Evidence on the relation between fear of war and mental health is insufficient. We carried out a prospective cohort study to find out whether fear of nuclear war is related to increased risk of common mental disorders.Within two months preceding the outbreak of Persian Gulf War in January 1991, 1518 adolescents [mean age 16.8 years, SD 0.9] filled in a self-administered questionnaire. Of the 1493 respondents, 47% gave their written informed consent to participate in the follow-up study. There were no material differences between those who chose to respond anonymously and those who volunteered to give their name and address for the follow-up study. In 1995, the response to the follow-up questionnaire was 92%. Common mental disorders were assessed by 36-item version of the General Health Questionnaire [GHQ]. A score 5 or higher was considered to indicate caseness. We excluded 23 cases which had used mental health services in the year 1991 or earlier and two cases with deficient responses to GHQ. This left 626 subjects for analysis [400 women].After adjusting for significant mental health risk factors in logistic regression analysis, the risk for common mental disorders was found to be significantly related to the increasing frequency of fear for nuclear war, high scores of trait anxiety and high scores of immature defense style. Elevated risk was confined to the group reporting fear of nuclear war once a week or more often [odds ratio 2.05; 95% confidence interval 1.29–3.27].Frequent fear of nuclear war in adolescents seems to be an indicator for an increased risk for common mental disorders and deserves serious attention. Risks of war and terrorism are threatening our health, both directly in actual life and also indirectly by the increasingly violent content of video games and other forms of entertainment. How does this affect mental health? Earlier during the cold war period, fear of war was found to be common among adolescents, and more prevalent among girls than boys -5. LittlBetween December 4, 1990, and January 16, 1991, 1518 adolescents from five high schools in Helsinki and five in Jyväskylä, Finland, representing a cross-section of school entrance requirement levels filled in a self-administered questionnaire during an ordinary classroom hour. Of the 1493 respondents, 709 (47%) gave their written informed consent to participate in the follow-up study. There were no significant differences between those who chose to respond anonymously and those who volunteered to give their name and address for the follow-up study with respect to baseline predominance of mature, immature or neurotic defence styles, trait anxiety, trait depression, the number of positive and that of negative life-events, self-esteem, coherence of future, or availability of social support. Anonymous respondents reported less somatic symptoms than those who gave informed consent to follow up. The absolute difference in the symptom score was not very large, however. The mean scores (SE) were 21.8 (0.22) and 22.8 (0.29) for men, 24.2 (0.23) and 25.0 (0.23) for females, respectively . Of the We excluded 23 cases who reported having used mental health services in the year 1991 or earlier and two cases with deficient responses to GHQ. This left 626 subjects for analysis, of whom 400 were women. At the baseline, the age range was 15 to 19 years (mean 16.8, SD 0.9).Frequency of fearing nuclear war during past four weeks (scores in parentheses) was assessed by a question with six options: not at all (0), less than once a week (0.5), 1–2 times a week (6), 3–5 times a week (16), almost daily (22) and daily (28).The Defence Style Questionnaire (DSQ) consisted of 72 statements assessing possible conscious derivatives of 20 defences. It is based on the 88-item version of the Bond's Defense Style Questionnaire . AndrewsThe Trait Anxiety Inventory was used to measure trait anxiety as a general tendency of feeling . Trait aDepressive trait  was asseAn abbreviated version of the Life Event Checklist  consisteThe Somatic Symptom Score is an abbreviated 14-item version of an original 18-item score used earlier in Finnish studies on adults and adolescents [The self-esteem scale by Rosenberg  consistsCoherence of future was measured by three items (no. 11, 22 and 27) from the Sense of Coherence Scale  relatingSocial support was ascertained by asking "Do you have a significant other person with whom you may discuss your personal activities and problems?".Social class assessment was based on father's occupation or on mother's occupation when the father was not living in the family of the adolescent. Use of the City of Helsinki Social Group Classification divided the sample into four categories: (i) professionals, managers and higher administrative or clerical employees, (ii) lower clerical employees, (iii) skilled workers, and (iv) unskilled workers.The General Health Questionnaire (GHQ) ,22 is a Data were analysed with SPSS 11. Logistic regression was used to model the relationship between assumed risk factors and high GHQ score [5 or more]. Initial models included sex, social class, availability of social support [dichotomous variables], age, self-esteem, coherence of future, number of positive and that of negative life-events, neurotic, immature and mature defence styles, trait anxiety, trait depression, somatic symptom score [continuous variables]. Second-level interactions were studied by adding product terms to the models. Because of missing values, the number of cases was lower than 626 in some analyses.Only significant [p < 0.05] confounders remained in the final models. To evaluate relative risk, fear of nuclear war and the significant confounders were categorised and odds ratios with their 95% confidence interval were estimated.Of the 400 women, 27.5% reported having feared nuclear war once a week or more often in 1990. The respective figures for men were 226 and 13.7%. Thirty-six per cent of the women and 22.1 % of the men scored 5 or higher on GHQ. The initial full model included all putative confounders under study  in mental health at follow-up examination, i.e. incident problems. False positives might have included individuals with mild or transient psychological disturbance, which should have biased the association towards the null. Still, the relation was significant. However, some caveats should be discussed.Could the association be due to some confounding factors? We controlled for several potential confounders. Those, known to increase or decrease the risk of mental disorders, included neurotic, immature and mature defence styles -31, traiAdolescents not willing to answer a mental health questionnaire may have more mental health risk factors and problems than participants. However, we found no significant differences between the anonymous and identifiable respondents in possible mental health risk factors analysed except that anonymous respondents reported slightly more somatic symptoms than those who identified themselves. The difference was, however, small in absolute terms (data presented above in section on design). This suggests that subjects with high risk were not underrepresented in the present sample.The degree of perceived threat of nuclear war may depend on several factors, such as (i) actual presence and size of the nuclear weapon arsenal, (ii) actual political tensions and threats, (iii) media coverage of the former, (iv) mental, conscious and unconscious processing of information, and (v) psychological developmental influences specific to adolescence.detente period may be illustrative:Part of the fear may be based on realistic evaluation of the threat. Our baseline examination was carried out within two months before the outbreak of the Persian Gulf War in January 1991 and before the reductions in nuclear weapon arsenals in the United States and in Russia started. A quote from a novel describing the life experience of one teen-age girl during the pre-"One was obliged to think about something important. One was obliged to think about the crisis between China and Soviet Union. A war could break out, the World War III and nuclear fallout would burn everything. The familiar fear for war pressed me inside so that it was difficult to breathe."Laura Honkasalo. Sinun lapsesi eivät ole sinun. Jyväskylä: Gummerus, 2001, p. 132.But similar experiences were not unknown among boys either, as witnessed by a seasoned cook from New York:"I grew up thinking the Big One could come at any moment, and this country – or fear of it, the way my country reacted to the threat – radicalized, marginalized, and alienated me in ways that still affect me."Alan Bourdain. A cook's tour in search of the perfect meal. London: Bloomsbury, 2001. p. 80.Widespread media coverage on any potential danger may bring about considerable increase in perceived fear . Mass mePerceptions of the threat of nuclear war as well as other dangers are processed mentally. Conscious or unconscious intentions are often projected to or mixed with dangerous external events, and they may distort the association between the actual threat of war and perceived fear. There is growing evidence that violent films and video games may trigger fear, aggression and violence among adolescents vulnerable to such content , and perStudies on the prevalence of fearing or worrying about nuclear war during periods of low political tension suggest that this phenomenon is common in adolescence and disappears or at least diminishes later in life . CognitiGlobal threats may vary in time as well as in their appraisal. In addition to old risks of nuclear war and aircraft hijacking, international terrorism and biological warfare loom at present. How should we handle these risks? We might inquire into the fears of our patients, appraise the risks realistically, point out that widespread media coverage tends to exaggerate the risks, and, as Durodié and Wessely  point ouA clear positive association was found between fear of nuclear war and common mental disorders among adolescents. Fear of nuclear war may either be a risk indicator produced by an underlying vulnerability to psychopathological process or have a more direct causal role in the onset of mental disorder among adolescents. In either case, frequent fear of nuclear war in adolescence seems to be an indicator for an increased risk for common mental disorders that deserves serious attention.The authors declare that they have no competing interests.KP and JL planned and designed the study. KP wrote the study proposal and received funding. KP, TA-S, AT-H, MM and JL designed the follow-up, supervised the data collection, and interpreted data. KP analysed data and drafted the paper. KP revised it with contributions from all authors. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Pregnancy-related pelvic girdle and/or low back pain is a controversial syndrome because insight in etiology and prognosis is lacking. The controversy relates to factors eliciting pain and some prognostic factors such as the interpretation of pain at the symphysis. Recent research about treatment strategies also reflects those various opinions, in fact suggesting there is professional uncertainty about the optimal approach. Currently, physiotherapists often prescribe a pain-contingent treatment regime of relative rest and avoiding several day-to-day activities. Additionally, treatment more often includes an exercise program to guide rectification of the muscle imbalance and alignment of the pelvic girdle. Effectiveness of those interventions is not proven and the majority of the studies are methodologically flawed. Investigators draw particular attention to biomedical factors but there is growing evidence that important prognostic issues such as biopsychosocial factors appear to be even more important as point of action in a treatment program.This pragmatic randomized controlled trial is designed to evaluate the effectiveness of a tailor-made treatment program with respect to biopsychosocial factors in primary care. The effect of the experimental intervention and usual care are evaluated as they are applied in primary health care. The trial is embedded in a cohort study that is designed as a longitudinal, prospective study, which studies prevalence, etiology, severity and prognosis during pregnancy until one year after delivery. The present paper focuses on choices regarding recruitment procedures, in-/exclusion criteria and the development of a well-timed intervention.This section briefly discusses the actions taken to minimize bias in the design, the proper time-window for the experimental intervention and the contrast between the experimental intervention and usual care. Since 1962,4, the qCurrently, physicians and physiotherapists usually prescribe a pain contingent treatment regimen of relative (bed) rest and avoiding several day-to-day activities such as using the stairs, bending, twisting, lifting and cycling. Additionally, the usual treatment approach of a physiotherapist more often includes an exercise program to guide rectification of the muscle imbalance and alignment of the pelvic girdle . TherapiThere are several reasons for publishing a study protocol before obtaining research data. The main reason is to reflect on the study design independently of the results. Considerations and choices concerning methodology and treatment can be described more detailed. The present paper focuses on choices about recruitment procedures, in-/exclusion criteria and the development of a well-timed experimental intervention. We also present details about the enrollment of women with pregnancy-related pelvic girdle and/or low back pain in the controlled trial.The trial is embedded in a cohort study that is designed as a longitudinal, prospective study, which studies the prevalence, etiology, severity and prognosis of pregnancy-related pelvic girdle and/or low back pain until one year after delivery Figure . The preWe performed a randomized controlled trial in primary care to determine whether a tailor-made program with respect to biopsychosocial factors (intervention group) benefits women with pregnancy-related pelvic girdle and/or low back pain more in terms of effectiveness and costs than usual care would on a traditional pain contingent basis (control group).The medical ethics committee of the Maastricht University Hospital approved the intervention and cohort study. The study is performed in the Southeast of the Netherlands. Midwives and gynecologists recruited the women during early pregnancy (10–14 weeks). Participation of midwives and gynecologists in the recruitment of eligible pregnant women is of major importance for the success of the cohort and intervention study . We haveWomen are included in the cohort if they are at least 18 years old, pregnant and well versed in Dutch language. Women are given written information explaining the aims and contents of the cohort and intervention study before they decide to participate. Concerning the intervention study they are told that to current knowledge the two investigating treatment options are considered to be equally effective. The moment of inclusion for the intervention study lies at about three weeks after delivery. An individual woman enters the intervention study after signing informed consent for both the cohort and intervention study during early pregnancy and meets the in- and exclusion criteria of the intervention study three weeks after delivery. Women are included when having pain in the pelvic girdle and/or low back with an onset during pregnancy or just after delivery (cohort data), are restricted in their normal daily activities because of pelvic girdle and/or low back pain and if there is a delay in recuperation (not yet in the condition to participate satisfactory in housekeeping and care of children because of the complaints under investigation). The severity of symptoms must be varying with physical activities and time during the day. Women diagnosed with a relevant specific pathology (such as nerve root pathology, rheumatoid disorders, carcinoma, obstetric complications) that affects pain and activities of daily life are excluded. Exclusion also occurs in case of family related or psychosocial problems or when a disablement procedure is not yet finished.Final important aspects for in-/exclusion are the willingness of a woman to participate in the study or having a clear treatment preference. We onlyA basic principle for selection of eligible women in this study is that inclusion criteria must have a meaningful influence in goal setting for treatment. We therefore focused on criteria such as a delay in recuperation and restrictions in normal daily activities caused by pregnancy-related pelvic girdle and/or low back pain. However, other studies in this field formulated inclusion/exclusion criteria based on certain diagnostic classification strategies. Although rationales of these strategies greatly differ, they all attach great importance to the outcomes of particular  diagnostic tests. In the absence of a clear definition and reference standard to diagnose pregnancy-related pelvic girdle and/or low back pain, the outcomes of these procedures not only led to different selections of women having complaints, the prognostic and diagnostic importance of these subgroups also remain unclear .Exclusion of differential diagnoses is a major point of concern. For that reason we included a history taking and a physical examination protocol that focuses on differential diagnoses at first and then on the formulated inclusion criteria. The various specific physical examination tests to diagnose pregnancy-related pelvic girdle pain are left aside. For a better understanding of the complaints and tailoring treatment, application of these tests has no supplemental value .An experienced research-physiotherapist visited women at home, about three weeks after delivery. This visit is called for on the basis of a short self-administered questionnaire and/or initiated by midwives. A positive answer from a participating woman from the cohort and/or her midwife on the question: "Do you or does this woman need treatment?" took a central position in these questionnaires. In advance of a possible home visit, a short history taking by telephone took place about two weeks after delivery. History taking focuses on exclusion criteria such as: willing to participate in this part of the study, a diagnosis with relevant specific pathology, limitations in daily life caused by pregnancy-related pelvic girdle and/or low back pain and a delay in recuperation. During a home visit, a standardized history is taken and physical examination to exclude specific pathology is performed. Self-administered questionnaires are used to question the women about pain, limitations in activities, restrictions in participation, pain-related fear, pain catastrophizing, positive and negative affectivity, depression, expectancy of treatment result and quality of life. The questionnaires contain clear instructions for completion with no help or support from others. If a woman meets the selection criteria, she is informed about the aim and method of the intervention study and if she is willing to participate, the informed consent procedure is completed. The research-physiotherapist collecting the baseline data is trained in performing the measurements in a standardized way and is unaware of the women's treatment assignments.Randomization takes place after collecting the baseline data. In this study we used a block randomization (size of four). An independent research assistant (unaware of the baseline data) carried out the randomization procedure according to a random computer-generated list. When a woman is allocated to the intervention group, the participating physiotherapist in the environment of the woman is contacted and we ensured that treatment could start as soon as possible (within one week). Treatment is covered for all participants in the intervention group on a research-physician's referral. Women, allocated to the usual care group, are free to choose usual treatment by a (not participating) physiotherapist. Information about a possible guidance by a general practitioner and feasible treatments received after randomization is collected by means of questionnaires in the follow-up period.Women are blinded to a certain extent to the allocated treatment because they are kept naïve of the exact content of both treatment options. Participating physiotherapists are not blinded to the treatment option but not involved in the baseline and effect measurements. Researchers dealing with the baseline and outcome data are unaware of the treatment assignments.During a home visit a standardized history is taken  and physAfter history taking a short standardized clinical examination program is performed, which includes tests of nerve root radiation (exclusion). The resPrior to the trial, detailed information is gathered about the contents of traditional treatment options. Part of the information is collected by means of group discussions with experienced physiotherapists and occupational therapists and interviews on an individual basis with affected women out of the cohort. An independent rehabilitation specialist, specialized in pain treatment chaired the meetings with the therapists. Some subjects for discussion were: differences in clinical spectrum seen by the therapists, contents of treatment programs during pregnancy and after delivery, common knowledge by the therapists about etiology, prognosis and prevalence of the syndrome, the optimal time-window for treatment in the course of complaints and the therapist-patient relationship. Items that provided