Cytoplasmic islet cell antibodies recognize distinct islet antigens in IDDM but not in stiff man syndrome

Cytoplasmic islet cell antibodies are well-established predictive markers of IDDM. Although target molecules of ICA have been suggested to be gangliosides, human monoclonal ICA of the immunoglobulin G class (MICA 1-6) produced from a patient with newly diagnosed IDDM recognized glutamate decarboxylase as a target antigen. Here we analyzed the possible heterogeneity of target antigens of ICA by subtracting the GAD-specific ICA staining from total ICA staining of sera. This was achieved 1) by preabsorption of ICA+ sera with recombinant GAD65 and/or GAD67 expressed in a baculovirus system and 2) by ICA analysis of sera on mouse pancreas, as GAD antibodies do not stain mouse islets in the immunofluorescence test. We show that 24 of 25 sera from newly diagnosed patients with IDDM recognize islet antigens besides GAD. In contrast, GAD was the only islet antigen recognized by ICA from 7 sera from patients with stiff man syndrome. Two of these sera, however, recognized antigens besides GAD in Purkinje cells. In patients with IDDM, non-GAD ICA were diverse. One group, found in 64% of the sera, stained human and mouse islets, whereas the other group of non-GAD ICA was human specific. Therefore, mouse islets distinguish two groups of non-GAD ICA and lack additional target epitopes of ICA besides GAD. Longitudinal analysis of 6 sera from nondiabetic ICA+ individuals revealed that mouse-reactive ICA may appear closer to clinical onset of IDDM in some individuals

Determination of islet cell antibodies using an ELISA system with a preparation of rat insulinoma (RIN A2) cells

An enzyme-linked immunosorbent assay (ELISA) was established for the detection of islet cell antibodies in human sera. The antigen was prepared from rat insulinoma (RIN A2) cells. Cells were dissociated in lysis buffer and the lysate was centrifuged at 100,000 x g. The supernatant was used to coat microtiter ELISA plates (10 micrograms protein/ml in PBS pH 7.2). Non-specific binding sites on the plates were blocked with 2% PBS-BSA. Human test sera were preabsorbed on separate plates using 2% PBS-BSA and incubated on precoated plates at an optimal dilution of 1/10 in 60 mM PBS for 60 min at 37 degrees C. Phosphatase-labeled anti-human IgG serum and phosphatase substrate were applied and the reaction was stopped by adding 3 M NaOH. Out of 90 sera from type I diabetic patients, 47 (52.2%) reacted in the new ELISA whereas none of 15 type II diabetics, 50 sera containing non-islet specific antibodies or 100 normal controls were positive. In the same group of patients, ICA were positive in 63.3%. When both, the ELISA and conventional ICA testing were applied, the number of positives was increased to 83%. The ICA-ELISA with the above described antigen preparation provides a well standardized and reproducible test method which is highly specific for type I diabetes. It may therefore be useful for large screening procedures

Epidemiology and immunogenetic background of islet cell antibody - positive nondiabetic schoolchildren : Ulm-Frankfurt population study

Islet cell antibodies (ICAs) were determined in a large cohort of white nondiabetic schoolchildren (n = 4287) from a homogenous population in southern Germany. The prevalence of ICA levels greater than or equal to 5 Juvenile Diabetes Foundation (JDF) U was 1.05% (95% confidence interval 0.8-1.4%). Analysis of HLA-DR beta and -DQ beta alleles revealed that the specificities found to be increased in insulin-dependent (type I) diabetic subjects with the same ethnic background were also associated with ICA positivity in the nondiabetic schoolchildren. HLA-DR3 (P less than 0.01) and -DR4 (P less than 0.01) phenotypes and absence of Asp residue (P less than 0.01) at codon 57 of the HLA-DQ beta-chain were significantly increased in ICA+ compared with control subjects. High levels of ICAs, which were categorized as either greater than or equal to 17 or greater than or equal to 30 JDF U, were found to be associated with amino acids other than Asp at position 57 of the HLA-DQ beta-chain. No association of ICA level was found for HLA-DR phenotypes

