Irritable bowel syndrome is a common condition in general practice. It occurs in 10 to 20% of the population, but less than half seek medical assistance with the complaint.A questionnaire was sent to the 406 GPs listed on the West Sussex Health Authority Medical List to investigate their views of this condition and whether they felt hypnotherapy had a place in its management38% of general practitioners responded. The achieved sample shared the characteristics of target sample.Nearly half thought that irritable bowel syndrome (IBS) was a "nervous complaint" and used a combination of "the placebo effect of personal care," therapeutic, and dietary advice. There is considerable divergence in the perceived effectiveness of current approaches. Over 70% thought that hypnotherapy may have a role in the management of patients with IBS; though the majority (68%) felt that this should not be offered by general practitioners. 84% felt that this should be offered by qualified hypnotherapist, with 40% feeling that this should be offered outside the health service.General practitioners vary in their perceptions of what constitutes effective therapy in IBS. They are willing to consider referral to a qualified hypnotherapist. This study explores general practitioners' beliefs about irritable bowel syndrome (IBS), and whether they see hypnotherapy as an appropriate complementary therapy for its management; and if so, who should deliver it.IBS is estimated to occur in 10–20% of the population in most countries [Over a period of time of between 2 and 5 years, it is thought that there is a 30% turnover of patients having IBS [IBS is typical of many conditions seen in general practice. There is a risk that some of its symptoms may actually represent serious underlying illness and there are a number of conventional medical interventions, but for many patients the interest of their practitioner is paramount . The maiUK general practitioners are having a greater say in the management of the local health services through the organisation of local health services into Primary Care Trusts (PCT). The latter are responsible for commissioning the health services for their resident population. Via these, general practitioners have the potential to influence the development of services for the treatment of this condition. Most PCTs are financially constrained and cannot meet all the needs of every patient. Services have to be prioritised according to need. The study investigated general practitioners' opinions as to whether hypnotherapy should be provided by Primary Care Trusts, or provided privately.The possibility of hypnotherapy being used as a primary care level intervention for IBS has developed from the realisation in the 1980's that hypnotherapy was an effective treatment for intractable IBS in a hospital context ,8. TheseThere is a lack of literature about general practitioners' opinions and few validated questionnaires. A literature search identified two recent studies about IBS in primary care ,10 whichThere are few studies describing the experiences and attitudes of GPs towards IBS management in primary care, especially pertaining to the use of alternative or psychological treatments. Although it is very likely that treatments such as hypnotherapy and cognitive behaviour therapy would have a prolonged impact on IBS if they were commonly employed at an early stage in the illness, little is known about the attitudes of primary care teams in encouraging this sort of approach.We therefore conducted this study to discover how general practitioners perceive IBS and if they consider it to be managed effectively by current conventional therapy. We wanted to know if GPs consider hypnotherapy to be an appropriate intervention and whether GPs would refer there patients for it. Finally, we wanted to know whether such a service would be provided within general practice or another setting and if it should be funded by the NHS or be provided from the private sector.A survey was conducted of all general practitioners in West Sussex. The setting for the study is a mixed area. There are market towns and no large cities. Much of the population lives in sub-urban and rural areas. The area has a lower than average deprivation level than the regional norm, but in common with most of the south east of England, income is higher than the national average. As it is a rural area a larger number of practices dispense their own medicines, rather than send patients with a prescription to a local pharmacy .A questionnaire was designed and pre-tested with a pilot group for readability, validity, reliability, acceptability of layout and time taken to complete . The queThe sample was the 406 general practitioners listed as unrestricted principals on the Health Authority's list in 1997. This list included both full and part time general practitioners. The questionnaires were mailed to GPs using the Heath Authority's internal mail system and were returned via the same Health Authority post. At that time, the use of this service was free to the investigator. Once the first group of questionnaires had been returned, the questionnaire was repeated once to those practices that had not responded.The responses were mostly in the form of a Likert scale. This scale enables measurement of degrees of opinion, so increasing the sensitivity of the analysis. A central category was provided for a neutral response. The design purposefully did not force a polarised choice because it was thought that the treatment would be unlikely to be in common use. It was possible that many general practitioners would genuinely not have an opinion about some of the questions. To prevent acquiescence bias, the questions were worded so that expected responses varied unpredictably according to the direction of the scale . DemograQuestionnaire themes included GP perception of IBS, its management, and hypnotherapy as a treatment. It also included questions related to the funding of treatment and the acceptability of hypnotherapy as part of the management of IBS in primary care. The questions were grouped together using these themes. Piloting enabled assessment of face validity and content validity . The conWe analysed the data using SPSS . The target and achieved samples were compared using the Chi-square test of proportion . Where t155 (38%) general practitioners of the target sample returned questionnaires after the initial request and one further follow-up reminder.The characteristics of general practitioners in achieved sample were compared with that of the target sample to assess sample bias. No statistically significant differences were found between the achieved and target samples other than fewer GPs from non-training practices and from single-handed practices responded to the survey. With the exception of these factors, the characteristics of the achieved sample are not very different from that of the population of GPs in West Sussex. A comparison of the achieved and target sample are shown in Table 45.2% of the respondents agreed with the statement that "IBS is mainly a nervous complaint", and 40% felt that IBS "responds mainly to the placebo effect of personal care and attention". Although many respondents seem to categorise IBS as a 'nervous complaint', conventional medical management with drugs and dietary advice is seen to have a role to play with 45.2% agreeing with the statement that drug therapy works effectively, and 38.7% dietary advice works effectively. A striking finding is that a sizeable minority of GPs were unsure if IBS patients respond to placebo effect (at 37.4%) or to medical therapy (at 41.3%). Combining the 'unsure' and the 'disagree' categories show that the majority of GPs in this survey are uncertain if existing treatment regimes for IBS  are efficacious. These results are set out in Table Just under half of the respondents (45.2%) agreed that IBS requires more attention in primary care and less than a quarter (22.6%) disagreed with the statement that IBS required more attention. The majority (56.8%) of the respondents felt that it would be possible to manage IBS better in their practices. Only 12.9% disagreed with this statement. The vast majority (84.5%) felt that the present management of IBS is variable with less than 10% of practitioners believing that their care is effective. These results are summarised in Table Three quarters of general practitioners (75.5%) saw hypnotherapy as an alternative therapy. Notwithstanding this, a large majority of general practitioners in this survey agreed that hypnotherapy could help patients who suffer from both physical and psychological problems, 72.9% and 77.4% respectively. However just over a third of general practitioners (34.8%) saw hypnotherapy as potentially dangerous, with just under half feeling unsure (40.6%).Nearly 84% felt that hypnotherapy was the province of accredited therapists, and only 20% felt that they would be willing to receive training to provide hypnotherapy themselves. The statement that hypnotherapy should be available through an accredited hypnotherapist (83.9% agreed) and not to take this on as a general practitioner (68.4% with 10.3 unsure) were two of the most polarised responses.If a course of 8 × 30 minutes hypnotherapy were shown to be effective, there would be a willingness amongst most respondents (78.1%) to advise hypnotherapy for some, but definitely not all, IBS patients. 83% of those questioned felt that it was not something that should be offered to every patient with IBS. These results are set out in Table There is considerable uncertainty and divergence of opinions amongst the respondents on how hypnotherapy should be funded. Only 36.1% of general practitioners thought that more NHS resources should be used to give IBS sufferers better treatment, yet a slightly larger percentage (44.5%) thought that the money could be better spent elsewhere. The proportion of those answered 'unsure' in response to the above 2 statements are at large at 45.8% and 38.7% respectively.Whilst just under half (49%) would support their primary care organisation investing in hypnotherapy if it were shown to work, there was less support for providing it within the NHS, at 29%. Most general practitioners 56.8%) thought that hypnotherapy should be provided through private hospitals, with a substantial minority (40%) thinking that insurance companies should pay for it. Details of these responses are shown in Table 6.8% thouFinally despite the divergence and uncertainty on hypnotherapy for IBS sufferers, over three quarters of respondents, 76.3% (table not shown), said that they would be willing to refer patients into a study of hypnotherapy for the treatment of IBS in primary care if it were conducted.The principal findings of the study are that many general practitioners see IBS as a "nervous condition" to be treated with care and attention, as well as drugs and dietary change. There is considerable divergence in the perception of the effectiveness of current approaches, and a willingness amongst many GPs to refer to qualified hypnotherapists if it can be shown to be effective, even though this treatment is considered "alternative" and potentially dangerous. It is also acknowledged that this could not be made a priority for the NHS, but should be provided privately outside it.Unfortunately there were not sufficient resources available to perform telephone reminders, and this may have in part accounted for the low response rate. Comparison of the achieved sample with the target sample shows that fewer single-handed