DNA microarray-based screening of differentially expressed genes related to acute lung injury and functional analysis.

OBJECTIVES: The purpose of this study was to identify differentially expressed genes (DEGs) related to acute lung injury (ALI) induced by sepsis with DNA microarray. MATERIALS AND METHODS: Gene expression profile GSE10474 was downloaded from Gene Expression Omnibus (GEO) database which includes 34 samples, among which 13 patients with ALI + sepsis and 21 patients with sepsis alone. The DEGs were identified between ALI + sepsis and sepsis alone samples using R, which were further analyzed using bioinformatics methods. Firstly, HitPredict was used to search protein-protein interactions of the DEGs. Secondly, WebGestalt was adopted for functional enrichment analysis of genes in the interaction networks. Finally, DNA methylation was analyzed to explain the differential expression. RESULTS: A total of 12 genes were identified as DEGs by comparing chip data from ALI + sepsis samples and those from sepsis alone samples, among which occludin (OCLN) and major histocompatibility complex, class II, DQ beta 1 (HLA-DQB1) had 21 and 6 interactors, respectively. Functional enrichment analysis revealed several significantly over-represented terms: cellular component organization, macromolecular organization and biosynthesis, and response to stimulus. In addition, methylation was found in the promoters of OCLN and HLA-DQB1. CONCLUSIONS: We screened DEGs in septic ALI samples, and several interesting genes were obtained, especially OCLN and HLA-DQB1. They may be developed into marker genes for diagnosis or treatment of ALI.

Ventilatory chemoresponsiveness, narcolepsy-cataplexy and human leukocyte antigen DQB1*0602 status.

We hypothesised that hypocretin (orexin) plays a role in the determination of ventilatory chemosensitivity. 130 patients with narcolepsy-cataplexy (mean ± SD age 20 ± 10 yrs, 69% male) and 117 controls (22 ± 6.9 yrs, 62% male) were recruited and tested for human leukocyte antigen (HLA)-DQB1*0602 status, hyperoxia hypercapnic (change in minute ventilation (δV'(E))/carbon dioxide tension (δP(CO(2))) L·min(-1)·mmHg(-1)) and hypoxic (δV'(E) /change in arterial oxygen saturation measured by probe oximetry (δS(p,O(2))) L·min(-1) per %S(p,O(2))) responsiveness, and by spirometry. Hypocretin deficiency was determined either by measures of cerebrospinal fluid hypocretin-1 (37 patients) or by positive HLA-DQB1*0602 status. All patients and 49% of controls underwent polysomnography and multiple sleep latency testing. Despite similar spirometric values, patients had a higher apnoea/hypopnoea index (AHI) (2.8 ± 5.4 versus 0.8 ± 1.6 h(-1); p = 0.03) and lower minimal oxygen saturation during sleep (87% ± 7 versus 91 ± 4%; p = 0.0002), independent of age, sex and body mass index. Patients had depressed hypoxic responsiveness (0.13 ± 0.09 versus 0.19 ± 0.13 L·min(-1) per %S(p,O(2)); p<0.0001), independent of AHI, but hypercapnic responsiveness did not differ. Examined by HLA status, positive (26 out of 117) controls had lower hypoxic but similar hypercapnic responsiveness than those marker-negative (0.13 ± 0.08 versus 0.20 ± 0.14 L·min(-1) per %S(p,O(2)); p<0.0001). Thus, a lower hypoxic responsiveness in the narcolepsy-cataplexy group is a result of DQB1*0602 status rather than the clinical features of disease.

Body mass index-independent metabolic alterations in narcolepsy with cataplexy.

