Comprehensive flow cytometric reference intervals of leukocyte subsets from six study centers across Europe.

A group of European FOCIS Centers of Excellence adapted panels of the Human Immunophenotyping Consortium (HIPC) for whole blood analysis. Using four core panels [T/regulatory T cell/B/natural killer (T/Treg /B/NK) and myeloid cells] the main leukocyte populations were analyzed in a clinical-diagnostic setting in a harmonized manner across different platforms. As a first step, the consortium presents here the absolute and relative frequencies of the leukocyte subpopulations in the peripheral blood of more than 300 healthy volunteers across six different European centers.

A study of 51 subtypes of peripheral blood immune cells in newly diagnosed young type 1 diabetes patients.

Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing beta cells in pancreatic islets. Various immune cell populations are involved in disease development and natural course. However, to our knowledge, so far there are no comprehensive comparative investigations of all main immune cell populations and their most important subsets at the onset of disease. Therefore, in the current study, we analyzed 51 peripheral blood immune cell populations in 22 young T1D patients and in 25 age-matched controls using a comprehensive polychromatic flow cytometry panel developed for whole blood by the COST Action no. BM0907 ENTIRE (European Network for Translational Immunology Research and Education: From Immunomonitoring to Personalized Immunotherapy) consortium. We found that in T1D patients, frequencies and absolute counts of natural killer (NK) cells, dendritic cells (DC) and T cells, as well as their respective subsets, were significantly altered compared to controls. Further, we observed that changes in several cell populations (e.g. CD14+ CD16+ non-classical monocytes, plasmablasts) were dependent on the age of the patient. In addition to age-related changes, we also found that alterations in immune cell patterns were associated with parameters such as the presence of ketoacidosis and C-peptide serum levels. Our study provides a foundation for future studies investigating different cell lineages and their role in T1D and illustrates the value of polychromatic flow cytometry for evaluating all main peripheral immune cells and their subsets in whole blood samples.

Differences in B7 and CD28 family gene expression in the peripheral blood between newly diagnosed young-onset and adult-onset type 1 diabetes patients.

Type-1 diabetes (T1D) is a heterogeneous autoimmune disease, and there are pathogenetic differences between young- and adult-onset T1D patients. We hypothesized that the expressions of genes involved in costimulatory immune system pathways in peripheral blood are differently regulated in young- and adult-onset T1D. Study group I consisted of 80 children, adolescents, and young adults (age range 1.4-21.4 y; 31 controls and 49 T1D patients). Study group II consisted of 48 adults (age range 22.0-78.4 y; 30 controls and 18 T1D patients). The mRNA expression levels of CD86, CD28, CD25, CD226, CD40, BTLA, GITR, PDCD1, FoxP3, TGF-ß, ICOS, sCTLA4, flCTLA4, and CD80 were measured in peripheral blood. Genetic polymorphisms (HLA haplotypes; rs231806, rs231775, and rs3087243 in CTLA4; rs763361 in CD226; and rs706778 in CD25) and T1D-associated autoantibodies were analyzed. In group I, there was significantly lower expression of CD226 in T1D patients than in the controls. In group II, there were significantly higher expression levels of CD86 and TGF-ß in T1D patients than in the controls. In the T1D patients in group I, the upregulated CD80 expression correlated with the expression of both CTLA4 splice variants (sCTLA4 and flCTLA4). In contrast, in group II, upregulated CD86 correlated with TGF-ß and CD25. In group I, the inhibitory CD80-CTLA4 pathway was activated, whereas, in group II, the activation CD86-CD28 pathway and TGF-ß production were activated. These results emphasize the differences between young-onset and adult-onset T1D in the regulation of costimulatory pathways. These differences should be considered when developing novel treatments for T1D.

The prevalence and phenotypic characteristics of spontaneous premature ovarian failure: a general population registry-based study.

