Increased expression of BCL11B and its recruited chromatin remodeling factors during highly active antiretroviral therapy synergistically represses the transcription of human immunodeficiency virus type 1 and is associated with residual immune activation.

Persistence of human immunodeficiency virus 1 (HIV-1) latency and residual immune activation remain major barriers to treatment in patients receiving highly active antiretroviral therapy (HAART). In the present study, we investigated the molecular mechanisms of persistent HIV infection and residual immune activation in HAART-treated patients. We showed that the expression level of B-cell CLL/lymphoma 11B (BCL11B) was significantly increased in CD4+T cells from HIV-infected patients undergoing HAART, and this was accompanied by increased expression of BCL11B-associated chromatin modifiers and inflammatory factors in comparison to healthy controls and untreated patients with HIV. In vitro assays showed that BCL11B significantly inhibited HIV-1 long terminal repeat (LTR)-mediated transcription. Knockdown of BCL11B resulted in the activation of HIV latent cells, and dissociation of BCL11B and its related chromatin remodeling factors from the HIV LTR. Our findings suggested that increased expression of BCL11B and its related chromatin modifiers contribute to HIV-1 transcriptional silencing, and alteration of BCL11B levels might lead to abnormal transcription and inflammation.

Calcium/calcineurin synergizes with prostratin to promote NF-κB dependent activation of latent HIV.

Attempts to eradicate HIV have been thwarted by the persistence of a small pool of quiescent memory CD4 T cells that harbor a transcriptionally silent, integrated form of the virus that can produce infectious virions following an anamnestic immune response. Transcription factors downstream of T-cell receptor activation, such as NF-κB/Rel and nuclear factor of activated T cells (NFAT) transcription members, are considered important regulators of HIV transcription during acute HIV infection. We now report studies exploring their precise role as antagonists of HIV latency using cell and primary CD4 T cell models of HIV-1 latency. Surprisingly, RNA interference studies performed in J-Lat CD4 T cells suggested that none of the NFATs, including NFATc1, NFATc2, NFATc3, and NFAT5, played a key role in the reactivation of latent HIV. However, cyclosporin A markedly inhibited the reactivation response. These results were reconciled when calcium signaling through calcineurin was shown to potentiate prostratin induced activation of NF-κB that in turn stimulated the latent HIV long terminal repeat (LTR). Similar effects of calcineurin were confirmed in a primary CD4 T cell model of HIV latency. These findings highlight an important role for calcineurin in NF-κB-dependent induction of latent HIV transcription. Innovative approaches exploiting the synergistic actions of calcineurin and prostratin in the absence of generalized T-cell activation merit exploration as a means to attack the latent viral reservoir.

Endometrial epithelial cell response to semen from HIV-infected men during different stages of infection is distinct and can drive HIV-1-long terminal repeat.

OBJECTIVES: Although more than 60% of HIV transmission occurs via semen, little is known about the immune impact of seminal plasma on HIV susceptibility. Here, we examined the level of selected immunomodulatory factors in seminal plasma from HIV-uninfected and therapy-naive, HIV-infected men in acute and chronic stages; the cytokine response elicited by seminal plasma in genital epithelial cells (GECs); and whether any GEC response to seminal plasma could drive HIV replication in infected T cells. METHODS: A panel of nine cytokines and chemokines was measured in seminal plasma from HIV-uninfected and HIV-infected men and in primary GEC cultures following seminal plasma exposure. HIV-long terminal repeat (LTR) activation was measured in 1G5 T cells exposed to supernatants from seminal plasma-treated GECs. RESULTS: Pro-inflammatory cytokines and chemokines were present at significantly higher levels in seminal plasma from acute men, whereas transforming growth factor (TGF)-ß1 was significantly higher in seminal plasma from chronic men. Pro-inflammatory cytokine production by GECs was significantly decreased following incubation with seminal plasma from chronic men. Blocking the TGF-ß1 receptor in GECs prior to seminal plasma exposure enhanced pro-inflammatory cytokine production. Exposure to seminal plasma activated nuclear factor (NF)-κB in GECs and blocking it significantly reduced pro-inflammatory cytokine production. GEC responses to seminal plasma, especially from acute men, significantly activated HIV-LTR activation in 1G5 T cells. CONCLUSION: Immunomodulatory factors in seminal plasma vary, depending on presence and stage of HIV infection. Exposure to seminal plasma leads to NF-κB activation and pro-inflammatory cytokine production, whereas TGF-ß in seminal plasma may suppress pro-inflammatory cytokine production by GECs. GEC responses to seminal plasma can activate HIV-LTR in infected CD4(+) T cells.

