Induction of HIV-1 replication in latently infected CD4+ T cells using a combination of cytokines.

Although it has been demonstrated that certain cytokines, particularly proinflammatory cytokines, can enhance ongoing viral replication in peripheral blood mononuclear cells (PBMCs) of HIV-1-infected individuals, it is unclear what role these cytokines play in the induction of HIV-1 replication in latently infected, resting CD4(+) T cells. This study demonstrates that the in vitro combination of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha together with the immunoregulatory cytokine IL-2 are potent inducers of viral replication in highly purified, latently infected, resting CD4+ T cells derived from HIV-infected individuals who are antiretroviral therapy-naive as well as those who are receiving highly active antiretroviral therapy (HAART). Viral replication induced by this combination of cytokines was completely suppressed in the presence of HAART in vitro. Given that an array of cytokines, including IL-6, TNF-alpha, and IL-2, are copiously expressed in the microenvironment of the lymphoid tissues, which harbor the latent viral reservoirs, induction of HIV by this combination of cytokines may in part explain the commonly observed reappearance of detectable plasma viremia in HIV-infected individuals in whom HAART was discontinued. Moreover, since it is likely that these infected cells die upon activation of virus and that HAART prevents spread of virus to adjacent cells, the observation that this combination of cytokines can markedly induce viral replication in this reservoir may have important implications for the activation-mediated diminution of the latent reservoir of HIV in patients receiving HAART.

B cells of HIV-1-infected patients bind virions through CD21-complement interactions and transmit infectious virus to activated T cells.

The impact of HIV-associated immunopathogenesis on B cells has been largely associated with indirect consequences of viral replication. This study demonstrates that HIV interacts directly with B cells in both lymphoid tissues and peripheral blood. B cells isolated from lymph node and peripheral blood mononuclear cells (PBMCs) of 4 and 23 chronically infected patients, respectively, demonstrated similar capacities to pass virus to activated HIV-negative PBMCs when compared with CD4(+) cells from the same patients. However, in contrast to T cells, virus associated with B cells was surface bound, as shown by its sensitivity to pronase and the staining pattern revealed by in situ amplification of HIV-1 RNA. Cell sorting and ligand displacing approaches established that CD21 was the HIV-binding receptor on B cells, and that this association was mediated through complement-opsonized virus. These B cells were also found to express significantly lower levels of CD21 compared with HIV-negative individuals, suggesting a direct perturbing effect of HIV on B cells. These findings suggest that B cells, although they themselves are not readily infected by HIV, are similar to follicular dendritic cells in their capacity to serve as extracellular reservoirs for HIV-1. Furthermore, B cells possess the added capability of circulating in peripheral blood and migrating through tissues where they can potentially interact with and pass virus to T cells.

Effects of exercise training and amino-acid supplementation on body composition and physical performance in untrained women.

The purpose of this study was to determine the effects of 6 wk of essential amino acid (EAA) supplementation on body composition and exercise performance in untrained women (n = 21). Subjects were randomly assigned to a placebo (cellulose) or an EAA (average daily dose of 18.3 g of EAAs in pill form) group. Each subject participated in aerobic and heavy-resistance training three times per week. Body composition was assessed via dual x-ray absorptiometry analysis. Muscular endurance was determined via treadmill time to exhaustion, and strength was assessed by the total amount of weight lifted for one set to exhaustion at an estimated 12 repetitions maximum. No changes occurred in either group for body weight, lean body mass, fat mass, or bone mineral content. Treadmill time to exhaustion (TTE) improved significantly (P < 0.05) in the EAA group (mean +/- SD; pre-TTE = 13.15 +/- 3.67 min, post-TTE = 14. 73 +/- 4.26 min), whereas the placebo group did not change significantly. The total weight lifted at the subject's maximum 12 repetitions did not significantly change in either group. In previously untrained individuals, the ingestion of EAAs combined with aerobic and heavy-resistance training for 6 wk did not have a significant effect on body composition or muscular strength; however, aerobic muscular endurance increased significantly.

In vitro reactivation of human immunodeficiency virus 1 from latently infected, resting CD4+ T cells after bacterial stimulation.