STUDY QUESTION: What is the measured prevalence and phenotype of spontaneous premature ovarian failure (POF) in the general population? SUMMARY ANSWER: Spontaneous POF occurs in ∼1% of the general population with unique phenotype of post-menopausal ageing distinct from surgically induced premature menopause. WHAT IS KNOWN ALREADY: POF is multifactorial ovarian quiescence before the age of 40. The clinical features of POF are diverse and the population prevalence of POF is still not known. STUDY DESIGN, SIZE, DURATION: This population-depictive registry-based case-cohort study included 34 041 women from the Estonian Genome Center registered between 2003 and 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS: Spontaneous POF was selected retrospectively by excluding other causes for premature menopause under the age of 40 (N = 310) and women with surgically induced premature menopause participated as a reference group (N = 242). MAIN RESULTS AND THE ROLE OF CHANCE: The prevalence of spontaneous POF was 0.91% (0.81-1.02%) among women of the general population in Estonia. In women with POF, menarche occurred a few months later than in the reference group and a significantly higher number of live births during their reproductive life was recorded. Women with POF also consumed less alcohol and had smaller waist-to-hip ratios than those in the reference group, although both groups of women were similar in body mass index a decade after menopause. The prevalence of concomitant diseases was similar between two groups of women by their fifties, but the pattern of onset of these diseases was different. Surgically induced premature menopause associated with faster development of osteoporosis, hypertension, and connective tissue diseases, but slower development of allergies, compared with spontaneous POF. The age of menopause was determined by irregular menstrual cycles, but not by the length of regular menstrual cycles, the age of menarche, the number of pregnancies or live births, smoking or alcohol consumption, or the use of oral contraceptives for some time during the reproductive period. LIMITATIONS, REASONS FOR CAUTION: POF is rarely stated in medical records and cannot be diagnosed retrospectively by standard procedures. Therefore the data on all cases of women with primary amenorrhea or premature menopause before the age of 40 were requested from the registry and spontaneous POF was predicted retrospectively by excluding other extraovarian causes for premature menopause. Since the current study is retrospective registry-based data analysis, no genetic evaluation concerning possible candidate genes and no blood analysis concerning immunologic disorders could be performed to describe etiopathogenesis of POF. WIDER IMPLICATION OF THE FINDINGS: Spontaneous POF most likely comprises several diseases with different etiopathologies and there may be a unique phenotype of post-menopausal ageing distinct from that in surgically induced premature menopause. Irregular menstrual cycles may be a prospective risk for developing spontaneous POF. Compared with spontaneous POF, surgically induced premature menopause associates with faster development of age-related diseases. The data point to new ideas and hypotheses for further studies on etiopathologies and treatment options for spontaneous POF. STUDY FUNDING/COMPETING INTERESTS: The study was funded by grant SF0180044s09, SF0180027s10 and IUT20-43 from the Estonian Ministry of Education and Research, Enterprise Estonia, grant no EU30020, Eureka's EUROSTARS programme grant (NOTED, EU41564). No competing interests are declared.

Interferon and interferon-inducible gene activation in patients with type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease that is thought to be triggered by environmental factors in genetically susceptible individuals. Enteroviruses have been mentioned as the most probable induction component of the disease. Nevertheless, the literature is controversial regarding the association of T1D with viral infection and first-line antiviral defence components, for example type I interferons (IFNs). Our aim was to test the hypothesis that an abnormality in IFN-stimulated gene patterns may cause a failure in immunological tolerance and, thereby, initiate T1D as an autoimmune disorder. We studied material from 64 T1D and 36 control subjects, divided into two age groups: <10 years and ≥10 years old. Using a relative gene expression method, we observed a lower expression of interferon-induced helicase 1 (IFIH1) and other type I IFN-induced genes in the blood cells of T1D subjects, especially subjects under 10 years old, in spite of their higher IFN levels as measured by the pSTAT1-inducing capacity of their sera. Likewise, freshly purified CpG-stimulated cells from T1D patients showed significantly lower upregulation of IFN-induced genes, that is IFIH1 and CXCL10, compared to cells from the control group. The identified dysregulation in the IFN-α-induced antiviral response in T1D patients, especially in early childhood, could be one of the factors affecting T1D development.

The CD226 gene in susceptibility of type 1 diabetes.

