IA-2 autoantibodies restricted to the IgG4 subclass are associated with protection from type 1 diabetes.

The tyrosine phosphatase-like protein IA-2 is a major target antigen for autoantibodies in the preclinical period of type 1 diabetes. In this study, we examined whether immunoglobulin isotypes and IgG subclass specific autoantibodies directed at IA-2 discriminate between children at risk of type 1 diabetes who progressed to diabetes vs. those who remained diabetes-free. IgG1-4, IgA and the IgE-specific IA-2 antibody (IA-2A) were measured by radioligand assays in 50 patients with type 1 diabetes and 41 ICA-positive siblings of patients with type 1 diabetes who were followed for diabetes development. Of 41 siblings, 32 were positive for IA-2A; of these, 59 % had IA-2 IgG1, 59 % IgG4, 16 % IgG3, 9 % IgG2, 16 % IgA and 13 % IgE antibodies. IA-2 IgG1 was the dominant isotype in prediabetic children (n = 14, 86 % positive) and patients with type 1 diabetes (98 % positive) whereas only 7 of 18 (39 %) non-progressors had antibodies of this isotype. In subjects that remained diabetes-free, a significantly higher frequency of IA-2 IgG4 in the absence of IgG1 was observed (50 %) compared to progressors (7 %) and patients with type 1 diabetes (0 %). Life-table analysis revealed that IA-2A restricted to IgG4 correlated with protection from type 1 diabetes (p < 0.003). In contrast, IA-2 IgG2, IgG3, IgE and IgA did not differ significantly between study groups. Our findings suggest that the measurement of IA-2 IgG1 and IgG4 subclass antibodies can serve as surrogate marker to discriminate between antibody positive subjects at high or low risk for rapid development of diabetes.     

Autoantibodies predicting diabetes mellitus type I in celiac disease

Celiac disease (CD) and diabetes mellitus type I (DM-I) are both autoimmune diseases. Abnormal first-phase insulin response (FPIR) is associated with the prediabetic phase. Glutamic acid decarboxylase (GAD) and islet cell antibodies (ICAs) - especially the tyrosine phosphatase-like protein IA-2 antibodies - are considered to be serological markers of DM-I future development. The aim of this study is to investigate the presence of autoantibodies (GAD, IA-2) in individuals with CD, on a gluten-free diet, who have normal intestinal morphology. Thirty patients with CD (4-22, mean 15 years), 30 newly diagnosed diabetic children (2.5-16, mean 10 years) and 30 healthy subjects (7-35, mean 18 years) were investigated. Serum GAD and IA-2 autoantibodies were assessed by a quantitative enzyme-linked immunosorbent assay (ELISA) method in all patients and controls. Seven CD patients (23%), 28 diabetic children (93%) and none in the control group had positive GAD and IA-2 antibodies. The FPIR was normal in CD patients (>/=46 mU/l). Conclusions: GAD and IA-2 antibodies are detected in 23% of patients with CD. These patients may be at risk to develop DM-I. Regular follow-up and determination of FPIR for the early diagnosis of the prediabetic phase in patients with CD having circulating autoantibodies is recommended.     

High frequency of diabetes-specific autoantibodies in parents of children with type 1 diabetes. DENIS study group.

Strategies to identify subjects at risk for type 1 diabetes are largely based on the detection of autoantibodies directed to various beta cell autoantigens. Most previous studies only comprise siblings and children of patients with type 1 diabetes; only scare data are available on the antibody profile in older relatives. In this study, we examined the prevalence of cytoplasmic islet cell antibodies (ICA), antibodies to glutamic acid decarboxylase (GADA), antibodies to the protein tyrosine phosphatase IA-2 (IA-2A) and IA-2beta (IA-2betaA) in 531 unaffected parents of patients with type 1 diabetes, and compared the results with antibody frequencies in 2425 siblings. The frequency of ICA, GADA and IA-2A was substantially higher among siblings as compared to parents of patients with type 1 diabetes (8.0% vs. 4.5%, 8.0% vs. 4.3%, and 4.5% vs. 1.9%, respectively; p<0.01). However, subdividing the probands according to age revealed a high prevalence of ICA (5.5 %), GADA (5.9 %), and IA-2A (3.1%) among parents aged 31 -40 years which was similar to that observed in siblings above 20 years of age (6.4%, 6.4%, and 3.1%). In both cohorts, GADA and IA-2A were significantly associated with the presence of ICA. The combined screening for GADA and IA-2A identified 100% of parents and 91.9% of siblings at high risk for type 1 diabetes (>10 JDF-U). Furthermore, the analysis of antibody combinations revealed that among antibody positive individuals the percentage of subjects with two or three antibodies was even higher in parents (69.0%) than in siblings (58.2%). The present study shows a high frequency of single and multiple autoantibodies in unaffected parents of patients with type 1 diabetes. Our data indicate that GAD and IA-2 not only represent the major target of autoantibodies in young siblings but also in adult relatives. These findings may be important for the design of future intervention studies.     