important topics of conversation between the therapists were: the moment of taking up and finishing off treatment, the contents of education and advice given to the patient and the (lack of) compliance. The most striking characteristics of a traditional treatment were the character of the therapist-patient relation and the way of goal setting, focusing on disease management . There wHowever, interviews with affected women made clear that most of the women were irritated about this regimen in an increasing degree after starting the treatment sessions. The regimen was too strict and on a number of points not geared to the wishes and concerns of the women. These aspects caused a lack of compliance and an unremitting hesitation about a good prognosis and in particular about reassuming certain day-to-day activities after delivery. Therapists did not realize the nature of this problem although they did mention problems with compliance. Some women were not able to get a grip on their condition and left management of their pain and activities of daily life to the therapist. A larger part of the women was more or less uncertain about picking up their full range of activities again after delivery. Their beliefs and concerns about origin and prognosis of their complaints clearly bore the stamp of the introduced biomedical label. The relatively favorable prognosis after delivery was largely unknown to the women as well as to the physiotherapists.Women, allocated to the intervention group, are referred to a participating physiotherapist in their own neighborhood. These physiotherapists received an educational course about the treatment protocol prior and during the study. All physiotherapists were already experienced and specialized in treating women with pregnancy-related pelvic girdle pain prior to the study. The contents of the experimental therapy are based on the latest literature, results of interviews with affected women (participating in the cohort study) and group discussions with experienced physical and occupational therapists.A search procedure in literature resulted in various therapeutic interventions. However, effectiveness of those interventions remain unproven. An important common goal of these treatments is restoration of optimal biomechanics, although this is not based on established theoretical principles . The seaDuring development of the experimental intervention we focused on the following contradictions: patient-therapist relationship, education, and hesitation or avoiding of activities. Theoretical concepts of self-management ,17 and fComplaint-related problem solving is a key skill. The role of the therapist is to encourage women to identify possible causes of a problem, find a number of potential solutions, select one, then try it and finally evaluate the results and possibly adjust the solution. The second important key skill is action planning or goal setting. Often a plan must have been generally unacceptable (such as "go skiing") for a therapist in the usual care. Nevertheless, the protocol of the experimental intervention embraced the point of view that a woman is her own best judge of what is possible. Another major point of action planning is that a woman could not only receive but also give feedback on her own accomplishments. Endorsement by the therapist is very important for a woman to accept her new role. This way of collaborating with a therapist on short-term action planning enabled women to master new skills and to make changes that are realistic and feasible for them.Therapists also have a role in assisting women in understanding their symptoms. Knowledge of the course of the complaints during pregnancy and after delivery including pain flare-ups in the year after delivery, factors contributing to fluctuations in pain, evidence-based knowledge about etiology and the concept about pain-related fear are essential. Symptoms are explained as having many but not alarming causes, which offers the possibility to choose different actions by the concerning woman. Finally, therapists have a task in practicing social persuasion. A woman is more likely to change her behavior and have confidence in doing so if she perceives those around her, including the therapist to be supportive.A relationship in which the physiotherapist and the woman make health care decisions together is the basic assumption of the intervention. Generally, a time contingent policy is followed in which women set the pace by means of action plans. The expertise of the physiotherapists of the condition in general and of the women about their own specific condition and lives are equally important .Outcome measures Table  chosen tLimitations in activities are measured with the Dutch translation of the Roland Disability Questionnaire (RDQ)  and the Subjective measurements like global feeling of recovery  and severity of the main complaints (MC) reflecting a patient-specific approach are also selected. Global Perceived Effect (GPE) is measured by self-assessment on a 7-point scale (1 = completely recovered, 7 = worse than ever). The main complaints (MC) are selected by the woman in a standardized approach by selecting three activities, which are an essential and frequently performed part of her everyday life. However, the performance is difficult or impossible because of low back and/or pelvic girdle complaints at the moment of baseline measurement. Severity of a main complaint is rated on a visual analog scale (VAS). ,27.Pain is measured with two VAS-scales of the McGill Pain Questionnaire (MPQ-DLV) ,29 to reThe impact on participation and autonomy (IPA) is used to measure person-perceived restriction in participation and autonomy ,31. The Other important prognostic factors that can influence treatment results are fear of movement, pain catastrophizing, depression, negative and positive affect, expectancy of treatment result and pain behavior.Fear of movement is measured by the Dutch translation of the Tampa Scale for Kinesiophobia(TSK),33. We uPain catastrophizing is measured by the Pain Catastrophizing Scale (PCS),37.The Beck Depression Inventory (BDI)  measuresTo measure the experience of negative affect we used the 14-item Negative Emotionality Scale (NEM) . To measHealth status is evaluated by the Short-Form 36 (SF-36),43 and tA cost-diary  is used Expectancy of therapy result  is measuThe Pain Behavior Scale (PBS),15 is anWomen are asked to complete follow-up questionnaires at 12 weeks after randomization, 6 months after delivery and one year after delivery. Women who did not return their follow-up questionnaires were contacted by mail or phone and were asked to continue participation.The follow-up questionnaires ask all women how many treatment sessions they have followed in the previous period of time. Furthermore, information on contents, satisfaction and the aspects of the  treatment which benefited them most, is gathered. Co-interventions, medication, aids, additional medical consumption, recurrence of complaints, return to gainful employment and a possible subsequent pregnancy are also registered.Physiotherapists who treat the participants of the intervention group also answered questions about the number and contents of the treatment sessions after conducting the last meeting.Statistical analyses are carried out according to the "intention-to-treat" approach. The baseline status of the study groups is compared with respect to the distribution of all independent prognostic variables and the baseline values of the outcome variables. For the outcome measures recorded at baseline and at follow-up, we computed the difference between the baseline and the follow-up score for each woman. Differences between groups and 95%CI are calculated for each outcome measure according to the intention to treat approach. Primary analysis is done by means of analysis of an independent t-test (for continuous outcome variables) and chi-square test . In order to adjust for possible baseline differences a multiple linear regression analysis for continuous outcome measures is performed with the change scores as dependent variable, treatment option as independent variable and base line scores of the prognostic variables as co-variables. Missing data at the baseline-measurement are substituted by the "mean of series" imputation method. Longitudinal missing data are substituted with the "last value carried forward method". In all comparisons between the two treatment options a two-tailed p-value of 0.05 is considered to indicate statistical significance. Prognostic status at baseline for women with and without missing values for the outcome variables is compared for both groups. Analyses are done by using SPSS statistical software, version 12.0 (SPSS, Inc., Chicago, Illinois). Short term and long term effect analyses are performed separately.A cost-effectiveness analysis compares the costs and health effects of the experimental intervention to assess whether it is beneficial from an economic perspective. The costs of the intervention are calculated separately for the intervention group. For the whole study group all relevant health care costs, production loss and patient and family costs are measured by means of a cost-diary  and follDuring the study, 397 of the 7526 women (5%) signed only for the cohort study (n = 7526) and were therefore beforehand excluded for taking part in the intervention study. Throughout pregnancy, 73% of all women in the cohort reported pain in the lumbar/pelvic region leveling off to 35.9% three weeks after delivery . The research-physiotherapist dealing with the baseline measurement was therefore unaware of treatment allocation.Details about the enrollment out of the cohort into the trial also show that the start of the experimental intervention is well timed. Most women have complaints during pregnancy. However, a considerable drop in the number of women having persistent complaints in the first weeks after delivery is observed. Then again, numbers of women having one or more episodes of pain complaints remained stable in the year after delivery. We have seen similar trends of proportions of women with a request for treatment for their complaints during pregnancy (9%), just after delivery (4.8%) and in the year following delivery.The aim of the experimental intervention is to increase the level of activities. Therefore the primary outcome measure is limitations in activities. The contrast between both interventions is an important issue in this study. Major features that underscore the contrast are the character of the patient-therapist relationship, pain-contingent versus time-contingent treatment, compliance to a regime of avoiding and limiting activities versus action planning and personal goal setting by the women themselves. Among therapists, the approach of the experimental intervention is not