Islet Cell Autoantigen Research Articles

Do local immune-neuroendocrine disturbances initiate diabetes?

It has been suggested that there exists a local immune-neuroendocrine self-regulating system in the pancreas. The system consists of beta-cells, nerve ganglia, intercellular fluid, connective tissue, and endothelial and immunocompetent cells. The local immune-neuroendocrine system governs the background level of insulin production by intrinsic mechanisms both in normal conditions and in a recovery period after different kinds of stress. The activity of this system by a complex of metabolic, environmental, nerve, and nonspecific immune factors has been determined. The local immune-neuroendocrine system is partially autonomous as a result of local integrative nerve circuits, morphological and functional substrates. Increased or decreased synthesis and release of some cytokines or biologically active substances (neurotransmitters, neuropeptides, gamma-aminobutyric acid, metabolites, nitric oxide, ions, etc.) by various cell types in the local immune-neuroendocrine system above usual levels may result in disturbances of sensitivity and functions of beta-cells. If the capability of the local immune-neuroendocrine system is insufficient for their compensation, the islet cell autoantigens may occur, the specific immune mechanisms are involved, and the pathological process becomes irreversible. Some ways for prevention of disturbances in the local immune-neuroendocrine system during the early and late phases of diabetes are presented.

Do local immune-neuroendocrine disturbances initiate diabetes?

It has been suggested that there exists a local immune-neuroendocrine self-regulating system in the pancreas. The system consists of beta-cells, nerve ganglia, intercellular fluid, connective tissue, and endothelial and immunocompetent cells. The local immune-neuroendocrine system governs the background level of insulin production by intrinsic mechanisms both in normal conditions and in a recovery period after different kinds of stress. The activity of this system by a complex of metabolic, environmental, nerve, and nonspecific immune factors has been determined. The local immune-neuroendocrine system is partially autonomous as a result of local integrative nerve circuits, morphological and functional substrates. Increased or decreased synthesis and release of some cytokines or biologically active substances (neurotransmitters, neuropeptides, gamma-aminobutyric acid, metabolites, nitric oxide, ions, etc.) by various cell types in the local immune-neuroendocrine system above usual levels may result in disturbances of sensitivity and functions of beta-cells. If the capability of the local immune-neuroendocrine system is insufficient for their compensation, the islet cell autoantigens may occur, the specific immune mechanisms are involved, and the pathological process becomes irreversible. Some ways for prevention of disturbances in the local immune-neuroendocrine system during the early and late phases of diabetes are presented.

Vesicular autoantigens of type 1 diabetes.

Type 1 diabetes, also referred to as insulin-dependent diabetes mellitus (IDDM), is an autoimmune disorder resulting from the destruction of pancreatic beta-cells and insulin deficiency. In the last 10 years significant progress has been made in this field, primarily because of the identification of predisposing genes, the extensive investigation of animal models, and the characterization of major autoantigens. This review draws attention to how the study of beta-cell autoantigens may contribute insight into the pathogenesis of IDDM and provides an update on the cell biology of glutamic acid decarboxylase (GAD) and islet cell autoantigen 512, two major targets of autoimmunity in Type 1 diabetes on which I have focused my efforts. For reasons of space I have mostly considered here studies on GAD which have been published since 1994.

Anti-BSA Antibodies do not Cross-react with the 69-kDa Islet Cell Autoantigen ICA69