Early autoantibody responses in prediabetes are IgG1 dominated and suggest antigen-specific regulation.

The islet autoimmunity of preclinical type 1 diabetes remains poorly characterized in humans. In this paper, the IgG subclass response to the islet autoantigens insulin, glutamic acid decarboxylase, and IA-2 was studied sequentially from birth to diabetes onset or current follow-up in 26 autoantibody positive offspring of parents with diabetes. Islet autoantibody appearance was characterized by an early IgG1 peak response to one or more Ags, most commonly to insulin, at a median age of 2.2 yr (interquartile range, 2-2.9 yr). In five offspring, an acute fulminant beta-cell destruction and diabetes onset occurred during this initial Ab response. In the remainder, early Ab levels declined markedly, and Ab peaks against other beta cell Ags arose sequentially over several years suggesting regulation and spreading of autoimmunity. Second peak Ab responses to the same Ag were observed in only two offspring, both developing diabetes at this time. Two others developed diabetes with declining Ab levels. Abs of IgG1 subclass dominated against each Ag, and other subclasses, were usually only detected during peak IgG1 responses. The IgG4 response to insulin was exceptional, being dominant over IgG1 in four offspring and in five others appeared and/or persisted after IgG1 levels declined. These Th2-associated IgG4 responses were not correlated with protection from diabetes. The presence of IgG1-restricted responses to DA2 were associated with diabetes development. These findings suggest that type 1 diabetes has an early acute destructive phase of beta cell autoimmunity, which may be regulated and which spreads chronically until diabetes onset.     

Genome-wide association analysis of autoantibody positivity in type 1 diabetes cases

The genetic basis of autoantibody production is largely unknown outside of associations located in the major histocompatibility complex (MHC) human leukocyte antigen (HLA) region. The aim of this study is the discovery of new genetic associations with autoantibody positivity using genome-wide association scan single nucleotide polymorphism (SNP) data in type 1 diabetes (T1D) patients with autoantibody measurements. We measured two anti-islet autoantibodies, glutamate decarboxylase (GADA, n = 2,506), insulinoma-associated antigen 2 (IA-2A, n = 2,498), antibodies to the autoimmune thyroid (Graves') disease (AITD) autoantigen thyroid peroxidase (TPOA, n = 8,300), and antibodies against gastric parietal cells (PCA, n = 4,328) that are associated with autoimmune gastritis. Two loci passed a stringent genome-wide significance level (p<10(-10)): 1q23/FCRL3 with IA-2A and 9q34/ABO with PCA. Eleven of 52 non-MHC T1D loci showed evidence of association with at least one autoantibody at a false discovery rate of 16%: 16p11/IL27-IA-2A, 2q24/IFIH1-IA-2A and PCA, 2q32/STAT4-TPOA, 10p15/IL2RA-GADA, 6q15/BACH2-TPOA, 21q22/UBASH3A-TPOA, 1p13/PTPN22-TPOA, 2q33/CTLA4-TPOA, 4q27/IL2/TPOA, 15q14/RASGRP1/TPOA, and 12q24/SH2B3-GADA and TPOA. Analysis of the TPOA-associated loci in 2,477 cases with Graves' disease identified two new AITD loci (BACH2 and UBASH3A).     