STUDY OBJECTIVES: To contribute to the anthropometric and metabolic phenotyping of orexin-A-deficient narcoleptic patients, and to explore a possible risk of their developing a metabolic syndrome. DESIGN: We performed a cross-sectional study comparing metabolic alterations in patients with narcolepsy with cataplexy (NC) and patients with idiopathic hypersomnia without long sleep time. SETTING: University hospital. PATIENTS: Fourteen patients with narcolepsy with cataplexy and 14 sex and age-matched patients with idiopathic hypersomnia without long sleep time. INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: Metabolic parameters were evaluated by measuring body mass index (BMI), waist circumference (also with abdominal computed tomography), blood pressure, and daily calorie intake (3-day diary). Chronotypes were assessed through the morningness-eveningness questionnaire. Lumbar puncture for cerebrospinal fluid orexin-A determination and HLA typing were performed. Patients with narcolepsy with cataplexy (all HLA DQB1*0602 positive and with cerebrospinal fluid orexin-A levels < 110 pg/mL) had a higher BMI and BMI-independent metabolic alterations, namely waist circumference, high-density lipoprotein cholesterol, and glucose/insulin ratio (an insulin resistance index), with respect to patients with idiopathic hypersomnia without long sleep time (cerebrospinal fluid orexin-A levels > 300 pg/mL). Despite lower daily food intake, patients with narcolepsy with cataplexy displayed significant alterations in metabolic parameters resulting in a diagnosis of metabolic syndrome in more than half the cases. CONCLUSIONS: BMI-independent metabolic alterations and the relative hypophagia of patients with narcolepsy with cataplexy, as compared with patients with idiopathic hypersomnia without long sleep time, suggest that orexin-A influences the etiology of this phenotype. Moreover, considering that these dysmetabolic alterations are present from a young age, a careful metabolic follow-up of patients diagnosed with narcolepsy with cataplexy is mandatory.

[Gestational microchimerism in human diseases]. / Microchimérisme postgestationnel en pathologie humaine.

Microchimerism is defined as the persistence within an individual of a low level of cells or DNA derived from another individual. The most common source of microchimerism is pregnancy. Bidirectional transplacental materno-fetal cell trafficking occurs during most pregnancies and chimeric cells can persist in blood or tissues for decades after childbirth. It can leads to fetal (fetal-maternal transfer) or maternal (maternal-fetal transfer) microchimerism. Characterization of cells implied in microchimerism is incompletely known: it could be at least partly, fetal progenitors cells with ability of self renewal and specific differentiation according to the surrounding tissue. The transferred fetal cells can be recruited in various injured maternal tissues (auto-immune diseases, stroma of various tumours associated with pregnancy) but their precise biological role is uncertain. Microchimerism has been implicated in the pathogenesis of autoimmune diseases (especially systemic sclerosis) but current data suggest that fetal microchimeric cells may participate in maternal physiological response and injured tissue repair. Similar observations were made with maternal microchimerism (excepted with juvenile idiopathic inflammatory myopathies whose immunopathogenesis is probably related with transfer of maternal immune cells).

MHC loci affecting cervical cancer risk: distinguishing the effects of HLA-DQB1 and non-HLA genes TNF, LTA, TAP1 and TAP2.

Cervical cancer has been associated with specific human leukocyte antigen (HLA) haplotypes/alleles and with polymorphisms at the nearby non-HLA loci TNF, LTA, TAP1 and TAP2. Distinguishing effects of individual loci in the major histocompatibility complex (MHC) region are difficult due to the complex linkage disequilibrium (LD) pattern characterized by high LD, punctuated by recombination hot spots. We have evaluated the association of polymorphism at HLA class II DQB1 and the TNF, LTA, TAP1 and TAP2 genes with cervical cancer risk, using 1306 familial cases and 288 controls. DQB1 was strongly associated; alleles *0301, *0402 and (*)0602 increased cancer susceptibility, whereas *0501 and *0603 decreased susceptibility. Among the non-HLA loci, association was only detected for the TAP2 665 polymorphism, and interallelic disequilibrium analysis indicated that this could be due to LD with DQB1. As the TAP2 665 association was seen predominantly in non-carriers of DQB1 susceptibility alleles, we hypothesized that TAP2 665 may have an effect not attributable to LD with DQB1. However, a logistic regression analysis suggested that TAP2 665 was strongly influenced by LD with DQB1. Our results emphasize the importance of large sample sizes and underscore the necessity of examining both HLA and non-HLA loci in the MHC to assign association to the correct locus.

[Role of HLA-DR and HLA-DQ alleles in multibacillary leprosy and paucibacillary leprosy in the province of Chaco (Argentina)]. / Papel de las moléculas HLA-DR y HLA-DQ en la lepra multibacilar y paucibacilar en la provincia del Chaco; Argentina.