In contrast to several previously published reports, we demonstrate by a variety of antibody assays that bovine serum albumin (BSA) is not antigenically cross-reactive with the 69-kDa islet cell autoantigen (ICA69). Fast protein liquid chromatography purified BSA and highly purified recombinant human ICA69 were used to establish sensitive Western blot and ELISA assays in order to detect antibodies against these two proteins. The assays excluded BSA or powdered milk as blocking agents, since these would interfere with antibody binding. A panel of sera from diabetic individuals, first degree relatives, and normal controls showed that the majority (∼70%) of individuals from each group had antibodies against ICA69 as assayed by Western blots, whereas considerably fewer (∼13%) had anti-BSA antibodies on Western blots, and individuals with antibodies to both proteins occurred only rarely (2–3%). To explore this issue further we immunized a total of 16 individual rats, representing four different strains (bio-breeding diabetes resistant and diabetes prone, Wistar-Furth, and Sprague-Dawley), with either BSA (n=2 of each strain) or with recombinant ICA69 (n=2 of each strain), and for each animal pre- and postimmune antibody titres against BSA and against ICA69 were assayed separately by enzyme-linked immunoabsorbent assay. In rats immunized with BSA, anti-BSA titres increased about 100,000-fold over preimmune levels, whereas anti-ICA69 reactive antibodies were unchanged over preimmune levels. Similarly, in rats immunized with ICA69, anti-ICA69 titres rose about 100,000-fold over preimmune levels, whereas anti-BSA antibodies were unchanged over preimmune levels. Thus we find no evidence for the existence of antibody cross-reactivity between ICA69 and BSA, either in humans or in immunized rats. When our rat anti-BSA antisera were used to probe Western blots made from rat islets isolated in the strict absence of fetal calf serum, we were unable to detect a 69-kDa band, even when the islets were preincubated with γ-interferon, a treatment which has been reported to induce the BSA cross-reactive islet antigen. We conclude that BSA is not antigenically cross-reactive with ICA69 at the antibody level, and it is highly unlikely that BSA is antigenically cross-reactive with some other 69kD islet cell antigen.

IA-2 and IA-2beta: the immune response in IDDM.

Pancreatic islet cell autoantigens associated with insulin-dependent diabetes mellitus (IDDM) include a recently identified family of protein tyrosine phosphatase-like molecules, notably IA-2 and IA-2beta. IA-2 is a 979 amino acid transmembrane protein located on human chromosome 2q35, whereas IA-2beta is 986 amino acids long located on human chromosome 7q36. Comparison of human IA-2 and IA-2beta showed 74% identity within the intercellular domains, but only 27% indentify within the extracellular domains. These IA-2 molecules are expressed predominantly in cells of neuroendocrine origin, particularly pancreatic islets and brain. Radioimmunoprecipitation with recombinant IA-2 and IA-2beta has been used to measure autoantibodies to these molecules and their intracellular fragments. Autoantibodies to IA-2 are detected in the majority (60% to 80%) of newly diagnosed IDDM patients and in less than 2% of controls. The major antigenic determinants of both IA-2 and IA-2beta reside within the C-terminus of their intracellular domains. In first-degree relatives of IDDM patients, the presence of autoantibodies to IA-2 is predictive of IDDM and in combination with autoantibodies to glutamic acid decarboxylase (GAD) the positive predictive value is in the 50% range. The role of IA-2 and IA-2beta in the pathogenesis of IDDM is still unclear. Identification of these antigens has extended our ability to predict the disease and may be valuable in the search for antigen-specific therapies to prevent IDDM.

B-Cells Are Required for the Initiation of Insulitis and Sialitis in Nonobese Diabetic Mice

Nonobese diabetic (NOD) mice spontaneously develop an acute onset of hyperglycemia reminiscent of human type I diabetes. The disease is the end result of a mononuclear cell infiltration of pancreatic islets (insulitis), culminating in the selective destruction of islet beta-cells by autoreactive T-cells. NOD mice also exhibit defects in B-cell tolerance as manifested by the presence of autoantibodies against islet cell autoantigens. Based on the potential ability of B-cells to act as antigen presenting cells, we hypothesized that autoreactive B-cells of NOD mice may be necessary for the activation of islet reactive CD4+ T-cells. In the present study, we utilized an anti-mu antibody to induce in vivo depletion of B-cells and found that B-cell depletion completely abrogates the development of insulitis and sialitis in NOD mice. In contrast, control IgG-treated NOD mice developed insulitis and sialitis by 5 weeks of age. Additionally, the discontinuation of anti-mu chain antibody treatment led to the full restoration of the B-cell pool and the reappearance of insulitis and sialitis. Thus, we conclude that B-cells are a requisite cell population for the genesis of the inflammatory lesions of the islets of Langerhans. This finding suggests that autoreactive B-cells may act as the antigen presenting cells necessary for the initial activation of beta-cell-reactive CD4+ T-cells implicated in the pathogenesis of autoimmune diabetes.

