GAD65-reactive T cells are activated in patients with autoimmune type 1a diabetes

Insulin-dependent type 1 diabetes is an autoimmune disease mediated by T lymphocytes recognizing pancreatic islet cell antigens. Glutamic acid decarboxylase 65 (GAD65) appears to be an important autoantigen in the disease. However, T cells from both patients with type 1 diabetes and healthy subjects vigorously proliferate in response to GAD65 stimulation ex vivo, leading us to postulate that the critical event in the onset of human diabetes is the activation of autoreactive T cells. Thus, we investigated whether GAD65-reactive T cells in patients with diabetes functioned as previously activated memory T cells, no longer requiring a second, costimulatory signal for clonal expansion. We found that in patients with new-onset type 1 diabetes, GAD65-reactive T cells were strikingly less dependent on CD28 and B7-1 costimulation to enter into cell cycle and proliferate than were equivalent cells derived from healthy controls. We hypothesize that these autoreactive T cells have been activated in vivo and have differentiated into memory cells, suggesting a pathogenic role in type 1 diabetes. In addition, we observed different effects with selective blockade of either B7-1 or B7-2 molecules; B7-1 appears to deliver a negative signal by engaging CTLA-4, while B7-2 engagement of CD28 upregulates T cell proliferation and cytokine secretion.     

GAD65 autoantibody responses in Japanese latent autoimmune diabetes in adult patients

OBJECTIVE—To determine whether development of insulin requirement in patients with latent autoimmune diabetes in adults (LADA) is accompanied with the emergence of a type 1 diabetes–like autoimmune response.RESEARCH DESIGN AND METHODS—We correlated β-cell–specific autoimmunity reflected in autoantibodies to the 65-kDa isoform of GAD (GAD65) with insulin requirement. We determined GAD65Ab epitope specificities in type 1 diabetic patients, LADA patients without insulin requirement (nonprogressed), and LADA patients that had developed insulin requirement (progressed).RESULTS—Recognition of a type 1 diabetes–specific GAD65Ab epitope was more pronounced in type 1 diabetic patients than in nonprogressed (P < 0.001) or progressed (P < 0.01) LADA patients, with no significant differences between the two LADA cohorts. These differences were particularly pronounced in samples with GAD65Ab titers <1,000 units/ml, with no differences in epitope specificities in samples with higher GAD65Ab titers. Disease duration (initial diabetes diagnosis until sample collection or development of insulin requirement) in nonprogressed and progressed LADA patients, respectively, was not correlated with epitope specificity, suggesting lack of epitope maturation. This was supported by epitope analyses of longitudinal samples from LADA patients during progression to insulin requirement.CONCLUSIONS—First, the GAD65Ab-specific autoimmune reaction in type 1 diabetic patients with low and moderate GAD65Ab titers differs from that in LADA patients, irrespective of insulin requirement. Second, the GAD65Ab-specific autoimmune response in LADA patients does not change after their initial diabetes diagnosis. Finally, LADA patients with high GAD65Ab titers resemble type 1 diabetic patients in their GAD65Ab epitope specificity.Latent autoimmune diabetes in adults (LADA) consists of a subgroup (∼10%) of adult patients initially diagnosed with type 2 diabetes, who show signs of β-cell autoimmunity and eventually develop insulin requirement (1,2). Signs of β-cell autoimmunity, such as the well-characterized insulin autoantibodies, glutamate decarboxylase (GAD65), and the tyrosine phosphatase–like protein insulinoma-associated protein-2, indicate significant damage of the β-cells and subsequent development of insulin requirement in these patients (1). While autoantibodies to insulin and insulinoma-associated protein-2 antibody (Ab) are inversely correlated with age at onset, GAD65Ab shows no, and in some studies even a positive, correlation with age at onset and is therefore a particularly attractive marker for autoimmune diabetes in the adult population (3,4). Moreover, GAD65Ab can be detected years after the clinical onset of the disease, indicating that these autoantibodies may be permanent markers for the autoimmune response (5,6).Notably, not all LADA patients progress to insulin requirement, raising the possibility that the autoimmune response in these patients resembles that in autoantibody-positive healthy individuals, with no significant risk for development of insulin requirement (7,8). A better understanding of the autoimmune response is necessary to predict insulin requirement in LADA patients, which is important to prevent escalation of blood glucose levels and subsequent complications.In previous studies, we have investigated the humoral immune response toward GAD65 as a reflection of islet cell destruction (9). It remains unclear whether the autoimmune response in LADA patients and type 1 diabetic patients differs or whether only the duration of the prodomal period distinguishes between the two groups (10). Therefore, we compared the GAD65-specific humoral autoimmune response in type 1 diabetic patients with that in LADA patients who had or had not progressed to insulin requirement.     