widespread at all. The participating therapists are explicitly asked to not give any information about the contents of the experimental treatment to therapists who do not participate in the experimental intervention. It is necessary to interest physiotherapists in the trial for an efficient performing of the experimental treatment option, which can be achieved by a relevant research question and in practice applicable results.The author(s) declare that they have no competing interestsCHGB: first author, is involved in the design, data collection, statistical analyses, and the development of the experimental intervention.RAdB: participated in the design, coordination, statistical analyses, have made substantial contributions to the development of the experimental intervention, is involved in revising the article for important intellectual content.PMJCW: participated in the design, has made substantial contribution to the development of the experimental intervention and is involved in revising the article for important intellectual contentJWSV: has made substantial contributions to the development of the experimental intervention and is involved in revising the article for important intellectual contentJMB: participated in the design and statistical analyses, is involved in revising the article for important intellectual contentABAK: participated in the design and collecting of the dataAH: participated in the design, recruitment and collecting of the dataPvdB and GGME; participated in the design, have made substantial contributions to conception, design and experimental intervention and revising the article critically for important intellectual content.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycaemia. Increased oxidative stress and decreased antioxidant levels are the leading cause of diabetes and diabetic complications. So it is felt that supplementation of antioxidants may be useful in controlling the glucose levels and to postpone the occurrence of diabetic complications. The objective of our study is to find the influence of antioxidant supplementation (L-ascorbic acid) on tolbutamide activity in normal and diabetic rats.L- ascorbic acid/tolbutamide/L-ascorbic acid + tolbutamide were administered orally to 3 different groups of albino rats of either sex in normal and diabetic condition. Blood samples were collected from retro-orbital puncture at different time intervals and were analyzed for blood glucose by GOD-POD method. Diabetes was induced by alloxan 100 mg/kg body weight administered by I.P route.L-ascorbic acid/ tolbutamide produced hypoglycaemic activity in a dose dependant manner in normal and diabetic condition. In the presence of L-ascorbic acid, tolbuatmide produced early onset of action and maintained for longer period compared to tolbutamide matching control.Supplementation of antioxidants like L-ascorbic acid was found to improve tolbutamide response in normal and diabetic rats. Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycaemia. It requires life long treatment with drugs coupled with diet control and exercise. It may be due to decrease in the synthesis of insulin (Type-I diabetes) or due to decrease in the secretion of insulin from β-cells of islets of Langerhans of pancreas (Type-II diabetes). Insulin is the drug of choice in type – I diabetes and sulfonylureas are the drugs of choice in type II. Among sulfonylureas, tolbutamide is the drug of choice for geriatrics because of its short duration of action and lower incidence of hypoglycaemia in early hours of night.Diabetes is one of the stress related disorder. Diabetic subjects are shown to have increased oxidative stress and decreased antioxidant levels -3. It waad libitum. They were divided into 3 groups of five each. They were fasted for 18 h prior to the experiment allowing access to water only, and the water was withdrawn during the experiment. Blood samples were collected from the retro-orbital plexus of each rat at 0, 0.5, 1, 1.5, 2, 4, 6 hr after drug administration. Blood glucose levels were determined by using GOD – POD method [All animal experiments were performed in accordance with our institutional animal ethics committee. Albino rats of either sex (Mahaveer Enterprises, Hyderabad) weighing between 125 – 175 g were used in the study. They were housed five per cage at temperature 22 ± 2°C and 12/12 h light/dark under controlled environment. Rats were fed with standard pellet diet. (Mahaveer Enterprises, Hyderabad) and water Group I received L-ascorbic acid 60 mg / kg body weight, Group II received tolbutamide 20 mg/kg body weight and Group III was given L-ascorbic acid (60 mg / kg body weight) prior to the administration of tolbutamide 20 mg/kg body weight in normal rats. In clinical practice tolbutamide and vitamin C are administered orally hence in our study also this was administered orally.Albino rats of either sex weighing between 125 to 175 g were fasted overnight before injection with alloxan. Alloxan monohydrate was dissolved in saline solution and was administered at a dose of 100 mg/kg body weight intraperitonially. Animals were treated with 10% dextrose orally to combat the early phase of hypoglycaemia. Rats showing fasting blood glucose levels above 150 mg/dl were selected for the study. They were divided into 3 groups of five each. Group I received L-ascorbic acid 40 mg/kg body weight and Group II received tolbutamide 20 mg/kg body weight while Group III was given L-ascorbic acid 40 mg/kg prior to tolbutamide administration (20 mg/kg). L-ascorbic acid dose was fixed based on its response, which produced above 40%.The significance of blood glucose reduction produced by L-ascorbic acid with tolbutamide compared to tolbutamide control was determined by applying students unpaired t-test and the significance is indicated by * mark.in vitro studies. In normal rats L-ascorbic acid at the dose of 60 mg/kg body weight administered orally produced 50.91% blood glucose reduction at 0.5 h and 20 mg/kg body weight of tolbutamide produced 33% at 4 h as peak effects. In the presence of L-ascorbic acid (60 mg/kg), the action of tolbutamide was early in onset and maintained for 6 h. In diabetic rats, oral administration of L-ascorbic acid alone at the dose of 40 mg/kg body weight produced 42.53% blood glucose reduction at 1.5 h and tolbutamide 20 mg/kg body weight produced 45.09 at 4 h. Administration of L-ascorbic acid 40 mg/kg body weight prior to tolbutamide produced antidiabetic activity at 0.5 h and was maintained for 6 h. The percent blood glucose reduction with L-ascorbic acid / tolbutamide/ L-ascorbic acid + tolbutamide in normal rats and diabetic rats were given in table The presence of L-ascorbic acid upto 20 μg did not interfere with the blood glucose estimation when tested with different quantities in The drug interaction studies are usually conducted in animal models to find out the mechanism before they are conducted in humans. We have selected rat as animal model since it is one of the animal, which synthesize ascorbic acid and can be maintained easily in the laboratory conditions.Reactive oxygen species (ROS) are thought to be implicated in the pathogenesis of diabetes as well as other diseases. ReactivIt was reported that L-ascorbic acid levels were decreased in diabetic patients and rats. So it iThe study indicates that additive action of L-ascorbic acid on pharmacodynamic response of tolbutamide may be useful to improve the tolbutamide activity in insulin resistant cases and to postpone the occurrence of diabetic complications. However further work on human patients is required to confirm the observation in diabetic condition and usefulness of L-ascorbic acid as supplemental agent for improved control of blood glucose levels when administered along with sulfonylureas.The author(s) declare that they have no competing interests.SS Conceived of the study, participated in the design of the study, performed the statistical analysis and drafted the manuscript.EK Participated in the design and coordination and in the standardization of methods.RJ Carried out the study in normal rats.VA Carried out the study in diabetic rats.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Although the mechanisms that regulate development of the cerebral cortex have begun to emerge, in large part through the analysis of mutant mice , many qu A novel screen uncovers thirteen mutants with defects ranging from the establishment of epithelial polarity in the cortex to the invasion of axons from the thalamus Other important insights have come from the study of spontaneous mutations that cause radial migration defects and lead to lissencephaly and similar cortical defects in humans. Our understanding of radial migration and many other aspects of cortical development have also benefited enormously from the application of gene knockout approaches in mice. Despite this progress, many aspects of cortical development remain to be explored and would benefit enormously from additional mutant resources. The tangential migrations of cortical interneurons, for example, are regulated differently from the radial migrations of projection neurons, and only a few mutations have been described that disrupt interneuron migration.The cerebral cortex is the seat of consciousness and the means by which we carry out abstract reasoning. Understanding how the cortex is assembled during embryonic development will give deeper insights into how this marvelous machine functions and provide the basis for therapy and repair. Although a diversity of approaches will be needed to answer all of our questions, an important starting point in studying events in development is often the careful analysis of mutant phenotypes. Much of what we know about cortical development has emerged through the study of mutations in mice and humans. For example, spontaneous mutations in mice such as Drosophila, deletion strains and chromosomal inversions have been used to identify mutations within a specific region of the genome, and this approach has been elegantly adapted for use in the mouse  of the ganglionic eminences, in immature interneurons as they migrate tangentially through the intermediate zone (IZ) or marginal zone (MZ) of the cortex , and Circletail mice have a mutation in the scribble gene (Scrb1). Mapping of the line 90 mutation places it on Chromosome 15 in the region of Scrb1. Scrb1 encodes a protein of 1,665 amino acids with three leucine-rich repeat (LRR) domains near the amino terminus and four centrally located PDZ domains . This indicates quite clearly that the integrity of the LRR domains is critical for the tight coordination between Scrb1 and Ltap/Lpp1 in establishment of planar cell polarity.In escribed , 2003. Ahe mouse . Interes mutants . The int domains , is subsmegalin gene . Prolapse of the third ventricle choroid plexus