Virus-induced diabetes in mice: A quantitative evaluation of islet cell population by immunofluorescence technique

Summary The endocrine cell populations of pancreatic islets in encephalomyocarditis (EMC)-virus infected mice were assessed quantitatively by immunofluorescence using specific antisera against 4 islet hormones. A marked reduction of the volume of insulin-containing (B-) cells (up to one tenth of control values) was observed at all stages studied in the hyperglycaemic mice. This was accompanied by the inversion of the normal ratio between B- and non B-cells. The volume of the latter cell types was also modified at different time points after infection: glucagon-cells were augmented 14 days after infection; PP-cells were decreased 2–3 days and 21 days after infection; somatostatin-cells decreased to one-fourth of control values in hyperglycaemic animals 21 days after infection. The latter results suggest that non B-cells are also involved in islet reaction to virus infection.     

UV light-induced reactivation of herpes simplex virus type 2 and prevention by acyclovir.

UV B light is a potent stimulus for inducing reactivation of latent herpes simplex virus (HSV) infections. Patients were enrolled in a double-blind placebo-controlled crossover trial to determine whether acyclovir can prevent UV light-induced HSV-2 recurrences. Twenty-four patients with a history of recurrent infection of perigenital sites (e.g., buttock, thigh) were exposed one to four times with 4 minimum erythema doses of UV light. Patients were given acyclovir 200 mg orally five times daily or matched placebo beginning 1 day before each exposure and continuing for 5 days after exposure. There were 13 UV-induced recurrences among 36 placebo treatments and 3 after 38 acyclovir treatments (P = .004). The mean time to recurrence (+/- SE) was 4.8 +/- 0.3 days. HSV-2 lesions developed primarily at the site of UV exposure. The cutaneous distribution and timing of UV-induced recurrences was consistent with a neural localization (dorsal root ganglia) of latent viral infection. This UV light model permits direct examination of events leading to HSV-2 recurrences in humans and can be used to evaluate approaches to prevention.     

ELEVATED γ-GLOBULIN AND INCREASED ANTIBODY PRODUCTION IN MICE INFECTED WITH LACTIC DEHYDROGENASE VIRUS

Infection of mice with the lactic dehydrogenase virus (LDV) resulted in an elevated level of γ-globulin. Histologic examination of the spleen and lymph nodes revealed that the number of germinal centers was greatly increased. Immunization with human γ-globulin showed that the capacity of the virus-infected animal to produce anti-human γ-globulin was greatly enhanced and that the virus acted as an adjuvant. From these experiments it is concluded that a virus infection (LDV) can affect the immunologic response of the host to a heterologous antigen.     

Antibodies to IA-2 and GAD65 in type 1 and type 2 diabetes: isotype restriction and polyclonality

OBJECTIVE: To determine the isotypes and clonality of antibodies to GAD (GADA) and IA-2 (IA-2A) in patients with type 1 and type 2 diabetes. RESEARCH DESIGN AND METHODS: We studied the following consecutive series of patients who attended a diabetes center for antibodies to GADA and IA-2A: 52 newly diagnosed type 1 diabetic patients, 199 type 2 diabetic patients, 200 control patients, and a cohort of 34 nondiabetic identical twins of patients with type 1 diabetes (15 of whom developed diabetes) who were followed prospectively. RESULTS: GADA or IA-2A were detected in 37 (71%) type 1 diabetic patients compared with only 10 (5%) type 2 diabetic patients (P<0.0001). Both GAD and IA-2 antibodies, regardless of the type of diabetes, were usually subclass restricted to IgG1 and were polyclonal. IgM, IgG3, and IgE isotypes were also detected, but all isotypes of GADA and IA-2A were less prevalent than IgG1 (P<0.017 for either antibody). There was no evidence of spreading or switching of isotypes before the onset of type 1 diabetes. CONCLUSIONS: These observations suggest that the pathogenesis of antigen-specific antibodies in type 1 and type 2 diabetes is similar and probably involves a chronic nonrandom antigen-driven polyclonal B-cell activation that is consistent with a Th1-type immune response.     

