Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0011854 (type 1 diabetes)
 20,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutamic acid decarboxylase (GAD) is one major autoantigen involved in the pathogenesis of autoimmune insulin dependent diabetes mellitus (IDDM). Molecular mechanisms regulating GAD expression in pancreatic beta cell are still ill-defined. Here we investigated the effect of streptozotocin (STZ), a beta cell-specific toxin, on the expression of GAD67 in MIN6N8a mouse beta cell. A 5-6-fold increase in the expression GAD67 mRNA was found in cells treated with 1.25mM STZ for 12h. Addition of NAD+ to the incubation medium slightly reduced the STZ-induced upregulation of GAD67. STZ increased p53 levels that in turn up-modulated GAD67 expression. This effect was abolished upon addition of the antioxidant N-acetyl cysteine (NAC). STZ also activated NF-kappaB and blockade of NF-kappaB activation inhibited the STZ-mediated upregulation of GAD67 expression. As a whole these data show that low dose of STZ up-regulates GAD67 expression in mouse bate cell and that NF-kappaB activation through oxidative stress plays a key role in this phenomenon. They also suggest that various stimuli promoting NF-kappaB activation may up-regulate expression of GAD autoantigen in mouse beta cells.
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PMID:Streptozotocin upregulates GAD67 expression in MIN6N8a mouse beta cells. 1236 54

While both isoforms of glutamic acid decarboxylase (GAD) function as important autoantigens in autoimmune diabetes mellitus-GAD65 in humans and GAD67 in the NOD mouse-GAD67 is not synthesized in human pancreatic islets and is thought not to be an autoantigen in human diabetes. We have recently shown, however, that human islets contain a GAD67 splice variant: GAD25. Given the evidence that GAD67 could be a key diabetogenic autoantigen in the NOD mouse and the high prevalence of GAD65 autoantibodies in human type 1 diabetes, it became important to ask whether there is also immune reactivity to GAD25 in type 1 diabetes-possibly implicating it in the pathogenesis of the disease-and whether GAD25 reactivity could, like GAD65 reactivity, function as a clinically useful marker for the disease. We also hypothesized that the presence of autoantibodies to the smaller splice variant could be a cause of the up to 30% prevalence of GAD67 autoreactivity associated with type 1 diabetes. We therefore analyzed GAD25 reactivity in 105 newly-diagnosed children with type 1 diabetes and 74 control subjects. While 14 (13%) of the diabetic subjects were positive for GAD67 autoantibodies, only 3 (3%) were positive for GAD25 reactivity, none of which were GAD67 antibody-positive. Analysis of reactivity to a GAD67 chimera was consistent with GAD67 binding activity being due to cross-reactive GAD65 antibodies. Immunostaining confirmed the presence of GAD25 in human islets, revealing GAD25-positive cells to be sparse. Our results indicate that autoreactivity to GAD25 is rare in newly diagnosed type 1 diabetes and does not underlie GAD67 reactivity.
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PMID:Immune reactivity to GAD25 in type 1 diabetes mellitus. 1251 88

The smaller isoform of the enzyme glutamic acid decarboxylase (GAD65) is a major islet autoantigen in autoimmune type 1 diabetes mellitus (T1DM). Transgenic plants expressing human GAD65 (hGAD65) are a potential means of direct oral administration of the islet autoantigen in order to induce tolerance and prevent clinical onset of disease. We have previously reported the successful generation of transgenic tobacco and carrot that express immunoreactive, full-length hGAD65. In the present study, we tested the hypothesis that the expression levels of recombinant hGAD65 in transgenic plants can be increased by targeting the enzyme to the plant cell cytosol and by mediating expression through the potato virus X (PVX) vector. By substituting the NH2-terminal region of hGAD65 with a homologous region of rat GAD67, a chimeric GAD67(1-87)/GAD65(88-585) molecule was expressed in transgenic tobacco plants. Immunolocalization analysis showed that immunoreactive GAD67/65 was found in the plant cell cytosol. By using a radio-immuno assay with human serum from a GAD65 autoantibody-positive T1DM patient, the highest expression level of the recombinant GAD67/65 protein was estimated to be 0.19% of the total soluble protein, compared to only 0.04% of wild-type hGAD65. Transient expression of wild-type, full-length hGAD65 in N. benthamiana mediated by PVX infection was associated with expression levels of immunoreactive protein as high as 2.2% of total soluble protein. This substantial improvement of the expression of hGAD65 in plants paves the way for immunoprevention studies of oral administration of GAD65-containing transgenic plant material in animal models of spontaneous autoimmune diabetes.
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PMID:Improved in planta expression of the human islet autoantigen glutamic acid decarboxylase (GAD65). 1273 88

