Further refinements in the technique we currently use to isolate human islets are proposed. The procedure will test several solutions constructed to stabilize cells during collagenase digestion, and will introduce a hydroclone device to facilitate bulk separation of liquid from solids in the final digestion slurp. We will continue to modify a special centrifuge that will have capacity sufficiently large to hold the total digestion slurp in a discontinuous gradient composed of Eurocollins-Ficoll and/or Eurocollins-Albumin. The-gradient will be emptied by activating a hydraulic system that permits the key gradient layers to be harvested directly into a sterile plastic bag that is used to infuse the islet preparations. Clinical transplantation trials, already initiated, will be expanded and the effects on islet allograft survival of several different immunosuppression regimens will be compared. We will also generate a series of canine monoclonal antibodies directed against canine dendritic cells and 'macrophages, as well as to T cell subsets. CD4 and CD8 peptides will be synthesized based on sequence analysis data obtained from cloned cDNAs which encode these canine lymphocyte antigens. The peptides will be used to generate anti-CD4 and anti-CD8 MoAbs. The anti-dendritic cell and anti-macrophage MoAbs will be used to immunomodulate canine islets, in vitro. The anti-CD4 and anti-CD8 MoAbs will be used for recipient immunotherapy, alone, in combinations, and in association with low dose cyclosporine or FK506. Dogs with pancreatectomy-induced diabetes will be used to evaluate the potential value of deletion of CD4 and/or CD8 T cells in vivo for immunosuppression and/or tolerization to islet allografts.
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