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 (drug, dietary advice and placebo effect) 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 (HO-1, HO-2 and HO-3) 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 (Cys 264-Pro 265, Cys 281-Pro 282) [- 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. (Darmstadt, Germany). 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. (St-Constant, Que). 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 (1e/2e, 1e/3e...) 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 (based on our definition, Rfeature_i is a region with 100% discriminating efficiency for a specific 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 (based on our definition, Rfeature_i is a region with 100% discriminating efficiency for a specific tissue type), 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 (Table z-score, t-test) 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 (|s-1 gene . Furthers-1 gene . The hums-1 gene . It was s-1 gene .From the prostate dataset, the most cancer-correlated gene is the human hepatoma gene coding for serine protease hepsin. Brief literature search in PubMed showed that hepsin is a well-characterized transmembrane protease that is expressed at high level in tumor. Three separate studies identified hepsin as a significant cancer biomarker that can be used for cancer diagnosis . The secHomo sapiens cDNA and nuclear matrix protein NXP2), 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  (given tFor colon dataset, three axes for discriminating tissues are: 1) 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 [Subclinical hypothyroidism is characterized by an increase in serum thyrotropin (TSH) whilst serum levels of thyroxine (TH and T4 . HistoloH and T4 .HT has been shown to often coexist with other autoimmune diseases such as type 1 diabetes (T1D), celiac disease, rheumatoid arthritis, multiple sclerosis, vitiligo, etc -14. HT cIn common with probably all autoimmune disorders, the harmful interaction between internal (genetic) and external  factors is required to initiate Hashimoto's disease Fig. . EnvironThe development of the autoimmune failure of the thyroid is a multistep process, requiring several genetic and environmental abnormalities to converge before full-blown disease develops Fig. . At the In autoimmune thyroditis animal models, genetically determined immune defects have been suggestively linked to the breakdown of immunological self-tolerance that results in the presentation of host autoantigens and expansion of autoreactive lymphocyte clones. These immune defects are associated with the presence of particular MHC class II haplotypes, but other immune and immune regulatory genes (i.e., CTLA-4 and others) are also involved -32.Breakdown of the immune tolerance might occur in several ways including interrupting central tolerance (e.g. deletion of autoreactive T cells in the thymus), defects in maintaining peripheral tolerance (e.g. activation-induced T-cell death and suppressing activity of regulatory T lymphocytes) and anergy . Animal models genetically predisposed to develop an autoimmune disease, and patients with AITD, showed a lack of, or a deficiency in, a subpopulation of regulatory T cells with suppressive function -35.The mechanisms, whereby autoreactive T cells escape deletion and anergy, and become activated, remain uncertain. There is evidence that the thyroid cell itself, by "aberrantly" expressing MHC molecules, can play the role of "non-professional " APS and present disease-initiating antigen directly to the T cells ,37. The 3 and T4. Iodine can promote antithyroid immunity in a number of ways. Several studies suggest that iodination of Tg is crucial for recognition by Tg-reactive T cells [Iodine is a necessary component of normal thyroid hormonogenesis. Incorporation of iodine into thyrosine residues of Tg leads to the formation of mono-iodotyrosine and di-idothyrosine derivates that subsequently undergo an oxidative coupling event resulting in the producing of T T cells ,41. Iodi T cells . Therefo T cells . Excessi T cells . The sta T cells . A lower T cells .An influx of dendritic cells and macrophages to the thyroid may occur as a consequence of inflammatory events in the gland. Early non-specific necrosis of thyrocytes due to toxins (i.e. iodine, etc.), and perhaps viral or bacterial infection, can attract these cells to the thyroid. Moreover, these immune cells are normal constituents of the thyroid that are able to regulate the growth and function of thyrocytes via interleukin-1 (IL-1) and IL-6-mediated pathways .A central phase of HT is characterized by the recognition of presented autoantigens by the lymphocytes, followed by an apparent uncontrolled production of autoreactive CD4+ T cells, CD8+ cytotoxic T cells and immunoglobulin G (IgG) autoantibodies. Initially, the production of self-reactive cells and autoantibodies occurs in the draining lymph nodes Fig. . Later, Thyroglobulin, the main protein synthesized in the thyroid, serves both in the synthesis and in the storage of thyroid hormones. Human Tg molecules