The rs763361 single nucleotide polymorphism (SNP) within the CD226 gene has recently been reported as a novel susceptible locus for type 1 diabetes. The CD226 gene is implicated in the regulation of a number of cells involved in immune mechanisms leading to beta-cell destruction in type 1 diabetes. The aim of the present study was to confirm the association of the CD226 gene with type 1 diabetes in Estonian population. The TT genotype [odds ratio (OR) = 2.29, 95% confidence interval (CI) = 1.25-4.18, P = 0.0071) and the T allele (OR = 1.48, 95% CI = 1.11-1.98, P = 0.0084) of the rs763361 SNP were associated with the risk of type 1 diabetes. The current study replicates the novel association of the rs763361 SNP in susceptibility of type 1 diabetes and supports the CD226 gene as a susceptible candidate locus for type 1 diabetes outside the major histocompatibility complex region.

Cytotoxic T-lymphocyte antigen 4 gene polymorphisms are associated with latent autoimmune diabetes in adults.

BACKGROUND: Cytotoxic T lymphocyte antigen-4 (CTLA-4) molecule is an important regulator of T cell activation involved in the down-regulation of immune response. Polymorphisms within the CTLA-4 gene have been suggested to confer susceptibility to autoimmune endocrine disorders. METHODS: In order to evaluate the impact of allelic variants of the CTLA-4 gene in latent autoimmune diabetes in adults (LADA), the CT60 A/G SNP and the CTBC217_1 C/T SNP were studied in a population of Estonian origin, including 61 LADA patients and 230 controls. RESULTS: It was found that the CT60 GG genotype (p=0.004) and the CTBC217_1 TT genotype (p=0.007) were significant associated with LADA. CONCLUSIONS: Our investigation revealed that not only type 1 diabetes but also LADA is associated with CTLA-4 gene polymorphisms. The role of CTLA-4 gene in the pathogenesis of LADA is open and needs further investigations.

Protein tyrosine phosphatase non-receptor type 22 gene variants at position 1858 are associated with type 1 and type 2 diabetes in Estonian population.

Protein tyrosine phosphatase non-receptor type 22 (PTPN22) is considered an important regulator of T-cell activation. Polymorphisms within the PTPN22 gene have been suggested to confer susceptibility to autoimmune endocrine disorders. To evaluate the impact of a functional variation in the PTPN22 gene in type 1 (T1D) and type 2 diabetes (T2D), the PTPN22 C1858T single nucleotide polymorphism (SNP) was studied in the population of Estonian origin, including 170 T1D patients, 244 T2D patients and 230 controls. Using two methods for PTPN22 C1858T detection in parallel, we found that not only T1D but also T2D is associated with the PTPN22 1858T allele. The role of PTPN22 gene in the pathogenesis of T2D is yet unclear and needs further investigation.

Analysis of extended human leukocyte antigen haplotype association with Addison's disease in three populations.

OBJECTIVE: Addison's disease is an organ-specific autoimmune disorder with a polygenic background. The aim of the study was to identify non-class II human leukocyte antigen (HLA) susceptibility genes for Addison's disease. DESIGN AND METHODS: Addison's disease patients from three European populations were analysed for selected HLA-DR-DQ alleles and for 11 microsatellite markers covering approximately 4 Mb over the HLA region. Subjects were 69 patients with Addison's disease from Estonia (24), Finland (14) and Russia (31). Consecutively recruited healthy newborns from the same geographical regions were used as controls (269 Estonian, 1000 Finnish and 413 Russian). Association measures for HLA-DRB1, DQB1, DQA1 and 11 microsatellites between D6S273 and D6S2223 were taken. A low-resolution full-house typing was used for HLA class II genes, while microsatellite markers were studied using fluorescence-based DNA fragment sizing technology. RESULTS: We confirmed that the HLA-DR3-DQ2 and the DQB1*0302-DRB1*0404 haplotypes confer disease susceptibility. In Russian patients, we also found an increase of DRB1*0403 allele, combined with DQB1*0305 allele in three out of six cases (P<0.0001). Analysis of 11 microsatellite markers including STR MICA confirmed the strong linkage in DR3-DQ2 haplotypes but DRB1*0404-DQB1*0302 haplotypes were diverse. MICA5.1 allele was found in 22 out of 24 Estonian patients, but results from Finnish and Russian patients did not support its independent role in disease susceptibility. CONCLUSION: HLA-DRB1*0403 was identified as a novel susceptibility allele for Addison's disease. Additionally, we found no evidence of a non-class II HLA disease susceptibility locus; however, the HLA-DR3-DQ2 haplotype appeared more conserved in patient groups with high DR-DQ2 frequencies.