practices and more training practices took part in the survey, which may reflect a greater willingness of academically oriented practitioners to participate in research. It is beyond the scope of the present study to estimate how representative the sample is of other areas, especially inner cities and other more deprived areas where private hospital and insurance are simply not an option for most patients.Existing literature suggests that around 40% of patients in the UK have access to complementary therapy . This stOther studies show that doctors hold different 'public' and 'private' attitudes to IBS as an illness, and may classify patients informally into 'good' and 'bad' patients . This stAnother study concluded that 'Drug usage in the IBS is more than is justified and should, in our view, be minimised . The preMore research is needed into the spectrum of views of general practitioners about IBS. A qualitative study might have provided more information about general practitioner beliefs about IBS and what they feel should be done to improve services. Additional quantitative work might identify whether there are age-sex differences in practitioners attitudes.The general practitioners were clearly open to hypnotherapy to help manage IBS. It is unclear whether this was a specific attitude towards hypnotherapy, or a general willingness to promote any psychological or supportive therapy for this illness.It would also be interesting to investigate the opinions of gastroenterologists or psychosomatic therapists.The vast majority of general practitioners think that current management options for IBS are variable in their effectiveness. Many agreed that this condition needs improved treatment options in primary care; and general practitioners seem willing to consider hypnotherapy as one such treatment option if it can be shown to be effective.The authors declare that they have no competing interests.SC conceived the study and developed the questionnaire, TC and S de L analysed the data, S de L with contributions from the other authors wrote the paper.The pre-publication history for this paper can be accessed here:Appendix 1: The questionnaire Questionnaire used in the studyClick here for file


Like NO, CO has been accepted as an important cellular signaling molecule in biological systems. An up-regulation in both gene and protein expression of heme oxygenase-1 (HO-1) under oxidative/nitrosative stress has been well documented, and the protective role of HO-1 and HO-2 against oxidative damage is proposed. However, data on the direct effect of reactive oxygen/nitrogen species (ROS/RNS) on HO function is incomplete. Using gas chromatography to quantify carbon monoxide (CO) formation from heme oxidation, we investigated the effects of peroxynitrite  and diethylamine NONOate (DEA-NONOate). However, no additive effect was found following the application of NO donors and ONOO- together.Exposure to ONOO- may regulate HO-1 and HO-2 activities by mechanisms that involve different interactions with these proteins. It is suggested that while nitration of tyrosine residues and oxidation of sulfhydryl groups may be involved, consideration should be given to other facets of ONOO- chemistry. This inhibition of HO activity offers a mechanism for cross talk between the nitric oxide synthase and HO systems.These results indicate that ONOO Heme oxygenases HO, EC 1.14.99.3) are a highly conserved family of proteins that catalyse the oxidative cleavage of heme at the α-meso carbon to yield equimolar amounts of iron, carbon monoxide (CO) and biliverdin. Biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Three distinct isozymes of HO  have been identified. HO-1 (the inducible isoform) is predominantly expressed in the spleen, the primary site of heme catabolism but has been detected in many different tissues including the liver and the kidney. Several substances and conditions may induce the expression of HO-1. The involvement of HO and products of heme catabolism have been studied extensively with respect to oxidative stress, ischemia, hypoxia and protection against transplant rejection .14.99.3 -7. HO-3 in vivo. For example, an induction in the expression of HO-1 prevents superoxide associated endothelial cell sloughing in diabetic rats [-, a potent oxidizing agent generated by the interaction of NO and superoxide radical [- regulates the expression of HO-1 and that the heme oxygenase pathway contributes to protection against the cytotoxic effects of ONOO- which are due to its reactivity with cellular macromolecules such as the covalent modification of tyrosine, cysteine, methionine or tryptophan residues, oxidation of nucleic bases or the scavenging of cellular antioxidants such ascorbate and urate [--mediated HO-1 induction might occur via an interactive signalling mechanism that modulates oxidative stress responses but direct effects of ONOO- on HO activity have not been studied. In this work, we examined the effect of ONOO- on HO catalytic activity in rat spleen and brain microsomes respectively.Increasing amounts of evidence suggest that products of heme catabolism have cytoprotective roles such as anti-inflammation, anti-apoptosis and anti-proliferation. Biliverdin and bilirubin have anti-oxidant and anti-inflammatory properties -11, whiltic rats . Similar radical , causes  radical -24, as w radical . These snd urate . It is p- resulted in decreases of HO activity in a concentration dependent manner   Figure . The IC5- may affect protein targets by the oxidation of sulfhydryl groups as well as the nitration of tyrosine residues, we examined and compared the effect of other sulfhydryl modifying reagents on HO activity. Rat spleen or brain microsomal protein was treated with different sulfhydryl modifying reagents such as GSNO, DEA-NONOate, NEM and H2O2, and a combination of equimolar concentrations of ONOO- and GSNO or ONOO- and DEA-NONOate. We found that GSNO caused a concentration dependent inactivation of both HO-1 and HO-2. Consistent with the effect of ONOO-, GSNO was more active against HO-1 than HO-2  Table - and DEA-NONOate.To test whether effects of ONOO- in vivo is a consequence of oxidative and nitrosative stress, and ONOO--mediated tissue injury is thought to be involved in the pathogenesis of many conditions including atherosclerosis, ischaemia/reperfusion, shock, Alzheimer's disease, diabetes and multiple sclerosis [- causes a concentration-dependent increase in HO-1 protein expression suggesting that the HO pathway contributes to protection against the cytotoxic effects of ONOO- [- and the inhibitory effect of ONOO- on numerous enzyme systems, we sought to investigate the direct effect of ONOO- on the catalytic activity of two microsomal HO isozymes.Production of ONOOclerosis -30. Contclerosis ,9-11. ONof ONOO- -24. Most- inhibits the activity of both HO-1 and HO-2 in a concentration-dependent manner  decreased HO-1 activity by 50% while a much higher concentration (1.25 mM) was required to decrease HO-2 activity by the same magnitude. Generally, HO catalytic function is dependent on an accessory enzyme NADPH-cytochrome P450 reductase (CPR), which serves as a redox partner during the oxidative break down of heme and the conversion of NADPH to NADP+. It is possible therefore, that ONOO- may have altered HO activity indirectly by inactivating CPR. The ONOO- dependent inactivation of recombinant CPR in bacterial membranes has been noted [- on microsomal HO-1 and HO-2 (data not shown). This suggests that the effects of ONOO- on HO activity may be mediated by mechanisms that are independent of the effect of ONOO- on microsomal CPR activity. The differential effect of ONOO- on the catalytic activity of HO-1 and HO-2 may suggest a selective mechanism on the catalytic function of the inducible enzyme. This may have direct implications in biological systems where ONOO- is generated at high concentrations. The ONOO--mediated increase in HO-1 gene and/or protein expression may be a cellular response at the acute phase. Subsequently, total HO activity may then be maintained in certain ranges in order to retain intracellular oxidative responses and the balance of redox states for normal cellular function. This idea is consistent with other studies, which have shown that induction of HO-1 mRNA and protein expression is not always followed by a proportionate increase in catalytic function. For example, by using quantitative RT-PCR it was found that a 3.8-fold increase in sarcoma-induced HO-1 mRNA expression yields only 2.1-fold increase in total HO activity [--mediated regulation of the catalytic function of HO-1 and differences in the sensitivity of HO-1 and HO-2 may be attributed to disparity in the amino acid compositions and the structure of the two proteins.Rat spleen and brain microsomal fractions were used because of the predominant expression of HO-1 in the spleen and HO-2 in the brain. We have shown that ONOOen noted , but ouractivity . Overall- on protein function are mainly due to its reactivity and covalent modification of tyrosine and/or cysteine residues [--mediated inhibition of HO activity observed here, we examined the effects of TNM, another tyrosine-nitrating chemical compound on rat spleen and brain microsomal HO activity. Significant inhibition of HO catalytic activity by TNM was observed for spleen as well as brain microsomal fractions albeit at higher concentrations compared to ONOO-  that is/are important for conservation of HO-1 and HO-2 function. This is consistent with data from the analysis of the complete amino acid sequences of rat and human HO-1 [- could be due to modification of amino acid residues other than tyrosine. Oxidation of cysteine residues as a putative mechanism underlying ONOO--mediated inactivation of HO was investigated. Effects of GSNO, DEA-NONOate, NEM and H2O2, or a combination of equimolar concentrations of ONOO- and GSNO or DEA-NONOate on HO activity were tested. GSNO showed similar inhibitory effects to ONOO- but a combination of equimolar concentrations of ONOO- and DEA-NONOate or GSNO did not have any additive effect on the ONOO--mediated inactivation of HO-1 or HO-2. In addition, the inactivation of HO-1 by ONOO- and DEA-NONOate and HO-2 by ONOO- were reversible following prolonged pre-incubation time (60–120 minutes) Table -, GSNO, DEA-NONOate or NEM is suggestive of qualitative as well as quantitative differences in the distribution of target amino acids such as critical tyrosine and/or cysteine residues in the tertiary structures of these proteins. The amino acid sequences of human and rat HO-2 reveals a conserved core of cysteine residues  [- might exert its effects by interaction with the substrate as has been reported for the interaction of NO with heme [- is richer than is usually discussed; in their review Alvarez and Radi [- with a number of functional groups that are not often mentioned in the consideration of the interactions of ONOO- with various proteins. Thus, ONOO- inhibition of HO activity could be a result of an interaction with amino acids other than cysteine or tyrosine. This possibility of ONOO- interactions with other amino acids may also explain the reversibility of the inhibition of HO activity by ONOO-. Furthermore, it is possible that ONOO- inhibition of HO-1 and HO-2 occurs courtesy of different facets of ONOO- chemistry. Differences in the relative potency of DEA-NONOate, NEM, GSNO and ONOO- may also be attributed to differences in the chemical properties and the electrophilic potentials of these reagents. For instance, the nitric oxide