HIV-infected Peripheral Blood Mononuclear Cells Research Articles

Semen exosomes inhibit HIV infection and HIV-induced proinflammatory cytokine production independent of the activation state of primary lymphocytes.

Semen exosomes (SE) inhibit HIV infection. However, the effect of SE on cell activation and inflammation remains unknown. We characterized the response of peripheral blood mononuclear cells (PBMCs) from HIV-uninfected and antiretroviral therapy-suppressed HIV-infected (HIV+) subjects to SE. Quiescent PBMCs or T-cell receptor (TCR)-activated PBMCs from HIV- and HIV+ donors were stimulated with SE in the presence/absence of exvivo HIV infection. In HIV-infected PBMCs, SE did not reactivate HIV, did not induce lymphoblast development, nor increase CD69+/CD25+ numbers. Furthermore, SE inhibited denovo HIV infection without altering cell activation. SE also asynchronously downregulated HIV-inducible IL1β, IL8, and TNFα and upregulated CXCL10. These data suggest that SE inhibits HIV infection and production of HIV-induced proinflammatory cytokines while preserving lymphocyte activation.

A fully human antibody to gp41 selectively eliminates HIV-infected cells that transmigrated across a model human blood brain barrier.

Many HIV patients on combined antiretroviral therapy exhibit HIV-associated neurocognitive disorders because the brain becomes a viral reservoir. There is a need for therapeutics that can enter the central nervous system (CNS) and eradicate the virus. Radiolabeled human mAb 2556 to HIV gp41 selectively kills HIV-infected cells in vivo and in vitro. Here we tested the ability of 213Bi-2556 to cross a tissue culture model of the human blood brain barrier and kill HIV-infected peripheral blood mononuclear cells (PBMCs) and monocytes on the CNS side of the barrier. 2556 mAb isoelectric point was determined with isoelectric focusing. The ability of radiolabeled 2556 to penetrate through the barrier was studied by adding it to the upper chamber of the barriers and its penetration into the CNS side was followed for 5 h. To assess the ability of Bi-2556 to kill the HIV-infected cells on the CNS side of barrier, the HIV-infected and uninfected PBMCs and monocytes were allowed to transmigrate across the barriers overnight followed by application of Bi-2556 or control mAb Bi-1418 to the top of the barrier. Killing of cells was measured by TUNEL and Trypan blue assays. The barriers were examined by confocal microscopy for overt damage. The isoelectric point of Bi-2556 was 9.6 enabling its penetration through the barrier by transcytosis. Bi-2556 killed significantly more transmigrated HIV-infected cells in comparison to Bi-1418 and uninfected cells. No overt damage to barriers was observed. We demonstrated that Bi-2556 mAb crossed an in-vitro human blood brain barrier and specifically killed transmigrated HIV-infected PBMCs and monocytes without overt damage to the barrier.

HIV-1 Infected Peripheral Blood Mononuclear Cells Modulate the Fibrogenic Activity of Hepatic Stellate Cells through Secreted TGF-β and JNK Signaling

Background and AimsPatients with liver disease infected with the human immunodeficiency virus (HIV) exhibit accelerated progression of hepatic fibrosis and liver cirrhosis compared to uninfected individuals. We studied the effects of soluble factors secreted by HIV-infected peripheral blood mononuclear cells (PBMCs) on hepatic stellate cells (HSCs), which are central mediators of liver fibrosis.MethodsAn in vitro model was used in which a HSC line, LX2, was treated with culture supernatants of human PBMCs infected with macrophage tropic (R5) or T cell tropic (X4) strains of HIV-1. Quantitative reverse transcription PCR (qRT-PCR) and western blotting were used to assess the expression of fibrogenic and proinflammatory markers; LX2 proliferation and intracellular signaling pathways were also studied. A qRT-PCR based miRNome array was used for comparative miRNA profiling of LX2 cells treated with infected PBMC culture supernatants.ResultsPro-fibrogenic, angiogenic and proinflammatory markers, and proliferation of LX2 cells were increased following exposure to culture supernatants from HIV-1 infected PBMCs. The profiling of miRNAs in LX2 cells treated with culture supernatants from HIV-1 R5- or X4-infected PBMCs showed 66 and 22 miRNAs respectively, to be significantly altered compared to mock-treated LX2 cells. While different sets of miRNAs were altered in the two cases, bioinformatics analyses predicted these to be associated with common pathways, including TGF-β signaling and extracellular matrix receptor interaction pathways.ConclusionsHIV infection creates a favorable milieu for the activation of hepatic stellate cells and increased hepatic fibrosis. We identify some regulatory molecules important for these effects.