Two distinctly HLA-associated contiguous linear epitopes uniquely expressed within the islet antigen 2 molecule are major autoantibody epitopes of the diabetes-specific tyrosine phosphatase-like protein autoantigens

The related tyrosine phosphatase-like proteins islet Ag (IA)-2 and IA-2beta are autoantigens of type 1 diabetes in humans. Autoantibodies are predominantly against IA-2, and IA-2-specific epitopes are major autoantibody targets. We used the close homology of IA-2 and IA-2beta to design chimeras and mutants to identify humoral IA-2-specific epitopes. Two major IA-2 epitopes that are absent from the related autoantigens IA-2beta and IA-2Delta 13 splice variant ICA512.bdc were found contiguous to each other within IA-2 juxtamembrane amino acids 611-620 (epitope JM1) and 621-630 (epitope JM2). JM1 and JM2 are recognized by sera from 67% of patients with IA-2 Abs, and relatives of patients with type 1 diabetes having Abs to either JM epitope had a >50% risk for developing type 1 diabetes within 6 years, even in the absence of diabetes-associated HLA genotypes. Remarkably, the presence of Abs to one of these two epitopes was mutually exclusive of the other; JM2 Abs and not JM1 Abs were found in relatives with HLA DR3/4, DR4/13, or DR1/4 genotypes; and the binding of autoantibodies to the JM2 epitope, but not the JM1 epitope, markedly affected proteolysis of IA-2. This is a unique demonstration of HLA-associated B cell responses to epitopes within a single autoantigen in humans and is consistent with modification of Ag processing by specific Ab-influencing peptide presentation by HLA molecules.     

Autoantibodies against IA-2, GAD, and topoisomerase II in type 1 diabetic patients

Prevalence of autoantibodies against IA-2 (IA-2A), glutamic acid decarboxylase (GADA), and type II DNA topoisomerase (TopIIA) of Taiwanese type 1 diabetes mellitus (T1DM) patients was investigated. Correlations of these autoantibodies with patients' clinical manifestations were also analyzed. Prevalence of IA-2A, GADA, and TopIIA in our patients was 23.6%, 47.1%, and 55.2%, respectively. Eighty percent of the IA-2A recognized the carboxyl terminus of the IA-2 protein tyrosine phosphatase-like domain. Average disease duration of IA-2A+ patients was significantly shorter than that of IA-2A- patients [3.76+/-0.42 vs. 4.98+/-0.34 years, p = 0.028]. Presence of GADA was correlated with the mean age of onset [10.82+/-0.76 vs. 8.38+/-0.77 years for GADA+ and GADA- patients, p = 0.026]. Patients with adolescent onset have higher GADA prevalence and better residual beta-cell functions. TopIIA and GADA are suggested to be better markers for Taiwanese T1DM patients because of their higher prevalence and persistence.     

In vivo allogeneic effects: shift in the isotype profile of primary TI-2 responses in mice undergoing graft-vs-host reaction

We showed previously that primary responses to T-dependent (TD) and T-independent type 2 (TI-2) antigens were differentially affected by allogeneic effects induced in vivo during a graft-vs-host reaction (GVH). TD responses were greater than or equal to 80% suppressed, whereas the TI-2 responses were greatly enhanced, particularly the IgG component, which normally is very low. We have analyzed the IgG subclass distribution in primary responses of normal and GVH F1 mice in order to determine whether the strong T cell signals that occur during GVH reactions also induce shifts in the isotype profile. The effect of GVH on responses to TI-2 antigens was of particular interest because they are usually dominated by IgM and IgG3 classes in normal mice. We found a threefold to 10-fold increase in the PFC numbers of all four IgG subclasses in the response to TI-2 antigens, with an apparent shift from the usual IgG3 dominance to IgG1 in GVH mice. This IgG1 dominance was not found in serum antibodies where IgG3, IgG1, and IgG2b were equally expressed, although total IgG was increased greater than 20-fold. No isotype shift was found in either the TNP-KLH response, which was greater than or equal to 75% suppressed (IgG1 dominance was retained), or in the TI-1 response to TNP-Ba. The latter response was reduced (25 to 50%) in GVH mice and continued to be dominated by IgG2b/2a and IgG3. Unlike the unique isotype patterns found in primary responses, TNP-KLH primed mice challenged with TD, TI-1, or TI-2 antigens gave memory responses with identical isotype profiles that were dominated by IgG1 PFC. The role of T cells in B cell differentiation and isotype expression is discussed.     

Newly diagnosed latent autoimmune diabetes in adults (LADA) is associated with low level glutamate decarboxylase (GAD65) and IA-2 autoantibodies. Diabetes Incidence Study in Sweden (DISS).