radical (NO•) generated from DEA-NONOate is less reactive and unstable compared to NO+ produced from GSNO.Mechanistically, the effects of ONOOresidues . This ofresidues . To prob- Figure . This suman HO-1 ,35 and Hman HO-1 ,37. HowePro 282) ,39, but ith heme . This poand Radi  describe- may be important in the cross-talk between HO and NO systems. While tyrosine residues for HO-1 and/or sulfhydryl groups for HO-2 should be considered as potential targets for ONOO- interactions, other amino acids should be studied in the elucidation of mechanism(s) of ONOO- action on HO isozymes.Collectively, our data indicate that ONOO--mediated inactivation of HO-1 and HO-2. The IC50 for HO-1 was approximately 80-fold less than that for HO-2. While conserved tyrosine residues (HO-1) and/or sulfhydryl group(s) (HO-2) may play a critical role in maintaining the functional capacity of heme oxygenases, the consideration of other amino acids is suggested. In addition, the higher sensitivity of HO-1 to ONOO--mediated inactivation may indicate dual regulatory mechanisms on the catalytic function of the inducible enzyme under the conditions of oxidative/nitrosative stress.This study documented for the first time the ONOOHemin, ethanolamine, bovine serum albumin (BSA), β-NADPH, N-ethylmaleimide (NEM), tetra-nitromethane (TNM) and reduced glutathione (GSH) were purchased from Sigma Chemical Co. (St. Louis Mo.). Diethylamine NONOate (DEA-NONOate) was obtained from Calbiochem Inc. . All other chemicals were reagent grade from a variety of commercial sources.Adult male Sprague-Dawley rats (250–300 g) were obtained from Charles River Canada Inc. . The animals were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care. Microsomal fractions were prepared according to previously described procedures ,43. Sple- was synthesised from acidified nitrite and H2O2 according to the method of Beckman [- following the procedures described by Ji and Bennett [- at room temperature at indicated concentrations for 10 seconds by adding a small volume during vigorous mixing. HO activity was initiated by diluting the reaction mixture to a protein concentration of (0.5–1 mg/ml) in 100 mM phosphate buffer containing substrates . To control for the potential effect of nitrite and nitrate that may be formed during the incubation of ONOO-, ONOO- was allowed to decompose in phosphate buffer prior to incubation with microsomal protein and determination of enzyme activity for some of the reactions.ONOO Beckman  and micr Bennett . Brieflyet al., [2O2 in 100 mM potassium phosphate buffer, pH 7.4, at 37°C for 20 minutes. The reaction was stopped by dilution of the reaction mixture to a protein concentration of (0.5–1 mg/mL) in 100 mM phosphate buffer containing .GSNO was prepared by reacting equimolar amounts of sodium nitrite and GSH according to the method described by Ji et al., . Microso-1 protein. min-1) was linear with respect time and microsomal protein concentration. CO formation was monitored by gas chromatography according to the method described by Vreman and Stevenson [HO activity in rat spleen and brain microsomal fractions was determined by the quantitation of CO formed from the oxygen, CPR and NADPH-dependent degradation of methemalbumin as previously described ,46. The Data are presented as the mean ± SD from triplicate experiments and statistical analyses were performed by one-way ANOVA. P values ≤ 0.05 was considered to be significant.-, peroxynitrite; GSNO, S-nitrosoglutathione; DEA-NONOate, diethylamine NONOate; NEM, N-ethylmaleimide; TNM, tetra-nitromethane; ROS/RNS, reactive oxygen/nitrogen species; RT-PCR; reverse transcriptase polymerase chain reaction;  H2O2, hydrogen peroxide.HO, heme oxygenases; CPR, NADPH-cytochrome P450 reductase; CO, carbon monoxide; ONOORK, YJ and KN were involved in the experimental design and data collection. All authors read and approved the final manuscript.


The Mental Health First Aid training course was favorably evaluated in an uncontrolled trial in 2002 showing improvements in participants' mental health literacy, including knowledge, stigmatizing attitudes, confidence and help provided to others. This article reports the first randomized controlled trial of this course.Data are reported on 301 participants randomized to either participate immediately in a course or to be wait-listed for 5 months before undertaking the training. The participants were employees in two large government departments in Canberra, Australia, where the courses were conducted during participants' work time. Data were analyzed according to an intention-to-treat approach.The trial found a number of benefits from this training course, including greater confidence in providing help to others, greater likelihood of advising people to seek professional help, improved concordance with health professionals about treatments, and decreased stigmatizing attitudes. An additional unexpected but exciting finding was an improvement in the mental health of the participants themselves.The Mental Health First Aid training has shown itself to be not only an effective way to improve participants' mental health literacy but also to improve their own mental health. It is a course that has high applicability across the community. In 2000 we developed a Mental Health First Aid course in response to the findings of two large national mental health surveys in Australia ,2. TheseThe Mental Health First Aid course consists of three weekly sessions of three hours each. The content covers helping people in mental health crises and/or in the early stages of mental health problems. The crisis situations covered included suicidal thoughts and behavior, acute stress reaction, panic attacks and acute psychotic behavior. The mental health problems discussed included depressive, anxiety and psychotic disorders. The co-morbidity with substance use disorders is also covered. Participants learn the symptoms of these disorders, possible risk factors, where and how to get help and evidence-based effective help.The initial evaluation trial of the Mental Health First Aid course was an uncontrolled one with 210 members of the public with pre, post and 6-month follow-up. This trial showed that participants improved: their recognition of mental disorders, their beliefs about what treatments were helpful, attitudes towards people with mental illness, the amount of help provided to people with mental health problems, and their confidence in providing help to these people .The next step in our evaluation of this course was to conduct a randomised trial involving a wait-list control group. The present article reports this study, which was carried out in a workplace setting.Eligible participants (approximately 4800) were all Canberra-based employees of two Australian government departments: Health and Ageing, and Family and Community Services. The trial was advertised to staff by email. Participants had to agree to be randomly assigned to receive the training in either Month 1 or Month 6. Training was delivered and data collected at the worksite during office hours.The course content has been described in the Background and previously  and furtThe main objective was to assess whether Mental Health First Aid training improved mental health literacy and helping skills relative to a wait-list control. A secondary objective was to assess any benefits to the participants' own mental health.Outcomes were measured in the month before intervention (the pre-test assessment) and in the fifth month after intervention (the follow-up assessment). The intervention group received training in Month 1 (immediately after pre-test) and the wait-list control group received training in Month 6 (immediately after the follow-up).All outcomes were measured by self-completed questionnaires based on the ones used in the uncontrolled trial of Mental Health First Aid . The preTo score the items on beliefs about treatment, a scale was created showing the extent to which participants agreed with health professionals about which interventions would be useful. For depression, there is a professional consensus that GPs, psychiatrists, clinical psychologists, antidepressants, counseling and cognitive-behavior therapy are helpful . Thus, pThe questionnaire ended with the SF-12, which provided scales assessing the participant's mental and physical health . These sThe follow-up questionnaire was the same as the pre-test questionnaire except that it omitted the sociodemographic questions and asked about contact with anyone with a mental health problem over the 5 months since the last questionnaire (rather than 6 months).The questionnaires were sent out via internal departmental mail by a human resources staff member in each place of employment. The questionnaires were completed anonymously with only an ID number and posted back to the researchers at the Centre for Mental Health Research. The IDs of any non-responders were sent back to the human resources staff member who sent out a reminder. The researchers were never told the names of individual respondents and the human resources staff member in the place of employment never saw any completed questionnaires or individually identifiable data.The study was planned to have a sample of 300. The sample size was determined by practical constraints: when it was convenient to run classes that fitted the employees' work schedule and the workload on the instructor. It was determined that this sample size had excellent power to detect medium effect sizes for both continuous and dichotomous outcomes . The tri website [A staff member in the human resources section of the place of employment kept a list of participants' names and ID numbers. The researchers only had access to the IDs. One of the researchers (Jorm) randomly assigned participants to training or control groups by ID number using the Random Integers option at the  website . After rEthical approval for the study was given by the Australian National University Human Research Ethics Committee.Repeated measures analysis of variance was used to analyze continuous measures, with two groups (intervention and control) and two time points (pre-test and follow-up). The principal interest was in the group × time interaction effect. Logistic regression was used to analyze change in dichotomous measures, with group and pre-test score as the predictors and follow-up score as the outcome. Place of employment was also investigated to see if there was a difference in the effects of training. However, no interaction effects involving place of employment were found, so this variable was dropped from all analyses reported below.The analysis was carried out according to intention-to-treat principles, so that all persons who completed a pre-test questionnaire were included, even if they subsequently dropped out. In such cases, the pre-test score was substituted for the missing value, so that no improvement was assumed.An email inviting participation was sent to all staff of the relevant departments based in Canberra. The email was sent out in May 2002 for the Department of Health and Ageing and March 2003 for the Department of Family and Community Services. In order to participate, staff had to send back a consent form and fill out a pre-test questionnaire before the start of classes.Figure In terms of sociodemographic characteristics, 78.1% of the participants were female, 49.2% were aged 18–39 years, 50.2% were aged 40–59 and 0.7% aged 60+ years. There were 60.6% with a university degree, 1.3% were aboriginal and 8.6% did not have English as their first language. 