Tissue Analysis

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PROJECT SUMMARY (See instructions): The Tissue Analysis Core of this U19 proposal will provide established histologic and molecular analyses of tissues obtained in each of the projects. Tissues will be processed at the time of collection with samples partitioned and stored according to standard protocols that will be developed and supplied by this Core. Drs. Schacker and Estes will develop specialized staining techniques (i.e., double or triple labels using either chromagenic or fluorescent antibodies) as needed over the duration of the grant period. They have appropriate expertise and laboratory support to accomplish these goals. The Core will provide quantitative analysis of specified features of tissues obtained by each of the projects. This will include quantitative analysis of the size of specific cell populations (identified by chromagenic or fluorescent antibody staining) in a specified anatomic location (e.g., the frequency of macrophages in the parafollicular T cell zone). Quantitative data from these analyses will be combined with other analyses to identify location and frequency of specific cell phenotypes not readily identified by immuno-histochemistry (e.g., combining the size of the total CD4+ T cell population in lymph node with flow cytometry data that measures the relative size of the naive subset of CD4+ T cells can provide an estimate of the absolute size of the naive CD4+ T cell in that tissue). The Core will provide specialized techniques for identifying the location and phenotype of cells that are either vDNA+ or vRNA+, including in situ hybridization (vRNA+ cells) and in situ PCR (vDNA+ cells). Combining either of these two in situ techniques with immunohistochemistry allows identification of the exact phenotype of cells that are infected. Whole organ analysis to identify locations of vRNA+ cells will be done in brain, lung, kidney, liver, adrenals, organs of the GU tract, and the entire gut (mouth to anus) to assist in identification of reservoirs of persistent replication. Molecular analyses of all tissues will be performed to measure the frequency of vDNA+ cells and the frequency of 2-LTR circles and integrated DNA. Collectively these data will be used to estimate 1) sites of persistent replication while on suppressive ARV and 2) size and location of reservoirs of cells harboring latent infection.