Early Stage High-Content HIV Diagnosis Based on Concurrent Monitoring of Actin Cytoskeleton, CD3, CD4, and CD8

Antiretroviral treatment can reduce the death rate of human immunodeficiency virus (HIV) infection, and its effectiveness is maximized at the early stage of HIV infection. The present study demonstrates an early stage high-content HIV diagnosis based on multicolor concurrent monitoring of CD4, CD8, and CD3 coreceptors and F-actin cytoskeleton using quantum dot (Qdot)-antibody conjugates at the single cell level. Artificial HIV infection of peripheral blood mononuclear cells (PBMCs) was achieved by the treatment of PBMCs with gp120 glycoproteins. Using the present system, it was determined that the CD4/CD8 ratios of normal PBMCs obtained from the blood samples of 11 adults were in the range of 1.04 to 1.52 as a result of the quantitative counting of single PBMCs while the CD4/CD8 ratios of artificial HIV-infected PBMCs were from 0.045 to 0.63. In addition, the structural changes of actin filament alignments in PBMCs bound to gp120 proteins were clearly observed by the multicolor single cell imaging system. This approach suggests a new model of accurate early stage HIV diagnosis simultaneously providing information on actin cytoskeleton and subtypes of PBMCs as well as their CD4/CD8 ratios.

Novel ‘Si[sbnd]C’ carbosilane dendrimers as carriers for anti-HIV nucleic acids: Studies on complexation and interaction with blood cells

Treatment of HIV infection by gene therapy is a promising tool for combating AIDS. One of the primary limitations of gene therapy is the effective delivery of nucleic acids to the target cells. Dendrimers are nanoparticles that are increasingly being used as nucleic acid vehicles. We have synthesized “SiC” amino-terminated carbosilane dendrimers [GnO3(NMe3)m]m+ functionalized with quaternary ammonium (NMe3+) terminal groups via hydrosilylation of allyl dimethylamine with the corresponding GnO3(SiH)m dendrimers and further addition of MeI. These dendrimers are soluble in water. Initially, complexation between these “SiC” dendrimers and anti-HIV nucleic acids (oligodeoxynucleotides ANTITAR and GEM91, siRNA siP24) was studied and molar ratios for complete complexation were determined. Then the charge and size of the dendriplexes (complexes of “SiC” dendrimers with nucleic acids) were analyzed and it was found that they possessed charges of +5 to +40mV and sizes of 60–600nm (zeta-size) or 50–100nm (atomic force microscopy) suitable for cell transfection. Stability studies showed that the dendriplexes were stable over time and were resistant to degradation by serum albumin. The effects of dendrimers and their dendriplexes on erythrocytes (isolated and in whole blood) revealed that the dendriplexes were significantly less cytotoxic than the pure dendrimers. The effects of dendrimers and their dendriplexes on peripheral blood mononuclear cells (the main target of HIV) were analyzed and it was found that the dendriplexes were 10 times less cytotoxic than the pure dendrimers. Finally, transfection experiments revealed that “SiC”-carbosilane dendrimers had a restricted ability to deliver long-chain double-stranded nucleic acids. The results indicate that these cationic carbosilane dendrimers are good candidates for delivering short-chain siRNA and oligodeoxynucleotide to HIV-infected peripheral blood mononuclear cells or lymphocytes.