Increased level of IL-32 during human immunodeficiency virus infection suppresses HIV replication.

Interleukin-32 was recently identified as a pro-inflammatory cytokine produced by T-lymphocytes, natural killer cells, epithelial cells, and blood monocytes. IL-32 is induced by IFN-gamma in a time-dependent manner suggesting a role for IL-32 in innate and adaptive immune responses. In this study we present evidence that Human immunodeficiency virus promotes interleukin-32 production at both mRNA and protein levels. Our results showed that there is a 74% increase in the serum levels of IL-32 among HIV patients as compared to healthy individuals. There was a three-fold increase in the promoter activity of the IL-32 in the present infections HIV clone. This increase in IL-32 promoter activity was substantiated by increased IL-32 mRNA and protein levels. We have also demonstrated that IL-32 suppresses HIV replication. Our results show that HIV LTR activity was increased by more than six-folds when endogenous IL-32 was knocked down by IL-32-specific siRNA whereas it decreased by one-fold when IL-32 was over expressed in the cells. Similarly a more than two-fold increase and a 50% decrease in HIV p24 values were noted when IL-32 was knocked down and when IL-32 was over expressed in the cells, respectively. Our present work shows that raised IL-32 levels in HIV infection may in turn hamper HIV replication; one of the protective mechanisms of nature.

A soluble factor secreted by an HIV-1-resistant cell line blocks transcription through inactivating the DNA-binding capacity of the NF-kappa B p65/p50 dimer.

The identity and activity of several anti-HIV soluble factor(s) secreted by CD8 and CD4 T lymphocytes have been determined; however, some of them still await definition. We have established an HIV-1-resistant, transformed CD4 T cell line that secretes HIV-1 resistance protein(s). Our studies indicate that this protein(s), called HIV-1 resistance factor (HRF), inhibits transcription of the virus by interfering with the activity of NF-kappaB. In the present report we identified the site at which HRF exerts this inhibition by evaluating a set of discrete events in NF-kappaB action. We tested the kappaB oligonucleotide binding activity in nuclei of resistant cells, nuclear translocation and binding to the HIV-1 long terminal repeat of p65 and p50 proteins from susceptible cells after exposure to HRF, and the binding of recombinant p50 to the kappaB oligonucleotide in vitro as affected by prior or simultaneous exposure to HRF. The results of this experimental schema indicate that HRF interacts with p50 after it enters the nucleus, but before its binding to DNA and that this interaction impedes the formation of an NF-kappaB-DNA complex required for the promotion of transcription. These findings suggest that HRF mediates a novel innate immune response to virus infection.

Inhibition of intra- and extra-cellular Tat function and HIV expression by pertussis toxin B-oligomer.

HIV-1-transactivating factor Tat contributes to virus replication and to the onset of AIDS-associated pathologies by targeting different infected and uninfected cell types. We previously demonstrated that the B-oligomer of pertussis toxin (PTX-B) inhibits HIV infection and replication in primary T cells and macrophages and Tat-dependent HIV-1 long terminal repeat (LTR) transactivation inT lymphoid Jurkat cells. Here we demonstrate that PTX-B inhibits Tat-dependent NF-kappaB activation and HIV-1 LTR-transactivation in non-permissive epithelial HL3T1 cells in a phosphatidylinositol 3'-kinase-dependent way. PTX-B exerts its inhibition both when Tat is produced endogenously in transfected cells and in cells incubated with the extracellular Tat protein. In this latter case, PTX-B does not interfere with extracellular Tat uptake by cells. PTX-B inhibited also interleukin-8 secretion and virus expression stimulated in chronically infected U1 promonocytic cells by intra- and/or extracellular Tat. The genetically modified holotoxin PT-9 K/129G retains the capacity to inhibit Tat transactivating activity and HIV replication in both HIV-permissive and non-permissive cells. Inconclusion, PTX-B acts as a "pleiotropic" inhibitor of Tat, and this may significantly contribute to the broad spectrum of anti-HIV-1 effects exerted by PTX-B in different cell types, and suggests PTX-B and its derivatives as prototypic for the development of anti-Tat drugs.