A cholera toxoid-insulin conjugate as an oral vaccine against spontaneous autoimmune diabetes

Mucosally induced immunological tolerance is an attractive strategy for preventing or treating illnesses resulting from untoward inflammatory immune reactions against self- or non-self-antigens. Oral administration of relevant autoantigens and allergens has been reported to delay or suppress onset of clinical disease in a number of experimental autoimmune and allergic disorders. However, the approach often requires repeated feeding of large amounts of tolerogens over long periods and is only partly effective in animals already systemically sensitized to the ingested antigen such as in animals already harboring autoreactive T cells, and thus presumably also in humans with an autoimmune disease. We have recently shown that oral administration of microgram amounts of antigen coupled to cholera toxin B subunit (CTB), can effectively suppress systemic T cell reactivity in naive as well as in immune animals. We now report that feeding small amounts (2-20 microg) of human insulin conjugated to CTB can effectively suppress beta cell destruction and clinical diabetes in adult nonobese diabetic (NOD) mice. The protective effect could be transferred by T cells from CTB-insulin-treated animals and was associated with reduced lesions of insulitis. Furthermore, adoptive co-transfer experiments involving injection of Thy-1,2 recipients with diabetogenic T cells from syngeneic mice and T cells from congenic Thy-1,1 mice fed with CTB-insulin demonstrated a selective recruitment of Thy-1,1 donor cells in the peripancreatic lymph nodes concomitant with reduced islet cell infiltration. These results suggest that protection against autoimmune diabetes can be achieved by feeding minute amounts of a pancreas islet cell autoantigen linked to CTB and appears to involve the selective migration and retention of protective T cells into lymphoid tissues draining the site of organ injury.

Immunological Aspects of Nutritional Diabetes Prevention in NOD Mice: A Pilot Study for the Cow's Milk–Based IDDM Prevention Trial

Human epidemiological studies delineated early exposure to intact dietary protein (e.g., most infant formulas) as an environmental risk factor for the development of IDDM. The Trial to Reduce IDDM in the Genetically at Risk (TRIGR), an international IDDM prevention trial, has been designed to determine if avoidance of intact dairy protein in high-risk infants < or =6 months of age can reduce the subsequent diabetes incidence. We here studied the casein hydrolysate-based trial diet (Nutramigen) in NOD mice. When given either continuously or for 10 weeks after weaning, the test diet was highly effective in preventing autoimmune diabetes (32-week incidence: 4.6 vs. 58.8%) and in preserving pancreatic insulin levels, with little effect on islet inflammation. Spleen cells from protected NOD mice failed to adoptively transfer diabetes into irradiated syngeneic recipients. When co-transferred with splenocytes from diabetic donors, cells from diet-protected mice inhibited adoptive diabetes transfer (incidence 50 vs. 94%, P < 0.001). T-cell reactivity to the islet cell autoantigens ICA69 (islet cell antigen 69) and GAD65 developed only in diabetic recipients of spleen cell grafts, indicating that diabetes protection extends to more than one autoantigen. In protected mice, ICA69 T-cell reactivity was not detectable spontaneously nor after priming with this autoantigen; however, priming with the cross-reactive non-self-antigen bovine serum albumin recruited T-cells responsive to ICA69. Thus, diabetes prevention with the clinical trial diet is effective in NOD mice, where it affects some T-cell repertoires and allows development of regulatory cells that interfere with destructive autoimmunity.

The insulin gene is transcribed in the human thymus and transcription levels correlated with allelic variation at the INS VNTR-IDDM2 susceptibility locus for type 1 diabetes.

Type 1, or insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease associated with loss of tolerance to several pancreatic islet cell molecules, including insulin, glutamic acid decarboxylase (GAD), ICA69 and the tyrosine phosphatase IA-2 (refs 1-3). Among several predisposing loci, IDDM2 maps to the insulin gene (INS) VNTR (variable number of tandem repeats) minisatellite on chromosome 11p15 (refs 4-9). Allelic variation at this VNTR locus correlates with steady-state levels of INS mRNA in pancreas and transfected rodent cell lines, but it is difficult to reconcile the association of lower INS mRNA levels in the pancreas with class III VNTRs that are dominantly protective from IDDM. We show that during fetal development and childhood, mRNAs for insulin and other islet cell autoantigens (GAD, ICA69, IA-2) are expressed at low levels in the human thymus. Critically, we also detect proinsulin and insulin protein. VNTR alleles correlate with differential INS mRNA expression in the thymus where, in contrast to the pancreas, protective class III VNTRs are associated with higher steady-state levels of INS mRNA expression. This finding provides a plausible explanation for the dominant protective effect of class III VNTRs, and suggests that diabetes susceptibility and resistance associated with IDDM2 may derive from the VNTR influence on INS transcription in the thymus. Higher levels of (pro)insulin in the thymus may promote negative selection (deletion) of insulin-specific T-lymphocytes which play a critical role in the pathogenesis of type-1 diabetes.