was described in a knockout allele of megalin . Interneurons are GABAergic and so express GAD67, an enzyme involved in GABA synthesis. Glutamatergic projection neurons do not express GAD67. Whole-mount in situ analysis of GAD67 at E15.5 showed that the distribution of interneurons was abnormal, as predicted by the aberrant expression of the transgene  pattern in the limb at early stages. Hoxd13 is ectopically expressed in the anterior portion of line 239 and line 251 mutant limbs , and Bmp4 are not distinguishable from wild-type (WT) (unpublished data). In contrast, an ectopic domain of FGF4 persists in the anterior apical ectodermal ridge (AER)  H–4J. ThuThe 239, 251, and 275 mutants have clusters of LacZ-expressing cells in the IZ and/or SVZ of the rostral cortex, whereas more caudally, defects appear milder . In WT aDlx-LacZ transgene and Gad67. As at E14.5, aggregation of +; LacZ+Gad67 cells in the IZ/SVZ is a prominent feature of the phenotype  animals . We focuDlx-LacZ transgene allowed us to identify. In line 412, the mutant phenotype was detectable in whole mount as a subtle but consistent defect near the cortical-subcortical boundary. Upon sectioning, this defect was revealed to be a delamination in the region of the external capsule  expression vector, and, recently, using a in mouse . These sin mouse . In contecursors  led to tecursors , in a precursors . These tarista-less homolog, Arx, cause profound defects in interneuron migrations, and mutations in another arista-less homolog, Alx4, cause anterior polydactyly. However, as the limbs of Alx4 mutants, unlike the 239, 251, and 275 mutants, have ectopic Shh expression in the anterior limb bud, it is unlikely that the mutations described here disrupt an arista-less pathway. Shh maintains the expression of Fgf4 in the posterior AER of WT limb buds in a process that requires gremlin. Genes that act in this process downstream of Bmp4 are plausible candidates for the line 239, 251, and 275 mutations.Three out of the four tangential migration defects also result in anterior polydactyly. The association of limb patterning and neuronal migration defects has not been previously reported and, given that a great deal more is known about limb patterning than about tangential migration, it is tempting to speculate on what this may imply about the migration mutants. One possibility is that shared mechanisms are used to pattern the telencephalon and the limb. It is equally possible that genes that regulate patterning in the limb regulate cell migration decisions more directly. Mutations in an The work described here demonstrates clearly that genetic screening strategies in the mouse need not be limited to general or broad-based phenotyping approaches; a focused genetic screening strategy can provide a powerful means of dissecting a specific aspect of mammalian biology. The strategy of broad-based approaches has been an effective one for the past several years as the collection of mutants using chemically induced mutations resurged in popularity. In other genetic systems, strategies have evolved from general toward focused screens that allow mutations affecting a specific process to be identified, and it is likely that mouse genetics will progress in a similar fashion. An additional concern with mice, however, is the cost of breeding and housing, which is higher than that for other model genetic organisms. We have shown, nonetheless, that a laboratory-based, focused screening strategy is a productive pursuit. The costs of such a screen could be shared by the careful combination of reporters so that screening for several distinct processes could be carried out at once. This general idea is highlighted by our isolation of mutations in which the growth and patterning of the cortex is defective. With the exception of line 90, the identification of these mutants benefited from the easy visualization of cortical size and structure by expression of the transgene. In several cases, the interneuron migration mutants being a good example, the identification of the mutant phenotype without the transgene would have been very unlikely. Independent reporters could be used to pursue the simultaneous identification of several different classes of mutants more directly. For example, reporters that label migrating neurons with GFP, and thalamocortical axons with β-galactosidase or alkaline phosphatase, would allow both migration and axonal pathfinding mutations to be identified in the same screen. Given the large number of reporter and indicator strains that have been made over the last few years and the powerful tools that exist for gene identification, it is clear that focused screens in the mouse will provide the resources to address many questions in the coming years.Dlx-LacZ transgene. Male offspring of this cross  were backcrossed to the transgenic line, and female offspring  were saved. For each line, one to six of the G2 females were backcrossed to their fathers to generate timed pregnancies. Embryos were harvested either 13 d (the first 100 lines) or 14 d  after the vaginal plug was identified.Male C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, Maine, United States) and treated with three intraperitoneal injections of 100 mg/kg ENU (Sigma, St. Louis, Missouri, United States) spaced at 7-d intervals. Eight weeks after the last injection, the ENU-treated males were set up in breeding pairs with FVB/NJ females homozygous for the 2, 0.01% sodium deoxycholate and 0.02% Nonidet P-40). Embryos were then stained for 48–72 h at room temperature on a rocking platform using X-gal as a substrate for the detection of β-galactosidase activity. Staining was terminated after visual inspection by repeated washing in PBS. Embryos were then fixed again and stored in 4% PFA in PBS until further examined. All litters were examined in whole mount and approximately one-half were selected for sectioning on a vibratome . For sectioning, the heads of all pups from a litter were separated from the torsos and mounted aligned in the same orientation in an agarose block. Sections 100 μm thick were collected in PBS and mounted in Kaiser's glycerol gelatin (Merck, Darmstadt, Germany) on slides. All phenotypes reported here were seen in more than ten litters resulting from both G1 male × G2 female and G2 male × G2 female crosses.Embryos were dissected in phosphate-buffered saline (PBS) and fixed for 45 min at room temperature in 4% paraformaldehyde (PFA) in PBS. Subsequently, embryos were washed in detergent rinse  and anti-L1 (Chemikon, Mönchengladback, Germany) antibodies was performed on 100-μm free-floating sections following the instructions and using the reagents of the Vectastain ABC Kit (Vector). Primary antibodies were used at a dilution of 1:500. Horseradish peroxidase activity from secondary antibodies was revealed by diaminobenzidine as a substrate in staining buffer (0.5 mg/ml diaminobenzidine, 20 mM sodium cacodylate, and 30 mN acetic acid).http://www.cidr.jhmi.edu/mouse/mouse.html). Markers were chosen that could be scored easily on agarose gels. All chromosomal assignments reported are the result of LOD scores significantly greater than three. The intervals that are listed in Initial linkage was established using 12 DNA samples from both carriers and mutant embryos. We scored a set of 82 simple sequence repeat markers. The panel of markers was selected from the set at the Center for Inherited Disease Research Website  Dorsal views of the cortex of WT (left) and line 152 mutant (right) embryos stained for expression of the Dlx-LacZ transgene. The line 152 mutation reduces the size of the cortex.(B and C) Coronal sections through the cortex of E14.5 WT (B) and line 351 mutant (C) embryos. The cortex has a characteristic high-domed shape and is thinner in the mutant.(D and E) Dorsal (D) and ventral (E) views of adult brains of WT (left) and line 351 mutant (right) brains. The cortex is overall larger in the mutant than in the WT. The olfactory bulbs are present in the mutant but are tucked under the cortex, making them less visible.(9.1 MB TIF).Click here for additional data file.Figure S2(A) Lateral view of an E14.5 WT embryo.(B) The cleft upper jaw of a line 366 mutant is visible at the left. The telencephalon, including the cortex, is significantly shortened along the rostrocaudal axis.(C and D) Lateral and front views of the cleft and reduced upper jaw of an E18.5 embryo homozygous for the line 366 mutation.(E and F) Lateral views of WT (E) and line 357 homozygote (F) embryos at E13.5 showing the reduced telencephalon and relatively expanded midbrain.(G) A front view of the embryo in (F) shows the cleft upper jaw.(H and I) Sagittal sections through E13.5 WT (H) and line 357 (I) embryos. The overgrown midbrain in the mutant embryo has forced the neuroepithelium into folds.(9.9 MB TIF).Click here for additional data file.Figure S3(A) Lateral view of an E14.5 mutant embryo showing edema and hemorrhage suggestive of vascular defects. Frontal views of WT (B) and mutant (C) embryos illustrate the narrowed frontonasal process, maxilla, and mandible of the mutant. (D) shows coronal hemisections in a WT embryo. (E) shows the accumulation of Dlx-LacZ–positive cells in a SVZ-like area dorsal to the LGE.(4.3 MB TIF).Click here for additional data file.http://www.ncbi.nih.gov/LocusLink, and are as follows: Alx4 (11695), Arx (11878), Bmp4 (12159), Dlx5/6 (13395/13396), Fgf4 (14175), Gad67 (14415), gremlin (23892), Hoxd13 (15433), L1 (16728), Lrp2 (14725), Ltap/Lpp1 (93840), Ptc1 (19206), Rfx4 (71137), Scrb1 (105782), scribbled (44448), Shh (20423), and Tag1 (21367).Accession numbers of the genes discussed in this paper are available at LocusLink (


