Characteristics of familial type 1 diabetes: effects of the relationship to the affected family member on phenotype and genotype at diagnosis.

AIMS/HYPOTHESIS: In previous studies, the risk of developing familial type 1 diabetes has been reported to be more than two times higher in the offspring of affected fathers than in those of affected mothers. We tested the hypothesis that index children with an affected father may have a more aggressive disease process at diagnosis than those with other affected first-degree relatives. METHODS: A cross-sectional, observational study was performed using the Finnish Pediatric Diabetes Register. Clinical and metabolic characteristics, beta cell autoantibodies and HLA class II genetics were analysed from index children in Finland diagnosed before the age of 15 years between January 2003 and December 2016. Information on the presence of type 1 diabetes in first-degree relatives was collected at diagnosis using a structured questionnaire. RESULTS: Out of 4993 newly diagnosed index children, 519 (10.4%) had familial type 1 diabetes. More than 5% (n = 253, 5.1%) had an affected father, 2.8% (n = 141) had an affected mother, 1.9% (n = 95) had an affected sibling and 0.6% (n = 30) had two or more affected family members. All clinical and metabolic variables were markedly poorer in children with sporadic vs familial diabetes. The index children with an affected father or mother were younger than those with an affected sibling (median age 7.59 vs 6.74 vs 10.73 years, respectively; p < 0.001). After age- and sex-adjusted analyses, index children with an affected father presented more often with ketoacidosis (9.7% vs 3.6%; p = 0.033) and had greater weight loss before diagnosis (3.2% vs 0%; p = 0.006) than those with an affected mother. Children with familial disease tested negative for all autoantibodies more often (3.5% vs 2.1%; p = 0.041) and had insulin autoantibodies more frequently (49.8% vs 42.2%; p = 0.004) than those with sporadic disease. Both major HLA risk haplotypes (DR3-DQ2 and DR4-DQ8) were more often lacking among children with sporadic vs familial disease (15.9% vs 11.2%; p = 0.006). The DR4-DQ8 haplotype was more frequent in the familial vs the sporadic group (75.7% vs 68.5%; p = 0.001) and especially among children with an affected father when compared with children with sporadic disease (77.5% vs 68.5%; p < 0.05). When comparing index children with affected parents diagnosed before or after the birth of the index child, a clear male preponderance was seen among the affected parents diagnosed before the birth of the index child (fathers 66.2% vs mothers 33.8%; p = 0.006), whereas the proportion of fathers and mothers was similar if type 1 diabetes was diagnosed after the birth of the index child. CONCLUSIONS/INTERPRETATION: The more severe metabolic derangement at diagnosis in children with sporadic type 1 diabetes compared with those with familial type 1 diabetes was confirmed. The higher frequency of diabetic ketoacidosis and increased weight loss at diagnosis in index children with an affected father compared with an affected mother support the hypothesis that paternal type 1 diabetes is associated with more severe disease in the offspring than maternal diabetes. The sex difference seen between affected parents diagnosed before and after the birth of the index child supports the hypothesis that maternal insulin treatment protects against type 1 diabetes.

Antibodies to post-translationally modified insulin in type 1 diabetes.

AIM/HYPOTHESIS: Insulin is the most specific beta cell antigen and a potential primary autoantigen in type 1 diabetes. Insulin autoantibodies (IAAs) are the earliest marker of beta cell autoimmunity; however, only slightly more than 50% of children and even fewer adults newly diagnosed with type 1 diabetes are IAA positive. The aim of this investigation was to determine if oxidative post-translational modification (oxPTM) of insulin by reactive oxidants associated with islet inflammation generates neoepitopes that stimulate an immune response in individuals with type 1 diabetes. METHODS: oxPTM of insulin was generated using ribose and various reactive oxygen species. Modifications were analysed by SDS-PAGE, three-dimensional fluorescence and MS. Autoreactivity to oxPTM insulin (oxPTM-INS) was observed by ELISA and western blotting, using sera from participants with type 1 or type 2 diabetes and healthy controls as probes. IAA was measured using the gold-standard radiobinding assay (RBA). RESULTS: MS of oxPTM-INS identified chlorination of Tyr16 and Tyr26; oxidation of His5, Cys7 and Phe24; and glycation of Lys29 and Phe1 in chain B. Significantly higher binding to oxPTM-INS vs native insulin was observed in participants with type 1 diabetes, with 84% sensitivity compared with 61% sensitivity for RBA. oxPTM-INS autoantibodies and IAA co-existed in 50% of those with type 1 diabetes. Importantly 34% of those with diabetes who were IAA negative were oxPTM-INS positive. Altogether, 95% of participants with type 1 diabetes presented with autoimmunity to insulin by RBA, oxPTM-INS or both. Binding to oxPTM-INS was directed towards oxPTM-INS fragments with slower mobility than native insulin. CONCLUSION/INTERPRETATION: These data suggest that oxPTM-INS is a potential autoantigen in individuals with new-onset type 1 diabetes.

