Pradeep B. Parab

Beneficial effect of supplementation with copper sulfate on STZ-diabetic mice (IDDM).

Trace element status is known to be altered in the diabetic state. Current evidence suggests that reactive oxygen intermediates (ROIs) do participate in the destruction of pancreatic beta cells in STZ-induced Type 1 diabetes. The copper and zinc status of diabetic patients and their descendants was also found to have changed. The aim of this study was to evaluate the potential usefulness of copper sulfate in the treatment of STZ-induced type I diabetes. We used C57BL6 female mice, in whom copper sulfate treatment was started at 6 weeks of age followed by an IP injection of 40 mg/kg body weight of STZ for five consecutive days. Its effects were evaluated at 10 weeks of age. The treatment with copper sulfate significantly decreased blood glucose levels, levels of lipid peroxidation and mRNA expression of the enzyme iNOS and the cytokines IFN-gamma and IL-4. Histological analysis of the pancreas revealed that, three out of five animals in the copper sulfate treated groups showed absence of mononuclear cell infiltration and no change in the shape and size of islets as compared to pancreas of the STZ-induced diabetic group of animals. In conclusion, our observations indicate that copper sulfate treatment can exert beneficial effects in diabetes with preservation of beta-cell function in vivo. Copper sulfate could be exerting these beneficial effects either by acting directly by reducing free radicals or through reduction in glucose levels.

Human Neutrophil-Expressed CD28 Interacts with Macrophage B7 to Induce Phosphatidylinositol 3-Kinase-Dependent IFN-γ Secretion and Restriction of Leishmania Growth

We previously showed that CD28 is expressed on human peripheral blood neutrophils and plays an important role in CXCR-1 expression and IL-8-induced neutrophil migration. In this work we demonstrate that Leishmania major infection of macrophages results in parasite dose-dependent IL-8 secretion in vitro and in IL-8-directed neutrophil migration, as blocked by both anti-IL-8 and anti-IL-8R Abs, toward the L. major-infected macrophages. In the neutrophil-macrophage cocultures, both CTLA4-Ig, a fusion protein that blocks CD28-CD80/CD86 interaction, and a neutralizing anti-IFN-γ Ab inhibit the anti-leishmanial function of neutrophils, suggesting that the neutrophil-macrophage interaction via CD28-CD80/CD86 plays an important role in the IFN-γ-dependent restriction of the parasite growth. Cross-linking of neutrophil-expressed CD28 by monoclonal anti-CD28 Ab or B7.1-Ig or B7.2-Ig results in phosphatidylinositol 3-kinase association with CD28 and in wortmannin-sensitive but cyclosporin A-resistant induction and secretion of IFN-γ. Whereas the neutrophils secrete IFN-γ with CD28 signal alone, the T cells do not secrete the cytokine in detectable amounts with the same signal. Thus, neutrophil-expressed CD28 modulates not only the granulocyte migration but also induction and secretion of IFN-γ at the site of infection where it migrates from the circulation.

Immunobiology of CD28 expression on human neutrophils. I. CD28 regulates neutrophil migration by modulating CXCR-1 expression.

CD28, described as a T cell costimulatory molecule so far, is expressed on human peripheral blood neutrophils, as shown by cell surface staining and immunoprecipitation with anti‐CD28 monoclonal antibody, and by reverse transcription PCR. The phorbol 12‐myristate 13‐acetate‐augmented expression of CD28 on these cells can be blocked by actinomycin D, an RNA transcription inhibitor, and staurosporin, a protein kinase inhibitor. Cross‐linking of CD28 results in an early increase in IL‐8 receptor A (IL‐8RA or CXCR‐1) expression and a concurrent increase in IL‐8‐induced chemotaxis. The expression of CXCR‐1 is down‐regulated by receptor internalization 3 h after CD28 cross‐linking with concurrent decrease in IL‐8‐induced chemotactic migration. Thus, our results demonstrate for the first time that CD28 is expressed on human peripheral blood neutrophils and that CD28 may play an important role in the regulation of IL‐8RA expression and migration of neutrophils in response to IL‐8.

A novel CD28 mRNA variant and simultaneous presence of various CD28 mRNA isoforms in human T lymphocytes.

