Novel considerations on the antibody/autoantigen system in type I (insulin-dependent) diabetes mellitus

Insulin dependent diabetes (IDDM) has an autoimmune pathogenesis. Included is the presence of antibodies to pancreatic islet cells. The first identified were islet cell antibodies (ICA), detected by indirect immunofluorescence, and which react with all cells within islets. Importantly, the autoantibodies are found several years prior to disease and although a pathogenic role for the autoantibodies is unclear, they have become useful markers of prediabetes. A number of studies of twins discordant for IDDM and of first degree relatives of IDDM patients have established that there is an increased risk for disease in individuals who have ICA, especially when ICA levels are high. This high predictive value of ICA decreases in the general population where the incidence of IDDM is lower than in first degree relatives, and both ICA and the disease risk associated with ICA, appear to be influenced by a genetic susceptibility. This has been sustained in a study of patients with endocrine autoimmunity and ICA (Polyendocrine Study) where the predictive value of very high levels of ICA is less than 50% in patients without a first degree relative with IDDM. Hence, there remain a substantial number of patients with ICA who do not develop disease. From these patients, it was demonstrated that ICA include at least two distinct specificities, one of which is beta cell specific and is not associated with a high risk for IDDM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel Considerations on the Antibody/Autoantigen System in Type I (insulin-dependent) Diabetes Mellitus

Insulin dependent diabetes (IDDM) has an autoimmune pathogenesis. Included is the presence of antibodies to pancreatic islet cells. The first identified were islet cell antibodies (ICA), detected by indirect immunofluorescence, and which react with all cells within islets. Importantly, the autoantibodies are found several years prior to disease and although a pathogenic role for the autoantibodies is unclear, they have become useful markers of prediabetes. A number of studies of twins discordant for IDDM and of first degree relatives of IDDM patients have established that there is an increased risk for disease in individuals who have ICA, especially when ICA levels are high. This high predictive value of ICA decreases in the general population where the incidence of IDDM is lower than in first degree relatives, and both ICA and the disease risk associated with ICA, appear to be influenced by a genetic susceptibility. This has been substantiated in a study of patients with endocrine autoimmunity and ICA (Polyendocrine Study) where the predictive value of very high levels of ICA is less than 50% in patients without a first degree relative with IDDM. Hence, there remain a substantial number of patients with ICA who do not develop disease. From these patients, it was demonstrated that ICA include at least two distinct specificities, one of which is beta cell specific and is not associated with a high risk for IDDM.

These data, and an increasing list of new autoantibodies in IDDM, have increased the search for the relevant autoantigens. That of classic ICA is suggested to be a sialoganglioside, and a number of protein autoantigens have been suggested as other potential autoantigens. Of particular importance has been the identification of glutamic acid decarboxylase (GAD), the biosynthesizing enzyme of the neurotransmitter gamma amino butyric acid, as the autoantigen corresponding to the 64K autoantibodies found in the majority of newly diagnosed IDDM patients. Despite the identification of GAD, specific assays developed so far have not detected anti GAD antibodies at high frequencies in newly diagnosed patients. Like ICA, it appears that there may be distinct specificities of autoantibodies to GAD and associated products. Enzyme digestion of the autoantigen yields 50K, 40K and 37K fragments which appear to be derived from distinct polypeptide chains. Patients may have autoantibodies to either the 50K fragment or the 40/37K fragment or all fragments. Importantly, studies in twins and in the polyendocrine patients indicate that autoantibodies to the 37K fragment are more strongly associated with the progression to disease.

In addition to these autoantigens, autoantibodies are detected against the hormone insulin and its precursor proinsulin in up to 40% of newly diagnosed patients. Heat shock protein 65, carboxypeptidase H and glucose transporter have also been reported as autoantigens relevant to IDDM. It is now clear that the autoimmune response associated with IDDM is heterogeneous and against several islet antigens. The search will continue to determine which of these is important in the pathogenesis and which will provide effective markers for prediction which will eventually allow safe intervention therapy for the prevention of IDDM.     

Islet-reactive T cells are a marker of preclinical insulin-dependent diabetes.

