Multiplex Analysis of AIDS by Laser Scanning Cytometry

描述(申请人提供)：对疾病致病过程的机械理解建立在对细胞形态、蛋白质表达、RNA转录的描述基础上，在某些情况下，还包括DNA分析。激光扫描细胞术是一种结合了流式细胞术、图像分析、荧光和光学显微镜的新技术，用于临床和研究，目的是在细胞和组织水平上确定致病事件。这些应用包括活检和血液学标本的多颜色免疫表型表征，荧光原位杂交分析，以及结合免疫表型和DNA/RNA含量分析。与其他方式相比，激光扫描细胞术的重要优势是能够分析非常小的细胞/组织样本和重新分析此类样本的能力(连续分析)，从而将多种表型与单细胞分辨率相关联。在这个项目中，我们将专注于发展激光扫描细胞术来研究猴-人类免疫缺陷病毒(SHIV)感染新生儿恒河猴，这是一种儿童艾滋病的非人灵长类动物模型，活检/尸检样本的数量和大小受到限制。假设：这项研究的假设是，激光扫描细胞术可以用来结合病毒基因表达(通过原位杂交测量)和白细胞群(通过细胞免疫表型测量)在感染SHV的动物中的特征。因此，这种方法将有助于确定导致新生儿中猴艾滋病的细胞机制(S)。这一假设将通过以下具体目标来解决：(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鉴定的方案，以及(2)开发和优化激光扫描细胞仪用于检测先前已被免疫表型表征的细胞中的病毒转录的方案。意义：这个项目意义重大，因为我们为激光扫描细胞术定义的基本方案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.