OBJECTIVES: Segregation analyses in several populations have suggested a relationship between specific human leukocyte antigen (HLA) class II alleles and the development of different types of leprosy. The aim of this study was to determine the frequency of HLA class II DR and DQ alleles among leprosy patients in Chaco province, northeast Argentina, in an effort to determine whether these alleles might be involved in the development of the multibacillary (MB) and paucibacillary (PB) forms of leprosy. PATIENTS AND METHODS: Samples from 89 leprosy patients (MB = 70, PB = 19) and 112 healthy control subjects were analyzed. The HLA-DRB1 and HLA-DQB1 alleles were determined by PCR amplification and reverse hybridization with sequence-specific oligonucleotide probes, and analyzed with the INNO-LiPA typing system and LiPA software. DQB1*0201/0202/0203 in patients with MB leprosy and DRB1*04 in patients with PB leprosy were detected at significantly lower frequencies as compared with the normal controls. RESULTS: These data indicate that DQB1* 0201/0202/0203 may be a protective factor in MB leprosy and DRB1*04 in PB leprosy. DISCUSSION: We attribute the differences between our findings and those of other authors to the fact that the Caucasian inhabitants of Chaco include a considerable mixture of South American natives (Guaraníes and Tobas).

A structural and immunological basis for the role of human leukocyte antigen DQ8 in celiac disease.

The risk of celiac disease is strongly associated with human leukocyte antigen (HLA) DQ2 and to a lesser extent with HLA DQ8. Although the pathogenesis of HLA-DQ2-mediated celiac disease is established, the underlying basis for HLA-DQ8-mediated celiac disease remains unclear. We showed that T helper 1 (Th1) responses in HLA-DQ8-associated celiac pathology were indeed HLA DQ8 restricted and that multiple, mostly deamidated peptides derived from protease-sensitive sites of gliadin were recognized. This pattern of reactivity contrasted with the more absolute deamidation dependence and relative protease resistance of the dominant gliadin peptide in DQ2-mediated disease. We provided a structural basis for the selection of HLA-DQ8-restricted, deamidated gliadin peptides. The data established that the molecular mechanisms underlying HLA-DQ8-mediated celiac disease differed markedly from the HLA-DQ2-mediated form of the disease. Accordingly, nondietary therapeutic interventions in celiac disease might need to be tailored to the genotype of the individual.

Spontaneous myocarditis mimicking human disease occurs in the presence of an appropriate MHC and non-MHC background in transgenic mice.

Most individuals have viral infections at some point in their life, however, only few develop autoreactivity to cardiac myosin following infection resulting in myocarditis suggesting a genetic predisposition. Most mouse models of myocarditis are induced by viral infection or by immunization with cardiac myosin. We generated HLA-DR3.Abetao and HLA-DQ8.Abetao transgenic mice in NOD and HLA-DQ8.Abetao in B10 background to study spontaneous autoimmunity. A high mortality was observed in NOD.DQ8 female mice 16 weeks or older. Echocardiography showed marked systolic dysfunction. Histopathology of various organs revealed an enlarged heart with mononuclear infiltrate consisting of CD4 and Mac-1+ cells and myocyte necrosis. The autoimmunity was associated with the presence of spontaneous autoreactive T cells and antibodies to cardiac myosin. Serologically, mice were negative for all known mouse viruses. NOD.DR3.Abetao, the transgene negative littermates, NOD, and B10.DQ8 Abetao mice had no gross or microscopic cardiac pathology. Spontaneous cellular and humoral response to cardiac myosin suggests that NOD.DQ8 may harbor autoreactive cells that can lead to spontaneous myocarditis and dilated cardiomyopathy. HLA-DQ8 is required for the predisposition to the spontaneous autoreactivity while NOD background influences onset and progression of disease. This model of myocarditis occurs predominantly in female mice and may provide insight into the pathogenesis of heart disease in women.

The spectrum of HLA-DQ and HLA-DR alleles, 2006: a listing correlating sequence and structure with function.