The destruction of pancreatic islet beta cells in insulin-dependent diabetes mellitus (IDDM) is thought to be T cell mediated. To directly identify islet-reactive T cells in asymptomatic, "preclinical" IDDM individuals with islet cell antibodies (ICA), proliferation of peripheral blood mononuclear cells (PBMC) was measured in the presence of sonicated fetal pig proislets. Stimulation indices (mean +/- SD) for [3H]thymidine uptake by PBMC cultured with sonicated proislets were: preclinical IDDM subjects (n = 22) 6.10 +/- 6.50, recent-onset IDDM subjects (n = 29) 3.66 +/- 3.35, Graves' disease subjects (n = 6) 2.17 +/- 0.93, scleroderma subjects (n = 4) 1.65 +/- 0.19 and normal control subjects (n = 14) 1.63 +/- 0.62. 68% (15/22) of preclinical IDDM, 41% (12/29) of recent-onset IDDM and 17% (1/6) of Graves' disease subjects had T cell reactivity greater than the mean + 2 SD of controls. T cell reactivity to proislets was tissue specific, and greater in magnitude and frequency than to human insulin. The majority of preclinical subjects with ICA greater than 20 Juvenile Diabetes Foundation (JDF) units (12/15, 80%) or antibodies to a 64-kD islet autoantigen (11/15, 73%) had significant T cell reactivity to proislets. ICA greater than 40 JDF units, a strong prognostic marker for progression to clinical IDDM, was an absolute index of T cell reactivity. Overall, the frequency of T cell reactivity in preclinical subjects, 68% (15/22), was comparable to that of ICA greater than 20 JDF units or 64-kD antibodies. Greater T cell reactivity to proislets in preclinical subjects accords with the natural history of autoimmune beta cell destruction. The direct assay of islet-reactive T cells in peripheral blood may have prognostic significance for the development of clinical IDDM and should facilitate identification of the primary target autoantigen(s).     

Development of IDDM after donating kidney to diabetic sibling

The goal of this study was to describe a patient who developed insulin-dependent diabetes mellitus (IDDM) after donating a kidney to his sibling and to suggest a possible solution to prevent such an occurrence. A 42-yr-old man was found to have islet cell autoantibodies (ICAs) as part of a screening program of first-degree relatives with IDDM. Two years previously, he had donated his kidney to his HLA-identical sibling with long-standing IDDM. Both oral and intravenous glucose tolerance tests demonstrated a gradual loss of insulin secretion and increasing glucose intolerance until the patient developed IDDM 6 yr after the nephrectomy. Whether the presence of ICA is an absolute contraindication to being a kidney donor could be debated. Nonetheless, ICA should be used as a screening test to identify individuals at risk for subsequent IDDM. For those found to be positive, counseling should be provided.     

Antibodies to a 64,000 Mr human islet cell antigen precede the clinical onset of insulin-dependent diabetes.

Antibodies in sera from newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients are directed to a human islet cell protein of relative molecular mass (Mr) 64,000. Since IDDM seems to develop after a prodromal period of beta-cell autoimmunity, this study has examined whether 64,000 Mr antibodies could be detected in 14 individuals who subsequently developed IDDM and five first degree relatives who have indications of altered beta-cell function. Sera were screened by immunoprecipitation on total detergent lysates of human islets and positive sera retested on membrane protein preparations. Antibodies to the 64,000 Mr membrane protein were consistently detected in 11/14 IDDM patients, and in all 5 first degree relatives. 10 IDDM patients were already positive in the first samples, obtained 4-91 mo before the clinical onset of IDDM, whereas 1 patient progressed to a high 64,000 Mr immunoreactivity, at a time where a commencement of a decline in beta-cell function was detected. 64,000 Mr antibodies were detected before islet cell cytoplasmic antibodies (ICCA) in two patients. In the control groups of 21 healthy individuals, 36 patients with diseases of the thyroid and 5 SLE patients, the 64,000 Mr antibodies were detected in only one individual, who was a healthy sibling to an IDDM patient. These results suggest that antibodies against the Mr 64,000 human islet protein are an early marker of beta-cell autoimmunity and may be useful to predict a later development of IDDM.     

Imogen 38: a novel 38-kD islet mitochondrial autoantigen recognized by T cells from a newly diagnosed type 1 diabetic patient.

