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Substance use during pregnancy often leads to involvement in the child welfare system, resulting in multiple social service systems and service providers working with families to achieve successful child welfare outcomes. The Vulnerable Infants Program of Rhode Island (VIP-RI) is a care coordination program developed to work with perinatal substance-users to optimize opportunities for reunification and promote permanency for substance-exposed infants. This paper describes services used by VIP-RI participants and child welfare outcomes.Data collected during the first four years of VIP-RI were used to identify characteristics of program participants, services received, and child welfare outcomes: closed child welfare cases, reunification with biological mothers and identified infant permanent placements.Medical and financial services were associated with positive child welfare outcomes. Medical services included family planning, pre- and post-natal care and HIV test counseling. Financial services included assistance with obtaining entitlement benefits and receiving tangible support such as food and clothing.Findings from this study suggest services that address basic family needs were related to positive child welfare outcomes. The provision of basic services, such as health care and financial assistance through entitlement benefits and tangible donations, may help to establish a foundation so mothers can concentrate on recovery and parenting skills. Identification of services for perinatal substance users that are associated with more successful child welfare outcomes has implications for the child welfare system, treatment providers, courts and families. According to the Substance Abuse and Mental Health Service Administration (SAMHSA), 5% of pregnant women used illicit drugs in the past month . The 200Maternal substance use raises concerns about a woman's capability to adequately care for her child. Risk factors associated with substance abuse such as co-occuring psychiatric problems, violence, difficulties in interpersonal relationships, limited social support, unstable employment histories, and medical problems raise additional concerns about parenting abilities -7. EstimWhen substance use during pregnancy results in child welfare involvement, multiple social service systems intervene to address the family's needs. There is limited information about specific services used by perinatal substance users with child welfare involvement. An examination of services received and child welfare outcomes can increase understanding about how best to intervene with perinatal substance users to achieve positive child welfare outcomes. The present study describes services received by women participating in the Vulnerable Infants Program of Rhode Island (VIP-RI) (described below) and three child welfare outcomes: 1) a closed child welfare case, 2) reunification with biological mother and 3) an identified permanent placement for the infant.The provision of comprehensive services is widely considered to improve treatment outcomes for pregnant and parenting substance-using women -18. WomeRecent research has examined substance abuse treatment services and child welfare outcomes. A study investigating reunification of children whose mothers participated in the California Treatment Outcome Project , reported factors associated with reunification were mother's length of time in treatment and participation in programs that addressed larger family needs such as employment and education . MothersThe Vulnerable Infants Program of Rhode Island (VIP-RI) began as a demonstration grant to provide care coordination services to mothers with an open child welfare case because of drug use during pregnancy. Enrollment in VIP-RI typically occurs during a mother's hospitalization following delivery. When prenatal substance exposure is identified either through maternal self-report or a positive toxicology screen, a hospital social worker informs the mother about VIP-RI and with her consent, makes a referral to the program. The VIP-RI care coordinator assesses maternal and infant needs and facilitates referrals to appropriate services. VIP-RI remains involved with families until a permanent placement for the infant has been identified. In some instances, because of the voluntary nature of the program, families may withdraw before a decision regarding permanency has been made.Mothers who participate in VIP-RI have the option of participating in the Rhode Island Family Treatment Drug Court (RI FTDC). RI FTDC is a specialized court for perinatal substance users that provides structure and support and takes an interactive, therapeutic approach that involves close monitoring and making referrals to substance abuse treatment and ancillary services.VIP-RI's model of closely collaborating with social service agencies expedites parents obtaining the services and supports they need. Services typically arranged by VIP-RI include substance abuse treatment, mental health, medical care, parenting, and help obtaining entitlement benefits. Such services are generally accepted as helpful for perinatal substance users, however, little is known about which services are actually utilized and their relevance to child welfare outcomes. The purpose of this paper is to describe the services used by mothers who participated in VIP-RI and the following child welfare outcomes: 1) status of the child welfare case 2) reunification with biological mother and 3) an identified permanent placement for the infant. To our knowledge this is the first study to describe services for perinatal substance users and child welfare outcomes.An analysis of data from the first four years of VIP-RI identified services used by mothers while participating in VIP-RI and if the child welfare case had been closed, if reunification had been achieved, and if a permanent placement for the infant had been established. The hospital Institutional Review Board granted approval for the pilot VIP-RI program.At enrollment, VIP-RI staff administered a semi-structured face-to-face psychosocial history interview that included measures of maternal characteristics and information about legal invovlement, substance abuse and treatment histories, trauma, and services received. The Substance Abuse Subtle Screening Inventory-3 was usedServices were divided into eight categories shown in Table Chi-Square analysis was performed comparing dichotomous variables and child welfare outcome variables .During the first four years, 70% of mothers referred to VIP-RI enrolled, for a total of 195 mothers. Reasons for not enrolling were active or passive refusal (64%) , ineligibility when a child welfare case was not opened after the initial CPS investigation (16%), and not being appropriate for reasons that included current incarceration, planning to place the child for adoption, psychiatric issues, not substance abuse, being identified as the primary problem (20%).Most participants in VIP-RI were Caucasian (56%), followed by African American (22%), Hispanic (14%), and other (8%). Primary drugs of choice were cocaine (46%), opiates (27%), marijuana (24%) and alcohol (3%). Maternal ages ranged from 17 to 43 years . At the time of enrollment in VIP-RI, 89% of the mothers were single and had an average of three children (range 1 - 9). Over one third of the sample had less than a high school education (37%), 61% had a high school diploma or equivalent, and 2% had a four-year college degree. Most mothers had no reliable source of income or received disability or entitlement benefits; only 6% were employed.A higher proportion of mothers with at least a high school education , fewer children , or only one child had closed child welfare cases. Maternal characteristics associated with having an open child welfare case were childhood physical abuse and a history of criminal conviction .Similar results were found when maternal characteristics and reunification outcomes were analyzed. A greater proportion of mothers with fewer children or only one child achieved reunification. A smaller proportion of mothers with less than a high school education were reunified with their children . A smaller proportion of mothers with a history of childhood physical abuse achieved reunification with their infants .At the time of hospital discharge, 32% of infants remained with biological parents, 32% were placed in kinship care, 32% were placed in non-relative foster care and 4% went to specilaized care. Infant placements at the time of discharge from VIP-RI were 56% living with biological parents, 22% in kindship care and 22% in non-relative foster care. There were no statistically significant associations between maternal characteristics and child welfare outcome measures and maternal race, income or age. There also were no statistically significant associations between maternal characteristics and infant permanent placements.As shown in Table As shown in Table As shown in Table Families with parental substance use and child welfare involvement are less likely to reunify and more likely to experience lengthy stays in foster care and higher rates of re-reporting ,33-36. IIn this study, the majority of services related to closed child welfare cases and reunification with biological mother were medical and financial services. Medical services included HIV pre/post test counseling, prenatal and postnatal care, primary medical care and family planning. Medical professionals should be aware of the positive impact they can have on the lives of substance-exposed infants and parents beyond the medical attention they provide. A strong connection with medical providers can serve families well in terms of following through with routine medical visits and providing a foundation of care for a mother to address concerns about her own and her children's health.Financial services that were associated with positive child welfare outcomes included assistance with entitlement benefits and obtaining food and clothing. Stress associated with not having essential needs met can interfere with a woman's ability to focus on treatment and may contribute to her questioning if she is able to adequately provide for her children. Ensuring that a family's basic needs are met may have the additional benefits of promoting a mother's ability to work on recovery and child welfare case plans by allaying worries about how to feed, clothe and shelter her children. Building in these services as part of child welfare, treatment, or court may contribute to more positive child welfare outcomes.Not all services were associated with favorable child welfare outcomes. Permanency services and residential drug treatment were less likely to be associated with reunification with the biological mother or identified permanent placements for infants. Permanency services included providing support to families who were voluntarily or involuntarily having parental rights terminated, which may account for these services being associated with infants not being reunified or having established permanent placements. Residential treatment may have been a marker for more severe addiction and related risk factors, which may have indicated a poorer prognosis.Involvement in child welfare because of drug use during pregnancy is a complicated issue and numerous factors affect child welfare outcomes. This study did not control for other factors that could have influenced child welfare outcome, such as social support networks, satisfaction with services, etc. This study did not correct for multiple comparisons because the purpose of the study was to describe services related to specific outcomes, as this had not been done previously. Adjustment for multiple comparisons protects against rejecting the null hypothesis when it is correct (type I error). However, the cost of this protection is to increase type II error that findings are attributable to chance when they are not.In addition, this study is limited to a population of perinatal substance users who participated in an innovative program, VIP-RI, which has an active partnership with the state's FTDC. There was no comparison group of mothers with open child welfare cases because of drug use during pregnancy who did not participate in VIP-RI.Services that addressed family health and financial needs were associated with favorable child welfare outcomes for perinatal substance users with child welfare involvement. When considering the range of services substance-using women often need, it is important to make sure that their basic needs are not overlooked. Alleviating health and financial pressures may have positive consequences for women struggling to address substance abuse while attempting to assume parenting responsibilities by allowing them to focus more on their recovery and parenting skills as they attempt to succeed with their child welfare case plans.The authors declare that they have no competing interests.KJM, JET & BML conceived of the study and wrote the manuscript. DC performed the statistical analysis. RS & LA coordinated VIP-RI and the collection of data. All authors have read and approved the final manuscript. |
The epidemic of bovine spongiform encephalopathy (BSE) in the United Kingdom, which began in 1986 and has affected nearly 200,000 cattle, is waning to a conclusion, but leaves in its wake an outbreak of human Creutzfeldt-Jakob disease, most probably resulting from the consumption of beef products contaminated by central nervous system tissue. Although averaging only 10-15 cases a year since its first appearance in 1994, its future magnitude and geographic distribution cannot yet be predicted. The possibility that large numbers of apparently healthy persons might be incubating the disease raises concerns about iatrogenic transmissions through instrumentation and blood and organ donations. Government agencies in many countries continue to implement new measures to minimize this risk. |
Gaucher disease is a progressive lysosomal storage disorder caused by the deficiency of glucocerebrosidase leading to the dysfunction in multiple organ systems. Intravenous enzyme replacement is the accepted standard of treatment. In the current report, we evaluate the safety and pharmacokinetics of a novel human recombinant glucocerebrosidase enzyme expressed in transformed plant cells (prGCD), administered to primates and human subjects. Short term (28 days) and long term (9 months) repeated injections with a standard dose of 60 Units/kg and a high dose of 300 Units/kg were administered to monkeys (n = 4/sex/dose). Neither clinical drug-related adverse effects nor neutralizing antibodies were detected in the animals. In a phase I clinical trial, six healthy volunteers were treated by intravenous infusions with escalating single doses of prGCD. Doses of up to 60 Units/kg were administered at weekly intervals. prGCD infusions were very well tolerated. Anti-prGCD antibodies were not detected. The pharmacokinetic profile of the prGCD revealed a prolonged half-life compared to imiglucerase, the commercial enzyme that is manufactured in a costly mammalian cell system. These studies demonstrate the safety and lack of immunogenicity of prGCD. Following these encouraging results, a pivotal phase III clinical trial for prGCD was FDA approved and is currently ongoing.NCT00258778ClinicalTrials.gov Since its introduction in 1991, glucocerebrosidase enzyme replacement therapy (ERT) has become the standard of care for patients with symptomatic Gaucher disease due to its safety and efficacy profile In an attempt to offer an alternative source for the production of the glucocerebrosidase enzyme, we have developed a biotechnological expression platform which is based on the industrial scale expression of human recombinant proteins in genetically engineered plant cells The protocol for this trial and supporting CONSORT checklist are available as supporting information; see 2 basis, respectively. The clinical dose of 60 units/kg, equivalent to approximately 1.8 mg/kg in humans, corresponds to 66 mg/m2 (using the conversion factor of 37 kg/m2 for humans), and corresponds to 5.6 mg/kg in cynomolgus monkeys (using the conversion factor of 12 kg/m2 for Cynomolgus monkeys). Studies were performed according to Good Laboratory Practice (GLP) at MPI Research . This facility maintains an Animal Welfare Assurance statement with National Institutes of Health, Office of Laboratory Animal Welfare. All experiments were performed in accordance with the guidelines of the Animal Care and Use Committee of the Hebrew University. Animals were subjected to clinical observations and assessed for body weight, hematology, coagulation, clinical chemistry and urinalysis evaluations. Animals were sacrificed at the end of treatment period and organ weights, macroscopic and microscopic pathology were also determined. Antibodies were measured on Day 1 and Day 28 in the acute study and on Day 1, followed by 1, 3, 6 and 9 months in the chronic study. Plasma concentrations and toxicokinetics were measured on Day 1 and Day 28 in the acute study and on Day 1, Week 9 and Week 39 in the chronic study.Two extensive safety toxicology studies were performed: an acute 4-week daily intravenous infusion study and a chronic 39-week intravenous infusion study in Cynomolgus Monkeys. In each study, twenty four (24) animals (4/sex/dose) were intravenously infused either daily (in the acute study) or once every 2 weeks (in the chronic study) over 1 hr with multiples of 1 or 5 times the clinical dose (60 units/kg) adjusted to animal body surface area. The doses of 5.6 and 27.8 mg/kg/day represent 1× and 5× the clinical dose on a mg/mnd of Nov 2005 and was completed 20th of March 2006).The phase I study was designed as a single-center, non-randomized, open label, safety and pharmacokinetic study of escalating single doses of prGCD administered as intravenous infusion (IV) to six healthy volunteers. This study was conducted according to FDA and European GCP guidelines, and was approved by the IRB of the Hadassah University Hospital and the Israeli Ministry of Health and under an FDA investigational new drug application (IND). The study was performed at the Phase I Unit at the Goldyne Savad Institute of Gene Therapy at the Hadassah Hebrew University Hospital. The study was approved by the Helsinki committee of Hadassah-Hebrew University Hospital Jerusalem, Israel, and all subjects gave written informed consent. Inclusion and exclusion criteria are presented in last, Tmax, Cmax and Cmin).A vehicle control placebo followed by three single escalating doses of prGCD were administered via intravenous (IV) infusions. The vehicle was administered at the baseline visit, followed by an initial prGCD dose of 15 units/kg administered on Day 8; 30 units/kg on Day 15, and 60 units/kg administered on Day 22. The infusion rate was 1.5 mL/minute (135 mL over 90 minutes), which was the same for all doses. Subjects were closely monitored for 8 hours from the time of the initiation of the infusion and returned after 24 hours from the time of initiation of infusion for blood sampling and additional safety assessments. A follow up visit was performed on day 29, at the end of the study. The issue of safety was the primary outcome in this study; safety measurements included adverse events, general infusion related toxicities, physical examinations including changes in vital signs and body weight, and laboratory tests. Pharmacokinetic parameters were the secondary outcomes in this study. Blood samples were collected prior to dosing (0) and at 5, 45, 80 and 90 minutes during the infusion and 100, 115, 130, 150, 180, 210 minutes and 24 hours from initiation of infusion, and the plasma was analyzed to provide a pharmacokinetic profile , and tabulated based on the incidence, severity, and causality to study treatment.Statistical analysis of the clinical trial data was performed by TechnoStat Ltd., Kfar Saba, Israel.Analysis of anti-prGCD antibodies was performed using a validated immunoassay we have developed . Controls and unknown samples were incubated in a 96-well microtiter plate coated with prGCD molecules and incubated with shaking at room temperature, allowing any anti-prGCD antibodies present to bind to the prGCD molecules. After incubation, the plate was washed to remove any nonreactive serum components. Biotinylated prGCD conjugate was added which enabled binding to anti-prGCD antibodies already bound to the prGCD solid phase. Detection was performed using a streptavidin-peroxidase conjugate. The plate was washed to remove any unbound protein and reagents followed by the addition of the substrate, tetramethylbenzidine (TMB) solution. After a 10-minute incubation, 2 M sulfuric acid was added and the absorbance was measured at 450 nm. The intensity of the color produced was proportional to the concentration of anti-prGCD antibodies in the sample. Antibody-positive samples were determined by comparing the optical density (OD) with a predetermined cutoff OD. An OD equal to or greater than the cutoff identifies a sample as antibody-positive. Samples that were presumed positive underwent immunodepletion testing by addition of prGCD. The addition of prGCD to samples containing anti-prGCD was expected to block the anti-prGCD antibodies from binding to the prGCD coat on the plate, thereby decreasing the OD readings. Any sample demonstrating a decrease of greater than 50%, after the addition of prGCD, was considered positive for anti-prGCD antibodies.Analysis of neutralizing antibodies was performed using a validated immunoassay we have developed. Controls and unknown samples were incubated in a 96-well microtiter plate coated with prGCD molecules and incubated with shaking at room temperature, allowing any anti-prGCD antibodies present to bind to the prGCD. After one hour of incubation at room temperature, the plate was washed to remove any non-reactive serum components. The substrate, p-nitrophenyl β-D-glucopyranoside, was mixed with the activity assay buffer and added to the wells. After a one-hour incubation at room temperature, 5 M sodium hydroxide was added to stop the reaction and enhance the intensity of the color developed. Control samples included neutralizing and non-neutralizing controls. A solution of prGCD, substrate and enzyme inhibitor conduritol B epoxide (CBE) which was added to the uncoated wells was used to perform enzyme inhibition. The control of pooled naive human serum was added to the coated wells to track the activity of the enzyme coated to the surface of the microtiter wells in the presence of normal human serum. A sample is considered positive for neutralizing activity if the OD obtained is less than or equal to the assay cutoff.Determination of prGCD plasma concentrations was performed using a validated immunoassay we have developed. Controls and unknown samples were incubated in a 96-well microtiter plate coated with chicken-anti-plant recombinant glucocerebrosidase (prGCD) antibodies and incubated with shaking at room temperature allowing any prGCD present to bind to the anti-prGCD antibodies. After two hours of incubation, the plate was washed to remove any non-reactive plasma components. Rabbit anti-prGCD antibodies were added and the plates were incubated 1.5 hours at room temperature to allow the rabbit anti-prGCD to bind to the prGCD. The plate was washed to remove any unbound protein and reagents followed by the addition of an alkaline phosphatase affinity-purified goat anti-rabbit IgG conjugate. After a 60-minute incubation at room temperature, the plate was washed and detection was performed with phosphatase substrate. The absorbance was measured at 405 nm and color