口服重组GAD65蛋白延缓NOD小鼠Ⅰ型糖尿病发生

目的研究重组GAD65蛋白对NOD小鼠Ⅰ型糖尿病的预防作用。方法6周龄NOD雌鼠3次/周灌胃重组GAD65蛋白,共6~56周,观测胰岛炎和糖尿病发病情况。结果重组GAD65蛋白能明显降低NOD小鼠糖尿病的发生和胰岛炎的严重程度。结论口服重组GAD65蛋白能预防NOD鼠糖尿病。     

Diamyd, an alum-formulated recombinant human GAD65 for the prevention of autoimmune diabetes

Diamyd Medical AB is developing Diamyd (GAD-65), an alum formulation of a full-length recombinant human glutamic acid decarboxylase 65 for subcutaneous injection, for the potential prevention and treatment of type 1 diabetes (T1DM) or latent autoimmune diabetes (LADA) in adults. Phase II clinical trials indicated that Diamyd was safe and well tolerated in patients with T1DM or LADA. Diamyd is currently in phase II/III and III clinical trials for T1DM.     

Antibodies to GAD65 and a tyrosine phosphatase-like molecule IA-2ic in Filipino type 1 diabetic patients

OBJECTIVE: Type 1 diabetes is more prevalent in Europeans than it is in Asians. The disease is associated with autoantibodies to GAD65 and a protein tyrosine phosphatase-like molecule (IA-2). The frequency of GAD antibodies in Asian patients with type 1 diabetes may be lower than that in Europeans. No data are available on IA-2 antibodies in Asians. We tested antibodies to GAD65 and IA-2ic (the intracellular fragment containing the antibody epitope) in Filipino diabetic patients because this population has mixed European and aboriginal racial origins. RESEARCH DESIGN AND METHODS: A cross-sectional study of antibodies to GAD65 and IA-2ic was performed on a consecutive series of 91 type 1 diabetic patients, 74 type 2 diabetic patients, and 100 control subjects attending a diabetes clinic in Manila, the Republic of the Philippines. All subjects were <40 years of age, with a mean age +/- SD of 24.8+/-9.8, 34.3+/-5.8, and 25.8+/-8.0 years, respectively. Diagnosis of type 1 diabetes was determined clinically and confirmed by baseline C-peptide. RESULTS: Of 91 type 1 diabetic patients, antibodies to GAD65 were detected in 25 (27.4%), but antibodies to IA-2ic were found in only 8 (8.8%) (P = 0.002); neither autoantibody was detected in either the type 2 diabetic or control subjects. Of the 25 recently diagnosed type 1 diabetic patients (disease duration <2.0 years), autoantibodies to GAD65 were detected in 14 (56%), but those to IA-2ic in only 4 (16%) (P = 0.007); GAD65 antibodies were detected in only 4 (6%) of 66 patients with a longer disease duration (P = 0.0004). Comparison with recently diagnosed European type 1 diabetic patients of age and disease duration similar to that of the Filipinos indicated that IA-2ic antibodies, unlike GAD antibodies, were significantly less prevalent in Filipino type 1 diabetic patients (P = 0.0007). CONCLUSIONS: This is the first study investigating the prevalence and pattern of humoral immune response in type 1 diabetic patients from the Philippines. Antibodies to IA-2ic, unlike GAD antibodies, were infrequent. Patterns of immune responses to type 1 diabetes-associated antigens may differ worldwide, with important implications for prediction of the disease and the potential for antigen-specific therapy.     