Cell-mediated autoimmune attack directed against islet proteins of approximately 38 kD in size has been associated with type 1 diabetes. A novel murine cDNA encoding an antigen of this size was cloned using a screening procedure based on the proliferative response of a human diabetic T cell clone (1C6) to a recombinant antigen epitope library. Membrane preparations from COS 7 cells transfected with the full-length 1,267-bp cDNA elicited a proliferative response from the reporter T cells comparable to that of the defined peptide epitope and native insulinoma antigen. In vitro translation and transfection experiments suggested that the protein is initially synthesized as a 44-kD protein and then processed to the native 38-kD form through the proteolytic removal of a 54-aa NH2-terminal mitochondrial targeting sequence. Differential centrifugation, Percoll density gradient centrifugation, and immunofluorescence studies confirmed localization of the antigen to mitochondria. Northern blot, Western blot, and 1C6 T cell proliferation assays showed that, although imogen 38 was more highly expressed in beta cell than alpha cell lines, it was also present in other tissues. It is concluded that imogen 38 may be a target for bystander autoimmune attack in diabetes rather than a primary autoantigen.     

Prediction of insulin-dependent diabetes mellitus in siblings of children with diabetes. A population-based study. The Childhood Diabetes in Finland Study Group.

An unselected population of 755 siblings of children with insulin-dependent diabetes mellitus (IDDM) was studied to evaluate the predictive characteristics of islet cell antibodies (ICA), antibodies to the IA-2 protein (IA-2A), antibodies to the 65-kD isoform of glutamic acid decarboxylase (GADA), insulin autoantibodies (IAA), and combinations of these markers. We also evaluated whether the histochemical ICA test could be replaced by the combined detection of other markers. 32 siblings progressed to IDDM within 7.7 yr of the initial sample taken at or close to the diagnosis of the index case (median follow-up, 9.1 yr). The positive predictive values of ICA, IA-2A, GADA, and IAA were 43, 55, 42, and 29%, and their sensitivities 81, 69, 69, and 25%, respectively. In contrast to the other three antibody specificities, GADA levels were not related to the risk for IDDM. The risk for IDDM in siblings with four, three, two, one, or no antibodies was 40, 70, 25, 2, and 0.8%, respectively. Combined screening for IA-2A and GADA identified 70% of all ICA-positive siblings, and all of the ICA-positive progressors were also positive for at least one of the three other markers. The sensitivity of the combined analysis of IA-2A and GADA was 81%, and the positive predictive value was 41%. In conclusion, combined screening for IA-2A and GADA may replace the ICA assay, giving comparable sensitivity, specificity, and positive predictive value. Accurate assessment of the risk for IDDM in siblings is complicated, as not even all those with four antibody specificities contract the disease, and some with only one or no antibodies initially will progress to IDDM.     

Prevalence of autoantibodies to the 65- and 67-kD isoforms of glutamate decarboxylase in insulin-dependent diabetes mellitus.

We investigated the presence of autoantibodies to baculovirus-expressed human recombinant 65- and 67-kD isoforms of glutamate decarboxylase (GAD65 and GAD67) in insulin-dependent diabetes mellitus (IDDM). In the immunoprecipitation test using [35S]methionine-labeled GADs antibodies to GAD65 were detected in 13/15 (87%) islet cell antibody (ICA)-positive and in 1/35 (2.9%) ICA-negative first-degree relatives of patients with IDDM, in 6/11 (54.5%) ICA-positive nondiabetic schoolchildren, and in 35/50 (70%) patients with newly diagnosed IDDM. GAD67 antibodies were positive only in five (33%) of the ICA-positive relatives (P < 0.05) and in nine (18%) IDDM patients at onset (P < 0.00001). After onset of IDDM antibodies to GAD65 and GAD67 declined but were still positive in 25 and 9.4% of subjects with long-standing IDDM (> 10 yr). In all study groups antibodies to GAD67 were only detected in GAD65 antibody-positive sera. An immunotrapping enzyme activity assay for GAD65 antibodies was positive in 64/75 (85.3%) of sera that were GAD antibody positive in the immunoprecipitation test (r = 0.870, P < 0.0001). In two (2.7%) sera GAD65 antibodies that block GAD enzyme activity were found. Our data suggest that antibodies to GAD65 but not to GAD67 represent sensitive markers for preclinical and overt IDDM. The immunotrapping assay here described represents a valuable technique for specific and sensitive screening for GAD antibodies.     

