Insulinoma-associated Protein 2 Research Articles

Autoantibodies to CCL3 are of low sensitivity and specificity for the diagnosis of type 1 diabetes

Type 1 diabetes (T1D) is a T cell-dependent tissue-specific autoimmune disease, characterized by the selective destruction of the β cells of the pancreatic islets of Langerhans. Recently, contradictory findings have been reported about the relationship of autoantibodies to CC chemokine 3 (CCL3) and T1D, which need to be confirmed by more investigations in larger cohorts. The aim of our research was to investigate whether autoantibodies to CCL3 are useful markers for T1D in a large cohort of Chinese patients. We analyzed autoantibodies to CCL3, glutamic acid decarboxylase(GADA), insulinoma-associated protein-2 (IA-2A), and zinc transporter-8 (ZnT8A) by a radioimmunoprecipitation assay in 290 T1D subjects, 200 subjects with type 2 diabetes (T2D), 210 subjects with other diseases, and 178 healthy control subjects. Results showed that the frequencies of autoantibodies to CCL3 in subjects with T1D, T2D, and healthy control subjects were similar [3.10% (9/290), 2.50% (5/200), and 0.56% (1/178), respectively, P = 0.189]. Autoantibodies to CCL3 were not significantly different between T1D patients with or without GADA, IA-2A, or ZnT8A antibodies (2.7% vs. 3.9%, P = 0.725). In contrast, patients with systemic lupus erythematosus and rheumatoid arthritis showed higher positivity for autoantibodies to CCL3 than healthy control subjects [15.6% (5/32) and 12.5% (8/64) vs. 0.56% (1/178), all P = 0.000], and higher titer of autoantibodies to CCL3 than T1D patients (median 0.9633 and 0.4095 vs. 0.0873, P = 0.012 and P = 0.034, respectively). We conclude that autoantibodies to CCL3 are of low sensitivity and specificity for T1D and cannot be used in the diagnosis of T1D.

Latent Autoimmune Diabetes in Adults: Autoimmune Diabetes in Adults with Slowly Progressive β-cell Failure

Type 1 diabetes (T1D) is caused by interactions of genetic predisposition, immunologic triggers, and environmental events leading to autoimmune-mediated pancreatic β-cell destruction and insulin deficiency, although some subjects show lack of autoantibodies to pancreatic islets. Regarding the time lag of complete β-cell mass loss in T1D, simple or unique patterns may not be identified, with some individuals progressing for more than several years. Contrary to T1D, type 2 diabetes (T2D), irrelevant of autoimmune-mediated β-cell destruction, is a heterogeneous disorder characterized by insidious onset and insulin resistance with relative insulin secretory dysfunction. Clinically, some diabetic patients exhibit autoimmune antibodies without insulin requirement. This biphasic type of diabetes is a special form of diabetes that is clinically similar to the early stage of T2D and pathophysiologically defined as a disorder of the autoimmune-mediated progressive β-cell dysfunction. To clarify different features from T1D and T2D, Zimmet [1] introduced the eponym autoimmune diabetes of (LADA) to describe this subgroup of adult phenotypic T2D patients positive for autoantibodies. Based on the natural history of LADA, in which secretory β-cell dysfunction is prominently aggravated depending on the presence of antibodies seen in T1D, Stenstrom et al. [2] suggested LADA is not always a latent disease. Therefore, autoimmune diabetes in adults with slowly progressive β-cell failure might be a more adequate concept. The Immunology of Diabetes Society (IDS) has proposed criteria to standardize the definition of LADA: 1) ≥35 years of age, 2) positive for at least 1 of the 4 antibodies commonly seen in T1D patients; islet cell autoantibodies (ICA), anti-glutamic acid decarboxylase (anti-GAD), Insulinoma-associated protein-2 antibodies (IA-2A), and insulin autoantibodies (IAA), and 3) not requiring insulin therapy within the first 6 months after diagnosis [3].

Sorting nexin 19 regulates the number of dense core vesicles in pancreatic β-cells.