The list of alleles in the HLA-DRB, HLA-DQA, and HLA-DQB gene loci has grown enormously since the last listing in this journal 8 years ago. Crystal structure determination of several human and mouse HLA class II alleles, representative of two gene loci in each species, enables a direct comparison of ortholog and paralog loci. A new numbering system is suggested, extending earlier suggestions by [Fremont et al. in Immunity 8:305-317, (1998)], which will bring in line all the structural features of various gene loci, regardless of animal species. This system allows for structural equivalence of residues from different gene loci. The listing also highlights all amino acid residues participating in the various functions of these molecules, from antigenic peptide binding to homodimer formation, CD4 binding, membrane anchoring, and cytoplasmic signal transduction, indicative of the variety of functions of these molecules. It is remarkable that despite the enormous number of unique alleles listed thus far (DQA = 22, DQB = 54, DRA = 2, and DRB = 409), there is invariance at many specific positions in man, but slightly less so in mouse or rat, despite their much lower number of alleles at each gene locus in the latter two species. Certain key polymorphisms (from substitutions to an eight-residue insertion in the cytoplasmic tail of certain DQB alleles) that have thus far gone unnoticed are highly suggestive of differences or diversities in function and thus call for further investigation into the properties of these specific alleles. This listing is amenable to supplementation by future additions of new alleles and the highlighting of new functions to be discovered, providing thus a unifying platform of reference in all animal species for the MHC class II allelic counterparts, aiding research in the field and furthering our understanding of the functions of these molecules.

DQB1*0301 and DQB1*0601 modulate narcolepsy susceptibility in Koreans.

The association of narcolepsy with HLA-DQB1*0602 is established in Japanese, African-Americans, European, and North American Caucasians. We examined DRB1, DRB3, DRB4, DRB5, DQA1, and DQB1 in 163 patients with centrally mediated daytime sleepiness (100 with narcolepsy) and 211 Korean controls. In this population, the DQB1*0602 association was always evident in the context of the DRB1*1501-DQA1*0102-DQB1*0602 haplotype. The DQB1*0602 association was highest in cases with hypocretin deficiency (100% vs 13% in controls), most of which had narcolepsy-cataplexy (81%). A weaker DQB1*0602 (45%) association was present in cases without cataplexy. No human leukocyte antigen (HLA) association was present in idiopathic hypersomnia or in cases with normal cerebrospinal fluid (CSF) hypocretin-1. As in other populations, DQB1*0602 homozygosity increased risk in cases with cataplexy and/or hypocretin deficiency (odds ratio = 2.0 vs heterozygotes). Non-DQB1*0602 allelic effects were also observed but could not be interpreted in the context of DQB1*0602 overabundance and linkage disequilibrium. We therefore next analyzed compound heterozygote effects in 77 subjects with either hypocretin deficiency or cataplexy and one copy of DRB1*1501-DQA1*0102-DQB1*0602, a sample constructed to maximize etiologic homogeneity. In this analysis, we found additional predisposing effects of DQB1*0301 and protective effects for DQA1*0103-DQB1*0601. Unexpectedly, the predisposing effects of DQB1*0301 were present in the context of various DQA1-bearing haplotypes. A predisposing effect of DQA1*0303 was also suggested. These results indicate a remarkable consistency in the complex HLA association present in narcolepsy across multiple ethnic groups.

A functional variant in the CD209 promoter is associated with DQ2-negative celiac disease in the Spanish population.

AIM: To address the role of CD209 in celiac disease (CD) patients. Non-human leukocyte antigen (HLA) genetic factors in CD predisposition are poorly understood, and environmental factors like infectious pathogens may play a role. CD209 is a dendritic and macrophage surface molecule involved in pathogen recognition and immune activation. Recently, a functional variant in the promoter of the CD209 gene (-336A/G) has been shown to affect the transcriptional CD209 activity in vitro and it has been associated with a higher susceptibility to/or severity of infection. METHODS: The study population was composed of two case-control cohorts of 103 and 386 CD patients and 312 y 419 healthy controls as well as a panel of 257 celiac families. Genotyping for the -336A/G CD209 promoter polymorphism was performed using a TaqMan 5' allelic discrimination assay. HLA-DQ was determined by hybridization with allele specific probes. RESULTS: Initially, the case-control and familial studies did not find any association of the -336 A/G CD209 genetic variant with CD susceptibility. However, the stratification by HLA-DQ2 did reveal a significant association of CD209 promoter polymorphism in the HLA-DQ2 (-) group (carrier A vs GG in DQ2 (-) vs DQ2 (+) patients (P = 0.026, OR = 3.71). CONCLUSION: The -336G CD209 allele seems to be involved in CD susceptibility in HLA-DQ2 (-) patients. Our results might suggest a possible role of pathogens in the onset of a minor group of CD patients.

Human leukocyte antigen-DQ8 transgenic mice: a model to examine the toxicity of aerosolized staphylococcal enterotoxin B.

