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Despite the efficacy of antiretroviral therapy (ART), HIV persists in a latent form and remains a hurdle to eradication. CD4+ T lymphocytes harbor the majority of the HIV reservoir, but the role of individual subsets remains unclear. CD4+ T cells were sorted into central, transitional, effector memory, and naive T cells. We measured HIV DNA and performed proviral sequencing of more than 1900 proviruses in 2 subjects at 2 and 9 years after ART initiation to estimate the contribution of each subset to the reservoir. Although our study was limited to 2 subjects, we obtained comparable findings with publicly available sequences. While the HIV integration levels were lower in naive compared with memory T cells, naive cells were a major contributor to the intact proviral reservoir. Notably, proviral sequences isolated from naive cells appeared to be unique, while those retrieved from effector memory cells were mainly clonal. The number of clones increased as cells differentiated from a naive to an effector memory phenotype, suggesting naive cells repopulate the effector memory reservoir as previously shown for central memory cells. Naive T cells contribute substantially to the intact HIV reservoir and represent a significant hurdle for HIV eradication.

After initiating antiretroviral therapy (ART), a rapid decline in HIV viral load is followed by a long period of undetectable viremia. Viral outgrowth assay suggests the reservoir continues to decline slowly. Here, we use full-length sequencing to longitudinally study the proviral landscape of four subjects on ART to investigate the selective pressures influencing the dynamics of the treatment-resistant HIV reservoir. We find intact and defective proviruses that contain genetic elements favoring efficient protein expression decrease over time. Moreover, proviruses that lack these genetic elements, yet contain strong donor splice sequences, increase relatively to other defective proviruses, especially among clones. Our work suggests that HIV expression occurs to a significant extent during ART and results in HIV clearance, but this is obscured by the expansion of proviral clones. Paradoxically, clonal expansion may also be enhanced by HIV expression that leads to splicing between HIV donor splice sites and downstream human exons.

Objectives: Human Immunodeficiency Virus (HIV) infection can induce neurocognitive complications classified as HIV-associated neurocognitive disorder (HAND). The chitinase family is associated with innate immunity cells and many infectious diseases., Methods: We analyzed microarray datasets obtained from NCBI in order to verify the expression of chitinase family genes in hippocampus of uninfected rhesus macaques versus those with histopathologic evidence of Simian Immunodeficiency Virus Encephalitis (SIVE). Moreover, we have analysed two human microarray datasets to verify the results obtained in macaques hippocampus affected by SIVE. For these studies, we have also used the open source tools Genome-scale Integrated Analysis of gene Networks in Tissues (GIANT) to identify the chitinase genes network., Results: CHIT1, CHI3L1 and CHI3L2 levels were significantly increased in SIVE hippocampus as compared to non-infected control specimens. Furthermore, we found a negative correlation between CHIA vs. Brain Viral Load (BVL). These data was confirmed partially in human brain section of HAD/HIVE subjects. Also, we showed that HIV-1 was able to modulate the expression of CHIT1, CHI3L1, CHI3L2 and CHID1 in human macrophages., Conclusions: These results suggest that chitinase gene expression is altered in SIVE and in HAD/HIVE brain sections and call for more studies examining whether this is a protective immunological reaction or a destructive tissue response to encephalitis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In an era when most individuals with treated HIV infection can expect to live into old age, clinicians should proactively review their patients' current and future treatment needs and challenges. Clinical guidelines acknowledge that, in the setting of virological suppression, treatment switch may yield benefits in terms of tolerability, regimen simplification, adherence, convenience and long-term health considerations, particularly in the context of ageing. In this paper, we review evidence from six key clinical studies on switching virologically suppressed patients to regimens based on integrase strand transfer inhibitors (INSTIs), the antiretroviral class increasingly preferred as initial therapy in clinical guidelines. We review these studies and focus on the virological efficacy, safety, and tolerability of switching to INSTI-based regimens in suppressed HIV-positive individuals. We review the early switch studies SWITCHMRK and SPIRAL [assessing a switch from a ritonavir-boosted protease inhibitor (PI/r) to raltegravir (RAL)-containing regimens], together with data from STRATEGY-PI [assessing a switch to elvitegravir (EVG)-containing regimens; EVG/cobicistat (COBI)/emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF) vs. remaining on a PI/r-containing regimen], STRATEGY-NNRTI [assessing a switch to EVG/COBI/FTC/TDF vs. continuation of a nonnucleoside reverse transcriptase inhibitor (NNRTI) and two nucleoside reverse transcriptase inhibitors (NRTIs)], STRIIVING [assessing a switch to a dolutegravir (DTG)-containing regimen (abacavir (ABC)/lamivudine (3TC)/DTG) vs. staying on the background regimen], and GS study 109 [assessing a switch to EVG/COBI/FTC/tenofovir alafenamide fumarate (TAF) vs. continuation of FTC/TDF-based regimens]. Switching to INSTI-containing regimens has been shown to support good virological efficacy, with evidence from two studies demonstrating superior virological efficacy for a switch to EVG-containing regimens. In addition, switching to INSTI regimens was associated with improved tolerability and greater reported patient satisfaction and outcomes in some studies. INSTI-based regimens offer an important contemporary switch option that may be tailored to meet and optimize the needs of many patients., (© 2016 British HIV Association.)

