Tumor necrosis factor receptor expression and signal transduction in HIV-1-infected cells.

OBJECTIVE: To examine the inter-relationship between HIV-1 infection and the cell surface receptors for tumor necrosis factor (TNF)-alpha, an immunoregulatory cytokine that can enhance HIV-1 replication. DESIGN: Infected promyelocytic and promonocytic cells were examined because they normally express both types of TNF receptors. METHODS: TNF receptor surface expression was determined by specific monoclonal antibody recognition and flow cytometry, and signal transduction was detected by gel shift analysis. HIV-1 activation and expression was quantitated by reverse transcriptase assay. RESULTS: In the OM-10.1 promyelocytic model of chronic infection, TNF-alpha-induced HIV-1 expression also resulted in a substantial increase in 75 kd TNF receptor (TR75) expression although 55 kD TNF receptor (TR55) levels were not dramatically altered. A series of uninfected parental HL-60 subclones all reduced TR75 surface expression in response to TNF-alpha treatment. Enhanced TR75 expression on OM-10.1 cells followed the same TNF-alpha-dose dependency as that observed for HIV-1 production. An increase in TR75 expression was also evident during the peak of an acute HIV-1 infection of HL-60 promyelocytes. Although TR55 expression was unaltered during TNF-alpha-induced HIV activation, this receptor was still involved in the viral activation process. Antibody cross-linking of TR55, in the absence of exogenous TNF-alpha, induced maximal HIV-1 expression, an up-modulation of surface TR75, and nuclear NF-kappa B activity in OM-10.1 cultures. Surprisingly, this was the case even when an antagonistic anti-TR55 antibody was used. Anti-TR55 antibody cross-linking in chronically infected U1 promonocytic cultures could only partially substitute for TNF-alpha-induced HIV-1 expression. CONCLUSIONS: Our results demonstrated that HIV-1 infection can selectively influence the surface expression of TNF receptors, potentially influencing its own expression and altering normal immunoregulatory signal transduction.

Sustained plasma TNF-alpha and HIV-1 load despite resolution of other parameters of immune activation during treatment of tuberculosis in Africans.

OBJECTIVE: To determine the impact of treatment of tuberculosis on plasma HIV-1 load in African subjects and to correlate viral load with response to treatment and changes in immune activation. DESIGN: Clinical and microbiological responses, immune activation parameters and plasma HIV-1 load were determined in 20 patients with pulmonary tuberculosis and HIV-1 coinfection in Ghana, West Africa during the first 3 months of anti-tuberculosis treatment. METHODS: Plasma HIV-1 load and markers of immune activation were determined by commercially available assays. Human leukocyte antigen (HLA)-DR incorporation into the HIV-1 envelope was measured by using an immunomagnetic capture technique. RESULTS: Treatment of tuberculosis resulted in significant improvements in weight and haemoglobin, a high sputum smear conversion rate and marked reductions in mean plasma tumour necrosis factor (TNF) receptor-1, interleukin-6 and C-reactive protein. Furthermore, incorporation of host HLA-DR into the HIV-1 envelope decreased; this also suggested a reduction in immune activation of the cells supporting viral replication. However, of importance with regard to AIDS pathogenesis, neither mean plasma TNF-alpha nor HIV-1 load decreased significantly. CONCLUSIONS: The failure of HIV-1 plasma load to decline significantly during the initial months of anti-tuberculosis treatment is associated with high, sustained systemic levels of TNF-alpha. The dissociation between the sustained levels of plasma TNF-alpha and the major reductions in other, diverse immune activation parameters may represent dysregulation of cytokine production in these African patients.

Application of latent HIV-1 infected cellular models to therapeutic intervention.

The 10-year period of clinical latency following infection with the human immunodeficiency virus type-1 remains as a tremendous opportunity for therapeutic intervention. To decipher the viral and cellular mechanisms involved in controlling active and nonproductive viral expression, chronically infected cell lines have been developed which mimic in vivo latency at a cellular level. This review compares these models of chronic infection, emphasizing the advantages and limitations of this approach to the understanding of AIDS progression. In addition, it accentuates the utility of these models of chronic infection in the development and testing of novel drugs aimed at altering the efferent component of the HIV--1 life cycle. It is this component of the viral life cycle that has remained largely unexplored and open to novel therapeutic strategies for the prevention of lethal immunosuppression.

