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this paper is the report on and balance of some thirty years of transcultural research on mood disorders . the aim of our work was to produce a hypothesis bringing together the different ways in which discomfort manifests itself , with special emphasis on discomfort viewed through mood symptoms . our goal was to see if behaviours defined as pathological and which produced maladjustment in the patient in determined contexts may produce adaptive effects in different contexts and , thanks to attenuated expressions , find in these circumstances the conditions for replication and thus become factors leading to social change . we searched for an interpretation of interactions between environment and behaviours , not from a simplistic perspective that interprets the modalities through which discomfort is expressed as the product of a culture , but by applying an evolutionary approach to see if certain behaviour patterns can be selected from the changing context ( since a static context would not modify the social significance of such patterns ) and finally to see if the very same behaviours can contribute to influencing the environment . in the case of mood disorders , this amounts to seeing how specific milieus can be adaptive for persons with temperaments or " subthreshold " depressive and/or expansive hyperergic symptoms . our investigation started from h. b. murphy 's hypothesis . in the 1970s he performed an historical analysis of the descriptions of depression found in europe , collecting mostly the contributions of physicians and members of the clergy over a period of some centuries . according to murphy , the symptomatology characterized by low self - esteem , feelings of guilt and helplessness supposedly arose " epidemically " in a specific area of 17th - century england . the phenomenon was already known at the time and was defined as the " english malady " . prior to that time the ( rare ) descriptions of depression had concentrated on a psychosomatic symptomatology and in particular on symptoms of tiredness and loss of hope . the changes in symptoms coincided with a period of profound social upheaval : the geographical area was that in which the first historical form of industrialization was taking place in textile manufacturing . the social change supposedly took place on two interacting levels . firstly , the rhythms of industrial work marked the end of the extended family typical of the rural world . this was followed by the loss of the close identification of individuals with their group or family and the impossibility of maintaining the close emotive relationship that the extended family could provide . the emphasis of the new society on individual responsibility was to represent the other decisive element . the emergence of middle class morals was accompanied by the conviction that individuals were the forgers of their own destinies . the understanding that people were able to manage and guide events emerged among the philosophers in that crucible of middle class culture that was renaissance florence ( cf . machiavelli il principe , reprint 2006 ; pico della mirandola , oratio de hominis dignitate , in cassirer 1974 ) but it developed as this new interpretation of reality , this new way of perceiving the world and one 's active and important role led the development of the middle class concept of moral responsibility . this prospect negated the idea that providence was the cause and driving force of events and saw individuals as the prime arbiters of their own destinies . since the reaching of one 's goals was to be considered the result of individual efforts , consequently defeats and failures were the result of an individual s ineptitude . divine indulgence , according to the new protestant ethic , which took hold with the changing economy , was the reward for the hard work of the good christian . many studies were carried out in the 1950s and 60s on depression in areas of sub - saharan africa in which cultural contacts with western societies had been limited . thus in this context depression could not be considered a " western " disorder . according to henry collomb , who had transformed the fann psychiatric hospital in dakar into a sort of therapeutic village , depression was to be considered the most significant element in african psychopathology , although clinically the disorder was characterized by ideas of reference , persecutory delusions and psychosomatic symptoms . the ideas of guilt , self - depreciation and suicidal behaviours contrasted with " western " depressive symptomatology . an element of extreme interest in this scenario was reported by field , who documented an epidemic of a depression - like disorder in an area of ghana inhabited by ashanti people . murphy interpreted the innovation as a sort of hybrid pathological manifestation combining the traditional psychosomatic discomfort and the innovative pathology apparently introduced by economic individualism represented by the emergence of guilt symptoms , although this was in a context in which these elements were not yet ascribable to a presumed individual conscience as opposed to a more " collective " one related to the concomitant social transformation and the beginning of cacao production on an industrial scale . the hypothesis advanced by the school of dakar was that the specificity of western africa in expressing discomfort depended on how the main mother - child and community - individual relationships brought about a sort of symbiotic personality . from this standpoint , the characteristics of the personality were considered to be directly correlated to the way of expressing discomfort , that is , feeling bad and being sad was tantamount to feeling split up , separated from the group the person belonged to and thus threatened and persecuted . the psychodynamic authors of the 1960s attributed the cause of this particular african depressive pathology to the absence of an individual super - ego : the constant presence of caregiving figures may have determined the development of a collective super - ego [ 9 , 10 ] . later , the sociologist zempleni - rabain pointed out that the education of children in western africa emphasized the sense of belonging . more recently , thanks to cooperation between the regional centre of traditional medicine of bandiagara and the university of berkeley , the extensive relationship between mother and child was studied by means of aetiological models derived from the strange situation in a dogon village in mali . a study performed by the school of dakar observed that senegalese emigrants who had moved to dakar from rural areas were found to be protected from the psychopathological risk connected with migration by the effect of social cohesion thanks to the associative structures of the islamic brotherhoods . these institutions were thought to represent the social manifestation of the community s attitude towards personality in a syncretic islamism still imbued with magic and religious elements . sylla and mbaye studied the modalities by which these networks modulated the need for group unity , the necessity to emigrate and psychological risk among western african populations , especially the wolofs of senegal . the departure to seek work the social significance of the ritual is that of creating a bond that places the individual under the control of the mother or wife in the mother country . since the family depends on the remittances that the emigrant sends from abroad , it is fundamental to have a ritual that modulates the effects of an experience that risks being strongly centrifugal with respect to the community . it is believed that the emigrant can be recalled thanks to a ceremony called wotal ; if he does not respond he will lose his mental health . thanks to this bond the family can be sure of receiving periodic remittances from the family member abroad : distance does not break the bond with the group to which the emigrant belongs . offers in money to the brotherhood also exonerate the emigrant from the obligation to remain at home . according to the koran , emigration to places where the true faith is not practised is allowed only for religious reasons . almost all senegalese emigrants with psychotic disorders observed starting from the 1990s in our centre were convinced that they were influenced by the wotal . the clinical pictures were characterized by bouffes with persecutory - like delusions and mental confusion . the onset of an episode was perceived as a breaking of the symbiotic bond with the group . it is thus not therapeutically surprising that on the return to senegal of a relative with a psychotic maladjustment a series of traditional rituals are performed by family and friends to remove the negative effects of the wotal . in a study performed in sardinia [ 15 , 16 ] , a random sample of senegalese street traders , whose work made community life possible , did not appear to be at risk of depression any more than sardinian controls . unexpectedly , a high frequency of anxiety and depressive disorders was found in the few senegalese who had found regular jobs and pay . a strong determinant associated with the onset of the psychopathology was , in this second case , the loss of contact with their fellow senegalese and the breaking away from the brotherhood . compared to a population of moroccan emigrants employed in similar jobs , the senegalese street traders presented a lower risk of depression as well as a lower incidence of psychopathological episodes in the first months after their arrival in sardinia . the observations on african emigrants encouraged our group to perform a series of in - the - field studies in africa . this is where a research centre funded by italian cooperation for development and the centre rgional de mdicine traditionnelle was set up . the cooperative project proposed to use the principles of dogon herbal medicines to produce low - cost drugs . the method of testing the efficacy and standardizing the active ingredients was introduced based on the traditional empirical grounds of western scientific culture . from this standpoint , a study conducted together with local medicine men was indispensable to compare traditional nosography with western disease classifications . it was necessary to understand which active ingredients could be useful and for which disease . it emerged that depressive and anxiety disorders were unknown and not taken into consideration by the medicine men . on the plateau they either did not exist or were not considered as falling within the scope of the system of treatment . laboriously , and with the help of many linguists , we translated some of the epidemiological instruments used in europe in epidemiological investigations of mental health . we then performed a study to verify the presence of depression on the dogon plateau . it was a study conducted using western nosological criteria but with the intention of comparing them to the interpretive modalities of the culture under examination , studied according to the approach that anthropologists and new transcultural psychiatrists would define as dialectic between " etic " and " emic " . the population studied lived in a very poor sahelian area in nomadic camps ( the peuls ) or villages ( the dogons ) with no electricity , organized transport or communications systems . thus we are dealing with a study of extreme interest owing to its singularity , albeit next to impossible to replicate . numerous observations in western africa appear , in faft , to indicate a progressive change , today , in psychopathology , especially in circles that are urban and literate in the western sense [ 22 , 23 ] . the prevalence of depressive disorders found the dogon plateau was lower than what have found in studies performed in europe with similar instruments or in africa in urban contexts or in conditions of war [ 24 , 25 ] . among the illiterates , who were a consistent part of the cohort , most depressed individuals also suffered from somatic disorders that had begun prior to the onset of the psychiatric disorder . the study showed the importance of somatic diseases as a risk factor for depression when the reproductive role ( as in the case of parasitic infections of the genitourinary tract ) or the social or family role ( as in the case of heart diseases and tuberculosis ) have been affected . thus , in the societies studied , we may find a behavioural pattern similar , but not identical , to our depressive patterns . but this is rare , probably because its onset requires a triggering condition of higher impact . moreover , in agreement with what has been demonstrated by other studies , depression is not considered a disease and thus does not fall within conventional or traditional treatment systems . a later work aimed at characterizing the syndromic aggregations by means of a sophisticated statistical method based on how a symptom is more likely to occur associated with one or more symptoms . the results suggest a non - superimposition of the depressive phenomenology most frequently found on the dogon plateau on the psychological profiles usually found in western contexts . it also appears to confirm the existence of two opposing ways of expressing the discomfort which are most likely " culturally determined " . one way is more frequent and characterized by psychoastenic symptoms with persecutory overtones , while the other , similar to depression as we in industrialized countries know it , is found in literate persons . the bandiagara study suggests that social factors influence the manifestation of the melancholy phenomenology and perhaps they may affect the risk of becoming depressed , but it does not allow us to formulate any hypothesis explaining how this may come about . some elements of interest on this subject are presented in the results of an investigation carried out in the namwera area in malawi , bordering on mozambique . in this region , inhabited by yao and chicewa peoples , the economy was prevalently based on farming and only at the time of the study had other occupations emerged . malawi was in fact the only african country that had maintained a strongly traditional political structure up to the 1990s ; urbanization had been opposed by the central government , which had officially acknowledged the village chief as its representative . it is one of the poorest countries in the world with the highest rate of hiv infections . however , at the time of the study , malawi had undergone profound transformations involving micro- and macrosocial aspects and which , in the following years , had led to the setting up of a democracy with different political parties determined by a referendum . in a world that had not changed for centuries , these changes had a strong emotive impact on most of the population . in the period just before the referendum , mc lachlan had observed and described an authentic epidemic of forms of hysteria in groups of young women . according to the only western psychopathologist present , these phenomena were to be considered directly related to the hyper - involvement caused by conflict in the choice between tradition and innovation . the hysterical crises invariably began with triggering events brought on by occurrences connected with the referendum , such as political rallies and public debates . in this context , starting from 1988 , an industrial apparel initiative planned and financed by italian cooperation had been set up in the village of namwera . at the beginning , the project foresaw that after a training period women could purchase the machines required for setting up an independent dressmaking activity in their villages of origin . in consideration of the peculiar condition of women in the yao and chicewa cultures , the sudden passage from a traditional role to one that was to some extent entrepreneurial appeared to be a situation appropriate for studying aspects concerning the relationship between transformation of the self and psychopathological modifications . the investigation concerned three cohorts of women matched by age : one included all the dressmakers , another with women employed in traditional occupations ( farmers / housewives ) and the third with nurses and midwives . the history of development , the presence of stressful events and other risk factors , such as the presence of psychopathological symptoms and evident psychiatric disorders , were studied ; interpretations of the causes of any discomfort found were defined . the choice of the kind of non - traditional work made by the young women was in line with their personal histories of development . in fact , those who were employed in innovative jobs ( dressmakers and nurses ) had suffered abandonment by the father at an early age ( in more than 80% of the two cohorts ) . in malawi , marriage is matrilocal : the man moves to his wife 's home , but he often abandons the family , leaving it in precarious economic conditions . in the young dressmakers and nurses the choice of an innovative occupation was probably dictated by economic needs and subsistence . satisfaction with their work was higher in the women who had chosen innovative occupations ( dressmakers and nurses ) but only the dressmakers ( and not the nurses ) manifested a series of social conflicts connected with their new roles , such as difficulty in finding a stable partner and frequent abandonment by husbands who refused to accept the wife 's work , and this is surprising in consideration of the economic stability that the new activity offered . on the level of psychopathological evaluation they presented more psychopathological symptoms and , among these , the number of cases of depression diagnosed on the basis of the dsm - iiir manual was higher . among the housewives , a statistical analysis of the distribution of the specific symptoms revealed more frequent " psychosomatic " symptoms ( headaches , lack of appetite , poor digestion ) , excessive tiredness , loss of interest and difficulty in concentrating , ideas of devaluation , conviction that others did not acknowledge the importance of their roles and that an insult by someone may have caused the problems . they thus showed a tendency to suffer from a lack of respect by others and to localize externally the causes of their malaise . the dressmakers more often felt tense , nervous and worried ; they experienced sensations of uselessness and more frequently expressed a wish to die . more than in the control groups , problems involving self - esteem and the more conventional symptoms of depression emerged . some interesting psychosocial studies suggest that cultural transmission tends to remain when the specific cultural institutions ( in the case of the namwera study the occupational role ) are perceived by the persons involved ( and by the other members of the group ) as an integral part of their own personal identity , but at the same time as capable of responding to new exigencies and needs ( " the evolving self " ) . in the case of the namwera study this interpretation would explain the absence of external and internal conflicts connected with the new role and , consequently , the nurses ' in integrating into it . they receive a good salary but maintain a social role similar to that of the female tradition . on the contrary , the dressmakers , who were involved in a more individualistic occupation that was farther removed from women s traditional role , appeared to suffer more from the burden of an activity completely different in terms of individual responsibility and one that was probably less acceptable in the social and family context . in interpreting the results we must take into consideration that a nurse 's work appears to allow a namwera woman to maintain an acceptable social role , one that is also perceived as being closer to women 's " traditional " roles , especially as concerns care for and support of the weak thus it is not surprising that nurses form the emotionally most stable group , since they have reached a satisfactory economic situation which provides them with independence and autonomy , while maintaining them in an individually and socially acceptable role which is also innovative . we can also hypothesize that extreme needs , such as those caused by abandonment by a father , can cause inclined individuals to assume the onus of the problems of the micro - group and take a " step forward " ( new occupation or emigration ) . the choice of the new activity brings with it the risk of a breakaway from tradition if not adequately mediated . the cultural change would thus offer opportunities for success of individuals who are " desperate , explorative and super - responsible " ( with " compulsive hyper - responsibilization " in bowlbian terms ) while in other circumstances " unperturbed " by the change , they would probably be destined to undergo social humiliation . the social change from an evolutionary standpoint may also form the substrate that favours , selects and amplifies the frequency of behaviour of this kind . at the same time , this exposes to decompensation : the step may be rewarded , but those who take it are more at risk : they are alone on a new road and do not have the support of the group , which no longer recognizes them and tends to isolate them . the model may explain not only the transformation of the symptoms ( centred on responsibility and guilt ) but also the supposed increase in depression in western society . the supposed increase in depression in western societies can not in fact be explained by " ingenuous " biological and genetic hypotheses : it is irrational if not interpreted from a complex socio - biological standpoint . if depression were simply caused by genetic vulnerability we could not explain why a condition that afflicts persons who live shorter lives and give birth to fewer children does not disappear , but according to some has seen a continuous increase from the 17th century to the present . some studies appear to demonstrate it : the swedish study repeated over time by lundby ; the analysis of data of the national study in the united states conducted in the 1980s which , however , is a cross - sectional study ; a study at the general practitioner level in the united kingdom covering about thirty years . however , there are studies that do not appear to confirm this trend , such as the well - known study repeated over time in stirling county , nova scotia [ 35 , 36 ] . more recently , the two studies performed on a population sample in the united states ( of what first evaluation in 1992 and repeated in 2002 ) , and the australian study in new south wales ( first evaluation in 1998 and repeated in 2008 ) , appear undoubtedly to confirm the hypothesis of an increase in the prevalence and incidence of depression at least in the last few decades . sardinians , apparently , go in the opposite direction : this emerges from the data of our latest study performed in a sardinian mining district with three estimates of the frequency of depression in the overall population in 1988 , 1998 and 2008 . the pervasiveness of depression clearly appears to be decreasing in the last twenty years and , at least in the last ten years , the comparison with the first investigation needs to be considered with more caution in the light of the different interviewing method used and the fact that the diagnostic system had changed . in the ten years preceding the first study the area had seen a 50% decrease in the population in forty years . we have asked ourselves whether this decrease against the general trend has been caused by selective migration , that is , the departure of persons who were in some way " vulnerable " to depression . a certain popular literature has underscored that migration may select persons with " hypomanic temperaments " . gartner , in his book the link between ( a little ) craziness and ( a lot of ) success in america advances the hypothesis that success in america is due to immigrants who are more ambitious , determined , creative and resistant to frustration than others . the author , a psychiatrist well - known to the media , argues that emigrants are more frequently persons with hypomanic traits . this is an apparently " naive " theory , but one that may confirm what we stated as concerns the results in the mining district . we must in fact consider that persons with a hypo - manic temperament are more exposed than others to depressive episodes . i defined gartner 's theory as naive because we can not argue that all emigrants leave for similar reasons and with the same determination to accept a challenge . it is sufficient to consider refugees who are forced to abandon their homes rather than choosing to leave them . to arrive at a better understanding of the dynamics of possible selective , we attempted to study the specificity of sardinian emigration . in the 1960s , nereide rudas studied attitudes towards migration in sardinian couples who were about to leave for germany , france and northern italy [ 41 , 42 ] . the decision to emigrate the wives often did not agree completely , but bowed to the will of their spouses . although in our culture women have a voice in decision - making within the family , generally speaking they are more respectful of traditions and probably for this reason they were less enthusiastic about leaving . on the average , the migrating women had less self - esteem than the men . a long time after the first study we performed research on sardinian immigrants in paris at the beginning of the 1990s . most of these persons came from the northern ogliastra and barbagia regions ; they had left in the 1960s and 70s from sheep raising areas of those regions which were poor and at that time with few prospects for the future . the study compared the frequency of psychopathological disorders in a cohort of sardinians residing in sardinia , in a cohort of sardinians residing in paris and in a cohort of parisians . the sardinians in sardinia were found to be more frequently anxious and the parisians more often depressed . the emigrants were in the worst condition : they were as anxious as the sardinians who had remained at home and there were more cases of depression ; the young males were those most prone to depression . the depressive symptoms , especially among the young males , were frequently associated with substance abuse and anxiety disorders and more often chronic compared to depressed parisians . the passage from a static society with few opportunities to one with many opportunities , but highly competitive , appeared to increase the risk , especially in males , and even more in young males . a similar study conducted in a completely different situation , that of the economic collapse in argentina , the investigation had been conducted at la plata and in the south of the province of buenos aires in december 2001 and in 2002 , showed that the young male sardinian immigrants were not the ones at risk . in these specific circumstances those at risk were the women over the age of forty - five , the group traditionally considered the most vulnerable to major depressive disorders . in conclusion , a condition offering many opportunities , especially for young men who are competitive and full of initiative , appears to place at risk some of the very youths that the condition should favour . this recalls the theory of goal striving stress described by kleiner and parker : a society that creates great expectations produces stress in those who fail to reach their goals . a society in full economic and social catastrophe represents a risk for adult women emigrants . these were women who had not chosen to emigrate and who , at the time of departure , had less self - confidence than the men if we hypothesize that mood disorders are increasing and that the forms of them we know have appeared as mass phenomena at least starting from the 17th century , we must also postulate that at least the " minor " forms must have an adaptive advantage ( otherwise the disorder would have disappeared ) . " compulsive hyper - responsabilization may represent an adaptive substrate in certain conditions of social change . after becoming entrepreneurs , many young namwera seamstresses improved their economic and social status in the 1990s , just as many sardinians who today have reached a solid social position in paris , but who in those years faced a difficult challenge in a world completely different from their traditional one . a certain attitude towards challenges and hyperactivity although social interactions may have caused , such as in the case of the sardinians pairs , the selection of women rated to pessimism and the sense of duty . at the same time new opportunities may also appear as risky : those who accept challenges can reach new goals , but are also exposed to a greater risk of defeat compared to those who remain in their traditional roles or do not emigrate . selected people also expressed their suffering in a new , more " individualistic " way . the social changes that favour compulsive hyper - responsibilization are those that disrupt the support " extended " to the individual : this social hypothesis comes from murphy s theories and , in agreement with the results of the namwera study , it is perfectly in line with researches on human ethology which anxious avoidant attachment to a greater exploratory attitude but also to an increased risk of depression . a similar model can explain how the accent on individualism and responsibility has changed not only the frequency , but also the phenomenology of mood disorders . what emerges is the increase in our culture of depression among young men ( not only in the paris investigation , but also in many european , north american and australian studies [ 37 , 38 , 48 ] ) ; the increase in forms of co - morbidity , especially with substance addiction and anxiety ( an element not isolated in our studies ) may give weight to the theory of the increase in bipolar disorders . because , is doubtable that common epidemiologic tools have ability to detect between bipolar depressive episodes and major depressive episodes [ 49 - 51 ] . the sociobiologically determined roles of gender and their interaction may also increase the level of complexity . a couple composed of a hyper - explorative male and a traditionally protective female undoubtedly presents adaptive advantages in particular contexts of tumultuous changes , such as those that occur in an unfamiliar world in a couple of migrants , as has been described in studies on sardinians . it is reasonable to expect that different social conditions are capable of triggering specific vulnerabilities in the two sexes and that the interaction between environment and hormonal structure may produce the different psychopathological expression . the socioeconomic context found in paris in our investigation recalls scenarios common to europe in the last forty years . the " argentine model " appears to reflect that of europe in the next ten years . there will be a new change in the phenomenology of mood disorders in the future ?

introduction : the objective of this paper is to see if behaviours defined as pathological and maladjusted in certain contexts may produce adaptive effects in other contexts , especially if they occur in attenuated form . interactions between environment and behaviour are studied from an evolutionary standpoint in an attempt to understand how new attitudes emerge in an evolving context.methodology : narrative review . following an historical examination of how the description of depression in western society has changed , we examine a series of studies performed in areas where great changes have taken place as well as research on emigration from sardinia in the 1960s and 70s and immigration to sardinia in the 1990s.results and conclusions : if we postulate that mood disorders are on the increase and that the epidemic began in the 17th century with the " english malady " , we must suppose that at least the " light " forms have an adaptive advantage , otherwise the expansion of the disorder would have been self - limiting . " compulsive hyper - responsabilization , as well as explorative behaviours , may represent a base for adaptation in certain conditions of social change . the social emphasis in individualism and responsibility may have changed not only the frequency , but also the phenomenology of mood disorders particularly the increases in bipolar disorders . from the sociobiological standpoint the conditions that may favour " subthreshold " bipolar or depressive features are to be considered in relation to the contextual role of gender and the different risks of the two disorders in males and females .

INTRODUCTION THE ENGLISH MALADY STUDIES ON DEPRESSION IN AFRICA COMMUNITY PERSONALITY AND PSYCHOPATHOLOGY IN SENEGALESE MIGRATION THE BANDIAGARA STUDY IN MALI THE NAMWERA STUDY IN MALAWI IS DEPRESSION ON THE INCREASE IN WESTERN SOCIETY? THE DECREASE IN DEPRESSION IN SARDINIA'S MINING DISTRICT: IS IT THE RESULT OF SELECTIVE MIGRATION? STUDIES ON SARDINIAN EMIGRATION CONCLUSIONS CONFLICT OF INTEREST

primary angiitis of the central nervous system ( pacns ) was identified as a distinct clinical entity in adults in 1959 , and only recently now in children as a cause of childhood stroke . the disease should be suspected in children presenting with newly acquired neurological or psychiatric deficits after exclusion of causes of secondary vasculitis . we present a relapsing course of a young boy who was diagnosed as childhood pacns ( cpacns ) and was managed accordingly . a 6-year - old boy presented with low - grade fever and frontal headache off and on for last 1 month . the central nervous system ( cns ) examination revealed normal higher mental functions with palsy of right third cranial nerve and bilateral sixth cranial nerve . investigations revealed normal hemoglobin and leukocyte count ; c - reactive protein ( crp ) was positive , and erythrocyte sedimentation rate ( esr ) was raised 34 mm/1 h. cerebrospinal fluid ( csf ) examination was normal . the magnetic resonance imaging ( mri ) of the brain and spine was also normal . the patient deteriorated on day 3 of hospitalization with seizures , altered sensorium , quadriparesis , and left upper motor neuron seventh nerve palsy . repeat mri - angiography ( mra ) of the brain revealed block in the terminal right internal carotid artery ( ica ) , left ica , and bilateral posterior communicating artery ( pca ) suggestive of vasculitis with normal brain parenchyma and normal meninges [ figure 1a ] . the workup for cause of vasculitis was noncontributory ( koch 's workup , procoagulants , anti - nuclear antibody , lupus anticoagulant , and viral serology ) , and serum complement was normal . echocardiography was normal , and there was no evidence of systemic vasculitis . within 48 h of methylprednisolone , a diagnosis of idiopathic cns vasculitis was made , and the patient was started on oral prednisolone after five doses of methylprednisolone . the patient was discharged with residual deficit of bilateral squint ( sixth nerve paresis ) with a plan to continue steroids for 6 weeks . magnetic resonance imaging and angiography ( a ) initial scan : occlusion in the terminal right internal carotid artery and bilateral posterior communicating artery ; ( b ) follow - up after month : with thrombosis of the right internal carotid artery the patient presented after 1 month with fresh left twelfth nerve palsy and residual bilateral sixth nerve paresis . quadriparesis had resolved completely . repeat mra of the brain revealed resolution of thrombosis and of thickening of the left ica and bilateral pca as compared to initial scan but persistent thrombosis of the right ica [ figure 1b ] . the digital subtraction angiography of cranial vessels reported complete obliteration of right ica distal to carotid bifurcation with good collateral formation ; rest of brain vasculature was normal . a diagnosis of medium vessel pacns ( mv - pacns ) was made . in light of partial response to steroids with features of steroid toxicity as evident on examination , the patient was shifted to azathioprine ( steroid - sparing drug ) as maintenance therapy . the patient is currently recovering and has mild residual right sixth nerve and left twelfth nerve paresis at 1 year of follow - up , with resolution of cushingoid features . primary angiitis of cns is a rare form of vasculitis with extremely low annual incidence of 24 cases per million patient - years . the disease affects the small and medium size vessels of the brain resulting in lymphocytic infiltration of vessel walls with granulomatous differentiation ( granulomas are absent in childhood forms ) . the diagnosis of pacns is challenging because of rarity of disease , unexplained etiology , varied clinical presentation , and lack of sensitive and specific tests . the diagnosis of pacns should be suspected in any patient with multifocal or diffuse cns involvement with a remitting or progressive course . laboratory tests which can exclude secondary causes of cns vasculitis [ as listed in table 1 ] should be undertaken before establishing a diagnosis of pacns . the acute phase reactants may be raised , and csf abnormalities in the form of increased protein concentration or mild pleocytosis may be seen in majority though few studies have reported normal csf findings . secondary causes of central nervous system angitis in children in cpacns , two distinct patterns can be recognized small vessel pacns ( sv - pacns ) which presents with cognitive decline , seizures , and behavioral changes or mv - pacns where headache , hemiparesis , and other focal deficits are common manifestations . beading or segmental narrowing in large , intermediate , or small arteries with interposed regions of ectasia or normal luminal architecture with collateral flow . brain biopsy was initially considered as gold standard but carries a mortality risk of 3.7% . it may be difficult to detect sv - pacns by angiography , where diagnosis is confirmed with a brain biopsy . the mri with mra of the brain is a noninvasive and safer alternative diagnostic modality with good sensitivity ( 90%95% ) . the abnormalities seen in subcortical white matter and deep gray matter are ischemic areas and infarcts with evidence of vasculitis . the diagnosis of pacns is based on modified calabrese and mallek criteria which are a newly acquired focal or diffuse neurological or psychiatric symptom in a patient 18 years of age plus characteristic angiographic and/or histopathological features of cns angiitis , in the absence of an underlying systemic disorder that explains or mimics the features . the treatment of pacns includes an induction phase and a maintenance phase . in general , cyclophosphamide pulses for 36 months may be used in conjunction with steroids to induce remission , but myelosuppression should be anticipated . safer alternatives such as azathioprine , mycophenolate , and methotrexate are given as maintenance therapy . mv - pacns has a more benign course and fewer relapses as compared to sv - pacns where relapse may occur in 25%30% patients . the diagnosis of relapse rests on clinical presentation and supportive radiography ; acute phase reactants have little role . thus , patients with sv - pacns need to be monitored for long and require immunosuppressive therapy for longer period ( 5 years ) as compared to mv - pacns where maintenance therapy for 23 years suffices . . early identification of this condition helps to initiate appropriate immunosuppressive therapy which reduces chances of complications and mortality .

childhood primary angiitis of the central nervous system ( cpacns ) is a rare and a potentially fatal cause of childhood stroke . the disease poses a diagnostic dilemma for the clinicians due to overlapping and varied clinical manifestations such as headache , focal acute neurological deficits , cognitive impairment , or encephalopathy . we report a young boy who presented with low - grade fever and headache but rapidly progressed to develop acute encephalopathy and quadriparesis with multiple cranial nerve palsies , masquerading as acute disseminated encephalomyelitis . the neuroimaging was suggestive of vasculitis . he was diagnosed as cpacns and recovered with immunosuppressive therapy .

Introduction Case Report Discussion Conclusion Financial support and sponsorship Conflicts of interest

over the last decades , health care systems have faced important challenges in dealing with pressures to increase the performance of the services they offer while the allocated resources to offer such care have remained stable or have decreased . integration has been presented as an efficient solution to improve service delivery by tackling fragmentation and efficiency issues . one of the many forms of service integration is inter - organizational networks , which could be defined from a sociometric perspective as a set of three or more organizations linked together using different strategies [ 1 , 2 ] . other theorists , however , suggest that it is the mode of governance , neither hierarchical or competitive , that defines a network [ 2 , 3 ] . despite their apparent simplicity , networks are complex organisations that vary greatly on many aspects . indeed , networks can involve various organizational levels of the organization , such as administration , governance , caseload , management , etc . . they may be created at various levels , bringing governmental agencies , regional or local facilities or programs to work together using one or many integration strategies , such as communication , resource sharing , joint programs , client transfer agreements and so on . depending on the intensity of the links created , networks can be cooperative , coordinated or collaborative [ 6 , 7 ] , the latter being the most intensive and the most demanding . according to the theoretical perspective fostered , networks can also be thought of as a source of knowledge and information , a system of power regulation or a social structure created by environmental tensions . a very common perspective of networks in the field of health services is that of the services perspective , where network partners work together to create a seamless continuum of care . because they are viewed by many service professionals , policy makers and researchers as a potential integration strategy that could reduce service fragmentation and improve the coordination of services and effectiveness , networks have become a popular way to reconfigure health service delivery systems [ 911 ] found in many countries . in addition , the challenging context in which health care systems must operate has generated a growing commitment to the evaluation of health services . the rising popularity of networks and the renewed importance of health services evaluation have bred new interest in the evaluation of integrated networks . the evaluation of integrated networks is complicated by the fact that the concept of network performance is poorly defined . this is due in part to the fact that the advantages of networks have been predominantly studied with regard to their effect on individual organizations , and not with regard to the whole network [ 3 , 10 ] . to date , network performance has been mainly appraised using performance indicators traditionally used for individual organizations . at best , this may lead to partial and inaccurate evaluations of network performance [ 10 , 11 ] . the lack of clarity about the concept of network performance is likely to induce difficulties in network performance evaluation , and consequently limits our ability to use evaluation results to improve network performance . in addition , the evaluation of integrated networks is complicated because networks involve many organizations that may have different values , cultures and mandates . consequently , network members could have different perspectives about what constitutes the performance of their network [ 11 , 13 , 14 ] . moreover , provan et al . stated that network stakeholders tend to see the network and its performance from the perspective of their own organization . huxham also mentioned that the variety of organizational and individual agendas that are present in collaborative situations make it difficult to agree in practice . this author further suggests that when partners do not completely agree on a shared purpose , they may not be able to agree on the next steps of a collaborative process . indeed , if they do not agree on what constitutes new network performance , network partners could be in conflict on the evaluation process and indicators and interpret differently the evaluation results . a lack of a common vision risks disintegrating the collaborative advantage of network . to address various stakeholders perspectives of organizational performance , this approach states that the multiple constituencies or stakeholders of an organization could hold different perspectives about what constitutes its performance , and that it is unjustified or arbitrary to choose one perspective over another for evaluation purposes . according to the multiple - constituency approach , these perspectives should be incorporated into a larger vision reflecting the sum of the evaluative criteria applied by the various stakeholders involved . several studies have used this approach to build performance frameworks for various organizations [ 12 , 1820 ] , most of them having found differences in perceptions and priorities among different types of stakeholders . however , the use of this approach within a performance evaluation process presents two major weaknesses . the first weakness is that the multi - constituency approach does not explicitly explain how to deal with divergent perceptions . without further interaction among stakeholders , each group maintains its own different understanding of performance despite being embedded within a larger framework . deciding upon what to include in a performance evaluation framework , based solely on a multi - constituency approach , will thus likely fail to harmonize the standards and values of the different stakeholder groups and may be unsuccessful in promoting collaboration and mutual trust between network members . a second challenge associated with using the multi - constituency approach for performance evaluation is that the methods frequently used within this approach ( written questionnaires ) provide only a limited and thus superficial understanding of the underlying rationale for the differing opinions of stakeholders . moreover , responding to a questionnaire does not provide stakeholders with an opportunity to share and to discuss their preferences to better understand their partners vision . many researchers [ 20 , 22 , 23 ] have emphasized the need to go beyond simply reporting differences in stakeholder perceptions and to engage stakeholders in meaningful discussions to arrive at a common understanding of an organization s performance . this may be particularly important when integrated network links partners from different settings with different values and priorities . however , few authors have proposed ways to do so . indeed , zinn and colleagues repeated the steps of a delphi technique to obtain consensus within homogenous stakeholder groups regarding laboratory management performance indicators . they found the technique useful for reaching a consensus within groups of stakeholders of a similar type ( e.g. hospital executives , managed care executives , referring physicians , laboratory regulators and laboratory managers ) . however , they did not explore whether the technique was effective in reconciling the perceptions across various stakeholder types . the delphi technique has certain benefits ( e.g. anonymity and consultation of persons over large distances ) but it does not provide opportunities for meaningful discussions between stakeholders . discussion could be useful especially when the subject under study is complex and abstract , such as organizational performance . studies of collaboration highlight the importance of a drafting process that is highly participatory and involves key stakeholders . the creation of a common vision between partners sets the foundations for collaborative efforts and enhances the collaborative advantage of network . however , traditional approaches integrating multi - constituency perspectives fail to reconcile partners visions and involve them only superficially in the process . to gain a better understanding of what constitutes network performance and to improve the performance evaluation process , there is a need to explore innovative approaches to reconcile stakeholders perceptions while giving them an opportunity to discuss and share ideas . falling short of this may result in fragmented network efforts , disinterest of network partners with regard to the vision , evaluation process and evaluation outcome triage , a consensus technique , could be a viable method to create a shared vision in a participatory way . the general goal of this study was to explore whether triage group discussions could reconcile the perspectives of network members with regard to the importance of performance dimensions . specifically , we 1 ) determined the initial degree of consensus existing among individual tbi network organization members with regard to importance of performance dimensions for performance evaluation , 2 ) explored the usefulness of consensus group sessions to reconcile these perceptions , by comparing the importance paid to the different dimensions of performance using questionnaires and group discussion . we conducted the present study from november 2006 to november 2008 during the accreditation process of network organizations providing services to persons with traumatic brain injury in the province of qubec ( canada ) . we used the triage consensus technique [ 28 , 29 ] , a method that combines an individual data collection phase using questionnaires with a group data collection phase involving group consensus sessions . in a previous study , we used a questionnaire to survey the representatives of 46 individual organizations ( i.e. acute care facilities , rehabilitation facilities , regional health authorities , the accreditation body and the provincial health ministry ) . we asked participants to determine the relative importance of the 16 dimensions included in the egipss ( evaluation globale et intgre de la performance des systmes de sant ) performance framework ( figure 1 ) for the evaluation of the performance of tbi network using a scale where 0% indicates not important at all and 100% indicates extremely important . for the present study , we re - analyzed these survey data ( excluding data from the accreditation body and the ministry ) by compiling the results for each specific network , where a network was composed of an acute care facility , one or more rehabilitation facilities and regional health authorities . then , for the group data collection , the accreditation process provided an opportunity to bring together network members who are otherwise geographically spread across each of the regions . the group participants were the clinical coordinators and the managers from provincially - funded tbi programs . the participants completed socio - demographic data and consents forms at the beginning of the session . the group animator then reminded participants of the study goals and proposed an agenda for the session . the discussions began with the dimensions of the egipss framework for which differences in the importance scores attributed in the individual data collection phase by members within the same network were inferior to 20% . for these consensual dimensions , a consensual score , based on the average of the importance scores attributed by the individual organizations , was attributed . in contrast , dimensions having a score difference 20% were discussed in depth . as per the triage technique , the names of these dimensions were written on cardboard sheets and stuck on the wall under the grouping section for viewing by the group . participants were invited to explain why their individual organization teams had rated the importance of a dimension the way they did . then , as network members , they discussed and agreed upon the importance score the network should attribute to the dimension . when consensus was attained , the dimension name ( and new importance score if different from zero ) was placed on the wall under the selection heading . dimensions rejected as being unimportant to the network ( i.e. scored as having zero importance ) were placed in a garbage section . dimensions for which consensus could not be reached quickly were put in a fridge section for further discussion at the end of the session . if a consensus could not be reached , even after these additional discussions , the dimensions could be placed in a veto section and subsequently submitted to an external expert committee . using this technique , a final list of dimensions , each with a consensus - based importance score , was created . these networks linked trauma centers designated to provide specialized trauma care with facilities providing in- and/or out - patient rehabilitation services and with regional health authorities . five of these the networks included each three organizations and operated on a regional basis ( e.g. member organizations were all located in the same socio - demographic region ) . the three other networks operated on a larger inter - regional level ensuring specialized trauma care and/or in - patient rehabilitation services availability to regions of qubec not having these services in their region . one inter - regional network was comprised of six organizations , while two others linked 11 and 13 different organizations . members of the accreditation body and representatives from the health ministry were excluded from the group sessions since they were technically members of each network . the minimum and maximum importance scores attributed to the dimensions of the egipss framework by the individual members of each network were compiled , and the differences between these scores were calculated to determine the dimensions to be discussed during the group sessions . for each dimension , we computed the median importance score and its mean rank using individual organisation and group data . whitney tests were used to examine differences between individual and group data for the 16 dimensions . pearson and mann whitney tests were also used to compare the characteristics of the participants involved in the individual and group data collection phases . statistical analyses were conducted with spss 16.0 software , using a p - value of 0.05 as the significance level . the consensus group sessions were taped and transcribed , and the verbatim was analysed using nvivo 8.0 software . in a previous study , we used a questionnaire to survey the representatives of 46 individual organizations ( i.e. acute care facilities , rehabilitation facilities , regional health authorities , the accreditation body and the provincial health ministry ) . we asked participants to determine the relative importance of the 16 dimensions included in the egipss ( evaluation globale et intgre de la performance des systmes de sant ) performance framework ( figure 1 ) for the evaluation of the performance of tbi network using a scale where 0% indicates not important at all and 100% indicates extremely important . for the present study , we re - analyzed these survey data ( excluding data from the accreditation body and the ministry ) by compiling the results for each specific network , where a network was composed of an acute care facility , one or more rehabilitation facilities and regional health authorities . then , for the group data collection , the accreditation process provided an opportunity to bring together network members who are otherwise geographically spread across each of the regions . the group participants were the clinical coordinators and the managers from provincially - funded tbi programs . the participants completed socio - demographic data and consents forms at the beginning of the session . the group animator then reminded participants of the study goals and proposed an agenda for the session . the discussions began with the dimensions of the egipss framework for which differences in the importance scores attributed in the individual data collection phase by members within the same network were inferior to 20% . for these consensual dimensions , a consensual score , based on the average of the importance scores attributed by the individual organizations , was attributed . in contrast , dimensions having a score difference 20% were discussed in depth . as per the triage technique , the names of these dimensions were written on cardboard sheets and stuck on the wall under the grouping section for viewing by the group . participants were invited to explain why their individual organization teams had rated the importance of a dimension the way they did . then , as network members , they discussed and agreed upon the importance score the network should attribute to the dimension . when consensus was attained , the dimension name ( and new importance score if different from zero ) was placed on the wall under the selection heading . dimensions rejected as being unimportant to the network ( i.e. scored as having zero importance ) were placed in a garbage section . dimensions for which consensus could not be reached quickly were put in a fridge section for further discussion at the end of the session . if a consensus could not be reached , even after these additional discussions , the dimensions could be placed in a veto section and subsequently submitted to an external expert committee . using this technique , a final list of dimensions , each with a consensus - based importance score , was created . these networks linked trauma centers designated to provide specialized trauma care with facilities providing in- and/or out - patient rehabilitation services and with regional health authorities . five of these the networks included each three organizations and operated on a regional basis ( e.g. member organizations were all located in the same socio - demographic region ) . the three other networks operated on a larger inter - regional level ensuring specialized trauma care and/or in - patient rehabilitation services availability to regions of qubec not having these services in their region . one inter - regional network was comprised of six organizations , while two others linked 11 and 13 different organizations . members of the accreditation body and representatives from the health ministry were excluded from the group sessions since they were technically members of each network . the minimum and maximum importance scores attributed to the dimensions of the egipss framework by the individual members of each network were compiled , and the differences between these scores were calculated to determine the dimensions to be discussed during the group sessions . for each dimension , we computed the median importance score and its mean rank using individual organisation and group data . whitney tests were used to examine differences between individual and group data for the 16 dimensions . pearson and mann whitney tests were also used to compare the characteristics of the participants involved in the individual and group data collection phases . statistical analyses were conducted with spss 16.0 software , using a p - value of 0.05 as the significance level . the consensus group sessions were taped and transcribed , and the verbatim was analysed using nvivo 8.0 software . one hundred and thirty - nine professionals from acute care facilities ( 25% ) , rehabilitation facilities ( 65% ) and regional health authorities ( 10% ) participated in the individual data collection ( questionnaires ) . participants had a mean professional experience of 17 years ( sd 9.7 ) and reported working in their current position for an average of eight years ( sd 6.1 ) . all existing networks ( n=8 ) agreed to participate in the study and 62 persons from these eight networks participated in the group sessions : 27% were from acute care facilities , 52% from rehabilitation facilities and 21% represented regional health authorities . group participants had a mean professional experience of 18 years ( sd 9.7 ) and reported working in their current position for eight years ( sd 8.8 ) . the participants of the individual and group data collection phases were similar in terms of work experience and types of organization . table 1 presents the minimum and maximum importance scores attributed by the individual participants within a network to the dimensions of the egipss framework ; differences 20% are highlighted in gray . overall , the differences in the importance scores within the networks ranged from 0% to 100% , with a mean range of 33.7% . in fact , for the majority of dimensions , the observed individual importance score differences exceeded 20% ( 66% , corresponding to 85/[8 networks16 dimensions ] ) . with regard to the variability of the importance scores across networks , we found the networks had on average 10.2/16 dimensions for which the score ranges were 20% . the extent of consensus on importance scores varied according to the network examined . for the inter - regional network a , there was a total absence of agreement in scores , while for regional networks g and h , the majority of the performance dimensions scored within the 20% range . three domains of the egipss model , i.e. the adaptation , values maintenance and production , had similar proportions ( 31.3% ) of dimensions with scores with ranges of < 20% ( results not included in table 1 ) . in contrast , the goal attainment domain showed greater consensus with 40.6% of its corresponding dimensions with ranges of 20% or less . , four dimensions were attributed a wide range of scores by 7/8 networks : ability to adapt to requirements and tendencies , ability to innovate and transform , productivity , and quantity of care and services . in contrast , three dimensions ( efficiency , quality and continuity ) were attributed a range of scores 20% by only half ( 4 ) of the networks and only one dimension ( capacity to attract the clientele ) was similarly scored by the majority of the networks . eighty - five discussions took place about the different dimensions . on nine occasions , because the group deemed them unimportant , dimensions were placed in the garbage and received an importance score of 0% . four groups rejected quantity of care and service , productivity was rejected by three , and quality and effectiveness by one each . on eight occasions , at least one dimension was temporarily placed in the fridge section . however , after discussion , the groups were able to establish a consensus about their importance such that no dimension was placed in the veto section . at the end table 2 details , in decreasing order , the median importance scores after group discussions , the inter - quartile ranges ( iqr ) and the mean rank of the egipss dimensions obtained in the individual and group data collection phases . the median importance scores attributed to the dimensions via the individual survey varied from 60% to 100% ( median of 85% ) , while those attributed through consensus varied from 20.0% to 96.7% ( median of 86.6% ) . overall , the differences between the scores attributed in the individual and group data collection phases were not statistically significant ( z=0.88 , p=0.378 ) . five dimensions received higher importance scores during consensus group sessions as compared to the individual data collection phase . the capacity to attract the clientele , continuity , and efficiency dimensions were consistently scored as the three most important dimensions , both during the individual and group production phases . the scores attributed through consensus group sessions were slightly lower for nine of the 16 dimensions . the ability to adapt to requirements and tendencies and quantity of care and services remained among the least important dimensions in the two data collection phases . the differences in importance scores were statistically significant only for the productivity and the quantity of care and services dimensions , for which the importance scores attributed by participants dropped dramatically during the group process . with regard to the mean ranks indicating the relative importance of performance dimensions , the group process did not greatly influence the order of importance of the dimensions , with the exception of the continuity dimension , which was scored as relatively more important during the group sessions . it is noteworthy that the dimensions with the highest importance scores ( e.g. capacity to attract the clientele , efficiency , quality , etc . ) group discussions provided the participants with an opportunity to justify their individual scoring and to agree as a group on a new importance score . many of the general comments pertained to the desired balance between the integration and the differentiation of network partners . indeed , while agreeing that some degree of integration is required to provide quality services , network members wish to remain distinct organizations with their own culture , values , and intervention methods . when discussing the dimensions related to the adaptation domain , participants perceived ability to adapt and meet the client s needs as the only one fundamental to a network . the other five dimensions of the domain were seen as additional activities going beyond the fundamentals of a network . because they require considerable resources and energy to be carried out , participants highlighted the need to carefully consider the impact of engaging in activities requiring the network to adapt and transform . indeed , working together gives network participants opportunities to examine their practice through the eyes of their partners , to share and to exchange new knowledge likely to improve their network activities . some network participants attributed less importance to the dimensions deemed as encompassing relations with the external environment , such as capacity to acquire resources and ability to mobilize community support . this was because they felt they had little control over their external environment and they did not want to be labeled as less performing if , for instance , they failed to mobilize community support : one could put a lot of energy into trying to mobilize partners in the community , but at the end it is the partners who decide whether or not to provide their support . the external environment was seen as more of a constraint than an opportunity for adaptation and transformation . however , a few participants mentioned that the difficulties encountered in the external environment are likely to bring individuals , organizations and networks to think outside the box and to find creative solutions to improve their performance . with regard to dimensions included in the value maintenance domain , group participants explained that the collaborative climate could facilitate or impede partnerships at the political or managerial levels , but in the end , it has a small effect on the quality of care provided to patients . they also mentioned that the consensus with fundamental values dimension was seen as a pre - requisite to any collaboration . however , because they felt it is already present in the current public health system , it was not deemed to be a sensitive indicator of network performance . with regard to the dimensions belonging to the production domain , continuity was described as a very fundamental dimension of the performance of the tbi networks . indeed , participants indicated that the collaborative links and coordination established between network participants aims primarily to enhance the coherence of services ( e.g. theoretical frameworks , discharge and admission criteria , etc . ) and their seamlessness ( e.g. absence of delays between inter - facility transfers , absence of service interruption ) . the quality of care and services dimension was seen as important , but was perceived as being embedded in other dimensions , such as continuity and satisfaction of clients and partners . members of one network decided to give a score of 0% ( not important at all ) to this dimension because they believe that the evaluation of quality is the exclusive responsibility of an organization , and not of a network . the two others dimensions , productivity and quantity of care and services , suffered a considerable drop in their mean importance scores when discussed in groups . productivity was also described as being under the strict responsibility of individual facilities not of the network s , while quantity of care and services was considered as useless if not associated with other measures such as the number of clients seen . finally , with regard to the dimensions related to the goal attainment domain , all participants agreed that it is fundamental for the network to reach its goals . furthermore , participants explained that efficiency is a more important performance dimension to appraise than effectiveness , ( you do what you have to do versus the former dimension was perceived as providing more information , while the latter was perceived as a manifestation of conformity toward authority expectations . since they perceived effectiveness as embedded in the efficiency dimension , one network decided to reject ( attribute a score of 0% importance ) the effectiveness dimension to avoid redundancy in the evaluation . the satisfaction of clientele and partners was described as central to client - centered care , but participants expressed concerns about the subjectivity of patient satisfaction evaluations as they can be influenced by external elements or unrealistic expectations . group participants explained that the very creation of integrated health care for persons with tbi could lead to an inequity in the service provision for other patients ; tbi networks require many resources possibly depleting them for remaining patient populations . one hundred and thirty - nine professionals from acute care facilities ( 25% ) , rehabilitation facilities ( 65% ) and regional health authorities ( 10% ) participated in the individual data collection ( questionnaires ) . participants had a mean professional experience of 17 years ( sd 9.7 ) and reported working in their current position for an average of eight years ( sd 6.1 ) . all existing networks ( n=8 ) agreed to participate in the study and 62 persons from these eight networks participated in the group sessions : 27% were from acute care facilities , 52% from rehabilitation facilities and 21% represented regional health authorities . group participants had a mean professional experience of 18 years ( sd 9.7 ) and reported working in their current position for eight years ( sd 8.8 ) . the participants of the individual and group data collection phases were similar in terms of work experience and types of organization . table 1 presents the minimum and maximum importance scores attributed by the individual participants within a network to the dimensions of the egipss framework ; differences 20% are highlighted in gray . overall , the differences in the importance scores within the networks ranged from 0% to 100% , with a mean range of 33.7% . in fact , for the majority of dimensions , the observed individual importance score differences exceeded 20% ( 66% , corresponding to 85/[8 networks16 dimensions ] ) . with regard to the variability of the importance scores across networks , we found the networks had on average 10.2/16 dimensions for which the score ranges were 20% . the extent of consensus on importance scores varied according to the network examined . for the inter - regional network a , there was a total absence of agreement in scores , while for regional networks g and h , the majority of the performance dimensions scored within the 20% range . three domains of the egipss model , i.e. the adaptation , values maintenance and production , had similar proportions ( 31.3% ) of dimensions with scores with ranges of < 20% ( results not included in table 1 ) . in contrast , the goal attainment domain showed greater consensus with 40.6% of its corresponding dimensions with ranges of 20% or less . , four dimensions were attributed a wide range of scores by 7/8 networks : ability to adapt to requirements and tendencies , ability to innovate and transform , productivity , and quantity of care and services . in contrast , three dimensions ( efficiency , quality and continuity ) were attributed a range of scores 20% by only half ( 4 ) of the networks and only one dimension ( capacity to attract the clientele ) was similarly scored by the majority of the networks . eighty - five discussions took place about the different dimensions . on nine occasions , because the group deemed them unimportant , dimensions were placed in the garbage and received an importance score of 0% . four groups rejected quantity of care and service , productivity was rejected by three , and quality and effectiveness by one each . on eight occasions , at least one dimension was temporarily placed in the fridge section . however , after discussion , the groups were able to establish a consensus about their importance such that no dimension was placed in the veto section . at the end table 2 details , in decreasing order , the median importance scores after group discussions , the inter - quartile ranges ( iqr ) and the mean rank of the egipss dimensions obtained in the individual and group data collection phases . the median importance scores attributed to the dimensions via the individual survey varied from 60% to 100% ( median of 85% ) , while those attributed through consensus varied from 20.0% to 96.7% ( median of 86.6% ) . overall , the differences between the scores attributed in the individual and group data collection phases were not statistically significant ( z=0.88 , p=0.378 ) . five dimensions received higher importance scores during consensus group sessions as compared to the individual data collection phase . the capacity to attract the clientele , continuity , and efficiency dimensions were consistently scored as the three most important dimensions , both during the individual and group production phases . the scores attributed through consensus group sessions were slightly lower for nine of the 16 dimensions . the ability to adapt to requirements and tendencies and quantity of care and services remained among the least important dimensions in the two data collection phases . the differences in importance scores were statistically significant only for the productivity and the quantity of care and services dimensions , for which the importance scores attributed by participants dropped dramatically during the group process . with regard to the mean ranks indicating the relative importance of performance dimensions , the group process did not greatly influence the order of importance of the dimensions , with the exception of the continuity dimension , which was scored as relatively more important during the group sessions . it is noteworthy that the dimensions with the highest importance scores ( e.g. capacity to attract the clientele , efficiency , quality , etc . ) group discussions provided the participants with an opportunity to justify their individual scoring and to agree as a group on a new importance score . many of the general comments pertained to the desired balance between the integration and the differentiation of network partners . indeed , while agreeing that some degree of integration is required to provide quality services , network members wish to remain distinct organizations with their own culture , values , and intervention methods . when discussing the dimensions related to the adaptation domain , participants perceived ability to adapt and meet the client s needs as the only one fundamental to a network . the other five dimensions of the domain were seen as additional activities going beyond the fundamentals of a network . because they require considerable resources and energy to be carried out , participants highlighted the need to carefully consider the impact of engaging in activities requiring the network to adapt and transform . indeed , working together gives network participants opportunities to examine their practice through the eyes of their partners , to share and to exchange new knowledge likely to improve their network activities . some network participants attributed less importance to the dimensions deemed as encompassing relations with the external environment , such as capacity to acquire resources and ability to mobilize community support . this was because they felt they had little control over their external environment and they did not want to be labeled as less performing if , for instance , they failed to mobilize community support : one could put a lot of energy into trying to mobilize partners in the community , but at the end it is the partners who decide whether or not to provide their support . the external environment was seen as more of a constraint than an opportunity for adaptation and transformation . however , a few participants mentioned that the difficulties encountered in the external environment are likely to bring individuals , organizations and networks to think outside the box and to find creative solutions to improve their performance . with regard to dimensions included in the value maintenance domain , group participants explained that the collaborative climate could facilitate or impede partnerships at the political or managerial levels , but in the end , it has a small effect on the quality of care provided to patients . they also mentioned that the consensus with fundamental values dimension was seen as a pre - requisite to any collaboration . however , because they felt it is already present in the current public health system , it was not deemed to be a sensitive indicator of network performance . with regard to the dimensions belonging to the production domain , continuity was described as a very fundamental dimension of the performance of the tbi networks . indeed , participants indicated that the collaborative links and coordination established between network participants aims primarily to enhance the coherence of services ( e.g. theoretical frameworks , discharge and admission criteria , etc . ) and their seamlessness ( e.g. absence of delays between inter - facility transfers , absence of service interruption ) . the quality of care and services dimension was seen as important , but was perceived as being embedded in other dimensions , such as continuity and satisfaction of clients and partners . members of one network decided to give a score of 0% ( not important at all ) to this dimension because they believe that the evaluation of quality is the exclusive responsibility of an organization , and not of a network . the two others dimensions , productivity and quantity of care and services , suffered a considerable drop in their mean importance scores when discussed in groups . productivity was also described as being under the strict responsibility of individual facilities not of the network s , while quantity of care and services was considered as useless if not associated with other measures such as the number of clients seen . finally , with regard to the dimensions related to the goal attainment domain , all participants agreed that it is fundamental for the network to reach its goals . furthermore , participants explained that efficiency is a more important performance dimension to appraise than effectiveness , ( you do what you have to do versus the former dimension was perceived as providing more information , while the latter was perceived as a manifestation of conformity toward authority expectations . since they perceived effectiveness as embedded in the efficiency dimension , one network decided to reject ( attribute a score of 0% importance ) the effectiveness dimension to avoid redundancy in the evaluation . the satisfaction of clientele and partners was described as central to client - centered care , but participants expressed concerns about the subjectivity of patient satisfaction evaluations as they can be influenced by external elements or unrealistic expectations . group participants explained that the very creation of integrated health care for persons with tbi could lead to an inequity in the service provision for other patients ; tbi networks require many resources possibly depleting them for remaining patient populations . in this study , we first observed that when obtained through individually answered questionnaires , the extent of consensus about the importance of performance dimensions is low and network members have different perceptions about the importance of at least 50% of the performance dimensions . this suggests that any methodology that does not involve interactions between stakeholders is not likely to produce consensual results , and that a performance framework based on survey results could fail to promote a shared vision among network stakeholders . we also observed that the extent of consensus varied across networks and according to the dimensions examined . several studies using a multi - constituency approach involved participants from multiple settings [ 18 , 20 , 25 ] . however , to our knowledge , our study is the first to examine the variability in the extent of consensus across integrated settings ( e.g. networks ) . larger networks exhibited more differences in stakeholder perceptions , while smaller ones were typically ( but not systematically ) more consensual . this is not surprising since networks linking a large number of participants are more likely to include heterogeneous organizations . our findings thus support huxham s theory , in that the complexity of partnerships seems to influence mutual understanding . however , it is also possible that participants of smaller networks had the opportunity to be in closer proximity to each other and developed stronger links . the small size of the network could thus facilitate the adoption of a common vision . even in smaller networks , we observed an absence of consensus for at least half of the dimensions surveyed . in combination , these results suggest that consensus group sessions could be beneficial for organizations of all sizes , but especially for larger ones , regrouping many members potentially holding different perceptions of what is important to consider for performance evaluation . the results of the survey also indicate that the dimensions deemed more important by network participants are more consensual than others . this result suggests that the participants intuitively agree on the higher importance of a core set of dimensions often qualified as fundamental to the networks when discussed in groups . the fact that a high importance was paid to dimensions such ability to attract the clientele ( accessibility ) and continuity supports the idea that the performance of an integrated network of care is different from the performance of a single organization [ 7 , 10 , 11 ] , but shares the goal of many integration initiatives . because the concept of network performance is relatively new and yet not fully shared by all network members , its specificity should be made explicit to all stakeholders before any evaluation process takes place to avoid different visions tainted by the characteristics of a single organization ( rather than of a whole network ) . another important result of our study is that overall , consensus group sessions did not influence the quantitative importance attributed to performance dimensions by the member participants . given the small changes in importance scores provided by group participants , one might question the usefulness of consensus group sessions over the simple computation of median importance scores compiled using an individual data collection phase . we argue that the added value of consensus group sessions is evident in the discussions made possible between participants . indeed , qualitative analysis of the group discussions showed that participants expose the reasons that motivated the initial and group scoring . the exchanges help other network members understand their visions and it helps sustain discussions leading to a shared understanding of network performance . such harmonization of values and standards helps increase mutual trust and confidence among partners , which are deemed essential to improve collaboration [ 6 , 14 ] and to increase network performance . a second important added value of the consensus group session is that it represents a bottom - up approach to evaluation , in that it involves stakeholders in the design of the evaluation process . because group discussion takes into account the values and interest of networks partners , they are likely to improve the responsiveness and the usefulness of the evaluation process as well as stakeholders adherence to the resulting performance framework . consensus group discussion can also help to empower network participants who usually have less power in the network , by giving them an opportunity to take part in the decision about the network performance . collaborative inertia that are often observed in collaborative efforts , such as networks . in general , the results suggest that consensus group sessions are a useful step in the process of developing a tbi network performance evaluation framework and that the reconciliation of stakeholder perspectives could be a logical complement to multi - constituency approach studies . they could be used in any type of network , but would be most suitable for service networks , such as the one studied here and they may be more useful to participants who already have minimal experience of working together . consensus group sessions may be useful to clarify network performance frameworks , but could also sustain goal or process clarification . as such , the triage technique may represent a tool for integrated care , to improve a common vision and language . for an example , it could help practitioners who work collaboratively in a joint program to explore their perceptions of the program s goals , and to discuss the ones to prioritize . it constitutes a collaborative and reflexive tool that taps into benefits from the multiple perspectives and empowers the participants into establishing a shared construction of a common vision . it could be beneficial to the effectiveness and to the climate of a network , two important dimensions of network performance . group sessions allow participants to decide upon objectives they deem important , while using minimal time and human resources . with regard to the effectiveness of triage , the technique allowed the participants in each network to discuss their perceptions and to agree upon the importance they attributed to the dimensions discussed . this surpasses the 80% cut - off typically used in consensus methods , such as the delphi technique . triage , however , requires travelling for some participants , which could be difficult when a network is geographically spread out . group discussions could be limited by time restrictions or influenced by the level of expertise or the personality of participants . we used an arbitrary cut - off of 20% to determine the dimensions requiring discussion during the sessions . using another cut - off could participate in the group sessions and we could not verify whether their participation was representative of the opinions expressed by all of their team members . the high participations rates of individual organizations and networks support the generalizability of the results . this study reports the usefulness of consensus group sessions to reconcile the perceptions of stakeholders about the important dimensions to include in an evaluation of network performance . since our study is the first to illustrate such a process , repeating similar experiences is required to determine the advantages and drawbacks of different processes to reconcile divergent stakeholder perspectives . moreover , it remains to be demonstrated how implicating stakeholders from an early stage affects the evaluation process . in the future , the exploration and reconciliation of stakeholder perspectives about performance evaluation could become an important step in the planning of any performance evaluation process . edgar borgenhammar , phd , mba , dsi , professor of health services management , sweden louis demers , prof , cole nationale d'administration publique [ national school of public administration ] , qubec , canada robyn keast , associate professor , school of management , faculty of business and research director , airport metropolis project , faculty of built environment and engineering , queensland university of technology , australia .

backgroundhaving a common vision among network stakeholders is an important ingredient to developing a performance evaluation process . consensus methods may be a viable means to reconcile the perceptions of different stakeholders about the dimensions to include in a performance evaluation framework.objectivesto determine whether individual organizations within traumatic brain injury ( tbi ) networks differ in perceptions about the importance of performance dimensions for the evaluation of tbi networks and to explore the extent to which group consensus sessions could reconcile these perceptions.methodswe used triage , a consensus technique that combines an individual and a group data collection phase to explore the perceptions of network stakeholders and to reach a consensus within structured group discussions.resultsone hundred and thirty - nine professionals from 43 organizations within eight tbi networks participated in the individual data collection ; 62 professionals from these same organisations contributed to the group data collection . the extent of consensus based on questionnaire results ( e.g. individual data collection ) was low , however , 100% agreement was obtained for each network during the consensus group sessions . the median importance scores and mean ranks attributed to the dimensions by individuals compared to groups did not differ greatly . group discussions were found useful in understanding the reasons motivating the scoring , for resolving differences among participants , and for harmonizing their values.conclusiongroup discussions , as part of a consensus technique , appear to be a useful process to reconcile diverging perceptions of network performance among stakeholders .

Introduction Method Procedures Participants Analysis Results Participants characteristics Consensus observed through questionnaire use Consensus observed through group sessions Comparison of the consensual scores Explanations provided through group discussions Discussion Conclusion Reviewers

this study was conducted during 14 months ( october 2014 to november 2015 ) on patients with ag ( n=39 ) and gc ( n=40 ) at two medical oncology and gastroenterology facilities in the northwestern region of algeria . infection with h. pylori was confirmed by histopathology of biopsies sampled during diagnostic endoscopy ( at least four biopsies of the antrum and body gastric mucosa should be available for each case ) and anti - hp igg serum antibody tests . the corresponding tissue sections were stained for slide reading in the anatomopathology department of the university hospital of sidi - bel - abbes , algeria . the existence of h. pylori dna in gastric biopsies and buccal swabs were analyzed by pcr amplification of the urec gene using primers as described by lage et al . ( 16 ) ( forward : 5-aagcttttaggggtgttaggggttt-3 and reverse : 5-aagcttactttctaacactaacgc-3 ) at a hybridization temperature of 55c . we employed a dna volume of 25 l according to pcr criteria , as described below . the amplified fragments were visualized by using the gelred nucleic acid gel stain , 10,000 x ( biotium , hayward , ca , usa ) , and 2.5% agarose gel . information about personal data , sociodemographic characteristics , educational level , blood type , rhesus factor , eating habits , smoking , and alcohol consumption were evaluated using a predetermined questionnaire . furthermore , all participants gave their consent after the study protocol had been explained to them . 387 ( article 25 ) dated 31 july 2006 about ethical trials in algeria , we obtained the required access authorizations to the concerned health facilities in order to accomplish our study protocol . furthermore , we obtained informed written consent from all participants and their treating physicians after the study protocol had been explained to them . il-1-31 and il-1-511 polymorphisms are single nucleotide changes in the promoter region , while the il-1rn polymorphism is a repetition of 86 bp in the second intron . the dna was extracted from 200 l whole blood samples , 20 mg of fresh gastric biopsy , or buccal swab in h. pylori transport medium using an extraction kit ( magazorbe genomic dna , promega , fitchburg , wisconsin , usa ) stored at 20c . genotyping of the two polymorphisms ( il-1-31 and il-1-511 ) was performed using the restriction fragment length polymorphism method ( pcr - rflp ) ( table 1 ) ( 6 , 10 ) . briefly , 2550 ng of dna was used in a volume of 25 l with 150 mm dntp , 6.25 pmol of each primer , 1 u of dna polymerase gotaq g2 hot start polymerase ( promega , fitchburg , wisconsin , usa ) , 5% dmso , 4 mm mgcl2 , and 5 l of 5x pcr buffer gotaq . the amplified / digested fragments were visualized with the gelred nucleic acid gel stain , 10,000 x ( biotium , hayward , ca , usa ) in 2.5% agarose gel . primers and conditions for analysis of the il-1 and il-1rn polymorphisms tm : melting temperature ; il-1-31 : cluster of gene encoding interleukin-1 in the promoter region of tata box at the 31 position ; il-1-511 : cluster of gene encoding interleukin-1 in the promoter region of tata box at the 511 position ; il-1rn : gene encoding the anti - inflammatory antagonist ( interleukin-1ra ) . variable numbers of tandem repeats ( vntr ) analysis in the second intron of the il-1rn gene was carried out by pcr . the different alleles were identified by the size of the pcr fragments and encoded in a conventional manner ( 6 , 13 ) as follows : allele 1 ( four repetitions ) , allele 2 ( two repetitions ) , allele 3 ( five repetitions ) , allele 4 ( three repetitions ) , and allele 5 ( six repetitions ) . according to previous studies ( 6 , 13 ) , as described in table 1 , the il-1rn alleles were dichotomized into two categories : long genotype ( l:>three repetitions ; alleles 1 , 3 , 4 , 5 ) and short genotype ( 2 : two repetitions ; allele 2 ) . data were processed and analyzed using spss 22.0 ( statistical package for the social sciences , ibm corporation ; chicago , il , august 2013 ) . chi - square test was used to evaluate differences in the distribution of polymorphisms between different groups . all allelic distributions were examined for the corresponding differences of their hardy - weinberg equilibrium . the relative risk was evaluated by multivariate logistic regression based on categorical reference predictor models , adjusting for sex and age with a confidence interval ( ci ) of 95% and statistical significance set at p<0.05 . allelic combinations of models of the three variants closely related polymorphisms of il-1 and il-1rn were analyzed . the study of the haplotypes and linkage disequilibrium between loci ( estimated by r and d ) was carried out via the web interface in the linkdos ( www.genepop.curtin.edu.au/linkc.html ) . this study was conducted during 14 months ( october 2014 to november 2015 ) on patients with ag ( n=39 ) and gc ( n=40 ) at two medical oncology and gastroenterology facilities in the northwestern region of algeria . infection with h. pylori was confirmed by histopathology of biopsies sampled during diagnostic endoscopy ( at least four biopsies of the antrum and body gastric mucosa should be available for each case ) and anti - hp igg serum antibody tests . the corresponding tissue sections were stained for slide reading in the anatomopathology department of the university hospital of sidi - bel - abbes , algeria . the existence of h. pylori dna in gastric biopsies and buccal swabs were analyzed by pcr amplification of the urec gene using primers as described by lage et al . ( 16 ) ( forward : 5-aagcttttaggggtgttaggggttt-3 and reverse : 5-aagcttactttctaacactaacgc-3 ) at a hybridization temperature of 55c . we employed a dna volume of 25 l according to pcr criteria , as described below . the amplified fragments were visualized by using the gelred nucleic acid gel stain , 10,000 x ( biotium , hayward , ca , usa ) , and 2.5% agarose gel . information about personal data , sociodemographic characteristics , educational level , blood type , rhesus factor , eating habits , smoking , and alcohol consumption were evaluated using a predetermined questionnaire . furthermore , all participants gave their consent after the study protocol had been explained to them . 387 ( article 25 ) dated 31 july 2006 about ethical trials in algeria , we obtained the required access authorizations to the concerned health facilities in order to accomplish our study protocol . furthermore , we obtained informed written consent from all participants and their treating physicians after the study protocol had been explained to them . il-1-31 and il-1-511 polymorphisms are single nucleotide changes in the promoter region , while the il-1rn polymorphism is a repetition of 86 bp in the second intron . the dna was extracted from 200 l whole blood samples , 20 mg of fresh gastric biopsy , or buccal swab in h. pylori transport medium using an extraction kit ( magazorbe genomic dna , promega , fitchburg , wisconsin , usa ) stored at 20c . genotyping of the two polymorphisms ( il-1-31 and il-1-511 ) was performed using the restriction fragment length polymorphism method ( pcr - rflp ) ( table 1 ) ( 6 , 10 ) . briefly , 2550 ng of dna was used in a volume of 25 l with 150 mm dntp , 6.25 pmol of each primer , 1 u of dna polymerase gotaq g2 hot start polymerase ( promega , fitchburg , wisconsin , usa ) , 5% dmso , 4 mm mgcl2 , and 5 l of 5x pcr buffer gotaq . the amplified / digested fragments were visualized with the gelred nucleic acid gel stain , 10,000 x ( biotium , hayward , ca , usa ) in 2.5% agarose gel . primers and conditions for analysis of the il-1 and il-1rn polymorphisms tm : melting temperature ; il-1-31 : cluster of gene encoding interleukin-1 in the promoter region of tata box at the 31 position ; il-1-511 : cluster of gene encoding interleukin-1 in the promoter region of tata box at the 511 position ; il-1rn : gene encoding the anti - inflammatory antagonist ( interleukin-1ra ) . variable numbers of tandem repeats ( vntr ) analysis in the second intron of the il-1rn gene was carried out by pcr . the different alleles were identified by the size of the pcr fragments and encoded in a conventional manner ( 6 , 13 ) as follows : allele 1 ( four repetitions ) , allele 2 ( two repetitions ) , allele 3 ( five repetitions ) , allele 4 ( three repetitions ) , and allele 5 ( six repetitions ) . according to previous studies ( 6 , 13 ) , as described in table 1 , the il-1rn alleles were dichotomized into two categories : long genotype ( l:>three repetitions ; alleles 1 , 3 , 4 , 5 ) and short genotype ( 2 : two repetitions ; allele 2 ) . data were processed and analyzed using spss 22.0 ( statistical package for the social sciences , ibm corporation ; chicago , il , august 2013 ) . chi - square test was used to evaluate differences in the distribution of polymorphisms between different groups . all allelic distributions were examined for the corresponding differences of their hardy - weinberg equilibrium . the relative risk was evaluated by multivariate logistic regression based on categorical reference predictor models , adjusting for sex and age with a confidence interval ( ci ) of 95% and statistical significance set at p<0.05 . allelic combinations of models of the three variants closely related polymorphisms of il-1 and il-1rn were analyzed . the study of the haplotypes and linkage disequilibrium between loci ( estimated by r and d ) was carried out via the web interface in the linkdos ( www.genepop.curtin.edu.au/linkc.html ) . the mean age of all participants was 45.017.67 years , and ranged from 16 to 86 years . in healthy individuals , the mean age was 35.812.72 years with a male - to - female ratio of 1:1 ( table 2 ) . however , in patients with ag and/or gc , mean ages were 49.319.35 ( male - to - female ratio of 0.56:1 ) and 59.314.00 years ( male - to - female ratio of 2.27:1 ) , respectively . comparison between groups according to gender ag : atrophic gastritis ; gc : gastric carcinoma ; hc : healthy controls comparison between the three groups using chi - square test . the frequencies of the tested genotypes were in hardy - weinberg disequilibrium according to the following results . linkage disequilibrium analysis between il-1-31 and il-1-511 for ag patients gave the following results : r=0.6804 , d=0.46294 ; moreover , analysis of linkage disequilibrium between il-1-31 and il-1-511 in gc patients : r=0.69357 , d=0.48 . considering the whole studied population , il-1rn was positively correlated ( r=0.424 , r=0.180 ) with il-1-31 ( p=0.023 ) and with il-1-511 ( p=0.006 ) . in patients with gc , we noticed weak positive correlations ( r=0.377 , r=0.142 ) of il-1rn with il-1-31 ( p=0.550 ) and il-1-511 ( p=0.056 ) , respectively . non - significant correlations ( r=0.454 , r=0.206 ) between il-1rn , on the one hand , and il-1-31 ( p=0.703 ) and il-1 -511 ( p=0.072 ) , on the other hand , were observed in patients with ag . nevertheless , the correlations were non - significant in healthy subjects ( p=0.134 for il-1-31 and p=0.058 for il-1-511 ) . as depicted in table 3 , the frequency of l / l wild - type pattern of il-1rn was 87.5% in controls compared to 62.5% in gc and 82.1% in ag patients . the il-1rn2 allele rates , for either heterozygous or homozygous , were 37.5% in gc , 17.9% in ag , and 12.5% in controls . distribution of the genotypes in the study population ag : atrophic gastritis ; gc : gastric carcinoma ; all : atrophic gastritis and gastric carcinoma ; hc : healthy controls ; il-1-31 : cluster in gene encoding interleukin-1 in the promoter region of tata box at the -31 position ; il-1-511 : cluster in gene encoding interleukin-1 in the promoter region of tata box at the 511 position ; il-1rn : gene encoding for the anti - inflammatory antagonist ( interleukin-1ra ) ; l : long genotype ( more than three repetitions ; alleles 1 , 3 , 4 , 5 ) ; 2 : short genotype ( two repetitions ; allele 2 ) ; c or t : single nucleotide polymorphisms . regarding the il-1-511 , the proportions of c / c wild - type pattern were 56.3% in healthy subjects , 47.5% in gc , and 23.1% in ag patients . individuals carrying the allele il-1-511 t are represented by 76.9% of ag , 52.5% of gc , and 43.8% of healthy controls . our results show that 53.1% of healthy subjects , 35.9% of patients with ag , and 25.0% of patients with gc have the t / t wild - type pattern of il-1-31 . as illustrated in table 4 , we investigated major genotypes implicated as risk factors for having gc and ag compared to controls . when multivariate logistic regression analysis was performed , our results showed that the c / c homozygous 4.614 ( 95% ci , 1.43714.817 ) and the c / t heterozygous 1.974 ( 95% ci , 0.5626.939 ) genotypes of the il-1-31 each indicated a strong contribution to gc . likewise , the same genotypes have non - significant risk of having ag ( c / c : 2.429 [ 95% ci , 0.7697.673 ] and c / t : 1.455 [ 95% ci , 0.4025.260 ] ) . however , the association between the two homozygous and heterozygous genotypes c / t and t / t of il-1 -511 was 4.286 ( 95% ci , 1.54411.898 ) times more at risk of having ag compared to controls . regression analysis of the distribution of the genotypes in the study ag : atrophic gastritis ; gc : gastric carcinoma ; hc : healthy controls ; or : odds ratio . gc * hc : regression analysis of combined gastric carcinoma and healthy controls , ag * hc : regression analysis of combined atrophic gastritis and healthy controls , ag * gc : regression analysis of combined atrophic gastritis and gastric carcinoma . il-1-31 : cluster in gene encoding interleukin-1 in the promoter region of tata box at the 31 position ; il-1-511 : cluster in gene encoding interleukin-1 in the promoter region of tata box at the 511 position ; il-1rn : gene encoding the anti - inflammatory antagonist ( interleukin-1ra ) ; l : long genotype ( more than three repetitions ; alleles 1 , 3 , 4 , 5 ) ; 2 : short genotype ( two repetitions ; allele 2 ) ; c or t : single nucleotide polymorphisms . concerning the heterozygous genotype l/2 of il-1rn , our findings show that the odds ratio of having gc was 4.200 ( 95% ci , 1.23014.337 ) . further analysis of combined genotype between il-1rn and il-1-31 ( table 5 ) and il-1rn and il-1-511 ( table 6 ) was performed using test . the association between il-1-31 and il-1rn was significant in ag patients ( p=0.028 ) and in controls ( p=0.021 ) . furthermore , the relationship between il-1rn and il-1-511 was significant for the same groups ( ag : p=0.014 , and hc : p=0.015 ) . distribution of genotypes among patient groups regression analysis of combined ( il-1rn vs. il-1-31 ) chi - square test . ag : atrophic gastritis ; gc : gastric carcinoma ; hc : healthy controls ; il-1-31 : cluster in gene encoding interleukin-1 in the promoter region of tata box at the 31 position ; il-1-511 : cluster in gene encoding interleukin-1 in the promoter region of tata box at the 511 position ; il-1rn : gene encoding for the anti - inflammatory antagonist ( interleukin-1ra ) ; l : long genotype ( more than three repetitions ; alleles 1 , 3 , 4 , 5 ) ; 2 : short genotype ( two repetitions ; allele 2 ) ; c or t : single nucleotide polymorphisms . distribution of genotype among patient groups regression analysis of combined ( il-1rn and il-1-511 ) chi - square test . ag : atrophic gastritis ; gc : gastric carcinoma ; hc : healthy controls ; il-1-31 : cluster in gene encoding interleukin-1 in the promoter region of tata box at the 31 position ; il-1-511 : cluster in gene encoding interleukin-1 in the promoter region of tata box at the 511 position ; il-1rn : gene encoding for the anti - inflammatory antagonist ( interleukin-1ra ) ; l : long genotype ( more than three repetitions ; alleles 1 , 3 , 4 , 5 ) ; 2 : short genotype ( two repetitions ; allele 2 ) ; c or t : single nucleotide polymorphisms . the paradigm of previous studies on gc with regard to different haplotypes of il-1rn and il-1 has been investigated since 2000 on scottish and polish populations ( 6 , 7 ) . the genotype carriers of homozygous il-1-31c and il-1rn2 have a high risk of hypochlorhydria as a result of h. pylori infection with evidence for genetic machado et al . ( 13 ) reported results on a portuguese population by studying the etiological role of il-1-511 t allele , il-1rn2 allele , and their association with an increased risk of developing intestinal gc . until now , polymorphisms in genes of the pro - inflammatory cytokines il-1 and tnf- constitute an increased risk of developing gc in different gastric anatomical sites when the patients ethnicity is not considered ( 1719 ) . several studies on the association between il-1 and the il-1rn genotype in asian and caucasian populations show that il-1-511 t is significantly associated with the risk of gc ( 20 ) . other studies showed that il-1-511 t and il-1rn2 were associated with an increased risk of developing gc in caucasians . similarly , a significantly increased risk was noticed when analyses showed positive associated with h. pylori infection with a moderate increase among hispanics , but not among asians ( 15 , 2123 ) . to the best of our knowledge , only one study on the il-1 and il-1rn polymorphisms and their association with gastric cancer was performed in an arab population , namely in oman . the authors found that the il-1rn2 polymorphism increases the risk of gc , but the il-1-31c allele was not associated with the risk of developing gc ( 24 ) . in africa , kimang'a studied the association of these polymorphisms with h. pylori infection with different pathologies of the gastric mucosa without considering gc . he reported a significant effect of the il-1-511 t allele on gastritis , gastrointestinal reflux and gastric ulcers ( 25 ) . in this study , we evaluated the association between il-1 and il-1rn vntr polymorphisms and the risk of ag and cg in northwestern algeria . according to globocan 2012 , gc is the fourth most common cancer ( both genders ) in algeria , with about 686 cases per year ( 26 ) . our results show that the il-1-31c allele is associated with an increased risk of developing gc ( or=4.614 ; p=0.01 ) . the il-1rn2 heterozygous allele type is significantly associated with ag ( or=4.2 ; p=0.022 ) . these findings are in concordance with results on caucasians but different from those of asian and arab populations in oman ( 24 , 27 ) . considering the il-1-511 t allele , no significant effect on gc was noticed in the studied patients . this result is in agreement with studies on japanese and korean populations ( 27 , 28 ) , but different from results from caucasian populations ( 7 , 13 ) . individuals carrying the il-1-511 t allele have more risk of developing ag ( or=4.286 ; p=0.005 ) than gc . contrariwise , in a recent study from mexico , individuals carrying il-1-511c and il-1-31 t alleles separately are more likely to develop ag and gastric ulcers ( 30 ) . moreover , this work investigated the association between h. pylori infection and polymorphisms in only two genes , il-1 and il-1rn , without invoking other carcinogenic factors ; this could be considered as another limitation . in conclusion , the presence of il-1 and il-1rn polymorphisms with h. pylori infection of the gastric mucosa contributes to the development of chronic ag and gcs in an algerian population . individuals carrying the il-1-31c and il-1rn2 alleles are more likely to have an increased risk of developing gc . on the other hand these results highlight the importance of ethnicity studies about the effect of il-1 and il-1rn polymorphisms associated with h. pylori infection on gastric carcinogenesis . thus , large investigations needed to gain a proper understanding of gastric mucosa carcinogenesis in our region . the authors have not received any funding or benefits from industry or elsewhere to conduct this study .

backgroundinfection with helicobacter pylori is considered a potential risk of developing gastric cancer in association with contributing host genetic factor . il-1 and il-1rn polymorphisms appear to maintain and promote helicobacter pylori infection and to stimulate neoplastic growth of the gastric mucosa.objective and methodsin order to elucidate the effect of these polymorphisms in combination with gastric cancer in a population from northwestern algeria , a case - control study was carried out on 79 patients infected with h. pylori with chronic atrophic gastritis and/or gastric carcinoma , and 32 subjects were recruited as case - control . il-1-31 bi - allelic and il-1-511 bi - allelic polymorphisms and il-1rn penta - allelic were genotyped.resultsil-1-31c was associated with an increased risk of developing gastric carcinoma ( or=4.614 [ 1.4314.81 ] , p=0.01 ) . however , il-1rn2 heterozygous allele type was significantly associated with chronic atrophic gastritis ( or=4.2 [ 1.233.61 ] , p=0.022 ) . il-1-511 t was associated with an increased risk of development of chronic atrophic gastritis ( or=4.286 [ 1.5411.89 ] , p=0.005).conclusionil-1 and il-1rn polymorphisms associated with h. pylori infection contribute to the development of chronic atrophic gastritis and gastric carcinomas in an algerian population . the alleles il-1-31c and il-1rn were associated with an increased risk of developing gastric carcinoma , and il-1-511 t with an increased risk of developing chronic atrophic gastritis with no significant association of developing gastric carcinoma .

Methods Patients Analysis of IL-1 Statistical analysis Results Discussion Conflict of interest and funding

, this definition probably not only applies to different symptoms and hence deserves further classification [ 1 , 2 ] ( for example , the distinction between spontaneous and provoked confabulations ) but also appeals to a broader spectrum of disorders ( psychosis , delirium , korsakoff disease , stroke ) and to multiple brain underpinnings . confabulatory syndromes arising from focal stroke and confined to specific brain areas are not infrequent . they include the corporeal confabulations associated with anosognosia of hemiplegia , hemianopia or blindness after stroke including strategic associative areas on the right hemisphere , delusional misidentification syndromes with right frontotemporal strokes , spontaneous or provoked confabulations after stroke within the frontal - subcortical circuits processing both executive functions and memory [ the limbic thalamus ( dorsomedial and anterior nuclei ) , and the orbitofrontal cortex ( with rupture of aneurysms of the anterior communicating artery ) ] . we describe the case of a patient who showed persistent confabulations after two consecutive strokes of the territory of the lenticulostriate arteries on both hemispheres . a 75-year - old right handed swiss woman , with 12 years of education , was transferred to the rehabilitation center 1 month after a right ischemic subcortical stroke , which manifested with delirium and transitory left facial - brachial paresis . a cardioembolic etiology was retained as the patient had chronic atrial fibrillation and the anticoagulation rate was below the therapeutic range . four years before , she had a left lenticulostriate artery territory stroke with behavioral symptoms ( confabulations and subcortical aphasia ) , which completely recovered after 2 weeks . the patient was a retired housekeeper who lived autonomously with no history of alcohol consumption , nutritional disorders or memory disturbances before the actual stroke ( iqcode under the cutoff score for dementia ) . brain mri , performed at 6 weeks after the onset of the last stroke , showed the two lesions : a subacute ischemic lesion in the territory of the right lenticulostriate artery ( involving the caudate head , putamen and part of the pallidum ) , and an ancient lesion in the territory of the left lenticulostriate artery ( involving the caudate head and the surrounding white matter with the ipsilateral frontal horn enlargement ) ( fig . the patient 's language was characterized by a continuous stream of spontaneous confabulations and delusional thoughts . each time , without relevance to the context , either with staff or visitors , the patient provided unrequested and unrealistic accounts on a variety of topics . i have a plant that is growing in the intestine because of the dietary supplements , which i am taking on medical advice . i saw that plant the first time in my bathroom at home. other ones occurred isolated as the marriage proposals from therapists , imaginary visits by relatives and friends , late at night always in bizarre circumstances and fantastic scenarios . in one occasion , she said to one of the examiners that , few hours before , a human voice originating from the near lake suggested me to behave affably with your brother , whom i knew personally some years before. on another circumstance the patient claimed that the brother of a famous italian politician had recently deceased , and that the patient 's sister was right at that moment traveling to rome by train for the funerals , dressed with religious clothes . such statements generally withstood against contradictions offered by the medical staff : faced to the evidence ( on the journals or tv ) that the brother of the italian politician had not died at all , she stated that the press was not updated yet . however , despite high emotional expression , only occasionally the patient seemed to behave according to the content and facts of the confabulations , giving the impression to be interested only in the verbal reports of such random thoughts . these confabulations persisted over the 6 weeks of hospitalization and until the 6 months of follow - up , despite several behavioral interventions and pharmacological treatments ( ssris , neuroleptics , acetylcholine - esterase inhibitors ) . standard neuropsychological assessment ( table 1 ) showed deficits in orientation to time and personal information , sustained attention , executive functions ( reduced mental flexibility , perseverative behavior , compromised control on automatic responses , incapacity of planning ) and a severe anterograde memory impairment in memory tests . however , there were some residual episodic learning capacities ( as documented by the galveston orientation and amnesia test , according to which the patient had a borderline performance , and by the ravlt delayed recall score which showed a partial long - term retention ) . this was also reflected by the fact that the patient was able to recognize names and faces of therapists , and with a little practice she learned and remembered the route from the offices back to her room . language and visuoperceptive skills were slightly affected : conversation was fluent without paraphasias ; comprehension of test instructions and object recognition ( including overlapped or degraded figures ) were good . a confabulation battery showed that confabulations were triggered by episodic memory cues and do not know questions ( either episodic or semantic ) , as usually found for confabulators , and not upon personal semantic and linguistic cues . the patient 's language was characterized by a continuous stream of spontaneous confabulations and delusional thoughts . each time , without relevance to the context , either with staff or visitors , the patient provided unrequested and unrealistic accounts on a variety of topics . some of them were recurrent as i have a plant that is growing in the intestine because of the dietary supplements , which i am taking on medical advice . i saw that plant the first time in my bathroom at home. other ones occurred isolated as the marriage proposals from therapists , imaginary visits by relatives and friends , late at night always in bizarre circumstances and fantastic scenarios . in one occasion , she said to one of the examiners that , few hours before , a human voice originating from the near lake suggested me to behave affably with your brother , whom i knew personally some years before. on another circumstance the patient claimed that the brother of a famous italian politician had recently deceased , and that the patient 's sister was right at that moment traveling to rome by train for the funerals , dressed with religious clothes . such statements generally withstood against contradictions offered by the medical staff : faced to the evidence ( on the journals or tv ) that the brother of the italian politician had not died at all , she stated that the press was not updated yet . however , despite high emotional expression , only occasionally the patient seemed to behave according to the content and facts of the confabulations , giving the impression to be interested only in the verbal reports of such random thoughts . these confabulations persisted over the 6 weeks of hospitalization and until the 6 months of follow - up , despite several behavioral interventions and pharmacological treatments ( ssris , neuroleptics , acetylcholine - esterase inhibitors ) . standard neuropsychological assessment ( table 1 ) showed deficits in orientation to time and personal information , sustained attention , executive functions ( reduced mental flexibility , perseverative behavior , compromised control on automatic responses , incapacity of planning ) and a severe anterograde memory impairment in memory tests . however , there were some residual episodic learning capacities ( as documented by the galveston orientation and amnesia test , according to which the patient had a borderline performance , and by the ravlt delayed recall score which showed a partial long - term retention ) . this was also reflected by the fact that the patient was able to recognize names and faces of therapists , and with a little practice she learned and remembered the route from the offices back to her room . language and visuoperceptive skills were slightly affected : conversation was fluent without paraphasias ; comprehension of test instructions and object recognition ( including overlapped or degraded figures ) were good . a confabulation battery showed that confabulations were triggered by episodic memory cues and do not know questions ( either episodic or semantic ) , as usually found for confabulators , and not upon personal semantic and linguistic cues . after two temporally separated , bilateral consecutive strokes in the lenticulostriate artery territories the patient showed , over at least 6 months , a deep and persistent behavioral change consisting of spontaneous confabulations and delusions associated with a severe impairment of the episodic memory . this clinical picture could not be explained uniquely by an attention deficit as in delirium ( e.g. forward span in the normal range and attention improvement in the early follow - up , absence of cognitive and vigilance fluctuations ) . hence , it should be considered as a dementia after strategic strokes . furthermore , and most importantly , the persistent confabulations and delusions dominated the clinical picture , precluding most rehabilitative efforts . persistent confabulations after localized bilateral caudate stroke should be considered an extremely rare clinical pattern as bilateral caudate strokes are rare and similar case studies were not previously reported . the pervasiveness and persistence of confabulations are probably related to the bilaterality of the lesions . the bilateral interruption of frontal - subcortical circuits ( and more specifically the connections of the caudate nucleus ) might be the putative mechanism of the behavioral changes . similar to thalamic post - stroke amnesia or wernicke - korsakoff disease , where memory deficits and confabulations arise after damage of mammillo - thalamic tracts or thalamic nuclei , it might be speculated that in our case , again , the bilaterality of damage ( and thus precluding compensatory mechanisms ) is the crucial factor leading to the persistence of the syndrome . for our patient , the peculiar aspects of the confabulation syndrome consisted of : the spontaneous character , the pervasiveness with bizarre concatenation of unrelated contents and ideas ( mosaic confabulations ) , the presence of fantastic , visionary or delusional themes with emotional involvement and weak autobiographical references , the association with severe executive dysfunction and anterograde memory deficits . the patient 's confabulations might fit better with the category of spontaneous confabulation as they consisted mostly of incoherent and context - free retrieval of memories and associations . however , as in provoked confabulations , she showed prominent intrusions and irrelevant memories during retrieval attempts in challenge to anterograde memory tests . furthermore , her confabulations showed all the general peculiarities that , according to dalla barba , are common to all the variants of the confabulatory syndromes : ( 1 ) general memories , habits and misplacements , ( 2 ) memory confusion , and ( 3 ) autoreferential contamination . according to kopelman 's view , spontaneous confabulations result from the superimposition of frontal dysfunction on an organic amnesia ( although this pattern of cognitive impairment may also be present in patients with provoked confabulations ) , while dalla barba and boisse emphasize the presence of different lesion sites ( within the frontal lobe systems ) and the integrity of the medial temporal lobe . to other authors , confabulations could reflect retrieval of irrelevant past memories or overlearned information in patients with shallow encoding strategies . the latter line of argumentation is also followed by moscovitch and melo who argued that confabulations result from a failure in the strategic retrieval processes involved in focusing the memory search and in monitoring the veracity of retrieved memories implying that a monitoring deficit is necessary for confabulation to occur . another view suggests that confabulations reflect the inability to distinguish a memory derived from the direct experience from one generated by imaginative acts , or the inability to handle ambiguous or metaphoric cues in thoughts or speech . according to others , emotional factors might be the most relevant mechanisms for confabulations to provide more positive self - representations than true memories and to protect from unacceptable aspects of the reality . although all these mechanisms can be advocated together for the confabulations found in our patient , they also share , together with the pattern of cognitive deficits ( attention , memory and executive deficits ) , many similarities with the thought disorder exhibited by patients with schizophrenia ( pseudologia fantastica ) . these similarities refer to the reduced communicability , the fragmentation , discontinuity and incoherence of arguments , the creation of false memories as well as perceptions or beliefs about the self or the environment . other similarities include the arousal of negative emotions , the idiosyncratic symbolization of a visionary world ( where nonhuman objects live ) , and the interpretation of fantastic scenarios as if the patients were governed by random external stimuli . this single - case study offers further evidence that different types of confabulations may arise even after bilateral damage of subcortical structures because of the probable disconnection of prefrontal and limbic regions involved in episodic memory processing , affect and mood regulation . subcortical spontaneous confabulations after bilateral caudate involvement seem to differ from the spontaneous confabulations due to ventromedial and orbitofrontal lesions , since they seem to have only weak connections with past real memories that should fail to undergo an appropriate reality monitoring . our case study indicates that fantastic or implausible confabulations after bilateral caudate nuclei involvement seem to share many similarities with delusions of schizophrenic patients and with psychotic - like symptoms infrequently reported in patients with huntington 's disase [ 18 , 19 ] . caudate confabulatory syndrome could still be the result of the damage of several interconnected diffusely distributed fronto - limbic cortico - subcortical circuits and might correspond more to an emotion - based models of delusions .

we describe the case of a 75-year - old woman who manifested persistent confabulations after two consecutive strokes encompassing the area of the lenticulostriate arteries territory on both hemispheres . findings reported on this rare clinical syndrome suggest that fantastic confabulations and delusional thoughts may arise after bilateral damage of subcortical nonthalamic structures .

Introduction Case Report General Behavioral and Cognitive Assessment Discussion

manual material handling ( mmh ) tasks are one of the common occupational factors ( 1 ) . in most industries , and even in non - occupational settings , manual handling and lifting of weights happens very frequently ; each of these tasks have their own specific requirements , and such tasks are one of the important reasons for the incidence of low back pain ( 2 ) . low back pain and other musculoskeletal disorders caused by work , ranked the second after cardiovascular diseases , are among the most important and common diseases and cause of patients referral to the physicians in iran ( 3 ) . moreover , 85% of the population would suffer from back pain at some points in their life time . on the other hand , 25% of days off from the work are due to back pain ( 4 ) . in recent years , low back pain related to the job or career has become a major concern . in many studies conducted so far on different jobs and employees ( such as posts staff , doctors , soldiers , construction workers and other groups ) the prevalence of low back pain has been reported from 15% to 84% ( 5 ) . low back pain can reduce the functional performance and lead to absenteeism around the world and it imposes a huge economic burden on the individual , family , community , industry , and government . until 10 years ago it was thought that the problem is limited to western countries , however , since then the increased number of researches has found that low back pain is also a big problem in low and middle income countries as well ( 6 ) . low back pain can be caused by mechanical , ergonomic , personal , and social factors . among the ergonomic and mechanical factors , we can note the followings : lack of adequate rest between the work shifts , doing work with hurry and top speed , improper body postures , high frequency and repetitive movements , and lifting or moving heavy objects . in addition , obesity , gender , age and other factors can be also introduced as the causes of low back pain ( 7 ) . based on the mentioned facts , back injuries associated with mmh tasks have been a rigid problem for both the person and the national economy . there are several different main types of the mmh tasks , such as lifting , holding , carrying , pushing , and pulling . nearly 50% of back injuries occur when lifting objects , whereas only 9% occur when holding , throwing , or carrying objects . therefore , as the most common mmh task associated with the prevalence of low back injuries is lifting tasks ( 8) . during lifting and bending forward , the trunk is placed in front of the lumbar spine and it leads to shear and compressive forces to be imposed on the disk . on the other hand , each person 's tolerance of the load on the spine depends on different factors such as body posture , gender , age , the weight of the imposed load , and the individual anatomical changes ( 9 ) . many studies have examined the lifting task through using a biomechanical approach , which estimated the stresses incurred in the body while lifting materials . the focus of these studies is primarily on the lower back , especially the l5/ s1 disc ( 10 , 11 ) . accordance of the statistical data , between 85% and 95% of all disc herniations with a repeat equal proportion occur at the l4/l5 and l5/s1 levels , respectively ( 12 ) . in all the methods designed for the assessment of the risk related to the lifting , the biomechanical factors are of great importance . biomechanical factors in the work place , such as the workload , distance , and location of the workstations are effective in the prevalence of musculoskeletal disorders . based on the previously conducted studies , the severity of the effects of each of these factors is quite variable and depends on the range of changes in the shear and compressive forces imposed on the lumbar discs and the incidence and prevalence of low back pain ( 13 ) . manual lifting tasks can be evaluated for back injury risk via the analysis of the back compressive force ( bcf ) ( 14 ) . there are several methods used to estimate the compressive force in the lower back , among which we can note the followings : direct measurement method , simulation models , finite element method , niosh lifting equation , 3d static strength prediction program ( 3dsspp ) , hand calculated bcf model ( hcbcf ) , and surface emg - based models . in this study the hcbcf is a simple hand - calculation method for bcf based on the original utah bcf calculation model . in a study by elfeituri and taboun , the same method was used to estimate the biomechanical force . of all the subjects , 54% of the workers were suffering from a force on their low back , which was more than the permissible limit ( 3400 newton ) . moreover , in this study , the researchers concluded that the workers lifted a weight that was proportional to their capacity ( 15 ) . the peak bcf for dynamic model on the lumbosacral joint was 33% to 60% higher than that in the static model at four different lifting techniques ( 16 ) . niosh work practices guide for manual lifting indicated , biomechanical bcf on the l5/s1 disc in most workers are not tolerable over 650 kg ( 1430 lb ) and a 350 kg ( 770 ib ) bcf can be tolerated by most young , healthy workers . these criteria from cadaver spine failure and cross - sectional epidemiologic studies of job loads associated with back injuries were established ( 17 ) . this study was aimed to determining the changes in back compressive force when measuring maximum acceptable weight of lift in iranian male students . fifteen young male students ( 2030 yr old ) were recruited from the total population of students at tehran university . the mean ( sd ) demographic data of the subjects were as follows : age 22.2 ( 2.1 ) yr , body weight 67.5 ( 7.4 ) kg and height 177.7 ( 5.3 ) cm . we were looking at after that examined to ensure they had no considerable cardiovascular and musculoskeletal problems and no previous history of significant regarding lower back pain . they participated in training sessions and familiarized using the experimental processes before collecting your experimental data . the reason for selecting students as the subjects was the availability in terms of schedule flexibility and experimental duration . prior to commencing the study , smoking habits , alcohol and carbonated liquids consumption , eating habits , physical activity , and normal sleep duration of students were considered . all participants signed a consent form before the test , and they were paid to them . the summary of data on the subjects ' age , height , and weight are presented in table 1 . demographic description of the subjects ( n=15 ) a height adjustable set up , shown in fig . 1 , similar to the device used by snook , was used to simulate the 18 different lifting conditions considered in this study . digital accurate scale was used for weighing body weight and determined maximum weight of lifting . two plastic boxes with external handles ( 4.2 cm thick and 17.8 cm long ) were used . the small box has the width , length , and depth of 33.4 cm , 56.2 cm , and 16.0 cm , respectively . the large box has the width , length , and depth of 76.1 cm , 56.5 cm , and 22.0 cm , respectively . the width of the box represents the horizontal distance between hands and the central axis of the body . box dimensions and handles were the same as those used in the snook and ciriello mmh lifting tables ( 22 ) . 1 ) . height - adjustable shelves and boxes for handling lifts the experiments , designed to study the effects of independent variables on the back compressive force ( table 2 ) . three different task variables including frequency and height of lifting and box size were used in this study . mmh task parameters at different levels these three variables are the important descriptors of manual lifting tasks . three levels of lifting frequency ( 1 lift / min , 4.3 lift / min , 6.67 lift / min ) were applied . three different lifting heights ( floor to knuckle ( f - k ) , knuckle to shoulder ( k - s ) , and shoulder to arm reach ( s - a ) were fixed . each subject determined the maximum acceptable weight of lift and randomly , started with either a very light or a heavy weight ( according to 10% ile and 90% ile male as per the snook tables and were allowed to adjust it to arrive at the maximum acceptable weight of lift ( 26 ) . the room temperature was kept in the range of 2224 c and the relative humidity was 4555% . the psychophysical methodology ( 18 , 22 , 23 ) was used in this experiment . the maximum amount of weight or force could handle for 8 h without symptoms of fatigue or muscle weakness were determined by subjects . prior to commencing the experiments , the participants rested for 10 min in prone lying position . the procedure of experiments and the purpose of the research were explained to the students . before the start of each series of tests next , the participant performed one of the eighteen possible experimental conditions ( three lifting frequencies , three lifting heights , and two box sizes ) . each person participating in the test was asked to lifting the box using a free - style posture . to the control the frequency of lifting a periodic sound that was broadcast by the mobile phone was used . once the participants heard the sound , they lifted the box and then waited for the next sound . the participants were allowed to added or subtracted inside the weight from the box between the lift trials . during the lifting , the subjects changed the weight of boxes by adding and subtracting the sand bags . the final weight at the end of each course as it was intended as the maximum acceptable weight ( mawl ) for that particular frequency . the condition of the experiments was run twice for each subject ( one replication ) . for a given lifting task condition , if the participant s mawl for the second adjustment was within 15% of the first adjustment , the average of the two adjustments was considered as the final mawl for that lifting condition . otherwise , the results were discarded and the relevant data were collected at the next time . the back compressive force evaluated with hcbcf method . fifteen young male students ( 2030 yr old ) were recruited from the total population of students at tehran university . the mean ( sd ) demographic data of the subjects were as follows : age 22.2 ( 2.1 ) yr , body weight 67.5 ( 7.4 ) kg and height 177.7 ( 5.3 ) cm . we were looking at after that examined to ensure they had no considerable cardiovascular and musculoskeletal problems and no previous history of significant regarding lower back pain . they participated in training sessions and familiarized using the experimental processes before collecting your experimental data . the reason for selecting students as the subjects was the availability in terms of schedule flexibility and experimental duration . prior to commencing the study , smoking habits , alcohol and carbonated liquids consumption , eating habits , physical activity , and normal sleep duration of students were considered . all participants signed a consent form before the test , and they were paid to them . the summary of data on the subjects ' age , height , and weight are presented in table 1 . 1 , similar to the device used by snook , was used to simulate the 18 different lifting conditions considered in this study . digital accurate scale was used for weighing body weight and determined maximum weight of lifting . two plastic boxes with external handles ( 4.2 cm thick and 17.8 cm long ) were used . the small box has the width , length , and depth of 33.4 cm , 56.2 cm , and 16.0 cm , respectively . the large box has the width , length , and depth of 76.1 cm , 56.5 cm , and 22.0 cm , respectively . the width of the box represents the horizontal distance between hands and the central axis of the body . box dimensions and handles were the same as those used in the snook and ciriello mmh lifting tables ( 22 ) . the experiments , designed to study the effects of independent variables on the back compressive force ( table 2 ) . three different task variables including frequency and height of lifting and box size were used in this study . mmh task parameters at different levels these three variables are the important descriptors of manual lifting tasks . three levels of lifting frequency ( 1 lift / min , 4.3 lift / min , 6.67 lift / min ) were applied . three different lifting heights ( floor to knuckle ( f - k ) , knuckle to shoulder ( k - s ) , and shoulder to arm reach ( s - a ) were fixed . each subject determined the maximum acceptable weight of lift and randomly , started with either a very light or a heavy weight ( according to 10% ile and 90% ile male as per the snook tables and were allowed to adjust it to arrive at the maximum acceptable weight of lift ( 26 ) . the room temperature was kept in the range of 2224 c and the relative humidity was 4555% . the psychophysical methodology ( 18 , 22 , 23 ) was used in this experiment . the maximum amount of weight or force could handle for 8 h without symptoms of fatigue or muscle weakness were determined by subjects . the students wore the normal clothes and flat - soled sport shoes . prior to commencing the experiments the procedure of experiments and the purpose of the research were explained to the students . before the start of each series of tests , next , the participant performed one of the eighteen possible experimental conditions ( three lifting frequencies , three lifting heights , and two box sizes ) . each person participating in the test was asked to lifting the box using a free - style posture . to the control the frequency of lifting a periodic sound that was broadcast by the mobile phone was used . once the participants heard the sound , they lifted the box and then waited for the next sound . the participants were allowed to added or subtracted inside the weight from the box between the lift trials . during the lifting , the subjects changed the weight of boxes by adding and subtracting the sand bags . the weight of the boxes was set in accordance with procedures . the final weight at the end of each course as it was intended as the maximum acceptable weight ( mawl ) for that particular frequency . the condition of the experiments was run twice for each subject ( one replication ) . for a given lifting task condition , if the participant s mawl for the second adjustment was within 15% of the first adjustment , the average of the two adjustments was considered as the final mawl for that lifting condition . otherwise , the results were discarded and the relevant data were collected at the next time . the back compressive force evaluated with hcbcf method . the mean and standard deviation of bcf for the small box at a frequency of one lift / min at different heights of f - k height , k - s height and s - a height , were 1001.02 ( 86.74 ) , 875.26 ( 86.81 ) , and 833.57 ( 72.64 ) ib . the obtained values for the same box at frequencies of 4.3 and 6.67 lift / min at the mentioned heights are presented in table 2 . in addition , the mean and standard deviation of the bcf for the large box at a frequency of one lift / min at different heights of f - k height , k - s height and s - a height , were 1210.57 ( 93.77 ) , 1166.57 ( 72.31 ) , and 1092.42 ( 84.69 ) ib . the obtained values for the same box at frequencies of 4.3 and 6.67 lift / min at the mentioned heights are presented in table 3 . mean of back compressive force ( ib ) for lifting various boxes at different heights and frequencies the results of the anova showed that there was a significant difference between mean bcf in terms of frequencies of lifts ( p=0.02 ) . turkey 's post hoc test showed that the mean bcf did not show a significant difference between the frequencies of 1 lift / min and 4.3 lift / min ( p>0.05 ) ; however , there was a statistically significant difference between the frequencies of 1 lift / min and 6.67 lift / min ( p=0.01 ) . there was no significant difference between the frequencies of 4.3 lift / min and 6.67 lift / min ( p>0.05 ) . as the frequency increased from 1 lift / min to 4.3 lift / min , the mean bcf declined by nearly 8.00% , from approximately 948.65 ib to 875.74 ib . a further decline of 6.20% from 875.74 ib to 822.16 ib was observed when the lifting frequency increased to 6.67 lift / min . the results of anova showed that there was no significant difference between mean bcf in terms of lifting heights ( p>0.05 ) . the results of t - test showed that there was a significant difference between mean bcf in terms of the sizes of the two boxes ( p=0.001 ) . when the box size increased from the small size to the large one , the mean bcf increased by approximately 10% from 837.47 ib to 926.98 ib . the main effect of various lifting parameters for back compressive force is shown in fig . 2 . effect of main lifting parameters on back compressive force ( bcf ) the results of pearson correlation test showed that there was a significant relationship between the bcf and maximum acceptable weight of lift in all test conditions ( p=0.001 ) . thus , with an increase in the maximum acceptable weight of lift , bcf also increased . the mean bcf had no significant difference in terms of the combined variables of lifting frequency , lifting height , and the size of the box ( p>0.05 ) , except for the simultaneous effects of frequency of lifts and height of lifts ( p=0.02 ) . the purpose of this study was to estimate changes bcf when subjects determined maximum acceptable weight of lifts during a 20 min lifting of the loads . chen , to estimate the peak bcf at level of the l5/s1 disc and reported that for the subjects with an average mass of 67 kg , a mean value of 3300370 n was generated when lifting a 5-kg load , a mean value of 4490520 n while lifting a 15-kg load , and a mean value of 5050500 n while lifting a 20-kg load . the mean of the bcf at l5/si ranged from 3272 to 5877 n and average subject 's mass of 67.5 kg , which is comparable as observed in chen study ( 24 ) . in this study , subjects significantly increased peak bcf at l5/s1 disc when they had to lift the heavy loads . this result supports previous studies ( 24 , 25 ) , where lifting the heavy loads led to significantly increase peak bcf at l5/s1 disc . the effects of magnitude and knowledge of loads on the l5/s1 compressive force during lifting the loads , showed that during lifting of light load whereas subject no knowledge of weight the load , subjects overestimated the load , and significantly the peak bcf at l5/s1 increased . knowledge of the mass did not significantly change the peak bcf while lifting a heavy load ( 25 ) . in addition , the results of this study were compared with the safety limit of bcf proposed by the niosh organization . the results showed that in all cases of lifting the big box the mean rate of bcf was higher than the safety limit . with reference to the small box , at a frequency of 6.67 lift / min at the heights of the k - s height and the s - a height mean rate bcf was lower than the safety limit while in other cases it was higher ( fig . 3 ) . compare mean of the back compressive force for all experimental conditions ( small and large boxes ) and for the compressive force limit ( niosh ) in a study , the force on the back ( back compressive force ) was more than the admissible rate . however , as the result of correction of the angle of load lift , the estimated biomechanical force was set back in the permissible range ( 26 ) . compressive and shear forces exerted on the disc l5/s1 , respectively , in 17.5% and 10.8% of the studied workers were higher than permissible rate ( 27 ) . this study was only conduced on students aged 20 to 30 yr , as they were easily available , therefore , it is recommended to conduct similar studies on workers with a wider age range , so that to achieve more accurate standards for the bcf and to reduce the risks of handling weights . there was a significant difference between mean bcf in terms of frequencies of lifts and size of the boxes , but the difference was not significant in terms of lifting height . besides , in 100% of cases of lifting the large box , bcf was larger than the safety limit while it 77.87% of cases it was true for the small box . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .

background : low back pain caused by work , ranked the second after cardiovascular diseases , are among the most common reasons of patients referral to the physicians in iran . this study aimed to determine the changes in back compressive force when measuring maximum acceptable weight of lift in iranian male students.methods:this experimental study was conducted in 2015 on 15 young male students were recruited from tehran university of medical science . each participant performed 18 different lifting tasks involving three lifting frequencies , with three lifting heights , and two box sizes . each set of experiments was conducted during the 20 min work period using free - style lifting technique . the back compressive force evaluated with hand - calculation back compressive force method . finally , pearson correlation test , analysis of variance ( anova ) and t - test were used for data analysis.results:the mean of back compressive force ( bcf ) for the small and large boxes at a frequency of 1lift / min at heights of f - k height , were 1001.02 ( 86.74 ) , 1210.57 ( 93.77 ) ib , respectively . there was a significant difference between mean bcf in terms of frequencies of lifts ( p=0.02 ) . the result revealed significant difference between frequencies of 1 lift / min and 6.67 lift / min ( p=0.01 ) . there was a significant difference between mean bcf in terms of the sizes of the two boxes ( p=0.001 ) . there was a significant relationship between the bcf and maximum acceptable weight of lift in all test conditions ( p=0.001).conclusion : bcf is affected by box size , lifting frequency and weight of load .

Introduction Materials and Methods Participants Equipment Experimental design Study procedure Results Discussion Conclusion Ethical considerations

during the seventeenth and eighteenth centuries , scurvy came to prominence as a disease of sailors and soldiers . there was general agreement that it was caused by putrefaction of the humours , the corruption of which was often attributed to a diet of noxious foods or prolonged exposure to cold and damp . by the late eighteenth century , however , the way in which scurvy was understood began to change , in common with many other diseases . the humoral theory was gradually abandoned and new insights were incorporated into medicine from nervous physiology and pneumatic chemistry . utilising these theories , medical authors began to portray scurvy as a disease of dietary deficiency , even though few could agree on what precisely was missing . from the mid-1790s , the view that scurvy was due to defective diet was bolstered by practical experience . the universal provision of citrus juices for naval vessels brought a dramatic reduction in cases of scurvy and gave the british fleet an advantage over its enemies . for this reason , the 1790s are often regarded as a watershed in the health of the navy and in the history of scurvy itself . thereafter , we are told , medical interest in the disease began to decline . it was as if the problem of scurvy had been solved . the reality was very different , for scurvy remained a complex and controversial disease through to the middle of the nineteenth century and beyond . this article offers an explanation of why such a diversity of views persisted beyond 1790 and why it was not until the mid - nineteenth century that a loose and rather fragile consensus began to emerge . the reasons , i suggest , owe more to prevailing doctrines of political economy and administrative expediency than to the force of scientific logic . after a survey of changing views on the physiology of scurvy during the eighteenth century , this article examines how the problem of scurvy was understood in the royal navy and in two other contexts in which it presented a problem : the territories of british india and the new penitentiaries constructed in britain . these were not the only settings in which scurvy gave cause for concern , but they suffice to demonstrate that its etiology remained unstable and that attempts were made to order the disease in different ways . scurvy did not suddenly appear in the eighteenth century , but it became far more common at that time . ships were able to remain at sea for longer because of the discovery of longitude and other aids to navigation . the global wars and trading interests of the eighteenth century also meant that many vessels were undertaking such voyages . although scurvy was by no means the principal medical problem facing the royal navy especially by comparison with typhus or ship fever it often combined with other diseases to reduce the morale and efficiency of crews . nor was scurvy solely a problem affecting mariners . it was widely recognised as a land disease , occurring among soldiers on active service and among all peoples subject to famine and deprivation . however , scurvy was not yet regarded as a disease of deficiency in any sense of the term . it was not simply that individuals differed in their understanding of why the disease occurred , but very few writers attributed it to any single factor . this diversity has been widely acknowledged in historical studies of the period and some writers particularly christopher lawrence have made much of the fact that even james lind ( who seemingly demonstrated the efficacy of citrus juices during a trial in 1747 ) saw scurvy as having many causes , including poor hygiene and discipline . indiscipline resulted in dirty ships and feeble bodies , he claimed , both of which were conducive to scurvy . discover that scurvy was a deficiency disease ; nor was his work unjustly neglected by the admiralty . his results were significant , but the methods he employed were not as persuasive as they are now . medical men using experiments and trials had to struggle for many years to establish the superiority of quantitative methods over more traditional sources of knowledge . like most other diseases at the time , scurvy continued to be understood within a familiar framework of causation which was derived from the natural histories of aristotle and which had been the stock in trade of european medical practitioners from ancient times . it had , however , undergone numerous refinements , incorporating new theories about how the body worked . renaissance anatomy and physiology , chemical and mechanical theories of disease , and theories of contagion all left their mark . most diseases were understood in terms of the inter - relationship between so - called proximate causes ( deranged organs , fluids and manifestations of disease in the body ) ; predisposing causes such as fatigue and depression ; and exciting causes such as vitiated diet , contagion and atmospheric influences . this complex but flexible framework allowed diseases to be understood in much the same way as other natural phenomena and they were classified like plants and animals into different genera and species . this exercise was generally known as nosology or nosography and many physicians devised their own classifications . in most of these , putrid diseases ; that is , as a disease characterised by putrefaction of the humours or four substances from which the fluid parts of the body were said to be comprised ( blood , phlegm , and black and yellow bile ) . corruption of the humours seemed to be indicated by many common symptoms of the disease such as swellings and other outward signs of decay , as well as by loss of teeth and foetid breath . james lind , for example , believed that it was caused by perspiration which had been thwarted by moisture in the air , combined with the effects of a diet composed chiefly of salted meat . alkalescent disposition of the body a term derived from the famous dutch physician herman boerhaave ( 16681738 ) , whose writings were indebted to the new chemical philosophy . boerhaave s work had a formative influence on teaching at the newly established medical faculty at the university of edinburgh ; lind studied there before entering the navy and afterwards completed an md at the same institution . however , in lind s view the action of dietary factors could be aggravated by indolence and filth , both of which predisposed the body to corruption . the putrid theory of scurvy was also evident in accounts of the disease as it occurred in the british army , most influentially john pringle s theory that the disease arose from gradually accumulated putrefaction caused by moist or vitiated air , lack of exercise , deficiency of fresh vegetables and rotten items of diet . salted meats , such as those used by sailors and sometimes on military expeditions , were liable to become putrid , he believed . pringle s medical annotations indicate that scurvy was attributed to a great variety of causes and that there were many supposed preservatives and treatments . some of these , such as malt extracts , are well known but there were many others , including tea and various types of acids . antiseptics , meaning that they were able to reverse or retard the process of putrefaction . sir john pringle ( 170782 ) was not merely a respected military physician , having been physician to the continental army in the war of austrian succession ( 17408 ) , but also president of the royal society of london between 1772 and 1778 . as such , his opinions had a formative influence on medical theory throughout the english - speaking world . however , the tenets of the putrid theory of scurvy are equally evident in continental writings on the disease . the austrian court physician baron gehard van swieten was typical in regarding scurvy as a disease brought about by humours that had become putrid , acrimonious and condensed . in his view , it was to be cured by attenuating the viscosity of the humours and in preventing or correcting putrefaction. this meant avoiding external causes such as noisome vapours , arising from marshy grounds and stagnating waters , inaction , scarcity of greens and vegetables , drinking of corrupted and stagnating waters , the use of salted and smoked flesh and fish damp and low lodgings. it also meant counteracting predisposing factors such as fear and sorrow and avoiding food which had gone bad . it was for this latter reason , he insisted , that persons trapped in besieged places commonly fell prey to the disease . this etiological framework remained relatively unchanged until the last two decades of the eighteenth century . authors placed different degrees of emphasis on factors such as diet , but most conceived of scurvy as a putrid disease and described it in the familiar vocabulary of humoral medicine . but from the 1780s , writings on scurvy began to be coloured by nervous theories of disease , the principal source of these in britain being the distinguished edinburgh professor , william cullen ( 171090 ) . cullen believed that the proximate cause of most diseases was a disorder of the nervous system , which could be brought about by either the action of external agents such as weather or poisons , or by personal dispositions and habits . nervous physiology had gained ground slowly through the middle decades of the century and by the 1780s it is clearly evident in works on scurvy , such as that of the physician francis milman . the oxford - educated milman moved in distinguished circles in london but he had little direct experience with the disease . nevertheless , his connections and his fellowship of the royal college of physicians ensured that his work was taken seriously . milman concurred with earlier writers that scurvy was a putrid disease , but he did not regard it as having originated from putrefaction of the humours . instead , he believed that scurvy arose from nervous debility a term derived from the writings of john brown , a rebellious pupil of cullen . brown was a controversial figure , but his influence can be detected beyond his immediate circle and contributed to a growing tendency to place the nervous system at the centre of medicine and intellectual life . in milman s view , scurvy was the consequence of several influences acting jointly on the nervous system . exciting causes such as indigestible diet and food lacking in nutrition , as well as predisposing causes such as indolence , fatigue , lack of exercise / sleep , cold , sorrow and melancholia . prevention lay in counteracting as many of these as possible , for example , by protecting bodies against the elements , providing better food , and insisting on cleanliness and discipline . thus , despite the fact that milman had abandoned humoralism for nervous theory , his causal framework remained practically the same : diet was merely one element albeit an important one among many . the only real differences lay in some of milman s recommendations for prevention and cure . instead of the common tendency to treat scurvy with a variety of substances reckoned to counteract putrefaction , milman , like other brunonians , recommended stimulants such as brandy and opium , which were supposed to act by raising the body s nervous tone . fixed air ( later known as carbon dioxide ) , betraying a debt to the pneumatic chemistry of joseph priestley and others . pneumatic medicine of the kind associated with priestley tended to appeal to the same people who were drawn to brunonianism : men outside the physician elite such as practitioners in the armed forces , some of them religious and political dissenters . but the majority of works on scurvy published in the decade after milman s enquiry were less radical in tone and of comparatively little consequence . they tended to combine the most acceptable and least controversial elements of older works with insights from newer ones , embracing both humoral and nervous theories of disease some were practical in nature , but others were clearly designed to appeal to all tastes and to serve as advertisements for their authors patent remedies . both these works were written by naval practitioners whose authority rested on claims to direct and extensive experience . one was written by thomas trotter ( 17601832 ) and the other by gilbert blane ( 17491834 ) . like many practitioners entering the armed forces , trotter joined the navy with an incomplete university education , in his case at edinburgh , to which he later returned to take a medical degree . a man of modest means but untiring assiduity , trotter worked his way up through the service to become second physician at haslar hospital in 1793 and physician to the channel fleet a year later . trotter shared milman s disdain for humoral pathology but he was more indebted to his former professor , william cullen , than to brown and he dismissed brown was regarded by many physicians as a mountebank who had spawned a host of imitators touting patent medicines which claimed to restore nervous vitality . however , trotter was also critical of some aspects of cullen s work , particularly his theory of scurvy , in which he detected vestiges of humoralism ( his notion that scurvy was characterised by a saline state of the blood ) . trotter attempted to distinguish his own work by appealing to his extensive experience at sea by comparison with landlubbers like milman . in his view , scurvy was caused primarily by the lack of fresh vegetables and not , as milman claimed , by a diet that was indigestible . trotter thought the juices of lemons and limes particularly effective , but at the time he wrote his first treatise on the subject , in 1786 , the problem of preserving them on long voyages had still to be overcome . trotter also differed from most previous writers on scurvy in that he rejected the view that it could be caused by the elements alone , although he admitted that dampness and cold could be contributing factors . other predisposing factors included indolence and depression , and trotter noted the tendency of scurvy to appear among those whom he termed skulkers. such feelings were often brought on by impressment , he argued , and this was one of the reasons why he condemned this arbitrary system of conscription . in the second edition of his observations on the scurvy ( 1792 ) trotter continued to denounce milman and other brunonians , but he embraced another medical fashion in the form of pneumatic chemistry . vital air ( oxygen ) and this explained why ships well provisioned with such victuals had been able to protect their crews from scurvy . trotter later admitted ( in response to criticism from the radical physician thomas beddoes ) that he had made certain mistakes in his rendering of chemical theories . but he did not depart from his main conclusion , contradicting critics such as beddoes and other writers who believed that he had placed too much emphasis on defective diet . as far as beddoes was concerned , anything which reduced the oxygen content of the blood was capable of causing scurvy , including vitiated air , and the disease could be prevented solely by keeping vessels well ventilated ( like captain cook on his famous voyages of discovery ) or simply by change of climate . likewise , any substance containing what beddoes believed to be large quantities of oxygen , including meat and elixirs of various acids , might work as well as citrus juices in restoring the depleted body to health . among trotter s other critics was dr seguin henry jackson , a physician of westminster general and st george s hospitals . phlogiston in the blood ( an element which was reckoned by some to impart combustibility to the air ) and could be relieved by any number of methods , such as the provision of pure drinking water . these critics lacked trotter s first - hand experience of the disease and were in some senses marginal figures . whereas beddoes s radicalism put him beyond the pale as far as many more orthodox practitioners were concerned , jackson s theories harked back to an earlier period of chemical philosophy and could be dismissed as anachronistic and idiosyncratic . nevertheless , trotter s opinion that scurvy was essentially a deficiency disease clearly provoked disagreement . soon , however , trotter would have the opportunity to put his theory to the test , for he was appointed physician to the channel fleet in 1794 . his experiences over the next 12 months appeared to confirm his earlier conclusions about the centrality of diet . in december 1794 while it lay at anchor no fresh meat was issued , only salt provisions , and vegetables were scarce because of the season . by january 1795 the fleet had moved to spithead , where fresh vegetables remained in short supply . over the coming months , cases of scurvy began to appear on every ship and 200 men were sent ashore to hospital . the epidemic did not abate until after the arrival of summer and ample supplies of fresh fruit . in trotter s view , these facts proved that his theories were true : the outbreak was unquestionably related to dietary deficiency and was remedied by fresh provisions . furthermore , the emphasis which beddoes had placed on impure air was shown to be wrong because ventilation and sanitary conditions on all the ships were excellent . but though trotter attached more importance to diet than many of his contemporaries , he remained convinced that scurvy was aggravated if not caused by other factors ; in this case , low temperatures , the abuse of hard liquor , and the debilitating effects of illnesses like the epidemic catarrh which swept the fleet at spithead during january and february 1795 . crosfield , who was confined among british prisoners of war in france , attributed an outbreak of scurvy among the captives to a combination of defective nutrition , cold , damp and low spirits . in his view , fresh vegetables were not in themselves capable of restoring the sick to health . this inclusive view of the causation of scurvy was shared by the other chief authority on the disease , the naval physician gilbert blane . blane was somewhat unusual among naval practitioners in that he entered the service already in possession of a medical degree . most of those who claimed the title physician took their degree , like trotter , after a period of service which left them with sufficient funds to do so . blane , however , hailed from a prosperous merchant family and was able to complete his medical education . his family connections also secured him a position as physician to lord rodney s fleet in the caribbean during the american war of independence . under rodney s patronage , blane acquired an enviable list of aristocratic clients and an appointment as head of the navy s medical board in 1796 . blane s first contribution to the literature on scurvy was his observations on the diseases incident to seamen ( 1785 ) . this early work may be regarded as a transitional text : its descriptive vocabulary is largely drawn from humoral pathology but its explanation of causation resembles milman s . but while it placed slightly more emphasis on diet than milman , blane s treatise still emphasised the importance of predisposing causes such as sloth , moisture , filth and cold . each new edition of the work was rooted more deeply in nervous physiology than the last . the third edition ( 1803 ) , for example , drew heavily on the theories of erasmus darwin , which in some respects resembled those of brown . accordingly , scurvy was regarded as a disease of debility rather than of putrefaction , and citrus juices were said to work because they administered a stimulus to the system . however , the growing emphasis on nervous physiology did not come at the expense of predisposing causes . while the notion of stimulus invited practitioners to look outward to the environment or to certain properties in foodstuffs , it acknowledged the importance of factors which had a depressing effect on the mind and body , such as poor weather or lack of mental and physical stimulation . indeed , the later editions of blane s work show progressively more emphasis upon the role of such factors in causing the disease . as blane put it : there seems to be something in the habits of life in a ship , whether at sea or in port , favourable to sea scurvy . the ships belonging to the channel fleet in 1794 and 1795 , were subject to scurvy even when at spithead , though the men were fed with fresh beef and drank beer . the difference of these situations consists , partly in the superior dryness , cleanliness , and ventilation of the latter ; but more , i apprehend , in the want of exercise and recreation on board a ship . there seems to be something in the habits of life in a ship , whether at sea or in port , favourable to sea scurvy . the ships belonging to the channel fleet in 1794 and 1795 , were subject to scurvy even when at spithead , though the men were fed with fresh beef and drank beer the difference of these situations consists , partly in the superior dryness , cleanliness , and ventilation of the latter ; but more , i apprehend , in the want of exercise and recreation on board a ship . this stress on the non - dietary causes of scurvy may be surprising in view of blane s crucial role in securing the standard issue of citrus juices to naval vessels in 1795 . it is well known that blane regarded that year as a watershed in the health of the navy and later produced statistics to illustrate the dramatic upturn in the navy s health . but he recognised that the issue of lemon juice was only one of a series of reforms which had improved the health of sailors , including new regulations on hygiene and cleanliness introduced in the 1790s and enforced through the new diet was seen as the principal factor in preventing scurvy but certainly not the only one , and the reforms of the 1790s addressed many of the hygienic and disciplinary concerns formerly raised by such practitioners as lind . scurvy was effectively brought under control by these actions , but naval views of scurvy remained relatively open and there was no administrative reason to insist that they be simplified . in some of the other contexts in which scurvy occurred , the situation was very different . as cases of scurvy in the royal navy fell to negligible proportions , the disease was becoming more common elsewhere . scurvy had occurred periodically among the east india company s troops throughout the eighteenth century , especially when its forces were besieged in fortresses by indian rulers and the french . but as the company began to establish itself as a major territorial power and to deploy its forces further afield , cases of scurvy began to increase . it was a particular problem during the campaign in burma in 18246 , when some 20,000 deaths were attributed to disease and starvation . many of these were said to be due to scurvy ( directly and indirectly ) and its familiar symptoms were present in many of those who died . in 1826 , for example , the disease appeared among european and indian troops in the vicinity of rangoon . after the city was captured , burmese forces compelled locals to move to the interior and cut off provisions , leaving the company s army without the means to replenish its dwindling supplies . heavy losses among both british and indian troops during the first burma war prompted a rethink of many aspects of military policy , including victualling and disease prevention . most of these have been discussed extensively elsewhere , but the incidence of scurvy has gone largely unnoticed , despite evidence of growing interest in this disease in anglo - indian medical circles . this is of particular significance because the observations of anglo - indian doctors do not accord with the findings of naval practitioners such as trotter . on the contrary , indian conditions led to the emergence of a distinct body of knowledge , some of which contradicted prevailing wisdom at the admiralty . writing of the outbreak at rangoon , the east india company surgeon dr g. waddell acknowledged the effects of poor diet in causing the outbreak ( the reliance of europeans on putrid salt meats and of all races on diets deficient in vegetable matter ) , but he placed equal weight on meteorological factors . the outbreak of scurvy was attributed as much to the inclement season in which it occurred as to the deficiency of rations . waddell likened the atmosphere then to be found in rangoon to that in some of the company s troopships , in which cases of scurvy were relatively common . in these vessels , he insisted , scurvy arose primarily from exposure to impure air. predisposing factors were also important ; hence in both the company s ships and in its armies it was the laziest and filthiest who were affected first . by the early 1800s , the east india company had also established institutions such as prisons , hospitals and asylums , of which many were plagued by diseases , including scurvy . the most likely explanation for the poor health of prisoners is that many had subsisted for a long period on a diet lacking in ascorbic acid , and this was especially true in periods of dearth during which prisons tended to reach their capacity . in view of the increasing visibility of scurvy , professional bodies such as the medical and physical society of calcutta began to call for more papers on the subject . however , explanations of scurvy in these institutions were no more likely to ascribe them to an impoverished diet than accounts of outbreaks in the army . in his observations on scurvy as it occurred at the murshidabad insane asylum in 1822 , dr burt saw many similarities with the outbreak at rangoon , which he had later witnessed as a military surgeon . in both cases , the disease appeared to be caused by a combination of poor diet and impurities of the air arising from the marshes which surrounded the city . the ill - effects of the location were evident in the stubborn nature of the complaints , for scurvy proved hard to eradicate even when the army had received supplies of fresh vegetables and lime juice . indeed , the disease only disappeared once the weather became drier . as burt put it : i am inclined to suppose , from its breaking out during the rains and cold weather , and generally disappearing in the hot and dry from its attacking the gloomy and dejected that humidity and depression of spirits are the principal , if not the only exciting causes [ emphasis added ] . these depress nervous energy , and induce a torpid action of the general functions of the body . the stomach and digestive organs partaking of this diminished action , do not secrete a sufficient supply of chyle [ bile ] to nourish the body ; hence scurvy is induced . i am inclined to suppose , from its breaking out during the rains and cold weather , and generally disappearing in the hot and dry from its attacking the gloomy and dejected that humidity and depression of spirits are the principal , if not the only exciting causes [ emphasis added ] . these depress nervous energy , and induce a torpid action of the general functions of the body . the stomach and digestive organs partaking of this diminished action , do not secrete a sufficient supply of chyle [ bile ] to nourish the body ; hence scurvy is induced . burt s account had the same physiological basis as works on scurvy in the navy and , like them , it continued to acknowledge the importance of predisposing factors such as melancholia and imbecility . however , it differs substantially in that dietary deficiency was not the only or even the most important external cause : on the whole , meteorological and other environmental conditions were regarded as more significant . in some respects this is only to be expected in view of the overwhelming dominance of climatic explanations of disease in anglo - indian medicine at this time and , indeed , in contemporary accounts of the constitutions and habits of the people . although scurvy did not affect indians exclusively , it seldom occurred among europeans except when they had been subjected to abnormal deprivation . but instead of concluding that indians must be more deprived than europeans , british medical practitioners tended to attribute the disease to exposure to noxious influences . the vulnerability of europeans increasingly disposed them to seek refuge from the climate , while indians went about barely clothed . while they were less frequently prey to acute diseases , those who inhabited the least salubrious environments tended to show the progressive effects of debility . it was widely held that such influences , passed down through many generations , accounted for the degraded state of many indians in relation to europeans and in relation to the more robust natives of low , marshy areas , such as parts of deltaic bengal , exhibited this tendency towards degeneration most markedly , it was claimed . dr d. macnab , in his observations on scurvy among sepoys at nusserbad , bengal , in 1836 , thus attributed the outbreak to depleted nervous energy , possibly due to poor nutrition ( adulterated grain ) but mostly to the effects of climate . climate has always been admitted to exercise important bias over the character of disease , as well as over the constitution itself of man, he opined . in his view , the atmosphere around the cantonment was deficient in four years later , dr j. bourchier also dismissed any notion that the outbreaks of scurvy he had witnessed were due to diet . writing of an epidemic among prisoners in the rutnagherry jail in the mid-1830s , he noticed that there were no cases among female prisoners despite them receiving the same food as the men . in his view , the most likely explanation was the scarcity and poor quality of water from the wells during the summer months . this appeared to explain why scurvy became more of a problem in times of drought . what these interpretations had in common was that they drew attention away from diet and the shortages caused by famine . first , it effectively naturalised scurvy , detracting from new methods of agricultural production and related issues such as the controversial revenue settlements recently imposed by the east india company in many parts of india . as macnab put it : there are thousands , year after year , living on in poverty and wretchedness , yet when have we heard of its giving birth to scorbutic disease , without a combination of other contingencies which are of rare occurrence. second , rations for the company s soldiers , and the bengal army in particular , had often provoked unrest among sepoys , not least during the barrackpore mutiny of 1824 , which was caused in part by the failure to provide appropriate items of diet . if poor diet was officially declared the principal cause of scurvy then the company could be held responsible for this , too . these factors may help to explain why its surgeons were reluctant to regard scurvy as due to deficiencies of diet . however , those who wrote about scurvy could not always agree on what , if not diet , was the most important factor . climatic explanations tended to dominate , but some also blamed what they saw as the dissolute habits of many indians . in his 1835 paper on scurvy among sepoys stationed near cachar , bengal , the east india company s surgeon j. hutchinson placed most emphasis on the widespread use of opium , which he claimed destroyed nervous power . such explanations were also common when accounting for the high incidence of scurvy and other diseases in indian jails . as one correspondent to the india journal of medical science put it : the prisoners are most of them of the very lowest caste . they are generally men of debauched habits , who have led very irregular lives the very scum of society enter our jails . and are not people of as depraved a nature particularly predisposed to disease on being subjected to the restraints of prison ? this tendency to ascribe the disease to factors other than diet did not really change until the 1840s , for reasons which will become clear in a moment . they are generally men of debauched habits , who have led very irregular lives the very scum of society enter our jails . and are not people of as depraved a nature particularly predisposed to disease on being subjected to the restraints of prison ? if the logic of administration in india suited a view of scurvy which was broadly environmental , quite the opposite was true in britain where the problem was most evident in new penal institutions such as the national penitentiary at millbank the reformatory designed by the utilitarian philosopher jeremy bentham . it was intended as an engine for the reform of miscreants rather than a repository for the refuse of society . these new institutions were supposed to be clean and free from disease , but their regime was also harsh and unyielding . well regulated labour and religious instruction. like the workhouses which would later be introduced by the new poor law , food and other comforts were intended to be attempts to reform prisons preceded the utilitarian - inspired reforms of the prison system and stemmed from the labours of philanthropists such as john howard and sir george onesiphorus paul . however , the idea of the clean prison , free from disease , was essential to the utilitarian project . reform of inmates could not be accomplished if they were sick , for disease impaired efficiency , but not long after millbank began to reduce its luxurious rations in line with benthamite principles , disease began to show itself in the form of scurvy . the recommendation to reduce rations was made by the prison s principal medical officer dr hutchinson , who had been asked to look into the matter by the prison s management committee . according to hutchinson , a general fullness of habit appeared to prevail among all the inmates of the penitentiary , particularly the women . a reduced scale of diet was therefore advocated , following the precedent set by dorchester prison . instead of the former allowance of three - and - a - half ounces of meat and one pound of potatoes , the inmates were to receive one - and - a - half ounces of bread , two pints of broth and one pint of gruel . however , the committee first sought the opinion of sir james mcgrigor , the director general of the army medical department , presumably on account of his responsibility for the health of military prisoners . mcgrigor agreed that the old diet was too full and approved the plan proposed . in july 1822 introduction of the new diet soon led to a serious deterioration in the inmates health and the matter was brought repeatedly before the management committee and the prison s board of visitors . no substantial alteration was made , however , and the decision to persist with the ration placed dr hutchinson at odds with his subordinate medical officer , mr pratt , who was resident at the penitentiary , as well as with the matron , nurses and other officers of the establishment . by january 1823 the first definite symptoms of scurvy appeared in one of patients seen by hutchinson , and the disease became more common over the coming months among prisoners of all types and both sexes . on 14 february , mcgrigor was brought in to inspect the prisoners and , having examined all the female and many of the male convicts , he reported that they were in reasonable health , the incidence of sickness being much as he would expect for the time of year . most importantly , mcgrigor concluded that none of the sickness was due to inadequate diet or length of confinement . but within a few days scurvy began to gain ground rapidly , increasing from 53 cases in the middle of february 1823 to 118 by the end of the month . this disturbed the committee and it sought a second medical opinion from two other physicians , drs latham and roget , who were asked whether the disease could be attributed to the new rations . latham and roget visited the prison on 1 march and found 110 inmates lying in the infirmary 44 males and 66 females . alongside dr hutchinson , they inspected each of the prison s pentagonal cellblocks and found that 448 of the total of 880 inmates showed signs of disease , often a combination of scurvy and dysentery . women were more badly affected than men , on the whole , as were those who had been longest in confinement . in the opinion of drs roget and latham , the high incidence of scurvy among the inmates at millbank was due primarily to their new diet , but they also concluded that cold , overwork , insufficient exercise and depression had been predisposing factors . their opinion was largely , though not completely , welcomed by the select committee of the house of commons which was appointed in 1823 to investigate the outbreak . the appointment of such a committee is worthy of note , for it shows the seriousness of scurvy in political terms . the high incidence of disease in millbank reflected badly not only on the management of that prison but also on the new system of penal reform and , by extension , the political and economic principles on which it was based . but the fact that the select committee was willing to entertain a report critical of the penitentiary s dietary regime may seem surprising . it did so almost certainly because such a conclusion did not require a radical alteration or rejection of the penitentiary system but merely a relatively modest alteration of rations . that the select committee had such an objective in mind is strongly suggested by the line of questioning to which roget , latham and other witnesses were subjected . while they had stressed the primacy of diet as a causative factor in scurvy , like most physicians at the time they continued to acknowledge the contribution of predisposing factors , mental and physical . their insistence on these predisposing factors was less welcome to the select committee than their comments concerning diet . accordingly , the questions asked of the doctors were such as to cast doubt on all factors other than the deficiency of rations . the select committee was particularly displeased with the assertion that cold weather had contributed to the outbreak , the implication being that many of the cells afforded inadequate protection from the elements . it pointed to the fact that prisoners who worked in the kitchens suffered less from scurvy and stated its opinion that this was due to additional food rather than to better heating : a point which dr roget later conceded . the committee also pointed to the fact that few other prisons had heated cells and yet did not suffer scurvy , forcing roget to admit this , too . having elicited the opinion it desired , the committee concluded that there was no need to provide artificial heating for the cells a provision which would clearly have been expensive . roget and latham s contentions that the scurvy had been due in part to depression and exhaustion were more problematic . these observations called into question the reformatory regime itself , for it was founded on lengthy sentences and rigorous , repetitive labour . the select committee questioned the assertion that these things were causes of scurvy by again referring to the absence of the disease in other jails . it seemed oblivious to the physicians argument that it was a combination of causes rather than any single factor which was operative . however , the committee did concede that the prisoners showed signs of low spirits and recommended more exercise and a wider selection of literature for them as an antidote not merely works which had been selected to promote christian knowledge . this small concession was the only one made by the select committee other than the need to improve the prisoners diet . it had effectively closed off all alternative or even supplementary explanations for the outbreak , thus leaving the penitentiary regime intact . in the committee s view , there was nothing wrong with it that could not be fixed by a simple alteration of diet . this was not the last time that parliament would hear of scurvy in prisons or , indeed , in millbank . throughout the 1820s and 1830s scurvy was still common in some prisons , especially those in london and east anglia . the inspectors who had been appointed in the wake of the parliamentary enquiries of the early 1820s carefully recorded the views of prison surgeons , most of whom drew attention to the inadequacy of diet but also , in some cases , the contributory affects of cold weather and hard labour . the emphasis on hard labour called into question the logic of penal reform and was clearly unwelcome to advocates of the penitentiaries , but some surgeons persisted in their opinions . in 1837 , for example , the inspectors of prisons noted that the surgeon of the springfield house of correction in essex believed that the combined effect of hard labour , with inadequate and insufficiently varied diet , is further evident from the fact that no such disorder existing in the old gaol at chelmsford , which is damp , and in local respects , less healthy than springfield , but where there is no tread - mill labour. but prison doctors had no power to compel their governors to alter rations . indeed , they were charged with managing scorbutic symptoms by dietary manipulation , allowing rations to be kept to the bare minimum compatible with health . by the 1840s , although there were still occasional references to dampness and other contributory factors , scurvy was represented medically and administratively as a deficiency disease . ordered in this way , the problem of scurvy could be solved with comparative ease , for a cheap and effective remedy was at hand . dr william baly , physician to the penitentiary at millbank and lecturer on forensic medicine at st bartholomew s hospital , was one of a number of medical men who recommended the addition of potatoes to prison rations and he did so after conducting an investigation of scurvy among military prisoners at millbank . at that time , military prisoners had a lower allowance of potatoes in their diet than civilian inmates and it was this that accounted for the high prevalence of scurvy among them . there were practically no cases among civilians , the disease having been eradicated after latham and roget were permitted to alter their diet by adding potatoes . whereas civilian prisoners were allowed five pounds of the vegetables weekly , the military prisoners were permitted only half a pound , and then only after six months imprisonment . baly also examined the reports of the inspectors of prisons with a view to ascertaining whether scurvy was due to the absence of fresh vegetables . this was clearly the case in two instances the oxford and northampton county jails in which few fresh vegetables had been served . conversely , in several prisons scurvy had completely disappeared after the addition of a few pounds of potatoes to the weekly ration . according to baly , the value of the potato as a preventive of scurvy lay in the fact that it contained a high concentration of organic acids , including the citric and tartaric varieties . potatoes thus appeared to contain the same active ingredients as other well - known antiscorbutics such as citrus fruits . however , they were far easier to obtain and store , as well as being considerably cheaper to supply . in view of this , he argued , they were not only well suited to prisons and other public institutions but might also be used by the merchant navy which continued to suffer badly from scurvy . in baly s opinion , parsimony and laziness had led the merchant marine to neglect the naval practice of storing lemon juice on vessels , but potatoes might offer a cheap and practicable alternative . having considered the situation in british prisons , it is instructive to return to the subject of scurvy in the royal navy . while scurvy was no longer an operational problem in the way that it had been during the eighteenth century , naval medical officers still regularly faced the disease on convict vessels and occasionally on other long voyages and expeditions , most obviously those to the arctic . this section concentrates largely on outbreaks of scurvy on convict ships , to which royal navy surgeons were appointed . it was on these vessels that the majority of cases seen by naval surgeons occurred and on which we might expect to find similar concerns to those arising in penitentiaries . a good deal is already known about the health of ships designated to transport convicts and immigrants from britain to australia . after numerous voyages with high death rates , conditions on board such vessels improved following the imposition of regulations which stipulated the attention of surgeons and better provisions and medical supplies . on convict vessels , the problems experienced with scurvy and other diseases were more often the legacy of malnourishment and infection prior to boarding rather than conditions on the vessels themselves . from the 1820s through to the 1840s , scurvy was among the most common diseases appearing on the prison hulks at woolwich , chatham and other locations , and many prisoners were probably borderline scorbutic cases before they were transported to australia . the following discussion confirms this impression , but its chief purpose is to determine how far naval medical lore concerning scurvy had changed since the days of trotter and blane . in particular , it seeks to ascertain whether the scandals in civilian jails had much impact on the way in which scurvy was conceived and treated on convict vessels . on the eve of the outbreak at millbank , the views of naval surgeons on the causation of scurvy do not seem to have altered to any significant extent . dietary deficiency appears to have been regarded as the principal cause of scorbutic complaints but by no means the only one , and in some cases it was expressly ruled out . in 1821 , for example , the surgeon of hm convict ship lady ridley attributed the numerous cases of scurvy and debility on his vessel to the bad quality of our water which has been very putred [ sic ] for some time. scurvy continued to be a regular feature of the convict voyages and it appeared so frequently that it was seldom the object of serious attention . but this is not to say that surgeons were uninterested in the disease or that they were unaware of what had been written about it in other contexts . some had clearly read the report by roget and latham on the millbank outbreak and compared their descriptions of the disease to that seen on convict vessels . latham and roget s conclusions were not dissimilar to those espoused by the majority of naval surgeons , whose views of scurvy remained flexible and inclusive . one of the more serious outbreaks of scurvy on a convict vessel occurred in 18334 towards the end of the southworth s voyage to van diemen s land . by the time the vessel had reached hobart there were 30 on the sick list , mostly cases of scurvy or scorbutic dysentery . the surgeon , william evans , gave the afflicted convicts citric acid and nitrate of potash ( which had come into vogue as a treatment for scurvy and scorbutic dysentery ) and paid close attention to the cleanliness of the prisoners . these actions suggest that he did not regard the outbreak as due solely to dietary deficiency , but he noted that most cases were cured after they landed and were given plenty to eat . evans commented that nothing short of liberal diet of fresh animal food and recent vegetables with soft bread , can affect that necessary change in the circulatory fluids so as to re - establish health. it would therefore seem that evans regarded diet as the principal factor in the causation of scurvy , but not the only one . furthermore , there is no evidence that he thought scurvy due to the absence of any particular dietary component or substance . in his opinion , this fresh diet seems to have acted principally as a stimulant which reinvigorated the body s vital power . while most cases of scurvy treated by naval surgeons were confined to convict vessels , there were occasional outbreaks on long naval voyages . one such instance occurred on hms atholl in 1827 , during operations off the african and arabian coasts . the crew was said to have a disposition towards scurvy and three of the most severe cases died during the voyage . of these men , two were said to have very bad habits of body prior to their showing evidence of scurvy and one had suffered several attacks of fever . the surgeon , william aitken , was unsure to what he should attribute the outbreak , except unwholesome water , bad biscuit , short allowance of spirits and provisions , and deficiency of vegetable diet , as the atmosphere was pure and the sky transparent. there is nothing in aitken s report to suggest that he regarded the outbreak as due to the deficiency of a single item of diet or some principle therein . indeed , although atmospheric causes were excluded in this instance , aitken s remarks indicate a willingness to entertain such an explanation . the persistence of a multi - factorial view of scurvy is further indicated by the fact that a range of preventive measures was enjoined , ranging from the distribution of lime juice to the cleansing of drinking water with lime . in fact , throughout the 1830s and 1840s it appears that citric acid was not highly regarded as a treatment for scurvy . nitrate of potash , sometimes mixed with vinegar and sugar , was reckoned by some to be more effective . all seem to have agreed on the restorative effects of diet , especially a diet abundant in beef and fresh vegetables , but there is no indication that the problem of scurvy could be solved by the addition of any single item such as the potato . there is a clear difference between the outlook of most naval surgeons and that of the parliamentary select committee which enquired into the outbreak at millbank . the same can be said of the select committee which later examined the transportation of offenders to australia . that committee s report in 1837 extolled the virtues of the potato as a cheap and nutritious item of diet and recommended that the proportion of this vegetable in the diet of prisoners working on chain gangs and penal settlements in australia be considerably increased . some surgeons on board prison hulks in british waters tried similar expedients using carrots , but their views on the causation of scurvy and similar diseases remained more diverse and they were prepared to entertain factors such as depression . indeed , on occasion they directly criticised the regime in some of the prisons from which the convicts had been drawn prior to boarding the hulks . in his medical report for 1842 , for example , archibald robertson declared that scurvy had been one of the principal complaints among the 436 patients sent from the convict hulk fortitude to the hospital ship wye . in his view , the remote and exciting causes of the disease were to be found in the previous prison discipline , solitary confinement , and low diet of the majority of our gaols , and which render very many of our convicts more fit for the hospital than for dockyard labour. the persistence of a reasonably broad view of scurvy thus presented few difficulties for the navy or , for many years , in india , but it posed a significant threat to the new penitentiary system in britain and parts of scurvy s etiological framework were excised as a result . ultimately the politics of confinement in britain were to have consequences for india , too . growing scrutiny of the indian prison system in the wake of the british enquiries forced the east india company to take more account of the rations given to the inmates in its prisons . jails were ordered to revise their dietary scales and medical officers were assigned to devise new ones . more stress was now placed on the provision of fresh vegetables and other foods known to possess antiscorbutic qualities . by the late 1840s , some indian jails were already showing signs of improvement and some had established their own gardens specifically to provide prisoners with fresh vegetables . medical officers who continued to preside over gaols with a high incidence of scurvy were removed . although indian soldiers were still permitted to obtain most of their own foodstuffs , the rations of british soldiers were altered in accordance with what had become the dominant view of scurvy in britain . but the principal reason was that the old environmental paradigm of scurvy had been found inconvenient . rather than abandoning remote stations previously reckoned to be prone to scurvy on account of their climate , an effort was made to cultivate vegetables locally and to make up for deficiencies with extra supplies . better rations were also provided for invalids proceeding home to britain , including on vessels which did not carry the east india company s flag . what these various initiatives show is that the natural history of scurvy was closely and inextricably linked to administrative considerations , be they the principles of penal reform or the strategic implications of withdrawal from remote but crucially important outposts of empire . by the middle of the nineteenth century there was a very loose consensus around the importance of dietary factors in the causation of scurvy , but this was more narrowly construed in british penitentiaries and indian prisons than in the navy or even on board the convict vessels manned by its surgeons . in prisons , political expediency dictated a very definite view of what caused scurvy and how it was to be remedied . lacking the constraints imposed by these administrative requirements , however , practitioners in other contexts continued to speculate widely over the causes of scurvy . some still attributed it to unfavourable weather , fatigue , mental states and poor hygiene , in addition to inadequate diet , while others were later to claim that it was the consequence of bacterial infection . these widely differing accounts of the genesis and therefore the prevention of scurvy persisted into the first world war , when mounting evidence was adduced from the campaign in mesopotamia to support the view that it was caused by the lack of a single substance , eventually to be known as vitamin c.

from the late eighteenth century , the ways in which scurvy was understood changed in consequence of the abandonment of humoral pathology and the adoption of a new causal framework informed by nervous physiology . although there was some narrowing of the etiological framework around dietary deficiency in the wake of the navy s success with the issue of citrus juices , this was rarely to the exclusion of predisposing causes such as fatigue , weather and flagging spirits . within the navy , the persistence of a multi - factoral framework was relatively unproblematic , for the standard issue of citrus juices and improvements in victualling occurred at the same time as other important reforms in naval health . but outside the navy it was a different matter . in other institutional settings , the continuing belief in the importance of factors other than diet created tensions between medical officers and administrators who found such inclusive views politically inconvenient.after a brief survey of the principal changes in the physiology of scurvy , this article examines how the problem of scurvy was understood , not only in the navy but also in the armies of the east india company and in british prisons . these were not the only contexts in which scurvy caused concern , but they serve to illustrate the fact that it remained a complex and controversial disease . the article shows how different medical cultures and institutional imperatives took the natural history of scurvy in different directions .

Introduction A multifaceted disease: scurvy in the eighteenth century Scurvy in India Penitentiaries Scurvy and the nineteenth-century navy Conclusion

aggressive angiomyxoma ( aa ) is a rare benign soft tissue tumour usually affecting the pelvis and perineum of young women . magnetic resonance imaging ( mri ) is crucial in the management of aa patients for its diagnostic contribution and for the preoperative assessment of the actual tumour extension . given the current development of less aggressive therapeutics associated with a higher risk of recurrence , close follow - up with mri is fundamental after treatment . in this context , diffusion - weighted ( dw ) imaging has already shown high efficacy in the detection of early small relapses in prostate or rectal cancer . we report here a case of pelvic aa in a 51-year - old woman examined with dynamic contrast enhancement and dw - mri , including apparent diffusion coefficient mapping and calculation . to our knowledge , this is the first description of dw - mri in aa reported in the literature . here , aggressive angiomyxoma ( aa ) is a benign soft tissue tumour with myxoid and vascular components that arises mostly in the lower pelvis and perineum in women of reproductive age . most of them ( > 90% ) occurred in young women , usually in the second to fourth decade . the risks of local extension and postoperative local relapse justify surgical excision of the tumour . imaging and particularly magnetic resonance imaging ( mri ) are fundamental for the clinical management of aa . precise preoperative assessment of local extension is crucial to plan the best surgical approach for tumour removal involving as little mutilation as possible . mri allows the monitoring of the response to possible preoperative treatment such as hormone therapy . finally , it is also crucial during the patient 's follow - up after initial treatment . for a few years now , classical mri coupled with diffusion - weighted ( dw ) sequences has developed in oncology , leading to improvements in the detection and characterization of tumours , treatment response monitoring , and detection of recurrence [ 3 , 4 ] . to our knowledge , we report here the first case of pelvic aa explored with dw - mri and dynamic contrast enhancement ( dce)-mri . in july 2011 , a 51-year - old woman presented to her general practitioner with an incidentally discovered renitent mass in the internal part of the left buttock . initial ultrasound ( us ) showed a well - circumscribed mass initially evoking a lipoma . due to the large volume of the mass , she was otherwise asymptomatic with a performance status score of 0 on the who scale . us showed a superficial , soft , hypoechoic , well - limited , solid mass . mri showed a bulky perineal and intrapelvic tumour displacing rather than infiltrating the vagina , anal canal , and rectal structures . the mass was well limited , hourglass - shaped and had a maximum diameter of 11 cm ( fig . t2-weighted imaging showed typical high signal intensity with layered strands of lower signal intensity and swirled architecture . a fat linear structure of high signal intensity on t1- and t2-weighted images was present inside the mass . its significance was unknown , perhaps representing neighbouring tissues trapped when the tumour was growing . after intravenous contrast injection , the mass was greatly and heterogeneously enhanced , exhibiting a layered pattern ( fig . regions of interest located in the layers demonstrated the same profile curves of progressive enhancement with variable intensity . apparent diffusion coefficient ( adc ) mapping showed a high value in the tumour , estimated at 2 10 ( 0.15 ) mm / sec . it also showed disease extension , notably through the pelvic diaphragm , which made a combined transperineal and laparoscopic abdominopelvic approach necessary . before surgery , the examination under general anesthesia showed that the mass was soft , renitent , and seemed to come from the pelvic cavity through the levator ani muscles . tumour excision was monobloc and macroscopically complete , without any rectal or vaginal resection or tumour capsular rupture . macroscopic examination of the surgical specimen showed a firm , partly circumscribed tumour measuring 16 12 6.5 cm . a cut section showed a homogeneous , glistening , and gelatinous appearance without any hemorrhagic or necrotic areas . microscopic analysis showed a monotonous hypocellular proliferation of small nonatypical stellate - shaped cells arranged in a myxoid stroma , along with numerous , variably sized vessels . no mitosis or necrosis could be seen . in its periphery , the proliferation infiltrated the nontumour adipose tissue , and resection margins were focally involved by the tumour cells . immunohistochemical analysis showed strong expression of cd34 , and estrogen and progesterone receptors by tumour cells , without expression of desmin , cd117 , -catenine , and mdm2 , nor epithelial ( pancytokeratin , ema ) , smooth muscle ( -smooth actin , h - caldesmon ) , and schwann cell ( ps100 ) markers . after surgery , no adjuvant treatment was given , and the patient was regularly monitored clinically and radiologically . at her last visit , in october 2012 , more than 1 year after the diagnosis , the patient was alive with an excellent performance status and without any recurrence of the disease on pelvic dw - mri . aa is a compliant tumour that grows slowly in the perineal or pelvic region and can cross the pelvic diaphragm . classically , the tumour shows finger - like projections at the periphery and into the surrounding tissue without infiltrating or invading adjacent organs . in general , the tumour is well limited and large ( often more than 10 cm in diameter ) at the time of diagnosis , and its consistency is soft or renitent [ 1 , 6 ] . histologically , aa is a benign hypocellular tumour , composed of scattered spindle or stellar cells , embedded in a loose myxoid matrix with a few collagen fibers and abundant vessels of varying sizes . frequently , tumour cells show positive immunohistochemical staining for desmin , smooth muscle actin , muscle - specific actin , and hormone receptors . rare and recent molecular analyses have identified translocation in the 12q1315 region involving the hmga2 gene . aggressiveness is related to the high rate of local recurrence after surgery because of frequent incomplete excision . this can be due to the absence of a preoperative diagnosis and estimation of the actual extent of the tumour , or to a particular strategic location of the mass near the urethra , vagina , anal sphincter and rectum , with frequent extension through the pelvic diaphragm , making complete resection difficult . the formerly high rate of relapse has decreased from ~75 to 1030% during the last 20 years thanks to improved imaging techniques , which allow a more complete surgical resection . aa in men is unusual and affects the genitourinary tract especially the scrotum and inguinal region [ 1 , 4 ] . most patients present with a slow - growing palpable or visible mass in the pelvi - perineal region . other signs may include discomfort or pain due to the mass , or signs related to pressure effects on adjacent organs such as dyspareunia and dysuria . morphological imaging like computed tomography ( ct ) checks for the absence of pelvic hernia , gynaecological prolapse or myoma . vaginal cysts , gartner cysts and bartholin cysts are frequent and easy differential diagnoses that do not show significant enhancement except if infected . in that case , mri will usually show peripheral enhancement of the wall of the cyst with variable thickness , which is totally different from the aa pattern of enhancement . the lack of macroscopic fat on ct and mri sequences with and without fat suppression can rule out infiltrating angiolipoma and lipoma or liposarcoma with or without myxoid component [ 7 , 8 ] . endometriomas and melanomas show typical high signal intensity on t1-weighted images without signal intensity dropout on fat - suppressed images . myxoma is mainly an intramuscular tumour that occurs in older patients , and whose enhancement does not show the same features as aa . mri is also a helpful diagnostic tool with in - phase and opposed - phase sequences showing microscopic fat inside the angiomyofibroblastoma . other diagnoses such as myxoid neurofibroma , myxoid - type malignant fibrous histiocytoma , myxofibrosarcoma , vaginal botryoid pseudosarcoma , and rhabdomyosarcoma are made by histological analysis of the tumour . in general , mri will help with the preoperative diagnosis by showing typical images of aa , as in our case . this is all the more important because , if the final diagnosis is obviously based on histology and immunohistochemistry , a preoperative diagnosis may be difficult to obtain from a percutaneous biopsy . typical images show the lesion as a well - limited mass , displacing rather than infiltrating adjacent organs and muscles , with content rich in water , loose myxoid matrix , and hypervascularity . finger - like projections into neighbouring adipose tissue can be present and are relatively characteristic of aa . classically , us without contrast injection shows a homogeneous and hypoechoic mass , sometimes cystic , that may be misleading . ct scan shows an enhanced mass , which is iso- or hypoattenuating relative to muscle [ 7 , 11 ] . mri shows very characteristic aspects : an isointense tumour ( more rarely hypointense ) relative to muscle on t1-weighted images , and hyperintense on t2-weighted images ( high water content and loose myxoid matrix ) [ 2 , 5 , 7 ] . the tumour enhances avidly and heterogeneously after intravenous gadolinium contrast administration , reflecting its inherent vasculature [ 2 , 5 , 7 , 8 ] . a typical mri feature of aa is the presence of an internal layered pattern and/or swirled architecture , which is described in 83% of cases on t2-weighted mri or on t1-weighted mri after intravenous contrast injection [ 2 , 5 , 6 , 7 , 8 , 9 ] . our case perfectly illustrated these mri findings , allowing us to suspect the aa diagnosis before surgery . preoperative mri is also crucial to determine the true extent of the disease and plan the most optimal surgical approach . the importance of accurate preoperative diagnosis and extent screening is clearly reported . in our case , an abdominoperineal approach was chosen as the tumour extended above the pelvic diaphragm on mri , allowing complete resection . during treatment finally , mri is very helpful in order to detect local relapses during follow - up . because of its high sensitivity in detecting tumour presence , its noninvasive nature , and its ability to provide valuable qualitative and quantitative information , dwi has become indispensable for characterizing lesions , and for monitoring the response to nonsurgical therapy and the patient 's follow - up after treatment . this is already well documented for the detection of prostate or rectal cancer relapse . to our knowledge the typical architecture of the tumour was seen on t2-weighted images and b0 sequences ; however , it disappeared both as the b value increased and on adc mapping imaging even in a mass more than 10 cm in diameter . dwi also showed a high value of tumour adc measurements in the region of 2 10 mm / sec , comparable with adc calculations in myxoid tumours . the persistence of high tumour signal intensity on dwi with a high b value suggests the possibility of easy and earlier relapse diagnosis using these types of sequences . furthermore , we can hypothesize that normal mri using dw sequences during patients follow - up is probably an effective way to confirm the absence of recurrence . however , aa can theoretically be polymorphous , and more patients in larger case series are needed to confirm these first observations . due to the rarity of this disease , wide surgical resection is the treatment of choice , sometimes after preoperative angiographic embolization to decrease the size of the tumour . but , because of its frequently large size and location in close proximity to vital organs ( urogenital tract and rectum ) , wide excision is not always possible and/or may cause significant morbidity in young patients . furthermore , the prognostic value of extensive surgical resection to obtain clear margins is questioned . a review of over 100 cases published in 2000 by chan et al . showed that patients with clear resection margins have similar chances of remaining disease free ( 50% at 10 years ) compared with those having tumour - involved resection margins ( 40% ) . these observations explain that incomplete resection is acceptable nowadays if followed by long - term follow - up and careful monitoring for timely identification and prompt resection of relapse . treatment involving chemotherapy and radiotherapy has been disappointing , probably due to the low mitotic activity of the tumours [ 1 , 11 ] . aa is likely hormone dependent as suggested by the reproductive age of the patients , the few cases reported during pregnancy , and the frequent expression of hormone receptors . thus , gonadotropin - releasing hormone agonists have been administered to aa patients in the postoperative as well as in the preoperative setting with dramatic responses , facilitating surgery or avoiding mutilating surgery [ 10 , 13 , 14 ] . one case report of aa treated exclusively with gonadotropin - releasing hormone agonists was published in 2003 . two cases of successful control of recurrent aa with relatively high doses of external radiotherapy have been reported , but a literature analysis shows a definite shift towards less morbid treatments . after treatment , long - term follow - up is required since late recurrences have been described up to 14 years following treatment . recurrence rates are variable in the literature and probably depend on when patients were treated ( related to surgical and mri quality ) . . found a recurrence rate in the region of 50% between 1984 and 1998 . . otherwise , 71% of recurrences occurred within the first 3 years , regardless of the initial tumour size and patient 's age . periodic clinical examination may be insufficient and imaging studies , such as mri , are fundamental . they may detect recurrence early , when the tumour is smaller in size , making surgery easier to perform or allowing the use of less morbid percutaneous ablative therapeutic strategies such as radiofrequency ablation or cryotherapy . mri is crucial in the management of aa patients for its diagnostic contribution and preoperative assessment of the actual extent of the tumour . during eventual neoadjuvant treatment , it allows monitoring of the tumour response and , after treatment , it is very useful for detecting recurrences . here , we presented the use of mri in a typical case of aa . however , this case report is also the first description in the literature of aa with complete dce and dwi analysis . we have updated the current knowledge on aa and , furthermore , we recommend the use of diffusion sequences in the follow - up of young patients treated for aa , regardless of what type of treatment they have previously had .

introductionaggressive angiomyxoma ( aa ) is a rare benign soft tissue tumour usually affecting the pelvis and perineum of young women . magnetic resonance imaging ( mri ) is crucial in the management of aa patients for its diagnostic contribution and for the preoperative assessment of the actual tumour extension . given the current development of less aggressive therapeutics associated with a higher risk of recurrence , close follow - up with mri is fundamental after treatment . in this context , diffusion - weighted ( dw ) imaging has already shown high efficacy in the detection of early small relapses in prostate or rectal cancer.case reportwe report here a case of pelvic aa in a 51-year - old woman examined with dynamic contrast enhancement and dw - mri , including apparent diffusion coefficient mapping and calculation.conclusionto our knowledge , this is the first description of dw - mri in aa reported in the literature . here , knowledge about imaging features of aa will be reviewed and expanded .

Introduction Case Report Conclusion Introduction Case Report Discussion Conclusion

the syndrome often presents in late middle life ; however , the incidence in the elderly has almost certainly been underestimated . in a recent pathologically ascertained series of 100 cases , the mean age of onset was 60.3 years with a range of 4079 years ( hodges et al . the features of the language disturbance are captured in the recent consensus criteria for sd ( gorno - tempini et al . typically , the patient complains of word - finding difficulty and will ask the meaning of familiar words ; on examination , there is profound anomia , impaired generation of words and categories , reduced comprehension of single words and regularisation errors on reading words aloud ( for example , sounding the english word yacht as yached ) , resulting from the inappropriate use of surface rules of sound print correspondence ( surface dyslexia ) . speech articulation , phonology , syntax and prosody are intact and repetition of words and phrases and comprehension of grammatical relations ( within the limits of single word comprehension ) are normal . initially , speech is generally copious and circumlocutory but the relentless erosion of vocabulary leads ( as in other progressive aphasia syndromes ) ultimately to mutism . during the course of sd , the meaning of sensory objects is generally also lost with supervening agnosia for visual , auditory ( bozeat et al . 2010 ) , tactile ( coccia et al . 2004 ) and chemosensory ( rami et al . 2010 ) stimuli : the deficit in these cases particularly affects object recognition , or the association of the sensory percept with meaning ( associative agnosia ) , while perceptual encoding and discrimination are relatively ( or entirely ) spared . in some patients , nonverbal deficits dominate the clinical presentation : of these nonverbal presentations , the best defined is selective impairment of face recognition ( progressive prosopagnosia ) , and such cases usually develop significant aphasia later in the course . nonverbal associative agnosias can be demonstrated within the respective sensory modalities ( i.e. after removing potentially confounding effects from verbal labelling or other cross - modal recognition procedures hodges and patterson 2007 ; goll et al . furthermore , analogous kinds of deficits appear in separate modalities ( hodges and patterson 2007 ) : thus , for nonverbal objects , as for words , recognition becomes heavily dependent on familiarity and typicality , with better recognition of superordinate categories than specific exemplars and a tendency to make typicalisation errors ( the patient may identify a peacock only as a bird or later , as an animal , and may also omit to draw its distinctive tail or may represent it with four legs when asked to draw a peacock from memory the nonverbal visual equivalent of surface dyslexia ) . in cognitive neuropsychological terms , this decline into typicality and generality could be characterised as a , the development of deficits extending across modalities in sd implies an underlying essentially pan - modal defect of semantic memory . integral to the sd syndrome is initially intact ( or relatively preserved ) performance in a range of other cognitive domains , including episodic ( autobiographical ) memory and perceptual , spatial , praxic and nonverbal executive functions ( warrington 1975 ; graham et al . 2000 ; hodges and patterson 2007 ) . although ( in contrast to the behavioural variant of frontotemporal dementia ) behavioural disturbances are not usually prominent at presentation in sd , these generally do become significant as the disease evolves and include obsessionality and mental rigidity , clock - watching , preoccupation with games and puzzles , irritability and disinhibition , alterations of eating behaviour with odd food preferences , and loss of emotional understanding and responsiveness ( edwards - lee et al . 1997 ; hodges and patterson 2007 ; rohrer and warren 2010 ) . these behaviours overlap closely with the spectrum of frontotemporal dementia and are likely to arise both from impaired understanding of social signals ( for example , culturally sanctioned food combinations piwnica - worms et al . other neurological ( extrapyramidal and rarely , amyotrophic ) features may emerge later in the course of sd ( hodges and patterson 2007 ; stberg p and bogdanovic 2010 ; kremen et al . 2011 ) though these are seldom evident at presentation . patients may remain in a state of dependency for a number of years ; the median clinical disease duration to death in the large series reported by hodges et al . the differential diagnosis of sd includes other forms of primary progressive aphasia , behavioural variant frontotemporal dementia and clinical alzheimer s disease ( kertesz et al . however , the striking and disproportionate involvement of semantic memory , with loss of word knowledge despite well - preserved language structure and fluency , generally allows sd to be distinguished from these other possibilities , at least in its earlier stages . while semantic impairment does accompany alzheimer s disease ( garrard et al . the clinical syndrome of sd is associated with a characteristic neuroanatomical profile , which usually secures the diagnosis ( see fig . 1 ) . structural volumetric mri typically reveals selective atrophy of the antero - inferior temporal lobe , usually bilateral but asymmetric , and frequently more severe in the left hemisphere . atrophy is most marked in temporal polar cortex , fusiform gyrus and mesial temporal structures ( chan et al . 2001 ) with a clear antero - posterior gradient ; knife - blade atrophy at the pole gives way to relatively normal cortex in more posterior temporal and peri - sylvian regions . 2002 ) and alzheimer s disease ( chan et al . 2001 ) . as might be anticipated in an essentially sporadic disease , little information is available concerning the very earliest anatomical changes in sd ; however , longitudinal imaging using mri volumetry ( czarnecki et al . 2009 ) and tensor - based morphometry ( brambati et al . 2009 ) has shown that disease evolution is associated with extension of atrophy to inferior frontal , insular and more posterior temporal lobe cortices and to the contralateral temporal lobe , so that the less affected temporal lobe catches up over the course of the disease ; post mortem data indicate a largely symmetric distribution of atrophy ( davies et al . variants of sd as a single coherent clinic anatomical syndrome ( seeley et al . atrophy rates during the phase of early established clinical disease are sufficiently large and uniform that either whole brain or regional lobar atrophy could feasibly serve as a biomarker in future clinical trials : in terms of projected sample sizes required to detect a moderate mri treatment effect , sd compares favourably with other ftld subtypes ( rohrer et al . 1representative coronal t1-weighted mri sections of the brain of a patient with semantic dementia ( clinical disease duration 3 years ) . the sections show key structures at the level of the temporal pole ( tp ) , anterior temporal lobe ( atl ) and posterior temporal lobe ( ptl ) ; the left hemisphere is shown on the left in all sections . the sections show a characteristic profile of asymmetric focal atrophy most markedly affecting the anterior and inferior temporal lobes representative coronal t1-weighted mri sections of the brain of a patient with semantic dementia ( clinical disease duration 3 years ) . the sections show key structures at the level of the temporal pole ( tp ) , anterior temporal lobe ( atl ) and posterior temporal lobe ( ptl ) ; the left hemisphere is shown on the left in all sections . the sections show a characteristic profile of asymmetric focal atrophy most markedly affecting the anterior and inferior temporal lobes more recently , mr volumetry has been complemented by other structural and functional imaging techniques and these provide a convergent picture of regional neural network breakdown in sd . regional resting metabolic changes overlap with ( but may extend beyond ) the zone of maximal cortical atrophy ( diehl et al . 2004 ; diehl - schmid et al . 2006 ; desgranges et al . 2007 ; acosta - cabronero et al . 2011 ) ; however , task - related pet and fmri has demonstrated more widespread alterations associated with verbal semantic processing ( mummery et al . 1999 ) , reading ( wilson et al . 2009 ) , topographical recognition ( cipolotti and maguire 2003 ) and autobiographical memory ( maguire et al . 2010 ) , suggesting distributed functional reorganisation involving networks coupled to the primary semantic network centred on the anterior temporal lobes . diffusion tensor tractography suggests possible anatomical substrates for these functional changes : sd is associated with signal alterations in major white matter tracts traversing the temporal lobe ( the inferior longitudinal , arcuate and uncinate fasciculi ) and the genu of the corpus callosum but sparing the fronto - parietal superior longitudinal fasciculus ( whitwell et al . resting - state fmri has demonstrated that atrophy in sd targets a leftward - asymmetric intrinsic connectivity network linking the temporal pole , amygdala and subgenual cingulate ( seeley et al . 2009 ) : these findings align sd with an emerging paradigm of neurodegenerative disease , according to which large - scale brain networks present in the healthy brain are selectively targeted by particular neurodegenerative pathologies ( seeley 2008 ; zhou et al . 2010 ) . to date , these networks are best understood for alzheimer s disease and the behavioural variant of frontotemporal dementia , but network disintegration is likely to be of general relevance as a pathophysiological paradigm potentially explaining a range of neurodegenerative diseases . particular features of the sd syndrome have been shown to correlate with the regional distribution of atrophy . verbal deficits are especially associated with left ( dominant ) temporal lobe involvement while nonverbal deficits ( including prosopagnosia , other forms of object agnosia and impaired knowledge of living things ) have been linked to right temporal lobe involvement ( joubert et al . 2003 ; thompson et al . 2003 ; snowden et al . 2004 ; mendez et al . 2010 ; mion et al . more specifically , atrophy of the right anterior fusiform gyrus has been linked to deficits of face recognition ( josephs et al . 2008 ; omar et al . behavioural disturbances too are often more prominent in association with predominantly right - sided atrophy ( seeley et al . 2005 ; hodges and patterson 2007 ) . rather than any strict partition of functions between hemispheres or within the temporal lobe , the balance of evidence suggests disruption of an integrated bi - temporal network that mediates semantic processing across sensory modalities , with relative anatomical selectivity for particular aspects of semantic knowledge or particular knowledge modalities within this network ( snowden et al . the temporal poles ( a site of particularly severe damage in sd ) may serve a key role , perhaps by instantiating a cross - modal or amodal network hub for integration of modality - specific information into coherent concepts ( lambon ralph et al . 2007 , 2010 ) , though other candidate regions for such a hub ( in particular , the anterior fusiform gyrus ) have been advanced ( mion et al . precise one - to - one mappings between regional atrophy and cognitive deficits are often difficult to substantiate : for example , the severity of hippocampal atrophy in sd is comparable to that seen in alzheimer s disease ( chan et al . 2001 ) , yet episodic memory deficits in sd ( in contrast to alzheimer s ) are generally not a significant clinical issue . the key to resolving this apparent paradox is likely to lie with the network basis of sd ( and other neurodegenerative disorders ) : thus , hippocampal atrophy in sd is associated with atrophy of an anterior network focussed on surrounding neocortical areas in the temporal and inferior frontal lobes , whereas hippocampal atrophy in alzheimer s is associated with breakdown of a posteriorly directed network including the posterior cingulate gyrus ( pereira et al . among the syndromes in the ftld spectrum , the histopathological substrate for sd is one of the best defined : at pathological examination , approximately 75% of cases have features conforming to subtype 1 in the original sampathu classification of the tdp-43 proteinopathies or type c in the recently proposed consensus classification system ( sampathu et al . this subtype is characterised by the presence of numerous , long tortuous dystrophic neurites staining positive for tdp-43 in superficial cortical laminae ( predominantly layers ii and iii ) with sparse neuronal cytoplasmic inclusions and few or no neuronal intranuclear inclusions . an important minority of cases of sd have alternative histopathological and molecular associations , including other tdp-43 proteinopathies ( stberg p and bogdanovic 2010 ) , mutations in the gene coding microtubule - associate protein tau ( bessi et al . 2010 ) , classical pick s disease ( hodges et al . the aphasia associated with mutations in the progranulin gene may resemble sd but can generally be distinguished clinically based on the presence of grammatical and phonological errors and associated parietal signs ( rohrer et al . 2010 ) . however , such cases underline the need for more precise diagnostic criteria , both to identify the core syndrome of sd and to distinguish it from overlap and halo syndromes . this issue is not merely of academic interest : while the relatively specific mapping between clinical syndrome , anatomical profile and tissue pathology makes sd an attractive target for future trials of disease modifying therapies , the success of such therapies will depend on accurate prediction of the target disease process ( kertesz et al . 2010 ) . here , we argue that the design and evaluation of rational therapies for sd will depend ultimately on a detailed characterisation of the pathophysiology of the syndrome , and that this in turn will require an understanding of the phenomenology of regional network dysfunction and the mechanisms that translate the molecular lesion of sd to a large - scale ensemble of neurones . at present , these remain elusive goals . from a phenomenological perspective , the essential characteristics of the sd syndrome are well understood but the cognitive basis for the syndrome has not been fully defined and unresolved problems remain . these chiefly concern the scope of the syndrome , the relations between the component deficits and the boundaries of the core deficit . thus , sd affects all modalities of object knowledge , yet certain self - contained , specialised symbolic systems ( in particular , mathematics and music ) may be relatively spared ( hailstone et al . ; julien et al . 2008 ; weinstein et al . 2011 ) , suggesting that the core deficit lies with world - based ( referential ) knowledge rather than truly global semantic failure . at least earlier in the course of sd , the erosion of semantic function is not uniform across modalities ( for example , verbal semantic deficits generally lead nonverbal semantic deficits : hodges and patterson 2007 ) nor always within modalities ( category effects , though uncommon , are documented lambon ralph et al . it is uncertain to what extent these effects reflect the testing procedures used to document the deficits , the intrinsic cognitive demands of processing in different semantic domains or the underlying disease process . however , this evidence suggests at least some neuropsychological differentiation within the damaged semantic system . despite the emphasis on semantic impairment , deficits of apperceptive processing ( structural representation of objects ) have been reported in sd for both the visual and auditory modalities ( joubert et al . 2010 ) : it is not clear whether such deficits simply reflect spread of disease to adjacent cortical areas mediating apperceptive processing or point to a more fundamental disruption of object analysis at the interface between perceptual and associative mechanisms . while the balance of evidence supports network dysfunction as a key mechanism in the pathogenesis of sd , the anatomical status of the putative culprit network is less well established than ( for example ) the default mode network implicated in the pathogenesis of alzheimer s disease . taken together with the neuropsychological data ( in particular , apparent disparities between verbal and nonverbal deficits ) , this has led to the suggestion that two or more separate networks are essentially involved in sd , with implications for the nosology of the syndrome in relation to the progressive aphasias ( mesulam et al . these issues may only be settled by joint neuropsychological and neuroimaging ( especially , functional imaging ) experiments designed to test specified hypotheses about the network organisation of object knowledge and semantic memory more broadly . from a molecular perspective , the mechanisms by which abnormal proteins lead to selective neuronal destruction remain largely unknown , though a number of candidates have been identified . axonal transport deficits might reduce growth factor supply to projection neurones resulting in spreading axonal degeneration ( salehi et al . 2009 ) , or abnormal proteins might themselves spread transynaptically between neurones ( as in the case of prion protein abnormally folded tau has been shown to induce abnormal folding in nearby tau molecules ( frost et al . information concerning the in vivo behaviour of tdp-43 remains limited ; however , it is likely to play a key role in gene transcription and splicing regulation , in addition to other cellular processes including apoptosis , cell division , mrna stabilisation , regulation of neuronal plasticity and nuclear these activities of tdp-43 could potentially have downstream effects via loss of function or toxic gain of function mechanisms . if a cortical convergence zone or hub were affected by the molecular process , this strategic localisation would tend to amplify its effects with rapid disintegration of an entire connected neuronal system ( seeley et al . taking these caveats and uncertainties into account , we outline a pathophysiological model of sd which makes certain key assumptions . , we assume that the sd syndrome arises from disintegration of a distributed neural network with specific intrinsic anatomical and functional connectivity . we further assume that semantic information about objects and concepts is distributed within this network . for present purposes , we do not assume a particular form of semantic network organisation amongst the several which have been proposed ( for a review see lambon ralph et al . however , the model should generate two key features of the sd syndrome : the progressive neuropsychological erosion of fine - grained object concepts with each of these features could arise if a gradient of disease - related damage were established following an initial stochastic insult ( or insults ) within the network . here , we make a key assumption that the pathological process acts trans - synaptically and spreads in an anatomically restricted manner via projection neurones linking regions within each temporal lobe as well as homologous regions in both temporal lobes . in this scenario , the most heavily connected network elements ( e.g. convergence zones in the anterior temporal lobe ) that are most likely to integrate detailed ( feature - rich ) semantic representations would be relatively more vulnerable to the effects of the instigating insult . in contrast , more remote network elements ( e.g. those in the posterior ipsilateral and contralateral temporal lobes ) would be relatively less vulnerable . this feature of the model is in line with recent empirical data in sd ( acosta - cabronero et al . the schematic represents a distributed neural network in both temporal lobes , linked via commissural fibres ( c ) ; the temporal lobes are outlined in coronal section ( compare fig . 1 ) and stylised left temporal lobe is the more affected ( atrophic ) but the model does not assume a particular lateralisation . an initial ( stochastic ) neurodegenerative event ( here indicated as cross - hatching at location i ) propagates trans - synaptically through the network . the schematic shows a single instigating neurodegenerative focus ; however , this could be multifocal . as it traverses successive synaptic relays ( here coded as grey within the initially maximally affected temporal lobe , and black in the contralateral temporal lobe ) : though the instigating molecular lesion has not been characterised precisely , such a dilution could in principle result from spread of a toxic molecule or loss of function effects ( e.g. synaptic dysregulation or loss of trophic support ) . trans - synaptic processes are assumed to act bidirectionally ( i.e. potentially both anterograde and retrograde ) ; however , in general there will be a net direction of effects according to whether the triggering event is local or remote from the involved network element . the overall result of the process is a gradient of neural damage across the network . at a given disease stage , areas with high synaptic convergence ( locations ii and iii ) are more severely affected by the neurodegenerative process ( represented as unfilled circles ) , establishing a gradient of atrophy within the temporal lobe . in addition , there is an inter - hemispheric gradient of damage manifesting as asymmetric temporal lobe atrophy . a precise mapping of commissural pathways between homologous areas in opposite temporal lobes and mirrored network connectivity patterns in each temporal lobe are together assumed to recapitulate the sequence of regional atrophy evolution in the initially less affected temporal lobe a pathophysiological model of semantic dementia . the schematic represents a distributed neural network in both temporal lobes , linked via commissural fibres ( c ) ; the temporal lobes are outlined in coronal section ( compare fig . 1 ) and stylised neurons represent different elements of the network . in this representation , the left temporal lobe is the more affected ( atrophic ) but the model does not assume a particular lateralisation . an initial ( stochastic ) neurodegenerative event ( here indicated as cross - hatching at location i ) propagates trans - synaptically through the network . the schematic shows a single instigating neurodegenerative focus ; however , this could be multifocal . diluted as it traverses successive synaptic relays ( here coded as grey within the initially maximally affected temporal lobe , and black in the contralateral temporal lobe ) : though the instigating molecular lesion has not been characterised precisely , such a dilution could in principle result from spread of a toxic molecule or loss of function effects ( e.g. synaptic dysregulation or loss of trophic support ) . trans - synaptic processes are assumed to act bidirectionally ( i.e. potentially both anterograde and retrograde ) ; however , in general there will be a net direction of effects according to whether the triggering event is local or remote from the involved network element . the overall result of the process is a gradient of neural damage across the network . at a given disease stage , areas with high synaptic convergence ( locations ii and iii ) are more severely affected by the neurodegenerative process ( represented as unfilled circles ) , establishing a gradient of atrophy within the temporal lobe . in addition , there is an inter - hemispheric gradient of damage manifesting as asymmetric temporal lobe atrophy . a precise mapping of commissural pathways between homologous areas in opposite temporal lobes and mirrored network connectivity patterns in each temporal lobe are together assumed to recapitulate the sequence of regional atrophy evolution in the initially less affected temporal lobe a critical ( and still undetermined ) feature of the model is the nature of the underlying molecular lesion that allows the putative tissue gradients to become established . this lesion could in principle act via a toxic gain of function or loss - of - function mechanism : axonal transfer of a toxic molecule , synaptic dysregulation or loss of trophic support could allow a gradual trans - synaptic spread of disease restricted to target projection elements within the network . in this regard , we note that cortical laminae ii and iii ( a major focus of pathology in the most common histopathological substrate of sd ) are key targets for inter - hemispheric cortico - cortical afferent fibres , and lamina iii is the principal source of cortico - cortical efferent fibres ( miller 1996 ) . we suggest that a disease process with these characteristics could produce a graded and anatomically selective atrophy profile like that found in sd . one strong ( and empirically testable ) prediction of this model is a precise sequential spread of atrophy mirrored between homologous subregions in each temporal lobe : empirical data , though limited , are consistent with such a longitudinal atrophy profile ( czarnecki et al . this mirroring process would be facilitated by inter - hemispheric disease spread ( i.e. via commissural pathways ) ; however , involvement of commissural pathways need not be the sole mechanism . if network connectivity is itself mirrored in each temporal lobe , this would ensure that the sequence of disease spread is recapitulated in the initially less affected temporal lobe without necessarily invoking inter - hemispheric effects . this principle could also account for a similar pattern of atrophy evolution arising from different kinds of molecular lesions ( i.e. different histopathlogical substrates ) , in line with the pathological heterogeneity of the sd syndrome . it follows that network morphology , rather than the precise histopathological insult , would be the key determinant of the macroscopic and clinical manifestations of sd . more speculatively , intrinsic asymmetries of network organisation in the left and right temporal lobes might account for the over - representation of cases with leftward asymmetric temporal lobe atrophy ( though this is potentially attributable to various factors , including ascertainment bias ) . aside from being a promising candidate target for future clinical trials and a compelling experiment of nature , sd is a key model system for understanding how neurodegeneration translates to regional brain dysfunction . the essential linkage in this pathophysiological cascade appears to be neural network disintegration . if indeed sd is a specific network - opathy , future progress in understanding and ultimately treating this focal dementia will require a detailed analysis of the nature of the network dysfunction and the mechanisms by which such specific dysfunction could arise within a neuronal ensemble . this enterprise will require a multidisciplinary approach drawing on neuropsychology , structural and functional brain imaging and molecular physiology . we anticipate that the fusion of these traditionally disparate levels of analysis will herald a paradigm shift in neurodegenerative disease research and a new era of rational therapies .

semantic dementia ( sd ) is a unique syndrome in the frontotemporal lobar degeneration spectrum . typically presenting as a progressive , fluent anomic aphasia , sd is the paradigmatic disorder of semantic memory with a characteristic anatomical profile of asymmetric , selective antero - inferior temporal lobe atrophy . histopathologically , most cases show a specific pattern of abnormal deposition of protein tdp-43 . this relatively close clinical , anatomical and pathological correspondence suggests sd as a promising target for future therapeutic trials . here , we discuss outstanding nosological and neurobiological challenges posed by the syndrome and propose a pathophysiological model of sd based on sequential , regionally determined disintegration of a vulnerable neural network .

Clinical Features Brain Anatomy: Structure and Function Histopathology Outstanding Difficulties A Pathophysiological Model of Semantic Dementia Future Directions

breast cancer is a heterogeneous group of diseases which deviate from each other in natural history , morphology , molecular phenotype , clinical and radiological manifestations , and prognosis . prognostic parameters are essential for predicting the outcome and response to therapy in individual cases . the long list of more or less powerful prognostic parameters that includes patient age , mode of detection , tumor size , histologic grade , lymph node status , and presence or absence of distant metastases was recently widened with molecular tumor phenotypes assessed with either genetic tests or immunohistochemistry . since the number of therapeutic options is rather limited , the parameters for which assessment is routinely required for therapeutic decisions are also few . whereas hormone receptor status , her-2 status , and proliferative activity are the major determinants of oncological therapy , proper characterization of the subgross morphology of breast carcinoma is essential for planning appropriate surgery and radiation therapy [ 14 ] . the prognostic significance of subgross parameters is also observed [ 1 , 4 , 5 ] . for correct subgross characterization of a case , the following parameters should be assessed : tumor size ( defined as the largest diameter of the largest invasive focus ) , lesion distribution ( unifocal , multifocal , or diffuse distribution of the invasive and in situ tumor components ) , disease extent ( corresponding to the tissue volume containing all the malignant structures within the breast ) , intratumoral or intertumoral heterogeneity , and the position of the tumor within the breast . these parameters can be assessed with radiological and histopathological methods , the most efficient being a combination of these methods in the form of detailed and systematic radiological - pathological correlation [ 510 ] . an applied histopathology method substantially influences the success rates of documenting and assessing this subgross morphological parameters and correlating them to radiological findings . the traditional small block sampling method is based on taking 1 - 2 cm sized samples from breast specimens , often under the control of only the pathologist 's naked eye and sometimes using radiological guidance . this way , the specimen is fragmented and the interrelationship of the different tumor components , which are not represented in the same block , is destroyed . taking large numbers of small blocks , sequential numbering of the blocks , and marking the sample placement on a macrophotograph of the specimen or in the specimen radiograph represent attempts to compensate for the obvious limitations of the sampling method . at the same time , these attempts are proof that such compensation is necessary . large - format histopathology is based on embedding and processing contiguous tissue slices representing the entire cross section of a segmentectomy specimen , preserving the interrelationships of the components of the tumor , and documenting them together in one plane . this advantage makes this method the best approach in correctly assessing the subgross morphological parameters , which also facilitates the detailed radiological - pathological correlation [ 5 , 6 , 810 ] . this technique has been successfully adapted to the needs of busy routine laboratories and the procedure has been repeatedly described in detail [ 5 , 6 , 1113 ] . the advantages of this method have also been observed in a recent cost - benefit analysis . defined as the tumor volume containing all the actual malignant structures within the breast , the extent of disease is the most important subgross parameter influencing the feasibility of breast - conserving surgery in an actual case . this is the volume of breast tissue the surgeon aims to remove within certain margins in order to prevent local recurrences . disease extent that is 40 mm in the greatest dimension is associated with an approximately three - fold risk of ipsilateral local recurrence after breast conserving surgery and irradiation compared with those cases with disease extent limited to a volume of < 40 mm [ 4 , 16 ] . in addition , patients with extensive disease ( 40 mm in the largest dimension ) have significantly decreased long - term disease - specific survival compared with those with tumors of limited extent . all this underlines the importance of correctly assessing the subgross morphological prognostic parameter . in everyday routine , the pathologist should begin the analysis of a case by recapitulating the radiological findings , including the radiological disease extent . the next step should be comparing the uncut specimen with the whole specimen radiograph and keeping the in vivo orientation of the specimen by inking it at its margins . breast cancer is a lobar disease most often involving parts of a single sick lobe [ 18 , 19 ] . the lobe is a pyramid - like structure , with the lactipherous duct opening in the nipple , branching in the direction of the pectoralis muscle , and ending up in a large number of terminal units . in order to demonstrate the largest cross - section of the involved lobe , the segmentectomy specimen has to be sliced into 3 - 4 mm slices parallel to the pectoralis fascia , but not perpendicular to it . the perpendicular slicing method leads to a substantial underestimate of the extent of the disease in the vast majority of ductal carcinoma in situ cases [ 20 , 21 ] . the space the malignant structures occupy in the breast rarely shows the regular shape of a geometric body ; it is almost always irregular . this means that the borders of this space are different at different levels of the specimen and in different projections . consequently , the area representing the cross section of this tissue space in the tissue slices of the specimen also varies . for correct visualization of the real disease extent , the slice with the largest disease area should be chosen ( based on the specimen radiograph and macroscopy ) , embedded , and processed ; but additional levels should also be embedded because some components of the disease may not be visible on imaging and macroscopy . approaching from the periphery of the section , the pathologist should mark the most peripheral malignant structures ( in situ or invasive ) and repeat the process from all directions . the result will be a marked area representing a cross section through the diseased tissue . summarizing the findings in adjacent tissue slices and/or tissue slices taken at different levels of the specimen is often necessary . the realistic aim of the disease extent assessment is an appropriate categorization of the tumor as of extensive ( occupying a space 40 mm ) or limited ( occupying a space < 40 mm ) extent rather than achieving millimetric concordance of the radiological and the histological extent . in a consecutive series of 1000 newly diagnosed breast cancers in our material ( central hospital falun , sweden , period dec 2007 to jun 2012 ) , 495 cases were extensive and occupied a tissue volume of 40 mm in the greatest dimension and 505 were nonextensive occupying smaller tissue volumes . purely in situ carcinomas together with microinvasive ( < 1 mm , 4 cases ) tumors comprised 14% ( 144/1000 ) of the series , and half of the cases were extensive ( 48% , 69/144 ) and half were nonextensive ( 52% , 75/144 ) . early invasive carcinomas ( 114 mm ) comprised 35% ( 349/1000 ) of the series ; 42% ( 146/349 ) were extensive , and 58% ( 203/349 ) were nonextensive . in more advanced cancers ( 15 mm in size , 50% of the series , 500/1000 ) , 55% ( 273/500 ) of the cases were extensive ( table 1 ) . after the extent of the disease is characterized as described in table 1 , the pathologist should judge whether the lesions within the tissue area are individual ( well demarcated and separate from each other ) or confluent ( inseparable ) . this judgment is easier if the invasive tumor component(s ) and the in situ component(s ) are assessed separately . a simple practice of encircling the separable invasive foci with one color and the separable in situ foci with another color is helpful . while characterization of the foci requires microscopic control , the judgment of lesion distribution after the individual lesions are marked must be carried out using a naked eye examination of the large - format histology sections , without using a microscope . on the preoperative tumor board , the pathologist should register the radiological lesion distribution and plan the dissection of the specimen on the basis of this information . radiologically unifocal lesions are usually properly represented in one or two large - format histology sections , provided that one of these contains the tumor at its largest cross - section . in radiologically multifocal cases , several slices should be embedded to visualize as many tumor foci as possible . in radiologically diffuse cases , the most important task is to visualize the correct extent of the disease and , if the diffuse component is in situ , to catch the radiologically or macroscopically evident invasive component(s ) . our previously published system is the only one that takes into account both the invasive and in situ components of the tumor and defines their distribution both individually and in combination . in addition , our system recognizes the diffuse distribution of both the in situ and invasive tumor components , in contrast with other systems described in publications on breast cancer multifocality [ 1 , 23 ] . in our system , invasive lesions are considered unifocal if only one invasive focus can be observed that is well delineated and may or may not contain an in situ component . multifocal invasive lesions are characterized by the presence of multiple , well - delineated , invasive tumor foci separated from each other by uninvolved breast tissue containing normal tissue , benign lesions , or in situ carcinoma , regardless of the distance between the invasive foci . tumors dispersed over a large area with no distinct tumor mass , for example , like a spider 's web , are classified as diffuse . in situ carcinomas are regarded as unifocal if they appear to involve a single terminal ductal lobular unit ( tdlu ) or several neighboring tdlus . in situ carcinomas are designated multifocal if they involve several distant tdlus with uninvolved breast ducts and tdlus in between and as diffuse if they mainly involve the larger ducts . the distribution of the invasive and in situ components is then combined so that a diffuse distribution of either the in situ or the invasive component qualifies the lesion to be categorized as diffuse . typical cases with unifocal , multifocal , and diffuse in situ and invasive breast carcinomas are illustrated in figure 1 . as shown in figure 2 , there are 17 different combined distribution patterns in breast carcinomas ( unifocal , multifocal , diffuse , or missing in situ component combined with the same invasive categories , plus a mixed category ) . although the combined pattern of lesion distribution in breast carcinomas is not always easy to assess , and higher levels of interobserver reproducibility may require substantial experience , the combinations reduce the 17 different pattern possibilities to 3 aggregate patterns . multifocality is often described in the literature as the presence of satellite tumors around and in the vicinity of a dominant mass . although this situation is common , the concept is erroneous because there are cases with multiple tumor foci of approximately the same size , without the presence of a dominant mass . these foci may be dispersed over a large area without the tendency to concentrate around one foci . with regard to their evolution , two different types of multifocal invasive cancer may exist : one with multiple individual invasive foci , which develops from in situ lesions at different parts of the same lobe simultaneously or with a time difference , and one in which the individual foci represent in transit metastases of a primary focus and are not related to an in situ component . the cases in our series of 1000 breast carcinomas showed the following combined lesion distribution : unifocal in 36% ( 366/1000 ) , multifocal in 35% ( 347/1000 ) , and diffuse in 28% ( 280/1000 ) , as shown in table 1 . in situ carcinomas , including 4 cases of microinvasive tumors , were unifocal in 31% ( 44/144 ) , multifocal in 28% ( 42/144 ) , and diffuse in 41% ( 58/144 ) of cases . the majority ( 68% , 236/349 ) of the early invasive cancers ( < 15 mm in size ) had a unifocal invasive component , but when the combined morphology of the in situ and invasive components was taken into account , the majority ( 60% , 209/349 ) were in fact multifocal or diffuse . approximately one - third of more advanced ( 15 mm in size ) breast carcinomas ( 36% , 182/500 ) had unifocal combined ( in situ plus invasive ) morphology , one - third ( 38% , 189/500 ) had multifocal , and the remainder ( 25% , 129/500 ) had diffuse - combined lesion distributions , mainly because the diffuse in situ component ( figure 3 ) . these data are in full agreement with our previously published results [ 22 , 2730 ] and are similar to the results of other studies based on an analysis of large - format histology slides [ 31 , 32 ] . testing the prognostic significance of the lesion distribution defined above has resulted in clear separation of the unifocal , multifocal , and diffuse tumors with regards to the invasive component , the in situ component , and the combined distribution . patients with multifocal or diffuse invasive carcinomas have a more than double risk of lymph node metastasis compared with unifocal tumors [ 22 , 2830 , 33 , 34 ] , and the differences are related to macrometastatic disease . differences in disease - specific survival are also evident ; patients with diffuse invasive or diffuse combined tumor growth patterns have a worse outcome , those with multifocal disease an intermediate outcome , and those with unifocal tumors have the best long - term outcome . a worse survival of patients with multifocal tumors was also observed in both early and recent studies [ 23 , 37 ] . by stereomicroscopic examination of large - format thick histological sections , foschini at al . demonstrated that the distance between the individual foci of some low - grade in situ carcinomas is more than 20 mm indicating the possibility that these foci are located within different lobes . although some breast lobes are large and widespread , synchronous or asynchronous development of a carcinoma in different lobes of the same breast is a real possibility . these multilobar / multicentric cases are regularly associated with multiplicity of tumor foci and with large disease extent . in practice , the above described rules of assessing the lesion distribution and disease extent are also applicable in the multilobar cases . tumor size is defined as the largest diameter of the largest invasive tumor focus and represents one of the most powerful prognostic parameters , a constituent of the tnm staging system . many studies document its prognostic significance , and the larger the tumor , the purer the prognosis . this represents the basis for the success of mammography screening by finding tumors at an earlier stage of their natural history when they are still small , improving the overall prognosis of breast cancer patients in the screened population . in addition to purely in situ carcinomas , microinvasive cancers , which have invasive foci < 1 mm , and invasive carcinomas < 15 mm belong to the category of early breast carcinomas [ 39 , 40 ] . patients with these tumors have an excellent , over 90% , 10-year disease - specific survival and , provided that they are detected by mammography screening , the overall survival of these patients does not differ from the survival of age - matched women in the general population . forty nine percent of cases in our material were classified in this category : 14% ( 144/100 ) were in situ and in situ with microinvasive cancers , and 35% ( 349/1000 ) were invasive carcinomas of < 15 mm . patients with these tumors have less favorable survival outcomes compared with early breast cancer cases [ 39 , 40 ] . the proportion of cases in our material classified in this category was 51% ( 500/1000 unifocal , multifocal , or diffuse cases plus 7 cases with mixed growth patterns ) ( table 1 ) . determining tumor size is a complex task . breast cancers are often irregular in shape , such that the largest diameter of their nongeometric body varies in different projections . during the dissection , the pathologist should attempt to slice the specimen so that the cross section with the largest diameter of the tumor can be visualized ( see figure 4 ) and to document it in its entirety in a large section , without fragmenting the tumor . embedding slices at different levels of the specimen and summarizing the findings in different slides are as important as in determining the extent of the disease . radiological methods , especially modern ultrasound and magnetic resonance imaging , provide an accurate measure of the size of the tumor in several projections . the main shortcoming of these otherwise very accurate measurement methods is that they do not always distinguish in situ and invasive parts of the same tumor ; because of this , the histologically verified tumor size may deviate from the radiological one . obvious additional discrepancies between the radiological and histological tumor size may be the result of preoperative neoadjuvant therapy , but can also be caused by an erroneous choice of the embedded slice during the dissection or result from a failure in the radiological - pathological correlation . there is no international consensus about measuring tumor size ; for example , as a size restricted to measuring the tumor body or including the invasive extensions ( spiculations ) . because the spiculations may be long but are usually thin , they contain invasive cancer representing only a minor part of the tumor burden . including such extensions when measuring tumor size may lead to an overestimate of the tumor burden . the aim of the tumor size measurement should be to categorize the case as early ( < 15 mm in size ) or more advanced , rather than to expect a millimetric concordance of radiological and histopathological findings . radiological - pathological correlation is essential for diagnosing breast carcinoma and in assessing the subgross morphological prognostic parameters listed above . a pathologist who is not familiar with the radiological findings when processing a preoperative biopsy or an operative specimen is more likely to make mistakes . testing the concordance between the radiological and histological findings is not a matter of just comparing the values provided by these methods . the breast is hanging during the magnetic resonance imaging examination and the antero - posterior axis of the breast becomes transiently longer than when the patient is in an upright position . during mammography , the breast is compressed to a certain level , and the cranio - caudal axis becomes shorter . the breast tissue is much softer than the tumor itself and is easily deformed when placed on the firm surface of a transport plate or the bottom of a formalin - filled dish . formalin fixation will cause shrinkage of the specimen , but deformation of the specimen during fixation in a dish of inadequate size may cause much more obvious discrepancies . the most common cause of discrepancies is , however , failure in the radiological - pathological correlation . it uses different imaging modalities for the same lesion , which when combined can compensate for the limitations of the results of the individual methods . tables 2 and 3 show our preliminary results regarding tumor size measurement with the imaging methods of mammography plus ultrasound versus magnetic resonance imaging as compared with the findings in large - format histological sections . as mentioned previously , it is not realistic to expect a perfect millimetric concordance of the radiological and the histological values ; rather , the findings should be categorized in clinically important groups , like early versus more advanced breast cancer , or nonextensive versus extensive tumors . the concordance analysis only means comparing the results without naming a gold standard method ; histopathology is as likely to underestimate or overestimate the subgross parameters as the radiological methods . concordant results were reached in at least 80% of our cases when the cases were categorized by tumor size into early and more advanced categories ( table 2 ) . similar levels of concordance were reached when diagnosing extensive tumors . however , a substantial proportion of cases characterized radiologically as nonextensive turned out to actually be extensive in the histological examination ( table 3 ) . these discrepant cases corresponded to radiologically occult , most often noncalcified , low - grade multifocal or diffuse in situ carcinomas ( 72/162 cases ) or to radiologically occult , most often < 5 mm in size , invasive tumor foci ( 78/162 cases ) . very rarely , large diffuse invasive breast carcinomas were radiologically occult or manifested with nonspecific signs . the magnetic resonance imaging - large - format histopathology correlation of a case of breast carcinoma with multifocal invasive and diffuse in situ components is shown in figure 5 . although up to 70% of invasive tumors have only an unifocal invasive component , most breast carcinomas have a complex morphology when the distribution of the in situ and invasive components are combined . half of breast cancer cases are extensive and occupy a tissue volume measuring 40 mm in the greatest dimension . tumor size , disease extent , and lesion distribution are essential parameters for planning appropriate therapy and also have very significant prognostic power . proper assessment of these parameters requires additional effort from the pathologists , including a detailed and systematic radiological - pathological correlation in every case of breast cancer .

breast cancer subgross morphological parameters ( disease extent , lesion distribution , and tumor size ) provide significant prognostic information and guide therapeutic decisions . modern multimodality radiological imaging can determine these parameters with increasing accuracy in most patients . large - format histopathology preserves the spatial relationship of the tumor components and their relationship to the resection margins and has clear advantages over traditional routine pathology techniques . we report a series of 1000 consecutive breast cancer cases worked up with large - format histology with detailed radiological - pathological correlation . we confirmed that breast carcinomas often exhibit complex subgross morphology in both early and advanced stages . half of the cases were extensive tumors and occupied a tissue space 40 mm in its largest dimension . because both in situ and invasive tumor components may exhibit unifocal , multifocal , and diffuse lesion distribution , 17 different breast cancer growth patterns can be observed . combining in situ and invasive tumor components , most cases fall into three aggregate growth patterns : unifocal ( 36% ) , multifocal ( 35% ) , and diffuse ( 28% ) . large - format histology categories of tumor size and disease extent were concordant with radiological measurements in approximately 80% of the cases . noncalcified , low - grade in situ foci , and invasive tumor foci < 5 mm were the most frequent causes of discrepant findings .

