Human immunodeficiency virus type 1 modulates telomerase activity in peripheral blood lymphocytes

The effect of human immunodeficiency virus type 1 (HIV-1) on telomerase activity in peripheral blood lymphocytes (PBL) was examined. Telomerase is an enzyme that is involved in mechanisms that control cell life span and replicative potential. HIV-1 reduced telomerase activity in in vitro-infected PBL and impaired enzyme activation upon cell stimulation. Telomerase activity was significantly lower in PBL from 23 HIV-1-infected patients than in PBL from healthy donors and significantly increased during highly active antiretroviral therapy (HAART) in 10 patients who had both a virological and an immunological response and in 5 and 8 patients with a virological or an immunological response, respectively. Further analyses of fractionated cells revealed that telomerase activity increased mainly in CD4(+) lymphocytes. Overall, these findings demonstrate that HIV-1 infection down-modulates telomerase activity and suggest that both the HIV-1 decline and immunorestoration in response to HAART contribute to increased telomerase activity in CD4(+) lymphocytes.     

CD8(+) T cell activation in women coinfected with human immunodeficiency virus type 1 and hepatitis C virus

Immune activation is a hallmark of human immunodeficiency virus type 1 (HIV-1) infection and impacts innate and adaptive immunity. Individuals coinfected with HIV-1 and hepatitis C virus (HCV) may have increased immune activation early in HIV disease because of a high HCV antigen load in tissues such as the liver. We evaluated T cell markers of activation and maturation in women with or without HIV-1 infection, by HCV antibody and HCV RNA status. We found increased percentages of activated CD8(+) T cells (i.e., CD8(+)HLA-DR(+)38(+) cells and CD8(+)CD28(+)HLA-DR(+) cells) but not of CD4(+) T cells among women who tested positive for HIV-1, HCV antibody, and HCV RNA, compared with HIV-1-positive women who tested negative for HCV antibody. Because CD8(+) T cell activation is related to HIV-1 disease progression, these data may have implications for the medical management of patients coinfected with HIV-1 and HCV.     

Pentoxifylline (Trental) decreases the replication of the human immunodeficiency virus type 1 in human peripheral blood mononuclear cells and in cultured T cells.

Pentoxifylline (Trental), used routinely for the treatment of intermittent claudication, has been shown previously to decrease the levels of tumor necrosis factors-alpha (TNF-alpha) RNA in cancer patients and to lead to a general improvement of well being. Increased TNF-alpha levels have been observed not only in cancer patients but also in cachectic patients with the acquired immunodeficiency syndrome (AIDS), and TNF-alpha is known to increase the expression of the human immunodeficiency virus type 1 (HIV-1) via activating its long terminal repeat (LTR). Moreover, TNF-alpha decreases the therapeutic efficacy of zidovudine (AZT). Here we show a significant decrease in HIV-1 replication by pentoxifylline in infected human peripheral blood mononuclear cells. The reduction was proportional to the downregulation of expression of a reporter gene, the bacterial gene for chloramphenicol acetyl transferase, linked to the HIV-1 LTR in human monocytoid cells. We conclude that patients with AIDS may benefit from pentoxifylline treatment because of its blockage of TNF-alpha-mediated HIV-1 upregulation, from increased efficacy of AZT, and also from improvement in TNF-alpha-induced cachexia.     

Clonal CD8+ T cell expansions in peripheral blood from human immunodeficiency virus type 1-infected children

The T cell receptor (TCR) repertoires of 24 human immunodeficiency virus (HIV) type 1-infected children were determined by flow cytometry in combination with sequencing of the highly variable TCR complementarity-determining region 3, permitting a quantitative and qualitative assessment of TCR repertoire. Expanded subsets of CD8(+) cells expressing a particular TCR beta-chain variable region were more commonly identified in HIV-1-infected children than in healthy control subjects (75% vs. 13.5%; P<.0001). Older age and lower percentage of CD4(+) cells were correlated with expansions. Oligoclonal populations occupied 71%-95% of each expanded subset, and predominant clones had high absolute counts. There was evidence of functional differentiation to CD28(-) effector cytotoxic T lymphocytes, and cells bearing identical TCRs were identified in both CD28(+) and CD28(-) cell populations. HIV-1 specificity was observed for expanded clones. Children with expansions were not more likely to have increased numbers of CD8(+) T cells, a finding consistent with the possibility that the CD8(+) TCR repertoire has limited diversity.     

Killing of primary CD4+ T cells by non-syncytium-inducing macrophage-tropic human immunodeficiency virus type 1.