Inhibition of HIV activation in latently infected cells by flavonoid compounds.

Acute HIV-1 infection of H9 and C8166 cultures has been shown to be suppressed by certain flavonoids, and evidence for inhibition of HIV-1 protease, integrase, and reverse transcriptase by flavonoids also exists. The present aim was to determine whether flavonoids inhibit HIV-1 activation in models of latent infection. By screening flavonoids from six different classes, three structurally related compounds (chrysin, acacetin, and apigenin) were identified that inhibited HIV expression in TNF-alpha-treated OM-10.1 cultures. The three compounds had favorable potencies against HIV activation in relation to their growth inhibitory effects (therapeutic index 5-10). Chrysin also inhibited HIV expression in response to PMA in OM-10.1 cells, in ACH-2 cells stimulated with either TNF-alpha or PMA, and in 8E5 cultures. Furthermore, return to viral latency in OM-10.1 cells previously exposed to TNF-alpha occurred over a shorter time interval when chrysin was added. The inhibition of HIV activation was not dependent on preincubation with flavonoids relative to TNF, and was characterized by a lack of HIV RNA accumulation by Northern analysis. Gel-shift experiments revealed that NF-kappa B activation after TNF-alpha treatment was not inhibited by these agents, suggesting that some other critical factor(s) needed for viral transcription was being affected. These findings indicate that flavonoids inhibit HIV-1 activation via a novel mechanism, and that these agents are potential candidates for therapeutic strategies aimed at maintaining a cellular state of HIV-1 latency.

Production of a novel viral suppressive activity associated with resistance to infection among female sex workers exposed to HIV type 1.

To investigate mechanisms of natural resistance to human immunodeficiency virus type 1 (HIV-1), we obtained blood samples from eight women who remained HIV-1 negative after > 3 years of high-risk sex work in Chiang Rai, Thailand. CD4+ T lymphocytes from these highly exposed, persistently seronegative (HEPS) women were readily infectable in vitro with HIV-1 subtypes B and E. Autologous CD8+ cell suppression of both HIV-1 subtypes was evident in HEPS infection cultures, but to an extent also observed in cultures from non-HIV-exposed individuals. Furthermore, production of beta-chemokines was not enhanced in HEPS cultures. However, HEPS cultures displayed significantly enhanced production of a soluble activity that suppressed postintegrated HIV-1 replication. This activity was the unique product of CD4+ T cell and monocyte cocultures. Therefore, although HEPS individuals are apparently susceptible to infection, the production of a postintegrated HIV-1 suppressive activity during monocyte-T cell interactions might protect against the establishment of infection by limiting viral dissemination.

Therapeutic targeting of human immunodeficiency virus type-1 latency: current clinical realities and future scientific possibilities.

Factors affecting HIV-1 latency present formidable obstacles for therapeutic intervention. As these obstacles have become a clinical reality, even with the use of potent anti-retroviral regimens, the need for novel therapeutic strategies specifically targeting HIV-1 latency is evident. However, therapeutic targeting of HIV-1 latency requires an understanding of the mechanisms regulating viral quiescence and activation. These mechanisms have been partially delineated using chronically infected cell models and, clearly, HIV-1 activation from latency involves several key viral and cellular components. Among these distinctive therapeutic targets, cellular factors involved in HIV-1 transcription especially warrant further consideration for rational drug design. Exploring the scientific possibilities of new therapies targeting HIV-1 latency may hold new promise of eventual HIV-1 eradication.

Spectrum of cdk-9 inhibitor activity against HIV-1 replication among various models of chronic and latent infection.