1. Introduction 2. Documenting the Extent of the Disease 3. Assessing Lesion Distribution 4. Documenting Tumor Size 5. Radiological-Pathological Correlation in the Multimodality Imaging Era 6. Conclusions

slx4 is a scaffold protein that binds to three dna repair endonucleases , mus81-eme1 , xpf - ercc1 , and slx1 ( andersen et al . , 2009 , , 2009 , muoz et al . , 2009 , saito et al . , 2009 , the slx4 complex is required for the efficient repair of dna interstrand crosslinks ( icls ) , ( fekairi et al . , 2009 , 2009 ) , and the available evidence strongly suggests a role in processing dna recombination intermediates during icl repair . the importance of slx4 for icl repair was underscored by the findings that biallelic mutations in slx4 in humans causes fanconi anemia ( fa ) ( kim et al . many dna repair proteins form subnuclear foci at sites of dna damage , but slx4 overexpressed in epitope - tagged form localizes to subnuclear foci even without dna damage ( svendsen et al . , 2009 ) . it was suggested that these foci correspond to telomeres , regions of repetitive dna at chromosome ends , which protect the ends from degradation ( svendsen et al . , 2009 ) . telomeres terminate in an overhang that is thought to invade adjacent duplex telomeric repeats to form a telomeric ( t ) loop so that the chromosome ends are not perceived as dna breaks . an additional layer of telomere protection is afforded by a multiprotein complex called shelterin , that binds to telomeric dna ( palm and de lange , 2008 ) . in normal somatic cells , telomeres shorten with every cell division , and telomere shortening contributes to organismal aging by limiting the proliferative capacity of adult stem cells ( blasco , 2007 ) . in immortalized cells and in cancers , telomere length is maintained by telomerase , a reverse transcriptase that can add telomere repeats with the aid of an associated rna template ( greider and blackburn , 1989 , mocellin et al . , 2013 ) . some other immortalized cells , cancer cells , and even normal somatic cells can lengthen telomeres in a telomerase - independent manner using the alt ( alternative lengthening of telomeres ) pathway , which probably involves recombination ( bryan et al . first , when overexpressed in a special clone of hela cells with abnormally long telomeres slx4 forms subnuclear foci that colocalize with shelterin ( svendsen et al . , 2009 ) . second , slx4 was identified in a global screen for proteins that bind telomeres ( djardin and kingston , 2009 ) . also , slx4 promotes telomere cleavage in cells lacking the rtel1 helicase ( vannier et al . , 2012 ) . in rtel1 cells , replisomes stall in telomeres leading to cleavage of t loops and the production of extrachromosomal t circles , which is mediated by the slx4 complex ( vannier et al . , 2012 ) . although these observations link the slx4 complex to telomeres , it is not known whether endogenous slx4 localizes at telomeres , if this is applicable in cells that differ in the mechanisms used to maintain telomere length , or how slx4 is recruited to chromosome ends . furthermore , in rtel1-proficient cells it remains to be determined if slx4 has a physiological role to play in telomere homeostasis . here we first tested if endogenous slx4 localizes at telomeres . using antibodies raised in - house , we found that endogenous slx4 forms subnuclear foci in u2os cells without addition of genotoxins ( figure 1a ) . these foci are specific for slx4 because they disappeared when cells were transfected with slx4-specific small interfering rnas ( sirnas ) ( figure s1a ) . endogenous slx4 foci did not appear to be restricted to any particular cell - cycle stage , and they did not increase in number or intensity after exposure of cells to genotoxins ( data not shown ) . to investigate if endogenous slx4 foci correspond to telomeres , we tested colocalization with subunits of shelterin . most ( around 80% ) , but not all , endogenous slx4 foci colocalized with trf2 in u2os cells ( figure 1a ) , and also with trf1 and with a peptide - nucleic acid ( pna ) probe specific for telomeric dna ( figure 1b ) . when signal intensity was measured along a straight line in a single z - section through the nucleus , the peaks corresponding to slx4 overlap with trf2 ( figure 1c ) . it is important to note that , whereas all trf2 foci in u2os cells contained slx4 , a proportion of slx4 foci did not overlap with trf2 ( figure 1a ) . these observations indicate that slx4 is found at all telomeres in u2os cells , but a proportion of the slx4 foci do not correspond to telomeres . slx4 associates with three separate structure - specific nucleases , xpf - ercc1 , mus81-eme1 , and slx1 . xpf - ercc1 was previously shown to localize at telomeres ( zhu et al . , 2003 ) , and in this light we found that all of the xpf - ercc1 foci in cells colocalize with slx4 ( figures s1b and s1d ) . mus81-eme1 was also shown to localize at telomeres ( zeng et al . , 2009 ) , but only around 5% of u2os cells show mus81 foci colocalizing with slx4 ( figures s1c and s1d ) . endogenous slx1 foci were only barely detectable with our anti - slx1 antibodies ( data not shown ) . slx4 controls the stability of slx1 ( muoz et al . , 2009 ) , and overexpression of a gfp - tagged form of slx4 increased the levels of endogenous slx1 ( data not shown ) . under these conditions , endogenous slx1 foci could be detected with anti - slx1 antibodies ( figure 1d ) , and most of these foci colocalize with trf2 ( data not shown ) . in contrast , slx1 foci could not be detected when we overexpressed a mutant form of slx4 ( c1805r ) that is incapable of interacting with slx1 ( figure 1d ) ( d.c . we also found that a gfp - tagged form of slx1 formed foci that colocalized with trf2 ( figure 1e ) . taken together , these data show that the endogenous slx4 complex localizes at telomeres . to confirm the association of slx4 with telomeres telomeric dna , but not alu repeat dna , was detected in slx4 immunoprecipitates of u2os cells using two separate anti - slx4 antibodies raised in sheep , but not when nonspecific sheep immunoglobulin ( ig ) g or anti - cenpa antibodies were used . the chip signal in slx4 precipitates was comparable to the signal in trf2 precipitates ( figure 2a ) . we also detected telomeric dna , but not alu dna , in anti - slx1 immunoprecipitates in u2os cells ( figure 2b ) . telomere length is maintained by the alt pathway in u2os cells , whereas in other cell lines telomere length can be maintained by telomerase ( cesare and reddel , 2010 ) . chip experiments showed that slx4 associates with telomeric dna , but not alu dna , in a549 and hela epithelial cells , two non - alt cell lines that are telomerase positive ( figures 2c and 2d , left panels ) . the chip signal in anti - slx4 immunoprecipitates from these cells was weaker than in u2os cells , probably because a549 and hela cell telomeres are shorter than in u2os cells ( lee et al . , 2008 ) . nonetheless , the chip signal in slx4 precipitates from a549 and hela cells was comparable to the signal in trf2 precipitates ( figures 2c and 2d , left panels ) . furthermore , slx4 foci in a549 cells and hela cells are considerably less intense than in u2os cells , but a significant proportion of the foci still colocalized with trf2 ( figures 2c and 2d , middle panels ) . when signal intensity along an arbitrary track in a single z - section of the nucleus was quantitated , the peaks corresponding to slx4 and trf2 in a549 and hela cells demonstrated good overlap ( figures 2c and 2d , right panels ) . we also observed slx4 foci in ccl-211 primary lung fibroblasts , and some of these colocalize with trf1 similar to hela cells ( figure s1e ) . although a significant proportion of telomeric foci in these cells do not contain slx4 , when signal intensity along an arbitrary track in a single z - section of the nucleus was quantitated , the peaks corresponding to slx4 demonstrated reasonably good overlap with trf1 ( figure s1e ) . taken together , these data show that the slx4 complex localizes at telomeres in a range of human cell lines that differ in the mechanisms used to maintain telomere length . both subunits of the trf2-rap1 shelterin subcomplex were found previously in slx4 immunoprecipitates ( muoz et al . , 2009 , svendsen et al . , 2009 ) . we next sought to determine whether slx4 is recruited to telomeres by either of these two proteins . yeast two - hybrid analysis , using xpf as a positive control , revealed that slx4 binds to trf2 , not rap1 ( figure 3a ) , and it seemed likely therefore that slx4 is recruited to telomeres by trf2 . to investigate this possibility , we sought mutations in slx4 that abolish the interaction with trf2 , and our search was aided by a previous report describing a motif found in a range of proteins that interact with trf2 ( f / y - x - l - x - p ; figure 3b ) ( chen et al . , 2008 ) . inspection of the primary amino acid sequence of human slx4 failed to identify a classical trf2-binding motif ( tbm ) , but we found a tbm - like motif ( h - x - l - x - p ) in which the first residue ( f / y ) is replaced by a histidine residue , which like f and y contains a planar aromatic ring structure ( figure 3b ) . this motif in slx4 lies after the btb domain in an unstructured region of the protein ( figure 3b , data not shown ) . we next tested the effect of mutating the key residues in the putative tbm in human slx4 on its interaction with trf2 . to this end , u2os cells were cotransfected with rfp - tagged trf2 and ha - tagged slx4 wild - type or ha - slx4 in which h1020 , l1022 , or p1024 were mutated to alanine . whereas wild - type ha - slx4 coprecipitated with rfp - trf2 , mutation of h1020 , l1022 , or p1024 caused a major reduction in the amount of ha - slx4 coprecipitating with rfp - trf2 ( figure 3c ) . similar results were obtained when gfp - tagged slx4 was immunoprecipitated from cells coexpressing rfp - trf2 ( figure 3d ) . importantly , mutating h1020 , l1022 , or p1024 had no effect on the ability of slx4 to interact with xpf - ercc1 , mus81 , or slx1 ( figure s2a ) . taken together , these data show that a tbm in human slx4 mediates interaction with trf2 . intriguingly , a clear tbm is found in slx4 in primates but not in mammals lower down the evolutionary tree ( figure s2b ) . slx4 orthologs in some mammals have a motif that is vaguely similar to the tbm in primates ( figure s2b ) , but none of the mammalian motifs would be expected to interact with trf2 based on previous work ( chen et al . , 2008 ) . we next tested if mutating the tbm in slx4 affects its localization at telomeres . to this end , gfp - tagged wild - type slx4 , or gfp - slx4-l1022a , was stably expressed in u2os cells and foci were analyzed . surprisingly , the overall number of foci formed by the slx4 l1022a mutant was similar to wild - type slx4 ( figures 3e and 3f ) . however , whereas almost 65% of wild - type gfp - tagged slx4 foci colocalized with trf2 , only around 10% of foci formed by the slx4 l1022a mutant coincided with trf2 , and more than 40% of the foci clearly did not ( figures 3e and 3f ) . the remaining 40% of slx4l1022a foci did not colocalize with trf2 , but were found adjacent to , but distinct from trf2 foci ( figures 3e and 3f ) . to further test the impact of mutating the slx4 tbm on telomere binding , we used chip analysis , which revealed that mutating slx4 l1022 to alanine caused an almost 80% reduction in the amount of telomeric dna associated with slx4 ( figures 3 g and 3h ) . we tested this idea further using a dominant - negative form of trf2 ( trf2bm ) that heterodimerizes with endogenous trf2 blocking its binding to dna ( van steensel et al . , 1998 ) . as shown in figure s2c , overexpression of an rfp - tagged form of trf2bm , but not wild - type trf2 , caused a substantial reduction in the number of slx4 foci in u2os cells ( figure s2c ) . some of the cells transfected with trf2bm did not express this protein , and slx4 foci were normal in these cells ( figure s2c ) . taken together , these data indicate that trf2 recruits slx4 to telomeres in human cells . we also tested if the slx4-trf2 interaction is required for association of slx1 with telomeres . we showed earlier that endogenous slx1 forms trf2-coincident foci in cells overexpressing wild - type slx4 ( figure 1d ) . slx1 also forms foci in cells overexpressing slx4-l1022a , but , although these foci colocalize with slx4-l1022a , they do not colocalize with trf2 ( figure s3a ) . slx4 is required for efficient dna repair . because trf2 has been shown to localize at sites of dna damage ( huda et al . , 2012 ) , and to promote dna repair ( huda et al . , 2009 ) , we tested the possibility that trf2 might recruit slx4 to dna damage sites as well as to telomeres . to this end , we induced dna damage using local laser microirradiation and then checked by confocal microscopy for the ability of slx4 to form laser stripes . time - lapse experiments revealed that gfp - tagged slx4 formed subnuclear stripes along the track of dna damage induced by laser irradiation visible within 12 min of irradiation , reaching a maximum intensity after 1 hr ( figure s3b ) . the slx4-l1022a mutant localized at tracks of laser - induced dna damage with similar kinetics and intensity to wild - type slx4 ( figure s3b ) . we conclude from these data that trf2 we also tested if the association of slx4 with trf2 is required for dna repair using genotoxin hypersensitivity as readout . to this end , we stably expressed untagged forms of slx4 and slx4-l1022a in cells from fanconi anemia patient eufa-1354 that express abnormally low levels of an n - terminally truncated form of slx4 ( stoepker et al . wild - type slx4 and slx4-l1022a are indistinguishable in their ability to rescue the mitomycin - c hypersensitivity of eufa-1354 cells . taken together , the data above indicate that neither the localization of slx4 at dna damage sites , nor the ability of slx4 to promote dna repair , requires binding to trf2 . because the l1022a slx4 mutant interacts with xpf - ercc1 , mus81-eme1 , and slx1 ( figure s2a ) but does not localize at telomeres ( figures 3e3h ) , we reasoned that it might function as a dominant negative by sequestering the associated nucleases away from telomeres . in this light , we made u2os cells that stably express gfp - tagged slx4 , or gfp - slx4-l1022a in a tetracycline - inducible manner . upon induction of slx4-l1022a , we noticed an increase in the proportion of cells with tifs ( telomere dysfunction - induced foci ) ( takai et al . , 2003 ) , defined as 53bp1 foci that colocalize with trf1 , compared with cells expressing wild - type slx4 ( figure 4a ) . in contrast , there was little difference between wild - type slx4 and the slx4-l1022a mutant in terms of the induction of 53bp1 foci that did not colocalize with trf1 ( figure 4a ) . consistent with these data , sirna - mediated depletion of slx4 from u2os cells increased the proportion of cells with tifs ( figures s4a and s4b ) . slx4-specific sirnas 3 and 4 resulted in more efficient depletion of slx4 than sirnas 1 and 2 , and consequently they were more potent at inducing tifs . intriguingly , the lack of an obvious trf2 binding motif in nonprimate mammalian orthologs of slx4 suggests that slx4 should not localize at telomeres in mouse cells , for example , and we next investigated this possibility . as shown in figure s4c , whereas human slx4 forms foci that colocalize with trf2 when ectopically expressed in mouse embryonic fibroblasts ( mefs ) , mouse slx4 forms foci but they do not colocalize with trf2 . this is reminiscent of the human slx4-l1022a mutant that forms foci that do not colocalize with telomeres ( figure 3e3h ) . these observations indicate that slx4 does not associate stably with telomeres in mouse cells , at least in fibroblasts . nonetheless , we reasoned that slx4 might have roles at telomeres that do not require stable association with chromosome ends . with this in mind , we analyzed telomeres in slx4 mice generated in this laboratory , which will be described in detail elsewhere ( d.c . first , we observed increased incidence of tifs in mouse embryonic fibroblasts ( mefs ) from slx4 mice ( figure s4d ) . second , telomeres in livers from slx4 mice were longer than in slx4 mice or wild - type mice ( figure 4b ) . telomeres were also longer in mefs from slx4 mice , but lengthening was reversed by expression of mouse slx4 in these cells so that telomeres were similar in length to wild - type mefs ( figure 4c ) . these data suggest that slx4 might play a role in trimming telomeres to prevent overlengthening . we also found that telomeres from slx4 mefs show increased fragility assessed by the frequency of multitelomeric signals ( mtss ) compared with slx4 mefs and wild - type cells ( figure 4d ) . the data above indicate that targeting of slx4 to telomeres prevents dna damage in human cells , and that slx4 also plays a role in telomere homeostasis in mice in a manner that does not appear to require the stable retention of the slx4 complex at telomeres . in this study , we showed slx4 is recruited to telomeres in human cells by trf2 . we identified a motif in human slx4 required for direct interaction with trf2 that is similar but not identical to the trf2-binding motif ( tbm ; f / y - x - l - x - p ) identified in proteins such as apollo ( chen et al . , 2008 ) . in the slx4 motif , however , the f / y residue is replaced by a histidine residue . this observation extends the consensus trf2-binding motif and raises the possibility that other proteins might interact with trf2 through tbms similar to that of slx4 . although slx4 is a rapidly evolving protein , it is somewhat surprising that the slx4 tbm - like motif is conserved in primates but not in mammals lower down the evolutionary tree ( figure s2b ) . the absence of a tbm , however , does not exclude the possibility that slx4 ( or an slx4-associated protein ) in nonprimate species associates with trf2 and/or telomeres by alternative mechanisms . however , we have been unable to detect endogenous slx4 at telomeres in mouse embryonic fibroblasts , and , whereas human slx4 overexpressed in mefs forms telomeric foci , mouse slx4 does not ( figure s4c ) . therefore , slx4 associates with trf2 and telomeres in primates but not in other mammals . in human cells , preventing localization of slx4 at telomeres caused signs of dna damage ( figure 4a ) . the precise nature of this damage is not yet clear , but it might result from difficulties in one of the many processing steps during and after dna replication necessary for establishing the correct structure at telomeres . it appears that not all of the telomere - related functions of the slx4 complex require stable association with telomeres , because telomeres are longer and more fragile in organs and cells from slx4 mice and telomere lengthening in slx4 mefs can be reversed by expression of wild - type slx4 ( figures 4b and 4c ) . from this perspective telomere trimming , a mechanism that has been proposed to prevent overlengthening of telomeres ( pickett and reddel , 2012 ) . it is not yet clear which of the slx4-associated nucleases are required to prevent telomere lengthening . it is unlikely that xpf - ercc1 is the relevant nuclease because telomere length is not affected in ercc1 mice ( zhu et al . , 2003 ) . the nuclease that promotes slx4-dependent telomere shortening could be mus81-eme1 and/or slx1 ; it will be interesting to test the relevant knockout mice for abnormally long telomeres . slx1 is a good candidate considering the boulton lab has shown that it is required for the production of t circles in cells lacking rtel1 ( vannier et al . , 2012 ) . the consequences of overlong telomeres for cell function and organism function are not clear , but this will be interesting to study . u2os cells expressing gfp - slx4 were preextracted in csk buffer : 10 mm pipes ( ph 6.8 ) containing 100 mm nacl , 300 mm sucrose , 3 mm magnesium chloride , 1 mm egta , and 0.5% [ v / v ] triton x-100 , fixed with 2% paraformaldehyde ( pfa ) ( ph 7.0 ) , permeabilized with pbs containing 0.5% ( v / v ) triton x-100 , blocked with bsa ( 3% w / v ) in pbs , and probed with primary antibodies for 1 hr at room temperature . after extensive washing , cells were incubated with secondary alexa antibodies for 2 hr at room temperature . wide - field image stacks of cells were acquired using a deltavision microscope ( applied precision ) and subjected to iterative deconvolution to remove out - of - focus light and were further processed in omero ( allan et al . , 2012 ) . for endogenous slx4 immunofluorescence , cells grown on poly - l - lysine - coated coverslips were preextracted in csk buffer or pbs containing 0.5% triton x-100 prior to fixation in 3% pfa in csk ( ph 7.0 ) . cells were permeabilized in pbs - containing 0.5% ( v / v ) triton x-100 and then blocked in antibody dilution buffer ( abdil ) : pbs containing 5% normal donkey serum , 0.1% fish skin gelatin , 0.1% triton x-100 , 0.05% tween-20 , and 0.05% sodium azide , before incubation with primary antibodies in abdil for 1 hr at room temperature or overnight at 4c . after washing , cells were incubated with the relevant secondary antibodies for 2 hr at room temperature , stained with dapi , and mounted on glass slides . cells grown to subconfluency on 15 cm plates were crosslinked with 1% ( v / v ) formaldehyde for 15 min , with 0.125 m glycine for 5 min , and then washed twice with pbs . cells ( 4 10 cells per ip ) were lysed in chromatin immunoprecipitation ( chip ) lysis buffer ( 1% sds , 10 mm edta , 50 mm tris - hcl [ ph 8.0 ] plus protease inhibitors ) , followed by sonication for 15 s at 50% amplitude and 30 s rest ; this was repeated eight times at 4c before cells were centrifuged at 12,000 rpm at 4c for 10 min . cell supernatants were diluted 1:10 in chip dilution buffer ( 1% [ v / v ] triton x-100 , 2 mm edta , 150 mm nacl , and 20 mm tris - hcl [ ph 8.0 ] ) and precleared with 2 g of sheared salmon sperm dna ( invitrogen ) and 20 l protein g - sepharose beads per equivalent ip volume ( 2 ml ) . the supernatant was incubated with the relevant antibodies overnight , before the addition of protein g - sepharose beads for 1 hr . after extensive washes , immune complexes were eluted with elution buffer ( 100 mm nahco3 and 1% sds ) and incubated overnight at 65c to reverse crosslinks . protein and rna were removed by incubation with proteinase k and rnase a at 37c for 1 hr . dna was purified by extraction with phenol / chloroform / isoamyl alcohol ( 25:24:1 ) , followed by centrifugation and retrieval of the aqueous supernatant . dna was precipitated with ice - cold ethanol ( 96% v / v ) , washed with 70% ethanol , and resuspended in 100 l of water . extended experimental procedurescell culture , fractionation , and lysisu2os cells were cultured in dmem growth medium ( gibco ) supplemented with 10% ( v / v ) fetal bovine serum , 100 units / ml penicillin , 100 g / ml streptomycin and 1% l - glutamate ( gibco , invitrogen ) . u2os cells stably expressing gfp - slx4 were generated using the flp - in t - rex system ( invitrogen ) as described previously ( muoz et al . , 2009 ) . cells were maintained in selective media containing 100 g / ml hygromycin and 15 g / ml blasticidin . protein expression was induced by adding tetracycline to 0.1 g / ml for 24 hr . for immunoprecipitations and western blotting , cells were lysed in 50 mm tris - hcl ph 7.4 containing 150 mm nacl , 1 mm edta ph 8 , 1 mm egta ph 8 , 1% ( v / v ) triton x-100 , 270 mm sucrose supplemented with -mercaptoethanol ( 0.1%v / v ) , benzonase ( 50mu/l ) and 1 roche protease inhibitor tablet per 50 ml of buffer.slx4 micethe generation of slx4 mice will be described elsewhere ( d.c . and j.r , unpublished data ) . animals were housed under specific pathogen free conditions in accordance with uk and eu regulations . all procedures were carried out in accordance with university of dundee and united kingdom home office regulations.isolation and immortalization of mefsday 13.5 embryos were derived from timed matings between heterozygous parents . after decapitation , the red organs were removed , the embryo was minced and resuspended in 1 ml trypsin and incubated at 37c for 15 min before the addition of 10 ml growth medium . cells were plated and allowed to attach over night before cells were washed with fresh medium to remove debris . when cells reached confluency they were split and re - plated and this was considered passage 1 . mefs were 3t3-immortalized.sirnacells were transfected with the relevant sirna duplex ( 30 nm ) using the calcium phosphate precipitation method or hiperfect ( qiagen ) according to the manufacturer s protocol . the slx4 mrna target sequences used in figures s4a and s4b were : slx4 - 1 gagaagaaccctaatgaaa dtdtslx4 - 2 gcacaagggcccagaacaa dtdtslx4 - 3 ggagaaggaagcagagaat dtdtslx4 - 4 aaacgtgaatgaagcagaa dtdtslx4 - 4 was used in figure s1a . the final concentration of sirna added to cells was 20 nm.antibodiesthe following primary antibodies were used in this study.western blottingmouse anti - human xpf diluted 1:5000 ( thermoscientific ms1381 ) ; rabbit anti - trf2 diluted 1:1000 ( cst # 2645 ) . sheep polyclonal slx4 antibodies were raised against a bacterially expressed protein fragment corresponding to anti - human slx4 ( amino acids 1535 - 1834 ) fused to gst . the antibodies were purified from sheep serum by affinity chromatography on ch - sepharose to which the peptide immunogen had been covalently coupled . sheep polyclonal slx1 antibodies were raised against bacterially expressed human slx1 ( full - length ) fused to gst . the antibodies were purified from sheep serum by affinity chromatography on ch - sepharose to which the peptide immunogen had been covalently coupled.immunoprecipitationsheep anti - human slx4 ( 1535 - 1834 ) as above ; sheep anti - gfp ( as sheep igg control , raised in the dstt ) ; mouse anti - trf2 ( millipore 41794 ) ; rabbit anti - rap1 ( bethyl a300 - 306a ) ; normal mouse igg ( cst # 5415 ) ; normal rabbit igg ( cst # 2729 ) . all antibodies were used at 4 g antibody per 2 mg of cell lysate.immunofluorescencemouse anti - trf1 diluted 1:200 ( abcam ab10579 ) ; rabbit anti-53bp1 diluted 1:500 ( a300 - 272a ) ; mouse anti - human xpf diluted 1:500 ( thermoscientific ms1381 ) ; mouse anti - trf2 diluted 1:400 ( millipore 4a794 ) ; anti - slx1 diluted 1:100 . all secondary antibodies were from invitrogen : donkey anti - mouse alexa 594 ( diluted 1:800 ) ; donkey anti - sheep alexa 594 ( diluted 1:800 ) ; donkey anti - mouse alexa 647 ( diluted 1:500 ) ; donkey anti - rabbit alexa 647 diluted 1:500 . sheep polyclonal slx4 antibodies for immunofluorescence were raised against a bacterially expressed protein fragment corresponding to anti - human slx4 ( amino acids 381 - 680 ) fused to gst . the antibodies were purified from sheep serum by affinity chromatography on ch - sepharose to which the peptide immunogen had been covalently - coupled . these antibodies were diluted 1:200.chipsheep anti - human slx4 ( 381 - 680 ) as above ; sheep anti - human slx4 ( 1535 - 1834 ) as above ; mouse anti - trf2 ( imgenex img124a ) ; mouse anti - cenpa ( abcam ab13939 ) ; sheep anti - gfp ; rabbit anti - gfp ( abcam ab290 ) ; normal mouse igg ( cst # 5415 ) ; normal rabbit igg ( cst # 2729 ) . all antibodies used at 3 g per chip.dot blotting and hybridizationchip dna was blotted onto hybond h membrane ( ge healthcare ) and cross - linked with 1200 j / m of uv - c light . membranes were pre - hybridized by incubation in church buffer ( 0.5 m sodium phosphate buffer ph7.2 , 1 mm edta , 1% bsa ) for 1 hr . membranes were then incubated with 800bp ( ttagggn ) telomere repeat probe radio - labeled with [ p]-dctp using klenow fragment , boiled before incubation in church buffer . alternatively , membranes were incubated with a poly - nucleotide kinase ( pnk ) gamma 32p end labeled alu repeat probe ( cggagtctcgctctgtcgcccaggctggagtgcagtggcgcga ) in church buffer . membranes were washed in 2 x ssc ( 300 mm nacl , 15 mm sodium citrate ) four times for 5 min each at 65c , and exposed to photographic film overnight at 80c.coupled immunofluorescence - fishin coupled immunofluorescence ( if)-fish experiments , immunofluorescence was carried out as normal with cells grown on coverslips . cells were then washed twice in pbs and fixed again with 3% formaldehyde ( 5 min ) before further washing . cells were then serially dehydrated in 70% , 95% and then 100% ethanol before drying in air for 2 min . cells were incubated with fitc labeled telomeric fish probe ( dako ) and dishes were gently covered with saranwrap to aid distribution of the probe . after 10 min at 80c , the coverslips were transferred to 4c and incubated overnight . after two 15 min washes in buffer ( 10 mm tris - hcl , ph7.4 ) containing 70% ( v / v ) formamide at 50c , coverslips were washed three times for 5 min each with pbs ; the second pbs wash included dapi at 1 g / ml . cells were then mounted for microscopic analysis.telomere fluorescence analyses of liver sectionsparaffin - embedded liver sections were hybridized with a pna - telomeric probe , and fluorescence intensity of telomeres was determined as previously described ( zijlmans et al . , 1997 ) . quantitative image analysis was performed on images obtained using a confocal microscope ( leica tcs - sp5 dobs ) and the definiens developer cell software ( version xd 1.2 ; definiens ag).telomere length and cytogenetic analysis using telomere q - fish on metaphase spreadsq - fish on metaphases from primary mef was performed as described previously ( herrera et al . , 1999 ) . where indicated , mefs were treated with aphidicolin at 0.5 m for 18 hr followed by 6 hr recovery . images were captured at 100x magnification using a cohu ccd camera on a leica dmra ( leica , heidelberg , germany ) microscope . telomere fluorescence signals were quantified by using the tfl - telo program ( gift from peter lansdorp , vancouver , canada ) ( zijlmans et al . , 1997).laser microirradiationcells were plated on glass - bottomed dishes and pre - sensitized with 10 mm 5-bromo-2-deoxyuridine ( brdu , sigma - aldrich ) for 24 h at 37c . dna damaged was induced using a 405 nm cw 30 mw diode laser scanned 50x at 100% power over the selected roi ( region of interest ) in one optical section corresponding to the middle of the nucleus . live cell imaging was carried out on a zeiss 710 confocal microscope equipped with a 37c environmental chamber and supplied with humidified 5% co2 . cells were imaged with a zeiss plan - apochromat 63x 1.4na oil immersion objective lens . u2os cells were cultured in dmem growth medium ( gibco ) supplemented with 10% ( v / v ) fetal bovine serum , 100 units / ml penicillin , 100 g / ml streptomycin and 1% l - glutamate ( gibco , invitrogen ) . u2os cells stably expressing gfp - slx4 were generated using the flp - in t - rex system ( invitrogen ) as described previously ( muoz et al . cells were maintained in selective media containing 100 g / ml hygromycin and 15 g / ml blasticidin . protein expression was induced by adding tetracycline to 0.1 g / ml for 24 hr . for immunoprecipitations and western blotting , cells were lysed in 50 mm tris - hcl ph 7.4 containing 150 mm nacl , 1 mm edta ph 8 , 1 mm egta ph 8 , 1% ( v / v ) triton x-100 , 270 mm sucrose supplemented with -mercaptoethanol ( 0.1%v / v ) , benzonase ( 50mu/l ) and 1 roche protease inhibitor tablet per 50 ml of buffer . the generation of slx4 mice will be described elsewhere ( d.c . and animals were housed under specific pathogen free conditions in accordance with uk and eu regulations . all procedures were carried out in accordance with university of dundee and united kingdom home office regulations . the red organs were removed , the embryo was minced and resuspended in 1 ml trypsin and incubated at 37c for 15 min before the addition of 10 ml growth medium . cells were plated and allowed to attach over night before cells were washed with fresh medium to remove debris . when cells reached confluency they were split and re - plated and this was considered passage 1 . cells were transfected with the relevant sirna duplex ( 30 nm ) using the calcium phosphate precipitation method or hiperfect ( qiagen ) according to the manufacturer s protocol . the slx4 mrna target sequences used in figures s4a and s4b were : slx4 - 1 gagaagaaccctaatgaaa dtdtslx4 - 2 gcacaagggcccagaacaa dtdtslx4 - 3 ggagaaggaagcagagaat dtdtslx4 - 4 aaacgtgaatgaagcagaa dtdtslx4 - 4 was used in figure s1a . slx4 - 1 gagaagaaccctaatgaaa dtdt slx4 - 2 gcacaagggcccagaacaa dtdt slx4 - 3 ggagaaggaagcagagaat dtdt slx4 - 4 aaacgtgaatgaagcagaa dtdt slx4 - 4 was used in figure s1a . the final concentration of sirna added to cells was 20 nm . the following primary antibodies were used in this study . mouse anti - human xpf diluted 1:5000 ( thermoscientific ms1381 ) ; rabbit anti - trf2 diluted 1:1000 ( cst # 2645 ) . sheep polyclonal slx4 antibodies were raised against a bacterially expressed protein fragment corresponding to anti - human slx4 ( amino acids 1535 - 1834 ) fused to gst . the antibodies were purified from sheep serum by affinity chromatography on ch - sepharose to which the peptide immunogen had been covalently coupled . sheep polyclonal slx1 antibodies were raised against bacterially expressed human slx1 ( full - length ) fused to gst . the antibodies were purified from sheep serum by affinity chromatography on ch - sepharose to which the peptide immunogen had been covalently coupled . sheep anti - human slx4 ( 1535 - 1834 ) as above ; sheep anti - gfp ( as sheep igg control , raised in the dstt ) ; mouse anti - trf2 ( millipore 41794 ) ; rabbit anti - rap1 ( bethyl a300 - 306a ) ; normal mouse igg ( cst # 5415 ) ; normal rabbit igg ( cst # 2729 ) . all antibodies were used at 4 g antibody per 2 mg of cell lysate . mouse anti - trf1 diluted 1:200 ( abcam ab10579 ) ; rabbit anti-53bp1 diluted 1:500 ( a300 - 272a ) ; mouse anti - human xpf diluted 1:500 ( thermoscientific ms1381 ) ; mouse anti - trf2 diluted 1:400 ( millipore 4a794 ) ; anti - slx1 diluted 1:100 . all secondary antibodies were from invitrogen : donkey anti - mouse alexa 594 ( diluted 1:800 ) ; donkey anti - sheep alexa 594 ( diluted 1:800 ) ; donkey anti - mouse alexa 647 ( diluted 1:500 ) ; donkey anti - rabbit alexa 647 diluted 1:500 . sheep polyclonal slx4 antibodies for immunofluorescence were raised against a bacterially expressed protein fragment corresponding to anti - human slx4 ( amino acids 381 - 680 ) fused to gst . the antibodies were purified from sheep serum by affinity chromatography on ch - sepharose to which the peptide immunogen had been covalently - coupled . sheep anti - human slx4 ( 381 - 680 ) as above ; sheep anti - human slx4 ( 1535 - 1834 ) as above ; mouse anti - trf2 ( imgenex img124a ) ; mouse anti - cenpa ( abcam ab13939 ) ; sheep anti - gfp ; rabbit anti - gfp ( abcam ab290 ) ; normal mouse igg ( cst # 5415 ) ; normal rabbit igg ( cst # 2729 ) . chip dna was blotted onto hybond h membrane ( ge healthcare ) and cross - linked with 1200 j / m of uv - c light . membranes were pre - hybridized by incubation in church buffer ( 0.5 m sodium phosphate buffer ph7.2 , 1 mm edta , 1% bsa ) for 1 hr . membranes were then incubated with 800bp ( ttagggn ) telomere repeat probe radio - labeled with [ p]-dctp using klenow fragment , boiled before incubation in church buffer . alternatively , membranes were incubated with a poly - nucleotide kinase ( pnk ) gamma 32p end labeled alu repeat probe ( cggagtctcgctctgtcgcccaggctggagtgcagtggcgcga ) in church buffer . membranes were washed in 2 x ssc ( 300 mm nacl , 15 mm sodium citrate ) four times for 5 min each at 65c , and exposed to photographic film overnight at 80c . in coupled immunofluorescence ( if)-fish experiments , cells were then washed twice in pbs and fixed again with 3% formaldehyde ( 5 min ) before further washing . cells were then serially dehydrated in 70% , 95% and then 100% ethanol before drying in air for 2 min . cells were incubated with fitc labeled telomeric fish probe ( dako ) and dishes were gently covered with saranwrap to aid distribution of the probe . after 10 min at 80c , the coverslips were transferred to 4c and incubated overnight . after two 15 min washes in buffer ( 10 mm tris - hcl , ph7.4 ) containing 70% ( v / v ) formamide at 50c , coverslips were washed three times for 5 min each with pbs ; the second pbs wash included dapi at 1 g / ml . paraffin - embedded liver sections were hybridized with a pna - telomeric probe , and fluorescence intensity of telomeres was determined as previously described ( zijlmans et al . , 1997 ) . quantitative image analysis was performed on images obtained using a confocal microscope ( leica tcs - sp5 dobs ) and the definiens developer cell software ( version xd 1.2 ; definiens ag ) . q - fish on metaphases from primary mef was performed as described previously ( herrera et al . , 1999 ) . where indicated , mefs were treated with aphidicolin at 0.5 m for 18 hr followed by 6 hr recovery . images were captured at 100x magnification using a cohu ccd camera on a leica dmra ( leica , heidelberg , germany ) microscope . telomere fluorescence signals were quantified by using the tfl - telo program ( gift from peter lansdorp , vancouver , canada ) ( zijlmans et al . , 1997 ) . cells were plated on glass - bottomed dishes and pre - sensitized with 10 mm 5-bromo-2-deoxyuridine ( brdu , sigma - aldrich ) for 24 h at 37c . dna damaged was induced using a 405 nm cw 30 mw diode laser scanned 50x at 100% power over the selected roi ( region of interest ) in one optical section corresponding to the middle of the nucleus . live cell imaging was carried out on a zeiss 710 confocal microscope equipped with a 37c environmental chamber and supplied with humidified 5% co2 . cells were imaged with a zeiss plan - apochromat 63x 1.4na oil immersion objective lens .

summaryslx4 , a scaffold for structure - specific dna repair nucleases , is important for several types of dna repair . many repair proteins bind to sites of dna damage , resulting in subnuclear foci , but slx4 forms foci in human cells even without dna damage . using several approaches , we show that most , but not all , slx4 foci localize to telomeres in a range of human cell lines irrespective of the mechanisms used to maintain telomere length . the slx1 holliday - junction - processing enzyme is recruited to telomeres by slx4 , and slx4 , in turn , is recruited by a motif that binds to the shelterin subunit trf2 directly . we also show that trf2-dependent recruitment of slx4 prevents telomere damage . furthermore , slx4 prevents telomere lengthening and fragility in a manner that appears to be independent of telomere association . these findings reveal that slx4 plays multiple roles in regulating telomere homeostasis .

Introduction Results Discussion Experimental Procedures

crotoxin ( ctx ) is the main toxic component of the venom of the south american rattlesnake , crotalus durissus terrificus [ 1 , 2 ] . the toxin is a heterodimeric complex consisting of basic and toxic phospholipase a2 and an acidic , nontoxic , and nonenzymatic component named crotapotin . in addition to its toxic properties , several experimental observations indicated that ctx also has immunomodulatory , anti - inflammatory , antimicrobial , analgesic , and antitumor effects for review . several studies have shown antitumor effects of snake venoms or their isolated components [ 39 ] . ctx has been shown to inhibit proliferation of various cell lines ( in vitro ) and growth of various tumors in vivo . as reported by cura and colleagues , ctx is toxic to several tumor cell lines in vitro and , in some of them , via epidermal growth factor receptors . the antitumoral effects of ctx have also been reported in patients with lung and mammary carcinoma . evidence has been accumulated that ctx presents inhibitory effects on inflammatory response [ 13 , 14 ] . mentioned , the mechanisms involved in the antitumor effects of the ctx still remain to be determined . increased formation of prostaglandins pge2 and pgd2 occurs in the beginning of an inflammatory response . afterwards , the profile of lipid mediators activates the expression of 15-lox in leukocytes , which switches the mediator profile of these cells from ltb4 to lipoxins ( lxs ) . lipoxin a4 ( lxa4 ) and lipoxin b4 ( lxb4 ) are synthesized by transcellular metabolism of aa due to an interaction among neutrophils , endothelial cells , fibroblasts , and platelets localized in the inflammatory exudate . afterwards , the profile of lipid mediators switches from proinflammatory eicosanoids to lipoxins ( lxs ) that bind to g - protein - coupled lxa4 receptor ( formyl peptide receptor 2-fpr2/alx ) and triggers the proinflammatory termination signal . lxs are produced from arachidonic acid via 5-lipoxygenase ( 5-lo ) and 15-lipoxygenase ( 15-lo ) pathways . acetylation of cyclooxygenase-2 ( cox-2 ) by aspirin leads to biosynthesis of 15-epi - lipoxins , the 15-epimers carbon of native lxs . 15-epi - lxa4 has more potent and longer lasting effects than does the native lxa4 that is less rapidly inactivated [ 19 , 20 ] , for review . the native lxs and their stable analogues regulate cell functions through activation of g - protein - coupled lxa4 receptor ( formyl peptide receptor , fpr2 , also termed alxr ) . as fprs are potentially important therapeutic targets , studies have been focused on identification of natural and synthetic compounds having the ability to interact with these receptors or interfere with the frp - involved pathways [ 25 , 26 ] . lxs are involved in the development of pathological conditions such as rheumatoid arthritis , asthma , sepsis , diabetes , and tumor [ 16 , 19 , 27 ] . administration of lxs and their natural analogue 15-epi - lxa4 causes inhibition of disease - related inflammation and suppresses tumor growth and cancer - associated pain [ 20 , 2831 ] . ctx treatment promotes release of lxa4 and 15-epi - lxa4 in cultured macrophages and macrophages cocultivated with tumor cells , which may contribute to the antiproliferative activity of these leukocytes . we tested herein the hypothesis that ctx treatment reduces tumor growth through formyl peptide receptors ( fprs ) and production of lxa4 and 15-epi - lxa4 . to investigate this hypothesis injection of walker 256 carcinoma cells results in the development of inflammation , cell proliferation , and tumor tissue growth , angiogenesis and hyperalgesia . over 70% of anticancer compounds are either natural products or natural product - derived compounds . the discovery of new drugs for different types of cancer is a hot area of investigation since many tumors still remain unresponsive to any existing treatment . evidence is presented herein that crotoxin may be a new therapeutic drug to be clinically investigated so as to treat cancer . it has been shown in clinical trials that the lxa4 analogues present efficacy and safety , expanding the pharmacological perspectives herein proposed . male wistar rats , weighing between 160 and 180 g , were used throughout the study . the rats were housed in an animal care facility and taken to the testing room 2 days before the experiment . all experiments and assays were carried out in accordance with the guidelines for the ethical use of conscious animals in pain research , published by the international association for the study of pain . the institutional animal care committee of the butantan institute approved the procedures used in this study ( ceuaib , protocol number 359/2006 ) . ctx was obtained from lyophilised venom of crotalus durissus terrificus supplied by the laboratory of herpetology , butantan institute , so paulo , brazil , and maintained at 20c . crude venom solution was subjected to anion - exchange chromatography as previously described by , using a mono - q hr 5/5 column in an fplc system ( pharmacia , uppsala , sweden ) . the fractions ( 1 ml / min ) were eluted using a linear gradient of nacl ( 0 - 1 mol / l in 50 mmol / l tris - hcl , ph 7.0 ) . three peaks ( p1 , p2 , and p3 ) were obtained : p2 corresponded to the pure ctx fraction ( about 60% of the crude venom ) ; peaks 1 and 3 included the other cdtv toxins . prior to pooling , the fractions containing ctx were tested for homogeneity by nonreducing sodium dodecyl sulphate - polyacrylamide gel electrophoresis ( 12.5% ) and the phospholipase a2 activity was assessed by a colorimetric assay using a synthetic chromogenic substrate . ctx was subcutaneously injected ( 18 g per rat in 300 l of the saline ) , daily , for 5 days . the dose of ctx was based on previous work and did not cause clinical signs of crotalus durissus terrificus envenomation , such as neurotoxic faces , external and internal ophthalmoplegia , and respiratory paralysis . other rats received lxa4 ( 2.0 g per rat/300 l saline , subcutaneously ) , based on von der weid et al . . the same volume of saline was subcutaneously administered to the respective reference groups . to investigate the involvement of fprs in the ctx effect , rats were treated with boc-2 , a selective fprs antagonist , butoxycarbonyl - phe - leu - phe - leu - phe , from phoenix pharmaceutical inc . , usa , in a dose of 5 g per rat , intraperitoneally , in 1 ml saline containing 1% dimethyl sulfoxide . thirty minutes later , the animals were subcutaneously injected with ctx or lxa4 or saline , in the same volume . the results were compared to two reference groups ; the first group received saline by the same route used for tumor cell inoculation and the other received lxa4 . on the fifth day of the injection of tumor cells , the animals were submitted to analysis of tumor growth through increase in tumor volume , mechanical hyperalgesia and allodynia , in addition to plasma collection for measurement of the lxa4 and 15-epi - lxa4 . after 5 days of intraperitoneal injection of walker 256 carcinoma cells ( 1 10/2 ml ) , ascitic liquid from the peritoneal cavity was collected to obtain fresh tumor cells and the percentage of viable cells was determined by using 1% trypan blue aqueous solution in a neubauer chamber . cells were harvested and a suspension of 10 cells per ml was obtained by dilution with phosphate - buffered saline ( pbs , ph 7.4 ) . antibiotic ( benzylpenicillin , 120,000 units in 10 ml of cell suspension , benzetacil ; eurofarma , brazil ) was added to cell suspension to avoid microbial contamination . tumor cells ( 100 l ) were then subcutaneously injected into the plantar region of the rat right hind paw ; pbs ( 100 l ) injection into the contralateral hind paw was used as reference for tumor growth assessment . tumor growth was assessed with the aid of a pachymeter ( mitutoyo , japan ) or by measurement of the volume increase ( edema ) of paws up to the tibiotarsal articulation . the measurements were carried out before the injection of tumor cells and pbs ( in the contralateral paw ) and at chosen time intervals thereafter according to brigatte et al . . the difference between values obtained for both paws was used as a measure of edema volume increase and so tumor growth . on the fifth day of the experiment , after the last measurement of the paw volume , the animals were anesthetized with ketamine ( 100 mg kg , 0.5 ml / kg , vetbrands brasil ltda . , brazil ) and xylazine ( 10 mg kg , 0.5 ml / kg , vetbrands brasil ltda . , brazil ) , intraperitoneally injected , and blood samples were obtained from the abdominal aorta in tubes containing disodium salt of ethylene diamine tetra acetic acid ( edta ) as anticoagulant . lxa4 and 15-epi - lxa4 were measured in plasma by immunoenzymatic assays [ 32 , 45 ] using specific kits for each lx ( neogen , lexington , ky , usa ) . plasma samples were acidified with 1 n hcl to ph 3.43.6 and passed slowly through an octadecylsilyl silica column ( c18 sep - pak column , waters corporation , usa ) , prewashed with 10 ml absolute ethanol and 10 ml water . after activation of the column with 10 ml water , 2 ml absolute ethanol , and 2 ml water again , the eicosanoids were eluted from the column with 1 ml water , 1 ml ether , and 2 ml methyl formate and the samples dried under a stream of nitrogen . the animals were euthanized in a co2 chamber , on the fifth day after tumor cell injection . samples were embedded in paraffin , sectioned into 5 m sections , and stained with monastral blue . micrographs were taken in an olympus bx 51 microscope ( usa ) and measurements were carried out using the axio vision 4.8 program . statistical analysis of the differences between groups was performed according to glantz by using the graphpad instat software version 3.01 ( graphpad software inc . , san diego , ca , usa ) . one - way anova followed by bonferroni 's test was also used to prepare dose - response curves for a single time point . p < 0.05 was considered for differences to be significant . the alpha level ( significance level related to the probability of rejecting a true hypothesis ) significant differences were then compared using bonferroni 's test with a significance coefficient of 0.05 . the injection of tumor cells caused a significant and progressive increase in paw volume as compared to the values obtained before cell inoculation ( figure 1 ) . the edema was measurable already on the second day ( 9% ) after cell injection and reached up to 40% increase on the fifth day ( figure 1 ) . daily subcutaneous administration of ctx , for 5 days , caused significant decrease of paw volume from the third day of tumor cell inoculation ( third day : 49% ; fourth day : 30% ; fifth day : 30% ) ( figure 1 ) . a representative histological slide of a normal rat paw is shown in brigatte and colleague . after 5 days of treatment with ctx , the number of vessels was significantly lowered ( 47% ) as compared to pbs treated animals ( figures 2(a ) , 2(c)(1 ) , and 2(c)(2 ) ) . also , vessel diameters ( figures 2(b ) and 2(c)(3 ) and 2(c)(4 ) ) were significantly smaller ( 37% ) in ctx treated rats . plasma lxa4 concentration was assessed on the fifth day of ctx treatment ( figure 3(a ) ) . treatment of control ( nt ) rats with ctx induced a significant increase of lxa4 plasma levels ( 74% ) when compared to saline treated animals . walker 256 tumor - bearing animals showed low plasma concentrations of both lxa4 and 15-epi - lxa4 . the treatment of tumor - bearing rats with the toxin , under the same experimental conditions described above , induced an increase of plasma levels of lxa4 and 15-epi - lxa4 ( 38% ) when compared to saline ( figure 3(a ) ) . ctx induced a significant increase in plasma concentration of the stable analogue 15-epi - lxa4 ( by 1.65-fold ) as compared to animals without tumor ( nt ) and injected with saline ( figure 3(b ) ) . similar increase was observed in walker 256 tumor - bearing rats that received subcutaneous injection of the toxin ( 42% ) , daily , for five days as compared to saline injected tumor - bearing animals ( figure 3(b ) ) . the treatment of rats with no tumor ( nt ) with boc-2 thirty minutes before the subcutaneous injection of ctx blocked the increase in plasma concentration of both lxa4 and 15-epi - lxa4 when compared to treatment with saline . these results show that the fprs mediated the effects of ctx on production of both lipid mediators . the boc-2 per se did not cause marked changes in plasma levels of lxa4 and 15-epi - lxa4 when compared to saline injected rats ( figures 3(a ) and 3(b ) ) . to evaluate the participation of lxa4 and 15-epi - lxa4 in the antitumoral effects of ctx , boc-2 was i.p . administered , daily , 30 minutes before the subcutaneous injection of ctx for 5 days , from the 1st day of walker 256 tumor cell injection . the results showed that , on the fifth day of the tumor inoculation , both ctx and lxa4 ( a fprs agonist ) inhibited walker 256 tumor growth ( by 63% and 67% , resp . ) it is noticeable that , on the 5th day of treatment , boc-2 completely abolished the reducing effect of the toxin on tumor growth as compared to saline ( figure 4 ) . the pretreatment with boc-2 also blocked the inhibitory effect of ctx on formation of new vessels ( figure 5(a ) ) . the treatment with lxa4 did not interfere with the formation of vessels as compared to saline . on the other hand , the diameter of the vessels was decreased by both ctx and lxa4 and this effect was totally abolished by boc-2 ( figures 5(a ) and 5(b ) ) . tissue histological slides of the paws ( figure 5(c ) ) were obtained from nt animal ( 1 ) and walker 256 tumor - bearing rats treated with saline ( 2 ) ; ctx ( 3 ) ; lxa4 ( 4 ) ; boc-2 + saline ( 5 ) ; boc-2 + ctx ( 6 ) ; and boc-2 + lxa4 ( 7 , 8) . together , the data presented here suggest that the ctx induced increased plasma levels of lxa4 and its analogue being probably released by leukocytes into the deep dermis that migrated from the systemic circulation , which might inhibit both tumor growth and formation of new vessels via fprs . tumor growth induced by intraplantar inoculation of walker 256 carcinoma cells in rats was assessed by the increase in volume of the glabrous region of the hind paw . the volume of the cell inoculated paw started to increase on the second day after inoculation , and thereafter it increased progressively up to the fifth day as observed in our previous work . the features of cancer pain symptoms ( including hyperalgesia and allodynia ) and of tissue morphological changes observed herein were also reported in our previous study . ctx significantly inhibited tumor growth from the second day and this inhibition remained until the fifth day of study . this effect was accompanied by a decrease in formation of new vessels and in the diameter of the vessels , suggesting that ctx interferes with tumor growth by impairing angiogenesis . recent results have shown that ctx inhibits proliferation of human leukemic jurkat t - cell line ( sandra coccuzzo sampaio and yara cury , unpublished data ) and llc wrc 256 tumor cells ( odair jorge faiad and sandra coccuzzo sampaio , unpublished data ) . ctx also inhibits t.end.1 cell function , indicating direct action of this toxin on endothelial cells . in addition to this direct activity of the toxin on tumor and endothelial cells , ctx raises the production of reactive oxygen and nitrogen species and lxa4 and its analogue 15-epi - lxa4 in macrophages cocultured with llc wrc 256 tumor cells , reducing tumor cell proliferation . ctx significantly inhibited tumor growth from the second day and this inhibition remained until the fifth day of study . this effect was accompanied by a decrease in formation of new vessels and in the diameter of the vessels , suggesting that ctx inhibition of tumor growth involves impairment in angiogenesis . angiogenesis has been associated with the development of several diseases such as rheumatoid arthritis , psoriasis , and cancer . tumor growth depends on a persistent neovascularization [ 50 , 51 ] and is proportional to the extent of angiogenesis . tumor angiogenesis is a combination of angiogenesis and vasculogenesis and the mainstay of tumor blood vessels derived from preexisting ones , although the circulating endothelial precursor cells contribute to the growth of endothelial cell mass . active proliferation of tumor cells , which usually accompanies the initial phase of tumor growth , is balanced by the cell death caused by the withdrawal of blood supply to the tumor . rapid and exponential tumor growth requires neovascularization whereas angiogenesis is paralleled to the process of metastasis [ 55 , 56 ] . we demonstrated in previous studies that ctx inhibits secretory activity and endothelial cell function , evidencing direct action of this toxin on endothelial cells . in addition , macrophages treated with ctx inhibit in vitro angiogenic events and the consequent formation of capillary structures by endothelial cells in 3d matrix . monocytes are recruited from the systemic circulation into tumor tissue , in response to chemokines secreted by tumor cells , and differentiate into macrophages [ 58 , 59 ] . tumor - associated macrophages modulate tumor cell migration , extravasation , and also angiogenesis [ 60 , 61 ] . activated macrophages exert tumoricidal effect by secreting molecules such as hydrogen peroxide ( h2o2 ) , nitric oxide ( no ) , and lxs [ 20 , 6264 ] . various leukocytes including monocytes , polymorphonuclear cells , and macrophages secrete lxa4 and 15-epi - lxa4 [ 20 , 32 , 62 ] . lxs are biosynthesised and rapidly inactivated , whereas related compounds , such as 15-epi - lxa4 , are more stable . acetylation of cyclooxygenase-2 induced by aspirin or other endogenous substrates ( cytochrome p450 and reactive oxygen species ) leads to stereoselective formation ( 40% r and 60% s form ) of 15-epi - lipoxins that are more potent and longer acting than the native 15-s containing lx form [ 16 , 18 , 66 ] . treatment with ctx caused a significant increase in plasma concentrations of lxa4 and its stable analogue in both control and tumor - bearing rats . similar results were previously published [ 67 , 68 ] . despite the direct actions of ctx on cells of the tumor microenvironment , such as tumor and endothelial cells , production of lxa4 and its analogue by macrophages might also be involved in the inhibitory action of the toxin on tumor growth and angiogenesis herein reported . the actions of these lipid mediators were mediated by fprs since pretreatment with boc-2 , antagonist of the fpr2/alx , and fpr1 [ 67 , 68 ] completely blocked it . only ctx decreased the number of vessels whereas both ctx and lxa4 decreased vessel diameters . chen and colleagues reported an inhibitory effect of lxa4 and 15-epi - lxa4 on primary tumor growth . however , the analogue was able to induce toxicity of tumor cells whereas lxa4 did not cause antiproliferative effects . ctx induced formation of the analogue in large quantities , which probably led to inhibition of angiogenesis and so tumor growth . the inhibitory action of ctx and lxa4 on paw volume increase , observed at the fifth day after injection of tumor cells , was completely abolished by boc-2 . lipoxins and their stable analogues exert biological actions , such as anti - inflammatory and antiangiogenic properties , by binding to fprs and then interfering with cell proliferation and tumor growth [ 31 , 37 , 6971 ] . the reduction of tumor mass induced by treatment with the toxin was associated with a decrease of the hyperalgesic response ( 40% ) ( data not shown ) . this antinociceptive effect of ctx was not mediated by opioids , since naloxone , a nonspecific opioid receptor antagonist , did not modify it . evidence has been accumulated that lxs and their analogues promote analgesic effects in bone cancer through reduction in proinflammatory mediators . nogueira - neto and colleagues showed that ctx induces a long - lasting antinociceptive effect in neuropathic pain , induced by transection of rat sciatic nerve , via central muscarinic , -adrenergic , and serotonergic receptors . therefore , the analgesic effect observed on day 5 may be a result of both reduction of the tumor mass per se and the analgesic activity described for lxs . this proposition is reinforced by data demonstrating the ability of lxs to modulate the events involved in tumor growth and cancer pain [ 2931 ] . in conclusion , ctx , the main neurotoxic component of crotalus durissus terrificus venom , reduced walker 256 tumor growth possibly due to an antiangiogenic effect . the fprs played a key role in the effect of the ctx on tumor growth . these receptors mediated the increase in plasma levels of lxa4 and 15-epi - lxa4 and also the actions of the lipid mediators .

we investigated the effects of crotoxin ( ctx ) , the main toxin of south american rattlesnake ( crotalus durissus terrificus ) venom , on walker 256 tumor growth , the pain symptoms associated ( hyperalgesia and allodynia ) , and participation of endogenous lipoxin a4 . treatment with ctx ( s.c . ) , daily , for 5 days reduced tumor growth at the 5th day after injection of walker 256 carcinoma cells into the plantar surface of adult rat hind paw . this observation was associated with inhibition of new blood vessel formation and decrease in blood vessel diameter . the treatment with ctx raised plasma concentrations of lipoxin a4 and its natural analogue 15-epi - lxa4 , an effect mediated by formyl peptide receptors ( fprs ) . in fact , the treatment with boc-2 , an inhibitor of fprs , abolished the increase in plasma levels of these mediators triggered by ctx . the blockage of these receptors also abolished the inhibitory action of ctx on tumor growth and blood vessel formation and the decrease in blood vessel diameter . together , the results herein presented demonstrate that ctx increases plasma concentrations of lipoxin a4 and 15-epi - lxa4 , which might inhibit both tumor growth and formation of new vessels via fprs .

1. Introduction 2. Material and Methods 3. Results 4. Discussion

postmortem dismemberment cases , sometimes referred to in the media as corpse mutilation , abuse of corpse or criminal dismemberment , present a challenge to investigators attempting to identify the tool used in the dismemberment . if multiple tools are recovered , examinations could be conducted to link or eliminate each of the tools used in the dismemberment . postmortem dismemberment is typically accomplished with a knife , axe , saw or a combination of these cutting tools . when these types of cutting tools are used , they can leave tool marks on cartilage and bone . sometimes these marks can be used to match or eliminate the tool [ 2 , 3 ] . even if the cutting tool can not be matched or eliminated , an examination of the tool marks can provide some information about the general characteristics of the tool [ 4 , 5 ] . cutting tools leave two types of characteristics , individual and class , that can be used to analyze tool marks . individual characteristics are random imperfections along the leading edge of the cutting tool that produce striations on a substrate ; therefore , these striations can be used to match a cutting tool to a specific tool mark . class characteristics include similarities that can place the tool in a category or eliminate it from a group [ 68 ] . for example , a saw blade that is 6.350 mm in thickness could be eliminated from a cut mark that is 3.175 mm in width . moreover , in any form of physical comparative analysis , class characteristics are examined first to determine if they are the same or similar . if a tool can be eliminated based on a difference in class characteristics , it is not necessary to search for individual characteristics . however , if the class characteristics are the same or similar , a physical match or elimination can be achieved based on the agreement or disagreement of individual characteristics . when saws are used to cut bone , false starts occur when the saw blade comes into contact with bone briefly , removes some of the bone but then stops , skips or restarts in a new position usually close to the initial cut and continues to cut the bone . the characteristic examined in this study was the kerf width of a saw cut into compact bone . the kerf mark widths were measured to determine which possible saw blade could be eliminated based on the kerf width . ten sections of bovine bone were cut into ~22.86 cm by ~5.08 cm sized pieces and used as a substrate to collect kerf marks from five human - powered and five mechanical - powered saws . the human - powered saws included : a hand saw , hack saw , meat saw , bow saw and molding cut - off saw . the mechanical - powered saws included : a reciprocating saw , circular saw , cut - off saw , portable band saw , and jig saw . of the mechanical - powered saws , the circular saw , cut - off saw and portable band saw used cutting blades with a rotating motion . kerf marks were produced on the diaphyseal sections by sawing into the cortical bone with each of the saws . the depth of each saw cut was approximately the thickness of the compact bone . figures 1 , 3 , 5 , 7 , 9 , 11 , 13 , 15 , 17 , and 19 illustrate the saw blades used and figs . 2 , 4 , 6 , 8 , 10 , 12 , 14 , 16 , 18 , and 20 illustrate the kerf for each of the blades respectively.fig . ( for boxplot values , the top line represents the maximum width , next down the 3rd quartile , median , 1st quartile and lastly the minimum width . also some boxplots have an outlier which is indicated by o ) portable band saw blade portable band saw blade kerf molding cut - off blade molding cut - off blade kerf reciprocating blade kerf circular saw blade kerf cut - off saw blade kerf boxplot of kerf widths . ( for boxplot values , the top line represents the maximum width , next down the 3rd quartile , median , 1st quartile and lastly the minimum width . also some boxplots have an outlier which is indicated by o ) dial calipers were used to measure the thickness of each saw blade . before recording measurements first , the dial caliper jaws were closed and the pointer setting was at 0 . second , using the thumbwheel , the dial caliper jaws were opened to the maximum distance and then third , they were closed . when closed , the pointer setting returned to 0 . the thickness of the blade and the teeth are reported in table 1 . the width of the teeth on mechanical - powered saw and human - powered saws can be the same size as the blade or wider than the blade . the teeth on the blades from the human - powered saws ranged from 0.025 mm to .0457 mm in size while the teeth on the mechanical saws ranged from 0.025 mm to 0.762 mm in size.table 1blade and teeth width ( mm ) characteristics for non - powered and electrical sawssaw typesbladesteethdifferenceshand saw0.9401.4000.460hack saw0.6100.6600.050meat saw0.6350.9140.279bow saw0.8890.9140.025 m . band saw0.6350.6600.025jig saw0.9401.1430.203 blade and teeth width ( mm ) characteristics for non - powered and electrical saws ten saw types were used to cut kerfs into ten separate sections of bone . each kerf was examined with a stereoscopic boom microscope equipped with a digital camera and measuring software . next , the digital camera measuring software was calibrated according to the manufacturer s instructions using a calibration disk with a 7,500 m black calibration circle that was supplied with the digital camera and measuring software . the measurement data was then subjected to statistical analysis for comparison of saw kerf widths and saw blade thicknesses.table 2saw kerf and teeth width characteristics ( mm ) for non - powered and electrical sawssaw typeskerf rangeaverage kerfteeth widthdifference between av . kerf and teethhand saw1.3971.8291.4101.3970.013hack saw0.7111.0200.8130.6600.153meat saw1.0671.2451.1181.0670.051bow saw0.9141.0410.9650.9140.051 m . cut - off0.8891.1430.9910.8890.102reciprocating1.3971.5751.4481.3970.051skill saw2.2103.1502.7182.0820.636cut - off saw2.6672.8702.7942.6670.127p . band saw0.6600.8640.7370.6600.077jig saw1.2701.4731.3461.143.203 saw kerf and teeth width characteristics ( mm ) for non - powered and electrical saws two cumulative logit models using statistical analysis system ( sas ) software were used to analyze the data in this study [ 11 , 12 ] . in statistics , logit models provide estimated probabilities given the realization of covariates compared with statistical descriptive analysis such as mean , median , standard deviation , interquartile range and range . therefore , the logit models were used to indicate the likelihood of similar categories of saw types . first , as suggested by fig . 1 , the average kerf width is an indicator of the saw type , and incorporating the order of kerf widths improves the accuracy of the model . second , there is one slope for each covariate which facilitates interpreting the effect of the covariate and provides the proportional odds property needed for analysis . lastly , in analyzing the kerf data , the quasi - complete separation problem for the data was avoided . model # 1 is\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \log { \text{it}}[p ( y \le j ) ] \,=\,\alpha_{j } + \beta x_{j } , $ $ \end{document}where the covariate x is the kerf width and j is the ascending order of the 10 saw kerf widths ( table 3 ) . the y axis in model # 1 represents the probability of saws based on kerf widths . the sas results in table 3 suggest the average kerf width is significant compared to the kerf width of other saws with a p - value less than 0.01%.table 3sas results for the cumulative regression model # 1testing global null hypothesis : beta = 0testchi - squaredfpr > chisqlikelihood ratio241.75321<0.0001score78.10151<0.0001wald68.77591<0.0001parameterdfestimatestandard errorwald chi - squarepr > chisqintercept 1111.89831.620453.9194<0.0001intercept 2113.90351.773361.4725<0.0001intercept 3115.45941.896666.4385<0.0001intercept 4117.08542.044469.8408<0.0001intercept 5119.53952.412865.5813<0.0001intercept 6121.97732.671767.6682<0.0001intercept 7123.77592.784572.9106<0.0001intercept 8130.90984.330550.9476<0.0001intercept 9144.21375.389067.3120<0.0001kerf mean ( mm)115.87591.914368.7759<0.0001analysis of maximum likelihood estimates : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \log { \text{it}}[p(y \le j)]\,=\,\alpha_{j } + \beta x. $ $ \end{document } sas results for the cumulative regression model # 1 analysis of maximum likelihood estimates : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \log { \text{it}}[p(y \le j)]\,=\,\alpha_{j } + \beta x. $ $ \end{document } the second logit model considered the kerf widths and saw teeth widths . model # 2 is\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \log { \text{it}}[p(y \le j ) ] \,=\,\alpha_{j } + \beta_{1 } x_{1 } + \beta_{2 } x_{2 } , $ $ \end{document}where x1 is the kerf width and x2 is the saw tooth width . the y axis in model 2 represents the probability of kerf widths for the middle sections . the sas results in table 4 indicate the p - value is less than 0.01% ; thus , the hypothesis that both covariates are negligible is not rejected . therefore , the maximum likelihood estimates are significant.table 4sas results for the cumulative regression model # 2testing global null hypothesis : beta = 0testchi - squaredfpr > chisqlikelihood ratio351.66292<0.0001score82.65852<0.0001wald28.66522<0.0001parameterdfestimatestandard errorwald chi - squarepr > chisqintercept 1132.73906.596324.6339<0.0001intercept 2138.53498.115022.5493<0.0001intercept 3143.21658.915423.4971<0.0001intercept 4145.11969.002325.1202<0.0001intercept 5151.466510.625423.4615<0.0001intercept 6161.623912.831423.0647<0.0001intercept 7167.039513.703623.9328<0.0001intercept 8185.7253798.40.01150.9145intercept 91118.929.464816.2826<0.0001kerf mean ( mm)112.71983.648412.15480.0005teeth134.62669.466713.37910.0003analysis of maximum likelihood estimates : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \log { \text{it}}[p(y \le j)]\,=\,\alpha_{j } + \beta_{1 } x_{1 } + \beta_{2 } x_{2 } , $ $ \end{document } sas results for the cumulative regression model # 2 analysis of maximum likelihood estimates : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \log { \text{it}}[p(y \le j)]\,=\,\alpha_{j } + \beta_{1 } x_{1 } + \beta_{2 } x_{2 } , $ $ \end{document } figure 21 shows the boxplots of the kerf widths from the ten saws considered . when the kerf width is approximately 2.6 mm , the probability that the saw is a cut - off saw or a circular saw is approximately 0.50 . it suggests that the cut - off saw and circular saw share similar medians ; however , the kerf widths for the circular saw are more varied than the cut - off saw . this variation is possibly due to single or multiple factors . some factors that could account for the differences in range of widths between the two saws are hand , wrist or arm movement while sawing which may affect the lateral movement of the blade , cutting rate , and blade vibration . also , wear on the bearings could account for differences in the range of widths between kerfs produced by the two saws . the cut - off saw is attached to a steel frame and the saw movement is controlled by a hinge . figure 1 suggests that there is a linear pattern among the other eight saws . in order to estimate the prediction error of the models , repeated stratified therefore , regular cross - validation might not be sufficient , while repeated cross - validation provides more evidence about the prediction error of the models . tables 5 and 6 contain the simulation results of the repeated stratified 10-fold cross - validation , where table 5 is generated from model # 1 and table 6 is generated from model # 2 . the simulation in r was run with a random seed number 68 to ensure that the results were reproducible . the replication time was 100.table 5prediction results for model # 1 using stratified 10-fold cross validation with 100 replicationssaw typepredicted from model # 1hand sawjig saw ( 22% ) reciprocating ( 30% ) and hand saw ( 48%)hack sawportable band ( 39% ) hack saw ( 35% ) bow saw ( 19% ) and molding cut - off ( 7%)meat sawmeat saw ( 89% ) and jig saw ( 11%)bow sawbow saw ( 54% ) and molding cut - off saw ( 46%)molding cut offhack saw ( 8% ) bow saw ( 52% ) molding cut - off saw ( 25% ) and meat saw ( 15%)reciprocatingjig saw ( 5% ) reciprocating saw ( 60% ) hand saw ( 35%)skill sawskill saw ( 60% ) and cut off - saw ( 40%)cut - off sawskill saw ( 31% ) and cut off - saw ( 69%)portable bandportable band saw ( 76% ) and hack saw ( 24%)jig sawjig saw ( 82% ) reciprocating saw ( 12% ) hand saw ( 6%)table 6prediction results for model # 2 using stratified 10-fold cross validation with 100 replicationssaw typepredicted from model # 2hand sawreciprocating ( 52% ) and hand saw ( 48%)hack sawportable band ( 39% ) hack saw ( 61%)meat sawmeat saw ( 100%)bow sawbow saw ( 34% ) and molding cut - off saw ( 47% ) meat saw ( 19%)molding cut offbow saw ( 83% ) molding cut - off saw ( 10% ) and meat saw ( 7%)reciprocatingreciprocating saw ( 65% ) hand saw ( 35%)skill sawskill saw ( 100%)cut - off sawcut off - saw ( 100%)portable bandportable band saw ( 88% ) and hack saw ( 12%)jig sawjig saw ( 100% ) prediction results for model # 1 using stratified 10-fold cross validation with 100 replications prediction results for model # 2 using stratified 10-fold cross validation with 100 replications comparisons were made between the two statistical logit models . model # 1 was used to calculate possible saw blade eliminations and likelihood similarities to other saws based on kerf width measurements . based on this model , there is a 48% chance that the hand saw kerf originated from the hand saw , a 22% chance that the hand saw kerf was from the jig saw and a 30% chance that it was from the reciprocating saw . the least accurate predictions for model # 1 were with the hack saw and molding cut - off saw . there was only a 35% chance that the kerf from the hack saw originated from the hack saw and a 39% chance that kerf was from the portable band saw . for the molding cut - off saw , there was a 25% chance that the molding cut - off saw kerf originated from the molding cut - off saw . there was a 52% chance that the molding cut - off saw kerf was from the bow saw . model # 2 was used to calculate possible saw blade eliminations and likelihood similarities to other saws using kerf widths as well as saw teeth widths . all saws were eliminated from the meat saw , circular saw , cut - off saw and jig saw . however , the bow saw kerf was predicted to have a 34% chance of originating from the bow saw and a 47% chance of originating from the molding cut - off saw . also , the molding cut - off saw kerf width had only a 10% chance of originating from the molding cut - off saw and an 83% chance of originating from the bow saw . in summary , statistical model # 2 is a more accurate model for predicting the possible saw types based on kerf widths used in this study . the logit models have limitations when some kerf marks can not be separated . therefore , the logistic regression provides an estimated probability of each category and it is possible that two or more categories may share similar probabilities given a specific covariate value , namely , the kerf width . when the categories are not separable , there may be a higher false probability rate . for example , in model # 1 the bow saw and molding cut - off saw present limitations due to similarities of the kerf marks . also , in model # 2 the kerf marks produced by the bow saw and molding cut - off saw present limitations because they can not be separated . saw characteristics affecting the kerf width and bone surface adjacent to the kerf include style and design of the teeth , width of the teeth , teeth per inch ( tpi ) , degree of wear on the teeth , saw cutting speed , blade vibration , defects in the blade or erratic sawing motion . also , a worn bushing in mechanical - powered saws can affect kerf widths . with the exception of band saw type saws , mechanical - powered saws generally require thicker blades than human - powered saws due to saw power and operating speeds . consequently , these saws produce broader kerf widths than human - powered saws [ 1315 ] . for example , fire can result in a loss of structural integrity causing bone to easily fracture . it can obscure some of the individual and class characteristics and also cause shrinkage in bone [ 1618 ] . postmortem dismemberment cases are sometimes referred to in the media as corpse mutilation , abuse of corpse and criminal dismemberment.the style and design of the teeth , width of the teeth , teeth per inch ( tpi ) , degree of wear on the teeth , saw cutting speed , blade vibration , defects in the blade , and erratic sawing motion are factors that affect the kerf . accurate microscopic measurements may be more difficult when the kerf shoulders are fibrous.analyzing kerf mark measurements can be an effective method for predicting and eliminating possible saw blades by comparing the width of the blade to the width of the kerf.statistical model # 2 provides more accurate predictions of possible saw types based on kerf widths than model # 1 . however , both models have limitations ; when the categories can not be separated , there may be higher false probability rates.the boxplot of kerf widths in fig . 21 provides the investigator with a reference guide for eliminating saw types based on kerf width range calculations . postmortem dismemberment cases are sometimes referred to in the media as corpse mutilation , abuse of corpse and criminal dismemberment . the style and design of the teeth , width of the teeth , teeth per inch ( tpi ) , degree of wear on the teeth , saw cutting speed , blade vibration , defects in the blade , and erratic sawing motion are factors that affect the kerf . analyzing kerf mark measurements can be an effective method for predicting and eliminating possible saw blades by comparing the width of the blade to the width of the kerf . statistical model # 2 provides more accurate predictions of possible saw types based on kerf widths than model # 1 . however , both models have limitations ; when the categories can not be separated , there may be higher false probability rates . the boxplot of kerf widths in fig . 21 provides the investigator with a reference guide for eliminating saw types based on kerf width range calculations .

saw marks on bone have been routinely reported in dismemberment cases . when saw blade teeth contact bone and the bone is not completely sawed into two parts , bone fragments are removed forming a channel or kerf . therefore , kerf width can approximate the thickness of the saw blade . the purpose of this study is to evaluate 100 saw kerf widths in bone produced by ten saw types to determine if a saw can be eliminated based on the kerf width . five measurements were taken from each of the 100 saw kerfs to establish an average thickness for each kerf mark . ten cuts were made on 10 sections of bovine bone , five with human - powered saws and five with mechanical - powered saws . the cuts were examined with a stereoscopic microscope utilizing digital camera measuring software . two statistical cumulative logistic regression models were used to analyze the saw kerf data collected . in order to estimate the prediction error , repeated stratified cross - validation was applied in analyzing the kerf mark data . based on the two statistical models used , 7090% of the saws could be eliminated based on kerf width .

Introduction Methods Statistical analysis Results Discussion Key Points

despite advancements in airway management , treatment of fractures in the maxillofacial region under general anesthesia remains a unique anesthetic challenge . we reviewed the pattern of airway management in patients with maxillofacial fractures and assessed those challenges associated with the different airway management techniques employed . the anesthetic chart , theatre and maxillofacial operations records of patients who had reduction and immobilization of various maxillofacial fractures over a 2-year period were reviewed . information obtained included the patient demographics , mechanisms of injury , types of fractures and details about airway management . statistical package for social sciences , spss version 17.0 was utilized for all data analysis . mask ventilation was easy in 8090% of the patients , 80% had mallampati three or four , while 4 ( 7.8% ) had laryngoscopy grading of 4 . there was no statistically significant difference between the fracture groups in terms of the laryngoscopy grading ( p = 0.153 ) but there was statistical significant difference in the technique of airway management ( p = 0.0001 ) . nasal intubation following direct laryngoscopy was employed in 64.7% of the patients , fiber - optic guided nasal intubation was utilized in only 7.8% . laryngoscopic grading and not adequacy of mouth opening predicted difficult intubation in this group of patients in the immediate preoperative period . despite the distortions in the anatomy of the upper airway that may result from maxillofacial fractures , nasal intubation following direct laryngoscopy treatment of fractures in the maxillofacial region , just like in other parts of the body , is by reduction and immobilization either under local or general anesthesia . the maxillofacial region being a shared field between the surgeon and the anesthetist is naturally a challenge for both specialists . in addition to this , when traumatic maxillofacial injury results in the disruption of the normal anatomy from edema or structural damage to tissues , bleeding , unstable skeletal structures , and foreign bodies that can interfere with instrumentation or easily get dislodged , a situation that requires sound and experienced anesthetic judgment is usually presented . these injuries could present in various ways and patterns , each with its own unique anesthetic challenges , requiring different modes of airway management based on their merit . hence , we reviewed airway management techniques in 51 patients with varied forms of maxillofacial fractures who had reduction and immobilization under general anesthesia over a 2-year period to determine the pattern of airway management employed in our center and challenges associated with each technique . the anesthetic chart , theater and maxillofacial operations records of patients who had reduction and immobilization of various maxillofacial fractures at the university college hospital , ibadan from december 2009 to november 2011 were reviewed . information obtained included the patient demographics , mechanisms of injury , types of fractures , and details about airway management . all variables were expressed as the number of cases / percentages or means with standard deviations . statistical package for social sciences ( spss ) version 17.0 , chicago il . was utilized for all data analysis . a total of 51 patients had reduction and immobilization of facial fractures under general anesthesia during the 2-year period . age range was 4 - 58 years with a mean of 29.35 ( 12.07 ) years . majority of the patients were in the age group of 20 - 29 years ( 21 patients , 41.2% ) with only seven patients ( 13.7% ) above 40 years [ table 1 ] . furthermore , 43 ( 84.3% ) and 8 ( 15.7% ) were males and females , respectively . motor vehicular and motor cycle crashes were responsible for maxillofacial fractures in 39 ( 76.5% ) and 8 ( 15.7% ) of patients , respectively [ table 1 ] . patient characteristics the most common type of maxillofacial fracture requiring treatment under general anesthesia was mandibular fracture ( 16 patients , 31.4% ) , followed by panfacial fracture ( 15 patients , 29.4% ) , with the least being nasal complex fracture ( three patients , 5.9% ) [ table 1 ] . mallampati grading were as follows ; 5 ( 9.8% ) patients each had grade 1 and 2 , 16 ( 31.4% ) had grade 3 , and 25 ( 49.0% ) had grade 4 . mask ventilation was easy in 80 - 90% of patients in the different fracture groups . furthermore , 43 ( 84.3% ) of patients in this review had laryngoscopy grading 1 and 2 . however , one patient each in the mandibular and zygomatic fracture groups ( 6.7% , 16.2% ) , and two patients in the maxillary fracture group ( 18.2% ) had laryngoscopy grading of 4 [ table 2 ] . there was no statistically significant difference between the fracture groups in terms of the laryngoscopy grading ( p = 0.153 ) . laryngoscopy grading in the different fracture groups nasal intubation following direct laryngoscopy and orotracheal intubation were employed in 33 ( 64.7% ) and 14 ( 27.5% ) of patients across the different fracture groups respectively [ table 3 ] . there was no statistical significant difference in the use of nasotracheal intubation in the different fracture groups ( p = 0.120 ) . fiber - optic guided nasal intubation was utilized only in those patients with laryngoscopy grading of 4 that is , two patients with maxillary fracture , and one in the zygomatic and mandibular fracture groups , respectively ( p = 0.001 ) [ figure 1 ] . there was statistical significant difference in the technique of airway management for the different fracture groups ( p = 0.0001 ) laryngoscopic grading and airway management technique types of fractures related to mask ventilation , laryngoscopy grading , and airway management techniques road traffic injuries are becoming increasingly common in developing countries due to fast vehicular traffic on the highways and poor traffic management . it is of great importance to the society because young productive lives are often involved , and there is a male predominance , our review and some others show this . in this study , this ratio is more than that reported in other countries ( 3:1 in england , france , and jordan ; 2.8:1 in the united states ) . the majority of patients in these studies were between 20 and 29 years of age , as was the case in our study . maxillofacial fractures are commonly caused by road traffic crashes ( rtc ) , assaults , sports , industrial accidents , and warfare . in this study , rtc involving cars and motorcycles were the most common cause of these fractures , comprising 92% of the etiology of the fractures . this figure was 40% in one study from the united states , 24.7% in one study from england , 48% in a study from france , and 55.2% in a study from jordan . the trauma usually disrupts the facial anatomy with associated edema and bleeding in the oral cavity . it may be difficult for the mask to be properly close - fitted to the face , to enable effective mask ventilation as seen in some of our patients . in these patients , endotracheal tube was passed through one of the nostrils up to the nasopharynx , the patient 's mouth and nostrils were closed with the fingers and thumbs of both hands , while another person manually ventilated the patient using a breathing system connected to the endotracheal tube . the challenge in performing endotracheal intubation arises mainly from the difficulty in visualizing the vocal cords with conventional direct laryngoscopy . as observed in this review , 80% had mallampati grading of 3 or 4 , however , only 4 ( 7.8% ) of them could not be intubated using direct laryngoscopy . this shows that pain is mainly responsible for the limited mouth opening seen in this group of patients and as such effective analgesia and muscle relaxation prior to laryngoscopy provided adequate mouth opening for intubation . from the foregoing , laryngoscopic grading and not adequacy of mouth opening predicted difficult intubation in this group of patients in the immediate preoperative period . an adequate preoperative airway assessment and well thought out plan of action for airway management are mandatory before reduction and immobilization of facial fracture under anesthesia . this includes , but not limited to radiological investigations to reveal temporomandibular joint injury / comminuted facial fractures and other means of providing an emergency airway if the primary airway technique fails . although there are many options available for performing endotracheal intubation in patients with a facial fracture presenting for reduction and immobilization , a lot depends on patient 's situation , expertise and facilities available . a full range of difficult airway management tools is prerequisite to safe airway management in this group of patients . this includes , but not limited to ; laryngeal mask airways , combitube , gum elastic bougie , video laryngoscopes , and tools for providing emergency surgical airway . in our center , because of the additional cost of care and success often recorded with endotracheal intubation following a direct laryngoscopy in most of our patients , fiberoptic bronchoscopic assistance is usually reserved for those few cases of failed intubation . this is in contrast to the practice of routinely employing fiberoptic - assisted endotracheal intubation for patients with significant limited mouth opening . proponents of the later claim there is a higher success with the use of fiberoptic bronchoscope if employed electively . the relative success we have with endotracheal intubation may not be unconnected to the early presentation of our patients after injury that is , before fracture healing . furthermore , the presence of minimally displaced fractures in most of our patients [ figure 2 ] may be responsible for the success with direct nasotracheal intubation , as posteroinferior displacement of a fractured maxilla parallel to the inclined plane of the skull base often blocks the nasopharyngeal airway . nasotracheal intubation in a patient with multiple facial fractures in the absence of fiberoptic bronchoscope , blind nasal intubation is a common simple technique employed and often successful in the hands of experienced anesthetists . however , it has two major drawbacks : infrequent success on the first pass and increased trauma with repeated attempts , precipitating complete airway obstruction that necessitates emergency cricothyrotomy . the use of gum elastic bougie for patients with laryngoscopic grading 2 and 3 helped with endotracheal intubation and prevented the use of blind nasal intubation and/or fibreoptic guided intubation in about 70% of our patients . this supports the need to be equipped with the full range of airway management tools during anaesthetic care in this group of patient . in an attempt to avoid both tracheostomy and nasotracheal intubation in patients with basilar skull fracture , orotracheal intubations can be used in some patients especially when the procedure precludes maxillomandibular fixation ( mmf ) . in cases where mmf is required , the passage of an armored endotracheal tube through the floor of the mouth ( submental ) has been advocated [ figure 3 ] . both anterior and lateral submandibular routes have been described in the surgical management of severe panfacial or maxillary fractures . the technique , in its various forms , is said to be relatively simple and safe to perform and produces a cosmetically acceptable scar . it is further claimed that it may be safely used for elective ventilation for periods of up to 10 days . in a review , submental intubation was found to be safe , but increased tracheal pressure , as a result , of deviation and compression of tube was observed . orotracheal tube tunneled submentally in view of availability of video laryngoscopes , it is now uncommon to give surgical airway to patients with maxillofacial fracture coming for reduction and immobilization , except for patients with severe maxillofacial injuries or failed intubation . despite the distortions in the anatomy of the upper airway that may result from maxillofacial fractures , nasal intubation with direct visualization of the vocal cords may still be possible in many of patients with maxillofacial fractures , especially when the injury spares the temporomandibular joint . however , some patients with maxillofacial fractures will require more advanced form of airway management like fiberoptic - guided intubation , especially those with displaced fractures . though nasal intubation allows for surgical access and reduces the chances of dislodgement of the tracheal tube , the aim must be to employ the best method that effectively secures and maintains the airway . the presence of a skillful anesthetist and collaboration with maxillofacial and ent surgeons are mandatory to ensure the best form of airway management and a successful outcome .

background : despite advancements in airway management , treatment of fractures in the maxillofacial region under general anesthesia remains a unique anesthetic challenge . we reviewed the pattern of airway management in patients with maxillofacial fractures and assessed those challenges associated with the different airway management techniques employed.materials and methods : the anesthetic chart , theatre and maxillofacial operations records of patients who had reduction and immobilization of various maxillofacial fractures over a 2-year period were reviewed . information obtained included the patient demographics , mechanisms of injury , types of fractures and details about airway management . statistical package for social sciences , spss version 17.0 was utilized for all data analysis.results:fifty-one patients were recruited during the 2-year study period . mask ventilation was easy in 8090% of the patients , 80% had mallampati three or four , while 4 ( 7.8% ) had laryngoscopy grading of 4 . there was no statistically significant difference between the fracture groups in terms of the laryngoscopy grading ( p = 0.153 ) but there was statistical significant difference in the technique of airway management ( p = 0.0001 ) . nasal intubation following direct laryngoscopy was employed in 64.7% of the patients , fiber - optic guided nasal intubation was utilized in only 7.8% . none of the patients had tracheostomy either before or during operative management.conclusion:laryngoscopic grading and not adequacy of mouth opening predicted difficult intubation in this group of patients in the immediate preoperative period . despite the distortions in the anatomy of the upper airway that may result from maxillofacial fractures , nasal intubation following direct laryngoscopy may be possible in many patients with maxillofacial fractures .

Background: Materials and Methods: Results: Conclusion: I M R D

certain colorectal conditions , such as ulcerative colitis and diffuse polyposis of the colon and rectum , and congenital intestinal abnormalities , such as total colonic aganglionosis , require a proctocolectomy and some type of intestinal restoration 1 . although these procedures are technically demanding for surgeons , they are more challenging for the patients following the restoration of intestinal continuity . frequency of bowel evacuations typically increases dramatically postsurgery , contributing to induced metabolic derangements 2,3 . we present a case of juvenile polyposis coli , initially treated with a subtotal colectomy and ileostomy , followed by a proctectomy , ileal - j - pouch and serial transverse enteroplasty ( step ) of the distal ileum 4 . an eight - year - old female patient was referred to our department with a chronic history of unremitting bloody diarrhea , weight loss , anemia and intractable rectal prolapse . there was no other significant past medical history or family history and her vaccinations were up to date . the initial clinical examination revealed a pale child who was underweight for age with mild clubbing of the fingernails . the abdominal examination was unremarkable , and the rectal examination was unnecessary because the cause of the prolapsed rectum was obvious , with a presentation of multiple polyps ( fig1 ) . blood tests confirmed clinical evident anemia with a hemoglobin of 7 g / dl , and her albumin level was 19 rectal polyposis was ratified by sight ( fig1 ) , and total colonic involvement was suspected on the contrast enema ( fig2 ) and was substantiated by colonoscopy ( fig3 ) and by the subtotal colectomy specimen ( fig4 ) . the histological diagnosis of hamartomatous juvenile polyposis was confirmed from the colonoscopic biopsies and the operative specimen . despite the initial temporizing approach of subtotal colectomy and ileostomy , anemia and protein - losing enteropathy persisted . the patient was discharged with dietary supplementation , and she had regular outpatient follow - up . the child s nutritional status improved over time , and she had albumin levels consistently over 36 a proctectomy was performed to remove the residual rectal polyps , and the ileostomy was taken down and fashioned as a j - pouch ( fig5 ) . multiple serial enteroplasties ( fig6 ) starting just above the pouch over a length of approximately 80 cm was performed , followed by a hand sewn ileo - anal anastomosis . the postoperative course was uneventful , and the patient was discharged 2 weeks later . at that stage , she was passing two soft stools per day without any medication or dietary restrictions . the follow - up at 3 months postsurgery confirmed a well - child who had gained 1.5 kg . she had returned to school and continued to pass one to two soft stools per day . restorative proctocolectomy has replaced disparate surgical options such as a hartmann s procedure and permanent ileostomy or ileo - rectal anastomoses because of their technical inadequacy , inability to improve quality of life and persistent cancer risk 5,6 . it removes the diseased intestine , thereby improving quality of life and reducing cancer risk 3 . intestinal leaks and pouchitis are major morbidities that might result in a long term or permanent ileostomy . an increased frequency and volume of enzyme - rich liquid stools although dietary restrictions and opioids might temporarily reduce the symptoms , they fail to cure these patients 7,8 . in these cases , the postoperative quality of life is indisputably not better than the pre - operative status . it is well reported that patients affected by severe short bowel syndrome benefit from the step procedure , because such an intervention can significantly reduce the frequency of stooling 913 . such large volumes must therefore have time and an appropriate intestinal length for water , electrolytes and other nutrients to be absorbed . this process is expected to occur within the j or other pouch configurations following restoration 14,15 . these pouches are quickly filled , and they easily surge ; the anal sphincter does not effectively counteract and contain the volume , which is usually controlled by the ileocaecal valve . large amounts of nutrients , electrolytes and water are wasted because evacuation is inevitable and continence training is not possible . step is a technique for lengthening the intestine in short bowel syndrome by interrupting the intestinal lumen thereby also slowing peristalsis . this procedure allows time for the nutrients , water and electrolytes contained within the intestinal lumen ( and distant from the distal anal anastomosis ) to be absorbed 4,11 . the outcome of our technical option was rewarding because the stool frequency and consistency were immediately in an acceptable range of two to three times per day and a soft consistency , respectively . this outcome was achieved without the use of opioids or the need for dietary restrictions . the child tolerated an unrestricted hospital diet and was discharged after 2 weeks when we were certain of her bowel habits and that she had maintained her physiological parameters . at 3 months follow - up , she was growing well and maintained a stool frequency of one to two soft stools per day . stool frequency can be crippling following proctocolectomy , and although opioids might provide temporary relief of the symptoms , they can not prevent relapses . we believe that step alone , performed in an adequate length of bowel , can control the stooling of such patients .

key clinical messagewe present a pediatric case of medically unmanageable juvenile colonic polyposis , initially treated with subtotal colectomy and an ileostomy followed by a proctectomy , ileal - j - pouch and serial transverse enteroplasties ( step ) of the distal ileum . the step procedure in an adequate length was able to control stooling of our patient .

Introduction Clinical case Discussion Conflict of Interest

children between 3 and 10 years of age with type 1 diabetes for at least 6 months and healthy controls subjects were recruited . all procedures were approved by the institutional review board and all parents or guardians signed informed consents . children over 7 years of age signed assents ( clinicaltrials.gov study nct # 00449891 ) . diabetes history , time - weighted average hba1c ( calculated using the trapezoid rule for all hba1c values from the time of diagnosis ) , cognitive testing , and mri training were completed as previously described ( 12 ) . subjects completed the wechsler preschool and primary scale of intelligence iii ( wppsi - iii ) or wechsler intelligence scale for children iv ( wisc - iv ) based on their age . all subjects were included in the full - scale intelligence quotient ( fsiq ) scores analysis , but the younger subjects were excluded from the wisc - iv score analyses . the time - weighted hba1c value is used for all data analyses . bg values were between 80 and 250 mg / dl before the start of any neurocognitive testing or mri scan . we specifically examined neurocognitive skills associated with wm function using tasks specifically designed for children , including subtests from the wisc - iv ( block design digit span , picture concepts , vocabulary , letter - numbering sequencing , matrix reasoning , and comprehension ) and the developmental neuropsychological assessment ( nepsy ) ( design copy , phonological processing , memory of faces , tower , auditory attention and response , speeded naming , arrows , delayed memory of faces , comprehension of instructions , imitating hand positions , and narrative memory ) . subjects underwent high - resolution structural mri scans using a general electric signal 1.5 tesla imaging system . coronal three - dimensional volumetric spoiled grass gradient recalled series were acquired with the following scan parameters : repetition time ( tr ) 35 ms , echo time ( te ) 6 ms , flip angle 45 , 1.5 mm thick , 0-mm gap , number of excitations 1 , field of view ( fov ) 24 cm , and a 256 192 matrix size for 124 contiguous slices ( scan time : 14 min ) . imaging parameters for the diffusion - weighted sequence were as follows : fov , 24 cm ; matrix size , 128 128 ; tr / te 5,400/min ; 43 axial - oblique slices ; slice thickness , 3.2 mm / no skip . diffusion gradient duration was = 32 ms and diffusion weighting was b = 850 s / mm , and b = 0 as reference images . diffusion - weighted images were corrected for eddy current distortions and head motion using an affine transformation of automated image registration ( 16 ) . we excluded six subjects ( four with diabetes , two control subjects ) from further analysis because of significant artifacts . the remaining images were averaged and the pixel intensities of the multiple diffusion - weighted images were then fitted to obtain the six elements of the symmetric diffusion tensor . scalars such as fractional anisotropy ( fa ) , axial diffusivity ( ad ) , and radial diffusivity ( rd ) were calculated using dtistudio ( https://www.mristudio.org/ ) . anisotropy within a given wm voxel is determined by fiber diameter and density , degree of myelination , extracellular diffusion , interaxonal spacing , and intravoxel fiber -tract coherence . ad is the diffusivity of water molecules along the main axis of diffusion within a voxel ( vector with the largest eigenvalue ) , and rd is the mean of the diffusivities perpendicular to the vector with the largest eigenvalue . ad is thought to reflect fiber coherence , whereas rd is thought to represent fiber integrity and myelination ( 17,18 ) . first , fa images from each subject were aligned into a common space using nonlinear and linear registrations . a skeletonization algorithm was applied to the group mean fa image to define a group template of the lines of maximum fa , thought to correspond to centers of wm tracts . the original registration parameters of the fa were then applied to the ad and rd images . fa , ad , and rd data projected onto the skeleton were fed into voxel - wise cross - subject statistics ( p < 0.05 ) using randomise ( v. 2.1 in fsl4.1 ) , a permutation program used for inference ( thresholding ) on statistic maps when the null distribution is not known ( 19 ) . all analyses were corrected for multiple comparisons ( family - wise error ) and used threshold - free cluster enhancement ( 20 ) with default parameters . statistical analyses of behavioral and cognitive data were performed using spss software version 13 ( spss inc . , index and specific subtest scores were compared between diabetes and hc subjects using t tests . among those with diabetes , linear correlations were performed between wisc - iv and nepsy subtest scores and hba1c and number of seizures all scores are reported as mean values and standard deviation . we further analyzed correlations between cognitive data , time - weighted hba1c , age at diagnosis , duration of illness , history of seizures , and wm structure ( fa , ad , and rd ) using tbss . diffusion - weighted images were corrected for eddy current distortions and head motion using an affine transformation of automated image registration ( 16 ) . we excluded six subjects ( four with diabetes , two control subjects ) from further analysis because of significant artifacts . the remaining images were averaged and the pixel intensities of the multiple diffusion - weighted images were then fitted to obtain the six elements of the symmetric diffusion tensor . scalars such as fractional anisotropy ( fa ) , axial diffusivity ( ad ) , and radial diffusivity ( rd ) were calculated using dtistudio ( https://www.mristudio.org/ ) . anisotropy within a given wm voxel is determined by fiber diameter and density , degree of myelination , extracellular diffusion , interaxonal spacing , and intravoxel fiber -tract coherence . ad is the diffusivity of water molecules along the main axis of diffusion within a voxel ( vector with the largest eigenvalue ) , and rd is the mean of the diffusivities perpendicular to the vector with the largest eigenvalue . ad is thought to reflect fiber coherence , whereas rd is thought to represent fiber integrity and myelination ( 17,18 ) . first , fa images from each subject were aligned into a common space using nonlinear and linear registrations . a skeletonization algorithm was applied to the group mean fa image to define a group template of the lines of maximum fa , thought to correspond to centers of wm tracts . the original registration parameters of the fa were then applied to the ad and rd images . fa , ad , and rd data projected onto the skeleton were fed into voxel - wise cross - subject statistics ( p < 0.05 ) using randomise ( v. 2.1 in fsl4.1 ) , a permutation program used for inference ( thresholding ) on statistic maps when the null distribution is not known ( 19 ) . all analyses were corrected for multiple comparisons ( family - wise error ) and used threshold - free cluster enhancement ( 20 ) with default parameters . statistical analyses of behavioral and cognitive data were performed using spss software version 13 ( spss inc . , chicago , il ) . index and specific subtest scores were compared between diabetes and hc subjects using t tests . among those with diabetes , linear correlations were performed between wisc - iv and nepsy subtest scores and hba1c and number of seizures all scores are reported as mean values and standard deviation . we further analyzed correlations between cognitive data , time - weighted hba1c , age at diagnosis , duration of illness , history of seizures , and wm structure ( fa , ad , and rd ) using tbss . we approached 81 subjects with type 1 diabetes : 35 by e - mail or phone and 46 directly in the clinic . likewise , 30 healthy control subjects , mostly siblings or friends of our subjects , were directly approached in person . forty - five subjects ( n = 27 diabetes , n = 18 healthy control subjects ) completed the neurocognitive testing , and 42 ( n = 26 diabetes , n = 16 healthy control subjects ) of them completed dti scans . six subjects ( n = 4 diabetes , n = 2 healthy control subjects ) were excluded from further analysis because of significant image artifacts . the data presented in this manuscript are restricted to the subset of subjects who completed both the neurocognitive testing and the dti scans ( table 1 ) . wppsi - iii and wisc - iv results were very similar ( p = 0.38 ) for fsiq score for subjects with diabetes ( wppsi - iii 109 4.2 ; wisc - iv 108.4 14.7 ) and healthy control subjects ( wppsi - iii 107 11.0 ; wisc - iv 112.7 13.5 ) . there were no statistically significant differences in general cognitive ability in regard to executive function , processing speed , memory / attention , and motor domains between subjects with diabetes and healthy controls ( all p values > 0.05 ) . however , calculations of the effect size are approximately 0.30 ( cohen s d ) , suggesting that there may be a modest difference between the two groups wisc scores . there were no significant between - group differences on nepsy scores ( all p values > 0.05 ) . within the diabetes group , higher hba1c levels were correlated with lower overall intellectual functioning measured by fsiq ( r = 0.215 , p = 0.03 ) ( fig . 1 ) . there was a nearly statistically significant relationship between greater seizure occurrence and lower wisc - iv verbal comprehension index scores ( r = 0.173 , p = 0.054 ) , as well as with hba1c levels and wisc - iv digit span ( r = 0.214 , p = 0.053 ) . lastly , greater seizure number was predictive of lower wisc - iv processing speed index scores ( r = 0.402 , p < 0.01 ) . greater hba1c levels predicted lower overall intellectual functioning measured by fsiq in children with type 1 diabetes ( r = 0.215 , p = 0.03 ) . compared with controls , children with diabetes had lower ad values ( p = 0.046 covaried for age and gender ) in the internal capsule , body of the corpus callosum ( cc ) , right cingulate gyrus , right thalamus , right superior temporal gyrus , and posterior parietal lobe ( fig . 2 ) . there were no significant differences between groups in fa and rd . within the diabetes group , there was a statistically significant , positive correlation between time - weighted hba1c and rd ( p = 0.028 ) ( fig . specifically , this correlation was seen in the inferior fronto - occipital fasciculi , uncinate fasciculi , subgenual wm , anterior forceps , right internal capsule , superior middle and inferior temporal gyri , splenium of the cc , superior longitudinal fasciculi , and occipital wm . a negative correlation between hba1c values and fa approached significance ( p = 0.057 ) in the right internal capsule , the anterior forceps , inferior fronto - occipital fasciculi , and splenium of the cc . regions of significant reductions in ad ( shown in yellow ) in children with type 1 diabetes as compared with healthy controls subjects , shown in serial images in the axial orientation . ( a high - quality digital representation of this figure is available in the online issue . ) regions of significant positive correlation between hba1c values and rd ( shown in light blue ) within the type 1 diabetes group , shown in serial images in the axial orientation . group differences were thickened for visualization purposes ( shown in dark blue ) . ( a high - quality digital representation of this figure is available in the online issue . ) within - group analysis of those with type 1 diabetes detected a positive correlation between fa and wisc coding ( p = 0.034 ) and digit span ( p = 0.028 ) subtests , and positive correlations between fa and wisc fsiq ( p = 0.064 ) and fa and wisc ( p = 0.058 ) processing speed approached significance . higher fa values correlated with the subjects abilities to focus attention and quickly scan , discriminate between , and sequentially order visual information . there was a significant negative correlation between rd and wisc fsiq ( p = 0.022 ) and rd and nepsy auditory attention score ( p = 0.02 ) . the negative correlation between rd and nepsy attention / executive function approached statistical significance ( p = 0.067 ) . wppsi - iii and wisc - iv results were very similar ( p = 0.38 ) for fsiq score for subjects with diabetes ( wppsi - iii 109 4.2 ; wisc - iv 108.4 14.7 ) and healthy control subjects ( wppsi - iii 107 11.0 ; wisc - iv 112.7 13.5 ) . there were no statistically significant differences in general cognitive ability in regard to executive function , processing speed , memory / attention , and motor domains between subjects with diabetes and healthy controls ( all p values > 0.05 ) . however , calculations of the effect size are approximately 0.30 ( cohen s d ) , suggesting that there may be a modest difference between the two groups wisc scores . there were no significant between - group differences on nepsy scores ( all p values > 0.05 ) . within the diabetes group , higher hba1c levels were correlated with lower overall intellectual functioning measured by fsiq ( r = 0.215 , p = 0.03 ) ( fig . 1 ) . there was a nearly statistically significant relationship between greater seizure occurrence and lower wisc - iv verbal comprehension index scores ( r = 0.173 , p = 0.054 ) , as well as with hba1c levels and wisc - iv digit span ( r = 0.214 , p = 0.053 ) . lastly , greater seizure number was predictive of lower wisc - iv processing speed index scores ( r = 0.402 , p < 0.01 ) . greater hba1c levels predicted lower overall intellectual functioning measured by fsiq in children with type 1 diabetes ( r = 0.215 , p = 0.03 ) . compared with controls , children with diabetes had lower ad values ( p = 0.046 covaried for age and gender ) in the internal capsule , body of the corpus callosum ( cc ) , right cingulate gyrus , right thalamus , right superior temporal gyrus , and posterior parietal lobe ( fig . 2 ) . there were no significant differences between groups in fa and rd . within the diabetes group , there was a statistically significant , positive correlation between time - weighted hba1c and rd ( p = 0.028 ) ( fig . specifically , this correlation was seen in the inferior fronto - occipital fasciculi , uncinate fasciculi , subgenual wm , anterior forceps , right internal capsule , superior middle and inferior temporal gyri , splenium of the cc , superior longitudinal fasciculi , and occipital wm . a negative correlation between hba1c values and fa approached significance ( p = 0.057 ) in the right internal capsule , the anterior forceps , inferior fronto - occipital fasciculi , and splenium of the cc . regions of significant reductions in ad ( shown in yellow ) in children with type 1 diabetes as compared with healthy controls subjects , shown in serial images in the axial orientation . ( a high - quality digital representation of this figure is available in the online issue . ) regions of significant positive correlation between hba1c values and rd ( shown in light blue ) within the type 1 diabetes group , shown in serial images in the axial orientation . group differences were thickened for visualization purposes ( shown in dark blue ) . ( a high - quality digital representation of this figure is available in the online issue . ) within - group analysis of those with type 1 diabetes detected a positive correlation between fa and wisc coding ( p = 0.034 ) and digit span ( p = 0.028 ) subtests , and positive correlations between fa and wisc fsiq ( p = 0.064 ) and fa and wisc ( p = 0.058 ) processing speed approached significance . higher fa values correlated with the subjects abilities to focus attention and quickly scan , discriminate between , and sequentially order visual information . there was a significant negative correlation between rd and wisc fsiq ( p = 0.022 ) and rd and nepsy auditory attention score ( p = 0.02 ) . the negative correlation between rd and nepsy attention / executive function approached statistical significance ( p = 0.067 ) . neuroimaging techniques have been used to further our understanding of how medical diseases affect brain structure ( 21,22 ) . in our study , we examined how type 1 diabetes affects wm microstructure in young children and the resultant effects on neurocognitive performance . although there were no differences in cognitive test scores between the two groups , we did find that in those with diabetes , higher hba1c levels were correlated with lower fsiq scores . also , we describe differences in wm microstructure between those with diabetes and controls , especially in the frontal and temporal regions , and that these differences were more apparent among those with higher hba1c levels . neuroimaging studies in young children with type 1 diabetes have been limited to date ( 12,23,24 ) , and understanding the impact of diabetes on neurodevelopment is still based largely on inferences drawn from adult neurocognitive and neuroimaging data ( 25 ) . dti provides a valuable tool to determine the relationship between wm connectivity and cognitive performance and allows additional , fine - grained investigation of wm microstructure . based on adult studies , abnormal wm ( lower fa ) has been reported in four major brain regions and is thought to be responsible for neurocognitive deficits involving mental processing speed , attention , and executive functioning skills such as mental flexibility ( 13,26 ) . however , it is unknown when these wm differences developed or whether they are directly attributed to the course of the diabetes . although we did not find significant differences in fa between those with diabetes and controls , this may be due to the young age of our participants , resulting in lack of diabetes - related complications and shorter duration of the disease . despite this , we did see a negative correlation between hba1c and fa ; although this did not reach statistical significance , larger future studies may indicate a clearer connection . ad and rd are indices that complement fa in providing additional information and increased understanding about the underlying wm and neuronal integrity . in this study , children with type 1 diabetes had lower ad values , a measure associated with less axonal coherence ( 27,28 ) . within this group , there also was a significant , widespread positive correlation between hba1c and rd , suggesting that higher bg values may affect fiber myelination or the permeability of axonal membranes ( 14 ) . parents and providers fear hypoglycemia in young children at the expense of hyperglycemia ( 29 ) and accept chronically elevated bg values as reflected by the american diabetes association guidelines where there is a higher hba1c target in young children ( 30 ) . hypoglycemia has been traditionally thought to be the cause of the neurocognitive deficits ; however , two longitudinal pediatric studies show that history of severe hypoglycemia alone could not explain the neurocognitive differences from healthy controls ( 31,32 ) . although we found a near - significant relationship between history of seizure occurrence and selected neurocognitive measures , the parents reaction to the hypoglycemia , resulting in a period of chronic hyperglycemia , may be a confounding factor . therefore , the discrepancy between the between - group analysis ( which revealed significant differences in ad ) and the within - group correlation of wm structure ( as reflected by rd and hba1c levels ) could possibly be explained by other diabetes - related factors such as hyperglycemia , perhaps leading to increased nonenzymatic glycosylation . hyperglycemia has a number of proposed mechanisms to cause wm changes , including nonenzymatic glycosylation and activation of the sorbitol pathway . myelin has lysine residues that are specifically susceptible to nonenzymatic glycosylation ( 33 ) . in animal models of diabetes , advanced glycosylation end products and the receptor for advanced glycosylation end products are both increased in the hypothalamus ( 34 ) and associated with significant loss of wm ( 35 ) . activation of the sorbitol pathway has been seen in 1-month - old rats when subjected to chronic hyperglycemia , leading to reductions in dendritic branching and spine density , and subsequent significant increases in brain sorbitol and inositol , when compared with rats with hypoglycemia ( 36 ) . therefore , the effect of hyperglycemia in young children may be particularly relevant to wm microstructure and in the resulting neurocognitive implications . a history of diabetic ketoacidosis ( dka ) may be another cause of axonal injury and be reflected in changes in ad . ( 37 ) of children between ages 7 and 16 years found that children with a history of type 1 diabetes and dka perform poorly on tests of memory function compared with children with a history of type 1 diabetes but not dka . the memory deficits seem to be specifically related to the hippocampus , a region of the brain that is particularly sensitive to episodes of hypoxia or ischemia . in our study , seven of the children with diabetes had presented with dka at onset and no one experienced dka after diagnosis . we did not observe a significant correlation between those who experienced dka and fa or ad or rd , although our small number of subjects do not allow for valid inferences . between - group differences in ad and rd occurred most prominently in the temporal and parietal regions of the brain in our study . positron emission tomography studies in healthy adults show that the temporal and frontal regions are most vulnerable to hypoglycemia and hyperglycemia ( 5,28 ) . young children with diabetes frequently experience glycemic variability ( 3 ) , resulting in more fluctuations in brain glucose levels and impacting brain glucose metabolism . therefore , it is not surprising to find that the most prominent areas of wm microstructural changes in children with type 1 diabetes also occur in these regions . although the study did not find any particular neurocognitive variation that correlated with either the temporal or frontal lobe function at this time , it will be intriguing to see if the microstructural changes on dti predict temporal and frontal lobe related neurocognitive changes over time . perhaps decreased episodes of glycemic variability in these regions prevent memory loss , improve attention span , or decrease the comorbidity of depression in children with diabetes as they age . it will also be important to elucidate if wm microstructural patterns predict adverse neurocognitive changes over time , and to develop clinical interventions to prevent such changes . both the small sample size and the high heterogeneity in performance on neurocognitive testing in this age group may have not allowed for detection of the cognitive differences . the study was not adequately powered to detect subtle or even moderate wm neurocognitive measures between those with type 1 diabetes and controls . also , subjects in this study completed different wechsler intelligence scales because of their ages . although not ideal , there is still a strong correlation between the two wechsler intelligence scales . the correlation between the two fsiq scores ( r = 0.89 ) ( 38 ) is nearly as high as the wisc - iv fsiq test - retest correlation and wppsi - iii fsiq test - retest correlations ( r = 0.93 and r = 0.92 , respectively ) , suggesting they measure similar constructs . finally , because of the age range of our sample , the subjects in the study are undergoing brain development at varying rates . however , the examination of wm microstructure variations in this age range is important and makes this study novel . wm changes and neurocognitive differences reported in adults with long - term type 1 diabetes likely begin in childhood . an association between early age of onset of disease ( generally before the age of 6 years ) and neurocognitive deficits has been the most consistent finding in the pediatric literature ( 1,25 ) . as northam et al . ( 25 ) recently summarized , chronic hyperglycemia , occurrence of diabetic ketoacidosis , bg fluctuations , and hyper- or hypoinsulinism all may impact the neurodevelopment of young children with type 1 diabetes . we suggest that there are wm microstructure differences between young children with type 1 diabetes and healthy controls after a short duration of diabetes , and support larger , longitudinal studies to confirm these changes . knowledge of when these changes begin will impact our understanding of diabetes and the brain , identify the mechanisms of neuronal injury , and modify management of diabetes to minimize the chances of injury . traditionally , the management of type 1 diabetes in young children has been to avoid hypoglycemia ; however , we may need to tolerate less hyperglycemia during childhood as well .

objectiveto detect clinical correlates of cognitive abilities and white matter ( wm ) microstructural changes using diffusion tensor imaging ( dti ) in young children with type 1 diabetes.research design and methodschildren , ages 3 to < 10 years , with type 1 diabetes ( n = 22 ) and age- and sex - matched healthy control subjects ( n = 14 ) completed neurocognitive testing and dti scans.resultscompared with healthy controls , children with type 1 diabetes had lower axial diffusivity ( ad ) values ( p = 0.046 ) in the temporal and parietal lobe regions . there were no significant differences between groups in fractional anisotropy and radial diffusivity ( rd ) . within the diabetes group , there was a significant , positive correlation between time - weighted hba1c and rd ( p = 0.028 ) . a higher , time - weighted hba1c value was significantly correlated with lower overall intellectual functioning measured by the full - scale intelligence quotient ( p = 0.03).conclusionschildren with type 1 diabetes had significantly different wm structure ( as measured by ad ) when compared with controls . in addition , wm structural differences ( as measured by rd ) were significantly correlated with their hba1c values . additional studies are needed to determine if wm microstructural differences in young children with type 1 diabetes predict future neurocognitive outcome .

RESEARCH DESIGN AND METHODS Diffusion-weighted scans preprocessing Tract-based spatial statistics (TBSS) analysis Statistical analysis RESULTS Cognitive testing TBSS analysis DTI and neurocognitive correlations CONCLUSIONS

diseases that were prevalent are now increasingly rare because of the widespread use of vaccines . next to clean water , no single intervention has had such an overwhelming effect on reducing mortality from childhood diseases as the use of vaccines . the smallpox vaccine has eradicated a disease that had a 30 % fatality rate . after over two decades of intensive efforts , the global polio eradication initiative is approaching its final stage aided by the two polio vaccines developed by jonas salk and albert sabin . an important field in the development of next - generation vaccines is the development of vaccines suitable for mucosal immunization . most viral and bacterial infections start at the mucosal surfaces ; thus , immunity against infective agents may depend on the induction of a mucosal immune response . as a result , for certain infectious agents , the mucosal route is the most appropriate method of immunization because it has been reported to induce both mucosal and systemic immune responses [ 4 , 5 ] . mucosal vaccination can be achieved via a number of routes including oral , intranasal , pulmonary , rectal , or vaginal . of these , the nasal route is the most straightforward and is suitable for vaccine administration . the prime inductive site for mucosal immunity in the nasopharyngeal tract in rodents is the nasal - associated lymphoid tissue ( nalt ) [ 79 ] . nalt is considered the rodent equivalent of waldeyer s ring ( fig . 1 ) , the lymphoid tissues ( tonsils ) present in humans . waldeyer s ring forms a protective site at the opening of the pharynx to provide immunity . protection at this mucosal surface is correlated with secretory immunoglobulin - a ( siga ) antibodies which , alongside other innate defence mechanisms , provide additional protection from pathogens . murine nalt is a functional analogue to human tonsils , and animal research has provided insight into human immunology . however , human immunity and biological composition can differ from those of animal models . for nasal vaccine development , development of appropriate in vitro models has provided a reproducible approach in which phenotypic and physiological features of the nalt can be simulated . techniques to collect and analyse mucosal secretions and cell types have also provided a rational approach for evaluation of vaccine efficacy .table 1advantages and disadvantages of intranasal vaccinationadvantagesdisadvantagesneedle - free thus patient compliancerapid clearancenon - invasiveinefficient uptakesmall antigenic doselack of human compatible mucosal adjuvantinduction of systemic and mucosal immunityimmunity at primary and distal mucosal sitesfig . it is formed by the lymphoid tissues near the opening of the respiratory and digestive systems . it consists of the adenoid , tubal , palatine and lingual tonsils advantages and disadvantages of intranasal vaccination waldeyer s ring is an induction site for mucosal responses . it is formed by the lymphoid tissues near the opening of the respiratory and digestive systems . it consists of the adenoid , tubal , palatine and lingual tonsils nalt comprises a organised structure of cells involved in the induction of an immune response , including dendritic cells , t cells and b cells , which are covered by an epithelial layer of cells containing distinctive cells called m cells . m cells in the nalt are the sites of antigen uptake for induction of mucosal immunity . whilst small soluble antigens are able to penetrate the nasal epithelium , particulate antigens are mainly taken up by m cells in the nalt . antigen is actively transported by m cells , to reach dendritic cells , macrophages and b cells , for processing and presentation . consequent activation of antigen - specific cd4 + t helper cells ( th cells ) interact with b cells which develop into iga committed ( iga+ ) . iga+ b cells move to effector sites such as the nasal passage where they differentiate into iga - producing plasma cells and secrete iga in dimers . dimeric iga then become s - iga by binding to the polymeric ig receptor which transports iga to effector sites . an overview of the immune response in nalt is summarized in fig . 2 . s - iga are able to bind toxins , bacteria or viruses and neutralize their activity , thus preventing entry into the body or reaching the internal organs . whilst this can prevent infection through the mucosal epithelium , certain pathogens such as group a streptococcus can concurrently infect through the skin . the advantage of vaccination at the mucosal surface by intranasal administration is the induction of mucosal and systemic immune responses , whereas traditional parenteral administration generally only results in systemic immune responses . therefore , in the context of group a streptococcus and other pathogens which concurrently infect through the mucosa and systemic sites , an ideal vaccine could be an intranasally administered vaccine eliciting neutralizing iga preventing colonization of the throat and systemic igg antibodies facilitating clearance from systemic sites .fig . 2scheme of immunological pathway for induction of local mucosal response in nalt scheme of immunological pathway for induction of local mucosal response in nalt an effective vaccine formulation for intranasal delivery maintains the antigen in a stable form , ensures the antigen , remains in the nasopharyngeal region long enough for the antigen to interact with the lymphatic system and stimulates the immune system with or without additional adjuvants to provide long - term immunity . this review discusses the leading strategies in delivery systems and adjuvants currently under investigation for the development of nasal vaccines . nasally delivered influenza vaccines based on live - attenuated strains are currently under investigation to prevent seasonal and pandemic influenza . live - attenuated vaccines have the advantage of mimicking natural infection by presenting influenza antigens in their native conformation to the nasal mucosal tissues , without inducing flu - like symptoms . an example of this type of vaccine is flumist by astra - zeneca , the first nasal trivalent vaccine for seasonal influenza [ 20 , 21 ] . when compared with an injectable trivalent influenza vaccine , the flumist vaccine conferred a longer duration of protection , better cross - protection , enhanced efficacy and both mucosal and systemic immunity . despite the promise of the live - attenuated influenza vaccine strategy , adverse side effects have been reported including safety concerns for patients with allergy or asthma as well as irregular side effects similar to bell s palsy . most antigens have little to no affinity for the nasal epithelium and tend to be removed quickly by mucociliary clearance [ 22 , 23 ] . extending the nasal residence time by co - administering the antigen with mucoadhesives is a logical approach to enhance absorption and residence time to facilitate interaction with the immune system [ 16 , 24 ] . significant results with biodegradable , mucoadhesive polymeric carriers have highlighted their use for mucosal vaccine delivery . polymers such as polylactide - co - glycolide ( plga ) , chitosan , alginate and carbopol have been explored for the delivery of antigens via the intranasal routes . hydrophilic polymers , like sodium alginate and carbopol , absorb to the mucus by forming hydrogen bonds , consequently enhancing nasal residence time [ 25 , 26 ] . sodium alginate is a linear copolymer and consists of 14-linked -d - mannuronic acid and 14-linked -l - guluronic acid residues . sodium alginate is biocompatible and mucoadhesive and has been used for controlled delivery of drugs following intranasal administration [ 27 , 28 ] . in vivo studies showed that the therapeutic efficacy of a drug - sodium alginate formulation significantly improved in comparison to nasal administration of drug alone . good biocompatibility , low cost and ease of preparation are the major advantages of using alginate polymers for the delivery of an antigen . extensive use of sodium alginate as an antigen delivery system to improve the efficacy of mucosal immunization in livestock has been carried out . for example , induction of systemic and mucosal immune response in cattle by intranasal administration of antigen in alginate microspheres has been reported . when mixtures of starch and carbopol have been used as carriers of influenza virus antigen , systemic antigen - specific igg responses were induced after intranasal delivery . the level of igg and the immune response kinetics were improved by the presence of carbopol . carbopol has been used experimentally as a delivery system to enhance adhesion to mucosal surfaces and may facilitate enhanced protection of peptides and proteins against enzymatic degradation . carbopol has received considerable attention delivery of proteins , and witschi and mrsny showed ( using bovine serum albumin as model antigen ) that carbopol gels can be used for nasal protein delivery . chitin is a naturally occurring polymer found in the exoskeletons of insects and crustaceans . as a cationic polymer , mucociliary clearance is decreased , and a transient effect on the paracellular transport of antigens has been observed . in addition to being a mucoadhesive , chitosan has also been reported to have adjuvant properties that enhance humoral and cellular immune responses [ 4143 ] via opening of intercellular tight junctions , which favours the transport of antigens . combination of transient opening of tight junctions and mucoadhesive properties most likely enables the interaction of the chitosan - antigen formulation to interact with the lymph nodes of the nasal cavity , leading to improved immunological responses . for controlled vaccine release , chitosan nanoparticles are reported to be effectively taken up by antigen presenting cells and induce strong mucosal and systemic immune responses against antigens [ 36 , 46 , 47 ] . chitosan - based formulations have been used to improve the delivery and immunogenicity of a variety of antigens . luteinizing hormone - releasing hormone ( lhrh ) displays poor immunogenicity and requires the presence of an adjuvant . chitosan - based formulations were studied as a potential adjuvant for a vaccine against lhrh . a reduction in animal steroidogenesis and spermatogenesis was observed when chitosan was used as an adjuvant , showing the ability of this delivery system to neutralize lhrh . the influenza a , matrix protein 1 ( m1 ) , is highly conserved and can form the basis of a vaccine . mice intranasally administered m1 and chitosan were challenged with lethal doses of h9n2 , h1n1 and h5n1 viruses . the results showed chitosan had an improvement over the efficacy of the m1-based vaccine alone . additionally , nasal immunization with n - trimethyl chitosan ( tmc)-based nanoparticles increased the nasal residence time of ovalbumin , a model antigen . after intra - mucosal administration , slow antigen - releasing tmc nanoparticles did not induce detectable antibody titres whereas fast antigen - releasing tmc nanoparticles showed high siga levels and serum antibody titres . it was found that these nanoparticles were mucoadhesive and stimulated the maturation of dendritic cells . furthermore , a formulation of chitosan and dna encoding a viral protein from coxsackievirus b3 was shown to induce high levels of mucosal siga and serum igg . following intranasal challenge with lethal cvb3 , significant reduction of viral load chitosan was reported to slow down the nasal mucociliary clearance and prolong the contact period between the nalt and the chitosan these examples with chitosan illustrate that the co - administration of antigens with mucoadhesive agents can enhance efficacy , potentially reduce antigen dose and generally facilitate the development of mucosal vaccine delivery systems . since most vaccines alone are not sufficiently taken up after mucosal administration , co - administered with penetration enhancers , adjuvants or encapsulation in particles immunogenicity enhancing properties of chitosan due to combination of transient opening of tight junctions and mucoadhesive properties makes it promising for mucosal vaccine delivery . particulate delivery systems used for nasal administration of vaccines include liposomes , immune - stimulating complexes ( iscoms ) and polymeric particles including virosomes [ 19 , 5355 ] . particulate antigens use the transcellular route to reach the lymphoid tissues and target m cells . m cells , which are part of the nalt , act as portals of entry for antigens to enter regions containing professional antigen presenting cells ( dendritic cells ) , b cells and t cells , thereby contributing to both humoral and cellular immune responses [ 15 , 17 , 56 , 57 ] . particulate systems have the capacity to present multiple copies of the antigen and tend to have a similar size to pathogens , mimicking natural infection . liposomes are particulate vesicles composed of different ratios of lipids and cholesterol enclosing an aqueous core , enabling the incorporation of a wide variety of antigens . the immunogenicity conferred by liposomes is due to : their ability to accommodate multiple copies of antigens , preferential uptake by macrophages , protection within the biological environment and effects on the intracellular processing of antigen [ 19 , 58 ] . surface - modified ( glycol chitosan or oligomannose coated ) liposomes have been shown to elicit humoral and cellular immune responses that were significant ( when compared to antigen alone ) following intranasal administration [ 59 , 60 ] . fusogenic liposomes encapsulated with antigens have been demonstrated to effectively stimulate a mucosal immune response . iscoms are made up of saponin , as an adjuvant , along with lipids and an antigen , and are generally held together by hydrophobic interaction between the constituents . the essential components to form iscoms are cholesterol and saponin . their inherent particulate nature , multimeric antigen presentation and potent immunostimulatory activity several studies have shown that iscom - incorporated antigens induce specific local and systemic immune responses following intranasal administration , conferring protection against influenza , respiratory syncytial virus and hepatitis b [ 6669 ] . virosomes are an alternative particulate delivery form composed of extracted glycoproteins from virus particles and mimic viral structures . the viral glycoproteins present on virosomes have a high affinity for the mucosal surfaces of the respiratory tract . virosomes enable efficient induction of humoral and cellular responses and target dendritic cells [ 7176 ] . they have been demonstrated to be efficient nasal delivery systems for several antigens , including dna , and influenza [ 77 , 78 ] and hiv proteins . particulate systems are promising for the nasal delivery of vaccines , enhancing uptake by antigen - presenting cells , conferring a depot effect and protecting the antigen from degradation . further research in the physicochemical properties of particulates that influence immunogenicity will contribute to the development of nasal vaccine delivery systems . lipopeptides of bacterial origin , or their synthetic derivatives , represent potent immunostimulants when given in combination with peptide or protein antigens . examples of lipid moieties include tri - palmitoyl - s - glyceryl cysteine ( pam3cys ) , di - palmitoyl - s - glyceryl cysteine ( pam2cys ) , single / multiple - chain palmitic acids and lipoamino acids ( laas ) ( fig . 3 ) . the desirable immunological activity of these lipid moiety conjugates arises from their intrinsic adjuvant properties , such as b cell and macrophage activators , ability to induce maturation of dendritic cells and promote an inflammatory response [ 8185 ] . generally , this occurs via signalling through receptors of the immune system that recognise these lipid moieties as pathogen - associated molecular patterns [ 8689].fig . 3the structure of the lipid moieties used to enhance immunogenicity of weak antigens the structure of the lipid moieties used to enhance immunogenicity of weak antigens lipopeptides containing pam3cys and pam2cys have been evaluated for development of immunocontraceptive vaccines . when administered intranasally , the constructs were highly immunogenic , capable of inducing high titres of antibodies and efficiently sterilized female mice when administered alone in saline . a pam2cys - based vaccine against streptococcus pyogenes had the ability to induce mucosal and systemic antibodies following intranasal immunization and conferred protection against bacterial challenge . for influenza , pam2cys - based lipopeptides lipopeptide vaccines based on a palmitic acid moiety may present an ideal strategy against pathogens that infect mucosal surfaces . intranasal administration of a palmitoylated lipopeptide vaccine for human cytomegalovirus ( a herpes virus ) elicited both systemic and local mucosal cellular responses when administered intranasally . herpes simplex virus type 2 peptide epitopes conjugated to a palmitic acid moiety have shown the ability to prevent transmission and/or limit the severity of diseases . a library of lipopeptide vaccine candidates composed of a s. pyogenes b cell epitope , a t helper epitope and a lipid moiety based on laas ( fig . the orientation of each component of the lipopeptide was optimized to elicit a strong immune response following intranasal immunization . the antibody titres elicited in response to these lipopeptides provided important information for the design of an effective , lipopeptide - based mucosal vaccine [ 95 , 96 ] . interestingly , the lipopeptides were self - adjuvanting , negating the use of additional adjuvants , which could easily be applied to other peptide - based vaccines . an important consideration for lipopeptide vaccines is ensuring that the lipid moiety is easy to synthesize , couple to the antigen , purify and formulate . as a result , laas are an attractive option because they can be synthesized and coupled to peptide antigens using standard peptide synthesis methods .fig . 4structures of lipopeptides with a t helper epitope ( p25 ) , s. pyogenes b cell epitope ( j14 ) and a lipid moiety based on laas structures of lipopeptides with a t helper epitope ( p25 ) , s. pyogenes b cell epitope ( j14 ) and a lipid moiety based on laas lipopeptides provide several advantages for nasal vaccine development . firstly , they negate the requirement for adjuvant , which has the potential to induce adverse side effects . secondly , lipopeptide vaccine candidates can induce cellular and antibody responses , allowing control over the desired immune response . current pre - clinical and clinical trials with lipopeptides suggest they are effective , non - toxic and can be synthesized using current methods of peptide synthesis to a high purity and yield . the lipopeptide - based approach for the nasal delivery of vaccines induces effective mucosal and systemic immune responses . dna - based vaccines where antibodies raised to specific proteins involved in immunity to infection have shown promise in animal models . the composition and negative charge of dna generally hinders the entrapment efficiency and stability of vaccine formulations . several investigators have used cationic components such as chitosan and polyethylenimine ( pei ) to complex with dna antigens in the form of nano-/microparticles for intranasal delivery . plasmid dna containing chitosan nanoparticles for nasal immunization against nucleocapsid ( n ) protein of severe acute respiratory syndrome coronavirus ( sars - cov ) showed elicitation of mucosal iga as well as systemic igg against n protein . sars dna vaccine complexed with other cationic polymers effectively delivered the plasmid dna to induce antigen - specific humoral and cellular immune responses . pei is of particular interest for intranasal delivery due to reported many - fold increase in gene transfer in the respiratory tract . using pei formulation , intranasal vaccination with dna - encoding influenza a h5n1 or h1n1 antigens , high levels of antibodies cationic plga particles have also been used as an intranasal delivery system for administering foot and mouth disease ( fmdv ) vaccine encoding the fmdv capsid protein . intranasal delivery of the cationic plga particles containing fmdv dna vaccine formulations enhanced protective immunity against fmdv . whilst dna vaccines have been shown promise for intranasal vaccine delivery , the technology requires further research to develop effective vaccines . improving the immunogenicity of antigens through the use of adjuvants ( which can also act as delivery systems ) is a rational approach for vaccine development . nevertheless , achieving a potent adjuvant effect whilst avoiding reactogenicity or toxicity is a major challenge . however , existing licensed adjuvants , such as alum and to a lesser extent mf59 , are not suitable as adjuvants for mucosal vaccine administration and in general do not induce mucosal antibodies . unlike the systemic immune system , mucosal surfaces regularly encounter an extensive range of foreign material . to accommodate this , the mucosal immune system must be selective in responding to antigens in order to avoid undesirable immune responses and excessive activation of the immune system . thus , induction of mucosal immunity is more difficult , and novel strategies are critical to the successful development of mucosal adjuvants . dendritic cells and m cells are the major cell types to be targeted by a mucosal adjuvant . cpg oligodeoxynucleotides ( cpg odn ) flt3 ligand and monophosphoryl lipid a ( mla ) represent dendritic cell targeting ligands , whilst flt3 is a growth factor reported to stimulate dendritic cells . cpg odn mimics the immunostimulatory effects of bacterial dna and is known to target plasmacytoid dendritic cells for their activation , maturation . mucosal administration of cpg odn with model antigens , including influenza virus and tetanus toxoid , effectively elicited antigen - specific immunity [ 107 , 108 ] . furthermore , flt3 has been demonstrated as a safe adjuvant with nasal dendritic cell targeting properties that confer protection against fatal pneumococcal pneumonia in mice . to this end , cpg odn and flt3 represent mucosal dendritic cell targeting adjuvants for the induction of antigen - specific , protective mucosal immune responses . chemical modifications to alleviate its toxic effects resulted in the identification of mla ( fig . the clinical grade mla formulation corresponds to the main active component of corixa s mpl adjuvant . in numerous preclinical and clinical studies , it has been used extensively as an adjuvant in human vaccine trials for several infectious diseases and cancer . effectiveness of mpl as a mucosal adjuvant was investigated following intranasal administration of a formulation of mpl added to soluble antigen or liposomes encapsulating antigen . the liposomal antigen formulation with mpl resulted in iga responses that were consistently higher than seen in mice immunized with liposome antigen or free antigen without mpl . these results demonstrated the effectiveness of mpl as an adjuvant for potentiating mucosal immune responses.fig . 5chemical structure of mla chemical structure of mla m cells provide a portal of entry for pathogens , through which they can invade the host . identification of the molecules that are required for bacterial or viral entry through m cells may provide a rational basis for developing an effective mucosal adjuvant / delivery system . for example , reoviruses initially infect through m cells using their surface protein sigma-1 ( p1 ) . an m cell - targeting dna vaccine formulation consisting of plasmid dna and the reovirus p1 induced significant mucosal and systemic immunity . this suggests that m cell targeting could be a useful approach for the development of a mucosal vaccine . the above examples represent a sample of the strategies that could be used to facilitate the development of novel mucosal adjuvants . however , mucosal adjuvants must still overcome the two major hurdles : effectiveness and safety . these are both relatively more difficult objectives compared with the development of vaccines that elicit systemic immunity , due to the uniqueness and complexity of the mucosal immune system . the development of novel , mucosally active , intranasally administered vaccines has the potential to provide immunity against a myriad of infectious diseases . mucosal administration of vaccines presents an ideal strategy against many pathogens that infect via mucosal surfaces . a suitable combination of adjuvants targeting m cells or dendritic cells and delivery systems that are mucoadhesive could facilitate the development of effective nasal vaccines . the use of the non - invasive , needle - free nasal route is advantageous for vaccination programmes , since it can enhance patient compliance and reduce need to be administered by specialised healthcare workers .

the vast majority of human pathogens colonize and invade at the mucosal surfaces . preventing infection at these sites via mucosally active vaccines is a promising and rational approach for vaccine development . however , it is only recently that the stimulation of local immunity at the mucosal surfaces has become a primary objective in addition to inducing systemic immunity . this review describes vaccine formulations designed for mucosal delivery to the nasal - associated lymphoid tissue , via intranasal administration . the association of antigens with mucosal adjuvants and delivery systems is emphasised .

Introduction Attenuated intranasal vaccines Enhancement of mucoadhesion Particulate delivery systems Lipopeptide-based delivery systems DNA-based vaccines Mucosal adjuvants Conclusion

carcinoma of the prostate is the most common malignancy in men with increased incidence rates owing to the population ageing and the improvement of diagnostic procedures . the early detection of the bone metastases is of value in making decision regarding the treatment plan , which may vary extremely according to the bone status . the likelihood of the bone metastases at the first diagnosis varies with the histological score and serum level of prostate - specific antigen , and it is possible to some extent to estimate the degree of tumor spread in the light of the psa value . bone involvement is much less frequently involved with low psa level , so that the bone staging is not recommended with psa less than 10 ng / ml , except if there are known bone disorders which may later cause false positive findings [ 13 ] . however , in patients with psa relapse after primary therapy it is not easy to be guessed based on psa values whether the bone involvement or another involvement is behind a psa rising . in established skeletal involvement , there is a need of effective imaging method to monitor the status progress . bone scan is the widely used screening technique for assessing the bone status in the most malignancies , and it shows mostly high sensitivity in detecting the bone involvement . pet / ct with f - fluorodeoxyglucose ( fdg ) is known to have general limitation in prostate cancer . pet with radiolabeled choline such as f - fech - pet was found to be an effective method in diagnosis of primary and recurrent pca tumors as well [ 4 , 5 ] . two possible mechanisms have been proposed to explain the increased choline uptake in prostate cancer cells . the first is increased cell proliferation in tumors , because choline is a precursor for the biosynthesis of phosphatidylcholine and other phospholipids , the major components of the cell membrane . the second explanation is the overproduction of choline kinase in cancer cells , which was experimentally confirmed in human - derived prostate cancer [ 68 ] . although , f - fech - pet is being increasingly used primarily in patients with prostate cancer and for followup , its efficacy in detecting the bone involvement still needs further investigations in comparison with the established method ( bone scan ) . to our knowledge we aim in this study mainly to assess the value of f - fech - pet / ct in detecting the bone metastases and to compare the results with that of bone scan . 37 patients ( mean age 69 7 ) had been referred in our department for re - staging purposes due to biochemical recurrence , psa median 2.6 ng / dl ( range 0.321 ) . gleason 's score ranges among 3 and 9 : gs.7 ( n = 12 ) , gs.8 ( n = 8) , gs.9 ( n = 7 ) , gs . 3 ( n = 1 ) , and gs 5 ( n = 1 ) . the patients underwent a wide variety of initial therapies including radical prostatectomy ( n = 11 ) , radiotherapy ( n = 7 ) , and pure hormonotherapy in patients with locally advanced disease ( n = 3 ) . a number of patients underwent combined therapy due to previous slight psa increase such as radical prostatectomy followed by salvage radiotherapy ( n = 12 ) , radical prostatectomy followed by salvage antihormonal therapy ( n = 3 ) , or radical prostatectomy followed by hifu ( n = 1 ) . the study was approved by the ethics committee of the heidelberg university , and a written consent was obtained from all patients . bone scintigraphy was performed using a modern double - head gamma - camera ( ecam , siemens medical solution ) equipped with low - energy , high - resolution collimators . whole - body images were obtained 2 - 3 h after intravenous injection of 700 mbq ( 19 mci ) of tcm - mdp at the scan speed of 15 cm / min in the anterior and posterior projection ; additional imaging were acquired , when required . all pet studies were carried out using a biograph 6 pet / ct ( siemens / cti ) . imaging was started as dynamic imaging immediately parallel to administration of a standard of 250 mbq of f - ethylcholine for 10 min before proceeding to whole body imaging . for attenuation correction of the pet scan , a low - dose ct ( 130 kev , 30 mas ; care dose ) without contrast medium was done . static emission scans , corrected for dead time , scatter , and decay were acquired from the vertex to the proximal legs requiring eight bed positions , 4 minutes each . the images were iteratively reconstructed with the osem algorithm using four iterations with eight subsets and gauss filtering to an in - plane spatial resolution of 5 mm at full - width half - maximum . the delay between both modalities ranged between 2 and 45 days ( average 14 days ) . the images were evaluated visually by two skilled nuclear medicine physicians in presence of a skilled radiologist after viewing the images in different planes . the image assessment was achieved in combination with the clinical information and close correlation with other available imaging studies . the findings concerning the bone status were assessed as positive in presence of typical findings validated with followup and/or by correlation with other modality and as negative in absence of the pathological bone uptake ( there was no need to prove the negativity ) , or false positive / negative ( if proved the contrary ) . sensitivity , specificity , npv , and ppv are the statistical indicators utilized in assessing the universal outcome . in the patients with confirmed bone metastases ( n = 18 ) , the skeleton was divided into five regions ( skull , ribs , pelvis , vertebra , and extremities ) to simplify the comparison between the matching regions in both examinations . the metastases had been drawn as black points on a separate skeleton outline ( figure 1 ) . each imaging was evaluated separately , so that the interpretation would not be influenced . the anona test was used to compare the extent of metastatic spread in both examinations in the different regions . suvs were acquired by manually drawing a volume of interest over the pathologic bone lesions that had been identified on visual analysis of static emission images . by using attenuation - corrected pet data , suvs were calculated as the ratio of regional radioactivity concentration ( becquerel / milliliter ) divided by the injected amount of radioactivity ( becquerel ) normalized to body weight in gram . ct reading was based on characteristic patterns of morphological change ; all detectable lesions on ct were categorized by a radiologist as sclerotic , lytic , or mixed metastases . the quantitative radiodensity of sclerotic lesions was measured by means of the hounsfield unit ( hu ) . for quantitative assessment of mdp uptake , we considered an uptake score based on the visual assessment and graduated from 0 till 4 ( 0 no uptake , 1 decent uptake , 2 moderate uptake , 3 high uptake , 4 extremely severe uptake ) . so the intensity of choline uptake was once compared with the radio - density represented by hu and once with mdp uptake ( evaluated visually ) . lesions detected in ct and evaluated as high suspected were encompassed in this comparison , regardless whether they showed a positive choline uptake or not . some limitations should be kept in mind such as the absence of pathological verification of the bone metastases . thus , the patients were confirmed as having bone metastases just depending on clinical followups in correlation with other diagnostic tools , for example mri . the average clinical followup duration to confirm the final diagnosis was 1 year . the characteristics of the patients are summarized in table 1 . generally , 18 patients out 37 patients referred with biochemical relapse were confirmed to have bone involvement . since the pathological confirmation was not available , the followup and correlation with other modalities were adopted in our verification . ( followup was available in 11 patients ; in the remaining seven patients , the verification was based on a typical findings in pet , assessed by two nuclear medicine physicians matching with morphological images such as ct or mri . ) in patients with positive bone findings , the psa median was 4 ng / dl ( range 121 ) . by contrast , the patients with negative bone findings had a psa median value of 1 . 5 ng / dl ( range 0.319 ) . statistically there was a significant difference between both groups ( p = 0.02 ) ( figure 2 ) . in patients with negative bone findings the psa relapse was attributed to lymph node metastases ( n = 5 ) , local recurrence ( n = 2 ) , and lung metastases ( n = 1 ) . confirmed bone involvement cases , f - fech - pet / ct identified correctly the bone involvement in 15 cases with a sensitivity of 83.3% . no false positive was identified ; consequently its specificity was 100% , npp 86.3% , and ppv 100% . on the other hand , bone scan showed the presence of the metastases in 17 out of 18 confirmed cases with sensitivity of 94.4% . from 19 negative findings , the bone scan showed two false positive cases due to paget disease and trauma . as mentioned earlier , the skeleton had been divided into multiple regions to simplify the matching of findings . totally , the patients with positive findings have 122 lesions . f - fech - pet was capable to identify 101 lesions with a sensitivity 82.7% versus 109 identified using bs ( bone scan ) with sensitivity of 89.4% . the sensitivity of both modalities differs extremely with the anatomical region ( table 2 ) . indeed this superiority of bone scan was demonstrated in ribs ( 71% in f - fech - pet versus 94% in bs ) and cranium . in remaining regions , f - fech - pet was superior in identifying more metastases , in vertebrae ( 92% versus 85% ) and pelvis ( 91% versus 89% ) . in the extremities f - fech - pet shows also superiority over bone scan ( 90% versus 50% ) in spite of limited field of view ( fov ) figure 3 . statistically using anova test there was no significant difference between both modalities ( p > 0.1 ) in the number of detected metastases in all anatomical regions . degenerative changes : f - fech - pet / ct versus bs ( figure 4)whereas the bone scan demonstrates an intensive uptake in this degenerative change , the f - fech - pet / ct shows a negative choline uptake . whereas the bone scan demonstrates an intensive uptake in this degenerative change , the f - fech - pet / ct shows a negative choline uptake . trauma : f - fech - pet / ct versus bs ( figures 5 and 6)these images show a discrepancy between mdp uptake and choline uptake in a compression fracture and rib fractures . these images show a discrepancy between mdp uptake and choline uptake in a compression fracture and rib fractures . a total of 94 out of 122 with the following distribution : ( 3 in cervical spine , 10 in lumbar spine , 35 in pelvis , 7 in ribs , 4 in sternum , 15 in thoracic spine , 12 in the extremities and 7 in other regions ) were selected to make a further quantitative comparison between f - fech - pet and both ct and bone scan using the above mentioned quantitative parameters . 28 lesions were excluded from this analysis , for example , in ribs due to neighborhood to liver ( false high suv through partial volume effect ) or due to technical limitations . as previously mentioned , the quantitative assessment in bone scan was based on visual score escalating from 0 till 4 . based on the radiological portrayal in ct , lesions were divided into osteoblastic lesions ( n = 42 ) , osteolytic ( n = 19 ) , and mixed ( n = 16 ) . 16 lesions of positive choline uptake : ( 11 in the spine7 thoracic and 4 lumbar2 in the pelvis and 3 in the extremities ) were lacking morphological alterations . they were characterized with high choline uptake ( suv 5 , 95 1 , 5 ) and mostly with decent or absent mdp uptake . they were attributed to bone marrow involvement ( figure 7 ) 13 of sclerotic lesions ( 6 in pelvis , 3 in thoracic spine and 4 in ribs ) ( hu mean 739 216 ) were of negative choline uptake but mostly of intensive mdp uptake ( qmdp score 3 ) ( qmdp quantitative mdp evaluated visually ) . the decline or absence of choline uptake in sclerotic metastasis was noted in particular in patients receiving systemic therapy ( figures 8 and 11 ) . in contrast , the osteolytic metastases ( 4 in the pelvis and 1 in the humerus ) were characterized with high choline uptake ( 7 2 , nadir 5.5 ) and wide extent of mdp uptake ( 4 showed negative uptake , 4 showed intensive uptake and 2 showed moderate uptake ) . a significant negative correlation was demonstrated between tracer uptake suv(max ) and the density of sclerotic lesions assessed by hu ( r = 0.58 , p < 0.01 ) ( figure 9 ) . on the other hand , numerical score of mdp was correlated negatively with suv(max ) value ( r 0.01 ) and positively with hu ( r 0.34 ) . however , they were statistically not significant . in few mixed metastases it was demonstrated that the choline uptake was solely concentrated in the osteolytic part evading the sclerotic part ( figure 10(c ) ) . in pure sclerotic metastases it was shown that uptake concentrates at the rim of metastases , where there is a transitional region with a moderate sclerosis ( figures 10(a ) and 10(b ) ) . examples of the effects of ongoing systemic therapy on pet findingssee figures 11 and 12 . it is known that the skeleton is a favorable place for metastasis in prostate cancer . it is the second most common site of metastatic disease after lymph nodes and considered as the main cause of morbidity and mortality in prostate cancer patients and mostly related to poor prognosis . although bone scan is ranked first in assessing bone status in many malignancies including pca , it still lacks specificity . the comparison between pet modality and bone scan in detecting bone involvement was a concern of many studies , mostly involving f - fdg pet / ct [ 911 ] . cheng et al . showed , in their review of six studies , that f - fdg pet / ct has both higher sensitivity and specificity than bone scintigraphy . in patients with pca , tiwari et al . proved that f - fdg - pet / ct can play a complimentary role to the conventional skeletal scintigraphy , particularly in detection of bone marrow disease . whether or not f - fech - pet / ct is capable to assess the bone status in an adequate way or requires supplementary test is still blurred . according to our knowledge , there is lack of studies concerning the comparison of f - fech - pet / ct with standard bs . . showed that f - choline- pet / ct had lower sensitivity than f fluoride pet - ct for detection of bone metastases ( f - fluoride is bone seeking pet radiopharmaceutical ) . bone scan showed superiority over f - fech - pet / ct in patient - based results ( sensitivity 81.2% versus 93.7% ) . however , the high sensitivity of bone scan encounters a high likelihood of false positive findings . by contrast , f - fech - pet / ct appears to be less affected by such unspecific lesions . the benefit of associated diagnostic ct ( occasionally with contrast media ) should be kept in mind in minimizing the false positive cases . yet , ct is not the sole factor affecting the increase of the specificity of f - fech , mccarthy et al . suggested that f - fech - pet can separate benign conditions such as trauma and arthropathy from malignancy and may be useful as an assistant to bone scan in equivocal cases . basically , there is need to perform bone scan in case of negative f - fech - pet / ct finding if there is clinical suspicion , under suggestion that f - fech - pet is of less sensitivity . however , bone scan can be reserved and an unnecessary radiation exposure can be avoided , if proven otherwise . so whether or not bone scan can be abandoned as a complementary test to f - fech - pet / ct is of a high clinical value . we found that f - fech - pet / ct showed less sensitivity compared with bs ; however , two out of three patients demonstrating false negative pet findings were under systemic therapy , suggesting a potential impact of systemic therapy in minimizing the sensitivity . in our patient group , looking at the findings of both modalities in the various anatomical regions , we observed better lesion detection efficiency of bs in the ribs with sensitivity of 98% versus 73% . on the other hand , the f - fech - pet / ct was more effective in pelvis ( 93% versus 87% ) and in extremities in spite of the presence of few metastases outside the field of view . in vertebra the f - fech - pet / ct was also more accurate than bs . because of poor spatial resolution of bs , an uptake in the spine could be incorrectly attributed to degenerative changes that may explain the low sensitivity of bs in spine compared with f - fech - pet / ct . moreover spect ( single photon emission computed tomography ) can minimize the shortcoming of planar bs in the assessment of the spine ; however it is not routinely applied . in this regard , it should be mentioned that spinal metastases have typical locations , mostly in the posterior part of vertebrae due to the many short intraosseous arteries [ 16 , 17 ] , and they can be easily recognized and differentiated from degenerative changes in f - fech - pet / ct in the light of morphological view provided by ct component . in other anatomical regions f - fech - pet / ct showed advantage over bs ; this can be attributed to the superior spatial resolution . of course the associated ct contributed to a certain degree in increasing the sensitivity of f - fech - pet / ct through detecting the bone metastases of a negative choline uptake . in the same topic , we found that fech uptake decreases with increasing sclerosis ( negative correlation between hu and suv(max ) r = 0.58 , p < 0.01 ) . this issue emphasizes the importance of ct in compensating the deficiency of f - fech - pet in patients under aht . further benefit of ct is the validating the pet finding through excluding other benign changes that may share the same findings with the metastases . since the bone marrow is associated with minimal morphological changes and unusual to cause bone reaction , it remains undetectable or can be easily overlooked using morphological modalities or using bone seeking nuclides . kato and coworkers reported a case of bone marrow metastasis finally detected by marrow biopsy , without any abnormality on bone scintigraphy or computed tomography . generally , the bm aspiration and biopsy remain the procedures of choice to detect bm involvement . f - fdg pet is supposed to have special value in detecting bm involvement ; aydin et al . described a case of bone marrow metastasis detected by f - fdg pet and missed by bone scintigraphy in widespread melanoma . f - fech - pet / ct in prostate cancer patients shares this property , whereas there is no role of ct or bs . described a positive choline bone marrow metastasis in the thoracic spine without any ct abnormality . in our results , pet positive findings without morphological changes in spine , pelvis and proximal extremities were attributed to bone marrow involvement . bs was not of further benefit in identifying them ; they were either of negative or decent mdp uptakes . that raises the following question : what is the real clinical benefit of bs over f - fech - pet / ct , if the deficiency of choline - pet is mainly in osteoblastic metastases , which are simply to be visualized using associated ct ? in other words , if ct can adequately compensate the deficiency of f - fech - pet , still bone scan offers further diagnostic information ? indeed , in order to validate its further performance as complementary test , bone scan is required to show a superiority over f - fech - pet / ct both components pet and ct . just looking at lesion - based results , there is no real justification for additional bone scan , since the gain in detecting more lesions is not valuable in patients with multiple metastases , who should anyway undergo systemic therapy . however , in patient - based results we notice that associated ct failed in identifying three false negative cases in f - fech - pet / ct , which were ultimately detected using bs . more advantage of bone scan is emphasized in its availability and ease to repeat for followup . moreover , in multiple bone metastases if pain radiotherapy is indicated , bone scan is necessary because mdp distributes in a similar way as bone seeking therapy - tracer . in f - fech - pet / ct , choline uptake is supposed to be linked with presence of tumor cells which can transport and metabolize it . in contrast , bs displays the bone metastases indirectly through demonstrating the structural alterations . for this reason , any alteration concerning the tumor focus ( whether progress or regress ) is supposed to be first detected in choline - pet . this fact and the possibility to perform a quantitative assessing grant the f - fech - pet a unique value in therapy monitoring . subsequent to therapy onset , a drop in suv is an appropriate indicator for response . by contrast , in bone scan the therapy response may lead to transient increasing mdp uptake due to an amplified osteoblastic response in the so - called flare phenomenon . this can be mistaken with progress and typically lasts about 6 months after therapy [ 2123 ] . for this reason , the early response ct is also of negligible role in therapy monitoring , since the morphological alterations occur in delay . to understand the weakness of bs in therapy monitoring , we ought to return to the pathology of the bone metastases in prostate cancer . in this type of bone metastasis , intramembranous ossification takes place in regions of fibrous stroma and after therapy administration ; the ossification process stays for a certain time running in the manufactured stroma despite damaging the causing tumor cells and evoked mediators . in other words , the ongoing mineralization processes , thus mdp uptake , would not be inhibited after therapy onset even if the therapy is successful . compared with bs , as mentioned earlier , choline uptake in f - fech - pet / ct is supposed to reflect directly the tumor focus , and the reduction in suv early after initiation of therapy can distinguish responders from nonresponders . however , does uptake decline always indicate response ? in some cases , it was demonstrated that shortly after treatment onset , the f - fech - pet / ct turned to be negative just in osteoblastic metastases , while osteolytic metastases were preserved . this effect of aht in the sclerotic metastases was already described by beheshti et al . . it may also explain the lower total sensitivity of f - fech - pet / ct compared with bs in the area - based results ( 6 patients were under ongoing aht in our study ) . in another case of our study under aht , the followup shows a dramatic uptake decline in bone metastases therefore this uptake decline in bone metastases is likely to reflect a temporal diminishing of uptake rather to be an indicator for real response . the loss of choline uptake was also observed in osteoblastic metastases in patients undergoing no therapy . as mentioned earlier , choline uptake declines with increasing sclerosis , probably due to the drop in blood supply thus in nuclide availability . . found that a hu level above 825 is associated with an absence of metabolic activity . due to the lack of long - term followup , it is still unknown whether or not these metastases are still alive if reaching the stage of negative choline uptake . obviously , even if the metastasis center reaches this stage , the rim where there is a moderate sclerosis remains active . ultimately , the potential role of f - fech - pet / ct in therapy monitoring seems to be limited in case of bone involvement , because choline uptake is likely to be affected by local factors such as the level of sclerosis . no significant gain in sensitivity was achieved using bone scan compared with f - fech - pet / ct . in lesion - based results , the diagnostic potential of both modalities varies in the different anatomical regions and shows f - fech - pet / ct mostly of superior value . that was attributed to the higher spatial resolution and the additional benefit of accompanied ct except for its value in detecting the bone marrow involvement .

18f - fech - pet / ct has been proved to be an imaging agent for prostate carcinoma . however , its role in detecting the bone metastases is still blurred owing to the lack of related studies . the purpose of our study was to assess the efficacy of pet with 18f - ethylcholine in assessing the bone status and to compare the results with that of conventional bone scan findings . for this purpose , we selected 37 patients ( mean age 69 7 ) , who had been referred for restaging purposes due to biochemical recurrences and underwent both 18f - fech - pet / ct and bone scan in a short interval . generally 18 patients out 37 patients referred with biochemical relapse were confirmed to have bone involvement . from 18 confirmed bone involvement cases , 18f - fech - pet / ct identified correctly the bone involvement in 15 cases with overall sensitivity of 83.3% . on the other hand , bone scan identified 17 out of 18 confirmed cases with overall sensitivity of 94.4% . the lesion - related results show that the sensitivity of each investigation differs with the anatomical regions , and by comparing both results , 18f - fech - pet / ct was mostly superior to bone scan ; however , without a statistical significance ( p > 0.1 ) . in conclusion , no significant gain in sensitivity was achieved using bone scan compared with 18f - fech - pet / ct .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

actinomycosis , a rare chronic granulomatous infection induced by actinomyces species , colonizes the mouth , colon , and vagina ( 1 ) . mucosal disruption may lead to infection at virtually any anatomic site of immunocompromised body , such as head and neck , chest , abdomen , and pelvis region . in literature reports , about 50% of all cases appear in the head and neck region , with relatively rare appearance at other sites ( 2 ) . f - fluorodeoxyglucose ( fdg ) is a highly sensitive but not specific radiotracer that can accumulate at various inflammation and infection sites . only a few cases of actinomycosis appearance on f - fdg positron emission tomography / computed tomography ( pet / ct ) have been documented recenlty ( 23456 ) . here , we describe a rare appearance of pulmonary actinomycosis with intrapulmonary disseminated distribution and abscess formation on f - fdg pet / ct . we found that the involved bones demonstrated increased focal uptake of radiotracer on mtc - methylene diphosphonate ( mdp ) bone scintigraphy , which has not been reported to the best of our knowledge . a 41-year - old man was admitted to our hospital with complaints of cough for 8 months . in addition , he had right thoracodorsal and shoulder discomfort as well as wrist swelling pain for 4 months . physical examination found a palpable abscess at both right upper backside and right wrist . blood routine examination and serum tumor marker analysis revealed no abnormality . a chest ct scan revealed a 7.8 5.0 cm lung mass in the right upper lobe with spiculated margins and irregular chest wall infiltration , suggesting peripheral lung carcinoma . subsequently , a tc - mdp whole bone scintigraphy was performed to determine whether there were bone metastasis . 1a , posterior ) showed that the uptake of radiotracer was increased at the right third and fourth posterior ribs ( small black arrows ) as well as right multiple carpal bones ( large black arrow ) . regional chest and right wrist ( fig . 1b : b1 , single photon emission computerized tomography [ spect ] ; b2 , ct ; b3 , spect / ct fusion ) tomography imaging examinations found no osseous abnormality . the patient underwent f - fdg pet / ct for further evaluation since this imaging method had improved sensitivity compared to tc - mdp bone scintigraphy . 1d : d1 , pet ; d2 , ct ; d3 , pet / ct fusion ; fig . 1i , sagittal ) demonstrated a 7.8 5.0 cm mass in the upper lobe of the right lung with soft - tissue density and spiculated margins similar to results of the previous ct scan . the mass had intense f - fdg accumulation with maximal standardized uptake value ( suvmax ) of 13.3 . the infiltrative mass extended to adjacent chest wall , thoracodorsal muscle , and subcutaneous dermal tissues . the soft tissues at the right upper backside was swelling with a high activity ( suvmax = 9.2 ) . additionally , some opaque mottled shadows were distributed in multiple lobes of the right lung and the inferior lobe of the left lung ( fig . 1e2 , f2 , ct ; fig . 1e3 , f3 , pet / ct fusion ) . a ct - guided lung puncture biopsy was implemented to confirm the nature of the pulmonary mass . several days later , 4 - 5 ml yellowish pus was leaked out of the previous puncture pore . a little pus was collected by squeezing the puncture pinhole after sterilization . in the meantime , 0.5 finally , the patient received treatment with intravenous antibiotics ( piperacillin - sulbactam combined with levofloxacin ) followed by oral antibiotics ( cefuroxime combined with levofloxacin ) for 14 days . actinomycosis is a rare , chronic , and slowly progressive bacterial infection caused by several members of actinomyces . among six pathogenic species of actinomyces spp . , actinomyces israelii is the most common human pathogen . in our case , a less common species ( actinomyces odontolyticus ) was found . actinomycosis induces suppurative and granulomatous inflammation characterized by swelling with suppuration , sinus tract formation , and purulent discharge containing yellowish sulfur granules in severe cases . although it is usually involved in oral and cervicofacial infections , other sites of immunocompromised body can be infected . it is mainly caused by aspiration of oropharyngeal or gastrointestinal secretions into the respiratory tract ( 7 ) . the most common clinical symptoms of pulmonary actinomycosis are chest pain , productive cough , and dyspnea . these non - specific clinical presentations make pulmonary actinomycosis difficult to be diagnosed , often leading to misdiagnosis of malignancy rather than an infective disease ( 7 ) . only a few authors have recently reported pulmonary actinomycosis appearance on f - fdg pet / ct ( 4891011 ) . because there is not much difference in pet / ct findings between actinomycosis and malignancy , utilization of pet / ct to rule out malignancy seems unnecessary . therefore , pulmonary actinomycosis should be kept in mind for differential diagnosis of cases with intensive activity on f - fdg pet / ct . microbiological and pathological examinations with demonstration of actinomyces are indispensable for correct diagnosis of actinomycosis . in our case , ct - guided lung puncture biopsy was implemented and abscesses were punctured to culture the actual bacteria . to avoid contaminations with other external bacteria , the specimen should be collected prior to antimicrobial therapy and carefully transported to the laboratory in anaerobic media . in addition , histopathologic finding of yellowish sulfur granules within pus is often necessary for differential diagnosis of actinomycosis ( 12 ) . regarding treatment options , intravenous administration of penicillin should be given followed by oral penicillin or amoxicillin ( 13 ) . in cases of penicillin allergy or resistance , ceftriaxone , hyperbaric oxygen therapy may be used as an adjunct to conventional therapy when the disease process is refractory to antibiotics .

here we report a case of 41-year - old man with a soft tissue density mass at right upper lung and palpable abscesses at right upper backside and right wrist . 18f - fluorodeoxyglucose positron emission tomography / computed tomography demonstrated a 7.8 5.0 cm mass with soft - tissue density in the upper lobe of the right lung with high metabolic activity . the infiltrative mass extended to adjacent chest wall soft tissue . final diagnosis of pulmonary actinomycosis with multiple abscesses was made . the patient responded well to antibiotics treatment .

INTRODUCTION CASE REPORT DISCUSSION

muscle contraction is due to cyclic interactions between myosin ii motor proteins and actin filaments powered by the cellular fuel adenosine-5-triphosphate ( atp ) . in in vitro assays one uses surface adsorbed myosin motors , or preferentially myosin motor fragments such as heavy meromyosin ( hmm ) , to propel fluorescent actin filaments . whereas such assays have inspired a range of nanotechnological applications , actin and myosin have not previously been interfaced with vertical nanowire arrays . we chose gap as our material system because of the established biocompatibility of gap nanowires and the generally advantageous optical properties of the iii v semiconductor materials . nanowires were produced by gold - particle - seeded metal organic vapor phase epitaxy ( movpe ) ( figures 1a and s1 ) . because nanowire growth is initialized by gap assembly at the interface of the substrate to the gold particle , the wire size and array geometry ( figures 1a and 2a ) can be defined by electron beam lithography ( ebl)-defined gold patterning . the use of regular nanowire spacing enables the growth of arrays with uniform wire length . after testing several surface derivatization procedures ( see supporting information , results section ) with respect to actomyosin motility quality ( speed and fraction of mobile actin filaments ) we chose atomic layer deposition of al2o3 on nanowires ( figure 1a ) and surrounding areas , creating suitable surface contact angles for motility without the need for prior silanization . together with the gap nanowire core diameter ( 3050 nm in different arrays ) , the 60 nm of al2o3 coating resulted in a total nanowire diameter of 150171 nm ( 160 nm unless otherwise stated ) . the wire length was either 0.96 0.03 or 5 0.2 m ( mean standard deviation ) in different arrays . in vitro motility studies were performed by infusing hmm ( 120 g / ml ) into flow cells formed by a chip with nanowire arrays ( figure 1 ) as the ceiling and a coverslip as the floor . irrespective of the actual geometry , top and bottom in the text below are defined as indicated in figure 1b . the hmm - induced sliding of alexa fluor 488 phalloidin labeled actin filaments was observed after infusion of assay solution ( 1 mm mgatp ; 2225 c ) . we used a nikon eclipse te300 inverted microscope with hg - lamp illumination , nikon 100 1.4 na oil immersion objective , fitc filter set [ excitation , 465495 nm ; dichroic mirror cutoff , 505 nm ; emission barrier filter , 515555 nm ] and a hammamatsu emccd camera . further details of the experimental methods are given in the supporting information , methods section , e.g. , quantitative analysis of the motility quality of filaments moving up and down wires ( supporting figures s2s3 ) . principle of experiments . a , nanowires are grown from gold seed particles ( i ) by metal organic vapor phase epitaxy ( ii ) and coated with al2o3 by atomic layer deposition ( iii ) . b , schematic illustration of fluorescently labeled actin filaments ( red ) that are propelled by hmm motors adsorbed to the al2o3 surface of nanowires . note , illustration not to scale . nanowire arrays . a , scanning electron microscopy ( sem ) image of nanowires in 50 50 m array with 1 m interwire distance and 5 m nanowire length . b , fluorescence micrograph from in vitro motility assay experiment with the same type of array geometry as in panel a before the addition of atp . actin filaments ( bright fluorescent lines ) are aligned with nanowire arrays being attached to hmm on the tip of the nanowires . strongly fluorescent spots on nanowires attributed to light - guiding from fluorescent actin filaments interacting with the wires along their length . occasional fluorescent spots outside the array in panel b ( arrows ) are attributed to actin filaments attached to nanowires spuriously formed by parasitic growth ( examples of such wires also indicated in panel a ) and are therefore in focus . we found that actin filaments can be bound to and propelled by hmm adsorbed to al2o3-coated gap nanowires . first , we infused fluorescent actin filaments in the absence of atp where hmm is expected to bind the filaments but not propel them . under these conditions , filaments attached themselves horizontally across the top ( figure 1b ) of a nanowire array , apparently binding to multiple nanowire tips ( figure 2 ) . in addition , several strongly fluorescent spots were associated with individual wires in sparse ( interwire distance 1 m ; figure 2 ) arrays and along the edges in denser arrays ( interwire distance < 300 nm ; not shown ) . we attribute these spots to filaments that are attached vertically along individual wires ( see below ) . upon addition of atp , we observed actin motility on areas surrounding the nanowire arrays ( figure 3a ) as well as on top of arrays having interwire distance of 1 m ( figure 3b ; supporting information , movie 1 ) and < 300 nm ( figure 3c ; supporting information , movie 2 ) . the filaments on the top moved from tip to tip , and , ultimately , some of them were propelled vertically down the wires ( figure 3d ) . occasionally , filaments were also observed climbing up wires from the surrounding surface ( figure 3e ) . however , observation of the latter phenomenon with 5 m long nanowires required refocusing of the microscope because the underlying substrate and the top of the nanowire array were located in different focal planes ( figure 3f ) . this fact was important as it also enabled us to unambiguously identify the plane in which filaments were moving ( see also supporting information , figure s4 and movie 3 ) . a , motility on al2o3 surface outside autofluorescent array illustrated by maximum pixel intensities over 50 consecutive images at 2.5 s frame rate . b , the maximum pixel intensity in sequence of 220 images at 2.5 s frame rate for array with 1 m interwire distance and 5 m nanowire length . arrows show starting points for some trajectories of actin filaments sliding on top of the array . c , image sequence of array with circular and square nanowire patterns connected by zigzag lines with nanowires . scanning electron micrograph in inset showing area on another array similar to that in circle in main figure ) . d , e , time sequence of filaments climbing down and up a nanowire , respectively . for dashed rectangles in panel d , see figure 4c . the crosses in panel e indicate tracking of the trailing filament end to estimate the length of the filament segment that is vertically attached along the nanowire ( fluorescent spot of increasing intensity ) at each given point in time . f , fluorescence images where focus changed from the bottom surface ( left ) to the top of the wires ( right ) . see also supporting information movie 3 and figure s4 for similar effects of change in focus on another area . a climbing event , whether up or down a wire , was associated with an initial rapid increase in intensity of a fluorescent spot centered on the wire ( figure 3 ; supporting information , movie 1 ) , followed by rapid decline in fluorescence of this spot ( see further below ) . such intensity variations ( blinking ) were not observed in the absence of atp , and they were seen in all cases where the filament transition from horizontal to vertical movement could be directly observed . by observing individual actin filaments first approach and then climb up or down individual nanowires , we could identify and quantify light guiding of fluorescent emission from actin filaments specifically bound to nanowires . on a sparse array ( nanowire length 5 m , interwire distance 1 m ) , filaments that climbed up or down wires were visible from the top as strongly fluorescent spots ( figure 3b ; also figure 2b , figure s2 and supporting information , movie 1 ) with a time - dependent intensity proportional to the fraction of the filament length attached vertically along the wire via hmm . importantly the number of fluorophores along the wires , at each given point in time , is directly proportional to the filament length along the wire in a well characterized way . this follows because there is precisely one binding site for the aph fluorophore per actin monomer . further , the distance between the monomers is well - known giving 362 aph binding sites per m ( see further supporting information , methods ) . thus , taking advantage of this homogeneous fluorescence labeling , we were able to estimate , with good accuracy , the number of fluorophores on the filament segments along the wire by tracking the trailing end of individual filaments whose leading end progressed up or down the wire ( figure 3e ) . results of such tracking for three filaments climbing 5 m long nanowires are illustrated in figure 4a , together with intensity measurements that show progressive linear increase in intensity ( itip ) emitted from the nanowire tip as the filament length along the wire increases . further , this intensity saturated at low values ( figure 4b ) for nanowires with length of less than 1 m ( supporting information , figure s5 ) , whereas the maximum intensity itip for filaments moving along 5 m long nanowires increased in proportion to the maximum intensity ( ih ; proportional to length ) measured when the filaments moved horizontally . the results for the two wire lengths show that only the part of the filament held close to the wire surface contributes significantly to the fluorescence signal . more importantly , however , the results in figure 4a , b for 5 m long wires can not be explained by , for example , the projection of fluorescence of vertically aligned filaments because the depth of focus of our objective ( < 0.6 m ; figure 3f ; supporting information , figure s4 and movie 3 ) , was much too limited to collect light from all along the nanowire . instead , light - collection and light - guiding by the nanowire is necessary to integrate the filament fluorescence intensity into a nearly diffraction limited spot , in the focal plane of the wire tip . this view is further supported by the effects of changes in focus on the intensity ( supporting information , figure s4 ) measured within a 0.5 0.5 m roi centered on a nanowire . a , average background - subtracted pixel fluorescence intensity from the tip of nanowires ( 0.5 0.5 m region of interest ( roi ) ; 16 bit images ; intensity units , iu16 ) plotted against the distance that three different filaments ( different symbols ) have moved down a wire of 5 m length . the nonzero intercept on the vertical axis is due to the tip of horizontally moving filaments entering the roi without movement of the trailing filament end that is tracked . this may occur for filaments moving on top of a sparse nanowire array as the number of strongly attached actin propelling heads on the wire tips vary with time . thus , the leading filament end would temporarily move faster than the tracked trailing end due to buckling caused by previous faster sliding of the trailing end . b , total background - subtracted fluorescence intensity ( itip ; average intensity number of pixels ) observed from nanowire tip due to nanowire - climbing filaments plotted against the total intensity ( ih ) of the same filaments when sliding on top of the array ( for 5 m high nanowires ; filled circles ) or on the surface between nanowires ( 0.96 m wires ; open squares ) . measurements using matlab after conversion to 8 bit images corresponding to units , iu8 . c , time course of the fluorescence signal at three different locations as indicated in figure 3d , illustrating the potential to use the light guiding effect for fluorescence detection with increased signal / noise ratio . measurement using imagej and 16 bit images showing average intensity per pixel . for details of imagej and matlab - based analyses , interestingly , the behavior was different in denser nanowire arrays ( < 300 nm spacing ) where similar apparent light - guide effects were mainly observed at edges ( indicated by strong fluorescent spots in figure 3c ; see also supporting information , movie 2 ) . quantitatively , we estimate that 50% of the light emitted from fluorescent actin filaments attached vertically along wires in sparse ( 1 m interwire distance ) arrays is emitted from their tip . we draw this conclusion , first , from the measured slope near unity ( 1.04 ; 95% ci [ 0.91 , 1.17 ] ) of the observed linear relationship ( r 0.91 ) between itip and ih for nanowires of 5 m length ( figure 4b ) . second , we reasonably assume that less than 50% of the light from a filament ( moving in an optically homogeneous environment across the nanowire tips ) is available for collection by the objective . we further note that our experimental setup does not fulfill any geometrical conditions for intensity enhancement ( neither of itip nor ih ) by fluorescence interference contrast effects . our results are thus consistent with the notion that the entire emitted fluorescence intensity from a fluorescence labeled actin filament , held by hmm along a nanowire in a sparse array , couples into the wire and that 50% of the light intensity is emitted at the wire tip , whereas 50% enters the surface substrate . in this context it is important to note that zincblende gap has a direct band gap of 2.78 ev , corresponding to 450 nm light wavelength . this should be compared to excitation wavelengths in the range 465495 nm and emission in the range 500600 nm ( observed in range 515555 nm with the current microscope filter set ) . therefore , the gap nanowires and the underlying gap substrate should absorb and reflect light only to a minor degree . our observations of efficient coupling of light into the gap nanowires and light guiding along these wires are consistent with previous findings using other semiconductor materials . in those prior studies , photonic confinement was shown for a range of nanowires ( e.g. , zno , cds , their high refractive index and the surrounding dielectric material allow for wave - guiding with amplified output . furthermore , enhanced light trapping and emission has been observed using light coupled into tips of iii v semiconductor nanowires that are grown in ordered arrays and coated with a dielectric material with a smaller refractive index than the core ( e.g. , al2o3 ) . the degree of enhancement appeared to depend on fine - tuning of the dimensions of the iii this is consistent with our observations of different degrees of light guiding for different interwire distances and suggests that there is appreciable room for optimization of light collection and emission in future experiments . our demonstration of light - guiding of gap nanowires in a biological buffer has a range of interesting applications in biosensing ( see also ref ( 5 ) ) . thus , the nanowires would perform well as optical sensors that capture analyte molecules via recognition elements ( e.g. , antibodies or oligonucleotides ) immobilized on their large surface areas . if fluorescent secondary recognition molecules are sandwiched via the analytes to the nanowires , light intensity proportional to the number of such recognition molecules would be emitted from the wire tip . in the following , we illustrate and quantify the advantages of this approach using a specific example where the velocity and fraction of motile actin filaments are evaluated . high - throughput assays with this capability are of considerable interest per se , e.g. , to evaluate effects of drugs on actomyosin motility . the efficient detection of actin filaments is also important in motor - driven diagnostics and biocomputation devices . the achievable s / n ratio for the detection of actin filament speed using nanowire arrays is much higher than that achievable on flat substrates and automated read - out is greatly simplified ( see below ) . we quantify the fraction of motile filaments by the ratio of the numbers of time - varying and time - invariant fluorescent spots ( figure s2 , supporting information ) and determine the filament velocity from the rate of change of the fluorescence intensity ( figure 4a ; see also figure s3 , supporting information ) . the fraction of motile filaments and velocity for nanowire - climbing actin filaments determined in this way was 81 3.8% ( mean standard error of the mean ; se ; n = 136 filaments ) and 2.18 0.38 m / s ( n = 10 filaments ) , respectively , compared to 84 3.9% ( n = 83 ) and 5.16 0.15 m / s ( n = 22 ) , respectively , for a standard trimethylchlorosilane ( tmcs)-derivatized flat surface . crucially , however , the time - varying fluorescence intensity for a filament moving down a nanowire was detected with considerably higher s / n ratio than for a horizontally moving filament ( figure 4c ) . to exemplify , for two different filaments the s / n ratio was 13 and 17 , respectively , when moving down a wire ( using a region of interest [ roi ] of 0.5 0.5 m ; figure 4c ) compared to less than 4 when the filaments moved on top of the nanowire array ( whether using small , 0.5 0.5 m or large rectangular rois ; figure 3d ) . qualitatively similar data were observed for several more filaments and also for filaments that first moved on the flat underlying substrate and then climbed up wires . under the present , far from optimized conditions ( see below ) , the limit of detection ( lod ) was 50100 fluorophores on individual wires as suggested by observation of the photobleaching of stationary actin filaments attached via hmm to the wires , assuming 362 fluorophores per m of an actin filament ( see above and supporting information , methods ) . here , the lod was defined as the number of fluorophores that could be detected with s / n ratio of 3 or greater . the number of 50100 fluorophores per wire corresponds to the amount of protein accumulating on the surface of a nanowire with 100 nm diameter in less than 1 h at 1 pm solution concentration . however , importantly the lod is probably somewhat lower than 50100 fluorophores as there is most likely less than 362 monomers per m of the filament , e.g. , due to prior photobleaching events . averaging the signal over a large number of wires ( nwire ) would increase the s / n ratio in proportion to ( nwire ) . for instance , a 100-fold increase in s / n ratio would be achieved by averaging over 10 000 nanowires , which could be readily fitted into < 100 100 m area . this would allow detection of analyte at 1 pm concentration in minutes or 10 fm concentration in an hour . unlike in the present study where actin filaments were bound to only a small fraction of the nanowires , recognition elements and analytes in biosensor applications the averaging should therefore be readily automated , e.g. , by appropriately adjusting array interwire distance , objective magnification , and ccd pixel size to each other . several additional improvements would markedly increase the s / n ratio and further lower the lod . this includes oblique illumination of the observation area together with reduced flow - cell height for reduced out - of - focus background and use of laser illumination instead of an hg lamp to increase excitation intensity at 480 nm wavelength . furthermore , a major source of noise , absent in a general biosensor application , is attributed to fluorescent actin filaments floating in solution . these unbound model analytes were not eliminated by rinsing as would be implemented for any other analyte , following specific binding to recognition molecules on the wires . finally , it is likely that a theoretical analysis of the underlying optical mechanisms would allow optimization of the in- and out - coupling of light , further enhancing s / n ratio . thus , the fluorescence wavelength , the nanowire material , length , diameter , spacing , and the thickness of the oxide shell might affect coupling of light into the wire . this coupling may also depend on the distance of the fluorophore to the nanowire surface ( 40 nm for hmm - actin ) , the position of the emitting fluorophores along the wire , and the polarization of fluorescence . a second key result of our study is the unique method to unambiguously demonstrate specific analyte detector interaction using molecular motor propelled cytoskeletal filaments . this was enabled by ( i ) measurement of the length of overlap between a fluorescence labeled filament and the long axis of the nanowire , ( ii ) a well - defined number of fluorophores per m of the filament , and ( iii ) molecular - motor driven variation of the filament nanowire overlap . here we used heavy meromyosin propelled actin filaments labeled with a maximum of one fluorophore per monomer ( 362 monomers/m ) via the strong phalloidin actin interaction . however , other motor systems , such as the kinesin - microtubule system , may also be used , enabling wide applicability due to different dependence of motor function on surface chemistry ( cf . ref ( 36 ) ) for the two systems . to the best of our knowledge , the present results also constitute the first demonstration of actomyosin motility on al2o3 substrate and on vertical nanowire arrays , opening for a range of opportunities . first , the observed climbing of filaments along nanowires enables three - dimensional motor driven lab - on - a - chip devices . second , the observation of actin filament propulsion across the tips of 160 nm wide nanowires at 1 m interwire spacing opens for unique studies of actomyosin function . for instance , it will allow testing of hypotheses about the motility of actin filaments over a sparse layer of motors with minimal interference from surface they are also of interest for studying contractile events at low myosin densities on actin filaments as occurs physiologically at low activation levels in muscle cells when only a fraction of the regulatory units along the filament are turned on to allow myosin binding . for developments of functional assays it may be beneficial to make the vertical parts of the nanowires resistant to hmm binding in analogy to a previous study where kinesin propelled microtubules were observed moving between tips of micrometer wide poly(dimethylsiloxane ) ( pdms ) pillars . we have demonstrated light guiding with high efficiency along al2o3 coated gap nanowires of appreciable relevance for biosensing applications . with further improved s / n ratio as discussed , sensor elements based on individual vertical nanowires could achieve fm pm sensitivity , and arrays of such nanowires could perform appreciably better . the use of molecular motor driven propulsion of fluorescent filaments along the nanowires to detect specific molecular interactions may have broad applicability in the evaluation of biosensor performance . in addition , our findings lay the foundations for novel types of fundamental studies and nanotechnological applications of molecular motors .

semiconductor nanowire arrays offer significant potential for biosensing applications with optical read - out due to their high surface area and due to the unique optical properties of one - dimensional materials . a challenge for optical read - out of analyte - binding to the nanowires is the need to efficiently collect and detect light from a three - dimensional volume . here we show that light from fluorophores attached along several m long vertical al2o3 coated gallium phosphide nanowires couples into the wires , is guided along them and emitted at the tip . this enables effective collection of light emitted by fluorescent analytes located at different focal planes along the nanowire . we unequivocally demonstrate the light - guiding effect using a novel method whereby the changes in emitted fluorescence intensity are observed when fluorescent cytoskeletal filaments are propelled by molecular motors along the wires . the findings are discussed in relation to nanobiosensor developments , other nanotechnological applications , and fundamental studies of motor function .

Actomyosin Motility Interfaced with Nanowire Arrays Light-Guiding by Nanowires Applications of Light-Guiding New Method for Evaluating Biosensor Performance Further Implications of Results Conclusions Supplementary Material

this study was conducted in a school center that had been the focus of intense public concern over 2 years because of suspected mold and health problems . because several attempts to find solutions to the problem within the community were not satisfactory , outside specialists were needed for support in solving the problem . the study group consisted of experts in civil engineering , indoor mycology , and epidemiology . the studies were conducted in close cooperation with the city administration . structures at risk were opened , moisture and temperature were measured , and the causes of damage were analyzed . microbial samples were taken from the air , surfaces , and materials . health questionnaires were sent to the schoolchildren and personnel . information on the measurements and their results was released regularly to school employees , students and their parents , and to the media . repairs were designed on the basis of this information . moisture damage was caused mainly by difficult moisture conditions at the building site , poor ventilation , and water leaks . fungal genera ( concentrations < 200 colony - forming units ( cfu)/m(3 ) , < 3000 cfu / cm(2 ) ) typical to buildings with mold problems ( e.g. , aspergillus versicolor , eurotium ) were collected from the indoor air and surfaces of the school buildings . where moisture - prone structures were identified and visible signs of damage or elevated moisture content were recorded , the numbers of microbes also were high ; thus microbial results from material samples supported the conclusions made in the structural studies . several irritative and recurrent symptoms were common among the upper secondary and high school students . the prevalence of asthma was high ( 13% ) among the upper secondary school students . during the last 4 years , the incidence of asthma was 3-fold that of the previous 4-year period.imagesfigure 1

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poly(vinylidene fluoride ) ( pvdf ) is a widely studied and used polymer , especially for industrial application due to its excellent chemical , mechanical , and uv stability properties . pvdf membranes have been extensively used in ultrafiltration ( uf ) and microfiltration ( mf ) applications for separation processes and wastewater treatment and currently are explored as contactors and in bioseparation applications . an interesting new application is involving the use of pvdf blends to make artificial muscles . in addition , there is increasing interest in the modification of pvdf membranes with environmentally sensitive functionalities for many diverse applications such as drug delivery , cell encapsulation , electronic devices , sensors , and water softening . to meet the needs of the desired application , various methods are currently used such as coating , adsorption , the grafting of functional groups or graft polymerization to the membranes , and chemical modification of membrane materials . in our previous work , we described methods for obtaining pvdf membranes with ph and temperature responsive properties via in situ hydrogel cross - linking or in situ polymerization in solvent phase . these functionalized membranes were subsequently used as a support to immobilize fe or fe / pd nanoparticles to remove toxic chloro - organics from groundwater . in this work , we extend previous studies and present a new method to form covalently attached polymers onto the pvdf membranes involving chemical treatment of the pristine membrane with alkaline solutions to obtain dehydrofluorinated pvdf membranes . the reaction mechanism of alkaline degradation of pvdf is shown as follows : where x = na , k , or li . the dehydrofluorinated pvdf polymer forms conjugated double bonds or a polyene structure which can be attacked by specific reactants to introduce functional groups to the membrane , such as acrylic acid ( aa ) . the introduction of covalent bonding can eliminate the concerns on the stability of poly(acrylic acid ) chains on the pvdf membrane prepared by the in situ hydrogel cross - linking method . as mentioned above , the pvdf membrane support is further used to immobilize metal nanoparticles for a dechlorination reaction . as expected , a higher metal loading leads to a higher reaction rate , and a higher surface area of the support pvdf membrane is required to achieve a greater degree of metal loading . therefore , in this work , another improvement was achieved through the modification of pristine pvdf structure . pvdf membranes are usually made by a phase inversion method induced by immersion of a cast solution in a polymer nonsolvent bath . the membrane morphology , porosity , flux and retention properties can be altered by changing the casting and immersion parameters . in this work , a sponge - like pvdf membrane is designed to have higher ion adsorption capacity which results in higher nanoparticle loading . the dehydrofluorination of pvdf membranes has been reported . however , the application of dehydrofluorinated pvdf for covalent attachment of different polymers for reaction and separation has been reported very little . the main goals of the present study are to ( 1 ) create dehydrofluorinated membranes for the introduction of proper functional groups through covalent bonding ; ( 2 ) investigate the effect of alkaline treatment time on the membrane structure ; ( 3 ) study the ph responsive behavior of functionalized pvdf membranes ; and ( 4 ) prepare sponge - like pvdf membranes with high surface area and metal loading . sodium hydroxide ( naoh ) , acrylic acid ( aa ) , ammonium persulfate ( aps ) , ferrous chloride tetrahydrate ( fecl24h2o ) , sodium borohydride ( nabh4 ) , trichloroethylene ( tce ) , n , n - dimethylfomamide ( dmf ) , and lithium chloride ( licl ) were purchased from sigma - aldrich . deionized ultrafiltered water ( diuf ) was purchased from fisher scientific . the pvdf powder was a commercial product ( kynar 761 ) ( mw = 350 000 g / mol ) kindly offered by ultura inc . ultrahigh purity ( uhp ) nitrogen gas used in flux experiments was purchased from scott specialty gases . a piece of pvdf membrane was soaked in 15 wt % naoh solution in 40 ml diuf for 10 min . then , the membrane was sandwiched between two glass plates and placed in an oven at 70 c to react for 1 , 3 , and 22 h. the final membrane was washed with diuf until the ph became neutral . the dehydrofluorinated pvdf membrane was functionalized with paa by in situ polymerization with acrylic acid . the polymerization solution contained 11.1 wt % acrylic acid ( monomer ) and 0.4 wt % of aps ( initiator ) . the def - pvdf membrane was dipped in the polymerization solution for 5 min , sandwiched between two glass plates , and placed in an oven at 90 c for 2 h. the undehydrofluorinated pvdf membrane was also functionalized with paa as a control by using the same pore - filling method . the casting solution was made of 20 wt % pvdf , 2 wt % pvp , 2.25 wt % licl , and 75.75 wt % dmf and heated to 50 c . a film of pvdf solution was first cast on a glass plate ( 2325% humidity ) and then immersed in diuf water at 50 c for coagulation . after 10 s , the formed membrane was put in pure diuf water at 23 c to wash and let it dried in an oven at 70 c . the full - scale spvdf was also fabricated under the same conditions by ultura inc . attenuated total reflectance fourier transform infrared ( atr - ftir ) spectroscopy ( varian 7000e ) was used to determine the presence of functional groups in dehydrofluorinated pvdf and functional membranes . the samples were placed on the diamond crystal , and the spectrum was obtained between 500 and 4000 cm for 32 scans at a resolution of 8 cm . the surface and cross - section morphology of the blank pvdf , def - pvdf , functionalized membranes , and spongy pvdf were studied using a hitachi s-4300 scanning electron microscope ( sem ) . the samples were mounted on sample studs , and a thin layer of gold was sputtered on the sample surface for imaging purpose . the sem measurements were performed at an accelerating voltage of either 10 or 3 kv . the surface composition of membrane was characterized using an x - ray photoelectron spectroscopy ( thermo scienctific k - alpha ) with al / k ( h = 1486.6 ev ) anode mono x - ray source . the sample was directly mounted on a sample holder then transferred into the analyzer chamber . the entire spectra of all the elements were recorded with very high resolution using avantage software . the water permeability was measured at different ph levels to study the ph responsive flux behavior of paa - def - pvdf . the tested membrane was mounted in a stirred cell ( millipore ) which contains feedwater of varying ph . once the membrane flux reached steady - state , volume flux was measured in triplicates by recording the volume passed through the membrane in a given time interval . na2so4 rejection experiments were performed using a stirred membrane cell provided by millipore with a membrane cross - sectional area of 13.2 cm which included a stirring device to minimize the effects of concentration polarization . the permeate sodium concentration was measured with a varian aa220 series atomic absorption spectrophotometer . the method for the synthesis of nanoparticles was developed by our group and described previously . the fe / pd nanoparticles were made by an ion - exchange with fe , the reduction with sodium borohydride , and a post - palladium coating . the dechlorination was conducted in a 40 ml vial loaded with nanoparticles and fed a tce solution at 30 mg / l . the tce concentration was determined after a certain time interval by extracting the sample with pentane and analyzing by gas chromatography ( hp series ii model 58590 ) with a mass spectrometer ( gc - ms ) . the pvdf membranes were treated with 15 wt % of sodium hydroxide for different treatment times . after treatment , a color change was observed for all the membranes from white to a light yellow or deep yellow or even black as shown in figure 1 . the atr - ftir spectra of blank pvdf , def - pvdf , and paa - def - pvdf membranes are shown in figure 2 . the characteristic peaks at 1403 and 1600 cm are corresponding to ch2 and c = c , respectively ( figure 2b d ) . this is due to the formation of polyene in the membrane as the fluorocarbon groups of pvdf are changed by the treatment ( 10001250 cm ) ( figure 2a ) . as the treatment time is increased , the intensity of the carbon double bond response increases . it should be noted that the low absorbance intensity at 1600 cm is due to the inactive c = c bond in infrared . the hydrocarbon at peaks 13501450 and 800900 cm do not change significantly with treatment . change in color of the pvdf membranes after treatment with 15 wt % of naoh for different hours at 70 c : ( a ) 0 , ( b ) 1 , ( c ) 3 , ( d ) 22 h. atr - ftir spectrum of blank pvdf , def - pvdf , and paa - def - pvdf membranes . ( a ) blank pvdf ; ( b ) def - pvdf with 1 h naoh treatment ; ( c ) def - pvdf with 3 h naoh treatment ; ( d ) def - pvdf with 22 h naoh treatment ; ( e ) paa - def - pvdf ( paa functionalization of sample c ) . the percentage of transmission scales is varied in the range 10040% . as for the paa functionalized def - pvdf membranes , the appearance of an obvious peak at 1720 cm belongs to the cooh stretching vibration . to prove the covalent attachment of paa with def - pvdf this result together with the atr - ftir spectra obviously demonstrates that the paa was successfully covalently grafted onto the surface of the pvdf membrane . the morphologies of the blank and functionalized pvdf membranes were characterized by sem as shown in figure 3 . a pristine pvdf membrane ( figure 3a ) shows fairly porous structure with mostly circular shape and uniform pore size . the effect of treatment time with naoh on the structure of membrane was also investigated by sem , and the results can be seen in figure 3b and c. when treated for a short amount of time ( such as 1 or 3 h ) , no change in the membrane surface was observed as shown in figure 3b . on the other hand , large pores are formed on the membrane surface ( figure 3c ) after being treated for 22 h. it was also found that the membrane was easy to crack with less mechanical force due to the loss of fluorine by the dehydrofluorination treatment . additionally , from figure 3d , it can be clearly seen that paa is grafted on the membrane surface and also has little effect on the structure of the pvdf membrane . sem image of blank pvdf ( a : sample a in figure 2 ) , dehydrofluorinated pvdf-3h ( b : sample c in figure 2 ) and -22h ( c : sample d in figure 2 ) , and paa - def - pvdf ( d : sample e in figure 2 ) . the surface compositions of the blank membrane , def - pvdf , and paa - def - pvdf membranes were investigated by xps . compared with a blank pvdf membrane ( figure 4a ) , the dehydrofluorinated pvdf membrane ( figure 4b ) shows a higher level of noise due to the change in surface roughness after treatment with naoh solution . the dehydrofluorination is proved by a reduction of fluorine intensity , and relative increase of carbon intensity . the decrease in fluorine concentration and the increase in carbon concentration with time are also shown in figure 5 , which indicate that most of the dehydrofluorination happens in the first hour of the treatment , and it is also seen that the rate of reaction slows down as treatment time increases . as shown in figure 4c , the increase of carbon and oxygen intensity indicates that the paa was grafted on the membranes . figure 6 show the respective c 1s core - level spectra of the blank pvdf ( figure 6a ) and paa - def - pvdf ( figure 6b ) membranes . the blank pvdf membrane can be fitted with two main peaks at 286 ev for ch2 groups and 290.6 ev for cf2 groups . on the other hand , the c 1s spectra of the paa - def - pvdf membrane can be fitted with five chemical species . the peak with binding energy of 288.5 ev belongs to the c = o species of the grafted acrylic acid ( aa ) groups . the peak with binding energy at 286.4 ev can be assigned to c o of aa groups and the peak with binding energy at 284.6 ev is assigned to ch2 groups from the grafted paa polymer . xps survey spectra of pvdf ( a : sample a in figure 2 ) , def - pvdf ( b : sample c in figure 2 ) , and paa - def - pvdf ( c : sample e in figure 2 ) . changes in xps elemental percentages of fluorine and carbon with different treatment time using15 wt % naoh . xps c 1s core - level spectra of pristine pvdf ( a : sample a in figure 2 ) and paa - def - pvdf ( b : sample e in figure 2 ) membranes . figure 7 shows the dsc curves of the pristine pvdf , def - pvdf , and paa - def - pvdf membranes . it is well - known that pristine pvdf membrane is a partially crystalline polymer because of its symmetrical structure and a melting point of about 173 c . after dehydrofluorination , the melting point for the def - pvdf membrane does not change as seen in figure 7b . however , after the covalent binding of paa on the dehydrofluorinated pvdf membranes , the symmetrical structure of the pristine pvdf is partially destroyed resulting in the decrease of the melting point to 168 c ( figure 7d ) . on the other hand , the pvdf membranes functionalized with paa by pore - filling methods show no obvious change in the melting point ( figure 7c ) due to the formation of polymer blends . thus , the dsc results suggest that the grafting of paa chains on the pristine pvdf membrane structure should impart the membranes with enhanced and stable functionalities . dsc thermogram of ( a ) pristine pvdf ( sample a in figure 2 ) , ( b ) def - pvdf ( sample c in figure 2 ) , ( c ) paa - pvdf by the pore - filling method , and ( d ) paa - def - pvdf ( sample e in figure 2 ) . since paa was present in the functionalized membranes , a response of water permeation through the membrane with ph would be expected . the ph effect on the water permeability of paa - def - pvdf functionalized membranes is shown in figure 8 . for comparison , the permeability of a pristine pvdf membrane and def - pvdf membrane are 964 and 1474 l / mhbar , respectively . as expected , when the membrane was functionalized with paa , the flux decreases due to the filling of pores with paa polymer . it is also seen that as the ph of the feed solution increases , the water flux decreases . this is consistent with previous findings where similar tests were done with paa functionalized pvdf membrane by the pore - filling method . the change in flux as a response to the change in ph can be caused by the conformational change of acrylic acid polymer chains in the membrane . the linear relationship between water flux and applied pressure resulting in constant flux at ph 4 for different experimental runs indicate that the paa functionalization on the membrane is very stable . the water permeability data can be fitted with the following equations to obtain the pka:12where , lp , max and lp , min are the maximum permeability ( 760 l / mhbar ) and minimum permeability ( 29.1 l / mhbar ) when the ph is increased from 3 to 8.5 . the experimental data were fitted with both equations and the pka was found to be 5.2 , which is consistent with the literature reported pka value for paa . ph effect on the pure water permeability of the paa - def - pvdf functionalized membrane . the amount of cooh groups in the functionalized membrane was characterized by the amount of ca entrapped by the membrane due to the high affinity of the free cooh groups to ca . to do this , 100 ml of a 1000 mg / l ca solution the feed and permeate samples were collected , and the ca concentrations were analyzed using a varian aa220 series atomic absorption spectrophotometer . based on ion exchange principles , the assumption is that 1 mol ca can be bound to 2 mol carboxyl groups . therefore , the calculated cooh quantity is 0.03 mmol / cm . our previous publication showed that for paa - pvdf membranes functionalized by pore - filling methods , ca pick - up can range from 0.008 to 0.014 mmol / cm for different degrees of cross - linking . here , the proper amount of cross - linker is required to keep the paa functionalization stable without washing out . on the other hand , it has been found that too much cross - linking will block membrane pores and increase the mass transfer resistance . the covalent binding of paa onto the membrane surface offers a solution to avoid this problem . the effect of ph on the ion exclusion of the paa functionalized def - pvdf membrane is evaluated , and the results are shown in figure 9 . with a ph of 8 , meanwhile at lower ph ( around ph 4 ) , the observed rejection is decreased to 10% . this dramatic decrease of solute rejection is because of the helix coil transitions of the paa chain in response to the ph change . at lower ph , the paa chain is protonated , which can reduce the overall charge and thus decrease the electrostatic interaction between the membrane and permeate ions . when the ph increases to ph 8 , the paa is ionized with enhanced electrostatic interaction and thus a higher rejection is seen . effect of feed solution ph on the ion exclusion ( p = 0.7 bar ) of a 100 mg / l na2so4 solution through paa - def - pvdf membrane . the relationship between na2so4 rejection and permeate flux for a paa functionalized pvdf membrane is shown in figure 10 . the inset figure shows the linear relationship between the pressure and flux , which indicates the absence of membrane fouling . the observed decline in rejection at a higher flux is in direct contrast to experimental trends generally observed for ion separations in dense media such as reverse osmosis ( ro ) and nanofiltration ( nf ) . for nf type membranes , it is seen that the solution rejection increases with increasing flux due to the reduced effect of diffusion . the microporous membranes used in this study show a nonuniform distribution within the pore geometry . this is primarily due to the incomplete pore coverage resulting from nonuniform functionalization or a low chain length to pore ratio . at higher applied pressures , there is enhanced flow through the region of the membrane that is not covered by paa chains due to the reduction in hydrodynamic thickness of the paa layer . commercial nanofiltration membranes are reported to have a 95 to 99% na2so4 rejection . however , these membranes have a pore size of about 1 nm and are operated at pressures ranging from 10 to 40 bar . therefore , the development of much more effective membranes will require much higher paa loading and the limitation of the core region . effect of operating pressures on the ion exclusion of a 100 mg / l na2so4 solution at ph = 6.5 . the inset is the water flux ( ph = 6.5 ) dependence on pressure . nanoparticles were used for the dechlorination of trichloroethylene ( tce ) , a ubiquitous pollutant in groundwater . figure 11 shows the successful degradation of tce with time by fe / pd nanoparticles . this reaction can be modeled as a pseudo - first - order heterogeneous reaction:3where c is the tce concentration in water ( mg / l ) ; ksa is the surface area normalized reactivity ( l / mh ) ; s is the specific surface area of nanoparticles ( m / g ) ; m is the mass concentration of nanoparticles ( g / l ) , and t is the time ( h ) . under the reaction conditions leading to the results in figure 11 , ksa is calculated to be 0.11 l / mh which is consistent with the literature reported dechlorination reactivity by fe / pd nanoparticles . tce dechlorination by fe nanoparticles immobilized in paa - def - pvdf membrane : vol = 43 ml , initial ph = 6.8 , initial tce concentration = 30 mg / l , iron loading amount = 0.2 g / l ( pd = 1.4 wt % of fe ) , temperature = 25 c . as discussed in previous session , the effective dechlorination or ion separation require high loading of paa into the membrane pores . therefore , spongy pvdf membranes were first prepared by the phase - inversion method in lab - scale . the full - scale spvdf was later developed to assess the feasibility of continuously manufacturing these membranes . it is clearly seen that the overall structure of the membrane cross - section is asymmetrical consisting of a skin surface ( figure 12a ) on the top layer supported by a macroporous spongelike sublayer ( figure 12b d ) . these macrovoids became more accentuated and extended throughout the entire cross - section . the void volume of the spongelike pvdf membrane can be estimated as follows:4where vm and vp are the membrane volume and volume occupied by pvdf polymer respectively , l is the pvdf membrane thickness ( cm ) , a is the membrane area ( cm ) , wm is the membrane mass ( g ) , and p is the density of pvdf ( about 1.78 g / cm ) . the properties of the spvdf membranes were compared with our previous pvdf membranes from millipore as shown in table 1 . the spvdf membrane shows high porosity at about 78.6% and almost 4 times higher surface area compared with that of pvdf ( millipore ) . although the spvdf showed less permeability , the skin layer can enhance antifouling properties due to the prevention of large particles deposition into membrane pores . while the highly porous structure below the skin layer without tortuosity can reduce the residence time of solute and eliminate the mass transfer resistance . sem images of cross - section spongy pvdf morphology under different magnification : ( a ) whole picture , 300 ; ( b ) skin surface and macroporous matrix , 4000 ; ( c ) interconnected fibrous structure in the microporous matrix , 10 000 ; ( d ) microporous matrix under skin surface , 10 000 . supplied by manufacturer . with more surface area , a higher grafting yield will be expected . therefore , these two membranes were functionalized with acrylic acid under the same conditions , and the results are shown in table 2 . as expected , the spvdf shows high grating yield at about 81.1 wt % compared to 13% for pvdf ( millipore ) . the ion exchange capacity was evaluated by ca pickup on these two functionalized membranes . the adsorption capacity of the paa - spvdf increases in the first hour and then remains relatively constant at 3.2 mg / cm ( 265.1 mg / g ) . the paa - spvdf membrane shows high ion - exchange performance with an adsorption capacity that is 10 times higher than paa - pvdf ( millipore ) as shown in table 2 . the sem - eds spectra shows the elemental composition and distribution of species and gives the atom ratio of 3.3 ( oxygen per calcium ) ( figure 13 ) . the pvdf membrane treated with alkaline solution leads to a modified membrane ( def - pvdf ) with conjugated double bonds which were confirmed by atr - ftir and xps spectra . the introduced double bonds make the membrane suitable for the covalent attachment of paa polymer . the paa - def - pvdf membrane shows ph responsive behavior for both the hydraulic permeability and solute retention . this porous medium offers less resistance to solvent transport , allowing for higher permeation rates at much lower operating pressures with less energy consumption comparing to traditional nf membrane . the paa - def - pvdf membrane was also utilized as a support to immobilize metal nanoparticles to remove toxic chlorinated compounds ( such as , tce ) from water . asymmetric membranes with a macroporous sponge - like structure were successfully prepared by the phase inversion method in both lab - scale and large - scale . the modified pvdf membrane shows high surface area , high porosity and higher yield of paa functionalization compared to traditional microfiltration pvdf membrane . the ion - exchange capacity of ca is 3.2 mg / cm ( 265.1 mg / g ) , which is 10 times higher than that reported in our previous publications . the paa - spvdf membrane can be used to remove heavy metal ions from water due to its good ion - exchange properties . these two modifications of pvdf can extend the development of functionalized membranes with advanced applications .

poly(vinylidene fluoride ) ( pvdf ) was modified by chemical treatments in order to create active double bonds to obtain covalent grafting of poly(acrylic acid ) ( paa ) on membrane . the attenuated total reflectance fourier transform infrared ( atr - ftir ) spectrum confirms the formation of conjugated c = c double bonds with surface dehydrofluorination . the membrane morphology was studied by scanning electron microscopy ( sem ) . the surface composition was characterized by x - ray photoelectron spectroscopy ( xps ) . the thermal stability of the dehydrofluorinated membrane ( def - pvdf ) and functionalized membranes were investigated by differential scanning calorimetry ( dsc ) analysis . the influence of covalently attached paa on def - pvdf membrane has been investigated to determine its effect on the transport of water and charged solute . variations in the solution ph show an effect on both permeability and solute retention in a reversible fashion . metal nanoparticles were also immobilized in the membrane for the degradation of toxic chlorinated organics from water . in addition , pvdf membranes with an asymmetric and sponge - like morphology were developed by immersion - precipitation phase - inversion methods in both lab - scale and large - scale . the new type of spongy pvdf membrane shows high surface area with higher yield of paa functionalization . the ion - capacity with ca2 + ions was also investigated .

Introduction Experimental Section Results and Discussion Conclusion

the first - line regimen for the treatment of helicobacter pylori ( h. pylori ) infection in japan is triple therapy with a proton pump inhibitor ( ppi ) , amoxicillin and clarithromycin [ 1 , 2 ] . failure of eradication of h. pylori infection with this first - line therapy has been reported in approximately 20% of infected patients [ 3 , 4 ] . with the increase in the frequency of clarithromycin - resistant h. pylori , there is rising concern about the potential decline in the eradication rate of this infection . although 7-days therapy with ppi - amoxicillin - metronidazole has been found to be effective as a second - line regimen in patients showing failure of the first - line regimen , approximately 10% of patients fail to respond to even the second - line treatment [ 6 , 7 ] . we recently developed a useful predictor of the response to metronidazole - containing second - line regimens based on the minimal inhibitory concentrations ( mics ) of both amoxicillin and metronidazole , and the results of the urea breath test . since a high eradication resistance index signals a poor response to second - line eradication therapy , patients with a high eradication resistance index may benefit from a change to other treatment regimens selected based on the results of drug susceptibility testing . currently , a standard third - line therapy still remains to be established , and european guidelines recommend culture before the selection of a third - line treatment based on the microbial sensitivity to the antibiotics . h. pylori isolates after two eradication failures are often resistant to both metronidazole and clarithromycin . therefore , these two drugs are not recommended for inclusion in third - line regimens . the alternative candidates for third - line therapy are quinolones , tetracycline , rifabutin , furazolidone ; high - dose ppi / amoxicillin therapy is also promising [ 10 , 11 ] . quinolones show good oral absorption , no major side effects and marked activity against h. pylori . a recent in vitro study showed synergistic effects of a quinolone and ppi against h. pylori strains . some studies have evaluated the efficacy of quinolones for use in a third - line regimen . herein , we discuss the usefulness of quinolone - based therapy as a third - line regimen . primary resistance of h. pylori to quinolones has been reported to range between 2%22% in different countries or regions [ 1421 ] . the prevalence of quinolone resistance is higher in japan , korea and italy ( 1522% ) , and the prevalence is very low in china and egypt ( about 2% , fig . 1 ) . we reported ahigh resistance rate ( 47.9% ) togatifloxacin ( 8-methoxy quinolone ) after eradication failure in japan . resistance to quinolones is easily acquired , and the resistance rate is relatively high in countries with a high consumption rate of these drugs . this enzyme , in addition to relaxing supercoiled dna , introduces negative supercoils into the dna causing the bacterial chromosome to be maintained in a negatively supercoiled state . the bacterial enzyme gyrase is a tetramer consisting of two a and two b subunits encoded by the gyra and gyrb genes , respectively . quinolones exert their antimicrobial activity at the level of the a subunit of the dna gyrase . this subunit , responsible for dna cleavage and rejoining , is also the site of action of quinolones . several studies have shown that the quinolone resistance - determining region ( qrdr ) of the gyra gene plays a critical role in the resistance of h. pylori to quinolones . h. pylori does not possess a gene encoding topoisomerase iv , an important quinolone target in other bacteria . we recently demonstrated a significant association between the mics of gatifloxacin equal to or greater than 1 g / ml against h. pylori and mutations of the qpdr of the gyra . furthermore , we recently designed a rapid test based on an allele - specific polymerase chain reaction ( pcr ) to detect gyra mutations . because the traditional culture test for bacterial susceptibility to antibiotics is expensive and requires 1014 days , this test is not feasible in routine clinical practice . however , the genotyping by allele - specific pcr takes less than 34 h and the allele - specific - pcr method is useful for easy identification of quinolone - resistant strains of h. pylori . a few years ago , we showed the in vitro development of gatifloxacin resistance of h. pylori strains in gatifloxacin - containing agar culture . in these studies , four gatifloxacin and clarithromycin - susceptible strains were serially plated onto gatifloxacin - containing agar or clarithromycin - containing agar , respectively , of increasing agar density . none of the strains plated on the clarithromycin - containing agar developed resistance to clarithromycin , until the 10th generation of repeated culture . in contrast , all four strains plated on the gatifloxacin - containing agar developed gatifloxacin resistance , and three of these four strains had mutations of gyra . these data suggest that resistance to quinolones is easily acquired and widespread prescription of quinolones may lead to the spread of resistance of h. pylori to quinolones . zullo et al . evaluated the efficacy of a combination of levofloxacin - amoxicillin in 36 patients with a history of failure of two or more therapeutic attempts . a 10-day regimen of rabeprazole ( 20 mg b.i.d . ) , levofloxacin ( 250 mg b.i.d . ) , and amoxicillin ( 1 g b.i.d . ) h. pylori was successfully eradicated in 30 patients , representing an eradication rate of 83.3% ( 95% confidence interval , 71.295.5 ) and 88.2% ( 95% confidence interval , 77.499 ) by intention - to - treat ( itt ) and per protocol ( pp ) analysis , respectively . this study demonstrated that levofloxacin - amoxicillin triple therapy administered for 10 days is a successful third - line therapeutic approach for h. pylori eradication ( table 1 ) . in a prospective multicenter study , managed 100 patients with a history of two consecutive h. pylori eradication failures with a third - line levofloxacin - based regimen . patients with failure of a first trial of omeprazole - clarithromycin - amoxicillin and a second trial of omeprazole - bismuth - tetracycline - metronidazole ( or ranitidine bismuth citrate with these antibiotics ) were enrolled . a 10-day regimen consisting of omeprazole ( 20 mg b.i.d . ) , levofloxacin ( 500 mg b.i.d . ) , and amoxicillin ( 1 g b.i.d . ) was administered . the eradication rates determined by pp and itt analyses were 66% ( 95% confidence interval , 5675% ) and 60% ( 95% confidence interval , 5070% ) , respectively , suggesting that levofloxacin - based rescue therapy may represent an encouraging empirical third - line strategy after multiple previous h. pylori eradication failures . furthermore , gisbert et al . compared rifabutin and levofloxacin rescue regimens in patients with two consecutive h. pylori eradication failures . patients who failed two eradication attempts received 10 days treatment with either rifabutin ( 150 mg b.i.d . ) or levofloxacin ( 500 mg b.i.d . ) , plus amoxicillin ( 1 g b.i.d . ) and omeprazole ( 20 mg b.i.d . ) . the cure rate obtained by per - protocol analysis was 45% ( 95% confidence interval , 2666% ) in the rifabutin group and 81% ( 5793% ) in the levofloxacin group ( p<0.05 ) . the eradication rates obtained by itt analysis were 45% ( 2666% ) and 85% ( 6495% ) , respectively ( p<0.01 ) . this study demonstrated that a triple levofloxacin - based rescue regimen administered for 10 days is more effective than a rifabutin - based triple regimen after two previous h. pylori eradication failures . hsu et al . designed the prospective study to assess the efficacy of levofloxacin , amoxicillin , bismuth and rabeprazole quadruple therapy as a third - line treatment for h. pylori infection . the patients were 37 consecutive h. pylori - infected patients with a history of failure of standard first - line and second - line treatments and received a 10-day quadruple therapy comprising rabeprazole ( 20 mg b.i.d . ) , bismuth subcitrate ( 300 mg q.d.s . ) , amoxicillin ( 500 mg q.d.s . ) and levofloxacin ( 500 mg o.d . ) . h. pylori was successfully eradicated in 84% of the patients , as determined by both itt analysis and pp analysis . this study suggested that a 10-day regimen of levofloxacin and amoxicillin - based quadruple therapy is well tolerated and yields a high eradication rate as a third - line empirical treatment regimen for h. pylori infection . recently novel quinolones were developed , and superior in vitro activity of gatifloxacin over that of levofloxacin against h. pylori was reported . furthermore , garenoxacin ( des - fluoro(6 ) quinolone ) and sitafloxacin show activities four - fold or greater than those of gatifloxacin [ 2931 ] . evaluated the efficacy of a 7-day regimen of gatifloxacin ( 400 mg o.d . ) , amoxicillin ( 1 g b.i.d . ) and rabeprazole ( 20 mg b.i.d . ) in 45 patients as a second - line treatment . h. pylori was successfully eradicated in 84.4% of the patients , as determined by both itt analysis and pp analysis ( 95% confidence interval , 7495% ) . although the meta - analysis of gisbert et al . showed that 10-day regimens were more effective than 7-day regimens in the case levofloxacin - based triple therapy , ( 81% vs 73% ; p<0.01 ) , gatifloxacin - based triple therapy was sufficiently effective even when administered for only 7 days . we recently investigated the efficacy of gatifloxacin - based triple therapy as a third - line treatment for h. pylori eradication , administered after assessment of the susceptibility of the organisms to gatifloxacin and the presence of gyra mutations . a 7-day regimen of gatifloxacin ( 400 mg o.d . ) , amoxicillin ( 500 mg q.d.s . ) and rabeprazole ( 10 mg q.d.s . ) was administered in 11 patients . successful eradication of h. pylori was achieved in 63.6% of the patients , as assessed by both itt analysis and pp analysis . the eradication rate was 100% in the patients infected with gatifloxacin - susceptible bacteria and/or bacteria without gyra mutations , but only 33.3% in those infected with gatifloxacin - resistant bacteria or bacteria with gyra mutations . this difference in the eradication rate between patients infected with gatifloxacin - susceptible and gatifloxacin - resistant bacteria was statistically significant ( p<0.05 ) . our data suggest that the selection of gatifloxacin for third - line therapy should be based on the results of drug susceptibility testing or gyra analysis . gisbert et al . reported that adverse effects occurred in 25% of the patients ( 23/91 ) administered the 10-day regimen of levofloxacin , rabeprazole and amoxicillin , consisting mainly of metallic taste ( 8% ) , nausea ( 8% ) , myalgia / arthralgia ( 5% ) , and diarrhea ( 4% ) . reported that adverse effects occurred in 8.9% of the patients ( 4/45 ) treated with a 7-day regimen of gatifloxacin , rabeprazole and amoxicillin mainly consisting of nausea , headache and mild diarrhea , none of which necessitated discontinuation of therapy . however , they mention that clinicians should be aware of possible alterations in the blood glucose levels , which finally leads to the withdrawal of gatifloxacin , qtc interval prolongation , seizures and phototoxicity with the use of fluoroquinolones , especially in patients with other risk factors for these conditions . quinolone - based rescue therapy serves as an encouraging third - line strategy after multiple previous h. pylori eradication failures . novel quinolones , including sitafloxacin or garenoxacin , are more potent against h. pylori than levofloxacin and gatifloxacin , and switching to these quinolones may improve third - line eradication efficacy . european guidelines recommend culture before the selection of a third - line treatment based on microbial sensitivity to the antibiotics . the selection of quinolones for third - line therapy should be based on the results of drug susceptibility testing or gyra analysis . as other alternatives for third - line rescue therapy , rifabutin - based triple therapy [ 36 , 37 ] , high - dose ppi / amoxicillin therapy and furazolidone - based therapy , if available , have been suggested .

currently , a standard third - line therapy for helicobacter pylori ( h. pylori ) eradication remains to be established . quinolones show good oral absorption , no major side effects , and marked activity against h. pylori . several authors have studied quinolone - based third - line therapy and reported encouraging results , with the reported h. pylori cure rates ranging from 60% to 84% . resistance to quinolones is easily acquired , and the resistance rate is relatively high in countries with a high consumption rate of these drugs . we recently reported a significant difference in the eradication rate obtained between patients infected with gatifloxacin - susceptible and gatifloxacin - resistant h. pylori , suggesting that the selection of quinolones for third - line therapy should be based on the results of drug susceptibility testing . as other alternatives of third - line rescue therapies , rifabutin - based triple therapy , high - dose proton pump inhibitor / amoxicillin therapy and furazolidone - based therapy have been suggested .