Objectives: T-cell repertoire dysfunction characterizes human immunodeficiency virus type 1 (HIV-1) infection, but the pathogenic mechanisms remain unclear. Disease progression is probably due to a profound dysregulation of Th1, Th2, Th17 and Treg patterns. The aim of this study was to analyze the features of CD4+ T cells in HIV-positive patients with different viroimmunological profile., Methods: we used a gene expression dataset of CD4+ T cells from healthy donors, HIV+ naive patients and Elite Controllers (EC), obtained from the NCBI Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/, accession number GSE18233)., Results: Principal Component Analysis (PCA) showed an almost complete overlap between the HIV-infected and EC patients, which cannot easily explain the different responses to HIV infection of these two group of patients. We have found that HIV patients and the EC showed an upregulation of the Th1 pro-inflammatory cytokines and chemokines, compared to the controls. Also, we have surprisingly identified IL28B, which resulted downregulated in HIV and EC compared to healthy controls. We focused attention also on genes involved in the constitution of the immunological synapse and we showed that HLA class I and II genes resulted significantly upregulated in HIV and in EC compared to the control. In addition to it, we have found the upregulation of others syncytial molecules, including LAG3, CTLA4, CD28 and CD3, assisting the formation of syncytia with APC cells., Conclusions: Understanding the mechanisms of HIV-associated immunological chaos is critical to strategically plan focused interventions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is known to suppress NF-kB activity by interfering with its pathways. The aim of this study was to investigate the ability of 1,25(OH)2D3 in reducing the reactivation of the HIV virus J-LAT cells, an established model of latently infected cells, which were treated with TNFalpha (100 ng/ml) for 2 h with or without 24 h 1,25(OH)2D3 (100 nM) pretreatment. Reactivation of HIV RNA in J-LAT was evaluated in terms of green fluorescent protein (GFP) expression. The same experimental setting was repeated on T cells from HIV-infected patients. Treatment with TNFalpha was associated with a 16 % increase in GFP+ cells and a five-fold increase in unspliced HIV RNA expression (p < 0.04). Pretreatment of J-LAT cells with 1,25(OH)2D3 for 24 h followed by TNFalpha (100 ng/ml) for 2 h reduced the percentage of GFP+ cells by 8 %; moreover, a 2.4-fold decrease in unspliced HIV RNA expression was observed (p < 0.002). In T cells from patients, treatment with TNFalpha significantly increased unspliced HIV RNA expression (sixfold increase, p < 0.02), whereas prestimulation with 1,25(OH)2D3 reduced its expression (2.5-fold decrease, p < 0.02) compared to controls.1,25(OH)2D3 is able to reduce the ability of TNFalpha to upregulate the transcription of HIV RNA from latently infected cells. These data provide further understanding of the pathogenic mechanisms regulating viral reactivation from latent reservoirs, along with new insight in viral internalization.

