A. L. Notkins

Congenital rubella syndrome as a model for Type 1 (insulin-dependent) diabetes mellitus: increased prevalence of islet cell surface antibodies

SummaryAn increased prevalence of Type 1 (insulin-dependent) diabetes has been reported in patients with congenital rubella. Rubella virus multiplies in the pancreas, and we have hypothesized that studies of children with congenital rubella would be of great importance in following the development of Type 1 diabetes in a defined, susceptible population. Two hundred and forty-one children with congenital rubella (mean age 17.4±0.3 years; 65% black and hispanic) have been evaluated, 30 of whom already have diabetes and 17 of whom have borderline glucose tolerance. In these latter two groups, HLA-DR3 is significantly increased and HLA-DR2 significantly decreased. Pancreatic islet cell cytotoxic surface antibodies are found in 20% of the total congenital rubella population, including in more than 50% in the time period before they develop diabetes and are not related to any specific HLA type. In addition, anti-microsomal and anti-thyroglobulin antibodies are found in 34% of this population. The data demonstrate that Type 1 diabetes developing in congenital rubella patients has the genetic and immunological features of classical Type 1 diabetes, namely the presence of HLA-DR3, the absence of HLA-DR2, islet cell surface antibodies before decompensation and an increased prevalence of anti-thyroid antibodies. Patients with non-diabetic congenital rubella represent an easily identifiable group in whom other immunological factors associated with Type 1 diabetes can be elucidated and possibly modified.

Prevention of ultraviolet-light-induced herpes labialis by sunscreen

Sunlight exposure is reported by some patients to precede onset of recurrent herpes labialis. Ultraviolet (UV) B light is known to be a stimulus for the reactivation of herpes simplex virus (HSV) infections. We assessed the effect of a sunblocking agent on UV-light-induced reactivation of recurrent herpes labialis in a double-blind, placebo-controlled crossover trial. 38 patients were exposed on two separate occasions to four minimum erythema doses of UV light at an area of previous labial herpes recurrence. A solution containing sunscreen was applied to the lips before one exposure and a matched placebo before the other. After placebo and UV exposure, herpes labialis developed in 27 (71%) of the 38 patients, with a mean time to recurrence of 2.9 (SEM 0.2) days. In contrast, when sunscreen was applied before UV exposure, no lesions developed, but 1 of the 35 patients shed virus at the exposure site. We conclude that UV light is a potent stimulus for inducing reactivation of herpes labialis, and that application of sunscreen may be effective in the prevention of sunlight-induced recurrent infection.

The IA-2 gene family: homologs in Caenorhabditis elegans, Drosophila and zebrafish

Aims/hypothesis. IA-2 and IA-2β are major autoantigens in Type I (insulin-dependent) diabetes mellitus and are expressed in neuroendocrine tissues including the brain and pancreatic islets of Langerhans. Based on sequence analysis, IA-2 and IA-2β are transmembrane protein tyrosine phosphatases but lack phosphatase activity because of critical amino acid substitutions in the catalytic domain. We studied the evolutionary conservation of IA-2 and IA-2β genes and searched for homologs in non-mammalian vertebrates and invertebrates.¶Methods. IA-2 from various species was identified from EST sequences or cloned from cDNA libraries or both. Expression in tissues was determined by transfection and in situ hybridization.¶Results. We identified homologs of IA-2 in C. elegans, Drosophila, and zebrafish which showed 46, 58 and 82 % identity and 60, 65 and 87 % similarity, respectively, to the amino acids of the intracellular domain of human IA-2. Further studies showed that IA-2 was expressed in the neural tissues of the three species. Comparison of the genomic structure of the intracellular domain of human IA-2 with that of human IA-2β showed that they were nearly identical and comparison of the intron-exon boundaries of Drosophila IA-2 with human IA-2 and IA-2β showed a high degree of relatedness.¶Conclusion/Interpretation. Based on these findings and sequence analysis of IA-2 homologs in mammals, we conclude that there is an IA-2 gene family which is a part of the larger protein tyrosine phosphatase superfamily. The IA-2 and IA-2β genes represent two distinct subgroups within the IA-2 family which originated over 500 million years ago, long before the development of the pancreatic islets of Langerhans. [Diabetologia (2001) 44: 81–88]

Deletion of Ia-2 and/or Ia-2β in mice decreases insulin secretion by reducing the number of dense core vesicles

Aims/hypothesisIslet antigen 2 (IA-2) and IA-2β are dense core vesicle (DCV) transmembrane proteins and major autoantigens in type 1 diabetes. The present experiments were initiated to test the hypothesis that the knockout of the genes encoding these proteins impairs the secretion of insulin by reducing the number of DCV.MethodsInsulin secretion, content and DCV number were evaluated in islets from single knockout (Ia-2 [also known as Ptprn] KO, Ia-2β [also known as Ptprn2] KO) and double knockout (DKO) mice by a variety of techniques including electron and two-photon microscopy, membrane capacitance, Ca2+ currents, DCV half-life, lysosome number and size and autophagy.ResultsIslets from single and DKO mice all showed a significant decrease in insulin content, insulin secretion and the number and half-life of DCV (p < 0.05 to 0.001). Exocytosis as evaluated by two-photon microscopy, membrane capacitance and Ca2+ currents supports these findings. Electron microscopy of islets from KO mice revealed a marked increase (p < 0.05 to 0.001) in the number and size of lysosomes and enzymatic studies showed an increase in cathepsin D activity (p < 0.01). LC3 protein, an indicator of autophagy, also was increased in islets of KO compared with wild-type mice (p < 0.05 to 0.01) suggesting that autophagy might be involved in the deletion of DCV.Conclusions/interpretationWe conclude that the decrease in insulin content and secretion, resulting from the deletion of Ia-2 and/or Ia-2β, is due to a decrease in the number of DCV.