A quantitative assay with microSepharose was used to determine GAD65Ab and IA-2Ab levels in 771 population-based patients diagnosed with diabetes mellitus at 15 to 34 years of age, and in 828 matched controls. Among the patients, 587 (76%) were classified with type I, 108 (14%) with type II, and 76 (10%) with unclassifiable diabetes. The levels above normal demonstrated a prevalence of GAD65Ab in 66% of type I diabetes, 50% of type II diabetes and 54% of unclassifiable patients and for IA-2Ab in 40%, 17% and 21%, respectively. Among the autoantibody-positive sera, the LADA patients had a lower GAD65Ab index (median 0.19, p < 0.0001) and IA-2Ab index (median 0.28, p < 0.0001) than the type I patients (median 0.37 and 0.66). Patients with unclassifiable diabetes had a GAD65Ab (median 0.43) or IA-2Ab (median 0.63) index which was not different from the type I diabetes patients. Our data demonstrate that young adult new-onset LADA patients have low level GAD65Ab and IA-2Ab. The low-level autoantibodies may signify a less aggressive beta-cell autoimmunity, which may explain why these patients are often classified with type II or non-insulin-dependent diabetes.     

Gamma 3 gene-disrupted mice selectively deficient in the dominant IgG subclass made to bacterial polysaccharides. II. Increased susceptibility to fatal pneumococcal sepsis due to absence of anti-polysaccharide IgG3 is corrected by induction of anti-polysaccharide IgG1

Bacterial polysaccharides (PS) are type 2 T-independent Ags that elicit Abs restricted in isotype to IgM and predominantly IgG2 in humans and IgM, and IgG3 in mice. Humans with IgG2 subclass deficiency are susceptible to sinus and pulmonary infections with PS-encapsulated bacteria. We previously developed an IgG3-deficient mouse by disrupting the gamma3 H chain constant region gene via targeted mutagenesis. Mutant mice lacking IgG3 were backcrossed for 10 generations to wild-type (WT) BALB/c mice to generate BALB/c mice that have complete absence of IgG3. WT mice immunized with type 3 Streptococcus pneumoniae capsular PS made anti-PS IgM, IgG3, and small quantities of IgG1, which opsonized S. pneumoniae for killing by polymorphonuclear leukocytes. These mice were protected against death from lethal doses of type 3 S. pneumoniae. In contrast, IgG3(-/-) mice made similar titers of anti-PS IgM and IgG1 as WT mice but no IgG3, and had poorly opsonic sera with significantly increased mortality after S. pneumoniae challenge. Immunization of IgG3(-/-) mice with type 3 S. pneumoniae PS conjugated to carrier protein CRM(197)-elicited IgM and high-titer IgG1 Abs, restored serum opsonization, and gave protection from mortality after S. pneumoniae, challenge comparable to WT mice. We conclude that mice lacking the dominant IgG3 subclass made to bacterial PS are more susceptible to fatal S. pneumoniae sepsis than WT mice, but that IgG1 induced by a S. pneumoniae glycoconjugate can adequately protect against S. pneumoniae sepsis. This model suggests that IgG subclass of anti-PS Ab is an important component of immunity to encapsulated bacteria.     

Mapping of epitopes for autoantibodies to the type 1 diabetes autoantigen IA-2 by peptide phage display and molecular modeling: overlap of antibody and T cell determinants

IA-2 is a major target of autoimmunity in type 1 diabetes. IA-2 responsive T cells recognize determinants within regions represented by amino acids 787-817 and 841-869 of the molecule. Epitopes for IA-2 autoantibodies are largely conformational and not well defined. In this study, we used peptide phage display and homology modeling to characterize the epitope of a monoclonal IA-2 Ab (96/3) from a human type 1 diabetic patient. This Ab competes for IA-2 binding with Abs from the majority of patients with type 1 diabetes and therefore binds a region close to common autoantibody epitopes. Alignment of peptides obtained after screening phage-displayed peptide libraries with purified 96/3 identified a consensus binding sequence of Asn-x-Glu-x-x-(aromatic)-x-x-Gly. The predicted surface on a three-dimensional homology model of the tyrosine phosphatase domain of IA-2 was analyzed for clusters of Asn, Glu, and aromatic residues and amino acids contributing to the epitope investigated using site-directed mutagenesis. Mutation of each of amino acids Asn(858), Glu(836), and Trp(799) reduced 96/3 Ab binding by >45%. Mutations of these residues also inhibited binding of serum autoantibodies from IA-2 Ab-positive type 1 diabetic patients. This study identifies a region commonly recognized by autoantibodies in type 1 diabetes that overlaps with dominant T cell determinants.     