Of the two homologous forms of glutamic acid decarboxylase, GAD65 and GAD67, only GAD65 is a common target of autoimmunity. Epitope profiles of autoantibodies to GAD65 (GADA) in 140 type 1 diabetes, adult-onset diabetes mellitus (AODM), and thyroid diseases (TD) were studied. Probes were GAD65, GAD65/67 hybrids (displaying separately GAD65 residues 1-95, 96-444, and 445-585), delta GAD65 (a truncated GAD65 spanning residues 69-585), and GAD67. delta GAD65 and GAD65 detected 137 and 125 positive patients, respectively. The hybrids reacted with 113 sera and in 3 cases disclosed cryptic epitopes. Eighteen patients reacted with GAD67, indicating GAD65-GAD67 cross-reactivity. Most patients recognized both middle and C-terminal epitopes, had low reactivity against N-terminal epitopes, and seldom displayed reactivity limited to the N or C terminus. Compared with type 1 and AODM, TD patients showed a greater prevalence of multiple reactivity and higher incidence of GAD67 positivity.
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PMID:Engineered variants of human glutamic acid decarboxylase (GAD) and autoantibody epitope recognition. 1286 69

Ketosis-prone diabetes is heterogeneous. Its causes could include novel beta-cell functional defects. To characterize such defects, 103 patients with diabetic ketoacidosis were evaluated for beta-cell autoimmunity and human leukocyte antigen (HLA) class II alleles, with longitudinal measurements of beta-cell function and biochemical and clinical parameters. They were classified into four A beta groups, based on the presence of glutamic acid decarboxylase (GAD)65, GAD67, or IA-2 autoantibodies (A+ or A-) and beta-cell functional reserve (beta+ or beta-). The group distribution was: 18 A+beta-, 23 A-beta-, 11 A+beta+, and 51 A-beta+. Collectively, the two beta- groups differed from the two beta+ groups in earlier onset and longer duration of diabetes, lower body mass index, less glycemic improvement, and persistent insulin requirement. HLA class II genotyping showed that the A-beta- group differed from the A+beta- group in having lower frequencies of two alleles strongly associated with autoimmune type 1 diabetes susceptibility: DQA*03 and DQB1*02. Similarly, the A-beta+ group differed from the A+beta+ group in having a lower frequency of DQB1*02. Ketosis-prone diabetes comprises at least four etiologically distinct syndromes separable by autoantibody status, HLA genotype, and beta-cell functional reserve. Novel, nonautoimmune causes of beta-cell dysfunction are likely to underlie the A-beta+ and A-beta- syndromes.
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PMID:Ketosis-prone diabetes: dissection of a heterogeneous syndrome using an immunogenetic and beta-cell functional classification, prospective analysis, and clinical outcomes. 1460 30

Induction of specific immunological unresponsiveness by feeding protein antigens is termed oral tolerance and may be a potential therapy for autoimmune diseases. Whereas oral tolerance therapy may be both simple and effective, the requirement for large amounts of protein will limit clinical testing of autoantigens, which are difficult to produce. We have previously demonstrated transgenic plant production and direct oral delivery of a beta cell autoantigen murine GAD67 to prevent autoimmune diabetes in nonobese diabetic mice. Mucosal adjuvants such as cholera toxin B subunit may lower the level of autoantigen required, but the development of neutralizing mucosal antibody responses may limit usefulness in enhancing long-term oral tolerance. IL-4, being an endogenous protein, would avoid this result and possibly enhance oral tolerance but has not been tested as a mucosal adjuvant. In this study, human GAD65 (hGAD65), as well as murine IL-4, was expressed in transgenic plants for feeding trials. Both IL-4 and hGAD65 plant tissue were required to protect nonobese diabetic mice from diabetes, and no benefit was found if either was used alone. Combined therapy enhanced levels of IgG1 anti-GAD antibodies, increased splenocyte IL-4/IFN-gamma cytokine responses, and produced protective regulatory T cells. These results demonstrate that orally administered plant IL-4 remains biologically active and is synergistic when given with hGAD65 in inducing robust oral immune tolerance. Using transgenic plants expressing IL-4 and GAD65 may be a novel clinical approach to the prevention of human type 1 diabetes by oral tolerance.
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PMID:Induction of oral tolerance to prevent diabetes with transgenic plants requires glutamic acid decarboxylase (GAD) and IL-4. 1505 61

Glutamic acid decarboxylase (GAD) is an autoantigen in stiff man syndrome (SMS) and type 1 diabetes (T1DM). Different GAD autoantibody characteristics in these disorders have suggested distinct underlying mechanisms of autoimmunity. Here, it is shown that increased prevalence of autoantibodies to GAD65 amino terminal and GAD67 epitopes and autoantibodies of IgG2, IgG3, or IgG4 subclass in patients with SMS (P < 0.001 vs. T1DM) are secondary to the markedly higher autoantibody titers in SMS patients (P < 0.0001) and that autoantibody epitopes and subclasses were similar when patients were matched for autoantibody titer. Exposure to autoantigen in the disorders is likely to involve similar humoral antigenic determinants, but different B cell regulation.
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PMID:Humoral autoimmune responses to glutamic acid decarboxylase have similar target epitopes and subclass that show titer-dependent disease association. 1602 42