contain at least four thyroid hormone synthesis sites from the iodinated tyrosine residues at positions 5, 2553, 2567 and 2746 . The horThyroid peroxidase is another significant autoantigen in the thyroid of patients affected with HT and AITD. This enzyme catalyses the oxidation of iodine to an iodinating species that forms iodotyrosines in a Tg molecule and subsequently iodotyronines . TPO antAutoantibodies against other thyroid-specific antigens such as thyrotropin receptor and sodium iodide symporter were also found in serum of HT patients. However, these antibodies occur at low frequency and do not appear to contribute any diagnostic power for HT ,57.H1)-mediated mechanisms involving production of IL-12, tumor necrosis factor-α (TNF-α) and interferon-γ play a major role in the destruction of thyrocytes, rather than TH2 type mechanisms directed by IL-4 and IL-10 [H1-mediated mechanisms [In a final, destructive step of Hashimoto's thyroiditis, the autoreactive T cells diffusely accumulate in large numbers and infiltrate thyroid parenchyma Fig. . In the nd IL-10 . The infchanisms . Fas andchanisms .H1 or TH2 cytokines might regulate thyrocyte survival through the induction of pro-apoptotic and anti-apoptotic proteins. TH1-mediated mechanisms lead to thyrocyte depletion in Hashimoto's thyroiditis through the involvement of death receptors and cytokine-regulated apoptotic pathways [Autoimmune responses against specific antigens are primary determinants in thyroid autoimmunity. Other molecular mechanisms including cell apoptosis may play a role in determining the opposite phenotypic outcomes of AITD such as thyroid destruction in HT and thyroid hyperplasia in GD. T-helper lymphocytes produce cytokines that influence both immune and target cells at several levels. The predominance of Tpathways ,62.The normal thyroid gland has been shown to act as an immune privileged site having carefully regulated mechanisms of cell death and self-protection against attack by infiltrating activated T-cells induced by apoptosis ,64. CellImmune-mediated apoptosis of thyrocytes is directed by CD8+ cells. Receptors on the target cell are triggered by lymphocyte ligands and/or released soluble factors are delivered to the target cell . ReceptoThe common apoptotic pathway consists of subsequent activation of specific intracellular proteases known as caspases. These caspases are themselves activated by specific proteolytic cleavage or may be activated by cleavage performed by other caspases. The caspase cascade ultimately induces enzymes that progressively destroy the cell and its genetic material, finally lead to cell death. The apoptosis, or programmed cell death, can be initiated by binding death ligands, such as TNF, TNF-related apoptosis-induced ligand (TRAIL) and FasL, to the cell surface. This in turn starts intracellular signal cascading of caspases .Several apoptosis signalling pathways, initiated by molecules such as FasL and TRAIL, have been shown to be active in thyrocytes and may be involved in destructive thyroiditis . Fas-medThus, the rate of thyrocyte apoptosis dictates the clinical outcome of thyroid autoimmunity. Though rare in normal thyroid, it markedly increases during HT, but not in GD. Therefore, regulation of thyrocyte survival is a crucial pathogenic determinant.S), that is the ratio of the prevalence of disease in siblings to the prevalence in the general population, can be used as a quantitative measure of the genetic contribution to the disease. Usually, a λS of more than five indicate a significant genetic contribution to the disease development. Based on historical data, the λS for AITD is estimated to be greater than 10, supporting a strong case of genetic influence on disease development [S value is about 28 for HT [Abundant epidemiologic data (population-based and family-based studies, twin studies) suggest a strong genetic contribution to the development of HT. The disease clusters in families ,73. Thyrelopment . Using H8 for HT .In Danish twin study, the concordance rates for Hashimoto's disease were 38% for monozygotic (MZ) twins and 0 for dizygotic (DZ) twins . For HT,Animal models of AITD still hold immense promise for the discovery of pathways, genes and environmental factors that determine the development of thyroid autoimmunity. Animals affected by experimental autoimmune thyroiditis (EAT) provide a unique opportunity to uncover disease-associated pathways, which are complicated to define in man.h4 spontaneously develops iodine-induced autoimmune thyroiditis but not diabetes [One of the oldest inbred models is the obese strain chicken (OS), which develops goitrous lympholytic thyroiditis with the subsequent atrophic lympholytic thyroiditis followed by a rapid onset of hypothyroidism . The biodiabetes . In partdiabetes .in vitro activated T cells from Tg-immunized mice [EAT can be induced in mice by injecting with murine or human Tg,  and in nzed mice . The indzed mice ,82.h4 mouse is induced by dietary iodine that supports epidemiologic data on human populations. In addition, the iodinified mouse represents high levels of IgG2b