Staphylococcal enterotoxins (SEs) belong to a large group of bacterial exotoxins that cause severe immunopathologies, especially when delivered as an aerosol. SEs elicit the release of lethal amounts of cytokines by binding to major histocompatibility complex (MHC) class II and cross-linking susceptible T-cell receptors. Efforts to develop effective therapeutic strategies to protect against SEs delivered as an aerosol have been hampered by the lack of small animal models that consistently emulate human responses to these toxins. Here, we report that human leukocyte antigen-DQ8 (HLA-DQ8) transgenic (Tg) mice, but not littermate controls, succumbed to lethal shock induced by SEB aerosols without potentiation. Substantial amounts of perivascular edema and inflammatory infiltrates were noted in the lungs of Tg mice, similar to the pathology observed in nonhuman primates exposed by aerosol to SEB. Furthermore, the observed pathologies and lethal shock correlated with an upsurge in proinflammatory cytokine mRNA gene expression in the lungs and spleens, as well as with marked increases in the levels of proinflammatory circulating cytokines in the Tg mice. Unlike the case for littermate controls, telemetric evaluation showed significant hypothermia in Tg mice exposed to lethal doses of SEB. Taken together, these results show that this murine model will allow for the examination of therapeutics and vaccines developed specifically against SEB aerosol exposure and possibly other bacterial superantigens in the context of human MHC class II receptors.

Seasonality in clinical onset of type 1 diabetes in belgian patients above the age of 10 is restricted to HLA-DQ2/DQ8-negative males, which explains the male to female excess in incidence.

AIMS/HYPOTHESIS: Type 1 diabetes arises from an interplay between environmental and genetic factors. The reported seasonality at diagnosis supports the hypothesis that currently unknown external triggers play a role in the onset of the disease. We investigated whether a seasonal pattern is observed at diagnosis in Belgian Type 1 diabetic patients, and if so whether seasonality varies according to age, sex and genetic risk, all known to affect the incidence of Type 1 diabetes. METHODS: The seasonal pattern at clinical diagnosis was assessed in 2176 islet antibody-positive diabetic patients aged 0 to 39 years diagnosed between 1989 and 2000. Additional stratification was performed for age, sex and HLA-DQ genotype. RESULTS: Overall, a significant seasonal pattern at clinical diagnosis of diabetes was observed (p<0.001). More subjects were diagnosed in the period of November to February (n=829) than during the period of June to September (n=619) characterised by higher averages of maximal daily temperature and daily hours of sunshine. However, the seasonal pattern was restricted to patients diagnosed above the age of 10 (0-9 years: p=0.398; 10-19 years: p<0.001; 20-29 years: p=0.003; 30-39 years: p=0.015). Since older age at diagnosis is associated with a male to female excess and a lower prevalence of the genetic accelerator HLA-DQ2/DQ8, we further stratified the patients aged 10 to 39 years (n=1675) according to HLA-DQ genotype and sex, and we found that the seasonal pattern was largely restricted to male subjects lacking DQ2/DQ8 (n=748; p<0.00 vs all others: n=927; p=0.031). CONCLUSIONS/INTERPRETATION: In a subgroup of male patients diagnosed over the age of 10, the later stages of the subclinical disease process may be more driven by sex- and season-dependent external factors than in younger, female and genetically more susceptible subjects. These factors may explain the male to female excess in diabetes diagnosed in early adulthood.

A spontaneous model for autoimmune myocarditis using the human MHC molecule HLA-DQ8.

Genome-wide analyses have shown that the MHC class II region is the principal locus that confers susceptibility to a number of human autoimmune diseases. Due to the high degree of linkage disequilibrium across the MHC, it has been difficult to dissect the contribution of individual genes to disease susceptibility. As a result, intensive efforts have been made to generate mice transgenic for human class II molecules as models of autoimmune disease. However, in every case, additional manipulations-such as immunization with Ag in adjuvant, expression of immunostimulants on target tissues, or coexpression of TCR transgenes-have been required to induce disease. In this study, we show that expression of the human HLA-DQ8 (DQA1*0301/DQB1*0302) molecule alone in three lines of transgenic nonobese diabetic murine class II-deficient (mII(-/-)) mice results in the spontaneous development of autoimmune myocarditis. The disease shares key features of human myocarditis and was characterized by lymphocytic infiltrates in the myocardium and cardiac myocyte destruction, circulating IgG autoantibodies against cardiac myosin heavy chain, and premature death due to heart failure. We demonstrate that myocarditis could be transferred into healthy HLA-DQ8(+)RAG-1(-/-)mII(-/-) nonobese diabetic recipients with lymphocytes, but not sera. It has been widely thought that autoimmune myocarditis is of infectious etiology, with the immune responses arising secondary to cardiac damage from pathogens. These studies provide direct experimental evidence that spontaneous autoimmune myocarditis can occur in the absence of infection and that expression of HLA-DQ8 confers susceptibility to this organ-specific autoimmune disease.