Aims/Introduction: Insulinoma-associated protein 2 (IA-2) regulates insulin secretion and the number of dense core vesicles (DCV). However, the mechanism of regulation of DCV number by IA-2 is unknown. We examined the effect of sorting nexin 19 (SNX19), an IA-2 interacting protein, on insulin secretion and the number of dense core vesicles (DCV). Stable SNX19 knockdown (SNX19KD) MIN6, a mouse pancreatic β-cell line, and stable SNX19-reintroduced SNX19KD MIN6 were established. Quantification of DCV, and lysosomes was carried out using electron micrographs. The half-life of DCV was detected by pulse-chase experiment. Insulin secretion and content were decreased in stable SNX19KD MIN6 cells compared with those in control MIN6 cells. Electron micrographs showed that DCV number in SNX19KD cells was decreased by approximately 75% and that DCV size was decreased by approximately 40% compared with those in control cells, respectively. Furthermore, when SNX19 was reintroduced in SNX19KD cells, insulin content, insulin secretion and DCV number were increased. The half-life of DCV was decreased in SNX19KD cells, but was increased in SNX19KD cells in which SNX19 was reintroduced. The number of lysosomes and the activity of lysosome enzyme cathepsin D were increased by approximately threefold in SNX19KD cells compared with those in control cells. In contrast, they were decreased to approximately half to one-third in SNX19-reintroduced SNX19KD cells. SNX19 regulates the number of DCV and insulin content by stabilizing DCV in β-cells. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00138.x, 2012).

The three ZNT8 autoantibody variants together improve the diagnostic sensitivity of childhood and adolescent type 1 diabetes

Aims: We tested whether autoantibodies to all three ZnT8RWQ variants, GAD65, insulinoma-associated protein 2 (IA-2), insulin and autoantibodies to islet cell cytoplasm (ICA) in combination with human leukocyte antigen (HLA) would improve the diagnostic sensitivity of childhood type 1 diabetes by detecting the children who otherwise would have been autoantibody-negative.Methods: A total of 686 patients diagnosed in 1996–2005 in Skåne were analyzed for all the seven autoantibodies [arginin 325 zinc transporter 8 autoantibody (ZnT8RA), tryptophan 325 zinc transporter 8 autoantibody (ZnT8WA), glutamine 325 Zinc transporter 8 autoantibody (ZnT8QA), autoantibodies to glutamic acid decarboxylase (GADA), Autoantibodies to islet-antigen-2 (IA-2A), insulin autoantibodies (IAA) and ICA] in addition to HLA-DQ genotypes.Results: Zinc transporter 8 autoantibody to either one or all three amino acid variants at position 325 (ZnT8RWQA) was found in 65% (449/686) of the patients. The frequency was independent of age at diagnosis. The ZnT8RWQA reduced the frequency of autoantibody-negative patients from 7.5 to 5.4%—a reduction by 28%. Only 2 of 108 (2%) patients who are below 5 years of age had no autoantibody at diagnosis. Diagnosis without any islet autoantibody increased with increasing age at onset. DQA1-B1*X-0604 was associated with both ZnT8RA (p = 0.002) and ZnT8WA (p = 0.01) but not with ZnT8QA (p = 0.07). Kappa agreement analysis showed moderate (>0.40) to fair (>0.20) agreement between pairs of autoantibodies for all combinations of GADA, IA-2A, ZnT8RWQA and ICA but only slight ( < 0.19) agreement for any combination with IAA.Conclusions: This study revealed that (1) the ZnT8RWQA was common, independent of age; (2) multiple autoantibodies were common among the young; (3) DQA1-B1*X-0604 increased the risk for ZnT8RA and ZnT8WA; (4) agreement between autoantibody pairs was common for all combinations except IAA. These results suggest that ZnT8RWQA is a necessary complement to the classification and prediction of childhood type 1 diabetes as well as to randomize the subjects in the prevention and intervention of clinical trials.