Introduction Quinolone Resistance and Quinolone-Based Triple Therapy Adverse Effects Conclusion

diabetes mellitus ( dm ) is a popular lifelong metabolic disease , and probably will remain the most frequent cause of death in the following decades . diabetes as a concerned epidemic problem is increasing all over the world.[210 ] statistical estimations show that the number of diabetic individuals will be 65% of the world population ( 380 millions ) by 2025 . the prevalence of dm is as much as nearly 5% of the world population and it has doubled during the recent 20 years . it is the fifth cause of death in the usa and one of the four causes of non - communicable disorders in different age groups . data show that almost 1520% of population in different areas may have either dm or impaired glucose tolerance ( igt ) in 2025 . lifestyle modification could reduce the risk of diabetes by 3067% , and surprisingly , this reduction maintains even after removing the lifestyle modifications . these modifications consist of physical activity and changes in dietary habits , which are 27% more useful than taking various medications . most of the assessments revealed that broad preventive population - based strategies are critical to challenge the pandemic of dm . dm is a multi - factorial disorder in which both genetic and environmental factors have major roles . besides urbanization and technology development , the rapid nutritional and lifestyle transition faced by us doubles the burden of diseases . poor quality of diet and nutritional habits have potential effects on the obesity - based chronic diseases.[22023 ] diet has a major role in preventing and managing hyperglycemia and dm . previous focus of nutrition science was on the amount and distribution of nutrients and food components . however , this approach has been replaced by a focus on a combination of nutrients and considering foods and food components together . different unknown components and various interactions of food components provide the idea of focusing on dietary patterns instead of food components or nutrients . dietary pattern approach helps in assessing the entire diet , which is also more understandable compared to just focusing on nutrient- or food - based approach to make recommendation for population . dietary pattern as a new focus in nutritional epidemiology is a suitable approach that covers biological native interactions of nutrient components in different food groups in relation with various disorders . dietary pattern reflects an individual 's food consumption and its change during the life span , so it provides comprehensive information about dietary habits and identifies more effective recommendations to manage different chronic diseases . the complex interactions are shown in numerous dietary patterns such as mediterranean , dietary approaches to stop hypertension ( dash ) , prudent , western , traditional , and healthy dietary patterns . therefore , we aimed to assess the relationship between different dietary pattern and the prevalence of type 2 dm and also assess the effect of different eating habits on managing type 2 dm in this review paper . we reviewed all the study designs ( prospective cohort study , clinical trials , cross - sectional studies ) and focused on the available sources between 1992 and 2011 , including journals , electronic books , seminars , and symposium contexts . the search focused on databases published in cochrane central register of controlled trials databases , pubmed , iran medex , and magiran using the keywords : diabetes mellitus , insulin resistance , metabolic syndrome , and dietary pattern . we also studied food , insulin resistance ( ir ) , fasting blood glucose ( fbg ) , and homeostasis model assessment of insulin resistance ( homa - ir ) as the basic related features of dm and metabolic syndrome . the relevance of surveys was found with a hierarchical approach on the basis of titles , abstracts , and full text of articles . our search was limited to english materials we excluded letters , and studies focusing on single nutrients and food items , children , adolescents , and animal studies . the information had been summarized based on fixed protocol : lead author , country , year of publication , sample size , sex and age of subjects , assay method , follow - up duration , outcome measure , race / ethnic , dietary pattern , effect size measurements [ odds ratio ( or)/relative risk ] , and confounding factors adjustments . in the initial search , we found more than 569 articles in pubmed on dm and dietary pattern , 23,296 entries on dm and diet , 344 articles on dietary pattern and ir , 10,359 entries on diet and ir , and 12,879 articles on dm and food . after scanning the articles , 46,709 of the extracted studies were excluded on the basis of the title , abstract , and their major purposes which were limited to the single nutrients and food items . this food habit emphasizes on good source of monoun - saturated fatty acids ( mufa ) from olive oil , accompanied with intake of vegetables , nuts , seeds , fruits , and whole grains , and reduction in the consumption of red meat and unhealthy fats . high content of fiber , magnesium , polyphenol , and antioxidants makes this diet as a useful approach to control hyperglycemia and weight maintenance . the results of a large spanish cohort study among 13,380 men and women showed that adherence to the mediterranean diet could decrease the risk of type 2 dm by 83% . this inverse association between mediterranean dietary pattern and the prevalence of type 2 dm was observed in various multiethnic studies . several epidemiological studies explained lower odds of having dm and inverse relation between glucose level,[3537 ] insulin,[3139 ] and homa - ir ( as one of the basic features of metabolic syndrome and type 2 dm)[3133364042 ] following adherence to the mediterranean dietary pattern even after controlling different confounding agents.[4348 ] higher adherence to the mediterranean dietary pattern score in normoglycemic persons decreased fasting glucose , insulin , and ir . however , this reduction was not significant after multiple adjustments were made for diabetic patients and subjects with impaired fasting glucose ( ifg ) . the aforementioned findings are observed in elderly group and high - risk persons with cardiovascular risk factors and in also those with history of myocardial infarction.[315052 ] another cohort study on 7751 subjects in different ethnics compared four dietary patterns : 1 . mediterranean - like diet containing rice , pasta , vegetables , fruits , and wine ; 2 . healthy pattern that includes whole bread , fruits , vegetables , low - fat dairies , and little alcohol ; 3 . sweet pattern rich in white bread , processed meat , and high - fat dairy products ; and 4 . unhealthy eating pattern high in white bread , processed meat , fries , and full - cream milk . results showed that healthy diet is the best pattern in lowering the incidence of dm . observed a lower or of diabetes among greek adults with high score of adherence to the mediterranean dietary pattern . the comparison of the effects of mediterranean hypocaloric diet and very - low - carbohydrate hypocaloric diet on fasting plasma glucose , serum insulin level , and insulin 2 h post - load concentration and homa - ir did not show a significant difference even after 8 weeks weight loss in obese women . a longitudinal clinical trial for 54 months revealed that consuming a mediterranean diet could decrease homa - ir and serum glucose and serum insulin levels in persons with metabolic syndrome . as patients with metabolic syndrome are at high risk for developing type 2 diabetes , following the mediterranean diet can have a protective effect in this regard . table 1 shows the different studies which evaluate the effects of mediterranean dietary pattern on type 2 dm . characteristics of various studies that evaluated the effects of mediterranean dietary pattern on diabetes mellitus type 2 several studies have focused on the association between lower risk of type 2 dm and adherence to the prudent pattern . prudent pattern is another dietary pattern which is characterized by higher consumption of whole grains , vegetables , fruits , poultry / sea foods , legumes , and coffee . in an american prospective cohort study among 42,504 men , higher adherence to the prudent pattern reduced the risk of dm by 16% , however , after 12 years of follow - up . western dietary pattern contains red and processed meat , butter , french fries , refined grains , deserts , potatoes , sweets , high - fat dairy , and soda . the results were similar to the findings of the atherosclerosis risk in communities ( aric ) study and al so , the observations of fung et al . in the nurses health study ( nhs ) following 12 years . in another study , the conservative and prudent patterns were compared . conservative eating pattern , which contains butter , potatoes , and whole milk , has been correlated positively with the risk of diabetes by 50% while adherence to the two cross - sectional studies of villegas et al . defined usual dietary pattern of adults in three clusters , which correspond to traditional , prudent , and alcohol and convenience diets . traditional pattern had high percent of fat in total calorie intake ; predominantly fat intake was in the forms of saturated fatty acids ( sfa ) and mufa , in contrast to prudent diet which was characterized as a pattern rich in polyunsaturated fatty acids ( pufa ) , high p / s ratio , fiber , and antioxidant vitamin sources . alcohol and convenience diet included the highest intake of alcohol , protein , cholesterol , vitamin b - complex , iron , phosphorus , selenium , zinc , and the lowest amount of pufa and antioxidant vitamins . analysis showed the lowest ir and hemoglobin a1c ( hba1c ) level in subjects adhering to the prudent dietary pattern . an iranian nested cross - sectional study among 425 subjects with igt ( 2535 years old ) showed the association between plasma glucose as one of the major features of metabolic syndrome and five eating patterns : 1 . western pattern ( rich in sweets , butter , soda , mayonnaise , sugar , cookies , tail of a lamb , hydrogenated fat , and eggs ) ; 2 . prudent pattern ( contains fish , peas , honey , nuts , juice , dry fruits , vegetable oil , liver and organic meat , and coconuts ) ; 3 . vegetarian pattern ( includes potatoes , legumes , fruits , rice , green leafy vegetables , and fruits ) ; 4 . high - fat dairy pattern ( high in fatty dairies ) ; and 5 . chicken and plant pattern ( rich in chicken , fruits , green leafy vegetables , and mayonnaise ) . after controlling various confounding factors , vegetarian diet can reduce blood glucose level significantly . different studies that assessed the effects of prudent and western patterns among type 2 dm are shown in table 2 . characteristics of various studies that evaluated the effects of prudent and western dietary patterns on diabetes mellitus type 2 food items of similar dietary patterns are different , based on geographic and ethnic characters . traditional pattern is characterized by higher consumption of refined grains , potato , tea , whole grains , hydrogenated fats , legumes , and casserole , or it can be defined as a pattern rich in high - fat sandwich spreads , red meat , potatoes , butter and lard , low - fat fish , sandwich meat , and sauces . healthy diet usually contains higher intake of whole meal bread , fruit and vegetables , and polyunsaturated margarine , besides lower consumption of red meat , sweet foods , and wine and beer , or food rich in fruits , vegetables , tomato , poultry , legumes , tea , fruit juices , and whole grains . pattern rich in red meat , processed meat , french fries , high - fat dairy products , refined grains , and sweets and desserts , or sweets , butter , soda , mayonnaise , sugar , cookies , tail of a lamb , hydrogenated fat , and eggs , and refined grains , red meat , butter , processed meat , high - fat dairy , sweets and desserts , pizza , potato , eggs , hydrogenated fats , and soft drinks is called as a western dietary pattern . in a cross - sectional study of 486 iranian women between 40 and 60 years of age , healthy pattern defined by frequent consumption of fruits , green leafy and cruciferous vegetables , tomatoes , legumes , tea , and poultry ; 2 . ready - to - use food items as the main footsteps of modernization or so - called as western dietary pattern ; and 3 . iranian traditional pattern consists of whole and refined grains , potatoes , legumes , tea , hydrogenated fats , and broth . the highest or of ir and syndrome x was observed in people with the adherence to the highest quintile of western diet . this result is similar to the result of fung et al . who observed a positive association between western pattern adherence and insulin level . pattern can reduce risk of ir by 45% , while adherence to the western dietary pattern increases this risk to 15% . mcnaughton et al . in an australian cohort study ( whitehall ii ) observed higher incidence of type 2 diabetes and higher value of homa - ir by adherence to the highest quartile of a diet rich in soft drinks , sugar - sweetened beverages , burgers and sausages , snacks , and white bread , and low intake of fiber food sources , by 2.95 times . a multiethnic hawaiian survey revealed that higher ors for type 2 dm were associated with animal foods , local ethnic dishes , and animal and local ethnic dishes included high intake of cabbage , rice , shell fish , corned beef plus hawaiian and filipino dishes . modern dietary pattern had a lower postprandial plasma glucose , after adjusting the confounding factors in the analysis in brief more consumptions of vegetables , vegetable oil / vinegar dressing , fruits , cereals , rice , pasta , and poultry against traditional diet ( contains red meat , high - fat meat sandwich , butter and lard , potatoes , low - fat fishes , and sauces ) . this study showed that eating pattern can affect 2 h plasma glucose independent of subject 's tolerance . analysis of dietary intake among 1508 samoan and american samoan subjects derived two major diets as neo - traditional pattern and modern pattern . neo - traditional pattern is rich in local dishes such as coconut products , lobster , starchy vegetables , crab , and low consumptions of soda and chips , while modern dietary pattern contains processed and ready - to - use foods like cake , butter , eggs , and chips . in contrast with neo - traditional pattern , modern pattern represented lower serum glucose concentration and metabolic syndrome in samoan men and women . the effects of healthy , western , and traditional dietary patterns among type 2 dm were evaluated in various studies whose characters are reflected in table 3 . characteristics of various studies that evaluated the effects of healthy and western and traditional dietary patterns on diabetes mellitus type 2 besides the first known effect of dash on blood pressure control,[7177 ] the other useful potent roles of its main components , such as low - fat dairies and fiber - based foods , were observed on lipid profile and features of metabolic syndrome . dash can also reduce the coronary heart disease . in recent years , several surveys assessed dash effects on blood glucose and incidence of type 2 dm . in the insulin resistance atherosclerosis study ( iras ) of 862 subjects from three different ethnics ( hispanic , non - hispanic white and black ) , dash diet lowered the risk of diabetes incidence by as much as 36% in whites against black and hispanic participants . the effect of dash diet on fbg was shown in two randomized clinical trial studies . in one study , these diets consisted of weight reducing diet , dash diet , and control diet . the weight reducing diet is defined as a diet with lower calorie contents and more consumption of healthy food items ( such as vegetables , fruits , and low - fat dairies ) subjects in the control group followed their usual eating habits . the other cross - over randomized clinical trial ( rct ) assessed the effects of dash diet on hba1c and fasting blood sugar ( fbs ) levels in diabetic patients . reduction in fbg and hba1c following adherence to the dash diet showed that dash eating pattern is a useful strategy in controlling blood glucose among type 2 diabetic patients . characteristics of various studies that evaluated the effects of dash dietary pattern on diabetes mellitus type 2 in panagiotakos study , a positive correlation between red meat intake and homeostasis model assessment of insulin . resistance and insulin secretory capacity ( homa - ir , homa - b ) , insulin , and blood glucose levels however , there was no significant relationship between healthy food groups such as vegetables , legumes , and fruits consumption and glycemic control after controlling for potential confounders . reverse association between dm incidence and dietary pattern rich in meat and milk was observed in another study . white rice was the dominant food item in all three diets . but noodle and dumpling in korean pattern and processed meat products in meat and alcohol eating habit showed significantly different effect on fbg . meat and alcohol dietary pattern increased the risk of high blood glucose by 33% . furthermore , data from the puerto rican elder inhabitants with rice and bean rich diet were studied in a longitudinal investigation among 1167 subjects . analysis of their eating habits extracted three main patterns : meat and french fries , traditional pattern ( rice , beans , and oils ) , sweets , sugary beverages , and dairy desserts . after controlling the confounders , just a cross - sectional study among 984 iranian women revealed that the clusters of diet containing fish , vegetables , legumes , cereals , and fruits , and diet rich in dairy products and eggs could decrease fbs . however , diet rich in red or white meat , meat products , and potatoes , pasta and sweet patterns had unfavorable effects . a reverse association of habitual diet with hyperglycemia and hyperinsulinemia was also observed in a nested cross - sectional survey of six dietary patterns among sweden adults . data showed lower hyperinsulinemia in women with higher milk - fat based food habits against taking white bread items , in common . he et al . derived four dietary patterns after analysis of semi - quantitative food frequency questionnaire among 20,210 chinese adults : ( high vegetables and low animal food style in the southeast area ) , new affluence ( rich in animals and soy products which is used by well - to - do individuals ) , and yellow earth [ high carbohydrate ( cho ) and low fruits , vegetables , and animal dishes , which is common in the northwest area ] . comparing green water consumer , the prevalence of igt in yellow earth and new affluence escalated to ratios of 1.22 and 2.05 , in sequence . risk of dm in caucasian , japanese , american , and native hawaiian participants in a 14-year follow - up survey showed a significant difference according to sex , and their three dietary designs included : fat and meat , vegetables , and fruit and milk dietary patterns . the hazard ratios for type 2 diabetes among women / men who adhered to the fifth quartiles of mentioned diets were 1.22/1.40 , 1.02/0.86 , and 0.85/0.92 , respectively . these ratios were significant in all ethnics , except for native hawaiian subjects . another multiethnic population - based cohort study among 6814 white , black , hispanic , and chinese subjects , which was conducted during 5 years , revealed two patterns which were defined as : 1 . beans , tomatoes , refined grains , high - fat dairy , red meat and 2 . whole grain , fruit , nuts / seeds , green leafy vegetables , low - fat dairy , and low - risk food patterns as the common dietary habits . adherence to the first diet was accompanied with 18% higher risk ratio , while the others reduced the risk by 15% . dietary pattern had decreased diabetes incidence by almost 22% , in contrast to meat , fruit , and vegetable rich pattern which increased the relative risk to 1.05 . all the mentioned studies in this review paper had used factor analysis and cluster analysis . these analyses detect dietary patterns from the existing data with no any prior assumption of disease . besides these posteriori methods , another method for evaluating the overall diet is based on our previous knowledge about the effects of dietary components on health and disease . which is a prior method . this method is based on scoring different components of a diet according to the scientific evidence regarding diet high diet quality index which is defined based on dietary variety , and high intake of whole grain , lean protein , low - fat dairy , and high - fiber food items was associated with or of type 2 dm by 0.38 in men and or for pre - diabetes by 0.66 in women . alternative healthy eating index ( ahei ) is defined based on its association with various disorders , which consists of nine chief components of diet quality such as intake of vegetables , nuts , fruits , soybean , the white - to - red meat ratio , trans fatty acids , polyunsaturated to saturated fatty acids ratio ( p / s ratio ) , alcohol drinking , and taking multivitamin , and it is a suitable substitute for hei . in a prospective study , fung et al . data show that high index can lower the incidence of type 2 diabetes by 36% during 18 years . the cluster analysis on dietary intake of 1052 italians derived five main components which included : ( low in fat but rich in mufa and cho ) , animal products ( high in animal protein , sfa , and cholesterol ) , starch ( rich in vegetable protein and starch ) , vegetal / fat(high in olive oil and seeds ) , and vitamin / fiber(rich in fruits and vegetables ) . observed the lowest igt prevalence by adherence to starch pattern , in contrast to animal food pattern . in the four dietary patterns derived from population - based cohort study , a negative association between commonly consumed fiber - based foods and white meat in diet 1 and diet rich in cake , vegetable , and cheese pattern named as diet 2 with igt was seen . there was also a 28% reduction in diabetes incidence following adherence to the diet containing fiber - based foods and white meat . sex difference in relation to dietary pattern and features of metabolic syndrome was reflected significantly in malmo diet and cancer study of swedish adults . risk of hyperglycemia accompanying dietary pattern rich in meat and dairies increased by 64% among men , while it was not significantly associated with hyperglycemia among women however , heidemann revealed that those in the highest quartiles of diet rich in fruits and poor in meat , beer , and soft drink had lower incidence of type 2 dm by 0.27 . in framingham offspring study , fruits , reduced fat dairy , and whole grains , refined grains and sweets , beer and soda diets were extracted . data revealed that adherence to soda pattern is accompanied by higher fasting insulin level . a pattern high in fruits , vegetables , whole grains , and reduced fat dairy protects against insulin resistance compared to sweet food pattern and soda pattern which are associated with insulin resistance . table 5 shows the different surveys that assess the effects of various dietary patterns among type 2 dm patients . characteristics of various studies that evaluated the effects of different dietary patterns on diabetes mellitus type 2 this food habit emphasizes on good source of monoun - saturated fatty acids ( mufa ) from olive oil , accompanied with intake of vegetables , nuts , seeds , fruits , and whole grains , and reduction in the consumption of red meat and unhealthy fats . high content of fiber , magnesium , polyphenol , and antioxidants makes this diet as a useful approach to control hyperglycemia and weight maintenance . the results of a large spanish cohort study among 13,380 men and women showed that adherence to the mediterranean diet could decrease the risk of type 2 dm by 83% . this inverse association between mediterranean dietary pattern and the prevalence of type 2 dm was observed in various multiethnic studies . several epidemiological studies explained lower odds of having dm and inverse relation between glucose level,[3537 ] insulin,[3139 ] and homa - ir ( as one of the basic features of metabolic syndrome and type 2 dm)[3133364042 ] following adherence to the mediterranean dietary pattern even after controlling different confounding agents.[4348 ] higher adherence to the mediterranean dietary pattern score in normoglycemic persons decreased fasting glucose , insulin , and ir . however , this reduction was not significant after multiple adjustments were made for diabetic patients and subjects with impaired fasting glucose ( ifg ) . the aforementioned findings are observed in elderly group and high - risk persons with cardiovascular risk factors and in also those with history of myocardial infarction.[315052 ] another cohort study on 7751 subjects in different ethnics compared four dietary patterns : 1 . mediterranean - like diet containing rice , pasta , vegetables , fruits , and wine ; 2 . healthy pattern that includes whole bread , fruits , vegetables , low - fat dairies , and little alcohol ; 3 . sweet pattern rich in white bread , processed meat , and high - fat dairy products ; and 4 . unhealthy eating pattern high in white bread , processed meat , fries , and full - cream milk . results showed that healthy diet is the best pattern in lowering the incidence of dm . observed a lower or of diabetes among greek adults with high score of adherence to the mediterranean dietary pattern . the comparison of the effects of mediterranean hypocaloric diet and very - low - carbohydrate hypocaloric diet on fasting plasma glucose , serum insulin level , and insulin 2 h post - load concentration and homa - ir did not show a significant difference even after 8 weeks weight loss in obese women . a longitudinal clinical trial for 54 months revealed that consuming a mediterranean diet could decrease homa - ir and serum glucose and serum insulin levels in persons with metabolic syndrome . as patients with metabolic syndrome are at high risk for developing type 2 diabetes , following the mediterranean diet can have a protective effect in this regard . table 1 shows the different studies which evaluate the effects of mediterranean dietary pattern on type 2 dm . characteristics of various studies that evaluated the effects of mediterranean dietary pattern on diabetes mellitus type 2 several studies have focused on the association between lower risk of type 2 dm and adherence to the prudent pattern . prudent pattern is another dietary pattern which is characterized by higher consumption of whole grains , vegetables , fruits , poultry / sea foods , legumes , and coffee . in an american prospective cohort study among 42,504 men , higher adherence to the prudent pattern reduced the risk of dm by 16% , however , after 12 years of follow - up . western dietary pattern contains red and processed meat , butter , french fries , refined grains , deserts , potatoes , sweets , high - fat dairy , and soda . the results were similar to the findings of the atherosclerosis risk in communities ( aric ) study and al so , the observations of fung et al . in the nurses health study ( nhs ) following 12 years . in another study , conservative eating pattern , which contains butter , potatoes , and whole milk , has been correlated positively with the risk of diabetes by 50% while adherence to the two cross - sectional studies of villegas et al . defined usual dietary pattern of adults in three clusters , which correspond to traditional , prudent , and alcohol and convenience diets . traditional pattern had high percent of fat in total calorie intake ; predominantly fat intake was in the forms of saturated fatty acids ( sfa ) and mufa , in contrast to prudent diet which was characterized as a pattern rich in polyunsaturated fatty acids ( pufa ) , high p / s ratio , fiber , and antioxidant vitamin sources . alcohol and convenience diet included the highest intake of alcohol , protein , cholesterol , vitamin b - complex , iron , phosphorus , selenium , zinc , and the lowest amount of pufa and antioxidant vitamins . analysis showed the lowest ir and hemoglobin a1c ( hba1c ) level in subjects adhering to the prudent dietary pattern . an iranian nested cross - sectional study among 425 subjects with igt ( 2535 years old ) showed the association between plasma glucose as one of the major features of metabolic syndrome and five eating patterns : 1 . western pattern ( rich in sweets , butter , soda , mayonnaise , sugar , cookies , tail of a lamb , hydrogenated fat , and eggs ) ; 2 . prudent pattern ( contains fish , peas , honey , nuts , juice , dry fruits , vegetable oil , liver and organic meat , and coconuts ) ; 3 . vegetarian pattern ( includes potatoes , legumes , fruits , rice , green leafy vegetables , and fruits ) ; 4 . high - fat dairy pattern ( high in fatty dairies ) ; and 5 . chicken and plant pattern ( rich in chicken , fruits , green leafy vegetables , and mayonnaise ) . vegetarian diet can reduce blood glucose level significantly . different studies that assessed the effects of prudent and western patterns among type 2 dm are shown in table 2 . characteristics of various studies that evaluated the effects of prudent and western dietary patterns on diabetes mellitus type 2 food items of similar dietary patterns are different , based on geographic and ethnic characters . traditional pattern is characterized by higher consumption of refined grains , potato , tea , whole grains , hydrogenated fats , legumes , and casserole , or it can be defined as a pattern rich in high - fat sandwich spreads , red meat , potatoes , butter and lard , low - fat fish , sandwich meat , and sauces . healthy diet usually contains higher intake of whole meal bread , fruit and vegetables , and polyunsaturated margarine , besides lower consumption of red meat , sweet foods , and wine and beer , or food rich in fruits , vegetables , tomato , poultry , legumes , tea , fruit juices , and whole grains . pattern rich in red meat , processed meat , french fries , high - fat dairy products , refined grains , and sweets and desserts , or sweets , butter , soda , mayonnaise , sugar , cookies , tail of a lamb , hydrogenated fat , and eggs , and refined grains , red meat , butter , processed meat , high - fat dairy , sweets and desserts , pizza , potato , eggs , hydrogenated fats , and soft drinks is called as a western dietary pattern . in a cross - sectional study of 486 iranian women between 40 and 60 years of age healthy pattern defined by frequent consumption of fruits , green leafy and cruciferous vegetables , tomatoes , legumes , tea , and poultry ; 2 . ready - to - use food items as the main footsteps of modernization or so - called as western dietary pattern ; and 3 . iranian traditional pattern consists of whole and refined grains , potatoes , legumes , tea , hydrogenated fats , and broth . the highest or of ir and syndrome x was observed in people with the adherence to the highest quintile of western diet . this result is similar to the result of fung et al . who observed a positive association between western pattern adherence and insulin level . pattern can reduce risk of ir by 45% , while adherence to the western dietary pattern increases this risk to 15% . mcnaughton et al . in an australian cohort study ( whitehall ii ) observed higher incidence of type 2 diabetes and higher value of homa - ir by adherence to the highest quartile of a diet rich in soft drinks , sugar - sweetened beverages , burgers and sausages , snacks , and white bread , and low intake of fiber food sources , by 2.95 times . a multiethnic hawaiian survey revealed that higher ors for type 2 dm were associated with animal foods , local ethnic dishes , and animal and local ethnic dishes included high intake of cabbage , rice , shell fish , corned beef plus hawaiian and filipino dishes . modern dietary pattern had a lower postprandial plasma glucose , after adjusting the confounding factors in the analysis in brief more consumptions of vegetables , vegetable oil / vinegar dressing , fruits , cereals , rice , pasta , and poultry against traditional diet ( contains red meat , high - fat meat sandwich , butter and lard , potatoes , low - fat fishes , and sauces ) . this study showed that eating pattern can affect 2 h plasma glucose independent of subject 's tolerance . analysis of dietary intake among 1508 samoan and american samoan subjects derived two major diets as neo - traditional pattern and modern pattern . neo - traditional pattern is rich in local dishes such as coconut products , lobster , starchy vegetables , crab , and low consumptions of soda and chips , while modern dietary pattern contains processed and ready - to - use foods like cake , butter , eggs , and chips . in contrast with neo - traditional pattern , modern pattern represented lower serum glucose concentration and metabolic syndrome in samoan men and women . the effects of healthy , western , and traditional dietary patterns among type 2 dm were evaluated in various studies whose characters are reflected in table 3 . characteristics of various studies that evaluated the effects of healthy and western and traditional dietary patterns on diabetes mellitus type 2 besides the first known effect of dash on blood pressure control,[7177 ] the other useful potent roles of its main components , such as low - fat dairies and fiber - based foods , were observed on lipid profile and features of metabolic syndrome . , several surveys assessed dash effects on blood glucose and incidence of type 2 dm . in the insulin resistance atherosclerosis study ( iras ) of 862 subjects from three different ethnics ( hispanic , non - hispanic white and black ) , dash diet lowered the risk of diabetes incidence by as much as 36% in whites against black and hispanic participants . the effect of dash diet on fbg was shown in two randomized clinical trial studies . in one study , these diets consisted of weight reducing diet , dash diet , and diet is defined as a diet with lower calorie contents and more consumption of healthy food items ( such as vegetables , fruits , and low - fat dairies ) subjects in the control group followed their usual eating habits . the other cross - over randomized clinical trial ( rct ) assessed the effects of dash diet on hba1c and fasting blood sugar ( fbs ) levels in diabetic patients . reduction in fbg and hba1c following adherence to the dash diet showed that dash eating pattern is a useful strategy in controlling blood glucose among type 2 diabetic patients . characteristics of various studies that evaluated the effects of dash dietary pattern on diabetes mellitus type 2 in panagiotakos study , a positive correlation between red meat intake and homeostasis model assessment of insulin . resistance and insulin secretory capacity ( homa - ir , homa - b ) , insulin , and blood glucose levels was observed among 2832 subjects without cardiovascular disorders . however , there was no significant relationship between healthy food groups such as vegetables , legumes , and fruits consumption and glycemic control after controlling for potential confounders . reverse association between dm incidence and dietary pattern rich in meat and milk was observed in another study . noodle and dumpling in korean pattern and processed meat products in meat and alcohol eating habit showed significantly different effect on fbg . meat and alcohol dietary pattern increased the risk of high blood glucose by 33% . furthermore , data from the puerto rican elder inhabitants with rice and bean rich diet were studied in a longitudinal investigation among 1167 subjects . analysis of their eating habits extracted three main patterns : meat and french fries , traditional pattern ( rice , beans , and oils ) , sweets , sugary beverages , and dairy desserts . a cross - sectional study among 984 iranian women revealed that the clusters of diet containing fish , vegetables , legumes , cereals , and fruits , and diet rich in dairy products and eggs could decrease fbs . however , diet rich in red or white meat , meat products , and potatoes , pasta and sweet patterns had unfavorable effects . a reverse association of habitual diet with hyperglycemia and hyperinsulinemia was also observed in a nested cross - sectional survey of six dietary patterns among sweden adults . data showed lower hyperinsulinemia in women with higher milk - fat based food habits against taking white bread items , in common . he et al . derived four dietary patterns after analysis of semi - quantitative food frequency questionnaire among 20,210 chinese adults : ( high vegetables and low animal food style in the southeast area ) , new affluence ( rich in animals and soy products which is used by well - to - do individuals ) , and yellow earth [ high carbohydrate ( cho ) and low fruits , vegetables , and animal dishes , which is common in the northwest area ] . comparing green water consumer , the prevalence of igt in yellow earth and new affluence escalated to ratios of 1.22 and 2.05 , in sequence . risk of dm in caucasian , japanese , american , and native hawaiian participants in a 14-year follow - up survey showed a significant difference according to sex , and their three dietary designs included : fat and meat , vegetables , and fruit and milk dietary patterns . the hazard ratios for type 2 diabetes among women / men who adhered to the fifth quartiles of mentioned diets were 1.22/1.40 , 1.02/0.86 , and 0.85/0.92 , respectively . these ratios were significant in all ethnics , except for native hawaiian subjects . another multiethnic population - based cohort study among 6814 white , black , hispanic , and chinese subjects , which was conducted during 5 years , revealed two patterns which were defined as : 1 . beans , tomatoes , refined grains , high - fat dairy , red meat and 2 . whole grain , fruit , nuts / seeds , green leafy vegetables , low - fat dairy , and low - risk food patterns as the common dietary habits . adherence to the first diet was accompanied with 18% higher risk ratio , while the others reduced the risk by 15% . dietary pattern had decreased diabetes incidence by almost 22% , in contrast to meat , fruit , and vegetable rich pattern which increased the relative risk to 1.05 . all the mentioned studies in this review paper had used factor analysis and cluster analysis . these analyses detect dietary patterns from the existing data with no any prior assumption of disease . besides these posteriori methods , another method for evaluating the overall diet is based on our previous knowledge about the effects of dietary components on health and disease . which is a prior method . this method is based on scoring different components of a diet according to the scientific evidence regarding diet high diet quality index which is defined based on dietary variety , and high intake of whole grain , lean protein , low - fat dairy , and high - fiber food items was associated with or of type 2 dm by 0.38 in men and or for pre - diabetes by 0.66 in women . alternative healthy eating index ( ahei ) is defined based on its association with various disorders , which consists of nine chief components of diet quality such as intake of vegetables , nuts , fruits , soybean , the white - to - red meat ratio , trans fatty acids , polyunsaturated to saturated fatty acids ratio ( p / s ratio ) , alcohol drinking , and taking multivitamin , and it is a suitable substitute for hei . in a prospective study , fung et al . data show that high index can lower the incidence of type 2 diabetes by 36% during 18 years . the cluster analysis on dietary intake of 1052 italians derived five main components which included : ( low in fat but rich in mufa and cho ) , animal products ( high in animal protein , sfa , and cholesterol ) , starch ( rich in vegetable protein and starch ) , vegetal / fat(high in olive oil and seeds ) , and observed the lowest igt prevalence by adherence to starch pattern , in contrast to animal food pattern . in the four dietary patterns derived from population - based cohort study , diet 1 and diet rich in cake , vegetable , and cheese pattern named as diet 2 with igt was seen . there was also a 28% reduction in diabetes incidence following adherence to the diet containing fiber - based foods and white meat . sex difference in relation to dietary pattern and features of metabolic syndrome was reflected significantly in malmo diet and cancer study of swedish adults . risk of hyperglycemia accompanying dietary pattern rich in meat and dairies increased by 64% among men , while it was not significantly associated with hyperglycemia among women however , heidemann revealed that those in the highest quartiles of diet rich in fruits and poor in meat , beer , and soft drink had lower incidence of type 2 dm by 0.27 . in framingham offspring study , fruits , reduced fat dairy , and whole grains , refined grains and sweets , beer and soda diets were extracted . pattern is accompanied by higher fasting insulin level . a pattern high in fruits , vegetables , whole grains , and reduced fat dairy protects against insulin resistance compared to sweet food pattern and soda pattern which are associated with insulin resistance . table 5 shows the different surveys that assess the effects of various dietary patterns among type 2 dm patients . characteristics of various studies that evaluated the effects of different dietary patterns on diabetes mellitus type 2 dietary intakes have important roles against insulin resistance , and in the prevention and management of hyperglycemia . epidemiological studies revealed that dietary patterns high in fiber - rich food items such as vegetables , fruits , whole grains , seeds and nuts , plus white meat sources like poultry and fish could have protective effects against the incidence of dm . however , dietary patterns rich in processed meat and red meat , refined cereals , and sfas are associated with higher risk of dm . healthy patterns , mediterranean , and prudent dietary patterns also are protective dietary patterns against dm . however , adherence to western dietary pattern is associated with higher risk of diabetes . these results are in line with iranian dietary pattern surveys which emphasize on adherence to healthy pattern to manage abnormal glucose homeostasis . it is suggested to see the relationship of different dietary patterns and the incidence of type 2 diabetes in cohort studies among different populations in future studies .

objective : to investigate the role of dietary patterns in prevention and management of type 2 diabetes mellitus.materials and methods : a systematic review of databases which were published in isi , cochrane central register of controlled trials databases , pubmed , iran medex , and magiran was performed . diabetes and dietary pattern were used as the keywords.results:a total of 58 studies which aimed to focus on diabetes mellitus , insulin resistance , metabolic syndrome , dietary pattern , and other related key words were reviewed . more than 47,447 articles were found and 46,709 entries of the extracted studies were excluded on the basis of the title and abstracts . the major dietary patterns were : healthy , western , traditional , prudent , unhealthy , mediterranean , modern , and dietary approach to stop hypertension ( dash ) diets . comparison of the effects of different diets revealed that dietary patterns containing fiber - rich foods have a protective role in managing diabetes mellitus . healthy , mediterranean , prudent , and dash dietary patterns were associated with lower risk of hyperglycemia.conclusions:the adherence to the mediterranean , prudent , or dash diets could control hyperglycemia . the higher intake of vegetables , fruits , nuts , whole grains , and lower intake of red meat could reduce the risk of type 2 diabetes mellitus .

INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION Mediterranean dietary pattern Prudent and Western dietary pattern Healthy, Western, and Traditional dietary patterns DASH dietary pattern Other dietary habits CONCLUSION

tissues are diverse not only in their structure and function but also in the dynamics of their constituent cells ( leblond & walker , 1956 ) . sc turnover rates in adult mouse lineages vary by orders of magnitude ( fig 1 ) . epithelial and male germ scs cycle rapidly , while in tissues such as liver , kidney and muscle , proliferation is scarcely detectable . in homoeostasis , on average , one sc and one differentiating cell are produced per sc division , and the rate of sc proliferation is matched with the rate of loss of cells from the differentiated lineage(s ) it supports . following damage , however , scs must rapidly and reversibly switch to produce an excess of proliferating cells to regenerate the lost tissue . here , we will define scs in purely functional terms as cell populations that sustain longterm tissue turnover and/or contribute to tissue repair after injury . we first survey sc behaviour in high turnover and quiescent tissues in homoeostasis , then consider how scs respond to injury and conclude by considering the physical and molecular signals which switch sc behaviour to meet the requirements of the organism . there are two cellular mechanisms by which scs may maintain homoeostasis ( watt & hogan , 2000 ) . a population of scs may behave so that on average , 50% of their progeny are themselves scs and 50% differentiating cells . population asymmetry may result from the cell autonomous properties of scs or external regulation , for example by a niche of restricted size in which scs can only divide when a cell differentiates and leaves the niche . advances in lineage tracing and live imaging reveal population asymmetry emerges as the predominant mechanism in tissues with a high rate of turnover ( alcolea & jones , 2013 ; rompolas & greco , 2014 ) . we will focus on tissues in mice where robust evidence on sc behaviour is available , before turning to other species . some of the first insights into sc dynamics in vivo came from studies of squamous epithelia , which cover the skin , mouth and oesophagus and predominantly consist of layers of keratinocytes . proliferation is confined to the deepest , basal cell layer , which forms the sc niche ( fig 2a ) . when dividing cells commit to differentiation , they withdraw from the cell cycle and migrate out of the basal layer ( alcolea & jones , 2014 ) . largescale lineage tracing has revealed that cellular homoeostasis is achieved by a single population of keratinocyte scs in the basal cell layer ( clayton et al , 2007 ; doupe et al , 2010 , 2012 ; mascre et al , 2012 ; lim et al , 2013 ) . the outcome of individual cell divisions is unpredictable , generating two scs , two differentiating cells or one cell of each type ( alcolea & jones , 2013 ) ( fig 2a ) . however , the probabilities of each division outcome are balanced , so an equal number of scs and differentiating cells are generated across the basal layer , an example of population asymmetry ( watt & hogan , 2000 ; clayton et al , 2007 ) . the random element in sc fate appears at odds with histological features such as the ordered columns of differentiating cells seen in mouse epidermis , but these arise simply from the physical packing properties of suprabasal cells rather than reflecting deterministic sc behaviour ( doupe et al , 2010 ) . analysis of lineagetracing data argues that the balanced stochastic fate of squamous scs is a cellintrinsic property rather than a consequence of external regulation , but the molecular machinery that achieves this is unknown ( clayton et al , 2007 ; jones & simons , 2008 ) . ( a ) squamous epithelia consist of layers of keratinocytes , scs ( green ) reside in the basal cell layer , along with postmitotic cells waiting to stratify ( red ) . when a differentiating cell leaves the niche ( arrow ) , a nearby sc divides with one of the three division outcomes shown to maintain constant cell density in the niche . the probabilities of each outcome ( expressed as per cent , oesophageal epithelium shown ) are balanced , so equal numbers of scs and differentiated cells are produced across the population . ( b ) the hair follicle cycles between a resting stage ( telogen ) and expansion of the lower follicle ( anagen ) . multiple scs have been identified , in the junctional zone ( purple ) , the bulge ( green ) and the hair germ ( blue ) , which lie in contact with the mesenchymal cells of the dermal papilla ( blue ) . the hair shaft ( black ) is surrounded by concentric layers of inner root sheath cells that have been omitted for clarity . in the transition into anagen , hair germ and upper bulge cells selfduplicate . in the bulge , divisions are aligned parallel with the axis of the hair shaft ( inset ) . lower bulge cells contribute differentiating progeny to greatly expand the root sheath . later in anagen , hair germ cells assemble around the dermal papilla and then generate inner root sheath cells ( arrows ) . selfrenewal and differentiation are balanced , so the numbers of scs in each compartment is maintained at a constant level across multiple hair cycles . ( c ) intestinal epithelium contains four lineages sustained by scs ( green ) that lie between paneth cells in the crypt base . differentiating cells migrate through a progenitor compartment in the upper crypt from which postmitotic cells populate the villus , from which they are shed . inset shows a simplified topdown view of the niche . as a differentiating sc exits the niche , dormant scs ( green ) lie close to arterioles , receiving paracrine signals from endothelial cells ( red ) , perivascular cells expressing ng2 and sympathetic nerve endings ( blue ) . on activation , transplantation of daughter cells of a single sc reveals selfduplicating and asymmetric divisions : dark green : longtermreconstituting scs ; light green : short or intermediatetermreconstituting scs ; yellow : megakaryocyte progenitor ; and blue : common myeloid progenitor . lineage tracing in homoeostasis suggests that myeloid lineages may be maintained by selfsustaining progenitor cells ( yellow ) exhibiting population asymmetry by generating equal proportions of progenitor and differentiating cells ( grey ) ( see inset ) . haematopoietic scs ( green ) make negligible contribution to myelopoiesis in homoeostasis , but function as reserve cells . ( g ) male germ cell scs are diverse in appearance but functionally equivalent . male germ cells expressing gfr1 reside in the outermost layer of the seminiferous tubule . scs ( green ) exist as singles or 24 cell syncytia connected by cytoplasmic bridges , which may selfduplicate ( blue arrows ) to generate two scs or undergo fragmentation ( short red arrows ) . upon differentiation ( pink arrow ) into ngn3positive cells ( red ) , the same behaviour continues , but ngn3 cells are unlikely to revert to gfr1 scs in homoeostasis . once in the ngn3 compartment , syncytia larger than 4 cells form which may undergo further differentiation into ckitexpressing cells ( not shown ) , which are even less likely to revert to gfr1positive cells . quiescent neural scs ( light green ) reside in the subventricular zone niche , extending processes into cerebrospinal fluid ( blue ) that fills the lateral ventricles ( lv ) and underlying endothelial cells ( red ) lining blood vessels ( bv ) . cells ( pink ) and neural precursor cells ( orange ) lie adjacent to the scs . ( i ) division outcomes of neural scs in the dentate gyrus inferred from lineage tracing . neural scs interconvert between quiescent ( light green ) and proliferating ( dark green ) states . division of scs has a range of symmetric and asymmetric outcomes as shown , generating neural scs , neuroblasts ( orange ) or differentiated astrocytes ( blue ) . the epidermis is punctuated by appendages in the form of hair follicles ( hfs ) and sweat ducts , each a selfmaintaining miniorgan containing multiple cell lineages ( schepeler et al , 2014 ) . hfs are split into functionally discrete compartments ( page et al , 2013 ) . the lower hair follicle undergoes cyclical expansion ( anagen ) and contraction ( catagen ) followed by a period of quiescence ( telogen ) ( fig 2b ) . the hf is homoeostatic in sc numbers , and tissue organisation remains the same at telogen phase of each successive hair cycle . hfs are sustained by sc populations in a region known as the bulge and the adjacent hair germ ( rompolas & greco , 2014 ) . lineage tracing and cell culture studies have revealed a bewildering range of apparently overlapping candidate scs in hfs , though it is unclear whether variations in gene expression mark functionally distinct sc populations ( rompolas & greco , 2014 ; schepeler et al , 2014 ) . the hf bulge , which marks the upper limit of the lower follicle , was first identified as a distinct sc niche as it contains slowcycling cells ( cotsarelis et al , 1990 ; tumbar et al , 2004 ) ( fig 2b ) . bulge scs differentiate into multiple lineages when sorted and transplanted , and undergo both symmetric and asymmetric divisions ( blanpain et al , 2004 ; waghmare et al , 2008 ; zhang et al , 2009 ) . more recently , lineage tracing and intravital imaging have shown that scs in the hair germ are the first cells to be mobilised as hfs enter anagen , undergoing apparently symmetric selfduplicating divisions aligned with the long axis of the hf ( greco et al , 2009 ; rompolas et al , 2012 ) . only when the exponentially expanding hair germ has morphed to envelop a bud of mesenchyme called the dermal papilla ( dp ) scs differentiate into transit amplifying cells that will form the inner root sheath that surrounds the hair shaft ( rompolas et al , 2012 ) . signals from the dp are essential for the progression of the hair cycle ; indeed , the number of cells in the dp determines hair size and shape ( rompolas et al , 2012 ; chi et al , 2013 ) . in the bulge , scs remain in slow cycling or quiescent , until a proportion of cells are recruited into division by signals from ta cells . these induce scs to first contribute to the outer root sheath and then undergo selfrenewal until the bulge is repopulated with scs , when quiescence resumes ( hsu et al , 2014 ) ( fig 2b ) . hf expansion is followed by involution ( catagen ) , and by the time the telogen phase is reached , bulge and hair germ scs are restored , achieving homoeostasis over the entire cycle ( hsu et al , 2014 ) . the glandular epithelium that lines the intestine is also rapidly turned over but has a distinctive tissue organisation that contributes to sc regulation . cell replacement is achieved by scs and progenitor cells located in pits known as crypts ( clevers , 2013 ) ( fig 2c ) . cycling scs lie at the crypt base among cells expressing the wnt target gene lgr5 , maintaining four lineages of differentiated cells via the lineagecommitted progenitor cells that populate the upper crypt ( barker et al , 2007 ) . differentiated paneth cells lie next to scs and secrete signals to sustain scs in the niche ( sato et al , 2011b ) . the requirement for paracrine signals restricts the size of the sc pool and ensures homoeostasis . lineage tracing indicates that as a sc differentiates , it migrates out of the niche and is replaced by the selfduplicating division of a neighbouring sc ( fig 2c ) ( lopezgarcia et al , 2010 ; snippert et al , 2010 ) . it was initially thought that all lgr5 cells were cycling functionally equivalent scs , but a recent study has shown only 5 of the 1415 lgr5 cells in each crypt base are proliferating at any one time ( kozar et al , 2013 ) . live imaging further suggests the likelihood of a sc differentiation depends on its location , increasing in the uppermost part of the niche ( ritsma et al , 2014 ) . analysis of spontaneous clones carrying mitochondrial mutations argues similar sc behaviour maintains human colonic epithelium ( baker et al , 2014 ) . intestinal homoeostasis is thus achieved by linking differentiation to selfduplicating sc division within a niche of restricted size . it has long been argued that the prodigious cell turnover of the haematopoietic system is sustained by rare scs , found both in the circulation and in a specific niche environment in the bone marrow . heamopoietic scs ( hscs ) are assayed by their ability to reconstitute the blood system in the long term following transplantation into irradiated host animals ( babovic & eaves , 2014 ; ema et al , 2014 ) . highly purified hscs expressing cell surface identical markers may be cycling or dormant , but only the latter group has longterm transplant potential ( fig 1 ) ( wilson et al , 2008 ; foudi et al , 2009 ; catlin et al , 2011 ; anjosafonso et al , 2013 ; oguro et al , 2013 ) . dormant hscs reside in a distinct niche environment , next to arterioles , and migrate to lie close to venous sinusoids on activation ( fig 2d ) ( kunisaki et al , 2013 ; mendelson & frenette , 2014 ) . transplant assays argue that haematopoiesis is a hierarchy , with hscs continually generating progenitors that become progressively restricted in both lineage and selfrenewal potentials as they differentiate , but recent discoveries are challenging this view . transplantation of daughters from a single hsc division reveals selfduplicating and asymmetric divisions which generate a variety of differentiating progenitors directly from hscs , challenging the view of an ordered differentiation programme ( yamamoto et al , 2013 ) ( fig 2e ) . more remains to be discovered about the apparent heterogeneity among hscs with longterm transplant potential . some of this may result from hscs commuting between different states that vary in their ability to survive the rigours of transplantation ( ema et al , 2014 ; van galen et al , 2014 ) . recently , transposonbased lineage tracing has revealed native haematopoiesis is very different from that in transplanted animals . rather than myeloid lineages deriving from a few hscs , as is seen after transplantation , numerous shortlived progenitor clones are observed , consistent with myelopoiesis being maintained by a selfsustaining progenitor pool exhibiting population asymmetry ( fig 2f ) ( sun et al , 2014b ) this argues for a separation of roles in haemopoiesis , with a pool of progenitors maintaining the myeloid lineage in homoeostasis and quiescent hscs functioning as a reserve , mobilised to expand the progenitor population when the demand for myeloid cells rises ( wilson et al , 2008 ; sun et al , 2014b ) . spermatogenesis continues throughout life , sustained by a population of scs , which reside in the basal cell layer of the seminiferous tubule , express gfr1 and are negative for the differentiation marker ngn3 . spermatogonia undergo incomplete cell division , generating syncytial clusters of cells linked by cytoplasmic bridges ( yoshida , 2012 ) ( fig 2 g ) . it was thought that only single gfr1 cells were scs , but combined live imaging and lineage tracing have revealed that all gfr1 cells , whether single or in syncytia of up to 4 cells , are functionally equivalent scs ( hara et al , 2014 ) . quantitative modelling argues scs divide symmetrically producing two sc daughters , while the stochastic differentiation of a sc into a ngn3 cell appears linked to syncytial fragmentation by an as yet unknown mechanism . in combination , these mechanisms achieve population asymmetry as sc division is balanced with differentiation across the gfr1 compartment ( klein et al , 2010 ; hara et al , 2014 ) . ngn3 cells have a high probability of undergoing differentiation into ckitexpressing cells , but infrequently revert back to gfr1 sc . the spermatogenesis paradigm supports the concept that sc function may reside in a pool of heterogeneous cells which interconvert between different states , so that there are multiple reversible paths by which a given sc may differentiate ( nakagawa et al , 2010)(hara et al , 2014 ) . turning to tissues with a low rate of turnover , it is now clear that both rodent and human brains , once thought to be postmitotic , continue to produce new neurons throughout life which contribute to specific forms of learning and memory and responding to stress ( snyder et al , 2011 ; spalding et al , 2013 ; aimone et al , 2014 ; ernst et al , 2014 ) . adult neural scs are found in the subventricular zone ( svz ) lining the lateral ventricles and in the dentate gyrus ( dg ) of the hippocampus and are largely quiescent ( fig 2h ) . lineage tracing in transgenic mice reveals that proliferating dg scs are capable of both asymmetric and symmetric divisions generating two scs or two differentiating astroglial or neural progenitor daughters ( bonaguidi et al , 2011 ) ( fig 2i ) . after division , some scs return to a quiescent state , negative for proliferation markers , paralleling the transition between dormant and active states seen in heamatopoietic scs . even establishing the existence of scs in adult tissues with a very low rate of turnover studies in mouse and human support a slow rate of turnover of differentiated hepatocytes in the liver and glomerular and tubular cells in the kidney , though the existence of a discrete stem cell population in these tissues is controversial ( magami et al , 2002 ; rodins et al , 2002 ; fellous et al , 2009 ; kusaba et al , 2014 ; miyajima et al , 2014 ) . in contrast , adult skeletal muscle contains a welldefined quiescent sc population , residing among the satellite cells that lie beneath the plasma membrane of terminally differentiated multinucleate muscle fibres ( brack & rando , 2012 ) . reserve role of scs , actively maintained in a quiescent state until they are mobilised following muscle injury ( cheung et al , 2012 ; cheung & rando , 2013 ) . in summary , there are two cellular mechanisms of population asymmetry in mammalian tissues with a high turnover ( watt & hogan , 2000 ; klein & simons , 2011 ) . open plan niche with an apparently cellintrinsic mechanism which balances the probabilities of generating scs or differentiating progeny ( clayton et al , 2007 ; alcolea & jones , 2014 ) . an alternative strategy seen in the intestine is having a niche of limited size in which selfduplicating sc division occurs as a differentiating cell exits the niche ( lopezgarcia et al , 2010 ; vermeulen & snippert , 2014 ) . in both these examples , the robust linkage of division and the exit of a nearby cell another common theme is that while the fate of an individual sc is unpredictable , homoeostasis is achieved across the sc population . the male germ cell lineage illustrates the unreliability of using marker gene expression or cellular morphology to define scs , as such characteristics may vary within a functionally equivalent pool of scs . likewise , active scs may coexist with those within a dormant state in the hair follicle , bone marrow and intestinal crypt . while the discussion above has focussed on mammalian systems , population asymmetry has also been observed in drosophila , where neutral competition for a niche of fixed size is seen in ovarian scs , while a balanced three way division outcome reminiscent of mammalian squamous epithelia is a feature of midgut scs ( de navascues et al , 2012 ; kronen et al , 2014 ) . intriguingly transgenic lineage tracing reveals that neural retina scs of the medaka fish , orizias latipes , have fixed asymmetric cell division in homoeostasis , the first observation of such behaviour in an adult tissue ( centanin et al , 2014 ) . in summary , for the mammalian and drosophila lineages where sc dynamics has been resolved , cellular homoeostasis is achieved by population asymmetry , which may be cell intrinsic or niche specified . the fate of individual cells is unpredictable , but the probabilities of selfrenewal and differentiation are balanced across the sc population , so equal proportions of scs and differentiating cells are generated . evolution has also sampled another mechanism of tissue maintenance , fixed asymmetric sc division , but to date , this has only been observed in fish retina . tissue injury is an inevitable part of life . it is becoming clear that following damage , scs and their differentiating progeny exhibit hitherto unexpected plasticity in their behaviour ( doupe & jones , 2013 ) . we first consider how cycling sc populations respond to injury before turning to the activation of quiescent scs in low turnover tissues . wounding is frequent in surface epithelia . in the squamous epithelium of the oesophagus and the epidermis of the paw , scs adjacent to a wound rapidly and reversibly switch to producing an excess of scs , reverting to homoeostatic behaviour once the defect is closed ( fig 3a ) ( doupe et al , 2012 ; lim et al , 2013 ) . the need for such flexibility may be one of the reasons for three division outcomes in squamous epithelial scs . pure asymmetric division achieves cellular homoeostasis but is inflexible , while adjusting the probability of the two types of symmetric division allows rapid modulation of cell production . next to the wound ( w ) exit the cell cycle and migrate towards the defect ( yellow area and inset ) . behind this migrating front , cycling progenitors undergo divisions heavily biased to selfduplication ( green area and inset ) , expanding the progenitor compartment to generate the excess cells required to repair the epithelium . once the wound has closed , progenitor behaviour switches back to homoeostasis . ( b ) wound repair in epidermis . in the epidermis , progenitor cells change their behaviour as in the oesophagus , but wound repair is supported by a flux of cells into the epidermis from hair follicles ( purple arrows ) and , in tail skin , mobilisation of quiescent reserve cells in the epidermis . migration explains the appearance of radial clones around a healed wound seen in lineagetracing experiments . following ablation of scs in the bulge ( green ) of telogen hfs , cells in the upper follicle ( purple ) migrate and proliferate ( purple arrows ) to reconstitute the bulge sc population , enabling the hair cycle to proceed normally . following ablation of lgr5expressing scs in the crypt base ( green ) , paneth cell precursors ( blue with pink border ) dedifferentiate and colonise the crypt base niche with scs functionally equivalent to the original population . ( e ) injury response of male germ scs . following treatment with the cytotoxic agent busulphan , the gfr1 sc population is depleted ( grey outlines ) but then reconstituted by surviving ngn3 cells reverting into gfr1 cells ( green arrows ) and a decrease in the probability of gfr1 cells transferring to the ngn3 compartment ( pink dotted arrows ) . dormant scs are recruited into cycle by treatment after damage to the haematopoietic system from a cytotoxic drug ( 5fu ) or cytokines , such as gcsf , released after infection . ( g ) neural sc responses to stroke . following a cns stroke , which results in cell death due to ischaemia , lineage tracing argues these derive both from mobilisation of neural scs in the svc followed by migration to the site of injury ( orange with green border ) and from differentiated astrocytes within the area of the stroke entering a neurogenic programme ( orange with red borders ) . most of the epidermis has a dense array of hfs , which normally make negligible contribution to homoeostasis . following injury , however , the progeny of bulge and upper follicle stem cells migrate into the epidermis and contribute extensively to wound repair ( ito et al , 2005 ; levy et al , 2005 ; page et al , 2013 ) ( fig 3b ) . in the specialised tail epidermis , which is sparse in hfs , rare , reserve scs are mobilised , while in hairless skin , sweat ductderived cells contribute to wound healing ( lu et al , 2012 ; mascre et al , 2012 ; roshan & jones , 2012 ) . in humans , similar principles apply , but sweat ducts make a larger contribution to epidermal repair than hfs ( rittie et al , 2013 ) . the infiltration of specialised gamma delta t cells following epidermal injury activates wnt signalling via fgf9 signalling ( gay et al , 2013 ) . wnt has been shown to alter the axis of division of bulge stem cells to generate a new hair follicle alongside an existing one ( deschene et al , 2014 ) . recent developments in live imaging combined with laser ablation to delete cell subpopulations have also revealed unexpected plasticity in hfs . following ablation , bulge scs are replaced by the progeny from cells in the upper hf which normally make no contribution to the hair cycle ( fig 3c ; rompolas et al , 2013 ; rompolas & greco , 2014 ) . only destruction of the niche cells surrounding the bulge or separation of the dp from the hair germ halts hair regeneration , indicating the niche plays an essential role in hair regeneration . following ablation of the lgr5positive cells in the intestinal crypt , differentiating lineagecommitted precursor cells above the crypt base dedifferentiate into functional scs which repopulate the crypt ( fig 3d ) ( van es et al , 2012 ; buczacki et al , 2013 ; ritsma et al , 2014 ) . in lung and gastric epithelia , reversion of postmitotic differentiated cells into scs is observed following injury ( stange et al , 2013 ; tata et al , 2013 ) . in nonepithelial tissues , evidence for dedifferentiation is more limited , but following cytotoxic drug treatment , the depleted male germ cell gfr1 sc pool is regenerated by the combination of decreasing the probability of reversion of gfr1 differentiation and reversion of ngn3 cells , most of which differentiate in homoeostasis , back into gfr1 scs ( fig 3e ; nakagawa et al , 2010)(hara et al , 2014 ) . tissue damage or stress mobilises slowcycling or quiescent scs , which function as reserve cells , such as dormant haematopoietic scs that are rapidly recruited into cycle following treatment with the cytotoxic drug 5 fluorouracil or the cytokine gcsf , the latter also triggering sc migration into the circulation ( wilson et al , 2008 ; cheung & rando , 2013 ) ( fig 3f ) . the liver is an intriguing organ , capable of extensive regeneration after a variety of insults ( miyajima et al , 2014 ) . lineage tracing in different models of injury indicates surviving hepatocytes reenter the cell cycle to restore their own lineage ( schaub et al , 2014 ; tarlow et al , 2014 ; yanger et al , 2014 ) . tissue repair can thus be achieved by dedifferentiation alone in the absence of a reserve stem cell population . following vascular stroke in mouse brain , lineage tracing provides evidence for differentiated astrocytes generating proliferating neuroblasts , supplementing neuroblasts derived from mobilised neural scs in the svz which have migrated into the zone of injury ( magnusson et al , 2014 ) ( fig 3 g ) . the ultimate test of the regenerative potential of a sc is to reconstitute a tissue from a single cell , a feat that requires an exponential increase in sc number and the generation of differentiated cells . transplantation into immunesuppressed mice has long been a standard sc assay , but more recently , sc culture methods have been developed which reveal the ability of a single sc to reconstitute a tissuelike organoid in defined culture conditions ( sato et al , 2009 ; sato & clevers , 2013 ) . this was first demonstrated with human epidermal scs that generate large colonies containing multiple scs and differentiating keratinocytes , which fuse into stratified sheets ( rheinwald & green , 1975 ; jones et al , 1995 ) . when this cultured epidermis is grafted back onto burnt patients , it persists over many years ( gallico et al , 1984 ; compton et al , 1989 ) . more recently , 3dimensional culture systems were established for intestinal and prostatic scs from both mice and humans ( sato et al , 2009 , 2011a ; chua et al , 2014 ; karthaus et al , 2014 ) . disaggregation and reculturing reveal each organoid contains selfrenewing scs capable of multiple rounds of organoid formation ( sato et al , 2009 ) . the ability of scs and their progeny to assemble themselves into a niche within a tissuelike array of differentiated cells in the absence of external spatial cues is remarkable and is most graphically illustrated in the formation of complex brainlike structures in 3d cell culture ( lancaster et al , 2013 ; lancaster & knoblich , 2014 ) . if this technology can be used to expand sc populations ex vivo which successfully reconstitute and sustain damaged tissues in vivo , it will have great potential for regenerative medicine ( huch et al , 2013a , b ; sato & clevers , 2013 ; dorrell et al , 2014 ) . in combination with genome editing , ex vivo stem cell expansion may be particularly powerful for treating genetic diseases with tissuerestricted phenotypes , where engrafted scs can compete with host scs on at least even terms ( schwank et al , 2013 ) . the factors required to culture organoids give insight into the nature of niche signals for sc survival and proliferation , an area we will explore below ( sato et al , 2011b ) . to summarise , advances in cell culture are further revealing the selforganising ability of scs and their progeny in generating organlike structures in the absence of tissue cues . we now turn to examine recent insights into the regulation of sc dynamics in homoeostasis and regeneration , critical in tuning cell production to the requirements of the tissue and whole organism . potentially adjustable parameters include the rate of sc division and the balance of the resulting sc and differentiating progeny . in tissues with minimal cell turnover , scs we will consider the relative contributions of cellintrinsic factors , paracrine signalling in the sc microenvironment , the physical characteristics of the niche and remote signals such as hormones and neural regulation in switching sc behaviour . for squamous scs , population asymmetry requires balancing the probabilities of three potential division outcomes ( clayton et al , 2007 ; doupe et al , 2012 ) . this process requires notch signalling , which was long known to drive the differentiation of postmitotic keratinocytes ( lowell et al , 2000 ; rangarajan et al , 2001 ; nicolas et al , 2003 ; blanpain et al , 2006 ) . transgenic inhibition of notch signalling in individual oesophageal scs accelerates the rate of cell division and blocks the symmetric differentiation division outcome ( alcolea et al , 2014 ) . as a result , intriguingly , as this occurs , the basal layer becomes crowded and the symmetric differentiation division outcome is restored ( alcolea et al , 2014 ) . in culture , cell crowding promotes keratinocyte differentiation , suggesting the reinstatement of homoeostasis may be a consequence of increased cell density within the basal layer ( watt et al , 1988 ) . regulation of cell density is a robust and widespread phenomenon , and potentially is a defence against scs carrying any mutation that enhances proliferation and results in crowding in the niche ( eisenhoffer et al , 2012 ; marinari et al , 2012 ; frede et al , 2014 ) . the regulation of squamous sc proliferation also involves highly conserved cell cell and paracrine signalling pathways . epidermal scs secrete both wnt ligands and wnt inhibitory dkk proteins , resulting in the activation of wnt target genes in basal cells ( choi et al , 2013 ; lim et al , 2013 ) . blockade of wnt signalling in scs inhibits their proliferation , resulting in a thinned epidermis ( choi et al , 2013 ; lim et al , 2013 ) . autocrine wnt signalling is thus an essential part of the squamous sc niche , providing permission to proliferate ( frede & jones , 2013 ) . the remodelling of hfs during the hair cycle makes dissection of sc regulation in the fastchanging niche especially challenging , but cell autonomous factors and paracrine signalling again emerge as crucial sc regulators . expression of the transcription factor sox9 in hf scs is essential for their survival and normal differentiation , but this is achieved at least in part by regulation of paracrine activin / tgf signalling by sox9 target genes ( kadaja et al , 2014 ) . if wnt signals are inhibited , hf scs enter a state of suspended animation in which they persist long term but do not proliferate , reentering the cell cycle when the wnt activity is reinstated ( choi et al , 2013 ) . in contrast , activating the wnt pathway triggers sc expansion and tilts the axis of cell division to generate new hfs ( deschene et al , 2014 ) . as with the epidermis , there is evidence for paracrine signalling , as scs with high wnt activity recruit adjacent scs by secreting wnt ligands ( deschene et al , 2014 ) . hedgehog signalling mediates the activation of quiescent scs by differentiating ta cells ( hsu et al , 2014 ) . the short range of wnt signals from paneth cells , essential for scs remaining in an undifferentiated state , restricts the number of scs in the intestinal niche ( sato et al , 2011b ) . expansion of the paneth cell population is a feature of benign intestinal tumours , whose formation is driven by wntactivating apc mutations ( schepers et al , 2012 ) . apcmutant scs eventually colonise the crypt through their enhanced fitness over wildtype cells , as a differentiating cell is more likely to be replaced by a mutant than a wildtype cell ( vermeulen et al , 2013 ) . loss of lrig1 , which negatively regulates erbb family growth factor signalling , causes crypt expansion , while activation of ras , which lies downstream of erbb receptors , in intestinal scs can trigger a process called crypt fission in which a mutant crypt splits into two allowing a mutant stem cell clone to spread through the intestine ( wong et al , 2012 ; amoyel et al , 2014 ) . thus , while limiting niche size is a simple means of achieving homoeostasis , it is not invulnerable to mutations that confer a clonal fitness advantage on scs . the morphology of the niche has long been recognised as of pivotal importance in plants , whose architecture is determined by the spatial arrangement of the meristem ( heidstra & sabatini , 2014 ) . the size and shape of hair can be manipulated by altering the number of cells in the dermal papilla that forms the niche for hair germ scs ( chi et al , 2013 ) . within the bone marrow , hscs reside in close proximity to stromal cells adjacent to blood vessels and the bone surface ( mendelson & frenette , 2014 ) . each of these cell types makes a distinct paracrine contribution to the niche ( ding et al , 2012 ) . for example , the chemokine cxcl12 , which is essential for maintaining hscs , derives primarily from the perivascular stromal cells that lie directly adjacent to quiescent hscs ( ding & morrison , 2013 ; greenbaum et al , 2013 ) . paracrine niche signals also regulate neural scs in the svz , which extend cellular processes into the cerebrospinal fluid ( csf ) that fills the ventricles and forms contacts with underlying endothelial cells ( fig 2h ) ( silvavargas et al , 2013 ; codega et al , 2014 ) . these contacts allow regulation of neural scs via both secreted factors in the csf and signals from the vasculature . for example , endotheliumderived ntf3 and g proteincoupled receptor ligands in the csf promote sc quiescence ( codega et al , 2014 ; delgado et al , 2014 ) . seemingly at odds with scs residing in an ordered niche is their ability to recapitulate the morphology of a tissue in culture ( rheinwald & green , 1975 ; sato et al , 2009 ; stange et al , 2013 ; karthaus et al , 2014 ) . feeder cells or exogenous growth factors and ligands in the media are able to substitute for the paracrine environment provided by the niche , but in isotropic 3dimensional culture systems , morphogenic gradients are initially absent . a common feature of many protocols is the requirement for the rock kinase inhibitor y26732 during establishment of the culture . this suggests that manipulation of cytoskeletal signalling is required for isolated scs to enter a state that allows survival and promotes proliferation in the absence of niche cells ( sato et al , 2009 ; huch et al , 2013a , b ; karthaus et al , 2014 ) . rather than being determined by scs , the resemblance of organoids to their parent tissue may rest on the ability of differentiating cells to selfassemble into a tissuelike array by virtue of cell shapedefined packing considerations and differential adhesion ( watt & green , 1982 ; honda et al , 1996 ) . thus , recent studies are revealing more of the microarchitecture of the sc niche and the highly conserved paracrine signals , such as wnt , which regulate cell behaviour across a wide range of lineages . while the niche may not micromanage the outcome of each individual sc division , it has an essential role in sustaining sc populations in a proliferating or quiescent state . understanding how cellintrinsic factors , autocrine signals and exogenous factors combine to sustain scs in culture will likely give new understanding of the niche in vivo . stem cell behaviour must not only match local requirements in the tissue , but also needs across the organism ( fig 4 ) . for example in the mouse oesophagus , 10fold more scs undergo mitosis in the early diurnal than in the early nocturnal phase ( burns et al , 1976 ) . in the epidermis , hair follicle bulge scs vary in their transcription of the per1 gene , arguing they are in different phases of the circadian cycle . cells high in per1 expression have increased clonogenicity in vitro , suggesting variation in circadian phase may contribute to functional heterogeneity in bulge scs ( janich et al , 2011 ) . in the hematopoietic system , scs regularly leave the bone marrow and enter the circulation before returning to their niche . this migration also has a circadian rhythm with twice as many circulating scs in the diurnal than in the nocturnal phase . the exit of scs is controlled centrally from the suprachiasmatic nucleus in the brain , via release of noradrenaline from nerve endings in the bone marrow , which in turn regulates the transcription of cxcl12 , a chemokine that directs hsc migration ( mendezferrer et al , 2008 ) . sex hormone regulation of reproductive and mammary scs is well documented , but recently has been found to extend to scs in other tissues . for example , hscs are subject to positive regulation of proliferation and selfrenewing cell divisions by oestrogen in female mice . in pregnancy , oestrogen levels rise , the number of cycling hscs increases and more erythroid progenitor cells are produced , meeting the physiological requirement of the animal for increased oxygen delivery to tissues ( nakada et al , 2014 ) . another example is the hair cycle , which is arrested during in pregnancy by prolactin driving hf scs into a quiescent state ( goldstein et al , 2014 ) . a significant evolutionary pressure on organisms and scs is starvation , which induces a bodywide adaptive response that includes effects on scs ( longo & mattson , 2014 ; mihaylova et al , 2014 ) . the midgut of fasted drosophila expands dramatically after feeding , underpinned by a switch in the mode of sc division from balanced cell production to selfduplication in response to insulin ( o'brien et al , 2011 ) . in the murine small intestine , fasting reduces mtorc1 signalling in the paneth cells that form the sc niche ( yilmaz et al , 2012 ) . in turn , this increases paracrine signalling to scs via cyclic adp ribose , resulting in an increased efficiency of organoid formation in cultures from fasted animals ( yilmaz et al , 2012 ) . while not definitive , this result suggests starvation may expand the sc population by acting on the niche . starvation also impacts haematopoiesis , acting via igf1 signalling to increase the number of transplantable hscs consistent with an enhanced probability of selfrenewing division ( cheng et al , 2014 ) . another route for physiological sc regulation is via the nervous system , recently revealed as having a key role in hsc regulation . chronic psychological stress induces increased levels of leucocytes . as with circadian control , this effect is mediated by noradrenaline release from sympathetic neurons in the bone marrow , which decreases production of cxcl12 in niche cells triggering hsc proliferation ( heidt et al , 2014 ) . the importance of this mechanism is illustrated in the neoplastic myeloproliferative disorder , which is caused by jak2 mutations in hscs ( arranz et al , 2014 ) . secretion of interleukin 1b by the mutant hscs induces neuropathy in bone marrow , decreasing cxcl12 levels and triggering hsc proliferation and disease progression . the development of the disease is halted by restoring adrenergic signalling by treatment with 3adrenergic agonists ( arranz et al , 2014 ) . hscs also illustrate how systemic signals interact with cellintrinsic factors to promote the generation of a specific lineage from a multipotent sc population . for example , the cytokine mcsf , which is released in high levels during infection , induces the master myeloid transcription factor pu1 , increasing the probability of hscs differentiating into myeloid progenitors to meet the increased requirement for leucocytes ( mossadeghkeller et al , 2013 ) . drawing these studies together reveals a new aspect of sc biology that goes beyond single lineage or tissue . understanding how neural , endocrine and cytokine signals regulate cell production to meet the requirements of an organism is a key challenge for sc research . maintaining the quiescence of scs is an active process ( cheung & rando , 2013 ) . for example , muscle satellite cells are maintained in a quiescent state by specific mirnas and transcription factors downstream of notch , loss of which results in depletion of the sc population ( fukada et al , 2011 ; cheung et al , 2012 ; crist et al , 2012 ) . in the liver , the hepatocytes that fulfill the role of reserve scs are actively maintained in a quiescent and differentiated state by hippo signalling acting via the notch pathway ( yimlamai et al , 2014 ) . entry into cycle may require specific transcription factors , such as ascl1 , which is required for neural scs to exit the quiescent state ( andersen et al , 2014 ) . , the rapid upregulation of translation is achieved by posttranslational mechanisms involving the sequestration of regulatory mirnas in mrnp granules ( crist et al , 2012 ) . following a remote injury , circulating factors acting via hgf signalling and the mtorc1 signalling node prime quiescent scs enhancing their regenerative potential . once activated , some scs contribute to differentiated muscle , while others reenter the quiescent state to maintain the sc population ( shea et al , 2010 ) . return to quiescence requires multiple factors including sprouty1 , a negative regulator of receptor tyrosine kinase signalling and active notch signalling ( shea et al , 2010 ; gopinath et al , 2014 ) . it is becoming clear that the quiescent state is far from being a protected state as used to be thought . while cell competition results in the loss of mutant scs in proliferating tissues , quiescent scs accumulate epigenetic and genetic damage , resulting in impaired function as they age ( liu et al , 2013 ; martins et al , 2014 ; sun et al , 2014a ) . in quiescent hscs , the errorprone nonhomologous endjoining dna repair alterations progressively accumulate ( mohrin et al , 2010 ; beerman et al , 2014 ) . recruitment of hscs into cycle is accompanied by induction of a broad range of dna repair genes mobilised to remedy genomic alterations ( beerman et al , 2014 ) . as well as intrinsic factors , ageassociated inflammatory signalling acting via the jak / stat pathway impairs muscle sc function ( price et al , 2014 ; tierney et al , 2014 ) . intriguingly , administration of a single circulating growth factor , gdf11 , seems able to reverse the defects in ageing muscle scs ( sinha et al , 2014 ) . gdf11 also benefits neurogenesis by ageing neural scs , possibly acting via effects on the vasculature in the svz ( katsimpardi et al , 2014 ) . understanding how quiescent scs accumulate molecular damage and the ways in which this may be reversed is a central question in the biology of ageing . adult scs have evolved a diverse repertoire of sc behaviours to meet tissuespecific requirements . in cycling tissues , homoeostatic balance between selfduplication and differentiation may be achieved by cellintrinsic mechanisms , a spatially constrained niche , or exceptionally invariant asymmetric cell division . in each case , sc division is restrained by the requirement to maintain a constant cell density within the niche . repair is also achieved by diverse strategies , with mobilisation of reserve scs , plasticity of sc behaviour and dedifferentiation emerging as common themes . while specific growth factors may bias cellintrinsic lineage selection , a shared paracrine signalling toolkit sustains scs across multiple lineages ( clevers et al , 2014 ) . the application of powerful imaging and transgenic tools to reveal the dynamics of scs within their niche offers the prospect of integrating molecular and cellular mechanisms to gain a broader perspective on the functional plasticity of scs in the context of the whole organism .

abstractadult organisms have to adapt to survive , and the same is true for their tissues . rates and types of cell production must be rapidly and reversibly adjusted to meet tissue demands in response to both local and systemic challenges . recent work reveals how stem cell ( sc ) populations meet these requirements by switching between functional states tuned to homoeostasis or regeneration . this plasticity extends to differentiating cells , which are capable of reverting to scs after injury . the concept of the niche , the microenvironment that sustains and regulates stem cells , is broadening , with a new appreciation of the role of physical factors and hormonal signals . here , we review different functions of scs , the cellular mechanisms that underlie them and the signals that bias the fate of scs as they switch between roles .

Introduction The functional repertoire of SCs Regeneration: using all the options Autonomy and instruction: regulating SC states Remote control: integrating SC dynamics and whole body physiology Sleeping, waking and sleeping again: regulating SC quiescence Conclusion Conflict of interest

diseases of the heart continue to be the leading cause of death in the united states [ 1 , 2 ] and accounted for 24.6% of all deaths in 2009 , down only 1.4% from 2006 despite several national campaigns and research strategies to reduce mortality [ 1 , 2 ] . african americans have poorer health outcomes compared to their white counterparts [ 1 , 2 ] , and african american women disproportionately suffer from heart disease , with nearly half ( 45% ) of african american women having some type of cardiovascular disease compared to only 32% of white women [ 3 , 4 ] . cardiometabolic risk factors , such as high blood pressure and resting heart rate , elevated cholesterol and glucose levels , and high body fat percentage , are associated with cardiovascular diseases and may result from lifestyle choices , such as physical inactivity and poor dietary habits [ 6 , 7 ] . the prevalence of high blood pressure , or hypertension , among african americans in the united states has increased from 35.8% to 41.4% between 1988 and 2002 and is particularly high among african american women ( 44.8% in 2006 ) compared to white women ( 31.1% in 2006 ) [ 8 , 9 ] . among african americans , the prevalence of high ( 200 mg / dl ) and elevated ( 240 mg / dl ) cholesterol is higher among women than men , with 54.9% of african american women having high or elevated cholesterol compared to 51.1% of african american men . body composition and obesity are also directly linked to cardiovascular diseases and other health compromising conditions , such as diabetes and cancer . over one - third ( 39.2% ) of african american women are obese or have a body mass index ( bmi ) 30 kg / m , compared to only 21.8% of white women , 25.4% of white men , and 31.6% of african american men . african american women also have greater adiposity or body fat compared to caucasians [ 12 , 13 ] . regularly performed physical activity improves body composition and nearly all known health conditions ; yet , self - reported measures suggest nearly 50% of the adult population fails to meet minimum physical activity recommendations , while objective measurement shows only 5% meet recommendations , suggesting that sedentary time comprises the largest portion of most people 's days . african american women are less physically active than white women , putting them at greater risk for chronic health conditions related to physical inactivity , including cardiovascular diseases , and leading to rising health care costs , which exceeded $ 11 billion among morbidly obese adults in 2000 . several studies have looked at the relationship between sedentary behavior and disease risk , but few have looked at specific measured sedentary behavior , defining sedentary time as low physical activity during leisure time [ 1820 ] . for example , increased sedentary time has been associated with increased bmi , mortality rates , high glucose levels , and insulin resistance , regardless of physical activity level among both men and women [ 2125 ] . however , there have also been recent studies that did not demonstrate these relationships [ 2628 ] . in addition , a recent literature review suggested that although there is not sufficient evidence to support significant relationships between sedentary behavior and body weight gain and sedentary behavior and cardiovascular disease biomarkers , there is evidence in the literature to support a strong relationship between sedentary behavior and mortality , suggesting that , while important , these relationships are not well documented or described . inconsistencies in the reported relationships between sedentary behavior and cardiometabolic risk factors and body composition suggest that specific population or measurement characteristics of studies may be contributing to findings . for example , one study showed that 27.3% to 95.9% of the association between sedentary behavior and health outcomes ( e.g. , blood pressure , cholesterol ) was explained by bmi or waist circumference . most of the literature has included a majority of white participants [ 22 , 27 , 28 , 30 ] , and the relationship of sedentary behavior to cardiometabolic risk factors and body composition in ethnic minority women remains unclear . one study suggested that decreased occupational sitting time may decrease bmi and promote healthy behaviors among women , but few have explored the direct relationship between sitting time that includes both weekday and weekend , occupational and leisure time , or have distinguished between occupational and leisure sitting time and its associations with cardiometabolic risk factors and body composition . the purpose of this study was to investigate the relationship of weekday and weekend sitting time to cardiometabolic risk factors , including blood pressure , resting heart rate , cholesterol , triglycerides and glucose , and body composition among overweight and obese african american women . we hypothesized that increased sitting time would be associated with higher rates of cardiometabolic risk factors , obesity , and increased body fat and explored the issue of whether weekday ( occupational ) sitting time or weekend ( recreational ) sitting time would be more important in contributing to cardiometabolic outcomes . the current study was a secondary analysis using data from the health is power ( hip ) study ( 1r01ca109403 ) . four hundred ten community dwelling , african american ( n = 263 ) , and hispanic or latina ( n = 147 ) women participated in hip , a multisite , longitudinal , community - based , randomized controlled trial to increase physical activity [ 3142 ] in houston and austin , texas . eligible participants , self - identified as african american or hispanic or latina , were between the ages of 25 and 60 years old , able to read , speak , and write in english or spanish , not pregnant or planning to become pregnant within the next 12 months , a harris or travis county resident , not planning on moving in the next 12 months , physically inactive or doing fewer than 30 minutes of physical activity per day on 3 or more days per week , and free from health conditions that would be aggravated by physical activity . a subsample of houston african american participants ( n = 135 ) completed laboratory assessments at baseline , time 1 ( t1 ) [ 31 , 41 ] . all hip study assessments , measures and procedures were approved by the committee for the protection of human subjects at the university of houston , and women provided written informed consent prior to participation . women who met inclusionary criteria gave informed consent and completed a t1 health assessment , where they completed an interviewer administered questionnaire measuring physical activity and demographics , measures of blood pressure and resting heart rate , and anthropometric measures of bmi and body fat [ 3638 , 42 ] . the laboratory assessment included a venous blood sample and a whole body dual - energy x - ray absorptiometry ( dxa ) scan [ 31 , 41 ] . women completed these measures after fasting for 8 or more hours and wore metal - free clothing . systolic and diastolic blood pressures were measured using manual aneroid sphygmomanometry by a trained research team member using established protocols . participants were asked to sit quietly during measurement with their left arm bared and supported at heart level and their feet flat on the floor . two readings were obtained , separated by two minutes , and averaged for use in analyses . if the first two readings differed by more than 5 mmhg , a third reading was obtained and averaged . a venous blood sample was collected from a peripheral arm vein into vacutainers pretreated with either sodium heparin or k2 edta ( vacutainer ; becton - dickinson , franklin lakes , nj ) after 8 or more hours of fasting and analyzed for plasma total cholesterol , high - density lipoprotein ( hdl ) , low - density lipoprotein ( ldl ) , triglyceride , and glucose concentrations using separate enzymatic assays in triplicate as described by the manufacturer ( pointe scientific , canton , mi ) . a ratio of total cholesterol to hdl ( total cholesterol / hdl ) was also used in analyses . anthropometric measures of bmi and body fat were collected by trained personnel using established protocols [ 3638 , 41 , 42 ] . individual height was measured using a standard stadiometer apparatus with participants ' shoes being removed . body weight and percent body fat were measured twice using bioelectrical impedance analysis ( bia ) using a tanita tbf-310 body composition analyzer ( tanita , arlington heights , illinois ) . all measures were collected twice , and the average of the two measurements was used in analyses . dxa measurements were completed by a trained staff member between 6:00 and 8:00 am and took 1015 minutes per participant . dxa scans were used to measure whole body fat mass , lean mass , bone mass and total percent body fat , as previously described [ 31 , 41 ] . only total percent body fat was used in current analyses . sitting time was measured using items from the international physical activity questionnaire ( ipaq ) long form administered at the baseline health assessment . the ipaq long form is typically used to measure work - related , transportation , domestic and leisure - time physical activity . in addition , the instrument measures time spent sitting over the last seven days by time spent sitting in a motor vehicle and time spent sitting during the week and weekend . sitting time adhering to the ipaq protocol , data were cleaned and missing or spurious data were excluded from any analyses . items assessing ethnicity , household income , and education were adapted from the maternal infant health assessment ( miha ) survey , derived from the cdc 's pregnancy risk assessment monitoring system ( prams ) questionnaire . all statistical analyses were conducted in spss version 19.0 ( ibm spss statistics for windows , ibm corporation , somers , ny ) . the current study is limited to a subsample of african american women enrolled in houston , tx , who were offered a laboratory assessment ( n = 135 ) at baseline t1 . only participants with complete data for a particular measure were included in all analyses , which varied by assessment procedure / measure . women in the subsample were slightly older ( m = 46.6 years , sd = 8.9 ) than the total african american sample ( m = 42.9 years , sd = 9.6 ; t = 3.141 , p = .002 ) but were similar in education , income , bmi , and percent body fat . bivariable correlations were conducted among cardiometabolic risk factor variables and body composition variables and between cardiometabolic risk factor and body composition variables . simultaneous linear regression models were used to estimate the effect of weekday and weekend sitting time on cardiometabolic risk factors , including systolic and diastolic blood pressure , resting heart rate , total cholesterol , hdl , ldl , triglycerides , the ratio of total cholesterol to hdl , and glucose , and on body composition , including bmi , bia percent body fat , and dxa percent body fat , controlling for age , education , and income . african american women were middle aged ( m = 46.6 years , sd = 8.9 ) and obese ( m bmi = 34.9 kg / m , sd = 9.5 ) . over half ( 52.7% ) had graduated from college , and the majority ( 56.7% ) reported an income 401% or greater above the federal poverty level or an income greater than $ 82,807 . mean ( and sd ) cardiometabolic risk factors and body composition are presented in table 1 . triglycerides varied by education ( f(1,26 ) = 5.650 , p = .025 ) ; women who had not graduated from college had higher triglyceride values than women with a college education ( m = 73.2 versus 37.9 ) . there were no other significant differences in cardiometabolic factors or body composition by education or income . age was significantly positively correlated with systolic blood pressure ( r = .219 , p = .011 ) and glucose ( r = .288 , p = .036 ) . total cholesterol was significantly negatively correlated with weekend sitting time ( r = .374 , p = .050 ) and total sitting time ( r = .376 , p = .049 ) . ldl was also significantly negatively correlated with weekend sitting time ( r = .425 , p = .027 ) . sitting time was not correlated with any other cardiometabolic risk factors or body composition variables . there were no significant linear associations between weekday sitting time and either cardiometabolic factors or body composition . in contrast , linear regression models suggest a moderate association between weekend sitting time and glucose ( = .266 , t = 1.960 , p = .056 ) , which may be significant with increased power . linear regression models mimicked bivariable correlations and indicated a surprising negative linear association for both weekend ( = .374 , t = 2.058 , p = .050 ) and total ( = .376 , t = 2.069 , p = .049 ) sitting time to total cholesterol , suggesting that greater sitting time was associated with lower total cholesterol levels . greater sitting time during the weekend was also associated with lower ldl levels ( = .425 , t = 2.347 , p = .027 ) . also of interest based on previous research , we expected to find that greater time spent sitting was associated with poorer cardiometabolic indicator values . we found some limited support for this hypothesis with glucose ; however , reduced sample size limited our power to detect this effect . it may be that since this sample was already overweight and obese , time spent sitting had little impact on this outcome . in contrast to our hypotheses , we found that greater time spent sitting on the weekend was associated with better cholesterol levels , suggesting that in this sample , there is some advantage to time spent sitting that may counteract the sedentary nature of sitting . the curious relationship that we found between sitting and improved cholesterol suggests that something unexpected is driving this relationship . perhaps women who have more sitting time are more relaxed and have more leisure time in general . the variables used to measure sedentary time included time spent sitting while driving . in this sample , in sprawling houston , we have found an inverse association between car ownership and physical activity ( results not shown ) ; perhaps more time spent sitting while driving led to more time doing physical activity either because the participant commuted to their physical activity destination or because the increased driving time led to a desire to be more active , which may have impacted these cardiometabolic factors . finding is that nearly all of the relationships reported here were between weekend sedentary time , rather than weekday time . during the week , perhaps time is more carefully scripted by work and family responsibilities , while weekends have more discretionary time . future studies should continue to specify time spent during weekends and weekdays separately in terms of both measurement and intervention . in contrast , people may feel that weekends are a time for rest and relaxation , that is , sedentary time . thus , intervention strategies that decrease sedentary and sitting time during the week might be more sustainable , as they get integrated along with already ritualized weekday responsibilities . previous research exploring the relationship between sitting time and other cardiometabolic risk factors has yielded similar findings . found that sitting time was positively associated with fasting insulin , c - reactive protein , and insulin resistance in women after adjusting for physical activity . however , no other studies have found a relationship between sedentary time and cholesterol or lipoprotein measures in women [ 27 , 50 ] , warranting further investigation to elucidate study findings . in our sample of community volunteers , we found slightly elevated blood pressure , with 60.1% of the sample exceeding normal ranges for both systolic and diastolic , somewhat higher than the national prevalence [ 8 , 9 ] . this sample had relatively poor cholesterol levels , with most women having too low values of hdl and too high levels of ldl , similar to national samples . most of the sample was overweight or obese , which likely reflects not only very high prevalence of high body fat in the population [ 1113 ] but also the nature of the study recruitment , which sought volunteers to enroll in a study to increase physical activity or improve dietary habits . this study is among the first to investigate the relationship of weekday and weekend sitting time to cardiometabolic risk factors in african american women and includes a sizeable sample of african american women , who are most vulnerable to obesity and chronic health conditions . this study includes the use of validated and reliable measures for this population , including dxa- and bia - measured percent body fat . accelerometry is considered the gold standard of physical activity and sedentary behavior measurement and may have enhanced study findings . in addition , this study was limited to the relationship between weekday and weekend sitting time and cardiometabolic risk factors and did not explore the relationship between physical activity and these risk factors , for which there is a known strong relationship . the use of cross - sectional versus longitudinal data limits us from making assumptions about causality in this study , and due to study population characteristics , findings may not be generalized to other non - african american populations . missing data for laboratory assessments and measures also limits findings and may explain differences in findings between the current study and established literature . although this sample was generally representative of african american women in terms of health status , this sample was of higher socioeconomic status , as is often the case with community volunteers in health promotion studies . future studies should investigate larger samples that represent the entire community and continue to account carefully for sedentary and sitting time . as others have suggested , these findings suggest that simply decreasing sedentary time may not be sufficient to improve cardiometabolic risk and body composition . these findings may have produced more questions than they answered but underscore the complexity of the relationships between sedentary behavior and health outcomes , particularly in more vulnerable groups in the population .

findings from previous research linking sedentary time with cardiometabolic risk factors and body composition are inconsistent , and few studies address population groups most vulnerable to these compromising conditions . the purpose of this paper was to investigate the relationship of sitting time to cardiometabolic risk factors and body composition among african american women . a subsample of african american women ( n = 135 ) completed health and laboratory assessments , including measures of blood pressure , resting heart rate , cholesterol , triglycerides , glucose , body mass index , body fat , sitting time , and demographics . simultaneous , adjusted regression models found a positive association between weekend sitting time and glucose and an inverse association between weekly sedentary time and cholesterol ( ps < .05 ) . there were no significant associations between sedentary behavior and body composition . the unexpected relationship between sedentary time and cholesterol suggests that the relationship of sedentary behavior to cardiometabolic risk factors may depend on existing characteristics of the population and measurement definition of sedentary behavior . results suggest distinctly different relationships between weekend and weekday sitting time , implicating a need for careful measurement and intervention that reflects these differences .

1. Introduction 2. Materials and Method 3. Results 4. Discussion

for efficacy studies , up to 3 female horses at a time were housed in single pens under bsl-4 conditions meeting the victorian bureau of animal welfare code of practice for the welfare of horses ( www.dpi.vic.gov.au/agriculture/about-agriculture/legislation-regulation/animal-welfare-legislation/codes-of-practice-animal-welfare/code-welfare-of-horses ) . one of the sides of each pen was able to be moved in toward the horse on a ratchet mechanism , allowing staff close access to the horses , as required , over the side of the pen without the need for them to enter the pen itself ( 13 ) . room temperature was maintained at 22c with 15 air changes / h ; humidity ranged from 40% to 60% . horses were fed a mixture of lucerne ( alfalfa ) and grass hay , concentrates , and specified fruit and vegetables . on the day before hev exposure , an indwelling jugular catheter was sutured in position , and an intrauterine temperature data - logger was placed into each horse . all vaccinated horses were euthanized electively on day 7 , 8 , or 9 after challenge ; unvaccinated horses were euthanized upon reaching a predetermined humane endpoint ( 69 days after vaccination ) . the humane end point was defined as fever for up to 48 h accompanied by increased respiratory rate , dyspnea , depression , ataxia , or pressing the head against the side of the stall . euthanasia was conducted by intravenous injection of a barbiturate following sedation with intravenous detomidine and butorphanol . ferrets and guinea pigs used as controls in efficacy studies to confirm pathogenicity of the inoculum were housed in pairs in the bsl-4 facility , given species - appropriate dry rations and dietary treats , and provided with water ad libitum . for virus challenge and sampling , they were immobilized by intramuscular injection of a mixture of ketamine hydrochloride ( 3 mg / kg ) and medetomidine ( 30 g / kg ) . the effects of medetomidine were reversed by intramuscular injection of atipemazole ( 15 g / kg ) . while in the bsl-4 animal room , staff wore fully encapsulated suits with an external air supply . as appropriate , animal studies were endorsed by the csiro australian animal health laboratory animal ethics committee and/or commonwealth serum laboratories /zoetis animal ethics committee . work using gamma - irradiated hev soluble g ( hevsg ) glycoprotein produced in chinese hamster ovary ( cho ) cells was done under australian quarantine and inspection service in vivo permit number 2012/012 , and work using non - gamma irradiated hevsg glycoprotein produced in 293f human embryonic kidney was done under australian quarantine and inspection service in vivo permit number 2010/027 . all clinical trials were conducted under the australian pesticides and veterinary medicines authority research permits per 7250 , per 13169 , per 13247 , and per 13418 . a subunit vaccine containing recombinant hevsg glycoprotein ( 14 ) hevsg glycoprotein was produced by using a chinese hamster ovary ( cho ) or a 293f human embryonic kidney cell expression system ( 15 ) with 1 of 2 different hevsg glycoprotein preparations : 1 ) affinity - purified sg glycoprotein ( 293f cells ) or 2 ) clarified sg containing cell culture supernatant ( cho cells ) . vaccines for initial efficacy studies in target species were formulated with 50 g or 100 g of affinity - purified sg glycoprotein . all subsequent vaccines were formulated with clarified cho cell culture supernatant that was then gamma irradiated . the change of the expression system from 293f cells to cho cells was driven by the need for higher antigen yields , and equivalence was supported by laboratory analysis of the expressed antigens from the 2 systems and a comparison study in ferrets . vaccine formulations used in efficacy studies are summarized in table 1 . * hek , human embryonic kidney cells ; cho , chinese hamster ovary cells . all immunizations comprised two 1-ml doses administered intramuscularly 3 weeks apart , unless stated otherwise . in the efficacy studies , 7 horses ( v1 , v2 , and v6v10 ) received vaccine containing 100 g of hevsg glycoprotein / dose and 3 horses ( v3v5 ) received 50 g of hevsg glycoprotein / dose ( table 1 ) . horses in the efficacy studies were exposed oronasally to 2 10 50% tissue culture infectious doses of a low - passage hev isolate ( hendra virus / australia / horse/2008/redlands ) . horses v1v7 were challenged 28 days after the second vaccination , and horses v8v10 were challenged 194 days after the second vaccination . horses v8v10 were selected from 29 vaccinated horses in a larger field efficacy and safety study on the basis of temperament and for having the lowest serum neutralization titers in the group at the time . overall , 4 efficacy tests were completed ; 2 vaccinated horses were used in the first test , 3 were used in the second , 2 were used in the third , and 3 were used in the fourth . for the 4 tests , a pathogenicity control for the inoculum was provided by 1 horse ( test 1 ) , 4 guinea pigs ( test 2 ) , 2 ferrets ( test 3 ) , and 2 ferrets ( test 4 ) . guinea pigs and ferrets each received 50,000 50% tissue culture infectious doses of the same virus preparation that was used in the horses ; guinea pigs received the dose by intraperitoneal injection , and ferrets received the dose by the oronasal route . experience has shown that these doses and routes of administration were expected to be lethal in > 25% of guinea pigs and 100% of ferrets . exposure conditions for 3 additional unvaccinated control horses were equivalent to those used in both vaccinated horses and the inoculum - control horse and have been described ( 6 ) . during efficacy studies , nasal , oral , and rectal swab samples ; urine and feces samples ; and blood samples ( in edta ) were collected from the horses before virus exposure and then daily until the animals were euthanized . swab samples were collected in duplicate into 1 ml of phosphate - buffered saline for virus isolation or into 800 l of magmax lysis / binding solution ( ambion , austin , tx , usa ) for rna extraction . for urine and edta blood samples , 100 l of fluid was added to 260 l of the lysis / binding solution . at postmortem examination , the following tissues were collected for viral genome detection , virus isolation , histopathology , and immunohistochemistry according to ( 15 ) : adrenal gland , bladder , brain ( including olfactory pole ) , cerebrospinal fluid , guttural pouch , heart , kidney , large intestine , liver , lung , lymph nodes ( bronchial , inguinal , intermandibular , mandibular , renal ) , meninges , nasal turbinates , ovaries , pharynx , small intestine , spinal cord , spleen , sympathetic nerve , trigeminal ganglion , and uterus . the following analyses were conducted as described ( 15 ) : quantitative reverse transcription pcr for the detection of the hev n gene , histology , immunohistology , serum neutralization test , and virus isolation . for efficacy studies , up to 3 female horses at a time were housed in single pens under bsl-4 conditions meeting the victorian bureau of animal welfare code of practice for the welfare of horses ( www.dpi.vic.gov.au/agriculture/about-agriculture/legislation-regulation/animal-welfare-legislation/codes-of-practice-animal-welfare/code-welfare-of-horses ) . one of the sides of each pen was able to be moved in toward the horse on a ratchet mechanism , allowing staff close access to the horses , as required , over the side of the pen without the need for them to enter the pen itself ( 13 ) . room temperature was maintained at 22c with 15 air changes / h ; humidity ranged from 40% to 60% . horses were fed a mixture of lucerne ( alfalfa ) and grass hay , concentrates , and specified fruit and vegetables . on the day before hev exposure , an indwelling jugular catheter was sutured in position , and an intrauterine temperature data - logger was placed into each horse . all vaccinated horses were euthanized electively on day 7 , 8 , or 9 after challenge ; unvaccinated horses were euthanized upon reaching a predetermined humane endpoint ( 69 days after vaccination ) . the humane end point was defined as fever for up to 48 h accompanied by increased respiratory rate , dyspnea , depression , ataxia , or pressing the head against the side of the stall . euthanasia was conducted by intravenous injection of a barbiturate following sedation with intravenous detomidine and butorphanol . ferrets and guinea pigs used as controls in efficacy studies to confirm pathogenicity of the inoculum were housed in pairs in the bsl-4 facility , given species - appropriate dry rations and dietary treats , and provided with water ad libitum . for virus challenge and sampling , they were immobilized by intramuscular injection of a mixture of ketamine hydrochloride ( 3 mg / kg ) and medetomidine ( 30 g / kg ) . the effects of medetomidine were reversed by intramuscular injection of atipemazole ( 15 g / kg ) . while in the bsl-4 animal room , staff wore fully encapsulated suits with an external air supply . as appropriate , animal studies were endorsed by the csiro australian animal health laboratory animal ethics committee and/or commonwealth serum laboratories /zoetis animal ethics committee . work using gamma - irradiated hev soluble g ( hevsg ) glycoprotein produced in chinese hamster ovary ( cho ) cells was done under australian quarantine and inspection service in vivo permit number 2012/012 , and work using non - gamma irradiated hevsg glycoprotein produced in 293f human embryonic kidney was done under australian quarantine and inspection service in vivo permit number 2010/027 . all clinical trials were conducted under the australian pesticides and veterinary medicines authority research permits per 7250 , per 13169 , per 13247 , and per 13418 . a subunit vaccine containing recombinant hevsg glycoprotein ( 14 ) was formulated in a proprietary adjuvant ( zoetis ) . for vaccine formulation , hevsg glycoprotein was produced by using a chinese hamster ovary ( cho ) or a 293f human embryonic kidney cell expression system ( 15 ) with 1 of 2 different hevsg glycoprotein preparations : 1 ) affinity - purified sg glycoprotein ( 293f cells ) or 2 ) clarified sg containing cell culture supernatant ( cho cells ) . vaccines for initial efficacy studies in target species were formulated with 50 g or 100 g of affinity - purified sg glycoprotein . all subsequent vaccines were formulated with clarified cho cell culture supernatant that was then gamma irradiated . the change of the expression system from 293f cells to cho cells was driven by the need for higher antigen yields , and equivalence was supported by laboratory analysis of the expressed antigens from the 2 systems and a comparison study in ferrets . vaccine formulations used in efficacy studies are summarized in table 1 . * hek , human embryonic kidney cells ; cho , chinese hamster ovary cells . all immunizations comprised two 1-ml doses administered intramuscularly 3 weeks apart , unless stated otherwise . in the efficacy studies , 7 horses ( v1 , v2 , and v6v10 ) received vaccine containing 100 g of hevsg glycoprotein / dose and 3 horses ( v3v5 ) received 50 g of hevsg glycoprotein / dose ( table 1 ) . horses in the efficacy studies were exposed oronasally to 2 10 50% tissue culture infectious doses of a low - passage hev isolate ( hendra virus / australia / horse/2008/redlands ) . horses v1v7 were challenged 28 days after the second vaccination , and horses v8v10 were challenged 194 days after the second vaccination . horses v8v10 were selected from 29 vaccinated horses in a larger field efficacy and safety study on the basis of temperament and for having the lowest serum neutralization titers in the group at the time . overall , 4 efficacy tests were completed ; 2 vaccinated horses were used in the first test , 3 were used in the second , 2 were used in the third , and 3 were used in the fourth . for the 4 tests , a pathogenicity control for the inoculum was provided by 1 horse ( test 1 ) , 4 guinea pigs ( test 2 ) , 2 ferrets ( test 3 ) , and 2 ferrets ( test 4 ) . guinea pigs and ferrets each received 50,000 50% tissue culture infectious doses of the same virus preparation that was used in the horses ; guinea pigs received the dose by intraperitoneal injection , and ferrets received the dose by the oronasal route . experience has shown that these doses and routes of administration were expected to be lethal in > 25% of guinea pigs and 100% of ferrets . exposure conditions for 3 additional unvaccinated control horses were equivalent to those used in both vaccinated horses and the inoculum - control horse and have been described ( 6 ) . during efficacy studies , nasal , oral , and rectal swab samples ; urine and feces samples ; and blood samples ( in edta ) were collected from the horses before virus exposure and then daily until the animals were euthanized . swab samples were collected in duplicate into 1 ml of phosphate - buffered saline for virus isolation or into 800 l of magmax lysis / binding solution ( ambion , austin , tx , usa ) for rna extraction . for urine and edta blood samples , 100 l of fluid was added to 260 l of the lysis / binding solution . at postmortem examination , the following tissues were collected for viral genome detection , virus isolation , histopathology , and immunohistochemistry according to ( 15 ) : adrenal gland , bladder , brain ( including olfactory pole ) , cerebrospinal fluid , guttural pouch , heart , kidney , large intestine , liver , lung , lymph nodes ( bronchial , inguinal , intermandibular , mandibular , renal ) , meninges , nasal turbinates , ovaries , pharynx , small intestine , spinal cord , spleen , sympathetic nerve , trigeminal ganglion , and uterus . the following analyses were conducted as described ( 15 ) : quantitative reverse transcription pcr for the detection of the hev n gene , histology , immunohistology , serum neutralization test , and virus isolation . vaccine efficacy in immunized horses was assessed against the clinical , virologic , and pathologic features of hev infection in 4 unvaccinated control horses . infection characteristics for 3 of these unvaccinated animals have been described ( 6 ) ; data from the fourth control animal was gathered as part of the current work . in that fourth control , onset of fever accompanied by a rising heart rate was noted on postchallenge day 6 . on postchallenge day 7 , the horse became clinically depressed , its temperature and heart rate continued to rise , and it was euthanized . gross postmortem findings included pleural thickening and moderate dilation of the lymphatic vessels on the ventral 10 cm of the cardiac lung lobes . histologic examination revealed systemic vasculitis affecting the lung ( figure 1 , panel a ) , spleen , kidney , nasal epithelium , lymph nodes , and brain ; alveolitis ; and lymphadenitis . hev antigen was identified in endothelial cells and vascular walls within lung , brain ( figure 1 , panel b ) , nasal epithelium , lymph nodes , spleen , kidney , liver , myocardium , salivary gland , pharynx , small intestine , uterus , ovary , and adrenal gland , as well as in myocardial fibers and glomeruli . histologic and immunohistologic findings in hendra virus infected horse tissue . a ) hematoxylin and eosin b ) immunohistologic examination , using polyclonal rabbit anti - nipah n protein , indicates hendra virus antigen in a blood vessel in the brain . was detected in nasal swabs collected on postchallenge day 3 ( table 2 ; summarized in table 3 ) and also in blood collected immediately before the onset of fever . after onset of fever , but before development of other clinical signs of illness , hev rna was also detected in the oral swab sample . on the day of euthanasia , genome was detected in oral and nasal swab samples , blood , rectal swab , and urine samples ; however , virus was not reisolated from any sample collected before postmortem examination . reisolation of virus was attempted for all tissues : hev was recovered from lung , submandibular lymph node , small intestine , large intestine , and adrenal gland . * duplicate samples were obtained and tested by reverse transcription pcr . cycle threshold values were converted to relative copy numbers by using a standard curve of a sample with a known copy number . n gene data for horses 24 are unpublished data from a previous study ( 6 ) . in a series of vaccine efficacy studies , 10 horses were immunized with hevsg glycoprotein and then exposed to an otherwise lethal dose of hev by the oronasal route . in the first of these , the pathogenicity control was the fourth control horse described above . together with historical data gathered from 3 horses following their exposure to hev under equivalent experimental conditions ( 5 ) , data from this horse completed the requirements of the australian pesticides and veterinary medicines authority for defining the horse infection model . in subsequent studies , guinea pigs or ferrets were used as pathogenicity controls to maximize the number of vaccinated horses that could be accommodated in the bsl-4 facility . these animals duly displayed signs , lesions , tissue antigen and viral genome distribution , and virus reisolation data consistent with acute hev infection . in contrast to unvaccinated control horses , vaccinated horses remained clinically healthy during the observation period after exposure to hev . following elective euthanasia at the time of predicted peak viral replication , there was no gross or histologic evidence of hev infection in vaccinated horses ; all tissues examined were negative for viral antigen by immunohistochemistry ; and viral genome was not recovered from any tissue , including nasal turbinates , pharynx , and guttural pouch ( table 3 ) . for 9 of 10 vaccinated horses , viral rna was not detected in daily nasal , oral , or rectal swab specimens or from blood , urine , or feces samples collected before euthanasia , and virus was not reisolated from any of these clinical samples . for 1 ( v9 ) of 3 horses exposed to hev 6 months after completing the vaccination course , low viral gene copy numbers were detected in nasal swab samples collected on postchallenge days 24 and 7 ( figure 2 ) ; this finding was consistent with self - limiting local replication . scatter plot showing quantitation of the hendra virus n gene in nasal swab samples from 1 vaccinated horse ( v9 ) and 4 control horses ( c1c4 ) ; controls were challenged but not vaccinated . serum neutralization titers before hev challenge ranged from 128/256 to > 4,096 for horses v1v7 when challenged 21 days after the second vaccination and from 16 to 32 for horses v8v10 when challenged 6 months after the second vaccination ( table 3 ) . at the time of euthanasia , no rise in antibody titer was detected in any vaccinated horse following exposure to hev . the formal launch of the hev horse vaccine in november 2012 represents the culmination of multiple studies conducted in several animal infection models over the course of many years . studies using nipah virus in cats ( 16,17 ) and monkeys ( 18 ) and hev in ferrets ( 15 ) provided strong evidence that a hevsg glycoprotein subunit based vaccine could prevent not only disease but often infection in animals exposed to otherwise lethal doses of nipah virus or hev . where evidence of low - level virus replication did occur in secretions , it was transient and unaccompanied by the development of clinical illness , and virus was not isolated from the secretions . the henipavirus surface - expressed g glycoprotein has the critical role of initiating infection by binding to receptors on host cells , and antibodies directed against this protein can neutralize virus ( 19 ) . earlier reports have shown that passive immunotherapy with antibody to the g or f glycoprotein of hev or nipah virus alone can prevent fulminating disease ( 20 ) : g glycoprotein specific human monoclonal antibody prevented nipah virus disease in ferrets ( 21 ) and hev infection in african green monkeys ( 22 ) ; and f or g glycoprotein specific monoclonal or polyclonal antibodies prevented hev and nipah virus disease in hamsters ( 2325 ) . thus it is likely that , as seen for other paramyxoviruses with a viremic infection phase ( e.g. , measles and mumps ) , antibodies to the g and f glycoproteins play a major role in protection provided by hevsg glycoprotein vaccination ( 2628 ) . in the studies reported here , we show that 2 doses of a commercially formulated hevsg glycoprotein subunit based vaccine prevented infection in 7 of 7 horses exposed to hev at least 21 days after the second vaccine dose ; this finding is in contrast to that for unvaccinated control horses . similar results were obtained for 2 of 3 horses exposed to hev 6 months after vaccination . in the third horse , which also remained clinically healthy , evidence of hev replication was limited to low - level transient detection of viral genome ( but not virus ) from the nasal cavity . in assessing the field significance of this observation , the following must be noted : the experimental horses were exposed to considerably higher levels of hev than have been recovered from flying foxes ( 1 ) , higher levels of viral genome were routinely found in the nasal secretions of nonimmunized horses , and all human infections have been acquired from animals in which clinical disease developed . it is reasonable to suggest that the higher transmission risk that is clearly associated with such horses is a consequence of not only increased viral load but also of the illness itself : it is the clinically ill horse that promotes increased human we conclude that the level and pattern of virus replication in the 1 vaccinated horse do not meet the epidemiologic criteria presently associated with transmission of infection to humans . in previous henipavirus vaccine efficacy studies in cats and ferrets , a neutralizing antibody titer of 32 was shown to be protective against the development of clinical disease ( 17 ) . in the horse efficacy studies , the 3 horses with prechallenge antibody titers of 16 or 32 were similarly protected from clinical illness . however , we caution that any correlation between antibody titer at the time of exposure to virus and levels of subsequent protection against infection and disease is unlikely to be linear ; it is possible that animals with even lower titers will have epidemiologically meaningful protection against hev exposure occurring in the field , not least because of stimulation of immunological memory . additional studies assessing the duration of protection are planned , and the outcome of these will further inform recommendations regarding booster vaccination . as expected , initial uptake of the hevsg glycoprotein subunit based vaccine was strongest in the area with the highest perceived risk for hev infection , namely coastal queensland , australia . in other regions where hev infection of horses has not been reported , there is understandably more uncertainty regarding the value of vaccination as part of horse preventative health programs . any reluctance to vaccinate horses against hev that is based on assessment of risk is probably exacerbated by several factors , including the novelty of the vaccine roll - out process to the australian horse industry , a ( mistaken ) perception that fast - tracking vaccine release involved overlooking key safety and efficacy issues , the lack of published data on safety in pregnant mares , reluctance of certain industry sectors to vaccinate because of import restrictions on hev - seropositive horses , and cost . although it is likely that each of these barriers will diminish over time , our experiences may assist the development of road maps to guide the future release of vaccines against bsl-4 pathogens that are associated with highly sporadic disease events and where the decision to vaccinate is in the hands of the persons whom vaccination was designed to protect . several recently emerged zoonotic viruses , including hev , nipah , ebola , and marburg viruses , are classified as bsl-4 agents because of their ability to cause severe illness or death in humans and because there have been no effective vaccines or postexposure treatments to protect against the diseases they cause . the vaccine against hev ( equivac hev ) is a commercially deployed vaccine developed against a bsl-4 agent and is the only licensed treatment for henipavirus infection . development of vaccines against bsl-4 agents for use in humans requires that the us food and drug administration implement the animal rule , which requires that such vaccines first be tested for efficacy in at least 2 animal models ( 29 ) . as a veterinary vaccine , equivac hev did not need to meet this requirement , and it was both cheaper and faster to produce than a vaccine intended for human use . at the same time , the vaccine is expected to provide a substantial health benefit to humans . in so doing , this vaccine encapsulates the spirit of a one health approach , not just in terms of the interconnectedness of human and animal health but also with respect to environmental health . one consequence of the recent hev outbreaks was a move to eradicate bat populations , despite their crucial environmental roles in pollination and reduction of the insect population . successful deployment of the hev vaccine , with a targeted reduction in the risk for acute disease events in horses and humans , should help reduce the current momentum toward the setting of control policies with potential adverse effects on the environment . furthermore , the increasing evidence for henipaviruses and henipa - like viruses in bats in other areas ( 3032 ) raises the possibility of future henipavirus outbreaks . the current hevsg glycoprotein vaccine technology provides a platform for the rapid development of related vaccines to counter future emergent threats .

in recent years , the emergence of several highly pathogenic zoonotic diseases in humans has led to a renewed emphasis on the interconnectedness of human , animal , and environmental health , otherwise known as one health . for example , hendra virus ( hev ) , a zoonotic paramyxovirus , was discovered in 1994 , and since then , infections have occurred in 7 humans , each of whom had a strong epidemiologic link to similarly affected horses . as a consequence of these outbreaks , eradication of bat populations was discussed , despite their crucial environmental roles in pollination and reduction of the insect population . we describe the development and evaluation of a vaccine for horses with the potential for breaking the chain of hev transmission from bats to horses to humans , thereby protecting horse , human , and environmental health . the hev vaccine for horses is a key example of a one health approach to the control of human disease .

Materials and Methods Animals, Accommodation, Handling, and Biosafety Vaccine Preparation Immunization Animal Infection Sample Collection and Analysis Results Discussion

in vivo studies using the animals provide invaluable information about various disease processes and help in development of treatment strategies . virtually every medical achievement in the twentieth century relied on the use of animals in some way.1 the institute for laboratory animal research of the u.s . national academy of sciences argue that even sophisticated computers are unable to model interactions between molecules , cells , tissues , organs , organisms and the environment , making animal research necessary in many areas.2 animal studies have been used in the past and shall continued to be used in the future for a number of applications in the field of orthopedics . human testing can not be done for any novel treatment unless adequate safety has been ensured . any new implant material should match various safety standards in terms of biocompatibility , mechanical stability , lack of local and systemic toxicity . in vitro studies are quintessential initial step in testing new implant materials but results from in vitro studies are hard to apply to the in vivo situation . many aspects of biocompatibility and safety like tissue reaction to implant , systemic toxicity , long term safety , late carcinogenicity , effect of controlled physiologic loading etc . , can not be tested in an in vitro situation.3 hence , extensive in vitro , as well as in vivo studies [ animal studies ] are essential , prior to clinical application in humans . animal studies bridge the knowledge gap between successful in vitro testing and safe human use . innumerable advances in the field of orthopedics and allied sciences have been critically dependent on animal experiments . advances in internal fixation of fractures , newer implant materials , chemotherapeutic drugs for various neoplastic conditions , use of stem cells in nonunion of fractures and cartilage defects etc . by law , any new drug has to pass through a series of toxicological analysis in animals before being introduced to clinical research and usage . the results from animal studies can be extrapolated to human since the internal milieu of humans are similar to animals.5 after a successful in vitro tests of a new treatment strategy [ stem cells , drugs etc . ] , it has to show its effect in an animal model before it is introduced into clinical research . healing of bone gaps by stem cells , characterization of new implant materials all have to undergo animal trials prior to human application . healing potential of tissue engineered stem cells , chondrogenic construct has to be tested in cartilage defect model in rabbits or dogs to demonstrate its efficacy prior to human trials.6 biodegradable materials are analyzed in vitro and in vivo to determine various characteristics of the material like rate of degradation , enzymatic reactions , immunological reactions , effect of physiological loading etc . , larger animals [ sheep , goat ] are used for testing of joint replacement components and surface and to study the innovative methods of fixation of bone fragments . an ideal animal model is one in which the anatomy and physiology match the specific study design . the pathogenesis and disease progression should parallel that of humans and so should the histopathology response.4 the in vivo milieu should closely mimic the human clinical situation . invertebrate animals are preferred over vertebrate animals for experiments ; however , commonly used animals for experiments are rats , mice , rabbits , goats , sheep , pigs , dogs , cats and primates.7 rabbits are commonly used animal in tissue engineering research and in orthopedic practice . they are vertebrate animals with an appropriate size for surgical operation , hence are suitable for studies on healing of gap nonunion in bones , repair of damaged articular cartilage , reconstruction of ligaments and tendons , and spinal fusion.811 rat is also in use for tissue engineering research due to its low cost and easier maintenance.4 it is used for studies on fracture healing,12 biocompatibility studies.13 and repair of bone defects.14 mouse , a small rodent has been extensively been used as its genome can be easily manipulated and because of its ease of handling . regular mice or immuno compromised nude mice have been used in studies on bone15 and cartilage healing.16 generally , larger animals impose more housing and handling difficulties than small animals such as rats and rabbits , and are more expensive . the factors involved in selection of the animal model includes the study design , cost , ability of the facility to handle the animals , resistance of the animal to diseases [ specially in long term studies ] and transportation . an ideal animal model is one in which the anatomy and physiology match the specific study design . the pathogenesis and disease progression should parallel that of humans and so should the histopathology response.4 the in vivo milieu should closely mimic the human clinical situation . invertebrate animals are preferred over vertebrate animals for experiments ; however , commonly used animals for experiments are rats , mice , rabbits , goats , sheep , pigs , dogs , cats and primates.7 rabbits are commonly used animal in tissue engineering research and in orthopedic practice . they are vertebrate animals with an appropriate size for surgical operation , hence are suitable for studies on healing of gap nonunion in bones , repair of damaged articular cartilage , reconstruction of ligaments and tendons , and spinal fusion.811 rat is also in use for tissue engineering research due to its low cost and easier maintenance.4 it is used for studies on fracture healing,12 biocompatibility studies.13 and repair of bone defects.14 mouse , a small rodent has been extensively been used as its genome can be easily manipulated and because of its ease of handling . regular mice or immuno compromised nude mice have been used in studies on bone15 and cartilage healing.16 generally , larger animals impose more housing and handling difficulties than small animals such as rats and rabbits , and are more expensive . the factors involved in selection of the animal model includes the study design , cost , ability of the facility to handle the animals , resistance of the animal to diseases [ specially in long term studies ] and transportation . before an animal experiment is designed there must be reasonable expectation that it will significantly advance the present knowledge and understanding about the subject and lead to improvement in care.5 public opinion polls have consistently shown that a majority of people approve the use of animals in biomedical research that does not cause pain to the animal and leads to new treatments and cures.2 animals should be used in research only when there are no suitable alternatives . the designing of research projects should be such that minimal required number of animals should be used and in a manner that conform to established standards of animal care . animal care and ethical committee should be formed in all institutes and its due approval should be made essential prior to starting the animal studies . many national bodies have been formed in various countries for humane use and care of animals used for experiments through recognition of ethical principles and scientific responsibilities . international council for laboratory animal science ( iclas ) , with a membership of about 100 countries is the apex body . the iclas draws guidelines on animal husbandry , experimental procedures , teaching and training of researchers and professionals in the field world over.17 iclas is dedicated to advancing human and animal health by promoting the ethical care and use of laboratory animals in research worldwide . iclas promote high standards of animal care and use in education , research , testing and diagnosis , to promote good science and foster humane practices in scientific research . the aims of iclas are compatible with the highest possible standards of animal research internationally . indian national science academy ( insa ) issued such guidelines in 1992 and recently revised it in 2000 . indian council of medical research ( icmr)5 also set up a unit called laboratory animal information service ( lais ) at the indian cancer research centre now called cancer research institute , mumbai . this unit subsequently got shifted to national institute of nutrition , hyderabad , wherein in 1986 it received additional support from department of biotechnology , and developed into a national centre for laboratory animal sciences ( nclas ) . india enacted its first animal law as early as 1960 , called the prevention of cruelty to animals act. it was subsequently amended in 1982 , which provided for the prevention of cruelty to animals in general . chapter 4 of act deals with control of experimentation on animals . the act also empowers committee formed under it to take care of the legal and ethical aspects of experimental animals being used in research and enact preventive actions when there is violation of the law.5 the ministry of social justice and empowerment , new delhi , issued a notification18 on fifteenth february , 2001 , where some guidelines were defined . in this guidelines , the word experiment means any program / project involving use of an animal for the acquisition of knowledge of a biological , psychological , ethological , physical or chemical nature . the establishment on registration for the purpose of performing experiments on animals shall comply with the conditions as may be specified at the time of registration by the committee and every registered establishment shall maintain a register of particulars about the animals used for conducting experiments . a detailed specifications for housing , feeding and maintenance of various species to be used in animal experimentation as notified by the committee , it also specifies that the experiments shall be performed in every case by or under the supervision of a person duly qualified , that is , degree holders in medicine or veterinary science , post graduate and above in life sciences / pharmaceutical sciences or any other natural sciences , degree or diploma holders in pharmacy , diploma or certificate in laboratory animal techniques sciences from a recognized institution as identified by committee . it also mentions that experiments shall not be performed for the sole purpose of attaining or retaining manual skill . the committee for the purpose of control and supervision on experiments on animals ( cpcsea ) guidelines19 are well defined and is a must read document for any one likes to carry out research with animal facilities . it provides the guidelines regarding the animal procurement , animal care , requirement of physical facilities and the environmental issues . the guidelines also deal with the issues of their food , water , sanitation , and transportation . the guidelines regarding anesthesia , standard operating procedures , euthanasia are helpful for any orthopedic surgeon or research institutions if they wish to carry the research using the animals . in order to reduce the impact of research on animals , the principles of three rs as advocated by russel and burch ( 1959)20 are still considered as good laboratory practice . refining the experiment to reduce their suffering and whenever possible , replacing experiments on animals with alternative techniques . in india animals the animal experimentations are in primitive stage in india due to poor infrastructure in most institutes , lack of expertise in handling animals , dearth of adequate funding and lack of interest in basic research among orthopedic surgeons . most experiments involve small laboratory - bred animals and are approved by the institutional animal ethics committee ( iaec ) . these committees are formed as per the guidelines of the indian national science academy , and include a nominee of the cpcsea . the current rules ( 2000 ) empowers iaecs to allow experiments only on small laboratory bred animals- guinea pigs , rabbits , rats , mice , hamsters , and invertebrates.21 for all other animals , permission must be sought from a subcommittee of the cpcsea.19 the medical council of india ( 2010 ) has allowed medical colleges to use software instead of animals for teaching and training undergraduate students . the condition can be improved by more focus on animal experiments at medical colleges , better awareness about need and methodology of animal experiments , more funding , stringent laws regarding unnecessary harm to animals and their strict implementation . though animal experiments have contributed immensely towards our understanding of various disease processes and in developing better treatment methods , the opposition to use of animals for research purposes has always existed . organizations like peta [ people for ethical treatment of animals ] , ifaw [ international fund for animal welfare ] etc . , have protested against animal experiments . appropriate permissions from animal welfare boards , maintenance of highest standards of care of animals and being abreast with the current law on animals and their use is essential to avoid trouble from the animal rights activists . the set up for conducting animal studies is costly and faculty must be trained in handling animals well . in the west , 1 or 2 year diploma courses are available to train biology , medical and veterinary graduates . in india , short term courses ranging from six to twelve weeks are run by national bodies for those involved in animal husbandry and animal studies . national centre for laboratory animal sciences ( nclas ) at hyderabad under the icmr and central drug research institute ( cdri ) , lucknow under the council for scientific and industrial research ( csir ) host such training programs on a regular basis.5 the animal experiments are relevant to an average orthopedic surgeon . an average orthopedic surgeon may not come into direct contact with animal studies but he should know the importance of animal studies in his day to day practice . new drugs for osteoporosis , osteoarthrosis , antibiotics , cancer chemotherapy etc . , have been derived from animal research . new implant materials for fracture fixation , bearing surfaces for joint replacement , biodegradable interference screws for ligament reconstruction , stem cell therapy for nonunion of bones are only a few examples to show the bearing animal studies have on the day to day practice of an average orthopedic surgeon . thus , all of us as the orthopedic community should be aware of the issues associated with animal studies , recent developments , legal and ethical aspects associated with the same . all of us should pay due respect and be deeply indebted to the animal kingdom , our fellow species for making our daily practice of orthopedics so much more better and this world a much better place to live in . many national bodies have been formed in various countries for humane use and care of animals used for experiments through recognition of ethical principles and scientific responsibilities . international council for laboratory animal science ( iclas ) , with a membership of about 100 countries is the apex body . the iclas draws guidelines on animal husbandry , experimental procedures , teaching and training of researchers and professionals in the field world over.17 iclas is dedicated to advancing human and animal health by promoting the ethical care and use of laboratory animals in research worldwide . iclas promote high standards of animal care and use in education , research , testing and diagnosis , to promote good science and foster humane practices in scientific research . the aims of iclas are compatible with the highest possible standards of animal research internationally . indian national science academy ( insa ) issued such guidelines in 1992 and recently revised it in 2000 . indian council of medical research ( icmr)5 also set up a unit called laboratory animal information service ( lais ) at the indian cancer research centre now called cancer research institute , mumbai . this unit subsequently got shifted to national institute of nutrition , hyderabad , wherein in 1986 it received additional support from department of biotechnology , and developed into a national centre for laboratory animal sciences ( nclas ) . india enacted its first animal law as early as 1960 , called the prevention of cruelty to animals act. it was subsequently amended in 1982 , which provided for the prevention of cruelty to animals in general . chapter 4 of act deals with control of experimentation on animals . the act also empowers committee formed under it to take care of the legal and ethical aspects of experimental animals being used in research and enact preventive actions when there is violation of the law.5 the ministry of social justice and empowerment , new delhi , issued a notification18 on fifteenth february , 2001 , where some guidelines were defined . in this guidelines , the word experiment means any program / project involving use of an animal for the acquisition of knowledge of a biological , psychological , ethological , physical or chemical nature . the establishment on registration for the purpose of performing experiments on animals shall comply with the conditions as may be specified at the time of registration by the committee and every registered establishment shall maintain a register of particulars about the animals used for conducting experiments . a detailed specifications for housing , feeding and maintenance of various species to be used in animal experimentation as notified by the committee , shall be adhered to by the registered establishment . it also specifies that the experiments shall be performed in every case by or under the supervision of a person duly qualified , that is , degree holders in medicine or veterinary science , post graduate and above in life sciences / pharmaceutical sciences or any other natural sciences , degree or diploma holders in pharmacy , diploma or certificate in laboratory animal techniques sciences from a recognized institution as identified by committee . it also mentions that experiments shall not be performed for the sole purpose of attaining or retaining manual skill . the committee for the purpose of control and supervision on experiments on animals ( cpcsea ) guidelines19 are well defined and is a must read document for any one likes to carry out research with animal facilities . it provides the guidelines regarding the animal procurement , animal care , requirement of physical facilities and the environmental issues . the guidelines also deal with the issues of their food , water , sanitation , and transportation . the guidelines regarding anesthesia , standard operating procedures , euthanasia are helpful for any orthopedic surgeon or research institutions if they wish to carry the research using the animals . in order to reduce the impact of research on animals , the principles of three rs as advocated by russel and burch ( 1959)20 are still considered as good laboratory practice . refining the experiment to reduce their suffering and whenever possible , replacing experiments on animals with alternative techniques . the animal experimentations are in primitive stage in india due to poor infrastructure in most institutes , lack of expertise in handling animals , dearth of adequate funding and lack of interest in basic research among orthopedic surgeons . most experiments involve small laboratory - bred animals and are approved by the institutional animal ethics committee ( iaec ) . these committees are formed as per the guidelines of the indian national science academy , and include a nominee of the cpcsea . the current rules ( 2000 ) empowers iaecs to allow experiments only on small laboratory bred animals- guinea pigs , rabbits , rats , mice , hamsters , and invertebrates.21 for all other animals , permission must be sought from a subcommittee of the cpcsea.19 the medical council of india ( 2010 ) has allowed medical colleges to use software instead of animals for teaching and training undergraduate students . the condition can be improved by more focus on animal experiments at medical colleges , better awareness about need and methodology of animal experiments , more funding , stringent laws regarding unnecessary harm to animals and their strict implementation . though animal experiments have contributed immensely towards our understanding of various disease processes and in developing better treatment methods , the opposition to use of animals for research purposes has always existed . organizations like peta [ people for ethical treatment of animals ] , ifaw [ international fund for animal welfare ] etc . , have protested against animal experiments . appropriate permissions from animal welfare boards , maintenance of highest standards of care of animals and being abreast with the current law on animals and their use is essential to avoid trouble from the animal rights activists . the set up for conducting animal studies is costly and faculty must be trained in handling animals well . in the west , 1 or 2 year diploma courses are available to train biology , medical and veterinary graduates . in india , short term courses ranging from six to twelve weeks are run by national bodies for those involved in animal husbandry and animal studies . national centre for laboratory animal sciences ( nclas ) at hyderabad under the icmr and central drug research institute ( cdri ) , lucknow under the council for scientific and industrial research ( csir ) host such training programs on a regular basis.5 the animal experiments are relevant to an average orthopedic surgeon . an average orthopedic surgeon may not come into direct contact with animal studies but he should know the importance of animal studies in his day to day practice . new drugs for osteoporosis , osteoarthrosis , antibiotics , cancer chemotherapy etc . new implant materials for fracture fixation , bearing surfaces for joint replacement , biodegradable interference screws for ligament reconstruction , stem cell therapy for nonunion of bones are only a few examples to show the bearing animal studies have on the day to day practice of an average orthopedic surgeon . thus , all of us as the orthopedic community should be aware of the issues associated with animal studies , recent developments , legal and ethical aspects associated with the same . all of us should pay due respect and be deeply indebted to the animal kingdom , our fellow species for making our daily practice of orthopedics so much more better and this world a much better place to live in .

in vivo studies using the animals are helpful in developing the treatment strategies as they are important link between the successful in vitro testing and safe human use . various research projects in the field of fixation of fractures , development of newer biomaterials , chemotherapeutic drugs , use of stem cells in nonunion of fractures and cartilage defects etc . , have hugely depended on animal experimentation . the employment of animals in experiments is both scientific and ethical issue . there must be reasonable reasons to show that it will significantly advance the present knowledge and lead to improvement in care . the regulatory bodies exist for humane use and care of animals used for experiments e.g. , international council for laboratory animal science , council for international organizations of medical sciences , international union of biological sciences , international committee on laboratory animals . in india , indian national science academy , indian council of medical research , national centre for laboratory animal sciences promote high standards of laboratory animal quality , care and health . the committee for the purpose of control and supervision on experiments on animals guidelines are well defined and is a must read document for any one interested to carry out research with animal facilities .

I S Selection of an animal model E Guidelines for care of animals The Indian scenario Challenges in the field

these tumors are rarely reported in infants . these lesions present as slowly progressive masses that can either compress to the adjacent vital structures or interfere with normal physiology . we report a case of giant , solitary nf presenting as a progressive mass in parotid region in an infant and review the relevant literature . an 11-month - old female child was admitted in surgery with a progressively enlarging swelling in the right parotid region of two months duration . there was a large ( 7 8 cm ) , firm , non - tender mass present in right parotid and infra - temporal region with a normal overlying skin [ figure 1 ] . there were no external features suggestive of nf-1 / nf-2 like macules , lich nodules . her hemoglobin was 12 gm % and total leucocyte count and erythrocyte sedimentation rate ( esr ) were 6200 / cm m and 20 respectively . the routine urine examination , c - reactive protein , and kidney function test were also within normal limits . routine x - ray and ultrasonography ( usg ) of her chest and abdomen and computed tomography ( ct ) scan of head were normal . the doppler study of the neck showed a homogenous and hyper - vascular solid mass , situated within the subcutaneous plane of right parotid region . ct with contrast suggested a large well defined homogenous , enhancing mass reaching up to the lateral pharyngeal wall without infiltrating the surrounding structures [ figure 2 ] . fine needle aspiration cytology ( fnac ) of the neck was inconclusive . in view of persisting confusion over the diagnosis , the tumor was exposed and excised completely with extended blair 's incision [ figure 3 ] . on exploration the tumor was situated within the subcutaneous plane and the parotid gland was shifted superiorly and well separated from the mass . facial nerve and its branches the patient is doing well at two - year follow - up . clinical photograph showing large mass in the right parotid region ct scan showing details of the extent of mass and the lesion was enhancing after contrast administration intra - operative photograph well defi ned plane of cleavage and complete excision of the tumor histopathology showing features of neurofi broma ( h & e , 40 ) schwannomas or nfs are most common with a common origin the schwann cell . in the head and neck , about 25% of all nfs are found but solitary nfs are rare in this region . generally nfs show autosomal dominant pattern , occurring either due to spontaneous mutation or from the passing of infected gene with complete penetrance and variable expression . they are well - differentiated , non - encapsulated nerve sheath tumor , composed of shawn cells , perineural - like cells , fibroblasts , and transitional cells and arise within the endo - neurium . the nerve fibers are incorporated within the tumor therefore can not be separated and resection may sacrifice the nerve of origin . nfs have to be differentiated from schwannomas and malignant peripheral nerve sheath tumors ( mpnst ) . schwannomas are encapsulated tumors of nerve sheath and grow eccentrically without infiltrating the nerve and may be dissected out successfully from the nerve . they have a typical palisade pattern of the nuclei , and tumor cell density is higher compared with that in nfs . mitosis and a mucoid extra cellular matrix with only scattered tumor cells ( the indicator of malignancy ) are common in schwannoma . histopathologicaly , type - a tissue of antoni ( the narrow cylinders with tapering ends ) - compact and composed bundles of long bipolar spindle cells with oval or rod - shaped nuclei and type b tissue - a loose texture ; and polymorphism of the tumor cells are highly specific structures of neurilemomas . nfs are seen to be slowly growing asymptomatic soft tissue masses and often do not have any neurological deficits . the diagnostic workup includes ruling out the presence of other nfs and any possibility of neurofibromatosis nf1 and nf2 . the diagnostic criteria for nf1 includes family history , six or more caf - au - lait macules , neurofibromas , lisch nodules , tumor on the optic nerve and skeletal abnormalities , of which two must be present in a patient to be diagnosed as nf1 . the diagnostic criteria for nf2 is bilateral vestibular schwannomas ( vs ) or family history of nf2 ( first degree family relative ) plus unilateral vs < 30 years or any two of the meningioma , glioma , schwannoma and juvenile posterior sub capsular lenticular opacities/ juvenile cortical cataract . usg is not very specific and both schwannomas and nfs are commonly homogeneous and hypo echoic and produce posterior acoustic enhancement . ct with contrast in nfs usually shows an ovoid , well - demarcated , homogeneous and iso - attenuation relative to muscle . heterogeneous contrast enhancement , as demonstrated in the present case , has been described to be typical for nfs . immuno - histochemically , many of the spindle cells are positive for common neural markers , with patchy staining for cd34 and epithelial membrane antigen . immuno - reactivity for s-100 protein , neuron - specific enolase , and vimentin is characteristic for nfs . the transformation of a nf into a mpnst has been observed in 2 - 5% of cases with nf-1 . to summarize , solitary nfs are rare in infants , especially in head and neck region . they may mimic parotid tumor and even with careful evaluation preoperative diagnosis may be difficult . the present case is unique in its age of presentation ( 11 months ) , site and size ( about 8 15 cm ) , without any symptoms or neurological deficit and could be managed successfully with good outcome .

solitary neurofibroma is a rare tumor of the head and neck region . it is more common in viscera , where it is associated with features of nf1 . it occurs most often between the third and fourth decade . these lesions are extremely rare in infants . we report a case of giant , solitary neurofibroma presenting as a progressive mass in parotid region in an infant . this case is unique in its age of presentation ( 11 months ) , site , size ( about 8 15 cm ) without any symptoms or neurological deficit .

Introduction Case Report Discussion

advances in our understanding of the genetic alterations underlying clear cell renal cell carcinoma ( rcc ) has led to a proliferation of clinically active agents for this disease , and since 2005 , a total of seven agents ( sorafenib , sunitinib , temsirolimus , bevacizumab , everolimus , pazopanib , and axitinib ) have been approved for the treatment of metastatic kidney cancer . while kidney cancer was once considered to be a single entity , extensive research over the past 3 decades has resulted in classification of rcc into distinct subtypes based on disparate genetic and molecular alterations . most of the fda approved agents have been tested in a fairly uniform patient population with predominantly clear cell tumors . extrapolating the benefit of these agents to the other common subtypes of kidney cancer including papillary and chromophobe tumors , while common in clinical practice , is not supported by robust scientific and clinical evidence . thus , clinical trial participation should be encouraged for patients with non - clear cell histology , a recommendation endorsed by the nccn.1 several small phase ii trials have looked at the use of targeted agents in the patients with papillary and other non - clear cell variants of rcc and have not demonstrated an overwhelming benefit.2,3 the clinical utility of inhibitors of the vegf or mtor pathways in patients with chromophobe rcc remains unclear with limited evidence acquired from retrospective analyses and expanded access trials . choueiri and colleagues described their experience with vegf pathway antagonists in a multi - institutional , retrospective series , reporting 3 partial responses in 12 patients.4 data from the expanded access program for sorafenib also suggests some activity in chromophobe rcc as 90% of patients ( n=18 ) had some measure of disease control ( 1 partial response and 17 stable disease).5 for mtor agents , the results are more uncertain with no large case series or prospective studies available . post hoc subgroup analysis of the arcc study with temsirolimus demonstrated a potential benefit in non - clear cell rcc ; however the incidence of chromophobe rcc in the non - clear group is not reported.6 in order to better understand the potential role of mtor , we evaluated the literature and our institutional experience with patients with metastatic chromophobe rcc . additionally we sought to better understand the burden of metastatic chromophobe rcc in the united states using the surveillance epidemiology and end result ( seer)-17 program . we reviewed the literature for published reports of chromophobe rcc treated with mtor therapy from 2006 - 2011 . all manuscripts were reviewed for patients with metastatic chromophobe rcc who had received systemic ' targeted therapy ' including sirolimus , everolimus , or temsirolimus . reports selected had details outlining the agent used , indication , line of therapy , prior treatment , response , and patient outcome . our institutional database was reviewed to determine cases of metastatic chromophobe rcc from 2006 - 2011 treated with mtor therapy . only one patient was identified and we reviewed the patient 's presentation , surgical management , systemic therapy , and clinical response . a brief review of the surveillance epidemiology and end result ( seer)-17 program provided an estimate of the deaths due to chromophobe rcc each year . the seer program now records cancer statistics from approximately 28% of the united states population . from 2003 to 2008 , all patients listed as having cancer of the kidney and renal pelvis were selected . cases with icd - o-3 histology codes believed to represent rcc ( 8140 , 8260 , 8270 , 8290 , 8310 , 8312 , 8316 , 8317 - 8320 ) were chosen for further review . patients dying of rcc from 2003 - 2007 were reviewed to determine the number of deaths attributed to chromophobe rcc ( histology codes 8270 and 8317 ) . the annual death rates per 100,000 was calculated for rcc overall and for chromophobe histology using seer*stat ( version 7.05 ) . review of our institutional database revealed a single patient with metastatic chromophobe rcc treated with targeted therapy . in february , 2000 , a 45 year - old woman presented with fatigue , weight loss , and a large palpable abdominal mass . a ct scan of the abdomen demonstrated an 18 cm left renal mass ( figure 1 ) . she underwent a left sided radical nephrectomy and final pathology demonstrated a t2 , chromophobe renal cell carcinoma ( rcc ) with areas of sarcomatoid transformation . she was followed closely with periodic surveillance and was free of disease for 6 years . in june 2006 , she presented with multiple small liver nodules ; laparoscopic resection of a hepatic lesion confirmed the presence of metastatic chromophobe rcc . once recovered , she was placed on sunitinib ( 50 mg orally on a four - weeks on , two - weeks off schedule ) and had regression in some of the liver lesions . following five cycles ( approximately 7 months ) , she demonstrated progressive disease , with enlargement of several liver lesions ( largest approximately 2.5 cm ) . sunitinib was discontinued after june of 2007 and temsirolimus was recommended ( 25mg / kg iv every week ) . the patient was unable to promptly initiate therapy and started temsirolimus around the time of repeat imaging in october 2007 at which time the largest liver lesion had increased to 3.2 cm . on therapy , multiple liver lesions regressed , and the patient achieved a partial response per recist . in february 2008 , a chest ct obtained for restaging revealed mild interstitial changes . however , the patient had no accompanying symptoms and pulmonary function testing was normal ; temsirolimus was therefore continued with close monitoring for respiratory symptoms . during the next two years the liver lesions remained stable on therapy . however , in august , 2009 , she developed several small bilateral sub - centimeter pulmonary nodules , consistent with lung metastases . fortunately , the lung and liver lesions have remained fairly stable with minimal interval growth of some lesions . currently , she remains on temsirolimus with excellent performance status , alive 5 years after the development of metastatic disease . figure 2 summarizes her treatment history and clinical course . in the past year , a small number of interesting case reports have described the activity of mtor agents in chromophobe rcc ( table 1).7 - 9 the first report from paule and colleagues described a 57 year - old man who had an early retroperitoneal recurrence after a left radical nephrectomy for a t2 fuhrman grade 2 - 3 chromophobe rcc.8 the patient had initial treatment with subcutaneous interferon - alpha followed by sorafenib but eventually progressed . temsirolimus therapy lead to disease stabilization for 26 months and the patient had a partial resection of the retroperitoneal mass . the last imaging study showed a significant response of the residual retroperitoneal mass per author assessment . zardavas and colleagues report a case of a 27 year - old female who underwent a radical nephrectomy for a t2n1m0 chromophobe rcc.9 the patient presented three years later with metastatic disease in the liver and received sunitinib and then sorafenib therapy without an objective response . the patient was placed on temsirolimus and had a partial response by recist criteria and remains on therapy after a year with excellent performance status . the final report from larkin and colleagues described a 53 year - old man who underwent a right radical nephrectomy for a 6 cm , t3a , fuhrman grade iii chromophobe rcc.7 the patient was found to have an unresectable retroperitoneal recurrence three years later and was placed on sunitinib . after a partial response everolimus was started and the patient experienced a partial response by recist criteria , still remaining on mtor therapy after 2 years . from 2003 - 2008 , the annual adjusted death rate per 100,000 persons for rcc overall ranged from 2.670 to 3.091 . for chromophobe histology , the death rate was extremely low , ranging from 0.015 to 0.034 per 100,000 persons ( figure 3a ) . the number of seer cases of deaths from chromophobe rcc identified was between 4 - 9 cases / year and accounted for approximately 1% of total deaths from rcc ( figure 3b ) . however , based on this data , the number of total chromophobe deaths a year in the us population would be roughly 14 - 32 per year . review of our institutional database revealed a single patient with metastatic chromophobe rcc treated with targeted therapy . in february , 2000 , a 45 year - old woman presented with fatigue , weight loss , and a large palpable abdominal mass . a ct scan of the abdomen demonstrated an 18 cm left renal mass ( figure 1 ) . she underwent a left sided radical nephrectomy and final pathology demonstrated a t2 , chromophobe renal cell carcinoma ( rcc ) with areas of sarcomatoid transformation . she was followed closely with periodic surveillance and was free of disease for 6 years . in june 2006 , she presented with multiple small liver nodules ; laparoscopic resection of a hepatic lesion confirmed the presence of metastatic chromophobe rcc . once recovered , she was placed on sunitinib ( 50 mg orally on a four - weeks on , two - weeks off schedule ) and had regression in some of the liver lesions . following five cycles ( approximately 7 months ) , she demonstrated progressive disease , with enlargement of several liver lesions ( largest approximately 2.5 cm ) . sunitinib was discontinued after june of 2007 and temsirolimus was recommended ( 25mg / kg iv every week ) . the patient was unable to promptly initiate therapy and started temsirolimus around the time of repeat imaging in october 2007 at which time the largest liver lesion had increased to 3.2 cm . on therapy , multiple liver lesions regressed , and the patient achieved a partial response per recist . in february 2008 , a chest ct obtained for restaging revealed mild interstitial changes . however , the patient had no accompanying symptoms and pulmonary function testing was normal ; temsirolimus was therefore continued with close monitoring for respiratory symptoms . during the next two years the liver lesions remained stable on therapy . however , in august , 2009 , she developed several small bilateral sub - centimeter pulmonary nodules , consistent with lung metastases . fortunately , the lung and liver lesions have remained fairly stable with minimal interval growth of some lesions . currently , she remains on temsirolimus with excellent performance status , alive 5 years after the development of metastatic disease . in the past year , a small number of interesting case reports have described the activity of mtor agents in chromophobe rcc ( table 1).7 - 9 the first report from paule and colleagues described a 57 year - old man who had an early retroperitoneal recurrence after a left radical nephrectomy for a t2 fuhrman grade 2 - 3 chromophobe rcc.8 the patient had initial treatment with subcutaneous interferon - alpha followed by sorafenib but eventually progressed . temsirolimus therapy lead to disease stabilization for 26 months and the patient had a partial resection of the retroperitoneal mass . the patient resumed therapy and remained on treatment for over two years . the last imaging study showed a significant response of the residual retroperitoneal mass per author assessment . zardavas and colleagues report a case of a 27 year - old female who underwent a radical nephrectomy for a t2n1m0 chromophobe rcc.9 the patient presented three years later with metastatic disease in the liver and received sunitinib and then sorafenib therapy without an objective response . the patient was placed on temsirolimus and had a partial response by recist criteria and remains on therapy after a year with excellent performance status . the final report from larkin and colleagues described a 53 year - old man who underwent a right radical nephrectomy for a 6 cm , t3a , fuhrman grade iii chromophobe rcc.7 the patient was found to have an unresectable retroperitoneal recurrence three years later and was placed on sunitinib . after a partial response everolimus was started and the patient experienced a partial response by recist criteria , still remaining on mtor therapy after 2 years . from 2003 - 2008 , the annual adjusted death rate per 100,000 persons for rcc overall ranged from 2.670 to 3.091 . for chromophobe histology , the death rate was extremely low , ranging from 0.015 to 0.034 per 100,000 persons ( figure 3a ) . the number of seer cases of deaths from chromophobe rcc identified was between 4 - 9 cases / year and accounted for approximately 1% of total deaths from rcc ( figure 3b ) . however , based on this data , the number of total chromophobe deaths a year in the us population would be roughly 14 - 32 per year . unlike clear cell rcc , the natural history of metastatic chromophobe rcc is not well characterized and therefore , it may be difficult to quantify the therapeutic benefit associated with targeted therapeutic agents in this population . several series investigate the survival of patients with metastatic chromophobe rcc and survival appears to vary widely with median survival ranging from 7 to 29 months . 10,11 the low incidence of metastatic chromophobe rcc , the limited insight into disease biology , and the fact that most targeted agents were mainly developed for and evaluated in patients with clear cell rcc are some factors that make characterization of the natural history and development of a standard therapeutic approach for this condition challenging.12 a better understanding of the biology of chromophobe rcc can enhance our ability to develop rational targeted strategies directed against specific pathways thought to contribute to this disease . understanding the biochemical consequences of vhl loss in hereditary and subsequently , sporadic forms of clear cell rcc was a critical step in the development of both vegf- and mtor - pathway antagonists.13 similarly , emerging data from studies of a hereditary form of chromophobe rcc , the birt - hogg - dube syndrome ( bhd ) , may shed light on the biology of the sporadic form of the disease . patients with bhd are at risk for the development of bilateral , multifocal renal tumors and have germline inactivating mutations in the flcn gene . hasumi and colleagues from our institution demonstrated that folliculin loss leads to development of tumors with highly active pi3k / mtor pathways.1 baba and colleagues have demonstrated that mtor inhibition with rapamycin could reverse the phenotype of polycystic kidneys and renal failure in a conditional flcn knockout mouse model.14 although the role of folliculin in non - hereditary forms of chromophobe rcc remains to be fully elucidated , these data provide a reasonable basis for the evaluation of the mtor pathways and inhibitors of the pi3k / akt / mtor axis in chromophobe rcc . moving forward , it may be nearly impossible to conduct a large randomized trial with metastatic chromophobe patients due to the rarity of this disease , as demonstrated by our seer analyses . a reasonable approach might be to investigate agents whose use in chromophobe rcc is supported by preclinical studies , in carefully designed , well - coordinated multi - center phase 2 studies . studies including patients with all types of non - clear cell histologies may also provide insight , as long as subtype stratification is performed . one such trial , aspen , a phase ii trial of everolimus vs. sunitinib in metastatic , non - clear cell tumors , may provide some data on the activity of these agents in chromophobe rcc ( nci trial i d : nct01108445 ) . in conclusion , we support the evaluation of mtor - directed therapy in chromophobe rcc with cautious enthusiasm ; this approach is supported by both interesting anecdotal evidence and preclinical rationale . due to the rarity of this disease , uncertainty about the biology and disease course , and lack of a coordinated approach to the management of this and other rare entities , there are many unanswered questions . we hope institutions across the globe will work together to determine the optimal systemic treatment for this challenging kidney cancer population .

chromophobe kidney cancer accounts for approximately 5% of cases of renal cell carcinoma ( rcc ) . while the genetics of clear cell rcc has been a major focus of research , little is known about the biology of chromophobe tumors . there is ample preclinical rationale for the use of targeted therapy in clear cell tumors , and agents targeting the vhl / hif pathway are now widely used in clinical practice . however , there is limited experience with targeted agents in non - clear cell tumors . recently , a few case reports have emerged which report the use of mtor inhibitors in chromophobe tumors . here , we report our experience with targeted therapy in a patient with advanced chromophobe rcc who had a durable partial response to temsirolimus . we also include a literature review summarizing the published experience with targeted therapeutic approaches in chromophobe rcc . additionally , the preclinical rationale for the use of mtor inhibitors in this population based on our characterization of the hereditary form of chromophobe kidney cancer , birt - hogg - dube syndrome , is discussed .

Introduction Methods Results Case Report Published Reports SEER Population Estimates Discussion

silver is a naturally occurring element with various physical properties , such as high thermal and electrical conductivity , ductility , hardness , malleability , and even antimicrobial ability . silver was known and used by ancient people as early as 4000 bc , along with gold and copper.1 silver now has a wide variety of applications , including in photography , soldering , metallurgy , medicine and dentistry , water sanitation , jewelry , coins , silverware , and mirrors.2,3 silver nitrate ( agno3 ) is a soluble silver salt used in medications to treat intestinal and oral ulcers as well as to control gingival bleeding . another relatively common use of silver nitrate is water purification , as in the case reported here . silver - containing compounds should be used with caution , given the risk of toxicity.3 generalized argyria is a rare dermatological disorder caused by silver granule deposits in the skin and mucous membranes , due to chronic exposure of patients to silver - containing compounds.4 persistent respiratory , topical , or oral exposure can lead to deposition of silver in the eye and periocular tissue , and a condition known as ocular argyrosis.5 this pathology has been described previously.58 the clinical presentation includes gray - blue discoloration of the periorbital skin , bulbar and palpebral conjunctiva , and lacrimal sac , as well as cornea opacities and cataracts.58 the latter are the main causes of specialist referrals.8 findings at the posterior pole include a leopard spot pattern of the macula and a dark choroid , observed by means of red - monochromatic fundus pictures and fluorescein angiography , respectively.9 although there are no visual symptoms in most reported cases , there are a few case reports in which patients complained of nyctalopia.5 however , patients undergoing electrophysiologic testing did not demonstrate functional deficits in scotopic or photopic electroretinography , nor were there any abnormalities on color vision testing.5 in the following case report , we describe a patient who developed ocular argyrosis after chronic ingestion of silver nitrate in his personal water supply . in order to expand the available data regarding this condition an 86-year - old caucasian male with a previous medical history remarkable for normal pressure hydrocephalus after ventricular shunt placement , early dementia , and skin argyria ( 2 years ) was seen at the ophthalmology clinic , complaining of slowly decreasing visual acuity and nyctalopia in both eyes ( ou ) . according to the primary caretaker , the patient had been drinking water from a well and purifying it with silver nitrate compounds for a period of at least 13 years , on the basis of advice from an alternative medicine professional , who recommended their use as antimicrobial agents and as a water purification method . the patient was subsequently advised by dermatology to suspend his silver nitrate intake 2 years prior to presentation to us . during the initial consultation , the patient s appearance was remarkable for a generalized gray - bluish hue of the skin and fingernails , more pronounced on the face , periorbital area , and forearms ( figure 1 ) . best corrected visual acuity was right eye ( od ) 20/40 and left eye ( os ) 20/50 . there was no afferent pupillary defect and confrontational vision fields were full in both eyes . conjunctiva had a purple - blue hue , but were normally vascularized and showed no epithelial defect . the palpebral portion of the main lacrimal gland also had a purple - bluish hue . fundus examination was remarkable for retinal pigment epithelium ( rpe ) changes and drusen formation on macula ou , consistent with age - related macular degeneration ( figure 2 ) . fluorescein angiography showed choroidal blockage with completely dark choroid in later phases in both eyes . areas of dot hyperfluorescence in the macula were present , corresponding to the drusenoid deposits . spectralis domain optical coherence tomography showed multiple excrescences of rpe in both eyes , most likely representing drusen from age - related macular degeneration ( figure 3 ) . an electroretinogram was obtained because of the patient s symptoms of nyctalopia , and revealed normal scotopic and photopic amplitudes and latency , as well as normal oscillatory potentials and 30 hz flicker testing ( data not shown ) . in this case report , we describe the clinical and ancillary test findings in a patient with ocular argyrosis . it is important to mention that , although there are previous reports and clinical descriptions of the anterior segment alterations and periocular skin deposits , there are relatively few descriptions of involvement of the posterior pole . the interesting finding of dark choroid , which occurs when the normal choroidal background fluorescence is blocked , was made in our patient on fluorescein angiography . silent choroid is most commonly associated with stargardt s disease and other posterior retinal dystrophies.9 the pathophysiology in patients with silver accumulation which leads to this angiographic finding is not understood as yet . wislocki and ladman conducted an experiment in albino rats involving giving them water containing silver nitrate for a period of 6 months to a year and trying to demonstrate the blood - ocular barrier . they reported histopathologic data showing that silver accumulated in two regions of the eye , ie , the ciliary processes and bruch s membrane.10 they also noted that the longer silver nitrate was administered , the greater the amount that accumulated . spencer et al performed a histopathologic examination in a patient with systemic argyrosis and found that brown - black silver pigment was present in three locations of the eye , ie , bruch s membrane , bowman s membrane , and descemet s membrane , with the majority of deposition occurring in bruch s membrane.11 therefore , the question arises as to how systemic argyrosis causes findings of dark choroid on fluorescein angiography , as in our patient . one of the mechanisms proposed is that accumulation of silver pigment deposits in large amounts in bruch s membrane causes absorption of short - wavelength light during fluorescein angiography , which could be responsible for the phenomenon of dark choroid.911 furthermore , in the same study series , red light monochromatic pictures were taken , which utilize a long wavelength to bypass rpe and visualize the choroidal vasculature in normal subjects . however , the choroidal vasculature could not be visualized in any of the six cases from that study utilizing red light pictures , meaning that the long wavelengths were blocked as well . thus , it has been proposed that silver deposits block all light , from short to long wavelengths.911 given the results above , it is reasonable to question whether silver might accumulate not only in bruch s membrane but also in rpe cells . however , a previous histopathologic study in albino rats did not show any accumulation of silver deposits in rpe cells,10 and furthermore , the fundus autofluorescence images from our patient did not show any signs of rpe damage . to our knowledge , there is only one previous report describing choroidal and retinal changes in patients with ocular argyrosis by means of fundus photographs and fluorescein angiography.9 in this case report , we expand the available information regarding changes in the retina and rpe . our patient underwent noninvasive evaluation for retinal pigment abnormalities with fundus autofluorescence , which uses a confocal scanning laser ophthalmoscope to detect accumulations , typically of lipofuscin granules , in rpe cells . . ophthalmologist and retinal specialists should be aware that conditions other than stargardt s disease can cause findings of dark choroid on fluorescein angiography .

this case report describes the clinical , autofluorescence , and optical coherent tomography findings in a patient with panocular argyrosis secondary to chronic intake of diluted silver nitrate salts in his water supply . an 86-year - old caucasian male with a distinctive gray - bluish hue of the skin presented to our clinic , having developed a slow decrease in visual acuity in both eyes and nyctalopia for the past 2 years . based on the patient s history of chronic intake of silver nitrate salts and a positive skin biopsy ( performed by the dermatology department , data not shown ) , a diagnosis of panocular argyrosis was made . fluorescein angiography showed choroidal blockage with a completely dark choroid . fundus autofluorescence was within normal limits . optical coherent tomography showed multiple excrescences of retinal pigment epithelium in both eyes . although the drusen - like changes on fundus examination and retinal pigment epithelium changes may account for the diminished vision , the presence of concomitant nyctalopia suggests underlying damage of the photoreceptors .

Introduction Case report Discussion

schwannomas are infrequent tumors originating from the neural crest cells and usually interpreted as slow - growing benign nerve sheath tumors [ 1 , 2 ] . visceral sites of this tumor include the gastrointestinal tract , liver , pancreas , kidney , brain , heart and retroperitoneum . retroperitoneal schwannomas account for only 13% of all schwannomas and for only 1% of all retroperitoneal tumors . the literature reports schwannomas detected using computed tomography ( ct ) and magnetic resonance imaging ( mri ) , but we could not find any case of schwannoma detected using positron emission tomography ( pet)-ct imaging . herein , we report a case of large adrenal schwannoma which was diagnosed incidentally and review the cases previously reported in an attempt to clarify the inconsistent features of this rare disease . a 32-year - old female presented with new onset of intermittent right flank pain . during examination , a left adrenal mass was found incidentally on abdominal ultrasonography and she was referred to our institution for additional evaluation . her medical and family histories were unremarkable . on physical examination the abdomen was soft , blood pressure was 130/85 mm hg and pulse rate was 72 beats / min ; sinus rhythm was normal . a heterogeneously enhancing soft tissue mass on the medial aspect of the left adrenal gland measuring 65 95 mm was visualized and no additional mesenteric or retroperitoneal lymphadenopathy was identified on the abdominal ct scans . as the mass was approximately 10 cm in size , [ f ] fluorodeoxyglucose ( fdg ) pet - ct was also performed . it showed intense uptake within the left adrenal nodule with a standardized uptake value ( suvmax ) of 9.5 , indicative of malignancy ( fig . 1 ) . endocrinological examinations including serum electrolytes , cortisol , urinary metanephrine , normetanephrine , adrenalin , noradrenalin and vanillyl mandelic acid , as well as plasma renin and aldosterone were within normal range . also , a dexamethasone suppression test showed values within the normal range at onset and at 30 min . due to the large size of the tumor , standard transperitoneal adrenalectomy postoperative pathological evaluation revealed schwannoma in the adrenal gland , a hard , well - circumscribed , encapsulated mass 4 8 10 cm in overall dimensions that was grossly compressing the adrenal parenchyma . histologically , the tumor consisted of spindle cells with alternating areas of compact hypercellularity with irregular streams and without atypia or mitosis . immunohistochemical analysis demonstrated rare ki67-positive cells ( < 5% ) while sma , desmin , cd34 and cd117 staining was negative in tumor cells . in contrast to these results , s-100 and vimentin staining were diffusely positive across the tumor . thus , the evidences corresponded to a benign adrenal schwannoma type antoni a ( fig . schwannomas are slow - growing benign nerve sheath tumors whose principal components originate from crest cells , the vestibulocochlear nerve being the most frequent site [ 1 , 4 ] . they are most often found within the head , the neck as well as the upper and lower extremities , and with a lesser frequency on the trunk , in the gastrointestinal tract , liver , pancreas , kidney , brain , heart and retroperitoneum . these rare tumors account for only 15% of retroperitoneal masses and comprise only 13% of all schwannomas [ 6 , 7 , 8 ] . these tumors are usually characterized by a benign course , are encapsulated and vary from firm solitary masses to fluctuant cysts . schwannomas were initially described by verocay in 1908 ; after this description antoni had performed histological subclassification into two patterns . histologically , these tumors consist of compact hypercellularity with irregular streams ( antoni type a ) and loose hypocellularity with cystic spaces ( antoni type b ) . investigators believe that schwann cells of nerve fibers innervating the medulla are the source of these adrenal lesions [ 11 , 12 , 13 ] . functional adrenocortical tumors can be easily revealed by characteristic hormone - related symptoms , blood chemistry or urinary tests . however , non - functional adrenocortical tumors present no characteristic lesions . due to their non - secreting and asymptomatic properties , adrenal schwannomas are often diagnosed incidentally . moreover , increasing medical examination health screening and advances in imaging modalities give rise to an increased incidental detection of retroperitoneal tumors . ct scan shows a well - circumscribed , homogeneous , round - oval mass with cystic degeneration or calcification . mri findings are usually non - specific , but helpful in the differential diagnosis of solid non - functional tumors . solid tumors with a low signal intensity on t1wi and heterogeneously high intensity on t2wi are revealed on mri . fdg pet has previously been suggested as valuable in discriminating benign from malignant adrenal masses . the concept of fdg pet is based on an increased glucose uptake by malignant lesions . the quantitative analysis of fdg uptake is performed using standardized suvs or by qualitative visual evaluation with respect to liver uptake . the sensitivity of fdg - pet in identifying malignant lesions varied between 93 and 100% with a specificity between 80 and 100% [ 16 , 17 , 18 , 19 , 20 ] . the sensitivity of fdg pet imaging is only moderate , however , for the diagnosis of small lesions , and false - positive results have to be considered . in our case , the huge size of the tumor and the location and also the characteristics of the lesion on imaging were strongly suspicious of a malignant adrenal neoplasm . whereas some of the characteristics of retroperitoneal schwannomas , including heterogeneity and degeneration , may mimic malignancy on radiological interpretation , a preoperative diagnosis of adrenal schwannoma is almost impossible and usually should be confirmed by histopathological examination as seen in our patient . it should not be forgotten that the malignant form is frequently associated with von recklinghausen disease and neurofibromatosis . in consequence of significant risks , fine needle aspiration should be performed only in patients with a history of carcinoma or suspected metastasis . furthermore , physicians should consider the possibility of local recurrence and malignant formation in benign schwannomas despite a prior benign diagnosis . a non - secreting adrenal mass larger than 4 cm should be removed surgically with the added benefit of a definitive diagnosis . in cases of huge tumors as our case , the conventional approach can be preferred to laparoscopic resection . histologically , neoplastic cells that simulate the appearance of differentiated schwann cells organized as cellular areas with nuclear palisading ( antoni a ) and loose , hypocellular , myxoid lesions with microcystic spaces ( antoni b ) are seen . in our case the specimen consisted of spindle cells with alternating areas of compact hypercellularity with irregular streams and without atypia or mitosis . immunohistochemically , these lesions stain positively for s-100 antigen , vimentin , collagen iv and laminin and negatively for keratin , desmin , actin , cd34 and cd117 [ 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 23 ] . fine et al . demonstrated that a positive stain for calretinin , the same protein family as s-100 , which expressed for schwannoma but not for neurofibroma , can be used for the discrimination of these two diseases . we report the case of an incidentally discovered schwannoma difficult to diagnose in preoperative evaluation . because of the lack of definitive non - histological diagnostic modalities in this rare tumor , surgical excision can provide the benefit of a definitive diagnosis .

adrenal schwannoma is an extremely uncommon cause of incidentaloma . it originates from neural sheath schwann cells of the adrenal gland . we report the case of a left adrenal schwannoma incidentally discovered in a 32-year - old woman during examination of bloated feeling and stomach ache . the patient was incidentally found to have a left adrenal mass of 9 cm on abdominal ultrasonography . computed tomography ( ct ) of the abdomen and [ 18f ] fluorodeoxyglucose positron emission tomography ( pet ) were also performed . metabolic evaluation was unremarkable . due to the large size of the tumor , left adrenalectomy was performed . the postoperative course was uneventful . histological examination established the diagnosis of schwannoma . this diagnosis was supported by immunohistochemistry of s-100 and vimentin positivity . in conclusion , adrenal schwannoma is an extremely rare entity and can grow considerably in size . the present case report emphasizes that clinicians should be aware of the possibility of retroperitoneal schwannoma . total excision of benign schwannoma is associated with a favorable outcome . to our knowledge , there are case reports of schwannoma with ct and magnetic resonance imaging findings in the literature , although this is the first schwannoma case with pet - ct imaging .

Introduction Case Report Discussion Conclusion

reports from the world health organization ( who ) reveal that 17.6 million persons contracted whooping cough in 2003 resulting in 279000 deaths ; however , some authorities estimate the real figures of cases with pertussis to be around 50 million with 90% living in developing countries . according to the report of who regional office for the eastern mediterranean , 19000 deaths have occurred in under 5-year - old children because of pertussis . immunization against pertussis is included in the expanded program of immunization developed by the who and has been implemented in iran since 1950 . whole cell pertussis vaccine is used in combination with diphtheria and tetanus toxoids ( as dwpt ) to vaccinate infants and children against these three deadly diseases . in spite of a coverage of > 95% for dwpt young children continue to contract whooping cough and are placed at risk for complications and sometimes mortality associated with this vaccine preventable disease . occurrence of pertussis in vaccinated individuals has raised questions about the protection afforded by the whole cell vaccine ; numerous researchers have suggested different approaches for assessment of vaccine efficacy including estimating the prevalence of pertussis in vaccinated populations . since pertussis may mimic other respiratory diseases such as adenovirus , influenza and mycoplasma infections , resulting in the so - called pertussis - like syndrome , relying solely on the clinical presentation would not be a true measure for documenting the disease , as many cases would be clinically mislabeled as pertussis . current diagnostic procedures include culture , polymerase chain reaction ( pcr ) , and a rise in antibodies through enzyme - linked immuno - assay ( elisa ) . isolation of bordetella pertussis which is the gold standard for diagnosis is a difficult and time consuming procedure , making it impractical for epidemiologic studies . detecting the organism by pcr is rapid and sensitive but sensitivity decreases with time and with antibiotic treatment . serology , however , appears to be an easily available and reliable technique to document definite infection with bordetella pertussis ; a rise in igg antibodies against pertussis toxin ( igg - pt ) is seen in > 90% of individuals exposed to b. pertussis either through a natural infection or through vaccination . serum iga , however , does not rise after vaccination and is detectable only in children who acquire natural infection . in vaccinated children because of the anamnestic response of the immune system after immunization , a rapid increase in anti - pertussis antibodies is seen which prevents a significant difference in antibody concentrations between the acute and recovery sera . therefore , in vaccinated individuals , detection of anti - pertussis iga , single values of igg antibodies above a certain level , and single high values of igg antibodies 2 to 3 standard deviations exceeding the mean value in vaccinated uninfected individuals have been used to diagnose natural infection . we aimed to determine the prevalence of pertussis in vaccinated infants and children at different ages ranging from 2 months to 6 years by measuring the anti - pertussis igg and iga antibodies . we aimed to provide an estimate of the protection afforded by the whole cell pertussis vaccine incorporated in the dwpt vaccine currently used in iran for routine immunization of children . this cross - sectional study was done in 6 health facility centers affiliated to tehran and shahid beheshti universities of medical sciences , tehran , iran . the protocol of this study was approved by the ethics committee of shahid beheshti university of medical sciences , tehran , iran . we included disease - free and afebrile infants and children aged 2 , 4 , 6 , 12 , 18 and 72 months with a valid vaccination record ( card ) , referring to centers for dwpt vaccination . children with incomplete or poorly documented vaccination records , those with a history of blood transfusion , immune - compromised children or those receiving immunosuppressive drugs were excluded from our study . the sample size was estimated to be 100 samples from each age group ( power=80% , confidence interval=95% ) . the children s vaccination cards showed that their vaccination status was up - to - date . after documenting the relevant data , 2 ml venous blood was collected from each child and sent to the laboratory where the sample was centrifuged and the serum stored at -70c . samples were then tested by elisa for the presence of anti - pertussis iga ( anti - pertussis toxin , anti - filamentous hemaglutinin , and anti - lipopolysaccharides antibodies ) and igg ( anti - pertussis toxin , anti - filamentous hemaglutinin , and anti - lipopolysaccharides antibodies ) using the kit supplied by the ibl company , germany ( reference no : re56131 and re 56141 ) . serum igg and iga levels were measured in 2 , 4 , 6 , 12 , 18 and 72-month - old children before administering the scheduled dwpt vaccine , imported from the serum institute of india and is routinely administered at 2 , 4 , 6 , 18 , and 72 months of age . the antibody levels were recorded at different ages and compared with baseline levels at 2 months . in further analysis , the geometric mean titer ( gmt ) were classified sequentially for both igg and iga at ages 2 , 4 , 6 , 12 , and 18 months as the baseline levels and compared with the gmt of the two antibodies at higher ages . a natural pertussis infection was determined through any of the following : 1- a positive iga titer , 2- to have an igg level above the mean+2sd level . in each age group , after excluding iga positive individuals , as naturally infected persons in the remaining children , assumed as being uninfected , the mean+2sd level of igg was assigned as the upper limit of vaccine induced antibody and also as a cut - off ( threshold ) . any rise above this level of igg 3- to have an igg level 100 iu / ml . categorical variables were reported as frequency and percentage and for quantitative variables were presented as meanstandard deviation ( sd ) . antibody gmts and related standard errors ( se ) were calculated by logarithmic transformation of data . linear or logistic regression analyses were done according to the type of dependent variable . to evaluate the level of antibodies against burdetella pertussis between the groups , analysis of variance ( anova ) and for pair wise comparisons bonferroni test have been used . to evaluate the association of categorical data with each other , p<0.05 was considered as statistically significant . in case of performing multiple comparisons to evaluate a single hypothesis , we included 725 children aged 2 , 4 , 6 , 12 , 18 , and 72 months . the most collected samples ( n=182 ) were from the 6-year - old group . mean ( sd ) igg levels ( gmts ) at 2 , 4 , 6 , 12 , 18 , and 72 months were 8.43 ( 1.07 ) , 6.31 ( 1.22 ) , 8.29 ( 1.04 ) , 8.58 ( 1.08 ) , 7.35 ( 1.11 ) , 14.4 ( 1.06 ) u / ml , respectively . the mean ( sd ) iga levels at the same ages were 1.48 ( 1.21 ) , 1.43 ( 1.23 ) , 1.45 ( 1.32 ) , 2.66 ( 1.21 ) , 2.24 ( 1.19 ) , 2.03 ( 1.1 ) u / ml , respectively ( tables 1 and 2 ) . comparison of iga geometric meansd titers at different ages with 2 , 4 , 6 , 12 , and 18 months considered sequentially as bases for comparison with higher ages gmt : geometric mean titer ; * statistically significant the estimated frequency of natural pertussis infection using different variables the gmts for igg showed a slight fall after 2 months and a definite rise in children aged 72 months ( figure 1 ) . the gmts for igg shows a slight fall after 2 months and a definite rise in children aged 72 months . gmt : geometric mean titer this figure shows the gmt for iga at different ages of 2 , 4 , 6 , 12 , 18 and 72 months . gmt : geometric mean titer 1% of 2 , 4 , and 6-month - old infants , 6% of the 12 and 18-month - olds and 12% of 6-year - old children had igg levels above the determined cut - off ( derived from mean+2sd ) . 1% of the 2 , 4 , and 6month - old infants , 5% of the 12 and 18month - olds and 10% of the 6-year - old children had igg levels 100 iu / ml . gmts of serum iga were compared between different age groups , which showed significantly higher gmts at certain ages ( table 2 ) . gmts for iga reached the highest levels at 12 and 18 months of age ( figure 2 ) . iga levels above the assay cut - off were detected in 5 , 9 , 6 , 23 , 11 , and 8% of the children at the ages of 2 , 4 , 6 , 12 , 18 and 72 months , respectively . considering the equivocal results of iga as well as the positive ones ( iga8 u / ml ) , the frequency of natural infection were 5 , 9 , 6 , 23 , 11 , and 8% at the ages of 2 , 4 , 6 , 12 , 18 and 72 months , respectively . our findings revealed that igg titers declined slightly after 2 months , and then remained constant until 18 months of age . however , a sharp rise in the igg gmt was seen at 72 months , i.e. before receiving the pre - school booster . the plateau in the gmt until 18 months can be explained by the repeated vaccinations including the primary series at 2 , 4 and 6 months and the first booster given between 12 and 18 months of age . however , the unexpected sharp rise in igg before the preschool booster i.e. between 4 - 5 years after the previous immunization implies recent contact with bordetella pertussis , which could only be explained through the acquirement of natural infection . in france , 360 children were tested for pertussis serology 0.5 to 158 months after complete whole cell pertussis vaccination . they concluded that unrecognized pertussis is common in france despite massive and sustained immunization in infants and that vaccinated children become susceptible to infection more than 6 years after their last vaccination . although a rise in serum iga is observed only after a natural infection , all infections are not associated with an iga response . although , detection of iga against bordetella pertussis has a low sensitivity with a negative predictive value of 61% , it is highly specific for a definite diagnosis of pertussis with a positive predictive value of almost 100% . in our study serum iga level was elevated in 6 - 12% of infants < 2years of age and in 4% of 6-year - old children . it has been reported that iga against the pt antigen rises in 20 - 40% and iga against the anti - filamentous hemaglutinin is increased in 30 - 50% of natural infections . in this study we measured iga against three of bordetella pertussis antigens ( anti - pertussis toxin , anti - filamentous hemaglutinin and anti - lipopolysaccharides antibodies ) . therefore , we presumed that the sensitivity of iga in our study would be higher than the quoted figures for the measurement of separate antigens . besides , as the half - life of iga antibodies is considerably shorter than igg , the presence of this antibody denotes a recent infection . based solely on iga levels , we estimated the prevalence of natural infection in our studied population of vaccinated children at ages of 4 , 6 , 12 , 18 , and 72 months to be between 9 - 11% , with the highest percentage was at 18 months . in infants aged 2 months , yet to receive their dwpt vaccination , 5% revealed evidence of recent exposure to bordetella pertussis . because of the low sensitivity of iga , these records are believed to be only a part of the real figures . some investigators have used cut - off points for single serum samples derived from the mean+2sds of anti - pertussis igg to document natural infection . in our study we used a similar strategy to estimate the frequency of the naturally infected vaccinated children through measuring anti - pertussis igg . then , a cut - off point of mean+2sd of the anti - pertussis igg was assumed in the remaining samples ( uninfected group ) as the maximum level of vaccine induced antibody . even these figures are an underestimation of naturally infected individuals , because inclusion of iga negative but igg positive individuals in the uninfected group would increase the mean igg levels . consequently , the cut - off point of mean+2sd would rise , resulting in the underestimation of truly infected children . the protective effect of the dwpt vaccine is reported to last for a varying period from 4 - 12 years . moreover , only about 52% of children would have a protective level of antibodies 4 years after receiving the vaccine . in a recent study from australia it was noticed that the peak rate of pertussis had shifted from the age of 8 - 9 years to 12 - 13 years , after the 5 dose of the dwpt vaccine was introduced as a pre - school booster in 4 - 5 year - old children . the authors concluded that the protection provided by the dwpt vaccine declines 6 - 9 years after the last dose . immunity against pertussis , either after vaccination or even after natural infection does not bear a direct relationship with the antibody levels , and an individual may be protected against the disease even if the antibodies are undetectable in the blood . on the other hand , antibody levels may rise after an asymptomatic infection in vaccinated individuals . the who reports 98 proven cases of pertussis from iran in 2004 , and 125 cases in 2005 . these figures have grossly underestimated the true numbers and result from under - diagnosis and under - reporting , both of which stem from a lack of definite clinical diagnostic criteria and appropriate laboratory methods . our figures vary widely from those of ghanaie and colleagues from iran who quote an incidence of 318/100000 in 2008 in tehran among school children between the ages of 6 - 14 years . this disparity could be explained by the differences in the age of the study population in the 2 studies as well as the use of different criteria used for the diagnosis of pertussis ; theirs being clinical manifestation plus a positive pcr . a considerable percentage of children had high levels of anti - pertussis igg antibodies , positive anti - pertussis iga , and most importantly a significant rise of anti - pertussis igg gmt at 6 years of age , depicting a natural infection in vaccinated children . further surveys need to be done to study the medium and long - term protection afforded by the current vaccine , in order to prevent the disease in these age group .

background : it seems that the incidence of pertussis - like illnesses is considerably increasing despite the wide coverage of immunization with the whole cell pertussis vaccine . we aimed to investigate the occurrence of pertussis in vaccinated children by measuring anti - pertussis antibodies . methods : in this cross - sectional study , blood samples were taken from vaccinated children aged 2 , 4 , 6 , 12 , 18 , and 72 months . anti - pertussis igg and iga were measured by elisa . p<0.05 was considered significant . results : 725 children were enrolled in the study . geometric mean titers for igg that showed a slight decease after 2 months of age and increased distinctly in children aged 72 months . the frequency of the individuals whose igg was above the determined cut - off ( derived from mean+2sd ) was observed in 1% of the 2 , 4 , and 6-month - old infants , 6% of the 12 and 18-month - olds and 12% of the 6-year -old children . positive iga titers were detected in 5 , 9 , 6 , 23 , 11 , and 8% of children aged 2 , 4 , 6 , 12 , 18 , and 72 months , respectively . conclusion : since a considerable percentage of children had high levels of anti - pertussis igg antibodies ( 2 sd ) , positive anti - pertussis iga , and most importantly an increased level of anti - pertussis igg geometric mean titer at 6 years of age , further investigations regarding the protection provided by the presently used pertussis vaccine seems necessary .

Introduction Subjects and Methods Results Discussion Conclusion None

pilar cysts are common , and benign lesions and malignant transformation in them is extremely rare . trichilemmal cyst or pilar cyst is defined as a cyst containing keratin and its breakdown products . it is usually situated on the scalp with a wall resembling external hair root sheath . it is seen mainly in the at middle age and is inherited in an autosomal dominant fashion . in this case report , we present a 55-year - old man with multiple giant pilar cysts that were present since childhood . one of the cyst on the chest was transformed to squamous cell carcinoma ( scc ) . a 55-year - old man was referred to us because of multiple cystic lesions located on the body , some of them measuring several centimeters [ figures 16 ] . these lesions were present since childhood , and during this period the patient had no complaint except cosmetic concerns . patient had ignored these lesions for many years until one of the lesions on the chest became ulcerated and then infected [ figure 5 ] . pathology of the lesion located on the chest showed neoplastic proliferation of the epithelial cells . pathology of the five other lesions showed a cystic lesion covered with stratified squamous epithelium without granular layer containing homogenous laminated keratin . mohs surgery was performed to excise large scc of the chest , and the site of operation was repaired by split - thickness skin graft technique . the patient visited every month for 1 year and during this period he showed no sign of recurrence in the operation site . also , no change was observed in the remaining pilar cyst of the patient and he is now followed every 6 months . one of the lesions on the chest was transformed to scc a closer view of the figure 1 close up of lesions on the trunk pilar lesions on the back of patient a close view of the scc showing large infected ulcer pilar cyst of the scalp pathology of the lesion on the chest showing squamous nests in well - differentiated scc pathology of pilar cyst showing trichilemmal pattern of keratinization ( 400 ) pilar or trichilemmal cysts contain keratin and its breakdown products , lined by a wall resembling the external ( outer ) hair root sheath . the patient had these lesions since childhood , although most patients with this lesions are affected in middle age . although pilar cyst are usually observed on the scalp , the giant cystic lesions of our lesions were scattered over the whole body . one of these lesions on the chest was transformed to squamous cell carcinoma that was excised using mohs surgery . according to our literature review , up to now the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . pilar cysts may rarely transform to scc and also may occur on many parts of the body . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . pilar cysts may rarely transform to scc and also may occur on many parts of the body . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . pilar cysts may rarely transform to scc and also may occur on many parts of the body .

trichilemmal cyst or pilar cyst is defined as a cyst containing keratin and its breakdown products . it is usually situated on the scalp with a wall resembling external hair root sheath . in this case report we present a 55-year - old man with multiple giant pilar cysts that were distributed over the whole body since childhood . one of the cyst on the chest was transformed to squamous cell carcinoma ( scc ) .

Background Case Report Discussion None Declaration of patient consent

gastrointestinal stromal tumors ( gists ) , which arise primarily in the gut wall , are most commonly mesenchymal neoplasms closely related to the interstitial cells of cajal . gists typically carry gain - of - function mutations in genes encoding the kit receptor tyrosine kinase ( cd117 ) or platelet - derived growth factor receptor a , both of which are involved in cell survival , development and proliferation . although gists can arise at any location in the gastrointestinal tract , they are found most often in the stomach ( 60 - 70% ) , followed by the small intestine ( 20 - 30% ) , colon and rectum ( 5% ) , and esophagus ( < 5% ) . in most cases in the stomach , gists show the appearance of a submucosal tumor , with a broad base and smooth surface within normal gastric mucosa , and sometimes have central delle or depression . in larger cases , central ulceration or necrosis presenting with gastrointestinal bleeding the gist of the stomach described in this report was a rare case , with narrow stalk - like based , uneven protruding mass presenting with severe acute anemia despite small size . a 56-year - old woman was admitted to fujita health university with epigastric pain and nausea during the previous one month . laboratory evaluation on admission showed severe anemia ( red blood cell count 138 10/l , hemoglobin level 4.3 g / dl , hematocrit 13.7% ) ( table 1 ) . esophagogastroduodenoscopy revealed a narrow stalk - like based , hemorrhagic and uneven protruding lesion in the lesser curvature of the gastric upper corpus ( fig . 1 ) . endoscopic ultrasonography showed a low echoic mass , measuring 2.0 cm in diameter , within the fourth layer , suggesting that the tumor originated from the intrinsic muscle layer , and showed growth to the mucosal side ( fig . the labeling index ( li ) for mib-1 , determined by counting positively stained nuclei among 1,000 tumor cells , was 1% . although the histological findings suggested that the tumor was probably a benign gist , laparoscopy - assisted local resection was performed after 2 weeks because the patient had continuous severe anemia and epigastric pain . the resected tumor was 1.8 1.5 1.0 cm in size within the locally resected surgical speciemen . the tumor was elastic but hard in consistently , and its surface was uneven and irregular ( fig . the tumor cells originated from the intrinsic muscle layer and showed growth to the mucosal side , cropping out to the surface in most areas of the protruding lesion . histological assessment revealed that the tumor was composed of elongated spindle - like cells , containing rounded or oval , relatively uniform nuclei without apparent atypia ( fig . the li for mib-1 , determined by counting positively stained nuclei among 1,000 tumor cells , was about 1% ( fig . immunohistochemical staining of the tumor by the avidin - biotin peroxidase complex method showed that most of the tumor cells demonstrated immunoreactivity for kit ( fig . the tumor described in this report showed immunoreactivity for kit and cd34 , but not for asma , s100 and desmin , implying that the tumor was pathologically a gist with a lack of differentiation toward smooth muscle , and neural elements . most gists in the stomach generally show the appearance of submucosal tumors , with a broad base and smooth surface within normal gastric mucosa , and sometimes have small central delle or depression . in larger cases , central ulceration or necrosis presenting with gastrointestinal bleeding however , the gist of the stomach described in this report showed narrow stalk - like based , uneven protruding mass presenting with severe acute anemia despite the small size of less than 2 cm , and local resection was needed due to continuous severe anemia and epigastric pain . although the association between the patient 's gastric symptoms and this small submucosal tumor is not fully understood , the considerable amount of blood in the stomach due to continuous bleeding may have led to the gastric symptoms such as epigastric pain and nausea . histological assessment showed that the tumor cells showed growth to the mucosal side , cropping out to the surface in most areas of the protruding lesion , and only a small part of the tumor was within nontumoral gastric mucosa , despite the tumor cells originating from the intrinsic muscle layer . thus the case of our report may be a rare case for its morphological appearance as well as clinical course . metastasis and/or recurrence after surgery may be observed for several gists , despite an initial diagnosis of a benign tumor . therefore , it is recommended to apply risk classification based on tumor size and mitoic count under a hpf . the mib-1(ki-67 ) li or the presence or absence of tumor necrosis is also reported to reflect proliferation activities and malignant outcome . the gist described in our report was less than 2 cm in diameter without necrosis , and had low mitotic activity ( 0/50 hpf ) and li for mib-1 ( about 1% ) . in this context , our gist may have a very low malignant potential and thus metastasis and/or recurrence may not be observed in the future . actually , the patient in this report has been disease - free 18 months after surgery .

we report the case of a 56-year - old woman who had a gastrointestinal stromal tumor ( gist ) of the stomach . she was admitted to our hospital for epigastric pain , nausea , and severe acute anemia ( hemoglobin level 4.3 g / dl ) . esophagogastroduodenoscopy revealed a narrow stalk - like based , hemorrhagic and uneven protruding lesion in the lesser curvature of the gastric upper corpus . although the tumor was less than 2 cm in diameter and was probably a benign gist according to histology , laparoscopy - assisted local resection was needed because the patient had continuous severe anemia and epigastric pain . histological assessment showed that the elongated spindle - like tumor cells originated from the intrinsic muscle layer , and was shown with growth to the mucosal side , cropping out to the surface in most areas of the protruding lesion . only a small part of the tumor was within nontumoral gastric mucosa . most of the tumor cells demonstrated immunoreactivity for kit and cd34 in the cytoplasm but not for sma , s100 , and desmin . mitotic activity ( 0/50 high power field ) and the labeling index for mib-1 ( about 1% ) were low . the gist of the stomach described in this report was a rare case with a narrow stalk - like based , uneven protruding mass presenting with severe acute anemia despite small size .

Introduction Case Report Discussion

for this cross - sectional study , we recruited men with type 2 diabetes ( with and without cna ) , and attempted to age - match them with healthy control subjects . inclusion criteria for the diabetic participants ( with and without cna ) included ambulatory men aged between 40 and 80 years with established type 2 diabetes for more than 5 years and diabetes - derived nerve damage . exclusion criteria included a documented history of vitamin d deficiency , thyroid disease , renal disease , or other metabolic diseases known to affect bone , as well as prior oral or intravenous antiresorptive drug use . charcot patients had to be ambulatory and in the nonacute , convalescence stage ( stage 2 ) of the disease , which is different from acute stage ( stage 1 ) that is characterized by osseous resorption , soft - tissue swelling , and joint effusions ( 16 ) . this study was approved by the eastern virginia medical school institutional review board , and all subjects provided written consent before participating in this study . the inclusion criteria for the study were met by 80 men , comprising 30 healthy control subjects , 30 type 2 diabetic patients without charcot , and 20 type 2 diabetic patients with stage 2 cna . many of the cna patients had , however , already undergone stabilization of the fractured or dislocated joints with beaming screws and other means of metallic fixation . subjects evaluation consisted of an analysis of fasting blood chemistries ; a complete neurologic examination , including quantitative sensory , autonomic nerve function , and nerve conduction tests ; a bone density assessment ; and quantitative ultrasound imaging of bilateral calcanei . continuous data are expressed as mean sd ( range ) , and categoric data as n. * p < 0.05 vs. control subjects . lower limb nerve conduction parameters were measured bilaterally by surface stimulation of the sural , peroneal , and tibial nerves . measurements included peroneal and tibial motor nerve amplitude , conduction velocity , distal onset latency , and f wave . all motor amplitudes were measured baseline to peak , and latencies were taken as onset latency . motor conduction velocity was calculated as total conduction velocity ( not segmental ) using onset latencies throughout . sensory amplitude was taken as baseline to peak , latency was reported as onset latency , and conduction velocity was calculated across the single segment using onset latency ( neuromax , excel - tech ltd . , quantitative sensory testing was performed using the medoc device according to previously published methods ( 17 ) . warm and cold thermal thresholds and heat and cold pain were tested using the tsa2001/vsa3001 computer - driven sensory measuring device ( medoc , durham , nc ) . vibration perception threshold was quantified using the medoc device and the method of limits protocol , where repeated , increasing vibratory stimulation is applied to the plantar aspect of the dominant great toe . autonomic function was assessed by three tests using the anx-3.0 autonomic monitor : heart rate variability during deep breathing at six breaths per min ( e / i ratio ) and r / r variation in response to the valsalva maneuver and postural change ( ansar medical technologies , inc . , bmd scans of the whole body , bilateral proximal femurs , and bilateral feet were obtained using dual - energy x - ray absorptiometry ( ge lunar prodigy advance , ge healthcare , waukesha , wi ) . scans were performed according to the manufacturer s protocols , with the exception of the feet , for which no standardized protocol exists . mode , where a subject sat on the edge of the scan bed with the knee bent at 90 and the foot flat on the bed . the laser marker was positioned at the midline of the ankle , at the level of the lateral malleolus . during analysis , the region of interest was selected manually and included only the metatarsals and phalanges to exclude any metal fixators . test - retest reliability for this measure is high ( r = 0.96 , n = 31 ) , and the coefficient of variation is < 1% for our laboratory . this corresponds well to other published studies of reproducibility of bmd using the hand mode ( 20 ) , and is also comparable with other regional analyses of bone , where coefficients of variation typically range from 0.5 to 1.2% . because no reference database currently exists for scans of the feet , diabetic patients were compared with sex- and age - matched control subjects . calcaneal stiffness was determined using quantitative ultrasound imaging ( ge achilles express , waukesha , wi ) . bone stiffness is automatically computed using an algorithm supplied by the manufacturer : stiffness index = 0.67 bua 0.28 sos 420 , where bua is broadband ultrasound attenuation and sos is the speed of sound through the tissues ( ge healthcare ) . because bone stiffness predicts fracture independent of bmd , it is considered to be a measure of bone quality that also accounts for factors related to fracture that might not be detected by a measure of bmd alone . fasting blood samples were obtained by antecubital venipuncture into serum separator tubes and separated immediately by refrigerated centrifugation . the serum was stored at 80c according to instructions required by the manufacturers of the enzyme - linked immunosorbent assay ( elisa ) kit . samples were subsequently evaluated in duplicate for srage by the quantikine rage enzyme - linked immunoassay ( r&d systems , minneapolis , mn ) ; cross - linked n - telopeptides of type 1 ( ntx ) collagen , as a measure of bone resorption ( osteomark ntx elisa / eia , wampole laboratories , princeton , nj ) ; and osteocalcin , as a measure of bone formation ( alpco diagnostics , salem , nh ) . group differences on each dependent measure were evaluated using one - way anova with a least significant differences post hoc analysis . a multiple linear stepwise regression analysis was run to help identify factors that may distinguish those who develop cna from those who do not . all analyses were run using spss 18.0 software ( spss inc . , chicago , il ) with significance set at p < 0.05 . lower limb nerve conduction parameters were measured bilaterally by surface stimulation of the sural , peroneal , and tibial nerves . measurements included peroneal and tibial motor nerve amplitude , conduction velocity , distal onset latency , and f wave . all motor amplitudes were measured baseline to peak , and latencies were taken as onset latency . motor conduction velocity was calculated as total conduction velocity ( not segmental ) using onset latencies throughout . sensory amplitude was taken as baseline to peak , latency was reported as onset latency , and conduction velocity was calculated across the single segment using onset latency ( neuromax , excel - tech ltd . , quantitative sensory testing was performed using the medoc device according to previously published methods ( 17 ) . warm and cold thermal thresholds and heat and cold pain were tested using the tsa2001/vsa3001 computer - driven sensory measuring device ( medoc , durham , nc ) . vibration perception threshold was quantified using the medoc device and the method of limits protocol , where repeated , increasing vibratory stimulation is applied to the plantar aspect of the dominant great toe . autonomic function was assessed by three tests using the anx-3.0 autonomic monitor : heart rate variability during deep breathing at six breaths per min ( e / i ratio ) and r / r variation in response to the valsalva maneuver and postural change ( ansar medical technologies , inc . , bmd scans of the whole body , bilateral proximal femurs , and bilateral feet were obtained using dual - energy x - ray absorptiometry ( ge lunar prodigy advance , ge healthcare , waukesha , wi ) . scans were performed according to the manufacturer s protocols , with the exception of the feet , for which no standardized protocol exists . mode , where a subject sat on the edge of the scan bed with the knee bent at 90 and the foot flat on the bed . the laser marker was positioned at the midline of the ankle , at the level of the lateral malleolus . during analysis , the region of interest was selected manually and included only the metatarsals and phalanges to exclude any metal fixators . test - retest reliability for this measure is high ( r = 0.96 , n = 31 ) , and the coefficient of variation is < 1% for our laboratory . this corresponds well to other published studies of reproducibility of bmd using the hand mode ( 20 ) , and is also comparable with other regional analyses of bone , where coefficients of variation typically range from 0.5 to 1.2% . because no reference database currently exists for scans of the feet , diabetic patients were compared with sex- and age - matched control subjects . calcaneal stiffness was determined using quantitative ultrasound imaging ( ge achilles express , waukesha , wi ) . bone stiffness is automatically computed using an algorithm supplied by the manufacturer : stiffness index = 0.67 bua 0.28 sos 420 , where bua is broadband ultrasound attenuation and sos is the speed of sound through the tissues ( ge healthcare ) . because bone stiffness predicts fracture independent of bmd , it is considered to be a measure of bone quality that also accounts for factors related to fracture that might not be detected by a measure of bmd alone . fasting blood samples were obtained by antecubital venipuncture into serum separator tubes and separated immediately by refrigerated centrifugation . the serum was stored at 80c according to instructions required by the manufacturers of the enzyme - linked immunosorbent assay ( elisa ) kit . samples were subsequently evaluated in duplicate for srage by the quantikine rage enzyme - linked immunoassay ( r&d systems , minneapolis , mn ) ; cross - linked n - telopeptides of type 1 ( ntx ) collagen , as a measure of bone resorption ( osteomark ntx elisa / eia , wampole laboratories , princeton , nj ) ; and osteocalcin , as a measure of bone formation ( alpco diagnostics , salem , nh ) . group differences on each dependent measure were evaluated using one - way anova with a least significant differences post hoc analysis . a multiple linear stepwise regression analysis was run to help identify factors that may distinguish those who develop cna from those who do not . all analyses were run using spss 18.0 software ( spss inc . , chicago , il ) with significance set at p < 0.05 . we evaluated 80 adult men , aged 55.3 9.0 years , consisting of 30 healthy control subjects , 30 type 2 diabetic patients , and 20 type 2 diabetic patients diagnosed with stage 2 cna . according to the classification scheme of sanders and frykberg ( 16 ) to describe neuropathic osteoarthropathy patterns of joint involvement , 28% of cna patients displayed pattern 2 ( tarsometatarsal / lisfranc s joint involvement ) , 45% displayed pattern 3 ( midtarsal and naviculocuneiform joint involvement ) , and 27% displayed pattern 4 ( ankle and subtalar joint involvement ) . no patients were classified as pattern 1 ( forefoot ) or pattern 5 ( calcaneus ) , which might have otherwise interfered with the measurement of bmd or calcaneal stiffness , or both . cna patients had similar duration of diabetes but higher hba1c levels than their counterparts with diabetes ( 8.3 1.6 vs. 7.0 2.9% , table 1 ) . fasting serum blood urea nitrogen and creatinine values for all diabetic and cna patients were within the normal reference ranges . cna and diabetic patients were slightly older , had a greater bmi , and a higher waist - to - hip ratio than control subjects . diabetic and cna patients showed more peripheral nerve damage than control subjects , and cna subjects exhibited the highest degree of sympathetic and parasympathetic dysfunction and small and large fiber neuropathy compared with the other study groups ( table 2 ) . peripheral and autonomic neuropathy tests continuous data are expressed as mean sd ; categoric data as n. * p < 0.05 vs. control subjects . p < 0.05 vs. diabetic and control subjects . bmd of the proximal femur was slightly higher at the femoral neck and greater trochanter for diabetic patients than for cna or control individuals , but bmd of the feet was not significantly different across groups ( table 3 ) . calcaneal stiffness was similar for diabetic and control subjects but was markedly reduced in the cna patients ( p < 0.01 ) . these results indicate that ultrasound imaging may detect features that are not detectable by bmd scans using dual - energy x - ray absorptiometry . blood biomarkers , bone density , and stiffness data are presented as mean sd . * p < 0.05 vs. diabetic and control subjects . p < 0.05 vs. control subjects . cna patients displayed srage values more than seven times lower than control subjects and more than three times lower than diabetic patients ( p < 0.05 ) . across all subjects , there was a significant positive correlation between the calcaneal bone stiffness index and srage ( r = 0.35 , p < 0.01 ) and a negative correlation between stiffness index and osteocalcin ( r = 0.39 , p < 0.001 ) . these results demonstrate a cross - sectional relationship between an impaired age defense , increased bone turnover , and reduced bone stiffness . in multiple linear stepwise regression analysis , when measures of large and small fiber neuropathy , autonomic neuropathy , blood measures , bmd , and bone stiffness were entered into the model , 74.5% of group membership was predicted by vibration perception , pressure perception , and srage ( r = 0.745 , p < 0.001 ) . similarly , vibration perception and sympathetic valsalva response accounted for 50.3% of the variability in bone stiffness ( r = 0.503 , p < 0.001 ) , indicating a possible relationship of both large fiber and autonomic neuropathies in the progression to cna . the two most important findings from this study were 1 ) an indication that charcot patients have an impaired rage defense mechanism with an accompanying increase in serum markers of bone turnover , and 2 ) reduced bone stiffness in the calcaneus , not accompanied by reductions in bone mineral density . in this study , srage values in cna patients were reduced by 86% , but the diabetic patients had a 50% reduction compared with control subjects . hyperglycemia generates higher levels of age , but without adequate srage to bind age , it and other ligands tend to accumulate in tissues with slow turnover , such as tendon , skin , bone , amyloid plaques , and cartilage ( 21 ) . accumulation of age has been shown to reduce osteoblastic activity via an increase in rage that increases rankl activation that leads to enhanced osteoclastogenesis and impaired matrix mineralization ( 10,11,22 ) . although our biomarker for bone resorption ( ntx ) was not different between groups , this does not preclude the possibility that bone may be inadequately mineralized and not contributing to bone strength . studies have demonstrated that bone toughness and stiffness are affected by age cross - link formation , and the elastic modulus is independent of other determinants of bone strength such as bone density and microarchitecture ( 23,24 ) . our finding that bone stiffness detected by quantitative ultrasound imaging was markedly lower in cna subjects , despite bmd values in the feet and proximal femur within normal reference ranges , supports the notion that bone stiffness is not necessarily correlated with bmd in certain disease states . elevated bone formation activity in charcot patients is likely the result of an accumulation of microfractures , and an excessive attempt at bone repair to maintain bone integrity of the foot . so taken together , our finding that cna patients display higher levels of bone formation markers and reduced srage may suggest that cna patients are susceptible to age - mediated osteoblast apoptosis , thereby limiting their ability to repair bone . this represents another possible pathway linking cna to bone loss besides the rankl / osteoprotegerin pathway . it would be valuable to investigate whether certain drugs that raise rage levels ( e.g. , ace inhibitors , statins , and glitazones ) ( 25 ) , may also have a role in the prevention of charcot progression in patients with severe neuropathy . we have shown , along with others , that charcot patients display significantly worse autonomic neuropathy ( both sympathetic and parasympathetic ) than diabetic or control subjects . this is an important finding , because it has recently been suggested that the presence of cna may coincide with a loss of sympathetic activity that allows shunt vessels to open , which increases perfusion of the feet and may speed bone resorption ( 12 ) . simple autonomic function tests may indeed be useful tools to detect an increased predisposition to developing cna in those with diabetes . a limitation of our study was the slightly older age of the diabetic and cna patients compared with control subjects , despite an attempt to age - match subjects . in addition , bone stiffness and rage levels were still different between diabetic and cna individuals , despite the presence of diabetes for a similar duration . therefore , we were not concerned that these comparisons were confounded by the 5-year difference in average age between the diabetic and cna subjects and the control group . although all of our cna patients were in the chronic stage 2 of the disease , we did include patients who had recently undergone operative stabilization . this type of stabilization , however , does not represent a cure or even improvement of the underlying disease process . it is possible , however , that postsurgical cna patients may possess a different biochemical profile than presurgical patients . none of our cna patients had involvement of the calcaneus or metatarsals , so we were confident that our measures of bone stiffness and bmd were accurate . lastly , we did not evaluate classic inflammation or oxidative stress markers , which would have added significantly to the argument that reductions in srage promote inflammation or oxidative stress , or both . in summary , we show that there is a spectrum of srage deficiency wherein diabetes reduces srage levels by 50% , suggesting a role in the complications of diabetes . furthermore , cna patients who have progressed to stage 2 have an 86% reduction in srage and extremely advanced somatic and autonomic neuropathy that may prevent a normal defense against oxidative stress and may predispose them to reductions in bone integrity and strength . to further elucidate the deleterious effects of cna on collagen and bone quality , future studies could evaluate components of bone quality such as bone microarchitecture and mineralization from calcaneal biopsy specimens of cna patients . in addition , it would be very valuable to fully explore the protective potential of medications that elevate rage on bone formation pathways in this vulnerable population .

objectivethis study investigated the relationship between circulating soluble receptor for advanced glycation end products ( srage ) and parameters of bone health in patients with charcot neuroarthropathy ( cna).research design and methodseighty men ( aged 55.3 9.0 years ) , including 30 healthy control subjects , 30 type 2 diabetic patients without charcot , and 20 type 2 diabetic patients with stage 2 ( nonacute ) cna , underwent evaluations of peripheral and autonomic neuropathy , nerve conduction , markers of bone turnover , bone mineral density , and bone stiffness of the calcaneus.resultscna patients had worse peripheral and autonomic neuropathy and a lower bone stiffness index than diabetic or control individuals ( 77.1 , 103.3 , and 105.1 , respectively ; p < 0.05 ) , but no difference in bone mineral density ( p > 0.05 ) . cna subjects also had lower srage levels than control ( 162 vs. 1,140 pg / ml ; p < 0.01 ) and diabetic ( 162 vs. 522 pg / ml ; p < 0.05 ) subjects , and higher circulating osteocalcin levels.conclusionscna patients had significantly lower circulating srage , with an accompanying increase in serum markers of bone turnover , and reduced bone stiffness in the calcaneus not accompanied by reductions in bone mineral density . these data suggest a failure of rage defense mechanisms against oxidative stress in diabetes . future studies should determine if medications that increase srage activity could be useful in mitigating progression to cna .

RESEARCH DESIGN AND METHODS Nerve conduction studies Quantitative sensory tests Autonomic function tests BMD Calcaneal stiffness Blood measures Statistical analysis RESULTS CONCLUSIONS

study area - blood and serum collections were carried out during four expeditions , three of which were conducted in the rainy season , while one was carried out in the dry season . six riverside communities along the border of ac and am were studied : mapinguari ( 0924'08"s 6808'51.6"w ) and andara ( 0917'29.8"s 06745'44.0"w ) , on the antimary river , porto acre ( 935'18 's 6731'57''w ) and vila antimary ( 0904'01.0"s 06723'50.1"w ) , on the acre river , and two localities located on opposite edges of the purus river - monte verde ( 0868'13 ' 's 6739'94.8''w ) and praia do gado ( 0874'12 ' 's 6739'94.4''w ) , in the municipality of boca do acre . the number of inhabitants in the selected communities were as follows : mapinguari , 142 , andara , 10 , porto acre , 14,309 , vila antimary , 156 , praia do gado , 7,550 and monte verde , 236 [ data provided by the technology foundation of acre , department of basic health actions / state department of health of acre , national health foundation ( funasa)/boca do acre and brazilian institute of geography and statistics in 2008 ] . in these areas , with the exception of porto acre and boca do acre , the inhabitants live in scattered , hidden and very isolated communities . most of these inhabitants are sustained by subsistence farming and occasional hunting and in some communities they are provided with some basic schooling and primary health care . survey procedure - a local radio station advertised the visit of a research team to the area to the inhabitants of the selected communities from the antimary and acre rivers as well as the purposes of the study and the risk factors for simuliid - transmited diseases . characteristic symptoms such as skin diseases ( pruritus , erythema ) , articular pain , lymphadenopathy and headache were also provided . the communities along the purus river were contacted through the funasa / boca do acre and clinical evaluations and blood collections were scheduled in advance . the nature of our parasitological analysis and the methods involved were explained to the population of each locality and individuals who agreed to be volunteers received and signed a terms of consent form . personal and clinical data , such as the presence / absence of any of the described symptoms , were recorded during the clinical evaluations . a convenience sampling method was adopted in the area and the sample size was determined by assuming an expected prevalence of 50% with an error of 5% and a confidence interval ( ci ) of 95% . a total of 355 volunteers from six different communities were enrolled in the study . all enrolled volunteers were 18 years of age or older and , in the case of women , were not pregnant or breast feeding . the study design was approved and registered by the ethical committee of oswaldo cruz foundation ( fiocruz ) ( 281/05 ) . data on the prevalence of m. ozzardi were obtained through venous blood samples collections performed using 10 ml sterile , disposable syringes . one millilitre of venous blood was deposited in a polystyrene tube with 10 ml of a 2% formalin solution ( knott 1939 ) . after a period of 12 h , thin films were prepared with the deposited sediment , then fixed with methanol and stained with giemsa . to circumvent problems due to the preparation of material in the field , two samples consisting of thin films were prepared from each volunteer . prevalence data were evaluated according to the age , sex and occupational activity of the participants . to analyse the disease prevalence in terms of economic activity , the study subjects were split into three different groups based on whether such activities were carried out in primarily indoor , outdoor or mixed environments . group a included all volunteers who were enrolled in outdoor activities such as agriculture , cattle raising and fishing , whereas group b consisted of those individuals whose daytime activities consisted of primarily indoor activities , including nurses , policemen , cooks and drivers . participants involved in activities of a mixed nature , such as housewives , motorboat drivers , teachers , students and salesmen , were placed in group c. the results of clinical assessments of symptoms were compared between infected and uninfected individuals . elisa for o. volvulus proteins were performed according to the method described by bradley et al . the recombinant proteins included in a cocktail for elisa were ovmbp/10 ( expressed during the l3 , l4 and semi - adult stages ) , ovmbp/11 ( expressed in all developmental stages ) and ovmbp/16 ( expressed only in adult worms ) from o. volvulus . the recombinant proteins were provided by dr mario rodriguez - prez from the centre of genomic biotechnology / national politechnic institute , mexico . plasma samples were obtained from blood collected in heparin - treated tubes , followed by centrifugation for 10 min at 400 g to separate the pellets containing packed erythrocytes from the plasma . a total of 355 plasma samples were identified and kept in a container with liquid nitrogen until transportation to laboratory of simuliidae and onchocerciasis / oswaldo cruz institute ( lso / ioc)-fiocruz in the state of rio de janeiro ( rj ) , where they were stored at -20c . ten plasma samples from onchocerciasis and mansonellosis - nave donors from rj were selected and used as negative controls in each experiment . in addition , each elisa plate contained duplicates of blank and reference positive ( n = 3 , strongly reacting plasma from onchocerciasis patients ) and negative ( n = 3 , nave non - endemic donors ) controls . the cut - off values for seropositive samples were calculated as the mean optical density ( od ) plus three standard deviations obtained from the sera of negative controls from rj ( n = 10 ; igg cut - off value = 0.116 ) . to standardise the od data obtained in different experiments , an od index was calculated as the ratio of the observed od / cut - off values . the anti - maltose binding protein from escherichia coli ( mbp ) responses of all analysed sera were determined in parallel . all elisa od values were corrected by subtracting the anti - mbp od values from each serum specimen prior to determining the cut - off values . statistical analysis - all data obtained in this study were tested for significance via ( 2-tailed ) fisher 's exact modification of the 2 x 2 chi - squared test with a 95% ci and p < 0.05 indicating statistical significance . the mantel - haenszel test was applied using epi info7 software and the results are shown in tables i , ii . fischer 's exact test was performed with graphpad prism software , version 4.0 for windows ( graphpad.com ) . table iprevalence of mansonella ozzardi infections in six riverside communities of antimary , acre and purus rivers bordering region of the states of acre and amazonas , brazil ( 2006 - 2008 ) m. ozzardi prevalence riversacre antimary purus community porto acre np / n ( % ) vila antimary np / n ( % ) mapinguari np / n ( % ) monte verde np / n ( % ) praia do gado np / n ( % ) total np / n ( % ) sex males 4/39 ( 10.3 ) 17/55 ( 31 ) 1/6 ( 16.7 ) 0/39 ( 0 ) 14/19 ( 73.7 ) 7/12 ( 58.3 ) 43/170 ( 25.7 ) females 1/90 ( 1.1 ) 6/33 ( 18.2 ) 0/4 ( 0 ) 0/29 ( 0 ) 10/19 ( 52.6 ) 5/10 ( 50 ) 22/185 ( 11.7 ) total community 5/129 ( 3.9 ) 23/88 ( 26.1 ) 1/10 ( 10 ) 0/68 ( 0 ) 24/38 ( 63.2 ) 12/22 ( 54.5 ) 65/355 ( 18.3 ) river 28/217 ( 13 ) 1/78 ( 1.3 ) 36/60 ( 60 ) - n : number of subjects examined ; np : number of positive subjects . table iidistribution of mansonella ozzardi infected individuals according to economic activities in six riverside communities of acre and purus rivers bordering region of the states of acre and amazonas , brazil ( 2006 - 2008 ) occupation groups m. ozzardi prevalence np / n ( % ) a 37/152 ( 24.3 ) b 4/54 ( 7.4 ) c 24/149 ( 16.1 ) total 65/355 ( 18.3 ) group a : individuals enrolled in outdoor activities such as agriculture , fishing , cattle raising ; group b : individuals enrolled in indoor activities such as nurses , policemen , cookers , drivers ; group c : individuals enrolled in activities of mixed nature such as students , teachers , housewives , motor boat drivers ; n : number of subjects examined ; np : number of positive subjects . group a : individuals enrolled in outdoor activities such as agriculture , fishing , cattle raising ; group b : individuals enrolled in indoor activities such as nurses , policemen , cookers , drivers ; group c : individuals enrolled in activities of mixed nature such as students , teachers , housewives , motor boat drivers ; n : number of subjects examined ; np : number of positive subjects . as shown in table i , a total of 355 blood films prepared according to the knott ( 1939 ) test were examined and 65 ( 18.3% ) were found to be positive for m. ozzardi microfilariae . monte verde , which is located on the left bank of this river , exhibited 63% positive samples , whereas on the opposite side of the river , 54.5% of the volunteers in the locality of praia do gado presented blood microfilariae of m. ozzardi ( p > 0.05 ) . only one volunteer from andara , located along the antimary river , was positive for microfilariae and all of the individuals from mapinguari presented blood samples that were negative for microfilariae . vila antimary and porto acre , situated along the acre river , both presented some infected individuals , with vila antimary displaying a higher prevalence rate ( p < 0.05 ) . when the rates of infection were compared between men and women , a slight difference could be detected only in porto acre , whereas in all other communities , the rates of infections were evenly distributed between the two sexes . the prevalence rates were higher among individuals who primarily performed outdoor tasks ( group a ) ( p < 0.05 ) compared to those performing mostly indoor activities ( group b ) ( table ii ) . no differences were observed between groups a and c. all previously described clinical aspects were evaluated and it was found that symptoms such as headache and articular pain were the prevalent symptoms in infected individuals ( table iii ) . table iiicomparative analysis of clinical aspects of mansonella ozzardi infections between infected and uninfected individuals from six riverside communities of acre and purus river bordering region of the states of acre and amazonas , brazil ( 2006 - 2008 ) m. ozzardi infections subjects positive ( n = 65 ) np ( % ) negative ( n = 290 ) np ( % ) porci symptomatic individuals coldness in legs 19 ( 29 ) 80 ( 28 ) 0.7623 1.084 0.5990 - 1.963 headache 43 ( 66 ) 96 ( 33 ) < 0.0001 3.950 2.236 - 6.978 articular pain 37 ( 57 ) 94 ( 32 ) 0.0003 2.755 1.591 - 4.772 lymphadenopathy 4 ( 6 ) 52 ( 18 ) 0.023 0.3001 0.1045 - 0.8623 asymptomatic individuals 21 ( 32 ) 182 ( 63 ) < 0.0001 0.2832 0.1599 - 0.5018 ci : confidence interval ; n : total examined ; np : number of positive individuals ; or : odds ratio . ci : confidence interval ; n : total examined ; np : number of positive individuals ; or : odds ratio . furthermore , division of the subjects according to age / sex revealed that among volunteers under 40 years of age , the prevalence of infection was higher in males ( p < 0.05 ) , whereas among those aged 40 - 50 and over 50 years , the prevalence of infection was distributed evenly between the two sexes ( p > 0.05 ) . m. ozzardi infections were generally more prevalent among males in the study area ( p < 0.05 ) ( table iv ) . table ivprevalence of mansonella ozzardi infected individuals according to age and gender in six riverside communities of acre and purus rivers bordering region of the states of acre and amazonas , brazil ( 2006 - 2008 ) age group males np / n ( % ) females np / n ( % ) total np / n ( % ) < 40 20/95 ( 21 ) 05/109 ( 4.6 ) 25/204 ( 12 ) 40 - 50 05/25 ( 20 ) 07/41 ( 17 ) 12/66 ( 18 ) > 50 18/47 ( 38 ) 10/38 ( 26 ) 28/85 ( 33 ) total 43/167 ( 26 ) 22/188 ( 12 ) 65/355 ( 18 ) n : number of subjects examined ; np : number of positive subjects . when the results regarding prevalence were compiled , it was possible to note a gradient from the communities where m. ozzardi infections are more prevalent in am to those located in the border regions of am and ac , where transmission foci may have been recently established ( figure ) . the results obtained via elisa using the recombinant proteins ov10 , ov11 and ov16 coupled to mbp revealed that igg responsiveness to the cocktail was negative in 354 of the 355 samples tested , with the one exception coming from porto acre . the surveyed localities were chosen based on previous field studies revealing the presence of simulium species such as s. amazonicum and simulium oyapockense in this region , which are potential vectors of filarial parasites ( nascimento et al . 2009 ) and could serve as risk factors for simuliid - transmitted diseases in the population . m. ozzardi infections are well known along the purus river and its tributaries ( medeiros et al . 2008 , 2009 , 2011 ) , but no epidemiological studies aimed at determining the presence and dispersion of this species through strategic points on the border of am and ac have been performed previously . the results of the present survey demonstrated varying rates of human infection at the studied sites . the thin films obtained from all volunteers from mapinguari , which is situated along the antimary river , were negative , suggesting that transmission was likely not occurring at this point along the river , although breeding sites of simuliids had been detected ( nascimento et al . a city bordering the acre river , low but detectable rates of m. ozzardi infection were observed . indeed , as porto acre is moderately developed compared to other riverine communities , most of the volunteers from this site were involved in indoor activities such as nursing , teaching and domestic care , reducing their exposure to simuliid bites . the geographical proximity of riverine communities to this particular city makes it very attractive in terms of economic activities such as trade , fishing and agriculture . porto acre is still very important to individuals searching for medical assistance , resulting in frequent human migration to the city . the results obtained in monte verde and praia do gado support the contention that m. ozzardi infections are scattered along the purus river and that the infected individuals in these areas exhibit high parasitic loads on average ( data not shown ) . indeed , the infection rates observed in monte verde were much higher than have previously been described ( medeiros et al . 2009 ) , possibly due to differences in methodology , such as the use of the knott ( 1939 ) method , which improves sensitivity . it is of note that the rates of m. ozzardi infection in this area ( the purus river ) have been the subject of various studies and appear to be increasing with time . in 1975 , shelley studied riverine communities along the purus river and reported an index of 4.4% . ( 2009 , 2011 ) found prevalence rate of 20% to almost 40% depending of the locality . in general , m. ozzardi infections are observed at prevalence rates of approximately 20 - 47% in endemic areas such as those in bolivia , venezuela and colombia ( kozek et al . the present work was conducted in localities bordering the antimary , acre and purus rivers , which have never been surveyed previously . these areas were presumed to display prevalence rates of approximately 50% , which is the value employed when there is an absence of conclusive data . instead , the actual global prevalence rate recorded was 18.3% and by examining each community , the highest rates were shown to occur along the purus river . in studies conducted in communities along the pauini and purus rivers as well as in lbrea , medeiros et al . as some localities along the purus river are very important economically , they appear to be exporting infected individuals to previously uninfected areas , such as areas of ac . when the prevalence of m. ozzardi infections was compared between males and females , the frequency of infection was observed to be higher in males , in agreement with previously reported data ( tavares 1981 , medeiros et al . 2011 ) . indeed , our data showed that for individuals involved in outdoor and mixed indoor - outdoor activities , there was an enhanced probability of exposure to infective bites . farmers , fishermen , housewives and teachers are examples of occupations that are frequently reported to exhibit high prevalence rates ( batista et al . 1960b , medeiros et al . 2009 , 2011 ) . additionally , the infection rate increased with age and became more evident from 40 years of age onward , with very similar rates being detected between men and women , as reported for other areas ( moraes et al . 1978 , medeiros et al . 2008 ) . since 1960 , when batista et al . described the most frequent symptoms presented by infected individuals with a high parasitic load , mansonellosis , as a disease , has been a subject of controversy . briefly , restrepo and ochoa ( 1986 ) carried out clinical trials and observed symptomatology similar to that reported previously in brazil . in argentina , jorg ( 1983 ) described a unique case of mansonellosis in an immunocompromised patient . however , in studies carried out in bolivia , no correlation was found between infections and clinical signs ( bartoloni et al . additionally , the suspected involvement of m. ozzardi in ocular impairment was studied in volunteers from a endemic area along the solimes river in am ( cohen et al . 2008 ) and , more recently , an association with corneal lesions was found ( vianna et al . the clinical analysis indicated a significant relationship between infection and two of the traditional symptoms described in the literature : headache and articular pain . the physiological reasons for these symptoms have not been well studied , but circulating immune complexes associated with m. ozzardi infections have previously been described and could serve as an explanation for the articular pain observed in infected individuals ( godoy 1998 ) . furthermore , asymptomatic individuals were present in significant numbers , some of whom exhibited high parasitaemia levels ( data not shown ) , which may be a result of a well - balanced host / parasite relationship . finally , 40% of the serum samples collected previously from subjects in vila antimary and tested at lso / ioc - fiocruz were found to be positive when subjected to elisa against a cocktail of recombinant proteins from o. volvulus ( shelley et al . 2001 ) , which is considered suggestive of exposure to this parasite ( bradley & unnasch 1996 ) . the use of non - invasive methods such as serological tests has increased , but although such methods are more sensitive , extensive cross - reaction between different nematodes has been observed , compromising their specificity ( cabrera et al . controversial findings and variations in sensitivity and specificity depending on the geographical area and the parasite proteins employed have been reported ( bradley & unnasch 1996 ) . the antigens used in the present work were selected due to showing promising results in other studies and despite one inconclusive positive result from an uninfected volunteer from porto acre , the elisa applied here using the ov10 , ov11 and ov16 recombinant proteins showed high specificity ( 99.7% ) , with no cross - reactivity with m. ozzardi - infected individuals being detectable . 2001 ) in which cross - reaction with serum samples from m. ozzardi - infected individuals was detected . however , these previous assays employed ov29 rather than ov16 . furthermore , blood microfilarias similar to those of o. volvulus in the cephalic space , but with a posterior region morphology similar to m. ozzardi have been found in the study area ( adami et al . it is possible that the presence of these parasites in blood samples could be responsible for the previously observed elisa cross - reactivity . however , even in infected volunteers with atypical microfilarias , the serological profile has not changed . atypical microfilarias have also been found in peru and a molecular analysis performed on these samples clustered the atypical microfilarias into the mansonella group , which is indistinguishable from m. ozzardi ( arrspide et al . other factors , such as the preparation of materials and variations in protocols , may have had some effect on the obtained results , as a different combination of recombinant proteins was employed in our cocktail . in conclusion , epidemiological surveillance should be sustained through border regions of am and ac to prevent the critical dispersion of m. ozzardi infections to non - endemic areas where simuliid species are present and can act as competent vectors . as long as mansonellosis remains a poorly understood filarial disease and scattered infections continue in human populations situated along the purus river and some of its tributaries , further studies will be needed to increase our understanding of its pathology and to develop safe treatment strategies .

mansonella ozzardi infections are common in the riverside communities along the solimes , negro and purus rivers in the state of amazonas ( am ) . however , little is known about the presence of this parasite in communities located in regions bordering am and the state of acre . the prevalence rate of m. ozzardi infections was determined in blood samples from volunteers according to the knott method . a total of 355 volunteers from six riverine communities were enrolled in the study and 65 ( 18.3% ) were found to be infected with m. ozzardi . as expected , most of the infections ( 25% ) occurred in individuals involved in agriculture , cattle rearing and fishing and an age / sex group analysis revealed that the prevalence increased beginning in the 40 - 50-years - of - age group and reached 33% in both sexes in individuals over 50 years of age . based on the described symptomatology , articular pain and headache were found to be significantly higher among infected individuals ( 56 and 65% prevalence , respectively , p < 0.05 ) . sera from volunteers were subjected to elisa using a cocktail of recombinant proteins from onchocerca volvulus to evaluate the specificity of the test in an endemic m. ozzardi region . no cross - reactions between m. ozzardi - infected individuals and recombinant o. volvulus proteins were detected , thus providing information on the secure use of this particular cocktail in areas where these parasites are sympatric .