M48U1 CD4 mimetic has a sustained inhibitory effect on cell-associated HIV-1 by attenuating virion infectivity through gp120 shedding

BackgroundHIV-1 infected cells can establish new infections by crossing the vaginal epithelia and subsequently producing virus in a milieu that avoids the high microbicide concentrations of the vaginal lumen.FindingsTo address this problem, here, we report that pretreatment of HIV-infected peripheral blood mononuclear cells (PBMCs) with a 27 amino acid CD4-mimetic, M48U1, causes dramatic and prolonged reduction of infectious virus output, due to its induction of gp120 shedding.ConclusionsM48U1 may, therefore, be valuable for prophylaxis of mucosal HIV-1 transmission.

Why are the neurodegenerative disease-related pathways overrepresented in primary HIV-infected peripheral blood mononuclear cells: a genome-wide perspective

We demonstrate for the first time that the genome-wide profiling of HIV-infected peripheral blood mononuclear cells (PBMCs) from HIV-patients free of neurologic disease show overrepresentation of neurodegenerative pathways (Alzheimer’s, Parkinson’s, ALS, Huntington’s and Prion Disease, etc.) in genome-wide microarray analysis, which suggests that this genome-wide representation of neurodegenerative diseases-related pathways in PBMCs could possibly be a subcellular manifestation of neurologic interference by HIV. Further, the cell-tagging analysis attested this belief showing the large majority of genes tagged with cells of monocyte and macrophage lineage, which are implicated in neuronal dysfunction in both viral and non-viral neurodegenerative diseases. Together, these findings suggest that the genomic interference of HIV with neurodegenerative pathways is not by chance, but may be an early sign of HIV-mediated sub-genomic and sub-cellular manifestation of neurologic disease. Moreover, these findings signify the utility of PBMC and genome-wide mapping of the host gene expression as a powerful tool in predicting possible early events in neurologic deterioration in HIV patients.

HIV-1 Infection Ex Vivo Accelerates Measles Virus Infection by Upregulating Signaling Lymphocytic Activation Molecule (SLAM) in CD4 + T Cells

Measles virus (MV) infection in children harboring human immunodeficiency virus type 1 (HIV-1) is often fatal, even in the presence of neutralizing antibodies; however, the underlying mechanisms are unclear. Therefore, the aim of the present study was to examine the interaction between HIV-1 and wild-type MV (MVwt) or an MV vaccine strain (MVvac) during dual infection. The results showed that the frequencies of MVwt- and MVvac-infected CD4(+) T cells within the resting peripheral blood mononuclear cells (PBMCs) were increased 3- to 4-fold after HIV-1 infection, and this was associated with a marked upregulation of signaling lymphocytic activation molecule (SLAM) expression on CD4(+) T cells but not on CD8(+) T cells. SLAM upregulation was induced by infection with a replication-competent HIV-1 isolate comprising both the X4 and R5 types and to a lesser extent by a pseudotyped HIV-1 infection. Notably, SLAM upregulation was observed in HIV-infected as well as -uninfected CD4(+) T cells and was abrogated by the removal of HLA-DR(+) cells from the PBMC culture. Furthermore, SLAM upregulation did not occur in uninfected PBMCs cultured together with HIV-infected PBMCs in compartments separated by a permeable membrane, indicating that no soluble factors were involved. Rather, CD4(+) T cell activation mediated through direct contact with dendritic cells via leukocyte function-associated molecule 1 (LFA-1)/intercellular adhesion molecule 1 (ICAM-1) and LFA-3/CD2 was critical. Thus, HIV-1 infection induces a high level of SLAM expression on CD4(+) T cells, which may enhance their susceptibility to MV and exacerbate measles in coinfected individuals.