Expression and function of HLA-DR3 and DQ8 in transgenic mice lacking functional H2-M.

H2-M or HLA-DM are non-classical class II molecules encoded by the MHC and play an important role during antigen presentation. They catalyze exchange of CLIP (Class II-associated invariant chain peptide) or other low-affinity peptides bound to class II molecules for peptides capable of more efficient binding. The phenotype of mice lacking H2-M is determined by the allotype of the MHC class II molecules expressed. In general, H2-M deficiency does not affect the surface expression of mature class II molecules. The class II molecules in such cases predominantly contain CLIP in their peptide-binding groove. In some mice strains, H2-M deficiency results in defective CD4+ T-cell development accompanied by defective responses to conventional antigens and superantigens. Even though the HLA class II molecules show similar dependency for HLA-DM for presenting antigens in vitro, their interaction in vivo is not known. By using transgenic approach we show here that DQ8 and DR3 are expressed at normal levels in H2-M-deficient mice and the CD4+ T-cell development is unaltered. However, the ability of DQ8 molecules to present peptide antigens is compromised in a H2-M-deficient state. Presentation of exogenous bacterial superantigens by both DQ8 and DR3 is unaffected in H2-M-deficient mice. Unexpectedly, Staphylococcal Enterotoxin B-induced systemic IFN-gamma production was significantly higher in H2-M-deficient DQ8/DR3 transgenic mice and these mice were susceptible to SEB-induced toxic shock at doses that are non-lethal to H2-M-sufficient counterparts.

Autoimmune diabetes in HLA-DR3/DQ8 transgenic mice expressing the co-stimulatory molecule B7-1 in the beta cells of islets of Langerhans.

The major predisposing genetic component in type 1 diabetes (T1D) maps to the MHC locus in both mice and humans. To better understand the HLA class II association with disease pathogenesis, we bred mice expressing HLA-DQ8 and -DR3, either alone or in combination, to transgenic mice expressing the co-stimulatory molecule B7-1 in the beta cells of islets of Langerhans. Spontaneous diabetes occurred only in RIP-B7-1 transgenic mice expressing transgenic HLA-DR3 or -DQ8 molecules and the incidence of diabetes was comparable between the two (approximately 30% in either sex up to 50 weeks of age). Presence of DR3 and DQ8 together only marginally elevated the overall incidence of spontaneous disease (38%). Non-specific activation of T cells by superantigen and provision of concomitant co-stimulation through 4-1BB (CD137) by an agonistic antibody did not accelerate the incidence of diabetes over a short period of time. Neither the antibody-mediated depletion of CD25+ T cells nor sublethal, whole-body irradiation of young, naive HLA transgenic mice expressing RIP-B7-1 resulted in diabetes. However, administration of only two doses of the beta cell toxin streptozotocin (STZ; 40 mg/kg) induced autoimmune diabetes in 85% of mice within 7 weeks after STZ treatment only when B7-1 was expressed on the pancreatic beta cells. This effect was HLA dependent as none of the STZ-treated RIP-B7-1 transgenic mice lacking HLA class II developed diabetes. In conclusion, this study confirmed the diabetogenic potential of HLA-DQ8 and established the role of HLA-DR3 in the pathogenesis of T1D.

High GAD65 autoantibody levels in nondiabetic adults are associated with HLA but not with CTLA-4 or INS VNTR.

