Non-HLA type 1 diabetes genes modulate disease risk together with HLA-DQ and islet autoantibodies.

The possible interrelations between human leukocyte antigen (HLA)-DQ, non-HLA single-nucleotide polymorphisms (SNPs) and islet autoantibodies were investigated at clinical onset in 1-34-year-old type 1 diabetes (T1D) patients (n=305) and controls (n=203). Among the non-HLA SNPs reported by the Type 1 Diabetes Genetics Consortium, 24% were supported in this Swedish replication set including that the increased risk of minor PTPN22 allele and high-risk HLA was modified by GAD65 autoantibodies. The association between T1D and the minor AA+AC genotype in ERBB3 gene was stronger among IA-2 autoantibody-positive patients (comparison P=0.047). The association between T1D and the common insulin (AA) genotype was stronger among insulin autoantibody (IAA)-positive patients (comparison P=0.008). In contrast, the association between T1D and unidentified 26471 gene was stronger among IAA-negative (comparison P=0.049) and IA-2 autoantibody-negative (comparison P=0.052) patients. Finally, the association between IL2RA and T1D was stronger among IAA-positive than among IAA-negative patients (comparison P=0.028). These results suggest that the increased risk of T1D by non-HLA genes is often modified by both islet autoantibodies and HLA-DQ. The interactions between non-HLA genes, islet autoantibodies and HLA-DQ should be taken into account in T1D prediction studies as well as in prevention trials aimed at inducing immunological tolerance to islet autoantigens.

Prevalence of obesity was related to HLA-DQ in 2-4-year-old children at genetic risk for type 1 diabetes.

OBJECTIVES: Body size is postulated to modulate type 1 diabetes as either a trigger of islet autoimmunity or an accelerator to clinical onset after seroconversion. As overweight and obesity continue to rise among children, the aim of this study was to determine whether human leukocyte antigen DQ (HLA-DQ) genotypes may be related to body size among children genetically at risk for type 1 diabetes. METHODS: Repeated measures of weight and height were collected from 5969 children 2-4 years of age enrolled in The Environmental Determinants of Diabetes in the Young prospective study. Overweight and obesity was determined by the International Obesity Task Force cutoff values that correspond to body mass index (BMI) of 25 and 30 kg m(-)(2) at age 18. RESULTS: The average BMI was comparable across specific HLA genotypes at every age point. The proportion of overweight was not different by HL A, but percent obesity varied by age with a decreasing trend among DQ2/8 carriers (P for trend=0.0315). A multivariable regression model suggested DQ2/2 was associated with higher obesity risk at age 4 (odds ratio, 2.41; 95% confidence interval, 1.21-4.80) after adjusting for the development of islet autoantibody and/or type 1 diabetes. CONCLUSIONS: The HLA-DQ2/2 genotype may predispose to obesity among 2-4-year-old children with genetic risk for type 1 diabetes.

Next-generation sequencing for viruses in children with rapid-onset type 1 diabetes.

AIMS/HYPOTHESIS: Viruses are candidate causative agents in the pathogenesis of autoimmune (type 1) diabetes. We hypothesised that children with a rapid onset of type 1 diabetes may have been exposed to such agents shortly before the initiation of islet autoimmunity, possibly at high dose, and thus study of these children could help identify viruses involved in the development of autoimmune diabetes. METHODS: We used next-generation sequencing to search for viruses in plasma samples and examined the history of infection and fever in children enrolled in The Environmental Determinants of Diabetes in the Young (TEDDY) study who progressed to type 1 diabetes within 6 months from the appearance of islet autoimmunity, and in matched islet-autoantibody-negative controls. RESULTS: Viruses were not detected more frequently in plasma from rapid-onset patients than in controls during the period surrounding seroconversion. In addition, infection histories were found to be similar between children with rapid-onset diabetes and control children, although episodes of fever were reported less frequently in children with rapid-onset diabetes. CONCLUSIONS/INTERPRETATION: These findings do not support the presence of viraemia around the time of seroconversion in young children with rapid-onset type 1 diabetes.

The association between the PTPN22 1858C>T variant and type 1 diabetes depends on HLA risk and GAD65 autoantibodies.