was allowed to develop at room temperature for 30 to 45 minutes or until the optical density (OD) was 1.0 or greater. The intensity of the color produced was proportional to the concentration of prGCD in the sample. The prGCD levels were quantified according to a standard curve generated by measuring purified recombinant prGCD in a 4% cynomolgus monkey plasma or human plasma matrix utilizing a four-parameter curve fit equation.Plasma analysis data were analyzed by Noncompartmental pharmacokinetic analyses (NCA) using WinNonlin®, version 5.0.1 software and Microsoft Office Excel 2003. Plasma prGCD concentration versus time data for individual animals and human subjects were analyzed for maximum concentration (Cmax), the time at which Cmax occurred (Tmax), and for area under the plasma concentration versus time curve from the start of infusion to 24 hours post-infusion, using the linear trapezoid rule . All AUC calculations were based on the time interval from start of infusion to the last measurable plasma concentration (AUClast). , Clearance (CL) and the volume of distribution at steady-state (Vss) were determined.No treatment-related side effects on survival or any clinical signs were observed during both the acute and chronic studies in primates. There was no effect of prGCD on electrocardiogram parameters, ophthalmological, hematological, coagulation, clinical chemistry, or urinalysis values. Organ weights were not affected and there were no investigational drug-related macroscopic or histopathology findings. All changes were considered to be within normal limits for cynomolgus monkeys. Based on these data, a no-observed-adverse-effect-level (NOAEL) of 27.8 mg/kg/day, the highest dose tested, was identified.In the acute study, repeated daily dosing of monkeys with prGCD for 28 consecutive days did not result in production of any anti-prGCD antibodies or neutralizing antibodies. In the chronic study, samples from 5 of the 24 animals were found positive for anti-prGCD antibodies, 3 out of 8 animals receiving 5.6 mg/kg/day and in 2 out of 8 animals receiving 27.8 mg/kg/day. All antibodies were determined to be negative for neutralizing antibody activity.All animals treated with prGCD had significant plasma exposure to the investigational drug. The prGCD plasma exposure profiles were similar at both dose levels. Plasma concentrations of prGCD increased with increasing dose of prGCD.Seventeen volunteers were assessed for eligibility. Six volunteers were enrolled and allocated for the study. All six had completed all study steps and their data was available for analysis. None had dropped out during the study period. The volunteers, 3 males and 3 females, were all Caucasian aged 19–36.Six healthy volunteers were administered with the vehicle at the baseline visit, followed by an initial prGCD dose of 15 units/kg administered on Day 8; 30 units/kg on Day 15, and 60 units/kg on Day 22. Safety results indicated that prGCD was well tolerated with only non-drug- related, minor side effects that were self limited and resolved with no treatment. No serious adverse events were attributable to prGCD administered intravenously once a week at a dose of up to 60 units/kg. Laboratory tests for kidney and liver function tests were all within normal range. One volunteer had measurements of Bilirubin up to1.4 mg% during the follow-up period and a second volunteer had measurements of Bilirubin up to 1.1 during the follow-up period ; all the rest had normal Bilirubin levels within normal limit all along the study.. Immunological laboratory tests were all within normal limits. The Complete Blood Counts (CBC) differentials revealed that one volunteer had at screening and at follow-up a slight elevation in eosinophils, up to 7% ; and the second volunteer had a single measurement of 6% of eosinophils . Complement Component 3 (C3) measurements: 5 measurements were found to be slightly lower than normal range. One subject had lower values at screening and at Visit 1, which were normal afterwards. One subject had three values lower than normal: at Visit 3, Visit 4 and follow-up. None of these results were clinically significant. IgE measurements were within normal range in five volunteers and only one exhibited a slightly high value, which was similar to his baseline and the vehicle control dosing period. Adverse events were classified by body system using MedDRA, and tabulated based on the incidence, severity, and causality to study treatment . Adminislast). Inspection of the NCA results also confirmed no obvious difference between males and females for Cmax and AUClast for these small group sizes. A mean half-life (t½) of approximately 15 min (range: 8–32 min) was determined and is based on all subjects in the 30 and 60 units/kg dose groups. At 30 and 60 units/kg, individual CL values ranged from 0.8 to 3.4 mL/min/kg. Volume of distribution (Vss) estimates ranged from 34 to 94 mL/kg at 30 and 60 units/kg, which is consistent with the size of the human plasma compartment.Comparison of prGCD concentration versus time for males versus females at each dose level revealed no clear difference in exposure between genders. Combining prGCD concentration for males and females and plotting mean concentration versus time for all three doses indicate dose-dependence in exposure to prGCD. The pharmacokinetic profile, combined for 5 patients at 30 units/kg and 60 units/kg is presented in The primary objective of this Phase I clinical trial was to determine the safety of recombinant plant cell expressed glucocerebrosidase (prGCD) in healthy volunteers. The safety results confirmed that there was no clinical or laboratory evidence of any significant innate or humoral immune reactions of this investigational drug with any clinically significant adverse events. prGCD administered IV once a week up to 60 mg/kg was shown to be safe and non immunogenic. This data was strongly supported by pre-clinical toxicological studies in animals including primates, performed with prGCD. Based on these results, the US Food and Drug Administration (FDA) allowed prGCD to proceed to a Phase 3 pivotal clinical trial, which is currently ongoing. An interesting finding in this study comes from the pharmacokinetic results, wherein the half life of prGCD was prolonged relative to that of Cerezyme® , the commercially available enzyme, reported as 3.6–10.4 min Development of antibodies to the current commercially available recombinant human glucocerebrosidase, expressed in CHO cells, has been reported in approximately 15% of the treated patients tested and antibody formation was reported following 3–12 months of treatment There is a public concern regarding the high cost of the recombinant GCD enzyme approved for Gaucher's disease It remains to be determined in larger scale clinical studies, as required in any new protein drug development process, whether plant-derived biopharmaceutical glycoproteins are associated with any excessive immunogenicity effects or excessive neutralizing antibody formation, beyond the standard rate seen for recombinant therapeutic proteins, which may limit their use. However, the current regulatory viewpoint, based on data accumulated in a number of clinical trials involving different plant-expressed proteins, is quite promising in this regard Currently, several biotechnology companies have used plant biotechnology to produce protein pharmaceuticals, such as glucocerebrosidase to treat Gaucher disease in our case, lipase to treat cystic fibrosis, alpha-interferon, lactoferrin, and others, which are under evaluation in human studies Checklist S1CONSORT Checklist(0.06 MB DOC)Click here for additional data file.Protocol S1Trial Protocol(1.72 MB DOC)Click here for additional data file. |
Sexual dysfunction is common in systemic sclerosis (SSc). Male erectile dysfunction (MED) has been reported in around 80% of subjects and more than half of female patients fulfill criteria for diagnosis as female sexual arousal Disorder (FSAD). While some evidence supports a role for cavernosal fibrosis, abundant data suggest that MED is yet another clinical feature of SSc related to vasculopathy. The contribution of vasculopathy to the more complex issues of female sexual dysfunction is less clear. Inhibitors of Type V phosphodiesterase are effective in men with MED secondary to SSc. Limited study in women suggests inconsistent effects on behavior (frequency) but not on measures related to perfusion. Sexual activity is an important component of quality of life and an important domain for the caregiver to address; it is not clear that it warrants primary consideration as a consistent measure of scleroderma-related vasculopathy. Any comprehensive hypothesis concerning the pathogenesis of systemic sclerosis must account for the varying contributions of vascular damage, extravascular tissue fibrosis, and inflammation . While cComplications from scleroderma do have a negative impact on sexual function and in turn on the overall quality of life. In spite of the 80% female predominance of SSc , most ofMED is defined as the consistent inability to attain and maintain an erection sufficient to permit satisfactory sexual performance . The firSeveral studies have ruled out a neurological basis for ED in SSc , 21. RecPenile vascular damage in SSc patients was assessed using Duplex ultrasonography , 23 showPhosphodiesterase type-5 (PDE5) is an enzyme which degrades cGMP. PDE-5 inhibitors enhance erectile function by blocking degradation of cGMP leading to an increase in intracellular cGMP in the corpus cavernosum and penile vasculature. The result is an increase in the relaxation of the smooth muscle leading to an increased blood flow and penile erection . There aThere is an inequality in the number of studies focusing on male versus female sexual dysfunction (FSD) in the general population and the Changes associated with SSc can have a negative impact on female sexuality and sexual functioning. Symptoms such as skin tightening around the vaginal introitus, joint contractures, muscle weakness, changes in skin around the breasts and breast muscle, and joint pain have been found to be associated with lower levels of sexual functioning, desire, arousal, lubrication, and satisfaction , 6, 30. The number of women with SSc reporting sexual dysfunction is higher than those reported in the general population and also higher than those reported in studies on other chronic conditions –13. BhadWhile there are many studies on the use of PDE-5 inhibitors in the treatment of ED there are few studies on their use in the treatment of female sexual arousal problems and their effectiveness is in question . Our undWhile there are few studies on the effectiveness of PDE-5 inhibitors for FSD in the general population, no studies were found in SSc patients. The paper by Chivers and Rosen regardinP = .021; 2-tailed) as was the change from baseline . The only other statistically significant difference in this study was found in a change from baseline in the sexual desire domain of the FSFI for drug although this was not statistically different from the change in the placebo group. A total of 39 patients met all inclusion criteria, agreed to attempt sexual activity at least once weekly and successfully completed all 5 visits. When comparing change from baseline between drug and placebo on the six domains of the FSFI no statistically significant differences were found although patients reported greater change when on drug versus when on placebo. A significant difference was found between drug and placebo in the number of sexual activities with SSc patients reporting more frequent sexual activities when receiving drug than when on placebo . This difference was statistically significant in spite of a host of physical and psychological difficulties associated with their disease. Complete care should consider agents for vaginal lubrication, advice about positioning and attention to sexual adverse events of therapies. |
Sarcodon imbricatum wildly grown in the Black Sea Region of Turkey were investigated in this study. Antioxidant activities were evaluated in terms of total antioxidant activity, reducing power, metal chelating ability, inhibition of linoleic acid peroxidation, superoxide, peroxide and hydrogen peroxide scavenging effects. Various antioxidant activities were compared to references antioxidants such as α-tocopherol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and trolox. In total antioxidant (12674.45 μmol α-tocopherol/g of extract), superoxide scavenging (53.74%) and peroxide scavenging activity (45.73%), the methanol extract of Sarcodon imbricatum showed stronger activity patterns than that of references antioxidants. Reducing power, metal chelating activity and free radical (DPPH•) scavenging activity was increased with the increasing concentration. The contents of total phenolic, flavonoid, anthocyanin, ascorbic acid, β-carotene and lycopene of Sarcodon imbricatum were determined and found to be noteworthy.The antioxidant activities of the methanol extract of The mixture was shaken vigorously and kept at room temperature for 30 min. Then the absorbance was measured at 517 nm. The decrease in the absorbance of the DPPH• solution indicates an increasing of DPPH• radical-scavenging activity. This activity was calculated by the following equation:The effect of of Blois. wherein • scavenging effect (%) = [(A0-A1)/A0 × 100],DPPH0 is the absorbance of control and A1 is the absorbance of mushrooms or standards.where Aet al.Hydrogen peroxide scavenging activity, (%) = (V0 is the volume of Na2S2O3 solution hydrogen peroxide (without extract), V1 is the volume of Na2S2O3 solution mixed with the extract or standard antioxidants.where V4 and H2O2. The reaction mixture contained 1 ml FeSO4 (1.5 mM), 0.7 ml H2O2 (6 mM), 0.3 ml sodium salicylate (20 mM) and sample . After incubation for 1 h at 37°C, the absorbance of the hydroxylated salicylate complex was measured at 562 nm. The percentage scavenging effect was calculated as follows:Peroxide scavenging activity was measured according to a modified method of Smirnoff and Cumbes. Peroxide1-A2)/Ao] × 100 where A0 is the absorbance of the control (without extract or standards) and A1 is the absorbance including the extract or standard, A2 was the absorbance without sodium salicylate.The peroxide scavenging activity (%)= [1- or standard errors (SE). The results were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's HSD test with α= 0.05. This treatment was carried out using SPSS v.16.0 software.Sarcodon imbricatum.The amount of extractable compounds was 234 mg/g dry plant material for methanol extract of r2=0.9904) and expressed in gallic acid equivalents (GAE). The total phenolic content per gram of crude extract was found 30.20±1.38 mg GAE/g DW. The greater efficiency of methanol in extracting the phenolic compounds would be expected to result in higher antioxidant activity. This value was higher than that reported research.[Sarcodon imbricatum extract might account for the better results found in its antioxidant assays.The content of extractable phenolic compounds in extract, determined from regression equation of calibration curve and expressed in catechin equivalents. The total flavonoid content of methanol extract from Sarcodon imbricatum was found 0.260±0.009 mg quercetin/g of DW. Flavonoids are very important plant constituents because of active OH.− and show antioxidant activity.[The total flavonoid content of methanol extract from activity.Sarcodon imbricatum extract was 0.13 ± 0.03 as mg cyanidin 3-glucoside/g dry weight. It is to be expected that several activities might be related to a possible antioxidant action from anthocyanosides like polyphenol compounds. The extracts of edible plant were most abundant in phenolics were also most abundant in anthocyanins . It has been found that polyphenolic compounds are one of the most effective antioxidant constituents in plant foods, including fruits, vegetables and grains.[The content of total anthocyanosides in d grains.Sarcodon imbricatum extract was 0.013±0.002 mg ascorbic acid/g DW, 2.08±0.09 μg caratenoid/ g DW, and 2.03±0,05 μg lycopene / g DW, respectively. These values were found in valuable amounts, which were in agreement with other reports concerning ascorbic acid, β-carotene and lycopene quantification in different mushrooms.[The content of ascorbic acid, β-carotene and lycopene in the methanolic extract of ushrooms.–14max), 327 (λmax), and 356 (λmax).[max of extract 409 and 513 nm may be due to the presence of flavonoids and anthocyanins, respectively. It was found that flavonoids and anthocyanins exhibit comparable special bands to the values reported in the literature.[Sarcodon imbricatum extract and chemical contents.[The UV–Vis absorption 200-800 nm) spectra of the mushrooms extracts were assessed for the characterization of phenolic compounds, flavonoid and anthocyanins . Phenoli6 (λmax).39 λmax oterature.–40 These0-800 nm Sarcodon imbricatum and references antioxidants at 100 μg/mL concentration as measured by phosphomolybdenum method is presented in Sarcodon imbricatum and reference antioxidants exhibited the following order: BHA > Sarcodon imbricatum > BHT > trolox. These results were statistically significant, (P < 0.05). The results of the three plants are considered to be noteworthy when compared to the findings of other studies concerning medicinal plants in Turkey.[The phosphomolybdenum method usually detects antioxidants such as ascorbic acid, some phenolics, α-tocopherol, and carotenoids. The antin Turkey. The antin Turkey.–16Sarcodon imbricatum inhibited linoleic acid peroxidation. The results obtained for extract lower than the standards α-tocopherol, BHA, and trolox, but higher than BHT. In conclusion, we can infer that this mushroom extract competes with α-tocopherol, BHA, BHT, and trolox, but not significantly. In previous studies, the inhibition of linoleic acid of methanolic extraction of several commercial and medicinal mushrooms have been reported.[reported.–422.−, which is a reduced form of O2, has been implicated in the initiating oxidation reactions associated with aging. In the PMS/NADH-NBT system, superoxide anion derived from dissolved oxygen by PMS/NADH coupling reaction reduces NBT. Antioxidants are able to inhibit the blue NBT formation.[Sarcodon imbricatum had strong superoxide radical scavenging activity, higher than that of references antioxidants at the same concentrations, (P < 0.05). Superoxide radical scavenging activity of those samples followed the order: Sarcodon imbricatum > BHA > α-tocopherol > BHA > trolox. The results were found statistically significant (P<0.05). It was reported that the superoxide anion scavenging activity could be due to the action of a free hydroxyl group.[The production of highly reactive oxygen species such as superoxide anion radicals, hydrogen peroxide, and hydroxyl radicals is also catalyzed by free iron through Haber-Weiss reactions. O2.−, whormation. The decryl group.Sarcodon imbricatum extract did not show good hydrogen peroxide scavenging activity against H2O2, the lowest being that of reference antioxidants. H2O2 scavenging activity of those samples followed the order: BHT > BHA > trolox > α-tocopherol > Sarcodon imbricatum. These results were statistically significant, (P< 0.05). It is well established that hydrogen peroxide is not dangerous as it is, but may well be because of its ability to form the hydroxyl radical, thereby emphasizing on the importance of its elimination. Indeed, it has previously been proven that dietary phenols protect mammalian and bacterial cells from cytotoxicity induced by hydrogen peroxide,[2O2 scavenging activity of our edible mushrooms could be due to the presence of phenols.peroxide, indicatiSarcodon imbricatum had stronger scavenging ability against hydroxyl radicals than the antioxidants (P < 0.05) [Sarcodon imbricatum > α-tocopherol > BHA > BHT > trolox and these results were statistically significant, (P< 0.05). Previous studies had reported two types of antioxidant mechanism: suppression against hydroxyl radical generation, and cleaning hydroxyl radical generated.[The methanol extract of < 0.05) . Peroxidenerated. The formenerated.K3Fe(III)(CN)6+Ph−OH→Kx(Fen(III)Fe(II)(CN)63+Ph−O•+H+2+ concentration.[Sarcodon imbricatum was lower than that of the reference antioxidants. But, the reducing power of the extract increased with concentration significantly, P< 0.05. It was reported that the reducing power of mushrooms might be due to their hydrogen-donating ability.[Therefore, measuring the formation of Perl's Prussian blue at 700 nm can monitor the Fentration. The redu ability.+2 accelerates lipid peroxidation by breaking down hydrogen and lipid peroxides forms by Fenton free radicallic reaction; +2 ion can form complexes with ferrozine. In the presence of chelating agents, the complex formation is prevented, resulting in a decrease in the red color of the complex. Measurement of color reduction allows determination of metal chelating activity. Measurement of the rate of color reduction allows estimation of the chelating activity of the coexisting chelator.[Sarcodon imbricatum and the reference antioxidants interfered with the formation of ferrous-ferrozine complex, suggesting that it has chelating activity and captures ferrous ion before ferrozine. Sarcodon imbricatum > BHT > BHA > α-tocopherol > trolox, with percentage chelations of 98.90, 80.20, 80.00, 75.00, 69.50, and 64,00%, respectively. In parallel to the data obtained from some edible mushrooms that exhibited the methanolic extract of mushroom.[Iron has the most important lipid pro-oxidant. It is known that FeOH−+OH•). Fe+2 ionchelator. In this mushroom.• free radicals can be used to evaluate the antioxidant activity of specific compounds or extracts in a short time. The radical scavenging of mushroom extract was tested using a methanolic solution of the “stable” free radical, DPPH. Sarcodon imbricatum showed appreciable DPPH free radical scavenging activities at the selected scope of concentrations. The methanolic extract of mushroom tested showed much stronger scavenging activities against DPPH compared to antioxidant such as α-tocopherol, BHA, and BHT [Sarcodon imbricatum and reference antioxidants on the DPPH· radical were of the following order: trolox > Sarcodon imbricatum > BHA > BHT > α-tocopherol, with the percentage scavenging values of 98.80, 93.30, 86.00, 71.30, and 61.65%, respectively, at the concentration of 500 μg/ml. These results were statistically significant, P<0.05. The methanolic extracts from other mushrooms showed a moderate increase in DPPH free radical scavenging activities. In the previous studies, the same situations were reported for the methanolic extracts of wild grown mushrooms.[Free radical scavenging is one of the known mechanisms by which antioxidants inhibit lipid oxidation. The method of scavenging DPPH and BHT . The scaushrooms.Sarcodon imbricatum widely consumed in Giresun of Turkey. The results underline that the extract of mushroom has possessed significant antioxidant properties. Therefore, mushroom extract could be a potential source of natural antioxidants. The consumption of Sarcodon imbricatum might give certain level of health protection against oxidative damages. With the established antioxidant activity of this mushroom extract, the specific chemical characteristics and separation of the antioxidative components in the methanol extract should be further investigated.On the basis of the results obtained, we have herein demonstrated the antioxidant activities of the methanol extract of |