Insulin VNTR I/III genotype is associated with autoantibodies against glutamic acid decarboxylase in newly diagnosed type 1 diabetes.

BACKGROUND: In type 1 diabetes (T1D), the influence of age at diagnosis and of the IDDM1 and IDDM2 genetic susceptibility loci on the profile of beta-cell autoantibodies has been demonstrated. We studied these associations in a group of 92 patients (children, adolescents and adults, aged 2-62 years) with newly diagnosed T1D. METHODS: The prevalence of the HLA-DQB1*02 and *0302 alleles and of the classes of variable number of tandem repeats (VNTR) of the insulin gene (INS), and of beta-cell autoantibodies (GADA, IA-2A, ICA and IAA) was determined. Statistical analysis was performed using linear and logistic regression models. RESULTS: The presence of IAA, IA-2A and ICA, but not of GADA, was negatively associated with age at diagnosis. Younger patients were more likely to have multiple autoantibodies. There was a tendency of a higher prevalence of IAA in patients with the HLA-DQB1*02/0302 genotype or with the DQB1*0302 allele compared to patients lacking these markers. As a novel observation, the INS VNTR I/III genotype was significantly associated with the presence of GADA (OR = 4.79; p = 0.018). CONCLUSION: The association between the INS VNTR I/III genotype and GADA may suggest that in patients with T1D lacking the INS VNTR I/I genotype, the effect of other susceptibility factors prevails, which promotes the development of autoimmunity to beta-cell antigens other than insulin.

Allergic sensitization and microbial load--a comparison between Finland and Russian Karelia.

Epidemiological data have indicated that some infections are associated with a low risk of allergic diseases, thus supporting the idea (hygiene hypothesis) that the microbial load is an important environmental factor conferring protection against the development of allergies. We set out to test the hygiene hypothesis in a unique epidemiological setting in two socio-economically and culturally markedly different, although genetically related, populations living in geographically adjacent areas. The study cohorts included 266 schoolchildren from the Karelian Republic in Russia and 266 schoolchildren from Finland. The levels of total IgE and allergen-specific IgE for birch, cat and egg albumen were measured. Microbial antibodies were analysed against enteroviruses (coxsackievirus B4), hepatitis A virus, Helicobacter pylori and Toxoplasma gondii. Although total IgE level was higher in Russian Karelian children compared to their Finnish peers, the prevalence of allergen-specific IgE was lower among Russian Karelian children. The prevalence of microbial antibodies was, in turn, significantly more frequent in the Karelian children, reflecting the conspicuous difference in socio-economic background factors. Microbial infections were associated with lower risk of allergic sensitization in Russian Karelian children, enterovirus showing the strongest protective effect in a multivariate model. The present findings support the idea that exposure to certain infections, particularly in childhood, may protect from the development of atopy. Enterovirus infections represent a new candidate to the list of markers of such a protective environment. However, possible causal relationship needs to be confirmed in further studies.

Insulin gene VNTR, CTLA-4 +49A/G and HLA-DQB1 alleles distinguish latent autoimmune diabetes in adults from type 1 diabetes and from type 2 diabetes group.

Recent research has underlined the need to explore pathogenic, genetic and clinical spectrum of adult onset autoimmune diabetes, also known as latent autoimmune diabetes in adults (LADA). We aimed to investigate whether genetic factors that are associated with type 1 diabetes (T1D) susceptibility, namely HLA-DQB1 alleles, cytotoxic T-lymphocyte antigen 4 gene (CTLA-4) and insulin gene (INS) polymorphisms, are also associated with an atypical subset of patients diagnosed with type 2 diabetes (T2D). The case-control study included 70 T1D, 305 T2D and 252 nondiabetic controls. The T2D group was divided into atypical T2D (LADA, n = 61) or typical T2D (n = 244) subgroups based on the presence of at least one pancreas-specific antibody. Our data suggested that HLA-DQB1 alleles of all three risk classes, INS variable number of tandem repeat (VNTR) I/I and CTLA-4 +49 GG or AG genotypes, were independent risk factors for developing LADA and could be used as a diagnostic tool to discriminate between LADA and T2D. Additionally, there was an increased association between LADA and CTLA-4 diabetes-susceptibility genotypes and decreased association with INS VNTR and high-risk HLA-DQB1 alleles, compared with T1D. Our study suggested the need for further investigation into the genetic background and functional genomics of LADA in comparison with T1D and T2D.