13.0% described themselves as mental health consumers, 9.6% as carers for a person with a mental health problem, and 6.3% as health service providers. When asked their reason for doing the course, 27.2% cited reasons relating to their workplace, 11.7% reasons relating to family or close friends, 4.9% reasons relating to their own mental health status, 20.5% cited duty as a citizen, 29% said they were just interested, and 6.7% wanted more accurate or updated information on mental health. 165 (54.8%) of the participants worked at the Department of Health and Ageing and 136 (45.2%) at the Department of Family and Community Services.The data were analyzed according to intention-to-treat principles, so that all persons who completed a pre-test questionnaire were included, even if they subsequently dropped out. For every analysis, there were 146 participants analyzed in the intervention group and 155 in the control group.Participants were asked about whether they themselves had ever experienced a mental health problem or whether anyone in their family had. Table Table Table Table Table Table Given that an educational intervention was evaluated with a non-clinical sample, there was no justification for a systematic inquiry into adverse events. Informally, no adverse events were reported.This trial has found a number of benefits from Mental Health First Aid training. Relative to the control group, the intervention group showed greater confidence in providing help to others, greater likelihood of advising people to seek professional help, improved concordance with health professionals in beliefs about treatment, decreased social distance from people suffering from depression, and improved mental health of the participants themselves. Recognition of disorders in vignettes did not improve, but there was a very high recognition at pre-test, limiting the scope for improvement.A potential criticism of Mental Health First Aid training is that it will lead to excessive labeling of life problems as mental disorders by members of the public. To check this possibility we asked participants about mental health problems in themselves and family members. Although a high prevalence rate was reported, we found that the course had no effect on these rates.A surprising effect was that the course improved the participants' scores on the SF-12 mental health scale. We included this scale to explore whether there was any impact on mental health, but did not have any strong expectation that it would. The course is not aimed at the participants' own mental health and does not include any therapy. Furthermore, only 5% of participants cited their own mental health as a reason for doing the course. Nevertheless, the participants' mean score on the mental health scale was around half a standard deviation below Australian population norms , showingThe data analysis involved a conservative intention-to-treat strategy in which participants who failed to complete the whole course were included and those who failed to respond to the follow-up questionnaire were assumed to show no change. A particular limitation in the present study is that participants in the intervention group showed a poorer response to the follow-up questionnaire than controls. The reason for this poorer response is unknown, but we believe it occurred because the intervention group had already received the course and had nothing to gain by filling out a further questionnaire. By contrast, the controls were still waiting to receive their training and may have believed that filling out the questionnaire would assist this. Whatever the reason, the poorer response in the intervention group meant that more of them were assumed to show no change, thus minimizing any benefits of the training. It is likely that the true effects of Mental Health First Aid training are greater than the present data indicate.The present trial evaluates efficacy rather than effectiveness. The trial was carried out in a workplace setting with well-educated employees who were allowed to do the course during working hours. There was only one instructor, who was the developer of the Mental Health First Aid course, limiting the generalizability of the findings to other instructors. Further research is needed to evaluate the course as taught by other instructors in more typical settings. We are currently engaged in an effectiveness trial with members of the public in a large rural area, with local health service staff trained to run the courses.The Mental Health First Aid training evaluated in this trial was 9 hours long. Based on feedback from participants that the course needed to be longer, we now routinely run the course over 12 hours. This longer course expands on each of the topics covered, especially substance use disorders. Whether this longer course has additional benefits remains to be evaluated. However, our expectation is that it would produce greater effects on beliefs about treatment, confidence in providing help and actual help to provided to others.Mental Health First Aid training appears to be effective in improving some aspects of mental health literacy, confidence in providing help to others, and the type of help provided. The training also benefits the mental health of participants. The course is highly acceptable in a workplace setting and could be widely applied. Over 100 Mental Health First Aid instructors have now been trained and the course is available throughout much of Australia and in Scotland, Hong Kong and New York State, USA. Dissemination in other localities is planned in the near future.The authors were the developers of the Mental Health First Aid course.BAK co-designed the study and the evaluation questionnaire, taught the Mental Health First Aid courses, and co-wrote the manuscript.AFJ co-designed the study and the evaluation questionnaire, analyzed the data, and co-wrote the manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Pre-test questionnaire. questions used in pre-test questionnaireClick here for file


Precise classification of cancer types is critically important for early cancer diagnosis and treatment. Numerous efforts have been made to use gene expression profiles to improve precision of tumor classification. However, reliable cancer-related signals are generally lacking.Using recent datasets on colon and prostate cancer, a data transformation procedure from single gene expression to pair-wise gene expression ratio is proposed. Making use of the internal consistency of each expression profiling dataset this transformation improves the signal to noise ratio of the dataset and uncovers new relevant cancer-related signals (features). The efficiency in using the transformed dataset to perform normal/tumor classification was investigated using feature partitioning with informative features (gene annotation) as discriminating axes (single gene expression or pair-wise gene expression ratio). Classification results were compared to the original datasets for up to 10-feature model classifiers.82 and 262 genes that have high correlation to tissue phenotype were selected from the colon and prostate datasets respectively. Remarkably, data transformation of the highly noisy expression data successfully led to lower the coefficient of variation (CV) for the within-class samples as well as improved the correlation with tissue phenotypes. The transformed dataset exhibited lower CV when compared to that of single gene expression. In the colon cancer set, the minimum CV decreased from 45.3% to 16.5%. In prostate cancer, comparable CV was achieved with and without transformation. This improvement in CV, coupled with the improved correlation between the pair-wise gene expression ratio and tissue phenotypes, yielded higher classification efficiency, especially with the colon dataset – from 87.1% to 93.5%. Over 90% of the top ten discriminating axes in both datasets showed significant improvement after data transformation. The high classification efficiency achieved suggested that there exist some cancer-related signals in the form of pair-wise gene expression ratio.The results from this study indicated that: 1) in the case when the pair-wise expression ratio transformation achieves lower CV and higher correlation to tissue phenotypes, a better classification of tissue type will follow. 2) the comparable classification accuracy achieved after data transformation suggested that pair-wise gene expression ratio between some pairs of genes can identify reliable markers for cancer. The completion of the Human Genome Project and advances in DNA-array technology have allowed highly parallel genetic analyses to take place on a genome-wide scale. They have revolutionized the way tumors are studied, and promised to provide a better and more thorough understanding of the underlying mechanisms for tumorigenesis. Eventually, they will lead to more comprehensive diagnosis/prognosis of tumor with more effective therapeutic interventions.Tumor development is a process in which gene expression is modified, causing abnormal cell behaviour . Many teDespite its advantages, the DNA-array technology poses three major challenges that render the interpretation of expression data less efficient than expected. Firstly, the gene expression data is inherently variable due to various factors that either depend on biological factors that remain difficult to control , or depend on difficulties in setting up of the experiment (RNA extraction) . These dIn this study, we introduced a novel data transformation meant to investigate relationships between pair-wise gene expression ratios and tissue phenotype within a given experiment. With this procedure, we aimed to discover strong cancer-related signals (features) that exist in the form of pair-wise ratios (or higher order relationship when we extend to N-feature model classifier for N>2) in a given sample, while improving the signal to noise ratio of the dataset by minimizing its coefficient of variation (CV). The underlying concept for adopting pair-wise gene expression ratios as the discriminating axes for tissue type classification is that an experiment is self-consistent . With this approach we could "subtract" correlated variations by considering the sample as a whole, without making inferences such as those needed for normalization. Basically, we avoided studying gene expression in an absolute term because this requires robust normalization method to account for arrays from different experiments, different platforms and different profiling technologies. By resorting to analyze features in the form of ratios, we attempted to minimize the effect of normalization and look for co-varying signals in each experiment..The 62 colon cancer sample dataset is composed of measurements for 1,988 gene probes, of which 40 were labelled as tumor and 22 were labelled as normal. The samples were collected from patients, their RNAs were extracted and hybridised to Affymetrix Hum6000 arrays. Please refer to paper [.The 102 prostate cancer sample dataset is composed of measurements for 12,600 gene probes, of which 52 were labelled as tumor and 50 were labelled as normal. The samples were collected from patients, their RNAs were extracted and hybridised to Affymetrix U95Av2 arrays. Please refer to paper [Both datasets were pre-processed to eliminate those probe pairs that showed significant fluctuation in their hybridisation signals  than their corresponding perfect match probe cells (PM); these probe pairs indicate non-specific hybridisation by background