The primary transcript of the human CD28 gene in T lymphocytes, encoding for a costimulatory molecule, is known to undergo alternative splicing, and different small sets of variant isoforms have been reported. This report presents the novel simultaneous presence of eight different mRNA isoforms, all observed together in normal human T cells; this is an interesting finding in the study of CD28 mRNA structural variants. A similar pattern was found in a total of four individuals. In addition, we also report the occurence and sequence of a new CD28 mRNA isoform, one of the above eight, which is a novel variant generated by the use of a new combination of splice donor and acceptor sites.

The Th1-Specific Costimulatory Molecule, M150, Is a Posttranslational Isoform of Lysosome-Associated Membrane Protein-1

In an earlier report, we had shown a 150-kDa protein termed as M150, isolated from the surface of activated macrophages, to possess costimulatory activity for CD4+ T cells. Significantly, this protein was found to specifically elicit Th1 responses. In this study, we characterize M150, which belongs to a unique subset of the lysosome-associated membrane protein-1 glycoprotein. Interestingly, the costimulatory activity of M150 depends on its posttranslational modification, which has a distinct glycosylation pattern restricted to macrophages. Furthermore, it has been demonstrated that in addition to stimulating Th1-specific responses, M150 is also capable of driving differentiation of naive CD4+ T cells into the Th1 subset. This altered posttranslational modification of housekeeping protein appears to represent a novel pathway by which APCs can additionally regulate T cell responses.

Regulation of GAD65 expression by SMAR1 and p53 upon Streptozotocin treatment.

BackgroundGAD65 (Glutamic acid decarboxylase 65 KDa isoform) is one of the most important auto-antigens involved in Type 1 diabetes induction. Although it serves as one of the first injury markers of β-islets, the mechanisms governing GAD65 expression remain poorly understood. Since the regulation of GAD65 is crucial for the proper functioning of insulin secreting cells, we investigated the stress induced regulation of GAD65 transcription.ResultsThe present study shows that SMAR1 regulates GAD65 expression at the transcription level. Using a novel protein-DNA pull-down assay, we show that SMAR1 binding is very specific to GAD65 promoter but not to the other isoform, GAD67. We show that Streptozotocin (STZ) mediated DNA damage leads to upregulation of SMAR1 and p53 expression, resulting in elevated levels of GAD65, in both cell lines as well as mouse β-islets. SMAR1 and p53 act synergistically to up-regulate GAD65 expression upon STZ treatment.ConclusionWe propose a novel mechanism of GAD65 regulation by synergistic activities of SMAR1 and p53.

An in vitro model for screening oral hypoglycemics.

Dear Editor: The majority of noninsulin-dependent type II diabetics are treated with oral hypoglycemics which induce insulin secretion from [3-cells and thus control the hyperglycemia. Therefore, usually while developing these drugs, studies are carried out in whole animals. We have explored the possibility of developing an in vitro model with isolated islets for testing insulin secretagogue activities of oral hypoglycemics. Most of the oral hypoglycemics available in the market and the few of those being developed (3,4,8-10) are insulin secretagogues with exception of biguanides (10). The model has been validated with well established hypoglycenfic drugs such as tolbutamide and glibenclamide and was also tested on a new potential hypoglycemic drug, linogliride (8,9). For in vivo studies, Sprague-Dawley male rats weighing 150 to 200 g were obtained from Haffkine Institute, Bombay (India). The animals were fasted overnight and were given either saline (control group) or glucose solution (1 g per kg body wt) orally. Thirty rain after saline or glucose feeding, experimental groups were given 1 ml of 10% suspension of either tolbutamide (groups II and V, Table 1) or linogliride (groups III and VI, Table 1) per kg body wt in normal saline, i.e., 100 mg per kg body wt. Half an h after giving oral hypoglycemics (maximum response to oral hypoglycemics, results not shown), blood was taken from the tail vein for estimation of glucose by the glucose oxidase method (7) and insulin by radioimmunoassay (RIA) (1). The effect of two oral hypoglycemics was also studied on clonidine-induced hyperglycemia (Table 2). For in vitro studies, islets were isolated from Sprague-Dawley rat pancreases by an adaptation of the method of Gotoh et al. (5). Briefly pancreases were distended by injecting 0.1% eollagenase with