An Islet-Cell Protein Tyrosine Phosphatase Is a Likely Precursor to the 37-kDa Autoantigen in Type 1 Diabetes: Human and Macaque Sequences, Tissue Distribution, Unique and Shared Epitopes, and Predictive Autoantibodies

We sought to identify novel islet-cell autoantigens to better understand the pathogenesis, prediction, and immunotherapy of type 1 diabetes. Macaque and human islet cDNA libraries expressed in mammalian cells were screened with human diabetes sera. A positive clone was sequenced directly and after 5' rapid amplification of cDNA ends (RACE). Northern blotting and in situ hybridization revealed the tissue distribution of the corresponding protein. Antigen, expressed by in vitro translation, and tryptic peptides were analyzed by SDS-PAGE. For the immunoprecipitations, 183 diabetic, 60 prediabetic, and 91 control sera were used. Truncated antigens were used in immunoprecipitations for epitope mapping. Recombinant antigen expressed in transfected fibroblasts was used in competition assays. Sequencing yielded an 111-kDa, 1,013 amino acid, transmembrane protein (M1851) containing consensus protein tyrosine phosphatase (PTPase) sequence. M1851 was 77% identical in the intracellular domain, but only 31% identical extracellularly, to the islet-cell autoantigen ICA512. mRNA localized to brain, prostate, pancreatic islets, and adrenal medulla. After limited trypsinization, the in vitro translated antigen was 37 kDa. M1851 was recognized by 47% of prediabetes sera, 31% of new diabetes sera, but only 1% of healthy controls. Only 1/73 sera binding M1851 failed to bind ICA512, whereas 42/114 binding ICA512 did not bind M1851. M1851 reactivity was not fully displaced by ICA512 in 24/49 sera. Removing the C-terminal 27, 80, or 160 amino acids of M1851 decreased reactivity by 70%, 90%, and 100%, respectively. This new islet-cell PTPase is likely to be the precursor to the 37-kDa tryptic fragment antigen. It is structurally related to ICA512 but has distinct diabetes autoantibody epitopes located at the C terminus.

Value of ICA512 antibodies for prediction and diagnosis of type 1 diabetes

Islet cell autoantigen (ICA) 512 of type I diabetes is a receptor tyrosine phosphatase-like protein associated with the secretory granules of neurons and endocrine cells including insulin-secreting β-cells of the pancreas. Here we show that in a yeast two-hybrid assay its cytoplasmic domain binds β2-syntrophin, a modular adapter which in muscle cells interacts with members of the dystrophin family including utrophin, as well as the signaling molecule neuronal nitric oxide synthase (nNOS). The cDNA isolated by two-hybrid screening corresponded to a novel β2-syntrophin isoform with a predicted molecular mass of 28 kDa. This isoform included the PDZ domain, but not the C-terminal region, which in full-length β2-syntrophin is responsible for binding dystrophin-related proteins. In vitro binding of the β2-syntrophin PDZ domain to ICA512 required both ICA512′s C-terminal region and an internal polypeptide preceding its tyrosine phosphatase-like domain. Immunomicroscopy and co-immunoprecipitations from insulinoma INS-1 cells confirmed the occurrence of ICA512-β2-syntrophin complexes in vivo. ICA512 also interacted in vitro with the PDZ domain of nNOS and ICA512-nNOS complexes were co-immunoprecipitated from INS-1 cells. Finally, we show that INS-1 cells, like muscle cells, contain β2-syntrophin-utrophin oligomers. Thus, we propose that ICA512, through β2-syntrophin and nNOS, links secretory granules with the actin cytoskeleton and signaling pathways involving nitric oxide.