Identifying the molecular basis for the impressive regenerative capacities of some organisms may help us to devise effective methods for enhancing regeneration in mammals The regeneration of lost body parts and injured organs has captured the human imagination since the time of the ancient Greeks. The deep-seated roots of this early fascination can be seen in Greek mythology. The many-headed Hydra nearly defeated the hero Heracles by growing two new heads for every one that Heracles cut off, and the liver of Prometheus, devoured by a ravenous eagle each night, regenerated every morning. Aristotle, who lived from 384–322 BC, noted that the tails of lizards and snakes, as well as the eyes of swallow-chicks, could regenerate . This faDuring the nineteenth century and for most of the twentieth century, regeneration research primarily focused on the phenomenology of regeneration and its cellular basis. Many important discoveries were made during this period, which led in part to the general conclusion that progenitor cells are required for most regenerative processes. However, the origin of these progenitor cells varies between regenerating systems. In some cases, such as the regeneration of skin, blood, muscle, and bone in mammals and the replacement of lost tissues in the flatworm planarian, the progenitor cells pre-exist as reserve cells or stem cells that only need to be activated in response to injury or tissue depletion. In other cases, the progenitor cells can be created de novo through a process in which fully differentiated cells reverse their normal developmental processes and revert to proliferating progenitor cells. This latter process, known as cellular dedifferentiation, is especially prominent in vertebrates with exceptional regenerative abilities, such as salamanders. For example, during salamander limb regeneration, cells from muscle, bone, cartilage, nerve sheath, and connective tissues participate in the dedifferentiation process to form a pool of proliferating progenitor cells known as the regeneration blastema  Chalkle. It has An Essay on Animal Reproductions, with a series of questions— which, except for the antiquated language, could be asked by citizens of the twenty-first century: But if the abovementioned animals, either aquatic or amphibious, recover their legs, even when kept on dry ground, how comes it to pass, that other land animals, at least such as are commonly accounted perfect, and are better known to us, are not endued with the same power? Is it to be hoped they may acquire them by some useful dispositions? [A]nd should the flattering expectation of obtaining this advantage for ourselves be considered entirely as chimerical?The public has recently exhibited a renewed interest in regeneration research, due in large part to stem cell research, which has provided promising avenues for the field of regenerative medicine. In addition, celebrities such as Christopher Reeve and Michael J. Fox have given a human face to the many people who could benefit from effective regenerative therapies. The political, ethical, and religious controversies surrounding the use of human embryonic stem cells for therapeutic purposes have only served to increase the public's awareness of the promising potential of regenerative medicine. But this interest in using scientific knowledge to enhance the regenerative capacity in humans is not new. Spallanzani closed his 1768 monograph on regeneration, Although most of the current interest in regenerative medicine focuses on the potential benefits of either embryonic or adult stem cells, there are several investigators who are now taking an entirely different approach to the problem. These researchers think that although stem cells may offer some benefits in the relatively near future, a more comprehensive approach will be required to meet all of our regenerative needs. To achieve this goal, they must first learn how nature has already solved the problem of regeneration and then use this information to enhance the regenerative capacity in mammals. These studies seek to understand the biology of regeneration, especially the cellular and molecular mechanisms that govern regenerative processes. The experimental systems range from the unicellular protozoa to complex vertebrates, such as salamanders and mice.Msx1, BMP4, and Notch1. These genes encode, respectively, a transcriptional repressor, a signaling ligand, and a signaling cell surface receptor.With the technological advances that followed the advent of molecular biology, researchers acquired the basic tools to begin to unravel the molecular basis for cellular plasticity and regeneration. However, progress in this arena has been slow, given that most organisms with marked regenerative abilities are not yet amenable to routine genetic manipulation. Recent advances, such as the application of mutagenic screens to study fin regeneration in zebrafish  and the Msx1-deficient mice exhibit impaired fetal digit-tip regeneration, a phenotype that can be rescued in ex vivo cultures in a dose-dependent manner by application of exogenous BMP4 (Xenopus tadpoles are unable to regenerate their tails during a refractory period of development between stages 45 and 47 (BMP4, Msx1, and Notch-1, are not expressed. However, transgenic frogs carrying a hyperactive form of Msx1 or constitutively active ALK3 (a receptor for BMP4) are able to regenerate their tails during the refractory period. Transgenic frogs carrying a constitutively active Notch-1 receptor will regenerate their notochords and spinal cords but exhibit little or no muscle regeneration, suggesting that Notch-1 signaling alone cannot rescue complete regenerative capacity in frog tadpoles , coupled with serum stimulation, can induce these multinucleated cells to reduce their levels of myogenic proteins and undergo a cell cleavage process that produces proliferating mononucleated cells (a process known as cellularization)  . Clonal or cells .Msx1 appear to have a similar effect on mouse myoblasts, although no reports have yet addressed whether reversine might induce dedifferentiation of multinucleated myotubes.Cellularization of myotubes and myoblast dedifferentiation can also be induced by at least two synthetic trisubstituted purines. Myoseverin is a trisubstituted purine that binds to and disassembles microtubules, leading to the cellularization of multinucleated myotubes . The resPLoS Biology, Msx1 function to microtubule disassembly during the process of salamander myofiber cellularization and fragmentation . Their results suggest that Msx1 expression induces microtubule disassembly, which then leads to myofiber cellularization or fragmentation. If Msx1 function is markedly reduced in salamander myofibers by preventing the efficient synthesis of the Msx1 protein, cellularization or fragmentation of the myofiber is inhibited, suggesting that Msx1 is required for this process. Thus, this study complements previous work  accession numbers of the genes discussed in this paper are ALK3 (MGI: 1338938), BMP4 (MGI: 88180), Msx1 (MGI: 97168), and Notch1 (MGI: 97363). The GenBank (http://www.ncbi.nih.gov/GenBank/) accession numbers of the genes discussed in this paper are deltaC (NM 130944), notch1b (Y10352), Ambystoma mexicanumMsx1 (AY525844), Danio rerio msxb , D. rerio msxc (NM 131272), Homo sapiensALK3 (Z22535), and Mus musculusALK3 (Z23154).The Mouse Genome Informatics (


Autoimmune hepatitis (AIH) is a chronic necroinflammatory disease of the liver characterized by hypergammaglobulinemia, characteristic autoantibodies, association with HLA DR3 or DR4 and a favorable response to immunosuppressive treatment. The etiology is unknown. The detection of non-organ and liver-related autoantibodies remains the hallmark for the diagnosis of the disease in the absence of viral, metabolic, genetic, and toxic etiology of chronic hepatitis or hepatic injury. The current classification of AIH and the several autoantibodies/target-autoantigens found in this disease are reported. Current aspects on the significance of these markers in the differential diagnosis and the study of pathogenesis of AIH are also stated. AIH is subdivided into two major types; AIH type 1 (AIH-1) and type 2 (AIH-2). AIH-1 is characterized by the detection of smooth muscle autoantibodies (SMA) and/or antinuclear antibodies (ANA). Determination of antineutrophil cytoplasmic autoantibodies (ANCA), antibodies against the asialoglycoprotein receptor (anti-ASGP-R) and antibodies against to soluble liver antigens or liver-pancreas (anti-SLA/LP) may be useful for the identification of patients who are seronegative for ANA/SMA. AIH-2 is characterized by the presence of specific autoantibodies against liver and kidney microsomal antigens (anti-LKM type 1 or infrequently anti-LKM type 3) and/or autoantibodies against liver cytosol 1 antigen (anti-LC1). Anti-LKM-1 and anti-LKM-3 autoantibodies are also detected in some patients with chronic hepatitis C (HCV) and chronic hepatitis D (HDV). Cytochrome P450 2D6 (CYP2D6) has been documented as the major target-autoantigen of anti-LKM-1 autoantibodies in both AIH-2 and HCV infection. Recent convincing data demonstrated the expression of CYP2D6 on the surface of hepatocytes suggesting a pathogenetic role of anti-LKM-1 autoantibodies for the liver damage. Family 1 of UDP-glycuronosyltransferases has been identified as the target-autoantigen of anti-LKM-3. For these reasons the distinction between AIH and chronic viral hepatitis  is of particular importance. Recently, the molecular target of anti-SLA/LP and anti-LC1 autoantibodies were identified as a 50 kDa UGA-suppressor tRNA-associated protein and a liver specific enzyme, the formiminotransferase cyclodeaminase, respectively. Anti-ASGP-R and anti-LC1 autoantibodies appear to correlate closely with disease severity and response to treatment suggesting a pathogenetic role of these autoantibodies for the hepatocellular injury. In general however, autoantibodies should not be used to monitor treatment, predict AIH activity or outcome. Finally, the current aspects on a specific form of AIH that may develop in some patients with a rare genetic syndrome, the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED) are also given. Autoantibodies against liver microsomes (anti-LM) are the specific autoantibodies detected in AIH as a disease component of APECED but also in cases of dihydralazine-induced hepatitis. Cytochrome P450 1A2 has been identified as the target-autoantigen of anti-LM autoantibodies in both APECED-related AIH and dihydralazine-induced hepatitis. The latter may indicate that similar autoimmune pathogenetic mechanisms can lead to liver injury in susceptible individuals irrespective of the primary defect. Characterization of the autoantigen-autoantibody repertoire continues to be an attractive and important tool to get access to the correct diagnosis and to gain insight into the as yet unresolved mystery of how hepatic tolerance is given up and AIH ensues. The estimated prevalence of AIH in Northern European countries is approximately 160–170 patients/10abitants . The disabitants -5. The oabitants . Howeverabitants . Althougabitants .Liver histology is not pathognomonic for AIH and there is no single serologic test of sufficient specificity for the diagnosis of AIH as for the diagnosis of viral hepatitis A to E. Although the presence of autoantibodies is one of the distinguishing features of AIH, there is no single autoantibody with the diagnostic significance and specificity that antimitochondrial autoantibodies (AMA) demonstrate for the diagnosis of primary biliary cirrhosis (PBC). For this reason, autoantibodies can not be employed as a single marker for the diagnosis of AIH. It is rather a diagnosis reached by the exclusion of other factors leading to chronic hepatitis that include viral, toxic, genetic and metabolic causes . Under tIn recent years however, significant progress has been made in the characterization of liver-related