Why do cells die in HIV infection? Potential mechanisms inducing programmed cell death/apoptosis

This work reviews the suggested mechanisms which result in programmed cell death in human HIV infection. Here we present state-of-the-art scientific information related to the newly rediscovered phenomenon of Apoptosis, and to its biological relevance in the pathogenesis of HIV disease. General features of this phenomenon are reviewed, as well as available evidence for its occurrence and possible role in AIDS pathogenesis. A complex series of cellular and molecular events leading to cellular apoptosis are also reviewed and discussed. They include events which take place at the cell membrane level and those which occur at the intramembrane level and cytoplasmic locations, which result from the immunological activation of affected cells. Cellular events which follow and occur within the mitochondrial space and at the nuclear level are also discussed. The biological significance of all these phenomena is summarized in a theoretical scheme, which attempts to integrate all cellular events leading a primed cell into its HIV-induced programmed death

Multifunctional immunological monitoring of HIV positive patients: a novel staging system

A tri-functional in vitro evaluation has been utilized to analyze peripheral blood mononuclear cells (BMNC) from HIV-infected patients, which allows for the classification of these individuals into convenient stages, according to the number of in vitro parameters affected. The classifying functional parameters are: the mitochondrial metabolic activity of freshly isolated BMNC, measured by an MTT reduction assay, the detection of apoptosis in 72 hour cultures of these cells assessed by propidium iodide staining and dual parametric flow cytometric analysis, and their proliferative response to pokeweed mitogen. Our results indicate that HIV-infected patients at different stages of their clinical disease, can present dysfunctions in one, two or three of the above-mentioned parameters. Based on these results, patients can be classified into four newly-described stages which are Stage 0, including uninfected controls and all patients with unaffected parameters, and Stages 1, 2 and 3, including patients having one, two or all three parameters affected, respectively. This type of immunological evaluation and classification of HIV-infected patients has the potential of becoming a predictive tool in the longitudinal follow-up of their HIV infection

Effect of heat-shock on rhesus monkey lymphocytes and IL-2-dependent cells

Treatment of Rhesus monkey peripheral blood lymphocytes and IL-2 dependent cell lines with heat prior to incubation with mitogens or IL-2, respectively, induces significant cell changes at the nuclear level, detected by DNA staining with Vindelov's propidium iodide and the simultaneous measurement of its red fluorescence and 90 degrees light scatter. These changes are an increase in their nuclear granularity and in apparently fragmented DNA which shows less fluorescence intensity than DNA from nuclei in the G0G1 phase, a phenomenon suggestive of apoptosis. Treated cells also show an increased number of nuclei in G1 or early S phase, with a reduction in those reaching the G2 or M phases. After heat-shock treatment, CTLL-2 cells show an increase in their response to low doses of recombinant IL-2 and an impaired ability to proliferate at higher IL-2 concentrations. These results provide further evidence for the regulatory role of stress-induced events

Breve historia del SIDA en los primates no humanos y su contribución al estudio del síndrome de inmunodeficiencia adquirida / Brief history of AIDS in non-human primates and its contribution to the study of acquired imunodeficiency syndrome

Infection of the rhesus monkey (Macaca mulatta) with retroviruses originating from African non human primates (SIV) induces in this species an acquired immunodeficiency syndrome (SAIDS) closely resembling AIDS in humans. Analogies between the SIV-rhesus system and AIDS in humans are described in this work, analyzing the close relationship existing between the HIV and SIV viruses, and the similarities between SIV disease in the rhesus and HIV disease in humans. A review of current advances in AIDS vaccine research, using the SIV-rhesus model, is also included

Multiparametric analysis of nuclei from activated B and T lymphocytes of rhesus monkeys

Los parámetros físicos de tamaño y granularidad nuclear, y los biológicos de contenido proteico y de ADN nuclear se midieron simultáneamente a través del análisis flujocitométrico de linfocitos provenientes de monos Rhesus activados com mitógenos. El propósito de estos ensayos fué el establecimiento de valores comparables entre el modelo simio y el ya definido modelo humano de activación mitogénica de linfocitos. Se observaron respuestas proliferativas significativas con los mitógenos PHA, PWM, Con A y LPS. Las determinaciones simultáneas de: contenido de ADN/tamaño, contenido protéico/granularidad, contenido de ADN/granularidad; y contenido protéico/contenido de ADN, demostraran la existencia de correlaciones positivas entre estos parámetros. Estos resultados ilustran la validez de su medición en estudios in vitro de activación de linfocitos de simios, y su valor potencial en la evaluación de cambios en función linfocítica, causados por algunas infecciones virales en este modelo experimental

Analysis by fluorescence microscopy and flow cytometry of Monoclonal antibodies produced against cell surface antigens

La reactividad de siete anticuerpos monoclonales contra antígenos de superficie de las células de mieloma Sp2/O-Ag14 fue analizada simultáneamente por microscopía de fluorescencia y por citometría de flujo. Se incubaron 1 x 10**6 células de Sp2/O-Ag14 con 200 micronl de anticuerpo monoclonal a 4-C por 30 min. Luego de lavar 2 veces con PBS las células fueron incubadas a 4-C por 30 minutos con una dilución 1:400 de anticuerpos de carnero anti-immunoglobulinas de ratón, conjugados a isotiocianato de fluoresceína. Luego de lavar tres veces con PBS, las células se utilizaron para el análisis. Por microscoía de fluorescencia se detectaron diferentes patrones de reactividad con los anticuerpos monoclonales. Estos patrones fueron denominados: homogéneo anular, parchos anulares, parchos putiformes, difuso y homogéneo. Estos patrones podrían representar diferentes espítopes reconocidos monoclonales contra los antígenos de superficie de Sp2/O-Ag14 varió entre un 79 a un 90% al analizarse por cimetría de flujo con el sistema EPICS V. Al compararla con la microscopía de fluorescencia, la citometria de flujo determinó de una forma mas rápida, sensitiva y cuantitativa la reactividad de los diferentes anticuerpos monoclonales contra antígenos de superficie de las células Sp2/O-Ag14

Production of monoclonal antibodies to Sp 2/0-AG14 myeloma cells

La técnica de hibridomas, desarrollada por Kohler y Milstein, permite la obtención de anticuerpos de especificidad única (p.e. anticuerpos monoclonales). Esta metododlogía fue introducida en el Laboratorio de Immunología del Departamento de Microbiología de la Escuela de Medicina, Recinto de Ciencias Médicas, UPR en 1986. La producción de anticuerpos monoclonales contra Sp2/0-Ag 14 que aquí se reporta, describe la metodología utilizada y es de importancia porque constituye el primer trabajo sobre anticuerpos monoclonales realizado en el Recinto de Ciencias Médicas y en Puerto Rico