SUBJECTS, MATERIALS AND METHODS RESULTS DISCUSSION

to identify risk variants for childhood acute lymphoblastic leukemia ( all ) we conducted a genome - wide association study of 2 case - control series , analyzing the genotypes of 291,423 tagging snp genotypes in a total of 907 all cases and 2,398 controls . we identified risk loci for all at 7p12.2 ( ikzf1 , rs4132601 ; or = 1.69 , p = 1.20 x 10 - 19 ) , 10q21.2 ( aridb5 , rs7089424 ; or = 1.65 , p = 6.69 x 10 - 19 ) and 14q11.2 ( cebpe , rs2239633 ; or = 1.34 , p = 2.88 x 10 - 7 ) . the 10q21.2 ( aridb5 ) risk association appears to be selective for the subset of b - cell precursor all with hyperdiploidy . these data show that common low - penetrance susceptibility alleles contribute to the risk of developing childhood all and provide novel insight into disease causation of this hematological cancer ; notably all 3 risk variants map to genes involved in transcriptional regulation and differentiation of b - cell progenitors .

Supplementary Material

it is a common tumor of male paratesticular tissues ( epididymis , tunica or spermatic cord ) and has also been described in females ( uterus , fallopian tube , ovary and paraovarian tissues ) . the majority of these tumors are asymptomatic and presents as a mass in the genital area , which is worrisome to both patient and clinician and requires a definitive diagnosis to rule out malignancy ( 2 ) . fnac is a rapid , reliable and cost effective diagnostic tool for preoperative diagnosis of paratesticular swellings . we report a case of epididymal adenomatoid tumor diagnosed on aspiration cytology , which was confirmed on histopathology and immunohistochemistry . a 24 year old male presented with progressively increasing painless swelling of the right epididymis for 2 months . local examination revealed a firm , mobile , mildly tender mass measuring 1.5x1.5 cm in diameter in the right epididymis . ultrasonography of the swelling revealed a well - defined hypoechoic lesion of 18x11 mm , with increased flow on color doppler ( figure 1 ) . well defined hypoechoic lesion of size 18x11 mm in right epididymis ( white arrow ) and normal right testis ( black arrow ) fine needle aspiration of the swelling was performed using a 23-gauge needle and 10 ml syringe . examination of the smears revealed cells arranged in loose aggregates in a vaguely glandular pattern and singly scattered at places along with stromal fragments and a few inflammatory cells in a haemorrhagic background . the cells have ovoid , vesicular nuclei , finely granular and evenly distributed chromatin , small nucleoli and abundant cytoplasm with vacuolization at places ( figure 2 ) . stromal cells ( romanowsky x 200 ) the patient underwent a conservative testis sparing surgery with the excision of epididymal nodule . the nodule measured 1.5x1.2 cm in diameter and the cut section was gray - white . histopathology revealed some slit like or glandular spaces lined by flattened cells with intracytoplasmic vacuoles in an abundant fibromuscular stroma and few inflammatory cells . on immunohistochemistry tumor cells were positive for mesothelial marker calretinin and negative for cd34 , factor - viii and cea . photomicrograph showing a ) histopathological features of adenomatoid tumor consisting of dilated tubular structures lined by cuboidal to flattened epithelial cells ( h and e x 200 ) . adenomatoid tumor(at ) . is the most common benign tumor of paratesticular tissue and accounts for 32% of all tumors in this location ( 3 ) . the term adenomatoid tumor was first coined by godman and ash in 1945 ( 4,5 ) . besides the epididymis , adenomatoid tumor can be located in the spermatic cord , prostrate , ejaculatory ducts and scrotal capes and in females it can be located in uterus , fallopian tubes and ovaries ( 6 ) . it is common in the 3rd and 4th decades of life ( 4,6,7 ) . most of these are asymptomatic and are noticed incidentally by the patient or by the physician on physical examination , as a painless intrascrotal mass . it presents as a relatively small , well demarcated uncapsulated nodule , usually less than 2 cm in diameter . the histogenesis of at has been a matter of debate for many years with the proposed cells of origin being endothelial , mesothelial , mesonephric , mullerian and epithelial ( 8) . though the structural and immunohistochemical studies support the mesothelial origin , the usual coexistence of chronic inflammatory cells and fibrosis suggests mesothelial hyperplasia ( 6,8 ) . the first cytological description of at was described by perez - guillemro et al in 1989 ( 2 ) . adenomatoid tumors have been described briefly in textbooks on fna cytology and only a few cases of this entity diagnosed by fnac are recorded in the literature ( 2,5 ) . on fine needle aspiration cytology adenomatoid tumors reveal abundant cellularity and comprise sheets and multilayered clusters of monotonous cells with round or ovoid , eccentric nuclei containing small central nucleoli . paranuclear clearing with a pink colouration ( giemsa stain ) . or a clear vacuole like area ( papanicolaou stain ) with a background of naked nuclei and stromal fragments ( 2 ) . the cytological differential diagnosis include reactive mesothelial hyperplasia , papillary cystadenoma , spermatic granuloma , malignant mesothelioma and metastatic adenocarcinoma ( 4,5 ) . reactive mesothelial cells with hyperplasia can be seen in hydrocoele fluid , the cells show anisocytosis and pleomorphism and have a moderate amount of pale cytoplasm . they may not have definite arrangement of cells , a feature seen distinctly in adenomatoid tumors . cytologically papillary cystadenoma comprises of papillary structures and single isolated cells without nuclear atypia and with cytoplasmic vacuoles in a mucoid background . malignant mesothelioma show a diffuse cell pattern with cells having ruffled borders with dense cytoplasm , nuclear enlargement , macronucleation and multinucleation . metastatic adenocarcinoma show cytological features of malignancy and cells are positive for mucicarmine ( 4,5 ) . on histopathology , the tumor has a plethora of microscopic appearances , with proliferation of cells ranging from cuboidal to flattened , which forms solid cords with an epithelial appearance alternating with channels having dilated lumina simulating vascular structures . the prominent intervening stroma may contain abundant smooth muscles and elastic fibres ; it may also have a reactive desmoplastic quality and be infiltrated by inflammatory cells . on immunohistochemistry these cells are positive for mesothelial related markers ( calretinin , hmbe1 ) . and negative for cea and factor viii related antigen ( 6,7 ) . ultrasonography along with cytological features helps to make a definite diagnosis and to evolve an organ sparing surgical approach ( 4 ) the preoperative cytology may be used as an important diagnostic tool to evaluate the paratesticular masses . pathologist should be aware of cytological features of these common lesions to avoid aggressive or unnecessary surgical procedures .

adenomatoid tumor is a benign mesothelial neoplasm of the paratesticular region in males but can also occur in female genital tracts . the epididymis is the most common site of involvement and constitutes 32% of paratesticular neoplasms.we diagnosed a case of adenomatoid tumor on cytopathology in 24 years old male , who presented with mass lesion right epididymis since 2 months . the diagnosis was confirmed on histopathology and immunohistochemistry after surgical excision of the nodule.fnac is a rapid , reliable and cost effective diagnostic tool for preoperative diagnosis to take appropriate surgical decisions . pathologists should be aware of the cytological features of such lesions so as to differentiate it from other paratesticular lesions .

INTRODUCTION CASE REPORT DISCUSSION

appalachia is a region of over 530,000 square kilometers in the eastern united states of america that roughly corresponds to the area surrounding the appalachian mountains . it is largely rural and has been geographically isolated in the past because of mountainous terrain , as well as transportation systems that have hampered easy travel . nevertheless , appalachia is heterogeneous and includes urban areas : pittsburgh , located in allegheny county , pennsylvania , is its largest city ( 2013 city population 306,000 ; county population 1,233,000 ) . figure 1 shows the extent of appalachia , as defined by the appalachian regional commission , a partnership of federal , state , and local governments established in 1965 to promote community and economic development in the region . over 25 million people historically , they have been burdened by relatively high levels of poverty . in 1960 , for example , 31% of the population was living below the poverty level . this figure has declined to about 17% in the period 20092013 , in comparison with 15.4% for the usa as a whole . nevertheless , in some parts of appalachia , poverty and attendant issues such as food insecurity are quite prevalent . in 2013 , for example , the per capita market income for appalachia was almost us$10,000 less than the national average ( us$27,979 versus us$37,127 ) . chronic poor oral health , with increased rates of dental caries , gingivitis , and periodontitis that can ultimately result in edentulism , has an unusually high prevalence in appalachia . in west virginia , the only state located entirely within appalachia , two - thirds of adults over 65 have lost 6 or more teeth and one - third are completely edentulous , compared to 40% and 17% , respectively , for the usa . caries rates in 1619-year - olds are also increased in west virginia over the general population ( 84% versus 67% [ 5 , 6 ] ) . these oral health problems occur in the context of numerous other overall health issues ( e.g. , diet and nutrition , physical activity , obesity , diabetes , cardiovascular disease , and substance abuse ) and are associated with a substantial regional health and economic burden . unlike many cultural differences in the usa that are defined by race and ethnicity , the geographical region also boasts a unique , yet heterogeneous , appalachian identity , with its distinctive topography , foods , music , values , and behavior patterns . , many of those living in rural appalachia have been regarded as economically disadvantaged and undereducated . however , in spite of real and perceived health , economic , and social problems , people from appalachia share numerous strengths , including independence , self - reliance , humility , modesty , sense of community and place , spirituality , patriotism , and ( often self - effacing ) humor . the center for oral health research in appalachia ( cohra ) was established in 2000 as a collaboration between the university of pittsburgh and west virginia university to study the high rates of oral health problems seen in northern appalachia . since then , cohra has embarked upon multiple research programs to collect and study samples from this region . the first cohra research program ( cohra1 , 20002010 ) studied multiple genetic and environmental factors in families with children ages 118 living in central west virginia and western pennsylvania . a high prevalence of caries was confirmed , even in the very young children in the study families . in particular , 5% of the cohra1 two - year - olds , 21% of the three - year - olds , 35% of the four - year - olds , and 51% of the five - year - olds had some degree of decay . cohra1 findings also suggest that individuals in northern appalachia have high rates of untreated caries even with increased use of sealants and have dental fear and anxiety that may be transmitted across generations . in response to these findings , a second major study ( cohra2 , 2011present ) was initiated to investigate oral health in pregnant women and their babies , specifically the factors predisposing a relatively high proportion of very young northern appalachian children to dental caries . cohra2 was designed to test the hypothesis that the mechanisms leading to oral health disparities develop very early within the first two years of life and include a complex interplay among behavioral / environmental , microbiological , and genetic factors . to this end , cohra2 is collecting extensive oral health , demographic , medical , dietary , behavioral , genetic , and microbiological data on a large sample of women and their babies from west virginia and southwestern pennsylvania over multiple time points and has reached 70% of its recruitment goal . in this report , we describe the cohra2 protocol and the oral health of the sample of cohra2 pregnant women recruited from 2011 to 2015 , including their oral health behaviors , personal and household demographics , and social behaviors . under the cohra2 study protocol , we collect data on women during their pregnancy and follow them and their babies longitudinally through the early years of the baby 's life . as detailed in the cohra2 study design ( figure 2 ) , data are collected concerning the mother 's general and oral health and the household and individual environment as well as the infant 's general and oral health . multiple microbial samples are taken from the mothers and babies at different time points , and human dna is extracted from saliva . several focused working groups ( oral / dental health , medical history , diet , behavior , genetics , and microbiology ) comprised of collaborators and outside experts designed the study . most in - person visits can be completed in an hour or less and telephone interviews in 45 minutes or less , minimizing participant burden and boosting retention . healthy us caucasian women who are in the 12th to 29th weeks of pregnancy are potentially eligible for the study . in west virginia , women who have not reached the 12th week of their pregnancy can be enrolled if necessary for scheduling appointments . women must also be at least 18 years old , be relatively fluent in english , have a singleton pregnancy and can not have tuberculosis or be immunocompromised . women may also be excluded if they do not think they will remain in the general regions of west virginia or southwestern pennsylvania for the duration of the study , or if they do not have a reliable telephone contact . cohra2 focuses on factors affecting normal healthy women and children , so if a woman delivers prematurely , that is , before the 35th week of her pregnancy , or if she or her baby develops a serious medical condition , they are withdrawn from the study . if a low birth weight baby is delivered , cohra2 consulting pediatric physicians determine on a case - by - case basis if there are additional health problems necessitating removal of the mother - baby pair from the study . recruitment is limited to caucasians to minimize the potential biases that ethnic heterogeneity can introduce into genetic analyses . future studies are planned that will focus on women of different ethnicities from the same northern appalachian regions . cohra2 has two enrollment teams , one operating out of the university of pittsburgh ( dr . mary l. marazita , co - pd / p.i . ) and the other out of west virginia university ( dr . daniel w. mcneil , co - pd / p.i . ) , and a third team at the university of michigan focusing on microbial ecology ( dr . the pittsburgh team recruits women who , for the most part , deliver at magee - womens hospital of upmc , pittsburgh , pa , one of the largest hospitals in southwestern pennsylvania with over 10,000 deliveries annually . in contrast , 2022,000 babies are born annually in the entire state of west virginia , necessitating statewide recruitment by the west virginia team . the university of pittsburgh site is also the study coordinating center , receiving and analyzing data and samples from all sites . pittsburgh and west virginia each plan to recruit at least 500 women , for a total initial sample size of over 1000 mother - baby pairs . in pittsburgh , recruitment began in january 2012 and is being conducted primarily through the clinics and outreach offices of magee - womens hospital . flyers and brochures are distributed throughout the magee health network and at other locations in the greater pittsburgh area . on - site cohra2 research staff and/or the magee clinical and translational research center ( ctrc ) explain the study to potentially eligible women receiving prenatal care through physicians affiliated with magee . interested women provide their contact information , and the research staff contacts them with more details about the study . they are scheduled for an initial appointment before the end of their 29th week of pregnancy . in west virginia , recruitment began in november 2011 , with women recruited throughout the state . recruitment is facilitated with the help of 42 partnering health and dental clinics , health department offices , community centers , and hospitals . in addition , ob - gyn offices , wic offices , birth - to - three offices , the west virginia perinatal initiative , early head start , and community service organizations recruit for the study . print , radio , and television ads as well as brochures and posters are distributed periodically . the research team promotes the project through participation in health and pregnancy fairs , conferences , and interactive online presentations . interested women contact the research staff , who screen for eligibility , provide details about the study , and schedule the initial appointments before the end of the 29th week of pregnancy . all potential participants have the study explained to them in detail and are sent copies of the consent forms before their initial appointments . at the first visit , the study is explained again , questions are answered , and the women sign consent forms prior to any research assessments . the in - person assessments consist of ( 1 ) an extensive dental assessment that includes collection of saliva and microbial samples ( described in detail below ) ; ( 2 ) physical measurements , including mother 's and baby 's height and weight , baby 's head circumference , and mother 's leg length [ 11 , 12 ] ; ( 3 ) an interview that includes basic demographics , pregnancy and medical history including antibiotic use , and questions specific to some time points ; ( 4 ) an inventory of current medications ; ( 5 ) the dental fear and anxiety scale , a 29-item scale that includes the 20-item dental fear survey plus the 9-item fear of pain questionnaire ; ( 6 ) a portion of the child behavior checklist ; and ( 7 ) a household water sample for determination of fluoride concentration . in pittsburgh , 3d facial photographs and 2d hand scans are also taken to document growth parameters . the dental assessment for mothers has several components , performed in the following order : ( 1 ) blood pressure measurements ; ( 2 ) oral rating index , a visual measure of gingival health and oral hygiene ( ori ) ; ( 3 ) a modified version of the whole mouth fluorosis score ; ( 4 ) unstimulated salivary flow rate ; ( 5 ) salivary ph ; ( 6 ) microbial sampling of saliva , gingiva , and plaque ; ( 7 ) soft tissue inspection ; ( 8) malocclusion examination ; ( 9 ) caries assessment ; and ( 10 ) saliva collection for dna extraction using oragenediscover kits ( ogr-500 , dna genotek ) . microbial samples are collected using omnigenediscover kits ( om-501 or 505 , dna genotek ) . gingival swabs are taken from the maxillary and mandibular anterior buccal regions and the mandibular right posterior lingual region and pooled into an omnigene vial . plaque is taken with a stimudent or curette from three intact tooth surfaces ( 8-buccal , 24-buccal , 31-occlusal , or nearby surfaces if these are not intact ) and pooled into an omnigene vial . finally , plaque is taken from tooth surfaces with untreated lesions , including enamel hypoplasia , white spots , and cavitation of the enamel or dentin . up to three surfaces presenting the same type of lesion the caries assessment follows the phenx toolkit dental caries experience prevalence protocol ( http://www.phenxtoolkit.org/ , protocol number 080300 ) , modified to be as simple as possible for evaluation of the dentition in two - year - olds . tooth surface codes include a set of enamel hypoplasia codes following oliveira et al . and codes for active and inactive white spots . the tooth code for fluorosis has been replaced by the whole mouth fluorosis score , which facilitates the assessment of other tooth surface conditions . the modified tooth codes are compatible with the codes used in the cohra1 study and allow decayed , missing , and filled tooth ( dmft ) and surface ( dmfs ) scores to be calculated in two ways , including ( d1mft / s ) and excluding ( d2mft / s ) white spots . the child dental assessment does not include the unstimulated salivary flow rate or the malocclusion exam and is greatly simplified when very few teeth have erupted . the clinical protocol is performed by a licensed dentist or dental hygienist and a research assistant , using a dental chair with appropriate lighting . participants are asked not to eat or brush their teeth for two hours before the dental assessment . dental examiners and research assistants are trained in working with babies and young children , including use of the knee - to - knee position . the dental assessment does not include periodontal probing or any procedure that might induce bleeding . if women or children have oral health issues requiring treatment , appropriate referrals are made . in pittsburgh , research visits take place primarily at the university of pittsburgh center for craniofacial and dental genetics ( dr . mary l. marazita , director ) , with birth visits conducted at magee - womens hospital of upmc by either a member of the cohra2 research staff or the magee ctrc nurses . the research suite of the ccdg includes two fully equipped dental cubicles plus additional rooms for interviews and physical measurements . in west virginia , assessments are performed by the research staff at the two main study coordinating sites in morgantown and summersville , which have fully equipped dental cubicles , as well as at the 42 statewide partner sites described above . in practice , some of the more remote sites are not utilized often , because it is not cost - effective for the research team to drive several hours for a single appointment for which a participant may not show . most of the demographic and behavioral data is collected via a telephone questionnaire that is administered multiple times to the mothers from both sites by the university of pittsburgh center for social and urban research ( ucsur , dr . this 3045-minute interview collects data on mother and baby , including demographics ( education , ethnicity , household composition , medical and dental insurance , and income ) , detailed food and beverage intake over the past week , dietary habits ( frequency of meals , food anxiety , and purging ) , breastfeeding and bottle feeding routines , oral hygiene , medical and dental histories ( developmental delays , hospitalizations , and use of dental services ) , social behaviors and exposures ( smoking , alcohol , and recreational drug use ) , the center for epidemiologic studies depression scale ( ces - d ) , the perceived stress scale ( 10-item pss ) , a single omnibus overall fear item from the dental fear survey , caregivers inside and outside the home , and baby 's temperament , tooth eruption history , and sleeping habits , including the brief infant sleep questionnaire ( bisq ) . if women score 27 or higher on the ces - d scale , they are sent a letter advising that they may have symptoms of depression , along with a list of mental health resources including contact information specific to their place of residence . as summarized in table 1 , participants complete in - person assessments at 46 time points : ( 1 ) prenatal visit ( between 12 and 29 weeks of pregnancy ) ; ( 2 ) birth visit ( magee - womens hospital , pittsburgh only ) ; ( 3 ) pretooth visit ( baby is 2 - 3 months old ) ; ( 4 ) first - tooth visit ( one month after the eruption of baby 's first tooth , pittsburgh only ) ; ( 5 ) 1-year visit ; and ( 6 ) 2-year visit . the ucsur telephone questionnaire is administered once prenatally and five times postnatally about every six months through the course of the study . in the future , this timeline may be extended to older children . not all parts of the protocol are performed at every visit ( table 1 ) . in particular , the birth visit ( pittsburgh only ) has an abbreviated protocol and serves mainly to collect microbial samples before baby goes home from the hospital . pittsburgh also performs an in - person assessment within one month after the eruption of baby 's first tooth , in order to compare baby 's microbial community before and shortly after a tooth erupts . the research team in pittsburgh conducts a monthly short phone interview ( spi ) that includes questions about breastfeeding , baby 's diet and beverage intake , health issues , including doctor visits and antibiotic use , and any tooth eruptions . the purpose of the spi is to get baseline information on a frequent basis and to schedule the first - tooth visit . after the first tooth erupts , the spi is performed every 2 - 3 months . logistical issues involving extensive travel to research facilities statewide prevent the west virginia team from conducting the birth visit , first - tooth visit , and the spi . the telephone interview is conducted six times once prenatally and five times postnatally , when the baby is about 10 weeks old , 6 months old , 12 months old , 18 months old , and 24 months old . the uniformity and quality of the questionnaire data are maintained across sites , freeing site research staff to focus on recruiting , scheduling , and conducting study appointments . john j. warren , teresa a. marshall , and jeff murray who have contributed their expertise in pediatric dental research , dietary assessments , and genetics , respectively . oversight is provided by the nidcr through a clinical study oversight committee ( csoc ) that monitors scientific issues and progress in sample acquisition and periodic site visits by the clinical research operations and management support ( croms ) system . all members of the pittsburgh and west virginia research teams are trained and certified in the conduct of human subject research ( citi modules ) . the dental examiners have been trained and calibrated on the tooth codes from the caries assessment on a regular basis since 2011 , before data collection began . west virginia examiners conducted an initial training / calibration in march 2012 , at which 10 adults were assessed by four raters twice on two consecutive days . on day 1 , interrater reliability scores ( cohen 's kappa ) for sound , decayed , and filled groups of tooth surface codes ranged from 71.8 to 86.1 . following training and analysis of day 1 , formal training / calibration sessions are conducted on a regular basis for examiners from all sites , using adults , older children , and cohra2 participants . staff are compared to a gold standard dental examiner , following the guidelines of the early childhood caries collaborative centers ( ec4 , http://oralhealthdisparities.ucsf.edu/ ) , which was provided by dr . john j. warren ( protocol for the training and calibration of dental examiners , unpublished ) during a two - day training and calibration session in pittsburgh , pa . to calibrate raters , tooth surface codes are collapsed into two categoriessound and decayed / filled teeth which are necessary to calculate dmft scores . cohen 's kappas are calculated to determine the reliability of this distinction . by this measure , the two most experienced raters ( jgz , u pitt ; lb , wvu ) have an excellent kappa of 87.8 , ensuring good reliability across sites and making it possible for both of them to serve as gold standards for training and calibrating newer assessors at each site . in general , the average kappa across all staff ( compared to jgz ) is 70.8 , indicating substantial agreement . kappas for individual staff members range from 46.1 to 80.6 . during a calibration session , discrepant tooth scores are discussed immediately , so that initially moderate agreement is improved at subsequent sessions . for the telephone questionnaires administered by ucsur , all telephone interviewers are trained in general survey interviewing techniques via a standard three - day ucsur protocol and receive project - specific training , including detailed question - by - question instructions , from the pittsburgh site investigators . raw data for categorical variables were collapsed for cells with very small numbers , and missing / unknown responses ( less than 1% unless noted ) were removed . since several women completed their prenatal in - person visit but were withdrawn before their prenatal telephone interview , the total available responses differed for different variables . data were analyzed using the r statistical environment ( r foundation for statistical computing , vienna , au ) . means for three variables ( age , d1mft , and d2mft ) were compared using the nonparametric wilcoxon - mann - whitney test due to significantly nonnormal distributions . comparisons of categorical variables were performed using the chi - square test with appropriate degrees of freedom , with the yates continuity correction for 2 2 tables . fisher 's exact test was employed in the event that any set of data for a categorical variable did not satisfy the conditions for the chi - square test . as of january 31 , 2015 , the cohra2 study has enrolled 744 pregnant women , 368 from pittsburgh and 376 from west virginia . seventeen of these women have been enrolled twice , during two separate pregnancies , in order to conduct preliminary microbiological studies of siblings , so 727 independent women have completed the first prenatal in - person visit . of these 727 women , 153 subsequently have been withdrawn at different stages of the protocol ( 52 from pittsburgh , 101 from west virginia ) , for an overall retention rate of 79% ( 574/727 ) . first , premature delivery ( less than 35 weeks ' gestational age ) resulted in 13 women being withdrawn ( 8 from pittsburgh , 5 from west virginia ) , for a premature birth rate of 1.8% ( 13/727 ) . other reasons participants became ineligible include babies and/or mothers developing serious health problems ( n = 8) , mothers losing custody or not living with their children ( n = 8) , mothers moving out of the region ( n = 6 ) , baby deaths ( n = 5 ) , and miscarriages ( n = 2 ) , for a total of 42 women withdrawn because they became ineligible . these data were taken from the first in - person assessment and the first telephone interview , that is , during pregnancy , and divided by site . data from the 153 women who later were withdrawn are included in these tables . tables 2 and 3 summarize the women 's oral health status and behaviors , respectively . caries status is summarized in table 2 by the d1mft and d2mft scores , which differ based on the inclusion or exclusion of white spots . both dmft scores are significantly elevated in west virginia , compared to pittsburgh , indicating higher rates of caries in west virginia . gingival health is documented in table 2 by the ori score and a self - reported frequency of gingival bleeding during tooth brushing . in particular , 75% of the women in pittsburgh have ori scores of excellent or good , compared to 55% of the women from west virginia . tooth brushing and dental flossing habits are roughly the same between sites ( table 3 ) . over 70% of the women at both sites brush their teeth at least twice a day . more women sampled in pittsburgh floss ( 75% versus 67% , p = 0.02 ) , but they floss less frequently than the flossers in west virginia ( p = 0.03 ) . women in west virginia visit the dentist less frequently than women in pittsburgh ( p < 0.00001 ) . for example , 79% of the pittsburgh sample reported a visit to the dentist within one year , compared to 57% of the west virginian women . approximately 38% of the women in the study are first - time moms , and 97% of the women have some type of medical insurance . women in the west virginia sample are significantly younger than the pittsburgh sample ( average age 27.0 years versus 29.8 years , p < 0.00001 ) . they have significantly less education and higher rates of unemployment , and fewer of them have dental insurance ( 42% versus 87% , p < 0.00001 ) . finally , more of the west virginian sample self - reports their general health to be fair or poor , compared to pittsburgh . for the women living with at least one other person , the households in west virginia are significantly larger , due to increased numbers of children in the household ( p = 0.0002 ) . women in west virginia live in households that have significantly lower income ( p < 0.00001 ) and significantly more anxiety about food . 23% of households in the west virginia sample occasionally or often ran out of food in the past year , versus 12% from pittsburgh ( p = 0.001 ) . significantly more pregnant women in the pittsburgh sample reported drinking in the period beginning three months prior to the pregnancy and continuing into the second trimester ( 75% versus 53% , p < 0.00001 ) , while more women in the west virginia sample reported smoking ( 42% versus 32% , p = 0.02 ) . among the women who drank immediately before the pregnancy , a higher percentage in west virginia stopped drinking in the first trimester ( 73% versus 61% , p = 0.01 ) and the second trimester ( 94% versus 84% , p = 0.005 ) . with regard to smoking , 23% of the smokers at both sites did not smoke in the first trimester of pregnancy , and about 46% did not smoke in the second trimester . caries develops from an imbalance between demineralization and remineralization of tooth surfaces , when diet , oral hygiene , and the intrinsic features of the oral cavity create an environment favorable for cariogenic bacteria . in addition to the direct pathogenesis , however , multiple components operating on individual , family , and community levels influence caries risk [ 2426 ] , including factors that are specific to certain geographical regions or stages of life , such as pregnancy or young childhood [ 2730 ] . the cohra2 study is examining many of these factors in pregnant women and their babies over the early years of life , sampled from west virginia and the greater pittsburgh region . here , we describe the protocol and present summary data from 727 pregnant women enrolled to date . women who delivered at a gestational age of 35 - 36 weeks were retained in the study , even though the standard definition of full term delivery begins at 37 weeks . review of the literature determined that a very high percentage of babies born at gestational ages of 35 - 36 weeks are healthy , allowing us to retain these women and resulting in relatively low exclusion rates based on premature delivery . we observe that our samples of pregnant women recruited into cohra2 in west virginia , when compared to the sample recruited in pittsburgh , are younger , have worse oral health and similar brushing and flossing habits , see the dentist less often and have less dental insurance , less education , and more unemployment , live in households with more children and less income , drink less , and smoke more . additional work by the cohra2 research team relates pregnant women 's depression status to site differences , with possible clinical implications for dental practitioners . recruiting practices between the pittsburgh and west virginia sites may account for some of these observations . pregnant women are approached about the study and asked if they are interested ; only a few answer more general ads or respond to brochures . in contrast , there is no centralized source of pregnant women in largely rural west virginia , and a statewide health partnership network has been built , along with extensive advertising , to facilitate recruiting . these divergent recruiting strategies although necessary given the nature of the populations may result in different types of samples and may reflect an overall urban / rural difference between the two sites . limitations . the cohra2 study is still in the process of recruiting participants ; thus , sample sizes will be larger in the future . however , with 70% of the target sample size completed , the sample is sufficiently large for initial description of the data . increasing the sample should increase power but not fundamentally alter the nature of the observations presented in tables 26 . the cohra2 study was not designed to investigate differences between women sampled in west virginia versus pittsburgh . both pittsburgh and west virginia are part of northern appalachia , where oral health disparities are known to occur . pittsburgh , located about 120 kilometers from west virginia university in morgantown , has some of the same geographical qualities that are characteristic of west virginia , that is , a mountainous terrain broken up by river valleys . however , the observed demographic differences between subjects enrolled in the pittsburgh and west virginia sites reveal that northern appalachia is not one large demographically homogeneous region and serve to validate the design of the cohra2 study in selecting sites from different regions in northern appalachia . these demographic differences also underscore the importance of stratifying by site when studying genetic , environmental , and microbial factors contributing to poor oral health in the appalachian region . samples of pregnant women from the cohra2 study in west virginia and pittsburgh show different levels of oral health problems and have several different demographic properties . however , as the cohra2 study looks for associations between risk factors and dental caries , including the interactions between genetics , microbiology , and diet and other exposures , the associations should be more broadly generalizable , even if the prevalence of these factors is specific to the two study sites . the cohra2 study provides an invaluable wealth of data for understanding the oral health issues facing this region .

background . chronic poor oral health has a high prevalence in appalachia , a large region in the eastern usa . the center for oral health research in appalachia ( cohra ) has been enrolling pregnant women and their babies since 2011 in the cohra2 study of genetic , microbial , and environmental factors involved in oral health in northern appalachia . methods . the cohra2 protocol is presented in detail , including inclusion criteria ( healthy , adult , pregnant , us caucasian , english speaking , and nonimmunocompromised women ) , recruiting ( two sites : pittsburgh , pennsylvania , and west virginia , usa ) , assessments ( demographic , medical , dental , psychosocial / behavioral , and oral microbial samples and dna ) , timelines ( longitudinal from pregnancy to young childhood ) , quality control , and retention rates . results . preliminary oral health and demographic data are presented in 727 pregnant women , half from the greater pittsburgh region and half from west virginia . despite similar tooth brushing and flossing habits , cohra2 women in west virginia have significantly worse oral health than the pittsburgh sample . women from pittsburgh are older and more educated and have less unemployment than the west virginia sample . conclusions . we observed different prevalence of oral health and demographic variables between pregnant women from west virginia ( primarily rural ) and pittsburgh ( primarily urban ) . these observations suggest site - specific differences within northern appalachia that warrant future studies .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

further improvements in sequencing techniques have allowed sequences to be produced on a large scale and inserted into a computer . appropriate programs , capable of processing data , have been developed to facilitate access and make all of the information available throughout the process ( morozova and marra , 2008 ) . after dna sequencing is necessary to re - build the complete genome from the fragments obtained . genome assembly uses programs that read the sequences , in some cases find redundancies between them and merge the sequences , forming larger continuous consensus sequences . multiple rounds of assembly are sometimes required for find the best genome assembly sets . in the end of assembly process is necessary ordering the contigs between them or by anchoring in a reference genome to obtain the genome scaffold ( fagin et al . , 1992 ) . this entire process requires a large processing capacity , due to enormous amount of sequences generated by modern equipments . even a simple organism requires many hours of processing and memory for its complete assembly . the increasing availability of computers with massive computational power connected to high - speed networks has enabled the aggregation of geographically dispersed resources for the execution of large - scale applications . grid computing is a model that proposes the use of computing resources of several machines located in various places , even on separate continents , to solve problems that require massive computational power , such as data mining , weather forecasting , computational biology , and medical image processing ( foster , 2001 ; foster et al . , 2002 ; berman et al . , 2003 ) grids are currently being used as an alternative for clusters for achieving large - scale processing capacity . grid computing differs from cluster computing because of the heterogeneity of their resources ( which may consist of computers with different architectures , operating systems , and processing capabilities ) and due to the dynamic character of the same . cluster computing is defined as the sharing of resources working cooperatively and managed by a single global system synchronized and centralized . in grids , on other hand , each node has its own manager and resource allocation policy , which is not so visible ( foster et al . , 2008 ) . programs are composed of small pieces with specific responsibilities and clearly defined , called tasks . each task has a set of attributes , among them the priority that should be assigned according to their importance . each task is performed independently , but they need to interact with each other so that the system meets its objectives . in order for rules to be applied to a set of tasks that may use information from the various computers , it is necessary to adopt appropriate scheduling policies for each application . applications of the type bag - of - tasks ( bot ) facilitates scaling because they are composed of independent tasks , allowing the use of policies based on data from only a few systems . they do not require information on grid infrastructure , such as bandwidth , network topology , and network latency . policies can be called static or dynamic , depending on how the schedule is set ( foster and kesselman , 2004 ) . in spite of the bot applications are simple , it is not easy job to make the scheduling in a heterogeneous and dynamic environment such as grid computing . the scheduling of independents tasks in grid is still difficult due to the use of resources that are shared and due restraint created by other applications , which are running simultaneously . to obtain a good performance in this type of situation requires the use of good information to make scheduling more efficient . in order to achieve this purpose , we developed a dynamic algorithm for bot applications on grid environments because it has a better match and do not require detailed information ( cirne et al . the employment of an efficient algorithm for managing resources is crucial to reduce the time needed to finish tasks in a grid . here , we propose a task scheduling algorithm that takes grid characteristics into account and can be implemented within abyss , software used for parallelized de novo genome assembly , in order to optimize the performance of the genome assembly stage and consequently improve the efficiency of this process as a whole ( bittencourt and madeira , 2006 ; simpson et al . , 2009 ) . the main goal of our experiments was to evaluate the performance of abyss using the scheduling algorithm that we developed compared to the default scheduler bundled with abyss . the abyss is a software used for the de novo genome assembly and can be used in grids by using message passing interface ( mpi ) for the communication between nodes ( pacheco , 1996 ) . reads are distributed among the nodes to form a distributed graph , such that each node knows where the rest of the graph is . the proposed algorithm acts only at this stage of assembly of the distributed graph ( simpson et al . , 2009 ) . these experiments allowed us to evaluate the influence of the heterogeneity of networks ( different speeds ) , heterogeneity of the tasks ( size variation ) , and the granularity of tasks ( number of tasks per machine ) . we used genomic data available from the institute of biological sciences , federal university of para , consisting in a set of 33 millions short reads with fixed length ( 25 bp ) and 110 coverage of corynebacterium pseudotuberculosis i19 genome . this is a structured set of functions implemented in c language for the simulation driven to events , using an extendend mackup language ( xml ) file as input to define the network topology and the characteristics of resources and responsibilities . in these simulations , the network transfer times are negligible , because the focus of this analysis to verify the processing efficiency of the cpus using the proposed task scheduler ( casanova , 2001 ) . in order to run a simulation in simgrid , it is necessary to perform the following steps ( legrand et al . , 2003 ) : model the file of deployment of application , where is specified the location of the creation and allocation of agents . the experiments were conducted with six heterogeneous machines , i.e. , with different operating systems , hardware , and processing power . only the k - mer ( length hash ) parameter was changed to 17 , such value has proved satisfactory . the proposed scheduler does not change the mode of action of abyss , just the way as the tasks are distributed among the nodes . thus , the final assembly was not altered , because the main goal is not to improve the quality of the assembly using the abyss , but minimize the processing time that this step demands . the scheduler developed in the c language provides abyss with the capacity to distribute his tasks according to the rules above . all the files ( the scheduler itself , the configuration files for simgrid , the dataset used , and the detailed instructions ) are available on http://sourceforge.net/p/abyssgrid . the trend , which can be observed in figure 1 , is that in situations with a greater number of tasks per machine and less granularity , performance improves when there are many tasks . the situation changes only at the end of the execution due to load imbalance , which degrades performance . another point to consider is the execution time of tasks , which is relatively long because of the large computational load in the case of genomic data . figure 2 shows the impact of the heterogeneity of the machines in the grid . each point shows the levels of heterogeneity of tasks . we can conclude that the scheduler responds effectively in terms of processing time , even when machines are very heterogeneous . as shown in figure 2 , we can observe that processor 3 failed to perform well , because of the considerable heterogeneity of the machines . the possibility of a slow machine performing a large task is considerable , though it has only a small impact on the execution time of tasks , as was the case for processors 4 and 5 . figure 3 shows the impact of heterogeneity on the performance of the tasks scheduling algorithm . we can conclude that the heterogeneity of the tasks did not have a significant impact on the performance of the task scheduler that we proposed , demonstrating its efficiency in this context figure 4 show that using abyss without our algorithm spent more time spent to execute all the tasks in the same environment . this is due to the fact that the abyss uses , to distribute its tasks , the first in , first out ( fifo ) algorithm , which does not take into account the differences between the available resources in the environment or its load . grid computing has greatly advanced because of the demand for scientific applications that require processing , storage , and manipulation of large amounts of information . fields that require such processing power include molecular biology , astronomy , and earthquake prediction . genome sequencing techniques are also advancing and generating very large amounts of data that must be processed using resources with great computational power . computational grids can be used to address this demand for execution of parallel or distributed applications . though computers have become much more powerful , the genome assembly stage continues to confront limitations because of the type and volume of data generated by next - generation sequencers . increasing amounts of data exponentially rise the processing time , which can overwhelm the assembly process , so that it becomes necessary to implement techniques that will minimize these limitations . future work will be based on improvements in the scheduler so the distribution of tasks is made by first checking if the processors available have enough computing power to speed up processing . based on the experiments , the obtained results were considered satisfactory , since the assembly can be completed , and when such results are compared to the assembly without the use of the scheduler developed , they proved to be faster . the fact of the machines has used different configurations , and the tasks have different sizes , such fact did not affect significantly the completion and assembly performance . the simulations also showed that the scheduling algorithm submitted to testing can be considered scalable in its entirety , since it reached a minimum efficiency , and for any tested task , there was a minimum number of processors available or imminently available to be perform the processing . we proposed a scheduling algorithm for computer grids , using the de novo assembly software abyss . the algorithm proved being very effective in tests with heterogeneous tasks , and there was no impact on processor performance . however , when granularity increased , we identified a small imbalance , causing decreased processor performance . the runtime of the tasks did not affect processor performance in the tests that we run . when the tests were performed with a heterogeneous group of machines , the scheduler responded effectively to the processing challenge . even when there was deterioration in performance due to a slow machine that was allocated a large task , other processes ended up compensating for the slight loss of time in the task . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

improvements in genome sequencing techniques have resulted in generation of huge volumes of data . as a consequence of this progress , the genome assembly stage demands even more computational power , since the incoming sequence files contain large amounts of data . to speed up the process , it is often necessary to distribute the workload among a group of machines . however , this requires hardware and software solutions specially configured for this purpose . grid computing try to simplify this process of aggregate resources , but do not always offer the best performance possible due to heterogeneity and decentralized management of its resources . thus , it is necessary to develop software that takes into account these peculiarities . in order to achieve this purpose , we developed an algorithm aimed to optimize the functionality of de novo assembly software abyss in order to optimize its operation in grids . we run abyss with and without the algorithm we developed in the grid simulator simgrid . tests showed that our algorithm is viable , flexible , and scalable even on a heterogeneous environment , which improved the genome assembly time in computational grids without changing its quality .

Introduction Materials and Methods Results Discussion Conclusion Conflict of Interest Statement

the clinical implications of left axis deviation ( lad ) in patients with left bundle branch block ( lbbb ) have not been clearly elucidated . some studies have reported that patients with lad do not have a significantly different prognosis compared with patients with a normal qrs axis,1)2)3 ) while others have shown that patients with lad have a higher incidence of conduction abnormalities and a poor prognosis , including cardiovascular death.4)5)6 ) regardless of the differences in prognosis , previous studies have proposed that diseased myocardium is a plausible mechanism for axis deviation.3)4 ) the selvester qrs scoring system evaluates myocardial scarring based on 12-lead electrocardiogram ( ecg ) readings and has been updated for extensive application : it can be used to evaluate patients with bundle branch block and also those with pacing rhythm.7)8)9 ) the 12-lead ecg has valuable clinical significance and has been validated with cardiac magnetic resonance imaging.10)11 ) furthermore , since the 12-lead ecg is a widely used diagnostic tool that is highly accessible , the myocardial scar score derived from ecg may be valuable in clinic environments . in this study , the association between the qrs axis and the myocardial scar score was investigated in patients with lbbb . in addition , the importance of a left axis deviation concomitant with lbbb was evaluated . this was a retrospective , observational cohort study . from october 2004 to june 2014 , 829 patients were evaluated by 12-lead ecg and diagnosed with lbbb at seoul national university hospital ( seoul , korea ) . one hundred five patients were excluded because they did not have ecg results available for review , or they had been misdiagnosed with lbbb . an additional 410 patients were excluded due to one or more of the following : severe valvular heart disease , presence of an implantable cardiac defibrillator or cardiac resynchronization therapy ( crt ) device , history of cardiac surgery or intervention , ischemic heart disease documented by imaging studies , symptoms of ischemic heart disease or heart failure , or previously proven cardiomyopathy or myocarditis ( fig . we obtained demographic data , data on the presence of underlying diseases , clinical presentation , laboratory test results , and the initial diagnosis of lbbb from the hospital electronic medical records . blood sampling and tests were performed as part of routine practice by laboratories certified by the korean association of quality assurance for clinical laboratory . the criteria for diagnosing lbbb by 12-lead ecg were defined as : qr or rs in leads v1 or v2 ; mid - qrs notching or slurring in 2 of leads v1 , v2 , v5 , v6 , i , and avl ; and qrs duration 140 ms ( male ) or 130 ms ( female).12)13 ) normal axis was defined as -30 r axis < 90. in addition to information on gender and age , a myocardial scar score was calculated according to the previously reported selvester scoring protocol , which was applicable for lbbb patients.8)10 ) the primary endpoint was any major adverse cardiovascular event ( mace ) , defined as a composite of sustained ventricular tachycardia or ventricular fibrillation , pacemaker implantation due to complete atrioventricular block , crt implantation , hospital admission due to heart failure , or cardiovascular death . data are described as meanstandard deviation for continuous variables and as numbers and frequencies for categorical variables . the test or fisher 's exact test was applied for categorical variables , and an unpaired student 's t - test was used for continuous variables . a receiver - operating - characteristics curve was used to evaluate the predictive power of the myocardial scar score on lad . kaplan - meier estimates were applied to evaluate the chronological inclination of outcomes , and the log - rank test was used to analyze the differences between groups ( subjects with lad , subjects with normal axis ) . a multivariable cox proportional - hazards regression model variables associated with mace with p<0.1 in the univariate analysis were included as confounding factors in the multivariate analysis . this was a retrospective , observational cohort study . from october 2004 to june 2014 , 829 patients were evaluated by 12-lead ecg and diagnosed with lbbb at seoul national university hospital ( seoul , korea ) . one hundred five patients were excluded because they did not have ecg results available for review , or they had been misdiagnosed with lbbb . an additional 410 patients were excluded due to one or more of the following : severe valvular heart disease , presence of an implantable cardiac defibrillator or cardiac resynchronization therapy ( crt ) device , history of cardiac surgery or intervention , ischemic heart disease documented by imaging studies , symptoms of ischemic heart disease or heart failure , or previously proven cardiomyopathy or myocarditis ( fig . we obtained demographic data , data on the presence of underlying diseases , clinical presentation , laboratory test results , and the initial diagnosis of lbbb from the hospital electronic medical records . blood sampling and tests were performed as part of routine practice by laboratories certified by the korean association of quality assurance for clinical laboratory . the criteria for diagnosing lbbb by 12-lead ecg were defined as : qr or rs in leads v1 or v2 ; mid - qrs notching or slurring in 2 of leads v1 , v2 , v5 , v6 , i , and avl ; and qrs duration 140 ms ( male ) or 130 ms ( female).12)13 ) normal axis was defined as -30 r axis < 90. in addition to information on gender and age , a myocardial scar score was calculated according to the previously reported selvester scoring protocol , which was applicable for lbbb patients.8)10 ) the primary endpoint was any major adverse cardiovascular event ( mace ) , defined as a composite of sustained ventricular tachycardia or ventricular fibrillation , pacemaker implantation due to complete atrioventricular block , crt implantation , hospital admission due to heart failure , or cardiovascular death . data are described as meanstandard deviation for continuous variables and as numbers and frequencies for categorical variables . the test or fisher 's exact test was applied for categorical variables , and an unpaired student 's t - test was used for continuous variables . a receiver - operating - characteristics curve was used to evaluate the predictive power of the myocardial scar score on lad . kaplan - meier estimates were applied to evaluate the chronological inclination of outcomes , and the log - rank test was used to analyze the differences between groups ( subjects with lad , subjects with normal axis ) . a multivariable cox proportional - hazards regression model was used to investigate the independent prognostic indicators of mace . variables associated with mace with p<0.1 in the univariate analysis were included as confounding factors in the multivariate analysis . statistical tests were performed using ibm spss statistics version 22 ( spss inc . , chicago , il , usa ) . the mean age was 68 years , 105 patients ( 33% ) were male , 107 patients ( 34% ) had hypertension , 89 patients ( 28% ) had diabetes mellitus , and 32 patients ( 10% ) had dyslipidemia . when classified according to qrs axis , a total of 91 patients ( 29% ) were classified with lad , and the remaining 71% had a normal axis . the meanstandard deviation myocardial scar score was 3.62.0 for the normal axis group and 5.12.0 for the lad group . the lbbb patients with normal qrs axis were younger and had comparable prevalence of hypertension , diabetes mellitus , and dyslipidemia to those with left axis deviation . there was a significant inverse correlation between the myocardial scar score and the qrs axis ( r=-0.356 , p<0.001 ) ( fig . the myocardial scar score was also significantly associated with age ( r=0.184 , p=0.001 ) and hemoglobin level ( r=-0.165 , p=0.007 ) ; it was not correlated with body mass index ( r=-0.028 , p=0.627 ) , total cholesterol level ( r=-0.039 , p=0.526 ) , or serum creatinine level ( r=0.040 , p=0.504 ) . in the roc curve analysis , the area under the curve of the myocardial scar score to predict lad was 0.700 ( 95% ci 0.639 - 0.761 , p<0.001 ) ( fig . the median follow - up duration was 30 months ( interquartile range : 6 to 63 months ) . during the follow - up period , 10 of 314 patients died ( including two cardiovascular deaths caused by ventricular fibrillation ) . four patients underwent pacemaker implantation due to complete atrioventricular block , and two patients were admitted to the hospital due to de novo acute heart failure ( table 2 ) . stratification according to the qrs axis indicated that patients in the lad group were older and demonstrated a higher incidence of mace compared with patients with a normal axis ( 5.5% vs. 1.3% , log - rank p=0.010 ) ( fig . lad was a significant prognostic indicator of mace in the univariate analysis [ hazard ratio ( hr ) 5.400 ; 95% confidence interval ( ci ) 1.281 - 22.758 ; p=0.022 ] , while myocardial scar score failed to show prognostic implication ( hr 0.877 ; 95% ci 0.619 - 1.243 ; p=0.460 ) . the lad predictive value was marginally attenuated in multivariate analysis ( hr 4.117 ; 95% ci 0.955 - 17.743 , p=0.058 ) after adjusting for the confounding variables of age , sex , and diabetes mellitus ( table 3 ) . the mean age was 68 years , 105 patients ( 33% ) were male , 107 patients ( 34% ) had hypertension , 89 patients ( 28% ) had diabetes mellitus , and 32 patients ( 10% ) had dyslipidemia . when classified according to qrs axis , a total of 91 patients ( 29% ) were classified with lad , and the remaining 71% had a normal axis . the meanstandard deviation myocardial scar score was 3.62.0 for the normal axis group and 5.12.0 for the lad group . the lbbb patients with normal qrs axis were younger and had comparable prevalence of hypertension , diabetes mellitus , and dyslipidemia to those with left axis deviation . there was a significant inverse correlation between the myocardial scar score and the qrs axis ( r=-0.356 , p<0.001 ) ( fig . the myocardial scar score was also significantly associated with age ( r=0.184 , p=0.001 ) and hemoglobin level ( r=-0.165 , p=0.007 ) ; it was not correlated with body mass index ( r=-0.028 , p=0.627 ) , total cholesterol level ( r=-0.039 , p=0.526 ) , or serum creatinine level ( r=0.040 , p=0.504 ) . in the roc curve analysis , the area under the curve of the myocardial scar score to predict lad was 0.700 ( 95% ci 0.639 - 0.761 , p<0.001 ) ( fig . the median follow - up duration was 30 months ( interquartile range : 6 to 63 months ) . during the follow - up period , 10 of 314 patients died ( including two cardiovascular deaths caused by ventricular fibrillation ) . four patients underwent pacemaker implantation due to complete atrioventricular block , and two patients were admitted to the hospital due to de novo acute heart failure ( table 2 ) . stratification according to the qrs axis indicated that patients in the lad group were older and demonstrated a higher incidence of mace compared with patients with a normal axis ( 5.5% vs. 1.3% , log - rank p=0.010 ) ( fig . lad was a significant prognostic indicator of mace in the univariate analysis [ hazard ratio ( hr ) 5.400 ; 95% confidence interval ( ci ) 1.281 - 22.758 ; p=0.022 ] , while myocardial scar score failed to show prognostic implication ( hr 0.877 ; 95% ci 0.619 - 1.243 ; p=0.460 ) . the lad predictive value was marginally attenuated in multivariate analysis ( hr 4.117 ; 95% ci 0.955 - 17.743 , p=0.058 ) after adjusting for the confounding variables of age , sex , and diabetes mellitus ( table 3 ) . the study analysis indicated that the qrs axis of lbbb patients was associated with the myocardial scar score , and lad concomitant with lbbb was a prognostic indicator of mace . the clinical prognostic importance of lad in lbbb patients has been reported in numerous studies ; however , the results remain controversial.1)2)3)4)5)6)14)15 ) a recent study designed to include subjects without clinical cardiovascular disease reported that lad was associated with a higher risk of mortality and cardiovascular disease regardless of concomitant lbbb.16 ) the reason for the different prognostic impact between studies , including ours , may be explained by the different study populations , definition of lad , and study period ( table 4 ) . although the exact mechanism for developing lad is still uncertain , it has been suggested that comorbidities such as hypertension and diabetes mellitus exert an influence and result in conduction disturbance of fascicles and the main left bundle.3)16 ) in addition to prolonged and asynchronous contraction due to concomitant lbbb , this could also cause poor clinical outcomes . myocardial scarring is generated by both ischemic and nonischemic damage.17)18)19 ) the electrical depolarization initiates from the free and septal walls of the right ventricle in lbbb patients , but this vector may be interrupted and recorded differently in an ecg if myocardial scarring is present.8 ) because myocardial scarring is considered to be a cumulative indicator reflecting myocardial damage , we speculated that the qrs axis is related to the myocardial scar score . the selvester qrs score , a quantitative scoring system calculated from 12-lead ecg , can be used to estimate the myocardial scar amount in the left ventricle . the scoring system was established to estimate the myocardial infarct size and has been improved and updated for applications in various conditions , including lbbb.7)8)9)10 ) in lbbb patients , the myocardial scar calculated by the selvester qrs score and that measured by cardiac magnetic resonance imaging were significantly correlated ( r=0.80 , p<0.001 ) and showed a mean difference of 7.6% overestimation . in regard to reproducibility , absolute differences of 0.4 and 0.6 myocardial scar scores were observed in intraobserver and interobserver examination , respectively . the intraobserver and interobserver agreement were =0.96 and =0.86 , respectively , in a previous report.10 ) in this study , we measured the myocardial scar with the selvester qrs scoring system based on the accuracy and reproducibility of the test . our data was similar to previous studies that investigated the prognosis of patients with lbbb and lad and demonstrated a significant negative correlation between the myocardial scar score and the qrs axis in patients with lbbb . despite the significant inverse relationship between myocardial scar score and qrs axis , a high myocardial scar score was not a statistically significant prognostic indicator , in contrast to the presence of lad in lbbb patients ( table 3 ) . results have indicated that lad , not the myocardial scar score , was a prognostic indicator in lbbb patients , and that lad might be partially influenced by myocardial scarring as well as by other confounding pathologic factors . this was a retrospective cohort study , rather than a prospective cohort study ; therefore , there could be unmeasured confounding variables that influenced the results . additionally , although we only included asymptomatic patients who did not have abnormal cardiac imaging studies , we were not able to completely eliminate patients had asymptomatic structural or functional heart disease who therefore did not undergo a cardiac evaluation before the recruitment . finally , the r / s or r / q ratio at lead ii was included in the selvester qrs scoring system , suggesting that patients with lad had higher myocardial scar scores . however , the myocardial scar score difference between the two groups was larger than the scoring point of lead ii . concomitant lad is a prognostic indicator that can predict poor prognosis for patients with lbbb and may be associated with greater myocardial scarring . there was a significant inverse correlation between the qrs axis and the myocardial scar score based on 12-lead ecg results . this was a retrospective cohort study , rather than a prospective cohort study ; therefore , there could be unmeasured confounding variables that influenced the results . additionally , although we only included asymptomatic patients who did not have abnormal cardiac imaging studies , we were not able to completely eliminate patients had asymptomatic structural or functional heart disease who therefore did not undergo a cardiac evaluation before the recruitment . finally , the r / s or r / q ratio at lead ii was included in the selvester qrs scoring system , suggesting that patients with lad had higher myocardial scar scores . however , the myocardial scar score difference between the two groups was larger than the scoring point of lead ii . concomitant lad is a prognostic indicator that can predict poor prognosis for patients with lbbb and may be associated with greater myocardial scarring . there was a significant inverse correlation between the qrs axis and the myocardial scar score based on 12-lead ecg results .

background and objectivesleft bundle branch block ( lbbb ) with left axis deviation ( lad ) has a worse prognosis than lbbb with a normal axis , and myocardial dysfunction has been suggested as a cause of left axis deviation . this study investigated the prognostic significance of the qrs axis in patients with lbbb and analyzed its relationship with the amount of myocardial scarring.subjects and methodsa total of 829 patients were diagnosed with lbbb at seoul national university hospital from october 2004 to june 2014 . of these , 314 who were asymptomatic and had no previous history of cardiac disease were included in the present study . myocardial scarring was calculated using the selvester qrs scoring system , and lad was defined as a qrs axis between -180 and -30.resultsof the total patients , 91 ( 29% ) had lad , and patients were followed for a median of 30 months . during follow - up , two patients were hospitalized for de novo heart failure , four had pacemaker implants , and 10 died . there was a significant inverse correlation between myocardial scar score and the qrs axis ( r=-0.356 , p<0.001 ) . patients with concomitant lad had a higher rate of major cardiac adverse events compared with patients with a normal axis ( 5.5% vs. 1.3% , log - rank p=0.010 ) ; the prognostic value was attenuated in multivariable analysis ( hazard ratio 4.117 ; 95% confidence interval 0.955 - 17.743 ; p=0.058).conclusionconcomitant lad is an indicator of poor prognosis for patients with lbbb and may be associated with greater myocardial scarring .

Introduction Subjects and Methods Patients and data collection Endpoints Statistical analysis Results Baseline characteristics of the study population Association between myocardial scar score and QRS axis Clinical outcomes Discussion Limitations Conclusion

rheumatoid arthritis ( ra ) and other connective tissue disorders are frequently associated with certain types of corneal or / and scleral pathology such as peripheral ulcerative keratitis , dry eye syndrome , and scleritis . it is also known that these conditions may lead to perforation of the eye by compromising the integrity of its outermost layers . keratoconus ( kc ) is generally regarded as an isolated condition with no apparent ophthalmic or systemic associations at the time of diagnosis . however , elevated levels of corneal proinflammatory cytokines such as interleukin ( il)-1 have been linked to corneal thinning , implying the involvement of the immune system in the pathogenesis of kc . only a small number of autoimmune diseases ( e.g. , ra , asthma , inflammatory bowel disease ) have been related to kc , while others have not ( e.g. , systemic lupus , crohn s disease , myasthenia gravis , and multiple sclerosis ) . cell - mediated mechanisms are suggested to play a pivotal role in the pathogenesis of ectatic corneal disorders and kc itself . further , confocal microscopy has revealed that in patients with ra , the central corneal and stromal thicknesses are significantly lower compared with controls , and these patients have significantly higher numbers of hyperreflective stromal cells . the activation of these keratocytes is mediated by proinflammatory cytokines , such as il-1 and il-6 . we report a case of paracentral corneal perforation in a patient with keratoconus and a previously undiagnosed connective tissue disorder . a 53-year - old man presented in the emergency room complaining of severe ocular pain with sudden onset and a significant decrease in visual acuity in his left eye . regarding his ocular history , the patient noted that his ophthalmologist had been modifying his spectacle prescription annually over the last 13 years because of the increasing myopic shift . the patient reported no systemic drug intake except for analgesics for constant lumbar back pain . the patient s best corrected visual acuity was measured cf at 10 cm for the left and 20/60 for his right eye . his spectacle prescription at the time of the examination was : 1.00/5.5 100 os and 4.5/4.5 70 od . slit lamp biomicroscopy revealed a paracentral corneal perforation , 1 2 mm , accompanied by iris prolapse , corneal thinning , and a markedly shallow anterior chamber in the left eye . the right eye displayed clinical features of moderate keratoconus , which was confirmed by corneal topography ( magellan mapper , nidek ; figs . 1 and 2).fig . steep corneal curvature on biomicroscopy raised suspicions of a coexistent keratoconus topography image demonstrating significant keratoconus on the right eye the patient was immediately prepared for surgical intervention in order to preserve the integrity of the affected eye . amniotic membrane ( am ) was primarily available and transplanted in an attempt to seal the perforation and promote corneal healing ( fig . cryopreserved am was attached over the perforation in multiple layers and covered with an epithelial - side - up , limbal - sutured sheet . the surgical technique involved scraping of residual corneal epithelium and 360 limbal peritomy of the conjunctiva prior to am transplantation . the outermost of the multiple layers was sutured with 8/0 vicryl on the cornea whereas the overlaid transplant was sutured at the limbus with a running 10/0 nylon . a bandage contact lens was applied at the end of the operation in order to promote reepithelialization of am and to prevent mechanical rubbing of the transplant on blinking.fig . 3photo of left eye on second post - op day after amniotic membrane transplantation in multiple layers in an attempt to maintain the eye s anatomical integrity photo of left eye on second post - op day after amniotic membrane transplantation in multiple layers in an attempt to maintain the eye s anatomical integrity further blood tests revealed a significantly elevated rheumatoid factor at 2,170 iu / ml ( reference value < 20 iu / ml ) . specific blood tests in addition to detailed clinical examination carried out by an orthopedic surgeon and immunologist confirmed the diagnosis of ra , and the patient was referred to rheumatologists for further assessment . the operated on eye displayed a well - formed anterior chamber , without any leakage , over the 3 post - op weeks . visual acuity was slightly improved to cf at 50 cm . however , reepithelialization failed to cover am completely whilst subsequent tissue thinning resulted in re - leakage at week 4 postoperatively . one month after corneal grafting and while on systemic immunosuppressants , the patient developed endophthalmitis with unfavorable outcome . kc is a noninflammatory corneal disorder in which stromal thinning and ectasia result in an atypical corneal shape . although the etiology is not completely understood , there is evidence suggesting that kc probably represents a multifactorial process . kc with acute hydrops can be complicated with spontaneous corneal perforation ; however , this has been reported mainly in cases with advanced kc . nevertheless , spontaneous perforation in mild to moderate kc cases has also been reported recently . ophthalmic involvement in autoimmune disorders such as ra , systemic lupus erythematosus , and sjgren syndrome has been extensively described . however , many patients experience ocular sequelae during the course of undiagnosed immunological disorders . reduced tear secretion , chronic inflammation of the ocular surface , as well as ultrastructural abnormalities are believed to play a major part in decompensating the ocular surface . this condition may lead to spontaneous perforation in sporadic cases . in our case , am transplantation was used as a first - line solution since previous studies showed that urgent penetrating keratoplasties in perforated eyes displayed higher rates of graft failure compared to those performed several weeks to months following the perforation [ 10 , 11 ] . multilayer am transplantation has been reported to promote epithelial healing and reduce inflammation , neovascularization , and scarring in a diversity of external eye disorders . am - related local anti - inflammatory effects contribute to faster and more efficient reepithelialization . furthermore , am serves as a biological scaffolding in cases where normal tissue is lost and promotes reinforcement or even sealing of small perforations . am has been used in patients with ra - related epithelial defects as a temporizing solution with the aim for delayed reconstruction . however , as solomon and colleagues reported , am transplantation showed poorer outcomes in patients with systemically undertreated ra compared to other autoimmune conditions like ocular pemphigoid or even sjogren s . other authors have proposed cyanoacrylate and fibrin glue adhesives as an efficacious solution in cases where corneal perforations occur [ 13 , 14 ] . in our case , am transplantation was preferred over other treatment modalities mainly due to severe inflammation of the ocular surface and the small size of the perforation . besides , the diagnosis of ra had not yet been established . furthermore , penetrating keratoplasty does not represent a first - choice operation in our country due to the relatively restricted number of centers authorized to perform corneal transplantations and to limited availability of corneal grafts . in our patient , although the am transplant initially provided a sufficient corneal sealing , it failed as a long - term solution due to poor reepithelialization and the progressive tissue degradation . recurrent aqueous humor leak necessitated an emergent penetrating keratoplasty , which took place in another center . there are quite a few studies that extensively describe the relationship between ra and ocular surface disorders that may permanently affect vision [ 15 , 16 ] . kc is another thoroughly studied condition that , apart from major refractive alterations , may occasionally cause corneal perforation when undiagnosed or / and untreated . to the best of our knowledge , this is the first report of a case with corneal perforation arising from coexistent kc and ra . it seems possible that the impact of these conditions is more severe when they interact . we should therefore raise awareness and either rule out or efficiently treat underlying connective tissue disorders in patients with kc . this article is distributed under the terms of the creative commons attribution license which permits any use , distribution and reproduction in any medium , provided the original author(s ) and source are credited .

purposethe purpose of this study is to report a case of corneal perforation in a patient with undiagnosed keratoconus and underlying rheumatoid arthritis.methodsthis is a retrospective case study based on the patient s medical records and followed by a brief review of the literature.resultsa 53-year - old patient was referred to our department for acute pain and sudden decrease of visual acuity in his left eye . corneal perforation was detected and attributed to a previously undiagnosed and untreated keratoconus . additional laboratory work - up and clinical examination revealed a coexisting rheumatoid arthritis . amniotic membrane was originally transplanted in order to maintain the structural integrity and promote healing of the perforated eye . subsequently , the patient underwent a penetrating keratoplasty , though with unfavorable results due to postoperative endophthalmitis.conclusionsrheumatoid arthritis may be associated with higher risk of corneal perforation in patients with ectatic degenerative diseases such as keratoconus .

Introduction Case report Discussion Conclusion Conflict of interest Open Access

pushing the boundaries of surgical options in the curative treatment of hepatic malignancies has recently led to the development of a new surgical technique for patients in whom a conventional major liver resection ( 3 liver segments ) is not feasible . the technique , known as alpps ( associating liver partition and portal vein ligation for staged hepatectomy ) , is a two - stage procedure encompassing portal vein ligation and in situ split of the liver parenchyma during the first phase and completion of the resection by removal of the deportalized liver after an interval of 12 weeks in the second phase ( fig . alpps is indicated in patients in whom insufficient volume of remnant liver is anticipated carrying a high risk of postoperative liver failure . this technique induces an accelerated hypertrophy response of the future remnant liver ( frl ) following contralateral ligation of the portal vein , while transection of the parenchyma allows the deportalized liver to contribute to liver function until the second stage . however , since the introduction of alpps , there has been an ongoing discussion regarding its safety , as the morbidity ( i.e. , clavien - dindo grade iiib ) and 90-day mortality rates reported in the literature reach up to 59 and 29% , respectively . one of the key issues in alpps is the timing of the second stage at the point sufficient hypertrophy response has been achieved . this modality enables volumetric measurement of the total liver , frl and tumor volumes . in patients with healthy liver parenchyma , frl volume and function can be considered equal . however , in the majority of cases , the quality of the liver remains uncertain and may be affected by longstanding chemotherapy or fibrosis . tc - mebrofenin hepatobiliary scintigraphy ( hbs ) with spect - ct is a quantitative liver function test measuring hepatic uptake of this liver - specific tc - labeled iminodiacetic acid derivative . firstly , hbs is performed together with spect - ct allowing not only the measurement of the total uptake of mebrofenin in the whole liver , but also regional measurements in the liver segments , e.g. frl . secondly , one single cutoff value of 2.7%/min / m has been validated for both patients with compromised and with noncompromised liver parenchyma . since the validation of the cutoff value for the prediction of postoperative liver failure , hbs has become part of standard patient care at our center for patients who require major liver resection . hbs has also proven useful in the assessment of the hypertrophy response of the frl after preoperative portal vein embolization and selective internal radiation therapy [ 5 , 7 ] . as accurate assessment of the frl function rather than its volume is crucial in performing the alpps procedure , hbs could play a prominent role in monitoring frl function after the first stage of alpps , giving guidance in the timing of the second stage . we herein report the sequential changes in frl function measured using hbs in a patient who underwent alpps procedure . a 79-year - old female patient was referred to our surgical department with metachronous colonic liver metastases . the patient was diagnosed with 3 lesions suspicious of metastases in segments 4a , 7 and 8 , and she was scheduled for metastasectomy of the aforementioned lesions . however , at examination , additional lesions in segments 3 , 5 and 6 were found necessitating extended right hemihepatectomy ( i.e. resection of segments 48 ) with concomitant metastasectomy of the lesion in segment 3 . this procedure was not considered feasible at the time of examination as the frl volume was 19.6% , which was below the volumetric cutoff value for safe resection ( 25% of total liver volume ) . furthermore , the frl uptake function of segments 1 , 2 and 3 was only 1.5%/min / m , which is also below the cutoff value for safe resection ( 2.7%/min / m ; table 1 ) . therefore , it was decided to apply the alpps procedure , and the first stage was carried out in order to induce hypertrophy of segments 2 and 3 . the study has been approved by the institutional review board , and the need for written informed consent was waived . ct imaging with ct volumetry and hbs were performed before and after each stage of the alpps . hbs was performed after a 4-hour fast , as food consumption stimulates hepatic function and bile flow , which might influence test results . the patient was put in a supine position on the imaging table and a large fov spect - ct camera ( symbia t16 ; siemens ) was positioned over the liver and heart region . the spect - ct camera was equipped with low - energy high - resolution collimators . after intravenous administration of 200 mbq ( 5.41 mci ) freshly prepared tc - mebrofenin ( bridatec ; ge - amersham health ) , dynamic acquisition was obtained ( 36 frames of 10 s / frame , 128 128 matrix ) , which was used for the calculation of the hepatic mebrofenin uptake rate . subsequently , a fast spect acquisition was performed ( 60 projections of 8 s / projection , 256 256 matrix ) , centered on the peak of the hepatic time - activity curve , which was used for the 3-dimensional assessment of liver function and the calculation of functional liver volume . immediately after spect , a low - dose non - contrast - enhanced ct scan was obtained for attenuation correction and anatomical mapping . in order to evaluate biliary excretion , a second dynamic acquisition ( 15 frames of 60 the hbs parameters related to mebrofenin uptake in the total liver and frl were calculated as described before [ 5 , 9 ] . a cut - off value of 2.7%/min / m was used to discriminate the normal from decreased frl uptake rate as was validated in a previous study . ct imaging with ct volumetry and hbs were performed before and after each stage of the alpps . hbs was performed after a 4-hour fast , as food consumption stimulates hepatic function and bile flow , which might influence test results . the patient was put in a supine position on the imaging table and a large fov spect - ct camera ( symbia t16 ; siemens ) was positioned over the liver and heart region . the spect - ct camera was equipped with low - energy high - resolution collimators . after intravenous administration of 200 mbq ( 5.41 mci ) freshly prepared tc - mebrofenin ( bridatec ; ge - amersham health ) , dynamic acquisition was obtained ( 36 frames of 10 s / frame , 128 128 matrix ) , which was used for the calculation of the hepatic mebrofenin uptake rate . subsequently , a fast spect acquisition was performed ( 60 projections of 8 s / projection , 256 256 matrix ) , centered on the peak of the hepatic time - activity curve , which was used for the 3-dimensional assessment of liver function and the calculation of functional liver volume . immediately after spect , a low - dose non - contrast - enhanced ct scan was obtained for attenuation correction and anatomical mapping . in order to evaluate biliary excretion , a second dynamic acquisition ( 15 frames of 60 s / frame , 128 128 matrix ) was obtained . the hbs parameters related to mebrofenin uptake in the total liver and frl were calculated as described before [ 5 , 9 ] . a cut - off value of 2.7%/min / m was used to discriminate the normal from decreased frl uptake rate as was validated in a previous study . figure 2a shows the ct image after the first stage of the alpps procedure . on postoperative day ( pod ) 3 , the frl volume had increased from 19.6 to 26.2% of the total liver volume , while the frl function had increased from 1.5 to 2.0%/min / m . the frl volume was measured again on pod 6 showing a further increase in volume up to 26.5% ( table 1 ) . on pod 8 , the frl function had increased above the cutoff value to 2.9%/min / m ( fig . the functional and volumetric increase in the frl after 1 week was comparable to volumetric and functional measurements performed on pod 90 in a historic cohort of patients who underwent major liver resection at our institute . the postoperative course was complicated by ascites due to thrombosis of the left hepatic vein , which resolved after anticoagulation therapy . nearly all of the patients reported after alpps showed sufficient volume hypertrophy after the first stage of the procedure and went on to the second stage . however the morbidity and mortality rates after alpps are high when compared to liver resection after preoperative portal vein embolization or in the setting of a conventional two - stage liver resection with an interval of 46 weeks . the timing of the second stage of the procedure is largely determined by the degree of the hypertrophy response . in this paper , we demonstrate a marked increase in the frl function measured with hbs in a patient who underwent both stages of the procedure , showing that only 8 days after transection of the liver parenchyma , the mebrofenin uptake rate of the frl had nearly doubled and was comparable with the average functional increase documented 90 days after regular major liver resection . furthermore , we were able to show a substantial difference between the increase in frl volume and function . just 3 days after the first stage of the resection , the frl volume had increased from 19.6 to 26.2% indicating that the patient was ready to undergo the next step of the operation as the cutoff value for safe resection is approximately 25% . however , the frl function at that time point was 2.0%/min / m , which is below the validated cutoff value for safe resection ( 2.7%/min / m ) . five days later , the frl function had sufficiently increased in order to proceed with the next step of resection . hence , early after the first stage of alpps , the volume of the frl might overestimate the function . however , in previous studies , we have shown differences in the volumetric and functional assessment of the frl , and we were able to establish a cutoff value irrespective of liver parenchymal quality for the effective prevention of postoperative liver failure . our previous experiences with hbs in risk assessment of patients considered for extended liver resections corroborate the value of hbs in the setting of alpps . in conclusion , hbs with spect - ct is a quantitative test enabling monitoring of frl function in patients undergoing alpps procedure . hbs is a useful tool in the timing of the second stage of alpps and may contribute to the improvement of the procedure - related morbidity and mortality rates . the authors declare that there are no conflicts of interest regarding the publication of this article . none of the authors has received funding related to this subject from the following organizations : national institutes of health ; wellcome trust ; howard hughes medical institute and other(s ) .

alpps ( associating liver partition and portal vein ligation for staged hepatectomy ) is a new surgical technique for patients in whom conventional treatment is not feasible due to insufficient future remnant liver ( frl ) . during the first stage of alpps , accelerated hypertrophy of the frl is induced by ligation of the portal vein and in situ split of the liver . in the second stage , the deportalized liver is removed when the frl volume has reached 25% of total liver volume . however , frl volume does not necessarily reflect frl function . 99mtc - mebrofenin hepatobiliary scintigraphy ( hbs ) with spect - ct is a quantitative test enabling regional assessment of parenchymal uptake function using a validated cut - off value for the prediction of postoperative liver failure ( 2.7%/min / m2 ) . this paper describes the changes in frl function and frl volume in a 79-year - old patient diagnosed with metachronous colonic liver metastases who underwent alpps . we have observed a substantial difference between the increase in frl volume and frl function suggesting that hbs with spect - ct enables monitoring of the frl function and could be a useful tool in the timing of resection in the second stage of the alpps procedure .

Introduction Case Report Imaging Results Discussion Statement of Ethics Disclosure Statement

this is an interesting case of a child who , following a minor head injury a few weeks earlier , was initially found to have a thin chronic subdural haematoma on plain computed tomography ( ct ) imaging . following burr hole surgery for evacuation of the subdural haematoma his post - operative scan revealed an underlying arachnoid cyst . it is established that intra - cystic bleeding can ensue following minor head injury in patients with this benign developmental malformation . often the haemorrhage is obvious on initial imaging but this is not always the case . a 13-year - old boy presented with a 1-month history of worsening global headache with associated nausea and vomiting . the symptoms developed following an injury sustained by falling and hitting his head on a chair . he had no significant past medical history , met all his developmental milestones and had no significant family history . on examination he had normal higher mental and cranial nerve function and no focal motor deficits . a plain ct head [ figure 1 ] revealed a thin isodense subdural collection overlying the left cerebral convexity with 4 mm of midline shift . sulcal effacement and distortion of the frontal horn of the left lateral ventricle could also be seen . minor head trauma often precedes complications causing rupture of small bridging vessels between the dura and outer membrane of the cyst . , intra- and extra - cystic bleeding had occurred simultaneously causing an isodense arachnoid cyst with an accompanying subdural collection . the child was managed surgically first with a left - sided burr hole evacuation of the chronic subdural hematoma . a second ct head scan was , therefore , performed [ figure 2 ] , revealing the left middle cranial fossa arachnoid cyst causing the residual midline shift . this was also managed surgically with a temporal craniotomy and marsupialization of the high - pressure cyst [ figures 3 and 4 ] . axial computed tomography brain : postburr hole drainage : left temporal arachnoid cyst revealed photograph : craniotomy to reveal membrane of arachnoid cyst photograph : evacuation of high - pressure cystic hematoma the majority of arachnoid cysts are incidental findings on brain imaging and do not require treatment treatment options vary and include microscopic or endoscopic cystocisternostomy , cyst wall resection , and shunt insertion . in this case , the cyst was located in the cerebral convexity and had displaced deeper brain structures ; therefore , craniotomy for cyst wall resection / marsupialization was indicated . there is increasing evidence to suggest that fenestrations made endoscopically are as safe and effective as open surgical techniques . . magnetic resonance imaging should show the cyst as a well - defined nonenhancing mass that is isointense to cerebrospinal fluid and should , therefore , be considered in any child presenting with a subdural collection .

we present an unusual case of a 13-year - old child who following minor head injury presented with what appeared to be a thin chronic subdural hematoma on plain computed tomography imaging . the child was found to have an underlying arachnoid cyst . intra- and extra - cystic bleeding had occurred simultaneously causing an isodense cyst with an accompanying subdural collection . this radiographically occult pathology should be excluded using magnetic resonance imaging in any child presenting with a subdural collection .

Introduction Clinical Background Discussion Conclusion Financial support and sponsorship Conflicts of interest

like other mammals , cattle possess a large amount of single nucleotide polymorphisms ( snps ) in their genome . the current ncbi dbsnp collection contains more than 2.2 million bovine snps ( http://www.ncbi.nlm.nih.gov/projects/genome/guide/cow/ ) . among them , ~2.1 million snps were contributed by the baylor college of medicine on behalf of the bos taurus sequencing project using a single breed , hereford cattle . the same team also provided an additional 114,958 snps discovered by comparing random shotgun reads from individuals of six diverse cattle breeds to the hereford genome assembly . based on 126,800 bac ( bacterial artificial chromosome)-end sequences and 1,091,070 bac shotgun reads released by the encode project 1 and the bovine hapmap project 2 , matukumalli and colleagues 3 identified 89,832 unique putative snps for cattle . expressed sequence tags ( ests ) derived from different sources of samples were also used to mine putative snps representing coding regions of the bovine genome 4 . in order to further enhance broad discovery of snps for wide utility , van tassell and colleagues 5 developed a deep sequencing approach on reduced representation libraries derived from different target populations . the approach relies on next generation sequencing , which was performed on an illumina genome analyzer . using dna samples of 66 cattle representing three populations , the authors produced nearly 50 million sequences , which identified 62,042 putative snps and estimated their allele frequencies in cattle . all of these publicly available snp data made it possible for the illumina scientists and collaborators to develop an informative and high - density snp genotyping microarray for community use 3 . , san diego , ca ) is comprised of 54,001 snp probes . after testing a panel of 576 animals , including 392 animals from 14 taurine dairy and beef breeds , 73 animals from three breeds of predominantly indicine background , 48 animals from two breeds that are taurineindicine composites , and 45 animals from two african breeds , matukumalli et al . 3 found that the average call rate for individual samples was greater than 97.5% and 85% of samples had call rates above 98.8% . the authors also observed that 51,383 ( ~95% ) of the 54,001 called snps were polymorphic among the 558 cattle with an average minor allele frequency of 0.26 . to date , the bovinesnp50 beadchip has been used in cattle for genomic evaluation 6 - 7 , identification of copy number variation 8 , paternity validation 9 , and whole genome association studies for mycobacterium avium subsp . the chip has also been used to estimate genetic variability between american and european bison 13 . catttle ( bos taurus ) and water buffalo ( bubalus bubalis ) belong to the subfamily bovinae . the former species was domesticated between 8,000 and 10,000 years ago 14 , while domestication of the latter species occurred at least 7,000 years ago 15 . for centuries , both animals have been heavily used for production of power , milk and meat to benefit human society . although both species shared a common ancestor approximately 16.9 million years ago 16 , their phenotypic divarication has continued to present time . for example , a cow can produce 5 to 10 times more milk than a buffalo , but buffalo milk contains more total solids ( 18 - 23 % as compared to 13 - 16% in cow milk ) ( ftp://ftp.fao.org/docrep/fao/010/ah847e/ah847e.pdf ) . water buffalo meat flavor is similar to beef , but contains about one - half the amount of cholesterol , and less than one - fourth the amount of fat , particularly saturated fat . water buffaloes are able to subsist on a low quality , high roughage diet and are resistant to most of the internal and external parasites that affect cattle . therefore , the primary goal of this study was to test and evaluate the cross - species transferability of snps from cattle to buffalo in a mammalian model system . in order to do so our report here represents the largest snp transferability study between these two species at the whole genome level . in particular , characterization of cross - species snp transferability might provide new insights into understanding molecular mechanisms of snp divergence and genome evolution associated with the phenotypic divergence between these two species . basic information on the 54,001 snps on the illumina bovinesnp50 beadchip ( illumina inc . , san diego , ca ) , including snp name , chromosome , and map location was downloaded from the illumina website ( illumina inc . , san diego , ca ) . among these snps , 1,672 remain unassigned to any bovine chromosome , while 74 snps have information on chromosome , but without specified locations . in the present study , thereafter , dna was extracted from 200 l of whole blood with the genelute blood genomic dna extraction kit ( sigma , st . ten water buffalo dna samples were genotyped at geneseek ( lincoln , ne ) on the bovinesnp50 genotyping beadchip . geneseek ( lincoln , ne ) performed a quality control check on our water buffalo samples for their illumina bovinesnp50 beadchip genotypes . a custom - generated ( using > 500 holstein cattle ) cluster file was applied and then the markers were filtered by a specific set of criteria . call rates below 80% , ab r mean below 0.1 , cluster separation below 0.13 , ab t mean below 0.2 or above 0.8 , gentrain score below 0.3 , or het excess below -0.7 or above 0.7 usually cause a marker to be examined , unless there is a valid explanation for excluding one or more criteria . because of the small number of samples this was applied as completely as possible . it should be understandable that there are noticeable differences between individual breeds of cattle and surely there are even more differences between cattle and water buffalo . fortunately , we only observed three snps in our water buffalo data set that had a cluster separation score of less than 0.13 . bovine reference genome assembly ( btau_4.0 ) . the bovine genome sequencing and analysis consortium 17 has led to genome sequencing and its assembly for cattle . the current assembly ( btau4.0 ) combined both bac and whole - genome shotgun ( wgs ) sequences , which were then placed on chromosomes by employing different mapping methods . the consortium estimated that the bovine genome size is ~2.87 gbp , and the current assembly covers at least 92% of the genome . as the snp map locations are based on the btau_4.0 assembly , we downloaded the gene information from the cow genome resources at national center for biotechnology information ( ncbi ) ( bovine genome resources ) . the information includes gene symbol , start position , stop position , orientation on the chromosome , and gene description . , kernel density estimation method was used to describe the distribution of the number of snp markers along the bovine genome . ~ f be an independent and identically - distributed sample of a random variable x , where xi is the observable location , say in bp , of the i - th snp marker . then , its kernel density estimator is : where h>0 is a smoothing parameter called the bandwidth ( bw ) , and k( ) is some kernel , taken to be a standard gaussian function with mean zero and variance 1 in the present analysis . in the above , h is a free parameter , which may exhibit a strong influence on the resulting kernel density estimates ( plots ) . the kernel density plots showed similar patterns with bw = 1 m or less , but the density plots tend to be over - smoothed with bw=5 m and beyond . this also corresponds to the fact that the 54,000 snps on the illumina bovine50kbeadchip have an average spacing of 51.5 kb on the bovine genome . call frequency plots.let xi be a map position pertaining to snp i , and yi be a measure of call frequency , for i=1, ,n .. lowess ( locally weighted scatterplot smoothing ) was used to portray the relationship between xi 's and yi 's , as follows : where g is a smoothing function and i is a random variable with mean 0 and constant scale . lowess is a non - parametric curve- or function - fitting technique 18 - 19 , in which the fit at point x is made using only points in the neighborhood of x. thus , this method has weaker assumptions about the form of the relationship , as compared to parametric methods , and allows the relationship to be described more closely to its true form , as revealed by the data . in this analysis , lowess starts with a local polynomial ( a k - nn type fitting ) least squares fit and then uses robust methods to obtain the final fit . first , a polynomial regression is fit in a neighborhood of x. this is equivalent to finding r that minimizes where wki(x ) denote k - nn weights . then , the residuals and the scale parameter are computed , and robustness weights are defined as : finally , the analysis fits the polynomial regression in ( 4 ) but with weights iwki(x ) . a noteworthy feature of the above procedure is that we do not need to define a global function of any form to fit a model to the data , but the fit is made locally only using a segment of the data . local is defined by the distance represented as the largest integer no greater than fn , where f is the smoother span . the value of f gives the proportion of points in the plot which influence the smooth at each value . thus , the good choice of f is a value which is as large as possible that minimizes the variability in the smoothed points without distorting the pattern in the data . by this standard the current annotation of the bovine genome ( btau_4.0 ) indicates that the gene count on bovine chromosomes ranges from 273 genes on chromosome 27 to 1,535 genes on chromosome 3 , while the number of snps on the illumina bovinesnp50 beadchip varies from 740 snps on chromosome x to 3,339 snps on chromosome 1 ( figure 1 ) . overall , there are far more illumina snps than genes on most chromosomes , but the difference between snp and gene counts on chromosomes 18 , 19 and 23 is not dramatic . the number of illumina snps is less than the number of genes on chromosome x. our analysis indicated that 19,294 snps ( 37% ) are intragenic ( i.e. , located within bovine genes ) and 32,961 snps ( 63% ) are intergenic ( i.e. , located between bovine genes ) . the number of snps by genes and by intergenic regions was extrapolated from downloaded data and is shown in figure 2 . over 14,000 genes in the bovine genome have zero intragenic snps and the remaining genes have between 1 and 44 intragenic snps . in the latter category , most genes have between 1 and 6 intragenic snps : around 6,000 bovine genes have only 1 intragenic snp ; more than 1,500 genes have 2 intragenic snps ; 1,459 genes have between 3 and 6 intragenic snps ; the remaining genes contain between 7 and 44 intragenic snps . on the other hand , every intergenic region is covered by at least 1 snp : over 4,500 intergenic regions have only 1 snp , nearly1,500 include 2 snps ; 2,719 have between 3 and 10 snps ; the remaining intergenic regions have between 11 and 58 snps ( figure 2 ) . the kernel density plots showed similar patterns with bw = 1 m or less , but the density plots tend to be flat with bw=5 m and beyond ( data not shown ) . as illumina claimed that these 54,001 snps have an average spacing of 51.5 kb , we choose to present the results obtained with bw=0.05 m ( 50 kb ) . the means for the whole genome average and by each chromosome , and two times the standard deviations of the chromosome mean were plotted on each kernel density plot ( bw=0.05 m ) . empirically , we defined a snp - rich region along the chromosome as the region at which the computed kernel density value was above two times the standard deviation of the chromosome snp density mean . likewise , a region along the chromosome was considered snp - poor if the point fell below two times the standard deviation of the chromosome snp density mean . as shown in figure 3 , bovine chromosomes 3 , 11 , 13 , 16 , 17 , 19 , 20 , 22 , and 28 have almost no snp - rich regions , but still possess various numbers of snp - poor regions . when the low point of the snp - poor regions approaches zero , it indicates that this region has no snps placed on the illumina bovinesnp50 beadchip . chromosome x shows the greatest snp density dynamics , because it harbors more than 30 snp - rich regions and also has at least 20 regions with no snps ( figure 3 ) . call frequency of the bovine 54,001 snps on water buffalo samples . among 54,001 snps on the illumina bovinesnp50 beadchip , 41,870 ( 77.5% ) were successfully genotyped on all 10 water buffalo dna samples ( call frequency = 1 ) , while 5,360 ( 9.9% ) had no calls ( call frequency = 0 ) ( table 1 ) . the remaining 6,771 ( 12.6% ) snps had calls on various numbers of water buffalo samples , including 733 bovine snps on one animal , 560 on two animals , 512 on three , 470 on four , 458 on five , 546 on six , 703 on seven , 972 on eight , and 1,817 on nine water buffalo samples , respectively ( table 1 ) . we also observed that 78.27% of the intragenic snps across the genome had a call frequency of 1.0 , whereas 77.13% of intergenic snps had a call frequency of 1.0 . the chi - squared test showed that the difference ( 78.27%-77.13%=1.13% ) is significantly different from zero ( p = 0.0059 ) . however , 9.79% of all intragenic snps across the genome had no calls , while 10% of intergenic snps had a call frequency of 0 . on the basis of chromosomes , the percentage of snps with a call frequency of 1 ranged from 74.3 on bovine chromosome 27 ( bta27 ) to 81.1 on chromosome x ( btax ) , while the percentage with a call frequency of 0 varied from 4.7 on btax to 11.5 on bta27 . most interestingly , each bovine chromosome can be divided into cluster regions of snps with high or low call frequencies ( figure 4 ) . for example , bta8 contains seven cluster regions of snps with a call frequency of 1 and seven cluster regions of snps that had calls on water buffalo samples ranging from 0 to 9 animals . polymorphic status of the bovine 54,001 snps on water buffalo samples . among 41,870 bovine snps that were successfully genotyped on all 10 water buffalo dna samples , only 926 ( 2.2% ) are polymorphic in water buffalo , including 254 , 132 , 95 , 80 , 82 , 58 , 59 , 52 , 60 , and 54 snps with minor allele frequencies of 0.05 . 0.10 , 0.15 , 0.20 , 0.25 , 0.30 , 0.35 , 0.40 , 0.45 and 0.50 , respectively ( supplemental table 1 ) . however , 386 of these 926 snps called on all 10 water buffalo dna samples have minor allele frequencies 0.10 and are probably too rare to be useful in water buffalo . while only 926 snps were polymorphic in all ten samples , there were 6,771 bovine snps that had calls on one to nine water buffalo samples ; of these , 2,277 ( 33.6% ) are polymorphic markers in the species . interestingly , 1,398 of these snps were heterozygous on all called animals ( table 1 ) . although the heterozygous status decreased from 100% when all 413 snps were called as heterozygotes on one animal to 22.75% when 53 of 233 snps were heterozygous on all nine called water buffalo samples , the observed figure significantly exceeds the expectation ( chi square = 5.924 , p=0.0149 for the former case and chi square = 44.053 , p=0.0000 for the latter case ) . however , among 41,870 snps that had calls on all ten samples , only 32 were heterozygotes without significant bias from the expected number of 41 ( chi square = 1.111 , p=0.2919 ) . approximately , 97% of the snps on the bovinesnp50 beadchip have map locations on the current bovine reference genome assembly ( btau_4.0 ) , which can be downloaded in order to characterize snps at a whole genome level . while all of the snps are evenly distributed across the bovine genome , the number of snps on each chromosome is not correlated with the number of genes on each chromosome ( figure 1 ) . as a consequence , not all genes have intragenic snps ; in fact , most genes do not have a snp ( figure 2 ) . of the genes that do have intragenic snps , most have only 1 . in contrast , all intergenic regions have at least 1 snp and there are many more intergenic snps with a 1:1.71 intragenic to intergenic snp ratio . these intragenic snps may be used as targets for future functionality research when utilizing the bovinesnp50 beadchip to transfer markers to water buffalo . the intergenic snps can also be transferred from cattle to water buffalo . while they may not be primary targets for functionality research as they fall in non - coding or junk dna regions , these snps might provide secondary targets for research regarding regulation of gene expression . research has shown that aside from the coding regions , evidence exists that another 80% of the bases in the genome are expressed , which may indicate that junk dna is not actually junk 20 . the non - coding regions contain genetic switches that do not encode proteins but regulate where and when genes are expressed 21 and may encode signals that are functionally important to chromosome assembly , dna replication , and gene expression 22 . subirana and messeguer 23 found that non - coding dna may play a role in maintaining structure and function of eukaryotic chromosomes ; this includes roles in centromere function , chromosome condensation , axis formation , and chromosome pairing during meiosis . variations in non - coding dna may also influence many complex conditions and diseases 24 . the emerging data regarding function in non - coding regions makes intergenic snps ideal targets for research on disease expression and structural functionality . snp density plots evaluate snp distribution dynamics across the genome and identify snp - poor and snp - rich regions ( figure 3 ) . we observed that most regions fall within 2 standard deviations of the chromosome snp density mean . the fact that the 54,001 snps on the illumina bovine50k beadchip have an average spacing of 51.5 kb ( i.e. , at least 1 snp per every 50 kb ) prompted us to generate snp density plots with a bw=0.05 m ( 50 kb ) for each chromosome . these density plots might not be most ideal to portray the distribution of these snps on these chromosomes , but they sufficiently depicted some snp - poor and a few snp - rich regions . for example the most dynamic chromosome in terms of snp distribution is chromosome x , which has more than 30 snp - rich regions and at least 20 regions with zero snps ( figure3 ) . therefore , further research in snp identification should focus on snp - poor regions in order to provide even coverage along the entire bovine genome , which can be used on future snp beadchips . to date , the illumina bovinesnp50 beadchip has been used to genotype various breeds and species in the tribe bovini . matukumalli and colleagues 3 used the chips to genotype 576 animals and found that the number of snps with minor allele frequencies of 0.05 ranged from 31,633 to 42,711 among 14 taurine breeds , from 41,720 to 42,594 between two taurineindicine composite breeds , from 28,823 to 35,425 between two african breeds and from 23,284 to 30,139 among three indicine breeds ( figure 5 ) . however , the figure further reduced to 6,352 , 2,506 , 1,604 , 1,429 , 1,262 , and 949 snps when the 54,001 bovine snps were tested on samples of lowland anoa ( bubalus depressicornis ) , gaur ( bos gaurus ) , north american bison ( bison bison ) , banteng ( bos javanicus ) , cape buffalo ( syncerus caffer ) , and yak ( bos grunniens ) , respectively 3 . pertoldi and colleagues 13 also genotyped the same set of 54,001 bovine snp probes on bison samples of three sub - species and found only 929 cattle snps remain polymorphic in european bison , 1,524 in american wood bison and 1,403 in american plain bison , respectively . when we combine the snps that were successfully genotyped on both nine and ten water buffalo samples , a total of 1,159 bovine snps remained polymorphic in the species ( figure 5 ) . these results clearly indicate that polymorphic status of cattle snps is variable within and between species . therefore , the present study provides insight into the snp evolutionary process , and helps to understand within- and between - species biodiversity , phylogenetics and adaption to environmental changes . in recent years , the water buffalo research community has been working hard to generate genome resources in terms of cytogenetic characterization , whole genome mapping and whole genome sequencing ( reviewed by michelizzi et al . the present study added more than 1,000 useful snps as genetic markers to the water buffalo genome resources ( supplemental table 1 ) . for example , this set of genetic markers can be used to advance sustainable water buffalo production systems for higher productivity through manipulating the variation within and between breeds , in order to realize more rapid and better - targeted gains in breeding value . this process would help water buffalo breeders and farmers to overcome challenges and problems such as poor reproductive efficiency , sub - optimal production potential , high incidence of infertility diseases , low rates of calf survival and high costs of feeding . no doubt , phenotypic data is required to estimate the effects associated with the snp so accurate recording systems are as necessary as marker discovery . the functions of many of these genes and the importance of these snps have been investigated in cattle and they can provide insight into potential functions of the transferable snps in water buffalo . therefore , this research makes it possible to identify potential economically important genes in water buffalo based on known functionality of these genes in cattle . in fact , our present work was performed on the illumina bovinesnp50 beadchip v1 . since then , illumina has improved the beadchip and made the bovinesnp50 v2 with a total of 54,609 bovine snps ( http://www.illumina.com/products/bovine_snp50_whole-genome_genotyping_kits.ilmn ) . now , the next - generation high - density bovine beadchip ( bovinehd ) array is also available , featuring a total of 777,962 snps ( http://www.illumina.com/products/bovinehd_whole-genome_genotyping_kits.ilmn ) . on the other hand , for example , using the celera assembler , the center for bioinformatics and computational biology at university of maryland has reassembled the bovine genome and released the data to the public 26 . the umd3.0 release involves 36.82 million reads into a 2.649 billion bp genome out of which 2.640 billion ( 99% ) bp were placed on chromosomes ( http://www.cbcb.umd.edu/research/bos_taurus_assembly.shtml ) . in addition , several groups have started to sequence the water buffalo genome ( reviewed by michelizzi et al . , we recently sequenced a male sample of water buffalo and obtained over 46 gb of sequences for the species . all these new resources present powerful high - throughput solutions for us to pursue a larger whole - genome snp transferability study between cattle and water buffalo in the near future . for example , with the whole genome assembly available in both species , we would be able to further examine how sequence conservation causes the scored snp sites , how sequence divergence causes the partially scored or non - scored snp sites and what sequence contexts make snps remain polymorphic or result in copy number variations in water buffalo .

the illumina bovinesnp50 beadchip features 54,001 informative single nucleotide polymorphisms ( snps ) that uniformly span the entire bovine genome . among them , 52,255 snps have locations assigned in the current genome assembly ( btau_4.0 ) , including 19,294 ( 37% ) intragenic snps ( i.e. , located within genes ) and 32,961 ( 63% ) intergenic snps ( i.e. , located between genes ) . while the snps represented on the illumina bovine50k beadchip are evenly distributed along each bovine chromosome , there are over 14,000 genes that have no snps placed on the current beadchip . kernel density estimation , a non - parametric method , was used in the present study to identify snp - poor and snp - rich regions on each bovine chromosome . with bandwidth = 0.05 mb , we observed that most regions have snp densities within 2 standard deviations of the chromosome snp density mean . the snp density on chromosome x was the most dynamic , with more than 30 snp - rich regions and at least 20 regions with no snps . genotyping ten water buffalo using the illumina bovinesnp50 beadchip revealed that 41,870 of the 54,001 snps are fully scored on all ten water buffalo , but 6,771 snps are partially scored on one to nine animals . both fully scored and partially / no scored snps are clearly clustered with various sizes on each chromosome . however , among 43,687 bovine snps that were successfully genotyped on nine and ten water buffalo , only 1,159 were polymorphic in the species . these results indicate that the snps sites , but not the polymorphisms , are conserved between two species . overall , our present study provides a solid foundation to further characterize the snp evolutionary process , thus improving understanding of within- and between - species biodiversity , phylogenetics and adaption to environmental changes .

Introduction Materials and Methods Results Discussion Supplementary Material

the purpose of this article is to report a case of central toxic keratopathy in a patient post transepithelial photorefractive keratectomy ( transprk ) , followed immediately by corneal collagen cross - linking . this article describes the case of a 26-year - old male after bilateral aberration - free , transprk laser ( schwind amaris 750s ) . the procedure was performed for compound myopic astigmatism in november 2015 , followed immediately by accelerated corneal collagen cross - linking for early keratoconus . from day 3 post - op , tear film debris underneath both contact lenses with corneal haze and early , progressive central anterior stromal opacity formation only in the left eye were noted . at 2 weeks post - op , the left eye was noted to have a significant hyperopic shift with central corneal thinning in the anterior stroma . a central anterior stromal dense opacity had formed in the left eye with the surrounding superficial stromal haze . as of month 2 the hyperopic shift peaked at 2 months and continued to improve , largely due to epithelial compensation with a gradual recovery of stromal thickness . the question remains as to what provokes the typical central corneal necrosis / thinning in central toxic keratopathy . we hypothesize that the space between the contact lens and the corneal surface post transprk is prone to a pseudo - interface pathology that could mimic diffuse lamellar keratitis - like pathology . suboptimal lid hygiene , resulting in tear film combinations of bacteria , inflammatory cells , matrix metalloproteinases and other proteolytic enzymes , contributes to the degradation of vulnerable , exposed collagen stromal tissue post transprk or any surface corneal ablation . refractive surgeons should maintain a healthy lid margin and tear film , especially in contact lens wearers , to prevent potential complications in refractive surgery procedures . refractive surgeries are currently performed routinely all over the world . in the vast majority of cases , there are insignificant complications . however , there are rarely cases in which the postoperative complications can be quite severe . one of the most dreadful yet less understood postoperative complications is known as central toxic keratopathy ( ctk ) . ctk is a rare , self - limited , noninflammatory , post - refractive laser condition that presents with central corneal opacification and a significant hyperopic shift.14 the cause of ctk is unknown , although it has been hypothesized that photoactivation of some substance , possibly povidone - iodine , by the laser induces keratocyte apoptosis and stromal necrosis.5 the inciting factor , however , is not the microkeratome itself as this condition could occur after photorefractive keratectomy ( prk ) as well as after femtosecond laser - created flaps.5 the hypothesis that this reaction could be an intrinsic patient response to laser ablation is also unlikely since it does not recur after enhancements.5 the purpose of this article is to report a case of ctk post transepithelial photorefractive keratectomy ( transprk ) with accelerated corneal collagen cross - linking ( ccl ) . to date , ctk has not been reported in a refractive laser - ablated eye post transprk with ccl . this case may also help to exclude some previously hypothesized etiologies in ctk , e.g. , equipment sterilization method , iodine usage and glove powder . written informed consent was obtained from the patient for this case report , as well as all the images herein , to be published . the patient was noted to have blepharitis preoperatively with some debris noted within the tear film . the right eye refraction was 2.50/0.75 175 , with the best spherical corrected visual acuity ( bscva ) 1.0 and uncorrected distance visual acuity ( udva ) 0.1 ( visual acuity [ va ] in a decimal format ) . the left eye ( in which the ctk incident occurred ) refraction was 2.00/2.75 170 , with bscva 1.0 and udva 0.1 . the steepest keratometry value was 46.2 and the pachymetry value was 469 m ( figure 1 ) . the patient underwent single - step transprk treatment in both eyes using the amaris 750s laser platform ( schwind eye - tech - solutions gmbh , kleinostheim , germany ) aiming for plano refraction . the optical zone was 6.50 mm in both eyes with a transition zone of 1.14 mm and 0.82 mm in the right and left eyes , respectively , and the residual stromal thickness was 366 m and 344 m in the right and left eyes , respectively . the accelerated ccl has been shown to penetrate less corneal stroma than the standard dresden protocol . the authors , however , recommend that proven cornea thickness safety profiles are adhered to when performing ccl . the accelerated ccl protocol included saturation of the post - ablative exposed stroma with riboflavin ( vibex rapid ; avedro inc . , the ccl was performed using the kxli uv - a source ( avedro inc . ) . on the right eye , the settings were 30 mw 80 seconds = 2.4 j / cm and on the left eye , 30 mw 120 seconds = 3.6 j / cm . a higher ccl energy was used on the left eye due to the slightly higher k reading pre - op and because more corneal tissue ablation was planned in comparison to the right eye due to the higher astigmatism ; hence , a thinner residual stromal bed was expected . no mitomycin c was used intraoperatively . a bandage contact lens ( acuvue oasys ; jacksonville , fl , usa ) of base curve 8.8 mm and diameter 14 mm was inserted in both eyes at completion of ccl . postoperative treatment protocol was commenced on day 0 , which included topical antibiotic ( ofloxacin , qid ) , topical steroid ( maxidex , qid ) and topical tear lubricant hourly ( thealoz duo , thea , france ) . on day 1 , there was no corneal opacity or haze and there was a healing epithelial defect in both eyes . on day 3 , debris was noted beneath the left contact lens within the tear film . fine corneal haze with anterior stromal opacity had begun to appear in the left eye with a healing epithelial defect . a mild anterior chamber inflammatory reaction of 1 + cells was noted . on day 5 the left eye still had a very small healing epithelial defect of < 1 mm , the steroid was stopped in the left eye and regenerating agent ( rgta ) ( glycosaminoglycan mimetic / matrix therapy agent to promote epithelial healing , cacicol ; thea , clermont - ferrand , france ) was added 4 times daily only in the left eye . at 1 week post - op , the right eye was healing well ; however , some stromal haze ( grade 1 fantes scale ) was present . the left eye epithelium had healed , but the eye had developed a diffuse anterior stromal haze with a dense central anterior stromal opacity . at 2 weeks post - op , the left eye was noted to have a significant hyperopic shift ( + 5 , 25 d ) with central corneal thinning of the superficial stroma , and a corneal optical coherence tomography ( oct ) was performed to confirm this . a central corneal opacity had increased in density at the central anterior stroma with surrounding diffuse superficial stromal haze ( figure 2 ) . autologous serum q6h and zovirax q6h were added to the current topical post - op treatment in the left eye . at 1 month post - op , vertical stromal striae at the center of the dense opacity were appreciated . corneal thickness on orbscan of the right eye pred forte tds , autologous serum q6h and minocycline 50 mg bd by mouth ( po ) were commenced . ganciclovir gel bd was commenced in the left eye as there was uncertainty around the underlying pathology at this stage and a reactivated necrotic viral infection could not be excluded . at 9 weeks post - op , the left corneal opacity with vertical striae and fine iron line remained visible at 4 months post - op ( figure 3a and b ) . the opacity gradually continued to improve in size and density at 6 months post - op . the vision and hyperopic refraction continued to slowly improve over 6 months post - op , and the central opacity still gradually continued to improve in size and density ( table 1 ) . at 2 weeks post - op , the right eye had healed well with an uncorrected visual acuity ( ucva ) of 1.0 ; however , fine anterior stromal reticular haze had formed centrally , which remained visible 6 months post - op ( figure 4 ) . a corneal oct comparison of 2 weeks and 6 months post - op of the left eye showed how the epithelium compensated largely for the stromal thinning and hyperopic shift ( figure 5a and b ) . there was no anterior stromal thinning or epithelium thickening in the right eye on corneal oct at 6 months post - op ( figure 6 ) . this may be the first documented report of a patient who developed ctk in a cornea post - transprk with accelerated ccl . although the possibility of ccl as a cause for the ctk could not be entirely excluded , the authors deemed the ccl treatment an unlikely etiology based on the time of onset and depth of the pathology . ctk usually starts within 39 days after laser refractive surgery.2,3,57 the inciting procedure may be prk , laser - assisted in situ keratomileusis ( lasik ) or intra - lasik . the opacity clears gradually , usually within 1218 months.5 it is rare for the opacity to persist beyond 18 months.5 recurrence of ctk after enhancement surgeries has not been observed.5 the etiology of ctk is still under contention ; however , some of the proposed etiologies may perhaps be excluded from this report . no iodine was administered in this case preoperatively as the patient had given a history of iodine allergy . powder - free gloves were used , and other than the speculum , no sterilized instrumentation touched the eye intraoperatively . cases of deep stromal scarring post - ccl have been reported , although these cases tend to present much later postoperatively ( ~3 months ) and the scarring is deep stromal , unlike in this case.8 the authors could not find any literature or case reports of ccl resulting in corneal pathology of this similar nature . tu and aslanides9 reported on 3 patients with bilateral ctk post lasik where 2 of the 3 patients received early surgical intervention in the form of flap lift and irrigation . in 1 eye imaging confirmed early stromal loss posterior to the flap with the stroma regaining some thickness over the following months . the authors concluded that early surgical intervention in the form of flap lift and irrigation could at least arrest part of the process of progressive corneal thinning / necrosis.2,3,9,10 this successful intervention of early flap lift and irrigation may contribute to the hypothesis that an overload of bacteria , proteolytic enzymes and/or inflammatory cells within the tear film , with trapping of the cellular components underneath the postoperative contact lens , may contribute to the formation of ctk and possibly even diffuse lamellar keratitis ( dlk)-like pathology . there has been some controversy as to whether ctk may be considered an advanced form of dlk . dlk is generally accepted as an interface pathology occurring post lasik flap creation.7 we would like to hypothesize that perhaps the space between the contact lens and corneal surface post transprk could be prone to a pseudo - interface pathology that could mimic dlk - like pathology . until the pathology of ctk is better understood , the optimal intervention options for this dreaded condition may remain unclear . perhaps microbiological / cytology studies of early ctk corneas and/or tear film may help to provide some clarity on the subject . interestingly , in a study by larkin and leeming,11 contact lens wearers were found to have a significantly higher number of commensal bacterial species at the lid margin and within the tear film than controls . significant quantitative changes were identified on the lid margin and tear film , with particularly high bacterial counts in some lens wearers.1214 quantitative / osmolarity changes in the tear film are thought to be secondary to changes at the lid margin , for which no explanation is apparent.11 the authors conclude that suboptimal lid hygiene with high concentrations or osmolarity of tear film bacteria , inflammatory cells , matrix metalloproteinases and/or other proteolytic enzymes contributes to the degradation of the vulnerable , exposed collagen stromal tissue , potentially resulting in stromal degradation and opacity formation . particular attention needs to be drawn to a healthy lid margin and tear film pre - ablation treatment , especially in contact lens wearers . perhaps this may contribute to a decline of this dreaded postoperative dilemma for the refractive surgeon .

purposethe purpose of this article is to report a case of central toxic keratopathy in a patient post transepithelial photorefractive keratectomy ( transprk ) , followed immediately by corneal collagen cross-linking.methodsthis article describes the case of a 26-year - old male after bilateral aberration - free , transprk laser ( schwind amaris 750s ) . the procedure was performed for compound myopic astigmatism in november 2015 , followed immediately by accelerated corneal collagen cross - linking for early keratoconus.resultsfrom day 3 post - op , tear film debris underneath both contact lenses with corneal haze and early , progressive central anterior stromal opacity formation only in the left eye were noted . at 2 weeks post - op , the left eye was noted to have a significant hyperopic shift with central corneal thinning in the anterior stroma . a central anterior stromal dense opacity had formed in the left eye with the surrounding superficial stromal haze . as of month 2 , the opacity gradually started to improve in size and density . the hyperopic shift peaked at 2 months and continued to improve , largely due to epithelial compensation with a gradual recovery of stromal thickness.conclusionthe question remains as to what provokes the typical central corneal necrosis / thinning in central toxic keratopathy . we hypothesize that the space between the contact lens and the corneal surface post transprk is prone to a pseudo - interface pathology that could mimic diffuse lamellar keratitis - like pathology . suboptimal lid hygiene , resulting in tear film combinations of bacteria , inflammatory cells , matrix metalloproteinases and other proteolytic enzymes , contributes to the degradation of vulnerable , exposed collagen stromal tissue post transprk or any surface corneal ablation . refractive surgeons should maintain a healthy lid margin and tear film , especially in contact lens wearers , to prevent potential complications in refractive surgery procedures .

Purpose Methods Results Conclusion Introduction Case report Discussion

reactive oxygen ( ros ) and nitrogen ( nos ) species ( rons ) normally regulate many of the intracellular signaling cascades that govern multiple cellular activities . however , when the amount of rons exceeds the cell s ability to metabolize / eliminate them , the cell becomes stressed and acquires genetic and epigenetic aberrations . thus , antioxidation of excess levels of rons is crucial for reducing the risk of developing cancer . the primary rationale for this conclusion is the marked increased risk of developing cancer in patients with ulcerative colitis and crohn s disease [ 1 , 2 ] . a strong case has also been made for a major role of inflammation and rons in the carcinogenesis of the more prevalent sporadic form of colon cancer . specifically , mclean and associates [ 4 ] recently reported an extensive immunohistochemical study that clearly showed significant infiltration of proinflammatory cells , including macrophages expressing inducible nitric oxide synthase ( nos2 ; also known as inos ) in adenomatous lesions . nos2 is an enzyme that converts arginine to citrulline plus the radical nitric oxide ( no ) . the radical itself is oxidative , and also because it generates the radical peroxynitrite ( oono ) when it combines with o2 . other support for a role of inflammation and rons in colon carcinogenesis is that nsaids reduce the risk of developing this disease , and the frequently noted overexpression of nadph oxidase enzymes ( nox ) that produce superoxide ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{o}}_2^ { \bullet - } $ $ \end{document } ) and h2o2 , which dissociates to the ho radical . thus , inflammatory - associated rons are a key player in the genesis of this disease . chemopreventative foods and their active constituents have been shown to decrease the number and size of colon cancer in animal models and recently in humans as well . the paramount feature of the vast majority of these chemopreventive substances is their ability to antagonize proinflammatory cytokines and chemokines and reduce the level of rons . therefore , it is expected that molecular pathways most commonly affected by chemopreventive agents would be those that are dysregulated by proinflammatory factors and free radicals . the primary condition that initiates stress to epithelial cells is an inflammatory microenvironment [ 1 , 2 , 3 , 4 , 714 ] , wherein infiltrating lymphocytes and macrophages raise the level of rons to amounts that exceed the ability of the cell to eliminate them by its antioxidant phase ii enzymes ( eg , superoxide dismutase , catalase , glutathione peroxidase , and peroxiredoxins , glutathione - s - transferase , quinone oxidoreductase , epoxide hydrolase , and others ) . the inflammatory microenvironment of colon lesions contains many rons - producing cell types , including neutrophils , monocytes , t and b lymphocytes , macrophages , and natural killer cells . some of these cells release a number of growth factors and cytokines ( eg , transforming growth factor- [ tgf- ] ) , and chemokines ( eg , ccl2 , ccl7 , ccl3 , ccl4 , cxcl12 , and interleukin [ il]-8 ) , along with tumor necrosis factor ( tnf)-. these factors chemoattract monocytes and macrophages . when chronically activated , this inflammatory / oxidative environment becomes a relentless cycle that results in genetic and pathological damage to the apposition epithelium . some of the procarcinogenic activity of the infiltrating lymphocytes is caused by their release of ros as they phagocytize fusobacterium nucleatum bacteria . previously known as a periodontal pathogen , this microbe is a rare member of the fecal microbiota of noncancerous patients . however , it is prevalent in polyps and is a major resident in tumor tissue . when tested with peripheral blood lymphocytes , the oxidative burst produced by phagocytizing f. nucleatum was significantly greater than those from other species of bacteria . at the same time , this microbe indirectly caused differential expression of 208 genes in the lymphocyte . seven percent of these genes were transcription regulators , 13 % were involved in signal transduction , and 14 % were ros - response genes . the primary enzyme that produces these radicals is the nox family gene nadph oxidase 2 ( nox2 ) , which produces \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{o}}_2^ { \bullet - } $ $ \end{document } [ 15 , 16 ] . one natural dietary constituent ( ndc ) that may affect the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{o}}_2^ { \bullet - } $ $ \end{document } produced by nox2 infiltrating proinflammatory cells is dark cocoa , which antagonizes nox2 activation . cocoa contains relatively high amounts of the flavonoid epicatechin and has been found to have nearly twice the antioxidant content of red wine and up to three times that of green tea when assessed in vitro . one mechanism is that flavonoids can bind directly to some protein kinases and thereby antagonize the consequences of ros - inactivated phosphatases ( see below ) . fish oil diet has also been shown to reduce inflammation in a rat colon carcinogenesis model . the suspected target is nox2 , but the mechanism has not been elucidated . regarding inflammatory cytokines , the ndc astaxanthin suppresses colon carcinogenesis in mice , partly through inhibition of the expression of inflammatory cytokines . the combination of -cryptoxanthin , a carotenoid , and hesperidin , a flavonoid extracted from the mandarin orange , also suppresses the expression of proinflammatory cytokines , but the mechanism has not been described . other ndcs , such as the antioxidant curcumin , a polyphenol extracted from turmeric , act through the inhibition of phosphorylation of the inhibitor of b ( ib ) , which in turn reduces the nuclear translocation of nuclear factor-b ( nf-b ) and thereby silences expression of proinflammatory cytokines cxcl1 and cxcl2 [ 21 , 22 ] . these unique cells are localized to the crypts and express the nox family member nox1 . presumably , these normal cells use this enzyme for defense against the fecal microbiota . on the negative side , when activated , these adherent lymphocytes are postulated to be involved in the pathogenesis of inflammatory bowel disease , and they may also be involved in initiating and maintaining the micro - inflammatory environment associated with sporadic colon cancer . another are the infiltrating activated macrophages and monocytes that upregulate nos2 activity in response to cytokines [ 13 , 14 ] . upregulated nos2 underpins chronic inflammation in general and colon carcinogenesis in particular . the stat1 signaling cascade is one of the major pathways that upregulates nos2 . one antioxidant ndc that antagonizes nos2 in colon inflammatory lesions and cells is the green tea constituent epigallocatechin gallate ( egcg ) . this compound inhibits activation of stat1 by suppressing nox - produced phosphorylation of the pro - stat1 protein . these antioxidant anthocyanidins are found in grape , blackberry , blueberry , cherry , cranberry , elderberry , and raspberry as well as apple , plum , red cabbage , and red onion . they work by attenuating nf-b activity , which regulates nox2 transcription through its binding site in the promoter of nos2 . eight other stat - inhibiting ndcs , including capsaicin and curcumin , are discussed by trecul and coauthors . one way colon cells produce ros is via xanthine oxidase ( xo ) , which converts hypoxanthine to xanthine and then uric acid in the presence of molecular oxygen to yield \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{o}}_2^ { \bullet - } $ $ \end{document } and h2o2 . there is some evidence that the xo - derived ros normally play a role in antimicrobial defense . 1-acetoxychavicol acetate ( aca ) , an ndc present in members of the ginger plant family , is an inhibitor of xo enzymatic activity . another source of ros is the mitochondria , wherein 1 % to 2 % of the o2 electrons used for producing adenosine triphosphate ( atp ) leak from the electron transport chain as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{o}}_2^ { \bullet - } $ $ \end{document } and h2o2 . family members nox1 and nox4 produce abundant amounts of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{o}}_2^ { \bullet - } $ $ \end{document } and h2o2 in colon cells [ 6 , 34 ] . they are expressed in the crypts and probably play a role in the innate response by the normal colon to fecal microbiota [ 35 , 36 ] . nox-1 is activated in colon cells by the proinflammatory factor tumor necrosis factor ( tnf)- . one of the earlier reports showing an important relationship between nox1 and colon cancer cells was by szanto et al . . these authors showed that antisense oligonucleotides of nox1 not only lowered ros production but at the same time reduced the proliferation rate of the cells . unfortunately , the literature is lacking information on any ndcs that can directly diminish the activity of nox1 or nox4 . on the other hand , potentially all the antioxidant ndcs may be able to scavenge the ros produced by these enzymes before they can produce damage to the cell . the binding of growth factors ( ie , tgf- ) to their receptors initiates signal transduction , which activates ras . rac1 is a key downstream target of pi3k , and one of a number of the protein targets of pi3k kinase activity is a member of the nox complex , noxa1 . this growth factor - ras - rac1-noxa1 signaling pathway partially explains why colon tumor cells that harbor a mutant k - ras gene , or exhibit overexpression of growth factors and/or their receptors have constitutively activated nox1 activity [ 38 , 4244 ] . the downstream targets of the pi3k akt pathway are often abnormally activated in colon because of gain - of - function mutations that lead to oncogenicity . as a result , bcl2-antagonist of cell death ( bad ) is inhibited and there is increased cell survival . recent studies show that flavonoids such as quercetin and epicatechin can bind directly to some protein kinases , including akt / protein kinase , janus kinase 1 ( jak1 ) , mitogen - activated protein kinase kinase 1 ( mek1 ) , pi3k , mitogen - activated protein ( map ) kinase kinase 4 ( mkk4 ) , and raf1 , and alter their phosphorylation state and their ability to regulate multiple cell - signaling pathways . the polyphenol antioxidant ndc ellagic acid ( obtained from blackberries , cranberries , pecans , pomegranates , raspberries , strawberries , walnuts , wolfberry , grapes , and peaches ) also prevents pi3k / akt activation [ 48 , 49 ] . the proinflammatory nf-b , which is a commonly overactive gene in colon cancer , participates in the control of transcription of more than 3,000 target genes , including nos2 , cyclooxygenase 2 ( cox-2 , also called ptgs2 ) , nox1 , nox4 , nox2 , interleukins , akt1 , tnf , egfr , gadd45b , map2k6 ( mek6 ) , nqo1 , stat3 , tp53 , bcl2a1 , bcl - x , p21 , birc2 , gadd45b , nqo1 , pdgfb , plau , sod2 , rel , and myc . thus , nf-b is involved in the inflammation , formation of dna adducts , cell cycle regulation , apoptosis , angiogenesis , and invasion / migration / metastasis aspects of carcinogenesis [ 2 , 8 , 33 , 50 , 51 ] . briefly , in the canonical pathway of activation , nf-b is bound to ib. the ikappab kinase ( ikk ) complex phosphorylates ib , thereby releasing it from nf-b . it has been shown that tnf- and il-1 activate nox4 ( which does not have noxa1 or rac1 within the complex ) with a concomitant rise in ros . interacts with ib in a redox - dependent manner . in cells exposed to tnf- or h2o2 , lc8 dissociates from ib. the released ib interestingly , nf-b and the other nadph oxidase in colon cells ( nox1 ) regulate each other . this is brought about because the promoter of the nox1 gene has a consequence sequence for nf-b binding [ 41 , 53 ] . thus , there is a positive feedback loop in which nf-b activation by nox1-derived o2 and h2o2 leads to further induction of nox1 , with ever greater production of o2 and h2o2 and ever - increasing levels of damage to the cells . one of the means of cross - talk between infiltrating cells and epithelial cells is the stat3-nf-b axis of transcription [ 54 ] . activated infiltrating cells release proinflammatory cytokines that activate production of stat3 and nf-b in the epithelial cells . in turn , the epithelial cells exhibit increased proliferation , survival , and angiogenesis . included within this group is lycopene ( from tomatoes , carrots , red bell peppers , watermelons , and papayas ) [ 55 ] . it affects nf-b through mapk / erk and pi3k / akt signaling pathways that are plausibly dysregulated by rons . thus , lycopene inhibited cell proliferation of human colon cancer cells via suppression of the akt signaling pathway and downstream targeted molecules . other antioxidant ndc affecters of nf-b are curcumin , the carotenoid astaxanthin , and sulforaphane . sulforaphane is obtained from cruciferous vegetables such as broccoli , brussel sprouts , cabbages , garlic , and onions . the anthocyanidin family , represented by delphinidin ( from cranberries , concord grapes , and pomegranates ) , also works by inhibiting nf-b activation . pterostilbene , which is chemically related to resveratrol and is found in blueberries and grapes , reduced nf-b activation by inhibiting the phosphorylation of pkc , and decreased downstream target gene expression , including nos2 , cox-2 , and nuclear factor ( erythroid - derived 2)-like 2 ( nrf2 ) signaling . nos2 has a major role in the activation of cox-2 , which is an immediate early response proinflammatory gene that is upregulated in nearly all colon cancers . cox-2 indirectly regulates gene expression through metabolizing arachidonic acid ( aa ) to prostaglandin e2 ( pge2 ) . the receptors regulate several signal transduction pathways ( eg , pkc to erk , and epidermal growth factor receptor [ egfr ] to akt ) . these pathways affect cell proliferation , invasion , survival , and angiogenesis [ 2 , 59 ] . interestingly , in liver cells , nox1 activation has been shown to upregulate cox-2 expression and activity , which in turn induced nox4 expression and ros production . taken together , these results indicate that the nox proteins in colon and cox-2 are reciprocally regulated [ 60 , 61 ] . liberation of arachidonic acid by cytosolic phospholipase a(2 ) ( cpla ) is often the initial and rate - limiting step in leukotriene ( lt ) and pg biosynthesis . because cox-2 enhances the formation of a cpla(2)alpha - inos binding complex , it appears that cox-2induced augmentation of cpla(2)alpha s - nitrosylation is mediated at least in part through increased association between nos2 and cpla(2)alpha . therefore , therapy aimed at disrupting this interplay may represent a promising strategy to effectively inhibit pge2 production and thereby prevent inflammation and carcinogenesis . some of these work indirectly through inhibiting nf-b . included in this list are curcumin , olive oil , resveratrol , egcg , the flavone apigenin ( from leafy vegetables [ eg , parsley , artichoke , basil , celery ] ) , the isoflavone genistein ( from fava beans and soy beans ) , the flavonoid kaempferol ( from tea , broccoli , grapefruit , and apple ) , the flavonoid chrysin ( from honey ) , and sulforaphane [ 9 , 63 ] . some of these may actually be affecting nf-b expression in the activated macrophages of the inflammatory lesion . the enzyme and its metabolites are elevated in adenomas and are even higher in cancer tumors . lts are potent inflammatory mediators that act by stimulating the g protein coupled receptors blt1 and blt2 that in turn induce nf-b , tnf- , and ils in the activated macrophages and leukocytes within the inflammatory lesion . red ginger has the potential to decrease eicosanoid levels , perhaps by inhibiting their synthesis from arachidonic acid . the sequence of some of the genetic changes that correlate with the observed pathological transformations that occur during the initiation and promotion / progression stages of colon carcinogenesis was identified more than 25 years ago [ 1 , 2 , 65 ] . it begins with loss of apc function , as the tissue changes from normal appearing into an early adenomatous lesion . aneuploidy and methylation events arise in the early adenomatous lesion , and microsatellite instability , k - ras mutations , and upregulation of cox-2 develop as the early adenoma evolves into an intermediate adenoma . as this lesion progresses to the late adenoma stage , there is loss of heterozygosity of the genes deleted in colorectal carcinoma ( dcc ) and smad family member 4 ( smad4 ) . lastly , just before the onset of clinical cancer , the p53 gene is mutated . these genetic aberrations are associated with the formation of ros- and nos - caused 8-hydroxy - deoxyguanosine ( 8-oh - dg ) adducts in the dna . the result is a gcta base pair transversion and/or an apurinic / apyrimidinic site if not repaired before dna replication occurs . thus , 8-oh deoxyguanosine bases frequently result in mutations in genes ( eg , k - ras and p53 ) . in addition , rons cause single- and double - strand breaks , with the result of loss and gain of genetic information . radicals also cause dna - protein cross - links and pyrimidine and purine lesions . these adversely affect the integrity of the genome , with genomic instability as one consequence . another effect of rons is reduction of 5-methyl - deoxycytosine content in dna [ 66 , 67 ] . the presence of oxidized deoxyguanosine reduces ability of the dna to serve as a substrate for dna methyltransferase ( dnmt ) . hence , the enzyme does not transfer the methyl group from methionine to deoxycytosines during dna replication , and the level of 5-methyl - cytosines becomes progressively reduced . this hypomethylation of the cell s genome correlates with the onset of genomic instability , microsatellite instability , and increased mutation rate . dna hypomethylation also causes activation of genes normally transcriptionally downregulated by hypermethylation of their promoter region ( ie , the oncogenes h - ras and c - myc , which are frequently overexpressed by colon cancer cells ) . excess rons also leads to shutting off tumor suppressor genes by hypermethylation of their promoter . it activates nrf2 ( see below ) signaling , which in turn induces phase ii enzymes . others are sulforaphane , indole-3-carbinol , and phenethyl isothiocyanate ( peitc ) ( from cruciferous vegetables ) . the coffee constituents 5-o - caffeoylquinic acid and n - methylpyridinium have also been identified as inducers of the nrf2-regulated detoxifying pathways . antioxidant ndcs also target the epigenome . among these are the polyphenols from green tea , apples and coffee , genistein , soy isoflavones , curcumin , ellagitannin , indole-3-carbinol , lycopene , sulforaphane , peitc , and phenylhexyl isothiocyanate ( phitc ) . also effective is resveratrol , which affects the activity of histone deacetylase sirt1 , which regulates the activity of dnmt1 . others are inhibitors of histone acetyl transferases ( anacardic acid from cashew nut ) , garcinol ( from mangosteen ) , and ursodeoxycholic acid ( a metabolic byproduct of intestinal bacteria ) . finally , the relatively unexplored modulators of histone lysine methylation ( chaetocin , a fungal metabolite , and n-3 polyunsaturated fatty acids ) may be effective in inhibiting changes in the epigenome . also effective in targeting the epigenome we recently demonstrated that daily oral administration of brbs for 4 weeks resulted in protective modulation of multiple epigenetic biomarkers ( sfrp2 , sfrp5 , pax6a , and wif1 ) of the wnt pathway that was modulated in a protective direction by brbs in healthy tissues and in colorectal tumors in cancer patients . low levels of rons serve as second messengers to regulate multiple signaling pathways as a way to maintain cellular homeostasis . rons accomplish this by using switching proteins that are reversibly oxidized by h2o2 and returned to their normal state by reducing enzymes . one switch position is brought about through cysteines ( cys ) on the surface of these proteins . the thiol moiety ( -sh ) of these cys is highly , and in some proteins specifically , sensitive to oxidation , with the results of the formation of a sulfenyl moiety that can form a disulfide bond with another oxidized surface cys . consequently , the protein acquires a structural change and is either inactivated or indirectly activated ( see ask1 below ) . the switch is flipped back by reduction of the cys by thioredoxin ( trx ) , thereby establishing on and off states of molecular pathways regulated by rons [ 7679 ] . with respect to carcinogenesis , foremost among the ros inactivation switch proteins are members of the large family of ptps [ 7679 ] . for example , ptps have an essential role in downregulating receptor tyrosine kinase ( rtk)-mediated signaling by dephosphorylating rtks . on the other hand , ptp inactivation by nox - derived ros enhances rtk tyrosine phosphorylation which leads to amplified activity of the erk pathway , suggesting that modulation of rtk signaling via ptp inactivation by nox - derived ros may have a significant contribution in mediating the activation of erk / mapk pathway . normally , pten dephosphorylates phosphoinositide substrates , thereby negatively regulating the intracellular levels of phosphatidylinositol-3,4,5-trisphosphate . alternatively , inactivation of pten constitutively activates the akt pathway , a major player in colon cancer carcinogenesis . however , ros may also be a player , as when cys on pten are oxidized , the enzyme is inactive , and the akt pathway is upregulated and cell survival is augmented . another ros - targeted phosphatase activity is the gene dual specificity protein phosphatase ( dusp ) [ 7679 ] . dusp negatively regulates the activity of c - jun n - terminal kinase ( jnk ) and p38 mitogen - activated protein kinase ( p38 ) stress signaling pathways in a way analogous to pten . when dusp protein is oxidized , its phosphatase activity is inactivated and stress signaling pathways are upregulated . thus , by oxidizing dusp and activating jnk and p38 , several important cellular functions , including cell growth , differentiation , survival , and apoptosis , are dysregulated . the cdc25 phosphatases ( a , b , and c ) that participate in cell cycling are also oxidized and inactivated by ros . central to the cell - cycling process are the cdks and their catalytic partners , the cyclins . the cdks are negatively regulated by cdk inhibitors ( eg , p16 , p21 , and p27 ) and positively activated by cdk - activating kinases . cdc25 regulates the extent of phosphorylation of cdks at differing times of the cell cycle . accordingly , when cdc25 is oxidized and inactive , the cdks are fully activated throughout the cycle and the cells proliferate faster . meeran and katiyar have discussed the ndcs that affect the cell - cycling pathway . the apoptosis signal - regulating kinase 1 ( ask1 ) , which is a member of the mapk family , is indirectly activated by rons [ 7679 ] . ask1 elicits numerous cellular responses to stress through its activation of the jnk and p38 pathways . , it binds to ask1 and inhibits its kinase activity . under oxidative stress conditions , the two cysteine residues in the active site of trx undergo reversible oxidation to form a disulfide bond with each other . thus , the conformation of trx protein is changed , ask1 dissociates from trx , and its kinase activity is turned on . nrf2 is retained in the cytoplasm and destroyed in the proteasome when bound to keap1 . reactive species oxidize keap1 , thereby permitting nrf2 to translocate into the nucleus and activate a myriad of oxidative response genes , whose resultant enzymes extinguish the negative effects of the elevated levels of rons . many antioxidant ndcs increase nrf2 activity . nox4 have been implicated in this inappropriate upregulation . in this pathway , the wnt factor causes the translation of the transcription factor -catenin ( -ctn ) into the nucleus . the primary means of activation of the wnt pathway in colon cancer is through deletions in the adenomatous polyposis coli ( apc ) gene . mutations of apc are considered to be early events in colon carcinogenesis , thereby explaining why inappropriate expression of the wnt pathway is one of the earliest events leading to colon cancer . in normal cells , apc forms a complex consisting of -ctn , casein kinase-1- , glycogen synthase kinase-3 ( gsk3 ) , and axis inhibitor . gsk3 phosphorylates -ctn , thereby priming it for degradation by the proteasomes . when the wnt factor binds and activates its receptor frizzled , the signal is passed to disheveled ( dvl ) . -ctn then translocates into the nucleus to activate its target genes . among these are cd44 , c - myc , c - jun , fos - related antigen-1 , cyclin - d1 , transcription factor-1 , and cox-2 . if apc is inactivated , phosphorylation of -ctn does not occur and it translocates into the nucleus , even in the absence of wnt . its reduced state , nrx binds to dvl . when nrx is oxidized , it releases dvl to bind with the -ctn - apc complex . with dvl in the complex , the kinase activity of gsk3 is blocked and -ctn translocates into the nucleus to activate specific target genes . the source of ros that oxidizes nrx in the colon has been shown to be nox1 . it has been speculated that notch activation might be an essential event triggering colorectal cancer . although the molecular mechanism of notch activation is incompletely understood , a strong role for nox1 has been implicated . quercetin , an ndc available from black and green tea , capers , apples , onions , red grapes , citrus fruit , tomato , broccoli , and a number of berries ( including raspberry , cranberry , chokeberry , buckthorn , and the fruit of the prickly pear cactus ) is a strong disruptor of dysregulation of the wnt pathway . others are egcg , curcumin , sulforaphane , soy isoflavone , epigallocatechin-3-gallate , resveratrol , lycopene , and piperine ( from black pepper ) [ 55 , 87 , 88 ] . micrornas ( mirnas ) are short , noncoding rnas that regulate cell homeostasis by inhibiting translation or causing degradation of the mrna of target genes . when inappropriately expressed , they can affect the expression of tumor suppressor genes and oncogenes . in addition , through their actions on mrnas , they can cause epigenetic modifications of the dna , genomic amplifications and deletions , cellular stress , and inflammation [ 8991 ] . it has recently been reported that some mirna expression is governed by the binding of the proinflammatory nf-b transcription factor to the consequence sequence of the mirna promoter region . inflammation also elicits changes in expression of mirna , primarily through the actions of proinflammatory cytokines . specifically , proinflammatory signaling cytokines il-1 , il-6 , and tnf- alter the expression profile of mirnas and the promoter region of mir-146a , which has a consequence sequence region for nf-b . colon cancer cells exhibit numerous changes in their mirna profile as compared with that of the normal cells / tissues that dysregulate expression of numerous genes that are involved in all aspects of colon carcinogenesis . included in this collection are egcg , apple polyphenols , coffee polyphenols , ellagic acid , genistein , parthenolide ( from chrysanthemum ) curcumin , ellagitannin , and indol-3-carbinol . a major target of mirnas is regulation of nf-b . excellent reviews discussing ndcs and mirnas relationships were recently published by li et al . [ 94 ] and parasamka et al . [ 95 ] . a steady state of elevated rons and proinflammatory factors is a formative driver of colon carcinogenesis ( fig . 1 ) . the intention of this review is to assess the thesis that colon cancer chemopreventive foods and their active constituents work through ameliorating the molecular pathways that are dysregulated by proinflammatory factors and free radicals . extensive research has revealed that continued oxidative stress leads to chronic inflammation , which in turn mediates most chronic diseases , including colon cancer . oxidative stress activates a variety of transcription factors , including nf-b , ap-1 , p53 , -ctn - wnt , and nrf2 , which results in the expression of more than 5,000 different genes , including those for growth factors , inflammatory cytokines , chemokines , and cell cycle regulatory molecules [ 24 , 96 ] . the information in this review clearly implicates the role of rons in different phases of inflammation and carcinogenesis . therefore , targeting redox - sensitive pathways and transcription factors with antioxidative ndcs offers great promise for cancer prevention and therapy . we have outlined numerous agents that can interfere with redox - regulated cell signaling pathways . however , npcs that antagonize the enzymes responsible for much of the endogenous production of ros ( ie , the nox family members 1 , 2 , and 4 ) are rare . in addition , triantafyllou et al . reported that whereas tnf- significantly increased the cellular superoxide production by nox1 and 2 , mastic gum completely abolished this stimulation . furthermore , mastic gum inhibited the activity of pkc activity in cell homogenate , suggesting that ndc inhibited phosphorylation of noxa1 by pkc . thus , mastic resin may antagonize activation of these two nadph oxidases . on the other hand , the increase of nox4 in colon epithelial cells would not be affected . we hope continued research will soon identify several and they may be used to reduce the incidence of colon cancer.fig . 1a steady - state of elevated rons and pro - inflammatory factors is a formative driver of colon carcinogenesis . the thesis of this review is that colon cancer chemopreventative foods and their active constituents work through ameliorating the molecular pathways that are dysregulated by a continous cycle of pro - inflammatory factors and free radicals ( depicted by the red arrows ) . oxidative stress activates a variety of transcription factors including nf-b , p53 , -ctn - wnt , and nrf2 which results in the expression of over 5,000 different genes , including those for growth factors , inflammatory factors , and cell cycle regulatory molecules . the paramount feature of the vast majority of chemopreventive substances is their ability to antagonize pro - inflammatory factors and reduce the level of rons . therefore , it is expected that molecular pathways most commonly affect by chemopreventive agents would be those that are dysregulated by pro - inflammatory factors and free radicals . green arrow signifies up - regulation and blue arrows signify progression of processes a steady - state of elevated rons and pro - inflammatory factors is a formative driver of colon carcinogenesis . the thesis of this review is that colon cancer chemopreventative foods and their active constituents work through ameliorating the molecular pathways that are dysregulated by a continous cycle of pro - inflammatory factors and free radicals ( depicted by the red arrows ) . oxidative stress activates a variety of transcription factors including nf-b , p53 , -ctn - wnt , and nrf2 which results in the expression of over 5,000 different genes , including those for growth factors , inflammatory factors , and cell cycle regulatory molecules . the paramount feature of the vast majority of chemopreventive substances is their ability to antagonize pro - inflammatory factors and reduce the level of rons . therefore , it is expected that molecular pathways most commonly affect by chemopreventive agents would be those that are dysregulated by pro - inflammatory factors and free radicals .

there has been increasing interest lately in understanding how natural dietary antioxidants affect chemoprevention , and recently , there has been a merging of information about antioxidants , endogenous and exogenous reactive oxygen and nitrogen species ( rons ) , and inflammation . rons normally serve the cells as second messengers to regulate many of the intracellular signaling cascades that govern multiple cellular activities . however , when the amount of rons exceeds the cell s ability to metabolize / eliminate them , the cell becomes stressed and acquires genetic and epigenetic aberrations and dysregulated intracellular signaling cascades . in addition , there has been a better understanding of the role of tissue inflammation in the carcinogenesis process . herein we integrate these fields to explain where rons arise and how natural dietary antioxidants are principally working through refurbishing pathways that use rons as second messengers .

Introduction The Inflammatory Microenvironment ROS and NOS Production by Colon Cancer Cells RONS and Mutagenesis RONS-Regulated Signaling Pathways Micrornas, RONS, and Inflammation Conclusions

pulmonary embolismis a major global public health concern , with an estimated 900,000 venous thromboembolic events occurring annually in the united states alone . massive pulmonary embolism in non - surgical patients is relatively common and presents a high degree of mortality ( 25 - 65% ) . the incidence of massive intra - operative intra - cardiac or pulmonary emboli ( ic / pe ) is low but these intra - operative events have a high degree of mortality ( 68% ) . there is a lack of outcomes - based evidence to establish guidelines for intra - operative diagnosis and management of these rare events . current recommendations and data emanates from case reports , case series , expert opinion , and extrapolation from other patient populations , with many practitioners relying on supportive care alone . additionally , as there is no consensus for management of intra - operative ic / pe , the utility of several diagnostic tools remains unclear . thus , this review evaluated four commonly used diagnostic modalities under the primary end - point of in - hospital mortality to identify whether the use of more invasive modalities prior to the ic / pe event improved mortality and whether post - intervention improvements in the physiologic parameters for each modality correlated with improvement in patients outcomes . therefore , we conducted this systematic review with the primary intention of evaluating mortality outcomes for four intervention groups in the management of thrombotic ic / pe : surgical embolectomy [ 5 , 6 ] ; thrombolysis [ 2 , 7 , 8 ] ; anticoagulation [ 9 , 10 ] ; or supportive care alone . we hypothesized that either surgical or medical thrombectomy offers survival benefit in massive intraoperative ic / pe and also evaluated the use of more invasive techniques for early detection of ic / pe along with the improvement of physiologic parameters for each modality . to identify the relevant literature , we used computerized literature searches of the ovid and national library of medicine s pubmed databases from 1957 to january 2012 , in addition to the google scholar s database , limited to articles in or translated - to english . the search strategy was set up using the words : pulmonary embolism , pulmonary emboli , thromboembolism , thrombosis , thrombus , embolus , intra - cardiac thrombosis , or cardiac thrombi , in combination with intra - operative , operative , or surgery . four broad therapeutic intervention groups were selected and each of them assessed under the primary end - point of in - hospital mortality : surgical embolectomy ( encompassing both open surgical techniques and percutaneous embolectomy ) [ 5 , 6];thrombolysis ( with variability in choice of thrombolytic agent , dosing , mode of delivery , and frequency of administration ) [ 2 , 7 , 8];anticoagulation ( typically heparin , but at variable doses and duration of therapy ) [ 4 , 9 , 10 ] supportive care alone . surgical embolectomy ( encompassing both open surgical techniques and percutaneous embolectomy ) [ 5 , 6 ] ; thrombolysis ( with variability in choice of thrombolytic agent , dosing , mode of delivery , and frequency of administration ) [ 2 , 7 , 8 ] ; anticoagulation ( typically heparin , but at variable doses and duration of therapy ) [ 4 , 9 , 10 ] supportive care alone . additionally , four diagnostic modalities were assessed under the primary end - point of in - hospital mortality in two different ways : whether the use of more invasive modalities prior to the ic / pe event improved mortality and whether post - intervention improvements in the physiologic parameters for each modality correlated with improvement in patients outcomes . the four diagnostic modalities were : invasive and non - invasive monitoring of vital signs ; pulmonary artery catheter ( pac ) or central venous monitoring device ( cvp ) ; end - tidal carbon dioxide monitoring ( etco2 ) ; and trans - esophageal echocardiography ( tee ) . a subgroup analysis for thrombotic type emboli invasive monitoring was defined as use of the pac , cvp and arterial line monitoring . the primary end - point of mortality was used for each diagnostic and therapeutic modality . all data were derived from case reports or case series outlined in the reference section . inclusion criteria required that major embolic disease was present , but the diagnosis criteria were not objectively defined . diagnosis was instead left to the discretion of the practitioner and institution , and in some cases the diagnosis was made post - mortem . all reports for extra - operative embolic events were excluded , as were reports of emboli or thromboembolic that did not result in hemodynamic compromise or were not classified as major embolic disease by the authors . there were 7 cases that met the above - mentioned inclusion and exclusion criteria , but were ultimately excluded because the presenting sign of ic / pe was sudden death , which did not allow for any of the four primary interventions assessed in this review to be initiated . additionally , non - thrombotic emboli s and neoplastic related emboli s were not included in the assessment of therapeutic modalities and mortality outcome . data were entered into a microsoft excel worksheet and transferred to a ncss datasheet version 2007 ( salt lake , ut ) . univariate analyses of categorical data ( either binomial or multinomial ) were done using chi - squared and fisher s exact test and logistic regression to assess the impact of the four primary interventions on mortality . odds ratios ( ors ) were reported with 95% confidence interval and pvalues less than 0.05 were considered statistically significant . for the analysis of each treatment modality , the supportive care group was used as the reference . age was the only numerical data that was used in multivariate logistic regression as an independent factor to predict patient mortality . the results for numerical variables were reported as mean standard deviation and were analyzed using one - way analysis of variance ( anova ) . data from a total of 78 cases meeting inclusion and exclusion criteria were reviewed ( table 1 ) but one case of amniotic fluid embolism and 7 patients who presented with sudden death were excluded for analysis of therapeutic intervention ( table 1 ) . overall mortality was not statistically different between women ( 44% ) and men ( 38% ) , with 50% of patients being female ( table 1 ) . although the number of the reported cases of intraoperative ic / pe increased over past 40 years , the mortality rate for this uncommon event has remained stable ( 40.9% ) as depicted in figure 1 . the mortality rate of intra - operative intra - cardiac or pulmonary embolism over time . different types of emboli were not statistically significant predictors of mortality . among all the cases reported , the thrombotic etiology ( 61.5% ) was the most common type of emboli followed by gaseous ( 17.9% ) [ 5 , 10 , 11,12,13,14,15,16,17,18,19,20,21 ] and neoplastic emboli ( 16.7% ) . there were also two cases of fat emboli and one case of amniotic fluid embolism ( table 1 ) . the mortality rates were similar among those with thrombotic and neoplastic etiologies ( 43.5% and 40.0% , respectively ) which were significantly worse than those with air embolism ( 27.3% , p<0.05 ) . although there were reports of incidental diagnosis of ic / pe by use of tee , the preoperative use of this tool did not alter mortality . however , the use of pac prior to the embolic event was associated with improved mortality ( or=0.3[0.1 - 0.8 ] , p=0.035 , table 1 ) . for post - intervention monitoring , improvements in physiologic parameters for each of the four diagnostic modalities correlated significantly with survival , but it is worthwhile noting that 13 - 16% patients that showed early improvement of vital signs did not survive ( table 2 ) . mortality outcomes per improvement in post - intervention physiologic parameters . of note , to assess the efficacy of each therapeutic intervention , we excluded 7 patients who manifested with sudden cardiac death that precluded them from further treatment modality . overall , therapeutic interventions resulted in a better outcome compared to those received supportive care . regardless of the cause of the embolus , 32 out of 47 patients ( 70% ) survived when all types of therapeutic intervention were aggregated ( e.g. , surgical embolectomy , thrombolysis or conventional anticoagulation ) while only 14 out of 31 patients ( 45% ) survived with instituting a supportive care , or=0.38[0.15 - 0.98 ] , p=0.04 . univariate analysis of primary interventions with death as a primary outcome resulted in a lack of significantly different outcomes ( p=0.08 ) , but some striking findings should be noted . after separating the type of the interventions , 9 out of 11 patients ( 82% ) in the anticoagulation intervention group and 21 out 29 patients ( 72% ) in the surgical embolectomy group survived , compared to 2 out of 7 patients ( 29% ) in the thrombolytic group and 13 out of 23 patients ( 57% ) in the supportive care group ( table 3 ) . however , the total number of patients was too low to provide sufficient power to show a significant difference among the groups . since the usefulness of the therapeutic interventions examined were most relevant to thrombotic emboli through decreasing the size of the embolus by preventing clot formation or by its lysis / removal , we reexamined their effect on mortality in thrombotic subtype alone ( n=48 ) . again , there were no significant differences in outcome among the treatment groups in comparison to supportive care ( p=0.10 ) . we confirmed that massive intra - cardiac and pulmonary emboli are rare events in the operating room but are associated with high rates of morbidity and mortality . to our knowledge , this is the first study to provide evidence - based recommendations for management of intraoperative ic / pe . the presence of a triad of inflammation , venous stasis and hypercoagulability promotes thrombus formation , which may in turn result in massive ic / pe . during intra - operative period application / release of tourniquet or esmarch band has been reported to cause massive ic / pe . although no statistically significant mortality differences were identified among the intervention groups , some striking findings should not be ignored . primarily , mortality was far greater in the thrombolytic intervention group ( 71% ) than in any of the other groups ( surgical embolectomy , 28% ; anticoagulation , 18% ; supportive care , 43% ) . these results did not reach statistical significance because the data available from published reports resulted in an underpowered sample , but the degree of difference in mortality raises significant concerns about the safety of the use of thrombolytics for management of intraoperative ic / pe . subsequent review of this topic with additional cases may yield adequate power to confirm this trend , but in the interim period it may be prudent for practitioners to consider thrombolysis as a therapy associated with a greater degree of mortality . the finding that thrombolytic therapy is associated with worse outcomes contradicts recommendations from several sources , including the aha / acc guidelines for management of massive pulmonary emboli outside of the operating room environment . this discordance of results may be because use of thrombolytics is contraindicated for patients whom have undergone recent surgery , suggesting that intraoperative use of thrombolytics may be contraindicated altogether . the use of thrombolytics in the non - operative environment for treatment of pulmonary embolus in patients with contraindications has been assessed and has been reported to be high ( 40% ) , but included a high incidence of complications ( major bleeding in 92 of 478 patients and cerebral bleeding in 5 patients ) . use of pac prior to the occurrence of the event significantly correlated with improvements in survival , which may be explained by allowing for early detection of changes in hemodynamic variables that would otherwise not have been monitored . although early use of tee ( prior to the embolic event ) has improved the diagnosis of ic / pe in some cases the routine perioperative use of this device was not associated with an improved outcome . when intraoperative embolic events were suspected , however , tee was often initiated intraoperatively , proving to be a useful diagnostic and prognostic tool . in addition to visualization the mass of thrombus or tumor , tee was generally felt to increase the detection of wall motion abnormalities and post - intervention improvements in right ventricular wall motion that correlated positively with improved survival . in fact , for each of the diagnostic modalities ( vital sign monitoring , etco2 monitoring , pac , and tee ) post - intervention improvements in physiological parameters were positively correlated with an improvement in survival , but it is well worth noting that 13 - 16% of patients with improvement in these parameters subsequently died . this is cause for caution to each practitioner who might see improvement post - intervention , as there is still a high incidence of mortality for an undefined subset of patients . as this study is a systematic review of previously published literature , it is inherently burdened with multiple limitations but , although a greater evidence grade would be preferable upon which to establish guidelines , it is currently impossible to perform a prospective randomized trial for management of massive intra - operative ic / pe . aside from the standard limitations of a retrospective review ( publication bias , recall bias of past reports , and inconsistent data reporting ) , perhaps the most obvious limitations in this particular study were the relative infrequency of events , the lack of standardization between cases , and the relatively low level of evidence , as all data were extracted from previous case reports or series . secondly , in a study of liver transplant patients whose procedures were complicated by pulmonary emboli and/or intra - cardiac thrombi , the authors compared subgroups and reported that pulmonary embolus alone was associated with a greater mortality rate as compared to combination pulmonary emboli and intra - cardiac thrombi . our review did not include such a subgroup analysis evaluating the effectiveness of each intervention based on anatomical distribution of emboli or thromboemboli . additionally , there was inter - operator variability in the technique and/or delivery of each therapeutic intervention , and the resultant variation may have significant clinical implications that can not be effectively accounted for with the current available data . in regards to diagnostic modalities , there was a lack of consistency in reporting of the use of each diagnostic tool , predisposing this study s resultant statistical analysis to reporting bias . the use of intra - operative tee has often been reported and is thought to be an effective diagnostic modality as it has the capacity to both directly visualize an ic / pe and , indirectly , to establish cardiac strain as indirect evidence of hemodynamic compromise caused by mass effect but its use prior to a massive embolic event was not associated with improved mortality outcomes . amniotic fluid embolism was not analyzed in this review , which was in itself conducted under the assumption that mass effect contributes most significantly to the pathophysiology of massive intra - operative ic / pe events , but it has been postulated that immune modulatory pathways play a significant role in the pathophysiology of amniotic fluid emboli , perhaps more so than with other causes of ic / pe . hence , it would have been inappropriate to assess whether therapeutic modalities such as surgical embolectomy ( one of the four intervention groups ) result in better or worse outcomes , considering that mass excision may not be possible for amniotic fluid emboli . prior to this review , recommendations for management of massive intra - operative ic / pe were limited to supportive care measures and analyses from data extrapolated from other patient populations . in this systematic review , anticoagulation and surgical embolectomy resulted favorable therapies for management of intra - operative ic / pe , while a trend toward harm from use of thrombolytic agents was noted . when the size of the embolus is large enough to produce acute hemodynamic derangement , the surgical removal of the thrombus or tumor clearly offers early survival benefit and may decrease the mortality in the long run . of note , not all institutions have the capacity to do surgical embolectomy or support a patient on cardiopulmonary bypass and , for those that do , it is worth emphasizing that anticoagulation resulted in an equally good outcome in those with thrombotic emboli , without introducing the morbidity associated with cardiac surgery . it is of further value to reemphasize that 13 - 16% of patients died despite post - intervention improvement in vital signs and other physiological measures , irrespective of the intervention utilized , and practitioners should remain vigilant in the post - intervention period as a significant as - of - yet undefined subset of patients appear to remain at higher risk for mortality . further studies are necessary to obtain a higher level of evidence to strengthen recommendations for management of these often - catastrophic intra - operative events .

introductionthe management of massive intra - operative embolism remains controversial . our hypothesis was that either surgical or medical thrombectomy offers survival benefit in these patients.methodspublished case reports were reviewed for intra - operative intra - cardiac or pulmonary embolism and outcomes for the following four intervention groups were evaluated for mortality benefit : surgical embolectomy ; thrombolysis ; anticoagulation ; supportive care alone . we also assessed whether the use of diagnostic modalities prior to each embolism event resulted in a mortality benefit and , separately , whether post - intervention improvement in physiologic parameters resulted in improvement in outcomes . univariate analyses and logistic regression were performed to assess the impact of the four primary interventions on mortality , the primary outcome.resultsseventy-eight cases were reviewed and therapeutic interventions resulted in improved survival ( 70% ) compared to supportive care ( 45% ) , odds ratio=0.38[0.15 - 0.98 ] , p=0.04 . univariate analysis of primary interventions with death as a primary outcome resulted in a lack of significantly different outcomes ( p=0.08 ) . mortality rates were 71% in the thrombolytic ; 28% in surgical embolectomy ; 18% in anticoagulation and 43% in the supportive care groups . the routine pre - event use of trans - esophageal echocardiography was not related with improved outcomes ( p=0.36 ) but the use of pulmonary artery or central venous catheters was ( p=0.035 ) . post - intervention improvements in the physiologic parameters of each diagnostic modality were associated with an improvement in mortality ( p<0.05).conclusionsour data present some important trends among the intervention groups , raising significant concerns about the safety for the use of thrombolytics in the management of intra - operative embolism .

Introduction Methods Results Discussion Conclusion

the capacity to produce antimicrobial peptides ( amps ) is widespread among gram - positive bacteria . these amps are directed against competitive microorganisms in order to generate a selective advantage for the producer organism . class i consists of the so - called lantibiotics , which are posttranslationally modified peptides containing ( methyl-)lanthionines , and have a typical size of < 5 kda . class ii comprises heat - stable , nonmodified peptides of 3758 amino acids ( typical size of < 10 kda ) with the leader peptide , as for class i amps , being removed during maturation . class iii contains amps that are heat labile and that are generally larger in size ( roughly 30 kda ) . especially heat - stable peptides secreted by lactic acid bacteria have been studied extensively because of their potential use as natural preservatives in the food industry . one of the best characterized amps is nisin , which is secreted by lactococcus lactis and is a member of the class i amps ( for comprehensive reviews , see [ 2 , 3 ] ) . although nisin has been used as a food preservative for more than fifty years , no significant bacterial resistance against nisin has been reported . nisin , encoded by the nisa gene , is a ( methyl-)lanthionine ring containing peptide that is ribosomally synthesized as a prepeptide consisting of 57 amino acids . the nisa prepeptide is modified posttranslationally by the dehydratase nisb , which selectively dehydrates serine and threonine residues , and by the cyclase nisc , which catalyzes lanthionine ring formation . nist finally transports the modified prenisin across the cell membrane , and mature , biologically active nisin is produced upon cleavage of the leader peptide by the extracellular , membrane - anchored protease nisp . mature nisin harbors three dehydrated amino acids ( one dehydrobutyrine and two dehydroalanines ) , one lanthionine ring , and four methyl - lanthionine rings . in particular , these intramolecular rings are important for the biological activity of nisin . it is worth mentioning that nisin induces its own synthesis via interaction with the two - component regulatory system nisrk . firstly , nisin inhibits cell - wall synthesis by binding to lipid ii , an essential membrane - anchored cell - wall precursor , and secondly , nisin permeabilizes the target membrane . the binding of nisin to lipid ii induces membrane integration of nisin resulting in the formation of a pore , likely composed of eight nisin and four lipid ii molecules . this highly specific interaction with lipid ii is reflected by the low nano- to micromolar concentrations of nisin , sufficient to permeabilize the membrane of the target cells . the bactericidal activity of amps is generally measured by quantifying growth inhibition of an amp - sensitive target organism , grown either on agar plates or in liquid culture . on agar plates , zones of growth inhibition of the indicator organism can be easily visualized , and these so - called halo assays allow determination of the minimum inhibitory concentration ( mic ) of the tested amp . alternatively , bacterial growth can be monitored in liquid culture in dependence of the amp concentration , by measuring the optical density . this method allows the simple determination of both the mic and the ic50 , that is , the concentration of amp that inhibits cell growth by 50% . nisin from l. lactis , like almost all naturally produced amps , can be purified directly from the culture medium [ 10 , 11 ] . it is a cationic peptide , and therefore , commonly purified using cation exchange chromatography ( ciex ) at acidic ph , using high salt concentration for elution , typically a single - step elution with 1 m nacl [ 1216 ] . by using nisin purified via such a method , the ic50 and mic values were determined for a variety of bacteria , such as various l. lactis strains , enterococcus faecium , bacillus anthracis , bacillus subtilis , staphylococcus aureus , and streptococcus mutans [ 9 , 17 ] . whereas e. faecium and s. mutans were equally sensitive to nisin exhibiting an ic50 of 5 m and mic of 12.5 m , l. lactis hp was much more sensitive to nisin , with an ic50 of 14 nm and mic of 32 nm . nisin shows promising activity towards clinical isolates of the methicillin - resistant staphylococcus aureus ( mrsa ) bacterium , streptococcus pyogenenes , and several of the most severe human pathogens , including the multiresistant streptococcus pneumoniae and vancomycin - resistant e. faecium or e. faecalis , against which new effective antibiotics are most urgently needed [ 1820 ] . in all these , studies purified nisin was used and the bactericidal activity of nisin was measured by determining the mic or ic50 . here , we describe a rapid and easy nisin purification protocol , optimized to yield active , monomeric nisin . for this purpose , nisin was either produced from l. lactis strain nz9700 or purchased in the form of a lyophilized powder that contains 2.5% ( w / w ) nisin . nisin from both sources was purified and tested for bactericidal activity using the nisin - sensitive l. lactis nz9000 strain . during the purification low molecular weight contaminants are removed , which results in purified nisin with high specific activity . a culture of the l. lactis strain nz9700 was grown overnight in m17 medium containing 0.5% ( w / v ) glucose ( gm17 ) at 30c . next , 100 ml of the overnight culture was used to inoculate 2 l of gm17 , and cells were grown at 30c . at an od600 of 0.8 , the culture was supplemented with nisin ( sigma ) to a final concentration of 1 ng / ml cell culture to ensure nisin production and growth was continued over night . the supernatant was used and diluted with 1 l of 50 mm lactic acid ph 3 resulting in a 3 l solution with a ph of 5 . this solution was loaded on a 5 ml hitrap sp hp cation exchange ( ciex ) column ( ge healthcare ) using a flow rate of 4 ml / min . due to the large volume , this step is optimally performed overnight . unfortunately , it is not possible to detect nisin at 280 nm , because it does not contain any aromatic amino acids ; therefore , the 215 nm was chosen as wavelength . the column was subsequently washed with 50 mm lactic acid ph 3 until a stable baseline was reached to remove nonspecifically bound material . peptides were eluted by increasing the nacl concentration stepwise using a flow rate of 1 ml / min , resulting in elution fractions with 200 mm ( step i ) , 400 mm ( step ii ) , 600 mm ( step iii ) , 800 mm ( step iv ) and 1 m ( step v ) nacl . to remove nacl , protein in the elution fractions was precipitated with 20% ( v / v ) trichloroacetic acid ( tca ) overnight at 4c . precipitated protein was washed two times with ice - cold acetone to remove residual tca . finally , the protein pellet was suspended in 50 mm lactic acid ph 3 . nisin concentrations were determined by a colorimetric assay ( pierce bca protein assay kit , thermo scientific ) by measuring the absorbance at 584 nm according to the protocol of the manufacturer . commercial nisin ( sigma ) is available as a lyophilized powder containing 2.5% ( w / w ) nisin . 1.3 g of nisin powder ( corresponding to 32.5 mg nisin ) was dissolved in 100 ml 50 mm lactic acid ph 3 and filtered through a 0.45 m membrane filter ( pall corporation ) . the nisin solution was applied to a 5 ml hitrap sp hp cation exchange column ( ge healthcare ) at a flow rate of 2 ml / min , whereas elution was performed at a flow rate of 1 ml / min . tricine - sds - page was essentially carried out as described in . for analysis , 16 l sample was supplemented with 4 l 5x sds sample buffer ( 0.2 m tris - hcl , ph 6.8 , 10% ( w / v ) sds , 40% ( v / v ) glycerol , 0.02% ( w / v ) bromophenol blue , and 10 mm dtt ) and loaded on a tricine gel consisting of a stacking gel containing 5% acrylamide and a separation gel containing 16% acrylamide . the gel was run at 100 v for 2 hours , and proteins were detected via silver staining . for all purification fractions , 3.2 g of total protein was analyzed . samples obtained from ciex chromatography were precipitated with tca and washed with acetone as described above . the protein pellets were then dried for 15 min . at 30c in a vacuum concentrator ( eppendorf concentrator plus ) . dried pellets were stored at 20c until analysis . for mass spectrometric analysis , the samples were dissolved in water with 1% ( v / v ) formic acid . measurements were performed using a maldi - tof instrument ( voyager - de str , applied biosystems ) with a nitrogen laser ( = 337 nm ) operating in reflector mode with 25 kv acceleration voltage . the samples were prepared by the standard dried - droplet procedure , by applying 0.5 l of 2,5-dihydroxybenzoic acid ( dhb ) matrix solution ( 10 mg in 1 ml water ) plus 0.5 l of sample solution . an external calibration with the monomer ion of des - pro - bradykinin , sub p , bombesin , and melittin was used . the spectrum was obtained by averaging 200 laser shots . to investigate the antimicrobial activity of nisin , the nisin sensitive l. lactis strain nz9000 was grown in a 96-well plate in gm17 medium . the total volume in each well was 200 l , consisting of 50 l sample and 150 l gm17 containing l. lactis nz9000 cells ( starting od600 = 0.1 ) . samples were prepared by diluting nisin obtained from various ciex elution fractions in 50 mm lactic acid ph 3 to yield final protein concentrations ranging from 0.15 nm to 300 nm in the wells . cells were grown at 30c , and the optical density was monitored at 620 nm every 20 min for a period of 8 hours ( 96 plate reader bmg ) . to determine the ic50 values , the optical density was normalized and plotted against the log of the nisin concentration . data were evaluated according to ( 1)y = od min + ( od max od min ) 1 + 10((log ( ic50)x)slope ) . y stands for the normalized optical density value , and x represents the logarithmic concentration of the peptide . the ic50 value is calculated as the value of the peptide concentration used where the growth inhibition ( or od600 ) is 50% . the antimicrobial activity of the different nisin preparations was assessed by means of a halo assay . purified nisin obtained from the different ciex elution fractions were supplemented ( 16 l ) with 4 l of 5x sds sample buffer and tricine - sds - page was carried out as described above . after electrophoresis the gel was incubated for 30 min in an aqueous solution containing 20% ( v / v ) isopropanol and 10% ( v / v ) acetic acid . subsequently , the gel was washed two times for 30 min in ddh2o . at this stage , finally , the gel was overlaid with gm17-agar ( 0.5% w / v agar ) containing l. lactis nz9000 cells at an od600 of 0.1 . after solidification , the overlaid gel was incubated overnight at 30c to allow for bacterial growth . the bactericidal activity of nisin is readily visualized by the presence of clear zones ( halos ) resulting from growth inhibition . to determine the antimicrobial activity of commercially available nisin , we used a lyophilized powder , which contains 2.5% ( w / w ) nisin . to further purify nisin , we initially performed sp sepharose cation exchange chromatography using 1 m nacl to elute the bound nisin . subsequent sds - page analysis of the eluate revealed a major protein band corresponding to a peptide with a molecular mass of about 3.5 kda ( data not shown ) in line with the calculated mass of 3354 da for mature nisin . it is of note that these contaminants are not readily visualized by coomassie brilliant blue staining , whereas these impurities are clearly detected by silver staining . nisin purified via this method showed antimicrobial activity against l. lactis nz9000 exhibiting an ic50 of 30 12 nm . we first tested elution with a linear gradient ( 50 times the column volume ) ranging from 01 m nacl . this approach , however , resulted in a broad peak eluting throughout the nacl gradient and further analysis revealed no improvement when compared to the single - step 1 m nacl elution ( data not shown ) . in contrast , a substantial improvement was achieved when a five - step nacl step gradient was used to elute nisin from the ciex column ( figure 1(a ) ) . bound protein eluted at every step as evidenced by the elution profile and subsequent tricine - sds - page analysis ( figure 2(a ) ) . the step ii elution fraction contained the bulk of nisin as evident by the major protein band with a corresponding molecular mass of 3.5 kda , whereas substantially lower amounts of nisin were detected in elution fractions i , iii , and iv . in the step v fraction the step i and ii elution fractions contained exclusively nisin , while elution fractions iii v contained predominantly higher molecular weight compounds ( ranging from 6 kda70 kda ) . in the latter fractions compounds with molecular weights of 8 the total protein concentration of the elution fractions was determined to be : 2.1 mg / ml for step i ( 200 mm nacl ) , 7.5 mg / ml for step ii ( 400 mm nacl ) , 1.9 mg / ml for step iii ( 600 mm nacl ) , 0.5 mg / ml for step iv ( 800 mm nacl ) and 0.4 mg / ml for step v ( 1 m nacl ) ( table 1 ) . in step ii , 60% of the total purified nisin eluted . thus , it appears that 400 mm nacl is sufficient to elute the vast majority of the nisin molecules . an alternative to purchasing nisin is to produce it in the laboratory , since l. lactis strains that secrete nisin in large amounts are readily available . we used l. lactis nz9700 grown in gm17 medium to produce nisin ( see section 2 ) . culture supernatant containing nisin was subjected to ciex chromatography using the same five - step nacl elution gradient as described above . here , a high absorbance at 215 nm occurred at the step i elution ( figure 1(b ) ) , but this did not correspond to nisin or other proteins as evidenced by silver - staining following sds - page analysis ( figure 2(b ) , lane 2 ) . likely , this high absorbance is due to ingredients from the growth medium , which contains large amounts of peptone , tryptone , and yeast extract . this was confirmed by a run with only gm17 media ( data not shown ) . the remainder of the elution profile is similar to that of the lyophilized nisin purification , with two major absorbance peaks observed for the step ii and iii fractions ( figure 1(b ) ) . analysis of the protein content of the different elution fractions revealed that nisin is exclusively found in the 400 mm nacl elution fraction ( figure 2(b ) ) . the total protein concentration of the 400 mm elution fraction was 2.9 mg / ml . however , whereas nisin is most prominent in this fraction , components with a mw 6 kda , 10 kda , and 12 kda are also present . the 0.61 m elution fractions on the other hand contained compounds with molecular weights ranging from 8 kda70 kda , similar to those observed for the purification of lyophilized nisin ( figures 2(a ) and 2(b ) ) . when compared to the purification of lyophilized nisin , nisin purified from gm17 medium still contained contaminants . this difference in purity may relate to differences in the loaded material . the lyophilized nisin powder ( also containing denatured milk solids ) was dissolved in 50 mm lactic acid , whereas nisin produced by l. lactis nz9700 was applied to the cationic exchange column as a 1 : 2 mixture of gm17 culture medium and 50 mm lactic acid , respectively . we considered the possibility that the compounds with molecular weights of 68 kda may represent the unprocessed form of nisin . immature nisin , that is , nisin still containing the leader sequence , has a molecular weight of 5.9 kda and may arise if inefficient leader cleavage by the protease nisp occurs . we therefore performed western - blot analysis using a polyclonal antibody raised against the nisin leader sequence . purified prenisin secreted by a l. lactis strain lacking nisp was used as a positive control for western - blot analysis . for all elution fractions , no signals were observed suggesting that the observed compounds are not derived from prenisin ( data not shown ) . to test the biological activity of the purified nisin , we used l. lactis nz9000 as indicator organism . l. lactis nz9000 is a derivative of the plasmid - cured l. lactis mg1363 and contains the nisrk genes inserted in the chromosomal pepn locus . this strain is commonly used as the host for nisin - induced expression system ( nice ) purposes . however , since this strain lacks the nisin immunity genes nisifeg , it is sensitive to nisin . the antimicrobial activities associated with the different elution fractions obtained as described above were tested in a so - called halo assay . for this , the various fractions containing nisin were analyzed by tricine - sds - page and the tricine gel was overlaid with gm17-agar containing nisin sensitive bacteria ( see section 2 ) . the biological activity of nisin is visualized by the growth inhibition zones ( halos ) at the position where nisin is present . the results for the lyophilized and the laboratory produced nisin are shown in figures 3(a ) and 3(b ) , respectively . after overnight incubation at 30c halos were observed for elution step i iv for the lyophilized nisin , while for the produced nisin , a halo was only observed for elution fraction ii . for both purifications , the highest level of growth inhibition importantly , the zones of inhibition are located only at the position of the 3.5 kda nisin peptide ( figure 3 ) . thus , purified nisin was biologically active and no growth inhibitory activity is associated with the higher molecular weight compounds . to assess and confirm the presence of nisin in the individual ciex elution fractions ( step ii v ) of the lyophilized nisin purification , we applied maldi - tof mass spectrometry . the 400 mm nacl elution fraction contained only one peptide with a molecular mass of 3355.09 da ( figure 4 ) , which is in agreement with the calculated mass of 3354.07 da for nisin . peak integration of the total mass spectrum revealed that the 400 mm elution fraction contains > 98% of nisin , indicating that this fraction is essentially devoid of contaminants . nisin was also found in the 600 mm and 800 mm elution fractions , whereas nisin was not detected in the 1 m nacl fraction . these results are in agreement with tricine - sds - page analysis ( figure 2(a ) ) . however , the 0.61 m nacl elution fraction contained several peptides with higher molecular masses . subsequent tandem ms analysis of these peptides yielded in - sequence tags of eight amino acids and six amino acids , which unfortunately could not be assigned to specific proteins . this was due to the fact that the obtained sequence tags were too short and when blasted gave multiple different protein hits ( data not shown ) . nevertheless , the obtained sequence tags did not match with the primary sequence of ( pre)nisin . therefore , it can be excluded that the peptide contaminants with molecular masses of 68 kda are derived from prenisin . to quantitatively assess the growth inhibitory activity of nisin obtained from the different purification fractions , a liquid culture assay was performed using l. lactis nz9000 as reporter organism . the optical density of the l. lactis nz9000 cultures after 5 hours of growth was plotted against the total protein concentration of the nisin purification fractions . results are shown for both , the lyophilized ( figure 5(a ) ) and the produced nisin ( figure 5(b ) ) , respectively . the resulting growth curves and the calculated ic50 values are shown in figure 5 and table 1 , respectively . for lyophilized nisin , all ciex elution fractions exhibited growth inhibitory activity , however , with very distinct ic50 values ( figure 5(a ) and table 1 ) . the step ii elution fraction displayed the highest bactericidal activity with an ic50 of 2.6 0.1 nm . the other elution fractions showed substantially higher ic50 values . whereas the step v elution fraction had only an inhibitory effect at the highest tested concentrations , the step i , iii , and iv elution fractions exhibited an ic50 value of 35.1 0.1 nm , 6.9 0.2 nm , and thus , the 400 mm nacl elution fraction contains not only the bulk of nisin , it also contains nisin that displayed the highest specific activity . a similar observation can be made for nisin purified from the medium ( figure 5(b ) ) . however , here only the 400 mm elution fraction shows bactericidal activity , which is in agreement with tricine - sds - page analysis ( figure 2(b ) ) and the halo assay ( figure 3(b ) ) . the calculated ic50 of 11.2 0.3 nm is , however , 4-fold higher than that of the corresponding fraction obtained from the purification of lyophilized nisin . we attribute this difference to the contaminants that are still present ( figure 2(b ) , lane ii ) . nevertheless , for both purifications the highest bactericidal activity is associated with the fraction that contains the highest amount of nisin ( figures 2 - 3 and 5 ) . taken together the data indicate that nisin obtained from the step ii elution fraction has the highest specific activity . to determine whether nacl used for elution has an effect on nisin activity , we repeated the experiment and adjusted the concentration of nacl after elution in every fraction to 500 mm either by dilution with buffer without salt or by adding buffer and salt . precipitated protein from these fractions was subsequently used for growth experiments as described above . in all cases , the ic50 values of the salt experiment were slightly higher than when measured directly after elution , indicating that more nisin is needed to inhibit cell growth by 50% ( data not shown ) . this indicates that residual salt does not have a major influence on the activity of nisin . it can , therefore , be excluded that the differences in ic50 values of the various nisin containing elution fractions are induced by the amounts of nacl used to elute nisin from the column . their uniqueness of especially the lantibiotic group of amps , resides in the posttranslational modifications , such as dehydration of amino acids and intramolecular thioether bridges . one of the best - characterized amp is nisin , a compound used for more than 40 years in up to 80 countries as an effective agent to combat food - borne pathogens . commonly , nisin and other cationic amps are purified using a single 1 m nacl elution step , from a ciex column at an acidic ph [ 1214 , 16 , 26 ] . the activity of amps is strictly dependent on the target organism . for example , the ic50 value of nisin ranges from 14 nm for l. lactis hp to 5 m for vancomycin - resistant e. faecium , with respective mics of 32 nm and 12.5 m . in all these studies , it is noticeable that there is a large variation in the sensitivity to nisin between isogenic strains of gram - positive bacteria , whereas some bacteria are inherently resistant to nisin . the most prominent example is the enzymatic destruction or modification of the antibiotic , thereby rendering it ineffective . a second important mechanism of resistance is shielding of the target such that the antibiotic can not get access to it for example , by cell - surface alterations ( capsules s - layers ) or by active extrusion by efflux pumps . moreover , the resistance of the amp producer organism towards its secreted amp ( autoimmunity ) is typically based on atp - binding cassette ( abc ) transporters which expel the amp from the membrane . in l. lactis nz9700 , cells autoimmunity is mediated by the scavenger protein nisi and the abc transporter nisfeg . l. lactis cells lacking the nisifeg defense system ( e.g. , l. lactis nz9000 ) are sensitive to nisin and can be used as indicator organism to measure the biological activity of nisin . the nisi and nisfeg genes are part of the nis operon and are expressed in concert with the genes involved in nisin production and secretion . recently , in nisin - non - producing l. lactis , nisin resistance was shown to be conferred by a specific nisin resistance gene ( nsr ) , which encodes a 35 kda nisin resistance protein ( nsr ) . nsr proteolytically removes the last six amino acids of nisin , thereby reducing its bactericidal activity by a factor of 100 . the level of intrinsic resistance and the employed mechanisms of antibiotic resistance may differ greatly among microorganisms . it is , therefore , difficult , if not impossible , to directly compare ic50 values for a given amp when comparing strains . moreover , the purity of the amp preparation is of great importance to determine accurately the bactericidal activity of the amp . in this study , we optimized the purification of the amp nisin and determined its ic50 values against the nisin - sensitive l. lactis strain nz9000 . nisin typically purified via a 1 m nacl one - step elution yields high levels of active nisin ( see above and ) but contains a substantial amount of contaminants . we show that these contaminants , which are mainly proteinaceous in nature , are largely removed by using a five - step nacl elution . notably , 400 mm nacl ( step ii ) was sufficient to elute the bulk of the nisin molecules , while the majority of contaminants remain bound to the column . in this manner when tested against the nisin - sensitive l. lactis nz9000 , this highly active nisin exhibited an ic50 of 2.6 0.1 nm , which is a 10-fold improvement as compared to the nisin obtained via the one - step elution . the potent bactericidal activity of nisin against l. lactis nz9000 lacking nisi and nisfeg suggests that these autoimmunity proteins are of great importance for l. lactis cells that produce nisin ( e.g. , l. lactis nz9700 ) . amps get more and more into the focus of biochemical , biophysical , and medical studies due to their antimicrobial activity against a wide variety of bacteria . here , we report an easy and rapid protocol for the purification of highly active nisin , purified either directly from the culture medium or from a commercially available lyophilized powder . a pure , homogenous , and biologically active preparation will ensure reliable determination of the efficacy of amps towards their microbial target(s ) . due to the similar chemical and biophysical properties of lantibiotics , our manner of purification may also apply to amps other than nisin .

nisin is an antimicrobial peptide produced and secreted by several l. lactis strains and is specifically active against gram - positive bacteria . in previous studies , nisin was purified via cation exchange chromatography at low ph employing a single - step elution using 1 m nacl . here , we describe an optimized purification protocol using a five - step nacl elution to remove contaminants . the obtained nisin is devoid of impurities and shows high bactericidal activity against the nisin - sensitive l. lactis strain nz9000 . purified nisin exhibits an ic50 of ~3 nm , which is a tenfold improvement as compared to nisin obtained via the one - step elution procedure .

1. Introduction 2. Material and Methods 3. Results 4. Discussion

dental caries , periodontal disease , and opportunistic infections are among the common diseases of the oral cavity caused by the accumulation of pathogenic microorganisms , inappropriate nutritional habits , and improper oral hygiene [ 14 ] . oral streptococci are the first isolated species to play a role in the formation of dental plaque and development of caries [ 57 ] . measures taken to clinically inhibit plaque accumulation , namely , tooth brushing , dental flossing , and use of mouth rinses ( as an adjunct ) , are highly effective for preventing the supragingival plaque and gingivitis and decreasing the number of microorganisms . however , despite extensive antimicrobial activity , it has side effects such as altered sense of taste and staining of tooth surfaces and restorations [ 911 ] . many individuals residing in developing countries strongly believe in the efficacy of herbal medications for primary care [ 12 , 13 ] . considering the increasing resistance of microorganisms to chemical synthetic drugs and their side effects , researchers are becoming more and more interested in finding alternative herbal medications and their active components [ 1418 ] . the pomegranate with the botanical name of punica granatum linn . is a fruit - bearing deciduous shrub or small asian tree from the family of punicaceae growing between 5 to 8 meters tall . it is native to iran and northern india and has long been cultivated in the asian mediterranean region , europe , and africa . punica granatum is rich in bioactive compounds , which are used for treatment of cancer , cardiovascular diseases , diabetes mellitus , dental diseases , bacterial infection , antibiotic resistance , skin conditions due to uv radiation , diarrhea , bloody diarrhea , and hemorrhoids . moreover , p. granatum is used as a mouth rinse for treatment of some types of sore throat . parts of the plant used for medicinal purposes include its flowers , trunk skin , fruits , roots , and seeds [ 1921 ] . the punica granatum flowers are odorless but colorful red or reddish , 3.5 to 7 cm in length , and campanulate or cylindrical . the unfertilized flower is with smaller , barren , and short - styled , short - stamened petals , in which the stigma is far below the anthers . trunk skin and roots are used for parasitic infections , dried flowers are beneficial for treatment of bronchitis , diarrhea , and bloody diarrhea , and the brewed pomegranate is used for treatment of inflammation of the throat and oral cavity . pomegranate has long been recommended as a hemostatic agent and for treatment of diabetes mellitus in ancient greek medicine [ 1921 ] . the antimicrobial properties of p. granatum have been recently noticed [ 19 , 2330 ] . the ethanol , water , methanol , and acetone extracts of p. granatum have shown strong antimicrobial properties against gram - positive and gram - negative nonoral microorganisms [ 14 , 19 , 22 , 24 , 31 ] . however , a few studies have evaluated the antibacterial properties of this plant on oral bacteria [ 14 , 3133 ] . however , the mentioned studies are very limited and mostly lack an appropriate microbiological method . search of the literature only yielded one previous study on the effect of water extract of p. granatum flower ( petal ) on oral microbial pathogens , which showed its greatest antimicrobial effect on s. sanguinis , although menezes et al . reported that hydroalcoholic extract of p. granatum was very effective against biofilm forming microorganisms in the mouth of patients . considering the high prevalence of oral and dental diseases due to oral pathogens and the recent public interest in medicinal plants , this study aimed to assess the effect of hydroalcoholic extract of p. granatum petal on s. mutans , s. sanguinis , s. salivarius , s. sobrinus , and e. faecalis in vitro . p. granatum flowers were obtained from the last harvest in darab city and its purity was confirmed in a pharmacognosy laboratory . powdered petals were precisely measured by a digital scale and poured into an erlenmeyer flask . the erlenmeyer flasks were capped with aluminum foil and stored in the dark for 10 days . next , the flasks were placed on a shaker ( gfl 3017 ) operating at 90 rpm for 24 hours . the filtered solution was poured into a sterile glass container and capped by aluminum foil . a few holes were perforated in the foil and the glass container was placed in bain - marie at 90c to dry . standard strains of s. mutans ( atcc 35668 , ptcc 1683 ) , s. sanguinis ( atcc 10556 , ptcc 1449 ) , s. salivarius ( atcc 9222 , ptcc 1448 ) , s. sobrinus ( atcc 27607 and ptcc 1601 ) , and e. faecalis ( atcc 11700 , ptcc 1393 ) were obtained in lyophilized form from the persian type culture collection center . bacteria were activated by inoculation in the brain heart infusion agar ( bhia , merck , germany ) culture medium followed by 24 hours of incubation at 37c . for preparation of microbial suspension , a 24-hour culture was used . the concentration of microorganisms in the microbial suspension was adjusted to 0.5 mcfarland standard using a spectrophotometer at a wavelength of 625 nm ( a mcfarland standard is a chemical solution with a turbidity comparable to that of microbial suspension . using this suspension , number of bacteria per each milliliter of the suspension can be estimated , which is equal to 1.5 10 cfu / ml ) . primary assessment of the antimicrobial effect of extracts was done using the cup - plate technique . 500 l of each microbial suspension at 0.5 mcfarland standard concentration was cultured in bhia ( swabbed on the plate ) . then , wells measuring 8 mm in diameter were created on the agar surface . different concentrations of the extracts were prepared by serial dilution ( dilution by one - half ) using sterile distilled water solvent ; 100 l of each concentration of extract was poured into each well . this process was repeated in triplicate and the mean diameter of the growth inhibition zone was calculated for different concentrations of the extract . the mic is defined as the lowest concentration of an antimicrobial agent that inhibits the growth of a microorganism ( 0.5 mcfarland standard in this study ) . it is the minimum concentration of the extract that completely prevents visible growth and proliferation of bacteria compared to the negative control group . to determine mic , macrodilution method according to the standard technique described by the clinical and laboratory standards institute ( 2012 ) different concentrations of the extract were prepared by serial dilution ( dilution by one - half ) in bhi broth medium . using this medium , the 0.5 mcfarland standard suspension was diluted 1 to 150 to obtain a bacterial count of 10 cfu / ml . microbial suspension was then diluted by one - half using the culture medium and 1 ml of it was added to the tubes containing serially diluted extract . the negative control tube only contained the culture medium and extract with no microbial suspension . after 24 hours of incubation at 37c , growth and proliferation of microorganisms were evaluated and the mic value of the extract for each bacterial strain was determined . , 20 l of the suspension in the tube containing mic of the extract and tubes showing no bacterial growth were cultured on plates containing bhia . after 24 hours of incubation at 37c , the plates were evaluated for growth of microorganisms . the concentration with no bacterial growth the effect of 0.2% chx ( shahr daru , iran ) and ampicillin on the microorganisms was also evaluated using the cup - plate method and the mic and mbc values of chx and ampicillin for the microorganisms were determined as well . the tests were repeated in triplicate and the mean and standard deviation ( sd ) of the growth inhibition zone diameter in cup - plate method as well as the mic and mbc of the extract , chx , and ampicillin were determined . the hydroalcoholic extract of p. granatum showed inhibitory effects on the growth and proliferation of all five bacteria using the cup - plate method . the largest and the smallest diameter of growth inhibition zone belonged to s. sanguinis and e. faecalis , respectively . the mean ( and sd ) diameter of the growth inhibition zone due to the effect of hydroalcoholic extract of p. granatum on different microorganisms is shown in table 1 . the mic and mbc of hydroalcoholic extract of p. granatum were determined using serial dilution method . the highest antibacterial effect of p. granatum extract was on s. mutans with mic and mbc values of 3.9 mg / ml . the mic and mbc values of this extract for different microorganisms are presented in tables 2 and 3 . the cup - plate method showed that 0.2% chx had antimicrobial effect on all five bacterial strains . the mean and sd of the diameter of growth inhibition zone due to the effect of 0.2% chx on the five bacterial strains are shown in table 4 . assessment of the mic and mbc values revealed that chx had the highest effect on s. sanguinis . tables 2 and 3 show the mic and mbc of chx for the five bacterial strains . the cup - plate method showed that ampicillin had inhibitory effects on the growth and proliferation of all five bacterial strains . the greatest diameter of growth inhibition zone belonged to s. mutans and s. sanguinis due to the effect of ampicillin . the mean and sd of the diameter of growth inhibition zone due to the effect of ampicillin on different microorganisms are shown in table 5 . assessment of the mic and mbc values revealed that ampicillin had the highest effect on s. sanguinis . tables 2 and 3 show the mic and mbc of ampicillin for the five bacterial strains . this study was the first to experimentally assess the antimicrobial effect of hydroalcoholic extract of p. granatum petal on s. mutans , s. sanguinis , s. salivarius , s. sobrinus , and e. faecalis in vitro using the cup - plate method . the mic , mbc , and growth inhibition zone diameter values of the extract for different microorganisms were also calculated and compared to chx and ampicillin . based on the results , the hydroalcoholic extract of p. granatum petal had inhibitory effect on the growth and proliferation of all five bacterial strains . evaluated the mic of pomegranate fruit gel and compared it with miconazole against adhesion of s. mutans , s. mitis , and c. albicans . they showed that pomegranate fruit gel was more effective than miconazole in preventing the adhesion of streptococci to glass . dahham et al . assessed the antimicrobial effect of ethanolic extract of seed , fruit , peel , and juice of pomegranate on specific bacteria and reported that the pomegranate peel extract had the greatest antimicrobial activity . stated that only at concentrations of 8 mg / ml and 12 mg / ml methanolic extract of p. granatum peel ( mepgp ) was effective against l. acidophilus , s. mutans , and s. salivarius . furthermore , no concentrations of mepgp inhibited a. viscosus and c. albicans . in present study hydroalcoholic extract of p. granatum petal was effective against s. mutans in lower concentration than mepgp in abdollahzadeh et al . therefore , it seems that hydroalcoholic extract of petal against s. mutans was more effective than mepgp . it should be considered that s. mutans is main microorganism of dental plaque and caries . also , in current study , both mic and mbc were considered . furthermore , both mic and mbc values against s. mutans were similar ( 3.9 mg / ml ) . the mic and mbc values of p. granatum showed the best antibacterial effect of this extract on s. mutans , while the lowest effect was noticed on e. faecalis . the only previous study on the antimicrobial effect of methanolic extract p. granatum flower on s. mutans was conducted by haghighati et al . . also , they showed no significant difference between the efficacy of p. granatum and chx against s. mutans ; these findings show that the method used was not proper and therefore their results are not reliable . in a study by vahid - dastjerdi et al . , the effect of water extract of p. granatum flower ( petal ) on the same microorganisms as in our study was evaluated . the highest antimicrobial effect was seen on s. sanguinis compared to the mic and mbc values obtained in the current study . therefore , it appears that the solvent containing alcohol releases more amount of the antimicrobial agent and subsequently exerts a greater antimicrobial effect on s. mutans . the composition of extract is influenced by the geographical location of the plant , season of harvesting , age of plant , growth stage , method of drying , and extraction technique . also , extracts of different parts of the plant have variable level of antimicrobial activity and bacteria have variable sensitivity to different extracts . also , isolated components of an extract show greater antimicrobial effects than the extract itself . alcoholic extract of p. granatum has well known isolated components such as gamma terpinene , borneol , camphor , carvacrol methyl ether , and methyl palmitate . a previous study isolated the constituents of rosemary and evaluated their effects on oral pathogenic microorganisms . camphor had inhibitory effects on s. mutans , s. sanguinis , s. salivarius , s. sobrinus , and e. faecalis . thymol is an isomer of carvacrol , showing its antimicrobial activity by making the bacterial membrane permeable . chlorhexidine mouthwash has extensive antimicrobial activity and is more effective on gram - positive than gram - negative bacteria . it is known as the most effective mouthwash and the gold standard of antibacterial activity . the mic and mbc values and inhibitory effects obtained in our study for chx were in accord with the results of specific microbiological assessments on chx and ampicillin , which confirmed the accuracy and precision of the laboratory phases in our study . one limitation of our study was that we only evaluated the p. granatum flowers harvested at a specific time from a specific location . also , standardized microbial strains were used and microorganisms collected and purified from the oral cavity were not assessed . future studies are required to confirm the results in normal biological environment ( oral cavity ) . also , future studies must focus on the isolated constituents of different extracts and the antimicrobial agent in their composition . given that the effects of these constituents on other microorganisms such as l. acidophilus , a. viscosus , and c. albicans are confirmed , they may be used in the form of pure extract , mouth rinse , or other antimicrobial products in clinical trials . within the limitations of this study , the following conclusions were drawn : hydroalcoholic extract of p. granatum had a significant antibacterial effect on common oral bacteria , namely , s. sanguinis , s. mutans , s. salivarius , s. sobrinus , and e. faecalis.hydroalcoholic extract of p. granatum had maximum antibacterial effect on s. mutans with mic and mbc of 3.9 mg / ml . hydroalcoholic extract of p. granatum had a significant antibacterial effect on common oral bacteria , namely , s. sanguinis , s. mutans , s. salivarius , s. sobrinus , and e. faecalis . hydroalcoholic extract of p. granatum had maximum antibacterial effect on s. mutans with mic and mbc of 3.9 mg / ml .

objectives . this study aimed to assess the effect of hydroalcoholic extract of punica granatum linn . ( p. granatum ) petal on streptococcus sanguinis , streptococcus mutans , streptococcus salivarius , streptococcus sobrinus , and enterococcus faecalis . materials and methods . in this in vitro study , p. granatum extract was prepared using powdered petals and water - ethanol solvent . antibacterial effect of the extract , chlorhexidine ( chx ) , and ampicillin was evaluated on brain heart infusion agar ( bhia ) using the cup - plate method . by assessing the diameter of the growth inhibition zone , the minimum inhibitory concentration ( mic ) and minimum bactericidal concentration ( mbc ) of the extract were determined for the above - mentioned bacteria . results . hydroalcoholic extract of p. granatum petal had inhibitory effects on the proliferation of all five bacterial strains with maximum effect on s. mutans with mic and mbc of 3.9 mg / ml . the largest growth inhibition zone diameter belonged to s. sanguinis and the smallest to e. faecalis . ampicillin and chx had the greatest inhibitory effect on s. sanguinis . conclusions . hydroalcoholic extract of p. granatum had a significant antibacterial effect on common oral bacterial pathogens with maximum effect on s. mutans , which is the main microorganism responsible for dental plaque and caries .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

research from the last decades clearly shows that zinc has a vital role in neonatal development . zinc is an essential trace element in humans and animals and is involved in countless metabolic and signaling pathways within the body . however , a particular role of zinc in the immune system and brain has been reported . zinc is one of the most prevalent metal ions in the brain and participates in the regulation of neurogenesis , neuronal migration , and differentiation , thereby shaping cognitive development and maintaining healthy brain function . zinc deficiency during pregnancy results in specific impairments in the offspring , which have been observed in animal models but might also be present in humans . intriguingly , among individuals with autism spectrum disorders ( asd ) , the incidence rate of zinc deficiency has been reported to be significantly increased compared to age matched healthy control subjects . the occurrence of zinc deficiencies in asd is particularly pronounced in very young age [ 4 , 5 ] , where a rate of almost 50% was reported in the age group of 03 years . these low levels of zinc often occur along with copper overload and the cu / zn ratio was reported to correlate with the severity of symptoms associated with autism [ 68 ] . this early occurrence of zinc deficiency with decline later in life and the manifestation of some of the core features of asd , such as impaired social behavior and language and communication problems in prenatal zinc deficient mice , have recently put maternal zinc status in the focus as a possible environmental factor in the etiology of asd . thus , maintaining adequate zinc status during pregnancy might be a promising approach to prevent cognitive and neurobehavioral deficits later in life . two major pools of zinc can be found within the body : a slowly zinc exchanging pool that contains about 90% of the body 's zinc and a pool that rapidly exchanges zinc with the plasma . the latter , which contains the other 10% of zinc , is the one that is especially reactive to the amount of absorbed zinc and is the first to be depleted under conditions of zinc deficiency . plasma zinc is also the source of the embryo 's zinc supply . in order to maintain proper zinc levels during pregnancy , both endogenous losses and the increased demand resulting , for example , from synthesis of novel tissue thus , while the metabolic zinc requirement of 2.5 mg / d for an adult woman is generally met when consuming daily 10 to 15 mg zinc , due to the additional need for zinc during pregnancy , an additional 510 mg zinc per day must be consumed to meet the increasing demand of 0.08 , 0.24 , 0.53 , and 0.73 mg of metabolic zinc per day for the four quarters of pregnancy . similarly , during lactation , the metabolic daily requirement increases by another 2.5 mg per day . meeting these requirements is challenged by several factors . first , it is not uncommon for women of childbearing age to consume low zinc diets . second , zinc status of women may be compromised due to increased intake of dietary constituents that reduce the availability of zinc . impact of low zinc status of the mother can be magnified depending on time and severity of the deficiency , ranging from teratogenic effects with severe deficiency to functional impairments acting , for example , on brain development with mild deficiency . in particular , teratogenic effects have been reported in rodent models [ 11 , 12 ] as well as in humans , where women with acrodermatitis enteropathica , a genetic disorder resulting in impaired zinc absorption , show a high incidence of birth defects . in general , although the brain seems most vulnerable , all organ systems are affected by systemic zinc deficiency in times of active proliferation and differentiation . thus , although mild zinc deficiency does not lead to gross morphological malformations in the offspring , the reported behavioral impairments might result from a combination of alterations in brain development and other organ systems . this novel vista on the role of zinc deficiency in asd broadens the focus from the action of zinc within the brain to other organs such as the gi system . proper zinc status is necessary for healthy gut development and both pre- and perinatal zinc deficiency might affect the neonate and potentially trigger downstream events that contribute to pathological processes . these processes may , among others , include inflammation due to increased intestinal epithelium permeability and immune system abnormalities including the generation of autoantibodies . another consequence of impaired or delayed gut development will be lowered trace metal absorbance , which might contribute to the slow normalization of biometals in children with asd after birth . gi discomfort , changes in gut microbiome , food aversion , and an increased intestinal permeability have been shown to correlate with the severity of behavioral symptoms in individuals with asd [ 1521 ] . given that inflammatory cytokines and other immune signaling molecules originating from the gi tract interact with the hypothalamic - pituitary - adrenal gland ( hpa ) stress axis , prenatal stress itself can be integrated in this pathomechanism , targeting the same structures . thus , some of the major environmental risk factors for the development of asd are linked in this model . taken together , maternal zinc deficiency might impair the gut development of the offspring and thereby increase the risk for gi problems , inflammatory events , abnormal immune signaling , trace metal imbalances , and ultimately altered brain function . a well - orchestrated sequence of highly specialized processes is required for the development of the intestine from the embryonic gut tube to a complex organ responsible for food digestion and absorption of essential nutrients . particularly the sophisticated intestinal epithelium is strongly dependent on a proper development in order to fulfill its widespread functions ranging from defense of antigens to absorption of important nutrients . these processes are dependent on the correct sequence of cell proliferation , differentiation , and apoptosis . given that these processes require a plethora of zinc dependent enzymes , it is quite obvious that zinc deficiency especially during the embryonic development of the gut might lead to alterations in intestinal morphology and cell composition resulting in possible functional alterations ( figure 1 ) . unfortunately , only very limited data is available on the precise effects of prenatal zinc deficiency during fetal development and differentiation of the small intestine . intriguingly , researchers found that feeding sows an additional 250 ppm zinc from zinc amino acid complex during the last trimester of pregnancy resulted in improved intestinal development of pigs . the offspring of sows fed the additional zinc had increased villous height and villus / crypt ratio in the jejunum and higher goblet cell counts in the ileum . furthermore , intestinal defenses of these pigs against pathogens appeared to have been improved as indicated by an increased number of intraepithelial lymphocytes in the duodenum and ileum . however , most of the available data on the role of zinc in gut development originate from induced zinc deficiency in immature and mature animals . several studies have shown fatal consequences of acute and chronic zinc deficiency on the structure and function of the small intestinal epithelium . for example , zinc is crucial for the maintenance of the small mucosal integrity [ 2426 ] and zinc deficiency accompanied with mucosal necrosis and ulceration as well as increased mucosal apoptosis , inflammation , oedema , and structural alterations of villi . thus , it is not surprising that zinc supplementation has been shown to have beneficial effects on mucosal integrity in many pathophysiological and inflammatory conditions of the small intestine [ 26 , 27 ] . further , individuals with acrodermatitis enteropathica who suffer from severe zinc deficiency showed villus atrophy and gut necrosis . when zinc deficient diet is fed to immature male rats for 28 days , a significant reduction of small intestinal length and further morphological changes in the jejunum , including shortening and narrowing of the villi , reduction in absorptive surface , and an increased number of villi per unit area of serosa , were reported that could be restored by zinc supplementation [ 29 , 30 ] . moreover , ultrastructural changes on a cellular level , such as appearance of membrane - bound autophagic vacuoles , pyknotic nuclei , and dilated nuclear periphery can be observed in zinc deficient rats . additionally , a study in zinc deficient rats and sheep revealed an altered composition of intestinal mucin hinting towards functional alterations in mucin - secreting goblet cells . a factor contributing to this impairment might be an increase in the number of apoptotic cells in villi and crypts , especially in the midzone of the crypts that serves as the zone of renewal of the intestinal epithelium [ 33 , 34 ] . this hints towards a reduced renewal capacity of the intestinal epithelium that is required for its proper function . a positive effect of zinc supplementation on the repair capacity of the small intestine , especially the third segment , has been reported in mice . these mice showed a higher intestinal epithelium cell production rate and shorter duration of mitosis compared to their control littermates . thus , besides the effects of zinc deficiency on morphology of the small intestine , the maintenance and repair capacity of the epithelium are affected . during development , small intestine maturation is measured by indicators like increased cell proliferation and differentiation as well as an altered activity of brush border disaccharidases like lactase and sucrase due to changing nutritional demands . lactase and sucrase serve as markers of enterocyte maturity and functional capacity as well as villus height and crypt depth . several alterations in the activities of brush border enzymes have been reported to result from zinc deficiency . chronic zinc deficiency , for example , reduces the activity of disaccharidases like sucrase , trehalase , lactase , leucine aminopeptidase , alkaline phosphatase , and maltase by 3050% at the brush border of the small intestine [ 34 , 36 , 37 ] . correct function of intestinal disaccharidases is inevitable for proper digestion of carbohydrates and absorption of saccharides . given that zinc is crucial for maintenance of membrane structure and function , the loss of brush border integrity due to zinc deficiency might lead to the dysfunction of these enzymes and thus altered gut maturation . additionally , many genes regulating the differentiation into intestinal epithelium in adults as part of a self - renewal process of the epithelium by intestinal stem cells localized in the base of crypts also play a crucial role in the regionalization of the gut during the development . zinc dependent transcription factors are highly involved in the regulation of these genes and their dysfunction has severe consequences on intestinal development . for example , the transcription factors gata4 and gata6 are involved in the proximal - distal specification of the intestine as well as epithelial cell differentiation . gata4 seems to regulate sucrase - isomaltase and lactase transcription hinting towards a role in maturation of the enzymatic brush border composition [ 39 , 40 ] and the loss of gata4 leads to decreased absorption of cholesterol and fats . furthermore , the conditional knockout of gata4 and gata6 results in reduced promotion of enteroendocrine cell differentiation . a further zinc dependent transcription repressor , b lymphocyte - induced maturation protein 1 ( blimp1 ) , is required to delay the final maturation of suckling to weaning intestinal epithelium allowing the dietary transition from mother 's milk to solid diet and is therefore specifically expressed in developing and postnatal intestine . blimp1 knockout mice are born with features resembling an adult intestine such as more serrated appearance of villi and accelerated development of paneth cells [ 43 , 44 ] . during suckling period , the expression of disaccharidases , typically expressed in postweaning periods , is upregulated in blimp1 knockout mice whereas the expression of disaccharidases important for lactose digestion is lost [ 43 , 44 ] . additional zinc dependent transcription factors like the growth factor independent 1 ( gfi-1 ) and mtgr1 are involved in secretory cell differentiation [ 45 , 46 ] . although zinc is bound within zinc finger transcription factors with high affinity and only potent zinc chelators are able to resolve zinc binding , it might be possible that , along normal protein turnover , severe zinc deficiency leads to less stable and/or functional transcription factors . further , zinc binds with less affinity to enzymes like class i histone deacetylases that have been reported to be involved in the regulation of intestinal epithelium differentiation . additionally , activity of the zinc binding matrix metalloproteinase 9 ( mmp-9 ) seems to influence the number of goblet cells and by that increases secretion of the mucin muc-2 . however , it has to be mentioned that despite the beneficial effects of zinc supplementation on small intestinal epithelial structure , excessive amounts of zinc can lead to damage in vitro . thus , the appropriate zinc status during development is crucial for a healthy functional intestine . taken together , zinc deficiency in animals and humans has strong effects on the intestinal epithelium structure and function ( figure 1 ) . due to these severe consequences it is likely that zinc deficiency during embryologic development might lead to morphological alterations resulting in functional impairment of the small intestine . these impairments might include malabsorption of essential nutrients leading to malnutrition , diarrhea , and inflammation in the immature gut . a growing amount of research indicates that at least a portion of the dysfunctions associated with asd is related to gi problems . however to date it remains unclear whether gi problems are comorbidities or a causative pathomechanism of asd [ 50 , 51 ] . it has been repeatedly reported that children with asd frequently suffer from gi problems such as diarrhea , constipation , bloating , abdominal pain , and gastroesophageal reflux [ 52 , 53 ] . gi problems ( based on parents ' reports ) were identified in 42% of children and 12% of controls , with constipation ( 20% ) and chronic diarrhea ( 19% ) being the most common symptoms . furthermore , altered intestinal barrier function has been found in subjects with asd along with an increased intestinal permeability . another contributing factor to gi problems in individuals with asd might be an abnormal composition of gut microbiota . in the gi flora of autistic children , using stool samples , lower levels of beneficial bifidobacter species and higher levels of lactobacillus species were found compared to controls . other studies stated altered clostridium species numbers and types in children with asd [ 5358 ] and differences concerning the phylum level with an increase in bacteroides and a decrease in firmicutes in the asd group [ 5961 ] . it has been stated that gi disturbances correlate with the severity of asd . the stronger the gi symptoms are , the more likely children presented severe autistic symptoms . the gi tract plays an important neurological function and therefore sometimes is referred to as the the second brain . via enteric nerves and networks , the gi tract is able to affect the brain and vice versa [ 62 , 63 ] . intriguingly , in healthy human subjects , modulation of the gut microbiome was shown to have the potential to alter brain responsiveness to an emotion recognition task . besides the gi disorders often found in asd patients , the gut - brain interaction seems to play a role in other neurological disorders as well . investigating the prevalence of depression and anxiety disorders as comorbidity in inflammatory bowel disease ( ibd ) , individuals with crohn 's disease and ulcerative colitis , two types of ibd , are more likely to suffer from psychiatric disorders like depression and anxiety disorders in comparison to the general population [ 6668 ] . when comparing the comorbidity of the gi disorders ibd and irritable bowel syndrome ( ibs ) , significantly more subjects were diagnosed with depression ( ibs : 61% ; ibd : 16% ) , generalized anxiety disorder ( ibs : 54% ; ibd : 11% ) , panic disorder ( ibs : 61% ; ibd : 11% ) , and agoraphobia ( ibs : 25% ; ibd : 25% ) , with a higher prevalence for a lifetime diagnosis of the aforementioned comorbidities in comparison to the general population [ 68 , 69 ] . intriguingly , along with the core features of asd , comorbidities occur frequently in asd patients such as seizures , depression , and anxiety disorders that have been associated with zinc deficiency before . thus , the susceptibility to infections is increasing with a decreasing zinc status as reported from animal models and human studies . the vulnerability to infections is associated with an impaired t and b lymphocyte development and differentiation and their reduced activity [ 7072 ] whereby t lymphocytes seemed to be more seriously affected by zinc deficiency . studies on prenatal zinc deficient animals have shown that zinc deficiency , even a marginal one , results in smaller lymphoid organs and less immunoglobulins . beneficial effects of zinc supplementation in diseases include reduced incidence and duration of acute and persistent diarrhea [ 7476 ] , reduced incidence of acute lower respiratory infections , and reduced duration of the common cold . a role of abnormal immune system function in asd has long been hypothesized . in postmortem brains of individuals with asd , similar to some animal models , activation of astroglia and microglia furthermore , a relationship between familial autoimmune disorders and anti - inflammatory / immune - modulating drug in asd has been reported . usually the organism fight against pathogens is initiated by the activation of the complement system as well as natural killer cells and polymorphonuclear leukocytes . all these defending mechanisms are depressed by zinc deficiency [ 8487 ] resulting in a prolonged inflammation . the disruption of these processes is also associated with diarrhea or inflammatory bowel disease , both also consequences of zinc deficiency . inflammation in the gi tract can lead to intestinal permeability , commonly called leaky gut . here , increased spaces present between cells in the small intestine may result in incompletely broken down foods and other toxins entering the blood stream , which may lead to an immune system response triggering the release of antibodies . this process may result in chronic inflammation that might influence microbe proliferation in the gi tract and cause vitamin and mineral deficiencies , as well as food allergies and autoimmune diseases such as celiac disease . furthermore , a direct effect on the brain causing behavioral , cognitive , and psychiatric impairments may occur [ 89 , 90 ] . additionally , gut inflammation and zinc deficiency are also linked to physiological and psychological stress . animal studies have shown that psychological stress decreased serum zinc levels . reduced zinc levels and psychological stress both increase the release of glucocorticoids [ 92 , 93 ] . increase levels of glucocorticoids in turn have been associated with thymic atrophy and reduced b lymphocyte numbers [ 92 , 94 , 95 ] . furthermore , persistent high levels of glucocorticoids might lead to resistance of glucocorticoid receptors and in turn lead to failure of immune system downregulation . this downregulation is necessary to avoid chronic inflammatory processes . taken together , gi abnormalities , immune system dysfunction , stress , and zinc deficiency are highly linked processes ( figure 2 ) . the final result may be an altered signaling to and within the developing brain , possibly contributing to the development of asd . due to the multifaceted effect of zinc on gut development and morphology , pre- and perinatal zinc deficiency might affect gut development of the neonate and potentially mitigate many of the dysfunctions shared between asd and other neurological disorders . based on this hypothesis , a model emerges ( figure 3 ) that might serve as starting point for future studies . zinc is taken up from our dietary sources and/or supplements in the proximal small intestine , either the distal duodenum or proximal jejunum [ 96 , 97 ] . within enterocytes , intracellular transporters and zinc buffering proteins such as metallothioneins ( mts ) influence the transport and release of zinc in the blood stream . however , various agents can decrease zinc absorption . for example , it is not uncommon for women of childbearing age to consume calcium supplements for the prevention of osteoporosis or drink water naturally high in calcium . similar to copper which has an antagonistic relationship with zinc [ 99102 ] , calcium might interfere with the absorption of zinc , though this effect is not as well established [ 103 , 104 ] . research has shown decreased zinc absorption when different forms of calcium have been ingested following consumption of a zinc dose , suggesting an antagonist relationship between the minerals . ingestion of high concentrations of iron might also affect zinc uptake [ 106108 ] . additionally , folic acid is a nutrient commonly prescribed during pregnancy and supplied at higher levels in prenatal supplements . folic acid has been shown to increase fecal zinc losses , indicating decreased zinc absorption [ 109111 ] . other dietary constituents that influence zinc availability include phytate and high fructose corn syrup ( hfcs ) . inositol hexaphosphates and pentaphosphates , the phytate forms that bind to zinc and reduce its availability , are present in staple foods such as wheat , corn , and rice [ 112 , 113 ] . hfcs is commonly used in the us to sweeten food and drinks with estimated yearly per capita consumption in the us being 12.3 kg in 2012 [ 114 , 115 ] . consumption of alcohol leads to a reduced placental zinc transport and it was hypothesized that the consequences of fetal alcohol syndrome may unfold not only through the effects of ethanol but also through zinc deficiency . for example , ace inhibitors used to treat high blood pressure may decrease blood zinc levels similar to thiazide diuretics , the anticonvulsant valproic acid ( vpa ) that was already reported to increase the risk for autism upon prenatal exposure , tetracycline antibiotics , corticosteroids , acid blockers such as histamine-2 receptor antagonists ( h2-blockers ) , and many neuropsychiatric drugs such as fluoxetine ( prozac ) , paroxetine ( paxil ) , sertraline ( zoloft ) , citalopram ( celexa ) , and venlafaxine ( effexor ) . once absorbed , zinc passes into portal blood and is transported bound to proteins . placental transport of zinc is a fast process and influenced by the number and size of fetuses present . however , due to low zinc diets or compromised absorption due to increased intake of dietary constituents that reduce the availability of zinc , a zinc deficiency of the embryo may occur . zinc deficiency might influence embryonic and fetal development through several mechanisms including abnormal nucleic acid metabolism , reduced protein metabolism , reduced rates of tubulin polymerization , high rates of cellular oxidative damage , higher rates of apoptosis , impaired cell migration , and reduced binding of transcription factors and hormones that , among others , affect lymphocytes . at least 50 intestinal epithelial differentiation genes have been implicated in development and differentiation of the intestinal epithelium , and many of them have a direct relationship with zinc . among them , adenomatous polyposis coli ( apc ) is a crucial determinant of cell fate in the murine intestinal epithelium . loss of apc perturbs differentiation along the enterocyte , goblet , and enteroendocrine lineages and promotes commitment to the paneth cell lineage through -catenin / tcf4-mediated transcriptional control of specific markers of paneth cells , the cryptdin / defensin genes . conditional deletion promotes paneth cell differentiation at the expense of enterocyte , goblet , and enteroendocrine cell differentiation . furthermore , pr domain zinc finger protein 1 also known as blimp-1 has an effect on postnatal epithelial maturation , mediating the transition of neonatal intestinal epithelium to adult intestinal epithelium . caudal - related homeobox ( cdx ) regulates intestinal development , differentiation , and maintenance . both variants , cdx1 and cdx2 , contain a zinc finger motif at their n - terminus . gata is another family of zinc finger transcription factors thought to regulate genes involved in embryogenesis . gata4 , -5 , and -6 are expressed in various mesoderm and endoderm derived tissues such as heart , liver , lung , gonad , and gut where they play critical roles in regulating tissue - specific gene expression . gata4 , -5 , and -6 have been implicated in the regulation of epithelial cell differentiation [ 123 , 124 ] . indian hedgehog ( ihh ) is expressed by mature colonocytes and regulates their differentiation in vitro and in vivo . ihh binds zinc ions stabilizing the protein and mediating protein - protein interactions [ 125 , 126 ] . similarly , kruppel - like factor 4 ( klf4 , formerly gklf ) is a zinc finger transcription factor expressed in the epithelia of the gi tract and several other organs . in vitro and in vivo studies have suggested that klf4 plays an important role in cell proliferation and/or colonic epithelial cell differentiation . matrix metalloproteinases ( mmps ) are a family of zinc binding extracellular matrix degrading enzymes . mmp-9 is a zinc dependent endopeptidase , synthesized and secreted in monomeric form as zymogen and contributes to gut microbe homeostasis [ 128 , 129 ] . furthermore , myc - associated zinc finger ( maz ) protein has been implicated as a critical target of the canonical wnt pathway , which is essential for formation and maintenance of the intestinal mucosa [ 130 , 131 ] . the notch signaling pathway promotes proliferative signaling during neurogenesis and is activated in the progenitor domain of the gastrointestinal epithelium influencing binary fate decisions of cells that must choose between the secretory and absorptive lineages in the gut . it is thus likely that insufficient zinc supply will affect development of the fetal gi tract contributing to many of the reported gi problems associated with asd such as metallothionein dysfunction , plasma cu / zn inversion , heavy metal overload [ 4 , 83 , 134136 ] , candida and clostridium overgrowth , constipation and/or diarrhea [ 51 , 53 ] , leaky gut , food sensitivities and allergies , inefficient processing of gluten and casein [ 54 , 55 , 65 , 137139 ] , enzyme deficiency [ 51 , 140142 ] , vitamin and mineral malabsorption [ 51 , 143145 ] , inefficient fat digestion and metabolism , and esophagitis and gi ulcers . these gi symptoms can give rise to behavioral difficulties , ranging from inattentive or irritable behaviors to self - injury . several human disorders with gi problems like including inflammatory bowel disease ( including crohn 's disease ) , irritable bowel syndrome , and obesity have a modulatory influence on social , emotional , and anxiety - like behaviors . changes in behavior thereby might be based on both acute alterations in brain function as well as alterations during brain development [ 147149 ] . for example , vagal afferent signaling has been implicated modulating mood and affect , including distinct forms of anxiety and fear . moreover , although the gi symptoms might be transient , long lasting behavioral changes have been reported . in rats , neonatal gastric irritation leads to increase in depression- and anxiety - like behaviors , increased expression of crf in the hypothalamus , and an increased sensitivity of hpa axis to stress in adults . thus , it is possible that shared comorbidities such as increased anxiety in adhd , mood disorders , and asd correlate with abnormal gi development caused by zinc deficiency or other factors . the presented model does not exclude the possibility that the gi symptoms are the consequence of altered brain to gut signaling or the consequence of altered gut regulation by the enteric nervous system , which might occur in parallel . synaptic genes affecting excitatory and inhibitory neurotransmission might lead to alterations in neural or endocrine elements of the enteric nervous system . however , given that a central pathway at synapses related to asd , neurexin - neuroligin - shank signaling has also been shown to depend in part on the availability of zinc , a link between zinc deficiency and brain to gut signaling can not be excluded . supplementing women of childbearing age with an effective source of zinc might help mitigate the negative effects of dietary constituents and nutrients in prenatal supplements on zinc availability , helping women attain and maintain adequate zinc status . zinc amino acid complexes might be advantageous to zinc oxide and zinc sulfate based on better absorption . a combination of the inorganic and amino acid complexed zinc might also be advantageous due to different absorption pathways . additionally , research has shown that zinc antagonists such as phytate and fiber reduced the bioavailability of zinc from zinc sulfate more than that from a zinc amino acid complex . provided the amino acid remains complexed to the zinc , interaction of the mineral with dietary components such as phytate and fiber preabsorption can be minimized and zinc can be absorbed into the enterocyte via amino acid transporters versus metal transporters , reducing competition for absorption between zinc in the zinc amino acid complex and other dietary metals ( figure 4 ) . although measures to prevent maternal zinc deficiency would be most desired , further treatment strategies emerge from this concept for young children with asd . for example , the use of probiotics in asd has been suggested [ 60 , 154 , 155 ] . however , probiotics have been used with variable efficacy and data on the effectiveness of probiotics is currently just emerging with more studies and meta - analyses needed in future . intriguingly , treatment of the offspring of maternal immune activation ( mia ) mice that are known to display features of asd with the human commensal bacteroides fragilis corrected gut permeability , altered microbial composition , and ameliorated defects in communication , stereotypic- as well as anxiety - like and sensorimotor behaviors [ 65 , 156 ] ( figure 4 ) . additionally , a gluten and milk protein - free diet ( exclusion of the protein compound gluten found in wheat products and casein contained in dairy ) was reported to potentially be beneficial to improve some behaviors in individuals with asd and reduce intestinal permeability [ 157 , 158 ] . elimination of cow 's milk protein from the diet of asd children via restrictive diet improved autistic behavior , while the oral challenge with milk protein seemed to have an opposite effect . when evaluating iga , igg , and igm specific antibodies , autistic children had significantly higher serum levels of iga antibodies , high levels of igm antibodies specific for lactalbumin , and igg and igm levels for casein [ 159 , 160 ] . however in a small sample size study with 15 autistic children investigating the effects of a gluten - casein free diet , no significant differences between individuals on the restrictive diet and nontreated controls could be found although some of the parents claimed to have noticed an improvement regarding the child 's language , the occurrence of tantrums , and the level of hyperactivity . thus , although some studies show inflammation of the gut or a leaky gut in asd and some studies report that gluten and casein showed beneficial effects , given that other scientific publications did not indicate significant improvements , more research is needed to make a recommendation . in general , the gut microbiome might have great impact on brain development early in life . in an altered microbiome , bacterial metabolites such as 4-ethylphenylsulphate ( 4eps ) or the neurotransmitter -aminobutyric acid ( gaba ) produced from intestinal bacteria might affect brain development and , ultimately , behavior later in life . serotonin ( 5-ht ) signaling is not only important in the brain , but also in the gi tract . the 5-ht(1a ) receptor plays an important role in the developing brain but is additionally expressed in the gut . 5-ht is released from gut enterochromaffin cells and might contribute to 5-ht signaling in the brain . however , the gut and the brain are not the only sites of action for 5-ht . its receptors are also present in the immune system where 5-ht signaling may mediate both innate and adaptive responses . it remains to be established whether 5-ht3 antagonists ( e.g. , ramosetron ) or 5-ht4 agonists can have a modulatory effect in asd . moreover , the intestinal tract has a very important immune function . besides markers for inflammation , enhanced levels of cytokines and chemokines have been detected in the brain and in the cerebrospinal fluid of children with autism [ 166 , 167 ] . therefore , therapeutics used to treat inflammatory events caused by abnormal gi function , such as in inflammatory bowel disease ( anti - inflammatory , immune - modulating , and microbiome - modulating therapies ) , might be a potential source for novel treatment strategies . prenatal stresses , such as depressive illness , anxiety disorders , and posttraumatic stress disorders , are a risk factor for asd [ 83 , 169 ] thus , stress is also connected to abnormalities in the gi tract , zinc signaling , and the immune system [ 170 , 171 ] . many studies support the influence of the corticotropin - releasing factor ( crf ) system in stress response . the use of crf receptor antagonists suggested a significant effect against stress - related behavior , but also hyperalgesia , colonic secretion , and motility [ 172 , 173 ] . thus , medications acting on crf1 and crf2 receptors that are involved in neuroendocrine , autonomic , behavioral , and visceral responses to stress , such as nbi 27914 and astressin-2b , respectively , might provide new treatment approaches . finally , zinc supplementation might also be useful in young children with asd . given that younger individuals with asd have an especially high risk of zinc deficiency , zinc supplementation will help to overcome some impairments associated with acute zinc deficiency . for example , diarrhea has been linked to zinc deficiency [ 174 , 175 ] and zinc supplementation was reported to significantly reduce the symptom as well as increase immune function [ 1 , 177 ] and ameliorate neurosensory deficits associated with zinc deficiency ( figure 4 ) . given that asd is a heterogeneous group of disorders and zinc deficiency or increased intestinal permeability only present in a subset of patients , unless clinical trials use patient populations that are enriched based on this particular clinical history , clinical benefits of any possible treatment will be hard to demonstrate . additionally , asd is a neurodevelopmental disorder . thus , the pathomechanisms already act in utero , leading to alternative modeling of the brain . if therapies are to prevent or correct such changes , they may have to be implemented in the perinatal period and may be ineffective in an individual with asd later in life . taken together , we conclude that due to multifaceted effect of zinc on gut development and morphology improving zinc status of the pregnant mother as well as the offspring has the potential to improve gut development of the neonate and potentially mitigate dysfunctions associated with asd .

a growing amount of research indicates that abnormalities in the gastrointestinal ( gi ) system during development might be a common factor in multiple neurological disorders and might be responsible for some of the shared comorbidities seen among these diseases . for example , many patients with autism spectrum disorder ( asd ) have symptoms associated with gi disorders . maternal zinc status may be an important factor given the multifaceted effect of zinc on gut development and morphology in the offspring . zinc status influences and is influenced by multiple factors and an interdependence of prenatal and early life stress , immune system abnormalities , impaired gi functions , and zinc deficiency can be hypothesized . in line with this , systemic inflammatory events and prenatal stress have been reported to increase the risk for asd . thus , here , we will review the current literature on the role of zinc in gut formation , a possible link between gut and brain development in asd and other neurological disorders with shared comorbidities , and tie in possible effects on the immune system . based on these data , we present a novel model outlining how alterations in the maternal zinc status might pathologically impact the offspring leading to impairments in brain functions later in life .