The single nucleotide polymorphism 1858C>T in the PTPN22 gene is associated with type 1 diabetes (T1D) in several populations. Earlier reports have suggested that the association may be modified by human leukocyte antigen (HLA), as well as by islet autoantibodies. In a large case-control study of Swedish incident T1D patients and controls, 0-34 years of age, we tested whether the odds ratio (OR) measure of association was dependent on HLA or autoantibodies against the islet autoantigens glutamic acid decarboxylase 65 kDa autoantibodies (GADA), insulin, islet antigen-2, or islet cell. The association between the carrier status of 1858C>T allele in PTPN22 (PTPN22(CT+TT)) and T1D was modified by HLA. In addition, in GADA-positive T1D, the OR was 2.83 (2.00, 3.99), whereas in GADA-negative T1D, the OR was 1.41 (0.98, 2.04) (P for comparison=0.007). The OR of association between PTPN22(CT+TT) and GADA-positive T1D declined with increasing HLA-risk category from 6.12 to 1.54 (P=0.003); no such change was detected in GADA-negative T1D (P=0.722) (P for comparison=0.001). However, the absolute difference in risk between PTPN22(CC) and PTPN22(CT+TT) subjects with high-risk HLA was five times higher than that for subjects with low-risk HLA. We hypothesize that the altered T-cell function because of the PTPN22(1858C>T) polymorphism is exclusively associated with GADA-positive T1D at diagnosis.

Genetic association of HLA DQB1 with CD4+CD25+(high) T-cell apoptosis in type 1 diabetes.

Type 1 diabetes (T1D) has a strong genetic component and the major locus lies in the HLA DQB1 region. We found earlier an increased apoptosis with decreased viability and function of the CD4+CD25+(high) T-cell subset (Treg) in human subjects with recent-onset T1D and in multiple autoantibody-positive, high at-risk individuals. Tregs normally inhibit or delay onset of T1D in animal models and increased Treg apoptosis could bring on or accelerate disease from effector T-cell-mediated destruction of insulin-producing beta cells. In this study, we test the hypothesis that HLA DQB1 genotypes are associated with increased CD4+CD25+(high) T-cell apoptosis. HLA DQ-based genetic risk status was significantly associated with CD4+CD25+(high) T-cell apoptosis, after adjustment for age, gender and phenotypic status (n=83, F=4.04 (d.f.=3), P=0.01). Unaffected, autoantibody-negative high risk HLA DQB1 control subjects showed increased CD4+CD25+(high) apoptosis levels compared with low risk HLA DQB1 control subjects (n=26, P=0.002), confirming that the association precedes disease. The association of specific HLA DQB1 genotypes with Treg apoptosis was also tested, showing significance for HLA DQB1*0302, DQB1*0201 and HLA DQB1*0602 alleles. Our study shows an association of HLA DQB1 genotypes with CD4+CD25+(high) T-cell apoptosis, which implicates CD4+CD25+(high) T-cell apoptosis as a new intermediate trait for T1D.

Hepatic glucagon metabolism. Correlation of hormone processing by isolated canine hepatocytes with glucagon metabolism in man and in the dog.

We have found that canine and rat hepatocytes convert (125I)iodoTyr10-glucagon to a peptide metabolite lacking the NH2-terminal three residues of the hormone. The peptide is released into the cell incubation medium and its formation is unaffected by a variety of lysosomotropic or other agents. Use of specific radioimmunoassays and gel filtration demonstrated in both normal subjects and in chronic renal failure patients a plasma peptide having the properties of the hormone fragment identified by cell studies. Studies of the dog revealed a positive gradient of the fragment across the liver and no differential gradient of the fragment and glucagon across the kidney. We conclude that the glucagon fragment arises from the cell-mediated processing of the hormone on a superficial aspect of the hepatocyte, the glucagon fragment identified during experiments in vitro represents the cognate of a peptide formed during the hepatic metabolism of glucagon in vivo, and measurement of the fragment by COOH-terminal radioimmunoassays could lead to an understimulation of hepatic glucagon extraction.

Glutamate decarboxylase-, insulin-, and islet cell-antibodies and HLA typing to detect diabetes in a general population-based study of Swedish children.