RNAs). Both datasets used average intensity as quantitative measurements of the level of gene expression. Base-10 logarithmic transformations were performed for each dataset.ri (Equation 1) for each gene i using the full dataset, and ranked the genes according to their correlation coefficient ri. For the calculation of r, we assigned a number to each tissue phenotype: 1 for normal tissue and 10 for cancer tissue. After obtaining the correlation coefficients for all genes, we used a simple threshold value (|r|>0.4) to select the set of cancer-related genes. There were two reasons for set the threshold value at 0.4. When lower thresholds were used, we incorporated many genes that were not known to be cancer-related (data not shown). Furthermore, too many genes will later cause computer tractability problem when we calculate their pair-wise gene expression ratio for each tissue sample and later the N-feature model classifier. At |r|>0.4, we were able to account for most of previously known cancer related genes.For downstream classification analysis, we extracted only the genes whose expression pattern correlated strongly to the tissue phenotype. To achieve this, we first calculated the correlation coefficient V1 is a vector representing the gene expression pattern for gene #1; sample Vis the dichotomous representation of tissues; V1Sand sample Sstandard deviation of 1V, sampleV; ,  are the mean of 1V, sampleV.where 1e, 2e...1988e), there are 1988C2 combinations  of pair-wise gene expression ratios , in order to discriminate between the normal/tumor tissues based on specific feature i (single gene expression), the first step is to determine the threshold value, iT, that can optimally splits all the tissue samples into tumor and normal tissue. The FPM algorithm has a recursive version [iTs are derived. Nonetheless, they are very intuitive and non-parametric in nature. Also, they restrict no priori distribution patterns for features used. We adopted the simple FPM for tissue classification where each feature was treated individually. There are two criteria for deriving a valid threshold value i Tfor each feature. First, it has to delineate correctly (discriminating efficiency = 100%) the one-dimensional region (Rfeature_i) for either all the normal/tumor tissues using all tissue samples. Secondly, it has to minimize the percentage of false prediction for the other tissue type. Take gene #1659 for example. To fulfill the two aforementioned criteria, it was determined that the region greater than 63.7 (R#1659) incorporates all the tumor samples  were determined.Regarding the  version , in whics Figure . It clasiTs  were recruited. The outcome of the tissue class will be determined depending on whether one/both the expression values of the unknown sample fall completely in either the normal/cancer region (Rfeature_i). This is to say that if any of the two features from the unknown sample meets the criteria (Rfeature_i) to be either normal/tumor tissue type , the unknown sample will be assigned to be normal/tumor respectively. This is repeated exhaustively for all possible combinations constituting of any two features. The procedure will be repeated for all tissue samples to evaluate the overall classification accuracy for 2-feature model classifier. In total, we evaluated the classification of tissue samples based on different combinations of N genes and investigated the classifiers up to 10-feature model classifier.Now, to classify an unknown sample using 2-feature model classifier, a combination of any two features and their corresponding pre-determined threshold values M. Take the ratio #1537/#1831 for example. To fulfill the two aforementioned criteria, it was determined that the region greater than 0.755 (R#1537/#1831) incorporates all the tumor tissue samples  were recruited. The outcome of the tissue class will be determined depending on whether one/both the expression values of the unknown sample fall completely in either the normal or cancer region (Rfeature_i). This is to say that if any of the two features (pair-wise gene expression ratio) from the unknown sample meets the criteria (Rfeature_i) to be either normal/tumor , the unknown sample will be assigned to be normal/tumor respectively. This is repeated exhaustively for all possible combinations constituting of two features. The procedure will be repeated for all tissue samples to evaluate the overall classification accuracy for 2-feature model classifier. In total, we evaluated the classification of tissue samples based on different combinations of N genes and investigated the classifiers up to 10-feature model classifier.Now, to classify an unknown sample using 2-feature model classifier, a combination of any two features (pair-wise gene expression ratio) and their corresponding pre-determined threshold values r (Equation 1) for all pair combinations of the top 25 genes listed in Table D. Each entry in the pair-wise distance matrix was measured by the value of (1-r). Each row/column corresponds to a specific gene and an entry at the intersection of row X and column Y corresponds to the distance of gene expression between gene #X and gene #Y. Such matrix has a diagonal entry of value 0. Only the lower/upper triangular matrix of D is required to construct the relationship tree. After obtaining lower/upper triangular matrix of D, the neighbor-joining method (NJ) algorithm was used to construct the relationship tree [We calculated the cross correlation coefficient hip tree . with 24 CPUs (each running with a clock speed of 900 MHz) was employed throughout this study. The computation of correlation coefficient and classification procedures were implemented using the Matlab Technical Programming language (Matlab programs can be downloaded at .A Sun Fire 6800 Server r|>0.4) were selected from the colon and prostate dataset for downstream analysis  in a comparable analysis. The heavy chain of non-muscle myosin, denoted as the embryonic smooth muscle myosin heavy chain (SMemb), was found to be down-regulated in cancer. It was also determined experimentally to be a target for the protein encoded by the metastasis-related mts-1 gene [After initial gene selection, respectively 82 and 262 genes , which revealed informative pair-wise gene interaction in relation with their corresponding tissue phenotypes. They reflected how cell adjusts to their pair-wise product in response to physiological changes. Based on these observations, we found that the relative abundance between the numerator and denominator exhibited a strong mutual dependency, and had strong correlation to tissue phenotype. For pair-wise gene expression ratio #119/#54, the elongation factor 1-delta is involved in a sequence of events during the decoding of mRNA on the ribosome [cDNA of mouse that incorporates proteins involved in chromosome partitioning and cell decision [To date, reliable markers with low coefficient of variation (CV) are generally lacking. Discovering robust cancer marker is crucial for the purpose of successful cancer diagnosis. We investigated the CV between samples after data transformation: the lowest CVs decreased to 16.5% in the colon dataset while it increased to 25.8% for the prostate dataset Table . Toppingribosome . For thedecision .Prior to data transformation the lowest coefficients of variations for single gene expression were 45.3% and 24.5% for colon and prostate datasets respectively. When using the data transformation we proposed, significant improvement was achieved in the colon dataset. Interestingly, this was followed by an improved data correlation to the tissue phenotype as well as to the classification efficiency. We did not observe a similar improvement of the CV, data correlation to tissue classes or classification efficiency in the prostate dataset.r| >0.4 from the bulk (Table vs #119 in colon cancer (r = 0.95306) and gene #7775 vs #10749 in prostate cancer (r = 0.92922)). It should be pointed out that the two humps in the probability density function are not zero-centered, but concentrated at non-zero correlation r. For colon dataset, positive correlation was the dominant type. For prostate dataset, a balanced distribution in their gene correlation was observed. We determined that some improvement in tissue classification is achieved when pair-wise gene expression ratio was used as discriminating axes instead of using a single gene expression , others (Human gene for heterogeneous nuclear ribonucleoprotein (hnRNP), P24480 CALGIZZARIN, Human mitochondrial matrix protein P1, Human mRNA for aldose reductase and human adipsin) have not been identified from in-silico studies of tissue DNA-array expression data. The cancer related genes for colon and prostate cancer were ranked according to their discriminating predictive power. The list should provide hints for researchers during selection of molecular target for diagnostic, prognostic or attempts to cure the disease. Overall classification results and accuracies for each N-feature model classifier across two datasets were reported in Table Several previous studies have already endeavored to identify correlations between specific gene expression and cancerous transformation ,13,23. IFor the sake of brevity, we will discuss three single gene expressions and two pair-wise gene expression ratios from colon cancer. For prostate cancer, two single gene expressions and two pair-wise gene expression ratios will be discussed.For colon cancer single gene expression, three axes for discriminating tissue types are: 1) Human monocyte-derived neutrophil-activating protein (MONAP); 2) Human desmin gene and 3) Human cysteine-rich protein (CRP) gene. Their threshold values were determined to be 62.73, 2787.0 and 749.4 respectively.For colon cancer pair-wise gene expression ratio, the two axes for discriminating tissue types are: 1) #1831/#1537 and 2) #753/#768. Their threshold values were reported to be 1.32 and 1.85 respectively.For prostate cancer individual gene expression, the two axes for discriminating tissue types are: 1) Human hepatoma mRNA for serine protease hepsin and 2) Human adipsin. Their threshold values were reported to be 115.0 and 182.0 respectively.For prostate cancer pair-wise gene expression ratio, the two axes for discriminating tissue types are: 1) #6185/#5840 and 2) #6185/#6749. Their threshold values were reported to be 2.69 and 2.55 respectively.To illustrate graphically the result of tissue classification, two examples, each based on three genes or pair-wise gene expression ratios that altogether yielded the optimum classification efficiency for the prostate cancer are shown Figure .B. subtilis and E. coli) and successfully determined the co-regulated operon gene structures (supplementary file #1).The relationship tree for top 25 genes listed in Table As the expression profiling technologies mature, the identification of significant cancer-related signals from noisy datasets (characterized by a high CV) remains a major challenge. In particular, a robust normalization method is critical to ascertain that arrays from two experiments are comparable with minimum noise prior downstream analysis. However, the existing normalization methods pose limitations due to the lack of good models to account for sources of experimental and biological variations . HoffmanHere, instead of resolving to single gene expression, that depends heavily on normalization, for tissue classification, we presented a transformation method that uses pair-wise gene expression ratios within the same experiment as the discriminating axes. By