergoing or are committed to sexual development than is detectable by microscopy. Several genes encoding membrane proteins, including SERA and rifin genes, were also found to be differentially expressed. These genes are members of multigene family that encode surface proteins which are thought to be involved in immune evasion [23,24]. The observed increase in transcription for these genes in vivo could be due either to genetic or transcriptional modulation of the parasite's defense repertoire or geographic variation. Differences between the in vivo and in vitro expression of SERA antigens may be due to differences in the in vivo stage of development as this gene family is transcribed at later stages in the in vitro life cycle [2,4,25]. There was overall higher hybridization intensity of the in vitro samples due to higher parasite counts and a subset of genes were found to be overexpressed in vitro after normalization. This analysis focussed on genes overexpressed in vivo as these results would not be influenced by normalization algorithms. Overall, this data demonstrates that P. falciparum transcripts can be detected from in vivo samples, and that there are potentially important differences between trancription of in vivo samples and that of the 3D7 in vitro trancription profile. In summary, this study provides evidence that whole genome gene expression in P. falciparum can be studied in vivo from a small blood sample of an infected patient. The in vivo sample however contains human RNA, whose quantity may vary from sample to sample and therefore differenes in parasite transcript level between samples must be reported relative to a reference transcript. Despite the abundance of human RNA the genomes are sufficiently different with resultant probes specificity. Predictably, there was a high correlation of in vivo expression with the in vitro ring stage 3D7 transcriptome [2]. Importantly these data also suggest differences between in vivo and in vitro expression levels in genes typically found in transmissable forms and encoding variant surface proteins. Evaluation of trancription of genes specific for gametocyte development in specific patient populations may uncover the in vivo conditions that favor development of transmissable forms. Similarily, a whole genome analysis can comprehensively characterize expression of multigene families that encode variant surface proteins under in vivo conditions. Further exploration of the in vivo biology of P. falciparum using specific probes to all annotated genes will be undertaken to confirm and explore other important biological differences. 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Part III: Microarray experiments and data analysis Mayo Clin Proc 2002 77 927 940 12233926 Spielmann T Fergusen DJ Beck HP Etramps, a new Plasmodium falciparum gene family coding for developmentally regulated and highly charged membrane proteins located at the parasite-host cell interface Mol Biol Cell 2003 14 1529 1544 12686607 10.1091/mbc.E02-04-0240 Blisnick T Morales Betoulle ME Barale JC Uzureau P Berry L Desroses S Fujioka H Mattei D Braun Breton C Pfsbp1, a Maurer's cleft Plasmodium falciparum protein, is associated with the erythrocyte skeleton Mol Biochem Parasitol 2000 111 107 121 11087921 10.1016/S0166-6851(00)00301-7 Miller SK Good RT Drew DR Delorenzi M Sanders PR Hodder AN Speed TP Cowman AF de Koning-Ward TF Crabb BS A subset of Plasmodium falciparum SERA genes are expressed and appear to play an important role in the erythrocytic cycle J Biol Chem 2002 277 47524 47532 12228245 10.1074/jbc.M206974200 Abdel-Latif MS Khattab A Lindenthal C Kremsner PG Klinkert MQ Recognition of variant Rifin antigens by human antibodies induced during natural Plasmodium falciparum infections Infect Immun 2002 70 7013 7021 12438381 10.1128/IAI.70.12.7013-7021.2002 Aoki S Li J Itagaki S Okech BA Egwang TG Matsuoka H Palacpac NM Mitamura T Horii T Serine repeat antigen (SERA5) is predominantly expressed among the SERA multigene family of Plasmodium falciparum, and the acquired antibody titers correlate with serum inhibition of the parasite growth J Biol Chem 2002 277 47533 47540 12244052 10.1074/jbc.M207145200

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==== Front Biomed Eng OnlineBioMedical Engineering OnLine1475-925XBioMed Central London 1475-925X-3-271529870810.1186/1475-925X-3-27ResearchThermal modeling of lesion growth with radiofrequency ablation devices Chang Isaac A [email protected] Uyen D [email protected] Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, U.S. Food and Drug Administration, Rockville, Maryland, USA2 Department of Biomedical Engineering, Catholic University of America, Washington DC, USA2004 6 8 2004 3 27 27 26 4 2004 6 8 2004 Copyright © 2004 Chang and Nguyen; licensee BioMed Central Ltd.2004Chang and Nguyen; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Temperature is a frequently used parameter to describe the predicted size of lesions computed by computational models. In many cases, however, temperature correlates poorly with lesion size. Although many studies have been conducted to characterize the relationship between time-temperature exposure of tissue heating to cell damage, to date these relationships have not been employed in a finite element model. Methods We present an axisymmetric two-dimensional finite element model that calculates cell damage in tissues and compare lesion sizes using common tissue damage and iso-temperature contour definitions. The model accounts for both temperature-dependent changes in the electrical conductivity of tissue as well as tissue damage-dependent changes in local tissue perfusion. The data is validated using excised porcine liver tissues. Results The data demonstrate the size of thermal lesions is grossly overestimated when calculated using traditional temperature isocontours of 42°C and 47°C. The computational model results predicted lesion dimensions that were within 5% of the experimental measurements. Conclusion When modeling radiofrequency ablation problems, temperature isotherms may not be representative of actual tissue damage patterns. ==== Body Introduction The mitigation of primary and metastatic tumors by radiofrequency ablation is a developing research area. The goal of ablation is to necrose treatment volumes by raising the temperature of targeted tissues. Ablation probes are inserted percutaneously, laparoscopically, or during surgery into cancerous tumors. Once positioned, high frequency alternating current (450–550 kHz) is delivered through an uninsulated electrode into the surrounding tissues to a dispersive ground pad that is applied to the patient. The electromagnetic energy is converted to heat by resistive heating. While the usage of radiofrequency ablation devices is well established, efforts to optimize treatment strategies are ongoing. An important consideration in optimizing ablation is determining what treatment volumes are necessary and acceptable. In liver ablation, for example, treatment volumes generally extend a centimeter beyond the dimensions of a tumor [1-3]. Since the liver possesses regenerative characteristics, it is more critical to insure that necrosis is achieved in 100% of the cancerous cell volume than to minimize damage to healthy tissues. In contrast, a centimeter margin in cardiac ablation is generally unacceptable since many vital substructures are in close proximity. The growth of ablation lesions remains a central issue in the development of radiofrequency ablation devices. Knowing the expected shape of lesions is essential for treatment planning and procedure optimization. To date, many approaches have been attempted to characterize lesion size. The results have varied widely. Ablation lesions generated in vitro and in vivo in animal models show wide variations, since many of the key parameters (i.e. tissue perfusion) cannot be controlled [4,5]. In addition, the boundaries of lesions in animal models are often "fuzzy" and are subject to interpretation. Computational modeling is a valuable tool in the optimization process, since it allows the systematic examination of the various parameters affecting the outcome of ablation. However, most computational models fail to capture essential physiologic phenomena. Many computational studies have been reported in the literature to predict the growth of lesion size during ablation [6-19]. However, the majority of these models do not directly calculate lesion size. Surrogate endpoints, such as temperature [20-22] and thermal dosing [23] are calculated and are interpreted as being equivalent to lesion size. In many cases, these surrogate endpoints do not correlate well with clinical outcome and vary considerably. The microwave hyperthermia literature, for example, cites 42 degrees Celsius as the point at which thermal damage occurs to tissues [24,25]. In the cardiac ablation literature, 47 degrees Celsius is generally accepted as the onset of tissue damage [23,24,26,27]. Neither of these values can be derived directly from gross histological measurements of lesion size, since the tissue pathology does not provide a record of temperature. Many computational studies justify these surrogate endpoints by showing a high correlation between temperature isotherms and lesion size. However, temperature isotherms and lesion size have never actually been shown to be equivalent. Several investigators have demonstrated that tissue damage is a function of both temperature and time [28-30]. As tissue temperature is increased, the amount of time necessary to achieve a threshold of damage decreases. Tissue damage can be characterized using the Arrhenius equation which relates temperature and exposure time using a first order kinetics relationship. Data from experimental studies, where tissues are exposed to uniform temperatures for controlled time intervals, are fit to the Arrhenius equation to determine the frequency factor A(s-1), and the activation energy ΔE (J mol-1). Arrhenius parameters have been determined in skin [31-35], artery [36,37], blood [38-40], pancreas [41,42], heart [43], cornea [44-46], muscle [47], prostate [48], ovary [49], kidney [50-52], and liver [30,52,53]. For a specified exposure temperature and time, the fit parameters A and ΔE determine the amount of cell damage incurred for a specific tissue type. In combination with computational modeling techniques, it is then possible to calculate the distribution of cell damage surrounding ablation probes. In this study, we compare the temperature distribution and tissue necrosis patterns for a hepatic ablation probe at body temperatures. At each time step, the specific absorption rate (SAR), temperature, and the tissue damage are calculated. The level of tissue perfusion is varied for the models to determine the maximum variation in lesion size resulting from a typical ablation. These data are validated experimentally using an ablation probe in liver tissue. Methods Radiofrequency ablation probes operate between 460–550 kHz. At these frequencies, the wavelength of the electromagnetic energy is several orders of magnitude larger than the size of the ablation electrodes. Thus, the primary mode of energy transfer is through electrical conduction and can be modeled as a coupled quasistatic electrical conduction and heat conduction problem. The electric field is solved by using Laplace's equation, ∇·[σ(T)∇V] = 0     (Eq.1) where ∇ is the gradient operator, σ (T) is the temperature-dependent conductivity (Siemens/meter), and V is the electric potential (Volts). Temperature is solved by using a modified Pennes bioheat equation [54], where ρ is the density, 1060 kg/m3 [55]; C is the heat capacity of tissues, 3600 J/kg-K [55]; k is the heat conduction coefficient, 0.502 W/K-m [55]; ρb is the density of blood, 1000 kg/m3 [9]; Cb is the heat capacity of blood, 4180 J/kg-K [9];α is a tissue state coefficient; ω is the blood perfusion coefficient, 6.4 × 10-3 sec-1 [9];Tamb is the ambient body temperature, 37°C; and Qm is the metabolic heat source term. For all cases, we assumed that the metabolic heat source was insignificant. The tissue state coefficient (α) ranges from 0–1 depending on the local level of tissue damage At each time step, the cumulative damage integral is computed using the well established Arrhenius equation where Ω(t) is the degree of tissue injury, c(t) is the concentration of living cells, R is the universal gas constant, A is a "frequency" factor for the kinetic expression (s-1), and ΔE is the activation energy for the irreversible damage reaction (J-mol-1) [50]. The kinetic parameters account for morphologic changes in tissue relating to the thermal degradation of proteins [56]. The parameters A and ΔE are dependent on the type of tissue and have been characterized for liver tissues by Jacques et. al. (A = 7.39 × 1039 s-1 and ΔE = 2.577 × 105 J-mol-1) [52]. In the context of finite element modeling of tissue damage, a damage integral of Ω = 1, corresponds to a 63% percent probability of cell death at a specific point. A damage integral of Ω = 4.6, corresponds to 99% percent probability of cell death at a point in the model. The significance of Ω = 1 has been reported as the point at which tissue coagulation first occurs [36]. Once tissue coagulation occurs, tissue perfusion ceases. This corresponds to a tissue state coefficient of α = 0. Intermediate levels of the tissue state coefficient are calculated as α = 1/exp(Ω). Figure 1 shows a diagram of a typical needle ablation electrode used in clinical practice for hepatic tumor ablation. The probe is 6.0 cm long with a diameter of 0.15 cm. The distal 2.0 cm of the probe is uninsulated and the proximal 4.0 cm of the probe is covered with a thin electrically insulating material. Figure 2 shows a three-dimensional representation of the axisymmetric two-dimensional geometry of the model. The active portion of the probe is situated in the center of a cylindrical model that is 6.0 cm in radius and 12.0 cm in height. Electrical and thermal properties of liver are used in the model to simulate a fully-embedded insertion of the needle electrode. The electrical properties of tissue are assumed to be temperature dependent and solved according to Chang [57], where the electrical conductivity appears as Figure 1 Ablation probe geometry diagram of a single needle ablation electrode that is used for hepatic tumor ablation. Therapeutic treatment is achieved by applying a source voltage to the conducting tip. A conducting pad applied to the patient skin serves as an electrical ground return. Figure 2 Model geometry three dimension representation of the axisymmetric two-dimensional finite element model. All external surfaces of the cylindrical model serve as the electrical ground and are at body temperatures (37°C). The entire ablation probe is assumed to be thermally insulating. σ(T,N) = σ(25,N) {1.000-1.962 × 10-2Δ + 8.08 × 10-5Δ2 - NΔ [3.020 × 10-5 + 3.922 × 10-5Δ + N(1.721 × 10-5Δ - 6.584 × 10-6Δ)]}     (Eq.4) where σ (25,N) = N [10.394-2.3776 N + 0.68258 N2 - 9.13538 N3 + 1.0086 × 10-2 N4] ;N is the normality of an electrically equivalent sodium chloride solution, N = 0.0111; and Ä = 25-T, which produces an equivalent electrical conductivity of liver tissues at 37°C (approximately 0.134 S/m). The thermal properties of liver used in the model were acquired from Tungjitkusolmun et al.[9] and Duck [55]. A source voltage (Vo) is applied to the conducting tip of the ablation probe. The outer surface of the model serves as an electrical ground return (V = 0). An electrically insulating boundary condition is applied to the non-conducting portions of the probe such that n·(σ∇ V) = 0; where n is the unit vector normal to the surface, σ is the electrical conductivity, and V is the voltage at the insulating surface. A thermal boundary condition of T = Tamb is applied to the outer surfaces of the model to simulate ambient temperature. Since the thermal mass of the probe is small compared to the surrounding tissue, we assumed that heat conduction into the probe itself was minimal. Thus, all other surfaces of the ablation probe are considered to have a thermally insulating boundary condition such that n·(k ∇ T) = 0. A hybrid finite element model was developed using Femlab (Comsol, Burlington MA, USA) and Matlab (Mathworks, Natick MA, USA) to calculate temperature and tissue damage. While conventional finite element models effectively solve field solutions using a nonuniform geometrical mesh, tissue exposure calculations are integrated at each point in the model over the course of ablation and are more easily calculated using uniform rectilinear grids. As shown in Figure 3, Femlab is used to solve the coupled electromagnetic and heat conduction equations simultaneously at each timestep. This is done to insure that the temperature-dependent electrical conductivity is updated with each iterative calculation of temperature for a given timestep. The converged temperature is mapped from the finite element mesh into a rectilinear grid, which is passed into the Matlab environment. The amount of tissue damage occurring at each timestep is calculated using the Arrhenius equation and tracked at each point in the model. Once the level of damage exceeds 63% cell damage, it is assumed that tissue coagulation has occurred, causing a cessation in tissue perfusion. The 63% cell damage point is historically used because it corresponds to the earliest onset of visible tissue coagulation. A rectilinear grid containing the perfusion characteristics at each point in the model is mapped back into the finite element mesh and used in subsequent Femlab calculations. The rectilinear grid of temperature is also used to calculate the change in the electrical conductivity which is an explicit function of temperature. This data is also mapped back into the finite element mesh and is used to change the electromagnetic sourcing characteristics. Augmented matrices are used to insure that calculations made on the geometric borders of the Femlab model are interpolated correctly. Figure 3 Computational technique diagram of data flow used in a hybrid finite element model implemented in Femlab/Matlab to calculate temperature and tissue damage. The electric field and temperature are solved simultaneously in Femlab (blue blocks). The data structure is changed from finite element meshing to rectilinear gridding so that the resulting temperature can be used to calculate tissue exposure and electrical conductivity change in Matlab (orange blocks). A tissue damage level of 63% corresponds to the onset of tissue necrosis and is associated with a cessation in local blood flow. The Matlab results are then imported into Femlab as inputs for calculation at the next time step. Given the axial symmetry of the problem, we used a 2D-axisymmetric mesh consisting of 13,641 nodes and 26,880 elements. The Femlab 'Fldaspk' ordinary differential equation solver was used to achieve convergence. This is a robust variant of the traditional ODE15s stiff differential equation solver used in solving finite element problems in Matlab. Ablations were simulated at source voltages of 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, and 30 volts. For each of the source voltages, we varied the initial level of tissue perfusion at 0%, 20%, 40%, 60%, 80%, and 100% normal tissue perfusion (6.4 × 10-3 cubic meters of blood/ cubic meter of tissue/ second) [9]. Ambient tissue temperature was assumed to be 37°C. The model simulates a 15 minute ablation and updates tissue parameters at 2 second timestep intervals. Once the 15 minute ablation has ended, the model continues to solve solutions for 15 minutes post-ablation. For each simulation, the electric field (E), the current density (J), the temperature (T), and the tissue damage (D) were calculated. All calculations were implemented on a Dual 3.02 GHz Xeon processor workstation with 4 GByte RAM. Each simulation takes approximately 3 hours to run. Experimental Validation To validate the computational model, experimental measurements were made in 6 freshly excised porcine liver sections. A single needle ablation probe with a 2 cm uninsulated tip was inserted 3 cm into each liver tissue. Since commercial RF ablation generators operate using either constant temperature or constant power feedback algorithms, an experimental constant voltage RF generator (500 kHz) was used [5]. Tissue samples were allowed to equilibrate to room temperature (approximately 22°C) prior to the start of ablation. Two samples were ablated at 20 volts for 15 minutes. After allowing the tissue to cool for an additional 15 minutes, the probes were removed and the tissue was bisected to expose the lesion. The tissues were immediately placed in a 1% 2,3,5-triphenyltetrazolium chloride (red) solution for 20 minutes to stain for tissues containing active dehydrogenase, an indicator of cell viability [58,59]. This stains healthy tissue brick red, leaving the ablated region a pale grey color. The maximum width and depth of the macroscopically pale ablated regions were measured. This procedure was repeated for two samples at 25 volts and for the last two samples at 30 volts. Computational model calculations were made at 20, 25, and 30 volts following the same experimental protocol. Ambient temperature for these calculations was 22°C instead of the 37°C temperature used in the main simulations. The calculated lesion sizes were directly compared with the measurements in tissue. Results Table 1 shows the maximum temperatures attained in tissue for the computational models for a range of voltages (2.5–30 Volts) and tissue perfusion rates (0–100% normal tissue perfusion.). The table shows a nonlinear relationship between the source voltage and the maximum temperature that results from the use of a temperature-dependent electrical conductivity. The maximum variation in the temperature data for a given source voltage did not exceed 17%. The data show that the rate of temperature increase accelerates as a function of the source voltage. As the level of tissue perfusion increases, tissue temperature decreases. Table 1 Maximum Temperature (Degrees Celsius)1 Values represent the maximum temperature attained in tissue for the computational models. Source Voltage (Volts) 0%2 Perfusion (0.0 × 10-3 mb3/mt3/s) 20%2 Perfusion (1.3 × 10-3 mb3/mt3/s) 40%2 Perfusion (2.6 × 10-3 mb3/mt3/s) 60%2 Perfusion (3.8 × 10-3 mb3/mt3/s) 80%2 Perfusion (5.1 × 10-3 mb3/mt3/s) 100%2 Perfusion (6.4 × 10-3 mb3/mt3/s) 2.5 37.3 37.3 37.2 37.2 37.2 37.2 5.0 38.6 38.5 38.4 38.3 38.2 38.2 7.5 41.0 40.6 40.3 40.1 40.0 39.9 10.0 44.3 43.6 43.1 42.8 42.5 42.3 12.5 48.8 47.7 46.9 46.3 45.8 45.4 15.0 54.6 52.9 51.6 50.7 50.0 49.4 17.5 62.0 59.5 57.7 56.2 55.2 54.3 20.0 71.1 67.9 65.5 63.4 61.9 60.7 22.5 82.4 78.6 75.4 73.0 70.7 69.0 25.0 96.1 91.8 88.0 84.8 82.1 79.7 27.5 112.73 107.83 103.43 99.5 96.1 93.4 30.0 132.53 126.93 121.83 117.43 113.43 109.93 1 – The maximum temperature for the case of 0% perfusion was located along the center of the conducting electrode. In all other cases, the maximum temperature occurred at the tip of the probe; 2 – The units for tissue perfusion are cubic meters of blood (mb3) per cubic meter of tissue (mt3) per second; 3 – These temperatures do not account for energy loses associated with tissue desiccation or gas formation. Table 2 shows the maximum electrical conductivity in the tissue after heating for 15 minutes at a variety of source voltages. All tissues initially have an electrical conductivity of 0.144 S/m at 37°C. The data show that tissue electrical conductivity is primarily a function of the source voltage, changing 320% over the course of a 15 minute ablation using a 30 volt source. With normal tissue perfusion (6.4 × 10-3 mb3/ mt3/s), the electrical conductivity changes as much as 260% using a 30 volt source. The electrical conductivity is indirectly a function of tissue perfusion since tissue perfusion is zero in the necrosed treatment volume. Tissue perfusion lowers the tissue temperature outside the treatment volume which helps to conduct heat away from temperatures within the ablated area.. Table 2 Maximum Electrical Conductivity (Siemens/meter)1 Values represent the maximum electrical conductivity attained in tissue for the computational models. Source Voltage (Volts) 0%2 Perfusion (0.0 × 10-3 mb3/mt3/s) 20%2 Perfusion (1.3 × 10-3 mb3/mt3/s) 40%2 Perfusion (2.6 × 10-3 mb3/mt3/s) 60%2 Perfusion (3.8 × 10-3 mb3/mt3/s) 80%2 Perfusion (5.1 × 10-3 mb3/mt3/s) 100%2 Perfusion (6.4 × 10-3 mb3/mt3/s) 2.5 0.144 0.144 0.144 0.144 0.143 0.143 5.0 0.147 0.147 0.146 0.146 0.146 0.146 7.5 0.153 0.152 0.151 0.151 0.150 0.150 10.0 0.162 0.160 0.159 0.158 0.157 0.156 12.5 0.173 0.170 0.168 0.167 0.165 0.164 15.0 0.189 0.185 0.181 0.179 0.177 0.175 17.5 0.210 0.203 0.198 0.194 0.191 0.189 20.0 0.238 0.228 0.221 0.215 0.210 0.207 22.5 0.274 0.262 0.251 0.244 0.237 0.232 25.0 0.321 0.306 0.293 0.282 0.273 0.265 27.5 0.383 0.364 0.348 0.334 0.321 0.312 30.0 0.463 0.440 0.419 0.401 0.385 0.372 1 – The maximum electrical conductivity for the case of 0% perfusion was located along the center of the conducting electrode. In all other cases, the maximum temperature occurred at the tip of the probe; 2 – The units for tissue perfusion are cubic meters of blood (mb3) per cubic meter of tissue (mt3) per second. Table 3 shows the maximum SAR computed for a range of voltages and tissue perfusion rates. The SAR is defined as SAR = σ /ρ*|E|2, where σ is the electrical conductivity, ρ is the tissue density, and |E| is the magnitude of the electric field. The data shows that the SAR is highest with increasing source voltage with no tissue perfusion. Initially, this seems counterintuitive as one would expect a higher maximum SAR for perfused flows, where a greater amount of power is needed to compensate for the convective heat loss. This observation can be explained by the large changes in the electrical conductivity (Table 2). Since higher temperatures are achieved for cases with no tissue perfusion, the change in the electrical conductivity is highest with no tissue perfusion. Since, at a given point, the density and the magnitude of the electric field are essentially constant (<0.02% change), the SAR will vary as a function of the electrical conductivity only. Table 3 Maximum Specific Absorption Rate (Watts/kg)1 Values represent the maximum specific absorption rate (SAR) attained in tissue for the computational models. Source Voltage (Volts) 0%2 Perfusion (0.0 × 10-3 mb3/mt3/s) 20%2 Perfusion (1.3 × 10-3 mb3/mt3/s) 40%2 Perfusion (2.6 × 10-3 mb3/mt3/s) 60%2 Perfusion (3.8 × 10-3 mb3/mt3/s) 80%2 Perfusion (5.1 × 10-3 mb3/mt3/s) 100%2 Perfusion (6.4 × 10-3 mb3/mt3/s) 2.5 645.6 645.3 645.1 644.9 644.8 644.8 5.0 2608 2603 2600 2598 2596 2595 7.5 5966 5940 5924 5912 5903 5896 10.0 10850 10770 10720 10680 10650 10630 12.5 17450 17260 17120 17030 16950 16900 15.0 26030 25620 25330 25120 24970 24860 17.5 36940 36140 35600 35190 34900 34680 20.0 50590 49180 48230 47480 47000 46620 22.5 67470 65210 63520 62510 61610 60990 25.0 88170 84890 82360 80440 78940 77910 27.5 113300 108900 105300 102400 100100 98390 30.0 143400 137700 133000 129000 125700 123000 1 – The maximum current density was always located at the tip of the ablation probe. Therefore, all values listed above are comparable with each other; 2 – The units for tissue perfusion are cubic meters of blood (mb3) per cubic meter of tissue (mt3) per second. Figure 4 shows the tissue temperature and the cell death penetration into tissue for a 15 minute ablation using a 30 volt constant voltage source for perfusion rates ranging from no perfusion to 100% normal tissue perfusion. The data show that cell death decreases more rapidly than tissue temperature. At the center of the active electrode, temperatures decrease as a function of the inverse of the radius squared (1/r2), whereas cell damage exhibits an S-shaped curve. Figure 4 shows that 63% tissue damage is roughly correlated with the 60°C isotherm for liver tissues. Conventional temperature isotherms for tissue damage for hyperthermia (42°C) and radiofrequency ablation (47°C) substantially overestimate the size of the lesions. Figure 4 Tissue temperature and cell death penetration for a 15 minute ablation using a 30 volt constant voltage source. Simulation results for a 15 minute ablation using a 30 volt constant voltage source measured from the center of the active electrode. The graph shows temperature (solid) and cell death (dotted) penetration into liver tissue for a range of tissue perfusion rates. The units for tissue perfusion are cubic meters of blood (mb3) per cubic meter of tissue (mt3) per second (s). Figure 5 shows a plot of tissue temperature and cell damage calculated at a distance of 4 millimeters from the center of the active electrode for a 15 minute ablation using a 30 volt constant voltage source. Temperature decrease and cell damage that occurs after the ablation is monitored for an additional 15 minutes. The data show that near the electrode, tissue damage will reach 100% well within the first few minutes of energy application. For cases of no tissue perfusion, 100% tissue damage occurs after 5 minutes at a distance of 4 millimeters. For cases with normal tissue perfusion, 100% tissue damage occurs approximately 8 minutes into the ablation. At a distance of 10 millimeters from the center of the active electrode under the same conditions (Figure 6), the data show that tissue damage will not always reach 100%. For the case of no perfusion, 100% cell damage is reached a minute after the termination of radiofrequency energy. In cases with varying levels of tissue perfusion, cell damage is significantly reduced and, in some cases, insignificant. Although the overall temperatures are lower at 10 millimeters than at 4 millimeters, temperatures near 60°C are reached but do not result in complete tissue damage because the length of time in which the tissue is exposed is not sufficient. Figure 5 Tissue temperature and cell death at a distance of 4 millimeters from the center of the active electrode using a 30 volt constant voltage source. Ablation simulation results attained 4 millimeters from the center of the active electrode for a 15 minute ablation using a 30 volt constant voltage source. The graph shows temperature (solid) and cell death (dotted) penetration into liver tissue for a range of tissue perfusion rates. The units for tissue perfusion are cubic meters of blood (mb3) per cubic meter of tissue (mt3) per second (s). Figure 6 Tissue temperature and cell death at a distance of 10 millimeters from the center of the active electrode using a 30 volt constant voltage source. Ablation simulation results attained 10 millimeters from the center of the active electrode for a 15 minute ablation using a 30 volt constant voltage source. The graph shows temperature (solid) and cell death (dotted) penetration into liver tissue for a range of tissue perfusion rates. The units for tissue perfusion are cubic meters of blood (mb3) per cubic meter of tissue (mt3) per second (s). Figure 7 and 8 show comparisons of temperature distribution and lesion size development with no tissue perfusion (Figure 7) and with normal tissue perfusion (Figure 8) for a 30 volt constant voltage source ablation at 1, 3, 5, 10 and 15 minutes. The data demonstrate that the shapes of the temperature isotherms do not correlate well with tissue damage profiles. Tissue perfusion greatly affects the size of the resulting ablated region. At 15 minutes, lesion volumes are 267% larger without perfusion than with tissue perfusion. Figures 9 and 10 show a comparison of the temperature distribution and lesion size development with no tissue perfusion (Figure 9) and with normal tissue perfusion (Figure 10) at 1, 3, 5, 10, and 15 minutes following a 15 minute constant 30 volt ablation. In the case of no tissue perfusion, the lesion size continues to grow 14% within the first 5 minutes after radiofrequency energy is terminated. The lack of tissue perfusion prolongs the time needed to conduct the heat away from tissues near the surface of the ablation electrode. For cases with normal tissue perfusion, heat is quickly dissipated by tissue perfusion causing the lesion volume to stabilize in less than 2 minutes. By definition, the area of coagulative necrosis has no tissue perfusion. This accounts for the residual heating pattern within the ablated region as seen up to 3 minutes following the ablation. Figure 7 Comparison of temperature and lesion size development with no tissue perfusion for a 30 volt constant voltage source ablation. Ablation simulation results for a 30 volt constant voltage source ablation with no tissue perfusion. The results on the top half of the figure represent the temperature distribution surrounding the ablation probe in degrees Celsius. The results on the bottom half of the figure represent the percent tissue damage. The numbers listed at the bottom are the lesion volume sizes computed from the 63% cell damage isocontours at each time interval shown. Figure 8 Comparison of temperature and lesion size development with normal tissue perfusion (6.4 × 10-3 mb3/mt3/s) for a 30 volt constant voltage source ablation. Ablation simulation results for a 30 volt constant voltage source ablation with normal tissue perfusion (6.4 × 10-3 mb3/mt3/s). The results on the top half of the figure represent the temperature distribution surrounding the ablation probe in degrees Celsius. The results on the bottom half of the figure represent the percent tissue damage. The numbers listed at the bottom are the lesion volume sizes computed from the 63% cell damage isocontours at each time interval shown. Figure 9 Comparison of temperature and lesion size development post ablation with no tissue perfusion for a 30 volt constant voltage source ablation. Ablation simulation results following a 15 minute ablation without perfusion for a 30 volt constant voltage source ablation. The results on the top half of the figure represent the temperature distribution surrounding the ablation probe in degrees Celsius. The results on the bottom half of the figure represent the percent tissue damage. The numbers listed at the bottom are the lesion volume sizes computed from the 63% cell damage isocontours at each time interval shown. Figure 10 Comparison of temperature and lesion size development post ablation with normal tissue perfusion (6.4 × 10-3 mb3/mt3/s) for a 30 volt constant voltage source ablation. Ablation simulation results following a 15 minute ablation with normal tissue perfusion (6.4 × 10-3 mb3/mt3/s) for a 30 volt constant voltage source ablation. The results on the top half of the figure represent the temperature distribution surrounding the ablation probe in degrees Celsius. The results on the bottom half of the figure represent the percent tissue damage. The numbers listed at the bottom are the lesion volume sizes computed from the 63% cell damage isocontours at each time interval shown. A comparison of lesion volumes with no tissue perfusion computed using 63% and 100% iso-damage threshold contours and 42°C, 47°C, 60°C, and 90°C isothermal contours is presented for the cases of no tissue perfusion (Table 4) and normal tissue perfusion (Table 5). The sensitivity of the cell damage function (Figure 4) results in less than 10% differences in the size of lesions calculated using tissue damage thresholds of 63% and 100% cell damage. In contrast, volume sizes based on isothermal contours varies considerably at each temperature. When using traditional isothermal contours of 42°C and 47°C, the calculated lesion volumes are grossly overestimated by 500% and 167%, respectively. In both the case of no tissue perfusion and normal tissue perfusion, the 60°C isothermal contour resembles the lesion sizes calculated using the iso-damage contours. Table 4 Lesion Volume with No Tissue Perfusion Values represent the total volume of tissue necroses calculated over the course of the simulated ablation using various cell damage thresholds (D) and isothermal temperatures (IT). Source Voltage (Volts) D = 63% (mm3) D = 100% (mm3) IT = 42°C (mm3)1 IT = 47°C (mm3)1 IT = 60°C (mm3) IT = 90°C (mm3) 2.5 0 0 0 0 0 0 5.0 0 0 0 0 0 0 7.5 0 0 0 0 0 0 10.0 0 0 89 0 0 0 12.5 0 0 444 13 0 0 15.0 0 0 942 216 0 0 17.5 9 3 1649 526 6 0 20.0 121 67 2508 915 87 0 22.5 314 242 3549 1414 296 0 25.0 577 495 4860 2070 547 6 27.5 923 809 6452 2866 870 55 30.0 1388 1228 8386 3830 1243 202 1 – The 42°C and 47°C isothermal volumes were chosen specifically because they are frequently used to establish damage thresholds in hyperthermia and radiofrequency ablation, respectively. Table 5 Lesion Volume with 100% Normal Tissue Perfusion. Values represent the total volume of tissue necroses calculated over the course of the simulated ablation using various cell damage thresholds (D) and isothermal temperatures (IT). Source Voltage (Volts) D = 63% (mm3) D = 100% (mm3) IT = 42°C (mm3)1 IT = 47°C (mm3)1 IT = 60°C (mm3) IT = 90°C (mm3) 2.5 0 0 0 0 0 0 5.0 0 0 0 0 0 0 7.5 0 0 0 0 0 0 10.0 0 0 1 0 0 0 12.5 0 0 47 0 0 0 15.0 0 0 185 6 0 0 17.5 0 0 358 68 0 0 20.0 4 2 513 177 1 0 22.5 23 19 784 287 15 0 25.0 131 89 1068 488 107 0 27.5 251 222 1502 759 241 3 30.0 433 364 2014 1064 423 24 1 – The 42°C and 47°C isothermal volumes were chosen specifically because they are frequently used to establish damage thresholds in hyperthermia and radiofrequency ablation, respectively. Table 6 shows a comparison of lesion width and depth computed using 63% and 100% iso-damage threshold contours and 42°C, 47°C, 60°C, and 90°C isothermal contours. The data show overestimations of 30–77% in the width and 18–54% in the depth of lesions when using traditional isothermal temperatures for a 15 minute ablation with no perfusion. Table 7 shows that in cases with normal tissue perfusion, calculations using traditional isothermal contours results in overestimations of 25–88% in the width and 15–41% in the depth of lesions. Table 6 Lesion Dimensions with no Tissue Perfusion Values represent the maximum lesion width and depth calculated over the course of the simulated ablation using various cell damage thresholds (D) and isothermal temperatures (IT). Width (mm) Depth (mm) Source Voltage (Volts) D = 63% D = 100% IT = 42°C1 IT = 47°C1 IT = 60°C IT = 90°C D = 63% D = 100% IT = 42°C1 IT = 47°C1 IT = 60°C IT = 90°C 2.5 0 0 0 0 0 0 0 0 0 0 0 0 5.0 0 0 0 0 0 0 0 0 0 0 0 0 7.5 0 0 0 0 0 0 0 0 0 0 0 0 10.0 0 0 8 0 0 0 0 0 19 0 0 0 12.5 0 0 16 0 0 0 0 0 26 0 0 0 15.0 0 0 22 8 0 0 0 0 31 13 0 0 17.5 6 4 28 14 4 0 7 5 35 24 6 0 20.0 10 8 32 18 10 0 21 16 39 28 18 0 22.5 14 12 36 22 14 0 25 24 43 31 24 0 25.0 18 18 40 26 18 4 28 26 45 34 28 6 27.5 22 22 44 30 22 8 31 30 49 37 30 14 30.0 26 26 46 34 26 12 33 33 51 39 33 23 1 – The 42°C and 47°C isothermal volumes were chosen specifically because they are frequently used to establish damage thresholds in hyperthermia and radiofrequency ablation, respectively. Table 7 Lesion Volume with 100% Normal Tissue Perfusion Values represent the maximum lesion width and depth calculated over the course of the simulated ablation using various cell damage thresholds (D) and isothermal temperatures (IT). Width Depth Source Voltage (Volts) D = 63% D = 100% IT = 42°C1 IT = 47°C1 IT = 60°C IT = 90°C D = 63% D = 100% IT = 42°C1 IT = 47°C1 IT = 60°C IT = 90°C 2.5 0 0 0 0 0 0 0 0 0 0 0 0 5.0 0 0 0 0 0 0 0 0 0 0 0 0 7.5 0 0 0 0 0 0 0 0 0 0 0 0 10.0 0 0 2 0 0 0 0 0 2 0 0 0 12.5 0 0 8 0 0 0 0 0 16 0 0 0 15.0 0 0 10 0 0 0 0 0 25 0 0 0 17.5 0 0 14 6 0 0 0 0 27 7 0 0 20.0 4 4 16 8 2 0 5 4 29 23 2 0 22.5 6 6 20 10 6 0 10 8 31 25 8 0 25.0 10 8 24 14 8 0 23 23 33 27 23 0 27.5 12 12 26 18 12 4 25 25 36 29 25 5 30.0 16 14 30 20 16 6 27 27 38 31 27 9 1 – The 42°C and 47°C isothermal volumes were chosen specifically because they are frequently used to establish damage thresholds in hyperthermia and radiofrequency ablation, respectively. To validate the computational model, ablation experiments were performed at room temperature (22°C) in excised porcine liver tissue using 20, 25, and 30 volt constant voltage radiofrequency sources (500 kHz). Ablations were made for a 15 minute exposure time. Figure 11 shows that no visible lesion can be seen in tissues where the 20 volt constant voltage ablation was performed, as predicted by the computational simulation. A lesion that was approximately 10 millimeters in width and 22 mm in depth resulted from the 30 volt constant voltage ablation. Table 8 shows a high correlation between the computational data calculated at 22°C and the experimental results. Figure 11 Experimental validation radiofrequency ablation lesions in excised porcine liver tissue produced by a 20 volt (left) and 30 volt (right) constant voltage radiofrequency generator (500 kHz) for a 15 minute exposure time. The lesions were produced using a single needle ablation probe with a 2 centimeter uninsulated tip. Table 8 Comparison of Computational Data to Experimental Validation Data at 22°C. Lesion Width Lesion Depth Source Voltage (Volts) D = 63% (mm) Experimental (mm) D = 63% (mm) Experimental (mm) 20.0 0 0 0 0 22.5 0 -- 0 -- 25.0 4 5 5 6 27.5 8 -- 9 -- 30.0 10 10 21 22 Simulated lesion dimensions with no tissue perfusion at an ambient temperature of 22°C using a 63% cell damage threshold (D) were compared to experimental measurements made at 20, 25, and 30 volts using an experimental constant voltage radiofrequency ablation source. Lesions were generated with a single needle ablation probe with a 2.0 cm active electrode. All lesion measurements were measured visually under a micrsope at 10× magnification. Discussion To date, several computational studies have been performed to described the rate of lesion growth in radiofrequency ablation applications. In many cases, these studies use surrogate endpoints such as temperature isotherms and thermal dosing to calculate equivalent expressions for lesion size. While many models exists that account for far-more elaborate parameters such as tissue perfusion through large blood vessels, the interpretation of such models is difficult since most do not account for transient changes in tissue properties and often report tissue temperature only [6-9,12,14-19,56,57,60]. Several studies have identified that both exposure time and temperature contribute to tissue damage, however, few have actually calculated tissue damage. Those that do, have not allowed tissue damage to transiently influence the electrical and thermal properties of tissues [6,56]. In this study, we created a computational simulation that tested some of the basic assumptions made in modeling lesion growth problems. We developed a model where tissue perfusion and the electrical conductivity are allowed to vary at each time step and spatial position as a function of tissue damage and temperature. These simulations are significantly more time-consuming since gross simplifications to heating mechanisms are not made. Although our model geometry is simpler than others that appear in the literature, we chose to ignore large vessels since their position and impact are highly variable. We chose a simpler geometry so that the impact of damage-dependent tissue perfusion and temperature-dependent electrical conductivity could be assessed more directly. The damage-dependent tissue perfusion accounts for physiological observations of tissue coagulation and local cessation of blood flow. Unlike thermal dosing, where thermal injury is calculated globally over the entire duration of an ablation, tissue damage is calculated at every time step. The intermediate tissue damage that results at every timestep influences the local tissue perfusion and creates a moving boundary condition which changes the local heat sink properties. Ignoring the intermediate timesteps causes tissue perfusion to remain constant throughout the entire ablation, which results in an underestimation of the true lesion size. The use of temperature-dependent electrical conductivity greatly affects modeling results, as the electrical conductivity has been shown to increase dramatically over the course of tissue heating [57]. When constant electrical conductivity is used, the SAR is grossly underestimate, which also results in an underestimation in lesion size. An important outcome of this study is the demonstration that, temperature isotherms and tissue damage patterns are not synonymous. Traditional use of temperature isotherms that are used to define lesion size rely on coagulation temperature for protein (42–47°C) and grossly overestimate lesion dimensions. Our studies show that temperature decrease is gradual, while tissue damage decreases rapidly as a function of distance. It is this sharp decrease in tissue damage that causes lesion boundaries to appear fuzzy, as predicted by our model. The results also demonstrate that ablation lesions continue to grow after the applied power is terminated. Lesions continue to grow while temperature envelopes collapse after ablation since sufficiently high temperature are present to accrue tissue damage. In nearly all cases, lesions continued to grow several minutes following the ablation. A comparison of the resulting lesion dimensions between fully perfused and non-perfused tissues show that the lesion width decreases 38–46% and the lesion depth decreases 18–20% when tissue perfusion is accounted for in the model. Previous studies have shown that tissue perfusion can account for as much as 50% change in the size of the lesions generated during ablation [59]. An important observation in this study is the resemblance of the 60°C isocontour to lesion size. While the 42°C and the 47°C isotemperature contours are poor indicators of lesion size, 60°C is highly correlated with the lesion volumes. Seemingly, this would suggest that time-intensive tissue damage calculations need not be made since a critical temperature of 60°C can be used to identify lesion size. However, this is only true if the calculated temperature is a function of both transient changes in tissue perfusion and the electrical conductivity. In the absence of either of these phenomena, lesion sizes calculated at 60°C would underestimate lesion size. The validation data demonstrate that the model accurately accounts for the behavior of lesion growth in tissue. There are, however, a few limitations to this model. First, it is well established that temperature elevation of tissues results in the denaturing of proteins, which may drastically change the electrical conductivity of tissue in a nonlinear fashion [51,57]. Preliminary data suggests that the electrical conductivity substantially increases, which would likely increase the rate of tissue damage. The results of this study show that as a first order approximation the conductivity of equivalent sodium chloride solutions produces results that are within 5% of the experimental measurements. Although the phenomena described in this reporting are applicable to different tissues, the resulting lesion dimensions and temperature profiles in this study apply only to liver tissue. Similar studies can be made in other tissues, but were not pursued in this study. A second limitation in our model is that it is only valid for temperatures below 100°C. At temperatures above 100°C, tissues begin to boil and generate gas. When this occurs, some of the energy that contributes to temperature increase is used to change the water content of tissues into gas. At substantially higher temperatures, the composition of gas may be highly complex as tissue begins to burn and break down. Although gas generation is commonly seen in clinical use of radiofrequency ablation, impedance rises due to tissue charring limit the progressive rise in temperature. The complexity of multi-phasic ablation was beyond the scope of this study. 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==== Front Cardiovasc UltrasoundCardiovascular Ultrasound1476-7120BioMed Central London 1476-7120-2-121530789010.1186/1476-7120-2-12HypothesisTissue Doppler echocardiography – A case of right tool, wrong use Thomas George [email protected] Department of Cardiology, Indira Gandhi Co-operative Hospital and the Co-operative Medical College, Kochi 682 020, India2004 12 8 2004 2 12 12 18 7 2004 12 8 2004 Copyright © 2004 Thomas; licensee BioMed Central Ltd.2004Thomas; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The developments in echocardiography or ultrasound cardiography (UCG) have improved our clinical capabilities. However, advanced hardware and software capabilities have resulted in UCG facilities of dubious clinical benefits. Is tissue Doppler echocardiography (TDE) is one such example? Presentation of the hypothesis TDE has been touted as advancement in the field of echocardiography. The striking play of colors, impressive waveforms and the seemingly accurate velocity values could be deceptive. TDE is a clear case of inappropriate use of technology. Testing the hypothesis To understand this, a comparison between flow Doppler and tissue Doppler is made. To make clinically meaningful velocity measurements with Doppler, we need prior knowledge of the line of motion. This is possible in blood flow but impossible in the complex myocardial motion. The qualitative comparison makes it evident that Doppler is best suited for flow studies. Implications of the hypothesis As of now TDE is going backwards using an indirect method when direct methods are better. The work on TDE at present is only debatable 'research and publication' material and do not translate into tangible clinical benefits. There are several advances like curved M-mode, strain rate imaging and tissue tracking in TDE. However these have been disappointing. This is due to the basic flaw in the application of the principles of Doppler. Doppler is best suited for flow studies and applying it to tissue motion is illogical. All data obtained by TDE is scientifically incorrect. This makes all the published papers on the subject flawed. Making diagnostic decisions based on this faulty application of technology would be unacceptable to the scientific cardiologist. EchocardiographyDoppler studiesTissue DopplerFlow Dopplerusesmisuse ==== Body Background Echocardiography or Ultrasound Cardiography (UCG) is a key investigation in cardiology. Its non-invasive nature makes it a widely acceptable and safe form of investigation. In many cases it has surpassed cardiac catheterisation in diagnostic yield and has become the investigation of choice. The rapid development of UCG has made available to the clinician newer insights into the anatomy and physiology of the heart. With the availability of digital technology it is possible to manipulate raw data in different ways. This has also spawned UCG facilities of dubious clinical benefits. Tissue doppler echocardiography (TDE) is one such example. When we consider UCG we are interested in 2 distinct aspects of the heart: 1. Structure 2. Blood flow. Both these ultimately indicate cardiac function. B-mode/M-mode imaging or simply Ultrasound Imaging (USI) studies structure. Blood flow is studied by Doppler studies (DS). In each stream every development should incrementally improve our understanding of the heart, its structure and function. Or the development should improve the ease of operation or permit better documentation. In USI we had the following developments and each development added to the diagnostic yield. M-mode – temporal resolution; 2D – spatial resolution; Harmonic imaging – improvement in image quality; B-color – enhanced tissue perception; Ultrasonic tissue characterization and acoustic quantification – quantitative analysis of tissue; Omni-plane M-mode – temporal resolution at different segments and 3D imaging – another dimension of imaging. In DS too we had many developments, each improving our perception. Continuous wave (CW) – detected and measured high velocity flows; Pulse wave (PW) – located the abnormal flow; Color flow mapping (CFM) – made it possible to 'image' blood flows and power amplitude Doppler – to study vascular flow. Contrast, trans-oesophageal and intravascular UCG are the clinical extensions and exploitation of these developments What we see in TDE is a retrograde development. In the context of UCG, DS is an indirect method to study blood flow since USI in its present state cannot image fast moving blood cells. Spontaneous echo contrasts could mark the beginning of USI of blood cells. By using TDE we are using an indirect method where direct USI methods are better. Besides, TDE is an inappropriate application of the Doppler methodology. Presentation of the hypothesis TDE is a by-product of color flow mapping (CFM) technology. In CFM, tissue signals are suppressed and flow signals are analyzed. The reverse is true in TDE. Doppler velocities associated with tissue motion are slower than blood flow. Flow signals are eliminated on the basis of signal amplitude. The amplitude of tissue motion is about 40 db greater than the corresponding flow amplitude. Blood flow imaging applies a high pass filter to exclude the strong but low frequency tissue signals and other 'noise' before the signal is input into an autocorrelator that estimates velocity.[1] Erroneous filter settings could cause the autocorrelator to include components of tissue signals so that tissue velocities become encoded. This principle has been legitimized to color code tissue motion and we get an 'image' which is entirely Doppler information. In this discussion TDE includes both pulse tissue Doppler and color tissue Doppler (also known as Doppler tissue imaging or tissue velocity imaging). The first report on the use of TDE appeared in 1989.[2] However the real development and the widespread use of this technique started after the publication of the validation work of TDE setting the 'scientific' basis for the quantitative analysis of myocardial velocities in real time.[3] There are several papers on the use of TDE.[4,5] Various values and indices are already in place.[6,7] Over the past few years technological advances in TDE like curved M-mode, strain rate imaging and tissue tracking have been developed. These are in addition to modalities like the phase imaging, amplitude imaging and acceleration imaging.[8] Text books on TDE has also been added to the number of books already available on UCG.[9] Even now there are a number of papers on tissue Doppler echocardiography (TDE) appearing in leading journals.[10] Is TDE methodology correct? Is it in agreement with the Doppler principles? Has TDE made any tangible improvement in the already available UCG techniques? Has TDE improved our diagnostic yield? This article presents the hypothesis that TDE is a flawed application of Doppler and hence data collection with TDE is erroneous. Testing the hypothesis The article explains the flaws in tissue Doppler echocardiography. As the concept itself is flawed, all data using this modality is flawed. You cannot even think of designing a study to prove the point because the mensuration technique itself is wrong. So we have to use the scientific methods of comparison and reductio ad absurdum to verify it. Any new modality of diagnosis or treatment should be compared with the existing systems. In clinical trials we usually employ quantitative comparison with statistical methods. In this case, since the data acquisition is flawed we have to use a qualitative comparison. This is accepted practice in clinical medicine – It is like comparing a weak limb with the strong one in neurology. One also needs to understand the Doppler methodology to test the hypothesis. The Doppler methodology To estimate clinically significant peak Doppler velocity the following steps are required: 1. You must have prior knowledge of the line of motion of the object. Only if you know this line of motion, you can apply the interrogating signal along the path of motion. This is because of the directional bias of Doppler. 2. Next you will know the direction of motion – whether the object is moving towards the interrogating signal or away. 3. Only after the first two steps you can measure the clinically significant peak velocity. Based on the above discussion it is clear that the following information can be derived from Doppler: 1. Is there motion? Does the object move? This is a random application of Doppler. This is one aspect of tissue Doppler. But this information is redundant as we can already see the movement better by ultrasound imaging. 2. What is the direction of motion? Is it moving towards or away from the interrogating signal? This is also redundant information in tissue Doppler for the reason given above. 3. To measure the peak significant velocity we must know the clear-cut line of motion. Application of Doppler along this line enables us to measure the meaningful peak velocity. Thus the primary step is to know the line of motion. This is possible in flow Doppler but impossible in tissue Doppler. For example we know for sure that the blood moves from left atrium to left ventricle through the mitral valve. At the mitral valve this is a unique free linear motion towards the apex. This produces a clinically significant velocity. That is why we place the sample volume at the mitral valve from apical views so as to align the beam in the line of anticipated flow. That is also why we do not measure the mitral flow velocity from the parasternal views. Next we come to learn whether the blood is flowing towards or away from the transducer. After all these steps it is possible to measure velocity. In tissue Doppler we can measure several velocities in several directions. But we can never know which is the clinically significant peak velocity. Flaws in TDE TDE is a clear case of misuse of technology. To understand this, a comparison between flow Doppler and tissue Doppler is in order. CFM allows us to 'see' what we cannot see with ultrasonic 'eyes' hence its value is great. In color TDE we see more or less what we already see by USI hence its value is marginal. In CFM the anatomic landmarks are intact as the color is superimposed on the B-mode image. In color TDE the B-mode is eliminated and the entire 'picture' is Doppler information. It is difficult to determine the different anatomic regions on TDE. For example it would be very difficult to delineate the blood-endocardial boundary. In fact, the word 'tissue Doppler' is a misnomer and this is one of the reasons for the prevailing confusion. It gives the impression that only myocardial tissues are studied. The appropriate term would have been low velocity Doppler. Any movement in the low velocity range will be detected by 'tissue Doppler'. Myocardial tissue movement is just one among them. The discriminatory power of this modality is unsatisfactory. Myocardial motion is very complex and not amenable to Doppler studies. In the cardiac motion there are translational, rotational and deformational movements. Besides many tissues near the heart move – due to transmitted cardiac motion, vessel pulsation, respiratory motion and involuntary muscle movements and these interact with cardiac motion further and cause false Doppler shifts.[1] Velocity is a vector quantity and so Doppler interrogation at one point will determine the velocity of the resultant of all these movements projected in the line of the Doppler beam with angle correction. Similarly at a particular point there are movements in several axes and we can never predict the sum resultant vector. Even if known, the resultant is accurately recorded only if it is in the line of the Doppler beam. This is due to the inherent problem of directional bias. It is like measuring the length of a twisted rod directly with a straight rigid ruler. In the case of strain rate imaging, the protagonists have substituted a vector quantity (velocity) for a scalar quantity (length) in the original formula of strain calculation.[11] This would be mathematically unacceptable. Cardiac motion becomes more complicated in the presence of wall motion abnormalities. Blood flow is simple and suitable for Doppler study. In flow Doppler at a particular point the linear projectile motion of free moving blood cells are studied. At the current interrogation points there is a unique unidirectional flow in one part of the cardiac cycle. For example in mitral valve Doppler interrogation, the unique directional signal is obtained only in diastole. If there is a signal in the in systole, it becomes abnormal and this information has great value. In TDE the to- and- fro motion (systole and diastole) of a tethered interconnected syncytium of myocytes is imaged. Such information is useless. This can be even otherwise seen and analyzed by USI. In flow Doppler there are definite 'points of interrogation', which are the normal and abnormal orifices. In TDE there are no such definite points. While in flow Doppler higher velocities are studied, TDE studies lower velocities i.e. the study of hypo functioning myocardial segments. Lower velocities are difficult to appreciate. The higher velocities are easier to appreciate due to aliasing and variance. There are no such indicators for low velocity. Thus hypo function is difficult to analyze. The derivations from flow Doppler are based on accepted hydrodynamic formulae (like Bernoulli's equation) and allows us to get orifice size, amount of flow and pressure gradients, which are clinically of great importance. The derivations from TDE are useless. Thus Doppler in its present form, is best suited to study flow. Implications of the hypothesis TDE has shown some promise in the Wolff-Parkinson-White syndrome.[12] But even here by having a high frame rate or by using the omni plane M-mode, we could be able to locate the pre-excited tissue by USI. Diastolic dysfunction in pseudo-normalized mitral Doppler spectrum and in atrial fibrillation are other areas where TDE is found useful.[13,14] Here the benefits are marginal as there are other parameters to study diastolic dysfunction.[15] Besides, the determination of diastolic dysfunction by Doppler in clinical practice may not be sacrosanct.[16] It has been used as a method to differentiate between constrictive pericarditis and restrictive cardiomyopathy.[17] Here its role is supplementary and does not provide critical distinction. The main role of TDE as a method of detecting regional wall motion abnormalities has been stressed.[18] Here again it is complementary to routine USI. TDE is being used in the study of cardiac transplant rejection.[19] In this situation the results are not clear and there are other better UCG methods to study rejection.[20,21] TDE has been advocated in planning and follow-up of cardiac re-synchronization therapy.[22,23] Here again the conventional methods are adequate[24,25] and the false-positive data (see below) may be confounding. Besides several issues regarding the therapy need to be settled.[26] Mitral annular tissue velocities have been used in the determination of left ventricular filling pressures.[27] Here again an 'hydrodynamic' information (fluid pressure) is derived from non-hydrodynamic (tissue velocity) measurement. Mitral annulus is a circular area. As mentioned earlier here also we cannot define the precise point of interrogation. An infinite number of values can be obtained all around the mitral annulus. It is important to note that in all the above papers the data acquired is flawed due to the basic problem in technology application. Hence the correlations obtained in these papers are spurious or non-sense correlations. One paper presents a misuse of statistics when tissue Doppler velocities have been correlated with myocardial interstitial fibrosis and myocyte interstitial beta-adrenergic receptor density.[28] By analogy, using a little statistical jugglery, we could find correlation between blood cholesterol levels and flow Doppler indices. All these papers are mentioned just for completeness. Accepting the data in these papers would be tantamount to rejecting the Doppler principle and methodology. As of now TDE is going backwards using an indirect method where direct methods are available. The work on TDE at present is only debatable 'research and publication' material and does not translate into tangible clinical benefits. The prime use of TDE is to study regional wall motion abnormalities. It is claimed that it is possible to quantify wall motion at rest and during stress. However with the above-mentioned limitations it is unlikely to prove more effective than USI. The measurements are ultra sensitive and represent gross movements rather than myocyte contractions. With the advent of omni-plane M-mode it is possible to get temporal resolution in inaccessible planes and study wall motion abnormalities in a better way. The Doppler methods use mathematical formulae to indirectly study motion that cannot be directly measured. Examples are movements of stellar bodies and in the present context movements of blood cells. We use the indirect method based on Doppler principle because we cannot 'image' blood cells by USI. When the imaging of blood cells become possible, Doppler studies would probably be relegated to the background. Color TDE would have been valuable if direct USI was not available. Tissue Doppler has been a disappointing modality in clinical ultrasound cardiology. This is due to the basic flaw in the application of the principles of Doppler. Doppler is best suited for flow studies and applying it to tissue motion is not acceptable. In flow Doppler we suppress the tissue 'noise' and display flow.[1] In TDE it is the other way round. Besides TDE is ultra sensitive and so the information gathered is almost useless (too many false positive information). In fact excellent cardiac waveforms can be obtained by placing the sample volume just outside the cardiac region! This is also the reason why it is useless for even studying the temporal aspects of the cardiac cycle and waveform analysis. Once the foundation of a modality is wrong, all derivations tend to be wrong. However the author is of the view that TDE could probably have some role in diagnostic UCG. What is required is that Doppler should be used for the purpose it was intended i.e. to study blood flow. Thus we will have 2 types of Doppler studies: High velocity flow Doppler (HVFD) studies and Low velocity flow Doppler (LVFD) studies. Conventional Doppler would be HVFD. TDE would be LVFD and we would be imaging and analyzing very low velocity flows. The possible uses of color LVFD studies would be the study of low flow thrombogenic states, flow in the atria, the blood-endocardial/endothelial interface, low velocity turbulences and blood flow in organs. Pulse wave spectral LVFD could be useful for the detection of low velocity turbulences as a cause for murmurs (not detectable with HVFD). These are potential areas for investigation and research. It is time to look at TDE on more realistic terms. As a new modality of imaging it appears exciting. But its real clinical utility is doubtful. TDE does not give any additional information over the conventional modalities. In fact due to the above-mentioned deficiencies it could give misleading information. Making diagnostic and therapeutic decisions based on this faulty application of technology would be unacceptable to the scientific cardiologist. List of abbreviations used CFM Color Flow Mapping CW Continuous Wave DS Doppler Studies HVFD High Velocity Flow Doppler LVFD Low Velocity Flow Doppler PW Pulse Wave TDE Tissue Doppler Echocardiography UCG Ultrasound Cardiography USI Ultrasound Imaging Competing interests None declared. ==== Refs Maslak SH Freund JG Lanzer P Color Doppler Instrumentation. In Vascular Imaging by Color Doppler and Magnetic Resonance 1991 Springer-Verlag Berlin Heidelberg 87 123 Isaaz K Thompson A Ethevenot G Cloez JL Brembilla B Pernot C Doppler echocardiographic measurement of low velocity motion of left ventricular posterior wall. Am J Cardiol 1989 64 66 75 2741815 10.1016/0002-9149(89)90655-3 McDiken WN Sutherland GR Moran CM Gorden LN Color Doppler velocity imaging of the myocardium. Ultrasound Med Biol 1992 18 651 654 1413277 10.1016/0301-5629(92)90080-T Sutherland GR Stewart MJ Groundstroem KW Moran CM Fleming A Guell-Peris FJ Color Doppler myocardial imaging: a new technique for the assessment of myocardial function. J Am Soc Echocardiogr 1994 7 441 458 7986541 Rambaldi R Poldermans D Vletter WB ten Cate FJ Roelandt JR Fioretti PM Doppler tissue imaging in the new era of digital echocardiography. G Ital Cardiol 1997 27 827 839 9282289 Palka P Lange A Fleming AD Sutherland GR Fenn LN McDicken WN Doppler tissue imaging: Myocardial wall motion velocities in normal subjects. J Am Soc Echocardiogr 1995 8 659 668 9417209 Pai RG Gill KS Amplitudes, durations and timings of apically directed left ventricular velocities: Their normal pattern and coupling to ventricular filling and ejection. J Am Soc Echocardiogr 1998 11 105 111 9517548 Lars-Åke Brodin van der Linden Jan Björn Olstad Echocardiographic functional images based on tissue velocity information. Herz 1998 23 491 498 10023583 Garcia-Fernandez MA Zamorano J Azvedo J Eds Doppler Tissue Imaging Echocardiography 1998 McGraw-Hill/Interamericana de Espana, Madrid Sengupta PP Mohan JC Mehta V Kaul UA Trehan VK Effects of percutaneous mitral commissurotomy on longitudinal left ventricular dynamics in mitral stenosis: Quantitative assessment by tissue velocity imaging. J Am Soc Echocardiogr 2004 17 824 829 15282484 Pislaru C Abraham TP Belohlavek M Strain and strain rate echocardiography. Current Opinion in Cardiology 2002 17 443 454 12357119 10.1097/00001573-200209000-00002 Nakayama K Miyatake K Uematsu M Tanaka N Kamakura S Nakatani S Application of tissue Doppler imaging technique in evaluation early ventricular contraction associated with accessory atrioventricular pathways in Wolff-Parkinson-White syndrome. Am Heart J 1998 135 99 106 9453528 Barros MV da Costa Rocha MO Ribeiro AL Machado FS Tissue Doppler imaging enables the identification of diastolic dysfunction of pseudonormal pattern in Chagas' disease. J Am Soc Echocardiogr 2001 14 353 359 11337680 10.1067/mje.2001.111155 Thamilarasan M Grimm RA Rodriguez LL Sun JP Odabashian JA Agler DA Left ventricular diastolic dysfunction in lone atrial fibrillation determined by Doppler tissue imaging of mitral annular motion. Am J Cardiol 2000 86 1026 1029 11053722 10.1016/S0002-9149(00)01144-9 Nishimura RA Tajik AJ Evaluation of diastolic filling of left ventricle in health and disease: Doppler echocardiography is the clinician's Rosetta Stone. J Am Coll Cardiol 1997 30 8 18 9207615 10.1016/S0735-1097(97)00144-7 Fyrenius A Wigstrom L Bolger AF Ebbers T Ohman KP Karlsson M Pitfalls in Doppler evaluation of diastolic function: insights from 3-dimensional magnetic resonance imaging. J Am Soc Echocardiogr 1999 12 817 826 10511650 Rajagopalan N Garcia MJ Rodriguez L Murray RD Apperson-Hansen C Stugaard M Comparison of new Doppler echocardiographic methods to differentiate constrictive pericardial heart disease and restrictive cardiomyopathy. Am J Cardiol 2001 87 86 94 11137840 10.1016/S0002-9149(00)01278-9 Derumeaux G Loufoua J Pontier G Cribier A Ovize M Tissue Doppler imaging differentiates transmural from nontransmural acute myocardial infarction after reperfusion therapy. Circulation 2001 103 589 596 11157727 Mankad S Murali S Kormos RL Mandarino WA Gorcsan J 3rd Evaluation of the potential role of color-coded tissue Doppler echocardiography in the detection of allograft rejection in heart transplant recipients. Am Heart J 1999 138 721 730 10502219 Mooradian SJ Goldberg CS Crowley DC Ludomirsky A Evaluation of a non-invasive index of global ventricular function to predict rejection after paediatric cardiac transplantation. Am J Cardiol 2000 86 358 360 10922454 10.1016/S0002-9149(00)00935-8 Segovia E Perez JE Characterization of myocardial tissue by ultrasound backscatter. Rev Esp Cardiol 1999 52 708 715 10523882 Santos JF Caetano F Parreira L Madeira J Cardoso P Fonseca N Tissue Doppler echocardiography for evaluation of patients with ventricular resynchronization therapy. Rev Port Cardiol 2003 22 1363 1371 14768491 Penicka M Bartunek J De Bruyne B Vanderheyden M Goethals M De Zutter M Improvement of left ventricular function after cardiac resynchronization therapy is predicted by tissue Doppler imaging echocardiography. Circulation 2004 109 978 983 14769701 10.1161/01.CIR.0000116765.43251.D7 Molhoek SG Van Erven L Bootsma M Steendijk P Van Der Wall EE Schalij MJ QRS duration and shortening to predict clinical response to cardiac resynchronization therapy in patients with end-stage heart failure. Pacing Clin Electrophysiol 2004 27 308 313 15009855 10.1111/j.1540-8159.2004.00433.x Braunschweig F Kjellstrom B Gadler F Linde C Optimization of cardiac resynchronization therapy by continuous hemodynamic monitoring. J Cardiovasc Electrophysiol 2004 15 94 6 15028081 Mehra MR Greenberg BH Cardiac resynchronization therapy: caveat medicus! J Am Coll Cardiol 2004 43 1145 1148 15063420 10.1016/j.jacc.2003.10.050 Ommen SR Nishimura RA Appleton CP Miller FA Oh JK Redfield MM Clinical utility of Doppler echocardiography and tissue Doppler imaging in the estimation of left ventricular filling pressures: A comparative simultaneous Doppler-catheterization study. Circulation 2000 102 1788 1794 11023933 Shan K Bick RJ Poindexter BJ Shimoni S Letsou GV Reardon MJ Relation of tissue Doppler derived myocardial velocities to myocardial structure and beta-adrenergic receptor density in humans. J Am Coll Cardiol 2000 36 891 896 10987616 10.1016/S0735-1097(00)00786-5