1. Introduction 2. Zinc and Gut Formation 3. Gut-Brain Interaction in ASD and Other Neurological Diseases 4. Zinc, the GI Tract, Stress, and the Immune System 5. Conclusions

the deficiency of vitamin d3 is commonly associated with chronic kidney disease ( ckd ) , and the prevalence of this hypovitaminosis increases as kidney function declines [ 1 , 2 ] . several factors , such as aging , loss of appetite , and other factors affecting cutaneous synthesis , such as low sun exposure and skin pigmentations , have consistently been associated with low 25-hydroxyvitamin d [ 25-(oh ) d3 ] levels in the general population . therefore , it is common in the elderly , malnourished individuals , and some societies . even though there is growing evidence to suggest that vitamin d3 status is associated with the development and progression of cardiovascular disease [ 5 , 6 ] , diabetes , and immune system disorders , there is limited information about the association of 25-(oh ) d3 deficiency and inflammation in the general population without ckd and in patients with ckd . studies examining the association between low 25-(oh ) d3 levels and inflammation infection are still popular . there are studies suggesting a relationship between a lack of vitamin d3 and morbidity and also mortality as well [ 912 ] . randomized controlled trials of vitamin d3 supplementation have shown incompatible results , with some trials suggesting a decrease [ 13 , 14 ] and other studies concluding no effect on inflammatory biomarkers . the potential relationship between the deficiency of vitamin d3 and infection - inflammation remains poorly understood . therefore , the aim of present study is to examine the association between the level of plasma 25-(oh ) d3 and inflammatory markers in the general population without chronic kidney disease and in patients with ckd . present study was conducted between january 1 , 2008 and april 25 , 2012 in bulent ecevit university hospital and 1897 patients with 25-(oh ) d3 levels and inflammatory markers measured simultaneously were included . patients whose age under 18 years , patients with primary hyperparathyroidism and hypoparathyroidism , were excluded . the relationship between 25-(oh ) d3 levels and serum creatinine , parathormone ( pth ) , sensitive c - reactive protein ( crp ) , erythrocyte sedimentation rate ( esr ) , leucocyte count , platelet count , and hemoglobin concentrations were evaluated as retrospectively in this study population . serum 25-(oh ) d3 levels vary depending on season ; we categorized patients into two groups according to serum 25-(oh ) d3 levels . g / l ) population , and group 2 was composed of vitamin d3 normal group ( > 21 g / l and 20 g / l were excluded from the analysis in order to avoid the effects of seasonal changes . patients with known levels of crp were grouped categorically as normal ( crp < 6 mg / l ; there was no inflammation or infection ) and as abnormal ( crp > 30 mg / l ; there was important inflammatory or infectious status ) . likewise , to examine the relationship between renal failure and 25-(oh ) d3 levels , participants were divided into categorical groups : the patients with and without renal failure . study cases with serum creatinine levels above 1.3 mg / dl for more than 3 months were considered as patients with ckd . moreover , the participants in this study were also divided into two groups : ambulatory patients and hospitalized patients . finally , vitamin b12 levels were measured in vitamin d3 deficient and vitamin d3 normal group , and then these two groups were divided into subgroups of their own . primary endpoints are as follows : determining the 25-(oh ) d3 level in the general population and in patients with ckd;comparing the clinical and laboratory data regarding inflammation with levels of 25-(oh ) d3;evaluation of whether low and normal 25-(oh ) d3 levels and inflammation could explain this potential association ; determining the 25-(oh ) d3 level in the general population and in patients with ckd ; comparing the clinical and laboratory data regarding inflammation with levels of 25-(oh ) d3 ; evaluation of whether low and normal 25-(oh ) d3 levels and inflammation could explain this potential association ; 25-hydroxyvitamin d3 levels were measured by high performance liquid chromatographic analysis performed with using a zivak hplc system ( gebze , turkey ) using a commercial 25-oh vitamin d3 kit ( recipe , munich , germany ) . l for summer seasons . a deficiency in 25-(oh ) d3 level was considered as below 10 serum vitamin b12 and plasma pth levels were measured with chemiluminescence method by immulite 2000 ( diagnostic products corp . , c - reactive protein was assayed with dade behring bn prospec system using a nephelometric method . leukocyte count , platelet count , and hemoglobin concentrations were measured by beckman coulter lh 780 hematology analyzer . continuous variables were expressed as median ( minimum - maximum ) and categorical variables as frequency and percent . continuous variables were compared with the mann - whitney u test and categorical variables were compared using pearson 's chi - square test . patients that measured 25-(oh ) d3 levels under 10 g / l were 598 ( 31.5% ) , the number of those between 10 and 21 g / l was 751 ( 39.5% ) , and the number of those over 21 g / l was 550 ( 28.9% ) , respectively , in the study group ( figure 1 ) . the difference between male and female in 25-(oh ) d3 levels was statistically significant ( p < 0.001 ) , and 25-(oh ) d3 levels were significantly lower in female [ 16.1 12.8 ( 3121 ) ] than in male [ 19.2 12.9 ( 3201 ) ] . for this reason , male and female patients were divided into groups according to the presence of renal failure . there was no significant correlation between age and vitamin d3 deficiency in our study population . there were lower serum albumin levels in patients with vitamin d3 deficiency , but this was not statistically significant ( table 3 ) . median serum creatinine levels were less in patients with vitamin d3 deficiency without renal failure than in participants with normal vitamin d3 levels without renal failure ( table 4 ) . serum albumin , crp , esr , and wbc levels had no significant relationship in groups that vitamin d3 deficiency and vitamin d3 normal in male and female patients without renal failure ( tables 14 ) . there was no difference in the levels of albumin , crp , esr , and wbc in women with renal insufficiency , but there was significant difference between levels of serum albumin and esr in male patients . the inflammatory status measured by crp showed no difference with respect to the 25-(oh ) d3 ( p = 0.318 ) . in crp variable that was categorized as < 6 mg / l and 6 mg / l , there was no significant difference between crp categories and 25-(oh ) d3 levels ( p = 0.728 ) . the results are summarized in table 5 . in crp variable that was categorized as < 6 mg / l and 30 mg / l , there was no significant difference between these crp categories and 25-(oh ) d3 levels ( p = 0.635 ) ( table 6 ) . then , all participants were included in the study , both 25-(oh ) d3 and crp variables taken as a numerical , and correlation analysis was performed . there was no correlation between the two groups ( r = 0.03 , p = 0.335 ) . the difference between men and women crp levels was statistically significant ( p = 0.013 ) . crp and vitamin d3 levels in men and women were different ; therefore , similar analyses were repeated in men and women groups . median crp in vitamin d3 deficient group and vitamin d3 normal group showed no significant difference in the male and female patients . there was a weak positive correlation between age and crp ( r = 0.21 , p < 0.001 ) . when 25-(oh ) d3 and crp variables are taken as categorical variables and analyzing the relationships between variables , no significant correlation was found ( p = 1.000 ) . the prevalence of vitamin d3 deficiency in patients with renal failure had a higher number ( p = 0.000 ) ( table 6 ) . this frequency were not statistically significant in male patients except advanced - stage renal failure ( creatinine < 3.8 mg / dl ) , ( p = 0.148 ) . the frequency of vitamin d deficiency was evaluated between outpatient and hospitalized patients groups in this study population . the incidence of vitamin d3 deficiency in all - cause hospitalized patients was more frequent ( p = 0.000 ) . the prevalence of vitamin d3 deficiency in outpatients was 46.3% ( n = 236 ) and in hospitalized patients was 67.5% ( n = 77 ) . ( n = 274 ) and for hospitalized patients was 32.4% ( n = 37 ) , respectively ( table 6 ) . moreover age , esr , wbc , and crp medians had higher levels in hospitalized patients . the prevalence of low vitamin b12 ( < 160 pg / ml ) was 68% ( n = 30 ) , and the prevalence of normal vitamin b12 level ( > 160 pg / ml ) was 32% ( n = 14 ) in the group with vitamin d3 deficiency , whereas the prevalence of low vitamin b12 was 51.4% ( n = 108 ) , and the prevalence of normal vitamin b12 ( number 30 ) was 48.6% ( n = 102 ) in the normal vitamin d3 group . vitamin b12 deficiency was more frequently seen in patients with vitamin d3 deficiency ( p = 0.043 ) ( table 6 ) . present study did not reflect the true incidence of vitamin d3 deficiency because patients who are thought to lack of vitamin d3 were included in this study . a limited number of studies conducted in turkey have shown that vitamin d3 deficiency is a common issue during the fall and winter in individuals , particularly for elderly . vitamin d3 deficiency rates are 8084% in the middle east , 6065% in asia , 5055% in europe , and 50% in latin america [ 1618 ] . female constitutes the majority of patients may be due to less exposure to the sun and the higher prevalence of osteoporosis . there was no significant correlation between age and vitamin d deficiency and that may be due to individual characteristics of the studied population . but many studies did not mentioned the relationship between age and vitamin d levels . in present study we did not find a relationship between vitamin d3 deficiency and inflammatory markers , such as crp , esr , and leukocyte counts . some other studies measured crp was found the relationship but in these studies the relatively small number of participants were the limiting factor [ 20 , 21 ] . in several studies were unknown accompanying diseases , and hospitalization rates [ 9 , 22 ] . there were no studies evaluating esr , and leukocyte counts were evaluated in 25-(oh ) d3 deficiency . sensitive crp that was not measured is the limitation of the study . to resolve this drawback was categorized patients according to the levels of crp firstly , we counted the number of patients with and without vitamin d3 deficiency in crp normal group . secondly , we separated the study population into crp normal and significantly high crp groups . the reason for this classification was to evaluate the frequency of vitamin d3 deficiency in out - patients with important high level of crp . finally , we applied correlation analysis between the level of crp and 25-(oh ) d3 . but a relationship between the level of crp and 25-(oh ) d3 was not found in all our analyses . in other words , we did not observe an association between vitamin d deficiency and crp levels anyway . patients ' age , serum albumin , crp , and esr levels , leukocyte counts , and creatinine values were significantly different between ambulatory and hospitalized patients . the medians of inflammatory markers of hospitalized patients were higher compared to those of ambulatory patients except albumin levels . in addition , the frequency of 25-(oh ) d3 deficiency was higher once again in hospitalized patients . these also mean that 25-(oh ) d3 deficiency aggravates all - cause diseases , which is associated with the course of inflammation and infection but not crp levels . the prevalence of vitamin d3 deficiency in patients with ckd was more common at all stages in female patients ; however , it was more common at advanced stage in male patients . this could be explained by a combination of factors , such as poor nutrition or a lack of skin synthesis due to low sun exposure . in ckd patients , dietary restriction and loss of appetite due to uremia or high levels of fibroblast growth factor 23 may be stronger determining factors for 25-(oh ) d3 deficiency . in groups without renal failure , higher pth values were known and expected to be higher among the vitamin d3 deficient patients . however , the low level of creatinine was not been described previously , and this difference was statistically significant . in our study population , the levels of albumin were lower in vitamin d3 deficient patients than in vitamin d3 normal participations . it has been reported decreased level of albumin in a large scaled study of melamed et al . . in groups without renal failure , low creatinine and albumin levels might be associated with a nutritional disorder or other comorbid inflammatory - infectious status . it is known that deficiency of vitamin d3 and malnutrition were related to each other . multivitamin deficiency was common in malnourished and elderly patients , but there was no study that tested vitamin b12 levels in vitamin d3 deficient patient in the literature . for more accurate assessment it is necessary to know other factors that trigger inflammation and infection in studies examining the relationship between vitamin d and inflammatory markers . for example , when hospitalized patients are included in our analysis ; all inflammatory markers gained significance statistically . in addition , the reason for the deficiency of 25-(oh ) d3 should be known in similar studies . however , there may be no relationship in encountered 25-(oh ) d3 deficiency due to low sunlight exposure , and it could be expected in patients with 25-(oh ) d3 deficiency due to malnutrition . according to the results of our study , high levels of crp in vitamin d deficient patients might be related to other factors such as infectious , inflammatory status , malnutrition , cachexia , or multivitamin deficiency . these factors and others may affect high morbidity and mortality in patients with vitamin d3 deficiency . therefore , replacement of vitamin d alone could be corrected only in patients with vitamin d deficient patients in the foreground .

although some studies revealed a positive relationship between vitamin d3 deficiency and inflammatory markers , there have been also many studies that failed to find this relationship . the aim of this large scaled study is to determine the association between the level of plasma 25 hydroxy vitamin d3 [ 25-(oh ) d3 ] and inflammatory markers in the general population without chronic kidney disease ( ckd ) and in patients with ckd . participants with simultaneously measured inflammatory markers and 25-(oh ) d3 levels were retrospectively analyzed ( n = 1897 ) . the incidence of all - cause inflammation infection , hospitalization , chronic renal failure , and vitamin b12 deficiency was evaluated . the medians of serum creatinine levels in subjects without renal failure were lower in 25-(oh ) d3 deficient group . patients with ckd were more likely to have vitamin d3 deficiency compared with normal gfr . 25-(oh ) d3 levels were associated with a greater incidence of all - cause hospitalization , hypoalbuminemia , and vitamin b12 deficiency . however , there was no relationship between inflammatory markers and vitamin d3 levels . in 25-(oh ) d3 deficient patients , inflammatory markers can be related to other inflammatory and infectious status such as malnutrition and cachexia . we believed that there must be a relationship between vitamin deficiency and inflammatory markers due to other causes than low 25-(oh ) d3 status .

1. Introduction 2. Methods 3. Results 4. Discussion

the online version of this article ( doi:10.1007/s00415 - 012 - 6498 - 2 ) contains supplementary material , which is available to authorized users . patients with an acute basilar artery occlusion ( bao ) have a high risk of long - lasting disability and death [ 1 , 2 ] . although higher age , analyzed as a continuous variable , has been associated with poor functional outcome after bao , only limited data are available on functional outcome in elderly patients [ 38 ] . one small case series suggested that all patients 75 years have poor functional outcome . in another study , we analysed data from the basilar artery international cooperation study ( basics ) to determine outcomes in patients with bao 75 years . basics is a prospective , observational , registry of 619 consecutive patients 18 years with an acute symptomatic bao [ 2 , 9 ] . the protocol was approved by the ethics committee of the university medical center utrecht , the netherlands . embolic bao was defined as complete recanalization on follow - up and no indication of dissection , or maximum deficit from onset and cardiac or vertebral source of embolism , or maximum deficit from onset with complete absence of other atherosclerotic cerebrovascular lesions . atherosclerotic bao was defined as known symptomatic basilar artery stenosis ( > 50 % ) prior to occlusion , or residual stenosis after recanalization and no evidence of cardiac or vertebral artery source of embolism , or prior tias or stroke in the basilar artery territory and no evidence of cardiac or vertebral artery source of embolism . primary outcome was poor functional outcome after 1 month [ predefined as modified rankin scale ( mrs ) score 46 ] . secondary outcomes were death , insufficient vessel recanalization [ defined as thrombolysis in myocardial infarction ( timi ) score 01 ] and symptomatic intracranial hemorrhage ( sich ) . we also investigated if our conclusions changed if poor functional outcome was defined as an mrs of 36 . sich was not predefined by the registry , and the reporting of sich was done on the basis of each investigator s judgment . for the purpose of this study , patients were divided into four age - groups , based on quartiles : 1854 , 5564 , 6574 , and 75 years . . risk ratios ( rr ) and corresponding 95 % confidence intervals ( ci ) were calculated . variables that affected the crude risk ratio most were used simultaneously in poisson regression analyses to calculate adjusted risk ratios ( arr ) . missing baseline data ( finally , we explored the incidence of poor functional outcome in patients 7579 , 8084 , 8589 , and 90 years or older . baseline characteristics are listed in table 1 . in total , 162 patients ( 26 % of total cohort ) were 75 years . in this group of patients , the most common cause of stroke was embolism and 64 % had an nihss score > 20 . treatment of any kind was initiated in 148 patients ( 91 % ) .table 1baseline characteristics according to age groupvariableall patientsn = 6191854 yearsn = 1535564 yearsn = 1336574 yearsn = 17175 yearsn = 162male sex390 ( 63 % ) 94 ( 61 % ) 105 ( 79 % ) 116 ( 68 % ) 75 ( 46 % ) hypertension383 ( 62 % ) 41 ( 27 % ) 87 ( 65 % ) 133 ( 78 % ) 122 ( 75 % ) diabetes mellitus135 ( 22 % ) 13 ( 8 % ) 31 ( 23 % ) 49 ( 29 % ) 42 ( 26 % ) hyperlipidemia167 ( 27 % ) 30 ( 20 % ) 45 ( 34 % ) 55 ( 32 % ) 37 ( 23 % ) atrial fibrillation133 ( 21 % ) 3 ( 2 % ) 20 ( 15 % ) 42 ( 25 % ) 68 ( 42 % ) coronary artery disease109 ( 18 % ) 12 ( 8 % ) 21 ( 16 % ) 35 ( 20 % ) 41 ( 25 % ) location of basilar artery occlusion distal third202 ( 33 % ) 42 ( 27 % ) 30 ( 23 % ) 55 ( 32 % ) 75 ( 46 % ) middle third143 ( 23 % ) 37 ( 24 % ) 29 ( 22 % ) 46 ( 27 % ) 31 ( 19 % ) proximal third274 ( 44 % ) 74 ( 48 % ) 74 ( 56 % ) 70 ( 41 % ) 56 ( 35 % ) median nihss score ( iqr)22 ( 1230)20 ( 930)20 ( 1230)22 ( 1328)25 ( 1630)nihss score > 20336 ( 54 % ) 73 ( 48 % ) 66 ( 50 % ) 93 ( 54 % ) 104 ( 64 % ) type of treatment antithrombotics183 ( 30 % ) 47 ( 31 % ) 33 ( 25 % ) 52 ( 30 % ) 51 ( 31 % ) iv tpa or iv tpa / iat121 ( 20 % ) 25 ( 16 % ) 37 ( 28 % ) 24 ( 14 % ) 35 ( 22 % ) iat only288 ( 47 % ) 78 ( 51 % ) 59 ( 44 % ) 89 ( 52 % ) 62 ( 38 % ) no treatment27 ( 4 % ) 3 ( 2 % ) 4 ( 3 % ) 6 ( 4 % ) 14 ( 9 % ) time to treatment 03 h179 ( 29 % ) 41 ( 27 % ) 39 ( 29 % ) 49 ( 29 % ) 50 ( 31 % ) 46 h190 ( 31 % ) 45 ( 29 % ) 37 ( 28 % ) 58 ( 34 % ) 50 ( 31 % ) 79 h84 ( 14 % ) 22 ( 14 % ) 19 ( 14 % ) 26 ( 15 % ) 17 ( 10 % ) 1012 h57 ( 9 % ) 14 ( 9 % ) 14 ( 11 % ) 16 ( 9 % ) 13 ( 8 % ) 1324 h55 ( 9 % ) 17 ( 11 % ) 15 ( 11 % ) 9 ( 5 % ) 14 ( 9 % ) > 24 h27 ( 4 % ) 11 ( 7 % ) 5 ( 4 % ) 7 ( 4 % ) 4 ( 2 % ) cause of stroke embolism224 ( 36 % ) 42 ( 27 % ) 38 ( 29 % ) 59 ( 35 % ) 85 ( 52 % ) atherosclerosis215 ( 35 % ) 39 ( 25 % ) 57 ( 43 % ) 72 ( 42 % ) 47 ( 29 % ) dissection32 ( 5 % ) 24 ( 16 % ) 5 ( 4 % ) 3 ( 2 % ) 0 ( 0 % ) other6 ( 1 % ) 2 ( 1 % ) 1 ( 1 % ) 2 ( 1 % ) 1 ( 1 % ) unknown142 ( 23 % ) 44 ( 29 % ) 30 ( 23 % ) 31 ( 18 % ) 27 ( 17 % ) nihss national institutes of health stroke scale , iqr interquartile range , iv intravenous , tpa tissue plasminogen activator , iat any intra - arterial treatment ( either intra - arterial tpa , mechanical clot disruption , or both ) baseline characteristics according to age group nihss national institutes of health stroke scale , iqr interquartile range , iv intravenous , tpa tissue plasminogen activator , iat any intra - arterial treatment ( either intra - arterial tpa , mechanical clot disruption , or both ) modified rankin scale scores for all age groups are presented in fig . 1 . patients 75 years had a higher risk of poor functional outcome [ arr 1.33 ( 1.141.55 ) , table 2 ] and death [ arr 2.47 ( 1.753.51 ) , table 3 ] than patients aged 1854 years . nevertheless , 35 patients ( 22 % , 95 % ci 1528 % ) of those 75 years had a good functional outcome . no significant differences between age groups were observed for insufficient recanalization [ patients 75 vs. 1854 years arr 1.69 ( 0.953.03 ) ] and sich [ patients 75 vs. 1854 years rr 1.77 ( 0.774.06 ) ] . since sich occurred in only 42 ( 7 % ) patients , no further analyses were performed for this outcome measure.fig . 1modified rankin scale scores according to age group . mrs modified rankin scale scoretable 2poor functional outcome ( mrs 46 ) according to age - group1854 years5564 years6574 years75 yearstotal153 ( 25 % ) 133 ( 21 % ) 171 ( 28 % ) 162 ( 26 % ) poor outcome91/153 ( 60 % ) 85/133 ( 64 % ) 126/171 ( 74 % ) 127/162 ( 78 % ) unadjusted11.08 ( 0.901.29)1.24 ( 1.061.45)1.32 ( 1.131.54)adjusted male sex11.06 ( 0.881.28)1.23 ( 1.051.45)1.33 ( 1.141.55 ) location of occlusion11.06 ( 0.891.27)1.26 ( 1.081.47)1.37 ( 1.181.60 ) nihss11.06 ( 0.901.25)1.22 ( 1.061.41)1.23 ( 1.061.41 ) time to treatment ( h)11.08 ( 0.901.30)1.26 ( 1.071.49)1.33 ( 1.131.56 ) type of treatment11.07 ( 0.891.29)1.24 ( 1.051.46)1.32 ( 1.121.55 ) 3 factors11.05 ( 0.891.24)1.24 ( 1.071.43)1.28 ( 1.111.48 ) 5 factors11.06 ( 0.901.25)1.28 ( 1.101.49)1.33 ( 1.141.55)reference group . data are number ( % ) or risk ratio ( 95 % ci)adjustment for national institutes of health stroke scale ( nihss ) score , sex , and location of occlusionadjustment for nihss score , sex , location of occlusion , time to treatment , and type of treatmenttable 3mortality according to age group1854 years5564 years6574 years75 yearstotal153 ( 25 % ) 133 ( 21 % ) 171 ( 28 % ) 162 ( 26 % ) mortality33/153 ( 22 % ) 43/133 ( 32 % ) 80/171 ( 47 % ) 84/162 ( 52 % ) unadjusted11.50 ( 1.022.21)2.17 ( 1.543.05)2.40 ( 1.723.37)adjusted male sex11.48 ( 1.0022.19)2.16 ( 1.533.04)2.43 ( 1.733.40 ) location of occlusion11.47 ( 0.9982.16)2.22 ( 1.583.11)2.55 ( 1.833.56 ) nihss11.46 ( 1.0082.12)2.13 ( 1.552.94)2.16 ( 1.572.98 ) time to treatment ( h)11.56 ( 1.042.35)2.28 ( 1.593.27)2.41 ( 1.683.46 ) type of treatment11.55 ( 1.032.34)2.24 ( 1.563.21)2.42 ( 1.683.47 ) 3 factors11.47 ( 1.022.12)2.22 ( 1.613.05)2.31 ( 1.683.17 ) 5 factors11.56 ( 1.052.31)2.41 ( 1.713.39)2.47 ( 1.753.51)reference group . data are number ( % ) or risk ratio ( 95 % ci)adjustment for national institutes of health stroke scale ( nihss ) score , sex and location of occlusionadjustment for nihss score , sex , location of occlusion , time to treatment , and type of treatment modified rankin scale scores according to age group . mrs modified rankin scale score poor functional outcome ( mrs 46 ) according to age - group reference group . data are number ( % ) or risk ratio ( 95 % ci ) adjustment for national institutes of health stroke scale ( nihss ) score , sex , and location of occlusion adjustment for nihss score , sex , location of occlusion , time to treatment , and type of treatment mortality according to age group reference group . data are number ( % ) or risk ratio ( 95 % ci ) adjustment for national institutes of health stroke scale ( nihss ) score , sex and location of occlusion adjustment for nihss score , sex , location of occlusion , time to treatment , and type of treatment if poor functional outcome was defined as an mrs of 36 , the proportion of patients with poor functional outcome in each age group was 108/153 ( 71 % ) in patients 1854 years of age , 101/133 ( 76 % ) in patients 5564 years , 142/171 ( 83 % ) in patients 6574 years , and 137/162 ( 85 % ) in patients 75 years . also when this definition was used , patients 75 years had a higher risk of poor functional outcome [ arr 1.21 ( 1.071.36 ) ] than patients aged 1854 years . baseline characteristics of patients 75 years and their relationship with functional outcome are shown in table 4 . the following variables were associated with an increased risk of poor functional outcome after 1 month : male sex ( rr 1.18 , 95 % ci 1.001.38 ) , location of occlusion ( middle third vs. distal third : rr 1.21 , 95 % ci 1.021.44 ) , nihss score > 20 on presentation ( rr 1.36 , 95 % ci 1.101.67 ) , type of treatment ( no treatment vs. intra - arterial thrombolytic therapy ( iat ) only : rr 1.32 , 95 % ci 1.151.52 ) , sich ( rr 1.21 , 95 % ci 1.031.43 ) , and insufficient recanalization ( rr 1.38 , 95 % ci 1.071.77).table 4characteristics of patients 75 years and risk of poor functional outcomevariablerisk of poor functional outcome ( mrs 46)rr ( 95 % ci)characteristic presentcharacteristic absentmale sex64/75 ( 85 % ) 63/87 ( 72 % ) 1.18 ( 1.001.38)hypertension98/122 ( 80 % ) 29/40 ( 73 % ) 1.11 ( 0.901.37)diabetes mellitus29/42 ( 69 % ) 98/120 ( 82 % ) 0.85 ( 0.681.05)hyperlipidemia27/37 ( 73 % ) 100/125 ( 80 % ) 0.91 ( 0.741.13)atrial fibrillation57/68 ( 84 % ) 70/94 ( 74 % ) 1.13 ( 0.96 - 1.32)coronary artery disease35/41 ( 85 % ) 92/121 ( 76 % ) 1.12 ( 0.961.32)location of basilar artery occlusion proximal third43/56 ( 77 % ) 56/75 ( 75 % ) 1.03 ( 0.851.25 ) middle third28/31 ( 90 % ) 56/75 ( 75 % ) 1.21 ( 1.021.44)nihss score > 2090/104 ( 87 % ) 37/58 ( 64 % ) 1.36 ( 1.101.67)type of treatment antithrombotics38/51 ( 75 % ) 47/62 ( 76 % ) 0.98 ( 0.791.22 ) iv tpa or iv tpa / iat28/35 ( 80 % ) 47/62 ( 76 % ) 1.06 ( 0.851.31 ) no treatment14/14 ( 100 % ) 47/62 ( 76 % ) 1.32 ( 1.151.52)time to treatment 46 h35/50 ( 70 % ) 38/50 ( 76 % ) 0.92 ( 0.731.17 ) 79 h13/17 ( 76 % ) 38/50 ( 76 % ) 1.01 ( 0.741.37 ) 10 h27/31 ( 87 % ) 38/50 ( 76 % ) 1.15 ( 0.931.41)cause of stroke embolism65/85 ( 76 % ) 38/47 ( 81 % ) 0.95 ( 0.791.14)symptomatic intracranial hemorrhage14/15 ( 93 % ) 113/147 ( 77 % ) 1.21 ( 1.031.43)insufficient recanalization18/20 ( 90 % ) 32/49 ( 65 % ) 1.38 ( 1.071.77)distal occlusion was taken as reference and is recorded under characteristic absent characteristics of patients 75 years and risk of poor functional outcome distal occlusion was taken as reference and is recorded under characteristic absent the proportion of patients with poor functional outcome in age subgroups 75 years was as follows : 64/82 ( 78 % ) in patients 7579 years of age , 40/49 ( 82 % ) in patients 8084 years , 16/23 ( 70 % ) in patients 8589 years , and 7/8 ( 88 % ) in patients 90 years or older . the oldest patient with good functional outcome was 91 years of age , despite an nihss score of 21 on admission . this patient was treated with intravenous recombinant tissue plaminogen activator ( rtpa ) only , and had an mrs score of 2 at 1 month follow - up . the basics study shows that patients 75 years with bao have an increased risk of poor functional outcome and death compared with younger patients , despite comparable recanalization rates . in contrast with a small previous study , our data show that a substantial group of patients 75 years survives with good functional outcome . previously , it has been suggested that the increased risk of poor functional outcome in elderly patients resulted from a higher prevalence of atherosclerotic occlusions and consequently lower recanalization rates . however , in our study population patients 75 years were more likely to have an embolic rather than an atherosclerotic cause of bao , mainly due to a higher prevalence of atrial fibrillation . patients 75 years with an embolic cause of bao had a similar risk of poor functional outcome compared with patients in this age group with an atherosclerotic cause of bao . sufficient recanalization was achieved in 71 % of patients in this age group . in patients 75 years , several baseline- and treatment - related characteristics were associated with an increased risk of poor functional outcome . a recent large case series of patients with bao , in which only a minority of patients was 75 years , identified similar risk factors for poor functional outcome and death . the strength of this study is that basics was a prospective registry of consecutive patients , and therefore our results are representative for daily practice . a limitation of this study is that this was a post hoc analysis of non - randomized data , and therefore the data regarding treatment - dependent outcomes are prone to bias . due to the prospective collection of detailed data , we were able to perform poisson regression analyses to adjust for important confounding baseline characteristics . we conclude that a substantial group of elderly patients survives with a good functional outcome . this study can not answer the question which treatment option is superior in elderly patients , nor can it define an upper age limit above which treatment is no longer effective . these and other questions may be answered in the recently started basics trial in which patients with bao of up to 85 years of age are randomized between intravenous thrombolysis ( ivt ) alone vs. ivt followed by additional intra - arterial therapy ( http://www.trialregister.nl/trialreg/admin/rctview.asp?tc=2617 ; accessed february 1 , 2012 ) . supplementary material 1 ( doc 23 kb ) supplementary material 1 ( doc 23 kb ) this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

patients with an acute basilar artery occlusion ( bao ) have a high risk of long - lasting disability and death . only limited data are available on functional outcome in elderly patients with bao . using data from the basilar artery international cooperation study , we aimed to determine outcomes in patients 75 years . primary outcome measure was poor functional outcome ( modified rankin scale score 46 ) . secondary outcomes were death , insufficient vessel recanalization ( defined as thrombolysis in myocardial infarction score 01 ) and symptomatic intracranial hemorrhage ( sich ) . patients were divided into four age - groups , based on quartiles : 1854 , 5564 , 6574 , and 75 years . outcomes were compared between patients 75 years and patients aged 1854 years . risk ratios with corresponding 95 % confidence intervals ( ci ) were calculated and poisson regression analyses were performed to calculate adjusted risk ratios ( arr ) . we included 619 patients [ 1854 years n = 153 ( 25 % ) , 5564 years n = 133 ( 21 % ) , 6574 years n = 171 ( 28 % ) , and 75 years n = 162 ( 26 % ) ] . compared with patients aged 1854 years , patients 75 years were at increased risk of poor functional outcome [ arr 1.33 ( 1.141.55 ) ] and death [ arr 2.47 ( 1.753.51 ) ] . nevertheless , 35/162 ( 22 % , 95 % ci 1528 % ) of patients 75 years had good functional outcome . no significant differences between age groups were observed for recanalization rate and incidence of sich . although patients 75 years with bao have an increased risk of poor outcome compared with younger patients , a substantial group of patients 75 years survives with a good functional outcome.electronic supplementary materialthe online version of this article ( doi:10.1007/s00415 - 012 - 6498 - 2 ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Introduction Methods Results Discussion Electronic supplementary material Conflicts of interest Open Access

babesiosis is caused by a haemotropic protozoal parasite of the genus babesia , member of the phylum apicomplexa and transmitted by the bite of an infected tick ( 1 ) . babesiosis can occur without producing symptoms , but also be severe and sometimes fatal due to the intraerythrocytic parasite development ( 1 ) . the disease can cause fever , inappetence , increased respiratory rate , muscle tremors , anemia , jaundice , body weight loss and hemolytic anemia lasting from several days to several months ( 1 , 2 ) . all babesial parasites described to date are transmitted by ixodid ticks to their vertebrate hosts , and replicate in the vertebrate host s red blood cells ( 3 ) . in babesiosis , there is an increase in the erythrocyte membrane permeability and phagocytosis of the erythrocytes by activated macrophages . in conclusion , a possible role of the reactive oxygen and nitrogen species ( ros and rns ) in the pathogenesis of parasitic infections has been an active area of research in recent years ( 5 , 6 ) . in addition , the mechanisms by which cellular defense kills microorganisms have been the subject of intense research . numerous studies have demonstrated that a variety of inflammatory cells are induced or activated by various oxidant - generating enzymes to kill intra - cellular and extra - cellular parasites ( 7 ) . the reactive species are produced primarily to attack invading microorganisms by nitration , oxidation and chlorination reactions . however , excess amounts of ros and rns can cause an injury to host cells and lead to tissue damage ( 7 - 9 ) . in fact , there are some studies determined the effects of babesiosis on oxidative markers and antioxidants ( 10 , 11 ) , but there is no report available on dna damage in goats infected with babesia spp . therefore , in this study , we investigated the role of ros and rns generating by macrophages as a first line of defense during phagocytosis in parasitic infections on dna strand breaks , total antioxidant activity ( aoa ) , reduced glutathione ( gsh ) , protein carbonyl ( pco ) , nitric oxide ( nox ) and malondialdehyde ( mda ) in babesia infected goats . vanadium ( iii ) chloride ( vcl3 ) , sodium chloride ( nacl ) , ethylenediaminetetraacetic acid disodium ( edta - na2 ) , sodium hydroxide ( naoh ) , thiobarbituric acid ( tba ) , trichloroacetic acid ( tca ) , hydrochloric acide ( hcl ) , sulfanilamide ( sulf ) , and n-(1-naphthyl ) ethylenediamine dihydrochloride ( nedd ) were obtained from merck ( darmstadt , germany ) . dinitrophenlyhydrazine ( dnph ) was purchased from fluka ( sigma - aldrich chemical co. , st . ten anatolian black goats naturally infected with b. ovis , at the age of 2 - 3 years were used in the study . ten healthy anatolian black goats aged 2 - 3 years from the same area served as controls . they were not exposed to babesia infection and none had treatment with either mineral or vitamin drugs . the study protocol was performed in accordance with the helsinki declaration as revised in 1989 . on clinical examination , the infected goats showed signs of babesiosis including rise in rectal temperature , dyspnea , tachycardia , anorexia , muscle tremors , signs of anaemia in pale mucous membranes and brownish urine . blood smears of all the selected animals were prepared aseptically on grease free sterile glass slides directly from the ear vein . the smears were air dried , fixed in methanol and stained with freshly prepared 5% giemsa stain for 45 minutes . after being stained with giemsa , the smears were examined for b. ovis under a research microscope and 100x objective . blood samples were taken from each animal by puncture of the jugular vein into heparinized tubes . two milliliters of blood was immediately pipetted into another tube to measure mda , gsh and dna damage . plasma samples were stored at -30c for the analysis of pco , aoa and nox . the endogenous lymphocyte dna damage was analyzed by alkaline comet assay using a similar method described by singh et al . samples were processed within 2 h and lymphocyte isolation for the comet assay was performed by using the histopaque 1077 . an amount of 1 ml blood was carefully layered over 1 ml histopaque and centrifuged for 35 min at 500g and 25 c . the interface band containing lymphocyte was washed with phosphate buffered saline ( pbs ) and then collected by 15-min centrifugation at 400g . membrane integrity was assessed trypan blue exclusion method and revealed membrane integrity in 95% of cells . ten microliters of fresh lymphocyte cell suspension was mixed with 80 l of 0.7% low - melting - point agarose in pbs at 37 c . subsequently , 80 l of this mixture was layered onto slides that had previously been coated with 1.0% hot ( 60 c ) normal - melting - point agarose , and then covered with a coverslip at 4 c for at least 5 min to allow the agarose to solidify . after removing the coverslips , the slides were submerged in freshly prepared cold ( 4 c ) lysing solution ( 2.5 m nacl , 100 mm edta - na2 ; 10 mm tris - hcl , ph 10 - 10.5 ; 1% triton x-100 and 10% dmso were added just before use for at least 1 h ) . the slides were then immersed in freshly prepared alkaline electrophoresis buffer ( 0.3 mol / l naoh and 1 mmol / l etda - na2 , ph 13 ) at 4 c for 30 min and then electrophoresed ( 25 v/300 ma , 25 min ) . after electrophoresis , the slides were then neutralized ( 0.4 m / l tris - hci , ph 7.5 ) for 5 min . the dried microscope slides were stained with ethidium bromide ( 2 g / ml solved in distilled h2o ; 70 l / slide ) , covered with a coverslip , and analyzed using a fluorescence microscope ( olympus , japan ) . each image was classified according to the intensity of the fluorescence in the damaged nuclei and was given a value of 0 , 1 , 2 , 3 , or 4 so that the total scores of the slide could be between 0 and 400 arbitrary units ( au ) ( 7 ) . mda levels , an index of lipid peroxidation , were measured by the double heating method of draper and hadley ( 14 ) . the method is based on spectrophotometric measurement of the purple color generated by the reaction of tba with mda . for this purpose , 2.5 ml of tca solution ( 10% , w / v ) was added to 0.5 ml whole blood in each centrifuge tube . the tubes were then placed in a boiling water bath for 15 min . after cooling to room temperature , the tubes were centrifuged at 1000g for 10 min and 2 ml of each sample supernatant was transferred to a test tube containing 1 ml of tba solution ( 0.67% , w / v ) . each tube was then placed in a boiling water bath for 15 min . after cooling to room temperature , the absorbance was measured at 532 nm by using the shimadzu uv 1601 spectrophotometer . the concentration of mda was calculated based on the absorbance coefficient of the tba mda complex ( =1.56105 cm / m ) . briefly , 15 l of plasma was placed in each of the two glass tubes . then 0.5 ml of 10 mm dnph in 2.5 m hcl was added to one of the tubes , while 0.5 ml hcl ( 2.5 mm ) was added to the second tube . samples were vortexed every 15 min . then 0.5 ml tca ( 20% , w / v ) was added and the tubes were left on ice for 5 min followed by centrifugation for 5 min to collect the protein precipitates . the pellet was then washed three times with 2 ml ethanol ethyl acetate ( 1:1 , v / v ) . the final precipitate was dissolved in 1 ml of guanidine hydrochloride solution ( 6 m ) and was incubated for 15 min at 37c while mixing . the carbonyl content was calculated based on the molar extinction coefficient of dnph ( =2.2104 cm / m ) . the assay measures the capacity of the serum to inhibit the production of tba reactive substances ( tbars ) from sodium benzoate under the influence of the oxygen - free radicals derived from fenton s reaction . the antioxidants from the added sample suppress the production of tbars , and the inhibition of color development is defined as aoa . briefly , 0.2 ml of whole blood was added to 1.8 ml distilled water . precipitating solution of 3 ml ( 1.67 g metaphosphoric acid , 0.2 g edta , and 30 g nacl in 100 ml distilled water ) the mixture was allowed to stand for approximately 5 min , filtered ( whatman no . 42 ) , and 2 ml of the filtrate was transferred into another tube , after which 8 ml of the phosphate solution ( 0.3 m disodium hydrogen phosphate ) and 1 ml dtnb were added . a blank was prepared with 8 ml of phosphate solution , 2 ml of diluted precipitating solution ( three parts to two parts distilled water ) , and 1 ml of dtnb reagent . the optical density was measured at 412 nm in the spectrophotometer nitric oxide decomposes rapidly in aerated solutions to form stable nitrite / nitrate products ( nox ) . plasma nitrite / nitrate concentration was measured by a modified method of griess assay , described by miranda et al . ( 18 ) . the principle of this assay is the reduction of nitrate by vanadium combined with detection by the acidic griess reaction . the serum was added to 96% cold ethanol at 1:2 ( v / v ) and then vortexed for 5 min . after incubating for 30 min at 4c , the mixture was centrifuged at 8000g for 5 min and the supernatants were used for the griess assay . one hundred microliters of filtrated plasma was mixed with 100 l of vcl3 and was rapidly followed by the addition of the griess reagents , which contain sulf 50 l and nedd 50 l . the absorbance was measured by a microplate reader ( multiskan spectrum , thermo labsystems , finland ) at 540 nm . all data were presented separately as mean se for the infected and the healthy groups . the comparisons of parameters were performed with student s t - test and the correlation analyses by pearson s correlation test . data were analyzed using the spss for windows computing program ( version 10.0 ) and p<0.05 was considered statistically significant ( 19 ) . vanadium ( iii ) chloride ( vcl3 ) , sodium chloride ( nacl ) , ethylenediaminetetraacetic acid disodium ( edta - na2 ) , sodium hydroxide ( naoh ) , thiobarbituric acid ( tba ) , trichloroacetic acid ( tca ) , hydrochloric acide ( hcl ) , sulfanilamide ( sulf ) , and n-(1-naphthyl ) ethylenediamine dihydrochloride ( nedd ) were obtained from merck ( darmstadt , germany ) . dinitrophenlyhydrazine ( dnph ) was purchased from fluka ( sigma - aldrich chemical co. , st . ten anatolian black goats naturally infected with b. ovis , at the age of 2 - 3 years were used in the study . ten healthy anatolian black goats aged 2 - 3 years from the same area served as controls . they were not exposed to babesia infection and none had treatment with either mineral or vitamin drugs . the study protocol was performed in accordance with the helsinki declaration as revised in 1989 . on clinical examination , the infected goats showed signs of babesiosis including rise in rectal temperature , dyspnea , tachycardia , anorexia , muscle tremors , signs of anaemia in pale mucous membranes and brownish urine . blood smears of all the selected animals were prepared aseptically on grease free sterile glass slides directly from the ear vein . the smears were air dried , fixed in methanol and stained with freshly prepared 5% giemsa stain for 45 minutes . after being stained with giemsa , the smears were examined for b. ovis under a research microscope and 100x objective . blood samples were taken from each animal by puncture of the jugular vein into heparinized tubes . two milliliters of blood was immediately pipetted into another tube to measure mda , gsh and dna damage . plasma samples were stored at -30c for the analysis of pco , aoa and nox . the endogenous lymphocyte dna damage was analyzed by alkaline comet assay using a similar method described by singh et al . samples were processed within 2 h and lymphocyte isolation for the comet assay was performed by using the histopaque 1077 . an amount of 1 ml blood was carefully layered over 1 ml histopaque and centrifuged for 35 min at 500g and 25 c . the interface band containing lymphocyte was washed with phosphate buffered saline ( pbs ) and then collected by 15-min centrifugation at 400g . membrane integrity was assessed trypan blue exclusion method and revealed membrane integrity in 95% of cells . ten microliters of fresh lymphocyte cell suspension was mixed with 80 l of 0.7% low - melting - point agarose in pbs at 37 c . subsequently , 80 l of this mixture was layered onto slides that had previously been coated with 1.0% hot ( 60 c ) normal - melting - point agarose , and then covered with a coverslip at 4 c for at least 5 min to allow the agarose to solidify . after removing the coverslips , the slides were submerged in freshly prepared cold ( 4 c ) lysing solution ( 2.5 m nacl , 100 mm edta - na2 ; 10 mm tris - hcl , ph 10 - 10.5 ; 1% triton x-100 and 10% dmso were added just before use for at least 1 h ) . the slides were then immersed in freshly prepared alkaline electrophoresis buffer ( 0.3 mol / l naoh and 1 mmol / l etda - na2 , ph 13 ) at 4 c for 30 min and then electrophoresed ( 25 v/300 ma , 25 min ) . after electrophoresis , the slides were then neutralized ( 0.4 m / l tris - hci , ph 7.5 ) for 5 min . the dried microscope slides were stained with ethidium bromide ( 2 g / ml solved in distilled h2o ; 70 l / slide ) , covered with a coverslip , and analyzed using a fluorescence microscope ( olympus , japan ) . each image was classified according to the intensity of the fluorescence in the damaged nuclei and was given a value of 0 , 1 , 2 , 3 , or 4 so that the total scores of the slide could be between 0 and 400 arbitrary units ( au ) ( 7 ) . mda levels , an index of lipid peroxidation , were measured by the double heating method of draper and hadley ( 14 ) . the method is based on spectrophotometric measurement of the purple color generated by the reaction of tba with mda . for this purpose , 2.5 ml of tca solution ( 10% , w / v ) was added to 0.5 ml whole blood in each centrifuge tube . the tubes were then placed in a boiling water bath for 15 min . after cooling to room temperature , the tubes were centrifuged at 1000g for 10 min and 2 ml of each sample supernatant was transferred to a test tube containing 1 ml of tba solution ( 0.67% , w / v ) . each tube was then placed in a boiling water bath for 15 min . after cooling to room temperature , the absorbance was measured at 532 nm by using the shimadzu uv 1601 spectrophotometer . the concentration of mda was calculated based on the absorbance coefficient of the tba mda complex ( =1.56105 cm / m ) . briefly , 15 l of plasma was placed in each of the two glass tubes . then 0.5 ml of 10 mm dnph in 2.5 m hcl was added to one of the tubes , while 0.5 ml hcl ( 2.5 mm ) was added to the second tube . samples were vortexed every 15 min . then 0.5 ml tca ( 20% , w / v ) was added and the tubes were left on ice for 5 min followed by centrifugation for 5 min to collect the protein precipitates . the pellet was then washed three times with 2 ml ethanol ethyl acetate ( 1:1 , v / v ) . the final precipitate was dissolved in 1 ml of guanidine hydrochloride solution ( 6 m ) and was incubated for 15 min at 37c while mixing . the carbonyl content was calculated based on the molar extinction coefficient of dnph ( =2.2104 cm / m ) . the assay measures the capacity of the serum to inhibit the production of tba reactive substances ( tbars ) from sodium benzoate under the influence of the oxygen - free radicals derived from fenton s reaction . the antioxidants from the added sample suppress the production of tbars , and the inhibition of color development is defined as aoa . gsh concentration of erythrocyte was measured using the method described by beutler et al . ( 17 ) . briefly , 0.2 ml of whole blood was added to 1.8 ml distilled water . precipitating solution of 3 ml ( 1.67 g metaphosphoric acid , 0.2 g edta , and 30 g nacl in 100 ml distilled water ) the mixture was allowed to stand for approximately 5 min , filtered ( whatman no . 42 ) , and 2 ml of the filtrate was transferred into another tube , after which 8 ml of the phosphate solution ( 0.3 m disodium hydrogen phosphate ) and 1 ml dtnb were added . a blank was prepared with 8 ml of phosphate solution , 2 ml of diluted precipitating solution ( three parts to two parts distilled water ) , and 1 ml of dtnb reagent . nitric oxide decomposes rapidly in aerated solutions to form stable nitrite / nitrate products ( nox ) . plasma nitrite / nitrate concentration was measured by a modified method of griess assay , described by miranda et al . ( 18 ) . the principle of this assay is the reduction of nitrate by vanadium combined with detection by the acidic griess reaction . the serum was added to 96% cold ethanol at 1:2 ( v / v ) and then vortexed for 5 min . after incubating for 30 min at 4c , the mixture was centrifuged at 8000g for 5 min and the supernatants were used for the griess assay . one hundred microliters of filtrated plasma was mixed with 100 l of vcl3 and was rapidly followed by the addition of the griess reagents , which contain sulf 50 l and nedd 50 l . the absorbance was measured by a microplate reader ( multiskan spectrum , thermo labsystems , finland ) at 540 nm . all data were presented separately as mean se for the infected and the healthy groups . the comparisons of parameters were performed with student s t - test and the correlation analyses by pearson s correlation test . data were analyzed using the spss for windows computing program ( version 10.0 ) and p<0.05 was considered statistically significant ( 19 ) . the results of whole blood mda , dna damage , gsh , pco , serum aoa and plasma nox levels in controls and infected groups are summarized in table 1 . as seen in table 1 , the amount of endogenous mononuclear leukocyte dna damage and levels of mda , pco and plasma nox concentrations were higher in the infected goats than in the healthy goats ( p<0.05 ) . furthermore , aoa and gsh concentrations were lower in the infected goats as compared to the healthy goats . the correlation coefficients obtained between biochemical parameters in the infected goats are presented in table 2 . mda levels were positively correlated with dna strand breaks , pco and nox in the babesia group ( r=0.766 , p < 0.05 , r=0.759 , p < 0.05 and r=0.886 , p < 0.05 , respectively ) . besides these data , mda levels were negatively associated with concentrations of aoa and gsh ( r=-0.792 , p < 0.05 and r=-0.763 , p < 0.05 , respectively ) . gsh levels in the infected group were positively correlated with aoa ( r=0.796 , p < 0.05 ) . however , gsh levels were negatively correlated with dna strand breaks and nox ( r=-0.769 , p < 0.05 and r=-0.801 , p < 0.01 , respectively ) . aoa was negatively correlated with pco and dna strand breaks in the infected group ( r=-0.823 , p < 0.05 , r=0.797 , p < 0.05 , respectively ) . the correlations of nox and dna strand breaks in the infected group with pco were positive ( r=0.723 , p < 0.05 and r=0.803 , p < 0.05 , respectively ) . there was significantly positive correlation between nox and dna strand breaks in the infected group ( r=0.735 , p < 0.05 ) . parasitic infections cause an activation of inflammatory cells which play an important role in the host defense ( 4 , 9 ) . activation of inflammatory cells induces and activates various oxidant - generating enzymes . the induction and activation of these enzymes in inflammatory cells are also regulated by many pro - inflammatory cytokines including tnf- il-1 il-6 and others ( 7 ) . in babesiosis caused by b. bovis , the infection involves production of il-1 il-12 , ifn- and tnf- ( 20 , 21 ) . the enhanced production of such cytokines might increase cancer risk by inducing or activating enzymes involved in the production of inflammatory cytokines because these enzymes produce large amounts of highly toxic molecules , such as ros including superoxide anion , hydrogen peroxide and hydroxyl radicals , and rns , including nitric oxide ( 7 , 20 , 21 , 22 ) . excess ros and rns productions are capable of degrading numerous biomolecules , including dna , carbohydrates , lipids and proteins ( 23 , 24 ) . ( 7 ) demonstrated that cutaneous leishmaniasis infection could induce dna damage through the production of free radicals generated by polymorphonuclear cells . kim et al . ( 25 ) studied the mutagenicity of ros and rns produced by stimulated leukocytes and observed that stimulated human promyelocytic leukemia cells and lps / ifn - g - stimulated murine macrophages induced 8-ohdg formation in transgenic chinese hamster ovary cells mediated by ros and rns . the results in the present study showed that dna damage increased significantly in infected goats with b. ovis . this result is the first report on the assessment of dna strand breaks in babesia infection . in the cells of hosts infected with different species of parasites , the amount of reactive oxygen species ( ros ) , thereby causing cell and tissue damage ( 9 , 26 ) . ros induce the oxidation of polyunsaturated fatty acids in biological systems and lead to the formation of lipid peroxidation ( lp ) products . one of the most frequently used ros biomarkers , providing an indication of the overall lp levels , is mda , one of several from products of lp ( 27 ) . in the present study , this result is an agreement with the result in cattle infected with b. bigemina ( 4 ) and indicates that lp increases in babesia infection . ( 28 ) reported that lp is closely related to the formation of cancer and degradation products of hydroxyl aldehydes , which indirectly damage dna and stimulate cancer gene expression . in the present study , there was also a positive correlation between mda levels , pco content and dna damage in infected goats . considering the close relationship between dna damage and mda , it could be stated that an elevated level of dna damage in goats infected with b. ovis might depend on the oxidative radicals generated during the course of the disease . free radicals react with proteins and modify amino acid residues by oxidation , nitrosation , and carbonylation . the oxidative inactivation of enzymes and the oxidative modification of proteins cause the formation of protein carbonyl derivatives ( 15 ) . in the present study , this result suggests that babesia infection causes the alterations in structure and function of proteins induced by free radicals . nitric oxide ( no ) is an important mediator of both physiological and pathophysiological processes . macrophages , neutrophils and mast cells have all been shown to be major producers of this molecule ( 29 ) . the no levels increased a tissue dysfunction or injury in the organism ( 30 ) . in the present study this result is an agreement with the results of studies reported that nitrate concentration , one of no metabolites , increases in blood of animals infected with parasites ( 31 ) . the antioxidant defense system includes small molecular antioxidants , antioxidant enzymes , and metal chelating agents . the total aoa of body fluids suggests a simultaneous interaction between various antioxidants and is crucial for the maximum suppression of a free radical reaction in extracellular compartments . such an activity appears to indicate the antioxidant characteristics of only one antioxidant , whereas total antioxidant activity represents the aggregate antioxidant characteristics of all antioxidants found in the plasma . in addition to aoa and reduced gsh exhibit the major defense against ros - induced cellular damage . gsh is a nonenzymatic antioxidant and serves as a reductant in oxidation reactions resulting in the formation of gssg . therefore , gsh can protect cells against the damage from ros and free radicals that arise during conditions of oxidative stress ( 32 , 33 ) , and thus reduced gsh levels may reflect a depletion of the antioxidant reserve . as a consequence of gsh deficiency , a number of related functions may be impaired protein biosynthesis , immune function , accumulations of lipid peroxidation products and detoxification capacity ( 34 , 35 ) . ( 36 ) expressed that parasites damage the cells which synthesis the molecules carrying the anti - oxidative agents . in the present study , this result is agreement with the results reported that the gsh concentration fell in animals infected with parasite species ( 9 , 37 - 39 ) and suggests that babesia infection causes a significant depletion of the antioxidant reserve of the host . babesia infection increased dna damage , mda , pco and concentration of nox from the oxidative stress markers and decreased aoa and gsh concentrations from the antioxidant markers in the present study . these results indicate that overproduction of reactive oxygen and nitrogen species by activated neutrophils and macrophages due to babesia infection increases oxidative stress , and thus leads to the acceleration of lipid peroxidation , dna damage and protein oxidation in the host . the positive relationship between dna damage and oxidants as well as the negative relationship with antioxidants supports our hypothesis , which claims that the oxidants generated in babesiosis could cause a significant damage in dna .

backgrounda reactive oxygen and nitrogen intermediate produced during an inflammatory response is the important part of host - defense strategies of organisms to kill the parasite . however , it is not well known whether these intermediates cause dna damage and oxidative stress in goats infected with babesia ovis . the purpose of this study was to clarify the effects of babesiosis on basal levels of dna damage and oxidative status of goats naturally infected with b.ovis.methodsdna damage and antioxidant parameters were determined in b. ovis infected goats . ten infected anatolian black goats with b. ovis diagnosed via clinical signs and microscopic findings and ten healthy were used in the study.resultsthe babesia infection increased the levels of dna damage , malondialdehyde ( mda ) , protein carbonyl content ( pco ) and plasma concentration of nitric oxide metabolites ( nox ) , and decreased total antioxidant activities ( aoa ) and reduced glutathione ( gsh ) . a significant positive correlation between dna damage , mda , pco , and nox concentrations was found in the infected goats . dna damage showed a negative association with aoa and gsh concentrations in the infected goats.conclusionthe babesia infection increases oxidative stress markers and dna damage and decreases aoa in goats . these results suggest that the increases in the production of free radicals due to babesia infection not only contribute to host - defense strategies of organisms to kill the parasite but also induce oxidative damage in other cells .

Introduction Materials and Methods Chemicals Subjects and parasitological examination Sample collection and biochemical estimation DNA Determination of malondialdehyde levels Measurement of protein carbonyl content Determination of plasma total antioxidant activity Determination of blood-reduced glutathione levels Estimation of plasma nitric oxide levels Statistical analysis Results Discussion Conclusion

herpes simplex virus-1 ( hsv-1 ) and hsv-2 which are dna viruses of alphaherpesvirinae subfamily and herpesviridae family , are the aetiological agent of hsv infections . these infections are among the infections most frequently encountered by humans , worldwide because humans are their only natural reservoirs , and no vectors are involved in their transmission . although , these viruses can infect both orofacial and pubic areas , hsv-1 predominates in the orofacial region while hsv-2 in the pubic region . it is a common worldwide affliction on which neither public health procedures , vaccines , nor antiviral chemotherapy have yet had a significant clinical impact . herpes labialis is a benign infective condition associated with the formation of vesiculo - ulcerative around the mouth which is often preceded by prodromal symptoms including pain , tingling and burning . although herpes labialis is generally associated with mild morbidity , frequent and/or severe recurrences remains a significant problem that impairs quality of life and impacts on healthcare consumption . rare complications like perioral scarring and depigmentation after healing of lesional areas may also occur . herpes labialis is usually caused by reactivation of the nonreplicating dormant hsv-1 and sometimes hsv-2 in the trigeminal nerve ganglia . however , it is the amplification of the preexisting activation that makes the condition , clinically visible . it has been reported that approximately one - third of patients who had initial hsv-1 viral infection undergo these recurrences at a rate of 14 recurrences per year which decrease after 35 years . on reactivation , the dormant virus in the trigeminal ganglion resumes multiplication then moves down the trigeminal nerve to the site of initial inoculation to infect the epithelial cells causing a herpes labialis and this is considered as ganglion trigger theory . the other , the skin trigger theory proposed by hill and blythe , holds that virus is continuously shed from neuronal endings and lesions develop when the susceptibility of the skin is sufficiently permissive for the development of a clinically apparent infection . this attests to the existing controversy concerning the mechanism of development of recurrent hsv infections . investigators have not fully elucidated the triggers that allow the virus to emerge from its dormant condition to cause a recurrence . nevertheless , several factors have been identified , such as ultraviolet light of both natural and artificial source as in sunlight and tanning beds , lip chapping , lip trauma or abrasion , fever , menstruation , fatigue , overexposure to wind , extremes of temperature , immunosuppression , dental procedures , iatrogenic trauma , dietary factor , upper respiratory tract infection , digestive problems , traveler 's diarrhea , decompression of the trigeminal nerve , pregnancy , physical and emotional stress . the diagnosis of herpes labialis is usually based on case history , clinical appearance and the location of the lesions in immunocompetent patients while confirmatory laboratory diagnosis in form of viral culture , polymerase chain reaction , serology , direct fluorescent antibody testing , or tzanck test may be necessary in immunocompromised patients . however , prompt recognition of herpes simplex infection and early initiation of therapy are of utmost importance in the management of the disease as prompt topical or oral antiviral therapy are quite effective in decreasing the severity and duration of herpetic episodes and contributes to the prevention of recurrence of herpes labialis . the review of the literature did not reveal any report of the prevalence of herpes labialis infection among both undergraduate and postgraduate dental healthcare providers . hence the objective of this study was to determine the prevalence and risk factors of self - reported herpes labialis among nigerian dental health providers the protocol for this study was reviewed and approved by the ethics and research committee of university of benin teaching hospital , benin city , nigeria . this cross - sectional study was conducted among nontobacco using final year dental students and dentists undergoing postgraduate training ( house officers and residents ) at university of benin teaching hospital , benin city , nigeria in june 20014 . all the final nontobacco dental students and dentists undergoing postgraduate training were included because of their small number . however , it was only those present during data collection that were assessed . the demographic information , lifetime and period ( previous year ) experience of the herpes labialis , perceived triggers and action taken during the last episode were obtained using a self - administered questionnaire . the questionnaires were administered to the undergraduates and postgraduates at a regular scheduled class and postgraduate seminar respectively . the data obtained were subjected to descriptive and binary regression statistics using statistical package for the social sciences ( spss version 17.0 , chicago , il , usa ) . in the binary regression analysis , lifetime experience of herpes labialis was the dependent variable while demographic characteristics were the independent variables . the protocol for this study was reviewed and approved by the ethics and research committee of university of benin teaching hospital , benin city , nigeria . this cross - sectional study was conducted among nontobacco using final year dental students and dentists undergoing postgraduate training ( house officers and residents ) at university of benin teaching hospital , benin city , nigeria in june 20014 . all the final nontobacco dental students and dentists undergoing postgraduate training were included because of their small number . the demographic information , lifetime and period ( previous year ) experience of the herpes labialis , perceived triggers and action taken during the last episode were obtained using a self - administered questionnaire . the questionnaires were administered to the undergraduates and postgraduates at a regular scheduled class and postgraduate seminar respectively . the data obtained were subjected to descriptive and binary regression statistics using statistical package for the social sciences ( spss version 17.0 , chicago , il , usa ) . in the binary regression analysis , lifetime experience of herpes labialis was the dependent variable while demographic characteristics were the independent variables . the majority of the participants were > 28 years , male , single , postgraduate healthcare workers , those without self - reported chronic medical condition and negative family history of herpes labialis [ table 1 ] . the lifetime prevalence was significantly associated with marital status , professional status and family history of herpes labialis . however , in binary regression , it was only marital status and family history of herpes labialis that were the determinants of this lifetime prevalence [ table 2 ] . a total of 6 ( 85.7% ) of participants that reported herpes labialis in the last 12 months had it , once while 1 ( 14.3% ) of them had it , 4 times [ table 3 ] . the participants reported more than one perceived trigger factors and the most common trigger factors reported for the last episode of herpes labialis were fever ( 61.9% ) , malaria ( 28.6% ) , fatigue ( 28.6% ) and stress ( 28.6% ) [ table 4 ] . the actions taken by participants for the last episode of herpes labialis were using drugs without prescription ( 14.3% ) , application of lubricant ( 23.8% ) , nothing ( 57.1% ) and could not remember ( 4.8% ) [ table 5 ] . demographic characteristics of the participants lifetime prevalence of herpes labialis among the participants the frequency of herpes labialis among the participants in the last 12 months perceived triggers for the last episode of herpes labialis among the participants action taken by the participants during the last episode of herpes labialis the prevalence of hsv infection worldwide has increased over the last several decades making it , a major public health concern . in this study , the lifetime prevalence was 22.1% . this falls within the lifetime seroprevalence of 2080% . this is lower than 52.5% reported among turkish nursing and midwifery students , 30.2% among ghanaian and south african health science undergraduates about four decades ago and 26.4% among jordanian university students . this lower lifetime prevalence in present study may be due to the fact that all the dental healthcare providers in this study were nontobacco users . the socioeconomic status , race , and culture which are correlated with hsv infection may have differed in participants in this study with the compared studies thereby explaining the difference in the lifetime prevalence . the lifetime prevalence was significantly associated with marital status , professional status and family history of herpes labialis . however , the determinants of lifetime prevalence of herpes labialis were only marital status and family history of herpes labialis . this indicates that marital relationship and relationship in family increases contact and possibly the infectivity potential as both herpes viruses are transmitted across epithelial mucosal cells , as well as through skin interruptions . they then migrate to nerve tissues , where they persist in a latent state until activation by appropriate trigger . the higher prevalence of fever and malaria in married participants and those with positive family history of herpes labialis which were the dominantly reported triggers in this study may be an additional explanation [ table 4 ] . a noted common characteristic of herpes labialis is recurrence which may be frequent or infrequent . it has been reported that approximately one - third of patients who had initial hsv-1 viral infection undergo recurrences at a rate of 14 recurrences per year which then decrease after 35 years . in this study , the annual prevalence of herpes labialis was 7.4% and the majority experienced it once while a few , more than once in the last 12 months . report that stated that most patients experience < 2 episodes of herpes labialis per year while 510% may experience 6 occurrences per year . this also indicates that herpes labialis occurs only in a subset of patients that have suffered primary hsv infection . antibodies to hsv-1 and hsv-2 increase with age starting in childhood and adolescence respectively reaching 5080% in different socioeconomic status at the age of 30 years . in the absence of cure and vaccine to prevent or eliminate the herpes infection at the moment , recurrence of herpes viral infection , most commonly as herpes labialis will occur in the presence of appropriate triggers because herpes virus have unique ability the virus to establish latency and undergo subsequent recurrence . the most common trigger factors reported by the participants for the last episode of herpes labialis in this study were fever , malaria , fatigue and stress . it is not surprising that fever topped the reported trigger factors as infections that manifest as fever are very common in developing country like nigeria . malaria which is a specific and common fever causing condition was the second most common reported trigger among the participants . it has been reported that malaria can contribute to hsv-1 reactivation in the oral cavity by profoundly affecting the host immune system by mediating at the same time immunosuppression and immune hyper - activation . the rating of fatigue and stress as the third and fourth common triggers respectively were noted and thereby confirms the widely documented stressful nature of dentistry . the higher state anxiety and increased daily hassles and stressful life events have been found to influence herpes labialis and the suggested biologic mechanism is through the modulations of t - lymphocyte function . treatments are available to reduce viral reproduction and shedding , prevent the virus from entering the skin , and alleviate the severity of symptomatic episodes . the treatment can be episodic or suppressive , depending upon the frequency and severity of episodes . in episodic therapy , it is essential that patients recognize prodromal symptoms for immediate self - medication . in this study , although further assessment of the drugs were not done but they are likely analgesics and antibiotics which are the most common self - medicated drugs among dental healthcare providers which would have been taken to control associated pain and prevent secondary bacterial infection . the use of lubricant will help in reducing discomfort associated with the condition and may also be helpful in preventing perioral scarring which is a rare complications of herpes labialis . however , the usual mildness of symptoms and the self - limiting nature of the condition may explain why more than half ( 51.7% ) of the participants that had herpes labialis did nothing . although the findings of this study may be limited by the fact that it is self - reported and could have be influenced by recall bias , assessment of prevalence using this method is acceptable in diseases with classical clinical presentation such as herpes labialis and can serve as baseline data which be compared with findings of other epidemiological studies that used the same study model . data from this study revealed herpes labialis as a significant problem among the studied population . the reduction of fever inducing infections , stress and fatigue which were major triggers will help decrease herpes labialis among this group .

introduction : the epidemiology of herpes labialis has been relatively neglected . the objective of this study was to determine the prevalence and risk factors of self - reported herpes labialis among nigerian dental health providers.materials and methods : this cross - sectional study of final year dental students and dentists undergoing postgraduate training at university of benin teaching hospital , benin city , nigeria was conducted in june , 2014 . the demographic information , lifetime and period ( previous year ) experience of the herpes labialis , perceived triggers and action taken during the last episode were obtained using a self - administered questionnaire.results:the annual prevalence of herpes labialis was 7.4% while the lifetime prevalence was 22.1% . the lifetime prevalence was significantly associated with marital status , professional status and family history of herpes labialis . however , in binary regression , it was only marital status and family history of herpes labialis that emerged as the determinants of this lifetime prevalence . the most common trigger factors reported by the participants for the last episode of herpes labialis were fever , malaria , fatigue and stress . the actions taken by participants for the last episode of herpes labialis were using drugs without prescription ( 14.3% ) , application of lubricant ( 23.8% ) , nothing ( 57.1% ) and could not remember ( 4.8%).conclusion : data from this study revealed that one out of fourteen and one out of five every studied dental healthcare providers had experienced herpes labialis in the last 12 months and their lifetime respectively . the reduction of fever inducing infections , stress and fatigue which were major triggers will help decrease herpes labialis among this studied group .

INTRODUCTION MATERIALS AND METHODS Ethical clearance Study sample and sampling technique Questionnaire Data collection Statistical analyses RESULTS DISCUSSION CONCLUSION

bile leaks can result from penetrating injury , such as gunshot ( gsw ) or knife wounds , or from blunt trauma , such as motor vehicle ( mva ) or motorcycle accidents ( mca ) . the incidence of bile leaks following hepatobiliary trauma ranges from 0.5 to 21% depending on the criteria and methods used to diagnose the bile leak [ 1 , 2 , 3 ] . following blunt or penetrating abdominal trauma , there can be significant damage to the biliary tract with severe injuries ( grades iii vi ) leading to an intrahepatic biloma , intraperitoneal leakage of bile , hemobilia and bilhemia , and intra - abdominal abscess . there is no consensus on the treatment of traumatic bile leaks and decisions are often based on extent and mechanisms of injury , associated organ injuries and local expertise . regardless of the etiology , bile leaks can be a considerable cause of morbidity in afflicted patients . unstable patients , as well as those with a penetrating injury , require laparotomy in conjunction with less invasive techniques . hemodynamically stable patients with blunt hepatic trauma can be managed nonoperatively which has become the treatment of choice over the past two decades . in earlier years , however , the treatment of bile leaks has evolved with improvements in technology and procedural techniques . the management of surgical bile leaks with therapeutic endoscopic retrograde cholangiopancreatography ( ercp ) is well - established [ 5 , 6 ] . however , the diagnosis of traumatic bile leaks can often be delayed and difficult . it has been suggested that posttraumatic bile leaks require more therapeutic procedures , patients have longer hospital courses , and higher hospital charges . ercp has been found to be a useful tool for the diagnosis and treatment of posttraumatic bile leaks , but data on outcome after therapeutic ercp for traumatic bile leaks is limited [ 1 , 3 , 8 , 9 ] . several case reports and a case series in the surgical literature report that ercp can be useful in the management of traumatic bile leaks . a variety of endoscopic techniques have been used to manage bile leaks . these include biliary sphincterotomy alone , biliary stenting with or without sphincterotomy , and nasobiliary drainage with or without sphincterotomy . these methods share the common goal of decreasing or eliminating the pressure gradient between the bile duct and duodenum , allowing preferential flow of bile from the duct into the duodenum instead of bile exiting the leak site . the absence of ongoing bile flow through the leak site allows the defect to heal [ 1 , 8 ] . we conducted a retrospective case review to evaluate the role of ercp in the management of patients who present with a bile leak after blunt or penetrating abdominal trauma . the prospectively recorded endoscopy database at the university of texas health science center , houston , tex . , usa , was reviewed to identify all patients who underwent ercp for the management of traumatic bile leaks between january 2004 and march 2009 . all patients who developed a posttraumatic bile leak were included , regardless of the mechanism of injury ( blunt vs. penetrating ) or the initial method of management ( operative vs. nonoperative ) . all patients were admitted to the trauma service and were initially managed in the surgical intensive care unit . two of the 14 patients were initially managed conservatively with 1 of these 2 patients eventually undergoing interventional radiology computed tomographic - assisted aspiration of biloma and drain placement . a bile leak was suspected when there was persistent drainage of bile via the surgical wound or through an intra - abdominal drain placed at surgery , after percutaneous drainage of a perihepatic fluid collection , or a positive hepatoiminodiacetic acid scan or when a computed tomography ( ct ) scan revealed a subhepatic fluid collection . ercp was performed with standard therapeutic duodenoscopes ( olympus tjf-100/130/140/160 ; olympus america corporation , inc . , patients underwent ercp in the endoscopy unit with the assistance of anesthesia , using a combination of diprivan , meperidine , and midazolam or in the operating room if patient remained intubated . once a bile leak was demonstrated on cholangiogram , a 7.0- , 8.5- or 10-f transpapillary biliary stent ( cook endoscopy , winston - salem , n.c . , usa ) was placed . after the ercp , surgically or percutaneously placed drains were removed after there was decreased or absent output from the drains . a repeat ercp was performed 416 weeks later to evaluate for resolution of a bile leak . the time interval between ercps varied among patients because most of them had other associated injuries , were critically ill , and required other interventions . on repeat ercp , the patients were monitored daily after ercp for 48 h to asses for any complications and again in clinic 12 weeks after discharge . endoscopic therapy was considered successful if the bile leak had resolved at the time of repeat ercp . failure of endoscopic therapy was defined as persistence of a bile leak at follow - up ercp , persistence of biliary drainage through the percutaneous drain or persistence of biloma on ct that necessitated additional interventions to manage the bile leak . to assess the long - term outcome , follow - up data were obtained by telephone contact with the patients , who were questioned specifically about any evaluation or treatment of a recurrent bile leak , as well as complications , subsequent to their last endoscopic treatment . a total of 14 patients ( 9 men , age range 1661 years ; 5 women , age range 1145 years ) , between january 2004 and march 2009 , underwent ercp for a bile leak secondary to traumatic injury to the abdomen . eleven cases were because of blunt trauma : mva in 8 , and mca in 3 ; the remaining 3 were because of penetrating trauma from a gsw . injuries were graded according to predefined criteria and were grade iii ( <3 cm parenchymal laceration ) in 1 patient , grade iv ( 2575% parenchymal disruption ) in 10 patients , and grade v ( > 75% parenchymal disruption with or without involvement of major hepatic vasculature ) in 3 patients . bile leaks were located in the common bile duct ( cbd ) in 1 patient , right ductal system in 8 patients , and in the left ductal system in 5 . other associated injuries included lacerations to the spleen , diaphragm , kidney or major abdominal vessels , and hemothorax . details on patient demographics and the nature of bile duct injury with location , interventions , and outcomes are shown in table 1 . two patients were managed conservatively until bile leak was suspected based on ct scan findings . one of these 2 patients subsequently underwent ct - guided biloma drainage and percutaneous drain placement . all 8 patients eventually underwent ercp with a bile leak found in the right hepatic system in 4 and in the left hepatic system in 3 . of the 3 patients with mca , all were initially managed with surgery with subsequent ercp with endobiliary stent placement . follow - up ercp done in all patients with blunt trauma revealed resolution of the bile leak and the stent was removed . three patients had a penetrating injury ( grade v in all 3 ) from a gsw and required emergent surgery . all 3 patients underwent ercp because of continued bilious output from the surgically placed drain . two of the 3 patients were found to have a bile leak from the left ductal system on cholangiogram , the third patient was found to have a bile leak from the right ductal system . all 3 patients had endobiliary stents placed . of note , 2 of the 3 patients had delayed presentation of the bile leak . one of the 2 patients developed bilious output from the surgically placed drain on postoperative day 11 . the other patient developed fever and pain on postoperative day 18 and ct scan revealed a perihepatic fluid collection which prompted the referral for ercp . at follow - up ercp , all 3 patients had resolution of the bile leak and the endobiliary stents were removed . this study showed that ercp with sphincterotomy and endobiliary stent placement is effective in the treatment of patients with major bile duct damage after hepatobiliary trauma . in our series , the resolution of a bile leak was seen in 100% of patients who underwent endobiliary stent placement and sphincterotomy . this data is supported by a recent study reported by lubezky et al . , where ercp with biliary sphincterotomy and stent placement was found to be effective in all 11 patients who developed a traumatic bile leak . also , in a study by bridges et al . , resolution of a traumatic bile leak was seen in 90% of patients who underwent endobiliary stent placement . biliary ductal injuries may not be seen on initial imaging or laparotomy and can therefore have an insidious presentation . the biliary injuries that result from hepatic trauma may be simple bile leaks into the lacerated liver , peritoneal cavity or pleural cavity or may result in biliary - vascular fistulas . bile leaks usually present several days after the initial trauma , with the patient complaining of abdominal pain , increasing abdominal distention , and ascites . in the setting of penetrating abdominal trauma such as gsw , presentation of bile leak can be delayed up to two weeks as was reported previously . the biliary injury can be intrahepatic , extrahepatic or both . in our series , 13 of 14 patients had injuries to the intrahepatic ductal system . the other 4 cases involved the left intrahepatic ductal system and 1 involved the cbd . in previous series , the right intrahepatic ducts were the predominant sites of bile leaks following abdominal trauma [ 1 , 9 , 12 ] . whether or not the location of the bile leak can predict treatment outcome is still unclear and can not be elicited from our study or others . surgery has been the conventional treatment for posttraumatic bile leaks but can be difficult to perform due to a combination of adhesions , inflammation , damaged liver parenchyma or the patient being a poor surgical risk . the most important factors that influence survival in these patients are the associated injuries and the nature and the extent of liver injury . it has been shown that liver - related complications appear to be less frequent in patients managed without surgery than in those managed surgically [ 8 , 9 , 10 ] . nonoperative management of bile duct injury is effective with success rate ranging from 90 to 94% . in our series the success rate of therapeutic endoscopic intervention ranges from 90 to 100% [ 1 , 8 , 9 , 12 ] . reported healing of bile leaks in 5 of 6 patients after therapeutic ercp . in a series by bajaj et al . , therapeutic ercp was successful in 8 of 9 ( 89% ) patients for managing posttraumatic bile leaks . bridges et al . reported healing of bile leaks in 9 of 10 ( 90% ) patients after biliary stenting . hence , an ercp is both of diagnostic and therapeutic benefit in these patients . in the present series , resolution of bile leak was documented on repeat ercp which was anywhere from 4 to 16 weeks after initial ercp . sharma et al . documented resolution of bile leak after 8.5 days ( range 330 days ) of endoscopic interventions . in a series by singh et al . , bile leaks closed in 15.76 days in patients who had undergone sphincterotomy , and in 12.14 days in patients without sphincterotomy . reported resolution of bile leaks in 67 days ( range 510 days ) after ercp . bajaj et al . reported time to resolution of bile leak after ercp as 7.4 days ( range 217 days ) . we routinely perform biliary sphincterotomy and endobiliary stent placement for the management of a bile leak . there are , however , several studies that clearly demonstrate the efficacy of biliary stent placement alone in this cohort of patients . other series support our combined approach of endoscopic sphincterotomy and stenting for management of bile leaks following hepatic trauma [ 8 , 9 , 12 ] . in major leaks it is unlikely that sphincterotomy alone can seal the leak . the ideal length of time for stenting is not known . follow - up in our patients ranged from 54 to 175 days ( mean of 85.6 days ) . none of our patients showed evidence of post - ercp complications . in a study by sugiyama et al . on long - term follow - up , there were no late complications after endoscopic treatment . in the series by sharma et al . , after a mean follow - up of 33 months , there were no complications and all the patients remained asymptomatic . the limitations of this study include the retrospective case series design as well as the small number of subjects included in the series . this particular cohort is small in the general population , and thus these procedures are rarely undertaken . as was the case with previously published case series , the management of bile leaks was not standardized . the follow - up was also short compared to other studies , and thus some long - term complications from ercp could have occurred . in summary , this study demonstrated the efficacy of ercp with sphincterotomy and endobiliary stent placement in the management of patients with extensive traumatic hepatic injury with intrahepatic bile leaks . the severity of an injury and the site of a bile leak do not preclude endotherapy as a management option in these patients . the results of our series and those of previously published series suggest that ercp should be considered as a first - line therapy for traumatic bile leaks . matthew p. spinn and frank j. lukens have no conflicts of interest or financial ties to disclose .

traumatic bile leaks often result in high morbidity and prolonged hospital stay that requires multimodality management . data on endoscopic management of traumatic bile leaks are scarce . our study objective was to evaluate the efficacy of the endoscopic management of a traumatic bile leak . we performed a retrospective case review of patients who were referred for endoscopic retrograde cholangiopancreatography ( ercp ) after traumatic bile duct injury secondary to blunt ( motor vehicle accident ) or penetrating ( gunshot ) trauma for management of bile leaks at our tertiary academic referral center . fourteen patients underwent ercp for the management of a traumatic bile leak over a 5-year period . the etiology included blunt trauma from motor vehicle accident in 8 patients , motorcycle accident in 3 patients and penetrating injury from a gunshot wound in 3 patients . liver injuries were grade iii in 1 patient , grade iv in 10 patients , and grade v in 3 patients . all patients were treated by biliary stent placement , and the outcome was successful in 14 of 14 cases ( 100% ) . the mean duration of follow - up was 85.6 days ( range 54175 days ) . there were no ercp - related complications . in our case review , endoscopic management with endobiliary stent placement was found to be successful and resulted in resolution of the bile leak in all 14 patients . based on our study results , ercp should be considered as first - line therapy in the management of traumatic bile leaks .

Introduction Patients and Methods Results Discussion Disclosure Statement

giant intracranial aneurysms have a lower propensity to bleed than small and medium - sized lesions and they may present with symptoms secondary to mass effect . while the diagnosis of a giant aneurysm can be readily made on radiology if there is any circulating component within the aneurysmal sac , establishing a pre - operative diagnosis becomes difficult if the aneurysm is completely thrombosed . the absence of a circulating component and the visualization of a large thrombus would lead to consideration of various other differentials depending on the location of the lesion . a 45-year - old male presented with complaints of progressivelyworsening vision in his left eye and headache since 6 months . he also complained of gait ataxia and occasional social incontinence of urine . on examination , visual acuity in mri brain revealed a large left basifrontallesion extending superiorly and distorting the corpus callosum [ figure 1 ] . the lesion was hyperintense on t1-weighted images ( wi ) [ figure 1a ] and hypointenseon t2wi[figure 1b ] . the lesion did not enhance on gd - contrast sequences and no blood flow was demonstrated within the lesion on contrast mr angiography ( mra ) . ct angiography ( cta ) demonstrated displacement of left anterior and middle cerebral arteries ; there was no enhancement within the lesion [ figure 1d ] . ( a ) t1-weighted coronal image showing a t1 hyperintense lesion . ( b ) t2wi demonstrating the hyppointense capsule as well as contents . ( d ) ct angiogram demonstrating the distortion of the left mca and acas based on the location and presence of a large thrombus , adifferential of giant cavernous hemangioma was considered . however , the mri appearance of a well - delineated sac with a thrombus within it was not entirely concordant with this diagnosis . however , digital subtraction angiogram ( dsa ) did not reveal any filling of contrast within the sac of the lesion . the dsa also revealed that the left middle cerebral artery was displaced inferiorly by the lesion [ figure 2a ] . the left a1 was not well visualized ; both a2s were filling on right carotid injection and a round shift of the a2s was noted [ figure 2b ] . since dsa did not demonstrate any contrast within the lesion , a possibility of intracerebral intravascular papillary endothelial hyperplasia ( masson 's tumor ) was also considered , although this is a rare lesion within the brain . the a2s are shifted to the right due to the mass effect of the lesion . no contrast is seen within the lesion a left pterional craniotomy was made and the lesion approached via the trans - sylvianroute . there was a good plane of cleavage and capsular resection was performed in a piecemeal manner . , a portion of the capsule was found to be significantlyadherent to the left a1-a2 junction . there were areas of calcification in this area that further rendered dissection from the a1-a2 complex difficult [ figure 3a ] . multiple perforators were found arising from the a1a2 junction and traversing posteriorly ; these were adherent to the capsule . postoperative contrast - enhanced ct and mri revealed no residual capsule[figure 3b ] and subsequent dsa revealed preserved flow in all vessels and no fresh abnormality [ figure 3c ] . histopathology of the resected specimen was diagnostic of aneurysmal wall with thrombus [ figure 3d ] . follow - up dsa is planned to ensure that no regrowth of the aneurysm has occurred . ( a ) intraoperative image showing the optic chiasm ( star ) ; the lesion ( horizontal arrow ) is closely adherent to the left a1 ( vertical arrow ) . ( c ) postop angiogram demonstrating the right internal carotid artery supplying both a2 arteries across a patent acoma . ( d ) photomicrograph of the aneurysm wall composed of fibrocollagenous wall , congested blood vessels , lymphocytic infiltrate and hemosiderin laden macrophages ( h and e 400 ) spontaneousand complete thrombosis of giant intracranial aneurysm is reported to occur in 3 - 20% of cases . the majority of giant aneurysms that have been reported to have undergone spontaneous thrombosis arose from the cavernous segment of the internal carotid artery . there are singlecase reports of giant posterior cerebral artery , superior cerebellar artery and pericallosal artery aneurysmsthat underwent spontaneous thrombosis . there exist no previous reports of giant anterior communicating artery aneurysms undergoing spontaneous thrombosis . in the present instance , aneurysm was not considered as the top preop differential since the lesion did not enhance on contract administration . intra - operatively , the only clues to the nature of the lesion werethe presence of a multi - lamellatedthrombuswithin the cavity and the attachment to the anterior communicating artery . totally thrombosed giant aneurysms can be confused with other tumors like cavernous hemangiomas , masson 's tumor , oligodendrogliomas and occasionally meningiomas . there are several hypotheses to explain the phenomenon of spontaneous thrombosis in giant intracranial aneurysms . the fundus of a giant aneurysm may itself compress the parent artery , resulting in stenosis and a low - flow state . this may lead to intra - aneurysmal clot formation ultimately resulting in thrombosis of the aneurysm . a large aspect ratio ( indicative of a small neck ) could also result in a low - flow state . extension of the thrombus to the neckwould result in a further reduction of blood flow within the sac resulting in total thrombosis . it is possible that in the present case similar mechanisms could have been responsible for complete isolation of the aneurysmal sac from the circulation . the formation of a neo - intimal layer in the narrow neckcould explain the absence of bleedingdespite close dissection of the sac from the a1a2 junction . pre - operative dsa is an absolute imperative to establish the diagnosis of large thrombosed intracranial mass lesions . this lesion was not recognized as an aneurysm at surgery owing to the complete absence of bleeding and the calcification at the neck ; thus , the neck was not clipped . however , it is generally recommended that a clip be placed across the neck since there have been several reports of regrowth of giant aneurysms that have not been clipped and completely excluded from circulation . spontaneous thrombosis of giant intracranial aneurysms is a rare phenomenon . but aneurysms need to be considered in the differential diagnosis of mass lesions located in close proximityto cerebral vasculature . the aim of surgery would be to decompress the surrounding neural structures . a clip application across the neck may not be necessary if there is no filling of the sac while dissecting close to the neck .

giant anterior communicating artery aneurysms are rare . apatient presented with visual dysfunction , gait ataxia and urinary incontinence . mri showed a giant suprasellar mass . at surgery , the lesion was identified as being an aneurysm arising from the anterior communicating artery . the difficulty in preoperative diagnosis and relevant literature are reviewed .

Introduction Case Report Discussion Conclusions

our ancestors knew that human beings , like plants , seeds , and other living things in the universe , were immersed in an evolutionary cycle . thus , since the beginning of civilization , our ancestors have turned their gaze to the sky for answers and found that certain astronomical cycles overlap each other , just like the seasons , day and night , migratory movements of animals , etc . , so they used these celestial cycles as the background for deciding , among other things , the times that were more conducive to hunting , planting , and harvesting . the term climate in traditional chinese medicine ( tcm ) , from the point of view of chinese medicine , refers to agents that cause disease ; tcm believes that man is a reflection upon the universe , like the existence of a microcosm within the macrocosm . each of these climate forces has a specific action on the body , depending upon their characteristics . hence humans must follow the laws of the universe to accomplish harmony and total health . tcm uses a unique terminology to diagnose and treat a wide range of health problems . patients or modern practitioners who are not familiar with chinese medicine have a look of concern or smile in a humoring manner when an acupuncturist or herbalist tells them they are suffering from a devil wind strike , stagnation of liver qi , or blood deficiency ; they often erroneously assume that something is physically wrong with their liver or blood . the theory of chinese medicine uses the names of the organs to help illustrate related patterns of physical and psychological problems . when a traditional chinese doctor diagnoses a patient with a problem , from a historic chinese medicine perspective , the doctor is usually talking about a problem with the hepatic system . this system involves the physical liver , acupuncture meridian liver - related disharmonies ( or patterns ) , and liver diseases ( western medicine ) such as hepatitis and cirrhosis . wind is one of the most difficult terms for modern doctors to understand , particularly when they approach alternative medicine . it considers the human body as a whole and attributes disease to an imbalance between the different elements it considers . not only is what happens within the body important , but what happens throughout the body and how that is manifested in response to external and environmental stimuli are also important . thus , tcm treatments , rather than being aimed at healing a particular symptom , focus on restoring the body s balance , emphasizing the need to have a healthy , nutritious lifestyle , with plenty of relaxation and breathing exercises . wind is one of five climates that characterize the five seasons according to a chinese philosophical principle . heat occurs in summer , humidity in late summer , drought in autumn , and cold wind in winter and spring . while wind is present in all seasons , its manifestation will be stronger in the season that matches it . wind in the body resembles the wind in nature ; thus , it generates both movement and movement in what would otherwise remain motionless . it produces change and acceleration in what otherwise would be steady and slow and causes things to appear and disappear quickly . it affects the body in the same way as moving branches and leaves on a tree affect the tree ; consequently , wind is a yang phenomenon . when feng xie , or wind nefarious , attacks the body by penetrating the skin and the pores , an important result in tcm is the emergence of imbalances of external origin caused by climatic aggression pathogenic factors . wind is associated with spring , but a disharmony characterized by wind can occur in any season . feng xie is a corrupting influence that rarely appears alone , usually being accompanied by some other external pernicious influence , such as cold and damp weather . wind that is directed toward the upper body has a pernicious influence on yang . because wind is light and airy , the huangdi neijing says , the damage inflicted by wind affects primarily the top . wind is thought to be initially manifested in the highest parts of , and more outside , the body , especially the face , skin and sweat glands , and lungs . when the body is invaded from outside , its defensive capabilities are weakened , causing a mismatch in the opening and the closing of the pores in the entire body , leading to the invasion of other pathogenic factors causing diseases with symptoms such as a headaches , nasal obstruction , painful and itching throat , facial edema , abnormal aversion to wind , and perspiration . cold wind is an aversion to wind and chill and is accompanied by fever , headaches and generalized aches , a runny nose , and a cough . hot wind is accompanied by fever , sweating , headaches , red eyes , sore throat , photosensitivity , thirst , a cough with yellow and dense sputum , respiratory problems , constipation , and epistaxis . damp wind has effects similar to those of the common cold , with sore limbs , listlessness , nausea , anorexia , and diarrhea and can cause diseases like arthritis . an endogenous wind attacks the liver and causes dizziness , spasms , convulsions and even coma . as wind is associated with movement , its attack is varied and fast and is often recognized by signs like pain that moves from one place to another , itching or rashes that change location , spasms , tremors , tics , and lightheartedness . feng xie is often associated with cold , humidity , dryness and heat , forming complex pathogenic factors such as wind - cold , wind - humidity , wind - dryness and wind - heat . a characteristic of winds as harmful exterior influences is the speed with which that characteristic appears , as is the case with all other external pernicious influences . winds often include a fever , which is a sign of conflict between an exterior influence and normal qi , sweating , sudden headaches , nasal obstruction , itching , and throat irritation . as wind is typically accompanied by another harmful influence , it contains the signs of the other baneful influence . the inner feng xie generally accompanies chronic disharmonies , and often these disharmonies involve the liver , which is responsible for regular movement within the body and , therefore , are sensitive to irregular movement . signs of unpromising wind include dizziness , ringing in the ears , heaviness or numbness of the limbs , tremors , and seizures . heavenly stems and earthly branches are collectively known as stem - branch or gan - zhi . the 10 heavenly stems are jia , yi , bing , ding , wu , ji , geng , xin , ren and gui . the 12 earthly branches are zi , chou , yin , mao , chen , si , wu , wei , shen , you , xu and hai . each heavenly stem is paired with an earthly branch to form the gan - zhi sexagenary cycle that starts with jia - zi . six excesses ( wind , cold , heat , dryness , moisture , and heat of summer ) originated thousands of years ago and may sound primitive and unscientific , but are precisely associated with many diseases and disorders and with how they behave in the body ( table 1 ) for example , in nature , wind often breaks out quickly without warning , coming and going . similarly , the symptoms of winds in the body are often characterized by coming and going , sometimes suddenly . have you ever had a sudden headache that is passed as mysterious as to why it appeared ? that was a wind attack . if during the headache , you were red - faced , sweating , and hot , those symptoms indicate that the condition was caused by a wind - heat . wind is one of the six external factors of disease ( six qi or six yin / six excesses ) . these climates can attack the body , enter the meridians , and cause external diseases ; e.g. , cold wind can cause a cold . wind is a climatic condition that is observed everywhere , but is given a heightened importance in tcm . a healthy body is the result of a balance between yin and yang and is supported by a network of activities and complementary forces that generate and put limits on one another . thus , qi moves the blood , but at the same time keeps it in place ; heart stores the shen ( spirit ) and moves the blood ; gallbladder regulates the ascension ; stomach regulates the descent ; liver controls distribution and kidneys regulate storage ; lungs regulate the flow of qi descent ; kidney qi governs fixation . an energy imbalance occurs as a result of breakage of the relationship between real power and pathogenic power ( zheng qi - xie qi ) . when the balance is disturbed , the yin and the yang no longer fit , and the body becomes vulnerable to the damaging effects of a pernicious influence . for tcm , the root of most diseases is a serious imbalance of the various energy systems of the body organs and the lack of overall functional harmony of the entire structure , which occur unfailingly when an organic imbalance exists . the human body has the capacity to resist different pathogenic factors and to maintain a relative balance inside the body and between it and the outside world . this ability to resist zheng qi ( true power ) is the ability of the body to resist pathogens . it is a term of tcm that is preserved in the kidneys , is inspired by nature , and comes from consumed water and rice . it is the quintessence of action and the ability to maintain the physiological functions of the tissues and the organs throughout the body . keep in mind that zheng qi , real power , is the sum of energy inherited and acquired . in pathogenesis , tcm pays attention to physical functions , believing that if a person is full of positive force , a malicious energy will not attack . xie qi or malicious energy is another term used in tcm and refers broadly to all pathogenic factors . the appearance of disease results from a struggle between these two factors , zhengqi - xie qi . zheng qi - xie qi has no resistance , so the natural balance between yin and yang in the body is lost . the factors that tend to break this relative equilibrium are called xie qi or pernicious influences . if the zheng qi is strong because the person has inherited a proper zheng qi , good contributions provided later by nutrition and breathing are maintained , and no emotional disturbances and no environmental and traumatic assault occur . a xie- qi single attack or unbalance occurs in extreme circumstances and is an extreme response . the body will always be positive even with acute symptoms such as fever , headache , and erratic pain . when the xie - qi factor reaches the interior , overcoming the various levels of defense , it affects the viscera and the bowels , causing an imbalanced yin- yang , and can attack the normal functions of rising and falling qi , organic liquids , and blood . if zheng qi is weak , xie qi will always be present , causing a person to be sickly , with a latent and prolonged aspect , the signs of which may appear as weakness , asthenia , chills , and clinical chronic conditions that are exacerbated by any factor affecting the body , whether dietary , emotional or climatologic . a malicious power vacuum leads to infiltration , and the sky and the earth remain closed and dark . mists and clouds can not rise , and rain will not fall . the above and below the conflict between zheng qi and xie qi can be outlined as follows : normality when zheng qi is stronger than xie qi , fullness when zheng qi is durable and is equal to or slightly less than xie zheng , empty when zheng qi is weak but less weak than xie qi , chronicity when xi -qi is weak but less than zheng qi , gravity when zheng qi is real and much lower than xie qi . when the body is weakened by an imbalance of yin and yang , a weather phenomenon can invade and become a destructive influence . a harmful influence is a natural event that becomes destructive only when the body has an inappropriate relationship with that influence in this state ; the body is subjected to a conflict between the injurious influence and normal qi . if the qi protector is durable , the destructive influence is expelled , and the person recovers . however , if the qi is weak or the pernicious influence is very strong , an imbalance develops and penetrates deeper , engaging the internal organs more . the imbalances generated by some of the pernicious influences that invade the body are produced suddenly and are characterized by an aversion to the specific influence that generated those imbalances , for example , fear of cold , aversion to wind , fever , chills , body aches and malaise . these symptoms are the result of an attempt to shed the influence that is affecting normal qi and the qi protector . when a hurtful influence invades the body in this way from the outside , it is called an external pernicious influence . in ancient china , people were very concerned with wind . at that time , people believed that the world was full of benevolent spirits and evil spirits , both of which affected human events . if a person became ill , especially with a sudden onset , that illness may have been attributed to an evil spirit that had entered the person s body . for example , if someone had a stroke or heart attack , an unexpected or severe pain , or loss of consciousness or motor abilities or had died , an evil spirit was thought to have possessed the person s body . in africa and the middle east , zr is used as a term for malevolent spirits or demons that are thought to possess individuals , mostly women , and cause discomfort or illness . the superstitious belief that demons travelled with the wind led to the use of words like evil wind stroke , and this explanation for the causes of disease has continued to the present . diseases caused by erratic wind , which are predominant in the spring , exist throughout the year . leprosy is the best example for illustrating the ambiguity of the epistemological status of contagion in the middle ages . at that time , leprosy was considered to be a disease of the soul , the consequence of sin and divine punishment . when classifying different types of infection diseases , avicenna in the canon says , pass from one to another and mentions that leprosy , scabies , smallpox , pestilential fevers and some eye diseases are transmitted by wind . leprosy is a chronic infectious disease that mainly affects the skin and peripheral nerves ; severe cases can involve deep tissue and visceral organs . although leprosy rarely causes death , it can lead to physical disability and deformity , so that patients are lost to the labor force . leprosy can occur at any age , and both men and women may be affected . in explanation of leprosy , tcm uses many terms such as apathetic wind ( ma feng ) and releasing the wind ( fang feng ) , scabies gale , wind scabies , wind in blood , also known as harsh wind , and pandora wind / pandora s ulcers . all above terms are associated with the release of all the evils of humanity into the world , just like pandora opened her box . pandora wind is a toxic pathogenic wind that causes apathetic feeling of chronic skin diseases . releasing the wind is followed by sickness , fatigue , madness , leprosy and plague , sorrow , poverty , and crime ; all the world s ills are spreading through the land . when we mean that any of the acts we perform in life will bring us new evils and unknown misfortunes . from antiquity to the renaissance , physicians explained the mechanisms for the spread of disease on the basis of direct contact with poison or mediation of the foul air between a sick agent and a healthy patient . as the medical ideas moved away from devil - caused diseases to nature - caused diseases , the concept soon developed into the six qi and six yin , and wind became specifically associated with the spring season , even though spring is definitely not the season in which there is more wind , as we imagine wind today . the exposure of a person to the wind after perspiring or sleeping in a place with drafts promotes the development of wind syndromes . wind generates humidity , dryness and sometimes heat , which invade the body as a result of wind . by its nature its direction is upward ; thus , it affects the upper body ( face and head ) and the body s surface , causing a mismatch between the openings and the closings of the pores of the skin . clinical manifestations of wind include fever , aversion to cold , headache , sore throat , sweating , feeling of hyperthermia , cough , rhinal obstruction , rhinorrhea , nasal pruritus , a tongue with a thin white coating , and a floating or moderate pulse . wind invades the body from the outside , and defensive qi loses its ability to protect the body against foreign pathogens . in this initial struggle , manifestations of the openings of the pores of the skin and attempted expulsions of external pathogens are observed . thus , the patient is seen to be sweating , with the temperature of the body beginning to rise , and to have both an aversion to cold and a headache . that is to say , the location of wind changes , thus , for example , causing pain in various joints . likewise , wind causes movement disorders , such as dizziness , tremors , muscle spasms , convulsions , a stiff neck , and facial paralysis . as wind s attack is fast , diseases can change abruptly , with symptoms sometimes appearing and disappearing rapidly . for example , conditions such as hives or itching can suddenly appear and then suddenly disappear , and rheumatism can migrate from one joint to another . other morbific factors mostly attach themselves to wind to invade the human body , as in wind - cold syndrome , wind - heat syndrome , and wind - dampness syndrome . . an evil - excess will attack the body only when the defense system is weak , and the protective qi is deficient somewhere along the surface of the body . one of the aims of preventive medicine is to keep the body invulnerable to such attacks from outside the body . diseases of the six evils are most likely to appear under abnormal weather conditions when the body is prepared for the powers of the present season but is suddenly faced with an opposite force . sudden cold emerges in the mid - summer and , for example , often causes epidemics of grippe . correspondingly , people who go from a cool , dry place to a warm , damp place are more likely to be exposed to an invasion by local meteorological excesses than natives of the region . because , plague was a major problem during ancient times , practitioners of tcm learned about it long ago and blamed it on pestilential evils . attacks of pestilential evils are usually related to bizarre climates , such as droughts , floods , and extreme heat , as well as environmental conditions such pollution . epidemics occur abruptly , with the victims having severe symptoms and the disease being highly contagious . for example , wind diseases occur in spring , summer - heat diseases in summer , clammy diseases in late summer and early autumn , dry diseases in autumn , and cold diseases in winter . in addition , people who live for a long time in a humid environment tend to be easily attacked by the humid evil , and those who work extensively in a high - temperature environment are freely attacked by the dry - heat evil or fire evil . evils can work alone or in groups of two or more in attacking the body ; syndromes such as the common cold ( wind - cold type ) , damp - heat diarrhea , and wind - cold - damp blockage are examples of medical problems caused by a unification of evils . during the early stages of a disease , any one of the six evils can influence the others and can transform into another kind of evil under particular conditions . for example , the cold evil that enters the body can be transformed into the heat evil , and the prolonged summer - heat with dampness evil can be transformed into the dryness evil . the six climatic evils penetrate the body and cause disease , mostly through the spaces located between the skin and the muscles or through openings such as the nose and the mouth . wind is one of five climates that represent the five seasons [ 15 , 16 ] ( table 2 ) . external wind comes from outside , enters the body through the acupuncture meridians , and causes what are known as colds , flu , and virus . wind is said to be the spearhead as it promotes the opening of the pores and the penetration of other evil summer will be more conducive to heat - wind penetration , causing sunstroke or heatstroke . winter will be more conducive to cold wind penetration ( colds , flu ) , and spring is typically associated with allergic rhinitis . the harmful action of wind penetrates into the muscle layers in the meridians of the triple burner and the small intestine , which blocks the normal flow of qi , causing muscle stiffness . cases of occasionally or permanently weakened defensive energy or immunity are associated with insomnia or overeating , leading to an attack of an external pathological wind in unregulated seasons . in other words , our bodies follow the rhythm of the energies of the seasons and do not have the ability to adapt to changes . the following is a summary of some key points : allopathic practitioners who begin to study tcm are surprised to hear about wind , heat , humidity , dryness , and cold . tcm believes that man is a reflection of the universe , a microcosm within the macrocosm . each of these climate forcers has a specific action in the body , depending on their characteristics . a person is clear and calm , the flesh and the interstice structures are firmly closed up and resist . even though there is a strong wind which is a violent poison , it will be unable to harm that person . diseases involving excess movement of the body are epilepsy ( rare convulsions ) and parkinson s disease , diseases involving symptoms appearing in various parts of the body at different times are early stage rheumatism involving differing joints or skin rashes that appear in multiple places . diseases involving loss of movement are stroke , paralysis , tetany , and coma . various kinds of pain and numbness , as well as spastic syndromes , are sometimes referred to as bi syndromes and include a headache , a toothache , limb numbness , tendon spasms , arthritis , and deep bone pain . diseases that are acute are the common cold , influenza , a sinus infection , a skin eruption , a sore throat , a cough , and eye disorders . diseases that affect the surface of the body ( skin or flesh , rather than viscera ) are chronic eczema , leprosy , scrofula , and hair loss . in western medicine , acute cerebrovascular disease refers to a non - traumatic serious cerebral blood supply disorder caused by focal neurological damage . in tcm , zhong feng terms is refers to human body targeted ( struck ) by an external pathogenic wind and internal wind . zhong refers to a sudden onset of this condition because the character zhong conveys the idea of an arrow hitting its target . a wind strike in chinese medicine corresponds to all of the following medical conditions in western medicine : brain hemorrhage , cerebral thrombosis and vessel spasm . clinical characteristics of cerebrovascular disease are a sudden onset and rapid progress . in tcm , this is a type of bi - syndrome . a vicious wind , chill , wet or heat invasion of the body leads to a blockage of the meridians . working or living in a chilled and damp environment , walking in the rain , working long - term underwater , or drastic changes in climate are other reasons that wind - cold - dampness evil invades the body and causes disease . it is a common problem of old age because deficiencies of yin naturally develop with aging , giving rise to liver yang agitation , which can produce interior wind . facial paralysis after a stroke is due to internal wind while bell s palsy is attributable to external wind . wind strike can be summarized in four words : wind - phlegm - fire - stasis . all of these may or may not be present , but at least three of them must be present to produce blowing wind . they can also be present to different degrees of intensity , which can give rise to many various kinds of wind strikes . wind causes the sudden loss of self - awareness of an acute attack , i.e. , wind strike and its subsequent hemiplegia . when holes are clogged , phlegm causes aphasia or slurred speech . when fire damages the yin , subsequent yin fails to nourish the tendons and meridians , which occurring meridians malnutrition and hemiplegia . blood stasis affects the meridians and joints , causing stiffness and pain in the extremities ; this is an observed consequence of wind strike . naturally , it is a pathogenic factor listed in the wind blowing . in addition , some deficiencies of qi , blood or yin , especially yin kidney and/or yin liver , are observed . overwork : working long hours under stressful conditions without adequate rest lead to kidney yin deficiency . powerful stress : a combination of overwork and stress factors is the most common cause of kidney yin deficiency in industrialized societies . liver yang , especially in the elderly , often results in liver wind , and liver wind is a causative agent of stroke , coma , mental clouding , and paralysis . internal wind and external wind 3 . eating irregularly or ingesting amounts of fat , dairy products , fatty foods , fried foods , and/or sugars weakens the spleen and leads to phlegm , which predisposes the body to obesity . phlegm causes numbness in the extremities , mental clouding , slurred speech or aphasia , and a swollen tongue with a sticky coating . extreme physical work , including excessive exercise and sports , also weakens the spleen and can lead to spleen deficiency , which , in turn , causes phlegm . marrow fails to nourish the blood , and eventually this can lead to blood stasis . extravagant physical activity and inadequate rest , such as lifting heavy objects or taking part in too many sports or excessive exercise , weakens the spleen , muscles , and meridians . spleen deficiency lead to a failure to produce sufficient blood , so blood deficiency develops in the meridians . in qi and blood deficiency on the other hand , exposure to external wind interacts with internal wind meridians , leading to paralysis of the limbs . gout is one of the most painful forms of arthritis and is due to hyperuricemia caused by the accumulation of monosodium- urate crystals . drop is derived from gutta ; drop in latin , as in the middle ages , was believed to be due to a drop of evil humor that someone had deposited in the joints . gout is also called rich man s disease because it occurs most often in the upper classes . in tcm , white tiger joint disease is the ancient name for gout or disease of the toes . gout , the so - called white tiger disease , produces pain like that of a tiger gnawing on one s toes . white tiger joint disease , which is a bi - syndrome , is a disorder resulting from obstruction of the meridians and sluggishness of qi and blood flow after an attack of pathogenic wind , cold , dampness or heat . it is characterized by pain ; insensibility and heaviness of muscles , tendons and joints or edema ; hotness with limitations on joint movement . in tcm , gout is believed to be caused by excessive intake of fatty and sweet food , alcohol abuse , overstrain , tension , or contraction of wind , cold , dampness and heat , resulting in obstruction of the meridian qi due to stagnation of qi and blood , as well as retention of phlegm and blood in the joints . the pathological changes are as follows : pathogenic wind - heat coupled with dampness creates a predisposition for the disease . in addition , predominance of yang , hyperactivity of the liver , and excessive intake of alcohol and food can all lead to retention of pathogenic wind , dampness and heat , or pathogenic wind , phlegm and heat in the meridians and joints , obstructing the flow of qi and blood and bringing about a bi - syndrome due to wind , dampness and heat . furthermore , pathogenic wind - cold combined with dampness can invade the meridians and obstruct the flows of qi and blood , bringing about a bi - syndrome due to wind , dampness and cold . if the bi - syndrome persists , the flows of qi and blood will become even more sluggish , giving rise to an obstinate adhesion of turbid phlegm and blood stasis in the joints and meridians , which is marked by pricking pain , nodules , and even deformities of the joints . the prolonged lingering of pathogens will inevitably impair the healthy qi , so a deficiency of spleen yang and kidney yang and fatigue or weakness often occur in the late stages of the disease due to unchecked discharge of essence . cancer metastasis is the spread of cancer cells to tissues and organs beyond where the tumor originated , resulting in the formation of new tumors . the formation of metastases is a major problem in clinical oncology because it is one of the main causes of death in most cancer patients . classical chinese medicine has no specific concept of cancer ; however , experts in tcm are studying the causes and treatments of metastasis . tcm doctors believe the causes of cancer are multiple , including toxins and other environmental factors , called external causes , as well as internal causes such as blood and qi stagnation , emotional stress , bad eating habits , wastes accumulated from food , and damaged organs . they also believe that internal wind of hepatic origin is not only one of the causes of malignant tumors but also the main cause for the formation of metastases . an analysis of the causes of internal wind in patients with malignant tumors suggests that removing wind is the fundamental method of tcm for the treatment of patients with malignant tumors and metastases ; thus , drugs that eliminate wind are important prescriptions for treating patients with such tumors . three main patterns appear in patients with cancer , which are phlegm ( tan ) , toxic heat - fire ( re - huo ) and wind - cold causing qi stagnation and to which are attributed , from the western viewpoint , the concepts of hyperplasia , neoplasia , and metastasis , respectively . the pathogenesis of metastasis caused by pathogenic wind is as follows : tcm holds that pathogen wind is ranked as external wind and internal wind , the first being the most important of the six climatic factors and the second being a pathological manifestation of a disturbance in yang qi . the inner wind that is due to a yin and yang imbalance is called internal wind that rises in the liver and is characterized by change and movement . it is not as strong as external wind and attacks internal organs only when they are weak , resulting in symptoms of internal involvement . pathological changes in the liver can propagate along the meridians and collaterals and attack other internal organs , which is the reason the liver is considered in tcm to be the source of all diseases . in tcm , tumors are believed to be the result of zheng qi failure , internal organ dysfunction , and pathogen accumulation in the meridians , collaterals , and internal organs , which can cause an imbalance between yin and yang , qi stasis , and blood stasis , the production of tan , the accumulation of toxins and , eventually , the formation of a tumor . tumor formation is accompanied by internal wind , which , in turn , helps liver wind , leading to blood stasis , phlegm , and internal organ toxins , resulting in the formation of metastases in the lungs , liver , brain , lymphatic system , bone , or skin . diseases caused by wind are also characterized by a rapid onset and change , which explains why cancer patients are already in middle or an advanced stage of the disease when they receive the diagnosis . a small portion of patients with cancer in an early phase can quickly move to end - stage cancer with the formation of metastases in a short time . the imbalance of qi , blood , yin , and yang is the leading cause of metastasis . stasis qi , blood stasis , phlegm generation , and retention of toxins are the basic factors of metastasis while the movement of the liver inner wind is the condition under which metastases are formed . drugs that eliminate wind have an anti - cancer effect . in short , in the treatment of cancer , the practitioner needs to pay attention both to the pathogenesis of liver internal wind and to the origin of metastases produced by the liver wind . drugs that disperse wind are an important part of treatments for cancer . according to studies in allopathic medicine for that reason , research both in the field of modern medicine and tcm is focused on finding effective methods for preventing and treating cancer and metastases , on providing new methods for preventing and treating metastases , and on establishing a theoretical basis for the application of drugs that eliminate wind to treat cancer . chinese medicine is useful in all stages of the disease to augment the benefits of conventional treatments , to prevent recurrence and metastasis in early stages of breast cancer , and to promote health , improve the quality of life and prolong life in advanced stages [ 26 - 38 ] . a study on human melanoma cells reported that treatment with essential oils induced deoxyribonucleic acid ( dna ) damage in cancer cells , which is an indicator of apoptosis . modern pharmacological studies have shown that the following drugs eliminate wind and have an anticancer effect [ 40 - 42 ] : dang gui ( radix angelicae sinensis ) , angelica pubescens ( du huo ) , bupleurum chinese ( chai hu ) , pueraria lobata ( ge gen ) , vitex rotundifolia ( ma jing zi ) , arcticum lappa ( niu bang zi ) , stephania tetandra ( fang ji ) , aristolochia mollissima ( xun gu feng ) , pinus tabuleaformis ( song jie ) , viscum coloratum ( sang ji sheng ) , acanthopanax gracilistylus ( wu jia pi ) , lagenaria chaenomeles ( mu gua ) , erodium stephanianum ( lao guan cao ) , chloranthus glaber ( zhong jie feng ) , agkistrodon acutus ( bai hua she ) , scorpio ( quan xie ) , scolopendra ( wu gong ) , gekko japonicus ( bi hu ) , agkistrodon halys ( fu she ) , smilax glabra ( tu fu ling ) , solanum lyratis ( bai ying ) , arisaema consanguineum ( tian nan xing ) , acorus grass ( shi chang pu ) , sophora flavescens ( ku shen ) , dictamnus dasycarpus ( bai xian pi ) , polygonum multiflorum ( he shou wu ) , ligusticum chuanxiong ( chuan xiong ) , zanthoxylum nitidum ( lian mian zhen ) , campsis grandiflora ( ling xiao hua ) , dwarf musk deer ( she xiang ) , taurus domesticus ( niu huang ) , tribulus terrestris ( bai ji li ) , pheretima aspergilum ( di long ) , paris polyphylla ( chong lou ) , amyda sinensis ( bie jia ) and bombyx mori ( bai jian can ) . among compounds derived from plants , essential oils from aromatic plants have been reported to possess anticancer properties . all tcm practitioners should be very familiar with the herbs and acupuncture points related to treating patients with the conditions caused by wind . table 3 gives a partial listing of well - known herbs that are reputed to dispel wind . the actions and the indication of the herbs are from the practical dictionary of chinese medicine with only those aspects that are directly or indirectly related to the concept of wind included . the uses of wind - dispelling herbs are various , but mainly include the treatment of patients with pain , stroke and paralysis , spasms , skin disorders , and a variety of acute diseases involving infection or inflammation affecting the upper part of the body . table 4 gives a partial listing of acupuncture points well - known to be wind - dispelling points . the concepts of wind in traditional medicine books have gone beyond the old established manuscript boundaries and are widely incomprehensible . this study intended to provide information , context , and guidance for a collection of all important subjects and simplification concepts for wind . this new vision for understanding earlier chinese medicine is applicable for public health specialists , traditional and complementary medicine practitioners , and those who are interested in historical medicine and provides a theoretical basis for herbal drugs or acupuncture administration to eliminate wind in order to treat various diseases .

the use of folk medicine has been widely embraced in many developed countries under the name of traditional , complementary and alternative medicine ( tcam ) and is now becoming the mainstream in the uk and the rest of europe , as well as in north america and australia . diversity , easy accessibility , broad continuity , relatively low cost , base levels of technological inputs , fewer side effects , and growing economic importance are some of the positive features of folk medicine . in this framework , a critical need exists to introduce the practice of folk medicine into public healthcare if the goal of reformed access to healthcare facilities is to be achieved . the amount of information available to public health practitioners about traditional medicine concepts and the utilization of that information are inadequate and pose many problems for the delivery of primary healthcare globally . different societies have evolved various forms of indigenous perceptions that are captured under the broad concept of folk medicine , e.g. , persian , chinese , grecian , and african folk medicines , which explain the lack of universally accepted definitions of terms . thus , the exchange of information on the diverse forms of folk medicine needs to be facilitated . various concepts of wind are found in books on traditional medicine , and many of those go beyond the boundaries established in old manuscripts and are not easily understood . this study intends to provide information , context , and guidance for the collection of all important information on the different concepts of wind and for their simplification . this new vision for understanding earlier chinese medicine will benefit public health specialists , traditional and complementary medicine practitioners , and those who are interested in historical medicine by providing a theoretical basis for the traditional medicines and the acupuncture that is used to eliminate wind in order to treat various diseases .

1. Introduction 2. Pernicious influence factor pathogens 3. Reason pernicious influence factor pathogens are called evils? 4. Zhong Feng (Wind Strike) 5. Tong Feng (painful Wind; white tiger arthritis) or gout 6. Wind and cancer metastasis in traditional Chinese medicine 7. Conclusion

in recent years , advances in neuroimaging and surgical interventions have significantly decreased neurosurgical mortality and morbidity . however , postoperative central nervous system infection ( pcnsi ) remains one of the main reasons for poor prognosis in patients after neurosurgery . interestingly , the american national nosocomial infections surveillance system assessed a total of 43,135 neurosurgical cases between 1992 and 2004 and found the prevalence of intracranial infection to be about 3%.1 indeed , pcnsi can worsen the underlying disease , delay postoperative recovery of the neurological function , and even cause disability or death . another study in the people s republic of china determined the incidence of postoperative intracranial infection after neurosurgery to be between 2.89% and 5.35%.2 bacterial meningitis represents the main central nervous system infection , and it is associated with relatively high mortality and morbidity.3 postneurosurgical bacterial meningitis ( pnbm ) is a potentially life - threatening complication of neurosurgery.4 it has been shown to display the highest incidence among all pcnsis in epidemiological studies.5,6 importantly , bacterial meningitis is one of the most dangerous postneurosurgical complications;4 to some degree , mortality and morbidity due to this infection are affected by the pathological characteristics of the primary lesion , diagnosis time , and administration of specific antimicrobial drugs.7 previous studies have focused mainly on risk factors for pnbm , including the site of the lesion , history of meningitis , surgery that entered a sinus , and increased american society of anesthesiologists score,8,9 rather than the effects of patients clinical characteristics on the pnbm treatment course . in this study , we retrospectively analyzed the clinical data of patients who underwent neurosurgery in our hospital in people s republic of china and carried out a series of statistical analyses to identify the risk factors affecting the duration of the pnbm treatment course , in order to obtain new insights that may prove beneficial for pnbm prevention and treatment . a total of 7,458 patients underwent neurosurgery between january and december 2012 in the neurosurgery department of beijing tiantan hospital , affiliated hospital of capital university of medical sciences , beijing , people s republic of china . of these patients , 283 ( 3.8% ) underwent neurosurgery for benign or malignant tumors and were confirmed as having pnbm ; there were 158 males ( 55.8% ) and 125 females ( 44.2% ) aged 42.77.1 years ( range : 874 years ) . this study was approved by the ethics committee of beijing tiantan hospital , affiliated hospital of capital university of medical sciences , beijing , people s republic of china . written informed consent was obtained from each patient included in the study . included in this study were 1 ) patients who underwent neurosurgery in the neurosurgery department of beijing tiantan hospital between january and december 2012 and 2 ) patients with intracranial infection showing clinical manifestations and auxiliary examination features , including high fever , headache , vomiting , and positive meningeal irritation signs . patients with a body temperature of up to 38c were assessed within 24 hours by lumbar puncture ; routine blood examinations ; bacterial , fungal , and viral cultures of blood and cerebrospinal fluid ( csf ) ; and specific pathogen antibody titration assays of blood and csf samples . intracranial infection was diagnosed based on csf white blood cell ( wbc ) count > 100/mm , neutrophil granulocyte percentage > 80% , csf glucose < 40 mg / dl , csf protein > 100 mg / dl , and peripheral wbc count > 1010/l.10 regular lumbar punctures and laboratory examinations were carried out during the course of intracranial infection treatment . patients who had 1 ) incision infections , 2 ) brain or subdural abscesses , 3 ) head or spinal injuries , and 4 ) preoperative fever or elevated peripheral blood wbc count were excluded from this study . the following characteristics were obtained before intracranial infection treatment was decided : 1 ) normal body temperature , no headaches , no vomiting , and no meningeal irritation signs ; 2 ) normalization of csf wbc count as well as protein and glucose levels ; and 3 ) normalization of peripheral blood wbc count . the treatment duration was defined as the time elapsed between diagnostic confirmation of intracranial infection and treatment termination . in order to assess the patient characteristics that were associated with the requirement for prolonged treatment duration of intracranial infection , the 283 patients included in this study ( and confirmed as having pnbm ) were subdivided into two groups for analysis : patients requiring long - term treatment , defined as 15 days , and those requiring short - term treatment , defined as 14 days . the use of 14 days as the cutoff point to differentiate between short- and long - term treatments was based on our observation that the mean treatment duration in the 283 patients was 13.52.1 days and on previous studies of intracranial infections that utilized 14 days to distinguish between treatment durations.11,12 statistical techniques were employed to determine the effects of sex , age , history of hypertension , history of diabetes mellitus , csf culture , csf pressure , csf wbc count , csf protein content , csf glucose level , csf culture results , postoperative drainage , and the nature of the lesion on treatment duration . the data were analyzed using the spss 17.0 software ( spss inc , chicago , il , usa ) . categorical data were compared using the test , while intragroup comparisons of continuous data were made using the student s t - test . initially , one - way analysis of variance was used to identify factors showing significant differences between patient groups . these factors were then subjected to multivariate stepwise logistic regression analysis to determine the independent predictors of prolonged treatment duration . a backward selection method was used to identify variables with a p - value < 0.1 for inclusion into the model , and the standardized regression coefficient was calculated . the treatment duration ( 14 days input as 1 , 15 days as 2 ) was considered the dependent variable , and the risk factors were treated as independent variables . a total of 7,458 patients underwent neurosurgery between january and december 2012 in the neurosurgery department of beijing tiantan hospital , affiliated hospital of capital university of medical sciences , beijing , people s republic of china . of these patients , 283 ( 3.8% ) underwent neurosurgery for benign or malignant tumors and were confirmed as having pnbm ; there were 158 males ( 55.8% ) and 125 females ( 44.2% ) aged 42.77.1 years ( range : 874 years ) . this study was approved by the ethics committee of beijing tiantan hospital , affiliated hospital of capital university of medical sciences , beijing , people s republic of china . written informed consent was obtained from each patient included in the study . included in this study were 1 ) patients who underwent neurosurgery in the neurosurgery department of beijing tiantan hospital between january and december 2012 and 2 ) patients with intracranial infection showing clinical manifestations and auxiliary examination features , including high fever , headache , vomiting , and positive meningeal irritation signs . patients with a body temperature of up to 38c were assessed within 24 hours by lumbar puncture ; routine blood examinations ; bacterial , fungal , and viral cultures of blood and cerebrospinal fluid ( csf ) ; and specific pathogen antibody titration assays of blood and csf samples . intracranial infection was diagnosed based on csf white blood cell ( wbc ) count > 100/mm , neutrophil granulocyte percentage > 80% , csf glucose < 40 mg / dl , csf protein > 100 mg / dl , and peripheral wbc count > 1010/l.10 regular lumbar punctures and laboratory examinations were carried out during the course of intracranial infection treatment . patients who had 1 ) incision infections , 2 ) brain or subdural abscesses , 3 ) head or spinal injuries , and 4 ) preoperative fever or elevated peripheral blood wbc count were excluded from this study . the following characteristics were obtained before intracranial infection treatment was decided : 1 ) normal body temperature , no headaches , no vomiting , and no meningeal irritation signs ; 2 ) normalization of csf wbc count as well as protein and glucose levels ; and 3 ) normalization of peripheral blood wbc count . the treatment duration was defined as the time elapsed between diagnostic confirmation of intracranial infection and treatment termination . in order to assess the patient characteristics that were associated with the requirement for prolonged treatment duration of intracranial infection , the 283 patients included in this study ( and confirmed as having pnbm ) were subdivided into two groups for analysis : patients requiring long - term treatment , defined as 15 days , and those requiring short - term treatment , defined as 14 days . the use of 14 days as the cutoff point to differentiate between short- and long - term treatments was based on our observation that the mean treatment duration in the 283 patients was 13.52.1 days and on previous studies of intracranial infections that utilized 14 days to distinguish between treatment durations.11,12 statistical techniques were employed to determine the effects of sex , age , history of hypertension , history of diabetes mellitus , csf culture , csf pressure , csf wbc count , csf protein content , csf glucose level , csf culture results , postoperative drainage , and the nature of the lesion on treatment duration . the data were analyzed using the spss 17.0 software ( spss inc , chicago , il , usa ) . categorical data were compared using the test , while intragroup comparisons of continuous data were made using the student s t - test . initially , one - way analysis of variance was used to identify factors showing significant differences between patient groups . these factors were then subjected to multivariate stepwise logistic regression analysis to determine the independent predictors of prolonged treatment duration . a backward selection method was used to identify variables with a p - value < 0.1 for inclusion into the model , and the standardized regression coefficient was calculated . the treatment duration ( 14 days input as 1 , 15 days as 2 ) was considered the dependent variable , and the risk factors were treated as independent variables . patients clinical characteristics , csf culture results , and data obtained by pathological examination of the intracranial lesions are shown in table 1 . the initial clinical manifestations of pnbm included fever , headache , vomiting , decreased consciousness , seizures , and signs of meningeal irritation . laboratory examinations of csf obtained by lumbar puncture identified the causative organisms in 35 of the 283 included patients ( 12.4% ) as methicillin - resistant staphylococcus aureus , acinetobacter baumannii , pseudomonas aeruginosa , escherichia coli , and staphylococcus epidermidis . after pnbm diagnosis , 60 patients were treated with a single antibiotic , while 223 participants received a combination of two or more antibiotics . in patients in whom the causative organism was identified , the antibiotic regimen was adjusted according to the csf culture results . successful treatment of intracranial infection was achieved in 282 of the 283 patients , with a treatment duration of 13.52.1 days . one patient died ( a mortality rate of 0.4% ) ; subsequent pathologic examinations revealed intracranial metastasis of small cell lung cancer and the cause of death was respiratory failure after the surgery . to determine which characteristics were associated with pnbm treatment duration , patients were subdivided into two groups that had received treatment for 15 days ( long treatment ) and 14 days ( short treatment ) , respectively . this grouping was based on the mean treatment duration of 13.52.1 days obtained for these patients and it was consistent with those in previous studies of bacterial meningitis.11,12 as shown in tables 2 and 3 , single - factor analysis revealed significantly higher proportions of males ( p=0.042 ) and patients with malignant lesions ( p<0.001 ) in the long - term treatment group compared with those who received short - term treatment . however , age , history of hypertension , history of diabetes , csf culture , csf pressure , csf wbc count , csf protein content , csf glucose level , postoperative drainage , and lumbar cistern drainage were not significantly different between both groups . patient sex , the nature of the lesion ( benign or malignant ) , csf pressure , and csf culture were selected as independent variables for the multivariate logistic regression analysis . as shown in table 4 , the nature of the lesion constituted the risk factor significantly affecting the treatment duration ( odds ratio : 2.5962 , 95% confidence interval : 1.10926.6134 ) , with the presence of malignant lesions requiring a long treatment course . patients clinical characteristics , csf culture results , and data obtained by pathological examination of the intracranial lesions are shown in table 1 . the initial clinical manifestations of pnbm included fever , headache , vomiting , decreased consciousness , seizures , and signs of meningeal irritation . laboratory examinations of csf obtained by lumbar puncture identified the causative organisms in 35 of the 283 included patients ( 12.4% ) as methicillin - resistant staphylococcus aureus , acinetobacter baumannii , pseudomonas aeruginosa , escherichia coli , and staphylococcus epidermidis . after pnbm diagnosis , 60 patients were treated with a single antibiotic , while 223 participants received a combination of two or more antibiotics . in patients in whom the causative organism was identified , the antibiotic regimen was adjusted according to the csf culture results . successful treatment of intracranial infection was achieved in 282 of the 283 patients , with a treatment duration of 13.52.1 days . one patient died ( a mortality rate of 0.4% ) ; subsequent pathologic examinations revealed intracranial metastasis of small cell lung cancer and the cause of death was respiratory failure after the surgery . to determine which characteristics were associated with pnbm treatment duration , patients were subdivided into two groups that had received treatment for 15 days ( long treatment ) and 14 days ( short treatment ) , respectively . this grouping was based on the mean treatment duration of 13.52.1 days obtained for these patients and it was consistent with those in previous studies of bacterial meningitis.11,12 as shown in tables 2 and 3 , single - factor analysis revealed significantly higher proportions of males ( p=0.042 ) and patients with malignant lesions ( p<0.001 ) in the long - term treatment group compared with those who received short - term treatment . however , age , history of hypertension , history of diabetes , csf culture , csf pressure , csf wbc count , csf protein content , csf glucose level , postoperative drainage , and lumbar cistern drainage were not significantly different between both groups . patient sex , the nature of the lesion ( benign or malignant ) , csf pressure , and csf culture were selected as independent variables for the multivariate logistic regression analysis . as shown in table 4 , the nature of the lesion constituted the risk factor significantly affecting the treatment duration ( odds ratio : 2.5962 , 95% confidence interval : 1.10926.6134 ) , with the presence of malignant lesions requiring a long treatment course . the following main findings were obtained in this analysis : methicillin - resistant s. aureus , a. baumannii , and p. aeruginosa were the three most common pathogens identified in csf cultures from patients with pnbm ; the group of patients treated for 15 days contained more males and showed higher incidence of malignant lesions compared with the short - term treatment group ; the presence of a malignant lesion was an independent predictor of the requirement for a long - term treatment course ( 15 days ) in pnbm . thus , clinicians should be aware that postneurosurgery , intracranial infections in patients with malignant lesions may require longer duration of antibiotic therapy . csf culture is considered the gold standard for the diagnosis of bacterial meningitis.3 according to a study by pizon et al13 csf staining and culture provide the diagnostic basis in 60%90% of patients with meningitis . previous studies have shown that prophylactic use of antibiotics can effectively reduce the incidence of pcnsi.14,15 however , such prophylaxis will also decrease the rate of successful growth and identification of organisms in blood and csf cultures.16 all the patients assessed herein received antibiotic prophylaxis prior to surgery , which likely explains the low rate of organism growth in csf cultures ( 12.4% ) . hence , in situations where antibiotic prophylaxis is used , other indices can help in diagnosing bacterial meningitis , including csf glucose level , lactate content , and cell classification.17 in addition , 99% of patients with pnbm show clinical manifestations such as headache , fever , signs of meningeal irritation , and decreased consciousness.18 therefore , the patient s clinical symptoms and signs , combined with laboratory results , provide sufficient evidence for the diagnosis of bacterial meningitis.19 pnbm differs from community - acquired bacterial meningitis in terms of the susceptible population and pathogen characteristics.20,21 we have shown herein that methicillin - resistant s. aureus , a. baumannii , and p. aeruginosa are the three most common strains in csf cultures , consistent with previous findings.22 for patients with negative csf culture results , the antibiotic regimen selection for empirical therapy should be done in accordance with the epidemiological characteristics of the pathogens . therefore , cefazolin , ceftriaxone , or meropenem , combined with vancomycin or linezolid , would be expected to effectively cover the common bacterial spectrum of pnbm . the patients assessed herein were closely monitored after surgery for signs of fever , decreased consciousness , or meningeal irritation . any patient suspected with pnbm was assessed within 24 hours by lumbar puncture and laboratory tests , which included csf cytology , biochemistry , staining , culture , and antibody assays . patients in whom the causative organism could not be identified were treated with empirical antibiotic therapy , according to clinicians experience and relevant research data.23 the treatment options included single - antibiotic ( third - generation cephalosporin or meropenem ) or combination therapy ( third - generation cephalosporin or meropenem combined with vancomycin ) . during the course of treatment , the regimen was adjusted according to the etiology ( ie , identification of the causative organism ) . several studies have shown that early diagnosis and treatment can significantly improve the prognosis of bacterial meningitis.24,25 the clinicians involved in this study were fully aware that bacterial meningitis is an important potential postoperative complication and that once pnbm is diagnosed , antibiotic therapy should be administered as soon as possible ; this would have effectively improved the prognosis of the patients in this study.26 this may also explain the low mortality rate observed ( only one patient died , due to respiratory insufficiency ) . the average duration of treatment in this study was 13.52.1 days , comparable to values reported previously in similar studies involving bacterial meningitis.11,12 therefore , we selected 14 days as the cutoff point and subdivided the patients into two groups , one composed of patients treated beyond average duration ( 15 days ) , and the other made up of individuals treated with average duration or less ( 14 days ) . our statistical analyses revealed that the treatment duration required in patients with pnbm was longer in those with intracerebral malignant lesions compared with individuals having benign lesions . a possible explanation is that there may be differences in postoperative immune states between patients with malignancies and those with benign lesions . studies of postoperative immune function in patients with meningioma or glioma have demonstrated that all patients show immunosuppression after neurosurgery ; however , although the immune function of patients with benign lesions began to recover 23 days after surgery ( characterized as increased lymphocyte activity ) , patients with malignant lesions exhibited postoperative immunosuppression for 710 days.27 in addition , lymphocyte activity was significantly lower after surgery compared with the preoperative period , despite a notably increased count.28 t - helper cell and eosinophil levels were found to be lower in patients with intracranial malignant tumors ( world health organization grade ii iv glioma ) than in those with benign lesions , while immunoglobulin ( ig ) m and neutrophil levels were higher.29 previous studies have also reported that postoperative serum iga and igg levels were significantly reduced in patients with intracranial malignant tumors.30 thus , the requirement for a long duration of pnbm treatment observed in patients with malignant tumors may be secondary to the malignancy s immunosuppressive effects . immunosuppression in patients with intracranial malignant tumors may be related to increased levels of cytokines with immunosuppressive function , including interleukin-10 and transforming growth factor-1 , along with decreased interleukin-12 level.31,32 the secretion of prostaglandin e2 is increased in patients with intracranial malignant tumors , possibly mediating the inhibitory effects of monocytes and macrophages on t - cells . brain barrier may also be reduced by tumor cells , further suppressing the immune function in patients with malignant intracranial lesions.33 interestingly , studies at the molecular level have suggested that the postoperative immune function of patients with glioma was significantly lower in those whose tumors retained chromosome 10q than in individuals with allelic 10q loss ; importantly , patients without 10q allelic loss were also more likely to have postoperative infection.34 the present study has described the common pathogens associated with pnbm , outcomes , and factors associated with treatment duration ; these findings may prove beneficial for disease prevention and treatment . several studies have reported the clinical characteristics of pnbm.35,36 however , we assessed a larger sample size ( 283 cases ) , systematically investigated the characteristics of postneurosurgery meningitis , and analyzed the factors affecting the duration of treatment . in addition , this study was carried out in a chinese population , in mainland people s republic of china ; to our knowledge , no previous reports exist on clinical characteristics and treatment of pnbm in a chinese population , although a few studies in taiwanese patients have been published.35,36 furthermore , we found for the first time that intracranial malignant lesions represent an independent risk factor for prolonged treatment duration . pnbm is an important complication of neurosurgery , but the prophylactic use of antibiotics decreases the rate of positive csf cultures in patients with intracranial infections . therefore , pnbm diagnosis requires a comprehensive consideration of the signs , symptoms , and results from laboratory tests of the csf . furthermore , intracranial malignant lesions represent an independent risk factor for the prognosis of pnbm treatment .

aimour aim was to study the clinical features of postoperative meningitis after neurosurgery and identify the factors affecting the duration of treatment.methodsthis retrospective study assessed 283 patients with postoperative bacterial meningitis in the neurosurgery department of beijing tiantan hospital , beijing , people s republic of china , between january and december 2012 . patients clinical data were reviewed , and multivariate logistic regression analysis was used to identify the factors associated with a prolonged treatment course.resultsthe mortality rate was 0.4% in these patients , of whom 12.4% were found with pathogens in the cerebrospinal fluid . among the three most common pathogens detected were methicillin - resistant staphylococcus aureus , acinetobacter baumannii , and pseudomonas aeruginosa . the mean treatment course was 13.52.1 days . interestingly , the treatment duration for postoperative meningitis was significantly longer in patients with intracranial malignant tumors than in those with benign lesions . single - factor analysis showed that male sex ( p=0.042 ) and malignant ( rather than benign ) lesions ( p<0.001 ) were significantly associated with prolonged treatment duration . multivariate analysis further confirmed that malignant intracranial lesions represented an independent risk factor for prolonged treatment duration ( odds ratio : 2.5962 ; 95% confidence interval : 1.10926.6134).conclusionthe nature of the intracranial lesion is an independent risk factor for the duration of treatment in postoperative meningitis after neurosurgery .

Introduction Materials and methods Patients Inclusion criteria Exclusion criteria Determination of treatment duration Statistical analysis of the factors associated with prolonged treatment duration Results Patient characteristics Single-factor analysis of characteristics associated with PNBM treatment duration Multivariate logistic regression analysis of the factors associated with PNBM treatment duration Discussion Conclusion

larsenico ( as ) un metalloide associato a diverse rocce , specialmente a minerali contenenti solfuro , rivelandosi molto mobile nellambiente : lerosione delle rocce converte infatti i solfuri di as in ossido arsenioso che entra nel ciclo ambientale contaminando i corpi idrici e , attraverso la catena alimentare , distribuendosi diffusamente in tutto il regno vegetale e animale . si tratta quindi di un elemento ubiquitario , gi naturalmente presente nei suoli , nelle acque , nei sedimenti e negli organismi . dallavvento della metallurgia , ai livelli di fondo dellas ambientale si sono aggiunti quelli antropogenici , in quanto esso rappresenta anche un sottoprodotto nellestrazione dei metalli , mentre il contributo antropogenico recente alle concentrazioni di as ambientale derivato soprattutto dalluso di pesticidi a base di suoi composti . va perci sottolineato come la concentrazione di as nella crosta terrestre presenti notevoli variazioni geografiche , anche dipendenti dal tipo di rocce , ed nota limportanza di conoscere i processi che controllano la mobilit e la speciazione dellas nei suoli , nelle acque e nel biota . spesso , infatti , la forma o specie chimica di un determinato elemento chimico importante quanto la sua quantit e ci vale in particolar modo per las , in quanto la pericolosit per luomo delle sue diverse forme varia in modo davvero considerevole ( sharma e sohn , 2009 ) . la valenza sanitaria del problema ancora pi evidente dal quadro che ci viene fornito dalla dinamica delle diverse forme chimiche dellas in ambiente acquatico marino . las disciolto in acqua di mare si trova infatti prevalentemente in forma di arsenato [ as(v ) ] , mentre larsenito [ as(iii ) ] , potenzialmente pi tossico e cancerogeno , rappresenta raramente pi del 20% ( neff , 1997 ) dellarsenico totale ( astot ) . oltre alle forme inorganiche , las in ambiente marino presente in varie forme organiche , a partire da quelle mono- , di- e tri - metilate ( figura 1 ) . le alghe marine assorbono facilmente larsenato dallacqua ed in esse si riscontra la presenza di diversi composti organici arseno - ribosidici . tali composti , spesso indicati col termine generico di arsenozuccheri ( figura 1 ) , rappresentano un anello importante nel ciclo biochimico dellas in ambiente marino ( kirby et al . , 2005 ) . costituiscono infatti la forma prevalente di as nelle alghe ( circa della frazione di as ) , ma sono presenti in quantit significative anche nei molluschi marini , in particolare nei gasteropodi erbivori ( circa della frazione di astot ) e , viceversa , sono presenti in bassissime concentrazioni nei pesci . gli studi sulla tossicit degli arsenozuccheri risultano quindi numerosi e di grande interesse anche in ambito europeo , per il crescente uso alimentare delle alghe , consolidato invece per tradizione nei paesi asiatici . tuttavia , nonostante la notevole mole di dati bibliografici , non ci sono ancora informazioni definitive sulla eventuale tossicit di questi composti ( francesconi , 2010 ) . gli animali acquatici marini ( pesci , crostacei e molluschi acquatici ) hanno invece una capacit limitata di bioconcentrare arsenico inorganico ( asinorg ) dallacqua marina , mentre possono bioaccumulare e biotrasformare tutti i tessuti , sia degli invertebrati che dei pesci marini , contengono infatti concentrazioni molto elevate di as ( neff , 1997 ) , ma questo si trova per lo pi nella forma organica . gli organismi marini sembra infatti che abbiano affrontato evolutivamente il problema sfruttando la ricca chimica organica dellas , per trasformare le forme inorganiche in una gamma di composti organici , fondamentalmente atossici ( francesconi e edmonds , 1998 ) . il composto pi abbondante larsenobetaina , con quantit residuali di arsenocolina ( figura 1 ) . in tutta la copiosa bibliografia scientifica disponibile ( francesconi , 2010 ) , larsenobetaina e larsenocolina sono considerate non tossiche , sebbene la prima sia stata molto pi studiata e ne sia stata verificata linerzia metabolica , mentre sullarsenocolina ( che , comunque costituisce generalmente meno dell1% dellastot presente in organismi marini ) esistono ancora lacune conoscitive . appare chiaro perci come le determinazioni delle sole concentrazioni dellastot nelle diverse matrici ambientali ( sedimento , acqua e soprattutto biota ) non possano , da un lato , costituire un elemento sufficiente per poter valutare il possibile effetto sugli organismi e la dinamica di tale metalloide nellambiente marino - costiero considerato ( srs et al . , 2003 ; fattorini et al . , 2004 ; 2006 ) e , dallaltro , non essere sufficienti per lo svolgimento di una corretta valutazione del rischio umano per esposizione alimentare a prodotti della pesca contaminati da tale metalloide ( de gieter et al . bench le forme inorganiche dellas siano pi tossiche di quelle organiche , la quasi totalit dei dati sulla presenza di as negli alimenti , raccolti nel quadro dei controlli ufficiali in ambito europeo , riportano solo lastot , senza fare distinzioni tra le sue varie forme . poich , proprio nei prodotti della pesca las prevalentemente presente in forme organiche , stata evidenziata la necessit di disporre di dati di speciazione ( efsa , 2009 , 2014 ) , anche in considerazione del fatto che un risk assessment che non tenesse conto di ci , condurrebbe a sovrastimare notevolmente il rischio sanitario . gli organismi marini presentano infatti concentrazioni notoriamente molto alte di astot , ma con una prevalenza di composti innocui ( come larsenobetaina in pesci , crostacei e molluschi ) , o ritenuti tali ( quali gli arsenozuccheri in molluschi ed alghe ) , bioaccumulati secondo un complesso ciclo bio - geochimico ( figura 2 ) . i composti dellas presenti negli ambienti acquatici marini sono quindi diverse decine ed nota la variabilit della capacit dei diversi organismi vegetali e animali , anche in relazione al livello trofico da questi occupato ( kirby e maher , 2002 ) , di bioaccumulare alcuni di questi composti in luogo di altri ( rahman et al . , 2012 ) . in particolare quindi , lanalisi selettiva delle forme inorganiche da quelle organiche dellas a rivestire unimportanza cruciale nella valutazione del rischio legato allesposizione a tale elemento attraverso la dieta , in quanto tali diverse forme chimiche presentano notevolissime differenze nella tossicit ( neff , 1997 ; francesconi , 2010 ) . i composti inorganici dellas sono infatti potenti cancerogeni per luomo e , inoltre , ampiamente dimostrato che lesposizione cronica ad essi determina un aumento nel rischio dinsorgenza di numerose altre patologie non tumorali , come ipercheratosi , ipertensione , diabete , nonch disturbi respiratori , neurologici e cardiovascolari ( saha et al . , 1999 ) . il suo stato di ossidazione : dati di letteratura indicano infatti che las sotto forma di arsenito [ as(iii ) ] da 10 a 60 volte pi tossico della forma di arsenato [ as(v ) ] e da 70 a 100 volte pi tossico delle specie organiche metilate ( borak e hosgood , 2007 ) . quali forme chimiche siano implicate , se organiche o inorganiche , rappresenta quindi il fattore determinante per quanto riguarda la valutazione della tossicit , oltre che la biodisponibilit , dellas ( moreda - pieiro et al . , 2012 ) . ad oggi sarebbero pi di cinquanta le forme di as organico identificate in organismi marini ( francesconi , 2010 ) , nei tessuti dei quali , oltre a tutta la gamma finora descritta , si aggiungono pure i composti lipidici ( sele et al . , 2012 ) . negli oli di pesce stata infatti da tempo rilevata una serie di acidi grassi saturi con la porzione terminale dimetil - arsinoil-(figura 1 ) . la presenza di arsenolipidi in oli di pesce non sembra tuttavia sollevare problemi di tossicit nelluomo : bench essi vengano assorbiti dal tratto gastrointestinale , las infatti rapidamente escreto sotto forma di metaboliti organici idrosolubili ( prevalentemente composti dimetilati ed arsenobetaina ) . in sintesi , per quanto riguarda le forme organiche dellas , sufficientemente assodato come larsenobetaina , che la forma predominante nei prodotti della pesca , non desti alcuna preoccupazione dal punto di vista tossicologico e che , relativamente agli arsenozuccheri e agli arsenolipidi ( poich vengono nelluomo generalmente metabolizzati in dimetilarsinato ) , non siano attualmente disponibili sufficienti informazioni sulleventualit e grado di una qualche loro tossicit residuale . in un precedente studio ( piras e mulas , 2011 ) sul contenuto di metalli pesanti e metalloidi in molluschi bivalvi , crostacei e pesci lagunari campionati nellarea dindagine , la laguna di boi cerbus nella parte sud - occidentale della sardegna ( sulcis - iglesiente ) , erano stati rilevati elevati livelli di as ed erano state avanzate stime per la valutazione del rischio da esposizione alimentare . seguendo lapproccio metodologico armonizzato europeo , che utilizza il parametro tossicologico del provisional tolerable weekly intake fissato dal joint fao / who expert committee on food additives , le concentrazioni di astot riscontrate avrebbero contribuito di 2,15 volte la dose accettabile nella classe det dei bambini e di 1,26 volte in quella dei giovani . trattato ovviamente di stime molto prudenziali , nellipotesi estrema che tutto las rilevato avesse gli effetti tossici dellasinorg . nello scenario di ricerca che si aperto , apparso quindi necessario poter disporre di tecniche analitiche capaci di discriminare , in una consistente gamma di prodotti della pesca , lasinorg dal complesso di composti riferibili alle forme organiche . i metodi disponibili per lanalisi di speciazione si basano fondamentalmente sulla combinazione di tecniche di separazione con quelle di rivelazione . , 2002 ; srs et al . , 2003 ; francesconi e kuehnelt , 2004 ) le tecniche di separazione attualmente pi diffuse sono quelle cromatografiche hplc in combinazione ( o hyphenated applications ) con rivelatori di massa icp . lanalisi di speciazione dellas in campioni marini stata per generalmente eseguita per ricerche ad hoc e finalit scientifiche ( comunque non nel contesto di piani di monitoraggio / sorveglianza ) , ponendosi come obiettivo soprattutto la differenziazione multipla delle specie organiche dellelemento ( hirata et al . , 2006 ; moreda - pieiro et al . , con un approccio che meglio si presta ad obiettivi ed operativit della sanit pubblica , nel presente studio si invece adottata una particolare procedura di preparazione del campione che permette , sempre attraverso tecnica cromatografia liquida ad alta prestazione - spettrometria di massa a plasma accoppiato induttivamente ( high performance liquid chromatography - inductively coupled plasma mass spectrometry ; hplc - icpms ) , unanalisi di speciazione semplificata , ovvero finalizzata alla sola determinazione dellasinorg . originariamente sperimentata in campioni di pesci marini ( larsen et al . , 2005 ) , dopo unulteriore semplificazione e perfezionamento , stata applicata , anche sui molluschi bivalvi e crostacei ( sloth et al . , 2005 ) ed stata recentemente implementata presso il laboratorio controllo chimico e biomonitoraggio della sezione di ancona dellizs - um , dove sono state svolte le analisi di speciazione del presente studio . si quindi voluta sviluppare la ricerca nellarea costiera indagata , con lo scopo di rilevare le frazioni tossiche dellas ( come asinorg / astot x 100 ) su organismi marini sentinella , rappresentati da una diversificata gamma di specie marino - lagunari edibili , ponendo particolare attenzione ad evidenziare eventuali differenze interspecifiche o tra taxa , oltre che tra organismi bentonici ( o a contatto col sedimento ) vs quelli nectonici , ed approfondire le conoscenze sul comportamento dellas in organismi marino - costieri con habitat prossimo ad aree minerarie , come quelle del sulcis - iglesiente , e valutarne limpatto sulla sicurezza alimentare . utilizzando la sopra riferita procedura di speciazione basata su tecnica accoppiata hplc / icp - ms ( sloth et al . , 2005 ) , si studiata la distribuzione dellasinorg ( e sua quantificazione proporzionale sullastot ) in n 80 campioni , riferibili a n 14 specie acquatiche marine eduli ( tabella 1 ) della laguna di boi cerbus pescate / raccolte nel corso di tre calate del : 21 febbraio , 29 aprile e 6 luglio del 2013 . gli organismi acquatici sono stati campionati nella sola parte edibile ed immediatamente refrigerati a + 4c per massimo 3 giorni e infine trasportati in continuit termica al laboratorio dellizs - um , dove sono stati prontamente congelati a -18c ed in breve tempo liofilizzati . la stabilit delle specie chimiche durante la movimentazione e lo stoccaggio del campione prima delle analisi infatti un prerequisito fondamentale per ottenere risultati affidabili , in particolare per le analisi di speciazione , dovendosi evitare , anche in questa fase , che intervengano modifiche nella ripartizione delle varie forme chimiche . il congelamento in laboratorio dei campioni e la loro liofilizzazione in attesa dellanalisi di speciazione stata valutata ( dahl et al lo schema di analisi di speciazione utilizzato ha previsto due procedure autonome : quella per la determinazione della concentrazione di astot ( con tecnica icp - ms ) e , in parallelo , quella per la speciazione dellas ( con tecnica combinata hplc - icp - ms ) . si quindi proceduto alle analisi con una dotazione strumentale specifica costituita da : una pre - colonna ed una colonna hplc ( icsep ion-120 , 4,6 mm , lunghezza 120 mm ) , uno strumento per hplc ( perkin elmer flexar liquid chromatography ) ed uno strumento per icpms ( perkin elmer elan drc - ii ) . laccoppiamento online dellhplc con licpms , attraverso connessione del capillare in uscita dalla colonna cromatografica con la camera di nebulizzazione dellicp - ms , stato gestito tramite pc dotato di specifico software ( chromera ) . per lanalisi dellastot , i sub - campioni sono stati sottoposti a digestione in microonde con 6 ml di hno3 concentrato e 2 ml di h2o2 al 30% . per lestrazione e lanalisi dellasinorg , i sub - campioni sono stati miscelati con una soluzione 9/1 di hcl prima della separazione cromatografia gli estratti sono stati centrifugati ( circa 4000 rpm per 10 minuti ) e filtrati ( 0,45 mm ) . tutti i campioni sono stati analizzati in doppio ( il valore riferito quindi quello medio ) . relativamente allanalisi statistica dei dati , per i confronti temporali stato utilizzato il test non parametrico di kruskal - wallis ( o one - way anova by ranks ) e per i confronti tra gruppi di organismi marini ( per habitat e per taxon ) il test , ancora non parametrico , di mann - witney ( o wilcoxon rank - sum test ) . la tecnica di accoppiamento hplc - icp - ms secondo la procedura della quale ci si avvalsi ( sloth et al . , 2005 ) , applicata anche ad organismi marini eduli della laguna di boi cerbus , ha confermato che i composti chimici dellas presenti in concentrazione di gran lunga superiore sono quelli organici . le concentrazioni delle forme inorganiche sono cos basse da non essere rilevabili o , se lo sono , rappresentano frazioni percentuali sullastot che arrivano al massimo all1% , com il caso dei molluschi bivalvi . come si pu rilevare anche dalla tabella 1 , le concentrazioni relativamente maggiori per lasinorg si hanno nei cuori ( cerastoderma glaucum ) , cui seguono i granchi da moleca ( carcinus aestuarii ) , mentre tutte le altre specie presentano concentrazioni inferiori e tra loro confrontabili . i risultati del presente studio sono coerenti con quanto riporta la letteratura ( neff , 1997 ; de gieter et al . , 2002 ; srs et al . , 2003 ; larsen et al . , 2005 ; sloth et al . , 2005 ; greene e crecelius , 2006 ) che , come si pi volte rimarcato , riferisce valori assoluti e percentuali molto bassi di asinorg per i prodotti della pesca . relativamente allanalisi di speciazione dellas adottata in questo studio , va sottolineato come la fase di estrazione rivesta unimportanza decisiva , considerata la necessit di mantenere la distribuzione originale delle specie chimiche presenti nel campione . ogni conversione delle specie organiche dellas , nettamente predominanti nei prodotti della pesca , in specie inorganiche durante la procedura di pretrattamento del campione comprometterebbe infatti la precisione riferita alla proporzione dellasinorg sul totale . tuttavia , relativamente alla specifica procedura utilizzata ed allobiettivo sotteso , da ritenersi ammissibile la conversione di composti organici dellas in altri composti organici del medesimo elemento . la procedura adottata garantisce infatti sul fatto che , dai dati sperimentali disponibili ( sloth et al . , 2005 ) , non sia stata osservata alcuna conversione delle specie madri di as organico in asinorg , mentre sono state rilevate possibili inter - conversioni tra alcuni dei composti organici dellas , precisando che tale eventualit non influenza il contenuto originario di asinorg . inoltre , col termine di arsenico inorganico ( asinorg ) si intende la somma di as(iii ) e as(v ) poich , durante il trattamento a microonde , avviene la conversione quantitativa della prima forma nella seconda e la frazione di as(iii ) dellacido arsenioso viene tutta ossidata in as(v ) dellacido arsenico . si consideri a tal proposito che la separazione delle specie inorganiche bersaglio [ somma di as(iii ) + as(v ) ] da quelle organiche rappresenta proprio lobiettivo di speciazione che ci si posti , come informazione fondamentalmente utile per le finalit del risk assessment in sanit pubblica . relativamente allanalisi statistica dei dati , lutilizzo del test di kruskal - wallis , al quale si ricorsi per la valutazione di eventuali trends stagionali , non ha evidenziato differenze statisticamente significative , n sulle concentrazioni di astot ( p=0,2549 ) , n sulle percentuali medie di asinorg sul totale ( p=0,0817 ) , fra le tre calate di pesca / raccolta . al fine di valutare leventuale influenza degli habitat , le 14 specie sono state raggruppate in due cluster : gli organismi bentonici ( rappresentati da cuori , murici , granchi , ghiozzi , bavose e sogliole ) vs quelli nectonici ( con latterini , salpe , cefali , lotregani , orate e spigole ) . attraverso lutilizzo del test non parametrico di mann - whitney , la differenza dei livelli medi di astot tra gli organismi bentonici ( 38,535,21 ) vs quelli nectonici ( 4,890,80 ) risultata statisticamente significativa ( p<0,0001 ) . le stesse specie sono state raggruppate in base al taxon in due nuovi cluster : gli invertebrati ( cuori , murici e granchi ) vs i vertebrati ( le restanti specie , rappresentate da pesci teleostei ) . sempre ricorrendo al test non parametrico di mann - whitney , anche la differenza dei livelli medi di astot tra gli invertebrati ( 52,567,46 ) vs i vertebrati ( 9,751,33 ) risultata statisticamente significativa ( p<0,0001 ) . i dati al momento disponibili , non stato per possibile scomporre il peso attribuibile alla differenza di habitat piuttosto che a quella di taxon ma , plausibilmente , entrambi i fattori sono da ritenersi dei determinanti causali significativi . relativamente ai livelli medi percentuali di asinorg sul totale tra gli organismi bentonici ( 0,210,04% ) vs quelli nectonici ( 0,020,01% ) , sempre ricorrendo al test non parametrico di mann - whitney , la differenza non risultata statisticamente significativa ( p=0,1038 ) , mentre la differenza dei livelli medi percentuali di asinorg sul totale tra gli invertebrati ( 0,310,06% ) vs i vertebrati ( 0,030,01% ) risultata statisticamente significativa ( p=0,0002 ) . tale ultimo riscontro , considerata lampia premessa svolta sulla chimica e sullecologia dellas negli ambienti marini , pu trovare una plausibile ipotesi giustificativa sempre nelle differenze intrinseche , biologiche e filogenetiche , esistenti tra gli invertebrati acquatici ed i pesci , e verosimilmente trovare spiegazione , pur non disponendo col presente studio di evidenze con una sufficiente significativit statistica , anche nelle marcate differenze di habitat tra i due gruppi di organismi indagati ( che vede gli invertebrati occupare permanentemente il benthos , mentre i pesci si presentano naturalmente pi mobili e tendenti ad occupare , seppur in modo differenziato , tutta la colonna dacqua , dominio del necton ) . si consideri a tale proposito che la maggior attitudine allaccumulo , in termini quantitativi , dellasinorg in ambienti marini da parte degli invertebrati rispetto ai vertebrati stata descritta ed indagata gi da tempo , presupponendo il fatto che lelemento possa essere accumulato a partire sia dallacqua , che dal cibo o dai sedimenti , secondo meccanismi differenti a seconda dei taxa . infatti , non solo le alghe , ma anche i molluschi ed i crostacei ( ma scarsamente o per nulla i pesci ) sono ritenuti capaci di accumulare arseniato direttamente dallacqua di mare ( maher e butler , 1988 ) , secondo modelli di assorbimento proporzionale alla sua concentrazione , almeno fino ad un valore soglia ( oltre il quale lassorbimento di arseniato sarebbe inibito o , comunque , indipendente dalla sua concentrazione nellacqua marina ) . quando invece lelemento si accumula attraverso il cibo , le differenze di concentrazione tra le diverse specie marine ( e nelle stesse specie in ambienti diversi ) possono essere direttamente correlate alla dieta , tenuto conto che lassorbimento dellastot per via alimentare risulta essere la modalit pi efficiente e che , in tale forma di assunzione , i composti organici dellelemento vengono assimilati a preferenza dellasinorg ( maher e butler , 1988 ) . le forme organiche dellarsenico , come ampiamente stato riferito nella premessa , si ritiene siano infatti il prodotto della mesofauna associata ad alghe e bioti nei loro habitat naturali ( edmonds e francesconi , 1987 ) . un altro aspetto da considerare , anche se il fenomeno del bioaccumulo dellelemento si verifica a tutti i livelli trofici , il fatto che non viene rilevata la sua biomagnificazione verso i livelli alti della catena alimentare ( maher e butler , 1988 ) . a ci si aggiunga come siano i sedimenti marini a rappresentare il pi grande serbatoio di asinorg nei sistemi acquatici e che le concentrazioni di tale forma chimica dellelemento possano diventare elevate in alcuni estuari e aree costiere adiacenti a zone industriali o ad aree minerarie e metallurgiche ( maher e butler , 1988 ) . nei sedimenti marini las si trova infatti soprattutto in forma inorganica , come arsenato o arsenito , associato principalmente agli ossidi / idrossidi di ferro e manganese ( sharma e sohn , 2009 ) e , anche nei bassi fondali lagunari , i sedimenti non sono stati descritti capaci di svolgere un ruolo primario nel condizionare la qualit dellacqua sovrastante in relazione ai tenori di asinorg . per contro , le correlazioni delle concentrazioni di asinorg nei tessuti di organismi bentonici , bivalvi in particolare , rispetto a quelle dei sedimenti hanno consentito di dimostrare ( maher e butler , 1988 ) la capacit di tali organismi acquatici di ingerire una frazione di particolato legata allasinorg nei sedimenti medesimi . i risultati di questo preliminare studio che , ai fini di un corretto risk assessment , rappresentano un utile contributo alla conoscenza dei livelli naturali e della speciazione chimica dellas negli organismi marini eduli presenti in zone costiere prossime a siti industriali e ad aree minerarie dismesse , nel contempo suggeriscono la necessit di uno sviluppo delle indagini sulla portata del fenomeno e sulle sue implicazioni nelle dinamiche ambientali e sul destino metabolico dellarsenico inorganico nei diversi organismi marini eduli ivi presenti .

the boi cerbus lagoon , facing a mining and industrial site in sardinia ( italy ) , is an important fishing area for the local population . previous studies showed high concentrations of total arsenic ( astot ) in fish , molluscs and crustaceans sampled in the lagoon , and a possible exceeding of the provisional tolerable weekly intake set by the joint fao / who expert committee on food additives by some local consumer groups . however , the percentage of inorganic as ( asinorg ) should be known for a correct assessment of potential risk , as its toxicity is much higher than that of the organic forms . eighty samples of 14 different species of fish , molluscs and crustaceans , sampled in the boi cerbus lagoon in 3 different seasons ( winter , spring and summer ) , were analysed for astot by inductively coupled plasma mass spectrometry ( icp - ms ) and asinorg by high performance liquid chromatography - icp - ms . all the data obtained from the analysis were statistically processed to evaluate significant differences based on season , taxon and habitat , in preparation for a subsequent risk assessment .

Introduzione Materiali e Metodi Risultati Discussione Conclusioni

exposure of the esophagus to caustic material can lead to necrosis , ulceration , fibrosis , and ultimately stricture . balloon and bougie dilation have similar efficacies for treating these strictures , although stricture recurrence after balloon dilation can occur in up to 30% of these cases . furthermore , a long - term response can not be guaranteed in cases of non - peptic strictures , and symptomatic recurrence frequently occurs within 1 year after the initial dilation . thus , caustic esophageal strictures occasionally require a long - term treatment option , and one case report described endoscopic incisional therapy for a patient with a refractory caustic esophageal stricture . we report a case of an intractable caustic esophageal stricture that was successfully managed with sequential treatment that comprised incision with an insulated - tip knife , balloon dilation , and an oral steroid . a 58-year - old woman presented with severe swallowing difficulty , because of an esophageal stricture that was caused by accidental exposure to anhydrous acetic acid at infancy . the patient reported that she had to manually compress the site of the stricture to swallow solid foods , such as meat , and she had grade 2 dysphagia . she was originally treated using bougie dilation 22 years before the present admission , and we had attempted four balloon dilations during the last 10 years , although the esophageal stricture remained intractable ( figs . 2 , 3 ) . therefore , we performed incisional therapy parallel to the longitudinal axis of the esophagus using an insulated - tip electrosurgical knife ( itknife nano , kd-612l / u ; olympus , tokyo , japan ) . the electrical current was generated using an icc200 unit ( erbe elektromedizin gmbh , tubingen , germany ) , which was set to the pure cut current ( endocut mode ; effect 1 , 60 w ) . this treatment dilated the lumen ( figs . 4 , 5 ) , and her symptoms markedly improved on the next day . thus , we added two sessions of balloon dilation , and the balloon was inflated to a diameter of 9 mm at 4.5 atm for 30 seconds . however , her symptoms worsened , and 2 days later , she could not swallow water because of odynophagia . we performed esophagogastroduodenoscopy and chest computed tomography , which revealed severe wall thickening at the stricture . therefore , we started oral prednisolone ( 20 mg / day ) , and her symptoms markedly improved by the 2-month follow - up ( grade 1 dysphagia ) . approximately one - third of patients who experience a caustic esophageal injury subsequently develop an esophageal stricture and the resulting dysphagia can occur 2 weeks after the injury . the cardinal feature of a caustic stricture is dysphagia because of esophageal narrowing or motility abnormalities , which are closely related to the scarred segment . more than 80% of caustic strictures can be successfully treated with dilation , and bougie and balloon dilators are equally effective for treating benign lower esophageal strictures . however , approximately one - third of these cases subsequently develop recurrent dysphagia within 1 year after dilation . consequently , patients with caustic strictures require a median of five sessions to achieve adequate dilation , compared to three sessions for patients with peptic strictures . unfortunately , a long - lasting response after dilation can not be guaranteed for non - peptic strictures , and the treatment s effect and re - treatment frequency often depends on the stricture s etiology , length , and severity . we classify esophageal strictures as simple or complex , and the complex strictures are more difficult to treat and tend to be refractory or recurrent , despite dilation therapy . caustic strictures typically progress to the complex type , and strictures that are long ( > 2 cm ) , angulated , irregular , or severely narrowed tend to be refractory or recurrent , despite dilation therapy . incisional therapy has been reported as a treatment for anastomotic stricture after gastrointestinal surgery , peptic esophageal stricture , schatzki s ring , esophageal caustic stenosis , esophageal stricture after chemoradiotherapy or endoscopic submucosal dissection , and in combination with another treatment modality . the success rate of incisional therapy is 80.6% for anastomotic strictures after gastrointestinal surgery , and incisional therapy was also successful in 16 of 20 peptic stricture cases ( 80% ) . incision and bougie dilation for schatzki s rings , and reported more favorable outcomes for incisional therapy , which provided a longer symptom - free period and a greater reduction in dysphagia scores at 1 month . described successful incisional therapy for a refractory esophageal caustic stricture in a 44-year - old man , who was free from dysphagia at the 1-year follow - up . evaluated the usefulness of incisional therapy combined with balloon dilation for cicatricial anastomotic strictures , and reported that five of the six patients exhibited both subjective and objective improvements for > 4 weeks.in the present case , the patient s odynophagia occurred 2 days after the treatment , and we believe that the odynophagia might be related to marked edema and narrowing caused by the electric currents from the incisional therapy , rather than the bougienage after the incision . there is a report that local steroid injection therapy after endoscopic balloon dilation for benign esophageal stricture can decrease the recurrence of stricture . furthermore , kim et al . recently reported that oral steroid therapy after endoscopic submucosal dissection for an esophageal stricture can help prevent relapse after endoscopic balloon dilation . therefore , in the present case , we used oral steroid therapy to help ameliorate the esophageal inflammatory response and prevent stricture recurrence . in conclusion , we report a rare case of an intractable caustic esophageal stricture that was successfully managed with sequential treatment that comprised incision with an insulated - tip knife , balloon dilation , and an oral steroid . however , additional studies are needed to confirm the usefulness of this procedure for caustic esophageal strictures .

bougie or balloon dilation is a good short - term treatment for caustic esophageal strictures , although recurrence after dilation occurs in approximately 30% of these cases . therefore , long - term treatment options are required in some cases , and endoscopic incisional therapy has been used for patients with an anastomotic stricture in the gastrointestinal tract . a 58-year - old woman presented with severe swallowing difficulty because of a caustic esophageal stricture , which was caused by accidental exposure to anhydrous acetic acid at infancy . she had undergone several previous bougie and balloon dilations but the stricture did not improve . we performed sequential treatment comprising incision with an insulated - tip knife , balloon dilation , and an oral steroid , which resulted in the patient s symptoms markedly improving . thus , we report this case of an intractable caustic esophageal stricture , which was successfully treated using combined endoscopic sequential treatment .

INTRODUCTION CASE REPORT DISCUSSION

familial hypobetalipoproteinemia ( fhbl ) is a rare autosomal co - dominant genetic disorder of lipid metabolism . it is characterized by serum low - density lipoprotein ( ldl ) cholesterol or apolipoprotein b ( apob ) levels 5th percentile . apob is the largest of the apolipoproteins and is important in the formation and secretion of very - low - density lipoprotein ( vldl ) and chylomicrons . these two isoforms are derived from differential splicing of rna from a single apob gene . apob-48 is synthesized by enterocytes and is a key component of chylomicrons , while apob-100 is synthesized by the liver and is the key protein component of vldl and ldl particles ; b-100 also contains the binding site for hepatocyte uptake . the apob gene is found on chromosome 2p24 and is unlinked to other apolipoprotein gene clusters . since the discovery of the first kindred with fhbl , many subsequent kindreds have been described , leading to the identification of numerous single nucleotide substitutions , gene deletions , splicing variations and linkage abnormalities in the apob gene that can lead to defects in vldl and chylomicron production and secretion . persons with fhbl can be heterozygotes or homozygotes , with severity of the clinical phenotype related to the number of defective apob alleles . heterozygotes often have no clinical manifestations other than very low serum apob - containing lipoprotein levels ; these low levels of atherogenic lipoprotein particles may actually confer cardiovascular protection . in contrast , fhbl homozygotes can have a range of clinical symptoms , including fat malabsorption , neurologic symptoms , red cell acanthocytosis , retinitis pigmentosa and/or nonalcoholic fatty liver disease . in fact , the clinical presentation of homozygous fhbl can be indistinguishable from that of abetalipoproteinemia ( abl ) due to mutation in mttp , which is the gene that encodes microsomal triglyceride transfer protein . we describe here a case of nonalcoholic steatohepatitis ( nash ) that led to the diagnosis of fhbl . a 44-year - old man was referred to the vancouver general hospital division of gastroenterology for advice regarding management of nash . he was a shop owner , a life - long nonsmoker and drinking about half a bottle of wine a week . , he was told that ast ( normal 535 u / l ) , alt ( normal 756 u / l ) and ggt ( normal 878 u / l ) were all about 2-fold above the upper limits of normal . a liver biopsy was recommended , however the patient declined the procedure and elected to initiate a weight loss program with dietary modification and increased exercise for presumed nash . about 3 years before presentation , he was found to have elevated transaminases on blood work done for an insurance application , with alt 130 u / l and ast 111 u / l . after about 6 months of strict diet and exercise , as well as a 10-pound intentional weight loss , his liver enzymes normalized with alt 44 u / l and ast 41 u / l . he reintroduced mild alcohol intake and reduced the amount of exercise , and 6 months later his liver enzymes were again elevated with alt 94 u / l and ast 80 u / l . other causes for elevated liver enzymes , including anti - nuclear antibody , ceruloplasmin and alpha-1 antitrypsin , were normal or negative . however , he was found to have an elevated ferritin at 737 g / l ( normal 18250 g / l ) . subsequent testing revealed him to be heterozygous for the c282y mutation in the hfe gene for hemochromatosis . , he was a stocky male with a body mass index of 32 kg / m . review of his blood tests from 3 years before revealed ldl cholesterol 0.24 mmol / l ( normal 1.83.5 mmol / l ) , high - density lipoprotein cholesterol 1.29 mmol / l ( normal 0.91.5 a liver biopsy had been done in 2009 by the referring physician and had shown normal liver architecture . there was very mild portal and pericellular centrilobular fibrosis , mild focal mononuclear cell infiltration and moderate macrovesicular steatosis . she was obese , had type 2 diabetes and had recently been diagnosed with esophageal varices . his mother ( i-2 ) and several maternal relatives were known to have a very low cholesterol level . apob immunoblotting on fasting plasma showed only a band at the expected apob-100 position , arguing against truncation mutations . dna was extracted from plasma samples from the patient and subsequently from all first - degree relatives and sent to the cardiovascular genetics lab in london , ontario . direct automated sequencing revealed two different mutations in the apob gene , namely r463w and v703i . these were found to be on opposite chromosomes , so the proband is a compound heterozygote . family relationships , cholesterol levels and apob mutation analysis are shown in figure 1 and table 1 . our patient was advised to return to regular exercise and to maintain a healthy low - fat diet . his weight decreased by about 5 kg over 6 months and his alt normalized to 25 u / l . rare monogenic disorders have been very informative for identifying key components of the metabolism of apob - containing lipoproteins and triglycerides . these disorders include abl due to mutation of the mttp gene , hypobetalipoproteinemia due to mutation of the apob gene , familial combined hypolipidemia due to mutation of the angptl3 gene and pcsk9 deficiency due to mutations in the pcsk9 gene [ 10 , 11 ] . in addition to being important in metabolism , some of the gene products causing these disorders are now targets for new drugs designed to lower ldl cholesterol . for instance , mipomersen is an anti - sense rna biologic that reduces ldl cholesterol by targeting the apob gene , while lomitapide reduces ldl cholesterol by inhibiting microsomal triglyceride transfer protein [ 12 , 13 ] . there are also numerous new biologic drugs in development that lower ldl cholesterol by targeting pcsk9 . while these new drugs seem to be very effective at lowering ldl cholesterol , there is concern about potential long - term side effects , such as hepatosteatosis . clinical evaluation of experiments of nature such as fhbl patients who carry a germ - line mutation in apob can provide clues of the potential long - term benefits and adverse reaction of pharmacologic therapies that target these key metabolic pathways . about 45 different truncated forms of apob have been discovered to date , ranging from apob-2 to apob-90 [ 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 ] . the numbers represent estimated apob mass based on polyacrylamide gel electrophoresis , with apob-100 being normal . truncated apob can form varying sizes and densities of lipoprotein particles depending on the length of truncation . truncated apob particles have a lower production rate and higher clearance rate , resulting in low plasma levels of apob [ 19 , 40 , 41 , 42 ] . apob smaller than apob-27.6 is undetectable in plasma due to rapid clearance . in fhbl heterozygotes with apob truncation , the average plasma concentration of apob is around 2530% of normal [ 43 , 44 ] . fhbl due to angptl3 gene linked to chromosome 3p21 is associated with about a 50% reduction in apob levels compared to normal individuals , as well as an elevated rate of catabolism of vldl apob [ 10 , 45 , 46 , 47 ] . at least some individuals with this subtype of fhbl have gain - of - function mutations of pcsk9 . this is a serine protease that destroys ldl receptors in the liver and thereby controls the level of ldl in plasma . mutations that increase pcsk9 activity cause hypercholesterolemia , whereas mutations that inactivate pcsk9 have the opposite effect . in fhbl , there have been several reports of nonalcoholic fatty liver disease in fhbl subjects as detected by ultrasound or liver biopsy [ 28 , 49 , 50 , 51 , 52 ] . analyzed liver fat by magnetic resonance spectroscopy , body mass index , waist circumference and glucose tolerance of 21 individuals with fhbl due to apob truncations compared with 14 controls . despite similar mean values for obesity and insulin indexes , there was a 5-fold increase in liver fat in fhbl subjects . similar studies with fhbl subjects due to chromosome 3p21 linkage or pcsk9 mutations revealed normal hepatic fat content , an important phenotypic difference between these mutations and apob truncation [ 7 , 46 ] . the proband in this report is a 44-year - old man with nash secondary to fhbl . genetic analysis revealed compound heterozygosity for two mutations , each of which results in amino acid substitution . as shown in figure 1 , he inherited the r463w mutation from his mother ( subject i-2 ) . heterozygosity for this mutation is associated with hypobetalipoproteinemia , while homozygosity has a more severe phenotype . interestingly , the r463w mutation was first reported in an extended christian lebanese family , which is also the ethnic background of our proband 's mother . this is a novel mutation , and analysis using the polyphen program predicts the effect of this amino acid substitution as possibly damaging to apob function . although the two v703i heterozygotes in this kindred , subjects i-1 and ii-1 , had normal ldl cholesterol levels when the second allele encoded normal apob-100 , it is possible that this mutation , in the context of a defective form of apob-100 , such as the r463w mutation , contributes to increased intracellular degradation of apob . this might act to compound the impairment of vldl secretion in subject ii-2 who had one copy of r463w , as evidenced by his lower ldl cholesterol level compared to the two r463w simple heterozygotes ( subjects i-2 and ii-3 ) . although nash is common in fhbl patients , the long - term effects of fhbl are still unclear [ 28 , 49 , 51 , 54 ] . interestingly , harada et al . reported a patient with recurrent nash and cirrhosis due to fhbl post liver transplantation . this suggests that at least in some forms of fhbl , the intestinal defect may be sufficient to cause the phenotype . however , his sister ( subject ii-3 ) has developed esophageal varices , possibly due to liver cirrhosis from fhbl compounded by diabetes mellitus . diet and exercise continue to be the mainstay of treatment for patient with nash induced by fhbl . as in our patient , fat malabsorption can be managed through diet modification by limiting intake of fats containing long - chain fatty acids as well as supplementing fat - soluble vitamins . reports have suggested that the cellular membranes of patients with fhbl are relatively deficient in vitamin e ; indeed severe vitamin e deficiency in both homozygous fhbl and abl is responsible for the development of neurologic signs and symptoms [ 55 , 56 ] . while the most common neurologic symptom is decreased or absent deep tendon reflexes , ataxia , proprioceptive deficits and pain have been described in both homozygous fhbl and abl [ 57 , 58 , 59 ] . there are reports of success with vitamin e treatments for neurologic symptoms in fhbl and abl [ 57 , 60 ] . there is some evidence that combination therapy of vitamin a and e can slow the progression of retinal degeneration and may lead to restoration of vision if started early [ 61 , 62 , 63 ] . our patient was started on vitamin e and multivitamin supplementation for primary prevention , as he does not currently have any neurological , hematological or ocular abnormalities . fhbl is an uncommon autosomal co - dominant disorder of lipid metabolism that is often overlooked because low levels of ldl cholesterol may not attract the attention of health care providers . in the present case liver enzyme abnormalities patients with fhbl and nash can progress to end - stage liver failure , therefore monitoring and risk reduction is important . finally , patients with such rare disorders can provide insight into the possible long - term consequences of new ldl - lowering biologic therapies .

familial hypobetalipoproteinemia ( fhbl ) is a rare genetic disorder of lipid metabolism that is associated with abnormally low serum levels of low - density lipoprotein ( ldl ) cholesterol and apolipoprotein b. it is an autosomal co - dominant disorder , and depending on zygosity , the clinical manifestations may vary from none to neurological , endocrine , hematological or liver dysfunction . nonalcoholic fatty liver disease is common in persons with fhbl , however progression to nonalcoholic steatohepatitis is unusual . we describe here a patient with a novel apob mutation , v703i , which appears to contribute to the severity of the fhbl phenotype . he had liver enzyme abnormalities , increased echogenicity of the liver consistent with steatosis , very low ldl cholesterol at 0.24 mmol / l ( normal 1.83.5 mmol / l ) and an extremely low apolipoprotein b level of 0.16 g / l ( normal 0.61.2 g / l ) . apob gene sequencing revealed him to be a compound heterozygote with two mutations ( r463w and v703i ) . apob r463w has previously been reported to cause fhbl . genetic sequencing of his first - degree relatives identified the apob v703i mutation in his normolipidemic brother and father and the apob r463w mutation in his mother and sister , both of whom have very low ldl cholesterol levels . these results suggest that the apob v703i mutation alone does not cause the fhbl phenotype . however , it is possible that it has a contributory role to a more aggressive phenotype in the presence of apob r463w .