Toll-like receptor 2 (TLR2) and TLR9 signaling results in HIV-long terminal repeat trans-activation and HIV replication in HIV-1 transgenic mouse spleen cells: implications of simultaneous activation of TLRs on HIV replication.

Opportunistic infections are common in HIV-infected patients; they activate HIV replication and contribute to disease progression. In the present study we examined the role of Toll-like receptor 2 (TLR2) and TLR9 in HIV-long terminal repeat (HIV-LTR) trans-activation and assessed whether TLR4 synergized with TLR2 or TLR9 to induce HIV replication. Soluble Mycobacterium tuberculosis factor (STF) and phenol-soluble modulin from Staphylococcus epidermidis induced HIV-LTR trans-activation in human microvessel endothelial cells cotransfected with TLR2 cDNA. Stimulation of ex vivo spleen cells from HIV-1 transgenic mice with TLR4, TLR2, and TLR9 ligands (LPS, STF, and CpG DNA, respectively) induced p24 Ag production in a dose-dependent manner. Costimulation of HIV-1 transgenic mice spleen cells with LPS and STF or CpG DNA induced TNF-alpha and IFN-gamma production in a synergistic manner and p24 production in an additive fashion. In the THP-1 human monocytic cell line stably expressing the HIV-LTR-luciferase construct, LPS and STF also induced HIV-LTR trans-activation in an additive manner. This is the first time that TLR2 and TLR9 and costimulation of TLRs have been shown to induce HIV replication. Together these results underscore the importance of TLRs in bacterial Ag- and CpG DNA-induced HIV-LTR trans-activation and HIV replication. These observations may be important in understanding the role of the innate immune system and the molecular mechanisms involved in the increased HIV replication and HIV disease progression associated with multiple opportunistic infections.

The maturation of dendritic cells results in postintegration inhibition of HIV-1 replication.

Maturation of dendritic cells (DC) is known to result in decreased capacity to produce HIV due to postentry block of its replicative cycle. In this study, we compared the early phases of this cycle in immature DC (iDC) and mature DC (mDC) generated from monocytes cultured with GM-CSF and IL-4, trimeric CD40 ligand (DC(CD40LT)), or monocyte-conditioned medium (DC(MCM)) being added or not from day 5. Culture day 8 cells exposed to X4 HIV-1(LAI) or R5 HIV-1(Ba-L) were analyzed by semiquantitative R-U5 PCR, which detects total HIV DNA. CXC chemokine receptor 4(low) (CXCR4(low)) CCR5(+) iDC harbored similar viral DNA amounts when exposed to either strain. HIV-1(LAI) entered more efficiently into DC(CD40LT) or DC(MCM) with up-regulated CXCR4. CCR5(low) DC(CD40LT) still allowed entry of HIV-1(Ba-L), whereas CCR5(-) DC(MCM) displayed reduced permissivity to this virus. Comparing amounts of late (long terminal repeat (LTR)-gag PCR) and total (R-U5 PCR) viral DNA products showed that HIV-1(Ba-L) reverse transcription was more efficient than that of HIV-1(LAI), but was not affected by DC maturation. Southern blot detection of linear, circular, and integrated HIV DNA showed that maturation affected neither HIV-1 nuclear import nor integration. When assessing virus transcription by exposing iDC to pNL4-3.GFP or pNL4-3.Luc viruses pseudotyped with the G protein of vesicular stomatitis virus (VSV-G), followed by culture with or without CD40LT or MCM, GFP and luciferase activities decreased by 60-75% in mDC vs iDC. Thus, reduced HIV replication in mDC is primarily due to a postintegration block occurring mainly at the transcriptional level. We could not relate this block to altered expression and nuclear localization of NF-kappa B proteins and SP1 and SP3 transcription factors.

Phosphorothioate backbone modification modulates macrophage activation by CpG DNA.

