Further refinements in the technique we currently use to isolate human islets are proposed. The procedure will utilize standard enzymatic digestion combined with chemical and mechanical disruption of the exocrine pancreas to liberate islets from the dense fibrous stoma of the pancreas. We will generate additional monoclonal antibodies against human pancreatic acinar and ductal cells. These reagents will be used to lyse cells that generally contaminate isolated human islets, in order to obtain highly purified islet preparations that can be transplanted into the liver of patients with IDDM. Clinical transplantation trials, already initiated, will be expanded. In dogs we will expand our current effort to impair or kill the immunostimulatory cells within islets using anti-Ia monoclonal antibodies and newly generated anti-dendritic cell monoclonal antibodies. The immunomodulatory effects of these reagents as well as ultra-violet light irradiation will be tested in vitro in lymphocyte-islet lymphoproliferative assays and in vivo following islet allografting in pancreatectomized and spontaneously diabetic dogs. A putative autoimmune canine model of diabetes will be developed for these allotransplantation studies. We will continue to explore the mechanism of tolerance to islet alloantigens induced by cyclosporine in dogs. In vitro, we will study secretory kinetics and structural integrity of canine, porcine, and human islets that have been maintained in microcapsules in tissue culture for long periods of time. In vivo we will evaluate the metabolic responses of microencapsulated islet allografts and porcine xenografts in pancreatectomized and spontaneously diabetic dogs. We will also examine whether down-regulation of putative glucoreceptors, the mass of islet transplanted, or the site of transplantation influence long term functional survival of islet allografts in dogs. Finally, we will also define the relative importance of islet Ia-bearing monocytes/macrophages, dendritic cells, and Ia-bearing capillary endothelium on the immunostimulatory effects of human islets. Thus, the current proposal will address the potential problems that may limit the application of islet transplantation in humans namely (a) improvement in islet isolation and preservation, (b) prevention of rejection by immunomodulation of the graft and/or recipient, or by isolation of allografted islets from the host immune system, and (c) the possibility of recurrence of the autoimmune process in transplanted islets.
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