Tissue Analysis

组织分析

• 批准号: 8376044
• 负责人: Timothy W Schacker
• 金额: $ 73.65万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8376044
• 关键词: Acquired Immunodeficiency Syndrome; Address; Adrenal Glands; Anatomy; Antibodies; Anus; Architecture; Biological Assay; Blood; Brain; CCL19 gene; CCL20 gene; CD4 Positive T Lymphocytes; CXCL10 gene; Cell Size; Cells; Cellular Structures; Collaborations; Collection; DNA; Data; Data Analyses; Flow Cytometry; Fluorescent Antibody Technique; Frequencies; Goals; Grant; HIV; HIV Infections; Hematoxylin and Eosin Staining Method; Histocytochemistry; Histologic; Histology; Image Analysis; Immunohistochemistry; In Situ; In Situ Hybridization; Infection; Instruction; Interferon Type I; Interleukin-1; Interleukin-10; Interleukin-15; Interleukin-7; Kidney; Label; Laboratories; Lead; Letters; Ligands; Liver; Location; Lung; Macrophage Colony-Stimulating Factor; Measurement; Measures; Methods; Minnesota; Molecular; Molecular Analysis; Oral cavity; Organ; Pathologic; Pathology; Phenotype; Population; Process; Protocols documentation; RNA; Relative (related person); Research; Research Personnel; Research Support; SIV; Sampling; Site; Specific qualifier value; Staining method; Stains; Structure; Surveys; T-Lymphocyte; Techniques; Technology; Time; Tissues; Trichrome stain; Tryptophan 2,3 Dioxygenase; Universities; antiretroviral therapy; chemokine; collaboratory; human tissue; innovation; latent infection; latent persistent infection; lymph nodes; macrophage; performance site; viral DNA; viral RNA

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.

项目摘要（参见说明）： 该 U19 提案的组织分析核心将为每个项目中获得的组织提供既定的组织学和分子分析。将在收集时对组织进行处理，并根据该核心开发和提供的标准协议对样本进行分区和存储。博士。 Schacker 和 Estes 将在资助期内根据需要开发专门的染色技术（即使用显色或荧光抗体的双重或三重标记）。他们拥有适当的专业知识和实验室支持来实现这些目标。核心将对每个项目获得的组织的特定特征进行定量分析。这将包括定量 分析特定解剖位置中特定细胞群的大小（通过显色或荧光抗体染色来识别）（例如，滤泡旁 T 细胞区中巨噬细胞的频率）。 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易识别的特定细胞表型的位置和频率（例如，将淋巴结中总 CD4+ T 细胞群的大小与测量幼稚 CD4+ T 细胞子集的相对大小可以提供幼稚 CD4+ T 细胞绝对大小的估计 该组织中的细胞）。该核心将提供专门技术来识别 vDNA+ 或 vRNA+ 细胞的位置和表型，包括原位杂交（vRNA+ 细胞）和原位 PCR（vDNA+ 细胞）。将这两种原位技术与免疫组织化学相结合，可以鉴定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（口腔到肛门）中进行全器官分析，以确定 vRNA+ 细胞的位置，以协助识别持续复制的储存库。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。 总的来说，这些数据将用于估计 1) 抑制性抗逆转录病毒药物持续复制的位点，以及 2) 潜伏感染细胞库的大小和位置。