Detection of GAD65-reactive T-Cells in type 1 diabetes by immunoglobulin-free ELISPOT assays

OBJECTIVE: To investigate the prevalence of beta-cell autoantigen-reactive peripheral T-cells in type 1 diabetes, we developed an immunoglobulin-free enzyme-linked immunospot (ELISPOT) assay and assessed its usefulness for diagnosing this disease. RESEARCH DESIGN AND METHODS: Cellular immune responses to beta -cell autoantigens were studied both by immunoglobulin-free proliferation assays and ELISPOT assays in 33 patients with type 1 diabetes and 15 patients with type 2 diabetes, compared with 23 healthy control subjects. Autoantibodies against GAD65 and IA-2 were measured by radioimmunoassay. RESULTS: Significant proliferative responses to GAD65 were observed in 10 of 31 (32.3%) type 1 diabetic patients (P < 0.05), whereas GAD65-reactive gamma-interferon (IFN-gamma)-secreting cells were detected in 22 of 33 patients (66.7%) by ELISPOT assay (P < 0.001). Of patients negative for both GAD65 and IA-2, five of six (83.3%) showed IFN-gamma positivity in ELISPOT and two of five (40.0%) showed significant proliferation against GAD65. CONCLUSIONS: Using a newly developed ELISPOT assay, GAD-reactive T-helper 1 cells in PBMC of type 1 diabetic patients could be identified at a higher frequency than by the proliferation assay. Therefore, the immunoglobulin-free ELISPOT assay is an excellent tool for detecting T-cell reactivity to autoantigens with greater specificity and, in combination with beta-cell autoantibody determination, will improve the diagnosis of type 1 diabetes.     

Antibodies to IA-2 and GAD65 in type 1 and type 2 diabetes: isotype restriction and polyclonality

OBJECTIVE: To determine the isotypes and clonality of antibodies to GAD (GADA) and IA-2 (IA-2A) in patients with type 1 and type 2 diabetes. RESEARCH DESIGN AND METHODS: We studied the following consecutive series of patients who attended a diabetes center for antibodies to GADA and IA-2A: 52 newly diagnosed type 1 diabetic patients, 199 type 2 diabetic patients, 200 control patients, and a cohort of 34 nondiabetic identical twins of patients with type 1 diabetes (15 of whom developed diabetes) who were followed prospectively. RESULTS: GADA or IA-2A were detected in 37 (71%) type 1 diabetic patients compared with only 10 (5%) type 2 diabetic patients (P<0.0001). Both GAD and IA-2 antibodies, regardless of the type of diabetes, were usually subclass restricted to IgG1 and were polyclonal. IgM, IgG3, and IgE isotypes were also detected, but all isotypes of GADA and IA-2A were less prevalent than IgG1 (P<0.017 for either antibody). There was no evidence of spreading or switching of isotypes before the onset of type 1 diabetes. CONCLUSIONS: These observations suggest that the pathogenesis of antigen-specific antibodies in type 1 and type 2 diabetes is similar and probably involves a chronic nonrandom antigen-driven polyclonal B-cell activation that is consistent with a Th1-type immune response.     

