Integrated morphologic, immunohistologic and molecular analyses of blood and tissue samples will aid the 3 individual projects and facilitate the development of the entire Program, as a result of the ongoing analysis and critical evaluation of data by an experienced investigator/pathologist. In each area, the Core will provide state-of-the-art support and seek project advancement by application of innovative techniques and technologies. We propose 3 ways in which the Core will support the program: 1. We hypothesize development of chronic rejection will be linked with distinct morphologic and immunologic features, and that modulation of their expression is of value in monitoring the efficacy of new therapeutic approaches. Host responses will be studied by histologic and immunohistologic methods, with analysis of fibrosis, vasculopathy, IgG deposition, and leukocyte infiltration; and associated expression of pro- and anti-inflammatory cytokines, chemokines/chemokine receptors, CTL proteins, signaling molecules and apoptotic and anti-apoptotic (""""""""protective"""""""") genes. 2. We hypothesize that morphologic and immunologic parameters preceding development of chronic rejection (specific aim 1) can be monitored by mRNA analysis of serial graft and blood samples. Real-time PCR (""""""""TaqMan"""""""") studies will be used to provide rapid, quantitative and specific data concerning circulating leukocyte and biopsy levels of a series of candidate genes whose expression is linked experimentally, or clinically, with the pathogenesis of alloantigen-dependent or independent graft injury and development of chronic rejection, and site of gene expression will be determined by in situ hybridization. 3. We hypothesize that important changes in gene expression may be detected in a subset of cells isolated by laser-capture microdissection (LCM), whereas such changes may be masked if additional cell types or whole biopsy samples were included in the analysis. LCM will be used to analyze the range and nature of genes expressed by vessel wall (endothelial and smooth muscle cells) vs. graft-infiltrating cells harvested in protocols in which serial analysis, or comparison of infiltrates in well-functioning vs. rejecting grafts, would be advantageous. The power of this application is unheralded since, in contrast to real-time PCR or other techniques, the precise site of gene expression wan be determined on morphologic grounds or through selecting cells of a specific phenotype based upon prior antibody labeling (e.g. picking just IL-2R+ cells). Collectively, functions of the Core will complement and integrate aspects of each project, such that ongoing and serial morphologic, immunopathologic and molecular studies will be undertaken efficiently and expertly.
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