Most autoimmune diabetes occurs in those without a diabetic relative, but few cases are identifiable prospectively. To model general population prediction, 491 consecutive newly diabetic children from all of Sweden were tested for autoantibodies to glutamate decarboxylase (GAD65ab), insulin (IAA), and islet cells (ICA), and for HLA-DQ genotypes by PCR; 415 matched control children were tested in parallel. GAD65ab sensitivity/specificity was 70/96%, versus 84/96% for ICA, 56/97% for IAA, 93/93% (any positive), 39/99.7% (all positive), and 41/99.7% (GAD65ab plus IAA). The latter's 25% predictive value was not improved by requiring concomitant high-risk HLA genotypes. GAD65ab were associated with DQA1*0501/B1*0201 (DQ2; P = 0.007) but not DQA1*0301/B1*0302 (DQ8), and IAA with DQA1*0301/B1*0302 (DQ8; P = 0.03) but not DQA1*0501/B1*0201 (DQ2). GAD65ab were more prevalent in females than males (79 vs. 63%; P < 0.0001) but did not vary with onset age nor season. Combining the three antibody assays yielded sufficient sensitivity for screening. GADab were relatively sensitive/specific for diabetes, but even with HLA marker combinations yielded predictive values insufficient for early immunointervention in the low-prevalence general population.

Quantitative assay using recombinant human islet glutamic acid decarboxylase (GAD65) shows that 64K autoantibody positivity at onset predicts diabetes type.

At and before onset, most insulin-dependent diabetics (IDDM) have islet GAD65 autoantibodies (GAD65Ab). Since IDDM also occurs in older patients where non-insulin-dependent diabetes is common, we studied GAD65Ab at onset to classify diabetes type. Our quantitative immunoprecipitation assay uses recombinant human islet GAD65 stably expressed in hamster fibroblasts. Electrophoretic mobility was identical to native islet GAD65. Like native antigen, recombinant GAD65 migrated as two bands during electrophoresis, but converted to one under stronger reduction. Immunoprecipitation was linear with respect to antibody or antigen concentration. In 120 population-based diabetic patients of all ages grouped by treatment at onset and after 18 mo, GAD65Ab were present in 70% on insulin (n = 37), 10% on oral agent (n = 62, P < 0.0001), 69% changing from oral agent to insulin (n = 16, P < 0.001), and 1 of 33 controls. 65% with GAD65Ab, versus 8% without, changed from oral agent to insulin (P < 0.01). The GAD65Ab quantitative index was remarkably stable, and only 2 of 32 patients changed antibody status during follow-up. Concordance between GAD65Ab and islet cell antibodies was 93%. Quantitative correlation was approximate but significant. This highly sensitive, quantitative, high capacity assay for GAD65Ab reveals treatment requirements better than clinical criteria, perhaps guiding immunomodulatory therapy.

A large sample of finnish diabetic sib-pairs reveals no evidence for a non-insulin-dependent diabetes mellitus susceptibility locus at 2qter.

In the first reported positive result from a genome scan for non-insulin-dependent diabetes mellitus (NIDDM), Hanis et al. found significant evidence of linkage for NIDDM on chromosome 2q37 and named the putative disease locus NIDDM1 (Hanis et al. 1996. Nat. Genet. 13:161-166). Their total sample was comprised of 440 Mexican-American affected sib-pairs from 246 sibships. The strongest evidence for linkage was at marker D2S125 and best estimates of lambdas (risk to siblings of probands/population prevalence) using this marker were 1.37 under an additive model and 1.36 under a multiplicative model. We examined this chromosomal region using linkage analysis in a Finnish sample comprised of 709 affected sib-pairs from 472 sibships. We excluded this region in our sample (multipoint logarithm of odds score /= 1.37. We discuss possible reasons why linkage to 2q37 was not found and conclude that this region is unlikely to be playing a major role in NIDDM susceptibility in the Finnish Caucasian population.

Recombinant glutamic acid decarboxylase (representing the single isoform expressed in human islets) detects IDDM-associated 64,000-M(r) autoantibodies.