Patterns of ß-cell autoantibody appearance and genetic associations during the first years of life.

We analyzed demographic and genetic differences between children with various diabetes-associated autoantibodies reflecting the autoimmune process. In a prospective birth cohort comprising children with HLA-conferred susceptibility to type 1 diabetes (T1D), the pattern of autoantibody appearance was analyzed in 520 children with advanced ß-cell autoimmunity associated with high risk for disease. In 315 cases, a single biochemical autoantibody could be identified in the first positive sample as insulin (insulin autoantibody [IAA]) in 180, as GAD (GAD antibody [GADA]) in 107, and as IA-2 antigen (IA-2 antibody [IA-2A]) in 28. The age at seroconversion differed significantly between the three groups (P = 0.003). IAA as the first autoantibody showed a peak time of appearance during the second year of life, whereas GADA as the first autoantibody peaked later, between 3 and 5 years of age. The risk-associated insulin gene rs689 A/A genotypes were more frequent in children with IAA as the first autoantibody compared with the other children (P = 0.002). The primary autoantigen in the development of ß-cell autoimmunity and T1D seems to strongly correlate with age and genetic factors, indicating heterogeneity in the initiation of the disease process.

Associations of polymorphisms in non-HLA loci with autoantibodies at the diagnosis of type 1 diabetes: INS and IKZF4 associate with insulin autoantibodies.

OBJECTIVE: More than 50 loci outside the human leukocyte antigen (HLA) region have been confirmed to affect type 1 diabetes (T1D) risk but their effect on ß-cell autoimmunity is poorly defined. We analyzed the association of 35 single nucleotide polymorphism (SNP) markers previously associated with T1D with the presence of disease-predictive autoantibodies at the time of T1D diagnosis. SUBJECTS AND METHODS: The study cohort comprised 1554 children diagnosed with T1D before the age of 15 yr. The associations between various genotypes and positivity for antibodies against islet cells [islet cell antibodies (ICA)], insulin [insulin autoantibodies (IAA)], glutamic acid decarboxylase (GADA), islet antigen 2 (IA2A), and zinc transporter 8 (ZnT8A) were analyzed. RESULTS: INS gene polymorphism rs689 and IKZF4 polymorphism (rs1701704) were strongly associated with IAA positivity at the time of T1D diagnosis (p = 0.000004 and 0.00044, respectively). The presence of the T1D-risk conferring INS AA genotype was associated with IAA. In contrast, the presence of the susceptible C allele of the IKZF4 marker was inversely associated with IAA. The INS and IKZF4 polymorphisms were not significantly associated with ICA, GADA, IA2A, or ZnT8A positivity. CONCLUSIONS: Both INS and IKZF4 polymorphisms modified the probability of IAA positivity at time of T1D onset but the inverse association of IKZF4 risk allele with IAA suggests that the IKZF4 polymorphism is involved in a pathway of ß-cell autoimmunity alternate to the route characterized by IAA and development of T1D in early childhood. The IKZF4 gene encodes Eos, which is implicated to play an important role in Treg programming where this gene might exert its influence on T1D risk.

Genetic association of zinc transporter 8 (ZnT8) autoantibodies in type 1 diabetes cases.