Baculovirus-Mediated Expression of Human 65 kDa and 67 kDa Glutamic Acid Decarboxylases in SF9 Insect Cells and Their Relevance in Diagnosis of Insulin-Dependent Diabetes Mellitus

cDNAs coding for the full-length human 65 and 67 kDa glutamic acid decarboxylases (GAD65 and GAD67) were amplified from pancreas and hippocampus cDNA libraries by polymerase chain reaction, respectively. Both cDNAs were inserted into a baculovirus vector which mediated highly efficient expression of the human GAD65 and GAD67 with histidine-hexapeptides as affinity ligands at their C-termini in Spodoptera frugiperda (Sf9) cells. The recombinant GAD proteins were purified to homogeneity by affinity chromatography using a metal-chelating matrix. The infected Sf9 insect cells expressed the recombinant human GAD65 and GAD67 with natural-like conformations, as confirmed by measurement of their enzyme activities as well as their fully restored autoantigenicities. Immunoprecipitation of metabolically labeled infected Sf9 cells demonstrated the autoantigenic potential of the recombinant GAD proteins. The practicability of using recombinant GAD65 and GAD67 derived from the baculovirus expression system for the development of an immunoassay for the diagnosis of insulin-dependent diabetes mellitus is discussed

Risk for cardiovascular events responds nonlinearly to carotid intima-media thickness in the KORA F4 study

Background and aims: Risk assessment studies on the impact of carotid intima-media thickness (CIMT) on cardiovascular events (CVEs) often apply a linear relationship in Cox models of proportional hazards. However, CVEs are mostly induced through rupture of plaques driven by nonlinear mechanical properties of the arterial wall. Hence, the risk response might be nonlinear as well and should be detectable in CVE incidence data when associated with CIMT as surrogate variable for atherosclerotic wall degeneration.Methods: To test this hypothesis, we investigate the KORA F4 study comprising 2580 participants with CIMT measurements and 153 first CVEs (86 strokes and 67 myocardial infarctions). CIMT is only a moderate predictor of CVE risk due to confounding by attained age. Biological evidence suggests that age-related CIMT growth is not entirely connected with atherosclerosis. To explore the complex relations between age, CIMT and CVE risk, we apply linear and nonlinear models of both CIMT and dnCIMT, defined as deviation from a sex and age-adjusted normal value.Results: Based on goodness-of-fit and biological plausibility, threshold and logistic step models clearly reveal nonlinear risk response relations for vascular covariables CIMT and dnCIMT. The effect is more pronounced for models involving dnCIMT as novel risk factor, which is not correlated with age.Conclusions: Compared to the standard approach of risk assessment with linear models involving CIMT, the application of excess dnCIMT with nonlinear risk responses leads to a more precise identification of asymptomatic high risk patients, especially at younger age

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

Different effects of lifestyle intervention in high- and low-risk prediabetes

Lifestyle intervention (LI) can prevent type 2 diabetes, but response to LI varies depending on risk subphenotypes. We tested if prediabetic individuals with low risk benefit from conventional LI and individuals with high risk benefit from an intensification of LI in a multi-center randomized controlled intervention over 12 months with 2 years follow up. 1105 prediabetic individuals based on ADA glucose criteria were stratified into a high- and low-risk phenotype, based on previously described thresholds of insulin secretion, insulin sensitivity and liver fat content. Low-risk individuals were randomly assigned to conventional LI according to the DPP protocol or control (1:1), high-risk individuals to conventional or intensified LI with doubling of required exercise (1:1). A total of 908 (82%) participants completed the study. In high-risk individuals, the difference between conventional and intensified LI in post-challenge glucose change was -0.29 mmol/l [CI:-0.54;-0.04], p=0.025. Liver fat (-1.34 percentage points [CI:-2.17;-0.50], p=0.002) and cardiovascular risk (-1.82[CI:-3.13-0.50],p=0.007) underwent larger reductions with intensified than with conventional LI. During a follow up of 3 years, intensified compared to conventional LI had a higher probability to normalize glucose tolerance (p=0.008). In conclusion, it is possible in high-risk individuals with prediabetes to improve glycemic and cardiometabolic outcomes by intensification of LI. Individualized, risk-phenotype-based LI may be beneficial for the prevention of diabetes