Identification of Autoantibody-Negative Autoimmune Type 2 Diabetic Patients

OBJECTIVEIslet autoimmunity has long been recognized in the pathogenesis of type 1 diabetes and is becoming increasingly acknowledged as a component in the pathogenesis of type 2 diabetes. Islet reactive T cells and autoantibodies have been demonstrated in type 1 diabetes, whereas islet autoimmunity in type 2 diabetes has been limited to islet autoantibodies. In this study, we investigated whether islet reactive T cells might also be present in type 2 diabetic patients and how islet reactive T cells correlate with β-cell function.RESEARCH DESIGN AND METHODSAdult phenotypic type 2 diabetic patients (n = 36) were screened for islet reactive T-cell responses using cellular immunoblotting and five islet autoantibodies (islet cell antibody, GADA, insulin autoantibody, insulinoma-associated protein-2 autoantibody, and zinc transporter autoantibody).RESULTSWe identified four subgroups of adult phenotypic type 2 diabetic patients based on their immunological status (Ab−T−, Ab+T−, Ab−T+, and Ab+T+). The Ab−T+ type 2 diabetic patients demonstrated T-cell responses similar to those of the Ab+T+ type 2 diabetic patients. Data were adjusted for BMI, insulin resistance, and duration of diabetes. Significant differences (P < 0.02) were observed among groups for fasting and glucagon-stimulated C-peptide responses. T-cell responses to islet proteins were also demonstrated to fluctuate less than autoantibody responses.CONCLUSIONSWe have identified a group of adult autoimmune phenotypic type 2 diabetic patients who are Ab−T+ and thus would not be detected using autoantibody testing alone. We conclude that islet autoimmunity may be more prevalent in adult phenotypic type 2 diabetic patients than previously estimated.

Persistently autoantibody negative (PAN) type 1 diabetes mellitus in children

Autoantibody-negative children diagnosed with type 1 diabetes might have unrecognized monogenic or type 2 diabetes. At diagnosis of type 1 diabetes (between ages 0.5 and 16.3 yr, n = 470), autoantibodies [glutamic acid decarboxylase (GAD), insulinoma-associated protein 2 (IA2), insulin autoantibodies (IAA), and/or islet cell antibody (ICA)] were positive (ab+) in 330 and negative in 37 (unknown in 103). Autoantibody-negative patients were retested at median diabetes duration of 3.2 yr (range 0.9-16.2) for autoantibodies (GAD, IA2, ZnT8), human leukocyte antigen (HLA) typing, non-fasting C-peptide, and sequencing of HNF4A, HNF1A, KCNJ11, and INS. Nineteen (5% of 367) remained persistently autoantibody negative (PAN), 17 were positive on repeat testing (PORT), and 1 refused retesting. No mutations were found in PORT. One PAN was heterozygous for P112L mutation in HNF1A and transferred from insulin to oral gliclazide. Another PAN transferred to metformin and the diagnosis was revised to type 2 diabetes. The remaining 17 PAN were indistinguishable from the ab+ group by clinical characteristics. HLA genotype was at high risk for type 1 diabetes in 82% of remaining PAN and 100% of PORT. After excluding patients with diabetes duration <1 yr, C-peptide was detectable more frequently in the remaining PAN (7/16) and PORT (6/17) than in a random selection of ab+ (3/28, p = 0.03). The diagnosis of type 1 diabetes should be reevaluated in PAN patients, because a subset has monogenic or type 2 diabetes. The remaining PAN have relatively preserved C-peptide compared with ab+, suggesting slower β-cell destruction, but a very high frequency of diabetogenic HLA, implying that type 1B (idiopathic) diabetes is rare.

MPO-ANCA陽性両側進行性難聴を呈した抗IA-2抗体単独陽性の1型糖尿病の1例

MPO-ANCA陽性両側進行性難聴を呈した抗IA-2抗体単独陽性の1型糖尿病の1例

Hyperglycaemic clamp test for diabetes risk assessment in IA-2-antibody-positive relatives of type 1 diabetic patients

The aim of the study was to investigate the use of hyperglycaemic clamp tests to identify individuals who will develop diabetes among insulinoma-associated protein-2 antibody (IA-2A)-positive first-degree relatives (IA-2A(+) FDRs) of type 1 diabetic patients. Hyperglycaemic clamps were performed in 17 non-diabetic IA-2A(+) FDRs aged 14 to 33 years and in 21 matched healthy volunteers (HVs). Insulin and C-peptide responses were measured during the first (5-10 min) and second (120-150 min) release phase, and after glucagon injection (150-160 min). Clamp-induced C-peptide release was compared with C-peptide release during OGTT. Seven (41%) FDRs developed diabetes 3-63 months after their initial clamp test. In all phases they had lower C-peptide responses than non-progressors (p < 0.05) and HVs (p < 0.002). All five FDRs with low first-phase release also had low second-phase release and developed diabetes 3-21 months later. Two of seven FDRs with normal first-phase but low second-phase release developed diabetes after 34 and 63 months, respectively. None of the five FDRs with normal C-peptide responses in all test phases has developed diabetes so far (follow-up 56 to 99 months). OGTT-induced C-peptide release also tended to be lower in progressors than in non-progressors or HVs, but there was less overlap in results between progressors and the other groups using the clamp. Clamp-derived functional variables stratify risk of diabetes in IA-2A(+) FDRs and may more consistently identify progressors than OGTT-derived variables. A low first-phase C-peptide response specifically predicts impending diabetes while a low second-phase response may reflect an earlier disease stage. ClinicalTrials.gov NCT00654121 The insulin trial was financially supported by Novo Nordisk Pharma nv.