Regional Cortical Thinning Associated with Detectable Levels of HIV DNA

High levels of human immunodeficiency virus (HIV) DNA in peripheral blood mononuclear cells (PBMCs), and specifically within CD14+ blood monocytes, have been found in HIV-infected individuals with neurocognitive impairment and dementia. The failure of highly active antiretroviral therapy (HAART) to eliminate cognitive dysfunction in HIV may be secondary to persistence of HIV-infected PBMCs which cross the blood-brain barrier, leading to perivascular inflammation and neuronal injury. This study assessed brain cortical thickness relative to HIV DNA levels and identified, we believe for the first time, a neuroimaging correlate of detectable PBMC HIV DNA in subjects with undetectable HIV RNA. Cortical thickness was compared between age- and education-matched groups of older (>40 years) HIV-seropositive subjects on HAART who had detectable (N = 9) and undetectable (N = 10) PBMC HIV DNA. Statistical testing revealed highly significant (P < 0.001) cortical thinning associated with detectable HIV DNA. The largest regions affected were in bilateral insula, orbitofrontal and temporal cortices, right superior frontal cortex, and right caudal anterior cingulate. Cortical thinning correlated significantly with a measure of psychomotor speed. The areas of reduced cortical thickness are key nodes in cognitive and emotional processing networks and may be etiologically important in HIV-related neurological deficits.

Evaluation of a Quantitative Real-Time PCR Assay to Measure HIV-Specific Mucosal CD8+ T Cell Responses in the Cervix

Several candidate HIV vaccines aim to induce virus-specific cellular immunity particularly in the genital tract, typically the initial site of HIV acquisition. However, standardized and sensitive methods for evaluating HIV-specific immune responses at the genital level are lacking. Therefore we evaluated real-time quantitative PCR (qPCR) as a potential platform to measure these responses. β-Actin and GAPDH were identified as the most stable housekeeping reference genes in peripheral blood mononuclear cells (PBMCs) and cervical mononuclear cells (CMCs) respectively and were used for normalizing transcript mRNA expression. HIV-specific cellular T cell immune responses to a pool of optimized CD8+ HIV epitopes (HIV epitope pool) and Staphylococcal enterotoxin B (SEB) superantigen control were assayed in HIV infected PBMC by qPCR, with parallel assessment of cytokine protein production. Peak HIV-specific mRNA expression of IFNγ, IL-2 and TNFα occurred after 3, 5 and 12 hours respectively. PBMCs were titrated to cervical appropriate cell numbers to determine minimum required assay input cell numbers; qPCR retained sensitivity with input of at least 2.5×104 PBMCs. This optimized qPCR assay was then used to assess HIV-specific cellular T cell responses in cytobrush-derived cervical T cells from HIV positive individuals. SEB induced IFNγ mRNA transcription was detected in CMCs and correlated positively with IFNγ protein production. However, qPCR was unable to detect HIV-induced cytokine mRNA production in the cervix of HIV-infected women despite robust detection of gene induction in PBMCs. In conclusion, although qPCR can be used to measure ex vivo cellular immune responses to HIV in blood, HIV-specific responses in the cervix may fall below the threshold of qPCR detection. Nonetheless, this platform may have a potential role in measuring mitogen-induced immune responses in the genital tract.

Interferon-γ-mediated pathways andin vitroPBMC proliferation in HIV-infected patients