High prevalence of coeliac disease: need for increasing awareness among physicians.

BACKGROUND: Coeliac disease is a wheat gluten and related prolamines-induced disease with a prevalence that may be underestimated in many geographical regions and populations. AIM: To investigate the prevalence of coeliac disease in a population of schoolchildren of Estonia using tissue transglutaminase antibodies for screening. SUBJECTS AND METHODS: The study was designed as cross-sectional. Serum samples from 1160 randomly selected schoolchildren (636 female and 564 male, aged 9 or 15 years) were studied using a novel tissue transglutaminase antibody immunoassay (EliA Celikey IgA assay). Antibody-positive subjects were investigated for coeliac disease. RESULTS: A total of five subjects had antibodies. Four of them agreed for further investigations. By small-bowel biopsy they all were confirmed to have active coeliac disease, including three subjects with symptoms that were not considered by their family doctors. The prevalence of coeliac disease is at least 1 case per 290 (0.34% with CI 0.09-0.88%) in Estonia. It is much higher than that in our previous screening studies but is comparable with data from other European countries. CONCLUSION: The prevalence of coeliac disease might have increased during the last decade in Estonia. This study clearly shows that the awareness of coeliac disease among physicians is low. Thus, there is a need for more epidemiological studies and education related to coeliac disease.

Serum antibodies to enterohepatic Helicobacter spp. in patients with chronic liver diseases and in a population with high prevalence of H. pylori infection.

BACKGROUND: Enteric Helicobacter species might be a risk factor for chronic liver and biliary tract diseases. AIMS: To analyse serum antibody levels to three enteric Helicobacter species in patients with various biliary tract and chronic liver diseases and compare results with corresponding parameters for an adult population group, known to have a high prevalence of Helicobacter pylori infection, and with healthy blood donors, to explore a possible association of enteric Helicobacter with chronic liver diseases. SUBJECTS: Sera of 90 patients with various chronic liver diseases, 121 Estonian adult persons and 68 blood donors were analysed. METHODS: Sera, previously tested for H. pylori were analysed for IgG to Helicobacter hepaticus, Helicobacter bilis and Helicobacter pullorum. ELISA was initially used for screening and exclusion of negative cases. Sera with positive ELISA results were further analysed by immunoblot. To remove cross-reactive antibodies between H. pylori and the enteric species, sera were pre-absorbed with lysed H. pylori cells. RESULTS: Liver patients showed a significantly higher seroprevalence to H. hepaticus and H. bilis, compared with the adult population group (p=0.0001 and 0.04, respectively), and to H. hepaticus, compared with blood donors (p=0.01). Patients with autoimmune hepatitis showed no significant antibody reactivity to the enteric Helicobacter spp. in contrast to patients with other chronic liver diseases. CONCLUSION: Patients with chronic liver diseases, except autoimmune hepatitis patients, showed increased antibody levels to H. bilis/H. hepaticus compared with the population and blood donors indicating a possible role of enteric Helicobacter in the natural course of chronic liver diseases. Immunoblot seems to be a promising method for serodiagnosis of infections with these fastidious pathogens.

Relationship between the incidence of type 1 diabetes and maternal enterovirus antibodies: time trends and geographical variation.

AIMS/HYPOTHESIS: We have previously observed an inverse correlation between the incidence of type 1 diabetes and enterovirus infections in the background population. The aim of this study was to analyse whether maternal enterovirus antibody status, which reflects both the frequency of enterovirus infections and the protection conferred by the mother on the offspring, also correlates with the incidence of type 1 diabetes. METHODS: Maternal enterovirus antibodies were analysed from serum samples taken from pregnant women between 1983 and 2001 in Finland and Sweden using enzyme immunoassay and neutralisation assays. Comparable samples were also taken between 1999 and 2001 in countries with a lower incidence of diabetes (Estonia, Germany, Hungary, Israel, Lithuania, Russia). RESULTS: A clear decrease was observed in maternal enterovirus antibody levels over the past 20 years (p<0.0001). The frequency of enterovirus antibodies was higher in countries with a low or intermediate incidence of type 1 diabetes compared with high-incidence countries (p<0.0001). CONCLUSIONS/INTERPRETATION: These findings are in line with our previous observations supporting the hypothesis that a low frequency of enterovirus infection in the background population increases the susceptibility of young children to the diabetogenic effect of enteroviruses.