One of the least-appreciated advances in pediatric rheumatology over the past 25 years has been the delineation of the many ways in which children with rheumatic disease differ from adults with the same illnesses. Furthermore, we are now learning that paradigms that are useful in evaluating adults with musculoskeletal complaints have limited utility in children. Nowhere is that more true than in the use of commonly used laboratory tests, particularly antinuclear antibody (ANA) and rheumatoid factor (RF) assays. This short review will provide the practitioner with the evidence base that supports a more limited use of ANA and RF testing in children. Order an ANA and a rheumatoid factor." It's common on adult wards, but it's used with disturbing frequency on pediatric wards as well. One of the least appreciated advances in pediatric rheumatology over the past 20 years has been the realization that models used to evaluate adults with musculoskeletal complaints do not serve children well . Thus, itologist ,4. This tologist . In thatThis short report will summarize some of the evidence base that supports a more limited use of ANA and rheumatoid factor tests by primary care physicians evaluating children for musculoskeletal complaints and/or suspected rheumatic disease. It is intended as a review for primary care physicians who wish to better understand how to use and interpret these two commonly-ordered tests.did have positive RF tests could be easily identified on the basis of the history and physical examination; the test did nothing to establish the diagnosis. McGhee and colleagues [Rheumatoid factor (RF) tests remain a standard assay for screening adult patients with musculoskeletal complaints and, depending on the population studied, may be positive in as many as 85% of adults with rheumatoid arthritis and fewer than 10% of healthy adult individuals . This tethere is never a reason to request a rheumatoid factor assay as a diagnostic test on a child. Pediatric rheumatologists will, however, continue to use rheumatoid factor testing as a prognostic biomarker until better indicators of prognosis emerge.Although the prevalence rate of RF-positive JRA/JIA is higher in certain populations , the diAntinuclear antibody (ANA) assays have the opposite shortcoming of RF assays: they are commonly positive in healthy children ,10) and Does this child have systemic lupus? It would be reasonable to propose, therefore, that a request for ANA testing in a child be accompanied by requests for a complete blood count and differential, urinalysis, and serum C3 and C4 levels, all of which have a high likelihood of being abnormal in childhood-onset systemic lupus [The limits of ANA in evaluating children with suspected JRA/JIA was corroborated in the earlier McGhee study , as showic lupus . Not surIt should be evident from the foregoing discussion that autoantibody testing has a much more limited utility in the evaluation of children with musculoskeletal complaints or suspected rheumatic disease than it does in adults. This is not to say that the laboratory is not helpful. A complete blood count and differential may, for example, provide the diagnosis in a child with severe musculoskeletal pain and refusal to walk, as musculoskeletal pain, and even frank arthritis, are common presentations of children with leukemia ,15. Simidistinctive clinical presentations of rheumatic disease in children, including the typical signs and symptoms, age range of the affected children, and diseases that mimic those under primary consideration.The reader will note that, in each of the above examples, the utility of the laboratory derives from the ability of the clinician to formulate a differential diagnosis based on the history and physical examination. Having a mental category called "rheumatic disease" is not particularly helpful in assessing children, as the different rheumatic diseases display a broad spectrum of presentations, affect children of different ages, and are characterized by physical findings that show only limited overlap between the different disease entities. This means, therefore, that the practitioner needs to be familiar with the children aren't just adults.It is reasonable for clinicians to desire a simple "test" that will allow them to consider or exclude "rheumatic disease" as a broad category in children. Unfortunately, such a test doesn't exist, any more than there's a "test" that excludes metabolic disease or endocrine disease (children with defects in steroid synthesis present at different ages and with different symptoms compared with children with type 1 diabetes). There is no reason to believe that such a test will ever emerge. We are therefore left with what good primary care physicians have always relied on: a good history, focused physical examination, and broad knowledge base. After all, |
To evaluate changes in intraocular pressure (IOP) after clear corneal phacoemulsification (CCP) in normal patients.th day, lst, 2nd, 3rd month and 6 months after surgery.A prospective study including 273 normal patients selected for cataract extraction by CCP. Intraocular pressure was recorded on the 15For statistical analysis, Epi Info was used to determine the statistical significance of changes in IOP.P = 0.12), but significantly correlated with change in anterior chamber depth (ACD) (P = 0.002). The postoperative IOP was inversely related to preoperative ACD (P = 0.012). Age, sex and axial length were not significantly related to IOP reduction (P = 0.2–0.5)The mean age of 96 women and 177 men was 71 ± 12 years. The mean IOP before surgery was 14.18 ± 3.4 mmHg. Our patients showed a mean decrease in IOP of 2.25 mmHg (16%) compared to preoperative values. Change in IOP was not related to lens thickness (CCP was associated with a statistically significant reduction in IOP. The exact mechanism by which cataract surgery results in IOP reduction is unclear. CCP can be performed with the intent of achieving better IOP control. Cataract extraction surgery, independent of the technique used, induces variations in intraocular pressure (IOP). Although an elevation of the IOP in the early postoperative stage may be noted, many studies have reported a reduction in IOP.‐3 Some s4This was a prospective study including 273 normal patients selected for cataract extraction by phacoemulsification using a 3.2 mm clear corneal incision between June 2003 and January 2006. The study was approved by the Hassan II University Ethics Committee.A prospective analysis was performed using clinical charts focusing on patient age and sex, size of the capsulorhexis, and pre- and postoperative IOP. The axial length, lens thickness and anterior chamber depth (ACD) were measured during preoperative assessment three weeks before surgery using ultrasound A scan by contact technique. Central corneal thickness was not assessed. IOP was measured by Goldmann applanation tonometer by the same examiner preoperatively, on the 15th day, and subsequently one, two, three and six months after surgery. The mean change in IOP after surgery was calculated. The mean follow-up period was six months.Patients with history of ocular surgery, trauma, preoperative IOP greater than 21 mmHg, on ocular medication and who developed postoperative complication were excluded from the study.For statistical analysis, Epi Info was used to determine the statistical significance of changes in IOP. The statistical significance between the groups was estimated using Chi test. A valueAll surgeries were performed by the same surgeon.Most of the patients underwent surgery under peribulbar anesthesia using Lidocaine.Surgery involved a 3.2 mm superior clear corneal tunnel incision, injection of viscoelastic material into the anterior chamber, capsulorhexis of 5 mm, hydrodissection, in the bag phacoemulsification using phaco-chop technique, cortex aspiration, additional injection of viscoelastic material and insertion of foldable hydrophobic intraocular lens (IOL) in the capsular bag. The viscoelastic material was then removed. The corneal incision was closed by stromal hydration.Postoperatively, all patients were treated with topical combination of dexamethasone and Neomycin eyedrops during four weeks, and topical nonsteroidal antiinflammatory eyedrops four times daily for eight weeks.P = 0.012), after 1 month 11.98 ± 3.1 mmHg (P = 0.015), after two months 11.92 ± 2.4 mmHg (P = 0.005), after 3 months 11.84 ± 1.4 mmHg and after six months 11.82 ± 1.3 mmHg (P = 0.005).Two hundred and seventy three eyes of 273 patients were recruited for the study. The mean age of the 96 women and 177 men was 71 ± 12 years. The mean IOP before surgery was 14.18 ± 3.4 mmHg. Mean preoperative ACD was 2.96 mm and postoperative ACD was 4.09 mm. The mean lens thickness was 4.24 mm and axial length was 23 mm. A postoperative reduction of IOP was found as shown in The postoperative reduction in IOP in mmHg and in percentage is shown in Figures P = 0.011). Lens thickness was not significantly related to change in IOP (P: 0.12); however, it was significantly related to change in ACD (P: 0.002). The postoperative IOP was inversely related to preoperative ACD (P: 0.012). Age, sex, axial length were not significantly related to IOP reduction range of P: 0.2–0.5.The reduction of IOP measured after 15 days was 2.1 mmHg, after 1 month 2.26 mmHg, after three months 2.34 mmHg and after six months 2.36 mmHg. Our group had a mean decrease in IOP of 2.25 mmHg (15.77%) compared to preoperative values (Numerous studies have shown that cataract surgery by phacoemulsification with posterior chamber IOL induces a mid-and long-term lowering of IOP.‐10 Altho7IOP reduction has been shown to be more prominent after CCP than after phacoemulsification with sclerocorneal tunnel.14 Howeve20et al reported that the size of the capsulorhexis had an effect on the IOP after phacoemulsification; they showed that a capsulorhexis of 4 mm had a greater IOP lowering effect than a capsulorhexis of 6 mm.[The exact mechanism by which cataract surgery improves IOP is unclear. Many hypotheses have been presented in the literature, namely 1 of 6 mm. In our sStudies on patients with open-angle glaucoma have demonstrated a pressure lowering effect of CCP.1823 In e182In conclusion, cataract surgery by CCP induces reduction of IOP in normal patients. The pathogenic mechanisms are still unclear. CCP can be performed with the intent of achieving better IOP control. |
Eight patients with angioimmunoblastic lymphadenopathy have been studied by a variety of immunological and pathological techniques. They exhibited a spectrum of immunological reactivities that, in this small series, could be roughly correlated with survival. Those patients with relative B-cell predominance as shown by cell marker studies, histologically showed large numbers of plasma cells, and this pattern was associated in 3 of our patients with a survival of 3 years or more. T-cell predominance or both B- and T-cell depletion was associated histologically with large numbers of blast cells and eosinophils, but with few plasma cells. These patients responded poorly to therapy and had short survival times. One patient with B-cell predominance subsequently died of a histiocytic lymphoma. |
Grid cells in the rat entorhinal cortex display strikingly regular firingresponses to the animal's position in 2-D space and have beenhypothesized to form the neural substrate for dead-reckoning. However, errorsaccumulate rapidly when velocity inputs are integrated in existing models ofgrid cell activity. To produce grid-cell-like responses, these models wouldrequire frequent resets triggered by external sensory cues. Such inadequacies,shared by various models, cast doubt on the dead-reckoning potential of the gridcell system. Here we focus on the question of accurate path integration,specifically in continuous attractor models of grid cell activity. We show, incontrast to previous models, that continuous attractor models can generateregular triangular grid responses, based on inputs that encode only therat's velocity and heading direction. We consider the role of thenetwork boundary in the integration performance of the network and show thatboth periodic and aperiodic networks are capable of accurate path integration,despite important differences in their attractor manifolds. We quantify the rateat which errors in the velocity integration accumulate as a function of networksize and intrinsic noise within the network. With a plausible range ofparameters and the inclusion of spike variability, our model networks canaccurately integrate velocity inputs over a maximum of ∼10–100meters and ∼1–10 minutes. These findings form aproof-of-concept that continuous attractor dynamics may underlie velocityintegration in the dorsolateral medial entorhinal cortex. The simulations alsogenerate pertinent upper bounds on the accuracy of integration that may beachieved by continuous attractor dynamics in the grid cell network. We suggestexperiments to test the continuous attractor model and differentiate it frommodels in which single cells establish their responses independently of eachother. Even in the absence of external sensory cues, foraging rodents maintain anestimate of their position, allowing them to return home in a roughly straightline. This computation is known as dead reckoning or path integration. Adiscovery made three years ago in rats focused attention on the dorsolateralmedial entorhinal cortex (dMEC) as a location in the rat's brain wherethis computation might be performed. In this area, so-called grid cells firewhenever the rat is on any vertex of a triangular grid that tiles the plane.Here we propose a model that could generate grid-cell-like responses in a neuralnetwork. The inputs to the model network convey information about therat's velocity and heading, consistent with known inputs projectinginto the dMEC. The network effectively integrates these inputs to produce aresponse that depends on the rat's absolute position. We show that sucha neural network can integrate position accurately and can reproducegrid-cell-like responses similar to those observed experimentally. We thensuggest a set of experiments that could help identify whether our suggestedmechanism is responsible for the emergence of grid cells and for pathintegration in the rat's brain. Since the discovery of grid cells in the dorsolateral band of the medial entorhinalcortex (dMEC) These ideas differ radically from each other, but they share a common assumptionabout the nature of the input feeding into dMEC, namely, that the input conveysinformation primarily on the rat's velocity and heading. Within all thesemodels, grid cell activity must then arise from precise integration of therat's velocity.Grid cell firing exhibits remarkable accuracy: The periodic spatial tuning patternremains sharp and stable over trajectories lasting 10's of minutes, with anaccumulated length on the order of hundreds of meters in vitro intracellularrecordings in vivo extracellularrecordings How do theoretical models measure up, in estimating position from input velocitycues? The theta-oscillation model of grid cells For continuous attractor models, we previously showed Conceptually, the existence of an integrating apparatus seems pointless if it iscompletely dependent on nearly continuous corrections coming from an external sourcethat specifies absolute position. Thus, it seems reasonable to require thattheoretical models of path integration in dMEC, if using faithful velocity inputs,have the ability to reproduce stable grid cell patterns for trajectories lasting afew minutes.Our aim, therefore, is to establish whether it is possible for model grid cells toaccurately integrate velocity inputs. We restrict our analysis specifically tocontinuous attractor networks. As will become clear, the precision of velocityintegration can strongly depend on various factors including network topology,network size, variability of neural firing, and variability in neural weights. Herewe focus on three of these factors: boundary conditions in the wiring of the network(periodic vs. aperiodic), network size, and stochasticity in neural activityWe quantify path integration accuracy in both periodic and aperiodic recurrentnetwork models of dMEC, and demonstrate that within a biologically plausible rangeof parameters explored, such networks have maximum attainable ranges of accuratepath integration of 1–10 minutes and 10–100 meters. Larger, lessnoisy networks occupy the high end of the range, while smaller and more stochasticnetworks occupy the low end. We end with suggestions for experiments to quantifyintegration accuracy, falsify the continuous attractor hypothesis, and determinewhether the grid cell response is a recurrent network phenomenon or whether itemerges from computations occurring within single cells.In our model, each neuron receives inhibitory input from a surrounding ring of localneurons. The entire network receives broad-field feedforward excitation (To reproduce the regular single-neuron (SN) lattice patterns observed in experiment,the pattern formed in the neural population must be coupled to the rat'svelocity. This coupling is arranged in such a way neuronsarranged in a square sheet. Neurons close to each edge of the sheet formconnections with neurons on the opposite edge, such that the topology of thenetwork is that of a torus. We simulate dynamics in a network of neurons driven by velocity inputs obtainedfrom recordings of a rat's trajectory neurons also performs well enough to produce coherent SN grids over the sametrajectory, A deterministic periodic network of only 40The presence of a clear spatial grid in the SN response to velocity inputs aloneis a good indication of the accuracy of integration. If the rat'sinternal estimate of position were to drift by half a grid period, the neuronwould fire in the middle of two existing vertices rather than on a vertex. Asthe rat traveled over its trajectory, the neuron would fire at various“wrong” locations, with the resulting SN response becomingprogressively blurred until no grid would be discernible. This would happen evenif the population pattern remained perfectly periodic throughout.Therefore, the following properties are equivalent: (1) Coherent grids in the SNresponses, (2) Accurate path integration of the full trajectory over which theSN responses are visualized, with errors smaller than the grid period. Anexample of this equivalence is given in Next, because the population pattern phase accumulates errors whenever thepattern slips relative to rat motion, another equivalent condition for accuratepath integration is (3) Linear relationship between network flow velocity andinput velocity over the input velocity range, independent of direction.These equivalent conditions for accurate integration apply to both periodic andaperiodic network models of grid cells (discussed next).It is unclear whether a torus-like network topology, in which neurons alongopposite edges of the network are connected to form periodic boundaryconditions, exists in the rat's brain. Even if such connectivityexists, it may require, at an earlier stage of development, an initiallyaperiodic network also serve to stabilize the orientationof the population pattern (data not shown), suggesting that the undesirablecoupling of velocity inputs to rotation is also related to the existence ofthe boundaries.2 (∼104) neurons decay as the pattern relaxes to its preferred orientation.Similarly, distorting the pattern by stretching it, adding noise, or by removingblobs from the pattern will generate an unstable state, which will rapidly decayto a steady state within the attractor manifold.aperiodic network, translations of a steady state patternare similar but not exactly equivalent, because the phase of the activitypattern relative to the boundary affects the energy of the state. Strictlyspeaking then, these states do not form a continuous attractor manifold, In the A stable population pattern state can be rotated around the center of a circularaperiodic neural sheet to obtain another stable state that is identical inenergy to the original one. Hence, rotations correspond to a flat direction inthe energy surface, In the network models described here, the structure of the attractor manifolde.g., iscomplSo far we have considered errors in integration that occur in the absence ofnoise. Unlike in the noise-free case, neural noise can induce the populationpattern to flow or rotate even when velocity inputs are absent. To assess hownoise influences the precision of the network's response, we presentresults from spiking neural networks with the same connectivity as in the ratebased models. Dynamics in these networks are noisy due to the stochasticity ofdiscrete spiking events.For the same network parameters as in Integration can be decomposed into two elements: a memory that holds onto thestate of the integrator, and a mechanism that correctly increments the stateof the integrator in response to inputs. The linearity of the velocityresponse of the network, described earlier for noise-free networks, may beviewed as an assessment of the accuracy of the increment mechanism, whilethe degree of drift in the network state in the absence of velocity inputsand external corrective cues is a quantification of the network'sability to hold onto its current state. Therefore, a way to assess theeffect of noise on integration accuracy is to examine the drift in thepopulation state when external velocity inputs are absent.As shown in 4 neurons and CV = 1,roughly in agreement with our observations from Quantitatively, the drift in the phase of the population pattern appearsdiffusive to achieve a similarperformance, even though the two networks showed similar performance in thenoise-free case.Assuming that the translational drift in the aperiodic network is similar tothat measured in the periodic network we conclude that, in the aperiodicnetwork, rotations are the more severe source of noise-driven decoherence ofthe SN response. This conclusion is in agreement with the observation thatthe 128Motivated by the result that sub-Poisson spiking statistics are important foraccurate integration in the grid-cell network, we analyzed spike recordingsfrom neurons in dMEC For various reasons, it is not possible to exactly compare the CV used in oursimulations and the CV of the recorded cells in dMEC. For example, dMECcontains numerous cell types, each of which may have different CVs. Also,the effects of individual neural variability on integration performance areameliorated by averaging over the network population, but the size of theactual dMEC network may not be the same as in our simulations, and theactual network may contain correlations not included in our model, so thateven if we were able to pick the “correct” CV forindividual neurons, the net effect on integration performance may bedifferent in the model from that in dMEC. Finally, the CV is alow-dimensional measure that does not fully characterize the spikingstatistics of a neuron: even if we could match the size of the dMEC networkand the CV of each neuron type, the statistics of our model neurons couldgreatly differ from those in the rat.Despite these caveats, our results suggest that a significant blurring of theSN response is expected to occur on a time scale ranging between a fewminutes to a few tens of minutes, within a reasonable range of estimates forthe number of neurons in the network and the variability of neuralspiking.Armed with the proof-of-concept results that a continuous attractor network modelcan integrate velocity inputs accurately enough to produce SN grids, we nextseek to explore testable predictions of the continuous attractor hypothesis inthe grid cell system and contrast them with the properties of