Proinflammatory cytokines are believed to participate in the loss of functional islet mass as well as graft rejection following transplantation. Also complicating human islet transplantation is the lack of sufficient islet mass due, in part, to islet cell apoptosis following isolation. The broad goals of this research are to elucidate the biochemical mechanisms by which cytokines mediate pancreatic beta-cell destruction and to identify the mechanisms that mediate the loss of islet mass following isolation from cadaver donors. Endoplasmic reticulum (ER) stress activates an unfolded protein response (UPR) that is known to induce apoptosis. Recently, we have shown that nitric oxide, a primary mediator of the inhibitory actions of interleukin-1 (IL-1) and interferon-gamma(IFN-gamma) on rodent and human beta-cell function, is an activator of the UPR in beta-cells. This proposal will address the hypothesis that prolonged UPR activation in response to cytokine or nitric oxide treatment or stress during human islet isolation results in the apoptotic loss of pancreatic beta-cells. There are two specific aims: 1. To test the hypothesis that nitric oxide activates the UPR (or ER stress) pathway in human islets and that prolonged activation of this pathway results in the apoptotic loss of (-cells. Experiments proposed will evaluate the effects of cytokines and nitric oxide donors on UPR activation and determine if UPR inhibition protects beta-cells from cytokine-mediated death. 2. To test the hypothesis that islet damage during isolation induces ER stress and UPR activation, and that UPR activation is one mechanism responsible for the loss of islet mass during isolation. Proposed experiments will examine the effects of human islet isolation on UPR activation and determine whether UPR inhibition prevents or attenuates the loss of islets following isolation. A number of biochemical, molecular biological, immunological, and histochemical techniques will be utilized to investigate the role of UPR activation as one potential mediator of beta-cell apoptosis following cytokine treatment or following human islet isolation. It is hoped that insights into the mechanisms of cytokine-mediated beta-cell apoptosis, and the mechanisms associated with the loss of islet mass following isolation gained from these studies will influence the design of therapeutic strategies aimed at the attenuation of islet graft rejection and increase the functional mass of islets available for transplantation.
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