Although glutamic acid decarboxylase (GAD) has been implicated in IDDM, there is no direct evidence showing GAD-reactive T cells are diabetogenic in vivo. To address this issue, 3-wk-old NOD mice received two injections of purified rat brain GAD; one mouse rapidly developed diabetes 3 wk later. Splenocytes from this mouse showed a proliferative response to purified GAD, and were used to generate a CD4+ T cell line, designated 5A, that expresses TCRs encoding Vbeta2 and Vbeta12. 5A T cells exhibit a MHC restricted proliferative response to purified GAD, as well as GAD65 peptide 524-543. After antigen-specific stimulation, 5A T cells secrete IFNgamma and TNFalpha/beta, but not IL-4. They are also cytotoxic against NOD-derived hybridoma cells (expressing I-Ag7) that were transfected with rat GAD65, but not nontransfected hybridoma cells. Adoptive transfer of 5A cells into NOD/SCID mice produced insulitis in all mice. Diabetes occurred in 83% of the mice. We conclude that GAD injection in young NOD mice may, in some cases, provoke diabetes due to the activation of diabetogenic T cells reactive to GAD65 peptides. Our data provide direct evidence that GAD65 autoimmunity may be a critical event in the pathogenesis of IDDM.
Islet isolation is a time-consuming process. Islet yields vary, and previous in vitro studies suggest that Ficoll may be an islet toxin. Here, we describe an alternative, Ficoll-free method to purify murine islets by filtration through a cell strainer. Collagenase digestion of pancreata was carried out using standard procedures. The pancreatic digest was divided into aliquots and purified either by Ficoll or by filtration. Following filtration, islets were intact and separated from nondigested tissue. Purity was similar to that achieved using Ficoll. However, purification by filtration was faster, increased islet yield, and resulted in higher insulin secretion in vitro. Moreover, when syngeneic diabetic hosts were transplanted with a marginal islet mass, islets purified by filtration restored normoglycemia significantly faster than those isolated by Ficoll. This suggests that Ficoll exposure negatively impacts islet function. In conclusion, islet filtration is a simple and rapid procedure for purification of islets that demonstrate improved functional mass.
Whereas NF-B has potent antiapoptotic function in most cell types, it was reported that in pancreatic  cells it serves a proapoptotic function and may contribute to the pathogenesis of autoimmune type 1 diabetes. To investigate the role of  cell NF-B in autoimmune diabetes, we produced transgenic mice expressing a nondegradable form of I B␣ in pancreatic  cells (RIP-mI B␣ mice).  cells of these mice were more susceptible to killing by TNF-␣ plus IFN-␥ but more resistant to IL-1 plus IFN-␥ than normal  cells. Similar results were obtained with  cells lacking I B kinase , a protein kinase required for NF-B activation. Inhibition of  cell NF-B accelerated the development of autoimmune diabetes in nonobese diabetic mice but had no effect on glucose tolerance or serum insulin in C57BL/6 mice, precluding a nonphysiological effect of transgene expression. Development of diabetes after transfer of diabetogenic CD4 ؉ T cells was accelerated in RIP-mI B␣/nonobese diabetic mice and was abrogated by anti-TNF therapy. These results suggest that under conditions that resemble autoimmune type 1 diabetes, the dominant effect of NF-B is prevention of TNF-induced apoptosis. This differs from the proapoptotic function of NF-B in IL-1-stimulated  cells.
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