GAD is an autoantigen in IDDM. Molecular cloning and specific antibodies allowed us to demonstrate that only the lower M(r) GAD64 isoform is expressed in human islets, in contrast to human brain, rat islets, and rat brain, all of which express both GAD64 and GAD67. Expression of the human islet GAD64 isoform in COS-7 and BHK cells resulted in an enzymatically active rGAD64, which is immunoreactive with diabetic sera comparable with that of the islet 64,000-M(r) autoantigen. Immunoprecipitation analyses showed that 21/28 (75%) IDDM sera had rGA D64 antibodies compared with only 1/59 (1.7%) of the healthy control sera. In immunoblot analyses, an SMS serum--but only 1/10 randomly selected IDDM sera--recognized the blotted rGAD64 without relation to immunoprecipitation titers. In conclusion, only the GA D64 isoform is expressed in human islets, in contrast to rat islets, which also express the GAD67 isoform. The immunological properties of human rGAD64 are comparable with the native 64,000-M(r) islet autoantigen, allowing further studies of the immunopathogenesis of IDDM.

Differential expression of glutamic acid decarboxylase in rat and human islets.

The GABA synthesizing enzyme GAD is a prominent islet cell autoantigen in type I diabetes. The two forms of GAD (GAD64 and GAD67) are encoded by different genes in both rats and humans. By in situ hybridization analysis of rat and human pancreases, expression of both genes was detected in rat islets, whereas only GAD64 mRNA was detected in human islets. Immunocytochemical analysis of rat and human pancreatic sections or isolated islets with antibodies to GAD64 and GAD67 in combination with antibodies to insulin, glucagon, or SRIF confirmed that a GAD64 and GAD67 expression were beta-cell specific in rat islets. In contrast, only GAD64 was detected in human islets and was, in addition to beta-cells, also surprisingly localized to some alpha-cells, delta-cells, and PP-cells. In long-term (4 wk) monolayer cultures of newborn rat islet cells, GAD64 expression remained beta-cell specific as observed in vivo, whereas GAD67 was localized not only to the beta-cells but also in the alpha-cells and delta-cells. A small but distinct fraction of GAD positive cells in these monolayer cultures did not accumulate GABA immunoreactivity, which may indicate cellular heterogeneity with respect to GABA catabolism or GAD enzyme activity. In a rat insulinoma cell line (NHI-6F) producing both glucagon and insulin depending on the culture conditions, GAD64 expression was detected only in cultures in which the insulin producing phenotype dominated. In conclusion, these data demonstrate that the two GAD isoforms are differentially expressed in rat and human islets but also that the expression differs according to culture conditions. These findings emphasize the need to consider both the species and culture conditions of islets.

Autoantibodies in IDDM primarily recognize the 65,000-M(r) rather than the 67,000-M(r) isoform of glutamic acid decarboxylase.

Glutamic acid decarboxylase autoantibodies may aid in rapid screening strategies predicting IDDM before clinical onset. Rat islets contain GAD65 and GAD67 autoantibody targets, but human islets express only GAD65, now confirmed by direct immunoprecipitation from radiolabeled rat and human islets. Because human IDDM involves beta-cell-specific autoimmunity, we tested 190 new IDDM patients and 51 healthy control subjects for antibodies to recombinant human islet GAD65, rat islet GAD67, or human insulinoma/cerebellum GAD67, each expressed separately in hamster fibroblasts. By using immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and densitometric fluorogram scanning, 132 of 190 (70%) of new IDDM patients had GAD65 autoantibodies, whereas only 17 of 190 (9%) had antibodies to rat GAD67 (P < 0.001). Of healthy control subjects, 2 of 51 (3.9%) and 1 of 51 (1.9%) had antibodies to GAD65 and GAD67, respectively. All 17 GAD67 antibody-positive patients also had GAD65 antibodies; 14 of 17 with greater GAD65 than GAD67 index. Control studies showed comparable reactivity between recombinant rat and human GAD67 and between different subcellular preparations of recombinant GAD67 of either species. In conclusion, only GAD65 is expressed in human islets, the autoantibody response is primarily to this isoform, and GAD67 antibodies add little to IDDM detection.

Immunogenetic analysis suggests different pathogenesis for obese and lean African-Americans with diabetic ketoacidosis.