doing so, we aimed to minimize the influence of different normalization methods considering that an experiment is self-consistent with the same factors affecting all genes in the same fashion. The rationale is that even when the normalization methods differ between two array experiments, their pair-wise gene expression ratios within the same experiment will remain relatively stable. If reliable cancer-related signal, exist in the form of pair-wise gene expression ratio, were indeed discovered successfully, they will be relatively independent from the normalization method used on a dataset.100C10= 1.731030945644000 × 1013 different combination of features). As a result, more computation time will be required to search all possibilities. As an example, the discriminating axes that accounted for the optimum accuracy in 1 to 3-feature model classifier are reported in Table The improvement in CV Table  and overiT, in the feature selection procedure. In other word, instead of adopting the more conservative classification accuracy test where only a subset of tissue samples are used to derive a set of classification criteria , we adjusted our methodology to use all tissue samples so that our results are unbiased (when comparing the outcome from single gene and pair-wise gene ratio) and in-line with our objective that is to compare the classification efficiency between single gene and pair-wise gene ratio. Admittedly, we have a noisy dataset whereby selecting a subset of tissue samples that are a representable population for the entire dataset remains a challenge [Regarding the high classification accuracy reported in Table hallenge   Human monocyte-derived neutrophil-activating protein (MONAP); 2) Human desmin gene and 3) Human cysteine-rich protein (CRP) gene. The association of the first two genes and cancer biology had been discussed earlier. We will discuss the Human cysteine-rich protein gene. The expression and induction of this protein has been associated with protection against DNA damage, oxidative stress and apoptosis . In the For colon cancer pair-wise gene expression ratio, the two axes for discriminating tissues are: 1) #1831/#1537 and 2) #753/#768. Using these two axes, 98.4% of the tissue samples can be classified correctly. The expression ratio between #1831 (gelsolin precursor) and #1537  was able to discriminate 93.6% of the total tissue data. The vascular endothelial growth factor was determined recently to be a plausible biomarker for colon cancer . GelsolimRNA for serine protease hepsin, and 2) Human adipsin. The first gene was discussed in the previous paragraph. For the second gene, adipsin had also been suggested by Chow et al. [For prostate cancer single gene expression, the two axes for discriminating tissues are: 1) Human hepatoma w et al.  as a goomRNA for serine protease hepsin) and #5840 (Homo sapiens mRNA for KIAA1109 protein) was able to discriminate 92.2% of total tissues. The human hepatoma mRNA for serine protease hepsin had been determined to be an important marker for cancer cell development [mRNA for serine protease hepsin) and #6749 (Homo sapiens mRNA for KIAA1055 protein) was able to discriminate 90.10% of total tissues. The human hepatoma mRNA for serine protease hepsin was discussed in the previous section. The KIAA1055 protein is an unknown protein in human chromosome 15 [cDNA that had been found to function as a cancer inhibiting protein [For prostate cancer pair-wise gene expression ratio, the two axes for discriminating tissues are: 1) #6185/#5840 and 2) #6185/#6749. Using these two axes, all tissue samples can be classified correctly. The expression ratio between #6185  improved from 87.1% to 93.55% in colon dataset. In prostate dataset, nine out of the top 10 discriminating axes showed significant improvement. The mean accuracy for 1-gene classifier improved from 76.8% to 91.2% and 75.8% to 81.9% in both datasets.4. The comparable classification accuracy achieved after data transformation suggested that there exist some cancer-related signals in the form of pair-wise gene expression ratio, especially prominent in the colon dataset.5. Through the single gene analysis, we identified key biomarkers that agree with the findings by other researchers. In addition, study on gene-to-gene correlation and the classification outcome based on the pair-wise gene expression ratio suggested that genetic network within a cluster of cancer-related genes should be explored further.The authors declare that they have no competing interests.YLY proposed the idea, participated in the design, performed the statistical analysis and wrote the first draft of the manuscript.AD participated in the design and overall coordination of this study as well as in the writing of the manuscript.XWZ participated in the design of the study.YCW, XHW and MTL participated during the revision phase of this study.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Hashimoto's thyroiditis (HT) is an organ-specific T-cell mediated disease. It is a complex disease, with a strong genetic component. To date, significant progress has been made towards the identification and functional characterization of HT susceptibility genes. In this review, we will summarize the recent advances in our understanding of the genetic input to the pathogenesis of HT. Hashimoto's thyroiditis (HT) is one of the most common human autoimmune diseases responsible for considerable morbidity in women . It is aGoitrous autoimmune thyroiditis, or Hashimoto's thyroiditis is a common form of chronic autoimmune thyroid disease (AITD). The disorder affects up to 2% of the general population . In the 4) and triiodothyronine (T3) remain normal. The overt disease is defined by the dramatic loss of thyroid follicular cells (thyrocytes), hypothyroidism, goitre, circulating autoantibodies to two primary thyroid-specific antigens, thyroglobulin (Tg), thyroid peroxidase (TPO), and lowered concentrations of serum TSH and T4 , complement components  and heat shock proteins (HSP) . Both TNHSP70 gene cluster consists of three genes encoding HSP70-1, HSP70-2 and HSP-Hom proteins. They are expressed in response to heat shock and a variety of other stress stimuli . HSPs are also important for antigen processing and presentation . GeneticOther genes crucial to the immune response, including TAP (transporters associated with antigen processing), LMP , DMA and DMB genes are located within the HLA class II region . ProteinDMA and DMB genes are involved in the assembly of HLA class II peptides. These genes encode subunits of a functional heterodimer that is critical for class II antigen presentation ,162. BasThe CTLA-4 gene is the most frequently studied of the immune modulatory genes located outside the HLA region, in relation to the genetics of HT. This gene encodes a costimulatory molecule, which suppresses T-mediated immune response and is crucial in the maintenance of peripheral immunological self-tolerance . An inveFamily studies showed linkage between CTLA-4 and GD , thyroidThis investigation has recently been performed involving about 600 AITD families and more than 1300 affected patients . The CTLin vitro expreriments, the Ala17 (G49) allele was found to represent a translation product, which was not glycosylated in one of two N-linked glycosylation sites [It may be that the amino acid change at codon 17 of the signal peptide could alter the function of the signal peptide to direct intracellulat trafficking of CTLA-4. In on sites . This abon sites . These fon sites ,178.n repeat polymorphism at the 3'-UTR of the CTLA-4 gene [The codon 17 single nucleotide polymorphism (SNP) is shown to be in tight linkage disequilibrium with another SNP situated at position (-318) of the CTLA-4 promoter region and with the (AT)A-4 gene ,179-181.A-4 gene . Since tn repeat polymorphism at the 3'-UTR of the CTLA-4 gene has been shown to affect the expression of this costimulatory molecule [n alleles have shorter half-lives and, hence, are more unstable [n microsatellite in the 3'-UTR influences the mRNA stability. Additionally, the CTLA-4 AT-repeat polymorphism was recently shown to alter the inhibitory function of CTLA-4. The long AT-repeat allele is associated with reduced control of T-cell proliferation and thus contributes to the pathogenesis of GD [The (AT)molecule . Adenylamolecule ,184. Theunstable ,185. Indis of GD .et al. [n repeat polymorphism [The AT-repeat may also affect splicing of one or more of the alternative CTLA-4 transcripts but this should be clarified. Ueda et al.  showed tmorphism . Howevermorphism ,188.It should be noted that the genomic region 2q33 linked to autoimmune disease contains cluster of three genes encoding costimulatory molecules CTLA-4, CD28 and inducible costimulator (ICOS) . HoweverThe CTLA4 gene should be recognised as the first major known non-HLA locus of human autoimmunity and that its role in the pathogenesis of HT is rather general and non-specific ,153. AssIn initial phases of AITD, oligoclonal expansion of T lymphocytes occurs in the thyroid gland. These T cells are restricted by their T cell receptor V gene use ,90. TherAnother likely candidate among immune-related genes was the IGH gene because HT individuals commonly produce Tg-autoantibodies restricted by IgG class . Early iet al. [Cytokines are crucial in the regulation of immune and inflammatory responses. Multiple investigations showed the important role of these regulatory molecules in directing autoimmune and apoptotic pathogenic processes, of particular, in central and late stages of the development of HT ,80,199. et al.  evaluateet al. . Blakemoet al.  failed tThe autoimmune regulator (AIRE1) gene is known to contribute to the pathogenesis of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a rare monogenic autoimmune disease with endocrine components including T1D, adrenal failure, and thyroid dysfunction, with major autoantibodies directed against adrenal, pancreas, and thyroid tissue . However3 2D3) is the most active natural vitamin D metabolite that effectively prevents the development of autoimmune thyroiditis in an animal model [The vitamin D-mediated endocrine system plays a role in the regulation of calcium homeostasis, cell proliferation and (auto) immunity. 