Human Immunodeficiency Virus Reservoir Research Articles

Poly (propyleneimine) dendrimer based nanocontainers for targeting of efavirenz to human monocytes/macrophages in vitro

Cells of the mononuclear phagocytic system, in particular monocytes/macrophages (Mo/Mac) serve as a reservoir for human immunodeficiency virus (HIV) and are believed to be responsible for its dissemination throughout the body and especially into the brain. Treatment of HIV infection, therefore, must reach these cells in addition to the lymphocytes. The purpose of the present study is to develop poly(propyleneimine) (PPI) dendrimer-based nanocontainers for targeting of efavirenz (EFV) to Mo/Mac. Fifth generation PPI dendrimer, t-Boc–glycine conjugated PPI dendrimer (TPPI) and mannose conjugated dendrimers were synthesized and characterized. While the haemolytic activity and cytotoxicity of PPI dendrimer was found to be very high, the toxicity of t-Boc–glycine conjugated dendrimer and mannose conjugated dendrimers were found to be negligible. The entrapment efficiency of mannose conjugated dendrimer was found to be 47.4%, followed by that of PPI dendrimer (32.15%) and t-Boc–glycine conjugated dendrimer (23.1%). The in vitro drug release profile shows that while PPI dendrimer releases the drug by 24 h, the dendrimer-based nanocontainers prolong the release rate up to 144 h (83 ± 0.4% in case of t-Boc–glycine conjugated dendrimer and 91 ± 0.3% in mannose conjugated dendrimer). The cellular uptake of EFV was found to be both concentration and time dependent. Significant increase in cellular uptake of EFV by Mo/Mac cells were observed in case of mannose conjugated dendrimer which is 12 times higher than that of free drug and 5.5 times higher than that of t-Boc–glycine conjugated dendrimer. While mannose conjugated dendrimer was taken up by the lectin receptors of the cells, phagocytosis of t-Boc–glycine conjugated dendrimer might be responsible for its enhanced uptake. Results suggest that the proposed carriers hold potential to increase the efficacy and reduce the toxicity of antiretroviral therapy.

The effectiveness of modified sperm washes in severely oligoasthenozoospermic men infected with human immunodeficiency and hepatitis C viruses

Men who are seropositive for human immunodeficiency virus (HIV) or hepatitis C virus now can father children safely, thanks to sperm washing and nested polymerase chain-reaction techniques, followed by intracytoplasmic sperm injection. However, in a small percentage of such patients, it is impossible to recover spermatozoa after said procedures because of their highly impaired spermatogenesis. We have established that less rigorous methods, such as repeated centrifugation, yield nested polymerase chain-reaction HIV- and hepatitis C virus-negative specimens, even in sperm samples from men with severe oligoasthenozoospermia.

CD163 Identifies Perivascular Macrophages in Normal and Viral Encephalitic Brains and Potential Precursors to Perivascular Macrophages in Blood

Perivascular macrophages are uniquely situated at the intersection between the nervous and immune systems. Although combined myeloid marker detection differentiates perivascular from resident brain macrophages (parenchymal microglia), no single marker distinguishes perivascular macrophages in humans and mice. Here, we present the macrophage scavenger receptor CD163 as a marker for perivascular macrophages in humans, monkeys, and mice. CD163 was primarily confined to perivascular macrophages and populations of meningeal and choroid plexus macrophages in normal brains and in brains of humans and monkeys with human immunodeficiency virus or simian immunodeficiency virus (SIV) encephalitis. Scattered microglia in SIV encephalitis lesions and multinucleated giant cells were also CD163 positive. Consistent with prior findings that perivascular macrophages are primary targets of human immunodeficiency virus and SIV, all SIV-infected cells in the brain were CD163 positive. Using fluorescent dyes that definitively and selectively label perivascular macrophages in vivo, we confirmed that dye-labeled simian perivascular macrophages were CD163 positive and able to repopulate the central nervous system within 24 hours. Flow cytometric studies demonstrated a subset of monocytes (CD163(+)CD14(+)CD16(+)) that were immunophenotypically similar to brain perivascular macrophages. These findings recognize CD163(+) blood monocytes/macrophages as a source of brain perivascular macrophages and underscore the utility of this molecule in studying the biology of perivascular macrophages and their precursors in humans, monkeys, and mice.

