AIRE-Deficient Patients Harbor Unique High-Affinity Disease-Ameliorating Autoantibodies.

APS1/APECED patients are defined by defects in the autoimmune regulator (AIRE) that mediates central T cell tolerance to many self-antigens. AIRE deficiency also affects B cell tolerance, but this is incompletely understood. Here we show that most APS1/APECED patients displayed B cell autoreactivity toward unique sets of approximately 100 self-proteins. Thereby, autoantibodies from 81 patients collectively detected many thousands of human proteins. The loss of B cell tolerance seemingly occurred during antibody affinity maturation, an obligatorily T cell-dependent step. Consistent with this, many APS1/APECED patients harbored extremely high-affinity, neutralizing autoantibodies, particularly against specific cytokines. Such antibodies were biologically active in vitro and in vivo, and those neutralizing type I interferons (IFNs) showed a striking inverse correlation with type I diabetes, not shown by other anti-cytokine antibodies. Thus, naturally occurring human autoantibodies may actively limit disease and be of therapeutic utility.

Significant associations between bone mineral density and vascular calcification in patients with different stages of chronic kidney disease.

INTRODUCTION: Chronic kidney disease-mineral and bone disorders (CKD-MBD) is characterised by generalised vascular calcification (VC) and impaired bone health. We aimed to investigate the relationship between VC and bone mineral density (BMD) in CKD patients. METHODS: We performed a cross-sectional study of patients with different stages of CKD. For assessment of VC of abdominal aorta lateral lumbar X-rays (Kauppila score), the ankle-brachial index (ABI) and echocardiography were used. Total body densitometry provided BMD. RESULTS: Ninety patients (41% male, median age 64 years (range 29-87)) were included, of whom 41.1% had a Kauppila score > 1. Evidence of peripheral VC as measured by ABI was detected in 23.3% of cases. Lesions of the heart valves were found in 46.7% of patients. There was a significant association between high ABI and lesions of the heart valves. In the multivariate regression model to analyse the independent determinants of abdominal aorta calcification (AAC) and ABI, the BMD of the femoral neck was identified as significant for both (p = 0.001, p = 0.001). The total spine BMD was found to be significant for AAC (p = 0.001), and the BMD of spine L1-L4 and the ribs were found to be significant for ABI (p = 0.01, p = 0.002 respectively). In factorial regression analysis, where BMD was independent determinant, valvular calcification was significant for BMD of femur, femoral neck and total BMD. Age and tALP were inversely correlated with the BMD of femur and femoral neck. CONCLUSIONS: Our work highlighted clinically important relationships between VC and bone mineral density (BMD) in CKD patients. We detected inverse relationships between AAC, high ABI and BMD. Secondly, BMD at certain bone sites (femur, femoral neck) and total BMD were associated with important lesions of heart valves. Thirdly, a significant association between a high ABI and lesions of the heart valves. We believe that the results of our study will help in the planning of future research and in current clinical practice for the early diagnosis, further monitoring and management of CKD-MBD. Additionally, these results may have treatment implications on use of different CKD-MBD medications.

Clinical signs of type 1 diabetes are associated with type 2 diabetes marker transcription factor 7-like 2 polymorphism.