AIMS/HYPOTHESIS: Autoantibodies to zinc transporter 8 (ZnT8A) are associated with risk of type 1 diabetes. Apart from the SLC30A8 gene itself, little is known about the genetic basis of ZnT8A. We hypothesise that other loci in addition to SLC30A8 are associated with ZnT8A. METHODS: The levels of ZnT8A were measured in 2,239 British type 1 diabetic individuals diagnosed before age 17 years, with a median duration of diabetes of 4 years. Cases were tested at over 775,000 loci genome wide (including 53 type 1 diabetes associated regions) for association with positivity for ZnT8A. ZnT8A were also measured in an independent dataset of 855 family members with type 1 diabetes. RESULTS: Only FCRL3 on chromosome 1q23.1 and the HLA class I region were associated with positivity for ZnT8A. rs7522061T>C was the most associated single nucleotide polymorphism (SNP) in the FCRL3 region (p = 1.13 × 10(-16)). The association was confirmed in the family dataset (p ≤ 9.20 × 10(-4)). rs9258750A>G was the most associated variant in the HLA region (p = 2.06 × 10(-9) and p = 0.0014 in family cases). The presence of ZnT8A was not associated with HLA-DRB1, HLA-DQB1, HLA-A, HLA-B or HLA-C (p > 0.05). Unexpectedly, the two loci associated with the presence of ZnT8A did not alter risk of having type 1 diabetes, and the 53 type 1 diabetes risk loci did not influence positivity for ZnT8A, despite them being disease specific. CONCLUSIONS/INTERPRETATION: ZnT8A are not primary pathogenic factors in type 1 diabetes. Nevertheless, ZnT8A testing in combination with other autoantibodies facilitates disease prediction, despite the biomarker not being under the same genetic control as the disease.

[Association of GHRd3 variant of growth hormone receptor gene with autoimmunity in type 1 diabetes]. / Asociación de la variante GHRd3 del receptor de la hormona de crecimiento con autoinmunidad en la diabetes tipo 1.

BACKGROUND: Growth Hormone Receptor (GRH) is expressed in the liver, pancreas, stomach and small intestine. A high expression of GHR mRNA in the mucosal gut suggests a possible role of this receptor on digestive and immune functions. AIM: To investigate the putative effects of the GHRd3 variants on the cytokine profile and distribution of auto-antibodies in children with type 1 diabetes (T1D). MATERIAL AND METHODS: Unrelated unaffected controls (n =192) and incident cases of children with T1D (n =127) were analyzed for GHRd3 polymorphism, cytokine profile and a panel of auto-antibodies. RESULTS: The allele frequency for d3 was 24.8% in type 1 diabetics and 34.1% in controls (p =NS). Among type 1 diabetic children, the carriers of the GHRd3 polymorphism had significantly higher levels of interleukin-lB than homozygous for the wild type genotype (5.7 and 17.7, pg/ml respectively p <0.015). Carriers of d3 variant had a higher frequency of positive anti-insulin antibodies (anti-IAA) than children without this variant (39.6 and 17.7% respectively, p <0.01). CONCLUSIONS: The observed frequency of the GHR d3/d3 genotype was comparable to other reports. A relationship between d3 variant and anti-IAA antibodies and interleukin-lss was observed.

Autoantibodies to zinc transporter 8 and SLC30A8 genotype stratify type 1 diabetes risk.

AIMS/HYPOTHESIS: Our aim was to determine the relationships between autoantibodies to zinc transporter 8 (ZnT8), genotypes of the ZnT8-encoding gene SLC30A8 and type 1 diabetes risk. METHODS: ZnT8 autoantibodies (ZnT8A) were measured in sera of 1,633 children with a first-degree family history of type 1 diabetes and who were prospectively followed from birth. Antibodies were measured by Protein A-based radiobinding assays and COOH-terminal (R325, W325 or Q325 variants) or NH(2)-terminal constructs of human ZnT8. SLC30A8 genotyping at single-nucleotide polymorphism (SNP) rs13266634 was performed on 1,170 children. RESULTS: Antibodies against COOH-terminal ZnT8 constructs (ZnT8A-COOH) developed in 58 children as early as 9 months of age (median 3 years). They were detected in 55 of 128 (43%) children with autoantibodies to insulin, GAD and/or insulinoma-associated protein 2 and 34 of 42 (81%) who progressed to diabetes. The additional presence of ZnT8A-COOH stratified diabetes risk in islet autoantibody-positive children (p < 0.0001). SLC30A8 genotype strongly influenced ZnT8A type and diabetes risk in ZnT8A-COOH-positive children. Antibody binding against the ZnT8 R325 variant was strictly correlated with the number of the corresponding SLC30A8 R325-encoding alleles, whereas binding against the W325 variant was highest in children who had SLC30A8 W325-encoding alleles (p = 0.001). Moreover, ZnT8A-COOH-positive children who carried homozygous SLC30A8 SNP rs13266634 genotypes progressed faster to diabetes than those who were heterozygous (59% [95% CI 42.3-75.7%] vs 22% [95% CI 0-44.3%] within 5 years; p = 0.01). CONCLUSIONS/INTERPRETATION: Autoimmunity against the COOH-terminal region of ZnT8 is a highly relevant prognostic feature in childhood type 1 diabetes. Risk stratification in ZnT8A-COOH-positive children is further improved by SLC30A8 genotyping.