Microbial coinfections have been associated with transient bursts of human immunodeficiency virus (HIV) viremia in patients. In this study, we have investigated whether microbial coinfections can induce replication of HIV-1 in latently infected CD4(+) T cells derived from HIV-infected patients who are receiving highly active antiretroviral therapy and in whom plasma viremia is undetectable by sensitive assays. We demonstrate that supernatants from macrophages exposed to the bacterial product lipopolysaccharide can induce in vitro activation of HIV-1 from latently infected, resting CD4(+) T cells obtained from HIV-infected individuals. Depletion of proinflammatory cytokines from the supernatant markedly reduced-whereas depletion of ss chemokines increased--the ability of the supernatant to induce replication of HIV-1. Our results suggest that coinfection with microbial pathogens such as bacteria may induce viral replication in the latent viral reservoirs in vivo.

Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy.

Although highly active antiretroviral therapy (HAART) in the form of triple combinations of drugs including protease inhibitors can reduce the plasma viral load of some HIV-1-infected individuals to undetectable levels, it is unclear what the effects of these regimens are on latently infected CD4+ T cells and what role these cells play in the persistence of HIV-1 infection in individuals receiving such treatment. The present study demonstrates that highly purified CD4+ T cells from 13 of 13 patients receiving HAART with an average treatment time of 10 months and with undetectable (<500 copies HIV RNA/ml) plasma viremia by a commonly used bDNA assay carried integrated proviral DNA and were capable of producing infectious virus upon cellular activation in vitro. Phenotypic analysis of HIV-1 produced by activation of latently infected CD4+ T cells revealed the presence in some patients of syncytium-inducing virus. In addition, the presence of unintegrated HIV-1 DNA in infected resting CD4+ T cells from patients receiving HAART, even those with undetectable plasma viremia, suggests persistent active virus replication in vivo.

Effect of immune activation on the dynamics of human immunodeficiency virus replication and on the distribution of viral quasispecies.

Virus replication in a human immunodeficiency virus (HIV)-infected individual, as determined by the steady-state level of plasma viremia, reflects a complex balance of viral and host factors. We have previously demonstrated that immunization of HIV-infected individuals with the common recall antigen, tetanus toxoid, disrupts this steady state, resulting in transient bursts of plasma viremia after immunization. The present study defines the viral genetic basis for the transient bursts in viremia after immune activation. Tetanus immunization was associated with dramatic and generally reversible shifts in the composition of plasma viral quasispecies. The viral bursts in most cases reflected a nonspecific increase in viral replication secondary to an expanded pool of susceptible CD4(+) T cells. An exception to this was in a patient who harbored viruses of differing tropisms (syncytium inducing and non-syncytium inducing [NSI]). In this situation, immunization appeared to select for the replication of NSI viruses. In one of three patients, the data suggested that immune activation resulted in the appearance in plasma of virus induced from latently infected cells. These findings illustrate certain mechanisms whereby antigenic stimulation may influence the dynamics of HIV replication, including the relative expression of different viral variants.

Suppression of HIV replication in the resting CD4+ T cell reservoir by autologous CD8+ T cells: implications for the development of therapeutic strategies.

CD8+ T cell-mediated antiviral activity against HIV has been described consistently in infected individuals; however, the role of this activity in controlling replication of HIV in the latently infected, resting CD4+ T cell reservoir is unclear. By using an ex vivo system, we show that replication of HIV in this viral reservoir is effectively suppressed in coculture by autologous CD8+ T cells in long-term nonprogressors (LTNPs) and in patients whose viremia was controlled by highly active antiretroviral therapy (HAART), but not in therapy-naive patients who had substantial levels of plasma viremia. This antiviral activity was largely independent of cytotoxic CD8+ T lymphocytes (CTL). When the role of soluble CD8+ T cell-derived factors was examined, we found that CC-chemokines played a major role in inhibition of viral replication in the latent viral reservoir in some LTNPs and patients receiving HAART, but not in chronically infected patients who were not receiving antiretroviral therapy. Potent antiviral activity, independent of CC-chemokines, was found mainly in patients in whom HAART was initiated shortly after the acute phase of HIV infection. These results indicate that CD8(+) T cells provide potent suppressive activity against HIV replication in the latent viral reservoir via direct cellular contact in patients who are naturally LTNPs or in those who are treated with HAART. Furthermore, the profound antiviral activity exerted by non-CC-chemokine soluble factors in infected patients who began HAART early in HIV infection suggests that preservation of this HIV-suppressive mechanism by early initiation of therapy may play an important role in the containment of viral replication in infected patients following interruption of therapy.