AIMS/INTRODUCTION: We aimed to assess the distribution of transcription factor 7-like 2 gene TCF7L2 (rs7903146) polymorphism and to find possible associations between TCF7L2 and the characteristics of type 1 diabetes. MATERIALS AND METHODS: We studied 190 newly diagnosed type 1 diabetes patients (median age 12.7 years, range 2.0-72.5) and 246 controls (median age 23.8 years, range 1.4-81.5) for TCF7L2 single nucleotide polymorphism. We determined anti-islet autoantibodies, random C-peptide levels, diabetes associated HLA DR/DQ haplotypes and genotypes in all patients. RESULTS: There were no differences in the distribution of TCF7L2 single nucleotide polymorphism between patients and controls. However, patients with in type 1 diabetes, after adjusting for age and sex, subjects carrying C allele were at risk for a C-peptide level lower than 0.5 nmol/L (OR 5.65 [95% CI: 1.14-27.92]) and for zinc transporter 8 autoantibody positivity (5.22 [1.34-20.24]). Participants without T allele were associated with a higher level of islet antigen-2 autoantibodies (3.51 [1.49-8.27]) and zinc transporter 8 autoantibodies (2.39 [1.14-4.99]). CONCLUSIONS: The connection of TCF7L2 polymorphism with zinc transporter 8 and islet antigen-2 autoantibodies and C-peptide levels in patients supports the viewpoint that TCF7L2 is associated with the clinical signs and autoimmune characteristics of type 1 diabetes. The mechanisms of the interaction between the TCF7L2 risk genotype and anti-islet autoantibodies need to be studied further.

IgA-Type Enterovirus Antibodies Are Increased among Adults and Children with Recently Diagnosed Type 1 Diabetes.

Enteroviruses (EV) are among the leading environmental triggers of childhood-onset type 1 diabetes (T1D). Our aim was to determine the prevalence of antibodies against EV and their association with T1D in different age groups (n = 62), including young adults, and to compare these data with results from HLA-matched control participants (n = 62). IgA, IgG, and IgM antibodies against EV were detected. IgA EV antibodies were present in 46.8% of participants with T1D (median level 10.9 EIU) and in 11.3% of controls (median level 3.4 EIU). IgA EV positivity and higher level of IgA EV antibodies were both significant risk factors for T1D (odds ratio (OR) 8.33; 95% confidence interval (CI) 2.52-27.6; p = 0.0005 and OR 1.04; 95% CI 1.01-1.06; p = 0.0105, respectively). Importantly, the prevalence of IgA EV antibodies in the subgroups of both children and young adults was also significantly different between participants with T1D and their matched controls (p = 0.0089 and p = 0.0055, respectively). Such differences were not seen for IgG and IgM EV antibodies. However, IgG EV antibodies were associated with 65 kDa glutamic acid decarboxylase antibodies, but not with zinc transporter 8 and protein tyrosine phosphatase IA2 antibodies. The genotype frequency of PTPN22 (rs2476601) and IFIH1 (rs1990760) was not associated with EV positivity. This study showed that EV infections may be an important disease-promoting factor of T1D not only in childhood-onset but also in adult-onset T1D. However, to further confirm this association, direct virological studies are needed in the latter T1D group.

Symptomless celiac disease in type 1 diabetes: 12-year experience in Estonia.

BACKGROUND: We aimed to determine the prevalence and characteristics of celiac disease in children with type 1 diabetes in Estonia, a country with a formerly low frequency of both diseases. METHODS: Altogether, 271 patients with diabetes were studied over 12 years (1995-2006): 122 at diagnosis and 149 patients 0.1-14.8 years after diagnosis. In addition, 73 patients were followed up over 1-6 years. Immunoglobulin A type endomysium and tissue transglutaminase antibodies were determined. Patients with antibodies and/or with celiac-disease-related symptoms were invited for a small-intestinal biopsy. RESULTS: At the primary screening, celiac disease was histologically confirmed in nine patients (all without symptoms), that is, in 3.3% (95% confidence interval: 1.63-6.42) of type 1 diabetes cases. At follow up, celiac disease was additionally detected in two (2.7%) of 73 diabetic patients, that is, in 0.016 (95% confidence interval: 0-0.072) celiac disease cases per follow-up year. CONCLUSION: The prevalence of celiac disease among type 1 diabetes patients in Estonia is similar to that in countries with a high incidence of celiac disease and type 1 diabetes. As celiac disease is mostly symptomless, all children with type 1 diabetes, irrespective of their geographic origin, should be regularly screened for celiac disease.

Thyroid autoimmunity and treatment response to escitalopram in major depression.