models in whichthe grid responses emerge independently in each cell relationship between cells, defined by whetherneurons are co-active or active at different phases. The stability of theattractor manifold and the instability of states outside it have a number ofimplications for experiment.As described earlier, the low-dimensional structure of the attractor meansthat only a very small subset of possible states of the network, defined bystrict inter-relationships in neural activity (population patterns), arestable, while other states quickly decay away. The quantity conserved acrosspattern translations and therefore across the attractor manifold is thephase Due to the stability of the attractor manifold, phase relationships in theperiodic network should be stable over the time-scale of days (because thepattern does not rotate), regardless of inevitable drifts in the absolutephase of individual neurons. Even in aperiodic networks, we expect phaserelationships to persist over 1–10 minutes, but possibly notlonger due to the possibility of rotations. Under similar conditions inmodels where the grid is generated separately by individual neurons(“independent neuron models”), like temporalinterference models Because the attractor dynamics are restoring, small perturbations of state without a componentalong the attractor manifold should not produce lasting changes in thestates of these neurons or the network. Network interactions should restorethe state to the original state that preceded the perturbation: thus, boththe absolute phases of cells and their phase relationships should beunchanged by the perturbation. This statement also applies to largeperturbations, if they have no appreciable projection along the attractormanifold . By contrast, following small or large perturbations tosubsets of cells in independent neuron models, the absolute activity statesof those cells, as well as their relative phase relationships withunperturbed neurons should change, due to the absence of restoring networkinteractions.Perturbations that have a large component along the attractor manifold shoulddrive a coherent transition to the point on the attractor manifold that isclosest to the perturbed state. Because the new state will be on theattractor manifold, phase relationships between neurons should be unchanged.Head direction cells provide a means to induce such a perturbation:Stimulating a subset of head direction cells should drive a rigid (coherent)and lasting translation of the entire population pattern, producing the sameshift in phase in all cells, regardless of whether or not they receiveddirect head direction input. By contrast, similar inputs provided only tosubsets of cells in independent neuron models should produce changes inphase only in the stimulated cells.The continuous attractor model predicts that all cells in the network musthave identical orientations, and all phases must be equally represented inthe population every cell in the network must display the sameirregularity, up to a global shift in phase. Indeed, our preliminaryanalysis of data from Further, in the continuous attractor model, if any cell's gridresponse contains a reproducible irregularity of any kind , it follows thatIn experiments where a familiar enclosure is resized, the SN response isobserved to rescale along the rescaled dimension of the enclosure, at leasttemporarily To explain why these rescaling experiments are consistent with a continuousattractor model of grid cells, it is important to stress the differencebetween the population-level and the SN responses. The attractor manifoldconsists of the steady states of the population response, which consists oftranslations of a canonical pattern.Thus, stretching and rotation of the population pattern are forbidden(unstable) and cannot be invoked within the continuous attractor models toexplain the experimental observations.The SN response, on the other hand, is not directly subject to constraintsimposed by the attractor manifold on the population pattern, because it is afunction of both the instantaneous population pattern and the velocityresponse of the pattern in time. If the pattern were to flow more slowlyalong one dimension than the other, for equivalent rat speeds, the SNresponse would be a stretched version of the regular underlying populationgrid, with the stretched dimension corresponding to the slow flow dimension.Hence, stretching of the SN response can be explained in the continuousattractor model by an amplitude modulation of head direction inputs tuned tothe relevant head direction, without inflicting such a deformation on thepopulation pattern . If the How can experiments distinguish between these two possibilities? Thecontinuous attractor model predicts that the phase relationships betweenneurons must remain unchanged upon stretching of the SN response . This prIn contrast, if the SN stretching is due to a similar stretching in thepopulation response, there should be little to no change in the amplitude ofvelocity modulation of the cells. In summary, changes in the phaserelationships between cells, or no change in the velocity modulation of thehead direction inputs to dMEC, when the SN responses have been stretched,would be evidence against the attractor model.Similarly, a rotation Lidocaine, or another blocker of spiking activity, applied locally to dMECwithout affecting inputs to dMEC should abolish periodic spatialresponsiveness in the subthreshold activity of grid cells. This is becauseall periodic patterning in the continuous attractor model arises fromrecurrent interactions within dMEC. By contrast, individual-neuron models,where the computation is performed within each neuron, may continue to showspatially periodic responses under such a manipulation.Given that both periodic and aperiodic continuous attractor network models ofdMEC are capable of accurate integration of rat velocity inputs, how mightit be possible to experimentally distinguish between the two possibilities?A periodic network shows no pinning, and rotations of the population responseare forbidden. Thus, phase relationships between neurons should beabsolutely stable over very long times even in the absence of any sensoryinputs. By contrast, aperiodic networks should be pinned for sufficientlylow velocity inputs, and in the absence of external corrective cues, areexpected to rotate on slow timescales (minutes to 10's of minutes).A population-wide rotation will be manifest in altered phase relationshipsbetween single neurons, or it could be probed by looking at differential(relative) rotations in the orientation of quickly estimated SN grids versusthe head direction cell population.Next, in an aperiodic network, neurons at the boundaries must receive fadinginput, meaning that their maximal activity is substantially lower than thatof neurons in the bulk; thus, the distribution of maximal rates across gridcells of the same type in an aperiodic network should be wide. If themaximal firing rate of every cell (of the same type) in the network isroughly the same, it would be inconsistent with an aperiodic network. Theconverse need not be true .We emphasize that the boundaries of the neural population are not related tophysical boundaries in space. Hence the neurons at the boundaries, discussedabove, are not expected to bear a relationship to the recently discoveredcells in dMEC whose receptive field encodes the rat's proximity toboundaries in the environment all SN responses would incidentally be strong evidenceof a continuous attractor network.Finally, if defects exist in the single neuron response, they may helpdistinguish between a periodic and an aperiodic network. By defects, here weonly mean those arising spontaneously from the pattern formation process ina network whose connectivity is itself defect-free. Defects arising fromimperfections in the weights will not flow in response to velocity inputs,and are therefore not expected to produce a systematic defect in the SNresponse. In the aperiodic case, any defect in the SN response must beeliminated if the rat returns to the area where the defect was observedafter first moving in one direction until the defect has flowed off thepopulation pattern. Conversely, if the defect persists upon return to thevicinity of the defect location even after long excursions, the lattice hasperiodic boundaries. The Presence of a stable defect which is present inThe last two predictions can help to distinguish even a well-tuned aperiodicnetwork, which may show relatively little rotation or pinning, from aperiodic network.The three main contributions of this work are:A demonstration through modeling that under reasonable conditions grid cellscan be good velocity integrators, and more specifically, that continuousattractor models are capable of accurate path integration.By ‘good’ integration, we mean that if the model networkis given accurate velocity inputs, it produces an accurate estimate of ratposition over comparable distance and time-scales to those probed inbehavioral assays. Within a plausible range of estimates for network sizeand neural stochasticity, higher accuracy was reached in larger andrelatively noise-free networks, sufficient to reproduce coherent grid cellpatterns in response to the full trajectories from Furnishing good upper bounds on idiothetic path integration accuracy withindMEC.A notable finding is that even noise-free, large networks (periodic andaperiodic) have only finite integration accuracy, and this level of accuracyis only a factor of 10–100 larger than known behavioral abilities.We provide estimates of integration accuracy in the presence of neuralnoise, which are in the range of 1–10 minutes. Integrationperformance in a fixed-size periodic network is not expected to vary greatlywith parameter tuning; aperiodic networks are more sensitive to parametertuning, and we have not optimized all parameters. However, aperiodicnetworks are upper-bounded in their performance by the correspondingperiodic network. Thus, we expect our estimates to serve as reasonable upperbounds on integration accuracy in dMEC, within the continuous-attractorpicture.Providing predictions that can falsify the continuous attractor hypothesisand help distinguish between the possibilities that grid responses aregenerated through continuous attractor networks or through independent cellcomputations.So far, the predictions of continuous attractor models are consistent with the fullcorpus of grid cell data, and explanatory of many results from experiment,suggesting, when combined with conclusion (1), that continuous attractor dynamicsare a viable, relevant mechanism for grid cell activity and path integration.Accurate behavioral dead reckoning is a cascaded result of accurate velocityinput (relative to the rat's motion) and accurate integration of thatinput. Our interest in this work was in assessing how well continuous attractormodels of dMEC can integrate their inputs. Thus, we did not focus on potentialinaccuracies (noise or biases) in the velocity inputs themselves. Even if thenetwork were a perfect integrator, errors in the input would produce anincorrect position estimate. Such errors are likely to play a role in reducingthe behavioral range over which rats display accurate dead-reckoning.A strength of attractor networks is that responses are self-averaging over thefull network: if the velocity inputs are unbiased estimators of rat movements,but are noisy, or if the velocity inputs to the network are not perfectlybalanced in number for all directions, the full network will average all itsinputs, and the net pattern flow will only reflect this average. For accurateposition estimation, however, it is important and therefore likely that inputsto the network are well tuned.Another factor that could degrade integration performance is inhomogeneity orstochasticity in the recurrent network weights. While stochasticity in neuralactivity causes the network state to drift along the attractor manifold,variability in network connectivity modifies the structure of the attractormanifold itself. If recurrent connectivity deviates significantly from thetranslation-invariant form needed to ensure that all translations of the patternare accessible without crossing over energy barriers, the activity pattern canbecome pinned at particular phases Because knowledge about synaptic strengths in the brain is exceedingly limited,it is unclear what level of variability should be expected in dMEC weights, andwhether this amount is sufficient to cause significant pinning. A question fortheory, not addressed in this work, is to estimate the amount of variability inthe network weights that would be sufficient to reduce the accuracy ofintegration below that observed in dead reckoning behavioral experiments. Forexperiments, the difficult challenge is to obtain an estimate of variability indMEC connectivity.3–104 neurons) may be viewed as awasteful proposed use of neurons, but it is broadly consistent with estimatesfor the total number of neurons in the entorhinal cortex The network size estimate in our continuous attractor model: each neuron would simply have spatially restrictedcenter-surround interactions with its neighbors. This has prompted theobservation that such a topographic network could serve as a starting point forthe development of a network with a less topographical layout and periodicboundaries We showed that the population pattern in a deterministic aperiodic network fullyequipped with a translational velocity shift mechanism and driven by purelytranslational velocity inputs, tends to rotate within a few minutes. This is theshort end of the time-scales over which plasticity mechanisms for networkdevelopment would act. If the network is entirely driven by noise and lacks aspecific velocity shift mechanism as in , the proThe problem of pattern rotations over the time scale of learning is pertinent forany effort to produce a periodic network from an initially aperiodic one in theabsence of anchoring sensory inputs and a velocity coupling mechanism.The concept of low-dimensional continuous attractors has influenced ourunderstanding of neural systems and produced successful models of a number ofneural integrators The dynamics of rate-based neurons is specified by:τ = 10 ms. The time-stepfor numerical integration isdt = 0.5 ms.The neural transfer function We assume that neurons are arranged in a 2-d sheet. Neuron The preferred directions are restricted to N,S,E,W for convenience in modeling; inthe rat, these preferences might span the continuum The recurrent weight matrix isThe feedforward input to neuron , e.g., .If The envelope function For the network with periodic boundary conditions, the envelope function is 1everywhere. For the aperiodic network,m-th spike and discarding all the other spikes. Thisprocedure generates a spike train with rate To simulate a Poisson process , ineach time-step Aperiodic network: initially network activity is low; neurons receive externalinput with The network is initialized to the exact same initial template state at thebeginning of each step . Each step consists of a constantvelocity input, with one of four directions . (A) Instantaneous activity within the neural sheet (colorrepresents the firing rate: black corresponds to vanishing rate). (B) Gridcell response: average firing rate of a single neuron (located at theelectrode tip in panel A), as a function of the rat's positionwithin the enclosure. (C) Velocity integration in the network. Top: Actualdistance of the rat from a fixed reference point (black), compared to thenetwork's integrated position estimate, obtained by tracking theflow of the pattern in the population response (blue). The reference pointis at the left-bottom corner of the square in which the circular enclosureis inscribed. Bottom: Accumulated difference between the integrated positionestimate and the actual position.Path integration and generation of grid cells in a small periodic network.Simulation of network response, with velocity inputs corresponding to arat's recorded trajectory in a 2 m circular enclosure (0.73 MB EPS)Click here for additional data file.Figure S2Population pattern in an aperiodic network with a modulation of weights. Thesteady-state pattern in a network where the strengths of the outgoingweights from each neuron are modulated based on the neuron'slocation in the sheet, according to the envelope function of Equation 5. Theexternal input is spatially uniform. All parameters are identical to thesimulation of (1.00 MB EPS)Click here for additional data file.Figure S3Path integration in periodic and aperiodic stochastic spiking networks.Simulation of network response, with velocity inputs corresponding to arat's recorded trajectory in a 2 m circular enclosure (3.43 MB EPS)Click here for additional data file.Figure S4v|<vcutoff = 8cm/s, which are of duration larger thanTv = 4 s. Wefound no blocks where the integrated displacement was more than λ/4cm, meaning that the intervals represented traverses of approximately oneblob diameter or less, with the typical distance being much shorter. Thus,the rat is likely to be either on or off a blob for the entire duration of ablock, and should have a roughly constant underlying firing rate. (2)Identify high-rate blocks where the rate is higher than some upper cutoffthreshold (to locate on-blob episodes), withrISI(t)>rhighfor each time in the block. Only those high-rate blocks of duration longerthan Tr were retained.rISI is the instantaneous firing rate,computed as the reciprocal of the inter-spike interval of adjacent spikes.rhigh = 10Hz was chosen to be large enough to exclude all intervals except those wherethe rat is clearly on a blob for the recorded cell. In all the above, theminimum interval durationTr = 5 s waschosen to eliminate random (non)spike events that momentarily change therate without reflecting an actual change in the underlying firing rate ofthe cell, while capturing as many intervals as possible for ISI analysis. Ineach of methods (1) or (2), we compute μ(ISI) and σ(ISI) foreach block as a single data-point. Next, we bin together data points withthe same rate (in bins of 1 Hz), pooling across all cells . The two methods (1) and (2) are complementary in the sense thatinterval sampling is based in the first case on rat velocity, and in thesecond case by rate-based on-blob or off-blob considerations. Neither methodguarantees that the underlying firing rate within one interval is constant.However, the two methods yield consistent results, and thus add a measure ofconfidence to the analysis.Stochasticity of recorded dMEC neurons. (A) Standard deviation (σ) ofthe inter-spike interval (ISI) distribution plotted against the mean ISI,for various values of the mean ISI. Data points from multiple simultaneouslyrecorded cells (from a single electrode) in dMEC (0.68 MB EPS)Click here for additional data file.Figure S5Deviations from a perfect triangular lattice in existing measurements. (A)Comparison of grid correlation functions from three simultaneously recordedcells, adapted from (0.59 MB EPS)Click here for additional data file. |
Previous investigations have shown that, during metazoan radiation, the exon-intron patterns of serpin superfamily in lineages pre- and postdating the split of vertebrates. Multiple intron gains were detected in a group of ray-finned fishes, once the canonical groups of vertebrate serpins had been established. In two genes, co-occurrence of non-standard introns was observed, implying that intron gains in vertebrates may even happen concomitantly or in a rapidly consecutive manner. DNA breakage/repair processes associated with genome compaction are introduced as a novel factor potentially favoring intron gain, since all non-canonical introns were found in a lineage of ray-finned fishes that experienced genomic downsizing.Here we investigated intron dynamics in the serpin genes of a lineage of ray-finned fishes, but not in any other vertebrates, suggesting that insertion rates for introns may be episodically increased. The co-occurrence of non-standard introns within the same gene discloses the possibility that introns may be gained simultaneously. The sequences flanking the intron insertion points correspond to the proto-splice site consensus sequence MAG↑N, previously proposed to serve as intron insertion site. The association of intron gains in the serpin superfamily with a group of fishes that underwent genome compaction may indicate that DNA breakage/repair processes might foster intron birth.Multiple intron acquisitions were identified in Spliceosomal introns are key attributes of most eukaryotic genes. Their origin is still unclear, though descent from group II self-splicing introns seems to be likely ,2. All cvice versa, was proposed to foster functional diversification of serpins [The serpins are a superfamily of proteins that cover a highly divergent spectrum of functions ,13. The serpins ,15.serpin genes are often arranged in tandem arrays and they constitute a substantial fraction of mammalian genomes. During diversification of vertebrates the superfamily has undergone considerable expansion [Serpin genes of basal metazoans, in contrast, are not generally intron-rich, and their exon-intron structures are not conserved along the lineages leading to vertebrates. Sporadic investigations of various species revealed radically different intron patterns in serpin genes, indicating that, during diversification of eumetazoans, massive changes in gene architectures have occurred [serpin genes from various vertebrates and Branchiostoma floridae (B. floridae), representing a group of extant cephalochordates (phylum: Chordata). Experimental approaches disclosed several genes and cDNAs coding for serpins in B. lanceolatum, further superfamily members were detected by mining the genome of the closely related B. floridae [serpin genes (L1–L3) with distinctly different intron patterns were observed . The L2 genes contain introns at positions ~86b, 151c, 223b, 283c and 339c. The L3 genes exhibit common introns at positions 73b, 125b, ~175c and 339c, however, there are also introns that are unique to individual group members . Another intron in Bflor_Spn10 is located outside the conserved serpin scaffold (gene-specific numbering of position: 29a). There are several additional serpin-like sequences in the B. floridae genome, but it is currently not discernible whether they represent intact genes (not shown); however, it is clear that serpin genes from lancelets and vertebrates differ largely with respect to their exon-intron organizations. In fact, just a single intron location (position 339c) is shared. Apparently, major changes affecting the exon-intron patterns of serpin genes have occurred since the cephalochordate/vertebrate split.We first studied floridae . Altogetn genes L–L3 with n genes L–L3 with serpins in vertebrates, we turned to lampreys, a group of basal, jawless fishes. Lampreys, in sharp contrast with lancelets, depict at least four of the six canonical groups of vertebrate serpins. A survey of cDNA and genomic sequences from Lampetra fluviatilis and from Petromyzon marinus revealed representatives of groups V2, V4 and V6 . Members of groups V3 and V5 were not identified.Having established lancelets as appropriate outgroup for evaluating evolution of L. fluviatilis and P. marinus , among other features, definitely reveals this protein as member of the serpin superfamily.Inspection of lamprey serpin sequences disclosed the presence of angiotensinogen and heparin cofactor II (HCII), two prominent members of group V2. All known angiotensinogen proteins depict a conserved decapeptide sequence close to the N-terminus that, after controlled enzymatic cleavage, gives