(taken from the application) Cytokine-mediated pancreatic beta cell toxicity is likely to play an important role in the pathogenesis of Type 1 diabetes and islet transplantation rejection. The mechanisms by which cytokines induce damage in beta cells are starting to be elucidated, and free radical/lipid hydroperoxide formation appears to be an important factor. Our work has focused on the 12-lipoxygenase (12-LO) enzyme pathway that is found specifically in islet beta cells, but not alpha or delta cells. 12-LO is induced by cytokines and oxidant stress, and leads to the generation of lipid hydroperoxides in pancreatic beta cells. In addition, metabolites of the 12-LO pathway can act as intracellular signals that regulate the redox-sensitive transcription factor NF-kappa-B and also may be involved in programmed cell death. In this proposal, we will test the hypothesis that inhibition of the 12-LO pathway with a novel molecular tool called a ribozyme will protect pancreatic beta cells from cytokine-induced dysfunction and cytotoxicity. We will compare the efficiency of adenovirus (AdV) versus adeno-associated virus (AAV) in transferring a 12-LO ribozyme to pancreatic islets and beta cells. We will first test the efficacy of AdV and AAV vectors in transferring the reporter genes beta-galactosidase and placental alkaline phosphatase. We will then examine the effects of viral vector based delivery of the 12-LO ribozyme (12-LORz) and a catalytically inactive ribozyme (12-LOMRz) to islets and beta cells. In addition, porcine pancreatic islets will be treated with 12-LORz and 12-LOMRz and implanted into the kidney subcapsular space of streptozotocin-induced diabetic rats. Islets implanted will be compared for degree of acute graft rejection. As an alternative approach to understanding the effects of 12-LO inhibition on cytokine-induced islet cytotoxicity, we will utilize a 12-LO knockout mouse model. Islets will be purified from knockout mice and controls and compared fo sensitivity to cytokines and activation of stress-induced kinases. Finally, we will overexpress 12-LO in MIN6 cells with a tetracycline-inducible promoter in order to determine the effects of excess 12-LO products on pancreatic beta cel function and cytotoxicity. The data generated in this grant will help to determine whether AAV can efficiently transfer genes to pancreatic islets and provide evidence supporting the role for the 12-LO pathway in immune mediated islet cell destruction.
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