Asociación de la variante GHRd3 del receptor de la hormona de crecimiento con autoinmunidad en la diabetes tipo 1 / Association of GHRd3 variant of growth hormone receptor gene with autoimmunity in type 1 diabetes

Background: Growth Hormone Receptor (GRH) is expressed in the liver, pancreas, stomach and small intestine. A high expression of GHR mRNA in the mucosal gut suggests a possible role of this receptor on digestive and immune functions. Aim: To investigate the putative effects of the GHRd3 variants on the cytokine profile and distribution of auto-antibodies in children with type 1 diabetes (T1D). Material and Methods: Unrelated unaffected controls (n =192) and incident cases of children with T1D (n =127) were analyzed for GHRd3 polymorphism, cytokine profile and a panel of auto-antibodies. Results: The allele frequency for d3 was 24.8 percent in type 1 diabetics and 34.1 percent in controls (p =NS). Among type 1 diabetic children, the carriers of the GHRd3 polymorphism had significantly higher levels of interleukin-lB than homozygous for the wild type genotype (5.7 and 17.7, pg/ml respectively p <0.015). Carriers of d3 variant had a higher frequency of positive anti-insulin antibodies (anti-IAA) than children without this variant (39.6 and 17.7 percent respectively, p <0.01). Conclusions: The observed frequency of the GHR d3/d3 genotype was comparable to other reports. A relationship between d3 variant and anti-IAA antibodies and interleukin-1ß was observed.

Islet autoantibodies are associated with HLA-DQ genotypes in Han Chinese patients with type 1 diabetes and their relatives.

The objective of this study was to explore the relationship between islet autoantibodies of glutamic acid decarboxylase (GADA), islet antigen-2A (IA-2A), insulin autoantibody (IAA), and human leukocyte antigen (HLA)-DQ genotypes in type 1 diabetes (T1D) patients and their first-degree relatives (FDRs). Cross-sectional and case-control study. Four hundred and ninety-five T1D patients, 419 FDRs, and 376 control subjects in Han Chinese populations were recruited and tested for GADA and IA-2A, while 71 cases, all FDRs and 300 controls were tested for IAA. The 338 T1D patients (including 187 antibody-positive and 151 antibody-negative patients), 173 FDRs and 278 controls were genotyped for HLA-DQ with polymerase chain reaction sequencing-based method. Compared with the control, the frequency of DQA1*03-DQB1*0303, DQA1*05-DQB1*0201, and DQA1*03-DQB1*0401 haplotypes was higher (P < 0.05-0.01) but DQA1*0102-DQB1*0602 haplotype was lower (P < 0.01) in T1D patients. DQA1*03 allele was less in the FDRs than in their probands (P < 0.05). GADA was more prevalent in T1D patients carrying DQA1*05-DQB1*0201 or DQA1*03-DQB1*0401 haplotype (55.8% vs 41.0%, 65.5% vs 40.3%, P < 0.05-0.01), whereas IA-2A presented more in the patients carrying DQA1*03-DQB1*0303 haplotype (27.0% vs 7.9%, P < 0.05-0.01), both GADA and IA-2A showed frequently in the patients with DQA1*03-DQB1*0303/DQA1*05-DQB1*0201 haplotypes (34.5% vs 9.7%, P < 0.01). GADA positivity was lower in the patients with DQA1*0102-DQB1*0602 haplotype (16.7% vs 45.9%, P < 0.05). The frequency of IAA was not different between patients with and without susceptible DQ haplotypes (P > 0.05). GADA, IA-2A or IAA presented frequently in FDRs with DQA1*03-DQB1*0303 haplotype. The findings in the study indicate that some of specific HLA-DQA1/-DQB1 genotypes and haplotypes not only confer susceptibility to T1D but also are associated with the presence of the islet autoantibodies in the Han Chinese population.

The effect of HLA class II, insulin and CTLA4 gene regions on the development of humoral beta cell autoimmunity.