Introduction Case Report Discussion Conclusion

airway inflammation and tissue remodeling that leads to peribronchial fibrosis are characteristic of copd , and contribute to the relentless progress of the disease.12 inhaled glucocorticoids are very effective anti - inflammatory agents in asthma , but have limited benefits in copd . reduced expression of histone deacetylase 2 ( hdac2 ) in alveolar macrophages and airway epithelial cells in copd has been suggested to contribute to the limited ability of glucocorticoids to inhibit the inflammation present in copd.3,4 however , hdac2 expression in lung fibroblasts and its role in mediating the effect of glucocorticoids on lung fibroblast functions are unclear . histone acetyltransferases and histone deacetylases ( hdacs ) play important roles in regulating gene expression.5 acetylation of histone core structure results in opening up of the chromatin allowing transcription factors and ribonucleic acid ( rna ) polymerase to bind to dna and initiate gene transcription . there are eleven hdac isoenzymes that deacetylate histones within the nucleus and specific hdacs may regulate different groups of genes.6 expression of inflammatory genes is regulated by acetylation of histone 4 and hdac2.7,8 in copd alveolar macrophages and airway biopsy specimens , histones that are associated with the promoter region of inflammatory genes are highly acetylated and the degree of acetylation increases with disease severity.3 this increased acetylation of inflammatory genes seems to be not due to increased activity of acetyltransferase but rather due to the reduced expression of hdac2 in human alveolar macrophages and lung tissue.3 lung fibroblasts play a key role in airway remodeling and tissue repair following inflammatory injury of small airways.2 the expression of hdac2 and its role in mediating glucocorticoid anti - inflammatory effect in fibroblasts has not been investigated . the current study , therefore , was designed to investigate hdac2 expression in human lung fibroblasts from control and copd subjects and to determine the role of hdac2 in regulating the effect of budesonide on the release of inflammatory mediators by lung fibroblasts . to accomplish this , human lung fibroblasts were treated with interleukin ( il)-1 and tumor necrosis factor ( tnf)- , the two well - known pro - inflammatory cytokines that are believed to play a role in copd and that stimulate human lung fibroblasts to synthesize and release several inflammatory mediators including il-6 and il-8 , matrix metalloproteinase ( mmp)-1 and mmp-3.911 the role of hdac2 in mediating budesonide inhibition of the release of il-6 , il-8 , mmp-1 , and mmp-3 was then assessed using a pharmacologic inhibitor and rna interference . human fetal lung fibroblasts ( hfl-1 ) , obtained from the american type culture collection ( manassas , va , usa ) , were cultured with dulbecco s modified eagle s medium ( dmem ; life technologies , carlsbad , ca , usa ) supplemented with 10% fetal calf serum ( fcs ) ( life technologies ) , 50 u / ml penicillin , 50 g / ml streptomycin ( life technologies ) and 1 g / ml amphotericin b ( x - gen , big flats , ny , usa ) . fibroblasts were trypsinized ( 0.05% trypsin , 0.481 mm ethylene diamine tetraacetic acid [ edta ] ; life technologies ) , plated into tissue culture plates , grown to near confluence and used for assays . normal human bronchial epithelial cells ( hbecs ) were acquired from bronchial biopsies under a research protocol approved by the university of nebraska institutional review board using a previously published method with slight modifications.12 briefly , the biopsy tissues were allowed to attach to the type i collagen - coated 60 mm tissue culture dishes under serum - free conditions using a 1:1 mixture of lhc-9/rpmi-1640 ( laboratory of human carcinogenesis-9/roswell park memorial institute-1640 ) medium ( life technologies ) without addition of cortisol.13 medium was changed every other day . after 710 days culture , hbecs were trypsinized and plated on collagen ( purecol ; advanced biomatrix , san diego , ca , usa ) coated tissue culture dishes ( falcon ; becton - dickinson labware , lincoln park , nj , usa ) at 37c in a humidified , 5% co2 atmosphere . cells between the 3rd and 10th passage were characterized by staining for pancytokeratin and vimentin and used for experiments . monocytic cell line cells ( thp-1 ) , obtained from the american type culture collection , were cultured in rpmi-1640 supplemented with 10% fcs ( life technologies ) . primary human lung fibroblasts were cultured from lung tissues obtained from subjects with or without clinical and functional signs of copd as described earlier.14 all subjects were undergoing surgery and acquisition of samples was approved by the human studies committee of the medical board of the state of schleswig - holstein ( germany ) . fibroblasts from six normal subjects ( forced expiratory volume in 1 second [ fev1 ] : 89.4%5.7% predicted [ mean + standard error of the mean ] , range 75.6%115.4% ) and six copd subjects ( fev1 : 57.0%1.6% predicted [ mean standard error of the mean ] , range 53.2%63.1% ) were used in the current study . il-6 was quantified by enzyme linked immunosorbent assay ( elisa ) as described previously15 briefly , 96-well plates were coated with capturing antibodies specific for il-6 ( r&d systems , minneapolis , mn , usa ) at 4c overnight . samples and standard ( r&d systems ) were added and allowed to react for 1 hour . after washing , the second antibody ( calbiochem , emd millipore , billerica , ma , usa ) was applied and incubated for 1 hour at room temperature . after washing , horseradish peroxidase ( hrp)-conjugated anti - rabbit antibody ( rockland immunochemicals , gilbertsville , pa , usa ) was applied for 1 hour at room temperature . o - phenylenediamine ( opd ) substrate was applied and absorbance read at 495 nm wavelength . il-8 was quantified by using human il-8 duoset kit ( r&d systems ) following the manufacturer s instruction . to evaluate mmp-1 and -3 , conditioned media ( 2 ml ) collected from the monolayer culture of the fibroblasts were concentrated by ethanol precipitation and re - suspended in distilled h2o ( 50 l).16 the samples ( 15 l ) were then added to the same volume of 2x concentrated electrophoresis sample buffer ( 0.5 m tris ph 6.8 , 2% sodium dodecyl sulfate [ sds ] , 20% glycerol , 0.1 % bromophenol blue ) and heated for 5 minutes at 95c . as mmps were evaluated in culture medium in which there are no reference proteins , care was taken to load equal volumes and quantitative results are expressed relative to control cells treated only with cytokines ( il-1 and tnf- ) . thirty microliters of each sample was loaded into each lane of a 10% sds - polyacrylamide gel electrophoresis ( page ) gel and electrophoresed . the proteins were transferred to a polyvinylidene difluoride ( pvdf ) membrane with a semi - dry transfer apparatus . the membrane was blocked in 5% milk in phosphate buffered saline ( pbs)-tween at room temperature for 1 hour , then exposed to primary antibodies ( mouse anti - human mmp-1 or mmp-3 from r&d systems ) , which were subsequently detected using hrp - conjugated rabbit anti - mouse immunoglobulin g ( igg ) in conjunction with an enhanced chemiluminescence ( ecl ) detection system . for hdac , whole cell lysate proteins were extracted with 100 l per 60 mm dish of cell lysis buffer ( 35 mm tris - hcl , ph 7.4 , 0.1% triton x-100 , 0.4 mm ethylene glycol tetraacetic acid [ egta ] , 10 mm mgcl2 ) containing a protease inhibitor cocktail ( sigma - aldrich , st louis , mo , usa ) . cells were briefly sonicated followed by centrifugation at 10,000 g for 10 minutes at 4c and the supernatant was subjected to immunoblotting for hdac2 ( 1:200 dilution ; abcam , cambridge , ma , usa ) , with -actin ( 1:4000 dilution ; sigma - aldrich ) as loading control . sds - page electrophoresis , protein transfer and immunoblotting were conducted as described above . to inhibit hdac , a non - selective hdac inhibitor , trichostatin a ( tsa ; cell signaling , danvers , ma , usa ) was used . after incubation of hfl-1 , hbecs and thp-1 cells with various concentrations of tsa for 30 minutes , varying concentrations of budesonide ( astrazeneca , lund sweden ) and il-1 plus tnf- ( r&d systems ) were added to the final concentrations of il-1 plus tnf- , 1 ng / ml each . after 24 hours , media were harvested for quantification of il-6 and il-8 by elisa , as well as mmp-1 and mmp-3 by immunoblotting . cells were trypsinized and counted with a coulter counter ( beckman coulter , brea , ca , usa ) . levels of the cytokines were normalized by cell number for each experiment . to selectively silence hdac2 , rna interference was performed . briefly , cells were seeded in 6-well dishes at a cell density of 2 10 cells per well . the next day , cells were transfected with small interfering ( si)rna targeting hdac2 or nontargeting control sirna ( final concentration of sirna was 50 nm ; santa cruz biotechnology inc , santa cruz , ca , usa ) in opti - mem ( invitrogen , carlsbad ca , usa ) using lipofectamine 2000 ( invitrogen ) . after 16 hours transfection , media were changed to 10% fcs - dmem for hfl-1 cells or lhc-9/rpmi for hbecs . after 24 hours , the cells were treated with cytokines ( il-1 and tnf- ) and/or budesonide ( astrazeneca ) . media were harvested and the cell lysates were extracted on day 4 and the efficacy of rna interference was assessed by immunoblotting . cse was prepared with a modification of the method reported previously17 briefly , the smoke from one 84 mm cigarette ( research cigarette 3r4f ; university of kentucky , lexington , ky , usa ) without filter was bubbled through 15 ml of deionized water at a speed of 50 cc / minute . the filtered solution with a 0.22 m pore filter ( lida manufacturing , kenosha , wi , usa ) was considered to be 100% cse and applied to fibroblast cultures within 30 minutes of preparation . in the current study , fibroblasts were exposed to 5% cse diluted with serum free dmem . results were always confirmed by repeating each experiment on separate occasions at least three times . group data were analyzed by one - way analysis of variances ( anova ) followed by tukey s test or two - way anova followed by bonferroni test using the graphpad prism 4 software ( graphpad software , inc . , la jolla , ca , human fetal lung fibroblasts ( hfl-1 ) , obtained from the american type culture collection ( manassas , va , usa ) , were cultured with dulbecco s modified eagle s medium ( dmem ; life technologies , carlsbad , ca , usa ) supplemented with 10% fetal calf serum ( fcs ) ( life technologies ) , 50 u / ml penicillin , 50 g / ml streptomycin ( life technologies ) and 1 g / ml amphotericin b ( x - gen , big flats , ny , usa ) . fibroblasts were trypsinized ( 0.05% trypsin , 0.481 mm ethylene diamine tetraacetic acid [ edta ] ; life technologies ) , plated into tissue culture plates , grown to near confluence and used for assays . normal human bronchial epithelial cells ( hbecs ) were acquired from bronchial biopsies under a research protocol approved by the university of nebraska institutional review board using a previously published method with slight modifications.12 briefly , the biopsy tissues were allowed to attach to the type i collagen - coated 60 mm tissue culture dishes under serum - free conditions using a 1:1 mixture of lhc-9/rpmi-1640 ( laboratory of human carcinogenesis-9/roswell park memorial institute-1640 ) medium ( life technologies ) without addition of cortisol.13 medium was changed every other day . after 710 days culture , hbecs were trypsinized and plated on collagen ( purecol ; advanced biomatrix , san diego , ca , usa ) coated tissue culture dishes ( falcon ; becton - dickinson labware , lincoln park , nj , usa ) at 37c in a humidified , 5% co2 atmosphere . cells between the 3rd and 10th passage were characterized by staining for pancytokeratin and vimentin and used for experiments . monocytic cell line cells ( thp-1 ) , obtained from the american type culture collection , were cultured in rpmi-1640 supplemented with 10% fcs ( life technologies ) . primary human lung fibroblasts were cultured from lung tissues obtained from subjects with or without clinical and functional signs of copd as described earlier.14 all subjects were undergoing surgery and acquisition of samples was approved by the human studies committee of the medical board of the state of schleswig - holstein ( germany ) . fibroblasts from six normal subjects ( forced expiratory volume in 1 second [ fev1 ] : 89.4%5.7% predicted [ mean + standard error of the mean ] , range 75.6%115.4% ) and six copd subjects ( fev1 : 57.0%1.6% predicted [ mean standard error of the mean ] , range 53.2%63.1% ) were used in the current study . il-6 was quantified by enzyme linked immunosorbent assay ( elisa ) as described previously15 briefly , 96-well plates were coated with capturing antibodies specific for il-6 ( r&d systems , minneapolis , mn , usa ) at 4c overnight . samples and standard ( r&d systems ) were added and allowed to react for 1 hour . after washing , the second antibody ( calbiochem , emd millipore , billerica , ma , usa ) was applied and incubated for 1 hour at room temperature . after washing , horseradish peroxidase ( hrp)-conjugated anti - rabbit antibody ( rockland immunochemicals , gilbertsville , pa , usa ) was applied for 1 hour at room temperature . o - phenylenediamine ( opd ) substrate was applied and absorbance read at 495 nm wavelength . il-8 was quantified by using human il-8 duoset kit ( r&d systems ) following the manufacturer s instruction . to evaluate mmp-1 and -3 , conditioned media ( 2 ml ) collected from the monolayer culture of the fibroblasts were concentrated by ethanol precipitation and re - suspended in distilled h2o ( 50 l).16 the samples ( 15 l ) were then added to the same volume of 2x concentrated electrophoresis sample buffer ( 0.5 m tris ph 6.8 , 2% sodium dodecyl sulfate [ sds ] , 20% glycerol , 0.1 % bromophenol blue ) and heated for 5 minutes at 95c . as mmps were evaluated in culture medium in which there are no reference proteins , care was taken to load equal volumes and quantitative results are expressed relative to control cells treated only with cytokines ( il-1 and tnf- ) . thirty microliters of each sample was loaded into each lane of a 10% sds - polyacrylamide gel electrophoresis ( page ) gel and electrophoresed . the proteins were transferred to a polyvinylidene difluoride ( pvdf ) membrane with a semi - dry transfer apparatus . the membrane was blocked in 5% milk in phosphate buffered saline ( pbs)-tween at room temperature for 1 hour , then exposed to primary antibodies ( mouse anti - human mmp-1 or mmp-3 from r&d systems ) , which were subsequently detected using hrp - conjugated rabbit anti - mouse immunoglobulin g ( igg ) in conjunction with an enhanced chemiluminescence ( ecl ) detection system . for hdac , whole cell lysate proteins were extracted with 100 l per 60 mm dish of cell lysis buffer ( 35 mm tris - hcl , ph 7.4 , 0.1% triton x-100 , 0.4 mm ethylene glycol tetraacetic acid [ egta ] , 10 mm mgcl2 ) containing a protease inhibitor cocktail ( sigma - aldrich , st louis , mo , usa ) . cells were briefly sonicated followed by centrifugation at 10,000 g for 10 minutes at 4c and the supernatant was subjected to immunoblotting for hdac2 ( 1:200 dilution ; abcam , cambridge , ma , usa ) , with -actin ( 1:4000 dilution ; sigma - aldrich ) as loading control . to inhibit hdac , a non - selective hdac inhibitor , trichostatin a ( tsa ; cell signaling , danvers , ma , usa ) was used . after incubation of hfl-1 , hbecs and thp-1 cells with various concentrations of tsa for 30 minutes , varying concentrations of budesonide ( astrazeneca , lund sweden ) and il-1 plus tnf- ( r&d systems ) were added to the final concentrations of il-1 plus tnf- , 1 ng / ml each . after 24 hours , media were harvested for quantification of il-6 and il-8 by elisa , as well as mmp-1 and mmp-3 by immunoblotting . cells were trypsinized and counted with a coulter counter ( beckman coulter , brea , ca , usa ) . briefly , cells were seeded in 6-well dishes at a cell density of 2 10 cells per well . the next day , cells were transfected with small interfering ( si)rna targeting hdac2 or nontargeting control sirna ( final concentration of sirna was 50 nm ; santa cruz biotechnology inc , santa cruz , ca , usa ) in opti - mem ( invitrogen , carlsbad ca , usa ) using lipofectamine 2000 ( invitrogen ) . after 16 hours transfection , media were changed to 10% fcs - dmem for hfl-1 cells or lhc-9/rpmi for hbecs . after 24 hours , the cells were treated with cytokines ( il-1 and tnf- ) and/or budesonide ( astrazeneca ) . media were harvested and the cell lysates were extracted on day 4 and the efficacy of rna interference was assessed by immunoblotting . cse was prepared with a modification of the method reported previously17 briefly , the smoke from one 84 mm cigarette ( research cigarette 3r4f ; university of kentucky , lexington , ky , usa ) without filter was bubbled through 15 ml of deionized water at a speed of 50 cc / minute . the filtered solution with a 0.22 m pore filter ( lida manufacturing , kenosha , wi , usa ) was considered to be 100% cse and applied to fibroblast cultures within 30 minutes of preparation . in the current study , fibroblasts were exposed to 5% cse diluted with serum free dmem . results were always confirmed by repeating each experiment on separate occasions at least three times . group data were analyzed by one - way analysis of variances ( anova ) followed by tukey s test or two - way anova followed by bonferroni test using the graphpad prism 4 software ( graphpad software , inc . in order to study the effect of budesonide on release of cytokines and mmps by fibroblasts , hfl-1 cells were treated with il-1 plus tnf- ( 1 ng / ml , each ) in monolayer culture to assess il-6 and il-8 as well as mmp-1 and mmp-3 . as expected il-1 plus tnf- significantly stimulated il-6 , il-8 , mmp-1 , and mmp-3 release by hfl-1 cells ( figure 1a d ) . budesonide ( from 10 to 10 m ) significantly inhibited il-1 plus tnf--induced release of all four mediators ( figure 1a d , p < 0.01 ) . tsa ( 1010 m ) , a nonselective hdac inhibitor , did not block budesonide inhibition of cytokine or mmp release ( figure 2a d ) in human lung fibroblasts . interestingly , in the presence of il-1 plus tnf- , tsa ( 10 m ) significantly inhibited mmp-1 release ( figure 2c ) , but significantly stimulated mmp-3 release ( figure 2d ) . in contrast to the lack of effect of tsa on budesonide inhibition of mediator release in fibroblasts , tsa significantly blocked the inhibitory effect of budesonide ( 10 m ) on il-8 release in hbecs ( figure 3a , tsa at 10 m ) and in the thp-1 cell line ( figure 3b , tsa at 10 m ) . similarly , tsa also significantly blocked the inhibitory effect of budesonide on cytokine - induced il-6 release by hbecs ; budesonide ( 10 m ) reduced release to 59.0% 9.5% ( mean standard error of the mean ) of cytokine - stimulated cells ( 100% ) and tsa blocked this effect resulting in 98.2% 10.2% release ( mean standard error of the mean ; control non - stimulated cells release was 45.7% 2.9% of cytokine - stimulated cells ) . these results demonstrate that budesonide can inhibit fibroblast mediator release via an hdac independent mechanism , while the effect on other cells requires hdac . in order to further investigate the role of hdac in mediating budesonide inhibition of cytokine and mmp release by fibroblasts , hfl-1 cells were transfected with an sirna specifically targeting hdac2 , followed by treatment with budesonide . hdac2 was significantly suppressed by the sirna ( figure 4a ; p < 0.01 ) , however , despite suppression of hdac2 , budesonide ( 10 m ) still significantly inhibited cytokine - stimulated il-6 , il-8 , mmp-1 , and mmp-3 ( figure 4b d , f and g , p < 0.01 ) . in order to ensure that the sirna was active , the effect of sirna suppression of hdac2 was investigated on budesonide ( 5 10 m ) inhibition of il-6 release by hbecs . the sirna targeting hdac2 was found to significantly abolish the effect of the high concentration of budesonide in hbecs ( figure 4e and h , p < 0.05 ) . finally , since hdac2 expression has been reported to be reduced in alveolar macrophages from copd patients,3 hdac2 protein expression was examined in lung fibroblasts cultured from copd patients and compared to that in fibroblasts obtained from normal control subjects . while the expression of hdac2 was numerically lower in copd fibroblasts than in normal lung fibroblasts , the difference was not statistically significant ( figure 5a ; 24% reduction , 4.0 1.5 versus 5.2 1.4 [ mean standard error of the mean , n = 6 ] ) . furthermore , budesonide at low concentration ( 10 m ) inhibited cytokine - induced il-6 , il-8 , mmp-1 and mmp-3 from both normal and copd fibroblasts to a similar degree ( figure 5b e ; p < 0.05 ) . interestingly , hdac2 protein was significantly decreased to 54.0% 6.1% in the hfl-1 cells exposed to 5% cse for 72 hours compared to the cells without cse exposure ( figure 6a , p < 0.05 ) . consistent with previous reports,18,19 cse stimulated il-6 and il-8 release by human lung fibroblasts under control conditions as well as release of mmp-1 . cytokines ( il-1 plus tnf- ) markedly stimulated il-6 , il-8 , mmp-1 , and mmp-3 in both cse - pretreated and control fibroblasts . budesonide at concentrations of 10 and/or 1 0 m significantly inhibited cytokine - induced il-6 , il-8 , mmp-1 , and mmp-3 from both control and cse - pre - exposed fibroblasts to a similar degree , suggesting that any inhibition of hdac2 that may result from smoke exposure did not alter the ability of budesonide to inhibit mediator release ( figure 6b e ) . in order to study the effect of budesonide on release of cytokines and mmps by fibroblasts , hfl-1 cells were treated with il-1 plus tnf- ( 1 ng / ml , each ) in monolayer culture to assess il-6 and il-8 as well as mmp-1 and mmp-3 . as expected il-1 plus tnf- significantly stimulated il-6 , il-8 , mmp-1 , and mmp-3 release by hfl-1 cells ( figure 1a d ) . budesonide ( from 10 to 10 m ) significantly inhibited il-1 plus tnf--induced release of all four mediators ( figure 1a d , p < 0.01 ) . tsa ( 1010 m ) , a nonselective hdac inhibitor , did not block budesonide inhibition of cytokine or mmp release ( figure 2a d ) in human lung fibroblasts . interestingly , in the presence of il-1 plus tnf- , tsa ( 10 m ) significantly inhibited mmp-1 release ( figure 2c ) , but significantly stimulated mmp-3 release ( figure 2d ) . in contrast to the lack of effect of tsa on budesonide inhibition of mediator release in fibroblasts , tsa significantly blocked the inhibitory effect of budesonide ( 10 m ) on il-8 release in hbecs ( figure 3a , tsa at 10 m ) and in the thp-1 cell line ( figure 3b , tsa at 10 m ) . similarly , tsa also significantly blocked the inhibitory effect of budesonide on cytokine - induced il-6 release by hbecs ; budesonide ( 10 m ) reduced release to 59.0% 9.5% ( mean standard error of the mean ) of cytokine - stimulated cells ( 100% ) and tsa blocked this effect resulting in 98.2% 10.2% release ( mean standard error of the mean ; control non - stimulated cells release was 45.7% 2.9% of cytokine - stimulated cells ) . these results demonstrate that budesonide can inhibit fibroblast mediator release via an hdac independent mechanism , while the effect on other cells requires hdac . in order to further investigate the role of hdac in mediating budesonide inhibition of cytokine and mmp release by fibroblasts , hfl-1 cells were transfected with an sirna specifically targeting hdac2 , followed by treatment with budesonide . hdac2 was significantly suppressed by the sirna ( figure 4a ; p < 0.01 ) , however , despite suppression of hdac2 , budesonide ( 10 m ) still significantly inhibited cytokine - stimulated il-6 , il-8 , mmp-1 , and mmp-3 ( figure 4b d , f and g , p < 0.01 ) . in order to ensure that the sirna was active , the effect of sirna suppression of hdac2 was investigated on budesonide ( 5 10 m ) inhibition of il-6 release by hbecs . the sirna targeting hdac2 was found to significantly abolish the effect of the high concentration of budesonide in hbecs ( figure 4e and h , p < 0.05 ) . finally , since hdac2 expression has been reported to be reduced in alveolar macrophages from copd patients,3 hdac2 protein expression was examined in lung fibroblasts cultured from copd patients and compared to that in fibroblasts obtained from normal control subjects . while the expression of hdac2 was numerically lower in copd fibroblasts than in normal lung fibroblasts , the difference was not statistically significant ( figure 5a ; 24% reduction , 4.0 1.5 versus 5.2 1.4 [ mean standard error of the mean , n = 6 ] ) . furthermore , budesonide at low concentration ( 10 m ) inhibited cytokine - induced il-6 , il-8 , mmp-1 and mmp-3 from both normal and copd fibroblasts to a similar degree ( figure 5b e ; p < 0.05 ) . interestingly , hdac2 protein was significantly decreased to 54.0% 6.1% in the hfl-1 cells exposed to 5% cse for 72 hours compared to the cells without cse exposure ( figure 6a , p < 0.05 ) . consistent with previous reports,18,19 cse stimulated il-6 and il-8 release by human lung fibroblasts under control conditions as well as release of mmp-1 . cytokines ( il-1 plus tnf- ) markedly stimulated il-6 , il-8 , mmp-1 , and mmp-3 in both cse - pretreated and control fibroblasts . budesonide at concentrations of 10 and/or 1 0 m significantly inhibited cytokine - induced il-6 , il-8 , mmp-1 , and mmp-3 from both control and cse - pre - exposed fibroblasts to a similar degree , suggesting that any inhibition of hdac2 that may result from smoke exposure did not alter the ability of budesonide to inhibit mediator release ( figure 6b e ) . the current study demonstrates that budesonide inhibits release of cytokines ( il-6 and il-8 ) and mmps ( mmp-1 and mmp-3 ) from il-1 plus tnf--stimulated human lung fibroblasts ( hfl-1 and primary cells ) , hbecs , and cells from a human monocytic cell line ( thp-1 ) . human lung fibroblasts express hdac2 , but neither inhibition of hdac by a pharmacologic inhibitor tsa nor suppression with hdac2 specific sirna affected budesonide inhibition of mediator release from fibroblasts . this contrasts with hbecs and thp-1 cells , in which the inhibitory effect of budesonide was significantly blocked by hdac inhibition . protein expression in lung fibroblasts from copd patients was slightly but not significantly lower than that in normal lung fibroblasts whereas it was significantly decreased by nearly 50% in the lung fibroblasts ( hfl-1 ) exposed to cse compared to nonexposed cells . nevertheless , budesonide inhibited production of il-6 , il-8 , mmp-1 , and mmp-3 in both normal and copd lung fibroblasts or cse - exposed and nonexposed cells similarly . taken together , these results demonstrate that budesonide can inhibit fibroblast mediator release via an hdac independent mechanism and that fibroblasts from copd patients or cse - exposed fibroblasts are sensitive to this inhibition . acetylation of core histones by histone acetyltransferase results in opening the chromatin structure to allow transcription factors and rna polymerase binding to dna thus , initiating gene transcription.5 histone acetylation is reversed by hdacs , and by this mechanism gene expression is turned off . there are 11 hdac isoenzymes described and , of these , hdac2 has been reported to be critically important in mediating glucocorticoid suppression of airway inflammation.4,8 budesonide is a glucocorticoid with potent anti - inflammatory effects and limited systemic bioactivity due to extensive ( 90% ) first - pass hepatic metabolism by cytochrome p450 enzymes.20 inhalation of glucocorticoids has been shown to be effective in suppressing airway inflammation in asthmatics but is generally less effective in copd . reduction of hdac2 expression in airway macrophages and epithelial cells in copd patients is believed to account for the inflammation in copd being less responsive to glucocorticoid therapy.3 this resistance poses a paradox , as glucocorticoids have well - demonstrated therapeutic benefits in copd , including improvement in airflow and reduction in the frequency of exacerbations . because the clinical benefits develop slowly over a matter of weeks , an anti - inflammatory action has been suggested , despite the reports that macrophages and epithelial cells are resistant to the anti - inflammatory effects of glucocorticoids in copd.3,4,8 consistent with previous reports , the current study demonstrated that budesonide inhibited il-6 and il-8 release by human bronchial epithelial cells,21 lung fibroblasts,11 and monocytic cells.22 moreover , also consistent with previous reports,23,24 inhibition of hdac activity by tsa blocked the inhibitory effect of budesonide on il-8 and il-6 release by hbecs and thp-1 cells . in contrast , the inhibitory effect of budesonide on the lung fibroblasts was not sensitive to tsa . interestingly , in the presence of il-1 plus tnf- , tsa alone inhibited mmp-1 release but stimulated mmp-3 release indicating that it had an effect on the fibroblasts , although it did not block budesonide - mediated inhibition . consistent with this , tsa has been reported to up - regulate mmp-11 expression in colon cancer cells,25 but tsa inhibited il-1-stimulated mmp-1 , mmp-3 , and mmp-13 in human articular chondrocytes.26 the mechanisms for tsa stimulation or inhibition of mmps , however , remains to be determined . the current study , however , confirms previous reports that histone deacetylation plays a key role in the glucocorticoid - mediated inhibition of mediator release by hbecs and monocytes / macrophages , and demonstrates that glucocorticoids may still modulate the inflammatory response by inhibiting the release of mediators from lung fibroblasts , which is not hdac dependent . this approach had a clear effect in blocking budesonide - mediated inhibition of mediator release in hbecs , but had no effect in lung fibroblasts . sirna knockdown was confirmed by western blotting . as is usually the case , knockdown was not complete in either cell type , although hdac2 expression was reduced by 75% in lung fibroblasts and was similarly reduced in hbecs when normalized to -actin . our experiments can not exclude the possibility of an effect of residual hdac2 in fibroblasts , and it is possible that the amount of hdac2 activity required may vary among cell types . importantly , however , the results with the sirna and the tsa consistently support no role for hdac2 in mediating the effect of budesonide in lung fibroblasts . they are also the major cell type present in the subepithelial tissue in the airways and comprise up to 25% of alveolar cells , although the markedly attenuated structure of the alveolus makes them difficult to recognize on routine histology.27 in both locations , fibroblasts are believed to be the major source of extracellular matrix macromolecules . these include growth factors for epithelial and endothelial cells , and enzymes that can mediate the degradation of connective tissue . however , fibroblasts , because of their presence in large numbers in normal tissues , may be particularly important in mediating chronic inflammatory processes . in this context , fibroblasts respond vigorously to the early inflammatory response mediators il-1 and tnf-.28,29 these mediators induce a number of responses in fibroblasts including release of the cytokines il-6,10 il-8,30 and mmps.16 thus , fibroblasts may participate in the amplification of il-1 and tnf--induced inflammation . the quantitative production of mediators by fibroblasts may be less than that of monocytes / macrophages or epithelial cells , however , this does not exclude a role for fibroblasts in amplifying and sustaining an inflammatory response . the current study provides evidence that the mechanism of glucocorticoid inhibition of lung fibroblast - mediated inflammation differs from that of monocytes / macrophages and airway epithelial cells , which may help to explain the clinical benefits of inhaled glucocorticoids in copd . we also examined expression of hdac2 in the lung fibroblasts from normal and copd subjects as well as in hfl-1 cells exposed to 5% cse for 72 hours . in contrast to the reduced hdac2 expression reported in alveolar macrophages from copd patients,3 there was no statistically significant difference in hdac2 expression between normal and copd lung fibroblasts . nevertheless , the slight numerical difference observed ( 24% reduction in copd lung fibroblasts , n = 6 ) in the current study does not exclude the possibility that evaluation of a larger number of samples , or samples from more severely affected copd patients , might show a statistically significant difference . based on the data obtained a study of 25 subjects per group would be required to have an 80% chance of showing a statistically significant difference of the size observed in the current study . thus , a modest inhibition of hdac2 in lung fibroblasts from copd patients can not be excluded . in support of this concept , however , in both the normal and copd fibroblasts as well as in hfl-1 cells exposed or not exposed to cse , the release of il-6 , il-8 , mmp-1 , and mmp-3 in response to il-1 and tnf- was significantly inhibited by budesonide to a similar degree . thus , the reduction of hdac activity appeared to have no effect on glucocorticoid inhibition of mediator release from lung fibroblasts , further supporting our finding that in human lung fibroblasts budesonide inhibition of inflammatory mediator release is independent of hdac2 level . budesonide inhibits release of il-6 , il-8 , mmp-1 , and mmp-3 from human lung fibroblasts stimulated by il-1 plus tnf. suppression of hdac2 by either a pharmacologic inhibitor ( tsa ) or an sirna targeting hdac2 does not block the inhibitory effects of budesonide in fibroblasts , which contrasts with hdac - dependent inhibition in hbecs and monocytic cells . the demonstration that the anti - inflammatory effects of budesonide in lung fibroblasts are hdac - independent suggests that budesonide has the potential to modulate fibroblast - mediated tissue remodeling following airway inflammation in copd and provides a mechanism for the therapeutic benefits of glucocorticoids in copd .

objective and designreduced expression of histone deacetylase 2 ( hdac2 ) in alveolar macrophages and epithelial cells may account for reduced response of chronic obstructive pulmonary disease ( copd ) patients to glucocorticoids . hdac2 expression and its role in mediating glucocorticoid effects on fibroblast functions , however , has not been fully studied . this study was designed to investigate whether hdac2 mediates glucocorticoid effects on release of inflammatory cytokines and matrix metalloproteinases ( mmps ) from human lung fibroblasts.methodshuman lung fibroblasts ( hfl-1 cells ) were stimulated with interleukin ( il)-1 plus tumor necrosis factor ( tnf)- in the presence or absence of the glucocorticoid budesonide . cytokines ( il-6 and il-8 ) were quantified by enzyme linked immunosorbent assay ( elisa ) and mmps ( mmp-1 and mmp-3 ) by immunoblotting in culture medium . the role of hdac2 was investigated using a pharmacologic inhibitor as well as a small interfering ribonucleic acid ( sirna ) targeting hdac2.resultswe have demonstrated that budesonide concentration - dependently ( 1010107 m ) inhibited il-6 , il-8 , mmp-1 , and mmp-3 release by hfl-1 cells in response to il-1 plus tnf-. while an hdac inhibitor significantly blocked the inhibitory effect of budesonide on human bronchial epithelial cells ( hbecs ) and monocytes ( thp-1 cells ) , it did not block the inhibitory effect of budesonide on release of cytokines and mmps from hfl-1 cells . similarly , an hdac2-sirna blocked budesonide inhibition of cytokine release in hbecs , but it did not block the inhibitory effect of budesonide on hfl-1 cytokine and mmp release . furthermore , budesonide significantly blocked release of cytokines and mmps to a similar degree in normal and copd lung fibroblasts as well as in hfl-1 cells exposed or not exposed to cigarette smoke extract.conclusionthese findings suggest that , in contrast to airway epithelial cells and monocytes / macrophages , hdac2 is not required for budesonide to inhibit mmp and cytokine release by lung fibroblasts and this inhibitory pathway appears to be intact in cultured fibroblasts from copd patients . these results also suggest that budesonide has the potential to modulate fibroblast - mediated tissue remodeling following airway inflammation in copd , which is mediated via an hdac2 independent pathway .

Introduction Material and methods Cell culture Cytokine quantification by ELISA Immunoblotting HDAC inhibition Selective inhibition of HDAC2 by RNA interference Preparation of cigarette smoke extract (CSE) Statistical analysis Results HDAC inhibitor blocked budesonide effect in HBECs but not in HFL-1 cells HDAC2 suppression by siRNA blocked the effect of budesonide in HBECs but not in HFL-1 cells Budesonide effect was not impaired in COPD lung fibroblasts and in HFL-1 cells exposed to cigarette smoke Discussion Conclusion

specificity is one of the foundational components of training success . in sport performance training , one of the initial steps in the design of an exercise regimen is the identification of movements specific to the needs of the individual , which may include joint angles , and muscle actions and recruitment ( 1 , 4 ) . movement analysis is an integral step in the selection of exercises that mimic sport - specific actions and transfer to competition . furthermore , the intensity and movement velocity ( explosiveness ) utilized in the performance of an exercise can be a vitally important component of specificity ( 1 , 4 ) . sprinting , and the acceleration phase in particular , is characterized by the explosive extension of the hip , knee , and ankle joints ( 3 ) , with a greater overall contribution of the hip joint particularly as speed increases ( 2 ) . the kettlebell swing ( kbs ) is similarly characterized by activation of the posterior chain muscles , particularly in relation to the hip , in the horizontal plane that occurs during the sprint motion ( 3 , 8 , 11 , 16 ) thereby indicating movement specificity . additionally , the rapid concentric phase and ballistic nature of the kbs ( 8 , 10 ) may align well with sprint performance , which also requires rapid force production . comparisons between the kbs and a traditional one - repetition maximum back squat resulted in a greater impulse demand in the kbs , pointing towards the potential for a large rate of force development despite differences in load , and demonstrating the importance of the velocity of this exercise ( 8) . thus , it is likely that this velocity specificity would also align well with sprinting . despite the popularity and potential applicability of kettlebell training in multiple domains ( 7 , 10 , 13 ) , the sport performance community has not responded with an appropriate depth of rigorous scientific studies into how kettlebell training may transfer into sprinting . in 2012 , lake and lauder reported six weeks of two - handed kbs exercise progressing to 60% of maximal loads , improved maximal and explosive strength ( 7 ) . conversely , results from a comparison study suggested that kbs at 60% of maximal loads are not sufficient to develop lower body maximal and explosive strength , and concluded that the kbs may be best used as adjuvant training during a strength and conditioning program ( 8) . additionally , otto et al . reported that six weeks of traditional weightlifting induced significantly greater improvements in strength compared with kbs training , however , both were effective in increasing muscular strength and power in recreationally - active men ( 13 ) . regardless of training modality , it is understood that volume plays an important role in the degree of muscular strength and power improvement . it is plausible that different training volumes used in the aforementioned studies may have resulted in ambiguous conclusions about the efficacy of kbs on power performance . moreover , though hypothesized , it has yet to be determined whether kbs training can have positive effects on sprint performance ( 7 ) . therefore , the present study examined the effect of an eight - week kbs program versus a program of equal intensity , volume , and movement specificity using the stiff - leg deadlift ( sdl ) on sprint performance . the sdl was chosen as it utilizes very similar muscle recruitment as the kbs , while allowing a slower movement velocity ( 1 , 4 ) . in this fashion , it is possible to utilize similar movement specificity and volume , while addressing the additional velocity specificity of the kbs . therefore , it was hypothesized that the eight - week kbs program would improve sprint performance to a greater degree than an eight - week sdl program . twenty ( n=20 ) healthy college - age female students ( 1825 yrs ) were recruited for the current investigation . approval by the slippery rock university institutional review board was acquired and all participants completed informed consent . participants were instructed to maintain their current aerobic exercise and dietary programs throughout the duration of the study . no participants had extensive experience with kettlebell exercise or completed sprint or resistance training in the previous 6 months as a means to limit extraneous variables from prior training history . study investigators , however , were trained in the safe and effective implementation of resistance exercise , and completed specific sessions on coaching the kbs and sdl . participants were excluded from the present study if they were not cleared by their physician for vigorous exercise , were currently utilizing a resistance training program , were outside of the age range ( 1825 ) , or were unable attend the scheduled sessions . participants were assigned into one of two groups ( kbs or sdl ) based on their initial sprint performance , to ensure that there were participants of similar ability in each cohort . following the group assignment all participants participated in one instructional session on their assigned exercise prior to data collection . technique mastery was not necessary during the single instructional session as the participants were observed ( i.e. appropriate cueing ) throughout the duration of the training intervention for both safety and effectiveness . each group underwent an 8-week ( twice - weekly ) exercise intervention consisting of sixteen training sessions . quite simply , this schedule was chosen as it fulfilled training frequency guidelines for beginners ( i.e. 23 sessions per week ) , and fit within the time constraints of a university academic semester . each training session began with a 5 min light ( <3 mets ) aerobic warm - up on a treadmill followed by two 15 meter striders with a 30 second rest . all training sessions were performed using a tabata interval timer ( 20 seconds of exercise ; 10 seconds of rest ; 8 rounds ) based on previous work demonstrating a large anaerobic component to this training approach ( 5 , 15 ) . the participants were required to elect two non - consecutive days each week to exercise under the supervision of a project coordinator . a hardstyle kettlebell swing emphasizing maximal hip recruitment and minimal knee flexion was utilized in this study ( 7 ) . training started with a consistent prescribed weight ( ~9.1 kg ) with the potential for a ~2.3 kg ( next kettlebell weight increment ) increase following week three , and another ~2.3 kg increase at week six , if the participant maintained proper form throughout each session . form was monitored by trained instructors throughout the sessions , and included emphasis on maintaining an explosive concentric phase during maximal hip recruitment . training started with a consistent barbell weight ( ~27.3 kg ) , with the potential for a ~4.5 kg increase following week 3 , and another ~4.5 kg increase at week 6 , if the participant maintained proper form throughout each session . form checks for the sdl group included the maintenance of a two - second up / two - second down exercise tempo and maximal hip recruitment . each participant completed both a 30-meter sprint test and countermovement vertical jump test on a consistent , indoor track . identical post - testing was completed between 48 and 72 hours of the final exercise session . if a participant performed the vertical jump test before the sprint test in pre - testing , they were asked to perform the sprint test first for post - testing . each participant performed the same warm - up during the pre- and post - training testing . this warm - up consisted of a 400-meter jog followed by a 40-meter interval of the following drills : high knees , butt kicks , walking knee - to - chest stretch , walking quad stretch , toe touches , hamstring swings , carioca , and striders . participants were given the option to add additional warm - up striders before performing their maximal sprint ability . thirty meter sprint performances were timed using a tc - photogate ( brower timing systems , draper , ut ) . participants used a standing position and began the maximal sprint individually without a formal start . the tc - photogate recorded when the participants crossed the start and the finish line . each participant performed two sprint trials with a two - minute rest in between attempts , with the fastest sprint recorded . a vertical jump measurement device ( vertec , jump usa , sunnyvale , ca ) was used to measure maximal vertical jump height . participants reached to maximum vertical height on the vertec with their dominant arm while their feet were flat on the ground . each participant performed two countermovement jumps with arm swing and displacement of the highest vane was determined . to calculate maximum vertical jump height , all analyses were performed using a standard statistical software program ( ibm spss statistics for windows , version 21 , 2012 ) . pre- and post - testing differences among groups were assessed using an independent t - test . a repeated measures anova was utilized to assess potential differences in pre- and post - testing differences across groups . an a - priori -significance level of 0.05 was accepted as a reflection of differences in the mean . twenty ( n=20 ) healthy college - age female students ( 1825 yrs ) were recruited for the current investigation . approval by the slippery rock university institutional review board was acquired and all participants completed informed consent . participants were instructed to maintain their current aerobic exercise and dietary programs throughout the duration of the study . no participants had extensive experience with kettlebell exercise or completed sprint or resistance training in the previous 6 months as a means to limit extraneous variables from prior training history . study investigators , however , were trained in the safe and effective implementation of resistance exercise , and completed specific sessions on coaching the kbs and sdl . participants were excluded from the present study if they were not cleared by their physician for vigorous exercise , were currently utilizing a resistance training program , were outside of the age range ( 1825 ) , or were unable attend the scheduled sessions . participants were assigned into one of two groups ( kbs or sdl ) based on their initial sprint performance , to ensure that there were participants of similar ability in each cohort . following the group assignment , all participants participated in one instructional session on their assigned exercise prior to data collection . technique mastery was not necessary during the single instructional session as the participants were observed ( i.e. appropriate cueing ) throughout the duration of the training intervention for both safety and effectiveness . each group underwent an 8-week ( twice - weekly ) exercise intervention consisting of sixteen training sessions . quite simply , this schedule was chosen as it fulfilled training frequency guidelines for beginners ( i.e. 23 sessions per week ) , and fit within the time constraints of a university academic semester . each training session began with a 5 min light ( <3 mets ) aerobic warm - up on a treadmill followed by two 15 meter striders with a 30 second rest . all training sessions were performed using a tabata interval timer ( 20 seconds of exercise ; 10 seconds of rest ; 8 rounds ) based on previous work demonstrating a large anaerobic component to this training approach ( 5 , 15 ) . the participants were required to elect two non - consecutive days each week to exercise under the supervision of a project coordinator . a hardstyle kettlebell swing emphasizing maximal hip recruitment and minimal knee flexion was utilized in this study ( 7 ) . training started with a consistent prescribed weight ( ~9.1 kg ) with the potential for a ~2.3 kg ( next kettlebell weight increment ) increase following week three , and another ~2.3 kg increase at week six , if the participant maintained proper form throughout each session . form was monitored by trained instructors throughout the sessions , and included emphasis on maintaining an explosive concentric phase during maximal hip recruitment . training started with a consistent barbell weight ( ~27.3 kg ) , with the potential for a ~4.5 kg increase following week 3 , and another ~4.5 kg increase at week 6 , if the participant maintained proper form throughout each session . form checks for the sdl group included the maintenance of a two - second up / two - second down exercise tempo and maximal hip recruitment . each participant completed both a 30-meter sprint test and countermovement vertical jump test on a consistent , indoor track . identical post - testing was completed between 48 and 72 hours of the final exercise session . if a participant performed the vertical jump test before the sprint test in pre - testing , they were asked to perform the sprint test first for post - testing . each participant performed the same warm - up during the pre- and post - training testing . this warm - up consisted of a 400-meter jog followed by a 40-meter interval of the following drills : high knees , butt kicks , walking knee - to - chest stretch , walking quad stretch , toe touches , hamstring swings , carioca , and striders . participants were given the option to add additional warm - up striders before performing their maximal sprint ability . thirty meter sprint performances were timed using a tc - photogate ( brower timing systems , draper , ut ) . participants used a standing position and began the maximal sprint individually without a formal start . the tc - photogate recorded when the participants crossed the start and the finish line . each participant performed two sprint trials with a two - minute rest in between attempts , with the fastest sprint recorded . a vertical jump measurement device ( vertec , jump usa , sunnyvale , ca ) was used to measure maximal vertical jump height . participants reached to maximum vertical height on the vertec with their dominant arm while their feet were flat on the ground . each participant performed two countermovement jumps with arm swing and displacement of the highest vane was determined . to calculate maximum vertical jump height , all analyses were performed using a standard statistical software program ( ibm spss statistics for windows , version 21 , 2012 ) . pre- and post - testing differences among groups were assessed using an independent t - test . a repeated measures anova was utilized to assess potential differences in pre- and post - testing differences across groups . an a - priori -significance level of 0.05 was accepted as a reflection of differences in the mean . one individual was removed due to an unrelated ankle injury ; and following the eighth week of training , another individual withdrew due to an appendectomy . pre- and post - testing data were recorded for eight kbs participants , and ten sdl participants . kbs had 95% adherence to the training intervention , with a total training volume of 15,850 swings . the average number of repetitions performed was 124 , with an average training session volume - load of ~1378.2 kg . however , only 6,746 sdls were completed ( due to the intentionally slower repetition velocity ) , with the average number of repetitions per training session at 43 . despite lower repetitions , there were no significant differences noted in the pre - training 30 m sprint times between the kbs and sdl cohorts ( table 1 , p>0.05 ) . likewise , there were no significant differences in the post - training 30 m sprint times between the kbs and sdl groups ( table 1 , p>0.05 ) . in kbs or sdl pre- to post - testing sprint times were not significantly different ( table 1 , p>0.05 ) . there were no significant differences noted in the pre - training vertical jump performances between the kbs and sdl groups ( table 1 , p>0.05 ) . likewise , there were no significant differences in the post - training vertical jump performance between the kbs and sdl groups ( table 1 , p>0.05 ) . however , across groups , pre- to post - testing vertical jump performances improved by approximately 4% ( table 1 , p<0.05 ) , with no effect of training group . to the knowledge of the authors , this is the first study to determine the effect of an 8-week kbs program versus a program of similar volume - load , and movement specificity using sdl on sprint and counter movement vertical jump performance . the primary finding in the current investigation was kbs training ( 8 week ; 16 sessions ) did not significantly increase sprinting performance when compared to sdl . additionally , vertical jump performance was improved with training . due to the design of the study , no differences existed in pre - intervention sprint times or vertical jump heights between groups . the current investigation measured recreationally - active females who did not partake in resistance training activities . it is known that training status and modality can influence determinants of power ( e.g. sprinting ) . our recreationally - active cohort was not unusual in that they demonstrated similar average 30 meter sprint times for females when compared to those studied by mangine and colleagues ( 5.20.2 v. 5.50.5 seconds ) ( 9 ) . interestingly , no differences in the post - intervention sprint performances between kbs and sdl were observed . while both cohorts increased training loads and volumes over the course of the eight - week intervention , likely indicating adaptation , kbs training provided no additional transfer to the actions of sprinting , though a modest increase in countermovement vertical jump performance was observed . to our knowledge , few investigators have attempted to quantify the efficacy of kettlebell exercise with equivocal findings . kbs has been reported to improve strength and power ( 7 , 10 ) , while another study has reported no additional benefit over traditional training ( 13 ) . it is likely that improvements with kbs may be due to the cyclical , eccentric loading phase of the exercise combined with the rapid reversal of force necessary to transition to the concentric phase . furthermore , the mechanical demand of the kbs is dictated by both a vertical and horizontal mechanical output ( 8) , with a ballistic component provided by the long lever arm and variable center of mass of the participant / kettlebell system . therefore , considering the movement- and velocity - specificity between kbs and sprinting , additional explanations for a lack of observed transfer in our findings must be considered . the multifaceted nature of sprint performance must be considered first and foremost in the discussion of the current findings . based on the duration of the training intervention , neural adaptations would likely account for a majority of the participants ability to adapt to the training regimen and increase training volume ( 1 , 12 ) . neural adaptations , while important , may not account for the spectrum of underlying changes necessary for improved sprint performance . it is likely that structural adaptations including muscle hypertrophy ( 3 ) , increases in stride frequency and length , and enhanced sprint technique ( 12 ) are critical components of sprint performance , but were not addressed in this intervention . the results of this study suggest that kbs training alone may not provide a sufficient stimulus to improve sprint performance in recreationally - active females even with a focus on movement and velocity specificity . it is likely that the present study may not have supplied a great enough training volume - load and intensity for improved sprint performance . in comparison to a study that demonstrated increases in explosive strength ( vertical jump ) in athletes using kbs ( 7 ) , our training volume was likely inadequate . assuming similar kbs movement and velocity specificity , when extrapolating an average training session from our study ( < 70 kg subject ; ~9.1 kg kettlebell ; 15 swings/20 seconds ; eight work intervals ) to training parameters of similar work in athletes ( < 70 kg subject ; 12 kg kettlebell ; ~22 swings/30 seconds ; twelve work intervals ) , our trainees received only one - third of the average training session volume ( 7 ) . additionally , it is likely that the work to rest ratio ( 2:1 ) of the tabata protocol resulted in muscular fatigue which limited participants from generating maximum power outputs throughout the exercise sessions . thus , the drop in power output and relative intensity could limit the transferability of the intervention to sprint performance , which utilizes maximum power output . it is likely that an altered work to rest ratio ( e.g. 1:4 or 1:5 ) would preserve movement quality and explosiveness to a greater degree than the tabata protocol . while athletes may respond differently to training , and pose greater challenges than their recreationally - active counterparts ( 12 ) , it is probable that the volumes and intensities used in this investigation were underestimated . a conservative starting load was chosen for the kbs group , as the best current guidelines for power programming highlight the need for rapid force development ( 5 ) . consistent load assignment was deemed for this experiment in order to ensure similar training volume across participant groups , though it is likely that some means of individual strength or power testing in order to more accurately assign participant loads may have improved measured outcomes . currently , however , specific load , intensity and volume recommendations for the various potential applications of the kbs are not readily available . with the effectiveness of kbs training on vertical jump performance documented ( 7 , 13 ) , vertical jump was used as a comparable benchmark to gauge participant responsiveness to training . as countermovement vertical jump height changed significantly in both training cohorts ( approximately 4% improvement ) it is more likely that a lack of emphasis on sprint mechanics and technique may have had a larger impact on the lack of transfer to sprint performance than a simple lack of training volume . though correct form and adherence to the hardstyle kettlebell swing and stiff - legged were coached across all 16 training sessions , nonconsecutive training days per week was chosen purely out of a need for freedom in scheduling between the participants and investigators , a more rigorous schedule format may have altered the findings of the study . it is plausible that both sprint and jump performances are sensitive to diurnal variation ( 14 ) . in the current study , scheduling conflicts resulted in a difference between pre- and post - testing time of day ( 12:002:00 pm and 7:009:00 am , respectively ) . furthermore , changes in the time of testing may have also impacted variables related to food consumption , hydration , body temperature , motivation , and sleep patterns . in conclusion , an eight - week kbs program was not shown to improve measures of sprint performance when compared to an sdl program . training volumes are an important consideration for effectiveness in interventions designed to improve sprint outcomes , however , an equal consideration must be placed on mechanics and technique . due to movement- and velocity - specificity , the kbs should not be excluded from resistance training programs designed to accompany sprint training , especially given the limited volume of literature currently available . recently , it has been reported that a two - handed kbs is appropriate for the training of ballistic or explosive outcomes ( 6 ) . it is plausible that an appropriate kbs prescription as part of a comprehensive training program ( e.g. teaching sprint technique ) can improve sprint performance . however , data from the current investigation do not confirm or support the efficacious use of kbs in sprinting . therefore , future research is still needed to determine the value of kbs in sprint - training programs and provide volume and intensity recommendations .

the kettlebell swing ( kbs ) , emphasizing cyclical , explosive hip extension in the horizontal plane , aligns with movement- and velocity - specificity of sprinting . the present study examined the effect of an eight - week kbs intervention on sprinting in recreationally - active females , in comparison to an eight - week intervention using the stiff - legged deadlift ( sdl ) . following a pre - testing session measuring 30 meter sprint and countermovement vertical jump performance , participants were divided evenly by sprint time into kbs ( n=8 ) and sdl ( n=10 ) cohorts . following familiarization with the exercises , kbs met twice weekly to perform swings using the tabata interval ( 20s work , 10s rest , 8 rounds ) , stressing a rapid , explosive tempo . in contrast , the sdl group performed their tabata stiff - legged deadlifts at a conventional resistance training tempo ( 2 seconds concentric , 2 seconds eccentric ) . following eight weeks and greater than 95% training adherence , the sdl group only had a slightly greater average training volume ( ~3% ) than kbs . no significant differences in pre - test values , or changes were noted in sprint performance from pre- to post - intervention in either group . an improvement in vertical jump performance was noted across groups . potential explanations for the lack of sprint improvement compared to previous studies include differences between recreationally - active and athletic females , and low exercise volume ( ~46% of a comparable study with improvements in vertical jump ) . future studies should seek to determine the appropriate volume and intensity for kbs components of sprint programming .

INTRODUCTION METHODS Participants Protocol Statistical Analysis RESULTS DISCUSSION

in central and eastern european and central asian regions , countries report high rates of abortion , while at the same time displaying higher levels of secondary infertility ( females with previous life births ) . additionally , the prevalence of primary infertility ( females with no previous life births ) is significantly higher in ukraine than in other countries of the region , reaching a level of 3% of the female population.1 despite an increasing medical demand for infertility treatments , public funding challenges for in vitro fertilization ( ivf ) exist in a number of jurisdictions . while some countries ( such as france , spain , and israel ) provide full coverage of ivf treatments as a matter of policy , others either partially cover expenses ( eg , portugal , sweden , turkey ) , or fail to cover it at all ( eg , india , the people s republic of china).2 meanwhile , when coverage for ivf is absent or incomplete , as is the case throughout the usa , it may lead to cases where ivf treatments are unaffordable to couples who need it most . moreover , and of particular interest from a governmental perspective , a number of economic studies have concluded that there are long - term financial benefits to be gained from creating new citizens who will eventually become future taxpayers . the cost - efficiency of state investments in ivf is assessed by calculation of net income , usually expressed through taxes and other state revenues received from the working population . economic evaluations revealed that there were net tax benefits of ivf financing in both high - income countries ( eg , the usa , uk , denmark , sweden)37 and medium - income countries like brazil.8 net income gained from the ivf populations in all countries studied was positive ; however , the largest gain was found for the uk ( 109,939 ) , while the smallest gain was found for brazil ( us$ 61,428 ) . while encouraging , these results may not be easily used in the decision - making process in other countries,9 such as those of the central and eastern european region . besides putative differences in health care systems and population characteristics , the country s wealth must be taken into account when considering the efficiency of medical technologies . for example , in lower - income countries , costly medical technologies potentially may be less cost - efficient than they would be in higher - income countries . countries of the central and eastern european region have , on average , a much lower gross domestic product ( gdp ) than their western european or north american counterparts ; thus , the generalizability may be questionable for economic studies on ivf subsidies from high - income countries ( like the usa and uk ) to lower - income jurisdictions . finally , the economic impact for the population ( expressed as the difference between state spending and economic benefits ) , may not be directly proportional to the gdp , depending more on internal policy of the country , such as tax level , social contributions , net revenues from public enterprises , and so on . thus , financing of ivf also may be cost - efficient for governments of countries with relatively low gdp per capita level . for example , while having universal health care coverage and free access to medical procedures for their respective populations , governments in former soviet union countries ( such as ukraine , belarus , kazakhstan ) do not consider ivf a priority , so provide only limited , insufficient funds for its coverage . however , economic analysis may be a justification for reexamining their policies where ivf coverage is concerned . with the above discussion as a rationale , the present study was conducted to : assess the economic cost and benefits of financing ivf technologies ( one cycle per woman ) in ukraine , belarus , and kazakhstan from the governmental perspective ; explore relationships between gdp per capita and level of financial impact on the population ; and address transferability of the received results to other countries of the central and eastern european region . similar to previous studies , an economic model using the methodology of generational accounting was developed to estimate for ukraine , belarus , and kazakhstan whether publicly funded ivf treatments result in a financial benefit , by calculating the net revenue gained from a child conceived via ivf in each country.38 generational accounting evaluates whether there will be sufficient tax revenue in the future to pay for current investments into ivf programs by calculating the net present value ( npv ) of lifetime net taxes ( gross taxes minus financial expenditures of the government on population ) . because taxation remains the main source of revenue for most states,5 using this applied approach provides an appropriate assessment of rationality for ivf investments by the governments . in the model we defined five stages during which populations have different expenses and revenues : prenatal ; early childhood ( from birth until school ) ; late childhood ( period when individual receives education including high school ) ; employment ; and retirement . the cohort is a receiver of financial flows from the state that consists of social support , sick leave payments , medical help , and education financing . during the employment period , the population provides revenue to the state in the form of tax payments , but also receives unemployment support and medical help . after retirement , the employment rate decreases , likewise tax contribution , but pension and health care are provided until the end of life . for each age category , state spending and income from population are calculated . similar to connolly et al46 and krger and ejzenberg,8 the following formula was applied for assessing the npv of lifetime net taxes : npv=t=0t(rtet(1+r)t)k0where rt is the sum of the governmental revenues from individual s age t ; et is the sum of the governmental expenditures from individual s age t ; r is the discount rate ; t is life expectancy ; and k0 is the direct costs of ivf . as npv is used to estimate how much future returns from the investment are worth today , npv > 0 represents profitable investment , npv = 0 represents investment that is neither profitable nor unprofitable , and npv < 0 represents unprofitable investment.7 applying ivf success rates , the costs per ivf - born cohort and cost per live birth is calculated . because belarus - specific data for this parameter were not available , it was assumed in the base case that the success of ivf was equal in all three countries to 31.9% on average ( success rate of ivf for 35- to 37-year - old women based on the data from the society for assisted reproductive technology summary report on 154,412 cycles conducted in 2011).10 the percentage of boys born as a result of intervention was assumed to be equal to the naturally born cohort and was assessed from data on sex ratios ( 51.70% , 51.5% , and 48.5% boys for born in ukraine , belarus , and kazakhstan , respectively).1113 country - specific state - registered prices were used to assess costs of drugs and medical supplies , while the need in quantities of the defined medical products per one ivf cycle was assessed from the national state reproductive program in ukraine ( and considered to be similar for belarus and kazakhstan).1416 despite all three countries providing universal free health care , country - specific pregnancy - related costs are unknown and , for this reason , medical care expenditures were accounted using costs per outpatient ( during ivf procedures and pregnancy ) and inpatient ( delivery ) visits by who - choice estimates.17 the average number of visits during one ivf cycle was calculated assuming the following referrals : first appointment ( one visit ) , preparation ( one visit ) , initiation ( one visit ) , visits during treatment with fertility medications ( five visits ) , egg harvesting ( one visit ) , embryo transfer ( one visit ) , consultation ( one visit ) . nine monthly visits to gynecologists and three hospitalization days were accounted for during pregnancy and delivery . although ivf procedures frequently result in multiple births , only one - child pregnancy was assumed in the base - case scenario . moreover , while during the early perinatal stage ivf children may require more medical assistance , in the life - duration model used here children conceived with ivf were considered to be comparable with those conceived naturally . ivf success data and rates of multiple deliveries due to ivf were used to understand if these country - specific parameters have a significant impact on the results . the following assumptions were used in this scenario : the birth rate from the ivf procedure was calculated from data on ivf success rate and multiple pregnancies from the study conducted by kupka et al18because of the low incidence of triple births ( 1% for both ukraine and kazakhstan)18 this parameter was accounted together with dual birthsbecause no belarus - specific data were available , the rate of births due to ivf was calculated as an average in russia , belarus , and kazakhstan18negative health impact and higher costs associated with multiple pregnancies included the following : higher probability of dying during neonatal the period ( 6.4 times);19 higher medical costs during the 1st year of life ( the calculated cost ratio between single and dual births , 3.29);19 and doubled payments for maternity support because of multiple births . the birth rate from the ivf procedure was calculated from data on ivf success rate and multiple pregnancies from the study conducted by kupka et al18 because of the low incidence of triple births ( 1% for both ukraine and kazakhstan)18 this parameter was accounted together with dual births because no belarus - specific data were available , the rate of births due to ivf was calculated as an average in russia , belarus , and kazakhstan18 negative health impact and higher costs associated with multiple pregnancies included the following : higher probability of dying during neonatal the period ( 6.4 times);19 higher medical costs during the 1st year of life ( the calculated cost ratio between single and dual births , 3.29);19 and doubled payments for maternity support because of multiple births . in all three analyzed countries , the first - child allowance was accounted in all of the cases , as it was considered that ivf is applied by childless families . it was also considered that one parent is not employed , thus receives monthly financial support for 3 years in belarus ( $ 82.10 per month ) and ukraine ( $ 16.26 per month ) and for 1 year in kazakhstan ( $ 214.2 per month ) as ensured by state policies in these countries ( estimation was made by the data of the labor informational resource [ http://mojazarplata.by ; http://mojazarplata.kz ; mojazarplata.com.ua ] ) . basing estimates on data from accounting departments in three companies ( two in ukraine and one in kazakhstan , 485 employees total ) , it was considered that mothers spend 15 sick leave days paid by the government annually per child aged 36 years and 10 days per child aged 712 years . in the model , people receive health care services throughout their lives and educational services from 0 to 19 years . average expenses of governmental support for unemployed were calculated using official unemployment rates among population of working age , coverage with financial support for unemployed , duration of unemployment , and the amount of financial support . from retirement until death , the population receives a pension from the government . because data on disability prevalence were unavailable , no additional costs besides those associated with regular health care were accounted . government revenue accrues from income tax on population of working age or from the aged population who continue to work and pay taxes . as no age - stratified income is available for the study countries , average salary and tax rates were applied for the entire lifetime of the cohort . though governments receive additional revenue from other sources ( eg , land taxes , business and enterprise payments , social contributions ) , income taxes are argued to be the largest part of state revenues ; therefore , the impact of population increase on state income was assessed as direct taxes from salaries on official employment . no country - specific data were available bearing on a relationship between age and income , thus average salaries , taxes , and employment rates were applied to calculate the income from the working - age population . moreover , a percentage of the retired population who are officially employed was used to calculate additional income from this group . life expectancy at birth for children born in 2009 was applied.20 all costs provided in the national currencies were transferred into us$ according to the national bank exchange rates on november 22 , 2014 . rate per 1 $ was equal to 15.096 uah ( ukraine ) , 10,780.00 byr ( belarus ) , and 180.87 kzt ( kazakhstan ) . state expenses before birth of the ivf cohort were adjusted to current prices ( 2014 ) using the inflation index for consumer prices.21 governmental payments and incomes were assumed to grow annually with the rate of annual gdp growth22 while being discounted at 3% in the deterministic model . the complete list of model input parameters together with a full reference list are provided in table s1 . the parameters possible for state regulation were varied in one - way sensitivity analysis with break - even costs calculation . subcategory analysis for women of different age categories presenting variations in ivf success rates was conducted to analyze an impact of ivf success rate on the results of economic analysis and , moreover , to assess the possible implication of limiting financing for different groups . as drug costs are frequently negotiable , probabilistic sensitivity analysis ( psa ) on the most influential parameters with 5,000 simulations was conducted to ensure the validity of the calculation . because the aim of psa was to assess the uncertainty related to long duration of the model ( cohort lifetime ) , the prenatal parameters which potentially can be controlled by the government ( ie , age of mother and ivf costs ) were excluded from this assessment . moreover , the ivf success rate was varied in the psa to address the uncertainty of the success of the procedure . costs included in the early childhood period were not varied in psa because of the low impact on a lifetime model and no data on deviation parameters . a number of prognostic factors ( such as a possible tax decrease in ukraine , prolongation of the retirement age to the average in the organisation for economic co - operation and development region for all three countries ) were assessed from publicly available information sources describing current political trends that may affect model results . because health care expenditures already are relatively low in the study countries , it was assumed they may only increase from current values or remain the same . if multiple data sources were available , the source providing the largest data deviation was used . the complete range of parameters and distributions used in the psa are presented in table s1 . similar to previous studies , an economic model using the methodology of generational accounting was developed to estimate for ukraine , belarus , and kazakhstan whether publicly funded ivf treatments result in a financial benefit , by calculating the net revenue gained from a child conceived via ivf in each country.38 generational accounting evaluates whether there will be sufficient tax revenue in the future to pay for current investments into ivf programs by calculating the net present value ( npv ) of lifetime net taxes ( gross taxes minus financial expenditures of the government on population ) . because taxation remains the main source of revenue for most states,5 using this applied approach provides an appropriate assessment of rationality for ivf investments by the governments . in the model we defined five stages during which populations have different expenses and revenues : prenatal ; early childhood ( from birth until school ) ; late childhood ( period when individual receives education including high school ) ; employment ; and retirement . the cohort is a receiver of financial flows from the state that consists of social support , sick leave payments , medical help , and education financing . during the employment period , the population provides revenue to the state in the form of tax payments , but also receives unemployment support and medical help . after retirement , the employment rate decreases , likewise tax contribution , but pension and health care are provided until the end of life . for each age category , state spending and income from population are calculated . similar to connolly et al46 and krger and ejzenberg,8 the following formula was applied for assessing the npv of lifetime net taxes : npv=t=0t(rtet(1+r)t)k0where rt is the sum of the governmental revenues from individual s age t ; et is the sum of the governmental expenditures from individual s age t ; r is the discount rate ; t is life expectancy ; and k0 is the direct costs of ivf . as npv is used to estimate how much future returns from the investment are worth today , npv > 0 represents profitable investment , npv = 0 represents investment that is neither profitable nor unprofitable , and npv < 0 represents unprofitable investment.7 applying ivf success rates , the costs per ivf - born cohort and cost per live birth is calculated . because belarus - specific data for this parameter were not available , it was assumed in the base case that the success of ivf was equal in all three countries to 31.9% on average ( success rate of ivf for 35- to 37-year - old women based on the data from the society for assisted reproductive technology summary report on 154,412 cycles conducted in 2011).10 the percentage of boys born as a result of intervention was assumed to be equal to the naturally born cohort and was assessed from data on sex ratios ( 51.70% , 51.5% , and 48.5% boys for born in ukraine , belarus , and kazakhstan , respectively).1113 country - specific state - registered prices were used to assess costs of drugs and medical supplies , while the need in quantities of the defined medical products per one ivf cycle was assessed from the national state reproductive program in ukraine ( and considered to be similar for belarus and kazakhstan).1416 despite all three countries providing universal free health care , country - specific pregnancy - related costs are unknown and , for this reason , medical care expenditures were accounted using costs per outpatient ( during ivf procedures and pregnancy ) and inpatient ( delivery ) visits by who - choice estimates.17 the average number of visits during one ivf cycle was calculated assuming the following referrals : first appointment ( one visit ) , preparation ( one visit ) , initiation ( one visit ) , visits during treatment with fertility medications ( five visits ) , egg harvesting ( one visit ) , embryo transfer ( one visit ) , consultation ( one visit ) . nine monthly visits to gynecologists and three hospitalization days were accounted for during pregnancy and delivery . although ivf procedures frequently result in multiple births , only one - child pregnancy was assumed in the base - case scenario . moreover , while during the early perinatal stage ivf children may require more medical assistance , in the life - duration model used here children conceived with ivf were considered to be comparable with those conceived naturally . ivf success data and rates of multiple deliveries due to ivf were used to understand if these country - specific parameters have a significant impact on the results . the following assumptions were used in this scenario : the birth rate from the ivf procedure was calculated from data on ivf success rate and multiple pregnancies from the study conducted by kupka et al18because of the low incidence of triple births ( 1% for both ukraine and kazakhstan)18 this parameter was accounted together with dual birthsbecause no belarus - specific data were available , the rate of births due to ivf was calculated as an average in russia , belarus , and kazakhstan18negative health impact and higher costs associated with multiple pregnancies included the following : higher probability of dying during neonatal the period ( 6.4 times);19 higher medical costs during the 1st year of life ( the calculated cost ratio between single and dual births , 3.29);19 and doubled payments for maternity support because of multiple births . the birth rate from the ivf procedure was calculated from data on ivf success rate and multiple pregnancies from the study conducted by kupka et al18 because of the low incidence of triple births ( 1% for both ukraine and kazakhstan)18 this parameter was accounted together with dual births because no belarus - specific data were available , the rate of births due to ivf was calculated as an average in russia , belarus , and kazakhstan18 negative health impact and higher costs associated with multiple pregnancies included the following : higher probability of dying during neonatal the period ( 6.4 times);19 higher medical costs during the 1st year of life ( the calculated cost ratio between single and dual births , 3.29);19 and doubled payments for maternity support because of multiple births . in all three analyzed countries , child benefits are provided to the families of newborns . the first - child allowance was accounted in all of the cases , as it was considered that ivf is applied by childless families . it was also considered that one parent is not employed , thus receives monthly financial support for 3 years in belarus ( $ 82.10 per month ) and ukraine ( $ 16.26 per month ) and for 1 year in kazakhstan ( $ 214.2 per month ) as ensured by state policies in these countries ( estimation was made by the data of the labor informational resource [ http://mojazarplata.by ; http://mojazarplata.kz ; mojazarplata.com.ua ] ) . basing estimates on data from accounting departments in three companies ( two in ukraine and one in kazakhstan , 485 employees total ) , it was considered that mothers spend 15 sick leave days paid by the government annually per child aged 36 years and 10 days per child aged 712 years . in the model , people receive health care services throughout their lives and educational services from 0 to 19 years . average expenses of governmental support for unemployed were calculated using official unemployment rates among population of working age , coverage with financial support for unemployed , duration of unemployment , and the amount of financial support . from retirement until death , the population receives a pension from the government . because data on disability prevalence were unavailable , no additional costs besides those associated with regular health care were accounted . applying ivf success rates , the costs per ivf - born cohort and cost per live birth is calculated . because belarus - specific data for this parameter were not available , it was assumed in the base case that the success of ivf was equal in all three countries to 31.9% on average ( success rate of ivf for 35- to 37-year - old women based on the data from the society for assisted reproductive technology summary report on 154,412 cycles conducted in 2011).10 the percentage of boys born as a result of intervention was assumed to be equal to the naturally born cohort and was assessed from data on sex ratios ( 51.70% , 51.5% , and 48.5% boys for born in ukraine , belarus , and kazakhstan , respectively).1113 country - specific state - registered prices were used to assess costs of drugs and medical supplies , while the need in quantities of the defined medical products per one ivf cycle was assessed from the national state reproductive program in ukraine ( and considered to be similar for belarus and kazakhstan).1416 despite all three countries providing universal free health care , country - specific pregnancy - related costs are unknown and , for this reason , medical care expenditures were accounted using costs per outpatient ( during ivf procedures and pregnancy ) and inpatient ( delivery ) visits by who - choice estimates.17 the average number of visits during one ivf cycle was calculated assuming the following referrals : first appointment ( one visit ) , preparation ( one visit ) , initiation ( one visit ) , visits during treatment with fertility medications ( five visits ) , egg harvesting ( one visit ) , embryo transfer ( one visit ) , consultation ( one visit ) . nine monthly visits to gynecologists and three hospitalization days were accounted for during pregnancy and delivery . although ivf procedures frequently result in multiple births , only one - child pregnancy was assumed in the base - case scenario . moreover , while during the early perinatal stage ivf children may require more medical assistance , in the life - duration model used here children conceived with ivf were considered to be comparable with those conceived naturally . ivf success data and rates of multiple deliveries due to ivf were used to understand if these country - specific parameters have a significant impact on the results . the following assumptions were used in this scenario : the birth rate from the ivf procedure was calculated from data on ivf success rate and multiple pregnancies from the study conducted by kupka et al18because of the low incidence of triple births ( 1% for both ukraine and kazakhstan)18 this parameter was accounted together with dual birthsbecause no belarus - specific data were available , the rate of births due to ivf was calculated as an average in russia , belarus , and kazakhstan18negative health impact and higher costs associated with multiple pregnancies included the following : higher probability of dying during neonatal the period ( 6.4 times);19 higher medical costs during the 1st year of life ( the calculated cost ratio between single and dual births , 3.29);19 and doubled payments for maternity support because of multiple births . the birth rate from the ivf procedure was calculated from data on ivf success rate and multiple pregnancies from the study conducted by kupka et al18 because of the low incidence of triple births ( 1% for both ukraine and kazakhstan)18 this parameter was accounted together with dual births because no belarus - specific data were available , the rate of births due to ivf was calculated as an average in russia , belarus , and kazakhstan18 negative health impact and higher costs associated with multiple pregnancies included the following : higher probability of dying during neonatal the period ( 6.4 times);19 higher medical costs during the 1st year of life ( the calculated cost ratio between single and dual births , 3.29);19 and doubled payments for maternity support because of multiple births . in all three analyzed countries , child benefits are provided to the families of newborns . the first - child allowance was accounted in all of the cases , as it was considered that ivf is applied by childless families . it was also considered that one parent is not employed , thus receives monthly financial support for 3 years in belarus ( $ 82.10 per month ) and ukraine ( $ 16.26 per month ) and for 1 year in kazakhstan ( $ 214.2 per month ) as ensured by state policies in these countries ( estimation was made by the data of the labor informational resource [ http://mojazarplata.by ; http://mojazarplata.kz ; mojazarplata.com.ua ] ) . basing estimates on data from accounting departments in three companies ( two in ukraine and one in kazakhstan , 485 employees total ) , it was considered that mothers spend 15 sick leave days paid by the government annually per child aged 36 years and 10 days per child aged 712 years . in the model , people receive health care services throughout their lives and educational services from 0 to 19 years . average expenses of governmental support for unemployed were calculated using official unemployment rates among population of working age , coverage with financial support for unemployed , duration of unemployment , and the amount of financial support . from retirement until death , the population receives a pension from the government . because data on disability prevalence were unavailable , no additional costs besides those associated with regular health care government revenue accrues from income tax on population of working age or from the aged population who continue to work and pay taxes . as no age - stratified income is available for the study countries , average salary and tax rates were applied for the entire lifetime of the cohort . though governments receive additional revenue from other sources ( eg , land taxes , business and enterprise payments , social contributions ) , income taxes are argued to be the largest part of state revenues ; therefore , the impact of population increase on state income was assessed as direct taxes from salaries on official employment . no country - specific data were available bearing on a relationship between age and income , thus average salaries , taxes , and employment rates were applied to calculate the income from the working - age population . moreover , a percentage of the retired population who are officially employed was used to calculate additional income from this group . life expectancy at birth for children born in 2009 was applied.20 all costs provided in the national currencies were transferred into us$ according to the national bank exchange rates on november 22 , 2014 . rate per 1 $ was equal to 15.096 uah ( ukraine ) , 10,780.00 byr ( belarus ) , and 180.87 kzt ( kazakhstan ) . state expenses before birth of the ivf cohort were adjusted to current prices ( 2014 ) using the inflation index for consumer prices.21 governmental payments and incomes were assumed to grow annually with the rate of annual gdp growth22 while being discounted at 3% in the deterministic model . the complete list of model input parameters together with a full reference list are provided in table s1 . the parameters possible for state regulation were varied in one - way sensitivity analysis with break - even costs calculation . subcategory analysis for women of different age categories presenting variations in ivf success rates was conducted to analyze an impact of ivf success rate on the results of economic analysis and , moreover , to assess the possible implication of limiting financing for different groups . as drug costs are frequently negotiable , the impact of changes in ivf expenses on npv was assessed . probabilistic sensitivity analysis ( psa ) on the most influential parameters with 5,000 simulations was conducted to ensure the validity of the calculation . because the aim of psa was to assess the uncertainty related to long duration of the model ( cohort lifetime ) , the prenatal parameters which potentially can be controlled by the government ( ie , age of mother and ivf costs ) were excluded from this assessment . moreover , the ivf success rate was varied in the psa to address the uncertainty of the success of the procedure . costs included in the early childhood period were not varied in psa because of the low impact on a lifetime model and no data on deviation parameters . a number of prognostic factors ( such as a possible tax decrease in ukraine , prolongation of the retirement age to the average in the organisation for economic co - operation and development region for all three countries ) were assessed from publicly available information sources describing current political trends that may affect model results . because health care expenditures already are relatively low in the study countries , it was assumed they may only increase from current values or remain the same . if multiple data sources were available , the source providing the largest data deviation was used . the complete range of parameters and distributions used in the psa are presented in table s1 . the observed cost variation for different schemes of ivf cycles applied resulted in a difference in average cost per cycle , with the lowest one observed in ukraine ( $ 732 per cycle ) and the highest one in belarus ( $ 1,607 per cycle ) . using a similar ivf success rate , the average cost of one ivf birth from the governmental perspective varied from $ 2,599 in ukraine to $ 5,509 in belarus ( table 1 ) . projected lifetime net revenues from the ivf cohort are illustrated in figure 1 , where changes in npv depending on the age of the ivf cohort are observed . while in the early stages of an individual s life , net monetary flows are negative for the government since education , health and social support are provided to the family by state without financial returns , during the working years , the individual s financial balance tips positive for the government , as state revenue is collected as tax payments and lower social expenses are paid . advancing in age , individuals begin to provide less revenue to the state while simultaneously receiving increased social spending , primarily because of pension payments . because the net revenue positions for an ivf - conceived and a naturally - conceived child follow similar trajectories , where the only difference between the two is the additional cost of ivf investment required for conception , figure 1 presents the npv for an ivf - conceived individual only . in light of the observed difference in expenses on population in the three study countries , ivf may be considered an attractive economic option in ukraine , belarus , and kazakhstan , with discounted npvs of $ 9,839 , $ 21,702 , and $ 2,295 , respectively ( table 2 ) . when country - specific ivf birth rates were applied in the model considering both ivf success rate and multiple pregnancies ( table 2 ) , the results did not change significantly . a possible explanation for the small impact of higher - cost ivf children stems from the positive economic impact of the individual in general . as such , higher frequency of multiple births in ivf population compensates for the additional expenses related to ivf newborns and the higher mortality during neonatal stage . the psa based on 5,000 simulations shows that the average npv per person remains positive : $ 1,894$7,619 , $ 27,925$12,407 , and $ 17,229$24,637 in ukraine , belarus , and kazakhstan , respectively . at the same time , the ranges and standard deviations for ukraine and kazakhstan indicate that under some circumstances ( meaning of inputs ) , financing of ivf can become negative for these countries . the results of one - way sensitivity analysis ( table 3 ) show a positive npv until the mother s age is over 42 years in ukraine and belarus , while in kazakhstan the break - even age of the mother ( age at which financing ivf remains economically beneficial for the government ) is 3840 years . the costs of ivf drugs and supplies impact significantly the results of the economic analysis . a negative npv was obtained using a discount rate of 10% in all three countries , as well as with a discount rate of 0% in belarus and kazakhstan . in the psa , with a fixed 0% discount rate , a negative npv was obtained for ukraine and kazakhstan ( $ 19,962$33,263 and $ 44,084$89,815 , respectively ) and positive for belarus ( $ 24,328$63,580 ) . the instability of these results is indicated by the value of the standard deviation , which exceeds the average value . the results presented in this paper show how public financing of ivf in three former soviet union countries ( belarus , kazakhstan , and ukraine ) will generate a positive return to the state in future tax contributions . understanding the financial benefits from medical technologies not related to life - saving technologies is especially important in jurisdictions where financial resources are limited , such as in low - income and middle - income countries like ukraine , belarus , and kazakhstan . economic evidence in terms of cost minimization or budget impact techniques may be applied to rationalize financing a limited number of ivf cycles , or to define an intent - to - treat patient population . while in belarus nearly 600 children were born via state - financed ivf treatments,23 ukraine,16 and kazakhstan together report about 600 ivf cycles to be state - financed annually ( personal communication ) , although in kazakhstan this number is expected to rise in 2015 . the present research shows that financing ivf may have a positive npv , not only in high - income but also in lower - income countries . based on the average cost per child conceived with ivf in a state clinic and using current levels of financial flows between populations and governments of ukraine , belarus , and kazakhstan , the discounted returns to state all were positive over the projected lifetime of an individual , with higher uncertainty of results for ukraine and kazakhstan . a higher npv from financing ivf in ukraine , belarus , and kazakhstan can be achieved by limiting coverage of the procedure to women of younger age and by negotiating lower prices with ivf drug suppliers . in western european countries and brazil , the discounted npv of ivf ranged from $ 61,428 ( brazil ) to $ 177,002 ( uk ; exchange rate 1 pound = us$ 1.61 on september 9 , 2014 ) , while in the countries of the central and eastern european region studied here the financial returns to the state were significantly lower , although still positive.38 however , an interesting observation from this study is that the npv derived from an ivf population may not always be proportional to the income level of the country , expressed in gdp per capita . for example , the lowest financial return in the present study was observed in the country with the highest gdp per capita , ie , kazakhstan . it should be noted that gdp per capita is not always the best approach for evaluating the income of a country s population , as wealth can be distributed unequally , an especially common case in countries with a developing economy . meanwhile , it also may be assumed that state income from population may not be related linearly to the country s wealth in general in cross - country comparisons , because of differences in taxation policies and government spending . another interesting conclusion resulted from the probabilistic model applied , in which we tried to account for possible changes of the input parameters that may be expected during the long run of the model ( lifetime of the ivf cohort ) . while applying the individual prognostic factors for each country , it appeared that the npv in the probabilistic model may differ from the deterministic model , a finding explained by differences in economic forecast for a long time horizon . moreover , opposite the deterministic model , the npv of the ivf population may be higher in kazakhstan than in ukraine , if changes in the taxation policy ( which are currently under political discussion ) are applied in the future . because of the long horizon of generational accounting models , we suggest that it is obligatory to apply psa in order to define the stability of the received results under conditions of possible political and economic change . the sensitivity analysis with the 0% discount rate has shown the inaccuracy of using this value in a life - duration model based on generational accounting . the assumption in the model that annual expenses increase proportionally to gdp makes the expenses on the retired population inaccurately higher than on the working population in countries with larger values for pensions and gdp growth . the generational accounting framework from the governmental perspective used in this model assesses costs and benefits attributed to conceiving an ivf child as an investment required to achieve a live birth with consequent long - term economic returns . this economic model was used to assess the cost efficiency of state investments in countries with nationally funded health services ( ukraine , belarus , and kazakhstan ) where both investments ( financing ivf procedures ) and returns ( tax received ) will present a financial flow between two stakeholders , population , and state . this model may be potentially applied to other countries with similar political , economic , and health care structures , where major state revenues are expected to come from tax payments ( such as russia , georgia , or azerbaijan ) . while the results of the current study show a positive economic balance with stability of the received results by psa in ukraine , belarus , and kazakhstan , the transferability of the model to other countries of the region may be assessed in the future . in most western european countries , a complete ivf treatment consists of a maximum of three ivf cycles , where treatment choices for each cycle can differ . in a cost - effectiveness analysis reflecting the real - world situation conducted in the netherlands , it was found that combining several transfer policies was not cost - effective , and so the single - choice treatment option should be preferred , ie , elective single embryo transfer , standard treatment policy , or double embryo transfer.24 a cost - effectiveness analysis of replacing one , two , or three embryos per cycle of ivf in specific populations of women ( < 38 years , 38 years ; one cycle , two cycles , and three or more cycles ) has shown that the most cost - effective and least cost - effective scenarios occurred , respectively , with younger and older women who received three or more cycles , in the move from one embryo transfer to two embryo transfers.25 meanwhile , in the current study , we assessed only two scenarios : expenses and incomes related to one birth only , with the ivf success rate aiming at the minimum budget impact for the intervention financing ; expenses and incomes related to ivf birth according to the current countries data on multiple births and cycle success rate . taking into consideration cost - efficiency of the different ivf procedures in future studies may show increases in state benefits from ivf financing . while taxation - based income may represent a reasonably accurate means of estimating future economic benefits for the state , it should be noted that population also contributes to other financial governmental flows , such as trade and enterprise development , which was not accounted for in the current model . this may particularly affect the results of economic studies in belarus , where the unemployment rate is assessed by the number of people receiving unemployment support from the government . the model accounts for linear increases in spending and earning , based on average values from a retrospective historical assessment of the countries input parameters . as a consequence , the impact of unpredictable economic crises or growth also was not accounted for in the evaluation . another model limitation is that the calculation applied average earnings in the population , ignoring the possibility of wealthier generations in the future . while the return of state investments was assessed from the narrow governmental perspective using only future net tax contributions , we may consider that with broader assessment of net marginal contributions from individuals , the net state benefit from ivf - conceived children will present an even more attractive economic option . while taxation - based income may represent a reasonably accurate means of estimating future economic benefits for the state , it should be noted that population also contributes to other financial governmental flows , such as trade and enterprise development , which was not accounted for in the current model . this may particularly affect the results of economic studies in belarus , where the unemployment rate is assessed by the number of people receiving unemployment support from the government . the model accounts for linear increases in spending and earning , based on average values from a retrospective historical assessment of the countries input parameters . as a consequence , the impact of unpredictable economic crises or growth also was not accounted for in the evaluation . another model limitation is that the calculation applied average earnings in the population , ignoring the possibility of wealthier generations in the future . while the return of state investments was assessed from the narrow governmental perspective using only future net tax contributions , we may consider that with broader assessment of net marginal contributions from individuals , the net state benefit from ivf - conceived children will present an even more attractive economic option . the results of this study may have implications for ivf reimbursement policy not only in ukraine , belarus , and kazakhstan , but in other settings with comparable populations and financial flows between population and governments , particularly those which may be considering universal coverage for fertility treatments . while income from a population may not be directly proportional to gdp per capita , it appears that financing ivf technologies collectively may represent a promising potential for state financial returns . input parameters in deterministic and probabilistic models assumption 22.8% difference in male / female salaries according to state statistics report ( ukraine);2 assumption 61.9% difference in male / female salaries according to state statistics report ( kazakhstan);14 average monthly support received for 12 months ; average monthly support received for maximum 4 months ( under the law on obligatory social insurance of the republic of kazakhstan ) ; assumed that retirement age will not be lowered from existing .

backgroundan economic value calculation was performed to estimate the lifetime net present value of in vitro fertilization ( ivf ) in ukraine , belarus , and kazakhstan.methodsnet lifetime tax revenues were used to represent governmental benefits accruing from a hypothetical cohort of an ivf population born in 2009 using the methodology of generational accounting . governmental expenses related to this population included social benefits , education and health care , unemployment support , and pensions . where available , country - specific data referencing official sources were applied.resultsthe average health care cost needed to achieve one additional birth from the governmental perspective varied from $ 2,599 in ukraine to $ 5,509 in belarus . the net present value from the population born using ivf was positive in all countries : for ukraine ( $ 9,839 ) , belarus ( $ 21,702 ) , and kazakhstan ( $ 2,295 ) . the break - even costs of drugs and supplies per ivf procedure is expected to be $ 3,870 , $ 8,530 , and $ 1,780 , respectively . probabilistic sensitivity analyses based on 5,000 simulations show that the average net present value per person remains positive : $ 1,894$7,619 , $ 27,925$12,407 , and $ 17,229$24,637 in ukraine , belarus , and kazakhstan , respectively.conclusionfinancing ivf may represent a good investment in terms of governmental financial returns , even in lower - income countries with state - financed health care systems such as ukraine , belarus , and kazakhstan .

Introduction Materials and methods Model design IVF costs and outcome Base-case scenario Country-specific scenario Government transfers State income Other input parameters of the model Validity of the model Results Discussion Limitations Conclusion Supplementary materials

every year , more than 1 billion people are infected and more than 1 million die from vector - borne diseases . world health organization ( who ) has highlighted the serious and increasing threat of vector - borne diseases with the theme preventing vector - borne diseases and also with the slogan small bite , big threat for the year 2014 . among vector - borne diseases , malaria poses the biggest threat with about 40% of the world 's population at risk of infection . in 2013 , 97 countries had ongoing transmission of malaria . malaria decreases economic growth by more than one percentage point per year in endemic countries . malaria transmission season generally coincides with the harvesting season and brief periods of illness exact a high cost on the world 's poorest regions . in spite of various recent advancements in diagnostic and treatment modalities , malaria still remains a public health problem in developing countries and changing environmental and climatic conditions are considered as the biggest challenge in fighting against the scourge of malaria . malaria is an entirely preventable and treatable illness caused by parasites of plasmodium species and transmitted exclusively by the bites of anopheles mosquito . although preventable and treatable , malaria causes significant morbidity and mortality , particularly in resource poor regions . there were an estimated 207 million cases of malaria and an estimated 627 000 deaths in 2012 . in south east asia , which is the second most affected region by malaria in the world , india has the maximum number of cases with an estimated 24 million cases per year . unlike africa , where most of the deaths are reported in infants and children , in india , it is seen that malarial mortality is maximum in the economically productive age groups of 15 to 44 years . a malaria - stricken family spends an average of over one quarter of its income on malaria treatment , as well as paying prevention costs and suffering loss of income . due to the severe health impact of malaria , there is a growing need for methods that will allow forecasting and early warning with timely case detection in areas of unstable transmission , so that more control measures can be implemented effectively . studies of malaria epidemics have shown their association with climatic conditions like rainfall patterns , temperature , and humidity . in many places , malaria infections are often more common during rainy seasons because of increase in number of breeding sites . optimal conditions for malaria transmission occur when the temperature is between 20c and 30c and the mean relative humidity is at least 60% . water temperatures regulate the duration of aquatic breeding cycle of the mosquito vector and high relative humidity increases mosquito longevity . although there has been marked reduction in the number of malaria cases in india under national vector borne disease control programme ( nvbdcp ) , malaria still is the leading cause of infectious diseases with the development of drug resistant plasmodium species and insecticide resistant mosquitoes . forecasting of malaria cases allows for allocation of appropriate resources to target prevention and treatment of malaria and also to plan for eventual elimination . malaria incidence in a particular month can be predicted by rainfall , temperature , and relative humidity . therefore , the present study was designed with objectives of developing a temporal model for forecasting malaria cases using climatic factors such as rainfall , relative humidity , and mean maximum temperature as predictors in delhi , india . the study was conducted at the rural health training centre ( rhtc ) , najafgarh , which is a field practice area of the department of community medicine , vardhman mahavir medical college and safdarjung hospital . the study area is situated at an altitude of 216 metres above sea level and has a monsoon influenced humid subtropical climate with high variation between summer and winter temperature and precipitation . the total number of monthly cases of malaria slide positives from january 2006 to december 2013 was taken from the register maintained at the malaria clinic located at rhtc najafgarh . this is the only malaria clinic located in the catchment area of rhtc najafgarh , new delhi , and it captures almost all malaria cases in that area . the data for mean rainfall , relative humidity ( at 08:30 ist ) , and mean maximum temperature for the corresponding months were taken from regional meteorological centre , delhi . since the data was collected from official registers and no personal information of any kind was obtained , ethical clearance was not sought . 21 software was used to fit the best suitable model for the time series data . the stationarity of the data was checked by autocorrelation function ( acf ) and partial autocorrelation function ( pacf ) . seasonal adjusted factor ( saf ) was used to determine the peak of seasonal variation . the ljung - box ( modified box - pierce ) test was used to determine if the model was correctly specified . to address the confounding factors , forecasting of the incidence of monthly malaria cases the total number of monthly laboratory confirmed cases of malaria showed a declining trend from the year 2006 to 2011 ( table 1 ) . but it increased during the years 2012 and 2013 due to increase in the amount of rainfall received as shown in figure 1 . exploration of the monthly malaria infections , rainfall pattern , mean maximum temperatures , and relative humidity from 2006 to 2013 shows no clear trend and suggests a seasonal dependency in the series . the significant peaks in the series created by monthly malaria infections appeared to be separated by more than a few months showing a cyclical seasonal pattern as the peak of malaria infections follows a similar pattern with an interval of few months between the peaks ( figure 1 ) . the autocorrelation function ( acf ) for the series by monthly malaria infections showed a significant peak at a lag of 12 months ( autocorrelation = 0.675 ; box - ljung statistics ( p = 0.000 ) ) suggesting the presence of a seasonal component in the data . partial autocorrelation function ( pacf ) also showed a significant peak at a lag of 12 which confirmed the presence of seasonal component in the time series data . table 2 shows that from the month of june to september , the seasonal adjustment factor ( saf ) of malaria was more than 1 ; that is , in these months the malarial infections were more above the typical months . august had the highest saf of 3.14 followed by september which had saf of 3.1 . this shows that the peak of malaria transmission occurs during these months where the risk is 3.14 times and 3.1 times higher than a typical month . 21 was used to find the best fit model for forecasting malaria cases using mean rainfall , relative humidity , and mean maximum temperature , all lagged at one month as covariates . it suggested autoregressive integrated moving average model , arima ( 0,1,1 ) ( 0,1,0 ) as the best fit statistical model for this time series data . it was found that only two climatic factors , mean rainfall ( p value = 0.004 ) , and relative humidity ( p value = 0.001 ) both lagged at one month were significant predictors of malaria infections in the study area . the actual observed values and the predicted values matched reasonably well ( figure 2 ) . the ljung - box ( modified box - pierce ) statistics indicated that the model was correctly specified ( table 2 ) . although the time series modeler offers a number of different goodness - of - fit statistics , here stationary r - squared value was used since it provides an estimate of the proportion of the total variation in the series that is explained by the model . it is preferable to ordinary r - squared when there is a trend or seasonal pattern as is the case here . larger values of stationary r - squared ( up to a maximum value of 1 ) indicate better fit . a value of 0.725 meant that the model could explain 72.5% of the observed variation in the series ( table 3 ) . the forecasting model proposed , arima , provides a comprehensive set of tools for univariate time series model identification , parameter estimation , and forecasting , and it offers great flexibility in analysis , which has contributed to its popularity in several areas of research and practice . a seasonal arima model is represented by arima ( p , d , q)(p , d , q ) where p and p- are the autoregressive and seasonal autoregressive , respectively , d and d- are the nonseasonal differences and seasonal differencing , respectively , q and q- are the moving average parameters and seasonal moving average parameters , respectively , and s represents the length of the seasonal period . arima ( 0,1,1 ) ( 0,1,0 ) model was used to forecast the monthly malaria cases for the future from january 2014 to december 2015 . this model also included the significant predictors for malaria cases , rainfall , and relative humidity which were lagged at one month . the forecasted model predicted that there will be a total of 41 cases of malaria in 2014 and 53 cases in 2015 . the forecasted cases also show a seasonal pattern with significant peaks during rainy season ( figure 3 ) . arima models are useful in modeling the temporal dependence structure of a time series as they explicitly assume temporal dependence between observations and a useful tool in epidemiological surveillance . arima ( 0,1,1 ) ( 0,1,0 ) model developed in this study attempts to provide a simple tool to predict the expected number of malaria cases per month in the future based on observed malaria cases over the years and combination of climatic factors ; in this study , rainfall and relative humidity were found to be significant predictors . these climatic covariates were lagged at one month because anopheles vector takes two weeks to complete their life cycle and additional two more weeks for the generation of parasites in the new host . have also found arima ( 2,1,1 ) ( 0,1,1 ) to be the best possible model for predicting malaria cases in bhutan . arima model was also used for forecasting malaria cases in sri lanka and ethiopia . malaria transmission was found to occur between the months of july and september with a peak in the month of august . this shows that the malaria incidence in delhi has a strong correlation with rainfall in the preceding month . it is also interesting to find that the maximum number of cases occur during the months of august and september and decreases thereafter . continuous and heavy rainfall washes off the vector breeding places . though rainy season in delhi begins in late june , it receives heavy rainfall during august and september . malaria transmission also depends on relative humidity since mosquitoes have a limited range of tolerable relative humidity . the high surface area to volume ratio of mosquitoes makes them especially sensitive to desiccation at low humidity levels . transmission of malaria occurs when the relative humidity is at least 60% . in the present study , relative humidity in delhi remains above 60% in the peak transmission season except during the summer months of april and may during which the transmission of malaria was also found to be low . apart from rainfall and relative humidity , temperature also plays a significant role in malaria transmission . temperature rise is expected to increase transmission and prevalence of malaria by shortening the incubation period of the parasite in the mosquitoes . sporogonic cycles take about 9 to 10 days at temperatures of 28c but higher than 30c and below 16c have negative impact on parasite development . but in the present study , mean maximum temperature was not found to be a significant predictor of malaria incidence unlike studies done in bhutan and rwanda . the mean maximum temperature during summers in delhi is above 40c which is unsuitable for vector breeding unlike bhutan where the mean maximum temperature is around 35c . apart from climatic factors , various other factors like the type of vector , the type of parasite , population movement and migration , urbanization , the level of immunity to malaria in the human hosts , insecticide resistance in mosquitoes , and drug resistance in parasites all play a significant role in affecting the severity and incidence of malaria . the statistical model developed in this study assumes that these factors remain constant over a period of time , taking only climatic predictors to forecast malaria incidence . based on the results of the present study , we believe that malaria transmission in delhi depends on rainfall and relative humidity . the results also indicate that the malaria cases will continue to occur in the near future if appropriate actions are not initiated on time . the potential implication of this study is that by developing forecasting models for predicting the expected number of malaria cases in advance , timely prevention and control measures can be effectively planned like eliminating vector breeding places , spraying insecticides , and creating public awareness months before the peak season . the study also provides a model to foresee and allocate appropriate resources during peak transmission seasons . the time series model used in this study can also be used to inform travelers about malaria risk and screening and to take necessary precautionary measures . the study assumes that the reporting and registering of monthly malaria cases remain the same throughout the study period . since the study was done by collecting data from a single centre , it is difficult to generalize the results in the actual population . the study can be done on a wider area and further research can be done to evaluate the effectiveness of integrating the forecasting model into the existing disease control program in terms of its impact in reducing the disease occurrence . a seasonal pattern was observed in malaria incidence in delhi with variable amplitudes of fluctuation with peak during the months of august and september . arima ( 0,1,1 ) ( 0,1,0 ) model was found to be the best fit statistical model for predicting malaria cases in delhi . rainfall and relative humidity were found to be strong predictors of malaria infection in the study area . this information will also be useful for administrators in effectively implementing preventive and control measures for malaria .

background . malaria still remains a public health problem in developing countries and changing environmental and climatic factors pose the biggest challenge in fighting against the scourge of malaria . therefore , the study was designed to forecast malaria cases using climatic factors as predictors in delhi , india . methods . the total number of monthly cases of malaria slide positives occurring from january 2006 to december 2013 was taken from the register maintained at the malaria clinic at rural health training centre ( rhtc ) , najafgarh , delhi . climatic data of monthly mean rainfall , relative humidity , and mean maximum temperature were taken from regional meteorological centre , delhi . expert modeler of spss ver . 21 was used for analyzing the time series data . results . autoregressive integrated moving average , arima ( 0,1,1 ) ( 0,1,0)12 , was the best fit model and it could explain 72.5% variability in the time series data . rainfall ( p value = 0.004 ) and relative humidity ( p value = 0.001 ) were found to be significant predictors for malaria transmission in the study area . seasonal adjusted factor ( saf ) for malaria cases shows peak during the months of august and september . conclusion . arima models of time series analysis is a simple and reliable tool for producing reliable forecasts for malaria in delhi , india .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

quality of services is one of the most important factors for growth , success , and the presences of organizations , and it was considered as an effective and comprehensive strategy in order to improve their performances throughout the world ( 1 , 2 ) . in the health sector compared with other sectors , the quality of health services provided is more than important because the health sector has a significant association with the length of people s lives and their well - being . because of the importance of health services , the quality of health services provided by health organizations is becoming more and more crucial , and the demands for quality control , management , and improvements are increasing ( 3 , 4 ) . hospitals , as the main providers of healthcare services in all countries , should be moved toward its main objectives , including providing healthcare services with high quality and meeting the needs and expectations of patients ( 4 , 5 ) . the main ways to improve the quality of healthcare services is to monitor and evaluate the quality of the healthcare provided over time by using valid tools and models for the measurement of quality . one of the best and most extensively used models for evaluating of the quality of the healthcare services is the servqual ( service quality ) model , which was introduced by parasuraman et al . in 1980 ( 68 ) . this model also is recognized as the gap analyzer model , and it measure the gap between the expectations and perceptions of the patients regarding the quality of services provided by hospitals . however , in this model , the quality of healthcare services is defined as the difference between performance ( what is ) and expectations ( what should be ) ( 4 , 7 , 9 ) . some researchers used the servqual model to measure the quality of healthcare services in various countries ( 6 , 7 , 1016 ) . showed that , in hospitals in egypt , patients expectations concerning the quality of the healthcare services were affected on their stratification from types of services and hospitals ( public or private ) ( 18 ) . a study by miranda and et al . on 644 patients injured by vehicle accidents in peru in 2013 indicated that 95% of patients in public hospitals and 78% of patients in private hospitals were dissatisfied with the quality of the healthcare services provided by these hospitals ( 19 ) . showed that there are significant differences between patients expectations and their perception of the actual quality of healthcare . they also concluded that patients ranked the assurance and responsiveness domains as the most and least important , respectively ( 20 ) . according to the importance of measuring quality in the health sector and its role in health promotion , this study aimed to determine the different dimensions of the quality of the services being provided in educational hospitals in western iran and to evaluate the quality of the hospitals services from the patients perspectives . this cross - sectional study was conducted during the summer of 2015 in educational hospitals in kermanshah in western iran . the research population of this study included patients who were hospitalized at least two days in seven educational hospitals , i.e. , farabi , taleqani , imam reza , imam khomeini , mohammad kermanshahi , imam ali , and moetazedi hospitals . the sample size for the study was determined to be 400 patients . the patients were selected randomly in each hospital , and the questionnaires were given to them on the day they were discharged . the first part includes 7 questions regarding the socio - demographic information of the patients , and the second part includes 22 items in 5 service quality dimensions , i.e. , tangibility ( 4 items ) , reliability ( 4 items ) , responsiveness ( 4 items ) , assurance ( 5 items ) , and empathy ( 5 items ) . the reliability and validity of the questionnaire were confirmed by previous studies ( 10 , 12 ) , and the persian version was available . a five - point likert scale , varying from strongly disagree ( 1 ) to strongly agree ( 5 ) , was used to measure the expectations and perceptions of then patients about level of service quality . we asked the patients to rank the five dimensions of service quality according to the importance of the dimension . the significance of the difference between the mean score of the patients expectations and their perceptions was checked by the wilcoxon test . the kruskal - wallis test was used to compare the mean score of the patients expectations and their perceptions in different socio - demographic levels . a p - value less than 0.05 was considered to be statistically significant . this study was approved by the ethics committee of the deputy of research , kermanshah university of medical sciences . the first and last names of the patients , the name of the hospital , the name of the physician , the name of the ward in which the patients were hospitalized , and the number of international code were considered confidential to ensure the ethics of this research . this cross - sectional study was conducted during the summer of 2015 in educational hospitals in kermanshah in western iran . the research population of this study included patients who were hospitalized at least two days in seven educational hospitals , i.e. , farabi , taleqani , imam reza , imam khomeini , mohammad kermanshahi , imam ali , and moetazedi hospitals . the patients were selected randomly in each hospital , and the questionnaires were given to them on the day they were discharged . the first part includes 7 questions regarding the socio - demographic information of the patients , and the second part includes 22 items in 5 service quality dimensions , i.e. , tangibility ( 4 items ) , reliability ( 4 items ) , responsiveness ( 4 items ) , assurance ( 5 items ) , and empathy ( 5 items ) . the reliability and validity of the questionnaire were confirmed by previous studies ( 10 , 12 ) , and the persian version was available . a five - point likert scale , varying from strongly disagree ( 1 ) to strongly agree ( 5 ) , was used to measure the expectations and perceptions of then patients about level of service quality . we asked the patients to rank the five dimensions of service quality according to the importance of the dimension . the significance of the difference between the mean score of the patients expectations and their perceptions was checked by the wilcoxon test . the kruskal - wallis test was used to compare the mean score of the patients expectations and their perceptions in different socio - demographic levels . a p - value less than 0.05 was considered to be statistically significant . this study was approved by the ethics committee of the deputy of research , kermanshah university of medical sciences . the first and last names of the patients , the name of the hospital , the name of the physician , the name of the ward in which the patients were hospitalized , and the number of international code were considered confidential to ensure the ethics of this research . four hundred patients were included in the study of which 63% ( 252 ) were males and 37% ( 148 ) were females . the results showed the average age of the respondents was 38.5 years with a standard deviation of 23.6 . ( the ages varied from 1 to 89 . ) in addition , 6.6% of the patients were illiterate ; 94.4% of the patients had insurance coverage . table 2 presents the mean scores of the patients expectations , their perceptions , and quality gap in the five dimensions of service quality based on the servqual model . based on our findings , the overall mean scores of their perceptions and their expectations of service quality were 3.81 and 4.58 , respectively . also , the score gap between overall mean score of perceptions and expectations was 0.78 , and this gap was statistically significant . the study showed that the highest and lowest perception score was related to the responsiveness ( 3.96 ) and reliability ( 3.65 ) , respectively . also , the highest and lowest expectation score was related to the assurance ( 4.7 ) and reliability ( 4.43 ) , respectively . our empirical analysis indicated that there was a statistically significant difference between the mean score of perception and their expectation of the patients in all dimensions . however , there was a gap between perception and expectation of the service quality of educational hospital in kermanshah in western iran ( table 2 ) . the status of quality gap in all of the dimensions between expectations and perceptions of the patients about service quality is shown in figure 1 . based on this figure , the highest quality gap was related to assurance ( 0.88 ) , whereas the lowest quality gap was associated with responsiveness ( 0.56 ) . our findings about the important five dimensions from the patients perspectives are shown in figure 2 . the majority of patients ( more than 80% ) rated all of the dimensions of service quality as important and very important , i.e. , responsiveness ( 91% ) , tangibility ( 75% ) , reliability ( 86% ) , assurance ( 79% ) , and empathy ( 83% ) ( figure 2 ) . the results of kruskal - wallis test indicated that there was no association between the mean score of expectation of the patients and their perception in different age ( p - value = 0.76 ) , gender ( p - value = 0.31 ) , education level ( p - value = 0.064 ) , and marital status ( p - value = 0.092 ) . the main objective of the study was to measure the gap of the service quality provided based on expectation of patients and their perception in educational hospitals in kermanshah in western iran in 2015 . furthermore , to help health policy - makers and hospital managers in suitable programming to allocate financial resources for improving performance that have affected the perceptions of the patients of service quality ( 10 , 21 ) . based on the results of our study , there are negative gaps in all of dimensions concerning the quality of services provided by the hospitals . the results also indicated that there are significant difference between perception and expectation of patients of the service quality provided . this result is similar with the study conducted by tabibi et al . in hospitals in tehran , iran , in 2011 ( 7 ) , the study by anbari and tabaraie in arak , iran , in 2012 ( 22 ) , the study by brahmbhatt et al . in hospitals in india in 2011 ( 23 ) , the study of lim and tang in singapore hospitals in 2000 ( 24 ) , and the study of huseyin et al . in hospitals in greece ( 21 ) . the study showed that the highest and lowest mean score of perception were related to responsiveness and reliability , respectively , whereas the highest and lowest mean score of expectation were associated with assurance and reliability , respectively . this result was consistent with the results of studies conducted by aghamolaei and et al . ( 25 ) , lim and tang in singapore hospitals ( 24 ) , and karydis et al . in dental healthcare in greece ( 26 ) . in a study by aghamolaei et al . in shahid mohammadi hospital in bandar abbas in hormozgan province in southern iran , it was reported that the highest expectation was related to the assurance dimension ( 25 ) . as the results showed , assurance had the highest negative gap among all of the dimensions , whereas the lowest negative gap was related to responsiveness . these results paralleled the results of previous studies ( 7 , 27 , 28 ) . bahadori et al . in the armed forces hospital of hamadan reported the responsiveness ( 0.22 ) and assurance ( 0.43 ) had the highest and the lowest gap of quality in all of dimensions , respectively ( 7 ) . also , in a study conducted by havasbigi el al . in public hospitals in kermanshah and ilam , western iran , the highest and lowest negative gap in all of dimension based on servqual model was found to be assurance ( 1.36 ) and tangibility ( 0.87 ) , respectively ( 28 ) . first , the study conducted in the educational hospitals in kermanshah city in western iran and other studies must be piloted in other hospitals , such as private hospitals and social security hospitals to increase the generalizability of the results of the study . another limitation was that the majority of the patients lived in urban areas and had insurance coverage . thus , based on the limitations of the study , our findings must be used with caution , and generalization of the results was restricted to the actual population of kermanshah province , especially people who live in the rural areas without insurance coverage and the hospitals of other towns in kermanshah province . the current study indicated that there were negative gaps in all of the dimensions in the studied hospitals . however , the expectations of the patients concerning the quality of the services provided were higher than their perceptions of the quality of those services . based on our study , it is suggested that the planners and managers of hospitals must improve the timeliness of the delivery of care and enhance the communication skills of staff members in order to increase the quality of the services provided . the authors recommend that future studies be conducted in private and social security hospitals and that the results be compared to the results of this study .

introductionthe main mission of hospitals in any health system is to deliver high quality healthcare for patients and meet their needs and expectations . the aim of the current study was to assess the quality of the service of educational hospitals affiliated with kermanshah university of medical sciences in 2015 , from the perspective of patients.methodsin this cross - sectional study , the perspectives of 400 patients were assessed about the quality of the services provided by educational hospitals in kermanshah ( western iran ) in 2015 . the quality was assessed by the servqual questionnaire with five dimensions , i.e. , tangibility , reliability , responsiveness , assurance , and empathy . in addition , the wilcoxon test and the kruskal - wallis test were used to explore any association between the dependent variable and explanatory variables . the data were analyzed using stata v.12 software.resultsthere were negative gaps in all five dimensions . the highest and lowest gaps in the mean score were found in the assurance ( 0.88 ) and responsiveness ( 0.56 ) dimensions . the patients ranked responsiveness as the most important dimension of the quality of healthcare.conclusionthere were gaps between the patients perceptions and their expectation about the five dimensions that were studied based on the servqual model . also , it is recommended that improving the quality of healthcare is possible by various policies , such as good responsiveness , access to health workers , and delivering healthcare in less time .

1. Introduction 2. Material and Methods 2.1. Study design and setting 2.2. Sampling 2.3. Survey instrument 2.4. Data analysis 2.5. Research ethics 3. Results 4. Discussion 5. Conclusions

anaplastic thyroid cancer ( atc ) is one of the most aggressive tumors in human medicine . therefore , the prognosis of atc patients is one of the worst among the malignant tumors in general . despite multimodal treatment , which includes surgery , its annual incidence is about 2 per million residents and its share in the structure of patients with thyroid cancer is decreasing and now stands at 1 - 2% [ 1 , 2 ] . atc occurs most frequently in the elderly but has been described in all age groups [ 3 , 4 ] . as one of the most aggressive tumors , atc draws a lot of attention , but it has not been sufficiently researched . due to its low incidence , there are not many institutions that have single series with more than 100 patients . analysis of prognostic factors is possible only in this larger series because most patients unfortunately die in the first few weeks or months after diagnosis , so one might get wrong impression that there is no significant survival in patients with atc . in larger series , significant survival was recorded , which enables analysis of prognostic factors associated with longer survival in patients with atc . the aim of this study was to determine the role and importance of age in survival of patients with atc . we analyzed retrospectively the data on 150 patients with atc ( 96 women and 54 men ) diagnosed in the period from 1995 to 2006 . in 85 operated - on patients , the diagnosis was obtained on the basis of definitive histopathological finding , and in 65 nonoperated - on patients diagnosis was based on cytological findings after fine needle aspiration . patients with histologically proven preexisting papillary thyroid carcinoma ( 49 ) were also included in the study . reviewing medical records , we collected data on demographic characteristics of patients ( age , sex , place of residence , and life on endemic goiter area ) , duration of disease before diagnosis , whether the patients knew of goiter before and if so for how long back , and whether the operation was done or just fine needle aspiration biopsy . in the operated - on patients , the following has been analyzed : the type of surgery that was done , then the size of the tumor , its multicentricity , the involvement of one or both lobes of the thyroid gland , the presence of regional lymph node metastases , or distant metastases proven . special attention was focused on the histopathological findings in the operated - on patients and the presence of preexisting well - differentiated thyroid cancer , as well as the degree of lymphocytic infiltration of the tumor . data on whether in the operated - on patients radiotherapy ( preoperative or postoperative ) , chemotherapy , or radioactive iodine therapy was implemented has been gathered . some other diseases in patients with anaplastic thyroid cancer which could have an impact on survival were taken into consideration , other malignant tumors , diabetes , and hypertension . data on whether patients were still alive or when they died were gathered through interviews of patients or their families . the kaplan - meier method and log - rank test were used to determine the overall survival . the largest number of patients with anaplastic thyroid carcinoma was in the seventh decade of life ( more than half of the patients ) and then in the sixth and eighth decade of life ( almost 20% ) . under 40 years of age , anaplastic carcinoma was detected in just two affected ( 1.3% ) and in younger than fifty in 11 patients ( 7.3% ) . the average age of patients was 64 years ( standard error 8.5 years ) . table 3 and figure 1 show the survival rates of patients with anaplastic carcinoma of the thyroid gland in relation to the age . the patients were divided into three age groups : the first consisted of patients younger than 51 years , the second consisted of patients aged 51 to 70 years , and the third consisted of patients older than 70 years . the three age groups < 50 ys , 5170 ys , and > 70 were arbitrarily chosen . the longest survival in all periods was found in the youngest age group and the lowest survival in the oldest group of patients in which no one had a survival of more than 3 years . in the youngest age group , one - year survival was observed in more than half of the patients , whereas in both the older age groups one - year survival was 3 - 4 times less . during the first month from diagnosis of anaplastic thyroid carcinoma , almost 30% of patients older than 70 years died and less than 10% in the two younger age groups . five - year survival rates were three times more often in the youngest age group than in the middle one . in the oldest group the middle and the oldest age group were compared for other characteristics described in material and methods section , but the observed differences did not affect the survival according to multivariate analysis . mean survival and median survival in patients with anaplastic carcinoma of the thyroid gland in relation to age are presented in table 4 . in the youngest age group the median survival was 62 weeks and in the middle age group 16 weeks , which was the median for all patients with anaplastic thyroid cancer overall , while the shortest median of 12 weeks was found in the oldest age group of patients . this difference was statistically highly significant according to the log - rank test ( p = 0.008 ) . according to univariate cox regression analysis , among the factors that influence the survival of patients with anaplastic thyroid cancer , patients age was significantly statistically associated with longer survival ( p = 0.000 , or = 0.96 , 95% ci = 0.940.99 ) . all the factors associated with longer survival in patients with atc at the level of statistical significance ( p < 0.1 ) according to univariate model were then included in the model of multivariate analysis : patients age , presence of longstanding goiter before the appearance of atc , surgery , type of surgery , tumor multicentricity , presence of distant metastases at the time of diagnosis , histologically proven preexistent papillary carcinoma , radioiodine therapy , and postoperative radiotherapy . according to multivariate analysis , after eliminating the confound effect , besides surgery ( p = 0.000 , or = 0.43 , 95% ci = 0.290.63 ) , only patients age ( p = 0.023 , or = 0.68 , 95% ci = 0.490.95 ) was independent prognostic factor of favorable survival in patients with atc . the average age of patients with atc in our study was 64 years , and more than half of the patients were in their seventh decade of life . only two affected were younger than 40 years of age , and the youngest patient was a 35-year - old woman . our oldest patient with anaplastic thyroid cancer was an 89-year - old man . according to the literature , the largest number of patients with anaplastic carcinoma of the thyroid gland is in the seventh and eighth decade of life . the average age of patients ranges from 65 to 72 years , and there is no significant difference between men and women in relation to the age at which anaplastic thyroid cancer usually occurs [ 6 , 7 ] . more than 90% of patients in all the major series are older than 50 years [ 5 , 8 ] . its appearance is quite rare and has been described only in few cases in the world . unfortunately , in the childhood , it has the same fatal outcome , as well as in adult patients [ 1012 ] . age of patients with anaplastic thyroid cancer at the time of diagnosis , according to our results , is an independent factor that was significantly associated with longer survival ( p < 0.05 , rr = 0.65 , 95% ci = 0.49 to 0.95 ) . in other words , survival is significantly longer in younger than in older patients . however , it should be noted that in the youngest age category of patients with atc ( younger or at the age of 50 ) in whom the best survival was found , there is only 11 patients . almost half of the patients died during the first year ( 5/11 ) ; a five - year survival was observed only in two patients . according to the literature , data on the impact of age on survival in patients with atc are controversial , although longer survival can be expected in younger patients , and it is known that anaplastic thyroid cancer usually occurs in older patients . point out that in patients with anaplastic thyroid cancer , who are older than 70 years , the risk of shorter survival is 1.5 times higher than in patients younger than 70 years , but the age is not an independent predictor according to the multivariate regression analysis . . found that survival was significantly longer in patients younger than 65 years , as a reason for cited comorbidity that is present in the elderly . giuffrida and gharib and kebebew et al . found longer survival in patients with anaplastic thyroid cancer who are younger than 60 years . . found that one - year survival in patients with anaplastic thyroid cancer declines in older patients . li and colleagues by analyzing 12 patients with atc under the age of 55 found that the prognosis is better than in older patients but only if the remaining of histologically proven preexistent papillary carcinoma exists . sugitani et al . in the largest of all studies of prognostic factors in atc , which included 677 atc patients from 38 institutions , in multivariate regression analysis among other factors independently associated with a worse prognosis state also the age over 70 years unlike the most , according to the results by haigh et al . , years of age had no influence on survival in patients with anaplastic thyroid carcinoma . according to our study , besides patient age , only surgery was independent prognostic factor of favorable survival in patients with atc . among the nonoperated - on patients , no one survived longer than one year , and among the operated - on ones the mean survival rate was seven times higher . however , the independent predictor significantly associated with survival is the type of operation performed , that is , how radical the operation was . as many as one - half of the patients that underwent radical surgery lived longer than one year and one - fourth lived longer than five years . . found a significant link between surgical treatment and the length of survival . in their series , 68% of the patients underwent surgical treatment , and half of them had complete removal of the tumor . the results of haigh et al . prove that radical surgical treatment is an independent survival predictor , and it was possible to carry it out in 31% . according to literature , radical surgical treatment is an independent survival predictor [ 6 , 20 ] . the best results are obtained by multimodal treatment [ 5 , 8 , 13 ] . however , some of the authors did not find that multimodal treatment had an impact on the survival , where the only positive prognostic factor was the complete resection of the tumor [ 7 , 21 ] . their study , like ours , showed that none of the nonoperated - on patients reached the point of one - year survival . multiannual survival in patients with complete resection of the tumour occurs irrespective of the extent of operation on the thyroid , that is , irrespective of the fact whether a total thyroidectomy or only hemithyroidectomy was performed , the only condition being that the tumour was totally removed . according to venkatesh et al . , surgery is vital but not sufficient enough for longer survival , as one - year survival is noted in operated - on patients but almost never in nonoperated - on ones , and radical operation without multimodal treatment does not significantly prolong the survival . age is one of the most important factors in the survival of patients with atc . the best prognosis has patients younger than 50 years , but the smallest number of patients is in this age category . the limitation of our study is relatively small number of patients although it is one of the single institution series with the greatest number of patients in the world . nevertheless , the results of this study should be verified as many cases as possible by conducting multicenter study out of different centers in the world .

background . anaplastic thyroid cancer ( atc ) is one of the tumors with the shortest survival in human medicine . aim . the aim was to determine the importance of age in survival of patients with atc . material and methods . we analyzed the data on 150 patients diagnosed with atc in the period from 1995 to 2006 . the kaplan - meier method and log - rank test were used to determine overall survival . prognostic factors were identified by univariate and multivariate cox regression analysis . results . the youngest patient was 35 years old and the oldest was 89 years old . according to univariate regression analysis , age was significantly associated with longer survival in patients with atc . in multivariate regression analysis , patients age , presence of longstanding goiter , whether surgical treatment is carried out or not , type of surgery , tumor multicentricity , presence of distant metastases , histologically proven preexistent papillary carcinoma , radioiodine therapy , and postoperative radiotherapy were included . according to multivariate analysis , besides surgery ( p = 0.000 , or = 0.43 , 95% ci = 0.290.63 ) , only patients age ( p = 0.023 , or = 0.68 , 95% ci = 0.490.95 ) was independent prognostic factor of favorable survival in patients with atc . conclusion . age is a factor that was independently associated with survival time in atc . anaplastic thyroid cancer has the best prognosis in patients younger than 50 years .

1. Introduction 2. Material and Methods 3. Results 4. Discussion 5. Conclusion

low back pain ( lbp ) is a representative musculoskeletal disease with a current prevalence of 6090% ; more than 80% people will experience lbp at least once in their lifetime . lbp occurs when a poor lifestyle weakens the muscular strength of the waist and excessive loads on and tensions of the muscles create pain . mediating muscle weakening to stabilize the trunk is an important aspect in the recovery of body function in patients with lbp2 . structural changes of the waist muscles and histomorphological changes lead to chronic lbp ; in particular , weakening and atrophy is more common in the muscle fibers that stabilize the waist3 . in addition , if pain develops in the muscles , weakening and atrophy will occur , the ligaments and joints will become stiff , and activity will decrease . a vicious circle of worsening pain repeats by muscle rigidity and sprain4 . therefore , it is important to maintain mechanical alignment of the trunk muscles to prevent lbp . it is necessary to keep an upright pattern of muscle activity as well as proper muscle length and strength5 . intra - abdominal pressure that is produced by the connection of trunk stabilization and respiratory muscles affects waist stability and protects the trunk muscles6 . effective mediation is reportedly accomplished using simultaneous breathing exercises to relax excessive muscle tension7 . in addition , if intra - abdominal pressure increases by diaphragm stimulating activity by performing breathing exercises , even the pelvic floor muscles will be activated . as such , contraction of the abdominal muscles during breathing can easily activate even the deep stabilization muscles8 . clinicians recognize that stabilization exercises to strengthen the waist and abdominal muscles are very important . mediating breathing exercise activates the trunk muscles more effectively and decreases lbp . as such , this study intended to induce activity of the trunk muscles that contribute to spinal stabilization and increase intra - abdominal pressure by performing exhalation exercises as well as to investigate the effects on trunk muscle activity and oswestry disability index ( odi ) by comparing spinal stabilization and exhalation exercises . we intend to utilize this exercise method to effectively treat lumbar pain in the future . subjects were 20 male patients ( age 3145 years ) with chronic lbp who visited b hospital located in jeollanamdo , south korea , and complained of lbp over 12 weeks . patients with acute pain , continuous severe pain , spondylolysis , or spondylolisthesis were excluded from the study . those who had paresthesia , myoparalysis , or psychological problems as well as those who had difficulty in performing exercise due to lack of comprehension were also excluded . subjects who understood the purpose of this study and consented to participate were selected ( table 1table 1.subjects characteristicsitemsexperimental group(n=10)control group(n=10)age ( years)42.5 5.340.1 5.3height ( cm)168.5 5.7166.3 4.9weight ( kg)64.3 9.865.4 8.2odi ( points)26.2 4.225.3 3mean sd , shapiro - wilk test . odi : oswestry disability index this study was approved by the bioethics committee of sehan university center ( approval number : 2015 - 7 ) . this study included 20 male patients with chronic lbp , and 10 subjects each were randomly allocated to the exhalation exercise group ( experimental group ) and trunk stabilization group ( control group ) . the intervention program consisted of daily 30-minute sessions once daily 4 times a week for 6 weeks . the trunk muscle activities and odi were calculated , compared , and analyzed before versus after the intervention program by using surface electromyography ( semg ) . a surface electromyography ( emg ) mp 100 system ( biopac system inc , goleta , usa ) was used to measure trunk muscle activities , with a sampling rate of 1,000 hz . the frequency band - pass filter was set at 20450 hz . each subject s hair was removed and dead skin cells were removed using a thin sandpaper to minimize skin resistance to the emg signal . two ag / agcl surface electrodes were attached to the belly of each muscle in a direction parallel to the muscular fiber at 2-cm intervals . standard electrodes were attached to the spinous process of the 12th thoracic vertebrae and scapula spine . the emg signals of the right rectus abdominis , transverse abdominis , external oblique abdominis , and erector spine muscles were collected . the sampling rate for the emg signal collection was set to 1,000 hz , the notch filter was set to 60 hz , and the band pass filter was set to 20450 hz . the signals were converted to root mean square ( rms ) values , and an emg signal analysis was conducted using acqknowledge 3.9.1 software ( biopac ) . emg data were collected while the subjects were performing the stabilization exercise and 6 of the 10 seconds were used . maximal voluntary isometric contraction ( mvic ) was used to standardize the emg signal during the stabilization exercise9 . the mvic of the rectus abdominis muscle was measured in the knee - bent position , with both arms on the chest and the trunk bent at 45 at maximum resistance . the mvic of the transverse abdominis muscle was measured in the knee - bent position , with both arms on the chest and during sit - ups , twisting the trunk from left to right at maximum resistance10 . the mvic of the external oblique abdominis muscles was measured while rotating the trunk to opposite side with the trunk bending , while the mvic of the erector spinae muscles was measured in a prone position , with both hands on back of the head and the trunk extended at maximum resistance11 . mvic was measured when the researcher made the subjects perform isometric contraction maximally while a beep was played for 5 seconds . the first and last seconds of the measurements were excluded ; thus , a 3-second measurement was analyzed . the researcher continuously encouraged subjects to maintain a maximum contraction and the percentage value of the muscle was the highest average value of 3 measurements . the subjects were given a 30-second rest after the mvic of each muscle was measured12 . the odi was measured using the korean version of the odi to quantify physical difficulties in daily life due to lbp ( cronbach s alpha=0.92 ) . the original odi is composed of 10 items evaluated as 05 points ( low to high severity ) . in the korean odi , considering the korean culture , the item about sex life was excluded , so a total of 9 items were evaluated ( for a highest possible score of 45 ) . the total score was divided by number of items 5 and was expressed as a percentage13 . a trunk stabilization exercise , the modifying exercise method of urquhart et al.14 and steven et al.15 , which consists of 3 stages16 , was used . the exercise was performed for 30 minutes a session 4 times a week for 6 weeks at an intensity acceptable to each subject ( table 2table 2.trunk muscle stabilization intervention exercise programstagetimecontentsprogramwarming - up exercise5 minflexibility whole - body exerciseupper - lower extremity stretching and joint moving exercisemain exercise20 mintrunk muscle stabilization exercisestage 11 ) pelvic posterior tilting in a supine position with both knees flexed2 ) abdominal draw - in in a supine position with both knees flexed3 ) pelvic anterior tilting in a supine position with both knees extendedstage 24 ) abdominal draw - in in a supine position with both knees extended5 ) bridging in supine positionstage 36 ) ball bridge7 ) unstable bridging : bridging with extension of the left / right legcool - down exercise5 minaerobic exercisebreathing with abdominal muscles ) . the bracing exercise was performed for 20 minutes a session , 5 times a week , for 6 weeks in the group training the exhalation muscles17 . in this exercise method , the subjects bent their knees at 90 and adjusted the alignment of their waist and pelvis in supine position . a biofeedback system was then attached to the waist and pressure was maintained at 40 mmhg . the pressure of the manometer increased from 40 mmhg to 70 mmhg and was maintained by quick contractions of the abdominal muscles by the application of abdominal strain . this exercise was intended to be performed with the feeling that the trunk muscles spread outward and the rectus abdominis muscles did not bulge while the subjects were able to breathe easily17 . spss 18.0 for windows was used to process the data and the shapiro - wilk test was used to test the normality of the subjects general characteristics , respiratory muscle activities , and body functions of the groups . a paired t - test was used to compare intragroup changes , while analysis of covariance was used to compare intergroup changes . there were significant differences in the rectus abdominis , transverse abdominis , external oblique abdominal , and erector spinae muscle activation and in the odi within the experimental group ( p<0.05 and < 0.001 , respectively ) . there were significant differences in the rectus abdominis muscle , external oblique abdominal muscle , erector spinal muscle and odi within control group ( p<0.05 ) . in addition , there was a meaningful difference in the transverse abdominis muscle alone on comparing the change in trunk muscle activity and in odi between the groups ( p<0.05 ) ( table 3table 3.comparison of change of muscle activity and odi within each group and between the groupsgroupprepost%rvcraexperimental group 21.3 5.125.6 4.2*control group21.6 5.225.8 4.3*taexperimental group 16.2 2.419.4 3.1*control group14.3 3.215.7 3.6eaoexperimental group 16 2.520.2 2.1*control group17.3 3.221 3.5*esexperimental group 23 6.4928.2 6*control group26 5.1929.1 4.9*odiexperimental group 27.1 2.723.2 1.9**control group28.2 2.726.2 1.7**p<0.05 , * * p<0.001 . ra : rectus abdominis muscle ; ta : transverse abdominis muscle ; eao : external abdominal oblique muscle ; es : erector spinae muscle ; odi : oswestry disability index ) . ra : rectus abdominis muscle ; ta : transverse abdominis muscle ; eao : external abdominal oblique muscle ; es : erector spinae muscle ; odi : oswestry disability index recently , interest in the use of breathing exercises such as abdominal respiration and yoga breathing to improve vertebral joint stability and motor control is increasing18 . hodges et al.3 found that the respiratory muscles are an important factor affecting spinal stabilization . in particular , if respiratory and abdominal muscle activities are uncertain , spinal stabilization will decrease due to decreased intra - abdominal pressure . urquhart et al.19 reported that activities of the deep abdominal muscles should be evoked using a combination of breathing and stabilization exercises rather than stabilization exercises alone . marshall et al.20 classified 20 patients with non - idiopathic lbp into a stabilization exercise group with abdominal respiration exercise ( n=10 ) and a general exercise group ( n=10 ) and compared their muscle activity and range of motion . the muscle activities of the abdominal rectus , external oblique abdominis , and erector spinae muscle increased , as did range of motion of the waist . this study s findings support advanced research by drawing a conclusion that the activities of the rectus abdominis , transverse abdominis , external oblique abdominis , and erector spinae muscles increased in the experimental group performing breathing exercises . there was a meaningful intergroup difference in the transverse abdominis alone , in terms of the change of trunk muscle activities . the transversus abdominis muscle is located in the deepest part of the abdomen and is called the corset muscle because it plays a role in stabilizing the waist and creating abdominal pressure owing to being attached to the thoracolumbar fascia . of all of the abdominal muscles , the transverse abdominis has the widest and closest attachment to the thoracolumbar fascia . ki and heo21 classified 24 patients with chronic lbp into a spinal stabilization exercise with feedback respiratory exercise group and a trunk stabilization exercise group that they performed 3045 minutes a session , 3 times a week , for 6 weeks . the spinal stabilization exercise with feedback respiratory exercise increased stability of the vertebral joints and flexibility of the thoracic vertebrae more than the spinal stabilization exercise only . ferreira et al.22 reported that improvement in transverse abdominis muscle activity was higer after postural control exercise than after conventional physiotherapy , targeting 34 patients with chronic lbp . in particular , those findings supported ours since they showed that the transverse abdominis muscle plays an important role in controlling vertebral shearing force . yoon et al.23 studied 10 patients with chronic lbp who performed 3 types of stabilization exercises in the quadrupedal position for 20 minutes and then compared trunk muscle activities . the activities of the back muscles of the trunk showed a higher increase in the group in which a hand and a foot were asymmetrically lifted in the quadrupedal position than in the group in which a hand was lifted in the quadrupedal position or in the group which a foot was lifted in the quadrupedal position . however , advanced studies have limited ability to induce abdominal muscle contractions because most agonists were back muscles of the trunk when the stabilization exercise was performed in various quadrupedal positions . niemisto et al.24 found that odi in the trunk stabilization exercise group decreased meaningfully when trunk stabilization exercise and conventional physiotherapy were mediated in 203 subjects with chronic lbp for 312 months . sung25 found that odi decreased meaningfully after mediating spinal stabilization exercise for 4 weeks in 16 subjects with chronic lbp . kang and jeong26 found that odi decreased in a spinal stabilization exercise group as a result of mediating spinal stabilization exercises for 34 patients with chronic lbp for 4 weeks . this study drew a conclusion supporting advanced research because there were meaningful differences in odi . the reason for this is that the delivery of stimulation on pain receptor tissues such as those around the spine and articular capsules and ligaments decreases as trunk stabilization and breathing exercises strengthen the deep stability muscles and it affects abdominal muscle functional recovery . we found that they are effective to functional disability of lbp by decreasing the delivery of stimulation to the pain receptor tissue such as that around the spine and articular capsules and ligaments . son27 found a disability index change , center of body gravity change , and area decrease in both a spinal stabilization exercise group with abdominal respiration and a spinal stabilization exercise group ; however , no intergroup change was seen . regardless , there was a meaningful difference in this study in direct opposition to that of advanced research . abdominal respiration mediation in advanced research is a type of breathing exercise used in the resting state . on the other hand , exhalation exercise is more effective at reducing odi because compensation in the lumbar region decreases largely as back and waist mobility increase . breathing exercises effectively increase muscle activity due to training of the gross and fine muscles of the trunk . since it strengthens the muscles that stabilize the body , the breathing exercise proved to be a very important element as it relieves lbp , prevents lumbar pain , and prevents recurrence .

[ purpose ] this study investigated the effect of exhalation exercises on trunk muscle activity and oswestry disability index by inducing trunk muscle activity through increasing intra - abdominal pressure and activating muscles , contributing to spinal stability . [ subjects and methods ] this intervention program included 20 male patients with chronic low back pain . a total of 10 subjects each were randomly assigned to an exhalation exercise group as the experimental group and a spinal stabilization exercise group as the control group . [ results ] there were significant differences in the activities of the rectus abdominis , transverse abdominis , external oblique abdominal , and erector spinae muscles as well as in the oswestry disability index within the experimental group . there were meaningful differences in the activities of the rectus abdominis , external oblique abdominal , and erector spinae muscles and in the oswestry disability index within the control group . in addition , there was a meaningful intergroup difference in transverse abdominis muscle activity alone and in the oswestry disability index . [ conclusion ] the breathing exercise effectively increased muscle activity by training gross and fine motor muscles in the trunk . moreover , it was verified as a very important element for strengthening body stability because it both released and prevented low back pain .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

low - grade fibromyxoid sarcoma ( lgfms ) is a distinctive variant of fibrosarcoma , which was first described as a pathologic entity by evans in 1987 . lgfms presents as a rare soft tissue tumor with a high metastasizing potential , despite the benign histologic appearance . these tumors usually occur in the lower extremities , followed by the trunk , groin , upper extremities , thorax , buttocks and abdominal wall ; the maxillofacial region being rarely involved . the vast majority of lgfms occur in a subfascial location ; rarely , the subcutis or dermis may be affected . lgfms typically involves young or middle - aged adults , but a large number of pediatric cases have also been reported . here , a 35-year - old male patient reported to the department of oral medicine and radiology with a complaint of swelling in the mandibular left posterior region since 6 months [ figure 1 ] . history revealed that it was initially peanut sized , 6 months back but had gradually increased to the present size . there was no history of pain , trauma , paraesthesia or anesthesia , or pus discharge associated with the swelling . on examination , there was a solitary well - defined sessile swelling seen on the left body of the mandible , measuring approximately 3 cm 2 cm in dimension . the swelling was nontender , firm in consistency , noncompressible , nonfluctuant and possessed no mobility . intraoral examination revealed a mild vestibular obliteration in the region of 34 , 35 and 36 [ figure 2 ] . root stumps were present in relation to 18 , 27 and 37 . based on the clinical findings , the differential diagnoses considered were peripheral nerve sheath tumor , soft tissue fibroma and soft tissue sarcoma . extra orally , a well - defined ovoid sessile swelling seen on the body of the mandible on the left side intra oral examination revealed vestibular obliteration in the region of 34 , 35 and 36 and root stumps in relation to 37 orthopantomograph showed root stumps in relation to 37 and a subtle change in the trabecular pattern in the periapical region of 35 , 36 and 37 [ figure 3 ] . occlusal radiograph showed a well - defined soft tissue outline of the tumor on the buccal aspect with erosion of the buccal cortex in the region of 35 [ figure 4 ] . cropped orthopantomograph showed root stumps wrt 37 and a subtle change in the trabecular pattern in the peripaical region of 35 , 36 and 37 occlusal radiograph showed a well - defined soft tissue outline of the tumour on the buccal aspect with erosion of the buccal cortical plate in the region of 35 ultrasonography ( usg ) revealed an ill - defined hyperechoic soft tissue lesion in the deep planes of the left submandibular region abutting the body of the mandible . the tumor revealed ill - defined margins with erosion of the underlying bone and insignificant vascularity . marginally enlarged lymph nodes were noted in the left cervical region , largest measuring 1.1 cm in dimension . computed tomography ( ct ) showed a well - defined encapsulated fusiform soft tissue mass with a density of 2030 hu measuring 3 cm 1.8 cm 2.8 cm adjacent to the left body of the mandible . there was minimal appreciable enhancement and the tumor was causing scalloping of the adjacent bone with thinning of the buccal cortex [ figure 5 ] . computed tomography showed a well - defined encapsulated fusiform soft tissue mass with a density of 2030 hu , minimal appreciable enhancement , causing scalloping of the adjacent bone and thinning of the buccal cortex based on the radiological features , a diagnosis of benign nerve sheath tumor was made . histopathological examination of core biopsy revealed loosely arranged fascicles of spindle cells with loose myxoid stroma . the features were suggestive of a benign nerve sheath tumor . surgical excision of the tumor was done under general anesthesia . the tumor was seen encasing the mental nerve which was subsequently resected at the level of the mental foramen [ figure 6 ] . cut section of the tumor showed a solid , gray - white mass with myxoid areas [ figure 7 ] . the mental nerve was resected at the level of the mental foramen cut section shows solid , gray - white mass with myxoid areas histopathological examination showed a myxoid tumor with hypocellular areas in nodules merging with collagenised areas . immunohistochemical analysis showed strong positivity for vimentin and negativity for s100 protein , desmin , smooth muscle actin ( sma ) , cd34 , cd31 , cd68 , cytokeratin ( ae1/ae3 ) and epithelial membrane antigen ( ema ) [ figures 9 and 10 ] . based on the histopathological and immunohistochemical findings , a final diagnosis of lgfms was given . bland spindle cells with no atypia in short fascicles and whorls in the myxoid zone ( h&e stain , 40 ) positivity of vimentin in tumour cells ( ihc stain , 100 ) s100 negative in tumour cells ( ihc stain , 40 ) lgfms is a rare soft tissue neoplasm described first by evans in 1987 . in his original paper , evans had described the tumor histopathologically as a bland appearing soft tissue neoplasm associated with aggressive behavior and high degree of local recurrence or local metastasis . in his second paper in 1993 , evans reported 12 cases of lgfms , of which nine patients had local recurrence and seven patients experienced distant metastasis and died from lgfms . folpe et al . in 2000 , described 54 cases of lgfms and reported that only 9% of the cases showed local recurrence and 3% showed distant metastasis . the tumor mainly affects the young and middle - aged with an age range of 2050 years having no gender predilection . the most frequent sites of occurrence of this tumor are the lower extremities , followed by the trunk and groin , upper extremities and buttocks . lgfms involving the maxillofacial region is a very rare entity with only six cases reported in english literature till date [ table 1 ] . reported cases of low grade fibromyxoid sarcoma in the maxillofacial region it is extremely difficult to make a diagnosis of lgfms before surgical intervention because of the relatively benign clinical nature and nonspecific findings of imaging modalities such as usg , ct and magnetic resonance imaging ( mri ) . contrast enhanced ct shows heterogeneous enhancement , but sometimes the enhancement is not obvious , as in our case . mri shows homogeneous signal intensity similar to muscle on t1-weighted images and heterogeneous low to high signal intensity on t2-weighted images . morphologic and molecular overlap of hyalinizing spindle cell tumor ( hsct ) with giant rosettes and sclerosing epithelioid fibrosarcomas with lgfms suggests the possibility of considering them as variants . evans described the typical histological findings of lgfms as contrasting fibrous and myxoid areas with moderate to low cellularity , deceptively benign - appearing spindle cells with no or slight nuclear pleomorphism and rare mitotic figures and a swirling , whorled growth pattern . in addition to these features , two cases of lgfms with heterotopic ossification and giant rosettes have also been reported . although there is no specific marker for lgfms immunohistochemistry is essential to exclude the possibility of tumors with similar histopathologic findings . muc4 is now considered highly specific and quite sensitive marker helpful in distinguishing lgfms from mimics . markers including s100 , sma , desmin , cd34 , cd31 , cd68 , ema and cytokeratin ( ae1/ae3 ) have been used to distinguish between a number of tumors . hsct presents with varying number of large , rosette - like structures which arise from hyalinization zones or emerge abruptly from the spindled stroma . on immunohistochemistry , both the entities are strongly positive for vimentin , but there are differences between hsct and lgfms for other antibodies . investigators have reported that giant rosettes of hsct are positive for s100 , occasionally positive for cd34 , leu-7 and neuron - specific enolase and negative for desmin , sma and ema . in contrast , the tumor cells of lgfms are occasionally positive for desmin , sma , ema and cd34 but are negative for s100 , leu-7 and neuron - specific enolase . an important cytogenetic tool which will be helpful in the diagnosis of both these has been the recognition of a characteristic balanced translocation t(7 ; 16 ) ( q34:p11 ) and the finding of a novel fusion gene fus / creb3 l2 . techniques include local excision , radical surgery , wide en bloc resection , compartmental resection . the role of chemotherapy and radiotherapy as adjuncts to surgical intervention is unclear . in a study conducted by tang et al . in 2010 , data of 273 patients diagnosed with hsct or lgfms were collected , of which follow - up data were available for 184 patients . local recurrences and distant metastases were observed in 54 ( 29.34% ) and 34 ( 18.48% ) patients , respectively . metastasis was seen mainly in the lung followed by other soft tissues and very rarely in bone . due to the relatively high local recurrence rate and late metastasis , lgfms is a very rare low - grade malignancy occurring in the maxillofacial region , with nonspecific histopathological findings . diagnosis is not possible with clinical and imaging studies , rather a meticulous histopathological , immunohistochemical examination and cytogenetics is required to arrive at a diagnosis , differentiating from its mimics . long - term follow - up is required in the management of these cases because of late metastasis and high recurrence rate .

low - grade fibromyxoid sarcoma ( lgfms ) is a distinctive variant of fibrosarcoma with a high metastasizing potential and is characterized by a long interval between tumor presentation and metastasis . lgfms involving the maxillofacial region is a very rare entity with only six cases reported till date . lgfms is characterized by its benign histological appearance , with spindle cells in whorling pattern , and collagenized and myxoid areas . the heterogeneous histological appearance makes the diagnosis challenging . immunohistochemical staining has been reported by a number of authors , with some conflicting results , showing positivity with vimentin , but no immunoreactivity with antibodies to keratin , desmin , actin , s100 or epithelial membrane antigen . we present a case of a 35-year - old male patient who developed a mass on the left mandibular body region . the tumor was excised along with mental nerve at the level of the mental foramen . the tumor on pathologic and immunohistochemical evaluation was diagnosed as lgfms .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

monocortical miniscrew - type temporary anchorage devices have been widely used to move a single tooth or segments of teeth.1,2 in addition , some studies have proposed simultaneous distalization of the entire dentition by applying force vector(s ) from interradicular miniscrews to the main archwire.3 the underlying concept is that a distalizing force applied to the anterior segment might be transmitted to the posterior segment along the continuous archwire and proximal contact points , leading to distalization of the molars as well as the incisors . these reports are encouraging in terms of the efficiency of correcting a class ii relation showing class ii molars and canines and an increased overjet , in contrast to extraoral appliances or intermaxillary elastics which require patient compliance , or intraoral distalizers which cause forward movement of the anterior segment.4 since an elastic chain connecting the miniscrew head and the archwire hook delivers a relatively constant single line of force , it is conceivable that the initial force system may be a strong determinant of the resultant displacement of the target segment . previous studies have mainly focused on control of the anterior segment in cases of bicuspid extraction,5,6 as if respective anterior and posterior segments were independent . along a continuous archwire tied to a non - extraction model , however , displacement of the anterior segment may affect the placement of the posterior segment due to the rigid connection between the segments . it is known that the resultant displacement pattern of an object is a function of the relationship between the center of resistance and the line of force . however , to date little is known about the effect of varying force vectors on the displacement of overall dentition , specifically with regard to the anteroposterior and vertical displacement of each segment . for instance , it is not yet clear whether a single force vector applied to the anterior segment would induce translation or rotation of the posterior segment . in addition to the clinical evidence , experimental investigation of the displacement pattern in response to force vectors with varying angulations would clarify aspects of simultaneous total arch movement . the experimental methods that have been utilized for the interpretation of force systems and the resultant displacement patterns include the photoelasticity method,7 laser holography method,8 electrical resistance straingauge method,9 and finite element method ( fem).10 the fem is a powerful tool , as it enables quantitative visualization of an object in three dimensions . it also allows simple and objective manipulation of geometrical configurations , material properties , loading conditions , and boundary conditions . in order to observe the differential movement pattern within the maxillary arch , the purpose of this study was to investigate the stress distribution and displacement pattern of the entire maxillary arch , with regard to various distalizing force vectors applied by interdental miniscrews , to assess the possibility and predictability of movement of the entire arch . a finite element model was constructed via laser scanning of a maxillary dentition from a nissin dental model ( nissin dental products , kyoto , japan ) , according to the average teeth dimensions of asian adults with normal occlusion.11 the constructed teeth were aligned and leveled using a broad arch form ( ormco , glendora , ca , usa ) as a template , while referring to the andrews12 prescription for inclination and angulation . the thickness of the periodontal ligament was assumed to be uniform ( 0.25 mm),13 and the alveolar bone crest was constructed to follow the curvature of the cementoenamel junction ( cej ) 1 mm apical to the cej ( figure 1).14 the dimensions of micro - arch brackets ( tomy , tokyo , japan ) were simulated as attachments to the teeth . the interface between the teeth and the bracket was completely joined , to omit the intervention of the composite bonding material . the distance from the incisal edge of the maxillary central incisor to the bracket slot was 4.5 mm ( perpendicular to the occlusal plane ) , 11 mm to the labial cej , and 11.8 mm to the labial alveolar crest . the main archwire was modeled according to the dimensions of a 0.017 0.025-inch ( in ) stainless steel archwire , and it was assumed that there was no play between the brackets and the archwire . at the interfacial nodes between the archwire and the brackets , translational degrees of freedom along the axial direction of the archwire were not constrained , to simulate free sliding of the archwire.15 the retraction hook was modeled using a rigid ( 0.036-in ) stainless steel wire , in order to reduce the deflection when retraction force was applied . the midpoints of incisal edges , buccal cusp tips , and root apices were used as landmarks for the assessment of displacement , and the occlusal plane was defined by connecting the midpoint of the central incisal edge and the mesiobuccal cusp of the first molar ( figure 1a ) . the miniscrew position was set at 8 mm apical to the archwire , at the midpoint between adjacent brackets ( figure 1b ) . the teeth , alveolar bone , brackets , periodontal ligament , and archwire were all constructed using fine tetrahedron solid elements , and were all assumed to be isoparametric and homogeneous linear elastic bodies . altogether , the model was constructed with 53,665 nodes and 272,118 elements . due to the large number of elements , teeth and bone were approximated as uniform structures , without differentiation into enamel / dentin or cortical / trabecular bone respectively.16 each tooth contacted the adjacent tooth at the contact point as an individual element . at the interface between the archwire and the brackets , transitional degrees of freedom were not constrained and the friction in the interface was ignored.15 other flexural directions of the archwire were coupled at the connected nodes at the junction , to eliminate possible bracket - wire play . the young 's modulus and poisson 's ratio of the elements were obtained from previous studies ( table 1).15,17 in view of the displacement of the dentition within the basal bone , the model was constrained at the nasal floor side of the alveolar bone in all directions . in order to investigate the influence of the height of the retraction hook on the displacement pattern and stress distribution , a retraction hook was located between the maxillary lateral incisor and canine . a single force vector ( 200 g ) was applied by a miniscrew positioned between the maxillary second premolar and first molar to retraction hooks extending 0 , 2 , 4 , 6 , 8 , and 10 mm apical to the archwire ( figure 2a ) . the length of the hook was set at 0 mm , and a single postero - superior force ( 200 g ) was applied from the same miniscrew position to each interbracket retraction hook , resulting in increasingly vertical angulations ( figure 2b ) . a standard coordinate system was constructed with the x - axis corresponding to the bucco - palatal direction , the y - axis the antero - posterior direction , and the z - axis the superior - inferior direction . a + x value was defined as the medial direction , + y as the anterior direction , and + z as the apical direction ( figure 3 ) . the displacements of the teeth were calculated by applying the x , y , and z coordinates at the midpoints of the incisal edges of the central and lateral incisors , the cusp tip of the canine , the buccal cusp tips of the premolars , the mesiobuccal cusp tips of the molars , and the root apices of each tooth . furthermore , the von mises stress distribution along the periodontal ligament was calculated and visualized in the contour plot , as h = ( 1 + 2 + 3)/3 , where represents the principal stresses in each dimension . , canonsburg , pa , usa ) was used to calculate and visualize the results . a finite element model was constructed via laser scanning of a maxillary dentition from a nissin dental model ( nissin dental products , kyoto , japan ) , according to the average teeth dimensions of asian adults with normal occlusion.11 the constructed teeth were aligned and leveled using a broad arch form ( ormco , glendora , ca , usa ) as a template , while referring to the andrews12 prescription for inclination and angulation . the thickness of the periodontal ligament was assumed to be uniform ( 0.25 mm),13 and the alveolar bone crest was constructed to follow the curvature of the cementoenamel junction ( cej ) 1 mm apical to the cej ( figure 1).14 the dimensions of micro - arch brackets ( tomy , tokyo , japan ) were simulated as attachments to the teeth . the interface between the teeth and the bracket was completely joined , to omit the intervention of the composite bonding material . the distance from the incisal edge of the maxillary central incisor to the bracket slot was 4.5 mm ( perpendicular to the occlusal plane ) , 11 mm to the labial cej , and 11.8 mm to the labial alveolar crest . the main archwire was modeled according to the dimensions of a 0.017 0.025-inch ( in ) stainless steel archwire , and it was assumed that there was no play between the brackets and the archwire . at the interfacial nodes between the archwire and the brackets , translational degrees of freedom along the axial direction of the archwire were not constrained , to simulate free sliding of the archwire.15 the retraction hook was modeled using a rigid ( 0.036-in ) stainless steel wire , in order to reduce the deflection when retraction force was applied . the midpoints of incisal edges , buccal cusp tips , and root apices were used as landmarks for the assessment of displacement , and the occlusal plane was defined by connecting the midpoint of the central incisal edge and the mesiobuccal cusp of the first molar ( figure 1a ) . the miniscrew position was set at 8 mm apical to the archwire , at the midpoint between adjacent brackets ( figure 1b ) . the teeth , alveolar bone , brackets , periodontal ligament , and archwire were all constructed using fine tetrahedron solid elements , and were all assumed to be isoparametric and homogeneous linear elastic bodies . altogether , the model was constructed with 53,665 nodes and 272,118 elements . due to the large number of elements , teeth and bone were approximated as uniform structures , without differentiation into enamel / dentin or cortical / trabecular bone respectively.16 each tooth contacted the adjacent tooth at the contact point as an individual element . at the interface between the archwire and the brackets , transitional degrees of freedom were not constrained and the friction in the interface was ignored.15 other flexural directions of the archwire were coupled at the connected nodes at the junction , to eliminate possible bracket - wire play . the young 's modulus and poisson 's ratio of the elements were obtained from previous studies ( table 1).15,17 in view of the displacement of the dentition within the basal bone , the model was constrained at the nasal floor side of the alveolar bone in all directions . in order to investigate the influence of the height of the retraction hook on the displacement pattern and stress distribution , a retraction hook was located between the maxillary lateral incisor and canine . a single force vector ( 200 g ) was applied by a miniscrew positioned between the maxillary second premolar and first molar to retraction hooks extending 0 , 2 , 4 , 6 , 8 , and 10 mm apical to the archwire ( figure 2a ) . the length of the hook was set at 0 mm , and a single postero - superior force ( 200 g ) was applied from the same miniscrew position to each interbracket retraction hook , resulting in increasingly vertical angulations ( figure 2b ) . a standard coordinate system was constructed with the x - axis corresponding to the bucco - palatal direction , the y - axis the antero - posterior direction , and the z - axis the superior - inferior direction . a + x value was defined as the medial direction , + y as the anterior direction , and + z as the apical direction ( figure 3 ) . the displacements of the teeth were calculated by applying the x , y , and z coordinates at the midpoints of the incisal edges of the central and lateral incisors , the cusp tip of the canine , the buccal cusp tips of the premolars , the mesiobuccal cusp tips of the molars , and the root apices of each tooth . furthermore , the von mises stress distribution along the periodontal ligament was calculated and visualized in the contour plot , as h = ( 1 + 2 + 3)/3 , where represents the principal stresses in each dimension . , canonsburg , pa , usa ) was used to calculate and visualize the results . when a posterior single force ( 200 g ) was applied to the retraction hook between the lateral incisor and canine , initial displacement of the teeth mainly occurred in the anterior segment . distalizing force was also expressed in the posterior segment , albeit a small amount , resulting in mild distal tipping of the molars ( figures 4 and 5a ) . the von mises stress was highly concentrated on the anterior teeth , especially at the lateral incisor and canine close to the retraction hook . the stress decreased in the posterior segment and showed relatively uniform distribution over the entirety of the root surfaces ( figure 5c ) . applying force to lower level retraction hooks ( conditions 1 and 2 ) led to greater lingual and coronal displacement of the incisal edges of anterior teeth than was apparent in the rest of the conditions , due to lingual rotation of the incisors . bodily movement of the anterior teeth was likely to occur when force was applied to 4 mm or 6 mm retraction hooks ( conditions 3 and 4 ) . when force was applied to higher level hooks that extended more apically , lingual root movement and labial crown movement of the anterior teeth occurred , as well as intrusion of the anterior teeth and extrusion of the posterior segment , resulting in a flattening rotation of the occlusal plane ( conditions 5 and 6 ) . the rotational and vertical changes were greater in the anterior segment according to the change of vector angulation , in contrast to the constant distalization in the posterior segment evident in all conditions ( figures 4 and 5 ) . the amount of molar distalization increased slightly as the length of the lever arm increased , due to the greater horizontal vector of the distalizing force ( figure 4b ) . as the retraction hook point moved posteriorly ( conditions 7 and 8) , the molars , in contrast , showed less distal tipping and even slight mesial tipping in condition 8 , due to the elastic bowing of the archwire between the second premolar and first molar ( figure 6a ) . when viewed from the front , greater vertical angulation of the vector induced more buccal tipping in all teeth , possibly due to the increased vertical component of the force on the buccal side ( figure 6b ) , leading to broadening of the arch form . broadening of the arch was a uniform finding in conditions 1 , 7 , 8 , and 9 possibly due to the mere buccal component of force created by the miniscrew positioned on the buccal side ( figure 6b ) . within the boundary of this study , minimal buccal displacement was found when the hook was positioned distal to the canine ( condition 7 ) , indicating effective distalization with minimal buccal tipping of the posterior segment . figure 7 shows the rotation of the occlusal plane in conditions 1 to 9 , presented in descending order of the degree of rotation . as the lever arm shortened , the amount of rotation became smaller , resulting in conversion of the direction of rotation between conditions 2 and 3 . as the point of force application moved posteriorly , the occlusal plane became steeper ( figure 7 ) . when a posterior single force ( 200 g ) was applied to the retraction hook between the lateral incisor and canine , initial displacement of the teeth mainly occurred in the anterior segment . distalizing force was also expressed in the posterior segment , albeit a small amount , resulting in mild distal tipping of the molars ( figures 4 and 5a ) . the von mises stress was highly concentrated on the anterior teeth , especially at the lateral incisor and canine close to the retraction hook . the stress decreased in the posterior segment and showed relatively uniform distribution over the entirety of the root surfaces ( figure 5c ) . applying force to lower level retraction hooks ( conditions 1 and 2 ) led to greater lingual and coronal displacement of the incisal edges of anterior teeth than was apparent in the rest of the conditions , due to lingual rotation of the incisors . bodily movement of the anterior teeth was likely to occur when force was applied to 4 mm or 6 mm retraction hooks ( conditions 3 and 4 ) . when force was applied to higher level hooks that extended more apically , lingual root movement and labial crown movement of the anterior teeth occurred , as well as intrusion of the anterior teeth and extrusion of the posterior segment , resulting in a flattening rotation of the occlusal plane ( conditions 5 and 6 ) . the rotational and vertical changes were greater in the anterior segment according to the change of vector angulation , in contrast to the constant distalization in the posterior segment evident in all conditions ( figures 4 and 5 ) . the amount of molar distalization increased slightly as the length of the lever arm increased , due to the greater horizontal vector of the distalizing force ( figure 4b ) . as the retraction hook point moved posteriorly ( conditions 7 and 8) , lingual inclination of the anterior teeth became more pronounced . the molars , in contrast , showed less distal tipping and even slight mesial tipping in condition 8 , due to the elastic bowing of the archwire between the second premolar and first molar ( figure 6a ) . when viewed from the front , greater vertical angulation of the vector induced more buccal tipping in all teeth , possibly due to the increased vertical component of the force on the buccal side ( figure 6b ) , leading to broadening of the arch form . broadening of the arch was a uniform finding in conditions 1 , 7 , 8 , and 9 possibly due to the mere buccal component of force created by the miniscrew positioned on the buccal side ( figure 6b ) . within the boundary of this study , minimal buccal displacement was found when the hook was positioned distal to the canine ( condition 7 ) , indicating effective distalization with minimal buccal tipping of the posterior segment . figure 7 shows the rotation of the occlusal plane in conditions 1 to 9 , presented in descending order of the degree of rotation . as the lever arm shortened , the amount of rotation became smaller , resulting in conversion of the direction of rotation between conditions 2 and 3 . as the point of force application moved posteriorly , the occlusal plane became steeper ( figure 7 ) . unlike predicting the movement of a single unencumbered tooth or a segment of teeth , it is difficult to predict the movement of an individual tooth tied to a continuous archwire , because inherently the system is a large segment , which has been characterized as being statically indeterminate.18 therefore , instead of mathematical calculations , the type of modeling described herein may predict the resultant displacement patterns more accurately , thus providing better clinical guidelines for the manipulation of the entire dental arch . as the finite element model in this study involved multiple interfaces between each material , i.e. , tooth , bone , periodontal ligament , bracket , and wire , the material properties and each interface were approximated according to previous reports , to simulate the clinical situation within the practical limitations of the experiment . cattaneo et al.19 claimed that load transfer did not differ between homogenous and density - based alveolar bone . since the present study focused more on the overall displacement pattern of a large segment , rather than the specific stress distribution in a small area , a homogenous model was considered appropriate . the material properties of the periodontal ligament have historically been rather controversial , with the elastic modulus ranging from 0.01 mpa to 100 mpa.17,20,21 while some insist on the non - linearity of its elastic modulus,16 it has been shown that the non - linearity mainly affects the magnitude of the stress , and not the actual movement pattern of the teeth.19 using an iterative calculation method , andersen et al.22 claimed that the material properties did not significantly reverse the resulting displacement pattern . since the effect of the force system is exerted by direct contact with juxtaposed elastic materials , any voids or plays were not defined at each interface between the bracket and wire , or between the bracket and tooth.15 although it was anticipated that archwire deformation would cause flexure of the long teeth segment , an archwire with indefinite strength was considered unrealistic,23 so the archwire in the present study was defined as an elastic material . in the present model , the point of force application in the alveolus was set 8 mm from the archwire , after considering the average crown height and the level of the mucogingival junction in healthy subjects.24,25 the displacement pattern of the dentition was analyzed when 200 g of distalizing force was applied at varying points of force application along the archwire . when the distalizing force was applied to the retraction hook between the lateral incisor and canine at various heights , stress and the initial displacement of the teeth however , the stress also spread to the second molar , leading to its posterior movement and distal tipping . this finding may be valid evidence in support of the assumption that when distalizing force is applied to an anteriorly located hook , movement of the total arch is effectively induced . when force was applied to lower level hooks , initial displacement of the anterior segment led to lingual inclination and downward displacement of the incisal edges , as well as intrusion of the posterior segment , including the premolars and molars , which resulted in steepening of the occlusal plane . in contrast , when force was applied to higher level hooks , lingual root movement and labial crown movement of the anterior teeth and extrusion of the posterior segment occurred , resulting in flattening of the occlusal plane . although these occlusal plane rotations were somewhat exaggerated by the wire deflection in the anterior segment , they can be explained by changes in the relationship between the center of resistance of the entire dentition and the line of action . jeong et al.23 reported that the center of resistance of the full maxillary dentition was 11.0 mm apical to and 26.5 mm posterior to the incisal edge of the upper central incisor when using the same finite element model employed in this study . thus , the force applied to lower level retraction hooks has a line of action that passes below the center of the resistance that causes a steepening rotation of the occlusal plane , and vice versa for forces applied to higher - level retraction hooks . these findings are not concordant with those of billiet et al.,8 who reported that translation of the maxillary dentition was observed when the force vector applied by the head - gear passed through the area of the key - ridge . this is because they applied extraoral force from outside the maxillary complex , while the distalizing force in this study was from miniscrews placed within the maxillary complex , resulting in the displacement of maxillary dentition relative to the maxilla , with the periodontal ligament as an interface . thus , the results of this study appear to be more applicable to the clinical situation using miniscrews,26 and the displacement pattern of the entire arch may be a function of the relationship between the line of action and the center of resistance of the maxillary dentition , which can be applied to arbitrary movement of the entire arch ( figure 8) . the lingual root movement of the anterior teeth when force is applied to higher - level hooks can also be explained by the line of action passing above the center of resistance of the anterior teeth . however , deflection of the wire may also affect displacement , exaggerating the labial lifting of the incisors.5 therefore , a long retraction hook can be effectively applied in cases that call for lingual root movement of the anterior teeth , while being cautious of extrusion of the posterior segment . deflection of the archwire can be minimized by loading the orthodontic force periodically to provide sufficient time for rebounding of the archwire , or by using a more rigid archwire . in contrast to the significant conversion in the displacement pattern of the anterior segment associated with the length of the retraction hook , the posterior segment showed relatively consistent and mild displacement in the distal direction . this result is encouraging , as conventional intraoral distalizers have shown a tendency to cause significant distal tipping of the molars.4 choy et al.27 suggested that the s ( variance of distribution of stress ) value increased with blunting of the root , and that a tooth with a large s value might be less prone to tipping , as the axis of rotation is more manageable . applying this concept , the posterior segment can be described as a long object antero - posteriorly , relative to the line of action , presumably with a larger s value and greater resistance to tipping than the anterior segment , which can be described as a short object antero - posteriorly . therefore , highly accurate appliance design , such as lever arm design , is more important for control of the anterior segment than the posterior segment . a relatively uniform distalization pattern , regardless of the level of the line of action , encourages distalization of the molars using a continuous archwire , suggesting a fail - safe distalization system . when postero - superior distalizing force was applied to the main archwire , the vertical component of the force , which increased as the retraction hook was located more posteriorly , led to intrusion and buccal tipping of the molars . this finding is reasonable , since the force was applied to buccal surfaces of the teeth , which generated a moment of force tipping them buccally . this can be clinically relevant when an intrusive force is applied from a miniscrew placed in buccal interdental alveolar bone to treat an anterior open bite , as buccal tipping of the molars can cause extrusion of the palatal cusps , which in turn might worsen the open bite.28,29 however , a single distalizing force on the buccal side may not have to be balanced by another force on the lingual side , as long as the vertical component of force remains minimal.3 the buccal component ( x - axis ) of force also partially contributed to the buccal tipping tendency , especially when the retraction hook was located between the lateral incisor and canine . schematic guidelines taking these findings into consideration are proposed in figure 8 , in terms of distalization and/or intrusion of the entire maxillary arch . these may still be theoretical , but there have already been substantial clinical reports and studies that support them.26,30 overall , arbitrary control of the entire maxillary dentition , encompassing rotation and translation , is expected to be feasible . firstly , the study intended to theoretically visualize the initial displacement and stress distribution of forces during full arch distalization using a fem , so the results may not reflect exact clinical outcomes , which are influenced by the cumulative effects of continuous bone reactions and rebounding of the archwire related to secondary displacement of the teeth . secondly , the thickness of the periodontal ligament was assumed to be uniform ( 0.25 mm ) , whereas in reality , it has a hour - glass shape with the narrowest zone at the mid - root level.17 furthermore , other material properties assumed in this study may differ from those found in the corresponding natural structures . for instance , the exact thicknesses of the cortical bone and trabecular bone were not incorporated into the fem model , since they were not considered determinants of the initial displacement . lastly , there are other factors that were not taken into consideration , such as deformation of the bone tissue and brackets , occlusal force , and soft tissue pressure from perioral muscles and gingival fibers . nonetheless , when supplemented with clinical , cytological , and biochemical research , the findings of this study may be effectively applied to clinical situations . 1 . a single distalizing force applied at the archwire level induced initial lingual inclination of the anterior segment , and slight intrusive distal tipping of the posterior segment . in contrast , applying force from a retraction hook at a higher level resulted in initial lingual root movement of the anterior segment , and extrusive distal translation of the posterior segment . , deflection of the archwire increased as the retraction hook was located more posteriorly , increasing the tendency for lingual inclination and extrusion of the anterior segment . also , the vertical component of the force led to intrusion and buccal tipping of the molars . the rotation of the occlusal plane was anticipated according to the relationship between the line of force and the possible center of resistance of the entire arch .

objectivethe purpose of this study was to observe stress distribution and displacement patterns of the entire maxillary arch with regard to distalizing force vectors applied from interdental miniscrews.methodsa standard three - dimensional finite element model was constructed to simulate the maxillary teeth , periodontal ligament , and alveolar process . the displacement of each tooth was calculated on x , y , and z axes , and the von mises stress distribution was visualized using color - coded scales.resultsa single distalizing force at the archwire level induced lingual inclination of the anterior segment , and slight intrusive distal tipping of the posterior segment . in contrast , force at the high level of the retraction hook resulted in lingual root movement of the anterior segment , and extrusive distal translation of the posterior segment . as the force application point was located posteriorly along the archwire , the likelihood of extrusive lingual inclination of the anterior segment increased , and the vertical component of the force led to intrusion and buccal tipping of the posterior segment . rotation of the occlusal plane was dependent on the relationship between the line of force and the possible center of resistance of the entire arch.conclusionsdisplacement of the entire arch may be dictated by a direct relationship between the center of resistance of the whole arch and the line of action generated between the miniscrews and force application points at the archwire , which makes the total arch movement highly predictable .

INTRODUCTION MATERIALS AND METHODS Model creation and boundary conditions Simulation of force vectors RESULTS The effects of distalizing force application to variable lengths of lever arm The effect of distalizing force application on the main archwire with variable points of force application Rotation of the occlusal plane according to the line of action of the distalizing force DISCUSSION CONCLUSION

gestational diabetes mellitus ( gdm ) is a common medical complication of pregnancy defined as any degree of glucose intolerance with onset or first recognition during pregnancy [ 1 , 2 ] . the initial criteria for diagnosis were established more than 40 years ago ; however , these criteria did not necessarily identify pregnancies with increased risk of adverse pregnancy outcome . the hyperglycaemia and adverse pregnancy outcome ( hapo ) study was conducted to clarify the associations between maternal hyperglycaemia and adverse outcomes . the study showed associations between increasing levels of fasting blood glucose ( fbg ) , 1-hour and 2-hour plasma glucose obtained following an oral glucose tolerance test ( ogtt ) , and birthweight > 90th centile and cord - blood serum c - peptide level > 90th centile . the secondary outcomes of premature delivery , shoulder dystocia or birth injury , admission to intensive neonatal care unit , hyperbilirubinemia , and preeclampsia were also increased by maternal hyperglycaemia . the consideration of hapo data led to a recommendation in 2010 by the international association of diabetes and pregnancy study groups ( iadpsg ) for the fbg and 1 h and 2 h glucose levels to diagnose gdm . the diagnostic threshold values were the average glucose values at which the odds for birthweight > 90th centile , cord c - peptide > 90th centile , and percent body fat > 90th centile reached 1.75 times the estimated odds of the outcomes at mean glucose values [ 4 , 5 ] . however , concern has been expressed that adoption of the new diagnostic criteria would lead to a dramatic increase in the incidence of gdm . one australian study reported that the change in diagnostic criteria from the previously utilised australasian diabetes in pregnancy society ( adips ) 1999 criteria to the new iadpsg 2010 criteria would increase the prevalence of gdm from 9.6% to 13.0% . national debate continues on the workforce implications of the revised criteria and their clinical impact [ 9 , 10 ] . the aim of this study was to audit the impact of the change from the adips 1999 criteria to the iadpsg 2010 diagnostic criteria within a geographically defined region . a prospective audit of all pregnancies diagnosed with gdm commenced from july 1 , 2010 , following publication of hapo and the iadpsg recommendations . the institutional ethics committee determined that the project fulfilled the criteria of an audit project as pregnancy outcomes were being audited and no intervention other than routine care according to existing clinical protocols was planned . all pregnant women greater than 20-week gestation referred for public maternity care who resided within the postcodes 60016007 , 6147 , 6148 , 6151 , 6152 , and 6155 within the north metropolitan health service of the western australian department of health between july 1 , 2010 , to june 30 , 2014 , were included in the audit . women with a history of preexisting diabetes mellitus ( type 1 or 2 ) were specifically excluded from the project . all women had an ogtt between 24 and 28 weeks of gestation in accordance with the existing clinical protocol . the adips 1999 criteria were used to diagnose gdm in the period from july 1 , 2010 , to december 31 , 2011 . women had a 75-gram ogtt with glucose samples taken after an overnight fast and at 2 hr postprandially . gdm was diagnosed if the fasting glucose was 5.5 mmol / l ( 100 mg / dl ) and/or the 2 h glucose was 8.0 mmol / l ( ~145 mg / dl ) . from january 1 , 2012 , to june 30 , 2014 , women had a 75-gram ogtt with glucose samples taken after an overnight fast and at 1 hr and 2 hr postprandially . one or more abnormal values were needed for a diagnosis of gdm to be made : fbg > 5.0 the majority of ogtt in the audit period was performed at the western diagnostics pathology laboratories . a small number ( 2.8% ) all patients had their weight ( kg ) and height ( m ) recorded at their booking visit to calculate their body mass index ( bmi ) . patients with a bmi greater than 40 had their antenatal care at the local maternity hospital but were referred for delivery to the regional tertiary hospital . all pregnancies diagnosed with gdm across the audit period received identical clinical care according to a written protocol . this involved an initial consultation with a diabetic educator , dietician , and obstetric doctor . patients commenced self - monitoring of blood sugar levels and adopted a diabetic diet . a review visit a fortnight delivery outcomes were entered into a computerized database called meditec by attending midwifery staff as part of routine practice . delivery outcomes were subsequently extracted from meditec , case note audit , and a postnatal clinical service for all women with gdm conducted by one author ( julie quinlivan ) . these were mode of delivery , elective or emergency caesarean section , estimated blood loss , and 3rd or 4th degree perineal tear . these were gestational age at birth , birthweight , birthweight > 90th centile adjusted for gestational age , apgar at 1 and 5 minutes , umbilical artery and vein ph , admission to special care unit , and serious perinatal complications such as stillbirth , neonatal death , or birth trauma including shoulder dystocia . a power calculation assumed that the change in incidence of gdm would be 30% , a conservative estimate based on the previous australian and new zealand studies [ 7 , 8 ] . the baseline rate of gdm in the audit region was approximately 3.5% . assuming a power of 80% and alpha error of 0.05 , a sample of 10,994 women was required across the audit period to detect a change in incidence from 3.5 to 4.6% . descriptive statistics of predefined clinical outcomes were compared using student 's t - test for continuous variables and chi square test or fisher exact test for discrete data . of 10,296 women delivering in the audit period , antenatal ogtt results could be traced for 10,277 women ( 99.8% ) . the remaining 19 ( 0.2% ) of these women , 5 attempted an ogtt and were unable to complete the test due to nausea and/or vomiting . the other 14 women either presented for care too late for testing or declined testing . the overall incidence was not significantly different with 3.4% diagnosed under the adips 1999 criteria and 3.5% under the iadpsg 2010 criteria . in the subgroup of 342 women with a bmi > 40 ( representing 3.3% of the study population ) the incidence of gdm was 3.7% using adips 1999 criteria and 8.5% using iadpsg 2010 criteria . this difference was not statistically significant ( p = 0.11 ) ; however , the audit was not adequately powered to detect a difference in the subgroup of women with high bmi . across the audit period the proportion of women with gdm who required management with medication ( metformin or insulin ) in addition to diet was not significantly different ( 25% in women diagnosed by adips 1999 criteria and 28% in women diagnosed by iadpsg 2010 criteria , resp . ) . delivery data for 10,277 ( 98% ) women were available for audit through meditec , case note audit , or the postnatal clinical service . babies born to women diagnosed with gdm according to the iadpsg 2010 criteria had significantly higher umbilical artery ph ( 7.28 versus 7.21 ; p = 0.01 ) . they had a significant lower birthweight ( 3360 gms versus 3470 gms ; p = 0.02 ) and birthweight above the > 90th centile adjusted for gestational age ( 11% versus 18% ; p = 0.04 ) . the audit found no significant difference in the incidence of gdm before and after the introduction of the iadpsg 2010 criteria , with the overall incidence being low at 3.5% . our results differ from the previous australian and new zealand studies [ 7 , 8 ] . one explanation may be that prevalence of gdm in our region is low compared to many other sites . our rate of 3.5% contrasts higher background rates in the sites involved in the hapo trial where incidences ranged from 8 to 25% . however , hapo study sites were specifically included because of their high rates of gdm . they were tertiary sites where women with high bmi and other pregnancy complications were referred for antenatal and delivery management [ 5 , 12 ] . our study was based upon a geographical region rather than a hospital cohort and thus captured women of all risk levels , including a majority who were of normal risk , unlike the patient population within a tertiary centre . a second explanation for the observed difference in outcome between our study and previous ones may be the racial mix of the population . although our geographic maternity cohort reflected the wider australian public maternity cohort in terms of maternal age and parity , racial background was overwhelmingly english speaking caucasian . our geographic catchment has a low prevalence of overweight and obese patients compared to many sites . lower obesity levels mean that the underlying risk of metabolic hyperglycaemia is lowered . of note , in our subgroup of women with a bmi > 40 the incidence of gdm rose from 3.7% to 8.5% under the iadpsg 2010 criteria , more in line with studies elsewhere [ 7 , 8 ] . as a secondary consideration , the adoption of the iadpsg 2010 criteria did not adversely impact upon our predefined maternal and newborn outcomes . there was a significant improvement in three newborn outcomes , being an increase in umbilical artery ph and a reduction in birthweight and birthweight > 90th centile adjusted for gestational age . firstly , data were extracted from a defined geographic region before and after implementation of the iadpsg 2010 diagnostic criteria . thirdly , there was high compliance with screening for gdm ( 99.8% ) and ascertainment of outcome ( 98% of women ) . a study limitation is the low background incidence of gdm that limits generalizability to regions where incidence rates are higher . a second limitation is that only 3.3% of women presented with a bmi > 40 . in this subgroup of women , centres where the obstetric population has a higher incidence of obesity may report an increase in the incidence in gdm utilising the new diagnostic criteria . however , it is likely that this reflects a genuine increase in metabolic pathology , as obesity is a major risk factor for gdm and adverse pregnancy outcome . the iadpsg used a consensus process to redefine gdm based on its association with adverse pregnancy outcomes . however , in our audit study of 10,296 women , we observed no significant increase in the incidence of gdm .

background . controversy surrounds the decision to adopt the international association of diabetes and pregnancy study groups ( iadpsg ) criteria for the diagnosis of gestational diabetes mellitus ( gdm ) as fears that disease prevalence rates will soar have been raised . aims . to investigate the prevalence of pregnancy complicated with gdm before and after the introduction of the iadpsg 2010 diagnostic criteria . materials and methods . a prospective audit of all women who delivered from july 1 , 2010 , to june 30 , 2014 , in a predefined geographic region within the north metropolitan health service of western australia . women were diagnosed with gdm according to australian diabetes in pregnancy society ( adips 1991 ) criteria until december 31 , 2011 , and by the iadpsg 2010 criteria after this date . incidence of gdm and predefined pregnancy outcomes were audited . results . of 10,296 women , antenatal oral glucose tolerance test ( ogtt ) results and follow - up data were obtained for 10,103 women ( 98% ) , of whom 349 ( 3.5% ) were diagnosed with gdm . the rate of gdm utilising adips criteria was 3.4% and the rate of utilising iadpsg criteria was 3.5% ( p = 0.92 ) . conclusion . iadpsg diagnostic criteria did not significantly increase the incidence of gdm in this low prevalence region .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusion

neuregulin 1 ( nrg1 ) is a ligand for the neu / erbb2 protooncogene and closely related to cell - cell signal interactions required for the growth and development of multiple organ systems . nrg1 exerts its effect on the epithelium , cardiovascular system , and central nervous system ( cns ) . in early embryogenesis , nrg1 is expressed on neural tissue , respiratory epithelium , and endocardium , and in later stage predominantly expressed in neural tissue . genetic factors which promote neuronal development and modulate synaptic plasticity may influence the development and symptoms of schizophrenia . nrg1 plays a role in antipsychotic treatment of schizophrenia and may affect dopamine receptors ( d2 and d3 ) . nrg1 is involved in the abnormal gamma - aminobutyric acid ( gaba ) neurotransmission in schizophrenia , together with erbb4 , which is synaptic receptor of nrg1 and regulates synaptic maturation . furthermore , n - methyl - d - aspartate ( nmda ) receptor functional change is related to schizophrenia . nrg1 polymorphisms were reported to be associated with susceptibility to schizophrenia in iceland population , and replication studies of scottish and chinese populations . in another scottish population study , , we investigated the relationship between nrg1 polymorphisms and the development of schizophrenia in korean population . a total of 221 schizophrenia patients and 359 control subjects ( 44.26.3 years ) were recruited . the schizophrenia group consisted of 122 males and 99 females , and the control group was comprised of 180 males and 179 females . schizophrenia patients were selected among participants who visited at the departments of neuropsychiatry in the east - west neomedical center and kyung hee medical center , seoul , korea . patients were diagnosed with schizophrenia by two psychiatrists according to the diagnostic and statistical manual of mental disorders ( dsm - iv ) . subjects with other psychiatric disorders , neurological diseases , and any severe diseases were excluded . controls subjects were recruited among participants who assessed as mentally healthy through a general health examination program . this study was conducted in accordance with the guidelines of the helsinki declaration and approved by the ethics review committee of medical research institute , kyung hee university medical center , seoul , korea . we searched the promoter and coding regions of the nrg1 gene in the snp database of the national center for biotechnology information ( http://www.ncbi.nlm.nih.gov/snp , build 135 ) , and selected two promoter snps ( rs7014762 , -1174 a / t and rs11998176 , -788 a / t ) and one missense snp ( rs3924999 , arg253gln ) among the nrg1 snps . peripheral blood sample of each subject was collected in heparin or edta tubes . dna isolation kit for cells and tissues ( roche , indianapolis , in , usa ) was used for extracting genomic dna . polymerase chain reactions ( pcrs ) were performed as the following condition : 35 cycles at 94 for 30 sec , 58 for 30 sec , 72 for 30 sec , and 1 cycle at 72 for 5 min for the final reaction . the primer sequences for pcrs were as following : rs7014762 ( sense , tgccaacttgcagaatcttggg ; anti - sense , aatgggcgatagatccacactg ) , rs11998176 ( sense , cagtgtggatctatcgcccatt ; anti - sense , aacgctctctctccttgcagcg ) , and rs3924999 ( sense , gatccattttcgctcatccattt ; anti - sense , cccaaagagctgggattacagtt ) ( table 1 ) . the pcr products were processed through direct sequencing ( macrogen , seoul , korea ) , and genotypes of each snp were analyzed with seqmanii software ( dnastar , madison , wi , usa ) . we applied multiple logistic regression models in analysis of genotype data : codominant1 ( major allele homozygotes vs. heterozygotes ) , codominant2 ( major allele homozygotes vs. minor allele homozygotes ) , dominant ( major allele homozygotes vs. heterozygotes+minor allele homozygotes ) , recessive ( major allele homozygotes+heterozygotes vs. minor allele homozygotes ) , and log - additive ( major allele homozygotes vs. heterozygotes vs. minor allele homozygotes ) models . odd ratios ( or ) , 95% confidence intervals ( ci ) , and p - values were calculated with snpstats ( http://bioinfo.iconcologia.net/index.php?module=snpstats ) and spss 18.0 ( spss inc . , haploview version 4.2 ( daly lab , cambridge , ma , usa ) was used in determining linkage disequilibrium ( ld ) block and haplotypes . a total of 221 schizophrenia patients and 359 control subjects ( 44.26.3 years ) were recruited . the schizophrenia group consisted of 122 males and 99 females , and the control group was comprised of 180 males and 179 females . schizophrenia patients were selected among participants who visited at the departments of neuropsychiatry in the east - west neomedical center and kyung hee medical center , seoul , korea . patients were diagnosed with schizophrenia by two psychiatrists according to the diagnostic and statistical manual of mental disorders ( dsm - iv ) . subjects with other psychiatric disorders , neurological diseases , and any severe diseases were excluded . controls subjects were recruited among participants who assessed as mentally healthy through a general health examination program . this study was conducted in accordance with the guidelines of the helsinki declaration and approved by the ethics review committee of medical research institute , kyung hee university medical center , seoul , korea . we searched the promoter and coding regions of the nrg1 gene in the snp database of the national center for biotechnology information ( http://www.ncbi.nlm.nih.gov/snp , build 135 ) , and selected two promoter snps ( rs7014762 , -1174 a / t and rs11998176 , -788 a / t ) and one missense snp ( rs3924999 , arg253gln ) among the nrg1 snps . peripheral blood sample of each subject was collected in heparin or edta tubes . dna isolation kit for cells and tissues ( roche , indianapolis , in , usa ) was used for extracting genomic dna . polymerase chain reactions ( pcrs ) were performed as the following condition : 35 cycles at 94 for 30 sec , 58 for 30 sec , 72 for 30 sec , and 1 cycle at 72 for 5 min for the final reaction . the primer sequences for pcrs were as following : rs7014762 ( sense , tgccaacttgcagaatcttggg ; anti - sense , aatgggcgatagatccacactg ) , rs11998176 ( sense , cagtgtggatctatcgcccatt ; anti - sense , aacgctctctctccttgcagcg ) , and rs3924999 ( sense , gatccattttcgctcatccattt ; anti - sense , cccaaagagctgggattacagtt ) ( table 1 ) . the pcr products were processed through direct sequencing ( macrogen , seoul , korea ) , and genotypes of each snp were analyzed with seqmanii software ( dnastar , madison , wi , usa ) . we applied multiple logistic regression models in analysis of genotype data : codominant1 ( major allele homozygotes vs. heterozygotes ) , codominant2 ( major allele homozygotes vs. minor allele homozygotes ) , dominant ( major allele homozygotes vs. heterozygotes+minor allele homozygotes ) , recessive ( major allele homozygotes+heterozygotes vs. minor allele homozygotes ) , and log - additive ( major allele homozygotes vs. heterozygotes vs. minor allele homozygotes ) models . odd ratios ( or ) , 95% confidence intervals ( ci ) , and p - values were calculated with snpstats ( http://bioinfo.iconcologia.net/index.php?module=snpstats ) and spss 18.0 ( spss inc . , haploview version 4.2 ( daly lab , cambridge , ma , usa ) was used in determining linkage disequilibrium ( ld ) block and haplotypes . the age of the schizophrenia group was 42.010.6 ( mean agestandard deviation ) years and that of the control group was 44.26.3 years ( data not shown ) . table 2 shows the genotype and allele frequencies of the three examined snps ( rs7014762 , -1174 a / t ; rs11998176 , -788 a / t ; rs3924999 , arg253gln ) of nrg1 in the control group and the schizophrenia group . the genotype distributions of the three tested snps were consistent with the hardy - weinberg equilibrium ( hwe ) in the control group ( rs7014762 , hwe p=0.91 ; rs11998176 , hwe p=0.71 ; rs3924999 , hwe p=0.78 , data not shown ) . the genotype ( t / t : a / t : a / a genotypes ) distributions of rs7014762 in the control and schizophrenia groups were 43.9%:44.5%:11.6% and 44.1%:45.5%:10.4% , respectively . the allele ( t : a alleles ) distributions of rs7014762 in the control and schizophrenia groups were 66.1%:33.9% and 66.8%:33.2% . the genotype ( a / a : a / t : t / t ) distributions of rs11998176 in the control and schizophrenia groups were 86.1%:13.4%:0.5% and 82.8%:16.7%:0.5% , respectively . the allele ( a : t ) distributions of rs11998176 in the control and schizophrenia groups were 92.8%:7.2% and 91.2%:8.8% , respectively . the genotype and allele distributions of two snps ( rs7014762 and rs11998176 ) were not significant between controls and schizophrenia patients ( p>0.05 , table 2 ) . however , the genotype ( a / a : a / g : g / g ) distributions of rs3924999 in the control and schizophrenia groups were 55.2%:38.7%:6.1% and 64.7%:32.1%:3.2% , respectively . the differences were significant association ( or=0.67 , 95% ci=0.47 - 0.95 , p=0.022 in the dominant model and or=0.69 , 95% ci=0.51 - 0.93 , p=0.013 in the log - additive model ) . the allele ( a : g ) distributions of rs3924999 in the control and schizophrenia groups were 74.5%:25.5% and 80.8%:19.2% . the difference of allele distributions showed significant association ( or=0.70 , 95% ci=0.52 - 0.93 , p=0.014 ) ( table 2 ) . ld block was determined using haploview 4.2 , and one ld block between rs7014762 and rs11998176 was constructed ( fig . 1 ) . in order to investigate association between the haplotypes of ld block snps and schizophrenia , there were three haplotypes ( ta , 0.585 ; aa , 0.336 ; tt , 0.078 in frequency ) . the haplotypes showed no significant difference between the control group and the schizophrenia group ( table 3 ) . nrg1 is well - known schizophrenia - related gene , and there have been association studies of nrg1 snps with schizophrenia susceptibility from a decade ago [ 10 - 13 ] . a genome - wide association study ( gwas ) used 33 families consisted by 110 patients in iceland was performed in the year 2002 and the researchers identified nrg1 as a schizophrenia candidate gene . they concluded that the ' at - risk ' haplotypes at the 5 ' end of the nrg1 gene is responsible for the analysis result . replication study was conducted in scottish population , and they found that the ' at - risk ' haplotype and rs35753505 snp were still significant . however , in another replication study conducted in han chinese populations , they did not show significance , instead , they found a new haplotype ' hap china 3 ' consisted by three groups of microsatellite markers . in another scottish population study , rs6988339 and rs3757930 snps , which were in near the 3 ' region of the nrg1 gene and not previously studied in iceland population , were associated with bipolar disorder as well as schizophrenia . such differences suggest that various snps of nrg1 may contribute or not to the schizophrenia development in certain populations . in our study results , two promoter snps ( rs7014762 , -1174 a / t and rs11998176 , -788 a / t ) selected from the nrg1 gene were not statistically associated with risk of schizophrenia , and one missense snp ( rs3924999 , arg253gln ) was associated with the development of schizophrenia . the rs7014762 snp was once reported to be associated with bipolar disorder , however , not with schizophrenia . and there was no prior study of rs11998176 ( promoter , -788 a / t ) . however , the study result showed no association between rs11998176 and schizophrenia development in korean population . the rs3924999 snp has been frequently reported in schizophrenia - related researches . in a nrg1 genetic study in chinese population another chinese population study showed that rs3924999 was associated with schizophrenia risk , especially in the subgroup of patients with positive symptom . in the other study of chinese population , rs3924999 was associated with the prepulse inhibition ( ppi ) , which is a measurable schizophrenia phenotypic feature . on the contrary , association of rs3924999 with auditory p300 in schizophrenia was reported . in our study result , the g allele of rs3924999 , which is minor allele , showed less odds ratio in the schizophrenia group than that of the control group in the dominant model ( or=0.67 , 95% ci=0.47 - 0.95 , p=0.022 ) and the log - additive model ( or=0.69 , 95% ci=0.51 - 0.93 , p=0.013 ) . allelic distribution of rs3924999 was significantly different between schizophrenia and controls ( or=0.70 , 95% ci=0.52 - 0.93 , p=0.014 ) . these results were slight association , however , it might be suggested from the result that the variant allele g may have a dominant effect and more protective effect from schizophrenia than the majority allele a of rs3924999 , or the a allele of rs3924999 might be an allele with increased risk of schizophrenia development . the g allele of rs3924999 changes original 253th amino acid arginine to variant amino acid glutamine , and its change might have modified the cell signaling of nrg1 . in conclusion , this study results showed that a missense snp rs3924999 ( arg253gln ) of nrg1 was weakly associated with the development of schizophrenia in korean population . two other snps of nrg1 ( rs7014762 and rs11998176 ) did not show significant associations . the results suggest that rs3924999 of the nrg1 gene may contribute to schizophrenia development in korean population .

neuregulin 1 ( nrg1 ) is associated with the pathogenesis of schizophrenia through controlling activation and signaling of neurotransmitter receptors . influence to schizophrenia development by the nrg1 gene may differ in individuals , and genetic polymorphism is one of the factors affecting their differences . association between three single nucleotide polymorphisms ( snps ) ( rs7014762 , -1174 a / t ; rs11998176 , -788 a / t ; rs3924999 , arg253gln ) of nrg1 and the development of schizophrenia was analyzed in 221 schizophrneia and 359 control subjects . polymerase chain reaction and direct sequencing were performed to obtain genotype data of nrg1 snps of the subjects . in analysis of genetic data , multiple logistic regression models ( codominant1 , codominant2 , dominant , recessive , and log - additive model ) were applied . snpstats and spss 18.0 were used to calculate odds ratio ( or ) , 95% confidence interval ( ci ) , and p - value of each model . the genotype distributions of rs3924999 were associated with schizophrenia development ( or=0.67 , 95% ci=0.47 - 0.95 , p=0.022 in the dominant model and or=0.69 , 95% ci=0.51 - 0.93 , p=0.013 in the log - addtive model ) and allelic distributions also showed significant association ( or=0.70 , 95% ci=0.52 - 0.93 , p=0.014 ) . the results suggest that rs3924999 of the nrg1 gene may be associated with schizophrenia susceptibility .

INTRODUCTION MATERIALS AND METHODS Schizophrenia and control subjects SNP selection and genotyping Statistical analysis RESULTS DISCUSSION

a total of 220 crc samples were obtained from patients who had undergone surgery between 2003 and 2004 at chonbuk national university hospital . we obtained clinical data from the patients ' records , and survival data were collected in august 2011 . this study received a local ethics committee approval from the institutional review board of chonbuk national university hospital . the mean age of the patients at the time of surgery was 62.2 years ( range , 28 to 84 years ) . hematoxylin and eosin ( h&e ) stained slides were reviewed and were graded according to the world health organization classification.21 pathologic staging was reviewed based on the tumor , node , metastasis staging system described by the american joint committee on cancer.22 the patients were grouped according to their age , gender , location , tumor size , differentiation , lymphovascular invasion , pathologic stage ( i and ii vs iii and iv ) , and preoperative serum carcinoembryonic antigen ( cea ) level . formalin - fixed paraffin - embedded tissue blocks of 220 cases were obtained , and three cores were taken from every case . each case was represented by one normal core and two tumor cores with a cross - sectioned diameter of 2 mm . the tissue microarray slides from original paraffin - embedded specimens were stained uniformly with h&e . each pathological diagnosis was reviewed by two experienced pathologists ( h.s.park and y.n.kim ) who were blind to each other 's evaluation . paraffin - embedded tissue samples for immunohistochemistry were provided by the chonbuk national university hospital , a member of the national biobank of korea , which is supported by the ministry of health , welfare and family affairs . tissue microarray sections were cut into 4-m thick tissue sections , and endogenous activity was quenched by incubation with 3% hydrogen peroxidase for 30 minutes after deparaffinization and hydration . tissue sections were treated with a microwave antigen retrieval procedure in 0.01 m sodium citrate buffer ( ph 6.0 ) for cortactin and with a high pressure cooker antigen retrieval procedure in 0.01 m sodium citrate buffer ( ph 9.0 ) for fak . after blocking endogenous peroxidase , sections were incubated with protein block serum - free ( dako , carpinteria , ca , usa ) at room temperature for 10 minutes to block non - specific staining . the sections were then incubated at 4 overnight with monoclonal anti - cortactin ( 1:100 , clone 30 , bd bioscience , san jose , ca , usa ) and polyclonal anti - fak ( 1:50 , cell signaling technology , danvers , ma , usa ) . after incubation with the appropriate biotin - conjugated secondary antibody and subsequently with a streptavidin solution , color development was done using 3-amino-9-ethylcarbazole as a chromogen and the tissues were counterstained with hematoxylin . an immunohistochemical analysis was done by four authors ( y.n.kim , j.e.choi , k.y.jang , and h.s.park ) without knowledge of the clinicopathologic information . each case was evaluated by multiplying score of staining intensity and stained area of cortactin and fak . staining intensity was determined as negative , 0 ; weak , 1 ; moderate , 2 ; and strong , 3 . stained area was divided into four groups : negative , 0 ; 25% of cells , 1 ; > 25% and 50% of cells , 2 ; > 50% of cells , 3 . in addition , immunoreactivity was divided into two groups on the basis of the multiplying score ; 0 - 2 was defined as negative , 3 was defined as positive immunoreactivity.7 of the 220 cases , 2 cases for cortactin and 3 cases for fak were missed during immunostaining . eventually , 218 cases for cortactin and 217 cases for fak were evaluated for immunohistochemical analysis . the points of interest were the relapse - free survival and the overall survival of patients . the point that required follow - up was the last date of contact or the patient 's date of death until august 2011 . overall survival was calculated as the time from diagnosis to the date of death or last contact . patients who were alive at last contact were treated as censored for overall survival analysis . relapse - free survival was calculated from the time of diagnosis to the date of recurrence , distant metastasis , death , or last contact . patients who were alive at the last contact and who had not experienced recurrence were treated as censored for relapse - free survival analysis . the associations between the expression of cortactin or fak and other clinicopathologic factors were analyzed using pearson 's chi - square test . a univariate and multivariate cox proportional hazard regression analysis was done to estimate the impact of clinicopathologic factors and the expression of each marker on a relapse - free survival and overall survival . kaplan - meier survival curves were constructed to further illustrate the impact of overall survival when indicated . spss ver . 18.0 ( spss inc . , chicago , il , usa ) was used for statistical analysis , and a p - value of < 0.05 was considered statistically significant . a total of 220 crc samples were obtained from patients who had undergone surgery between 2003 and 2004 at chonbuk national university hospital . we obtained clinical data from the patients ' records , and survival data were collected in august 2011 . this study received a local ethics committee approval from the institutional review board of chonbuk national university hospital . the mean age of the patients at the time of surgery was 62.2 years ( range , 28 to 84 years ) . hematoxylin and eosin ( h&e ) stained slides were reviewed and were graded according to the world health organization classification.21 pathologic staging was reviewed based on the tumor , node , metastasis staging system described by the american joint committee on cancer.22 the patients were grouped according to their age , gender , location , tumor size , differentiation , lymphovascular invasion , pathologic stage ( i and ii vs iii and iv ) , and preoperative serum carcinoembryonic antigen ( cea ) level . formalin - fixed paraffin - embedded tissue blocks of 220 cases were obtained , and three cores were taken from every case . each case was represented by one normal core and two tumor cores with a cross - sectioned diameter of 2 mm . the tissue microarray slides from original paraffin - embedded specimens were stained uniformly with h&e . each pathological diagnosis was reviewed by two experienced pathologists ( h.s.park and y.n.kim ) who were blind to each other 's evaluation . paraffin - embedded tissue samples for immunohistochemistry were provided by the chonbuk national university hospital , a member of the national biobank of korea , which is supported by the ministry of health , welfare and family affairs . tissue microarray sections were cut into 4-m thick tissue sections , and endogenous activity was quenched by incubation with 3% hydrogen peroxidase for 30 minutes after deparaffinization and hydration . tissue sections were treated with a microwave antigen retrieval procedure in 0.01 m sodium citrate buffer ( ph 6.0 ) for cortactin and with a high pressure cooker antigen retrieval procedure in 0.01 m sodium citrate buffer ( ph 9.0 ) for fak . after blocking endogenous peroxidase , sections were incubated with protein block serum - free ( dako , carpinteria , ca , usa ) at room temperature for 10 minutes to block non - specific staining . the sections were then incubated at 4 overnight with monoclonal anti - cortactin ( 1:100 , clone 30 , bd bioscience , san jose , ca , usa ) and polyclonal anti - fak ( 1:50 , cell signaling technology , danvers , ma , usa ) . after incubation with the appropriate biotin - conjugated secondary antibody and subsequently with a streptavidin solution , color development was done using 3-amino-9-ethylcarbazole as a chromogen and the tissues were counterstained with hematoxylin . an immunohistochemical analysis was done by four authors ( y.n.kim , j.e.choi , k.y.jang , and h.s.park ) without knowledge of the clinicopathologic information . each case was evaluated by multiplying score of staining intensity and stained area of cortactin and fak . staining intensity was determined as negative , 0 ; weak , 1 ; moderate , 2 ; and strong , 3 . stained area was divided into four groups : negative , 0 ; 25% of cells , 1 ; > 25% and 50% of cells , 2 ; > 50% of cells , 3 . in addition , immunoreactivity was divided into two groups on the basis of the multiplying score ; 0 - 2 was defined as negative , 3 was defined as positive immunoreactivity.7 of the 220 cases , 2 cases for cortactin and 3 cases for fak were missed during immunostaining . eventually , 218 cases for cortactin and 217 cases for fak were evaluated for immunohistochemical analysis . the points of interest were the relapse - free survival and the overall survival of patients . the point that required follow - up was the last date of contact or the patient 's date of death until august 2011 . overall survival was calculated as the time from diagnosis to the date of death or last contact . patients who were alive at last contact were treated as censored for overall survival analysis . relapse - free survival was calculated from the time of diagnosis to the date of recurrence , distant metastasis , death , or last contact . patients who were alive at the last contact and who had not experienced recurrence were treated as censored for relapse - free survival analysis . the associations between the expression of cortactin or fak and other clinicopathologic factors were analyzed using pearson 's chi - square test . a univariate and multivariate cox proportional hazard regression analysis was done to estimate the impact of clinicopathologic factors and the expression of each marker on a relapse - free survival and overall survival . kaplan - meier survival curves were constructed to further illustrate the impact of overall survival when indicated . chicago , il , usa ) was used for statistical analysis , and a p - value of < 0.05 was considered statistically significant . the immunohistochemical staining of cortactin and fak in microarray tissue of normal colorectal mucosa and crc are shown in fig . 1 . expression of cortactin and fak was found in epithelial cells , endothelial cells and smooth muscle cells , but not in other stromal cells . the intensity of cytoplasmic expression of cortactin and fak were only weak to moderate in normal colorectal mucosal epithelium , and were moderate to strong in crcs . in crcs , there was no difference in staining intensity of cortactin and fak whether or not the tumor cells were located in the center of tumor nests . as shown in table 1 , cortactin expression was seen in 84% ( 184 of 218 cases ) of crcs and 9% ( 20 of 218 cases ) of normal colorectal mucosa , respectively , while fak expression was seen in 61% ( 133 of 217 cases ) of crcs and 11% ( 24 of 217 cases ) of normal colorectal mucosa , respectively . the positive expression rate of cortactin and fak was significantly higher in crcs than in normal colorectal mucosa . , cortactin expression was correlated with poor differentiation ( p=0.011 ) and positive expression of fak ( p<0.001 ) . other clinicopathologic variables including age , gender , location , tumor size , lymphovascular invasion , pathologic stage and preoperative cea had no association with cortactin expression . however , among the 217 cases of crcs , fak expression was correlated with lymph node metastasis ( p=0.032 ) but not with other clinicopathological variables such as age , gender , location , tumor size , differentiation , lymphovascular invasion , pathologic stage and preoperative cea . the univariate cox proportional hazard analysis of the expression of cortactin and fak and their association with overall survival and relapse - free survival are shown in table 3 . patient 's age over 60 years ( p=0.011 ) , tumor size larger than 4.3 cm ( p=0.011 ) , poor differentiation ( p<0.001 ) , advanced pathologic stage ( p<0.001 ) , preoperative cea level over 5.0 ng / ml ( p=0.002 ) and cortactin expression ( p=0.021 ) predicted shorter overall survival . tumors located in the left colon ( p=0.033 ) predicted longer overall survival than those in the right colon . on the other hand , a tumor size larger than 4.3 cm ( p=0.008 ) , a tumor with poor differentiation ( p=0.006 ) , an advanced pathologic stage ( p<0.001 ) , a preoperative cea level over 5.0 ng / ml ( p=0.001 ) and a cortactin expression ( p=0.005 ) predicted shorter relapse - free survival . however , fak expression in crcs predicted neither shorter overall nor shorter relapse - free survival . kaplan - meier survival curves for overall survival in relation to the patient 's age , pathologic stage , preoperative cea level , and cortactin expression in crcs are presented in fig . in addition , kaplan - meier survival curves for relapse - free survival in relation to tumor size , pathologic stage , preoperative cea level and cortactin expression in crcs are also presented in fig . a multivariate analysis was done in 209 patients with complete information for clinicopathologic factors including age , tumor size , pathologic stage , preoperative cea and cortactin expression . from the multivariate analysis , pathologic stage , preoperative cea and cortactin expression were independent prognostic factors that were significantly associated with both overall and relapse - free survival ( table 4 ) . an advanced stage ( iii / iv ) was an independent prognostic factor which was significantly associated with poor overall ( adjusted hazard ratio [ hr ] , 3.700 ; 95% confidence interval [ ci ] , 2.170 to 6.309 ; p<0.001 ) and relapse - free survival ( adjusted hr , 2.786 ; 95% ci , 1.755 to 4.424 ; p<0.001 ) . a preoperative cea over 5 ng / ml was an independent prognostic factor significantly associated with poor overall ( adjusted hr , 1.899 ; 95% ci , 1.121 to 3.216 ; p=0.017 ) and relapse - free survival ( adjusted hr , 1.833 ; 95% ci , 1.145 to 2.933 ; p=0.012 ) . cortactin expression was an independent prognostic factor significantly associated with poor overall ( adjusted hr , 3.075 ; 95% ci , 1.111 to 8.514 ; p=0.031 ) and relapse - free survival ( adjusted hr , 3.565 ; 95% ci , 1.433 to 8.868 ; p=0.006 ) . in addition , an age of over 60 years was also an independent prognostic factors significantly associated with poor overall ( adjusted hr , 2.537 ; 95% ci , 1.440 to 4.469 ; p=0.001 ) , but not relapse - free survival . the immunohistochemical staining of cortactin and fak in microarray tissue of normal colorectal mucosa and crc are shown in fig . 1 . expression of cortactin and fak was found in epithelial cells , endothelial cells and smooth muscle cells , but not in other stromal cells . the intensity of cytoplasmic expression of cortactin and fak were only weak to moderate in normal colorectal mucosal epithelium , and were moderate to strong in crcs . in crcs , there was no difference in staining intensity of cortactin and fak whether or not the tumor cells were located in the center of tumor nests . as shown in table 1 , cortactin expression was seen in 84% ( 184 of 218 cases ) of crcs and 9% ( 20 of 218 cases ) of normal colorectal mucosa , respectively , while fak expression was seen in 61% ( 133 of 217 cases ) of crcs and 11% ( 24 of 217 cases ) of normal colorectal mucosa , respectively . the positive expression rate of cortactin and fak was significantly higher in crcs than in normal colorectal mucosa . , cortactin expression was correlated with poor differentiation ( p=0.011 ) and positive expression of fak ( p<0.001 ) . other clinicopathologic variables including age , gender , location , tumor size , lymphovascular invasion , pathologic stage and preoperative cea had no association with cortactin expression . however , among the 217 cases of crcs , fak expression was correlated with lymph node metastasis ( p=0.032 ) but not with other clinicopathological variables such as age , gender , location , tumor size , differentiation , lymphovascular invasion , pathologic stage and preoperative cea . the univariate cox proportional hazard analysis of the expression of cortactin and fak and their association with overall survival and relapse - free survival are shown in table 3 . patient 's age over 60 years ( p=0.011 ) , tumor size larger than 4.3 cm ( p=0.011 ) , poor differentiation ( p<0.001 ) , advanced pathologic stage ( p<0.001 ) , preoperative cea level over 5.0 ng / ml ( p=0.002 ) and cortactin expression ( p=0.021 ) predicted shorter overall survival . tumors located in the left colon ( p=0.033 ) predicted longer overall survival than those in the right colon . on the other hand , a tumor size larger than 4.3 cm ( p=0.008 ) , a tumor with poor differentiation ( p=0.006 ) , an advanced pathologic stage ( p<0.001 ) , a preoperative cea level over 5.0 ng / ml ( p=0.001 ) and a cortactin expression ( p=0.005 ) predicted shorter relapse - free survival . however , fak expression in crcs predicted neither shorter overall nor shorter relapse - free survival . kaplan - meier survival curves for overall survival in relation to the patient 's age , pathologic stage , preoperative cea level , and cortactin expression in crcs are presented in fig . in addition , kaplan - meier survival curves for relapse - free survival in relation to tumor size , pathologic stage , preoperative cea level and cortactin expression in crcs are also presented in fig . a multivariate analysis was done in 209 patients with complete information for clinicopathologic factors including age , tumor size , pathologic stage , preoperative cea and cortactin expression . from the multivariate analysis , pathologic stage , preoperative cea and cortactin expression were independent prognostic factors that were significantly associated with both overall and relapse - free survival ( table 4 ) . an advanced stage ( iii / iv ) was an independent prognostic factor which was significantly associated with poor overall ( adjusted hazard ratio [ hr ] , 3.700 ; 95% confidence interval [ ci ] , 2.170 to 6.309 ; p<0.001 ) and relapse - free survival ( adjusted hr , 2.786 ; 95% ci , 1.755 to 4.424 ; p<0.001 ) . a preoperative cea over 5 ng / ml was an independent prognostic factor significantly associated with poor overall ( adjusted hr , 1.899 ; 95% ci , 1.121 to 3.216 ; p=0.017 ) and relapse - free survival ( adjusted hr , 1.833 ; 95% ci , 1.145 to 2.933 ; p=0.012 ) . cortactin expression was an independent prognostic factor significantly associated with poor overall ( adjusted hr , 3.075 ; 95% ci , 1.111 to 8.514 ; p=0.031 ) and relapse - free survival ( adjusted hr , 3.565 ; 95% ci , 1.433 to 8.868 ; p=0.006 ) . in addition , an age of over 60 years was also an independent prognostic factors significantly associated with poor overall ( adjusted hr , 2.537 ; 95% ci , 1.440 to 4.469 ; p=0.001 ) , but not relapse - free survival . in the present study , we examined the immunohistochemical expressions of cortactin and fak in human crcs and their prognostic significance . we have demonstrated that the positive expression rates of cortactin and fak were significantly higher in crcs than in normal colorectal mucosa . it also served an independent prognostic factor and predicted shorter overall and relapse - free survival . additionally , cortactin expression showed slight correlation with a high preoperative cea level of more than 5 ng / ml . however , it correlated with lymph node metastasis and albeit slightly , with an advanced pathologic stage . that the expression of cortactin was higher and more intense in crcs than in normal colorectal epithelia , for instance , lee et al.10 found higher expression of cortactin in the crc and tubular adenoma than in the normal colorectal epithelia . on the other hand , cai et al.7 reported that cortactin expression is a significant prognostic factor for overall survival in rectal cancer patients . they also mentioned that the expression of cortactin was associated with tumor invasion , histologic grade and preoperative cea level in stage ii & iii crc patients.7 additionally , hirakawa et al.23 demonstrated that overexpression of cortactin and its localization at the cell periphery were critical factors for the progression of cancer . they also suggested that the association of zo-1 and cortactin plays an important role in regulating cell adhesion and spreading.23 in contrast , other investigators argued that cortactin expression was not a predictor for overall survival , although it was associated with more advanced cancer stages.10 moreover , zhang et al.24 reported that cortactin expression was negatively correlated with tumor - node - metastasis staging and lymphatic invasion status in crc . fak expression was found to be higher in invasive tumors and metastatic tumors of the colon rather than in adenomatous tissues.25 this suggests that fak expression may result in changes in the signaling pathways involved in tumor cell invasion . cance et al.16 also showed up - regulation of fak in tumor and relative lack of expression in normal colon tissue . furthermore , murata et al.26 suggested that high expression of fak predicts the recurrence of colorectal cancer . contrastingly , studies found that fak expression is not a prognostic predictor in certain carcinomas such as colonic adenocarcinoma , small cell lung cancer , pancreatic cancer and squamous cell carcinoma of head and neck.27 - 30 theocharis et al.27 reported that fak expression had no association with any clinicopathologic parameters including age , gender , location of tumor , stage and grade in the colonic adenocarcinoma . the discordant findings on the prognostic role of fak expression may be attributed to differences in tissue types or tumor progression stages . furthermore , it may also be caused by differences in methodologies ( e.g. , different antibodies used to evaluate fak expression ) and to the fact that fak expression does not reflect its enzymatic activity . we demonstrated that fak expression was higher and more intense in crcs than in normal colorectal epithelia indicating its potential role in the development of crcs . in addition , fak expression was significantly correlated with cortactin expression and showed a tendency to be associated with the pathologic stage . recent studies revealed that cortactin acts as a bridging molecule between actin filaments and focal adhesions and is recruited by fak into focal adhesions where it is phosphorylated by the fak - src complex.15,20 although cortactin and fak have been reported to be important factors in tumor migration , invasion , and metastasis , the relationship between expression of these proteins and tumor progression has not been clearly established . in this study , we observed lower expression of cortactin and fak in normal colorectal epithelia than in crcs . this indicates ongoing migratory events of normal colorectal epithelial cells , and moreover , the possibility that normal colorectal epithelia adjacent to the invasive cancers contain genetic abnormalities that could have an influence on the expression of cortactin and fak . , our results demonstrated that the cortactin expression was associated with differentiation , and was an independent factor for prognosis in crc . although multiple factors contribute to tumor progression , our findings suggest that cortactin expression is associated with fak expression and may play an important role in tumor progression . moreover , expression of cortactin is a satisfactory biomarker to predict tumor progression and survival in crc patients .

backgroundcortactin and focal adhesion kinase ( fak ) are two important components among actin cross - linking proteins that play a central role in cell migration.methodsthe aims of this study were to evaluate the expression of cortactin and fak in normal colorectal mucosa and colorectal adenocarcinoma ( crc ) using tissue microarray of 2 mm cores to correlate their expression with other clinicopathological factors and , investigate their prognostic significance.resultstwenty ( 9% ) and 24 cases ( 11% ) of normal colorectal mucosa were immunoreactive for cortactin and fak . in addition , 184 ( 84% ) and 133 cases ( 61% ) of crcs were immunoreactive for cortactin and fak , respectively . cortactin expression was associated with histologic differentiation and fak expression . cortactin , but not fak expression was also correlated with poor overall and relapse - free survival and served well as an independent prognostic factor for poor survival.conclusionscortactin expression , in association with fak expression , may plays an important role in tumor progression . furthermore , it may also be a satisfactory biomarker to predict tumor progression and survival in crc patients .