Islet cell autoantibodies: pathobiology and clinical applications

Autoimmunity directed against pancreatic islet cells results in slowly progressing beta-cell destruction, culminating over years in clinically manifested insulin-dependent diabetes mellitus (IDDM). Circulating serum autoantibodies directed against the endocrine cells of the islets of Langerhans are an important hallmark of this disease. Assays for these islet cell antibodies (ICA) have facilitated the investigation and understanding of several facets in the pathogenesis of autoimmune diabetes. Their applications have begun to extend into clinical practice and have opened new avenues for early preclinical prediction and preventive prophylaxsis in IDDM.     

Immunological aspects of diabetes mellitus: prospects for pharmacological modification

It is now well known that insulin-dependent diabetes is a chronic progressive autoimmune disease. The prolonged prediabetic phase of progressive beta-cell dysfunction is associated with immunological abnormalities. A prediabetic period is suggested by the appearance of islet cell antibodies, anti-insulin antibodies, and anti-insulin receptor antibodies. The existence of activated T lymphocytes and abnormal T cell subsets are also other markers. There is still no concensus about the use of the immunosuppression superimposed upon conventional insulin therapy in early diagnosed IDDM and the follow-up of the relatives of IDDM patients who share the genetic predisposition and serological markers for the risk of future onset of IDDM. Treatment in the prodromal period cannot be justified because a link between the disease and early markers such as ICA has not been established with certainty (Diabetes Research Program NIH, 1983). Many immunopharmacological manipulations were reported to be effective in animal models. However, most of them are not readily applied to human subjects. Moreover, IDDM patients are now believed to be heterogeneous, with a complex genetic background. HLA-DR, and more recently DQ, are closely related to the genetic predisposition to IDDM but those genes are not themselves diabetogenic. The contribution of autoimmunity does not appear to be uniform, and in some cases, the contribution of virus is considered more important. There is a lack of a marker for the future onset of IDDM. ICA and ICSA were found after mumps infection, but the existence of those autoantibodies and even the co-existence of HLA-DR3 do not always indicate the future trend to insulin dependency. More precise markers will be disclosed through the biochemical analysis of the target antigens on pancreatic beta-cell for islet antibodies and effector T cells. Much safer and more effective immunopharmacological treatment will be developed through animal experimentation using rat and mouse models. The recent development and interest in this field will further facilitate the attainment of the goal for the complete prevention of IDDM.     

Anti-sympathetic ganglia antibodies and postural blood pressure in IDDM subjects of varying duration and patients at high risk of developing IDDM

We examined the sera of 94 subjects with insulin-dependent diabetes mellitus (IDDM) for the presence of complement-fixing sympathetic ganglia (CF-SG) antibodies. In a cross-sectional analysis (duration 0-43 yr), 22% had detectable CF-SG antibodies. Subjects at high risk for IDDM were also studied. Four groups were studied: group 1 (aged 4-64 yr) islet cell antibody-positive (ICA+) prediabetic subjects, 10 of 19 (53%) were CF-SG+; group 2 (aged 6-14 yr) ICA- prediabetic subjects (first-degree relatives of IDDM subjects with either transient hyperglycemia, impaired oral glucose tolerance, and/or first-phase insulin release after intravenous glucose tolerance testing), 4 of 9 (44%) were CF-SG+ (2 of the 4 ICA- CF-SG+ subjects have progressed to IDDM); group 3 (aged 1.5-43 yr) ICA+ IDDM subjects (less than or equal to 1 yr duration) 6 of 10 (60%) were CF-SG+; and group 4 (aged 8-59 yr) ICA- IDDM subjects (less than or equal to 1 yr duration), 2 of 11 (18%) were CF-SG+. All groups had increased CF-SG compared with controls. Postural blood pressure and simultaneous CF-SG antibody measurements were performed in 28 IDDM subjects. The drop in systolic blood pressure was greater in the CF-SG+ subjects (P less than .05), and the frequency of CF-SG was greater in the mean to -2SD group (P less than .03) when data were analyzed within mean +/- 2SD of the normal blood pressure response.     