Background: The innate immunity plays a predominant role in the early control of HIV infection, before the induction of adaptive immune responses. The cytokine secretion operated by the CD4(+) T helper cells is able to induce a response in the innate immunity cells and significantly affect HIV-1 persistence and replication. One of the pathways activated by monocytes to restrain viral infection is the 2' -5' -oligoadenylate synthetase (OAS)/RNase L pathway. OAS is activated by dsRNA and IFNs to produce 2' -5' oligoadenylates, which are activators of RNase L. This enzyme degrades viral and cellular RNAs, thus restricting viral infection., Materials and Methods: We analyzed a microarray dataset obtained from the NCBI Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) databank (accession number GSE18464) in order to verify the modulation of the OAS gene family in CD14 (+) monocytes isolated from 55 subjects, 22 with HIV-1 HVL (high viral load), and 22 with HIV-1 LVL (low viral load), as well as in 11 HIV-1 seronegative controls. We have validated the data on the expression levels of the OAS genes by performing real-time PCR on monocyte from a cohort of HIV infected patients (n = 20), with clinical characteristics similar to those of the patients recruited in the study present in the microarray., Results: Microarray analysis showed that OAS gene family are significantly upregulated in monocyte of HIV-1 patients with HVL, as compared to LVL patients and to healthy donors. Furthermore, we showed a significant correlation between the OAS gene family and the log2 viral load and CD4 count. These results were confirmed by the in vitro validation., Conclusions: Data from this study suggest an involvement for the OAS gene family in the control of HIV-1 infection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

After starting highly active antiretroviral therapy (HAART), HIV-infected patients may experience what is termed immune reconstitution inflammatory syndrome (IRIS). IRIS is characterized by a paradoxical inflammatory response to either previously or recently treated infections or unmasked subclinical infections, when the patient regains the ability to mount a suitable immune response against specific antigens or pathogens. Cryptococcal IRIS (C-IRIS) is thought to be mediated by recovery of Cryptococcus-specific immune responses, resulting in exaggerated host inflammatory responses. In HIV-positive subjects, two distinct modes of presentation of C-IRIS are recognized, "paradoxical" and "unmasking" C-IRIS. "Paradoxical" C-IRIS presents as worsening or recurrence of treated cryptococcal disease following HAART initiation, despite microbiological treatment success. In the "unmasking" form, patients with no prior diagnosis may develop acute symptoms of cryptococcosis, such as meningitis or necrotizing lymphadenopathy, after starting HAART. Here, we present the case of an HIV-positive man, who developed cryptococcal meningitis two months after having started HAART and experienced several meningeal relapses and a "paradoxical" C-IRIS during the following year.

Sirtuins are a family of NAD+-dependent protein deacetylases, which regulate cell survival and energy metabolism, inflammation and cancer. Recent studies have shown that sirtuin-1 (SIRT1) modulates Human Immunodeficiency Virus (HIV)-1 transcription. The HIV-1 Tat protein is a substrate for the deacetylase activity of SIRT1; SIRT1 recycles Tat to its unacetylated form, catalyzing a fundamental step to start new cycles of viral transcription. Moreover, Tat has been reported to promote T-cell hyperactivation by suppressing SIRT1 activity. In fact, Tat blocks the ability of SIRT1 to deacetylate lysine 310 in the p65 subunit of nuclear factor- κB (NF- κB) by interacting with the deacetylase domain of SIRT1. This mechanism leads therefore to the hyperactivation of NF- κB proinflammatory pathway and may likely contribute to the chronic immune activation state of HIV-infected individuals. In the present review we first briefly describe the biological functions of sirtuins, then we delineate the interplay between SIRT1 and HIV-1 and discuss the potential role of SIRT1 as a pharmacological target of HIV-1 replication.