ERK1-/- mice exhibit Th1 cell polarization and increased susceptibility to experimental autoimmune encephalomyelitis

Activation of MAPK ERK1/2 has been shown to play an important role in Th1/Th2 polarization and in regulating cytokine production from APCs. The ERK family consists of two members ERK1 and ERK2, which share approximately 84% identity at the amino acid level and can compensate for each other for most functions. Despite these features, ERK1 and ERK2 do serve different functions, but there is very little information on the contribution of individual forms of ERK on innate and adaptive immune responses. In this study, we describe that ERK1(-/-) mice display a bias toward Th1 type immune response. Consistent with this observation, dendritic cells from ERK1(-/-) mice show enhanced IL-12p70 and reduced IL-10 secretion in response to TLR stimulation. Furthermore, serum from ERK1(-/-) mice had 100-fold higher total IgG2b and 10-fold higher total IgG2a and IgG1 Ab isotype titers, and enhanced levels of Ag-specific IgG2b Ab titers, compared with wild-type mice. Consistent with this enhanced Th1 bias, ERK1(-/-) mice showed enhanced susceptibility to myelin oligodendrocyte glycoprotein (MOG)35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE) and developed EAE earlier, and with increased severity, compared with wild-type mice. Importantly, there was a profound skewing toward Th1 responses in ERK1(-/-) mice, with higher IFN-gamma production and lower IL-5 production in MOG35-55-primed T cells, as well as an augmentation in the MOG-specific IgG2a and IgG2b Th1 Ab isotypes. Finally, increased infiltrating cells and myelin destruction was observed in the spinal cord of ERK1(-/-) mice. Taken together, our data suggest that deficiency of ERK1 biases the immune response toward Th1 resulting in increased susceptibility to EAE.     

Heritability of Antibody Isotype and Subclass Responses to Plasmodium falciparum Antigens

Background

It is important to understand the extent to which genetic factors regulate acquired immunity to common infections. A classical twin study design is useful to estimate the heritable component of variation in measurable immune parameters.

Methodology/Principal Findings

This study assessed the relative heritability of different plasma antibody isotypes and subclasses (IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE) naturally acquired to P. falciparum blood stage antigens AMA1, MSP1-19, MSP2 (two allelic types) and MSP3 (two allelic types). Separate analyses were performed on plasma from 213 pairs of Gambian adult twins, 199 child twin pairs sampled in a dry season when there was little malaria transmission, and another set of 107 child twin pairs sampled at the end of the annual wet season when malaria was common. There were significantly positive heritability (h ²) estimates for 48% (20/42) of the specific antibody assays (for the seven isotypes and subclasses to the six antigens tested) among the adults, 48% (20/42) among the children in the dry season and 31% (13/42) among the children in the wet season. In children, there were significant heritability estimates for IgG4 reactivity against each of the antigens, and this subclass had higher heritability than the other subclasses and isotypes. In adults, 75% (15/20) of the significantly heritable antigen-specific isotype responses were attributable to non-HLA class II genetic variation, whereas none showed a significant HLA contribution.

Significance

Genome-wide approaches are now warranted to map the major genetic determinants of variable antibody isotype and subclass responses to malaria, alongside evaluation of their impact on infection and disease. Although plasma levels of IgG4 to malaria antigens are generally low, the exceptionally high heritability of levels of this subclass in children deserves particular investigation.     

Isotype concentrations of human antibodies to Haemophilus influenzae type b polysaccharide (Hib) in young adults immunized with the polysaccharide as such or conjugated to a protein (diphtheria toxoid)

Antibody responses of young adults to Haemophilus influenzae type b polysaccharide (Hib) or its protein conjugate were studied with special attention to the isotype composition of the antibodies. Three conclusions of interest can be made: 1) Immunoglobulin G (IgG) antibodies in polysaccharide-immunized volunteers displayed the subclass pattern previously found in antibodies to meningococcal type A polysaccharide. IgG1 was the predominant subclass in IgG antibodies of some individuals, IgG2 in others. Still others had the two subclasses in varying but more even proportions. 2) The conjugate vaccine induced a geometric mean response 2 to 3 times higher and an IgG response 4 times higher to Hib than the polysaccharide vaccine. 3) Anti-Hib antibodies induced by the conjugate vaccine still had essentially the same IgG subclass composition as anti-Hib antibodies induced by the polysaccharide. This composition was strikingly different from the composition of the anti-diphtheria toxoid response induced by the same conjugate vaccine.     