OBJECTIVE: When presenting with diabetic ketoacidosis (DKA), lean and obese patients differ in their subsequent clinical course. Although lean patients tend to remain insulin dependent, most obese patients recover endogenous insulin secretion and discontinue insulin therapy. The aim of this study was to determine whether obese African-American patients with DKA could be determined to have type 1 or type 2 diabetes based on insulin secretion or the presence of immunological and genetic markers. RESEARCH DESIGN AND METHODS: This was a prospective study that analyzed the clinical characteristics, insulin secretion indices, immunological markers (islet cell, GAD, ICA512, and insulin autoantibodies), and HLA susceptibility genes (DR/DQ) in 131 patients with DKA (77 obese and 54 lean), 51 obese patients with hyperglycemia but no DKA, and 25 nondiabetic subjects. All subjects were African-American. Beta-cell function was evaluated by the C-peptide response to glucagon (1 mg i.v.) within 48 h of resolution of DKA or hyperglycemia. RESULTS: The acute C-peptide response was lower in obese DKA patients (1.0+/-0.1 ng/ml) than in obese patients with hyperglycemia (1.7+/-0.2 ng/ml, P < 0.01), but was higher than that in lean DKA patients (0.2+/-0.1 ng/ml, both P < 0.01). The overall prevalence of autoantibodies in obese subjects with DKA (17%) and obese subjects with hyperglycemia (16%) was lower than that in lean subjects with DKA (65%, P < 0.01). Obese patients with hyperglycemia and positive autoantibodies had lower rates of insulin secretion than those without antibodies. Regardless of body weight, all DKA patients with GAD autoantibodies carried the DQB1*0201 allele. However, there were no significant differences in HLA distribution between the three patient groups. CONCLUSIONS: Our results indicate that most obese African-American patients with DKA have type 2 diabetes characterized by higher insulin secretion, the absence of autoimmune markers, and a lack of HLA genetic association. In contrast, most lean African-American patients with DKA have metabolic and immunological features of type 1 diabetes. At presentation, assessment of beta-cell function and determination of autoimmune markers allow for correct classification of diabetes in African-Americans with hyperglycemic crises.

Islet cell and glutamic acid decarboxylase antibodies present at diagnosis of diabetes predict the need for insulin treatment. A cohort study in young adults whose disease was initially labeled as type 2 or unclassifiable diabetes.

OBJECTIVE: To clarify the predictive value of islet cell antibody (ICA) and GAD65 antibody (GADA) present at diagnosis with respect to the need for insulin treatment 6 years after diagnosis in young adults initially considered to have type 2 or unclassifiable diabetes. RESEARCH DESIGN AND METHODS: The patient material was representative of the entire Swedish population, consisting of patients who were 15-34 years old at diagnosis of diabetes in 1987-1988 but were not considered to have type 1 diabetes at onset. At follow-up, 6 years after the diagnosis, it was noted whether the patient was treated with insulin. The presence of ICA was determined by an immunofluorescence assay, and GADAs were measured by a radioligand assay. RESULTS: Six years after diagnosis, 70 of 97 patients were treated with insulin, and 27 of 97 patients were treated with oral drugs or diet alone. At diagnosis, ICAs and GADAs were present in 41 (59%) of 70 patients and 41 (60%) of 68 patients, respectively, of those now treated with insulin, compared with only 1 (4%) of 26 patients and 2 (7%) of 27 patients who were still not treated with insulin. For either ICA or GADA, the corresponding frequencies were 50 (74%) of 68 for patients who were later treated with insulin and 3 (12%) of 26 for those who were still not treated with insulin, respectively. The sensitivity for later insulin treatment was highest (74%) for the presence of ICA or GADA, and the specificity was highest (100%) for ICA and GADA. The positive predictive value was 100% for the combination of ICA and GADA, 98% for ICA alone, and approximately 95% for GADA alone. CONCLUSIONS: Determination of the presence of ICA and GADA at diagnosis of diabetes improves the classification of diabetes and predicts the future need of insulin in young adults.

Mapping genes for NIDDM. Design of the Finland-United States Investigation of NIDDM Genetics (FUSION) Study.