The risk of developing disease for siblings of patients with insulin dependent diabetes mellitus

We analyzed the risk of developing insulin-dependent diabetes mellitus (IDDM) in 411 siblings of patients with IDDM. We found that siblings who had a positive test for antibodies against islet cells (ICA) at the time of diagnosis of the index case had a higher risk of developing IDDM than did those who had negative tests. However, of the ten siblings who developed IDDM, only four were positive at the initial testing. The period of time elapsing from a negative test at screening to a positive test at diagnosis varied but was less than one year in one child. Two of the ten siblings who developed IDDM had negative tests both at screening and at diagnosis. Amongst siblings who were negative at the initial screening, those in whom the index case was diagnosed at a young age had a higher risk of developing IDDM than did those in whom the index case was diagnosed at an older age. The age of the sibling at the time of screening, the sex of the sibling, and a positive family history (one which includes in addition to the index case one or more first-degree relatives with IDDM) did not confer increased risk. Our data suggest that screening for ICA will have to be done often and will have to be continued into adult life in order to identify the 70-80% of diabetics who will be positive at some time in the evolution of their disease.     

Aerosol Insulin Induces Regulatory CD8 γδ T Cells That Prevent Murine Insulin-dependent Diabetes

Cellular immune hyporesponsiveness can be induced by the presentation of soluble protein antigens to mucosal surfaces. Most studies of mucosa-mediated tolerance have used the oral route of antigen delivery and few have examined autoantigens in natural models of autoimmune disease. Insulin is an autoantigen in humans and nonobese diabetic (NOD) mice with insulindependent diabetes mellitus (IDDM). When we administered insulin aerosol to NOD mice after the onset of subclinical disease, pancreatic islet pathology and diabetes incidence were both significantly reduced. Insulin-treated mice had increased circulating antibodies to insulin, absent splenocyte proliferation to the major epitope, insulin B chain amino acids 9–23, which was associated with increased IL-4 and particularly IL-10 secretion, and reduced proliferation to glutamic acid decarboxylase, another islet autoantigen. The ability of splenocytes from insulin-treated mice to suppress the adoptive transfer of diabetes to nondiabetic mice by T cells of diabetic mice was shown to be caused by small numbers of CD8 γδ T cells. These findings reveal a novel mechanism for suppressing cell-mediated autoimmune disease. Induction of regulatory CD8 γδ T cells by aerosol insulin is a therapeutic strategy with implications for the prevention of human IDDM.     

Elevated proinsulin levels related to islet cell antibodies in first-degree relatives of IDDM patients.

OBJECTIVE--To assess whether proinsulin levels are elevated in first-degree relatives of insulin-dependent diabetes mellitus (IDDM) patients and whether there is a relationship between proinsulin levels and the occurrence of immunological markers. RESEARCH DESIGN AND METHODS--Fasting proinsulin concentrations were measured in 85 first-degree relatives (54 siblings, 20 parents, 11 children) of IDDM patients and in 90 age- and weight-matched control subjects with no family history of diabetes mellitus. RESULTS--Fasting proinsulin levels (median, 25th, and 75th percentiles) were 8 pM (range 3.2-14 pM) in first-degree relatives and 1.7 pM (range 1.7-4 pM) in control subjects (P less than 0.0001). Proinsulin was significantly elevated in siblings (7.2 pM, range 3.8-15 pM; P less than 0.0001), parents (9.8 pM, range 6.4-13 pM; P less than 0.0001), and children (6.6 pM, range 1.8-12 pM, P = 0.04) compared with control subjects but without differences between these groups. Islet cell antibody positive (ICA+) IDDM relatives had significantly higher proinsulin levels than ICA- (16 pM; range 7.2-25 vs. 6.9 pM, range 3.1-12 pM; P = 0.02). There was no difference between individuals with and without insulin autoantibodies. No difference in proinsulin levels was observed if the relatives were subdivided according to HLA-DR sharing with the diabetic proband. CONCLUSIONS--Fasting proinsulin concentrations were raised not only in siblings but also in parents and children of IDDM patients. Because proinsulin is more elevated in ICA+ than in ICA- subjects, increased proinsulin levels could reflect minor beta-cell damage due to previous immunological attack.     

Slowly progressive IDDM

A prospective study over 10 years on the natural history of beta-cell function in islet cell antibody (ICA)-positive NIDDM disclosed a characteristic findings of slowly progressive IDDM distinct from those of acute onset IDDM. The characteristics includes: (1) the clinical phenotype at the onset of diabetes is non-insulin requiring, (2) the onset is late age, (3) beta-cell failure progresses slowly over several years with persistently positive low-titer ICA and high titer of GAD antibodies, (4) pathological study on autopsied pancreas demonstrated that beta-cell loss is incomplete, and exocrine pancreas is often atrophied with cell infiltration of CD8+ T lymphocytes, (5) higher family history of NIDDM, and (6) association with some genetic predisposition including HLA-DQA1*0301-DQB1*0401 and/or mitochondrial gene mutation at nucleotide pair 3243, and a lack of association with HLA-A24. These results are sharply contrast with acute onset IDDM, suggesting a presence of distinct subtype of IDDM in Japanese population. We also demonstrated that small dose of insulin therapy was effective for the suppression of immunological beta-cell failure.     

