CORE--IMMUNOPATHOLOGY

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The purpose of Core A is to provide common facilities and expertise for the immunopathological studies in all projects of the Program Project. The primary role of the pathological analyses in these tolerance protocols is to document and characterize the status of the graft (presence or absence of rejection, the status of chimerism (cell type, distribution) and the systemic effects of the protocols (toxicity, complications). In addition, the immunopathological analysis will give insights into the mechanisms of graft rejection and acceptance, with protocol biopsies in all of the large animal and patient studies and provide essential data for the analysis of mechanisms of xenograft rejection. The techniques that will be utilized include routine histology, immunochemistry, immunofluorescence, in situ hybridization, electron microscopy, PCR, cell culture, and flow cytometry. Cryostat sections will be stained using immunoperoxidase techniques with a panel of mAbs to distinguish the infiltrating cell types, adhesion and cytokine molecules and receptors, and activation markers. Immunofluorescence will be used to localize the deposition of immunoglobin isotypes, complement and clotting components. In situ hybridization will be used to identify the site of synthesis of key cytokines. Cell injury will be assessed by markers of apoptosis/DNA fragmentation (TUNEL). Culture of graft cells will be used to correlate with the functional studies in the blood. Chimerism in the human and primate studies will be detected by PCR and flow cytometry techniques. The pathology features will be assessed and graded by the same group pathologists for all studies, which we consider an important asset of the Program Project. The panel of antibodies and cytokine probes will also be comparable, whenever possible. This should promote cross fertilization among the projects by extension of novel findings and provide insights into the general significance of the results in one project by comparing and contrasting grafts in different species and between different organs.