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==== Front Health Qual Life OutcomesHealth and Quality of Life Outcomes1477-7525BioMed Central London 1477-7525-2-411529195910.1186/1477-7525-2-41ResearchThe ECOS-16 questionnaire for the evaluation of health related quality of life in post-menopausal women with osteoporosis Badia Xavier [email protected]íez-Pérez Adolfo [email protected] Raquel [email protected]án Luis [email protected]és Xavier [email protected] Jordi [email protected] Health Outcomes Research (HOR) Europe, Plató 6, 1° 5a, 08021 Barcelona, Spain2 Departament d'Epidemiologia I Salut Pública de l'Hospital de la Santa Creu i Sant Pau, Barcelona, Spain3 Servei de Medicina Interna, Hospital Ntra. Sra. del Mar, Universitat Autònoma de Barcelona, Barcelona, Spain4 Unidad Docente de Medicina de Familia, Castellón, Spain5 Novartis Farmacéutica, Barcelona, Spain2004 3 8 2004 2 41 41 9 12 2003 3 8 2004 Copyright © 2004 Badia et al; licensee BioMed Central Ltd.2004Badia et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The aim of this study is to validate the questionnaire ECOS-16 (Assessment of health related quality of life in osteoporosis) for the evaluation of health related quality of life (HRQoL) in post-menopausal women with osteoporosis. Methods An observational, prospective and multi-centre study was carried out among post-menopausal women with osteoporosis in primary care centres and hospital outpatient clinics. All patients attended 2 visits: at baseline and at 6 months. In addition, the subgroup of outpatients attended another visit a month after the baseline to assess the test-retest reliability. The psychometric properties of the questionnaire were evaluated in terms of feasibility, validity (content validity and construct validity) and internal consistency in baseline, and in terms of test-retest reliability and responsiveness to change in visit at month and visit at 6 months, respectively. In all visits, ECOS-16, EUROQoL-5D (EQ-5D) and four 7-point items about health status (general health status, back pain, limitation in daily activities and emotional status) were administered, whereas only outpatients were given MINI-OQLQ (Mini Osteoporosis Quality of Life Questionnaire), besides all clinical variables; and sociodemographic variables at baseline. Results 316 women were consecutively included, 212 from primary care centres and 104 from hospital outpatient clinics. Feasibility: 94.3% of patients answered all items of the questionnaire. The mean administration time was 12.3 minutes. Validity: factor analysis suggested that the questionnaire was unidimensional. In the multivariate analysis, patients with vertebral fractures, co-morbidity and a lower education level showed to have worse HRQoL. Moderate to high correlations were found between the ECOS-16 score and the other health status questionnaires (0.47–0.82). Reliability: internal consistency (Cronbach's α) was 0.92 and test-retest reliability (ICC) was 0.80. Responsiveness to change: ECOS-16 scores increased according to change perceived by the patient, as well as the effect size (ranges between 1.35 to 0.43), the greater the perception of change in patients' general health status, the greater the changes in patients' scores. The Minimal Clinically Important Difference (MCID) suggested a change of 0.5 points in the ECOS-16 score, representing the least improvement in general health status due to their osteoporosis: "slightly better". Conclusion ECOS-16 has been proven preliminarily to have good psychometric properties, so that it can be potentially a useful tool to evaluate HRQoL of post-menopausal women with osteoporosis in research and routine clinical practice. ==== Body Background Osteoporosis is characterised by low bone mass and a deterioration in bone tissue micro-architecture, leading to increasing bone weakness and consequently risk of fracture. The most common clinical complications of osteoporosis are hip fracture, vertebral deformity and wrist fracture. According to bone densitometry values, osteoporosis affects approximately 2 million women in Spain [1,2]. The most frequent symptom of osteoporosis is low back pain resulting from vertebral fractures. This pain can have a considerable impact on the ability to carry out usual activities of daily living. Patients are unable to work normally, are limited in their social and leisure activities, and may be severely affected emotionally [3]. To date, clinical trials on osteoporosis have been based on outcomes measured by imaging tests. But these measurements do not adequately reflect the extent to which the patient is affected in their usual daily activities, and are not appropriate to assess patients' disability and symptoms [4]. Nevertheless, recently some specific Health Related Quality of Life (HRQoL) questionnaires such as OPAQ (Osteoporosis Assessment Questionnaire) have been used as the main outcome in clinical studies on osteoporosis [5]. Several generic HRQoL questionnaires, such as the Sickness Impact Profile (SIP), SF-36 or the Nottingham Health Profile (NHP), have been used more frequently to assess the impact of osteoporosis on HRQoL [6]. These questionnaires are applicable to any population and disease, thereby enabling comparison between subjects suffering from different diseases. However, they have serious limitations given the fact that they fail to explore in detail the specific aspects of osteoporosis. For instance, some studies have shown that certain aspects, such as the fear of falling and suffering a bone fracture, the inability to adequately carry out domestic tasks, the ability to dress oneself adequately without help and despair about an uncertain future are all stressful for these patients [7]. These items are not included in generic questionnaires and their omission could lead to an incomplete or biased evaluation of HRQoL of patients with osteoporosis. Disease specific questionnaires for osteoporosis are available, such as the Osteoporosis Quality of Life Questionnaire (OQLQ) [8] or the Quality of Life Questionnaire of the European Foundation for Osteoporosis (QUALEFFO) [9]. However, their limited applicability due to their length and time for administration have restricted their use to clinical trials and highlighted the need for the development of questionnaires which are easier to administer in routine clinical practice. In order to expand their use in clinical practice, it is necessary to develop valid, short, easy to administer and comprehensible questionnaires. For this reason, a specific short form HRQoL questionnaire for women with osteoporosis was developed [10]. Its items were obtained from the Spanish versions of the OQLQ and QUALEFFO questionnaires, and were then reduced by using the Rasch analysis to obtain a total of 16 items; 12 from the QUALEFFO and 4 from the OQLQ. These questionnaires were selected because they were the only questionnaires already validated in Spain. More information regarding the development process of the ECOS-16 has been published elsewhere [11,12]. The aim of the present study is to evaluate the psychometric properties of the ECOS-16 in post-menopausal women with osteoporosis. Methods 316 post-menopausal women with primary osteoporosis attended in Primary Care Centres or in outpatient clinics were included in the study. Diagnosis was confirmed by a Bone Mineral Density (BMD) using Dual Energy X-Ray Absorptiometry (DEXA). Furthermore, outpatients should have at least one prevalent vertebral fracture confirmed by Genant's radiological criteria due to osteoporosis, a requirement which was not essential in patients from Primary Care Centres. 212 patients from 49 Primary Care Centres and 104 patients attended in outpatient clinics from 14 hospitals were consecutively selected and evaluated from March 2000 to August 2001. All patients attended two visits, a baseline and a follow-up visit 6 months after the inclusion. In order to evaluate test-retest reliability, outpatients were also attended in another follow-up visit one month after the baseline. All patients received the study information and gave their informed consent. Study design An observational, prospective and multi-centre study was carried out for the validation of the ECOS-16 in post-menopausal women with vertebral fractures due to osteoporosis in conditions of clinical practice. At the baseline, feasibility together with the content and construct validity of the ECOS-16 were evaluated. At the visit after 6 months, responsiveness to change with regard to ECOS-16 was evaluated. Outpatients also attended another visit a month after the inclusion so that the test-retest reliability of the ECOS-16 could be evaluated. In the baseline, data on the patients' sociodemographic characteristics (age, education level) and clinical variables (weight, height, body mass index, age at onset of menopause, BMD, presence and site of vertebral and non-vertebral fractures, concomitant chronic diseases and received treatment) were collected as well as the ECOS-16, the EUROQoL-5D and four 7-point items which refer to general health status, back pain, limitation in daily activities and emotional status. These items were used in a previous study and showed its validity [10]. Outpatients were also administered the Spanish version of the MINI-OQLQ questionnaire [3]. In the two follow-up visits, any modifications to the specific treatment for osteoporosis prescribed in the baseline, the number of concomitant treatments, and patients' withdrawals causes were registered. Moreover, the ECOS-16, EUROQoL-5D and the four 7-point change items were again administered. Outpatients were again administered the MINI-OQLQ questionnaire. In the present study, the EQ-5D and MINI-OQLQ questionnaires were used in order to assess the validity of the ECOS-16. Health related quality of life questionnaires ECOS-16 The ECOS-16 (Please see additional file 1 ([appendix]) was developed with the aim of measuring HRQoL in postmenopausal women with osteoporosis. It is based on the combination of two disease-specific HRQoL questionnaires for women with osteoporosis: the Osteoporosis Quality of Life Questionnaire (OQLQ) [8] and the Quality of Life Questionnaire of the European Foundation for Osteoporosis (QUALEFFO) [9]. The development process consisted in five phases: Phase I-Search for common structures; Phase II-Independent OQLQ and QUALEFFO item reduction using Rasch analysis; Phase III-OQLQ and QUALEFFO item equating; Phase IV-Quantitative reduction of equated items; Phase V-Qualitative reduction [11]. This newly questionnaire consists of 12 items from the QUALEFFO and 4 from the OQLQ (see Annex 1). All items have five possible response options, although the response options differ from one item to another. The 16 items in the new questionnaire are divided qualitatively into four dimensions. The nature of the four dimensions also suggests that they can be further combined to produce two summary scores that would include Physical Function and Pain in one Physical score, and another one that would include Fear of Illness and Psychosocial Function in a Mental score. These two summary scores could, in turn, be combined to provide an overall score for the questionnaire. However, although the 16 items can be classified qualitatively into four dimensions, this is an unidimensional questionnaire, according to the quantitative analysis [11]. The score of each item ranges from 1 to 5. ECOS-16 generates a single summary score obtained from the arithmetic mean of the answered items, so the total score ranges from 1 (best HRQoL) to 5 (worst HRQoL). The time frame for the questionnaire was one week. All items have the same weight on the overall questionnaire score and the overall score is calculated as the mean score of all the response items. It is a self-administered questionnaire, apart from some special cases (eyesight difficulties or illiteracy) where it was acceptable for the questionnaire to be administered by health care personnel or experienced interviewers. EUROQoL-5D The EUROQoL-5D (EQ-5D) is a generic HRQoL questionnaire. The EQ-5D consists of two parts: a descriptive system and a Visual Analogue Scale (VAS) [13]. The descriptive system contains 5 health status dimensions: Mobility, Self-Care, Usual activities, Pain/Discomfort and Anxiety/Depression. These dimensions are always presented in the same order, each one with 3 degrees of severity: no problems, some or moderate problems, and extreme problems, given a value of 1, 2 and 3, respectively. For each dimension, the respondent should mark the degree of severity which best describes their actual health status. The VAS of the EQ-5D is a vertical scale divided into millimetres along a 20 centimetres long thermometer where the two ends are labelled "worst imaginable health state" and "best imaginable health state" with a score of 0 and 100, respectively. The respondent should mark the point on the thermometer which, in their opinion, best describes their actual overall health status. MINI-OQLQ The MINI-OQLQ is a specific HRQoL questionnaire for women with vertebral fractures due to osteoporosis. The MINI-OQLQ is based on a selection of the two highest impact items from each of the five domains of the Osteoporosis Quality of Life Questionnaire (OQLQ). The MINI-OQLQ is, therefore, composed of 10 items grouped into the same five HRQoL dimensions (symptoms, physical function, activities of daily living, emotional function and leisure) [3]. Each item has seven response options ranging from 1 (worse HRQoL) to 7 (better HRQoL). The scoring is obtained per dimension, by calculating the mean score of the response items for each dimension, so that the higher the score in each dimension, the better the resulting HRQoL. 7-point change items (general health status, back pain, limitation in daily activities and emotional status) due to osteoporosis Changes in four health status items were assessed through four different items regarding the change in the patient's overall health status: the change in the patient's general health status due to osteoporosis, the change in the patient's back pain, the change in the patient's limitation in daily activities and the change in the patient's emotional status, all of them due to osteoporosis, with reference to the baseline. The items have seven possible response options, ranging from "Much better" to "Much worse" and including the category "More or less the same". These items were designed to be self-administered and validated in previous studies [10,14]. Statistical analysis Double data entry was carried out with a subsequent validation to guarantee the quality and consistency of the data. A statistical significance level of p < 0.05 was used in all statistical tests performed. The statistical program SPSS® for Windows version 10.0 (SPSS, Inc., Chicago, Illinois) was used to carry out the entire data analysis. Previously the statistical analysis, a Kolmogorov-Smirnov test was conducted to assess the distribution of the variables in order to use a parametric or non-parametric tests. With the aim to describe the study sample characteristics and to evaluate differences among patients with and without vertebral fractures a descriptive and comparative analysis was done on patients' sociodemographic characteristics (age, education level) and clinical variables (Body Mass Index (BMI), number of years with menopause, presence or absence of non-vertebral fractures, concomitant diseases and received treatment during the previous year) according to vertebral fracture presence. In order to compare the two groups, the chi-squared test was used, given that the variables were categorical, and the Bonferroni correction for multiple tests. The feasibility of the ECOS-16 was analysed on the basis of missing data, time and method of administration. In order to evaluate the missing data, the number and percentage of missing response items in the whole questionnaire as well as the number and percentage of patients who failed to respond to any of the questionnaire's items were both calculated. The time spent administering the questionnaire was evaluated according to the method of administration (self-administered or administered by an interviewer). The floor (percentage of patients with the lowest score) and ceiling (percentage of patients with the highest score) effect were calculated for each one of the ECOS-16 items and for the overall score. An exploratory factor analysis was used to assess the content validity of the ECOS-16, using the scores obtained during the baseline by all patients for all 16 questionnaire items. Factors were extracted using principal-axis factoring method and varimax rotations. The adequacy of the factor analysis was assessed with the Kaiser-Meyer-Olkin measure and the Bartlett's test of sphericity [15]. To evaluate the construct validity, the correlations between the ECOS-16 scores and the patients' sociodemographic and clinical characteristics (bivariate analysis) were analysed. Pearson's correlation coefficient was used for continuous variables and the analysis of variance (ANOVA) for categorical variables. A multivariate analysis was also carried out taking the ECOS-16 score as a dependent variable and the sociodemographic and clinical variables that were significant in the bivariant analysis as independent variables, so that any possible confounding factors could be controlled. Secondly, the relationship between the ECOS-16 and the EQ-5D scores, the four 7-point items (general health status, back pain, limitation in daily activities and emotional status) and the MINI-OQLQ (only outpatients) were all analysed using Spearman's correlation coefficient, apart from the VAS which was analysed using Pearson's correlation coefficient. Higher correlations were expected between dimensions that measure the same HRQoL aspects. Because the lowest score in ECOS-16, in EQ-5D and in 7-point general health status item represent the best HRQoL, the expected correlations among their dimensions would be positive. The opposite applies to the dimensions of MINI-OQLQ and the other three 7-point items (back pain, limitation in daily activities and emotional status), where the lowest score represent the worst HRQoL and, therefore, the expected correlations between ECOS-16 would be negative. The reliability of the ECOS-16 was evaluated in terms of internal consistency and test-retest reliability. Internal consistency was calculated by Cronbach's α coefficient using the baseline scores of all questionnaire items. Test-retest reliability was evaluated only for outpatients who did not perceive a change in their general health status due to osteoporosis after a month, as shown by the change in 7-point general health status item (response category: 'More or less the same'). The Intraclass Correlation Coefficient (ICC) between the scores for both visits was used for this analysis. The hypothesis that the standard psychometric recommendations for Cronbach's α and ICC were greater than or equal to 0,7 was taken as a starting point for both internal consistency and test-retest reliability [16]. Longitudinal validity of the ECOS-16 was evaluated by analysing the correlations among changes registered in the ECOS-16, and changes in the EQ-5D, changes in the MINI-OQLQ and changes in the four 7-point change items from baseline to visit at 6 months. For this purpose the Spearman's correlation coefficient was used. The expected correlations are the same as in the construct validity. To assess responsiveness to change, first of all, attention has been drawn to whether the questionnaire detects the changes which are perceived by patients between the baseline and the visit at 6 months. In order to do so, the Student's t-test for paired data was used. To assess the magnitude of changes, the effect size was calculated, thus it was establish that the changes in the patients' scores increased at the same time that the changes perceived by patients. In order to calculate the effect size, the change in 7-point general health status item is used [17]. The Minimal Clinically Important Difference (MCID) has been defined as the smallest difference between the scores in a questionnaire that the patient perceives to be beneficial [14]. The MCID was calculated for those patients who, at visit at 6 months, declared changes "slightly better" in the general health status item (difference between the scores from baseline and the visit at 6 months). Results Table 1 shows the patients' sociodemographic and clinical characteristics evaluated according to the presence of vertebral fractures. Table 1 Patients' sociodemographic and clinical characteristics according to the presence of vertebral fracture With vertebral fracture Without vertebral fracture Total Age‡  ≤ 65 years 42 (32.8%) 92 (51.4%) 134 (43.6%)  > 65 years 86 (67.2%) 87 (48.6%) 173 (56.4%)  Total 128 (100.0%) 179 (100.0%) 307 (100.0%) Education level  No formal education 35 (28.2%) 46 (25.7%) 81 (26.7%)  Primary school 78 (62.9%) 101 (56.4%) 179 (59.1%)  Secondary school 11 (8.9%) 27 (15.1%) 38 (12.5%)  University --- 5 (2.8%) 5 (1.7%)  Total 124 (100.0%) 179 (100.0%) 303 (100.0%) BMI†  ≤ 30 94 (73,4%) 153 (83,6%) 247 (79,4%)  > 30 34 (26,6%) 30 (16,4%) 64 (20,6%)  Total 128 (100,0%) 183 (100,0%) 311 (100,0%) Years with menopause‡  ≤ 20 years 59 (46.8%) 115 (65.3%) 174 (57.6%)  > 20 years 67 (53.2%) 61 (34.7%) 128 (42.4%)  Total 126 (100.0%) 176 (100.0%) 302 (100.0%) Non-vertebral fractures  Presence 24 (18.7%) 21 (11.4%) 45 (14.4%)  Absence 104 (81.3%) 163 (88.6%) 267 (85.6%)  Total 128 (100.0%) 184 (100.0%) 312 (100.0%) Concomitant diseases‡  Presence 81 (62.8%) 144 (79.1%) 225 (72.3%)  Absence 48 (37.2%) 38 (20.9%) 86 (27.7%)  Total 129 (100.0%) 182 (100.0%) 311 (100.0%) Received treatment In the previous year‡  Yes 92 (70.2%) 71 (38.4%) 163 (51.6%)  No 39 (29.8%) 114 (61.6%) 153 (48.4%)  Total 131 (100.0%) 185 (100.0%) 316 (100.0%) †p < 0.05 ‡ p < 0.007 Statistically significant differences (p < 0.01) were found for age, concomitant diseases (patient-reported), and length of time with menopause, it being the case that those patients with vertebral fractures were older and had had menopause for a longer time. In addition, those patients with vertebral fracture were those that had received more treatments during the previous year (p < 0.01) and also had a greater BMI (p < 0.05). However, those patients without a vertebral fracture presented concomitant diseases more frequently (p < 0.01). Only 14.4% of patients showed some type of non vertebral fracture and about 70% had completed at least primary education level. After the Bonferroni correction, the analysis that were still significant (p < 0,007) were age, years with menopause, concomitant disease and previous treatment. At baseline, all osteoporotic patients were prescribed or changed their treatment by the physicians, some having changed his previous treatment but others having received no treatment before. The mean (SD) administration time of the questionnaire was 12.3 (7.8) minutes for all patients, with a median of 10 minutes. In 55.1% of cases, the questionnaire was self-administered, the remainder being administered by health care personnel (due to eyesight difficulties or illiteracy). No statistically significant differences were observed in administration times according to the type of administration. The 99.7% of the patients answered all items of the questionnaire. Only one patient failed to respond to any item. A third of the items had a floor effect greater than or equal to 20%, 38.3% in item "Do you have problems with dressing?". The maximum ceiling effect was observed in 55.7% of the patients in item "How often have you had back pain in the last week?". In the remaining items, the ceiling effect was lower, ranging from 1.3% to 20.3%, the highest being in item "Are you afraid of getting a fracture?". Two patients (0.7%) recorded the highest scores for all the items (ceiling effect of the overall score). In the factor analysis, the Kaiser-Meyer-Olkin measure was 0.916 indicating a good sampling adequacy. The Bartlett's test of sphericity (p < 0.001) made it possible to accept the identity of the matrix correlations for the ECOS-16 items, thus indicating the suitability of the factor analysis. Table 2 shows the relationship between the patients' sociodemographic and clinical characteristics and the ECOS-16 score. In the bivariate analysis was observed a worse HRQoL in women with greater BMI (p < 0.05), a lower education level (p < 0.01) and concomitant chronic diseases (p < 0.05). Table 2 ECOS-16 scores according to patients' clinical and sociodemographic characteristics N ECOS-16 mean (SD) score Age  ≤ 65 years 134 2.79 (0.77)  > 65 years 173 2.92 (0.82)  Total 307 2.87 (0.80) Education level‡  No formal education 81 3.15 (0.73)  Primary school 179 2.84 (0.81)  Secondary school 38 2.46 (0.63)  University 5 1.94 (0.58)  Total 303 2.86 (0.80) BMI†  ≤ 30 247 2.81 (0.79)  > 30 64 3.06 (0.81)  Total 311 2.86 (0.80) Years with menopause  ≤ 20 years 174 2.80 (0.78)  > 20 years 128 2.95 (0.83)  Total 302 2.86 (0.81) Vertebral fractures  Presence 131 2.94 (0.83)  Absence 185 2.81 (0.78)  Total 316 2.86 (0.80) Non-vertebral fractures  Presence 45 2.88 (0.71)  Absence 267 2.87 (0.81)  Total 312 2.87 (0.80) Lumbar BMD* 298 0.024 Lumbar T-score * 308 -0.004 Neck BMD* 257 -0.042 Neck T-score* 264 0.090 Concomitant diseases†  Presence 225 2.91 (0.81)  Absence 86 2.71 (0.72)  Total 311 2.85 (0.79) Received treatment in the previous year  Yes 163 2.87 (0.77)  No 153 2.86 (0.83)  Total 316 2.86 (0.80) *Pearson's correlation coefficient † p < 0.05 ‡ p < 0.01 A multivariate analysis was carried out to identify patients' characteristics that were related to the ECOS-16 score (the variables entered into the multivariate analysis were age, education level, BMI, years with menopause, non-vertebral fractures, concomitant diseases and received treatment in previous years, all were codified as in table 1). The results showed that the variables of education level, number of concomitant diseases and the presence of vertebral fractures were related to the ECOS-16 score. Nevertheless, the percentage of variance explained by the variables included in the multivariate model was low, 11.1%. Table 3 shows the relationship between the ECOS-16 score and each one of the EQ-5D's dimensions, the four 7-point items and the MINI-OQLQ. All the EQ-5D's dimensions showed a statistically significant correlation with the ECOS-16 score, the dimensions with the greatest correlation being 'Mobility', 'Self-Care' and 'Pain/Discomfort'. The VAS was also statistically significant, with a Pearson's correlation coefficient of 0.61. The four 7-point items showed high correlations (greater than 0.7) with the ECOS-16 score, the item 'Limitation in daily activities' being the highest correlated item (0.82). The MINI-OQLQ dimensions showed moderate but statistically significant correlations (range: 0.47–0.73) with the ECOS-16, the 'Symptoms' and 'Leisure' dimensions having the highest correlations (0.71 and 0.74, respectively). Table 3 Correlations between the ECOS-16 scores and the EUROQoL-5D, the four 7-point items scores and the MINI-OQLQ N Spearman's correlation EUROQol-5D  Mobility 315 0.643  Self-care 315 0.623  Usual activities 315 0.594  Pain/Discomfort 314 0.608  Anxiety/Depression 315 0.555  VASa 312 -0.610 General health status 316 0.712 Back pain 313 -0.741 Limitation in daily activities 313 -0.822 Emotional status 313 -0.791 MINI-OQLQ b  Symptoms 104 -0.711  Physical function 104 -0.670  Activities of daily living 104 -0.455  Emotional function 104 -0.473  Leisure 104 -0.736 aPearson's Correlation Coefficient; bOutpatients only; All correlations are statistically significant at the 0.001 level The observed correlations between the changes among ECOS-16 questionnaire and the changes among the dimensions of the HRQoL questionnaires administered: EQ-5D, MINI-OQLQ and the four 7-point change items were similar to those of the construct validity (range: 0.33–0.76). All these correlations were also statistically significant. The internal consistency of the ECOS-16 was very high, with a Cronbach's α coefficient of 0.92. Test-retest reliability was analysed for 44 outpatients who declared that their general health status due to osteoporosis had not changed after a month, with an Intraclass Correlation Coefficient of 0.80 and a mean (SD) score change of 0.2 (0.5) points. Figure 1 shows the ECOS-16 patient scores for baseline and at visit at 6 months, as well as the effect size (ES) according to the changes in 7-point general health status item perceived by the patient after 6 months. The greater the perception of change in patients' general health status, the greater the changes in patients' scores. The same happened with the effect size as the patients who declared a 'much better' change in their general health status due to their osteoporosis at visit at 6 months had an effect size of 1.35, compared to those patients who declared a 'quite better' change having an ES of 1.23. The Minimal Clinically Important Difference (MCID) represented a mean change (SD) in the ECOS-16 score of 0.69 points, taking the category representing the least improvement in general health status due to their osteoporosis: 'slightly better'. Figure 1 ECOS-16 scores according to perceived changes in general health status Discussion The measurement of HRQoL has attracted increasing attention as a clinically relevant outcome of research and clinical practice. HRQoL questionnaires reflect the impact of health care interventions on health aspects such as physical, mental and social well-being. However, either in clinical research and in practice, a lengthy questionnaire is problematic for both the health care personnel and the patient. Shortish measure attempt to minimize time and effort as well as to increase patient interest [18]. Thus, shortish questionnaires need to be sufficiently psychometrically robust, proving that they are truly measuring what they set out to (validity), that they measure in a reliable way (reliability) and that they are capable of detecting real changes in perceived health status among patients with osteoporosis (responsiveness to change). The ECOS-16 originates from the reduction of two validated and widely used HRQoL questionnaires in osteoporosis patients with vertebral fracture [11]. However, in the present study, the ECOS-16 was administered to as many patients with fracture as those without, in order to establish its general applicability to patients with osteoporosis. Although the ECOS-16 requires a short time to be administered, self-administration was not possible in a high percentage of patients who needed help from health care personnel under conditions of usual clinical practice. In the future, it would be necessary to assess whether scores obtained through the questionnaire are maintained once the administration method changes, which would give more consistency to the questionnaire. Almost all the patients responded to all the questionnaire items. The presence of a floor effect for one third of the questionnaire items could lead to the conclusion that the questionnaire detects changes only when the severe disease status occurs. However, according to a previous qualitative division [12], the floor effect is concentrated both in the items belong to physical and psychosocial dimensions as for the mobility and self-care EQ-5D dimensions -the most conceptually equivalent- leading to think that the study sample has a certain clinical stability. As some previous studies, this study shows that the variables usually used to evaluate patients with osteoporosis, such as Bone Mineral Density (BMD) and the presence of vertebral fractures, have low or no correlation with HRQoL scores. This finding is not new [19,20] and suggests that HRQoL scores could be influenced by other factors such as personal, clinical and sociodemographic characteristics. In this study, the fact that patient's education level is the most significantly correlated variable draws particular attention. However, this observation has already been made in previous studies in patients with musculoskeletal problems [21,22]. The presence of concomitant diseases and/or a higher Body Mass Index (BMI) is scarcely correlated with HRQoL. This finding is interesting when taking into account studies evaluating HRQoL in patients with osteoporosis, whether they be descriptive or experimental in its design. The outcomes between compared groups could be underestimated if potential characteristic differences, particularly the education level, are not monitored. In this respect, the ECOS-16 apparently remains scientifically robust in its ability to discriminate among different education levels. In this study, the fact that the presence of vertebral fractures does not have a negative effect on HRQoL (bivariate analysis) deserves special attention. There seems to be some discrepancy regarding this issue in literature. Several studies have shown that HRQoL progressively deteriorates in relation to the presence and number of vertebral fractures [23,24]. However, other studies in the same area failed to find such a relationship [25], or have only found it when vertebral deformity is severe, while failing to find a relationship with any other fractures [26,27]. Recently, a significant number of studies have highlighted the importance of the site of the vertebral fracture and its effect on HRQoL. In this regard, it seems that the site of vertebral fracture has a much greater effect on HRQoL than the presence and number of vertebral fractures [28-30]. This difference could be explained by the relative rigidity of the thorax column in relation to the lumbar column, in the sense that mobility is more restricted when a lumbar rather than a thoracic region fracture occurs [31]. Moreover, lumbar column deformities have probably a greater impact on postural stability than alterations to the thoracic column. When the severity and site of the fracture is taken into account, fractured vertebrae in the transitional thoracolumbar region have a negative impact on HRQoL, at a Genant's degree greater than 1 [32]. Nevertheless, prospective studies addressing this issue should be conducted in the future assessing the impact of the time of the fracture and the site. Although in the present study, the bivariate analysis does not discriminate between patients with and without vertebral fracture, the multivariate model shows that the presence of fractures is indeed significant even if the percentage of explained variation is small. This is not surprising given the fact that such analysis is dealing with prevalent fractures in a short term observational study [28]. The inclusion of the time spent since the fracture occurred could improve the model, since it is well known that pain and disability due to a fracture progressively diminish over time [33]. However, among other health problems, a small percentage of explained variance was also found [33]. Nevertheless, the variables were entered into the multiple regression analysis dichotomously, as in table 1, an this may reduce the likelihood of finding a relationship. Therefore, it is likely that administering a HRQoL questionnaire in conjunction with the analysis of clinical variables could provide a better overall picture of the osteoporosis impact on patients. The results obtained for internal consistency and test-retest reliability showed high levels of homogeneity among questionnaire items and good reproducibility over time. Moreover, the outcomes, have also been shown that the new questionnaire effectively detects changes in patients' perceived health status due to osteoporosis. Expressing responsiveness to change is an important characteristic of the new instrument, one which will doubtless allow its use in clinical research. The high correlation between the ECOS-16 and generic (EQ-5D) and specific (MINI-OQLQ) HRQoL questionnaires corroborates this hypothesis. It also highlights the new questionnaire's validity by demonstrating that it measures concepts which are closely related to already validated HRQoL questionnaires [3,13]. The mean change in score per question corresponding to the effect size in general and to the MCID in particular is consistent, in terms that the larger the change assessed by the ECOS-16, the larger the effect size. Moreover, MCID is consistent with the results of other HRQoL questionnaires, and it is useful to compare the magnitude of changes detected between them. MCID will also be useful in the planning of new trials, as sample size depends on the magnitude of the difference investigators consider clinically important and are not willing to risk failing to detect [34]. The potential limitations of this study are mainly due to it being unable to rely on certain variables which have shown a clear influence on HRQoL. In particular, the "time spent since the fracture occurred" [35] was not analysed even though the objective of the study were women with established osteoporosis. The inclusion of prevalent fractures and exclusion of the incidence fractures means that a smaller variability among the patients in this study was established and, possibly, it also means that vertebral fractures had less influence on HRQoL. A further possible limitation is the limited sample size, which was relatively low to obtain statistical significance for more than one fracture site and for a certain fracture severity. This was a prospective, observational study with a limited follow-up time, but under conditions of usual clinical practice it served to prove the short-term good responsiveness of the questionnaire and test its remaining psychometric properties. Nevertheless long term follow-up studies will be necessary in the future. The low education level of the study sample must also be taken into account. It is consistent with previous studies [8,9], and as in other chronic diseases, education level has an impact on the prevalence of osteoporosis [36] and on the preferences of patients [37]. Furthermore, the impact of osteoporosis on HRQoL, the site of the vertebral fracture, coupled with the time spent since its onset, are extremely important variables which must be taken into account, since traditional clinical variables -i.e. bone densitometry- do not have a remarkable relationship with HRQoL [38]. Conclusions In conclusion, the ECOS-16 is a HRQoL questionnaire which is short, easy to administer (although some women need aid) and with adequate preliminary psychometric properties. This makes the ECOS-16 potentially very useful during routine clinical practice or/and research for the treatment and follow-up of post-menopausal women with osteoporosis. Nevertheless, its actual potential must be proven in future clinical trials in order to recommend its use in research and clinical practice. Supplementary Material Additional File 1 ECOS Appendix1.doc, the ECOS-16 questionnaire Click here for file Acknowledgements This study was supported by an unrestricted grant from Novartis-Spain. ==== Refs Díaz-Curiel M Carrasco JL Honorato J Pérez Cano R Rapado A Ruiz I On the behalf of the Multicenter Research Project on Osteoporosis. 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==== Front Nucl ReceptNuclear Receptor1478-1336BioMed Central London 1478-1336-2-41531223410.1186/1478-1336-2-4ResearchEndotoxin leads to rapid subcellular re-localization of hepatic RXRα: A novel mechanism for reduced hepatic gene expression in inflammation Ghose Romi [email protected] Tracy L [email protected] Sundararajah [email protected] Saul J [email protected] Texas Children's Liver Center, Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA2004 16 8 2004 2 4 4 2 7 2004 16 8 2004 Copyright © 2004 Ghose et al; licensee BioMed Central Ltd.2004Ghose et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Lipopolysaccharide (LPS) treatment of animals down-regulates the expression of hepatic genes involved in a broad variety of physiological processes, collectively known as the negative hepatic acute phase response (APR). Retinoid X receptor α (RXRα), the most highly expressed RXR isoform in liver, plays a central role in regulating bile acid, cholesterol, fatty acid, steroid and xenobiotic metabolism and homeostasis. Many of the genes regulated by RXRα are repressed during the negative hepatic APR, although the underlying mechanism is not known. We hypothesized that inflammation-induced alteration of the subcellular location of RXRα was a common mechanism underlying the negative hepatic APR. Results Nuclear RXRα protein levels were significantly reduced (~50%) within 1–2 hours after low-dose LPS treatment and remained so for at least 16 hours. RXRα was never detected in cytosolic extracts from saline-treated mice, yet was rapidly and profoundly detectable in the cytosol from 1 hour, to at least 4 hours, after LPS administration. These effects were specific, since the subcellular localization of the RXRα partner, the retinoic acid receptor (RARα), was unaffected by LPS. A potential cell-signaling modulator of RXRα activity, c-Jun-N-terminal kinase (JNK) was maximally activated at 1–2 hours, coincident with maximal levels of cytoplasmic RXRα. RNA levels of RXRα were unchanged, while expression of 6 sentinel hepatic genes regulated by RXRα were all markedly repressed after LPS treatment. This is likely due to reduced nuclear binding activities of regulatory RXRα-containing heterodimer pairs. Conclusion The subcellular localization of native RXRα rapidly changes in response to LPS administration, correlating with induction of cell signaling pathways. This provides a novel and broad-ranging molecular mechanism for the suppression of RXRα-regulated genes in inflammation. liverinflammationendotoxinRXRJNKnuclear exportnuclear receptor ==== Body Background LPS, a major constituent of the outer membrane of gram-negative bacteria, potently stimulates host innate immune response [1]. LPS-induced activation of monocytes/macrophages leads to the release of proinflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNFα) in addition to other mediators such as cysteinyl leukotrienes [2]. LPS and LPS-induced cytokines have been implicated in the pathogenesis and progression of a variety of liver diseases, including cholestasis, as well as being principal mediators of the negative hepatic APR [3]. The cholestatic effect of LPS is primarily due to cytokine-mediated inhibition of the function and expression of hepatic genes encoding critical proteins involved in bile formation and transport (reviewed in [4]). These hepatocellular transporters include the basolateral sodium/taurocholate cotransporter (Ntcp/Slc10a1) and organic anion transporting proteins (Oatp1/Slc21a1), as well as the canalicular multispecific organic anion exporter (Mrp2/Abcc2) and the bile salt export protein (Bsep/Abcb11). Transcriptional down-regulation of the principal hepatic bile acid importer, Ntcp contributes to the reduction in bile acid uptake by hepatocytes in inflammation, whereas reduced Mrp2 expression leads to impaired excretion of conjugated bilirubin, glutathione and other organic anions into bile [5,6]. Recent reports have provided insights into the link between inflammation-mediated cell signaling and regulation of bile acid homeostasis in the liver. Geier et al. showed that LPS-mediated suppression of Ntcp RNA was almost completely blocked by pre-treatment with anti-IL-1β specific antibodies, indicating that the cholestatic effects of LPS on the expression of this gene may be primarily mediated by the cell signaling pathways initiated by this one cytokine [7]. We have shown that IL-1β treatment of HepG2 cells, or primary rat hepatocytes, leads to JNK-dependent repression of nuclear binding activity of the Ntcp transactivator, RXRα:RARα, with consequent down-regulation of Ntcp promoter activity [8]. Finally, LPS, cytokines, and activated JNK have been linked to reduced expression of the rate-limiting enzyme in the bile acid biosynthetic pathway, cholesterol 7α-hydroxylase (CYP7A1), thus linking inflammatory signaling in the liver to the known and coordinated suppression of both bile acid import and synthesis [9-11]. How activated JNK leads to reduced RXRα function is not known, but is likely to involve direct phosphorylation of RXRα [8]. Phosphorylation of nuclear receptors (NRs) is a rapid and potentially powerful means of regulating NR activity, that, depending upon the NR, can affect transcriptional activity, protein stability, sub-cellular localization, protein-protein interactions or DNA binding activity [12,13]. Phosphorylation of transfected RXRα was reported to alter its transactivation properties in vitro, however, a definite functional role for native RXRα phosphorylation remains controversial. Both enhanced and reduced proteasome-mediated RXRα degradation have been associated with RXRα phosphorylation [14,15]. Hyperphosphorylation of RXRα by JNK was reported by Adam-Stitah et al [16] and the phosphorylation sites were mapped to several residues (serines 61 and 75 and threonine 87) in the N-terminal region and serine 265 in the ligand binding domain of mouse RXRα. However, JNK-mediated hyperphosphorylation of RXRα did not affect the transactivation properties of transfected RXRα homodimers or RXRα:RARα heterodimers in cultured cells [16]. In contrast, we and others have demonstrated that phosphorylation of RXRα by JNK signaling pathways is associated with reduced RXRα-dependent promoter activity [8,17]. Clearly, the consequences of RXRα phosphorylation are complex and poorly understood. NR ligands and extracellular signal-mediated pathways can alter subcellular NR localization, some of which involves phosphorylation-dependent mechanisms [12,13,18-20]. The xenobiotic receptors, CAR (NR1I3) [19] and PXR (NR1I2) [20] are localized in the cytoplasm of mouse hepatocytes and translocated into the nucleus after administration of their respective ligands. GR and VDR are well-known to undergo ligand-dependent nuclear import in transfected cells [12,13,18,21]. In contrast to the well-described events leading to NR nuclear import, little is known about NR nuclear export, including RXRα. Perhaps the best understood example of cell signaling targeting of NR nuclear export is JNK-mediated phosphorylation of GR, as a means of terminating GR-mediated transcription [18]. Such a mechanism for RXRα has never been shown, although a reduction in nuclear RXRα protein levels has been demonstrated in an animal model of obstructive cholestasis induced by bile duct ligation, raising the possibility of nuclear export [22]. In these studies, we sought to determine whether alterations in RXRα-dependent hepatic gene expression seen in inflammation may be related to nucleo-cytoplasmic re-distribution of RXRα. LPS treatment resulted in the activation of hepatic JNK coinciding with marked reduction in nuclear RXRα levels, and the rapid appearance of RXRα in the cytosol. RNA levels of RXRα and six of its heterodimeric partners highly expressed in liver were analyzed after LPS treatment: RXRα, RARα, FXR (farnesoid X receptor) and PPARα (peroxisome proliferator-activated receptor) RNA levels were stable, whereas CAR (constitutive androstane receptor) and PXR (pregnane X receptor) RNA levels were markedly suppressed and LXR (liver X receptor) RNA was elevated. Hepatic RNA levels of multiple RXRα target genes whose expression depends upon adequate nuclear levels of RXRα were significantly reduced by LPS. This is likely to be a consequence of reduced nuclear binding activity of RXRα heterodimer pairs. Notably, the reduction in RXRα nuclear protein levels (~50%) quantitatively correlated with the reduction in RNA levels of RXRα target genes. Taken together, these studies indicate that post-translational modification and cellular re-distribution of RXRα coinciding with induction of cell signaling is a novel, broad-ranging, and rapid mechanism contributing to the negative hepatic APR phenotype in the inflamed liver. Results LPS activates hepatic JNK In order to determine the in vivo role for LPS-induced activation of hepatic JNK, we first established a time course for JNK activation, by measuring phospho-JNK and phospho-c-Jun levels. Liver whole cell extracts were prepared at various time points from 1–16 hours after injection with either LPS (2 μg/g bw) or vehicle (0.9% saline) (Fig. 1A). Phosphorylated JNK levels were maximal at 1–2 hours, and significantly higher than saline-injected animals at all time points studied. Total JNK levels did not vary between vehicle and LPS-treated samples. c-Jun is a direct substrate for phospho-JNK, and phospho-c-Jun levels are a well-described indicator of JNK activity [23]. LPS treatment led to maximal c-Jun phosphorylation at 1 to 2 hours, with a slight reduction at 4 and 6 hours, and was undetectable by 16 hours. Thus, in an animal model, LPS administration activates JNK signaling in the liver as early as 1 hour, with evidence for prolonged JNK activity lasting at least 6 hours. Figure 1 LPS activates JNK and leads to rapid nuclear export of RXRα. C57BL/6 male mice were injected IP with 0.9% saline (Sal) or 2 μg/g bw of Salmonella LPS. Livers were isolated at the indicated time-points and whole cell extracts were prepared. A. Phosphorylation of c-JUN (P-cJUN) and JNK (P-JNK) was determined by immunoblotting cell lysates with phospho-c-JUN and phospho-JNK antibodies respectively. Total JNK levels in the liver tissue (JNK) are shown in the lower panels. This data is representative of three animals per treatment group. B. Nuclear (Nuc) and cytosolic (Cyt) extracts were analyzed by immunoblotting with antibodies to RXRα and RARα to determine subcellular localization of RXRα. The extracts from 4 animals were combined to account for inter-animal variability. Note the high molecular weight smear in LPS-treated extractions (most evident at 1 h). Data quantified and normalized to saline-injected samples (set at 1.0). C. Immunofluorescent analysis of formalin-fixed mouse liver tissues after 1 h of saline or LPS treatment. The blue color indicates DAPI staining of the nuclei, the green color indicates RXRα detected with FITC-labeled secondary antibody, DAPI/FITC are the merged images. The saline and LPS-treated samples are represented in the left and right panels, respectively. LPS treatment leads to the rapid reduction of nuclear RXRα protein levels concomitant with the appearance of RXRα in the cytoplasm Treatment of HepG2 cells or primary rat hepatocytes with IL-1β leads to JNK-dependent repression of RXRα :RARα nuclear binding activity, with the consequent down-regulation of target gene expression [8]. However, whether such changes are observed in RXRα activity after LPS challenge in an animal model is unknown. As early as one hour after LPS treatment, the maximal point of JNK activation, nuclear RXRα levels were significantly reduced compared to control, and remained so for at least 16 hours after LPS treatment (Fig. 1B). RXRα was not present in the cytoplasmic fraction at any time point after saline treatment, yet was robustly evident within one to two hours after LPS treatment, and decreased thereafter. Interestingly, immunoblot analysis of nuclear RXRα revealed a slower migrating species after LPS treatment (most evident in the 1 hour LPS sample), suggestive of LPS-induced post-translational modification. Notably, hepatic RARα levels in both nuclear and cytoplasmic compartments were unchanged by LPS. To confirm the intracellular localization of RXRα in liver, immunofluorescence staining was carried out in formalin-fixed liver tissues prepared from mice 1 h after saline or LPS injection (Fig. 1C). In LPS-treated mouse livers, RXRα was clearly observed in both the nucleus and cytoplasm of hepatocytes, whereas it remained exclusively nuclear in saline-treated controls. Thus, there is a rapid, dramatic, and specific subcellular re-distribution of hepatic RXRα in response to LPS-administration, coinciding with induction of cell signaling. Effect of LPS on steady-state mRNA levels of RXRα and its partners in the liver One possible explanation for reduced nuclear RXRα levels could be suppression of hepatic RXRα RNA expression, as seen in response to higher doses of LPS [24]. We investigated whether low dose LPS administration had an effect on the RNA levels of RXRα, six of its heterodimeric partners and SHP–all known to be involved in hepatic gene expression [25] (Fig. 2A). At 16 h after LPS administration, RNA levels for RXRα, RAR, FXR and PPARα were unchanged, whereas PXR and CAR RNA levels were reduced and LXRα RNA levels were increased (Fig. 2A). LPS-mediated down-regulation of PXR and CAR RNA levels in mice have been reported by Beigneux et al[26], however our results do not support the reduction in RNA levels of RXRα, FXR and LXR and PPARα seen by others, perhaps due to differences in the experimental model, LPS dose or mouse strain [24,27]. Figure 2 Effects of LPS on RNA levels of NRs and RXRα target genes. C57BL/6 mice were injected with 0.9% saline (white bars) or 2 μg/g bw of Salmonella LPS (black bars) for 16 hours (n = 6 per group). RNA was prepared from the livers and analyzed for A. NRs and B. RXRα target genes by real-time PCR. All data were presented as mean ± SD and standardized for GAPDH RNA levels. Expression in the saline-treated control animals was set to 1. The asterisks indicate significant difference (p < 0.05). See supplemental information for primers and probes. Hepatocyte-selective RXRα-null mice have impaired metabolic function, with reductions in CAR, FXR, LXRα, PPARα, and PXR target gene expression [28]. As examples of genes regulated by RXRα and its partners, we studied RNA expression of six sentinel hepatic genes regulated by various RXRα heterodimer pairs: Ntcp (RARα), Bsep (FXR), Mrp2 (CAR, FXR, PXR), Cyp3A11 (CAR, PXR), Abcg5 (LXRα) and Lfabp (PPARα) (Fig. 2B). RNA levels of all of these hepatic RXRα-regulated genes were significantly reduced by LPS treatment. Ntcp, Bsep and Abcg5 RNA levels decreased by 50–60%, Cyp3A11 RNA by 80%, while Lfabp and Mrp2 expression were each reduced approximately 60–70% after LPS treatment. The comparatively greater reduction in Cyp3A11 gene expression can be attributed to the combined effects of diminished PXR and CAR RNA expression along with reduced nuclear RXRα protein levels; both PXR & CAR activate Cyp3A11 gene expression (reviewed in [29]). The orphan nuclear receptor SHP (small heterodimer protein, NR0B2) is known to repress the activities of RXRα and other NRs [11,29]. One possibility is that LPS-mediated suppression of hepatic genes could be mediated by the activation of the repressor, SHP [9]. However, this is unlikely, since LPS treatment dramatically reduced SHP RNA levels (Fig. 2A). Taken together, these studies indicate that the effect of LPS on hepatic RXRα-dependent gene expression is not due to reduced RXRα RNA levels or increased SHP–rather it appears to be a consequence of post-translational modification and rapid LPS-induced subcellular re-distribution of RXRα protein. This is in agreement that SHP-1 is a FXR/RAR target gene [11]. Effect of LPS on DNA binding activity of Type II nuclear receptor pairs in liver In order to determine if reduced nuclear RXRα protein levels leads to impaired DNA binding activity of RXRα and its partners, electrophoretic mobility shift analyses were performed. Nuclear extracts were prepared from livers of saline or LPS-treated mice and incubated with oligonucleotides containing canonical DNA elements scanning Type II NR binding sites–direct repeats of the hexad AGGTCA, separated by 1 to 5 nucleotides (DR1-5), or an inverted repeat separated by 1 nucleotide (IR1) [30,31]. As Type II NRs, RXRα partners with either RARα, PPARα, PXR, CAR, LXR or FXR to bind to one or more of these sites (reviewed in [25]). At 16 h after LPS treatment, binding to all 6 RXRα-containing canonical sequences was significantly reduced in hepatic nuclear extracts from LPS-treated animals (~45–70% reduction) (Fig. 3), consistent with a diminished nuclear RXRα. Since the expression of PXR and CAR was reduced upon LPS administration (Fig. 2A), there was a more dramatic decrease in binding to their recognition elements, DR3 and DR4 (~70% reduction). Binding to the consensus AP1 DNA sequence was increased (~70%) upon administration of LPS; this serves as a positive control for JNK-mediated activation of hepatic inflammation as well as an indication of specificity of suppression of RXRα-heterodimer pair DNA binding (Fig. 3). Figure 3 LPS reduces binding activities of RXRα-containing heterodimer pairs to canonical DNA elements. Electrophoretic mobility shift assay analysis of hepatic nuclear extracts prepared from C57BL/6 mice injected with control saline or 2 μg/g bw LPS for 16 h. Radiolabeled double-stranded DR1, DR2, DR3, DR4 and DR5 elements or a consensus AP1 element were employed (see Materials and Methods). The samples were electrophoresed through a 6% non-denaturing polyacrylamide gel, dried and analyzed by autoradiography. Discussion The negative hepatic APR is characterized by suppression of hepatic gene expression in response to inflammation and is well-modeled by LPS administration [32,33]. We hypothesized that reduced nuclear levels of RXRα after LPS administration would be manifested by broad alterations in RXRα-dependent gene expression across diverse physiological processes [28]. Our results demonstrate that LPS signaling induces rapid and profound reduction of hepatic nuclear RXRα protein levels, concomitant with appearance of RXRα in the cytoplasm, leading to subsequent reduction in the expression of RXRα-dependent hepatic genes. Recent studies have led to a broader understanding of the molecular basis for the role of LPS in intracellular signaling and hepatic function [24,26,34]. Activation of monocytes/macrophages by LPS leads to the secretion of a number of proinflammatory cytokines such as TNFα, IL-1β, and IL-6 [2]. LPS-induced activation of Kupffer cells, the resident hepatic macrophages, triggers several crucial intracellular signaling pathways in hepatocytes, including stress-activated mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), JNK and p38 mitogen-activated protein kinase (p38 MAPK) [35]. Stress-activated protein kinases, mitogen-activated protein kinase kinase-4 (MKK4/SEK1) and its downstream mediator JNK was shown to directly phosphorylate RXRα[8,17]. Previous studies by our group[8] demonstrated that inhibition of the JNK signaling pathway completely blocked IL-1β-mediated suppression of RXRα-dependent Ntcp gene expression, thus implicating JNK to be a central player in inflammation-induced cholestasis. Most evident in the 1 hour sample are high molecular weight forms of RXRα, consistent with covalent post-translational modification (Fig. 1B), although the actual nature of this high molecular weight species is currently unknown and under investigation. One plausible interpretation of these data is that LPS-induced activation of JNK leading to phosphorylation and likely further modification of RXRα, triggering its transport from nucleus to cytoplasm, where it may be targeted for degradation. Phosphorylation has been shown to be involved in the degradation of RXRα :RARα heterodimers by proteasomes, thus providing a mechanism for JNK-mediated inhibition of RXRα-dependent target gene transactivation[14,15] RNA levels of RXRα were not affected by LPS, further supporting nuclear export of RXRα as a primary mechanism of suppression of hepatic genes during negative hepatic APR. The interrelationship and roles played by JNK and phospho-RXRα are neither readily nor definitively explored in an in vivo model, especially using such a broadly-acting inflammatory agent like LPS. Hepatocytes and liver-derived HepG2 cells in culture respond to LPS-induced cytokines like TNFα and IL-1β by suppressing the expression of negative hepatic APR genes [8,24,26,36]. Recent work in our laboratory indicates that treatment of HepG2 cells with IL-1β leads to RXRα nuclear export, dependent upon JNK-mediated phosphorylation of select residues in RXRα (Zimmerman et al., manuscript in preparation). In transfected cells, nerve growth factor (NGF)-induced phosphorylation of the orphan nuclear receptor NGFI-B (Nur77) resulted in the translocation of RXR-NGFI-B complex out of the nucleus, indicating that distribution of RXR in these cells was regulated by NGFI-B [37]. The data presented here are the first to indicate that inflammation-mediated cell signaling leads to rapid subcellular redistribution of native RXRα, changing the previous impression of RXRα as a stable nuclear resident [38]. Finally, these findings indicate significant cross-talk between JNK-signaling and NR-mediated gene expression. Conclusions Overall, we conclude that RXRα is rapidly exported out of the nucleus in response to LPS. RXRα, as an obligate heterodimer with other class II NRs, regulates the expression of a broad array of genes involved in critical metabolic pathways in the liver, many of which are impaired during the negative hepatic APR. This helps explain how inflammation-induced signaling can lead to rapid, diverse and multiple alterations in hepatic gene expression, which has implications for future therapeutic targets of both acute and chronic liver diseases. Materials and methods Materials LPS (Salmonella typhimurium) was purchased from Sigma Chemical Co. (St. Louis, MO) and freshly diluted to the desired concentration in pyrogen-free 0.9% saline before injection. Anti-JNK (#9252), phospho-JNK (#9251) and phospho-cJUN antibodies (Ser 63) (#9261) (Cell Signaling, Beverly, MA); anti-RXRα (D-20) (#sc-553) and anti-RARα antibodies (#sc-551) (Santa Cruz Biotechnology, Santa Cruz, CA) were used according to manufacturer's instructions. [γ-32P]ATP was obtained from PerkinElmer Life Sciences (Boston, MA). Oligonucleotides were obtained from Sigma Genosys and Synthegen, Houston, TX. All reagents for real-time PCR were purchased from Applied Biosystems (Foster City, CA). Animals Adult male (8–10 weeks) C57BL/6 mice (20–25 g) were purchased from Charles River Laboratories, (Wilmington, MA). The animals were maintained in a temperature- and humidity-controlled environment and were provided with water and rodent chow ad lib. Mice were given intraperitoneal injection with 2 μg/g body wt LPS (Salmonella typhimurium; Sigma Chemical Co., St. Louis, MO) in saline or saline alone. LPS in this dose range has been shown previously to induce cholestasis, maximally inhibit bile acid uptake, and significantly reduce Ntcp mRNA from 12 to 16 hours after injection, while not inducing hepatic damage [6,39]. Livers were removed at the time indicated in the figure legends (1 to 16 hours) after treatment. All animal protocols were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee. Experiments were performed in triplicate and repeated three to four times. Preparation and analysis of nuclear and cytoplasmic and whole cell extracts Nuclear and cytoplasmic extracts were prepared according to Itoh et al[18] Whole cell extracts were prepared according to Li et al [8]. Protein concentration was determined by BCA assay according to the manufacturer's protocol (Pierce, Rockford, IL). These fractions were analyzed by immunoblotting. Signals were developed by a standard enhanced chemiluminescence method following the manufacturer's protocol (Perkin Elmer Life Sciences, Boston, MA) and quantified by a densitometer using ImageQuant software. Immunofluorescent analysis Livers were isolated from saline and LPS injected mice after 1 hour of treatment, fixed in 10% buffered neutral formalin overnight at 4°C and then stored in 70% ethanol. Fluorescent detection was performed by using anti-RXRα (D-20) antibody and fluorescein isothiocyanate (FITC)-labeled secondary antibody and nuclei was stained with 4'-6-diamidino-2-phenylindole (DAPI). Visualization was performed with a Deltavision Spectris Deconvolution Microscope System (Applied Precision, Inc.). Electrophoretic gel mobility shift assays Nuclear extracts were prepared according to Timchenko et al.[40] with some modifications. Double-stranded oligonucleotide probes were end-labeled and purified according to standard procedures [41]. 10 μg of nuclear extracts were incubated on ice for 30 min with 32P end-labeled oligonucleotide as described previously [41]. The oligonucleotide sequences are provided in Table 1. After binding, the samples were electrophoresed through a non-denaturing 6% polyacrylamide gel, dried and exposed to x-ray film. In addition, gels were exposed to a PhosphorImager screen and quantified using a PhosphorImager and ImageQuant software. Real time quantitative PCR analysis Total RNA was isolated from mouse liver tissues using the RNaesy kit from Qiagen. cDNA was synthesized from 7.5 μg of total RNA using the ProSTAR™ First-Strand RT-PCR Kit (Stratagene, La Jolla, CA). Real time quantitative PCR (RTQ-PCR) was performed using an ABI PRISM 7700 Sequence Detection System instrument and software (Applied Biosystems, Inc., Foster City, CA). Briefly, each amplification reaction (50 μl) contained 40–200 ng of cDNA, 300 nM of forward primer, 300 nM of reverse primer, 200 nM of fluorogenic probe and 25 μl of TaqMan® Universal PCR master mix. PCR thermocycling parameters were 50°C for 2 min, 95°C for 10 min and 40 cycles of 95°C for 15 s, and 60°C for 1 min. Quantitative expression values were extrapolated from standard curves and were normalized to GAPDH. The sequences of the primers and probes were obtained from the literature [42] or purchased from Applied Biosystems, and are listed in Table 2. Abbreviations The abbreviations used are: RXR, retinoid X receptor; RAR, retinoic acid receptor; FXR, farnesoid X receptor; PPAR, peroxisome proliferator-activated receptor; PXR, pregnane X receptor; CAR, constitutive androstane receptor; LXR, liver X receptor; SHP, small heterodimer partner; NR, nuclear receptor; GR, glucocorticoid receptor; PR, progesterone receptor; VDR, vitamin D receptor; JNK, c-Jun N-terminal kinase; AP-1, activator protein-1; Ntcp, sodium/taurocholate cotransporting polypeptide; Bsep, Bile salt export pump; Mrp2, multidrug resistance associated protein 2; Lfabp, liver fatty acid binding protein; Cyp3A11, cytochrome P450 3A11; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; APR, acute phase response; DR, Direct Repeat; IR, Inverted Repeat; FITC, fluorescein isothiocyanate; DAPI, 4'-6-diamidino-2-phenylindole. Acknowledgements The authors thank Dorene Rudman of the Molecular Core of the Texas Gulf Coast Digestive Diseases Center for assistance with immunofluorescent analysis. A portion of this work was presented at the AASLD, October 2003. 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==== Front Genet Vaccines TherGenetic Vaccines and Therapy1479-0556BioMed Central London 1479-0556-2-71529401810.1186/1479-0556-2-7ResearchA dual function fusion protein of Herpes simplex virus type 1 thymidine kinase and firefly luciferase for noninvasive in vivo imaging of gene therapy in malignant glioma Söling Ariane [email protected]ß Christian [email protected] Stephanie [email protected] Nikolai G [email protected] Molecular Neurooncology Laboratory, Dept. Neurosurgery, Martin-Luther-University Halle-Wittenberg, 06097 Halle, Germany2 Dept. Neurological Science, University of Liverpool, Liverpool 9L 7LJ, United Kingdom2004 4 8 2004 2 7 7 5 4 2004 4 8 2004 Copyright © 2004 Söling et al; licensee BioMed Central Ltd.2004Söling et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Suicide gene therapy employing the prodrug activating system Herpes simplex virus type 1 thymidine kinase (HSV-TK)/ ganciclovir (GCV) has proven to be effective in killing experimental brain tumors. In contrast, glioma patients treated with HSV-TK/ GCV did not show significant treatment benefit, most likely due to insufficient transgene delivery to tumor cells. Therefore, this study aimed at developing a strategy for real-time noninvasive in vivo monitoring of the activity of a therapeutic gene in brain tumor cells. Methods The HSV-TK gene was fused to the firefly luciferase (Luc) gene and the fusion construct HSV-TK-Luc was expressed in U87MG human malignant glioma cells. Nude mice with subcutaneous gliomas stably expressing HSV-TK-Luc were subjected to GCV treatment and tumor response to therapy was monitored in vivo by serial bioluminescence imaging. Bioluminescent signals over time were compared with tumor volumes determined by caliper. Results Transient and stable expression of the HSV-TK-Luc fusion protein in U87MG glioma cells demonstrated close correlation of both enzyme activities. Serial optical imaging of tumor bearing mice detected in all cases GCV induced death of tumor cells expressing the fusion protein and proved that bioluminescence can be reliably used for repetitive and noninvasive quantification of HSV-TK/ GCV mediated cell kill in vivo. Conclusion This approach may represent a valuable tool for the in vivo evaluation of gene therapy strategies for treatment of malignant disease. gliomabioluminescence imaginggene therapyherpes simplex virus type 1 thymidine kinaseluciferase ==== Body Background Treatment with the suicide gene/ prodrug activating system herpes simplex virus type I thymidine kinase/ ganciclovir (HSV-TK/ GCV) is highly efficient in animal models of malignant glioma [1-3]. In contrast, clinical trials employing the HSV-TK/ GCV system and a retroviral vector have demonstrated only a limited effect in glioblastoma patients [4-8], implying that transfer and distribution of the transgene in human brain tumors were very low in vivo and differed obviously from the findings in animal experiments. Presently, the standard method for assessing delivery of therapeutic transgenes to tumors relies on ex vivo analysis of explanted tumor tissue [7,9,10]. Time course analysis of transgene expression thus requires a multitude of animals to be sacrificed. In the past few years, noninvasive imaging techniques such as positron emission tomography (PET), magnetic resonance imaging, and optical imaging methods using fluorescence and bioluminescence were introduced and increasingly used for temporal and spatial monitoring of transgene expression [11-13]. Bioluminescence imaging (BLI) using luciferase (Luc) from the North American firefly Photinus pyralis as a reporter has several advantages compared to other imaging methods: (1) the technique is very sensitive (possibly 10-15 – 10-17 mole of luciferase/L are detectable in vivo, [13]) and detects tumor cells at a stage where radiography and PET cannot [14,15], (2) bioluminescence imaging using a cooled CCD camera does not require great technical expertise and (3) it is faster and less expensive than many other imaging techniques. Furthermore, in contrast to fluorescence imaging, where autofluorescence may interfere with the signal of interest [13], background luminescence is negligible. This study aimed at generating a sensitive tool for noninvasive in vivo monitoring of the activity of a therapeutic transgene by fusing the bioluminescent reporter gene Luc to the bioactivating "suicide" gene HSV-TK. We investigated whether this fusion construct could be used to monitor HSV-TK mediated cytotoxicity in malignant glioma by serial optical imaging in vivo. Noninvasive real time evaluation of localization, activity and persistence of a therapeutic gene in living animals may represent an important step towards optimization of gene therapy protocols. Methods Vector construction The HSV-TK cDNA from the retroviral vector G1Tk1SvNa ([16], kind gift from E. Otto, GTI Inc., Gaithersburg, MD) and the "humanized" firefly luciferase (Luc) gene from the pGL3 vector (Promega) were ligated into pCDNA 3.1(-) (Invitrogen). For the fusion construct, EGFP in the pEGFPLuc vector (BD Biosciences) was exchanged for HSV-TK cDNA, which had been amplified from G1Tk1SvNa by PCR. The resulting HSV-TK-Luc fusion gene contained a humanized form of the firefly Luc gene to ensure high expression in mammalian cells [17]. The full length HSV-TK cDNA was inserted in frame upstream of the Luc cDNA, and both genes were separated by a linker sequence of 33 nucleotides. All transgenes were expressed under the control of the CMV promoter. The correct sequence of the fusion construct HSV-TK-Luc was confirmed by DNA sequencing. Cell culture and transfection The human glioblastoma cell lines U87MG, T98G, LN18, U343, LN-Z308 and human embryonic kidney 293 cells were cultured under standard conditions. Cells were seeded in 6-well plates at a density of 3 – 5 × 105 cells/ well 16 to 24 h prior to transfection. Cells were transfected under serum-free conditions with the indicated amounts of DNA and Lipofectamine (Invitrogen) according to the manufacturer's protocol. For selection of stable clones transfected cells were replated at low density 48 h after transfection and incubated with 1 mg/ml (final concentration) geneticin (Calbiochem, Bad Soden, Germany) for 4 weeks. Colonies were picked and analyzed for transgene expression. Cytotoxicity assay Transiently or stably transfected U87MG cells were seeded at 4 × 103 cells/ well in a 96-well plate. GCV was added at final concentrations of 0 – 10 μg/ml and cells were incubated at 37°C/ 5% CO2 for 4 days. MTT (Sigma, Deisenhofen, Germany) was added at a final concentration of 0.5 mg/ml for 2 h. Absorbance was measured in a microplate reader (Victor2, Perkin Elmer Life Sciences, Turku, Finland) at 590 nm (reference 660 nm). Experiments were performed in quadruplicates and repeated at least twice. Results are reported along with the standard deviation (SD). Cell culture assays for luciferase activity Transiently transfected U87MG cells were lysed in CCLR lysis buffer (Promega) 2 days after transfection. Stably transfected cells were lysed in the same buffer when they had reached ~90% confluence. Protein content of all cell lysates was determined by the Bradford Protein assay (Bio-Rad, Munich, Germany). Equal amounts of protein were analyzed luminometrically for luciferase activity with a microplate reader (Victor2) using the Luciferase Assay System reagent (Promega). All experiments were repeated at least twice and mean values are reported along with the SD. For bioluminescence imaging of intact cells HSV-TK-Luc expressing U87 glioma cells were transferred to a black microtiter plate in order to minimize light scattering, and MTT assay was performed in quadruplicates as described above. On day 4 after addition of GCV, D-Luciferin was added to a final concentration of 500 μM to the culture medium. Cells were placed in a dark box and light emission was imaged using a cooled CCD camera (Visiluxx Imager, Visitron). Light emitted from a region of interest (ROI) drawn over each well was quantified and mean values from quadruplicate measurements were compared with MTT results. Immunohistochemistry and Hematoxylin-Eosin (HE) staining Immunohistochemistry on paraffin sections using a rabbit polyclonal anti-Luc antibody (CR2029RAP, Europa Bioproducts) was performed essentially as described by Lee et al [18]. HE staining was performed according to standard protocols. Animal experiments All animal protocols were approved by the Animal Care and Use Committee at Martin-Luther-University Halle-Wittenberg. Six week old male NMRI nu/nu mice (Charles River) were injected s.c. at four sites, each with 2 × 106 human U87MG glioma cells stably expressing the HSV-TK-Luc fusion protein. When xenografts had reached a size of ~5 mm in diameter, in general on days 7 to 9 post tumor implantation GCV therapy was initiated. Mice were injected twice daily i.p. with 30 mg/ kg GCV for 14 days. Control mice with xenografts (n = 3) received saline injections. Tumor size was measured every 2 to 4 days by caliper. Tumor volume was calculated according to the formula 0.52 × width2 × length. Bioluminescence imaging For BLI animals were anesthetized with ketamine/xylazine and injected i.p. with 150 mg/ kg D-Luciferin. Approximately 8 minutes after D-Luciferin injection mice were placed in a dark box and a grayscale image was acquired at low light (exposure time 2 seconds). Bioluminescence was measured in the dark by a CCD camera cooled to -120°C (VisiLuxx Imager), using an acquisition time of 15 min and binning 6. Bioluminescent signals were displayed in pseudocolors and superimposed on the grayscale image using Metamorph software (Visitron). Mice receiving GCV were imaged at least on days 7, 15, 22, 29, and 56 post tumor implantation (corresponding to start and day 8 of GCV therapy, as well as days 1, 8, and 35 after end of GCV therapy), while untreated control animals were subjected to BLI on days 7, 22, 29 and 35. In each animal a region of interest (ROI) was drawn over a single tumor or over all tumors as indicated in the text. Integrated as well as maximum light units (= counts) within this area were calculated after background subtraction. Final values are reported as the mean of the integrated or maximum counts obtained from all mice within one group. The CCD camera in use has a quantum efficiency approaching 90% at wavelengths between 550 and 770 nm, indicating that one photon is converted to ~0.9 electrons. One photoelectron corresponds to 4.52 counts. For serial quantification of light emission the conditions for image acquisition (e.g. exposure time, time between D-Luciferin application and image acquisition, stage position) were kept constant. Statistics Statistical analysis was performed using the ANOVA and Student's t test (SPSS and Microcal Origin Software). A p value of <0.05 was considered significant. Results Characterization of the HSV-TK-Luc fusion construct To achieve a strictly equimolar coexpression of a therapeutic and a reporter gene, the HSV-TK cDNA was fused in frame with the Luc cDNA in 2 ways: one fusion protein contained HSV-TK N-terminally, in the other construct Luc preceded the HSV-TK moiety. Both constructs were expressed under control of the CMV promoter. Several human glioma cell lines as well as 293 cells were transiently transfected with these constructs. In general, Luc activity was found to be up to 50-fold higher in cells expressing the HSV-TK-Luc construct compared to cells expressing the Luc-HSV-TK construct (data not shown). Therefore, all further studies were performed with the HSV-TK-Luc fusion construct. In order to characterize this fusion construct more thoroughly, both transient and stable transfection experiments were performed using the human U87MG glioma cell line (Figures 1 and 2). For transient transfection experiments, cells were transfected with 50 ng (~11.2 fmol) – 2 μg (~450 fmol) of plasmid DNA harboring either HSV-TK, Luc, or HSV-TK-Luc transgenes, respectively. As all 3 vectors were of equal size, equal amounts of DNA corresponded to equimolar amounts of plasmid. Cytotoxic activity as measured by MTT assay was compared to luminometrically determined light production and found to be tightly correlated in HSV-TK-Luc transfected cells (Figure 1, R2 = 0.99; p < 0.0001). Photon emission above background levels was not detectable in cells that had been transfected with HSV-TK only, while no cytotoxic activity was conferred to cells expressing only Luc. Figure 1 Cytotoxic and bioluminescent activity in U87MG glioma cells transiently transfected with different amounts of HSV-TK-Luc plasmid. (A) Cytotoxic activity as measured by MTT assay and (B) luciferase activity as determined luminometrically in cell lysates. Results are displayed as counts per second (cps)/ μg protein. (C) Linear regression analysis of cytotoxic activity plotted against luciferase activity for the different amounts of plasmid DNA. Results from 3 independent experiments were used. Figure 2 Comparison of the enzymatic activities of the HSV-TK-Luc fusion protein, HSV-TK, and Luc. U87MG cells were transiently transfected with 0.05 – 2 μg of HSV-TK-Luc, Luc, or HSV-TK plasmid. Cells transfected with equimolar amounts of DNA were analyzed for cytotoxic and bioluminescent activity. (A) Cytotoxic activity of cells transfected with HSV-TK-Luc or HSV-TK. For reasons of clarity only the graphs for 0.1 μg and 2 μg of transfected DNA are shown. (B) Luciferase activity in U87MG cells transfected with HSV-TK-Luc or Luc only. Results represent 3 independent experiments. Cytotoxic and Luc activity in cells transiently transfected with the HSV-TK-Luc fusion construct were also compared to the respective activities in cells transiently transfected with equimolar amounts (50 ng – 2 μg DNA) of HSV-TK or Luc alone. The overall cytotoxic activity of the fusion construct proved to be 60% of that measured in cells transfected with HSV-TK alone. Representative curves for 2 μg and 0.1 μg of transfected DNA are shown in Figure 2A. Luc activity of the fusion protein was also lower compared with native Luc: light production in cells transfected with HSV-TK-Luc was 22% of that seen in cells transfected with Luc only (Figure 2B). A tight linear correlation of bioluminescence to cell kill was achieved with the fusion protein, suggesting that light emission can indeed be used as a measure for the cytotoxic effect of transgenic HSV-TK. Stable expression of the HSV-TK-Luc fusion gene in human glioma cells Having demonstrated in transient transfection experiments that Luc could be employed as a reporter for monitoring the therapeutic effect of HSV-TK, U87MG cell clones stably expressing the HSV-TK-Luc fusion protein were generated by selection of transfected cells with geneticin. Comparison of 18 of these clones for Luc and cytotoxic activity revealed a good correlation between both enzymatic activities (R2 = 0.79; p < 0.001, data not shown). Enzymatic activity in the U87MG clone with both the highest Luc and HSV-TK activity was compared with U87MG clones expressing unfused HSV-TK or Luc. Cells stably expressing HSV-TK did not luminesce upon addition of D-Luciferin while Luc expressing cell clones were resistant to GCV mediated cell killing (data not shown). The dual function fusion protein compared favorably to the respective clones with the highest HSV-TK or Luc activity. Light production in the HSV-TK-Luc expressing cell clone was ~41% of that seen in Luc expressing U87MG cells while cytotoxic activity of the HSV-TK-Luc labeled U87MG clone was ~84% of that seen with the most active HSV-TK expressing U87MG clone (data not shown). Photon emission determined luminometrically was found to be linearly correlated with cell number over a range of at least 5 orders of magnitude (R2 = 0.99; p < 0.001, data not shown). Photon emission from as few as 500 intact cells expressing the fusion construct was detectable by the CCD camera while the lower detection limit for the Luc expressing U87MG cell clone was 125 cells. We further examined whether the cytotoxic activity of the HSV-TK moiety could be visualized by monitoring light emission from intact cells that had been treated with GCV at different concentrations. Signals captured by the CCD camera showed a close correlation to the cytotoxic effect as measured by MTT assay (Figure 3, R2 = 0.94; p = 0.029). These data demonstrate that both enzyme activities were also preserved in U87MG cells stably expressing the HSV-TK-Luc fusion construct. Figure 3 Correlation of light emission with cytotoxicity in intact U87MG glioma cells stably expressing the HSV-TK-Luc fusion construct and treated with GCV. (A) Cytotoxic activity as determined by MTT assay. (B) Bioluminescence imaging of quadruplicates of intact U87MG cells treated with the indicated amounts of GCV or left untreated (control). (C) Linear regression analysis of photon emission detected by the CCD camera plotted against cytotoxicity (R2 = 0.94; p = 0.029). Correlation of HSV-TK with luciferase activity in vivo The above high expresser U87MG cell clone was used for xenograft experiments in nude mice. For sensitivity testing, 2 × 103, 2 × 104 and 2 × 105 cells were injected s.c. on the back of the animals. Although not palpable, 2 × 104 cells expressing the fusion construct were detected by the CCD camera immediately after injection (= day 0), either when injected alone or mixed with 1.8 × 105 (90%) non-luminescent parental U87MG cells prior to injection, while 2 × 103 cells injected s.c. were not seen (data not shown). This high level of detectability by BLI proves the usefulness of the HSV-TK-Luc construct as a highly sensitive reporter in vivo. For therapeutic studies, mice received injections with 2 × 106 HSV-TK-Luc labeled U87MG cells at four different sites on the back and the flanks, respectively (Figure 4). When tumors had reached a size of ~5 mm in diameter, a bioluminescence image was acquired and GCV therapy was initiated (n = 7). GCV treatment did not cause any significant toxicity and treated mice displayed normal patterns of food intake and physical activity. Control animals (n = 3) received saline injections. Initial tumor volumes in all mice were 309 ± 37 mm3 and light intensity units (= counts) measured on day 7 were 122961 ± 22155. Serial measurements (during and after GCV therapy) of tumor volumes and integrated light intensity units within a region of interest (ROI) including all tumors were plotted against each other (Figure 5). Within the 2 weeks of GCV treatment, all 7 mice showed a rapid decline in photon emission from their tumors (mean decrease: 92 ± 7%, Figure 5A), which was accompanied by a somewhat slower decrease in tumor volume (65 ± 19%, Figure 5B). A linear regression analysis of mean tumor volumes in treated mice on days 7, 15, 22, 29, and 56 post tumor implantation plotted against the respective mean integrated light units is displayed in Figure 6A. Light emission and tumor volumes correlated closely with each other (R2 = 0.93; p = 0.008), thus confirming our cell culture data (Figures 1 and 3). Figure 4 Bioluminescence imaging of nude mice carrying HSV-TK-Luc expressing U87MG gliomas. (A) Serial images from a mouse with 4 s.c. xenografts treated with GCV from day 7 to day 21 post tumor implantation and (B) saline treated control mouse, sacrificed on day 35 post tumor implantation due to massive tumor growth. The largest tumor in this mouse on the upper right back has already become necrotic. The day 35 image is also displayed at a broader grayscale range for better visualization of tumor localization. Note that control tumors also showed decreased light emission and a reduction in tumor size within the first 3 weeks post tumor implantation. Figure 5 Comparison of (A) bioluminescence signals detected by the CCD camera and (B) tumor volumes in GCV-treated mice (n= 7, M1 – M7) harboring HSV-TK-Luc tagged U87MG glioma xenografts. GCV (60 mg/kg per day) was administered for 14 days starting at day 7 post tumor implantation. All mice were imaged at least on days 7, 15, 22, 29, and 56 post tumor implantation. BLI signals and tumor volumes at the beginning of therapy were set as 100%. Identical symbols in both graphs correspond to identical animals. Figure 6 Linear regression analysis. Light emission as determined by the CCD camera was plotted against tumor volume. Mean values for all animals in a group along with the S.E.M are reported. (A) GCV treated mice: mice (n = 7) were imaged at start of therapy, after 1 and 2 weeks of GCV treatment, and 1 and 5 weeks after end of GCV therapy (R2 = 0.93, p = 0.008). In some mice additional images were acquired (Figure 5), but these were not included in this plot. (B) Saline treated control mice: these mice (n = 3) were imaged on days 7, 22, 29, and 35 and were sacrificed after the last BLI due to massive tumor growth (R2 = 0.98, p = 0.010). Regarding therapeutic efficacy in these mice on an individual basis, photon emission and tumor volumes showed a significant correlation, with R2 values ranging from 0.78 to 0.96 and p ranging from 0.004 to 0.047, except for one mouse (R2 = 0.731; p= 0.065). In this mouse a significant correlation between light emission and tumor volume could be demonstrated, when tumor volumes were plotted against the maximum light emission within a ROI (R2 = 0.81; p = 0.037) instead of integrated light units. In general, integrated light units within a ROI correlated closely with maximum light emission from this ROI: correlation coefficients (R2) for all tumors in treated mice varied between 0.97 and 0.99, all p values were <0.003. Five weeks after end of GCV therapy (day 56) light emission was no longer detectable in 5 of the 7 GCV-treated mice, while in 4 of them small residuums at the tumor site were still visible. Two mice still showed very weak light emission from one of their flank tumors which also disappeared in subsequent imaging studies. All GCV treated mice survived and tumor recurrence was not observed until closure of the study at day 90 post tumor implantation. The 3 untreated control mice were imaged on days 7, 22, 29, and 35 post tumor implantation and had to be sacrificed on weeks 5 to 6 post cell injection due to massive tumor growth. Although 2 tumors with relatively strong light emission on day 7 post tumor implantation regressed within 4 weeks in one of the control mice, overall tumor growth in all control animals plotted against light emission from these tumors still showed a tight correlation (R2 = 0.98; p = 0.010; Figure 6B). Within 4 weeks, tumor volumes increased by ~11-fold while photon emission concomitantly rose ~6-fold. When tumors had become very large (>12–15 mm in diameter) further increase in light emission was much less than the increase in volume. This was mirrored by a less stringent linear correlation of BLI signal and tumor size in large tumors. The control mouse shown in Figure 4B serves as an example: on day 35 the tumor on the right upper back shows an attenuated bioluminescent signal although being the largest tumor. While linear regression analysis demonstrated a very good correlation of tumor volume to light emission for the other 3 tumors (R2 = 0.99 and p = 0.002 for tumors on the back and the right flank; R2 = 0.98 and p = 0.011 for the tumor on the left flank), R2 was 0.90 (p = 0.050) for this large tumor. When serially determined maximum light emission was used for quantification in this particular tumor, R2 dropped to 0.37 (p = 0.4). When control mice were sacrificed, tumors were explanted, and immediately reimaged. Bioluminescence imaging confirmed reduced light emission from hemorrhagic and necrotic areas in large tumors (Figure 7A). Immunohistochemical analysis of these tumor regions using a polyclonal anti-Luc antibody also showed a relatively scarce positive staining of necrotic areas as compared to areas with strong photon emission (Figure 7B). Figure 7 Ex vivo bioluminescence imaging and histological analysis of a large HSV-TK-Luc tagged U87MG glioma in a control mouse. (A) Bioluminescence image (exposure time 10 min) of the freshly explanted tumor after D-Luciferin injection in the mouse prior to sacrifice. The tumor was cut in the middle and placed with the cut side facing the CCD camera. One part of the tumor was obviously hemorrhagic while the other part looked vital. Photon emission displayed in pseudocolors precisely reflected the macroscopic findings. (B, a and b) HE staining of paraffin sections from the same tumor as seen in (A) vital tumor region (a); hemorrhagic and necrotic tumor area (b); (B, c and d) immunohistochemistry on corresponding sections using a polyclonal anti-Luc antibody; vital tumor area (c) and hemorrhagic and necrotic region (d) with only scarce positive staining. Original magnification × 400. Discussion This study demonstrates that firefly luciferase is a valuable tool for monitoring noninvasively the efficacy of the prodrug activating system HSV-TK/ GCV in cell culture and in vivo. The HSV-TK-Luc fusion protein was successfully used in a brain tumor animal model for serial and sensitive real time quantification of the cytotoxic effect of HSV-TK by BLI. Correlation of enzymatic activities Fusion of two enzymes is the only way to guarantee stoichiometric, and thus correlated expression of both fusion partners. We chose this approach because coexpression of two separate transgenes from either one or separate promoters has been reported to result in severely impaired gene expression, e.g. due to inefficient internal ribosome entry site (IRES)-mediated translation [19] or to promoter interference [20]. The HSV-TK protein contains several nuclear targeting signals and is usually located predominantly in the nucleus [21]. The enzyme may form a homodimer, and it has been proposed that only dimeric HSV-TK is transported to the nucleus [21]. On the other hand, in the commercially available firefly Luc we have used, the peroxisomal targeting sequence present in wild type Luc has been removed to ensure strict cytosolic compartmentalization, and thus reliable reporter function [17]. Fusing both enzyme moieties together resulted in a protein with predominant localization in the cytosol, as shown by the immunohistochemical analysis of HSV-TK-Luc expressing glioma cells (Figure 7B). In contrast, we have shown previously that fusion of the 27 kDa protein EGFP to HSV-TK allowed for predominant nuclear transfer of the enzyme while resulting in only minor loss of cytotoxic activity [22], suggesting that cytosolic localization and/ or reduced homodimer formation of the HSV-TK-Luc fusion protein may have some impact on its cytotoxic activity. A decrease in HSV-TK activity of up to 80% compared to unfused HSV-TK has also been observed by Ray et al. when fusing the enzyme to Renilla Luc [23]. N2A neuroblastoma cells harboring the fusion construct could be detected by PET and BLI in nude mice, but the cytotoxic activity of HSV-TK was not examined in the study. Renilla Luc activity in the above construct was found to be ~6 – 8-fold higher than seen with its unfused counterpart. As this enzyme is structurally unrelated to firefly Luc and has a lower molecular weight (36 kDa vs. 62 kDa), the study cannot be directly compared to our data. Notably, the authors mention that their attempt to fuse HSV-TK to firefly Luc resulted in a "poorly active" fusion protein [23]. Recently, the generation of several triple fusion proteins for imaging with different modalities was reported by two groups [24,25]. These triple reporters consisted of wild type or mutated HSV-TK, a fluorescent protein (EGFP, DsRed2, or monomeric red fluorescent protein (mRFP)) and firefly Luc [24,25] or Renilla Luc [25], respectively. Both groups showed that such triple fusion constructs could be used for simultaneous imaging in vivo with bioluminescence, fluorescence, and PET. Cytotoxicity generated by enzymatic conversion of prodrug by HSV-TK was however not measured in either of the above studies. Ponomarev et al. [24] did not present data on the correlated expression of the three reporters within the triple fusion protein (HSV-TK-EGFP-Luc), nor were the activity levels of the different fusion partners compared to those of their unfused counterparts. Despite these limitations, the study confirms that Luc remains functional if fused N-terminally to other proteins, and that enzymatic activity is sufficient for in vivo BLI. Ray et al. [25] compared the enzymatic and fluorescent activities of several triple fusion constructs transiently transfected into 293 cells to the respective activities of the unfused proteins. The described fusion proteins contained either Renillla or firefly Luc at the NH2-terminus, followed by a fluorescent protein and a mutated HSV-TK enzyme. This orientation of the fusion partners as well as the use of mutated HSV-TK optimized for use with PET limits the direct comparison of the presented data to our results. Bioluminescent activity of the 4 fusion constructs containing firefly Luc was reduced to 22 – 63% of the activity of unfused Luc, which is similar to our findings when expressing HSV-TK-Luc in U87MG glioma cells. One of the 4 constructs (Luc-mRFP-mutant HSV-TK) fully retained HSV-TK PET reporter activity while in the others HSV-TK activity (as assessed by intracellular radiotracer accumulation) was reduced to 30 – 61% of the activity of the corresponding unfused enzyme. This is in line with our findings when expressing HSV-TK-Luc transiently in U87MG glioma cells. Although it seems attractive to perform BLI with different luciferase enzymes, the following facts argue in favor of firefly Luc instead of Renilla Luc: (1) light emission of Renilla Luc peaks at 480 nm and thus shows only limited tissue penetration, (2) coelenterazine, the Renilla Luc substrate is prone to autoluminescence, resulting in high background if injected i.p. [26], (3) coelenterazine transport (and thus the bioluminescent signal) is modulated by the multidrug resistance MDR1 P-glycoprotein, a protein known to be overexpressed in cancer cells [27], and (4) coelenterazine is much more expensive than D-Luciferin. Iyer et al. [28] examined noninvasive imaging using PET and BLI in CD-1 mice after simultaneous i.v. delivery of the HSV-TK and Luc genes residing on different plasmids. The time point of peak activity of both reporters differed by ~19 hours, most likely due to differences in half lives of the two enzymes. This finding supports our approach of expressing both enzymes as one molecule as this should greatly diminish differences in protein stability. Attenuation of both enzymatic activities is most likely a result of steric hindrance and might be substantially reduced by selecting another linker sequence. Longer intervening sequences as well as introduction of flexible polyglycine linkers may contribute to an increase in enzyme activity [23,29]. Recently, De et al. [15] introduced into a lentiviral vector a mutant HSV-TK (optimized for PET imaging) and firefly Luc, separated by an IRES sequence. N2a neuroblastoma cells stably transduced with this construct and implanted s.c. into nude mice showed correlated expression of both enzymes as verified by PET and BLI (R2 = 0.86). In contrast to our study, GCV treatment in vivo resulted in a decrease in light emission while the tumors continuously grew in size. Most likely, this reflects the relatively poor cytotoxic activity of the mutant HSV-TK used in these experiments, implying that engineered HSV-TK optimized for use as a PET reporter may not retain its full cytotoxic activity when substrates such as GCV are used. Data on the cytotoxic potential of the virus construct in cell culture were not presented. Cytotoxic effects of the fusion construct We show here for the first time that a HSV-TK-Luc fusion protein in conjunction with GCV treatment can confer a curative effect on glioma bearing animals. While HSV-TK-Luc expressing glioma cells in culture were not killed completely when using GCV concentrations of up to 10 μg/ml, xenografts consisting of these cells were fully eliminated in all GCV treated mice. It has been shown by several groups that HSV-TK expressing tumor cells can elicit an antitumor immune reaction even in immunocompromised animals such as nude mice, most likely mediated by natural killer (NK) cells, activated in vivo by GCV induced cell killing [30,31]. We suggest that such an immune response may also have contributed to the elimination of HSV-TK-Luc expressing U87MG glioma cells in GCV-treated mice. This issue could be further addressed by in vivo depletion of NK cells through administration of appropriate antibodies [30]. Optical detection of transgene expression Our study used a subcutaneous glioma model for "proof of concept" to allow for simultaneous bioluminescence imaging and measurement of tumour size by caliper. HSV-TK-Luc expressing U87MG glioma cells were also detected by the CCD camera after inoculation of 2 × 106 cells intracerebrally in nude mice (data not shown), confirming the high sensitivity of BLI. Indeed, it has already been demonstrated in a murine orthotopic pituitary tumor model that bioluminescent light can travel through skull [32]. The cooled CCD camera system we used allows for quantification of emitted light. Some authors suggested that the level of transgene expression could be more reliably quantified by maximum light emission than by integrated light units within a ROI [33]. Although quantification is important if strategies for transgene delivery are to be examined, a systematic comparison of these two parameters in BLI has not been published until now. This prompted us to analyze these parameters in more detail in our study. If maximum and integrated light units within a ROI over single tumors were compared, we consistently found that both parameters tightly correlated with tumor size in tumors up to ~1 cm in diameter. In larger tumors (maximum diameter examined = 2 cm) the increase in size was in general less closely correlated with both integrated and maximum light units, but light emission from a ROI drawn over the entire tumor was still far more accurately mirroring tumor growth than maximum light units emitted from this region. With the increase in tumor thickness, maximum light emission from the tumor core is reduced due to necrosis, light scattering, and reduced supply of oxygen and D-Luciferin to tumor cells. On the other hand, the increase in tumor length and width is better reflected by light signals integrated over the entire tumor area, while a concomitant change in maximum light signal does not necessarily have to occur. Therefore, integrated light signals emitted from tumor ROIs seem to be the measure of choice for serial imaging of transgene expression in growing tumors. The fact that tumor volume and photon emission are less tightly correlated in large tumors as compared to smaller ones implies that photon emission reflects mainly the presence of viable tumor cells within a tumor and is a more precise measure for cytotoxic efficacy than tumor size, as has also been suggested by others [34,35]. Herpes simplex virus type I thymidine kinase has also been used as a reporter gene for monitoring therapeutic success with PET [11,36]. However, using optical imaging methods to quantify transgene expression has several advantages. The much greater sensitivity of BLI as compared to PET (10-15 – 10-17 vs. 10-11 – 10-12 mole/L of reporter probe are detectable, [13]) in combination with very low background signals render this imaging method particularly attractive for studying therapeutic strategies in animal models of cancer. In addition, high costs, the need for a radiopharmacy and considerable technical experience currently preclude widespread application of PET in experimental gene therapy of cancer. Conclusions We showed that therapeutic efficacy of a suicide gene/ prodrug activating system can be accurately monitored in vivo by BLI, when the bioluminescent reporter luciferase is fused in frame to the therapeutic gene HSV-TK. We used a clonal human glioma cell line stably expressing the HSV-TK-Luc fusion construct, thus guaranteeing high level transgene expression. Despite the somewhat attenuated activity of both fusion partners, a high degree of cytotoxicity by HSV-TK mediated GCV bioactivation as well as strong bioluminescent signals upon administration of D-luciferin were consistently demonstrated. In order to mirror more closely in vivo gene therapy of malignant brain tumors, experiments are underway to insert the HSV-TK-Luc fusion gene (and an improved version of it) into appropriate viral vectors and subsequently use them for treatment of orthotopically established gliomas in mice. Serial assessment of transduction levels, transgene localization and time course of fusion gene expression in living animals by BLI can aid in developing more potent gene therapy vectors for treatment of malignant glioma. List of abbreviations CCD, charged coupled device; CMV, cytomegalovirus; cps, counts per second; EGFP, enhanced green fluorescent protein; GCV, ganciclovir; HSV-TK, herpes simplex virus type 1 thymidine kinase; Luc, luciferase; mRFP, monomeric red fluorescent protein, PET, positron emission tomography; ROI, region of interest; SD, standard deviation, SEM, standard error of the mean. Competing interests None declared. Authors contributions AS constructed the plasmids, performed the animal experiments together with CT and set up the in vitro assays. CT and SJ participated in the cell culture experiments and enzymatic assays. SJ and AS carried out the immunohistochemical studies. NGR and AS designed the experiments and evaluated the data. All authors have read and approved the manuscript. Acknowledgements We thank Hans-Dieter Söling for continuous support and critical comments on the manuscript. The assistance of Roland Jacob in preparation of the manuscript and Cornelia Gottschalk and Martina Hennicke in animal care is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft (grant Ra 705/4-1 to NGR) and by the NBL3/ Wilhelm-Roux program (grant FKZ 2/36 to NGR and AS). ==== Refs Ram Z Culver KW Walbridge S Blaese RM Oldfield EH In situ retroviral-mediated gene transfer for the treatment of brain tumors in rats Cancer Res 1993 53 83 88 8380127 Izquierdo M Cortes M de Felipe P Martin V Diez-Guerra J Talavera A Perez-Higueras Long-term rat survival after malignant brain tumor regression by retroviral gene therapy Gene Ther 1995 2 66 69 7712334 Mizuno M Yoshida J Colosi P Kurtzman G Adeno-associated virus vector containing the herpes simplex virus thymidine kinase gene causes complete regression of intracerebrally implanted human gliomas in mice, in conjunction with ganciclovir administration Jpn J Cancer Res 1998 89 76 80 9510479 Ram Z Culver KW Oshiro EM Viola JJ DeVroom HL Otto E Long Z Chiang Y McGarrity GJ Muul LM Katz D Blaese RM Oldfield EH Therapy of malignant brain tumors by intratumoral implantation of retroviral vector-producing cells Nat Med 1997 3 1354 1361 9396605 Shand N Weber F Mariani L Bernstein M Gianella-Borradori A Long Z Sorensen AG Barbier N A phase 1 – 2 clinical trial of gene therapy for recurrent glioblastoma multiforme by tumor transduction with the herpes simplex thymidine kinase gene followed by ganciclovir. GLI 328 European-Canadian study group Hum Gene Ther 1999 10 2325 2335 10515452 10.1089/10430349950016979 Rainov NG A phase III clinical evaluation of herpes simplex virus type 1 thymidine kinase and ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with previously untreated glioblastoma multiforme Hum Gene Ther 2000 11 2389 2401 11096443 10.1089/104303400750038499 Harsh GR Deisboeck TS Louis DN Hilton J Colvin M Silver JS Qureshi NH Kracher J Finkelstein D Chiocca EA Hochberg FH Thymidine kinase activation of ganciclovir in recurrent malignant gliomas: a gene-marking and neuropathological study J Neurosurg 2000 92 804 811 10794295 Sandmair AM Loimas S Puranen P Immonen A Kossila M Puranen M Hurskainen H Tyynela K Turunen M Vanninen R Lehtolainen P Paljarvi L Johansson R Vapalahti M Yla-Herttuala S Thymidine kinase gene therapy for human malignant glioma, using replication-deficient retroviruses or adenoviruses Hum Gene Ther 2000 11 2197 2205 11084677 10.1089/104303400750035726 Rainov NG Kramm CM Aboody-Guterman K Chase M Ueki K Louis DN Harsh GR 4thChiocca A Breakefield XO Retrovirus-mediated gene therapy of experimental brain neoplasms using the herpes simplex virus-thymidine kinase/ ganciclovir paradigm Cancer Gene Ther 1996 3 99 106 8729908 Kruse CA Roper MD Kleinschmidt-DeMasters BK Banuelos SJ Smiley WR Robbins JM Burrows FJ Purified herpes simplex virus thymidine kinase retrovector™ particles. 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Ross BD Rehemtulla A Lawrence TS The potential of 5-fluorocytosine/cytosine deaminase enzyme prodrug gene therapy in an intrahepatic colon cancer model Gene Ther 2002 9 844 849 12080378 10.1038/sj.gt.3301706 Mendel DB Laird AD Xin X Louie SG Christensen JG Li G Schreck RE Abrams TJ Ngai TJ Lee LB Murray LJ Carver J Chan E Moss KG Haznedar JO Sukbuntherng J Blake RA Sun L Tang C Miller T Shirazian S McMahon G Cherrington JM In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/ pharmacodynamic relationship Clin Cancer Res 2003 9 327 337 12538485 Jacobs A Voges J Reszka R Lercher M Gossmann A Kracht L Kaestle C Wagner R Wienhard K Heiss WD Positron-emission tomography of vector-mediated gene expression in gene therapy for gliomas Lancet 2001 358 727 729 11551583 10.1016/S0140-6736(01)05904-9

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==== Front Genet Vaccines TherGenetic Vaccines and Therapy1479-0556BioMed Central London 1479-0556-2-81530168710.1186/1479-0556-2-8ResearchAttenuation of dengue virus infection by adeno-associated virus-mediated siRNA delivery Zhang Weidong [email protected] Rajeswari [email protected] Gary [email protected] Xiaoyuan [email protected] Homero San [email protected] Richard F [email protected] Shuen-Ju [email protected] Kevin [email protected] Shyam S [email protected] Division of Allergy and Immunology-JMC Airway Disease Research Center, Department of Internal Medicine, University of South Florida; VA Hospital Tampa, FL, USA2 Viral Diseases Department, Naval Medical Research Center, Silver Spring, Maryland, USA2004 9 8 2004 2 8 8 26 2 2004 9 8 2004 Copyright © 2004 Zhang et al; licensee BioMed Central Ltd.2004Zhang et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The need for safe and effective treatment of dengue virus (DEN), a class A agent that causes dengue hemorrhagic fever/dengue shock syndrome, has been a critical global priority. An effective vaccine for DEN is not yet available. In this study the possibility of attenuating DEN infection using adeno-associated virus (AAV)-encoded short interfering RNAs (siRNA) was examined in Vero cells and human dendritic cells (DCs). Methods A cassette encoding siRNA targeted to a 3' untranslated sequence common to all DEN serotypes was designed and tested for its ability to attenuate DEN infection by use of AAV delivery. Results Vero cells or DCs infected with AAV-siRNA showed a significant, dose-dependent reduction in DEN infection. Treatment of DCs with AAV-siRNA also decreased the DEN-induced apoptosis of DCs and did not induce significant inflammation. Conclusion These results demonstrate that AAV-mediated siRNA delivery is capable of reducing DEN infection in cells and may be useful in decreasing DEN replication in humans. dengue virussiRNAgene expressionadeno-associated virus ==== Body Introduction The need for a safe and effective prophylaxis or treatment for dengue virus (DEN) infection, a category A mosquito-borne human pathogen, is a critical global priority. DEN causes dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which is associated with heterologous secondary DEN infection and affects thousands of people worldwide. The incidence of DHF/DSS is increasing in the western hemisphere and, although many different approaches are being tried to develop prophylactic DEN vaccines, none have been licensed for public health and there are no specific antiviral treatments available. DEN belongs to the family of Flaviviruses and is an enveloped single plus-stranded RNA virus with four distinct serotypes. The DEN genome of approximately 11,000 nucleotides encodes a polyprotein (C-prM-E-NS1-NS2a-NS3-NS4a-NS4b-NS5) consisting of three structural proteins (C, prM and E) and seven nonstructural proteins. The open reading frame is flanked by a 100 nucleotide-long noncoding region (NCR) at the 5' end and a 400 to 600 nucleotide-long NCR at the 3'end [1]. Although the mechanism of DEN pathogenesis is unclear, DEN typically appears to replicate locally in skin or blood dendritic cells (DCs) and may also involve monocytes and macrophages. Secondary infection is usually more serious because of antibody-dependent enhancement (ADE). We reasoned that an effective antiviral approach aimed at attenuating the DEN virus burden might protect infected subjects from DHF/DSS and, therefore, we examined the potential of an in vivo gene-silencing approach using short interfering RNA (siRNA) to decrease DEN replication. RNA interference is triggered by dsRNA that is cleaved by an RNAse-III-like enzyme, Dicer, into 21–25 nucleotide fragments with characteristic 5' and 3' termini [2]. These siRNAs act as guides for a multi-protein complex, including a PAZ/PIWI domain containing the protein argonaute2, that cleaves the target mRNA [3]. These gene-silencing mechanisms are highly specific and can potentially inhibit the gene expression of different viruses [4,5]. This approach was found to be effective in blocking DEN replication in insect cells [6,7]. Plasmid DNAs or adenoviruses encoding appropriate DNA sequences allow transient siRNA expression in cells and in vivo leading to specific gene silencing. However, plasmids transfect mammalian cells poorly, and adenoviruses produce an acute inflammatory response and an immune response to viral vector-encoded antigens [7,8]. We therefore developed an adeno-associated virus (AAV) system capable of expressing siRNA cassettes, and tested this vector with a siRNA cassette composed of a nucleotide sequence from the 3' NCR of the DEN genome (siDEN3UT), which is common to all four serotypes. The results obtained in Vero cells and human DCs infected with AAV-siDEN3UT show significant decreases in DEN infection and DEN-induced apoptosis. Methods Plasmid constructs The pCMV-MCS plasmid (Stratagene) was digested with Not I and the larger fragment was ligated to the synthetic adapter containing in order, Not I-Kpn I-Apa I-Xho I-Hind III-EcoR I-Bam HI-Sac II-Sac I-Cla I-Sal I-Bgl II-Not I. The U6 promoter was obtained by PCR amplification, using specific primers with the desired restriction sites from the template pSilencer 1.0-U6 (Ambion), and inserted into the adaptor at the Kpn I and Apa I sites to get a novel plasmid pCMV-U6. Pairs of oligos were synthesized to develop siRNA constructs. The nucleotide sequence for each siRNA is as follows: siEGFP: 5'-GGC GAT GCC ACC TAC GGC AAG CTT CTC GAT TCG AAG CTT GCC GTA GGT GGC ATC GCC CTT TTT G-3' [10]; siRSVNS1:5'-GGC AGC AAT TCA TTG AGT ATG CTT CTC GAA ATA AGC ATA CTC AAT GAA TTG CTG CCT TTT TG-3'; siDENPrM: 5'-GGA AGA CAT AGA TTG TT G GTG CAC TCG AGT CAA CGT GCA CCA ACA ATC TAT GTC T TC CCT TTT TG-3'; siDEN3UT:5'-GGA AAA ACA GCA TAT TGA CGC TGC TCG AGT CAA CGC AGC GTC AAT ATG CTG TTT TTC CCT TTT TG-3'. Each pair of oligos was annealed and then inserted into pCMV-U6 digested with Apa I/Xho I and Xho I/EcoR I respectively. The modified pCMV-U6 plasmid was then redigested with Not I and the smaller fragment was ligated to the 2.9 kb fragment of pAAV-MCS (Stratagene) obtained following its Not I digestion to generate the corresponding si-vector for EGFP, RSV and DEN. HEK-293 cells were cotransfected with the helper plasmid and the si- plasmid to generate recombinant AAV. Cell culture and viral packaging HEK293 cells were cultured with DMEM (Cellgro) plus 10% FBS (Cellgro) and cotransfected with pSMWZ-siDEN, pHelper and pAAV-RC (Stratagene) by standard calcium phosphate transfection. Cells were harvested 48 hr post-transfection and the cell pellets were stored at -80°C. Purification of recombinant adenoassociated viruses Cells were lysed by 5 cycles of freezing and thawing to release the virus. Crude viral lysate were collected by centrifugation at 27,000 × g for 30 min, and the supernatants were harvested and put onto a CsCl gradient (density 1.20/1.50) in fresh tubes and centrifuged for 16 h at 100,000 × g. Opalescent bands were collected after ultracentrifugation. Titers of purified AAVs were measured using an AAV titration ELISA kit (Progen Biotechnik, Germany). Isolation and culture of dendritic cells from human peripheral blood Conditions were similar to those described previously [11]. Buffy coats were diluted with one volume of DMEM (Cellgro) and PBMCs were isolated by density-gradient centrifugation using Histopaque-1077 (SIGMA) according to the instructions. The PBMC layer was harvested, washed twice with DMEM, resuspended in DMEM supplemented with 10% FBS, and then seeded into six-well culture plates. After 2 h at 37°C/5%CO2 the nonadherent cells were removed and the adherent cells were cultured with fresh DMEM supplemented with 10% FBS (Cellgro), 200 ng/ml IL-4 (BD-Pharmingen) and 50 ng/ml GM-CSF (BD-Pharmingen) for 7 days prior to infection with DEN. Blocking dengue virus infection in vitro 1 × 105 Vero cells or DCs were seeded into six-well tissue culture plates and infected with different numbers of recombinant AAV carrying the DEN-siRNA silencing cassette. After 2 days the cells were infected with DEN-2 virus (strain16803) at an MOI of 0.1. Flow cytometry Cells were harvested and centrifuged for 10 min at 150 × g. The cell pellets were washed with PBS and resuspended with antibody to DEN-2 virus envelope protein, (Microbix Biosystems Inc, Clone No 3H5) on ice for 40 min. The cells were centrifuged and pellets were washed with PBS and then resuspended with secondary antibody conjugated with FITC (Sigma) for an additional 30 min. The number of infected cells was measured by flow cytometry 5 days post-infection. DCs were also stained with CD11c antibody conjugated with PE (BD-Pharmingen). Plaque assay The supernatants from DEN-2-infected DCs were collected at day 5 post-infection and 10-fold serial dilutions were allowed to adsorb to monolayers of Vero cells in six-well culture plates for 2 h. The medium was then removed and replaced by an agarose-containing overlay (2X DMEM, 10%FBS, non-essential amino acids (Gibco BRL), 1% low melting-point agarose (Gibco BRL) and the plates were incubated at 37°C/5% CO2 for 5 days. Afterwards, 1% neutral red (Sigma) was added to each well and the plaques were counted 48 h later. Apoptosis assay Infected-DCs were harvested on day 5 of infection with DEN-2 virus. Aliquots of DCs were put onto slides using a Cytospin and fixed with 4% paraformaldehyde. Apoptotic DCs were determined using the terminal dUTP nick end-labeling assay (TUNEL, Promega, Madison WI) and the annexin V apoptosis detection kit (BD Biosciences, CA). Cytometric bead array and ELISA analysis Supernatants from infected-DCs were harvested at 24 h, 48 h, 72 h and 96 h post-infection. Cytokine concentrations were measured by cytometric bead array (CBA) and ELISA (BD-Pharmingen) following instructions in the manuals. Statistical analysis Data were expressed as arithmetic mean ± SEM. Levels of significance of the differences between groups were determined by the student t test. Values of p < 0.05 were considered statistically significant. Results Development of an AAV-siRNA system for gene silencing In order to develop an AAV-siRNA system, a plasmid pSMWZ-1 was engineered that comprised a mouse U6 promoter linked to a siRNA cassette (Fig. 1A). To test whether this plasmid was functional and capable of suppressing gene expression, HEK293 cells were cotransfected with pEGFP, a plasmid expressing green fluorescent protein, and pSMWZ-siEGFP. The percentage of cells expressing EGFP was determined and the results showed that there was a dose-dependent silencing of EGFP expression (Fig. 1B). In contrast, control cells cotransfected with siRSVNS1 (targets the NS1 gene of human respiratory syncytial virus) in place of siEGFP did not show any reduction in EGFP expression. To test various siDEN candidates, Vero cells were transfected with either pSMWZ-siDENpreM (siDENpreM) or pSMWZ-siDEN3UT(siDEN3UT), then two days post-transfection infected with DEN-2 (strain 16803) at an MOI of 0.1. At five days post-infection, the numbers of DEN-2 virus-infected cells were quantified by fluorescence microscopy using antibody to DEN-2 envelope protein and FITC-conjugated secondary antibody. The results showed that siDEN3UT was better than siDENpreM in suppression of DEN-2 infection (Fig. 1C). The AAV-siRNA system was similarly tested using HEK293 cells which were infected with AAV-siEGFP and then transfected with pEGFP. The decrease in the percentage of cells expressing EGFP showed that there was a silencing of EGFP expression in a dose-dependent and sequence-specific manner (Fig. 1D). Figure 1 Construction and characterization of the siDEN suppressor. (A) Diagram of the construction of the plasmid vector, pSMWZ-1, capable of expressing a DEN infection suppressor cassette. Abbreviations: N*, Not I; K, Kpn I; A, Apa I; E, EcoR I; SUP-1, Suppressor cassette; (B) Co-transfection with pSMWZ-siEGFP and pEGFP inhibits the expression of EGFP in cultured cells. HEK293 cells were transfected with different concentrations of plasmid DNA and three days later, EGFP-positive cells were counted by fluorescence microscopy. Results are expressed as mean ± SEM. *p < 0.05 compared to control (siRSVNS1, an unrelated siRNA construct against respiratory syncytial virus). (C) pSMWZ-siDEN suppression of DEN-2 virus replication. Vero cells were transfected with pSMWZ-siDEN3UT or pSMWZ-siDENpreM plasmid. Two days later, the cells were infected with DEN-2 virus (MOI of 0.1) and 5 days later, the numbers of DEN-2 virus infected cells were counted by fluorescence microscopy. Data are mean ± SEM. *p < 0.05 compared to control DEN-2. (D) AAV-siEGFP inhibits the expression of EGFP in cultured cells. HEK293 cells were infected with different concentrations of AAV-siEGFP, and three days later the cells were transfected with pEGFP. EGFP-positive cells were counted by fluorescence microscopy. Statistically significant differences, **p < 0.01, when compared to pEGFP plasmid control, AAV-siRSV (107) and AAV-siEGFP (108) group, respectively. To test whether AAV-siDEN-2 expression decreases DEN-2 virus infection in cultured Vero cells, cells were infected with recombinant AAV carrying siDEN-2 (MOI 10) or siEGFP (MOI ~1000) silencing cassettes. After 2 days the cells were infected with DEN-2 virus at an MOI of 0.1. Five days later, the numbers of DEN-2 virus infected cells were quantified by flow cytometry using anti-DEN-envelope protein. Cells pre-infected with AAV-siDEN3UT, but not AAV-siEGFP, showed a significant reduction in DEN infection, and the reduction was dose dependent (Fig. 2). Figure 2 AAVsiDEN expression decreases DEN-2 virus infection in cultured Vero cells. Cells were infected with different amounts (PFU/ml) of AAV carrying the siDEN3UT silencing cassette and after 2 days the cells were infected with DEN-2 virus (MOI of 0.1). Five days later, the numbers of DEN-2 virus infected cells were measured by flow cytometry. siRNA suppresses DEN infection in human dendritic cells DEN is transmitted through Aedes aegypti mosquito bites, and resident skin DCs are regarded as the targets of DEN infection [12]. DCs are thought to be 10-fold more permissive for DEN infection than monocytes or macrophages [13]. We therefore tested the ability of AAV-siDEN3UT to attenuate DEN infection in human DCs. DCs were isolated from human blood and cultured in the presence of IL-4 and GM-CSF for 5 days to generate immature DCs (iDCs). These DCs were then infected with 109 PFU/ml of recombinant AAV carrying siDEN3UT or siEGFP (control) silencing cassette. After 2 days the cells were infected with DEN-2 at an MOI of 0.1. Five days later, the numbers of DEN-2 infected cells were quantified by flow cytometry using DEN-2 antibody. Cells preinfected with AAVsiDEN3UT showed a 50% reduction in the number of infected cells (Fig. 3A). To test whether the reduction in the number of infected DCs involved a reduction in DEN titer, the culture supernatants were examined using a Vero cell-based plaque assay. AAV-mediated siDEN3UT expression significantly decreased DEN-2 virus titer compared to control (Fig. 3B). These results indicate that AAV-siDEN3UT can significantly decrease DEN titers in human DCs. Figure 3 Suppression of DEN-2 replication in DCs by siDEN. (A) DCs were isolated from human peripheral blood and cultured in DMEM medium supplemented with FBS, IL-4 and GM-CSF. Non-adherent DCs were harvested on day 7, infected with AAVsiDEN, and two days later the cells were infected with DEN-2 at 0.1 MOI. DCs were harvested 5 days after DEN-2 infection and DEN-2 titers were measured by flow cytometry. (B) Supernatants from DEN-infected DCs were collected and added to culture plates containing confluent Vero cell monolayers. After virus adsorption, the Vero cells were overlaid with agarose and stained with 1% neutral red. Viral plaques were counted 48 h after neutral red overlay. Data are the averages of two independent experiments. *p < 0.05 compared to control. Silencing DEN-2 genes inhibits apoptosis in dendritic cells It has been reported that DEN infection induces apoptosis of DCs [11] leading to an immunosuppressed condition. To examine the effect of AAV-mediated siRNA delivery in DCs, apoptosis was investigated in infected DCs using the TUNEL assay. The results showed that a small percentage of DCs undergo apoptosis naturally during culture, but DEN infection causes much more apoptosis. The AAV-siRNA-treated DEN-infected DCs showed significantly fewer apoptotic cells compared to DEN-infected cells without AAV-siRNA (Fig. 4). Figure 4 AAVsiDEN reduces apoptosis in human DCs infected with DEN-2. DCs were isolated from human peripheral blood and infected with AAVsiDEN followed by DEN-2. Five days after infection, DCs were put onto slides and apoptosis was determined using the terminal dUTP nick end-labeling assay (TUNEL). Nuclei were stained with diamidinophenylindole (DAPI). Representative fields were visualized by fluorescence microscopy. Differential expression of cytokines by infected dendritic cells The supernatants of infected DCs were collected at different time points and cytokines were measured using cytokine bead array (CBA) and ELISA assays. As showed in Figure 5, cultured DCs spontaneously produced increased IL-1b. A variety of cytokines including IFN-γ, TNF-α, IL-8, IL-6, IL-12 were measured (data not shown). In the presence of AAV-siRNA infection, the production of IFN-γ, TNF-α, IL-8, IL-6, and IL-12 did not change significantly compared with cultured DCs. IL-1b secretion at 72 h post-infection was increased, however. These results indicate that in our system, AAV-siRNA delivery does not induce acute inflammation in DCs, in vitro, Figure 5 Differential expression of cytokines in the supernatants of infected DCs. Supernatants from DEN-2-infected DCs with or without siDEN treatment were harvested at the indicated time points and analyzed by CBA and ELISA to measure the concentrations of cytokines. Data are the averages of two independent experiments. **p < 0.01 in comparison with the value of DCs within individual group. Discussion The significant findings of this report include the development of an adeno-associated virus-based siRNA approach for downregulating gene expression. A recombinant AAV-siDEN3UT was utilized to induce significant decreases in DEN infection compared to control in both Vero cells and human DCs. The results indicate that siRNAs may be used to attenuate DEN infection in human DCs and may have therapeutic value. Interference of gene expression by siRNAs is a novel strategy to knock down specific genes in cells or tissues, and the specific silencing of pathogen genes using siRNA is a very attractive approach for the clinical treatment of infectious diseases. Long dsRNAs (of >30 nt in length) activate a dsRNA-dependent protein kinase and 2', 5'-oligoadenylate synthetase in mammalian cells, which leads to a non-specific reduction in levels of mRNAs [14]. The endogenous expression of siRNAs from introduced DNA templates is thought to overcome some limitations of exogenous siRNA delivery, in particular its transient effects on silencing specific genes and loss of phenotype [15]. AAV vectors have been proven to be safe and efficacious in Phase I clinical trials for gene therapy of cystic fibrosis and hemophilia B and are regarded as a potential alternative to retroviral and adenoviral vectors for gene therapy in humans. The AAV vectors have a number of advantages over other vectors. They are not pathogenic and do not induce production of neutralizing antibodies that could reduce transgene function. They possess a broad-range of tissue tropism and the capability of inducing long-term transgene expression [16]. In this study, we utilized a novel AAV system to deliver DEN siRNA into mammalian cells and estimated its anti-DEN effect in vitro. In this AAV system, we incorporated the mouse U6 promoter, which is important for transcription and folding of the suppressor RNA, into a plasmid pCMV-U6. The choice of appropriate target genes is necessary for the success of the siRNA strategy, and two siRNAs derived from either the pre-M or the 3' NCR region of DEN-2 were used in our study. An internal deletion of 3' NCR nucleotide sequences was found to be lethal for DEN virus replication in an in vivo study [17]. The 3' NCR of the flavivirus genome, which presumably functions as a promoter for minus-strand RNA synthesis, is predicted to form a stem-and-loop secondary structure. Computational analyses have revealed that there is conserved sequence in all flaviviruses within the 3' end [18,19]. Thus, two siRNA cassettes were tested in this study that included the 3'NCR sequence common to all four DEN serotypes. The other siRNA cassette is from the gene encoding the preM protein which is important for maturation of the virus into an infectious form. Our test of anti-DEN efficiency showed that siDEN3UT attenuated DEN Infection better than siDENpreM. Knocking down viral genes at the earlier stage of the viral multiplication cycle rather than in the structural protein synthesis phase may provide better antiviral protection, although the limited plasmid transfection ratio appeared to influence the suppression efficiency of siDEN to DEN-2 infection in Vero cells in the present study (Fig. 1C). The other DEN serotypes will be investigated with our 3'NCR cassette. DEN is transmitted through Aedes aegypti mosquito bite, and resident skin DCs are an early target of DEN infection [12]. Immature DCs are the most permissive for DEN infection and serve as a source of DEN replication and production [20]. Replication in the early target cells may be essential for dengue pathogenesis in the human host. In this study, we also utilized peripheral blood iDCs as a cell model to test our AAV system. Similar to results in Vero cells, AAV-mediated siDEN3UT delivery down-regulated DEN-2 protein expression in iDCs. However, the magnitude of suppression in iDCs at the same infectious titer of AAV-siDEN was less compared to that found in Vero cells. Previous data showed that variations in the efficiency of transduction among DCs derived from different normal blood donors is between 2% and 50% [21], and we found that the infectious ratio for AAVEGFP is about 45%~50% in Vero cells. That may be due to limited expression of the AAV receptor or differential activation of the mouse U6 promoter in Vero cells compared to DCs [22]. Increasing the AAV infection titer or utilizing a more effective promoter within the AAV vector backbone might elevate the suppression for DEN replication in iDCs. Nevertheless, DCs treated with recombinant AAV showed a significant reduction in DEN virus titer compared to control. This is important as viral titer is the gold standard for measuring antiviral activity. DCs are one of the most powerful of APCs. After infection with virus in the periphery, iDCs process viral antigens, then differentiate into mature DCs and migrate from peripheral tissues to lymph nodes where they prime naïve CD4 and CD8 T lymphocytes to maintain protective antiviral cytotoxic T cell memory [23,24]. Thus, DCs play an important role in the initiation of antiviral immunity and provide a crucial step in the development of adaptive antiviral immunity. Previous data showed that DEN infection induces apoptosis of DCs [11], which leads to a state of temporary immune-suppression during DEN fever. An important observation in our study is that AAV-siDEN treatment resulted in a significant decrease in apoptotic iDCs. The attenuation of apoptosis in iDCs following AAV-mediated siRNA delivery suggests that AAV-siRNA may be immunologically protective. After the primary DEN infection, most patients appear viremic in the early febrile phase, but the viruses are quickly cleared from the blood system after defervescence [25]. The activation of both a humoral and cellular immune response is considered to be involved in DEN clearance. The most severe outcome in DEN infection is development of DHF/DSS, which is associated with secondary infections by heterotypic DEN serotypes. It is postulated that the preexisting, cross-reactive, adaptive immune response leads to excessive cytokine production, complement activation, and the release of other inflammatory factors that produce DHF/DSS [20]. Therefore, it should be imperative for prophylaxis of DHF/DSS to eliminate DEN infection by different serotypes in the early target cells. Attenuation of DEN infection in DCs and protection of infected DCs from apoptosis would be a benefit for the elimination of the early DEN infection and the development and maintenance of antiviral innate/adaptive immune response in vivo. One of the important features of AAV vectors is the lack of inflammation following infection. We failed to detect significant IFNγ or IL-12 production in the supernatants of AAV-siDEN-infected DCs. This is in accordance with previous data [26-28], which demonstrated our AAV delivery system did not induce significant acute inflammatory responses and, therefore, is useful in gene therapy for DEN infection in humans. In conclusion, we developed a novel AAV-mediated siRNA delivery system. Our results demonstrate significant downregulation of DEN protein expression in Vero cells and human DCs, which strongly suggest that our AAV vector can be useful for siRNA delivery and that this AAV system may be applied in clinical settings to attenuate DEN infection. List of abbreviations AAV, adeno-associated virus; DCs, dendritic cells; DEN, dengue virus; DHF/DSS, dengue hemorrhagic fever/dengue shock syndrome; MOI, multiplicity of infection; pEGFP, enhanced green fluorescent protein; siRNA, small interfering RNA; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling. Competing interests None declared. Authors' contributions WDZ constructed the si-plasmids, performed cell culture, isolated and administered the virus and isolated and cultured dendritic cells. RS assisted with virus handling and cell culture, and performed flow cytometry. GH did TUNEL assays. XK did cytometric bead array assays and ELISAs. HSJ measured virus titer by plaque assay. RFL, SW, KP and SSM designed and implemented the experiments, performed troubleshooting, and did the analysis and interpretation of the data. Acknowledgements This study was supported by grants from the VA Merit Review Award to SSM and the Joy McCann Culverhouse Endowment of the Division of Allergy and Immunology. ==== Refs Lindenbach B Rich CM Knipe DM, Howley PM Flaviviridae: Fields Virology 2000 1 4 Wolters Kluwer Co., Lippincott Williams & Wilkins 991 1003 Provost P Dishart D Doucet J Frendewey D Samuelsson B Radmark O Ribonuclease activity and RNA binding of recombinant human Dicer EMBO J 2002 21 5864 5874 12411504 10.1093/emboj/cdf578 Hammond SM Boettcher S Caudy AA Kobayashi R Hannon GJ Argonaute2, a link between genetic and biochemical analyses of RNAi Science 2001 293 1146 1150 11498593 10.1126/science.1064023 Fire A RNA-triggered gene silencing Trends Genet 1999 15 358 363 10461204 10.1016/S0168-9525(99)01818-1 Blair CD Adelman ZN Olson KE Molecular strategies for interrupting arthropod-borne virus transmission by mosquitoes Clin Microbiol Rev 2000 13 651 661 11023962 10.1128/CMR.13.4.651-661.2000 Adelman ZN Blair CD Carlson JO Beaty BJ Olson KE Sindbis virus-induced silencing of dengue viruses in mosquitoes Insect Mol Biol 2001 10 265 273 11437918 10.1046/j.1365-2583.2001.00267.x Caplen NJ Zheng Z Falgout B Morgan RA Inhibition of viral gene expression and replication in mosquito cells by dsRNA-triggered RNA interference Mol Ther 2002 6 243 251 12161191 10.1006/mthe.2002.0652 Kumar M Behera AK Matsuse H Lockey RF Mohapatra SS Intranasal IFN-gamma transfer of prevents respiratory syncytial virus infection in BALB/c mice Vaccine 1999 18 558 567 10519947 10.1016/S0264-410X(99)00185-1 Donnelly JJ Ulmer JB Liu MA DNA vaccines Dev Biol Stand 1998 95 43 53 9855413 Sui G Soohoo C Affar el B Gay F Shi Y Forrester WC Shi Y A DNA vector-based RNAi technology to suppress gene expression in mammalian cells Proc Natl Acad Sci USA 2002 99 5515 5520 11960009 10.1073/pnas.082117599 Ho LJ Wang JJ Shaio MF Kao CL Chang DM Han SW Lai JH Infection of human dendritic cells by dengue virus causes cell maturation and 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J Infect Dis 1997 176 322 330 9237696 Bueler H Adeno-associated viral vectors for gene transfer and gene therapy Biol Chem 1999 380 613 622 10430026 Kay MA Glorioso JC Naldini L Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics Nature Med 2001 7 33 40 11135613 10.1038/83324 Zaiss A Liu Q Bowen GP Wong NC Bartlett JS Muruve DA Differential activation of innate immune responses by adenovirus and adeno-associated virus vectors J Virol 2002 76 4580 4590 11932423 10.1128/JVI.76.9.4580-4590.2002

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==== Front Int J Behav Nutr Phys ActThe International Journal of Behavioral Nutrition and Physical Activity1479-5868BioMed Central London 1479-5868-1-111527967410.1186/1479-5868-1-11Debate"Is there nothing more practical than a good theory?": Why innovations and advances in health behavior change will arise if interventions are used to test and refine theory Rothman Alexander J [email protected] Department of Psychology University of Minnesota Minneapolis, MN USA2004 27 7 2004 1 11 11 22 6 2004 27 7 2004 Copyright © 2004 Rothman; licensee BioMed Central Ltd.2004Rothman; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Theoretical and practical innovations are needed if we are to advance efforts to persuade and enable people to make healthy changes in their behavior. In this paper, I propose that progress in our understanding of and ability to promote health behavior change depends upon greater interdependence in the research activities undertaken by basic and applied behavioral scientists. In particular, both theorists and interventionists need to treat a theory as a dynamic entity whose form and value rests upon it being rigorously applied, tested and refined in both the laboratory and the field. To this end, greater advantage needs to be taken of the opportunities that interventions afford for theory-testing and, moreover, the data generated by these activities need to stimulate and inform efforts to revise, refine, or reject theoretical principles. ==== Body Background Even with the dramatic advances in our understanding of the biological processes that determine health and illness, it has never been more clear that rates of disease morbidity and premature mortality reflect people's behavioral practices. [1] The benefits, both for individuals and the societies in which they live, that would come from systematic improvements in diet, physical activity, and use of substances such as tobacco, alcohol, and illicit drugs are tantalizing and provide ample motivation to develop initiatives to elicit changes in health behavior. Yet, health behavior change has proven a worthy adversary. Despite the commitment of considerable time and effort, innovations and advances in our ability to improve health behaviors have been modest. In particular, the specification of methods that produce sustained improvements in behavior have been elusive [2-5]. At the same time, innovations in theories of health behavior have also been modest. Investigators continue to advocate for a broad range of theories and there has been limited progress in demonstrating the unique value of any specific theory. [6-8] Although there may be consensus in the professional community that there are considerable gaps in our understanding of health behavior change, critiques of the current state of affairs more often that not reflect the professional interests of the critic. Investigators who strive to specify the structural and psychological processes that regulate people's behavior lament the fact that too many interventions are not guided by a theoretical framework that specifies how they are supposed to elicit health behavior change. At the same time, investigators who design and implement health behavior interventions lament that the preponderance of theories of health behavior make it difficult to discern what factors are likely to be the most effective targets for intervention. Moreover, it is argued that theories are not sufficiently specified to determine when or how to modify factors that are to be targeted in an intervention. Of course, concerns regarding the link between theory and practice are not new and efforts to address this problem have taken several forms. Considerable effort has been given to provide practitioners with a comprehensive and concise understanding of the array of theories that have been developed to address health behavior. [9] Moreover, conceptual frameworks such as PRECEDE-PROCEED [10] and Intervention Mapping [11] have been developed to provide investigators with a structured process to improve the accuracy and ease with which theoretical concepts are used to address a practical problem. In both cases, these efforts have targeted improving how theoretical principles are applied and, in doing so, have relied on the assumption that current theories of health behavior are useful and productive. Is this assumption valid? Could the often repeated plea for investigators to ground their intervention efforts in theory be a sign that there are significant limitations to the practical principles that can be derived from current theories of health behavior? If so, merely improving how people use theories will not be sufficient. What is needed is a shift in how we engage the interplay between theory and practice, with an emphasis placed on developing initiatives that target opportunities to develop, test, refine health behavior theory. In this paper, I describe and advocate for a model of collaboration between basic and applied behavioral scientists. Although I recognize the value of improving the manner in which theoretical principles are matched to problems and methods, I propose that innovations in our understanding of and ability to promote health behavior change will not arise if theory is construed as a fixed entity that is delivered to interventionists for implementation. To date, although theories may fluctuate in their popularity, their properties have remained strikingly static over time. I believe greater attention must be paid to refining and, when necessary, rejecting theoretical principles. For this process to take shape, there needs to be an on-going series of exchanges between theorists and interventionists in which theory is treated as a dynamic entity whose value depends on it being not only applied and tested rigorously, but also refined based on the findings afforded by those tests. A fundamental implication of this perspective is that improvements in both health behavior theory and intervention methods depend on each other. If investigators are more receptive to the opportunities interventions afford for theory testing, there will be a dramatic increase in data that can reveal the adequacies and inadequacies of a given theory. These data will, in turn, enable theorists to improve the quality of the theoretical models available to guide subsequent intervention efforts. Discussion When is an intervention effective? Interventions are designed to address important practical problems (e.g., obesity) and thus their value is inextricably linked to their ability to alleviate the targeted problem. Interventions need to provide a meaningful return on the time, money, and effort invested such that the outcomes afforded by a intervention strategy are proportional to the resources utilized. Of course, determining what is a sufficient return on an investment can be a challenge. Small effects may be impressive if the intervention is directed at a construct or behavior that is considered difficult to move. [12] In addition, interventions can have minimal impact on an individual's behavior but when disseminated widely have a dramatic impact at the societal level. [13] What conditions are likely to facilitate a successful intervention? Broadly speaking, an intervention is most likely to be effective if it is appropriately grounded in the practical problem targeted. [11] For example, consider an intervention to promote healthy food choices. The intervention design team must possess a clear understanding of who is engaging in the targeted behavior (e.g., who is making unhealthy food choices), the underlying nature of the behavior (e.g., the frequency and function of food choices), and the context in which the behavior is performed (e.g., where and with whom do people make choices about food). In a similar manner, the intervention needs to be appropriately grounded in the biological, structural and psychological processes that shape and regulate people's behavioral practices. [14-16] For example, the expected value of altering a feature of the environment in which people make food choices (e.g., increasing the cost of high-fat foods) is predicated on the assumption that the intervention will directly, or indirectly through an intervening construct, influence people's food choice in that setting. Health behavior theories provide an explicit statement of the structural and psychological processes that are hypothesized to regulate behavior (e.g., increasing the cost of high-fat foods will curtail consumption of these foods by making it more aversive or, perhaps, more difficult to purchase them). If theories describe the factors that guide people's behavior and justify how an intervention is designed and implemented, interventionists depend on the quality and predictive value of a theory. What determines a theory's value? From the perspective of a theoretician, a theory's value rests on its ability to provide an accurate account of the factors that regulate people's behavior. [17] Although investigators may recognize that behavior is affected by factors at different levels of analysis (i.e., biological, psychological, social, environmental), a theory's value is not necessarily predicated on its ability to provide linkages across these levels. Because of this emphasis, theory testing tends to occur in controlled contexts, typically a laboratory setting, that afford the social and behavioral version of a Petrie dish. This approach allows investigators to observe the relation between a given set of constructs with greater precision, but it renders the generalizability and strength of the observed effect difficult to discern. For example, investigators may determine that focusing people's attention on the undesirable aspects of an object increases their interest in avoiding it, but be unable to specify the conditions under which this relation is and is not most likely to obtain. From the perspective of an interventionist, the accuracy of the relations specified in a theory is an important but not sufficient determinant of its value. Interventionists need theories that are accurate and applicable; that specify not only the relation between two constructs, but also whether that relation does or does not change across contexts (e.g., does the impact of risk perceptions on behavior differ whether one is examining decisions to test for radon or to start smoking?). Given a set of a factors hypothesized to regulate people's behavior, interventionists need to be able to discern which of these factors are the most appropriate targets for intervention. In fact, a common complaint regarding theories is that they are not useful (See Jeffery, this issue). A theory may specify a host of factors that regulate a person's behavior, but in the absence of information regarding the relative importance of each factor leave an interventionist unsure as to where to direct her or his resources. For example, the Theory of Planned Behavior [18] and Theory of Reasoned Action [19] propose that people's attitudes toward the behavior and their perceived subjective norm regarding the behavior are critical determinants of behavior (albeit mediated by behavioral intention), but the relative contribution of these constructs is allowed to fluctuate from setting to setting. In any given context, it is unclear how to determine a priori which set of constructs should be prioritized as a target for intervention. The interest interventionists have shown in stage-based models of health behavior may reflect the fact that the models attempt to specify the conditions under which specific constructs affect behavioral decisions. [8] Little guidance is also given as to how or even whether critical constructs can be manipulated. For example, my colleagues and I have proposed that satisfaction with the outcomes afforded by a pattern of behavior is a critical determinant of behavioral maintenance. [20,21] Claims such as this are typically predicated on evidence that measures of a construct, in this case satisfaction, uniquely predict a behavioral outcome. Yet, the observation that someone who is satisfied is more likely to sustain a pattern of behavior does not indicate what causes someone to be satisfied and, thus, little guidance is given as to what can be done to heighten the satisfaction people derive from changes in their behavior. In the absence of this type of information, interventionists may find little difference between developing intervention strategies that are or are not grounded in a health behavior theory. In fact, given these practical needs, it is not be surprising that interventionists are more likely to rely on health behavior theories (e.g., Social Cognitive Theory [22]) that specify the determinants of its primary constructs and thus provide guidance as to how to construct an intervention protocol. Breakdown in the evolution of health behavior theories If the design and implementation of intervention strategies rely on assumptions regarding the factors that regulate people's behavior, why haven't current theories of health behavior evolved in ways that would enable them to more effectively guide intervention development? I believe the critical problem is that there has been a breakdown in the relation between basic and applied scientists who study health behavior. [23] As scholars such as Kurt Lewin [24] have asserted, the development and specification of theories of human behavior depend upon an iterative series of research activities in which theoretical principles initially formulated by basic behavioral scientists are tested and evaluated by applied behavior scientists. These tests provide critical information that enables basic scientists to revise, refine, or reject their initial principles. Moreover, an applied setting can afford investigators the opportunity to assess the relative impact of different processes hypothesized to regulate people's behavior. It is through this on-going cycle of specification, application, and evaluation that accurate and applicable theoretical models arise. To the extent that behavioral theories are not tested in complex social settings such as those afforded by interventions to change health practices, the process by which theories develop is curtailed. Because the manner in which a theory is specified reflects, in part, the contexts in which it has been operationalized and tested, theories that are tested primarily in tightly controlled laboratory settings will likely be characterized by a rich description of the myriad of factors that could affect people's behavioral choices. The laboratory setting allows investigators to minimize noise and potential confounding or moderating factors and thus optimizes their ability to detect processes that can affect people's behavior without determining whether, in a more complex setting, they do affect behavior. [17] Thus, in the absence of initiatives that empirically test theoretical principles in complex social environments, investigators run the risk of developing a "hot house" theory of health behavior that has limited practical value. Interventions afford an invaluable opportunity to discern the context dependence of causal relations that have been revealed in the laboratory. Some factors may be shown to always be critical, whereas others may be critical only under certain conditions. [25] For example, self-efficacy may be a critical determinant of the decision to initiate a new pattern of behavior, but have a limited impact on the decision to maintain that behavior over time. [21] It is critical to understand that restricting the conditions under which a construct affects behavior does not mean that a given factor is not important. Information that would help delimit these conditions would enable theorists to develop more precise models. The case for why interventions should be more receptive to theory There are two sets of reasons why we must take better advantage of the opportunities interventions provide to implement and test theories of health behavior. One set focuses on what theory can do to improve the implementation and evaluation of an intervention, whereas the other set focuses on how interventions can be used to improve the accuracy and quality of prevailing health behavior theories. First, by grounding their work on theoretical principles regarding processes that regulate people's behavior, investigators can readily specify the critical assumptions that underlie their intervention protocol. These formal statements of cause and effect relations not only provide a clear justification for the proposed research activities (i.e., why an investigator believes a given intervention strategy will be effective), but also increase the likelihood that the proposed methodology will allow the investigator to detect whether and why the intervention had its intended effect. [10,11] When faced with unambiguous evidence of a successful intervention effect, investigators might be able to move forward without knowing why the intervention was effective. However, more often than not, investigators are faced with the task of determining why an intervention failed to produced the desired effect or why it worked under a limited set of conditions. An a priori set of theoretical principles can provide an important conceptual and analytic framework for determining why an intervention was ineffective. In particular, it increases the likelihood that investigators have not only identified the constructs that may determine whether an intervention will prove effective, but also assessed them at the appropriate points in the decision process. The second set of reasons why interventions should take advantage of opportunities to test theories of health behavior is that by providing a context in which some or all of the facets of a theory can be tested, interventionists are in a position to generate evidence that will enhance the accuracy and applicability of theory and thus, over time, improve the quality of the theories to which interventionists can turn. By systematically testing principles specified in health behavior theories, investigators are able to not only verify the accuracy of these predictions, but also develop a better understanding of their practical value. Across studies, evidence should accumulate that will allow investigators to differentiate between factors that should and should not be targeted for intervention. Because current theories of health behavior often provide a list of factors that may affect behavior, the set of potential mediating variables suggested by a theory may pose a daunting if not untenable measurement burden. However, the implementation of consistent and methodologically sound assessment of these factors should provide the empirical evidence needed to constrain and prioritize the variables on that list. The characteristics of intervention strategies that prove to be effective should also provide investigators with a better understanding of the determinants of a given construct. As was previously mentioned, theories may propose that a construct (e.g., satisfaction) is a critical determinant of decisions to maintain a new pattern of behavior, but provide limited guidance as to how to alter people's standing on that construct (e.g., how to help feel satisfied with the outcomes afforded by their new behavior). [21] An intervention protocol that is shown to successfully heighten people's satisfaction with process and outcomes associated with weight loss not only has clear practical value, but also can shed light on the process by which people determine whether they are satisfied with their experiences. If theorists can develop a more detailed account of the processes that shape the primary constructs identified in a health behavior theory, interventionists will find that theories can provide a more useful set of guidelines for how to develop strategies to target these constructs. Testing theoretical principles across a diverse array of settings and populations will also enable investigators to better specify the scope of a theory. Although interventions provide a wonderful opportunity to test theoretical principles in diverse samples and settings, formal and appropriately powered tests of moderators can put a considerable strain on sample size and resources. However, if investigators have appropriately assessed the critical constructs, systematic comparisons can be drawn across studies that taken together have tested a theoretical principle across a range of settings or people. The increase in public access to data sets should facilitate opportunities for this type of comparisons. With the information that is gleaned from these types of activities, it should be easier to determine which moderators are worth testing in a single, appropriately powered study design. The identification of situational or personal factors that moderate the impact of a theoretical principal can be indicative of a number of different scenarios. For example, what might one conclude if an intervention that promoted the health benefits of eating a balanced diet altered the eating habits of college students but not those of high school students? It could indicate that health benefits do not affect what high school students choose to eat. Alternatively, it might be that high school students are responsive to perceptions of the health benefits afforded by a balanced diet, but that other factors (e.g., control over access to food) preclude them from acting on those beliefs. The practical and theoretical conclusions that can be drawn from the identification of moderating factors are dramatically increased if investigators can identify the causal processes that underlie the observed impact of the moderator. In particular, can investigators discern whether the moderated effect was obtained because the moderator altered the ability of the intervention strategy to change the proposed mediating construct (e.g., the intervention raised perceptions of the health benefits held by college but not high school students) or because it altered the effect the mediator has on the primary outcome measure (e.g., perceived health benefits predicted the eating habits of college but not high school students)? Greater attention to the causal processes invoked by a moderator may also help investigators grapple with the daunting number of potential moderators. It is quite possible that moderators that differ at the level of description (e.g., gender, ethnicity) can be accounted for by the same underlying process. Finally, it is important to recognize that progress in theory development can arise from the failure to obtain evidence in support of a specific prediction. Empirical evidence that provides investigators with a better sense of the potential factors that do not affect health practices will allow them to reduce the number of constructs (and, in time, theories) invoked to predict and explain health behavior. What can be done to make interventions more theory-friendly? If one assumes that there is interest in rendering interventions more receptive to theory-testing, what can be done to enhance an intervention's ability to assess principles derived from current health behavior theories? One issue is the appropriate evaluation of the critical manipulation(s) imbedded in the intervention. Any conclusions that can be drawn from the intervention, regardless of whether it reveals the predicted pattern of results, is predicated on the success with which the independent variable was manipulated. To this end, investigators need to at least consider assessing several constructs: the degree to which the intervention was implemented (e.g., did the interventionists consistently provide participants with the intervention exercises?), the degree to which participants correctly identified the emphasis of the intervention (e.g., did participants assigned to the optimistic outcome condition report their was a greater emphasis on favorable outcomes than did those assigned to the control condition?), and finally the degree to which the intervention altered the targeted set of opportunities, thoughts or feelings (e.g., did those assigned to the optimistic outcome condition develop more favorable expectations regarding the benefits afforded by behavior change than did those assigned to the control condition?). Although it is important that interventionists explicitly specify the constructs that determine the influence of the intervention on participant behavior, the quality of the evidence that can be gathered depends on the assessment procedures that are utilized. The persuasiveness of any claims regarding the importance (or lack of importance) of a particular construct is contingent on the use of measures that have been shown to be reliable and valid. Given that many of the constructs specified in theories of health behavior are conceptually similar, it is difficult to draw strong conclusions regarding the specific contributions of different variables in the absence of well-designed measures. [26,27] In addition, the inclusion of a pool of potential mediators enables the investigators to make stronger claims as he or she can demonstrate that not only does the construct specified in the model serve as a mediator but that other factors do not operate as mediators. Adequately testing basic principles also depends on a well-timed assessment schedule. Assessments are often too infrequent to detect meaningful changes on the construct. This is particularly true if the constructs of interest are psychological states that both affect and are affected by behavioral practices. However, specifying the optimal time to assess the primary constructs can be difficult. To the extent that one wants to determine whether an intervention strategy (e.g., a tailored message about dietary changes) alters the predicted mediating variable (e.g., willingness to modify one's diet), one might consider minimizing the length of time between the delivery of the intervention and the assessment of the mediator. However, at the same time, interest in the association between the hypothesized mediator and the outcome variable (e.g., change in diet) would also benefit from a shorter window of time between the two assessments. In many cases, the length of these two time windows are inversely related to each other and thus efforts to improve the chance of detecting one relation may hinder effects to detect the other. Of course, there are practical constraints on an investigator's ability to adequately assess constructs. What is needed is for investigators to take advantage of the measurement and testing opportunities when they do arise. Although what can be concluded from any single assessment effort may be limited, the cumulative impact of well designed tests of a theoretical principle can be substantial. If investigators consistently wait for another time or another investigator to conduct the relevant assessments, innovations in theory and practice will continue to be slow. As interventionists specify the degree to which a given study can test all or a facet of a given theory, they are more likely to articulate the contribution a proposed study could make to the empirical literature. This process not only makes the justification for the intervention clear, but also improves the likelihood that investigators will recognize when their and their colleagues' efforts have focused consistently on a single or limited aspect of a given theory. Research activities motivated by the Transtheoretical Model [28] provide an excellent example of a domain where researchers have consistently relied on a limited number of methodological strategies and thus, despite an enormous amount of research activity, provided a very narrow test of the theory. [8] The commitment of time and effort to using interventions to test theoretical principles will in the end be for naught, if there is not an equal commitment to the dissemination of the findings generated by these activities. In particular, investigators who are engaged in the development of health behavior theories must take advantage of the information afforded by intervention activities and demonstrate that they are responsive to this information as they refine and revise their theories. Enhanced communication should also provide an opportunity for basic and applied behavioral scientists to recognize the strengths and weakness of current theories of health behavior and thus help formulate a fuller understanding of what needs to be done to improve the quality of our theories. Summary With an eye toward the future Although Lewin may have been right that there is "nothing more practical than a good theory" (p.169; [24]), his dictum rests on the assumption that good theories are available to address practical problems. The development of "good" theories – that is, theories that are both accurate and applicable – has been hindered by a breakdown in the on-going collaboration between basic and applied behavioral scientists. Research and professional activities that are able to foster a stronger sense of interdependence between these two groups are likely to provide a base for collaboration and, in turn, a opportunity for innovation. If critical advances in health behavior theory depend on an iterative process by which theoreticians and interventionists cooperate in the testing and evaluation of theoretical principles, individuals in both camps need to not only recognize the goals and values of each group, but also trust each other's ability to advance our understanding of both theory and practice. Competing Interests None declared. Acknowledgments The preparation of this paper has been supported in part by National Institute of Neurological Disorders and Stroke Grant 1R01-NS38441-01. ==== Refs Health people 2010 National health promotion and disease prevention objectives US Department of Health and Human Services 2001 Washington, D.C.: U.S. Government Printing Office Jeffery RW Drewnowski A Epstein LH Stunkard AJ Wilson GT Wing RR Hill DR Long-term maintenance of weight loss: Current status Health Psychol 2000 19 5 16 10709944 10.1037//0278-6133.19.Suppl1.5 Kumanyika SK Van Horn L Bowen D Perri MG Rolls BJ Czajkowski SM Schron E Maintenance of dietary behavior change Health Psychol 2000 19 42 56 10709947 10.1037//0278-6133.19.Suppl1.42 Marcus BH Dubbert PM Forsyth LH McKenzie TL Stone EJ Dunn AL Blair SN Physical activity behavior change: Issues in adoption and maintenance Health Psychol 2000 19 32 41 10709946 10.1037//0278-6133.19.Suppl1.32 Ockene JK Emmons KM Mermelstein RJ Perkins KA Bonollo DS Vorhees CC Hollis JF Relapse and maintenance issues for smoking cessation Health Psychol 2000 19 17 31 10709945 10.1037//0278-6133.19.Suppl1.17 Salovey P Rothman AJ Rodin J Gilbert D, Fiske S, Lindzey G Health behavior In Handbook of social psychology 1998 2 4 New York: McGraw-Hill 633 683 Weinstein ND Testing four competing theories of health-protective behavior Health Psychol 1993 12 324 333 8404807 10.1037//0278-6133.12.4.324 Weinstein ND Rothman AJ Sutton SR Stage theories of health behavior Health Psychol 1998 17 290 299 9619480 10.1037//0278-6133.17.3.290 Glanz K Rimer BK Lewis FM Health behavior and health education: Theory, research, and practice 2002 3 New York, N.Y.: Jossey-Bass Green LW Kreuter MW Health promotion planning: An educational and ecological approach 1999 3 Mountain View, CA: Mayfield Bartholomew LK Parcel GS Kok G Gottlieb NH Intervention mapping: Designing theory- and evidence-based health promotion programs 2001 Mountain View, CA: Mayfield Prentice DA Miller DT When small effects are impressive Psychol Bull 1992 112 160 164 10.1037//0033-2909.112.1.160 Glasgow RE Vogt TM Boles SM Evaluating the public health impact of health promotion interventions: the RE-AIM framework Am J Public Health 1999 89 1322 1327 10474547 Baranowski T Anderson Carmack C Mediating variable framework in physical activity interventions: How are we doing? How might we do better? Am J Prev Med 1998 15 266 297 9838973 10.1016/S0749-3797(98)00080-4 Baranowski T Lin LS Wetter DW Resnicow K Hearn MD Theory as mediating variables: Why aren't community interventions working as desired? Ann Epidemiol 1997 S7 S89 S95 Hansen WB McNeal RB Jr The law of maximum expected potential effect: Constraints placed on program effectiveness by mediator relationships Health Educ Res 1996 11 501 507 Mook DG In defense of external invalidity Am Psychol 1983 38 379 387 10.1037//0003-066X.38.4.379 Ajzen I The theory of planned behavior Organ Behav Hum Decis Process 1991 50 179 211 10.1016/0749-5978(91)90020-T Ajzen I Fishbein M Understanding attitudes and predicting social behavior 1980 Englewood Cliffs, NJ: Prentice Hall Rothman AJ Toward a theory-based analysis of behavioral maintenance Health Psychol 2000 19 64 69 10709949 10.1037//0278-6133.19.Suppl1.64 Rothman AJ Baldwin AS Hertel AW Vohs K, Baumeister R Self-regulation and behavior change: Disentangling behavioral initiation and behavioral maintenance In The handbook of self-regulation 2004 New York, NY: Guilford Press 130 148 Bandura A Self-efficacy: The exercise of control 1997 New York, NY: W.H. Freeman and Company Suls J Rothman AJ Evolution of the psychosocial model: Implications for the future of health psychology Health Psychol 2004 23 119 125 15008654 10.1037/0278-6133.23.2.119 Lewin K Field theory in social science: Selected theoretical papers 1951 New York, NY: Harper & Row McGuire WJ Gholson B, Shadish Jr WR, Neimeyer RA, Houts AC A perspectivist approach to the strategic planning of programmatic scientific research In Psychology of science: Contributions to metascience 1989 New York: Cambridge University Press 214 245 Cummings KM Jette AA Rosenstock IM Construct validation of the health belief model Health Educ Monogr 1978 6 394 405 299611 Harrison JA Mullen PD Green LW A meta-analysis of the health belief model with adults Health Educ Res 1992 7 107 116 10148735 Prochaska JO DiClemente CC Norcross JC In search of how people change: Applications to addictive behaviors Am Psychol 1992 47 1102 1114 1329589 10.1037//0003-066X.47.9.1102