STUDIES ON THE MULTIPLICATION AND THE PROPERTIES OF THE LACTIC DEHYDROGENASE AGENT

The procedure used to determine the infective titer of the LDH agent, the reproducibility of this assay, and the relationship between virus dose and plasma enzyme activity were described. Multiplication of the LDH agent began within 6 hours after infection and reached 10^10.8 ID₅₀/ml of plasma within 24 hours. The titer rapidly decreased over the next 72 hours but viremia persisted for at least 16 months with titers as high as 10^5.2 ID₅₀/ml. The appearance of the LDH agent in the circulation preceded the first noticeable rise in plasma LDH activity by close to 24 hours. After 10 months, when the plasma titer of the LDH agent had decreased nearly one millionfold, the plasma enzyme LDH had decreased by less than 50 per cent. The LDH agent is inactivated by ether but withstands lyophilization, and freezing and thawing. It is stable at low temperatures. Ultracentrifugation at 105,000 G for 2 hours leaves less than 0.1 per cent of the LDH agent in the supernatant fluid and filtration through gradocol membranes suggesst that the upper size of the LDH agent is about 55 mµ. Spread of the LDH agent from infected to uninfected mice kept in the same cage and transmission from mothers (infected prior to mating) to their offspring was relatively low.     

Kinetics of sensitization of herpes simplex virus and its relationship to the reduction in the neutralization rate constant

Incubation of herpes simplex virus (HSV) with rabbit anti-HSV for as little as 2.5 minutes rendered 75% of the surviving fraction neutralizable by anti-rabbit-gamma-globulin antiserum. The degree of this sensitization increased with time and at 20 minutes over 99.8% of the surviving virus was sensitized. Neutralization kinetics showed that as the degree of sensitization increased, the neutralization rate constant decreased. Incubation of HSV with anti-HSV for 5, 20, and 120 minutes resulted, respectively, in a 23%, 59%, and 87% reduction in the neutralization rate constant. These findings suggest that sensitization is an important factor in the inhibition of neutralization and the formation of the persistent fraction.     

Immunological heterogeneity in Type I diabetes: presence of distinct autoantibody patterns in patients with acute onset and slowly progressive disease

Summary Type I diabetes mellitus may represent a heterogeneous disorder with a distinct pathogenesis in patients with young and adult onset of the disease. To investigate whether serological markers directed to different autoantigens have the potential to distinguish acute onset from slowly progressive Type I diabetes we analysed antibodies to tyrosine phosphatases IA-2/ICA512 (IA-2A) and IA-2β/phogrin (IA2βA), antibodies to GAD65 (GADA) and cytoplasmic islet cell antibodies (ICA) in a non-selected group of diabetic patients clinically classified as having Type I or Type II diabetes at diagnosis. Both IA-2A and IA-2βA were found to be positively associated with onset before the age of 20 years and the presentation of classical features of Type I diabetes. In Type I diabetes 56 % (112/200) of patients were positive for IA-2A and 38 % (76/200) for IA-2βA. In contrast, only 1 of 785 (0.1 %) patients with Type II diabetes had IA-2A and all of them were negative for IA-2βA (p < 0.001). Among the patients with Type II diabetes 7.6 % (n = 60) were ICA positive and 2.8 % (n = 22) had GADA suggesting the presence of slowly progressive Type I diabetes. GADA were found in 8 of 60 (13.3 %) ICA positive subjects which was lower than the percentage detected in patients with acute onset of diabetes (115/157 73.2 %) (p < 0.001). Blocking of double antibody positive sera showed that only 3 of 8 (37.5 %) patients with slowly progressive diabetes had ICA restricted to GAD or IA-2 whereas ICA were completely inhibited in 12 of 20 (60.0 %) patients with Type I diabetes. Among 193 patients with Type II diabetes available for follow-up, 35 % of ICA positives, 58 % of GADA positives and 60 % of those positive for both markers required insulin by 3 years. However, using strict criteria for the switch to insulin treatment the corresponding sensitivity of each marker was only low (9 %, 10 % and 5 %). We show that clinical subtypes of Type I diabetes are associated with distinct humoral autoimmunity. IA-2A and GADA were associated with classical features of Type I diabetes whereas GADA and an uncharacterized ICA subspecificity indicate slowly progressive disease. [Diabetologia (1998) 41: 891–897] Received: 15 January 1998 and in revised form: 10 April 1998     

IA-2 and IA-2β: 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-2β. IA-2 is a 979 amino acid transmembrane protein located on human chromosome 2q35, whereas IA-2β is 986 amino acids long located on human chromosome 7q36. Comparison of human IA-2 and IA-2β 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-2β 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-2β 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-2β 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. © 1998 John Wiley & Sons, Ltd.     

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). Materials and Methods: 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. Results: 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. Conclusions: 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)