ICA 512, a Receptor Tyrosine Phosphatase-Like Protein, Is Concentrated in Neurosecretory Granule Membranes

Publisher Summary Islet cell autoantigen (ICA) 512 is a specific membrane marker of neurosecretory granules. It is the first member of the receptor protein tyrosine phosphatases (RPTPs) family found to be resident in an intracellular compartment. Interestingly, ICA 512 contains only one protein tyrosine phosphatase (PTP) “core domain”, rather than two, as usually found in receptor PTPs (RPTPs). Moreover, ICA 512 expressed in bacteria do not display PTP activity when tested with several common PTP substrates. Thus, the function of ICA 512 is still unknown. By Northern blot, ICA 512 transcripts have been detected in pancreatic islets, brain, and pituitary, suggesting an enrichment of ICA 512 in neuroendocrine tissues. To begin addressing the structure and function of ICA 512, its tissue distribution and intracellular localization using rabbit antibodies directed against either its recombinant extracellular domain or recombinant cytoplasmic domain. Confocal microscopy on rat tissues demonstrated that ICA 512 is expressed in virtually all neuroendocrine cells, including neurons of the autonomic nervous system; chromaffin cells of the adrenal medulla; α-, β-, and δ-pancreatic islet cells; cells in the anterior and intermediate pituitary, as well as neurons of the autonomic nervous system and of the hypothalamus and the amygdala in the brain. The strongest ICA 512 immunoreactivity has been found in the posterior pituitary, which contains the nerve endings of neurons located in the hypothalamus and the highest concentration of neurosecretory granules in the entire body. Thus ICA 512 and, thus, tyrosine phosphorylation–dephosphorylation plays a role in the life-cycle of neurosecretory granules.

Treatment with GAD65 or BSA Does Not Protect Against Diabetes in BB Rats

The Mr 65,000 isoform of glutamic acid decarboxylase (GAD65) has been implicated as the initiating islet cell antigen in the pathogenesis of diabetes, primarily based on studies in non-obese diabetic (NOD) mice. To test the role of this islet cell autoantigen in the pathogenesis of spontaneously occurring diabetes in another animal model, purified recombinant human islet GAD65 was injected i.v. at 200 μg/animal into 18-day-old diabetes-prone BB rats. For controls, bovine serum albumin (BSA), which has also been implicated in the pathogenesis of diabetes, or buffer alone was injected into age matched BB rats. At 210 days of age there were no differences in diabetes incidence in the 3 groups, i.e. 73% (11 of 15) in the GAD65-treated, 81% (13 of 16) in the BSA-treated and 65% (11 of 17) in the buffer-treated animals, or in the median age at onset of disease, i.e. 79 days (range 65-111), 87 days (range 60–107) and 86 days (range 74–109), respectively. The lack of protection against diabetes following GAD65 treatment could hypothetical be explained by no or by an aberrant expression of GAD in BB-rat islet cells. However, immunohistochemistry of pancreata and immunoblot-ting analysis of isolated islets showed that the expression of GAD65 and GAD67 was similar in BB and Lewis rats.In conclusion, these data indicate that neither GAD65 nor BSA autoimmunity is important for the development of diabetes in BB rats, in contrast to the situation in NOD mice, and further emphasizes that extrapolation from only one animal model to autoimmune diabetes in general may not be appropriate.

Association of Exposure to Cow's Milk Protein and β-Cell Autoimmunity

To the Editor. —In another retrospective epidemiologic study of possible links between insulin-dependent diabetes mellitus (IDDM) and early infant-feeding history, Dr Norris and colleagues 1 have used a novel approach in analyzing not diabetic children, but 18 probands considered to be at risk for diabetes development as defined by the presence of 1 (n=14) or more (n=4) antibodies to insulin (78%), glutamic acid decarboxylase (GAD 65 ), or islet cell autoantigen (ICA) 512 combined with insulinoma-associated islet tyrosine phosphatases autoantibody (IA-2). The authors compared retrospective questionnaires to obtain early infant-feeding histories and found no differences between the probands and 153 unrelated comtrols. They conclude that diabetes risk is not linked to any nutritional antigens derived from either plant or animal sources. Based on the analysis of their very small cohort of children that may (or may not) develop diabetes, the authors reject similar studies that collectively analyzed more than 1000 diabetic