Limited joint mobility in childhood diabetes: family studies

We examined 204 persons with insulin-dependent diabetes mellitus (IDDM), aged 7-23 yr, and 336 of their first-degree relatives, to determine whether there is a genetic component to the development of limited joint mobility. Simple and multiplex pedigrees with IDDM were studied along with normal controls. Only 1 of 90 normal controls had joint stiffness. Among 225 nondiabetic parents of children with IDDM, 7 (3%) had joint limitation, compared with 42 (21%) of children and youth with IDDM. Only 1 of 108 nondiabetic siblings of diabetic probands had limitation. Three parents had adult-onset diabetes and all had limited joint mobility. None of the 8 nondiabetic relatives with joint limitation had diabetic probands with joint involvement; 5 of these 8 tested were negative for islet cell auto-antibodies. There were 11 IDDM multiplex families with at least one member having joint limitation. The concordance rate for limited joint mobility of persons with diabetes for more than 5 yr who were over 12 yr of age was 56%, not different from the 48% frequency in patients with IDDM who met these age and duration criteria. Thus, evidence that limited joint mobility is a metabolic consequence of diabetes includes the virtual absence of limitation among first-degree relatives of probands, including probands with joint stiffness, and that the frequency of joint involvement is not increased in first-degree relatives of patients with IDDM. Furthermore, two brothers with pancreatic hypoplasia and a non-HLA-associated form of IDDM were affected with limited joint mobility. Nonetheless, the expression of this complication must be influenced by host factors, since not all persons with IDDM develop it, and those who do have variable ages of onset without correlation to control measures.     

Cytotoxic T cells specific for glutamic acid decarboxylase in autoimmune diabetes

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease that results in the destruction of the pancreatic islet beta cells. Glutamic acid decarboxylase (GAD) has been recently indicated as a key autoantigen in the induction of IDDM in nonobese diabetic mice. In human diabetes, the mechanism by which the beta cells are destroyed is still unknown. Here we report the first evidence for the presence of GAD-specific cytotoxic T cells in asymptomatic and recent diabetic patients. GAD65 peptides displaying the human histocompatibility leukocyte antigen (HLA)-A*0201 binding motif have been synthesized. One of these peptides, GAD114-123, binds to HLA-A*0201 molecules in an HLA assembly assay. Peripheral blood mononuclear cells from individuals with preclinical IDDM, recent-onset IDDM, and from healthy controls were stimulated in vitro with the selected peptide in the presence of autologous antigen-presenting cells. In three cases (one preclinical IDDM and two recent-onset IDDM), we detected specific killing of autologous antigen-presenting cells when incubated with GAD114-123 peptide or when infected with a recombinant vaccinia virus expressing GAD65. These patients were the only three carrying the HLA-A*0201 allele among the subjects studied. Our finding suggests that GAD- specific cytotoxic T lymphocytes may play a critical role in the initial events of IDDM.     

Demonstration of GAD-65 as the main immunogenic isoform of glutamate decarboxylase in type 1 diabetes and determination of autoantibodies using a radioligand produced by eukaryotic expression.

Plasmids containing cDNA for the rat 67- and 65-kD isoforms of glutamate decarboxylase (GAD-67 and GAD-65) were expressed in COS-cells, and lysates of [35S]methionine-labeled cells were used for immunoprecipitations. Sera from 38 patients with type 1 (insulin-dependent) diabetes mellitus, which precipitated a 64-kD antigen from rat islets, reacted with recombinant GAD-65 in relation to their anti-64-kD titers. The eight strongest sera also precipitated recombinant GAD-67, suggesting that certain epitopes are common to both isoforms. Subsequently, [35S]methionine-labeled GAD-65 was purified from COS cell lysates and employed in a binding assay with 50 sera of patients with recent onset of type 1 diabetes mellitus. 38 sera (76%) precipitated labeled GAD-65 with titers that correlated with islet cell antibodies (ICA), determined in a standard immunofluorescence assay. 2 sera were GAD positive but ICA negative, 4 were positive only for ICA, and 6 were negative for both GAD and ICA, as were the sera of 20 controls. The data illustrate that antibodies against GAD-65 are present in a majority of patients with type 1 diabetes mellitus and that autoantibodies against other islet cell antigens also exist. The radioligand-binding assay, which is convenient and sensitive for detecting GAD antibodies, will facilitate the screening of individuals with autoimmune islet cell disease.     