rise to formation of peptides (angiotensin I-IV) involved in blood pressure regulation and other important physiological processes . ClearlyP. marinus (see below). We also recognized a lamprey serpin exhibiting the exon-intron pattern of group V6 . Characteristic features of all HCII sequences are the highly conserved Arg/Lys-rich helix D that is involved in GAG binding and the acidic N-terminal extension that mediates GAG accelerated thrombin inhibition ,26. Thesollagens ,27. A haollagens and its serpin genes, the dynamics of their exon-intron patterns was analyzed. The list of species investigated included: man, chicken , the clawed frog , and several fishes including Danio rerio (D. rerio), Oryzias latipes (O. latipes), Gasterosteus aculeatus (G. aculeatus), Tetraodon nigroviridis (T. nigroviridis), and Takifugu rubripes (T. rubripes). Several serpin genes, though clearly members of one of the six canonical groups, were found to deflect from the standard organizations. Like their counterparts in lampreys, angiotensinogen genes of tetrapods and D. rerio exhibit the canonical gene architecture of group V2, the T. rubripes orthologue, however, contains two extra introns that split the exonic sequences at positions 77c and 233c, respectively . Though also found in O. latipes and G. aculeatus, this intron here is not considered any further, due to its location outside the serpin scaffold. Examination of lamprey HCII also revealed unique introns. The 83c intron (α1-antitrypsin numbering) is embedded in a well-conserved region; its origin, however, is difficult to evaluate. The others (correctly predicted?) map to the N-terminal extension (gene-specific numbering of positions: 38b and 118c).The mammals ,30, chicSpn_94a, with a surplus intron at position 94a . The origin of these genes is unclear; the unique surplus intron suggests that they share a common ancestor , all of which are equipped with the standard set of introns , which probably arose as a consequence of genome duplication events in the stem lineage of ray-finned fishes [Dan_HSP47_1, as indicated by the conserved gene order . In G. aculeatus, a second intact HSP47 homologue, Gast_HSP47_2, was identified. Dan_HSP47_2 and Gast_HSP47_2 are orthologous to each other, since they share a set of flanking markers ; Gast_HSP47_2, in contrast, merely possesses the default introns. These findings suggest that the novel introns, which are restricted to HSP47_1 orthologues, were acquired by group V6 during evolution of ray-finned fishes after divergence of the D. rerio lineage, though an intron loss scenario cannot be excluded. Two intron gains or a single, coupled intron gain event are/is sufficient to explain the exon-intron patterns found in group V6; the intron removal scenario, in contrast, requires multiple intron loss events. If such losses had occurred, they must have affected several taxa, including lampreys, tetrapods and fishes. Moreover, this scenario also demands that the same two introns (positions 36b and 102c) were always deleted in parallel, while all other introns were unaffected. The parallel emergence of introns at positions 77c and 233c in angiotensinogen genes , the only member of group V5, is a potent thrombin inhibitor in the presence of heparin. Among other characteristic features, AT orthologues are easily discernible through a highly conserved sequence centering around helix D . Intron 262c is a standard attribute of group V1 that is believed to share a common ancestor with group V5 [AT genes of tetrapods. We were not able to identify this gene in P. marinus; further tracing of the 262c intron was therefore not possible.In contrast with several intron gains, we detected a single case of probable intron loss during evolution of vertebrate group V5 . We therserpins from groups V3 and V4, deviations from the standard structures were not observed (not shown). All genes from group V3 analyzed featured introns at positions ~86a-90a, 167a, 230a, 290b, 323a, 352a, and 380a. The intron pattern characteristic of group V4 was also conserved.In T. rubripes [Spn_94a orthologues cannot be aligned without introducing gaps , referred to as proto-splice sites [serpins of ray-finned fishes were purchased from the Alfred-Wegener-Institut für Polar- und Meeresforschung, Helgoland, Germany. European river lampreys (L. fluviatilis) were obtained from the Bundesforschungsanstalt für Fischerei, Hamburg, Germany.Adult lancelets (The isolation of poly(A)-RNA and genomic DNA, the synthesis of cDNA, PCR amplification of serpin cDNA fragments using various sets of degenerate primers, and cloning of 5'- and 3'-cDNA ends followed published procedures ,51. DNA serpins from human, chicken [X. tropicalis [ D. rerio [ G. aculeatus [O. latipes [T. rubripes [T. nigroviridis [P. marinus [). Sequences from the B. floridae genome were gathered from the JGI genome browser . EST and cDNA data mining included searches in the NCBI trace archive and in the UCSC genome browser [Genomic data for chicken , X. tropopicalis , D. reriD. rerio , G. aculculeatus , O. lati latipes , T. rubrrubripes and T. noviridis were extoviridis , or in t marinus , from Pr browser , applyin1-antitrypsin as described [i.e. positions 33 to 394 of human α1-antitrypsin) were considered.All intron positions predicted by gene models were examined visually, corrected and amended manually, if necessary. Whenever cDNA or EST sequences were available, intron positions were checked by means of GENEWISE . Proteinescribed . All int)) and with RepBase Censor [Sequences of non-canonical introns were searched for repetitive elements with the RepeatMasker package (version 3.2.6; (ensor using dePhylogenetic analysis was performed using the Neighbor-Joining method conducteHR designed the study, performed part of data analyses, and wrote the paper. AK accomplished data analyses. KK, YW, CB, MAF, NP and OK generated data and contributed to data evaluation.Mapping of intron positions to aligned serpin sequences. Figure depicting intron positions of serpin genes mapped onto the aligned amino acid sequences.Click here for fileserpin genes analyzed in this study and their accession numbersList of . Table listing accession numbers of genes, cDNAs and ESTs investigated in this study.Click here for fileSpn_94a genesChromosomal gene order reveals orthology of . Figure showing chromosomal synteny of Spn_94a genes.Click here for fileserpin genesSequences of non-canonical introns from vertebrate . Figure depicting the sequences of non-standard introns.Click here for file |
Both minimally invasive surgery (MIS) and computer-assisted surgery (CAS) for total hip arthroplasty (THA) have gained popularity in recent years. We conducted a qualitative and systematic review to assess the effectiveness of MIS, CAS and computer-assisted MIS for THA.An extensive computerised literature search of PubMed, Medline, Embase and OVIDSP was conducted. Both randomised clinical trials and controlled clinical trials on the effectiveness of MIS, CAS and computer-assisted MIS for THA were included. Methodological quality was independently assessed by two reviewers. Effect estimates were calculated and a best-evidence synthesis was performed.Four high-quality and 14 medium-quality studies with MIS THA as study contrast, and three high-quality and four medium-quality studies with CAS THA as study contrast were included. No studies with computer-assisted MIS for THA as study contrast were identified. Strong evidence was found for a decrease in operative time and intraoperative blood loss for MIS THA, with no difference in complication rates and risk for acetabular outliers. Strong evidence exists that there is no difference in physical functioning, measured either by questionnaires or by gait analysis. Moderate evidence was found for a shorter length of hospital stay after MIS THA. Conflicting evidence was found for a positive effect of MIS THA on pain in the early postoperative period, but that effect diminished after three months postoperatively. Strong evidence was found for an increase in operative time for CAS THA, and limited evidence was found for a decrease in intraoperative blood loss. Furthermore, strong evidence was found for no difference in complication rates, as well as for a significantly lower risk for acetabular outliers.The results indicate that MIS THA is a safe surgical procedure, without increases in operative time, blood loss, operative complication rates and component malposition rates. However, the beneficial effect of MIS THA on functional recovery has to be proven. The results also indicate that CAS THA, though resulting in an increase in operative time, may have a positive effect on operative blood loss and operative complication rates. More importantly, the use of CAS results in better positioning of acetabular component of the prosthesis. Total hip arthroplasty (THA) is considered to be one of the most successful orthopaedic interventions of the past 40 years, with 10-year survival rates exceeding 90% . In receDespite the increase in use of MIS THA, its risks and benefits are still an ongoing debate issue in the orthopaedic society. Proponents of MIS THA claim that it results in less soft-tissue trauma , reduced blood loss and fewer blood transfusion requirements. Postoperative benefits include less pain, shorter hospital stay, quicker return to function and better cosmetic appearance ,5. OpponProper positioning of the hip prosthesis is essential for improving the long-term success of THA. Higher rates of pelvic osteolysis, asymmetric polyethylene wear and component migration have been observed when the acetabular component is malpositioned . LewinneAs a result, the interest in computer navigation systems for orientation of the hip prosthesis is increasing, since it may be the solution for the aforementioned problems related to proper prosthetic positioning. Moreover, CAS is not only aimed at an improved alignment of the hip prosthesis, it also provides instant information and feedback to the surgeon, which may make the surgical technique easier to perform and may result in better clinical outcomes. The imaging systems that are used during CAS can be roughly divided into image-based and imageless systems. Image-based systems require the collection of morphological information by preoperative CT scans or MRI, or by means of intraoperative fluoroscopy. Imageless systems use a virtual anatomical model which is embedded in the software and is supplemented by intraoperative registration data of anatomical landmarks .CAS in THA is not very common nowadays, due to the fact that current CAS systems may involve longer operation times and the introduction of new equipment in the operating room. Other factors that limit the broad application of CAS are costs and complexity of computer navigation systems . SeveralThe use of CAS may be the solution to the limited visibility of anatomical landmarks during MIS THA . Some evFollowing the recommendations of the Cochrane collaborations, an extensive computerised literature search of PubMed, Medline, Embase and OVIDSP was conducted on all studies published between 1995 and May 2009. We used database-appropriate terms, including hip arthroplasty(ies)/replacement(s), minimally invasive/MIS/mini-incision, and/or computer-assisted/navigation/CAS/CAOS. The search strategy was formulated by an experienced medical librarian. To find more studies, the reference lists of all relevant studies were reviewed for potential articles.A study was included in the review if 1) a randomized controlled trial or a clinical controlled trials was conducted; 2) the study was published in English, Dutch or German; 3) the study was a full-length published article or fully-written published report; 4) the study population comprised patients aged 18 years or older who were undergoing a THA; 5) the study group and control group were similar at baseline with respect to age, gender and BMI; 6) the study contrast was minimally invasive total hip arthroplasty, computer-assisted total hip arthroplasty or a combination of both; and 7) at least one of the following outcome measures was assessed: operative outcome including blood loss and operative time; length of hospital stay; adverse events including intraoperative and postoperative complications; radiographic outcomes including number of outliers of acetabular components outside the desired alignment range; and/or one of the Outcome Measures in Rheumatology Clinical Trials (OMERACT) : pain, sThe procedure for inclusion of studies was based on the recommendations described by Van Tulder et al. The studThe methodological quality of all articles was independently assessed by two reviewers (IHFR and BPH) using a criteria list . This liAnalysis of the extracted data from the included articles was conducted in line with guidelines for systematic reviews from the Cochrane Collaboration Back Review Group . For conEfforts to retrieve raw data or means and their standard deviations to compute effect sizes or odds ratios by contacting the authors of articles where these data were not reported, were unsuccessful. We therefore chose to summarise the results by means of a qualitative analysis using a rating system that consists of five levels of scientific evidence, taking into account the methodological quality and the outcome of the original studies (best-evidence synthesis) Table 20]..20].Since the search strategy for MIS, CAS and computer-assisted MIS for THA contained similar components, the results of these search strategies overlapped. After removing double citations, 1841 citations remained. A flow chart of the results of the selection procedure after selection based on title, abstract and full text is shown in Figure Eventually, 25 articles were included. In 18 of these articles the study contrast was minimally invasive THA ,17,22-37None of the included articles had computer-assisted minimally invasive THA as study contrast. The characteristics of the included studies are presented in Additional file The results of the methodological quality assessment of the included articles are presented in Table Operative time was reported in 16 studies with MIS THA as study contrast Table . Two higOperative time was reported in four studies with CAS THA as study contrast Table . Except Intraoperative blood loss was reported in 14 studies with MIS THA as study contrast was reported in 13 studies with MIS THA as study contrast Table . The higAll studies with CAS THA as study contrast reported on the number of acetabular outliers Table . Five stIn order to evaluate physical functioning after THA, several physician-based and self-reported questionnaires are in use. Furthermore, objective assessment of physical function can be done by means of gait analysis. In total, thirteen studies with MIS THA as study contrast reported on physical functioning outcome measures. None of the studies with CAS as study contrast assessed physical functioning of patients after THA.Ten studies with MIS THA as study contrast reported on physician-based physical functioning outcome measures Table . In thesFive studies with MIS THA as study contrast reported on patient-reported physical functioning by means of two disease-specific outcome measures, namely the Western Ontario McMaster University Osteoarthritis Index (WOMAC) ,34,36,37Four studies with MIS THA as study contrast reported gait analysis data to evaluate physical function after THA Table . All fouFive studies with MIS THA as study contrast reported on pain Table . One stuCompared to conventional THA, strong evidence was found for a decrease in operative time and operative blood loss after MIS THA. The evidence for a shorter length of stay was moderate. Strong evidence was also found for no difference in complication rates and position of the acetabular component. Moderate to strong evidence was found for no difference in physical functioning six weeks and six months after surgery. The evidence of a positive effect of MIS THA on physical functioning three months postoperatively was conflicting, as was the evidence for less pain after MIS THA six weeks postoperatively. The evidence for no differences in pain levels three and six months after surgery was strong.Strong evidence was found for a positive effect of CAS THA on the position of the acetabular component. The evidence for a positive effect on operative blood loss was limited. Strong evidence was found for an increase in operative time and for no significant difference in complication rates after CAS THA.We have reviewed the current literature evaluating the effectiveness of MIS, CAS and computer-assisted MIS for THA. The extensive literature search resulted in 18 articles with MIS THA as study contrast, and seven with CAS THA as study contrast, yet no study with computer-assisted MIS for THA as study contrast was discovered. The results of this systematic review indicate that there were no significant differences in operative complications and acetabular component positioning between MIS THA and the conventional procedure. Furthermore, MIS THA resulted in a reduction in blood loss, operative time and reduced length of stay. The added value of MIS THA over the conventional procedure in terms of a faster functional recovery however remains to be proven. Computer-assisted THA results in better positioning of the acetabular component. It may also have a positive effect on operative blood loss and complications despite an increased operative time.Contrary to what proponents of MIS THA stated, this review showed that MIS THA had no effect on physical functioning, as measured by questionnaires as well as gait analysis. Since the main purported benefit of MIS THA is a decrease in the amount of soft-tissue (muscle) damage, it can be postulated that a difference in improvement of physical functioning and pain will only be seen in the early postoperative period. Only eight studies reported data on physical functioning at six weeks postoperatively ,31,34,37The results for CAS THA demonstrate an increase in operative time and limited evidence for a decrease in operative blood loss, but CAS THA had no effect on operative complication rates. Additionally, the use of CAS during THA had a positive effect on the outliers of the acetabular component position outside the desired range. These results justify use of computer navigation during THA. With improved surgery patients should benefit from having lower morbidity rates, better functional outcome and greater longevity of implants . Wines aDespite efforts to get an ample overview of the available literature on MIS and CAS for THA, no articles with computer-assisted MIS for THA as study contrast were discovered. Some of the studies included compared computer-assisted MIS for THA with either MIS THA ,39 or CASome critical remarks can be made on the included studies. First, a wide variety of surgical approaches was used in them. We chose to analyse all surgical approaches together, since the aim of this systematic review was to assess the effectiveness of minimally invasive THA, but not of any specific minimally invasive THA approach. Second, the surgical approaches were too heterogeneous and often poorly described to perform subgroup analyses. Studies on image-based and imageless navigation systems were also analysed together, since research has shown that imageless navigation is as reliable as image-based navigation for positioning the acetabular component . Third, Some limitations of this review and its conclusions need to be addressed. In this systematic review, a highly sensitive comprehensive search was conducted following the recommendations of the Cochrane collaboration in order to identify articles of interest. For practical reasons though, only studies published in English, Dutch or German were included in the final review, which might have led to selection bias. Additionally, in order to get a broad overview of all the literature on MIS, CAS and computer-assisted MIS for THA, we chose to include not only RCTs but also CCTs. Shrier et al. stated tThe results of this systematic review indicate that MIS THA is a safe surgical procedure, without increases in operative time, blood loss, operative complications and component positioning when compared to the conventional procedure. However, the surplus value of MIS THA over the conventional procedure in terms of a faster functional recovery remains to be proven. The results of this review also indicate that computer-assisted THA, despite an increased operative time, may have a positive effect on operative blood loss and complications. More importantly, the use of CAS during THA results in better positioning of the acetabular component of the prosthesis. Since minimally invasive THA and the use of computer navigation are becoming increasingly popular in orthopaedics, combining 'the best of both worlds' would be a sensible next step to take. With respect to future research, well-designed studies on MIS THA, CAS THA and especially computer-assisted MIS THA are needed, in which the used definitions, surgical technique, study population, outcome measures and study end-points are adequately described.The authors declare that they have no competing interests.IHFR co-coordinated the review, contributed to the literature search, and performed the data extraction, statistical analyses and drafting of the manuscript. BPH contributed to the literature search, data extraction and drafting of the manuscript. MS and WZ participated in the study design and have been involved in, together with SKB, JWG, ALB and RW, critically revising the manuscript. All authors read and approved of the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/11/92/prepubStudy characteristics. Characteristics of the included studies.Click here for file |
