Important note: Information in this article was accurate in 1990. The state of the art may have changed since the publication date.
IN SITU HYBRIDIZATION: A HISTOMOLECULAR APPROACH TO THE DETECTION AND STUDY OF HUMAN IMMUNODEFICIENCY VIRUS INFECTION
HIV Detection by Genetic Engineering Methods. Luciw PA and Steimer KS, eds. New York, Marcel Dekker, p. 209-42, 1989.. Unique Identifier : AIDSLINE ICDB/90660263 Busch MP; Beckstead JH; Hollander H; Vyas GN; Irwin Memorial Blood Bank, San Francisco, CA
Abstract:
The adaptation of hybridization techniques to the detection of specific nucleic acid sequences in preparations of cells, tissues, or chromosomes is termed in situ hybridization (ISH). Although the general approach of ISH was described in 1969, broad application of this technique has been achieved only recently. A variety of applications of ISH to the analysis of HIV replication in human cells and tissues is described. Two crucial unresolved questions are discussed: (1) which immunologic and/or viral mechanisms mediate the downregulation of HIV gene expression in latently infected cells, and (2) what are the stimuli and mechanisms that result in subsequent viral activation and cytopathology? Topics include source and preparation of clinical samples, cell and viral culture methods, ISH analysis, comparative assays, standardization of ISH for HIV, direct detection of HIV in cells and tissues, and detection and quantification of HIV in cultured cells. Peripheral blood mononuclear cells (PBMCs) were obtained aseptically from 30 HIV antibody-positive individuals and 20 HIV antibody-negative blood donors; lymph node biopsy samples were obtained from 3 anti-HIV+ homosexual men; bone marrow samples were obtained from 20 anti-HIV+ patients (pts); and lymphoma and testicular samples were obtained at autopsy from a pt with AIDS who had developed an aggressive B-cell lymphoma. Direct analysis of ex vivo cells and tissues by ISH and immunocytochemistry consistently showed that detectable HIV RNA and protein were present in only 1:10,000 to 1:100,000 cells. This low-level viral replication in vivo was localized to cells with lymphoid and monocytoid morphologic features. HIV RNA and protein were not detected in myeloid, erythroid, or megakaryocytic precursors in bone marrow. Viral replication was restricted to only rare cells in lymph nodes and was not detected in B-lymphoma cells or testicular parenchymal cells of a pt with AIDS. In contrast to the scarcity of HIV+ cells detected by direct ISH analysis of ex vivo material from infected individuals, the number of cells actually harboring replication-competent HIV appears to be much higher, probably by 10- to 1000-fold. This conclusion is based on two observations: (1) the rapid emergence of HIV+ cells from anti-HIV+ PBMC preparations following stimulation with phytohemagglutinin in vitro, and (2) the correlation of direct ISH analysis of PBMC with limiting-dilution culture analysis. (80 Refs)
Keywords: Acquired Immunodeficiency Syndrome/DIAGNOSIS AIDS-Related Complex/DIAGNOSIS Biopsy Bone Marrow/PATHOLOGY Cell Line, Transformed Cytopathogenic Effect, Viral/*GENETICS *DNA Probes *Gene Expression Regulation, Viral Human HIV/*GENETICS/PATHOGENICITY HIV Infections/*DIAGNOSIS Lymph Nodes/PATHOLOGY Male Virus Activation/*GENETICS Virus Replication/GENETICS MONOGRAPH 900530
M9051019
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Important note: Information in this article was accurate in 1990. The state of the art may have changed since the publication date.