Antiretrovirals: Need for an effective drug delivery

Human Immunodeficiency Virus (HIV) is a retrovirus that causes irreversible destruction of the immune system, leading to the occurrence of opportunistic infections and malignancies. During the last decade, even though attempts were being made to eradicate HIV, it was found that eradication of HIV is highly unlikely, and effective antiretroviral therapy is required on a long-term basis to maintain viral suppression and reduce disease progression. During this decade, effective therapies aimed at continued suppression of HIV replication and targeted at resting HIV reservoirs such as brain, lymphatic systems will be critical to prolong survival and renewing hopes for a cure. Currently available antiHIV drugs can be classified into three categories: nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and protease inhibitors. Most of these drugs bear some significant drawbacks such as relatively short half-life, low bioavailability, poor permeability and undesirable side effects. Efforts have been made to design drug delivery systems for antiHIV agents to: a) reduce the dosing frequency, b) increase the bioavailability and decrease the degradation/metabolism in the gastrointestinal tract, c) improve the CNS penetration and inhibit the CNS efflux, and d) deliver them to the target cells selectively with minimal side effects. This article is an attempt to compile all major research work towards drug delivery for AIDS therapy and channel future attempts in the area of more effective controlled delivery of antiHIV agents.

Where does HIV live?

This survey of the cellular and anatomical sites of infection by human immunodeficiency virus (HIV) discusses the mechanisms of attachment of the virus to cells, the cellular receptors through which the virus enters cells, and the all-important reservoir of HIV, which persists despite antiretroviral therapy.

Human immunodeficiency virus reservoir might be actively eradicated as residual malignant cells by cytotoxic chemotherapy

A unique characteristic of human immunodeficiency virus (HIV) infection is that the virus must incorporate its cDNA into the host genomic DNA for replication. Once the virus gets into the host genome and becomes a part of the host genetic materials, elimination of the virus without killing the infected cells is virtually impossible. The use of highly active antiretroviral therapy (HAART) can result in a substantial decline in viremia. However, HAART does not eradicate HIV. The progressive HIV infection will unavoidably rebound after a cessation of the treatment. Searching for a new combination therapeutic strategy with cytotoxic agents that eliminate or significantly reduce the HIV reservoir is a potential way for better control of the disease. Theoretically, the HIV reservoir can be gradually eradicated by long-term use of certain antimetabolic cytotoxic drugs coupled with proper activation of latently infected cells, if viral replication is completely blocked by antiretroviral chemotherapy to protect uninfected, susceptible cells.

Long-term survival and virus production in human primary macrophages infected by human immunodeficiency virus.

The role of macrophages in the pathogenesis and progression of human immunodeficiency virus (HIV)-related infection is substantiated by in vitro and in vivo evidence. The unique ability to survive HIV infection and produce viral particles for long periods is postulated. Detailed studies of this phenomenon are lacking. The dynamics of HIV-1 replication and cumulative virus production was studied in long-term cultures of macrophages in the presence or in the absence of antiviral drugs. Multiply spliced and unspliced HIV-RNA production was assessed by quantitative PCR, and the number of infected cells was monitored by FACS analysis. Cumulative HIV-1 production was determined by a trapezoidal equation, including such parameters as times of collection and experimental values of genomic-RNA and p24 gag antigen. Unspliced and multiply spliced HIV-RNA increased linearly after macrophage infection; reached levels of 1.5 x 10(8) and 2.8 x 10(5) copies/10(5) cells, respectively, at day 10; and then remained stable throughout the course of the experiment. Cumulative production of genomic-RNA and p24 gag antigen was 10(10) copies/10(6) cells and 10(7) pg/10(6) cells, respectively, with an average of >200 virus particles produced daily by each macrophage. AZT decreased the cumulative production of both genomic-RNA and p24 gag antigen down to 2.5 x 10(9) copies and 1.1 x 10(6) pg/10(6) cells (73.8% and 88.9% inhibition, respectively) up to day 50 without virus breakthrough. Ritonavir had a limited, but consistent, efficacy on the release of mature virus proteins (about 40% inhibition), but not on HIV-RNA production. In conclusion, the long-term dynamics and the high cumulative virus production that characterize HIV-1 infection of macrophages underscore the peculiar role of these cells as a persistently infected reservoir of HIV.