The cellular transcription elongation factor, P-TEFb, and its kinase component, cdk-9, have been implicated in the regulation of HIV-1 transactivation and transcription. We tested a panel of demonstrated cdk-9 kinase inhibitors for the ability to block HIV-1 expression in a variety of cell models representing chronic and latent/inducible infection. These agents induced cellular toxicity, in accordance with their potency for cdk-9 inhibition, with more pronounced toxicity in cultures of T-cell lineage. These agents also inhibited HIV-1 expression, in accordance with their potency for cdk-9 inhibition, in several latent models tested (representing T-lymphocytic, promonocytic and promyelocytic lineages) and using various extracellular stimuli that activate HIV-1 expression via distinct intracellular pathways. Such was the case even though some of these cell models of latent/inducible HIV-1 infection harbour viral defects in the HIV-1 transactivation mechanism. Two additional cell models of latent/inducible HIV-1 infection, both derived from Jurkat T-lymphocytes, were relatively resistant to inhibition of viral expression by these agents. This apparent lack of effect was most likely due to the narrow therapeutic range of these agents in T-cell cultures. Inhibition of HIV-1 replication by these agents was also observed in two cell models representing constitutive viral expression in cells of T-lymphocytic and promyelocytic lineages. Overall, the observed pattern of viral inhibition with these compounds suggests that cdk-9 enzymatic activity is important for HIV-1 expression irrespective of cell lineage or cellular pathway of viral activation. However, because of the non-selective nature of these inhibitors, other cellular pathways must also be considered. Agents that target cellular components essential for HIV-1 expression may provide new therapeutic approaches to limit viral replication, especially when combined with potent antiretroviral regimens.

Isoquinolinesulphonamide derivatives inhibit transcriptional elongation of human immunodeficiency virus type 1 RNA in a promyelocytic model of latency.

Using the OM-10.1 promyelocytic model of inducible human immunodeficiency virus type 1 (HIV-1) infection, we tested a panel of known protein kinase inhibitors for an ability to block tumour necrosis factor-alpha-induced HIV-1 expression. Among the compounds tested, the broad-spectrum protein kinase inhibitor H-7 uniquely blocked HIV-1 expression at the level of viral transcription, but did not inhibit nuclear factor kappaB activation or function. In structure-activity analysis this inhibitory activity of H-7 on HIV-1 expression corresponded with the known structural requirements for the interaction of H-7 with the ATP-binding region of protein kinase C, suggesting that it was indeed related to the kinase inhibitory properties of H-7. The mechanism of H-7 transcriptional inhibition did not involve chromatin remodelling at the HIV-1 long terminal repeat promoter, as shown by nuc-1 disruption, and appeared to involve HIV-1 RNA elongation but not initiation. Therefore, H-7 and related isoquinolinesulphonamide analogues are most likely inhibiting a kinase target essential for HIV-1 transcriptional elongation whose identity may provide new therapeutic targets for intervention.

Evaluation of Compounds That Prevent Reactivation of HIV-1 in OM-10.1 Cells.

It is critically important that new therapeutic compounds and targets for therapeutic intervention be identified in the battle against the human immunodeficieny virus type 1(HIV-1). Many of the currently existing therapeutic approaches target virus-specific factors involved in steps along the HIV-1 life cycle prior to proviral integration. These preintegrative or afferent therapeutic approaches initially appeared quite promising (like reverse transcriptase [RT] inhibitors and soluble CD4) but have shown disappointing clinical benefit. With the recent appreciation of suppression of HIV-1 fusion by selected chemokines, more development of afferent inhibitors is certainly on the horizon.

Assessment of lymphocyte function during vitamin A deficiency.

Two variables of immune status were measured in vitamin A-deficient (A-), vitamin A-sufficient pair-fed (A + PF), and healthy control (A+) rats to examine the mechanisms of immune regulation by vitamin A. Parallel samples were run in bovine fetal serum (BFS)-containing medium and serum-free medium to control BFS-origin vitamin A in the cultures. Splenocytes derived from A- rats showed significantly depressed blastogenic response to all lectins tested in both mediums when compared with responses of A + PF and A+ rats. The splenocyte blastogenic response of A + PF rats was significantly lower than that of A+ (control) rats only when cultured in BFS medium and stimulated by lentil lectin, lipopolysaccharide, or concanavalin A mitogens. Thymic lymphocyte blastogenic transformation assays were equivocal. Splenic immunosuppression could not be linked to significant reductions in surface glycoprotein receptor availability, since splenocytes of A- rats, as well as thymocytes, were capable of binding fluorescein isothiocyanate-conjugated lectins in the same capacity as splenocytes of A+ and A + PF rats. The combination of depressed cellular function and adequate lectin binding to viable cells implies that the regulation of immunohomeostasis by vitamin A was achieved through intracellular mechanisms, possibly selective genomic expression.

Survey of veterinary conference attendees for evidence of zoonotic infection by feline retroviruses.