Immunotherapy for the Prevention and Treatment of Type 1 Diabetes

In the past 15 years, multiple clinical trials have attempted to find prevention for type 1 diabetes. The accompanying article by Bresson and von Herrath (1) reviews basic mechanisms underlying immunoprevention and immunotherapy of type 1 diabetes as well as selected human trials in the context of data from animal models. The second part of this mini-symposium provides an overview of the recent or ongoing human trials. Immunotherapy for prevention of type 1 diabetes or to ameliorate the course of the disease after clinical diagnosis is currently restricted to research studies. References are provided to the clinical.trials.gov database or other sources where the reader can find additional information. Type 1 diabetes is an autoimmune disease caused by interplay of genetic and environmental factors. Figure 1 summarizes the main stages in the development of type 1 diabetes and examples of prevention trials at these stages. The initial step—development of islet autoimmunity marked by the presence of autoantibodies to insulin, GAD (GAD65), insulinoma-associated protein 2 (IA-2), and tyrosine phosphatase or zinc transporter (ZnT8)—is believed to be driven by environmental trigger(s) (2). Over the past 40 years, the incidence of childhood type 1 diabetes worldwide has increased by 3–5% annually (3–6). Elimination of the environmental trigger(s) responsible for this epidemic would be the most efficient approach to primary prevention. However, there is lack of consensus regarding which environmental factor(s) initiates islet autoimmunity. The National Institutes of Health have established The Environmental Determinants of type 1 Diabetes in the Young (TEDDY) consortium to evaluate the leading candidates (7,8). Figure 1 Natural history of type 1 diabetes and prevention opportunities. After initiation of islet autoimmunity, most patients have a long preclinical period (9–12) that offers opportunity for secondary prevention—halting progression to clinical diabetes (Fig. 1). Large randomized trials initiated …

Differences in Baseline Lymphocyte Counts and Autoreactivity Are Associated With Differences in Outcome of Islet Cell Transplantation in Type 1 Diabetic Patients

OBJECTIVEThe metabolic outcome of islet cell transplants in type 1 diabetic patients is variable. This retrospective analysis examines whether differences in recipient characteristics at the time of transplantation are correlated with inadequate graft function.RESEARCH DESIGN AND METHODSThirty nonuremic C-peptide–negative type 1 diabetic patients had received an intraportal islet cell graft of comparable size under an ATG-tacrolimus–mycophenolate mofetil regimen. Baseline patient characteristics were compared with outcome parameters during the first 6 posttransplant months (i.e., plasma C-peptide, glycemic variability, and gain of insulin independence). Correlations in univariate analysis were further examined in a multivariate model.RESULTSPatients that did not become insulin independent exhibited significantly higher counts of B-cells as well as a T-cell autoreactivity against insulinoma-associated protein 2 (IA2) and/or GAD. In one of them, a liver biopsy during posttransplant year 2 showed B-cell accumulations near insulin-positive β-cell aggregates. Higher baseline total lymphocytes and T-cell autoreactivity were also correlated with lower plasma C-peptide levels and higher glycemic variability.CONCLUSIONSHigher total and B-cell counts and presence of T-cell autoreactivity at baseline are independently associated with lower graft function in type 1 diabetic patients receiving intraportal islet cells under ATG-tacrolimus–mycophenolate mofetil therapy. Prospective studies are needed to assess whether control of these characteristics can help increase the function of islet cell grafts during the first year posttransplantation.