AIMS/HYPOTHESIS: The aim of this study was to explore the contribution of genetic factors to the emergence of beta-cell-specific humoral autoimmunity. SUBJECTS AND METHODS: We analysed the effect of HLA class II, insulin (INS; -23 HphI variant) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4 [+49 and CT60]) genes on the appearance of beta-cell-specific autoantibodies in a large population-based birth cohort recruited in Finland. Infants carrying increased risk HLA DQB1 genotypes were monitored for the appearance of autoantibodies (islet cell autoantibodies [ICA], insulin autoantibodies [IAA], glutamic acid decarboxylase autoantibodies [GADA] and islet antigen 2 antibodies [IA-2A]). Those who developed beta-cell-specific autoantibodies were studied (n=574, mean follow-up time: 4.9 years; range 0.5-9.3). RESULTS: IAA emerged at a higher rate in children with the -23 HphI AA INS genotype than in those carrying AT or TT variants (hazard ratio 2.1, 95% CI 1.4-2.9, p<0.001). This effect of the INS locus was present in both HLA DQB1 risk groups. The appearance of IAA showed a strong association also with the HLA DRB1*0401 allele (hazard ratio 13.1, 95% CI 1.8-93.4, p<0.001). The development of IA-2A was also somewhat accelerated by the DRB1*0401 variant (p=0.03). Isolated ICA positivity was independent of the HLA and INS genotypes. None of the humoral immune markers showed association with the CTLA4 gene. CONCLUSIONS/INTERPRETATION: The INS and the DRB1 loci appear to contribute to the pathogenesis of type 1 diabetes by initiating/modifying insulin-specific autoimmunity. The emergence of IAA represents a crucial step in the development of beta cell autoimmunity in young children, in whom the appearance of GADA and IA-2A is linked to IAA.

Multiple germline kappa light chains generate anti-insulin B cells in nonobese diabetic mice.

The highly selective nature of organ-specific autoimmune disease is consistent with a critical role for adaptive immune responses against specific autoantigens. In type 1 diabetes mellitus, autoantibodies to insulin are important markers of the disease process in humans and nonobese diabetic (NOD) mice; however, the Ag-specific receptors responsible for these autoantibodies are obscured by the polyclonal repertoire. NOD mice that harbor an anti-insulin transgene (Tg) (V(H)125Tg/NOD) circumvent this problem by generating a tractable population of insulin-binding B cells. The nucleotide structure and genetic origin of the endogenous kappa L chain (Vkappa or IgL) repertoire that pairs with the V(H)125Tg were analyzed. In contrast to oligoclonal expansion observed in systemic autoimmune disease models, insulin-binding B cells from V(H)125Tg/NOD mice use specific Vkappa genes that are clonally independent and germline encoded. When compared with homologous IgL genes from nonautoimmune strains, Vkappa genes from NOD mice are polymorphic. Analysis of the most frequently expressed Vkappa1 and Vkappa9 genes indicates these are shared with lupus-prone New Zealand Black/BINJ mice (e.g., Vkappa1-110*02 and 9-124) and suggests that NOD mice use the infrequent b haplotype. These findings show that a diverse repertoire of anti-insulin B cells is part of the autoimmune process in NOD mice and structural or regulatory elements within the kappa locus may be shared with a systemic autoimmune disease.

Uncoupling of anergy from developmental arrest in anti-insulin B cells supports the development of autoimmune diabetes.

Loss of tolerance is considered to be an early event that is essential for the development of autoimmune disease. In contrast to this expectation, autoimmune (type 1) diabetes develops in NOD mice that harbor an anti-insulin Ig transgene (125Tg), even though anti-insulin B cells are tolerant. Tolerance is maintained in a similar manner in both normal C57BL/6 and autoimmune NOD mice, as evidenced by B cell anergy to stimulation through their Ag receptor (anti-IgM), TLR4 (LPS), and CD40 (anti-CD40). Unlike B cells in other models of tolerance, anergic 125Tg B cells are not arrested in development, and they enter mature subsets of follicular and marginal zone B cells. In addition, 125Tg B cells remain competent to increase CD86 expression in response to both T cell-dependent (anti-CD40) and T cell-independent (anti-IgM or LPS) signals. Thus, for anti-insulin B cells, tolerance is characterized by defective B cell proliferation uncoupled from signals that promote maturation and costimulator function. In diabetes-prone NOD mice, anti-insulin B cells in this novel state of tolerance provide the essential B cell contribution required for autoimmune beta cell destruction. These findings suggest that the degree of functional impairment, rather than an overt breach of tolerance, is a critical feature that governs B cell contribution to T cell-mediated autoimmune disease.