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==== Front J Transl MedJournal of Translational Medicine1479-5876BioMed Central London 1479-5876-2-281530169410.1186/1479-5876-2-28CommentaryConflicts of interest in translational research Parks Malcolm R [email protected] Mary L [email protected] Office of Research, University of Washington, Seattle, WA, USA2 Tumor Vaccine Group, University of Washington, Seattle, WA, USA2004 9 8 2004 2 28 28 27 7 2004 9 8 2004 Copyright © 2004 Parks and Disis; licensee BioMed Central Ltd.2004Parks and Disis; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Translational research requires a team approach to scientific inquiry and product development. Translational research teams consist of basic and clinical scientists who can be members of both academic and industrial communities. The conception, pre-clinical testing, and clinical evaluation of a diagnostic or therapeutic approach demands an intense interaction between investigators with diverse backgrounds. As the barriers between industry and academia are removed, issues of potential conflict of interest become more complex. Translational researchers must become aware of the situations which constitute conflict of interest and understand how such conflicts can impact their research programs. Finally, the translational research community must participate in the dialogue ongoing in the public and private sectors and help shape the rules that will govern conflicts that arise during the evolution of their research programs. ==== Body Introduction By its nature, translational research crosses boundaries between basic science and clinical application. It places researchers in new contexts and ushers in a range of new contacts and relationships. Crossing these boundaries contributes directly to the creativity and social impact of translational medicine. But crossing these boundaries also gives rise to new and often conflicting obligations between researchers, their employers, and their industry sponsors. The public is rightfully concerned that the financial interests of researchers, their institutions, and their corporate sponsors may bias research. Yet history also teaches us that industry collaboration is often essential in realizing the promise of translational research. Industry collaboration has figured prominently in many translational research successes including recombinant growth hormone, angioplasty, stenting for coronary artery disease, and many new medications and diagnostic devices [1]. Translational researchers must, therefore, understand what financial conflicts of interest are and how they are managed. Their industry partners must understand the constraints placed on researchers by federal and university policies as well as state laws. Relevant policies in the United States include the regulations issued by the Public Health Service and published as part of the Code of Federal Regulations (42 CFR 50.601–50.607) and in the National Science Foundation Grant Policy Guide (Section 510) [2,3]. Laws governing the use of state-owned resources may also be relevant for those working at or with public universities. What triggers financial conflicts of interest? Conflicts may arise whenever researchers' outside, personal financial interests have the potential to compromise an investigator's professional judgment and independence in the design, conduct, or publication of research. The most commonly regulated financial interests include consulting fees or compensation for personal services, equity or other ownership interests, royalties, and intellectual property rights. A researcher may, for example, receive consulting income or equity in exchange for service on a scientific advisory board of a company that then sponsors clinical research in her lab. Another researcher may be paid for talks to physician groups about an approved medication while simultaneously conducting research on potential off-label uses of the drug. The investigator or an immediate family member may hold stock in the research sponsor. These examples are all common cases and most institutions have relatively standardized ways of managing such common conflicts. Greater challenges are created when the financial relationships between commercial interests and investigators are either ambiguous or complex. Ambiguity can result in a number of ways, but one of the most frequent occurs when investigators approach consulting as an extension of discussions among academic colleagues. Thinking that talking to a corporate representative is the same as talking to an academic colleague, for example, may lead the investigator to make inappropriate disclosures that compromise intellectual property rights or contractual obligations. Often it is not any one financial interest, but rather the combination of multiple financial interests that makes a situation unmanageable. It is extraordinarily difficult, for example, to manage situations in which an investigator is the founder of a startup company, an inventor on a patent licensed to the company, a consultant to the company, and the recipient of other government and industry grants for closely related research. How institutions manage potential conflicts of financial interest Spurred by a combination of bad experiences and new regulations, most research institutions now have policies in place for the management of some aspects of personal financial interests in research. One strategy is simply to prohibit personal financial interests in research. The Association of American Universities, for example, advocates outright prohibition in cases involving research on human subjects unless there are "compelling circumstances" that justify an exception [4]. Prohibition forces financially interested researchers to either divest their interests or to remove themselves from the research. Although effective, prohibitions are blunt tools and in our opinion should be used only as a last resort. We say this not only because prohibiting financial interests may leave any number of other equally biasing interests in place [5], but also because, when properly managed, financial interests may play a positive role in the development of a translational research program. Access to company resources and sharing investigator knowledge are often critical to timely translations of basic science to clinical practice. The complex research enterprise needed to develop clinical products is simply beyond the scope of what many investigators can achieve on their own in academic institutions. Fortunately, there are usually less draconian alternatives to outright prohibition. These strategies seek to ensure the integrity of the research, guarantee public scrutiny and access, and, of course, to protect human participants [6]. One of most common is to assign key research activities such as recruiting, consenting, and data analysis to team members who have no financial stake in the results. Multi-center designs ensure that the biases of any one investigator are less likely to influence the final results. Independent data safety monitoring boards or other oversight committees may also check the influence of personal financial interests. So, too, will requirements to disclose financial interests to publishers, conference organizers, and institutional review boards. Research integrity is further protected by a vigilant stance regarding publication restrictions. Industry partners have a legitimate interest in protecting proprietary information, but this can usually be honored by providing a short period for review prior to submitting a manuscript for publication. No contract or agreement, however, should give the sponsor the right to control publication. Work that requires such control is more appropriately done in industry rather than in academic laboratories. The close attention to publication restrictions is particularly important when a researcher may have a student working on an industry sponsored project. Junior and student scientists working in a research program, who may not have any relationship with a company, must be able to have freedom in pursuing aspects of projects outside the bounds of the research agreement and publishing data in a timely fashion. Translational research that results in the development of a new company presents particular opportunities and challenges. Because of the importance of small companies to economic growth, public research universities often view the number of university-related startups as an index of their contribution to the state economy. More specific institutional interests are created when universities take equity in startups through licensing agreements. Unlike more established firms, start-up companies are often highly dependent on obtaining favorable research outcomes from a particular project. In many cases, prohibition may be the only way to manage the tangle of institutional and individual interests than can result in this situation. Universities may create "firewalls" between the management of equity and researchers [7]. They may require divestiture or bar researchers from receiving grants back from companies to which their inventions have been licensed. Consulting and other company contacts may be restricted when the investigator or the university has a significant financial interest in the startup. Although universities are still coming to grips with their own institutional financial interests, there is an emerging consensus that they should not conduct clinical research on their own inventions unless a strong case for locating the research at the university can be made on clinical grounds [8,9]. For novel biologic therapies, the investigator who invented the approach may be ideally suited to complete the clinical translation. The failure of many novel agents to demonstrate activity in Phase I may be linked to a disconnect between the scientist developing the agent and the physician running the clinical trial. Translational research is defined as that person being one and the same. Recently, the American Association of Medical Colleges published suggested guidelines for the management of conflict of interest based on input from a number of medical schools [10]. Such documents not only encourage dialogue concerning these issues, but also provide some guidance for individual institutions establishing their internal policies. How financial conflicts of interest can affect your research program Unrecognized and unmanaged conflicts of financial interest represent major risk factors for programs of translational research. Even when properly disclosed and managed, however, outside financial interests may limit your research activities in a variety of ways, ranging from mild to severe. If nothing else, time and staff resources must be allocated to institutional and extra-institutional review processes. Because approval is required before funding is released or human subjects are enrolled, the pace of research is slowed by outside financial interests. Scientists with personal financial interests in the research will usually find themselves barred from participating in sensitive research activities, especially those involving direct contact with human subjects. This increases the costs of research as additional staff or consultants are hired to do the work that the financially interested party would have otherwise performed. But even this strategy may prove difficult if key staff or alternative expert consultants also have financial interests. There is a tipping point beyond which so many potential team members are financially involved that the interests simply can not be managed and outright prohibition becomes the only option. In the most extreme circumstances it is possible for researchers to research themselves out of a job. Their personal financial interests in a research sponsor may be so great or so complex that their employers are unable to accept further funding from that sponsor. A line of translational research may simply end for a researcher when he or she is barred from carrying the work into the clinical arena as a result of individual or institutional financial interests. This can occur even when the researcher is not actively seeking financial gain. Early basic science work, for example, may lead to an invention that the university decides to patent and license. In most universities the researcher is entitled to a share of whatever revenue is produced. The researcher now has a personal financial interest that requires management and may disqualify her or him from participation in later clinical work designed to translate the basic science into practice. This is an extreme case, of course, but it does happen and it illustrates the often unseen and unintended implications of how financial interests are usually managed. Dealing intelligently with financial interests in research Public concern about personal and institutional interests in research is not going to go away. Nor is the need for industry collaboration in translational research. Indeed the need for collaborations between industry and academia is only going to grow as we move beyond a sequential "bench to bedside" model to acknowledge the benefits of combining clinical and basic biological studies [11]. If financial interests can not be avoided, we can at least be more thoughtful about how we manage them-as individual researchers, as industry collaborators, and as academic research institutions. Individual investigators should recognize that disclosure of personal financial interests, while perhaps uncomfortable, is vital to continued public confidence in science. They must also balance their wish for personal gain against the additional oversight and management of their activities that will inevitably result. They should recognize that some financial interests are more easily managed than others. Consulting income paid as cash, for example, is much more easily managed than consulting income paid with equity in the company. The latter creates a long-term financial interest and may imply management influence. The understandable desire to "share the wealth" with team members by assisting them in obtaining outside financial interests of their own backfires when it then becomes necessary to remove them from the tasks that they were hired to do in the first place. The decision to create a company in order to further a translational research agenda is appealing, often appropriate, but always more complex than researchers typically appreciate. It is a risky conceit to believe that one's success in obtaining research grants and managing a research team is adequate preparation to launch a business. This is borne out by the fact that technologies licensed to companies founded by faculty inventors are generally less successful than those licensed to companies with non-academic founders [12]. Even when the researcher has the requisite business experience, however, company formation may result in profound conflicts of commitment, inappropriate use of university resources in support of the company, and confusion over intellectual property. These difficulties combine to increase costs and slow, if not block, progress on translational research efforts. In spite of a growing entrepreneurial spirit within academia, the culture gap between industry and academia remains large. Industries seeking to partner with academic researchers must understand that universities are not simply laboratories for hire. They should not assume that they will own or control publication of the research they support. Universities are not set up to guarantee the same level of security and secrecy than can be obtained in industry laboratories. The open character of universities is in fact an asset- it is the foundation on which the creative engine rests. Finally it is useful to appreciate that "indirect costs" are real costs for universities. Indeed, even at the full rate, universities subsidize research contracts [13]. Efforts to avoid indirect costs, like efforts to negotiate contracts that contain publication restrictions and overreaching intellectual property clauses, ultimately have the effect of disrupting working relationships and slowing the pace of research. By the same token, industry collaborators should recognize that efforts to "build relationships" with academic researchers and their staff by providing extra financial incentives and benefits are often counterproductive, not only because they fail to buy loyalty, but also because they create unacceptable financial conflicts of interest for academic personnel. Universities, too, have much to learn about managing financial interests and collaborating creatively with their industry partners. Although a consensus regarding institutional conflicts of interest is emerging, universities still have much to do in terms of implementation, particularly with regard to credible external review of clinical research opportunities in which the institution holds a financial interest. Managing or avoiding institutional conflicts of interest will also require a more realistic view of technology transfer opportunities. Although "big hits" do occur, they occur only rarely and revenue from technology transfer operations is typically only a tiny fraction of revenue from sponsored research. Universities run the risk of disrupting their larger research mission by overly aggressive efforts to capture and commercialize intellectual property. Open publication and teaching should always be the most significant "knowledge transfer" functions of a research university. The increasing complexity of translational research also challenges universities to devise new kinds of collaboration with industry. Mankoff and her colleagues, for example, recently proposed the creation of centers that obtain revenue from existing therapies, while simultaneously providing material for biological studies and supporting experimental therapies [11]. Before these more complex collaborations can occur, however, there is much work to be done to simplify material transfer practices between laboratories and to create more straightforward ways for facilities and personnel to be shared. Individual researchers, industries, and universities have much to learn about the management of financial interests. Given the potential for financial interests to disrupt or even end programs of translational research, we believe that all parties would benefit from greater attention and greater creativity in the management of conflicts of interest. A dialogue must be established between the individual investigator, industry, academic institutions, and the public to define the issues and develop rational solutions. Such a dialogue could be initiated by the development of a focus on such topics at national meetings, by individual organizations whose membership is involved in translational research developing interdisciplinary panels to discuss the issues and attempt to develop guidelines, and by an integration of conflict of interest topics in the training of junior scientists. Solutions should enhance, not impede, translational research. ==== Refs Martin JB Kasper DL In whose best interest? Breaching the academic-industrial wall New England Journal of Medicine 2000 343 1646 1649 11096177 10.1056/NEJM200011303432213 Foundation National Science Grant Policy Manual 2002 Public health policies of general applicability Code of Federal Regulations 2000 Title 42: Part 50.601-50.607 Universities Association of American Report on Individual and Institutional Financial Conflict of Interest 2001 Washington (DC) Brown WA Financial gain: Just one of many motives The Scientist 2001 15 39 Koski Greg To protect those who serve New England Journal of Medicine 2000 343 1643 1645 11096176 10.1056/NEJM200011303432212 Johns Michael M.E. Barnes M Florencio PS Restoring balance to industry-academia relationships in an era of institutional financial conflicts of interest Journal of the American Medication Association 2003 298 741 746 10.1001/jama.289.6.741 Emanuel EJ Steiner D Institutional conflict of interest New England Journal of Medicine 1995 332 262 267 7808495 10.1056/NEJM199501263320412 Research Task Force on Financial Conflicts of Interest in Clinical Protecting subjects, preserving trust, promoting progress II: Principles and recommendations for oversight of an institution's financial interests in human subjects research 2002 Washington, DC, Association of American Medical Colleges Mankoff SP Brander C Ferrone S Marincola FM Lost in translation: Obstacles to translational medicine Journal of Translational Medicine 2004 2 2 14 14728728 10.1186/1479-5876-2-14 Shane Scott Selling university technology: Patterns from MIT Management Science 2002 48 122 137 10.1287/mnsc.48.1.122.14281 Goldman CA Williams T Adamson DM Rosenblatt K Paying for university research facilities and administration

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J Transl Med. 2004 Aug 9; 2:28

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J Transl Med

10.1186/1479-5876-2-28

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==== Front BMC BiolBMC Biology1741-7007BioMed Central London 1741-7007-2-171529870610.1186/1741-7007-2-17Research ArticleExpression of PEG11 and PEG11AS transcripts in normal and callipyge sheep Bidwell Christopher A [email protected] Lauren N [email protected] Allison C [email protected] Tracy S [email protected] Diane E [email protected] Noelle E [email protected] Department of Animal Sciences, Purdue University, 125 South Russell Street, West Lafayette, IN 47907-2042 USA2 Department of Animal, Dairy and Veterinary Sciences, College of Agriculture, Utah State University, 4800 Old Main Hill, Logan UT 84322-4800 USA2004 6 8 2004 2 17 17 17 3 2004 6 8 2004 Copyright © 2004 Bidwell et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The callipyge mutation is located within an imprinted gene cluster on ovine chromosome 18. The callipyge trait exhibits polar overdominant inheritance due to the fact that only heterozygotes inheriting a mutant paternal allele (paternal heterozygotes) have a phenotype of muscle hypertrophy, reduced fat and a more compact skeleton. The mutation is a single A to G transition in an intergenic region that results in the increased expression of several genes within the imprinted cluster without changing their parent-of-origin allele-specific expression. Results There was a significant effect of genotype (p < 0.0001) on the transcript abundance of DLK1, PEG11, and MEG8 in the muscles of lambs with the callipyge allele. DLK1 and PEG11 transcript levels were elevated in the hypertrophied muscles of paternal heterozygous animals relative to animals of the other three genotypes. The PEG11 locus produces a single 6.5 kb transcript and two smaller antisense strand transcripts, referred to as PEG11AS, in skeletal muscle. PEG11AS transcripts were detectable over a 5.5 kb region beginning 1.2 kb upstream of the PEG11 start codon and spanning the entire open reading frame. Analysis of PEG11 expression by quantitative PCR shows a 200-fold induction in the hypertrophied muscles of paternal heterozygous animals and a 13-fold induction in homozygous callipyge animals. PEG11 transcripts were 14-fold more abundant than PEG11AS transcripts in the gluteus medius of paternal heterozygous animals. PEG11AS transcripts were expressed at higher levels than PEG11 transcripts in the gluteus medius of animals of the other three genotypes. Conclusions The effect of the callipyge mutation has been to alter the expression of DLK1, GTL2, PEG11 and MEG8 in the hypertrophied skeletal muscles. Transcript abundance of DLK1 and PEG11 was highest in paternal heterozygous animals and exhibited polar overdominant gene expression patterns; therefore, both genes are candidates for causing skeletal muscle hypertrophy. There was unique relationship of PEG11 and PEG11AS transcript abundance in the paternal heterozygous animals that suggests a RNA interference mechanism may have a role in PEG11 gene regulation and polar overdominance in callipyge sheep. ==== Body Background The mutation responsible for the callipyge trait is located within an imprinted gene cluster on the distal end of ovine chromosome 18 [1-3]. The callipyge phenotype is associated with an altered carcass composition including a 30–40% increase in muscle mass, a 6–7% decrease in carcass fat, decreased organ weights, and a more compact skeleton, all without a net affect on animal growth [4-7]. There is a pronounced hypertrophy of muscles in the loin and pelvic limbs and a lesser degree of hypertrophy in muscles of the thoracic limbs [6-8]. The callipyge phenotype is inherited in a non-Mendelian mode termed polar overdominance [9,10] in which only animals that inherit a normal allele (wild type; +) from the dam and the mutant callipyge allele from the sire (CLPGPat) exhibit the callipyge phenotype. Maternal heterozygotes (CLPGMat/+Pat) and callipyge allele homozygotes (CLPGMat/CLPGPat) have muscling and carcass compositions that are similar to normal sheep (wild type homozygotes; +/+). A physical contig spanning the region containing the callipyge mutation was constructed using overlapping ovine bacterial artificial chromosomes [11] and 215 kb of sequence was obtained from the contig [3]. Comparisons of the ovine sequence to the human genome sequence and to expressed sequence databases indicated the presence of at least six transcribed genes with allele-specific expression [3] (Figure 1). The gene order along the contig was found to be Delta-like 1 (DLK1), DLK associated transcript (DAT), gene-trap locus 2 (GTL2), paternal expressed gene 11 (PEG11 / PEG11AS) and maternal expressed gene 8 (MEG8). The same conserved gene order was also found as an imprinted domain on human chromosome 14 and mouse chromosome 12 [12-15]. The DLK1 locus (also referred to as PREF-1, Zog-1 and pG2) encodes a transmembrane protein that contains epidermal-growth factor repeats [16-19]. Cleavage of the extracellular domain of DLK1 produces the circulating protein fetal antigen-1 [20]. DAT is a short non-coding RNA that has been proposed to be a cleavage product of extended DLK1 transcripts [21]. Both DLK1and DAT are expressed from the paternal allele [3,13-15]. GTL2 (also referred to as MEG3) and MEG8 genes express non-coding RNA from the maternal allele [3,13-15]. The PEG11 gene contains an intronless open reading frame of 1333 amino acids in sheep [3]. The human and mouse orthologues known as retrotransposon-like 1 (RTL1/rtl1) encode 1358 and 1745 amino acids, respectively. RNA transcripts were detected from the opposite strand of the same gene, referred to as PEG11AS (formerly known as antiPEG11) [3]. In the mouse, two maternally expressed microRNAs have been identified with perfect complementarity to mouse Rtl1 [22]. The causative mutation for callipyge is a single base transition of A (wild type; +) to G (CLPG) in the intergenic region located between DLK1 and GTL2 [23] (Figure 1). This mutation has been shown to be 100% concordant with all animals of the +Mat/CLPGPat genotype based on haplotype analysis [23]. Analysis of sheep from 19 different breeds as well as 13 mammalian species revealed a highly conserved 12 base sequence that includes the single nucleotide polymorphism [24]. The G polymorphism is unique to direct descendents of the first known callipyge animal, a ram named "Solid Gold". This animal was mosaic for the mutation [24], providing strong evidence that this single nucleotide polymorphism is the causative mutation. Initial results indicate that the mutation alters the expression of several of the genes within the imprinted cluster [25,26] when they are inherited in cis without altering their parent-of-origin-specific expression [25]. In this study, we analyzed the expression of five genes within the callipyge cluster in the muscles of lambs of all four genotypes. Quantitative analysis of gene expression using a series of orthogonal contrasts showed that DLK1, PEG11 and MEG8 exhibited a polar overdominant pattern of gene expression. The expression of PEG11 and PEG11AS transcripts in the muscles of paternal heterozygous callipyge lambs (+Mat/CLPGPat) was different from the other three genotypes. A sense/antisense interaction of PEG11 and PEG11AS, such as an RNA interference mechanism, would be consistent with a trans interaction between reciprocally imprinted genes that has been previously proposed as a mechanism for polar overdominance [27,28]. Results Northern blot analysis Muscle samples were collected from 12- and 8-week-old lambs, when muscle hypertrophy is well established in animals with the callipyge phenotype. Total RNA was extracted from three muscles that undergo hypertrophy including longissimus dorsi (loin), semimembranosus, and gluteus medius (pelvic limb), and one muscle that does not undergo hypertrophy, the supraspinatus, (thoracic limb). Strand specific probes were used to analyze PEG11 and PEG11AS expression. Hybridization of longissimus dorsi, semimembranosus and gluteus medius northern blots with a PEG11 probe indicated expression of a 6.5 kb PEG11 transcript in paternal heterozygotes (+Mat/CLPGPat) that was not readily detectable in the other three genotypes (Figure 2). Two smaller PEG11AS transcripts of 1.7 kb and 0.8 kb were detected in the two genotypes with maternally inherited callipyge alleles (CLPGMat/+Pat and CLPGMat/CLPGPat) by a probe from the complementary strand. PEG11 and PEG11AS transcripts were not detected in the supraspinatus. The expression of DLK1 was detectable at various levels in each of the four muscles and all four genotypes, although DLK1 appeared to be up-regulated in the loin and pelvic limb muscles of +Mat/CLPGPat and CLPGMat/CLPGPat animals (Figure 2). GTL2 transcripts of around 2.4 kb were evident in the CLPGMat/+Pat and CLPGMat/CLPGPat genotypes. A distinct 1.9 kb GTL2 transcript was consistently detected in the +Mat/CLPGPat genotype and no GTL2 transcripts were detected in the +/+ genotype. A 1.8 kb MEG8 transcript was expressed to a lesser degree than GTL2 in the CLPGMat/+Pat and CLPGMat/CLPGPat genotypes and was also detectable in the +Mat/CLPGPat genotype. The expression pattern of MEG8 was the least consistent in the northern blot analysis of individuals of the four genotypes. PEG11/PEG11AS Ribonuclease protection assays were performed using five riboprobes to map the PEG11 and PEG11AS transcripts to the contig sequence (Figure 3A). The results for riboprobe P confirm the expression of PEG11 transcripts in the gluteus medius of +Mat/CLPGPat animals and showed a very low level of PEG11 in the gluteus medius of CLPGMat/CLPGPat animals. PEG11AS transcripts were readily detectable in animals of the +/+, CLPGMat/+Pat and CLPGMat/CLPGPat genotypes but were slightly above background levels in the +Mat/CLPGPat animals (Figure 3B). Two other PEG11 probes from the open reading frame (E) and from the 3'UTR (C) showed equivalent results to riboprobe P in the gluteus medius (Figure 3B). The same expression pattern was seen for PEG11 and PEG11AS transcripts in the supraspinatus, but signal from the protected riboprobes was just above background level. Two probes from upstream of the PEG11 coding sequence (riboprobes F and G) did not detect any PEG11 transcripts, indicating that transcription of the 6.5 kb PEG11 was initiated within 350 bp of the start codon of the open reading frame. Riboprobe F detected PEG11AS transcripts in the gluteus medius and supraspinatus of all four genotypes, with the lowest expression in the paternal heterozygous (+Mat/CLPGPat) animals. The PEG11AS riboprobe G shows three protected fragments, suggesting variable splice junctions or transcription termination sites for PEG11AS transcripts. Quantitative analysis of the effect of genotype on gene expression Gene expression was measured in the gluteus medius and supraspinatus of 8 week-old animals (Figure 4) using quantitative PCR. The expression of glyceraldehyde-3-phosphate dehydrogenase was not significantly different across the four genotypes in the gluteus medius and supraspinatus (Table 1), indicating that equivalent amounts of RNA were used for cDNA synthesis and quantitative PCR. The effect of the callipyge mutation on genotype-specific expression of DLK1 and PEG11 in gluteus medius was the same (Table 1) although the magnitude of the response was greater for PEG11 than DLK1 (Figure 4A and 4B). The paternal heterozygous (+Mat/CLPGPat) animals had the highest transcript abundance (p < 0.05), followed by CLPGMat/CLPGPat animals, which had significantly greater transcript abundance (p < 0.05) than CLPGMat/+Pat or +/+ animals. The mRNA abundance of DLK1 in the gluteus medius was 6-fold and 2.5-fold greater in +Mat/CLPGPat lambs and CLPGMat/CLPGPat lambs respectively, relative to normal lambs (+/+; Figure 4A). PEG11 mRNA abundance in the gluteus medius was 200-fold and 13-fold greater in +Mat/CLPGPat lambs and CLPGMat/CLPGPat lambs respectively, relative to normal lambs (+/+; Figure 4B). The effect of the callipyge mutation on DLK1 and PEG11 expression in the supraspinatus was different (Table 1). No differences in DLK1 transcript abundance in the supraspinatus were found among the four genotypes (Figure 4C). Although not statistically analyzed, the level of DLK1 expression was similar between the gluteus medius and supraspinatus (Figure 4A and 4C). PEG11 expression in the supraspinatus had a different genotype-specific pattern than in the gluteus medius. PEG11 expression was elevated 150-fold in +Mat/CLPGPat animals (p < 0.05) but was not significantly changed in the other three genotypes (Figure 4D). The transcript abundance of PEG11 in supraspinatus was generally much lower than in gluteus medius of the same genotype. PEG11 expression in the supraspinatus was 12-fold lower than in the gluteus medius of paternal heterozygous animals (Figure 4B and 4D). Maternal inheritance of the callipyge allele significantly altered the expression of MEG8 but not PEG11AS in the gluteus medius (Table 1). Transcript abundance of MEG8 in gluteus medius was 6-fold greater (p < 0.05) in the CLPGMat/+Pat and CLPGMat/CLPGPat animals relative to the +Mat/CLPGPat and +/+ animals (Figure 4A). Expression of MEG8 in the supraspinatus was also affected by the callipyge mutation but to a lesser degree (Figure 4C). The homozygous callipyge animals had significantly higher MEG8 transcript abundance than paternal heterozygotes, but those two genotypes were not significantly different from maternal heterozygotes and homozygous wild type lambs. Orthogonal contrasts were used to analyze different models of gene action for the genes that had a significant effect for genotype (Table 1). The polar overdominance contrast was significant for transcript abundances of DLK1, PEG11 and MEG8 in gluteus medius and for PEG11 and MEG8 in the supraspinatus. The additive contrasts were also significant for DLK1, PEG11 and MEG8 in gluteus medius but were only significant for PEG11 in supraspinatus. The maternal dominance contrast was significant for DLK1 and PEG11 gluteus medius. Discussion Our results show a clear pattern of increased expression for genes within the imprinted callipyge cluster in muscles that become hypertrophied, whereas expression of these genes was either reduced or absent in a muscle that does not become hypertrophied. The increased gene expression occurred when the mutation was inherited in cis and was dependent on each gene's imprinting status, consistent with previous reports [25,26]. These results support the hypothesis that the mutation has disrupted a long range control element [27]. Two paternally expressed genes, DLK1 and PEG11, had significantly increased transcript abundance when a callipyge allele was inherited from the sire. One maternally expressed gene, MEG8, showed significantly increased transcript abundance when the callipyge allele was inherited from the dam. The changes in gene expression were sustained until 12 weeks of age, when the differences in growth and body composition between callipyge and normal lamb are established and are subsequently maintained [7]. Northern blot analysis suggests that expression of GTL2 and PEG11AS was increased by maternal inheritance of the callipyge allele. Quantitative analysis was not done for GTL2 in this study due to the expression of numerous alternatively spliced transcripts that have been reported for mice and sheep [26,29]. The 2.4 kb GTL2 mRNA seen in this study consisted of a heterogeneous population of alternatively spliced mRNAs. Similarly, two PEG11AS transcripts were detected over a 5.6 kb area extending from beyond the 5' end of the PEG11 transcript to the 3'UTR. The 1.7 and 0.8 kb PEG11AS transcripts detected by northern blot analysis using probe P would not be protected by probes C or G unless the PEG11AS transcripts undergo intron splicing or there are other transcripts that were not detected in the northern blots. Therefore, the effect of the callipyge mutation on GTL2 and PEG11AS will require a more extensive analysis to fully elucidate their expression patterns in the four genotypes and determine their role in the callipyge model. Due to their paternal allele-specific expression, the DLK1 and PEG11 genes are both candidates for an effector gene that is responsible for the skeletal muscle hypertrophy exhibited by paternal heterozygous (+Mat/CLPGPat) animals. In this study, both genes showed a polar overdominant expression pattern in that paternal heterozygotes had significantly higher levels of gene expression than the other three genotypes. The major differences between DLK1 and PEG11 were the magnitude and the muscle specificity of the up-regulation. DLK1 was readily detectable in all muscles and genotypes by northern blot, but the up-regulation in paternal heterozygous animals was restricted to muscles of the loin and pelvic limb. The quantitative results showed a 6-fold increase in DLK1 transcript abundance in the gluteus medius and no change of DLK1 abundance in the supraspinatus. This pattern of gene expression is consistent with studies on individual muscle growth that show significant muscle hypertrophy in the gluteus medius but not in the supraspinatus [6,8]. DLK1 transcripts were significantly increased (2.5-fold) in the gluteus medius of CLPGMat/CLPGPat animals, which do not exhibit muscle hypertrophy. The lack of a phenotype in CLPGMat/CLPGPat animals could be due to a threshold effect that requires more than a 2.5-fold increase in DLK1 transcript abundance to change muscle growth. The expression of PEG11 was very low in the gluteus medius and supraspinatus of normal sheep and was induced in both muscles of callipyge lambs. Northern blot analysis, ribonuclease protection assay and quantitative PCR results all show that the expression of PEG11 and PEG11AS transcripts was much lower in the supraspinatus than the other muscles. The high level of expression of PEG11 in the gluteus medius relative to the supraspinatus of callipyge lambs indicates that PEG11 could also be the gene responsible for muscle hypertrophy if there is a threshold level required to change muscle growth. The PEG11 gene has a long intronless open reading frame but it is not known if a protein is produced or what function it may have. The overdominant nature of the callipyge phenotype through the lack of muscle hypertrophy in animals with the CLPGMat/CLPGPat genotype is one of the more intriguing aspects of the trait and has led to a hypothesis of trans effects by other reciprocally imprinted genes in the callipyge region [25,27]. If the PEG11 gene has a direct role in muscle hypertrophy, either alone or in concert with DLK1, then PEG11AS may have a role in a trans effect on PEG11 expression. In +Mat/CLPGPat animals, PEG11 transcripts were 14-fold and 4-fold more abundant than PEG11AS transcripts in the gluteus medius and supraspinatus respectively. PEG11AS transcripts were more abundant than PEG11 transcripts in the other three genotypes for both muscles. Therefore, the relative abundance of PEG11 transcripts to PEG11AS transcripts was unique in paternal heterozygous animals. MicroRNAs are a central component of RNA interference mechanisms. In the mouse, two antisense microRNA have been identified for the orthologous rtl1 locus [22]. RNA interference mechanisms have been shown both to repress transcription by inducing heterochromatin formation [30-32] and to cause post-transcriptional silencing through nuclear retention or targeted degradation [33-35]. MicroRNAs may be produced from post-transcriptional processing of PEG11AS RNA and be involved in normal regulation of the locus and in generating overdominance [28]. Expression of PEG11AS may normally cause repression of the paternal PEG11 locus since very little PEG11 mRNA was detectable in the muscles of normal animals. In the animals with a paternally inherited callipyge allele, (+Mat/CLPGPat and CLPGMat/CLPGPat), the normal repression of the PEG11 locus has been disrupted by the mutation in a putative long range control element [27]. PEG11 transcripts only accumulate in significant excess of PEG11AS in the paternal heterozygous animals, whereas in the CLPGMat/CLPGPat animals the PEG11AS transcripts remain in excess and may prevent the accumulation of the threshold level of PEG11 mRNA required to produce a muscle hypertrophy phenotype. Although the expression of PEG11AS was not affected by genotype in quantitative PCR, the northern blots and transcript mapping indicate there are multiple transcripts that are likely to undergo different intron splicing. Further analysis of PEG11AS expression will be necessary to determine its role in the PEG11 locus regulation. Conclusions The effect of the callipyge mutation has been to increase transcript abundance of four genes, DLK1, GTL2, PEG11 and MEG8, within the imprinted cluster in skeletal muscles that become hypertrophied. The increase in transcript abundance was consistent with each gene's parental allele-specific expression. The DLK1 and PEG11 genes were both expressed at their highest levels in paternal heterozygous animals and exhibited polar overdominant gene expression patterns. Therefore, both genes are candidates for causing muscle hypertrophy. DLK1 expression was only elevated in muscles that undergo hypertrophy, so its muscle-specific increase was consistent with the callipyge phenotype. PEG11 was 12-fold more abundant in hypertrophied muscle than non-hypertrophied muscle and only paternal heterozygous animals had PEG11 transcript levels in excess of PEG11AS transcript levels. The unique relationship of PEG11 and PEG11AS in paternal heterozygous animals suggests that an RNA interference mechanism may have a role in regulating the PEG11 locus and polar overdominance in callipyge sheep. Methods Sample collection A series of planned matings were conducted to produce the four possible callipyge genotypes. The genotypes of all lambs were verified using the single nucleotide polymorphism [23,24] and several markers that flank the callipyge region on chromosome 18. Lambs were slaughtered in accordance with humane practices approved by the Utah State University Institutional Animal Care and Use Committee. Samples were collected from the longissimus dorsi, semimembranosus, gluteus medius and supraspinatus, and preserved in RNAlater (Ambion Inc., Woodlands, TX USA). The tissue samples were homogenized in 4 M guanidinium thiocyanate, 25 mM sodium citrate, 50 mM EDTA, 1% sodium-N-lauroyl-sarcosine and total RNA was sedimented by ultracentrifugation of the homogenate on a cushion of 5.7 M CsCl, 50 mM EDTA [36]. Purified RNA was quantified by spectrophotometry and the use of a constant mass of RNA for each quantitative assay was based on absorbance at 260 nm. Total RNA was treated with DNase I using DNA free™ reagents (Ambion Inc.) to remove trace genomic DNA prior to use in the ribonuclease protection assay and quantitative PCR. Northern blot analysis Northern blots were prepared using denaturing formaldehyde gel electrophoresis (NorthernMax™; Ambion Inc) of 10 μg of total RNA and transferred to positively charged nylon membranes using standard methods [37]. Primer sequences used to amplify probes from the callipyge region are given in Charlier et al. [25], and the PCR products were verified by DNA sequencing. Strand specific DNA probes were synthesized by 40 cycles of asymmetric PCR with Strip-EZ™ nucleotides (Ambion Inc.) and 50 μCi of α-[32P]-dATP (Amersham-Pharmacia, Piscataway, NJ USA). Unincorporated nucleotides were removed by spin column chromatography (BioSpin P30; Bio-Rad Inc., Hercules, CA USA). The probes were hybridized to the membranes without denaturation using Ultrahyb™ (Ambion Inc.) at 42°C overnight. After hybridization, the membranes were washed in 2X SSC (0.3 M sodium chloride, 0.03 M sodium citrate)/0.5% SDS followed by 3 washes in 1X SSC/ 0.1% SDS at 65°C for 30 min and a final high stringency wash in 0.1X SSC/ 0.1%SDS at 65°C for 30 min. The northern blots were exposed to Kodak XAR autoradiography film for 18 to 72 h at -80°C. After autoradiography, the probe was degraded and removed from the membranes using Strip-EZ™ reagents (Ambion Inc.). Ribonuclease protection assay Templates for synthesizing strand specific RNA probes for the ribonuclease protection assay (RPA) were generated from PCR products (Table 2) by ligation of double stranded oligonucleotides containing T7 or SP6 promoter sequences and re-amplification with an adapter and gene specific primer (Lig'n Scribe™, Ambion Inc.). Labeled RNA probes were synthesized using MAXIscript™ reagents (Ambion, Inc.) and 50 μCi of α-[32P]-UTP (Amersham-Pharmacia). The RPA were conducted using RPA III™ reagents (Ambion Inc.) and standard urea/acrylamide gel electrophoresis methods [37]. Dried gels were exposed to phosphorimaging screens and images were collected using a Cyclone Storage Phosphorimager (Packard Instrument Co., Meriden, CT USA). Quantitative PCR Complementary DNA was synthesized from 3.2 μg of total RNA using random hexamer priming and MMLV reverse transcriptase reagents (Invitrogen, Carlsbad, CA USA) with RNase inhibitor supplementation (Superase Inhibitor, Ambion Inc). The cDNA samples were diluted with water and aliquoted so that the quantification was based on 213 ng of total RNA for analysis of glyceraldehyde-3-phosphate dehydrogenase, DLK1 and MEG8 transcripts. PEG11 and PEG11AS transcripts were measured using gene-specific priming of cDNA synthesis from 1.6 μg of total RNA. The cDNA was diluted with water and aliquoted for quantification based on 400 ng of total RNA. Each cDNA sample was amplified in triplicate using the SYBR Green Jump Start™ system (Sigma-Aldridge, St. Louis, MO USA). Quantification standards were composed of aliquots of plasmids containing target PCR products in 10-fold serial dilutions ranging from 108 to 102 molecules. The standards were used to calculate a regression of threshold cycle on molecule copy number to determine a log value of starting abundance for each of the cDNA samples based on their threshold cycle. The PCR reactions were run for 40 cycles in an iCycler Real-Time PCR Detection System (Bio-Rad Inc.). The log value of starting abundance for each gene was analyzed by analysis of variance using the PROC MIXED procedure of SAS [38]. The analysis model included genotype as a fixed effect and animal within genotype as a random effect. The number of animals representing each genotype (6 to 8) is given in Table 3. Orthogonal contrasts were used to evaluate different models of gene action if the effect of genotype on log value of starting abundance was significant. Initially, traditional additive, dominance and reciprocal heterozygote effects were evaluated (see Table 3 for contrasts). If the reciprocal heterozygote effect was significant (p < 0.05), a second set of orthogonal contrasts was used to test additive, maternal dominance and polar overdominance effects as previously described by Freking et al. [10] (Table 3). Authors' contributions CB participated in planning the study and developing the experimental design, conducted the northern blot analysis and ribonuclease protection assays, and wrote the drafts of the manuscript. LK and AP isolated RNA and performed the quantitative PCR assays. TH collected the muscle samples and genotyped the animals used in this study. DM participated in experimental design and performed the statistical analysis. NC participated in planning the study, set up a series of matings to generate all four genotypes and provided the experimental animals. All authors read and approved the final manuscript. Acknowledgements This project was supported by funds from the USDA/NRI (2002-35205-11602) and the Purdue University Agricultural Research Program (paper No. 17430). The authors acknowledge Michel Georges and Carole Charlier, University of Liège, Belgium, for providing DNA sequence and PCR primer data and thank Christine Bynum, Anne Danielsson and David Forrester for their assistance with this experiment. Figures and Tables Figure 1 The callipyge region of ovine chromosome 18. A diagram of the callipyge region [3] based on GenBank accession No. AF345168 is shown. Six known transcripts are indicated along with the direction of transcription (arrows). Transcripts expressed from the paternal allele are shown as orange arrows and those expressed from the maternal allele are shown as black arrows [3]. A blue line indicates the position of the causative mutation. Alleles with an A are wild type (+) and the mutant callipyge allele (CLPG) has a G at this position [23, 24]. Figure 2 Northern blot analysis of skeletal muscle RNA from 12-week-old lambs. Expression of five genes from the callipyge region is shown for the four possible genotypes in 12-week-old lambs. Each lane contains total RNA from an individual animal. The genotypes are given with the maternal allele first followed by the paternal allele. The callipyge allele (CLPG) has been abbreviated to C and the paternal heterozygote that has the callipyge phenotype is indicated with an asterisk (*). Strand specific probes are indicated on the left and transcript sizes are indicated on the right. The skeletal muscles shown are the longissimus dorsi (LD), semimembranosus (SM), gluteus medius (GM) and the supraspinatus (SP). The blots were hybridized with an 18S RNA probe to show the equivalence of RNA loading and transfer. Figure 3 Mapping of PEG11 and PEG11AS transcripts by ribonuclease protection assay. A) A diagram of the PEG11 open reading frame (black arrow) and the position of the five riboprobes are shown. The PEG11 open reading frame extends from base 191859 to 187858 on the complementary strand of AF354168. B) Ribonuclease protection assays using PEG11AS (left column) and PEG11 (right column) are shown using total RNA from the gluteus medius (GM) and the supraspinatus (SP) muscles for each of the four possible genotypes. The genotypes are given with the maternal allele first followed by the paternal allele. The CLPG allele has been abbreviated to C and the paternal heterozygote that demonstrates the callipyge phenotype is indicated with an asterisk (*). Yeast RNA with RNase treatment (Y+) and without RNase treatment (Y-) are shown as controls. A black arrow indicates the RNA fragments protected by each of the five RNA probes. Some full-length probes for C and E that could not be completely eliminated by RNase digestion are present in both muscle RNA samples and the yeast RNA control (Y+). Figure 4 Quantitative reverse transcriptase PCR analysis of transcript abundance in the gluteus medius and supraspinatus muscles. Least square means and standard errors for transcript abundance by genotype are shown for the gluteus medius (GM), and supraspinatus (SP). The genotypes are given with the maternal allele first followed by the paternal allele and the CLPG allele has been abbreviated to C. Quantification of DLK1 and MEG8 transcripts (A, C) was based on random primed cDNA synthesis using 213 ng of total RNA. Quantification of PEG11 and PEG11AS (B, D) transcripts was based on gene-specific priming of cDNA synthesis using 400 ng of total RNA. Different superscripts indicate significant differences (p < 0.05) between genotypic means for a given mRNA transcript and muscle. Numerical values for the log starting abundance are given for each mRNA transcript in a table below the genotypes. Table 1 Statistical analysis of gene expression by quantitative PCR Orthogonal Contrast P-Values Muscle Gene Effect of Genotype (p-values) Additive Maternal Dominance Paternal Polar Overdominance GM DLK1 0.0001 0.0001 0.0261 0.0001 MEG8 0.0005 0.0009 0.4083 0.0047 PEG11 0.0001 0.0001 0.0001 0.0001 PEG11AS 0.2622 G3PD 0.1393 SP DLK1 0.1865 MEG8 0.0325 0.1398 0.9665 0.0094 PEG11 0.0001 0.0179 0.3315 0.0001 PEG11AS 0.1438 G3PD 0.3626 Table 2 Primer sequences for ribonuclease protection assay probes and quantitative PCR Sizea Tm Locationb Primer Sequence bp °C PEG11/PEG11AS C F AGGAACACCGCTGTGGAGGTAGAA 135 59 4337 R ACAGCAGAGGCAGCCAAGCA E F GGTGACGCCCGTCTGCAAGT 180 61 2427 R GTGGAACGGTTCGCCGACAT Pc F ACAGCTCAACAGTGGAGGTCATG 199 55 996 R ATCAGCTGGCAGAGCACGATGAAC F F TTCCCCCATGGCTGTGAGAAAT 175 58 -390 R CCTCTGTGACCTTCTGGTGACCAA G F AGGCTGAATTGACAGAGATGT 124 49 -1193 R GTTAAATGGCTCAAGAACGA DLK1 F CCCGTCCTCTTGCTCCTGCT 116 58 R GGCTGGCACCTGCACACACT MEG8 F CCCAGGGAGTGTGAGGCTCTTCT 100 56 R GGACCCACGGCTGACCTGTT G3PD F TGAGTGTCGCTGTTGAAGT 150 58 R CCTGCCAAGTATGATGAGAT aSize of the PCR product and protected fragment for RPA bLocation for the PCR product is relative to the first base of the PEG11 open reading frame. cCharlier et al. [3] Table 3 Statistical methods for genetic models Orthogonal Contrast Values by Genotype Genetic Model +/+ CLPGMat/+Pat +Mat/CLPGPat CLPGMat/CLPGPat Additive -1 0 0 1 Dominance -1 1 1 -1 Reciprocal Heterozygote 0 -1 1 0 Maternal Dominance -1 2 0 -1 Paternal Polar Overdominance -1 -1 3 -1 Number of Animals 6 7 8 8 ==== Refs Cockett NE Jackson SP Shay TL Nielsen D Moore SS Steele MR Barendse W Green RD Georges M Chromosomal localization of the callipyge gene in sheep (Ovis aries) using bovine DNA markers Proc Natl Acad Sci USA 1994 91 3019 3023 8159698 Fahrenkrug SC Freking BA Rexroad CE IIILeymaster KA Kappes SM Smith TPL Comparative mapping of the ovine clpg locus Mamm Genome 2000 11 871 876 11003702 10.1007/s003350010150 Charlier C Segers K Wagenaar D Karim L Berghmans S Jaillon O Shay T Weissenbach J Cockett N Gyapay G Georges M Human-ovine comparative sequencing of a 250-kb imprinted domain encompassing the callipyge (clpg) locus and identification of six imprinted transcripts: DLK1, DAT, GTL2, PEG11, antiPEG11, and MEG8 Genome Res 2001 11 850 862 11337479 10.1101/gr.172701 Jackson SP Green RD Miller MF Phenotypic characterization of rambouillet sheep expressing the callipyge gene: I. Inheritance of the condition and production characteristics J Anim Sci 1997 75 14 18 9027543 Jackson SP Miller MF Green RD Phenotypic characterization of rambouillet sheep expressing the callipyge gene: II. Carcass characteristics and retail yield J Anim Sci 1997 75 125 132 9027557 Jackson SP Miller MF Green RD Phenotypic characterization of rambouillet sheep expression the callipyge gene: III. Muscle weights and muscle weight distribution J Anim Sci 1997 75 133 138 9027558 Freking BA Keele JW Nielsen MK Leymaster KA Evaluation of the ovine callipyge locus: II. Genotypic effects on growth, slaughter, and carcass traits J Anim Sci 1998 76 2549 2559 9814893 Koohmaraie M Shackelford SD Wheeler TL Lonergan SM Doumit ME A muscle hypertrophy condition in lamb (callipyge): characterization of effects on muscle growth and meat quality traits J Anim Sci 1995 73 3596 3607 8655433 Cockett NE Jackson SP Shay TL Farnir F Berghmans S Snowder GD Nielsen DM Georges M Polar overdominance at the ovine callipyge locus Science 1996 273 236 238 8662506 Freking BA Keele JW Beattie CW Kappes SM Smith TP Sonstegard TS Nielsen MK Leymaster KA Evaluation of the ovine callipyge locus: I. Relative chromosomal position and gene action J Anim Sci 1998 76 2062 2071 9734855 Segers K Vaiman D Berghmans S Shay T Meyers S Beever J Cockett N Georges M Charlier C Construction and characterization of an ovine BAC contig spanning the callipyge locus Anim Genet 2000 31 352 359 11167521 10.1046/j.1365-2052.2000.00676.x Miyoshi N Wagatsuma H Wakana S Shiroishi T Nomura M Aisaka K Kohda T Surani MA Kaneko-Ishino T Ishino F Identification of an imprinted gene, Meg3/Gtl2 and its human homologue MEG3, first mapped on mouse distal chromosome 12 and human chromosome 14q Genes Cells 2000 5 211 220 10759892 10.1046/j.1365-2443.2000.00320.x Schmidt JV Matteson PG Jones BK Guan XJ Tilghman SM The Dlk1 and Gtl2 genes are linked and reciprocally imprinted Genes Dev 2000 14 1997 2002 10950864 Takada S Tevendale M Baker J Georgiades P Campbell E Freeman T Johnson MH Paulsen M Ferguson-Smith AC Delta-like and Gtl2 are reciprocally expressed, differentially methylated linked imprinted genes on mouse chromosome 12 Curr Biol 2000 10 1135 1138 10996796 10.1016/S0960-9822(00)00704-1 Wylie AA Murphy SK Orton TC Jirtle RL Novel imprinted DLK1/GTL2 domain on human chromosome 14 contains motifs that mimic those implicated in IGF2/H19 regulation Genome Res 2000 10 1711 1718 11076856 10.1101/gr.161600 Laborda J Sausville E Hoffman T Notario V Dlk, a putative mammalian homeotic gene differentially expressed in small cell lung carcinoma and neuroendocrine tumor cell line J Biol Chem 1993 268 3817 3820 8095043 Smas CM Sul HS Pref-1, a protein containing EGF-like repeats, inhibits adipocyte differentiation Cell 1993 73 725 734 8500166 10.1016/0092-8674(93)90252-L Okamoto M Takemori H Halder SK Nonaka Y Osamu H Implication of Zog protein (zona glomerulosa-specific protein) in zone development of the adrenal cortex Endocr Res 1998 24 515 520 9888532 Helman LJ Sack N Plon SE Israel MA The sequence of an adrenal specific human cDNA, pG2 Nucleic Acids Res 1990 18 685 2308864 Jensen C Krogh T Hojrup P Clausen P Skjodt K Larsson L Enghild J Teisner B Protein structure of fetal antigen 1 (FA1). A novel circulating human epidermal-growth-factor-like protein expressed in neuroendocrine tumors and its relation to the gene products of dlk and pG2 Eur J Biochem 1994 225 83 92 7925474 Paulsen M Takada S Youngson NA Benchaib M Charlier C Segers K Georges M Ferguson-Smith AC Comparative sequence analysis of the imprinted Dlk1-Gtl2 locus in three mammalian species reveals highly conserved genomic elements and refines comparison with the Igf2-H19 region Genome Res 2001 11 2085 2094 11731499 10.1101/gr.206901 Seitz H Youngson N Lin S-P Dalbert S Paulsen M Bachellerie J-P Ferguson-Smith AC Cavaillé J Imprinted microRNA genes transcribed antisense to a reciprocally imprinted retrotransposon-like gene Nat Genet 2003 34 261 262 12796779 10.1038/ng1171 Freking BA Murphy SK Wylie AA Rhodes SJ Keele JW Leymaster KA Jirtle RL Smith TPL Identification of the single base change causing the callipyge muscle hypertrophy phenotype, the only known example of polar overdominance in mammals Genome Res 2002 12 1496 1506 12368241 10.1101/gr.571002 Smit M Segers K Carrascosa LG Shay T Baraldi F Gyapay G Snowder G Georges M Cockett N Charlier C Mosaicism of Solid Gold supports the causality of a noncoding A-to-G transition in the determinism of the callipyge phenotype Genetics 2003 163 453 456 12586730 Charlier C Segers K Karim L Shay T Gyapay G Cockett N Georges M The callipyge mutation enhances the expression of coregulated imprinted genes in cis without affecting their imprinting status Nat Genet 2001 27 367 369 11279514 10.1038/86856 Bidwell CA Shay TL Georges M Beever JE Berghmans S Cockett NE Differential expression of the GTL2 gene within the callipyge region of ovine chromosome 18 Anim Genet 2001 32 248 256 11683710 10.1046/j.1365-2052.2001.00776.x Georges M Charlier C Cockett N The callipyge locus: evidence for the trans interaction of reciprocally imprinted genes Trends Genet 2003 19 248 252 12711215 10.1016/S0168-9525(03)00082-9 Lin S-P Youngson N Takada S Seitz A Reik W Paulsen M Cavaille J Ferguson-Smith AC Asymmetric regulation of imprinting on the maternal and paternal chromosomes at the Dlk1-Gtl2 imprinted cluster on mouse chromosome 2 Nat Genet 2003 35 97 102 12937418 10.1038/ng1233 Schuster-Gossler K Bilinski P Sado T Ferguson-Smith A Gossler A The mouse Gtl2 gene is differentially expressed during embryonic development, encodes multiple alternatively spliced transcripts, and may act as an RNA Dev Dyn 1998 212 214 228 9626496 10.1002/(SICI)1097-0177(199806)212:2<214::AID-AJA6>3.0.CO;2-K Volpe TA Kidner C Hall IM Teng G Grewal SIS Martienssen RA Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi Science 2002 297 1833 1837 12193640 10.1126/science.1074973 Hall IM Shankaranarayana GD Noma K-i Ayoub N Cohen A Grewal SIS Establishment and maintenance of a heterochromatin domain Science 2002 297 2232 2237 12215653 10.1126/science.1076466 Verdel A Jia S Gerber S Sugiyama T Gygi S Grewal SIS Moazed D RNAi-mediated targeting of heterochromatin by the RITS complex Science 2004 303 672 676 14704433 10.1126/science.1093686 Hannon GJ RNA interference Nature 2002 481 246 251 Cerutti H RNA interference: traveling in the cell and gaining functions? 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==== Front RetrovirologyRetrovirology1742-4690BioMed Central London 1742-4690-1-201531040510.1186/1742-4690-1-20ReviewRole of Tax protein in human T-cell leukemia virus type-I leukemogenicity Azran Inbal [email protected] Yana [email protected] Mordechai [email protected] Department of Microbiology and Immunology and Cancer Research Center, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel2004 13 8 2004 1 20 20 26 6 2004 13 8 2004 Copyright © 2004 Azran et al; licensee BioMed Central Ltd.2004Azran et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. HTLV-1 is the etiological agent of adult T-cell leukemia (ATL), the neurological syndrome TSP/HAM and certain other clinical disorders. The viral Tax protein is considered to play a central role in the process leading to ATL. Tax modulates the expression of many viral and cellular genes through the CREB/ATF-, SRF- and NF-κB-associated pathways. In addition, Tax employs the CBP/p300 and p/CAF co-activators for implementing the full transcriptional activation competence of each of these pathways. Tax also affects the function of various other regulatory proteins by direct protein-protein interaction. Through these activities Tax sets the infected T-cells into continuous uncontrolled replication and destabilizes their genome by interfering with the function of telomerase and topoisomerase-I and by inhibiting DNA repair. Furthermore, Tax prevents cell cycle arrest and apoptosis that would otherwise be induced by the unrepaired DNA damage and enables, thereby, accumulation of mutations that can contribute to the leukemogenic process. Together, these capacities render Tax highly oncogenic as reflected by its ability to transform rodent fibroblasts and primary human T-cells and to induce tumors in transgenic mice. In this article we discuss these effects of Tax and their apparent contribution to the HTLV-1 associated leukemogenic process. Notably, however, shortly after infection the virus enters into a latent state, in which viral gene expression is low in most of the HTLV-1 carriers' infected T-cells and so is the level of Tax protein, although rare infected cells may still display high viral RNA. This low Tax level is evidently insufficient for exerting its multiple oncogenic effects. Therefore, we propose that the latent virus must be activated, at least temporarily, in order to elevate Tax to its effective level and that during this transient activation state the infected cells may acquire some oncogenic mutations which can enable them to further progress towards ATL even if the activated virus is re-suppressed after a while. We conclude this review by outlining an hypothetical flow of events from the initial virus infection up to the ultimate ATL development and comment on the risk factors leading to ATL development in some people and to TSP/HAM in others. ==== Body Introduction Human T-cell leukemia virus type-I (HTLV-1) is the first discovered human retroviral pathogen [1]. It has been firmly implicated with the etiology of an aggressive malignancy known as adult T-cell leukemia (ATL) and of a neurological progressive inflammatory syndrome called tropical spastic paraparesis or HTLV-1 associated myelopathy (TSP/HAM). In addition, there are indications that it might be also associated with certain other clinical disorders [2,3]. In culture HTLV-1 can infect a wide variety of cell types from different species. However, in natural human infections this virus targets mainly mature CD4+ helper T-cells [4-6], resulting in benign expansion the infected cells [7]. Clonal or oligoclonal expansion of the infected CD4+ cells is mostly associated with development of ATL and 90–96% of the HTLV-I DNA is, indeed, found to segregate with CD4 cells in the peripheral blood of ATL patients [4], whereas CD4/CD8 double-positive leukemic cells are detected in rare cases [8]. CD8+ T-cells might also be infected [9,10], but their expansion is rather polyclonal and frequently occurs in asymptomatic carriers. Therefore, their disease association is unclear yet [11]. Shortly after infection the virus enters into a latent state, rendering the infected individuals asymptomatic seropositive carriers. About 5% of these individuals develop one of the viral associated diseases 10 to 40 years after infection. During latency the viral gene expression in the peripheral blood lymphocytes (PBLs) of such carriers is very low. Viral RNA is undetectable by Northern blot analysis in most of the infected cells (i.e. viral DNA harboring cells) freshly isolated from their peripheral blood [5], although it can be detected in some carriers by the highly sensitive RT/PCR analysis [12]. Furthermore, very little or no viral proteins are detectable in the carriers' PBLs [12,13]. Notably, despite this low virus expression, healthy carriers contain antibodies against viral antigens. They also display anti HTLV-1 specific cytotoxic T-lymphocytes (CTL) activity at variable levels that seem to be determined by hosts' genetic determinants, particularly by those associated with their HLA antigens [3,14,15]. Experimental evidence has been reported, pointing to the critical role of these two anti HTLV-1 immune response arms in keeping this low viral expression. It has been repeatedly shown that PBLs isolated from such carries start eliciting high viral gene expression within few hours of growing in culture [10,13,16]. However, Tochikura et al. have noted that addition of sera from HTLV-1 carriers or patients to the culture medium reduces this viral expression at an efficiency which correlates to their titer of anti HTLV-1 antibodies and that removal of these antibodies by protein A abolishes this inhibition. No such inhibition has been observed with sera of uninfected control donors [13]. Other workers have analyzed the level of HTLV-1 expression in PBLs grown in whole blood samples of various infected individuals and found that depletion of CTLs from these samples remarkably increases in the number of virus-expressing CD4+ cells compared to that found in the same samples without CTL-depletion [10,16]. Furthermore, these authors have demonstrated a similar increase by blocking the CTL-mediated cytolytic activity with concanamycin A. These data strongly suggest that anti HTLV-1 CTL activity, mounting in infected individuals, eliminate cells with high level of viral antigens and keep, thereby, the overall virus expression in the carriers' PBLs at low level. In view of this low virus expression, the viral load in HTLV-1 infected individuals has been noted to expand primarily through proliferation of the proviral DNA-harboring cells rather than through repeated cycles of cell-to-cell infection of new uninfected cells [17]. As discussed in our recent review article [18], this expansion pattern is widely considered to account for the maintenance of high sequence stability of the viral genome throughout the hundreds of thousands years of evolution since its emergence from its simian T-lymphotropic retrovirus origin. This stability is in striking contrast to the high genetic diversity of HIV-1 which is known to spread within the infected individuals through repeated infections of new cells by cell-free virions [19]. Although the mechanism of HTLV-1 pathogenicity is not fully understood yet, it is widely believed that a virally encoded transactivator protein, called Tax, plays a central role in this mechanism. It should, therefore, be noted that while the low level of the virus gene expression detected in latently infected carriers might be sufficient for maintaining their anti HTLV-1 seropositivity and CTL activity, the low Tax level, resulting from this reduced viral expression is, most likely, below its pathogenic threshold. This implies that generating an HTLV-1 related disease requires an activation of the dormant virus in order to elevate Tax to its pathogenic level. In this article we present a comprehensive review of the wide range of Tax molecular interactions and biological effects that might be closely relevant to the mechanism of ATL genesis and summarize this information by proposing hypothetical flow of a stepwise pathway leading to this malignancy or to TSP/HAM. HTLV-1 genomic structure and gene expression HTLV-1 is a complex retrovirus that, in addition to the two long terminal repeats (LTRs) and the gag, protease, pol and env genes, which are typical to most other retroviruses, its genome contains an additional region called pX, which resides between the env gene and the 3'-LTR,. This region includes four partially overlapping reading frames (ORFs), of which the most investigated ones are ORFs III and IV that encode for the viral regulatory Rex and Tax proteins respectively (see illustration in Fig. 1A). The gag, protease and pol precursor polypeptide is translated from the full genomic length viral RNA, whereas the env precursor polypeptide is translated from a singly spliced viral RNA. These precursor polypeptides are cleaved into the mature functional proteins by the viral protease. Tax and Rex are translated from a doubly spliced viral RNA, using two alternative translational initiation codons as illustrated in Fig. 1B. Figure 1 Schematic illustration of the HTLV-I genome organization (A) and its various mRNA species with their specific splicing and encoded protein products (B) (See the text for detailed explanation). Tax is present predominantly in the nucleus due to its nuclear localization signal (NLS) residing at its amino terminus [20,21]. However a substantial portion of Tax is present also in the cytoplasm due to its newly identified nuclear export signal (NES) [22]. Tax, which acts as a dimer [23], was originally discovered as a transactivator of viral RNA transcription from a promoter located at the 5'-LTR [24], but later proved to modulate the synthesis or function of a wide range of cellular regulatory proteins [25-27]. Rex, on the other hand, acts to promote the export of the unspliced and singly spliced viral RNAs species from the nucleus to the cytoplasm [28] by binding to a Rex responsive element (RxRE) residing in the 3' R region of the viral RNA [29]. In addition, there are some indications that Rex may also inhibit splicing and degradation of the viral RNAs [30]. Thus at high level of Rex there is a preferential export of the gag-protease-pol- and of the env-encoding RNA species and low export of the Tax/Rex-encoding RNA. This leads to a decline in the level of Rex and Tax proteins and consequently to a reduced viral RNA transcription. As a result, the Tax/Rex-encoding RNA is preferentially exported from the nucleus. In this manner Rex maintains these different RNA species at an optimal balance required for the virus production. Consistent with this notion Ye et al. [31] have shown that cells harboring proviral DNA with defective Rex reading frame produce high level of the doubly spliced tax/rex encoding mRNA and high level of functional Tax protein, but low level of p19 Gag protein and undetectable Rex protein. An alternatively spliced RNA encodes for another protein from ORF III, termed p21Rex, but its biological function is unclear [32]. More recently interest has been focused also on ORF I that encodes for p12 and p27 and ORF II that encodes for p13 and p30 proteins [33]. In contrast to Tax and Rex, which are encoded by a bicistronic pX mRNA formed by double splicing of the viral RNA [21,34,35], the other four accessory proteins are encoded by different pX mRNAs formed by alternative splicing events [33,36]. Pique et al. [37] have detected CTL activity in HTLV-I infected individuals against specific peptides from each of these ORF I and ORF II proteins, indicating that each of them is produced natural human infections. The functional role of these accessory proteins is not completely clear yet. Certain studies have demonstrated that deletions within frame I and II do not affect the replication and infectivity of HTLV-1 [36] nor its capacity to immortalize primary T-cells [36,38]. In contrast, by using molecular HTLV-1 clone, the group of Albrecht and Lairmore has provided evidence for the critical role of these accessory proteins in the viral replication and pathogenesis [33]. It has been shown that ablation of frame I markedly reduced the virus ability to infect quiescent peripheral blood lymphocyte (PBLs) [39] and to replicate in a rabbit model [40]. The explanation suggested by these investigators for the discrepancy between theirs and the others' results regarding p12 is that the other groups examined the role of this protein in IL-2/mitogen-activated PBLs, whereas their own data indicate that p12 is required for HTLV-1 infection in quiescent PBLs, since when they added a mitogen and IL-2 to their cultures the p12-defective HTLV-1 clone became highly infective [33,41]. Notably, p12 localizes to the endoplasmic reticulum (ER) and is associated with two ER-resident proteins; calerticulin and calnexin. Calerticulin is a calcium-binding protein that participates in calcium signaling and linked to activation of the transcription factor nuclear factor of activated T-cells (NFAT) [42]. In this manner, p12 can activate the HTLV-1 DNA clone-harboring quiescent PBLs and provide the physiological requirements for its infectivity, or vice versa, mitogen/IL-2 activation of the PBLs can override the deficiency imposed by the p12-defective clone. Since HTLV-1 targets quiescent T-cells in natural infection, these findings suggest an important role of p12 protein for the virus in vivo infectivity. The frame II encoded p30 protein has been shown to localize to the nucleus and to function as a transcription factor. Transient transfection experiments have demonstrated that this protein can modulate the expression of various promoters and to activate HTLV-I LTR expression independently of Tax [43]. It was also shown to interact with the transcriptional co-activators CREB-binding protein (CBP) and p300 [44]. Together, these and other data indicate that p30 may account for the activation of several genes in HTLV-1 infected cells [44] and play an important role in the virus replication [45] and maintaining high viral load in in-vivo infection [33,39,46]. In contrast, a recent study by Nicot et al. [47] have shown that p30 rather inhibits HTLV-I expression by binding to the tax/rex-encoding doubly spliced viral RNA and retaining it in the nucleus. In this manner p30 prevents the synthesis of Tax and Rex proteins and interferes, thereby, with the production of viral particles. Furthermore, high level of p30 has been found to interfere with Tax-induced activation of HTLV-I LTR [44]. In view of these data it has been suggested that by reducing HTLV-I expression high level of p30 protects the infected cells from the anti HTLV-I immune response and contribute, in this manner to the virus persistence [33]. The other frame II-encoded protein, p13 localizes in the mitochondria and alters its morphology and function [48]. This protein has been shown to be also essential for maintaining high viral load in rabbit [45,46]. It has been also demonstrated that p13 interferes with the phosphorylation of the guanine nucleotide exchanger Vav protein in T-cells [49]. Fig. 1 describes schematically the viral genome organization, its various mRNA species and the encoded proteins. Since Tax protein is widely regarded as a key element in the HTLV-1 related leukemogenic process. We will discuss in the following sections the molecular activities and biological effects of Tax that seem to contribute to its oncogenic potential. Modulation of viral and cellular gene expression by Tax Tax-mediated activation of CREB/ATF-dependent gene expression As noted before, Tax was initially discovered as a transactivator of the HTLV-1 gene expression [24]. It activates the viral LTR through three imperfectly conserved 21 bp repeats called Tax responsive elements (TxRE) [50], which contain a centered sequence TGACG(T/A)(C/G)(T/A) that is imperfectly homologous to the consensus cAMP responsive element (CRE; TGACGTCA) [51]. This element, which is also referred to as domain B of the TxRE, is flanked by a short G-rich stretch (AGGC) at its 5' side, termed domain A and a C-rich stretch (CCCC) at its 3' side, termed C domain C [27,51] (Fig. 2A). Although several basic leucine zipper (bZIP)-containing proteins, belonging to the CRE-binding/activating transcription factor (CREB/ATF) family, can bind to this viral CRE [52] only few of them can efficiently mediate the Tax-induced transactivation of HTLV-1 LTR [53-56]. A recent investigation of the effect of negative transdominant constructs against various bZIP proteins of this family has provided evidence that CREB is the most prominent factor that cooperates with Tax in activating HTLV-1 LTR expression [53]. Numerous earlier studies have demonstrated that in the absence of Tax, CREB forms unstable complex with the viral CRE, whereas Tax acts to stabilize this complex. By interacting with the bZIP region of CREB Tax enhances CREB dimerization and increases, thereby, its affinity to CRE [54,57-59]. This Tax-CREB-TxRE complex is further stabilized by direct binding of Tax to domains A and C of the TxRE through its N-terminus [60,61] (Fig. 2A). This stabilized binding enables Tax to recruit to the ternary Tax-CREB-TxRE complex the co-activators CREB binding protein (CBP) and its homologous protein p300 by binding to their KIX domain through its kinase-inducible domain (KID) [62] and the p300/CBP-associated factor (P/CAF), which binds through it carboxy terminus to a distinct site located around amino acid 318 to 320 of the Tax protein [63]. These three co-activators exert their effect by histone acethylation, which induces chromatin conformational modification at the site of the target promoter and facilitates, thereby, the interaction of the enhancer-bound transcriptional activators with the TATAA box-associated basal transcriptional factors [27] (Fig. 2A). Interestingly, however, Jiang et al. have shown that P/CAF can bind Tax without CBP or p300 and enhances its stimulatory effect on HTLV-1 LTR transcriptional expression independently of histone acetylation [63]. In contrast, several other studies have indicated that CREB2 (called also ATF-4), a member of another bZIP protein family, plays a more central role in Tax activation of HTLV-1 gene expression. These studies show that while in the absence of Tax, CREB can activate HTLV-1 LTR expression only if phosphorylated by protein kinase A (PKA), CREB2 can markedly activate the viral LTR without phosphorylation and that this protein mediates a much stronger activation of the viral LTR by Tax than CREB does [64-66]. Figure 2 Schematic illustration of the DNA elements and the activator and co-activator proteins involved in Tax-induced transcriptional activation of (A) HTLV-I LTR and (B) SRF-dependent promoters (See the text for detailed explanation). Of particular note are also the recent observations that when two copies of the TxRE are placed upstream to TATAA boxes from HTLV-1 LTR or from other promoters, the strongest activation by Tax is detected with the TATAA box of the HTLV-1 LTR, indicating that this TATAA box contains a specific Tax responsive element. Furthermore, these studies have also revealed that beside of the enhancing effect Tax on the association of the TATAA-box binding protein (TBP) to the TATAA site, Tax has an additional stimulatory effect that is directed towards a step occurring after the assembly of the basal transcriptional factors onto the TATAA box [53]. Many cellular genes contain in their promoters a consensus CRE element and are activated by signals that elevate the cellular cAMP level. The elevated cAMP activates PKA to phosphorylate CREB which, in turn, binds to CRE and to CBP/p300. However, there is a substantial controversy on whether Tax can activate only the viral CRE in its context with the CG-rich flanking domains in the viral LTR [25,61], or also CRE located in cellular promoters [67,68]. In addition, there are data demonstrating that Tax uses the CREB/ATF factors to repress the expression of certain genes, like the cyclin A [69], p53 [70] and c-myc [71]. This CRE-dependent effect of Tax on such cellular genes may contribute to the initiation of an oncogenic process by impairing the cell cycle and growth control. Tax mediated activation of SRF-dependent gene expression HTLV-1 infected and Tax-expressing T-cell lines display increased expression of immediate early genes such as c-Fos, c-Jun, JunB, JunD and Fra-1, which are components of the dimeric transcription factors AP1, Egr-1 and Egr-2 [72], fra-1 [73], Krox-20 and Krox-24 [74]. Formation of these transcription factors is normally activated by the serum responsive factor (SRF) in response to various mitogenic signaling agents like serum, lysophosphatidic acid (LPA), lipopolysaccharide (LPS), 12-O-tetradecanoylphorbol-13-acetate (TPA), cytokines and tumor necrosis factor-α (TNFα). SRF acts through an SRF responsive element (SRE) residing in the promoters of these genes [75]. The SRE region actually contains two binding sites; a CArG box [CC(A/T)6GG], and an upstream Ets box [GGA(A/T)]. After binding to the CArG box, SRF protein interacts with the ternary complex factors (TCFs), which consequently bind to the upstream Ets box. In addition, SRF requires for its transcriptional activity the CBP/p300 and p/CAF co-activators [76]. Tax activates these immediate early genes by interacting with SRF [77,78] and with TFCs, CBP/p300 and P/CAF [76] (Fig. 2B). Moreover, AP-1, which is highly expressed in HTLV-1 infected T-cells [79], regulates the expression of multiple genes essential for cell proliferation, differentiation and prevention of apoptosis [80], so that by activating SRF, Tax can also indirectly induce a wide variety of such cellular genes. Thus, constitutive activation of such genes in HTLV-1 infected T-cells independently of specific external signals might be a trigger for initial steps in the oncogenic transformation of HTLV-1 infected T-cells in culture as well as in human infection. Tax-mediated activation of NF-κB-dependent gene expression A substantial part of Tax oncogenic potential is attributed to its ability to activate transcription factors of the NF-κB family, since these factors regulate the expression of numerous cellular genes [81] associated with diverse biological processes, such as embryonic development, immune and inflammatory responses, cell growth, apoptosis, stress responses and oncogenesis [25,82-84]. The NF-κB factors are functionally related to the c-Rel proto-oncogene and include the p50(NF-κB1), p52(NF-κB2), p65(RelA), RelB and c-Rel proteins, which act in various combinations of homo- and heterodimers displaying distinct specificities. They share a common domain of 300 amino acids, termed Rel homology domain (RHD), which is involved in their dimerization, DNA binding and nuclear localization. The p65:p65 and p65:p50 κB are the most prominent dimers involved in NF-κB-dependent transcriptional activation, whereas the p50:p50 dimer is rather inhibitory [85]. In non-activated state NF-κB factors are trapped in the cytoplasm, tightly associated with inhibitory proteins called IκBs, primarily with IκBα and IκBβ. These inhibitors contain ankyrin repeats through which they bind to the RHD of the NF-κB factors and mask their nuclear localization signal (NLS) [86]. In addition, these complexes contain the catalytic subunit of protein kinase A (PKAc) which binds in the cytoplasm to both IκBα and IκBβ and is held there in an inactive state [87] (see illustration in Fig. 3 No. 1). NF-κB factors are activated in response to a wide variety of inflammatory cytokines and mitogens, such as TNF-α, IL-1, IL-6, IL-8, GM-CSF, bacterial lipopolysaccharide (LPS) and stress-inducing factors [81,83,84] (see Fig. 3, No. 2a and 3a). This activation proceeds in two phases, one taking place in the cytoplasm and the other in the nucleus. Figure 3 Schematic illustration of the factors and the molecular interactions associated with the release the NF-κB factors from their IκB inhibitors in the cytoplasm by external signaling stimuli and by HTLV-I Tax (See the text for detailed explanation). The cytoplasmic phase includes phosphorylation of IκBα on serine32 and serine36 and of IκB on serine19 and serine23 (Fig. 3, No. 6), which is followed by their ubiquitination and subsequent proteosomal degradation [88] (Fig. 3, No. 7). The release from IκBs, activates the associated PKAc, which phosphorylates the free p65(RelA) factor at its serine276 (Fig. 3, No. 8). As will be discussed later in more details, this phosphorylation is essential for the transcriptional activity the p65(RelA)-containing dimers [87]. In addition, degradation of the IκBs releases the sequestered NF-κB dimers to translocate to the nucleus [88] (Fig. 3, No. 9). The phosphorylation of IκBs is carried out by an IκB kinase (IKK) complex comprised of two catalytic subunits, IKK and IKK and a regulator subunit, IKK which is called also NF-κB essential modulator (NEMO) [89,90] (Fig. 3, No. 2a and 3a). IKKα and IKK share a 52% amino acid identity and a similar domain structure that includes amino-terminal kinase domain, a dimerization leucine zipper domain, and helix-loop-helix motifs, which are involved in regulating their kinase activity [89,90]. The phosphorylating function of the IKK complex is activated by upstream kinases such as the NF-κB inducing kinase (NIK) (Fig. 3, No. 2b), the mitogens-activated protein kinase/ERK kinase kinase-1 (MEKK1) (Fig. 3, No. 3b) and certain other signal-activated kinases [91]. NIK phosphorylates mainly the IKKα subunit (Fig. 3, No.2b), whereas MEKK1 activates both IKKα and IKKβ [92] (Fig. 3, No. 3b). Activation of IKKα results from its phosphorylation at serine176 and serine180, whereas IKK is activated by its phosphorylation at serine177 and serine181 [93,94]. Despite their high homology, IKKβ is much more active than IKKα in phosphorylating the IκBs [93,95,96]. This predominant activity of IKKβ over IKKα may be partially explained by the observation that in addition to the phosphorylation of IKKβ by MEKK1, IKKβ is directly phosphorylated also by IKKα, [97,98] (Fig. 3, No. 2b). A recent study has suggested an additional function for IKKα by showing that p65(RelA) needs to be phosphorylated by this kinase at serine536 in order to be transcriptionally active [99]. The third subunit, IKKγ/NEMO is devoid of kinase activity. Its role is to serve as a universal scaffold which connects between the two catalytic IKK subunits and their upstream activating factors into a large IKK complex [100,101] (Fig. 4, No. 2b and 3b). Iha et al., [102] have shown that these various factors assemble to the IKK complex through different domains of the IKKγ/NEMO protein, which could be selectively inactivated, thus attenuating certain NF-κB activating signals without affecting others. Figure 4 Schematic illustration of the factors and molecular interactions occurring in the nucleus which are involved in regulating the transcriptional competence of the NF-κB factors after reaching the nucleus and the function of HTLV-I Tax in this regulation (See the text for detailed explanation). Recently, much interest has been attracted to the nuclear regulation of the NF-κB transcriptional competence. It has been shown that after reaching the nucleus p65(RelA) can bind the CBP/p300 and P/CAF coactivators which are essential for the transcriptional competence of p65(RelA):p65(RelA) and p65(RelA):p50 dimers [103]. This binding depends on p65(RelA) phosphorylation at serine276 by PKA and certain other signal activated serine kinases [85,87,104-108] (see illustration in Fig. 5, No. 1a and 1b). This phosphorylation is blocked by an NF-κB-inducible protein termed SINK, which binds to p65(RelA). This binding does not affect the nuclear localization of p65(RelA), nor its binding to the target DNA sites. Instead, by inhibiting p65(RelA) phosphorylation SINK prevents its association with the CBP/p300 and P/CAF co-activators, thus creating a negative feedback control of p65(RelA) transcriptional activity [109]. Another inhibitor protein, called RelA-associated inhibitor (RAI), has been identified in the nucleus of certain cell types where it can interact with p65(RelA) and inhibit its transcriptional activity by blocking its DNA binding. It has been proposed that this protein provides an alternative cell-type specific control of NF-κB-dependent gene expression [110]. Figure 5 Schematic presentation of Tax biological effects which contribute to its oncogenic potential. In addition to its cytoplasmic inhibitory function IκBα plays an important regulatory role in the nucleus too. IκBα has an NLS signal which enables its translocation to the nucleus where it is protected from the signal-induced degradation described above [111]. Within the nucleus IκBα binds to the nuclear p65(RelA) and abrogates its transcriptional activity by inhibiting its DNA-binding [112]. IκBα has also a nuclear export signal (NES) which mediates the export of the p65(RelA):IκBα complex back to the cytoplasm via its interaction with the nuclear exporting protein CRM1 [113] (see Fig. 4, No. 2a, 2b, 2c and 2d). It has been proposed that as long as the signal-induced cytoplasmic degradation of the NF-κB-associated IκBα is active, induction of corresponding NF-κB-dependent gene expression can keep going on, whereas upon termination of this signal the export of the p65(RelA):IκBα complex from the nucleus may serve as an immediate terminator of this gene expression. However, the nuclear association of IκBα with p65(RelA) has been noted to depend on p65(RelA) acetylation status. The nuclear p65(RelA) can be acetylated by p300 and this acetylation avoids the binding of p65(RelA) to IκBα, thus preserving its transcriptional activity [114]. On the other hand, the nuclear p65(RelA) can bind to specific isoforms of histone deacetylase (HDAC) which deacetylate it and inhibit, thereby, its transcriptional activity by facilitating its association to IκBα [115]. (see Fig. 4. No. 2e). In contrast to this nuclear IκBα function, it has been noted that signals imposing persistent NF-κB activation, do so by enhancing the level of unphosphorylated IκBβ, which binds to p65(RelA) in the cytoplasm without masking its NLS or interfering with its DNA binding [116] (Fig. 4, No 3a, 3b and 3c). It has been proposed that under such conditions IκBβ escorts p65(RelA) to the nucleus, where it protects it from the inhibitory effect of the nuclear IκB and maintains, in this manner, a persistent NF-κB transcriptional activation [116]. IKK has also been found to have an important role in the nucleus (Fig. 4, No 4a) where it seems to affect the NF-κB transcriptional activity in several different ways. In one study the nuclear IKKα has been shown to bind CBP and p65(RelA) and to recruit, in this manner, the CBP co-activator to NF-κB-responsive promoters, where it acetylates histone H3 and facilitates, thereby, the expression of these promoters [117] (Fig. 4, No. 4b). Another study has shown that the nuclear p65(RelA)-associated IKKα stimulates the NF-κB-responsive promoters by directly phosphorylating histone H3 with its kinase activity [118] (Fig. 4, No 4c), and a third study has demonstrated that the nuclear IKKα phosphorylates the nuclear p65(RelA) and facilitates, thereby, its association with CBP/p300 [99] (Fig. 4, No 4d). IKKγ/NEMO too has been noted to translocate to the nucleus where it regulates the NF-κB transcriptional activity by competing with the nuclear p65(RelA) and IKKα for CBP/p300 [119] (Fig. 4, No. 5a and 5b correspondingly). In contrast to the transient NF-κB activation by external signals, NF-κB factors are constitutively activated by HTLV-1 Tax protein in Tax-expressing and HTLV-1-infected cells. Reported studies suggest that Tax may exert this activation in three ways: a) The most widely accepted concept is that Tax associates with the IKK complex through the adaptor IKKγ/NEMO subunit. Tax also binds to the upstream kinases, MEKK1 and NIK and enhances their kinase activity. In this manner Tax connects these activated kinases to IKKγ/NEMO and recruits their kinase activity to phosphorylate IKK and IKKβ [25,102,120-122] which, in turn, phoshphorylate IκBα and IκBβ (see Fig. 3, No. 4a, 4b and 6). A recent study have proposed that IKKγ/NEMO assembles into the large IKK complex as a homodimer or homotrimer and that its binding to Tax enhances its oligomerization [123]. b) Tax can bind directly to IKK and IKK and activates their kinase activity independently of their phosphorylation by the upstream signal-induced kinases [124] (Fig. 3, No. 5 and 6), c) Tax can bind directly to the IκBs and induce their proteosomal degradation independently of their phosphorylation by IKK [90,125] (Fig. 3, No. 10a, 10b and 10c). Thus, Tax induces phosphorylation-dependent or independent degradation of both of the IκBs and enables, thereby, the nuclear translocation of the released NF-κB factors independently of exogenous signals (Fig. 3, No. 9). A number of studies indicate that the nuclear Tax plays an important role in establishing the transcriptional activity of the NF-κB factors reaching to the nucleus. Bex et al. [126] have demonstrated that the nuclear Tax localizes in transcriptionally active structures containing the NF-κB factors p50 and p65(RelA), RNA polymerase II, nascent RNA and splicing factors. Other studies have shown that Tax physically binds to the NF-κB factors p65(RelA) [127], c-Rel [127], p50 [128] and p52 [129] and enhances, thereby, their dimerization [130], which is essential for their binding to the NF-κB responsive element in the target promoters [127,128]. Tax has noted also to associate with these factors when they are already bound to their DNA targets and facilitates their transcriptional activity [127,128]. In contrast, our own experiments, to be published elsewhere (manuscript in preparation), indicate that the binding of Tax to the free p65(RelA) factor occurs already in the cytoplasm and then the two proteins translocate to the nucleus together (see Fig. 3, No. 11a and 11b). In addition, it has been demonstrated that by its ability to bind the NF-κB factors [127-129] on one hand and the CBP/p300 [62,131] and P/CAF [63] co-activators on the other hand, Tax recruits these co-activators to the NF-κB factors independently of the above mentioned serine276 phosphorylation on p65(RelA) (see illustration in Fig. 4, No 6). However, a recent study has indicated that in order to be transcriptionally active, p65(RelA) needs to be phosphorylated by IKKα at serine536 even when activated by Tax [99]. This phosphorylation is mediated by Tax [99] through its capacity to physically bind IKK and IKKβ and induce their kinase activity [124]. Tax biological effects contributing to its oncogenic potential Enhancing T-cell proliferation There is ample of literature, reviewed in ref. [2,3,21,132,133], which demonstrate a modulation of expression of a wide range of cellular genes by Tax. cDNA profile analyses have detected several hundreds of Tax modulated cellular genes [82,134]. Some of them are directly involved in activation of T-cells proliferation, such as interleukin 2 (IL-2) [135] and the α subunit of its receptor (IL-2Rα) [136], which together establish an autocrine loop [137], IL-15 [138] and its receptor (IL-15R) [139], granulocyte-macrophage colony stimulating factor (GM-CSF) [140], tumor necrosis factor-α (TNF-α) [141], the MAD1 [142] and others [21]. Tax also activates cyclin D2 [143], cyclin D3 [144] and cdk6 [145], which are involved in the cell cycle progression, and inactivates p16INKA4 [146] which acts to restrain excessive cycle progression. In addition, Tax affects the functions of many regulatory proteins by physical binding to them. A recent protein profile analysis has revealed that Tax can form complexes with 32 different proteins. Many of them belong to the signal transduction and cytoskeleton pathways and transcription/chromatin remodeling [147]. Constitutive deregulation of such regulatory factors in HTLV-1 infected T-cells can set the cells into uncontrolled continuous proliferation. Induction of such a continuous proliferation of mature T-cells is likely one of the first steps in the initiation of the ATL leukemogenic process since it renders the cells more accessible to spontaneous and exogenously induced mutagenesis. Induction of genetic instability Enhancing mutagenesis via telomerase inhibition Telomeres are specialized nucleoprotein structures located at the ends of each chromosome. In human they consist of up to 15 kb long double stranded tracts of tandem TTAGG repeats, ending with a 3' single-stranded overhangs and are associated with a number of functional proteins [148]. These structures prevent chromosomes from fusing end-to-end with each other on one hand and protect them from degradation by exonucleases on the other hand. They also enable the cells to distinguish between ends of broken DNA and natural chromosomal ends and prevent these natural ends from initiating DNA damage-specific checkpoint or repair cascades [148]. Telomeres are formed by telomerase, which are present in germ and embryonal cells and in many cancers but not in normal adult somatic cells [149]. Hence, in the absence of telomerase activity the telomeres of normal somatic cells are progressively shortened in each cell division until they reach a critical length, at which point the cells enter a quiescent viable state and are subsequently eliminated by apoptosis. However, the same shortening process may abrogate the telomere's protective effects and allow, thereby, end-to-end chromosomal fusion that forms dicentric and multimeric chromosomal structures. Such structures can break during mitosis at variable points, resulting in aneuploidy and extensive non-reciprocal chromosomal translocations and rearrangements. This chromosomal instability can lead to accumulation of various mutations, including such that inactivate important checkpoints or induce a telomere-restoring mechanism, which may result in immortalization and carcinogenesis of the cells [150]. This implies that telomerase inactivation may, actually, play an important role in tumor initiation. Consistent with this notion many established human cancers maintain stabilized telomere length either due to mutational telomerase reactivation [149], or activation of other alternative mechanisms [151]. Of note in this context is, that unlike other types of human leukemia and lymphoma, ATL cells display numerous unique chromosomal aberrations [152,153] resembling those resulting from telomere dysfunction, frequently seen in solid tumors [149]. Moreover, HTLV-1 Tax has been recently found as capable of inactivating telomerase in a variety of cells [154], suggesting that telomerase inhibition in infected cells of HTLV-1 carriers might be one of the mechanisms by which Tax initiates the ATL-related leukemic process. This possibility is supported by data demonstrating that infection of primary peripheral blood T-lymphocytes with HTLV-1 results in an initial decline of the cell viability which parallels with reduction in telomerase activity and that this decline is subsequently followed by an outgrowth of selected immortal survivors displaying increased telomerase activity [155]. Additional support comes from the close correlation observed between telomerase activity in ATL cell and the clinical stage of the disease. Leukemic cells of acute ATL patients display the highest telomerase activity, whereas patients with less severe clinical stage, whose leukemic cells elicit high telomerase activity, were noted to rapidly progress to the acute form, suggesting that the increased telomerase activity is not a side result of the acute ATL conditions, but rather one of the causes leading to this stage [156]. Interference with DNA repair As noted above, HTLV-1 infected T-cells show high frequency of chromosomal abnormalities [152,153]. The first indication that Tax is associated with cellular genetic aberration came from the observation that Tax represses the expression of polymerase-β which is involved in DNA repair [157]. This notion was later substantiated by demonstrating the capacity of Tax to enhance mutation rate [158] and other types of genetic instability [159] via impairing the chromosomal segregation fidelity and interfering with several modes of DNA repair such as the, mismatch repair (MMR), base excision repair (BER) and nucleotide excision repair (NER) [160-165]. Particular interest has been focused in the last few years on Tax interference with NER, since this mode of repair is regarded as a major mechanism of maintaining the genome stability and its abrogation has been linked to increased cancer incidence [166]. However, there are several unresolved questions regarding the role of some factors in these DNA repair pathways. For example, Tax has been shown to enhance the expression of PCNA [167], an essential cofactor of DNA polymerase-δ and , which are involved with DNA replication and repair [168]. Mutagenesis analysis have suggested that the ability of Tax to stimulate PCNA correlates with its ability to inhibit NER [162]. Based on this observation it has been proposed that the increase of PCNA molar ratio over polymerase-δ interferes with the DNA repair activity of this polymerase without affecting its function in DNA replication [160,162]. However, this explanation seems to over-simplify the complex role of PCNA in coordinating polymerase-δ activities between DNA replication and DNA repair. Of note in this context is that moderate levels of p53 also stimulates PCNA [169], but yet NER is rather enhanced in these conditions [161]. Another explanation has been based on p53 ability to elevate the level of p21WAF-1 which binds to PCNA [170]. It has been proposed that this binding of p21WAF-1 directs PCNA towards inhibition of DNA replication without affecting NER [168] and that Tax can prevent this pathway by its capacity to inhibit p53 transcriptional activities [70,171,172]. However, we [173] and others [174] have shown that Tax also elevates p21WAF-1 and therefore, should accordingly, be anticipated to enhance NER rather than to inhibit it. Furthermore, other studies have demonstrated that p21WAF-1 is not needed for NER [175] or may even inhibit it [176]. Therefore, more intensive studies are needed to resolve these conflicts. Of note however, p53 has been found to act also at the early damage-recognition step of NER [177]. It would be interesting to find out whether Tax directs its inhibitory effect towards this early step of NER. In addition, p53 has been proved to be also directly involved in BER [178], suggesting that Tax interference with BER [164] might also be exerted through its inhibitory effect on p53 function. At any rate, this genetic destabilization by Tax is certainly an important element of Tax oncogenic potential. Inhibition of topoisomerase I Topoisomerase I (Topo-I) is involved in DNA synthesis and maintenance of the genome stability by participating in DNA repair and chromosome condensation. It alters DNA topology by transiently breaking one strand of the DNA, passing the other strand through the break and finally resealing the break [179]. Tax has been found to bind to topo-I and inhibit its activity [180], whereas Topo-I activity has been shown to be stimulated by p53 [181]. Thus Tax may interfere with Topo-I activity also through its effect on p53. This might be another way for Tax to destabilize the cellular genome, but more intensive investigation is required to further substantiate this possibility. Tax-mediated protection of HTLV-1 infected T-cells from stress-induced cell cycle arrest and apoptosis All the above effects of Tax which enhance mutations and other chromosomal aberrations and interfere with DNA repair should be expected to induce cell cycle arrest or apoptosis. This, in turn, should prevent further progression of the leukemogenic process in the infected cells, unless such cells can, somehow, escape the cell cycle arrest and apoptosis. There is a substantial controversy over the influence of Tax on the cell response to stress insults. While many studies have demonstrated that Tax protects cells from stress-induced cell cycle arrest or apoptosis [182-186], others have shown that it enhances the cell sensitivity to these stress-induced effects [186-190]. Indeed, cDNA microarray analysis of HTLV-1 Tax expressing cells exposed to DNA damage stress signal revealed elevated expression of pro- as well as of anti-apoptosis genes [191]. However, results from our [182] and other laboratories reviewed in ref. [186,191], suggest that in HTLV-1 producing T-cells the anti-apoptotic effects of Tax override its potential pro-apoptotic effects. Besides, Tax has been shown to suppress a wide range of factors participating in the apoptosis cascade on one hand and to stimulate factors acting as apoptosis inhibitors on the other hand [144,185,192,193]. A possible explanation for the above noted controversy is that in most of the studies presenting Tax pro-apoptotic effect, Tax was over-expressed through highly potent promoters. Excessive levels of Tax may, reasonably, sensitize the cells to apoptosis. However, these experimental conditions do not reflect the situation in HTLV-1 expressing T-cells, in which Tax cannot exceed the optimal level required for its replication due to the Rex-mediated fine regulation of the balance between the viral RNA species encoding for the gag, pol, prot and env proteins and those which encode for the Tax and Rex proteins [28,29] described earlier in this review. In addition, Tax has been shown to enhance the cell cycle progression and to release cells from stress-induced cell cycle arrest [26,145,194]. Experimental models for Tax oncogenicity Numerous studies have been focused on investigating Tax oncogenic potential in cultured cells and animal models. Most of them used plasmids expressing w.t. Tax or various Tax mutants under the control of HTLV-1 LTR or other different promoters. It was only few years ago that an infectious clone of the entire HTLV-1 genome was constructed and appropriate cell culture techniques were developed to introduce this clone into human primary PBLs. This clone was found as capable of propagating in cultured mammalian cells and to transform primary human T-lymphocytes [195-197]. This clone opened an opportunity to study the oncogenic potential of Tax and of various Tax mutants in the context of the entire viral genome. Some of the studies to be discussed in this section have been performed with such clones. Transformation of rodent cells Tax has been shown to induce neoplastic transformation of the rat fibroblast Rat-1 [198-203] and the mouse fibroblast NIH/3T3 [201] cell lines. Tax has been also shown to cooperate with the ras oncogene in transforming primary embryo fibroblasts [203]. Transformation was determined by formation of foci of morphologically transformed cells, colony formation in soft agar and tumor formation in nude mice. Several mechanisms have been proposed to mediate the transformation of Rat-1 cells: 1) Involvement of NF-κB [199], 2) involvement of CREB/ATF [68], 3) involvement of phosphoinositide-3 kinase-PKB/Akt [202] and 4) Stimulation of p21WAF-1 which prevents apoptosis and enhances the replication of the transformed cells [204]. The cooperation of Tax with ras in transforming primary embryo fibroblasts is postulated to be mediated by SRF through the CArG elements of SRF responding genes [198]. It should be emphasized, however, that maintenance of this transformation phenotype requires the continuous presence of active Tax and that no genetic mutation can be identified in these transformed cells [200]. Therefore, the process leading to this transformation is unlikely to reflect the entire pathway leading to ATL because the leukemic ATL barely express Tax [3] and they are characterized by intensive chromosomal aberration [152,153,165]. For the most, this transformation may reflect only the very early steps of the initiation of the ATL process. Immortalization and transformation of primary human T-lymphocytes A closer insight into the ATL leukemogenesis has been gained through studies using primary human T-lymphocytes. Such experiments have revealed that after infection in culture with HTLV-1 or permanent transfection with Tax, primary human T-cells undergo two stages of cellular changes. In the first stage the cell become immortalized but still remain dependent on IL-2 for their growth [205]. This immortalization has been shown to result from Tax-induced stimulation of the G1 phase-specific cyclin-dependent kinases CDK4 and CDK6, increased expression of signal transduction genes like cyclin G1, c-fgr, hPGT [206] and p21WAF-1 [204] and to be associated with mutations conferring increased telomerase activity [155]. Studies with different Tax mutants, deficient of CREB/ATF- or NF-κB-activation, have yielded conflicting results as to which of these two major regulatory pathways is involved in this Tax-mediated immortalization. While certain studies have shown that this immortalization depends on Tax ability to activate NF-κB [207,208], others have demonstrated that Tax mutants deficient of NF-κB activation still retain their capacity to induce this immortalization [209]. On the other hand, experiments with an infectious molecular clone of the entire HTLV-1 genome have shown that disruption of Tax ability to activate CREB results in preferential immortalization of CD8+ lymphocytes, rather than preferential immortalization of CD4+ lymphocytes seen with the wild-type infectious clone [210]. In addition, it has been found that disruption of Tax capacity to interact with CBP/p300 does not affect its immortalizing potential [195,210]. In the second stage few IL-2-independent clones of transformed cell emerge. Such transformed cells display an IL-2-independent constitutive activation of the IL-2 receptor (IL-2R) signaling pathway that includes the Janus kinases JAK1 and JAK3, and the signal transducers and activators of transcription STAT3 and STAT5, which are constitutively active in such cells [206,211-213]. Other studies have shown in such transformed cells a constitutive high expression of the growth factor independence-1 (Gfi-1) which is also involved in coffering their IL-2-independent growth [214]. In addition, intensive studies have been recently focused on factors participating in a negative regulation of the IL-2R associated pathway in HTLV-1 transformed T-cells. One of these factors is the SH2-containing tyrosine phosphatase SHP1. A gradual loss of this phosphatase has been noted to correlate with the progression of HTLV-1 infected primary T-cells from the immortalization (IL-2 dependence) to the transformation (loss of IL-2 dependence) stage [213,215]. Changes in other negative regulators of Jak/STAT/IL-2R pathway have been noted to vary between different transformed clones and, therefore, their role in acquiring the IL-2-indepence is unclear yet [213]. Also notable is that in contrast to the rodent cells, the HTLV-1 transformed primary human T-cells display high mutation rate [158] and other genetic aberrations [159,165,216]. Of particular interest in this context is the observation that exposure of HTLV-1 infected primary T-cells to carcinogens enhances a stepwise progression from their IL-2-dependent immortalized state to the autonomous transformed state [217]. Also interesting is the association noted between chromosome changes in such cells and their growth potential [216]. Many of the above changes observed in the HTLV-1/Tax immortalized and transformed primary human T-cells are quite analogous to those found in ATL cells. However, while fresh leukemic cells from ATL patients, as well as cell lines derived from these leukemic cells, are successfully engrafted in SCID mice and their leukemic infiltration to various organs is similar to that seen in ATL patients [218], primary human T-cells immortalized or transformed in culture by HTLV-1 infection, Tax transfection or intruding a molecular clone of the entire HTLV-1 genome, do not show such tumorigenicity in these mice [196,219]. This observation can be explained by postulating that during their progression through multiple selection steps in the infected patient the ATL leukemic cells accumulate selected genetic changes conferring their tumorigenic phenotype, whereas under culture conditions there is no selective pressure for preferential accumulation of such particular mutations. Tumor induction in transgenic mice Tax transgenic mice have been widely used as models for investigating the oncogenic effects of Tax in-vivo, hoping to get closer insight to the ATL leukemogenic process in human [220]. A wide range of different tumors have been described in such animals and it appears that the promoter used to express Tax determines at least partially the type of the developing tumors. Transgenic mice expressing Tax through HTLV-1 LTR were found to develop neurofibrosarcomas [221], mesenchymal tumors [222] or skeletal bone abnormalities [223], but not leukemias or lymphomas. Mice expressing Tax through the promoter of CD3ε were found to develop mesenchymal tumors at wound sites and salivary and mammary adenoma [224]. Only mice expressing Tax through the granzyme B promoter showed Tax expression in mature T-lymphocytes and developed large granular lymphocytic leukemia [225]. These studies suggest that Tax alone is capable of inducing tumors in various tissues, including lymphoid cells. A possible explanation for the failure of HTLV-1 LTR-Tax to induce leukemia in such animals may be provided by the observation that expression of this construct can be detected in various non-lymphoid organs like the brain, saliva glands, spleen, thymus, skin, muscle, bones and mammary glands [223,226] but not in the bone marrow [223]. It has been proposed that activation of HTLV-1 LTR expression in lymphoid cells requires the cooperation of the accessory proteins encoded by ORF1 and/or ORF II of the pX region with Tax protein. Therefore, Tax alone cannot activate the expression of the HTLV-1 LTR-Tax construct in these cells, whereas this cooperation is not needed in other organs. [3,33,226] Various modes of tumor induction by the transgenic Tax have been noted so far. Hall et al. [224] have shown that the mesenchymal and the mammary adenomal tumor induced by the CD3ε-Tax transgene displayed high levels of apoptosis which is associated with high levels of Myc, Jun and p53. In contrast Portis et al. [227] have demonstrated a Tax mediated functional inactivation of p53 in the early stage of the large granular lymphocytic tumor formation by the granzyme B-Tax transgene and p53-inactivating mutations in a later stage of the tumor progression. Other studies have demonstrated the importance of Tax-mediated activation of NF-κB in the induction of both the lymphoid [228] and non-lymphoid [229] tumors. As noted before, continuous Tax expression is required for maintaining the neoplastic phenotype of Rat-1 cells transformed by Tax in culture [200]. In contrast, suppression of Tax expression in transformed fibroblasts derived from tumors of Tax transgenic mice did not affect their growth rate and ability to form tumors in animals [230], indicating that Tax was involved only in the initiation of the in-vivo tumorigenic process, after which the cells continued to progress through several genetic changes rendering their neoplastic phenotype independent of Tax. Conclusive comments about the pathways leading to ATL and TSP/HAM In view of the above described pleiotropic effects of Tax, which are summarized in Fig. 5, it is widely accepted that the viral Tax protein is a key element in ATL genesis [2]. This implies that generating this malignancy requires active viral gene expression in the infected T-cells of HTLV-1 carriers in order to keep Tax protein at an effective level. However, as noted before, shortly after establishing the host immune response against the viral antigens, HTLV-1 virus expression is kept very low and is the level of Tax. This low Tax level accounts, likely, the "carrier" state of the infected individuals by supporting a limited but continuous expansion of the infected CD4+ cells [17] and for their anti HTLV-1 seropositive states. However, this low Tax level, plausibly, is insufficient for exerting all the above described oncogenic effects leading to ATL. Therefore, over 95% of the infected individuals do not develop this malignancy, or other HTLV-1 related clinical disorders, during their entire life [2,3]. Thus, generating this malignancy would, plausibly, require activation of the dormant virus in order to elevate Tax to its oncogenic threshold. Our previous studies [182,231-233] indicate that this activation can be induced by a variety of stress agents which are widely present in the daily human surrounding. However, such agents normally induce also cell cycle arrest or apoptosis, which could be expected to prevent the subsequent progression towards ATL. This paradoxical conflict was resolved by our observation that Tax protects HTLV-1 producing human T-cells from stress-induced apoptosis [182], implying that the Tax protein, emerging after activation of the latent virus, can rescue the host cells from the stressed-induced apoptosis. After this activation step there may, actually, be a progression to either ATL or TSP/HAM. Intensive studies, reviewed in ref. [3,234], indicate that genetic factors of the host, mainly those associated with the HLA histocompatibily complex class I, are the major factors determining whether the progression will proceed towards ATL or TSP/HAM [235]. Of note is that TSP/HAM is characterized by high virus expression [236-238]. Such high virus expression is widely considered to be a predisposing factor for TSP/HAM development [239]. We [240] and others [235] discussed in details in earlier review articles, how this high virus expression accounts for most of the pathological and immunological manifestations of this syndrome and correlates with its severity [238]. On the other hand, no or very little Tax can be detected in the leukemic cells of ATL patients [3,12,13,16,234]. It has been proved that anti Tax CTLs mounted in these patients eliminate the rare cells with high Tax expression and keep, thereby, Tax at very low level [10,16]. This difference seems to be determined by the HLA type of the host [14]. It can be postulated that the immune response of people with HLA types of high risk for TSP/HAM, permits permanent high expression of the activated virus, whereas the immune response of people with other HLA types probably act to re-suppress the activated virus. Therefore, progression towards ATL can, presumably, proceed only if a mutation, that abrogates one of the important cellular checkpoints, occurs before the activated virus is re-suppressed. This speculation is supported by reports showing that the leukemic cells of most ATL patients carry one or more mutations which deregulate the formation or function of cellular factors associated with T-cell replication, cell cycle arrest or apoptosis, such as the IL-2 receptor [241], the JAK/STAT proteins [242], the growth factor independence 1 (Gfi) [214], p53 [243-245], p15INK4B and p16INK4A [246], p27KIP1 [247], p16 (CDKN2) [248], pRb [249], surviving [193], Fas (Apo1/CD95) [250] and caspases [251]. Interestingly, the leukemic cells of most ATL patient are defective in the mitotic spindle checkpoint [252], which likely accounts for the frequent clastogenic and aneugenic chromosomal abnormalities detected in these cells [153]. However, no mutation in mitotic checkpoint genes has been identified in such cells [252]. Instead, the viral Tax protein has been shown, in one study, to inactivate the function of the mitotic spindle checkpoint protein MAD1 [142]. In another study the MAD1 and MAD2 checkpoint proteins, which normally reside in the nucleus, have been found in HTLV-1 infected cells to localize predominantly in the cytoplasm [252]. Since Tax is hardly detected in circulating ATL cells, it is rather unlikely to ascribe this checkpoint loss in the ATL cells to Tax activity. It seems more reasonable to speculate that this loss results from mutations in other genes which might indirectly affect the proper subcellular localization of these proteins. It is also reasonable to assume that this and the other mutations in the above mentioned regulatory genes are, most likely, acquired in the pre-leukemic stage during a certain time-gap when the cells are highly susceptible to mutagenesis. We like to propose that this time-gap is the time when Tax is still highly active in a large number of circular T-cells due to the putative activation of the latent virus. It is plausible to assume that the level of the anti Tax antibodies existing in latent HTLV-1 carriers is sufficient to repress Tax expression in the few high virus-expressing CD4+ cells [13] existing before activation of the latent virus and that existing level of anti Tax CTLs in such carriers is sufficient to eliminate these cells [10,16], but neither of these Immune response arms is sufficient to handle the overwhelming number of such high virus-expressing cells resulting from the virus activation. However, this situation is likely temporary and may plausibly last until the anti Tax antibodies and CTLs boosted to mount to a sufficiently higher level that can overcome this large number of high virus-expressing cells. This is the time-gap during which we thing that the pre-leukemic cells are most susceptible to mutagenesis and can acquire one or more of the above mentioned checkpoint-abrogating mutations. If, after the occurrence of such mutations, Tax expression is re-suppressed by the mounting level of the anti Tax antibodies and cells that still remain with high virus expression are eliminated by the mounting CTLs, this will not stop the remaining mutant cells to further accumulate additional mutations and progress towards ATL. This re-suppressed Tax expression will avoid exposure of the progressing cells to anti Tax CTLs. However, since the probability for such particular mutations to occur during this limited time-gap might be very low, it is quite possible that multiple episodes of such virus activation and re-suppression may occur in HTLV-1 carriers before progression to ATL can be turned on. Such flow of events, which is illustrated in Fig. 6, may explain why ATL usually develops after much longer clinical latency than TSP/HAM. Figure 6 Schematic hypothetical flow of the events occurring between the initial infection with HTLV-I and ATL or TSP/HAM development (See the text for detailed explanation). Authors' contributions Authour 1; (A.I) prepared the Fig.ures and together with author 2 (S-K.Y) covered the cited publications and prepared the draft of this review. Author 3 (A.M) designed the outlines of the review and together with the other two authors prepared the final version for submission. All authors read and approved the final manuscript. 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Immunopathogenesis of human T cell lymphotropic virus type I-associated neurologic disease J Infect Dis 2002 186 S187 192 12424696 10.1086/344269 Yoshida M Osame M Kawai H Toita M Kuwashaki N Nishida Y Hiraki Y Takahashi K Nomura K Sonada S Eiraku N Ijichi S Usuku K Increased replication of HTLV-I in HTLV-I-associated myelopathy Ann Neurol 1989 26 331 335 2802532 Gessain A Louie A Gout O Gallo RC Franchini G Human T-cell leukemia virus type-I (HTLV-I) expression in fresh peripheral blood mononuclear cells from patients with tropical spastic paraparesis/HTLV-I associated myelopathy J Virol 1991 65 1628 1633 1995955 Yamano Y Nagai M Brennan M Mora CA Soldan SS Tomaru U Takenouchi N Izumo S Osame M Jacobson S Correlation of human T-cell lymphotropic virus type 1 (HTLV-1) mRNA with proviral DNA load, virus-specific CD8+ T cells, and disease severity in HTLV-1-associated myelopathy (HAM/TSP) Blood 2002 99 88 94 11756157 10.1182/blood.V99.1.88 Nagai M Usuku K Matsumoto W Kodama D Takenouchi N Moritoyo T Hashiguchi S Ichinose M Bangham CR Izumo S Osame M Analysis of HTLV-I proviral load in 202 HAM/TSP patients and 243 asymptomatic HTLV-I carriers: high proviral load strongly predisposes to HAM/TSP J Neurovirol 1998 4 586 593 10065900 Gold E Schavinsky-Khrapunsky Y Ben-Aroya Z Aboud M Huleihel M Molecular aspects in HTLV-I pathogenesis Current Topics in Virol 2003 3 53 67 Baba H Yamada Y Mori N Hayashibara T Harasawa H Tsuruda K Sugahara K Soda H Takasaki Y Tawara M Hirakata Y Tomonaga M Kamihira S Multiple gammac-receptor expression in adult T-cell leukemia Eur J Haematol 2002 68 362 369 12225394 10.1034/j.1600-0609.2002.00653.x Takemoto S Mulloy JC Cereseto A Migone T-S Patel BKR Matsuoka M Yamaguchi K Takatsuki K Kamihira S White JD Leonard WJ Waldmann T Franchini G Proliferation of adult T cell leukemia/lymphoma cells is associated with the constitutive activation of JAK/STATproteins Proc Natl Acad Sci U S A 1997 94 13897 13902 9391124 10.1073/pnas.94.25.13897 Cesarman E Chadburn A Inghirami G Gaidano G Knowles DM Structural and functional analysis of oncogenes and tumor suppressor genes in adult T-cell leukemia/lymphoma shows frequent p53 mutations Blood 1992 80 3205 3216 1361372 Nishimura S Asou N Suzushima H Okubo T Fujimoto T Osato M Yamasaki H Lisha L Takatsuki K p53 gene mutation and loss of heterozygosity are associated with increased risk of disease progression in adult T cell leukemia Leukemia 1995 9 598 604 7723391 Sakashita A Hattori T Miller CW Suzushima H Asou N Takatsuki K Koeffler HP Mutations of the p53 gene in adult T-cell leukemia Blood 1992 79 477 480 1730092 Yamada Y Hatta Y Murata K Sugawara K Ikeda S Mine M Maeda T Hirakata Y Kamihira S Tsukasaki K Ogawa S Hirai H Koeffler HP Tomonaga M Deletion of p15 and/or p16 genes as a poor-prognosis factor in adult T-cell leukemia J Clin Oncol 1997 15 1778 1785 9164185 Morosetti R Kawamata N Gombart AF Miller CW Hatta Y Hirama T Said JW Tomonaga M Koeffler HP Alterations of the p27KIP1 gene in non-Hodgkin's lymphomas and adult T-cell leukemia/lymphoma Blood 1995 86 1924 1930 7655021 Uchida T Kinoshita T Watanabe T Nagai H Murate T Saito H Hotta T The CDKN2 gene alterations in various types of adult T-cell leukaemia Br J Haematol 1996 94 665 670 8826890 10.1046/j.1365-2141.1996.d01-1841.x Nakayama K Yamada Y Koji T Hayashi T Tomonaga M Kamihira S Expression and phosphorylation status of retinoblastoma protein in adult T-cell leukemia/lymphoma Leuk Res 2000 24 299 305 10713327 10.1016/S0145-2126(99)00186-1 Maeda T Yamada Y Moriuchi R Sugahara K Tsuruda K Joh T Atogami S Tsukasaki K Tomonaga M Kamihira S Fas gene mutation in the progression of adult T cell leukemia J Exp Med 1999 189 1063 1071 10190897 10.1084/jem.189.7.1063 Kamihira S Yamada Y Hirakata Y Tomonaga M Sugahara K Hayashi T Dateki N Harasawa H Nakayama K Aberrant expression of caspase cascade regulatory genes in adult T-cell leukaemia: survivin is an important determinant for prognosis Br J Haematol 2001 114 63 69 11472346 10.1046/j.1365-2141.2001.02902.x Kasai T Iwanaga Y Iha H Jeang KT Prevalent loss of mitotic spindle checkpoint in adult T-cell leukemia confers resistance to microtubule inhibitors J Biol Chem 2002 277 5187 5193 11729202 10.1074/jbc.M110295200