Maternal BMI Before Pregnancy, Maternal Weight Gain During Pregnancy, and Risk of Persistent Positivity for Multiple Diabetes-Associated Autoantibodies in Children With the High-Risk HLA Genotype

OBJECTIVETo assess whether maternal BMI before pregnancy and weight gain during pregnancy predicted the risk of islet autoimmunity in genetically susceptible children.RESEARCH DESIGN AND METHODSOf 46,939 newborns screened for the high-risk HLA genotype DR4-DQ8/DR3-DQ2, 1,003 were positive and 885 were followed with serial blood samples tested for autoantibodies to insulin, GAD, and insulinoma-associated protein 2 (IA2). The end point was defined as repeated positivity for two or three autoantibodies or the onset of type 1 diabetes (islet autoimmunity).RESULTSThirty-six children developed islet autoimmunity, of whom 10 developed type 1 diabetes. Both maternal BMI ≥30 kg/m2 before pregnancy and maternal weight gain ≥15 kg predicted the increased risk of islet autoimmunity (hazard ratio [HR] 2.5, P = 0.023, and HR 2.5, P = 0.015, respectively), independent of maternal diabetes.CONCLUSIONSMaternal weight may predict risk of islet autoimmunity in offspring with a high genetic susceptibility for type 1 diabetes.

Insulinoma-associated protein 2-deficient mice develop severe forms of diabetes induced by multiple low doses of streptozotocin

Insulinoma-associated protein 2 (IA-2) is the major autoantigen that contributes to the pathogenesis of type 1 diabetes (T1D). IA-2-deficient (IA-2-/-) mice showed impaired insulin secretion after intraperitoneal injection of glucose as well as elevated glucose level in a glucose tolerance test. Despite the fact that 70% of newly diagnosed T1D patients have an antibody against IA-2, the role of IA-2 in the pathogenesis of T1D is largely unknown. In this study, the sensitivity to diabetes induced by streptozotocin (STZ) of IA-2-/- mice was compared with that of wild-type (WT) mice. STZ injection to IA-2-/- mice caused significant elevation of blood glucose and depressed insulin concentration in the pancreas. Furthermore, abnormal ultrastructure in the beta cells of the IA-2-/- mice was revealed by electron microscopy, showing a decreased number of insulin containing vesicles and dilation of the ER-Golgi complex. These results demonstrated that IA-2-/- mice had higher sensitivity to STZ, suggesting a role of IA-2 not only in the secretion but also in the production of insulin.

Localization of insulinoma associated protein 2, IA-2 in mouse neuroendocrine tissues using two novel monoclonal antibodies

Aims Insulinoma-associated protein 2 (IA-2) is a member of the protein tyrosine phosphatase family that is localized on the insulin granule membrane. IA-2 is also well known as one of the major autoantigens in Type 1 diabetes mellitus. IA-2 gene deficient mice were recently established and showed abnormalities in insulin secretion. Thus, detailed localization of IA-2 was studied using wild-type and IA-2 gene deficient mice. Main methods To localize IA-2 expression in mouse neuroendocrine tissues, monoclonal antibodies were generated against IA-2 and western blot and immunohistochemical analyses were carried out in IA-2 +/+ mice. IA-2 −/− mice served as a negative control. Key findings Western blot analysis revealed that the 65 kDa form of IA-2 was observed in the cerebrum, cerebellum, medulla oblongata, pancreas, adrenal gland, pituitary gland, muscular layers of the stomach, small intestine, and colon. By immunohistochemical analysis, IA-2 was produced in endocrine cells in pancreatic islets, adrenal medullary cells, thyroid C-cells, Kulchitsky cells, and anterior, intermediate, and posterior pituitary cells. In addition, IA-2 was found in somatostatin-producing D-cells and other small populations of cells were scattered in the gastric corpus. IA-2 expression in neurites was confirmed by the immunostaining of IA-2 using primary cultured neurons from the small intestine and nerve growth factor (NGF)-differentiated PC12 cells. Significance The IA-2 distribution in peripheral neurons appeared more intensely in neurites rather than in the cell bodies.

