The objective of this proposal is to develop techniques for the immune alteration of endocrine tissues which will allow their transplantation to allogeneic recipients without immunosuppressive therapy. To accomplish this goal, we propose studies utilizing hyperbaric oxygen culture (HOC) to modulate graft immunogenicity and antigenicity. The study consists of four interrelated parts: 1) optimization of beta cell growth and differentiation with growth factors, 2) development and optimization of HOC conditions which maintain human fetal pancreas proislet (HFP) functional viability and eliminate tissue antigenicity, 3) development of the human PBL and fetal thymus reconstituted C.B-17 scid/scid model, and 4) characterization of the allograft response to HFP proislets and immunomodulation of the response by HOC. In part 1 we will use the growth factors IGF-II, human growth hormone, and prolactin in combination with IGF-I to enhance beta cell function during human fetal pancreas (HFP) proislet culture. Cellular viability will be determined by insulin release in response to challenge in vitro and in vivo (ability to reverse experimentally-induced diabetes in nude mice). In part 2 we will correlate oxidative stress during HOC with cell viability and reduced antigenicity. We will then use oxygen free radical scavengers to prevent cellular oxidative stress while maintaining reduced antigenicity. Cell viability will be determined as described above. Altered antigenicity and immunogenicity will be determined by cellular-ELISA, immunoperoxidase staining, CML, and MLC assay in vitro. Finally, in parts 3 and 4 we will develop the human-SCID mouse chimeric model. Reconstitution extent will be determined by human antibody production, response to T and B cell mitogens, human skin graft rejection, and human DNA dot blot analysis. We will then use the human-SCID mouse model to characterize the human allograft response to HFP proislet grafts and the ability of HOC to modify the response.
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