The purpose of the Pathology Core is to provide common facilities and expertise for the immunopathological studies in all three projects. The primary role of the pathological analyses in these allograft protocols is to document and characterize the status of the graft (nature of infiltrate, presence of acute or chronic rejection, including a humoral component) and the systemic effects of the protocols (toxicity, complications). In addition, the immunopathologic and molecular studies will give insights into the mechanisms of graft rejection and acceptance with protocol biopsies The techniques that will be utilized include routine histology, immunohistochemistry, immunofluorescence, electron microscopy, PCR, ELISPOT/cell culture, and flow cytometry. Immunoperoxidase techniques with a panel of mAbs will distinguish the infiltrating cell types, adhesion and cytokine molecules and receptors, and activation markers. Immunofluorescence will be used to localize the deposition of immunoglobulin and complement, as well as double/triple staining for cell markers using digital imaging. C4d stains will be used to detect humoral rejection. RT-PCR will be used to detect and quantify synthesis of key cytokines. Laser capture microdissection will be used for some of these studies in most of the projects to select the appropriate cells. Markers of apoptosis/DNA fragmentation (TUNEL) will assess cell injury. Culture of graft cells will be used with ELISPOT to correlate with the functional studies in the blood. Chimerism will be detected by flow cytometry techniques. The protocols are given in detail in each of the individual Projects. 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.
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