A New Luminescence Assay for Autoantibodies to Mammalian Cell–Prepared Insulinoma-Associated Protein 2

OBJECTIVE—Insulinoma-associated protein 2 (IA-2) is a major autoantigen in type 1 diabetes, and IA-2 autoantibodies are routinely detected by a liquid-phase radioimmunoprecipitation assay. The present experiments were initiated to develop a new assay that does not require the use of radioisotopes or autoantigens prepared in bacteria or by in vitro transcription/translation.RESEARCH DESIGN AND METHODS—IA-2 luciferase fusion protein was expressed in mammalian cells and assayed for autoantibodies by liquid-phase luciferase immunoprecipitation.RESULTS—Our study showed that there was no significant difference between the luciferase immunoprecipitation and the radioimmunoprecipitation assays in sensitivity and specificity, and comparison of the two assays revealed a high correlation coefficient (R2 = 0.805).CONCLUSIONS—The luciferase system offers a robust, inexpensive, nonradioactive method for the detection of autoantibodies to mammalian cell–prepared IA-2 and could be of practical value at the clinical level.

The Frequency and Immunodominance of Islet-Specific CD8+ T-cell Responses Change after Type 1 Diabetes Diagnosis and Treatment

Islet-reactive CD8(+) T-cells play a key role in the pathogenesis of type 1 diabetes in the NOD mouse. The predominant T-cell specificities change over time, but whether similar shifts also occur after clinical diagnosis and insulin treatment in type 1 diabetic patients is unknown. We took advantage of a recently validated islet-specific CD8(+) T-cell gamma-interferon enzyme-linked immunospot (ISL8Spot) assay to follow responses against preproinsulin (PPI), GAD, insulinoma-associated protein 2 (IA-2), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) epitopes in 15 HLA-A2(+) adult type 1 diabetic patients close to diagnosis and at a second time point 7-16 months later. CD8(+) T-cell reactivities were less frequent at follow-up, as 28.6% of responses tested positive at type 1 diabetes diagnosis vs. 13.2% after a median of 11 months (P = 0.003). While GAD and IA-2 autoantibody (aAb) titers were unchanged in 75% of cases, the fraction of patients responding to PPI and/or GAD epitopes by ISL8Spot decreased from 60-67 to 20% (P < 0.02). The previously subdominant IA-2(206-214) and IGRP(265-273) peptides were newly targeted, thus becoming the immunodominant epitopes. Shifts both in frequency and in immunodominance of CD8(+) T-cell responses occur more rapidly than do changes in aAb titers. These different kinetics may suggest complementary clinical applications for T-cell and aAb measurements.

On the Pathogenicity of Autoantigen-Specific T-Cell Receptors

Type 1 diabetes is mediated by T-cell entry into pancreatic islets and destruction of insulin-producing beta-cells. The relative contribution of T-cells specific for different autoantigens is largely unknown because relatively few have been assessed in vivo. We generated mice possessing a monoclonal population of T-cells expressing 1 of 17 T-cell receptors (TCR) specific for either known autoantigens (GAD65, insulinoma-associated protein 2 (IA2), IA2beta/phogrin, and insulin), unknown islet antigens, or control antigens on a NOD.scid background using retroviral-mediated stem cell gene transfer and 2A-linked multicistronic retroviral vectors (referred to herein as retrogenic [Rg] mice). The TCR Rg approach provides a mechanism by which T-cells with broad phenotypic differences can be directly compared. Neither GAD- nor IA2-specific TCRs mediated T-cell islet infiltration or diabetes even though T-cells developed in these Rg mice and responded to their cognate epitope. IA2beta/phogrin and insulin-specific Rg T-cells produced variable levels of insulitis, with one TCR producing delayed diabetes. Three TCRs specific for unknown islet antigens produced a hierarchy of insulitogenic and diabetogenic potential (BDC-2.5 > NY4.1 > BDC-6.9), while a fourth (BDC-10.1) mediated dramatically accelerated disease, with all mice diabetic by day 33, well before full T-cell reconstitution (days 42-56). Remarkably, as few as 1,000 BDC-10.1 Rg T-cells caused rapid diabetes following adoptive transfer into NOD.scid mice. CONCLUSIONS; Our data show that relatively few autoantigen-specific TCRs can mediate islet infiltration and beta-cell destruction on their own and that autoreactivity does not necessarily imply pathogenicity.