that is similar to HT patients expressing the predominance of IgG2 subclass, the human analog of murine IgG2b [h4 mice. Despite some differences between EAT and HT, these animal models have greatly contributed to the knowledge concerning the etiology and the pathogenesis of thyroid autoimmunity, most notably on the events occurring in the very early prodromal phases.Clinical features of EAT induced in the animal models mentioned above are similar to those of human HT. For example, autoimmune thyroiditis in the NOD-H2ne IgG2b . IgM clane IgG2b . HoweverMajor Histocompatibility Complex (MHC) molecules are thought to play an important role in the initial stages of the development of HT and AITD. MHC molecules, or Human Leukocyte Antigen (HLA) homologs, play a pivotal role in T-cell repertoire selection in the thymus and in antigen presentation in the periphery. Crystal structures of MHC molecules show a peptide-binding cleft containing the variable region of these molecules. Genetic polymorphism of the MHC molecule determines the specificity and affinity of peptide binding and T-cell recognition. Therefore, polymorphisms within MHC class I and class II loci can play a significant role in predisposition to autoimmune disease .g7) class II-knockout mice with EAT as a model for HT [A role of selected HLA class II genes susceptible to HT has been significantly clarified using transgenic NOD (H2Al for HT ,86. In ml for HT . A role l for HT . A role l for HT . In summl for HT ,87. Studl for HT . PolymorT cells recognize an antigenic peptide via interaction of their membrane T cell receptors (TcR) with antigen-MHC complexes presented on the surface of APC. Biased or restricted TcR gene use has been reported in a variety of human or murine autoimmune diseases . Biased 535–551) of the TPO amino acid sequence. This includes a cryptic epitope (TPO536–547) preferentially displayed after endogenous processing during inflammation [A new murine model that developed destructive thyroiditis with histological and clinical features comparable with human HT has been recently reported . The traammation . These rThe two-signal theory for T cell activation requires TcR engagement of its cognate antigen-MHC complex and CD28 binding to B7 ligands (B7-1 and B7-2) on APC. Activation of T cells results in increased expression of the cytotoxic T cell antigen-4 (CTLA-4) molecule that shares homology with CD28. Although B7-1 (CD80) and B7-2 (CD86) expressed on APC can bind to both CD28 and CTLA-4 (CD152), because of higher affinity, they preferentially bind to CTLA-4 on activated T cells and attenuate the T cell response .The importance of CTLA-4 in the down-regulation of T cell responses and in the induction of anergy and tolerance to alloantigens, tumors and pathogens, has been clearly demonstrated in experiments with CTLA-4 deficient mice. The mice developed a severe inflammatory disorder due to up-regulated proliferation of T cells ,96. CTLAGenes of the human MHC region are clustered on chromosome 6p21 and encode HLA glycoproteins and a number of additional proteins, which are predominantly related to immune response. The MHC locus itself contains three groups of genes: class I genes encoding HLA antigens A, B and C, class II genes encoding HLA-DR, DP and DQ molecules and class III genes .Previous studies in the early 1980s investigated the HLA locus in relation to the genetics of HT. Associations between HLA and HT have both been analysed by serologic typing of HLA and DNA typing using sequence-specific oligonucleotide probe analysis or restriction fragment length polymorphism. In Asians, HLA class I (A2, B16, B35, B46, B51, B54, C3) and HLA class II (DR2, DR9, DR53, DQ4) genes showed an association with the disease ,100-105.Early linkage, non-genome-wide studies of the HLA region have failed to detect linkage between the HLA locus and HT -129. UsiHowever, a linkage between the HLA region and HT was recently shown in the data set of 40 US multiplex families affected with AITD and type 1 diabetes . The linin vitro [The HLA region has been established to be involved in multiple autoimmune disorders . The mecin vitro  and in min vitro .Thyroid autoantigens need to occur in the thyroid or its draining lymph nodes in order for them to be presented by HLA molecules. It has been suggested that an aberrant intrathyroidal expression of MHC class II molecules by thyrocytes is necessary to initiate thyroid autoimmunity ,146. ThiGenetic contribution of HLA varies depending on the disease. HLA involvement in T1D, rheumatoid arthritis or multiple sclerosis is large and can constitute more than 50% of the genetic risk ,152. ConThe HLA class III region lies between class I and II genes and encodes important immunoregulatory proteins such as cytokines [tumour necrosis factor (TNF), lymphotoxin alpha (LT-α) and beta (LT-β)], complement components (C2, C4, properdin factor B) 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 (1,25(OH)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 (particularly, iodine intake and infection) 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