Multiple autoantibodies as predictors of type 1 diabetes in a general population

a Age when samples were collected b Initially autoantibody negative, but became autoantibody positive 3 years later Fig. 1. Autoantibodies in 55 ICA-positive subjects. Shaded areas show number of subjects who progressed to Type 1 diabetes in the presence of each of the autoantibodies Multiple autoantibodies as predictors of Type 1 diabetes in a general population At the initial screening, 55 children were ICA positive. These children were then tested for autoantibodies to insulin, GAD, IA-2 and IA-2β. Of the 55 children positive for ICA 13 also had antibodies to insulin, 18 to GAD, 13 to IA-2 and 8 to IA-2β (Fig. 1). Of the 55 ICA-positive children, 11 progressed to Type 1 diabetes. Of these 11 ICA-positive children, 6 had autoantibodies to insulin, 10 to GAD, 9 to IA-2 and 7 to IA-2β. During the course of the study, only one ICA-negative child developed Type 1 diabetes. Table 1 shows the autoantibody profiles of the 11 ICA-positive children who developed Type 1 diabetes. All had multiple autoantibodies at the initial screening. Clinical disease developed 3 months to 10 years later.

The IA-2 interactome

Aims/hypothesisIslet antigen-2 (IA-2), a major autoantigen in type 1 diabetes, is an enzymatically inactive member of the transmembrane protein tyrosine phosphatase (PTP) family. IA-2 is located in dense-core secretory vesicles and is involved in the regulation of insulin secretion. The present experiments were initiated to identify those proteins that interact with IA-2 (i.e. the IA-2 interactome) as a first step towards elucidating the mechanism(s) by which IA-2 influences insulin secretion and serves as an autoantigen.Materials and methodsTo determine the proteins with which IA-2 interacts, a yeast two-hybrid system was used to screen a human foetal library, and deletion mutants were used to determine the binding sites. Positive interactions were confirmed by immunoprecipitation pull-down experiments using cell lysate from transfected mammalian cell lines.ResultsSix new interacting proteins were identified by this approach: mitogen-activated protein kinase-activating death domain (MADD), the MADD isoform IG20, PTPρ, PTPσ, sorting nexin 19 (SNX19) and cyclophilin A. Using a series of IA-2 deletion mutants, we identified the regions on the IA-2 molecule to which five of the interacting proteins bound. Amino acids 744–979 of IA-2 were required for the maximum binding of MADD, IG20 and SNX19, whereas amino acids 602–907 of IA-2 were required for the maximum binding of PTPρ and PTPσ. Pull-down experiments with cell lysate from transfected mammalian cells confirmed the binding of the interacting proteins to IA-2.Conclusions/interpretationThe IA-2 interactome based on, pull-down experiments, currently consists of 12 proteins. The identification of these interacting proteins provides clues as to how IA-2 exerts its biological functions.

Overexpression of the autoantigen IA-2 puts beta cells into a pre-apoptotic state: autoantigen-induced, but non-autoimmune-mediated, tissue destruction.

IA‐2 is a major autoantigen in type 1 diabetes and autoantibodies to it have become important diagnostic and predictive markers. IA‐2 also is an intrinsic transmembrane component of dense core secretory vesicles and knock‐out studies showed that IA‐2 is a regulator of insulin secretion. Here we show that overexpression of IA‐2 puts mouse insulinoma MIN‐6 beta cells into a pre‐apoptotic state and that exposure to high glucose results in G2/M arrest and apoptosis. Molecular study revealed a decrease in phosphoinositide‐dependent kinase (PDK)‐1 and Akt/protein kinase B (PKB) phosphorylation. Treatment of IA‐2‐transfected cells with IA‐2 siRNA prevented both G2/M arrest and apoptosis and increased Akt/PKB phosphorylation. A search for IA‐2 interacting proteins revealed that IA‐2 interacts with sorting nexin (SNX)19 and that SNX19, but not IA‐2, inhibits the conversion of PtdIns(4,5)P2 to PtdIns(3,4,5)P3 and thereby suppresses the phosphorylation of proteins in the Akt signalling pathway resulting in apoptosis. We conclude that IA‐2 acts through SNX19 to initiate the pre‐apoptotic state. Our findings point to the possibility that in autoimmune diseases, tissue destruction may be autoantigen‐induced, but not necessarily immunologically mediated.