Macrophages respond to unmethylated CpG motifs present in nonmammalian DNA. Stabilized phosphorothioate-modified oligodeoxynucleotides (PS-ODN) containing CpG motifs form the basis of immunotherapeutic agents. In this study, we show that PS-ODN do not perfectly mimic native DNA in activation of macrophages. CpG-containing PS-ODN were active at 10- to 100-fold lower concentrations than corresponding phosphodiester ODN in maintenance of cell viability in the absence of CSF-1, in induction of NO production, and in activation of the IL-12 promoter. These enhancing effects are attributable to both increased stability and rate of uptake of the PS-ODN. By contrast, PS-ODN were almost inactive in down-modulation of the CSF-1R from primary macrophages and activation of the HIV-1 LTR. Delayed or poor activation of signaling components may contribute to this, as PS-ODN were slower and less effective at inducing phosphorylation of the extracellular signal-related kinases 1 and 2. In addition, at high concentrations, non-CpG PS-ODN specifically inhibited responses to CpG DNA, whereas nonstimulatory phosphodiester ODN had no such effect. Although nonstimulatory PS-ODN caused some inhibition of ODN uptake, this did not adequately explain the levels of inhibition of activity. The results demonstrate that the phosphorothioate backbone has both enhancing and inhibitory effects on macrophage responses to CpG DNA.

Transcriptional activation of HIV by Mycobacterium tuberculosis in human monocytes.

Recently it has been shown that infection with Mycobacterium tuberculosis increases the replication of HIV in mononuclear cells. The objective of this study was to investigate the mechanism(s) of up-regulation of HIV in primary human monocytes. Monocytes from healthy subjects were infected with HIV in vitro and then cultured with purified protein derivative (PPD) of M. tuberculosis. Culture supernatants were assessed for HIV p24 and cytokines. HIV expression was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). PPD induced HIV-infected monocytes to increased expression of HIV RNA and production of HIV p24. This effect correlated with production of tumour necrosis factor-alpha (TNF-alpha) in monocyte cultures. However, neutralizing antibody to TNF-alpha only partly abrogated the PPD-induced HIV p24 in these cultures. Also, PPD and culture filtrate of M. tuberculosis induced HIV mRNA expression. Further, using an adenovirus infection system containing an HIV long-terminal repeat (LTR) reporter plasmid, we showed that M. tuberculosis and its PPD induced HIV LTR. Therefore, the effect of M. tuberculosis and its PPD on HIV replication in monocytes is primarily one of transcriptional activation.

Cutting edge: a short polypeptide domain of HIV-1-Tat protein mediates pathogenesis.

HIV-1 encodes the transactivating protein Tat, which is essential for virus replication and progression of HIV disease. However, Tat has multiple domains, and consequently the molecular mechanisms by which it acts remain unclear. In this report, we provide evidence that cellular activation by Tat involves a short core domain, Tat21-40, containing only 20 aa including seven cysteine residues highly conserved in most HIV-1 subtypes. Effective induction by Tat21-40 of both NF-kappaB-mediated HIV replication and TAR-dependent transactivation of HIV-long terminal repeat indicates that this short sequence is sufficient to promote HIV infection. Moreover, Tat21-40 possesses potent angiogenic activity, further underscoring its role in HIV pathogenesis. These data provide the first demonstration that a 20-residue core domain sequence of Tat is sufficient to transactivate, induce HIV replication, and trigger angiogenesis. This short peptide sequence provides a potential novel therapeutic target for disrupting the functions of Tat and inhibiting progression of HIV disease.

Both PU.1 and nuclear factor-kappa B mediate lipopolysaccharide- induced HIV-1 long terminal repeat transcription in macrophages.