HAART has significantly changed the natural history of HIV infection: patients receiving antiretrovirals are usually able to control viremia, even though not all virological responders adequately recover their CD4+ count. The reasons for poor immune restoration are only partially known and they include genetic, demographic and immunologic factors. A crucial element affecting immune recovery is immune activation, related to residual viremia; indeed, a suboptimal virological control (i.e., low levels of plasma HIV RNA) has been related with higher levels of chronic inflammation and all-cause mortality. The sources of residual viremia are not yet completely known, even though the most important one is represented by latently infected cells. Several methods, including 2-LTR HIV DNA and unspliced HIV RNA measurement, have been developed to estimate residual viremia and predict the outcome of antiretroviral therapy. Considering that poor immunologic responders are exposed to a higher risk of both AIDS-related and non-AIDS-related diseases, there is a need of new therapeutic strategies, including immunomodulators and drugs targeting the latent viral reservoirs, in order to face residual viremia but also to "drive" the host immunologic responses.

Pentoxifylline, a caffeine-related compound, was shown to suppress human immunodeficiency virus type 1 (HIV-1) replication. This effect is thought to be mediated by inhibition of tumor necrosis factor-alpha (TNFalpha)-mediated long-terminal repeat (LTR)-driven expression. We now demonstrate that pentoxifylline efficiently inhibits transduction by HIV-1-based vectors. This latter effect is independent of LTR-driven expression, and correlates with a reduced efficiency of the completion of the integration process in infected cells. Finally, the effect of pentoxifylline is dramatically reduced in cells expressing a dominant negative ATR protein, and in primary human cells that exhibit low level of ATR activity, suggesting that the effect of pentoxifylline on HIV-1 transduction and replication is at least partly mediated by suppression of the ATR kinase., ((c) 2007 S. Karger AG, Basel.)

In the current study, we extended our previous works on natural endogenous reverse transcription (NERT) and further examined its potential as a virucide molecular target in sexual transmission of primate lentiviruses. HIV-1 and SIV virions were pretreated with select nucleoside (NRTIs) and nonnucleoside RT inhibitors (NNRTIs), either alone or in combination with NERT-stimulating substances. The effects of these antiretrovirals on virion inactivation were analyzed in human T cell lines and primary cell cultures. Pretreatment of HIV-1 virions with physiologic NERT-stimulants and 3'-azido-3'-deoxythymidine 5'-triphosphate (AZT-TP) or nevirapine potently inactivated cell-free HIV-1 virions and resulted in strong inhibition of the viral infectivity. Pretreatment of chimeric SHIV-RT virions with NERT-stimulating cocktail and select antiretrovirals also resulted in virion inactivation and inhibition of viral infectivity in T cell lines. Our findings demonstrate the potential clinical utility of approaches based on inhibiting NERT in sexual transmission of HIV-1, through the development of effective anti-HIV-1 microbicides, such as NRTIs and NNRTIs.

The interferon (IFN) system, including various IFNs and IFN-inducible gene products, is well known for its potent innate immunity against wide-range viruses. Recently, a family of cytidine deaminases, functioning as another innate immunity against retroviral infection, has been identified. However, its regulation remains largely unknown. In this report, we demonstrate that through a regular IFN-alpha/beta signal transduction pathway, IFN-alpha can significantly enhance the expression of apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) in human primary resting but not activated CD4 T cells and the amounts of APOBEC3G associated with a low molecular mass. Interestingly, short-time treatments of newly infected resting CD4 T cells with IFN-alpha will significantly inactivate human immunodeficiency virus type 1 (HIV-1) at its early stage. This inhibition can be counteracted by APOBEC3G-specific short interfering RNA, indicating that IFN-alpha-induced APOBEC3G plays a key role in mediating this anti-HIV-1 process. Our data suggest that APOBEC3G is also a member of the IFN system, at least in resting CD4 T cells. Given that the IFN-alpha/APOBEC3G pathway has potent anti-HIV-1 capability in resting CD4 T cells, augmentation of this innate immunity barrier could prevent residual HIV-1 replication in its native reservoir in the post-highly active antiretroviral therapy era.