OBJECTIVE: To examine exposure risks, possibility of zoonosis, and potential disease associations for feline retroviruses among a group of occupationally exposed individuals. DESIGN: Unlinked voluntary cross-sectional epidemiologic survey. SAMPLE POPULATION: 204 veterinarians, laboratory scientists, and other occupationally exposed individuals who attended a veterinary conference on feline geriatric medicine. PROCEDURE: Blood was collected from participants who also completed a 13-question survey requesting demographic, occupational, exposure, and health information. Blood specimens were fractionated into plasma and mononuclear cell components. Plasma was tested for antibodies against feline immunodeficiency virus (FIV) and feline foamy virus (FeFV), as well as p27 antigen of FeLV. Mononuclear cell lysates were tested for FeLV provirus. RESULTS: Subjects reported extensive duration of work with cats (mean, 17.3 years) and multiple high-risk exposures (eg, cat bites, scratches, and injuries with sharp instruments) per year. However, neither serologic nor molecular evidence of zoonosis with any of the 3 feline retroviruses was detected. CONCLUSIONS AND CLINICAL RELEVANCE: Veterinarians encounter occupational exposures to animal material that place them at high risk for zoonoses. For feline retroviruses, the risk of zoonosis among healthy adult humans appears to be extremely small. However, potential for retroviral zoonosis, especially for viruses such as FeLV and FeFV that can replicate in human cells, cannot be eliminated, and universal precautions to reduce potential exposures should be used when handling sick cats.

Cytokine involvement in viral permissiveness and the progression of HIV disease.

Many viruses have evolved novel means of exploiting host defense mechanisms for their own survival. This exploitation may be best exemplified by the interrelationships between certain viruses and the host cytokine networks. Many viruses, including the human immunodeficiency virus type-1 (HIV-1), rely on the liberation and cellular action of host immune cytokines to expand their host cell range, to regulate their cellular expression, and to maintain their dormant state until the proper extracellular conditions arise. As again exemplified by HIV-1, viruses may also take an active role regulating cytokine expression and cell surface cytokine receptors. Because the viral life cycle, and in particular the HIV-1 life cycle, is so intertwined with cytokine regulatory networks, these networks represent potential points for therapeutic intervention. As our understanding of cellular cytokine pathways involved in viral infection and replication continues to expand, so too will our ability to design rational anti-viral therapies to alter multiple steps along the viral life cycle.

Incorporation of HLA-DR into the envelope of human immunodeficiency virus type 1 in vivo: correlation with stage of disease and presence of opportunistic infection.

Human immunodeficiency virus type 1 (HIV-1) bearing HLA-DR in its envelope was detected in plasma from all patients with chronic HIV-1 infection (n = 16) and was present at higher levels in patients with active tuberculosis coinfection (n = 6). Intriguingly, however, HLA-DR was not detectable in HIV-1 from patients during primary viremia (n = 6), suggesting the possibility of virus replication in less-activated cells.

Regulation of HIV-1 expression by cytokine networks in a CD4+ model of chronic infection.

Using the CD4+ model of chronic HIV-1 infection, OM-10.1, we investigated the influence of TNF-alpha regulatory networks on induced viral expression. Previously, OM-10.1 cultures were characterized to respond to exogenous TNF-alpha, as nearly 100% of the cells were activated to express HIV-1 within 24 h. In this study, OM-10.1 cells were pulse-treated, by applying exogenous factors for short periods of time and then washing, to determine if autocrine TNF-alpha could sustain HIV-1 activation in the absence of additional exogenous stimulation. After a TNF-alpha pulse treatment, the progressive increase of HIV-1-expressing OM-10.1 cells was prevented by the continuous presence of anti-TNF-alpha mAb. The inductive activity of supernatant from TNF-alpha pulse-treated OM-10.1 cultures was completely removed by absorption on immobilized anti-TNF-alpha mAb. In addition, TNF-alpha pulse-treated OM-10.1 cells activated HIV-1 expression in untreated OM-10.1 cells when cultured across a permeable membrane indicating paracrine effects. Interestingly, if TNF-alpha pulse-treated OM-10.1 cells were further pulse-treated with anti-TNF-alpha mAb, a marked reduction in autocrine TNF-alpha was observed although the level of newly synthesized TNF-alpha mRNA remained unaffected. A similar degree of inhibition over autocrine TNF-alpha production was observed when soluble TNF receptors were used as the second pulse treatment in these experiments. Although the applicability of these results to in vivo chronically HIV-1-infected cells remains to be realized, these results do indicate that activated HIV-1 expression can be influenced by self-perpetuating mechanisms during periods of limited exogenous stimulation. Furthermore, physiologic mechanisms involving soluble cytokine receptors that counteract autocrine and paracrine activation of HIV-1 expression are shown here to play a regulatory role.