We recently reported that LPS stimulation of monocytic cells leads to the activation of PU.1, a member of the Ets family of transcription factors. Phosphorylation of PU.1 by protein kinase CK2 was found to up-regulate its trans-activation function, but not its DNA binding activity. Previous studies suggested that Ets proteins could bind to NF-kappa B motifs at the tetrameric core sequence TTCC. In macrophages, LPS-inducible HIV-1 gene expression is mediated in part by binding of NF-kappa B to identical tandem binding sites located within the long terminal repeat (LTR). Thus, we performed additional studies to determine whether PU.1 also played a role in regulating HIV-1 gene expression in macrophages. Our functional studies revealed that activation of the HIV-1 LTR in LPS-stimulated cells requires both NF-kappa B and PU.1. Extensive mutagenesis of the HIV-1 LTR revealed that PU.1-dependent activation requires the Ets motif within the upstream NF-kappa B site, whereas NF-kappa B itself binds to the downstream site. We also found that insertion of five additional nucleotides between the NF-kappa B sites abolished LPS inducibility, suggesting a direct interaction between factors that bind these sites. Lastly, we found that mutation of PU.1 at serine 148, which prevents its phosphorylation by CK2, blocked its ability to activate the HIV-1 LTR in response to LPS. These effects were promoter specific because PU.1 did not affect LPS-inducible activation of a distinct NF-kappa B-dependent promoter. While these data do not demonstrate direct binding of PU.1 to the HIV-1 LTR, they illustrate a novel role for PU.1 in activation of the HIV-1 LTR by LPS.

Plasmodium falciparum antigen-induced human immunodeficiency virus type 1 replication is mediated through induction of tumor necrosis factor-alpha.

Because malaria-stimulated cytokine production may have deleterious effects on human immunodeficiency virus type 1 (HIV-1) replication, the effects of Plasmodium falciparum antigens on HIV-1 replication were studied. Stimulation with malarial antigens significantly enhanced HIV-1 replication of HIV-1LAV and primary HIV-1 isolates (subtype A) in CD8-depleted peripheral blood mononuclear cells from naive donors. The malarial antigen-induced activation of HIV-1 was due to cellular activation as judged by the expression of cell activation markers and proliferative responses. While malarial antigen stimulation increased expression of tumor necrosis factor (TNF-alpha) and interleukin-6 (IL-6), only monoclonal antibodies (MAbs) to TNF-alpha inhibited malarial antigen-induced HIV-1 replication, whereas MAb to IL-6 had no effect. Malarial antigen increased HIV-1 replication by increasing viral mRNA expression and by activating long terminal repeat-directed viral transcription. These data suggest that P. falciparum infection can modulate HIV-1 pathogenesis by activating lymphocytes and stimulating viral replication through the production of cytokines.

CD8+ T cell supernatants of HIV type 1-infected individuals have opposite effects on long terminal repeat-mediated transcription in T cells and monocytes.

CD8+ T lymphocytes of HIV-1-infected individuals can efficiently suppress HIV-1 replication in CD4+ T lymphocytes via soluble factors. We compared the effect of CD8+ T cell-derived supernatants on HIV-1 LTR-driven gene expression in T cells and monocytic cell lines. Our results demonstrate that CD8+ T cell supernatants that suppressed HIV-1 LTR-driven gene expression in Jurkat T cells significantly enhanced expression in Tat-activated U38 monocytic cells in the presence and absence of mitogenic stimulation. Examination of a panel of CD8+ T cell-derived supernatants form HIV-infected individuals demonstrated that the extent of enhancement of transcription in U38 cells was mirrored in most cases by a similar level of suppression of transcription in Jurkat T cells. In latently infected U1 cells treated with TNF-alpha, culture with CD8+ T cell supernatants markedly enhanced virus production. In addition, the percentage increase in the enhancement of HIV-1 LTR-driven CAT expression by CD8+ T cell supernatants correlated strongly (r = 0.911) with the level of p24 detected. The level of LTR-mediated gene expression in U38 cells was not influenced by rhMIP-1 alpha rhMIP-1 beta, or rhRANTES over a wide range of chemokine concentration. Treatment of CD8+ T cell supernatant with a combination of antibodies to these chemokines resulted in a further augmentation of LTR-mediated CAT expression in U38 cells. Taken together, these results demonstrate that CD8+ T cell suppressive factors may have opposite effects on HIV-1 LTR-driven gene expression and replication dependent on target cell type and further suggest that the beta-chemokines do not influence HIV-1 LTR-mediated gene expression in monocytic cells.

Suppression of activation of the human immunodeficiency virus long terminal repeat by CD8+ T cells is not lentivirus specific.