HIV infection is characterized by progressive immunodeficiency: HIV-infected peripheral blood mononuclear cells (PBMCs) cannot properly react to stimulation with allo-antigens and mitogens. In this study, we examined interferon-gamma (IFN-gamma)-mediated pathways and the proliferative response of mitogen-stimulated HIV-infected PBMCs in vitro. PBMCs of 30 HIV-infected patients were stimulated with the mitogens concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweed mitogen (PWM). Mitogen stimulation induced expression of IFN-gamma, GTP cyclohydrolase I (GCH-I), and indoleamine (2,3)-dioxygenase (IDO) resulting in enhanced neopterin formation and tryptophan degradation by HIV-infected and control PBMCs. IFN-gamma concentrations correlated with neopterin levels and tryptophan degradation. Proliferative responses to PHA and PWM cytokine were lower in HIV patients, with IFN-gamma formation predicting proliferative responses. Higher mRNA expression of IFN-gamma, GCH-I and IDO after 6 h was related to better proliferative responses in HIV-infected PBMCs. In conclusion, induction of IFN-gamma and subsequent enzymes appears to importantly influence the proliferative response of HIV-infected PBMCs in vitro, suggesting a prominent role of the cytokine in the development of immunodeficiency.

Characterization of carbosilane dendrimers as effective carriers of siRNA to HIV-infected lymphocytes

One of the primary limitations of RNA interference as a technique for gene regulation is effective delivery of siRNA into the target cells. Dendrimers are nanoparticles that are increasingly being used as oligonucleotide and drug delivery vehicles. We have developed amino-terminated carbosilane dendrimers (CBS) as a means to protect and transport siRNA. Initially, stability studies showed that CBS bind siRNA via electrostatic interactions. Dendrimer-bound siRNA was found to be resistant to degradation by RNase. Cytotoxicity assays of CBS/siRNA dendriplexes with peripheral blood mononuclear cells (PBMC) and the lymphocytic cell line SupT1 revealed a maximum safe dendrimer concentration of 25 µg/ml. Next, utilizing flow cytometry and confocal microscopy, lymphocytes were seen to be successfully transfected by fluorochrome-labeled siRNA either naked or complexed with CBS. Dendriplexes with +/− charge ratio of 2 were determined to have the highest transfection efficiency while maintaining a low level of toxicity in these systems including hard-to-transfect HIV-infected PBMC. Finally, CBS/siRNA dendriplexes were shown to silence GAPDH expression and reduce HIV replication in SupT1 and PBMC. These results point to the possibility of utilizing dendrimers such as CBS to deliver and transfect siRNA into lymphocytes thus allowing the use of RNA interference as a potential alternative therapy for HIV infection.

Biphasic decay kinetics suggest progressive slowing in turnover of latently HIV-1 infected cells during antiretroviral therapy

BackgroundMathematical models based on kinetics of HIV-1 plasma viremia after initiation of combination antiretroviral therapy (cART) inferred HIV-infected cells to decay exponentially with constant rates correlated to their strength of virus production. To further define in vivo decay kinetics of HIV-1 infected cells experimentally, we assessed infected cell-classes of distinct viral transcriptional activity in peripheral blood mononuclear cells (PBMC) of five patients during 1 year after initiation of cARTResultsIn a novel analytical approach patient-matched PCR for unspliced and multiply spliced viral RNAs was combined with limiting dilution analysis at the single cell level. This revealed that HIV-RNA+ PBMC can be stratified into four distinct viral transcriptional classes. Two overlapping cell-classes of high viral transcriptional activity, suggestive of a virion producing phenotype, rapidly declined to undetectable levels. Two cell classes expressing HIV-RNA at low and intermediate levels, presumably insufficient for virus production and occurring at frequencies exceeding those of productively infected cells matched definitions of HIV-latency. These cells persisted during cART. Nevertheless, during the first four weeks of therapy their kinetics resembled that of productively infected cells.ConclusionWe have observed biphasic decays of latently HIV-infected cells of low and intermediate viral transcriptional activity with marked decreases in cell numbers shortly after initiation of therapy and complete persistence in later phases. A similar decay pattern was shared by cells with greatly enhanced viral transcriptional activity which showed a certain grade of levelling off before their disappearance. Thus it is conceivable that turnover/decay rates of HIV-infected PBMC may be intrinsically variable. In particular they might be accelerated by HIV-induced activation and reactivation of the viral life cycle and slowed down by the disappearance of such feedback-loops after initiation of cART.