OBJECTIVE: To map and identify susceptibility genes for NIDDM and for the intermediate quantitative traits associated with NIDDM. RESEARCH DESIGN AND METHODS: We describe the methodology and sample of the Finland-United States Investigation of NIDDM Genetics (FUSION) study. The whole genome search approach is being applied in studies of several different ethnic groups to locate susceptibility genes for NIDDM. Detailed description of the study materials and designs of such studies are important, particularly when comparing the findings in these studies and when combining different data sets. RESULTS: Using a careful selection strategy, we have ascertained 495 families with confirmed NIDDM in at least two siblings and no history of IDDM among the first-degree relatives. These families were chosen from more than 22,000 NIDDM patients, representative of patients with NIDDM in the Finnish population. In a subset of families, a spouse and offspring were sampled, and they participated in a frequently sampled intravenous glucose tolerance test (FSIGT) analyzed with the Minimal Model. An FSIGT was completed successfully for at least two nondiabetic offspring in 156 families with a confirmed nondiabetic spouse and no history of IDDM in first-degree relatives. CONCLUSIONS: Our work demonstrates the feasibility of collecting a large number of affected sib-pair families with NIDDM to provide data that will enable a whole genome search approach, including linkage analysis.

Regulation of glutamic acid decarboxylase diabetes autoantigen expression in highly purified isolated islets from Macaca nemestrina.

Macaca nemestrina, which may have larger and more numerous pancreatic islets than other species, was used for large scale islet isolation by ductal collagenase perfusion and Ficoll gradient centrifugation. The average yield was 51,000 islet equivalents per pancreas, or 8,750 islets equivalents per g. The average purity was 91%, often exceeding 95%. These are the highest reported size, purity, and yield per g of any nonautomated primate islet series. Perifusion with glucose, arginine, and isobutylmethylxanthine showed appropriate biphasic insulin secretion. Unlike that in the rat, human islet glutamic acid decarboxylase (GAD) isoform expression is restricted. However, glycemic regulation of GAD expression has been shown only in rats. We, therefore, tested hypotheses that M. nemestrina islets also have restricted GAD expression, that GAD expression in primates is stimulated by glucose, and that this stimulation remains restricted to the 64,000 mol wt (GAD65) isoform. Immunoprecipitation of labeled islet extracts showed that GAD65 expression increased 16.7 +/- 0.6-fold during high glucose in vitro culture. After controlling for observed increases in protein synthesis, specific glucose stimulation was still 4.2 +/- 0.2-fold. Specific antisera revealed no GAD67 expression under basal conditions, and isoform restriction was maintained during stimulation. Increased GAD65 synthesis thus accounts for glucose stimulation of 64K expression. These time- and concentration-dependent effects of glucose suggest that hyperglycemia increases autoantigenicity and may accelerate beta-cell destruction in primates, supporting a role for beta-cell rest in insulin-dependent diabetes mellitus prevention.

Islet cell antibodies, but not glutamic acid decarboxylase antibodies, are decreased by plasmapheresis in patients with newly diagnosed insulin-dependent diabetes mellitus.

The effects of plasmapheresis on islet autoantibody levels, C-peptide (beta-cell function), and hemoglobin-A1c (HbA1c, metabolic control) were tested in a prospective blinded study of 18 newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients randomly assigned to receive plasmapheresis (P), carried out as double filtration, or sham (S) treatment at diagnosis and 3 months thereafter. At diagnosis, 6 of 8 patients (75%) in group P and 9 of 10 patients (90%) in group S had islet cell antibodies (ICA), whereas 4 of 8 (50%) and 7 of 10 (70%) patients, respectively, had glutamic acid decarboxylase antibodies (GAD65-Ab), with no significant differences between the groups in ICA and GAD65-Ab levels. After 6 months, P patients showed significantly lower ICA levels than S patients (11 +/- 6 and 128 +/- 47 Juvenile Diabetes Foundation International Units, respectively; P < 0.02) due to an increase in ICA levels in 8 of 9 (88%) of the S patients not seen in P patients (P < 0.002). Concurrently, HbA1c stabilized in P, but not in S, patients and was significantly lower by 24 months (6.58 +/- 0.54% vs. 9.76 +/- 1.21%; P < 0.05). Moreover, fasting C-peptide increased significantly (214 +/- 11 pmol/L; P < 0.05) over the first 6 months in P. After the initial 6 months, ICA levels tended to decrease in all patients and were not detected after 60 months. GAD65-Ab levels were not influenced by plasmapheresis and, also in contrast to ICA, increased significantly (P < 0.05) in the whole study population after 60 months. In fact, 4 initially negative patients became GAD65-Ab positive after diagnosis (in 2 patients > 24 months after diagnosis). We conclude that plasmapheresis of newly diagnosed IDDM patients does not change subsequent GAD65-Ab levels, but ICA are significantly decreased with associated improved C-peptide and HbA1c levels.