target-autoantigens. This has led to the notion that some of the major target-autoantigens in AIH are active enzymes of the human hepatic and non-hepatic microsomal xenobiotic metabolism -16. The According to the pattern of autoantibodies detected in AIH patients, a subclassification of the disease into three types was proposed in 1994 . AIH typThe serological diversity of autoantibodies found in AIH supports the aforementioned subclassification and provides a framework for the scientific analysis of this heterogeneous disease group ,15. It aHowever, due to recent clinical, serologic and genetic findings, it has been suggested that anti-SLA seropositive patients do not define a subgroup of AIH, but rather belong to the AIH-1 group -32. For ANA and/or SMA are almost exclusively requisites for the diagnosis of AIH-1 ,14,15,30The most frequent and conventional method for ANA detection is the indirect immunofluorescence (IIF) assay on cryostat sections of rodent tissues and HEp-2 cells slides Figures  and 2 1,15,35,36SMA are detected by IIF on rodent liver and kidney, due to staining of vessel walls Fig. , and stoANA and/or SMA – usually in low titers – may occur in patients with chronic viral hepatitis B or C, but in most of these cases SMA lack F-actin specificity ,15,42,43These autoantibodies are directed against cytoplasmic constituents of neutrophil granulocytes and monocytes. Classically, they are detected by IIF using ethanol-fixed granulocytes as substrate . Using tSeveral recent studies however, have also documented the presence of high titers of p-ANCA in the sera of patients with AIH-1  and Western blotting followed by immunodetection can be performed ,63. UsinIn conclusion, the detection of ANCA may be a useful additional marker in searching for AIH-1, in particular in ANA/SMA/anti-LKM-1 negative cases of AIH. With the exception of a recent paper by Wu et al  the deteThe asialoglycoprotein receptor (ASGP-R) is a liver-specific glycoprotein of the cell membrane. Its main function is the internalization of asialoglycoproteins by binding a terminal galactose residue to coated pits. Anti-ASGP-R autoantibodies are detected in 88% of patients with AIH (both types) ,67. HoweThe ASGP-R is preferentially expressed on the surface of periportal liver cells where piecemeal necrosis is found as a marker of severe inflammatory activity in patients with AIH . This fiThe anti-SLA autoantibodies were described for the first time in 1987 . They caA scientific group from Tuebingen, Germany described for the first time the anti-LP autoantibodies in 1981 . The LP Regarding disease specificity, anti-SLA/LP autoantibodies have not been detected in patients with AIH-2, PBC, PSC, chronic viral hepatitis, alcoholic liver disease and non-hepatic autoimmune diseases by standardized ELISAs using reference autoantibody or recombinant antigen ,32,73,79Three types of anti-LKM autoantibodies have been identified ,63,72,85The major target-autoantigen of anti-LKM-1 autoantibodies in AIH-2 has been identified as the cytochrome P450 2D6 (CYP2D6) -89. It hIt is noteworthy to state here that depending on the geographic origin, 0–7% of patients with chronic hepatitis C – irrespective of the HCV genotype – develop anti-LKM-1 autoantibodies ,63,96,97The antigenic sites on CYP2D6 autoantigen recognized by anti-LKM-1 autoantibodies are different in AIH-2 and HCV/anti-LKM-1 positive cases ,101-104.35S-methionine-labelled CYP2D6 antigen obtained by in vitro transcription and translation synthesis [Recently, the development of a more sensitive and specific assay for the detection of anti-LKM-1 autoantibodies was achieved ,15. Thisynthesis ,99,104. ynthesis -83. The ynthesis ,106. Nowynthesis .From the clinical point of view, screening for anti-LKM autoantibodies is recommended before the initiation of interferon-alpha therapy in HCV patients and if found positive a careful monitoring appears reasonable because occasionally interferon-alpha may unmask, or provoke autoimmune hepatic reactions and even "true"AIH ,108-110.The mechanism(s) of the development and the pathogenic role of anti-LKM-1 autoantibodies in hepatocellular injury are still unclear. It has been suggested that viral infections by herpes simplex virus (HSV) and related viruses may trigger the autoantibody formation through molecular mimicry in at least some individuals with AIH-2 . Manns eBesides molecular mimicry, chemical modification of self-proteins and/or immunological cross-reactivity to homologous autoantigens may also provide potential triggers for autoimmune responses. The latter has been suggested by Choudhuri et al  who haveTwo possible mechanisms have been proposed for the involvement of anti-LKM-1 autoantibodies in the pathogenesis of liver injury. The first appears to be a direct binding of these autoantibodies to hepatocytes, leading to lysis of liver cells, while the second is associated with an anti-LKM-1 induction of activating liver-infiltrating T lymphocytes, which indicates the combination of B and T cell activity in the autoimmune process involved -117. A pSo far, anti-LKM type 2 autoantibodies (anti-LKM-2) have been detected only in some cases of drug-induced hepatitis caused by tienilic acid ,63. The Anti-LKM type 3 autoantibodies (anti-LKM-3) alone or in combination with anti-LKM-1 are also detected in about 5–10% of patients with AIH-2 ,119. In In 1988 a second autoantibody marker of AIH-2 was recognized . These fThe detection of anti-LC1 autoantibodies by IIF is obscured due to the anti-LKM-1 pattern that frequently found in most of the anti-LC1 positive sera. For these reasons other techniques such as, the ouchterlony double diffusion, immunoblot or counter-immunoelectrophoresis are required for their detection ,124-126.Anti-LC1 autoantibodies have been proposed as a more specific marker of AIH-2 than anti-LKM-1 autoantibodies, since in the original reports their presence was never associated with HCV infection ,124. HowChronic hepatitis as a disease component of APECED may develop in 10–18% of patients ,28,29,63Similar to AIH-2, hepatitis in APECED is associated with autoantibodies directed against cytochrome P450 proteins. In a large study with APECED patients, a typical LKM staining pattern and a predominant staining of the perivenous hepatocytes in the absence of staining of the kidney were observed . The lat35S-labeled CYP1A2 and CYP2A6.Indeed, screening of APECED sera with recombinant antigens using Western blots has shown reactivity against four different hepatic cytochromes P450: CYP1A1, CYP1A2, CYP2A6 and CYP2B6 -25,133. Contrary to a previous work in Sardinian patients with APECED , the detDalekos et al  using thAnti-LM autoantibodies are first described in dihydralazine-induced hepatitis . The majAnother hepatic autoantigen in APECED, the aromatic-L-amino acid decarboxylase (AADC) has also been identified recently ,138. ThiIn clinical practice the recognition of AIH is of great importance since most of the patients respond favorably to antiinflammatory and immunosuppressive treatment. In addition, recent novel findings dealing with the bone marrow hemopoietic progenitor cells and bone marrow stromal cells of patients with AIH suggests alternative therapeutic options even in refractory cases . DiagnosThe detection of non-organ specific autoantibodies remains the hallmark of AIH. A step by step diagnostic application of autoantibody tests is mandatory for the evaluation of acute or chronic hepatitis of unknown cause. ANA, SMA and anti-LKM-1 autoantibodies should be first tested in patients with acute or chronic elevation of aminotransferases when virologic tests are negative and there is no current or past history for drug or alcohol abuse. Determination of ANCA, which occur in up to 90% of patients with AIH-1, may be useful in the identification of individuals who are seronegative for the above conventional autoantibody markers but should be kept in mind that this autoantibody lacks specificity. Many target autoantigens of the non-organ specific autoantibodies have been identified, but the latter has not led to the characterization of specific subpopulations of patients or changes in the treatment strategies. In addition, most of the non-organ specific autoantibodies do not seem to be involved in the pathogenesis of liver injury in AIH. Anti-LKM-1 autoantibodies could be an exception to the above aspect since recent data have demonstrated the expression of CYP2D6 on the surface of hepatocytes, while AIH-2 has not been observed in individuals who are deficient for CYP2D6. These findings provide arguments for an antigen-driven autoimmune process. It is possible that mutations in the autoantigen itself can lead to alterations in the three dimensional structure of the antigen, which induces autoimmunity.Antibodies directed against liver-related antigens have had similar limitations. Anti-ASGP-R and anti-LC1 autoantibodies appear to correlate with disease severity and response to treatment suggesting a pathogenetic role to the hepatocellular damage. In general however, autoantibodies should not be used as a tool for monitoring of treatment or to predict AIH activity and outcome. Anti-SLA/LP autoantibodies have been considered as valuable and specific markers for the diagnosis of AIH-1. However, a recent study has shown that anti-SLA/LP autoantibodies can also be detected in AIH-2 and in children with PSC. Irrespective of the disease specificity of this marker, it is obvious that testing for anti-SLA/LP will help to reduce the group of cryptogenic liver disease, by recognizing previously misdiagnosed patients with AIH who were seronegative for ANA, SMA or anti-LKM-1.In APECED, autoantibodies are directed against specific cytochrome P450 enzymes (e.g. CYP1A2, CYP2A6, CYP21, CYP17, and CYP11A1), that are expressed in organs affected by the disease process. These observations argue against the idea that antibodies against cytochrome P450 complex are simply epiphenomena secondary to tissue damage and that they have no relation to the etiology and pathogenesis of APECED.It is not known what triggers autoimmunity in AIH. The hypothesis that different causes may lead to loss of tolerance against the same molecular target autoantigen seems attractive. For instance, CYP1A2 is the molecular target in dihydralazine-induced hepatitis and AIH as a component of APECED, CYP2D6 in AIH-2 and in some patients with HCV infection, CYP2A6 in APECED and in a proportion of patients with HCV infection and UGT1 in some cases of AIH-2 and chronic hepatitis D or C.Research protocols in order to define AIH pathogenesis, disease susceptibility, determinants of disease severity, and to understand the epidemiology of AIH are future challenges in the investigational and clinical arena of this disease -141.The author(s) declare that they have no competing interests.


