Characteristics of nitric oxide-induced apoptosis and its target cells in mitogen-stimulated peripheral blood mononuclear cells from HIV+ subjects.

The phenomenon of apoptosis observed in lymphoid cells from HIV+ subjects is an important factor contributing to their massive depletion. Several studies have identified nitric oxide (NO) as one of the molecules involved in the apoptosis phenomenon observed during HIV infection. It has been shown that HIV-derived gp120 enhances NO synthesis in cultured cells from HIV+ individuals. Therefore, we tested the potential of two nitric oxide synthase (NOS) inhibitors with different mechanisms of action as preventive agents of in vitro apoptosis, in peripheral blood mononuclear cells (PBMC) from HIV+ subjects. PBMC isolated from these patients always showed higher apoptosis levels than normal subjects, a fact that correlated with overproduction of NO and with reduction of mitochondrial transmembrane potential in these cells. We identified the CD8+ T lymphocyte sub-population as the major apoptosis target in PBMC cultures. Treatment with NO inhibitors N(G)-monomethyl-L-arginine (L-NMMA) and dexamethasone (DEX) inhibited spontaneous and mitogen-induced apoptosis, while reducing mitochondrial alterations in PBMC from both normal (30%) and HIV+ (70%) subjects. The development of apoptosis in target cells correlated with their mitochondrial transmembrane potential impairment and with increased expression of Fas (CD95) molecules. These results offer additional alternatives for the manipulation of cellular depletion in HIV disease.

Cyclin levels during TNF-alpha-induced apoptosis in HIV-infected cells.

Cyclins are cell cycle regulatory proteins. We compared the concurrent kinetics of apoptosis and cyclin expression between HIV-infected cells (J1.1), and uninfected Jurkat cells. Cells were cultured with TNF-alpha and harvested at 24, 48 and 72 hr to examine cyclin expression and DNA content. We found a decline in the levels of the mitotic B cyclin in Jurkat cells (16 to 2%, 48 hr), while in J1.1 cells it was observed in cyclin E (60 to 37%, 72 hr). Because cyclin B is mitotic, results suggest that Jurkat cells undergo apoptosis at G2, while J1.1 cells enter mitosis and then die by apoptosis, as no changes in cyclin B or DNA content at G2M were observed. G1 cyclin E decline in J1.1 cells also suggests that they die after entering mitosis. Based on differences in the cyclins involved, it seems that HIV-1 manipulates the cell cycle to protect J1.1 cells from apoptosis induction at G2, a critical cell cycle phase for HIV replication. Thus, cyclins are useful to characterize points in the cell cycle at which apoptosis is induced, and could become excellent tools to evaluate mechanisms of action of antiretroviral drugs in the cell cycle of HIV-infected cells.

Decreased levels of TNF-beta in cultured PBLs from HIV+ patients occur concomitantly to increased apoptosis and impaired PWM-induced proliferation and dehydrogenase activity.

The occurrence of apoptosis in cultured blood cells from HIV+ patients is well-documented. However, the relationship of this process to cytokine production is still undefined. We measured the production of TNF-beta by mitogen-stimulated PBLs from 33 HIV+ patients, while simultaneously assessing their development of apoptosis, dehydrogenase activity and proliferative responses to PWM and PHA; Only 3/33 patients had less than 30% apoptosis in PWM cultures (average value = 19.6%); patients were grouped in accordance with their having low (31-40%; average 34.8%), intermediate (41-50%, average 45.7%) or high levels (over 51%, average 53.6%) of apoptosis. Our results indicate that there is a quantitative correlation between the different degrees of apoptosis and the impaired production of TNF-beta, and support the hypothesis that PBLs from all HIV+ patients have defective immunological functions. In addition, our results show that TNF-beta production correlates with a stage classification of patients which reflects their PBL status of apoptosis, proliferative response to PWM and dehydrogenase activity in vitro.

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.

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

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.

The combined assessment of cellular apoptosis, mitochondrial function and proliferative response to pokeweed mitogen has prognostic value in SIV infection.

Using the assessment of the mitochondrial metabolic activity of freshly isolated blood mononuclear cells, the flow cytometric detection of apoptosis and of the proliferative responses to PWM, SIV-infected macaques were classified in: stage 0, which included all animals with unaffected parameters, and stages 1, 2, and 3, which included animals having one, two, or all three parameters affected, respectively. This novel three-parametric staging system (ISS) provides a new prognostic tool in the longitudinal study of SIV infection.

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

The physical parameters of nuclear size and granularity, and the biological parameters of nuclear protein and DNA content were simultaneously measured by the flow cytometric analysis of mitogen-activated Rhesus monkey lymphocytes. These experiments were performed with the purpose of establishing baseline values for mitogenic assays of Rhesus monkey blood lymphocytes, when examined by the flow cytometric analysis of their nuclear DNA and protein content, and their nuclear light scatter characteristics. Significant proliferative responses to the mitogens PHA, PWM, Con A, and LPS were observed in these experiments. Simultaneous value determinations of DNA content/size, protein content/granularity, DNA content/granularity and DNA/protein content, showed a direct correlation among them, for all mitogens utilized. These results demonstrate the validity of assessing those parameters in the study of in vitro simian lymphocyte activation and that these assays provide a promising tool for the evaluation of changes in lymphocytic function caused by some viral infections in this experimental model.

Assessment of cellular activation by flow cytometric methods.

The in vitro measurement of lymphoid cell activation is a valid correlate of immunological function in human subjects. This process can be evaluated by the assessment of the proliferative response or the cytokine synthesis and secretion by lymphoid cells upon their proper stimulation. The technology of flow cytometry provides unique conditions for the development of biological assays to achieve those purposes. In this work we describe the utilization of flow cytometric DNA analysis in the evaluation of: (1) the mitogenic response of lymphoid cells; (2) the cellular activity in mixed lymphocyte cultures; (3) the presence of interleukin-2 in culture supernatants, and (4) the cell cycle progression, simultaneous to the expression of a nuclear activation antigen among proliferating lymphocytes.