Despite dramatic reduction of the levels of human immunodeficiency virus type I (HIV-1) virions in blood and seminal plasma of infected patients, highly active antiretroviral therapy (HAART) does not eradicate HIV-1. Three patients, with less than 50 copies/ml of plasma viral RNA, were enrolled in this eradication protocol. Didanosine (DDI) and hydroxyurea (HU) were added to their baseline HAART and after a month of therapy, low dose OKT3, followed by a 2-week course of interleukin 2 (IL-2), was administrated. All antiretroviral therapy was then interrupted and the three patients developed viral rebound in the peripheral blood. The V3 loop region of the HIV-1 gp120 from cell-free viral RNA and proviral DNA in blood and seminal compartments was sequenced in one patient. The two major viral isolates in semen cells were macrophage- tropic (R5) and dual-tropic (R5X4), and these isolates were also present in the PBMCs. Six months after the viral rebound, we demonstrated a shift toward dual tropism in semen cell-associated HIV-1 proviral DNA, with the first appearance of a T-lymphotropic (X4) provirus solely in this compartment. The virus responsible for the blood plasma viral rebound was never found in the semen microenvironment. This study suggests viral compartmentalization of the semen microenvironment after an intensification and stimulatory HIV-1 eradication protocol, with evidence of viral evolution.

The use of exogenous cytokines is part of translational immune-antiretroviral approaches to induce immune reconstitution and possibly eliminate the persistence of human immunodeficiency virus type 1 (HIV-1) in virally suppressed infected individuals on highly active antiretroviral therapy (HAART). Recently, our laboratories demonstrated that interleukin-7 (IL-7) has significant efficiency in stimulating HIV-1 replication from proviral latency in CD4+ T lymphocytes of infected patients. The authors now investigated the possible role of IL-7 in HIV-1-associated dementia (HAD). The authors demonstrated that the IL-7 receptor is expressed on both human neurons (i.e., differentiated NT2 cells) and human astrocytes, with relatively higher mRNA levels in neurons. The translational protein levels of IL-7 receptor alpha were not proportional to those of the mRNA levels in these central nervous system (CNS)-based cell types. Exogenous IL-7 was observed to only slightly down-regulate IL-7 receptor alpha expression on both neurons and astrocytes, as assayed by Western blotting. Instead of promoting survival, surprisingly, exogenous IL-7 induced neuronal apoptosis, as detected by TUNEL assays. Furthermore, IL-7 augmented neuronal apoptosis induced by HIV-1 gp120. Human apoptosis genomic microarray analyses of IL-7-treated human neurons showed up-regulated expression of proapoptotic genes: protein kinases, caspase-10, FAST kinase, tumor necrosis factor (TNF) receptor, and BCL2-antagonist of cell death. These data suggest that IL-7 leads to neuronal apoptosis by a molecular mechanism(s) that occurs via Fas-mediated activation-induced cell death. These studies may therefore not only be key in evaluating the potential use of IL-7 in vivo as a therapeutic modality, but also suggest that IL-7, which is increased endogenously in HIV-1-infected individuals late in disease, may be involved in the neuronal apoptosis demonstrated during HAD.

Highly active antiretroviral therapy (HAART) does not lead to viral eradication, due to HIV-1 residual disease. We investigated whether the cervicovaginal tract serves as a viral reservoir. Seven out of eight cervicovaginal fluids were positive for cell-free HIV-1, by supersensitive reverse transcriptase-polymerase chain reactions (RT-PCR), with a detection limit of 1 copy/ml. No viral outgrowth, intracellular proviral DNA, or viral RNA was detected from cervicovaginal lavage and ecto- and endocervical cells. The cervicovaginal tract of patients on HAART is likely not a major solid tissue reservoir for HIV-1. Nonetheless, the presence of even low cell-free HIV-1 RNA in cervicovaginal secretions continues to suggest the importance of practicing protected sex, even in the era of HAART.