BACKGROUND: There is evidence that immune alterations play an important part in the pathogenesis of major depression. Thyroid autoimmunity has been found in association with major depression in several studies. AIM: 1) to examine whether the prevalence of anti-thyroid peroxidase autoantibodies (anti-TPO) in depressive patients differs from that in healthy controls; 2) to investigate the possible relationship between thyroid autoimmunity, total T3, free T3, free T4, thyroid-stimulating hormone (TSH), clinical status and treatment outcome in depression. METHOD: The study group consisted of 129 outpatients (69.8% female; mean age 31.7+/-12.0 years) with major depressive disorder with a Montgomery-Azsberg Depression Rating Scale total score of 22 or higher and 72 healthy controls (62.5% female; mean age 31.7+/-13.1 years). The patients were treated with escitalopram 10-20 mg/day for 12 weeks using open-label placebo non-controlled design. Anti-TPO, total T3, free T3, free T4 and TSH were measured before the treatment. RESULTS: The anti-TPO was found in eight (8.9%) depressive and two (4.8%) healthy females without statistical difference between these groups. Since anti-TPO was not seen in males, all further statistical analyses were carried out in females. At the end of week 12 of the treatment, 60 female patients (66.7%) were defined as responders and 30 depressive females (33.3%) showed insufficient response to treatment. Although there were no significant differences in the measurements between responders and non-responders, the last group showed a trend for a higher prevalence of anti-TPO compared with responders. CONCLUSION: Thyroid autoimmunity might be a factor predicting treatment response to antidepressants in depressive patients.

Demographic associations for autoantibodies in disease-free individuals of a European population.

The presence of autoantibodies usually precedes autoimmune disease, but is sometimes considered an incidental finding with no clinical relevance. The prevalence of immune-mediated diseases was studied in a group of individuals from the Estonian Genome Project (n = 51,862), and 6 clinically significant autoantibodies were detected in a subgroup of 994 (auto)immune-mediated disease-free individuals. The overall prevalence of individuals with immune-mediated diseases in the primary cohort was 30.1%. Similarly, 23.6% of the participants in the disease-free subgroup were seropositive for at least one autoantibody. Several phenotypic parameters were associated with autoantibodies. The results suggest that (i) immune-mediated diseases are diagnosed in nearly one-third of a random European population, (ii) 6 common autoantibodies are detectable in almost one-third of individuals without diagnosed autoimmune diseases, (iii) tissue non-specific autoantibodies, especially at high levels, may reflect preclinical disease in symptom-free individuals, and (iv) the incidental positivity of anti-TPO in men with positive familial anamnesis of maternal autoimmune disease deserves further medical attention. These results encourage physicians to evaluate autoantibodies in addition to treating a variety of patient health complaints to detect autoimmune-mediated disease early.

Demonstration of high prevalence of SS-A antibodies in a general population: association with HLA-DR and enterovirus antibodies.

Autoantibodies are helpful markers for diagnosing autoimmune diseases and there is a link between HLA-DR3 and the prevalence of SS-A antibodies in clinical groups. We aimed to study this association at the level of general adult population and to verify whether these antibodies are more common in persons with antibodies against enteroviruses as possible associates of Sjögren syndrome. The studied material included sera from 200 persons, randomly selected from a general population sample. The IgG type of SS-A/SS-B autoantibodies were measured by nuclear immunoblot, developed by us, and the results were compared to other results obtained by anti-SS-A immunoblot and ELISA. Enterovirus antibodies were detected by ELISA using common enterovirus antigenic peptide KEVPALTAVETGAT. Altogether 33 out of 200 sera showed SS-A and/or SS-B bands in immunoblot, including all seven ANA Profile 3 (Euroimmune) positive sera. One of the persons positive in these two tests showed also positive reaction on anti-SS-A ELISA (Euroimmune). None of the antibody-positive persons had Sjögren's syndrome or other rheumatic disease. Among 82 HLA typed persons, selected at random, the HLA-DRB1*03 and HLA-DRB1*11 allele carriers included significantly more persons with SS-A antibodies than the non-carries (p = 0.008). Antibodies against enterovirus peptide were present more frequently in persons with SS-A autoantibodies than in age- and sex-matched controls (p = 0.009). Summing up, our study showed that the prevalence of SS-A/SS-B antibodies in a general random population might be higher than thought previously being detected in up to 16.5% of persons including a significant number of those with HLA-DR3 or/and DR11 alleles and with antibodies against enteroviruses. Whether all these persons have the risk of developing rheumatic diseases should be evaluated by follow up studies.