1,25-Dihydroxi-vitamin Dal model  and inhial model . C/T polal model . Two polal model . Howeveral model . Meanwhial model , Addisonal model , multiplal model  and T1D al model .Thus, a wide variety of non-HLA immune regulatory genes located outside the HLA region showed no significant linkage or association with HT and AITD except for the CTLA4 gene. However, we still cannot estimate whether or not these genes significantly contribute to HT susceptibility due to a serious shortfall in information about their role in this disorder. It cannot be excluded that other genes in linkage disequilibrium with these genes are the susceptibility genes at these loci.Antibodies against thyroid peroxidase are one of the most specific features of HT . Therefon marker at intron 2 of the TSHR gene and a (CA)n marker that was mapped to approximately 600 kb of the TSHR gene, have been shown to be strongly associated with HT in Japanese patients [Within the other thyroid-specific gene, the TSHR gene, the T52P amino acid substitute was examined in US white and Thai populations but no association with HT was found ,218. Varpatients ,222. Theet al. [Tg-specific autoantibodies are common in AITD. The thyroglobulin gene makes a significant contribution to HT and AITD. Whole-genome scans in Japanese-affected sibling pairs have detected a HT susceptibility locus on chromosome 8q24, with a maximum linkage to marker D8S272 . This maet al.  identifiet al. ,227 and,et al. .The Tg gene spanning over 300 kilobases long is expected to harbour more than one haplotype block associated with AITD since the length of a linkage disequilibrium block of SNPs is shown to be less than 100 kilobases . It seemet al. [Recent investigation in Tunisians showed significant association of two polymorphic microsatellites (D7S496 and D7S2459) close to the PDS gene (7q31) with GD and HT, and one of them, D7S496, was linked to GD only . The PDSet al.  suggest +/I) symporter and paired box transcription factor-8, among others. They also need to be evaluated for any putative impact on HT.Finally, a role for other genes specifically expressed in the thyroid gland, has yet to be defined. These genes include those encoding thyrotropin-β, thyroid-specific factor-1, sodium iodide  and -β (ESR2), were examined but showed no linkage with HT . The ESRn repeat polymorphism in the intron 20 of the SEL1L gene with AITD [The SEL1L gene, encoding a novel transcription factor, was recently described . The genith AITD . This geith AITD . It liesith AITD .in vitro and this expression results in the decreasing expression of the truncated form of ZFAT (TR-ZFAT) [et al. found no association of the thyroglobulin gene with AITD when studying different ethnic groups [A new zink-finger gene designated ZFAT (a novel zink-finger gene in AITD susceptibility region) has been recently found on chromosome 8q24 . The T aTR-ZFAT) . This SNTR-ZFAT) . The disc groups . These rAt present, the CTLA-4 (chromosome 2q33), thyroglobulin (or ZFAT) (8q24) and likely HLA genes (6p21.3) are the only susceptibility loci for HT and thyroid autoimmunity to be mapped. Two HT-specific susceptibility loci that have been detected in mixed Caucasian families from USA and Europe, HT-1 (13q) near marker D13S173 and HT-2 on chromosome 12q in the vicinity of marker D12S351, are still not defined . HT-2 loAITD-1 locus located on chromosome 6p is very close yet distinct from the HLA region ,130. It A whole-genome scan in Japanese showed evidence for linkage with AITD on chromosome 5q31-q33 ,251. TheIn the Chinese, a whole-genome screening for AITD susceptibility found two chromosome regions (9q13 and 11q12) linked to AITD . SusceptIn a large Tunisian family affected with AITD, a susceptibility locus was mapped on 2p24  This locThese data suggest that both HT and AITD show genetic heterogeneity in different populations. Susceptibility loci differ in their chromosome location depending on the population being tested. The contributory value of these genes to the disease pathology varies significantly depending on the ethnic background. A gene, that has a major effect on the susceptibility to HT in one population, may contribute weakly in other population. To date, several regions of linkage to HT and AITD have been defined. Further studies are required to find a true susceptibility gene in these genomic regions to reveal the functional significance of disease-associated polymorphisms within these genes.AITD can be initiated in individuals genetically predisposed to AITD by non-genetic  triggers such as dietary iodine, infection, pregnancy, cytokine therapy Fig. . This inSequence variants of CTLA-4, associated with increased levels of the soluble form of this immune costimulator and with stability of CTLA-4 mRNA, could play a crucial role in the earliest stages of AITD (i. e. breakdown of self-tolerance and surviving autoreactive T lymphocytes). This role might be sufficient to regulate subsequent steps in the development of autoimmune responses to the production of thyroid autoantobodies .Environmental factors  could cause insult of the thyrocyte followed by abnormal expression of MHC class I and class II molecules, as well as changes to genes or gene products (such as MHC class III and costimulatory molecules) needed for the thyrocyte to become an APC . In thisH2-mediated switching of thyroid-infiltrating T cells. These induce the production of stimulating anti-TSHR antibodies by B cells and anti-apoptotic mechanisms that lead to clinical hyperthyroidism. In HT, preferential TH1 response initiates apoptosis of thyroid cells and results in clinical hypothyroidism [In later stages, thyroid autoimmunity could be switched towards GD or HT. GD is characterized by Tyroidism .It is clear that a number of loci and genes determine genetic predisposition to HT, with varying effects. These loci could be unique to HT or general for both HT and GD. Several whole-genome scans showed results suggesting that there is significant shared susceptibility to HT and GD ,134,135.The CD40 gene, an important immune modulator, appears to act as a GD-specific susceptibility gene. The gene is located within the 20q11 locus and shows significant linkage to GD, but not to HT, in UK Caucasians ,256,257.H1 or TH2 type mechanisms [H2 type mechanism, which can lead to hyperthyroidism [A probable susceptibility gene that could direct switching towards GD or HT is thought to be located within the 5q31 locus, which is linked to AITD and contains a cytokine gene cluster. Different sets of cytokines are known to regulate switching to Tchanisms . There mchanisms . IL-4 meyroidism .Another HT-specific susceptibility gene(s) may be an apoptotic gene. Apoptosis of thyroid follicular cells is the hallmark of HT and might be the primary cause of death of thyrocytes compared to T cell-mediated cytotoxity .Thus, it is necessary to identify additional susceptibility genes and disease-associated polymorphisms in apoptotic genes in AITD- and HT-linked loci by using a fine mapping approach and high-density panels of SNPs. Further functional analysis and search for correlations between genotype and phenotype will help to evaluate the role of these genes in the development of autoimmune thyroid disease. Susceptibility genes interact with thyroid autoimmunity ,130, andThe author(s) declare that they have no competing interests.ABFG


Registration of the fact of death is almost complete in the city of Chennai and not so in the rural Villupuram district in Tamilnadu, India. The cause of death is often inadequately recorded on the death certificate in developing countries like India. A special verbal autopsy (VA) study of 48 000 adult (aged ≥ 25 yrs) deaths in the city of Chennai (urban) during 1995–97 and 32 000 in rural Villupuram during 1997–98 was conducted to arrive at the probable underlying cause of death to estimate cause specific mortality.A ten day training on writing verbal autopsy (VA) report for adult deaths was given to non-medical graduates with at least 15 years of formal education. They interviewed surviving spouse/close associates of the deceased to write a verbal autopsy report in local language (Tamil) on the complaints, symptoms, signs, duration and treatment details of illness prior to death. Each report was reviewed centrally by two physicians independently. Random re-interviewing of 5% of the VA reports was done to check the reliability and reproducibility of the VA report. The validity of VA diagnosis was assessed only for cancer deaths.Verbal autopsy reduced the proportion of deaths attributed to unspecified and unknown causes from 54% to 23% (p < 0.0001) in urban and from 41% to 26% (p < 0.0001) in rural areas in Tamilnadu for adult deaths (≥ 25). The sensitivity of VA to identify cancer was 95% in the age group 25–69.A ten day training programme to write verbal autopsy report with adequate feed back sessions and random sampling of 5% of the verbal autopsy reports for re-interview worked very well in Tamilnadu, to arrive at the probable underlying cause of death reliably for deaths in early adult life or middle age (25–69 years) and less reliably for older ages (70+). Thus VA is practicable for deaths in early adult life or middle age and is of more limited value in old age. In developed countries, data on disease-specific mortality by age are readily available from national vital registration. In developing countries, where 80% of the world's deaths occur, estimation of cause of death is more difficult because the levels of coverage of vital registration and reliability of cause of death stated on the death certificate are generally low .A reliable assessment of disease-specific mortality rates is not yet possible in many parts of India, either because the underlying cause of the terminal illness was never known or because the relevant information was not recorded. For legal purposes death records do usually subdivide the causes of death into medical and non-medical  causes. But once non-medical causes have been excluded, specification of the underlying cause of a death from disease may be inaccurate, misclassified or missing for about 50% of adult deaths. For example, in Chennai, Tamilnadu, (south India) about half of those who died at home soon after the diagnosis of cancer  do not have cancer mentioned on their death certificate , and forElsewhere, 'Verbal Autopsy' i.e., 'systematic retrospective inquiry of family members about the symptoms and signs of illness prior to death' has been used to help determine the underlying medical cause of death, particularly in childhood ,3. For cA special study on 'verbal autopsy' of adult deaths was conducted in urban and rural areas in the state of Tamil Nadu, south India during 1998–2000. The aims of the study were (a) to develop a verbal autopsy instrument and test its utility to determine the underlying cause of death and (b) to estimate cause specific mortality using underlying cause of death arrived based on verbal autopsy reports. Now we report the type of training given to the field interviewers to interview and write the verbal autopsy report for adult deaths, the procedure followed to arrive at the probable underlying cause of death, the accuracy of the instrument developed and change in the proportions of cause of death based on the underlying cause of death arrived by reviewing 80 000 verbal autopsy reports.Male non-medical graduates with at least 15 years of formal education were selected. A ten days training was given on verbal autopsy interview techniques and writing verbal autopsy reports. There are four steps in training; 1). introduction to anatomy, signs and symptoms of various diseases, 2). mock interviews, 3). hands-on-training on writing verbal autopsy reports and 4). feed back session.This consisted of a basic three day introduction to anatomy, collecting data on history of past illness  from the respondent to write the verbal autopsy narrative report:• onset of illness prior to death: sudden or gradual• major symptom(s) and associated symptom(s) – in chronological order• If a symptom was present it was used as a filter to define what questions to be asked. For example, the filter symptom for heart attack was chest pain and the associated symptoms were breathlessness, sweating, vomiting and pain in the retrosternal area radiating to hand, shoulder, back etc. Cough for more than 4 weeks was a filter for lung cancer and tuberculosis. For each symptom, the duration should be recorded. Details of additional symptoms are built into the narrative in chronological order, by prompting, if necessary.• progress of the illness• any treatment received : Yes/No• If yes, type of treatment received• details of hospitalization prior to death:○ name of the hospital ,○ duration of hospitalization,○ whether discharged from the hospital against medical advice or not.