Structure of the Mature Ectodomain of the Human Receptor-type Protein-tyrosine Phosphatase IA-2

IA-2 (insulinoma-associated protein 2) is a protein-tyrosine phosphatase receptor located in secretory granules of neuroendocrine cells. Initially, it attracted attention due to its involvement in the autoimmune response associated to diabetes. Later it was found that upon exocytosis, the cytoplasmic domain of IA-2 is cleaved and relocated to the nucleus, where it enhances the transcription of the insulin gene. A concerted functioning of the whole receptor is to be expected. However, very little is known about the structure and function of the transmembrane and extracellular domains of IA-2. To address this issue, we solved the x-ray structure of the mature ectodomain of IA-2 (meIA-2) to 1.30A resolution. The fold of meIA-2 is related to the SEA (sea urchin sperm protein, enterokinase, agrin)) domains of mucins, suggesting its participation in adhesive contacts to the extracellular matrix and providing clues on how this kind of molecule may associate and form homo- and heterodimers. Moreover, we discovered that meIA-2 is self-proteolyzed in vitro by reactive oxygen species, suggesting the possibility of a new shedding mechanism that might be significant in normal function or pathological processes. Knowledge of meIA-2 structure should facilitate the search of its possible ligands and molecular interactions.

Late-onset Type 1 Diabetes Mellitus in an Elderly Japanese Woman with Single Positive Insulinoma-associated Tyrosine Phosphatase-like Protein-2 (IA-2) Antibody

Late-onset Type 1 Diabetes Mellitus in an Elderly Japanese Woman with Single Positive Insulinoma-associated Tyrosine Phosphatase-like Protein-2 (IA-2) Antibody

Immunization of HLA Class I Transgenic Mice Identifies Autoantigenic Epitopes Eliciting Dominant Responses in Type 1 Diabetes Patients

Type 1 diabetes (T1D) results from the autoimmune destruction of pancreatic beta cells. CD8(+) T cells have recently been assigned a major role in beta cell injury. Consequently, the identification of autoreactive CD8(+) T cells in humans remains essential for development of therapeutic strategies and of assays to identify aggressive cells. However, this identification is laborious and limited by quantities of human blood samples available. We propose a rapid and reliable method to identify autoantigen-derived epitopes recognized by human CD8(+) T lymphocytes in T1D patients. Human histocompatibility leukocyte Ags-A*0201 (HLA-A*0201) transgenic mice were immunized with plasmids encoding the T1D-associated autoantigens: 65 kDa glutamic acid decarboxylase (GAD) or insulinoma-associated protein 2 (IA-2). Candidate epitopes for T1D were selected from peptide libraries by testing the CD8(+) reactivity of vaccinated mice. All of the nine-candidate epitopes (five for GAD and four for IA-2) identified by our experimental approach were specifically recognized by CD8(+) T cells from newly diagnosed T1D patients (n = 19) but not from CD8(+) T cells of healthy controls (n = 20). Among these, GAD(114-123), GAD(536-545) and IA-2(805-813) were recognized by 53%, 25%, and 42% of T1D patients, respectively.

T-Cell Responses to Islet Antigens Improves Detection of Autoimmune Diabetes and Identifies Patients With More Severe β-Cell Lesions in Phenotypic Type 2 Diabetes

Latent autoimmune diabetes in adults or type 1.5 diabetes is considered to be a T-cell-mediated autoimmune disease. However, identification of patients is based commonly on autoantibody (Ab) detection. To determine whether measuring T-cell reactivity to islet proteins compared with measuring Abs improves detection of autoimmune diabetes and how beta-cell function correlates with T-cell reactivity compared with Ab positivity, we assessed the T-cell proliferative responses and Ab responses (islet cell autoantibodies, insulin autoantibodies, insulinoma-associated protein-2 autoantibodies, and GAD Abs) to islet proteins of 36 phenotypic type 2 diabetic patients. To be considered Ab(+) or T-cell(+), patients were required to be positive for a minimum of two consecutive time points. beta-Cell function was measured with fasting and glucagon-stimulated C-peptide. Independent of T-cell reactivity, Ab(+) and Ab(-) patients had comparable fasting and glucagon-stimulated C-peptide. Independent of Ab status, T-cell(+) patients demonstrated significantly lower glucagon-stimulated (P < 0.003) C-peptide compared with T-cell(-) patients. These data suggest that measuring T-cell responses to multiple islet proteins in phenotypic type 2 diabetic patients improves identification of patients with autoimmune diabetes and delineates those who have a more severe beta-cell lesion compared with Ab assessment alone.