Effects of prostratin on T-cell activation and human immunodeficiency virus latency.

Human immunodeficiency virus (HIV) replication is linked to cellular gene transcription and requires target cell activation. The latent reservoir of HIV-1 in quiescent T cells is thought to be a major obstacle to clearance of infection by highly active antiretroviral therapy (HAART). Thus, identification of agents that can induce expression of latent virus may, in the presence of HAART, allow elimination of the infected cells by the immune response. We previously used the SCID-hu (Thy/Liv) mouse model to establish that activation-inducible HIV can be generated at high frequency during thymopoiesis. Latently infected mature thymocytes can be exported into the periphery, providing an efficient primary cell model to determine cellular activation signals that induce renewed expression of latent virus. Here we characterized the effects of prostratin, a non-tumor-promoting phorbol ester, on primary human peripheral blood lymphocytes (PBLs) and assessed its ability to reactivate latent HIV infection from thymocytes and PBLs in the SCID-hu (Thy/Liv) model. Prostratin stimulation alone did not induce proliferation of quiescent PBLs; however, it could provide a secondary signal in the context of T-cell receptor stimulation or a primary activation signal in the presence of CD28 stimulation to induce T-cell proliferation. While prostratin alone was not sufficient to allow de novo HIV infection, it efficiently reactivated HIV expression from latently infected cells generated in the SCID-hu mouse. Our data indicate that prostratin alone is able to specifically reactivate latent virus in the absence of cellular proliferation, making it an attractive candidate for further study as an adjunctive therapy for the elimination of the latent HIV reservoir.

Eradication of human immunodeficiency virus type 1-infected cells by a combination of antimetabolic cytotoxic chemotherapy and antiviral chemotherapy in vitro: a pilot study.

Although highly active antiretroviral therapy against human immunodeficiency virus (HIV) type 1 reduces the mortality of persons with acquired immunodeficiency syndrome, it does not eliminate HIV reservoirs. In this study, which used a 6-thioguanine (6-TG) resistant clone (4C6) of the MT-2 cell line as a model, the combination of 6-TG with both reverse-transcriptase (RT) inhibitor and protease inhibitor or 6-TG with a protease inhibitor alone completely eradicated HIV-1-carrying cells from the culture and protected uninfected 4C6 cells from HIV-1 infection. The combination of 6-TG and a RT inhibitor, azidothymidine, provided partial protection. Protection was extended to human peripheral blood mononuclear cells. These results suggest that adding a cytotoxic drug in combination antiviral chemotherapy may reduce the establishment of virus reservoirs and prevent virus spread. The clinical value of this and similar strategies should be further evaluated in HIV-infected patients.

Induction of cell death in human immunodeficiency virus-infected macrophages and resting memory CD4 T cells by TRAIL/Apo2l.

Because the persistence of human immunodeficiency virus (HIV) in cellular reservoirs presents an obstacle to viral eradication, we evaluated whether tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) induces apoptosis in such reservoirs. Lymphocytes and monocyte-derived macrophages (MDM) from uninfected donors do not die following treatment with either leucine zipper human TRAIL (LZhuTRAIL) or agonistic anti-TRAIL receptor antibodies. By contrast, such treatment induces apoptosis of in vitro HIV-infected MDM as well as peripheral blood lymphocytes from HIV-infected patients, including CD4(+) CD45RO(+) HLA-DR(-) lymphocytes. In addition, LZhuTRAIL-treated cells produce less viral RNA and p24 antigen than untreated controls. Whereas untreated cultures produce large amounts of HIV RNA and p24 antigen, of seven treated CD4(+) CD45RO(+) HLA-DR(-) cell cultures, viral RNA production was undetectable in all, p24 antigen was undetectable in six, and proviral DNA was undetectable in four. These data demonstrate that TRAIL induces death of cells from HIV-infected patients, including cell types which harbor latent HIV reservoirs.