Influence of cell cycle on HIV-1 expression differs among various models of chronic infection.

Because of an inherent dependence on host cell second and third messenger signaling pathways for activation of HIV-1 expression, a potential exists for a relationship between the induction of latent HIV-1 and cell-cycle-related events. To investigate this potential relationship, cellular models of latent HIV-1 infection (OM-10.1 promyelocytes, ACH-2 T-lymphocytes, and U1 promonocytes) were chemically treated or gamma-irradiated to synchronize cultures at each cell cycle stage and then examined for constitutive and TNF-alpha-induced HIV-1 expression. Cell cycle synchronization alone had no effect on HIV-1 expression in OM-10.1 and U1 cultures; whereas enhanced constitutive HIV-1 expression was observed in ACH-2 cultures at G2 + M. A 2 hour TNF-alpha treatment of all synchronized OM-10.1 cultures activated HIV-1 expression to a similar extent as unsynchronized cultures. In contrast, the extent of TNF-alpha-induced HIV-1 expression in ACH-2 S and G2 + M cultures and in the U1 G0/G1 culture was greater than that in unsynchronized control cultures. However, no delay in the initial response was observed. Thus, the influence of cell cycle on constitutive and induced HIV-1 expression varied in each cellular model and, therefore, may further relate to the different molecular mechanisms maintaining viral latency.

Contribution of immune activation to the pathogenesis and transmission of human immunodeficiency virus type 1 infection.

The life cycle of human immunodeficiency virus type 1 (HIV-1) is intricately related to the activation state of the host cells supporting viral replication. Although cellular activation is essential to mount an effective host immune response to invading pathogens, paradoxically the marked systemic immune activation that accompanies HIV-1 infection in vivo may play an important role in sustaining phenomenal rates of HIV-1 replication in infected persons. Moreover, by inducing CD4+ cell loss by apoptosis, immune activation may further be central to the increased rate of CD4+ cell turnover and eventual development of CD4+ lymphocytopenia. In addition to HIV-1-induced immune activation, exogenous immune stimuli such as opportunistic infections may further impact the rate of HIV-1 replication systemically or at localized anatomical sites. Such stimuli may also lead to genotypic and phenotypic changes in the virus pool. Together, these various immunological effects on the biology of HIV-1 may potentially enhance disease progression in HIV-infected persons and may ultimately outweigh the beneficial aspects of antiviral immune responses. This may be particularly important for those living in developing countries, where there is little or no access to antiretroviral drugs and where frequent exposure to pathogenic organisms sustains a chronically heightened state of immune activation. Moreover, immune activation associated with sexually transmitted diseases, chorioamnionitis, and mastitis may have important local effects on HIV-1 replication that may increase the risk of sexual or mother-to-child transmission of HIV-1. The aim of this paper is to provide a broad review of the interrelationship between immune activation and the immunopathogenesis, transmission, progression, and treatment of HIV-1 infection in vivo.

Ligand passing by the p75 tumour necrosis factor receptor enhances HIV-1 activation.

Recently, a HIV-dependent upmodulation of the p75 tumour necrosis factor receptor (TNFr75) was observed using latently-infected OM-10.1 promyelocytes; although the participation of TNFr75 in HIV-1 activation remained undefined. Here, using receptor cross-linking by agonistic antibodies, no direct HIV-1 activation via TNFr75 was observed. Signalling via the p55 tumour necrosis factor receptor (TNFr55) accounted for the full extent of HIV-1 activation in OM-10.1 cultures. However, in tumour necrosis factor alpha (TNF-alpha) dose titration experiments, antibody blockade of TNFr75 decreased the dose response markedly, indicating a ligand passing function. TNFr75 blockade did not alter the dose response to agonistic TNFr55 antibody induction; verifying that the effect on the TNF-alpha dose response was not due to negative signalling or cytolysis. These results demonstrate that, although not directly involved in signal transduction resulting in HIV-1 activation, TNFr75 can serve a critical ligand passing function and permit continued HIV-1 expression during limited TNF-alpha availability.