The effects of a stable analogue of PGE1 on the IgG subclass response to particulate bovine tubular basement membrane in the brown-Norway rat

The IgG subclass and the IgM isotype response to immunization with particulate bovine tubular basement membrane (TBM) and adjuvants was studied in Brown-Norway rats receiving daily injections of a stable analogue of PGE1 (M-PGE1). M-PGE1 slightly reduced the average quantity of circulating TBM antibody as well as the average quantity of eluted IgG per gram of renal tissue as compared to controls. However, M-PGE1 did not qualitatively affect the distribution of the IgG subclass or IgM isotype response to TBM. The IgG response, which occurred predominantly in the IgG1 and IgG2a subclasses, increased from Days 8 to 14 after immunization, while the IgM response decreased over the same time period. The percentage of TBM antibody in the IgG2b subclass was markedly decreased as compared to the percentage of IgG2b antibody in total IgG. A substantial heterogeneity in the IgG subclass response was noted among individual rats with IgG1 constituting from 46 to 82% of circulating TBM antibody. Although no correlation between the IgG subclass response and the severity of tubulointerstitial nephritis was noted, heterogeneity in the IgG subclass response to autoantigens may, nevertheless, theoretically play an important role in the pathogenesis of autoimmune inflammatory phenomena.     

IgG subclass responses in childhood Helicobacter pylori duodenal ulcer: evidence of T-helper cell type 2 responses

BACKGROUND: Duodenal ulcer in adults chronically infected with Helicobacter pylori is associated with a polarized T-helper cell type 1 (Th1) mucosal immune response, with a predominantly immunoglobulin G2 (IgG2) systemic specific response. It has been suggested that children colonized by H. pylori also produce a mucosal Th1 response, but there are few studies that have measured IgG subclass responses in children with duodenal ulcer. MATERIALS AND METHODS: Seven children with endoscopically proven duodenal ulcer and H. pylori infection and 18 children with biopsy proven H. pylori infection but no duodenal ulcer had relative concentrations of IgG subclass responses (IgGsc) against H. pylori antigens measured by ELISA. Eighteen IgG seropositive adults acted as controls. The range of antigens recognised by IgG1 and IgG2 subclass responses were investigated by Western blots. RESULTS: There were no differences in mean IgGsc responses between children with or without duodenal ulcer. Adults produced an IgG2 predominant response. Western blots showed no qualitative differences in antigens recognised by IgG1 or IgG2. CONCLUSION: Children with duodenal ulcer, in contrast to adults, produce an IgGsc response consistent with a mucosal Th2 response to H. pylori regardless of the presence of duodenal ulceration. This suggests that disease causation amongst children with H. pylori associated duodenal ulceration may not be dependant upon a mucosal Th1 biased response.     

The combination of antibodies to GAD-65 and IA-2ic can replace the islet-cell antibody assay to identify subjects at risk of type 1 diabetes mellitus.

First-degree relatives of type 1 diabetic patients are at increased risk of developing diabetes and, until recently, islet cell antibodies (ICA) have represented the major risk marker used for identification of individuals at increased risk for subsequent progression to diabetes. In order to determine the value of antibodies to GAD-65 and IA-2ic to identify individuals at high risk for type 1 diabetes mellitus, we measured both autoantibodies and ICA in 1436 first-degree relatives of patients with type 1 diabetes. In addition, the sera were analyzed for thyroid, adrenal and gastric-parietal cell autoantibodies as markers for possible polyendocrine involvement. GAD-65 Abs were found in 135 out of 1436 (9.4%) first-degree relatives and in 57 of 98 (58.2%) ICA-positive subjects. IA-2ic were detected in 52 of 1436 (3.6%) first-degree relatives and in 44 of 98 (44.8%) ICA-positive relatives. IA-2ic and/or GAD-65 were detected in 73 of 98 (74.5%) ICA-positive relatives. Interestingly, antibodies to GAD-65 and/or IA-2ic were present in 91.2% of individuals with more than 20JDF-units. Anti-IA-2ic and GAD-65 were positively correlated with high levels of ICA. Anti-IA-2ic and GAD-65 were found in 19% and 48.5% of subjects with ICA levels of 5-20JDF-u but in 68.8% and 76.5% of individuals with ICA of 40JDF-u or more, respectively (p < 0.001), compared to subjects with ICA levels less than 5 JDF-u. When autoantibody frequencies among the relatives were analyzed according to relationship to the proband, the offspring and siblings had a higher frequency of ICA and IA-2ic (p<0.05) than the subgroup of parents. A significant association was observed between IA-2ic and thyroid antibodies. In addition, higher levels of IA-2ic were found in relatives with positive TPO antibodies (p < 0.001); this correlation was particularly strong in offspring and siblings (p < 0.01). Determination of GAD-65 and IA-2ic antibodies may be considered as an alternative to primary ICA-screening, enabling the screening of large populations.     