Human immunodeficiency virus type I (HIV-1) DNA integration is an essential step of viral replication. We have suggested recently that this stage of HIV-1 life-cycle triggers a cellular DNA damage response and requires cellular DNA repair proteins for its completion. These include DNA-PK (DNA-dependent protein kinase), ATR (ataxia telangiectasia and Rad3-related), and, at least in some circumstances, ATM (ataxia telangiectasia mutated). Host cell proteins may constitute an attractive target for anti-HIV-1 therapeutics, since development of drug resistance against compounds targeting these cellular cofactor proteins is unlikely. In this study, we show that an inhibitor of ATR and ATM kinases, caffeine, can suppress replication of infectious HIV-1 strains, and provide evidence that caffeine exerts its inhibitory effect at the integration step of the HIV-1 life-cycle. We also demonstrate that caffeine-related methylxanthines including the clinically used compound, theophylline, act at the same step of the HIV-1 life-cycle as caffeine and efficiently inhibit HIV-1 replication in primary human cells. These data reveal the feasibility of therapeutic approaches targeting host cell proteins and further support the hypothesis that ATR and ATM proteins are involved in retroviral DNA integration.

The persistence of HIV-1 in virally suppressed infected individuals on highly active antiretroviral therapy (HAART) remains a major therapeutic problem. The use of cytokines has been envisioned as an additional therapeutic strategy to stimulate latent proviruses in these individuals. Immune activation therapy using IL-2 has shown some promise. In the present study, we found that IL-7 was significantly more effective at enhancing HIV-1 proviral reactivation than either IL-2 alone or IL-2 combined with phytohemagglutinin (PHA) in CD8-depleted PBMCs. IL-7 also showed a positive trend for inducing proviral reactivation from resting CD4(+) T lymphocytes from HIV-1-infected patients on suppressive HAART. Moreover, the phylogenetic analyses of viral envelope gp120 genes from induced viruses indicated that distinct proviral quasispecies had been activated by IL-7, as compared with those activated by the PHA/IL-2 treatment. These studies thus demonstrate that different activators of proviral latency may perturb and potentially deplete only selected, specific portions of the proviral archive in virally suppressed individuals. The known immunomodulatory effects of IL-7 could be combined with its ability to stimulate HIV-1 replication from resting CD4(+) T lymphocytes, in addition to other moieties, to potentially deplete HIV-1 reservoirs and lead to the rational design of immune-antiretroviral approaches.

The induction of neuronal cell death in vivo has been recognized as a prominent feature of HIV type I (HIV-1) infection leading to HIV-1-induced encephalopathy. Viral and host cell products, released from HIV-1-infected cells, have been implicated as inducers of neuronal cell apoptosis. It is unclear which is more important in this process. Neuronal cells were treated with media bearing HIV-1 virions derived from infected T cells and macrophage or the same set of media depleted of virions. T cell media bearing virus induced high levels of apoptosis, whereas that depleted of virions did not. In contrast, neurons treated with media from infected macrophages induced cell death whether virions were present or depleted by ultracentrifugation. The former initiated a repeatedly and significantly higher degree of apoptosis. These data suggest that exposure of neurons to viral products is critical for the induction of apoptosis, in addition to putative host factors released from virally infected cells. Protein-array analyses identified host cell factors up-regulated from infected macrophages, versus their uninfected counterparts, and these host cell factors may be prime candidates for contributing to neuronal apoptosis. Gene-array analyses also identified mRNAs up-regulated in human neurons after treatment with purified HIV-1 gp120 envelope protein or virus-containing media from HIV-1-infected macrophages. These analyses suggest molecular mechanisms for the induction of apoptosis relating to the exposure of viral and host cell factors and rationally designed approaches toward neuroprotection.