Malaria enhances expression of CC chemokine receptor 5 on placental macrophages.

Malaria and human immunodeficiency virus (HIV) coinfections are common in pregnant women in sub-Saharan Africa. The current study shows that placentas of malaria-infected women contain 3 times as much CC chemokine receptor 5 (CCR5) RNA as placentas of women without malaria. By immunohistochemistry, CCR5(+) maternal macrophages were seen in placentas from malaria-infected women but not in placentas from malaria-uninfected women. In addition, CCR5 also was found on fetal Hofbauer cells in placentas from both groups. Thus, malaria infections increase the potential reservoir for HIV in the placenta by increasing the number of HIV target cells.

Targeting of 3'-azido 3'-deoxythymidine (AZT)-loaded poly(isohexylcyanoacrylate) nanospheres to the gastrointestinal mucosa and associated lymphoid tissues.

The aim of the studv was to evaluate the capacity of poly(isohexylcyanoacrylate) nanospheres to concentrate 3'-azido 3'-deoxythymidine (AZT) in the intestinal epithelium and associated immunocompetent cells, which are known to be one of the major reservoirs of the human immunodeficiency virus (HIV). The tissue concentration of 3H-radiolabeled AZT in the gastrointestinal (GI) tract was obtained 30 and 9() minutes after intragastric administration to rats at a dose of 0.25 mg AZT/100 g of body weight. The distribution along the intestine was determined. AZT concentrations in the lymph were obtained by lymphatic duct cannulation. Unlike the solution. nanoparticles did concentrate AZT very cfficiently in the intestinal mucosa, as well as in the Peyer's patches, and could simultaneously control the release of free AZT. Concentration in Peyer's patches was 4 times higher for nanoparticles, compared with the control solution. The tissue concentration was 30-45 microM, which was much higher than the reported IC50 of AZT (0.06-1.36 microM) and was regularly distributed along the gastrointestinal tract. Nanoparticles have been shown to be efficient in concentrating AZT in the intestinal epithelium and gut-associated lymphoid tissues, supporting the view that these particles may represent a promising carrier to treat specifically the GI reservoir of HIV.

Multiple drug resistance mutations in human immunodeficiency virus in semen but not blood of a man on antiretroviral therapy

The concept that the male reproductive tract harbors isolated reservoirs of human immunodeficiency virus (HIV) infection has now been widely accepted. The significance of semen viral burden to sexual transmission of HIV is obvious; however, its contribution to disease progression is unknown. We report a case study that demonstrates the emergence of resistance-conferring mutations to antiviral therapy in infected seminal leukocytes from a man with asymptomatic prostatitis associated with leukospermia. This finding demonstrates the potential importance of male reproductive tract organs to the development of therapy resistance in HIV-infected men.

Interleukin-2 up-regulates expression of the human immunodeficiency virus fusion coreceptor CCR5 by CD4+ lymphocytes in vivo.

Intermittent interleukin-2 (IL-2) therapy can substantially increase CD4+ T cell counts of human immunodeficiency virus (HIV)-infected subjects. Administration of IL-2 led to transient up-regulation of CCR5 on CD4+ T cells; up to 87% of CD4+ cells expressed CCR5 after a 5-day cycle, with return to baseline levels within 2 weeks. Unlike in vitro studies, CCR5 was coexpressed with CD45RA and CXCR4 on CD4+ T cells after IL-2 therapy. The observed increase in coreceptor expression was not associated with detectable increases in viral replication. IL-2 therapy induced CCR5 expression in >90% of circulating memory CD4+ T cells, determined to be a long-term reservoir of HIV, suggesting significant activation of these cells. These studies demonstrate that levels of expression of HIV coreceptors alone do not always correlate with HIV replication in vivo and that IL-2 therapy activates a majority of memory T cells in the circulation and likely throughout the immune system.