CD8+ T lymphocytes of HIV-1-infected individuals can efficiently suppress HIV-1 replication in CD4+ T lymphocytes. To elucidate the molecular events underlying this suppression, we have used the HIV-1 LTR directing the chloramphenicol acetyltransferase gene (CAT) in transient transfection assays using human Jurkat T cells. In addition to supernatants of patient CD8+ T lymphocytes (CD4+ > 350/microliters), supernatant of a T cell clone derived by Herpesvirus saimiri (HVS)-mediated transformation of CD8+ T lymphocytes of a patient demonstrating inhibition of virus replication were examined. Similar levels of inhibition of LTR-mediated gene expression in response to Tat or mitogenic activation with phorbol ester and calcium ionophore were observed by supernatants of both sources. The inhibitory effect of CD8+ T lymphocytes was not exclusive to lentiviral LTRs since transcription of both the HTLV-I LTR and RSV LTR in response to mitogen was effectively inhibited. In examination of the influence of CD8+ T cell-derived supernatant on NF kappa B-mediated activation, a dimer of the HIV-1 NF kappa B elements directing CAT was markedly inhibited by supernatants of both patient CD8+ lymphocytes and the HVS-derived CD8+ clone. Thus the inhibitory nature of CD8+ T lymphocytes appears not to be specific to lentiviral promoters and may mediate an inhibitory effect via the NF kappa B element.

Cryptosporidium sp. E isospora belli en pacientes con diarrea, infectados por el virus de inmunodeficiencia humana, Maracaibo 1993 / Cryptosporidium sp and isospora belli in patients with diarrhea infected with human immunodeficiency virus. Maracaibo 1993

Para investigar la prevalencia de Cryptosporidium sp e isospora belli entre pacientes con infección por el Virus de Inmunodeficiencia Humana (VIH) y diarrea, se estudiaron 20 pacientes VIH seropositivos con evacuaciones líquidas o semilíquidas, en números de 3 ó más por día, con 7 ó más días de evolución. A cada uno se le practicó examen de heces al fresco y con la coloración de Zielh Neelsen (ZN) modificada, realizándose estos examenes en forma seriada según los resultados, e individualizando cada caso para la solicitud de otros estudios. De los 20 pacientes, la mayoría fueron del sexo masculino y de 25 a 44 años de edad, de ellos 11 (55.5 por ciento) presentaron coccidios asociados a la diarrea. Entre los pacientes con coccidios, 8 presentaron Cryptosporidium sp y a 3 se les encontró Isospora belli correspondiéndole el 40 por ciento y 15 por ciento respectivamente del total. La observación del primer examen de heces al fresco y/o con la tinción de ZN modificada, permitió el diagnóstico en 7 de los pacientes con Cryptosporidium sp y en 2 de los 3 con Isospora belli. En algunos de los casos, Criptosporidiosis (75 por ciento) e Isosporiasis (100 por ciento) fueron la primera infección oportunista. Condiciones sanitarias inadecuadas parecen ser un factor de riesgo importante

Phagocytosis of Mycobacterium tuberculosis modulates human immunodeficiency virus replication in human monocytic cells.

Macrophage activation resulting from phagocytosis has the potential to modulate human immunodeficiency virus (HIV) replication. We have determined the effects of phagocytosis of particulate stimuli on transcription and release of HIV. Using THP-1 and Mono Mac 6 human monocytic cell lines transfected with HIV long terminal repeat sequence chloramphenicol acetyltransferase (LTR CAT) constructs we have demonstrated that phagocytosis of Mycobacterium tuberculosis enhanced HIV-1 and -2 LTR CAT expression. However phagocytosis of zymosan or inert latex beads had little or no effect on CAT expression. Enhancement of HIV LTR CAT expression was dependent upon intact NF-kappa B binding sites and was independent of tumour necrosis factor alpha secretion. M. tuberculosis strains of different degrees of virulence induced similar levels of enhanced CAT expression. In contrast, phagocytosis of M. tuberculosis by HIV-1-infected THP-1 cells reduced supernatant reverse transcriptase (RT) activity without suppression of p24 antigen release. Phagocytosis of zymosan granules or latex particles did not alter released RT activity. However, phagocytosis of either M. tuberculosis, zymosan granules or latex particles by HIV-1-infected human peripheral blood monocyte-derived macrophages reduced supernatant RT activity. These data indicate that phagocytosis of M. tuberculosis may enhance HIV transcription in monocytic cells although it may reduce release of intact HIV.