There is a common belief that most cancer prevention trials should be restricted to high-risk subjects in order to increase statistical power. This strategy is appropriate if the ultimate target population is subjects at the same high-risk. However if the target population is the general population, three assumptions may underlie the decision to enroll high-risk subject instead of average-risk subjects from the general population: higher statistical power for the same sample size, lower costs for the same power and type I error, and a correct ratio of benefits to harms. We critically investigate the plausibility of these assumptions.We considered each assumption in the context of a simple example. We investigated statistical power for fixed sample size when the investigators assume that relative risk is invariant over risk group, but when, in reality, risk difference is invariant over risk groups. We investigated possible costs when a trial of high-risk subjects has the same power and type I error as a larger trial of average-risk subjects from the general population. We investigated the ratios of benefit to harms when extrapolating from high-risk to average-risk subjects.a priori assumptions of constant relative risk across risk groups are not robust, limiting extrapolation of estimates of benefit to the general population. Second, a trial of high-risk subjects may cost more than a larger trial of average risk subjects with the same power and type I error because of additional recruitment and diagnostic testing to identify high-risk subjects. Third, the ratio of benefits to harms may be more favorable in high-risk persons than in average-risk persons in the general population, which means that extrapolating this ratio to the general population would be misleading. Thus there is no free lunch when using a trial of high-risk subjects to extrapolate results to the general population.Appearances here are misleading. First, the increase in statistical power with a trial of high-risk subjects rather than the same number of average-risk subjects from the general population assumes that the relative risk is the same for high-risk and average-risk subjects. However, if the absolute risk difference rather than the relative risk were the same, the power can be less with the high-risk subjects. In the analysis of data from a cancer prevention trial, we found that invariance of absolute risk difference over risk groups was nearly as plausible as invariance of relative risk over risk groups. Therefore Unless the intervention is targeted to only high-risk subjects, cancer prevention trials should be implemented in the general population. Some prevention trials are restricted to high-risk subjects. If the investigators are only interested in the effects of the intervention on subjects at increased risk  or if thHowever some investigators who are interested in studying the effect of the intervention in the general population may be tempted to design a "definitive" study to estimate the effect of the intervention in a high-risk group. Some investigators may believe that a trial of high-risk subjects would have greater power than a trial of the same size among average-risk subjects. Some examples of this type of thinking can be found in papers on risk prediction models ,3. Some pC = .02 in the control arm and pI = .01 in the study arm, and they believe that the difference in probabilities is clinically significant. Also suppose that due to the limited availability of the intervention, they can enroll at most n = 2000 study participants in each arm. The investigators compute power using the following standard formula [To crystallize our thinking about statistical power, we consider the following simple hypothetical and realistic example. Investigators want to estimate the effect of intervention in the general population, so they first consider designing a randomized trial among the general at-risk population. Suppose they anticipate that the cumulative probability of incident cancer over the course of the study is n is the sample size per arm, seNull is the standard error under the null hypothesis, and seAlt is the standard error under the alternative hypothesis. Let p = (pC + pI)/2. As discussed in [, sowhere NormalCDF is the cumulative distribution function for a normal distribution with mean 0 and variance 1, Δ is the anticipated difference one wants to detect, , soFor a study designed to estimate the relative risk, the statistic of interest is Applying these formulas to the above example and substituting either (2) or (3) into (1), the investigators obtain a power of .74 based on the absolute risk difference statistic and a power .76 based on a relative risk statistic see .pC = .04 and pI = .02 with n = 2000, the investigators compute a power of .96 using either the absolute risk difference or relative risk. Because the power is higher using high-risk subjects, the investigators plan the study for a high-risk population and will generalize the results to the general population.Suppose the investigators think this power is too low. To increase power they propose to restrict the study to a high-risk group in which the probability of cancer is .04. Also suppose the investigators make the typical assumption that if the intervention yields a relative risk of .5 in the general population, it would also yield a relative risk of .5 in the high-risk group. Applying (1–3) with high risk subjects for whom pC = .04, pI = .03, and n = 2000, the power (computed using either absolute risk difference or relative risk statistics) for the trial of high-risk subjects is only .41. The decreased power in a high risk group under a constant risk difference model is not surprising: if the risk difference pC - pI is the same, but pI is increasing, the variances, pC(1 - pC)/n and pI(1 - pI)/n, will increase as pC increases up to .5, which will reduce the power.Is there a free lunch? An underlying assumption in this example is that the relative risk is invariant between the general population and the high-risk group. There is no free lunch because the impact of violating this assumption could be substantial. For example, suppose instead that the absolute risk difference is invariant between the general population and the high risk group. Under this scenario the absolute risk difference in the general population is .01, so the absolute risk difference in the high-risk group is also .01. In this case for A crucial issue is whether or not the absolute risk difference or the relative risk is likely invariant between average-risk subjects in the general population and high-risk subjects. The answer depends on the cancer, the interventions, and the biology. To gain some appreciation of this issue, we analyzed published data  % confidence intervals and horizontal lines for  with 95% confidence intervals. We also presented the p-values corresponding to twice the difference in log-likelihoods for Varying RD versus Constant RD. Similarly, in the bottom row of Figure  with (100 - 5/k)% confidence intervals and horizontal lines for  with 95% confidence intervals. We also presented the p-value corresponding to twice the difference in log-likelihoods for Varying RR versus Constant RR. Out of 6 p-values (3 risk factors × 2 statistics) only one, for absolute risk difference under the risk factor of predicted risk had a small p-value . Based on these p-values and inspection of Figure Constant RD and Constant RR are both plausible, especially for age and family risk.To investigate the plausibility of the constant relative risk and constant risk difference models in this example, we plotted the estimates of s Figure . In the re  with 00 - 5/k re  with 00 - 5/k Constant RD is the true state of nature, the power will be lower in the high-risk group than the general population. However if Constant RR is the true state of nature, the power will be greater in the high-risk group than the general population. Thus there is high probability that the power could be reduced when studying high-risk subjects than when studying the general population. Therefore, there is no free lunch in terms of lowering statistical power.The trial designer does not know the true state of nature. If pC and pI are correctly chosen), the smaller trial of high-risk subjects may be more expensive than the larger trial of average-risk subjects from the general population. Consider the following two trials with a power of .90 and a one-sided type I error of .05. In the trial of high-risk subjects pC = .04 and pI = .02, and in the trial of average-risk subjects, pC = .02 and pI = .01. Suppose the statistic of interest is the absolute risk difference. To obtain sample size for each randomization group we use the standard sample size formula [Even if the model is correct (namely p = (pC + pI)/2, 1.644485 is the z-statistics corresponding to the 95th percentile of the normal distribution (for a one-sided type I error of .05) and 1.28155 is the z-statistics corresponding to the 90th percentile (for a power of .90). Based on (4), the sample size for a trial using average-risk subjects from the general population study is 2529 per group and the sample size for a trial of high-risk subjects is 1244 per group. Let CR denote the cost of recruitment per subject and CI denote the cost of intervention and follow-up per subject averaged over the two randomization groups. Suppose high risk subjects comprise a fraction f of the general population. The total cost of the trial for average-risk subjects from the general populations iswhere Cgeneral = 2(CR 2529 + CI 2529),    (5)and the total cost of the trial for high-risk subjects isChigh-risk = 2(CR 1244/f + CI 1244).    (6)Chigh-risk >Cgeneral) iswhere the factor of 2 is for the two randomization groups. The condition for the trial of high-risk subjects to cost more than the trial of average-risk subjects , which would then affect (5) and (6). In some settings, investigators may anticipate that high-risk subjects are more likely to comply with the intervention than average-risk subjects. To compensate for the anticipated increased compliance, study designers could reduce the sample size which would lower costs. However, in other situations, investigators may anticipate that subjects found to be at high-risk on a diagnostic test would likely seek the best therapy outside of the trial rather than chance randomization to standard or experimental therapy. To compensate for the anticipated dilution in treatment effect, investigators would need to increase the sample size which would increase the costs.One additional consideration is how noncompliance and contamination affect the intent-to-treat analysis. If noncompliance and contamination can be anticipated, the investigator can correspondingly adjust the sample size and costs. Mathematically the effect of noncompliance and contamination is to change the values of For the above reasons even if the probabilities under the alternative hypothesis are correctly specified, some trials of high-risk subjects may be more expensive than larger trials of average-risk subjects with the same power and type I error.When there is strong evidence prior to the trial of a high probability of harmful side effects due to the intervention, one would want to restrict the intervention to high-risk subjects. Otherwise, some investigators may be tempted to estimate the ratio of benefit to harms in the trial of high-risk subjects and extrapolate the ratio to average risk subjects. Unfortunately, even if the assumption of constant relative risk over risk categories were true, extrapolating the benefit-harm ratio from a high risk group to the general population could be misleading.Suppose that in a randomized trial involving average-risk subjects from the general population the probability of cancer is .02 in the control arm and .01 in the study arm. Also suppose that relative risk is same in the general population as in the high-risk group, so that in a randomized trial involving a high-risk group, the probability of cancer is .04 in the control arm and .02 in the study arm. Furthermore, suppose that the probability of harmful side effects is the same for high-risk subjects as for average-risk subjects in the general population, namely .015 in the control arm and .025 in the study arm. Based on these results, for every 1000 high-risk persons who receive the intervention, (.04 - .02) 1000 = 20 will benefit from the intervention and (.025 - .015) 1000 = 10 will be harmed by side effects, yielding a benefit-harm ratio of 20:10 = 2:1. Similarly for every 1000 average-risk person who receive the intervention, (.02 - .01) 1000 = 10 will benefit from the intervention and (.025 - .015) 1000 = 10 will be harmed by side effects yielding a benefit-harm ratio of 10:10 = 1:1. In this example it would be incorrect to extrapolate the high benefit-harm ratio estimated from the high-risk group to the general population for whom the benefit-harm ratio is much lower. For many cancer prevention interventions, the ratio of life-threatening disease avoided to life threatening harms would be favorable in the high-risk group but not favorable when extrapolated to the general population.There is no "free lunch" when using high-risk subjects in prevention trials design to make inference about the general population. Using high risk subjects instead of average-risk subjects from the general population may lower statistical power, increase costs, and yield a misleading ratio of benefit to harms than actually the case.Given the substantial costs of definitive randomized trials in cancer prevention, and the importance of accurately assessing the balance of benefit and harm when treating healthy and asymptomatic people, it is therefore important to conduct trials in the actual target population rather than try to conduct them in high-risk populations with the plan to extrapolate results to the general population.The authors declare that they have no competing interests.SGB wrote the initial draft, and BSK and DC made valuable improvements. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Appendix A, worked-out calculations of power.Click here for fileAppendix B, likelihood formulationsClick here for file