Type 1 diabetes is insulin -2221 MspI and CTLA-4 +49 A/G polymorphism dependent.

BACKGROUND: Several studies have demonstrated an association of type 1 diabetes with specific alleles of HLA class II molecules, as with polymorphisms of insulin gene region. The aim of our study was to evaluate the interaction of insulin -2221 MspI polymorphism to type 1 diabetes susceptibility in connection with autoimmunity associated gene--CTLA-4 polymorphism. MATERIALS AND METHODS: Insulin -2221 MspI C/T and CTLA-4 +49 A/G polymorphisms were detected by restriction fragment-length polymorphism analysis or oligonucleotide hybridization in type 1 (n = 69), type 2 diabetes (n = 301) patients and 158 healthy controls. Regression model adjusted for age, gender and gene polymorphisms was studied. RESULTS: C-allele of insulin -2221 MspI and G-allele of +49 CTLA-4 were significant risk factors for type 1 diabetes (crude OR 3.53 and 1.59, respectively) and this impact increased in the homozygous form of both alleles. The regression model supported the idea of insulin CC and CTLA-4 GG genotypes for an independent and clearly significant risk for developing type 1 diabetes. We could not detect any significant correlation between investigated polymorphisms and type 2 diabetes. CONCLUSIONS: There exists a significant association between the C-allele of -2221 MspI in the insulin gene and type 1 diabetes. The CTLA-4 G-allele is also positively correlated with type 1 diabetes. According to the regression model the investigated gene polymorphisms are independent risk factors for development of type 1 diabetes in the Estonian population. We propose that -2221 MspI is a good marker for evaluation of risk of insulin gene haplotype in type 1 diabetes patients.

Increased levels of IgA antibodies against desmin in children with coeliac disease.

BACKGROUND: In addition to IgA anti-endomysial antibodies, IgA anti-smooth muscle antibodies have been observed in a number of patients with coeliac disease (CoD), but only limited data exist on the frequency and antigen specificity of IgA against cytoskeletal proteins in CoD. METHODS: We evaluated the sera of 42 untreated CoD patients, follow-up sera of 26 CoD patients after treatment, and 116 control sera for IgA reactivity to cytoskeletal proteins from the human umbilical cord (HUC) by immunoblotting, and compared the results with anti-tissue transglutaminase IgA (anti-tTG IgA) ELISA and immunofluorescence results. RESULTS: Serum IgA from CoD patients most frequently recognized a 57-kD antigen in HUC cytoskeletal extract, identified as desmin using mass spectrometry and internal peptide sequencing. Increased IgA reactivity to the human desmin band was detected in 22 children with untreated CoD (52.4%), in 4 treated CoD patients (15.4%), and in 12 control subjects (10.3%) (p < 0.01); similar results were obtained with anti-chicken desmin IgA ELISA. Anti-tTG IgA levels (increased in 71.4% of untreated CoD patients) correlated significantly with anti-desmin IgA levels in untreated CoD, and both autoantibody reactivities decreased in response to a gluten-free diet. Pre-adsorption experiments and affinity purification of anti-desmin IgA antibodies further confirmed that desmin is an IgA autoantigen in CoD and is recognized by antibodies which are not cross-reactive with tTG. CONCLUSIONS: Anti-desmin IgA antibodies are frequently occurring if not the predominant cytoskeletal antibodies in children with untreated CoD and could be related to the disease process in the small intestine.

Comparison of the prevalence of glutamic acid decarboxylase (GAD65) and gliadin antibodies (AGA) in a randomly selected adult estonian population.