Understanding the mechanism by which human immunodeficiency virus type 1 (HIV-1) kills CD4+ T lymphocytes is important to the development of therapeutic and prophylactic strategies. Recent studies have indicated that, in some cases, progression to AIDS is associated with the appearance of syncytium-inducing, T cell line-tropic HIV-1 variants. Nevertheless, approximately 50% of subjects with AIDS harbor only non-syncytium-inducing, macrophage-tropic (NSI-M) variants of HIV-1. In most asymptomatic patients, NSI-M HIV-1 isolates are the predominant virus type found. We report here that cytopathicity of NSI-M HIV-1 for primary CD4+ T lymphocytes can be directly detected in vitro. The extent of CD4+ T-cell killing was not completely correlated with the rate of viral replication, suggesting that other characteristics of HIV-1 contribute to its cytopathicity. Our findings suggest that: (i) direct killing by NSI-M HIV-1 may contribute to CD4+ T-lymphocyte depletion in vivo, and (ii) the determinants of HIV-1 cytopathicity for CD4+ T lymphocytes and cell tropism or syncytia-forming ability are not necessarily tightly linked.     

Filarial infections increase susceptibility to human immunodeficiency virus infection in peripheral blood mononuclear cells in vitro

Because helminth infections and human immunodeficiency virus (HIV) coexist in areas where the spread of AIDS is most dramatic, their in vitro interaction was explored. Cryopreserved peripheral blood mononuclear cells (PBMC) from patients with filarial infections (n=24) and from unexposed control subjects (n=12) were depleted of CD8 T cells and were infected with macrophage (M)- and T cell-tropic viruses. A trend toward increased HIV replication in PBMC from filaria-infected patients was observed. Furthermore, PBMC from 6 filaria-infected patients before antifilarial treatment were significantly more susceptible to replication of M-tropic virus than their posttreatment PBMC (P=.03). No intergroup differences were found in the surface expression of HLA-DR, CD25, CCR5, CXCR4, CCR3 on CD4 T cells, or monocytes before infection. PBMC from filaria-infected patients produced less RANTES (P=.02) but more intracellular interleukin-4 than those of control subjects. Thus, PBMC from persons with filarial infections appear to have enhanced susceptibility to HIV-1 infection mediated by an undetermined mechanism.     

Trans-dominant inhibitory human immunodeficiency virus type 1 protease monomers prevent protease activation and virion maturation.

Production of infectious human immunodeficiency virus (HIV) requires proper polyprotein processing by the dimeric viral protease. The trans-dominant inhibitory activity of a defective protease monomer with the active site Asp-25 changed to Asn was measured by transient transfection. A proviral plasmid that included the drug-selectable Escherichia coli gpt gene was used to deliver the wild-type (wt) or mutant proteases to cultured cells. Coexpression of the wt proviral DNA (HIV-gpt) with increasing amounts of the mutant proviral DNA (HIV-gpt D25N) results in a concomitant decrease in proteolytic activity monitored by in vivo viral polyprotein processing. The viral particles resulting from inactivation of the protease were mostly immature, consisting predominantly of unprocessed p55gag and p160gag-pol polyproteins. In the presence of HIV-1 gp160 env, the number of secreted noninfectious particles correlated with the presence of increasing amounts of the defective protease. Greater than 97% reduction in infectivity was observed at a 1:6 ratio of wt to defective protease DNA. This provides an estimate of the level of inhibition required for effectively preventing virion processing. Stable expression of the defective protease in monkey cells reduced the yield of infectious particles from these cells by 90% upon transfection with the wt proviral DNA. These results show that defective subunits of the viral protease exert a trans-dominant inhibitory effect resulting from the formation of catalytically compromised heterodimers in vivo, ultimately yielding noninfectious viral particles.     

Human immunodeficiency virus 1 reservoir in CD4+ T cells is restricted to certain V beta subsets.

The human immunodeficiency virus 1 (HIV-1) replicates more efficiently in T-cell lines expressing T-cell receptors derived from certain V beta genes, V beta 12 in particular, suggesting the effects of a superantigen. The targeted V beta 12 subset was not deleted in HIV-1-infected patients. It was therefore possible that it might represent an in vivo viral reservoir. Viral load was assessed by quantitative PCR with gag primers and with an infectivity assay to measure competent virus. It was shown that the tiny V beta 12 subset (1-2% of T cells) often has a higher viral load than other V beta subsets in infected patients. Selective HIV-1 replication in V beta 12 cells was also observed 6-8 days after in vitro infection of peripheral blood lymphocytes from normal, HIV-1 negative donors. Viral replication in targeted V beta subsets may serve to promote a biologically relevant viral reservoir.     

Human immunodeficiency virus type 1 RNA in peripheral blood mononuclear cells of patients receiving prolonged highly active antiretroviral therapy.

The levels of early spliced mRNA and genomic RNA of human immunodeficiency virus (HIV) type 1 in peripheral blood mononuclear cells (PBMC) of 14 patients who were receiving highly active combination antiretroviral therapy for > or =116 weeks were determined. The level of viral genomic RNA was below the level of detection in the plasma of these patients (<50 copies/mL), but cell-associated viral tat, rev, and nef mRNA were detected in 86% (12 of 14) of the patients. Cell-associated viral genomic RNA was detected in 57% (8 of 14) of the patients. Early viral spliced mRNA was detected in the PBMC of all patients who had positive results of testing for HIV-1 genomic RNA, and the level of viral genomic RNA in these patients was 34-2214 copies per 10(6) cells.     