Enhanced in vivo human immunodeficiency virus-1 replication in the lungs of human immunodeficiency virus-infected persons with Pneumocystis carinii pneumonia.

The relationship of serum human immunodeficiency virus-1 (HIV-1) RNA levels to HIV-1 RNA levels in other compartments, such as the lungs, is not well characterized. The purpose of this study was to determine the viral burden of HIV-1 in the lungs by comparing HIV-1 RNA in cell-free bronchoalveolar lavage fluid (BALF) with that in serum. Specimens were examined from 77 HIV-seropositive adults (CD4(+) cell counts: 0 to 700 cells/mm(3); 48% receiving prescribed antiretroviral agents), comprising 43 asymptomatic individuals who were compared with 34 persons with active lung disease caused by Pneumocystis carinii (n = 26), bacteria (n = 3), Mycobacterium avium complex (n = 2), Nocardia sp. (n = 1), Aspergillus sp. (n = 1), or pulmonary Kaposi's sarcoma (n = 1). For serum HIV-1 RNA, the proportion of subjects with detectable levels and the mean values were similar for asymptomatic individuals and persons with active lung disease (85% versus 86%, respectively) (6.64 x 10(4) versus 1. 81 x 10(5) HIV-1 RNA copies/ml; p = 0.13). In contrast, HIV-1 RNA in BALF was more often detected (16% versus 62%; p = 0.001), and mean values were higher (1.04 x 10(5) versus 3.31 x 10(6) HIV-1 RNA copies/ml; p = 0.032), in subjects with active lung disease than in asymptomatic subjects, independent of early or advanced clinical stages of HIV-related disease. For both study groups, HIV-1 RNA levels in BALF exceeded those in serum in 56% of cases by up to 66-fold, and did not correlate with local levels of tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor, or interleukin-16. HIV-1 proviral DNA in cells from BALF was detected in up to 86% of subjects, more frequently in persons with advanced HIV disease (p = 0.0496), and often involved > 10% of BALF cells, but did not correlate with HIV-1 RNA detected in BALF. These data provide evidence for active HIV-1 replication in the lungs. HIV-1 replication is compartmentalized relative to serum, may be restricted, is independent of HIV-1 proviral DNA and clinical stage of HIV, and may be influenced by pulmonary disease such as P. carinii pneumonia or by other local or lung-specific factors. The lungs represent a large reservoir for HIV-1, and may present a source of persistent HIV-1 replication even during periods of apparent clinical latency of HIV-1 infection.

Re-emergence of HIV after stopping therapy.

A dormant reservoir of human immuno-deficiency virus (HIV) is established early on during primary infection1 which consists of latently infected, resting CD4+ T cells carrying replication-competent HIV. This pool can persist even in individuals who are receiving highly active antiretroviral therapy (HAART)2,3,4. Here we show that this pool rapidly re-emerges within weeks of discontinuing HAART in two patients, and that this re-emergence is associated with the appearance of HIV in the plasma (viraemia) of these patients. Both had been aviraemic while receiving HAART and intermittent treatment with interleukin-2 (ref. 5), and repeated attempts to isolate replication-competent HIV in this population of cells during therapy had been unsuccessful. This finding raises the possibility that there may be other tissue reservoirs of HIV that contribute to early plasma viral rebound following discontinuation of HAART in infected patients.

Level of human immunodeficiency virus DNA in peripheral blood mononuclear cells correlates with efficacy of antiretroviral therapy.

A novel colorimetric assay was developed and validated for accurate quantitation of human immunodeficiency virus (HIV) DNA in peripheral blood mononuclear cells (PBMCs). We tested 318 sequential samples from 56 subjects, 53 of whom were undergoing dual or triple therapy. Patients were considered responders when viremia levels were below 5, 000 HIV RNA copies/ml. The mean DNA copy numbers for untreated and responder subjects were similar (72 and 75, respectively), while it was 4.54-fold higher for nonresponders (339). This report provides strong evidence that HIV DNA levels in PBMCs correlate with therapeutic efficacy and suggests that DNA quantitation is a useful tool to monitor the decay of the HIV reservoir toward disease remission, especially when viremia is undetectable.