○ Status at the time of discharge from the hospital: alive/dead• history of similar episodes and treatment(s) givenif available• abstract information related to the illness prior to death from the investigation reports done for any illness close to the time of death (within 6 months prior to the death) / hospital discharge summary etc, • If a death certificate is available, copy the cause of death given on the death certificate .• While recording history of adults with long standing illness, the description should include details that occurred in the month preceding the death, with other information recorded in the past history section  in Additional file 1If the respondent is able to give the major symptoms and circumstances leading to death, then additional probing questions are asked about the associated symptoms using the symptoms/signs checklist (Appendix II(A & B) in ○ read out the filter symptom/sign of each module in the symptom/sign checklist○ check responses to each, and note down positive responses○ Where there is a positive response, additional details on that symptom and associated symptoms, if any, should be obtained.Thus, the methodology of collecting data in the open format using 'symptoms/signs checklist' is an interactive process, with the respondent taking the lead in providing the information, and the interviewer prompting where necessary for more details. The Field Interviewer gathers as much information as possible on the underlying cause of death from the respondent. It is imperative to get a logical and complete history of symptoms, signs, events, investigations and treatment, so that the medical reviewer gets sufficient information to assign a probable specific underlying cause of death.In the following two days, mock interviews were organized to illustrate techniques of probing a respondent to get the required information on cause of death as well as how to write the verbal autopsy report in local language (Tamil) in Appendix I in The third component of training included three days of hands on verbal autopsy training in the field. To limit distress over the terminal event, the field visit was carried out at least six months after death. Name of the deceased, father's name  or spouse name , age, gender, informant's name and address of the deceased at the time of death were given to field interviewers to locate the house of the deceased. The Field Interviewers carry Appendix I and II (symptoms/signs checklist) in The final component of training was feed back session for 2 days. This session involved teaching them how to include essential information in report writing. The feedback session mainly focused discussion on reports which did not have a specified underlying cause of death and reports with minimal information to arrive at the probable underlying cause of death; for example, a report may say that a person had a stroke ten days ago but did not specify the type of onset  or a report may say that the deceased had fever for ten days and died. It did not give details about the fever and other associated symptoms if any.This special verbal autopsy study was carried out in two areas in Tamilnadu. The Chennai city (urban) with a population of 4.2 million, and the Villupuram district , with a population of 2.5 million were chosen for this study. We have successfully traced 48 000 adult deaths (≥ 25 years at the time of death) in urban area and 32 000 adult deaths in rural Villupuram district and reviewed 80 000 verbal autopsy reports to arrive at the probable underlying cause of death.Information on deaths that occur in Chennai has been maintained manually by trained staff in Chennai Vital Statistics Department (VSD). The following data on deaths that occurred in Chennai during 1995 to 1997 were collected from the death registers in the Vital Statistics Department: deceased name, age, gender, marital status, father/spouse name, informant's name, occupation, place of death, address at the time of death, date of death and recorded cause of death . 72,000 deaths occurred during the study period of 1995–97. Of 72 000 found, 5000 deaths were attributed to external causes  in the death certificate, and were excluded. Of the remaining 67 000 deaths attributed in the VSD  to medical causes, 48 000 of the households were successfully visited during 1998–99 to try to assign cause of death by verbal autopsy.All formal and informal village records were to be sought to identify all deaths at any age during 1997–98. 41,000 such records were identified and 39 000 of the households were successfully visited during 1999–2000 to try to assign cause of death by verbal autopsy. Of 39 000 deaths, 7000 were before age 25.Feed back sessions were organized regularly throughout the study period to improve the quality of the verbal autopsy reports and 5% of the field visit reports were validated by re-interview one week after completion of the main interview, and blind to its results. This re-interviewing was done by one or other of two special interviewers because knowledge that a resurvey might well take place would ensure reliably motivated fieldwork at the initial survey, and also to check whether there were any systematic defects in the technique of any of the field workers: none were found. The underlying cause of death arrived based on re-interview data was not substantially different from the one arrived based on main interview data.th International Classification of Diseases, Injuries and Causes of Death [All verbal autopsy reports were centrally reviewed by two physicians independently in order to arrive at "probable underlying cause of death". Each made a diagnosis based on signs, symptoms and sequence of events prior to death given in the verbal autopsy report, which were then coded according to the 9of Death . The samIn Chennai city, the study was done in 1998–99 and the verbal autopsy reports were reviewed for 27 726 male deaths and 20 631 female deaths. Table In Villupuram district, verbal autopsy report was written for all deaths i.e., deaths due to medical and external causes. So verbal autopsy reports of 19 294 male deaths and 12 494 female deaths were reviewed. Deaths from unspecified medical causes and unknown causes decreased from 41% to 26% (p < 0.0001)  between the clinical diagnosis or clinical cause of death and postmortem findings -12 and m0–40% betWhere a cause recorded on the death certificate in the VSD differed from the underlying cause assigned by the VA, there was often no absolute way of knowing which was correct  except for cancer deaths which could be verified with the Chennai cancer registry records. Hence, the validity of VA diagnosis was assessed only for cancer deaths(ICD 9:140–208) by comparing with the stated cause of death in the VSD records and verifying with the Chennai cancer registry records and hospital medical records (only for cancer diagnosis). Chennai Population-Based Cancer Registry is a demographic registry in the network of the Indian Council of Medical Research, Govt. of India and has been functioning since 1982 at the Cancer Institute(WIA), Chennai. Cancer is not a notifiable disease in India. Hence registration has been done by active method. Cancer patients attending the Govt. hospitals are interviewed to collect data on age, sex, address, duration of stay in Chennai city, marital status, mother tongue and educational level. Interviews are done at the houses for those who have been missed by the registry staff during their (Govt.) hospital visit. The clinical data, such as date of cancer diagnosis, method of diagnosis, site of cancer, any spread of the disease, histology, treatment details and status  for all registered patients are abstracted from the hospital medical records. All data on cancer patients attending the private hospitals are abstracted from the hospital records. The mortality data available at the Vital Statistics Department are linked with the Chennai Cancer Registry data base. Therefore the cause of death arrived based on verbal autopsy report was compared with the hospital data on cancer patients available in the Chennai Cancer Registry data base.Table The Tamilnadu study on verbal autopsy ,16 used The verbal autopsy tool for adult deaths is an open narrative format uses the check list of symptoms and signs with filters to get more information on train of events or circumstances preceding the death. The sensitivity of this tool is 95% (94% compared to VSD records and 96% compared to Chennai population-based cancer registry) in the age group 25–69 during the study period. The validity of this verbal autopsy tool is influenced by the training given to the interviewers, on the immediate random checking of the 5% of interview data and reviewing of the field reports centrally by 2 physicians to arrive at the probable underlying cause of death which is better than that arrived by opinion-based algorithm . There iAs a result of using verbal autopsy method, adult deaths (≥ 25) from unspecified and unknown causes decreased from 54% to 23% (p < 0.0001) in urban and from 41% to 26% (p < 0.0001) in rural areas in Tamilnadu. Ten day training to write verbal autopsy reports followed by constant monitoring of the submitted reports resulted in arriving at the probable underlying cause of death for most of the deaths and to compute broad classification of the underlying causes of about 90% of deaths in early adult life or middle age: in old age, however, the proportion classifiable is substantially lower. The specific causes of death arrived based on verbal autopsy reports were used to estimate death rates for Chennai city  and to eThis methodology of arriving cause of death for adult deaths by verbal autopsy is now being adopted by the Registrar General of India, Govt. of India, for nationwide use in the Sample Registration System(SRS) that consists of 6671 units  spread across the country covering 1.1 million households and about 6 million population. The SRS is a large demographic survey of vital events occurring in a national random sample of urban and rural areas in India by the Registrar General of India (RGI) to provide annual estimates of age-specific birth and death rates at the national and state levels. This exercise on verbal autopsy for adult deaths along with the verbal autopsy questionnaire developed by the SRS collaborators for deaths that occur at ages less than 15 years and for maternal deaths is expected to yield reliable cause-specific death rates for India.A ten day training programme to write verbal autopsy report with adequate feed back sessions and random sampling of 5% of the verbal autopsy reports for re-interview worked very well in Tamilnadu, to arrive at the probable underlying cause of death reliably for deaths in early adult life or middle age (25–69 years) and less reliably for older ages (70+). Our experience shows that the open narrative, if well written, provides adequate information for assigning probable underlying cause of death for adult deaths.The authors declare that they have no competing interests.VG and RP participated in designing the study, analysis and preparation of the report. Development of verbal autopsy tool, training on verbal autopsy method, co-ordination of field work and data management were done by VG. Verbal autopsy review by VG and TS Kanaka, physician.The pre-publication history for this paper can be accessed here:Appendix I – used to write the history of past illness and verbal autopsy report for adult deaths (25 years or older) and in Appendix II – Symptoms/signs checklist for adult deaths (≥ 25 years) is givenClick here for file