Fibroblast growth factor-2 increases the renal recruitment and attachment of HIV-infected mononuclear cells to renal tubular epithelial cells

The role of circulating growth factors in the pathogenesis of childhood HIV-1-associated nephropathy (HIVAN) is not clearly understood. In previous studies, we found a significant accumulation of fibroblast growth factor-2 (FGF-2) in the circulation and kidneys of children with HIVAN. The purpose of this study was to determine whether circulating FGF-2 may play a role in the pathogenesis of HIVAN by increasing the renal recruitment and attachment of HIV-infected mononuclear cells to renal epithelial cells. Using in vitro cell adhesion assays, we showed that FGF-2 increased the attachment of peripheral blood mononuclear cells (PBMCs) to fibronectin-coated tissue culture dishes by approximately threefold through a mechanism that involved the alpha5 integrin subunit. In addition, we found that FGF-2 induces a similar increase in the attachment of HIV-infected PBMCs and monocytes/macrophages to plastic tissue culture dishes and to monolayers of primary renal tubular epithelial cells harvested from the urine of HIV-infected children with renal disease. Finally, we injected 16 adult C57Bl6/J male mice with recombinant adenoviral vectors carrying either the LacZ gene or a secreted form of human FGF-2 (5 x 10(8)pfu/mouse) and demonstrated that high levels of circulating FGF-2 can increase the renal recruitment of circulating inflammatory cells and induce transient tubulointerstitial injury in vivo. These data suggest that FGF-2 may have an immunomodulatory role in the pathogenesis of HIVAN by recruiting HIV-infected cells in the kidney.

Mannoproteins of Cryptococcus neoformans induce proliferative response in human peripheral blood mononuclear cells (PBMC) and enhance HIV-1 replication.

To investigate the possible role of Cryptococcus neoformans var. neoformans in HIV disease progression, and to identify the responsible cryptococcal components, an in vitro cell culture model was set up to study the C. neoformans-induced enhancement of HIV replication in HIV-1-infected PBMC. Similar to whole C. neoformans, cell-wall membrane fraction and mannoproteins induced proliferation of PBMC and enhancement of lymphotropic HIV replication in HIV-infected PBMC, while galactoxylomannan did not. MoAbs capable of interfering with MHC class II-mediated antigen presentation prevented the induction of cell proliferation by whole C. neoformans or cryptococcal mannoproteins. MoAb binding to adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) also inhibited C. neoformans-induced cell proliferation. In addition, anti-MHC class II MoAb inhibited the enhancement of HIV replication by C. neoformans. The results suggest that: (i) C. neoformans may accelerate HIV disease progression by stimulation of HIV replication through MHC class II-mediated antigen presentation; and (ii) cryptococcal mannoprotein may be one of the responsible components. The ability to enhance HIV replication in PBMC in vitro is not unique for C. neoformans. However, this is the first report to study in detail a yeast-induced enhancement of HIV replication in PBMC.

Role of neutralizing antibodies in HIV infection.

Role of neutralizing antibodies in HIV infection.

Structural requirements for potent anti-human immunodeficiency virus (HIV) and sperm-immobilizing activities of cyclohexenyl thiourea and urea non-nucleoside inhibitors of HIV-1 reverse transcriptase.