ICAAR, a Novel Member of a New Family of Transmembrane, Tyrosine Phosphatase-like Proteins

We have isolated a cDNA from human foetal brain cDNA library which encodes a putative transmembrane protein bearing an intracellular protein tyrosine phosphatase (PTPase) like domain. The PTPase like domain contains an alanine to aspartate amino acid change relative to other PTPases in the catalytic core domain. This amino acid change is found in only three other known proteins, islet cell autoantigens; human, murine and rat IA-2, murine IA-2b and its rat orthologue phogrin, which have a similar overall structure to ICAAR, and the recently identified X-linked myotubular myopathy (MTM1) gene. ICAAR, IA-2 and IA-2b clearly represent a new family of PTP-like proteins for which catalytic activity has yet to be demonstrated. An abundant ICAAR mRNA is detectable in the brain and pancreas but not in the other normal human tissues surveyed. We have localised ICAAR to human chromosome 7q36.

Immune reactivity of diabetes-associated human monoclonal autoantibodies defines multiple epitopes and detects two domain boundaries in glutamate decarboxylase.

Autoreactive islet cell Abs (ICA) accompany the pathogenic destruction of pancreatic beta cells in insulin-dependent diabetes mellitus (IDDM). Human monoclonal ICA (MICA 1-6), previously derived from a DR1/DR7-positive newly diagnosed diabetic patient, recognized the islet cell autoantigen glutamate decarboxylase 65 (GAD65) and defined two distinct conformational (MICA 1/3 and MICA 4/6) and one linear (MICA 2) autoimmune epitopes in this molecule. We have isolated 4 new ICA-reactive B cell lines, one from a DR4/DR11-positive newly diagnosed IDDM patient (MICA 7) and three from a DR3 homozygous patient with both IDDM and Graves' disease (MICA 8-10). Like MICA 1-6, MICA 7-10 are specific for GAD65, suggesting that GAD65-reactive B cells dominate the ICA response in IDDM. Comparative analysis of MICA 1-6 and MICA 7-10, using GAD65 mutants and blocking experiments, showed that MICA 7-10 define three novel conformational autoimmune epitopes in GAD65. Further structural analysis of the MICA 1-10 epitopes revealed two distinct and one overlapping region of epitope clusters. Thus, the C-terminal region, defined by amino acids 450 to 570, harbors the conformational MICA1/3 and MICA 7 epitopes as well as the linear epitope of MICA 2 (amino acids 506-531). The MICA 4/6 and MICA 10 epitopes are located in the middle region of the molecule defined by amino acids 245 to 449, whereas the N-terminal region contributes only to the MICA 8/9 epitopes (encompassed in amino acids 39-585). MICA 1-6, 7, and 8-10, derived from three IDDM patients of different HLA haplotypes, define six different epitopes in GAD65 and represent tools to determine the spectrum, possible HLA association, and temporal order of epitope recognition in IDDM.

Cloning and Characterization of Islet Cell Antigen-related Protein-tyrosine Phosphatase (PTP), a Novel Receptor-like PTP and Autoantigen in Insulin-dependent Diabetes

Cloning of the cDNA encoding a novel human protein- tyrosine phosphatase (PTP) called islet cell antigen-related PTP (IAR) predicts a receptor-like molecule with an extracellular domain of 614 amino acids containing a hydrophobic signal peptide, one potential N-glycosylation site, and an RGDS peptide which is a possible adhesive recognition sequence. The 376-amino acid intracellular region contains a single catalytic domain. Recombinant IAR polypeptide has phosphatase activity. Northern blot analysis shows tissue-specific expression of two IAR transcripts of 5.5 and 3. 7 kilobases, which are most abundant in brain and pancreas. The IAR PTP is homologous in its intracellular region to IA-2, a putative PTP that is an insulin-dependent diabetes mellitus (IDDM) autoantigen. IAR is also reactive with IDDM patient sera. IAR and IA-2 may distinguish different populations of IDDM autoantibodies since they identify overlapping but nonidentical sets of IDDM patients. Thus IAR is likely to be an islet cell antigen useful in the preclinical screening of individuals for risk of IDDM.

IA-2, a transmembrane protein of the protein tyrosine phosphatase family, is a major autoantigen in insulin-dependent diabetes mellitus.