CTLs are targeted to kill beta cells in patients with type 1 diabetes through recognition of a glucose-regulated preproinsulin epitope

The final pathway of β cell destruction leading to insulin deficiency, hyperglycemia, and clinical type 1 diabetes is unknown. Here we show that circulating CTLs can kill β cells via recognition of a glucose-regulated epitope. First, we identified 2 naturally processed epitopes from the human preproinsulin signal peptide by elution from HLA-A2 (specifically, the protein encoded by the A*0201 allele) molecules. Processing of these was unconventional, requiring neither the proteasome nor transporter associated with processing (TAP). However, both epitopes were major targets for circulating effector CD8⁺ T cells from HLA-A2⁺ patients with type 1 diabetes. Moreover, cloned preproinsulin signal peptide–specific CD8⁺ T cells killed human β cells in vitro. Critically, at high glucose concentration, β cell presentation of preproinsulin signal epitope increased, as did CTL killing. This study provides direct evidence that autoreactive CTLs are present in the circulation of patients with type 1 diabetes and that they can kill human β cells. These results also identify a mechanism of self-antigen presentation that is under pathophysiological regulation and could expose insulin-producing β cells to increasing cytotoxicity at the later stages of the development of clinical diabetes. Our findings suggest that autoreactive CTLs are important targets for immune-based interventions in type 1 diabetes and argue for early, aggressive insulin therapy to preserve remaining β cells.     

Time-resolved immunofluorometric dual-label assay for simultaneous detection of autoantibodies to GAD65 and IA-2 in children with type 1 diabetes

BACKGROUND: Autoantibodies to glutamic acid decarboxylase (GADAs), specifically the 65-kDa isoform GAD65, and autoantibodies to the protein tyrosine phosphatase-like molecule IA-2 (IA-2As) predict development of diabetes. Our aim was to develop a time-resolved immunofluorometric (TR-IFMA) dual-label assay method for the simultaneous detection of these autoantibodies and to evaluate the diagnostic sensitivity of the method compared with single-label TR-IFMA and fluid-phase radiobinding assay (RBA) in screening children with type 1 diabetes. METHODS: We incubated combined biotinylated GAD65 and IA-2 proteins, glutathione S-transferase (GST)-IA-2, europium-labeled GAD65, terbium-labeled anti-GST antibody, and serum sample or calibrator and transferred aliquots to a streptavidin-coated 96-well microtiter plate for a second incubation. After washing, we added Delfia Enhancement solution to each well and measured the fluorescence of Eu. We developed the Tb fluorescence signal by use of the Delfia Enhancer solution and measured it. We analyzed serum samples from a cohort of 100 children with newly diagnosed type 1 diabetes. RESULTS: The correlation coefficients between the autoantibody concentrations measured by dual- and single-label TR-IFMA assays were 0.962 for GADA and 0.874 for IA-2A. Among 100 children with newly diagnosed diabetes, 65 of them were GADA positive in the dual-label assay, 64 in the single-label assay, and 66 in the RBA GADA assay. Seventy-four of the children tested positive for IA-2A in both TR-IFMA assay types, and 79 in the RBA IA-2A assay. CONCLUSIONS: The novel dual-label immunofluorometric assay performed comparably to the separate, single-label GADA and IA-2A assays in screening for beta-cell autoimmunity in children with newly diagnosed type 1 diabetes.     

Clinical,autoimmune, and genetic characteristics of adult-onset diabetic patients with GAD autoantibodies in Japan (Ehime Study)