Screening for celiac disease in Down's syndrome patients revealed cases of subtotal villous atrophy without typical for celiac disease HLA-DQ and tissue transglutaminase antibodies.

AIM: To investigate the prevalence of celiac disease (CD) as well as CD marker antibodies and susceptibility HLA-DQ haplotypes in 134 karyotyped Down's syndrome (DS) patients. METHODS: Immunoglobulin A (IgA) and G (IgG) type anti-gliadin antibodies (AGA), IgA type anti-tissue transglutaminase (tTG) antibodies (anti-tTG) with antigen of guinea pig and human source were determined by enzyme-linked immunosorbent assay and endomysium antibodies (EMA) by indirect immunofluorescence test. HLA-DQA1*0501/DQB1*0201 (DQ2) was revealed by polymerase chain reaction. Celiac disease was diagnosed by revised ESPGHAN criteria. RESULTS: 41% of DS patients had AGA, 6.0% IgA anti-tTG with guinea pig antigen, and 3.0% IgA EMA (all positive for anti-tTG with human tTG). Subtotal villous atrophy was found in 5 out of 9 DS patients who had agreed to small bowel biopsy. One of them had DQA1*0501/DQB1*0201 and anti-tTG and EMA i.e. typical for CD markers (this case also fulfilled the ESPGHAN diagnostic criteria),but other four lacked these markers. Three non-biopsied DS patients had also most probably CD because DQA1*0501/DQB1*0201 and IgA anti-tTG (EMA) were detected. Thus, the prevalence of CD among our DS patients population is 3.0% (95 % of confidence interval [CI]: 0.1-5.9%). CONCLUSION: We confirm the increased frequency of CD among DS patients. In addition, we have revealed a subgroup of patients with subtotal villous atrophy but without characteristic for CD immunological and genetic markers. Whether these cases represent CD (with atypical immunopathogenesis) or some other immune enteropathy, requires further investigations.

Autoantibodies and hepatitis C virus genotypes in chronic hepatitis C patients in Estonia.

AIM: To determine the prevalence of several autoantibodies in chronic hepatitis C patients, and to find out whether the pattern of autoantibodies was associated with hepatitis C virus (HCV) genotypes. METHODS: Sera from 90 consecutive patients with chronic hepatitis C were investigated on the presence of anti-nuclear (ANA), anti-mitochondrial (AMA), anti-smooth muscle (SMA), anti-liver-kidney microsomal type 1 (LKMA1), anti-parietal cell (PCA), anti-thyroid microsomal (TMA), and anti-reticulin (ARA) autoantibodies. The autoantibodies were identified by indirect immunofluorescence. HCV genotypes were determined by a restriction fragment length polymorphism analysis of the amplified 5' noncoding genome region. RESULTS: Forty-six (51.1%) patients were positive for at least one autoantibody. Various antibodies were presented as follows: ANA in 13 (14.4%) patients, SMA in 39 (43.3%), TMA in 2 (2.2%), and ARA in 1 (1.1%) patients. In 9 cases, sera were positive for two autoantibodies (ANA and SMA). AMA, PCA and LKMA1 were not detected in the observed sera. HCV genotypes were distributed as follows: 1b in 66 (73.3%) patients, 3a in 18 (20.0%), and 2a in 6 (6.7%) patients. CONCLUSION: A high prevalence of ANA and SMA can be found in chronic hepatitis C patients. Autoantibodies are present at low titre (1:10) in most of the cases. Distribution of the autoantibodies show no differences in the sex groups and between patients infected with different HCV genotypes.