Expression of chemokine receptors CXCR4 and CCR5 in HIV-1-infected and uninfected individuals.

The chemokine receptors CXCR4 and CCR5 have been identified as major coreceptors for HIV-1 entry into CD4+ T cells. The majority of primary HIV-1 isolates in early disease use CCR5 as a coreceptor, whereas during disease progression with the emergence of syncytium-inducing viruses, CXCR4 is also used. We performed a cross-sectional study in which we evaluated the expression of two HIV-1 coreceptors, CCR5 and CXCR4, in whole blood samples taken from HIV-1-infected and uninfected individuals. We demonstrate that CXCR4 on CD4+ and CD8+ T cells, and CD14+ monocytes is significantly down-regulated, and CCR5 expression on CD4+ T cells is up-regulated in HIV-infected individuals compared with uninfected controls. Coreceptor expression correlated with the level of cellular activation in vivo in both HIV-infected and uninfected individuals, with CXCR4 being expressed predominantly on quiescent (HLA-DR-) T cells and CCR5 being expressed predominantly on activated (HLA-DR+) T cells. Lower expression of CXCR4 and higher expression of CCR5 on CD4+ T cells correlated with advancing disease. In addition, a tendency for greater activation of CXCR4+CD4+ T cells in patients with advanced disease was observed. Patients who harbored syncytium-inducing viruses, however, could not be distinguished from those who harbored nonsyncytium-inducing viruses based on the level of CD4+ T cell activation or chemokine receptor expression.

Both memory and CD45RA+/CD62L+ naive CD4(+) T cells are infected in human immunodeficiency virus type 1-infected individuals.

Cellular activation is critical for the propagation of human immunodeficiency virus type 1 (HIV-1) infection. It has been suggested that truly naive CD4(+) T cells are resistant to productive HIV-1 infection because of their constitutive resting state. Memory and naive CD4(+) T-cell subsets from 11 HIV-1-infected individuals were isolated ex vivo by a combination of magnetic bead depletion and fluorescence-activated cell sorting techniques with stringent criteria of combined expression of CD45RA and CD62L to identify naive CD4(+) T-cell subsets. In all patients HIV-1 provirus could be detected within naive CD45RA+/CD62L+ CD4(+) T cells; in addition, replication-competent HIV-1 was isolated from these cells upon CD4(+) T-cell stimulation in tissue cultures. Memory CD4(+) T cells had a median of fourfold more replication-competent virus and a median of sixfold more provirus than naive CD4(+) T cells. Overall, there was a median of 16-fold more integrated provirus identified in memory CD4(+) T cells than in naive CD4(+) T cells within a given patient. Interestingly, there was a trend toward equalization of viral loads in memory and naive CD4(+) T-cell subsets in those patients who harbored CXCR4-using (syncytium-inducing) viruses. Within any given patient, there was no selective usage of a particular coreceptor by virus isolated from memory versus naive CD4(+) T cells. Our findings suggest that naive CD4(+) T cells may be a significant viral reservoir for HIV, particularly in those patients harboring CXCR4-using viruses.

HIV-1 induces phenotypic and functional perturbations of B cells in chronically infected individuals.

A number of perturbations of B cells has been described in the setting of HIV infection; however, most remain poorly understood. To directly address the effect of HIV replication on B cell function, we investigated the capacity of B cells isolated from HIV-infected patients to respond to a variety of stimuli before and after reduction of viremia by effective antiretroviral therapy. B cells taken from patients with high levels of plasma viremia were defective in their proliferative responses to various stimuli. Viremia was also associated with the appearance of a subpopulation of B cells that expressed reduced levels of CD21. After fractionation into CD21(high)- and CD21(low)-expressing B cells, the CD21(low) fraction showed dramatically reduced proliferation in response to B cell stimuli and enhanced secretion of immunoglobulins when compared with the CD21(high) fraction. Electron microscopic analysis of each fraction revealed cells with plasmacytoid features in the CD21(low) B cell population but not in the CD21(high) fraction. These results indicate that HIV viremia induces the appearance of a subset of B cells whose function is impaired and which may be responsible for the hypergammaglobulinemia associated with HIV disease.