OBJECTIVES: To explore the relationship between genetic background and antibody levels in a nondiabetic population. We evaluated if high levels of autoantibodies against the 65 kDa isoform of glutamic acid decarboxylase (GAD65Ab), were associated with high-risk genes, i.e. HLA, CTLA-4 and INS VNTR genes. DESIGN AND SUBJECTS: Seventy-five (M/F 39/36) subjects exceeding the 95th percentile of GAD65 autoantibody index and 75 age and sex matched subjects below the 95th percentile, randomly selected amongst participants in the Västerbotten Intervention Programme. METHODS: The GAD65 Ab were measured in a radioligand-binding assay. HLA class II typing was performed by an oligoblot hybridization method. CTLA-4 repeat length was analysed and divided into short forms and long forms. Class I and class III alleles of INS VNTR were detected. Differences in distribution were tested by Pearson chi-square with Yates correction. Odds ratios (OR) were used to compare groups calculated with Cochran's and Mantel-Haenszel statistics. RESULTS: The DQB1*0201-DQA1*0501-DRB1*03 haplotype was increased in subjects with high GAD65Ab levels (P = 0.04). This increase seemed to be explained by a difference in haplotype frequencies amongst men (P = 0.01). Calculating OR showed a significant association between the DQB1*0201-DQA1*0501-DRB1*03 haplotype and elevated levels of GAD65Ab in all subjects (OR 2.2, 95% CI 1.02-4.9) as well as in men (OR 4.6, 95% CI 1.3-15.9). There was no association between high levels of GAD65Ab and either INS VNTR or CTLA-4 polymorphisms. CONCLUSION: Our study suggests that adult males with the DQB1*0201-DQA1*0501-DRB1*03 haplotype tend to develop high GAD65Ab titres. As none of these subjects have developed diabetes these data suggest that HLA may be important in GAD65Ab formation but that additional factors are required for the progression to overt type 1 diabetes.

HLA-DQ determines the response to exogenous wheat proteins: a model of gluten sensitivity in transgenic knockout mice.

We have investigated the genetic basis of the immune response to dietary gluten in HCD4/DQ8 and HCD4/DQ6 double transgenic mice. Mice were immunized with gluten i.p. or individual peptides s.c. and spleen or draining lymph node T cells were challenged in vitro. Strong proliferative responses to gluten were seen in the HCD4/DQ8 mice, whereas the HCD4/DQ6 mice responded to gluten poorly. A series of overlapping peptides spanning gliadin were synthesized. The HCD4/DQ8 mice reacted to many of the individual peptides of gliadin, while the HCD4/DQ6 mice were relatively unresponsive. T cells isolated from HCD4/DQ8 mice also responded well to modified (deamidated) versions of the gliadin peptides, whereas HCD4DQ6 mice did not. The T cell response to gluten was CD4 dependent and DQ restricted and led to the production of cytokines IL-6, TGF-beta, and IL-10. Finally, intestinal lymphocytes isolated from gluten-fed HCD4/DQ8 mice displayed an activated phenotype. These data suggest that this HLA class II transgenic murine model of gluten sensitivity may provide insight into the initiation of the MHC class II-restricted gluten sensitivity in celiac disease.

Susceptibility locus for non-obstructive azoospermia is localized within the HLA-DR/DQ subregion: primary role of DQB1*0604.

Non-obstructive azoospermia is a male infertility characterized by no or little sperm in semen as a result of a congenital dysfunction in spermatogenesis. Previous studies have reported a higher prevalence of particular human leukocyte antigen (HLA) antigens in non-obstructive azoospermia. As the expression of the RING3 gene located in the HLA class II region was predominant in the testis, mainly around spermatids and pachytene spermatocytes, it is tempting to speculate that RING3 is one of the strong candidate genes responsible for the pathogenesis of the disease. In this study, the genetic polymorphism in the RING3 gene was investigated by the direct sequencing technique. As a result, a total of 14 single nucleotide polymorphisms were identified. Among them, six were localized in the coding region but none of them was accompanied by an amino-acid substitution. No significant difference in the allelic distribution at these 14 polymorphic sites was observed between the patients and healthy controls, suggesting that the susceptible gene for non-obstructive azoospermia is not the RING3 gene. Then, in order to map the susceptibility locus for non-obstructive azoospermia precisely within the HLA region, 11 polymorphic microsatellite markers distributed from the SACM2L gene just outside the HLA class II region (187 kb telomeric of the DPB1 gene) to the OTF3 gene in the HLA class I region were subjected to association analysis in the patients. Statistical analysis of distribution in the allelic frequency at each microsatellite locus demonstrated that the pathogenic gene for non-obstructive azoospermia is located within the HLA-DR/DQ subregion. In fact, DRB1*1302 and DQB1*0604 were found to be strongly associated with non-obstructive azoospermia by polymerase chain reaction-based DNA typing. Further, haplotype analysis suggested that the DQB1*0604 allele may play a decisive role in the pathogenesis of non-obstructive azoospermia.