During embryonic development, signalling molecules known as morphogens act in a concentration-dependent manner to provide positional information to responding tissues. In the early zebrafish embryo, graded signalling by members of the nodal family induces the formation of mesoderm and endoderm, thereby patterning the embryo into three germ layers. Nodal signalling has also been implicated in the establishment of the dorso-ventral axis of the embryo. Although one can infer the existence of nodal gradients by comparing gene expression patterns in wild-type embryos and embryos in which nodal signalling is diminished or augmented, real understanding can only come from directly observing the gradients. One approach is to determine local ligand concentrations in the embryo, but this is technically challenging, and the presence of inhibitors might cause the effective concentration of a ligand to differ from its actual concentration. We have therefore taken two approaches to visualise a direct response to nodal signalling. In the first, we have used transgenic embryos to study the nuclear accumulation of a Smad2-Venus fusion protein, and in the second we have used bimolecular fluorescence complementation to visualise the formation of a complex between Smad2 and Smad4. This has allowed us to visualise, in living embryos, the formation of a graded distribution of nodal signalling activity. We have quantified the formation of the gradient in time and space, and our results not only confirm that nodal signalling patterns the embryo into three germ layers, but also shed light on its role in patterning the dorso-ventral axis and highlight unexpected complexities of mesodermal patterning. One of the earliest events in vertebrate embryonic development is the patterning of the embryo into three germ layers: the ectoderm, mesoderm, and endoderm. Morphogens are signalling molecules that act in a concentration-dependent manner to induce the formation of different cell types. Members of the nodal family are thought to form a morphogen gradient in the developing zebrafish embryo and to be essential for pattern formation. Mesoderm and endoderm are believed to develop due to high levels of nodal signalling, while cells experiencing the lowest concentrations of nodal signalling become ectoderm. Although this idea is widely accepted, the formation of a nodal morphogen gradient has never been observed directly, and we have therefore used two different approaches to visualise the intensity of nodal signalling within individual cells. Our approaches have allowed us to visualise a gradient of nodal signalling activity in the developing zebrafish embryo. Quantification of the levels of nodal signalling experienced by individual cells confirms that nodal signalling patterns the animal-vegetal axis of the zebrafish embryo and, in contrast to previous studies, also suggests that it plays a role in patterning the dorso-ventral axis of the zebrafish embryo. Gradients of nodal signalling in developing zebrafish embryos are visualized using a novel biofluorescence complementation reporter and quantified, demonstrating a role for nodal signalling in dorso-ventral patterning in addition to specifying the animal-vegetal axis. One exathe tail . Similarst lefty ,5.sqt and cyc in the zebrafish animal pole indicate that cyc acts only over short distances, whereas sqt functions as a morphogen and exerts its effects over long distances to induce target gene expression [goosecoid (gsc) expression, whereas lower levels activate no-tail (ntl). Thus, gsc is expressed in cells near a source of sqt, and ntl is expressed in cells further away.Misexpression of pression . High lentl is essential for patterning of the zebrafish embryo, because homozygous mutations in ntl disrupt mesoderm and notochord formation [Xenopus embryos lacking Brachyury (Xbra) [Xbra in isolated animal regions converts ectodermal cells into a mesodermal fate [ntl expression fails to initiate in embryos with diminished nodal signalling [The correct regulation of ormation . The samy (Xbra) , and ectmal fate . Consistgnalling –5.Xenopus embryo [Xenopus prevented a proper study of endogenous signalling events [Together, these experiments suggest that nodal family members form a gradient that induces target gene expression and specifies mesoderm and endoderm. Activation of the nodal signalling pathway within a cell results in the phosphorylation of Smad2, which then interacts with Smad4 . The ress embryo . Yolk aug events , but in gsc are only expressed in dorsal marginal cells. Our data also highlight the complexities of ntl regulation and of the formation of the border between dorsal mesendoderm and the neural plate.Our data allow us to follow in space and time the formation of a gradient of nodal signalling activity within the developing zebrafish embryo. The results illustrate the dynamics of gradient formation, and in contrast to previous studies , clearlyIn a preliminary attempt to investigate nodal signalling levels during zebrafish development, we generated transgenic embryos that express a Smad2-Venus fusion protein under the control of a ubiquitous promoter. During zebrafish development, marginal cells are thought to receive the highest levels of endogenous nodal signalling while cells at the animal pole experience low levels, if any . As predntl expression was unaffected in 90% of cases (n = 124), and in the remaining embryos, expression was normal in the marginal zone with weak ectopic expression in animal pole cells (unpublished data). These experiments demonstrate that our Smad BiFC constructs are suitable reagents for the analysis of endogenous nodal signalling.In an effort to improve the signal-to-noise ratio in such experiments, we turned to BiFC. When zebrafish embryos were injected with the N- and C- terminal halves of a modified form of the fXenopus embryo [Consistent with the experiments described above, we observed no nuclear BiFC fluorescence in animal pole cells of embryos injected with VNSmad2 and VCSmad4 D. As in s embryo , howevers embryo , strong s embryo . DeletioTogether, these experiments demonstrate that our Smad2-Venus transgenic embryos and Smad BiFC constructs report the activation of the TGF-β signal transduction pathway in the zebrafish embryo.ntl and experience endogenous nodal signalling [n = 25) .We first investigated endogenous nodal signalling in zebrafish embryos at 5–6 hours post fertilisation (hpf), when they express gnalling . Observagnalling D, or in Smad2-Venus transgenic embryos do not exhibit detectable nuclear fluorescence in the yolk syncytial layer (YSL) of the embryo A, and noWe went on to investigate the spatial and temporal patterns of Nodal signalling by allowing embryos to continue development after imaging and then noting the positions of the imaged cells relative to the shield. This analysis exploited the superior signal-to-noise ratio of the Smad2/4 BiFC technique see and 2. Intl and then studied the expression of gsc. Injected embryos became elongated were imaged. Following imaging embryos were incubated at 28 °C until 6–7 hpf. The agarose dish was then placed in hot water to melt the agarose, the embryos were removed from the agarose using forceps, and the positions of the imaged cells in relation to the shield was noted. Individual Z sections were used for the quantification of animal pole cells. Fluorescence intensity was quantified using Volocity software (Improvision). Individual nuclei were identified using a protocol to mark objects with intensities between 10 and 100% in the CFP (histone) channel. Quantifications were analysed using Microsoft Excel. For each image, the nuclei of the YSL were identified and the average distance and intensity of these nuclei was subtracted from all nuclei in that image. Video S1(23.19 MB AVI)Click here for additional data file.Video S2(40.67 MB AVI)Click here for additional data file. |
Over the past 30 years, benzimidazoles have increasingly been used to treat cystic echinococcosis (CE). The efficacy of benzimidazoles, however, remains unclear. We systematically searched MEDLINE, EMBASE, SIGLE, and CCTR to identify studies on benzimidazole treatment outcome. A large heterogeneity of methods in 23 reports precluded a meta-analysis of published results. Specialist centres were contacted to provide individual patient data. We conducted survival analyses for cyst response defined as inactive or as disappeared. We collected data from 711 treated patients with 1,308 cysts from six centres (five countries). Analysis was restricted to 1,159 liver and peritoneal cysts. Overall, 1–2 y after initiation of benzimidazole treatment 50%–75% of active C1 cysts were classified as inactive/disappeared compared to 30%–55% of CE2 and CE3 cysts. Further in analyzing the rate of inactivation/disappearance with regard to cyst size, 50%–60% of cysts <6 cm responded to treatment after 1–2 y compared to 25%–50% of cysts >6 cm. However, 25% of cysts reverted to active status within 1.5 to 2 y after having initially responded and multiple relapses were observed; after the second and third treatment 60% of cysts relapsed within 2 y. We estimated that 2 y after treatment initiation 40% of cysts are still active or become active again. The overall efficacy of benzimidazoles has been overstated in the past. There is an urgent need for a pragmatic randomised controlled trial that compares standardized benzimidazole therapy on responsive cyst stages with the other treatment modalities. Cystic echinococcosis (CE) is a parasitic infection of worldwide occurrence transmitted to humans by dogs. After infection cysts develop, mainly in the liver and lung. Ultrasound-based staging of cysts into active, transitional, and inactive has opened new venues for treatment and follow-up. Currently four treatment modalities are in use: (1) surgery, (2) percutaneous sterilization techniques, (3) chemotherapy with benzimidazoles, and (4) watch and wait for inactive cysts. However, evidence is insufficient for these four modalities, and determining individual treatment options for patients remains controversial. Medical treatment with benzimidazoles started in the 1970s. Important questions remain unanswered, however, such as efficacy stratified by cyst type and the duration of treatment. We therefore initiated EchinoMEDREV, a collaborative effort to collect individual patient data from patients treated with benzimidazoles and to analyze cyst outcome after initiation of benzimidazole therapy using a common analytical strategy across treatment centres. We found that the efficacy of benzimidazoles has been overstated in the past. Additionally, natural cyst decay has not been taken into account. Evidence from randomized controlled trials is urgently needed to determine the true efficacy of benzimidazoles. Our analysis will help to design benzimidazole trial arms on the basis of the currently available best evidence. Cystic echinococcosis is a parasitic disease of worldwide prevalence. Hydatid cysts occur mainly in the liver (70%) and the lung (20%). Clinical symptoms and signs depend on their localisation, size, and number. Currently four treatment modalities are in use: (1) surgery, (2) PAIR , (3) chemotherapy with albendazole (ABZ) or mebendazole (MBZ), and (4) watch and wait for inactive, clinically silent cysts. The evidence supporting any of the four treatment modalities, from carefully designed clinical studies, is insufficient, and choosing treatment options for patients remains controversial The use of benzimidazoles in CE treatment started in the 1970s with MBZ. In the early 1980s ABZ was introduced and since then has largely replaced mebendazole. The main advantages of ABZ are claimed to be a lower dosage and better intestinal absorption. In treatment centres MBZ and ABZ are given at the World Health Organisation (WHO) recommended dosages of Chemotherapy for the treatment of CE was initially recommended for inoperable patients and patients with multiple organ disease After more than 30 y of benzimidazole use, the following crucial question remains unanswered: what is the efficacy of benzimidazoles stratified by type and size of cysts, daily dose, and duration of treatment?This project started with a systematic review of the published literature on the efficacy of treating CE with benzimidazoles. We had to conclude, however, that we could not obtain a clear picture of the long-term outcome of benzimidazole treatment because inclusion criteria, treatment, outcome measures, and follow-up of published studies varied widely with substantial overlap of cohorts The main objectives of this collaborative study were to describe cyst outcome after initiation of benzimidazole treatment, with outcome defined by cyst stage determined by ultrasound following the WHO classification of 2001 A systematic search of MEDLINE, EMBASE, CCTR, and SIGLE was carried out from their inception until week 4 of 2004. The search was performed by a research librarian using the following search terms: echinococcosis, albendazole, mebendazole, hydatid disease, cystic echinococcosis. We also searched reference lists and asked researchers in the field for additional studies. No language restriction was used. Abstracts were screened for suitability by MS. The eligibility of studies was assessed independently by two investigators (TJ and MS). We included all types of study design with a minimum of 30 patients treated either with ABZ or MBZ. Studies in which drug treatment was an adjunct to surgery, PAIR, or a second drug were excluded.The studies identified in the literature search revealed that there were large differences in baseline assessment of cyst stages, interventions (dose and duration of chemotherapy), length of follow-up, and outcome measures between published trials. These differences precluded the possibility to perform a meta-analysis of published results. Therefore we decided to collect individual patient data from the identified centres and initiated the EchinoMEDREV project.Study centres that had conducted clinical studies on benzimidazole treatment of CE were contacted and asked to contribute published and unpublished individual patient data of benzimidazole-treated CE patients. Data extraction forms were developed, piloted, and revised. Data collection started in June 2005 and ended in December 2007. Data were extracted from patient charts at the individual treatment centres. Data collected were: demographic data ; treatment data ; imaging data . The forms were sent to the coordinating centre at the University Hospital in Heidelberg where data were electronically entered into a database with EpiData, using data entry checks. Accuracy in data entry was checked by double entry verification. A final dataset was created after correcting detected data entry errors and exported to Stata for statistical analysis. Patients with single or multiple hydatid cysts were eligible. Cyst stage had to be recorded at the beginning and at least once after completion of the initial treatment episode. The minimum follow-up period was 1 y after completion of initial treatment. Cyst activity had to be assessed by ultrasonography and classified according to WHO , Gharbi, Perdomo, or Caremani .The analysis presented here includes only liver and peritoneal cysts , which were assessed by ultrasonography, and excluded lung cysts as they are not usually assessed by ultrasonography.The cyst was used as the unit of analysis for a description of achieved outcomes, and the presence of multiple cysts was not taken into account. Data were analysed by intention-to-continue-treatment, ignoring treatment changes (MBZ/ABZ), interruptions, and subsequent treatment episodes.We analysed several endpoints. First, initial treatment success for a cyst was defined as transformation from an initially active or transitional stage to an inactive stage or disappearance of the cyst ; 25th percentile, lambda = p25/ln(1/0.75). We further assumed that the duration of the next phase is independent of the duration of the previous phase. For each simulated cyst we assessed the current stage at year 1 to year 5 after treatment initiation. All analyses were performed using Stata Version 10 (StataCorp).Out of 353 citations identified, 23 papers met the inclusion criteria . Three pAll 14 specialist centres identified were contacted: no reply was received from four centres The analysis presented here was restricted to patients with liver and peritoneal cysts, because of the reliability of ultrasound classification compared to other cyst locations. This restriction resulted in 1,159 cysts in 612 patients for analysis. Approximately 68% of data was obtained from one centre .p = 0.043), but not up to year one (p = 0.43), and a centre effect was noted from year one onwards .p = 0.006) and a strong centre effect was noted (p<0.0001).We further analysed the rate of inactivation/disappearance with regard to cyst size . OverallOverall, cysts that reached an inactive stage for the first time relapsed (returned into an A or T stage) in around 25% of cases 2 y after inactivation . Cysts tIn the simulation of hypothetical cysts, we estimated that 1 and 2 y after treatment initiation, 60% and 40% of cysts are still active or become active again.In a collaborative effort, individual data from patients with CE were pooled from six centres in five countries and outcomes of liver and peritoneal cysts treated with benzimidazoles were described. We found a strong association between cyst activity and response to treatment, with a better response in highly active CE1 cysts, and an association in treatment response depending on the size of the cyst at the beginning of treatment, with cysts <6 cm in diameter responding better. Thus, our data suggest that small highly active cysts show the best initial treatment response. Overall 25% of cysts reverted to active status within 1.5 to 2 y after having initially responded, and multiple relapses were observed. We estimated that 2 y after treatment initiation 40% of cysts are still active or become active again. Our results are biologically plausible because early in the disease host response resulting in an increasing thickness of the pericyst and consolidation of cyst content has not yet reached a degree that prevents the drug to reach its target There are several limitations to this study. The published data collected from participating specialist centres are from case series. Despite these limitations, to our knowledge, this study represents the largest CE dataset ever collected and analyzed in a uniform approach; further it is likely the only dataset obtained from the main international specialist groups. The recommendations on benzimidazole treatment of CE are currently based on the published results from these centres. Through the collection of individual patient data and the pooled analysis of these data we have managed to overcome some of the existing limitations present in the published literature.Does our study provide sufficient evidence to influence decisions for the treatment of CE? We think that our results are strong enough to cast doubts on overoptimistic expectations of the overall efficacy of benzimidazoles. When looking into substrata of the cyst population small CE1 cysts (diameter <6 cm) are a promising target for benzimidazole therapy, whereas stage CE2 and CE3 cysts respond poorly. The available evidence from this and other studies does not yet allow us, however, to formulate solid evidence-based drug treatment recommendations across all cyst stages, sizes, and locations. Our results highlight the urgent need to compare in a pragmatic randomised controlled trial the effect of standardized benzimidazole dose regimens on the individual active cyst stages substratified by cyst size. Such a trial would investigate as a primary outcome the proportion of cysts that become inactive (cyst stages CE4 and CE5) after treatment, and as a secondary outcome the yearly relapse rates up to 5 y after completion of treatment. The clarification of the efficacy of benzimidazoles in CE treatment is of paramount importance since benzimidazoles are the only drugs currently available to treat this neglected disease. Surgery as an alternative to benzimidazoles carries a significantly higher risk of adverse events, in particular intra- and postoperative morbidity and mortality and disseminated disease due to intraoperative spillage of viable hydatid material. Percutaneous fine needle techniques such as PAIR are only applicable to cyst stages CE1 and possibly CE3a, but not to CE2 and CE3b, which makes it necessary to explore large bore catheter techniques if albendazole turns out to be less effective in these cyst stages as suggested by our analysis.Checklist S1QUOROM checklist.(0.17 MB PDF)Click here for additional data file. |
The formation of metastasis is the most common cause of death in patients with lung cancer. A major implement to understand the molecular mechanisms involved in lung cancer metastasis has been the lack of suitable models to address it. In this study, we aimed at establishing a highly metastatic model of human lung cancer and characterizing its metastatic properties and underlying mechanisms.in vivo selection in NOD/SCID mice. After three rounds of selection, a new SPC-A-1sci cell line was established from pulmonary metastatic lesions. Subsequently, the metastatic properties of this cell line were analyzed, including optical imaging of in vivo metastasis, immunofluorescence and immunohistochemical analysis of several epithelial mesenchymal transition (EMT) makers and trans-well migration and invasion assays. Finally, the functional roles of fibronectin in the invasive and metastatic potentials of SPC-A-1sci cells were determined by shRNA analysis.The human lung adeno-carcinoma SPC-A-1 cell line was used as parental cells for developing of highly metastatic cells by in vitro, including increased potentials for cell spreading, migration and invasion. Importantly, fibronectin, a mesenchymal maker of EMT, was found to be highly expressed in SPC-A-1sci cells and down-regulation of it can decrease the in vitro and in vivo metastatic abilities of this cell line.A spontaneously pulmonary metastatic model of human lung adeno-carcinoma was established in NOD/SCID mice, from which a new lung cancer cell line, designated SPC-A-1sci, was isolated. Initially, the highly metastatic behavior of this cell line was validated by optical imaging in mice models. Further analyses showed that this cell line exhibit phenotypic and molecular alterations consistent with EMT. Compared with its parent cell line SPC-A-1, SPC-A-1sci was more aggressive s.c. mouse model can further be used to identify underlying mechanisms of lung cancer metastasis.We have successfully established a new human lung cancer cell line with highly metastatic potentials, which is subject to EMT and possibly mediated by increased fibronectin expression. This cell line and its reproducible According to the World Health Organization, lung cancer is responsible for more than 1.3 billion deaths worldwide annually. Despite advances in the treatment of primary tumours, recurrence and metastasis are the most common cause of death in patients with lung cancer. The current poor understanding of the molecular mechanisms involved in lung cancer metastasis is due, in large part, to the lack of suitable models for its study . AlthougEMT, a process whereby cells acquire molecular alterations that facilitate cell motility and invasion, has been shown to play an important role in tumour metastasis . More reIn the present report, we successfully develop a spontaneously metastatic model of human lung cancer that represents the full spectrum of metastasis, from which a highly metastatic human lung cancer cell line, termed SPC-A-1sci, was derived. This cell line exhibits typical changes in cellular phenotype similar to EMT. Moreover, fibronectin plays an important role in these alterations, and thus resulting in the highly metastatic potentials of this cell line.2 atmosphere in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum , 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were regularly certified as free of Mycoplasma contamination.The human lung adeno-carcinoma cell line SPC-A-1 was obtained from Cellular Institute of Chinese Academy of Science . This cell line was originally isolated from the surgical specimens of a Chinese man with advanced lung adeno-carcinoma by Shanghai Chest Hospital and Cellular Institute of Chinese Academy of Science in 1980. The cel6 of the SPC-A-1 cells were injected subcutaneously (s.c) into NOD/SCID mice. When the subcutaneous tumour developed, small pieces of tumour tissue were implanted into the s.c. sites of mice in the first generation of mouse models and the primary tumours were excised 4 weeks later. Those mice were sacrificed under deep anesthesia when they showed signs of distress, and visual lung metastases were isolated and s.c. implanted into the new recipient mice in the second generation of mouse models for in vivo selection. These procedures were repeated for three rounds. At the end of the selection, the lungs harboring massive metastatic lesions were isolated and s.c. implanted into new recipient mice, after which the primary tumour was removed to initiate in vitro primary culture.Five- to 6-week-old male congenitally immune-deficient nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice were maintained under specific pathogen-free (SPF) conditions. Mice were manipulated and housed according to protocols approved by the Shanghai Medical Experimental Animal Care Commission. To