IA-2 is a 105,847 Da transmembrane protein that belongs to the protein tyrosine phosphatase family. Immunoperoxidase staining with antibody raised against IA-2 showed that this protein is expressed in human pancreatic islet cells. In this study, we expressed the full-length cDNA clone of IA-2 in a rabbit reticulocyte transcription/translation system and used the recombinant radiolabeled IA-2 protein to detect autoantibodies by immunoprecipitation. Coded sera (100) were tested: 50 from patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM) and 50 from age-matched normal controls. Sixty-six percent of the sera from patients, but none of the sera from controls, reacted with IA-2. The same diabetic sera tested for autoantibodies to islet cells (ICA) by indirect immunofluorescence and glutamic acid decarboxylase (GAD65Ab) by depletion ELISA showed 68% and 52% positivity, respectively. Up to 86% of the IDDM patients had autoantibodies to IA-2 and/or GAD65. Moreover, greater than 90% (14 of 15) of the ICA-positive but GAD65Ab-negative sera had autoantibodies to IA-2. Absorption experiments showed that the immunofluorescence reactivity of ICA-positive sera was greatly reduced by prior incubation with recombinant IA-2 or GAD65 when the respective antibody was present. A little over one-half (9 of 16) of the IDDM sera that were negative for ICA were found to be positive for autoantibodies to IA-2 and/or GAD65, arguing that the immunofluorescence test for ICA is less sensitive than the recombinant tests for autoantibodies to IA-2 and GAD65. It is concluded that IA-2 is a major islet cell autoantigen in IDDM, and, together with GAD65, is responsible for much of the reactivity of ICA with pancreatic islets. Tests for the detection of autoantibodies to recombinant IA-2 and GAD65 may eventually replace ICA immunofluorescence for IDDM population screening.

Identification and characterization of glima 38, a glycosylated islet cell membrane antigen, which together with GAD65 and IA2 marks the early phases of autoimmune response in type 1 diabetes.

Immunoprecipitating IgG autoantibodies to glutamic acid decarboxylase, GAD65, and/or a tyrosine phosphatase, IA2, are present in the majority of individuals experiencing pancreatic beta cell destruction and development of type 1 diabetes. Here we identify a third islet cell autoantigen, a novel 38-kD protein, which is specifically immunoprecipitated with sera from a subset of prediabetic individuals and newly diagnosed type 1 diabetic patients. The 38-kD autoantigen, named glima 38, is an amphiphilic membrane glycoprotein, specifically expressed in islet and neuronal cell lines, and thus shares the neuroendocrine expression patterns of GAD65 and IA2. Removal of N-linked carbohydrates results in a protein of 22,000 Mr. Glima 38 autoantibodies were detected in 16/86 (19%) of newly diagnosed patients, including three very young children, who had a rapid onset of disease, and in 6/44 (14%) of prediabetic individuals up to several years before clinical onset. The cumulative incidence of GAD65 and glima 38 antibodies in these two groups was 83 and 80%, respectively, and the cumulative incidence of GAD65, glima 38, and IA2 antibodies in the same groups was 91 and 84%, respectively. GAD65, IA2, and glima 38 represent three distinct targets of immunoprecipitating IgG autoantibodies associated with beta cell destruction and type 1 diabetes.

ICA 512, an autoantigen of type I diabetes, is an intrinsic membrane protein of neurosecretory granules.

Islet cell autoantigen (ICA) 512 is a novel autoantigen of insulin-dependent diabetes mellitus (IDDM) which is homologous to receptor-type protein tyrosine phosphatases (++PTPases). We show that ICA 512 is an intrinsic membrane protein of secretory granules expressed in insulin-producing pancreatic beta-cells as well as in virtually all other peptide-secreting endocrine cells and neurons containing neurosecretory granules. ICA 512 is cleaved at its luminal domain and, following exposure at the cell surface, recycles to the Golgi complex region and is sorted into newly formed secretory granules. By immunoprecipitation, anti-ICA 512 autoantibodies were detected in 15/17 (88%) newly diagnosed IDDM patients, but not in 10/10 healthy subjects. These results suggest that tyrosine phosphorylation participates in some aspect of secretory granule function common to all neuroendocrine cells and that a subset of autoantibodies in IDDM is directed against an integral membrane protein of insulin-containing granules.