Dendritic cells (DCs) are potent antigen-presenting cells, and their physiological localization in tissues that interact with the external environment is important as a first barrier against pathogens such as human immunodeficiency virus type I (HIV-1). Several models have been proposed to explain the possible role of DCs as a reservoir for HIV-1 in patients on virally suppressive highly active antiretroviral therapy (HAART). However, the low yield of cell isolates has made this evaluation a difficult task. The present study analyzes whether peripheral blood DCs from HIV-1-infected individuals on virally suppressive HAART, with plasma HIV-1 RNA levels of less than 50 copies/ml, carry either HIV-1 provirus and/or HIV-1 virions. Peripheral blood DCs were isolated from a cohort of 10 HIV-1-seropositive men taking suppressive HAART. In five patients, plasmacytoid and myeloid dendritic cells were isolated to attempt to identify their respective roles in HIV-1 residual disease. Viral out-growth assays were performed in vitro, as well as gag and R/U5 polymerase chain reaction (PCR) amplification of viral RNA and DNA, respectively, from DC and peripheral blood mononuclear cell (PBMC) extracts. Fluorescence activated cell-sorting (FACS) data revealed cellular yields from 85.90 to 95.18%, of relatively pure DCs isolated from patients' PBMCs. Although HIV-1 RNA gag and DNA RU/5 were detected in all PBMC samples isolated from the patients, proviral DNA and viral RNA forms were not detected in any of the DC isolates. In addition, no replication-competent virus was demonstrated in DC coculture assays, while virus was isolated from each patients' CD8+ T-lymphocyte-depleted PBMC cocultures. Furthermore, HIV-1 gag proviral DNA was not detected in either plasmacytoid or myeloid DC subfractions. The current study suggests that in HIV-1-infected individuals treated with suppressive HAART, peripheral blood DCs do not carry HIV-1 proviral DNA or viral particles attached to their surface. These populations of peripheral blood DCs are likely not a major HIV-1 reservoir in patients on HAART with clinically undetectable plasma viral RNA.

Several studies have reported a crucial role for cholesterol-enriched membrane lipid rafts and cell-associated heparan sulfate proteoglycans (HSPGs), a class of molecules that can localize in lipid rafts, in the entry of human immunodeficiency virus type 1 (HIV-1) into permissive cells. For the present study, we examined the role of these cell surface moieties in HIV-1 entry into primary human brain microvascular endothelial cells (BMVECs), which represent an important HIV-1 central nervous system-based cell reservoir and a portal for neuroinvasion. Cellular cholesterol was depleted by exposure to beta-cyclodextrins and 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase inhibitors (statins), the loss of cholesterol was quantitated, and disruption of membrane rafts was verified by immunofluorescence. Nevertheless, these treatments did not affect binding of several strains of HIV-1 virions to BMVECs at 4 degrees C or their infectivities at 37 degrees C. In contrast, we confirmed that cholesterol depletion and raft disruption strongly inhibited HIV-1 binding and infection of Jurkat T cells. Enzymatic digestion of cell-associated HSPGs on human BMVECs dramatically inhibited HIV-1 infection, and our data from quantitative HIV-1 DNA PCR analysis strongly suggest that cell-associated chondroitin sulfate proteoglycans greatly facilitate infective entry of HIV-1 into human BMVECs. These findings, in combination with our earlier work showing that human BMVECs lack CD4, indicate that the molecular mechanisms for HIV-1 entry into BMVECs are fundamentally different from that of viral entry into T cells, in which lipid rafts, CD4, and probably HSPGs play important roles.

Highly active antiretroviral therapy (HAART) has led to significant changes in mortality and morbidity in the human immunodeficiency virus type 1 (HIV-1) epidemic. Nevertheless, because of molecular mechanisms of viral persistence, HAART does not eradicate HIV-1. Didanosine and hydroxyurea were added to the antiretroviral regimens of 3 HIV-1-infected men who were receiving stable HAART and who had HIV-1 RNA levels <50 copies/mL at the initiation of the study protocol, as a novel intensification to attack cryptic viral replication; low-dose OKT3 was then administered, followed by a course of interleukin-2, to stimulate latent provirus. Replication-competent virus was undetectable after treatment, and plasma viral RNA was either undetectable or <5 copies/mL. In trial periods during which no antiretroviral therapy was administered, the patients developed plasma viral rebound. This translational approach combines novel intensification and stimulation therapy to deplete residual HIV-1 reservoirs. Additional experimental approaches must be developed if HIV-1 eradication is to become possible in patients receiving virally suppressive HAART.

