Multiplex Analysis of AIDS by Laser Scanning Cytometry

描述（由申请人提供）：对疾病中致病过程的机制理解建立在对细胞形态、蛋白质表达、RNA转录以及在某些情况下DNA分析的描述基础上。激光扫描细胞术是一种新技术，它结合了流式细胞术，图像分析，荧光和光学显微镜的特点，旨在确定在细胞和组织水平的致病事件的临床和研究应用。这些应用包括活检和血液标本的多色免疫表型表征、荧光原位杂交分析以及联合免疫表型和DNA/RNA含量分析。激光扫描细胞仪相对于其他模式的重要优势是能够分析非常小的细胞/组织样本，并能够重新分析这些样本（系列分析），从而将多个表型与单细胞分辨率相关联，对于该项目，我们将专注于开发用于研究新生恒河猴的猴-人免疫缺陷病毒（SHIV）感染的激光扫描细胞术，用于儿科AIDS的非人灵长类动物模型，其中活检/尸检样品的数量和大小有限。假设：这项研究的假设是，激光扫描细胞术可以用于结合联合收割机的病毒基因表达（原位杂交测量）和白细胞群体的变化（细胞免疫表型测量）在SHIV感染的动物的特征。因此，这种方法将有助于确定解释新生儿中猿类艾滋病的细胞机制。该假设将通过以下特定目的来解决：（1）开发和优化使用激光扫描细胞术对猕猴淋巴细胞进行免疫表型表征的方案，以及（2）开发和优化激光扫描细胞术的方案，用于检测先前已进行免疫表型表征的细胞内的病毒转录。重要性：该项目意义重大，因为我们为激光扫描细胞仪定义的基本协议将直接适用于受数量和大小限制的细胞和组织标本的有效和全面的表型表征。

DESCRIPTION (provided by applicant): A mechanistic understanding of pathogenic processes in disease builds upon descriptions of cellular morphology, protein expression, RNA transcription, and, in some instances, DNA analysis. Laser scanning cytometry is a novel technology which combines features of flow cytometry, image analysis, and fluorescence and light microscopy for clinical and research applications aimed at defining pathogenic events at the cell and tissue level. Such applications include multi-color immunophenotypic characterization of biopsy and hematological specimens, analysis of fluorescence in situ hybridization, as well as combined immunophenotyping and DNA/RNA content analysis. Important advantages of laser scanning cytometry over other modalities are the ability to analyze very small cell/tissue specimens and the capability to reanalyze such specimens (serial analysis), thereby correlating multiple phenotypes with single-cell resolution, For this project, we will focus on developing laser scanning cytometry for the study of simian-human immunodeficiency virus (SHIV) infection of newborn rhesus macaques, a non-human primate model for pediatric AIDS where biopsy/necropsy samples are limited in amount and size. Hypothesis: The hypothesis for this research is that laser scanning cytometry can be used to combine characterization of viral gene expression (measured by in situ hybridization) and changes in leukocyte populations (measured by cellular immunophenotype) in SHIV-infected animals. Accordingly, this method will help define the cellular mechanism(s) that accounts for simian AIDS in newborns. This hypothesis will be addressed through the following Specific Aims: (1) to develop and optimize protocols for immunophenotypic characterization of macaque lymphocytes using laser scanning cytometry, and (2) to develop and optimize protocols for laser scanning cytometry for detection of viral transcription within cells that have previously been immunophenotypically characterized. Significance: This project is significant because basic protocols that we define for laser scanning cytometry will have direct applicability for efficient and comprehensive phenotypic characterization of cell and tissue specimens limited by amount and size.