Higher autoantibody levels and recognition of a linear NH2-terminal epitope in the autoantigen GAD65, distinguish stiff-man syndrome from insulin-dependent diabetes mellitus

The smaller form of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD65) is a major autoantigen in two human diseases that affect its principal sites of expression. Thus, destruction of pancreatic beta cells, which results in insulin-dependent diabetes mellitus (IDDM), and impairment of GABA-ergic synaptic transmission in Stiff-Man syndrome (SMS) are both characterized by circulating autoantibodies to GAD65. Anti-GAD65 autoantibodies in IDDM are predominantly directed to conformational epitopes. Here we report the characterization of humoral autoimmune responses to GAD65 in 35 SMS patients, of whom 13 (37%) also had IDDM. All SMS patients immunoprecipitated native GAD65 and the main titers were orders of magnitude higher than in IDDM patients. Furthermore, in contrast to the situation in IDDM, autoantibodies in 35 of 35 (100%) of SMS patients recognized denatured GAD65 on Western blots. Two major patterns of epitope specificity were identified on Western blots. The first pattern, detected in 25 of 35 SMS patients (71%), of whom 11 had IDDM (44%), was predominantly reactive with a linear NH2-terminal epitope residing in the first eight amino acids of GAD65. Nine of nine individuals who were HLA-haplotyped in this group carried an IDDM susceptibility haplotype and HLA-DR3, DQw2 was particularly abundant. The second pattern, detected in 10 of 35 patients (29%) of whom two had IDDM (20%), included reactivity with the NH2-terminal epitope plus strong reactivity with one or more additional epitope(s) residing COOH- terminal to amino acid 101. The second epitope pattern may represent epitope spreading in the GAD65 molecule, but may also include some cases of epitope recognition associated with IDDM resistant HLA- haplotypes. The principal NH2-terminal linear epitope in GAD65 distinguishes the reactivity of SMS and IDDM autoantibodies and may be a determinant of pathogenicity for GABA-ergic neurons. The greater magnitude and distinct specificity of the humoral response to GAD65 in SMS may reflect a biased involvement of the T helper cell type 2 (Th2) subset of CD4+ T cells and antibody responses, whereas IDDM is likely mediated by the Th1 subset of CD4+ T cells and cytotoxic T cell responses.     

Male sex increases the risk of autoimmunity but not type 1 diabetes

OBJECTIVE: The goal of this study was to explore the role of sex on the prevalence of autoantibodies, protective genetic subtypes, beta-cell function, and the incidence of type 1 diabetes in a population of first- and second-degree relatives of patients with type 1 diabetes (probands). We examined both the effect of the sex of the individual screened as well as the effect of the sex of the individual's proband on diabetes risk variables tested. RESEARCH DESIGN AND METHODS: The Diabetes Prevention Trial-Type 1 has screened 93,188 relatives of type 1 diabetic patients from February 1994 to January 2002. After observing that more men than women were islet cell autoantibody (ICA) positive for the group as a whole, we further explored the role of sex by detailed analysis of variables in this population. RESULTS: Our data suggest only an influence of sex on the type 1 diabetes disease process. After adjustment for race, age, and relationship to proband, male sex was associated with the appearance of autoimmunity, i.e., the presence of ICA and having two or more antibodies. There was no effect of sex on the presence of other autoantibodies, insulin secretion, results of oral glucose tolerance test, or development of diabetes. CONCLUSIONS: Our finding that male sex conveys an independent increased risk for development of ICA and multiple antibodies, while at the same time finding no difference with respect to the development of diabetes, suggests that male relatives with the known risk factor of ICA are less likely than comparable female relatives to progress to overt disease, that the pathogenesis of type 1 diabetes among men is slower compared with women, or that women develop diabetes manifesting different antibody responses.