The natural autoantibody repertoire of nonobese diabetic mice is highly active.

Analysis of spontaneous hybridomas generated from nonobese diabetic (NOD) mice indicates that the natural autoantibody repertoire of NOD mice is highly active compared with C57BL/6 and BALB/c mice. This property of increased B cell activity is present early in life (4 wk) and persists in older mice of both sexes. Even when selected for binding to a prototypic beta cell Ag, such as insulin, NOD mAb have characteristics of natural autoantibodies that include low avidity and broad specificity for multiple Ags. Analyses of the variable region of Ig H chain (V(H)) and variable region kappa L chain genes expressed by six insulin binding mAb show that V gene segments are often germline encoded and are identical with those used by autoantibodies, especially anti-dsDNA, from systemic autoimmune disease in MRL, NZB/W, and motheaten mice. V(H) genes used by four mAb are derived from the large J558 family and two mAb use V(H)7183 and V(H)Q52 genes. The third complementarity-determining region of Ig H chain of these mAb have limited N segment diversity, and some mAb contain DNA segments indicative of gene replacement. Genetic abnormalities in the regulation of self-reactive B cells may be a feature that is shared between NOD and conventional systemic autoimmune disorders. In NOD, the large pool of self-reactive B cells may fuel autoimmune beta cell destruction by facilitating T-B cell interactions, as evidenced by the identification of one mAb that has undergone Ag-driven somatic hypermutation.

Insulin autoimmunity: prediction/precipitation/prevention type 1A diabetes.

Type 1 diabetes of both the NOD mouse and man is associated with autoimmunity directed against insulin which is the only beta cell specific autoantigen identified to date. One can use autoantibodies to insulin to predict diabetes, use insulin peptides to create insulin autoantibodies, insulitis and diabetes, and use insulin or its peptides in animal models to prevent diabetes. An expanding set of resources are now available for the development and testing in man of therapies to prevent type 1 diabetes, and a number of trials utilizing insulin peptides are now underway.

Somatically mutated B cell pool provides precursors for insulin antibodies.

Antibodies to insulin are products of autoreactive B lymphocytes that escape inactivation or clonal deletion and are examples of "clonal ignorance." To understand the genetic origin of Abs from clonally ignorant B cells, the roles of somatic mutation and germ-line V(H) structures were examined for two murine IgG1 mAb that bind human and rodent insulin. Engineered mAb constructs that express germ-line or mutated V(H) genes show that somatic mutations introducing aspartic acid in or adjacent to CDRH2 play a key role in insulin binding. When either of the two anti-insulin V(H) regions is returned to its germ-line (unmutated) sequence, neither mAb binds insulin and the germ-line-encoded mAb are not polyreactive. Reconstruction of the somatic evolution of insulin binding in both mAbs shows that a single mutation in CDRH2 is sufficient to generate anti-insulin activity from a nonbinding precursor. When the role of somatic mutation in the binding of rodent insulin is examined, autoreactivity is associated with single mutations in both Abs. Together these findings indicate that, despite a low mutation frequency, IgG insulin Abs may not be derived directly from germ-line (unmutated) precursors. The requirement for somatic mutation as a prerequisite for measurable insulin binding suggests these Abs have their origin in a previously mutated B cell pool as a consequence of the individual's immune history. Low avidity interaction with endogenous insulin may play a role in selection of these B cells and contribute to the origin of clonal ignorance.

VH gene structure predicts a large potential anti-insulin repertoire.

The majority of insulin antibodies derived from immunization are IgG antibodies that cross-react extensively with the autologous hormone. To examine the relationship between VH genes expressed by such self-reactive antibodies and their germline (non-rearranged) counterparts, we used the polymerase chain reaction (PCR) to amplify and isolate the germline progenitors of anti-insulin VH genes derived from BALB/c mice immunized with beef or human insulin. Results indicate that two anti-insulin mAbs (123 and 124) express VH genes which arise from a small subset of the J558 gene family and are highly homologous to the VH gene used by the murine CD5 + B-cell tumor, BCL1. The anti-insulin IgG mAb 127 belongs to the VH-VIII (Vgam 3.2) family and the amplification and isolation of germline VH genes from this small family precisely identified only two somatic mutation events in the CDRH2 of mAb 127. Another anti-insulin mAb, 133, also shows two replacement substitutions in the CDRHs when compared to the germline encoded anti-dextran antibody 19.1.2. These findings indicate that the IgG response to this small self-protein uses multiple VH genes which are largely germline encoded with only a low level of somatic mutation in their CDRHs. Additionally, analysis of N-segment additions in CDRH3s indicates anti-insulin B cells may originate from both early (fetal) and adult repertoires. These data are consistent with the concept that the mechanisms of clonal anergy or deletion do not regulate anti-insulin B cells and indicate that there is a large potential VH gene repertoire for insulin.