Association study of the NRAMP1 gene promoter polymorphism and early-onset type 1 diabetes

Natural resistance-associated macrophage protein 1 (NRAMP1) has an important role in regulating macrophage functions that affect innate resistance as well as immune responses. We analyzed the microsatellite polymorphism in the promoter region of the human NRAMP1 gene in 206 type 1 diabetes patients and 200 normal children to determine whether this polymorphism might be associated with type 1 diabetes in the Japanese population. The frequency of allele 2 (180 bp) of the promoter microsatellite polymorphism of the NRAMP1gene was slightly lower in the early-onset population (2-10 years of age) of type 1 diabetes patients than in controls, although the difference did not reach statistical significance. The association study of the cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) gene, located near the NRAMP1 gene, and type 1 diabetes showed that the CTLA-4 gene significantly contributed to the development of type 1 diabetes, whereas NRAMP1 had an additional effect on the onset of type 1 diabetes in the young population.     

Human monoclonal antibodies isolated from type I diabetes patients define multiple epitopes in the protein tyrosine phosphatase-like IA-2 antigen

Protein tyrosine phosphatase-like IA-2 autoantigen is one of the major targets of humoral autoimmunity in patients with insulin-dependant diabetes mellitus (IDDM). In an effort to define the epitopes recognized by autoantibodies against IA-2, we generated five human mAbs (hAbs) from peripheral B lymphocytes isolated from patients most of whom had been recently diagnosed for IDDM. Determination and fine mapping of the critical regions for autoantibody binding was performed by RIA using mutant and chimeric constructs of IA-2- and IA-2beta-regions. Four of the five IgG autoantibodies recognized distinct epitopes within the protein tyrosine phosphatase (PTP)-like domain of IA-2. The minimal region required for binding by three of the PTP-like domain-specific hAbs could be located to aa 777-979. Two of these hAbs cross-reacted with the related IA-2beta PTP-like domain (IA-2beta aa 741-1033). A further PTP-like domain specific hAb required the entire PTP-like domain (aa 687-979) for binding, but critical amino acids clustered in the N-terminal region 687-777. An additional epitope could be localized within the juxtamembrane domain (aa 603-779). In competition experiments, the epitope recognized by one of the hAbs was shown to be targeted by 10 of 14 anti-IA-2-positive sera. Nucleotide sequence analysis of this hAb revealed that it used a V(H) germline gene (DP-71) preferably expressed in autoantibodies associated with IDDM. The presence of somatic mutations in both heavy and light chain genes and the high affinity or this Ab suggest that the immune response to IA-2 is Ag driven.     

Effect of vaccination against pneumococcal infection in children with type 1 diabetes mellitus

Vaccination with polysaccharide pneumococcal vaccine "Pneumo 23" (Sanofi Pasteur, France) was performed in 31 children with type 1 diabetes mellitus (DM1) as well as in 19 children with respiratory tract diseases (asthma, chronic pneumonia), which formed comparison group. Fourty-three unvaccinated children with DM1 were included in the control group. Dynamics of IgG levels to mixture of pneumococcal polysaccharides (PS) included in the vaccine as well as to PS of serotypes 3, 6B, 9N, 23F, and to cell wall polysaccharides of Streptococcus pneumoniae were assessed. Using ELISA method, significant increase of IgG levels to mixture of PS and to PS of pneumococcal serotype 3 was detected. Although intensity of immune response to vaccination in children with respiratory diseases was significantly higher compared to children with DM1 (mean geometric titer of antibodies, proportion of patients with high antibody titers, and with 4-fold seroconversion). Development of methods to strengthen immune response in children with DM1 vaccinated against pneumococcal infection is required.