Antinuclear antibodies in atopic dermatitis: a cross-sectional study on 346 children.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease that can be classified into an extrinsic or intrinsic type. A high percentage of patients, especially adults with the extrinsic type of AD, have been reported to show antibodies to antinuclear proteins (ANA). We aimed to study the prevalence of ANA in children with AD and to evaluate clinical differences between patients with ANA-positive and ANA-negative AD. METHODS: A total 346 serum samples from children with active AD (mean age 5.8 years) and 117 hospital controls without known skin, inflammatory, or immune-mediated disease (mean age 7.9 years) were tested for IgG ANA with indirect immunofluorescence on HEp-2 cells, total serum IgE levels, and IgE type antibodies to food allergen panels. RESULTS: In total, 47 patients with AD (13.6%) and 15 subjects in the control group (12.8%) were ANA positive at screening dilution 1:10 (P > 0.05). In patients with AD, ANA was found already at the age of 2 years, significantly more often in females (P < 0.005) and at slightly higher titers (up to 1:160). No differences were found in ANA positivity regarding the severity of AD or sensitization to food allergens. CONCLUSION: No significant differences were observed between AD and the control group, or between different subtypes of AD in ANA prevalence. In both groups, ANA frequency increased with age, but in patients with AD, ANA had a tendency to appear earlier. Therefore, active AD during the early years of life could dispose selected patients towards earlier development of systemic autoreactivity and stress the need for regular follow-up of patients with ANA-positive AD.

Development of a luciferase-based system for the detection of ZnT8 autoantibodies.

Appearance of autoantibodies represents the first detectable sign of autoimmune destruction of beta cells in type 1 diabetes (T1D). In addition, autoantibody levels represent an important predictive marker regarding the development of an autoimmune process. Recently, the zinc transporter (ZnT8) protein was identified as an autoimmune target in T1D; therefore, there is a need for reliable and simple methods for detection of ZnT8 autoantibodies. This report describes an assay designed to detect anti-ZnT8 autoantibodies in the serum of patients with T1D. This was carried out by generating a ZnT8 C-terminal dimer fused to the N-terminus of the Gaussia princeps luciferase that was overexpressed in insect cells using the baculovirus expression system. Recombinant protein was semi-purified and used as the target antigen in the liquid-phase luciferase immunoprecipitation system assay (LIPS), and results were compared to data obtained using a commercial ELISA designed to detect ZnT8 autoantibodies in T1D patient sera, particularly among adults. LIPS was less effective in detecting antibodies in children probably due to the relatively high prevalence of IgM anti-ZnT8 antibodies in children with T1D.

Expression of B7 and CD28 family genes in newly diagnosed type 1 diabetes.

Type-1 diabetes (T1D) is caused by T-cell mediated autoimmune reaction directed against the insulin-secreting beta cells. We hypothesized that in addition to antigen/MHC recognition the co-stimulatory B7 and CD28 pathway is also strongly affected in T1D. CD80, CD86, CD28, CTLA4, ICOS and CD25 mRNA expression was characterized in 49 newly diagnosed young T1D patients (mean age 11 ± 5 years, 25 male/24 women) and 31 controls (mean age 14 ± 7 years, 14 male/17 women). In addition, polymorphisms in CTLA4 (rs231806, rs231775, rs3087243) were genotyped and soluble CTLA4 plasma levels measured by ELISA. T1D patients presented with higher peripheral blood expression levels of CD80 (Mann-Whitney U-test, p=0.0001) and lower ICOS levels compared to healthy controls (Glm adjusted for age, p=0.0004). CD80 expression in T1D patients correlated with expression of two CTLA4 splice variants (Spearman's rank correlation, rho=0.56, p=0.0002 for sCTLA4; rho=0.61, p<0.0001 for flCTLA4). In controls, CD80 expression correlated with CD25 expression (Spearman's rank correlation, rho=0.57, p=0.002). A strong correlation was observed between sCTLA4 and flCTLA4 (Spearman's rank correlation, rho=0.94, p<0.0001). We also found a tendency that the CTLA4 +49A/G polymorphism influenced sCTLA4 mRNA expression in T1D individuals and was lowest in individuals with the GG genotype (Mann-Whitney U-test, p=0.039). However, we could not identify associations between gene expression and plasma levels of sCTLA4. To summarize, we expect that newly diagnosed T1D among children and adolescents is associated with activation of CD80 and CTLA4 in peripheral blood. Additional studies will be needed to elucidate the role of CD80/CTLA4 signaling in T1D.

