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. We also have a well annotated Cynomolgus tissue bank of frozen and paraffin embedded tissue available from our previous transplantation experiments (>5000 samples), which can be drawn upon to expand and refine our analysis. The techniques that will be utilized include routine histology, immunohistochemistry, immunofluorescence, electron microscopy and digital slide imaging morphometry. 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. Markers of apoptosis/DNA fragmentation (TUNEL) will assess cell injury. The protocols are given in detail in each of the individual Projects. The pathology studies in the Core are essential to evaluate the status of the organ allografts (heart, lung and kidney) and the effects of the intervention to reduce inflammation and parenchymal injury. The studies are critical to determine the nature and location of the cells infiltrating the graft, the role of antibodies in rejection and systemic toxicity of treatment. The pathology results will be correlated with functional and molecular studies done in the projects and other cores. In addition, complete necropsies will be done on all animals, with samples from all major organs analyzed by routine and special techniques as indicated. We will compare the inflammatory molecular and cellular events in allografts in the different protocols. Serial samples of the graft and lymph nodes, starting on the day of transplant, will be assessed to provide insights into mechanisms of action of the treatment and tolerance induction. The immunopathologic studies are designed with a minimum of tissue sample (2 small blocks, typically 2x2x2 mm) processed for light microscopy and immunohistochemistry with formalin/paraffin techniques and in glutaraldehyde/paraformaldehyde/epon for one-micron sections and electron microscopy.
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