Ex vivo and in vitro effect of human immunodeficiency virus protease inhibitors on neutrophil apoptosis

Polymorphonuclear leukocytes (PMNL) from human immunodeficiency virus (HIV)-infected patients exhibit accelerated apoptosis and impaired functional activity. HIV protease inhibitor-based therapy produces improvements in both acquired and innate immune responses. Ex vivo and in vitro effects of HIV protease inhibitors on apoptosis and chemotaxis of PMNL were evaluated. After therapy, there was a rapid and significant decrease of PMNL apoptosis, which correlated with increased chemotactic function. These findings were found both in patients with immunological and virological response and in control subjects who showed an increase in CD4(+) T cell counts but no concomitant decline in HIV load. After in vitro treatment with ritonavir or indinavir, apoptosis of both HIV-infected and -uninfected PMNL markedly decreased and correlated with significant enhancement of chemotaxis. These results suggest that HIV protease inhibitors may improve the PMNL function by reducing the apoptosis rate and that this effect may, at least in part, be independent of their antiviral activity.     

Interleukin-18 inhibits human immunodeficiency virus type 1 production in peripheral blood mononuclear cells

Interleukin (IL)-18 is an interferon (IFN)-gamma-inducing factor and contributes to the Th1 immune response. IL-18 added after infection of peripheral blood mononuclear cells (PBMC) with monocyte-tropic human immunodeficiency virus type 1 (HIV-1) inhibited p24 antigen production by a maximum of 72%. IFN-gamma levels in these cultures were increased, and a significant inverse relationship between HIV-1 production and IFN-gamma levels was observed. A neutralizing anti-IFN-gamma antibody reversed the IL-18 inhibitory effect. Preincubation of PBMC with IL-18 before infection inhibited p24 without additional IL-18 (64%). However, compared with the degree of IL-18 inhibition observed after a 4-day culture, no additional IL-18 inhibitory effect was observed during days 5-13. IL-18 also reduced cell surface expression of the HIV-1 receptor CD4. These results demonstrate that IL-18 inhibited HIV-1 production in PBMC through intermediate IFN-gamma. Furthermore, inhibition was present during the early stages of viral infection and was associated with reduced HIV-1 receptor expression.     

Human erythrocytes bearing electroinserted CD4 neutralize infection in vitro by primary isolates of human immunodeficiency virus type 1

Human erythrocytes bearing electroinserted full-length CD4 (RBC-CD4) can bind and fuse with a laboratory strain of human immunodeficiency virus type 1 (HIV-1) or with T cells infected by HIV-1. Here we show that RBC-CD4 neutralize primary HIV-1 strains in an assay of cocultivation of peripheral blood mononuclear cells (PBMC) from HIV-1- infected persons with uninfected PBMC. RBC-CD4 inhibited viral p24 core antigen accumulation in these cocultures up to 10,000-fold compared with RBC alone. Viral p24 accumulation was inhibited equally well when measured in culture supernatants or in call extracts. The inhibition was dose-dependent and long-lived. Two types of recombinant CD4 tested in parallel were largely ineffective. The neutralization of primary HIV- 1 by RBC-CD4 in vitro was demonstrated in PBMC cultures from 21 of a total of 23 patients tested at two independent sites. RBC-CD4 may offer a route to blocking HIV-1 infection in vivo.     

Biphasic decay of latently infected CD4+ T cells in acute human immunodeficiency virus type 1 infection

Latent infection of resting CD4(+) T cells represents a major barrier to eradication of human immunodeficiency virus type 1 (HIV-1). The establishment and rate of decay of latent HIV-1 in resting CD(+) T cells from 9 acute seroconverters, 7 of whom began to receive highly active antiretroviral therapy (HAART) shortly after presentation, were studied. Before the initiation of therapy, these patients had very high frequencies of latently infected CD4(+) T cells, with a median frequency of 205 infectious units per million resting CD4(+) T cells. These values are > or =1 log higher than those seen in chronically infected patients who are not undergoing HAART. The number of latently infected cells declined dramatically after initiation of HAART but then tended to level off at a low but stable level. The biphasic decay of latent HIV in resting CD4(+) T cells in acute seroconverters supports current models of pre- and postintegration latency.     

Human immunodeficiency virus can infect CD4-negative human fibroblastoid cells.

Certain human immunodeficiency virus (HIV) strains were shown to infect human fibroblastoid cells. Replication was demonstrated only by coculturing normal peripheral blood mononuclear cells or human T-lymphotropic virus type 1-transformed T-cell lines with the infected human cells. This infection of human fibroblastoid cells did not involve the CD4 molecule and did not have the properties of endocytosis. Human sera could be distinguished by their ability to neutralize HIV infection of the fibroblastoid versus human T-cell lines. These observations demonstrate further that other mechanisms for viral entry, besides CD4 binding, must be considered for HIV. They also indicate the wide cellular host range and heterogeneity of HIV strains. The possibility that fibroblastoid cells serve as a reservoir for the AIDS virus and are involved in connective tissue disorders of infected individuals merits attention.