OBJECTIVE: To characterize the clinical, autoimmune, and genetic features in Japanese adult-onset diabetic patients with GAD autoantibodies. RESEARCH DESIGN AND METHODS: GAD autoantibodies (GADab) were screened in 4,980 diabetic patients with age of onset >20 years in the hospital-based Ehime Study, and the GADab-positive (GADab(+)) patients were then divided into two groups according to their insulin secretion and compared with nondiabetic subjects. The insulin-deficient state was defined as <0.33 nmol/l serum C-peptide (CPR) at 2 h postprandial or 6 min after a 1-mg glucagon load. RESULTS: GADab was detected in 188 (3.8%) of the 4,980 diabetic patients tested. Of these patients, 72 (38.3%) were classified as insulin deficient, 97 (51.6%) were classified as non-insulin deficient, and 19 (10.1%) were unclassified. The GADab(+) insulin-deficient patients were characterized by young age at onset of diabetes, low BMI, low maximum BMI, and high levels of HbA(1c). The prevalence of IA-2 autoantibodies and thyrogastric autoantibodies in the GADab(+) insulin-deficient patients were significantly higher than those in the GADab(+) non-insulin-deficient patients (P < 0.05). GADab(+) patients with insulin deficiency had increased frequencies of HLA DRB1*0405-DQB1*0401, *0802-*0302, and *0901-*0303 haplotypes, whereas the frequency of only HLA DRB1*0405-DQB1*0401 was increased in the case of GADab(+) non-insulin-deficient patients. Of note is the fact that the GADab(+) non-insulin-deficient group did not differ from healthy control subjects with respect to type 1 diabetes protective haplotype HLA DRB1*1502-DQB1*0601. A total of 13% of the GADab(+) patients with diabetes had genotypes comprising the DRB1*1501-DQB1*0602 or *1502-*0601 and were characterized by old age at onset of diabetes, high BMI, resistance to the insulin-deficient state, low titer of GADab, and low frequency of other organ-specific autoantibodies. CONCLUSIONS: We conclude that GADab(+) non-insulin-deficient patients differ from GADab(+) patients with insulin deficiency with respect to clinical characteristics, humoral autoimmunity to other organ-specific autoantibodies, as well as HLA class II genes.     

Serum 1,5-anhydroglucitol levels in patients with fulminant type 1 diabetes are lower than those in patients with type 2 diabetes

ObjectivesWe investigated clinical relevance of serum 1,5-anhydroglucitol (1,5-AG) levels in fulminant type 1 diabetes mellitus (FT1DM) patients, because 1,5-AG is known to reflect short term glycemic control.Design and methodsSubjects comprised 7 patients with FT1DM and 32 patients with type 2 diabetes mellitus (T2DM) with HbA1c < 8.5%. All of them have never been treated for diabetes.ResultsHbA_1C showed no significant difference between both groups. On the other hand, serum 1,5-AG levels were significantly lower in the FT1DM patients than in the T2DM patients. Serum 1,5-AG levels were < 5.0 μg/ml in 6 of 7 (86%) FT1DM patients, compared with only 1 of 32 (3%) T2DM patients.ConclusionsSerum 1,5-AG levels were lower in the FT1DM patients than in the T2DM patients. Serum 1,5-AG, but not HbA_1C, reflects short-term exacerbation of glycemia in patients with FT1DM.     

Anti-oxidized low-density lipoprotein antibody levels are associated with the development of type 2 diabetes mellitus

Background  Anti-oxidized low-density lipoprotein (LDL) antibodies are associated with the oxidative capacity of plasma, but whether they protect or promote diabetes is unknown. We undertook a prospective study to determine the predictive capacity of anti-oxidized LDL antibodies for the onset of type 2 diabetes mellitus (T2DM).
Materials and methods  We selected 391 non-diabetic women aged 18–65 years. The subjects were classified as being normal (oral glucose test tolerance normal, OGTT-N), or having impaired glucose tolerance (IGT), impaired fasting glucose (IFG) or T2DM according to their baseline glucose levels and after an OGTT. The same subjects were studied six years later. The levels of anti-oxidized LDL antibodies were classified as above or below the 50th percentile.
Results  Of the women who were OGTT-N at the start of the study and who had anti-oxidized LDL antibody levels below the 50th percentile, only 65·1% were still OGTT-N after 6 years versus 79·5% of those who had anti-oxidized LDL antibody levels above the 50th percentile ( P  = 0·015). Women who had IGT or IFG at the start of the study whose anti-oxidized LDL antibody levels were below the 50th percentile had a relative risk of 9·79 (95% confidence interval, 1·40–68·45) of developing diabetes ( P  < 0·001). Logistic regression analysis showed that the variables predicting the development of a carbohydrate metabolism disorder in the women after 6 years were body mass index ( P  < 0·001) and the levels of anti-oxidized LDL antibodies ( P  = 0·042).
Conclusions  Levels of anti-oxidized LDL antibodies are independent predictors for the development of T2DM in women.