Tissue Analysis

项目总结(见说明)： 该U19提案的组织分析核心将提供在每个项目中获得的组织的已建立的组织学和分子分析。组织将在收集时进行处理，样本将根据标准协议进行分割和存储，该协议将由该核心开发和提供。戴维斯博士。 Schacker和Estes将在赠款期间根据需要开发专门的染色技术(即使用显色抗体或荧光抗体的双重或三重标记)。他们有适当的专业知识和实验室支持来实现这些目标。该核心将对每个项目获得的特定组织特征进行量化分析。这将包括数量 分析特定解剖位置的特定细胞群的大小(通过显色或荧光抗体染色识别)(例如，滤泡旁T细胞区中巨噬细胞的频率)。 来自这些分析的定量数据将与其他分析相结合，以确定免疫组织化学不容易识别的特定细胞表型的位置和频率(例如，将淋巴结中总的CD4+T细胞群的大小与测量初始的CD4+T细胞亚群的相对大小的流式细胞术数据相结合可以提供初始的CD4+T细胞的绝对大小的估计 该组织中的细胞)。核心将提供专门的技术来识别vDNA+或vRNA+细胞的位置和表型，包括原位杂交(vRNA+细胞)和原位聚合酶链式反应(vDNA+细胞)。将这两种原位技术中的任何一种与免疫组织化学相结合，可以识别被感染细胞的确切表型。将在脑、肺、肾、肝、肾上腺、胃肠道器官和整个肠道(口到肛门)进行全器官分析，以确定vRNA+细胞的位置，以帮助识别持久复制的储存库。将对所有组织进行分子分析，以测量VDNA+细胞的频率以及2-LTR环和整合DNA的频率。 总而言之，这些数据将被用来估计1)持续复制的位置，同时抑制ARV，以及2)潜伏感染的细胞储存库的大小和位置。

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.