isolate a highly metastatic cell line, briefly, 2.0 × 10s.c. injection of minced tumour tissue suspended in PBS using a 1 cc tuberculin syringe and an 18-gauge needle.Subcutaneous tumour implantation was performed as described . In brievia s.c. injection, cells (2 × 106 per mouse) were injected subcutaneously into the right upper flank region of NOD/SCID mice. Mice were monitored weekly for tumour size and evidence of morbidity related to the primary tumour or metastases. Tumour size was quantified in two dimensions using calipers. Tumour volume was calculated as follows: tumour volume (mm3) = L × W × W/2, where L represents length and W represents width. Nine weeks later, all mice were sacrificed, and the organs, including lungs and livers, were removed and processed for standard histological studies. For histological analysis, the primary tumours and mouse organs were harvested at necropsy and fixed in 10% formalin. The fixed samples were then embedded in paraffin, and three non-sequential serial sections were obtained per animal. The sections were stained with H&E and analyzed for the presence of metastases.For primary tumour growth assays and spontaneous metastasis BamHI/XhoI sites of a pWPXL vector (Addgene). Transductions of SPC-A-1sci and SPC-A-1 cells were performed with the aforementioned lentiviral vector according to instructions supplied by Addgene http://www.addgene.org and stable transfectants were further isolated by cell sorting on the basis of their EGFP expression.The GFP-Luc lentiviral vector encoding a fusion gene of GFP and luciferase was generated by inserting the GFP-Luc gene from the plasmid eGFP-2A-CBGr99 (kindly provided by Professor Hammerling) into the MluI/ClaI sites of a pLVTHM vector (Addgene) with the following oligonucleotides respectively: 5'-CGCGTCGGCCCGGTTGTTATGACAATTTttcaagagaAAATTGTCATAACAACCGGGCTTTTTTGGAAT-3'and 5'-CGATTCCAAAAAAGCCCGGTTGTTATGACA ATTTtctcttgaaAAATTGTCATAACAACCGGGCCGA-3' for Fibronectin, and 5'-CGCGTCGTAGCGACTAAACACATCAATTttcaagagaAATTGATGTGTTTAGTCGCTATTTTTTGGAAT-3' and 5'-CGATTCCAAAAAATAGCGACTAAACACAT CAATTtctcttgaaAATTGATGTGTTTAGTCGCTACGA-3' for the negative control. Lenti-virus generation and infection of SPC-A-1sci cells were performed as described above. For experimental metastasis in vivo, SPC-A-1sci cells (2 × 106 per mouse) stably expressing shRNA against fibronectin or negative control were injected into the tail vein of NOD/SCID mice (n = 8). Four weeks later, the mice were sacrificed and the lungs were removed and processed for histological examination.The lenti-virus shRNA vector was constructed as described previously . Brieflyin vivo bioluminescence imaging (in vivo BLI), the animals were injected i.p. with 150 mg luciferin per kg of body weight, anesthetized with pentobarbital (10 mg/ml) in sterile water, and then placed in the NightOwl LB 983 Molecular Light Imager. For ex vivo biofluorescence imaging (ex vivo BFI), mice lungs were excised after in vivo BLI and placed in the chamber of the NightOwl LB 983 Molecular Light Imager and imaged.We used a Berthold LB983 NightOwl System to monitor the primary tumour growth and distant metastasis of SPC-A-1sci and SPC-A-1 cells in mouse models as previously described ,18. For The experiments were performed as described previously . For indFor immunohistochemical analysis, all tissue samples were fixed in phosphate-buffered neutral formalin, embedded in paraffin, and cut into 5-μm-thick serial sections. Immunohistochemical staining with antibodies to E-cadherin , Vimentin was performed according to standard procedures. Results were observed and photographed with an Axioskop 2 microscope and DP70 Imaging system .2 for three weeks. The colonies formed were photographed with an Axioskop 2 microscope and DP70 Imaging system . Pictures of three random fields in each well were obtained from three replicate, and the number of colonies was counted.Colony formation in soft agar was assayed as described previously . BrieflyThe experiment was performed as described previously with little modification . Cells wCell migration and invasion assays were performed using 6.5-mm trans-well chambers as described previously with some modifications . Cells w5) were applied to wells for various time points. After each time point, the wells were washed twice with PBS, and then the adherent cells were visualized and photographed with a CKX41 microscope at 200 × magnification and DP20 Imaging system .The experiments were performed as previously described with somTotal RNA of cultured SPC-A-1sci and SPC-A-1 cells were isolated using Trizol reagents (Invitrogen) according to the manufacturer's instructions. First-strand cDNA synthesis and amplification were performed using Reverse Transcription Reagents (Takara) following the manufacturer's instructions. Real-time PCR was carried out using a 7300 Real-Time PCR System with SDS RQ Study software (Applied Biosystems) according to the manufacturer's instructions. cDNA templates were combined with SYBR Green premix with Rox (Takara) to perform quantitative-PCR reactions. Primers used for quantitative-PCR were as follows: E-cadherin forward: 5'-TGGCTTCCCTCTTTCATC-3'; E-cadherin reverse: 5'-GTTCCGCTCTGTCTTTGG-3'; Fibronectin forward: 5'-GGAGTTTCCTGAGGGTTT-3'; Fibronectin reverse: 5'-GCAGAAGTGTTTGGGTGA-3'; Vimentin forward: 5'-CTGAACCTGAGGGAAACTAA-3'; Vimentin reverse:5'-AGAAAGGCACTTGAAAGCT-3'; β-actin forward: 5'-AGTGTGACGTGGACATCCGCAAAG-3'; β-actin reverse: 5'-ATCCACATCTGCTGGAAGGTGGAC-3'. Gene expression was normalized to β-actin. All reactions were run in triplicate.Cells were lysed and proteins were detected as described previously . Immunobt-test. P < 0.05 was accepted as statistically significant.Data are presented as the means ± SD and were evaluated with Student's in vivo selection, we removed the s.c. primary tumour from tumour-bearing mice to allow sufficient time for distant micrometastases to grow into macrometastases in the first generation of mouse models. Through three rounds of in vivo serial selection, the incidence of lung metastasis reached 100% in the fourth generation of the mouse model using the poorly metastatic human cell line SPC-A-1 as a starting point Fig. . The metel Table and s.c.in vivo behaviors of this cell line, we engineered SPC-A-1sci and its parent SPC-A-1 cells with dual reporters of GFP and Luciferase and monitored them in NOD/SCID mice by optical imaging techniques. As shown in Fig. in vivo bioluminescence imaging weekly, but also observe the pulmonary metastases by ex vivo fluorescence imaging in one sacrificed animal at week 7 and week 8. Subsequently, numerous metastases could be detected at week 9 in all remaining mice of this group when they displayed signs of being moribund. This was significant compared with the SPC-A-1 group, in which no fluorescence signals of pulmonary metastasis could be monitored at the same end-point when they still displayed an active status (data not shown). Briefly, we successfully developed a highly metastatic cell line from a poorly metastatic lung cancer SPC-A-1 cell line. Whereas the parental line SPC-A-1 required 14 weeks (post-primary tumour resection) for the formation of visible metastatic nodules in lungs, the SPC-A-1sci required only 7 weeks for the formation of large macroscopic nodules in the lungs.To further characterize The generation of motile, invasive carcinoma cells was recently found to share some of the key morphologic and molecular characteristics of EMT . As shows.c. primary tumours of SPC-A-1sci cells also confirmed the remarkably reduced expression of E-cadherin (Fig. This morphological change is one of the hallmarks of EMT. To determine whether the molecular alterations of EMT occurred in these cells, we examined the localization of several adherent junction proteins, such as E-cadherin and ZO-1, by confocal immunofluorescence staining. The results showed that the two proteins almost disappear from the cell membrane in the SPC-A-1sci cells but show strong membrane staining in their parent SPC-A-1 cells Fig. . Howeverrin Fig. . In contrin Fig. .EMT could be a reversible, dynamic process and may be regulated by the tumour microenvironment, carcinoma cells that have undergone EMT during invasion seem to regain their epithelial characteristics at the metastatic sites . As showCell proliferation and colonization are among the necessary functions required for metastatic progression of tumour cells. However, the SPC-A-1sci cells are not more aggressive than the parental population in the proliferation in culture and formation of subcutaneous tumours Fig. . This suin vivo.We next took steps to characterize other cellular properties that might be relevant to metastasis. First, we employed wound healing assays to determine migratory abilities of SPC-A-1sci and SPC-A-1 cells on fibronectin coated or uncoated plates. As shown in Fig. in vivo metastasis assays demonstrated the similar results. As shown in Fig. As described above, fibronectin could promote the spreading of the parent SPC-A-1 cells. We therefore examined the spreading ability of SPC-A-1sci and SPC-A-1 cells after plating on fibronectin coated or uncoated plates and found that SPC-A-1sci cells spread significantly faster than SPC-A-1 cells did on both conditions Fig. . Importavia direct injection of cancer cells into the circulation of mice, which puts the focus on end-stage metastatic cells. These models are thus likely to miss the essential genes and pathways required for the early steps of metastasis, including EMT, migration, invasion, and intravasion. It is therefore necessary to develop spontaneous metastatic models that faithfully mimic the selection and evolution of metastasis in human tumours.Metastasis, the foremost cause of mortality in cancer patients, accounts for more than 90% of all deaths in solid tumour diseases, but the underlying mechanisms remain elusive. Recently, a combination of tumour model systems and microarray profiling technologies has been proved to be effective in identifying relevant genes and pathways for tumour metastasis ,25. Howes.c. tumour implantation has been widely used for the majority of human tumour models [s.c. transplantation models [in vivo selection combined with resection of primary tumours in mice.Human tumours rarely metastasize from a primary tumour site in immune-deficient mice when they are transplanted into an ectopic site for the particular type of tumour being analyzed. Orthotopic implantation has been proved to be a better approach for the development of metastasis models. Unlike r models ,28, and n models ,29. It in models ,31. Recen models . In thisin vitro. The potentials of cell motility, migration and invasion are greatly increased in SPC-A-1sci cells. These results suggested that SPC-A-1sci cells acquire the increased abilities to migrate and invade, which further demonstrated that SPC-A-1sci cells have undergone EMT.Growing evidence suggests that EMT has an important role in tumour metastasis ,25. The in vitro and in vivo metastatic abilities of those cells. Furthermore, fibronectin is also a mesenchymal maker, whose expression positively correlates with EMT [The complex interactions between tumour cells and extracellular matrix play important roles in mediating and regulating many processes during tumour metastasis, including cell migration, cytoskeleton reorganization and morphologic transition ,40. Fibrwith EMT . But thes.c. mouse model and isolated a highly metastatic human lung adeno-carcinoma cell line by in vivo selection in NOD/SCID mice. The new cell line, SPC-A-1sci, possessed highly metastatic potentials and undergone an EMT program, which may be associated with increased expression of fibronectin. To the best of our knowledge, it is the first in the literature that reports the presence of EMT in lung cancer and the functional roles of fibronectin in lung cancer cell invasion and metastasis based on an in vivo tumour metastasis model. This model may provide a platform to identify elements that are important in the process of metastasis and to learn how they contribute functionally to the biology of lung cancer metastasis.In summary, we have successfully established a reproducible The authors declare that they have no competing interests.DJ performed major experimental work, and drafted the manuscript. MY, LL, HK carried out the experiments in mice. MW participated in the Trans-well migration and invasion assays. XF performed FACS analysis. LL performed lentiviral transduction of tumour cells. XH helped to draft the manuscript. JL performed the immnuohistochemical experiment and helped to draft the manuscript. MY participated in the design of the study, supervised the laboratory work. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/364/prepub |
Reserves are the principal means to conserve forests and biodiversity, but the question of whether reserves work is still debated. In the Amazon, fires are closely linked to deforestation, and thus can be used as a proxy for reserve effectiveness in protecting forest cover. We ask whether reserves in the Brazilian Amazon provide effective protection against deforestation and consequently fires, whether that protection is because of their location or their legal status, and whether some reserve types are more effective than others.Previous work has shown that most Amazonian fires occur close to roads and are more frequent in El Niño years. We quantified these relationships for reserves and unprotected areas by examining satellite-detected hot pixels regressed against road distance across the entire Brazilian Amazon and for a decade with 2 El Niño-related droughts. Deforestation fires, as measured by hot pixels, declined exponentially with increasing distance from roads in all areas. Fewer deforestation fires occurred within protected areas than outside and the difference between protected and unprotected areas was greatest near roads. Thus, reserves were especially effective at preventing these fires where they are known to be most likely to burn; but they did not provide absolute protection. Even within reserves, at a given distance from roads, there were more deforestation fires in regions with high human impact than in those with low impact. The effect of El Niño on deforestation fires was greatest outside of reserves and near roads. Indigenous reserves, limited-use reserves, and fully protected reserves all had fewer fires than outside areas and did not appear to differ in their effectiveness.Taking time, regional factors, and climate into account, our results show that reserves are an effective tool for curbing destructive burning in the Amazon. A history of massive deforestation linked to large-scale infrastructure projects (notably roads) in the Brazilian Amazon, and government plans for more such projects, has spawned debate about ways to avoid repeating past trends In the Amazon, deforestation and fire are inextricably linked. Satellite-detected “hot pixels” are a good proxy for deforestation fires, and can thus effectively tell us whether reserves are protecting forest cover. In addition to biodiversity concerns, deforestation fires indicate that biomass has rapidly been released as carbon to the atmosphere, another important measure of reserve effectiveness. Our analyses concentrate on fires as a measure of human impact and on the ability of reserves to mitigate it.2 resolution GOES hot pixels for 1998 only. Finally, because Nepstad et al. Studies that have looked at fire in the Amazon We start by considering the factors that affect fire incidence. Because there is large year-to-year variation in climate and in fires de facto, a consequence of reserve location , or de jure, because legal protections are respected? (3) Are some reserve types more effective than others in preventing deforestation fires? We recognize that there will be confounding factors: (a) Given that severe droughts remove moisture limitations and thus promote the spread of fires, do different kinds of reserves offer varying levels of protection in El Niño Southern Oscillation (ENSO) years? (b) Do reserves prevent deforestation fires even when human access is possible through road networks? (c) Finally, given these other factors, do the answers vary from place to place across the Amazon?In short, we ask three key questions: (1) Do reserves actually protect Amazonian forests from deforestation and consequently fires? (2) Is protection In the Amazon, roads are the major conduits for deforestation and accompanying fires Theoretically, reserves may halt fire because of restrictions on land use (forests are less disturbed and fire is not used for management) or because of restricted access (fewer roads and fewer ignition sources). Different types of reserves in Brazil allow different land uses Whatever the mechanism, reserves clearly limit road building, deforestation and fire in many highly affected areas Climate patterns produce different spatial and temporal patterns of drought in different regions of the Amazon The leading edge of development in the Brazilian Amazon forms an arc from the southwestern to the southeastern Amazon. Here, seasonally dry forests Because processes affecting fire differ between these regions, we divided the Amazon using relevant political and geographical boundaries and analyzed forest areas in the two regions separately.We used 3 remotely-sensed data sources to track fires. The first provided the most years of data. The others ran for fewer years, detected more fires, and allowed us to test whether data source affected our results.2 resolution hot pixels from the European Space Agency's Ionia World Fire Atlas (WFA) We tracked fire patterns with monthly composites of nighttime 1-kmDetecting fire from space remains challenging and each sensor has advantages and disadvantages. Detection algorithm, overpass time and frequency, spatial resolution, land cover, and type of fire all affect which fires are detected 2, but these satellites have daytime and more frequent overpass times. They detect more fires than the WFA. In addition to analyzing the full ten years of WFA data, we also separated 2001–2003 WFA data and directly compared those years with the AVHRR and MODIS data.To confirm the general pattern of our results, we also analyzed 3-year data sets released by the Large-Scale Biosphere-Atmosphere Experiment (LBA) in Amazônia Because of rapid change in land cover over the large spatial and temporal scale of our study, we did not include detailed land cover data. Instead, we assigned designations of forest or savanna vegetation derived from ecoregions http://www.dpi.inpe.br/prodesdigital/prodesmunicipal.php, accessed January 31, 2008). Pará had approximately 19% deforestation in 2006, but the majority of that deforestation occurred east of the Xingu River. Therefore, areas in Pará east of the Xingu River we classified as high-impact and the areas north and west of the Xingu River we considered low-impact.Social and economic drivers of fire and deforestation, as well as environmental variables, vary across the Amazon We grouped reserves into fully protected parks , limited-use areas , and indigenous lands, on the basis of activities that they allow.2 unless they were adjacent to another reserve of the same type. To avoid double-counting areas that had two different designations, we excluded limited-use areas and protected parks that overlapped by more than half of their area with indigenous lands. Altogether, we included the forest ecoregion portions of 53 parks, 109 limited-use reserves, and 238 indigenous reserves, totaling 180,125 km2, 409,984 km2, and 936,819 km2, respectively. The combined area was 1,526,928 km2 or approximately 37% of the forest ecoregion area of the Brazilian Amazon. We used road data and the Legal Amazon boundary from IBAMA . Road data include state and federal roads, and some private roads, but omit many unofficial roads that are visible in Landsat Images. As the vast majority of hot pixels detected (∼90%) were ≤10 km of roads in our dataset, this omission should not significantly affect our results.World Wide Fund for Nature-Brazil compiled the shape files of reserves. The original sources were FUNAI , IBAMA , and the state secretaries of the environment (state protected areas). We excluded marine and mangrove reserves. To avoid co-registration errors, we excluded reserves of <100 km2 pixel in forest ecoregions, we recorded the distance to the nearest road and whether the pixel had burned in a given year. We used analysis of covariance to assess patterns of hot pixel frequency in different reserve types, land designations and distance to roads (binned to 10 km wide classes). We measured ENSO severity with the Multivariate ENSO Index (MEI) For each 1-km2 pixel. However, hot pixels are typically clustered at a scale of a few square kilometers . In any case, individual hot pixels were not independent observations. Consequently, we used regression analysis on the average number of hot pixels/100 km2, in each road distance class, and for each category . Here, the sample sizes were far smaller, but only the residuals about the model needed to be independent. At this scale, there is no reason to think that the residuals would be correlated. We restricted analyses to distance classes for which the total combined number of hot pixels in the 2 classes being compared was >50. Classes with <50 hot pixels were generally either very far from roads (very few hot pixels in huge remote areas) or had very little land area .The statistical analyses raise the issue of the independence of individual fires. Treating fires as independent observations would result in huge sample sizes. The sensors detect distinct fires — or clusters of fires — to the resolution of a 1-kmAs expected based on state deforestation statistics, the majority (88%) of hot pixels detected in forest ecoregions with a decade of WFA data were in high-impact forest Only 12%There were far fewer fires inside reserves than outside for both low- and high-impact forests improved the statistical fit over a model with a common slope. We expected that at large distances from roads, it should matter less whether or not forest was inside a reserve; so again, the test was one-tailed. These results were mixed: two results were significant at p<.05, two more were close, but all differences were in the expected direction ; row 3.Converging regression lines imply that there is some distance from roads beyond which there is no difference in fire frequencies between areas inside and outside of reserves. Treating each distance class as a separate variable in an ANOVA allowed us to ask at what distance from roads were fire frequencies statistically different inside versus outside reserves. For the eight sets of results in In addition, there were more fires (inside and outside of reserves) in high-impact than in low-impact forests , 3 and tThere were generally more hot pixels in years with high ENSO indices than in years with lower ones. This was true both close (<10 km) and far (>10 km) from roads and inside and outside reserves had no hot pixels in any given year. In reserves with hot pixels, the average number/100 kmSO years . A sligh factors as we wide facto or de jure.Our first question was whether reserves actually protect Amazonian forests from deforestation fires. Our analysis clearly shows that they do. There are caveats, however, that relate to the second question of whether that protection is Reserves had many fewer fires than areas outside, but protection differed between high- and low-impact areas. Overall, there are roughly 3 times more deforestation fires in high- than in low-impact areas. These regional differences have been obvious since at least the early-1970s These differences illustrate the differences in pressure on reserves in high-impact areas. Even correcting for greater area outside reserves, there were always consistently more hot pixels close to roads outside reserves than inside, in both low- and high-impact areas. This difference diminished with increasing road distance. Because hot pixels are a proxy for deforestation, fewer fires close to roads inside reserves may relate to a lack of available infrastructure or to protected status that discourages land uses conducive to deforestation and fire along roads. This suggests that reserves provide the greatest protection from fires where the likelihood of burning would otherwise be greatest, that is, close to roads. On the other hand, the difference between fire occurrence in high- and low-impact reserves also indicates that reserves may not always provide sufficient protection when the pressure on them becomes very great. In addition, reserves that do not suffer deforestation fires may be subject to less detectable disturbance such as illegal logging or understory fire There is no simple answer to our third question of whether some types of reserves are universally more effective. At the scale of the Brazilian Amazon, reserve type did not significantly affect fire frequency for a given distance to roads and region.Statistical issues made it difficult to deny any effect of reserve type, however. First, only ∼20% of reserves had any hot pixels in most years . In highTo illustrate regional differences, we examined 3 places where all the factors discussed were roughly equal, but where all 3 kinds of reserves were adjacent to each other . This wo2. Inside it was generally <2 hot pixels/km2.In Rondônia, a massively deforested area, the contrast between fires inside and outside reserves was striking . In the Along the BR-174 in Amazonas, large areas burned in 1997 . These fIn the eastern Amazon, in an area with many fires, no reserve has successfully kept fires at bay . For exaThese examples illustrate the importance of local factors to the success of any reserve in protecting forest Our results imply that the prevalently-held view that uninhabited reserves are the best kind for conservation may not be so clear cut, especially in the context of rapid infrastructure development and deforestation in the Brazilian Amazon. The fact that there is not a significant difference in deforestation fires in inhabited versus uninhabited reserves provides an immediate policy implication. Indigenous lands contain 5 times the land area of fully protected parks and form the majority of protected land in highly contested areas Debate about the Amazon's future has rightly focused on roads as one of the most important drivers of deforestation Although previous work has found correlations between ENSO and understory fires Most deforestation and fires have occurred in drier parts of the Amazon, but these processes already accompany roads built into more humid forests (notably BR-163). Even along roads within their borders, and even during ENSO-related drought, reserves of all types reduced fires that closely accompany roads throughout the Amazon. New and existing reserves should thus be an integral part of the planning process to mitigate the environmental impacts of roads Abstract S1Abstract in Portuguese(0.03 MB DOC)Click here for additional data file.Abstract S2Abstract in Spanish(0.03 MB DOC)Click here for additional data file. |
