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.
