Autoimmune-mediated pancreatic beta-cell damage is central to the development of type 1 diabetes (T1DM) and its recurrence in islet transplant recipients. Proinflammatory cytokines and lipid mediators are important contributors to beta-cell damage. We have focused on the role of arachidonic acid metabolism by 12-Lipoxygenase (12LO) in leading to immune-mediated beta-cell destruction. 12LO can be activated and induced by proinflammatory cytokines. 12LO metabolites can initiate intracellutar signaling cascades leading to beta-cell dysfunction and death. In this proposal, we will test the hypothesis that the 12LO pathway plays a key role in autoimmunity-mediated inflammatory responses leading to beta-cell inhibition and death in T1DM.
The specific aims are: 1) to characterize 12LO activation and induction by proinflammatory cytokines in human islets and in insulin-releasing beta-cells. Study the role and mechanism of how the 12LO pathway mediates cytokine-induced beta-cell dysfunction and death. The particular type of 12LO in human islets will be characterized. The effect of molecular inhibition of 12LO using a ribozyme or over-expression on cytokine signaling and action will be tested. 2) to define the role of 12LO in cytokine-mediated beta-cell destruction in autoimmune diabetes. 12LO expression and lipid peroxide production will be monitored in autoimmune diabetic NOD mice. Cytokine-susceptibility and cytokine signaling processes will be determined in a 12LO-null mouse. We will also further study 12-null mouse generated on NOD background. Diabetes incidence, severeness of insulitis and cytokine effects on isolated islets will be tested. Mechanism of 12LO gene targeting reduces autoimmune beta-cell destruction will be evaluated. We will also identify the inflammatory signaling pathways disrupted upon 12LO depletion in these mice. 3) Effect of 12LO gene targeting on improving islet graft survival will be studied utilizing spontaneously diabetic NOD mice as recipients. Donor islets will be from 12LO null mice as well as the wild type mouse islets-treated with the 12LO ribozyme. Graft survival and reduction in autoimmune damage will be studied. The results from this completed proposal will advance our understanding of the inflammatory processes causing autoimmune beta-cell destruction. The findings should provide new methods to preserve beta-cell mass ultimately leading to new therapeutic modalities to prevent T1DM and improve ability to reverse T1DM using islet transplantation.
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