Background: HIV-1-infected men on suppressive highly active antiretroviral therapy (HAART) have a reduction of viral replication in vivo, but HIV-1 RNA is still detectable by certain ultrasensitive reverse transcriptase-PCR assays in blood plasma. Replication-competent virus can also be isolated from both peripheral blood mononuclear cells (PBMC) and seminal cells of these patients. Despite HAART, on-going in vivo infection of HIV-1-seropositive patients' PBMC was demonstrated by the detection of episomal HIV-1 moieties, known as HIV-1 two-long terminal repeat (2-LTR) DNA circles., Methods: The present study analyzes whether new cellular infections occur in vivo in seminal cells of HIV-1-infected men on suppressive HAART. PBMC and seminal cells were isolated from a cohort of HIV-1-seropositive men taking suppressive HAART (< 50 copies HIV RNA/ml blood plasma). Viral growth assays were performed in vitro, as well as semi-quantitative PCR to detect HIV-1 2-LTR circular DNA in PBMC and seminal mononuclear cells., Results: Viral growth in vitro was demonstrated in 16 out of 28 (57%) patients' PBMC, and in five patients' seminal cells (18%). Although 18 patients' PBMC were positive for HIV-1 2-LTR DNA circles, importantly, 2-LTR circular DNA was not detected in any semen sample, even when replication-competent HIV-1 virus had been recovered from a patient's seminal cells by viral co-culture assays., Conclusions: The current study suggests that in HIV-1-infected men treated with suppressive HAART, new cellular infections occur in PBMC, but that new infections do not take place in seminal cells in vivo. Thus, these findings suggest that mainly latent HIV-1 occurs in seminal cells of men on suppressive HAART, which may be a compartment-specific mechanism of residual HIV-1 disease.

A significant percentage of human immunodeficiency virus type 1 (HIV-1)-infected persons treated with highly active antiretroviral therapy (HAART) will develop plasma HIV-1-specific virion RNA levels <50 copies/mL. HIV-1-infected persons receiving virally suppressive HAART were studied with a viral outgrowth assay of the patients' peripheral blood mononuclear cells (PBMC), and a quantitative polymerase chain reaction assay was used to analyze HIV-1 2-long terminal repeat (2-LTR) circular DNA in PBMC, which indicates new HIV-1 infections of cells in vivo. Viral outgrowth in vitro correlated inversely with the level of peripheral blood CD4(+) T lymphocytes. Detection and quantitation of 2-LTR circular DNA correlated strongly with viral outgrowth patterns and inversely with CD4(+) T lymphocyte counts. Relevant subgroups of HIV-1-infected subjects on suppressive HAART with residual viral disease and reservoirs can now be stratified.

Mononuclear phagocytic cells and CD4+ T lymphocytes represent the major targets for infection by HIV-1 in vivo. The most severe pathogenic features associated with HIV-1 infection can be attributed to malfunction or premature death of these cells that are of hematopoietic origin. Patients with acquired immunodeficiency syndrome (AIDS), suffer from many hematologic disorders, particularly those persons with long-term infection of HIV-1. These disorders include anemia, lymphocytopenia, thrombocytopenia and neutropenia. The mechanisms that lead to the induction of these disorders are multi-factorial. However, sufficient evidence has accumulated which suggests that HIV-1 infection of cells within the microenvironment of the bone marrow can lead to the induction of hematopoietic deficits. Most studies indicate that marrow-derived hematopoietic stem cells cannot be infected by HIV-1 until they undergo modest differentiation in order to express the appropriate receptors to enable virus entry and subsequent replication. Some cells within the mixed environment of the marrow stroma appear to support HIV-1 replication however. These cells include marrow microvascular endothelial cells, sometimes referred to as blanket cells, stromal fibroblasts, as well as mononuclear phagocytes. Our recent experiments suggest that the HIV-1 accessory protein, Vpr, plays some role in the activation of marrow-derived mononuclear phagocytes which appears to result in premature phagocytosis of non-adherent marrow cells present in the in vitro cultures. This phenomenon could account, in part, for the induction of cytopenias that are typical of individuals infected by HIV-1.