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Retrovirology. 2004 Aug 13; 1:20

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Retrovirology

10.1186/1742-4690-1-20

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==== Front BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-5-1071530169310.1186/1471-2105-5-107SoftwareSTING Millennium Suite: integrated software for extensive analyses of 3d structures of proteins and their complexes Higa Roberto H [email protected] Roberto C [email protected] Arnaldo J [email protected] Juliana CF [email protected] Igor KS [email protected] Paula R [email protected] Michel EB [email protected] Adauto L [email protected] Goran [email protected] Núcleo de Bioinformática, Centro Nacional de Pesquisa Agropecuária, Empresa Brasileira de Pesquisa Agropecuária, Campinas, SP, Brazil2 Laboratório de Bioinformática, Embrapa/Recursos Genéticos e Biotecnologia, Empresa Brasileira de Pesquisa Agropecuária, Brasília, DF, Brazil2004 9 8 2004 5 107 107 26 1 2004 9 8 2004 Copyright © 2004 Higa et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The integration of many aspects of protein/DNA structure analysis is an important requirement for software products in general area of structural bioinformatics. In fact, there are too few software packages on the internet which can be described as successful in this respect. We might say that what is still missing is publicly available, web based software for interactive analysis of the sequence/structure/function of proteins and their complexes with DNA and ligands. Some of existing software packages do have certain level of integration and do offer analysis of several structure related parameters, however not to the extent generally demanded by a user. Results We are reporting here about new Sting Millennium Suite (SMS) version which is fully accessible (including for local files at client end), web based software for molecular structure and sequence/structure/function analysis. The new SMS client version is now operational also on Linux boxes and it works with non-public pdb formatted files (structures not deposited at the RCSB/PDB), eliminating earlier requirement for the registration if SMS components were to be used with user's local files. At the same time the new SMS offers some important additions and improvements such as link to ProTherm as well as significant re-engineering of SMS component ConSSeq. Also, we have added 3 new SMS mirror sites to existing network of global SMS servers: Argentina, Japan and Spain. Conclusion SMS is already established software package and many key data base and software servers worldwide, do offer either a link to, or host the SMS. SMS (Sting Millennium Suite) is web-based publicly available software developed to aid researches in their quest for translating information about the structures of macromolecules into knowledge. SMS allows to a user to interactively analyze molecular structures, cross-referencing visualized information with a correlated one, available across the internet. SMS is already used as a didactic tool by some universities. SMS analysis is now possible on Linux OS boxes and with no requirement for registration when using local files. ==== Body Background A need to integrate, visualize and mine large amount of protein structure data is accelerating. In order to accommodate visualization of data originating from several sources and make analysis of protein structure and structural parameters easier, we developed Sting Millennium Suite (SMS). SMS is a web-based suite of programs and databases providing visualization and a complex analysis of molecular sequence and structure for the data deposited at the Protein Data Bank (PDB) [1]. Using SMS it is possible to analyze: sequence to structure relationships, quality of the structure, nature and volume of atomic contacts of intra and inter chain type, relative conservation of amino acids at the specific sequence position based on multiple sequence alignment, indications of Folding Essential Residue (FER) based on relationship of the residue conservation to the intra-chain contacts, Cα – Cα and Cβ – Cβ distance geometry etc.. Specific emphasis in SMS is given to Interface Forming Residues (IFR) – amino acids that define interactive portion of the protein surfaces. SMS may simultaneously display and analyze previously superimposed structures. Parsing of data from relevant Data Bases (PDB [1], HSSP [2,3], Prosite [4]) is one of the key features of integrated SMS environment for structure/function analysis. SMS also has its own built in data bases: Contacts, Interface Contacts, Surface Accessibility, Dihedral Angles and Secondary Structure Elements [5]. This article is intended to show how Sting Millennium Suite of programs can be useful in the study of protein structure and analysis of its function, emphasizing recent improvement introduced to SMS. The program has extensive built-in instructions and detailed easy-to-use help which user is invited to consult before and during SMS use. Results and Discussion SMS overview In addition to basic macromolecular visualization, SMS is capable of identifying and visualizing the macromolecular interfaces as well as showing and analyzing previously aligned structures. SMS also does visualization of amino acids conservation based on multiple sequence alignments, in the context of three-dimensional protein structure, identification of the nature and volume of atomic contacts of intra and inter-chain type, presentation of data about the quality of a given structure etc.. SMS provides number of modules (SMS components (some of which are to be described in details separately)) to conveniently visualize large amount of physical-chemical, structural and biological information about the proteins with known structure. Variety of one-click-away renderings and color schemes helps to visualize bonding interactions and locations of residues of interest, as well as to localize patterns of evolution/conservation. The interactions which occur in the protein or between protein and its inhibitor/substrate, can be analyzed in great details with SMS. Graphical contacts SMS offers to the user a graphical presentation of inter-atomic contacts established between amino acids in form of the fan. The base point of the fan is the selected amino acid. From the base point a user can detect number of colored lines connecting to other residues (presented by single letter code). Colors of the fan lines follow SMS code of contacts. A specific HTML table displays residue name and number, its pair in contact establishing, type of the contact, distance between contacting atoms and accessibility and entropy of two contacting residues. Such contacts are divided in number of classes: hydrogen bonds, hydrogen bonds with intermediary water molecules, hydrophobic contacts, aromatic ring stacking contacts, electrostatic (attractive and repulsive) contacts and finally disulphide bridges. A special table is built for those interactions across the interface (IFR Graphical Contacts). Both Graphical and IFR Contacts are fully integrated with SMS so that information about any particular amino acid is highlighted in simultaneous fashion across sequence, structure and contacts window. The diagram Ramachandran Plot [6], used for checking the quality of the structure, is presented in SMS using all advantages of Java programming language. Menu options on interactive SMS Ramachandran Plot allow for coupling of data displayed in the dihedral angle window with a window showing the 3D structure of a molecule. Number of subsets among amino acids can be highlighted for better correlation of a 3D structure position and a phi-psi spot. Full integration and data coupling makes this SMS component a breed apart from the similar public domain products. A user may also produce an image in the gif format which is more appropriate for printing of publication quality figures. Again, SMS Ramachandran Plot is fully integrated with other SMS windows, allowing a user to concomitantly see structure and sequence information highlighted according to selection done in Ramachandran plot or in the sequence window. The module Scorpion provides a graphical presentation for simple statistical data on a frequency of occurrence for given amino acid and also for amino acid local environment in terms of class of amino acids surrounding given central residue. The Protein Dossier module provides a graphical report of several important structural characteristics of the PDB entry. It offers a plot from PDB cartoon annotated with color coded scales representing for each amino acid a corresponding temperature factor, solvent accessibility of the chain in isolation and in a complex with other present chains in the PDB file, sequence conservation in (HSSP derived) multiple alignment (relative sequence entropy) and histograms representing the atomic contacts (as in the Graphical contacts module), as well as IFR residue identification and IFR contacts. In addition, comparison of the Secondary Structure annotated by PDB, by DSSP [7] and by STRIDE [8] is presented. With STINGpaint it is possible to paint amino acids within multiple alignment of sequences according to two optional color schemes: STING's scheme and William Taylor [9] color scheme. This has effect on how easily the user can grasp regions of sequence identity. In addition, the user is presented with an entropy bar which facilitates even further pinpointing highly variable positions. The ConSSeq presents a sequence for a given PDB file and a consensus sequence (as found in the HSSP). A consensus sequence is obtained from the sequence alignment of the sequence-wise homologous proteins. Above those two sequences, ConSSeq shows a graphic bars colored by scale of colors according to the sequence conservation. The height of graphic bars is reflecting relative entropy. ConSSeq also offers information about residues present in other homologous sequences, with their respective frequency. For fast inspection of data, this program also generates a sequence logo. Complete interactivity with both sequence and chime-structure frame/window of the SMS is now operational, offering much better conditions for the thorough analysis of structure and sequence (alignment) interdependence. The Java Cα-Cα [Cβ-Cβ] Distance Plot is a diagram where the distances between the α [β] carbon of one residue and all α [β] carbon atoms of other residues, within a single chain of the PDB file, are represented by colored squares in a symmetrical plot. All the above mentioned modules and some others available from SMS, can be accessed either from the STING Millennium's sequence window or entering through the independent entry web page. An extensive list of links is available to increment a volume of information on a protein under the study. In this new SMS release we introduced ProTherm [10] link, exceptionally important information on protein stability/mutations, provided by the web site of Dr. Akinori Sarai group. The Sting Millennium and some of the SMS components are now capable of importing local files in PDB format. Algorithm and implementation SMS is organized in two logical layers: SMS server and SMS client. The server side is responsible for updating regularly all relevant public domain databases used by SMS. At the same time, SMS server is also responsible for calculation of a number of macromolecular properties for each PDB structure. The SMS client side provides to a user a friendly graphical interface and communicates to the SMS server, sending user's requests and receiving SMS responses. SMS interactive interface has been mostly implemented in the Java programming language, taking advantage of its object oriented design and graphical representation capabilities. Most important Java classes in SMS are dedicated to sequence and structure parameter presentation, depiction and interaction. Additional classes are used for efficient data handling utilities. As it is known, the object oriented software design is suitable especially because of its ease in code reusability and also because it provides interfaces for linking new software modules, resulting in systems easily expandable and built with extended capabilities. In addition, the Java programming language is very attractive to users for reasons of portability a key feature in today's versatile computing world. SMS also make extensive use of the C++ programming language, mostly for complex calculation of specific parameters. SMS runs in the Netscape browser or in Microsoft Internet Explorer (for Microsoft Windows operating system) and in the Netscape/Mozila (for the Linux OS) and requires installation of Java Plugin 1.3.1 and CHIME. Some restrictions apply, so a user is invited to consult details of SMS Requirements. Users can run the SMS program by selecting a previously deposited structure in the Protein Data Bank, or using local files with pdb format. Input file format for SMS SMS accepts the PDB format files from RCSB/PDB repository and also accepts local files of the same format, at the client end. A user is able to see structure of the local file in chime/SMS structure window as well as a sequence corresponding to this particular structure. The sequence itself is presented in the separate sequence window. Additionally, some other SMS components will work fine with user's local files: Graphical Contacts, IFR Graphical Contacts, SMS Ramachandran Plot, Scorpion, Formiga, Ca-Ca and Cb-CB contacts and Protein Dossier (although the last one might not have all the usual components that it displays for public PDB files). Comparison to other software packages Increase in availability of molecular structure data during the last decade, urged the development of computer applications for sequence/structure analysis and visualization. Consequently, numerous approaches have been made to the problem of sequence/structure visualization and analysis, resulting in diverse software packages: Protein Explorer, Cn3D, Swiss PdbViewer and ProCheck [11-14]. Each of these products seems to have been developed primarily to accomplish specific tasks. Inevitably, these products have differential strengths in areas that they cover, making difficult the task of comparisons and definitely arbitrary to certain extent. SMS, as well as comparable software resources, come with intuitive user friendly GUIs, allowing for easy navigation through the vast amount of structural data. SMS main advantage is the clear presentation of sequence along with the structure in addition to number of visualizing tools for variety of structure describing parameters. In the input layer SMS uses data from public databases: PDB, HSSP, DSSP and SwissProt. Simultaneous display of computed features/parameters/descriptors along with available annotations from above Databanks provides a useful and reach environment, which may complement and in many cases substitute and exceed the already existing tools for sequence/structure/function analysis and visualization. Conclusions Structure analysis is a difficult task due to the large amount of possible parameters/descriptors that can be calculated and associated with the sequence and corresponding structure. The way in which structure data and structure descriptors are stored and displayed, represents a major challenge when interactivity of a user with the data dispersed among many resources is addressed. Several structure viewers already exist, each one of them better suited to different needs and research interests. SMS offers an easy to use computer environment, designed to facilitate concomitant display of as many parameters as possible, coupled in a consistent fashion to each other. Experimental data and calculated information are all embodied in a clear display that offers instantly an intuitive aspect of a given structure and a large amount of biological information at hand. Inspection of the SMS displayed information can lead to valuable conclusions and cover a wide variety of biology issues concerning entire protein families. SMS has already been applied as a didactic tool for learning details about sequence/structure/function relationship in several universities. Future plans to extend the software platform include the ability to handle ever more descriptors/parameters of protein structure with the simultaneous display and analysis including data extracted from the statistical elaboration of common features among members of certain protein families/folds. In order to achieve such goal, we count with most generic yet very usable tools: Chime viewer and JAVA programming language. In addition, we count on growing interest of other research groups in participating in this project, contributing with their data and benefiting from the resulting unification of data format and data display. Issues such as the geometrical increase in the volume of the disk space and available CPU time for updating such a large data base should be taken into account. SMS is available free and can be accessed through the web. A user has to be careful with proper configuration of IT components (Operating System, browser, Chime viewer, Java JER version, firewall warnings) so that SMS can be used to its fullest potential. The detailed online manual/help/tutorial for viewing and analyzing displayed data is available and recommended for frequent consultation. Availability and requirements Project Name: STING Millennium Suite Lab Home Page: Project Home Page: Operating System(s): Servers: Extensively tested on SGI IRIX 6.5, SUN Solaris7.0 and 8.0 and LINUX Red Hat 7.3, 8.0 Clients: MS Windows XP, NT, 2000 with Netscape 7.0 and IE 6.0 SP1, platform with Java Runtime Environment (JRE) 1.3.1 installed and Linux Rad Hat with Mozila/Netscape 7.0 and CrossOver plugin. Chime 2.6 SP3/SP4 (depending on OS and browser used) plugin is essential for structure presentation. Programming Language: JAVA, C++, Fortran, JavaScript Other requirements: Installation of JRE 1.3.1. License: Free for Academic use. Abbreviations SMS: Sting Millennium Suite IFR: Interface Forming Residues FER: Folding Essential Residue PDB: Protein Data Bank RCSB: Research Collaboratory for Structural Bioinformatics GUI: Graphical User Interface Authors' contributions RH created the Graphical User Interface and part of the data processing programming and general procedures for SMS mirror installation, RT worked on general GUI and statistics for SMS access, AJM worked on ConSSeq integration to SMS, JP worked on HTML design and SMS help pages, IO worked on SMS implementation on Linux OS, PK worked on general data interpretation and GUI suggestions, MY worked on mathematical algorithms for parameter calculation, AM carried out most of the data processing and programming, and GN coordinated the whole project, suggesting the general directions and innovating features of the application. All authors have read and accepted the final manuscript. Acknowledgements This work was supported in part by following grants: FAPESP 01/08895-0, FINEP 1945/01 and CNPq 521093/2001–5 (NV). Figures and Tables Figure 1 SMS sequence and structure window. This SMS example screen displays the pdb file 1cho.pdb, highlighting the residue Leu46 in chain E. SMS sequence frame, from where all the SMS modules and features can be accessed, is shown at the top of this figure. The sequence is colored according to physical-chemical properties of the amino acids. The blue and red lines underneath the amino acid sequence represent the secondary structure elements (beta strands and helices respectively), according to the pdb file annotation. As the user scrolls the mouse over the sequence, information about the residues appears in the "residue info" box. Pull-down menu on the top of the sequence frame is also shown, demonstrating choice of SMS modules and rendering. SMS structure frame (Chime wiondow) is shown at the bottom of this figure. This figure was centered on the Leu_46 (painted in red) of the E chain. The other residues drawn in stick presentation are the residues that make contacts with the Leucine, colored according to the contacts they make, corresponding to the first two lines of the Protein Dossier in Figure 2. Figure 2 SMS Protein Dossier. Protein Dossier module – The meaning of the colors are on the top part of the image. The internal and interface forming contacts are shown above the amino acid sequence cartoon, color coded according to the type of contact. The first row (red line) under the sequence highlights the residues at the interface. The next row (yellow line bellow the sequence stretch: VTAAHGC) indicates the PROSITE pattern PS00134. The following three rows indicate the secondary structure according to the annotations in PDB, DSSP and Stride, respectively. Another five rows are displayed color coded according to: temperature factor, relative entropy, accessibility in complex, accessibility in isolation and dihedral angles. Figure 3 Atomic contacts among residues within same chain. Internal residue contacts formed by the Leu_46, showing all amino acids, belonging to the same chain E, that make contacts with it. The fan above the residue is color coded according to the type of contact the residue is involved with. Figure 4 Sequence Conservation in SMS ConSSeq. ConSSeq window where the amino acid sequence of 1cho, chain E is aligned with a consensus sequence as found in HSSP and, entropy histogram color and size coded with respect to the calculated degree of conservation. The size of sequence letters in the "logo" follows the same degree of conservation, while color coding is the same as in SMS sequence frame. Leu_46 is highlighted by sequence number 46 colored red. Figure 5 SMS Ramachandran interactive plot. Ramachandran plot of 1cho.pdb, highlighting the position of Leu_46 (blue colored spot), chain E in the diagram, and coloring plotted areas according to the allowed regions for the phi and psi angles. This image shows also the area of the applet which is dedicated to sub setting of the amino acids according to their type and position in the allowed and disallowed areas. ==== Refs Berman HM Westbrook J Feng Z Gilliland G Bhat TN Weissig H Shindyalov IN Bourne PE The Protein Data Bank Nucleic Acids Research 2000 28:1 235 242 10592235 10.1093/nar/28.1.235 Sander C Schneider R Database of Homology-Derived Protein Structures and the Structural Meaning of Sequence Alignment Proteins: Struc Func and Genet 1991 9 56 68 Schneider R Sander C The HSSP database of protein structure sequence alignments Nucleic Acids Res 1996 24 201 205 8594579 10.1093/nar/24.1.201 Bucher P Bairoch A Altman R, Brutlag D, Karp P, Lathrop R, Searls D A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology 1994 AAAIPress, Menlo Park 53 61 Neshich G Togawa R Mancini AL Kuser PR Yamagishi MEB Pappas JG Torres WV Campos TF Ferreira LL Luna FM Oliveira AG Miura RT Inoue MK Horita LG de Souza DF Dominiquini F Álvaro A Lima CS Ogawa FO Gomes BG Palandrani JCF dos Santos GF de Freitas EM Mattiuz AR Costa IC de Almeida CL Souza S Baudet C Higa RH STING Millennium: a Web based suite of programs for comprehensive and simultaneous analysis of protein structure and sequence Nucleic Acids Res 31 3386 3392 12824333 10.1093/nar/gkg578 Ramachandran GN Ramakrishnan C Sasisekharan V Stereochemistry of polypeptide chain configurations J Mol Biol 1963 7 95 99 13990617 Kabsch W Sander C Dictionary of Protein Secondary Structure: Pattern Recognition of Hydrogen-Bonded and Geometric Features Biopolymers 1983 22 2577 2637 6667333 Frishman D Argos P Knowledge-Based Protein Secondary Structure Assignment Proteins: Struc Func and Genet 1995 23 566 679 Taylor WR Residual colors: a proposal for aminochromography Protein Eng 1997 10 743 746 9342138 10.1093/protein/10.7.743 Abdulla BK Michael GM Uedaira H Kitajima K Sarai A ProTherm, version 4.0: Thermodynamic Database for Proteins and Mutants Nucleic Acids Res 2004 32 D120 D121 14681373 10.1093/nar/gkh082 Martz E Protein Explorer: Easy Yet Powerful Macromolecular Visualization Trends in Bioch Sci 2002 27 107 109 10.1016/S0968-0004(01)02008-4 Hogue CW Cn3D: a new generation of three-dimensional molecular structure viewer Trends in Biochem Sci 1997 22 314 316 9270306 10.1016/S0968-0004(97)01093-1 Guex N Peitsch MC SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling Electrophoresis 1997 18 2714 2723 9504803 Laskowski RA MacArthur MW Moss DS Thornton JM PROCHECK: a program to check the stereochemical quality of protein structures J Appl Cryst 1993 26 283 291 10.1107/S0021889892009944

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2021-01-04 16:02:46

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BMC Bioinformatics. 2004 Aug 9; 5:107

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BMC Bioinformatics

10.1186/1471-2105-5-107

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==== Front BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-5-1051529651910.1186/1471-2105-5-105Research ArticleEvaluation of the suitability of free-energy minimization using nearest-neighbor energy parameters for RNA secondary structure prediction Doshi Kishore J [email protected] Jamie J [email protected] Christian W [email protected] Robin R [email protected] The Institute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station A4800, Austin, TX 78712-0159, USA2004 5 8 2004 5 105 105 1 2 2004 5 8 2004 Copyright © 2004 Doshi et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background A detailed understanding of an RNA's correct secondary and tertiary structure is crucial to understanding its function and mechanism in the cell. Free energy minimization with energy parameters based on the nearest-neighbor model and comparative analysis are the primary methods for predicting an RNA's secondary structure from its sequence. Version 3.1 of Mfold has been available since 1999. This version contains an expanded sequence dependence of energy parameters and the ability to incorporate coaxial stacking into free energy calculations. We test Mfold 3.1 by performing the largest and most phylogenetically diverse comparison of rRNA and tRNA structures predicted by comparative analysis and Mfold, and we use the results of our tests on 16S and 23S rRNA sequences to assess the improvement between Mfold 2.3 and Mfold 3.1. Results The average prediction accuracy for a 16S or 23S rRNA sequence with Mfold 3.1 is 41%, while the prediction accuracies for the majority of 16S and 23S rRNA structures tested are between 20% and 60%, with some having less than 20% prediction accuracy. The average prediction accuracy was 71% for 5S rRNA and 69% for tRNA. The majority of the 5S rRNA and tRNA sequences have prediction accuracies greater than 60%. The prediction accuracy of 16S rRNA base-pairs decreases exponentially as the number of nucleotides intervening between the 5' and 3' halves of the base-pair increases. Conclusion Our analysis indicates that the current set of nearest-neighbor energy parameters in conjunction with the Mfold folding algorithm are unable to consistently and reliably predict an RNA's correct secondary structure. For 16S or 23S rRNA structure prediction, Mfold 3.1 offers little improvement over Mfold 2.3. However, the nearest-neighbor energy parameters do work well for shorter RNA sequences such as tRNA or 5S rRNA, or for larger rRNAs when the contact distance between the base-pairs is less than 100 nucleotides. ==== Body Background The biological functions of 16S, 23S and 5S rRNAs, tRNAs, telomerase RNA, Group I and II introns, RNaseP, and other structural RNAs are dictated by their three-dimensional structures. Thus, an accurate depiction of an RNA's secondary and tertiary structure is fundamental for our understanding of the mechanisms and consequences of its function, and an accurate prediction of an RNA folding into its secondary and tertiary structure from its primary structure will have a significant effect on our study of molecular biology. This RNA folding problem is usually divided into two components: the first is the determination of an RNA's folding pathway, and the second is the accurate and reliable prediction of an RNA's secondary and tertiary structure from its primary structure. In this paper, we focus on the second aspect and in particular RNA secondary structure prediction, which is a difficult problem. It has been estimated that the number of secondary structures models is greater than 1.8n, where n is the number of nucleotides (nt) in the sequence[1]. For example, Saccharomyces cerevisiae Phe-tRNA is only 76 nt in length and has an estimated 2.5 × 1019 secondary structure models, while a larger RNA, such as the 16S rRNA from Escherichia coli, with 1542 nt, has an estimated total of 4.3 × 10393 possible secondary structure models. The most popular method for predicting RNA secondary structure from a single sequence is free energy minimization using a dynamic programming approach[2,3], based on energy parameters determined according to the nearest-neighbor model[4-10]. Programs based on this technique include Mfold[2,11], RNAStructure[12,13] and RNAFold[14]. Mfold is the most popular program in use today. By default, Mfold determines the optimal (minimum energy) structure and a set of suboptimal foldings that are within 12 kcal/mol (default setting) of the minimum energy structure. The set of suboptimal foldings exists and covers such a large energy range due to uncertainties in the thermodynamic data[2]. Mfold applies the following constraints: 1) only G:C, A:U and G:U base-pairs are formed (due to limitations of the energy parameters), 2) hairpin loops have at least three bases, and 3) no pseudoknotted structures are formed[15]. Attempts have been made to characterize the reliability of an RNA secondary structure prediction using dot plots[16]. Comparative analysis is another method for predicting RNA secondary and some tertiary structure[17-25]. Comparative analysis of RNA sequences and structures is a knowledge-based technique based on two fundamental assumptions: 1) different, homologous RNA sequences are capable of folding into the same secondary and tertiary structure, and 2) during the course of evolution, the secondary and tertiary structure of an RNA molecule remains mostly unchanged, while the primary structure can change significantly. The accuracy of the comparative method has recently been established using high-resolution crystal structures for the 30S and 50S ribosomal subunits[26,27]. Over 97% of the secondary structure base-pairs predicted by comparative analysis are present in the crystal structures[28]. The superior performance of the comparative method may lead one to incorrectly assume that the other methods for RNA secondary structure prediction are no longer necessary. Different methods for predicting RNA secondary structure are utilized in different situations and can have different objectives. Free energy minimization based RNA structure prediction methods are usually applied to a single RNA sequence. The most energetically stable RNA secondary structure(s) that are composed of canonical G:C, A:U, and G:U base-pairs and organized into standard helices are predicted. Non-canonical base-pairs and base-pairs not in standard helices cannot be predicted at this time. In contrast, RNA comparative sequence analysis methods predict a structure by searching an alignment for base-pairings that are common to all sequences in the dataset. This latter method can accurately predict canonical and non-canonical base-pairs that occur in secondary and tertiary structures. However, RNA comparative analysis is an iterative process requiring substantial sequence data, accurate sequence alignments, and the analysis of a structure that is common to all of the sequences in the dataset. In order to create an initial alignment, sequences must have significant identity to be aligned accurately while having sufficient variation (and covariation) to identify potential base-pairs and posit a structural hypothesis. The structural hypothesis is subsequently tested, refined, and expanded by the addition of more sequences to the alignment. Much of this process is still done manually, as computational tools to automate the process do not currently exist. The most recent comparative structure model for SSU rRNA is based on an alignment of 7,000 sequences[28]. The data was collected and the model was refined over a period of 20 years. The sequences included in this analysis are very diverse, spanning the entire tree of life. In 1995 and 1996, the Gutell Lab conducted two comprehensive studies that: 1) determined how well the optimal secondary structure model predicted with the program Mfold (version 2.3) matched the structure model determined with comparative analysis for a set of 16S and 23S rRNAs, and 2) examined other aspects of the folding, such as the prediction accuracy of "short-range" base-pairs (base-pairings where the 5' and 3' halves of the base-pair are separated by 100 nt or less), or the prediction accuracy for base-pairs in different loop environments, to learn more about differences between the thermodynamic and comparative models[29,30]. The most significant findings from those studies were: 1) the average accuracy of the optimal prediction for a 16S rRNA was 46% while the average accuracy for a 23S rRNA was 44%. 2) The accuracies of the predicted secondary structure models for at least one individual 16S or 23S rRNA sequence were as high as 80% and as low as 10%. 3) On average, the Archaeal rRNAs were predicted with the highest accuracy, followed by the (eu)-Bacterial and Chloroplast, then Mitochondrial and Eukaryotic Nuclear rRNAs. 4) Short-range pairings, which comprised 75% of the total comparative base pairings for both 16S and 23S rRNA, were predicted more accurately than long-range pairings (base-pairings where the 5' and 3' halves of the base-pair are separated by more that 100 nt). 5) Base-pairs closing hairpin loops were predicted more accurately than those closing internal and multistem loops. Since those studies were completed in 1995, four new developments have directly affected RNA structure prediction. 1) A new version of Mfold (3.1) was released with energy parameters revised to include sequence dependence and different secondary structure motifs[31]. 2) The accuracy of the comparative model for ribosomal RNA was established[28] with high-resolution crystal structure data from both the small and large ribosomal subunits[26,27]. 3) The number of available 16S and 23S rRNA secondary structures determined by comparative analysis increased significantly[24]. 4) Faster computers, which have significantly decreased the time it takes to fold an individual sequence, had become available to facilitate large-scale folding studies. These developments afforded us the opportunity to do a comprehensive re-evaluation of Mfold. For more than 20 years, the basic paradigm for RNA secondary structure prediction, from a single sequence, has essentially remained the same: global free energy minimization with refinements to the nearest-neighbor energy parameters and minimization algorithms in an attempt to improve prediction accuracy. Refinements to the energy parameters, summarized in multiple sources[31-33], included measures for effects such as base-pair mismatches, base-pair positioning in helices, internal, bulge and multistem loops, and coaxial stacking. Newer versions of the program Mfold included these refinements in energy parameters in addition to improvements in the folding algorithm[31,34,35]. We questioned whether the improvements in the energy parameters and the algorithms could result in dramatic improvements in the accuracy and reliability of RNA secondary structure prediction programs such as Mfold, or would the energy-based RNA folding approach need to be fundamentally altered. To begin to address this question, we analyzed the ability of Mfold 3.1 to predict RNA secondary structure models determined with comparative analysis. In addition, we compared the predictions and accuracies of Mfold 3.1 with its predecessor, Mfold 2.3, for a large set of phylogenetically diverse 16S and 23S rRNA sequences. All metrics considered in previous studies to evaluate the accuracy of Mfold 2.3[29,30] were revisited here. In addition, we analyzed the suboptimal population of predicted secondary structures and characterized metrics such as the number of suboptimals that were better (or worse) than the optimal structure prediction, the total number of comparative base-pairs observed, and the number of times a given base-pair is predicted in a set of optimal and suboptimal structure predictions. Only the most significant findings and metrics were discussed here; the reader is referred to the website[36] for a detailed presentation of all results from this analysis. Results Comparative structure database The dataset assembled for this study was significantly larger than previous studies comparing RNA structure models predicted by comparative analysis and the Mfold folding program[29-31,35]. In particular, the 1995 and 1996 studies conducted by the Gutell Lab with Mfold 2.3 analyzed only 56 16S[29] and 72 23S[30] rRNA sequences respectively, and the 1999 study by Mathews et al. with Mfold 3.1 analyzed a total of 151,503 nt and 43,519 comparatively predicted canonical base-pairs (i.e., G:C, A:U and G:U) from 955 sequences, which included 22 16S, 5 23S, and 309 5S rRNA sequences, 484 tRNA sequences 23 Group I and three Group II intron sequences, 91 SRP sequences, and 16 RNase P sequences[31]. For this study, our sequence set included all three types of rRNA (5S, 16S and 23S) and Type I tRNA. As shown in Table 1, we analyzed a total of 1,411 RNA sequences, encompassing 1,505,143 nt and 385,854 canonical secondary structure base-pairs. Of the 1,411 sequences analyzed, 569 were tRNA, 496 were 16S rRNA, 256 were 23S rRNA, and 90 were 5S rRNA. While the size of the comparative structure databases increased significantly between this study and previous Gutell Lab studies, only minor differences exist between the 1995 and 2004 versions of the 16S and 23S rRNA comparative structure models. For the 2004 version of the Haloferax volcanii 16S rRNA secondary structure model, 30 base-pairs were added, 17 base-pairs were removed, and 427 base-pairs remained unchanged, resulting in a net difference of approximately 3% in the total number of base-pairs in the model. Similar numbers were observed for the other comparatively predicted structures evaluated. The comparative structure database used by Mathews et al.(1999) utilized known modified nucleotide information in tRNA to limit the base-pairing potential for those nucleotides that are modified[31]. In this study, rRNA or tRNA base modifications were not taken into account. A simple analysis of our tRNA dataset shows that 70% of our tRNA sequences came from genomic DNA sequences: as a result, no modification data was available for those sequences. For the remaining 30%, the number of modifications that could prevent A-form helix formation was minimal; between only 1 to 5 modifications per sequence. Our dataset was extremely diverse in sequence and structure and included sequences from each of the three major phylogenetic domains, the Archaea, Bacteria, and Eucarya[37]. The eukaryotic dataset included sequences encoded in the Nucleus, Chloroplast and Mitochondrion. Since a dataset with sequences that were nearly identical would be less useful than a dataset with significant variation between the sequences, we characterized the diversity of the sequences in our dataset by calculating sequence identity for all pairs of 16S and 23S rRNA sequences within the different phylogenetic classifications of our dataset. For our 16S rRNA dataset, 75% of the Archaeal, 86% of the Bacteria, 71% of the Chloroplast, 99% of the Mitochondrial, and 94% of the Eukaryotic Nuclear sequence pairs had less than 80% sequence identity, while only 4% or fewer of the pairs in a given phylogenetic classification had 95% or more sequence identity (Table 1). Moreover, 79% of the Mitochondrial and 48% of the Eukaryotic Nuclear 16S rRNA sequence pairs had less than 50% sequence identity (Figure 1). The 23S rRNA dataset exhibited even more diversity than the 16S rRNA dataset, as 87% of the Archaeal, 94% of the Bacteria, 76% of the Chloroplast, 99% of the Mitochondrial, and 97% of the Eukaryotic Nuclear sequence pairs had less than 80% sequence identity, while 2% or fewer of the sequence pairs in a given phylogenetic classification had more than 95% sequence identity (Table 1). This demonstration of significant sequence variation between sequences in the same phylogenetic categories reveals the relative independence of the sequences within our dataset. RNA secondary structure prediction The most important parameters used to control RNA secondary structure prediction by Mfold are window size (W), percent suboptimality (P), and the inclusion or exclusion of additional energy calculations based on coaxial stacking (efn2). The percent suboptimality variable establishes the energy range for computed foldings. The range is ΔGmin to ΔGmin + ΔΔG, where ΔΔG is P% of ΔGmin. The window size variable establishes the difference between the suboptimal folds by requiring that given folding has at least W base-pairs that are at least a distance W from any base-pairs in the foldings already computed. The program efn2 is used to re-compute the energetics of each predicted structure based on coaxial stacking opportunities with the structure. The structures are then re-ordered by the modified ΔG and a new optimal structure is selected. Previous studies by the Gutell Lab, Konings et al.[29] and Fields et al.[30], with Mfold 2.3 used window sizes of 10 and 20, respectively, with no efn2 re-evaluation; the selection of window size was limited by the computational resources available at the time the studies were conducted. Mathews et al.[31] used Mfold 3.1 with a window size of 0, percent suboptimality of 20%, and efn2 re-evaluation. Each of the 1,411 sequences in our dataset was folded with Mfold 3.1, using a window size (W) of 1, percent suboptimality (P) of 5%, and maximum number of suboptimal foldings (MAX) of 750. The optimal, or minimum, free energy prediction and 749 suboptimal predictions were determined after re-ordering the structure predictions by the efn2 re-computed energetics. (Note: some sequences did not yield 749 suboptimal structure predictions under the folding conditions used in this study.) We configured the folding of our RNA sequences to: 1. maximize the number of structures predicted for any given sequence, 2. densely sample the suboptimal population close to the minimum free energy structure, since the structure with the lowest free energy (based on nearest-neighbor energetics) is expected to be most similar to the structure observed in nature for the free energy minimization techniques, and 3. to include coaxial stacking in the energy calculations with the efn2 option in Mfold 3.1. The only difference between the run parameters from the previous Gutell Lab studies and this study was the significantly smaller window size used in the current study. The difference in window size affects the number of suboptimal structures computed. Since the previous Gutell Lab studies did not include any energy re-computation and re-ordering of predicted structures for potential coaxial stacking, this difference would not have a significant impact on the results. The study by Mathews et al. used different values for percent suboptimality and window size in computing suboptimal structure predictions. The net result of the difference is that the Mathews et al. study considered suboptimal structures with energy values further away from the minimum free energy prediction than in our study. This difference could have an impact on the results since the Mathews et al. study may include a structure prediction for a given sequence that is not very energetically stable (and would be excluded from the suboptimal population in our study) but upon efn2 re-ordering becomes the minimum energy structure. If this predicted structure was more accurate than any other prediction in the population, the Mathews et al. study would reflect a higher prediction accuracy score for that given sequence. Overall accuracy for RNA structure prediction with Mfold 3.1 Given that 97–98% of the RNA secondary structure base-pairs predicted with comparative analysis were verified with the high resolution crystal structures[28], we scored the accuracy of the structures predicted with Mfold 3.1 by quantifying how well the optimal (minimum energy) structure prediction matched the comparative structure model for each sequence in our dataset. Results were only based on sequences folded in their entirety. We calculated accuracy by dividing the number of comparative base-pairs that were predicted exactly with Mfold by the total number of canonical base-pairs in the comparative model (excluding any base-pairs with IUPAC symbols other than G,C,A or U, see Prediction Accuracy Calculations in Methods). This method for calculating accuracy was the same as the previous Gutell Lab studies that utilized Mfold 2.3[29,30], with the exception that previous studies excluded comparative base-pairs that were pseudoknotted from consideration. In contrast, base-pairs predicted with Mfold 3.1 in the Mathews et al.[31] study were considered correct if: 1. they matched a comparatively predicted base-pair exactly or 2. either nucleotide of the Mfold predicted base-pair (X,Y where X and Y are the positions of the nucleotides in the sequence) is within one nucleotide of its comparatively predicted position (X, Y ± 1 or X ± 1,Y). While the Mathews et al. study included a measure of the percentage of comparative base-pairs considered pseudoknotted, we were unsure if those base-pairs were specifically excluded from their accuracy calculations. Based on these differences in the accuracy calculations, the Mathews et al. study is reporting higher accuracies than our study. Direct comparisons between the current study and the two previous Gutell Lab studies are meaningful due to the use of similar methodologies to calculate prediction accuracy. The only difference is the scoring method between the studies is the exclusion of pseudoknotted base-pairs from previous Gutell Lab studies. However, direct comparison of results between the current study and the Mathews et al. study are impacted by differences in the folding parameters and the scoring criteria. Raw folding accuracy The compilation of the accuracies for each sequence and the accuracy ranges for each RNA type and phylogenetic grouping were summarized in Table 2. The average folding accuracies per sequence for 5S rRNA and tRNA, the two smallest molecules in this study, were 71% and 69% respectively. The study by Mathews et al. reported average accuracy per sequence of 78% for 5S rRNA and 83% for tRNA[31]. Accuracies for our sets of 5S rRNAs and tRNAs were about 25% higher than the average accuracies for the 16S (41%) and 23S (41%) rRNAs. By comparison, the Gutell Lab's previous studies using Mfold 2.3 reported an average folding accuracy of 46% for 16S rRNA and 44% for 23S rRNA[29,30]. The study by Mathews et al. reported average accuracies (for folding complete RNA sequences) of 51% for a dataset of 22 16S rRNAs and 57% for a dataset of 5 23S rRNAs[31]. When considering only sequences analyzed in previous Gutell Lab studies, the average prediction accuracy with Mfold 3.1 was 45% for 16S rRNA and 43% for 23S rRNA (Table 2). Variation in observed folding accuracy To gauge the variation in accuracy score for the optimal structures predicted with Mfold, the percentages of scores greater than 60% and less than 20%, the median accuracy score, and the highest and lowest accuracy scores were identified for the four RNA types (Table 2). This analysis revealed a large range of accuracy scores with values significantly larger and smaller than the respective average value. For our current analysis, the highest accuracy score for the optimal structure for each RNA type was 100% for tRNA (i.e., at least one of the predicted tRNA structures had 100% of the base-pairs in the comparative model), 98% for 5S rRNA, 77% for 16S rRNA, and 74% for 23S rRNA. In contrast, at least one of the optimal folds for 5S rRNA or tRNA had a score of 0 (i.e., none of the base-pairs in the comparative structure model were predicted with Mfold). The lowest accuracy score was 5% for 16S rRNA and 1% for 23S rRNA. The median accuracy score observed for each RNA type was 70% for tRNA, 81% for 5S rRNA, 41% for 16S rRNA and 41% for 23S rRNA. For 16S and 23S rRNA the overwhelming majority (86% for 16S rRNA and 89% for 23S rRNA) of optimal structures predicted with Mfold had an accuracy score greater than 20% and less than 60%, a trend also observed in our previous studies (Table 2)[29,30]. The majority of optimal structures predicted for 5S rRNA (77%) had an accuracy score greater than 60% (Table 2). For the tRNA, 60% of the optimal structures were predicted with accuracy greater than 60% and 39% of the optimal structures predicted with accuracy between 20% and 60%. The percentage of predicted structures with an accuracy score below 20% was highest for 23S rRNA (6%); increased from 1% previously[30], followed by 16S rRNA (4%); decreased from 9% previously[29], 5S rRNA (4%), and tRNA (2%) (Table 2). Our website[36] contains a complete list of accuracy scores and secondary structure diagrams indicating base-pairs that were predicted correctly for all sequences in our dataset. Phylogenetic dependence in observed folding accuracy Our previous thermodynamic-based folding analysis of 16S rRNAs[29] and 23S rRNAs[30] also revealed significant variation in the accuracy scores within and between the five major phylogenetic groups. For our current 16S rRNA dataset, the Archaeal sequences had the highest average accuracy (62%), while the Mitochondrial sequences had the lowest average accuracy (30%). Between these two extremes were the Bacteria (49%), Chloroplast (46%), and Eukaryotic Nuclear (34%) sequences (Table 3). These results were consistent with our previous studies, except that the Archaeal and Bacterial accuracy scores were slightly lower in our newer analysis (62% vs. 68% and 49% vs. 56%)[29]. For 23S rRNA (Table 3), the Archaeal dataset again had the highest accuracy scores (58%), followed by the Bacterial (49%), Eukaryotic Nuclear (42%), Chloroplast (39%), and Mitochondrion (30%). These results were also consistent with the trends observed in the previous studies, although the accuracy values for the current study were slightly less than the earlier analysis. Direct comparison of structure predictions by Mfold 2.3 and Mfold 3.1 for specific RNA sequences To access specific differences between the optimal foldings from Mfold 2.3 and Mfold 3.1 for select 16S and 23S rRNA sequences, we mapped the base-pairs predicted with both versions of Mfold onto the comparative structure models for each sequence. Some of the base-parings were predicted correctly with both versions of Mfold, other base-pairings were predicted exclusively by one version, while a third set of base-pairings were not predicted correctly with either version. The Haloferax volcanii 16S rRNA (Figure 1A) and Thermococcus celer 23S rRNA (Figure 1 B.1 and B.2) sequences were generally predicted very well with both versions of Mfold. Meanwhile, Giardia intestinalis 16S (Figure 1C) and 23S (Figure 1 D.1 and D.2) rRNA sequences were predicted poorly with both versions of Mfold. The base-pairings in the comparative model that were missed by both versions of Mfold were generally longer range (see Accuracy and the RNA Contact Distance). This relationship between the comparative structure model for G. intestinalis 16S and 23S rRNA and the poor prediction of this structure with both versions of Mfold (Figure 1C, D.1 and D.2) was representative of other sequences predicted with low accuracy by Mfold 2.3. A total of 9 out of 10 16S sequences and 7 of the 8 23S sequences predicted with accuracy of 30% or less with Mfold 2.3 were still predicted with less than 30% accuracy with Mfold 3.1 (Table 4). Six significant results came from this section. 1) Direct comparison of previous Gutell Lab studies with the current study (in light of the significantly larger size and richness in sequence variation of the comparative structure database for the current study and the inclusion of comparative base-pairs in pseudoknots) suggests that refinements in the energy parameters and folding algorithm have not improved the accuracy of the Mfold program between versions 2.3 and 3.1 for the set of 16S and 23S rRNAs analyzed. 2) The discrepancies between the results of the Mathews et al. study and our study could be due to the different methods by which the sequences were folded and prediction accuracy calculated. 3) The accuracy scores for the majority of the 16S and 23S rRNA secondary structure models predicted with Mfold 2.3 and 3.1 were between 20% and 60%, while the accuracy scores for the majority of 5S secondary structure models predicted with Mfold were greater than 60%. 4) Some secondary structure models predicted with Mfold 2.3 and 3.1 have accuracy scores less than 20%. 5) The folding accuracy for Archaeal rRNAs was the highest, followed by Bacterial, Eukaryotic Chloroplast, Nuclear, and Mitochondrial rRNAs. 6) Sequences that were well-predicted with Mfold 2.3 tend to be well-predicted with Mfold 3.1, and sequences that were poorly-predicted using Mfold 2.3 tend to be poorly-predicted using Mfold 3.1. Accuracy and the RNA contact distance For a given protein, the average sequence separation between pairs of amino acids involved in non-covalent interactions is defined as the "Contact Order"[38]. Two similar topological descriptions for non-covalent interactions in RNA are: 1) "RNA Contact Distance" is the separation on the RNA sequence between two nucleotides that base-pair, and 2) "RNA Contact Order" is the average of the RNA Contact Distances for a given RNA sequence. We considered any base-pair with a contact distance of 100 nt or less to be "short-range," a contact distance of 101–501 nt to be "mid-range," and a contact distance of 501 or greater to be "long-range." The majority of base-pairs in the 16S and 23S rRNA secondary structure models predicted with comparative analysis were short-range (Table 5), and previous studies have established that short-range base-pairs are predicted more accurately than long-range base-pairs[29,30]. In this section, we: 1) compared the accuracies of the short-range interactions predicted with Mfold 3.1 and Mfold 2.3, 2) compared the number of short-, mid-, and long-range base-pairs in the comparative models with those predicted by Mfold 3.1, and 3) determined the relationship between the base-pair prediction accuracy and the contact distance for 16S rRNA. Accuracy of Short-range interactions The 496 16S rRNA comparative structure models in this study were comprised of 191,994 canonical base-pairs. A total of 145,058 (76%) of these base-pairs were short-range, and 75,763 (52%) of these base-pairs were predicted correctly by Mfold 3.1 (Table 5). The average accuracy for short-range base-pairs was 50% per sequence (Table 5) (see Per Sequence Averages in Methods for a discussion on how per sequence averages are computed). By comparison, in the 1995 study, an average accuracy of approximately 55% per sequence was observed for short-range base-pairs[29]. For the 23S rRNA dataset, the 256 comparative structures contained a total of 178,958 canonical base-pairs. 134,085 (75%) of the 23S rRNA comparative, canonical base-pairs were short-range, and 67,130 (50%) of those base-pairs were predicted correctly by Mfold 3.1 (Table 5). The average prediction accuracy for short-range base-pairs was 47% per sequence (Table 5). In the 1995 study, an average accuracy of approximately 53% per sequence was observed for short-range base-pairs[30]. Distribution by RNA contact distance of comparative and Mfold predicted base-pairs A total of 223,957 base-pairs were predicted with Mfold 3.1 for our 16S rRNA dataset (Table 5). This was 31,963 more than in the 16S rRNA comparative structure models (Table 5). Of the 223,957 base-pairs, 150,886 (67%) were short-range and 73,071 (33%) were mid- or long-range. Of the 150,886 short-range base-pairs, 75,763 (50%) were correct while only 6,171 (8%) of the mid- and long-range base-pairs were correct. A total of 29,573 long-range base-pairs were predicted with Mfold 3.1, while the comparative models contained only a total of 3,932 long-range base-pairs; in other words, 13% of the total number of 16S rRNA base-pairs predicted with Mfold 3.1 were long-range while only 2% of the comparatively predicted base-pairs were long-range. Finally, of the 29,573 long-range base-pairs predicted by Mfold 3.1, only 193 (0.7%) were correct. Similar results were observed for our 23S rRNA dataset (Table 5). A total of 218,908 base-pairs were predicted by Mfold 3.1; 137,780 (63%) were short-range, while 81,128 (37%) were mid- or long-range. 67,130 (49%) of the total short-range base-pairs predicted were correct, but only 10,758 (13%) of the total mid- and long-range base-pairs predicted were correct. Akin to the 16S rRNA dataset, a total of 36,989 (17%) 23S rRNA long-range base-pairs were predicted with Mfold 3.1, while only 7,752 (4%) of the comparatively predicted base-pairs were long-range. Only 1,317 of the 36,989 (4%) long-range base-pairs predicted by Mfold 3.1 were correct. Relationship between prediction accuracy and RNA contact distance These results prompted a more sophisticated analysis to quantify the relationship between the accuracy of base-pairs predicted with Mfold 3.1 and RNA contact distance. Figure 2A shows the distribution of contact distances for the 191,994 canonical base-pairs from the 496 16S rRNA comparative structure models in this study. The frequency of base-pairs observed decreases exponentially as contact distance increases. Based on this observation, we divided the 191,994 16S rRNA comparative base-pairs into seven somewhat equally-sized bins (within one order of magnitude of one another) by considering the contact distance values on a logarithmic scale instead of a linear scale. (see Logarithmic Binning of Base-pairs by Contact Distance for 16S rRNA in Methods). The Mfold prediction accuracy for each of these bins was determined. The prediction accuracy was 61% for base-pairs in the smallest contact distance bin, 3–8, 57% for base-pairs in the 9–19 contact distance bin, 47% for base-pairs in the 20–47 bin, 46% for the 48–117 bin, 15% for the 118–293 bin, 7% for the 294–733 bin, and 0% for the 734–1833 bin (Figure 2B). The approximately linear relationship obtained from plotting the accuracy for logarithmically-scaled bins revealed an exponential relationship between the accuracy of Mfold and the contact distance (Figure 2B). Five significant results were observed from the analysis in this section. 1) The accuracy of the predictions for short-range base-pairings was similar for Mfold 3.1 and Mfold 2.3. 2) More base-pairs were predicted with Mfold for any given sequence than in the corresponding comparative structure model. 3) Significantly more long-range base-pairs were predicted with Mfold 3.1 than in the comparative structure models. 4) The number of base-pairs in the comparative structure models decreases exponentially as the contact distance increases. 5) Base-pairs with a contact distance between 3 and 8 were predicted with the highest accuracy (61%) by Mfold, and accuracy values decreased exponentially as the RNA contact distance increases. The complete set of results for the prediction of 16S and 23S rRNA short-, mid-, and long-range base-pairs with Mfold 3.1 and comparisons with the comparative structure models are provided at our website[36]. Suboptimal foldings One of the features of the dynamic programming algorithm for free energy minimization employed by Mfold was the ability to provide a set of suboptimal structure predictions in addition to the minimum free energy or optimal structure prediction[2,12]. Mathews et al. included metrics which consider how suboptimal population may impact the prediction accuracy[31]. In this section, we introduce new metrics to continue the examination of the suboptimal population using the 496 16S rRNA sequences in our dataset. Due to the differences in the folding parameters and in the methods for computing accuracy, our survey of the suboptimal population should not be directly compared with the Mathews et al. study. Rather, our metrics provide a different perspective from which to excogitate the importance of the suboptimal structure predictions. In particular, we considered: 1) the amount of structural variation and the ΔΔG difference (before efn2 based re-evaluation) for pairs of structure predictions in the suboptimal population, 2) how many additional unique, canonical base-pairs in the comparative models were found in the suboptimal population, and how many incorrect base-pairs were predicted, and 3) which comparative base-pairs were predicted correctly in all, an intermediate number, or no structure predictions, in the set of suboptimal foldings. Structural variation and ΔΔG difference for structure predictions in the suboptimal population It has been previously noted that suboptimal structure predictions can be very similar or very different from one another[31]. Here we tested for a relationship between the ΔΔG (before efn2 based re-evaluation) and the structural variation score (see Suboptimal Structural Variation Score in Methods) for pairs of structure predictions within a suboptimal population. Higher structural variation scores indicate that two structures compared were more different from one another, while lower structural variation scores indicate that two structures were more similar. We analyzed the two 16S rRNA sequences with the highest and lowest optimal accuracy before efn2 re-evaluation and re-ordering in the Archaea dataset, Haloferax volcanii and Methanospirillum hungatei. The accuracy for H. volcanii based on the pre-efn2 minimum free energy structure prediction was 80%, while M. hungatei was predicted at 46% accuracy (Table 6). For the suboptimal population of each sequence, we calculated the structural variation and the difference in free energy for all possible pairwise comparisons. The total number of unique pairwise combinations for each sequence was 280,875, based on a total of 750 structure predictions (optimal plus 749 from the suboptimal population). For H. volcanii, 24,621 pairs (9%) of structure predictions had a structural variation score of 501 or higher, while 134,44 pairs (48%) had a score of 100 or less (Table 6). Thus, the majority of the structural predictions in the suboptimal population were more similar with one another. The observed ΔΔG range was the same for both categories, 0–11 kcal/mol (Table 6). More striking was the similarity in the average ΔΔG. For those pairwise comparisons with a structural variation score of 100 or less, the average ΔΔG was ~2.60 kcal/mol (weighted) (Table 6). For those pairwise comparisons with a structural variation score of 500 or higher, the average ΔΔG was 2.24 kcal/mol (Table 6). These results were summarized graphically in Figure 3A. In contrast with H. volcanii, our analysis for M. hungatei revealed that a significant number of the structure predictions were different from one another. A total of 103,462 pairs (37%) of structure predictions had a structural variation score of 501 or higher, while only 43,376 pairs (16%) had a score of 100 or less (Table 6). The observed ΔΔG range was slightly smaller than in H. volcanii, 0–8.6 kcal/mol, while the average ΔΔG values were similar for H. volcanii and M. hungatei. For the pairwise comparisons with a structural variation score of 500 or higher, the average ΔΔG was 1.82 kcal/mol (Table 6). For those pairwise comparisons with a structural variation score of 100 or less, the average ΔΔG was ~2.05 kcal/mol (weighted) (Table 6). These results were summarized graphically in Figure 3B. The most important observation from this section was that the ΔΔG between two structure predictions appeared to be independent of the similarity between the structure predictions. For example, H. volcanii pairwise comparisons with a structural variation score of 500 or higher had an average ΔΔG of 2.24 kcal/mol, while those pairwise comparisons where the score was 100 or less had an average ΔΔG of ~2.60 kcal/mol (weighted). In other words, free energy alone was not sufficient to adequately distinguish between different structure predictions. While the results suggest that suboptimal structural variation score could potentially be used as an indicator of the reliability of the structure prediction by Mfold, further investigation is required to evaluate the extent of this correlation. Number of comparative base-pairs in the suboptimal population For each individual 16S rRNA sequence, we identified the set of unique comparative base-pairs present from the collection of all base-pairs predicted in the suboptimal population. Since most comparative base-pairs were observed in more than one suboptimal structure, the total number of unique base-pairs observed was much lower than the total number of comparative base-pairs in the suboptimal population. Our entire 16S rRNA dataset of 496 comparative structure models contained a total of 191,994 unique canonical, comparative base-pairs (Table 7). 81,934 of these canonical base-pairs were predicted with Mfold 3.1 to be in a minimum free energy structure (after efn2 re-evaluation and re-ordering), an average accuracy of 41% per sequence (Table 7) (see Per Sequence Average in Methods). However, when considering the entire suboptimal population of structure predictions for each sequence, a total of 137,000 comparative canonical base-pairs were predicted correctly by Mfold, an average accuracy of 71% per sequence (Table 7). This represented a 30% increase in the average number of base-pairs in the comparative model that were predicted correctly per sequence. The average accuracy per sequence for an Archaeal, Bacterial, Eukaryotic Nuclear, Chloroplast, and Mitochondrial sequence increased by 21%-41% respectively (Table 7), and the largest increase for a single sequence (68%) was observed in the Mitochondrial dataset (Table 7). However, these dramatic improvements in accuracy were offset by a significant increase in the number of base-pairs predicted incorrectly; Mfold experienced a large drop in selectivity. The total number of unique incorrect base-pair predictions for the 496 optimal structure predictions was only 142,023, while the total number of unique incorrect predictions was 2,372,305 for the 496 sets of optimal plus 749 suboptimal structure predictions, a 1,664% increase in the number of incorrect predictions (Table 7). Two significant results came from this analysis. 1) When all of the base-pairs in the suboptimal population were included in the accuracy computation, we observed a 30% increase in average accuracy per sequence. 2) This same collection of suboptimal structures contained a 1,664% overall increase in the number of base-pairs that were not in the comparative model compared to the optimal structure prediction. In other words, a large decrease in selectivity was observed. Distribution of base pairs throughout a suboptimal population In the previous section, we considered the unique base-pairs predicted with Mfold 3.1. As mentioned earlier, the number of unique base-pairs is very small compared to the total number of base-pairs predicted within the top 750 predicted structures. A total of 166,690,139 base-pairs were predicted in the top 750 structure predictions for all 496 16S rRNA sequences in our dataset (some of the sequences did not yield 750 structure predictions with the Mfold folding parameters used in this study, see Counts of Suboptimal Predictions More or Less Accurate than the Optimal Structure Prediction for Different 16S rRNA Sequences under Additional Information at our website[36]). Of these, 59,454,137 were correct, while 107,236,002 were incorrect. In this section, we investigated 1) the frequency at which each base-pair in the comparative structure model appeared in the set of 750 structures (i.e., optimal + suboptimal population) predicted with Mfold 3.1 and 2) the relationship between this frequency and the RNA contact distance. The frequency of prediction with Mfold 3.1 for each base-pair in the comparative structure model was displayed for two Archaeal 16S rRNA comparative structure models in Figure 4. Given the analysis in the previous section on suboptimal structural variation, we selected H. volcanii and M hungatei for the panels in Figure 4. For both H. volcanii 16S rRNA (Figure 4A), and M. hungatei 16S rRNA (Figure 4B), some comparative base-pairs were predicted correctly in all 750 structure predictions, while others were predicted correctly in 600–749 structure predictions, 151–599 structure predictions, and 1–150 structure predictions. A few of the canonical base-pairs in the comparative structure model were not predicted in any of the 750 structure predictions. For H.volcanii (Figure 4A), the majority of the base-pairs predicted correctly were present in 600 to 749 structure predictions, with a significant number of base-pairs predicted correctly in all 750 structure predictions. Base-pairs predicted correctly in all 750 structure predictions were almost exclusively short-range (RNA contact distance less that 100 nt), while those predicted correctly in only 1–150 structure predictions were almost always long-range (RNA contact distance greater than 100 nt). This distribution was different for the M. hungatei 16S rRNA (Figure 4B). Here, smaller numbers of base-pairs were predicted correctly in all 750 structure predictions, and more base-pairs were predicted correctly in only 151 to 599 of the structure predictions. Similar to H. volcanii, the majority of base-pairs predicted correctly in all 750 structure predictions have small RNA contact distances. For both sequences, short-range and long-range base-pairs were observed that were predicted correctly in zero structure predictions. The distribution of comparatively predicted base-pairs, as observed from up to 750 structures of the suboptimal population, as a function of the RNA contact distance for all 496 16S rRNA sequences in our dataset, was summarized in Table 8. 76% of the base-pairs were short-range (RNA contact distance less than 101 nt), 22% were mid-range (RNA contact distance of 101–500 nt), and 2% were long-range (RNA contact distance greater than 500 nt). Of the comparative base-pairs predicted correctly in all 750 structure predictions for each 16S rRNA sequence in our data set, 98% were short-range, representing almost 15% (21,049 out of 137,000) of the total number of base-pairs predicted correctly. In contrast, only 2% of long-range base-pairs were predicted correctly in 600 or more structure predictions, representing <<1% (33 out of 137,000) of the total number of base-pairs predicted correctly. For comparative base-pairs predicted correctly in 600–749 structure predictions, 94% were short-range, for 151–599 structure predictions, 81% were short-range, and for 1–150 structure predictions, 70% were short-range. 80% (1,161 out of 1,460) of the long-range base-pairs predicted correctly appeared in 150 or fewer structure predictions. A total of 54,994 canonical, comparative base pairs were never predicted correctly, an average of 111 per 16S rRNA considered; 54% of these base pairs were short-range, 42% were mid-range, and 4% were long-range. Three important observations were presented in this section. 1) For a given sequence, some of the comparative base-pairs were predicted correctly in all 750 structures (optimal + suboptimal population). 2) A sequence with higher optimal accuracy contained a larger percentage of the comparative base-pairs predicted correctly in more of the structure predictions within the suboptimal population, compared to a sequence with lower optimal accuracy. 3) Base-pairs predicted correctly in more suboptimal structure predictions tend to have a smaller RNA contact distance. Conclusions In this paper, we evaluated how well the computer program Mfold 3.1[31], with the newest nearest-neighbor energy values, can predict the secondary structure base-pairs in comparative structure models for different RNAs. This study expands upon previous studies conducted by this lab in four ways. First, we analyzed 5S rRNA and tRNA sequences in addition to 16S and 23S rRNA sequences. Second, the number of comparative structure models in the current dataset was significantly larger, with a total of 1,411 RNAs (vs. 56 16S[29] and 72 23S[30] rRNAs studied previously), 1.5 million nucleotides and over 400,000 base-pairs, which covered all three phylogenetic domains and exhibited significant sequence variation (Table 1). Third, the increase in the speed of computers allowed us to analyze the best 749 suboptimal predictions in addition to the optimal prediction. Finally, the latest version of Mfold (version 3.1) was used. Our five most important conclusions are summarized hereunder. 1) The comparative structure models for most sequences are predicted with similar accuracy by Mfold 2.3 and Mfold 3.1 (Figure 1, Table 2,3,4) when the differences between the datasets for previous Gutell Lab studies and the current study (e.g., sample size, minor differences in comparative models, sequence variation within the dataset) are considered. The average folding accuracy for our current study is 41% for complete 16S rRNA sequences and 41% for complete 23S rRNA sequences, which is slightly less than in our earlier studies (Table 2). While the majority of optimal structure predictions for each RNA sequence still have an accuracy score between 20% and 60%, sequences with accuracy scores less than 20% are also observed (Table 2,4). On average, Archaeal and Bacterial 16S and 23S rRNA sequences still have higher accuracy scores than Eukaryotic Nuclear, Chloroplast, and Mitochondrial sequences (Table 3). 2) Base-pairs with smaller RNA contact distances are both abundant in comparatively predicted structures and predicted more accurately by both versions of Mfold, and base-pairs with large RNA contact distances which are abundant in Mfold predicted structures only, are frequently incorrect. The prediction accuracy for individual base-pairs decreases exponentially as RNA contact distance increases. Figure 2A shows that the number of comparative base-pairs decreases exponentially as the RNA contact distance increases. Using a logarithmic scale, we show that the base-pair prediction accuracy decreases in a linear fashion as contact distance increases (Figure 2B), which indicates an exponential relationship between base-pair prediction accuracy and RNA contact distance. In addition, many more long-range base-pairs (RNA contact distance of 501 or higher) are predicted than found in the corresponding comparative structure model, and the overwhelming majority of these predicted base-pairs are incorrect (Table 5). 3) While uncertainties in the energy parameters may play a small role, free energy (calculated in its current form) alone is insufficient to distinguish between different structural possibilities for the same sequence. The variation between any two structure predictions within the suboptimal population is not correlated with the ΔΔG between those two structure predictions (Table 6, Figure 3). We observe that two structures that are more similar with one another (structure variation score of less than 100), and very different from one another (structure variation score greater than 500), have very similar ΔΔG values. 4) Without prior knowledge of the correct structure model, analysis of the suboptimal structure models can not improve our ability to both predict correctly the base-pairs in the secondary structure and assemble them into a single secondary structure model. Our analysis of the accuracy of base-pairs predicted in the suboptimal population for our 16S rRNA dataset reveals that the entire population contained a higher percentage of base-pairs present in the comparative model than the optimal structure prediction alone (Table 7). When considering the suboptimal population, the free energy minimization method is able to identify an average of 71% or more of the comparatively predicted base-pairs for a given sequence (Table 7) vs. only 41% when considering just the optimal structure prediction. However, this same suboptimal population contains a significant increase in the number of incorrect base-pairs (Table 7). In other words, the increase in recall is offset by the inability of Mfold to consistently identify a single structure model containing a high percentage of comparative base-pairs. 5) The frequency of correctly predicted base-pairs in the suboptimal population is extremely variable, and base-pairs with a smaller RNA contact distance are more likely to be observed at a higher frequency. A qualitative analysis of Figure 4 shows that some base-pairs in the comparative structure model are predicted in all structure predictions within a suboptimal population, others are predicted in a subset, and yet others are not predicted at all. 98% of base-pairs predicted correctly in all structure predictions have an RNA contact distance less than 101 nt, while 80% of base-pairs with an RNA contact distance of 501 nt or more are only predicted correctly in 150 or less structure predictions (Table 8). Additionally, 63% (2,472 out of 3,932) of base-pairs with an RNA contact distance of 501 nt or more are never predicted correctly in the suboptimal population (Table 8). From our previous analysis with version 2.3 of Mfold, we had determined that free energy minimization does not consistently identify the correct base-pairs in the 16S and 23S rRNA comparative secondary structure models[29,30]. We arrive at the same conclusion with our analysis of the current version 3.1 of Mfold and a significantly larger set of rRNA comparative structure models. One explanation could be incorrect energy parameters for multi-stem loops. Especially with longer sequences such as 16S or 23S rRNA, many long-range base-pairs occur along with the formation of these multi-stem loops. As we have shown in Table 5, only 8% (6,171 out of 73,071) of 16S rRNA and 13% (10,758 out of 81,128) 23S rRNA base-pairs predicted by Mfold with an RNA contact distance 101 or more nucleotides are correct. A more accurate characterization of the energetics of multi-stem loops may lead to significantly better prediction accuracies. Mathews et al. have started to address this issue using experimental studies[39,40] and known RNA secondary structures[31] to generate multi-stem loop initiation parameters that can be used in energetic calculations. We believe that another potential reason for the inaccurate structures predicted with Mfold is that kinetics plays a role in RNA folding. Here, we suspect that nucleotide interactions with smaller contact distances will form more quickly, as suggested by Higgs[41], and will dominate the number of base-paired interactions formed. Presumably, these short-range interactions that form rapidly will be in equilibrium with other helices with a minimum contact distance (driven by nearest-neighbor energetics), and in the process, prevent energetically stable helices with larger contact distances from forming[41]. Several of our results support these ideas: 1) In the 16S and 23S rRNA comparative structure models, 75% of the predicted base-pairings have a contact distance of 100 or less; 2) Minimum free energy structures for 16S rRNA (as predicted by Mfold) have almost 10 times more long-range base-pairs than the comparative structure models (Table 5); 3) 92% (75,763 out of 81,934) and 86% (67,130 out of 77,888) of correctly predicted base-pairs have a contact distance of 100 or less for 16S and 23S rRNA (Table 5); 4) The higher prediction accuracy observed for short RNA molecules such as 5S rRNA or tRNA (Table 2). Knowledge-based approaches that incorporate comparative analysis and high-resolution crystal structure data have been successful in the prediction of protein structures[42-44]. With the recent increase in the number of high-resolution crystal structures for different RNA molecules, it has been suggested that similar approaches could be utilized to predict RNA structure[45]. We envision a knowledge-based RNA folding algorithm with three fundamental facets: 1) a kinetic model of RNA folding that includes cooperative formation of short-range base-pairs and helices, 2) a thermodynamic component provided by the nearest-neighbor model applied locally within different parts of the sequence, and 3) relationships between RNA sequence and structure elements and observed structural biases, for example tetraloops[46], AA:AG motifs at the ends of helices[47], a bias for unpaired adenosines in the secondary structure model [48], and Lone Pair Triloops[49]. Use of sequence-structure relationships requires evaluation of known two- and three- dimensional RNA structures, hence the "knowledge-based" facet of the algorithm. Some researchers in the field have begun to adopt this approach. In Mfold 3.1, free-energy bonuses are applied to certain classes of hairpin loops and the energetic parameters for multi-branch loops were tuned using comparatively predicted structures[31]. Other researchers have developed RNA secondary structure prediction algorithms that combine energetics and comparative sequence analysis [50-52]. We believe that a tuned algorithm of the form just described has the potential to predict RNA secondary and eventually tertiary structure more accurately and reliably than methods currently available. Methods RNA secondary structure prediction All 1,411 sequences in our dataset were folded using Mfold 3.1[31]. The optional parameters used for this study were window size (W) of 1, percent suboptimality (P) of 5% (default value), and maximum number of possible foldings (MAX) of 750. The efn2 program was used to re-compute the energetics for each predicted structure for a given sequence. The predicted structures were then ordered by the efn2 calculated free energies, and the minimum free energy structure reported was the lowest energy structure after efn2 re-evaluation. Only a single structure was selected as the minimum free energy structure, and we did not look for other foldings with the minimum free energy. The previous Gutell Lab studies[29,30] used window sizes (W) of 10 and 20 respectively and no efn2 re-evaluation. The Mathews et al.[31] study used a window size (W) of 0, percent suboptimality (P) of 20%, and efn2 re-evaluation. Prediction accuracy calculations The accuracy of an Mfold prediction for a sequence was determined by: 1) counting the number of canonical base-pairs (excluding any base-pairs with IUPAC symbols other than G,C,A, or U) in the comparatively-derived secondary structure model that appeared in the Mfold 3.1 prediction, and 2) dividing that value by the total number of canonical base-pairs in the comparatively-derived model. Any canonical, pseudoknotted base-pairs in a comparatively derived secondary structure model were included in the total number of comparatively predicted base-pairs for the given model. For example, in the 16S rRNA from Archaeoglobus fulgidus, the Mfold 3.1 optimal structure prediction contained 256 of the 448 comparatively predicted canonical base pairings in the A. fulgidus comparative structure model; thus, the accuracy was 65%. Although the comparatively-derived models included non-canonical pairing predictions (e.g., U:U), these were not considered in the accuracy measure, since Mfold 3.1 does not predict non-canonical pairings. Comparative structure database A total of 1,411 secondary structure models were determined with comparative analysis (Table 1) and used to benchmark the accuracy of Mfold 3.1. For our tRNA secondary structure models, 30% of the sequences contained at least one known modification that would prevent the nucleotide from participating in A-helix base-pairs. These modifications were not included as constraints for Mfold to use in the secondary structure prediction. 41% (349 out of 842) of the rRNA secondary structure models were currently available (as of January 2004) at The Comparative RNA Web (CRW) Site[24]. The other diagrams were not available at the CRW Site for the following reasons: 1) visual improvements were needed for the diagrams, 2) a small number of base-pairs needed to be changed in the structure models and 3) the sequence and/or the structure was not publicly available, pending submission of a manuscript. The secondary structure drawing program XRNA[53], on Sun Microsystems computers, was used to draw the comparative structure diagrams. Per sequence averages Some average values for statistics computed in this study, such as secondary structure prediction accuracy, were calculated on a per sequence basis. A per sequence average variant of a particular statistic was calculated by averaging the value of the statistic for each individual sequence in the dataset. For example, for the 16S rRNA dataset, the overall accuracy was calculated by first determining the accuracy of the Mfold optimal structure prediction for each individual sequence[36]. Then, the 496 accuracy values were averaged to calculate the overall accuracy score of 41%. Computational setup All 1,411 sequences were folded on a computer with dual AMD Athlon MP 1800 processors and 1GB of RAM, under the SuSE Linux 8.0 operating system[54]. In addition, four single-processor AMD Athlon computers (Thunderbird 1GHz processor), each with 512 MB of RAM and SuSE Linux 8.0[54], were used to prepare sequences for folding and to compress the results for storage. The sequences were folded in under 48 hours using a workflow-based system that was developed for automatically managing the folding runs. Even after compression, the aggregate set of folding results required over 150 GB of disk space. The raw results were parsed and imported into a database, managed by MySQL[55]. The database contained over 100 tables that held intermediate results from the folding runs. Intermediate results were then retrieved, using simple SQL queries, when required to calculate final results. Logarithmic binning of base-pairs by contact distance for 16S rRNA Figure 2A showed that the number of comparative base-pairs observed decreased exponentially as the contact increased; therefore, logarithmic binning was required to group the base-pairs into somewhat equally-sized bins based on contact distance. The shortest and longest contact distances observed in our 16S rRNA data set were 3 and 1833, respectively[36]. Therefore, the overall range of our logarithmic scale was from log10 (3) to log10 (1833). This range was divided into equal increments to define our contact distance bins. After evaluating many increment sets, with the requirement that the sizes of the bins be within one order of magnitude of one another, seven distance bins were established (Figure 2B). Suboptimal structural variation score The suboptimal structural variation score measures the agreement between two different secondary structure models for the same RNA sequence. We compute the score by comparing the paired or unpaired state of each nucleotide in the two structure models. We increment the score when either a given position is unpaired in one structure model and paired in the other or when the position is paired to different positions in the respective structure models. We do not increment the score when a given position is paired to the same position in both structure models or is unpaired in both structure models. The higher the structural variation score, the lower the level of agreement between the two secondary structure models. The structural variation score is zero for two identical structure models. For two structure models that are different at every position the structural variation score equals the number of nucleotides in the sequence. Authors' contributions KJD developed the workflow based management system for folding the sequences, constructed the database for analyzing the folding results, tabulated the results and drafted the manuscript. JJC developed the secondary structure models for the tRNA dataset, contributed ideas to the analysis and the figure and table generation, and edited the manuscript. CWC assembled the tRNA alignment, tabulated results, and edited the manuscript. RRG provided vision and direction and rewrote portions of the manuscript. All authors read and approved the final manuscript. List of abbreviations Nucleotide – nt Acknowledgements The authors thank Dmitrii Makarov, Brent Iverson, Chang-Yong Lee and Ray L. Marr for many helpful discussions during the study, Edison Morales for assistance in figure generation, and others for creating secondary structure diagrams used in this analysis. This project was supported by the National Institutes of Health (GM48207 and GM067317), the Welch Foundation (F-1427), start-up funds from The Institute for Cellular and Molecular Biology at The University of Texas at Austin, and the Dean's Boyer Fellow grant. KJD was funded under an Institutional Research Service Award from the National Institutes of Health (T32 GM08747) and an IGERT from the National Science Foundation (DGE-0114387). Figures and Tables Figure 1 Direct Comparison of Mfold 2.3 and Mfold 3.1 Folding Accuracy for Selected 16S and 23S rRNAs. Base-pairs marked in red are predicted correctly by both Mfold 2.3 and Mfold 3.1. Base-pairs marked in blue are predicted correctly only by Mfold 2.3, and base-pairs marked in green are predicted correctly only by Mfold 3.1. Black base-pairs are not predicted correctly by either version of Mfold. Only canonical base-pairs in the comparative models in the current study and previous Gutell Lab studies are considered. Non-canonical base-pairs in the comparative structure models are not counted. Full-sized versions of each annotated structure diagram are available at our website[36]. A: Archaea 16S rRNA Haloferax volcanii. B.1: Archaea 23S rRNA, 5' half, Thermococcus celer. B.2: Archaea 23S rRNA, 3' half, Thermococcus celer. C: Eukaryotic Nuclear16S rRNA, Giardia intestinalis. D.1: Eukaryotic Nuclear 23S rRNA, 5' half, Giardia intestinalis. D.2: Eukaryotic Nuclear 23S rRNA, 3' half, Giardia intestinalis. Figure 2 Accuracy of Comparatively Predicted Base-pairs from 496 16S rRNA Sequences and RNA Contact Distance. A. The RNA contact distance (the number of nucleotides in the RNA sequence that are separates the 5' and 3' base-paired) for all 191,994 base-pairs in comparative structure models is determined and plotted. B. The 191,994 comparatively predicted base-pairs are divided into seven RNA contact distance bins (see Logarithmic Binning of Base-pairs by Contact Distance for 16S rRNA in Methods) represented by columns. The accuracies for all base-pairs in each bin are also plotted as points. Figure 3 ΔΔG vs. Structural Variation for Pairwise Comparisons from the "Suboptimal Population". A set of 750 structure predictions (optimal + top 749 suboptimal) are compared, resulting in a total of 280,875 pairwise comparisons. The ΔΔG (pre efn2 re-evaluation) for two structure predictions is calculated by taking the absolute value of the difference between the ΔG of each structure prediction before efn2 re-evaluation. Structural variation for two structure predictions is calculated by counting the number of nucleotides in each structure prediction that either 1) have different pairing partners or 2) are paired in one structure prediction and unpaired in the other structure prediction (see Suboptimal Structural Variation Score in Methods). The shading within the figure indicates the number of pairwise comparisons that have the same values for both ΔΔG and structural variation score. A: Archaea 16S rRNA Haloferax volcanii. B: Archaea 16S rRNA Methanospirillum Hungatei. Figure 4 Frequency of Base-pair predictions within a "Suboptimal Population" for selected 16S rRNAs. The frequency of the prediction of each of the base-pairs in the comparative structure model in a set of 750 structure predictions (optimal + top 749 suboptimal) is displayed on the comparative structure model. Base-pairs marked in red are predicted correctly in all 750 structure predictions. Base-pairs marked in blue are predicted correctly in 600 to 749 structure predictions. Base-pairs marked in magenta are predicted correctly in 151 to 599 structure predictions, base-pairs marked in green are predicted correctly in only 1 to 150 structure predictions, and base-pairs marked in black are not predicted in any of the 750 structure predictions (some are non-canonical or occur in pseudo-knots, and thus are not expected to be predicted correctly). Full-sized versions of each annotated structure diagram are available at our website[36]. A: Archaea 16S rRNA Haloferax volcanii. B: Archaea 16S rRNA Methanospirillum hungatei. Table 1 Distribution of Comparatively Predicted Secondary Structure Models Analyzed 5S rRNA 16S rRNA 23S rRNA tRNA Total Structures 90 496 256 569 1,411 Total AGCU Nucleotides1 10,777 724,475 712,575 42,283 1,490,110 Total Nucleotides2 10,819 736,412 714,723 43,189 1,505,143 Total Comparative Pairings3 3,107 191,994 178,958 11,796 385,854 Average Sequence Length 120 1,485 2,792 76 - Average Pairings/Structure3 35 387 699 21 - Phylogenetic Distribution  Archaea 12 23 17 76 128  Bacteria 28 195 75 155 453  Eucarya   Nuclear 45 133 52 207 437   Chloroplast 4 33 31 131 199   Mitochondrion 1 112 81 - 194 Pairwise Sequence Identity4 Archaea Bacteria Nuclear Chloroplast Mitochondrion  16S rRNA   < 80% Identity 380 / 75% 32,456 / 86% 16,574 / 94% 746 / 71% 12,332 / 99%   < 50% Identity 0 / 0% 0 / 0% 8,500 / 48% 98 / 9% 9,852 / 79%   >= 95% Identity 16 / 3% 1,588 / 4% 212 / 1% 4 / 0.4% 12 / 0.1%   Total Pairs 506 37,830 17,556 1,056 12,432  23S rRNA   < 80% Identity 236 / 87% 5,214 / 94% 2,568 / 97% 710 / 76% 6,394 / 99%   < 50% Identity 0 / 0% 0 / 0% 1,960 / 74% 62 / 7% 5,830 / 90%   >= 95% Identity 6 / 2% 42 / 1% 8 / 0.30% 18 / 2% 8 / 0.1%   Total Pairs 272 5,550 2,652 930 6,480 tRNA Amino Acid Distribution5 Archaea Bacteria Nuclear Chloroplast Total  Alanine (Aln) 13 14 6 5 38  Arginine (Arg) 4 9 17 12 42  Asparagine (Asn) 3 9 10 3 25  Aspartic acid (Asp) 4 4 12 6 26  Cysteine (Cys) 2 5 3 5 15  Glutamine (Gln) 3 6 13 4 26  Glutamic acid (Glu) 4 8 23 8 43  Glycine (Gly) 6 18 17 9 50  Histidine (His) 3 6 10 7 26  Isoleucine (Ile) 3 16 8 10 37  Leucine (Leu) - - - - -  Lysine (Lys) 3 8 15 4 30  Methionine (Met) 4 7 7 9 27  Phenylalanine (Phe) 3 6 16 10 35  Proline (Pro) 5 10 12 8 35  Serine (Ser) - - - - -  Threonine (Thr) 7 14 6 10 37  Tryptophan (Trp) 1 6 3 10 20  Tyrosine (Tyr) 2 - 6 3 11  Valine (Val) 6 9 23 8 46 Total 76 155 207 131 569 1 Considers only A, G, C or U nucleotides. 2 Considers all nucleotides. 3 Includes only G:C, A:U and G:U base-pairings predicted with comparative analysis. 4 Average sequence identities for all pairwise comparisons between sequences. Number of pairwise comparisons equals (n2-n) where n is the number of sequences considered. 5 Only Type I tRNAs are considered. Table 2 Average Accuracy of the Optimal RNA Structure Predicted with Mfold 3.1† 5S rRNA 16S rRNA 23S rRNA tRNA M1 C2 P13 M C P24 M C M5 C Sequences 309 90 56 22 496 72 5 256 484 569 Accuracy6,7,8,9 78 ± 23 71 ± 24 46 ± 17 51 ± 16 41 ± 13 45 ± 16 44 ± 11 57 ± 14 41 ± 13 43 ± 12 83 ± 22 69 ± 24 High/Low10 98/0 81/10 77/5 74/19 74/1 100/0 Median 81 41 41 70 Distributions ≤ 20% acc11 4 9 4 1 6 2 ≥ 60% acc12 77 25 9 6 5 60 20%<acc<60%13 19 66 86 93 89 39 †All values are percentages unless otherwise indicated. All averages are per sequence averages for folding complete sequences as defined in the Per Sequence Averages section in Methods. C, Current Study; P1, Previous Study by Gutell Lab for 16S rRNA[29]; P2, Previous Study by Gutell Lab for 23S rRNA[30]; M, Previous Study by Mathews et al.[31]. Accuracies from all previous studies are for folding complete sequences. 1 All sequences from the Mathews et al. study (M) were folded with Mfold 3.1 using a window size (W) of 0, percent suboptimality (P) of 20%, maximum number of suboptimals (MAX) of 750 and efn2 re-evaluation and re-ordering. 2 All sequences in the current study (C) were folded with Mfold 3.1 using a window size (W) of 1, percent suboptimality (P) of 5% and efn2 re-evaluation and re-ordering 3 All sequences in the previous Gutell Lab study on 16S rRNA (P1) were folded with Mfold 2.3 using a window size (W) of 10 and no efn2 re-evaluation and re-ordering. 4 All sequences in the previous Gutell Lab study on 23S rRNA (P2) were folded with Mfold 2.3 using a window size (W) of 20 and no efn2 re-evaluation and re-ordering. 5 Bases modified in tRNA that are subsequently unable to fit into an A form helix were constrained to be single-stranded. 6 Comparative base-pairs that are pseudoknotted were excluded from the analysis in previous Gutell Lab studies (P1, P2), but were included in the current study. The Mathews et al. study included a measure of the percentage of pseudoknotted base-pairs in comparatively predicted structures they considered, but it was unclear if they were included in the analysis. 7 In all studies, only canonical, comparative base-pairs (excluding any base-pairs with IUPAC symbols) were considered. For both the current study (C) and previous Gutell Lab studies (P1, P2), a predicted base-pair was considered correct only if it matched a comparative base-pair exactly. In the Mathews et al. (M) study, a base-pair was considered if: 1. it matched a comparatively predicted base-pair exactly or 2. either nucleotide of the Mfold predicted base-pair (X,Y where X and Y are the positions of the nucleotides in the sequence) is within one nucleotide of its comparatively predicted position (X, Y ± 1 or X ± 1,Y). 8 Accuracy values in bold under the (C) columns for 16S and 23S rRNA represent average prediction accuracies in the current study for just the subset of sequences considered in the previous Gutell Lab studies.[29, 30]. The following sequences were considered in previous Gutell Lab studies, but excluded from the current study, Olisthodiscus luteus (16S rRNA, Chloroplast) and Sulfolobus solfataricus (23S rRNA, Archaea). 9 When the efn2 re-evaluation and re-ordering step was omitted from our study, the average prediction accuracy was 40 ± 13 for 16S rRNA, 40 ± 13 for 23S rRNA, 69 ± 24 for 5S rRNA, and 66 ± 24 for tRNA. For complete details, see our website[36]. 10 Accuracy scores for the best and worst predicted structures in each group. 11 Percentage of predicted structures with an accuracy of 20% or less. 12 Percentage of predicted structures with an accuracy of 60% or higher. 13 Percentage of predicted structures with an accuracy between 20% and 60%. Table 3 Average Accuracy of the Optimal RNA Structure Predicted with Mfold 3.1 Grouped by Phylogeny† 5S rRNA 16S rRNA 23S rRNA tRNA C P1 C P2 C C Archaea 79 / 98 / 29 68 / 81 / 55 62 / 77 / 51 59 / 74 / 51 58 / 74 / 40 73 / 100 / 32 Bacteria 62 / 94 / 18 56 / 69 / 39 49 / 68 / 21 53 / 66 / 45 49 / 66 / 31 74 / 100 / 0 Eucarya (n)1 75 / 94 / 0 30 / 47 / 10 34 / 50 / 15 41 / 60 / 23 42 / 63 / 21 61 / 100 / 0 Eucarya (c) 67 / 85 / 16 48 / 71 / 32 46 / 71 / 19 39 / 54 / 19 39 / 49 / 21 73 / 100 / 19 Eucarya (m) 31 / 56 / 17 30 / 60 / 5 38 / 57 / 24 30 / 61 / 1 Eucarya (m)2 31 / 60 / 5 Eucarya (m)3 33 / 60 / 16 †All values (average/high/low) shown as percentages unless otherwise indicated. The determination of the accuracy for the structures predicted with Mfold is described in the Methods section, RNA Secondary Structure Prediction and Prediction Accuracy Calculations. C, Current Study; P1, Previous study by the Gutell Lab for 16S rRNA[29]; P2, Previous study by the Gutell Lab for 23S rRNA[30]. 1 (n), Nuclear-encoded sequences; (c), Chloroplast-encoded sequences; (m), Mitochondrial-encoded sequences. 2 Based on comparative models with 100 or more canonical base-pairs only. 3 Based on comparative models with 300 or more canonical base-pairs only. Table 4 RNA Folding Accuracy of Specific 16S and 23S rRNA Sequences using Mfold 2.3 and 3.1† Previous[29, 30] Current 16S rRNA Eukaryotic Mitochondrion   Zea mays (X00794) 17 30   Ascaris summ (X54253) 17 13   Caenorhabditis elegans (X54252) 23 24 Eukaryotic Nuclear   Hexamita sp. (Z17224) 27 29   Giardia muris (X65063) 22 29   Giardia ardeae (G17210) 30 33   Giardia intestinalis (X52949) 10 23   Encephalitozoon cuniculi (X98467) 18 21   Vairimorpha necatrix (Y00266, M24612) 28 25   Babesia bigemina (X59064) 20 19 23S rRNA Eukaryotic Chloroplast   Astasia longa (X14386) 19 23 Eukaryotic Mitochondrion   C. elegans (X54252) 30 31   Gallus gallus (X52392) 28 25   Saccharomyces. cerevisiae (J01527) 27 20   Z. mays (K01868) 24 29 Eukaryotic Nuclear   E. gracilis (X53361) 23 21   G. intestinalis (X52949) 24 33 †All values are percentages unless otherwise indicated. The determination of the accuracy for the structures predicted with Mfold is described in the Methods section, RNA Secondary Structure Prediction and Prediction Accuracy Calculations. Genbank accession numbers are listed in parentheses for each sequence. Table 5 Accuracy of Base-pairs Predicted with Mfold 3.1 as a Function of RNA Contact Distance† 16S rRNA 23S rRNA RNA Contact Distance 496 Structures 256 Structures Comparative Total Base-pairs % of Total Total Base-pairs % of Total Total 191,994 100 178,958 100 2–100 145,058 76 134,085 75  2–50 121,170 63 106,534 60  51–100 23,888 12 27,551 15 101+ 46,936 24 44,873 25  101–500 43,004 22 37,121 21  501+ 3,932 2 7,752 4 Predicted with Mfold 3.1 Total 223,957 100 218,908 100 2–100 150,886 67 137,780 63  2–50 123,708 55 109,078 50  51–100 27,178 12 28,702 13 101+ 73,071 33 81,128 37  101–500 43,498 19 44,139 20  501+ 29,573 13 36,989 17 Correctly Predicted with Mfold 3.1 %C1 %M %C %M Total 81,934 43 37 77,888 44 36 2–100 75,763 52 50 67,130 50 49  2–50 64,651 53 52 54,898 52 50  51–100 11,202 47 41 12,232 44 43 101+ 6,171 13 8 10,758 24 13  101–500 5,978 14 14 9,441 25 21  501+ 193 5 0.7 1,317 17 4 Avg. Percent Correct2 Current Previous[29] Current Previous[30] 2–100 50 55 47 53  2–50 52 - 49 -  51–100 44 - 40 - 101–200 22 15 26 35 201–300 10 14 22 21 301–400 9 13 13 10 401–500 4 12 16 13 501+ 4 - 14 - †All base-pairs predicted in the comparative and the Mfold optimal structure predictions including those base-pairs predicted correctly (any base-pairs with IUPAC symbols other than A,G,C, or U are excluded) are grouped by RNA contact distance for 16S and 23S rRNA. RNA contact distance is defined as the number of nucleotides intervening between the 5' and 3' halves of a base-pair. The determination of the accuracy for the structures predicted with Mfold is described in the Methods section, RNA Secondary Structure Prediction and Prediction Accuracy Calculations. 1 %C, the percentage of comparatively predicted base-pairs; %M, the percentage of Mfold predicted base-pairs. 2 The Per Sequence Average (see Per Sequence Average in Methods) percentage of comparative base-pairs in each distance category predicted correctly in the Mfold optimal structure predictions. Table 6 Average, Minimum and Maximum ΔΔG Values for Pairwise Comparisons of Different Suboptimal Folds† Haloferax volcanii Methanosprillum hungatei Optimal Accuracy1 80% 46% Total Fold Predictions (Optimal + Suboptimal) 750 750 Total Pairwise Comparisons 280,875 280,875  Structural variation score of 1 to 50   Num of Pairwise Comparisons 32,378 12%2 11,016 4%   ΔΔG Min (kcal/mol) 0 0   ΔΔG Max (kcal/mol) 11 8.60   ΔΔG Average (kcal/mol) 2.84 2.19  Structural variation score of 51 to 100   Num of Pairwise Comparisons 102,071 36% 32,360 12%   ΔΔG Min (kcal/mol) 0 0   ΔΔG Max (kcal/mol) 11 8.60   ΔΔG Average (kcal/mol) 2.53 2.01  Structural variation score of 101 to 500   Num of Pairwise Comparisons 121,805 43% 134,037 48%   ΔΔG Min (kcal/mol) 0 0   ΔΔG Max (kcal/mol) 11 8.6   ΔΔG Average (kcal/mol) 2.24 1.74  Structural variation score of 501+   Num of Pairwise Comparisons 24,621 9% 103,462 37%   ΔΔG Min (kcal/mol) 0 0   ΔΔG Max (kcal/mol) 11 8.6   ΔΔG Avg (kcal/mol) 2.24 1.82 †Both sequences are 16S rRNAs. For each sequence, Mfold 3.1 predicts one optimal or minimum free energy fold and 749 suboptimal folds (750 total folds). Pairwise comparisons are grouped based on the structural variation between the two folds compared. For details on how structural variation between two folds is calculated see Materials and Methods. The range of ΔΔG values observed is 0–11 kcal/mol for H. volcanii and 0–8.60 kcal/mol for M. hungatei., and all ΔG values are pre-efn2 re-evaluation. 1 Without efn2 re-evaluation and re-ordering of predicted folds. 2 Percentage of total pairwise comparisons. Table 7 Distribution of 16S rRNA Base-pairs Predicted Correctly and Incorrectly† Overall Archaea Bacteria Eucarya (C)1 (M) (N) Comparative 191,994 10,211 83,385 13,406 29,979 55,013 Opt Correct2 81,934 6,376 41,032 6,105 9,459 18,962 Subopt Correct3 137,000 8,570 65,177 10,032 21,201 32,020 Opt Incorrect2 142,023 4,758 49,563 8,603 27,617 51,482 Subopt Incorrect3 2,372,305 101,253 947,197 161,397 472,614 689,844 Opt Accuracy2,4 41% 62% 49% 46% 30% 34% Subopt Accuracy3,4 71% 84% 78% 75% 71% 59% Avg Improvement5 30% 21% 29% 30% 41% 24% Best Prediction6 92% 91% 89% 92% 92% 90% Max Improvement7 68% 35% 54% 53% 68% 48% Min Improvement8 10% 10% 12% 12% 14% 11% †All 496 16S rRNA sequences are considered. Each sequence is folded for a population of one optimal and 749 suboptimal structure predictions. The determination of the accuracy for the structures predicted with Mfold is described in the Methods section, RNA Secondary Structure Prediction and Prediction Accuracy Calculations. Values are calculated by summing the number of unique base-pairs encountered for each sequence that satisfy each particular category (any base-pairs involving IUPAC symbols other than A,G,C, or U are excluded). For example, Subopt Correct is calculated by summing the number of unique, correctly predicted base-pairs encountered in the population of optimal plus suboptimal structure predictions for each of the 496 16S rRNA sequences. Prediction accuracy when including base-pairs predicted correctly in suboptimal structure predictions is also tabulated. 1 (c), Chloroplast-encoded sequences; (m), Mitochondrial-encoded sequences; (n), Nuclear-encoded sequences. 2 Considering only the optimal prediction. 3 Considering the optimal prediction plus up to 749 suboptimal predictions. 4 Averages calculated on per sequence basis. Please see Per Sequence Averages in Methods. 5 Average improvement in Mfold secondary structure prediction accuracy when pooling base-pairs from both the optimal prediction and suboptimal predictions. 6 The highest Mfold secondary structure prediction accuracy for an individual sequence when pooling base-pairs from both the optimal and suboptimal populations. 7 The largest improvement in Mfold secondary structure prediction accuracy for an individual sequence when pooling base-pairs from both the optimal and suboptimal populations. 8 The smallest improvement in Mfold secondary structure prediction accuracy for an individual sequence when pooling base-pairs from both the optimal and suboptimal populations. Table 8 Frequency of Comparative Base-pairs in 750 Structures Predicted with Mfold 3.1† RNA Contact Distance Frequency1 2–100 nt 101–500 nt 501+ nt 750 21,049 98% 18% 417 2% 2% 0 0% 0% 21,466 600–749 42,362 94% 37% 2,805 6% 14% 33 0% 2% 45,200 151–599 20,775 81% 18% 4,594 18% 23% 266 1% 18% 25,635 1–150 31,285 70% 27% 12,253 27% 61% 1,161 3% 80% 44,699 Correct 115,471 84% 20,069 15% 1,460 1% 137,000 Never 29,587 54% 22,935 42% 2,472 4% 54,994 Total 145,058 76% 43,004 22% 3,932 2% 191,994 †For all 496 16S rRNA sequences, a total of 750 structure models are predicted for each sequence (one optimal and 749 suboptimal structure predictions). Every base-pair (excluding any base-pairs involving IUPAC symbols other than A,G,C, or U) in the comparative structure model that appears in a set of 750 structure predictions for a particular sequence is categorized by 1) the number of structure predictions in which it appears and 2) the RNA contact distance. The four bold percentages for each of the three RNA contact distances each total 100%, and reveal the percentage of base-pairs predicted correctly for the four frequency ranges. For example, a total of 115,471 base-pairs with an RNA contact distance of 2–100 nt were predicted correctly. Of those base-pairs, 18% (21,049) were predicted in 750 structure predictions, 37% (42,362) were predicted in 600–749 structure predictions, 18% (20,775) were predicted in 151–599 structure predictions, and 27% (31,285) were predicted in 1–150 structure predictions. In contrast, the three italicized percentages for each of the four frequency ranges, and the "Correct", "Never", and "Total" categories total 100%. For example, 54,994 base-pairs were never predicted in 750 structure predictions. Of those base-pairs, 54% (29,587) have an RNA contact distance of 2–100 nt, 42% (22,935) have an RNA contact distance of 101–500 nt, and 4% (2,472) have an RNA contact distance of 501+ nt. 1 Frequency of prediction throughout a suboptimal population of up to 750 structure predictions. ==== Refs Zuker M Sankoff D Rna Secondary Structures and Their Prediction B Math Biol 1984 46 591 621 Zuker M On finding all suboptimal foldings of an RNA molecule Science 1989 244 48 52 2468181 Zuker M Waterman MS The use of dynamic programming algorithms in RNA secondary structure prediction Mathematical Methods for DNA Sequences 1989 CRC Press 59 184 Borer PN Dengler B Tinoco I., Jr. Uhlenbeck OC Stability of ribonucleic acid double-stranded helices J Mol Biol 1974 86 843 853 4427357 Tinoco I., Jr. Borer PN Dengler B Levin MD Uhlenbeck OC Crothers DM Bralla J Improved estimation of secondary structure in ribonucleic acids Nat New Biol 1973 246 40 41 4519026 Tinoco I., Jr. Uhlenbeck OC Levine MD Estimation of secondary structure in ribonucleic acids Nature 1971 230 362 367 4927725 Uhlenbeck OC Borer PN Dengler B Tinoco I., Jr. Stability of RNA hairpin loops: A 6 -C m -U 6 J Mol Biol 1973 73 483 496 4715013 Gralla J Crothers DM Free energy of imperfect nucleic acid helices. 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==== Front BMC Mol BiolBMC Molecular Biology1471-2199BioMed Central 1471-2199-5-91529196810.1186/1471-2199-5-9Research ArticleShort-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors Fish Richard J [email protected] Egbert KO [email protected] Division of Angiology and Haemostasis, Department of Internal Medicine, Geneva University Hospital and University Medical Centre, Geneva, CH-1211 Switzerland2004 3 8 2004 5 9 9 23 4 2004 3 8 2004 Copyright © 2004 Fish and Kruithof; licensee BioMed Central Ltd.2004Fish and Kruithof; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background RNA interference (RNAi) can potently reduce target gene expression in mammalian cells and is in wide use for loss-of-function studies. Several recent reports have demonstrated that short double-stranded RNAs (dsRNAs), used to mediate RNAi, can also induce an interferon-based response resulting in changes in the expression of many interferon-responsive genes. Off-target gene silencing has also been described, bringing into question the validity of certain RNAi-based approaches for studying gene function. We have targeted the plasminogen activator inhibitor-2 (PAI-2 or SERPINB2) mRNA using lentiviral vectors for delivery of U6 promoter-driven PAI-2-targeted short hairpin RNA (shRNA) expression. PAI-2 is reported to have anti-apoptotic activity, thus reduction of endogenous expression may be expected to make cells more sensitive to programmed cell death. Results As expected, we encountered a cytotoxic phenotype when targeting the PAI-2 mRNA with vector-derived shRNA. However, this predicted phenotype was a potent non-specific effect of shRNA expression, as functional overexpression of the target protein failed to rescue the phenotype. By decreasing the shRNA length or modifying its sequence we maintained PAI-2 silencing and reduced, but did not eliminate, cytotoxicity. ShRNA of 21 complementary nucleotides (21 mers) or more increased expression of the oligoadenylate synthase-1 (OAS1) interferon-responsive gene. 19 mer shRNA had no effect on OAS1 expression but long-term selective pressure on cell growth was observed. By lowering lentiviral vector titre we were able to reduce both expression of shRNA and induction of OAS1, without a major impact on the efficacy of gene silencing. Conclusions Our data demonstrate a rapid cytotoxic effect of shRNAs expressed in human tumor cell lines. There appears to be a cut-off of 21 complementary nucleotides below which there is no interferon response while target gene silencing is maintained. Cytotoxicity or OAS1 induction could be reduced by changing shRNA sequence or vector titre, but stable gene silencing could not be maintained in extended cell culture despite persistent marker gene expression from the RNAi-inducing transgene cassette. These results underscore the necessity of careful controls for immediate and long-term RNAi use in mammalian cell systems. ==== Body Background Gene silencing is a powerful tool with which to study protein function. Gene inactivations in mice have revolutionised the way we study both basic biology and a plethora of disease types [1,2]. Gene silencing in human cells has, until recently, proven difficult to achieve [3]. Research with plants, flies and worms recently uncovered a mechanism by which eukaryotic cells target mRNAs, and perhaps even genetic loci, for specific gene silencing. This process is termed RNA interference (RNAi). RNAi can also be induced in mammalian cells using double-stranded RNAs (dsRNAs), and has become the method of choice for targeted knock-down of gene expression in mammalian cells [4]. The apparent specificity of RNAi also enables allele-specific gene targeting [5]. Initial studies using RNAi in mammalian cells centred around transient knock-down of target gene expression, either using direct transfection of synthetic short interfering RNA (siRNA) [6], transfection of in vitro transcribed siRNA [7] or transient expression of short dsRNA via transfection of plasmid DNA bearing RNA Polymerase III promoter-driven expression cassettes [8,9]. Short dsRNAs of 19 to 29 base-paired nucleotides, complementary to the target mRNA, were expressed as 2 complementary RNAs or as a hairpin structure (shRNA), and resulted in knock-down of the target message. While these initial RNAi methods gave a rapid phenotypic read-out in vitro, stable knock-down of gene expression is required for monitoring long-term effects on cell function, for example, in developing tumors in vivo or in cell-based gene therapy approaches. Efficient delivery of RNAi-inducing dsRNA or expression cassettes is required for effective transient and long-term studies. Transfer of functional shRNAs using lentiviral vectors appears to be a valid approach for effective, stable construct delivery to both cell lines [10] and primary cells [11-13]. More recently, using several different expression systems and target cells, reports have highlighted the utility and specificity of the RNAi approach [14-17]. Maintaining RNAi-inducing dsRNA below 30 nucleotides in length was thought to avoid activation of the interferon-induced anti-viral response. PKR is a key anti-viral regulator and its expression can be induced by the interferon response [18]. PKR is activated when bound to dsRNA longer than 30 nucleotides, despite interacting with shorter dsRNA molecules [19]. Four recent reports have pointed towards limitations to using RNAi as a tool in mammalian cells. The first demonstrated off-target gene silencing [20], highlighting the redundancy of short nucleotide sequences in the human transcriptome. The second characterised the expression profile of genes as a result of lentiviral vector-mediated RNAi. Interferon regulated gene expression was increased even with dsRNAs as short as 19 nucleotides [21]. The third report demonstrated similar interferon response gene up-regulation, after transfection of cell lines with synthetic siRNAs as short as 21 nucleotides [22]. Finally, Scacheri et al documented significant siRNA sequence-dependent changes in the expression of non-targeted proteins [23]. In this work we used a simple approach for gene silencing in human tumor cell lines, using lentiviral vectors for stable delivery of shRNAs. We aimed to study the effects of targeting the plasminogen activator inhibitor-2 (PAI-2 or SERPINB2) mRNA on cell survival in the presence of pro-apoptotic stimuli. In addition to its inhibitory activity on the urokinase plasminogen activator, PAI-2 is thought to have anti-apoptotic properties [24,25]. Its molecular targets in this respect are unknown. A recent report demonstrated a functional interaction between PAI-2 and the retinoblastoma protein cell cycle regulator [26]. Using lentiviral vectors for delivery of RNAi-inducing expression cassettes we achieved potent PAI-2 gene silencing, accompanied by a rapid cytotoxic effect. The degree of cytotoxicity was proportional to shRNA length and induction of an interferon response gene could be detected when shRNA of 21 complementary base pairs or more was expressed. The phenotype was not target gene specific, as PAI-2 overexpression failed to rescue cytotoxicity and control hairpins were also cytotoxic. Using lower vector titre, reduced shRNA expression and interferon response induction was measured without compromising gene silencing. Using a 19 complementary base pair shRNA expression vector, which reduced PAI-2 expression and induced no initial cytotoxicity or interferon response, transduced cell marker gene expression was maintained but gene silencing lost in long-term cell culture. Our results highlight the need for careful controls to monitor specificity and maintenance of gene silencing when using RNAi for stable loss-of-function studies in mammalian cells. Results Efficient transfer of RNAi-inducing cassettes using lentiviral vectors Lentiviral vectors were generated which deliver an expression cassette for human U6 promoter-driven expression of short hairpin RNA (shRNA), with exact homology to the human PAI-2 mRNA. The vector expression cassette also bears the enhanced green fluorescent protein (GFP) gene under the control of the EF-1α promoter, and an internal ribosome entry site (IRES) sequence (see Figure 1A). This cassette allows permanent expression of GFP in transduced cells, and the possibility of concomitant overexpression of a further cDNA, between the EF-1α promoter and IRES sequences, not used here. The shRNA sequences were chosen from the PAI-2 mRNA to include a 5' guanosine at the U6 promoter transcriptional start site, to exclude the 5' and 3' 100 nucleotides of the PAI-2 open reading frame, and to be between 30 and 70 % guanosine/cytidine rich. As controls, we have used a vector leading to expression of GFP alone (EGFP), a vector with the U6 promoter and transcriptional termination signals but lacking a hairpin encoding sequence (U6PT), and vectors leading to expression of scrambled sequences of certain hairpins. Figure 1 demonstrates the efficient transduction of Isreco-1 (IS-1) human colorectal carcinoma cells with one such PAI-2 targeting vector (sh325). Sh325 is designed for expression of a shRNA with a 25 nucleotide double-stranded stretch (a 25 mer) to target the PAI-2 mRNA. As controls, we used U6PT and a scrambled sequence (sh325scr) vector. Four days after transduction, each cell population expressed high levels of GFP, as a marker for transduction (Figure 1B). Compared to non-transduced cells, or cells transduced with the U6PT control vector, we measured a clear knock-down of endogenous PAI-2 protein and mRNA in cells transduced with the sh325 vector (see figure 1C and 1D). Figure 1 Effective gene silencing using lentiviral vectors for RNAi. The gene transfer cassette common to each vector for RNAi is shown in A. Each construction for RNAi was designed for expression of a shRNA, homologous to the target mRNA or with a scrambled sequence, driven by the RNA polymerase III-controlled human U6 promoter and ending with a terminator (T) sequence. The shRNA is represented by two arrows which encode 19 to 25 nucleotide complementary sequences and are joined by an eight nucleotide loop (L). EGFP expression is via the EF-1α promoter, oriented in the opposite direction, driving an IRES sequence and the EGFP gene. Each cassette is flanked by the HIV long terminal repeats (LTR), of which the 3' LTR is modified to ensure that the vectors are self-inactivating upon integration (SIN). B shows flow cytometry analysis of non-transduced IS-1 cells and cells four days after transduction with the U6PT control vector, a vector for expression of shRNA complementary to a region of the PAI-2 mRNA (sh325) and a vector for expression of a shRNA with a scrambled sh325 sequence (sh325scr). C shows an immunoblot for detection of PAI-2 in the cell lysate of these cells. NS highlights a single non-specific band which is consistently detected in PAI-2 immunoblots using IS-1 cell lysates. In D, PAI-2 mRNA levels from the same samples are measured by QRT-PCR of cDNA, using the ΔCT method and hypoxanthine phosphoribosyl transferase (HPRT) as the control gene. Each target gene was detected in duplicate, error bars represent the standard deviation of mean values. However, the control vector with a scrambled sequence (sh325scr) also reduced PAI-2 mRNA and protein levels. Equal sample loading for immunoblots was confirmed by Ponceau S staining of nitrocellulose membranes (data not shown) and the presence of equal amounts of a PAI-2 monoclonal antibody-reactive non-specific band in each blot (NS in Figure 1C). Rapid cytotoxic effect of RNAi vectors Many of the initial loss-of-function studies using RNAi have measured the phenotypic effect of gene silencing in the immediate time frame after introduction of the siRNA or RNAi-inducing expression vector. As seen in Figure 1, four days after transduction with our vectors appears to be sufficient for efficient target gene silencing. Transduction with lentiviral vectors leads to stable long-term integration of the desired transgene cassette, a key advantage in their use compared to other transient or less stable expression systems. Thus we reasoned that transduction with RNAi-inducing cassettes, using lentiviral vectors, would also be stable unless the reduction in target gene expression gave transduced cells a significant growth disadvantage or cytotoxic phenotype. Four to five days after transduction, IS-1 cells bearing the sh325 construct or cells transduced with a scrambled sh325 sequence rapidly changed morphology, compared to U6PT-transduced control cells. Sh325-transduced cells began to disintegrate into small particles and detach from cell culture dishes. After 10 days most of the sh325-transduced cells were dead while the U6PT-transduced cells were growing like the parent cell line. The scrambled hairpin vector-transduced cells gave a weaker cytotoxic phenotype, with deteriorating cell morphology and some detachment of transduced cells. Figure 2A shows the morphology of IS-1 cells 6 days after transduction. To understand further this cytotoxic effect, we performed quantitative RT-PCR (QRT-PCR) on RNA isolated from IS-1 cells, 4 days after transduction with the same vectors, in order to measure the levels of the 2'5'-oligoadenylate synthetase-1 (OAS1) mRNA after transduction with each vector. The OAS1 gene is recognised as an interferon response gene and has also been monitored elsewhere when using RNAi [21]. We measured increases in OAS1 expression in both sh325 and scrambled sh325 vector-transduced cells, whereas control-transduced cells (U6PT) had equal OAS1 levels to non-transduced cells (see Figure 2B). Figure 2 Cytotoxicity and OAS1 induction with RNAi vectors. In A, morphology was observed using phase contrast microscopy of non-transduced IS-1 cells, or cells six days after transduction with vectors leading to expression of no shRNA (U6PT), a 25 mer shRNA targeting PAI-2 (sh325) and a scrambled 25 mer control shRNA (sh325scr). B shows comparison of OAS1 expression in non-transduced cells or cells four days after transduction with U6PT, sh325 and sh325scr vectors, by QRT-PCR. Each target gene was detected in duplicate, error bars represent the standard deviation of mean values. 19 mer shRNAs induce less cytotoxicity than longer hairpins and do not increase OAS1 expression As both the target gene-specific and the scrambled sequence 25 mer shRNAs, sh325 and sh325scr, induced the OAS1 interferon response gene, we generated further vectors for delivery of shRNAs with reduced hairpin length. We reduced the length of sh325, from 25 to 23, 21 and 19 nucleotides and named the novel vectors sh323, sh321 and sh319, respectively. The truncations were made at the 3' end of the 25 nucleotide sense strand and therefore the 5' of its complementary anti-sense sequence (see Table 1). Each vector was used to transduce IS-1 cells and the growth of GFP positive cells monitored at 4 and 10 days after transduction, compared to U6PT control-transduced cells (see Figure 3A). Targeting of the PAI-2 mRNA and protein was monitored, four days after transduction, by QRT-PCR and immunoblotting of cell lysates (Figure 3B and 3C). While each PAI-2 targeted vector successfully reduced PAI-2 mRNA and protein four days after transduction, a strong negative selection was seen for shRNA-expressing cells after a further six days of culture. This selective pressure on transduced cells was stronger with the 21 mer, 23 mer and 25 mer shRNAs than with the shorter sh319-derived 19 mer (Figure 3A). OAS1 mRNA levels were measured by QRT-PCR of transduced cell cDNA four days after transduction (Figure 3B). The cells transduced with the 21 mer, 23 mer and 25 mer shRNAs showed induction of OAS1 mRNA, however, contrary to our expectations, highest OAS1 levels were obtained with the 21 mer shRNA. Loss of GFP positive cells over time was comparable for 21 mer, 23 mer and 25 mer hairpin constructs. To determine whether the lack of OAS1 induction was specific to sh319 or common to other 19 mer shRNAs, further transductions and QRT-PCR analysis were performed on mRNA from IS-1 cells transduced with sh319, sh321 and sh319scr vectors. sh319scr encodes a shRNA with a scrambled sh319 sequence. This analysis also confirms the specificity of the PAI-2 silencing, by comparing sh319 to sh319scr. Figure 3D shows that no induction of OAS1 was measured using sh319 or sh319scr vectors and sh319scr had no effect on the PAI-2 mRNA level. Table 1 Construct details and shRNA sequences. Vector names and the shRNA sequences they encode. In comments, numbers are coding human PAI-2 mRNA nucleotides (adapted from accession number M18082). Hairpin sequence Name sense loop antisense Comments sh319 GCGCACACCUGUACAGAUG CAAGCUUC CAUCUGUACAGGUGUGCGC PAI-2 684–702 sh321 GCGCACACCUGUACAGAUGAU CAAGCUUC AUCAUCUGUACAGGUGUGCGC PAI-2 684–704 sh323 GCGCACACCUGUACAGAUGAUGU CAAGCUUC ACAUCAUCUGUACAGGUGUGCGC PAI-2 684–706 sh325 GCGCACACCUGUACAGAUGAUGUAC CAAGCUUC GUACAUCAUCUGUACAGGUGUGCGC PAI-2 684–708 sh319scr GUCAUACCGGCAAGGAUCC CAAGCUUC GGAUCCUUGCCGGUAUGAC scrambled sh319 sh325scr GGCCGGAGAUAAGUUCACUCAACUC CAAGCUUC GAGUUGAGUGAACUUAUCUCCGGCC scrambled sh325 sh119 GAAGACCAGAUGGCCAAGG CAAGCUUC CCUUGGCCAUCUGGUCUUC PAI-2 151–169 EGFP No hairpin, empty vector. none U6PT Human U6 promoter/terminator, no hairpin. none Figure 3 Shorter shRNA length reduced, but did not eliminate, cytotoxicity. A represents flow cytometry analysis of IS-1 cells 4 and 10 days after transduction with U6PT, sh319, sh321, sh323 and sh325 vectors. GFP expression is detected, and the percentage of GFP expressing cells was determined using the M1 gating shown (percentage GFP positive cells is shown in each histogram). B shows a comparative analysis of PAI-2 and OAS1 mRNA, in the samples described in A, 4 days after transduction. Data were generated by QRT-PCR and error bars are as described in previous figures. In C, cell lysates from samples of transduced cells described in A and B were subjected to immunoblotting with anti-PAI-2 monoclonal antibodies. Ponceau S staining served as a gel loading control, as did comparison of a single non-specific band (NS) in the immunoblot. D shows QRT-PCR analysis, as in B, for IS-1 cell mRNAs after transduction with or without U6PT, sh319, sh319scr and sh321 vectors. Cytotoxicity is not target gene specific To determine if all or part of the cytotoxic effect seen with our shRNAs was due to down-regulation of PAI-2, we generated an IS-1 cell line which overexpresses functional PAI-2. A lentiviral vector was produced which delivers the wild type PAI-2 cDNA, and used to transduce IS-1 cells. This resulted in a homogeneous population of cells which overexpress PAI-2 (see Figure 4A, IS-1 PAI-2 cells). Using immunoblotting of PAI-2/u-PA complexes, formed by mixing IS-1 PAI-2 cell lysates with low molecular weight u-PA, we demonstrated that this overexpressed protein was functional (see Figure 4B). We transduced these cells with the series of shRNA-delivering vectors described in Figure 3 (sh325, sh323, sh321 and sh319), to test whether functional PAI-2 overexpression could reverse the cytotoxic phenotype. As even endogenous PAI-2 is not completely silenced using these vectors we reasoned that the RNAi they deliver would not be capable of functionally silencing overexpressed PAI-2. As predicted, our PAI-2 targeting shRNAs were unable to completely reduce the overexpressed PAI-2 protein levels (see Figure 4C, compared to the relative non-specific band intensity in Figure 3C). However, the cytotoxic effect seen with the parent IS-1 cell line was also clearly apparent in the PAI-2 overexpressing cells. We monitored the loss of GFP positive cells in the transduced PAI-2 overexpressing cell populations and saw almost identical kinetics, compared to the parent cell line (compare Figure 3A and Figure 4D). These data show that the cytotoxic effect is not target gene specific. We also measured the level of PAI-2 mRNA in this experiment, by QRT-PCR. Despite a several-fold decrease in overexpressed PAI-2 protein level (see immunoblot in Figure 4C) we were unable to detect knock-down of the overexpressed mRNA (Figure 4E). In a similar manner to the IS-1 parent cell line, transduction of PAI-2 overexpressing cells with sh325, sh323, sh321, but not the sh319 vector, induced the OAS1 interferon response gene (Figure 4F). To exclude IS-1 cell-specific effects of our vectors we transduced IS-1 cells and HeLa cells with a GFP control, the sh319 and the sh319 scrambled sequence (sh319scr) vectors. We monitored the percentage of GFP positive cells at day 4, 8 and 11 after transduction and observed similar selective loss of GFP positive cells for the sh319 and sh319scr vectors, in both cell lines (Table 2). Figure 4 PAI-2-targeted RNAi with overexpression of functional PAI-2 in IS-1 cells. IS-1 cells were transduced with a lentiviral vector for delivery of the human PAI-2 cDNA under control of the CMV promoter. A shows a flow cytometry analysis for detection of PAI-2 in transduced IS-1 PAI-2, and non-transduced IS-1 cells. Both cell types were fixed, permeabilised and labelled with anti-PAI-2 monoclonal antibodies, then incubated with PE-labelled secondary antibodies. In B, the functional activity of overexpressed PAI-2 was assessed by immunoblotting of cell lysates, for PAI-2 expression, with or without the addition of 10U of u-PA. u-PA alone is included in lane 1. In C, PAI-2 protein levels were assessed in cell lysates from IS-1 PAI-2 cells, after transduction with U6PT, sh319, sh321, sh323 and sh325 vectors. NS in B and C highlights a single non-specific band which is consistently detected in PAI-2 immunoblots of IS-1 cell lysates. D represents flow cytometry analysis of IS-1 PAI-2 cells 4 and 10 days after transduction with U6PT, sh319, sh321, sh323 and sh325 vectors. GFP expression is detected, and the percentage of GFP expressing cells was determined using the M1 gating shown (percentage GFP positive cells are given in each histogram). E and F show comparative analyses of PAI-2 and OAS1 mRNA expression, respectively, in samples described in D. Samples were analysed 4 days after transduction. Data were generated by QRT-PCR, each target gene was detected in duplicate, error bars represent the standard deviation of mean values. Table 2 Percentage GFP positive cells over time in shRNA-expressing IS-1 and HeLa cells. Transduced cells were assessed for GFP expression by flow cytometry. GFP positive cells were gated equally for each cell type, 4, 8 and 11 days after transduction with EGFP, sh319 and sh319scr vectors. % GFP positive cells IS-1 HeLa Vector Day 4 Day 8 Day 11 Day 4 Day 8 Day 11 EGFP 95 95 93 92 94 93 sh319 86 70 56 79 68 50 sh319scr 90 76 59 82 64 45 Reducing lentiviral vector titre can reduce shRNA expression level and OAS1 induction, while maintaining gene silencing To understand whether lentiviral vector titre and resulting shRNA expression levels influence the non-specific effects described, IS-1 cells were transduced with the U6PT, sh319 and sh321 vectors as described above and also using a 10-fold reduction in vector titre (vector titre details are given in the figure 5 legend). Both sh319 and sh321 vectors effectively down-regulate PAI-2, but only the sh321 vector induces OAS1 expression (see Figure 3B). Using 10-fold lower vector titre had little impact on PAI-2 mRNA silencing, as seen using QRT-PCR (Figure 5A), and resulted in a small decrease in OAS1 mRNA induction (Figure 5B) in sh321-transduced cells. To assess whether lower vector titre resulted in lower shRNA expression, total RNA from cells transduced with different titres of sh321 vector was subjected to RNase digestion after hybridization with a 32-P labelled RNA probe, designed to protect the first 19 nucleotides of the sh325 shRNA. Upon hybridization, this "sh3" probe should protect short RNA expressed in sh321 vector-transduced cells from RNase digestion. As negative controls, RNA from U6PT-transduced cells was subjected to the RNase protection procedure, and the sh3 probe was treated with RNase without target RNA. As a positive control a probe for the Mir-16 miRNA was constructed and used to detect endogenous Mir-16 miRNA in U6PT-transduced cells using the same protocol (Figure 5C). Sh3 probe-protected RNA was detected in sh321-transduced cells. Reducing the viral titre clearly reduced the expression of shRNA and this correlated with reduced OAS1 induction. Sh3 probe-protected RNA was not detected in control-transduced cells and the sh3 probe was completely digested in the absence of target RNA (Figure 5C). This data demonstrates that vector-derived shRNA expression can be reduced without impacting gene silencing and that lower expression correlates with a reduced interferon response. Figure 5 Efficient target gene silencing with reduced OAS1 induction and lower shRNA expression. Total RNA was isolated from IS-1 cells (CTRL) or IS-1 cells 4 days after transduction with U6PT, sh319 or sh321 vectors. After reverse transcription, cDNA was analysed for expression of PAI-2 (A) and OAS1 (B). Data were generated by QRT-PCR, each target gene was detected in triplicate, error bars represent the standard deviation of mean values. 1 ml or 0.1 ml of each lentiviral vector stock was used, hence the designations 1 and 0.1. Vector titres were approximately 106 transducing units per ml resulting in a multiplicity of transduction of approximately 10 for 1 ml used or 1 for 0.1 ml used. In C, shRNA expression was detected in total cell RNAs using a modified RNase protection protocol. Total RNA was mixed with radiolabelled probes for hybridization and RNase protection. Samples were resolved on a 15 % Acrylamide/8M Urea/TBE gel and RNase protected probes detected by autoradiography. Lane 1 shows the Mir-16 probe without RNase digestion or target RNA, lane 2 is as lane 1 with RNase digestion and lane 3 as lane 2 with U6PT-transduced cell RNA as hybridization target. Lane 4 shows the sh3 probe without RNase digestion or target RNA, lane 5 as lane 4 with RNase digestion and lane 6 as lane 5 with U6PT-transduced cell RNA as hybridization target. In lane 7 sh321-transduced cell RNA was used as the hybridization target for the sh3 probe with RNase digestion, and lane 8 is as lane 7 except that cells were transduced with 10-fold less vector titre (sh321 1 and sh321 0.1). Known nucleotide lengths (Ntds.) for the probes and the protected Mir-16 endogenous RNA are marked. Loss of long-term gene silencing despite persistent transduction marker gene expression In an attempt to generate a cell line with stable PAI-2 mRNA silencing without interferon response induction, we generated additional vectors which deliver 19 to 25 nucleotide shRNAs targeting different regions of the PAI-2 mRNA. Of these, the sh119 construct reduced PAI-2 expression, did not induce OAS1 and had no effect on cell morphology one week after transduction (data not shown). In parallel, we transduced IS-1 cells with the sh119 vector or a GFP control vector. We achieved high percentage transduction rates which were monitored for over two weeks (Figure 6A). GFP positive cells, from control- and sh119-transduced populations were sorted twice, using flow cytometry, to further enrich the GFP positive population of each (EGFPs and sh119s). Sh119-transduced cells showed PAI-2 gene silencing 10 days after transduction but not after one month of cell culture (Figure 6B and 6C). GFP marker gene expression was maintained in both sorted cell populations. During the prolonged culture period we noticed that, while the percentage of GFP positive cells remained stable, the intensity of GFP detected was reduced in the sh119-transduced cells, compared to GFP alone controls (Figure 6A). This was accompanied by a significant reduction in integrated vector copies (data not shown). Thus, although the marker protein was maintained at a reduced expression level, the prolonged culture period selected against cells with effective gene silencing. This suggests the presence of subtle cytotoxic effects of short dsRNA expression which are not apparent in the initial post transduction period and are in the absence of interferon response gene induction. Overexpression of PAI-2 in the IS-1 cells did not reduce the long-term selective effect on transduced cells or GFP expression levels in sh119-transduced cells (see Table 3). Figure 6 Long-term gene silencing is not stable, despite persistent marker gene expression. In A, IS-1 cells transduced with EGFP control or sh119 vectors were analysed by flow cytometry 3 and 17 days after transduction, and compared to non-transduced cells. EGFP expressing cells, from both transduced cell populations, were selected by cell sorting and named EGFPs and sh119s. 39 days after transduction, these cells were analysed for EGFP expression. Percentage EGFP positive cells, assessed by the M1 gating shown, are given in each histogram. In B, PAI-2 expression in EGFP and sh119 vector-transduced cell lysates were analysed by immunoblotting, 10 days after transduction. C is the same PAI-2 immunoblot as B, performed using EGFPs and sh119s cell lysates, 33 days after transduction. Table 3 Percentage GFP positive cells and mean GFP fluorescence in sh119-transduced IS-1 and IS-1 PAI-2 cells. Transduced cells were assessed for GFP expression by flow cytometry. GFP positive cells were gated equally for each cell type, 5 and 31 days after transduction with the sh119 vector. Mean F is the mean fluorescence of gated GFP positive cells. % GFP positive cells Mean F of GFP positive cells Day5 Day31 Day5 Day31 IS-1 cells 85 40 146 83 IS-1 PAI-2 cells 82 46 180 96 Discussion Here we report the use of lentiviral vectors for the delivery of expression cassettes designed for RNAi-induced stable knock-down of gene expression. We undertook this approach because of the promise of RNAi to easily create cell lines that are specifically deficient in one protein component. By using lentiviral vectors for shRNA expression, high transduction efficiencies can be achieved, avoiding effects due to clonal selection of phenotypically different cells. We successfully targeted the PAI-2 mRNA with the aim of studying the effects of reducing PAI-2 activity on cell sensitivity to apoptosis-inducing stimuli. PAI-2 activity has previously been implicated in protection from apoptosis [24]. The cytotoxic effect we observed, in cells transduced with RNAi-inducing vectors, appeared to correlate well with the reduction in PAI-2 protein levels. However, rapid selective growth pressure on cells bearing shRNA constructs with scrambled sequences, having no complementarity to the PAI-2 mRNA, suggested non-specific effects rather than a PAI-2-related phenotype. Using GFP as a marker gene, delivered by all vectors, enabled very sensitive detection of selective effects on transduced cells even when initial cell culture suggested stable transduction and cell growth. We were able to detect increased expression of an interferon response gene, OAS1, in cells transduced with all hairpins of 21 or more base-paired nucleotides. A 21 mer hairpin induced the most potent OAS1 induction. These results, and those of others [21,22], suggest that dsRNAs of less than 30 nucleotides can induce an interferon response, even if they cannot directly activate protein kinase R [19]. The absence of OAS1 induction in cells transduced with 19 mer shRNAs implies that the search for appropriate hairpin sequences should be limited to stretches of this length or less. Overexpression of functional PAI-2 did not rescue the cytotoxic effects or OAS1 induction observed in cells transduced with a series of vectors for expression of different length shRNAs. This result, the cytotoxicity associated with scrambled sequence hairpin-encoding constructs, and the same selective pressure seen on the growth of transduced HeLa cells, which do not express detectable PAI-2 mRNA or protein (Table 2 and data not shown), lead us to conclude that the phenotype we have seen in IS-1 cells is not PAI-2-specific. Without the ability to track transduced cells, via GFP expression, this conclusion would have been more difficult to obtain. In cells engineered to overexpress functional PAI-2, our RNAi-inducing vectors clearly reduced PAI-2 protein levels but did not significantly reduce the overexpressed PAI-2 mRNA. This suggests that in the presence of high concentrations of targeted mRNA, the machinery necessary for RNAi-induced mRNA cleavage is saturated and mRNA down-regulation undetectable. As protein levels are nevertheless reduced, the shRNA may be functioning post-transcriptionally, perhaps in a similar manner to natural miRNA. This phenomenon has been described elsewhere for siRNAs [27]. IS-1 cell transduction, using one PAI-2 targeting vector (sh119), initially appeared stable, compared to control-transduced cells. PAI-2 protein levels were clearly reduced 10 days after transduction and selective pressure on cell growth appeared to be minimal, as the percentage of sh119 GFP positive cells was apparently stable at about 80 %, 17 days after transduction. However, after cell sorting of GFP positive cells and prolonged cell culture of over one month after transduction, the PAI-2 antigen measured in sh119s (s for selected) cells was restored to control-transduced cell levels. Despite maintenance of transduction marker expression, gene silencing was absent. The GFP expression levels in selected sh119s cells was reduced after one month of growth, compared to cells monitored three days after transduction. It is possible that we selected cells with a greatly reduced, non-cytotoxic shRNA expression level, as we have detected a reduced number of integrated vector copies. The negative selective effect on cells transduced with the sh119 vector was not due to the suppression of PAI-2 expression, as PAI-2 overexpressing cells showed the same negative selection. In all experiments in which a selective pressure on growth was apparent on shRNA-expressing cells, reduced percentage GFP positive cells and reduced GFP expression in transduced cells was measured over time. We hypothesised that very high expression levels of the various shRNAs is cytotoxic. This could occur via high numbers of transcriptionally active vector integration events or integration at transcriptionally active chromosomal regions. Both might be controlled using a tightly regulatable expression system, which has been described [15], but may require careful dosage in a gene- and cell-specific manner. Our data demonstrate the importance of appropriate controls for using RNAi, as proposed in a recent editorial [28]. These include suppression of the RNAi phenotype by target gene overexpression, use of scrambled dsRNAs, and monitoring of non-specific gene expression in particular of interferon-responsive genes such as OAS1. Also, if considering the use of stable RNAi, it is imperative that stable knock-down is demonstrated as well as stable marker gene expression. In the cell culture system we used, conclusions drawn from experiments several days after RNAi delivery mask effects which are only apparent days later by monitoring the percentage of transduced cells. Such effects are likely be present in experiments using regulatable RNAi systems or using exogenously added dsRNA, where the experimental data linked to gene targeting may be collected before other effects are seen. The molecular events which lead to the long-term effect we have documented may well be underway during this experimental period. In the light of our own data and other recent reports [20-22], solutions for the induced cytotoxic effects we describe here include testing a series of target sequences, using dsRNA of no more than 19 nucleotides at low effective vector doses, and careful monitoring of transduced cell phenotype with and without functional target gene overexpression. Long-term monitoring of gene silencing appears to be necessary in stable systems, even in the presence of marker gene expression. Conclusions Our study demonstrates vector-derived RNAi in tumor cell lines and points towards the necessity of careful, but clearly feasible, controls when using RNAi for stable gene suppression in short- and long-term experiments. Methods Cell lines Isreco-1 (IS-1) cells were a gift from Dr. B. Sordat (ISREC, Lausanne). 293T cells were a gift from Dr. D. Trono (Geneva University Medical Centre). HeLa cells were purchased from the European collection of cell cultures, ECACC number: 93021013. All cells were maintained in DMEM supplemented with 10 % Fetal Bovine Serum and 10 mM HEPES pH 7.4 (IS-1 medium) (purchased from Invitrogen). Plasmid constructions Gene transfer plasmids, for RNA interference using lentiviral vectors, were constructed using the backbone of ploxEWiresGFP, a gift from Dr. P. Salmon, Geneva University Medical Centre. The human U6 gene was amplified by PCR using HeLa cell genomic DNA as template and the oligonucleotides U6-ClaI-F 5' GATC ATCGATAAGGTCGGGCAGGAAGAGGGCCTATTTCCC 3' and U6-ClaI-R 5' GATCATCGATTGGTAAACCGTGCACCGGCGATAAACG 3'. The 483 base pair PCR product was digested with ClaI, inserted into the ClaI site of pTRE2 hyg (Clontech), and its sequence verified by DNA sequencing. The U6 promoter and gene sequence corresponded to nucleotides 65 to 527 of Genbank accession number M14486. This plasmid was used as template for PCR reactions to amplify U6 promoter-driven expression cassettes. Each PCR product included the U6 promoter with shRNA-encoding sequences beginning at the U6 +1 site, and a run of 6 or 7 thymidine bases for an RNA polymerase III transcription termination signal. Each PCR introduced flanking ClaI restriction sites. PCR products were directly cloned into the pGEM Teasy (Promega) plasmid and sequenced. ClaI fragments of positive clones were excised and ligated into the unique ClaI site of ploxEWiresGFP. The U6 promoter cassettes in the resulting lentiviral vector plasmids were verified by DNA sequencing. Each construct used for vector production had the same orientation of the U6 expression cassette with respect to the PEF-1α-iresGFP region of the plasmid. A schematic of the gene transfer cassette is given in Figure 1A. Details of shRNA sequences used for each construct are given in Table 1. The gene transfer plasmid for PAI-2 overexpression was constructed by replacing the EGFP gene from ploxCW-GFP (a gift from Dr. P. Salmon, Geneva University Medical Centre) with the type B human PAI-2 (SERPINB2) [29] open reading frame. Vector production and transduction Lentiviral vectors were produced by three plasmid co-transfection of 293T cells, essentially as described previously [30]. Vectors were harvested 48 hours after transfection, passed through 0.45 μm filters and used directly on target cells in a 1:1 ratio with IS-1 medium in a total volume of 2 ml. Transductions were performed in 3 cm diameter 6-well plates, on cells seeded the previous day at 5 × 104 or 1 × 105 cells/well. Upon addition of vectors, plates were centrifuged for one hour at 1000 g in the presence of 8 μg/ml polybrene (hexadimethrine bromide, Sigma). After 24 hours, target cells were washed twice with PBS and cultured in IS-1 medium until analysis. Where indicated, viral titre was determined by transducing cells with 10 μl of lentiviral vector conditioned medium from 293T cell producer cells and measuring the % of GFP positive cells by flow cytometry. We routinely achieve 106 effective transducing units per ml of producer cell conditioned medium, which results in a typical multiplicity of transduction of 10. Comparisons between cell populations transduced with different vectors is made by flow cytometry analysis of GFP positive cells. Antibodies Anti-human PAI-2 monoclonal antibody 3750 was purchased from American Diagnostica. PE-labelled goat anti-mouse antibodies were purchased from Pharmingen. HRP-conjugated goat anti-mouse antibodies were purchased from Bio-Rad. Flow cytometry Flow cytometry was performed using Becton Dickinson FACScan, FACStrack or FACScalibur instruments at the Geneva University Medical Centre flow cytometry facility. GFP was detected in cells detached and resuspended in FACS buffer comprising 1 % BSA in PBS supplemented with 0.05 % sodium azide. For the detection of intracellular PAI-2, a method adopted from Dr. M. Ranson (University of Wollongong, Australia) was used. Cells were detached and fixed in 0.25 % paraformaldehyde (PAF)/PBS for one hour on ice. Cells were permeabilised in 0.1 % saponin/PBS for 30 minutes at room temperature. Fixed, permeabilised cells were incubated for 30 minutes in PBS/0.5 % BSA/0.1 % saponin containing 2 μg/ml 3750 anti-PAI-2 monoclonal antibody, washed twice in PBS/0.1 % saponin and incubated for 30 minutes in 0.1 % saponin/0.5 % BSA/PBS/goat anti-mouse-PE antibodies. Finally, cells were washed twice in 0.1% saponin/PBS, twice in PBS and resuspended in 2.5 % PAF for flow cytometric analysis. Sorting of live GFP positive cells was performed using a FACStar+ instrument (Becton Dickinson). Western blotting of cell lysates Cells in suspension were lysed in 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 0.5 % NP-40, 3 mM MgCl2, 5 mM DTT and 1 mM PMSF for one hour on ice. Lysates were centrifuged at 16000 g for 5–10 minutes to remove nuclei and precipitates. Supernatant protein concentrations were measured using the Bio-Rad protein assay with BSA in lysis buffer as a standard. Cell lysates were separated by reducing SDS-PAGE and transferred to nitrocellulose membranes. Equal total protein lysate was used for each blot, between 2.5 and 10 μg depending on the assay. Membranes were blocked for 1 hour at room temperature in TBS-0.1 % Tween 20/5 % non-fat milk, and probed using antibodies in TBS-0.1 % Tween 20/5 % non-fat milk. The 3750 anti-PAI-2 antibody was used at a concentration of 1 μg/ml. Microscopy Phase contrast microscopy was performed using a Zeiss Axiovert 100 M instrument on live cells. Images were collected using a Hamamatsu CCD camera (ORCA-100). PAI-2 functional activity To measure PAI-2 functional activity, IS-1 and IS-1 PAI-2 cells were lysed in 1 % NP-40, 150 mM NaCl and 50 mM Tris pH 8.0. Thereafter, 6 μg of cleared total cell lysate was incubated with and without 10 U of low molecular weight urokinase (u-PA). 3 μg total cell lysate samples were then subjected to reducing SDS-PAGE and immunoblotting as for other PAI-2 immunoblots. Quantitative RT-PCR analysis of mRNA Total RNA was isolated from cells using RNA preparation kits from Qiagen or TRIZOL® reagent (Invitrogen). cDNA was generated using ImpromII® reverse transcriptase (Promega) and random hexamers, according to the manufacturers instructions, typically using 1 μg of total RNA per reaction. Quantitative PCR was performed using an Applied Biosystems Prism 7000 instrument using Applied Biosystems SYBR® green master mix reagent and oligonucleotide pairs to detect hypoxanthine phosphoribosyl transferase (HPRT), PAI-2 and oligoadenylate synthase-1 (OAS1) cDNA. 5' to 3' primer sequences were as follows: HPRT forward TATTGTAATGACCAGTCAACAG, HPRT reverse GGTCCTTTTCACCA GCAAG, PAI-2 forward GGGTCAAGACTCAAACCAAAG, PAI-2 reverse CCTTTGAAGTAGACAGCATTC, OAS1 forward AGGTGGTAAAGGGT GGCTCC and OAS1 reverse ACAACCAGGTCAGCGTCAGAT. Data were analysed using Applied Biosystems Prism software and the ΔCT method. Briefly, target gene expression was normalised to the HPRT endogenous reference gene for each sample. The difference between mean threshold PCR cycle values for target and control genes gave the ΔCT value. This was then calibrated to the control sample in each experiment to give the ΔΔCT value, where the control had a ΔΔCT value of 0. The fold target gene expression, compared to the calibrator value, is given by the formula 2-ΔΔCT. Error bars represent the standard deviation of each target gene value, after evaluating the expression 2-ΔΔCT+s and 2-ΔΔCT-s, where s is the standard deviation of the ΔΔCT value. All reactions were performed in duplicates or triplicates. ShRNA detection Expression of shRNAs was detected using the mirVana™ Probe Construction and miRNA Detection kits from Ambion (Austin), according to the manufacturers instructions. These kits employ in vitro transcription for radiolabelled probe generation and an RNase protection protocol for detection of small RNA expression, respectively. Briefly, radiolabelled RNA probes incorporating α-32P-UTP (Amersham) were constructed using T7 polymerase-driven transcription templates. Templates were designed to generate RNAs which hybridize to the endogenous Mir-16 miRNA, as a control, or the first 19 nucleotides of the sh325 shRNA (see table 1). The sh3 probe should therefore hybridize to any of the sh319/sh321/sh323 or sh325-derived shRNAs. Before hybridization, radiolabelled probes were purified by migration of the transcription reaction on a 12 or 15% Acrylamide/8 M Urea denaturing gel and elution of radioactive probe bands after detection by autoradiography. 3 μg of total cellular RNA was used for all hybridizations and RNase protections, supplemented with 2 μg yeast RNA as a carrier. Control reactions without target RNA but with or without RNase digestion included 5 μg yeast RNA. After hybridization and RNase digestion, protected probes were detected by autoradiography after migration in a 15% acrylamide/8 M Urea denaturing gel. Authors' contributions RF carried out all of the experiments in this study and contributed to its conception, design and description. EKOK conceived the study and participated in its design and description. Both authors approved the final manuscript. Acknowledgements The authors wish to thank the Swiss Federation Against Cancer (KFS 1059-09-2000) and the Swiss National Science Foundation (no. 32.061510.00) for financial support. ==== Refs Kuhn R Schwenk F Advances in gene targeting methods Curr Opin Immunol 1997 9 183 188 9099792 10.1016/S0952-7915(97)80133-1 Metzger D Feil R Engineering the mouse genome by site-specific recombination Curr Opin Biotechnol 1999 10 470 476 10508631 10.1016/S0958-1669(99)00012-9 Weiss B Davidkova G Zhou LW Antisense RNA gene therapy for studying and modulating biological processes Cell Mol Life Sci 1999 55 334 358 10228554 10.1007/s000180050296 McManus MT Sharp PA Gene silencing in mammals by small interfering RNAs Nat Rev Genet 2002 3 737 747 12360232 10.1038/nrg908 Brummelkamp TR Bernards R Agami R Stable suppression of tumorigenicity by virus-mediated RNA interference Cancer Cell 2002 2 243 247 12242156 10.1016/S1535-6108(02)00122-8 Harborth J Elbashir SM Bechert K Tuschl T Weber K Identification of essential genes in cultured mammalian cells using small interfering RNAs J Cell Sci 2001 114 4557 4565 11792820 Donze O Picard D RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase Nucleic Acids Res 2002 30 e46 12000851 10.1093/nar/30.10.e46 Paddison PJ Caudy AA Bernstein E Hannon GJ Conklin DS Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells Genes Dev 2002 16 948 958 11959843 10.1101/gad.981002 Yu JY DeRuiter SL Turner DL RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells Proc Natl Acad Sci U S A 2002 99 6047 6052 11972060 10.1073/pnas.092143499 Abbas-Terki T Blanco-Bose W Deglon N Pralong W Aebischer P Lentiviral-mediated RNA interference Hum Gene Ther 2002 13 2197 2201 12542850 10.1089/104303402320987888 Rubinson DA Dillon CP Kwiatkowski AV Sievers C Yang L Kopinja J Rooney DL Ihrig MM McManus MT Gertler FB A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference Nat Genet 2003 33 401 406 12590264 10.1038/ng1117 Qin XF An DS Chen IS Baltimore D Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5 Proc Natl Acad Sci U S A 2003 100 183 188 12518064 10.1073/pnas.232688199 Tiscornia G Singer O Ikawa M Verma IM A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA Proc Natl Acad Sci U S A 2003 100 1844 1848 12552109 10.1073/pnas.0437912100 Gupta S Schoer RA Egan JE Hannon GJ Mittal V Inducible, reversible, and stable RNA interference in mammalian cells Proc Natl Acad Sci U S A 2004 101 1927 1932 14762164 10.1073/pnas.0306111101 Wiznerowicz M Trono D Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference J Virol 2003 77 8957 8961 12885912 10.1128/JVI.77.16.8957-8951.2003 Chi JT Chang HY Wang NN Chang DS Dunphy N Brown PO Genomewide view of gene silencing by small interfering RNAs Proc Natl Acad Sci U S A 2003 100 6343 6346 12730368 10.1073/pnas.1037853100 Semizarov D Frost L Sarthy A Kroeger P Halbert DN Fesik SW Specificity of short interfering RNA determined through gene expression signatures Proc Natl Acad Sci U S A 2003 100 6347 6352 12746500 10.1073/pnas.1131959100 Meurs E Chong K Galabru J Thomas NS Kerr IM Williams BR Hovanessian AG Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon Cell 1990 62 379 390 1695551 10.1016/0092-8674(90)90374-N Manche L Green SR Schmedt C Mathews MB Interactions between double-stranded RNA regulators and the protein kinase DAI Mol Cell Biol 1992 12 5238 5248 1357546 Jackson AL Bartz SR Schelter J Kobayashi SV Burchard J Mao M Li B Cavet G Linsley PS Expression profiling reveals off-target gene regulation by RNAi Nat Biotechnol 2003 21 635 637 12754523 10.1038/nbt831 Bridge AJ Pebernard S Ducraux A Nicoulaz AL Iggo R Induction of an interferon response by RNAi vectors in mammalian cells Nat Genet 2003 34 263 264 12796781 10.1038/ng1173 Sledz CA Holko M de Veer MJ Silverman RH Williams BR Activation of the interferon system by short-interfering RNAs Nat Cell Biol 2003 5 834 839 12942087 10.1038/ncb1038 Scacheri PC Rozenblatt-Rosen O Caplen NJ Wolfsberg TG Umayam L Lee JC Hughes CM Shanmugam KS Bhattacharjee A Meyerson M Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells Proc Natl Acad Sci U S A 2004 101 1892 1897 14769924 10.1073/pnas.0308698100 Dickinson JL Bates EJ Ferrante A Antalis TM Plasminogen activator inhibitor type 2 inhibits tumor necrosis factor alpha-induced apoptosis. Evidence for an alternate biological function J Biol Chem 1995 270 27894 27904 7499264 10.1074/jbc.270.46.27894 Kumar S Baglioni C Protection from tumor necrosis factor-mediated cytolysis by overexpression of plasminogen activator inhibitor type-2 J Biol Chem 1991 266 20960 20964 1939146 Darnell GA Antalis TM Johnstone RW Stringer BW Ogbourne SM Harrich D Suhrbier A Inhibition of Retinoblastoma Protein Degradation by Interaction with the Serpin Plasminogen Activator Inhibitor 2 via a Novel Consensus Motif Mol Cell Biol 2003 23 6520 6532 12944478 10.1128/MCB.23.18.6520-6532.2003 Zeng Y Yi R Cullen BR MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms Proc Natl Acad Sci U S A 2003 100 9779 9784 12902540 10.1073/pnas.1630797100 Editorial Whither RNAi? Nat Cell Biol 2003 5 489 490 12776118 10.1038/ncb0603-490 Kruithof EK Baker MS Bunn CL Biological and clinical aspects of plasminogen activator inhibitor type 2 Blood 1995 86 4007 4024 7492756 Zufferey R Nagy D Mandel RJ Naldini L Trono D Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo Nat Biotechnol 1997 15 871 875 9306402 10.1038/nbt0997-871

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==== Front BMC NeurosciBMC Neuroscience1471-2202BioMed Central London 1471-2202-5-271529870010.1186/1471-2202-5-27Research Article3 dimensional modelling of early human brain development using optical projection tomography Kerwin Janet [email protected] Mark [email protected] James [email protected] Luis [email protected] Stephen C [email protected]ínez-de-la-Torre Margaret [email protected] Jose Luis [email protected] Guangjie [email protected] Richard [email protected] Tom [email protected] Duncan [email protected] Susan [email protected] Institute of Human Genetics, University of Newcastle upon Tyne, International Centre for Life, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK2 Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh, EH4 2XU, UK3 Department of Human Anatomy and Psychobiology, University of Murcia, Spain4 School of Surgical & Reproductive Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 4LP, UK2004 6 8 2004 5 27 27 26 4 2004 6 8 2004 Copyright © 2004 Kerwin et al; licensee BioMed Central Ltd.2004Kerwin et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background As development proceeds the human embryo attains an ever more complex three dimensional (3D) structure. Analyzing the gene expression patterns that underlie these changes and interpreting their significance depends on identifying the anatomical structures to which they map and following these patterns in developing 3D structures over time. The difficulty of this task greatly increases as more gene expression patterns are added, particularly in organs with complex 3D structures such as the brain. Optical Projection Tomography (OPT) is a new technology which has been developed for rapidly generating digital 3D models of intact specimens. We have assessed the resolution of unstained neuronal structures within a Carnegie Stage (CS)17 OPT model and tested its use as a framework onto which anatomical structures can be defined and gene expression data mapped. Results Resolution of the OPT models was assessed by comparison of digital sections with physical sections stained, either with haematoxylin and eosin (H&E) or by immunocytochemistry for GAP43 or PAX6, to identify specific anatomical features. Despite the 3D models being of unstained tissue, peripheral nervous system structures from the trigeminal ganglion (~300 μm by ~150 μm) to the rootlets of cranial nerve XII (~20 μm in diameter) were clearly identifiable, as were structures in the developing neural tube such as the zona limitans intrathalamica (core is ~30 μm thick). Fourteen anatomical domains have been identified and visualised within the CS17 model. Two 3D gene expression domains, known to be defined by Pax6 expression in the mouse, were clearly visible when PAX6 data from 2D sections were mapped to the CS17 model. The feasibility of applying the OPT technology to all stages from CS12 to CS23, which encompasses the major period of organogenesis for the human developing central nervous system, was successfully demonstrated. Conclusion In the CS17 model considerable detail is visible within the developing nervous system at a minimum resolution of ~20 μm and 3D anatomical and gene expression domains can be defined and visualised successfully. The OPT models and accompanying technologies for manipulating them provide a powerful approach to visualising and analysing gene expression and morphology during early human brain development. ==== Body Background Brain development, particularly in human, involves complex changes in shape and structure over time. During a period of approximately 4 weeks (from 26 to 56 days of development; Carnegie stages CS12 to CS23) the major subregions of the human brain are established and development proceeds from a simple neuroepithelial tube to a highly complex three dimensional (3D) structure [1]. For many years it has been recognized that three dimensional models are an important aid to interpreting these developmental changes. In the past, these have been based on labour intensive methods for reconstructing representations of physical sections (e.g. Born reconstructions [2]) or, more recently, on computer-based methods, although these are still labour intensive [3]. Non-invasive techniques have also been used and these have advantages of speed and lack of sectioning artifacts and, for example with MRI, the ability to generate in vivo images and good quality images for larger specimens [4]. Recently, a new, rapid and non-invasive 3D modelling method, Optical Projection Tomography (OPT; [5,6]), has become available, and we have used this method to generate computer-based 3D models from intact early human developmental specimens. OPT has the advantage over MRI in that detailed models can be produced from small samples. With MRI, low signal-to noise ratios make it more difficult to obtain high quality data from embryos younger than CS17 [7]. MRI is likely to be useful for specimens larger than CS23 where the size of the specimen and the density of the tissue are too great to allow penetration of the light. The OPT models are visualised and manipulated using MAPaint, a suite of software programmes developed as part of the Edinburgh Mouse Atlas Project ([8-10]). The software allows any OPT model to be digitally sectioned in any plane and several different planes can be viewed simultaneously. These planes can be selected at any arbitrary viewing orientation and position through the model. In addition anatomical regions can be defined and manually "painted", allowing the user to interactively assess developing anatomy. OPT reconstructions were made of human embryos for a number of different stages of development. In all cases, reconstructions were made from autofluorescent imaging – in other words, the intrinsic fluorescence from the fixed specimens was used as the basis for the histological contrast seen in the 3D model. One of the models (CS17; approx. 41 days of development) was examined in detail in order to test the resolving power of the OPT technology on these unstained embryos, in relation to structures in the developing nervous system and to determine the feasibility of using the model as a framework for mapping gene expression patterns. Digital OPT sections were compared with corresponding histological sections stained in 3 different ways: standard haematoxylin and eosin stain to visualise cell nuclei, cytoplasm and connective tissue [11] and immunocytochemistry to detect GAP43 or PAX6 expression. H&E staining distinguishes amongst the ventricular, intermediate and mantle layers in the central nervous system and cranial nerves and ganglia are clearly identifiable. Growth-associated-protein 43 (GAP43) is expressed in growing dendrites and axons [12] and is expressed in the peripheral nervous system and developing tracts of the central nervous system. A number of genes have been identified, particularly in mouse and chick, that are involved in the specification of different brain regions (e.g. reviewed in [13] and [14]). Such gene expression patterns can be used to identify specific brain regions and compare their relative extent in different species (reviewed in [15] and [16]). PAX6 expression is well characterized as defining several regions and boundaries in the developing mouse brain [17,18] and [19]. The effectiveness of using 2D section data to generate 3D expression domains was tested by examining two boundary regions in the CS17 model, the zona limitans intrathalamica (between the dorsal and ventral thalamus) and the midbrain-diencephalon boundary. Results and Discussion An OPT model was generated from an intact, unstained embryo which had been staged according to the Carnegie staging protocol [20] modified for use with individual embryos rather than in comparisons of many embryos simultaneously [21,22]. The embryo was staged as CS17, which is approximately 41 days of development. The developing central nervous system (CNS) is clearly visible even in the external view of the CS17 model (Figure 1 and the accompanying movie, additional file 1 1). Differences in autofluorescence within the CNS and among different organs are also apparent. There is very little detail in the developing liver, for example, compared to the CNS. Blood vessels, dorsal root ganglia and developing vertebrae are clearly visible. Figure 1 CS17 OPT model (a). still shot from movie of 3D OPT model of a CS17 human embryo (approximately 41 days of development). bv, blood vessel; drg, dorsal root ganglion; h, heart; H, hindbrain; l, liver; T, telencephalon; v, vertebrae. (b; Additional file 1) Mpeg movie of 3D CS17 OPT model. Following OPT the embryo was embedded in paraffin wax and sectioned using standard methods and then every fifth section was either stained with haematoxylin and eosin or immunocytochemically with antibodies against GAP43 or PAX6. The actual plane of sectioning was identified in the OPT model by manipulating the model in MAPaint. This permitted a matched series of digital and physical sections to be compared (examples are shown in Figure 2) in order to assess the resolution of internal structures within the CS17 OPT model. Different features are highlighted by the different staining techniques and some examples of these are also shown in Figure 2. The cranial nerve ganglia stain partially with GAP43 (e.g., Xg) and in the H&E sections (e.g., Vg) and are clearly visible in the OPT model (Figure 2a and 2g). Cranial nerves also stain for GAP43 (e.g., III) but are less visible in both the H&E section and the OPT model. Surprisingly the rootlets of cranial nerve XII do show up clearly in the OPT model (Figure 2a). In the CNS, the ventricular layer can be clearly distinguished as more darkly staining in the H&E sections and it is also visible as a darker layer in the OPT model (Figure 2a,2d and 2g). The core of the zona limitans intrathalamica (zli) is seen as a pale region in all three sections (2g,2h,2i). The GAP43 stained fibres in the floor of the hindbrain (2e) show clearly as a pale area in the OPT model (2d). As was visible in figure 1, blood vessels and dorsal root ganglia show up very clearly in the OPT model (2a and 2g). Fluid filled spaces are also clearly visible in the OPT model, such as the diencephalic, midbrain and hindbrain ventricles and the otic vesicle (2a and 2d). Some artefactual changes in shape seen in the physical sections (for example, a collapse of the fourth ventricle roof [r in fig. 2f]) are not present in the OPT model (compare 2d and 2f). The size of a variety of structures was measured in the H&E and/or GAP43 stained sections in order to determine the resolution in the CS17 model. When OPT is performed on specimens in which specific cells have been fluorescently-labelled, then these small structures can be detected (unpublished data). However, in cases where the specimen contains no tissue-specific dyes, a resolution of 5 to 10 micron has previously been found [5]. At the magnification used to generate this CS17 model, the smallest clearly measurable structures are the rootlets of the XII cranial nerve which are approximately 20 μm in diameter (Figure 2a). Figure 2 Comparison of digital OPT sections with histology sections from the same embryo Digital OPT sections of the CS17 model (a, d and g), viewed using MAPaint software, compared with sections stained using antibodies against GAP43 (b, e and h), and Haematoxylin and Eosin stained sections (c, f and i). In b, e and h, expression is demonstrated by the brown chromagen. Structures of 20 μm in diameter (for example the hypoglossal rootlets) are clearly identifiable, as are the differences amongst a variety of developing tissues. bv, blood vessel; D, diencephalon; drg, dorsal root ganglion (~150 μm); fp, floor plate; H, hindbrain; III, oculomotor nerve; M, midbrain; nh, neurohypophysis (~200 μm by ~50 μm); ov, otic vesicle; r, collapsed roof of 4th ventricle; sc, spinal cord; Vg, trigeminal ganglion (~300 μm by ~150 μm); vl, ventricular layer; X, vagus nerve; Xg, vagus ganglion; XIIroot, hypoglossal rootlets (~20 μm); zli; zona limitans intrathalamica (~100 μm, core is ~30 μm). Scale bars = 200 μm MAPaint, a UNIX-based software suite, allows the OPT models to be digitally sectioned in any plane and several planes to be viewed simultaneously. This is illustrated for the CS17 model in Figure 3a (additional file 2) which moves through a series of digital transverse sections with the corresponding position of each section shown on a sagittal section, followed by a series of sagittal sections and the corresponding position on a transverse section. The CS17 model is shown with some painted anatomical domains and Figure 3b shows a snapshot of all fourteen 3D domains with the position of the two transverse sections shown in Figure 3c indicated in white. The anatomical domains were painted in one plane and then checked and refined by corrections introduced in orthogonal planes. The ease of manipulation of the model means that the best digital plane (normally orthogonal to any boundary or set of them) can be selected for each anatomical region being painted. The authors who did these tracings actually found the experience intellectually rewarding, since it amounted to being able to check instantly any difficult point upon a collection of identical specimens sectioned in many different planes. Doubts that often remain unresolved could be resolved very convincingly. Figure 3 Painted anatomical domains. Fourteen regions of the central nervous system in the CS17 specimen have been defined and painted. Forebrain, red (secondary prosencephalon), dark orange (prosomere 3 including ventral thalamus), light orange (prosomere 2 including dorsal thalamus) and yellow (prosomere 1 including pretectum); midbrain, light green; hindbrain; isthmus, dark green; various shades of blue and purple indicate rhombomeres 1–6 and the caudal medulla oblongata; spinal cord, dark red. (a; Additional file 2) In the Mpeg movie sagittal and transverse views of the painted model are shown, together with a representation of the 3D domains. The model is first sectioned in the transverse plane. This section plane has been matched to that of the histology sections shown in fig 2. As the section is moved through the model the corresponding position is displayed in the 3D box, and by a line on the sagittal section. The model is then moved through the sagittal plane, and the position shown by a line on the transverse section. A snapshot of the fourteen 3D anatomical domains (b), and two examples of painted sections that intersect several anatomic domains (i.e., are topologically nearly horizontal to the reconstructed transverse boundaries) (c). The position of the two digital transverse sections is indicated by white lines on the 3D view. As described in the Methods section, and in the legend to Figure 2, paraffin sections were immunostained with either anti-GAP43 or anti-PAX6 antibodies at approximately 50 μm intervals throughout the head of the CS17 specimen and the data were captured and mapped to the CS17 model. Figure 4a illustrates a sagittal section through the model with digital GAP43 expression (in red) which has been generated from the data thresholded from transverse sections, an example of which is shown in Figure 4b. There is an unexpected apparent region of no GAP43 expression in the hindbrain on the sagittal section (Fig 4a, arrow). However, the ability to relate the sagittal and transverse sections in the model makes clear that the lack of expression is due to the sagittal section being "cut" obliquely and the section is passing through the floor plate region of the hindbrain at that point, where there is no GAP43 expression (arrowed in Figure 4b). A sagittal section with digital PAX6 expression shown in green also illustrates the lack of PAX6 expression in the same floor plate region in the hindbrain (Figure 4c). Comparing Figure 4a and 4c it can be seen that the boundary of the PAX6 expression in the caudal diencephalon is approximately the same boundary as that of GAP43 which stains the posterior commissure, just caudal to which is the boundary between the pretectum, the most caudal region of the developing diencephalon [23,24] and the midbrain. The lack of PAX6 and GAP43 expression in the region just caudal to the posterior commissure is shown (Figure 4d; upper 2 panels) while the expression of both GAP43 and PAX6 in the pretectum is shown in the lower two panels of figure 4d. The 3D PAX6 domain identifies both the diencephalon-midbrain boundary and the negative intrathalamic boundary or zli (as shown in Figure 5a,5b and the accompanying movie (Additional file 3). Additional limits of PAX6 expression visible in Figure 5b correspond to the basal telencephalon (the striatopallidal boundary) and the alar-basal boundary across the entire diencephalon [25,23,24]. Separate areas of PAX6 expression appear in the hindbrain (Figs. 5a,5b) Figure 4 CS17 OPT model showing 3D gene expression domains. (a) Digital sagittal section through the CS17 OPT model, with the GAP43 gene expression domain shown in red. The plane of this sagittal section is shown by a line on the corresponding transverse section in (b). The GAP43-negative region in the hindbrain floor plate is shown on both sections by an arrow. (c) The same digital sagittal section, with PAX6 gene expression displayed in green. (d) High power images of GAP43 and PAX6 expression near the diencephalon/midbrain boundary. The upper two panels correspond to the rostral midbrain, where there is no expression of GAP43 or PAX6. The lower two panels correspond to the caudal diencephalon, in the region of the posterior commissure. Here both genes are expressed (brown chromagen). Figure 5 3 dimensional gene expression domains. A surface rendered model of the 3D expression pattern of PAX6. Separate gene expression domains in the forebrain and hindbrain are shown in green. For reference the neural tube has been painted pale grey and the eye dark grey. The diencephalon/midbrain (D/M) boundary, the absence of staining in the zona limitans intrathalamica (zli), plus the forebrain alar-basal boundary and the striatopallidal boundary in the basal telencephalon can be seen by viewing the 3D model at various angles (a, frontal and b, lateral). (c; Additional file 3) Mpeg movie of the PAX6 expression domain. The period from CS12 (approximately 26 days of development) to CS23 (approximately 56 days of development) is important because during this time all the major regions of the developing brain are established [26,1] and most major congenital abnormalities can arise. Major changes take place in the size, shape and complexity of the developing brain during this time and we tested the feasibility of generating OPT models throughout this period. There is a more than 60 fold increase in volume between the CS12 embryo and the CS23 head, however, OPT models have been generated successfully throughout these stages. Figure 6 shows snapshots of models at each of the stages from CS12 to CS23. These are not to scale because each model is generated at the maximum magnification possible, which varies according to the size of the specimen. The changes in shape and complexity are clearly seen even in these static images. We currently have 54 OPT models, including a male and female at each stage CS12-CS20 and CS22. At CS17 we have twelve different OPT models and have assessed their natural variability for 3 specific neurodevelopmental features (development of the choroid plexus in the lateral ventricles, the zli and the floor plate in the hindbrain). At this stage, there was little variation in the features assessed (data not shown). Movies of all these models can be viewed at our website ([27]), which includes a database of gene expression. The models are too large to display fully or to manipulate online, but a selection of the full models are available on CD on request and a new viewer, the Java Atlas Viewer, has been developed which enables the models to be viewed and manipulated on many platforms (Burton, Feng, Hill and Baldock, paper in preparation). See additional files 4 and 5 for the request form and Academic use licence agreement. Figure 6 OPT models of CS12 to CS23. Still shots of the left lateral side of 3 dimensional OPT models of human embryos spanning the major period of organogenesis (CS12-CS23). The developmental stage (e.g. CS12), specimen number (e.g. N285) and karyotype for each model are given underneath. The movies for all of these models can be viewed at [27]. The full models for all stages are available on request. Conclusions Many structures within the developing nervous system can be identified in the CS17 OPT model with a minimum defined resolution of approximately 20 μm. The CS17 model also acted as a framework onto which anatomical domains were easily painted and gene expression patterns mapped. OPT models have been successfully generated from CS12 to CS23, and these models will provide a means of analyzing and relating changes in anatomy and gene expression both within individual developmental stages and across developmental time. In the long-term, our aim is to link the 3D models to an anatomical database and embed both of them within a custom-designed gene expression database in order to create an Electronic Atlas of the Developing Human Brain ([27]). Methods Embryo collection Human embryos were collected from termination of pregnancy material, with appropriate written consent, approval from the Newcastle and North Tyneside NHS Health Authority Joint Ethics Committee and following national guidelines [28]. Embryos were collected into cold PBS, separated from surrounding tissue and fixed overnight in 4% paraformaldehyde at 4°C before short-term storage at 4°C in 70 % ethanol. Placental tissue was sampled for karyotype analysis prior to fixation of the embryo tissue. OPT Intact specimens were rehydrated through a graded series of ethanol and embedded in a block of 1% low melting point agarose. They were then dehydrated through a graded series of methanol before being cleared using a mixture of benzyl alcohol and benzyl benzoate. 400 digital images were then captured while the now almost transparent specimens were rotated in a full circle, with 0.9° steps between each image. The signal corresponded to the weak autofluorescence originating from the paraformaldehyde-fixed tissue and was detected using a wideband FITC filter with excitation at 465–500 nm and emission from 515–560 nm. The images were then assembled to recreate the 3D shape of the embryo, using modified tomography algorithms [5]. Post OPT processing and histology After OPT scanning the CS17 embryo N365 was rehydrated through a graded series of methanol and was then removed from the agarose block by incubation in a 0.29 M sucrose solution at 55°C. It was then processed for paraffin wax embedding and 5 μm microtome sections were cut. Every 5th section was stained with haematoxylin and eosin, following standard procedures. Immunohistochemistry The remaining N365 sections were used for immunohistochemistry. Alternate one-in-five section series were stained with antibodies against GAP43 (GAP-7B10, Sigma), or PAX6 (PRB-278P, Covance) using standard techniques. The reaction was visualised with diamino benzidine and the sections lightly counterstained with toluidine blue. Gene Expression Mapping Images of the stained sections were captured through a ×2.5 objective (as viewed down the microscope at 25× magnification) using the Zeiss Axiovision system. For the PAX6 and GAP43 data, a modified warping interface of the MAPaint software was used to match each stained, physical section to the corresponding digital OPT section. Correspondences between the physical (source) and digital (target) images were identified and manually tie-pointed. The source image was then transformed to the shape of the target section, and the image transformation saved. The interface uses interactive thresholding to extract the expression signal from the source image and then applies the image transformation to map this signal into the space of the 3D OPT model. This was carried out for approximately 190 sections through the head of the CS17 embryo until the full 3D expression pattern was built up for each of the GAP43 and PAX6 data sets. Authors' contributions JK reconstructed the OPT data, carried out the immunocytochemistry, mapped the GAP43 gene expression patterns to the CS17 model and drafted the manuscript. MS optimised the OPT methodology for human specimens, scanned the embryos, carried out the sectioning and mapped the PAX6 gene expression. JS invented the OPT technique and established the methodology in Newcastle. LP, MdlT and JLF identified and painted anatomical regions of the CS17 neural tube. GF developed the Java Atlas Viewer. RB designed/authored the MAPaint software and with DD heads the Edinburgh Mouse Atlas Project. TS, SCR and SL co-ordinate and oversee the Human Developmental Biology Resource which provided the human material. SL and TS conceived of the study and participated in its design and coordination. All coauthors participated in drafting or refining the text. List of abbreviations 3D-three dimensional 4D-four dimensional bv-blood vessel CNS-central nervous system CS-Carnegie Stage D-diencephalon D/M-midbrain/ diencephalon boundary drg-dorsal root ganglion EADHB-Electronic Atlas of the Developing Human Brain fp-floor plate GAP43-Growth Associated Protein 43 h-heart H&E-haematoxylin and eosin H-hindbrain III-oculomotor nerve l-liver M-midbrain nh-neurohypophysis OPT-optical projection tomography ov-otic vesicle r-collapsed roof of 4th ventricle sc-spinal cord T-telencephalon v-developing vertebrae Vg-trigeminal ganglion X-Vagus nerve Xg-Vagus ganglion XIIroot-hypoglossal rootlets Zli-zona limitans intrathalamica Supplementary Material Additional File 1 CS17 OPT model.mpg. Mpeg movie of 3D CS17 OPT model. Click here for file Additional File 2 CS17 painted anatomy.mpg. Mpeg movie of anatomical domains painted on the CS17 model. Sagittal and transverse views of the painted model are shown, together with a representation of the 3D domains. Click here for file Additional File 3 PAX6 3D expression.mpg. Mpeg movie of the 3D PAX6 expression domain. Click here for file Additional File 4 JAtlasViewer request form.pdf Click here for file Additional File 5 Academic Licence Agreement.pdf Click here for file Acknowledgements The project is funded by the National Institutes of Health (USA) Human Brain Project. (NIMH and NICHD). The human tissue was provided by the Joint MRC-Wellcome Human Developmental Biology Resource at IHG, Newcastle upon Tyne. ==== Refs O'Rahilly R Muller F The embryonic human brain An atlas of developmental stages 1999 2 Wiley-Liss Desmond ME O'Rahilly R The growth of the human brain during the embryonic period proper. 1. Linear axes Anat Embryol (Berl) 1981 162 137 51 7283177 Kaufman MH Brune RM Davidson DR Baldock RA Computer-generated three-dimensional reconstructions of serially sectioned mouse embryos J Anat 1998 193 323 36 9877288 10.1046/j.1469-7580.1998.19330323.x Jacobs RE Ahrens ET Meade TJ Fraser SE Looking deeper into vertebrate development Trends Cell Biol 1999 9 73 6 10087623 10.1016/S0962-8924(98)01435-4 Sharpe J Ahlgren U Perry P Hill B Ross A Hecksher-Sorensen J Baldock R Davidson D Optical projection tomography as a tool for 3D microscopy and gene expression studies Science 2002 296 541 5 11964482 10.1126/science.1068206 Sharpe J Optical projection tomography as a new tool for studying embryo anatomy J Anat 2003 202 175 81 12647867 10.1046/j.1469-7580.2003.00155.x Smith BR Huff DS Johnson GA Magnetic resonance imaging of embryos: an Internet resource for the study of embryonic development Comput Med Imaging Graph 1999 23 33 40 10091866 10.1016/S0895-6111(98)00061-5 The Edinburgh Mouse Atlas Project Baldock RA Dubreuil C Hill B Davidson D Levotsky S The Edinburgh Mouse Atlas: Basic Structure and Informatics Bioinformatics Databases and Systems 1999 Kluwer Academic Press 102 115 Baldock RA Bard JB Burger A Burton N Christiansen J Feng G Hill B Houghton D Kaufman M Rao J Sharpe J Ross A Stevenson P Venkataraman S Waterhouse A Yang Y Davidson DR EMAP and EMAGE: a framework for understanding spatially organized data Neuroinformatics 2003 1 309 25 15043218 10.1385/NI:1:4:309 Bancroft JD Stevens A Gamble M Theory and Practice of Histological Techniques 2001 Churchill Livingstone Benowitz LI Routtenberg A GAP-43: an intrinsic determinant of neuronal development and plasticity Trends Neurosci 1997 20 84 91 9023877 10.1016/S0166-2236(96)10072-2 Rubenstein JLR Puelles L Homeobox gene expression during development of the vertebrate brain Current Topics in Developmental Biology 1994 29 1 63 7828435 Puelles L Rubenstein JL Forebrain gene expression domains and the evolving prosomeric model Trends Neurosci 2003 26 469 76 12948657 10.1016/S0166-2236(03)00234-0 Puelles L Kuwana E Puelles E Bulfone A Shimamura K Keleher J Smiga S Rubenstein JLR Pallial and subpallial derivatives in the embryonic chick and mouse telencephalon, traced by the expression of the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, and Tbr-1 J Comp Neurol 2000 424 409 38 10906711 10.1002/1096-9861(20000828)424:3<409::AID-CNE3>3.0.CO;2-7 Redies C Ast M Nakagawa S Takeichi M Martínez-de-la-Torre M Puelles L Morphologic fate of diencephalic prosomeres and their subdivisions revealed by mapping cadherin expression J Comp Neurol 2000 421 481 514 10842210 10.1002/(SICI)1096-9861(20000612)421:4<481::AID-CNE3>3.0.CO;2-H Walther C Gruss P Pax-6, a murine paired box gene, is expressed in the developing CNS Development 1991 113 1435 49 1687460 Stoykova A Gruss P Roles of Pax-genes in developing and adult brain as suggested by expression patterns J Neurosci 1994 14 1395 412 8126546 Warren N Price DJ Roles of Pax-6 in murine diencephalic development Development 1997 124 1573 82 9108373 O'Rahilly R Muller F Developmental stages in human embryos Carnegie Institute, Washington, Publication no 637 1987 Bullen P Wilson D Strachan T, Lindsay S, Wilson DI The carnegie staging of human embryos: a practical guide Molecular Genetics of Early Human Development 1997 Bios Scientific publishers Bullen PJ Robson SC Strachan T Human post-implantation embryo collection: medical and surgical techniques Early Hum Dev 1998 51 213 21 9692791 10.1016/S0378-3782(97)00118-7 Puelles L A segmental morphological paradigm for understanding vertebrate forebrains Brain, Behavior and Evolution 1995 46 319 337 8564469 Puelles L Thoughts on the development, structure and evolution of the mammalian and avian telencephalic pallium Philos Trans R Soc Lond B Biol Sci 2001 356 1583 98 11604125 10.1098/rstb.2001.0973 Shimamura K Hartigan D-J Martínez S Puelles L Rubenstein JLR Longitudinal organization of the anterior neural plate and neural tube Development 1995 121 3923 3933 8575293 Hochstetter F Deuticke F Beiträge zur Entwicklungsgeschichte des menschlichen Gehirnes Parts I and II 1929 Wien, Leipzig The Electronic Atlas of the Developing Human Brain Polkinghorne J Guidelines on the Research Use of Foetuses and Foetal Material HMSO Ref No CM762 1989

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==== Front BMC PharmacolBMC Pharmacology1471-2210BioMed Central London 1471-2210-4-141529402610.1186/1471-2210-4-14Research ArticleA study comparing the actions of gabapentin and pregabalin on the electrophysiological properties of cultured DRG neurones from neonatal rats McClelland David [email protected] Rhian M [email protected] Louise [email protected] Duncan J [email protected] Roderick H [email protected] Department of Biomedical Sciences, Institute of Medical Sciences, The University of Aberdeen, Foresterhill, Aberdeen AB25 2RL, Scotland, UK2004 4 8 2004 4 14 14 2 2 2004 4 8 2004 Copyright © 2004 McClelland et al; licensee BioMed Central Ltd.2004McClelland et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Gabapentin and pregabalin have wide-ranging therapeutic actions, and are structurally related to the inhibitory neurotransmitter GABA. Gabapentin, pregablin and GABA can all modulate voltage-activated Ca2+ channels. In this study we have used whole cell patch clamp recording and fura-2 Ca2+ imaging to characterise the actions of pregabalin on the electrophysiological properties of cultured dorsal root ganglion (DRG) neurones from neonatal rats. The aims of this study were to determine whether pregabalin and gabapentin had additive inhibitory effects on high voltage-activated Ca2+ channels, evaluate whether the actions of pregabalin were dependent on GABA receptors and characterise the actions of pregabalin on voltage-activated potassium currents. Results Pregabalin (25 nM – 2.5 μM) inhibited 20–30% of the high voltage-activated Ca2+ current in cultured DRG neurones. The residual Ca2+ current recorded in the presence of pregabalin was sensitive to the L-type Ca2+ channel modulator, Bay K8644. Saturating concentrations of gabapentin failed to have additive effects when applied with pregabalin, indicating that these two compounds act on the same type(s) of voltage-activated Ca2+ channels but the majority of Ca2+ current was resistant to both drugs. The continual application of GABA, the GABAB receptor antagonist CGP52432, or intracellular photorelease of GTP-γ-S had no effect on pregabalin-induced inhibition of Ca2+ currents. Although clear inhibition of Ca2+ influx was produced by pregabalin in a population of small neurones, a significant population of larger neurones showed enhanced Ca2+ influx in response to pregabalin. The enhanced Ca2+ influx evoked by pregabalin was mimicked by partial block of K+ conductances with tetraethylammonium. Pregabalin produced biphasic effects on voltage-activated K+ currents, the inhibitory effect of pregabalin was prevented with apamin. The delayed enhancement of K+ currents was attenuated by pertussis toxin and by intracellular application of a (Rp)-analogue of cAMP. Conclusions Pregabalin reduces excitatory properties of cultured DRG neurones by modulating voltage-activated Ca2+ and K+ channels. The pharmacological activity of pregabalin is similar but not identical to that of gabapentin. The actions of pregabalin may involve both extracellular and intracellular drug target sites and modulation of a variety of neuronal conductances, by direct interactions, and through intracellular signalling involving protein kinase A. ==== Body Background Gabapentin (Neurotonin®) and pregabalin (S(+)-3-isobutyl GABA) were both originally designed as GABA mimetics (Figure 1A), with the intention that they would be able to cross the blood-brain barrier and interact with GABAergic systems and enhance GABA mediated inhibition. Although gabapentin appears to have diverse therapeutic utility in the treatment of pain disorders [1], psychiatric illnesses [2] and epilepsy [3], there is controversy regarding its molecular mechanisms of action. Whether the actions of gabapentin and pregabalin are mediated through GABAergic mechanisms or GABA receptors remains particularly contentious [4-6]. However, high affinity binding sites for gabapentin and pregabalin on distinct α2δ subunits of voltage-activated calcium channels have been identified and characterised [7]. For this reason voltage-activated Ca2+ channels remain primary candidate sites of action for these novel anticonvulsant and antihyperalgesic drugs. Functional data from studies on gabapentin and pregabalin also support this contention. Specifically, both gabapentin and pregabalin inhibited hyperalgesia [8,9], attenuated evoked Ca2+ influx into brain slices and reduced evoked transmitter release [10]. Additionally, gabapentin and pregabalin inhibited multiple firing of action potentials evoked by 300 ms depolarising current commands in cultured sensory DRG neurones [5]. Figure 1 Pregabalin inhibits Ca2+ currents. A) Structure of GABA (γ-aminobutyric acid), gabapentin (1-(aminoethyl)cyclohexane acetic acid) and pregabalin (S(+)-3-isobutyl GABA). B & C) Traces of high voltage-activated Ca2+ currents evoked from a holding potential of -90 mV by a depolarising step command to 0 mV showing inhibition by pregabalin (2.5 μM). B) Shows that pressure ejection of choline chloride extracellular solution does not induce inhibition of the Ca2+ current but in the same neurone pregabalin does produce a response. Traces show a control Ca2+ current (Ctrl), the current unaffected by application of choline chloride recording solution (ChCl), inhibition of current by 3 minutes application of pregabalin (PGB) and the current at 5 minutes recovery (Rec). C) Traces show a control Ca2+ current (Ctrl), the inhibition of current after 3 minutes application of pregabalin (PGB) and full recovery of the current 5 minutes after removal of the drug pipette (Rec). D) Graph showing the distribution of inhibitory responses produced by 0.025 – 2.5 μM pregabalin. Each symbol represents a result from a different experiment. Our previous studies have focused on the inhibitory effects of gabapentin (0.25–25 μM) on whole cell voltage-activated Ca2+ currents and K+ stimulated Ca2+ entry measured with fura-2. The cellular model systems used were primary cultures of dorsal root ganglion (DRG) neurones from 1–4 day old rats and differentiated F-11 cells (embryonic rat DRG × neuroblastoma hybrid cell line) [11,12]. In this present study the effects of pregabalin and gabapentin on the electrophysiological properties of cultured neonatal rat DRG neurones were measured with particular reference to Ca2+ entry through high voltage-activated channels and enhancement of K+ conductances. We had two specific aims for this project. The first was to determine whether gabapentin and pregabalin have the same mechanisms of action, by examining whether saturating concentrations of gabapentin and pregabalin act in an additive manner to attenuate Ca2+ influx. The second aim was to further evaluate GABA receptors as target sites for these drugs in sensory neurones. This latter element to the study was conducted because it has been proposed that specific GABAB receptors with a gb1a-gb2 heterodimer composition are the sites of agonist activity of gabapentin. These specific receptors can be coupled to an inwardly rectifying K+ channel and / or voltage-activated Ca2+ channels to dampen neuronal electrical excitability [13-15]. However, in certain situations gabapentin and the GABAB receptor agonist, baclofen, have different actions. Weaver mutant mice (wv/wv) are insensitive to both gabapentin and baclofen, however in control littermates, Weaver control mice (+/+, wv/+), only baclofen evoked a K+ current [16]. Results and discussion Actions of pregabalin on voltage-activated calcium currents The firing of multiple action potentials in response to a sustained depolarising current command is a property of a sub-population (under 20 %) of cultured DRG neurones. Application for 3 minutes, of either pregabalin (PGB; 2.5 μM) or gabapentin (GBP; 2.5 μM) reduced the frequency of action potential spikes evoked by 300 ms depolarising current step commands applied every 30 s [5]. Gabapentin attenuated repetitive action potential firing as measured by a reduction in the mean number of evoked action potentials during 300 ms depolarisations from 7 to 2 (n = 3, p < 0.01). Pregabalin (2.5 μM) reversibly reduced the number of action potentials during 300 ms depolarisations from 8 to 3 (n = 5, p < 0.01). However, pregabalin and gabapentin did not significantly alter the properties (amplitude, duration & threshold) of single action potentials evoked by 5 ms depolarising current commands. Consistent with our previous work [11,12], whole cell voltage-activated Ca2+ currents (ICa) recorded from DRG neurones were reversibly attenuated (22 ± 11%; n = 7) by 2.5 μM gabapentin. Similarly, 3 minutes application of pregabalin (25 nM – 2.5 μM; Figure 1B &1C; table 1) inhibited the mean Ca2+ current amplitude, measured at the peak of the inward current, and at the end of a 100 ms voltage step command to 0 mV. The inhibitory actions of pregabalin were not accompanied by any shift in the voltage-dependence of activation for ICa or by any change in holding or leak currents. At least partial recovery of ICa was observed 5 minutes after removal of the pregabalin-containing perfusion pipette (Figure 1B &1C). A very low concentration of pregabalin (0.25 nM) failed to produce significant inhibition of ICa. However, no clear dose-dependent relationship was established for the inhibitory actions of pregabalin, and a major part of the voltage-activated Ca2+ current was insensitive to the drug. It was also apparent that there was considerable variability in the sensitivity of the DRG neurones to any given dose of pregabalin. Some neurones did not respond to 2.5 μM pregabalin while in a few neurones this same concentration produced 60 % inhibition of ICa (Figure 1D). Table 1 Inhibitory actions of pregabalin on voltage-activated calcium currents. Peak control current amplitude (nA) Pregabalin Concentration (μM) Peak current in the presence of pregabalin (nA) Percentage inhibition and n value -1.52 ± 0.13 0.025 -0.98 ± 0.12 ** 31 ± 4 % (n = 8) -1.2 ± 0.14 0.25 -0.93 ± 0.18 * 22 ± 9 % (n = 6) -1.19 ± 0.11 2.5 -0.94 ± 0.09*** 21 ± 3 % (n = 26) ***P < 0.001; **P < 0.01 and *P < 0.05. Each value is given ± S.E.M. Data shows inhibition but a lack of dose-dependent actions on ICa of a wide range of pregabalin concentrations. Although varied responses were observed it was clear that a major component of ICa was resistant to inhibition by pregabalin. Distinct voltage-activated Ca2+ channels, defined by their alpha 1 subunit, have been suggested to be selective target sites for gabapentin and related drugs like pregabalin. With this in mind, the 1,4-dihydropyridine L-type Ca2+ channel agonist Bay K8644 was used to determine whether pregabalin was inhibiting L-type channels. Previously, we found that in the continual presence of gabapentin, Bay K8644 enhanced ICa [12]. In this present study similar results were obtained. After inhibiting part of the current with pregabalin (2.5 μM), Bay K8644 (1 μM) was applied with pregabalin and the enhanced ICa was measured at its peak and at the end of a 100 ms voltage step command to 0 mV (Figure 2). The percentage increases in current seen with Bay K8644 in the presence of either gabapentin or pregabalin were slightly less than those seen under control conditions in cultured DRG neurones [17]. This is consistent with some L-type Ca2+ current modulation by pregabalin and gabapentin in DRG neurones. Taken together these data suggest that neither gabapentin nor pregabalin are selectively inhibiting L-type ICa in DRG neurones. This contrasts with the effects of gabapentin on cortical pyramidal neurones where inhibition of L-type Ca2+ channels appears to be the predominant mechanism of action [18]. Figure 2 Bay K8644 enhanced pregabalin-insensitive current suggesting that L-type Ca2+ channels are still available for modulation by the 1,4-dihydropyridine agonist. A) Bar chart shows data for mean calcium current amplitudes measured at the peak of the inward current (open bars) and at the end of a 100 ms voltage step command to 0 mV (solid bars, n = 6). Data under control condition (Con) in the presence of 2.5 μM pregabalin (PGB) and in the presence of both pregabalin (2.5 μM) and Bay K8644 (1 μM) are shown. B) The inset traces show voltage and Ca2+ current records under control conditions, inhibition in the presence of 2.5 μM pregabalin (PGB) and enhancement of the Ca2+ current during continued application of pregabalin with Bay K8644 present (Bay K & PGB). C) Shows the current inhibited by pregabalin, (obtained by subtracting the net current recorded in the presence of pregabalin from the net control current). D) Shows the additional current produced by Bay K8644, (obtained by subtracting the net current recorded in the presence of pregabalin from the net current recorded in the presence of both pregabalin and Bay K8644). Actions of pregabalin on calcium influx through voltage-activated channels, measured using fura-2 imaging Given the variable and rather modest but reversible inhibitory actions of pregabalin on ICa we tested the actions of pregabalin on K+-evoked Ca2+ influx using fura-2 imaging. An extracellular solution containing 30 mM K+ was used to depolarise the DRG neurones and activate three consistent Ca2+ transients [11,12]. Pregabalin (2.5 μM) was applied during the second K+-evoked depolarisation and it produced a mixture of reversible effects on the Ca2+ transients. In 4 of 24 neurones the Ca2+ flux was decreased by 54 ± 13 % (p<0.05) but in 20 neurones from the same cultures pregabalin evoked a mean increase in Ca2+ flux to 185 ± 20 % (p<0.05) of the control. Raising the pregabalin concentration to 25 μM increased the proportion of inhibitory responses (n = 21 out of 49 neurones) but enhancement of Ca2+ transients was still observed in the remaining 28 neurones (Figure 3). However, 250 μM pregabalin caused an increase in K+-evoked Ca2+ influx in all neurones studied (n = 31). Although in some cells good recovery from pregabalin actions was observed (figure 3B) the inhibitory effect was often not associated with recovery. This may reflect long lasting effects of pregabalin. Run-down in some cells can not be completely ruled out but under control conditions the mean level of run-down of three K+-evoked Ca2+ transients was only 8.4 ± 1.5 % (n = 39). Similarly, the mean control level of increase in K+-evoked Ca2+ transients was only 7.2 ± 1.3 % (n = 35). Figure 3 Pregabalin produced mixed actions on Ca2+ influx evoked by 30 mM K+. A) Bar chart showing inhibition of Ca2+ influx by pregabalin (25 μM). Data for the total Ca2+ fluxes was normalised with respect to the first control response to K+ (Ctrl). The second and third responses were obtained in the presence of pregabalin (PGB) and after washing away the pregabalin (Rec). B) Record of K+-evoked Ca2+ transients showing partially reversible inhibition produced by 25 μM pregabalin (PGB). The period of stimulation is shown with open bars and PGB application with the filled bar. C) Bar chart showing enhancement of Ca2+ influx by pregabalin (25 μM). Data for the total Ca2+ fluxes were normalised with respect to the first control response to K+ (Ctrl). The second and third responses were obtained in the presence of pregabalin (PGB) and after washing away the pregabalin (Rec). D) Record of K+-evoked Ca2+ transients showing reversible enhancement produced by 25 μM pregabalin (PGB). To investigate the different responses to pregabalin, the sizes of cell somas were measured and compared. Although there is overlap, figure 4 shows that enhancement of K+-evoked Ca2+ transients by pregabalin was mainly seen in neurones with larger cell somas and that in smaller neurones pregabalin produced inhibitory effects. Figure 4 Different responses to pregabalin were observed in different populations of cultured DRG neurones. Bar chart showing the distribution of neurones with different cell soma areas and the response to pregabalin. The distributions for intermediate and larger neurones where pregabalin (25 μM) increased K+-evoked Ca2+ influx are shown in black bars. The distributions for small and some intermediate neurones where pregabalin (25 μM) attenuated K+-evoked Ca2+ influx are shown in open bars. Ca2+-dependent conductances of DRG neurones are sensitive to ryanodine and Ca2+-induced Ca2+ release has been reported in these neurones. Modulation of either Ca2+-induced Ca2+ release and / or Ca2+ homeostatic mechanisms might provide a mechanism by which pregabalin enhanced Ca2+ transients in neurones with intermediate and large sized cell somas. This was investigated in two ways. Firstly, the actions of pregabalin on caffeine-evoked Ca2+ transients were evaluated in nominally Ca2+-free extracellular conditions (NaCl-based solution with no added CaCl2). Single caffeine (1 mM) responses were obtained from DRG neurones in either the absence or presence of 25 μM pregabalin. No differences in either amplitudes or durations of Ca2+ transients were seen when 8 control caffeine responses were compared with 4 caffeine responses obtained in the presence of pregabalin (Figure 5A,5B). The second approach was to explore a possible role of Na+ / Ca2+ exchange by bathing cells in choline chloride-based medium containing only 1 mM Na+. Under these conditions the contribution of the Na+ / Ca2+ exchanger to handling of intracellular Ca2+ loads will be minimal. In choline chloride-based medium pregabalin produced both enhancement and inhibition of total Ca2+ flux in 7 and 3 neurones respectively. Enhancement in Ca2+ flux by pregabalin under these experimental conditions, suggest that inhibition of Na+ / Ca2+ exchange is not the main mechanism by which pregabalin enhances K+-evoked Ca2+ flux (Figure 5C). However, detailed analysis was made difficult by poor recovery of even the first Ca2+ transient evoked in low extracellular Na+ which does suggest that Na+ / Ca2+ exchange is an important homeostatic mechanism in DRG neurones. In conclusion these results indicate that modulation of Ca2+-induced Ca2+ release and Ca2+ homeostatic mechanisms do not account for pregabalin-induced enhancement of K+-evoked Ca2+ flux in a population of DRG neurones. Figure 5 Pregabalin does not appear to modulate caffeine-evoked Ca2+ release or Ca2+ homeostatic mechanisms. A & B) Show example records of Ca2+ transient evoked by caffeine (1 mM) applied to DRG neurones bathed in nominally Ca2+-free medium and measured using fura-2. Under these conditions the Ca2+ transients are only due to mobilisation of Ca2+ from intracellular stores. A) Illustrates a control caffeine response and B) shows a similar response to caffeine recorded in the presence of 25 μM pregabalin (PGB). C) Example trace showing an increase in K+-evoked Ca2+ flux by 25 μM pregabalin (PGB) in a DRG neurone bathed with choline chloride-based extracellular medium. The periods of stimulation with 30 mM KCl are shown with open bars and pregabalin application is shown with a filled bar. D) Example trace showing an increase in K+-evoked Ca2+ flux induced by 5 mM TEA in a DRG neurone bathed with standard NaCl-based extracellular medium. The periods of stimulation with 30 mM KCl are shown with open bars and TEA application is shown with a filled bar. Inhibitory modulation of potassium conductances could result in increased K+-evoked Ca2+ transients. To test this alternative mechanism, a relatively low concentration, 5 mM, of tetraethylammonium (TEA) was applied with NaCl-based extracellular medium and DRG neurones were stimulated with 30 mM KCl. Attenuation of potassium conductances by TEA markedly increased the K+-evoked Ca2+ flux in all eight DRG neurones studied, mimicking in part the action of pregabalin (Figure 5D). Do pregabalin and gabapentin have additive effects on cultured dorsal root ganglion neurones? Pregabalin and gabapentin have related chemical structures and appear to have similar but usually modest inhibitory effects on Ca2+ currents. Both pregabalin and gabapentin at a concentration of 2.5 μM produced a maximum level of current inhibition. Simultaneous application of pregabalin (2.5 μM) and gabapentin (2.5 μM) produced modest but significant inhibition of ICa at the peak of the current and at the end of the stimulus (Figure 6A). However, the percentage inhibition of ICa produced was not significantly different for 2.5 μM gabapentin alone (22 ± 11%; n = 7), 2.5 μM pregabalin alone (21 ± 3%; n = 26) and 2.5 μM gabapentin and 2.5 μM pregabalin applied together (19 ± 2%; n = 9). Figure 6 Pregabalin and gabapentin do not have additive actions on cultured DRG neurones. A) Bar chart showing the mean Ca2+ current amplitude recorded at the peak of the inward current (Peak) and end of a 100 ms voltage step command (End). Data is shown for measurements made under control conditions (Control), after 3 to 5 minutes application of both pregabalin (2.5 μM) and gabapentin (2.5 μM) (PGB + GBP) and after 5 minutes recovery (Recovery). B) Bar chart showing normalised data from Ca2+ imaging experiments in which simultaneous application of both pregabalin (25 μM) and gabapentin (25 μM) (PGB & GBP) reversibly attenuated the total Ca2+ flux. C) Record of K+-evoked Ca2+ transients, showing the reversible inhibition produced by 25 μM pregabalin and 25 μM gabapentin (PGB + GBP, filled bar). In spite of the added complexity of mixed responses with pregabalin, experiments were carried out using fura-2 to measure K+ evoked Ca2+ flux and the effects of both ligands. Our previous experiments with gabapentin (25 μM) had only identified inhibitory effects using this experimental approach [11,12]. Simultaneous application of pregabalin and gabapentin (both at 25 μM) reversibly reduced the K+ evoked Ca2+ influx (Figure 6B &6C). Pregabalin (25 μM) and gabapentin (25 μM) together reduced the total Ca2+ flux to 75 ± 5 % (n = 14) of the control response to 30 mM K+. Consistent with the electrophysiological experiments, this level of inhibition was similar to the inhibitory effects of pregabalin and gabapentin applied separately. The electrophysiological and fura-2 Ca2+ imaging data show that no additive inhibitory effects were found during simultaneous application of saturating concentrations of gabapentin and pregabalin. The data therefore support the contention that both drugs have a closely related or a common site of inhibitory action. The data also indicate that in the modulation of Ca2+ channels these drugs do not act in an additive manner even though a substantial proportion of current is resistant to both drugs. It should be emphasized however that pregabalin can reversibly enhance K+-evoked Ca2+ transients, an effect not seen with gabapentin. Therefore there may be other additional actions of pregabalin that can be identified in Ca2+ imaging experiments when all membrane conductances are intact. Does GABA receptor modulation alter responses to pregabalin in cultured dorsal root ganglion neurones? In this section of the study two strategies were used to evaluate the possible roles of GABA receptors in pregabalin responses in cultured DRG neurones. Firstly, pregabalin actions were studied in the presence of a saturating concentration of GABA; this initially activated all types of GABA receptor and then caused desensitization of these receptors. Secondly, the effect of a potent and selective GABAB receptor antagonist, CGP52432 [19] on pregabalin responses was evaluated. GABA (100 μM) evoked inward currents in a sub-population of cultured DRG neurones and induced a transient rise in intracellular Ca2+ in 18 of 41 DRG neurones studied (Figure 7A &7B). The inward currents were due to the activation of GABAA receptor Cl- channels; with the equilibrium potential for Cl- under our recording conditions being close to 0 mV the chloride conductance results in an inward current. Although in vivo the equilibrium potential for Cl- in DRG neurones is variable, it is predicted to be between -40 mV and -20 mV because of Cl- loading into the intracellular environment (for review see [20]). Therefore GABAA receptor Cl- channel activation can produce a depolarisation of the resting membrane potential. Our data indicated that the GABA-evoked depolarisation was sufficient to result in voltage-activated Ca2+ channel activity and produce a transient rise in intracellular Ca2+. In both neurones that responded to GABA and those that did not, subsequent application of pregabalin, produced either enhancement or inhibition of K+-evoked Ca2+ influx (Figure 7A,7B,7C &7D; Table 2). These responses to pregabalin in the presence of GABA were no different in character to the pregabalin responses obtained in the absence of GABA. In neurones that showed a Ca2+ transient in response to a GABA-evoked depolarisation, there was an apparent increase (to 83%) in the proportion of neurones that were inhibited by pregabalin. It is not clear why this is but it may reflect the sensitivity of Ca2+ transients evoked in DRG neurones, which can only be evoked consistently in most DRG neurones by three depolarising stimuli. Thus the GABA-evoked responses may influence Ca2+ homeostatic mechanisms and subsequent stimulated Ca2+ entry. However, the critical observation from these experiments is that GABA receptor desensitisation does not prevent either Ca2+ transient enhancement or inhibitory actions of pregabalin in neurones that were sensitive or insensitive to GABA. Figure 7 Activation and subsequent desensitisation of GABA receptors fails to prevent the modulation of voltage-activated Ca2+ channels in cultured DRG neurones by pregabalin. A) Bar chart showing data obtained from neurones that responded to GABA (100 μM). All data are normalised with respect to the first Ca2+ transient evoked by 30 mM KCl. Open bars show the relative responses to 100 μM GABA in cells (n = 3) where 25 μM pregabalin enhanced the Ca2+ flux. Filled bars show responses in cells (n = 15) where 25 μM pregabalin inhibited the Ca2+ flux. Data are shown for GABA responses (GABA), K+-evoked Ca2+ transients under control conditions with GABA present (Ctrl), K+ evoked Ca2+ transients in the presence of GABA and 25 μM pregabalin (PGB) and on recovery in the continued presence of GABA (Rec). B) An example trace, showing a response to 100 μM GABA and Ca2+ transients evoked by K+ in the presence and absence 25 μM pregabalin. The long open bar shows the period of GABA application, the short open bars show K+ stimulation and the filled bar the period of pregabalin application. C) Bar chart showing data obtained from neurones that did not responded to GABA (100 μM) but were continually bathed with GABA for the duration of the experiment. All data are normalised with respect to the first Ca2+ transient evoked by 30 mM KCl. Data are shown for K+-evoked Ca2+ transients under control conditions with GABA present (Ctrl), increased (n = 10; open bars) and decreased (n = 13; filled bars) K+-evoked Ca2+ transients in the presence of GABA and 25 μM pregabalin (PGB) and on recovery (Rec). D) An example trace, showing no response to 100 μM GABA but responses to K+ in the presence and absence of 25 μM pregabalin. The long open bar shows the period of GABA application, the short open bars show K+ stimulation and the filled bar the period of pregabalin application. Table 2 Actions of pregabalin (25 μM) on K+-evoked Ca2+influx in the continual presence or absence of 100 μM GABA. GABA sensitivity Mean percentage of control Ca2+ influx Number of neurones Proportion of Neurones Responders 110 ± 2 % 3 of 18 17 % Responders 77 ± 2 % 15 of 18 83 % Non-responders 121 ± 4 % 10 of 23 43 % Non-responders 81 ± 3 % 13 of 23 57 % Control 169 ± 24 % 28 of 49 57 % Control 85 ± 2 % 21 of 49 43 % Responders were those neurones in which GABA evoked a transient rise in intracellular Ca2+. Non-responder showed no change in fura-2 fluorescence ratio in response to GABA. Controls where neurones that pregabalin was applied to without GABA being present. The amplitudes of the GABA responses did not vary with the inhibitory or enhancing responses to pregabalin. The possibility of pregabalin-evoked inhibition of ICa taking place through activation of G-protein coupled GABAB receptors, was then assessed. CGP52432 (10 μM) when applied alone had no effect on ICa. In the presence of the GABAB receptor antagonist CGP52432 (10 μM), pregabalin (2.5 μM) evoked a 27 ± 3 % (n = 9) inhibition of the voltage-activated Ca2+ current and complete recovery was observed 5 minutes after removal of the drug-containing pipette (Figure 8A). In Ca2+ imaging experiments 10 μM CGP52432 had no effect on the responses to 25 μM pregabalin. In the presence of CGP52432, 4 out of 16 neurones showed enhanced K+-evoked Ca2+ influx (to 161 ± 24 % (n = 4) of control) in response to pregabalin and 12 neurones showed inhibition of Ca2+ influx (64 ± 8 % (n = 12) of control) in response to pregabalin (Figure 8B &8C). Figure 8 The GABAB receptor antagonist CGP52432 had no effect on the actions of pregabalin. A) Bar chart showing the inhibitory effects of 2.5 μM pregabalin in the presence of 10 μM CGP52432 (PGB & CGP) on Ca2+ current amplitude measured at the peak inward current (Peak) and at the end of a 100 ms voltage step command to 0 mV (End). The inset traces show the inhibition of the Ca2+ current produced by 3 minutes application of pregabalin and CGP52432 (PGB + CGP) and partial recovery 5 minutes after removal of the drug perfusion pipette. B & C) Show example records of 25 μM pregabalin in the presence of 10 μM CGP52432 (PGB + CGP) modulating Ca2+ flux evoked by 30 mM KCl. The open bars show the period of stimulation with K+ and the filled bar the application of pregabalin in the presence of CGP52432 (PGB +CGP). Is there a role for G-proteins or G-protein coupled receptors in the responses to pregabalin in cultured dorsal root ganglion neurones? Previously, we found that the actions of gabapentin on ICa were attenuated by pre-treating DRG neurones with pertussis toxin, an effect that did not appear to involve metabotropic GABAB receptors [12]. In this study several different approaches were taken to examine the influence of receptor and G-protein function in pregabalin actions. Firstly, the effects of a novel thiadiazole compound, SCH-202676, which inhibits ligand binding to a variety of G-protein coupled receptors (opioid, adrenergic, muscarinic and dopaminergic) were investigated. The selective and reversible action of SCH-202676 appears to involve allosteric modulation of both agonist and antagonist binding to G-protein coupled receptors [21]. Secondly, the potential influences of intracellular flash photolysis of caged GTP-γ-S and subsequent G-protein activation on pregabalin responses were also examined. SCH-202676 (10 μM) was applied to the intracellular environment via the patch pipette solution. After 5 minutes equilibration the whole cell Ca2+ current had a mean amplitude of -0.54 ± 0.08 nA (n= 7). During this period there was a clear reduction in the inward current, although a steady state current level was reached. However, subsequent application of pregabalin (2.5 μM) resulted in a further significant reduction in Ca2+ current amplitude to -0.43 ± 0.07 nA (n = 7, p<0.05). This represents a mean inhibition of 22 ± 5 % by pregabalin, a value very similar to the percentage inhibition produced by pregabalin in the absence of SCH-202676 (21 ± 3%). Therefore SCH-202676 had no effect on the pregabalin responses, indicating that pregabalin was not acting through an SCH-202676-sensitive G-protein coupled receptor (data not shown). Preliminary Ca2+ imaging experiments were also conducted to determine whether extracellular SCH-202676 (10 μM, continually applied throughout the experiment) altered the effects of pregabalin (2.5 μM) on K+-evoked Ca2+ transients. Under these experimental conditions pregabalin was found to markedly enhance or inhibit K+-evoked Ca2+ transients (data not shown). This data again suggests that pregabalin was not acting through mechanisms that were sensitive to SCH-202676. Caged GTP-γ-S (100 μM) was applied to the intracellular environment via the patch pipette solution and after entering the whole cell recording configuration DRG neurones were left for 5 minutes to equilibrate. Once a stable control Ca2+ current was obtained, three 200 V flashes of intense near UV light were applied to the neurone to achieve intracellular flash photolysis of the caged GTP-γ-S. We estimate that approximately 15 μM GTP-γ-S was photoreleased by the three flashes. As previously observed intracellular flash photolysis of caged GTP-γ-S reduced the amplitude and slowed the activation of ICa [22]. The effects of photoreleased GTP-γ-S were attenuated by applying a large depolarising pre-pulse [23], which is consistent with voltage-dependent G-protein modulation of Ca2+ channels (Figure 9A). No recovery from photoreleased GTP-γ-S was observed during the period of the experiment. GTP-γ-S and pregabalin (2.5 μM) had additive inhibitory effects on ICa. These additive actions of GTP-γ-S and pregabalin were apparent regardless of the order of application. One set of experiments was performed in which GTP-γ-S was photoreleased in the intracellular environment and then after stabilisation of the response pregabalin was applied (Figure 9B). In another set of experiments pregabalin was applied first and after equilibration GTP-γ-S was photoreleased in the continual presence of pregabalin (Figure 9C). When pregabalin (2.5 μM) was applied first it produced a mean inhibition in ICa by 25 ± 5 % (n = 4), when applied after photorelease of GTP-γ-S, pregabalin produced a 20 ± 10 % (n = 5) inhibition of ICa. Figure 9 Intracellular photorelease of GTP-γ-S did not alter the sensitivity of DRG neurones to pregabalin. A) Traces showing a Ca2+ current attenuated and slowed by intracellular flash photolysis of caged GTP-γ-S (GTP-γ-S) and a Ca2+ current showing voltage-dependent partial recovery of GTP-γ-S-evoked inhibition (GTP-γ-S + PP {pre-pulse to +120 mV}). B) Bar chart showing control (Ctrl) data, the inhibitory effects of both intracellular photorelease of GTP-γ-S (GTP-γ-S) and subsequent application of 2.5 μM pregabalin (GTP-γ-S & PGB) on the mean peak Ca2+ current amplitude. Inset records show individual Ca2+ currents recorded under control conditions, after intracellular photorelease of GTP-γ-S (GTP-γ-S) and after subsequent extracellular application of pregabalin for 3 minutes (GTP-γ-S + PGB). C) Bar chart showing control (Ctrl) data, the inhibitory effects of both 3 minutes extracellular application of 2.5 μM pregabalin (PGB) and subsequent intracellular photorelease of GTP-γ-S in the continued presence of pregabalin (PGB & GTP-γ-S) on the mean peak Ca2+ current amplitude. Inset records show individual Ca2+ currents recorded under control conditions, after application of pregabalin for 3 minutes (PGB) and after intracellular photorelease of GTP-γ-S (PGB & GTP-γ-S). None of the currents in this figure have been leak subtracted. However, neither pregabalin nor GTP-γ-S altered the leak current. Actions of pregabalin on voltage-activated potassium currents It was clear from the Ca2+ imaging experiments that not all the cellular actions of pregabalin could be explained by the inhibition of voltage-activated Ca2+ channels because both inhibition and enhancement in K+ evoked Ca2+ flux was observed. To investigate the actions of pregabalin further, its' effects on voltage-activated K+ currents in DRG neurones were studied. Previously, Stefani and colleagues found that gabapentin modulated neuronal steady state non-inactivating K+ currents [18]. Three minutes application of pregabalin (2.5 μM) had no significant effect on the voltage-activated K+ current activated from a holding potential of -90 mV by a 100 ms voltage step command to 0 mV (Figure 10A). However, raising the pregabalin concentration to 250 μM resulted in significant modulation of K+ currents in DRG neurones. Pregabalin (250 μM) applied for 3 to 5 minutes produced an enhanced K+ current in 11 out of 21 neurones but a modest inhibition of the outward current in 10 out of 21 neurones (Figure 10B and 10C). DRG neurones are a heterogenous population of neurones and there is evidence that they express at least 6 diverse voltage-activated K+ channels and that this expression is in part determined by the type of DRG neurone [24]. To distinguish between the two responses experiments were carried out on DRG neurones held at -30 mV to inactivate a proportion of the outward current. Under these conditions pregabalin inhibited the outward current in 9 out of 11 DRG neurones but still produced enhancement in 2 neurones. The level of inhibition increased at a holding potential of -30 mV to 30 ± 7% (n = 9) compared to 15 ± 4 % (n = 10) at -90 mV, (data not shown). Figure 10 Pregabalin modulates voltage-activated potassium currents in cultured DRG neurones. A) Traces showing that 2.5 μM pregabalin failed to significantly alter the outward K+ current evoked by a 100 ms voltage step command from -90 mV to 40 mV. B) Current / voltage relationship showing enhancement of outward current following 3 minutes application of 250 μM pregabalin (filled circles = control data and open circles currents recorded in the presence of pregabalin (PGB); * = P < 0.05 & ** = P < 0.01). Inset traces show the control outward K+ current activated at +40 mV and the enhanced K+ current recorded in the presence of pregabalin (250 μM). C) Current / voltage relationship showing inhibition of outward K+ current following 3 minutes application of 250 μM pregabalin (filled circles = control data and open circles K+ currents recorded in the presence of pregabalin (PGB); * = P < 0.05). Inset traces show the control outward K+ current activated at +40 mV and the attenuated K+ current recorded in the presence of pregabalin (250 μM). Interestingly, in apparent contrast to pregabalin, 250 μM gabapentin was initially only found to inhibit K+ currents in DRG neurones (Figure 11A,11B). However, when studies into long-term (10–15 minutes) actions of gabapentin were investigated slowly developing outward current enhancement was identified. So similar to actions of pregabalin, biphasic responses to gabapentin were found with initial inhibition of K+ currents and then a delayed enhancement of the outward current (Figure 11C), [5]. Figure 11 Acute application of gabapentin produced modest inhibition of voltage-activated K+ currents in cultured DRG neurones but long-term measurement of K+ currents shows a delayed enhancement in outward current. A) Bar chart showing data for the acute (3–5 minutes) reversible inhibition of the mean K+ current by 250 μM gabapentin (GBP). B) Traces showing a control outward K+ current activated at 0 mV and the inhibited current activated at the same voltage after 3 minutes application of 250 μM gabapentin (GBP). C) Traces showing the biphasic response to 250 μM gabapentin. Illustrated are the control current, the inhibited current recorded after 5 minutes application of gabapentin (GBP) and the enhanced outward current measured 5 minutes after removal of the perfusion pipette containing gabapentin (Enhanced Current). Long-term modulation of K+ current by pregabalin was then investigated. Dramatic increases in outward current were observed after a delay. This effect of pregabalin persisted even as pregabalin was removed from the extracellular environment, which may implicate a metabolic or intracellular signalling event in this response. The mean K+ current amplitude at +40 mV increased from 3.37 ± 0.73 nA to 7.56 ± 1.10 nA (n = 11; p<0.01) 15 minutes after perfusion of 250 μM pregabalin. Figures 12A and 12B show an individual example record and trace of the delayed response to pregabalin. These responses did reverse but this took about 40 minutes with the response developing 3–10 minutes after the start of pregabalin application. No change in holding current or in the leak conductances were associated with the long-term effect of pregabalin and in the absence of pregabalin stable K+ currents were recorded from DRG neurones for 16 minutes (n = 10). Figure 12 Pregabalin produced delayed enhancement of K+ currents in cultured DRG neurones. A) Line graph showing a time course for the delayed action of pregabalin in a single neurone, the open bar shows the period of application of pregabalin (250 μM). Little effect of pregabalin was seen in this neurone until 5 minutes after removal of the pressure ejection pipette containing pregabalin. B) Inset traces show a control K+ current activated at +40 mV, modest enhancement of the K+ current after 5 minutes application of 250 μM pregabalin and the enhanced outward K+ current recorded 9 minutes after application of pregabalin. Apamin mimicked the inhibitory action of pregabalin on K+ current but did not prevent enhancement of the K+ current by acute application of 250 μM pregabalin. C) Bar chart showing the modest inhibitory effect of apamin (1 μM) on K+ current and the enhancement in K+ current when pregabalin was applied in the continued presence of apamin (Apamin & PGB). D) Traces showing a control K+ current, the inhibited K+ current in the presence of 1 μM apamin and the enhanced K+ current observed when apamin and pregabalin were applied together. Apamin prevented the inhibitory effect of pregabalin but not the enhancement of K+ current. Several pharmacological experimental approaches were taken to characterise the biphasic actions of pregabalin and to determine the possible mechanism of action associated with the long-term K+ current modulation. Apamin, a toxin from the honeybee, was used to block small conductance Ca2+-activated K+ currents. Apamin (1 μM) caused a reduction in outward current at +40 mV from 2.64 ± 0.33 nA to 2.37 ± 0.39 nA, subsequent application of 250 μM pregabalin enhanced the current to 3.02 ± 0.39 nA (n = 11; p<0.01). No inhibitory effects were seen with pregabalin after treatment with apamin, suggesting that it is the apamin-sensitive Ca2+-activated K+ channels that are inhibited by pregabalin (Figure 12C &12D). Gabapentin can be transported into cells via the L α-amino acid transporter and may achieve intracellular concentrations 10–20 times the levels in the extracellular environment [25]. Furthermore, actions through intracellular signalling pathways and specifically protein kinase A, have been proposed for gabapentin [12]. To examine whether the long-term enhancement of K+ currents by pregabalin might involve intracellular sites of action, we applied pregabalin to the intracellular environments of DRG neurones via the patch pipette solution. Intracellular pregabalin (250 μM) evoked an increase in the K+ current that started to develop within the first minute of entering the whole cell recording configuration. After an initial increase that could reflect equilibration with the KCl-based patch pipette solution containing pregabalin, a slow sustained significant increase in the outward K+ current continued to develop over a 15 minute period (n = 10; p< 0.01; Figure 13A,13B). No similar change in K+ current was observed in control studies carried out in the absence of pregabalin (Figure 13C,13D). Figure 13 Intracellular application of pregabalin enhanced K+ currents in cultured DRG neurones. For these experiments pregabalin was included in the KCl-based patch pipette solution at a concentration of 250 μM. A) Bar chart showing the mean amplitudes of the K+ current recorded immediately after entering the whole cell recording configuration (Initial IK) and the maximum outward current recorded within 16 minutes of entering the whole cell recording configuration (Max IK). B) Traces of the first K+ current recorded using a patch pipette solution containing 250 μM pregabalin (Initial) and from the same cell the maximum outward K+ current recorded with intracellular pregabalin (Max). C) Bar chart showing control data recorded from neurones not exposed to pregabalin. Illustrated are the mean amplitudes of the K+ current recorded immediately after entering the whole cell recording configuration (Initial IK) and the maximum K+ outward current recorded within 16 minutes of entering the whole cell recording configuration (Max IK). D) Traces of the first K+ current recorded using the standard KCl-based patch pipette solution (Initial) and from the same cell the maximum outward current recorded under control conditions, within 16 minutes of entering the whole cell recording configuration (Max). Pertussis toxin pre-treatment results in the uncoupling of sensitive G-proteins from effector mechanisms, including voltage-activated Ca2+ and K+ channels. Additionally, pertussis toxin pre-treatment has previously been found to influence gabapentin actions on ICa. Pertussis toxin pre-treatment (500 ng/ml; 16–18 hours) prevented pregabalin-induced long-term K+ current enhancement in cultured DRG neurones (Figure 14A &14B). Figure 14 Pertussis toxin pre-treatment and intracellular (Rp)-cAMP prevented enhancement of K+ current by pregabalin. A) Bar chart showing the mean amplitude of K+ current recorded from DRG neurones pre-treated with pertussis toxin for 16–18 hours with 500 ng/ml (PTX Control) and after application of 250 μM pregabalin (PTX PGB), long term, up to 15 minutes monitoring of the current. B) Traces showing outward K+ currents recorded from a DRG neurone pre-treated with pertussis toxin, prior to pregabalin application (PTX Control) and 10 minutes after application of 250 μM pregabalin. C) Bar chart showing mean data obtained from neurones containing (Rp)-cAMP (30 μM), which was applied to the intracellular environment via the patch pipette solution. Data shows the mean K+ current amplitude recorded 1 minute and 5 minutes after entering the whole cell recording configuration (1 min; 5 min), after 5 minutes application of 250 μM pregabalin (PGB) and 10 minutes after removal of the pressure ejection pipette containing pregabalin. D) Traces from a single experiment showing the outward K+ currents at 1 and 5 minutes after entering the whole cell recording configuration and allowing entry of 30 μM (Rp)-cAMP in the DRG neurones. Also shown are the K+ current inhibited by 5 minutes application of 250 μM pregabalin (PGB) and the recovery of the K+ current after the pressure ejection pipette containing pregabalin was removed. Intracellular (Rp)-cAMP prevented the delayed long-term enhancement of the K+ current evoked by pregabalin. The possible role of cAMP-dependent protein kinase A (PKA) in the pregabalin-induced enhancement of K+ current was then assessed using the inhibitor (Rp)-cAMP [26]. Intracellular application of 30 μM (Rp)-cAMP applied via the KCl-based patch pipette solution had no effect over a 5 minute equilibration period on voltage-activated K+ current (n = 9). This may indicate that PKA has little or no basal or tonic activity on K+ currents in DRG neurones in culture. However, when pregabalin was applied to DRG neurones loaded with (Rp)-cAMP no long-term enhancement of the outward current was observed. Under these recording conditions pregabalin still produced some inhibition of the K+ current (Figure 14C &14D). These data provide evidence that the long-term modulation of voltage-activated K+ channels by pregabalin is dependent on PKA-mediated phosphorylation. Conclusions In conclusion, these results indicate that pregabalin acts via the same basic mechanisms as gabapentin to inhibit voltage-activated Ca2+ channels and that these inhibitory actions are independent of GABA receptor activation. Some features of the actions of pregabalin on intermediate size and large DRG neurones appear not to be seen with gabapentin. However, these distinct responses involve enhanced K+-evoked Ca2+ transients by pregabalin rather than inhibition of Ca2+ channels. Alpha2δ subunits of voltage-activated Ca2+ channels remain a possible site of action for both pregabalin and gabapentin. Dooley and colleagues showed that both gabapentin and pregabalin attenuated K+-evoked norepinephrine release from rat neocortical slices by inhibiting P/Q-type Ca2+ channels [10]. In cortical pyramidal neurones gabapentin predominantly works through L-type Ca2+ channels [27]. Our work with Bay K8644 indicates that in cultured DRG neurones gabapentin [5] and pregabalin act predominantly independently of L-type channels. A recent study has also indicated that acting via G-protein coupled GABAB receptors, gabapentin selectively inhibited N-type Ca2+ channels in hippocampal pyramidal neurones [15]. Interestingly, this selectivity of gabapentin seen in the hippocampus is different from the Ca2+ channel modulation seen with the GABAB receptor agonist, baclofen. Our previous investigation using "toxityping" showed that gabapentin inhibited a variety of Ca2+ channels in DRG neurones [11]. We conclude from all this work that gabapentin and pregabalin may have allosteric interactions with promiscuous α2δ Ca2+ channel subunits. These α2δ subunits are not specifically combined with distinct pore forming Ca2+ channel subunits (α1) in all neurones and may therefore inhibit pharmacologically diverse Ca2+ channels depending on the expression of Ca2+ channel subunits in different neurones. However, transfection studies have shown that oocytes expressing Ca2+ channels (Cav2.2) containing β1b and α2δ-1 or α2δ-2 subunits are insensitive to acute application of 50 μM gabapentin [28]. This work raises the possibility that other mechanisms independent of α2δ – Ca2+ channel subunits are involved in the modulation of neuronal excitability by gabapentin. Additionally, the modulation of voltage-activated Ca2+ channels by gabapentin and pregabalin acting through indirect mechanisms has been suggested. Candidates for such indirect mechanisms include the control of Ca2+ channel functional expression [29,30] and metabotropic mechanisms linked to pertussis toxin sensitivity and PKA activation [12]. In hippocampal pyramidal neurones, uncoupling G-proteins from metabotropic receptors with N-ethylmaleimide prevents modulation of K+ and Ca2+ channels by gabapentin [15]. However, in DRG neurones it is not clear how pertussis toxin influences the actions of gabapentin and pregabalin but it may involve disruption of multi-protein complexes of G-proteins and ion channel subunits after ADP-ribosylation of Gα. The role of any G-protein coupled receptors in either gabapentin or pregabalin responses in DRG neurones are not supported by our investigations with SCH-202676. Furthermore, receptor and direct G-protein involvement in pregabalin effects on Ca2+ channels also appears unlikely in DRG neurones. This is because in this study we found that intracellular flash photolysis of GTP-γ-S had no influence on pregabalin-evoked current inhibition and pregabalin did not alter responses to GTP-γ-S. Our findings are in agreement with those made in a previous study on cells expressing GABAB1a/B2 or GABAB1b/B2 receptor subunits. In this study GABA and the GABAB receptor agonist baclofen evoked [35S]-GTP-γ-S binding responses but even at high concentrations both gabapentin and pregabalin did not [31]. The experiments designed to assess potential roles of GABA receptors in the responses to pregabalin showed that GABA receptor desensitization or the blockade of GABAB receptors did not attenuate pregabalin actions. These findings add to the published studies that indicate that gabapentin and pregabalin effects are independent of GABA receptor activation at least in some preparations [31,32]. Specifically, in cultured DRG neurones gabapentin was previously found not to activate a Cl- conductance to alter membrane potential and input resistance and the GABAB receptor antagonist saclofen did not influence the inhibitory action of gabapentin on ICa [12]. Pregabalin has a higher affinity for α2δ subunits than gabapentin and in a number of studies is more effective. When applied alone pregabalin produced enhancement in Ca2+ flux in some neurones, an effect not seen with gabapentin. So, it was surprising to see in the imaging experiments that when gabapentin and pregabalin were applied together they produced only inhibitory effects. This may reflect the allosteric interactions between these drugs and α2δ subunits of Ca2+ channels as well as indirect modulation of Ca2+ dependent conductances and interactions with components of cell signalling. There appear to be some inconsistencies in the pregabalin data when its actions on voltage-activated Ca2+ currents are compared with effects on K+-evoked Ca2+ transients. This may in part be due to effects of pregabalin on membrane conductances in addition to voltage-activated Ca2+ currents. These effects may not be detected under voltage clamp recording conditions where Na+ and K+ currents are blocked to isolate Ca2+ currents. In the imaging experiments all voltage-activated conductances are intact and may be modulated by pregabalin. Alternatively, pregabalin could potentially have a direct or indirect influence on Ca2+-induced Ca2+ release from intracellular stores and alter Ca2+ homeostatic mechanisms. These effects may be modulated differently in different sub-populations of DRG neurones and so produce the mixed responses recorded in small, intermediate and large neurones in this study. Thus different actions of pregabalin may be detected using fura-2 fluorescence imaging but these additional mechanisms may not influence the measurements of Ca2+ currents. Although mixed responses to gabapentin were not previously identified, neither were clear effects on the amplitude of K+-evoked Ca2+ transients apparent in DRG neurones [11], although they were seen in differentiated F-11 cells [12]. These apparent anomalies seen with pregabalin do not appear to be due to drug effects on Ca2+-induced Ca2+ release or Ca2+ homeostatic mechanisms such as Na+/Ca2+ exchange. This conclusion was reached because pregabalin did not influence Ca2+ transients evoked by caffeine and still enhanced K+-evoked Ca2+ transients in choline chloride-based (low Na+) extracellular solution. The mixed responses to pregabalin seen in the Ca2+ imaging experiments prompted us to investigate the actions of pregabalin on voltage-activated K+ currents, which could be modulated to cause an increase in Ca2+ influx. This speculation was supported by the finding that application of the K+ channel inhibitor, TEA, enhanced K+-evoked Ca2+ transients. Stefani and colleagues have reported inhibition of outward K+ currents by gabapentin [18]. Additionally, in rat hippocampal and human neocortical brain slices gabapentin has been shown to inhibit K+-evoked [3H]-noradrenaline release. The activation of KATP channels is implicated in these responses because not only does glibenclamide, a KATP channel antagonist, attenuate the gabapentin response but also pinacidil, a KATP channel agonist, mimics the response and did not have additive effects with gabapentin [33]. Mixed or biphasic responses to gabapentin and pregabalin were seen, with both inhibition and enhancement of K+ currents observed. The inhibitory actions of both gabapentin and pregabalin on K+ currents recorded from DRG neurones may not reflect a direct action of these drugs on K+ channels. The effects of apamin, which prevented the inhibition of outward current by pregabalin indicated that small conductance Ca2+-activated K+ channels were involved in the response. This is consistent with the effects of gabapentin seen in isolated cortical neurones [18]. The pregabalin inhibitory action on voltage-activated Ca2+ channels may underlie the inhibition of outward K+ current. A reduction in Ca2+ influx would lead to reduced activation of Ca2+-activated K+ channels and therefore a smaller outward current. If this indirect effect of pregabalin resulted in a disproportionate reduction in Ca2+-activated K+ conductances this may result in prolonged depolarisation. In turn a prolonged depolarisation may maintain activation of Ca2+ channels that are insensitive to pregabalin and an increase in Ca2+ influx as seen in a sub-population of larger DRG neurones in the Ca2+ imaging experiments. It is not clear why gabapentin, which produced modest inhibition of K+ currents, did not also produce enhancement of K+-evoked Ca2+ transients. It may be explained by a balance between inhibition of Ca2+ influx through voltage-activated channels and modulation of Ca2+ flux through inhibition of K+ conductance. Consistent with this hypothesis, gabapentin appears to be less effective than pregabalin at modulating K+-evoked Ca2+ transients in DRG neurones. This is supported by our previous study that showed that gabapentin did not significantly alter the peak Ca2+ transients [11]. The delayed enhancement of voltage-activated K+ currents by gabapentin and pregabalin is a powerful mechanism for reducing cell excitability. The slow development of this response and its gradual decline suggested that intracellular signalling events were involved. Pregabalin produces enhancement of K+ currents when applied either inside or outside DRG neurones. The delay in the development of these responses is less with intracellular application of pregabalin, suggesting that it may have an intracellular site of action. This effect may also depend on drug uptake into cells via L α-amino acid transporters and the presence and activity of these transporters may greatly influence cell sensitivity to gabapentin and pregabalin. Although it is not clear how gabapentin and pregabalin might activate PKA, it is a candidate target site. Our previous work showed that inhibition of Ca2+ currents by gabapentin was sensitive to cAMP analogues that activated or inhibited PKA [12]. Similarly, in this present study (Rp)-cAMP blocked enhancement of K+ currents by pregabalin. In the literature there are a number of reports of PKA altering neuronal excitability and modulating K+ conductances. PKA is involved in pain signalling, playing a role in prostaglandin-induced activation and sensitization of DRG neurones [34]. PKA activity attenuates KV3.2 channel currents [35] and A-type current [36] and is involved in the inhibition of K+ conductances by prostaglandin E-2 [37]. Our results are not explained by these findings but PKA has also been implicated in presynaptic inhibition of GABA release [38], large conductance calcium – and voltage-activated K+ channel activity [39] and cannabinoid receptor mediated modulation of ion channels [40]. In the context of our project, of particular interest is the biphasic modulation in retinal cones of voltage dependent K+ and Ca2+ channels by a synthetic cannabinoid receptor agonist and the sensitivity of these responses to Wiptide, a PKA inhibitor [40]. However, the G-protein pharmacology is different in the cone cells suggesting different PKA activation pathways in DRG neurones exposed to pregabalin. There appears to be a considerable number of apparently conflicting effects mediated by PKA, although a number of different preparations have been studied. The concept of conditional protein phosphorylation such that initial phosphorylation state affects the sensitivity of different effector targets to subsequent activation [39] could determine responses to gabapentin and pregabalin acting through PKA. A future challenge will be to determine how PKA plays roles in activation and sensitization of DRG neurones and also plays a role in gabapentin and pregabalin responses to dampen down electrical excitability in the same neurones. Perhaps answers will come when we have a better understanding of the changes in neuronal phenotype that develop with pain disorders? An interesting feature of some actions of gabapentin and pregabalin is that their effects often outlast the period of drug delivery. Maintained intracellular signalling effects of these drugs and the delayed responses involving PKA modulation of Ca2+ and K+ conductances may underlie this characteristic. Gabapentin has previously been shown to attenuate high frequency action potential firing both in CNS neurones [41] and in cultured DRG neurones [5,12]. Pregabalin produces the same effect, dramatically but reversibly reducing the number of action potentials fired in response to 300 ms depolarising current commands. The underlying mechanisms involved in these reductions in electrical excitability could involve inhibition of voltage-activated Ca2+ channels with resulting reduced activation of Ca2+-activated K+ currents and effects on voltage-dependent ion channel availability. Although the delayed enhancement of voltage-activated K+ channels could contribute, this effect appears to develop over a longer period. These actions may contribute both to anticonvulsant effects of gabapentin and pregabalin as well as their therapeutic actions in pain disorders. It is particularly worth noting that the inhibition of Ca2+ influx is most consistently seen in small diameter cultured DRG neurones that are likely to be the pain fibres. Our data adds to previous work that has assessed the actions of gabapentin and pregabalin on K+-evoked neurotransmitter release in CNS neurones [10] and the pharmacology of pain in whole animal models [8,9,42-44]. The present work and these previous studies suggest that gabapentin and pregabalin mostly act through the same basic mechanisms. These mechanisms in cultured DRG neurones appear to involve allosteric inhibitory modulation of Ca2+ channels through drug interactions with α2δ subunits, reduced activation of Ca2+-dependent conductances and modulation of both Ca2+ and K+ channels through metabotropic mechanisms that involve PKA. Interestingly, both gabapentin and pregabalin inhibit release of sensory peptides (substance P and CGRP) this may provide a mechanism of action down-stream of ion channel modulation. This effect is only observed after inflammation and may contribute to their analgesic properties of gabapentin and pregabalin [45]. Ca2+ channels in DRG neurones are targets for antinociceptive agents but the pharmacology of these channels remains to be fully exploited in pain therapy [46,47]. Further studies on changes in ion channel subunit expression and alterations in intracellular signalling pathways in pain disorders may in the future open up new therapeutic opportunities. Methods Cell culture One to four-day old Sprague-Dawley rats were decapitated and dorsal root ganglia removed. DRG neurones were dissociated enzymatically (0.125% collagenase for 13 minutes and 0.25% trypsin for 6 minutes) and mechanically (trituration). Primary cultures of DRG neurones were plated on lamin-polyornithine coated coverslips and bathed in Ham's F-14 culture medium (Imperial Laboratories) containing 10% horse serum (Gibco), low NGF (20 ng/ml; Sigma), NaHCO3 (14 mM), streptomycin (50 μg/ml) and penicillin (50 IU/ml). The cultures were maintained for up to two weeks at 37°C in humidified air with 5% CO2. Cultures were re-fed with fresh media after 5 days. In some experiments DRG neurones were pre-treated with pertussis toxin (500 ng/ml; for 18 hours) to ADP-ribosylate the α subunits of certain G-proteins. This prevents pertussis toxin-sensitive G-protein being activated through a range of G-protein coupled receptors and inhibits coupling to voltage-activated Ca2+ channels and other potential effectors [12,48]. Electrophysiology and calcium imaging The whole cell patch clamp recording method and fura-2 Ca2+ imaging were used to measure the inhibitory actions of pregabalin and gabapentin on Ca2+ entry through voltage-activated channels. Initially, multiple firing properties of a sub-population of DRG neurones were studied using a patch pipette solution containing in mM: KCl, 140; EGTA, 5; CaCl2, 0.1; MgCl2, 2.0; HEPES, 10.0; ATP, 2.0. The extracellular solution containing in mM: NaCl, 130; KCl, 3.0; CaCl2 2.0; MgCl2, 0.6; NaHCO3 1.0, HEPES 10.0 and glucose 5.0. For recording Ca2+ channel currents the patch pipettes were filled with CsCl-based solution containing in mM: 140 CsCl, 0.1 CaCl2, 5 EGTA, 2 MgCl2, 2 ATP, 10 Hepes. The pH and osmolarity of the patch pipette solutions were corrected to 7.2 and 310–320 mOsm.l-1 with Tris and sucrose. The extracellular bathing solution used to study Ca2+ currents contained in mM: 130 choline chloride, 2 CaCl2, 3 KCl, 0.6 MgCl2, 1 NaHCO3, 10 HEPES, 5 glucose, 25 tetrethylammonium chloride, 0.0025 tetrodotoxin (Sigma). The pH and osmolarity of this extracellular bathing solution was corrected to 7.4 and 320 mOsml-1 with NaOH and sucrose respectively. The recording solutions used in these experiments were designed to attenuate voltage-activated Na+ and K+ currents and isolate voltage-activated Ca2+ currents. The range of values for the series resistance under our recording conditions was from ~8 to 15 MΩ. Pregabalin and other drugs were applied to the extracellular environment by low-pressure ejection from a blunt pipette positioned about 50–100 μm away from the cell being recorded. Voltage-activated Ca2+ currents were evoked by 100 ms voltage step commands applied every 30 s. Similar protocols were used to study the actions of pregabalin on voltage-activated K+ currents but NaCl-based extracellular bathing solution and KCl-based patch pipette solutions were used. In some experiments GTP-γ-S was photoreleased inside DRG neurones. Caged GTP-γ-S (100 μM; Molecular Probes) was included in the CsCl-based patch pipette solution. After obtaining control Ca2+ currents intracellular flash photolysis was achieved by flashing the neurone being studied (three 200 V flashes to produce ~15μM GTP-γ-S) using a XF-10 xenon flash lamp with a UG11 bandpass filter (Hi-Tech Scientific; [49]) All voltage-activated Ca2+ and K+ currents had scaled linear leakage and capacitance currents subtracted to obtain values for the net inward Ca2+ current or net outward K+ current. Data are given as mean ± standard error of the mean (s.e.m.) values and statistical significance was determined using a paired or independent Student's t test as appropriate. For Ca2+ imaging in cultured DRG neurones the cultures were incubated for 1 hour in NaCl-based extracellular solution contained (in mM): NaCl, 130; KCl, 3.0; MgCl2, 0.6; CaCl2, 2.0; NaHCO3, 1.0; HEPES, 10.0; glucose, 5.0 and fura-2AM, 0.01; (Sigma, 1 mM stock in dimethylformamide). The pH was adjusted with NaOH to 7.4 and the osmolarity to 310–320 mOsm with sucrose. The cells were then washed for 10–20 minutes with NaCl-based extracellular solution to remove the extracellular fura-2AM and this period allowed cytoplasmic de-esterification of the Ca2+ sensitive fluorescent dye. The cells were constantly perfused with NaCl-based extracellular solution (1–2 ml/min) and viewed under an inverted Olympus BX50WI microscope with a KAI-1001 S/N 5B7890-4201 Olympus camera attached. Some Ca2+ imaging experiments were carried out using a Ca2+-free NaCl-based solution (as standard NaCl-based extracellular solution but with no added CaCl2) or choline chloride-based solution (as standard NaCl-based extracellular solution but with choline chloride in place of NaCl). The fluorescence ratiometric images from data obtained at excitation wavelengths of 340 nm and 380 nm were viewed and analysed using OraCal pro, Merlin morphometry temporal mode (Life Sciences resources, version 1.20). The DRG neurones were stimulated with NaCl-based extracellular solution containing high K+ (30 mM), which produced depolarisation, activation of voltage-gated Ca2+ channels and large transient increases in intracellular Ca2+. Three consistent transient increases in intracellular Ca2+ could be obtained in a single experiment on cultured DRG neurones [11]. The actions of pregabalin and gabapentin (2.5–250 μM) were investigated on the response to the second stimulus in DRG neurones. The actions of pregabalin and gabapentin on the Ca2+ transient amplitude, duration at 1/2 peak amplitude and total Ca2+ flux were measured. All experiments were conducted at room temperature and data are expressed as means ± s.e.m. List of abbreviations DRG, Dorsal root ganglion. cAMP, Cyclic adenosine monophosphate GABA, Gamma aminobutyric acid GBP, Gabapentin GTP-γ-S, Guanosine 5'-o(3-thio)triphosphate ICa, Calcium current NGF, Nerve growth factor PGB, Pregabalin PKA, Protein kinase A PP, Pre-pulse (Rp)-cAMP, (R)-adenosine, cyclic 3', 5'-(hydrogenphosphorothioate) triethylammonium Authors' contributions All the authors of this manuscript contributed to electrophysiological and Ca2+ imaging experiments. DM and RHS designed and conducted the experiments on K+ currents and wrote the first draft of this manuscript. Acknowledgements The authors thank Pfizer both in the USA and UK (formerly Parke-Davis) for support and Ms Jennifer Spratt for constructive comments on the manuscript. ==== Refs Backonja M-M Symptomatic treatment of chronic neuropathic pain with gabapentin clinical practice and research perspective Current Neuropharmacology 2003 1 199 202 Megna JL Iqbal MM Aneja A Pharmacology and therapeutics of gabapentin in the treatment of Psychiatric Disorders; Present and Future Perspectives Current Neuropharmacology 2003 1 187 97 Leiderman DB Gabapentin as add on therapy for refractory partial epilepsy: results of five placebo controlled trials Epilesia 35 s75 s76 Taylor CP Gee NS Su T-Z Kocsis JD Welty DF Brown JP Dooley DJ Boden P Singh L A summary of mechanistic hypotheses of gabapentin pharmacology Epilepsy Res 1998 29 233 49 9551785 10.1016/S0920-1211(97)00084-3 Scott RH Martin DJ McClelland D Cellular actions of gabapentin and related compounds on cultured sensory neurones Current 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==== Front BMC PharmacolBMC Pharmacology1471-2210BioMed Central London 1471-2210-4-161530169210.1186/1471-2210-4-16Research ArticleExtrinsic factors regulate partial agonist efficacy of strychnine-sensitive glycine receptors Farroni Jeffrey S [email protected] Brian A [email protected] Department of Medical Pharmacology & Toxicology, Texas A&M University System Health Science Center, College Station TX 77843, USA2 Department of Marine Biology, Texas A&M University at Galveston, Galveston TX 77553, USA3 Department of Physiology & Pharmacology, Wake Forest University School of Medicine, Winston-Salem NC 27157, USA2004 9 8 2004 4 16 16 3 6 2004 9 8 2004 Copyright © 2004 Farroni and McCool; licensee BioMed Central Ltd.2004Farroni and McCool; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Strychnine-sensitive glycine receptors in many adult forebrain regions consist of alpha2 + beta heteromeric channels. This subunit composition is distinct from the alpha1 + beta channels found throughout the adult spinal cord. Unfortunately, the pharmacology of forebrain alpha2beta receptors are poorly defined compared to 'neonatal' alpha2 homomeric channels or 'spinal' alpha1beta heteromers. In addition, the pharmacologic properties of native alpha2beta glycine receptors have been generally distinct from receptors produced by heterologous expression. To identify subtype-specific pharmacologic tools for the forebrain alpha2beta receptors, it is important to identify a heterologous expression system that closely resembles these native glycine-gated chloride channels. Results While exploring pharmacological properties of alpha2beta glycine receptors compared to alpha2-homomers, we found that distinct heterologous expression systems appeared to differentially influence partial agonist pharmacology. The β-amino acid taurine possessed 30–50% efficacy for alpha2-containing receptor isoforms when expressed in HEK 293 cells. However, taurine efficacy was dramatically reduced in L-cell fibroblasts. Similar results were obtained for β-alanine. The efficacy of these partial agonists was also strongly reduced by the beta subunit. There were no significant differences in apparent strychnine affinity values calculated from concentration-response data between expression systems or subunit combinations. Nor did relative levels of expression correlate with partial agonist efficacy when compared within or between several different expression systems. Finally, disruption of the tubulin cytoskeleton reduced the efficacy of partial agonists in a subunit-dependent, but system-independent, fashion. Conclusions Our results suggest that different heterologous expression systems can dramatically influence the agonist pharmacology of strychnine-sensitive glycine receptors. In the systems examine here, these effects are independent of both absolute expression level and any system-related alterations in the agonist binding site. We conclude that complex interactions between receptor composition and extrinsic factors may play a significant role in determining strychnine-sensitive glycine receptor partial agonist pharmacology. ==== Body Background It has been well established that the amygdala is important in the acquisition and maintenance of fear/anxiety-related behaviors [1]. Strychnine-sensitive glycine receptors have recently been found in the adult rat basolateral amygdala (BLA) using whole cell and intracellular electrophysiology [2,3]. Reverse transcription polymerase chain reaction on whole BLA tissue and single cells revealed a prominent expression of α2 mRNA; and these receptors are likely to be α2β heteromers due to their low picrotoxin sensitivity [4]. This finding is consistent with prominent BLA 'general' immunoreactivity for α/β subunit protein but no apparent α1-specific protein expression [3]. A similar enrichment of α2/β heteromers is also evident in striatal cholinergic interneurons [5]. It is quite possible then that the α2β strychnine-sensitive glycine receptors present in the adult BLA and other forebrain areas represents a receptor population that could be functionally distinguished from those found in the spinal cord. Because the BLA regulates a number of anxiety- or fear-related behaviors [6], it is possible that this population of strychnine-sensitive glycine receptors may represent a novel therapeutic target for anxiety disorders. To insure that novel α2β compounds possess an appropriate therapeutic index, the pharmacology of these forebrain glycine receptors must be elucidated and extensively compared with the spinal isoform. There have been conflicting reports regarding the details of glycine receptor pharmacology when expressed in heterologous systems. For example, taurine acts as a partial agonists (ca. 50% efficacy compared to glycine) for GlyRα1 expressed in Xenopus ooctyes [7] whereas it shows nearly full agonist efficacy for GlyRα1 expressed in HEK 293 cells [8]. Compared to GlyRα1, taurine efficacy is even weaker for GlyRα2 (ca. 5–10% efficacy) when expressed in Xenopus oocytes [7]. However, native GlyRα2β receptors expressed by BLA neurons possess >50% efficacy for taurine and almost full efficacy for β-alanine [2]. While these results might initially be dismissed as expression system-dependent phenomena, brain region-specific effects are also evident in the literature. Taurine has markedly different efficacies at glycine receptors expressed by isolated adult lateral/basolateral amygdala neurons [2], adult hypothalamic magnocellular neurons [9], and juvenile spinal cord neurons [10]. It is therefore possible that the mechanisms regulating brain region-specific effects are related to those governing the divergence among heterologous expression systems. However, such mechanisms have not been systematically investigated, despite their potential usefulness in understanding region-to-region pharmacologic heterogeneity evident for some native receptors. This study utilizes whole-cell patch clamp electrophysiology to examine the influence of distinct heterologous expression systems on the β-amino acid pharmacology of glycine receptors composed of distinct subunit combinations. We have focused on the α2 and α2β receptors since these appear to be the predominate isoforms found in the embryonic and adult forebrain, respectively. Our results provide potentially important insight into the types of mechanisms that may govern brain region-to-brain region variation in glycine receptor pharmacology. Several aspects of this work have appeared in abstract form [11,12]. Results Subunit- and system-dependent effects on glycine pharmacology Given the variation of glycine receptor partial agonist pharmacology in the literature, we specifically sought to identify any role that expression system may play in their pharmacological profiles. First, glycine concentration-response relationships were established for GlyRα2, and GluRα2/β in HEK-293 cells and in L-cell fibroblasts. Glycine-gated responses for each receptor isoform were elicited in a dose-dependent manner in both cell types (Fig. 1A). The apparent EC50 of glycine HEK cells was 221 μM and 269 μM for α2 (n = 4–6) and α2β (n = 7–8), respectively. GlyR subunits expressed in L-cells displayed a similar pharmacological profile. However, the apparent glycine EC50 of both GlyRα2 (446 μM, n = 5–7) and GlyRα2β (667 μM, n = 4–8) appeared lower than apparent affinities for the same subunits when expressed in L-cells. Two-way ANOVA on the Log (EC50) values (Table 1) indicated a significant effect of system (F = 20.01, P < 0.001). However, the presence of the β-subunit did not significantly affect glycine apparent affinity in either system nor was there a significant interaction between system and subunit composition. These results indicate that glycine is less potent for receptors expressed in L cells compared to HEK cells. Figure 1 Glycine receptors expressed show expression-system dependent agonist pharmacology. (A) Glycine has a reduced potency in L-cells compared to HEK cells. Glycine current responses were plotted versus the log concentration of glycine and normalized to a maximal concentration of glycine (3–10 mM). Data are presented as mean ± SEM; 4 ≤ n ≤ 9 cells for each concentration. Concentration response relationships for GlyRα2 (□; EC50 = 221 μM) and GlyRα2β (○; 269 μM) in HEK cells and in L-cells (GlyRα2, ■, 446 μM; GlyRα2β, ●; 667 μM) were derived from logistic equation fits to individual cells. (B) β-alanine has both reduced apparent affinity and efficacy for most glycine receptor isoforms transiently expressed in L-cells compared to HEK 293 cells. β-alanine apparent potency in HEK 293 cells was 717 μM for GlyRα2 (□, n = 6) and 560 μM for GlyRα2β (○, n = 7). For L-cells, β-alanine potency for GlyRα2 (■, n = 5–8) was 1.61 mM and 1.79 mM for GlyRα2β (●, n = 7). Current responses were normalized to a maximal concentration of glycine (10 mM). Note the reduced apparent efficacy of α2β receptors compared to the α2 homomeric isoforms. (C) Taurine has both reduced apparent affinity and efficacy to GlyRs transiently expressed in L-cells compared to HEK 293 cells. Taurine concentration-response relationship in HEK 293 for GlyRα2 (□; 442 μM, n = 5–6) and GlyRα2β (○; 1.25 mM, n = 3–5). Taurine concentration-response relationship in L-cells yielded GlyRα2 (■, n = 4) and GlyRα2β (●, n = 7) potencies estimated at ≥ 3 mM. Current responses were plotted versus the log concentration of taurine and normalized to a maximal concentration of glycine. Table 1 Agonist Pharmacology in HEK and L-cells. GlyR α2 GlyR α2β System log EC50 EC50 (mM) Efficacya log EC50 EC50 (mM) Efficacy Glycine HEK -3.67 ± 0.15 0.22 -- -3.55 ± 0.06 0.28 -- L-cells -3.36 ± 0.05 0.43 -- -3.17 ± 0.04 0.67 -- β-Alanine HEK -3.12 ± 0.05 0.76 0.80 ± 0.06 -3.24 ± 0.03 0.57 0.55 ± 0.07 L-cells -2.71 ± 0.08b 1.93 0.39 ± 0.08 -2.59 ± 0.13 2.59 0.25 ± 0.05 Taurine HEK -3.13 ± 0.12 0.74 0.48 ± 0.12 -2.67 ± 0.12 2.20 0.32 ± 0.05 L-cells ≥-3.00 ± 0.11c 1.00 0.06 ± 0.01 ≥-2.97 ± 0.20c 1.10 0.05 ± 0.01 a – efficacy relative to maximal glycine response b – p < 0.0001 for system but not subunit using two-way ANOVA c – estimate due to low efficacy Subunit- and system-dependent effects on β-Alanine pharmacology With the glycine pharmacological profile established, we next examined the pharmacology of the partial agonists, β-alanine and taurine. The efficacy and potency of these β-amino acids were compared to glycine by normalizing the current response at each concentration to a maximal glycine response in that same cell. In HEK cells (Fig. 1B), the average β-alanine EC50 values calculated from individual cells were 770 μM (n = 6) and 570 μM (n = 7) for α2 and α2/β receptors, respectively. In L-cells, β-alanine also elicited currents in a dose-dependent manner. And, like glycine, β-alanine appeared to be less potent in these cells compared to HEK cells. EC50 values for α2 and α2/β receptors were 2.0 mM (n = 8) and 2.9 mM (n = 7), respectively. Two-way ANOVA on LogEC50 values from these studies indicate a significant effect of the expression system on β-alanine potency (F = 43.52, P < 0.0001). There was a trend for the presence of the β-subunit to influence potency but this was not significant nor was there any significant interaction between expression system and subunit composition. β-alanine efficacy was also examined in these same experiments by normalizing the maximal β-alanine response as a fraction of a maximal glycine response. In HEK cells, the α2 and α2β isoforms had efficacies of 80 ± 6% and 55 ± 7% of the maximal glycine response, respectively. A similar trend was noted in L-cells with the α2 and α2β isoform with β-alanine efficacies being 39 ± 8% and 25 ± 5% of the maximal glycine response. Two-way ANOVA analysis of these data indicate that both expression system and subunit composition had a significant influence on β-alanine efficacy (F = 27.6, P < 0.0001 and F = 7.9, P < 0.01 respectively). There was no significant interaction between these variables. These data demonstrate that the presence of the β subunit reduced β-alanine efficacy of α2-containing receptors and that this efficacy was substantially smaller L-cells compared to HEK cells. Subunit- and system-dependent effects on taurine pharmacology Similar analysis of taurine pharmacology in HEK and L-cells revealed more dramatic effects of system and subunit on this partial agonist (Fig. 1C). In HEK cells, the apparent EC50 for taurine was 501 μM for GlyRα2 (n = 9) and 2 mM for GlyRα2β (n = 7). Because of its remarkably low efficacy in L-cells (see below), we can only provide estimates of taurine potency in this expression system. Regardless, apparent taurine affinity for both GlyRα2 and GlyRα2β expressed in L cells were ~3 mM for both isoforms (n = 4 and 7, respectively). We did not compare L cell data with that obtained from HEK cells due to the uncertainty surrounding the fits. However, there was no significant difference in apparent taurine potency between the α2 and α2β receptors expressed in HEK cells (P >> 0.05, t-test). Taurine efficacy was obviously quite different between the two expression systems. In HEK cells, taurine efficacy was 48 ± 12% of glycine for GlyRα2 and 32 ± 4% of glycine for the GlyRα2β isoform. Efficacy for these same receptors was reduced to approximately 6 ± 1% and 5 ± 0.7% of glycine when they were expressed in L-cells in these particular studies. The system difference was significant with two-way ANOVA (F = 17.4, P < 0.001) with no substantial effects of subunit composition or interactions between these variables. Expression level and system-dependent pharmacology The preceding results suggest that there may be a complex interaction between subunit composition and the expression system in which the receptor is produced. Specifically, the system-dependent agonist pharmacology could be related to differences in the relative expression levels between various systems. Expression level has clearly been demonstrated to influence agonist pharmacology for G protein-coupled receptors (e.g. [13]), where the levels of G-protein bound to receptor and thus the relative levels of high affinity receptor can vary from system to system. However, the influence of expression level on ligand-gated channel function has not been extensively explored (see Discussion). Unfortunately, it is problematic to compare expression levels between HEK and L-cells since the relative efficiency of transfection varied widely between these systems. Indeed, liposome-mediated transfection is remarkably efficient in HEK 293 cells (70–90% of cells based on GFP fluorescence) but only marginally effective in L-cells (10–20% of cells, not shown). To get around these differences in transfection efficiency, we examined the relative expression level of GlyRα2 protein using western analysis of total lysate derived from the same number of GFP+ HEK 293 or L cells from transfected cultures (Fig. 2A). For this experiment, cells were harvested under native conditions, GFP+ cells were counted, and volumes of lysate corresponding to equivalent numbers of GFP+ cells was loaded onto the gel. Western blots from two separate experiments demonstrate that transfected HEK 293 cells expressed 4- to 5-fold more GlyRα2 protein than transfected L-cells. The mean optical density from the two experiments was 83 ± 2 units for HEK cells and 17 ± 2 units for L-cells. Figure 2 Relative expression levels does not influence taurine efficacy. (A) HEK and L-cells were co-transfected with the GlyRα2 subunit and GFP. Relative expression levels of α2-protein were examined using western blot analysis of total lysate from equal numbers of GFP+ HEK and L-cells. α2 protein was 4- to 5-fold greater in GFP+ HEK cells than in GFP+ L-cells. (B) Maximal glycine conductance across all experiments was significantly lower in L-cells compared to HEK cells, although only by about 2-fold. This may indicate that a significant amount of α2 protein in HEK cells (A) is present in a non-functional form or not associated with the plasma membrane. (C) Glycine current density vs. taurine efficacy in different cell lines expressing α2 (open symbols), α2β glycine receptors (closed symbols), and in isolated neurons from the adult rat basolateral amygdala. The correlation coefficient between glycine current density and taurine efficacy was 0.14 and was not significantly greater than zero (P >> 0.05). There was also no correlation (R2 = 0.01 to 0.3) between glycine current density and taurine efficacy when comparing individual cells within each of these systems. Maximal conductance is an independent measure of functional expression and was also larger for both α2 and α2β receptors expressed in HEK cells compared to receptors expressed in L cells (Fig. 2B). Across all experiments where maximal glycine concentrations were assayed, the conductance of α2 receptors expressed in L-cells was 65 ± 11 nS and was 114 ± 21 nS in HEK cells. Similarly, L-cells expressed α2β receptors at 42 ± 9 nS while HEK cells expressed this isoform at 104 ± 17 nS. Two-way ANOVA using subunit and system as variables revealed a significant effect of system (F = 15.4, P < 0.001) but not subunit, nor was there a significant interaction between variables. Results from both westerns and functional experiments therefore indicate that relative expression levels of glycine receptor were different between HEK and L-cells. To further explore the interaction between expression level and partial agonist efficacy, both current density and taurine efficacy were compared for α2 and α2β glycine receptors in a number of different heterologous systems, as well as for native receptors expressed in rat lateral/basolateral amygdala. In addition to HEK and L-cells, the heterologous systems included mouse 3T3 fibroblasts and MDCK kidney cells. α2β receptors expressed in mouse 3T3 fibroblasts had twice the current density (121 ± 34 pA/pF) of the mouse L-cells (59 ± 19 pA/pF) but had a similar taurine efficacy (13 ± 8% of glycine in 3T3 cells versus 8 ± 1% in L-cells). Similarly, α2β receptors expressed in HEK293 cells had a current density similar to GlyRs expressed in 3T3 fibroblasts (115 ± 11 pA/pF) but had a taurine efficacy compared to glycine of 48 ± 3%. This efficacy was similar to glycine receptors expressed by acutely isolated adult rat basolateral amygdala neurons (46 ± 5% of glycine) although the current density in this native system was only 57 ± 14 pA/pF. Note that the channels expressed by these neurons are composed primarily of α2+β subunits [4]. Canine kidney MDCK cells expressed the lowest α2β current density (15 ± 5 pA/pF); yet the channels expressed by this system had the highest taurine efficacy of any cell tested (101+6%). For α2 GlyRs, the rank order of glycine receptor density was 3T3 (111 ± 21 pA/pF)> HEK cell (90 ± 11 pA/pF)> L-cell (50 ± 9 pA/pF); while the rank order of taurine efficacy for these same receptors was HEK (74 ± 9%)> 3T3 (23+7%)> L-cells (11 ± 2%). Across all subunit combinations and systems, there was no significant correlation (R2 = 0.14, P >> 0.05) between taurine efficacy and glycine current density (Fig. 5C). Indeed, no correlation between expression level and taurine efficacy was evident within any given population of cells whether the receptors were expressed in native or heterologous systems. For example, the correlation coefficients for α2β receptors between taurine efficacy and glycine current density in individual systems were 0.11, 0.13, 0.19, 0.05, and 0.31 for HEK, L-cells, 3T3 cells, MDCK cells, and amygdala neurons, respectively (P >> 0.05). Thus, while there is clearly a difference in expression level between both the systems as well as between individual cells in a given system, this particular characteristic cannot account for the apparent taurine efficacy. The agonist-binding site is not affected by expression system There are a variety of possible mechanisms to account for the disparities in partial agonist pharmacology between two expression systems. One way to address this is to examine competitive antagonist binding properties in HEK and L-cells. We therefore examined the potency of the glycine receptor competitive antagonist strychnine in both systems. Following a 30 second pretreatment with the antagonist [2], we co-applied strychnine and an EC50 concentration of glycine. The strychnine KB was estimated for HEK and L-cells expressing either the GlyRα2 or GlyRα2+β subunits using the Cheng-Prusoff relationship (see Methods). This relationship takes into account the divergent Hill-slope and potencies for glycine found in these two expression systems. Receptors composed of the GlyRα2 subunit (Fig. 4A) had very similar KB values when expressed in either HEK (KB = 49 ± 8 nM, n = 11) or L-cells (KB = 38 ± 7 nM, n = 16; Fig. 4C). The same was true for cells expressing the GlyRα2β subunits where strychnine apparent affinity was 32 ± 7 nM (n = 10) in HEK cells and 38 ± 8 nM (n = 10) in L-cells. Two-way ANOVA did not reveal any significant effect of either system or subunit composition. Since strychnine is a competitive antagonist and site-directed mutation studies suggests that strychnine and glycine interact with overlapping regions of the receptor [14-16], our results strongly suggest that functional strychnine affinity, and hence the general structure of the agonist binding pocket, was not substantially influence by expression system. Figure 4 The association of glycine receptors with the tubulin-cytoskeleton may influence partial agonist efficacy. (A) Tubulin depolymerization with colchicine decreased both taurine and β-alanine efficacy of α2β glycine receptors expressed in HEK 293 cells. Cells were treated with 100 μM colchicine or γ-lumicolchicine at 37°C for 30 minutes. The graph shows the partial agonist efficacy as a fraction of the maximal glycine response. For taurine (□), colchicine treatment reduced apparent efficacy from 33 ± 6% in control cells (n = 8) to 13 ± 3% in treated cells (n = 10). γ-lumicolchicine, an inactive analogue of colchicine, had no effect on taurine efficacy (29 ± 9%, n = 5, ** – P < 0.01 from ANOVA). For β-alanine (■), efficacy was reduced from 70 ± 7% in vehicle-treated cells (n = 8) or 67 ± 8% in γ-lumicolchicine-treated cells (n = 5) to 49 ± 6% in cholchicine-treated cells (n = 10, * – P < 0.05, ANOVA). (B) Colchicine treatment does not influence partial agonist efficacy of the GlyRα2 homomeric channels. Taurine (□) efficacy was 34 ± 15% in control GlyRα2 cells (n = 4) and was 38 ± 10% in colchicine-treated cells (n = 5, P >> 0.05 t-test). Similarly, β-alanine efficacy was 72 ± 12% and 86 ± 8% in the same control and treated cells, respectively (P >> 0.05, t-test). (C) Colchicine treatment decreases β-alanine efficacy in L-cells expressing GlyRα2β heteromeric channels (■), but not those expressing GlyRα2 homomeric channels (□). For the GlyRα2β channels, colchicine treatment significantly reduced efficacy from 23 ± 2% (n = 7) to 12 ± 3% (n = 3, P < 0.05 t-test). (D) Gephyrin-like immunoreactivity was detected in both cells lines using 20 and 40 μg of whole cell lysate. * – denotes expected gephyrin mobility (approx. 100 kD). Cytoskeletal components influence glycine receptor pharmacology A third possible mechanism for reduced efficacy in L-cells compared to HEK cells or neurons could be related to intracellular factors that influence channel gating [17]. This hypothesis was examined by disrupting the cytoskeletal protein tubulin, which has been shown to be important for glycine receptor localization [18]. Direct application of 100 μM colchicine did not elicit any membrane currents. Furthermore, acute application of 100 μM colchicine and an EC50 concentration of glycine (300 μM) did not significantly affect glycine-gated currents themselves. Glycine currents were 17.3 ± 2.3 pA/pF while glycine+colchicine currents were 16.7 ± 2.2 pA/pF (p > 0.5, paired two-tail t-test, n = 7). The relative efficacy of β-alanine and taurine was examined in HEK cells expressing α2β subunits following 30 min incubation with 100 μM colchicine at 37°C, enough time to allow irreversible tubulin disruption [19]. As an additional control, γ-lumicolchicine, an inactive analog of colchicine [20], was also used to treat α2β-expressing HEK cells (Fig 4A). These brief treatments had no obvious effect on the survival of untransfected cells. There was a trend for colchicine treatment to reduce the overall current density at 300 μM glycine, 56.7 ± 9.1 pA/pF in control cells (n = 8), 41.2 ± 2.3 pA/pF in colchicine-treated cells (n = 10), and 50.1 ± 9.9 pA/pF (n = 6) in γ-lumicolchicine-treated cells; however, this was not significant (p > 0.05, ANOVA) and was probably not related to any direct action of colchicine given that the glycine current density was also slightly reduced in α2β-expressing cells exposed to γ-lumicolchicine compared to controls. However, the efficacy of both taurine (p < 0.01, One-way ANOVA) and β-alanine (p < 0.05, ANOVA) were significantly decreased by colchicine but not γ-lumicolchicine treatment. Taurine efficacy was 33 ± 6% of glycine in controls, 13 ± 3% following colchicine, and 28 ± 3% following γ-lumicolchicine. Similarly, β-alanine efficacy was 70 ± 7% of glycine in controls, 49 ± 6% following colchicine, and 72 ± 7% following γ-lumicolchicine. Similar treatment of α2-expressing HEK cells with colchicine (Fig. 4B) did not reveal any significant effect on glycine current density (54 ± 14 pA/pF in controls, 60 ± 15 pA/pF in treated), on taurine efficacy (34 ± 16% in controls vs. 38 ± 10% in treated), or on β-alanine efficacy (71 ± 12% in controls vs. 86 ± 8% in treated). Cholchicine treatment also significantly reduced β-alanine efficacy in L-cells expressing GlyRα2β (23 ± 2% in controls vs. 12 ± 3% in treated, P < 0.05, t-test) but not in GlyRα2-expressing L-cells (Fig. 4C). We did not attempt to examine taurine in L-cells treated with colchicine given the exceptionally low efficacy of receptors expressed in this cell line. Because the glycine receptor- and tubulin-binding protein gephyrin provides an obvious link between the receptor and the tubulin cytoskeleton, we used western analysis of HEK and L-cell lysates with a gephyrin monoclonal antibody specific for the C-terminus. These experiments revealed that gephyrin-like immunoreactivity was expressed in both expression systems (Fig. 4D). Notably, a ca. 100 kD band dominated the HEK cell gephyrin immunoreactivity, while multiple bands of varying intensity could be seen in lysate from L-cells. When taken with our colchicine data, differences in glycine receptor pharmacology between α2β receptors expressed in HEK and L-cells may be partially due to distinct, system-dependent interactions with distinct isoforms of the cytoskeletal protein gephyrin. Discussion We have expressed several the 'embryonic' (α2 homomeric) and 'forebrain' (α2β heteromeric) isoforms in two distinct expression systems to understand the influence of endogenous and exogenous factors on receptor partial agonist pharmacology. Although the pharmacology of the 'embryonic' GlyRα2 isoform and the 'adult spinal' isoform (GlyRα1β) have been explored more frequently in the literature, the pharmacology of GlyRα2β receptors has remained largely unexplored. Despite this, there is strong evidence that the adult 'forebrain' isoforms, specifically in the rat basolateral amygdala, is indeed α2β [4]. The current study indicates a general trend for decreased apparent affinity and reduced relative efficacy of agonists when receptors consist of the α2β subunits compared to their homomeric α2 counter parts. We were particularly surprised to find that receptors expressed in different expression systems possessed markedly different partial agonist efficacies. The remainder of our study focused on identifying extrinsic factors that influence difference in ligand-gated receptor pharmacology in distinct expression systems. While differences in efficiency of cDNA expression/transfection between systems could explain such differences, the expression levels of GlyRα2β receptors measured by current density was not correlated with taurine efficacy across several different cell types or within any given system. Importantly, the efficacy of β-alanine and taurine in HEK cells agree with previous findings where cells expressing GlyRα2 show almost full efficacy for taurine and β-alanine [21]. Similarly, distinct ligand binding characteristics of receptors expressed in different expression systems seemed to be another possible mechanism governing agonist efficacy or potency. For the glycine receptor, the binding site for the competitive antagonist strychnine is believed to be adjacent to the agonist-binding site, sterically hindering agonist binding. A gross alteration in the agonist binding pocket, particularly one that hindered agonist binding, would most likely affect strychnine binding as well. To examine this, strychnine KB was calculated for GlyRα2 and GlyRα2β isoforms expressed in both HEK and L-cells. In order to decrease the error in estimating KB, a derivation of the Cheng-Prusoff equation was used that takes in account variations in the slopes of the inhibition curves [22]. Differences in KB were negligible between expression systems, indicating the strychnine binding site, and presumably the agonist binding site, was altogether similar in these different systems. Differences in pharmacology between systems therefore cannot be explained by substantial alterations in the agonist/competitive antagonist binding pocket. Receptor gating is another mechanism by which receptor function may be altered. Cytoskeletal elements have been shown to play a crucial role in neurotransmitter receptor clustering [17] and may have a role in receptor function as well. For example, cytoskeletal stabilization has been shown to reduce Ca++-dependent inactivation of Ca++ channels in snail ganglia [23]; and, actin has been shown to modulate several different types of membrane ion channel [24-26]. Cytoskeletal depolymerization has also been found to inhibit the function of GABAA receptors, which share significant sequence homology and functional characteristics with strychnine-sensitive glycine receptors [27]. And the tubulin-gephyrin-glycine receptor interaction is critical for establishing functional glycinergic synapses [28]. The current study suggests that cytoskeletal elements may play a functional role in α2β glycine receptor pharmacology as well. β-containing glycine receptors are intimately associated with the tubulin-associated protein gephyrin [29] via the gephyrin binding site that lies within the intracellular domain of this subunit [30]. In our studies, the efficacy of both taurine and β-alanine were reduced in cells expressing GlyRα2β subunits. This despite the finding that gephyrin-like immunoreactivity in L-cells was apparently distinct from that in HEK cells, suggesting that distinct cytoskeletal components in these systems may have profound influence over GlyR α2β pharmacology. This is further supported by suggestions that gephyrin may exist in multiple, tissue-specific isoforms with potentially distinct functional roles [31-33]. It should be noted however that colchicine treatment of α2β-expressing HEK cells did not suppress partial agonist efficacy to a level that approached that found in L-cells or 3T3-fibroblasts. Given that colchicine had no perceptible effect of α2-homomeric channels expressed in HEK cells, our results suggest that additional system-dependent factors may have a more pronounced influence on the partial agonist pharmacology of strychnine-sensitive glycine receptors. Conclusions It is of particular interest that the β subunit appears to play a functional role in the pharmacology of α2-containing receptors regardless of expression system. For example, the beta subunit decreased the apparent efficacy of the partial agonists. Since the β-subunit itself does not appreciably interact with the competitive antagonist strychnine [34], these results are at least consistent with some allosteric interaction between the β-subunit and the agonist binding site present the α subunit. This may indicate that α2β glycine receptors in the forebrain may be distinguishable from other receptor isoforms given the appropriate pharmacologic agent. Although cytoskeletal components potentially play some role for these 'forebrain' receptors, there appear to be other 'extrinsic' factors governing expression system-dependent effects on agonist pharmacology. Since it is conceivable that such factors may be differentially distributed between different forebrain regions, the large apparent differences between glycine receptor pharmacology reported by various studies may not necessarily depend upon differential expression of glycine receptor subunits per se. At the very least, our findings suggest that great care should be taken when utilizing different expression systems to develop screens for novel pharmacophores acting on this receptor. Methods Cell culture and transfection HEK 293 (TSA 201; gift from Michael J. Davis, Dept. Med. Physiol., Texas A&M Univ. Health Science Center, College Station, USA), mouse L-cells (NCTC-929; American Type Culture Collection), NIH/3T3 fibroblasts (ATCC), and MDCK cells (gift from Alan Parrish, Dept. Med. Pharmacol. & Toxicol., Texas A&M Univ. Health Sci. Center) were grown in Dulbecco's modified Eagle's medium (DMEM, SIGMA) with 10% fetal bovine serum (HyClone Laboratories, Logan, UT, USA) and 1X penicillin/streptomycin (Life Technologies) on Thermanox cover slips in 35 mm culture dishes. Cells were transfected during log-phase growth (30–50% confluent) using the Superfect reagent (Qiagen, Valencia, CA, USA), according to manufacturers instructions. Rat glycine receptor α2 and β subunits were cloned previously [4]. Briefly, a total of 2–3 μg of plasmid constructs was added to 100 μL serum-free media along with 10 μL Superfect reagent, vortexed for several seconds, and incubated for 10 minutes to allow DNA/liposome formation. Standard media (600 μL) was added and this mixture applied the cells and incubated for 2–3 hours at 37°C. The cells were subsequently washed twice with phosphate-buffered saline, fresh media was applied and cells were then incubated for 24–48 hours prior to recording. Cells were co-transfected with green fluorescent protein (pEGFP-C1, Clontech, Pal Alto, CA, USA) to identify transfected cells before recording. Mass ratios of 1:1:5 were used in the GFP:α2:β transfections. An equal mass of the cloning vector pCI (Promega Corp) replaced the β subunit when examining α2 homomeric channels. Electrophysiology Whole cell recordings were performed at room temperature using standard patch-clamp techniques and the axopatch-1D amplifier (Axon Instruments, Inc., Foster City CA, USA) in the voltage clamp mode. Gigaohm seals were formed using patch pipettes made from borosilicate glass (World Precision Instruments, Sarasota FL, USA). For most experiments, the internal solution contained (in mM): CsCl 100, EGTA 11, HEPES 10, CaCl2 1, Mg-ATP 4, pH 7.2 with methane sulfonic acid; adjusted to 290–295 mmol kg-1 with sucrose. Whole cell capacitance and series resistance was manually compensated after opening the cell. Cells were continuously bath perfused with a HEPES-buffered saline (in mM): NaCl 150, Glucose 10, HEPES 10, KCl 2.5, CaCl2 2.5 MgCl2 1.0, pH 7.4, 305–320 mmol kg-1 Data will be analyzed off-line using pClamp software (Axon). Numerical analysis was performed using commercially available software. Independent student's t-test and two-way ANOVA were used for comparisons where appropriate; and statistical significance was based on p < 0.05. Concentration-response curves were generated from fits of data to a standard logistic equation as previously described (McCool & Botting, 2000). To derive KB from functional IC50 and EC50 data, the Cheng-Prusoff equation was used: where S = hillslope of agonist curve, A = concentration of agonist, EC50 = half-maximal agonist concentration, and IC50 = half-maximal antagonist concentration. Drugs Stocks of glycine, taurine, β-alanine, strychnine (Tocris) and colchicine (SIGMA) were prepared fresh each day. Agonists and antagonists were applied for 4–10 sec from an array of eight HPLC-grade capillary tubes (150 μm i.d.; Hewlett Packard Analytical Direct) placed within 100 μm of the cell of interest. Western analysis Cells were cultured, transfected as stated above except in 10 cm Petri dishes, and harvested by scrapping with 500 μL of lysis buffer (10 mM Tris pH 7.5, 1% SDS). Proteins were quantified using the Bradford assay and loaded onto 8–10% SDS-polyacrylamide gels, separated, and transferred to a nitrocellulose membrane (Hybond C, Amersham). The membrane was blocked overnight TBS (200 mM NaCl, 10 mM Tris pH 7.5) containing 0.2% Tween-20 and 10% low-fat dry milk. Blots were then incubated in TBS/0.1% Tween-20 containing primary GlyR antibody (monoclonal GlyR4a antibody, 1:200, Alexis Biochemicals) and monoclonal anti-gephyrin antibody (1:2000, Transduction Laboratories) for two hours at room temperature. After several washes, the HRP-coupled rabbit anti-mouse secondary antibody (SIGMA) was added for one hour (1:2000). The detection was performed by the ECL method (Amersham). List of abbreviations GlyR – glycine receptor; BLA – lateral/basolateral amygdala; HEK – human embryonic kidney cells; MDCK – Manin-Darby canine kidney cells Author's contributions JF carried out the electrophysiological recordings and participated in the western analysis. BM conceived of the study, participated in its design and coordination, performed the western analysis and some electrophysiology experiments, and drafted the manuscript. All authors read and approved the final manuscript. Figure 3 Glycine receptors expressed in different expression systems have similar 'functional' strychnine binding. Cells were pre-treated for 30 seconds with strychnine alone then exposed to a strychnine admixture with an EC50 concentration of glycine. (A) Strychnine-mediated inhibition of glycine-gated currents of GlyRα2 homomers expressed in HEK 293 (□; IC50 = 78.2 ± 13.5 nM) and L-cells (■; 33.1 ± 6.3 nM). (B) Strychnine-mediated inhibition of glycine-gated currents of GlyRα2β heteromers expressed in HEK 293 (○; 37.9 ± 7.9 nM) and L-cells (●; 23.2 ± 4.6 nM). (C) Functional KB values were calculated from IC50 values for individual cells using the Cheng-Prusoff relationship and glycine affinity/Hillslope data represented in Figure 1. Average KB values are shown for each subunit combination in the two expression systems. There was no significant effect of subunit compositions or expression system. Acknowledgements This work was supported in part by a Research Starter Grant from the Pharmaceutical Research & Manufacturers of America Foundation, Inc., and by the Texas A&M University Center for Environmental and Rural Health. We thank Dr. Jerry Trzeciakowski for his helpful discussions on the strychnine KB analysis. ==== Refs Davis M Aggleton JP The role of the amygdala in conditioned and unconditioned fear and anxiety. 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Colchicine binding properties of sea urchin sperm tail outer doublet tubulin J Cell Biol 1973 58 709 719 4747924 10.1083/jcb.58.3.709 Banks P Till R A correlation between the effects of anti-mitotic drugs on microtubule assembly in vitro and the inhibition of axonal transport in noradrenergic neurones J Physiol 1975 252 283 294 53281 Rajendra S Lynch JW Pierce KD French CR Barry PH Schofield PR Mutation of an arginine residue in the human glycine receptor transforms beta-alanine and taurine from agonists into competitive antagonists Neuron 1995 14 169 175 7826634 10.1016/0896-6273(95)90251-1 Leff P Dougall IG Further concerns over Cheng-Prusoff analysis Trends Pharmacol Sci 1993 14 110 112 8516953 10.1016/0165-6147(93)90080-4 Johnson BD Byerly L Ca2+ channel Ca(2+)-dependent inactivation in a mammalian central neuron involves the cytoskeleton Pflugers Arch 1994 429 14 21 7708473 Wang WH Cassola A Giebisch G Involvement of actin cytoskeleton in modulation of apical K channel activity in rat collecting duct Am J Physiol 1994 267 F592 8 7943357 Prat AG Cunningham CC Jackson G. R., Jr. Borkan SC Wang Y Ausiello DA Cantiello HF Actin filament organization is required for proper cAMP-dependent activation of CFTR Am J Physiol 1999 277 C1160 9 10600767 Krupp JJ Vissel B Heinemann SF Westbrook GL Calcium-dependent inactivation of recombinant N-methyl-D-aspartate receptors is NR2 subunit specific Mol Pharmacol 1996 50 1680 1688 8967993 Grenningloh G Rienitz A Schmitt B Methfessel C Zensen M Beyreuther K Gundelfinger ED Betz H The strychnine-binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors Nature 1987 328 215 220 3037383 10.1038/328215a0 van Zundert B Alvarez FJ Tapia JC Yeh HH Diaz E Aguayo LG Developmental-dependent action of microtubule depolymerization on the function and structure of synaptic glycine receptor clusters in spinal neurons J Neurophysiol 2004 91 1036 1049 12968009 10.1152/jn.00364.2003 Betz H Kuhse J Fischer M Schmieden V Laube B Kuryatov A Langosch D Meyer G Bormann J Rundstrom N et al. Structure, diversity and synaptic localization of inhibitory glycine receptors J Physiol Paris 1994 88 243 248 7874085 10.1016/0928-4257(94)90087-6 Meyer G Kirsch J Betz H Langosch D Identification of a gephyrin binding motif on the glycine receptor beta subunit Neuron 1995 15 563 572 7546736 10.1016/0896-6273(95)90145-0 Ramming M Kins S Werner N Hermann A Betz H Kirsch J Diversity and phylogeny of gephyrin: tissue-specific splice variants, gene structure, and sequence similarities to molybdenum cofactor-synthesizing and cytoskeleton-associated proteins Proc Natl Acad Sci U S A 2000 97 10266 10271 10963686 10.1073/pnas.97.18.10266 Kawasaki BT Hoffman KB Yamamoto RS Bahr BA Variants of the receptor/channel clustering molecule gephyrin in brain: distinct distribution patterns, developmental profiles, and proteolytic cleavage by calpain J Neurosci Res 1997 49 381 388 9260749 10.1002/(SICI)1097-4547(19970801)49:3<381::AID-JNR13>3.0.CO;2-2 Hermann A Kneussel M Betz H Identification of multiple gephyrin variants in different organs of the adult rat Biochem Biophys Res Commun 2001 282 67 70 11263972 10.1006/bbrc.2001.4553 Grenningloh G Pribilla I Prior P Multhaup G Beyreuther K Taleb O Betz H Cloning and expression of the 58 kd beta subunit of the inhibitory glycine receptor Neuron 1990 4 963 970 2163264 10.1016/0896-6273(90)90149-A

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==== Front BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-4-251529871910.1186/1471-2334-4-25Research ArticleEpidemiology of the human immunodeficiency virus in Saudi Arabia; 18-year surveillance results and prevention from an Islamic perspective Madani Tariq A [email protected] Yagob Y [email protected] Mohammad H [email protected] Huzaim Naser S [email protected] Department of Medicine, King Abdulaziz University, Jeddah2 Ministry of Health, Riyadh, Saudi Arabia2004 6 8 2004 4 25 25 5 2 2004 6 8 2004 Copyright © 2004 Madani et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background data on HIV epidemiology and preventive measures in Islamic countries is limited. This study describes the results of 18-year of HIV surveillance in Saudi Arabia (SA) and the preventive measures implemented from an Islamic perspective. Methods surveillance for HIV has been underway in SA since 1984. Indications for HIV testing include clinical suspicion, screening of contacts of HIV-infected patients, and routine screening of blood and organ donors, prisoners, intravenous drug users, patients with other sexually transmitted infections, and expatriates pre-employment. This is a case series descriptive study of all confirmed HIV infections diagnosed in SA from 1984 through 2001. Results a total of 6046 HIV infections were diagnosed, of which 1285 (21.3%) cases were Saudi citizens. Over the 18-year surveillance period the number of HIV infections diagnosed annually among Saudi citizens gradually increased and, over the period 1997–2001, it reached to 84 to 142 cases per year. The number of cases per 100,000 population varied widely between regions with a maximum of 74 cases and a minimum of 2 cases. The infection was most common in the age group 20–40 years (74.6%) and predominantly affected men (71.6%). The modes of transmission among Saudi citizens and expatriates, respectively, were as follows: heterosexual contact, 487 (37.9%) and 1352 (28.4%) cases; blood transfusion, 322 (25.0%) and 186 (3.9%) cases; perinatal transmission, 83 (6.5%) and 19 (0.4%) cases; homosexual contact, 32 (2.5%) and 38 (0.8%) cases; intravenous drug use, 17 (1.3%) and 33 (0.7%) cases; bisexual contact, 10 (0.8%) and 14 (0.3%) cases; unknown, 334 (26.0%) and 3119 (65.5%) cases. The number of HIV infections transmitted by blood or blood products transfusion declined to zero by year 2001 and all such infections occurred due to transfusions administered before 1986. At HIV diagnosis, 4502/6046 (74.5%) patients had no symptoms, 787 (13.0%) patients had non-AIDS defining manifestations, and 757 (12.5%) patients had AIDS. A total of 514/1285 (40%) Saudi patients died by year 2001. Conclusions the number of HIV cases in SA is limited with heterosexual contact being the main mode of transmission. From an Islamic perspective, preventive strategies include prevention of non-marital sex and intravenous drug use with encouragement of "safe sex" through legal marriage. ==== Body Background The human immunodeficiency virus (HIV) continues to be a major health problem worldwide despite enormous efforts to control its spread. As of December 2002, the estimated number of people living with HIV is 42 millions [1]. In year 2002 alone, the HIV epidemic claimed more than 3 million lives and an estimated 5 million people acquired HIV [1]. Available data point to increasing HIV infection rates in the Middle East and North Africa, with an estimated 83000 people having acquired the virus, and 37000 having died, in 2002. This brings to 550000 the estimated number of people living with HIV in this region [1]. Sexual contact remains the main mode of transmission of the disease worldwide followed by intravenous drug use and perinatal transmission [1]. Transfusion of blood and blood products is no longer a significant risk factor for acquiring HIV infection since the introduction of routine pre-transfusion HIV screening in 1985–1986 in most countries. Saudi Arabia occupies most of the Arabian Peninsula with an area of about 2,240,000 sq km (figure 1). It comprises 13 administrative provinces, namely, Makkah province (which includes the holy city of Makkah, Jeddah and Tayef), Madinah province (which includes the holy city of Madinah), Riyadh province (which includes the capital city Riyadh), the Eastern province (which includes Dammam, Ahsa, and Hafr Albaten), Asir province (which includes Abha and Bisha), Joaf province (which includes Joaf and Qerayyat), Hudud Shamaliyah (North borders) province (which includes Arar), and Baha, Jizan, Najran, Hail, Qassim, and Tabook provinces. The capital and largest city is Riyadh. The population in Saudi Arabia is 24,293,844, including 5,576,076 non-nationals as estimated in July 2003. Approximately, 42.3% of the population is below 15 years of age, 54.8%, between 15 and 64 years, and 2.9%, above 64 years of age. Ninety nine percent of the population is Muslim and the country is governed according to the Islamic law. Information on HIV epidemiology in Saudi Arabia and other Islamic countries is limited. Islam prohibits non-marital sex, homosexuality, and intravenous drug use. Therefore, the prevalence and incidence of HIV and other sexually transmitted infections are expected to be low in Islamic communities. This study describes the results of HIV surveillance activities that have been underway in Saudi Arabia from 1984 through 2001 and the preventive measures implemented by the government. Methods Surveillance for HIV infections has been underway since 1984 when the first case of HIV was diagnosed in Saudi Arabia. HIV cases are detected by HIV testing for various indications including clinical suspicion, screening of contacts of HIV-infected patients, routine screening of blood and organ donors, and testing of all prisoners, intravenous drug users, patients with other sexually transmitted infections, and expatriates pre-employment. In addition to being HIV tested in their homeland as a compulsory pre-requisite for employment in Saudi Arabia, expatriates are routinely retested for HIV upon arrival to Saudi Arabia before they are allowed to work and then regularly every 2 years to have their legal residence permits renewed. Only HIV-negative expatriates are allowed to work in Saudi Arabia. Routine screening of blood, blood products, and organ donors for HIV has been a standard procedure in Saudi Arabia since 1986. Enzyme-linked immunosorbent assays (ELISA) are used for routine HIV1/HIV2 testing. Positive ELISA results are confirmed by Western blot test performed in selected referral laboratories. The expanded World Health Organization (WHO) case definition was used to define AIDS [2]. All HIV infections diagnosed in governmental or private health care facilities are notified to the Ministry of Health (MOH) using unique identifying codes. This policy is strongly enforced by law. In addition to notifying the MOH, the Ministry of Interior is also informed about expatriates infected with HIV. HIV-infected expatriates are treated for any acute or life threatening complications and they are then returned back to their homeland. Saudi patients are referred to tertiary care governmental HIV-specialized centers where highly active antiretroviral therapy (HAART) medications and essential laboratory tests, such as HIV viral load and CD4/CD8 counts, are available. Epidemiological data are collected from patients by the attending physicians on standardized data collection forms. The likely mode of transmission is determined by the attending physician after interviewing the patient and taking complete history regarding high risk behaviors. The mode of transmission is considered to be unknown if the patient fails to admit to any high risk behavior. Collected information is subsequently submitted to the Department of Preventive Medicine in the Central MOH office in Riyadh where all surveillance data are compiled. Annual reports are issued but they are only utilized internally by the concerned officials in the MOH and the Ministry of Interior and they are not made available for the public. Results From 1984 through 2001, a total of 6046 cases with HIV infection were diagnosed; 1285 (21.3%) cases among Saudi citizens and 4761 (78.7%) cases among expatriates. Table 1 shows the demographic data, risk factors, and mortality of the infected patients. The likely mode of HIV transmission among 340 Saudi infected female patients was as follows: blood transfusion, 86 (25.3%) patients; marital sex, 74 (21.8%) patients; maternal transmission to female babies, 27 (7.9%) patients; non-marital sex, 8 (2.4%) patients; intravenous drug abuse, 3 (0.9%) patients; and in the remaining 142 (41.7%) patients the mode of transmission was unknown. Among 1376 non-Saudi HIV infected female patients, the likely mode of transmission was as follows: non-marital sex, 209 (15.2%) patients; blood transfusion, 32 (2.3%) patients; marital sex, 4 (0.3%) patients; intravenous drug abuse, 3 (0.2%) patients; maternal transmission to female babies, 3 (0.2%) patients; and in the remaining 1125 (81.8%) patients the mode of transmission was unknown. A total of 157/1285 (12.2%) Saudi patients had AIDS at the time of HIV diagnosis whereas the rest had either no symptoms (884 patients, or 68.8%) or non-AIDS defining manifestations (244 patients, or 19.0%) such as generalized lymphadenopathy, oral or vaginal thrush, oral hairy leukoplakia, recurrent herpes simplex, herpes zoster, molluscum contagiosum, condyloma acuminata, thrombocytopenia, or aphthus ulcers. Similarly, 600/4761 (12.6%) non-Saudi patients had AIDS at the time of HIV diagnosis, 3618 (76.0%) patients had no symptoms, and 543 (11.4%) had non-AIDS defining manifestations. Among 4761 HIV-infected expatriates, 3771 (79.2%) patients were from African countries, 624 (13.1%) patients, from Asian countries, 352 (7.4%) patients, from Middle East countries, and 14 (0.3%) patients, from Western countries. Ninety two percent of patients were from 10 countries, namely, Ethiopia (2214 patients or 46.5%), Nigeria (343 patients or 7.2%), Chad (329 patients or 6.9%), Yemen (309 patients or 6.5%), Sudan (267 patients or 5.6%), Eritrea (248 patients or 5.2%), India (219 patients or 4.6%), Somalia (176 patients or 3.7%), Pakistan (152 patients or 3.2%), and Bangladesh (133 patients or 2.8%), and the remaining 371 (7.8%) patients were from 41 other countries. Table 2 shows the number of HIV cases per 100,000 population by region. HIV cases have been reported from virtually all regions of Saudi Arabia but the highest prevalence was observed in Jeddah and Makkah in the western province (figure 1). Table 3 shows the indications for HIV testing in the Saudi patients. Approximately, one third of cases were identified because of clinical suspicion and the rest of cases were asymptomatic subjects identified following HIV screening for the indications mentioned in table 3. Figure 2 shows the annually reported HIV infections among Saudi patients over the surveillance period 1984 through 2001. There has been a gradual increase in the number of cases reported annually but this seems to have plateaued in the period 1997 through 2001. Figure 3 shows the annually reported number of HIV infections transmitted by transfusion of blood or blood products by year of HIV diagnosis from 1984 through 2001. The number of these infections has declined to zero by year 2001. All such infections were due to transfusions administered to patients before 1986. Discussion The prevalence and the annually reported HIV infections in Saudi Arabia were limited. More than three quarters of HIV cases were expatriates. Expatriates are routinely tested for HIV upon arrival to Saudi Arabia and then regularly every 2 years to have their legal residence permits renewed. Therefore, one possible explanation for the higher prevalence of HIV infections among expatriates is that HIV testing rates might be higher among expatriates compared to Saudi citizens. The number of HIV infections diagnosed annually among Saudi citizens gradually increased to reach a plateau of 84 to 142 cases per year in the last 5 years of the study period. The infection was more prevalent in Jeddah than it was in other cities in Saudi Arabia. A possible explanation for this increased prevalence in Jeddah is that this city is the main air and sea port for expatriates who come to work in the western province of Saudi Arabia and for those who come to visit the holy places in Makkah and Madinah. Additionally, the population in Jeddah is multi-cultural and inhomogeneous from the religion point of view. HIV infection in Saudi Arabia, as the case worldwide, was more common in the age group 20–40 years and predominantly affected men. All modes of transmission were registered but the main mode was heterosexual contact. Even though blood transfusion caused one quarter of HIV cases among Saudi patients, all such infections occurred due to transfusion of blood or blood products before 1986. This risk factor was virtually eliminated since the introduction of routine HIV screening of donated blood and blood products in 1986. The vast majority of Saudi HIV-infected women with identifiable risk factors acquired the infection through blood transfusion or marital sex with their infected husbands. The large proportion of patients with "unknown" risk factors was likely due to deliberate concealment of the actual risk factors, likely to be illegal sex, by the infected patients as non-marital sex and homosexuality are prohibited in Islam, the religion of all Saudi citizens and the vast majority of expatriates living in Saudi Arabia. The international efforts to control the HIV pandemic have failed to control it on a global scale despite their partial success in many of the developed countries and some of the developing countries. Limitation of resources in underdeveloped countries makes health education and other preventive strategies difficult to implement. Additionally, the limited access to antiretroviral medications in the underdeveloped countries has compounded the problem and perhaps contributed to some extent to the spread of HIV from untreated patients to new victims. Substance abuse is an increasing problem in Saudi Arabia as it is in the rest of the world [3]. Substances abused include injectable drugs such as heroin and cocaine and non injectable drugs such as cannabis and amphetamine-type stimulants. The estimated annual prevalence of heroin and amphetamine abuse in Saudi Arabia in 2000 as percentage of the population aged 15 and above was 0.01% and 0.002%, respectively [3]. The number of drug abusers annually admitted to detoxification centers in Riyadh, Jeddah, Dammam, and Qassim from 1996 through 2001 ranged from 4740 to 6650 patients with an average annual increment of 5.1% (unpublished data). Several studies were conducted in Saudi Arabia to describe the socio-demographic characteristics, pattern of substance abuse, and prevalence of blood-borne infections among drug abusers. For instance, 799 drug abusers from a voluntary detoxification unit in Jeddah were studied [4]. Sixty eight percent of subjects were under 35 years of age and 64% initiated drugs before age 25. Eighty seven percent used heroin or alcohol and 14% were dependent on more than one drug. Among heroin users, 91% injected the drug. The prevalence of hepatitis C virus infection among these patients was 69% [4]. In another study of 349 drug abusers in Jeddah, 281 (80.5%) subjects were intravenous drug users. The prevalence of HBsAg, anti-HBs, and anti-HBc was 12.6%, 49.0%, and 53.6%, respectively, suggesting that sharing of needles was a common practice [5]. In a more recent study in Jeddah including 1321 drug abusers, 1038 (78.6%) subjects were intravenous drug users and the prevalence of HBsAg and anti hepatitis D virus was 6.1% and 0.8%, respectively [6]. The prevalence of confirmed HIV infection among 2628 drug abusers in Jeddah, of whom 80% were intravenous drug users, was found to be only 0.15% [7]. Among 116 drug users in the Eastern province, 83% of subjects were below 32 years of age, 52.6% were unemployed, and the majority were of intermediate education [8]. Eighty-four percent of the patients abused heroin either alone or in combination with other drugs, 31% used alcohol, 26% used cannabis, and 10% used stimulants. The use of other drugs was rare [8]. Non-marital sex is expected to remain the main risk factor for acquiring HIV infection in Saudi Arabia as it is worldwide for several reasons. The ever-decreasing religious values, the ever-increasing ease of international transportation and communication, and the increasing poverty and unemployment are the main driving forces for non-marital sex, not only in Saudi Arabia but all over the world. Intravenous drug use is also expected to play a more important role for HIV transmission in Saudi Arabia in the years to come as the population of intravenous drug users increases. Preventive strategies should thus be directed towards these risk factors. Some of the preventive strategies that are advocated and used in other non-Islamic countries such as "Safe Sex" and "Needle Exchange Programs" contradict the Islamic rules and values and as such can not be used as valid and acceptable strategies to prevent the spread of this infection in Islamic countries. The concept of "Safe Sex" basically promotes the use of condoms for non-marital sexual relations, considered in Islamic countries a way of promoting non-marital sex which is absolutely prohibited in Islam. Needle Exchange Programs, likewise, is viewed by Islam as a way of encouraging people engaged in intravenous drug use to continue this prohibited practice. Strategies to prevent HIV infection in Islamic countries have to abide by the Islamic rules and values. Proposed preventive strategies that can be used in Islamic countries include strengthening of both Islamic and health education, encouraging people to follow and implement the Islamic rules and values that prohibit adultery, homosexuality, and intravenous drug use, and to practice safe sex only through legal marriage. Encouragement of legal marriage is a multidisciplinary task that should involve both governmental and non-governmental charitable organizations and the population at large to cut the cost of marriage and support programs that help the youth to get married. There are several such programs in Saudi Arabia. For example the "Charitable project to help the youth to get married" in Jeddah has helped 2500 young men to get married at a cost of 10 million Saudi Riyals (2.7 million US dollars) collected from donations (personal communication). Other Islamic aspects that are important in preventing non-marital and irresponsible sexual relationships include the fact that men are allowed to be married to up to four women and the fact that there is no age limit for marriage, thus permitting adolescents to get married. Further, Islam obliges women to cover themselves with the so called "Hijab" or veil and to be segregated from men in educational institutes and other mass-gathering. places. Islam also fights poverty, a driving force for commercial sex and prostitution, through a well established system of obligatory charity, known as "Zakat", and voluntary charity, known as "Sadaqa", taken from the rich people and given to the poor and needy. Additionally, Islam obliges the rulers to eliminate all means and factors that are conducive for indulging in non-marital sex and intravenous drug use such as sex trade, prostitution, and drug smuggling and to implement the Islamic penalties on those involved in such illegal acts. It should be noted, however, that HIV preventive strategies in some Islamic countries do not necessarily abide by the Islamic doctrine and that knowledge, attitude, and practice of Muslims in various Islamic societies do not necessarily conform to Islamic norms. Unfortunately, there is little published data on the effectiveness of the Islamic preventive strategies in reducing HIV transmission in Muslim communities. According to the United Nations and the World Health Organization data on HIV prevalence in different countries, however, the prevalence of HIV infection in Islamic countries is strikingly low compared to other countries [9,10]. A recent study showed that among 38 sub-Saharan African countries, the percentage of Muslims within countries negatively predicted HIV prevalence [11]. A survey of published journal articles containing data on HIV prevalence and religious affiliation showed that six of seven such studies indicated a negative relationship between HIV prevalence and being Muslim [11]. Conclusions Even though the number of HIV cases in Saudi Arabia is still limited, there is a potential for a rapid spread of this virus particularly among the youth. Strategies to prevent the spread of this virus in Saudi Arabia as well as in other Islamic countries should conform to the Islamic rules and values to be successful. Competing interests None declared Authors' contributions TAM designed the study, analyzed the data, and wrote the manuscript. YYM, MHJ, and NSH supervised the data collection and revised the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Figures and Tables Figure 1 A map of Saudi Arabia showing the main regions and cities where HIV cases were identified. Figure 2 Annually reported HIV infections among Saudi citizens from 1984 through 2001. Figure 3 Number of HIV infections transmitted through blood transfusion by year of HIV diagnosis in Saudi Arabia from 1984 through 2001. Table 1 Demographic data, risk factors, and mortality of HIV infected patients diagnosed in Saudi Arabia from 1984 through 2001. Saudi (%) Non-Saudi (%) Total Number of patients 1285 (21.3) 4761 (78.7) 6046 Gender  Male 945 (73.5) 3385 (71.1) 4330 (71.6)  Female 340 (26.5) 1376 (28.9) 1716 (28.4) Age groups (year)  < 5 72 (5.6) 19 (0.4) 91 (1.5)  5 – 14 84 (6.5) 20 (0.4) 104 (1.7)  15 – 19 54 (4.2) 67 (1.4) 121 (2.0)  20 – 29 359 (28.0) 1666 (35.0) 2025 (33.5)  30 – 39 369 (28.7) 2118 (44.5) 2487 (41.1)  40 – 49 188 (14.6) 662 (13.9) 850 (14.1)  50 – 59 86 (6.7) 167 (3.5) 253 (4.2)  ≥ 60 73 (5.7) 42 (0.9) 115 (1.9) Mode of transmission  Heterosexual contact 487 (37.9) 1352 (28.4) 1839 (30.4)  Blood transfusion 322 (25.0) 186 (3.9) 508 (8.4)  Perinatal 83 (6.5) 19 (0.4) 102 (1.7)  Homosexual contact 32 (2.5) 38 (0.8) 70 (1.2)  Intravenous drug use 17 (1.3) 33 (0.7) 50 (0.8)  Bisexual contacta 10 (0.8) 14 (0.3) 24 (0.4)  Unknown 334 (26.0) 3119 (65.5) 3453 (57.1) Number of patients with AIDS at diagnosis 157 (12.2) 600 (12.6) 757 (12.5) Mortality 514 (40.0) Unknownb Not applicable a Defined as men who have sex with men and women b Non-Saudi patients were returned back to their homeland. Table 2 Number of HIV cases per 100,000 population by region from 1984 through 2001 Region No. of Saudi patients No. of non-Saudi patients Total Prevalence per 100,000 population Jeddah 495 1381 1876 74 Makkah 76 594 670 45 Jizan 42 350 392 39 Dammam 167 474 641 35 Riyadh 217 1075 1292 27 Ahsa 48 139 187 20 Madinah 22 244 266 20 Tayef 66 114 180 19 Baha 45 25 70 18 Joaf 0 31 31 14 Hafr Albaten 4 37 41 14 Hail 5 52 57 12 Najran 11 35 46 12 Abha 44 93 137 10 Qassim 16 72 88 10 Bisha 8 15 23 8 Arar 2 19 21 8 Tabook 17 9 26 4 Qerayyat 0 2 2 2 Table 3 Indications for HIV testing in 1285 Saudi HIV-infected patients diagnosed in Saudi Arabia from 1984 through 2001. Indications Number % Clinical suspicion 438 34 Contacts of patients 190 15 Blood donors 146 11 Hemophilia and other blood disorders 113 9 Before surgery 96 7 Prisoners 84 7 Intravenous drug users 56 4 Patients on hemodialysis 51 4 Pre-employment 46 4 Patients with tuberculosis 36 3 Self-request 19 1 Pregnancy 10 1 Total 1285 100 ==== Refs Joint United Nations Programme on HIV/AIDS (UNAIDS) and World Health Organization (WHO) AIDS epidemic update: December 2002 WHO 2002 1 40 World Health Organization (WHO) Weekly Epidemiological Record 1994 69 273 275 7986696 Global illicit drug trends 2003 Office on Drugs and Crime, United Nations United Nations Publication Sales no E03X15, ISBN 92-1-148156-2, ISSN 1020-9298 Iqbal N Substance dependence. A hospital based survey Saudi Med J 2000 21 51 57 11533751 Njoh J Prevalence of hepatitis B virus markers among drug-dependent patients in Jeddah Saudi Arabia East Afr Med J 1995 72 490 491 7588140 Njoh J Zimmo S Prevalence of antibody to hepatitis D virus among HBsAg-positive drug-dependent patients in Jeddah, Saudi Arabia East Afr Med J 1998 75 327 328 9803612 Njoh J Zimmo S The prevalence of human immunodeficiency virus among drug-dependent patients in Jeddah, Saudi Arabia J Subst Abuse Treat 1997 14 487 488 9437619 10.1016/S0740-5472(97)85722-4 Hafeiz HB Socio-demographic correlates and pattern of drug abuse in eastern Saudi Arabia Drug Alcohol Depend 1995 38 255 259 7555626 10.1016/0376-8716(95)90001-X United Nations Programme on HIV/AIDS (UNAIDS) Report on the Global HIV/AIDS Epidemic 2002 Lenton C Will Egypt escape the AIDS epidemic? Lancet 1997 349 1005 9100637 Gray PB HIV and Islam: is HIV prevalence lower among Muslims? Soc Sci Med 2004 58 1751 1756 14990375 10.1016/S0277-9536(03)00367-8

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==== Front BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-4-261530419910.1186/1471-2334-4-26Research ArticleEpidemiological evidence of higher susceptibility to vCJD in the young Boëlle Pierre-Yves [email protected] Jean-Yves [email protected] Alain-Jacques [email protected] INSERM U444, Assistance Publique Hôpitaux de Paris, Université Pierre et Marie Curie, Paris, France2 Immunité Anti-Infectieuse JE 2236, UFR de Médecine de Grenoble, Université Joseph Fourier, Grenoble, France2004 10 8 2004 4 26 26 12 2 2004 10 8 2004 Copyright © 2004 Boëlle et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The strikingly young age of new variant Creutzfeldt-Jacob disease (vCJD) cases remains unexplained. Age dependent susceptibility to infection has been put forward, but differential dietary exposure to contaminated food products in the UK population according to age and sex during the bovine spongiform encephalopathy (BSE) epidemic may provide a simpler explanation. Methods Using recently published estimates of dietary exposure in mathematical models of the epidemiology of the new variant Creutzfeldt Jacob disease (vCJD), we examine whether the age characteristics of vCJD cases may be reproduced. Results The susceptibility/exposure risk function has likely peaked in adolescents and was followed by a sharp decrease with age, evocative of the profile of exposure to bovine material consumption according to age. However, assuming that the risk of contamination was proportional to exposure, with no age dependent susceptibility, the model failed to reproduce the observed age characteristics of the vCJD cases: The predicted cumulated proportion of cases over 40 years was 48%, in strong disagreement with the observed 10%. Incorporating age dependent susceptibility led to a cumulated proportion of cases over 40 years old of 12%. Conclusions This analysis provides evidence that differential dietary exposure alone fails to explain the pattern of age in vCJD cases. Decreasing age related susceptibility is required to reproduce the characteristics of the age distribution of vCJD cases. ==== Body Background Owing to the lack of data concerning age dependent dietary exposure to bovine material in food in the UK, mathematical models used in the study of new variant Creutzfeldt-Jacob disease (vCJD) have postulated an age risk function for contamination where the influence of age dependent dietary exposure to contaminated material could not be separated from intrinsic age related susceptibility [1-3]. For example, we modelled exposure/susceptibility by a plateau during the first 15 years of age followed by an exponential decrease afterwards [2]. Using this approach we estimated the duration of the incubation period for vCJD to circa 15 years; and predicted that the epidemic had likely peaked in 2001–2002 which is now consistent with the observed data [2,3]. The simple representation of the age risk function implied that few parameters were necessary to describe it, a desirable property when estimation is based on limited data. By June 2003, 139 cases of vCJD had been reported to the vCJD surveillance unit in the UK, as compared to the 79 cases used in our original paper [2]. This warrants the inclusion of more detail in the age risk function, especially to investigate whether the constant risk assumption in children holds, as it led to predict a bimodal age distribution for cases in the coming years [3]. At the same time, detailed estimates of the dietary bovine material consumption in the UK population according to age and sex have been made available [4]. This offers the opportunity to try to disentangle the role of exposure from that of age-specific susceptibility. Indeed, the estimates for dietary exposure show that it peaked during adolescence and decreased with age afterwards, a pattern which is consistent with the finding that most vCJD cases are young and were therefore at most teenagers during the years when the bovine spongiform encephalopathy (BSE) epidemic was at its maximum. We therefore set out to estimate the age risk function with a versatile description based on step functions, and to investigate whether dietary exposure alone could explain the age distribution of cases. Methods Data We obtained the age, sex and date of onset for the 137 vCJD cases reported to the UK vCJD unit as of June 2003, all of which had onset before October 2002. Delays in reporting may reach 18 months (R Will, personal communication) and bias downward the incidence curve. Therefore, we included in our analysis only the 129 cases with onset before November 2001, consisting in 71 men and 58 women. Mortality by age and sex for the UK was obtained from the Office for National Statistics [5]. Model The model extends our previously described method [3]. The instantaneous risk of infection for vCJD, λ(a,t), was assumed to depend on date t and age a in a multiplicative manner, with λ(a,t) = f(a) g(t), where parallels the entry of infected cows in the human food chain estimated from back-calculation results in the UK [6] and r corresponds to the impact of the specified risk material ban in 1989; f(a) is a function varying with age only (see following paragraph). Occurrence of cases of vCJD of sex i (i = Male or Female) is then modelled by a Poisson process in the (age, time) plane, with intensity , so that the expected number of onsets of sex i in the period [t,t+dt) and age [a,a+da) is πi(a,t) da dt [2,7]. In the equation above, h is the distribution of the incubation time, Si(a,t) is the probability of survival at time t for individuals of sex i aged a calculated from census information, and βι is the intensity of a homogeneous Poisson process. The incubation period distribution h was taken to be lognormal, as the choice of a particular shape is not critical for the estimates [3]. This distribution was also assumed to be independent of sex, and of age at contamination. Estimates of the parameters were obtained by numerical maximization of the log-likelihood, defined as , where the first sum is on all observed cases where si is the sex of individual i, and the second is over both sexes (s) and requires integration of the intensity over the domain (in time and age) D = [0,100] × [1 Jan 1980,31 Nov 2001]. Custom FORTRAN code, using SLATEC and TOMS library for numerical routines, was used in this step. In all instances, our model allowed estimation of the mean and standard deviation of the incubation period, the shape of f(a), and β. The model predicted distribution of age for cases with onset before November 2001 was determined by integrating (a,t) over the period [1/1/1980, 1/11/2001] for consecutive age class of width 10 years. The χ2 distance was computed to measure agreement between the observed and the model calculated age distributions, but no formal test was carried out. Age Risk function Model "Susceptibility & Diet" To allow a versatile shape in the age risk function, we chose a non parametric description based on step functions rather than a mathematical function with few parameters. We wrote so that the shape of f(a) was unconstrained for a<25 and decreased exponentially after age 25. We then estimated fi, i = 1,...,6 and α at maximum likelihood. With this particular choice, it is possible to visually check the progression in risk with age during infancy (f1, f2, f3), the presence of a peak in risk in teenagers (f2,f3,f4), and whether the drop in risk after 20 is strong enough to be exponential (f4, f5, f6). Model "Diet Alone" To investigate whether dietary exposure to meat was sufficient to explain the young age of vCJD cases, we first determined the exposure to meat according to age and sex from UK data, using consumption of carcass meat [4]. In these data, it is seen that the quantity consumed by week, as well as the percentage of consumers, changes with age. We therefore defined e(a) as the product of mean consumption by the corresponding percentage of consumers in age class a, and wrote f(a) = f0 e(a), where f0 was estimated at maximum likelihood. Model "Susceptibility | Diet" Finally, to estimate the influence of age standardised on dietary exposure, we wrote and likewise estimated fi from the data. In model "Susceptibility & Diet" and "Susceptibility | Diet", we normalised the fi by the largest of the values in the graphical presentation of results, yielding risks relative to the highest risk age class. Confidence intervals Maximum likelihood estimators have a limiting normal distribution around the true parameters, with limiting variance/covariance matrix given by the inverse of the Fisher information [8], therefore the quadratic form , where θ stands for a vector of k parameters, for the estimates, and i(θ) for the Fisher information matrix, has a limiting chi-squared distribution with k degrees of freedom. We therefore determined 95% confidence intervals by finding the values of parameters so that Q(θ) was less than the 95th quantile of the corresponding chi-squared distribution. Results The estimated age risk function in model "Susceptibility & Diet" is presented in Figure 1, and shows that an increase during childhood, peak during adolescence and sharp decrease afterwards provided the best fit. In this model, the average incubation period was estimated at 13.2 years (CI95% [11.2, 15.8]), with standard deviation 2.0 years (CI95% [1.1,3.7]). The model predicted age for the cases showed good agreement with the observed distribution (χ2 = 2.45; Figure 2). The shape of the estimated age risk function in Figure 1 is evocative of the age profile of dietary exposure to bovine carcass meat in the 1980s in the UK, as shown in Figure 3, where an increase in consumption was noted during childhood and adolescence, and decreased afterwards. However, in model "Diet Alone", while the average incubation period was estimated at 12.1 (CI 95% [10.2, 14.2]) years with a standard deviation of 2.4 (CI95% [1.2, 4.1])years, close to that of the first model, the predicted age distribution of the cases was at odds with the observed distribution (χ2 = 58.4), as apparent in Figure 2. More precisely, the predicted percentage of cases aged over 40 was 48% with the "Diet Alone" assumption, when the observed percentage was only 10%; it was 12% with the "Susceptibility & Diet" model. When estimating the residual influence of age, once exposure has been taken into account, model "Susceptibility | Diet" led to a profile for the fi as shown in Figure 4. This profile retained the major characteristics of the age risk function obtained in the "Susceptibility & Diet" model, although with an earlier maximum susceptibility. With this model, the average incubation period established at 12.6 years (CI 95% [10.5, 14.7]) with standard error 1.8 years (CI95% [1.2, 3.5]), and the predicted age distribution of cases showed agreement similar to that of the "Susceptibility & Diet" model (χ2 = 2.36). Discussion Incorporating differential dietary exposure to BSE infected products according to age and sex, in a flexible age risk function for vCJD contamination, we found that exposure alone could not explain the young age of vCJD cases seen in the UK. Decreasing age related susceptibility had to be assumed to reproduce the characteristics of the age distribution of these cases. In all instances, the estimates for the epidemiological characteristics of vCJD were in line with those previously reported from comparable number of cases [3,9,10], and pointed to an epidemic of moderate size. We obtained a smaller point estimate for the mean incubation period when the age risk was allowed to change during childhood rather than assumed constant (13.2 yrs CI95% [11.2, 15.8] vs. 16.4 yrs CI95% [11, 24] [3]). All models considered here predicted that, by 2010, the epidemic should have ended, provided it is limited to individuals with the observed susceptible genotype (as of today, all vCJD cases are methionine homozygous at codon 129 of the PrP gene[11]). The average number of cases to come is predicted at 43, leading to a total size of 172 cases. From the model "Susceptibility & Diet", it appears that a previous assumption of a constant age risk in children and adolescents [2] has likely led to overestimate the risk of infection in young children. The age-risk profile estimated here leads to a smaller risk than previously found in children born after 1980, to fewer cases among the young and an overall smaller total size for the epidemic than reported before. Furthermore, since few young cases are expected in the future, the bimodality of the predicted age distribution of cases is not found anymore. The estimated profile of the susceptibility/exposure age risk function agreed with that selected in scenario analysis [9], although our maximal risk is among the 15–20 years old rather than in the 10–15 years old. However, even after adjustment for dietary exposure, susceptibility remains rapidly decreasing in adults, as found in the "Susceptibility | Diet" model. This finding confirms that obtained in a recent analysis based on scenario analysis, where it was found that exponential decrease in susceptibility in the oldest cohorts was desirable [10]. This is also found here by direct estimation, and moreover, the age susceptibility appears to increase among children up to age 5–10, be almost constant among teenagers and rapidly decreasing afterwards. Three hypotheses have been put forward to explain the young age of the vCJD cases: age dependent incubation period, age dependent exposure, and age dependent susceptibility. Age dependent incubation period has recently been revived by Cooper et al., because scenarios including longer incubation periods in old cohorts provided a better fit of the data[10]. However, the increase of the mean age of cases with time which should occur with age dependent incubation periods [2], is not supported by the data. The correlation between age at onset and calendar time remains indeed extremely small (Spearman correlation coefficient r = -0.025, n = 129). The potential role of differential dietary exposure was proposed early in the epidemic [12], because substantial differences in exposure existed in adolescents and adults. A disequilibrium in sex ratio towards males was for example predicted on this basis [13], but is not statistically established today (χ2 test, P = 0.49) although the proportion of males is 55%. Our analysis shows that, while the estimated age risk function for infection with vCJD exhibits a peak in the young that was also found in dietary exposure to potentially BSE infected products, differential dietary exposure alone does not account for the observed pattern in the age of the vCJD cases. Indeed, the relative exposure does not decrease rapidly enough with age to reduce the risk in older adults. Therefore, an additional effect of age is required to fully account for the age of the vCJD cases. Once exposure is taken into account, this effect appears to be peaking in children less than 10 years old, and decreasing afterwards. Contamination through BSE infected food is today the most plausible explanation for the occurrence of vCJD; this is the rationale for correcting the risk of infection by the level of dietary exposure. However, this explanation remains today hypothetical, because even if epidemiology and biochemistry favour a link between BSE and vCJD, this is not regarded as ultimately conclusive [14]. In this work, we examined the further possibility that, provided dietary exposure was the culprit, differences in age-related exposure could explain the age distribution of cases. This assumes that the risk of vCJD infection may have been larger in those consuming more bovine products; however population based models linking bovine products consumption to vCJD incidence were not conclusive in this respect [15]. Conclusions The reasons for an increased susceptibility in teenagers compared to young children and adults are today speculative. In many infectious diseases, a decrease in susceptibility with age is mediated through the immunological responses to repeated exposure since birth. However, for vCJD, most cohorts in the population have had the same duration of exposure to BSE infected material. Due to the age range considered, biological processes involved in the maturation of the immune system; in response to hormonal changes may be incriminated. For example, changes in the permeability of the intestinal barrier occurs with age with decreasing Peyer's patches [16]. Further experimental research is required to provide explanations for what remains a very unusual characteristic in an infectious disease. List of abbreviations vCJD: variant Creutzfeldt-Jacob Disease BSE : Bovine Spongiform Encephalopathy Competing interests None declared. Authors' contributions PYB designed the study, analysed the results and drafted the manuscript. JYC participated in the design and writing. AJV participated in the design and writing. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements Robert Will for allowing access to vCJD data. Jean Gagnon for helpful discussions. Figures and Tables Figure 1 Age dependent exposure/susceptibility risk function for vCJD infection. Risks are relative to the [15, 20] years old. Box and whiskers plots show the 50% and 95% confidence intervals. Figure 2 Observed age distribution of the 129 vCJD cases with onset before November 2001 and model predicted cumulated age distribution of cases for susceptibility proportional to meat consumption only (dashed) or with estimated age risk function (plain). Bars limit 95% confidence intervals. Figure 3 Average dietary exposure per person per week in the UK according to sex (plain : Male, dashed : Female). Data from Cooper & Bird [4]. Figure 4 Age dependent susceptibility risk function for vCJD infection adjusted on dietary exposure to bovine material. Risks are relative to the [5, 10] years old. Box and whiskers plots show the 50% and 95% confidence intervals. ==== Refs Ghani AC Ferguson NM Donnelly CA Anderson RM Predicted vCJD mortality in Great Britain Nature 2000 406 583 584 10949288 10.1038/35020688 Valleron AJ Boelle PY Will R Cesbron JY Estimation of epidemic size and incubation time based on age characteristics of vCJD in the United Kingdom Science 2001 294 1726 1728 11721058 10.1126/science.1066838 Boelle PY Thomas G Valleron AJ Cesbron JY Will R Modelling the epidemic of variant Creutzfeldt-Jakob disease in the UK based on age characteristics: updated, detailed analysis Stat Methods Med Res 2003 12 221 233 12828243 10.1191/0962280203sm329ra Cooper JD Bird SM UK bovine carcass meat consumed as burgers, sausages and other meat products: by birth cohort and gender J Cancer Epidemiol Prev 2002 7 49 57 12501955 10.1080/147666502321082719 Office for National Statistics Tables 6.1 - Deaths : age and sex Population Trends 2004 116 47 Donnelly CA Ferguson NM Ghani AC Anderson RM Implications of BSE infection screening data for the scale of the British BSE epidemic and current European infection levels Proc R Soc Lond B Biol Sci 2002 269 2179 2190 12427310 10.1098/rspb.2002.2156 Keiding N Age-specific Incidence and Prevalence: a Statistical Perspective J R Stat Soc Ser A Stat Soc 1991 154 371 412 Cox DR Hinkley DV Theoretical Statistics 1974 1 1 London, Chapman & Hall 511 Ghani AC Donnelly CA Ferguson NM Anderson RM Updated projections of future vCJD deaths in the UK BMC Infect Dis 2003 3 4 12716457 10.1186/1471-2334-3-4 Cooper JD Bird SM Predicting incidence of variant Creutzfeldt-Jacob disease from UK dietary exposure to bovine spongiform encephalopathy for the 1940 to 1969 and post 1969 birth cohorts Int J Epidemiol 2003 32 784 791 14559750 10.1093/ije/dyg248 Will RG Ward HJ Clinical features of variant Creutzfeldt-Jakob disease Curr Top Microbiol Immunol 2004 284 121 132 15148990 Gore SM Bingham S Day NE Age related dietary exposure to meat products from British dietary surveys of teenagers and adults in the 1980s and 1990s Br Med J 1997 315 404 405 9277606 Cooper JD Gore SM Should we anticipate male excess in vCJD cases? J Epidemiol Biostat 1999 4 61 63 10619052 Aguzzi A Polymenidou M Mammalian prion biology: one century of evolving concepts Cell 2004 116 313 327 14744440 10.1016/S0092-8674(03)01031-6 Cousens S Everington D Ward HJ Huillard J Will RG Smith PG The geographical distribution of variant Creutzfeldt-Jakob disease cases in the UK: what can we learn from it? Stat Methods Med Res 2003 12 235 246 12828244 10.1191/0962280203sm332ra Van Kruiningen HJ Ganley LM Freda BJ The role of Peyer's patches in the age-related incidence of Crohn's disease J Clin Gastroenterol 1997 25 470 475 9412954 10.1097/00004836-199709000-00017

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==== Front BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-4-471531039910.1186/1471-2407-4-47Research ArticleAncestry reported by white adults with cutaneous melanoma and control subjects in central Alabama Acton Ronald T [email protected] Ellen H [email protected] William W [email protected] Amy L [email protected] Rodney CP [email protected] James C [email protected] Immunogenetics Program and Departments of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA2 Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA3 Department of Epidemiology and International Health, University of Alabama at Birmingham, Birmingham, Alabama, USA4 Southern Iron Disorders Center, Birmingham, Alabama, USA2004 13 8 2004 4 47 47 1 4 2004 13 8 2004 Copyright © 2004 Acton et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background We sought to evaluate the hypothesis that the high incidence of cutaneous melanoma in white persons in central Alabama is associated with a predominance of Irish and Scots descent. Methods Frequencies of country of ancestry reports were tabulated. The reports were also converted to scores that reflect proportional countries of ancestry in individuals. Using the scores, we computed aggregate country of ancestry indices as estimates of group ancestry composition. HLA-DRB1*04 allele frequencies and relationships to countries of ancestry were compared in probands and controls. Results were compared to those of European populations with HLA-DRB1*04 frequencies. Results Ninety evaluable adult white cutaneous melanoma probands and 324 adult white controls reported countries of ancestry of their grandparents. The respective frequencies of Ireland, and Scotland and "British Isles" reported countries of ancestry were significantly greater in probands than in controls. The respective frequencies of Wales, France, Italy and Poland were significantly greater in controls. 16.7% of melanoma probands and 23.8% of controls reported "Native American" ancestry; the corresponding "Native American" country of ancestry index was not significantly different in probands and controls. The frequency of HLA-DRB1*04 was significantly greater in probands, but was not significantly associated with individual or aggregate countries of ancestry. The frequency of DRB1*04 observed in Alabama was compared to DRB1*04 frequencies reported from England, Wales, Ireland, Orkney Island, France, Germany, and Australia. Conclusion White adults with cutaneous melanoma in central Alabama have a predominance of Irish, Scots, and "British Isles" ancestry and HLA-DRB1*04 that likely contributes to their high incidence of cutaneous melanoma. ==== Body Background The incidence of cutaneous melanoma in Alabama is high (16.9 and 8.6 per 100,000 men and women, respectively, in 1998) [1]. The average annual incidence rate in Alabama during 1996 was 15.5 per 100,000 for men and 8.8 per 100,000 for women, indicating that the incidence in men is increasing. This is consistent with reports that cutaneous melanoma is one of few cancers the incidence and mortality of which is increasing in white Americans [2]. Lifestyle, sun exposure, fair skin, light eye color, poor ability to tan, Northern European or Celtic ethnicity, family history of melanoma, benign nevi, various major histocompatibility complex alleles, and mutations in the p16 gene have been reported as risk factors for cutaneous melanoma [3-16]. HLA-DRB1*04 is significantly associated with cutaneous melanoma in white persons in Alabama [9] and Australia [11,12]. In addition, HLA-DRB1*04 was reported positively associated with cutaneous melanoma in white patients who had a high index of Celtic ancestry [14]. It has been hypothesized that the high incidence of melanoma in Alabama, a Sunbelt state with a mild climate, is due to the settlement of Alabama by a predominance of persons of Irish and Scots descent [8-10]. In the present study, we sought to evaluate this hypothesis by using questionnaires to obtain information about the countries of ancestry of the grandparents of white adults in central Alabama with cutaneous melanoma and of white control subjects from the central Alabama general population. Using data from the questionnaires, we evaluated the frequency of country of ancestry reports in cutaneous melanoma and control participants and computed country of ancestry indices to permit quantification and comparison of group ancestry data, as previously described [17]. We also compared HLA-DRB1*04 frequencies in participants with melanoma who reported "Celtic" and "non-Celtic" ancestry. The rationale for using country of ancestry information and HLA-DRB1*04 to identify persons who have increased risk to develop cutaneous melanoma is discussed. Methods General criteria for selection of study subjects The performance of this work was approved by the Institutional Review Boards of the University of Alabama at Birmingham and Brookwood Medical Center. All subjects were adults (≥ 18 years of age) who were central Alabama residents; each identified himself/herself as white. Persons with melanoma were diagnosed in routine medical care in the interval 1981 – 2002, but were otherwise unselected. We excluded persons of sub-Saharan African or African American descent, because most of these persons have non-European ancestry and their incidence of cutaneous melanoma is much lower than in white persons in central Alabama [1,2]. Melanoma probands Cutaneous melanoma was diagnosed as previously described [9]. HLA-DRB1 typing was performed by either the microdroplet lymphocytotoxicity test using B-lymphocytes isolated by the nylon wool column procedure, or by low-resolution SSP using genomic DNA obtained from peripheral blood buffy coat, as previously described [9,18]. Further, we included only the first persons diagnosed to have cutaneous melanoma in respective families; they were designated as probands. Control subjects Adult control subjects residing in central Alabama were recruited in two groups. The first group consisted of 83 spouses of persons with cutaneous melanoma and randomly recruited subjects. The second group consisted of 260 unselected volunteers who completed the present questionnaire (described below); they were recruited from hospital workers, employees of two universities, spouses of patients who attended a hematology/medical oncology outpatient clinic, and members of the general public encountered in two retail shopping malls. In both groups, we excluded persons who were known to be relatives of other study participants. We did not evaluate medical histories or perform physical examinations in control subjects. These data were pooled to yield a group of 343 unrelated controls, of whom 24 were eliminated because they did not know the country of ancestry of any of their grandparents (as indicated below). This left a group of 319 control subjects whose data were deemed evaluable for the present study. HLA typing was performed on some control subjects as indicated below. Questionnaire and interview design A one-page questionnaire was designed to permit each study participant to indicate the countries of ancestry of his/her paternal and maternal grandparents. This method is identical to that previously validated in a study of the ancestry of hemochromatosis probands [17]; in part, this method was modified from previously reported methods by including only the country of birth of grandparents [6,14,19]. We defined aggregate country categories as the composite of reports from respective countries [17]. Reports based solely on association of family names with specific countries were tabulated as "Don't Know." Reports from participants for whom each of four grandparents were categorized as "Don't Know" were defined as inevaluable and were excluded from final analysis [17]. We did not evaluate relationships of country of ancestry to gender of the grandparents or to the paternal or maternal side of the participant's family [17]. Frequencies of countries of ancestry reports We tabulated the number of participants who reported specific countries of ancestry or aggregate country categories (as defined above) for one or more grandparents. This method is identical to that previously described [17]; in part, this method was modified from previously reported methods by including only the country of birth of grandparents [14,19]. Country of ancestry indices The questionnaire and interview reports from each participant were evaluated to yield individual country of ancestry scores that reflect proportional national ancestry, as previously described [17]. We also computed aggregate country of ancestry indices for cutaneous melanoma probands and control subjects as estimates of group ancestry composition. These indices were expressed as the quotient of total individual scores for respective countries and the total number of cutaneous melanoma probands or control subjects, as appropriate [17]. Index of "Celtic" ancestry An index of Celtic ancestry was computed in a manner similar to that used for country of ancestry indices. In the present analysis, persons who reported one or more grandparents whose country of origin was Ireland or Scotland were defined as "Celts." Other participants were defined as "non-Celts." We did not use grandparental surnames or maiden names to quantify the degree of Celtic ancestry as did previous investigators [14,19]. Review of HLA-DRB1*04 phenotype frequencies in europe and australia A manual and computerized literature search was performed to identify reports of HLA-DRB1*04 phenotype frequencies in white persons with or without melanoma in countries or areas of Europe and Australia that are known to have had Celt settlements The HLA-DRB1*04 phenotype frequency among white control subjects in central Alabama is approximately 0.2148, based on the combined data from two previous reports [9,20]. Thus, we tabulated only those reports from countries or regions in which the HLA-DRB1*04 phenotype frequency was > 0.2148. We excluded studies which reported HLA-DRB1*04 frequency estimates on control cohorts of fewer than 100 subjects. Statistical considerations The dataset consisted of observations on 90 cutaneous melanoma probands and two groups of controls (83 interview subjects and 261 questionnaire responders, respectively). A computer spreadsheet (Excel 2000, Microsoft Corp., Redmond, WA) and a statistical program (GB-Stat, v. 8.0 2000, Dynamic Microsystems, Inc., Silver Spring, MD) were used to perform the present analyses. In a preliminary evaluation, we determined that the proportions of men and women, mean ages, country of ancestry reports, and frequencies of country of ancestry reports were not significantly different in the two control groups. Therefore, we pooled data from the two groups for comparison with those of melanoma probands. Frequencies of men and women, clinical abnormalities, and countries of ancestry were counted. General descriptive data are presented as percentages or mean ± 1 S.D. Comparisons between groups were tested for statistical differences using chi-square analysis, Fisher exact test or two-tailed Student t test, as appropriate. However, Student t-test can not be used to compare country of ancestry data groups in which all values are 0 (no variability). Accordingly, we arbitrarily assigned a country of ancestry datum of 0.25 for one person in each proband country group for which there were no actual country of ancestry reports. Student t-test was then performed using this modified data group; estimated p values from these tests are displayed in parentheses. A p value < 0.05 was defined as significant. Odds ratios (OR) were calculated as described by Woolf [21]. Results Characteristics of cutaneous melanoma probands There were reports from 90 evaluable probands (46 men, 44 women). Their mean age at the time of participation in the present study was 49 ± 16 years (range 19 – 80 years). Characteristics of control subjects There were reports from 319 evaluable control subjects (128 men, 191 women). The percentages of men and women in the control group were similar to those in the cutaneous melanoma probands (p = 0.0621, chi square analysis). Their mean age at the time of participation in the present study was 43 ± 16 years (range 18 – 82 years). The mean age of the cutaneous melanoma probands was significantly greater than that of the control subjects (49 ± 16 years vs. 43 ± 16 years, respectively; p = 0.0016). General analysis of questionnaire and interview reports No person declined to participate in the study, and there were no incomplete, equivocal, or unintelligible questionnaire or interview reports. Some participants reported that they were unaware of their ancestry due to adoption, family estrangement, or disinterest in genealogy. Ninety of 110 cutaneous melanoma probands (81.8%) and 319 of 347 control subjects (91.3%) provided reports for at least one of four grandparents; these differences were significant (p = 0.0026). Data from these participants were included for further analysis. Thus, there were reports from 90 evaluable cutaneous melanoma probands and 319 evaluable control subjects. Among evaluable cutaneous melanoma probands, the mean number of countries reported was 2.2 ± 1.1 (range 1 – 5 countries). The mean number of countries reported by evaluable control subjects was similar (2.4 ± 1.1 countries (range 1 – 7 countries); p = 0.2246). Frequencies of country of ancestry reports These data are displayed in Table 1. Most participants reported European countries of ancestry. Frequencies of Ireland and Scotland country reports of ancestry obtained from cutaneous melanoma probands were significantly greater than those from control subjects (p = 0.0042, OR = 2.0 and p = 0.0015, OR = 2.2, respectively). The "Celtic" country reports of ancestry was also significantly higher in melanoma probands than in control subjects (Table 1). The frequency of "British Isles" and "Europe Not British Isles" ancestry reports tabulated in cutaneous melanoma probands were not significantly different than those in controls subjects. The respective frequencies of Wales, France, Italy, and Poland ancestry reported by cutaneous melanoma probands were significantly lower than those in control subjects (Table 1). 16.7% of country of ancestry reports from cutaneous melanoma probands and 23.8% in control subjects indicated "Native American" ancestry; these percentages were not significantly different (Table 1). The percentage of cutaneous melanoma probands who reported "Don't Know" for the countries of ancestry of one, two, or three grandparents was similar to that in control subjects (30.0% vs. 30.1%, respectively) (Table 1). Country of ancestry indices These data are displayed in Table 2. The respective Ireland and Scotland indices in cutaneous melanoma probands were significantly greater than those in control subjects. The "Celtic" ancestry index was significantly higher in melanoma probands than in control subjects. The aggregate "British Isles" index was also significantly greater in melanoma probands (Table 2). The Wales, France, Spain, Austria, Italy, Poland, Russia, Sweden and "Europe Not British Isles" indices in probands were significantly lower than those in control subjects (Table 2). There were no other significant differences. HLA-DRB1*04 frequencies HLA-DRB1 phenotypes were available for 63 of the 90 present cutaneous melanoma probands. We divided the probands for which we had both country of ancestry data and HLA-DRB1 phenotypes into "Celts" (n = 19) and "non-Celts" (n = 44), as defined above. There was no significant difference in the frequency of HLA-DRB1*04 in the two groups (0.5263 vs. 0.3636, respectively; p = 0.3551, OR = 1.9). Because this finding was unexpected and differed from that of previous reports [14] we then computed the frequencies of HLA-DRB1*04 in Alabama white cutaneous melanoma probands and in control subjects from two previous Alabama reports [9,20]. Thus, the frequency of HLA-DRB1*04 in 123 probands (38.2%) was significantly different from that in 340 controls (21.5%) (p = 0.0005, OR = 2.3). DRB1*04 phenotype frequencies > 0.2148 in Europe and Australia We identified HLA-DRB1*04 phenotype frequency estimates of > 0.2148 from England, Ireland, Scotland, Wales, Orkney Islands, Brest, Germany, and Australia, areas populated predominantly by persons of Celtic ancestry [9,11,13,20,22]. These data and the results of studies in which HLA-DRB1*04 phenotype frequencies were assessed in melanoma patients are displayed in Table 3. The frequencies of HLA-DRB1*04 in these countries or regions are significantly higher than that in Alabama subjects (p ≤ 0.005) (Table 3). Discussion The present results indicate that England, Ireland, Scotland and the aggregate "British Isles" are the countries of ancestry reported most frequently by cutaneous melanoma probands and control subjects in central Alabama. Germany is another country of ancestry often reported by the present study participants. These results are consistent with historical accounts of early migrations of persons of English, Irish and Scots descent into central Alabama [23-26], with the national associations of surnames recorded in Alabama Census returns for 1820 and 1830 [27], and with the present composition of the southern United States [25]. In U.S. Census 2000, country of ancestry information (maximum of two countries) was reported on a "long form" provided to one in six census participants, and was tabulated as numbers of country-specific reports [28]. Thus, the data of U.S. Census 2000 cannot be compared statistically with the results of the present study, but the percentages of European countries of ancestry of white Alabama residents compiled in both studies reveal similar trends. In the present study, the largest subgroups of reports in the "North, Central, and South American Countries" category are those of "U.S." or "American" ancestry. Some participants did not report or know the country of ancestry of their grandparents. These findings are also consistent with trends in the U.S. Census 1990 and U.S. Census 2000, in which the percentages of white Americans who report "American" ancestry are increasing, and the percentages of those who report various European countries of ancestry are decreasing [28,29]. "Native American" ancestry, especially Cherokee or Creek heritage, was reported by many of the present study participants, and this is consistent with accounts of early Alabama history [24,30-32] and with U.S. Census data on Alabama since 1820 [27-29]. However, the corresponding aggregate "Native American" frequency of country ancestry reports and country of ancestry indices were not significantly different in melanoma probands and control subjects. This supports the postulate that native American ancestry does not contribute significantly to the increased frequency of cutaneous melanoma in central Alabama. The percentages of men and women were not significantly different in cutaneous melanoma probands and control subjects. Analyses of the two study groups indicate that age is not significantly correlated with country of ancestry indices. The mean age of cutaneous melanoma probands was significantly greater than that of control subjects. However, the mean number of countries reported by cutaneous melanoma probands and control subjects did not differ significantly. Thus it appears that diagnosis of cutaneous melanoma or greater age is not associated with greater interest or knowledge in personal ancestry, although this is unproven. 30.0% of cutaneous melanoma probands and 30.1% of control subjects did not know the country of ancestry of any of their four grandparents and were thus declared inevaluable. Many others did not know the ancestry of some of their grandparents. Some participants reported that they were unaware of their ancestry due to adoption, family estrangement, or disinterest in genealogy. Other participants could have been incorrect in their reporting. The percentages of evaluable cutaneous melanoma probands and control subjects who reported grandparents in the "Don't Know" category were similar. Altogether, it is unlikely that exclusion of subjects who did not know the country of ancestry of each of their grandparents would significantly change the outcomes of the present study. The overall trends in frequency of country reporting and country of ancestry indices in "British Isles" and "Europe Not British Isles" categories in the present study were similar. This suggests that uncertainty of participants about the exact degree of country of ancestry of some of their grandparents was probably not a significant contributor to the major conclusions of the present study. It is possible that control subjects who were included also had cutaneous melanoma. Nonetheless, it is unlikely that identification of a presumably small number of undiscovered control subjects with cutaneous melanoma would significantly change the conclusions of the present study. Previous studies reported that Celtic ancestry is a risk factor for cutaneous melanoma, particularly when people of Celtic ancestry inhabit areas of high flux of ultraviolet radiation [4,6]. Persons of Celtic ancestry usually have fair skin, eyes and hair of light color, poor ability to tan, and tendency to burn easily after sun exposure [4,6]. Areas of the British Isles where persons have a high degree of Celtic ancestry include Wales, Cornwall, Scotland and Ireland [6]. We did not specifically ask about nor did we receive reports of Cornwall as a region of origin in the present study. Similarly, Wales was reported as a country of origin by none of the present probands and few of the control subjects. However, the frequency of country reports and ancestry indices for Ireland, Scotland, aggregate "British Isles," and combined Ireland and Scotland ("Celts") in the present melanoma probands was significantly greater than that in control subjects, consistent with the hypothesis that Celtic ancestry is a risk factor for cutaneous melanoma. The present observations are consistent with those of another study in Alabama in which white persons with hemochromatosis, a disease thought to be of Celtic origin [33,34], reported a significantly greater Scotland and "British Isles" ancestry than controls [17]. The countries of ancestry reported was also consistent with the relative high frequency of C282Y, the major allele associated with hemochromatosis, in Alabama hemochromatosis probands compared to controls [17]. Using an index of Celtic ancestry that included grandparental surnames, maiden names, and country of birth to evaluate persons in Wales, other investigators observed that "high-scoring Celts" were significantly more likely to have Fitzpatrick skin type I or II (poor ability to tan, and tendency to burn easily after sun exposure) [35] than non-Celtic subjects [14,19]. Moreover, the frequency of HLA-DRB1*04 was significantly greater in "high-scoring Celts." These authors concluded that the increased risk of cutaneous melanoma and other types of skin cancer in persons of Celtic ancestry in Wales is due not only to paler skin, but also to HLA-DRB1*04 and associated immunologic factors. Observations from previous studies demonstrate that the frequency of positivity for the HLA-DRB1*04 phenotype is significantly greater in Alabama cutaneous melanoma probands than in control subjects [8,9,20]. This is consistent with reports from Australia and Wales [11,12,14]. Further, previous reports from Alabama suggest that the subgroup of individuals who possess HLA-DRB1*04 are at increased risk for cutaneous melanoma, independent of eye color, hair color, amount of melanin in the skin, or ethnic origin [8]. However, the failure to reach statistical significance in the comparison of HLA-DRB1*04 phenotypes in the present 19 "Celts" and the 44 "non-Celts" is likely due to the small number of probands who reported Celtic ancestry and for whom HLA typing data were available. Investigators in Texas and England reported that the HLA genotype DQB1*0301 influences either susceptibility to and/or severity of melanoma [15,36-38]. This conclusion is consistent with previous studies in which HLA-DRB1*04 was associated with melanoma, because HLA-DQB1*0301 is in linkage disequilibrium with HLA-DRB1*04 [37]. The highest frequencies of HLA-DRB1*04 in Europe occur in England, Wales, Ireland, the Orkney Islands, and the Brest area of France [22]. These geographic results could explain the significantly increased frequency of HLA-DRB1*04 in cutaneous melanoma probands from central Alabama, because the present melanoma probands had significantly higher Ireland, Scotland, and "British Isles" country of ancestry indices than Alabama control subjects. Our observations and those of others suggest that certain HLA genotypes may be markers for Celtic ancestry [8,14]. It has been reported that HLA phenotypes other than HLA-DRB1*04 occur in association with melanoma in various white national or ethnic groups [13,15,36,39,40]. This could be explained in part by the variation of HLA phenotype frequencies in these white national or ethnic groups or by other genes within the major histocompatibility complex in linkage disequilibrium that also play a major role in mediation of immune responses. Thus, various HLA phenotypes could be markers for white national or ethnic groups that also possess certain physical characteristics and immune response genes that increase their risk for developing cutaneous melanoma. Conclusions The present results support our hypothesis, and are also consistent with the association of cutaneous melanoma with Ireland, Scotland and "British Isles" ancestry, HLA-DR phenotypes, and estimations of a northern European somatic phenotype (eye color, hair color, amount of melanin in the skin) previously reported in white persons who reside in central Alabama [3,4,8,9]. The present observations also suggest that targeting white persons with relatively high "Celtic" or "British Isles" country of ancestry indices and HLA-DRB1*04 for cutaneous melanoma prevention and early diagnosis efforts would be an effective strategy to decrease the morbidity and mortality of this type of malignancy. Competing interests None declared. Authors' contributions RTA assessed the patients and participated in assessing the control population, conceived the study, participated in data collection, laboratory evaluation of the patients and controls, statistical evaluation, and wrote part of the manuscript. EHB participated in assessed the control population and in data collection. WWH participated in assessed the control population and in data collection. ALD participated in assessed the patient and control populations, in data collection and laboratory evaluation of the patients and controls. RCPG participated in conceiving the study. JCB participated in conceiving the study, assessing the control population, statistical evaluation and wrote part of the manuscript. All authors approved the final version of the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements This work was supported in part by the Immunogenetics Program, Southern Iron Disorders Center, Southern Hematology & Oncology, P.C. and NCI training grant R25 CA 76023-04 UAB Cancer Research Experiences for Student (CaRES). Figures and Tables Table 1 Frequencies of Country of Ancestry Reports in Adult Whites in Central Alabama. * Country Melanoma Probands % (n) Control Subjects % (n) P Value† England 46.7 (42) 43.9 (140) 0.6394 Ireland 62.2 (56) 45.1 (144) 0.0042 Scotland 44.4 (40) 27.0 (86) 0.0015 Ireland + Scotland ("Celts") 67.8 (61) 55.8 (178) 0.0417 Wales 0 (0) 4.7 (15) 0.0223 British Isles 73.3 (66) 75.6 (241) 0.6679 Native American 16.7 (15) 23.8 (76) 0.1493 Germany 24.4 (22) 33.5 (107) 0.1009 France 5.6 (5) 14.1 (45) 0.0287 Norway 1.1 (1) 1.6 (5) 0.6059 Netherlands 6.7 (6) 9.1 (29) 0.4678 Lithuania 0 (0) 0.3 (1) 0.7800 Czechoslovakia 1.1 (1) 0.9 (3) 0.6315 Spain 0 (0) 2.2 (7) 0.1730 Austria 1.1 (1) 2.2 (7) 0.4442 Belgium 1.1 (1) 0.3 (1) 0.3921 Denmark 1.1 (1) 1.6 (5) 0.6059 Finland 0 (0) 0.3 (1) 0.7800 Greece 2.2 (2) 1.3 (4) 0.3941 Hungary 1.1 (1) 0.6 (2) 0.5265 Italy 0 (0) 5.6 (18) 0.0102 Poland 0 (0) 5.0 (16) 0.0172 Romania 0 (0) 0.6 (2) 0.6079 Russia 0 (0) 2.5 (8) 0.1343 Sicily 0 (0) 1.6 (5) 0.2866 Sweden 1.1 (1) 4.4 (14) 0.1211 Switzerland 0 (0) 1.3 (4) 0.3685 Europe Not British Isles 63.3 (57) 62.7 (200) 0.9120 North, Central, and South American Countries 4.4 (4) 2.2 (7) 0.2057 Near and Middle East Countries 1.1 (1) 0.9 (3) 0.6315 African Countries 0 (0) 0.3 (1) 0.7800 Don't Know 30.0 (27) 30.1 (96) 0.9863 *These data include all reports by each evaluable study participant; there were 90 evaluable melanoma probands and 319 evaluable control subjects. The category "British Isles" was defined as the reports in England, Ireland, Scotland, and Wales categories. The category "Europe Not British Isles" is comprised of the composite data from the corresponding individual countries displayed in the present table. Participants who indicated specific "Native American" ancestry most frequently reported Cherokee and Creek descent. The "North, Central, and South America Countries" category (other than "Native Americans") included Canada, U.S., Virgin Islands, Colombia, and Brazil. The category "Near and Middle East Countries" included reports about grandparents from Iran, Lebanon, and Syria. The single observation in "African Countries" represents the Republic of South Africa. The "Don't Know" categories include data from participants who reported this category for one, two, or three grandparents. †Chi-square or Fisher exact tests were used, as appropriate; p values < 0.05 were defined as significant. Table 2 Country of Ancestry Indices in Adult Whites in Central Alabama. * Country Melanoma Probands Control Subjects p Value† England 0.6559 0.7755 0.2896 Ireland 0.9573 0.6603 0.0150 Scotland 0.5924 0.3038 0.0031 Ireland + Scotland ("Celts") 1.5500 0.9640 0.0002 Wales 0 0.0674 (0.0005) British Isles 2.2057 1.8069 0.0032 Native American 0.3184 0.2998 0.8493 Germany 0.4370 0.4610 0.8081 France 0.0889 0.1888 0.0001 Norway 0.0222 0.0230 0.9770 Netherlands 0.0903 0.1089 0.6909 Lithuania 0 0.0031 (0.9323) Czechoslovakia 0.0037 0.0125 0.2967 Spain 0 0.0219 (0.0442) Austria 0.0028 0.0214 0.0486 Belgium 0.0111 0.0031 0.4951 Denmark 0.0073 0.0167 0.3996 Finland 0 0.0063 (0.6110) Greece 0.0203 0.0408 0.4491 Hungary 0.0222 0.0094 0.5863 Italy 0 0.1129 (0.0002) Poland 0 0.0512 (0.0006) Romania 0 0.0157 (0.3308) Russia 0 0.0439 (0.0111) Sicily 0 0.0392 (0.0667) Sweden 0.0111 0.0674 0.0186 Switzerland 0 0.0157 (0.1400) Europe Not British Isles 0.7169 1.2628 0.0001 North, Central, and South American Countries 0.1000 0.0533 0.4378 Near and Middle East Countries 0.0444 0.0408 0.9407 African Countries 0 0.0063 (0.6110) Don't Know 0.5778 0.5235 0.6126 * These data include all reports by each evaluable study participant. The questionnaire and interview reports from each participant were evaluated to yield individual country of ancestry scores which reflect proportional national ancestry. For this, each participant was assigned 4.00 points divided equally among each of his/her 4 grandparents. If a participant indicated 2 or more countries of ancestry for a grandparent, the 1.00 point for that grandparent was equally divided among the respective countries to formulate the scores of individual participants. We also computed aggregate country of ancestry indices for melanoma probands and control subjects as estimates of group ancestry composition. These indices were expressed as the quotient of total individual scores for respective countries and the total number of melanoma probands or control subjects, as appropriate. The category "British Isles" was defined as the reports in England, Ireland, Scotland, and Wales categories. The category "Europe not British Isles" is comprised of the composite data from the corresponding individual countries displayed in the present table. Participants who indicated specific "Native American" ancestry most frequently reported Cherokee and Creek descent. The "North, Central, and South America Countries" category (other than "Native Americans") included Canada, United States, Virgin Islands, Colombia, and Brazil. The category "Near and Middle East Countries" included reports about grandparents from Iran, Lebanon, and Syria. The single observation in "African Countries" represents the Republic of South Africa. The "Don't Know" categories include data from participants who reported this category for one, two, or three grandparents. † Student t test cannot be used to compare country of ancestry data groups in which all values were 0 (no variability). Accordingly, we arbitrarily assigned a country of ancestry datum of 0.25 for one person in each proband country group for which there were no actual country of ancestry reports. Student t test was then performed using this modified data group; estimated values of p from these tests are displayed in parentheses. Table 3 HLA-DRB1*04 Phenotype Frequencies in Central Alabama, European and Australian White Populations. Country (City or Region) HLA-DRB1*04 Frequency Melanoma Patients (n) Control Subjects (n) USA (Central Alabama) 0.3820 (123)‡ 0.2148 (340)‡ England (Manchester) NA 0.3257 (786)† Wales NA 0.3651 (7,165)† Ireland (Belfast) NA 0.3422 (5,000)† Ireland (Dublin) NA 0.3063 (1,910)† Orkney Island NA 0.3556 (104)† France (Brest) NA 0.3933 (150)† Germany (Munich) 0.2350 (98)# 0.2170 (847)# Australia (Melbourne) 0.5590 (34)¶ 0.3280 (210)¶ NA = Not available ‡Reported in references 9, 20 † Reported in reference 22. # Reported in reference 13 ¶ Reported in reference 11 ==== Refs Alabama Department of Public Health Alabama Statewide Cancer Registry Ries LA Wingo PA Miller DS Howe HL Weir HK Rosenberg HM Vernon SW Cronin K Edwards BK The annual report to the nation on the status of cancer, 1973–1997, with a special section on colorectal cancer Cancer 2000 88 2398 2424 10820364 10.1002/(SICI)1097-0142(20000515)88:10<2398::AID-CNCR26>3.0.CO;2-I Acton RT Balch CM Budowle B Go RCP Roseman JM Soong S-J Barger BO Ferrone S Immunogenetics of Melanoma Melanoma, Antigens and Antibodies 1982 New York: Plenum Publishing Corporation 1 21 Acton RT Balch CM Barger BO Budowle B Go RCP Soong S-J Roseman JM The occurance of melanoma and its relationship with host, lifestyle and environmental factors 1983 The Hague: Martinus Nijhoff Publishers 151 182 Holman CD Armstrong BK Pigmentary traits, ethnic origin, benign nevi, and family history as risk factors for cutaneous malignant melanoma J Natl Cancer Inst 1984 72 257 266 6582314 Long CC Marks R Increased risk of skin cancer: another Celtic myth? A review of Celtic ancestry and other risk factors for malignant melanoma and nonmelanoma skin cancer J Am Acad Dermatol 1995 33 658 661 7673500 10.1016/0190-9622(95)91289-4 Lee JK Factors associated with melanoma incidence and prognosis Semin Surg Oncol 1996 12 379 385 8914201 10.1002/(SICI)1098-2388(199611/12)12:6<379::AID-SSU2>3.0.CO;2-7 Acton RT Barger BO Go RCP Roseman J Soong S-J Balch C HLA-DR phenotypes predicts onset of melanoma in a subethnic group of caucasians Proc Am Assoc Cancer Res 1983 24 189 Barger BO Acton RT Soong S-J Roseman J Balch C Increase of HLA-DR4 in melanoma patients from Alabama Cancer Res 1982 42 4276 4279 6980704 Budowle B Barger BO Balch CM Go RC Roseman JM Acton RT Associations of properdin factor B with melanoma Cancer Genet Cytogenet 1982 5 247 251 6917774 10.1016/0165-4608(82)90031-0 Czarneck D Nicholson I Tait B Nash C HLA DR4 is associated with the development of multiple basal cell carcinomas and malignant melanoma Dermatology 1993 187 16 18 8324271 Czarnecki D Tait B Nicholson I Lewis A Multiple non-melanoma skin cancer: evidence that different MHC genes are associated with different cancers Dermatology 1994 188 88 90 8136554 Mueller-Eckhardt G Schendel DJ Hundeiker M Riedel T O'Neill GJ Riethmuller G Mueller-Eckhardt C Possible association between HLA-DR5 and superficial spreading melanoma (SSM) Int J Cancer 1984 34 751 755 6334654 Long CC Darke C Marks R Celtic ancestry, HLA phenotype and increased risk of skin cancer Br J Dermatol 1998 138 627 630 9640367 10.1046/j.1365-2133.1998.02173.x Lee JE Reveille JD Ross MI Platsoucas CD HLA-DQB1*0301 association with increased cutaneous melanoma risk Int J Cancer 1994 59 510 513 7960221 Monzon J Liu L Brill H Goldstein AM Tucker MA From L McLaughlin J Hogg Lassam NJ CDKN2A mutations in multiple primary melanomas N Engl J Med 1998 338 879 887 9516223 10.1056/NEJM199803263381305 Barton E Barton J Hollowell WW Acton RT Countries of ancestry reported by hemochromatosis probands and control subjects in central Alabama Ethn Dis 2004 14 73 81 15002926 Acton RT Dasanayake AP Harrison RA Li Y Roseman JM Go RC Wiener H Caufield PW Associations of MHC genes with levels of caries-inducing organisms and caries severity in African-American women Hum Immunol 1999 60 984 989 10566600 10.1016/S0198-8859(99)00088-9 Roberts D Is Celtic skin different? Br J Dermatol 1996 135 34 Alarcon GS Phillips RM Wasner CK Acton RT Barger BO DR antigens in systemic sclerosis: lack of clinical correlations Tissue Antigens 1985 26 156 158 3864290 Woolf B On estimating the relation between blood group and disease Ann Hum Genet 1955 19 251 253 14388528 Terasaki P Gjertson D HLA 1997. UCLA Tisssue Typing Laboratory Los Angeles, CA: UCLA Tissue Typing Laboratory 1997 Pickett AJ The English in Georgia History of Alabama and Incidentally of Georgia and Mississippi, from the earliest period 1851. Clerk's office of the District Court of the United States for the Middle District of Alabama 1962 Republished: Birmingham, Birmingham Book and Magazine Co. 258 269 Southerland H Brown J The Federal Road through Georgia, the Creek Nation, and Alabama, 1806–1836 1989 Tuscaloosa, The University of Alabama Press 1 198 Marcum J Williams MW Geography. Population 1989 Chapel Hill, The University of North Carolina Press 555 556 Miller R Wilson CR, Ferris W Ethnic Life. Germans Encyclopedia of Southern Culture 1989 Chapel Hill, The University of North Carolina Press 429 430 Alabama Census Returns 1820 and An Abstract of Federal Census of Alabama 1830 Alabama Historial Society Quarterly Fall Issue 1944 Montgomery, Alabama State Department of Archives and History 1944 333 315 US Census Bureau Accessed on June 27, 2002 US Census Bureau Accessed on July 7, 2002 Pickett A History of Alabama and Incidentially of Georgia and Mississippi, from the Earliest Period. 1851 Clerk's Office of the District Court of the United States for the Middle District of Alabama 1962 Republished: Birmingham, Birmingham Book and Magazine Co 1 685 Williams WL Wilson CR, Ferris W Ethnic Life. Cherokees Encyclopedia of Southern Culture 1989 Chapel Hill, The University of North Carolina Press 423 424 Paredes J Wilson CR, Ferris W Ethnic Life. Creeks Encyclopedia of Southern Culture 1989 Chapel Hill, The University of North Carolina Press 426 Lucotte G Dieterlen F A European allele map of the C282Y mutation of hemochromatosis: Celtic versus Viking origin of the mutation Blood Cells, Mol Dis 2003 31 262 267 12972035 10.1016/S1079-9796(03)00133-5 Byrnes V Ryan E Barrett S Kenny P Mayne P Crowe J Genetic hemochromatosis, a Celtic disease: is it now time for population screening? Genet Test 2001 5 127 130 11551098 10.1089/109065701753145583 Fitzpatrick TB The validity and practicality of sun-reactive skin types I through VI Arch Dermatol 1988 124 869 871 3377516 10.1001/archderm.124.6.869 Bateman AC Turner SJ Theaker JM Howell WM HLA-DQB1*0303 and *0301 alleles influence susceptibility to and prognosis in cutaneous malignant melanoma in the British Caucasian population Tissue Antigens 1998 52 67 73 9714476 Howell WM Bateman AC Turner SJ Theaker JM HLA genotype and increased risk of skin cancer Br J Dermatol 1998 139 1111 9990384 10.1046/j.1365-2133.1998.2576a.x Lee JE Lu M Mansfield PF Platsoucas CD Reveille JD Ross MI Malignant melanoma: relationship of the human leukocyte antigen class II gene DQB1*0301 to disease recurrence in American Joint Committee on Cancer Stage I or II Cancer 1996 78 758 763 8756369 10.1002/(SICI)1097-0142(19960815)78:4<758::AID-CNCR11>3.3.CO;2-4 Lombardi ML Mercuro O Pirozzi G Ionna F Lombari V Mozzillo N Manzo C Molecular analysis of HLA DRB1 and DQB1 polymorphism in Italian melanoma patients J Immunother 1998 21 435 439 9807738 Lulli P Grammatico P Brioli G Catricala C Morellini M Roccella M Mariani B Pennesi G Roccella F Cappellacci S Trabace S HLA-DR and -DQ alleles in Italian patients with melanoma Tissue Antigens 1998 51 276 280 9550328

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==== Front BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-4-501531039610.1186/1471-2407-4-50Research ArticleFrequency of cancer in children residing in Mexico City and treated in the hospitals of the Instituto Mexicano del Seguro Social (1996–2001) Juárez-Ocaña Servando [email protected]ález-Miranda Guadalupe [email protected]ía-Aranguré Juan Manuel [email protected]ón-Macías Mario Enrique [email protected]ínez-García María del Carmen [email protected]érrez Arturo [email protected] Unidad de Investigación Médica en Epidemiología Clínica, Hospital de Pediatría, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, México2 Registro de Cáncer en Niños, Hospital de Pediatría, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, México3 División de Promoción de Desarrollo, Coordinación de Investigación en Salud del Instituto Mexicano del Seguro Social, México2004 13 8 2004 4 50 50 18 2 2004 13 8 2004 Copyright © 2004 Juárez-Ocaña et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The objective of this article is to present the frequency of cancer in Mexican children who were treated in the hospitals of the Instituto Mexicano del Seguro Social in Mexico City (IMSS-MC) in the period 1996–2001. Methods The Registry of Cancer in Children, started in 1996 in the IMSS-MC, is an on-going, prospective register. The data from 1996 through 2001 were analyzed and the different types of cancer were grouped according to the International Classification for Cancer in Children (ICCC). From this analysis, the general and specific frequencies by age and by sex were obtained for the different groups of neoplasms. Also, the frequency of the stage of the disease that had been diagnosed in cases of children with solid tumors was obtained. Results A total of 1,702 new cases of children with cancer were registered, with the male/female ratio at 1.1/1. Leukemias had the highest frequency with 784 cases (46.1%) and, of these, acute lymphoblastic leukemias were the most prevalent with 614 cases (78.3%). Thereafter, in descending order of frequency, were tumors of the central nervous system (CNST) with 197 cases (11.6%), lymphomas with 194 cases (11.4%), germinal cell tumors with 110 cases (6.5%), and bone tumors with 97 cases (5.7%). The highest frequency of cancer was found in the group of one to four year-olds that had 627 cases (36.8%). In all the age groups, leukemias were the most frequent. In the present work, the frequency of Hodgkin's disease (~4%) was found to be lower than that (~10%) in previous studies and the frequency of tumors of the sympathetic nervous system was low (2.3%). Of those cases of solid tumors for which the stage of the disease had been determined, 66.9% were diagnosed as being Stage III or IV. Conclusions The principal cancers in the children treated in the IMSS-MC were leukemias, CNST, and lymphomas, consistent with those reported by developed countries. A 2.5-fold reduction in the frequency of Hodgkin's disease was found. Of the children, the stage of whose disease had been determined, two thirds were diagnosed as having advanced stages of the disease. Epidemiologycancer in childrendiagnostic stageHodgkin's diseaseneuroblastomas ==== Body Background The frequency of malignant neoplasms in children has been found to vary among countries. For example, in children in Canada, the United States, and Europe, the three most common cancers are leukemias, tumors of the central nervous system (CNST), and lymphomas [1-3], whereas in children in Latin America, the order of frequencies is distinct: leukemias are still in first place, with lymphomas being more common than are CNST [2,4-6]. In other countries such as Nigeria, Malawi, and Egypt, lymphomas are the principal neoplasias [2]. The percentage of cases of each type of neoplasm in relation to the total number of cancers is also different. In the developed countries, the percentages for leukemias range between 30 and 37%; for CNST, between 18 and 27%; and for lymphomas, between 7 and 12% [1-3]. In Latin America, the percentages for leukemias are between 27 and 44%; of lymphomas, between 13 and 22%; and of CNST, between 10 and 19% [2,4-6]. In African countries, the percentages of lymphomas range between 30 and 64% [2]. In Asiatic countries such as Japan and China, the percentages of leukemias have been found to be between 30 and 40%; of CNST, between 12 and 20%; and of lymphomas, between 10 and 20% [2]. The study of the frequency of cancer in children not only is of interest to the clinical physician because it helps him/her to establish the pre-testing probability for a child suspected of having cancer [7], but also is of interest to the personnel in charge of the planning and programming of the medical attention for these children, as pertains to the assignment of human resources (physicians, specialized nurses, social workers, and others) and to the allotment of financial resources (centers providing medical attention, laboratories, imaging facilities, medicines, etc.) that are necessary for treating the children [8]. In Mexico, there existed data only from retrospective studies on the epidemiology of cancer in children and those studies had been carried out prior to 1993 [9,10]. Therefore, it was necessary to use more recent data, especially those from prospective studies in which the under-reporting of cases was reduced. The objective of this paper is to present the frequency of malignant neoplasias in the child population residing in the area served by the Instituto Mexicano del Seguro Social (IMSS-MC). The data were obtained from the Registry of Cancer in Children which was started in 1996 at, and is maintained by the Hospital de Pediatría del Centro Médico Nacional "Siglo XXI" of the Instituto Mexicano del Seguro Social in Mexico City (IMSS-MC). Methods Type of study Observational, descriptive, and prospective hospital inquiry. Population studied The Instituto Mexicano del Seguro Social (IMSS) offers medical attention to the population of workers and their families, which comprises 50% of the Mexican population [10]. The IMSS divides the population that it attends into four regions: North, South, East, and West. The IMSS-Mexico City (IMSS-MC) attends the population of the Southern region, which includes the populations in various states in the country, in addition to that of Mexico City. With the objective of avoiding a bias in the selection of the population for the present study, we included for analysis only cases from those states in which we were sure that more than 90% of the cases presented had been registered. This would be true of cases from the states that are located closest to Mexico City and of the cases in which the children who developed cancer had to be sent to Mexico City for treatment. Therefore, the cases analyzed came from Mexico City and from the following selected states: State of Mexico, Morelos, Guerrero, and Chiapas. It should be mentioned that not only the cost of treatment, but also the cost of transportation to Mexico City is covered by IMSS. This financial support provided by IMSS to families helps to ensure that cases of cancer come to the attention of IMSS-MC and are duly registered. Newly diagnosed cases of malignant neoplasias in children less than 15 years of age treated in the IMSS-MC were included in this study. In 100% of these cases, the diagnosis was confirmed by histological tests and/or by aspiration of the bone marrow. Participating facilities The IMSS-MC has two hospitals that provide medical attention to children with cancer. The Departments of Hematology and of Pediatric Oncology of both the Hospital de Pediatría of the Centro Médico Nacional "Siglo XXI" (HP) and the Hospital General del Centro Médico Nacional "La Raza" (HR) participated in this study. Both facilities have the infrastructure (competent personnel and suitable technology) needed for the precise diagnosis of a cancer. Study period Included in this study were the cases attended from 1 January 1996 to 31 December 2001. Study variables Prior to carrying out the study, a form for recording the variables of interest was designed. For this article, the variables analyzed were the following: type of neoplasia, sex of the patient, age at the time of diagnosis, and the stage of the cancer for those children with solid tumors. Procedure At each hospital, a fulltime nurse was assigned to register all new cases of cancer. Prior to collecting the data, each nurse was instructed in procedure necessary for obtaining all the different variable of the study. The nurse interviewed the parents and reviewed the clinical record of each child in order to obtain all the necessary information. The nurse was also taught how to encode and to determine the stage of cases of solid tumors. The standardization of the coding and determination of the stage of the solid tumors was done by all personnel (three physicians and two nurses) concerned with the registry; an excellent concordance was obtained (unweighted Kappa of 0.85) [11]. In addition to other duties related to the Registry, each nurse spent three days a week in the oncology and hematology departments of the hospital (HP or HR), searching for cases of children who, being suspected of having cancer, had been registered in a specific file and boarded at the hospital. After reviewing the clinical record in each file, the nurse either included the case in the study (encoding the data if the diagnosis of cancer had been confirmed) or eliminated the case if the diagnosis was not confirmed. If for any reason, the patient was discharged from the hospital and the diagnosis was undetermined, the nurse reviewed the clinical record in the clinical archive of the hospital in order to learn what the final diagnosis was. To encode the different cases of cancer, topographical and morphological coding was used. The second edition of the "International Classification of Diseases for Oncology" (ICD-O-2) was used for the cases collected from 1996 through 1999; the third edition (ICD-O-3), for the cases collected from 2000 through 2001 [12,13]. For determining the stage of cases of lymphomas and carcinomas, the recommendations of the American Joint Committee on Cancer (AJCC) and the International Union Against Cancer (IUAC) were used [14]. The stages for tumors of the central nervous system (CNST), neuroblastoma, retinoblastoma, renal tumors (Wilms' tumor), and those of the liver, bones, soft tissues, and germinal cells (GCT) were determined following the recommendations of the Children's Oncology Group [15]. The Child-Check Program developed by the International Agency for Research on Cancer (IARC) [16] was used to evaluate the internal consistency of the individual registries of cancer and to convert the nomenclature of ICD-O-2 to the International Classification of Childhood Cancer (ICCC) [17]. This program made crosses between different variables in order to find inconsistencies among the collected data. The crosses that were made were sex-topography, sex-histology, age-tumor type, unlikely combinations of topography-morphology, errors between date of birth and diagnosis, and duplication of cases. The result was a list of combinations, although either improbable or of low probability, that were needed for review in order to verify data or to correct data by rechecking the records of the patients. Cases of the ICD-O-3 that were not included in the Child-Check Program were classified by using other procedures and the data entered manually. Statistical analysis Cases were grouped according to the ICCC [17] that has established 12 different groups of cancer in children. From these were calculated the absolute and relative frequencies, both in general and according to sex and to age, the latter category being divided into four subgroups: under one year; one to four year-olds; five to nine year-olds; and ten to 14 year-olds. Due to the fact that the procedure for the determination of the stage of the disease was initiated in 1 Jun 1998, such determinations were made in 658 of the case of solid tumors. Therefore, the frequencies of the diagnostic states were based on this number of cases of children with solid tumors. Results A total of 1,702 cases of malignant neoplasias were analyzed. Of these, in the order of the most frequently found, were the following types of tumors: leukemias, 784 cases (46.1%); CNST, 197 cases (11.6%); lymphomas, 194 cases (11.4%); germinal cell tumors (GCT), 110 cases (6.5%); and bone tumors (BT), 97 cases (5.7%); and the remainder of the neoplasias were found in low percentages (Table 1). Table 1 shows that, in examining the subtypes of the different groups of neoplasias, it was found that, of the leukemias, the most frequent were the acute lymphoblastic leukemia (n = 614; 78.3%); of the lymphomas, the non-Hodgkin lymphomas [Burkitt, non Burkitt, and nonspecific together (n = 129; 66.5%)]; of the CNST, the astrocytomas (n = 97; 49.2%); of the tumors of the sympathetic nervous system (SNST), neuroblastomas and ganglioneuroblastomas together (n = 36; 92.3%); of the renal tumors, nephroblastoma [Wilms' tumor (n = 62; 87.3%)]; of hepatic tumors, hepatoblastoma (n = 26; 86.7%); of BT, osteosarcoma (n = 70; 72.2%); of the sarcomas of the soft tissues, rabdomyosarcoma and embryonic sarcoma together (n = 49; 55.1%); of the GCT, gonadal tumors (n = 80; 72.7%); and of the carcinomas, adrenocortical, malignant melanoma and skin carcinoma together (n = 9; 50.1%). The percentages of the different neoplasias showed variations according to sex and age group. These findings modified the pattern of presentation and made it different from the overall pattern. In males, over 70% of the cases consisted of the following types of tumors: leukemias (n = 412; 46.6%); lymphomas (n = 138; 15.6%); and CNST (n = 84; 9.5%). In females, 73.6% of the cases consisted of leukemias (n = 372; 45.5%); CNST (n = 113; 13.8%); GCT (n = 61; 7.5%); and lymphomas (n = 56; 6.8%). Overall, the ratio of males to females was 1.1; however, this ratio varied for the different groups of neoplasias, most notably a high of 2.5 for lymphomas and a low of 0.8 for both hepatic tumors, BT and GCT (Table 2). Table 2 shows that, for all age groups, leukemias had the highest frequency, ranging between 27.9 to 50.5%. In second and third place for the different age groups were the following types of tumors: under one year of age, retinoblastoma (n = 15; 17.4%) and GCT (n = 13; 15.1%); 1–4 year-olds, CNST (n = 69; 11.0%) and retinoblastoma (n = 56; 8.9%); 5–9 year-olds, lymphomas (n = 79; 16.2%) and CNST (n = 59; 12.1%); and 10–14 year-olds, lymphomas (n = 66; 13.2%) and BT (n = 65; 12.9%). With respect to the neoplasias in patients from other states in the Mexican Republic, leukemias were also found to have the highest frequency, followed by lymphomas and/or CNST, with discrete variations in the frequencies as mentioned above. It should be noted that in one state (Chiapas), although retinoblastoma was only the fourth most frequent there, its frequency (8.9%) was one of the highest (Table 3). In regard to the spread of the disease at the time of diagnosis for those children the stage of whose solid tumors had been determined, 89 (13.5%) were Stage I; 129 (19.6%), Stage II; 242 (36.8%), Stage III; and 198 (30.1%), Stage IV or higher. Discussion This is the first report of data, covering the six year period from 1996–2001, taken from the on-going Registry of Cancer in Children that was started in 1996 in Mexico City by the Instituto Mexicano del Seguro Social (IMSS-MC). The strategy of having placed a nurse in each of the two hospitals involved with the Registry of Children with Cancer resulted in the great majority (more than 90%) of the new cases that were treated by the two hospitals being identified and duly registered. We therefore concluded that this Registry of Cancer in Children was one of the most complete of its kind undertaken in Mexico City. Having access to the clinical records of the patients served to improve the quality of the data that was registered because, since cases were registered as soon as they were diagnosed, few cases were overlooked. The quality of the data was also enhanced by the use of the Child-Check Program, with which the possible errors, not only of registry but also of capture, were reviewed and were eliminated upon rechecking the respective clinical record in the hospital files. As mentioned in Methods, in 100% of the cases, the histopathology reports for children with solid tumors, or the reports on the aspirated bone marrow for children with leukemia, were obtained. Therefore, the data that was obtained for the children with cancer who were residents in the area covered by IMSS-MC and treated in Mexico City were the most precise that have been gathered to date in Mexico. The frequency of a disease in a population is a method of obtaining the pre-test probability that a patient has before a diagnostic test is performed [7]. It is important that this value be known, because there is a direct correlation with the positive predictive value of the test. This, in turn, is the probability that an individual whose test result was positive has of having the suspected disease [7]. That is, in the case of the children who were attended in the tertiary health care hospitals in IMSS-MC, the pre-test probability for a child whose parents sought medical attention (without taking into account his/her symptomatology) is a 43.4% chance of having some form of leukemia, an 11.1% chance of lymphoma, or a 12.6% chance of CNST. This probability increases or decreases depending on the symptomatology that the child presents, on the test requested, and on the result (positive or negative) of said test. Knowing the frequency of diseases in general and, in this case, the frequency of the different types of cancer that Mexican children present is therefore an important aid in diagnosis. The same can be said for the frequency of cancer by age and by sex. As has been mentioned, knowing the frequency of diseases serves in the estimation of administrative needs, not only with respect to personnel but also to the equipment and supplies necessary for diagnosis and treatment, and for providing medical attention in general and in particular for children with cancer [8]. Given that, from the data, 46.1% of these children develop leukemia, appropriate provision has to be made for their treatment, as well as for the children with other forms of cancer. A more clinical aspect, particular to children with cancer, that indicated the spread of their disease was the stage of the disease at the time of diagnosis. It was established that 66.9% were in stages III or IV, a finding that generally means the prognosis for the patient is not good. This datum was consistent with, and more precise than the result of the previously reported retrospective study, in which it was found that 56.4% had been diagnosed as having an advanced stage (III or IV) of the disease [10]. This fact indicates that, for Mexican children that develop a cancer, programs for integrated medical attention must be designed such that early diagnosis is a priority. Although the early diagnosis of cancer in children as a factor in a good prognosis is controversial [18], it is probable that such a program would have a great impact in Mexico. For this reason, as has been pointed out in prior studies [19], the influence of various factors such as the patient's family (educational level, socio-economic status, etc.), the type of cancer, the age of the child, the health system, and the physicians that care for the child on intake must be taken into account. Although the results obtained for the different groups of cancer in Mexican children in this study showed general agreement with data previously reported, there were some differences. Consistent with data obtained in other studies [9,10], it was found that leukemia, CNST, and lymphoma were the principal neoplasias and that Mexican children have one of the highest percentages of leukemias in the world (46.1% vs. 27–44%) [2,20,21]. In contrast to previous studies in Mexico, the frequency of Hodgkin's disease was notably lower, thus reducing the overall frequency of lymphoma to slightly lower than that of CNST and putting it in the range reported for developed countries (11.4% vs. 7–12%) [2]. We consider this reduction in the frequency of Hodgkin's disease a noteworthy and probably real effect, not just an artifact of the quality of the registry. The previous studies, being retrospective, had a greater tendency to under-register the number of cases and therefore result in an lower frequency than the actual value; yet, here, with a more accurate study, the frequency obtained was lower still (10% a 3.8%) (Table 4) [9,10]. However, we consider it necessary to obtain the incidence rates to confirm this and will do so for a forthcoming study in which we will present exclusively the incidence of cancer in children who are residents of Mexico City. It should be mentioned that the same phenomenon (reduction in the HD) was shown by Linet et al. [22] in their study of children in the U.S. during the period 1974 to 1995. There has been no explanation of this phenomenon because no program attempting to reduce HD in Mexico or in other parts of the world has been established. However, should an infectious agent be one of the causes in the development of HD, it is possible that the indiscriminant use of antiviral and/or antibiotics in Mexico may play a role. Perhaps future studies will be able to explain the phenomenon. In this and the two prior studies in Mexico, the frequency of CNST was found to be less than that in developed countries (18–27%). However, the lowered frequency of lymphomas found in the present study changed the hierarchical pattern of tumors, with CNST now having just edged out lymphomas for second place. We do not consider that this population has, strictly speaking, the pattern of U.S.-Canada-Europe, nor that of Latin America. We can say that the pattern of neoplasias in Mexican children was found to be a phenomenon in transition. It was interesting that the pattern of presentation found in children in Mexico City was different to that of the children that live in the northern part of the country (data not shown). The pattern in children from the north was similar to that of children in the U.S. and Canada, in that the principal neoplasias were leukemias, CNST, and lymphomas and had very similar frequencies [23,24]. This finding suggested the possibility that factors which cause cancer in children in the north of Mexico may be different to those causing cancer in children in Mexico City. Data for children under one year of age showed that, whereas neuroblastoma was the principal tumor for this age group in developed countries [1,3], in our study it was leukemias (27.9%), with the frequency of neuroblastoma being much lower than in developed countries (10.5 vs. 27.4). This finding is one that should be followed up because there are two factors that would have affect the reported number of cases and, hence the frequency: Because Mexico does not have the screening programs for detecting children with neuroblastoma that other countries do [25], it was probable that cases of this disease were not being diagnosed. Also, it is known that some of these tumors do regress spontaneously [26]. Another interesting aspect of the present study was that one of the highest frequencies of GCT (6.5%) in the world was found, a frequency similar to that for some Asiatic countries, such as Japan and Singapore (6.8%) [2]. We do not have an explanation of this finding, but studies directed toward establishing the causes of this high frequency should be carried out. Finally, it should be mentioned that one state of the Mexican Republic, Chiapas, was found to have one of the highest frequencies of retinoblastoma (8.9%), a frequency very similar to that of countries of Africa (Zimbabwe, 9.6%) and in India (Madras; 9.4%) [2]. Chiapas is one of the poorest states in Mexico and, as has been suggested, it is possible that the state of nutrition may play a role in the development of this tumor [27]. However, as for Hodgkin's Disease, it will be necessary to calculate the incidence rates to make this observation more precise. Conclusions It may be concluded that, in the children residing in Mexico City that were included in this study, the principal neoplasias were leukemias, CNST, and lymphomas, findings that were consistent with previously published data. It was found that, in comparison to previous studies in Mexico City, there was a reduction in the frequency of lymphomas and especially of Hodgkin's disease, and that, of the children with solid tumors, two thirds were diagnosed as having advanced stages (III-IV) of the disease. Competing interest None declared. Authors' contributions S J-O analyzed the data and wrote the first draft of the manuscript. G G-M registered, recorded, and analyzed the data. JM M-A, ME R-M, and MC M-G conceived and the designed the study and analyzed the data. A F-G conceived and designed the study, analyzed the data, and provided guidance to all aspects of this project. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgments The authors thank Veronica Yakoleff for translating and revising the manuscript. Supported in part by a grant from the Fondo para el Fomento a la Investigación (FOFOI grant number IMSS FP-363). Figures and Tables Table 1 Frequency of cancer in children served by the Instituto Mexicano del Seguro Social and treated in hospitals in Mexico City (1996–2001). Diagnostic group Total frequency Frequency by group n % n % I. Leukemias 784 46.1 784 100.0 IA. Acute lymphoblastic leukemia 614 36.1 614 78.3 IB. Acute non-lymphoblastic leukemia 146 8.6 146 18.6 CI. Chronic myeloid leukemia 16 1.0 16 2.0 ID. Other specific leukemias 4 0.2 4 0.5 IE. Nonspecific leukemias 4 0.2 4 0.5 II. Lymphomas* 194 11.4 194 100.0 IIA. Hodgkin disease 65 3.8 65 33.5 IIB. Non-Hodgkin lymphoma 104 6.1 104 53.6 IIC. Burkitt lymphoma 21 1.2 21 10.8 IIE. Nonspecific lymphomas 4 0.2 4 2.1 III. Central Nervous System Tumors 197 11.6 197 100.0 IIIA. Ependymoma 29 1.7 29 14.7 IIIB. Astrocytoma 97 5.7 97 49.2 IIIC. Primitive neuroectodermal tumors 52 3.1 52 26.4 IIID. Other gliomas 9 0.5 9 4.6 IIIE. Other specific intracranial and intraspinal neoplasms 9 0.5 9 4.6 IIIF. Other nonspecific intracranial and intraspinal neoplasms 1 0.1 1 0.5 IV. Symphatetic Nervous System Tumors 39 2.3 39 100.0 IVA1. Ganglioneuroblastoma 6 0.3 6 15.4 IVA2. Neuroblastoma 30 1.7 30 76.9 IVB1. Meduloepitelioma 1 0.1 1 2.6 IVB3. Neuroepithelioma 1 0.1 1 2.6 IVB4. Olfactory neuroblastoma 1 0.1 1 2.6 V. Retinoblastoma 73 4.3 73 100.0 V1. Retinoblastoma 46 2.7 46 63.0 V2. Differentiated retinoblastoma 24 1.4 24 32.9 V3. Undifferentiated retinoblastoma 3 0.2 3 4.1 VI. Renal Tumors 71 4.2 71 100.0 VIA1. Nephroblastoma 62 3.6 62 87.3 VIA2. Rabdoid sarcoma 2 0.2 2 2.8 VIA3. Clear-cell sarcoma 6 0.3 6 8.5 VIB. Renal Carcinoma 1 0.1 1 1.4 VII. Hepatic Tumors 30 1.8 30 100.0 VIIA. Hepatoblastoma 26 1.5 26 86.7 VIIB1. Hepatic carcinoma 1 0.1 1 3.3 VIIB2. Hepatic carcinoma undifferentiated 1 0.1 1 3.3 VIIB3. Neuroendocrine carcinoma 1 0.1 1 3.3 VIIB4. Acinar adenocarcinoma 1 0.1 1 3.3 VIII. Bone Tumors 97 5.7 97 100.0 VIIIA. Osteosarcoma 70 4.1 70 72.2 VIIIB. Chondrosarcoma 4 0.2 4 4.1 VIIIC. Ewing sarcoma 18 1.1 18 18.6 VIIID. Other specific malignant tumors 3 0.2 3 3.1 VIIIE. Unspecified malignant bone tumors 2 0.2 2 2.1 IX. Soft-Tissue Sarcomas 89 5.2 89 100.0 IXA. Rhabdomyosarcoma and embryonal sarcoma 49 2.9 49 55.1 IXB. Fibrosarcoma, neurofibrosarcoma and others fibromatous neoplasms 21 1.2 21 23.6 IXD. Other specific soft tissue sarcomas 17 1.0 17 19.1 IXE. Unspecified soft tissue sarcomas 2 0.2 2 2.2 X. Germ Cell Tumors 110 6.5 110 100.0 XA. Intracranial and intraspinal germ cell tumors 15 0.9 15 13.6 XB. Other and unspecified non-gonadal germ cell tumors 13 0.8 13 11.8 XC. Gonadal germ cell tumors 80 4.7 80 72.7 XD. Gonadal carcinomas 2 0.2 2 1.8 XI. Carcinomas 18 1.1 18 100.0 XIA. Adrenocortical carcinoma 3 0.2 3 16.7 XIB. Thyroid carcinoma 1 0.1 1 5.5 XIC. Nasopharyngeal carcinoma 1 0.1 1 5.5 XID. Malignant melanoma 3 0.2 3 16.7 XIE. Skin carcinoma 3 0.2 3 16.7 XIF. Other and unspecified carcinomas 7 0.4 7 38.9 Total 1702 100.0 1702 100.0 * 40 cases of Histiocytosis were not included Note: Numbers in circles indicate the five highest frequencies, in descending order Table 2 Frequency of cancer, according to sex and to age group, in children served by the Instituto Mexicano del Seguro Social and treated in hospitals in Mexico City (1996–2001). Diagnostic group Age group (years) Sex < 1 1–4 5–9 10–14 Male Female Ratio N % n % n % n % n % n % M/F I. Leukemias 24 27.9 301 48.0 246 50.5 213 42.4 412 46.6 372 45.5 1.1 II. Lymphomas* 0 0.0 49 7.8 79 16.2 66 13.2 138 15.6 56 6.8 2.5 Hodgkin disease 0 0.0 7 1.1 26 5.3 32 6.4 43 4.9 22 2.7 1.9 Non-Hodgkin lymphoma 0 0.0 42 6.7 53 10.9 34 6.8 95 10.7 34 4.1 2.8 III. CNST 6 7.0 69 11.0 59 12.1 63 12.5 84 9.5 113 13.8 0.7 IV. SNST 9 10.5 21 3.3 5 1.0 4 0.8 20 2.3 19 2.3 1.1 V. Retinoblastoma 15 17.4 56 8.9 1 0.2 1 0.2 34 3.8 39 4.8 0.9 VI. Renal Tumors 7 8.1 44 7.0 15 3.1 5 1.0 39 4.4 32 3.9 1.2 VII. Hepatic Tumors 7 8.1 11 1.8 7 1.4 5 1.0 13 1.5 17 2.1 0.8 VIII. Bone Tumors 0 0.0 6 1.0 26 5.3 65 12.9 43 4.9 54 6.6 0.8 IX. Soft Tissue Sarcomas 5 5.8 29 4.6 23 4.7 32 6.4 43 4.9 46 5.6 0.9 X. Germ Cell Tumors 13 15.1 38 6.1 19 3.9 40 8.0 49 5.5 61 7.5 0.8 XI. Carcinomas 0 0.0 3 0.5 7 1.4 8 1.6 9 1.0 9 1.1 1.0 Total 86 100 627 100 487 100 502 100 884 100 818 100 1.1 * 40 cases of Histiocytosis were not included M: male; F: female Numbers in circles indicate the three highest frequencies, in descending order CNST: central nervous system tumors SNST: sympathetic nervous system tumors Table 3 Frequency of cancer in children* of different states of the mexican republic served by the Instituto Mexicano del Seguro Social and treated in hospitals in Mexico City (1996–2001). Diagnostic group Chiapas Mexico City Guerrero State of Mexico Morelos n % n % n % n % n % I. Leukemias 46 37.4 280 43.5 33 31.7 377 51.5 48 48.0 II. Lymphomas** 13 10.6 83 12.9 20 19.2 67 9.2 11 11.0 Hodgkin disease 4 3.3 21 3.3 6 5.8 32 4.4 2 2.0 Non-Hodgkin lymphoma 9 7.3 62 9.6 14 13.5 35 4.8 9 9.0 III. CNST 18 14.6 85 13.2 19 18.3 67 9.2 8 8.0 IV. SNST 1 0.8 17 2.6 7 6.7 12 1.6 2 2.0 V. Retinoblastoma 11 8.9 22 3.4 5 4.8 31 4.2 4 4.0 VI. Renal tumors 4 3.3 26 4.0 5 4.8 30 4.1 6 6.0 VII. Hepatic tumors 3 2.4 18 2.8 0 0.0 8 1.1 1 1.0 VIII. Bone tumors 9 7.3 31 4.8 7 6.7 43 5.9 7 7.0 IX. Soft tissue sarcomas 8 6.5 35 5.4 4 3.8 36 4.9 6 6.0 X. Germ cell tumors 6 4.9 42 6.5 2 1.9 55 7.5 5 5.0 XI. Carcinomas 4 3.3 4 0.6 2 1.9 6 0.8 2 2.0 Total 123 100 643 100 104 100 732 100 100 100 Note: Numbers in circles indicate the five highest frequencies in descending order *Children 0–14 years **40 cases of histiocytosis were not included CNST: central nervous system tumors SNST: sympathetic nervous system tumor Table 4 Comparison of the frequency of cancer in children* in Mexico City and in four selected countries Diagnostic Group Mexico City 1980–19919/ Mexico City 1992–199310/ Mexico City 1996–2001 USA-SEER White 1983–19922/ German Federal Republic 1985–19902/ France 1983–19923/ Cuba 1986–19905/ n % n % n % n % n % n % n % I. Leukemias 1706 34.9 78 39.2 784 46.1 1757 30.7 2422 34.4 746 29.4 454 30.7 II. Lymphomas** 888 18.2 34 17.6 194 11.4 616 10.7 781 11.1 310 12.2 271 18.3 Hodgkin disease 524 10.7 18 9.0 65 3.8 259 4.5 279 4.0 103 4.1 84 5.7 Non-Hodgkin lymphoma 364 7.5 16 8.6 129 7.6 357 6.2 502 7.1 207 8.1 187 12.6 III. CNST 496 10.2 25 12.6 197 11.6 1222 21.4 1392 19.8 538 21.2 223 15.1 IV. SNST 133 2.7 6 3.0 39 2.3 469 8.2 552 7.8 244 9.6 104 7.0 V. Retinoblastoma 420 8.6 5 2.5 73 4.3 172 3.0 208 2.9 61 2.4 40 2.7 VI. Renal Tumors 279 5.7 9 4.5 71 4.2 366 6.4 464 6.6 152 5.9 69 4.7 VII. Hepatic Tumors 70 1.4 1 0.5 30 1.8 83 1.5 82 1.2 23 0.9 21 1.4 VIII. Bone Tumors 321 6.6 9 4.5 97 5.7 267 4.7 359 5.1 129 5.1 87 5.9 IX. Soft Tissue Sarcomas 238 4.9 10 5.0 89 5.2 387 6.8 492 7.0 154 6.1 91 6.2 X. Germ Cell Tumors 256 5.2 17 8.6 110 6.5 165 2.9 222 3.2 71 2.8 34 2.3 XI. Carcinomas 55 1.1 3 1.5 18 1.1 193 3.4 55 0.8 108 4.3 55 3.7 XII. 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Lyon: IARC 43 47 Kramárová E Stiller CA The international classification of childhood cancer Int J Cancer 1996 68 759 765 8980180 10.1002/(SICI)1097-0215(19961211)68:6<759::AID-IJC12>3.3.CO;2-G Massey-Stokes M Lanning B Childhood cancer and environmental toxins: the debate continues Fam Community Health 2002 24 27 38 11772347 Fajardo-Gutiérrez A Sandoval-Mex AM Mejía-Aranguré JM Rendón-Macías ME Martínez-García MC Clinical and social factors that affect the time of diagnosis of Mexican children with cancer Med Pediatr Oncol 2002 39 25 31 12116075 10.1002/mpo.10100 MacNeil DE Coté TR Clegg L Mauer A SEER update of incidence and trends in pediatric malignancies: acute lymphoblastic leukemia Med Pediatr Oncol 2002 39 554 557 12376977 10.1002/mpo.10161 Monge P Wesseling C Rodríguez AC Childhood leukaemia in Costa Rica, 1981–96 Paediat & Perinat Epidemiol 2002 16 210 218 10.1046/j.1365-3016.2002.00422.x Linet M Ries LAG Smith MA Tarone RE Devesa SS Cancer surveillance series: recent trends in childhood cancer incidence and mortality in the United States J Natl Cancer Ins 1999 91 1051 1058 10.1093/jnci/91.12.1051 Fajardo-Gutiérrez A Mejía-Aranguré JM Juárez-Ocaña S González-Miranda G Carreón-Cruz R Rendón-Macías ME Martínez-García MC García-Carrillo B González-Figueroa M Córdova-Jiménez AM Godoy-Franco AC García-Peña MC, Reyes-Morales H, Viniegra-Velázquez L Epidemiología de las neoplasias malignas en niños derechohabientes del IMSS, atendidos en los diferentes Centros Médicos del IMSS In Las múltiples facetas de la investigación en salud: proyectos estratégicos del IMSS 2001 México: Instituto Mexicano del Seguro Social 221 240 Ries LAG Smith MG Gurney JG editors Cancer incidence and survival among children and adolescents: United States SEER Program 1975–1995 National Cancer Institute, SEER Program 1999 NIH Pub. No. 99-4649. Bethesda, MD 65 72 Parker L Powell J Screening for neuroblastoma in infants young than 1 year of age: review of the first 30 years Med Pediatr Oncol 1998 31 455 469 10.1002/(SICI)1096-911X(199811)31:5<455::AID-MPO12>3.0.CO;2-J Yamamoto K Hanada R Kikuchi A Spontaneous regression of localized neuroblastoma detected by mass screening J Clin Oncol 1998 16 1265 1269 9552024 Buning GR Pre and postconception factors associated with sporadic hereditable and non-hereditable retinoblastoma Cancer Res 1989 49 5730 5735 2790788

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==== Front BMC Pulm MedBMC Pulmonary Medicine1471-2466BioMed Central London 1471-2466-4-51531039410.1186/1471-2466-4-5Study ProtocolInterval exercise versus continuous exercise in patients with moderate to severe chronic obstructive pulmonary disease – study protocol for a randomised controlled trial [ISRCTN11611768] Puhan Milo A [email protected]üsching Gilbert [email protected] Evelien [email protected] Christian [email protected]ünemann Holger J [email protected] Martin [email protected] Horten Centre, University of Zurich, Switzerland2 Klinik Barmelweid, Barmelweid, Switzerland3 Experimental Cardiology Research Group, Dept. Research, University of Basel, Switzerland4 Department of Clinical Epidemiology and Biostatistics, McMaster University, Hamilton, Ontario, Canada5 Departments of Medicine and of Social & Preventive Medicine, University at Buffalo, Buffalo, New York, USA2004 13 8 2004 4 5 5 30 1 2004 13 8 2004 Copyright © 2004 Puhan et al; licensee BioMed Central Ltd.2004Puhan et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Physical exercise has become a cornerstone of management of chronic obstructive pulmonary disease (COPD) because it leads to clinically relevant improvements of exercise capacity and health-related quality of life (HRQL). Despite the scarcity of randomised trials directly comparing exercise protocols, current guidelines recommend high intensity continuous exercise for lower extremities as the probably most effective exercise modality. However, for patients admitted to inpatient respiratory rehabilitation programmes, it is often difficult to initiate such an exercise programme because they are severely limited by dyspnoea and leg fatigue and therefore unable to perform continuous exercise at higher intensities and for periods longer than 30 minutes. Interval exercise may be an attractive alternative for these COPD patients because it allows high intensity exercise with recovery periods. The aim of this study is to assess if interval exercise compared to high intensity continuous exercise is not of inferior effectiveness in terms of HRQL and exercise capacity improvements but associated with better exercise tolerance in patients with moderate to severe COPD at the beginning of a respiratory rehabilitation. Methods/Design We will assign patients with moderately severe to severe COPD to either continuous exercise or interval exercise using a stratified randomisation. Patients will follow 12–15 exercise sessions during a comprehensive inpatient respiratory rehabilitation. Primary end point for effectiveness is HRQL as measured by the Chronic Respiratory Questionnaire (CRQ) two weeks after the end of rehabilitation and secondary endpoints include additional clinical outcomes such as functional exercise capacity, other HRQL measures, patients' experience of physical exercise as well as physiological measures of the effects of physical exercise such as cardiopulmonary exercise testing. Including expected drop-outs, we will need 52 patients per group to show differences corresponding to the minimal clinically important difference of the CRQ. Outcome assessors and investigators involved in data analysis will be blinded to group assignment until analyses have been carried out. Discussion Clinicians and the scientific community need evidence on the benefits and tolerance of exercise protocols available in clinical practice. The proposed trial will provide important and needed data on interval and continuous exercise for decision making in clinical practice. ==== Body Background Impaired exercise capacity, dyspnoea and reduced health-related quality of life (HRQL) are common complaints of patients with chronic obstructive pulmonary disease (COPD). A major exercise-limiting factor in COPD is peripheral muscle dysfunction characterised by atrophic muscles and reduced fatigue resistance due morphological and metabolic alterations of peripheral muscles[1] As much as 70% of COPD patients may be affected by peripheral muscle dysfunction.[2] Respiratory rehabilitation with physical exercise improves exercise capacity and HRQL.[3] Although physical exercise is a mandatory component of respiratory rehabilitation programmes[4,5], there is an ongoing debate about what type of exercise at which exercise intensity patients should perform.[1,6] There is substantial variation in exercise protocols used in practice[7] as well as in clinical trials[3]. Current guidelines recommend continuous exercise at high intensity for lower extremities[4,5] because a study indicated that high intensity may be more effective than low or moderate intensity.[8] However, data on high intensity continuous exercise come from a trial that included 19 patients with mild COPD who were able to exercise for 45 minutes five times per week during an outpatient programme.[8] For patients who need to be admitted to inpatient programmes because of more severe COPD and/or unstable health state, it is difficult to perform high intensity exercise and exercise sessions longer than 30 minutes because they are limited by dyspnoea and leg fatigue. Less than 20% may be able to sustain high intensity continuous exercise throughout the whole rehabilitation programme[9] To find a realistically tolerable exercise programme for these patients, who often initiate exercise programmes for the first time, is challenging. A solution to this dilemma may represent interval exercise[6] where patients exercise alternatively at high intensity and at low intensity, which allows short periods of recovery. Consequently, interval training may be better tolerated than high intensity continuous training. In addition, patients may be able to achieve a greater training load during the relatively short exercise sessions they can sustain. Patients and clinicians will accept interval exercise to treat peripheral muscular dysfunction in COPD only if it is not of inferior effectiveness compared with continuous exercise and if it is indeed associated with better compliance resulting from less dyspnoea and leg fatigue during exercise. There is limited evidence from three randomised controlled trials comparing interval exercise and continuous exercise.[10-12] A summary of these trials can be found in table 1. Table 1 Trials on interval exercise in patients with COPD Population Exercise protocols and rehabilitation program Main results Coppoolse 1999 [10] 21 stable male COPD patients (mean age 65 years, FEV1 36.8% predicted) Group 1: CT ergometer cycling at 60% of Wmax Group 2: IT ergometer cycling at 90% of Wmax (1 min) and 45% of Wmax (2 min) 3 days/week plus CT ergometer cycling at 60% of Wmax 2 days/week 8 weeks inpatient rehabilitation with 5 exercise sessions per week of 30 min. No additional physical exercise. Significant increase of VO2 and decrease of minute ventilation with CT but no changes with IT. Significant increase of Wmax and decrease of leg pain during exercise with IT but not with CT. Only significant differences between CT and IT for VO2/Wmax favouring CT. 91% of patients with CT and 90% of patients with IT completed the exercise program. Vogiatzis 2002 [11] 45 stable COPD patients (62% males, (mean age 65 years, FEV1 34.1% predicted) Group 1: CT ergometer cycling at 50% of Wmax weeks 1–4, at 60% weeks 5–8 and at 70% weeks 9–12 Group 2: IT ergometer cycling at 100% of Wmax (30 sec) and 45% of Wmax (30 sec) weeks 1–4, at 120% weeks 5–8 and at 140% weeks 9–12 12 weeks outpatient rehabilitation with 2 exercise sessions per week of 40 min. No additional physical exercise. Significant improvements of CRQ scores and Wmax and reductions of minute ventilation during CWRT in both groups. No significant differences between groups. Attendance rate for exercise sessions 88% for CT and 90% for IT. Kaelin 2001 [12] 19 stable COPD patients (89% males) (mean age 67 years, FEV1 26.9% predicted) Group 1: CT walking on stepper (70 steps/minute) or treadmill (1.5 miles/hour). Increase of 1 MET every 2 weeks Group 2: IT walking on stepper (70 steps/minute) or treadmill (1.5 miles/hour) with active rest to ratio of 2:1. Increase of 1 MET every 2 weeks 6 weeks outpatient rehabilitation with 3 exercise sessions per week of 10–30 min. Additional resistance training and flexibility training. Larger improvements of 6-minute walking distance with IT (80 meters) compared with CT (39 meters). No data on compliance. CT = Continuous training; IT = Interval training; CRQ = Chronic Respiratory Questionnaire Wmax = Maximum exercise capacity, measured by usual incremental exercise test; CWRT = Constant work rate test; VO2 = Maximum oxygen consumption MET = Metabolic equivalent The studies indicated that both interval and continuous training improved exercise capacity, dyspnoea and HRQL and showed insignificant differences between interval and continuous exercise. However, insignificant differences do not allow concluding that interval or continuous are of clinically equivalent effectiveness[13] These trials were too small to show clinical equivalence or non-inferiority and they did not provide evidence on the tolerance of these two exercise modalities. From a methodological point of view, the trial had several shortcomings because, for example, they did not provide details on concealment of random allocation or blinding of outcome assessors. In addition, in the trial with an inpatient rehabilitation.[10], patients of the interval exercise group had a mixed intervention (3 days of interval and 2 days of continuous exercise per week) so that differences can hardly be attributed to different interventions if they are detected at all. The investigators did not use steep ramp tests to determine exercise loads but normal incremental exercise tests. For interval exercise, muscle strength and anaerobic capacity is relevant because of the short high intensity intervals, but this is not measured by normal incremental exercise tests. In addition, training load tolerated during interval exercise may be underestimated when normal incremental exercise tests are used.[14] Exercise tests to establish training intensity should consider the exercise mode. Meyer et al. studied interval exercise in several studies [14-16] in patients with chronic heart failure who show similar patterns of physical deconditioning in terms of clinical manifestation as well as morphological and metabolic abnormalities [17-19] Meyer et al. used a steep ramp test to determine short time muscular maximum exercise capacity[14], which reflects muscle strength and anaerobic capacity, both relevant for interval exercise. Meyer et al. also assessed different ratios of work/recovery phases (1:2; 1:4 and 1:6) and found that with these ratios and relatively short phases of high intensity exercise (10–30 seconds), lactate did not accumulate presumably because of lactate elimination during the recovery phases.[14] Interval exercise with these ratios was therefore recommended as high intensity aerobic exercise modes for patients who do not sustain continuous exercise. Because of the scarcity of evidence on the comparative effectiveness of interval exercise for COPD patients, additional trials are needed.[1,6] Our primary objective is to assess if interval exercise is not of inferior effectiveness compared to continuous exercise of high intensity to improve HRQL and exercise capacity in patients with moderately severe to severe COPD and the secondary objective is to evaluate if interval exercise is better tolerated by COPD patients. Methods Study design (see figure 1) Figure 1 Flow of the study from screening for eligible patients to the final outcome assessment. All consecutive patients admitted to a teaching rehabilitation clinic for an inpatient respiratory rehabilitation (Klinik Barmelweid, Barmelweid, Switzerland) will be assessed for study eligibility by senior staff physicians. If patients are deemed eligible after exercise testing, which is part of the usual rehabilitation program, senior physicians will inform patients about the study orally and in writing. If patients are willing to participate and provide written informed consent they will be randomly assigned to respiratory rehabilitation with either interval or high intensity continuous exercise. Both groups will perform 12–15 exercise sessions and follow the rest of the rehabilitation programme. Follow-up assessments will be done during, at the end of the rehabilitation programme as well as two and twelve weeks afterwards when patients are back in their home environment. The Ethikkommission of the Kantonsspital Aarau, Aargau, Switzerland, has approved the study protocol. Patients We defined the following in- and exclusion criteria: Patients with COPD as defined by FEV1/FVC < 70% predicted, FEV1 < 50 % predicted after bronchodilation, with or without chronic symptoms (cough, sputum production) corresponding to a GOLD (Global Initiative for Chronic Obstructive Lung Disease) stage III-IV[20] and German as first or daily language. Exclusion criteria are arrhythmia (atrial flutter and fibrillation, ventricular tachycardia, premature beats > 8 per minute), ischemia during exercise testing, clinically decompensated Cor pulmonale or heart failure, untreated neoplasia or neoplasia that needed treatment within the previous two years, lung surgery within the previous three months, orthopedic, rheumatologic, vascular or neurological disorders that inhibit ergometer training, gymnastic or guided walking tours, and patients unable to perform or complete the six-minute walk test or the incremental cycle test Randomisation A third party not involved in the conduction of the trial will provide online central randomisation (DatInf GmbH, Tuebingen, Germany). A computerised 'minimisation' procedure will be used to avoid chance baseline imbalances in prognostically important variables[21] Minimisation variables will be exercise capacity (< 300 or ≥ 300 meters in six-minute walk test), the presence of affective disorders (Hospital Anxiety Depression Scale scores < or ≥ 8), status of COPD (unstable COPD = In- or outpatient medical care in the last eight weeks due to exacerbation of COPD versus stable COPD = no in- or outpatient medical care in the last eight weeks due to exacerbation of COPD) and the need for oxygen at rest (yes = long term home oxygen therapy or paO2 < 55 mmHg or no = paO2 ≥ 55 mmHg). Every time the study coordinator has been informed about an enrolled patients, he will enter the randomisation web site[22] enter the patient data required for randomisation (patient identification and stratification variables) and obtain group allocation. The study coordinator will then inform responsible physical therapists about group allocation. No other medical staff will have knowledge about group allocation. The randomisation provider will also send an e-mail to the study coordinator for each randomised patient with details on randomisation. This will ensure correct verification of group allocation after data analysis. Separating patient enrolment and baseline assessments (physicians and physical therapists) from the randomisation procedure (study coordinator not involved in patient enrolment or rehabilitation programme) will ensure concealment of random allocation. Interventions The rehabilitation programme will start one day after baseline assessments and study enrolment. Exercise sessions and group lessons will take place five days a week and will consist of daily cycle ergometer training, breathing therapies (30 minutes per day), and guided walking of 15 to 30 minutes. Relaxation therapies (technique according to Jacobson) take place twice a week, patient education (information about COPD, coping strategies, inhalation techniques) three times a week, smoking cessation advice once a week or more if needed. Apart from physical exercise, the rehabilitation programme of approximately three weeks will be identical for both groups. Group performing continuous exercise The target workload for this group will be ≥ 70% of the maximum exercise capacity expressed in Watts and heart rate achieved during the incremental cycle ergometer test. Patients are usually not able to perform high intensity continuous exercise from the beginning and have to adapt to physical exercise. Physical therapists will increase training load as soon as possible to ≥ 70% of the maximum exercise capacity or as high as each individual patient tolerates. In each session (see figure 2), patients will have a warm-up period of two minutes at 20% of maximum exercise capacity, increase the exercise intensity within two minutes to the target intensity, exercise for 20 minutes at high intensity and then have a decreasing period of two minutes (gradual decrease from 70% to 0%). Pulse oxymetry will be used to supervise patients during exercise. If oxygen saturation falls below 90%, oxygen supplementation will be provided to maintain ≥ 90%. Figure 2 The upper graph shows the continuous exercise protocol for a patient who achieved a maximum exercise capacity of 100 Watts during a usual incremental exercise test. The lower graph shows the interval exercise protocol for a patient who achieved a short time muscular exercise capacity of 200 Watts during a steep ramp test. If patients cannot sustain the workload because of perceived dyspnoea or leg fatigue or because the heart rate exceeds the limits determined during exercise testing, physical therapists will let patients rest for one minute and then resume exercise. If patients have to rest more than twice per session, physical therapists will lower the workload by steps of 10% of baseline maximum exercise capacity. In turn, if patients or physical therapists consider the workload to be too low or if patients do not reach their target heart rate at 70% of the maximum exercise capacity, physical therapists will increase workload by steps of 10% of maximum exercise capacity until patients and physical therapists consider the workload to be appropriate or until the target heart rate is reached. Group performing interval exercise Patients assigned to this group will perform a steep ramp test to determine the short time muscular maximum exercise capacity.[14] The steep ramp test is an incremental cycle ergometer test where patients pedal unloaded for 2 minutes and then pedal with increments of 25 Watts every 10 seconds until they cannot maintain a pedaling frequency above 50 per minute or above the heart rate limit set by the normal incremental exercise test (which all patients perform irrespective of group assignment). With the steep ramp test measurement of muscular maximum exercise capacity is possible because the tests lasts only for 30 to 120 seconds and patients are not limited by symptoms. Measuring muscular maximum exercise capacity is important to set the exercise load for interval exercise because the high intensity interval requires also muscle strength beside overall exercise endurance.[14] Patients should improve both endurance and muscle strength during interval exercise because both are required in daily activities. We therefore chose a work/recovery ratio of 1:2 that prevents from high lactate accumulation.[14] From short time maximum exercise capacity, we will derive the initial work rate for interval exercise (in Watts), which is set at 50% of the short time muscular maximum exercise capacity as measured by the steep ramp test. This workload corresponds in Watts approximately to 90–100% of the workload as measured by the normal incremental exercise test.[16]. Patients will start with exercise the day after the steep ramp test. Patients will perform interval exercise for twelve to fifteen sessions with a cycle ergometer. In each session, they will have a warm up period of two minutes at 20% of the short time maximum exercise capacity (figure 2). Then they exercise for 20 minutes at high intensity intervals of 20 seconds at 50% and at low intensity intervals of 40 seconds at 20% of the short time maximum exercise capacity, i.e. with a work/recovery ratio of 1:2. Then patients have a slow down period of two minutes before completion of the training session. Pulse oxymetry will be used and oxygen supplementation will be provided as described above. If patients cannot sustain exercise intensity because the heart rate exceeds the limits determined after exercise testing or because of perceived dyspnoea or leg fatigue, physical therapists will let patients rest for one minute and then resume exercise. If patients have to rest more than twice per session, physical therapists will lower the workload from 50% of the short time maximum exercise capacity by steps of 10% while the length of intervals remains constant. They will increase the training load again as possible for the patient. In turn, if patients or physical therapists consider the workload to be too low, physical therapists will increase workload of the high intensity interval by steps of 10% until patients and physical therapists consider the workload to be appropriate while the length of intervals remains constant. Clinical outcome measures Chronic Respiratory Questionnaire (CRQ) We will use the CRQ[23] to measure HRQL changes during rehabilitation. The CRQ is a widely used disease-specific instrument to assess symptoms of COPD patients [24-26] We will use the self-administered German CRQ.[27,28] with standardised dyspnoea questions.[29] that we have developed and validated in earlier studies. Patients will complete the CRQ in the Klinik Barmelweid at baseline, at the end of the rehabilitation, two and 12 weeks thereafter when they have returned to their home environment. HADS (Hospital Anxiety Depression Score) Affective disorders are common in patients with COPD and contribute to reduced HRQL[30] The HADS has been developed to assess symptoms of anxiety and depression in patients with physical impairment.[31] There are seven items for each domain (anxiety and depression) with statements on emotions and emotional situations. Patients express their agreement with the statements on a scale from 0 to 3. Domain scores are calculated by summing up the scores for the seven domains resulting in scores from 0 (no depression or anxiety) to 21 (depression or anxiety very likely to be present). Scores ≥ 8 indicate that there is an increased probability for the presence of an affective disorder. We will use the validated self-administered German version of the HADS.[32] The HADS will be completed in the Klinik Barmelweid at baseline, at the end of the rehabilitation, two and 12 weeks thereafter when they have returned to their home environment. Feeling Thermometer (FT) We will use a validated visual analogue scale, the FT[33], an increasingly used instrument for a global estimate of the effect of interventions, including respiratory rehabilitation[29,34]. The FT is a visual analogue scale presented as a thermometer with 100 marked intervals. The worst (dead = 0) and best (perfect health = 100) health states are defined anchors and facilitate comparisons between individuals and groups.[35] We will ask patients to reflect in their score how they felt in the last 7 days. The FT will be completed in the Klinik Barmelweid at baseline, at the end of the rehabilitation, two and 12 weeks thereafter when they have returned to their home environment. Six-minute walk test We will use the six-minute walk test to assess functional exercise capacity according to established criteria.[36] At baseline, patients will perform the six-minute walk test twice one day apart. We will use the results of the second six-minute walk test because the first test tends to underestimate exercise capacity due to unfamiliarity with the test.[36]. At the end of the rehabilitation and two weeks after completion of rehabilitation programme patients will perform additional six-minute walk tests under supervision of physical therapists blinded to group assignment. In addition, we will use a paperboard with a modified Borg scale on from 0 to 10 with verbal labels for 0 (no dyspnoea at all), 1–5, 7 and 10 (maximal dyspnoea) to assess the intensity of perceived dyspnoea at the end of the six-minute walk test. Monitoring of exercise sessions Dyspnoea and leg pain during exercise In each session, we will use modified Borg scale as described above to assess the intensity of perceived dyspnoea and leg pain after five minutes of exercise and at the end of the exercise sessions. Subjective experience of exercise There is no instrument available to assess the subjective experience of COPD patients with exercise, which is likely to influence compliance. We have developed a questionnaire using established methodology[37] to assess how patients experienced the sessions. The questionnaire (Exercise Tolerance Questionnaire) consists of five questions addressing the exercise limitation by shortness of breath and difficulties with breathing, leg fatigue, fatigue in general and too high exercise load. In addition, one question asks patients how they experienced the exercise session in general (from very enjoyable to very unpleasant). The report on questionnaire development will be published elsewhere. Adherence to and tolerance of the prescribed cycle ergometer exercise Physical therapists will record for every cycle ergometer exercise session if patients exercised at all (yes or no), the performed workload (in Watts), if patients reached the target workload (in Watts), adjustments of workload and the requirement for oxygen. We define adherence to exercise as "full adherence" if patients follow at least 12 exercise sessions. We consider the training to be fully tolerated if patients are able to follow the exercise protocol for at least 9 exercise sessions for continuous exercise (taking into consideration the first three exercise session when patients increase training load up to 70% of maximum exercise capacity) and for at least 12 exercise sessions for interval exercise. Adverse events Previous trials of respiratory rehabilitation or physical exercise did not report any adverse events. Nevertheless, we will record any adverse events such as injuries, cardiac events or increase of respiratory symptoms as they happen during the inpatient respiratory rehabilitation. Cardiopulmonary exercise testing At baseline and end of the rehabilitation, all patients will perform an incremental cycle ergometer test to the limit of tolerance (symptom based) under the supervision of a senior physician blinded to group assignment. Patients pedal unloaded for three minutes to warm up at 20 Watts. Then exercise load is increased by 7.5 Watts per minute until patients have to stop because of dyspnoea, leg pain, or criteria for stop testing (atrial or ventricular tachycardia, ischemia, hypoxemia). At the limit of tolerance, we will draw capillary blood samples to measures lactate concentrations and we will set the maximum exercise capacity expressed by Watts. The upper limit for the heart rate during exercise is set as the heart rate measured by the electrocardiogram at the maximum exercise capacity. During testing, we will record gas exchange and ventilatory variables form calibrated signals derived from rapidly responding gas analyzers and a mass flow sensor. We will record breath by breath the following variables: Pulmonary oxygen uptake, pulmonary CO2 output, minute ventilation, tidal volume and respiratory frequency. All patients will perform a steep ramp test at the beginning and end of the rehabilitation programme as described above. Additional data to be collected In order to characterize the patient included in the study, we will record their age, gender, lung function (FEV1, FEV1/FVC, diffusion capacity DLCO/VA, weight, height, smoking status at the beginning and end of rehabilitation as well as two and 12 weeks afterwards, duration of disease (time since diagnosis), co-morbidities such as hypertension, heart diseases, endocrine disorders, chronic rheumatological disorders and psychiatric disorders. Data analysis The randomisation code will not be broken (investigators remain blinded to group assignment and will receive only codes, such as group 1 and 2) until a draft of the manuscript has been written. The authors will write two versions with alternative possible allocation patterns to avoid bias in the interpretation of the results. This approach is methodologically rigorous and limits introduction of bias during the interpretation of the data that some of the authors might have.[38] We will submit the appropriate manuscript regardless of the results of unmasking. After agreement on the final versions of the two articles, we will break the randomisation code and we will submit the correct version of the manuscript. Effectiveness Our null hypothesis is that high intensity continuous exercise is of clinically superior effectiveness compared with interval exercise to improve HRQL (δ ≥ 0.5 in CRQ domains scores). The alternative hypothesis is that interval exercise is not of clinically inferior effectiveness compared with high intensity continuous exercise. In the primary analysis, we will calculate the raw difference and 95% confidence intervals between groups in the mean follow-up score for the CRQ domains 2 weeks after completion of the respiratory rehabilitation programme. In an additional analysis, we will adjust for the base-line score and the four stratification variables using an analysis of covariance. We will use independent t-test to compare the change scores between groups. We will also use the confidence interval approach as recommended for equivalence and non-inferiority trials[13] We will establish non-inferiority of interval exercise if the point estimate and its 95% confidence interval for difference between the change scores of the continuous and interval exercise group is smaller than the a priori determined boundary of clinical equivalence (see figure 3). If the 95% confidence intervals lie outside the boundaries of clinical equivalence we will establish clinical superiority of one exercise protocol. Figure 3 Illustration of the confidence interval approach to interpret results from randomised trials. The horizontal line indicates the difference between CRQ change scores between study groups. ± 0.5 points represent the predefined boundaries of equivalence. If the whole confidence interval is on the right of 0.5 points, interval exercise is not inferior to continuous exercise. If the whole confidence interval is within boundaries the two exercise protocols are of clinically equivalent effectiveness (upper three confidence intervals). Note that there can be a statistically significant difference between study groups but without any clinical relevance. Most methodologists and statisticians agree that the boundaries of equivalence should be defined as the minimal important difference and below the differences observed in previous trials comparing active to control treatments.[13,39] The minimal important difference is "the smallest difference in score in the outcome of interest that informed patients perceive as important, either beneficial or harmful, and which would lead the clinician to consider a change in the management"[40]. Therefore, the minimal important difference of our two main outcome measures for effectiveness have been established empirically and are around 0.5 for the CRQ domains scores [41-44] and 53 meters for the six-minute walk test[45]. A recent meta-analysis showed that respiratory rehabilitation with physical exercise leads to improvements of 50 meters in the six-minute walk test. Therefore we lower the boundaries for equivalence to ± 45 meters in order to have boundaries that are below the differences observed in previous trials comparing active to control treatments.[13,39] We will repeat the analysis with calculations of raw and adjusted differences for all other clinical and physiologic outcome measures and test for significant differences between groups using independent t-test if data are distributed normally. We will use both intention to treat and per protocol analysis to show equivalence in either case as recommended by others[13,39,46,47] Exercise tolerance Our null hypothesis is that patients equally experience high intensity continuous exercise and interval exercise as measured by the Exercise Tolerance Questionnaire. The alternative hypothesis is that interval exercise is associated with the experience of less limiting symptoms compared with high intensity continuous exercise. We will use independent t-tests to compare the measures for exercise tolerance (Exercise Tolerance Questionnaire, Borg scales for dyspnoea and leg pain) between groups. Again, we will assess the raw differences between groups in the primary analysis and adjust for baseline scores and the four stratification variables in the additional analysis. Confounder variables and effect modifiers Factors, which interfere with outcome measures, can distort the results if unevenly distributed between study groups. We use three approaches to control for confounders: First, we use randomisation to allocate patients. Second, we strengthen the randomisation using a computerised minimisation procedure with factors that are likely to influence the outcome measures (exercise capacity, pulmonary state and presence and absence of affective disorders). Third, we will collect a number of variables at baseline (age, gender, lung function, time since diagnosis, cumulative dose of oral steroids in previous three months, medication, cardiovascular, musculoskelettal and endocrine co-morbidities) that may modify the effect of exercise. We will compare the distribution of these potential effect modifiers between groups and statistically assess their influence on the outcome measures with multiple linear regression models. Sample size For calculating the required sample size, we use the formula for comparison of 2 means: n = [A + B]2 * 2 * SD2/DIFF2, where n = the sample size required in each group (double for total sample), SD = standard deviation of the outcome variable, DIFF = size of desired difference between groups. A and B depend on the desired significance level and desired power, respectively. We base our sample size calculations on the CRQ. We used empirical data from our previous trial.[27], where we applied similar inclusion criteria for patients with COPD undergoing respiratory rehabilitation, to estimate variability of the CRQ (standard deviation of the CRQ domain scores between 0.8 and 1.2). A clinically sensible way to determine the size of desired difference between groups (DIFF in formula above) is based on the minimal important difference (0.5 on the scale from 1 to 7 for the CRQ)[41-43,48] A sample size of 44 patients in each group will allow showing a difference of 0.5 in CRQ scores between the groups, assuming a standard deviation of 0.8, with a power of 90% at a significance level of 5% (one-sided). Assuming a drop out rate of 15%.[27], the total minimal sample size increases to 104. This sample size will also allow detecting a difference of 45 meters in the six-minute walking test scores between the two treatment groups with a power of 95% at a significance level of 5% (one-sided) assuming a drop out rate of 15%. A priori sample size calculations usually provide only rough estimates. Therefore, we will recalculate sample size during the study when we have the data for 30 patients in each group. To this end, we will re-assess the standard deviation of the outcome variables and, if necessary, adjust the sample size accordingly (without breaking the randomisation code). Data collection and quality control We will implemented a series of measures to ensure high data quality. 1. Site investigators will collect the data using standardized forms. All data will be collected and entered into a single database in the Horten Centre by one investigator. A second investigator will validate completeness and accuracy of data extraction by checking 20% of extracted patient data. 2. Checklists with all the data to be collected will be provided for physical therapists and physicians. 3. Teaching sessions will be held regularly for physical therapists and physicians involved in data collection aiming at robust data collection. 4. Investigators meetings will be held monthly, or more frequently if needed, to discuss recruitment of patients, problems in conducting the study, acquisition of data, to check for consistency and completeness of data and for interim analyses. 5. E-mail will serve as the first line of non-urgent communication between research team members. 6. Monthly reports will be prepared by the principal investigator that will include the number of patients recruited, stage of follow up for each patient, notification regarding missing patient data and queries from data received. Discussion In the last 30 years, researchers made great efforts to study the effectiveness of respiratory rehabilitation compared to usual care. The meta-analyses of a recent systematic review[3] showed that respiratory rehabilitation with physical exercise leads to clinically significant improvements of HRQL as well as to significant improvements of functional and maximum exercise capacity. Research in respiratory rehabilitation should now focus on the evaluation of different exercise protocols. When an effective treatment such as respiratory rehabilitation is available, patients and clinicians are not confronted with the decision whether to start treatment or not, but with the decision on the most appropriate treatment. Therefore, clinicians need evidence from pragmatic randomised controlled trials directly comparing different exercise protocols at issue rather than evidence from (explanatory) trials comparing exercise with no exercise or usual care.[49] The proposed pragmatic trial will therefore provide important and needed guidance for decision-making in respiratory rehabilitation, in particular for those COPD patients who are severely impaired and initiate respiratory rehabilitation programmes. The evidence generated in the proposed clinical trial will also be relevant for the scientific community. From the 1970s up to the mid-1990s most investigators conducted explanatory clinical trials in order to better understand how and why physical exercise is effective in patients with COPD. After the recognition of its effectiveness the debate on the optimal exercise modality arose[6]. However, only few pragmatic trials have been conducted so far to advance the understanding of the relative benefit and downsides of different exercise protocols. With the proposed trial, we compare two clinically relevant interventions with the use of a clinical trial design that is useful for clinical decision making. Such trials are currently needed to gain consensus on the optimal exercise protocol. There is a need for randomised controlled trials to explore which exercise protocols are most effective for COPD patients. Another topic that has received little attention in respiratory rehabilitation trials so far is compliance to and subjective experience of physical exercise[9] Exercise protocols and adherence to it have not been described in detail in published studies[3] and therefore very little is known about the tolerance of different training modalities. This is despite the fact that physical exercise has long been considered unfeasible in patients with COPD. Beside the paucity of data on the effectiveness and tolerance of different exercise protocols, there is also a need for trials that are methodologically sound and rigorous. Most of the studies on exercise or respiratory rehabilitation in patients with COPD did not report on details of exercise tests and protocols. In addition, study design related issues that introduce bias such as description of the randomisation procedure, concealment of allocation, sample size calculations or blinding have not been addressed frequently. Therefore, we try to use strong epidemiological methods to minimize bias in our trial. Our study could make an important contribution to the understanding of physical exercise in patients with COPD and have a significant impact on the structure of respiratory rehabilitation and exercise programmes. Competing interests None declared. Abbreviations COPD = Chronic obstructive pulmonary disease HRQL = Health-related quality of life CRQ = Chronic Respiratory Questionnaire HADS = Hospital Anxiety Depression Score FT = Feeling Thermometer Authors' contributions MP drafted and revised the manuscript. All authors participated in development of research protocols and in the design of the study. MP, CZ and HS resolved statistical and methodological issues. All authors read and corrected draft versions of the manuscript and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: ==== Refs Skeletal muscle dysfunction in chronic obstructive pulmonary disease. A statement of the American Thoracic Society and European Respiratory Society 1 Am J Respir Crit Care Med 1999 159 S1 40 10194189 Hamilton AL Killian KJ Summers E Jones NL Muscle strength, symptom intensity, and exercise capacity in patients with cardiorespiratory disorders 1 Am J Respir Crit Care Med 1995 152 2021 2031 8520771 Lacasse Y Brosseau L Milne S Martin S Wong E Guyatt GH Goldstein RS Pulmonary rehabilitation for chronic obstructive pulmonary disease Cochrane Database Syst Rev 2002 CD003793 12137716 Pulmonary rehabilitation-1999. 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Barnard A Swank K Adams A Ponto Results of 6 Minute Ambulation and MET Tolerance of Patients With Severe Chronic Obstructive Pulmonary Disease (COPD) Utilizing 2 Different Aerobic Training Regimes: Interval Training Versus Continuous Training American Society of Exercise Physiology 1997 2nd Annual Meeting Jones B Jarvis P Lewis JA Ebbutt AF Trials to assess equivalence: the importance of rigorous methods BMJ 1996 313 36 39 8664772 Meyer K Samek L Schwaibold M Westbrook S Hajric R Lehmann M Essfeld D Roskamm H Physical responses to different modes of interval exercise in patients with chronic heart failure--application to exercise training Eur Heart J 1996 17 1040 1047 8809522 Meyer K Schwaibold M Westbrook S Beneke R Hajric R Gornandt L Lehmann M Roskamm H Effects of short-term exercise training and activity restriction on functional capacity in patients with severe chronic congestive heart failure Am J Cardiol 1996 78 1017 1022 8916481 10.1016/S0002-9149(96)00527-9 Meyer K Samek L Schwaibold M Westbrook S Hajric R Beneke R Lehmann M Roskamm H Interval training in patients with severe chronic heart failure: analysis and recommendations for exercise procedures Med Sci Sports Exerc 1997 29 306 312 9139168 Drexler H Reduced exercise tolerance in chronic heart failure and its relationship to neurohumoral factors 10 Eur Heart J 1991 12 Suppl C 21 28 1680684 Mancini DM Walter G Reichek N Lenkinski R McCully KK Mullen JL Wilson JR Contribution of skeletal muscle atrophy to exercise intolerance and altered muscle metabolism in heart failure 1 Circulation 1992 85 1364 1373 1555280 Sullivan MJ Green HJ Cobb FR Altered skeletal muscle metabolic response to exercise in chronic heart failure. 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Behnke M. Laschke A. Lichtenschopf O. Brandli G.H. Guyatt Schunemann HJ Self-administration and standardisation of the chronic respiratory questionnaire: A randomised trial in three German-speaking countries Respiratory Medicine 2004 98 342 350 15072175 10.1016/j.rmed.2003.10.013 Puhan MA Behnke M Frey M Grueter T Brandli O Lichtenschopf A Guyatt GH Schunemann HJ Self-administration and interviewer-administration of the German Chronic Respiratory Questionnaire: instrument development and assessment of validity and reliability in two randomised studies 1 Health Qual Life Outcomes 2004 2 1 14713317 10.1186/1477-7525-2-1 Schunemann Holger J. Griffith Lauren Jaeschke Roman Goldstein Roger Stubbing David Austin Peggy Guyatt Gordon H. A Comparison of the Original Chronic Respiratory Questionnaire With a Standardized Version Chest 2003 124 1421 1429 14555575 10.1378/chest.124.4.1421 van Manen JG Bindels PJE Dekker FW IJzermans CJ van der Zee JS Schade E Risk of depression in patients with chronic obstructive pulmonary disease and its determinants Thorax 2002 57 412 416 11978917 10.1136/thorax.57.5.412 Zigmond AS Snaith RP The hospital anxiety and depression scale 1 Acta Psychiatr Scand 1983 67 361 370 6880820 Hinz A Schwarz R [Anxiety and depression in the general population: normal values in the Hospital Anxiety and Depression Scale] 20 Psychother Psychosom Med Psychol 2001 51 193 200 11417357 10.1055/s-2001-13279 KJ Bennet Measuring health state preferences and utilities: rating scale, time trade-off, and standard gamble techniques. Quality of life and pharmacoeconomics in clinical trials 1996 Philadelphia, Lippincott-Raven 259 MA Puhan M Behnke PJ Devereaux VM Montori O Brandli M Frey HJ Schunemann Measurement of Agreement on Health-related Quality of Life Changes in Response to Respiratory Rehabilitation by Patients and Physicians A Prospective Study Respir Med 2004 Torrance GW Feeny D Furlong W Visual analog scales: do they have a role in the measurement of preferences for health states? 1 Med Decis Making 2001 21 329 334 11475389 10.1177/02729890122062622 ATS statement: guidelines for the six-minute walk test 966 Am J Respir Crit Care Med 2002 166 111 117 12091180 Streiner D Norman G Health measurement scales Oxford Medical Publications 1998 Second Oxford, Oxford University Press 60 65 Gotzsche PC Blinding during data analysis and writing of manuscripts 1 Control Clin Trials 1996 17 285 290 8889343 10.1016/0197-2456(95)00263-4 Fleming TR Design and interpretation of equivalence trials Am Heart J 2000 139 S171 S176 10740126 10.1016/S0002-8703(00)90067-X Guyatt GH Osoba D Wu AW Wyrwich KW Norman GR Methods to explain the clinical significance of health status measures 1 Mayo Clin Proc 2002 77 371 383 11936935 Jaeschke R Singer J Guyatt GH Measurement of health status. Ascertaining the minimal clinically important difference Control Clin Trials 1989 10 407 415 2691207 10.1016/0197-2456(89)90005-6 Jaeschke R Guyatt GH Keller J Singer J Interpreting changes in quality-of-life score in N of 1 randomized trials 1 Control Clin Trials 1991 12 226S 233S 1663858 Redelmeier DA Guyatt GH Goldstein RS Assessing the minimal important difference in symptoms: a comparison of two techniques J Clin Epidemiol 1996 49 1215 1219 8892486 10.1016/S0895-4356(96)00206-5 Schunemann Holger J. Griffith Lauren Jaeschke Roman Goldstein Roger Stubbing David Guyatt Gordon H. Evaluation of the minimal important difference for the feeling thermometer and the St. George's Respiratory Questionnaire in patients with chronic airflow obstruction Journal of Clinical Epidemiology 2003 56 1170 1176 14680667 10.1016/S0895-4356(03)00115-X Redelmeier DA Bayoumi AM Goldstein RS Guyatt GH Interpreting small differences in functional status: the Six Minute Walk test in chronic lung disease patients Am J Respir Crit Care Med 1997 155 1278 1282 9105067 Greene WL Concato J Feinstein AR Claims of equivalence in medical research: are they supported by the evidence? Ann Intern Med 2000 132 715 722 10787365 McAlister FA Sackett DL Active-control equivalence trials and antihypertensive agents Am J Med 2001 111 553 558 11705432 10.1016/S0002-9343(01)00900-7 Wyrwich KW Tierney WM Wolinsky FD Further evidence supporting an SEM-based criterion for identifying meaningful intra-individual changes in health-related quality of life J Clin Epidemiol 1999 52 861 873 10529027 10.1016/S0895-4356(99)00071-2 Tunis SR Stryer DB Clancy CM Practical clinical trials: increasing the value of clinical research for decision making in clinical and health policy 1 JAMA 2003 290 1624 1632 14506122 10.1001/jama.290.12.1624

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==== Front BMC BiotechnolBMC Biotechnology1472-6750BioMed Central London 1472-6750-4-151527967810.1186/1472-6750-4-15Methodology ArticleInvestigating the utility of combining Φ29 whole genome amplification and highly multiplexed single nucleotide polymorphism BeadArray™ genotyping Pask Rebecca [email protected] Helen E [email protected] Bryan J [email protected] Sarah [email protected] Deborah J [email protected] Meera [email protected] Rebecca CJ [email protected] Anne [email protected] Alex C [email protected] Luc J [email protected] Neil M [email protected] John A [email protected] Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Cambridge, CB2 2XY, UK2004 27 7 2004 4 15 15 13 4 2004 27 7 2004 Copyright © 2004 Pask et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Sustainable DNA resources and reliable high-throughput genotyping methods are required for large-scale, long-term genetic association studies. In the genetic dissection of common disease it is now recognised that thousands of samples and hundreds of thousands of markers, mostly single nucleotide polymorphisms (SNPs), will have to be analysed. In order to achieve these aims, both an ability to boost quantities of archived DNA and to genotype at low costs are highly desirable. We have investigated Φ29 polymerase Multiple Displacement Amplification (MDA)-generated DNA product (MDA product), in combination with highly multiplexed BeadArray™ genotyping technology. As part of a large-scale BeadArray genotyping experiment we made a direct comparison of genotyping data generated from MDA product with that from genomic DNA (gDNA) templates. Results Eighty-six MDA product and the corresponding 86 gDNA samples were genotyped at 345 SNPs and a concordance rate of 98.8% was achieved. The BeadArray sample exclusion rate, blind to sample type, was 10.5% for MDA product compared to 5.8% for gDNA. Conclusions We conclude that the BeadArray technology successfully produces high quality genotyping data from MDA product. The combination of these technologies improves the feasibility and efficiency of mapping common disease susceptibility genes despite limited stocks of gDNA samples. ==== Body Background In order to locate the disease variants involved in complex common disease it is now generally accepted that very large sample numbers will be required [1-4]. Not only do the sample collections need to provide high quality gDNA, for the purpose of accurate genotyping, they also need to be sustainable. If, for example, one million SNPs were to be genotyped in a whole genome association scan and only 1 ng were required per SNP genotype, 1 mg of DNA would be required from each clinical sample. Given that the gDNA yield from a typical blood sample of 8 ml is approximately 200 μg, and that the typical yield from a mouth-swab is just 10 μg, there is clearly a short-fall in available quantities unless other means are employed to amplify the DNA resource. Moreover, many existing and effectively irreplaceable DNA sample collections, which have been used in previous studies and are now depleted, may consist of only nanogram quantities of gDNA. At present, the gold standard method for generating gDNA from whole blood samples is through the process of immortalisation by transformation of the peripheral blood lymphocytes with Epstein-Barr Virus (EBV) [5]. Although this method of transfecting EBV creates an unlimited resource of gDNA, the procedure is costly, lengthy and not applicable to existing collections for which the gDNA has already been extracted. If there was a reliable method to enzymatically amplify the whole genome from nanogram-levels of gDNA and directly from clinical samples to microgram amounts then this would enable the use of archived gDNA in future studies, as well as providing an accelerated route to full use of newly collected clinical samples for high-throughput genotyping. Molecular Staging, Inc. (MSI) (New Haven, CT, USA) have developed a method for whole genome amplification by Φ29 polymerase Multiple Displacement Amplification (MDA). It has been reported by the company that this method can reliably amplify the whole genome from gDNA, whole blood and other clinical samples [6-8]. Each DNA sample should give similar yields of product in all reactions with little dependency on the quantity of starting template [6,7]. Moreover, the MDA reaction should give complete coverage of the genome with little regional bias [6], which is critical when the product is to be used for high-throughput SNP genotyping. We set up a series of experiments with MSI in order to validate their claims that MDA product from gDNA is a viable alternative template to un-manipulated gDNA in SNP genotyping. Recent studies have been conducted using MDA product from Amersham [9,10], and report the high level of accuracy achieved when these products are genotyped using TaqMan or multiplex, four-colour fluorescent minisequencing with six and 45 SNPs, respectively. However, without DNA resource limitations, a genotyping bottleneck exists mostly as a result of time- and assay set-up costs and hence, in order to achieve large-scale genotyping, highly multiplexed assays are required. In such multiplexed assays, there is greater potential for erosion of genotyping quality, due to reduced substrate integrity. The validation of the use of amplified DNA resources with such highly multiplexed methods is, therefore, essential. The BeadArray genotyping platform of Illumina™ Inc. (San Diego, CA, USA) offers a high-throughput, highly multiplexed and highly automated genotyping service facility [11]. The BeadArray platform is highly miniaturised, using fibre optic bundles as a substrate for a high-density microarray [12]. It is the combination of this miniaturisation with an ability to multiplex up to 1,536 SNP assays [11] that makes BeadArray an attractive potential solution to the genotyping bottleneck. A recent study by Barker and colleagues, with 2,320 SNPs and five samples, found 99.86% concordance between MSI MDA product and gDNA [13]. However, since only five samples were studied it was not possible to evaluate accurately the efficacy of BeadArray on MDA product template, including estimation of sample exclusion and failure rates. In the present report we have, therefore, studied 86 MDA product samples and 384 SNPs using BeadArray, allowing comparison with the single-plex methods TaqMan® (Assays-by-DesignSM, Applied Biosystems, Foster City, CA, USA) and Invader® (Third Wave Technologies, Inc., Madison, WI, USA) with gDNA. Results MDA yield We selected and sent to MSI 20 ng of 88 gDNA samples for amplification, from which an average of 200 μg of MDA product was yielded in 100 μl reactions. The yields ranged from 85 μg to 280 μg, with 61% of samples yielding between 100 μg and 200 μg. The HLA-DRB1 genotype of each MDA sample was entirely concordant with the corresponding gDNA template, verifying the identity of each MDA sample and ruling out the possibility of contamination. When 100 ng of 448 gDNA samples were amplified using reagents supplied by MSI in kit form and the amplification carried out in-house, an average of 155 μg of MDA product was yielded in 150 μl reactions. The yields ranged from 31 μg to 260 μg, with 80% of samples yielding between 100 μg and 200 μg. Compatibility of MDA product with TaqMan and Invader Of 88 MDA products and their corresponding gDNAs tested at 95 SNPs using the TaqMan method of genotyping there were no samples that consistently failed to produce any data. This confirmed that, for all samples, amplification had been sufficiently successful for the TaqMan chemistry to perform at most SNPs. Genotype concordance rates between MDA product and gDNA and genotype failure rates are given in Table 1. These results demonstrate that the use of MDA product as a template for the TaqMan assay produces accurate data comparable to that from gDNA. We observed that, for the majority of TaqMan assays, the clustering of data points was less distinct when MDA product was used as a template, compared to gDNA. Example data from an assay in which deterioration in clustering was observed are shown in Figure 1. For a single SNP of the 95 tested, the insulin gene (INS) -23HphI (rs689), an allelic bias was observed in the MDA process, which resulted in the merging of the heterozygote cluster with the homozygote cluster of the major allele, making the correct assignments of genotype impossible, shown in Figure 2 [see additional file 1]. MDA product used as a template for the Invader genotyping method at this SNP produced similarly un-useable data. Using gDNA template at this SNP, however, both the TaqMan and Invader methods produced acceptable results, shown in Figure 2 [see additional file 1], indicating that the allelic bias was occurring at the MDA stage and not during subsequent genotyping. Interestingly, allelic bias has previously been observed at two other SNPs at INS in PCR reactions designed for the Pyrosequencing method (Pyrosequencing AB, Uppsala, Sweden) [14]. These results may indicate that INS may be situated in a sequence region that is predisposed to such allelic bias and the INS variable number of tandem repeats polymorphism, only 580 bp 5' to the -23HphI SNP, is a candidate for such an effect. For 13 additional SNPs for which Invader genotyping was performed, comparison of genotypes generated from MDA product with those from gDNA are shown in Table 1. Two SNPs were genotyped by TaqMan on the 448 samples amplified using reagents supplied by MSI in kit form (and the amplification carried out in-house), and on the corresponding gDNAs. The gDNA samples used for amplification had been extracted from whole blood. Genotype concordance rates between MDA product and gDNA and genotype failure rates are given in Table 1. Validation of BeadArray genotyping technology with gDNA template We commissioned Illumina to conduct a large-scale project using BeadArray genotyping technology involving 3,036 samples (2,950 gDNA samples and 86 MDA products) and 384 SNPs i.e. >1.1 million genotypes. In the first instance, 757 SNP sequences were sent to Illumina for in silico assay design. These SNPs were selected for their relevance to a range of ongoing projects in our laboratory, located at genes of strong functional candidacy and within regions of linkage to type 1 diabetes e.g. the putative IDDM10 locus on chromosome 10p14-11. All SNPs were validated, having been identified either from empirically confirmed SNPs in dbSNP or from our own re-sequencing efforts. Based on ranking from the in silico design criteria [15], 404 SNPs from 757 (53%) were suggested as most suitable for assay development, from which 384 were chosen. Thirty-nine of these failed to be converted into a viable assay (10.2%), leaving a total of 345 working assays. As well as excluding SNPs that fail to produce robust genotypes, the Illumina protocol excludes samples that do not consistently perform. Of the total number of samples 10.5% were excluded and as a consequence very few data points were missing from the data set, resulting in an apparently low genotype failure rate (Table 2). Within the 2,781 successfully genotyped gDNA sample set, 26 were duplicate samples. Of these 52 samples at 345 SNPs, the genotypes of 23 duplicates did not match each other and 19 data points were missing, giving a discordance rate (error rate) of 0.26% (23 of 8,951 data points). As our samples were family-based, a quality control check of misinheritance rates was possible using PedCheck [16]. Of the 345 SNPs, 20 displayed ≥ 10 misinheritances in the 742 families genotyped. For ten of these SNPs, TaqMan genotyping was attempted in the same samples in order to verify the results. It was possible to design TaqMan assays to only seven of the ten SNPs and, of these, only three produced interpretable data. At these three SNPs the numbers of misinheritances were 41, four and eight, respectively, by TaqMan, compared to 17, 22 and 14, respectively, by BeadArray. The number of SNPs with <10 or <5 misinheritances from the BeadArray experiment is shown in Table 3, along with our previous year's TaqMan results considering SNPs with allele frequencies >1%. The poor performance of both Illumina and TaqMan at the ten SNPs compared in detail, as well as the lab misinheritance rate for TaqMan (Table 3), indicates that the high misinheritance rates observed for some SNPs in the Illumina experiment is not a technology-specific failing. Within the panel of 384 SNPs attempted by Illumina, 17 were controls for which we had already produced genotyping data by either TaqMan or Invader methods, enabling an evaluation of the BeadArray data for concordance. Two of these 17 control SNPs failed to be converted to a working assay, giving 15 SNPs and a maximum of 2,503 samples that were genotyped in common. Excluding failed duplicates noted above, comparison of BeadArray genotypes with existing data revealed a concordance rate of 99.6% (129 discordant in 34,219 comparisons), indicating the compatibility of the non-excluded gDNA samples with BeadArray, and the quality of existing data. Of the 15 control SNPs, 11 had been genotyped using TaqMan and four using Invader. The concordance rates for each platform were 99.7% using TaqMan (104 discordant in 25,203 comparisons) and 99.6% using Invader (25 discordant in 9,016 comparisons) when compared with BeadArray data, showing no significant difference between the two platforms. Compatibility of BeadArray with MDA product Within the BeadArray experiment described above were 86 MDA products and their corresponding gDNA samples. These data were directly compared for sample failure rate and genotype failure rate as shown in Table 2. BeadArray genotype concordance rate between MDA product and gDNA are given in Table 1. These results provide evidence for the compatibility of the non-excluded MDA products with BeadArray technology. Evaluation of the Illumina's quality scores revealed no significant difference between the MDA and gDNA samples for any SNP (t-test P-value >0.05 for every SNP). Discussion In this study we have evaluated the Φ29 polymerase MDA whole genome amplification method from MSI by assessing the compatibility of its product with the established TaqMan and Invader genotyping chemistries and with the highly multiplexed BeadArray genotyping platform. We have also evaluated Illumina's BeadArray genotyping platform for a large-scale experiment using gDNA. At 95 SNPs, comparison of TaqMan genotypes generated from MDA product and gDNA templates revealed a very good concordance rate but a higher failure rate for MDA product compared to gDNA. This would need be estimated in a sample size larger than the current n = 88 in order to be confirmed. This result is comparable to the smaller study by Tranah et al. [9], in which six SNPs were genotyped by TaqMan on 172 samples, resulting in 100% concordance of pre- and post-MDA DNA. In the present study, the MDA product genotypes were slightly more difficult to assign, owing to more dispersed clusters. This was not observed by Lovmar et al. with fluorescent minisequencing on Amersham MDA products compared to gDNA [10]. One marker in our study, which may be unusually prone to allelic bias, was impossible to score using MDA product but was acceptable when using gDNA as a template (INS -23HphI, rs689). Compared to the yields indicated in Dean et al. [7], our average yield from in-house amplification using the reagents in kit form were in the order of five- to six-fold higher. This was probably due to differences in the two protocols: for example, our protocol used an increased reaction volume compared to the protocol used in Dean et al. [7]. Furthermore, the Dean et al. [7] protocol omitted the denaturation step, which is now standard practice. One other potential explanation for this variation is possible differences between laboratories in the quantitation of DNA using PicoGreen, the application of which requires a standard reference data set. We cannot at present fully resolve the differences in yields between studies but we can conclude that very large amounts of DNA are synthesized during the Φ29 reaction and that this is an excellent template for genotyping. MDA product should, therefore, be quantified and its concentration on completion of the MDA reaction not assumed to be consistent. Genotype failure rate, concordance rates with gDNA and the nature of genotype clustering showed similar patterns to service-generated MDA. However, a larger number of SNP markers would need to be genotyped on the MDA product using purchased kit reagents in order to verify these figures for in-house amplifications. In the evaluation of MDA product in conjunction with BeadArray technology, the high concordance rate between genotypes obtained from MDA product and gDNA templates is encouraging. A concordance rate of 99.86% has been reported by Barker et al. using 2,320 SNPs and five samples [13]. However, as our study used 86 samples, we were able to observe differences in genotype failure rate between the different templates, not noted in the previous study [11]. As with the TaqMan evaluation, BeadArray had a higher genotype failure rate for MDA product compared to gDNA (0.2% for MDA versus 0.06% for gDNA). We did not find any evidence for allele drop-out with MDA compared to gDNA. BeadArray genotyping excluded more MDA samples than gDNA samples (10.5% for MDA versus 5.7% for gDNA) indicating that gDNA is a superior genotyping template for BeadArray technology. This 2-fold exclusion rate for MDA is consistent with the approximately 2 to 3-fold genotype failure rate of MDA typically observed with TaqMan and Invader, compared to gDNA (unpublished data). The performance of MDA product is continuously being monitored in our laboratory. In a study blinded to all genotypers and database administrators, 288 family-based gDNA samples (prepared by the salting out method), were replaced with MDA product and left in continual use in our genotyping pipeline for 12 months. The change went undetected by all users. The failure rate for MDA was 3.34% for 15,921 genotypes, compared to 2.39% for 19,272 gDNA genotypes. Therefore, this improvement in the MDA performance for TaqMan is likely to be applicable to BeadArray, which improves the feasibility of mapping susceptibility loci in complex traits. When using a highly multiplexed, highly automated genotyping platform, slight reductions in the quality of template material are likely to have a greater adverse effect on data than in scenarios in which markers are assessed individually and manual scoring is undertaken. Our results indicate that MDA is an adequate solution for the vast majority of SNP markers, even in this highly multiplexed allelic assay platform. It is noted that 5.8% of markers that passed the Illumina acceptable scoring threshold were in fact showing high misinheritance rates in our family samples. This problem was at the same magnitude as TaqMan for individually genotyped markers. This highlights the importance of checking potential positive results with a second genotyping technology. MDA should allow the continuation of genetic analysis on archived DNA in researchers' freezers worldwide, providing the very necessary increases in sample sizes so urgently required [1,2,17]. Conclusions The combination of BeadArray high throughput, multiplex genotyping and amplified DNA (MDA product) successfully produced high quality genotype data thereby improving the feasibility and efficiency of mapping common disease susceptibility genes despite limited stocks of gDNA samples. Methods MDA product preparation For both the validation experiments (MDA product as a template for TaqMan and Invader genotyping) and for the combined experiment (MDA product as a template for BeadArray genotyping) the same MDA samples were tested. We sent to MSI 20 ng (5 μl at 4 ng/μl) of 88 gDNA samples for amplification, which was performed as a service according to the protocol for human gDNA with the omission of the denaturing step [7]. These gDNA samples had been extracted from cell pellets of EBV derived cell lines using a standard chloroform protocol that produces very high quality and stable gDNA [18]. The MDA product returned to us was quantified using PicoGreen dsDNA quantitation reagent (Molecular Probes Europe B.V., Leiden, the Netherlands). In order to verify the identity of each MDA-produced sample, genotyping was performed at HLA-DRB1 and comparison made with data generated from the corresponding gDNA. HLA-DRB1 genotyping was performed using the Dynal Auto RELI™ SSO HLA-DRB Test system (Dynal® Biotech, Wirrel, UK) for each gDNA sample and their MDA products. Although, these samples were amplified by MSI as a service, the reagents are also available from MSI in a kit form for amplification in-house. Following the amplification by MSI we have amplified 448 DNA samples by using the reagents in kit form and 100 ng (25 μl at 4 ng/μl) gDNA template in 100 μl reactions. In the interim, one major change to the MDA protocol had taken place, the inclusion of a denaturation of the DNA template prior to amplification. Previously no denaturation step took place. Two TaqMan markers were tested on these 448 samples and the genotype failure rate calculated. Evaluation of MDA product as a template for TaqMan and Invader genotyping SNP TaqMan assays were carried out for allelic discrimination, 8 ng of DNA (2 μl at 4 ng/μl) used in a 5 μl total reaction volume. TaqMan genotypes from the 88 MDA samples, described above, were compared with TaqMan genotypes generated from their corresponding gDNAs, at 95 SNPs, with a broad range of allele frequencies. The Invader method was used to genotype 13 additional SNPs on the same samples. Comparison was made between data generated from both templates by the measurement of genotype failure and genotype concordance rates. Evaluation of BeadArray genotyping technology and its compatibility with MDA product Of the 384 SNPs selected for genotyping 3,036 samples, 17 were control SNPs for which we had existing genotype data, generated by either TaqMan or Invader methods, with which comparison of genotype failure and genotype concordance rates were made. These 384 SNPs covered a broad range of allele frequencies. Incorporated into this experiment was an assessment of suitability and compatibility of the BeadArray genotyping method with MDA product. This involved 86 of the 88 amplified samples, described above, for which genotyping was attempted at all 384 SNPS. Concordance between genotypes generated from MDA product and gDNA templates, together with the genotype and sample failure rates of each template type were measured. In our laboratory we store genotyping data in a MySQL database on a Sun server. The volume of data expected from Illumina was the equivalent of 6 months' in-house genotyping. We separated phenotypic and pedigree information, which is associated with a sample, from genotype data, which is associated with a DNA plate and well position, with a link table to relate the two. Sample aliases are also supported, so that no recoding of identifiers is required, either to export or import Illumina data [19]. Authors' contributions RP participated in the design of the study, performed in-house genotyping experiments, and assisted in preparing the manuscript. HER prepared DNA and MDA amplified samples. BJB participated in the design of the study and assisted in the preparation of the manuscript. SN prepared DNA and MDA amplified samples. DS performed in-house genotyping experiments. MS prepared DNA and MDA amplified samples. RCJT participated in the design of the study and assisted in the preparation of the manuscript. AS participated in the design of the study. ACL and LJS performed bioinformatics. NMW managed the data and participated in its interpretation. JAT participated in the design of the study and assisted in the preparation of the manuscript. All authors read and approved the final version of the manuscript. Supplementary Material Additional File 1 Figure 2 TaqMan and Invader fluorescence data plotted for the INS -23 HphI SNP. Both the MDA product plots could not be scored. All plots represent the same individual samples with gDNA plots containing 8 additional samples. Click here for file Acknowledgments This work was funded by the Wellcome Trust and the Juvenile Diabetes Research Foundation International. We thank Vin Everett and Geoff Dolman for IT support, the DNA team for sample preparation and the Human Biological Data Interchange and Diabetes UK for family collections. We also thank Katherine Webster, Fawad Faruqi, Michael Egholm and Roger Lasken from MSI for customer support and David Barker from Illumina for useful discussion. Figures and Tables Figure 1 TaqMan fluorescence data plotted to compare the performance of MDA product with gDNA. This is a typical example of the clustering and scoring of TaqMan data for a common SNP in the evaluation of MDA product as a template for TaqMan genotyping. (a) End-point data from the 88 gDNA samples used in this evaluation and (b) the corresponding 88 MDA samples. In this example there are eight apparent failures for MDA, which are, in fact, empty wells. No genotypes were rejected for gDNA, and two genotypes were rejected for the MDA products due to poor clustering. Table 1 Genotype concordance and failure rates for MDA compared with gDNA across genotyping platforms. Genotype Concordance Rate (%) Genotype Failure Rate (%) Genotyping Platform Site of MDA production Number of SNPs Number of non-excluded samples gDNA and MDA product template gDNA template MDA product template TaqMan MSI 95 88 99.71 2.4 4.0 TaqMan In-house 2 448 99.4 0.7 1.9 BeadArray MSI 345 77 98.8 0.06 0.2 Invader MSI 13 88 99.9 0.9 2.6 Table 2 BeadArray sample exclusion and genotype failure rates for MDA product compared with gDNA. Template Original number of samples Number of excluded samples Exclusion rate Number of attempted genotypes Number of failed genotypes Genotype failure rate gDNA 2,950 169 5.7 959,445 590 0.06 MDA product 86 9 10.5 26,565 52 0.2 Table 3 Comparison of misinheritance rates in families genotyped by BeadArray and TaqMan platforms using gDNA. Genotyping Platform Number of families tested Number of SNPs tested Number of SNPs with <10 misinheritances Number of SNPs with <5 misinheritances BeadArray 742 300 280 (93.3%) 248 (82.7%) TaqMan 750 501 479 (95.6%) 409 (81.6%) ==== Refs Ioannidis JP Trikalinos TA Ntzani EE Contopoulos-Ioannidis DG Genetic associations in large versus small studies: an empirical assessment Lancet 2003 361 567 571 12598142 10.1016/S0140-6736(03)12516-0 Johnson GC Esposito L Barratt BJ Smith AN Heward J Di Genova G Ueda H Cordell HJ Eaves IA Dudbridge F Twells RC Payne F Hughes W Nutland S Stevens H Carr P Tuomilehto-Wolf E Tuomilehto J Gough SC Clayton DG Todd JA Haplotype tagging for the identification of common disease genes Nat Genet 2001 29 233 237 11586306 10.1038/ng1001-233 Risch N Merikangas K The future of genetic studies of complex human diseases Science 1996 273 1516 1517 8801636 Kruglyak L Prospects for whole-genome linkage disequilibrium mapping of common disease genes Nat Genet 1999 22 139 144 10369254 10.1038/9642 Packer RJ Bolton BJ Celis JE Immortilization of B lymphocytes by Epstein-Barr Virus Cell Biology: A Laboratory Handbook 1998 1 Academic Press 178 185 Hosono S Faruqi AF Dean FB Du Y Sun Z Wu X Du J Kingsmore SF Egholm M Lasken RS Unbiased whole-genome amplification directly from clinical samples Genome Res 2003 13 954 964 12695328 10.1101/gr.816903 Dean FB Hosono S Fang L Wu X Faruqi AF Bray-Ward P Sun Z Zong Q Du Y Du J Driscoll M Song W Kingsmore SF Egholm M Lasken RS Comprehensive human genome amplification using multiple displacement amplification Proc Natl Acad Sci U S A 2002 99 5261 5266 11959976 10.1073/pnas.082089499 Lasken RS Egholm M Whole genome amplification: abundant supplies of DNA from precious samples or clinical specimens Trends Biotechnol 2003 21 531 535 14624861 10.1016/j.tibtech.2003.09.010 Tranah GJ Lescault PJ Hunter DJ De Vivo I Multiple displacement amplification prior to single nucleotide polymorphism genotyping in epidemiologic studies Biotechnol Lett 2003 25 1031 1036 12889810 10.1023/A:1024173909401 Lovmar L Fredriksson M Liljedahl U Sigurdsson S Syvanen AC Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA Nucleic Acids Res 2003 31 e129 14576329 10.1093/nar/gng129 Illumina Oliphant A Barker DL Stuelpnagel JR Chee MS BeadArray technology: enabling an accurate, cost-effective approach to high-throughput genotyping Biotechniques 2002 Suppl 56 58 60-51 12083399 Barker DL Hansen MS Faruqi AF Giannola D Irsula OR Lasken RS Latterich M Makarov V Oliphant A Pinter JH Shen R Sleptsova I Ziehler W Lai E Two methods of whole-genome amplification enable accurate genotyping across a 2320-SNP linkage panel Genome Res 2004 14 901 907 15123587 10.1101/gr.1949704 Barratt BJ Payne F Rance HE Nutland S Todd JA Clayton DG Identification of the sources of error in allele frequency estimations from pooled DNA indicates an optimal experimental design Ann Hum Genet 2002 66 393 405 12485472 10.1046/j.1469-1809.2002.00125.x Illumina assay design criteria O'Connell JR Weeks DE PedCheck: a program for identification of genotype incompatibilities in linkage analysis Am J Hum Genet 1998 63 259 266 9634505 10.1086/301904 Dahlman I Eaves IA Kosoy R Morrison VA Heward J Gough SC Allahabadia A Franklyn JA Tuomilehto J Tuomilehto-Wolf E Cucca F Guja C Ionescu-Tirgoviste C Stevens H Carr P Nutland S McKinney P Shield JP Wang W Cordell HJ Walker N Todd JA Concannon P Parameters for reliable results in genetic association studies in common disease Nat Genet 2002 30 149 150 11799396 10.1038/ng825 Ueda H Howson JM Esposito L Heward J Snook H Chamberlain G Rainbow DB Hunter KM Smith AN Di Genova G Herr MH Dahlman I Payne F Smyth D Lowe C Twells RC Howlett S Healy B Nutland S Rance HE Everett V Smink LJ Lam AC Cordell HJ Walker NM Bordin C Hulme J Motzo C Cucca F Hess JF Metzker ML Rogers J Gregory S Allahabadia A Nithiyananthan R Tuomilehto-Wolf E Tuomilehto J Bingley P Gillespie KM Undlien DE Ronningen KS Guja C Ionescu-Tirgoviste C Savage DA Maxwell AP Carson DJ Patterson CC Franklyn JA Clayton DG Peterson LB Wicker LS Todd JA Gough SC Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease Nature 2003 423 506 511 12724780 10.1038/nature01621 Burren OS Healy BC Lam A Schuilenburg H Dolman GE Everett VH Laneri D Nutland S Rance HE Payne F Smyth D Lowe C Barratt BJ Twells RCJ Rainbow DB Wicker LS Todd JA Walker NM Smink LJ Development of an integrated genome informatics, data management and workflow infrastructure: A toolbox for the study of complex disease Human Genomics 2003 1 98 109

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==== Front Cardiovasc UltrasoundCardiovascular Ultrasound1476-7120BioMed Central London 1476-7120-2-81526876210.1186/1476-7120-2-8ReviewInsights into the pathogenesis of vein graft disease: lessons from intravascular ultrasound Murphy Gavin J [email protected] Gianni D [email protected] Bristol Heart Institute, University of Bristol, Bristol, BS2 8HW, UK2004 21 7 2004 2 8 8 24 6 2004 21 7 2004 Copyright © 2004 Murphy and Angelini; licensee BioMed Central Ltd.2004Murphy and Angelini; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The success of coronary artery bypass grafting (CABG) is limited by poor long-term graft patency. Saphenous vein is used in the vast majority of CABG operations, although 15% are occluded at one year with as many as 50% occluded at 10 years due to progressive graft atherosclerosis. Intravascular ultrasound (IVUS) has greatly increased our understanding of this process. IVUS studies have shown that early wall thickening and adaptive remodeling of vein grafts occurs within the first few weeks post implantation, with these changes stabilising in angiographically normal vein grafts after six months. Early changes predispose to later atherosclerosis with occlusive plaque detectable in vein grafts within the first year. Both expansive and constrictive remodelling is present in diseased vein grafts, where the latter contributes significantly to occlusive disease. These findings correlate closely with experimental and clinicopathological studies and help define the windows for prevention, intervention or plaque stabilisation strategies. IVUS is also the natural tool for evaluating the effectiveness of pharmacological and other treatments that may prevent or slow the progression of vein graft disease in clinical trials. Coronary artery bypass graftingintravascular ultrasoundsaphenous vein graft ==== Body Introduction The success of coronary artery bypass grafting (CABG), although the gold standard for the treatment of multivessel coronary artery disease, is limited by poor long-term vein graft patency [1]. Early vein graft thrombosis (within 1 month) occurs in up to 15% of vein grafts due to graft spasm or technical error [2,3], whilst late vein graft failure occurs as a consequence of early neointimal hyperplasia with later superimposed atheroma, so called 'vein graft disease' [1,4] and as many as 50% of all vein grafts are occluded at 10 years post surgery [5,6]. Despite the superiority of arterial graft patency over that of vein grafts, the multivessel nature of coronary artery disease and ready availability of saphenous vein still result in its use in over 70% of CABG procedures [7]. The alternative treatment modality for multivessel coronary atheroma, percutaneous coronary artery angioplasty and stenting (PCI), has traditionally suffered from even worse long-term results compared to CABG, due to high early restenosis rates; over 30% within 1 year [8]. This results in more frequent and more rapid return of symptoms and major adverse cardiac events (MACE) with PCI compared to CABG, necessitating more repeat revascularisation procedures [8]. The apparent success of new drug eluting stents has challenged this paradigm however. Rapamycin (a macrolide antibiotic) and the taxane, paclitaxel, two agents with potent antiproliferative properties, eluted from intracoronary stents, have dramatically reduced restenosis rates, MACE and reintervention rates in clinical trials [9,10], to the point where the superiority of CABG is now being seriously challenged [11]. This represents the clinical application of intensive research into the mechanisms of atherosclerosis and restenosis and strategies for their prevention over the last decade. Conversely, CABG has suffered from its apparent success, and with some exceptions [12], there have been comparatively few attempts to prevent or inhibit the progression of vein graft disease in CABG patients, a condition that must change. Vein graft disease differs from arterial atherosclerosis in that its natural history is much shorter and the date of onset is clearly defined, i.e. graft implantation. This process is therefore potentially amenable to strategies that may inhibit its progression. Although the cellular and molecular mechanisms underlying vein graft disease have been systematically investigated, the time course and development of this process in patients after coronary bypass has only recently been defined as a consequence of the increasing use of intravascular ultrasound (IVUS). Quantitative coronary angiography, traditionally the predominant imaging modality used to assess the severity of vein graft disease underestimates the severity of vein graft remodeling and athermanous plaque development by measuring the vessel lumen in only two dimensions [13,14]. In contrast, the tomographic IVUS image enables visualization of the full circumference of the vessel wall [15] allowing measurement of wall thickening, vein remodeling and atherosclerotic plaque size, distribution, and composition [15]. This results in the detection of diffuse atherosclerotic plaque, compensatory vessel enlargement and preservation of the luminal diameter even in angiographically normal vessels [13,14,16]. IVUS findings in vein grafts also show good correlation with histological findings in clinicopathological studies [17]. The purpose of this review is to summarize our current understanding of the natural history of vein graft disease from IVUS studies, correlate this with the findings of experimental and clinicopathological studies, and, finally to consider how this knowledge, may be used to target prevention or treatment strategies. Early changes in saphenous vein bypass grafts; wall thickening and adaptive remodelling Glagovian remodeling was first described as a radial enlargement of the entire cross sectional area of a vessel in response to intramural atheroma [18]. First identified in humans in post-mortem studies it was only with the widespread use of IVUS that its central role in atherosclerosis, post angioplasty restenosis, transplant vasculopathy and vein graft disease was realised [15]. Currently, the term not only applies to vessel enlargement, but also shrinkage, where in the presence of underlying plaque it becomes an important determinant of lumen loss [19]. In vein grafts, early after implantation, increases in overall vessel cross sectional area preserve luminal size despite significant increases in wall thickness [16]. IVUS measured parameters of vessel remodeling and wall thickening in vein grafts pre, or early post implantation versus later periods are described in Table 1. Vein graft dimensions within one month of implantation are remarkably similar to those in grafts prior to implantation [16,20,24], with significant wall thickening having occurred by six months, even in angiographically normal grafts [14,16]. Other studies have demonstrated wall thickening as early as 3 weeks to 3 months after CABG [23,24]. These changes are diffuse and concentric, and are observed from the aortic root to the coronary anastomosis [23,24]. Higuchi and colleagues compared IVUS measurements of 15 vein grafts performed within 1 month postoperatively with 14 vein grafts after 6 months postoperatively. This showed that significant wall thickening had occurred by 6-months, accompanied by compensatory enlargement, and preservation of the graft luminal diameter (Table 1), however wall thickening appeared to reach a plateau after 6 months with preservation of lumen area (Figure 1), suggesting that early remodeling responses may stabilize in the absence of atherosclerotic development [16]. Figure 1 Increases in wall thickness versus time after surgery. Wall area expressed as a percentage of total vessel area (%VWA) exceeded 40% and reached a plateau state after 6 months in angiographically normal vessels. Reproduced with permission from Higuchi et al, Heart Vessels 2002, 17:57–60, Springer – Verlag, Heidelberg, Germany [16]. Table 1 Early adaptive changes and neointima formation in saphenous vein grafts Study Reference Grafts / Patients Pre implantation to 1 month (mm2) >12 months (mm2) Lumen Wall Area Vessel CSA %wall area Lumen Plaque area Wall Area EEL area Vessel CSA % plaque area %wall area Nishioka et al 1996 [20] 43/42 16.5 ± 5.7 7.4 ± 2.1 23.9 ± 7.3 32.3 ± 7 8.9 ± 2.7 10.0 ± 5.3 15.2 ± 5.8 18.8 ± 7.5 24.0 ± 7.8 51 ± 10 63 ± 7 Ge et al 1999** [21] 43/43 12.6 ± 4.0 – 19.0 ± 9.7 64.5 ± 15.5 Hong et al 1999** [22] 104/93 12.0 ± 4.2 – 3.8 ± 1.9 7.2 ± 4.1 – 13.9 ± 4.9 10.0 ± 3.0 – 20.3 ± 6.5 16.7 ± .9 17.8 ± 6.1 20.8 ± 5.1 – 24.1 ± 7.8 30 ± 5 – 79 ± 9 45 ± 5 – 83 ± 7 Higuchi et al 2002τ [16] 47 16.2 ± 5.5 5.3 ± 2.0 21.6 ± 7.1 24.9 ± 5.0 12.8 ± 4.6 15.8 ± 5.2 28.8 ± 8.8 55.7 ± 6.8 **values represent range from reference segment to focal stenosis, τ angiographically normal vein. Vessel CSA, (cross sectional area) measured by tracing the outer border of the whole vein graft, Wall area, Vessel CSA minus lumen area. Percent wall area was calculated as the wall area divided by Vessel CSA. In situ veins do not have an external elastic membrane however; arterialized saphenous vein grafts develop a sonolucent zone, which has been reported to represent media. The EEL (external elastic membrane) area is measured by tracing the outer border of this sonolucent zone. Plaque area is calculated as external elastic membrane minus lumen area. Percent plaque area is calculated as plaque area divided by external elastic membrane area; this has also been called the plaque burden. Plaque burden and percent wall are closely correlated. IVUS changes correlate closely with the findings of experimental and clinical studies. In porcine saphenous vein bypass grafts, in the first week after grafting, adventitial medial and neointimal thickening occurs as a consequence of increased shear stress, surgical preparative injury and the subsequent activation of multiple growth factor and cytokine cascades. This is associated with the infiltration of inflammatory cells, medial smooth muscle cell proliferation and migration to form a neointima [25]. Adventitial myofibroblast proliferation and extracellular matrix deposition also results in the formation of a thick neoadventitia [26]. These myofibroblasts migrate through all the layers of the vessel wall, where subsequent extracellular matrix deposition contributes to overall wall thickening [27]. A similar distribution of cytoskeletal proteins characteristic of myofibroblasts is observed in explanted human saphenous vein grafts suggesting that similar mechanisms occur in man [27]. After the first week, wall thickening in porcine vein grafts occurs largely due to extracellular matrix deposition (fibrosis) and neointimal smooth muscle cell proliferation, however this thickening plateaus after one month [26]. The early changes seen in the vessel wall of vein grafts are similar to those seen during vessel remodeling in atherosclerotic coronary artery segments [19]. In normal arteries, remodeling is a homeostatic response to changes in flow and circumferential stretch, with compensatory enlargement and wall thickening normalizing shear stress and wall tension in response to higher blood pressures and flow velocities respectively. Outward remodeling in response to increased flow is largely dependent on shear-responsive endothelial production of nitric oxide and the gelatinase matrix metalloproteinases (MMPs) MMP-2 and MMP-9 [28,29]. MMPs are central to the turnover of the extracellular matrix, altering cell-cell interactions, modifying the extracellular milieu and permitting the movement and division of cells. Increased MMP production, with extracellular matrix degradation is a feature of the infiltration of inflammatory cells as well as the migration of smooth muscle cells and myofibroblasts [30,31], and this may also contribute to the remodeling process [19]. Late changes in vein grafts: atherosclerosis and pathological remodelling Early vein graft changes can be viewed as adaptive, however they also predispose the graft to later accelerated graft atherosclerosis [32]. Several components of the extracellular matrix that are abundant in diffuse fibrous intimal hyperplasia may increase the residence of atherogenic molecules, and promote the development of lipid-laden lesions [33,34]. Similarly, myofibroblasts are associated with contractile responses as part of wound healing [35] and it has been hypothesized that dissemination of these cells throughout all layers of the vein graft may be central to later inadequate or constrictive vessel remodeling [36,37]. Risk factors for, and the microscopic appearance of vein graft atherosclerosis are largely similar to those in coronary arteries and it is reasonable to suggest that similar pathological mechanisms are at work, however these occur over a much more rapid time course in vein grafts [1,4]. Atheromatous plaque is detected by IVUS as early as eight to ten months post grafting [38] in association with both expansive and constrictive remodelling [22] (Table 2). This is much earlier than originally suggested by angiography [5,6]. Early IVUS studies disagreed as to the nature of vein graft remodeling, with some studies reporting expansive remodeling [21,38] whilst others did not [20]. This confusion was most likely due to the small sample sizes in these early studies however (Table 2). Hong et al [22] used IVUS to assess the extent and direction of remodeling in 104 grafts in 93 patients, the largest analysis of diseased vein grafts published to date. In individual lesions, they defined remodeling by comparing the area within the EEL at the site of stenosis to that of a reference point. Positive remodeling was defined as a stenosis/ reference EEL area ratio >1.1, intermediate remodeling as a ratio 0.9 to 1.1, and negative remodeling as a ratio <0.9 [39]. All three processes were shown to occur, sometimes even within the same vessel. Overall plaque burden was greater in positively remodeled segments compared with intermediate or negative remodeling, whilst lumen area was preserved in all groups [22]. This is similar to changes that occur in atherosclerotic coronary arteries [40]. Mendellson and colleagues identified expansive remodeling in 98.5% of 24 vein graft lesions studied (Table 2). They showed that lumen area did not change with increasing percent area stenosis for vessels with ~30% of their area occupied by plaque (p = NS), however, for segments with >30% of the vessel area occupied by plaque, there was an inverse relation with the lumen area [38]. In this group (with >30% effective plaque area stenosis), the lumen area decreased as the percentage of vessel area occupied by plaque increased. Again these were the same changes as those noted in coronary arteries [41]. They suggest that "compensatory" enlargement mechanisms are very effective for early atherosclerotic lesions; but as lesions become more progressive, these mechanisms can no longer compensate, and lumen narrowing occurs. This does not explain constrictive or negative remodeling however and the mechanisms underlying this process remain unclear. In addition to the role of myofibroblasts, the formation of a dense neoadventitia with extensive collagen deposition has been implicated as a mechanical barrier to vessel enlargement, with later remodeling of this fibrous tissue ultimately resulting in vessel shrinkage [37]. In atherosclerotic arteries negative or inadequate remodeling is more common in insulin-using than non-insulin-using diabetics [42], more common in smokers compared with nonsmokers, and less frequent in patients with hypercholesterolaemia [43]. Mendellson et al showed that expansive vein graft remodeling was independent of graft age, insertion site, plaque eccentricity, patient age, or gender [38]. Altered local haemodynamics can also affect remodeling. Low shear predisposes the inner curves of tortuous segments to develop atheroma and may impair outward remodeling in a similar manner [44,45]. Alternatively, medial thinning as a consequence of atherosclerotic plaque development may result in bulging of the vessel wall due to diminished structural support at the site of the plaque [46]. Plaque volume often correlates with the level of inflammatory infiltrate, which again may contribute to expansion by promoting collagen lysis and deposition of loose myxoid extracellular matrix [47]. This is thought to underlie the propensity for such plaques to rupture and explain the association between expansive remodeling and unstable coronary symptoms [19]. Table 2 Late remodeling in atherosclerotic vein grafts Grafts / Patients Latency (years) Lumen area Intimal and medial(plaque) area External elastic lamina area Reference Stenosis Reference Stenosis Reference Stenosis Mendellson et al 1995 [38] 21/19 0.8–16 14.6 ± 7.5 7.1 ± 4.5 5.6 ± 3.4 18.3 ± 7.0 20.2 ± 8.5 25.4 ± 8.2 Nishioha et al 1996 [20] 43/42 3–12 15.7 ± 6.8 5.0 ± 1.5 3.2 ± 1.5 13.7 ± 6.3 18.9 ± 7.0 18.7 ± 7.3 Ge et al 1997 [21] 43/43 1–6 12.6 ± 4.0 19.0 ± 9.7 Hong et al 1999 [22] Negative remodeling 104/93 1.2–20.7 12.5 ± 4.0 3.6 ± 2.0 5.0 ± 2.4 - 10.9 ± 6.2 17.8 ± 7.9 14.2 ± 8.1 Positive remodeling 11.8 ± 3.5 4.0 ± 1.8 5.1 ± 1.4 15.3 ± 4.7 16.7 ± 4.6 19.4 ± 6.2 Remodeling can be defined as the ratio of the EEL at the site of the stenosis to that of the reference point. Positive remodeling is defined as a stenosis/ reference EEL area ratio >1.1, intermediate remodeling is defined as a ratio 0.9 to 1.1, and negative remodeling as a ratio <0.9. The future IVUS studies have clearly shown that early 'adaptive' or pathological changes occur within weeks of grafting and that occlusive atheroma, in susceptible individuals occurs within 1 year. IVUS studies have therefore defined the window in which strategies to inhibit vein graft disease might be effective. Furthermore, in addition to its advantages over coronary angiography, IVUS in vein grafts has been shown to be both accurate [12,16] and reproducible [20,22] making it the obvious investigational tool to explore the effectiveness of these strategies on in clinical trials. IVUS measurements correlate closely with clinicopathological findings [12] and can detect standardised differences of >1 in studies of saphenous vein grafts versus ungrafted vein as well as in vein grafts early post implantation (within 1 month) compared with later periods [16,20]. In addition, Nishioka and colleagues [20] demonstrated that for the measurement of lumen area, the mean difference between two observations by the same observer was 3.1%, with a range of 0% to 9.0%. Between two observers, the mean difference was 3.8%, with a range of 1.0% to 5.6%. This reproducibility facilitates not only accurate comparisons between groups of patients but also assessment of the effects of intervention on grafts in longitudinal studies. The ability to manipulate vein grafts ex vivo prior to implantation using pharmacological or other methods that may inhibit subsequent disease is a feature unique to vein graft disease. There are many examples of this being successfully achieved in experimental models. Pre-treatment with rapamycin [48], paclitaxel (GD Angelini, unpublished observations) and the intracellular calcium dependant ATPase inhibitor, thapsigargin [4], have been shown to significantly inhibit the progression of vein graft disease in experimental models in vivo. Oral agents, such as NO donating aspirins [49] and endothelin antagonists have also been shown to be effective in porcine vein grafts in vivo [50] as has the application of a porous external polyester stent which inhibits neointima formation and promotes expansive remodelling in the absence of vein wall thickening [51]. Targeted gene transfer is another attractive option, with viral transfer of E2F-decoy oligonucleotides inhibiting vein graft failure after peripheral arterial reconstruction in clinical trials [52]. Stabilisation of vein grafts that have undergone early adaptive changes, but have yet to develop a large plaque burden is also a possibility. Risk factor modification such as cessation of smoking and aggressive lipid lowering [62–64] has been shown to improve long-term graft patency on angiography. The effect of other interventions such as antiplatelet therapy on the progression of vein graft disease have not been evaluated in IVUS studies however. Identification of patients with a large plaque burden on IVUS, but otherwise angiographically normal vein grafts may also enable targeted plaque stabilisation therapy. Conclusion IVUS has significantly contributed to our understanding of vein graft failure. It also serves as the natural tool for the development of clinical strategies that may lead to significant improvements in vein graft patency and more importantly for better long-term quality of life and longevity for patients with coronary artery disease. The introduction of clinical trials to address this Achille's heel of coronary bypass surgery are long overdue. Competing interests None declared. Authors contributions G Murphy performed the literature review and prepared the manuscript. G Angelini conceived the idea for the manuscript and prepared the manuscript. ==== Refs Motwani JG Topol EJ Aortocoronary saphenous vein graft disease. 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NHLBI post coronary artery bypass graft clinical trial. Post CABG Trial Investigators Circulation 1999 99 3241 3247 10385497 Nase-Hueppmeier S Uebis R Doerr R Hanrath P Intravascular ultrasound to assess aortocoronary venous bypass grafts in vivo Am J Cardiol 1992 70 455 458 1642182 10.1016/0002-9149(92)91189-B Hozumi T Yoshikawa J Yoshida K Use of intravascular ultrasound for in vivo assessment of changes in intimal thickness of angiographically normal saphenous vein grafts one year after aortocoronary bypass surgery Heart 1996 76 317 320 8983677 Nissen SE Yock P Intravascular Ultrasound. 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An intravascular ultrasound study Int J Card Imag 1999 15 295 300 10.1023/A:1006125205217 Hong M Mintz GS Hong MK Intravascular ultrasound assessment of the presence of vascular remodelling in diseased human saphenous bypass grafts Am J Cardiol 1999 84 992 998 10569652 10.1016/S0002-9149(99)00486-5 Kobayashi T Makuuchi H Naruse Y Assessment of saphenous vein graft wall characteristics with intravascular ultrasound imaging Jpn J Thorac Cardiovasc Surg 1998 46 701 706 9785866 Yamada T Itoh T Nakano S Tokunaga O Time-dependent thickening of the intima in aortocoronary saphenous vein grafts: clinicopathological analysis of 24 patients Heart Vessels 1995 10 41 45 7730246 Angelini GD Bryan AJ Williams HMJ Mortgan R Newby AC Distension promotes platelet and leucocyte adhesion and reduces short-term patency in pig arteriovenous bypass grafts J Thorac Cardiovasc Surg 1990 99 433 439 2308361 Shi Y Pieniek M Fard A O'Brien J Mannion JD Zalewski A Adventitial remodeling following coronary arterial 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metalloproteinases in immunity J Immunol 1996 156 1 4 8598448 Schwartz SM deBlois D O'Brien ERM The intima: soil for atherosclerosis and restenosis Circ Res 1995 77 445 465 7641318 Camejo G Lopez A Lopez F Quinones J Interaction of low density lipoproteins with arterial proteoglycans: the role of charge and sialic acid content Atherosclerosis 1985 55 93 105 4004985 Vijayagopal P Srinivasan SR Radhakrishnamurthy B Berenson GS Lipoprotein-proteoglycan complexes from atherosclerotic lesions promote cholesteryl ester accumulation in human monocytes/macrophages Arterioscler Thromb 1992 12 237 249 1543697 Gabbiani G Ryan GB Majno G Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction Experientia 1971 27 549 550 5132594 Shi Y O'Brien J Fard A Mannion JD Wang D Zalewski A Adventitial myofibroblasts contribute to neointimal formation in injured porcine coronary arteries Circulation 1996 94 1655 1664 8840858 Zalewski A Shi Y Vascular Myofibroblasts: Lessons From Coronary Repair and Remodeling Arterioscler Thromb Vasc Biol 1997 17 417 422 9102158 Mendelsohn FO Foster GP Palacios IF Weyman AE Weissman NJ In vivo assessment by intravascular ultrasound of enlargement in saphenous vein bypass graft Am J Cardiol 1995 76 1066 1069 7484864 10.1016/S0002-9149(99)80299-9 Gussenhoven EJ Geselschap JH van Lankeren W Posthuma DJ van der Lugt A Remodeling of atherosclerotic coronary arteries assessed with intravascular ultrasound in vitro Am J Cardiol 1997 79 699 702 9068542 10.1016/S0002-9149(96)00849-1 Nishioka T Luo H Eigler N Berglund H Kim CJ Siegel RT Contribution of inadequate compensatory enlargement to development of human coronary artery stenosis, an in vivo intravascular ultrasound study J Am Coll Cardiol 1996 27 1571 1576 8636538 10.1016/0735-1097(96)00071-X Hermiller JB Tenaglia AN Kisslo KB In vivo validation of compensatory enlargement of atherosclerotic coronary arteries Am J Cardiol 1993 71 665 668 8447262 10.1016/0002-9149(93)91007-5 Kornowski R Mintz GS Lansky AJ Paradoxic decreases in atherosclerotic plaque mass in insulin-treated diabetic patients Am J Cardiol 1998 81 1298 1304 9631966 10.1016/S0002-9149(98)00157-X Tauth J Pinnow E Sullebarger JT Predictors of coronary arterial remodeling patterns in patients with myocardial ischemia Am J Cardiol 1997 80 1352 1355 9388115 10.1016/S0002-9149(97)00682-6 Lerman A Cannan CR Higano SH Coronary vascular remodeling in association with endothelial dysfunction Am J Cardiol 1998 81 1105 1109 9605050 10.1016/S0002-9149(98)00135-0 Wensing PJ Meiss L Mali WP Early atherosclerotic lesions spiraling through the femoral artery Arterioscler Thromb Vasc Biol 1998 18 1554 1558 9763526 Berglund H Luo H Nishioka T Highly localized arterial remodeling in patients with coronary atherosclerosis: an intravascular ultrasound study Circulation 1997 96 1470 1476 9315533 Libby P Current concepts of the pathogenesis of the acute coronary syndromes Circulation 2001 104 365 372 11457759 Schachner T Zou Y Oberhuber A Local application of rapamycin inhibits neointimal hyperplasia in experimental vein grafts Ann Thorac Surg 2004 77 1580 1585 15111146 10.1016/j.athoracsur.2003.10.008 Shukla N Angelini GD Ascione R Talpahewa S Capoun R Jeremy JY Nitric oxide-donating aspirins: novel drugs for the treatment of Saphenous vein graft failure Ann Thorac Surg 2002 75 1437 1442 12735559 10.1016/S0003-4975(02)04892-0 Wan S Yim AP Johnson JL Shukla N Angelini GD Smith FC Dashwood MR Jeremy JY The endothelin 1A receptor antagonist BSF 302146 is a potent inhibitor of neointimal and medial thickening in porcine saphenous vein-carotid artery interposition grafts J Thorac Cardiovasc Surg 2004 127 1317 1322 15115988 10.1016/j.jtcvs.2003.06.018 Izzat MB Mehta D Bryan AJ Reeves B Newby AC Angelini GD Influence of external stent size on early medial and neointimal thickening in a pig model of saphenous vein bypass grafting Circulation 1996 94 1741 1745 8840869 Mann MJ Whittemore AD Donaldson MC Ex-vivo gene therapy of human vascular bypass grafts with E2F decoy: the PREVENT single-centre, randomised, controlled trial Lancet 1999 354 1493 498 10551494 10.1016/S0140-6736(99)09405-2 Cavender JB Rogers WJ Fisher LD Gersh BJ Coggin CJ Myers WO Effects of smoking on survival and morbidity in patients randomized to medical or surgical therapy in the Coronary Artery Surgery Study (CASS): 10 year follow-up J Am Coll Cardiol 1992 20 287 294 1634662 Voors AA van Brussel BL Plokker T Smoking and cardiac events after venous coronary bypass surgery: a 15-year follow-up study Circulation 1996 93 42 44 8616939 The Post Coronary Artery Bypass Graft Trial Investigators The effect ofaggressive lowering of low-density lipoprotein cholesterol levels andlow-dose anticoagulation on obstructive changes in saphenous-vein coronary-artery bypass grafts N Engl J Med 1997 336 153 162 8992351 10.1056/NEJM199701163360301

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==== Front Cardiovasc UltrasoundCardiovascular Ultrasound1476-7120BioMed Central London 1476-7120-2-81526876210.1186/1476-7120-2-8ReviewInsights into the pathogenesis of vein graft disease: lessons from intravascular ultrasound Murphy Gavin J [email protected] Gianni D [email protected] Bristol Heart Institute, University of Bristol, Bristol, BS2 8HW, UK2004 21 7 2004 2 8 8 24 6 2004 21 7 2004 Copyright © 2004 Murphy and Angelini; licensee BioMed Central Ltd.2004Murphy and Angelini; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The success of coronary artery bypass grafting (CABG) is limited by poor long-term graft patency. Saphenous vein is used in the vast majority of CABG operations, although 15% are occluded at one year with as many as 50% occluded at 10 years due to progressive graft atherosclerosis. Intravascular ultrasound (IVUS) has greatly increased our understanding of this process. IVUS studies have shown that early wall thickening and adaptive remodeling of vein grafts occurs within the first few weeks post implantation, with these changes stabilising in angiographically normal vein grafts after six months. Early changes predispose to later atherosclerosis with occlusive plaque detectable in vein grafts within the first year. Both expansive and constrictive remodelling is present in diseased vein grafts, where the latter contributes significantly to occlusive disease. These findings correlate closely with experimental and clinicopathological studies and help define the windows for prevention, intervention or plaque stabilisation strategies. IVUS is also the natural tool for evaluating the effectiveness of pharmacological and other treatments that may prevent or slow the progression of vein graft disease in clinical trials. Coronary artery bypass graftingintravascular ultrasoundsaphenous vein graft ==== Body Introduction The success of coronary artery bypass grafting (CABG), although the gold standard for the treatment of multivessel coronary artery disease, is limited by poor long-term vein graft patency [1]. Early vein graft thrombosis (within 1 month) occurs in up to 15% of vein grafts due to graft spasm or technical error [2,3], whilst late vein graft failure occurs as a consequence of early neointimal hyperplasia with later superimposed atheroma, so called 'vein graft disease' [1,4] and as many as 50% of all vein grafts are occluded at 10 years post surgery [5,6]. Despite the superiority of arterial graft patency over that of vein grafts, the multivessel nature of coronary artery disease and ready availability of saphenous vein still result in its use in over 70% of CABG procedures [7]. The alternative treatment modality for multivessel coronary atheroma, percutaneous coronary artery angioplasty and stenting (PCI), has traditionally suffered from even worse long-term results compared to CABG, due to high early restenosis rates; over 30% within 1 year [8]. This results in more frequent and more rapid return of symptoms and major adverse cardiac events (MACE) with PCI compared to CABG, necessitating more repeat revascularisation procedures [8]. The apparent success of new drug eluting stents has challenged this paradigm however. Rapamycin (a macrolide antibiotic) and the taxane, paclitaxel, two agents with potent antiproliferative properties, eluted from intracoronary stents, have dramatically reduced restenosis rates, MACE and reintervention rates in clinical trials [9,10], to the point where the superiority of CABG is now being seriously challenged [11]. This represents the clinical application of intensive research into the mechanisms of atherosclerosis and restenosis and strategies for their prevention over the last decade. Conversely, CABG has suffered from its apparent success, and with some exceptions [12], there have been comparatively few attempts to prevent or inhibit the progression of vein graft disease in CABG patients, a condition that must change. Vein graft disease differs from arterial atherosclerosis in that its natural history is much shorter and the date of onset is clearly defined, i.e. graft implantation. This process is therefore potentially amenable to strategies that may inhibit its progression. Although the cellular and molecular mechanisms underlying vein graft disease have been systematically investigated, the time course and development of this process in patients after coronary bypass has only recently been defined as a consequence of the increasing use of intravascular ultrasound (IVUS). Quantitative coronary angiography, traditionally the predominant imaging modality used to assess the severity of vein graft disease underestimates the severity of vein graft remodeling and athermanous plaque development by measuring the vessel lumen in only two dimensions [13,14]. In contrast, the tomographic IVUS image enables visualization of the full circumference of the vessel wall [15] allowing measurement of wall thickening, vein remodeling and atherosclerotic plaque size, distribution, and composition [15]. This results in the detection of diffuse atherosclerotic plaque, compensatory vessel enlargement and preservation of the luminal diameter even in angiographically normal vessels [13,14,16]. IVUS findings in vein grafts also show good correlation with histological findings in clinicopathological studies [17]. The purpose of this review is to summarize our current understanding of the natural history of vein graft disease from IVUS studies, correlate this with the findings of experimental and clinicopathological studies, and, finally to consider how this knowledge, may be used to target prevention or treatment strategies. Early changes in saphenous vein bypass grafts; wall thickening and adaptive remodelling Glagovian remodeling was first described as a radial enlargement of the entire cross sectional area of a vessel in response to intramural atheroma [18]. First identified in humans in post-mortem studies it was only with the widespread use of IVUS that its central role in atherosclerosis, post angioplasty restenosis, transplant vasculopathy and vein graft disease was realised [15]. Currently, the term not only applies to vessel enlargement, but also shrinkage, where in the presence of underlying plaque it becomes an important determinant of lumen loss [19]. In vein grafts, early after implantation, increases in overall vessel cross sectional area preserve luminal size despite significant increases in wall thickness [16]. IVUS measured parameters of vessel remodeling and wall thickening in vein grafts pre, or early post implantation versus later periods are described in Table 1. Vein graft dimensions within one month of implantation are remarkably similar to those in grafts prior to implantation [16,20,24], with significant wall thickening having occurred by six months, even in angiographically normal grafts [14,16]. Other studies have demonstrated wall thickening as early as 3 weeks to 3 months after CABG [23,24]. These changes are diffuse and concentric, and are observed from the aortic root to the coronary anastomosis [23,24]. Higuchi and colleagues compared IVUS measurements of 15 vein grafts performed within 1 month postoperatively with 14 vein grafts after 6 months postoperatively. This showed that significant wall thickening had occurred by 6-months, accompanied by compensatory enlargement, and preservation of the graft luminal diameter (Table 1), however wall thickening appeared to reach a plateau after 6 months with preservation of lumen area (Figure 1), suggesting that early remodeling responses may stabilize in the absence of atherosclerotic development [16]. Figure 1 Increases in wall thickness versus time after surgery. Wall area expressed as a percentage of total vessel area (%VWA) exceeded 40% and reached a plateau state after 6 months in angiographically normal vessels. Reproduced with permission from Higuchi et al, Heart Vessels 2002, 17:57–60, Springer – Verlag, Heidelberg, Germany [16]. Table 1 Early adaptive changes and neointima formation in saphenous vein grafts Study Reference Grafts / Patients Pre implantation to 1 month (mm2) >12 months (mm2) Lumen Wall Area Vessel CSA %wall area Lumen Plaque area Wall Area EEL area Vessel CSA % plaque area %wall area Nishioka et al 1996 [20] 43/42 16.5 ± 5.7 7.4 ± 2.1 23.9 ± 7.3 32.3 ± 7 8.9 ± 2.7 10.0 ± 5.3 15.2 ± 5.8 18.8 ± 7.5 24.0 ± 7.8 51 ± 10 63 ± 7 Ge et al 1999** [21] 43/43 12.6 ± 4.0 – 19.0 ± 9.7 64.5 ± 15.5 Hong et al 1999** [22] 104/93 12.0 ± 4.2 – 3.8 ± 1.9 7.2 ± 4.1 – 13.9 ± 4.9 10.0 ± 3.0 – 20.3 ± 6.5 16.7 ± .9 17.8 ± 6.1 20.8 ± 5.1 – 24.1 ± 7.8 30 ± 5 – 79 ± 9 45 ± 5 – 83 ± 7 Higuchi et al 2002τ [16] 47 16.2 ± 5.5 5.3 ± 2.0 21.6 ± 7.1 24.9 ± 5.0 12.8 ± 4.6 15.8 ± 5.2 28.8 ± 8.8 55.7 ± 6.8 **values represent range from reference segment to focal stenosis, τ angiographically normal vein. Vessel CSA, (cross sectional area) measured by tracing the outer border of the whole vein graft, Wall area, Vessel CSA minus lumen area. Percent wall area was calculated as the wall area divided by Vessel CSA. In situ veins do not have an external elastic membrane however; arterialized saphenous vein grafts develop a sonolucent zone, which has been reported to represent media. The EEL (external elastic membrane) area is measured by tracing the outer border of this sonolucent zone. Plaque area is calculated as external elastic membrane minus lumen area. Percent plaque area is calculated as plaque area divided by external elastic membrane area; this has also been called the plaque burden. Plaque burden and percent wall are closely correlated. IVUS changes correlate closely with the findings of experimental and clinical studies. In porcine saphenous vein bypass grafts, in the first week after grafting, adventitial medial and neointimal thickening occurs as a consequence of increased shear stress, surgical preparative injury and the subsequent activation of multiple growth factor and cytokine cascades. This is associated with the infiltration of inflammatory cells, medial smooth muscle cell proliferation and migration to form a neointima [25]. Adventitial myofibroblast proliferation and extracellular matrix deposition also results in the formation of a thick neoadventitia [26]. These myofibroblasts migrate through all the layers of the vessel wall, where subsequent extracellular matrix deposition contributes to overall wall thickening [27]. A similar distribution of cytoskeletal proteins characteristic of myofibroblasts is observed in explanted human saphenous vein grafts suggesting that similar mechanisms occur in man [27]. After the first week, wall thickening in porcine vein grafts occurs largely due to extracellular matrix deposition (fibrosis) and neointimal smooth muscle cell proliferation, however this thickening plateaus after one month [26]. The early changes seen in the vessel wall of vein grafts are similar to those seen during vessel remodeling in atherosclerotic coronary artery segments [19]. In normal arteries, remodeling is a homeostatic response to changes in flow and circumferential stretch, with compensatory enlargement and wall thickening normalizing shear stress and wall tension in response to higher blood pressures and flow velocities respectively. Outward remodeling in response to increased flow is largely dependent on shear-responsive endothelial production of nitric oxide and the gelatinase matrix metalloproteinases (MMPs) MMP-2 and MMP-9 [28,29]. MMPs are central to the turnover of the extracellular matrix, altering cell-cell interactions, modifying the extracellular milieu and permitting the movement and division of cells. Increased MMP production, with extracellular matrix degradation is a feature of the infiltration of inflammatory cells as well as the migration of smooth muscle cells and myofibroblasts [30,31], and this may also contribute to the remodeling process [19]. Late changes in vein grafts: atherosclerosis and pathological remodelling Early vein graft changes can be viewed as adaptive, however they also predispose the graft to later accelerated graft atherosclerosis [32]. Several components of the extracellular matrix that are abundant in diffuse fibrous intimal hyperplasia may increase the residence of atherogenic molecules, and promote the development of lipid-laden lesions [33,34]. Similarly, myofibroblasts are associated with contractile responses as part of wound healing [35] and it has been hypothesized that dissemination of these cells throughout all layers of the vein graft may be central to later inadequate or constrictive vessel remodeling [36,37]. Risk factors for, and the microscopic appearance of vein graft atherosclerosis are largely similar to those in coronary arteries and it is reasonable to suggest that similar pathological mechanisms are at work, however these occur over a much more rapid time course in vein grafts [1,4]. Atheromatous plaque is detected by IVUS as early as eight to ten months post grafting [38] in association with both expansive and constrictive remodelling [22] (Table 2). This is much earlier than originally suggested by angiography [5,6]. Early IVUS studies disagreed as to the nature of vein graft remodeling, with some studies reporting expansive remodeling [21,38] whilst others did not [20]. This confusion was most likely due to the small sample sizes in these early studies however (Table 2). Hong et al [22] used IVUS to assess the extent and direction of remodeling in 104 grafts in 93 patients, the largest analysis of diseased vein grafts published to date. In individual lesions, they defined remodeling by comparing the area within the EEL at the site of stenosis to that of a reference point. Positive remodeling was defined as a stenosis/ reference EEL area ratio >1.1, intermediate remodeling as a ratio 0.9 to 1.1, and negative remodeling as a ratio <0.9 [39]. All three processes were shown to occur, sometimes even within the same vessel. Overall plaque burden was greater in positively remodeled segments compared with intermediate or negative remodeling, whilst lumen area was preserved in all groups [22]. This is similar to changes that occur in atherosclerotic coronary arteries [40]. Mendellson and colleagues identified expansive remodeling in 98.5% of 24 vein graft lesions studied (Table 2). They showed that lumen area did not change with increasing percent area stenosis for vessels with ~30% of their area occupied by plaque (p = NS), however, for segments with >30% of the vessel area occupied by plaque, there was an inverse relation with the lumen area [38]. In this group (with >30% effective plaque area stenosis), the lumen area decreased as the percentage of vessel area occupied by plaque increased. Again these were the same changes as those noted in coronary arteries [41]. They suggest that "compensatory" enlargement mechanisms are very effective for early atherosclerotic lesions; but as lesions become more progressive, these mechanisms can no longer compensate, and lumen narrowing occurs. This does not explain constrictive or negative remodeling however and the mechanisms underlying this process remain unclear. In addition to the role of myofibroblasts, the formation of a dense neoadventitia with extensive collagen deposition has been implicated as a mechanical barrier to vessel enlargement, with later remodeling of this fibrous tissue ultimately resulting in vessel shrinkage [37]. In atherosclerotic arteries negative or inadequate remodeling is more common in insulin-using than non-insulin-using diabetics [42], more common in smokers compared with nonsmokers, and less frequent in patients with hypercholesterolaemia [43]. Mendellson et al showed that expansive vein graft remodeling was independent of graft age, insertion site, plaque eccentricity, patient age, or gender [38]. Altered local haemodynamics can also affect remodeling. Low shear predisposes the inner curves of tortuous segments to develop atheroma and may impair outward remodeling in a similar manner [44,45]. Alternatively, medial thinning as a consequence of atherosclerotic plaque development may result in bulging of the vessel wall due to diminished structural support at the site of the plaque [46]. Plaque volume often correlates with the level of inflammatory infiltrate, which again may contribute to expansion by promoting collagen lysis and deposition of loose myxoid extracellular matrix [47]. This is thought to underlie the propensity for such plaques to rupture and explain the association between expansive remodeling and unstable coronary symptoms [19]. Table 2 Late remodeling in atherosclerotic vein grafts Grafts / Patients Latency (years) Lumen area Intimal and medial(plaque) area External elastic lamina area Reference Stenosis Reference Stenosis Reference Stenosis Mendellson et al 1995 [38] 21/19 0.8–16 14.6 ± 7.5 7.1 ± 4.5 5.6 ± 3.4 18.3 ± 7.0 20.2 ± 8.5 25.4 ± 8.2 Nishioha et al 1996 [20] 43/42 3–12 15.7 ± 6.8 5.0 ± 1.5 3.2 ± 1.5 13.7 ± 6.3 18.9 ± 7.0 18.7 ± 7.3 Ge et al 1997 [21] 43/43 1–6 12.6 ± 4.0 19.0 ± 9.7 Hong et al 1999 [22] Negative remodeling 104/93 1.2–20.7 12.5 ± 4.0 3.6 ± 2.0 5.0 ± 2.4 - 10.9 ± 6.2 17.8 ± 7.9 14.2 ± 8.1 Positive remodeling 11.8 ± 3.5 4.0 ± 1.8 5.1 ± 1.4 15.3 ± 4.7 16.7 ± 4.6 19.4 ± 6.2 Remodeling can be defined as the ratio of the EEL at the site of the stenosis to that of the reference point. Positive remodeling is defined as a stenosis/ reference EEL area ratio >1.1, intermediate remodeling is defined as a ratio 0.9 to 1.1, and negative remodeling as a ratio <0.9. The future IVUS studies have clearly shown that early 'adaptive' or pathological changes occur within weeks of grafting and that occlusive atheroma, in susceptible individuals occurs within 1 year. IVUS studies have therefore defined the window in which strategies to inhibit vein graft disease might be effective. Furthermore, in addition to its advantages over coronary angiography, IVUS in vein grafts has been shown to be both accurate [12,16] and reproducible [20,22] making it the obvious investigational tool to explore the effectiveness of these strategies on in clinical trials. IVUS measurements correlate closely with clinicopathological findings [12] and can detect standardised differences of >1 in studies of saphenous vein grafts versus ungrafted vein as well as in vein grafts early post implantation (within 1 month) compared with later periods [16,20]. In addition, Nishioka and colleagues [20] demonstrated that for the measurement of lumen area, the mean difference between two observations by the same observer was 3.1%, with a range of 0% to 9.0%. Between two observers, the mean difference was 3.8%, with a range of 1.0% to 5.6%. This reproducibility facilitates not only accurate comparisons between groups of patients but also assessment of the effects of intervention on grafts in longitudinal studies. The ability to manipulate vein grafts ex vivo prior to implantation using pharmacological or other methods that may inhibit subsequent disease is a feature unique to vein graft disease. There are many examples of this being successfully achieved in experimental models. Pre-treatment with rapamycin [48], paclitaxel (GD Angelini, unpublished observations) and the intracellular calcium dependant ATPase inhibitor, thapsigargin [4], have been shown to significantly inhibit the progression of vein graft disease in experimental models in vivo. Oral agents, such as NO donating aspirins [49] and endothelin antagonists have also been shown to be effective in porcine vein grafts in vivo [50] as has the application of a porous external polyester stent which inhibits neointima formation and promotes expansive remodelling in the absence of vein wall thickening [51]. Targeted gene transfer is another attractive option, with viral transfer of E2F-decoy oligonucleotides inhibiting vein graft failure after peripheral arterial reconstruction in clinical trials [52]. Stabilisation of vein grafts that have undergone early adaptive changes, but have yet to develop a large plaque burden is also a possibility. Risk factor modification such as cessation of smoking and aggressive lipid lowering [62–64] has been shown to improve long-term graft patency on angiography. The effect of other interventions such as antiplatelet therapy on the progression of vein graft disease have not been evaluated in IVUS studies however. Identification of patients with a large plaque burden on IVUS, but otherwise angiographically normal vein grafts may also enable targeted plaque stabilisation therapy. Conclusion IVUS has significantly contributed to our understanding of vein graft failure. It also serves as the natural tool for the development of clinical strategies that may lead to significant improvements in vein graft patency and more importantly for better long-term quality of life and longevity for patients with coronary artery disease. The introduction of clinical trials to address this Achille's heel of coronary bypass surgery are long overdue. Competing interests None declared. Authors contributions G Murphy performed the literature review and prepared the manuscript. G Angelini conceived the idea for the manuscript and prepared the manuscript. ==== Refs Motwani JG Topol EJ Aortocoronary saphenous vein graft disease. Pathogenesis, predisposition and prevention Circulation 1998 97 916 931 9521341 Bourassa MG Campeau L Lespérance J Grondin CM Changes in grafts and coronary arteries after saphenous vein aortocoronary bypass surgery: results at repeat angiography Circulation 1982 65 90 97 6979434 Rosenfeldt FL Guo-Wei H Buxton BF Angus JA Pharmacology of Coronary Artery Bypass Grafts Ann Thorac Surg 1999 67 878 888 10215263 10.1016/S0003-4975(98)01299-5 Angelini GD Jeremy JY Towards the treatment of vein bypass graft failure – a perspective of the Bristol Heart Institute Biorheology 2002 39 491 499 12122271 Lie JT Lawrie GM Morris G Aortocoronary bypass saphenous vein graft atherosclerosis Am J Cardiol 1997 40 907 914 Atkinson JB Forman MB Vaughn WK Robinowitz M Mcallister HA Virmani R Morphologic changes in long-term saphenous vein bypass grafts Chest 1985 88 341 348 3875453 Izzat MB West RR Bryan AJ Angelini GD Coronary artery bypass surgery: Current practise in the United Kingdom Br Heart J 1994 71 382 385 8198893 Hoffman SN TenBrook JA Wolf MP A meta-analysis ofrandomized controlled trials comparing coronary artery bypass graft with percutaneous transluminal coronary angioplasty: one- to eight-year outcomes J Am Coll Cardiol 2003 41 1293 1304 12706924 10.1016/S0735-1097(03)00157-8 Park SJ Shim WH Ho DS A paclitaxel-eluting stent for the prevention of coronary restenosis N Engl J Med 2003 348 1537 1545 12700373 10.1056/NEJMoa021007 Regar E Serruys PW Bode C Angiographic findings of the multicenter Randomized Study With the Sirolimus-Eluting Bx Velocity Balloon-Expandable Stent (RAVEL): sirolimus-eluting stents inhibit restenosis irrespective of the vessel size Circulation 2002 106 1949 1956 12370218 10.1161/01.CIR.0000034045.36219.12 Lemos PA Serruys PW van Domburg RT Unrestricted utilization of sirolimus-eluting stents compared with conventional bare stent implantation in the "real world": the Rapamycin-Eluting Stent Evaluated At Rotterdam Cardiology Hospital (RESEARCH) registry Circulation 2004 109 190 195 14691037 10.1161/01.CIR.0000109138.84579.FA Campeau L Hunninghake DB Knatterud GL Aggressivecholesterol lowering delays saphenous vein graft atherosclerosis in women, the elderly, and patients with associated risk factors. NHLBI post coronary artery bypass graft clinical trial. Post CABG Trial Investigators Circulation 1999 99 3241 3247 10385497 Nase-Hueppmeier S Uebis R Doerr R Hanrath P Intravascular ultrasound to assess aortocoronary venous bypass grafts in vivo Am J Cardiol 1992 70 455 458 1642182 10.1016/0002-9149(92)91189-B Hozumi T Yoshikawa J Yoshida K Use of intravascular ultrasound for in vivo assessment of changes in intimal thickness of angiographically normal saphenous vein grafts one year after aortocoronary bypass surgery Heart 1996 76 317 320 8983677 Nissen SE Yock P Intravascular Ultrasound. Novel Pathophysiological Insights and Current Clinical Applications Circulation 2001 103 604 616 11157729 Higuchi Y Hirayama A Shimizu M Sakakibara T Kodama K Postoperative changes in angiographically normal saphenous vein coronary bypass grafts using intravascular ultrasound Heart Vessels 2002 17 57 60 12541095 10.1007/s003800200044 Willard JE Netto D Demian SE Intravascular ultrasound imaging of saphenous vein grafts in vitro: comparison with histologic andquantitative angiographic findings J Am Coll Cardiol 1992 19 759 764 1545070 Glagov S Weisenberg E Zarins CK Stankunavicius R Kolettis G Compensatory enlargement of human atherosclerotic coronary arteries: N Engl J Med 1987 316 1371 1375 3574413 Ward MR Pasterkamp G Yeung AC Borst C Arterial Remodeling; Mechanisms and Clinical Implications Circulation 2000 102 1186 1191 10973850 Nishioka T Luo H Berglund H Eigler NL Kim C-J Tabak SW Siegel RJ Absence of focal compensatory enlargement or constriction in diseased human coronary saphenous vein bypass grafts: an intravascular ultrasound study Circulation 1996 93 683 690 8640996 Ge J Liu F Bhate R Does remodeling occur in the diseased human saphenous vein bypass grafts? An intravascular ultrasound study Int J Card Imag 1999 15 295 300 10.1023/A:1006125205217 Hong M Mintz GS Hong MK Intravascular ultrasound assessment of the presence of vascular remodelling in diseased human saphenous bypass grafts Am J Cardiol 1999 84 992 998 10569652 10.1016/S0002-9149(99)00486-5 Kobayashi T Makuuchi H Naruse Y Assessment of saphenous vein graft wall characteristics with intravascular ultrasound imaging Jpn J Thorac Cardiovasc Surg 1998 46 701 706 9785866 Yamada T Itoh T Nakano S Tokunaga O Time-dependent thickening of the intima in aortocoronary saphenous vein grafts: clinicopathological analysis of 24 patients Heart Vessels 1995 10 41 45 7730246 Angelini GD Bryan AJ Williams HMJ Mortgan R Newby AC Distension promotes platelet and leucocyte adhesion and reduces short-term patency in pig arteriovenous bypass grafts J Thorac Cardiovasc Surg 1990 99 433 439 2308361 Shi Y Pieniek M Fard A O'Brien J Mannion JD Zalewski A Adventitial remodeling following coronary arterial injury Circulation 1996 93 340 348 8548908 Shi Y O'Brien JE JrFard A Mannion JD Wang D Zalewski A Adventitial myofibroblasts contribute to neointimal formation in injured porcine coronary arteries Circulation 1996 94 1655 1664 8840858 Bassiouny HS Song RH Hong XF Singh A Kocharyan H Glagov S Flow regulation of 72-kD collagenase IV (MMP-2) after experimental arterial injury Circulation 1998 98 157 163 9679722 Wentzel JJ Kloet J Andhyiswara I Oomen JA Schuurbiers JC de Smet BJ Post MJ de Kleijn D Pasterkamp G Borst C Slager CJ Krams R Shear-stress and wall-stress regulation of vascular remodeling after balloon angioplasty: effect of matrix metalloproteinase inhibition Circulation 2001 104 91 96 11435344 Kenagy RD Vergel S Mattsson E Bendeck M Reidy MA Clowes AW The role of plasminogen, plasminogen activators, and matrix metalloproteinases in primate arterial smooth muscle cell migration Arterioscler Thromb Vasc Biol 1996 16 1373 1382 8911276 Goetzl EJ Banda MJ Leppert D Matrix metalloproteinases in immunity J Immunol 1996 156 1 4 8598448 Schwartz SM deBlois D O'Brien ERM The intima: soil for atherosclerosis and restenosis Circ Res 1995 77 445 465 7641318 Camejo G Lopez A Lopez F Quinones J Interaction of low density lipoproteins with arterial proteoglycans: the role of charge and sialic acid content Atherosclerosis 1985 55 93 105 4004985 Vijayagopal P Srinivasan SR Radhakrishnamurthy B Berenson GS Lipoprotein-proteoglycan complexes from atherosclerotic lesions promote cholesteryl ester accumulation in human monocytes/macrophages Arterioscler Thromb 1992 12 237 249 1543697 Gabbiani G Ryan GB Majno G Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction Experientia 1971 27 549 550 5132594 Shi Y O'Brien J Fard A Mannion JD Wang D Zalewski A Adventitial myofibroblasts contribute to neointimal formation in injured porcine coronary arteries Circulation 1996 94 1655 1664 8840858 Zalewski A Shi Y Vascular Myofibroblasts: Lessons From Coronary Repair and Remodeling Arterioscler Thromb Vasc Biol 1997 17 417 422 9102158 Mendelsohn FO Foster GP Palacios IF Weyman AE Weissman NJ In vivo assessment by intravascular ultrasound of enlargement in saphenous vein bypass graft Am J Cardiol 1995 76 1066 1069 7484864 10.1016/S0002-9149(99)80299-9 Gussenhoven EJ Geselschap JH van Lankeren W Posthuma DJ van der Lugt A Remodeling of atherosclerotic coronary arteries assessed with intravascular ultrasound in vitro Am J Cardiol 1997 79 699 702 9068542 10.1016/S0002-9149(96)00849-1 Nishioka T Luo H Eigler N Berglund H Kim CJ Siegel RT Contribution of inadequate compensatory enlargement to development of human coronary artery stenosis, an in vivo intravascular ultrasound study J Am Coll Cardiol 1996 27 1571 1576 8636538 10.1016/0735-1097(96)00071-X Hermiller JB Tenaglia AN Kisslo KB In vivo validation of compensatory enlargement of atherosclerotic coronary arteries Am J Cardiol 1993 71 665 668 8447262 10.1016/0002-9149(93)91007-5 Kornowski R Mintz GS Lansky AJ Paradoxic decreases in atherosclerotic plaque mass in insulin-treated diabetic patients Am J Cardiol 1998 81 1298 1304 9631966 10.1016/S0002-9149(98)00157-X Tauth J Pinnow E Sullebarger JT Predictors of coronary arterial remodeling patterns in patients with myocardial ischemia Am J Cardiol 1997 80 1352 1355 9388115 10.1016/S0002-9149(97)00682-6 Lerman A Cannan CR Higano SH Coronary vascular remodeling in association with endothelial dysfunction Am J Cardiol 1998 81 1105 1109 9605050 10.1016/S0002-9149(98)00135-0 Wensing PJ Meiss L Mali WP Early atherosclerotic lesions spiraling through the femoral artery Arterioscler Thromb Vasc Biol 1998 18 1554 1558 9763526 Berglund H Luo H Nishioka T Highly localized arterial remodeling in patients with coronary atherosclerosis: an intravascular ultrasound study Circulation 1997 96 1470 1476 9315533 Libby P Current concepts of the pathogenesis of the acute coronary syndromes Circulation 2001 104 365 372 11457759 Schachner T Zou Y Oberhuber A Local application of rapamycin inhibits neointimal hyperplasia in experimental vein grafts Ann Thorac Surg 2004 77 1580 1585 15111146 10.1016/j.athoracsur.2003.10.008 Shukla N Angelini GD Ascione R Talpahewa S Capoun R Jeremy JY Nitric oxide-donating aspirins: novel drugs for the treatment of Saphenous vein graft failure Ann Thorac Surg 2002 75 1437 1442 12735559 10.1016/S0003-4975(02)04892-0 Wan S Yim AP Johnson JL Shukla N Angelini GD Smith FC Dashwood MR Jeremy JY The endothelin 1A receptor antagonist BSF 302146 is a potent inhibitor of neointimal and medial thickening in porcine saphenous vein-carotid artery interposition grafts J Thorac Cardiovasc Surg 2004 127 1317 1322 15115988 10.1016/j.jtcvs.2003.06.018 Izzat MB Mehta D Bryan AJ Reeves B Newby AC Angelini GD Influence of external stent size on early medial and neointimal thickening in a pig model of saphenous vein bypass grafting Circulation 1996 94 1741 1745 8840869 Mann MJ Whittemore AD Donaldson MC Ex-vivo gene therapy of human vascular bypass grafts with E2F decoy: the PREVENT single-centre, randomised, controlled trial Lancet 1999 354 1493 498 10551494 10.1016/S0140-6736(99)09405-2 Cavender JB Rogers WJ Fisher LD Gersh BJ Coggin CJ Myers WO Effects of smoking on survival and morbidity in patients randomized to medical or surgical therapy in the Coronary Artery Surgery Study (CASS): 10 year follow-up J Am Coll Cardiol 1992 20 287 294 1634662 Voors AA van Brussel BL Plokker T Smoking and cardiac events after venous coronary bypass surgery: a 15-year follow-up study Circulation 1996 93 42 44 8616939 The Post Coronary Artery Bypass Graft Trial Investigators The effect ofaggressive lowering of low-density lipoprotein cholesterol levels andlow-dose anticoagulation on obstructive changes in saphenous-vein coronary-artery bypass grafts N Engl J Med 1997 336 153 162 8992351 10.1056/NEJM199701163360301

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PMC514614

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Int Semin Surg Oncol. 2004 Aug 11; 1:5

latin-1

Int Semin Surg Oncol

10.1186/1477-7800-1-5

oa_comm

==== Front World J Surg OncolWorld Journal of Surgical Oncology1477-7819BioMed Central London 1477-7819-2-281531040710.1186/1477-7819-2-28Case ReportPrimary small-cell neuroendocrine carcinoma of the duodenum – a case report and review of literature Sata Naohiro [email protected] Munetoshi [email protected] Masaru [email protected] Koji [email protected] Katsumi [email protected] Hideo [email protected] Tsutomu [email protected] Ken [email protected] Department of Surgery, Jichi Medical School, 3311-1 Yakushiji Minami-kawachi Tochigi, Japan2 Department of Pathology, Jichi Medical School, 3311-1 Yakushiji Minami-kawachi Tochigi, Japan2004 15 8 2004 2 28 28 6 5 2004 15 8 2004 Copyright © 2004 Sata et al; licensee BioMed Central Ltd.2004Sata et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Small-cell neuroendocrine carcinoma in the duodenum is an extremely rare neoplasm with poor prognosis. Case presentation A 57-year-old man presented with sudden onset gastrointestinal bleeding and fainting attacks. Duodenoscopy and hypotonic duodenography revealed a 3 × 3 cm protruding tumor with ulcerations situated opposite the ampulla of Vater in the second part of the duodenum. Local excision of the tumor was performed, followed by adjuvant chemotherapy with 5-fluoro uracil and leucovorin. Examination of the tumor by immunohistochemistry and electron microscopy indicated it to be neuroendocrine in nature, expressing synaptophysin and AE1/AE3, and containing dense core granules. The patient showed no sign of recurrence and has been disease-free for more than 48 months after surgery. Conclusions Most cases of small-cell neuroendocrine carcinoma in the duodenum show rapid progression of the disease, and even radical surgery with or without chemotherapy do not prevent death. We report a rare subtype of small-cell neuroendocrine carcinoma. This subtype appears to have a much better prognosis, and may be amenable to local excision, if the lesion is away from the ampulla of Vater. ==== Body Background Duodenal Neuroendocrine tumors constitute 5% of all gastrointestinal neuroendocrine tumors [1,2]. Most of these show well-differentiated features and are classified as carcinoids or somatostatinomas [3-6]. Occurrence of carcinoma is rare, and carcinomas with anaplastic character, which are classified as small-cell carcinomas, are even less frequent [7-12]. The most common small-cell neuroendocrine carcinoma (NEC) is the small-cell undifferentiated carcinoma of the lung [13,14]. Although the features of these pulmonary tumors are well defined, the characteristics of their extrapulmonary counterparts are still unknown. We report a case of small-cell NEC in the duodenum that had unique morphological features and exceptionally good clinical outcome. Case presentation A 57-year-old man presented with sudden gastrointestinal tract bleeding and episode of fainting. Duodenoscopy (Figure 1a) and hypotonic duodenography (Figure 1b) revealed a 3 × 3 cm protruding tumor with two ulcerations located opposite the ampulla of Vater in the second part of the duodenum. Laboratory data showed no abnormalities in blood chemistry, tumor markers (CEA, CA19-9, NSE, proGRP) and endocrine markers (somatostatin, gastrin, glucagons, serotonin, VIP) except a moderate anemia (9.5 g/dl hemoglobin). No abnormal findings were observed in the chest X-ray and computed tomography (CT). Figure 1 (a) Duodenoscopy showing a 3 × 3 cm protruding tumor with two ulcerations located opposite the ampulla of Vater in the second portion of the duodenum. (b) Hypotonic duodenography showing the donuts-shape tumor in the duodenum. A laparotomy was performed. As there was no serosal invasion or regional lymphadenopathy wide local excision of the tumor was performed. On gross examination, the tumor showed two ulcerations and two different morphological components (Figure 2a and 2b). One component (component A) was round in shape with a round ulceration on the top, and the other component (component B), which enclosed the round component, was crescent in shape with a spindle-shaped ulceration on the top. The two components showed different histopathological and immunohistochemical features (Table 1). The round component contained fibrous tissue, small nuclei, and clear nucleoli. Histopathologically, the crescent component had more anaplastic features typical of small-cell carcinoma, such as sheets of tightly packed anaplastic cells with round nuclei and scanty cytoplasm (Figure 2c, 2d). Neuroendocrine differentiation was investigated using immunohistochemical and ultrastructural techniques. Both components showed neuroendocrine features, with immunochemistry identifying synaptophysin and AE1/AE3 (Figure 3a and 3b), and electron microscopy identifying dense core granules (Figure 4). Immunochemistry also showed that the crescent component expressed less cytokeratin, vimentin and CD56, and more MIB-1 than the round component. Figure 2 Macroscopic and microscopic findings of the tumor. (a) Gross appearance of the tumor. The tumor was divided into two components, component A (round shape) and B (crescent shape). (b) Photomicrograph of the gross appearance of the tumor (Hematoxylin and eosin X 2). (c) Photomicrograph of the component A showing fibrous tissue, small nuclei, and clear nucleoli. (Hematoxylin and Eosin X 40). (d) Photomicrograph of the component B showing more anaplastic features typical of small-cell carcinoma, such as sheets of tightly packed anaplastic cells with round nuclei and scanty cytoplasm. (Hematoxylin and Eosin X 40). Table 1 Immunochemical characteristics of the two components of the tumor. Synaptophysin AE1/AE3 Vimentin CD56 chromogranin A MIB1 (A) Round component ++ ++ ++ + - 25% (B) Crescent component + - - - - 50% LCA, L26, UCHL1, CD3, ASMA, M-actin, desmin, CD34, NF, GFAP, and S100 were negative in both components. Figure 3 Immunostaining for AE1/AE3 showing (a) diffuse cytoplasmic positivity in the component A, and (b) no reactivity in the component B. Figure 4 Ultrastructural study showed cytoplasmic dense-core granules in the component A. The patient was discharged three weeks after operation with uneventful postoperative period. Four cycles of monthly adjuvant chemotherapy with 5-fluoro uracil (5-FU) (325 mg/m2) and leucovorin (20 mg/m2) were administered. The patient showed no sign of recurrence and is disease-free 48 months after surgery. Discussion Neuroendocrine carcinomas (NEC) in the duodenum are extremely rare, and are classified as either 'small-cell' or 'non small-cell' types. The small-cell NEC occurring in the duodenum and elsewhere in gastrointestinal tract are similar to the small-cell carcinoma of the lung [7,8]. Only eight cases of small-cell NEC in the duodenum have been reported, with the present case being the ninth (Table 2) [7-12]. Most cases occurred in middle-aged or geriatric males with lesions in the ampulla of Vater. Extra-ampullary small-cell NECs in the duodenum are extremely rare, with only two cases reported previously [7,8]. Table 2 Profiles of the cases of primary small-cell neuroendocrine carcinoma in the duodenum reported in the literature. Author Year Age/sex Clinical Manifestations Size (mm) Morphology Location Metastasis Surgery Chemotherapy Prognosis (month) 1 Swanson 1986 [7] 76 M Abdominal pain, anorexia, weight loss 15 ulceration adjacent to the ampulla* LN, liver biospy 5-FU, doxorubicin, mitomycin Dead (1.5) 2 Zamboni 1990 [8] 62 M jaundice, weight loss 25 polypoid the papilla of Vater LN PD - Dead (7) 3 Zamboni 1990 [8] 66 M jaundice, abdominal pain 20 ulceration the papilla of Vater LN PD - Dead (6) 4 Zamboni 1990 [8] 51 M jaundice, weight loss, abdominal pain 30 soft fungating mass the papilla of Vater LN PD - Dead (17) 5 Lee 1992 [9] 86 M jaundice, recurrent pancreatitis ? polypoid Peri ampullary ? - - (>5) 6 Sarker 1992 [10] 53 F jaundice, weight loss, back pain 35 mass with small ulceration the papilla of Vater LN PD 5-FU, TNF, interferon Recurrence + (>18) 7 Sato 1995 [11] 74 M jaundice 35 polypoid the papilla of Vater ? PPPD - ? 8 Shim 2000 [12] 54 M jaundice 30 ulceration the papilla of Vater liver PD cisplatin, etoposide, radiation Dead (8) 9 Sata 2004 present case 57 M GI Tract bleeding 30 mass with ulceration Peri ampullary - Local resection 5-FU leucovorin disease free (>48) * Hormone VIP; ? Don't know; M – male; F – Female; PD-pancreaticoduodenectomy; LN-lymph node; PPPD-pylorus preserving pancreaticoduodenectomy; 5 FU-f fluoro uracil; TNF – tumor necrosis factor The natural course of small-cell NECs in the duodenum is still not clear. Most cases reported in the literature show rapid progress of the disease, with radical surgery and/or chemotherapy not altering the clinical course, and thus a poor prognosis. In six of the previous eight cases, patients underwent pancreaticoduodenectomy for removal of the tumor, while the remaining two did not undergo surgery because of multiple liver metastasis or poor general condition. In spite of radical resection with or without adjuvant chemotherapy, most cases showed rapid recurrence and metastasis. Of the eight reported cases, one was unusual as it occurred in a middle-aged female with rapid progress of the disease but effective response to adjuvant chemotherapy using 5FU, tumor necrosis factor, and interferon this patient survived for more than eighteen months [10]. The present case was treated by a local excision of the tumor followed by adjuvant chemotherapy using 5-FU and leucovorin, and showed a distinctively unique clinical course, with the patient surviving for more than 48 months without any sign of recurrence. This case was presented with gastrointestinal bleeding, which contributed to early diagnosis, whereas the other previous cases in literature presented either with abdominal pain or jaundice. Hence, the good prognosis in present case could also be due to its earlier presentation. The lesion in the present case showed a different immunohistological character from that in other cases, such as no immunoreactivity to neuron-specific enolase (NSE) or chromogranin A. These differences too might partly explain the different character of this case. Conclusions This report identifies a new subtype of small-cell NEC in the duodenum. This subtype appears to have a much better prognosis, and may be amenable to local excision, if the lesion is away from the ampulla of Vater. Competing interests None declared. Authors' contributions NS, MT, MK, KY, KK, HN took part in the operation, performed the literature search and drafted the manuscript for submission. HN supervised the preparation of the manuscript and edited the final version for publication. TS, KS performed pathological investigations and contributed to the pathological content of the manuscript. All authors read and approved the manuscript. Acknowledgement The patient's consent was obtained for publication of his case records, duodenoscopy, barium, and histopathological pictures. ==== Refs Marcial MA Pinkus PS Scaring A Ampullary somatostatinoma Am J Clin Pathol 1983 80 755 761 6314801 Sanchez-Sosa S Angeles AA Orozco H Larriva-Sahd J Neuroendocrine carcinoma of the ampulla of Vater. A case of absence of somatostatin in a vasoactive intestinal polypeptide-, bombesin-, and cholecystokinin-producing tumor Am J Clin Pathol 1991 95 51 54 1670974 Capella C Riva C Rindi G Usellini L Chiaravalli A Solcia E Endocrine tumors of the duodenum and upper jejunum. A study of 33 cases with clinico-pathological characteristics and hormone content Hepatogastroenterology 1990 37 247 252 2160422 Feurle GE Anlauf M Hamscher G Arnold R Kloppel G Weihe E Xenin-immunoreactive cells and extractable xenin in neuroendocrine tumors of duodenal origin Gastroenterology 2002 123 1616 1626 12404236 Sawady J Katzin WE Mendelsohn G Aron DC Somatostatin-producing neuroendocrine tumor of the ampulla (Ampullary somatostatinoma) evidence of prosomatostatin production Am J Clin Pathol 1992 97 411 415 1371903 Rios A Fernandez JA Rodriguez JM Lujan JA Martinez E Parrilla P Massive upper gastrointestinal bleeding as a manifestation of somatostatinoma of the ampulla of Vater Dig Dis Sci 2001 46 2162 2165 11680591 10.1023/A:1011954713463 Swanson PE Dykoski D Wick MR Snover DC Primary duodenal small-cell neuroendocrine carcinoma with production of vasoactive intestinal polypeptide Arch Pathol Lab Med 1986 110 317 320 3006628 Zamboni G Franzin G Bonetti F Scarpa A Chilosi M Colombari R Menestrina F Pea M Iacono C Serio G Fiore-Donati L Small-cell neuroendocrine carcinoma of the ampullary region. A clinicopathologic, immunohistochemical, and ultrastructural study of three cases Am J Surg Pathol 1990 14 703 13 1696069 Lee CS Machet D Rode J Small cell carcinoma of the ampulla of Vater Cancer 1992 70 1502 1504 1325273 Sarker AB Hoshida Y Akagi S Hayashi K Murakami I Jeon HJ Takahashi K Akagi T An immunohistochemical and ultrastructural study of case of small-cell neuroendocrine carcinoma in the ampullary region of the duodenum Acta Pathol Jpn 1992 42 529 535 1384272 Sato T Yamamoto K Ouchi A Imaoka Y Tokumura H Matsushiro T Undifferentiated carcinoma of the duodenal ampulla J Gastroenterol 1995 30 517 519 7550864 Shim CS Moon JH Cho YD Lee MS Jeon HB Jin SY Lee HK Neuroendocrine carcinoma of the ampulla of Vater Gastrointest Endosc 2000 51 593 10805848 10.1067/mge.2000.103674 Bensch KG Corrin B Pariente R Spencer H Oat cell carcinoma of the lung: its origin and relationship to bronchial carcinoid Cancer 1968 22 1163 1172 5705778 Ibrahim NB Briggs JC Corbishley CM Extrapulmonary oat cell carcinoma Cancer 1984 54 1645 1661 6089995

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World J Surg Oncol. 2004 Aug 15; 2:28

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World J Surg Oncol

10.1186/1477-7819-2-28

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==== Front Reprod Biol EndocrinolReproductive biology and endocrinology : RB&E1477-7827BioMed Central London 1477-7827-2-601529651010.1186/1477-7827-2-60ResearchIn vitro fertilization and artificial activation of eggs of the direct-developing anuran Eleutherodactylus coqui Toro Esteban [email protected] Scott F [email protected] Department of Tropical Medicine, Box SL-17, Tulane University, New Orleans, LA 70112, USA2 Department of Developmental Biology, Stanford University, Stanford, CA 94045, USA2004 5 8 2004 2 60 60 22 3 2004 5 8 2004 Copyright © 2004 Toro and Michael; licensee BioMed Central Ltd.2004Toro and Michael; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Although much is known about the reproductive biology of pond-breeding frogs, there is comparatively little information about terrestrial-breeding anurans, a highly successful and diverse group. This study investigates the activation and in vitro fertilization of eggs of the Puerto Rican coqui frog obtained by hormonally induced ovulation. We report that spontaneous activation occurs in 34% of eggs, probably in response to mechanical stress during oviposition. Artificial activation, as evidenced by the slow block to polyspermy and the onset of zygote division, was elicited both by mechanical stimulation and calcium ionophore exposure in 64% and 83% of the cases, respectively. Finally, one in vitro fertilization protocol showed a 27% success rate, despite the fact that about one third of all unfertilized eggs obtained by hormone injection auto-activate. We expect these findings to aid in the conservation effort of Eleutherodactylus frogs, the largest vertebrate genus. ==== Body Background The study of reproduction and its artificial manipulation is important in many fields. For example, in sea urchins, an animal's testes can be dissected and sperm is activated by exposure to seawater. Eggs can be released by injecting KCl into the perivisceral cavity, and mixing eggs and sperm in vitro produces fertilization, as evidenced by the appearance of the fertilization membrane and subsequent development of embryos [1]. These simple techniques have been the basis for such dissimilar studies as those of Berdishev [1], dealing with the role of fatty acids and cannabinoids in fertilization, to investigation of the gene expression patterns of hybrids by Nielsen and coworkers [2]. Artificial reproduction has also been well-studied in mammals, and cloning of eutherians from somatic cells is now common [3-8]. Harvested eggs can be enucleated and merged with a somatic cell and the reconstructed embryos cultured in vitro before being implanted into surrogate mothers [8]. These methods have opened up new possibilities in both basic and applied science [e.g. [9]]. Importantly, artificial fertilization has been utilized as a means of assisting with the conservation effort of declining species [10,11]. Frogs have been favorite model organisms in reproductive and developmental biology for many years, mainly because of the ease with which they can be kept in captivity; their external fertilization; easily visible development in large, transparent eggs; and large numbers and ease of manipulation of their eggs. Consequently, research on frogs has often been in the vanguard of advancement in artificial reproduction techniques, and much is known about a few model species such as the African clawed frog, Xenopus laevis and the North American leopard frog, Rana pipiens [e.g. [12-15]]. Indeed, the first vertebrate cloned from a somatic nucleus was a frog [16]. Briggs and King injected female R. pipiens with male pituitary glands to induce ovulation and deposition of unfertilized eggs. The eggs were mechanically activated by pricking with a needle, a process which brings the pronucleus immediately under the surface of the animal pole. Taking advantage of this situation, the pronuclei were extruded, along with a small amount of cytoplasm, using a glass needle. In other species, such as the Xenopus or the axolotl, UV radiation can be used to destroy the female pronucleus instead [17,18]. Development was then directed by a somatic nucleus microinjected into the cytoplasm of the enucleated egg. In another group of experiments, Kroll and Amaya [19] developed an effective and reliable method for creating transgenic Xenopus: testes were macerated in solution and the sperm membranes partially dissolved, allowing access to the condensed chromosomes. Linearized bacterial plasmids containing genes of interest were mixed in with the sperm solution and recombinant ligase was used to covalently insert the bacterial plasmids into the sperm genomic DNA, resulting in the insertion of many copies of the plasmid construct into each genome. These nuclei were then microinjected into mature eggs, generating, under appropriate conditions, hundreds of nonmosaic, transgenic embryos. Such techniques allow the investigation of gene function in these species [e.g. [20]]. Clearly, there are enormous advantages to being able to manipulate a species' reproduction in the laboratory. However, despite the multiplicity of studies concentrating on anurans, to date all model species are aquatic breeders. Yet amphibians have the largest diversity of breeding strategies among terrestrial vertebrates, and it is to be expected that species with different reproductive strategies will require different methods for their manipulation in the laboratory. Therefore, many species remain experimentally intractable. Notably, terrestrial-breeding frogs, a very large and diverse group of organisms, are largely inaccessible to reproductive investigations. The neotropical frog genus Eleutherodactylus is characterized by terrestrial breeding and direct development without an aquatic larval stage. With more than seven hundred described species, this is the largest vertebrate genus [21,22]. There has been considerable experimental attention focused on Eleutherodactylus frogs, ranging from basic developmental biology [23-26]; to ecology [e.g. [27-30]]; to the evolution of development [23,31]. However, there are as yet no available techniques for performing in vitro fertilization in these frogs. The development of such techniques would allow additional investigations into the genetic regulation of direct development in these species and would also assist with conservation of declining populations, an important goal considering the fact that many species of Eleutherodactylus are declining, and several are already extinct [32,33]. Eleutherodactylus coqui are small tree frogs with internal fertilization and direct development [34]. This species is extremely common in the forests of Puerto Rico, and it has been found that their population size is limited by the availability of retreat sites, as opposed to food resources [35]. As with all other studied Eleutherodactylus species, E. coqui embryos develop directly into tiny froglets in terrestrial eggs, without a tadpole stage [36]. Protocols for the husbandry of these frogs have been reported, and it is possible to maintain them in the laboratory for multiple generations [37,38]. A method has also been developed to induce ovulation using an artificial form of luteinizing hormone-releasing hormone (LHRH) [39]. It is know in this species that sperm entry occurs at a small disc at the animal pole of the egg, and that polyspermy is apparently common but does not interfere with development [40]. Cortical granules and their exocitosis have also been described using electron microscopy [40], but the large (5 mm diameter), opaque and featureless eggs make it difficult to observe the rising of a fertilization membrane. As E. coqui is arguably the best-studied terrestrial-breeding frog, we have focused on this particular species as a model for the development of reproductive techniques. Materials and Methods Adult Eleutherodactylus coqui frogs were collected in Puerto Rico near El Verde Field Station in the Luquillo mountains and transported to Tulane University where they were housed and fed as previously described [37]. All animals were handled and experiments performed in accordance with the standards outlined in the NIH Guide for the Care and Use of Laboratory Animals. Natural matings were performed by placing a gravid female and a calling male together as described [37]. Mature, unfertilized eggs were obtained by injection of gravid females with 20 μg of des-Gly, D-Ala LHRH ethylamide (Sigma, St. Louis, MO. Catalog Number: L4513), as reported [39]. Hormonally induced females were placed in plastic bags and allowed to deposit unfertilized eggs. Eggs were experimentally manipulated without moving them from the surface of the plastic bag where they were deposited. Sperm was obtained from adult male frogs that were anesthetized by immersion in 5% benzocaine solution, decapitated and double-pithed. Testes were removed by dissection and macerated with fine forceps. In vitro fertilization (IVF) experiments using sperm in solution were carried out by macerating the testes from a single frog in 500 μL of sperm dilution buffer (SDB: 10 mM NaCl, 0.2 mM KCl, 0.1 mM CaCl2, 0.1 mM MgCl2, 0.5 mM Hepes pH 7.5), and adding this dropwise over the tops of the eggs or injecting it under the jelly coat using a tuberculin syringe (28.5 gauge, 13 mm length). Alternatively, small pieces of macerated testes were placed directly on top of each egg without the use of buffer solution. Incubation of sperm with egg jelly was accomplished by vigorously vortexing the jelly from one egg in 100 μl of SDB. Sperm was then incubated for 10 min in the jelly/buffer supernatant. Sperm preparations were checked for morphology, movement and membrane integrity by fluorescent microscopy with Live/Dead sperm stain (propidium iodide and SYBR 14) (Molecular Probes, Eugene, OR) using an Olympus BH-2 microscope [41]. Additionally, sperm were stained with 1 μM Lysosensor green fluorescent dye (Molecular Probes, Eugene, OR) and imaged with a Zeiss LSM 510 META laser-scanning confocal microscope [42]. For artificial activation experiments, mature eggs were either mechanically stimulated by gentle poking with fine forceps, taking care to penetrate the jelly coat but not the plasma membrane, or immersed in a solution containing 10 mM CaCl2 with 0.1 mM A23187 calcium ionophore. Scoring of activation was done by noting the appearance of the first cleavage furrow (see figure 1). Artificial activation experiments were also performed on oocytes dissected directly from the ovisac of gravid females. These oocytes were similarly treated by poking or exposure to 10 mM CaCl2 with 0.1 mM A23187 calcium ionophore, with or without pretreatment for 12 hrs. with 3 μM progesterone [43]. Figure 1 Early egg development. A) Untouched, unfertilized egg. The surface of the egg under the jelly coat is featureless. B) Sperm-activated egg at six hours post-fertilization at the four-cell stage. Note the straight, ordered cleavage pattern (arrow). C) Artificially activated egg pseudocleaving. Note the jagged and disorganized cleavage pattern (arrow). D) An egg pseudocleaving at 16 hours. This egg was not handled and did not cleave within the first ten hours after deposition. At ten hours it was poked, and started pseudocleavage shortly thereafter (arrow). Scale bar = 1 mm. Eggs were allowed to develop at room temperature in parafilm-sealed 60 or 100 mm polystyrene petri dishes and were moistened with an antibiotic solution consisting of 25 μg/ml amphotericin B, 10 U/ml penicillin and 10 ug/ml streptomycin. Eggs were scored as successfully fertilized following neurulation (stage 2 sensu Townsend and Stewart [36]; stage 14 sensu Gosner [44]). Results and Discussion Artificial activation Table 1 shows the activation effects of either mechanical stimulation or A23187 calcium ionophore exposure on the unfertilized eggs of E. coqui. This ionophore non-specifically activates the unfertilized eggs of a variety of species through stimulation of a calcium-dependent signalling cascade [45]. Ten hours after laying, 11 of 32 eggs (34%) pseudocleaved, even if left undisturbed. As unfertilized eggs do not posses a centrosome, if they are artificially activated, the mitotic apparatus cannot form properly and divisions are irregular. This well-described process is called pseudocleavage [[46]; figure 1]). Activation almost doubled to 28 of 44 (64%) when the eggs were poked with forceps. Further, 30 of 36 eggs (83%) exposed to 0.1 mM calcium ionophore pseudocleaved. These percentages include both eggs that would have auto-activated –presumably 34%- as well as eggs that were activated by the mechanical or chemical treatments. Table 1 Artificial activation of E. coqui eggs Egg origin Treatment Number cleaving at 10 hours/Number tested (%) Number cleaving at 16 hours, after being poked at 10 hours/Number tested (%) Eggs laid in response to hormone treatment Undisturbed 11/32 (34) 6/8 (75) Poked 28/44 (64) - A23187 30/36 (83) - Oocytes dissected directly from ovisac Undisturbed 0/40 - A23187 0/40 - Progesterone 0/40 - Progesterone & AA23187 0/40 - To test whether the pseudocleavage response was an effect of our stimuli and not a reaction tied to other uncontrolled variables, we examined eight eggs that had remained undisturbed and that had not started pseudocleavage ten hours after laying. At this time, we poked them with fine forceps, and 6 of 8 (75%) began pseudocleaving six hours later (table 1). This delay in activation as a response to a delay in the stimulus is a strong indication that our manipulation is in fact responsible for eliciting the onset of cell division. An interesting observation was that one third of all eggs deposited in response to hormone treatment activated of their own accord. This may be due to the mechanical stress to which the eggs are exposed during oviposition. Clearly, mechanical stimuli are able to activate the eggs, and stress incurred in during transit from the ovisac may be sufficient to cause activation. This would presumably not affect E. coqui during natural matings because this species undergoes internal fertilization, and the eggs will have already been fertilized prior to deposition [34]. In order to examine this hypothesis, we dissected oocytes directly from a female's ovisac, circumventing the passage through the oviduct and cloaca, and attempted to activate them with calcium ionophore (table 1). Controls were also performed with and without progesterone pretreatment in order to induce maturation. Since there is no information on the stage at which E. coqui eggs are arrested or what signal takes them out of their arrest, we followed procedures used in Xenopus [43]. However, none of these oocytes activated, regardless of the treatment. This may be because the oocytes did not respond to treatment with progesterone, and so never matured. Another possibility is that oocytes need to receive a signal from the oviducts and/or be coated in jelly before they can mature. The ability to initiate activation after a long delay was interesting as we suspected that E. coqui eggs might be sensitive to aging, as has been reported under certain conditions for the externally fertilizing X. laevis [47]. Because E. coqui has internal fertilization, we suspected that eggs laid unfertilized might degenerate rapidly, complicating the artificial manipulation of this species' reproduction. Consequently, we investigated the ability of eggs to be activated as a function of time. When we artificially activated eggs with the calcium ionophore at different time points and examined them six hours post treatment, the percentage that pseudocleaved was high even ten hours after being laid (Figure 2). Twenty-four hours after deposition, however, the eggs were no longer able to activate. Thus, there is an extended period in which it is possible to carry out experiments without concern for a decrease in activation potential. Figure 2 Artificial activation of E. coqui eggs in relation to time after deposition. Unfertilized eggs were treated with calcium ionophore to induce activation at 0 (n = 12), 1 (n = 12), 2 (n = 10), 3 (n = 12), 4 (n = 12), 5 (n = 12), 10 (n = 18), and 24 (n = 18) hours. Eggs were scored for activation by the presence of cleavage furrows at 6 hours post treatment. Note that for the 10 and 24 hour time points, six eggs at each time point had already auto-activated by 6 hours post deposition. At the 10 hour time point, nine additional eggs activated later in response to ionophore treatment, while at 24 hours, no additional eggs were observed to activate after ionophore treatment. In vitro fertilization The average fertilization efficiency for natural matings conducted in our laboratory was 72% (see table 2). As roughly one third of all unfertilized eggs laid in response to hormone treatment auto-activate (table 1), and so only approximately 66% of the eggs in a given clutch will actually be receptive to sperm. If we assume these two factors to be independent -because we hypothesize that the eggs auto-activate during laying, a problem that doesn't arise in natural matings- then only 48 of every hundred eggs will be available for IVF (100*0.66*0.72). However, despite these complications, we were able to obtain an in vitro fertilization efficiency of 27% -or, rather, 56% of all receptive eggs (27/0.48) – by simply mincing the testes and adding them directly over the eggs (see table 2). Other IVF techniques were not as successful. Using sperm diluted in SDB resulted in only a 12% total fertilization efficiency (table 3). Table 2 Natural mating fertilization percentages for E. coqui in captivity. Clutch Number of eggs laid Number that developed to neurula (%) 1 39 24 (62) 2 58 36 (62) 3 30 28 (93) 4 42 34 (81) Total 169 122 (72) Table 3 In vitro fertilization of E. coqui eggs Fertilization protocol Number that developed to neurula/Number tested (%) Sperm solution dripped over the eggs 5/63 (8) Testes minced directly over eggs 8/30 (27) Sperm solution injected under the jelly coat 0/12 Sperm incubated in jelly buffer and then injected under the jelly coat 1/10 (10) Poked, then fertilized 15 minutes later by mincing testes directly over the eggs 0/36 Sperm concentration may play a role in fertilization efficiency as the use of diluted sperm resulted in decreased fertilization. In support of this possibility, polyspermy has been observed in this species and is apparently not deleterious to fertilization and development [40]. A second possibility is that fertilization efficiency is linked to sperm capacitation and acrosome reaction. We attempted to study this possibility by examining the acrosomes of fresh and treated sperm using Lysosensor green fluorescent dye (Molecular Probes, Eugene, OR). This dye concentrates in low pH vesicles of living cells through an unknown mechanism and was shown to accumulate and preferentially stain the acrosome in X. laevis sperm [42]. However, we were unable to observe acrosome-specific staining in E. coqui sperm using this dye (data not shown). We also examined the possibility that a component of the egg jelly coat may be important for sperm capacitance. To test this, we incubated sperm with SDB and jelly, or SDB alone, and injected this under the jelly coat of eggs. None of 12 eggs were fertilized by sperm incubated with SDB alone, while pre-incubation of sperm with an extract of the jelly coat in SDB resulted in one fertilization out of 10 eggs (10%). This is considerably less than the 27% efficiency following direct placement of minced testes over the eggs, but suggests that interactions between sperm and the jelly coat may play a role in sperm capacitance and subsequent fertilization. To test the functional response of the eggs, we attempted to fertilize artificially activated eggs. As was explained above, we were able to achieve an IVF success rate of 27%. However, if we poked the eggs fifteen minutes prior to direct fertilization, none (0/36) developed (see table 3). If we assume our expected fertilization rate to be 25%, the possibility of this result being due to chance is (1-0.25)36 = 3.2 × 10-5, or less than one in ten thousand. This shows that fifteen minutes after being poked the eggs have established a block to polyspermy, one of the defining functional characteristics of activation. Hence, although the first visible indication of activation –the formation of the first cleavage furrow- will not be seen for six hours, we can conclude that the egg is undergoing the normal activation processes within minutes of being stimulated. Conclusions In an effort to conserve declining populations of animals, the development of protocols for the artificial manipulation of reproduction is of great interest. In the case of the neotropical frog E. coqui, we have observed that a large proportion of eggs that are laid unfertilized auto-activate. We showed that E. coqui eggs are easily activated by mechanical stimuli, leading to a need for careful manipulation of unfertilized eggs in all reproduction studies. Further, the cleavage pattern seen in mechanically activated eggs is similar to that of both auto-activated and chemically activated eggs, suggesting that mechanical stress, probably incurred in during oviposition, is responsible for the auto-activation mentioned above (table 1). Facile auto-activation of eggs has been reported in other species, complicating reproductive manipulation [48]. In E. coqui, this phenomenon may relate to internal fertilization, and it would be interesting to investigate auto-activation of unfertilized eggs in closely related, externally fertilizing species such as E. antillensis [38]. Some E. coqui eggs, however, remain intact and can be manipulated, showing signs of activation both at the morphological level, through the initiation of development, as well as the functional, through the slow block to polyspermy. By careful handling, we are now able to fertilize over half of the remaining, functionally viable eggs using a simple procedure. Finally, we have shown that E. coqui eggs do not degenerate rapidly and are capable of undergoing activation up to ten hours after deposition, thus creating a window of time for carrying out experimental procedures. Our results demonstrate efficient IVF in an internally fertilizing, terrestrial-breeding frog and help lay the foundation for future research and conservation possibilities in this unusually large genus of amphibians. Authors' contributions ET carried out the experiments and prepared the manuscript. SFM carried out preliminary experiments and supervised the manuscript. The study was conceived jointly. Both authors approved the final manuscript. Acknowledgements We gratefully acknowledge the assistance of the US Forest Service, Caribbean National Forest and the Puerto Rico Departamento de Recursos Naturales y Ambientales for providing permits. We also thank Alonso Ramirez, Paul Klawinski, Naomi Roots and the staff and students at El Verde for assistance collecting frogs. Three anonymous reviewers provided helpful comments on a previous draft of the manuscript. 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