In an attempt to immortalize immature T cells expressing a known T-cell antigenreceptor (TCR), Thy-1/c-myc transgenic mice were bred to αβ TCR transgenic mice (F5),and CD4+ CD8+ cell lines were established from thymic tumors in double-transgenic mice.These cells expressed high-level heat-stable antigen (HSA) and were able to undergoprogrammed cell death upon induction with steroids and CD3 cross-linking, but not withcognate peptide. In addition, one line had rearranged and transcribed endogenous TCR cand genes, in spite of the fact that transgenic α and β genes were also expressed.Furthermore, we show that Thy-1/myc transgenic mice deficient in recombination activatinggene-1 (RAG-1) do not develop tumors, in contrast to RAG-1-/- mice, which are alsotransgenic for both Thy-1/myc and the F5 TCR. This indicates that in order for thymocytesto be transformed by the Thy-myc transgene, they need to proceed to the double-positivestage.The c- |
In this paper, we studied expression, kinetics, chromatin remodeling of the CD3 gene at different time-points post HTLV-I infection.HTLV-I infected CD4CD3γ followed by the subsequent progressive reduction in CD3δ, then CD3ε and CD3ζ mRNA. Transient transfection experiments showed that the CD3γ promoter was still active in CD3- HTLV-I infected cells demonstrating that adequate amounts of the required transcription factors were available. We next looked at whether epigenetic mechanisms could be responsible for this progressive decrease in CD3 expression using DNase I hypersensitivity (DHS) experiments examining the CD3γ and CD3δ promoters and the CD3δ enhancer. In uninfected and cells immediately post-infection all three DHS sites were open, then the CD3γ promoter became non accessible, and this was followed by a sequential closure of all the DHS sites corresponding to all three transcriptional control regions. Furthermore, a continuous decrease of in vivo bound transcription initiation factors to the CD3γ promoter was observed after silencing of the viral genome. Coincidently, cells with a lower expression of CD3 grew more rapidly.The onset of this phenomenon coincided with a decrease of We conclude that HTLV-I infection initiates a process leading to a complete loss of CD3 membrane expression by an epigenetic mechanism which continues along time, despite an early silencing of the viral genome. Whether CD3 progressive loss is an epiphenomenon or a causal event in the process of eventual malignant transformation remains to be investigated. I stimulates calcium release from the endoplasmic reticulum, which induces NFAT transcription factors leading to T-cell activation[HTLV-I infection can lead to the development of adult T-cell leukemia/lymphoma (ATLL) in 2–5% of infected individuals depending upon geographic location and exposure to etiologic factors. It is currently thought that tumors develop from a persistently infected T-cell reservoir, which can be amplified by cytokine-induced activation leading to viral gene expression, cellular proliferation and survival of some expanded cells. Viral gene expression has been implicated in the disruption of various normal cellular processes, including activation, growth, and apoptosis, the latter allowing accumulation of abnormalities leading to cellular transformation. Several viral proteins have been shown to play an important role in tumor progression by modulating transcription factors. The pleiotropic viral protein Tax mediates the NF-κB activation resulting in abnormal cytokine and cytokine receptor expression. Sumoylactivation,5. The vctivation.+ lines and T-cells from patients with ATLL are characterized by a CD3- or CD3low phenotype [A keystone of the antigen-specific immune response is the T-cell receptor (TCR)/CD3 complex. Infected CD4henotype -9. In a henotype we have henotype . We deci+ T cells with HTLV-I was known to progressively downregulate CD3 genes transcripts, eventually leading to a CD3- surface phenotype after 200 days of in vitro infection [+ T cells with HIV-1 [- CD4+ T-cell lymphoma mediated hypereosinophilic syndrome [CD3γ gene transcripts. All T-lymphotropic viruses induce CD3 downregulation in the absence of a generalized suppression of host protein synthesis.Experimental infection of CD4nfection ,13; howeth HIV-1 -17, HIV-th HIV-1 , as wellsyndrome , all linCD3γ gene transcripts, ultimately leading to a CD3- phenotype. However, we show that the initial CD3γ transcripts decrease is followed by a subsequent progressive and sequential reduction in CD3δ, CD3ε and CD3ζ genes transcription, going on after early viral genes silencing. Our experiments also demonstrate that these phenomena occur through chromatin remodeling and progressive closure of the CD3 genes promoter sites and are not the results of transcription factors depletion. Finally, this loss of CD3 expression is timely associated with a growth advantage, but further experiments will be needed to determine whether there is a causal relationship between these two observations.The HTLV LTR responds to T cell-activation signals, which s+ T cell line[The WE17/10 cell line is a human IL-2 dependent CD4cell line that wascell line. WE17/10coRI (no cut into the HTLV-I provirus) or SacI (cut once into the HTLV-I LTR) and electrophoresed in an agarose gel then transferred to nylon membrane . The filters were hybridized with radiolabeled probe : a KpnI fragment[pro gene and ending in the env gene, at 65°C for 12 hours, washed in buffers, and then exposed to X-ray film at -80°C.We used a standard southern blot protocol. The genomic DNA was digested with E fragment, correspCells were analyzed for CD3 surface expression by flow cytometry as previously described. BrieflyWE17/10 cells (uninfected and HTLV-I infected) were transiently transfected using standard DEAE-dextran protocols with wild-type (pHγ3-wt) promoter construct as previously described.2, 10 mM Tris pH 7.4, 300 mM sucrose, 0.1 mM EGTA, and 0.1% NP-40) to isolate the nuclei. The nuclei were then resuspended in 1 ml of nuclear digestion buffer . Nuclei from 20 × 106 cells were digested for 3 minutes at 22°C using increments of DNase I (Roche Diagnostics) from 0 to 28 U/ml. The reaction was stopped by adding nuclear lysis buffer containing 0.1 mg/ml proteinase K and incubating for 5 min at 55°C then overnight at 37°C. Genomic DNA was subsequently isolated using standard phenol chloroform extraction techniques.Isolation and DNase I digestion of nuclei was performed using a method previously described . BrieflyCD3δ promoter, BamHI for the CD3δ enhancer and SacI for the CD3γ promoter prior to standard Southern blot analysis. Promoter probes were amplified by PCR using the following primer pairs:Genomic DNA was digested with BglI for the CD3γ promoter: forward, 5'-CACCTGCTGAAACTGAGCTG-3', reverse, 5'-TCCCAGACAGTGGAGGAGTT-3';CD3δpromoter: forward, 5'-GTTCCTCTGACAGCCTGAGC-3' and reverse 5'-TTTTAGGCCTGATGGCCTCT-3'.CD3δ enhancer was a BamHI digest of the human CD3δ cDNA (NCBI accession # BC070321).The probe used to detect the CD3 genes have been previously described[Total RNA was isolated from cells using the TriPure Isolation Reagent (Roche Applied Science) in a single-step extraction method. Standard reverse transcription was performed using 1 μg of total RNA at 42°C for 45 minutes and 50 ng of the resulting cDNA was used per PCR reaction. The primer pairs used to amplify the individual described,26 and aCD3γ: forward 5'-CATTGCTTTGATTCTGGGAACTGAATAGGAGGA-3', reverse 5'-GGCTGCTCCACGCTTTTGCCGGAGACAGAG-3';CD3δ: forward 5'-TTCCGGTACCTGTGAGTCAGC-3', reverse 5'-GGTACAGTTGGTAATGGCTGC-3'.et al[Real-time RT-PCR was performed using a TaqMan Gene Expression Assay for each of the individual CD3 genes . Eukaryotic translation elongation factor1 α(EF-1-α) and cancer susceptibility candidate 3 (MLN51) were used as CD4+ T cell specific endogenous reference genes as described by Hamalainen et al. Relativ7 cells, and EMSA experiments were performed as described previously[1/CD3γInr binding site: Spγ1/CD3γInrwt, 5'-GTGATGGGTGGAGCCAGTCTAG-3'[Nuclear extracts were prepared from 2 × 10reviously. The radGTCTAG-3'. The oli1, CD3γInr, and Spγ2 (205-bp product), forward, 5'-GGGTTCTTGCCTTCTCTCTCAA-3', reverse, 5'-CCCCTAGTAGGCCCTTACCTT-3'.The ChIP assay was performed as previously described using th32P-labeled PCR product was separated on a 6% acrylamide gel and detected by autoradiography.The amplified + T cell line WE17/10 infected by the HTLV-I viruses produced by the MT-2 cell line. The latter, used as virus source, contains 8 complete or defective proviral genomic integrations some defective proviral genomes being able to produce viral RNA transcripts. The most dominant species of unintegrated viral DNA was 3.7 kb in size; it hybridized to a full-length HTLV-1 DNA probe but not to a KpnI viral DNA fragment beginning in the pro gene and ending in the env gene[The cell lines were derived from the IL-2 dependent CD4 env gene that is EcoRI, which does not cut within the 9 kb of the HTLV-I genome, the complete provirus probe revealed a smear witnessing a polyclonal integration of the provirus in the WE17/10 infected cells decreased in a progression from CD3hi to CD3low to CD3-, similar albeit slower than that previously described for HIV-infected cells[hi expression.Cryopreserved cells from different stages of the primary infection were thawed and CD3 surface density was quantified in a parallel experiment to ensure that the detected changes were not attributable to variation in antibody labeling experiments Figure . A signited cells,15,18. TCD3γ, δ, ε and ζ) were lost after HTLV-I infection in vitro, but these experiments did not provide insight into the order of their loss. Our previous experiments have shown that TCR/CD3 surface receptors are down-modulated after infection with HIV-1[CD3γ gene transcripts. We therefore asked whether the CD3γ gene was also initially targeted after HTLV-I infection and found that its specific decrease of transcription precedes the progressive loss of surface CD3 expression on HTLV-I infected cells.A previous study found thith HIV-1,17 and Hith HIV-1 linked tCD3γ transcripts . Subsequently, a precipitous drop of about 80% in CD3γ transcripts appears while the density of the TCR/CD3 on the cell surface is ~70%. This erosion in CD3γ transcript numbers progresses until all of the cells are CD3γ and surface CD3 negative (± 9–12 mo. p.i.). This loss of CD3γ gene expression is followed by a steady decrease in CD3δ transcripts followed by a slower but also progressive reduction in CD3ε and CD3ζ transcripts. Maintained continuously in vitro, the HTLV-I infected cells eventually become negative for CD3δ as well as CD3γ transcripts. The level of CD3ε and CD3ζ transcripts remains ~25% in the CD3γ-δ- cells even after more than three years p.i. In MT-2 cells CD3γ, CD3δ and CD3ε transcripts are completely lost while the CD3ζ transcripts are still expressed but at a very low level (data not shown).A real time RT-PCR assay for quantification of all four CD3 gene transcripts revealed that the loss of TCR/CD3 complex at the cell surface occurs quite later than the loss of s Figure . InitialCD3γ promoter activity in the HTLV-I infected cells after the loss of CD3γ gene expression we used our previously described construct (pHγ3-wt)[CD3γ promoter activity was similar to that of uninfected WE17/10 cells. It was over 2.5 fold of the activity measured for the pGL3 plasmid basic vector (pGL3-BV). The CD3γ promoter cloned into a plasmid vector was active while the CD3γ gene transcripts are lost after HTLV-I infection. Thus, after HTLV-I infection, CD3γ gene silencing could not be explained by a lack of transcription factors but potentially by a restrained accessibility to its transcriptional regulation region.In an effort to investigate the full-length (pHγ3-wt) in a traCD3γ, CD3δ and CD3ε genes are located in a 50 kb cluster on chromosome 11q23, with CD3γ and CD3δ positioned head-to-head and separated by 1.6 kb. DNase I hypersensitivity experiments using probes designed to specifically detect the CD3γ promoter, CD3δ promoter or CD3δ enhancer (an enhancer for the CD3γ gene has not been identified yet) revealed that in uninfected (positive control) and HTLV-I infected CD3γ+δ+ cells all three DNase I hypersensitive sites (DHS) are readily discernible -binding motifs, with an Initiator (Inr) element present in the primary core promoter[1/CD3γInr[+ uninfected WE17/10 or from CD3- HTLV-I infected WE17/10 cells trichostatin A in association with the DNA-methylation inhibitor 5' deoxy-azacytidine rescued CD3γ promoter by comparing TCR/CD3+ uninfected, untreated and TSA/AZA treated TCR/CD3- HTLV-I infected WE17/10 cells /CD3 complex is the keystone of the antigen-specific immune response. Infection by HTLV-I has been shown to ultimately downregulate infection,13; howeinfection-17 and Hinfection associathenotype . The virhenotype in infecin vitro infection leads to progressive downmodulation of TCR/CD3 complexes from the cell surface following a pattern of decreasing surface density reminiscent of that observed for HIV-1[CD3γ gene. To ensure that this phenomenon was not restricted to our experimental setting and the utilized cell line, we have tested a number of well-established HTLV-I infected CD4+ cell lines and found a general down modulation of TCR/CD3 surface expression in comparison to their uninfected counterpart.In this study, we investigated proviral integration, viral gene expression, CD3 surface density, CD3 gene transcription and chromatin structure over a period of time of three years post HTLV-I infection of the WE17/10 cell line. We found that HTLV-I for HIV-1,15 and Hfor HIV-1, except CD3γ by HIV[CD3γ, 48% CD3δ, 62% CD3ε and 75% CD3ζ gene transcripts. This extensive loss of CD3γ transcripts prior to significant TCR/CD3 down-modulation was similar to what we have observed previously for TCR/CD3 loss after HIV-I infection[CD3γ parallels the receptor negative phenotype in cell lines infected with both viruses, CD3- HTLV-I infected cells continue to progressively loosing expression of the remaining CD3 genes, with CD3δ transcripts being absent at 29 months p.i and about ~25% CD3ε and CD3ζ transcripts being still expressed at 3 years p.i. In contrast, HIV-1 infected cells maintain CD3δ, CD3ε and CD3ζ transcripts at >75% of normal levels in the presence of steadily decreasing CD3γ transcripts. Our data thus reveal that while both HIV-1 and HTLV-I target the expression of the CD3 genes, remarkably they appear to accomplish this task with distinct kinetics.However in contrast to the selective targeting of 3γ by HIV,18, HTLVinfection. These dinfection. AlthougImportantly, we also observed that, in contrast with HIV infected cells, an in vitro selection of certain clones occurs, as demonstrated in Fig CD3γ, CD3δ and CD3ε genes, located together on chromosome 11q23, are highly homologous due to their common ancestry[CD3ζ gene is located on chromosome 1 and has no apparent sequence homology with the other CD3 genes. It is therefore remarkable that all four genes are sequentially targeted in HTLV-I infected cells. Previous studies investigating the role of individual CD3 chains in thymopoiesis suggest that a mechanism exists for controlling access to the CD3γ, CD3δ and CD3ε gene cluster. Disruption of the CD3ε gene by insertion of a neomycin cassette in place of either exon 5[-/- mice who did not only show a CD3ε deficiency, but also underwent a significant inhibition of CD3γ and CD3δ genes transcription. Expression of CD3γ and CD3δ could be restored in CD3ε-/- mice by deletion of the neomycin cassette using in vivo recombination but not by transgenic reconstitution of CD3ε protein expression[CD3γ [CD3δ [CD3 genes. It has been reported that the coding sequence of neo gene can act as a transcriptional silencer[neo insertion in CD3ε potentially functions as an insulator by separating CD3γ and CD3δ genes from a putative locus control region. Taken altogether, these data indicate the existence of a mechanism for the global control of the 11q23 CD3 genes cluster that is likely to be critical in modulating the expression of these genes during the early stages of T-cell commitment. Similar cellular factors may also be involved in controlling the CD3ζ gene to ensure its coordinate expression with the other CD3 genes during T-cell differentiation and development.The human ancestry, while ter exon 5, exons 5er exon 5 or the per exon 5 left CD3xpression. Furtherion[CD3γ or CD3δ 3γ [CD3δ genes ha silencer, which sCD3γ expression could be restored in HTLV-I infected cells lacking endogenous CD3γ expression. This demonstrates that the loss of CD3γ is not due to a defect in factors binding to the CD3γ promoter region and rather suggests a lack of accessibility of these factors to the promoter regions in HTLVI infected cells. We further demonstrated that the loss of CD3γ and CD3δ transcripts is associated with progressive closure of the CD3γ promoter DHS followed by the CD3δ promoter and enhancer DHS. Modification in the corresponding DHS occurred in tandem with the reduction and loss of CD3γ and CD3δ gene expression p.i.However, by transient transfection we observed that in vivo binding of Sp1, Sp3 and TFIID to the CD3γ core promoter region in CD3- HTLV-I infected WE17/10 cells in comparison with TCR/CD3+ uninfected cells, while the in vitro binding was not affected. It has been shown that Sp1 and Sp3 transcription factor binding to TRE-I repeat III participates in the regulation of HTLV-I viral gene expression[In addition, we showed a reduction xpression. On the xpression.CD3γ core promoter was more abundant in CD3- HTLV-I infected compared to CD3+ uninfected WE17/10 cells. As expected, treatment with the histone deacetylase inhibitor (HDAC) trichostatin A in association with the DNA-methylation inhibitor 5' deoxy-azacytidine reestablished the H4 hyperacetylation status and reduced the HDAC binding to the CD3γ core promoter and rescued the transcription of CD3γ and CD3δ in the CD3- HTLV-I infected. This result reemphasizes that an epigenetic mechanism is at work to downmodulate the four CD3 genes after HTLV-I infection. We recently started a study aiming at unraveling the molecular determinants that coordinate the successive downregulation of the four CD3 genes.Histone H4 hyper-acetylation is a typical feature of active transcription. Histone H4 hyperacetylation was reduced and binding of HDAC to the +cell line leads to progressive alteration of Ca++ influx that eventually results in loss of CD7 expression and activation of an antiapoptotic pathway involving AKT and BAD which paves the way for malignant transformation[In a previous work we have shown that HTLV-I infection of WE17/10 CD4formation the loss-, CD7- phenotype associated with perturbation of calcium fluxes and constitutive activation of PI3 kinase, which prevents apoptosis and augments growth of the infected cells. The mechanism by which these phenomena continue after the loss of viral gene expression will be the subject of further studies, as well as determining whether CD3 progressive loss is an epiphenomenon or a causal event in the process of eventual malignant transformation.We conclude that HTLV-I expression initiates a process leading to several phenomena, among which a progressive loss of TCR/CD3 by epigenetic mechanisms. These modifications persist after HTLV-I genes are silenced through a mechanism that we have started to investigate. This eventually leads to a CD3HTLV-I, human T-cell leukemia virus type I; ATL, adult T cell leukemia/lymphoma; NF-κB, nuclear factor kappa-B; NFAT, nuclear factor of activated T cell; HBZ, HTLV-I bZIP factor; TCR, T cell receptor; HIV, human immunodeficiency virus; DHS, DNase I hypersensitive site; EF-1-α, eukaryotic translation elongation factor1 α; MLN51, cancer susceptibility candidate 3.The author(s) declare that they have no competing interests.HA conceived this project and carried out most of experiments in Figs. - or CD3low phenotype.CD3 expression on the surface of HTLV-I-infected cells. We have tested the HTLV-I infected cell lines MT-2, C91, WE/HTLV and an ATLL derived cell line PaBe for their TCR/CD3 surface expression. All the cells had a CD3Click here for file |
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