Molecular cloning, expression and mutagenesis of an anti-insulin single chain Fv (scFv).

The immunoglobulin variable region genes of a murine anti-insulin IgG-producing hybridoma were rescued and cloned into a bacterial expression vector. The variable regions of the gamma heavy chain and the kappa light chain were expressed independently and together as a single chain antibody (scFv). The variable heavy chain alone demonstrated the ability to bind to insulin. The kappa light chain did not show any binding activity towards insulin. The scFv was constructed by PCR assembly using a (Gly4Ser)3 linker between the carboxyl end of the variable heavy chain and the amino terminus of the kappa light chain. The scFv bound insulin at an IC50 of 3.5 x 10(-8) M whereas the parent antibody bound insulin at 1.0 x 10(-8) M. Mutagenesis of the variable heavy chain complementarity determining regions (CDR) indicated that CDR1 and CDR3 were important for binding to insulin. Position 99 in CDR3 of the heavy chain was found to be a critical position for the ability of the scFv to bind to insulin.

Anti-insulin and regulatory anti-idiotypic antibodies use the same germ-line VHIX gene.

In humans and in BALB/c mice, immune responses to the hormone insulin use evolutionarily related VHV (human) and VHIX (murine) gene families. To determine if these structural relationships include regulatory elements, BALB/c mice were pretreated with autologous immunoglobulin G (IgG) monoclonal antibodies (mAb) that recognize shared idiotopes on human anti-insulin antibodies and the subsequent immune response to human insulin assessed. One mAb, Id227, was found to augment and accelerate the insulin response by inducing a human idiotype that is expressed on both insulin-binding and non-insulin-binding BALB/c antibodies. Analysis of VH gene utilization by Id227 shows that it expresses a VHIX gene similar to that of anti-insulin mAb 125, but the anti-Id has no anti-insulin activity. Using DNA amplification, four germ-line VHIX genes were isolated from BALB/c liver DNA and sequence analysis shows that the anti-insulin and anti-Id are derived from the same germ-line gene. Consistent with its role as a regulatory idiotype, IgG Id227 entirely preserves germ-line sequence in the complementary determining regions and contains only three mutations in framework regions. These studies show that both structural and regulatory features of immune responses to conserved self antigens extend beyond species boundaries.

Immunogenetic heterogeneity in type I (insulin-dependent) diabetes among the Tunisian population. HLA-DR antigens and organ-specific autoantibodies (family study).

The frequency of HLA-DR antigens, as well as the prevalence of islet cell insulin autoantibodies and other autoimmunity disorders, were investigated in Tunisian patients with insulin-dependent diabetes mellitus (IDDM) and were compared with family members (sibs) and healthy control subjects. Cytoplasmic islet cell autoantibodies (ICA) were found in 79 of 175 (45.1%) patients with IDDM, in 23 of 126 (18.25%) unaffected first degree relatives of type I diabetes patients and in only two of 146 (1.3%) control subjects. In 79 ICA positive patients with IDDM, 46.8% presented other evidence of autoimmunity by testing for specific autoantibodies. Insulin autoantibodies were found in 86.9% of healthy ICA-positive sibs. A good correlation between HLA-DR3/DR4 heterozygous phenotypes and the presence of ICA in patients with IDDM and their unaffected sibs was observed in the Tunisian population. In fact, this heterozygous phenotype is found in 63.3% of ICA-positive diabetic patients and in 44.4% of ICA-positive unaffected sibs, whereas, HLA-DR3/DR4 antigens were noted in only 22.9% of ICA-negative diabetic patients and in no ICA-negative unaffected sibs. In these studies, we also summarized the distribution of HLA-DR antigens in patients with IDDM who presented autoimmune disorders other than ICA.