A novel high-throughput method for accurate, rapid, and economical measurement of multiple type 1 diabetes autoantibodies.

Prediction of Type 1 diabetes for study of preventive therapies requires screening the general population, where 85% of new cases occur. Even with HLA-based prescreening, nearly 20% of all children will need multiple serum autoantibody testings. High-throughput, economical, and accurate methods are therefore essential. We have developed such a radiobinding method, using 96-well microtiter plates and a novel immune complex capture method via membrane-bound Protein A. Each microtiter plate contained a standard negative control serum, and low-, medium-, and high-level positive control sera. All sera were evaluated in triplicate. This readily allowed quality control criteria both for triplicates of individual sera and for each 96-well plate. Inter-assay coefficients of variation (CVs) were all

Autoimmunity of diabetes.

Insulin-dependent diabetes mellitus is associated with a growing number of immune abnormalities. At the time of clinical onset, most patients developing the disease as children or young adults have autoantibodies reactive with islet beta cells. Current autoantibody markers for IDDM are not sufficient to predict the disease in the general population. Studies in first-degree relatives indicate the presence of a subclinical disease characterized by beta cell dysfunction, which may or may not progress to overt IDDM. Although IDDM is genetically linked to certain HLA-DQ class II molecules, it needs to be clarified whether these molecules determine the propensity to react to certain antigens, the failure to maintain tolerance, or the ability to produce disease-associated autoantibodies. Circumstantial evidence suggests that yet another gene outside the HLA complex on chromosome 6 is more important. The interaction with the environment needs to be clarified, and the etiologic role of viruses has not been substantiated. An underlying systemic autoimmune propensity may influence environmental insults and perpetuate islet beta cell destruction. Until these mechanisms are understood, clinicians should periodically check their patients with IDDM for other organ-specific as well as non--organ-specific autoimmune diseases. Our understanding of these phenomena is poor, which may explain why clinical trials with immunosuppressive agents have been of limited success. Further studies on the molecular biology of the immune response against islet beta cell-specific antigens are necessary for the development of both predictive tests and novel measures to prevent IDDM.

Differential detection of rat islet and brain glutamic acid decarboxylase (GAD) isoforms with sequence-specific peptide antibodies.

We studied the distribution of the M(r) 65,000 and M(r) 67,000 isoforms of glutamic acid decarboxylase, GAD65 and GAD67, in rat islets and brain by immunocytochemistry. Synthetic peptides representing selected GAD65 or GAD67 sequences were used to produce sequence-specific antibodies, allowing differential immunocytochemical detection of the two isoforms. GAD-specific reactivity of each peptide antiserum was confirmed by ELISA, immunoblotting, and immunoprecipitation. Immunostaining specificity was verified by displacement with either immunizing or irrelevant peptide. Dual immunostaining with GAD isoform-specific antibodies and polyclonal antibodies to glucagon showed that GAD65 was primarily detected in rat pancreatic islet beta-cells, whereas alpha-cells had weak GAD65 staining. In contrast, GAD67 was detected primarily in alpha-cells. In rat brain, GAD65 and GAD67 were present in neuron cell bodies and processes. These data demonstrate that antibodies raised against the N-terminus of GAD allow differential immunocytochemical identification of GAD67 and GAD65. Differential expression of GAD isoforms within islet alpha- and beta-cells supports the role of GAD65 in autoimmune diabetes and stiff-man syndrome.