Newly-diagnosed pediatric epilepsy is associated with elevated autoantibodies to glutamic acid decarboxylase but not cardiolipin.

Glutamic acid decarboxylase autoantibodies (GADA) and anti-cardiolipin autoantibodies (ACA) have been detected in adult subjects with epilepsy, though the functional implications of these findings are a matter of debate. This study aimed to determine the prevalence of GADA and ACA and to investigate their clinical significance in pediatric subjects with newly-diagnosed epilepsy. For this purpose GADA and ACA were assessed by enzyme-linked immunosorbent assays in 208 pediatric patients with newly-diagnosed epilepsy and 128 controls. The clinical data (results of electroencephalography, magnetic resonance imaging, 6-month outcome etc.) was compared to antibody test results. Our study revealed GADA in 14 (6.7%) patients with epilepsy and in 1 (0.8%) control, which was a statistically significant difference (P=0.010; Chi-square test). The GADA-positive and -negative patients had similar clinical characteristics. The prevalence of ACA in patients with epilepsy (6.3%) was not significantly different than controls (2.6%). These results suggest that GADA is associated with epilepsy in a subgroup of newly-diagnosed pediatric patients. Further studies are required to determine the prognostic significance and pathogenic role of GADA among pediatric subjects with epilepsy.

IgG from patients with liver diseases inhibit mitochondrial respiration in permeabilized oxidative muscle cells: impaired function of intracellular energetic units?

The effect of IgG purified from the sera of healthy persons and patients with primary biliary cirrhosis (PBC) and chronic hepatitis (CH) on ADP dependent respiration (oxidative phosphorylation) in skinned muscle fibers from rat oxidative muscles (heart and M. soleus) and glycolytic skeletal muscle (M. gastrocnemius) was studied. The results show that IgG from three different sources inhibited the rate of respiration by 13, 44 and 42%, respectively, these effects being equally expressed in both types of oxidative muscles, whereas no inhibition was observed in glycolytic muscle. The following washout of unbound IgG did not abolish the inhibition of respiration suggesting that the specific interaction of IgG with antigens had taken place. Laser confocal analysis revealed binding of IgG predominantly to the sarcomeric structures such as Z-disk and M-lines in the cardiomyocytes. The staining of IgG within Z-disks and intermitochondrial space coincided throughout the muscle cells so that transversally serial spaces, each containing mitochondria and adjacent sarcomere, became clearly visible. When the IgG from a CH patient was incubated with the skinned myocardial fibers of the desmin knockout mice, its binding to Z-disks and the sarcomeric area was found to be similar to that in normal cardiac muscle. However, the transversal staining pattern was disintegrated, because of the slippage of the myofibrils in relation to each other and accumulation of mitochondria between them. These observations support the recent hypothesis that in oxidative muscles the mitochondria and adjacent sarcomeres form complexes, termed as the intracellular energetic units, ICEUs. Moreover, they indicate that human autoantibodies can be useful tools for localizing the proteins responsible for formation of ICEUs and modulation of their function. Thus, it appears that the proteins associated with the Z-disks and M-lines may participate in formation of ICEUs and that binding of IgG to these proteins decreases the access of exogenous adenine nucleotides to mitochondria, which manifests as decreased rate of ADP-dependent respiration.