MATERIALS AND METHODS Case selection and specimens Immunohistochemistry and interpretation Statistical analysis RESULTS Expression of cortactin and FAK and their association with clinicopathologic variables in CRCs Correlation of the expression of cortactin and FAK with overall survival and relapse-free survival in CRCs Expression of cortactin as an independent prognostic factor for worse survival outcome in CRCs DISCUSSION

the location and tissue characteristics on imaging studies are critical to narrow down the differential diagnosis of mediastinal masses . the most frequent lesions encountered in the mediastinum are thymoma , neurogenic tumours and benign cysts , altogether representing 60% of patients with mediastinal masses . neurogenic tumours , germ cell neoplasms and foregut cysts represent 80% of childhood lesions , whereas primary thymic neoplasms , thyroid masses and lymphomas are the most common in adults . the mediastinum is demarcated by the pleural cavities laterally , the thoracic inlet superiorly and the diaphragm inferiorly . anterior mediastinal tumours account for 50% of all mediastinal masses , including thymoma , teratoma , thyroid disease and lymphoma . masses of the middle mediastinum are typically congenital cysts while those arising in the posterior mediastinum are often neurogenic tumours . usual symptoms at presentation are cough , chest pain , fever / chills and dyspnea . localising symptoms are secondary to tumour invasion ( respiratory compromise ; paralysis of the limbs , diaphragm and vocal cords ; horner syndrome ; superior vena cava syndrome ) , while systemic symptoms are typically due to the release of excess hormones , antibodies or cytokines . many mediastinal reflections can be appreciated at conventional radiography ( cr ) , and their presence or distortion is the key to the interpretation of mediastinal abnormalities . however , computed tomography ( ct ) is the most important tool in the evaluation of a mediastinal mass . characterisation on ct is based on specific attenuation of air , fat , water and calcium ( fig . 1 ) . high - resolution multiplanar reformation images display the detailed anatomical relationship of the tumour with the adjacent structures . an excellent soft tissue contrast also designates magnetic resonance imaging ( mri ) as an ideal tool to evaluate tumours of the mediastinum . assessment of preoperative relationships with the pericardium , heart cavities , spinal cord and vascular involvement is a common indication . chemical - shift mri has been shown to be useful in distinguishing normal thymus and thymic hyperplasia from thymic neoplasms and lymphoma . diffusion - weighted mri ( dwi ) is another special application that unveils minute metabolic and biophysical differences between tissues . according to gumustas et al . , the mean apparent diffusion coefficient for the malignant mediastinal entities could be significantly lower than that for the benign diseases.fig . 1conventional radiograph can provide information pertaining to the size , anatomical location and density of a central mass . right : frontal chest radiograph shows a sharply defined area of increased opacity with a loss of the cardiac silhouette at the border of the right side of the heart ( * ) . contrast - enhanced ct scan reveals a thin - walled water - attenuation lesion ( * ) in the right cardiophrenic angle ( pericardial cyst ) . middle : lateral chest radiograph and contrast - enhanced ct scan show a unilocular , well - defined and homogeneously hypodense mass in the anterior mediastinum with peripheral calcification ( open arrows ) ( thymic cyst ) . left : contrast - enhanced ct scan demonstrates a multilobulated mass in the anterior mediastinum ( arrow ) , which accounts for the distortion of the ap window ( nodular sclerosis hodgkin disease ) conventional radiograph can provide information pertaining to the size , anatomical location and density of a central mass . right : frontal chest radiograph shows a sharply defined area of increased opacity with a loss of the cardiac silhouette at the border of the right side of the heart ( * ) . contrast - enhanced ct scan reveals a thin - walled water - attenuation lesion ( * ) in the right cardiophrenic angle ( pericardial cyst ) . middle : lateral chest radiograph and contrast - enhanced ct scan show a unilocular , well - defined and homogeneously hypodense mass in the anterior mediastinum with peripheral calcification ( open arrows ) ( thymic cyst ) . left : chest radiograph shows the aortopulmonary window with an abnormal convex border ( arrow ) . contrast - enhanced ct scan demonstrates a multilobulated mass in the anterior mediastinum ( arrow ) , which accounts for the distortion of the ap window ( nodular sclerosis hodgkin disease ) many entities that involve the mediastinum correspond to anatomical variants or masses arising from the spine or from the digestive tract , and should not be considered true mediastinal masses ( figs . 2 and 3 ) . 2 a contrast - enhanced ct scan shows a right - sided aortic arch ( arrow ) in an asymptomatic man with an absence of the aortic knuckle on the left . b contrast - enhanced ct scan demonstrates a soft tissue posterior mediastinal mass ( * ) in a 66-year - old woman with multiple myeloma diagnosis . note the mass effect to the descending aorta and left auricula by the mass and the bilateral pleural effusion ( open arrows)fig . b posterior mediastinal mass ( arrow ) in relation with squamous - cell carcinoma of the oesophagus . c oesophageal diverticulum ( arrow ) in a patient with oesophageal achalasia . note the thickened oesophagus . d hiatal hernia is a frequent incidental finding with or without air or air - fluid level ( open arrow ) . * pleural effusion a contrast - enhanced ct scan shows a right - sided aortic arch ( arrow ) in an asymptomatic man with an absence of the aortic knuckle on the left . b contrast - enhanced ct scan demonstrates a soft tissue posterior mediastinal mass ( * ) in a 66-year - old woman with multiple myeloma diagnosis . note the mass effect to the descending aorta and left auricula by the mass and the bilateral pleural effusion ( open arrows ) different masses arising from the digestive tract . b posterior mediastinal mass ( arrow ) in relation with squamous - cell carcinoma of the oesophagus . c oesophageal diverticulum ( arrow ) in a patient with oesophageal achalasia . note the thickened oesophagus . d hiatal hernia is a frequent incidental finding with or without air or air - fluid level ( open arrow ) . * pleural effusion lipomas predominantly occur in the anterior mediastinum and are reported to represent 1.62.3% of all primary mediastinal tumours . at ct , lipomas have homogeneous fat attenuation of approximately -100 hu . liposarcoma frequently occurs in the posterior mediastinum and it is usually symptomatic at the time of presentation , in contrast to lipoma . thymolipoma is a rare , benign , well - encapsulated thymic tumour that accounts for about 29% of thymic neoplasms tumours occur most frequently in the cardiophrenic angle of asymptomatic young adults without sex predilection . the fat content usually constitutes 5085% of the lesion but has been reported to account for as much as 95% of the tumour . associations with myasthenia gravis , graves disease and haematological disorders have been reported . at cr , thymolipomas may mimic cardiomegaly , excessive epicardial fat , diaphragmatic elevation , lobar collapse or a pericardial cyst . ct or mri is required to establish the diagnosis by showing a well - defined encapsulated mass that has extensive fat content and contains small amounts of solid areas and fibrous septa ( fig . axial and coronal multiplanar reconstruction of non - contrast - enhanced ct scan show a large and well - defined mass ( arrows ) that has extensive fat content and contains small amounts of thin fibrous septa thymolipoma in a 47-year - old asymptomatic man . axial and coronal multiplanar reconstruction of non - contrast - enhanced ct scan show a large and well - defined mass ( arrows ) that has extensive fat content and contains small amounts of thin fibrous septa mediastinal primary cysts represent 1520% of all primary mediastinal masses [ 1 , 15 ] . a smooth or oval mass with a homogeneous attenuation , with no enhancement of cyst contents and no infiltration of adjacent structures are the usual ct features of benign mediastinal cyst ( fig . any cyst may have a higher attenuation due to its calcic , proteinaceous , mucous or haemorrhagic content . true cystic lesions should be differentiated from the cystic degenerative changes observed in many solid tumours , in lymphomas before or after treatment , or in abscesses ( fig . 5a well - marginated mass with a homogeneous attenuation , usually in the range of water attenuation ( 020 hu ) and without an enhancement of the wall or infiltrative appearance are the typical features of benign mediastinal cysts . 6oblique - coronal multiplanar reconstruction of contrast - enhanced ct scan of a 33-year - old woman with a descending necrotising mediastinitis . hypodense para - aortic areas correspond with fluid - collections ( arrows ) that extend to the retroperitoneum . note the visceral space involvement ( arrowhead ) a well - marginated mass with a homogeneous attenuation , usually in the range of water attenuation ( 020 hu ) and without an enhancement of the wall or infiltrative appearance are the typical features of benign mediastinal cysts . probably thymic cystic ( a ) and pericardial cyst ( b ) oblique - coronal multiplanar reconstruction of contrast - enhanced ct scan of a 33-year - old woman with a descending necrotising mediastinitis . hypodense para - aortic areas correspond with fluid - collections ( arrows ) that extend to the retroperitoneum . note the visceral space involvement ( arrowhead ) bronchogenic cysts result from abnormal ventral budding or branching of the tracheobronchial tree during embryologic development [ 1517 ] . they are lined by respiratory epithelium and their capsule contains cartilage , smooth muscle and mucous gland tissue . approximately 40% of bronchogenic cysts are symptomatic , resulting in cough , dyspnea or chest pain . the bronchogenic cyst is commonly located in the near carina ( 52% ) and in the paratracheal region ( 19% ) . the anterior mediastinum is a rare location of the bronchogenic cyst ( fig . 7 ) air within the cyst is suggestive of secondary infection and communication with the tracheobronchial tree.fig . a non - contrast - enhanced ct scan shows a homogeneous anterior mediastinal mass with smooth contours and oval shape ( arrow ) . b t2-weighted mr image shows the same mass ( arrow ) with markedly high signal intensity . c although its localisation , a bronchogenic cyst was confirmed by histological examination after surgical resection bronchogenic cyst in a 37-year - old man . a non - contrast - enhanced ct scan shows a homogeneous anterior mediastinal mass with smooth contours and oval shape ( arrow ) . b t2-weighted mr image shows the same mass ( arrow ) with markedly high signal intensity . c although its localisation , a bronchogenic cyst was confirmed by histological examination after surgical resection duplication cysts are uncommon lesions lined by gastrointestinal tract mucosa and generally asymptomatic . however , if they contain gastric or pancreatic mucosa , there is the added risk of haemorrhage or rupture of the cyst from mucosal secretions . the majority of them are detected adjacent to or within the oesophageal wall ( fig . a contrast - enhanced ct scan shows a round well marginated mass ( arrow ) adjacent to the oesophagus with homogeneous water - attenuation . b the lesion ( arrow ) presents a bright signal intensity on t2-weighted fat - suppressed mr image duplication cyst in a 42-year - old asymptomatic man . a contrast - enhanced ct scan shows a round well marginated mass ( arrow ) adjacent to the oesophagus with homogeneous water - attenuation . b the lesion ( arrow ) presents a bright signal intensity on t2-weighted fat - suppressed mr image mediastinal neuroenteric cysts are anomalous protrusions of the leptomeninges through intervertebral foramen or defects in the vertebral body . they are associated with multiple vertebral anomalies and with neurofibromatosis [ 15 , 16 ] . most pericardial cysts are unilocular and commonly located in the right cardiophrenic space ( fig . 9 ) . however , they may occur anywhere in relation to the pericardium.fig . 9 a lateral chest radiograph of a 58-year - old smoker man allergic to iodine shows a well - defined mass ( * ) in the cardiophrenic space and a nodular lung opacity ( open arrow ) in the lower lung parenchyma . b , c non - contrast - enhanced ct scan confirms the presence of a fluid - attenuation mass ( * ) in the right cardiophrenic angle ( pericardial cyst ) and demonstrates a suspicious lung opacity ( open arrows ) in the right lower lobe of the lung ( squamous - cell carcinoma ) a lateral chest radiograph of a 58-year - old smoker man allergic to iodine shows a well - defined mass ( * ) in the cardiophrenic space and a nodular lung opacity ( open arrow ) in the lower lung parenchyma . b , c non - contrast - enhanced ct scan confirms the presence of a fluid - attenuation mass ( * ) in the right cardiophrenic angle ( pericardial cyst ) and demonstrates a suspicious lung opacity ( open arrows ) in the right lower lobe of the lung ( squamous - cell carcinoma ) thymic cyst represents 1% of all mediastinal masses . congenital cysts derive from remnants of the thymopharyngeal duct , they are typically unilocular and contain clear fluid ( fig . in contrast , acquired thymic cysts are much more common , tend to be multilocular ( fig . 11 ) and may arise in association with neoplasms such as thymomas , lymphomas or germ cell tumours . thymic cysts may also be seen in the anterior mediastinum after radiation therapy of hodgkin s disease , after inflammatory processes and occasionally in patients with aids , particularly in children .fig . contrast - enhanced ct scan demonstrates a unilocular unenhanced lesion in the anterior mediastinum which shows a homogeneous fluid - attenuation ( arrow)fig . a contrast - enhanced ct scan shows a well - defined water - attenuation multiloculated mass ( * ) in the anterior mediastinum . b sagittal t2-weighted mr sequence demonstrates a multiloculated mass with typical high signal intensitiy and fine internal septa within ( arrow ) congenital thymic cyst in a 47-year - old man . contrast - enhanced ct scan demonstrates a unilocular unenhanced lesion in the anterior mediastinum which shows a homogeneous fluid - attenuation ( arrow ) acquired thymic cyst in a 43-year - old man . a contrast - enhanced ct scan shows a well - defined water - attenuation multiloculated mass ( * ) in the anterior mediastinum . b sagittal t2-weighted mr sequence demonstrates a multiloculated mass with typical high signal intensitiy and fine internal septa within ( arrow ) lymphangioma is a rare benign lesion of lymphatic origin that represents 0.74.5% of all mediastinal tumours in adult population . lymphangiomas involve the neck or the axillary region in more than 80% of the cases and 12a cystic lymphangioma ( also referred to as hygroma ) in a 47-year - old woman with retroperitoneal disease . a posterior mediastinal mass with a homogeneous fluid - attenuation is identified on ct ( white arrow ) and on posteroanterior chest radiograph as a mass disrupting left paraspinal line inferiorly ( black arrows ) a cystic lymphangioma ( also referred to as hygroma ) in a 47-year - old woman with retroperitoneal disease . a posterior mediastinal mass with a homogeneous fluid - attenuation is identified on ct ( white arrow ) and on posteroanterior chest radiograph as a mass disrupting left paraspinal line inferiorly ( black arrows ) pancreatic pseudocyst can extend into the mediastinum via the oesophageal or aortic hiatus . ct shows a thin - walled , fluid - containing cyst within the posterior mediastinum which may be in continuity with the intrapancreatic or peripancreatic fluid collections ( fig . axial ( a , b ) and oblique - sagittal multiplanar reconstruction ( c ) ct scan show a thin - walled peri - oesophageal fluid - lesion ( white open arrows ) that comunicates with an intrapancreatic fluid - collection ( black open arrows ) through oesophageal hiatus by a duct fistula ( white arrows ) pancreatic pseudocyst in a 52-year - old man with a recurrent pancreatitis history . axial ( a , b ) and oblique - sagittal multiplanar reconstruction ( c ) ct scan show a thin - walled peri - oesophageal fluid - lesion ( white open arrows ) that comunicates with an intrapancreatic fluid - collection ( black open arrows ) through oesophageal hiatus by a duct fistula ( white arrows ) mediastinal goitre generally represents direct contiguous growth of a goitre into the anterior or superior mediastinum . typical features of mediastinal goitres are encapsulated and lobulated mass with inhomogeneous appearance with cystic areas , calcifications and marked contrast enhancement . an intrathoracic thyroid mass developing from heterotopic thyroid tissue without any connection to the thyroid in the neck is extremely rare ( fig . the presence of ill - defined margins , invasion of adjacent structures and nearby lymph node enlargement suggests the diagnosis of thyroid cancer ( fig . a coronal multiplanar reconstruction ct scan demonstrates an anterior mediastinal mass ( * ) arising from the thyroid more superiorly . b non - contrast - enhanced ct scan shows a unilateral mediastinal goitre with peripheral areas of calcification ( open arrow ) . c photograph of the resected surgical specimen shows a lobulated and heterogeneous appearance of the mass . d contrast - enhanced ct scan shows a well marginated posterior mediastinal goitre ( * ) with marked contrast enhancementfig . sagittal contrast - enhanced ct scan demonstrates a well - defined mass ( arrow ) located in the retrosternal space which shows an intense and heterogeneous contrast enhancement due to the presence of cystic areas ( * ) . no connection to the thyroid gland from the neck . b anaplastic thyroid carcinoma in a 64-year - old woman with respiratory failure . contrast - enhanced ct scan shows an anterior mediastinal soft tissue mass ( * ) which surrounds great vessels and oesophagus and compresses trachea . a coronal multiplanar reconstruction ct scan demonstrates an anterior mediastinal mass ( * ) arising from the thyroid more superiorly . b non - contrast - enhanced ct scan shows a unilateral mediastinal goitre with peripheral areas of calcification ( open arrow ) . c photograph of the resected surgical specimen shows a lobulated and heterogeneous appearance of the mass . d contrast - enhanced ct scan shows a well marginated posterior mediastinal goitre ( * ) with marked contrast enhancement a heterotopic mediastinal goitre . sagittal contrast - enhanced ct scan demonstrates a well - defined mass ( arrow ) located in the retrosternal space which shows an intense and heterogeneous contrast enhancement due to the presence of cystic areas ( * ) . no connection to the thyroid gland from the neck . b anaplastic thyroid carcinoma in a 64-year - old woman with respiratory failure . contrast - enhanced ct scan shows an anterior mediastinal soft tissue mass ( * ) which surrounds great vessels and oesophagus and compresses trachea . note the extension to the suprasternal fossa ( open arrow ) thymic hyperplasia can be divided into two distinct histological types [ 13 , 14 ] . true thymic hyperplasia is defined as enlargement of the thymus , which generally retains its normal shape . this disease entity is observed when a patient is recovering from some recent stress ( such as chemotherapy , corticosteroid therapy , irradiation or thermal burns ) . the phenomenon known as rebound hypeplasia is defined as a greater than 50% increase in thymic volume over baseline after such stress . among patients who undergo chemotherapy , approximately 1025% thymic lymphoid ( follicular ) hyperplasia of the thymus refers to the presence of an increased number of lymphoid follicles . it is commonly associated with autoimmune diseases , being seen in up to 65% of cases with myasthenia gravis , and it has been reported to occur in the early stages of human immunodeficiency virus infection . at ct it may appear normal ( 45% ) , enlarged ( 35% ) ( fig . 16thymic lymphoid hyperplasia in a 41-year - old woman with clinical diagnosis of myasthenia gravis . non - contrast - enhanced ct scan shows an enlarged thymic gland ( arrows ) without mass effect on adjacent structures thymic lymphoid hyperplasia in a 41-year - old woman with clinical diagnosis of myasthenia gravis . non - contrast - enhanced ct scan shows an enlarged thymic gland ( arrows ) without mass effect on adjacent structures it is important for radiologists to be able to distinguish thymic hyperplasia from neoplasm . diffuse symmetric enlargement of the gland , a smooth contour and normal vessels are the key morphological features of hyperplasia , whereas neoplasm tends to manifest as a focal mass with nodular contour and necrotic or calcified foci . detecting fat in the thymus is particularly relevant in these situations . according to takahashi et al . thymic hyperplasia reveals a relative signal loss on opposed - phase chemical - shift mri that it is different from no significant signal change between in - phase and opposed - phase chemical - shift mr images in patients with malignancy ( fig . 17 a c thymic hyperplasia in a 43-year - old woman ( arrows ) . a non - contrast - enhanced ct scan reveals a lobulated lesion with smooth margins in anterior mediastinum . b lesion appears slightly hyperintense on in - phase gradient - echo t1-weighted mr image . c opposed - phase gradient - echo t1-weighted mr image shows decreased signal intensity within the lesion , confirming presence of fat . f stage ii thymoma ( who type b2 ) in a 62-year - old woman ( arrows ) . d non - contrast - enhanced ct scan shows an anterior mediastinal soft tissue mass . e the lesion shows an intermediate signal intensity on in - phase gradient - echo t1-weighted mr image . f there is no substantial decrease in signal intensity relative to in - phase mr image on opposed - phase sequence a c thymic hyperplasia in a 43-year - old woman ( arrows ) . a non - contrast - enhanced ct scan reveals a lobulated lesion with smooth margins in anterior mediastinum . b lesion appears slightly hyperintense on in - phase gradient - echo t1-weighted mr image . c opposed - phase gradient - echo t1-weighted mr image shows decreased signal intensity within the lesion , confirming presence of fat . f stage ii thymoma ( who type b2 ) in a 62-year - old woman ( arrows ) . d non - contrast - enhanced ct scan shows an anterior mediastinal soft tissue mass . e the lesion shows an intermediate signal intensity on in - phase gradient - echo t1-weighted mr image . f there is no substantial decrease in signal intensity relative to in - phase mr image on opposed - phase sequence thymoma is the most common primary neoplasm of the anterior mediastinum but accounts for less than 1% of all adult malignancies [ 22 , 23 ] . thymomas typically occur in patients older than 40 years of age , being rare in children , and affecting men and women equally . between 30% and 50% of patients with a thymoma have myasthenia gravis , whereas 1015% of patients with myasthenia gravis have a thymoma . the updated histological classification elaborated by who in 2004 classified different types of thymomas ( table 1 ) on the basis of the morphology of the neoplastic epithelial cells together with the lymphocyte - epithelial cell ratio [ 23 , 24 ] . in contrast to histological classification , the stage of thymoma has clinical implications and it is a useful tool for management decisions . the masaoka - koga staging system is the most commonly used and describes thymomas in terms of the local extension of the tumour [ 14 , 2326 ] ( table 2 ) . the masaoka staging system is one of the two factors , including completeness of surgical resection , that most strongly correlates with prognosis of thymomas . the role of imaging is to initially diagnose and properly stage thymoma , with emphasis on the detection of local invasion and distant spread of disease . between 45 and 80% of thymomas on ct scans , thymomas usually appear as homogeneous solid masses with soft - tissue attenuation and well - demarcated borders , located anywhere from the thoracic inlet to the cardiophrenic angle . thymomas may be oval , round or lobulated and when they are large , cystic or necrotic degeneration may be shown ( fig . certain findings , such as encasement of mediastinal structures , infiltration of fat planes , irregular interface between the mass and lung parenchyma , and direct signs of vascular involvement are highly suggestive of invasion ( fig . pleural dissemination ( drop metastases ) manifests as one or more pleural nodules or masses , and they are almost always ipsilateral to the tumour ( fig . thymoma rarely presents with metastatic lymphadenopathy , metastatic pulmonary nodules or pleural effusion . at mri , thymomas commonly appear as homogeneous or heterogeneous masses with low to intermediate signal intensity on t1-weighted images and with high signal intensity on t2-weighted images ( fig . mri can prove useful in identifying the nodular wall thickening detected in cystic thymomas , absent from congenital cysts .table 1who classification for thymoma2004 who classificationdescriptiona ( spindle cell thymoma ; medullary thymoma)bland spindle / oval epithelial tumour cells with few or no lymphocytesab ( mixed thymoma)mixture of a lymphocyte - poor type a thymoma component and a more lymphocyte - rich type b - like component ( smaller and paler than those of b1 or b2 thymomas ) . lymphocytes are more numerous than in the type a component , but may be less numerous than in b1 thymomasb1 ( lymphocyte - rich thymoma ; lymphocytic thymoma ; organoid thymoma ; predominantly cortical thymoma)epithelial cells with a histological appearance practically indistinguishable from the normal thymus , composed predominantly of areas resembling cortex with epithelial cells scattered in a prominent population of immature lymphocytesb2 ( cortical thymoma)tumour cells closely resembling the predominant epithelial cells of the normal thymic cortex . a background population of immature t cells is always present and usually outnumbers the neoplastic epithelial cellsb3 ( epithelial thymoma ; squamoid thymoma)medium sized round or polygonal cells with slight atypia . the epithelial cells are mixed with a minor component of intraepithelial lymphocytesfrom travis et al . table 2masaoka - koga staging system of thymomastagedescriptionimacroscopically and microscopically encapsulated tumouriiamicroscopic invasion through the capsuleiibmacroscopic invasion into surrounding fatty tissueiiiinvasion into a neighbouring organ such as the pericardium , great vessels or lungivapleural or pericardial disseminationivblymphatic - haematogenous metastasesfrom references [ 14 , 2326]fig . 18 a , b stage ii thymoma ( who type b1 ) in a 33-year - old woman who presented with myasthenia gravis . frontal chest radiograph shows a hilum overlay sign ( arrow ) of a suggestive anterior mediastinal mass . contrast - enhanced ct scan confirms the presence of a low - heterogeneous anterior mediastinal mass ( arrow ) . c , d stage iii thymoma ( who type b2 ) in a 54-year - old woman . frontal chest radiograph reveals a lobulated mediastinal mass ( arrow ) on the right side . contrast - enhanced ct scan demonstrates an enhanced anterior mediastinal mass ( arrow ) with infiltration of surrounding fat ( open arrow)fig . a , b contrast - enhanced ct scan shows a well - circumscribed , flattened soft tissue lesion in the anterior mediastinum with calcification ( arrow ) . note the lobulated contour of the mass and the loss of the fat plane between the mass and the aorta . pleural seeding is identified as an enhancing pleura - based nodule ( open arrow ) . c irregular border between the mediastinal mass and the lung parenchyma ( arrowhead ) is observed as a sign of locally advanced disease . a contrast - enhanced ct scan reveals an anterior mediastinal mass ( arrows ) with irregular contours , homogeneous enhancement and peripheral and central calcification as well as a pleural nodule ( open arrow ) . b on an axial fdg - positron emission tomography ( pet ) image , the pleural nodule is fdg avid , confirming a drop metastasis . d photomicrograph ( haematoxylin - eosin stain ) of tissue from the lesion shows roughly equal numbers of epithelial cells ( white arrow ) and lymphocytes ( black arrow ) corresponding thymoma who type b2fig . 21axial ( a ) and coronal multiplanar reconstruction ( b ) of a non - contrast - enhanced ct scan of a 57-year - old man allergic to iodine with a thymoma . a solid lobulated thymic mass ( * ) with clumps of calcifications within ( arrowhead ) note the absence of a fat plane between the tumour and the aorta ( open arrow ) . d coronal t2-weighted mr image shows a typical signal hyperintensity of the tumour lesion ( * ) . c axial contrast - enhanced fat - suppressed t1-weighted mr image reveals a homogeneously enhanced solid tumour ( * ) which arises from the thymus . although mri demonstrates the presence of fat cleavage plane between ascending aorta and the tumour , a thymoma ( who type a ) with microscopic transcapsular invasion ( masaoka stage ii ) was confirmed after surgical resection who classification for thymoma from travis et al . masaoka - koga staging system of thymoma from references [ 14 , 2326 ] a , b stage ii thymoma ( who type b1 ) in a 33-year - old woman who presented with myasthenia gravis . frontal chest radiograph shows a hilum overlay sign ( arrow ) of a suggestive anterior mediastinal mass . contrast - enhanced ct scan confirms the presence of a low - heterogeneous anterior mediastinal mass ( arrow ) . c , d stage iii thymoma ( who type b2 ) in a 54-year - old woman . frontal chest radiograph reveals a lobulated mediastinal mass ( arrow ) on the right side . contrast - enhanced ct scan demonstrates an enhanced anterior mediastinal mass ( arrow ) with infiltration of surrounding fat ( open arrow ) ct is the imaging modality of choice for evaluating staging thymoma . a , b contrast - enhanced ct scan shows a well - circumscribed , flattened soft tissue lesion in the anterior mediastinum with calcification ( arrow ) . note the lobulated contour of the mass and the loss of the fat plane between the mass and the aorta . pleural seeding is identified as an enhancing pleura - based nodule ( open arrow ) . c irregular border between the mediastinal mass and the lung parenchyma ( arrowhead ) is observed as a sign of locally advanced disease . note the cellular bronchiolitis in the left lower lobe stage iva thymoma ( who type b2 ) in a 46-year - old man . a contrast - enhanced ct scan reveals an anterior mediastinal mass ( arrows ) with irregular contours , homogeneous enhancement and peripheral and central calcification as well as a pleural nodule ( open arrow ) . b on an axial fdg - positron emission tomography ( pet ) image , the pleural nodule is fdg avid , confirming a drop metastasis . d photomicrograph ( haematoxylin - eosin stain ) of tissue from the lesion shows roughly equal numbers of epithelial cells ( white arrow ) and lymphocytes ( black arrow ) corresponding thymoma who type b2 axial ( a ) and coronal multiplanar reconstruction ( b ) of a non - contrast - enhanced ct scan of a 57-year - old man allergic to iodine with a thymoma . a solid lobulated thymic mass ( * ) with clumps of calcifications within ( arrowhead ) note the absence of a fat plane between the tumour and the aorta ( open arrow ) . d coronal t2-weighted mr image shows a typical signal hyperintensity of the tumour lesion ( * ) . c axial contrast - enhanced fat - suppressed t1-weighted mr image reveals a homogeneously enhanced solid tumour ( * ) which arises from the thymus . although mri demonstrates the presence of fat cleavage plane between ascending aorta and the tumour , a thymoma ( who type a ) with microscopic transcapsular invasion ( masaoka stage ii ) was confirmed after surgical resection thymic carcinoma accounts for about 20% of thymic epithelial tumours with a mean age of 50 years . typical appearance is a multilobulated and heterogeneous mass that may contain areas of calcification or haemorrhage . . found that irregular contour , necrotic or cystic components , heterogeneous enhancement , lymphadenopathy and great vessel invasion strongly favoured thymic carcinoma . thymic carcinoids are rare , well - differentiated neuroendocrine tumours , which have a male predilection of 3:1 [ 13 , 14 ] . malignant lymphoma accounts for nearly 20% of all mediastinum neoplasms in adults and 50% in children . 22 ) represents approximately 5070% of mediastinal lymphomas , while non - hodgkin lymphoma comprises 1525% . frontal chest radiograph and contrast - enhanced ct scan show a homogeneous soft tissue mass at the level of the subcarina ( arrows ) . the right paratracheal stripe is not seen on frontal chest radiograph , having been most probably obliterated by a right paratracheal lymphadenopathy ( arrowhead ) . frontal chest radiograph and contrast - enhanced ct scan show a homogeneous soft tissue mass at the level of the subcarina ( arrows ) . the right paratracheal stripe is not seen on frontal chest radiograph , having been most probably obliterated by a right paratracheal lymphadenopathy ( arrowhead ) . * carina hodgkin disease ( hd ) has a bimodal distribution of incidence peaking in young adulthood and again after the age of 50 years . four subtypes of hd are described : nodular sclerosis ( by far the most common histological subtype ) ( fig . 23 ) , lymphocyte - rich , mixed cellularity and lymphocyte depleted hd [ 4 , 5 ] . cr is abnormal in up to 76% of patients with hd , often showing enlargement of the prevascular and paratracheal nodes . characteristic features on imaging are a homogeneous soft - tissue anterior mediastinal mass with mild to moderate contrast enhancement , irregular contours , surface lobulation , absence of vascular involvement , and high prevalence of associated mediastinal lymphadenopathy . frontal chest radiograph shows a large , well - defined mediastinal mass with increased density ( arrow ) . contrast - enhanced ct scan shows a bulky soft tissue mass ( arrows ) with homogeneous ct - attenuation value occupying prevascular space . photomicrograph reveals numerous neoplastic lacunar cells ( arrows ) in a background of small lymphocytes , histiocytes and eosinophils , which supports the diagnosis of nodular sclerosis type hodgkin lymphoma nodular sclerosis hodgkin lymphoma in a 44-year - old woman . frontal chest radiograph shows a large , well - defined mediastinal mass with increased density ( arrow ) . contrast - enhanced ct scan shows a bulky soft tissue mass ( arrows ) with homogeneous ct - attenuation value occupying prevascular space . photomicrograph reveals numerous neoplastic lacunar cells ( arrows ) in a background of small lymphocytes , histiocytes and eosinophils , which supports the diagnosis of nodular sclerosis type hodgkin lymphoma the two most common forms of mediastinal non - hodgkin disease ( nhd ) include diffuse large b - cell lymphoma and t - cell lymphoblastic lymphoma . the most common ct appearance is a large mediastinal mass representing thymic and lymph node enlargement , which compresses the airway and cardiovascular structures ( fig . primary mediastinal diffuse large b - cell lymphomas tend to occur in young to middle - aged adults with a mean age of 30 . it accounts for 7% of all cases of nhd and about 10% of all cases of high - grade nhd . the tumours appear as a large , smooth or lobulated , anterior mediastinal mass in nearly all patients . on ct the tumours show low attenuation areas , representing haemorrhage , necrosis or cystic degeneration in 50% of the cases and heterogeneous enhancement in about 40% of the cases ( fig . primary mediastinal b - cell lymphoma recurs in the chest . consequently , chest ct examination alone is sufficient for routine follow - up of these patients .fig . 24axial t1-weighted mr image of a 16-year - old man with a solid , large mass ( arrows ) in the anterior and superior mediastinum . supra - aortic trunks are almost completely surrounded by the lesion and trachea ( t ) is displaced to the contralateral side by the lesion . contrast - enhanced ct scan shows a bulky soft tissue mass in the anterior mediastinum ( arrowsin a ) , which shows heterogeneous ct - attenuation values with cystic changes within ( * ) . pericardial effusion is also observed ( open arrow in b ) axial t1-weighted mr image of a 16-year - old man with a solid , large mass ( arrows ) in the anterior and superior mediastinum . supra - aortic trunks are almost completely surrounded by the lesion and trachea ( t ) is displaced to the contralateral side by the lesion . pathological analysis demonstrated a t - cell lymphoblastic lymphoma primary mediastinal diffuse large b - cell lymphoma in a 19-year - old man . contrast - enhanced ct scan shows a bulky soft tissue mass in the anterior mediastinum ( arrowsin a ) , which shows heterogeneous ct - attenuation values with cystic changes within ( * ) . pericardial effusion is also observed ( open arrow in b ) germ cell tumours ( gcts ) mainly arise in gonads and in the midline of the body as well , the mediastinum being the most common extragonadal site . if a teratoma contains fetal tissue or neuroendocrine tissue , it is defined as immature and malignant with a poor prognosis . on ct , teratoma most commonly appears as a well - defined unilocular or multilocular cystic lesion containing fluid , soft tissue and fat attenuation ( fig . calcifications may be focal , rim - like or , in rare cases , representative of teeth or bone . common combinations of internal components of mature teratomas include soft tissue , fluid , fat and calcification in 39% ; soft tissue , fluid and fat in 24% ; and soft tissue and fluid in 15% . in 15% of the cases , mature teratomas appear as non - specific cystic lesions without fat or calcium . on mri , fat - fluid levels within the lesion are virtually diagnostic of teratoma . ruptured teratomas show an adjacent consolidation , atelectasis and pleural or pericardial effusion than do unruptured teratomas.fig . 26chest imaging shows well the highly heterogeneous contents of mediastinal teratomas . a mature cystic teratoma in a 40-year - old man . contrast - enhanced ct scan shows a heterogeneous anterior mediastinal mass with areas of fat ( open arrow ) , calcification ( arrow ) and fluid attenuation ( * ) . c contrast - enhanced ct scan of an asymptomatic 24-year - old woman demonstrates a well - defined uniloculated mass located in prevascular space which shows a cystic changes within ( * ) . d the mass was surgically removed and pathological examination confirmed a benign teratoma chest imaging shows well the highly heterogeneous contents of mediastinal teratomas . a mature cystic teratoma in a 40-year - old man . contrast - enhanced ct scan shows a heterogeneous anterior mediastinal mass with areas of fat ( open arrow ) , calcification ( arrow ) and fluid attenuation ( * ) . c contrast - enhanced ct scan of an asymptomatic 24-year - old woman demonstrates a well - defined uniloculated mass located in prevascular space which shows a cystic changes within ( * ) . d the mass was surgically removed and pathological examination confirmed a benign teratoma the non - teratomatous germ cell tumours ( ntgct ) are rare and malignant tumours which usually occur in young males and most frequently affect the anterosuperior mediastinum . these tumours grow rapidly and develop large , bulky , ill - circumscribed masses with lobulated shape . primary mediastinal seminomas comprise 2550% of malignant mediastinal gcts and occur almost exclusively in males during the period from the second to fourth decades of life . at imaging , areas of degeneration due to haemorrhage and coagulation necrosis may be present ( fig . non - seminomatous germ cell tumours include yolk sac tumours , endodermal sinus tumours , embryonal carcinomas , choriocarcinomas and mixed germ cell tumours , which present as large masses typically with marked heterogeneous attenuation . at diagnosis , afp and - hcg levels is important when making the diagnosis ( fig . contrast - enhanced axial ct scan demonstrates a large mass in the right side of the mediastinum with an obvious mass effect on great vessels and heart . the mass shows heterogeneous ct - attenuation values probably secondary to haemorrhage and coagulation necrosis . note also a right pleural effusion ( arrowhead ) and multiple lung metastasis ( arrows)fig . 28non - seminomatous malignant germ cell tumour of the anterior mediastinum in a 25-year - old man with chest pain and high serum level of -fetoprotein at admission ( 25.396 ng / ml ) . frontal chest radiograph shows a central mass ( * ) . the descending aorta is clearly seen ( arrows ) , indicating that the mass is not within the posterior mediastinum . a contrast - enhanced ct scan confirms a mass of low attenuation ( * ) in the anterior mediastinum that compresses the pulmonary artery . bilateral lung metastasis ( arrowheads ) and hilar and subcarinal lymphadenopathy is identified ( open arrows ) a 19-year - old man with seminoma . contrast - enhanced axial ct scan demonstrates a large mass in the right side of the mediastinum with an obvious mass effect on great vessels and heart . the mass shows heterogeneous ct - attenuation values probably secondary to haemorrhage and coagulation necrosis . note also a right pleural effusion ( arrowhead ) and multiple lung metastasis ( arrows ) non - seminomatous malignant germ cell tumour of the anterior mediastinum in a 25-year - old man with chest pain and high serum level of -fetoprotein at admission ( 25.396 ng / ml ) . frontal chest radiograph shows a central mass ( * ) . the descending aorta is clearly seen ( arrows ) , indicating that the mass is not within the posterior mediastinum . a contrast - enhanced ct scan confirms a mass of low attenuation ( * ) in the anterior mediastinum that compresses the pulmonary artery . bilateral lung metastasis ( arrowheads ) and hilar and subcarinal lymphadenopathy is identified ( open arrows ) neurogenic tumours represent approximately 20% of all adults and 35% of all paediatric mediastinal tumours and they are the most common cause of a posterior mediastinal mass . seventy to eighty percent of neurogenic tumours are benign , and nearly half are asymptomatic . these tumours are generally grouped into : peripheral nerve tumours , which are the most common ( 70% ) mediastinal neurogenic tumours and originate from spinal or proximal intercostal nerve ; however , they rarely arise from the vagus , recurrent laryngeal and phrenic nerve ( fig . schwannomas are the most common ( 50% ) mediastinal neurogenic tumours and frequently affect patients from 20 to 30 years old . they are usually solitary and encapsulated masses , but multiple schwannomas may be associated with neurofibromatosis type 2 . the tumour may grow through the adjacent intervertebral foramen and spinal canal to produce a dumbbell or hourglass configuration . neurofibromas are non - encapsulated soft tissue tumours and account for approximately 20% of mediastinal neurogenic tumours . a sudden increase in the size of a previously stable neurofibroma and these tumours are closely associated with neurofibromatosis and show more heterogeneous signal intensity and contrast enhancement on mri.fig . 29peripheral nerve tumours usually show a markedly convex mass arising from the mediastinum . a coronal multiplanar reconstruction of contrast - enhanced ct scan of an asymptomatic 59-year - old man with a mass in right superior mediastinum ( arrow ) . non - contrast - enhanced ct scan shows a well - defined and homogeneous paravertebral mass ( * ) peripheral nerve tumours usually show a markedly convex mass arising from the mediastinum . a coronal multiplanar reconstruction of contrast - enhanced ct scan of an asymptomatic 59-year - old man with a mass in right superior mediastinum ( arrow ) . non - contrast - enhanced ct scan shows a well - defined and homogeneous paravertebral mass ( * ) sympathetic ganglion tumours , which comprise 25% of mediastinal neurogenic tumours and arise from neuronal cells rather than from the nerve sheath . radiographically , the tumours are well - marginated , occurring along the anterolateral aspect of the spine and spanning three to five vertebrae . whorled appearance is due to curvilinear bands of low signal intensity that reflects collagenous fibrous tissue in the mass on t2-weighted images . ganglioneuroblastomas are the least common type of neurogenic tumour and show intermediate features in cellular maturity between neuroblastoma and ganglioneuroma . neuroblastomas are highly aggressive and readily metastasising tumours of neuroectodermal origin with a median age at diagnosis of 22 months . they are heterogeneous and non - encapsulated lesions , often exhibiting haemorrhage , necrosis , calcification or cystic degeneration ( fig . axial ( a ) and coronal ( b ) t2-weighted mr images , and sagittal ( c ) contrast - enhanced t1-weighted mr image demonstrate an expansive and heterogeneous mass in the left paravertebral space which shows cystic degeneration within ( * ) as well as an intensive enhancement ( in c ) . note the spinal involvement ( arrowhead in b ) neuroblastoma in a 20-year - old man . axial ( a ) and coronal ( b ) t2-weighted mr images , and sagittal ( c ) contrast - enhanced t1-weighted mr image demonstrate an expansive and heterogeneous mass in the left paravertebral space which shows cystic degeneration within ( * ) as well as an intensive enhancement ( in c ) . aortopulmonary paragangliomas are usually asymptomatic , while aortosympathetic paragangliomas ( along the sympathetic chain in the posterior mediastinum ) occur in symptomatic patients related to the functional activity of the tumour . a characteristic mri finding of paragangliomas is the presence of multiple curvilinear and punctate signal voids , which reflect high velocity flow in the intratumoral vessels , described as salt - and - pepper appearance.fig . a enhanced - ct scan shows a markedly enhancing lesion ( arrow ) located adjacent of the arch of aorta . paraganglioma ( white arrow ) ; descending aorta ( black arrow ) ; vagus nervus ( open arrow ) . c photomicrograph demonstrated a trabecular pattern of growth and scattered ganglion - like cells surrounded by fibrovascular septa ( arrows ) aortopulmonary paraganglioma in a 52-year - old woman . a enhanced - ct scan shows a markedly enhancing lesion ( arrow ) located adjacent of the arch of aorta . paraganglioma ( white arrow ) ; descending aorta ( black arrow ) ; vagus nervus ( open arrow ) . c photomicrograph demonstrated a trabecular pattern of growth and scattered ganglion - like cells surrounded by fibrovascular septa ( arrows ) clinical and radiological features of the most common mediastinal masses are detailed in table 3.table 3clinical and radiological features of the most common mediastinal massesanatomical locationimaging features ( ct)imaging features ( mri)manifestationslipomaanterior mediastinumencapsulated homogeneous fat attenuation ( -40 to -120 hu)homogeneous hyperintensity on t1-wiasymptomatic ( occasionally local compression of surrounding structures)liposarcomaposterior mediastinum- inhomogeneous fat attenuationinhomogeneous appearancesymptomatic at presentation- irregular areas of soft - tissue appearance- locally aggressive tumoursthymolipomacardiophrenic angle- fat - containing lesions ( up to 90% fat content)- homogeneous hyperintensity on t1-wi- asymptomatic ( occasionally local compression of surrounding structures)- variable component of soft - tissue elements- small amounts of solid areas and fibrous septa- a / w myasthenia gravis , graves disease and haematological disordersbronchogenic cystmiddle / posterior mediastinum- well - defined homogeneous fluid attenuation ( 020 hu)- homogeneous hyperintensity on t2-wi40% symptomatic at presentationduplication cyst- variable composition of fluid if complication or presence of protein or mucoid material- variable patterns of signal intensity on t1-wi because of variable cyst contentsasymptomaticpericardial cystcardiophrenic angleasymptomaticneuroenteric cystposterior mediastinum- well - defined homogeneous low - attenuation paravertebral masshomogeneous hyperintensity on t2-wia / w neurofibromatosis , vertebral and rib anomalies or scoliosis- enlargement of intervertebral foraminathymic cystanterior mediastinum congenital thymic cyst : unilocular well - defined fluid attenuation masshomogeneous hyperintensity on t2-wiasymptomatic acquired thymic cyst : multilocular well - defined fluid attenuation mass with a clearly seen wallhomogeneous hyperintensity on t2-wi with fibrous septaa / w thymic tumours , after thoracotomy or radiation therapy for hd or as sequelae of inflamatory processesmediastinal goitreanterior mediastinum- spontaneous hyperattenuation- heterogeneous appearance on t2-wiasymptomatic or airway / oesophageal compression- inhomogeneous density with cystic areas and calcifications- relatively hypointensity on t1-wi as compared with normal gland tissue , except foci of haemorrhage and cysts- markedly contrast - enhancementthymic hyperplasiaanterior mediastinum true thymic hyperplasia : enlargement of the thymus which remains normally organised- similar mr signals to those of normal thymusafter chemotherapy , corticosteroid therapy , irradiation or thermal burns lymphoid hyperplasia : normal , enlarged or as a focal thymic mass- apparent decrease in the signal intensity of the thymus at opposed - phase images in contrast to in - phase imagesa / w myasthenia gravis , rheumatoid arthritis , scleroderma , graves diseasethymomaanterior mediastinum- soft - tissue attenuation- low signal intensity on t1-wi- patients older than 40 years - old- mild to moderate contrast enhancement- relatively high signal intensity on t2-wi- asymptomatic vs pressure - induced symptoms- occasionally , focal haemorrhage , necrosis , cyst formation and linear or ring - like calcifications- complete obliteration of the adjacent fat planes highly suggests mediastinal invasion- a / w myasthenia gravis ( 30 - 50% ) , hypogammaglobulinaemia ( 10% ) and pure red cells apasia ( 5%)- pleural nodulesthymic carcinomaanterior mediastinum- ill - defined soft - tissue mass- similar features on ct- mean age of 50 years- necrotic or cystic component- absence of tumour capsule- symptomatic at presentation- heterogeneous contrast enhancement- lymphadenopathy and great vessel invasion- 5065% distant metastases at presentationlymphomaanterior mediastinum- homogeneous soft - tissue massvarious signal patterns on t1- and t2-wi- the most common cause of masses in the paediatric mediastinum- mild to moderate contrast enhancement- hd : bimodal distribution of incidence . pressure - induced symptoms- absence of vascular involvement- dlbcl : mean age of 30 years- mediastinal lymphadenopathy- pleural and pericardial effusionsteratomaanterior mediastinum- well - defined unilocular or multilocular cystic lesion containing fluid , soft tissue , and fat attenuation . calcifications may be present- heterogeneous signal intensityusually asymptomatic- 15% as nonspecific cystic lesion- fat - fluid levels within the lesion are virtually diagnostic of teratomantgctanterior mediastinum- heterogeneous ill - circumscribed large massheterogeneous signal intensity- symptomatic young males- pericardial and pleural effusion- tumour markers hcg and afp- involvement of great vessels- distant metastasesschwannomaposterior mediastinum- markedly convex masshomogeneous or heterogeneous high signal intensity on t2-wi- patients from 20 to 30 years old- dumbbell or hourglass configuration- a / w neurofibromatosis type 2 when multiple- cystic , haemorrhage and calcification elements are commonsgtposterior mediastinum- well - defined or ill - defined mass oriented along the anterolateral surface of several vertebraeheterogeneous high signal intensity on t2-wichildren and young adults- whorled appearanceparagangliomaapp : along great vesselshypervascular tumourssalt and pepper appearanceapp : asymptomatic patients older than 40 yearsasp : posterior mediastinumasp : younger adults . half of them present symptoms ntgct non - teratomatous germ cell tumours , sgt sympathetic ganglion tumours , afp alpha - fetoprotein , hcg beta human chorionic gonadotropin , hd hodgkin disease , tcll t - cell lymphoblastic lymphoma , dlbcl diffuse large b - cell lymphoma , app aortopulmonary paraganglioma , asp aortosympathetic paraganglioma , a / w association with , wi weighted imaging clinical and radiological features of the most common mediastinal masses ntgct non - teratomatous germ cell tumours , sgt sympathetic ganglion tumours , afp alpha - fetoprotein , hcg beta human chorionic gonadotropin , hd hodgkin disease , tcll t - cell lymphoblastic lymphoma , dlbcl diffuse large b - cell lymphoma , app aortopulmonary paraganglioma , asp aortosympathetic paraganglioma , a / w association with , wi weighted imaging parathyroid adenomas may be seen in ectopic locations , the mediastinum being the most commonly site . high - resolution ultrasonography ( us ) is recognised as a tool for detecting cervical parathyroid lesions . as it enlarges , an abnormal gland appears as a hypoechoic , and often anechoic , lesion , often posterior in location to the thyroid . colour doppler assessment of parathyroid lesions is a useful integration of grey - scale us and may be helpful in featuring parathyroid lesions . the colour doppler patterns termed parenchymal ( pattern iv , internal flow ) and vascular pole ( pattern ii , focal peripheral flow ) are typical of parathyroid lesions . the different colour doppler us patterns seem to be influenced by many factors as the location of the gland and the degree of vascularity . these tumours tend to be small and may contain calcifications at ct . technetium-99 sestamibi spect scans are more effective for their diagnosis ( fig . fibrosing mediastinitis is a dense fibrosis which progressively encases and eventually obliterates the lumen of the mediastinal vessels and airways ( fig . high attenuation of haematomas can be observed on unenhanced ct scans during the first 72 h ( fig . 34 ) . when the hematoma ages its attenuation decreases at ct in a centripetal fashion . haemangiomas in the mediastinum are rare and the may be associated with rendu - osler syndrome . sarcomas other than vascular or neural origin , including fibrosarcomas , osteosarcomas and chondrosarcomas , are also very uncommon . 32parathyroid adenoma in a 66-year - old man with hypercalcaemia , hypophosphataemia and elevated pth values . tc-99 m mibi scan shows a focus of hyperactivity ( black arrow ) adjacent to the lower pole of left thryoid lobule . a coronal multiplanar reconstruction and axial b constrast - enhanced ct scan show an infiltrating soft tissue mass ( white arrows ) in mediastinum encasing major vessels . punctate calcifications are observed ( black arrows ) as well as an elevation of the right hemidiaphragm ( open arrow ) secondary to the phrenic nerve involvementfig . 34 a contrast - enhanced ct scan of a man who had suffered a traffic accident . an infiltrative mediastinal haematoma is identified with subtle areas of high ct - attenuation values ( arrow ) . bilateral pleural effusion ( * ) and a sternum fracture ( open arrow ) are also observed . b iatrogenic mediastinal haematoma ( arrow ) in a 64-year - old man secondary to bronchoscopy with transtracheal biopsy . note the high attenuation value of the lesion compared with muscular tissue parathyroid adenoma in a 66-year - old man with hypercalcaemia , hypophosphataemia and elevated pth values . tc-99 m mibi scan shows a focus of hyperactivity ( black arrow ) adjacent to the lower pole of left thryoid lobule . enhanced - ct scan shows a superior mediastinal enhanced mass ( white arrow ) idiopathic fibrosing mediastinitis in a 64-year - old man . a coronal multiplanar reconstruction and axial b constrast - enhanced ct scan show an infiltrating soft tissue mass ( white arrows ) in mediastinum encasing major vessels . punctate calcifications are observed ( black arrows ) as well as an elevation of the right hemidiaphragm ( open arrow ) secondary to the phrenic nerve involvement a contrast - enhanced ct scan of a man who had suffered a traffic accident . an infiltrative mediastinal haematoma is identified with subtle areas of high ct - attenuation values ( arrow ) . bilateral pleural effusion ( * ) and a sternum fracture ( open arrow ) are also observed . b iatrogenic mediastinal haematoma ( arrow ) in a 64-year - old man secondary to bronchoscopy with transtracheal biopsy . note the high attenuation value of the lesion compared with muscular tissue lipomas predominantly occur in the anterior mediastinum and are reported to represent 1.62.3% of all primary mediastinal tumours . at ct , lipomas have homogeneous fat attenuation of approximately -100 hu . liposarcoma frequently occurs in the posterior mediastinum and it is usually symptomatic at the time of presentation , in contrast to lipoma . thymolipoma is a rare , benign , well - encapsulated thymic tumour that accounts for about 29% of thymic neoplasms [ 10 , 1214 ] . tumours occur most frequently in the cardiophrenic angle of asymptomatic young adults without sex predilection . the fat content usually constitutes 5085% of the lesion but has been reported to account for as much as 95% of the tumour . associations with myasthenia gravis , graves disease and haematological disorders have been reported . at cr , thymolipomas may mimic cardiomegaly , excessive epicardial fat , diaphragmatic elevation , lobar collapse or a pericardial cyst . ct or mri is required to establish the diagnosis by showing a well - defined encapsulated mass that has extensive fat content and contains small amounts of solid areas and fibrous septa ( fig . axial and coronal multiplanar reconstruction of non - contrast - enhanced ct scan show a large and well - defined mass ( arrows ) that has extensive fat content and contains small amounts of thin fibrous septa thymolipoma in a 47-year - old asymptomatic man . axial and coronal multiplanar reconstruction of non - contrast - enhanced ct scan show a large and well - defined mass ( arrows ) that has extensive fat content and contains small amounts of thin fibrous septa mediastinal primary cysts represent 1520% of all primary mediastinal masses [ 1 , 15 ] . a smooth or oval mass with a homogeneous attenuation , with no enhancement of cyst contents and no infiltration of adjacent structures are the usual ct features of benign mediastinal cyst ( fig . 5 ) . any cyst may have a higher attenuation due to its calcic , proteinaceous , mucous or haemorrhagic content . true cystic lesions should be differentiated from the cystic degenerative changes observed in many solid tumours , in lymphomas before or after treatment , or in abscesses ( fig . 5a well - marginated mass with a homogeneous attenuation , usually in the range of water attenuation ( 020 hu ) and without an enhancement of the wall or infiltrative appearance are the typical features of benign mediastinal cysts . 6oblique - coronal multiplanar reconstruction of contrast - enhanced ct scan of a 33-year - old woman with a descending necrotising mediastinitis . hypodense para - aortic areas correspond with fluid - collections ( arrows ) that extend to the retroperitoneum . note the visceral space involvement ( arrowhead ) a well - marginated mass with a homogeneous attenuation , usually in the range of water attenuation ( 020 hu ) and without an enhancement of the wall or infiltrative appearance are the typical features of benign mediastinal cysts . probably thymic cystic ( a ) and pericardial cyst ( b ) oblique - coronal multiplanar reconstruction of contrast - enhanced ct scan of a 33-year - old woman with a descending necrotising mediastinitis . hypodense para - aortic areas correspond with fluid - collections ( arrows ) that extend to the retroperitoneum . note the visceral space involvement ( arrowhead ) bronchogenic cysts result from abnormal ventral budding or branching of the tracheobronchial tree during embryologic development [ 1517 ] . they are lined by respiratory epithelium and their capsule contains cartilage , smooth muscle and mucous gland tissue . approximately 40% of bronchogenic cysts are symptomatic , resulting in cough , dyspnea or chest pain . the bronchogenic cyst is commonly located in the near carina ( 52% ) and in the paratracheal region ( 19% ) . the anterior mediastinum is a rare location of the bronchogenic cyst ( fig . air within the cyst is suggestive of secondary infection and communication with the tracheobronchial tree.fig . a non - contrast - enhanced ct scan shows a homogeneous anterior mediastinal mass with smooth contours and oval shape ( arrow ) . b t2-weighted mr image shows the same mass ( arrow ) with markedly high signal intensity . c although its localisation , a bronchogenic cyst was confirmed by histological examination after surgical resection bronchogenic cyst in a 37-year - old man . a non - contrast - enhanced ct scan shows a homogeneous anterior mediastinal mass with smooth contours and oval shape ( arrow ) . b t2-weighted mr image shows the same mass ( arrow ) with markedly high signal intensity . c although its localisation , a bronchogenic cyst was confirmed by histological examination after surgical resection duplication cysts are uncommon lesions lined by gastrointestinal tract mucosa and generally asymptomatic . however , if they contain gastric or pancreatic mucosa , there is the added risk of haemorrhage or rupture of the cyst from mucosal secretions . the majority of them are detected adjacent to or within the oesophageal wall ( fig . a contrast - enhanced ct scan shows a round well marginated mass ( arrow ) adjacent to the oesophagus with homogeneous water - attenuation . b the lesion ( arrow ) presents a bright signal intensity on t2-weighted fat - suppressed mr image duplication cyst in a 42-year - old asymptomatic man . a contrast - enhanced ct scan shows a round well marginated mass ( arrow ) adjacent to the oesophagus with homogeneous water - attenuation . b the lesion ( arrow ) presents a bright signal intensity on t2-weighted fat - suppressed mr image mediastinal neuroenteric cysts are anomalous protrusions of the leptomeninges through intervertebral foramen or defects in the vertebral body . they are associated with multiple vertebral anomalies and with neurofibromatosis [ 15 , 16 ] . . 9 ) . however , they may occur anywhere in relation to the pericardium.fig . 9 a lateral chest radiograph of a 58-year - old smoker man allergic to iodine shows a well - defined mass ( * ) in the cardiophrenic space and a nodular lung opacity ( open arrow ) in the lower lung parenchyma . b , c non - contrast - enhanced ct scan confirms the presence of a fluid - attenuation mass ( * ) in the right cardiophrenic angle ( pericardial cyst ) and demonstrates a suspicious lung opacity ( open arrows ) in the right lower lobe of the lung ( squamous - cell carcinoma ) a lateral chest radiograph of a 58-year - old smoker man allergic to iodine shows a well - defined mass ( * ) in the cardiophrenic space and a nodular lung opacity ( open arrow ) in the lower lung parenchyma . b , c non - contrast - enhanced ct scan confirms the presence of a fluid - attenuation mass ( * ) in the right cardiophrenic angle ( pericardial cyst ) and demonstrates a suspicious lung opacity ( open arrows ) in the right lower lobe of the lung ( squamous - cell carcinoma ) thymic cyst represents 1% of all mediastinal masses . congenital cysts derive from remnants of the thymopharyngeal duct , they are typically unilocular and contain clear fluid ( fig . in contrast , acquired thymic cysts are much more common , tend to be multilocular ( fig . 11 ) and may arise in association with neoplasms such as thymomas , lymphomas or germ cell tumours . thymic cysts may also be seen in the anterior mediastinum after radiation therapy of hodgkin s disease , after inflammatory processes and occasionally in patients with aids , particularly in children .fig . contrast - enhanced ct scan demonstrates a unilocular unenhanced lesion in the anterior mediastinum which shows a homogeneous fluid - attenuation ( arrow)fig . a contrast - enhanced ct scan shows a well - defined water - attenuation multiloculated mass ( * ) in the anterior mediastinum . b sagittal t2-weighted mr sequence demonstrates a multiloculated mass with typical high signal intensitiy and fine internal septa within ( arrow ) congenital thymic cyst in a 47-year - old man . contrast - enhanced ct scan demonstrates a unilocular unenhanced lesion in the anterior mediastinum which shows a homogeneous fluid - attenuation ( arrow ) acquired thymic cyst in a 43-year - old man . a contrast - enhanced ct scan shows a well - defined water - attenuation multiloculated mass ( * ) in the anterior mediastinum . b sagittal t2-weighted mr sequence demonstrates a multiloculated mass with typical high signal intensitiy and fine internal septa within ( arrow ) lymphangioma is a rare benign lesion of lymphatic origin that represents 0.74.5% of all mediastinal tumours in adult population . lymphangiomas involve the neck or the axillary region in more than 80% of the cases and the thorax in 10% of the cases ( fig . 12a cystic lymphangioma ( also referred to as hygroma ) in a 47-year - old woman with retroperitoneal disease . a posterior mediastinal mass with a homogeneous fluid - attenuation is identified on ct ( white arrow ) and on posteroanterior chest radiograph as a mass disrupting left paraspinal line inferiorly ( black arrows ) a cystic lymphangioma ( also referred to as hygroma ) in a 47-year - old woman with retroperitoneal disease . a posterior mediastinal mass with a homogeneous fluid - attenuation is identified on ct ( white arrow ) and on posteroanterior chest radiograph as a mass disrupting left paraspinal line inferiorly ( black arrows ) pancreatic pseudocyst can extend into the mediastinum via the oesophageal or aortic hiatus . ct shows a thin - walled , fluid - containing cyst within the posterior mediastinum which may be in continuity with the intrapancreatic or peripancreatic fluid collections ( fig . axial ( a , b ) and oblique - sagittal multiplanar reconstruction ( c ) ct scan show a thin - walled peri - oesophageal fluid - lesion ( white open arrows ) that comunicates with an intrapancreatic fluid - collection ( black open arrows ) through oesophageal hiatus by a duct fistula ( white arrows ) pancreatic pseudocyst in a 52-year - old man with a recurrent pancreatitis history . axial ( a , b ) and oblique - sagittal multiplanar reconstruction ( c ) ct scan show a thin - walled peri - oesophageal fluid - lesion ( white open arrows ) that comunicates with an intrapancreatic fluid - collection ( black open arrows ) through oesophageal hiatus by a duct fistula ( white arrows ) mediastinal goitre generally represents direct contiguous growth of a goitre into the anterior or superior mediastinum . typical features of mediastinal goitres are encapsulated and lobulated mass with inhomogeneous appearance with cystic areas , calcifications and marked contrast enhancement . an intrathoracic thyroid mass developing from heterotopic thyroid tissue without any connection to the thyroid in the neck is extremely rare ( fig . the presence of ill - defined margins , invasion of adjacent structures and nearby lymph node enlargement suggests the diagnosis of thyroid cancer ( fig . a coronal multiplanar reconstruction ct scan demonstrates an anterior mediastinal mass ( * ) arising from the thyroid more superiorly . b non - contrast - enhanced ct scan shows a unilateral mediastinal goitre with peripheral areas of calcification ( open arrow ) . c photograph of the resected surgical specimen shows a lobulated and heterogeneous appearance of the mass . d contrast - enhanced ct scan shows a well marginated posterior mediastinal goitre ( * ) with marked contrast enhancementfig . sagittal contrast - enhanced ct scan demonstrates a well - defined mass ( arrow ) located in the retrosternal space which shows an intense and heterogeneous contrast enhancement due to the presence of cystic areas ( * ) . no connection to the thyroid gland from the neck . b anaplastic thyroid carcinoma in a 64-year - old woman with respiratory failure . contrast - enhanced ct scan shows an anterior mediastinal soft tissue mass ( * ) which surrounds great vessels and oesophagus and compresses trachea . a coronal multiplanar reconstruction ct scan demonstrates an anterior mediastinal mass ( * ) arising from the thyroid more superiorly . b non - contrast - enhanced ct scan shows a unilateral mediastinal goitre with peripheral areas of calcification ( open arrow ) . c photograph of the resected surgical specimen shows a lobulated and heterogeneous appearance of the mass . d contrast - enhanced ct scan shows a well marginated posterior mediastinal goitre ( * ) with marked contrast enhancement a heterotopic mediastinal goitre . sagittal contrast - enhanced ct scan demonstrates a well - defined mass ( arrow ) located in the retrosternal space which shows an intense and heterogeneous contrast enhancement due to the presence of cystic areas ( * ) . no connection to the thyroid gland from the neck . b anaplastic thyroid carcinoma in a 64-year - old woman with respiratory failure . contrast - enhanced ct scan shows an anterior mediastinal soft tissue mass ( * ) which surrounds great vessels and oesophagus and compresses trachea . note the extension to the suprasternal fossa ( open arrow ) thymic hyperplasia can be divided into two distinct histological types [ 13 , 14 ] . true thymic hyperplasia is defined as enlargement of the thymus , which generally retains its normal shape . this disease entity is observed when a patient is recovering from some recent stress ( such as chemotherapy , corticosteroid therapy , irradiation or thermal burns ) . the phenomenon known as rebound hypeplasia is defined as a greater than 50% increase in thymic volume over baseline after such stress . among patients who undergo chemotherapy , approximately 1025% thymic lymphoid ( follicular ) hyperplasia of the thymus refers to the presence of an increased number of lymphoid follicles . it is commonly associated with autoimmune diseases , being seen in up to 65% of cases with myasthenia gravis , and it has been reported to occur in the early stages of human immunodeficiency virus infection . at ct it may appear normal ( 45% ) , enlarged ( 35% ) ( fig . 16thymic lymphoid hyperplasia in a 41-year - old woman with clinical diagnosis of myasthenia gravis . non - contrast - enhanced ct scan shows an enlarged thymic gland ( arrows ) without mass effect on adjacent structures thymic lymphoid hyperplasia in a 41-year - old woman with clinical diagnosis of myasthenia gravis . non - contrast - enhanced ct scan shows an enlarged thymic gland ( arrows ) without mass effect on adjacent structures it is important for radiologists to be able to distinguish thymic hyperplasia from neoplasm . diffuse symmetric enlargement of the gland , a smooth contour and normal vessels are the key morphological features of hyperplasia , whereas neoplasm tends to manifest as a focal mass with nodular contour and necrotic or calcified foci . detecting fat in the thymus is particularly relevant in these situations . according to takahashi et al . thymic hyperplasia reveals a relative signal loss on opposed - phase chemical - shift mri that it is different from no significant signal change between in - phase and opposed - phase chemical - shift mr images in patients with malignancy ( fig . 17 a c thymic hyperplasia in a 43-year - old woman ( arrows ) . a non - contrast - enhanced ct scan reveals a lobulated lesion with smooth margins in anterior mediastinum . b lesion appears slightly hyperintense on in - phase gradient - echo t1-weighted mr image . c opposed - phase gradient - echo t1-weighted mr image shows decreased signal intensity within the lesion , confirming presence of fat . d f stage ii thymoma ( who type b2 ) in a 62-year - old woman ( arrows ) . d non - contrast - enhanced ct scan shows an anterior mediastinal soft tissue mass . e the lesion shows an intermediate signal intensity on in - phase gradient - echo t1-weighted mr image . f there is no substantial decrease in signal intensity relative to in - phase mr image on opposed - phase sequence a c thymic hyperplasia in a 43-year - old woman ( arrows ) . a non - contrast - enhanced ct scan reveals a lobulated lesion with smooth margins in anterior mediastinum . b lesion appears slightly hyperintense on in - phase gradient - echo t1-weighted mr image . c opposed - phase gradient - echo t1-weighted mr image shows decreased signal intensity within the lesion , confirming presence of fat . d f stage ii thymoma ( who type b2 ) in a 62-year - old woman ( arrows ) . d non - contrast - enhanced ct scan shows an anterior mediastinal soft tissue mass . e the lesion shows an intermediate signal intensity on in - phase gradient - echo t1-weighted mr image . f there is no substantial decrease in signal intensity relative to in - phase mr image on opposed - phase sequence thymoma is the most common primary neoplasm of the anterior mediastinum but accounts for less than 1% of all adult malignancies [ 22 , 23 ] . thymomas typically occur in patients older than 40 years of age , being rare in children , and affecting men and women equally . between 30% and 50% of patients with a thymoma have myasthenia gravis , whereas 1015% of patients with myasthenia gravis have a thymoma . the updated histological classification elaborated by who in 2004 classified different types of thymomas ( table 1 ) on the basis of the morphology of the neoplastic epithelial cells together with the lymphocyte - epithelial cell ratio [ 23 , 24 ] . in contrast to histological classification , the stage of thymoma has clinical implications and it is a useful tool for management decisions . the masaoka - koga staging system is the most commonly used and describes thymomas in terms of the local extension of the tumour [ 14 , 2326 ] ( table 2 ) . the masaoka staging system is one of the two factors , including completeness of surgical resection , that most strongly correlates with prognosis of thymomas . the role of imaging is to initially diagnose and properly stage thymoma , with emphasis on the detection of local invasion and distant spread of disease . between 45 and 80% of thymomas are visible by chest radiography . on ct scans , thymomas usually appear as homogeneous solid masses with soft - tissue attenuation and well - demarcated borders , located anywhere from the thoracic inlet to the cardiophrenic angle . thymomas may be oval , round or lobulated and when they are large , cystic or necrotic degeneration may be shown ( fig . certain findings , such as encasement of mediastinal structures , infiltration of fat planes , irregular interface between the mass and lung parenchyma , and direct signs of vascular involvement are highly suggestive of invasion ( fig . 19 ) . pleural dissemination ( drop metastases ) manifests as one or more pleural nodules or masses , and they are almost always ipsilateral to the tumour ( fig . thymoma rarely presents with metastatic lymphadenopathy , metastatic pulmonary nodules or pleural effusion . at mri , thymomas commonly appear as homogeneous or heterogeneous masses with low to intermediate signal intensity on t1-weighted images and with high signal intensity on t2-weighted images ( fig . mri can prove useful in identifying the nodular wall thickening detected in cystic thymomas , absent from congenital cysts .table 1who classification for thymoma2004 who classificationdescriptiona ( spindle cell thymoma ; medullary thymoma)bland spindle / oval epithelial tumour cells with few or no lymphocytesab ( mixed thymoma)mixture of a lymphocyte - poor type a thymoma component and a more lymphocyte - rich type b - like component ( smaller and paler than those of b1 or b2 thymomas ) . lymphocytes are more numerous than in the type a component , but may be less numerous than in b1 thymomasb1 ( lymphocyte - rich thymoma ; lymphocytic thymoma ; organoid thymoma ; predominantly cortical thymoma)epithelial cells with a histological appearance practically indistinguishable from the normal thymus , composed predominantly of areas resembling cortex with epithelial cells scattered in a prominent population of immature lymphocytesb2 ( cortical thymoma)tumour cells closely resembling the predominant epithelial cells of the normal thymic cortex . a background population of immature t cells is always present and usually outnumbers the neoplastic epithelial cellsb3 ( epithelial thymoma ; squamoid thymoma)medium the epithelial cells are mixed with a minor component of intraepithelial lymphocytesfrom travis et al . table 2masaoka - koga staging system of thymomastagedescriptionimacroscopically and microscopically encapsulated tumouriiamicroscopic invasion through the capsuleiibmacroscopic invasion into surrounding fatty tissueiiiinvasion into a neighbouring organ such as the pericardium , great vessels or lungivapleural or pericardial disseminationivblymphatic - haematogenous metastasesfrom references [ 14 , 2326]fig . 18 a , b stage ii thymoma ( who type b1 ) in a 33-year - old woman who presented with myasthenia gravis . frontal chest radiograph shows a hilum overlay sign ( arrow ) of a suggestive anterior mediastinal mass . contrast - enhanced ct scan confirms the presence of a low - heterogeneous anterior mediastinal mass ( arrow ) . c , d stage iii thymoma ( who type b2 ) in a 54-year - old woman . frontal chest radiograph reveals a lobulated mediastinal mass ( arrow ) on the right side . contrast - enhanced ct scan demonstrates an enhanced anterior mediastinal mass ( arrow ) with infiltration of surrounding fat ( open arrow)fig . a , b contrast - enhanced ct scan shows a well - circumscribed , flattened soft tissue lesion in the anterior mediastinum with calcification ( arrow ) . note the lobulated contour of the mass and the loss of the fat plane between the mass and the aorta . pleural seeding is identified as an enhancing pleura - based nodule ( open arrow ) . c irregular border between the mediastinal mass and the lung parenchyma ( arrowhead ) is observed as a sign of locally advanced disease . a contrast - enhanced ct scan reveals an anterior mediastinal mass ( arrows ) with irregular contours , homogeneous enhancement and peripheral and central calcification as well as a pleural nodule ( open arrow ) . b on an axial fdg - positron emission tomography ( pet ) image , the pleural nodule is fdg avid , confirming a drop metastasis . d photomicrograph ( haematoxylin - eosin stain ) of tissue from the lesion shows roughly equal numbers of epithelial cells ( white arrow ) and lymphocytes ( black arrow ) corresponding thymoma who type b2fig . 21axial ( a ) and coronal multiplanar reconstruction ( b ) of a non - contrast - enhanced ct scan of a 57-year - old man allergic to iodine with a thymoma . a solid lobulated thymic mass ( * ) with clumps of calcifications within ( arrowhead ) note the absence of a fat plane between the tumour and the aorta ( open arrow ) . d coronal t2-weighted mr image shows a typical signal hyperintensity of the tumour lesion ( * ) . c axial contrast - enhanced fat - suppressed t1-weighted mr image reveals a homogeneously enhanced solid tumour ( * ) which arises from the thymus . although mri demonstrates the presence of fat cleavage plane between ascending aorta and the tumour , a thymoma ( who type a ) with microscopic transcapsular invasion ( masaoka stage ii ) was confirmed after surgical resection who classification for thymoma from travis et al . masaoka - koga staging system of thymoma from references [ 14 , 2326 ] a , b stage ii thymoma ( who type b1 ) in a 33-year - old woman who presented with myasthenia gravis . frontal chest radiograph shows a hilum overlay sign ( arrow ) of a suggestive anterior mediastinal mass . contrast - enhanced ct scan confirms the presence of a low - heterogeneous anterior mediastinal mass ( arrow ) . c , d stage iii thymoma ( who type b2 ) in a 54-year - old woman . frontal chest radiograph reveals a lobulated mediastinal mass ( arrow ) on the right side . contrast - enhanced ct scan demonstrates an enhanced anterior mediastinal mass ( arrow ) with infiltration of surrounding fat ( open arrow ) ct is the imaging modality of choice for evaluating staging thymoma . a , b contrast - enhanced ct scan shows a well - circumscribed , flattened soft tissue lesion in the anterior mediastinum with calcification ( arrow ) . note the lobulated contour of the mass and the loss of the fat plane between the mass and the aorta . pleural seeding is identified as an enhancing pleura - based nodule ( open arrow ) . c irregular border between the mediastinal mass and the lung parenchyma ( arrowhead ) is observed as a sign of locally advanced disease . note the cellular bronchiolitis in the left lower lobe stage iva thymoma ( who type b2 ) in a 46-year - old man . a contrast - enhanced ct scan reveals an anterior mediastinal mass ( arrows ) with irregular contours , homogeneous enhancement and peripheral and central calcification as well as a pleural nodule ( open arrow ) . b on an axial fdg - positron emission tomography ( pet ) image , the pleural nodule is fdg avid , confirming a drop metastasis . d photomicrograph ( haematoxylin - eosin stain ) of tissue from the lesion shows roughly equal numbers of epithelial cells ( white arrow ) and lymphocytes ( black arrow ) corresponding thymoma who type b2 axial ( a ) and coronal multiplanar reconstruction ( b ) of a non - contrast - enhanced ct scan of a 57-year - old man allergic to iodine with a thymoma . a solid lobulated thymic mass ( * ) with clumps of calcifications within ( arrowhead ) is identified . note the absence of a fat plane between the tumour and the aorta ( open arrow ) . d coronal t2-weighted mr image shows a typical signal hyperintensity of the tumour lesion ( * ) . c axial contrast - enhanced fat - suppressed t1-weighted mr image reveals a homogeneously enhanced solid tumour ( * ) which arises from the thymus . although mri demonstrates the presence of fat cleavage plane between ascending aorta and the tumour , a thymoma ( who type a ) with microscopic transcapsular invasion ( masaoka stage ii ) was confirmed after surgical resection thymic carcinoma accounts for about 20% of thymic epithelial tumours with a mean age of 50 years . typical appearance is a multilobulated and heterogeneous mass that may contain areas of calcification or haemorrhage . . found that irregular contour , necrotic or cystic components , heterogeneous enhancement , lymphadenopathy and great vessel invasion strongly favoured thymic carcinoma . thymic carcinoids are rare , well - differentiated neuroendocrine tumours , which have a male predilection of 3:1 [ 13 , 14 ] . malignant lymphoma accounts for nearly 20% of all mediastinum neoplasms in adults and 50% in children . 22 ) represents approximately 5070% of mediastinal lymphomas , while non - hodgkin lymphoma comprises 1525% . frontal chest radiograph and contrast - enhanced ct scan show a homogeneous soft tissue mass at the level of the subcarina ( arrows ) . the right paratracheal stripe is not seen on frontal chest radiograph , having been most probably obliterated by a right paratracheal lymphadenopathy ( arrowhead ) . frontal chest radiograph and contrast - enhanced ct scan show a homogeneous soft tissue mass at the level of the subcarina ( arrows ) . the right paratracheal stripe is not seen on frontal chest radiograph , having been most probably obliterated by a right paratracheal lymphadenopathy ( arrowhead ) . * carina hodgkin disease ( hd ) has a bimodal distribution of incidence peaking in young adulthood and again after the age of 50 years . four subtypes of hd are described : nodular sclerosis ( by far the most common histological subtype ) ( fig . 23 ) , lymphocyte - rich , mixed cellularity and lymphocyte depleted hd [ 4 , 5 ] . cr is abnormal in up to 76% of patients with hd , often showing enlargement of the prevascular and paratracheal nodes . characteristic features on imaging are a homogeneous soft - tissue anterior mediastinal mass with mild to moderate contrast enhancement , irregular contours , surface lobulation , absence of vascular involvement , and high prevalence of associated mediastinal lymphadenopathy . frontal chest radiograph shows a large , well - defined mediastinal mass with increased density ( arrow ) . contrast - enhanced ct scan shows a bulky soft tissue mass ( arrows ) with homogeneous ct - attenuation value occupying prevascular space . photomicrograph reveals numerous neoplastic lacunar cells ( arrows ) in a background of small lymphocytes , histiocytes and eosinophils , which supports the diagnosis of nodular sclerosis type hodgkin lymphoma nodular sclerosis hodgkin lymphoma in a 44-year - old woman . frontal chest radiograph shows a large , well - defined mediastinal mass with increased density ( arrow ) . contrast - enhanced ct scan shows a bulky soft tissue mass ( arrows ) with homogeneous ct - attenuation value occupying prevascular space . photomicrograph reveals numerous neoplastic lacunar cells ( arrows ) in a background of small lymphocytes , histiocytes and eosinophils , which supports the diagnosis of nodular sclerosis type hodgkin lymphoma the two most common forms of mediastinal non - hodgkin disease ( nhd ) include diffuse large b - cell lymphoma and t - cell lymphoblastic lymphoma . the most common ct appearance is a large mediastinal mass representing thymic and lymph node enlargement , which compresses the airway and cardiovascular structures ( fig . primary mediastinal diffuse large b - cell lymphomas tend to occur in young to middle - aged adults with a mean age of 30 . it accounts for 7% of all cases of nhd and about 10% of all cases of high - grade nhd . the tumours appear as a large , smooth or lobulated , anterior mediastinal mass in nearly all patients . on ct the tumours show low attenuation areas , representing haemorrhage , necrosis or cystic degeneration in 50% of the cases and heterogeneous enhancement in about 40% of the cases ( fig . primary mediastinal b - cell lymphoma recurs in the chest . consequently , chest ct examination alone is sufficient for routine follow - up of these patients .fig . 24axial t1-weighted mr image of a 16-year - old man with a solid , large mass ( arrows ) in the anterior and superior mediastinum . aortic trunks are almost completely surrounded by the lesion and trachea ( t ) is displaced to the contralateral side by the lesion . contrast - enhanced ct scan shows a bulky soft tissue mass in the anterior mediastinum ( arrowsin a ) , which shows heterogeneous ct - attenuation values with cystic changes within ( * ) . pericardial effusion is also observed ( open arrow in b ) axial t1-weighted mr image of a 16-year - old man with a solid , large mass ( arrows ) in the anterior and superior mediastinum . supra - aortic trunks are almost completely surrounded by the lesion and trachea ( t ) is displaced to the contralateral side by the lesion . pathological analysis demonstrated a t - cell lymphoblastic lymphoma primary mediastinal diffuse large b - cell lymphoma in a 19-year - old man . contrast - enhanced ct scan shows a bulky soft tissue mass in the anterior mediastinum ( arrowsin a ) , which shows heterogeneous ct - attenuation values with cystic changes within ( * ) . pericardial effusion is also observed ( open arrow in b ) germ cell tumours ( gcts ) mainly arise in gonads and in the midline of the body as well , the mediastinum being the most common extragonadal site . if a teratoma contains fetal tissue or neuroendocrine tissue , it is defined as immature and malignant with a poor prognosis . on ct , teratoma most commonly appears as a well - defined unilocular or multilocular cystic lesion containing fluid , soft tissue and fat attenuation ( fig . calcifications may be focal , rim - like or , in rare cases , representative of teeth or bone . common combinations of internal components of mature teratomas include soft tissue , fluid , fat and calcification in 39% ; soft tissue , fluid and fat in 24% ; and soft tissue and fluid in 15% . in 15% of the cases , mature teratomas appear as non - specific cystic lesions without fat or calcium . on mri , fat - fluid levels within the lesion are virtually diagnostic of teratoma . ruptured teratomas show an adjacent consolidation , atelectasis and pleural or pericardial effusion than do unruptured teratomas.fig . 26chest imaging shows well the highly heterogeneous contents of mediastinal teratomas . a mature cystic teratoma in a 40-year - old man . contrast - enhanced ct scan shows a heterogeneous anterior mediastinal mass with areas of fat ( open arrow ) , calcification ( arrow ) and fluid attenuation ( * ) . c contrast - enhanced ct scan of an asymptomatic 24-year - old woman demonstrates a well - defined uniloculated mass located in prevascular space which shows a cystic changes within ( * ) . d the mass was surgically removed and pathological examination confirmed a benign teratoma chest imaging shows well the highly heterogeneous contents of mediastinal teratomas . a mature cystic teratoma in a 40-year - old man . contrast - enhanced ct scan shows a heterogeneous anterior mediastinal mass with areas of fat ( open arrow ) , calcification ( arrow ) and fluid attenuation ( * ) . c contrast - enhanced ct scan of an asymptomatic 24-year - old woman demonstrates a well - defined uniloculated mass located in prevascular space which shows a cystic changes within ( * ) . d the mass was surgically removed and pathological examination confirmed a benign teratoma the non - teratomatous germ cell tumours ( ntgct ) are rare and malignant tumours which usually occur in young males and most frequently affect the anterosuperior mediastinum . these tumours grow rapidly and develop large , bulky , ill - circumscribed masses with lobulated shape . primary mediastinal seminomas comprise 2550% of malignant mediastinal gcts and occur almost exclusively in males during the period from the second to fourth decades of life . at imaging , areas of degeneration due to haemorrhage and coagulation necrosis may be present ( fig . non - seminomatous germ cell tumours include yolk sac tumours , endodermal sinus tumours , embryonal carcinomas , choriocarcinomas and mixed germ cell tumours , which present as large masses typically with marked heterogeneous attenuation . at diagnosis , afp and - hcg levels is important when making the diagnosis ( fig . contrast - enhanced axial ct scan demonstrates a large mass in the right side of the mediastinum with an obvious mass effect on great vessels and heart . the mass shows heterogeneous ct - attenuation values probably secondary to haemorrhage and coagulation necrosis . note also a right pleural effusion ( arrowhead ) and multiple lung metastasis ( arrows)fig . 28non - seminomatous malignant germ cell tumour of the anterior mediastinum in a 25-year - old man with chest pain and high serum level of -fetoprotein at admission ( 25.396 ng / ml ) . frontal chest radiograph shows a central mass ( * ) . the descending aorta is clearly seen ( arrows ) , indicating that the mass is not within the posterior mediastinum . a contrast - enhanced ct scan confirms a mass of low attenuation ( * ) in the anterior mediastinum that compresses the pulmonary artery . bilateral lung metastasis ( arrowheads ) and hilar and subcarinal lymphadenopathy is identified ( open arrows ) a 19-year - old man with seminoma . contrast - enhanced axial ct scan demonstrates a large mass in the right side of the mediastinum with an obvious mass effect on great vessels and heart . the mass shows heterogeneous ct - attenuation values probably secondary to haemorrhage and coagulation necrosis . note also a right pleural effusion ( arrowhead ) and multiple lung metastasis ( arrows ) non - seminomatous malignant germ cell tumour of the anterior mediastinum in a 25-year - old man with chest pain and high serum level of -fetoprotein at admission ( 25.396 ng / ml ) . frontal chest radiograph shows a central mass ( * ) . the descending aorta is clearly seen ( arrows ) , indicating that the mass is not within the posterior mediastinum . a contrast - enhanced ct scan confirms a mass of low attenuation ( * ) in the anterior mediastinum that compresses the pulmonary artery . bilateral lung metastasis ( arrowheads ) and hilar and subcarinal lymphadenopathy is identified ( open arrows ) neurogenic tumours represent approximately 20% of all adults and 35% of all paediatric mediastinal tumours and they are the most common cause of a posterior mediastinal mass . seventy to eighty percent of neurogenic tumours are benign , and nearly half are asymptomatic . these tumours are generally grouped into : peripheral nerve tumours , which are the most common ( 70% ) mediastinal neurogenic tumours and originate from spinal or proximal intercostal nerve ; however , they rarely arise from the vagus , recurrent laryngeal and phrenic nerve ( fig . schwannomas are the most common ( 50% ) mediastinal neurogenic tumours and frequently affect patients from 20 to 30 years old . they are usually solitary and encapsulated masses , but multiple schwannomas may be associated with neurofibromatosis type 2 . the tumour may grow through the adjacent intervertebral foramen and spinal canal to produce a dumbbell or hourglass configuration . neurofibromas are non - encapsulated soft tissue tumours and account for approximately 20% of mediastinal neurogenic tumours . a sudden increase in the size of a previously stable neurofibroma and these tumours are closely associated with neurofibromatosis and show more heterogeneous signal intensity and contrast enhancement on mri.fig . 29peripheral nerve tumours usually show a markedly convex mass arising from the mediastinum . a coronal multiplanar reconstruction of contrast - enhanced ct scan of an asymptomatic 59-year - old man with a mass in right superior mediastinum ( arrow ) . non - contrast - enhanced ct scan shows a well - defined and homogeneous paravertebral mass ( * ) peripheral nerve tumours usually show a markedly convex mass arising from the mediastinum . a coronal multiplanar reconstruction of contrast - enhanced ct scan of an asymptomatic 59-year - old man with a mass in right superior mediastinum ( arrow ) . non - contrast - enhanced ct scan shows a well - defined and homogeneous paravertebral mass ( * ) sympathetic ganglion tumours , which comprise 25% of mediastinal neurogenic tumours and arise from neuronal cells rather than from the nerve sheath . radiographically , the tumours are well - marginated , occurring along the anterolateral aspect of the spine and spanning three to five vertebrae . whorled appearance is due to curvilinear bands of low signal intensity that reflects collagenous fibrous tissue in the mass on t2-weighted images . ganglioneuroblastomas are the least common type of neurogenic tumour and show intermediate features in cellular maturity between neuroblastoma and ganglioneuroma . neuroblastomas are highly aggressive and readily metastasising tumours of neuroectodermal origin with a median age at diagnosis of 22 months . they are heterogeneous and non - encapsulated lesions , often exhibiting haemorrhage , necrosis , calcification or cystic degeneration ( fig . axial ( a ) and coronal ( b ) t2-weighted mr images , and sagittal ( c ) contrast - enhanced t1-weighted mr image demonstrate an expansive and heterogeneous mass in the left paravertebral space which shows cystic degeneration within ( * ) as well as an intensive enhancement ( in c ) . note the spinal involvement ( arrowhead in b ) neuroblastoma in a 20-year - old man . axial ( a ) and coronal ( b ) t2-weighted mr images , and sagittal ( c ) contrast - enhanced t1-weighted mr image demonstrate an expansive and heterogeneous mass in the left paravertebral space which shows cystic degeneration within ( * ) as well as an intensive enhancement ( in c ) . aortopulmonary paragangliomas are usually asymptomatic , while aortosympathetic paragangliomas ( along the sympathetic chain in the posterior mediastinum ) occur in symptomatic patients related to the functional activity of the tumour . a characteristic mri finding of paragangliomas is the presence of multiple curvilinear and punctate signal voids , which reflect high velocity flow in the intratumoral vessels , described as salt - and - pepper appearance.fig . a enhanced - ct scan shows a markedly enhancing lesion ( arrow ) located adjacent of the arch of aorta . paraganglioma ( white arrow ) ; descending aorta ( black arrow ) ; vagus nervus ( open arrow ) . c photomicrograph demonstrated a trabecular pattern of growth and scattered ganglion - like cells surrounded by fibrovascular septa ( arrows ) aortopulmonary paraganglioma in a 52-year - old woman . a enhanced - ct scan shows a markedly enhancing lesion ( arrow ) located adjacent of the arch of aorta . paraganglioma ( white arrow ) ; descending aorta ( black arrow ) ; vagus nervus ( open arrow ) . c photomicrograph demonstrated a trabecular pattern of growth and scattered ganglion - like cells surrounded by fibrovascular septa ( arrows ) clinical and radiological features of the most common mediastinal masses are detailed in table 3.table 3clinical and radiological features of the most common mediastinal massesanatomical locationimaging features ( ct)imaging features ( mri)manifestationslipomaanterior mediastinumencapsulated homogeneous fat attenuation ( -40 to -120 hu)homogeneous hyperintensity on t1-wiasymptomatic ( occasionally local compression of surrounding structures)liposarcomaposterior mediastinum- inhomogeneous fat attenuationinhomogeneous appearancesymptomatic at presentation- irregular areas of soft - tissue appearance- locally aggressive tumoursthymolipomacardiophrenic angle- fat - containing lesions ( up to 90% fat content)- homogeneous hyperintensity on t1-wi- asymptomatic ( occasionally local compression of surrounding structures)- variable component of soft - tissue elements- small amounts of solid areas and fibrous septa- a / w myasthenia gravis , graves disease and haematological disordersbronchogenic cystmiddle / posterior mediastinum- well - defined homogeneous fluid attenuation ( 020 hu)- homogeneous hyperintensity on t2-wi40% symptomatic at presentationduplication cyst- variable composition of fluid if complication or presence of protein or mucoid material- variable patterns of signal intensity on t1-wi because of variable cyst contentsasymptomaticpericardial cystcardiophrenic angleasymptomaticneuroenteric cystposterior mediastinum- well - defined homogeneous low - attenuation paravertebral masshomogeneous hyperintensity on t2-wia / w neurofibromatosis , vertebral and rib anomalies or scoliosis- enlargement of intervertebral foraminathymic cystanterior mediastinum congenital thymic cyst : unilocular well - defined fluid attenuation masshomogeneous hyperintensity on t2-wiasymptomatic acquired thymic cyst : multilocular well - defined fluid attenuation mass with a clearly seen wallhomogeneous hyperintensity on t2-wi with fibrous septaa / w thymic tumours , after thoracotomy or radiation therapy for hd or as sequelae of inflamatory processesmediastinal goitreanterior mediastinum- spontaneous hyperattenuation- heterogeneous appearance on t2-wiasymptomatic or airway / oesophageal compression- inhomogeneous density with cystic areas and calcifications- relatively hypointensity on t1-wi as compared with normal gland tissue , except foci of haemorrhage and cysts- markedly contrast - enhancementthymic hyperplasiaanterior mediastinum true thymic hyperplasia : enlargement of the thymus which remains normally organised- similar mr signals to those of normal thymusafter chemotherapy , corticosteroid therapy , irradiation or thermal burns lymphoid hyperplasia : normal , enlarged or as a focal thymic mass- apparent decrease in the signal intensity of the thymus at opposed - phase images in contrast to in - phase imagesa / w myasthenia gravis , rheumatoid arthritis , scleroderma , graves diseasethymomaanterior mediastinum- soft - tissue attenuation- low signal intensity on t1-wi- patients older than 40 years - old- mild to moderate contrast enhancement- relatively high signal intensity on t2-wi- asymptomatic vs pressure - induced symptoms- occasionally , focal haemorrhage , necrosis , cyst formation and linear or ring - like calcifications- complete obliteration of the adjacent fat planes highly suggests mediastinal invasion- a / w myasthenia gravis ( 30 - 50% ) , hypogammaglobulinaemia ( 10% ) and pure red cells apasia ( 5%)- pleural nodulesthymic carcinomaanterior mediastinum- ill - defined soft - tissue mass- similar features on ct- mean age of 50 years- necrotic or cystic component- absence of tumour capsule- symptomatic at presentation- heterogeneous contrast enhancement- lymphadenopathy and great vessel invasion- 5065% distant metastases at presentationlymphomaanterior mediastinum- homogeneous soft - tissue massvarious signal patterns on t1- and t2-wi- the most common cause of masses in the paediatric mediastinum- mild to moderate contrast enhancement- hd : bimodal distribution of incidence . pressure - induced symptoms- absence of vascular involvement- dlbcl : mean age of 30 years- mediastinal lymphadenopathy- pleural and pericardial effusionsteratomaanterior mediastinum- well - defined unilocular or multilocular cystic lesion containing fluid , soft tissue , and fat attenuation . calcifications may be present- heterogeneous signal intensityusually asymptomatic- 15% as nonspecific cystic lesion- fat - fluid levels within the lesion are virtually diagnostic of teratomantgctanterior mediastinum- heterogeneous ill - circumscribed large massheterogeneous signal intensity- symptomatic young males- pericardial and pleural effusion- tumour markers hcg and afp- involvement of great vessels- distant metastasesschwannomaposterior mediastinum- markedly convex masshomogeneous or heterogeneous high signal intensity on t2-wi- patients from 20 to 30 years old- dumbbell or hourglass configuration- a / w neurofibromatosis type 2 when multiple- cystic , haemorrhage and calcification elements are commonsgtposterior mediastinum- well - defined or ill - defined mass oriented along the anterolateral surface of several vertebraeheterogeneous high signal intensity on t2-wichildren and young adults- whorled appearanceparagangliomaapp : along great vesselshypervascular tumourssalt and pepper appearanceapp : asymptomatic patients older than 40 yearsasp : posterior mediastinumasp : younger adults . half of them present symptoms ntgct non - teratomatous germ cell tumours , sgt sympathetic ganglion tumours , afp alpha - fetoprotein , hcg beta human chorionic gonadotropin , hd hodgkin disease , tcll t - cell lymphoblastic lymphoma , dlbcl diffuse large b - cell lymphoma , app aortopulmonary paraganglioma , asp aortosympathetic paraganglioma , a / w association with , wi weighted imaging clinical and radiological features of the most common mediastinal masses ntgct non - teratomatous germ cell tumours , sgt sympathetic ganglion tumours , afp alpha - fetoprotein , hcg beta human chorionic gonadotropin , hd hodgkin disease , tcll t - cell lymphoblastic lymphoma , dlbcl diffuse large b - cell lymphoma , app aortopulmonary paraganglioma , asp aortosympathetic paraganglioma , a / w association with , wi weighted imaging parathyroid adenomas may be seen in ectopic locations , the mediastinum being the most commonly site . high - resolution ultrasonography ( us ) is recognised as a tool for detecting cervical parathyroid lesions . as it enlarges , an abnormal gland appears as a hypoechoic , and often anechoic , lesion , often posterior in location to the thyroid . colour doppler assessment of parathyroid lesions is a useful integration of grey - scale us and may be helpful in featuring parathyroid lesions . the colour doppler patterns termed ( pattern iv , internal flow ) and vascular pole ( pattern ii , focal peripheral flow ) are typical of parathyroid lesions . the different colour doppler us patterns seem to be influenced by many factors as the location of the gland and the degree of vascularity . these tumours tend to be small and may contain calcifications at ct . technetium-99 sestamibi spect scans are more effective for their diagnosis ( fig . fibrosing mediastinitis is a dense fibrosis which progressively encases and eventually obliterates the lumen of the mediastinal vessels and airways ( fig . high attenuation of haematomas can be observed on unenhanced ct scans during the first 72 h ( fig . 34 ) . when the hematoma ages its attenuation decreases at ct in a centripetal fashion . haemangiomas in the mediastinum are rare and the may be associated with rendu - osler syndrome . sarcomas other than vascular or neural origin , including fibrosarcomas , osteosarcomas and chondrosarcomas , are also very uncommon . 32parathyroid adenoma in a 66-year - old man with hypercalcaemia , hypophosphataemia and elevated pth values . tc-99 m mibi scan shows a focus of hyperactivity ( black arrow ) adjacent to the lower pole of left thryoid lobule . 33idiopathic fibrosing mediastinitis in a 64-year - old man . a coronal multiplanar reconstruction and axial b constrast - enhanced ct scan show an infiltrating soft tissue mass ( white arrows ) in mediastinum encasing major vessels . punctate calcifications are observed ( black arrows ) as well as an elevation of the right hemidiaphragm ( open arrow ) secondary to the phrenic nerve involvementfig . 34 a contrast - enhanced ct scan of a man who had suffered a traffic accident . an infiltrative mediastinal haematoma is identified with subtle areas of high ct - attenuation values ( arrow ) . bilateral pleural effusion ( * ) and a sternum fracture ( open arrow ) are also observed . b iatrogenic mediastinal haematoma ( arrow ) in a 64-year - old man secondary to bronchoscopy with transtracheal biopsy . note the high attenuation value of the lesion compared with muscular tissue parathyroid adenoma in a 66-year - old man with hypercalcaemia , hypophosphataemia and elevated pth values . tc-99 m mibi scan shows a focus of hyperactivity ( black arrow ) adjacent to the lower pole of left thryoid lobule . enhanced - ct scan shows a superior mediastinal enhanced mass ( white arrow ) idiopathic fibrosing mediastinitis in a 64-year - old man . a coronal multiplanar reconstruction and axial b constrast - enhanced ct scan show an infiltrating soft tissue mass ( white arrows ) in mediastinum encasing major vessels . punctate calcifications are observed ( black arrows ) as well as an elevation of the right hemidiaphragm ( open arrow ) secondary to the phrenic nerve involvement a contrast - enhanced ct scan of a man who had suffered a traffic accident . an infiltrative mediastinal haematoma is identified with subtle areas of high ct - attenuation values ( arrow ) . bilateral pleural effusion ( * ) and a sternum fracture ( open arrow ) are also observed . b iatrogenic mediastinal haematoma ( arrow ) in a 64-year - old man secondary to bronchoscopy with transtracheal biopsy . note the high attenuation value of the lesion compared with muscular tissue in assessment of mediastinal disease , cross - sectional imaging techniques allow excellent visualisation of the mediastinum . mri plays an increasing role in this disease due to the existence of new available mr techniques on mediastinum imaging . on each of ct and mr scanning , the size of tumour , contour , perimeter of capsule , septum , haemorrhage , necrotic or cystic component , homogeneity within tumour , presence of mediastinal lymphadenopathy , pleural effusion and great vessel invasion are assessed . in addition , presence of calcification are assessed on ct and signal intensities of the tumour are assessed on mri . imaging plays an essential role in the diagnosis , staging and follow - up of mediastinal disease . complete resection is the mainstay of treatment in many mediastinal tumours and the ability to accomplish a complete resection appears to be the most important prognostic factor . currently , ct is the modality most commonly used for follow - up after treatment . ct findings may serve as predictors of tumour invasiveness and of postoperative recurrence or metastases . in table 4 we summarise some teaching points and imaging pitfalls for the diagnostic approach to mediastinal masses before and after treatment.table 4teaching points and imaging pitfalls for the diagnostic approach to mediastinal masses before and after treatment ct is accurate in distinguishing mediastinal masses which usually differ in their appearance and the pattern of metastatic spread , both of which are readily detected by chest ct pericardial fat pads and lipomatosis are correctly interpreted as normal findings rather than possible pathological lesions when lipoma and liposarcoma are situated in the cardiophrenic space , the imaging findings are very similar to those of morgagni hernia mri more accurately distinguishes between cystic and solid lesions than ct soft - tissue components associated with cystic lesions can be related to a malignant process ( e.g. soft - tissue nodules in a cystic anterior mediastinal lesion suggest that the lesion is a cystic thymoma rather than a congenital cyst) non - neoplastic thymic enlargement must not be confused with thymoma . the normal thymus in young children and the hyperplastic thymus may mimic a mass when differentiation between non - neoplastic thymic enlargement and thymoma can not be achieved at ct or conventional mri , chemical - shift mri with in - phase and out - of - phase gradient - echo sequences can be helpful thymoma rarely manifests with lymphadenopathy , pleural effusions , or extrathoracic metastases the role of imaging is to initially diagnose and properly stage thymoma , with emphasis on the detection of local invasion and distant spread of disease , to identify candidates for preoperative neoadjuvant therapy late recurrence in thymoma is not uncommon . imaging of treated patients is directed at identifying resectable recurrent disease , since patients with completely resected recurrent disease have similar outcomes as those without recurrence findings associated with significantly more frequent recurrence and metastases of thymic tumours include lobulated or irregular contour , oval shape , mediastinal fat invasion or great vessel invasion and pleural seeding mediastinal lymphadenopathy , pleural effusions , and pulmonary metastases are characteristic of thymic carcinoma or non - teratomatous germ cell tumour lymphoproliferative disorders typically present pleural effusions , pericardial fluid , and mediastinal lymphadenopathy in many cases heterogeneous appearance ( due to necrosis , cystic change , or haemorrhage ) is typical of thymic carcinoma , lymphoma , sympathetic ganglion tumour , peripheral nerve tumour and non - teratomatous germ cell tumour . it can be seen in about one - third of thymomas a cystic anterior mediastinal mass with intrinsic fat attenuation typically represents a mature teratoma teaching points and imaging pitfalls for the diagnostic approach to mediastinal masses before and after treatment tumours of the mediastinum represent a wide diversity of disease state . the location and composition of a mass is critical to narrowing the differential diagnosis . the clinical spectrum of mediastinal masses can range from being asymptomatic to producing compressive symptoms . although many of these masses have similar imaging appearances , clinical history , anatomical position and certain details seen at ct and mri imaging allow correct diagnosis in many cases . this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

backgroundmultiple different types of mediastinal masses may be encountered on imaging techniques in symptomatic or asymptomatic patients . the location and composition of these lesions are critical to narrowing the differential diagnosis.methodsradiological compartmentalisation of the mediastinum helps in focusing the diagnosis of masses on the basis of their site . some diseases , however , do not occur exclusively in any specific compartment and can spread from one compartment to another.resultstissular components of the mass , the degree of vascularisation and the relationships with mediastinal structures assessed by computed tomography ( ct ) or magnetic resonance imaging ( mri ) are a leading edge of the radiological diagnosis . special applications at mri have been developed over the recent years in order to identify accurately tissular components of the mediastinal masses . the likelihood of malignancy of the mediastinal masses is influenced by the symptomatology and the age of the patient . this article reviews the most commonly encountered mediastinal masses considering clinical history and manifestations , anatomical position and certain details seen on different imaging modalities that allow correct diagnosis in many cases.conclusionfamiliarity with the radiological features of mediastinal masses facilitates accurate diagnosis , differentiation from other mediastinic processes and , thus , optimal patient treatment.teaching points ct and mri are important for the diagnosis of mediastinal masses. the location and tissue characteristics on imaging studies are critical to narrow down the differential diagnosis of mediastinal masses. symptomatology and patient age affect the likelihood of malignancy .

Teaching Points Introduction Imaging modalities Mediastinal masses Fatty masses Cystic masses Solid masses Uncommon mediastinal masses Follow-up Conclusion Open Access

a core principle of both financial investment and biology is that diversity leads to strength . in biology , hybrid vigor produces healthy organisms , because each parental genome contributes complementary strengths and balances the other 's weaknesses . in investing , diversity spreads risk , maximizing the chances of finding some big winners while reducing the chances of collapse based on sudden declines in a few companies ' fortunes . diversity at all levels from the kinds of science to the regions in which it is conducted to the backgrounds of the people conducting it strengthens the institute 's research portfolio and should lead to the best returns on the taxpayers ' investments . it is impossible to know where or when the next big advances will arise , and history tells us that they frequently spring from unexpected sources . it is also impossible to know what threads of foundational knowledge will be woven together to produce a new breakthrough . supporting a wide variety of lines of inquiry will improve the chances of important discoveries being made . this includes studying a diversity of organisms , because important and useful processes almost certainly remain to be discovered in areas of biology we have not yet explored . scientists ' past or current experiences have a significant impact on the problems they choose to study and on the ideas they have for approaching these problems . health burdens from specific diseases differ from state to state and population to population , and these variances can drive the kinds of questions researchers ask . in addition to variations in regional health burdens , other environmental factors such as local plants and animals can influence an investigator 's research directions . for example , baldomero olivera , now a prominent researcher at the university of utah , began studying deadly cone snail venom while he was working in the philippines , where the animals are endemic and people routinely died of snail stings ( telis , 2014 ) . olivera 's work on cone snail toxins has transformed neuroscience and has already led to one food and drug administration approved drug and several more in clinical trials . we also need to consider the identification and development of scientific talent when planning how we invest in biomedical research . for example , if cutting - edge biomedical research were only being conducted in 25 states , it would mean that high school and college students in the other 25 states could get research experience only if they were willing and able to move . the loss of talent to science in the united states caused by this research experience gap would be severe . in addition to the value of supporting a diverse research portfolio , it is important to recognize that the best ideas come from investigators themselves . although there are times when management and top - down direction can help break through systemic barriers or open up bottlenecks particularly when the development of new technologies is required fundamental research works best when investigators are following their noses and setting their own directions . during the national institutes of health ( nih ) budget - doubling period ( 19982003 ) , the fraction of the nigms portfolio dedicated to investigator - initiated research declined from 99 to 80% because of increasing use of programmatic initiatives ( figure 1 ) ; that is , funding targeted at specific scientific areas . with the budget doubling more than a decade behind us , it is time to return the institute 's focus to investigator - initiated research , to ensure that new scientific territory is opened for exploration by adventurous investigators . the fraction of the nigms portfolio committed to investigator - initiated research has declined over the past two decades . the blue bars ( left axis ) show the funds nigms committed to funding opportunity announcements ( foas ) targeted at specific areas of research in each fiscal year shown . the red line with triangles shows the change in the percentage of the nigms portfolio dedicated to investigator - initiated research . the analysis does not include fellowship , career development , and training awards ; programs transferred to nigms from the former national center for research resources ; and some other programs . a criticism of nigms ' renewed emphasis on investigator - initiated research has been that it will disadvantage team science and collaboration . but the dichotomy is not between investigator - initiated research and team science ; it is between investigator - initiated research and targeted or top - down research , in which funds are earmarked for specific scientific areas . team science aimed at studying fundamental biomedical problems can and usually should be investigator - initiated . interdisciplinary team science is undoubtedly extremely important and will become increasingly so as we delve deeper into the complexities of biological systems . one way to support team science is for independently funded principal investigators ( pis ) to form collaborations organically based on common interests and complementary skills , a method that , for fundamental research , seems likely to yield better results than collaboration borne of incentives such as targeted funding nih also has several mechanisms dedicated to supporting investigator - initiated team science , including multi - pi r01s and program project grants ( p01s ) . the centers of biomedical research excellence within the nigms institutional development award program also support team science , with a focus on developing the careers of junior investigators ( nigms , 2015 ) . moving forward , we plan to try to understand what kinds of teams benefit the most from unified grant mechanisms such as multi - pi r01s and p01s . press coverage , prizes , and renown all revolve around the concepts of discoveries and breakthroughs . eureka moments in which a paradigm shift occurs . and yet most major advances in science actually happen when a series of small steps coalesce into an important new understanding . for example , the discovery of restriction enzymes , a truly transformative advance that propelled biomedical research in the 1970s into the age of molecular biology and launched the biotechnology industry , began in the 1950s with the description of bacterial resistance to phage infection . dozens of papers published in various journals led to the insights that brought the nobel prize to warner arber , daniel nathans , and hamilton smith in 1978 ( loenen et al . , 2014 ) . discoveries arise from a complex web of knowledge , and without the network created by the steady progress of many researchers , they would not occur . as discussed earlier , it is impossible to know in advance where in this web the next big breakthroughs will arise or which strands of knowledge will be required to make them . the original observation of crispr sequences , for example , was made at the end of a 1987 journal of bacteriology paper that was otherwise devoted to reporting the sequence of the gene for the escherichia coli alkaline phosphatase isozyme converting protein iap ( ishino et al . describing the mysterious repeat sequences , the paper ends with the sentence , so far no sequence homologous to these has been found elsewhere in procaryotes , and the biological significance is not known . slowly , through years of careful characterization of the crispr pathway , our understanding of what this initial observation meant fueled the development of a novel technology that has dramatically improved our ability to replace genes in living cells , paving the way for advances in medicine and biotechnology . this and many other instances like it show how important it is for us to support as wide a web of research as possible . in 1985 , bruce alberts wrote a prescient commentary in which he laid out the inefficiencies created in the basic biomedical research enterprise when labs become too large ( alberts , 1985 ) . there are a number of reasons for these inefficiencies , but they mostly come down to bandwidth . for example , the more people a pi has in his or her lab , the less time he or she can devote to training and supervising each one . in addition , the bigger a lab gets , the more time the pi must spend on the grant - writing and administrative tasks needed to keep the operation running and the less time he or she has for actually doing research . for these reasons , alberts argued that the per capita output of a lab would generally diminish above a certain size and that nih and other agencies should cap pi funding , thereby limiting lab size and optimizing scientific productivity and quality . in 2010 , nigms conducted an analysis of the productivity and scientific impact of the research the institute funds as a function of each nigms investigator 's total direct - cost support from nih ( figure 2 ) . this study showed that , on average , these metrics increase only shallowly with funding above a moderate level ( $250,000300,000 ) and then actually decrease above $750,000 ( berg , 2010a , b ) . although a few investigators beat the averages and increase their productivity at a proportional or better rate as their funding increases , other well - funded investigators perform even worse than the averages . similar results have recently been published for chemical and biological research supported by other agencies ( fortin and currie , 2013 ; gallo et al . , 2014 ) and by the nih ( berg , 2015 ; danthi et al . , 2015 a 2010 analysis of researchers funded by nigms showed that , on average , productivity did not scale proportionally with funding beyond a relatively moderate direct - cost threshold . ( top panel ) average number of publications associated with nih grants of nigms - funded investigators as a function of their total nih direct costs ( red line with circles ) and the average impact factor of the journals in which each set of investigators published ( blue line with squares ) . ( bottom panel ) the unbinned data used to generate the averages shown in the top panel . jeremy berg , paul sheehy , and matt eblen ( nigms ) performed the data analysis . thinking about these data from the perspective of an investor of taxpayer funds , one can do a simple back - of - the - envelope calculation to determine what the best investment strategy for fundamental research should be . the first is from a talented established investigator and is for a new r01 , which would be his third , bringing him from $ 400,000 to $ 600,000 in total direct funding . in the first case , the additional $ 200,000 would buy taxpayers , on average , approximately one more paper during the funding period over what the established pi would have produced with $ 400,000 ( figure 2 ) . in the second case , the new pi would produce five papers on average in the funding period if she were awarded the grant . thus the choice seems obvious : taxpayers net four more papers by funding the new pi than by giving the established pi a third grant . , some biomedical research simply costs more money than the average , and this needs to be considered when grant budgets are set . and a difficulty here is that recent analyses have indicated that the peer - review process does not have sufficient resolution to accurately distinguish among the most promising applications , with at best modest correlations between score and productivity or impact for funded applications ( berg , 2011 , 2013 ; gallo et al . , 2014 ; so , for example , if the established pi 's application scored in the fifth percentile , and the new pi 's scored in the 15th percentile , could we really be confident that the former is likely to produce more important work than the latter ? along these same lines , as mentioned earlier , although we fund some pis who beat the average productivity versus funding curve , we also fund some who are below it , again indicating that picking the high performers is not easy , at least with the current process . these considerations suggest that we should be very selective in allowing pis to accumulate high funding levels and that , in general , funding more investigators at a moderate level rather than a few at a high level will yield the best payoffs . a question that at first glance may seem trivial but is , i believe , a significant one is whether our key metric for how we use the funds we invest in biomedical research should be the number of grants we award or the number of investigators we support . for example , we report to the scientific community and congress on the success rate for grant funding : how many grants we awarded in a year divided by the number of grant applications we received . in theory , focusing on grants tells us how many projects we are funding . however , because pis can have more than one nigms research grant , this focus distances our funding decisions from the key question of how many investigators are in our portfolio . if we instead used the number of pis we support as the key parameter to drive our funding and programmatic decisions , it would reduce the number of variables , allowing us to focus on the most important ones . for example , once we decided on the optimal number of nigms - supported investigators , we could then set a mean and median direct - cost target per investigator based on our total research budget . once we had established the total number of investigators who should be supported by the institute , we could determine how many new pis should enter and how many established pis should exit the system each year . we would also be better able to ensure the diversity and breadth of the institute 's research portfolio . overall this seemingly simple shift in how we view our mission could be a useful catalyst for reequilibrating the biomedical research enterprise to maximize its effectiveness , efficiency , and sustainability . promoting this shift is something we are working on at nigms , both as part of our new strategic plan and through a new funding mechanism , described in the following section . reequilibrating the biomedical research enterprise to make it more efficient and sustainable will require major changes in every part of the system . because we do not know a priori which changes will work best and because there is always a risk of unintended negative consequences , the soundest approach will be to experiment and to expand initiatives that succeed and abandon those that do not . this model requires us to define in advance the outcomes we hope to achieve and to collect the necessary data to measure them as the experiments progress . one such experiment is the nigms maximizing investigators ' research award ( mira ; preusch , 2015 ) . the mira program aims to transform how we support fundamental biomedical research , shifting away from the current paradigm of funding specific , predefined projects to one in which we focus on supporting the overall research program in each investigator 's lab . the goals of the mira pilot are to 1 ) increase funding stability for investigators to enhance their ability to take on ambitious research and approach problems creatively ; 2 ) increase flexibility for investigators to follow new directions as ideas and opportunities arise , which should help maximize the chances for breakthroughs ; 3 ) improve the distribution of funding , allowing the institute to support an optimally broad and diverse portfolio of investigators ; and 4 ) reduce the amount of time spent writing and reviewing grant applications , freeing up time to focus on research , training , and mentoring . each of these goals aims to help maximize the scientific returns on taxpayers ' investments . much discussion of the problems facing the biomedical research enterprise ( e.g. , ioannidis , 2011 ; bourne , 2013b , c ; alberts et al . , 2014 ) . among the many challenges , perhaps the most worrying is that junior scientists are becoming increasingly discouraged about their career prospects ( bourne , 2013a ; polka and krukenberg , 2014 ) , a growing crisis that could leave a serious deficit in the nation 's scientific capacity for years to come . but despite the many challenges we face , there are reasons to be optimistic about the future . scientifically , our deepening and expanding knowledge is opening up incredible new frontiers in research , and developments in technology are allowing us to address questions that a decade ago seemed completely inaccessible . although i have not discussed it here , renewed focus on improving scientific education , training , and career development seems likely to lead to an even more skilled , productive , and efficient workforce in the coming years . there is a growing recognition that we owe it to the nation and future generations to reoptimize the biomedical research enterprise ( lorsch , 2015 ) , and momentum is building in many sectors to make the changes this reoptimization will require . for example , the nih ( rockey , 2012 , 2015 ; maas , 2015 ) , members of congress ( harris and young , 2014 ; hearing on the fy 2016 national institutes of health budget request , 2015 ) , professional societies ( federation of the american societies for experimental biology , 2015 ) , and academic leaders ( daniels , 2015 ) are focusing attention on the challenges facing junior scientists and are trying to develop strategies to help this critically important group . if these trends continue , and all of the stakeholders take on their share of the responsibility , i am confident we can develop a more efficient and sustainable biomedical research enterprise and , in the process , accelerate advances in human health and prosperity .

the national institute of general medical sciences ( nigms ) at the u.s . national institutes of health has an annual budget of more than $ 2.3 billion . the institute uses these funds to support fundamental biomedical research and training at universities , medical schools , and other institutions across the country . my job as director of nigms is to work to maximize the scientific returns on the taxpayers ' investments . i describe how we are optimizing our investment strategies and funding mechanisms , and how , in the process , we hope to create a more efficient and sustainable biomedical research enterprise .

A diverse investment portfolio is a strong portfolio Emphasis on investigator-initiated research The value of team science Building the foundation for breakthroughs: the value of steady progress The optimal lab size Changing our funding metric A funding experiment: supporting research programs instead of specific projects Creating a bright future for biomedical research

the second generation polyamidoamine ( pamam ) dendrimer with amidoethanol surface groups and ethylenediamine core was purchased from sigma - aldrich ( poole , dorset , uk ) and used without further purification . the metal salts : agno3 , cucl22h2o , zncl2 , feso4 , and fecl3 were purchased from fisher scientific ( loughborough , uk ) , sigma - aldrich ( poole , dorset , uk ) , sigma chemical co. ( st . louis , mo ) , sigma chemical c. ( st . louis , mo ) , and bdh ltd . the pamam dendrimer was diluted to a concentration of 1 mm in methanol ( fisher scientific , loughborough , uk ) ; metal salts were dissolved in water ( j. t. baker , deventer , holland ) to a concentration of 1 mm . analytical solutions of pamam dendrimer and metal salts were prepared ( 1:1 ) in methanol : water ( 50:50 , vol / vol ) . a portion of this solution was diluted in methanol : water : acetic acid ( fisher scientific , loughborough , uk ) ( 49:49:2 , vol / vol ) to give a final concentration of 10 m pamam dendrimer and 10 m metal salt . all mass spectrometry experiments were performed on a thermo finnigan ltq ft mass spectrometer ( thermo fisher scientific , bremen , germany ) . samples were introduced to the mass spectrometer by an advion biosciences triversa nanomate electrospray source ( advion biosciences , ithaca , ny ) . xcalibur 2.0 software ( thermo fisher scientific ) was used for data acquisition and analysis . all mass spectra were acquired at a resolution of 100,000 at m / z 400 . automated gain control ( agc ) was used to accumulate sufficient precursor ions ( target value 1 10 ) . electrons were generated on the surface of an indirectly heated barium tungsten cylindrical dispenser cathode ( 5.1 mm diameter , situated 154 mm from the cell , 1 mm off - axis ) , ( heat wave labs , inc , watsonville , ca ) . the current across the electrode was 1.1 a. precursor ions were irradiated with electrons for 70 ms . all cid experiments were performed in the front - end linear ion trap and the fragments transferred to the icr cell for detection . cid experiments were performed with helium gas at normalized collision energy of 35% . each ecd and cid scan consisted of five co - added microscans . electrospray ionization of solutions containing equimolar concentrations of the second generation pamam dendrimer ( ethylenediamine core , 16 amidoethanol surface groups ; pamamg2oh ) and metal ions leads to the formation of multiply - charged metal - containing ions with the general formula [ pd + m + mh ] , where pd is the pamam dendrimer , m is the metal ion , n is the oxidation state of metal ion , and m is the number of protons . at higher concentrations of metal salt , complexes with the general formula [ pd + 2 m + mh ] and [ pd + 3 m + mh ] were observed , however the relative abundances of these complexes were much lower than those containing a single metal ion . the results presented here were obtained for [ pd + m + mh ] precursor ions , however similar fragmentation behavior ( both ecd and cid ) was observed for all charge states . ( for comparison , the ecd and cid mass spectra of [ pd + cu + 2h ] and [ pd + ag + 2h ] ions are shown in supplementary figures 14 , which can be found in the electronic version of this article ) . the notation system for representing fragmentation sites along the backbone of the pamam dendrimer is shown in scheme 1b . the system is based on that proposed by oh and coworkers , and follows the conventional nomenclature for peptide and protein fragment ions . assignments are given in the form gn(m ) , where subscript n refers to the generation in which fragmentation takes place and m denotes the type of fragmentation : ax , b / y , c / z . gn(in ) and gn(out ) refer to fragmentation that takes place core - side of the tertiary amines . l and j concern cleavages between carbon atoms in the amidoamine branches , and k refers to cleavage surface - side of the tertiary amines . fragment assignments were made on the basis of accurate mass measurement and verified by comparison of observed and expected isotope patterns . the dominant fragmentation channel for ecd of protonated pamam dendrimers is cleavage at the tertiary amines in the interior of the dendrimer . pronounced amide bond cleavage in the interior of the dendrimer is also observed . typically , the charge - reduced species [ m + nh ] constitutes a minor peak . the ecd ms / ms spectrum of [ pd + cu + 3h ] is shown in figure 1a and the fragments are detailed in supplementary table 1 . the results suggest that electron capture occurs either by the metal ion or by the dendrimer ligand . the base peak in this mass spectrum can be assigned to the charge - reduced complex , [ pd + cu + 3h ] , formed as a result of electron capture by the divalent copper ion . a series of peaks corresponding to fragment ions that arise from dissociation of this charge - reduced complex were also observed . for example , fragments formed as a result of cleavage within generation g0 of the dendrimer ligand , [ pd + cu g0(kout ) + h ] and [ pd + cu g0(x ) + h ] , were found . all of the cu - containing fragments derived from cleavages surface - side of the tertiary amines ( k cleavages , marked red on the mass spectrum ) or core - side of the amide ( a / x cleavage , marked blue on the mass spectrum ) . those cleavages are minor or non - existent in the ecd of protonated dendrimers . a series of fragment ions containing cu , e.g. , [ pd + cu g1(out ) ] and [ pd + cu g1(out ) + h ] , was also observed . the presence of cu - containing fragments resulting from cleavage surface - side of the tertiary amines ( k cleavages ) , e.g. , [ pd + cu g2(kout ) + h ] and [ pd + cu g2(kout ) ] , demonstrates the influence of the metal ion on fragmentation behavior : those cleavages are rare in the ecd of protonated pamam dendrimer ions . in addition , nonmetal - containing fragments resulting from cleavage at tertiary amine or amide bonds were observed . it is not possible to say whether these fragments are the result of electron capture by the metal or ligand , however , as they reflect the patterns observed for protonated species , we speculate they arise following electron capture by the ligand . the following fragments fall within this category : g1(out ) , g0(out ) , gcore(out ) , gcore(in ) , g1(y ) and g0(y ) . for comparison , the ecd mass spectrum of [ pd + cu + 2h ] is shown in supplemental figure 1 . cu - containing fragments deriving from cleavage surface - side of the tertiary amines ( k cleavage ) and core - side of the amide ( a / x cleavage ) , and cu - containing fragments deriving from k cleavage are observed . the ecd ms / ms spectrum of [ pd + fe + 2h ] complex is shown in figure 1b . intense peaks corresponding to the charge - reduced species formed following metal ion electron capture , and fragments thereof , are observed . the fe - containing fragments include [ pd + fe g0(x ) ] , [ pd + fe g0(kout ) ] , [ pd + fe g1(out ) ] , [ pd + fe g1(x ) ] , [ pd + fe g0(x ) + h ] , [ pd + fe g1(out ) + h ] , and [ pd + fe g1(x ) + h ] . no fe - containing fragments were observed , suggesting that the competition between electron capture by the metal ion and the ligand is weighted in the former 's favor . again , cleavages core - side of the amide ( a / x ) are prevalent ( marked blue on the mass spectrum ) . cleavage surface - side of the tertiary amine ( k ) was also observed ( marked red on the mass spectrum ) . unlike the cu complex , the ecd mass spectrum of the fe complex is dominated by fragments arising from cleavage at amide bonds : g0(y ) and g1(y ) , and at tertiary amines of dendrimer ligand : g0(out ) , g1(out ) , and g0(kout ) . it is not clear whether these fragments are the result of electron capture by the metal ion or the dendrimer ligand . the ecd ms / ms spectra ( see figure 2 ) for complexes of ag , zn , and fe with pamamg2oh dendrimer ligands are very similar . as seen for protonated pamamg2oh dendrimer ions , ecd of [ pd + ag + 4h ] , [ pd + zn + 3h ] , and [ pd + fe + 3h ] complexes are dominated by cleavage at the tertiary amines and amide bonds of pamamg2oh ligand . electron capture by the metal ions in the zn and fe complexes was not observed . a peak corresponding to [ pd + 4h ] was observed following ecd of [ pd + ag + 4h ] . that species must be the result of electron capture by ag . nevertheless , no dendrimer fragments containing ag(0 ) were observed . for comparison , the ecd mass spectrum of [ pd + ag + 2h ] is shown in supplemental figure 2 . peaks corresponding to fragments deriving from cleavage at tertiary amines and amide bonds in the inner generations dominate the spectrum . a peak corresponding to [ pd + 2h ] was also observed . the results suggest that the ecd behavior of these complexes depends strongly on the properties of the metal ions . these findings are supported by previous work , which showed that the nature of the metal cation dictates the ecd of peptide and lipid complexes [ 24 , 25 ] . in the present case , the electron can be captured either by the metal ion or by the pamamg2oh ligand . when the electron is captured by the pamamg2oh ligand , the ecd is due to protonation of the tertiary amines and the presence of amide bonds in the dendrimer backbone and can be explained on the basis of a charge solvation model [ 20 , 21 ] . when the electron is captured by the metal ( fe and cu ) , two main fragmentation channels are observed : a / x cleavage ( core - side of the amide ) and k cleavage ( surface side of the tertiary amine ) . on the basis of electron paramagnetic resonance ( epr ) experiments , tomalia and coworkers reported that in copper - dendrimer complexes the metal ion could be coordinated at various internal or external locations of the dendrimer . crooks and coworkers , however , suggested that dendrimers with amidoethanol surface groups form metal ion complexes with interior tertiary amines . a study of cu(ii ) complexes of a generation g0 pamam dendrimer by nolan et al . showed that for [ pd + cu + 4h ] , the metal was coordinated via the two core tertiary amines , and three amide oxygens ( two equatorial and one axial ) . the complex [ pd + cu + 2h ] was coordinated via the two core tertiary amines , one axial amide oxygen and two equatorial amide nitrogens . it is not clear which , if not both , coordination chemistries exist for the [ pd + cu + 3h ] complex studied here . as fe(iii ) also shows octahedral stereochemistry , it is reasonable to assume that [ pd + fe + 2h ] is also coordinated via the two core tertiary amines , one axial amide oxygen and either two amide nitrogens or two further amide oxygens . for both the fe and cu complexes , the pronounced a / x cleavage can be explained by coordination of the metal by amide nitrogens and/or oxygens . although the metal - containing fragments reveal a / x cleavage , complementary fragments are not observed . those fragments may arise from capture of the electron by the ligand ( see below ) or they may be the result of secondary cleavage of x ions via loss of co. in addition to the coordination chemistry described above , we hypothesize that for larger pamam dendrimers , the outer tertiary amines ( g0 , g1 , g2 ) may be involved in metal complexation , replacing the amide nitrogens / oxygens . no fragments were observed that contained the dendrimer core but not the metal ion , suggesting that the core is essential in coordination of the metal . in addition to the metal ion , the complexes studied also varied in the number of protons attached : the fe complex has two protons , the fe , zn , and cu complexes have three protons and the ag complex has four protons . we have observed previously that for the pamamg2oh dendrimer ecd behavior is independent of the number of protons attached . that finding was echoed by oh and coworkers in their work on the ecd of pamamg3oh . their results showed that dominant cleavage at the tertiary amines and production of y fragments was observed consistently over all charge states studied ( 4 + through 8 + ) . the results here suggest that ecd of metal complexes of pamamg2oh is independent of the number of the protons attached : ecd of [ pd + ag + 2h ] ( supplemental figure 2 ) reflects that of [ pd + ag + 4h ] and not that of [ pd + fe + 2h ] . the ecd of [ pd + cu + 2h ] ( supplemental figure 1 ) reflects that of [ pd + cu + 3h ] . the ecd of [ pd + cu + 3h ] ( figure 1a ) does not mirror that of [ pd + zn + 3h ] and [ pd + fe + 3h ] ( figure 2b and c ) . a study on the ecd of complexes of divalent metal ions and diacylglycerophosphocholine by o'hair and coworkers revealed that those metals with the highest second ionization energies stabilized the charge - reduced complexes via change in the oxidation state , whereas those metals with lowest second ionization energies promoted electron capture by ( and fragmentation of ) the ligand . we have found that , for those metals with 3d valence electrons , the ecd behavior of metal ion pamam complexes also tracks with the highest ionization energy ( ie ) : the fe ( ie3 30.6 ev ) and cu ( ie2 20.3 ev ) stabilize the charge - reduced complexes , via change in the oxidation state of the metal ion . ( in fact , no fe - containing fragments were observed ) . for metal ions with lower ionization energies , zn ( ie2 17.9 ev ) , and fe ( ie2 16.1 ev ) , formation of charge - reduced complexes and change in oxidation state of metal ion is not observed . instead , electron capture by , and fragmentation of , the pamamg2oh dendrimer ligand is promoted . for the ag complexes ( ie1 7.6 ev ; 4d valence electrons ) , the presence of [ pd + nh]species suggests that electron capture by the metal ion does occur . the cid mass spectrum of the [ pd + cu + 3h ] complex , shown in figure 3a , reveals four intense signals , which can be assigned to [ 1/2pd + cu ] , [ 1/2pd + cu + h ] , [ 1/2pd + 2h ] , and [ 1/2pd + 3h ] , respectively ( fragments are detailed in supplementary table 3 ) . these fragment ions are the result of cleavage between the two carbon atoms of the ethylenediamine core of the dendrimer ligand . other abundant fragments , including [ 1/2pd g2(lout ) + 3h ] , [ 1/2pd g0(z ) + h ] and [ 1/2pd g1(z ) + h ] also involve cleavage in the core of dendrimer ( marked red on the mass spectrum ) . ( for comparison , supplemental figure 3 shows the cid mass spectrum of [ pd + cu + 2h ] . similar fragmentation behavior was observed ) . that fragmentation behavior is particularly surprising considering the cid of protonated pamamg2oh dendrimers [ 19 , 21 ] , in which l and k cleavages , and combinations of the two cleavage types in various stoichiometries , from the outermost generation were the dominant processes . no core c c bond cleavage was observed in the cid of protonated pamam precursor ions . the results suggest that the cid of the [ pd + cu + 3h ] complex is linked with the coordination of the divalent copper ion to the pamamg2oh dendrimer ligand . as described above , it has been suggested that coordination of the cu ion involves the tertiary amines of the dendrimer core . that idea is supported by the fact that the cu - containing fragment [ pd + cu g0(kout ) + 2h ] , which arises through the loss of g0(kout ) from the innermost generation of the complex , is observed , but cu - containing fragments from the outer generations , e.g. , [ cu+ g2(kout ) + nh ] ) or [ cu + g1(kout ) + nh ] ) , are not . secondly , the abundance of fragments resulting from the cleavage of the core c c bond suggests that both core tertiary amines coordinate the metal ion , and as a result the c c bond is weakened . the cid ms / ms spectrum of [ pd + fe + 2h ] precursor ions is shown in figure 3b . the most abundant peaks correspond to [ 1/2pd + fe h ] , [ 1/2pd + fe ] , [ 1/2pd + 3h ] , and [ 1/2pd + 2h ] , respectively , and are formed as a result of the cleavage between carbon atoms in the ethylenediamine core ( marked red on the mass spectrum ) . the presence of metal - containing fragments such as [ pd + fe g2(lout ) + 2h ] suggest that the metal ion is coordinated to the interior of the dendrimer . it was postulated earlier that coordination of fe(iii ) also involves the core tertiary amines . we hypothesize that coordination of the fe ion weakens the core c c bond thus promoting its cleavage following vibrational excitation . the cid mass spectrum of the [ pd + ag + 4h]complex , shown in figure 4a , is dominated by peaks corresponding to fragments arising from l and k cleavages in the outer g1 and g2 generations ( i.e. , [ pd + ag g2(lout ) + 4h ] , [ pd + ag 2g2(lout ) + 4h ] , and [ pd g1(lout ) g1(kout ) + 4h ] ) . in addition , there are abundant peaks corresponding to [ gcore(out ) + 2h ] , [ gcore(out ) + ag+ h ] and g0(kout ) . most of the fragments observed for the [ pd + ag+ 4h]complex were also observed for the protonated [ pd + 6h ] ions in our earlier study . ( similar fragmentation was recorded following cid of [ pd + ag + 2h ] , see supplemental figure 4 ) . in all cases , similar fragmentation behavior has been reported for doubly charged [ pd + ag + h ] ions . thus , apparently , the cid of silver ion / pamamg2oh dendrimer complexes does not depend on the number of mobile protons . the presence of the silver - containing fragments [ gcore(out ) + ag + h ] and [ ag + g0(y ) ] , and absence of silver - containing fragments from generations g2 or g1 , suggest that the silver ion is bound close to the core of the dendrimer . however , cid of [ pd + ag + 4h ] does not result in any fragments deriving from cleavage of the core c c bond . according to our hypothesis that observation suggests that coordination of the ag ion does not involve both core tertiary amines . that idea is further corroborated by the presence in the mass spectrum of [ gcore(out ) + 2h ] and [ gcore(out ) + ag + h ] . in their study of ag(i ) complexes of a generation g0 pamam dendrimer , nolan et al the first species was [ pd + ag + 3h ] and the second was [ pd + 2ag ] . the structure proposed for the mononuclear complex involved trigonal planar coordination via the two core tertiary amines and an amide nitrogen ; and for the dinuclear complex , linear coordination of one of the core tertiary amines and a second , outer , tertiary amine , possibly accompanied by additional interaction of an amide oxygen . the cid results for [ pd + ag + 4h ] suggest that of the two possibilities , the latter coordination structure is more likely . the cid behavior of [ pd + zn+ 3h ] complex , shown in figure 4b , also reflects that observed for the protonated dendrimer . the majority of the fragments derive from l and k cleavages in generations g2 , g1 , and g0 . peaks corresponding to [ pd g0(kout ) + 3h ] , [ pd g0(kout ) g2(lout ) + 3h ] , [ g0(kout ) + zn ] , and the lack of zn - containing core fragments suggest that the zn is either not , or weakly , bound to the core of the dendrimer . exclusive loss of the zinc ion from the [ pd + zn + 3h]complex is not observed . fragments deriving from cleavage in generations g0 and g1 are observed both with and without the metal ion , e.g. , [ pd g1(lout ) g2(kout ) + h ] and [ pd + zn g1(lout ) g2(kout ) + h ] ; g0(kout ) and [ g0(kout ) + zn ] ; [ pd g0(kout ) + 3h ] and [ pd + zn g0(kout ) + h ] . the same is not true for fragments originating from cleavage in the outermost generation ( g2 ) : the fragments [ pd + zn g2(lout ) + 3h ] and [ pd + zn 2g2(lout ) + 3h ] are not accompanied by nonmetal - containing partners , suggesting the metal ion is not bound to g2 . similar overall cid behavior was observed for the [ pd + fe + 3h ] complex ( figure 4c , supplementary table 4 ) . the dominant pathways involved k and l cleavages in the outermost generation ( g2 ) : ( [ pd + fe g2(lout ) + 3h ] and [ pd + fe 2g2(lout ) + 3h ] are formed . fragments from g1 are observed both with and without fe , e.g. , [ pd g1(kout ) + 3h ] , and [ pd + fe g1(kout ) + h ] . it is not clear which is true in the present example , however the results suggest that the fe is at best weakly bound to only one of the core tertiary amines . note the marked difference between the cid of the fe complex and the fe complex ( figure 3b ) . we have investigated the electron capture dissociation ( ecd ) and collision - induced dissociation ( cid ) of complexes of the pamamg2oh dendrimer with different metal ions ( ag , cu , zn , fe , and fe ) . complementary information regarding the site and coordination of the metal ions can be obtained from the two techniques . for the cu complex and the fe complex , electron capture by the metal resulted in pronounced a / x cleavage suggesting that amide nitrogen atoms and/or oxygen atoms were involved in metal coordination . in addition , the pronounced k cleavage suggested that outer tertiary amines were also involved in metal coordination . the cid of these two complexes indicates that both the core tertiary amines are involved in metal coordination . the ecd of the zn and fe complexes mimicked that of protonated pamam dendrimer ions , i.e. , capture of the electron by the ligand was observed . fragments observed following ecd of the ag complexes were the result of electron capture by the ligand . for the fourth period metals ( 3d valence shells ) , the ecd behavior of the metal complexes tracked with the highest ionization energy : electron capture by the metal ion was observed for those with the highest ionization energy ( fe and cu ) and by the ligand for those with the lowest ( zn and fe ) . the cid behavior of the ag complex suggests that the metal is coordinated by one , but not both , of the core tertiary amines , while the cid of the zn and fe suggests that the metal is located in the interior of the dendrimer ( g0 , g1 ) but is not necessarily coordinated to the core . supplemental figure 1ecd ft - icr mass spectrum of [ pd + cu + 2h ] ions . k cleavages are marked in red ; a / x cleavages are marked in blue.supplemental figure 2ecd ft - icr mass spectrum of [ pd + ag + 2h ] ions . pd = pamam dendrimer.supplemental figure 3cid ft - icr mass spectrum of [ pd + cu + 2h ] ions . cleavages between carbon atoms in the ethylenediamine core are marked in red on the spectrum.supplemental figure 4cid ft - icr mass spectrum of [ pd + ag + 2h ] ions . pd = pamam dendrimer.supplementary table 1supplementary table 2supplementary table 3supplementary table 4 ecd ft - icr mass spectrum of [ pd + cu + 2h ] ions . k cleavages are marked in red ; a / x cleavages are marked in blue . ecd ft - icr mass spectrum of [ pd + ag + 2h ] ions . cid ft - icr mass spectrum of [ pd + cu + 2h ] ions . cleavages between carbon atoms in the ethylenediamine core are marked in red on the spectrum . cid ft - icr mass spectrum of [ pd + ag + 2h ] ions .

the electron capture dissociation ( ecd ) and collision - induced dissociation ( cid ) of complexes of polyamidoamine ( pamam ) dendrimers with metal ions ag+ , cu2 + , zn2 + , fe2 + , and fe3 + were determined by fourier transform ion cyclotron resonance mass spectrometry . complexes were of the form [ pd + m + mh]5 + where pd = generation two pamam dendrimer with amidoethanol surface groups , m = metal ion , m = 24 . complementary information regarding the site and coordination chemistry of the metal ions can be obtained from the two techniques . the results suggest that complexes of fe3 + and cu2 + are coordinated via both core tertiary amines , whereas coordination of ag+ involves a single core tertiary amine . the zn2 + and fe2 + complexes do not appear to involve coordination by the dendrimer core .

Experimental Results and Discussion Conclusions Supplementary data

the content of the paper is the responsibility of the authors alone . who may not necessarily agree . the content of the paper is the responsibility of the authors alone . who may not necessarily agree .

abstractend - user involvement in hiv guidelines development is often little , late or absent . other disciplines have long advocated handing over the stick ( i.e. power and control ) , as both ethical and strategic . women hiv activists have called this respectful engagement with , and learning from , communities miwa ( meaningful involvement of women living with hiv and aids ) .

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hepatocellular carcinoma ( hcc ) is one of the most common malignancies and has a high mortality rate worldwide . hcc usually develops in chronically damaged liver caused by an infection with the hepatitis b virus ( hbv ) or hepatitis c virus ( hcv ) , alcoholic abuse and nonalcoholic fatty liver disease . recent progress in diagnosis and new therapeutic approaches have improved the survival and prognosis of hcc . notably , antiviral therapy such as nucleoside analogs in hbv carriers and interferon administration in hcv carriers reduced the risk of hcc [ 3 , 4 ] . however , chronic infection with hbv is often associated with the development of hcc at a young age and in the absence of liver cirrhosis . patients with hcc in advanced clinical stages still have a dismal prognosis . not only tumor progression but also metastasis determines the prognosis of a wide range of malignancies including hcc . tumor infiltration of blood vessels at the primary tumor site is responsible for metastasis and causes the formation of metastatic lesions in distant organs . it has been believed that vascular invasion , which is one of the most significant features of hcc , contributes to intrahepatic or extrahepatic metastasis . extrahepatic metastases of hcc are frequently observed in distant organs such as the lungs , regional lymph nodes , kidneys and bone marrow . intracranial metastasis frequently develops in patients with advanced tumors , palliative therapy rather than aggressive treatment tends to be selected . therefore , both the benefits of treatment and the clinical course of intracranial metastasis of hcc remain to be elucidated . here , we describe a resected case of intracranial metastasis of hcc . surgical resection of the tumor resulted in a dramatic recovery of consciousness and enabled further therapeutic intervention against hcc . a 32-year - old man was referred to our hospital because of nausea and vomiting . he had been monitored for chronic liver disease caused by hbv infection 2 years before . as multiple hcc was detected in the hepatic right lobe 1 year prior , he underwent a right hepatic trisectionectomy . since his serum level of hbv - dna was more than 5 log copies / ml at that time , a nucleoside analog , entecavir , was administered . because of recurrence in the residual liver and lung metastasis detected 6 months after surgery , he was treated with an oral multikinase inhibitor , sorafenib . as adverse events such as liver dysfunction and eruption were observed 1 month after the administration of sorafenib , the dose was reduced from 800 to 200 mg / day . on admission , serum levels of hepatobiliary enzymes and markers for renal function such as blood urea nitrogen and creatinine were normal . although blood count tests showed slight anemia ( hemoglobin 11.2 g / dl ) and thrombocytopenia ( 12.6 10/l ) , his white blood cell count was normal ( 4,800/l ) . serological tests exhibited the following hbv markers : hepatitis b surface antigen ( hbsag ) positive , hepatitis b surface antibody ( hbsab ) negative , hepatitis b envelope antigen ( hbeag ) negative , hepatitis b envelope antibody ( hbeab ) positive , and hepatitis b core antibody ( hbcab ) positive . his serum level of hbv - dna measured by real - time polymerase chain reaction was 2.9 log copies / ml . anti - hcv antibody was negative . although the level of alpha - fetoprotein was elevated to 195,400 u / ml , that of pivka - ii was within the normal range ( 32 u / ml ) . of importance , the patient 's glasgow coma scale score was 11 points ( e3v4m4 ) , but no paresis was observed . however , the consciousness level gradually deteriorated and the glasgow coma scale score decreased to 6 points ( e2v2m2 ) on the second day of hospitalization . non - enhanced computed tomography ( ct ) revealed an intra - axial tumor with surrounding edema in the left frontal lobe ( fig . the tumor was found to be hyperdense on ct and hypointense on t2-star - weighted images of magnetic resonance imaging ( mri ) consistent with hemorrhage ( fig . a preoperative diagnosis of intracranial metastasis of hcc was made and a craniotomy was performed . macroscopic examination of resected specimens revealed that the tumor was grayish - white and accompanied by necrosis and hemorrhage ( fig . pathological examination revealed that it was a moderately differentiated hcc accompanied by necrosis and hemorrhage ( fig . follow - up ct conducted 1 week after the craniotomy revealed both successful removal of the tumor and improvement in the subfalcine herniation ( fig . contrast - enhanced ct showed not only multiple residual hccs with portal vein tumor thrombus ( fig . 3d ) . subsequently , an implanted port - catheter system was constructed and he was treated with hepatic arterial infusion chemotherapy with 5-fluorouracil and cisplatin . chronic infection with hbv is known to be a major risk factor for cirrhosis and hcc . lamivudine was the first nucleoside analog to be approved for the treatment of patients with chronic hbv infection , but drug resistance frequently develops during long - term lamivudine therapy . in contrast , treatment with entecavir , another nucleoside analog against hbv , rarely results in resistance . recent reports showed that both drugs reduced the risk of developing hbv - related hcc , although an unfavorable virological response to the treatment failed to decrease the incidence of hcc in patients with cirrhosis . in our case , the patient acquired hbv vertically and developed cirrhosis and hcc . when he started to take entecavir orally after right hepatic trisectionectomy , the hbv - dna decreased but did not disappear in the peripheral blood . extrahepatic metastases are detected in 1437% of hcc patients , although intracranial metastasis only accounts for a small percentage of extrahepatic metastases . since intracranial metastasis of hcc frequently causes serious complications such as intracranial hypertension , hemorrhage and cerebral edema , the prognosis of hcc is dismal . choi et al . reported that surgical resection and/or whole brain radiation therapy significantly prolonged survival compared with steroid treatment alone . it has been also documented that aggressive treatments such as resection and/or whole brain radiation therapy , single intracranial metastasis , and good liver reserve function serve as a prognostic factor . together , these findings indicate that sufficiently aggressive treatment may improve the prognosis for some patients with intracranial metastasis of hcc . in our case , there was only one metastatic lesion and the patient 's liver reserve function was good ( child - pugh a ) . successful resection of the intracranial tumor improved his level of consciousness dramatically and enabled further therapeutic approaches against hcc . together , it seems that surgical resection of intracranial metastasis of hcc contributed to the prolonged survival in this case . in conclusion , we report a rare case of intracranial metastasis of hcc resected successfully . considering that most extrahepatic metastases of hcc developed in patients with advanced intrahepatic lesions and vascular invasion , it is important to be aware of extrahepatic spread in patients with advanced hcc . in addition , it is necessary to determine whether aggressive treatment such as surgical resection improves the prognosis for intracranial metastasis of hcc . further examinations in large numbers of patients are necessary for both an understanding of the mechanisms underlying intracranial metastasis of hcc and the development of therapeutic approaches .

intracranial metastasis of hepatocellular carcinoma ( hcc ) is rare , but has an extremely poor prognosis . we report a case with successful surgical removal of intracranial metastasis of hcc . a 32-year - old man was admitted to our hospital with severe vomiting . he had been followed for liver cirrhosis due to hepatitis b virus infection and received a right hepatic trisectionectomy for hcc 1 year earlier . for the recurrence of hcc , sorafenib had been administered 6 months before admission . on admission , he exhibited consciousness disturbance , which gradually worsened . two days later , both computed tomography and magnetic resonance imaging revealed an intra - axial tumor with perifocal edema and hemorrhage in the left frontal lobe . the tumor was successfully removed by craniotomy and pathological examination revealed that it was composed of moderately differentiated hcc cells . the day after surgical resection of the tumor , his consciousness returned to normal . subsequently , he was treated with hepatic arterial infusion chemotherapy with 5-fluorouracil and cisplatin using an implanted port - catheter system . surgical resection of intracranial metastasis of hcc would be important and meaningful in some cases .

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nurses routinely encounter by many stressful working conditions . a well - defined stress factor for nursing caregiver this clinical condition ( agitation ) is also an early symptom of the acute withdrawal syndrome , especially following inappropriate use of naltrexone in addicted people , which may jeopardize health status of the addicts and also their close relatives . naltrexone is a well - known drug for the prevention of drug re - abuse in addicts , which competitively antagonizes opium euphoria due to its role in - and -receptor antagonism . the drug has a 10-h half - life and should be used 7 - 10 days after opium quit and 10 - 14 days after the last dose of methadone . severe withdrawal syndromes will appear if naltrexone is used accidentally or earlier than the above because of acute block of opioid receptor . agitation , delirium , yawning , severe muscular pain , tachycardia , diarrhea , and vomiting are common in this case , which may be much more dangerous in geriatric patients and also patients with underlying ischemic heart disease . in our setting , there are many admissions of addicts referred to the department of clinical toxicology just due to inappropriate use of naltrexone having severe agitation . as it is mentioned before , this agitation is so dangerous that the patient may hurt himself / herself or the health care professionals , and it can be managed by sedation . clonidine and propofol are believed to have a role in controlling these patients but using propofol needs icu admission and intubation . in addition , clonidine has a limited efficacy , which is not appropriate for acute and critical status of these patients . benzodiazepines are among the safest sedative drugs which their efficacy in similar situations are documented elsewhere . midazolam and diazepam are both available in parental dosage form , and both are used in acute - care settings in our department routinely . midazolam is a short - acting benzodiazepine with about 2 h half - life , which metabolizes in the liver and is used regularly for relieving preoperation anxiety and also agitation in critically ill patients . the onset of action of midazolam is about 2 min and is physically compatible with dextrose 5% and saline on administration . diazepam is a long - acting benzodiazepine with a 1 - 2 days half - life . one of the common causes of agitation in our department is misusing naltrexone in addicted patients . the routine medication in our department is diazepam bolus dose and infusion . although the different medications have been used for agitation induced by naltrexone in addicted patients , none of them had an acceptable efficacy on admission control of agitation . therefore this study was conducted to have a comparative evaluation of these two drugs for controlling agitation due to inappropriate use of naltrexone in addict patients . this double - blind randomized controlled clinical trial was carried out in 2005 in the department of clinical toxicology at noor general teaching hospital affiliated with isfahan university of medical sciences ( iran ) . eligible patients were naltrexone used agitated adult addicts who were not given or used any type of benzodiazepines in any dosage form ( oral , parenteral , or rectal ) , not on systematic central nervous system depressant drugs ( eg , tricyclic antidepressants , phenothiazines , buterophenones ) , without any known hypersensitivity to diazepam , midazolam , or any other component of their formulation . patients who required the endotracheal intubations were excluded . patients who were discharged from the ward with their own written consent were also excluded from the study due to ethical issues . families of eligible patients or their legally eligible companions were informed by one of the investigators about the whole study , and it was optional for the patients to take part in the investigation . so , all the included patients had a written informed consent form , which was signed without any hesitation after the interview . the consent form was in concordance with helsinki ii declaration and also tehran declaration for ethics in human researches . the study protocol was approved by isfahan university of medical sciences board of human studies . using ncss - pass ( jerry hintze , utah , 2004 ) the minimum patient number for at least 10% of difference in efficacy scoring of therapy ( = 0.05 ) and for a power of 80% , was calculated as 21 for each arm of the study ( diazepam and midazolam ) . assuming a dropout of about 20% of patients ( eg , if they met the exclusion criteria ) the total patient number needed for the study was at least 48 . in this study , 51 patients were included , from which 42 completed full course of study . patients were randomly allocated in 2 groups ( a and b ) , using the random number table after primary supportive measures . group a received an intravenous stat dose of 0.1 mg / kg diazepam and group b received 0.1 mg / kg stat dose of midazolam and 0.1 mg / kg / h of the drugs was infused in dextrose water 5% as the maintenance dose . patients were kept blinded using two types of coded vials , code 1 for midazolam vials and code 2 for diazepam vials . agitation was evaluated and scored by an attending physician who was unaware of the type of treatment groups . the patient s agitation score was measured using richmond agitation sedation scale . each patient was evaluated individually and was scored on admission at 30 , 60 , and 120 min . direct observation and judgment were needed to confirm and add up the results using the following definitions to describe the severity of agitation : mildly agitated ( score 1 - 3 ) , patient was quite calm and there was no need for any physical restriction ; moderate agitation ( score 4 - 6 ) , patient was more or less calm , muscular fasciculation was faded but he / she needed physical restriction ; severe agitation ( score 7 - 9 ) , patient was slightly sleepy after benzodiazepine administration with muscular fasciculation , and physical restriction was needed . finally , very severe agitation ( score 10 ) was observed when the patient was completely cooperative and had frequent involuntary organ movement and was firmly restricted to the bed . mann - whitney u test , student s t test , and t paired test were performed for statistical analysis using spss 11 statistical software . from 42 eligible patients ( 38 male and 4 female ) , 26 had taken naltrexone as 50 mg oral tablet and 3 patients were not sure about the naltrexone dosage form strength , but drug utilization was documented . a 72.8% of the patients had used opioid less than 10 h of admission and 27.3% of them 11 - 15 h before admission in hospital [ table 1 ] . intoxication and toxicologic characteristics of the study patients both groups had the agitation score of 10 on admission and 30 min after administrating benzodiazepines . comparing the mean agitation scores in midazolam and diazepam groups did not show a significant difference at 0 , 30 , and 60 min after medication administration . however , the difference was significant at 120 min ( p = 0.04 , table 2 ) . comparison of the mean agitation score in midazolam and diazepam groups the difference between the mean onset of action in the midazolam group ( at 67 min ) and diazepam group ( at 81 min ) was statistically significant ( p = 0.04 ) . correlation between the agitation score and naltrexone dose was not significant ( p = 0.020 ) . there was a negative and significant correlation ( r = - 0.09 p = 0.02 ) between agitation score and time interval from opioid used to taking naltrexone [ figure 1 ] . furthermore , there was a negative correlation between agitation score and time elapsed from naltrexone administration to admission . since many years , addict patients are considered as special population of ill people who need particular nursing care . nurse practitioners in the emergency wards all round the world are more or less familiar with the agitated addicts with withdrawal syndrome who need special nursing care . drug therapy is still considered as part of the treatment in these patients . to the best of our knowledge , this is the first comparative study on efficacy of midazolam and diazepam for controlling the agitation precipitated following naltrexone misuse . the results [ tables 1 and 2 ] showed that diazepam can not reduce agitation , and its mean onset of action is lower than midazolam , so midazolam acts more efficiently for controlling agitation , but on the base of our data and other studies , none of the drugs are suitable choices for controlling agitation because agitation management is important during first minutes that it is severe and patients may hurt himself / herself , companions , or medical staff . although propofol is a suitable alternative to benzodiazepines in severe acute withdrawal syndrome but has side effects , such as hypoventilation , causing atelectasis and respiratory arrest . our results indicate that , there is no relationship between naltrexone dose and the agitation score in the study patients . however , elongation of time elapsed between opioid usage and taking naltrexone , leads to a better response to treatment and lower agitation score , because of reduction of naltrexone half - life . based on our data [ tables 1 and 2 ] it is clear that severity of agitation depends on the amount of undetoxified opioid and it is in concordance with the results from other studies . only one patient responded to treatment very quickly , but it was confirmed that he had received a single dose of morphine in another clinic prior to the admission . it seems that that co - administration of benzodiazepines and a dose of morphine were effective in the management of agitation due to naltrexone inappropriate usage in that patient . a limitation of our study was the different number of males and females in the study sample . sex differences may influence the substance use disorders , response to opioid withdrawal , and rate of the response to the treatment or required dose of the drugs . also the type of the opioid that addicts had used was not mentioned in this study . opioids such as heroin and morphine have generally a short half - life , whereas methadone has a long one , and this issue can affect onset and severity of symptoms of withdrawal and also rate of the response to agitation treatment . further studies are needed to test the efficacy of combination therapy of benzodiazepines with opioid agonists , and also other sedative drugs , such as ultra - short - acting barbiturates ( eg , thiopental ) for controlling and management of agitation in these patients . regarding the increased reports of acute opioid withdrawal , which are precipitated by naltrexone , adequate patient counseling and family awareness seem to be necessary . in conclusion , the present study showed that despite the induction of sedation by midazolam and diazepam in their usage for the agitation due to inappropriate use of naltrexone , these agents are not drugs of choice in this issue . authors had no conflict of interest - including personal or financial relationships with organizations that might inappropriately influence , or be perceived to influence the work . this research was the result of a medical internship thesis project , which was supported financially by the vice chancellery for research and technology at the isfahan university of medical sciences .

background : agitation is an early symptom of the acute opioid withdrawal syndrome in addicts that may start by inappropriate use of naltrexone . the current drug interventions are not efficient or need critical care as well . this study compares the clinical role of midazolam and diazepam for the management of agitation due to inappropriate use of naltrexone.materials and methods : in this double - blind randomized controlled clinical trial , 44 agitated addicts , who did not use any type of benzodiazepine , not on systematic central nervous system depressant drugs , without any known hypersensitivity to diazepam , midazolam , or any other component of their formulation and had no evidence for the need of critical care , were enrolled . an i.v . stat dose of 0.1 mg / kg diazepam and 0.1 mg / kg stat dose of midazolam and a 0.1 mg / kg / h infusion of these drugs were administered for different groups of patients , respectively . agitation scores were recorded at 30 , 60 , 120 min after the start of drug administration using richmond agitation sedation scale score.results:a significant difference between the mean onset of agitation control in midazolam group ( at 67 min ) and diazepam group ( at 81 min ) was recorded . the difference of mean agitation score in the midazolam and diazepam group was only significant at 120 min . there was a negative correlation between agitation score and time elapsed from naltrexone administration to admission.conclusion:midazolam and diazepam may not be considered suitable and perfect pharmacologic agents for the initial controlling of agitation induced by naltrexone .

I M R D Conflict of interests Funding Sources

while the benefits of digital pathology in routine clinical use continue to inspire initiatives around the world , some european countries are arguably ahead of the curve . there are now a number of sites using digital pathology for primary diagnostic review , for example , in sweden , the netherlands , finland , lithuania , and denmark . the nordic symposium on digital pathology ( ndp ) was created in 2013 to promote exchange of state - of - the - art knowledge . the pioneering experiences in sweden have been a sounding board for the care providers moving in the same direction . in contrast to research use of digital techniques , ndp specifically targets the clinical adoption of whole - slide imaging ( wsi ) and other digital technologies . while there appears to be broad consensus on the opportunities that lie ahead , a foundational motivation for the symposium is that effective progress is dependent on tight collaboration between health care , industry , and academia . judging from the attendees evaluations from this and previous years , this type of venue with ample room for interactive discussions is highly appreciated and much needed . here , we provide a summary of the ndp 2016 , in the form of a meeting overview , results from the symposium 's integrated diagnostics workshop , speaker contributions , and finally brief conclusions . the 2016 ndp symposium took place on november 89 , 2016 , in linkping , sweden . a total of 170 attendees gathered , of which 47% listed health care as the primary affiliation , 35% industry , and 18% academia . pathologists constituted the lion share of health care representatives , with managers , laboratory technologists , and information technology ( it ) staff also being well - represented categories . the participants represented 13 different countries across europe , asia , and north america , with the nordic attendees being a large majority ( 88% ) . a large part of the program was devoted to invited talks and a workshop on integrated diagnostics . the contents of these sessions will be outlined in the sections below . in the science and innovation session , 12 posters were presented , with contributions from groups in linkping , bremen , tampere , uppsala , lund , and norrkping . the ndp also included an industrial exhibition with 12 vendors , showing a wide range of digital pathology products and services . figure 1 shows a snapshot from the symposium and the program details are available at the ndp website www.ndp2016.se . a central component of this year 's ndp was a workshop discussing collaboration between radiology and pathology and how digital tools relate to developments in this area . it is important to note that the survey respondents represent a biased selection among the pathology community . since only ndp participants were asked , this means that respondents are likely to be among the most positive to digital pathology and also among the most experienced . there is also strong geographical dominance from the nordics and , in particular , sweden . it is likely that the questions that require deep knowledge of clinical practice were dominated by pathologists as those questions to a substantial extent were skipped or received role distribution of survey respondents the survey first asked , today , to what degree do you use digital images of histology slides in your practice ? ( in percentage of all histology cases ) . the results are shown in figure 3 , for instance that a majority of respondents report substantial use of digital imaging in primary diagnostics ( another bias to note for these questions is that several people from the same institution may have responded ) . current use of digital pathology among survey respondents the main theme of the survey was integrated diagnostics , i.e. , the idea of closer collaborative work practices between pathology and radiology . such proposals have been voiced over the years and appear to be gaining traction in the respective communities , likely fueled by the increasing possibilities arising when pathologists also work in a primarily digital environment . to the end of forming a baseline , the survey asked pathologists , how often do you have direct interaction with a radiologist regarding a patient case ? figure 4 shows the results , painting the picture that multidisciplinary team meetings are where the interaction primarily happens but also that the direct interaction overall is low . one could argue that computer chat would be a very convenient and efficient way of interdisciplinary communication as it has proven to be within radiology settings . the low use may , however , merely be a sign that appropriate it tools are currently missing . interaction frequency between pathologist and radiologist , across different communication types the respondents were also asked to imagine well - crafted future collaboration support in the form of new it tools and workflows , and in that environment assess the potential : what impact will pathology - radiology collaboration have ? the impact within different areas is presented in figure 5 . overall , the ndp attendees were very positive about the potential , in particular , about the possibility for deeper discussion on diagnosis discordance . assessed impact of deepened pathology - radiology collaboration of different types , assuming well - crafted information technology tools and workflows are available . what impact will pathology - radiology collaboration have , in the area of the survey results were presented as a segway into a panel discussion . jeroen van der laak ( radboud university , nijmegen , the netherlands ) , and prof . some of the main points brought forward were as follows : the underlying technology is there , now we need to focus on integrating them into workflows and good user experiences ( prof . hamilton ) ; it is important to step out of the back office mindset and actively contribute to shaping the best possible care chain for the patient ( dr . devlin ) ; integrated diagnostics should also be seen in the broader perspective of regional / national integration ( prof . farstad ) ; it is vital to avoid misdirected diagnostic activities the diagnosticians should challenge any request they believe to be unnecessary ( dr . devlin ) ; an attractive concept is to have a matre d for diagnostics , a new role guiding diagnostic paths and providing effective single - point communication to referrers ( prof . the overall conclusion from the panel and the discussion with the ndp attendees is that closer collaboration between pathology and radiology is a positive and important development track going forward . levenson presented the latest achievements with slide - free microscopy technology based on ultraviolet ( uv ) excitation . the technology , dubbed microscopy with uv surface excitation , allows direct depiction of unsectioned specimens at full microscopic resolution . levenson demonstrated many case examples also including image features not represented in traditional staining and microscopy , including three - dimensional tissue information , features whose diagnostic value is now being explored . along another vein , levenson shared insights on using trained pigeons as unbiased proxies for human visual pattern recognition in pathology , work which was both humorous and thought - provoking . the second keynote speaker , associate prof . jeroen van der laak , radboud university , provided ample insight into state of the art in image analysis for wsi . among other topics , van der laak shared result details from the recent grand challenge , called camelyon16 , in lymph node metastasis detection . the research challenge encompassed a whole - slide task , in this sense closer to the clinical reality than previous challenges , and leading results demonstrated levels of accuracy in line with human assessment . of interest , however , was that machines were being compared with the ultimate human effort ( i.e. , the painstaking detailed microscopic review of every part of the lymph node , something not always feasible in clinical practice ) . in tandem with those promising results , van der laak emphasized the demanding efforts still needed to bring the algorithms into the everyday clinical context . johan hartman , karolinska institutet , presented work concluding that computational analysis outperformed manual assessment for breast cancer subtyping . for example , the image analysis algorithm showed higher sensitivity and specificity for the luminal b subtype , being considered a very challenging distinction . arvydas laurinavicius , vilnius university , spoke to the distinct prognostic value of bimodality of ki67 expression - an analysis requiring computational methods . instead of restricting proliferation measures to a hotspot , laurinavicius showed results where heterogeneity across the slide proved to be a more powerful survival indicator in breast cancer . he also shared his group 's experiences on running routine primary review using telepathology , with several colleagues reporting routine clinical work remotely . peter van ooijen , university medical center groningen , described the challenges an informatician will meet , ranging from overarching strategic efforts to mundane pragmatic problem - solving . a key takeaway was the importance of adopting and enforcing standards when building an effective and robust digital system . devlin , altnagelvin hospital , presented the journey to a fully digitized national radiology system in northern ireland . apart from advice on handling technology and workflow change , devlin emphasized the need to establish direct support from stakeholders in top leadership layers . bethany williams , leeds university , contributed to the ongoing efforts in wsi validation by presenting a systematic analysis of the type of cases where discordance with microscopic review has occurred and what can be learned from them . among the most challenging areas were diagnosis of dysplasia and finding small objects ( e.g. , micrometastases ) . tieto sweden had invited theo papaioannou from the vstra gtaland region , sweden , to present their ongoing large - scale digitization project . from philips , dr . hamilton presented computational imaging solutions and insights on remaining challenges for digital pathology in the context of workforce shortages and the need to collaborate . jesper molin from sectra presented key takeaways from his phd thesis , devoted to new opportunities for pathologist interaction in digital image review . finally , dr . per hertz from eizo nordic discussed the importance of high and controlled display performance in the context of diagnostic quality . finally , a seminar on implementing digital microscopy in medical education was hosted by clinicum , linkping university . the topics covered have evolved from entry - level concerns about how to scan slides properly , or whether complete digitization is possible , to mature discussions on how to design systems and workflows to accomplish quality and productivity gains gains that are undisputed . as in previous years , the attendees expressed the great value of this experience sharing across organizations , disciplines , and sectors . darren treanor is a member of the aperio / leica advisory board and the sectra advisory board . darren treanor is a member of the aperio / leica advisory board and the sectra advisory board .

the nordic symposium on digital pathology ( ndp ) was created to promote knowledge exchange across stakeholders in health care , industry , and academia . in 2016 , the 4th ndp installment took place in linkping , sweden , promoting development and collaboration in digital pathology for the benefit of routine care advances . this article summarizes the symposium , gathering 170 attendees from 13 countries . this summary also contains results from a survey on integrated diagnostics aspects , in particular radiology - pathology collaboration .

I M W S C Financial support and sponsorship Conflicts of interest

after ilizarov first reported successful distraction osteogenesis of the long bone , there was a report by mccarthy et al . ( 1 ) who were the first to perform a distraction osteogenesis of the human craniofacial mandible bone . thereafter numerous studies reported on craniofacial bone expansion . in order to successfully perform distraction osteogenesis in a craniofacial bone , both external and internal devices developed . ( 2 ) , who managed infant patients with craniofacial distraction by the direct application of helmets , which later gradually progressed to distraction techniques ( 3 ) . advantages of the external type device are that the direction of distraction can be determined 3 dimensionally even after surgery , and that it is easy to remove ( 4 ) . drawbacks of this method include the difficulty in maintaining the stability of the osteotomized segment , difficulty in applying it to a patient of less than 2 yr of age who has a thin calvarium , difficulty in activating , and the most problematic of all is the inability to maintain this device for a sustained period of time due to the bizarre morphological appearance of the patient ( 5 ) . the internal device was first utilized as a custom - made distractor since its first introduction in the mid-1990s , which was easier to apply as a modular type ( 6 , 7 ) . recently , a biodegradable or a device with a bendable plate has been introduced ( 8) . disadvantages of this device are the difficulty in determining the distraction direction , the inability to change the direction after surgical application , and the necessity of a secondary surgery for removal of the device ( 5 , 9 ) . to solve the problem of the lack of intracranial and intraorbital space during craniofacial dysostosis , fronto - orbital monoblock advancement with lefort iii osteotomy has been developed to simultaneously expand the frontal skull and mid - face bones . the combination of this technique with distraction osteogenesis further enhances the efficacy of the procedure . in our opinion , the application of either an external or internal device will both afford favorable results when combined with fronto - orbital monoblock advancement with lefort iii osteotomy , and although the internal device method is technically more difficult to employ , it may be applied for a longer period of time , and therefore seems to be advantageous in terms of osteogenesis and bone fusion . in korea , numerous reports have described the utilization of external devices for management of craniofacial dysostosis to date , but studies on the application of internal devices are not yet available ( 10 ) . we herein report the first case of a 7-yr - old male patient with pfeiffer syndrome in whom craniofacial monoblock distraction was successfully performed with an internal device . a 7-yr - old boy presented to our surgical center with a broad big toe and thumb , bulging skull on the anterior fontanelle , underdevelopment of the frontal bone , exophthalmos , small face , tongue protrusion , malocclusion with midfacial retrusion , mild respiratory difficulty due to minor upper airway obstruction , and developmental delay . physical examination showed that his head circumference was 55 cm ( more than 97th percentile ) in spite of the low body weight ( 10th percentile ) , and short height ( 3th percentile ) . he also demonstrated a low intelligence quotient ( korea wechsler preschool primary scale of intelligence ) of 70 , and anthrophobia with passive characteristics . we planned monoblock distraction osteogenesis to decrease the intracranial and intraorbital pressures , and to increase the nasal and pharyngeal airway spaces . using a bicoronal incision , a standard fronto - orbital osteotomy was performed . after separating the frontal bone flap , a forceful forward protrusion of the frontal lobe was noticed suggesting high intracranial pressure from the craniosynostosis . thereafter , the orbital bandeau was also separated with a pneumatic saw , and a lefort iii - osteotomy was performed . the osteotomy line ran across the zygomatic arch , from the sphenozygomatic suture along the floor of the orbit , behind the nasolacrimal duct , along the lacrimomaxillary suture to the nasal bone . the nasal septum was separated from the perpendicular plate of the ethmoid bone with a straight osteotome . the pterygoid plates were separated with a chisel placed into the pterygomaxillary fissure . the viscerocranium was mobilized . downward rotation of the facial skeleton was also possible when using rowe 's forceps for disjunction of the mid - face from the cranial base . the frontal bone flap and orbital bandeau were fixed into a fronto - maxillary monoblock bone flap with three micro - plates , and two modular midfacial distractors were fixed posteriorly to the temporal bone and anteriorly to the malar eminence of the zygomatic arch . bilateral activation cables were brought through a stab wound posterior to the coronal incision and anterior to the ear . the coronal incision was closed with continuous monofilament sutures subcutaneously , and staples in the skin . the total operating time was 380 min , and 740 ml of packed red cells was infused . the patient was kept on a mechanical ventilator for 10 hr , decannulated on the next morning after surgery , and stayed 1 day in the intensive care unit . postoperatively , a distance of approximately 1 cm appeared between the skull flap and the parietal skull resulting in increased intracranial volume , which was almost completely filled by expansion of the brain . we distracted the fronto - maxillary monoblock bone flap 1.0 mm everyday for 20 days beginning 1 week after the operation . the distractors were kept for 3 months of the consolidation phase to a final 20 mm displacement . follow - up 3-dimensional computed tomogram ( ct ) scans at 2 and 4 months after surgery showed increased intracranial space and sufficient expansion of the underlying brain with only a small free epidural space , and the patient also showed stable forward advancement of the fronto - orbito - maxillary faciocranial monoblock , resulting in improved bilateral eye and mouth closing and occlusion motions ( fig . 2 , 3 ) . we removed the two distraction bars 4 months after the initial establishment of the distraction system . at 12 months after surgery , the follow - up 3-dimensional ct scans showed proper ossification in the distracted area ( fig . the patient showed good cranial contours aesthetically , with upward elevation and forward displacement of the preoperatively depressed fronto - facial contour . he also showed a slightly increased intellectual quotient of 80 , and his character changed from passive to active , resulting in improved learning and rehabilitation exercise without any unexpected postoperative complications . surgical management by lefort iii osteotomy for children with craniofacial dysostosis syndrome was first reported by gillies in 1950 , after which this procedure was firmly established as the primary mode of treatment by tessier ( 11 ) . ( 12 ) developed the monoblock advancement with lefort iii osteotomy method whereby the fronto - orbital segment and the midface counterpart are advanced simultaneously . in the 1990s , this technique further advanced to distraction osteogenesis ( 13 , 14 ) . advantages of distraction osteogenesis are that distraction is less invasive since it is performed without a bone graft and therefore less blood loss occurs , the operative time is shorter , bone union is superior leading to longer bone formation or growth , the induced bone growth shows good bone fusion that is comparable to previous methods in terms of bone thickness and strength , and finally , favorable skin and subcutaneous soft tissue expansion allows repeat surgery ( 15 ) . the first operation for treatment of craniofacial dysostosis syndrome is usually necessary within months or within one year of birth , and additional surgery may be required at 2 - 3 yr of age . the age 5 - 6 yr , at which adenoid infections most commonly occurs and develop to further narrow the passageway leading to obstructive sleep apnea syndrome , is considered to be the optimum age for timing of the primary surgery of the mid - face ( 16 , 17 ) . additional craniofacial surgery is performed at 15 yr of age according to the status of the skull and facial bones . as our patient did not receive craniosynostosis surgery during the infancy , the increased intracranial pressure resulted in the horn - like central bony elevation of the anterior fontanelle , and the maxilla was underdeveloped leading to eyeball protrusion , malocclusion , and respiratory difficulty . therefore , the skull and maxilla were simultaneously expanded with distraction . in cases where the cranium and midface needs to be advanced simultaneously , it is advantageous to perform the monoblock advancement in which the midfacial segment and the fronto - orbital segment are advanced together , thus reducing the number of procedures to a single procedure . however , there exists a major drawback such as possible severe and fatal infection of the opened nasal cavity and frontal skull base ( 18 ) . in a recent study , it was suggested that since advancement for distraction osteogenesis is performed after nasal mucosal healing , opening the nasal cavity and frontal skull base would significantly decrease the possibility of a severe infection ( 19 ) . common and difficult problems when applying an internal device is the determination of the direction of the device and the inability to change the direction once it has been applied ( 5 , 9 ) . exceptional advancement employing an internal device during craniofacial monoblock distraction with lefort iii osteotomy frequently results in the soft tissue tension casing caudal rotation of the midfacial segment , which in turn displaces the zygomaticomaxillary complex more anteriorly than the dentoalveolus ( 20 ) . furthermore , lateral advancement forces leads to midline advancement , resulting in possible fractures of the zygomaticomaxillary junction , and worsening of the facial concavity ( 20 ) . to overcome these problems , authors expect that the zygomaticomaxillary complex balance with internal device distraction will prevent rotation , and also that sufficient bone separation during surgery will markedly decrease any lateral distraction of the zygomaticomaxillary complex . despite such inherent limitations of the internal device , the reason for this is that the external type is cosmetically unfavorable and uncomfortable for long - term application ( 21 , 22 ) . for bone formation induction and consolidation to take place after distraction osteogenesis , the distraction device needs to be in place for at least 1 - 3 months , which is very difficult with a poor patient 's compliance . hence , a shorter consolidation period ensues and the bone fusion rate decreases , and subsequently there is an increased chance of relapse . a review of previous reports showed that during the postoperative period , a latency period was applied for 0 - 7 days , the activation rate was 0.5 - 1.5 mm / day , and the activation period was 14 - 30 days ( 5 , 7 , 14 - 16 , 22 - 26 ) . also , the total distance of advancement has been reported to be 14 - 30 mm for cranial distraction , and 8 - 23 mm for facial distraction . the recommended consolidation period ranges from 3 weeks to 6 months ( 5 , 7 , 14 - 16 , 19 , 23 - 26 ) . as yet , there have been no definite conclusive statistical data in the literature that suggest which method is superior to the others , but it is generally recommended that activation and follow - up radiological studies such as 3-dimensional ct be conducted to assess bone fusion during the consolidation phase .

a 7-yr - old boy visited our surgical center with pfeiffer syndrome type 1 , presenting with macrocrania , broad big toe and thumb , exophthalmos , tongue protrusion , malocclusion with midfacial retrusion , mild respiratory difficulty due to minor upper airway obstruction , and developmental delay . he also exhibited anthrophobia with a passive character . the patient was treated with internal monoblock distraction osteogenesis to increase the intracranial and intraorbital volumes , and the nasal and pharyngeal airway spaces using two modular mid - facial internal distractors . for distraction , the latency period was 1 week , the daily activation of 1.0 mm was 20 days ( total advancement 20 mm at the midline ) , and the consolidation period was 3 months . the follow - up computed tomography 12 months after surgery showed expansion of the brain and proper ossification in the distracted area . the patient also showed aesthetically good cranial contours , improved tongue and eyeball protrusion , no respiratory difficulty , and improved learning . we suggest that the internal distraction may last longer than an external type , resulting in a better bone fusion rate and successful expansion of craniofacial bones .

INTRODUCTION CASE REPORT DISCUSSION

aminoglycoside - induced proteinuria may result from general renal damage or may reflect alterations in specific steps in the renal handling of proteins . to differentiate between the two possibilities , experiments were designed to quantify the effects of nephrotoxic doses of gentamicin , tobramycin and netilmicin in the intact rat , isolated perfused rat kidney ( ipk ) and kidney slices using the cationic low molecular weight protein lysozyme as a model protein . each aminoglycoside was administered ip to male wistar rats ( 15 or 30 mg / kg / day ) for 5 or 7 days . scanning and transmission electron microscopy indicated that gentamicin and tobramycin induced a decrease in the number and diameter of endothelial fenestrae and degranulation of the myoepithelioid cells of the juxtaglomerular apparatus . concurrently , gentamicin and tobramycin decrease the glomerular sieving coefficient of lysozyme from 0.8 to 0.6 and 0.5 , respectively . netilmicin did not affect the percentage reabsorption of lysozyme whereas gentamicin and tobramycin induced a 50% decrease in lysozyme reabsorption by the ipk . gentamicin and tobramycin decreased equally lysozyme degradation by the ipk ; this decrease was time- and dose - dependent when evaluated in slices from renal cortex . perfusion of rat kidneys with gentamicin induced a dose - dependent decrease in reabsorption and catabolism of lysozyme . in conclusion , these studies demonstrate that polycationic aminoglycosides alter ultrastructure and glomerular permeability , tubular reabsorption and intracellular digestion of proteins.imagesfigure 2.figure 3 . ( a)figure 3 . ( b )

Images

myasthenia gravis ( mg ) is more commonly identified in younger individuals , especially women , and is usually associated with thymus disease . in these patients , however , elderly - onset mg has been also reported among patients 65 years of age.14 thymus abnormalities are usually rare among patients with late - onset mg , aged 50 and above , and clinical presentation is also different from those with early - onset disease , aged less than 50 years old.5 differential diagnosis in elderly patients must consider cerebral stroke , motor neuron disease , and parkinson s disease as the majority present with bulbar symptoms . the treatment options for elderly patients will also differ from those with late- and early - onset mg . we summarize the cases of patients with early- , late- , and elderly - onset mg attending our clinic and detail three cases of elderly - onset mg that occurred in patients at a particularly advanced age ( 80 years of age ) . we report three patients with mg who had disease onset at an age of 80 years ( elderly onset at advanced age ) , whose characteristics are summarized in table 1 . ptosis developed at 80 years of age and she received bilateral eyelid lift surgery ( blepharoplasty ) following a diagnosis of age - related ptosis . however , she visited our hospital after feeling a worsening of weakness and fatigue in all limbs . ptosis , diplopia , and grade 4 proximal weakness in all limbs were noted ; gait was possible using a walker ; and myasthenia gravis foundation of america ( mgfa ) classification was iia . prednisolone ( psl ) dose was gradually increased , starting from 5 mg / day to a maximum of 20 mg / day by 5 mg / week gradually , and tacrolimus 3 mg / day was administered together with initial administration of psl . the tacrolimus dose was increased to 6 mg / day because of low plasma concentrations . repetitive nerve stimulation performed after the treatment revealed no decrementing response in abductor digiti minimi and upper trapezius . tuberculous pleurisy developed 2 years after treatment initiation and the tacrolimus dose was reduced to 2 mg / day . the tuberculous pleurisy improved after administration of anti - tuberculous medication using rifampicin , isoniazid , and pyrazinamide for about 1 year . osteoporosis - associated lumbar compression fracture occurred 3 years after treatment initiation and the patient temporarily became bedridden but recovered to a condition allowing housework . at 7 years after onset ( 6 years after treatment initiation ) , the anti - achr antibody level was lower than the detection limit under oral treatment with psl 5 mg / day and tacrolimus 1 mg / day , and there were minimal clinical signs . the patient is independent with respect to activities of daily living ( adl ) and she has not been hospitalized for recurrent mg . the patient is a japanese female currently 85 years of age , who had previously been healthy until developing a swallowing disorder at 80 years of age . treatment for cerebral infarction was administered by a physician but no improvement was achieved . as her weight loss muscle weakness in all limbs and easy fatigability were observed , which waned on repetitive stimulation testing . two weeks later , the patient was admitted as bulbar palsy symptoms worsened after the development of pneumonia . thymectomy ( resection of the thymus , pericardial membrane , and thyroid ) was performed as no major abnormality was noted with respect to cardiac or renal function , even though she was elderly . radiotherapy ( total dose 50 gy ) was applied to the region around the thymus after surgery . psl dose was gradually increased ( 5 mg / week ) to a maximum of 35 mg / day and then gradually decreased to 5 mg / day . tacrolimus 3 mg / day was administered concomitantly after surgery when the patient was receiving 20-mg / day dose of psl . nmol / l under treatment with psl 5 mg / day and tacrolimus 3 mg / day . the patient lives alone , works on a farm , and provides meals for her son living next door , as she had prior to the development of mg . the patient is a japanese female currently 87 years of age , who had previously been healthy . the patient was diagnosed with cardiac asthma and heart failure and was admitted to the cardiovascular department . as her anti - achr antibody was found to be positive ( 47.7 nmol / l ) , she was referred to our department . ptosis and distal dominant weakness in the muscles of all four limbs were present , tendon reflex was normal , and a swallowing disorder was noted . a maximum psl dose of 35 mg / day was administered and it was gradually reduced . when the psl dose was reduced to 30 mg the psl dose was eventually reduced to 5 mg / day and no recurrence of mg has occurred . adverse events were compression fracture due to a fall , cataract surgery , and the development of borderline diabetes . we also assessed different - onset of mg in 43 cases between 2002 and 2013 ( table 2 ) . the patients with early - onset mg ( 13 female , 6 male ) were in mgfa class i iiia , had an mg - adl score < 8 , and had a positive anti - achr antibody response except one patient . the antibody titer of 0.4 nmol / l and above is set as positive result in our hospital . all the cases started with diplopia and blepharoptosis , and two cases later developed dysphagia and respiratory distress , respectively . more than half of the 19 cases were treated with psl , and anti - cholinesterase inhibitor , and in addition to these medicines , 5 of the 19 cases were also treated with tacrolimus . among the cases with late - onset mg ( nine female , two male ) , three female patients ( mgfa class iia - iiib , mg - adl score 16 ) presented with generalized muscle weakness and received thymectomy along with intravenous immunoglobulin , psl , and tacrolimus . the other cases ( mgfa class i iii , mg - adl score 115 ) initially complained of diplopia and blepharoptosis and were treated with plasmapheresis , methyl psl 1000 mg / day for 3 days ( pulse - steroid therapy ) , oral psl , and a cholinesterase inhibitor without undergoing thymectomy . of the 13 cases with elderly - onset mg ( 6 female , 7 male ; mgfa class i v ; mg - adl score 224 ) , most initially reported dysphagia , respiratory distress , and/or generalized muscle weakness . one case was seronegative for anti - achr , muscle - specific kinase , and lipoprotein - related protein 4 antibodies and received treatment with plasmapheresis , pulse - steroid therapy , psl , and tacrolimus . the others were all anti - achr antibody positive and were treated with pulse - steroid therapy , psl , and tacrolimus . in this group , only 6 of the 13 patients ( 46.2% ) with a positive anti - achr antibody response underwent thymectomy . ptosis developed at 80 years of age and she received bilateral eyelid lift surgery ( blepharoplasty ) following a diagnosis of age - related ptosis . however , she visited our hospital after feeling a worsening of weakness and fatigue in all limbs . ptosis , diplopia , and grade 4 proximal weakness in all limbs were noted ; gait was possible using a walker ; and myasthenia gravis foundation of america ( mgfa ) classification was iia . prednisolone ( psl ) dose was gradually increased , starting from 5 mg / day to a maximum of 20 mg / day by 5 mg / week gradually , and tacrolimus 3 mg / day was administered together with initial administration of psl . the tacrolimus dose was increased to 6 mg / day because of low plasma concentrations . repetitive nerve stimulation performed after the treatment revealed no decrementing response in abductor digiti minimi and upper trapezius . tuberculous pleurisy developed 2 years after treatment initiation and the tacrolimus dose was reduced to 2 mg / day . the tuberculous pleurisy improved after administration of anti - tuberculous medication using rifampicin , isoniazid , and pyrazinamide for about 1 year . osteoporosis - associated lumbar compression fracture occurred 3 years after treatment initiation and the patient temporarily became bedridden but recovered to a condition allowing housework . at 7 years after onset ( 6 years after treatment initiation ) , the anti - achr antibody level was lower than the detection limit under oral treatment with psl 5 mg / day and tacrolimus 1 mg / day , and there were minimal clinical signs . the patient is independent with respect to activities of daily living ( adl ) and she has not been hospitalized for recurrent mg . the patient is a japanese female currently 85 years of age , who had previously been healthy until developing a swallowing disorder at 80 years of age . treatment for cerebral infarction was administered by a physician but no improvement was achieved . as her weight loss muscle weakness in all limbs and easy fatigability were observed , which waned on repetitive stimulation testing . two weeks later , the patient was admitted as bulbar palsy symptoms worsened after the development of pneumonia . thymectomy ( resection of the thymus , pericardial membrane , and thyroid ) was performed as no major abnormality was noted with respect to cardiac or renal function , even though she was elderly . radiotherapy ( total dose 50 gy ) was applied to the region around the thymus after surgery . psl dose was gradually increased ( 5 mg / week ) to a maximum of 35 mg / day and then gradually decreased to 5 mg / day . tacrolimus 3 mg / day was administered concomitantly after surgery when the patient was receiving 20-mg / day dose of psl . nmol / l under treatment with psl 5 mg / day and tacrolimus 3 mg / day . the patient lives alone , works on a farm , and provides meals for her son living next door , as she had prior to the development of mg . the patient is a japanese female currently 87 years of age , who had previously been healthy the patient was diagnosed with cardiac asthma and heart failure and was admitted to the cardiovascular department . as her anti - achr antibody was found to be positive ( 47.7 nmol / l ) , she was referred to our department . ptosis and distal dominant weakness in the muscles of all four limbs were present , tendon reflex was normal , and a swallowing disorder was noted . a maximum psl dose of 35 mg / day was administered and it was gradually reduced . when the psl dose was reduced to 30 mg / day , tacrolimus the psl dose was eventually reduced to 5 mg / day and no recurrence of mg has occurred . adverse events were compression fracture due to a fall , cataract surgery , and the development of borderline diabetes . we also assessed different - onset of mg in 43 cases between 2002 and 2013 ( table 2 ) . the patients with early - onset mg ( 13 female , 6 male ) were in mgfa class i iiia , had an mg - adl score < 8 , and had a positive anti - achr antibody response except one patient . the antibody titer of 0.4 nmol / l and above is set as positive result in our hospital . all the cases started with diplopia and blepharoptosis , and two cases later developed dysphagia and respiratory distress , respectively . more than half of the 19 cases were treated with psl , and anti - cholinesterase inhibitor , and in addition to these medicines , 5 of the 19 cases were also treated with tacrolimus . among the cases with late - onset mg ( nine female , two male ) , three female patients ( mgfa class iia - iiib , mg - adl score 16 ) presented with generalized muscle weakness and received thymectomy along with intravenous immunoglobulin , psl , and tacrolimus . the other cases ( mgfa class i iii , mg - adl score 115 ) initially complained of diplopia and blepharoptosis and were treated with plasmapheresis , methyl psl 1000 mg / day for 3 days ( pulse - steroid therapy ) , oral psl , and a cholinesterase inhibitor without undergoing thymectomy . of the 13 cases with elderly - onset mg ( 6 female , 7 male ; mgfa class i v ; mg - adl score 224 ) , most initially reported dysphagia , respiratory distress , and/or generalized muscle weakness . one case was seronegative for anti - achr , muscle - specific kinase , and lipoprotein - related protein 4 antibodies and received treatment with plasmapheresis , pulse - steroid therapy , psl , and tacrolimus . the others were all anti - achr antibody positive and were treated with pulse - steroid therapy , psl , and tacrolimus . in this group , only 6 of the 13 patients ( 46.2% ) with a positive anti - achr antibody response underwent thymectomy . we report details of three patients with elderly - onset mg at advanced age who were all 80 years of age at diagnosis . other autoimmune diseases were excluded as antibodies toward nuclear , sjgren s syndrome ( ss)-a , sjgren s syndrome ( ss)-b , thyroid peroxidase , and thyroglobulin antigens were all negative . in one patient , all the patients required the treatment with both oral psl and tacrolimus in order to control their severe disease conditions . the maximum dose of psl was 2035 mg / day , which was maintained 2 weeks and then followed by gradual dose reduction . no recurrence occurred while the patients were treated with maintenance psl 5 mg / day for about 1.5 years . an increase in the incidence of elderly - onset mg has been reported in publications over recent years,14 although development of the disease in patients 80 years of age represents a particularly advanced age of onset . the prevalence of mg in patients 80 years is unclear but the number of patients being diagnosed suggests an increase in incidence or an improvement in physicians diagnostic skills or both . eyelids droop slightly due to age - related skin extension and the muscle strength of all limbs decreases among patients 80 years of age even when no specific disease is present . when swallowing function is impaired , cerebral infarction is first suspected , which makes the diagnosis of mg difficult . the anti - achr antibody level appears to be high in both late- and elderly - onset mg6,7 and this should be actively utilized to assist diagnosis of the disease . the cholinesterase inhibitor is applied as the first choice of treatment for both ocular and generalized mg . however , immunosuppressive treatment should be supplemented as early as possible in generalized mg including bulbar form.5 although steroid therapy can cause major side effects , including diabetes , cataracts , osteoporosis , and infection , it is still considered as the most effective treatment option even in elderly mg patients.2 all three of our patients responded well to psl . psl dose was gradually increased to a moderate level for 2 weeks , which is a relatively short period , and it was then reduced gradually by about 10% every 2 weeks . tacrolimus was added midcourse , which allowed the total psl dose to be reduced and thereby minimize its adverse adrenocortical effects . the pharmaceuticals and medical devices agency of japan acknowledged tacrolimus and cyclosporine as standard immunosuppressive agents in the treatment of mg , but not mycophenolate and azathioprine . tacrolimus and cyclosporine are inhibitors of t - cell function through inhibition of calcineurin signaling . tacrolimus may have a steroid - sparing effect along and confirmed its safety and tolerability.8 about 4 years have passed since mg onset in all three patients : symptoms remain controlled on a combination of oral low - dose psl and tacrolimus without the recurrence of mg . during this period , infection , osteoporosis , compression fracture , and hyperglycemia have occurred ; however , all three patients maintain their adl independence and are able to return to the hospital for follow - up visits by themselves . the indication for thymectomy in late- and elderly - onset mg is less than that for early - onset mg and the combination of corticosteroids with immunosuppressive agents is recommended to reduce corticosteroid doses in late- and elderly - onset mg.5 thymectomy for cases of late- and elderly - onset mg is less recommended because they are susceptible to postoperative complications and other possible metabolic diseases , as the chance of lymphoid follicular hyperplasia is low and the thymus is likely to be atrophied in the elderly . moreover , the patients with late- and elderly - onset mg are less likely to benefit from thymectomy unless they have thymoma . although therapeutic plasmapheresis is widely used in myasthenic crises , it is not favorable for elderly patients to avoid possible cardiovascular events . in contrast , intravenous immunoglobulin is considered to be a suitable treatment in elderly patients not only in myasthenic crises but also for reducing psl dose . even though elderly - onset mg at advanced age can develop in patients 80 years of age , no specific recommendations are needed for their treatment . serious steroid - related adverse effects are likely to occur in patients of an advanced age and adl may be impaired due to treatment - associated complications . minimum necessary treatments are recommended to avoid adverse drug reactions and the general condition of each individual patient should be prioritized .

the number of patients with late - onset myasthenia gravis ( mg ) among patients 50 years has been increasing recently . we encountered three patients who developed elderly - onset mg at a particularly advanced age ( 80 years ) . all were female and positive for anti - acetylcholine receptor antibodies . about 4 years have passed since mg onset in all three patients and symptoms have been controlled without recurrence using a combination of oral low - dose prednisolone and tacrolimus . as many cases of elderly - onset mg do not require strong immunosuppression , we recommend minimum immunosuppressive treatment to avoid adverse events , particularly in patients at an advanced age of 80 years .

Introduction Case Reports Case 1 Case 2 Case 3 Discussion

enucleation was the standard treatment . in the early 1970s , radiotherapy and other treatment approaches , with the goal of preserving the eye , became increasingly popular . external beam irradiation using charged particles , such as protons may conserve a useful level of vision , depending on the size of the tumor and its location with respect to the optic disc and macula . protons deposit most of their energy near the end of their range , resulting in a uniform and highly localized dose being deposited largely in the tumor , sparing adjacent healthy tissue . furthermore , the liver is the most common site of metastatic disease ( einhorn et al 1974 ; char 1978 ; zakka et al 1980 ; rajpal et al 1983 ; diener - west et al 2001 ; kath et al 1993 ; bedikian et al 1995 ; coms 2001 , 2004 , 2005 ) . diagnosis of metastasis is often followed by rapid physical decline of the patient and treatment is generally ineffective ( albert et al 1992 ) . after diagnosis of choroidal melanoma , patients are screened for metastasis 6-monthly or annually , using liver function tests ( lfts ) and imaging studies . more recently routine screening by ultrasonography of the liver and a panel of lfts have been recommended ( donoso et al 1985 ; donoso et al 1985 ; pach and robertson 1986 ; coms 1995 ; hicks 1998 ; eskelin et al 1999 ) . our primary aim was to study the time interval between a diagnosis of melanoma and a diagnosis of metastasis , and the rates and sites of metastasis during follow - up time in our patients . a secondary aim was to record ocular complications after proton beam irradiation ( seddon et al 1986 ) . one hundred and fifty - two eyes of 152 patients with primary choroidal melanoma , whose primary treatment was either proton beam irradiation or enucleation , were included in the study . subjects with clinical evidence of metastatic disease at the time of treatment or prior to the treatment of their intraocular tumor were excluded . slit - lamp examination , biomicroscopy , indirect ophthalmoscopy , fundus photographs and a - scan and b - scan ultrasonography . tumor height was measured and used to classify a tumor as small ( 1 mm to 2.5 mm in apical height ) , medium ( > 2.5 mm to 10 mm ) or large ( > 10 mm in apical height ) . tumor location and associated features , such as secondary retinal detachment and hemorrhage were recorded . the initial evaluation also included fluorescein and indocyanine green angiography , computed tomography ( ct ) , and/or magnetic resonance imaging ( mri ) . complete blood count , liver function tests ( aspartate aminotranspherase [ ast ] , alanine aminotranspherase [ alt ] , alkaline phosphatase [ aph ] , gamma - glutamyl transpeptidase [ -gt ] , lactate dehydrogenase [ ldh ] , carcinoembryonic antigen [ cea ] ) . ultrasound and ct or mri evaluation of the abdominal organs ( especially the liver ) and chest radiograph . the patients from the proton beam irradiation group were admitted approximately two weeks before the scheduled date of irradiation . in the surgical suite , the tumor was localized using transillumination and indirect ophthalmoscopy , and four tantalum rings measuring 2.5 millimeters in diameter were sutured to the sclera to serve as references marking the tumor edges . tumors located too far anteriorly were diagrammed using precise measurements , without the tantalum rings . during the irradiation sessions , the patient was seated and the head was immobilized using a custom - molded plastic mask . lid retractors were used to remove the lid from the radiation field , and the patient was asked to fix his or her gaze on a light attached to the proton collimator . the eye was viewed on a closed circuit television camera using high magnification , to detect any deviation from the indicated direction of gaze . an imaging system was used to verify the eye s precise position during the treatment . provided with the necessary data , the computer generated a three - dimensional model of the eye and tumor , which could be viewed from any desired angle . at the time the model also calculated the best direction of gaze , ideal shape of the field defining apertures , proton range and range modulations , the structures lying in the path of the beam , and what the viewer should see in the alignment film used when aiming the beam . the rectus muscles were individually hooked and cut with scissors near their insertion into the sclera . the enucleation scissors were placed between tenon s capsule and the sclera , the optic nerve was cut and the eye removed . an orbital implant ( hydroxyapatite sphere ) following enucleation , eyes were processed for histopathologic confirmation of the diagnosis at the pathology laboratory of athens general hospital . the patients were re - examined 6-monthly for the first 5 years and annually thereafter . follow - up evaluation included : slit - lamp examination , biomicroscopy , indirect ophthalmoscopy , fundus photographs and a - scan and b - scan ultrasonography . complete blood count , liver function tests ( ast , alt , aph , -gt , ldh , cea ) . ultrasound evaluation of the abdominal organs ( especially the liver ) . in case of abnormal findings , ct or mri scan of the liver and/or biopsy or cytologic tests slit - lamp examination , biomicroscopy , indirect ophthalmoscopy , fundus photographs and a - scan and b - scan ultrasonography . tumor height was measured and used to classify a tumor as small ( 1 mm to 2.5 mm in apical height ) , medium ( > 2.5 mm to 10 mm ) or large ( > 10 mm in apical height ) . tumor location and associated features , such as secondary retinal detachment and hemorrhage were recorded . the initial evaluation also included fluorescein and indocyanine green angiography , computed tomography ( ct ) , and/or magnetic resonance imaging ( mri ) . complete blood count , liver function tests ( aspartate aminotranspherase [ ast ] , alanine aminotranspherase [ alt ] , alkaline phosphatase [ aph ] , gamma - glutamyl transpeptidase [ -gt ] , lactate dehydrogenase [ ldh ] , carcinoembryonic antigen [ cea ] ) . ultrasound and ct or mri evaluation of the abdominal organs ( especially the liver ) and chest radiograph . the patients from the proton beam irradiation group were admitted approximately two weeks before the scheduled date of irradiation . in the surgical suite , the tumor was localized using transillumination and indirect ophthalmoscopy , and four tantalum rings measuring 2.5 millimeters in diameter were sutured to the sclera to serve as references marking the tumor edges . tumors located too far anteriorly were diagrammed using precise measurements , without the tantalum rings . during the irradiation sessions , the patient was seated and the head was immobilized using a custom - molded plastic mask . lid retractors were used to remove the lid from the radiation field , and the patient was asked to fix his or her gaze on a light attached to the proton collimator . the eye was viewed on a closed circuit television camera using high magnification , to detect any deviation from the indicated direction of gaze . an imaging system was used to verify the eye s precise position during the treatment . provided with the necessary data , the computer generated a three - dimensional model of the eye and tumor , which could be viewed from any desired angle . at the time the model also calculated the best direction of gaze , ideal shape of the field defining apertures , proton range and range modulations , the structures lying in the path of the beam , and what the viewer should see in the alignment film used when aiming the beam . enucleation was performed at the department of ophthalmology of the university of athens . the surgical technique consisted of peritomy for 360 at the limbus . the rectus muscles were individually hooked and cut with scissors near their insertion into the sclera . the enucleation scissors were placed between tenon s capsule and the sclera , the optic nerve was cut and the eye removed . an orbital implant ( hydroxyapatite sphere ) was placed in the socket and the muscles were sutured to the implant . following enucleation , eyes were processed for histopathologic confirmation of the diagnosis at the pathology laboratory of athens general hospital . the patients were re - examined 6-monthly for the first 5 years and annually thereafter . follow - up evaluation included : slit - lamp examination , biomicroscopy , indirect ophthalmoscopy , fundus photographs and a - scan and b - scan ultrasonography . complete blood count , liver function tests ( ast , alt , aph , -gt , ldh , cea ) . ultrasound evaluation of the abdominal organs ( especially the liver ) . in case of abnormal findings , ct or mri scan of the liver and/or biopsy or cytologic tests were required to confirm or rule out metastatic disease . slit - lamp examination , biomicroscopy , indirect ophthalmoscopy , fundus photographs and a - scan and b - scan ultrasonography . tumor height was measured and used to classify a tumor as small ( 1 mm to 2.5 mm in apical height ) , medium ( > 2.5 mm to 10 mm ) or large ( > 10 mm in apical height ) . tumor location and associated features , such as secondary retinal detachment and hemorrhage were recorded . the initial evaluation also included fluorescein and indocyanine green angiography , computed tomography ( ct ) , and/or magnetic resonance imaging ( mri ) . complete blood count , liver function tests ( aspartate aminotranspherase [ ast ] , alanine aminotranspherase [ alt ] , alkaline phosphatase [ aph ] , gamma - glutamyl transpeptidase [ -gt ] , lactate dehydrogenase [ ldh ] , carcinoembryonic antigen [ cea ] ) . ultrasound and ct or mri evaluation of the abdominal organs ( especially the liver ) and chest radiograph . the patients from the proton beam irradiation group were admitted approximately two weeks before the scheduled date of irradiation . in the surgical suite , the tumor was localized using transillumination and indirect ophthalmoscopy , and four tantalum rings measuring 2.5 millimeters in diameter were sutured to the sclera to serve as references marking the tumor edges . tumors located too far anteriorly were diagrammed using precise measurements , without the tantalum rings . during the irradiation sessions , the patient was seated and the head was immobilized using a custom - molded plastic mask . lid retractors were used to remove the lid from the radiation field , and the patient was asked to fix his or her gaze on a light attached to the proton collimator . the eye was viewed on a closed circuit television camera using high magnification , to detect any deviation from the indicated direction of gaze . an imaging system was used to verify the eye s precise position during the treatment . a computer planning program was used to target the proton beam . provided with the necessary data , the computer generated a three - dimensional model of the eye and tumor , which could be viewed from any desired angle . at the time the model also calculated the best direction of gaze , ideal shape of the field defining apertures , proton range and range modulations , the structures lying in the path of the beam , and what the viewer should see in the alignment film used when aiming the beam . enucleation was performed at the department of ophthalmology of the university of athens . the surgical technique consisted of peritomy for 360 at the limbus . the rectus muscles were individually hooked and cut with scissors near their insertion into the sclera . the enucleation scissors were placed between tenon s capsule and the sclera , the optic nerve was cut and the eye removed . an orbital implant ( hydroxyapatite sphere ) following enucleation , eyes were processed for histopathologic confirmation of the diagnosis at the pathology laboratory of athens general hospital . the patients were re - examined 6-monthly for the first 5 years and annually thereafter . slit - lamp examination , biomicroscopy , indirect ophthalmoscopy , fundus photographs and a - scan and b - scan ultrasonography . complete blood count , liver function tests ( ast , alt , aph , -gt , ldh , cea ) . ultrasound evaluation of the abdominal organs ( especially the liver ) . in case of abnormal findings , ct or mri scan of the liver and/or biopsy or cytologic tests were required to confirm or rule out metastatic disease . tumor thickness according to b - scan ultrasonography , tumor location according to binocular indirect ophthalmoscopy , and goldmann triple mirror examination were recorded . in the enucleation group the affected eye was the right one in 18 ( 58% ) patients and the left one in 13 ( 42% ) . tumor thickness ranged from 4.6 mm to 13.4 mm with a mean value of 9.7 mm . tumor location was superior in 6 ( 19.4% ) patients , inferior in 4 ( 12.9% ) patients , temporal in 7 ( 22.6% ) patients , nasal in 4 ( 12.9% ) patients , superior / temporal in 2 ( 6.4% ) patients , inferior / temporal in 4 ( 12.9% ) patients , and inferior / nasal in 4 ( 12.9% ) patients ( figure 1 ) . in the irradiation group , the affected eye was the right one in 57 ( 47% ) patients and the left one in 64 ( 53% ) . tumor thickness ranged from 2.6 mm to 13.4 mm with a mean value of 7.3 mm . tumor location was superior in 22 ( 18.2% ) patients , inferior in 18 ( 14.9% ) patients , temporal in 20 ( 16.5% ) patients , nasal in 8 ( 6.6% ) patients , superior / temporal in 13 ( 10.7% ) patients , superior / nasal in 4 ( 3.3% ) patients , inferior / temporal in 26 ( 21.5% ) patients and inferior / nasal in 10 ( 8.3% ) patients ( figure 1 ) . of the152 patients in our study , 31 were treated by enucleation between 1992 and 2006 ( 16 males , 15 females ) . of the 31 patients , 6 ( 19.3% ) were diagnosed with metastasis ( 5 males , 1 female ) . the metastatic sites included : liver ( 5 patients , 16.1% of enucleation group , 83.3% of the patients with metastasis ) and skin ( 1 patient , 3.2% of enucleation group , 16.7% of the patients with metastasis ) ( figure 2 ) . the median interval time from diagnosis of choroidal melanoma to diagnosis of metastasis was 29.5 months ( 2.46 years ) . at 5 years , 5 patients were diagnosed with metastasis ( 16.1% of enucleation group , 83.3% of patients with metastasis ) . between 1992 and 2006 , 121 patients were treated by proton beam irradiation ( 53 males , 68 females ) . thirteen patients ( 10.7% ) were diagnosed with metastasis ( 7 males , 6 females ) . the metastatic sites included : liver ( 8 patients , 6.6% of irradiation group , 61.5% of the patients with metastasis ) , lung ( 2 patients , 1.6% of irradiation group , 15.4% of the patients with metastasis ) , brain ( 1 patient , 0.8% of irradiation group , 7.7% of the patients with metastasis ) , thyroid ( 1 patient , 0.8% of irradiation group , 7.7% of the patients with metastasis ) , and colon ( 1 patient , 0.8% of irradiation group , 7.7% of the patients with metastasis ) ( figure 2 ) . the median interval of time from diagnosis of choroidal melanoma to the diagnosis of metastasis was 44.8 months ( 3.7 years ) . at 5 years , 10 patients were diagnosed with metastasis ( 8.3% of irradiation group , 76.9% of patients with metastasis ) . ocular complications were recorded after proton beam irradiation , according to slit - lamp examination , biomicroscopy , indirect ophthalmoscopy , fundus photographs , and a - scan and b - scan ultrasonography . anterior segment complications were recorded in 99 ( 81.8% ) patients and posterior segment complications in 71 ( 58.7% ) patients . cataract was recorded in 59 ( 48.7% ) patients , iris neovascularization in 17 ( 14% ) patients , neovascular glaucoma in 14 ( 11.6% ) patients , and corneal opacification in 9 ( 7.4% ) patients . secondary retinal detachment was recorded in 25 ( 20.7% ) patients , retinopathy in 17 ( 14% ) patients , vitreous hemorrhage in 17 ( 14% ) patients and optic neuropathy in 12 ( 9.9% ) patients ( figure 3 ) . for more than a century , enucleation has been the standard treatment for choroidal melanoma . however , concerns about enucleation , especially possible tumor spread caused by manipulation of the eye during surgery , plus vision loss and the poor cosmetic outcome , have made irradiation therapy a popular alternative . when patients are deciding on their treatment for choroidal melanoma , the major influences on their decision are fear of death due to cancer , and concerns about the prognosis of vision in the affected eye . many ophthalmologists assume that better results can be achieved with irradiation therapy , because this form of treatment potentially allows the retention of some vision and avoids the perception of disfigurement . a 1986 study ( gragoudas et al 1987 ) showed a 20% cumulative risk of developing metastasis 5 years after irradiation , based on 128 patients followed for a median of 5.4 years . in a study ( gragoudas et al 1988 ) of 780 eyes ( which included many of the 128 noted above ) followed for a median of 2.2 years , 8% of that study population actually developed metastases , the median time to detection being 2.1 years . based on kaplan - meier probability curves , the same 5-year estimate of a 20% cumulative risk of metastasis was determined . eighty percent to 90% of metastases from uveal melanoma occurred in the liver in those patients , less common sites being the skin and lung . in our study we recorded the tumor location , the rates and sites of metastasis , and the time from diagnosis of melanoma to diagnosis of metastasis . in the enucleation group , the median time from diagnosis of choroidal melanoma to diagnosis of metastasis was 29.5 months ( 2.46 years ) . five of the 6 patients with metastasis ( 83.3% ) were diagnosed within the first 5 years after treatment ( enucleation ) . in the irradiation group , the median time from diagnosis of choroidal melanoma to diagnosis of metastasis was 44.8 months ( 3.7 years ) . ten of the 13 patients with metastasis ( 76.9% ) were diagnosed within the first 5 years after treatment ( irradiation ) . important prognostic factors for death from metastatic melanoma include the size of the tumor ( the larger the tumor , the worse the prognosis ) , the location of the tumor ( tumors within the ciliary body are associated with a poorer prognosis than those confined to the choroid ) , the age of the patient at the time of diagnosis ( the older the patient , the worse the short - term survival prognosis ) and extrascleral tumor extension ( augsburger and gamel 1990 ) . patients with choroidal melanoma are advised to undergo periodic examination , blood tests for liver enzyme levels and imaging studies , such as chest radiographs and ultrasonography or mri of the liver . we agree with other studies ( hicks 1998 ; eskelin et al 1999 ) that periodic screening for metastatic uveal melanoma is primarily beneficial , when effective treatment is available or when patients are eligible as candidates for clinical trials of promising treatment . such therapies are needed given the potentially frequent occurrence of micro - metastasis at the time of diagnosis of choroidal melanoma and the high incidence of metastasis detected clinically after diagnosis of the primary tumor . many treated eyes eventually lose a substantial amount of vision , as a consequence of complications after radiation therapy . factors that influence how much vision will be lost include the location of the posterior edge of the tumor relative to the optic disc and fovea , the visual acuity before treatment , the presence or absence of macular retinal detachment , and the thickness of the tumor . some systemic factors , such as diabetes mellitus , may also worsen a patient s prognosis for retention of useful vision ( seddon et al 1986 ) . retinal detachment , optic neuropathy , radiation retinopathy , neovascular glaucoma ( kim et al 1986 ; summanen et al 1996 ; daftari et al 1997 ) , vitreous hemorrhage , and cataract ( meecham et al 1994 ; gragoudas et al 1995 ) have emerged consistently as causes of visual loss in proton beam - irradiated eyes . establishing which of these conditions is most influential in any given case may cause difficulty , as they may occur in the same patient , in whom the presence of a cataract may further hamper a view of the fundus . in our study , irradiation - related complications involving the anterior segment , which contributed to a decline in visual acuity or eye loss , included cataract ( 59 patients ; 48.7% ) , neovascular glaucoma ( 14 patients ; 11.6% ) , and corneal neovascularization and opacification ( 9 patients ; 7.4% ) . gragoudas and colleagues ( 1995 ) showed that 42% of 383 patients who received proton beam irradiation for uveal melanoma , developed new posterior subcapsular opacities in direct proportion to the dose of radiation received by the lens in the irradiated eye . this result was determined by a computer - generated dose - volume assessment routinely calculated at the time of irradiation . cortical and sclerotic opacities were found to be correlated only with age in this study . cataract extraction is feasible in these patients and can restore vision in some ( augsburger and shields 1985 ; fish et al 1991 ; gragoudas et al 1992 ) . panretinal photocoagulation can substantially reduce the severity of neovascular glaucoma and reduce the risk of enucleation by half ( kim et al 1986 ) . in our study , posterior segment complications included secondary retinal detachment ( 25 patients ; 20.7% ) , radiation retinopathy ( 17 patients ; 14% ) , vitreous hemorrhage ( 17 patients ; 14% ) , and radiation neuropathy ( 12 patients ; 9.9% ) . maculopathy affects central vision ( gragoudas et al 1992 ; gunduz et al 1999 ; guyer et al 1992 ) , whereas papillopathy can lead to total loss of vision ( guyer et al 1992 ; gragoudas et al 1999 ) . patients who develop a blind , painful eye must undergo enucleation ( kellner et al 1993 ) . preservation of the eye and useful vision in many patients are the obvious advantages of conservative treatment with radiation compared with enucleation . however questions have been raised regarding the efficacy of this treatment in limiting metastatic spread of the tumor . observational studies ( egan et al 1989 ; seddon et al 1990 ; augsburger et al 1986 ) comparing irradiation with enucleation have indicated no significant differences in survival rates . in the collaborative ocular melanoma study ( adams et al 1998 ) , overall 5-year mortality rates were comparable in patients treated by enucleation and by plaque radio - therapy ( iodine 125 ) . many patients maintain some degree of function in the eye for long periods after treatment . overall rates of metastatic disease are comparable to those observed after enucleation ; thus , enucleation should be limited to patients with large tumors in whom the eye is unlikely to be salvaged by irradiation . doubtless continued refinement of current methods , and development of new techniques , will further improve the outlook of patients with uveal melanomas . in our series , 10.7% of the irradiation group and 19.3% of the enucleation group were diagnosed with metastasis . this difference is attributed to the larger tumor dimensions in patients of the enucleation group , given the observed correlation between increased metastasis rate and increased size of choroidal melanoma . effective methods are needed to prevent , diagnose , and treat metastasis from choroidal melanoma .

purposeto investigate tumor thickness and location , the interval of time between treatment for choroidal melanoma ( proton beam irradiation or enucleation ) and diagnosis of metastatic disease , and rates and sites of metastasis.designretrospective , interventional , noncomparative case series.methodsfollow-up of 152 patients with primary choroidal melanoma , between 1992 and 2006 ( 14 years ) . one hundred and twenty - one patients were treated with proton beam irradiation and 31 patients were treated with enucleation . baseline and annual or semiannual ophthalmic examination , b - scan ultrasonography , systemic and laboratory evaluations ( liver enzymes , chest x - ray , ultrasonography or magnetic resonance imaging of the liver ) were performed according to a standard protocol.resultsnineteen patients ( 12.5% ) were diagnosed with metastasis during follow - up time after treatment for choroidal melanoma . thirteen patients ( 10.7% ) of the irradiation group and 6 patients ( 19.3% ) of the enucleation group were diagnosed with metastasis . eight patients ( 6.6% ) of the irradiation group and 5 patients ( 16.1% ) of the enucleation group were diagnosed with liver metastasis . ocular complications after proton beam irradiation were recorded . fifty - nine patients ( 48.7% ) of the irradiation group presented with cataract . other complications were retinal detachment , retinopathy , vitreous haemorrhage , iris neovascularization , neovascular glaucoma , optic neuropathy , and corneal opacification.conclusionsin our series , 10.7% of the irradiation group and 19.3% of the enucleation group were diagnosed with metastasis . the liver was the most common site of metastasis in both groups . cataract was the most common complication in the irradiation group .

Introduction Methods Initial evaluation included Ophthalmic examination Systemic evaluation Ophthalmic examination Systemic evaluation Results Discussion Conclusion

a high demand for presbyopia correction at the time of cataract surgery has increased the implantation of multifocal intraocular lenses ( iols ) . residual astigmatism is a concern with these lenses , because refractive astigmatism of 1.0 d or more can negatively impact visual outcomes with multifocal iols.1 this amount of astigmatism may be encountered in ~40% of patients presenting for routine cataract surgery2 and must be managed in multifocal patients to reduce potential dissatisfaction with the visual outcome . two of the most common approaches to reduce astigmatism at the time of cataract surgery are using corneal relaxing incisions and/or toric iols . correcting astigmatism with corneal relaxing incisions can be more variable with lower effectivity , predictability , and stability than with toric iols.3,4 as such , astigmatic correction with a toric multifocal iol is a desirable alternative . some studies related to multifocal toric iols have been published in the literature , but they are limited to accommodative toric or bifocal toric designs , and we are only aware of one study discussing the clinical outcomes with an accommodative toric iol.5 studies of bifocal toric iols are more common . the refractive design has specific areas in the lens designed for either near vision or distance vision increasing their dependence on pupil size and need for proper alignment . the diffractive design splits incoming light , with one focus for distance images and the other for near images ; such designs have the potential to decrease contrast sensitivity . one of the most studied diffractive bifocal toric iols ( acrysof restor snd1 t ; novartis ag , basel , switzerland ) has been shown to be effective at significantly improving near vision while maintaining good distance vision and providing similarly reliable astigmatism correction when compared to an aspheric monofocal toric iol ( acrysof toric sn6at ; alcon , inc . ) . glare and halos are more common , and low - contrast sensitivity and low - contrast visual acuity ( lcva ) are reported to be poorer with multifocal iols compared to monofocal iols , but this is unlikely related to the addition of the toric correction to the multifocal iol.6,7 a primary concern with using a bifocal iol is that it does not provide an intermediate focal point ; a bench study has demonstrated that this can reduce the potential for adequate intermediate vision.8 with the proliferation of handheld devices , intermediate vision has become a significant concern for patients . the finevision iol ( physiol ; finevision toric , lige , belgium ) is a diffractive trifocal iol that provides an intermediate focus at 1.75 d and a near focus at 3.5 d ( both at the iol plane ) . this corresponds to a nominal intermediate add of approximately + 1.15 d and near add of about + 2.25 d at the corneal plane , depending on the geometry of the patient s eye . this intermediate focal point is expected to improve patient satisfaction relative to bifocal iols , since bifocal iols have a greater drop off in visual acuity ( va ) in the intermediate range.9 there is a concern that the addition of a third focal point may increase halos.10 however , a relatively small percentage of energy is dedicated to intermediate vision when compared to distance and near vision ; as such , photic phenomena and contrast sensitivity are unlikely to be more problematic with this diffractive trifocal iol than they are for any other diffractive bifocal iol.11,12 results in 198 eyes of 99 patients implanted with this diffractive trifocal iol show that the lens provides good near , intermediate , and distance vision under different illuminations with high patient satisfaction.13 a new diffractive trifocal toric iol is now available ( physiol , pod ay 26p f - t ; finevision toric ) ; it utilizes the same multifocal principles as the finevision trifocal iol and the same toric principles as the ankoris iol ( physiol ; ankoris , lige , belgium).14 the ankoris iol is a hydrophilic aspheric toric iol utilizing double - c loop quadripode haptics for stabilization.15 the purpose of the current study is to compare the refractive and va outcomes , including lcva , as well as the patient s visual function 3 months after implantation of this new diffractive trifocal toric iol with the restor snd1 t apodized diffractive bifocal toric iol . this study was a single - site , single - surgeon , randomized prospective study involving bilateral implantation of a trifocal toric iol in one group and a bifocal toric iol in the other group during one session . the regional committees for medical and health research ethics ( rek ) , norway reviewed and approved the study and patient informed consent . the study was powered sufficiently to detect a 0.5 line difference in logmar acuity based on data in the literature . subjects were eligible for inclusion if they were > 50 years old , had presented for uncomplicated cataract surgery , and were interested in reducing their dependence on glasses for daily life . they had to have regular corneal astigmatism and have a calculated iol power that was within the available range for each iol type . all subjects had to be willing and able to sign the informed consent for the study . the surgeon evaluated the interest of the patient in spectacle independence , their affect , and their expectations . if the surgeon felt there were factors that would be likely to affect the subject s postoperative vision ( eg , amblyopia and history of uveitis ) , the surgeon could exclude the subject . all subjects had a preoperative examination that included refraction , topography , keratometry , biometry , and a slit - lamp examination , after which , if eligible , they were asked to participate . toric iol calculations were performed using the standard calculation for the acry - sof lens ( www.acrysoftoriccalculator.com ) and for the physiol lens ( www.physioltoric.eu ) . all subjects were implanted bilaterally at a single visit using the standard phacoemulsification procedure at the site ; procedures were completed without any use of femtosecond laser systems . any surgical complications resulted in the subject being exited from the study , and replaced . at 3 months postoperatively , the subject s vision was tested both uncorrected and with his / her best distance correction at : distance ( 4 m ) , intermediate ( 63 cm ) , and near ( 40 cm ) . the examiner was not masked to the lens type , but the manifest refraction was determined for both groups using the standard methodology for the site , including the use of high - contrast logmar acuity charts . binocular defocus curves were measured with no correction and with the subject s best distance correction in place . quality of vision was measured using the national eye institute visual function questionnaire ( nei vfq)-25 . the nei - vfq was developed at the rand under the sponsorship of the nei ; it provides a self - reported measure of visual function.16 the data were imported into an ms access database for data checking , collation , and preliminary analysis ( microsoft corporation , redmond , wa , usa ) . statistical analyses were performed using the statistica data analysis software system , version 12 ( statsoft , inc . , www.statsoft.com ) . statistical testing was performed using analysis of variance on continuous variables and appropriate nonparametric tests on categorical data . a total of 22 patients were successfully enrolled , with no complications requiring any patients to exit the study . the average age in the trifocal group was 8 years lower than the bifocal group , a statistically significant difference ( p=0.02 ) . the trifocal group also had a statistically significantly higher average keratometry ( p=0.01 ) , but the average corneal astigmatism measured in both groups was similar ( p=0.58 ) . the surgically induced astigmatism ( sia ) was calculated for each eye based on the difference in the measured corneal astigmatism preoperatively and at 3 months postoperatively . there was no statistically significant difference in the measured magnitude of the sia between groups ( p=0.07 ) . the average sia magnitude was 0.49 d 0.36 d , while the calculated centroid ( the vector sum of all sia measures ) for both groups was between 0.10 d and 0.15 d. an intended lens orientation was available at the time of surgery , and lens orientation was measured at each follow - up visit . there were only two lenses with a measured change in orientation of > 5 between the 1-day and 1-month postoperative visits , one bifocal lens , and one trifocal lens ; the maximum measured change was 7. there was no statistically significant difference in the change of lens orientation between the 1-day and 1-month postoperative visits between iol groups ( p=0.07 ) . in three of the cases involving trifocal iols , a chart review showed a similar but slightly lower alignment error 1 day postoperatively suggesting an alignment error rather than actual iol rotation . these three lenses were reoriented to the intended angle between the 1-month and 3-month postoperative visits . with these manual exceptions , no change of lens orientation of > 5 was measured between the 1-month and 3-month visits for either lens . there was a statistically significant difference between the trifocal and bifocal groups in the change in lens orientation between the 1-month and 3-month postoperative visits ( p<0.01 ) ; the difference was < 2. the bifocal lenses appeared to rotate slightly more clockwise and the trifocal lenses appeared to rotate slightly more counterclockwise , with average measured rotation < 1 in both cases . there was no statistically significant difference in the absolute change in measured rotation between 1 month and 3 months postoperatively between the two iol groups ( p=0.98 ) . there was also no statistically significant correlation between change in lens orientation and axial length for either lens ( p>0.05 ) . at 3 months , average uncorrected logmar va in the trifocal group was about a half line better than in the bifocal group , which is presumably associated with the slightly lower ( 0.19 d ) residual refractive cylinder , but neither of these differences was statistically significant . the percentage of eyes with 0.5 d of refractive cylinder at 3 months was slightly higher in the trifocal group ( 82% , 18/22 ) relative to the bifocal group ( 68% , 15/22 ) , but the difference was not statistically significant ( fisher s exact test , two - tailed , p=0.48 ) . the distribution of uncorrected binocular va measured at the three test distances is shown in figure 1 . there was no statistically significant difference in the measured va at any distance between the groups . there was a higher number of subjects with 20/25 uncorrected va at 63 cm in the trifocal group ( ten of eleven vs seven of eleven in the bifocal group ) , but the difference was not statistically significant ( fisher s exact test , one - tailed , p=0.16 ) . similar results were obtained for the best distance - corrected va , with no statistically significant difference between groups at any distance . at 63 cm , 82% of eyes ( 9/11 ) with the trifocal toric iol had a best distance - corrected va equal to or better than 0.1 logmar ( 20/25 snellen ) , compared to 64% of eyes ( 7/11 ) with the bifocal toric iol ; the difference was not statistically significant ( fisher s exact test , one - tailed , p=0.18 ) . lcva was tested binocularly with the subject s best distance correction in place using a target at 4 m and a letter contrast of 10% . there was no statistically significant difference in the measured lcva between groups ( p=0.39 ) . figure 2a shows the uncorrected binocular defocus curve , and figure 2b shows the best distance - corrected binocular defocus curve for the two groups . results were not statistically significantly different at any test distances except + 2.0 ( not clinically relevant ) and 1.5 d , corresponding to a reading distance of ~67 cm . at both these levels of defocus , subjects implanted with the trifocal iol had statistically significantly better va . the questionnaire results from the nei - vfq were tabulated , and the subgroup scores for general vision , distance vision , near vision , and driving were compared between groups . figure 3 shows the distribution of these subgroup scores by the iol group . there were no statistically significant differences by the iol group for any of these subgroup scores ( p>0.26 in all cases ) in this study , statistically significant differences in preoperative average keratometry and patient age were found between groups , despite randomization , but the differences were not considered clinically significant . the small sample size is likely a contributing factor . more importantly , there was no statistically significant difference in the level of corneal astigmatism between groups . the bifocal toric iol is based on a modified l haptic design , while the trifocal toric iol uses a double c - loop quadripod.15 despite these design differences , the correction of astigmatism was effective and stable in both groups . three cases in the trifocal group required reorientation because of minor misalignment at the time of surgery . lens alignment was stable in both iol groups , varying only within a 5 range up to 3 months postoperatively . rotational stability has been documented previously for both iol groups ; the toric component of the trifocal iol has been shown to have a mean rotation of ~2.6,14 at 3 months , the postoperative astigmatism was slightly less ( 0.19 d ) in the trifocal toric group when compared to the bifocal toric group , but the difference was not statistically significant . the mean postoperative astigmatism in the bifocal toric group in this study was consistent with the average of the mean postoperative astigmatism reported in two previous studies using the same lens.6,7 the spherical equivalent refraction for the bifocal toric iol group in the current study was also the same as that reported in a larger study of the same lens.7 the mean postoperative astigmatism in the trifocal toric group was 0.3 d in the current study , which is slightly lower than the 0.4 d mean reported by a previous study of the same toric lens design.14 the va results were similar between the two groups at all distances . the mean uncorrected distance visual acuities for both groups were reasonably consistent with previously reported values for the bifocal toric group and the multifocal component of the trifocal toric group.7,13 this is not unexpected , as both lenses use a similar diffractive multifocal design . it is worth noting that the additional intermediate focal point in the trifocal lens did not appear to negatively impact distance or near vision . the binocular defocus curves in figure 2 show that the trifocal toric group had statistically significantly better va at 1.5 d ( corresponding to the vision of a target at 67 cm ) when compared to the bifocal toric group . a previous bench study comparing the multifocal component of both lenses found that the trifocal lens provides a true third intermediate focal point that is not found with the bifocal lens.8 clinical studies evaluating the bifocal toric iol and the multifocal component of the trifocal toric iol provided similar defocus curves to the one shown in the current study.7,13 these studies noted a similar drop in the intermediate vision with the bifocal iol relative to the trifocal iol . there is always some concern that the additional focal point provided by a trifocal iol may reduce visual quality more than bifocal iols , as a result of splitting the light into three foci.10 one objective measure of this is lcva . in the current study , previous studies have found lcva ( 10% ) to be better than reported here for the trifocal toric iol group and worse than reported here for the bifocal toric iol group , though different testing methods may have been used and the previous trifocal iol data did not include the toric component.6,13 another common measure of overall visual function is the nei - vfq questionnaire ; all subjects in the current study completed the questionnaire . the subscales for general vision , near vision , distance vision , and driving showed no statistically significant differences between the bifocal and trifocal iols , with median subscore values 80 in all cases , suggesting high satisfaction with postoperative vision . a high level of satisfaction related to near vision in subjects undergoing bifocal toric iol implantation has previously been reported.6 a high level of satisfaction based on the patients answer to whether or not they would have the same lens implanted again in those that underwent trifocal iol implantation has also been documented.13 in conclusion , the trifocal toric iol provided improved intermediate vision without negatively impacting distance or near va when compared to a bifocal toric iol . lcva and the patient s visual functions were comparable between the trifocal and bifocal toric iol groups . the trifocal toric lens demonstrated good rotation stability , leaving subjects with low postoperative refractive astigmatism .

purposethe aim of this study was to compare a new diffractive trifocal toric lens with an apodized diffractive bifocal toric lens in terms of refractive and visual acuity ( va ) outcomes , including low - contrast va ( lcva ) , as well as the patient s visual function 3 months after implantation.patients and methodsthis is a randomized prospective study involving bilateral implantation of a trifocal toric or a bifocal toric lens . at 3 months postoperatively , the subject s vision was tested both uncorrected and with his / her best distance correction at : distance ( 4 m ) , intermediate ( 63 cm ) , and near ( 40 cm ) . binocular defocus curves were measured with no correction and with the subject s best distance correction in place . quality of vision was measured using the national eye institute visual function questionnaire.resultsa total of 22 patients were enrolled ( eleven in each group ) . there was no statistically significant difference in the absolute change in measured rotation between 1 month and 3 months postoperatively between the two intraocular lens ( iol ) groups ( p=0.98 ) . at 3 months , the postoperative refraction and distance va by eye were similar between groups . there was no statistically significant difference in the measured lcva between groups ( p=0.39 ) . the defocus curve showed that at 67 cm , the trifocal toric lens had statistically significantly better va when compared to the bifocal toric lens . there were no statistically significant differences by group for any of the national eye institute visual function questionnaire scores ( p>0.26 in all cases).conclusionthe trifocal toric iol improved the intermediate vision without negatively impacting visual function and distance , near , or low - contrast va when compared to a bifocal toric iol . the toric component of the trifocal lens effectively reduced astigmatism and provided good rotational stability .