Glutamate decarboxylase (GAD), which has two isoforms, GAD65, and GAD67, is responsible for synthesis of the major inhibitory neurotransmitter, gamma-aminobutyric acid. GAD is expressed predominantly in the central nervous system; recent reports suggest that GAD is also expressed in non-neuronal organs including the pancreas. In the pancreatic islets, GAD serves as one of the autoantigens in type I diabetes mellitus. Recent flow cytometric analyses have shown that a variety of self-antigens, including GAD, are ectopically transcribed and expressed in particular cell populations of the thymus, although consensus concerning the cellular phenotype has not been obtained. The aim of this study was to clarify the localization and cellular phenotype of GAD67-expressing cells in the thymus at a cellular level with a novel approach using GAD67-green fluorescent protein (GFP) knock-in mice, in which GFP is expressed specifically in GAD67-positive cells. GFP-positive cells were detected in the thymic medulla and were identified as epithelial cells by immunohistochemistry. Almost all GFP-positive cells were positive for major histocompatibility complex (MHC) class II antigen staining and were positive for both cytokeratin and Ulex Europaeus Agglutinin I, markers of medullary thymic epithelial cells, but were negative for CD11c, Gr-1, and CD45, markers of dendritic cells, macrophages, and B-lymphocytes, respectively.
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PMID:Antigen-presenting cells expressing glutamate decarboxylase 67 were identified as epithelial cells in glutamate decarboxylase 67-GFP knock-in mouse thymus. 1657 56

Autoantibodies to the 65kDa isoform of glutamate decarboxylase (GAD65) are associated with type I diabetes and recognise highly conformational epitope(s) that remain to be defined. The human recombinant Fab from mAb b96.11 inhibits binding of most GAD65 antibody positive sera from patients and its epitope has previously been localized to the middle region of GAD65. Recent studies indicate that b96.11 antibody specificity predicts the risk of developing type 1 diabetes in prediabetic individuals. We describe the use homology modelling, protein-protein docking simulations and biopanning of random peptide phage displayed libraries with b96.11 to predict contact amino acids on the interface of GAD65/Fab b96.11 complex. Further analysis by in vitro mutagenesis of GAD65 followed by radioimmunoprecipitation refined the amino acids contributing to the b96.11 epitope. Our studies show an interface characterized by a protruding antibody-combining site centered on the long heavy chain CDR3 loop of Fab b96.11 establishing interactions with the critical residue Phe(344) in the core of the epitope on GAD65, surrounded by charged sites within (375)RK(376) and (305)DER(307). The epitope requires residues from both middle and the C-terminal domains, and is the first precise definition of an epitope on GAD65. The nature of the b96.11 epitope leads to considerations of potential structural variations for differences in antigenicity between the isoforms GAD65 and GAD67. The study shows the utility of using a combination of in silico techniques and experimental data for molecular characterization and localization of conformational epitopes for which crystal structures are lacking.
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PMID:Molecular characterization of a disease associated conformational epitope on GAD65 recognised by a human monoclonal antibody b96.11. 1693 Jul 8

The 65 kDa isoform of human glutamate decarboxylase (GAD65) is a major autoantigen in type 1 diabetes (T1D). In the present study, we have developed a sensitive sandwich time-resolved fluorescence immunoassay (TRFIA) for the quantification of GAD65 in cell extracts, cell media and serum. The monoclonal antibody GAD-6 is used to selectively capture GAD65 but not the slightly larger isoform GAD67, and the utilization of different detecting antibodies with distinct GAD65 epitope specificity allows modulating the specificity of the assay. To this effect we have biotinylated a recombinant antigen-binding fragment (rFab) with epitope specificity for the N-terminal region of rat and human GAD65 (rFab N-GAD65) and another rFab that selectively binds to the middle part of human GAD65 (rFab b96.11). In the assay the biotinylated rFabs are recognized by Europium labeled streptavidin. The obtained time-resolved fluorescence (TRF) is directly proportional to the concentration of GAD65 over a large measuring range (0.1 to >100 ng/mL). Based on total error estimation including both bias and imprecision, the lower limit of quantitation (LLOQ) of GAD65 in cell extracts is 0.33 ng/mL with the N-GAD65 TRFIA, and 0.10 ng/mL with the b96.11 TRFIA, but the latter is suitable for human GAD65 only, whereas the N-GAD65 TRFIA has equal sensitivity with rat and human GAD65. Specificity was further checked with GAD65/67 fusion proteins, confirming that the presence of intact capture as well as detection epitope on the analyte is a prerequisite for recognition in both assays. We show that the beta cell-specific marker GAD65 can be quantified in pancreatic cell extracts and in serum, allowing studies on discharge during cell death in vitro as well as in vivo.
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PMID:Species and epitope specificity of two 65 kDa glutamate decarboxylase time-resolved fluorometric immunoassays. 1721 Jan 61


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