Reduced number of Langerhans cells in oesophageal mucosa from AIDS patients.

The oesophageal mucosa is a frequent target of opportunistic infections in human immunodeficiency virus (HIV) infection. Langerhans cells (LC) are known as a target and reservoir of HIV in the skin. The aim of this study was to characterize oesophageal LC in HIV-infected patients. Thirty oesophageal biopsies were obtained from 29 patients (median age 35.5), all in stage IV of the HIV Center of Disease Control Classification. We performed histological assessment of the oesophageal mucosa and immunohistochemical detection of oesophageal LC using an anti-CD1a antibody, followed by morphometric analysis. Biopsies from 17 noninfected patients were studied using the same procedure. LC in oesophageal mucosa of the HIV positive patients showed a significantly and dramatically decreased number (LC(N) median = 5.85/mm2) and surface/epithelial surface (LC (S) ratio = 0.09) when compared with HIV-negative controls (LC(N) median = 29.7/mm2, LC(S) ratio = 1.83) with P = 0.003 for LC(N) and P < 0.0001 for LC(S). These data suggest that oesophageal LC are, like their epidermal counterparts, a preferential target for HIV infection. Their alterations may provide a clue to the pathogenesis of the decreased local oesophageal immunity and to the occurrence of opportunistic infections.

Ikappakappa mediates NF-kappaB activation in human immunodeficiency virus-infected cells.

Human monocytes and macrophages are persistent reservoirs of human immunodeficiency virus (HIV) type-1. Persistent HIV infection of these cells results in increased levels of NF-kappaB in the nucleus secondary to increased IkappaBalpha, IkappaBbeta, and IkappaBepsilon degradation, a mechanism postulated to regulate viral persistence. To characterize the molecular mechanisms regulating HIV-mediated degradation of IkappaB, we have sought to identify the regulatory domains of IkappaBalpha targeted by HIV infection. Using monocytic cells stably expressing different transdominant molecules of IkappaBalpha, we determined that persistent HIV infection of these cells targets the NH2 but not the COOH terminus of IkappaBalpha. Further analysis demonstrated that phosphorylation at S32 and S36 is necessary for HIV-dependent IkappaBalpha degradation and NF-kappaB activation. Of the putative N-terminal IkappaBalpha kinases, we demonstrated that the Ikappakappa complex, but not p90(rsk), is activated by HIV infection and mediates HIV-dependent NF-kappaB activation. Analysis of viral replication in cells that constitutively express IkappaBalpha negative transdominant molecules demonstrated a lack of correlation between virus-induced NF-kappaB (p65/p50) nuclear translocation and degree of viral persistence in human monocytes.

Virus replication begins in dendritic cells during the transmission of HIV-1 from mature dendritic cells to T cells

Background: To initiate immunity, dendritic cells (DCs) capture antigens or viruses at body surfaces, undergo maturation to express T-cell costimulatory molecules, and then migrate to lymphoid organs. DCs at body surfaces can capture human immunodeficiency virus 1 (HIV-1), but mature DCs do not support replication of the virus unless T cells are added. The initial site for HIV-1 replication remains unknown and it is unclear whether replication can take place in DCs or whether the virus must first be transmitted from DCs to T cells.Results: We generated mature DCs from monocyte precursors. Upon infection with HIV-1, reverse transcription was completed only when T cells were added. When the reverse transcriptase inhibitor azidothymidine was added to the DCs during exposure to HIV-1, the DCs remained fully infectious, as long as the drug was removed just before culturing the DCs with T cells. HIV-1 variants that were engineered to undergo only one cycle of replication were able to infect DCs and replicate once in these cells. When T cells were added, newly produced HIV-1 Gag protein was exclusively localized to the DCs. With wild-type virus, subsequent rounds of replication took place in T cells. Soluble CD40 ligand (CD40L) and CD40L-transfected fibroblasts stimulated HIV-1 replication in purified mature DCs.Conclusions: Mature DCs provide a drug-resistant reservoir for HIV-1. This reservoir is activated within DCs by CD40L and upon interaction with T cells, and the virus then spreads rapidly to other T cells.