Introduction Patients and methods Results Discussion Conclusion

a common setup for image - guided neurosurgery uses a tracked pointer on a preoperative magnetic resonance imaging ( mri ) scan to visualize the tumor and its critical surroundings for guidance during the surgery . this neuronavigation assists in tumor localization and resection and is a common practice in developed countries although typically only basic anatomic mri information is uploaded . it allows the integration and association of preoperative anatomic and functional data with intraoperative information . preoperatively , it includes the use of functional mri and diffusion tensor imaging ( dti ) in preplanning . during surgery , this approach includes the use of ultrasound , computed tomography ( ct ) , mri , and direct ( sub)cortical stimulation . the principle behind this approach is to use these modalities simultaneously and allow the intraoperative overlay of both anatomic and functional information to better handle the surgical approach and resection . functional mri measures brain activity by detecting associated changes in blood flow using the hemodynamic response of the blood - oxygen level - dependent ( bold ) signal . this technique relies on the fact that cerebral blood flow and neuronal activation are coupled . functional mri is a non - invasive method of brain - mapping to identify regions linked to critical functions ( figure 1 ) . tumors , however , can change the blood flow in ways not related to neural activity . novel methods that may improve both the spatial and time resolution of functional mri are being studied using markers , such as temperature , ph , calcium - sensitivity . dti is a diffusion - weighted mri that assesses the rate of diffusion of physiologic water and the preferred three - dimensional direction . dti enables the visualization of white matter tracts ( fibertractography ) and provides information about the relationship of these tracts to the tumor ( figure 2 ) . dti has provided excellent resolution of the major fiber pathways involved in speech , vision , and motor functions . recent studies in particular have demonstrated the value of dti for resections near the optic pathway and radiations . cystic lesion in the posterior parietotemporal region of the left hemisphere , closely related to the arcuate fasciculus and wernicke 's area . functional mri at a verbal fluency task shows activation in the broca 's area ( frontal region ) and the wernicke 's area ( parietotemporal region ) . marion smits , department of radiology , erasmus university medical centre , the netherlands ) a , tumor in the right primary motor cortex . b , on transverse & coronal color - coded diffusion tensor imaging ( dti ) maps , the corticospinal tract is blue . c , transverse & coronal fiber tracking of the corticospinal tract shows medial displacement of the corticospinal tract by the tumor . marion smits , department of radiology , erasmus university medical centre , the netherlands ) an obvious challenge of functional mri and dti is inaccurate image - to - patient mapping as well as brain shifts during surgery , which makes adjustments necessary during surgery to maintain accuracy . some modalities , such as intraoperative mri , are costly and require major modifications to the operating room . therefore , intraoperative ultrasound continues to be a pertinent alternative due to its ease of use and economy of cost . the ultrasound signal is typically tracked and superimposed on the preoperative mri used in a navigation system , allowing the surgeon to assess a misalignment or deformation with respect to the preoperative images . unfortunately , the comparatively poor image quality of ultrasound allows for the exposure of only a few anatomic structures , such as the tumor , ventricles , and falx . ongoing research is attempting to improve on corrections for brain shifts through the mapping of preoperative mri scans to intraoperative ultrasound images . recent techniques rely on the maximization of gradient orientation alignment in a reduced set of high - confidence locations of interest and apparently allows for fast , accurate , and robust registration . in addition to repeated landmark checks during surgery to improve clinical navigation accuracy , they advise limiting the craniotomy size to the minimum necessary to expose the tumor area and a narrow portion of the surrounding brain . furthermore , resection should be performed in a manner that safeguards the accuracy of neuronavigation . in cases of frontal tumors located near the cortico - spinal tract , resection began from the inferior - posterior border of the tumor ( figure 3 ) . similarly , in cases of parietal tumors , resection began from the anterior border ( figure 1 ) . however , even when these suggestions are followed , the risk of brain shift remains substantial , and the accuracy of the information provided by the neuronavigation system remains inadequate . in such cases , intraoperative mapping would be most beneficial . preoperative anatomic and functional imaging offers the opportunity to quickly identify the neurons and fibers associated with motor or language functions when used in combination with intraoperative data , such as ( sub)cortical mapping . the clinical relevance of this combined approach comes from the fact that it further enhances surgical safety by maintaining a very high rate of functional preservation while striving for supra - maximum resection . tumor located in the primary motor cortex , which is displaced inferiorly ( functional mri at fingertapping with both hands ) , and there is displacement of the corticospinal tract . awake craniotomies ( asleep - awake anesthesia ) are not novel although this approach is not yet widely used for routine glioma surgery. the routine of awake craniotomy may decrease postoperative neurologic deficits and allow for the extensive resection of tumor even in precarious areas of the brain . the patient 's verbal help is used for continuous clinical neurologic testing to detect early deficits in the sensory motor , language , or visual domains during tumor removal . indeed , knowledge that the patient 's neurologic function remains intact imbues the surgeon with the confidence to continue , resulting in a more extensive excision . naturally , surgeons who use awake craniotomies far away from risky areas of the brain will report the lowest deficits . additionally , direct intraoperative awake mapping is the gold standard for safe surgery in critical functional areas , , . ( sub)cortical bipolar stimulation depolarizes a very focal area , which in turn evokes certain responses in motor , language , or visual function . originating from the techniques developed in epilepsy surgery , large exposures of the cortex and the marking of positive functional sites was the rule until recently . in recent years , a negative sites strategy ( no clinical effect at stimulation ) however , negative mapping does not guarantee safe surgery , although safe surgery may be more problematic in low - grade glioma than in gbm . high - grade gliomas usually dislocate the tracts , whereas low - grade gliomas might infiltrate them . if the critical tissues that should be spared were identified , then supra - maximum resection could be feasible in gbm and could be even safer than in low - grade glioma . the rate of neurologic deficits will depend on how willing the surgeon is to approach the edge between the tumor and functional tissue . given that the risks should be in balance with survival benefits , a high - risk strategy appears less appropriate for gbm because the patient have little time to recover . the exceptions might be the neurologically and mentally intact younger patients in whom survival might be significantly protracted . cytoreduction is limited by the difficulty in distinguishing glioma infiltration from the normal brain during surgery and concerns over causing neurologic deficits . achieving supra - maximum resection is challenging when using only conventional white light microsurgical techniques ; mri - verified gross total resections are only achieved in 23% to 50% of all high - grade glioma resections , , . gross total resections might be achieved by better tumor localizing techniques as well as by better visualization and identification techniques . intraoperative mri has gained an evidence base with reports of increased complete resection rates and improved survival , . the proof of principle for better tumor visualization by fluorescence guidance was convincingly demonstrated in a large , multicenter phase iii randomized controlled trial of 270 patients with high - grade glioma , 88% of whom had gbm . after the oral administration of the pro - drug 5-amino - levulinic acid ( 5-ala ) , the fluorescent molecule protoporphyrin ix ( ppix ) accumulates in high - grade glioma , allowing the neurosurgeon to more easily detect and resect the tumor . in 65% of cases , a gross total resection was achieved compared with a 36% gross total resection rate when using conventional white light . intraoperative fluorescence imaging , especially near - infrared fluorescence imaging , has the potential to revolutionize neurosurgery by providing high sensitivity and real - time imaging guidance to surgeons to define glioma margins , . ppix fluorescence has been suggested to be better for defining tumor extent than gadolinium enhancement in intraoperative mri . in a novel application , ppix - positive coordinates were recorded intraoperatively and were used to correct mris for brain shift . in fluorescence - guided surgery using 5-ala , the photosensitizer ppix is still available in the remaining peripheral tumor extensions after the gross total resection ; therefore , photodynamic therapy ( pdt ) is possible . pdt is a two - stage therapy that makes use of nontoxic light - sensitive drugs that become toxic to targeted ( malignant ) cells when they are exposed to light . the application of light results in the production of free oxygen radicals ( type i pdt , caused through electron abstraction or transfer from a substrate molecule ) and singlet oxygen ( type ii pdt ) . pdt is recognized as a valuable treatment option for localized cancers , e.g. , cancers that have not spread far from where they started and are easily accessible , such as cancers of the skin , mouth , or gastrointestinal tract , . the major difference between different types of photosensitizers is the part of the cell that they target . unlike radiotherapy , where the damage targets the cell dna , most photosensitizers target other cell structures . for example , 5-ala has been found to localize in the mitochondria , other photosensitizers exhibit high affinity for endothelial cells , and so on . because local light application is necessary for the toxic effect pdt exhibits dual - selectivity ; the sensitizer accumulates more or less selectively in the tumor , and light irradiation is applied selectively to the tumor , . various strategies are conceivable in pdt . before closing , illumination of the resection cavity with the appropriate light so - called metronomic pdt involves prolonging the ( low - dose ) illumination by one or more light distributors in the tumor cavity and guiding them subcutaneously for intermittent pdt in the immediate postoperative days . one novel approach will be the use of an implantable telemetric light delivery and monitoring system for metronomic pdt . it is likely that the oral 5-ala will need to be administered multiple times during such a prolonged treatment . further novel intraoperative fluorescence imaging systems and probes , including fluorescein sodium , dye - containing nanoparticles , and targeted nano - probes , are being researched to improve the specificity and selectivity of intraoperative fluorescence . given that gbm is typically localized in one lobe , seldom metastasizes , and exhibits local recurrence , the disease is a proper candidate for local treatments . notably , the only double - blinded trials ever conducted on gbm studied the effect of carmustine wafers implanted in the resection cavity. these three studies reported a significant increase in survival after the local application of the impregnated polymer wafers during surgery . the largest study involved 240 patients with glioma , 86% of whom had gbm , and reported a median survival increase of 2.3 months . this result is consistent with the current standard based on the non - blinded trial on conventional radiotherapy and a concomitant adjuvant tmz regime . the latter involved 573 patients with glioma , 79% of whom had gbm , and reported a median survival increase of 2.5 months . the proof of principle for the efficacy of drug diffusion with locally high concentration is evident . obviously , the extra cytoreduction by local chemotherapy is well suited to bridge the unavoidable treatment gap between surgery and radiotherapy in these quickly dividing tumor cells . remarkably , randomized trials combining local chemotherapy wafers implanted at time of resection with standard radiotherapy and a concomitant tmz regime have never been performed . this multimodal approach has been evaluated in a retrospective analysis and a phase ii study as reported on only in congress abstracts of 2006 and 2008 , . the presented survival rate for patients with newly diagnosed malignant gliomas treated by this multimodal approach is 21 months , compared with 15 months for those treated by chemoradiotherapy alone . several other agents with activity against malignant tumors of the central nervous system are being evaluated in ( preclinical ) studies via local implants and gels , including taxanes , cyclophosphamide , adriamycin , and irinotecan , . nonetheless , in routine surgery , the immediate postoperative period largely remains a therapeutic hiatus . another local approach is convection - enhanced delivery of ( biological ) agents through low - flow microinfusion . this approach was explored in several trials , typically in combination with targeted ( immuno)toxins or gene therapy , both of which are experimental therapies . here , the challenge is to evenly distribute the toxic agent and cover the ( remnant ) tumor area . back flow along the microcatheters , the infusion rate , variable tissue pressure , routes , and areas of less resistance are all matters that must be better understood before widespread use . principally , the use of real - time imaging to follow the drug ( e.g. , by the co - infusion of a suitable tracer or more directly by stable labeling ) appears to be necessary but is not currently possible . after this challenge is surmounted , the heterogeneity problem of target expression and the potency of the drug must be resolved . the newest frontier of cutting - edge local treatment is gene therapy , the transfer of genetic materials into tumor cells for therapeutic purposes . however , gene therapy is similarly hindered by the problems of proper delivery and agent effectiveness that plague other approaches . in the context of gene therapy , several methods of delivery multiple injections into the ( remnant ) tumor have an intrinsic distribution flaw to an even greater degree than low - flow micro - infusion . synthetic vehicles , such as liposomes or nanoparticles , and viral transfer methods have emerged as more promising application techniques , . intelligent polymeric nanoparticles that dissolve , swell , or collapse in response to an internal stimulus ( e.g. , ph , glucose , reduction potential , and lysosomal enzymes ) or external stimulus ( e.g. , temperature , magnetic field , ultrasound , and light ) have been engineered to achieve improved drug release at the target site ( spatial control ) and/or at the right time ( temporal control ) . drug release in response to multiple stimuli , such as ph / temperature , ph / redox , ph / magnetic field , temperature / ph / magnetic , and ph / redox / magnetic , is another novel approach . programmable site - specific drug delivery methods are in the proof - of - principle phase , but such treatments may find application in targeted therapy , e.g. , in acidic or hypoxic tumor tissue . another possibility is the use of nanoparticles responsive to external stimuli , which could provide spatial , temporal , and dose control of drug release by an on / off switch . in vivo gene transfer makes use of non - replicating viral vectors and conditionally replicating oncolytic viruses . generally , the viral genome has been genetically modified to make it replication - incompetent and non - pathogenic as well as to make the insertion of a transgene possible . oncolytic viruses act selectively by self - replication in specific tumor cells , causing cell lysis , as well as by amplifying therapeutic genes . oncolytic viruses have also been tested , including conditionally replicating adenovirus , herpes simplex virus , retrovirus , poliovirus , newcastle disease virus , and measles virus . the transduction efficiency is higher with replication - competent viruses than with replication - deficient viruses . using various vectors , the gene therapy approaches for the treatment of malignant gliomas include pro - drug activation / suicide gene therapy , anti - angiogenic gene therapy , immunomodulatory therapy , correction of gene defects , inhibition of tumor invasion , apoptosis induction , gene therapy to enhance chemotherapy and radiotherapy , and antisense and rna interference - based strategies , as was recently reviewed , . gene therapy using viral vectors has been explored in several clinical gbm treatment trials with disappointing outcomes . failure is attributed to difficulties in achieving the uniform distribution of viral vectors in the tumor and in the sufficient transduction of gbm cells . the development of real - time imaging to visualize the gene transfer may address this problem . a novel treatment frontier is to use different types of autologous cell vectors , including neural stem cells , multi - potent stromal cells , hematopoietic progenitor cells , and skin - derived stem cells , as vehicles for transgenes , . implanting or injecting stem and progenitor cells that have been genetically modified to produce antitumor substances has several advantages over viral vector - mediated gene delivery . in contrast to viral vectors , stem cells are tumor - tropic and exhibit little tropism for normal neural cells ( non - neurotropic ) . however , the immune system may neutralize non - autologous vector cells , and autologous cell vectors will not be available at the time of the first surgery . given that gene delivery specific to the tumor cells appears feasible , the most significant treatment approach has yet to be realized . theoretically , stem cell - mediated delivery of oncolytic viruses has the best potential to target and eliminate tumor cells selectively . however , the common same issues of the choice of cell vector , the choice of therapeutic transgene , the optimal route of administration , and biosafety need to be addressed in a systematic way . an extensive review is provided by bexell et al .. currently , only preclinical studies have been performed . given the number of new agents , delivery variables , and all possible combinations , testing in classical clinical trials for the most powerful modality will be impractical . screening through unconventional phase ii trials in the framework of a working - party seems desirable . further testing can occur only in the event of an overwhelming survival benefit in a phase ii trial . overall , gene therapy has the potential to exhibit therapeutic improvements in well - designed clinical studies . to date , in addition to surgical cytoreduction , only carmustine wafers in three double - blinded controlled trials have met the common standard for local intracerebral therapies ; true advancements await clinical evidence .

the biggest challenge in neuro - oncology is the treatment of glioblastoma , which exhibits poor prognosis and is increasing in incidence in an increasing aging population . diverse treatment strategies aim at maximum cytoreduction and ensuring good quality of life . we discuss multimodal neuronavigation , supra - maximum tumor resection , and the postoperative treatment gap . multimodal neuronavigation allows the integration of preoperative anatomic and functional data with intraoperative information . this approach includes functional magnetic resonance imaging ( mri ) and diffusion tensor imaging in preplanning and ultrasound , computed tomography ( ct ) , mri and direct ( sub)cortical stimulation during surgery . the practice of awake craniotomy decreases postoperative neurologic deficits , and an extensive supra - maximum resection appears to be feasible , even in eloquent areas of the brain . intraoperative mri- and fluorescence - guided surgery assist in achieving this goal of supra - maximum resection and have been the subject of an increasing number of reports . photodynamic therapy and local chemotherapy are properly positioned to bridge the gap between surgery and chemoradiotherapy . the photosensitizer used in fluorescence - guided surgery persists in the remaining peripheral tumor extensions . additionally , blinded randomized clinical trials showed firm evidence of extra cytoreduction by local chemotherapy in the tumor cavity . the cutting - edge promise is gene therapy although both the delivery and efficacy of the numerous transgenes remain under investigation . issues such as the choice of ( cell ) vector , the choice of therapeutic transgene , the optimal route of administration , and biosafety need to be addressed in a systematic way . in this selective review , we present various evidence and promises to improve survival of glioblastoma patients by supra - maximum cytoreduction via local procedures while minimizing the risk of new neurologic deficit .

Presurgical Planning and Multimodal Neuronavigation Supra-maximum Resection The Immediate Postoperative Hiatus and Local Treatments Conclusions

biologics , which represent new developments in genetic engineering and biotechnology , include t - cell modulators as well as tumor necrosis factor ( tnf)-alpha ( tnf- ) antagonists ( eg , etanercept , infliximab , and adalimumab [ humira ( d2e7 ) ; abbott laboratories , abbott park , il , usa ] ) . these bioengineered proteins target specific steps in the pathogenesis of severe immune - mediated disorders including psoriasis ( ps ) , psoriatic arthritis , and rheumatoid arthritis ( ra ) , and of several inflammatory autoimmune diseases such as crohn s disease ( cd).13 the resulting promise that tnf- antagonists have shown in the effective control of these inflammatory autoimmune diseases has revolutionized the treatment of these diseases . however , there is the potential for systemic toxicity with these therapies , related to the immunosuppressive effects , including serious infections and an increased risk of malignancy.3 the debate as to whether or not these systemic treatments increase a patient s risk of malignancy remains largely unresolved . nevertheless , there has recently been considerable attention given to the growing evidence linking biological treatments with the occurrence of malignancies or the reactivation of latent malignancies , including malignant melanoma ( mm).47 the issues concerning the long - term safety of biologics remain to be clarified . adalimumab is a fully human recombinant immunoglobulin g1 ( igg1 ) monoclonal cytokine of the innate immune system that plays a key role in the surveillance of malignancies and the response to infections.8 the authors herein report the case of a patient who developed a primary mm after treatment with adalimumab for severe refractory cd . a 54-year - old woman of european caucasian ethnicity and greek nationality and suffering from severe cd presented at the surgical department of the university hospital of alexan - droupolis , greece , in february 2011 with an asymptomatic pigmented skin lesion just above the sternum . the lesion was 0.7 cm in diameter , with an irregular border and dark color variegation . the patient reported that she had a nevus at this site that had changed color and size during the previous year . she recalled this nevus being present in childhood and that it had a well - defined border and coloration . there were no risk factors for mm such as nevus phenotypic risk factors the patient had brown hair and eyes , she did not have freckling on the face as a child , she was able to tan easily and deeply , and she was resistant to burning . there was no high total nevus count on the body surface , no history of high environmental ultraviolet radiation exposure , and no personal or family history of mm or any dysplastic nevus syndrome . the patient s medical history included severe ileal cd ( involving approximately 35 cm of terminal ileum ) that was resistant to successive medical therapies . she had suffered from cd for the previous 8 years and had been treated with adalimumab ( 40 mg subcutaneously every other week ) for the previous 3 years . after the biological treatment , remission of active cd was achieved and no adverse events were observed . the patient was one of a total of 78 patients with severe cd treated with an anti - tnf regimen ( 47 of these patients were receiving adalimumab ) at this time in the inflammatory bowel diseases unit in the university hospital of alexandroupolis . the skin lesion was excised and histology of the biopsy specimen showed a mm ( breslow thickness of 1 mm , clark level iii ) , potentially arising on a preexisting nevus . adalimumab was discontinued and the patient was treated with budesonide ( budenofalk ; galenica , athens , greece ) ( enteric - coated capsules 3 mg ) 9 mg daily for 8 weeks , following consultation with a gastroenterologist . a wider excision of the lesion , with a 2.5 cm margin , was performed and this was followed by a bilateral axillary lymph node biopsy . the patient subsequently underwent a whole - body computed tomography scan , which was negative for metastatic disease . the patient received no other treatment for mm and was monitored closely . the clinical examination and the laboratory and imaging investigations performed at the 3- and 6-month follow - up appointments were negative for disease recurrence or metastatic foci . however , the patient presented with a relapse of cd with bowel obstruction within the 2-month period of receiving budesonide therapy ; surgical treatment was proposed and discussed in an oncologic multidisciplinary team meeting held at university hospital of alexan droupolis , greece . an uncomplicated right hemicolectomy with anastomosis of the proximal ileum and transverse colon was performed in september 2011 . the oncologic multidisciplinary team did not approve postoperative treatment with azathioprine , which was the proposed by gastroenterologist for cd , and therefore the patient received no medical treatment after the surgery . a total colonoscopy performed in december 2011 and again in july 2012 revealed no sign of active cd and the patient remains closely monitored . most of these nonrandomized studies concern ps and ra patients , including children , who developed hematologic malignancies , nonmelanoma skin cancer ( nmsc ) , or melanoma after treatment with tnf blockers.14 tnf- is a cytokine of the innate immune system that is critical in the surveillance of malignancies and infection . tnf- antagonists specifically bind to both the soluble and the transmembrane forms of tnf- and include infliximab , etanercept , and adalimumab . currently the main indications of these tnf- antagonists remain ra , ps , psoriatic arthritis , and cd . the adverse events are relatively mild , while serious infections such as tuberculosis , malignancies , and neurological disorders may complicate the therapy with biologic agents.13,9 tnf- antagonists have been shown to reactivate latent tuberculosis , which in most cases occurs within the first 90 days after therapy has commenced.10,11 although evidence for the promotion of tumorigenesis with biologics is still vague , and although the relevant studies reported in the literature lack the statistical power required for large clinical trials and long - term follow - up investigational periods , there have been various case reports that give support to the logical hypothesis that tnf- inhibitors can permit malignancies to occur.2,3,12,13 while the mechanism underlying the action of tnf- antagonists has been summarized in the dermatological literature , long - term studies concerning the malignancy risks associated with these agents have been more extensively explored in the ra population.3 for instance , a systematic review and meta - analysis of nine randomized controlled trials that assessed the adverse events with infliximab and adalimumab in patients with ra suggested the presence of increased risks of malignancies and severe infections.12,14 however , other investigators do not agree there is an association.15 furthermore , few of the reports involving patients with ra have concluded that there is an increased risk of nmsc and reactivation of latent malignancies ( eg , elanoma ) in patients receiving tnf- antagonists.1,3,1621 whether the findings extracted from ra studies translate to an increased risk in patients suffering from ps is still a matter of debate.3 studies on ps in the literature exploring the risk of cancer associated with tnf- antagonists similarly lack the statistical power required for large clinical trials . it has also become clear that ps itself places patients at risk of malignancy.3 however , there are several case reports that support the theory that anti - tnf- therapy can induce or reactivate latent malignancies.2,17,2225 furthermore , most studies examining the carcinogenic risk of tnf- inhibitors suggest that tnf- inhibitors can slightly increase the risk of cancer , including nmsc and hematologic malignancies.3 the relationship between lymphoma and tnf - blockers has also been documented in a few case reports of ps patients.26 girard et al26 reported a case of a patient with long - standing ps who was diagnosed with a lymphoma after initiation of a tnf- blocker . in 2007 , fulchiero et al2 first reported the late recurrence of eruptive locoregional metastatic melanoma in two patients after initiation of tnf- antagonists for ps in one of the patients and ra in the other . the association between lymphoproliferative disorders and t - cell modulators has been studied mostly in the ra and cd populations . it is well known that inflammation in cd is associated with high levels of tissue tnf- expression , and therapies directed against this cytokine have recently become the focus of interest . infliximab , a chimeric ( 75% human and 25% murine protein ) igg1 monoclonal antibody against tnf- , is the prototype anti - tnf- agent . shown to be effective for both induction and maintenance of fistula closure in approximately two - thirds of patients , infliximab has now become the cornerstone in medical therapy for fistulizing cd . however , some patients with fistulizing cd are resistant or intolerant to infliximab . recently , adalimumab , a fully human , subcutaneously delivered igg1 monoclonal antibody that binds with high affinity and specificity to tnf but not to lymphotoxin , has been proved to be a safe and effective treatment for induction and maintenance of remission in adult patients with moderate to severe cd ( luminal and/or fistulizing ) who are resistant to conventional therapy or intolerant to infliximab.27 debate on the association between the risk of lymphoma and tnf- antagonist therapy continues . diak et al9 reported cases of nongastrointestinal malignancies in children with juvenile idiopathic arthritis and inflammatory bowel disease treated with tnf- blockers , with only one case of mm that developed in a 14-year - old child with cd receiving infliximab.9 although it was concluded that therapy with tnf- blockers in children might be associated with the development of malignancy , a clear causal relationship was not established . several studies have found an increased incidence of nmsc in patients with ra.16,21 furthermore , the development of multiple keratoacanthomas and squamous cell carcinomas has also been reported in patients with ra and ps after the initiation of etanercept or infliximab therapy.17,19 also , it is well established that quiescent or previously treated melanoma can be reactivated by immunosuppressive therapy after solid organ transplantation.18,28 however , a causal relationship between tnf- inhibitors and the development and progression or the reactivation of mm has not yet been described . the authors are aware of few reports in the literature of an association between mm and tnf- blockers . fulchiero et al2 reported two cases of reactivated mm in patients treated with etanercept for ps and adalimumab for ra . gordon et al29 reported a case of primary mm in a patient receiving adalimumab for ps . kowalzick et al30 reported the occurrence of mm in a patient treated consecutively with infliximab , adalimumab , and etanercept for ps . khan et al31 reported another case of primary mm in a patient treated with infliximab for ra . finally , katoulis et al1 reported the development of two primary mms after treatment with adalimumab in a patient with ra . to the best of the present authors knowledge , this is the first reported case of primary mm occurring after treatment with a tnf- blocker ( adalimumab ) in an adult patient with cd . despite the controversial role of tnf- inhibition in the development of mm , there is strong evidence that the initiation of tnf- blockers in the treatment and control of any serious autoimmune condition potentially causes an increased risk for skin and other malignancies.32,33 therefore , patients receiving immunosuppressive therapy should be closely monitored , with follow - ups including skin examinations . in conclusion , therapy with tnf- blockers may be associated with the development of malignancies or the malignant transformation of benign lesions . although the risk seems low and is therefore difficult to uncover in clinical trials , clinicians must weigh the possibility of malignancy against the severity of the underlying disease . further long - term controlled clinical trials and registries are required to investigate this potentially serious association .

it is recognized that immunosuppression may lead to reduced immune surveillance and tumor formation . because of the immunosuppressive properties of tumor necrosis factor ( tnf)-alpha ( tnf- ) antagonists , it is plausible that these biologics may increase the risk of the occurrence of malignancies or the reactivation of latent malignancies . tnf- antagonists have gained momentum in the field of dermatology for treating rheumatoid arthritis and psoriasis , and they have revolutionized the treatment of other inflammatory autoimmune diseases such as refractory crohn s disease . however , there is accumulating evidence that tnf- inhibitors slightly increase the risk of cancer , including malignant melanoma ( mm ) . the authors herein report the case of a 54-year - old female patient who developed a primary mm during treatment with adalimumab for severe crohn s disease resistant to successive medical therapies . the patient had been receiving this tnf- blocker therapy for 3 years before the occurrence of mm . after wide surgical excision of the lesion and staging ( based on breslow thickness and clark level ) , evaluation with a whole - body computed tomography scan was negative for metastatic disease . the long duration of the adalimumab therapy and the patient s lack of a predisposition to skin cancer suggest an association between anti - tnf- drugs and melanocytic proliferation . the authors also review the literature on the potential association between anti - tnf regimens and the occurrence of malignancies such as melanocytic proliferations . there is a substantial hypothetical link between anti - tnf- regimens such as adalimumab and the potential for cancers such as melanoma . however , the risk of malignancy with biological therapy remains to be established , and most of the relevant studies have lacked the statistical power and randomization required for large clinical trials . further long - term controlled clinical trials and registries are required to investigate this potentially serious association .

Introduction Case report Discussion Conclusion

in spite of improvements in surveillance and clinical treatment strategies , the overall survival ( os ) of hepatocellular carcinoma ( hcc ) patients is unsatisfactory due to the high incidence of tumor recurrence and metastasis even after hepatectomy and lack of effective adjuvant therapy . identification of reliable biomarkers that can be used in prediction of tumor recurrence or prognosis of hcc patients ensures more effective clinical management after curative hepatectomy . cell division cycle and apoptosis regulator 1 ( ccar1 ) was originally identified as a peri - nuclear phospho - protein reguired for apoptosis signaling by chemotherapy drugs adriamycin and etoposide independent of the p53 status of the human breast cancer cells . ccar1 plays a dynamic role in regulation of cell growth and apoptosis by serving as a cofactor of steroid / thyroid nuclear receptors , -catenin , and p53 in a variety of cell types including different cancer cells . kim et al . reported that ccar1 promoted growth of mcf-7 human breast cancer cells in response to estradiol treatment . a recent study showed that ccar1 was required for growth of prostate cancer cells in part by functioning as a coactivator of androgen receptor transcription . another study reported that ccar1 was a binding partner of -catenin and mediated anchorage independent growth of colon cancer cells . however , whether ccar1 protein is overexpressed in hcc and the prognostic significance of ccar1 protein expression in hcc have not been reported . in 167 hcc patients with long - term follow - up , ccar1 protein expression was examined by immunohistochemistry to determine whether this protein can serve as a prognostic predictor . hcc tissues from 167 patients who underwent curative hepatectomy from july 2000 to may 2006 at samsung medical center , seoul , korea were analyzed . curative resection was defined as complete resection of all tumor nodules with clear microscopic resection margins and no residual tumors , as indicated by a computed tomography scan 1 month after surgery . tumor stages were classified according to both the american joint committee on cancer ( ajcc ) staging system and barcelona clinic liver cancer ( bclc ) staging classification . intrahepatic metastasis and multicentric occurrence were defined according to the previously reported criteria . using 2 years as the cut off , all patients were followed up every 3 months after surgery and serum -fetoprotein levels were assessed and three phase dynamic computed tomography scans or magnetic resonance imaging were performed . the median follow - up period was 119.1 months ( range , 24.0 to 151.4 months ) for survivors . recurrence - free survival ( rfs ) was defined from the date of surgery until the date of tumor recurrence , metastasis , or last follow - up . os was defined from the date of surgery until the date of death or last follow - up . hcc - related death was defined as : ( 1 ) tumor occupying more than 80% of the liver , ( 2 ) portal venous tumor thrombus proximal to the second bifurcation , ( 3 ) obstructive jaundice due to the tumor , ( 4 ) distant metastases , or ( 5 ) variceal hemorrhage with portal venous tumor thrombus proximal to the first bifurcation . hepatic failure was defined as the inability of the liver to perform its normal synthetic and metabolic function as part of normal physiology . epitope retrieval was performed with 0.01 mol / l citrate buffer at ph 6.0 for 30 minutes in a pressure cooker . the sections were incubated with rabbit polyclonal antibody to ccar1 ( hpa007856 , 1:100 , sigma - aldrich inc . , st . louis , mo ) for 60 minutes at room temperature . to validate the concordance between tissue microarrays and whole tumor sections , ccar1 expression was also detected for 40 corresponding whole tumor sections randomly chosen from the 167 cases . in negative controls , positive controls ( human normal kidney ) showed nuclear ccar1 expression in epithelial cells of convoluted tubules . immunoreactivity was evaluated independently by two blinded pathologists ( c .- k.p . and s.y.h . ) . nearly homogeneous nuclear immunostaining with moderate staining intensity was observed . to determine ccar1 protein expression , the percentage of tumor cells with positive staining was scored from 0% to 100% and each sample was rated from 0 to 4 ( 0 , < 5% ; 1 , 5%-25% ; 2 , 26%-50% ; 3 , 51%-75% ; and 4 , > duplicate tissue cores for each tumor showed high levels of homogeneity for the proportion of tumor cells with positive staining . chicago , il ) . the chi - square test or fisher exact test was used to examine possible correlations between ccar1 expression and clinicopathologic factors . survival curves were constructed using the kaplan - meier method , and differences in survival were evaluated using a log - rank test . univariate and multivariate analyses of survival were performed using the cox proportional hazards regression model . factors with prognostic significance in the univariate analysis were included in the subsequent multivariate analysis . hcc tissues from 167 patients who underwent curative hepatectomy from july 2000 to may 2006 at samsung medical center , seoul , korea were analyzed . curative resection was defined as complete resection of all tumor nodules with clear microscopic resection margins and no residual tumors , as indicated by a computed tomography scan 1 month after surgery . tumor stages were classified according to both the american joint committee on cancer ( ajcc ) staging system and barcelona clinic liver cancer ( bclc ) staging classification . intrahepatic metastasis and multicentric occurrence were defined according to the previously reported criteria . using 2 years as the cut off , all patients were followed up every 3 months after surgery and serum -fetoprotein levels were assessed and three phase dynamic computed tomography scans or magnetic resonance imaging were performed . the median follow - up period was 119.1 months ( range , 24.0 to 151.4 months ) for survivors . recurrence - free survival ( rfs ) was defined from the date of surgery until the date of tumor recurrence , metastasis , or last follow - up . os was defined from the date of surgery until the date of death or last follow - up . hcc - related death was defined as : ( 1 ) tumor occupying more than 80% of the liver , ( 2 ) portal venous tumor thrombus proximal to the second bifurcation , ( 3 ) obstructive jaundice due to the tumor , ( 4 ) distant metastases , or ( 5 ) variceal hemorrhage with portal venous tumor thrombus proximal to the first bifurcation . hepatic failure was defined as the inability of the liver to perform its normal synthetic and metabolic function as part of normal physiology . epitope retrieval was performed with 0.01 mol / l citrate buffer at ph 6.0 for 30 minutes in a pressure cooker . the sections were incubated with rabbit polyclonal antibody to ccar1 ( hpa007856 , 1:100 , sigma - aldrich inc . , st . louis , mo ) for 60 minutes at room temperature . to validate the concordance between tissue microarrays and whole tumor sections , ccar1 expression was also detected for 40 corresponding whole tumor sections randomly chosen from the 167 cases . in negative controls , positive controls ( human normal kidney ) showed nuclear ccar1 expression in epithelial cells of convoluted tubules . immunoreactivity was evaluated independently by two blinded pathologists ( c .- k.p . and s.y.h . ) . nearly homogeneous nuclear immunostaining with moderate staining intensity was observed . to determine ccar1 protein expression , the percentage of tumor cells with positive staining was scored from 0% to 100% and each sample was rated from 0 to 4 ( 0 , < 5% ; 1 , 5%-25% ; 2 , 26%-50% ; 3 , 51%-75% ; and 4 , > duplicate tissue cores for each tumor showed high levels of homogeneity for the proportion of tumor cells with positive staining . chicago , il ) . the chi - square test or fisher exact test was used to examine possible correlations between ccar1 expression and clinicopathologic factors . survival curves were constructed using the kaplan - meier method , and differences in survival were evaluated using a log - rank test . univariate and multivariate analyses of survival were performed using the cox proportional hazards regression model . factors with prognostic significance in the univariate analysis were included in the subsequent multivariate analysis . a p - value of < 0.05 was considered to indicate statistical significance . the study included 139 men and 28 women with an average age of 52.5 years ( range , 17 to 76 years ) . the adjacent non - tumor liver had liver cirrhosis in 88 cases ( 52.7% ) ( table 1 ) . tumor recurrence was detected in 122 patients ( 73.1% ) , early recurrence in 98 patients ( 58.7% ) , and late recurrence in 24 patients ( 14.4% ) . eight of these 14 deaths were due to hepatic failure , five were due to non - hepatic causes , and one was due to unknown cause . in hcc , immunoreactivity for ccar1 was observed in the nucleus of tumor cells with moderate staining intensity . weak ccar1 immunoreactivity was observed in the nucleus of bile duct epithelial cells , kupffer cells , and lymphocytes . ccar1 immunoreactivity was graded as low ( 0%-50% stained tumor cells regardless of staining intensity ) or high expression ( > 50% stained tumor cells ) . high ccar1 immunoreactivity was observed in 149 of the 167 hcc cases ( 89.2% ) ( fig . high ccar1 expression showed significant correlation with microvascular invasion ( p=0.002 ) , intrahepatic metastasis ( p=0.015 ) , and higher ajcc t stage ( p=0.017 ) . high ccar1 expression showed significant correlation with early recurrence ( p=0.001 ) , but not with late recurrence ( p=0.121 ) ( table 1 ) . in univariate analysis , early recurrence showed significant correlation with microvascular invasion ( p < 0.001 ) , intrahepatic metastasis ( p < 0.001 ) , higher ajcc t stage ( p < 0.001 ) , higher bclc stage ( p=0.002 ) , lower albumin level ( p=0.049 ) , viral etiology ( p=0.001 ) , and high ccar1 expression ( p=0.002 ) . to avoid potential bias , because ajcc t stage and bclc stage were associated with vascular invasion , multiple analyses with these variables were not performed . the significance of serum -fetoprotein level was not evaluated because of missing data ( n=161 ) . in multivariate analysis , intrahepatic metastasis ( p=0.010 ) and viral etiology ( p=0.019 ) were independent predictors of early recurrence ( table 2 ) . the rfs and os rates for 167 hcc patients were 35.7% and 71.7% at 3 years , 32.0% and 60.7% at 5 years , 27.1% and 51.1% at 7 years , and 26.0% and 46.6% at 9 years , respectively . in univariate analysis , larger tumor size , microvascular invasion , major portal vein invasion , intrahepatic metastasis , higher ajcc t stage , higher bclc stage , and lower albumin level showed unfavorable effects on both rfs and os . high ccar1 expression showed unfavorable effects on both rfs and os ( table 3 ) . the 5-year rfs rate of the high ccar1 expression group was significantly lower than that of the low ccar1 expression group ( 27.1% vs. 72.2% ) ( p=0.002 ) ( fig . the mean rfs was 42.5 months for the high ccar1 expression group and 100.9 months for the low ccar1 expression group . in subgroup analysis , among patients with -fetoprotein 20 ng / ml ( n=54 ) and patients with ajcc t stage 1 ( n=62 ) , significant differences in rfs were observed between high ccar1 expression groups ( n=41 and n=50 , respectively ) and low ccar1 expression groups ( p=0.015 and p=0.004 , respectively ) ( fig . 2b and c ) . in further analysis , among patients with tumor size 5.0 cm ( n=96 ) , no significant difference in rfs was observed between high ccar1 expression group ( n=86 ) and low ccar1 expression group ( p=0.189 ) ( fig . . the 5-year os rate of the high ccar1 expression group was significantly lower than that of the low ccar1 expression group ( 58.0% vs. 83.3% ) ( p=0.015 ) ( fig . the mean os was 86.9 months for the high ccar1 expression group and 126.1 months for the low ccar1 expression group . in subgroup analysis , among patients with -fetoprotein 20 ng / ml ( n=54 ) , significant difference in os was observed between high ccar1 expression group ( n=41 ) and low ccar1 expression group ( p=0.046 ) ( fig . 3b ) . in further analysis , among patients with ajcc t stage 1 ( n=62 ) , the high ccar1 expression group ( n=50 ) tended to show an unfavorable effect on os ( p=0.089 ) ( fig . 5.0 cm ( n=96 ) , no significant difference in os was observed between high ccar1 expression group ( n=86 ) and low ccar1 expression group ( p=0.448 ) ( fig . intrahepatic metastasis and lower albumin level were independent predictors of both shorter rfs and shorter os . high ccar1 expression tended to be an independent predictor of shorter rfs ( p=0.054 ) , but not shorter os ( p=0.348 ) . patients with high ccar1 expression were more likely to suffer from recurrence than those with low ccar1 expression ( hazard ratio , 2.184 ) ( table 4 ) . the study included 139 men and 28 women with an average age of 52.5 years ( range , 17 to 76 years ) . the adjacent non - tumor liver had liver cirrhosis in 88 cases ( 52.7% ) ( table 1 ) . tumor recurrence was detected in 122 patients ( 73.1% ) , early recurrence in 98 patients ( 58.7% ) , and late recurrence in 24 patients ( 14.4% ) . eight of these 14 deaths were due to hepatic failure , five were due to non - hepatic causes , and one was due to unknown cause . in hcc , immunoreactivity for ccar1 was observed in the nucleus of tumor cells with moderate staining intensity . weak ccar1 immunoreactivity was observed in the nucleus of bile duct epithelial cells , kupffer cells , and lymphocytes . ccar1 immunoreactivity was graded as low ( 0%-50% stained tumor cells regardless of staining intensity ) or high expression ( > 50% stained tumor cells ) . high ccar1 immunoreactivity was observed in 149 of the 167 hcc cases ( 89.2% ) ( fig . 1 ) . high ccar1 expression showed significant correlation with microvascular invasion ( p=0.002 ) , intrahepatic metastasis ( p=0.015 ) , and higher ajcc t stage ( p=0.017 ) . high ccar1 expression showed significant correlation with early recurrence ( p=0.001 ) , but not with late recurrence ( p=0.121 ) ( table 1 ) . in univariate analysis , early recurrence showed significant correlation with microvascular invasion ( p < 0.001 ) , intrahepatic metastasis ( p < 0.001 ) , higher bclc stage ( p=0.002 ) , lower albumin level ( p=0.049 ) , viral etiology ( p=0.001 ) , and high ccar1 expression ( p=0.002 ) . to avoid potential bias , because ajcc t stage and bclc stage were associated with vascular invasion , multiple analyses with these variables were not performed . the significance of serum -fetoprotein level was not evaluated because of missing data ( n=161 ) . in multivariate analysis , intrahepatic metastasis ( p=0.010 ) and viral etiology ( p=0.019 ) were independent predictors of early recurrence ( table 2 ) . the rfs and os rates for 167 hcc patients were 35.7% and 71.7% at 3 years , 32.0% and 60.7% at 5 years , 27.1% and 51.1% at 7 years , and 26.0% and 46.6% at 9 years , respectively . in univariate analysis , larger tumor size , microvascular invasion , major portal vein invasion , intrahepatic metastasis , higher ajcc t stage , higher bclc stage , and lower albumin level showed unfavorable effects on both rfs and os . high ccar1 expression showed unfavorable effects on both rfs and os ( table 3 ) . the 5-year rfs rate of the high ccar1 expression group was significantly lower than that of the low ccar1 expression group ( 27.1% vs. 72.2% ) ( p=0.002 ) ( fig . the mean rfs was 42.5 months for the high ccar1 expression group and 100.9 months for the low ccar1 expression group . in subgroup analysis , among patients with -fetoprotein 20 ng / ml ( n=54 ) and patients with ajcc t stage 1 ( n=62 ) , significant differences in rfs were observed between high ccar1 expression groups ( n=41 and n=50 , respectively ) and low ccar1 expression groups ( p=0.015 and p=0.004 , respectively ) ( fig . 2b and c ) . in further analysis , among patients with tumor size 5.0 cm ( n=96 ) , no significant difference in rfs was observed between high ccar1 expression group ( n=86 ) and low ccar1 expression group ( p=0.189 ) ( fig . the 5-year os rate of the high ccar1 expression group was significantly lower than that of the low ccar1 expression group ( 58.0% vs. 83.3% ) ( p=0.015 ) ( fig . the mean os was 86.9 months for the high ccar1 expression group and 126.1 months for the low ccar1 expression group . in subgroup analysis , among patients with -fetoprotein 20 ng / ml ( n=54 ) , significant difference in os was observed between high ccar1 expression group ( n=41 ) and low ccar1 expression group ( p=0.046 ) ( fig . 3b ) . in further analysis , among patients with ajcc t stage 1 ( n=62 ) , the high ccar1 expression group ( n=50 ) tended to show an unfavorable effect on os ( p=0.089 ) ( fig . 5.0 cm ( n=96 ) , no significant difference in os was observed between high ccar1 expression group ( n=86 ) and low ccar1 expression group ( p=0.448 ) ( fig . 3d ) . in multivariate analysis , intrahepatic metastasis and lower albumin level were independent predictors of both shorter rfs and shorter os . high ccar1 expression tended to be an independent predictor of shorter rfs ( p=0.054 ) , but not shorter os ( p=0.348 ) . patients with high ccar1 expression were more likely to suffer from recurrence than those with low ccar1 expression ( hazard ratio , 2.184 ) ( table 4 ) . ccar1 plays important roles in regulation of cancer cell growth in part by recruiting multiple mediators and confers optimal conformation for transcriptional functions of nuclear receptors [ 7 - 9 ] . apoptosis induction by ccar1 involves its binding with 14 - 3 - 3 protein and down - regulation of cell growth and cell cycle regulators c - myc , topoisomerase ii , cyclin b , and p21 rac1 . diminished expression of ccar1 in human breast cancers involved methylation - dependent gene silencing associated with hypermethylation of ccar1 promoter . ccar1 functional mimetics ( cfms ) are novel small molecule inhibitors of ccar1 interaction with anaphase promoting complex / cyclosome . cfms are potent inhibitors of a variety of cancer cells including drug ( adriamycin or tamoxifen)resistant breast cancer cells but not immortalized breast epithelial cells in part by activating apoptosis signaling and diminishing the levels of key cell cycle regulatory proteins . this study demonstrates , for the first time , that high ccar1 protein expression in hcc might be a marker of shorter rfs after curative hepatectomy . high ccar1 expression showed correlation with microvascular invasion , intrahepatic metastasis , and higher ajcc t stage , suggesting the involvement of ccar1 in the progression of hcc . in the current study , tumor recurrence was detected in 35 of 54 patients ( 64.8% ) with normal serum -fetoprotein levels and in 36 of 62 patients ( 58.1% ) with ajcc t stage 1 . identification of subpopulations with poor prognosis for early stage hcc patients is critical to the optimization of personalized treatment . ccar1 protein expression may be helpful in prediction of recurrence risk in patients with normal serum -fetoprotein levels and early stage hcc after curative hepatectomy . a nanolipid formulation of cfm-4 showed superior bioavailability and inhibited growth of xenografted triple - negative breast cancer and non - small cell lung cancer in nude mice , when administered orally . the absence of ccar1 protein expression in the normal liver enhances the potential of ccar1 as a treatment target in hcc . current treatment options for hccs are limited , thus discovery and development of molecules based on ccar1 signaling could yield novel tools for treatment of hccs . for the first time , the current study demonstrated that ccar1 protein could be a potential biomarker predicting rfs in hcc patients after curative hepatectomy . in addition , ccar1 had prognostic values in hcc patients with normal serum -fetoprotein levels or early stage hcc . patients with high ccar1 expression are considered candidates for adjuvant treatment after surgical resection . further study is needed to determine the underlying molecular mechanism of ccar1 in the development and progression of hcc .

purposecell division cycle and apoptosis regulator 1 ( ccar1 ) plays a dynamic role in regulation of cell growth and apoptosis by serving as a cofactor of steroid / thyroid nuclear receptors , -catenin , and p53 in a variety of cell types including different cancer cells . however , whether ccar1 protein is overexpressed in hepatocellular carcinoma ( hcc ) and the prognostic significance of ccar1 protein expression in hcc have not been reported.materials and methodsin 167 hcc patients with long - term follow - up , ccar1 protein expression was examined by immunohistochemistry.resultshigh ccar1 protein expression was observed in 149 of the 167 hcc cases ( 89.2% ) and showed significant correlation with microvascular invasion , intrahepatic metastasis , higher american joint committee on cancer ( ajcc ) t stage , and early recurrence . high ccar1 expression showed an unfavorable effect on recurrence - free survival ( rfs ) ( p=0.002 ) . in subgroup analysis , among patients with -fetoprotein 20 ng / ml ( n=54 ) and patients with ajcc t stage 1 ( n=62 ) , significant differences in rfs were observed between high ccar1 expression groups and low ccar1 expression groups ( p=0.015 and p=0.004 , respectively ) . high ccar1 expression tended to be an independent predictor of shorter rfs ( p=0.054 ) and showed an unfavorable effect on overall survival ( os ) ( p=0.015 ) . in subgroup analysis , among patients with -fetoprotein 20 ng / ml ( n=54 ) , significant difference in os was observed between high ccar1 expression group and low ccar1 expression group ( p=0.046).conclusionccar1 protein could be a potential biomarker predicting rfs in hcc patients after curative hepatectomy . in addition , ccar1 had prognostic values in hcc patients with normal serum -fetoprotein levels or early stage hcc .

Introduction Materials and Methods 1. Study subjects 2. Immunohistochemical analysis 3. Statistical analysis Results 1. Clinicopathologic patient characteristics 2. CCAR1 protein expression in HCC 3. Prediction of early recurrence in HCC 4. Association between CCAR1 expression and prognosis of HCC patients Discussion Conclusion

an 82-year - old japanese man was referred for detailed examination of hyperkeratotic erythematous plaques on his palms and soles for 6 months . two weeks before his first visit , he had undergone lung lobectomy for right lung squamous cell carcinoma ( scc ) . laboratory findings showed elevations of eosinophil counts , serum ige , thymus and activation - regulated chemokine , scc antigen , and soluble interleukin-2 receptor levels . histological results of a skin biopsy involving the left palm showed psoriasiform dermatitis . before lung lobectomy , the hyperkeratotic erythematous plaques on the palms and soles and the erythemas on the trunk and extremities were difficult to treat with topical steroids . after lobectomy , the skin symptoms dramatically and rapidly subsided with topical steroids . therefore , we diagnosed bazex syndrome ( bs ) , also known as acrokeratosis paraneoplastica , as a paraneoplastic cutaneous disease in lung scc . the mild eosinophilia subsided and levels of scc antigen , ige , and soluble interleukin-2 receptor were reduced . bs is a paraneoplastic cutaneous disease characterized by acral psoriasiform lesions associated with an underlying neoplasm . in a previous report , a shift to the th2 immune condition was found in patients with non - small cell lung cancer , as shown in our patient . epidermal growth factor receptor ( egfr ) is also known as tumor growth factor- receptor ; it is increased in psoriatic keratinocytes . in our case , egfr expression increased in lesional keratinocytes 2 weeks after surgery and decreased 4 weeks after surgery . we speculate that a shift to th2 immune reactions in lung scc may be the pathogenesis of bs , whereby lesional keratinocytes highly express egfr in parallel with disease activity .

Statement of Ethics Disclosure Statement

thus , these teeth often may require the use of an intracanal post for retention of the core 57,25,26 . the post fits into a space created by the removal of a portion of filling material and this procedure may affect the quality of the apical seal6,7 . during mechanical preparation of the post space , the root canal filling material may be dislodged creating voids in the obturation6 , and the filling material may be twisted or vibrated . several factors can affect the integrity of the apical seal7,14 while post space is prepared , such as length of gutta - percha to maintain the apical seal7,20,21 time of removal of the filling material3,7,8,15,19,22,26,29 and method of gutta - percha removal3,8,11,12,19,27,30 . furthermore , few studies have investigated the leakage of endodontic obturation techniques and materials related to post preparation and the effect of post space preparation on the integrity of the apical seal of teeth obturated initially by different material and obturation technique6,9,23,29 . grive and radford9 ( 1995 ) have suggested that root canal preparation for the placement of a post may cause displacement of remaining filling material , when this material is insufficient or not correctly condensed . the purpose of this study was to investigate the effect of immediate and delayed post space preparation on the integrity of the apical seal of root canals obturated with two commonly used root canal sealers each having different properties and root canal filling techniques using an electrochemical method . sixty - four recently extracted human maxillary incisors of approximately equal size with single root canals and mature apices were selected for this study . all teeth were stored in ionized water with thymol and were immersed in 5.25% sodium hypocholorite ( naocl ) for 8 h to remove surface - adhered organic material . the teeth were decoronated at the cementoenamel junction using water- cooled high - speed fissure bur ( prodottoda ; diamir srl loc . the canal lengths were measured by placing a size 15 k - file ( kerr / sybron , romulus , mi , usa ) into each root canal until the tip of the file was visible at the tip of the apical foramen . root canals were instrumented at the working length with a size 50 k - file ( kerr / sybron ) using a step - back technique . then , root canals were prepared from crown to apex using sizes 1 to 4 gates glidden drills ( man , inc . the roots were irrigated with 10 ml of 5.25% naocl , and then dried with paper points ( meta dental co. ltd . sixty roots were randomly assigned to 6 experimental groups of 10 teeth each and the remaining 4 roots were used as positive and negative controls of two specimens each . group 1 : the canals were filled with cold lateral condensation of gutta - percha ( dia dent group international inc . , korea ) and a calcium hydroxide - based sealer ( sealapex ; kerr / sybron ) . post space was prepared 30 days after root canal obturation ( ah26 , sealapex , and tubuliseal were partially set after 1 week and set was complete after 4 weeks 1 ) , using size 4 gates - glidden drill and leaving 5 mm of remaining filling . group 2 : root canal filling and post space preparation were done in the same manner as described for group 1 , except for the fact the post space was created 40 min after obturation ( sealapex with a bench setting time of 40 min become commercially available 18 ) . group 3 : root canal filling and post space preparation were done in the same manner as described for group 1 , except for the fact that a resin - based sealer ( diaket ; espe , seefeld , germany ) was used . group 4 : root canal filling and post space preparation were done in the same manner as described for group 3 , except for the fact the post space was created 40 min after obturation . group 5 : root canal filling and post space preparation were done in the same manner as described for group 3 , except for the fact that the warm vertical condensation technique was used . group 6 : root canal filling and post space preparation were done in the same manner as described for group 1 , except for the fact that the warm vertical condensation technique was used . in warm vertical condensation technique , due to the direct placement to the apical portion of root canal filling and because further post space preparation is unnecessary , experimental groups of immediate post space preparation were not designed . radiographs were taken to confirm the quality of root canal obturation using a dental x - ray equipment set at 70 kvp and 8 ma ( trophy - etx , cg 320 ; trophy radiologie , vincennes , cedex , france ) . after filling and post preparation were completed , the surfaces and coronal openings of all roots were coated with two layers of sticky wax ( cavex set up regular cavex holland bv , rw hearlem , the netherlands ) , except for the apical 2 mm . between phases of the experiment , the specimens used as positive and negative controls were instrumented and the two positive control roots were filled with a loosely fitted gutta - percha cone without endodontic sealer and the two negative controls were not obturated . the apical foramen of the positive control was completely covered with two layer of sticky wax , except for the apical 2 mm , whereas that of the negative control was completely covered with two layer of sticky wax . an insulated 1-mm - diameter copper wire ( alcatel , turkey ) one piece was inserted to maintain contact with gutta - percha ( 5 mm from the apical foramen , as confirmed radiographically ) , and extended to the outside , acting as one of two working electrodes . a1-mm - diameter stainless steel wire ( alcatel , turkey ) was also cut into 10-mm long pieces , which were used as the other working electrode , being immersed into the background electrolyte from the center of the bottle . a 1.0% nacl solution as the background electrolyte the electrical current between standard and experimental electrodes in canals was measured over a period of 10 days applying a conductivity meter ( cmd 750 wpa / linton cambridge - uk ) . on the first day , the electrical conductivity was measured at baseline and then 2 , 12 , 24 , 36 , 48 , 72 , 96 , 120 and 240 h. in addition , for each group , the electrical conductivity was measured by applying 50 hz 44 v electrical current constent for 10 s with a multimeter ( thurlby 1503 ; thandar ltd . , 2 gelebe rd , huntingdan , cambs , pe18,7dx , england ) after 10 days . the kruskal - wallis ( p=0.05 ) test was applied to the results to determine whether there was a significant difference in microleakage among the groups , and the mann- whitney u test ( p=0.01 ) was used for multiple comparison of grouping variables . table 1 shows the mean apical leakage ( in micro - siemens ) and standard deviations of the experimental and control groups with time . table 2 shows the variation of apical leakage at the time intervals according to the sealer , filling technique and time of filling removal . ec= the results suggest that there was a significant difference between the root canal filling techniques ( p<0.01 ) . there were no significant difference ( p>0.01 ) between the sealers and the times of filling removal for post space preparation ( delayed versus immediate ) . the restoration of endodontically treated teeth often requires intracanal post retention6,7 . in order to create space for post , some filling material is removed from the canal , and during the preparation of this space , the root filling material may be dislodged by reason of creating voids in the obturation14 . several studies have shown that the integrity of the reaming obturation after removal of root canal filling materials may be depend on a number of factors , and some of these factors are techniques and instruments used for removal3,8,11,12,19,27,30 the length of the remaining materials20,21,25 , endodontic obturation techniques6,8,9,23 , used cements and time of their setting1,15,18,24,26 and time of removal of root filling3,7,8,15,19,22,24,26,29 . in the present in vitro investigation , the effect of the guttapercha removal on the apical seal was evaluated , and various depths7,21 and techniques8,11,28,30 were recommended for the preparation of a post space . several studies8,12,19,27 have reported that no statistical difference in apical leakage could be observed before or after post preparation with various techniques . also , hiltner , et al.12 ( 1992 ) reported no significant difference in leakage when comparing guttapercha removal using flame - heated endodontic plugger , peeso reamer , gpx burs , and touch'n heat electrically heated spreader . however , mattison , et al.21 ( 1984 ) found little difference between mechanical removal with gates glidden burs and removal with a hot plugger . haddix , et al.11 ( 1990 ) found that removal with a warm plugger produced the least leakage . in the present study , rotary instrumentation was used to remove gutta - percha because it is a faster , easier and commonly performed method of creating post space13 . in the studies investigating the minimum amount of obturation material that must remain20,21,25 to avoid disruption of the apical seal in post space preparation , it is widely accepted that 5 mm of root canal filling should be left in the apical . in our study , in the apical section of the canal was remained at least 5 mm of root canal filling in conformity with studies20,21 . several studies have investigated the leakage of endodontic obturation techniques and materials related to post preparation6,9,23 . grive and radford9 ( 1995 ) have suggested that root canal preparation for the placement of a post may cause displacement of the remaining filling material , when this material is insufficient or not correctly condensed . in his study , neagly23 ( 1969 ) found that the apical seal was not significantly affected by the use of rotary instruments regardless of the type of filling material used , when the canals had been adequately obturated . many obturation techniques have been developed to minimize gap formation along the root canal and specifically to increase the quality of the apical seal17 . it is generally agreed that the use of gutta - percha cones with a sealer is one of the most reliable methods for filling the root canal system26 . currently , lateral condensation is widely used due to its acceptable long - term results , predictability controlled placement and relative ease of use16 . however , this technique produces many irregularities in the final mass of gutta - percha , and might not fill canal fins , cul - de - sacs and isthmuses . there is also inadequate dispersion of sealer , leaving voids in and around the gutta - percha cones16 . thus , a number of different plasticized gutta - percha techniques have been introduced with the aim of securing better sealing of the canal irregularities17 in this study the effect of immediate and delayed post space preparation on the integrity of the apical seal of the root canals obturated initially by commonly used the laterally condensed and warm vertical condensed of gutta - percha techniques was searched . the post space preparation probably caused the loss of apical integrity of the lateral condensation group , and differences in microleakage between techniques were found statistically significant . gutta - percha has no adhesive qualities or adaptability to dentin of the root canal wall , so it must be used in combination with sealer . if leakage occurs , it can take place at either the gutta - percha / sealer interface or the sealer / dentin wall interface4,26 . therefore , some of the physical properties of the sealer and the possible effect of a post preparation on the sealer become important15,26 . root canal sealers serve to fill voids and minor discrepancies of fit between the gutta - percha filling and root canal wall4,18 . grosman10 ( 1976 ) shrinkage , etc . ) and postulated that sealing ability may be related to those physical properties and that manipulation of the material during or after filling could affect the ability to produce a good seal . therefore , some of the physical properties of the sealer and the possible effect of a post preparation on the sealer become important . not only an endodontic sealer should help creating a root filling that seals the canal system effectively , but it must also allow the removal of the gutta - percha . for this reason , the ability of a sealer to bond to the tooth structure is of considerable importance26 . adhesion to dentin and the lack of tensile strength of some of sealers probably creates gaps and voids caused by crumbling of the set material15 . therefore , two different sealers were evaluated in the present study , but there were no significant differences in leakage between them . the electrochemical method used in the present study appears to be the most objective way to measure leakage in the canal . it is easily comparable and analyzed , and provides the opportunity to study leakage in a continuous time period , with the advantage of being able to record the time when maximum leakage occurs20 . another important question in post space preparation is : is it best to prepare post space at the time of canal obturation or wait until the sealer has set29 ? these steps may be performed individually or simultaneously , and there is no consensus on the time interval between the endodontic treatment and post preparation ; while some authors indicate an immediate preparation26,29 , others recommend different time intervals8,15 . before complete sealer setting is achieved , it is possible that the root filling become twisted or vibrate during mechanical post space preparation , in such a way to cause disruption of the apical seal14 . according to morgano , et al.22 ( 1994 ) , in the restoration of endodontically treated teeth , space for any posts required should be gained with rotary instruments at a subsequent visit after the complete setting of the sealer , and that the apical segment of the root filling can be condensed after the coronal gutta - percha has been removed . on the other hand , bishop and briggs2 ( 1995 ) stated the need for restoration immediately following completion of endodontic treatment in order to protect the treated tooth from microbial contamination . in addition , those authors stated that , at this time , the clinician is most familiar with the canal system anatomy and reference points . comparing delayed ( > 24 h ) versus immediate removal of gutta - percha , two studies19 found little or no difference on the apical seal , while another study17 found less leakage when immediate removal of gutta - percha was done . in the present study , delayed removal of gutta - percha for post space preparation produced numerically better results in apical leakage , but without statistical significance compared to immediate preparation . the quality of the existing obturation can effect the apical seal during post space preparation . for this reason , the selection of the obturation technique is important for teeth to be restored with intraradicular post . the warm vertical condensation technique should be prefered when the post space preparation is made , and care should be taken to avoid any distruption to the integrity of the apical seal while the post space is being prepared . if any doubt exists about the quality of the root filling before or after post space preparation , a new root canal filling should be placed . the quality of the root canal filling is important to maintain the integrity of the apical seal during post space preparation . according to the results , warm vertical condensation filling technique is better than cold lateral condensation filling technique to avoid disruption of apical seal integrity ; 2 . no significant difference was found between the sealers regarding the integrity of apical seal during post space preparation ; 3 .

during mechanical preparation of the post space , the root canal filling may be twisted or vibrated , depending on several factors associated with the preparation technique and quality of filling.objective:the purpose of this study was to investigate the effect of immediate and delayed post space preparation on the integrity of the apical seal.material and methods : sixty - four extracted human incisors were biomechanically prepared using the step - back technique . sixty roots were randomly assigned to 6 experimental groups of 10 teeth each and the remaining 4 roots served as positive and negative controls ( n=2 ) . the root canals in the different groups were obturated with cold lateral and warm vertical condensation of gutta - percha and one of two sealers ( sealapex and diaket ) . post space was prepared either individually or simultaneously . an insulated copper wire was cut into 10-cm - long pieces . in each canal , one piece was inserted to maintain contact with gutta - percha and extended to the outside as one of two working electrodes . a stainless steel wire with the same dimensions of those of the copper wire , used as the other working electrode , was immersed into the background electrolyte from the center of the bottle . the electrical current between standard and experimental electrodes in canals was measured over a period of 10 days applying a conductivity meter . the kruskal - wallis test ( p=0.05 ) determined whether there was a significant difference in microleakage among the groups and the mann - whitney u test ( p=0.01 ) was used for multiple comparison grouping variables.results:the results suggest that only the differences between the root canal filling techniques were statistically significant ( p<0.01 ) . there were no statistically significant difference in the sealers and the times of filling removal for post space preparation ( p>0.01).conclusion : the quality of the root canal filling is important for the integrity of the apical seal .

INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION CONCLUSION

annual influenza epidemics affect 1020% of the population resulting in substantial mortality and morbidity worldwide . the incidence of influenza in temperate areas of the northern and southern hemispheres is characterized by seasonal cycles with marked peaks in winter , hence , suggesting a role for climate . however , the year round pattern of epidemics observed in tropical countries implies nonuniformity in the factors governing epidemics . in addition , to environmental factors , such as temperature and humidity , fluctuations in host immune response throughout the year , seasonal changes in host behaviors , and overcrowding have also been implicated to play a role in seasonal variation . annual epidemics are attributed to the continuous evolution of influenza viruses resulting from point mutations in its antigenic determinants . despite the availability of extensive time series and developed surveillance systems the influence of interannual climate changes on the incidences and trends of a range of water and foodborne diseases caused by microbiological agents has been reported . , , el nio southern oscillation ( enso ) , which is associated with two extreme events , el nio ( warm ) and la nia ( cold ) , is the most wellstudied climate phenomenon known to have the largest effect on periodic climate variability . , thus , enso can serve as a marker to study the effect of climate variability on disease patterns . despite the strong seasonality , the timing of winter influenza epidemics changes from year to year . influenzalike illness in japan is mainly caused by influenza viruses and thus its incidence is highly representative of influenza activity . , in this study , we utilized influenzalike illness data from 25 epidemics to determine possible effect of enso on the timing of peak influenza activity . influenzalike illness report data were used as a marker of influenza activity . , under the national epidemiological surveillance of infectious disease in japan , clinically diagnosed influenzalike illness cases , defined as sudden fever 38c , respiratory symptoms , and myalgia , are electronically reported on a weekly basis to the infectious disease surveillance center ( idsc ) in the national institute of infectious diseases , tokyo . the number of sentinels is decided on the basis of the size of population of the health center area where they serve . influenzalike illness report data from 1983 through 2007 was obtained from the idsc s webpage ( http://idsc.nih.go.jp/index.html ) . the peak week for each influenza season was then defined as the week during which the most number of cases are reported . largescale epidemics were defined as those for which the peak was 38 cases per sentinel per week , which represents 70% of the largest peak observed during the study period , in 1994/1995 . data on circulating types and subtypes of influenza were also available from the idsc . in japan , enso cycles are identified using a 5 months moving average of the sea surface temperature anomalies . according to the definition of the japan meteorological agency ( jma ) el nio events are associated with positive sea surface temperature anomalies ( 05c ) , while la nia events are associated with negative anomalies ( 05c ) . statistical analyses were performed using fisher s exact probability test ( twotailed ) and scheffe s multiple comparison method . influenzalike illness report data were used as a marker of influenza activity . , under the national epidemiological surveillance of infectious disease in japan , clinically diagnosed influenzalike illness cases , defined as sudden fever 38c , respiratory symptoms , and myalgia , are electronically reported on a weekly basis to the infectious disease surveillance center ( idsc ) in the national institute of infectious diseases , tokyo . the number of sentinels is decided on the basis of the size of population of the health center area where they serve . influenzalike illness report data from 1983 through 2007 was obtained from the idsc s webpage ( http://idsc.nih.go.jp/index.html ) . the peak week for each influenza season was then defined as the week during which the most number of cases are reported . largescale epidemics were defined as those for which the peak was 38 cases per sentinel per week , which represents 70% of the largest peak observed during the study period , in 1994/1995 . in japan , enso cycles are identified using a 5 months moving average of the sea surface temperature anomalies . according to the definition of the japan meteorological agency ( jma ) el nio events are associated with positive sea surface temperature anomalies ( 05c ) , while la nia events are associated with negative anomalies ( 05c ) . statistical analyses were performed using fisher s exact probability test ( twotailed ) and scheffe s multiple comparison method . we analyzed approximately 147 million influenzalike illness cases in japan , which consist of weekly time series of disease incidence from 1983 through 2007 , including 25 influenza seasons . annual influenza seasons began between november and december , peaked between january and march , and returned to the baseline between april and june for the study period ( figure 1 ) . the peak influenzalike illness activity varied between 4th to 10th weeks ( late january and early march ) during 19831994 , between 4th and 5th weeks during 19952000 , and ranged up to the 11th week ( the middle of march ) during 20012007 . data from japan s infectious disease surveillance center . dominant influenza types or subtypes circulating during each season the dashed horizontal line indicates 38 cases per sentinel per week ( representing 70% of the largest epidemic in 1994/1995 ) . epidemics with peak greater than 38 cases per sentinel per week were defined as largescale epidemics . influenza a(h3n2 ) was dominantly circulating during the majority of seasons , followed by a(h1n1 ) and b. largescale epidemics were observed in seven seasons , namely the 1985/1986 , 1989/1990 , 1992/1993 , 1994/1995 ( largest ) , 1997/1998 , 2002/2003 and 2004/2005 seasons . major antigenic drift of a(h3n2 ) occurred in five of these epidemics ( figure 1 ) . the average peak week for the study period was 62 ( figure 2 ) . early peak was observed in six of the largescale epidemics ( figure 2 ) . yearly time series for peak week , defined as that during which the greatest number of influenzalike illness cases was reported , of influenza activity in japan from 1983 to 2007 ( data from infectious disease surveillance center ) . black boxes denote years during which el nio southern oscillation ( enso ) episodes happened ( e and l indicates el nio and la nia , respectively ) , while empty boxes resemble years with normal weather ( nonenso years ) . regarding enso , 10 episodes covering 11 influenza epidemics were identified during the study s period . the peak week of influenza epidemics was earlier than the average in 10 out of 11 enso years ( 909% ) compared to 6/14 ( 429% ) for nonenso years ( p = 003 by fisher s exact probability test , twotailed ) . the average peak week for enso years , 45 09 ( n = 11 ) , was significantly earlier than that for nonenso years ( average = 76 29 , n = 14 ; p = 001 by scheffe s multiple comparison method ) . no significant difference was found between the average peak weeks during el nio ( n = 5 ) and la nia ( n = 6 ) cycles , average = 48 13 and 42 04 , respectively ( p = 085 by scheffe s method ; figure 2 ) . seasonality in disease incidence can often infer an association with weather factors and climate variability . we present here an evidence for a role of interannual climate variability on the temporal dynamics of influenza infections in japan . the evidence is based on longterm timeseries analyses of the relationships between peak influenza activity and enso as a major climate index . el nio southern oscillation arises from fluctuations in sea surface temperature of the tropical eastern pacific ocean . it is well known for its wideranging and prominent consequences on weather around the world . it contributes to the likelihood of extreme weather events , such as strong winds , heavy rainfalls , and droughts . the association between enso and cholera patterns in bangladesh and malaria epidemics in parts of south asia and south america , , were documented . our data demonstrated that peak influenzalike illness activity occurred earlier in 16 out of 25 epidemics spanning 19832007 . early peaks were observed in 10 ( 909% ) out of 11 enso and six ( 857% ) out of seven largescale epidemics ( two of these occurred in enso cylces ) . thus , we conclude that early peak influenza activity occurs in association with enso year or / and largescale influenza epidemics . our observations demonstrated a strong association between the tendency to earlier peak activity and enso in japan . the human response to weather fluctuations involved by enso , as interannual climate variability , may be different from their adaptation to usually experienced weather conditions . changes in immunity , indoor crowding , and behavioral changes could set better conditions for virus transmission and consequently earlier peak influenza activity observed during enso cycles . future studies on what factors of enso correlate mostly to influenza activity could provide better insight on such association . moreover , we demonstrated an association between early peak of influenza activity and largescale epidemics mainly occurring because of a major antigenic drift of influenza a(h3n2 ) , which dominantly circulated during these seasons . although there was an exception in the 2004/2005 season in which influenza activity peaked late though being a largescale epidemic . in this season , both influenza b and a(h3n2 ) were codominantly circulating and their peak overlapped , which could explain high incidence at peak . in a previous study , we similarly reported that the size of epidemic was correlated to the change in antigenicity and that large epidemics were mostly observed with new antigenic variants of influenza a(h3n2 ) . furthermore , the greater the number of cases at peak week the shorter was the increasingtopeak period . thus , in case of a future pandemic we may expect large number of patients within short period , rapid speed of transmission , and early pandemic peak especially in winter season . reported that higher morbidity impact of influenza was shown during cold phases of enso ( la nia ) than in hot phases ( el nio ) . on the contrary , we found no significant difference in the incidence of influenzalike illness cases among the two phases . nevertheless , higher incidence of influenzalike illness was found to be associated with a major drift in a(h3n2 ) ( data not shown ) . influenza s association with winter in temperate regions could be partly attributed to the direct influence of cold weather on virus survival or on the defense mechanisms of the upper respiratory tract . we previously showed that influenzalike illness activity peaked first in westerncentral japan rather than eastern japan where the mean temperatures are lower , suggesting a minor role of temperature in triggering peak activity . however , in tropical countries , temperature and humidity were considered to play an important role in driving the timing of influenza epidemics . different patterns and timings possessed by influenza epidemics in the tropics and temperate areas highlight both its susceptibility to trend modification in response to changing climate and the diversity of its driving factors . the average peak week for the 25 influenza epidemics investigated in this study was in the winter season ( 6th week ) . yet , our data revealed that the peak of influenza activity was delayed until early spring during the 2000/2001 and 2006/2007 seasons . we have previously demonstrated a shift of peak rotavirus activity in japan from winter to early spring , which could be related to global warming . in contrast to the gradual shift observed in the case of rotavirus from 1983 through 2003 , peak influenza activity showed a prompt shift to early spring during two seasons only . the extent to which global warming might affect the timing of influenza epidemics should be carefully followed up in future studies . the importance of influenza as a human disease is well established , and concerns about future pandemics are clearly warranted . , , our study provides evidence of association between the timing of the peak influenzalike illness , as a marker of influenza activity , and enso and antigenic change of a(h3n2 ) , both which can be forecasted half to 1 year ahead and can therefore serve as tools for predicting early peak activity , and consequently improve our preparedness for annual seasonal epidemics . finally , there are clear complexities in trying to understand relations between climate change and disease patterns . knowing that climate change and enso effects on different locations of the globe are not uniform better understanding of global influenza patterns is paramount to improving our control strategies and mitigating the disease burden .

background seasonality characterizing influenza epidemics suggests susceptibility to climate variation . el nio southern oscillation ( enso ) , which involves two extreme events , el nio and la nia , is wellknown for its large effects on interannual climate variability . the influence of enso on several diseases has been described . objectives in this study , we attempt to analyze the possible influence of enso on the timing of the annual influenza activity peak using influenzalike illness report data in japan during 19832007 . materials influenza surveillance data for 25 influenza epidemics , available under the national epidemiological surveillance of the infectious diseases , was used in this study . enso data were obtained from the japan meteorological agency . results influenzalike illness peak week varied largely during the study period , ranging between 4th and 11th weeks ( middle of winter to early spring ) . the average of peak week during enso cycles ( n = 11 , average = 45 09 ) was significantly earlier than in nonenso years ( n = 14 , average = 76 29 ; p = 001 ) , but there was no significant difference in the peak timing between hot ( el nio ) and cold ( la nia ) phases . earlier peaks of influenza activity were observed in 16 , out of 25 , epidemics . these coincided with 10 ( 909% ) out of 11 enso and 6 ( 857% ) out of seven largescale epidemics . conclusion influenza activity peak occurred earlier in years associated with enso and/or large scale epidemics .

Introduction Materials and methods Influenza surveillance and data collection and analysis Climate data Statistical analyses Results Discussion

the children with idiopathic speech and language disorders are very heterogeneous by phenotype . the diagnosis of specific language impairment ( sli ) is generally made by applying exclusion and inclusion criteria according to icd 10 ( 1992 ) . sli is diagnosed , if the speech language development is significantly delayed , deviated or unclear and if the problem does not result from defective sense organs , any known neurological or neuropsychiatric disease , anomaly of oral apparatus or mental retardation ( icd 10 1992 ; barry et al 2007 ) . shriberg and colleagues ( 2005 ) have examined diagnostic and phenotype markers for genetically transmitted speech delay . . it is also difficult to evaluate the effect of recurrent early middle ear infections , particularly those with effusion , on speech and language development . it is generally thought that lengthened periods of otitis media with effusion impair acoustic - phonetic perception and persistent brain imprinting of speech sounds ( ptok and eysholdt 2005 ) . it is difficult to assess the prevalence and incidence of hereditary speech - language disorders because of the lack of clear criteria . the prevalence of familiar sli is reported to be about 20%80% in speech disordered children ( bishop and edmundson 1986 ; tallal et al 1989 ; barry et al 2007 ) . in typically developing children the respective estimation has been about 3%8% ( bishop and edmundson 1986 ; tomblin et al 1997 ; barry et al 2007 ) . the suggestions of causes resulting in idiopathic speech - language disorders have varied during decades and affected the terminology and diagnostic criteria . consistent articulation errors have been thought to originate from speech motor problems , whereas the label dysphonology has been attached to variable and unstable speech sound errors with no detectable physiologic or physical association . it has been argued that motor impairment can not explain that a child is capable of producing a specific sound in one condition , but not in another . the inconsistency of speech sound errors has been taken as a linguistic disorder and a deficiency to use the sounds as a part of the language system ( gibbon 1999 ) . because of the difficulty in recognizing the pathophysiology of the speech sound errors the term articulation / phonology disorder ( apd ) has been used . the omissions and substitutions of speech sounds have been suggested to represent phonological , ie , linguistic , disorders , while distortions indicate articulatory problems , ie , consistent dysfunction of articulating speech organs . these errors probably origin from undifferentiated lingual gestures , and thus reflect delayed or deviant control of functionally independent regions of the tongue . the lack of differentiation of articulators has been documented by electropalatography ( gibbon 1999 ) . the lack of differentiation of the tongue tip and blade from the movements of posterior part of the tongue , as well as the difficulty in controlling the lateral parts of the tongue in sagittal groove formation , is typical in apd . apd children also tend to lift the posterior part of the tongue , when only anterior lift is required . undifferentiated tongue movement reflects insufficient spatial acuity of the articulators that is typical in immature articulation . however , the motor variability in producing speech sounds may also result in linguistic impairment in terms of phonological inconsistency of the speech sound system of the language ( gibbon 1999 ) . considering developmental speech sound errors the term speech sound disorder ( ssd ) is currently preferred because this fairly common developmental disorder may have antecedents in both articulatory ( sensorimotor ) and phonological ( cognitive - linguistic ) domains ( lewis et al 2006 ; mcgrath et al 2007 ) . it is considered to be clinically useful to subtype the children with ssd into those that present with concomitant language disorder and to those without it ( lewis et al 2006 ) . the phonetico - phonological symptoms of apd and ssd share common speech characteristics with developmental verbal dyspraxia ( dvd ) , childhood apraxia of speech ( cas ) , or developmental apraxia of speech ( das ) . das and cas have been described as disorders of speech motor programming and control . in them , the behavioral characteristics are numerous , including inconsistent speech sound errors and dysprosody ( forrest 2003 ; nijland et al 2003 ; jacks et al 2006 ) . the later developing consonants ( eg , /l/ , /r/ , /s/ ) are typically omitted or substituted by early developing ones . the initial and final consonants are often omitted , the overall consonant accuracy is low and the production of consonant clusters and complex mono- and polysyllables is typically difficult ( jacks et al 2006 ) . the vowels may also be distorted , but the distortion is not always eliminated by advancing age ( davis et al 2005 ) . children with cas ( das / dvd ) often present with severe and long lasting unintelligibility of speech , and the treatment so far lacks generally accepted effective methods ( davis et al 1998 ; jacks et al 2006 ) . in summary , the developmental disorders apd , ssd , dvd , cas , and das share common characteristics and the terms seem to be used somewhat synonymously ( cf . gibbon 1999 ; forrest 2003 ; nijland et al 2003 ; jacks et al 2006 ; mcgrath et al 2007 ) . apd , ssd , dvd , cas , and das are idiopathic expressive speech disorders that may be included in developmental language impairment ( dli ) or specific language impairment ( sli ) . even though there is so far no consensus on the diagnostic inclusion or exclusion criteria and definition of dvd ( forrest 2003 ) , a consensus has been obtained about the definition of general developmental dyspraxia ( sanger et al 2006 ) . it is typical that the child has not yet achieved the skills to perform complex motor functions ( in the absence of muscular weakness , involuntary muscular movements , or deficient selective control of muscular functions or ataxic disability to activate correct motor patterns ) appropriate for age ( sanger et al 2006 ) . this definition fits also well in dvd , which may be taken as narrow band disorder among developmental dyspraxias . disorders of motor development associated with dli and sli are common , widely recognized and reported ( bishop et al 1987 ; robinson 1991 ; rintala et al 1998 ; hill 1998 ; webster et al 2006 ) . motor problems are found to be common also in association with reading disability , even in 60% of cases it is generally thought that the children start walking without support at the average age of 12 months . according to the who multicenter study ( 2006 ) , 50% of the children have started to walk unassisted by the age of 12 months , about 25% by the age of 11 months , and 3%10% by the age of 910 months . very little attention , if at all , has been paid on speedy motor development in association with speech - language disorders . even though speedy walking babies are less common in general population , they seem to be common among speech disordered children ( haapanen 2007 ) . according to clinical observations , early walking seems to be familial also among those speech - language disordered children , particularly in children with expressive disorders that have started walking at typical age . haapanen ( 2007 ) has described a case series of 10 children with a particular developmental behavioral triad . the children have started to walk unassisted earlier than typical ( at the latest by the age of 11 months ) , and they experience with expressive speech disorders resembling das and tongue movement dysfunction ( lingual dysfunction with undifferentiated lingual movements resulting in dentoalveolar ( ie , linguoalveolar ) sound disorder . to name and describe them shortly , these children are here called speedy babies ( vauhtiveikot in finnish ; haapanen 2007 ) with spee referring to speech and dy to developmental dyspraxia or speech - language disorder such as developmental dysphasia , and dysfunction of the tongue . the aim of the present case series that describes four children with unbalanced psychomotor development ( speedy babies ) is to draw attention to children who walk early and have speech - language disorders , so that professionals may recognize the problem without delay . the coexistence of particular psychomotor characteristics is discussed in terms of interpreting them as a syndrome . in addition , a rough preliminary analysis of relevant demographic and clinical data in a series of 60 consecutive cases was carried out in order to make a preliminary estimation of the frequency of early independent walkers among speech disordered children and to explore the distribution of the age at unassisted walking between two diagnostic groups , ie , children with mainly expressive and mainly receptive verbal problems . the children were referred to special medical consultation because of unclear speech , which caused social or emotional problems . all these four children were followed up and evaluated by a speech therapist several times each . the speech - language disorder was diagnosed on the basis of multidisciplinary evaluations , which included clinical examination performed by the medical speech pathologist and also individually administered , normative , and/or orienting tests and series of tasks that were performed by the speech therapist and the neuropsychologist . also , a series of 60 consecutive cases was preliminary examined . the data gathered included the diagnostic type of speech - language disorder ( ie , whether mainly expressive vs receptive ) , the age of the occurrence of walking without support and the familial occurrence of speedy walking and speech / language / reading disabilities . the children with intelligence deficiency , abnormality of orofacial structures ( except a minimal and nonsignificantly tongue tie , ie , short lingual frenulum ) , sense organ deficiency , psychiatric diagnosis or any recognizable neurological disease were excluded , as were also the children with a mother tongue some other than finnish , or with the age of more than 6 years . the data were analyzed by mann - whitney u - test and fisher s exact test . no sucking problems or dysphagia were ever reported . according to the parents reports and the carefully made baby clinic registrations , he had begun to walk without support at the age of 10 months , he had about five verbal symbols by the age of 1518 months , and he spoke short sentences by the age of 22.5 years . although the first words occurred at a normal age , his active vocabulary grew up slowly . the child was further referred to medical speech pathologist , ie , phoniatrician , at the age of 6 years for diagnosis and a comprehensive treatment plan . speech therapeutic intervention had been started at the age of 3 years because of unclear speech . speech and language development was then assessed during 16 sessions by the speech therapist . the child presented with unintelligible words , sound substitutions , assimilations and other articulatory / phonological disorders typical of verbal dyspraxia . when re - evaluated and tested at the age of 6 years , he performed within mean age level in the reynell speech comprehension test ( finnish version ; kortesmaa et al 2001 ) , but achieved results that were slightly below mean age level in a finnish normative sentence comprehension test ( korpilahti 1996 ) . psychological evaluations carried out at the age of 6 years were based on wppsi - r ( 1995 ) , the sentence and functional order comprehension items of the nepsy test ( korkman et al 1997 ) , ro ( rorschach test 1948 ) , free play observation and as well a parent questionnaire and kindergarten report analysis . he performed better in the performance items than in the language ones , albeit no significant discrepancy between them was found . he had some conceptual difficulties , particularly related to time , and some naming difficulty , albeit on the other hand he could describe some concepts better than what was typical for a child at his age . his attention tended to impair slightly in complicated tasks . by 6 years of age , many consonants were substituted by a sound auditively resembling a velar plosive /k/. he was still actively using the posterior part of the tongue in articulation . he also tended to speak with tongue tip and blade fixed to the bottom of the mouth . however , initial consonant deletions , consistent substitutions , omissions or reversals were no more detected , except some consonant cluster assimilations . yet , inconsistent sound substitutions and reversals typical of verbal dyspraxia were observed in semi sense sentences and nonsense word repetition tasks ( such as pa - ta - ka ) , implicating the presence of latent dyspraxia . the nonverbal tongue movements were considerable slow and groping , but he was able to protrude the tongue out of the mouth , and bend it on the upper lip . the child was found active and skilled in rough motor functions such as running , climbing , bicycling , and swimming . in kindergarten he was found impulsive and not very concentrated on issues that he was not interested in . but , no attentional problems were registered in association with issues and activities that he was interested in . according to kindergarten reports he was active , helpful , talkative , but he experienced some social problems with verbal communication because of unclear speech . no suspicion of attention - deficit hyperactivity disorder ( adhd ) was ever roused even though the child was lively . the child had had only one middle ear infection so far , and no allergies were recognized . his younger sister had begun to walk without support at the age of 9 months . the parents had not suffered from speech , reading , or other learning disabilities . for rehabilitative purposes , the child was recommended to be supported by parents and kindergarten stuff in social interaction and concentration . the speech therapist set the targets of individual intervention to improve abstract and basic concepts , as well as the clarity of the speech . the possibility of dyslexia could not be ruled out , and therefore the school stuff was recommended to organize the possibility of pedagogic rehabilitation by special teacher before hand . the follow up examinations of the child s development were scheduled to communal basic health care . a physically healthy male child was born after normal pregnancy and delivery . according to parents report and health care registrations he begun to walk without support at the age of 7 months , but spoke the first words not earlier than by the age of 2.5 years . the first sentences consisting of two words occurred at the age of 3 years . at the age of 5 years , the boy was referred to a local health care speech therapist because of unclear speech . he received 10 assessment and intervention sessions by the therapist that found the child talkative , yet presenting with the short sentences and inaccurate narrating . object naming assessed by the boston test ( kaplan et al 1997 ) was not appropriate for age . the child was referred to medical speech pathologist at the age of 6 years because of speech production difficulties , and for diagnosis and further rehabilitation plan . he was then still found to have articulatory errors and dysphonology typical of developmental verbal dyspraxia . sound was still palatalized in continuous speech , but no more in single words or in controlled speech production situations . assimilations of alveolar sounds /s/ and /r/ occurred when produced in association with other consonants . the child had difficulties in protruding the tongue and bending it up to the upper lip . movement of the tongue was not fully differentiated from the movement of the lower jaw . no velopharyngeal or labial dysfunction was recognized . in neuropsychological testing by wpps1-r ( 1995 ) and the items 3 , 7 , 15 , 16 , 30 of the nepsy test ( korkman et al 1997 ) carried out at the age of 6 years , his nonverbal performance was in the average level for the age . verbal performance was poorer compared to the nonverbal performance , and it was lower than the age average for his age . auditive short term memory span was more limited than normal as tested by digits and sentences . understanding of complicated sentences and instructed narrating of a heard story was age - appropriate , even though he scored a level of low normal . it was also found important to focus on phonological awareness to improve readiness for reading . according to health care recordings and information given by parents , he walked without support by the age of 1011 months . the first words occurred before the age of 12 months , but the speech development was slow and remained limited . he was examined by a local health care psychologist and assessed and rehabilitated by a speech therapist for 25 times . the following tests and assessment were carried out : auditive reasoning , understanding , and short term memory items of the itpa - test ( kuusinen and blfield 1974 ) , a finnish articulation test ( remes 1975 ) , a finnish test for active vocabulary ( odell 1953 ) , the reynell language comprehension items ( kortesmaa et al 2001 ) and a finnish sentence comprehension test ( korpilahti 1996 ) , wppsi - r ( 1995 ) , and nepsy ( linguistic items ) ( korkman et al 1997 ) , boehm test for basic concepts ( boehm 1993 ) , drawing tasks , and play observation . speech comprehension was below normal according to the reynell test at the age of 4 years , but had become age average at the age of 5 years when re - tested . at that age , he had no problems in color naming or in basic concepts , but the speech was still severely unclear including instable sound substitutions , assimilations , and other errors typical of verbal dyspraxia . narrating was somewhat limited . the child was referred to special health care at the age of 5 years and 4 months for diagnosis and treatment design . the tongue tip was a little deformed in a little heart - like form due somewhat short but a thin and flexible frenulum of the tongue . the very mild tongue tie was not regarded to explain the phonological errors or stable lingual dysfunction resulting in dentoalveolar sound distortions , substitution , omission or assimilations . visual perception was strongly above normal , albeit fine motor hand movements , such as using a pencil or binding the bands of the shoes were regarded clumsy . his parents regarded him skilled in different rough motor functions such as climbing and running , he had no allergies and only few otitis media . individual speech therapy recommended and assisting it with intraoral removable oral - plate treatment was considered . the kindergarten personnel was involved in rehabilitation of the child s speech and language development , and an assisting nurse was introduced . his first words occurred at the age of 18 months and he formed sentences by the age of 2 years and 5 months . the speech was already from the beginning very unclear , and he had received speech therapy for three years from the age of 2 years . in the beginning his active vocabulary was below age level , but grew to be average for at the age of 4 years , when also the reynell iii test ( finnish version ; kortesmaa et al 2001 ) results indicated normal speech comprehension . according to kindergarten reports , the boy was very active , lively , and daring . he was regarded to be skilled in rough motor functions . although the child spoke a lot and had long sentences , his speech was still unintelligible and he suffered from it . he was referred to medical speech pathologist at the age of 5 years , when neuropsychological testing was also carried out . both nonverbal performance and verbal performance was at the age - appropriate level , and no significant discrepancy was found between them . , /s/ , and /l/ sounds and he was not able to produce low vowels . a physically healthy male child was born after normal pregnancy and delivery . no sucking problems or dysphagia according to the parents reports and the carefully made baby clinic registrations , he had begun to walk without support at the age of 10 months , he had about five verbal symbols by the age of 1518 months , and he spoke short sentences by the age of 22.5 years . although the first words occurred at a normal age , his active vocabulary grew up slowly . the child was further referred to medical speech pathologist , ie , phoniatrician , at the age of 6 years for diagnosis and a comprehensive treatment plan . speech therapeutic intervention had been started at the age of 3 years because of unclear speech . speech and language development was then assessed during 16 sessions by the speech therapist . the child presented with unintelligible words , sound substitutions , assimilations and other articulatory / phonological disorders typical of verbal dyspraxia . when re - evaluated and tested at the age of 6 years , he performed within mean age level in the reynell speech comprehension test ( finnish version ; kortesmaa et al 2001 ) , but achieved results that were slightly below mean age level in a finnish normative sentence comprehension test ( korpilahti 1996 ) . psychological evaluations carried out at the age of 6 years were based on wppsi - r ( 1995 ) , the sentence and functional order comprehension items of the nepsy test ( korkman et al 1997 ) , ro ( rorschach test 1948 ) , free play observation and as well a parent questionnaire and kindergarten report analysis . he performed better in the performance items than in the language ones , albeit no significant discrepancy between them was found . he had some conceptual difficulties , particularly related to time , and some naming difficulty , albeit on the other hand he could describe some concepts better than what was typical for a child at his age . his attention tended to impair slightly in complicated tasks . by 6 years of age , his spontaneous speech was still unclear and he presented with some unintelligible words . many consonants were substituted by a sound auditively resembling a velar plosive /k/. he was still actively using the posterior part of the tongue in articulation . he also tended to speak with tongue tip and blade fixed to the bottom of the mouth . however , initial consonant deletions , consistent substitutions , omissions or reversals were no more detected , except some consonant cluster assimilations . yet , inconsistent sound substitutions and reversals typical of verbal dyspraxia were observed in semi sense sentences and nonsense word repetition tasks ( such as pa - ta - ka ) , implicating the presence of latent dyspraxia . the nonverbal tongue movements were considerable slow and groping , but he was able to protrude the tongue out of the mouth , and bend it on the upper lip . the child was found active and skilled in rough motor functions such as running , climbing , bicycling , and swimming . in kindergarten he was found impulsive and not very concentrated on issues that he was not interested in . but , no attentional problems were registered in association with issues and activities that he was interested in . according to kindergarten reports he was active , helpful , talkative , but he experienced some social problems with verbal communication because of unclear speech . no suspicion of attention - deficit hyperactivity disorder ( adhd ) was ever roused even though the child was lively . the child had had only one middle ear infection so far , and no allergies were recognized . his younger sister had begun to walk without support at the age of 9 months . for rehabilitative purposes , the child was recommended to be supported by parents and kindergarten stuff in social interaction and concentration . the speech therapist set the targets of individual intervention to improve abstract and basic concepts , as well as the clarity of the speech . the possibility of dyslexia could not be ruled out , and therefore the school stuff was recommended to organize the possibility of pedagogic rehabilitation by special teacher before hand . the follow up examinations of the child s development were scheduled to communal basic health care . a physically healthy male child was born after normal pregnancy and delivery . according to parents report and health care registrations he begun to walk without support at the age of 7 months , but spoke the first words not earlier than by the age of 2.5 years . the first sentences consisting of two words occurred at the age of 3 years . at the age of 5 years , the boy was referred to a local health care speech therapist because of unclear speech . he received 10 assessment and intervention sessions by the therapist that found the child talkative , yet presenting with the short sentences and inaccurate narrating . object naming assessed by the boston test ( kaplan et al 1997 ) was not appropriate for age . the child was referred to medical speech pathologist at the age of 6 years because of speech production difficulties , and for diagnosis and further rehabilitation plan . he was then still found to have articulatory errors and dysphonology typical of developmental verbal dyspraxia . sound was still palatalized in continuous speech , but no more in single words or in controlled speech production situations . assimilations of alveolar sounds /s/ and /r/ occurred when produced in association with other consonants . the child had difficulties in protruding the tongue and bending it up to the upper lip . movement of the tongue was not fully differentiated from the movement of the lower jaw . no velopharyngeal or labial dysfunction was recognized . in neuropsychological testing by wpps1-r ( 1995 ) and the items 3 , 7 , 15 , 16 , 30 of the nepsy test ( korkman et al 1997 ) carried out at the age of 6 years verbal performance was poorer compared to the nonverbal performance , and it was lower than the age average for his age . auditive short term memory span was more limited than normal as tested by digits and sentences . understanding of complicated sentences and instructed narrating of a heard story was age - appropriate , even though he scored a level of low normal . it was also found important to focus on phonological awareness to improve readiness for reading . a male child was born after normal pregnancy and delivery with normal physical health . according to health care recordings and information given by parents the first words occurred before the age of 12 months , but the speech development was slow and remained limited . he was examined by a local health care psychologist and assessed and rehabilitated by a speech therapist for 25 times . the following tests and assessment were carried out : auditive reasoning , understanding , and short term memory items of the itpa - test ( kuusinen and blfield 1974 ) , a finnish articulation test ( remes 1975 ) , a finnish test for active vocabulary ( odell 1953 ) , the reynell language comprehension items ( kortesmaa et al 2001 ) and a finnish sentence comprehension test ( korpilahti 1996 ) , wppsi - r ( 1995 ) , and nepsy ( linguistic items ) ( korkman et al 1997 ) , boehm test for basic concepts ( boehm 1993 ) , drawing tasks , and play observation . speech comprehension was below normal according to the reynell test at the age of 4 years , but had become age average at the age of 5 years when re - tested . at that age , he had no problems in color naming or in basic concepts , but the speech was still severely unclear including instable sound substitutions , assimilations , and other errors typical of verbal dyspraxia . narrating was somewhat limited . the child was referred to special health care at the age of 5 years and 4 months for diagnosis and treatment design . the tongue tip was a little deformed in a little heart - like form due somewhat short but a thin and flexible frenulum of the tongue . the very mild tongue tie was not regarded to explain the phonological errors or stable lingual dysfunction resulting in dentoalveolar sound distortions , substitution , omission or assimilations . visual perception was strongly above normal , albeit fine motor hand movements , such as using a pencil or binding the bands of the shoes were regarded clumsy . his parents regarded him skilled in different rough motor functions such as climbing and running , he had no allergies and only few otitis media . individual speech therapy recommended and assisting it with intraoral removable oral - plate treatment was considered . the kindergarten personnel was involved in rehabilitation of the child s speech and language development , and an assisting nurse was introduced . his first words occurred at the age of 18 months and he formed sentences by the age of 2 years and 5 months . the speech was already from the beginning very unclear , and he had received speech therapy for three years from the age of 2 years . in the beginning his active vocabulary was below age level , but grew to be average for at the age of 4 years , when also the reynell iii test ( finnish version ; kortesmaa et al 2001 ) results indicated normal speech comprehension . according to kindergarten reports , the boy was very active , lively , and daring . he was regarded to be skilled in rough motor functions . although the child spoke a lot and had long sentences , his speech was still unintelligible and he suffered from it . he was referred to medical speech pathologist at the age of 5 years , when neuropsychological testing was also carried out . both nonverbal performance and verbal performance was at the age - appropriate level , and no significant discrepancy was found between them . , /s/ , and /l/ sounds and he was not able to produce low vowels . table 1 shows the summary data of the distribution of the age at unassisted walking and speech data among 60 consecutive speech and/or language disordered children studied preliminarily . of those 60 children 23 ( 38% ) began to walk unassisted at the age of 11 months or earlier ( group a ) , and 37 ( 62% ) walked at the age of 12 months or later ( group b ) . sli ( with or without symptoms of verbal dyspraxia ) was diagnosed in a total of 49 children , out of which 12/49 ( 24% ) suffered from receptive / mixed type of sli . a total of 21/49 ( 43% ) children with sli belonged to group a. more precisely , 18/37 ( 48% ) of children with expressive type of sli and only 3/12 ( 25% ) of children with receptive or mixed type of sli begun to walk unassisted at the age of 11 months or earlier ( group a ) . the age of walking without support was not evenly distributed between the two diagnostic groups . expressive sli was significantly associated with earlier walking age according to mann - whitney u test ( u = 126.00 , p = 0.022 ) . in the total group of 60 children , the ones with expressive speech production disorders ( including children with mild ankyloglossia and impaired speech without diagnosed sli ) walked significantly earlier than the ones with speech comprehension problems ( mann - whitney u test , u = 195.5 , p = 0.013 ) . counting out the children who began walking at the age of 11 or 12 months , 10/16 ( 63 % ) children were left , who had expressive speech problems and walked without support before the age of 11 months . also 6/16 ( 37% ) children were left who had expressive speech impairment , and who walked at the age of 13 months or later . none of those children that had receptive or mixed sli belonged to the earlier walking group . the difference was statistically significant ( fisher s exact test , p = 0.035 ) . the children with expressive sli had significantly more often relatives with speedy walking than the children with mainly receptive sli ( fisher s exact test , p = 0.044 ) . the groups did not differ from each in respect to familial occurrence of speech / language or reading disabilities . multidisciplinary speech evaluation and longitudinal follow ups were carried out in all the four cases described in this study . it appeared that the children demonstrated characteristics typical of apd , ssd , dvd / das , or expressive sli ( dsm iv , icd 10 ) . the milestones of gross motor development are reported in terms of starting to walk without support . data of the age at which the first unassisted steps were made and when the occurrence of the first words and sentences occurred were obtained by the parents reports and the carefully made baby clinic recordings . however , parents typically note those milestones that are often emotionally charged so that they write them into baby books that are common in finnish families . parents tend to remember the developmental milestones of walking very well , even when the child is 3-years - old , and still fairly well , when the child is 5-years - old . however , recall of first words is less exact ( majnemer and rosenblatt 1994 ) . in these children , parents reports of developmental milestones although the children were reported skilled in many rough motor functions , such as climbing , running , bicycling , and various sports , data were not collected because of the difficulties of obtaining reliable data . a behavioral pattern of unbalanced motor and speech development is typical to the present children and similar to those described earlier by haapanen ( 2007 ) . the children presented with a triad of speedy unsupported walking ability associated with expressive speech impairment including inconsistent error characteristics typical of developmental verbal dyspraxia , as well as stable misarticulations . the consistent speech sound production errors were found to result from lingual dysfunction , in terms of the inability to lift and broaden the tip of the tongue on the upper lip or alveolar ridge and the deficiency in the control of lateral borders of the tongue . the present misarticulations resemble those due to undifferentiated lingual gestures that were described by gibbon ( 1999 ) . misarticulations of dentoalveolars and low vowels probably indicate difficulty in lowering the body and back of the tongue . typically consistent misarticulations occurred in the late developing alveolar , nonnasal continuous consonants ( /s/ , /l/ , /r/ ) . they were consistently distorted , but they also underwent inconsistent dysphonological processes such as omissions , substitutions , assimilations or reversals . no labial or velopharyngeal dysfunction occurred even though labials of velar stop consonants were inconsistently correctly produced / omitted / substituted or changed by other processes typical of verbal dyspraxia . in the case of two children , the neuropsychological discrepancy criteria that is used when diagnosing sli ( eg , dsm iv , icd 10 ) was not fulfilled , which is not , however , necessary in diagnosing verbal dyspraxia ( cf . it may not be possible to differentiate between dvd and ssd , that may occur with or without coexisting language disorder ( lewis et al 2006 ) . consistent lingual dysfunction that occurred in the present children and in those described by haapanen ( 2007 ) has not so far been reported as typical of verbal dyspraxia . however , the lingual dysfunction found in speedy babies may be similar to tongue motor difficulties , of which kent ( 1992 ) has stated that speech motor variability resulting in phonetic variability of speech sounds can be so great that it results in an apparent variability in phonology , ie , in dysphonology . murdoch and colleagues ( 1995 ) have found that children with dvd may have weaker lingual musculature and exhibit significantly reduced tongue strength endurance than the controls . even though the present children experienced with only a few consistent misarticulations , their speech disorder can be regarded as moderate or severe because of particularly inconsistent sound substitutions , omissions and assimilations . /s/ , and /l/ sounds are sounds that develop late and are often misarticulated in children even at the preschool age ( luotonen 1995 ) . the finnish /r/ sound is generally found very difficult to produce because of anterior tongue tip vibration necessary for normative /r/. the persisting /r/ sound disorders in finnish children have been taken as a marker of verbal dyspraxia ( qvarnstrm et al 1993 ) . the difference between the present speedy babies and typically developing children is , however , their dysphonology which is rare in the common population ( tomblin et al 1997 ) . an interesting finding is that the present speedy babies , as well as those described by haapanen ( 2007 ) seem to be physically healthy . they also can be taken as troublemakers because of their motor activity that results in demands for continuous attention to their physical safety , particularly in early infancy . since speedy rough motor development seems to be clustered in some children with idiopathic expressive speech impairment , particularly with speech characteristics typical of developmental verbal dyspraxia and consistent lingual dysfunction , it seems justifiable to consider those behavioral phenomena occurring as a syndrome . it is obvious that early walking does not explain the speech disorder , but there may be an inherited link between those developmental characteristics . interestingly , according to our preliminary observations , children that start walking early independently seem to be fairly common among speech disordered children , particularly among those with expressive speech - language disorders . in the present preliminary series of 60 consecutive cases , none of the children who had receptive sli walked before the age of 11 months . on the contrary , speedy motor development seems to be more often associated with delayed achievement of expressive speech considered normal by articulation or phonolgy . an interesting question arises : does biodiversity among human beings also appear in terms of bipolarity of the achievement of behavioral developmental milestones ? speedy walking development also seems to be common in the family members of both speedy babies and of those speech - language disordered children , who have started to walk at typical age . the children that start walking at typical age , but present with expressive sli seem to have significantly more often relatives with speedy walking than the children with receptive sli . this has not so far evoked any attention . instead , much attention has been paid on familial co - occurrence of speech / language and reading / writing disorders ( stormswold 1998 ; barry et al 2007 ) , which also are common in speedy babies , both in the present children and in those presented by haapanen ( 2007 ) . however , speech or reading and writing disorders do not seem to be significantly associated with either of the sli subtypes , whether expressive or receptive . it seems that dyslexia may not be as seemingly associated with early independent walking as expressive speech disorders seem to be . it is obvious that the association between the timetable of independent walking and speech and learning development warrants further studies . albeit developmental disorders of motor functions have been studied much ( hill 1998 ; webster et al 2006 ) , hardly any attention is paid on atypically speedy motor development in association with speech - language disorders . an unclear phenotypic diagnostics obscures the study populations and slow down accumulation of knowledge of this population ( sli consortium 2004 ) . in order to gather phenotypic and genotypic information and to design recognition and rehabilitation strategies of individuals with communication disorders , it is important to try to search for particular developmental and behavioral characteristics that seem to cluster . to delineate and sharpen the clinical picture of speedy babies , it is worth further studies to eg , examine how common speedy unassisted walking is among speech - language disordered population , how do the behavioral characteristics vary in different ages , how variable is their cognitive profile , what are their fine motor functions like . speedy rough motor and postural control development does not guarantee skilled fine motor functions , such as eg , eye - hand motor functions ( viholainen et al 2006 ) . it is noteworthy that the present children were fairly old , ie , 5- or 6-years - old , before they were referred to a medical specialist for further studies and arrangement of the financial recourses for their rehabilitation . it is emphasized that early recognition of the problems speedy babies is important , so that treatment strategies may be developed . early intervention on speech problems and appreciation of early gross motor skills will aid in sustaining appropriate self confidence in these often lively and active children . speedy babies require health care that recognizes and respects their motor skills and supports their needs for motor activities .

even though difficulties in motor development in children with speech and language disorders are widely known , hardly any attention is paid to the association between atypically rapidly occurring unassisted walking and delayed speech development . the four children described here presented with a developmental behavioral triad : 1 ) atypically speedy motor development , 2 ) impaired expressive speech , and 3 ) tongue carriage dysfunction resulting in related misarticulations . those characteristics might be phenotypically or genetically clustered . these children did nt have impaired cognition , neurological or mental disease , defective sense organs , craniofacial dysmorphology or susceptibility to upper respiratory infections , particularly recurrent otitis media . attention should be paid on discordant and unbalanced achievement of developmental milestones . present children are termed speedy babies , where speedy refers to rapid independent walking , spee and dy to dyspractic or dysfunctional speech development and lingual dysfunction resulting in linguoalveolar misarticulations . speedy babies require health care that recognizes and respects their motor skills and supports their needs for motor activities and on the other hand include treatment for impaired speech . the parents may need advice and support with these children .

Introduction Methods Case 1 Case 2 Case 3 Case 4 Preliminary data of 60 consecutive cases Discussion

it is always difficult to write something new in a review , but is even more so when a topic has been as extensively covered over the last few years as the role of natriuretic peptides ( nps ) in the management of heart failure ( hf ) . this review will present scientific evidence on nps , with a focus on n - terminal prohormone of brain ( or b - type ) np , generally known as nt - probnp . to help the willing and curious reader , we also list five recent publications that give , in our opinion , an informed and balanced view on different uses of np assays in hf and left ventricular ( lv ) dysfunction [ 15 ] . the incessant search for evidence makes the use of biomarkers in clinical practice difficult , and fosters a larger than ever number of publications focused on the clinical utility of different biomarkers . the strength of evidence has been evaluated by metaanalyses and selective bias has been found to be a common determinant of high associations with risk . however , it should be considered that even some traditional biomarkers such as prostate serum antigen ( psa ) , blood glucose and glycosylated hemoglobin ( hba1c ) remain under extensive scrutiny . upfront it is worth reminding the reader of the principles that hold in order for any biomarker to be used to guide patient management ( e.g. diagnosis , prognosis , treatment , monitoring ) , which have been summarized as follows : accurate , repeated measurements must be available to the clinician at a reasonable cost and with short turnaround times the biomarker must provide information that is not already available from a careful clinical assessment knowing the measured level should aid medical decision making . the finding that biomarkers can contribute to improved management of the individual patient should reassure the pragmatic clinician : there is no such a thing as the laboratory obscuring the brilliant clinical investigator . rather than replacing clinical judgment , nps can support and strengthen it . with this positive attitude , which may be called bias by some , we will summarize here the pros and cons of using np testing for managing patients with hf , focusing on the following three broad areas : dyspnea triage in the emergency room np - guided treatment of chronic hf management of patients with hf in primary care and nursing homes . nps are a family of hormones produced and secreted by the heart , with both the ventricular and atrial myocardium contributing to their release into the bloodstream where they are measured by different methods . the role of the atria in releasing atrial natriuretic peptide ( anp ) and the ventricles in releasing bnp is not so clear - cut and , therefore , nps are not chamber specific . in other words , both anp and bnp are elevated in the case of atrial or ventricular dilation . nps have important actions such as vasodilation , natriuresis , and anti - fibrosis which make them major players in the adaptive response of the body to a decrease ( over the short or long term ) in cardiac function . their actions are , in essence , beneficial , at variance with other biomarkers which play no role or a negative role in the index disease ( see for example the inflammatory markers , tumor necrosis factor [ tnf ] , c - reactive protein [ crp ] , etc ) , for this reason , np analogs , such as nesiritide , have been synthesized for use in the acute treatment of hf . nt - probnp is released from the myocardium in response to cardiac volume overload and consequent wall stress ; however , myocardial ischemia per se also induces release of nps , thus explaining ( at least in part ) the raised levels of np in acute coronary syndromes . based on these facts , the assay of circulating nps has gained a role in several clinical settings involved in the management of patients with acute and chronic hf , so that the topic has been extensively reviewed . although it goes beyond the scope of the present review , it is worth summarizing the features of point - of - care ( poc ) assays compared with traditional laboratory assays of nps in a few lines . clinicians need laboratory tests which can help in decision making with regard to diagnosis , prognosis , monitoring and treatment of the patient with hf . in an acute setting such as the emergency room ( er ) , there is a need for a rapid response following a test . poc measurement of cardiac biomarkers has the potential for faster turnaround times and consequent increases in the speed of diagnosis and subsequent triage of patients within the er . more specifically , poc testing can be helpful when transport to a central laboratory is inconvenient or untenable , when timely processing is difficult or unfeasible , or where immediate access to results may improve outcomes . after blood sampling , blood must be transported to the laboratory , processed to undergo analysis , and after the assay has been performed , the result must be validated before being transmitted to the clinician . by contrast , poc testing involves just blood collection , and assay time ( usually of the order of 10 - 20 minutes ) and the result is immediately available to the clinician . the overall decrease in turnaround time also reduces the chances of error in sample handling ( i.e. tube identification , tube loss ) . among the drawbacks that must be carefully considered before introducing a poc test in routine use are : assessment of the relative performance of the new test versus the standard laboratory - based test , the direct cost which is usually higher for poc , and the correct implementation of the new test system within the health care system , accounting also for the opposition by clinical chemists . the assessment of patients with suspected hf places a substantial burden on an er , and hf is diagnosed in the er with increasing frequency . moreover , so - called acute [ decompensated ] hf occurs in many instances as an acute exacerbation of chronic hf , and is a major cause of er and hospital admission in the united states and europe . dyspnea has been observed in about half of patients who received a primary diagnosis of hf in the er , although it is well known to the er physician that a number of other conditions present with dyspnea , including asthma , pulmonary edema , chronic obstructive pulmonary disease , pneumonia , and myocardial ischemia . rapid and accurate assessment of acute hf is , therefore , a priority when a patient presents with dyspnea in an er setting . in recent years , the measurement of bnp or nt - probnp has become increasingly established for the management of patients presenting to an er with dyspnea or other symptoms suggestive of hf to distinguish acute hf from these other conditions . the largest study using the triage poc in the er was the breathing not properly trial which enrolled 1586 patients presenting to the er with acute dyspnea . two independent cardiologists diagnosed hf in 744 of the patients ( 47% ) based on clinical data . the level of bnp was by itself the strongest predictor of a correct diagnosis of hf , with a cut - off of 100 ng / l ( sensitivity 90% , specificity 76% for distinguishing hf from other causes of dyspnea ) . based on the results of the breathing not properly trial , the us food and drug administration ( fda ) granted the indication for the triage poc as an aid in the differential diagnosis of congestive hf in patients presenting with shortness of breath . more studies along the same lines have been conducted since breathing not properly without substantial disagreements with the original results . therefore , the use of a quick and reliable assay of nps in the er for triage of the patient presenting with acute dyspnea is now part of european and us cardiology guidelines and part of the clinical routine . replayed by the same group with the biomarkers in acute heart failure ( bach ) trial , a prospective , multicenter , international study of 1641 patients presenting to the er with dyspnea . mid - region pro - atrial np ( mr - proanp ) ( 120 pmol / l ) proved non - inferior to bnp ( 100 pg / ml ) for the diagnosis of hf ( accuracy difference 0.9% ) . in tests of secondary diagnostic objectives , mr - proanp levels added to the utility of bnp levels in patients with intermediate bnp values and with obesity but not in patients with renal insufficiency , the elderly , or patients with edema . none of the biomarkers was able to predict re - hospitalization or visits to the er . in conclusion , mr - proanp appears to be as useful as bnp for acute hf diagnosis in dyspneic patients . smaller studies have been conducted on nt - probnp , suggesting that it may be of use in the acute setting to help diagnosis . although it seems reasonable to consider the different nps equivalent in diagnosing hf in the er , the evidence for nt - probnp is currently limited ; however , these data should be extended by the ongoing providing rapid out of hospital acute cardiovascular treatment ( proact ) study which is currently assessing the effects of nt - probnp level assayed in the ambulance by poc ( clinicaltrials.gov nct01634425 ) . the issue now is to make good use of the test in the acute setting , rather than repeatedly showing its usefulness . the management of hf in the er is not limited to its diagnosis at presentation but encompasses its treatment leading to discharge . one of the earliest studies showing that a decrease in nt - probnp could predict a safe discharge of patients admitted with acute hf was by bettencourt and collaborators . it is well accepted that many patients with chronic hf are not adequately treated and that a simple and effective way to improve the management of these patients is highly desirable . together with other approaches ( i.e. nursing - based strategies , telemedicine , implantable hemodynamic monitoring ) , the relatively simple approach based on np guidance for the treatment of hf has been proposed and studied for over 10 years . the rationale for using np level monitoring to guide treatment of hf stems from the observation that several drugs proven to be beneficial in hf ( i.e. angiotensin converting enzyme [ ace ] inhibitors , angiotensin receptor blockers , aldosterone antagonists , beta - blockers ) decrease circulating concentrations of nps . post - hoc analyses have shown that the decrease in nps over time was associated with improvements in symptoms and survival in patients with chronic hf . the earliest evidence comes from analysis of the valsartan heart failure trial ( val - heft ; figure 1 ) . these studies showed that not only high levels of np , but also rising concentrations of nps from low to high , were associated with poorer outcomes . in this particular case , nt - probnp performed somewhat better than bnp as a prognostic marker . patients enrolled in the val - heft trial were grouped according to the trajectory of baseline and 4-month concentrations of nt - probnp or bnp below or above their respective corresponding medians . the number of patients in each of the four groups is indicated on the left , and 23-month all - cause mortality rate on the right . redrawn from data from 18 and 49 . these studies and others led to prospective studies aimed at demonstrating the use of nps as a guide to optimize the management of hf . the areas of debate have been clearly outlined and the entire topic has been extensively reviewed with meta - analyses conducted . two issues related to biomarker - guided hf therapy are still controversial : the safety of the strategy and the potential for an interaction with age . the more convincing analysis of safety is that of the trial of intensified ( bnp - guided ) versus standard ( symptom - guided ) medical therapy in elderly patients with congestive heart failure ( time - chf ; the largest trial to date with 499 patients randomized ) , showing no differences in dropout rate or in adverse events between the nt - probnp - guided therapy or symptom - guided groups . in particular , worsening of renal function and hyperkalemia , two major concerns associated with intensification of hf therapy , occurred at the same incidence in both randomized groups . the two most representative studies in np - guided therapy of hf , the nt - probnp - assisted treatment to lessen serial cardiac readmissions and death ( battlescarred ) and time - chf randomized patients stratified by age : in both studies , np - guided therapy did better in younger patients . the lack of efficacy in older patients is an intriguing issue which does not appear to be related to a higher incidence of adverse effects in this age group . aiming at a resolution of the outstanding uncertainties regarding the role of nps in guiding the management of chronic hf , the us national heart lung blood institute has funded the new guiding evidence based therapy using biomarker intensified treatment ( guide - it ) trial . guide - it is a multicenter , randomized , parallel control group , unblinded trial comparing nt - probnp guided therapy with usual care in 1100 patients with hf and lv ejection fraction ( lvef ) < 40% at discharge from hospitalization for hf . overall , in the area of np - guided therapy of chronic hf with depressed lvef , evidence is largely based on nt - probnp : 1958 patients managed with nt - probnp compared with 629 patients with bnp . although there is no strong scientific basis for choosing one or the other , the two most frequently advocated reasons for preferring nt - probnp to bnp are : ( 1 ) a relatively longer plasma half - life of nt - probnp which would lead to a higher stability of its levels in the individual patient , and ( 2 ) suggestions from the val - heft trial that it may perform better than bnp overall in predicting outcomes . an outstanding issue is whether np - guided therapy may be beneficial in patients with hf and preserved ejection fraction ( hfpef ) . in this population that more frequently consists of elderly females with comorbidities , monitoring of nt - probnp has led to contradictory findings : while in the irbesartan in heart failure with preserved systolic function ( i - preserve ) trial the angiotensin receptor blocker irbesartan reduced outcomes only in patients with low nt - probnp at entry , in the perindopril in elderly people with chronic heart failure ( pep - chf ) trial , the ace inhibitor perindopril reduced the risk only in patients with higher nt - probnp at entry . based on these contradictory results , packer warns against relying on np measurement to improve management of a patient with hfpef . indeed , a post - hoc analysis of the 123 patients with hfpef in time - chf showed that ( 1 ) outcomes in hfpef were not better than in the 499 patients with hf and reduced ef ( hfref ) , and ( 2 ) the effects of nt - probnp - guided management in hfpef were opposite compared with hfref . the authors thus suggested that , in contrast to hfref , nt - probnp - guided therapy may not be beneficial in hfpef . epidemiological trends indicating an increasing incidence and prevalence of hf call for an effective preventive approach to hf . present strategies based on risk factor intervention in high - risk populations have shown some reductions in the incidence of new - onset hf , but also difficulties in achieving adequate risk factor control and suggesting they may not be as effective as desired . nps , in addition to elements of clinical history and physical examination , are useful for the diagnosis of hf in unselected patients suspected of having new - onset hf in primary care . recent meta - analyses have confirmed that nps have robust additional diagnostic value in primary care and improve discrimination between individuals with or without confirmed hf , on top of signs and symptoms . nt - probnp , but not the classical inflammatory marker high sensitivity crp ( hscrp ) , also improved the prediction of incident major cardiovascular events and death in 4775 primary care subjects enrolled in the diabetes cardiovascular risk evaluation targets and essential data for commitment of treatment ( detect ) study . nt - probnp was also a very effective predictor of cardiovascular mortality in elderly patients ( mean age 73 years ) with symptoms of hf in primary health care . therefore , the combination of np - based screening with traditional risk factors to refine risk prediction , identify patients at - risk of cardiovascular events and intensify their management , may provide an approach to prevention of hf in primary care . this hypothesis has recently been tested in the st vincent s screening to prevent heart failure ( stop - hf ) trial that recruited 1374 participants with cardiovascular risk factors in 39 primary care practices in ireland . patients were randomized to receive usual primary care or np screening , and for those with a bnp level 50 pg / ml , echocardiography and collaborative care between their primary physician and specialist cardiovascular care . the trial demonstrated a significant reduction of newly diagnosed hf , asymptomatic lv dysfunction , and also emergency cardiovascular hospitalizations in the intervention group using np - guided care in this large community cohort . the strategy was also associated with improved risk factor control , increased use of renin - angiotensin - aldosterone inhibitors , and increased use of cardiovascular diagnostic procedures . nt - probnp has been evaluated to guide treatment in patients with symptomatic hf in primary care . in the swedish intervention study guidelines and nt - probnp analysis in heart failure ( signal - hf ) , 252 patients in primary care with a diagnosis of chronic hf and elevated nt - probnp concentration ( > 800 ng / l for males , > 1000 ng / l for females ) were randomized to two treatment strategies according to guideline recommendations , clinical signs and symptoms , with or without nt - probnp monitoring . symptoms and signs guided the investigators in making the decision to intensify treatments in both arms ; in addition , a reduction of at least 50% from baseline in nt - probnp was targeted in the nt - probnp arm , if tolerated by the patient . nt - probnp - guided treatment did not result in important improvements in the primary outcome , a composite endpoint of days alive , days out of hospital and symptom score from the kansas city cardiomyopathy questionnaire , beyond what could be achieved by education and structured hf treatment according to guidelines . nursing or care homes represent another healthcare context in which nt - probnp is currently evaluated for the early identification of ( very ) elderly subjects with lv dysfunction and clinical hf [ 38 - 40 ] , for risk stratification , and for their clinical management . although nps did not seem to provide incremental accuracy for the differential diagnosis of hf beyond that achieved with a portable echocardiograph in 405 older institutionalized individuals ( mean age 84 years ) characterized by a high rate of undetected or misdiagnosed hf , integrated programs of management of hf in nursing homes are currently evaluating the clinical utility of nps in this particular setting . repeated readmissions after hospitalization for hf are common , with approximately one in four patients re - hospitalized within 1 month after discharge . this high rate places a considerable economic burden on health care systems , leading some of them to adopt this metric as an indicator of quality of care and as a criterion for reimbursement . reducing readmissions has therefore become a major objective through strategies that aim at improving in - hospital multidisciplinary care , post - discharge follow - up , home - based monitoring and at enhancing patients education and self - management . circulating nps may be helpful in this context for early detection of new acute decompensation in the absence of physical signs or imaging findings . they can predict which patients at discharge are at higher risk for readmission and require more intensive outpatient care , and detect early objective changes in wall stress that anticipate cardiac decompensation and require an adjustment of medical therapy . individualized post - discharge intensive management of high - risk patients guided by short - term changes in nt - probnp concentration in addition to multidisciplinary care reduced re - hospitalizations and improved outcome ( combined endpoint of death or hf re - hospitalization ) in a prospective , randomized pilot study . another pilot study has recently shown that daily home self - testing and telemonitoring of nps is feasible and safe after hospital discharge for acutely decompensated hf or with signs or symptoms of worsening hf . interventional trials with home testing of nps are now warranted to ascertain whether a tailored adjustment of therapy can prevent hospital readmissions and improve clinical outcomes . however , hospital readmissions for hf account for only approximately 35% of all 30-day readmissions after hospital discharge for hf . in addition , patients hospitalized for acutely decompensated hf are often elderly with several co - morbidities , and prone to readmissions for non - cardiovascular reasons . hence , cardiac markers are likely to pick up only a fraction of all hospital readmissions and integrated multidisciplinary strategies are needed . nt - probnp is a versatile biomarker that has been extensively studied in large cohorts of individuals in the general population , subjects at risk for developing lv dysfunction and cardiovascular events , and in patients with chronic or acutely decompensated hf . there is a general consensus that it is a powerful marker of risk across the spectrum of hf stages . how to translate this unique feature into the effective clinical management of patients with hf is the current challenge that faces physicians caring for patients at home or in primary care . bedside or poc devices for easy and fast np testing may play an important role in this situation . in the context of suboptimal medical therapy in hf , and although guided therapy with nps remains controversial ( in particular with regard to safety issues and the relationship with age ) , this strategy should be pursued to reduce unacceptably high mortality and the frequent re - hospitalizations that put a serious economic burden on healthcare systems .

n - terminal prohormone of brain natriuretic peptide ( nt - probnp ) is a versatile biomarker , that has been extensively studied in large cohorts of individuals in the general population , in subjects at risk for developing left ventricular dysfunction and cardiovascular events , and in patients with chronic or acutely decompensated heart failure ( hf).in this paper , the pros and cons of using natriuretic peptide testing to manage patients with hf are presented and discussed over 3 broad areas : ( 1 ) dyspnea triage in the emergency room , ( 2 ) natriuretic peptide - guided treatment of chronic hf , and ( 3 ) management of patients with hf in primary care and nursing homes .

INTRODUCTION NATRIURETIC PEPTIDES POINT-OF-CARE TESTING OF NPS DYSPNEA IN THE EMERGENCY ROOM NP-GUIDED TREATMENT OF CHRONIC HF HF MANAGEMENT WITH NT-PROBNP FOR PATIENTS IN PRIMARY CARE AND NURSING HOMES MONITORING OF NT-PROBNP TO REDUCE READMISSIONS AFTER HOSPITALIZATION FOR HF CONCLUSIONS

breast cancer is the most common cancer among us women and remains the number two cause of cancer death for us women . the recent figures and facts published in 2008 by the american cancer association indicate that 1 in 8 us women are at risk of developing breast cancer in a life time and that 1 in 35 us women are expected to die from this disease . one of the early stages of this disease is the manifestation of clustered microcalcifications and hence detection of such features can be a life saving mechanism in the war against breast cancer . mcs detection is a challenging task for both radiologists and computer - aided diagnostic ( cad ) systems due to minute and ubiquitous nature of microcalcifications . microcalcifications appear in a mammogram as bright regions of different shapes and sizes , and attain gray - level intensities and contrast that depend on their surrounding tissue . generally , microcalcifications can be characterized as follows : ( 1 ) malignant microcalcifications tend to exist in the form of clusters of irregular shapes and sizes while benign microcalcifications tend to appear as isolated spots mostly with regular , almost round , shapes , ( 2 ) the sizes of microcalcifications are very small , varying from 0.1 mm to 1 mm with an average size of about 0.3 mm . moreover , in some cases of isolated or benign mcs , they can appear with sizes of less than 0.1 mm . also , mcs present in a dense breast tissue mostly have low local contrast adding to the challenge of identifying them within the neighboring healthy tissue . computer - aided - diagnosis technology for breast cancer is mainly intended to be a second opinion that provides the radiologists with a preliminary diagnosis results , which has been demonstrated to increase the detection sensitivity ( number of detected breast cancer ) by an average of 10% . in a study by freer and ulissey by which they used cad technology to interpret 12,860 mammograms collected over a 12-month period , they reported that employing cad techniques increased the sensitivity of breast cancer detection by 19.5% , improved early detection of malignant cancer by 5% , and no significant increase of the recall rate was noticed . several commercial breast cancer cad systems have been fda approved and currently used by radiologists , examples are image checker ( r2 technologies , usa ) and second look ( icad systems , canada ) . although most cad systems achieved high sensitivity for detecting mcs and masses , they still provide low sensitivity for amorphous microcalcifications and other mammogram abnormalities such as architectural distortions . also , current cad systems can not be used as second reader because of the low specificity of the detection estimated at 1000 benign alarms for one cancer . boyer et al . presented a questions / answers review on the use of cad technology for breast cancer . using results from several relevant studies they addressed two important questions : ( 1 ) can a cad system replace the first or double reading ? and boyer reported that a cad system neither can be used as a first reading nor as a double reading . rather , cad systems can be used as an additional step to a double reading scheme , thus increasing the sensitivity of breast cancer diagnosis . in general , a cad system for mcs detection can be modeled as a two stages system . the first stage is a preprocessing phase to enhance the visual appearance of mcs using variety of enhancement methods and background suppression techniques . also , it is in this stage a detection of suspected regions can be accomplished . in literature , researchers have applied several image processing techniques for enhancing contrast in the region of microcalcifications such as global enhancement techniques ( contrast stretching , histogram equalizing ) , fixed and adaptive local enhancement , and region based enhancement aiming to adjust the contrast of rois in relation to their surround - ing areas . in fact , standard image enhancement methods failed to achieve a satisfactory enhancement of mcs which motivated the researcher to develop feature based enhancement methods such as locating features by extracting local statistical attributes of rois , fuzzy techniques , and multiscale analysis methods [ 12 , 13 ] . other enhancement methods focused on background subtraction to increase the appearance of mcs such as highpass filtering using wavelet reconstruction , fractal modeling , and morphological operators . aiming to detect the presence of mcs , the second stage is generally composed of several image segmentation , feature extraction and classification techniques . previous studies used standard image segmentation methods such as edge detection based methods and region growing based approaches . the fuzzy nature of clustered microcalcifications made its detection a challenging task forcing the researchers to integrate several tools of feature extraction and classifications to overcome this challenge . most detection schemes make use of statistical based feature extraction methods such as high - order statistics ( kurtosis and skewness ) parameters as in [ 17 , 18 ] and image modeling based methods [ 19 , 20 ] , spectral features based methods [ 4 , 18 , 21 ] , and textural based methods [ 19 , 22 ] . supervised learning machine based methods are also commonly used for feature classification and mcs detection [ 19 , 20 , 2226 ] . a typical learning machine examples of mcs detection using combined feature extraction and supervised learning methods are artificial neural network [ 19 , 23 ] based classifiers , stochastic discriminators such as bayesian classifier [ 19 , 22 , 24 , 25 ] ) , support vector learning machines methods [ 20 , 26 ] , fuzzy based approaches , and meta - heuristic methods such as genetic algorithm ( ga ) . a more comprehensive review of automated microcalcification detection can be found in [ 3 , 11 ] . stochastic bayesian classifier has been successfully employed for solving pattern recognition problems in many applications [ 19 , 22 , 24 ] ; a popular example is its use in multi - spectral remote sensing imagery . in this work , we propose a novel framework that uses linear bayesian classifier with simplified training stage for the segmenta - tion and detection of microcalcification clusters in digital mammograms . our proposed framework can be considered a single stage detection scheme consisting of two phases , namely , feature extraction and feature classification . in the feature extraction stage , four feature - images of a mammogram are constructed . these feature - images are stacked to form a multi - dimensional feature vector for each pixel of the original mammogram . in the learning stage , the parameters of the classifier 's decision function are estimated using synthetic model of mcs while in the classification stage , the processes of learning and testing are implemented . it is in this stage the breast tissue is classified into positive ( mcs ) or negative ( healthy ) via maximizing a posteriori probability method . we believe that this framework is novel for two reasons : ( 1 ) it uses the bayesian classifier ( bc ) as first stage to segment mcs as opposed to previous work which applied bc as second stage to classify suspicious regions into mcs or background and ( 2 ) it presents an unsupervised training by which it synthesizes the training set of mcs class and accomplishes the learning phase in an efficient and simplified method that does not require large training data set which is usually created by a tedious manually pre - labeling process [ 19 , 22 ] . instead , we propose to create models of the real mcs and produce synthetic learning samples to estimate the classifier 's decision function parameters . the remaining sections of this paper are organized as follows : related work and background on stochastic bayesian classifiers and wavelet transform are briefly discussed in section 2 . section 3 presents the details of the proposed detection method while the experimental results and conclusions are presented in sections 4 and 5 , respectively . bayesian classifier ( bc ) is a statistical method used for classification by maximizing a class a posteriori probability . the application of bayesian classifiers for pattern recognition assumes a prior knowledge of an analytical expression of the probability density functions of various classes . using a sufficient statistic sample patterns of each class mathematically , bayes classifier has a decision function of the form ( 1)dj(x)=p(x j)p(j ) , j=1,2, ,m , where p(x | j ) is the conditional probability density function of n - dimensional feature vector x belonging to a class j , p(j ) is the priori probability of class j , and m is the number of classes . assuming the probability density functions ( pdf ) of the measured features are gaussian [ 19 , 22 ] then , the n - dimensional gaussian density function can be expressed as ( 2)p(xj)=1(2)n/2|cj|1/2 e(1/2)(xmj)t cj1(xmj ) , where cj and mj are the covariance matrix and the mean vector of class j , respectively . the decision function given in ( 1 ) is monotonically increasing and because of the exponential nature of the gaussian density function of the conditional probability p(x | j ) , the decision function can be rewritten as ( 3)dj(x)=ln p(j)n2ln ( 2) 12ln |cj| 12 [ ( xmj)tcj1(xmj ) ] . since the term ( n/2)ln ( 2 ) is common for all classes and assuming that all classes are equally likely , the decision function dj(x ) reduces to ( 4)dj(x)= 12ln |cj|12[(xmj)tcj1(xmj ) ] . a feature vector x is assigned to a class with a minimum distance dj(x ) , . over the last two decades , wavelet theory and its multiresolution analysis ( mra ) ability has been recognized as the most powerful tools in signal processing . unlike fourier analysis , multiresolution representa - tion of wavelet transform provides spatial - frequency localization which enables the analysis of both local and global features of the processed signal . wavelet transforms are a set of basis functions derived by translation and dilation of a single function , the mother wavelet , which has the general form of ( 5)a , b(x)=1a(xba ) . equation ( 5 ) shows that the frequency and spatial resolution of the wavelet function a , b are functions of the translation and dilation parameters b and a , respectively . a special case of ( 5 ) is obtained when translation and dilation parameters are integers with a scaling parameter a as an integer of base 2 , resulting in the dyadic wavelet transforms and leading to the construction of orthonormal wavelet basis j , k : ( 6)j , k(x)=2j/2(2jxk ) . moreover , mra using wavelet transform is based on the existence of two unique functions called wavelet and scaling functions . the scaling function is defined as ( 7)j , k(x)=2j/2(2jxk ) , where the wavelet function is defined as given in ( 6 ) . an efficient algorithm for computing discrete wavelet transform of a given discrete signal is introduced in by which , each stage of wavelet decomposition process involves extracting an approximate ( lowpass ) version and a detail ( highpass ) version of the signal . this can be easily implemented using a set of finite impulse response ( fir ) filter banks followed by sub sampling as shown figure 1(a ) . the wavelet synthesis process as shown in figure 1(b ) is accomplished by first filtering the up - sampled c and d using the synthesis lowpass h and highpass g filters , respective - ly . then , given that the set of analysis and synthesis filters satisfying perfect reconstruction conditions , an original signal x^ is obtained by adding the output of each filter , h and g , . wavelet transforms are one dimensional in nature but easily extended to analyze 2-d discrete signals . separable 2-d wavelet transform of an image is constructed by applying 1-d wavelet transform along the image rows and columns . the 2-d wavelet and scaling functions derived from 1d wavelet (x ) and scaling (x ) functions , are expressed as ( 8)(x , y)=(x)(y),h(x , y)=(x)(y),v(x , y)=(x)(y),d(x , y)=(x)(y ) , where (x , y ) represents a 2-d separable lowpass filter applied along the horizontal x vertical y directions . (x , y ) , (x , y ) , (x , y ) are 2-d separable highpass filters extracting the signal details along the horizontal , vertical , and diagonal directions , respectively . variety of wavelet transforms have been proposed and used in the literature [ 24 , 29 , 30 ] in many applications . these transforms have different features such as regularity , number of vanishing moments , orthogonality , symmetry , and compact support . however , the selection of a wavelet transform with certain features is an application dependant . the gaussian nature of the mcs gray - level distribution and their wavelet representation [ 21 , 31 ] was used along with other textural features and with a bayesian classifier to identify the true mcs [ 19 , 22 ] . proposed a two stage scheme for detecting mcs by which they used wavelet based filtering and global thresholding to identify suspicious mcs regions . then , they employed bayes and back propagation neural network ( bpnn ) classifiers to identify mcs , that is , reduce the number of false signals resulted from wavelet filtering . also , they used statistical markov random field ( mrf ) modeling along with other image processing techniques to extract primary and secondary features of suspicious mcs and to serve as inputs of the classifiers . moreover , caputo et al . used bc and another mrf - based method , statistical spin - glass mrv ( sgmrv ) , to model the different regions within a mammogram . they followed it by a maximum a posteriori probability bayesian classifier and were able to demonstrate that their proposed approach outperformed both the back propagation artificial neural network and the nearest neighbor classifier detection systems . several wavelet based mcs detection [ 14 , 1719 , 21 , 31 ] and enhancement [ 12 , 13 ] schemes have been proposed in literature . strickland and hahn , for example , concluded that using an appropriate wavelet filter , one can easily detect and segment mcs within wavelet domain by thresholding the wavelet coefficients before the reconstruction process . modeling mcs as highpass local anomalies , wang and karayiannis also applied wavelet filtering , which is an elimination of an approximate wavelet subband and using detail subbands for reconstruction , they were able to detect mcs . following , several studies used this wavelet filtering method for detecting suspected mcs and to reduce false results . some studies demonstrated that least asymmetric daubechies are more suitable for enhancement of the mammogram images such as in microcalcification detection while other works demonstrated that the design of a spatial wavelet filter with high regularity is more successful in detecting microcalcifications than conventional wavelet filters such as the orthogonal daubechies db4 . moreover , the none - stationary nature of mammogram image texture motivated many researchers to design wavelet transforms using adaptive filters which has been reported to be more efficient than fixed or none - adaptive fir filter in the detection of low contrast mcs present in the denser breast tissue . learning machines for pattern recognition , such as artificial neural network , support vector machines and maximum a posteriori probability ( map ) classifiers consist of two phases : supervised learning and testing phases [ 19 , 20 , 22 , 25 , 26 ] . in the learning phase , a group of training samples that represents different objects or patterns to be extracted are selected manually to optimize the classifier 's decision function while in the testing phase , the trained classifier is used to classify features contained in new data sets or the independent samples . our proposed classification approach , figure 2 , follows the general structure of the classical learning machines but it uses a simplified learning stage denoted here as self - learning phase . such a process can be relatively described as an unsupervised learning since it does not require the huge number of training samples of mcs to be extracted in advance form different data mammograms as the case of classical supervised learning [ 19 , 22 , 24 ] and instead it synthesizes these samples and use them as training data . in this work , detection is modeled as a two - class pattern recognition problem where the first class , 1 , is the clustered mcs group and the second class , 2 , is the healthy breast tissue . modeling of microcalcifications : the training samples of mcs class are synthesized by blending a synthetic model of mcs with a mammogram image . feature extraction : linear and none - linear transforms are used to extract three features of each pixel of a mammogram image . these three feature images along with the graylevel mammogram image are registered spatially to form a 4d pattern vector x = [ x1 x2 x3 x4 ] of each class j as shown in figure 3 . in figure 3 , each pattern vector x is represented by a set of four components described as follows : graylevel or image intensity local maxima ranked using local histogram spectral feature extracted using wavelet transform singularity detection by detecting point discontinuity . learning phasethe proposed learning process estimates the classifier 's decision function parameters of each input mammogram , see figure 2 . unlike the classical method which collects the training sets from different mammograms , the proposed approach extracts the training samples of different classes from the input mammogram itself as follows : for mcs class , it models the mcs and creates synthetic training samples of mcs class for that mammogram , the locations of these samples are identified using the binary model of the synthetic mcs . for the healthy breast tissue class , the training data are collected from two rois chosen randomly within the breast region . the proposed learning process estimates the classifier 's decision function parameters of each input mammogram , see figure 2 . unlike the classical method which collects the training sets from different mammograms , the proposed approach extracts the training samples of different classes from the input mammogram itself as follows : for mcs class , it models the mcs and creates synthetic training samples of mcs class for that mammogram , the locations of these samples are identified using the binary model of the synthetic mcs . for the healthy breast tissue class , the training data are collected from two rois chosen randomly within the breast region . parameters estimation : pattern recognition using stochastic bc is based on the estimation of the probability density function of each class . assuming that the measured features of each class have a gaussian probability distribution , the classifier 's decision function can be computed as given in ( 4 ) requiring the estimation of the covariance matrix and the mean vector of each class . if the training set of each class is a sufficient statistically , one can efficiently estimate the distribution parameters ( i.e. , covariance matrix and mean vector ) of each class . further discussion of the parameter estimation for bayesian classifier used in this work is presented in section 3.4 . bayesian classification : the optimized classifier is applied to perform a pixel based classification of the breast region into microcalcification and healthy tissue . in this work , the classification results are binary 0 or 1 and they are used to create a binary image by assigning a binary 1 to pixels classified as class , 1 ( or mcs ) , while a binary 0 is assigned to pixels classified as class 2 ( or healthy breast tissue ) . post processing : the purpose of this step is to reduce the false classifications and to improve the classification results through the integration of some of the physiological traits of breast tissue and clustered microcalcifications . in this work , we use a method proposed in as an attempt to generate a model for real mcs as illustrated in figure 4 . in this method , a new mc model is derived from the standard model ( stdmodel ) , a binary model of synthetic mcs , using input image and the modeling constant k. that is , each gray level value from synthetic pixels is assigned initially a fraction that is proportional to the constant k of its corresponding mammogram pixel and through a blending process , a hybrid image is created from the original mammogram and the modified mcs model . such process is a pixel by pixel addition of the mcs model and mammogram followed by smoothing of the synthetic pixels using lowpass filter h , an example of the outcome of this scheme is shown in figure 5 . our experimental results indicate that k should be chosen based on the statistics of the breast tissue of the mammogram such as the mean and variance of the breast tissue intensity values . mcs synthesis method presented in this work has a significant difference from the one introduced in . this method has an explicit use of a modeling constant to control the synthesis of different types of mcs to ensure that a synthetic mc impersonates a real mc as much as possible . it is also worth noting that the purpose of using synthetic mcs in was to provide a testing material for the detection scheme , while it is employed in this work as a detection tool and a control parameter of the scheme . this work uses the general structure of pattern recognition using bayesian classifier which stacks and spatially registers a group of feature images . each mammogram is represented by a stack of four images ; ( 1 ) gray - level feature from original image , ( 2 ) feature image extracted using local maxima ranked using their local histogram , ( 3 ) highpass filtered image extracted using discrete wavelet transform , and ( 4 ) point singularity detected using euclidian distance ed8 . highpass filtering using discrete wavelet transform has proved to be a useful tool for detecting suspicious mcs [ 14 , 17 , 19 ] . in , the authors reported that orthogonal wavelet filters such db4 are more appropriate of detecting mcs since they have higher sensitivity to the presence of microcalcifications than other wavelet filters . also , the spike - like behavior of db4 wavelet transform justifies the successful use of this wavelet filter for detecting specious mcs in [ 17 , 19 ] . therefore , we decided to employ db4 to extract the spectral features of mcs and to use this feature as one input feature of the bayesian classifier . the basic idea behind this analysis is that , mcs represent highpass anomalies lay on a stationary lowpass background contributing to the detail subbands rather than to the coarse scale subbands of the wavelet multiresolution representation . in , the authors demonstrated that the features of mcs can be made more obvious after suppressing the background data , which is accomplished by eliminating the wavelet coefficients within coarse scale subband and reconstructing an image from detail subbands . microcalcifications also appear in intensities that are higher than that of the surrounding healthy tissue . therefore , a pixel belong to a microcalcification region is expected to experience a larger gray - level difference from its local neighborhood than that of a healthy one . one approach to extract this type of singularity is by employing a point detection kernel as shown in figure 7 . in this work , the point singularity feature ed8 of each pixel is defined as the sum of the absolute difference of a pixel graylevel and those of its 8-neighboures . example of this feature and other extracted features is demonstrated in figure 8 : ( 9)ed8ij=k2 k1|ii+k2,j+k1ii , j| , k1=0,1 , k2=0,1 . pattern recognition methods are in general supervised leaning machines [ 19 , 20 , 2226 ] , they partition the population data into training and validation sets . in such approaches , the training samples which are usually labeled manually , are employed to estimate the parameters of the classifier 's decision function [ 19 , 22 ] . our proposed approach can be considered an unsupervised method and thus it does not require the training set of mcs class to be extracted from real mammograms as in the supervised manner . instead , an adaptive and simple learning scheme is employed to estimate the classifier 's parameters . the advantage of the proposed training scheme over the classical one is the use of synthetic mcs as training samples for the mcs class rather than using real mcs extracted from mammograms as practiced in the supervised methods . training stage starts by extracting four features from the breast tissue ; these feature images are stacked to form a multidimensional feature vector of each pixel . the learning phase of the classifier is accomplished by blind , or unsupervised , selection of training data . such selection is done by employing the binary mcs model to identify the training samples for mcs region while two distinct regions randomly selected within the mammogram are used to locate the healthy breast tissue candidates . a drawback of this random selection of the training samples of healthy breast tissue is the possibility that these regions may lay over breast areas that have low probability of developing malignant microcalcifications such as fatty or background regions . the negative impact of this practice can be eliminated by having a preprocessing step in which , the user mark the two regions within the glandular breast area or by employing a preprocessing step to identify the glandular breast region . the learning process we propose has many advantages over the classical one ; first , is the simplicity of the process with respect to the size of learning data , second , training samples of all classes , including the mcs , were selected manually in [ 19 , 22 , 24 ] while the training set of the significant class , which is the mcs , is synthetically constructed in this work . another advantage of the proposed learning phase is that the training process of the classifier is adaptive to the breast tissue as the parameters of the classifier 's decisions function are estimated using self - learning method based on the input data . the first challenge occurs when the synthetic training samples are not statistically sufficient which may produce underestimation of the classifier 's parameters . this limitation can be mostly attributed to the simplicity of the proposed modeling itself , that may have add some constraints on the ability to generate mcs training set of sufficient statistics from a single mammogram . the other challenge occurs when regions representing the training samples of healthy tissue include members of the other class ( real mcs ) . while this work has not investigated the first problem , left it out as future work , the second challenge was addressed by using relatively large number of training samples of healthy ( or background ) class extracted from two different mammogram regions . the differencing in the sample size is significant due to the fact that mammogram texture is nonstationary and many samples of none - mcs class are available compared to the number of samples representing mcs that estimated to be no more than 1% of the whole mammogram . this work used about 4300 samples to represent the healthy ( or background ) class obtained from two distinct regions , which is about 50 times the size sample of mcs class . we investigated the effect of the sample size on the performance of the proposed detection scheme and the results indicated that a better detection can be obtained when two different regions used to extract the training samples of the healthy class than a single region . the results also indicated that the sample size of the healthy class must be larger , three times or more , than that of mcs class for better detection performance . assuming the two classes are equally likely and using the training pattern of both classes , our feature vectors , the decision function of the classifier is constructed by approximating the mean vector and covariance matrix for each class as given by ( 4 ) . the modeling constant , k , plays a significant role as it controls the appearance of synthetic mcs and their blending with the surrounding breast tissue . on approach that might be useful for selecting an appropriate value of the modeling constant prior to the training and classification stages is by measuring the difference between the corresponding components of the estimated mean vectors and the ratio of the corresponding diagonal entries ( feature variances ) of the estimated covariance matrices . our investigation of both measures concluded that interpreting the mean difference is more obvious , that is easier to make a conclusion , and can be employed for better detection results . analyzing the interclass mean difference leads to identifying two cases ; the first case occurs when a large value of the modeling constant is used , one that produces a large mean difference and leads to detect a single tone detail of the image , which might fail to detect the targeted mcs . this problem can be eliminated by adjusting the modeling constant to lower values before proceeding with training and segmentation stages . the second case occurs when a very small mean difference is used which decreases the discrimination power between classes and leads to an increase in false signals . testing the discrimination power of the classifier is usually accomplished by using the decision functions of the bc , ( 4 ) , which is computed using the estimated covariance matrix and mean vector to classify an independent set of samples followed by computing the misclassification rate . the segmentation results are interpreted , form the classification results , as a target ( microcalcification ) and represented by a binary 1 and a nontarget ( healthy tissue ) represented by a binary 0 . all mammograms used in this work are from a mini mammographic database provided by mammographic image analysis society ( mias ) , which includes 23 cases with 28 mcs . each mammogram from the database is a 1024 1024 pixels and with a spatial resolution of 200 m / pixel . these mammograms are already labeled with radiologist findings in terms of location and size of pathology results and tissue type which we found to be very useful when assessing experimental results . the proposed scheme starts by modeling of the mcs in each mammogram as explained in section 3.1 . the most significant step of this process is the selection of the modeling constant k , a typical value of k can be chosen between 0.1 and 1 . then , spatial , textural , and spectral features of all pixels are extracted and used as inputs to the bayesian classifier . the feature vector of each pixel is composed of the following : ( 1 ) brightness ( or graylevel ) , ( 2 ) local maxima ranked based on the tail ratio of their local histogram estimated within 9 9 neighborhoods , ( 3 ) highpass filtered image obtained from suppressing coarse ( or approximates ) of the 2-level wavelet representation ( db4 filters were used ) and reconstructing an image from detail subbands , and ( 4 ) point singularity values computed using ( 9 ) as presented in section 3.2.2 . stochastic bayesian classifier optimized by a simplified self - learning phase is used to segment ( or classify ) all image pixels into mcs or healthy ones . experimental results demonstrate that applying this proposed approach to the whole mammogram without extraction or prior knowledge of breast region produces more false positive signals than those resulting from using breast region extracted form the whole mammogram . this result is illustrated in figures 9(a ) and 9(c ) which also indicates that these false signals are mainly localized outside the breast region and can be significantly reduced using otsu 's thresholding . examples of this step results are demonstrated in figures 9(b ) and 9(d ) . in order to test the abilities of the proposed scheme for segmenting the whole mammogram and detecting the mcs , we used a simple thresholding scheme , otsu 's method , as a postprocessing instead of employing a prior breast region extraction or using some regional context within the detection process . another advantage of using otsu 's thresholding as postprocessing was eliminating all misclassifications occurring along the breast border and outside the breast region . such suppression process improved the detection performance by significantly reducing the overall number of false results , or misclassifications , while maintaining the detected mcs . this assessment would have been much more challenging without having the location and the size of true , real , mcs as documented by mias database . results indicate that synthesizing the training samples of mcs class and specifically the selection of the modeling constant k plays a significant role in the performance of the proposed classifier and the detection results . they also show that k values should be chosen based on statistics of the breast tissue characterized by the separation between the brightness of the region of mcs and that of the background tissue and the variance of the breast region . that is , the optimal k value is mostly correlated to the normalized mean difference ( nmd ) computed from the difference of the average graylevel ( brightness ) of the mcs and that of the of breast region . analyzing the breast regions of size 256 256 pixels extracted from each group indicates that a dense - glandular breast tissue has a larger intensity mean and variance than those of fatty breast tissue . moreover , mcs that may be present in a fatty breast tissue have a larger nmd than those of mcs in dense - glandular breast tissue . our results show that the modeling constant k can be adaptively chosen based on the type of breast tissue and the statistics of the breast region . therefore , from all experimental simulations on mammogram rois of size 256 256 pixels , we found that small values of k such as 0.10.4 are suitable for detecting mcs occur within dense - glandular breast tissue while larger values of k such as 0.51 are more appropriate for mcs in fatty breast tissue . such results are mostly due the fact that mcs present in denser mammogram tissue have lower local contrast than mcs occurring in fatty breast tissue . figure 10 illustrates the effect of the parameter k on the classification results , which shows that a large value of k ( k > 0.2 ) produces a detection of mcs with high specificity while low value of k as 0.1 or less leads to detection results with many false signals ( or low specificity ) as shown in figure 10(c ) . on the other hand , large values of k are more appropriate ( lower false signals ) for detecting mcs appearing in a region that has high nmd and local variance as shown in figure 11 . results show that no optimal k value produces the best detection results ( lowest fp and fn ) for all test data but some values such as k = 0.2 produces the best tp and fp rates . moreover , modeling mcs in dense breast tissue has shown more sensitivity to the value of k while the algorithm was more robust and allowed k to span a wider range for fatty breast tissue . figures 12(c ) and 12(f ) show that low values of k between 0.20.25 are suitable for detecting mcs within both fatty and dense breast tissue . furthermore , results indicated that mcs within fatty breast tissue can be modeled and detected using wider range of modeling values with lower fp results at larger values of k , as demonstrated in figures 11(b ) , 12(h ) , and 12(i ) . we believe that detection results can be further improved by fine tuning the selection of k if the breast region statistics were integrated into the algorithm . one approach to assess the performance of the detection scheme is by constructing a receiver operating characteristic ( roc ) curve which represents the false positive ( fp ) rates against the true positive ( tp ) rates obtained by varying a predefined control parameter such as k. furthermore , other studies have used other metrics such as specificity and sensitivity parameters . specificity and sensitivity parameters are defined in terms of tp , tn , and fp as follows : ( 10)specificity = tntn+fpsensitivity = tptp+fn where a true positive ( tp ) rate represents the probability of classifying a malignant tissue as target object while false positive ( fp ) refers to the probability of classifying healthy tissue as target one . also , true negative ( tn ) rate is defined as the probability of classifying a healthy tissue as none - malignant and a false negative ( fn ) represents the case when a malignant tissue being classified as a healthy one . in fact , mcs occur in mammogram in form of clusters rather than standalone . according to , a cluster of microcalcifications is defined as a group of three or more classification within a 1 cm area which is equivalent to a block of 50 50 pixels in mini mias mammograms ( digitized at 200 m edge resolution ) malignant calcifications also can only present in glandular breast tissue which is a fact that can also be used to eliminate any candidates ( segmented as mcs ) detected within dark regions . these physiological features are integrated in this work and mainly used in the computation of fp rates . moreover , the difficulty in counting precisely the number of real calcifications within the region of true mcs forced us to count tp signal and report the sensitivity rates in a method similar to the one used in . one reason for choosing tp per mammogram rather than per cluster is the nature of this proposed detection scheme , which applies a single model for segmenting all calcifications within the tested mammogram . before proceeding any further with the performance evaluation , a summary of the definition of tp , fp , fn , and tn as used in this study ought to be stated . tp is identified by visual inspection of the detection results at image locations corresponds to the real annotated mcs region per the mini - mias database . fn is identified when a mammogram region of size 50 50 pixels that belongs to real annotated mcs per the database is detected as a background class . fp is identified when a healthy or a background region of size 50 50 pixels included three or more image locations detected as mcs class . tn is identified when a healthy or background region of size 50 50 pixels is detected as background class or it included a maximum oftwo locations of isolated mcs . using these definitions , the total number of tn , fp of a given output binary image is calculated by dividing the segmented image into nonoverlapping regions of size 50 50 pixels excluding the region of the real ( actual ) mcs as identified and labeled by the database . table 1 demonstrates the average specific - ity and sensitivity of the detection scheme obtained using bc optimized via self - learning methods . per table 1 , the best sensitivity ( or tp rate ) is about 91.3% at k = 0.2 and the corresponding specificity ( fp rate ) is at 98.6% . although this proposed approach performs segmentation on a mammogram on a pixel level , it does estimate fp rates using region basis by utilizing physiological characteristics of clustered mcs , which is different form the previous work reported in . this fact makes it unreasonable to attempt to have a direct numerical comparison between our detection results and those obtained in [ 19 , 20 ] . however , in , the authors reported their results using 87 87 block sizes and used the total number of the true mc samples to be 25 mc regions . to ensure unbiased comparison with results reported in the literature , we decided to evaluate the performance of the detection results form this study in a similar manner . we used the same block size and counted fp to be the case when an mc is detected . however , tp is still evaluated by visually inspecting the detection results and comparing it with the real mcs as reported by the database . table 2 compared the specificity and the sensitivity or ( tp and fp rates ) of the proposed scheme with relevant works , which indicates that this proposed scheme produces lower tp and higher fp rates ( or lower specificity ) compared to those from but better than . in this paper , we proposed and implemented a new approach using stochastic bayesian classifier for segmenting a digital mammogram for the detection of microcalcification clusters . the proposed scheme models the image segmentation task as a two - class pattern recognition problem . this new framework accomplishes the learning phase of the classifier using a simple self - learning approach which synthesizes the training samples of mcs class in each mammogram . each image pixel , during both the learning and testing phases , is modeled using a four - feature vector extracted using spatial , statistical , and spectral via wavelet filtering methods . results demonstrated that synthetic patterns can be employed to simplify the supervised bayesian learning for mcs detection , which produces moderate detection performance . the relatively high fp and low tp rates can be related to the simplicity of mcs modeling used in this study as well as applying the detection scheme to the whole mammogram as opposed to regions continuing only breast tissue . other image processing techniques can be used , such as fuzzy k - mean clustering , to extract glandular breast regions and thus eliminating many of the misclassifications ( fp ) that are resulting at the breast borders and radiographic markers . another improvement may be suggested is to use a more sophisticated modeling of healthy breast tissue and mc regions which should be useful to overcome some of the classifier 's parameters ' estimation effects on results .

detection of clustered microcalcifications ( mcs ) in mammograms represents a significant step towards successful detection of breast cancer since their existence is one of the early signs of cancer . in this paper , a new framework that integrates bayesian classifier and a pattern synthesizing scheme for detecting microcalcification clusters is proposed . this proposed work extracts textural , spectral , and statistical features of each input mammogram and generates models of real mcs to be used as training samples through a simplified learning phase of the bayesian classifier . followed by an estimation of the classifier 's decision function parameters , a mammogram is segmented into the identified targets ( mcs ) against background ( healthy tissue ) . the proposed algorithm has been tested using 23 mammograms from the mini - mias database . experimental results achieved mcs detection with average true positive ( sensitivity ) and false positive ( specificity ) of 91.3% and 98.6% , respectively . results also indicate that the modeling of the real mcs plays a significant role in the performance of the classifier and thus should be given further investigation .

1. Introduction 2. Background 3. Segmentation Using Simplified Learning Bayesian Classifier 4. Experimental Results 5. Discussion and Conclusions

the belief that natural medicines are much safer than synthetic drugs has caused exceptional growth in human exposure to natural products , as plants , phytotherapeutic agents , and phytopharmaceutical products . this fact has lead to a resurgence of scientific interest in their biological effects . in most countries there is no universal regulatory system insuring the safety and activity of natural products and they had not been sufficiently investigated analytically or toxicologically . scientific research has shown that many plants used in traditional and folk medicine are potentially toxic , mutagenic , and carcinogenic [ 19 ] . carica papaya l. ( c. papaya l. ) is the most important species within the caricaceae genus , being widely cultivated for consumption as a fresh fruit , as juices , and as dried and crystallized fruit . biochemically , its leaves and fruits are complex , representing sources of several proteins and alkaloids with important pharmaceutical , medical , and industrial applications . it is used to treatment of infected wounds , malignant tumors , and burns . the juice of ripe papaya displayed in vivo and in vitro activities against oxidative stress [ 13 , 14 ] . it is an efficient scavenger of highly reactive hydroxyl radicals ( oh ) formed during co irradiation . the green ( unripe ) papaya , which is rich in papain , is used for dressing of ulcers . this treatment is described as effective and it is recommended in preference to other dressings for chronic skin ulcers . it has been used in many countries such as england , nigeria , ghana , gambia , india , and jamaica . in spite of its extensive use , the following disadvantages were described , as problems concerning the availability of green papaya and difficulties in preparing and storing papaya . the demonstration of the phytotherapeutic potential of a given species is a difficult task , since plant extracts consist of complex mixtures of major compounds , minor concomitant agents , and fibers , which can all be involved in the observed effects . thus , given the difficulties in determining the contribution of a specific substance in the biological effects exerted by whole extracts , the aim of this work was the study of papain isolated from c. papaya , which possesses vast application in medicine . papain , a purified protein extracted from the latex of the unripe papaya , is widely used by brazilian nurses in traditional medicine . it can be an alternative to green papaya and it can be used as phytotherapeutic agent in the treatment of pressure ulcers , gangrene , eschars , and as a debriding chemical agent to remove damaged or necrotic tissue . its benefits in these treatments are deemed effective , since more than 5000 patients at the pedro ernesto university hospital , at rio de janeiro / brazil , have undergone papain treatment and presented satisfactory results . despite its extensive use , there is little information about toxic , mutagenic , and antioxidant properties of papain itself or even unripe papaya , which contains high concentration of papain . short - term tests have been used to check compounds for their ability to induce lesions in dna , which may lead to genotoxicity , cytotoxicity , or mutagenicity . the experimental techniques using microbial cells such as escherichia coli ( e. coli ) and salmonella typhimurium ( s. typhimurium ) , as well as assays using dna as the target molecule , allowed the development of new tools to investigate toxic and mutagenic potentials of many physical and chemical agents and their correlation with the effects in eukaryotic systems [ 1 , 1720 ] . hydrogen peroxide ( h2o2 ) is a normal cell metabolite formed in several enzymatic and nonenzymatic reactions . h2o2 leads to oxidative stress , mutagenicity , loss of cell function , and ultimately apoptosis or necrosis [ 18 , 21 , 22 ] . in e. coli , a major component of the h2o2 toxicity is attributed to dna damage mediated by the fenton reaction , which generates reactive oxygen species ( ross ) , such as oh [ 2127 ] . e. coli possesses a number of antioxidant enzymes and dna repair activities encoded by several genes ( xtha , muty , oxyr , among others ) to counteract dna damage caused by oxidative stress . mutant strains lacking one or more of those genes are usually hypersensitive to h2o2 [ 18 , 21 , 22 , 26 ] . so , blanco and coworkers ( 1998 ) designed a series of e. coli wp2 tester strains ( ic203 up to ic207 , used in this study ) , which are useful for the screening of mutations resulting from oxidative stress as well in studies on antioxidants . it is well documented that oxidative damage has been implicated in various systemic chronic diseases such as cancer , alzheimer 's disease , rheumatoid arthritis , cardiovascular disease , cataracts , and other ageing processes . when generated in excess , ross in various active forms can damage tissues . in recent years , there has been a considerable interest in finding natural antioxidants from plant materials to replace synthetic molecules . data from both scientific reports and laboratory studies show that plants contain a large variety of substances that possess antioxidant activity . phytochemicals with antioxidant effects include some cinnamic acids , coumarins , diterpenes , flavonoids , lignans , monoterpenes , phenylpropanoids , tannins , and triterpenes . natural antioxidants occur in all higher plants and in all parts of the plant ( wood , bark , stems , pods , leaves , fruit , roots , flowers , pollen , and seeds ) [ 29 , 30 ] . the present work was carried out to evaluate the potential cytotoxic and mutagenic effects of papain using e. coli strains and plasmid dna . in addition , we have also investigated papain antioxidant and antimutagenic activities against oxidative stress induced by h2o2 . e. coli strains ab1157 ( wild type ) ; bh20 ( fpg ) ; pq65 ( rfa ) ; bw9091 ( xtha ) ; dh5f'iq ( puc 9.1 ) , wp2 ( wild type ) ; ic203 ( uvra oxyr pkm101 ) , ic204 ( uvra del[umudc ] ) , ic205 ( uvra del[umudc ] mutm ) , ic206 ( uvra del[umudc ] muty ) , and ic207(uvra del[umudc ] mutm oxyr ) were used in this work [ 18 , 2934 ] . in order to prepare cultures to evaluate cytotoxic , antioxidant , mutagenic effects of papain and to perform reverse mutation test , the strains were grown according to previously described methods [ 8 , 18 ] . samples ( ~10 cells ) of the e. coli strain stocks were taken from frozen vials ( 50% v / v glycerol ) and grown in liquid ( 10 ml ) lb medium at 37c , overnight with shaking ( reciprocal water bath shaker , model r76 , new brunswick , science co. inc , n.j . eua ) up to the stationary growth phase . from the overnight culture , a sample containing 100 l was added to 10 ml of fresh lb medium and incubated at 37c for 2 h , with shaking , to reach exponential growth phase ( 1 - 2 10 cells / ml ) . then , cells were collected by centrifugation ( 7,200 g in sorvall ss-34 rotor ) , washed twice with 0.9% nacl sterile solution , and resuspended in the same solution for each indicated treatment . bacto agar , bacto tryptone , and bacto yeast extract were purchased from difco laboratories , detroit , usa . papain powder ( from c. papaya l. , 30,000 usp - u / mg stabilized with sodium disulfite ) , sodium chloride , perhidrol ( 30% h2o2 ) , glucose , and tryptophan ( trp ) were from merck ( brazil ) ; streptomycin , chloramphenicol , and ampicillin were from sigma ( st . ultra pure water was obtained from a milli - q water system from millipore corp , bedford , ma , usa . papain powder , protected against ambient light , was suspended in 0.9% nacl sterile solution , vigorously shaken for 2 minutes at room temperature and immediately filtered through a sterile 0.22 m millipore cellulose acetate membrane to eliminate microbial contaminants . this solution was associated or not with sterile talc ( obtained after autoclavation , 121c , 30 minutes ) . this association ( talc and papain ) was prepared before each experiment . for the tests , the highest concentration of papain ( 500 g / ml ) to which the bacterial cultures were exposed is much greater than that used in the wounds of patients during treatment . the culture media , solutions , and cell growth were prepared as previously described [ 8 , 17 , 18 , 35 ] . dilutions of the chemical preparations and bacterial cultures were carried out with 0.9% nacl sterile solution , dimethyl sulfoxide ( dmso ) , or ultra pure milli - q water [ 8 , 17 , 18 ] . in order to evaluate the potential cytotoxic effect of papain , culture aliquots ( 1 ml ) of e. coli ab1157 , pq65 , bh20 , and bw9091 strains , in the exponential growth phase , were incubated under shaking for 60 minutes at 37c with different papain concentrations ( 0 , 50 , 250 , and 500 g / ml 0.9% nacl sterile solution ) . in order to evaluate the cytotoxic effect of papain associated with talc , aliquots ( 1 ml ) of e. coli ab1157 and bw9091 strains in exponential growth phase were incubated with papain in combination with talc ( 500 g / ml ) or talc alone ( 500 g / ml ) . a sample was incubated in 0.9% nacl sterile solution , under the same conditions , as a negative control . after the treatments , aliquots ( 100 l ) were taken , diluted with 0.9% nacl sterile solution , and spread , in duplicates , on lb - plates , following incubation at 37c for 24 hours . the colony - forming units were then scored and surviving fractions ( sf = n60/n0 ) were expressed as the mean of three experiments . in the growth inhibition test , 100 l ( ~10 cells ) of a fresh overnight e. coli culture ( in lb medium ) were added to 3 ml of molten top agar and poured onto lb plates supplemented with chloramphenicol ( 20 g / ml ) or ampicillin ( 20 g / ml ) , according to the tester strain . paper discs ( 5 mm diameter ) were impregnated with 10 l of 0.9% nacl sterile solutions containing one of the following amounts of papain ( 5 ; 12.5 ; 25 ; 50 ; 100 ; 125 ; 250 and 500 g / paper disc ) , papain ( 100 or 500 g / paper disc ) associated with h2o2 ( 300 g / paper disc ) or only h2o2 ( 300 g / paper disc ) as a positive control . the impregnated paper discs were placed on the center of plates and all plates were incubated overnight at 37c . inhibition halos were measured ( in mm ) and expressed after calculating the difference between the inhibition zone diameter and the disc diameter . ( 1988 ) ; e. coli ic203 and ic204 strains ( both trp ) were grown in nutrient broth liquid medium for ~16 hours at 37c in agitation . a suspension of the overnight - cultured strains ( 100 l ) was transferred to sterile screw - top tubes with 2.5 ml of 0.6% soft - agar at 45c and various concentrations of papain ( 0 , 12.5 , 25 , 50 , 125 , 250 , and 500 g / plate ) dissolved in 0.9% nacl sterile solution . the total tube content was spread immediately onto plates containing minimal et4 agar supplemented with tryptophan ( 0.01 mg / plate ) and incubated for 48 hours at 37c . the number of trp revertant colonies was determined and the reversion rate was compared to control ( negative and positive ) plates . in these experiments , each sample was assayed using duplicate plates and the data presented was the mean of three experiments . a sample was considered mutagenic when number of revertants were higher than 2 for at least one of the tested concentrations . agarose gel electrophoresis ( 0.8% ) was performed in order to separate different structural conformations of puc 9.1 plasmid dna after papain treatment : form i supercoiled ( sc ) native conformation , form ii open circle ( oc ) resulting from single strand breaks , and form iii linear ( l ) resulting from double strand breaks . plasmid dna aliquots ( 200 ng ) were treated with increasing concentrations of papain for 40 minutes at 25c and negative control was performed using ultra pure h2o ( milli - q ) . after treatments , each sample was mixed with loading buffer ( 0.25% xylene cyanol ff ; 0.25% bromofenol blue ; 30% glycerol in water ) and submitted to agarose gel electrophoresis in tris acetate - edta buffer , ph 8.0 at 6 v / cm . after electrophoresis , the gel was stained with ethidium bromide ( 0.5 g / ml ) and the dna bands were visualized by fluorescence in an ultraviolet dna transiluminator system [ 6 , 35 ] . the assay was repeated , at least three times , and the bands quantified with the gel pro analyzer 3.0 software ( media cybernetics , silver spring , md , usa ) . the results were analyzed by anova since ( i ) the data were normally distributed as verified by the method kolmogorov and smirnov and ( ii ) samples from populations had identical standard deviations ( sds ) , as verified by the bartlett method . anova was followed by the student newman keuls multiple comparison test using the statistical program instat version 3.01 ( graphpad software , san diego , ca , usa ) . these analyses compared the results obtained by the several treatments , at different papain concentrations , including the controls . the data collected by densitometry provided us with null events percentage ( no breaks = p(0 ; ) ) for each one of different papain concentrations tested . thus , the mean values of breaks per genome for each one of the concentrations using poisson distribution were obtained as follows : = ln p(0 ; ) . the results indicated that papain was not cytotoxic to e. coli strains ab1157 ( wild type ) , pq65 ( rfa ) , bh20 ( fpg ) , and bw9091 ( xtha ) at the tested concentrations ( table 1(a ) ) . statistical analysis indicated that there was no significant difference ( p > 0.05 ) among treated and untreated cells . this same methodoly was used to test papain in association with talc , as shown in table 1(b ) , and it was not cytotoxic , either in the wild - type or in the repair mutant ( xtha ) strain . since some reports preconize papain use in association with talc , we also decided to address if talc would alter cell viability . the results ( table 1(b ) ) showed that sterile talc was inert ; therefore the subsequent tests were performed with purified papain [ 10 , 15 ] . another methodology to investigate the toxic effect of papain comprised the use of growth inhibition test . the results concerning papain potential toxic effects obtained with e. coli ic203 , ic204 , ic205 , ic206 , and ic207 strains revealed no formation of inhibition halos at all tested concentrations . in fact , halos were produced only with the positive control h2o2 ( 300 g / disc ) ( p < 0.001 ) when compared to negative control ( 0.9% nacl ) , as shown in table 2 . the results obtained with wp2 mutoxitest showed that all the tested papain concentrations did not present mutagenic activity with e. coli ic203 ( wp2 uvra oxyr pkm101 ) and ic204 ( wp2 uvra del umudc ) . there was no significant difference ( p > 0.05 ) when papain treatment was compared to negative control ( 0.9% nacl ) ( table 3 ) . conformational changes in plasmid dna ( puc 9.1 ) after treatment with different concentrations of papain were also investigated , using agarose gel electrophoresis analysis . data showed ( figure 1 ) that papain treatment did not modify original plasmid dna conformational structure ( supercoiled form i ) . in patients from a brazilian university public hospital ( pedro ernesto hospital ) , papain , associated or not with talc , is used for the topical treatment of chronic skin ulcer . it is described as effective and recommended in preference to other dressings for the same purpose , as unripe papaya , in other countries . physicians from this hospital described the following advantages of using the papain in comparison with green papaya : dislodging of wounds , promotion of granulation tissue and healing , cost - effectiveness , standardized procedure , facility in its availability , preparing , and application . despite its extensive use in brazilian patients , then , as part of a continuous effort to understand papain effects , the aim of this work was to extend knowledge concerning papain toxicity mechanisms in bacterial systems and dna plasmid . although usually recommended , exogenous metabolic activation system ( s9 mix ) was not included in the tests , since papain treatment is only used topically , on chronic skin ulcer , and is not intended for internal use . the phytotherapeutic agent papain was not able to induce inactivation of all the e. coli strains tested . even e. coli pq35 was as resistant as the wild type strain ( table 1(a ) ) . e. coli pq65 is constitutively more permeable to bulky molecules than the others e. coli strains used in this work , due to one mutation ( rfa ) that causes partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria . therefore , two hypotheses are possible : ( i ) papain can not penetrate the cell wall and exert its toxic effect even in pq65 strain or ( ii ) it can penetrate the cell wall , but it is not toxic . . therefore , if papain induces dna damage , such as 8-oxoguanine , formamidopyrimidine ( fapy ) , or even ap sites , they may not be produced at levels high enough to cause cell lethality . besides , one should not discard other backup dna repair / tolerance systems that could take these lesions in charge , such as nucleotide excision repair ( ner ) and recombination . the antioxidant activity of papain against h2o2-induced damage was also assessed using growth inhibition test . when the cells were simultaneously treated with h2o2 ( 300 g / disc ) and papain ( 100 or 500 g / disc ) , there was observed a significant decrease in the growth - inhibition halo of e. coli ic203 ( wp2 uvra oxyr pkm101 ) ( p < 0.001 ) , ic204 ( wp2 uvra del umudc ) ( p < 0.01 ) , and ic207 ( wp2 uvra del umudc mutm oxyr ) ( p < alternatively , there was no significative decrease ( p > 0.05 ) in the growth - inhibition halos with the other studied strains , ic205 ( uvra umudc mutm ) and ic206 ( uvra umudc muty ) ( table 2 ) . it is important to note that the increase of papain to 500 g / disc was able to produce further protection to ic203 ( wp2 uvra oxyr pkm101 ) , but not to all other strains . this indicates that papain excess may prevent the production of dna lesions majorly repaired by nucleotide excision repair ( uvra ) and by the oxyr tolerance system . when base excision repair ( mutm and/or muty ) or umud and umuc proteins are absent , the excess of papain is unable to produce further cell protection . mutoxitest is a potent assay to assess the ability of a series of compounds to induce reversion of the trpe65 mutation in e. coli from auxotrophy to prototrophy and is currently accepted as a validated short - term genotoxicity test by international regulatory agencies . the results obtained with e. coli ic203 , ic204 , and ic207 strains treated in presence of h2o2 can be interpreted as indicative of an antioxidant property of papain . in this way , it can protect against genotoxic and/or mutagenic effects of h2o2 , when oxyr regulon and/or ner system are absent . on the other hand these results , at first sight , seem to contrast with those about the antioxidant potential of papain . however , they may also indicate that papain could not prevent the generation of certain premutagenic h2o2-induced lesions in dna , such as 8-oxoguanine . it was already observed that strains deficient in mutm or muty dna glicosilases are highly susceptible to sos - independent mutations promoted by these lesions . in fact , papain could be efficient in scavenging only certain specific ros generated by h2o2 treatment , eliminating hydroxyl and/or superoxide radicals , but not singlet oxygen , the major species responsible for the formation of 8-oxoguanine lesions in dna [ 22 , 39 , 40 ] . circular plasmid dna was used in vitro as target to study the induction of strand breaks in dna by compounds such as oxidant agents and natural products [ 6 , 8 , 41 , 42 ] . papain did not induce single or double strand breaks in dna in vitro . in the context of the methodology used in this work , these results reinforce the idea that the papain is neither a cytoxicity nor genotoxic agent . webman and coworkers ( 1989 ) and mehdipour and coworkers ( 2006 ) demonstrated an antioxidant effect of ripe c. papaya l juice , which is poor in papain content . in this case , the antioxidant properties found in papaya juice can not be unequivocally attributed to papain and may be due to other antioxidant substances . in fact , only unripe fruit contains papain . a problematic aspect in understanding potential toxicological events relevant to the medicinal use of c. papaya l. and many other medicinal plants is that the exact amounts of active chemicals are unknown . oloyede ( 2005 ) studied unripe pulp of c. papaya and its chemical compositon was determined . in general , the results from this phytochemical screening suggest the validity of therapeutical effect of aqueous extract of unripe pulp of c. papaya . but our results further support the notion that papain , the compound isolated from latex of unripe c. papaya l , is a promising source of potential antioxidant . a more detailed investigation of papain for the antioxidant activity is in progress using lower eukaryotic organisms , as yeast s. cerevisiae .

papain , a phytotherapeutic agent , has been used in the treatment of eschars and as a debriding chemical agent to remove damaged or necrotic tissue of pressure ulcers and gangrene . its benefits in these treatments are deemed effective , since more than 5000 patients , at the public university hospital at rio de janeiro , brazil , have undergone papain treatment and presented satisfactory results . despite its extensive use , there is little information about toxic and mutagenic properties of papain . this work evaluated the toxic and mutagenic potential of papain and its potential antioxidant activity against induced - h2o2 oxidative stress in escherichia coli strains . cytotoxicity assay , growth inhibition test , wp2-mutoxitest and plasmid - dna treatment , and agarose gel electrophoresis were used to investigate if papain would present any toxic or mutagenic potential as well as if papain would display antioxidant properties . papain exhibited negative results for all tests . this agent presented an activity protecting cells against h2o2-induced mutagenesis .

1. Introduction 2. Experimental Procedures 3. Results 4. Discussion

the beckwith wiedemann syndrome ( bws , omim 130650 ) is a congenital overgrowth disorder associated with dysregulation of imprinted genes located at chromosome 11p15.5 . children affected by bws show with variable expressivity several characteristic features , including macroglossia , gigantism , abdominal wall defects and embryonal tumors , particularly wilms tumor . the 11p15.5 imprinted gene cluster includes more than ten imprinted genes organized in two domains , each controlled by a separate imprinting centre region ( ic1 and ic2 , ref . ) . the igf2 and h19 genes are in the telomeric domain , and genes such as cdkn1c and kcnq1ot1 are in the centromeric domain . individuals with bws have heterogeneous molecular defects affecting one or both domains of the cluster , including paternal uniparental disomy ( upd ) or duplication , cdkn1c mutations and ic epimutations and microdeletions . bws cases with alterations in the telomeric domain or upd have a higher risk of developing wilms tumor than cases with other molecular abnormalities . the imprinting of the paternally expressed igf2 and maternally expressed h19 genes is controlled by ic1 , which is located upstream of the h19 transcription start site . on the maternal chromosome , ic1 binds the zinc finger protein ctcf that , by inducing higher order chromatin structures , insulates igf2 from the effects of downstream enhancers . on the paternal chromosome , the insulator function of ic1 is inactivated by dna methylation that inhibits ctcf binding . in bws , aberrant igf2 activation and h19 silencing result from gain of dna methylation at the maternal ic1 . in contrast , igf2 silencing and h19 activation are associated with loss of dna methylation at the paternal ic1 , in the growth - deficiency disorder , silver russell syndrome ( srs , ref . ) . in the majority of bws and srs cases , the ic epimution is not linked with sequence changes of the region , but in a few individuals the dna methylation defect is associated with in cis mutations . in particular , microdeletions in the ic1 sequence abolishing 13 ctcf - target sites ( cts ) have been described in bws cases [ 57 ] . the clinical phenotype associated with these mutations , however , is not uniform and we have previously proposed that a critical factor may be the spacing of the remaining ctss in the mutant allele . here the deletion is similar in size to previously reported ones , but removes different sets of ctss . when maternally transmitted , it is associated with ic1 hypermethylation and bws phenotype . overall , the results are consistent with the hypothesis that the spatial organization of the ctss is critical for the function of the imprinting regulatory element and that internal deletions resulting in long cts clusters are generally associated with hypermethylation and fully penetrant clinical phenotype . the propositus was born to unrelated parents after 37 weeks gestation and a pregnancy characterized by polyhydramnios . birth weight was 5080 g ( > 95th centile ) , length was 59 cm ( > 95th centile ) , and ofc was 36.1 cm ( 75th centile ) . the presence of macrosomia , macroglossia , ear pits , neonatal hypoglycaemia , rectum diastasis , umbilical hernia , hepatomegaly , nephromegaly and ureteral abnormalities were consistent with the clinical diagnosis of bws . the propositus mother was a 29 year - old woman , at the time of delivery . at birth , she was macrosomic ( birth weight 5400 g ) , displayed macroglossia and was affected by kidney disease ( renal congenital renal cysts and kidney stones ) . no signs typical of bws were evident in the other family members ( fig . 1 ) . all the genetic analyses were performed with the informed consent of the parents of the family members . pcr amplification of the ic1 region was obtained from leukocyte dna by using the primers : 5-cgctgtggctgatgtgtagtagag-3 and 5-catttccgtctccacagccacaac-3 and the taq bio - x - act long ( bioline ) as described . the fragments generated from the allele carrying the microdeletion were gel - purified and cloned onto pcr ii ( invitrogen ) . 11p15 microsatellite markers th , d11s4046 and d11s922 were amplified using fam - labeled and hex - labeled primers and detected using an abi 3100 capillary electrophoresis instrument . primers and pcr conditions for the microsatellite markers were obtained from the ncbi unists database ( http://www.ncbi.nlm.nih.gov/sites/entrez?db=unists ) . the level of dna methylation at ic1 ( cts1 and cts6 regions ) and ic2 was determined by cobra , as previously described . the allele - specific dna methylation profile of cts2 was determined by bisulfite genomic sequencing . dna was treated with sodium bisulfite using epitect bisulfite kit ( qiagen ) and pcr amplified with the following primer pairs : in the individuals carrying the 1.8 kb b5-b2 deletion : cts2_f 5-aggtgttttagttttttggatgata-3 with cts2_r 5-ctccaaaaatacacatatactatacaaaaac-3 for the deleted allele and cts2_f 5-aggtgttttagttttttggatgata-3 with cts2_r 5-caatttctatacctaccaaaaacc-3 for the wild - type allele . in the individual carrying the 1.4 kb b5-b3 deletion : cts2_f 5-aggtgttttagttttttggatgata-3 with cts2_r2 5-taaatatcctatccctaataac-3 for the deleted allele ( annealing temperature was 52 c with 1.5 mm of mgcl2 ) and cts2_f 5-aggtgttttagttttttggatgata-3 with cts2_r2 5-cacctaacttaaataacccaaaac-3 for the wild - type allele ( annealing temperature was 60 c with 2.0 mm of mgcl2 ) . the pcr products were gel - purified using qiaquick gel extraction ( qiagen ) , then cloned in topo pcrii vector ( invitrogen ) and individual clones sequenced by primm ( italy ) . the propositus , his unaffected brother and their parents were analysed for the presence of dna methylation defects at 11p15.5 . the results obtained by cobra demonstrated abnormal dna methylation at ic1 and normal dna methylation at ic2 in ii2 and iii1 . in particular , two ctcf - target sites ( cts1 and cts6 ) in the ic1 region were found hypermethylated when compared to controls ( table 1 ) . we previously demonstrated that a subset of bws patients with ic1 hypermethylation carry microdeletions in this regulatory region . dna purified from blood leukocytes of all family members was analysed by long - range pcr . a fragment carrying a 1.8 kb deletion was amplified in individuals ii2 and iii1 , while only a pcr product of normal length was obtained from the other family members . the deletion breakpoints were identified by sequencing the pcr products and found to be located between at chr11:2021870 and chr11:2023704 ( ncbi37/hg19 ) . the human ic1 is organized into two large repeated units , each composed of a 450 bp a - type repeat and several highly homologous 400 bp b - type repeats containing target sites for ctcf ( cts , see fig . 2 ) . the novel 1.8 kb deletion eliminates most of the b5 repeat together with the entire a2 , b4 , b3 and a small part of the b2 repeat . although the deletion has the same width of previously reported ones , it involves different b - repeats and ctss . indeed , the three previously reported 1.8 kb deletions abolish ctss 2 and 3 , while the novel deletion removes ctss 3 and 4 . in ii2 the ic1 microdeletion originated de novo . to determine the parental origin of the chromosome carrying the deletion in ii2 , we analysed the segregation of the 11p15 haplotype in the family , by typing for three microsatellite markers ( fig . 1 ) . the results showed that ii2 and iii1 inherited maternally the same 11p15 haplotype and that this was not shared by the unaffected ii1 and iii2 , indicating that ii2 , as well as iii1 , carries the ic1 microdeletion on her maternal chromosome . affected and unaffected individuals showed normal allelic balance at 11p15.5 . to further study the effect of the microdeletion on ic1 methylation , we determined separately the methylation status of the maternal and paternal ic1 alleles by bisulfite sequencing in ii2 and iii1 . for this purpose , we took advantage of the deletion and used allele - specific primers to amplify selectively the maternal and paternal cts2 sequence . as a control , we performed a similar analysis in an individual with normal phenotype previously shown to carry a 1.4 kb b5-b3 deletion on his paternal chromosome . the results demonstrated that both the paternal and the maternal ic1 alleles were methylated in ii2 and iii1 while the paternal allele only was methylated in the control ( fig . 3 and data not shown ) . this study reports a novel microdeletion of the ic1 region segregating with disease in a parent of origin - dependent dominant manner . the deletion abolishes two out of the seven ctcf target sites ( ctss ) and its maternal transmission is associated with hypermethylation of the residual ic1 sequence and the bws phenotype . in the wildtype ic1 , the ctss are arranged in two large repetitive units of three sites each , separated by about 1000 kb . the mutation described in this study further expands the list of molecular defects derived from recombination between repeats of the ic1 region , including both microdeletions and microduplications . comparison with previously reported ic1 mutations supports the hypothesis that the correct spacing rather than the number or the type of ctss is critical for ic1 function . indeed , in the alleles carrying the microdeletions , the spacing between the ctss changes and the two cluster of sites merge as a consequence of recombination between the repeats ( suppl . we observe that the cases in which clusters of 45 ctss are generated ( 6 mutations ) are all associated with hypermethylation of the maternal ic1 , while differential methylation is maintained in a case in which a longer deletion ( 2.2 kb , ref . ) generates a single cluster of three sites resembling those of the two large wild - type units . the mechanism by which some of the microdeletions result in gain of methylation of the residual ic1 sequence is unknown . point mutations found in a couple of bws cases implicate an oct1/oct4 binding site in the control of ic1 methylation . however , the loss of this site does not correlate with the dna methylation status of the deleted alleles ( fig . 2 ) . an alternative hypothesis is that the binding of ctcf to dna depends on the spacing of the ctss and that this protein binds the long clusters of the 1.41.8 kb alleles less efficiently than the wild - type and the 2.2 kb alleles . cts mutation results in hypermethylation of the h19 ic in the mouse , indicating that ctcf binding is required to maintain the insulator element hypomethylated and functional on the maternal chromosome . indeed , an incompletely penetrant bws phenotype was reported for the 2.2 kb deletion that maintains the normal differential methylation of the maternal ic1 . in contrast , this study demonstrated that the 1.41.8 kb deletions always result in ic1 hypermethylation and bws phenotype with complete penetrance . pre - natal genetic testing should be considered for such families , as the recurrence risk may be as high as 50% upon maternal transmission .

the overgrowth disorder beckwith wiedemann syndrome ( bws ) is associated with dysregulation of imprinted genes at chromosome 11p15.5 . the molecular defects are heterogeneous but most of the cases are associated with defective dna methylation at either one of two imprinting control regions ( ic1 and ic2 ) or uniparental paternal disomy ( upd ) at 11p15.5 . in rare cases , the bws phenotype has been found associated with maternal transmission of ic1 microdeletions . we describe a family with a novel 1.8 kb deletion that is associated with hypermethylation at ic1 . the mutation results from recombination between highly homologous sequences containing target sites for the zinc - finger protein ctcf ( ctss ) . this finding supports the hypothesis that the function of ic1 and the penetrance of the clinical phenotype depend on the spacing of the ctss resulting from recombination in the mutant allele .

Introduction Materials and methods Results Discussion

some heavy metals have been suspected of playing a role in the pathogenesis of nervous system diseases such as multiple sclerosis , amyotrophic lateral sclerosis , and alzheimer 's disease . in these disorders , autoantibodies against neural proteins are evident at some stage of the disease . lead is known to affect both the immune and nervous systems . work in our laboratory has shown that lead exposure leads to the production of autoantibodies against neural proteins , including myelin basic protein ( mbp ) and glial fibrillary acidic protein ( gfap ) . we hypothesize that lead aggravates neurological disease by enhancing the immunogenicity of nervous system proteins , including mbp and gfap . to test this hypothesis , lead - altered protein was prepared by incubating mbp or gfap with lead acetate for 24 hr . on days 0 , 14 , and 28 , mice received inoculations with either saline , native protein , or lead - altered protein . anti - mbp and anti - gfap , isotypes igm and igg , were measured in sera by elisa on day 38 . sera of mice treated with lead - altered mbp had statistically higher anti - mbp igg titers than both control and native mbp - immunized mice . an analogous response was seen in mice immunized with lead - altered gfap . supernatants from lectin - stimulated splenocytes were also examined for antibody titers and for interleukin 2 ( il-2 ) and interleukin 6 ( il-6 ) levels . a significant increase in il-6 production was seen in mice immunized with lead - altered mbp but not with lead - altered gfap . no changes were observed in the il-2 levels of mice immunized with either lead - altered protein.(abstract truncated at 250 words)imagesp1052-afigure 1.figure 2.figure 3.figure 4.figure 5.figure 6 .

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anti - mullerian hormone ( amh ) , a polypeptide , secreted by the granulosa cells of the preantral and early developing antral follicles , has been shown to be a predictor of ovarian activity . pcos patients show an increased number of antral follicles ; therefore they have higher circulating amh levels than the healthy women . the interactions between amh and the hormonal profile characteristics in pcos require a better assessment . the positive correlation between insulin resistance and serum amh levels suggests that insulin exerts an action on amh synthesis ; however this aspect is not yet fully understood . polycystic ovary syndrome ( pcos ) is a common endocrine disorder , affecting 610% of women in reproductive age . this syndrome is characterized by biochemical or clinical signs of hyperandrogenism , chronic anovulation , and polycystic ovaries . evidence suggests that insulin resistance and its compensatory hyperinsulinemia play an important role in pcos pathogenesis [ 5 , 6 ] . insulin is associated with hyperandrogenism ; it acts synergistically with luteinizing hormone to increase the androgen production of theca cells . inositol ( hexahydroxycyclohexane ) belongs to the vitamin b complex group ; it is a 6-carbon ring compound , having a hydroxyl group linked to each carbon of the ring , with nine possible stereoisomeric forms depending on the epimerization of the six hydroxyl groups . among them , myoinositol ( myo - ins ) is the mostly represented isoform , with very relevant biological functions . increasing evidence has demonstrated that myo - ins plays a key role in cell morphogenesis and cytogenesis , lipid synthesis , structure of cell membranes , and cell growth . myo - ins administration improves hormonal profile , oocyte maturation , and insulin resistance ; furthermore , it promotes the meiotic progression of germinal vesicle oocytes [ 11 , 12 ] . recent studies on pcos patients showed a decrease of androgen levels and an improvement in ovulation and metabolic parameters after treatment with myo - ins and d - chiro - inositol ( d - chiro - ins ) , which is another stereoisomeric form of inositol . it was highlighted that very promising results were achieved administering myo - ins plus d - chiro - ins at their physiological range in plasma ( i.e. , 40 : 1 ) to ensure better clinical results in the pcos therapy . it is important to investigate the possibility of using amh as a marker of some parameters : improved insulin resistance and decreased lh and androgen levels . combined oral contraceptive ( coc ) pills , especially containing antiandrogen , are commonly used in the treatment of pcos patients to suppress ovulation . in this study our primary outcome was to investigate the effect of myo - ins or coc on the clinical features , biochemical parameters , and amh levels in pcos patients . the secondary outcome of the study was to compare differences in changes after treatment with myo - ins or coc . this is a randomized prospective trial carried out at the department of obstetrics and gynecology ( dokuz eylul university , izmir , turkey ) between may 2013 and january 2014 . this study started after the approval by the clinical research ethics committee of the faculty of medicine , dokuz eylul university , and the turkish health ministry drug and medical device foundation . 137 patients diagnosed with polycystic ovary syndrome and admitted to dokuz eylul university faculty of medicine , department of obstetrics and gynecology , division of reproductive endocrinology and infertility outpatient clinic , were included in the study . the patients with odd numbers were allocated to coc group ( n = 60 ) , whereas the even ones to myo group ( n = 77 ) ( figure 1 ) . the coc used was 2 mg cyproterone acetate and 0.035 mg ethinylestradiol ( diane 35 ; schering ag , istanbul , turkey ) daily . the coc was given for 21 days and in the following 7 days no drugs were given . the product used contained 1 gram myoinositol and 100 gram folic acid ( inofolic ; lo.li . the drug was used twice a day continuously for 1216 weeks . in initial assessment , for patients who had regular menstrual cycle for both myo and coc group , the blood samples for hormonal assessment except progesterone were conducted on day 2 or 3 of the menstrual cycle . the assessment after three months was done when the treatment finished and the patients had her first menstrual cycle after three months of treatment . for both groups the patients were not under medication during the assessment of the hormone levels . in oligo / amenorrhoeic patients , blood samples were collected after withdrawal bleeding induced by oral progestin ( 5 mg medroxyprogesterone acetate twice a day ; tarlusal ; deva holding a. . , the diagnosis of pcos was made according to the rotterdam criteria ; as prescribed , two out of three features were detected in the patients : oligomenorrhea ( fewer than six menstrual periods in the preceding year ) and/or anovulation ; clinical and/or biochemical signs of hyperandrogenism ; presence of 12 follicles in each ovary measuring 29 mm in diameter ; and/or increased ovarian volume ( > 10 ml ) . smoking , hyperprolactinemia , hypogonadotropic hypogonadism , pregnancy , thyroid disease , congenital adrenal hyperplasia , androgen - secreting tumors , and cushing 's syndrome were ruled out during the screening phase . none among the enrolled patients had taken , at least in the previous six months , oral contraceptives , antiandrogens , or any drug that could influence carbohydrate metabolism . clinical evidence of hyperandrogenism was determined by the ferriman gallwey score 8 that reveals the presence of hirsutism and/or acne . initial physical examination included weight , height , and waist and hip circumferences , to calculate waist / hip ratio ( whr ) and body mass index ( bmi ) . the waist circumference was measured at the midpoint of lowest margin of 12th rib and the lateral iliac crest during the normal expiration . resting systolic and diastolic pressures were measured ; after 2 minutes , the second measurement was performed and the mean values were determined . serum levels of fasting plasma glucose and insulin , c - reactive protein ( crp ) , anti - mullerian hormone ( amh ) , apoprotein b ( apo b ) , apoprotein a1 ( apo a1 ) , high density lipoprotein ( hdl ) and low density lipoprotein ( ldl ) , total cholesterol , triglyceride , follicle stimulating hormone ( fsh ) , luteinizing hormone ( lh ) , total and free testosterone , dehydroepiandrosterone sulfate ( dheas ) , and sex hormone binding globulin ( shbg ) were measured . normal insulin sensitivity was defined by fasting serum glucose and insulin levels with homeostatic model of insulin resistance ( homa - ir ) . homa - ir was calculated by the formula : homa - ir = fasting blood glucose ( mg / dl ) fasting insulin ( iu / ml)/405 . fasting venous blood samples were taken between 08:00 am and 10:00 am after a 12-hour overnight fast . the blood samples were immediately centrifuged for 10 minutes and kept at 80c in eppendorf tubes until assayed . serum anti - mullerian hormone ( amh ) levels were analyzed with commercial kit according to manufacturer instructions based on the principle of competitive enzyme linked immunosorbent assay ( elisa ) method ( catalog number : csb - e12756h , cusabio biotech co. , usa ) . the microplate in the kit is precoated with goat - anti - rabbit antibody specific to amh . standards and undiluted samples are loaded into the appropriate microtiter plate wells with horseradish peroxidase ( hrp ) conjugated amh and antibody specific to amh . the competitive inhibition reaction was launched between hrp labeled amh and unlabeled amh with the antibody . following a wash to remove any unbound substances , substrate a and b solutions the color development is stopped and the intensity of the color is measured spectrophotometrically at a wavelength of 450 nm . a standard curve of known concentration ( 0 , 0.375 , 1.31 , 4.69 , 28.12 , and 150 ng / ml ) of amh was established and the concentration of analyte in the samples was calculated accordingly . the elisa assays of amh had a sensitivity of 0.375 ng / ml ; a detection range of 0.375150 ng / ml ; intra - assay coefficient of variation < % 10 , interassay coefficient of variation < % 15 , respectively . for transvaginal ultrasonography , the probes used were ev49/10ed center frequency 6.5 mhz and er49/10ed center frequency 6.5 mhz . for virgin patients transabdominal ultrasonography was used . the presence of 12 follicles in each ovary measuring 29 mm in diameter was recorded . longitudinal , transverse , and anteroposterior ovarian diameters were measured and multiplied by 0.5 to calculate the ovary volume . data were analyzed by using statistical program for social sciences ( spss , version 16 ) . the level of significance was accepted when p < 0.05 . in table 1 , the test used was independent t - test . in table 2 , the test used was independent t - test . in table 3 , the test used was paired samples test . in table 4 , the test used was paired samples test . 106 patients were analyzed , among them 54 patients were coc receivers ( group 1 ) , and 52 were myo - ins receivers ( group 2 ) . baseline demographic , clinical characteristics , and ultrasound results of all patients are presented in table 1 . the mean age was 22.79 4.13 years and 24.44 4.78 years for groups 1 and 2 , respectively . in group 1 , 41 ( 75.9% ) of 54 patients showed menstrual irregularity . at the end of the study period , patients in group 1 showed no menstrual irregularity , whereas the decrease of menstrual irregularity in group 2 was from 40 ( 76.9% ) to 8 ( 15.4% ) patients . the baseline biochemical and hormonal values showed that patient groups were matched in these parameters except sex hormone binding globulin ( shbg ) level which was higher in group 1 ( table 2 ) . increase in hdl and decrease in lh and lh / fsh ratio were determined statistically significant only in group 1 when compared with the baseline values ( for all p > 0.05 ) ( table 3 ) . in group 2 , the reduction of fasting glucose , ldl , dheas , total cholesterol , and prolactin levels was statistically significant ( for all p < 0.05 ) ( table 3 ) . other parameters , such as total testosterone , dheas , and fasting glucose , were statistically improved in group 2 , while no particular improvement was present in group 1 ( table 3 ) . after 3-month treatment , amh ( for group 1 p < 0.001 ; for group 2 p = 0.002 ) levels as well as ovarian volumes , ovarian antral follicle count , and total antral follicle counts showed a statistically significant decrease in both groups ( for all p < 0.001 ) ( table 3 ) . when we evaluate progesterone levels at the end of the treatment , group 1 showed a statistically significant decrease whereas group 2 showed a statistically significant increase ( for group 1 p = 0.014 ; for group 2 p < 0.001 ) . the lessening of ovarian volumes and amh levels was statistically significantly more in the myo group than coc group ( for amh ; p = 0.048 , for ovarian volumes p = 0.040 ) ( table 4 ) . this prospective study observed the change in clinical , biochemical parameters and anti - mullerian hormone levels after treatment with two different drug supplementations , myoinositol and combined oral contraceptives . study data demonstrated that myo - ins regimen in pcos patients positively affects metabolic parameters and modulates various hormonal factors deeply involved in the reproductive function and ovulation such as anti - mullerian hormone . it is well known that pcos is characterized by hyperandrogenism and irregular menstrual cycles . concerning menstrual irregularity , using myo - ins papaleo et al . and gerli et al . observed an improvement of 88% ( after six months ) and 70% ( after 14 weeks ) . in the current study , 76.9% of patients in group 2 , treated with myo - ins , had menstrual irregularity initially . use of myo - ins was associated with a significant improvement , and menstrual irregularity was strongly reduced in the study population . this effect may be explained by the specific protein phosphorylation processes via protein kinase c , which modulates various cellular processes as a second messenger system . anti - mullerian hormone is secreted primarily in the small antral follicles and amh measurements correspond to granulosa cell activity , total antral follicle count , and ovarian volume . in addition , several studies showed that amh might correlate with the severity of this syndrome . in recent researches , positive correlations between amh levels , ovarian volume , and total antral follicle count several studies in the literature support that coc treatment decreases ovarian volume and antral follicle [ 23 , 24 ] . our analysis showed significant reduction in total antral follicle count and ovarian volume after treatment with coc and myo - ins ( for both groups p < 0.001 ) . in line with our study , the two researches of genazzani demonstrated reduction in ovarian volume after myo - ins administration . however , they reported no changes in the total antral follicle count after myo - ins treatment [ 25 , 26 ] . although there are no previously published data about changes in serum amh levels during myo - ins therapy , there are several studies that observed amh changes after treatment with coc [ 2729 ] . examined 95 women in five different groups and they found no significant change in amh levels before and after treatment with combined oral contraceptives . in contrast , decrease in amh levels after coc was shown in previous studies and it has been confirmed in our study [ 28 , 29 ] . the decline in amh levels may be related to the suppression of ovarian function with coc . research has been mainly focused on coc 's activity and there are no clinical studies on myo - ins effect on amh levels . reported that myo - ins supplementation , in pcos patients , affected metabolic parameters such as insulin sensitivity and modulated positively hormonal factors like lh , fsh , and testosterone . there was one retrospective study which showed decreased amh levels after d - chiro - ins . to our knowledge , the present study is the first one in the scientific literature indicating statistically significant decrease in amh levels after 1216 weeks of myo - ins use . we believe that suppression in amh levels may be explained by the reduction of total antral follicle count and ovarian volume . however , the mechanism between decrease in total antral follicle count and myo - ins therapy is not clearly understood yet . we hypothesize that whereas hyperinsulinemia may stimulate the development of antral follicles and recover the sensitivity of granulosa cells to fsh , therefore leading to increase in the number of follicles , ovarian volume , and amh levels , myo - ins may improve clinical and hormonal features of pcos patients by enhancing insulin sensitivity that decreases hyperinsulinemia . in our trial , we compared the ovarian volumes , total antral follicle count , and serum amh levels after treatment with coc and myo - ins . patients in the study group showed significantly more relevant reduction in amh levels ( p = 0.048 ) and ovarian volume ( p = 0.040 ) than the coc group . the decrease in total antral follicle count for myo - ins and coc group was similar ( p = 0.356 ) . based on our data , we believe that myo - ins is more effective compared to coc in lowering amh levels and ovarian volumes . myoinositol can not replace coc for contraception and may be preferred for patients with infertility who desire to conceive pregnancy . also , myoinositol can be an option in patients with hirsutism , oligoanovulation , and symptoms of hyperandrogenism who have a contraindication to use coc . myoinositol is not a primary treatment ; it is an adjunct therapy to improve insulin resistance . in the present study , we also observed the effect of coc and myo - ins on fasting insulin , fasting glucose , and homa - ir . the results showed no significant change in insulin or homa - ir in both groups . only pcos patients , who received myo - ins , presented a significant reduction in glucose levels . however , we know that our study had a too short duration to be capable of detecting myo - ins effect on insulin and homa - ir . in contrast with our data , zacch et al . demonstrated that fasting insulin and homa - ir values were decreased after myo - ins treatment . reported that coc did not affect fasting insulin or fasting glucose levels or homa - ir . in minozzi et al . 's study , patients were divided into two groups as coc + myo - ins receivers and only coc receivers . the results showed a significant reduction in glucose levels only in coc + myo - ins group , whereas no significant change was observed in coc group . the results of minozzi et al . 's prospective study showed that the combination of coc and myo - ins was a more effective treatment for clinical symptoms of pcos and in controlling endocrine disorders and insulin resistance . many studies have been carried out in pcos patients with high serum androgen levels to determine the clinical implications . the results of this study showed that myoinositol treatment was able to improve some key parameters such as the total testosterone level . the reduction of testosterone is a key outcome in the management of pcos women , because of the typical symptoms related to the hyperandrogenic status , affecting the pcos pathway . these results could be due to the insulin sensitizing action of myoinositol and the sequent downregulation of the androgens production at ovarian level . the distinct effect of myoinositol on testosterone , amh , and ovarian volume is surprising and this could be explained by a reduction in hyperinsulinism . in our study , we did not observe significant reduction in insulin and homa - ir , but still there is a decrease in homa - ir blood levels . to observe the significant change demonstrated a decrease in free testosterone and total testosterone after myo - ins treatment , but no significant change in androstenedione levels . when we evaluate the changes in androgen levels in our study , we have seen that myo - ins treatment reduced dhea - s and total testosterone levels , but no significant differences in shbg and androstenedione were found . in the present study , whereas coc group showed a significant increase in shbg levels , statistical significance was not observed in the increase of dheas , free testosterone , and androstenedione and in the reduction in total testosterone levels . literature data indicate that if the treatment duration was extended , changes in androgen levels might become significant . therefore , it is more appropriate planning a long term therapy in the management of androgen excess , anovulation , and insulin resistance . this study shows that the combination of myoinositol plus folic acid should be considered in the treatment of pcos patients . considering previous studies , myo - ins studies in the literature indicate that myoinositol exerts positive effects on insulin resistance , but further researchers are still required to clarify the mechanism . the present study is the first one reporting that myoinositol is superior to coc in terms of lowering androgens levels . also ,

recently , myoinositol ( myo - ins ) and folic acid combination has gained an important role for treating polycystic ovary syndrome ( pcos ) , in addition to combined oral contraceptives ( coc ) . we aimed to examine myo - ins effects on anti - mullerian hormone ( amh ) levels and compare them with those ones obtained administering coc . in this prospective study , 137 pcos patients , diagnosed according to rotterdam criteria and admitted to the reproductive endocrinology and infertility outpatient clinic at dokuz eylul university ( izmir , turkey ) , were included . after randomization to coc ( n = 60 ) and myo - ins ( n = 77 ) arms , anthropometric measurements , blood pressure , modified ferriman gallwey scores were calculated . biochemical and hormonal analysis were performed , and lh / fsh and apo b / a1 ratios were calculated . data analysis was carried out in demographically and clinically matched 106 patients ( coc = 54 ; myo - ins = 52 ) . after 3-month treatment , increase in hdl and decreases in lh and lh / fsh ratio were statistically more significant only in coc group when compared with baseline ( in both cases p > 0.05 ) . in myo - ins group , fasting glucose , ldl , dheas , total cholesterol , and prolactin levels decreased significantly ( for all p < 0.05 ) . progesterone and amh levels , ovarian volume , ovarian antral follicle , and total antral follicle counts lessened significantly in both groups ( for all p < 0.05 ) . in pcos treatment , myo is observed more effective in reductions of total ovarian volume and amh levels .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusion