Tyrosine Phosphatase-like Protein IA-2 Research Articles

Anti-Islet Autoantibodies in Type 1 Diabetes.

Anti-islet autoantibodies serve as key markers in immune-mediated type 1 diabetes (T1D) and slowly progressive T1D (SPIDDM), also known as latent autoimmune diabetes in adults (LADA). Autoantibodies to insulin (IAA), glutamic acid decarboxylase (GADA), tyrosine phosphatase-like protein IA-2 (IA-2A), and zinc transporter 8 (ZnT8A) are currently employed in the diagnosis, pathological analysis, and prediction of T1D. GADA can also be detected in non-diabetic patients with autoimmune diseases other than T1D and may not necessarily reflect insulitis. Conversely, IA-2A and ZnT8A serve as surrogate markers of pancreatic β-cell destruction. A combinatorial analysis of these four anti-islet autoantibodies demonstrated that 93-96% of acute-onset T1D and SPIDDM cases were diagnosed as immune-mediated T1D, while the majority of fulminant T1D cases were autoantibody-negative. Evaluating the epitopes and immunoglobulin subclasses of anti-islet autoantibodies help distinguish between diabetes-associated and non-diabetes-associated autoantibodies and is valuable for predicting future insulin deficiency in SPIDDM (LADA) patients. Additionally, GADA in T1D patients with autoimmune thyroid disease reveals the polyclonal expansion of autoantibody epitopes and immunoglobulin subclasses. Recent advancements in anti-islet autoantibody assays include nonradioactive fluid-phase assays and the simultaneous determination of multiple biochemically defined autoantibodies. Developing a high-throughput assay for detecting epitope-specific or immunoglobulin isotype-specific autoantibodies will facilitate a more accurate diagnosis and prediction of autoimmune disorders. The aim of this review is to summarize what is known about the clinical significance of anti-islet autoantibodies in the pathogenesis and diagnosis of T1D.

Physical activity and progression to type 1 diabetes in children and youth with islet autoimmunity: The diabetes autoimmunity study in the young.

Physical inactivity may contribute to islet autoimmunity and progression to clinical type 1 diabetes. To test this hypothesis, we evaluated physical activity, assessed by accelerometer, as an independent risk factor for progression to clinical diabetes among genetically at risk for type 1 diabetes children and youth with islet autoimmunity. Accelerometer data were obtained for 95 children and youth participating in the diabetes autoimmunity study in the young who had islet autoimmunity. Islet autoimmunity was defined as the presence of islet autoantibodies to insulin, glutamic acid decarboxylase, tyrosine phosphatase-like protein IA-2, or zinc transporter 8. During prospective follow-up for up to 7 years, 13 of the 95 participants progressed to clinical diabetes. In multivariable survival analysis, none of the physical activity parameters examined predicted a higher risk of developing diabetes. In survival analysis with time-varying physical activity parameters, none of the physical activity parameters over time were associated with the risk of developing type 1 diabetes. It does not appear that low-physical activity is a risk factor for progression from islet autoantibodies to diabetes in children and youth at high-genetic risk for type 1 diabetes.

Correction: A Rapid Lateral Flow Immunoassay for the Detection of Tyrosine Phosphatase-Like Protein IA-2 Autoantibodies in Human Serum

Correction: A Rapid Lateral Flow Immunoassay for the Detection of Tyrosine Phosphatase-Like Protein IA-2 Autoantibodies in Human Serum

A rapid lateral flow immunoassay for the detection of tyrosine phosphatase-like protein IA-2 autoantibodies in human serum.

Type 1 diabetes (T1D) results from the destruction of pancreatic insulin-producing beta cells and is strongly associated with the presence of islet autoantibodies. Autoantibodies to tyrosine phosphatase-like protein IA-2 (IA-2As) are considered to be highly predictive markers of T1D. We developed a novel lateral flow immunoassay (LFIA) based on a bridging format for the rapid detection of IA-2As in human serum samples. In this assay, one site of the IA-2As is bound to HA-tagged-IA-2, which is subsequently captured on the anti-HA-Tag antibody-coated test line on the strip. The other site of the IA-2As is bound to biotinylated IA-2, allowing the complex to be visualized using colloidal gold nanoparticle-conjugated streptavidin. For this study, 35 serum samples from T1D patients and 44 control sera from non-diabetic individuals were analyzed with our novel assay and the results were correlated with two IA-2A ELISAs. Among the 35 serum samples from T1D patients, the IA-2A LFIA, the in-house IA-2A ELISA and the commercial IA-2A ELISA identified as positive 21, 29 and 30 IA-2A-positive sera, respectively. The major advantages of the IA-2A LFIA are its rapidity and simplicity.

Administering 25-hydroxyvitamin D3 in vitamin D-deficient young type 1A diabetic patients reduces reactivity against islet autoantigens

We investigated whether improving 25-hydroxyvitamin D status in young type 1A diabetic patients reduces reactivity of peripheral blood mononuclear cells against islet autoantigens and associates with beta-cell functional changes. Eight patients with 25-hydroxyvitamin D deficiency (<20ng/ml), out of 15 consecutive young type 1A diabetic subjects received 25-hydroxyvitamin D3 to achieve and maintain levels above 50ng/ml for up to one year. Peripheral blood mononuclear cell reactivity (Interferon-γ spots) against beta-cell autoantigens (glutamic acid decarboxylase 65-kD isoform, proinsulin and tyrosine phosphatase-like protein IA-2) and C-peptide during mixed meal were assessed before and after 25-hydroxyvitamin D3 replenishment. Target 25-hydroxyvitamin D blood levels were safely reached and maintained. Peripheral blood mononuclear cell reactivity against glutamic acid decarboxylase 65-kD isoform (3.8±4.0 vs. 45±16) and proinsulin (3.5±3.2 vs. 75±51) decreased significantly (p<0.001 and p<0.02) upon 25-hydroxyvitamin D3 replenishment, which was correlated with 25-hydroxyvitamin D concentrations. C-peptide values remained stable after one year of treatment. Safely restored and maintained 25-hydroxyvitamin D levels associated with reduced peripheral blood mononuclear cell reactivity against beta-cell autoantigens with no significant decrease of beta-cell function in this cohort of patients.

Early induction of GAD65‐reactive Th2 response in type 1 diabetic children treated with alum‐formulated GAD65

We have previously shown that two injections of 20 µg alum-formulated glutamic acid decarboxylase 65 (GAD(65)) (GAD-alum; Diamyd(®)) in children with recent-onset type 1 diabetes lead to preservation of residual insulin secretion. In vitro cytokine production at the 15 months' follow-up indicated immunomodulation. In the present study, we took advantage of peripheral blood mononuclear cells, cryopreserved during early follow-ups, to investigate whether the immunomodulatory effect of GAD-alum was apparent earlier after treatment, preceding the changes previously reported at 15 months. Peripheral blood mononuclear cells from 70 type 1 diabetic children, randomly assigned GAD-alum (n = 35) or placebo (n = 35), that had been frozen at baseline (n = 27) and after 1 (n = 58), 3 (n = 67) and 9 (n = 66) months, were stimulated in vitro with GAD(65), tyrosine phosphatase-like protein IA-2 peptide, insulin peptide, GAD-alum, alum formulation or phytohaemagglutinin. Interleukin (IL)-5, -6, -10, -12, -13, -17, tumour necrosis factor and interferon-γ were measured in cell supernatants and serum samples using Luminex. Expression of FOXP3 and transforming growth factor-β was determined by real-time reverse transcription polymerase chain reaction. Already 1 month after the first injection, GAD(65)-induced IL-5 and IL-13 together with FOXP3 were enhanced in GAD-alum-treated patients compared to those with placebo. The in vitro response at 3 and 9 months was characterized by a broader range of cytokines in the treated group. Notably, only the T-helper 2-associated cytokines IL-5 and IL-13 together with FOXP3 increased continuously over time. Treatment with GAD-alum in type 1 diabetic children induced an early T-helper 2 immune enhanced response to GAD(65), followed by a wider spectrum of cytokines at 3 and 9 months.

Humoral Autoimmunity against the Extracellular Domain of the Neuroendocrine Autoantigen IA-2 Heightens the Risk of Type 1 Diabetes

The objective of this study was to determine whether antigenic determinants localized within the extracellular domain of the neuroendocrine autoantigen tyrosine phosphatase-like protein IA-2 are targets of humoral responses in type 1 diabetes (T1DM). Previous studies indicated that the immunodominant region of IA-2 is localized within its intracellular domain (IA-2ic; amino acids 601-979). We analyzed 333 subjects from the Children's Hospital of Pittsburgh study, 102 of whom progressed to insulin-requiring diabetes (prediabetics). Autoantibodies from these individuals were initially assayed for ICA512bdc (Barbara Davis Center amino acids 257-556; 630-979), IA-2ic (amino acids 601-979), and IA-2 full-length (amino acids 1-979) in addition to islet cell antibody (ICA), glutamic acid decarboxylase, 65-kDa isoform, and insulin autoantibodies. We identified an autoantibody response reactive with the extracellular domain of IA-2 that is associated with very high risk of T1DM progression. Relatives with no detectable autoantibodies against ICA512bdc (or IA-2ic) exhibited antibody responses against the IA-2 full-length peptide (log rank, P = 0.008). This effect was also observed in first-degree relatives who were positive for glutamic acid decarboxylase, 65-kDa isoform (log rank, P = 0.026) or at least two islet autoantibodies but were negative for ICA512bdc (log rank, P = 0.022). Competitive binding experiments and immunoprecipitation of the IA-2 extracellular domain (amino acid residues 26-577) further lend support for the presence of autoantibodies reactive with new antigenic determinants within the extracellular domain of IA-2. In summary, the addition of measurements of autoantibodies reactive with the IA-2 extracellular domain to assays geared to assess the progression of autoimmunity to clinical T1DM may more accurately characterize this risk. This has considerable implications not only for stratifying high diabetes risk but also facilitating the search for pathogenic epitopes to enable the design of peptide-based immunotherapies that may prevent the progression to overt T1DM at its preclinical stages.

Screening for associated autoimmune disorders in Polish patients with Addison’s disease

Autoimmune Addison's disease (AAD) is the main reason of primary adrenal failure. More than a half of patients display additional autoimmune conditions, which represent a considerable clinical concern. This study aimed to investigate the prevalence of concomitant autoimmune disorders in 85 Polish AAD patients (61 females, 24 males). Mean age at AAD onset was 34.6 ± 12.6 years, significantly earlier in males (P < 0.001). Sixty-nine patients presented positive serum antibodies to 21-hydroxylase and shorter AAD duration than those with negative results (P = 0.027). Seventy-three subjects suffered from coexisting autoimmune disorders. Serum autoantibodies against thyreoperoxidase, thyroglobulin, TSH receptor, glutamic acid decarboxylase, insulin, tyrosine phosphatase-like protein IA2, parietal cell H(+)/K(+)-ATPase, intrinsic factor and tissue transglutaminase were detectable in 71.8, 41.2, 4.7, 21.0, 4.9, 2.5, 49.4, 12.0 and 3.5% of patients, respectively. Antinuclear antibodies were found in 12.5%. Thyroid autoimmunity was most common (46 subjects with lymphocytic thyroiditis, 19 with Graves' disease), followed by atrophic gastritis (29.4%), pernicious anaemia (11.8%), hypergonadotropic hypogonadism (8.2%), vitiligo (8.2%), type 1 diabetes (7.1%), celiac disease (3.5%) and alopecia (2.4%). Gender differences were observed only for thyroid autoimmunity. Current study confirms particular tendency of AAD patients to develop other autoimmune disorders. Active search for concomitant conditions is warranted to prevent serious complications.

A detailed investigation of maternally inherited diabetes and deafness (MIDD) including clinical characteristics, C‐peptide secretion, HLA‐DR and ‐DQ status and autoantibody pattern

This article presents a clinically characterization of the mitochondrial DNA mutation (A3243G) associated with maternally inherited diabetes and deafness (MIDD) syndrome in two families. Six patients with MIDD and one mutation-positive relative with normal glucose tolerance (NGT) were examined. Fasting serum C-peptide was measured in all subjects and compared with controls having NGT (n = 14). C-peptide response to an intravenous glucose tolerance test (IVGTT) was investigated in the diabetic patients not treated with insulin (n = 3) and in the mutation-positive healthy individual and compared with the controls. The A3243G heteroplasmy value varied between 5 and 30%. All A3243G carriers had HLA-DR1-DQ5 haplotype, and either the -DQ5 or the -DQ6 allele. The fasting and the serum C-peptide levels at 120 min during the IVGTT did not differ between the A3243G carriers and the controls. A missing first phase and a decreased total C-peptide response was detected in the mutation-positive diabetics compared with controls (p < 0.0001). The same abnormality was found in the A3243G carrier with NGT. Circulating islet cell antibody (ICA) was present in three patients with MIDD. Glutamic acid decarboxylase (GAD), tyrosine phosphatase-like protein IA-2 (IA-2) and mitochondrial antibodies were missing. The diagnosis of MIDD was delayed in each case. A missing first phase and a decreased total C-peptide response during an IVGTT was characteristic for the A3243G mutation. The fasting C-peptide level of the carriers did not differ from the controls. Circulating ICA was present in some patients, but GAD, IA-2 and mitochondrial antibodies were absent. All subjects had HLA-DR1-DQ5 haplotype, and either -DQ5 or -DQ6 alleles.

Prevalence and clinical associations of 10 defined autoantibodies in autoimmune polyendocrine syndrome type I.

The prevalence of autoantibodies against nine intracellular enzyme autoantigens, namely 21-hydroxylase, side-chain cleavage enzyme (SCC), 17 alpha-hydroxylase, glutamic acid decarboxylase 65, aromatic L-amino acid decarboxylase, tyrosine phosphatase-like protein IA-2, tryptophan hydroxylase (TPH), tyrosine hydroxylase, cytochrome P450 1A2, and against the extracellular calcium-sensing receptor, was assessed in 90 patients with autoimmune polyendocrine syndrome type I. A multivariate logistic regression analysis was performed for the presence of autoantibodies as independent predictors for different disease manifestations. Reactivities against 21-hydroxylase and SCC were associated with Addison's disease with odds ratios (ORs) of 7.8 and 6.8, respectively. Hypogonadism was exclusively associated with autoantibodies against SCC with an OR of 12.5. Autoantibodies against tyrosine phosphatase-like protein IA-2 were associated with insulin-dependent diabetes mellitus with an OR of 14.9, but with low sensitivity. Reactivities against TPH and, surprisingly, glutamic acid decarboxylase 65, were associated with intestinal dysfunction, with ORs of 3.9 and 6.7, respectively. TPH reactivity was the best predictor for autoimmune hepatitis, with an OR of 27.0. Hypoparathyroidism was not associated with reactivity against any of the autoantigens tested. No reactivity against the calcium-sensing receptor was found. Analysis of autoantibodies in autoimmune polyendocrine syndrome type I patients is a useful tool for establishing autoimmune manifestations of the disease as well as providing diagnosis in patients with suspected disease.

Stratification of type 1 diabetes risk on the basis of islet autoantibody characteristics.

Family history of type 1 diabetes and autoantibodies to the islet antigens insulin (IAA), glutamate decarboxylase (GADA), and the protein tyrosine phosphatase-like protein IA-2 (IA-2A) are strong predictors of type 1 diabetes, but the rate of progression to diabetes in multiple islet autoantibody-positive relatives varies widely. We asked whether detailed characterization of islet autoantibodies that included determination of titer, epitope specificity, and IgG subclass would improve diabetes prediction in a large cohort of autoantibody-positive relatives. The study shows a strong association between risk and high titer, broad antibody responses to IA-2 and insulin. The highest risks were associated with high-titer IA-2A and IAA, IgG2, IgG3, and/or IgG4 subclass of IA-2A and IAA, and antibodies to the IA-2-related molecule IA-2beta. Using models based on these antibody characteristics, autoantibody-positive relatives can be classified into groups with risks of diabetes ranging from 7 to 89% within 5 years.

Effect of proinflammatory cytokines on gene expression of the diabetes-associated autoantigen IA-2 in INS-1 cells.

Cytokines released from activated antigen-presenting cells and T-lymphocytes are crucially involved in the pathogenesis of type 1 diabetes. Previous studies have shown that proinflammatory cytokines play an important role in the induction of autoimmunity and beta-cell damage. Inhibition of insulin expression has been described, but their effects on other major target autoantigens, such as the tyrosine phosphatase-like protein IA-2, is not known. In the present study, we established sensitive real-time RT-PCR to measure IA-2, insulin, and inducible nitric oxide (NO) synthase (iNOS) mRNA expression. Rat insulinoma INS-1 cells were stimulated with IL-1beta, TNF-alpha, interferon (IFN)-gamma, and IL-2 as well as with two combinations of these cytokines (C1: IL-1beta + TNF-alpha + IFN-gamma; C2: TNF-alpha + IFN-gamma). Treatment with IL-1beta, TNF-alpha, or IFN-gamma alone caused a significant down-regulation of IA-2 and insulin mRNA levels in a time and dose-dependent manner, whereas IL-2 had no effect. Exposure to cytokine combinations strongly potentiates the inhibitory effects. Incubation of cells with C1 and C2 for 24 h induces a significant inhibition of IA-2 mRNA levels by 78% and 58%, respectively. Under these conditions, an up to 5 x 10(4)-fold increase of iNOS gene expression was observed. The hypothesis that the formation of NO is involved in IA-2 regulation was confirmed by the finding that the coincubation of C1 with 4 mM L-N(G)-monomethyL-L-arginine, an inhibitor of the iNOS, partly reversed the down-regulation of IA-2. Further, incubation with the synthetic NO-donor S-nitroso-N-acetyl-D-L-penicillamine significantly decreased IA-2 mRNA level to 51% of basal levels. In conclusion, we have demonstrated for the first time that IL-1beta, TNF-alpha, and IFN-gamma exert a strong inhibitory effect on expression of the diabetes autoantigen IA-2. The action of IL-1beta may be partly mediated by the activation of the NO pathway.

Application of phage display peptide library to autoimmune diabetes: identification of IA-2/ICA512bdc dominant autoantigenic epitopes.

Autoantigenic epitope mapping represents a critical issue in autoimmune diseases. The islet tyrosine phosphatase-like protein IA-2/ICA512bdc is a major autoantigen in type 1 diabetes (IDDM), but the epitopes responsible for autoantibody binding have been only partially defined. The aim of our study was to identify ICA512bdc epitopes, and in particular mini-epitopes, utilizing a novel strategy for autoimmune diseases. The study was performed in three sequential steps: (1) construction of a lambda-phage surface-displayed ICA512bdc cDNA library with the methodology of tagged random priming with peptides displayed as a fusion to the C terminus of the capsid protein D; (2) affinity selection of the resulting library, followed by immunoscreening, enzyme-linked immunosorbent assay and sequence analysis of positive clones, and (3) radioimmunoprecipitation to detect autoantibodies to the selected clones. This strategy resulted in the identification of two epitopes (IA-2 residues 761 - 964 and 929 - 979), which were recognized by 100 % and 62.9 % ICA512bdc-positive IDDM patients, respectively. Interestingly, the larger clone was detected also by a proportion (16.7 %) of new onset ICA512bdc-negative patients, thus suggesting that this region contains not only the main autoantigenic repertoire of ICA512bdc molecule, but is able to detect IA-2 autoantibodies in even higher percentages of patients. In addition, this study showed the existence of multiple epitopes located in the C-terminal domain of the IA-2 protein, one of which is formed by the 50 C-terminal amino acids, and provided evidence that the strategy used represents a valid tool for identification of epitopes within autoantigenic molecules.

IA-2 autoantibodies restricted to the IgG4 subclass are associated with protection from type 1 diabetes.

The tyrosine phosphatase-like protein IA-2 is a major target antigen for autoantibodies in the preclinical period of type 1 diabetes. In this study, we examined whether immunoglobulin isotypes and IgG subclass specific autoantibodies directed at IA-2 discriminate between children at risk of type 1 diabetes who progressed to diabetes vs. those who remained diabetes-free. IgG1-4, IgA and the IgE-specific IA-2 antibody (IA-2A) were measured by radioligand assays in 50 patients with type 1 diabetes and 41 ICA-positive siblings of patients with type 1 diabetes who were followed for diabetes development. Of 41 siblings, 32 were positive for IA-2A; of these, 59 % had IA-2 IgG1, 59 % IgG4, 16 % IgG3, 9 % IgG2, 16 % IgA and 13 % IgE antibodies. IA-2 IgG1 was the dominant isotype in prediabetic children (n = 14, 86 % positive) and patients with type 1 diabetes (98 % positive) whereas only 7 of 18 (39 %) non-progressors had antibodies of this isotype. In subjects that remained diabetes-free, a significantly higher frequency of IA-2 IgG4 in the absence of IgG1 was observed (50 %) compared to progressors (7 %) and patients with type 1 diabetes (0 %). Life-table analysis revealed that IA-2A restricted to IgG4 correlated with protection from type 1 diabetes (p < 0.003). In contrast, IA-2 IgG2, IgG3, IgE and IgA did not differ significantly between study groups. Our findings suggest that the measurement of IA-2 IgG1 and IgG4 subclass antibodies can serve as surrogate marker to discriminate between antibody positive subjects at high or low risk for rapid development of diabetes.

Autoantibodies to IA‐2 in Type 1 Diabetes

The tyrosine phosphatase-like protein IA-2 is an important islet autoantigen in type 1 diabetes. Although the radioligand binding assay with in vitro synthesized (35)S-labeled antigen has been used extensively for measuring autoantibodies to IA-2, disadvantages of both assays with respect to synthesis and handling of the radioactive antigen limit their use in routine laboratories. Therefore, we attempted to develop a nonradioactive ELISA for the simple detection of IA-2 autoantibodies. The biotinylated cytoplasmic domain of IA-2 expressed in Escherichia coli was used as an antigen. We evaluated two kinds of ELISA: ELISA with biotin-IA-2 directly captured on streptavidine-coated plates (solid phase) and ELISA with antigen-antibody preincubation in solution in which serum samples were reacted first with biotin-IA-2 and the mixture was transferred to streptavidine-coated plates (liquid phase). We compared their disease sensitivity and specificity with a conventional radioligand binding assay in 52 patients with recent-onset type 1 diabetes and 138 normal individuals. The radioligand binding assay had 61.5% sensitivity and 99.3% specificity. The liquid-phase ELISA showed relatively higher sensitivity (55.8%) and specificity (99.3%) than the solid-phase ELISA (sensitivity 53.8% and specificity 97.1%). Furthermore, the mean SD score in IA-2 autoantibody-positive serum samples measured by liquid-phase ELISA was significantly higher than the SD score obtained by solid-phase ELISA (P < 0.0001). We concluded that this liquid-phase ELISA is suitable for detecting IA-2 autoantibodies in patients with type 1 diabetes with a similar sensitivity and specificity to those of conventional radioligand binding assay.

Tyrosine Phosphatase-like Protein (IA-2) and Glutamic Acid Decarboxylase (GAD65) Autoantibodies: A Study of Chinese Patients with Diabetes Mellitus

Type 1 diabetes in most Asian populations may not have a salient autoimmune basis when assessed with single determinations of the major markers, islet cell antibodies (ICAs) and glutamic acid decarboxylase antibodies (GAD65ab). With the inclusion of antibodies to tyrosine phosphatase-like protein IA-2 (IA-2ab) as an additional major marker, we re-examined autoimmune diabetes in a group of Chinese patients. We studied 272 subjects at various stages of disease with blood samples procured for biochemical analysis. ICAs were measured by immunofluorescence, GAD65ab and IA-2ab by radioimmunoassay. Sixty-seven patients fulfilled clinical diagnosis of type 1 diabetes and the remaining 205 patients were type 2. Prevalence of single autoantibody type in recent-onset type 1 diabetes (<1 year duration; n =47 ) showed 10.6% with ICAs, 44.7% GAD65ab and 36.2% IA-2ab. GAD65ab account for more than two-thirds of the markers found in type 1 diabetes. Combined analysis further showed that 51.1% had at least one antibody type, 31.9% with two or more antibodies and 8.5% with all three antibodies. Islet autoimmunity presence in childhood-onset type 1 diabetes improved with the addition of IA-2ab, though less impact was seen in the adult-onset. Similarly, combined analysis for type 2 patients with recent diabetes showed a modest increase to 13% with islet autoimmunity compared to 8% when assessed by GAD65ab alone. Combining IA-2ab and GAD65ab assays results detected slightly more immune-mediated diabetes, compared to using a single GAD65ab determination. Non-autoimmune causes need to be considered in the pathogenesis of type 1 diabetes in Chinese, particularly in adults.

Expression of the protein tyrosine phosphatase-like protein IA-2 during pancreatic islet development.

A tyrosine phosphatase-like protein, IA-2, is a major autoantigen in Type 1 diabetes but its role in islet function is unclear. Tyrosine phosphorylation mediates regulation of cellular processes such as exocytosis, cell growth, and cell differentiation. To investigate the potential involvement of IA-2 in islet differentiation and insulin secretion, we analyzed by immunohistochemistry expression of IA-2 during islet development in fetal rats and during the maturation of insulin secretory responses after birth. In the fetus, IA-2 immunoreactivity was detected in primitive islets positive for insulin and glucagon at 12 days' gestation. Subsequently, IA-2 was only weakly detectable in the fetal pancreas. In neonatal rat, a progressive increase in IA-2 immunoreactivity was observed in islets from very low levels at 1 day of age to moderate labeling at 10 days. In the adult, relatively high levels of IA-2 were detected in islets, with heterogeneous expression in individual cells within each islet. IA-2 marks a population of endocrine cells that transiently appear early in pancreatic ontogeny. Islet IA-2 expression reappears after birth concomitant with the development of mature insulin secretory responses, consistent with a role for this protein in regulated hormone secretion.

Autoantibodies to ICA12 (SOX-13) are not specific for Type I diabetes.

The majority of patients with Type I (insulin-dependent) diabetes mellitus has autoantibodies to insulin, glutamic acid decarboxylase and the tyrosine phosphatase-like protein IA-2. Some patients with Type I diabetes, in particular with adult onset diabetes, have, however, only cytoplasmic islet cell antibodies that could be directed to as yet unknown beta cell autoantigens. Recently, one potential antigen, ICA12, was identified as the high mobility group (HMG) box transcription factor SOX-13. Our aim was to evaluate the diagnostic sensitivity and specificity of autoantibodies to SOX-13 for autoimmune diabetes. Full-length SOX-13 was cloned from human brain mRNA, expressed by vitro transcription/ translation and used to detect autoantibodies by immunoprecipitation and radioligand assay in patients suffering from autoimmune diabetes or non-organ-specific autoimmune diseases. We found SOX-13 antibodies to be present in 11 of 125 (8.8%) patients with newly diagnosed Type I diabetes and in 3 of 43 (7.0%) patients with latent autoimmune diabetes in adults. There was no association with age, sex and a particular pattern of diabetes-specific autoantibodies. Similar frequencies of SOX-13 antibodies were found in 84 patients with rheumatic diseases (4.0-11.4%) and 211 healthy control subjects (5.2 %). Our data indicate that SOX-13 represents a minor target of autoantibodies in patients with autoimmune diabetes. Because SOX-13 antibodies were found not to be disease-specific, we assume they are not helpful in improving the diagnosis and prediction of Type I diabetes.

Identification of IL-1β-induced messenger RNAs in rat pancreatic beta cells by differential display of messenger RNA

Interleukin-1beta is a putative mediator of pancreatic beta-cell dysfunction and damage in Type I (insulin-dependent) diabetes mellitus. To better understand the molecular mechanisms involved in IL-1beta effects, we carried out a differential display of mRNA by RT-PCR to identify novel cytokine-regulated genes. Fluorescence activated cell sorting-purified rat pancreatic beta-cells were exposed for 6 or 24 h to IL-1beta. Differentially expressed cDNA bands were cloned and then identified by comparing their sequences with data from the GenBank. Differential gene expression was confirmed by RT-PCR using specific primers. Interleukin-1beta increased the expression of adenine nucleotide translocator-1, phospholipase D-1 and cytokine-induced neutrophil chemoattractant-1 and decreased expression of the protein tyrosine phosphatase-like protein IA-2. Interleukin-1beta-induced differential expression of these genes in beta cells was confirmed by RT-PCR. In additional studies, IL-1beta was shown to induce chemokines other than cytokine-induced neutrophil chemoattractant-1, including cytokine-induced neutrophil chemoattractant-3 and monocyte chemotactic protein-1. Our observations indicate that IL-1beta modifies the expression of several genes in pancreatic beta cells. These genes may affect both function, viability and beta-cell recognition by the immune system. Functional characterization of the mRNAs which have been identified could facilitate a better understanding of the mechanisms leading to beta-cell destruction in Type I diabetes. [Diabetologia (1999) 42: 1199-1203]

Autoantibodies Predicting Diabetes mellitus Type I in Celiac Disease

Celiac disease (CD) and diabetes mellitus type I (DM-I) are both autoimmune diseases. Abnormal first-phase insulin response (FPIR) is associated with the prediabetic phase. Glutamic acid decarboxylase (GAD) and islet cell antibodies (ICAs) – especially the tyrosine phosphatase-like protein IA-2 antibodies – are considered to be serological markers of DM-I future development. The aim of this study is to investigate the presence of autoantibodies (GAD, IA-2) in individuals with CD, on a gluten-free diet, who have normal intestinal morphology. Thirty patients with CD (4–22, mean 15 years), 30 newly diagnosed diabetic children (2.5–16, mean 10 years) and 30 healthy subjects (7–35, mean 18 years) were investigated. Serum GAD and IA-2 autoantibodies were assessed by a quantitative enzyme-linked immunosorbent assay (ELISA) method in all patients and controls. Seven CD patients (23%), 28 diabetic children (93%) and none in the control group had positive GAD and IA-2 antibodies. The FPIR was normal in CD patients (≥46 mU/l). Conclusions: GAD and IA-2 antibodies are detected in 23% of patients with CD. These patients may be at risk to develop DM-I. Regular follow-up and determination of FPIR for the early diagnosis of the prediabetic phase in patients with CD having circulating autoantibodies is recommended.