We aimed to test the hypothesis that gluten might be associated with the development of islet cell autoimmunity. A random sample of 200 persons (87 males, mean age 42.4 years) from Estonia including one patient with type I diabetes mellitus was studied. IgG-type glutamic acid decarboxylase (GAD65) antibodies were determined using radioligand-binding assay and IgG/IgA-type gliadin antibodies (AGA) by enzyme-linked immunosorbent assay. Generic HLA-DRB1* alleles were analyzed using a polymerase chain reaction. Although our results revealed the highest GAD65Ab index and a high IgA-type AGA in a person with diabetes, no correlation between GAD65Ab and AGA values was revealed among the other 199 persons (p > 0.05). There were also no differences between test values among persons with and without different HLA-DRB1* alleles (p > 0.05). In the GAD65Ab assay, one person (0.5 %; 95 % CI: 0 - 1.5) out of 199 exceeded the 99(th) centile of the GAD65Ab index. In summary, the present study does not confirm the possibility that there is a relationship between the immune reactivity against GAD65 and gliadin, at least in persons without type I DM.

Autoantibody studies of female patients with reproductive failure.

The aim of this study was to investigate the prevalence and character of autoimmune derangements in women with reproductive failure. A total of 108 females (age range 17-43, mean 27.5 years), including 16 with primary menstrual cycle disturbances and polycystic ovaries (PCO), 20 with polycystic ovary syndrome (PCOS), 38 with endometriosis (E), and 34 with chronic anovulation, luteal phase insufficiency, subfertility or unexplained infertility (INF) were investigated. A control group of 392 women was formed from an unselected population sample (age range 17-43, mean 31.0 years). All sera were tested by indirect immunofluorescence method to assess common autoantibodies: nuclear (ANA), smooth muscle (SMA), parietal cell (PCA), thyroid microsomal (TMA), reticulin (ARA), mitochondrial (AMA) and liver/kidney microsomal autoantibodies (LKMA). Enzyme-linked immunosorbent assay was used to detect antibodies against beta2-glycoprotein I (anti-beta 2GPI) and carbonic anhydrase (anti-CA). Our results showed that 40.7% of patients' sera and 14.8% of control sera contained one or more common autoantibodies, ANA and SMA were most frequently detected (difference between two groups P<0.005). Anti-beta 2GPI were found in eight cases (7.4%), including two patients with INF but without other autoantibodies. Anti-CA were revealed in nine cases (8.3%) including patients' PCOS, E and INF. A comparison of patients' clinical data with antibody assay results did not reveal any significant associations. Our results indicate a high prevalence of autoimmune reactions in women with reproductive failure due to the most common causes PCO, PCOS and E as well as in unexplained infertility. This might reflect the propensity to develop autoimmune reactions in such patients, including pathogenic autoimmune reactions to specific target antigens.

The follow-up of asymptomatic persons with antibodies to pyruvate dehydrogenase in adult population samples.

PURPOSE: The presence of antimitochondrial antibodies (AMA), the hallmark of primary biliary cirrhosis (PBC), precedes the clinical manifestation of the disease for many years. The main mitochondrial autoantigen is the E2 component of the pyruvate dehydrogenase complex (PDC). The aim of this study was to identify anti-PDC-positive persons from two Estonian populations by different methodologies and to follow up the positive cases. METHODS: Enzyme-linked immunosorbent assay (ELISA) tests for antibodies to native PDC and recombinant PDC-E2 fusion protein were performed in 1461 persons (age range, 15-95 years) from Karksi-Nuia (plus 104 volunteers from the neighborhood) and to native PDC in 497 persons (age range, 50-91 years) from Abja-Paluoja (plus 28 volunteers from that neighborhood). Positive cases were tested with an enzyme inhibition assay. RESULTS: We identified 14 asymptomatic persons with antibodies to native PDC and/or recombinant PDC-E2 from these two population samples. Eight of the 14 were available for follow-up. Three of the 8 developed abnormal liver biochemical test results by the ninth year of follow-up. These persons also had, or developed, during the follow-up, a positive AMA immunofluorescence test, inhibitory antibodies to PDC, and anti-PDC of at least IgG and IgA class. Five of the 8 persons with low levels of anti-PDC, of only one immunoglobulin class reacting with only one PDC preparation, did not show any signs of cholestasis or changes in their immunoreactivity during follow-up. CONCLUSIONS: A significant number of asymptomatic patients found to have antibodies to PDC are at high risk of developing primary biliary cirrhosis.