The current pandemic of sexually transmitted human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) has created an urgent need for a new type of microbicide, one that is both a spermicide and a virucide. In a systematic effort to identify a non-detergent-type antiviral spermicide, we have rationally designed and synthesized a series of cyclohexenyl thiourea (CHET) nonnucleoside inhibitors (NNIs) of HIV-1 reverse transcriptase (RT) with sperm-immobilizing activity (SIA). To gain further insight into the structural requirements for the optimal activity of these dual-function NNIs, we compared the effects of thiazolyl, benzothiazolyl, and pyridyl ring substitutions and functionalization with electron-donating and electron-withdrawing groups as well as the importance of thiourea and urea moieties of 15 heterocyclic ring-substituted NNIs. RT activity and p24 antigen production in HIV-infected peripheral blood mononuclear cells were used as markers of viral replication. Computer-assisted sperm analysis was used for evaluating SIA of CHET compounds. The rabbit model was used for evaluation of in vivo mucosal toxicity and contraceptive activity of the lead NNIs. Three CHET-NNIs with a bromo, chloro, or methyl substitution at the 5 position of the pyridyl ring exhibited potent anti-HIV activity at nanomolar concentrations (IC(50) = 3-5 nM) and SIA at micromolar concentrations (EC(50) = 45-96 micro M). The dual-function CHET-NNIs were potent inhibitors of drug-resistant HIV-1 strains with genotypic and phenotypic NNI resistance. Upon substitution of the sulfur atom of the thiourea moiety with an oxygen atom, the most striking difference noted was a 38-fold reduction in time required for 50% sperm immobilization (T(1/2)). A quantitative structure-activity relationship (QSAR) analysis was used in deriving regression equations between 20 physicochemical properties and SIA of NNIs. QSAR analysis showed that the T(1/2) values positively correlated with values for molecular refractivity (r = 0.88), hydrophobicity (r = 0.72), atomic polarizability (r = 0.70), and principal moment of inertia (r = 0.63) of spermicidal NNIs. A stepwise multiple regression model to describe the relationship of T(1/2) values with these four regressors provided excellent predictability (r = 0.93). Exposure of semen to thiourea/urea NNIs either alone or in combination at the time of artificial insemination led to marked or complete inhibition of pregnancy in rabbits as assessed by the number of embryo implants versus corpora lutea on Day 8 of pregnancy. Repeated intravaginal application of a gel-microemulsion with and without 0.5%, 1%, and 2% CHET-NNI or its urea analog either alone or in combination did not induce mucosal toxicity. We hypothesize that the gain of spermicidal function by CHET-NNIs is due to their metabolic oxidation to urea analogs by sperm. Three reaction pathways are discussed. The extremely rapid SIA of the urea analog as well as the broad-spectrum anti-HIV activity of spermicidal CHET-NNIs together with their lack of mucosal toxicity and the marked ability to reduce in vivo fertility is particularly useful for the clinical development of a dual-function spermicidal microbicide. The cyclohexenyl pyridyl NNIs, especially N-[2-(1-cyclohexenyl)ethyl] N'-[2-(5-bromopyridyl)]-thiourea in combination with the urea analog, show unique clinical potential as anti-HIV spermicides aimed at curbing the sexual transmission of multidrug-resistant HIV-1 while providing effective fertility control for women.

Effects of norepinephrine, HIV type 1 infection, and leukocyte interactions with endothelial cells on the expression of matrix metalloproteinases.

The expression of matrix metalloproteinases (MMPs) associated with AIDS-related cardiomypathies and cocaine abuse was examined in an in vitro coculture model. Human peripheral blood mononuclear cells (PBMCs), HIV infected or uninfected, were placed in coculture with primary human cardiac microvascular endothelial cells (HMVEC-C) in the presence or absence of the cocaine-inducible catecholamine norepinephrine (NE). Culture supernatants were assayed for MMP-1, -2, -3, -7, -9, and -13, and for tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-2, by enzyme-linked immunosorbent assay. Low levels of constitutively expressed MMP-1 and -2 were detected in individual cultures of HMVEC-C and PBMCs. NE did not induce MMP or TIMP expression by HMVEC-C and caused modest increases (3- to 4-fold) in MMP-1 and -2 by uninfected PBMCs. Increased levels of NE-induced MMP-1 (5-fold) and MMP -2 (15-fold) were detected in cocultures of HMVEC-C and uninfected PBMCs. HIV infection enhanced MMP-1 (46-fold) and MMP-2 (48-fold) and active MMP-7 (33-fold) and MMP-9 (50-fold) by PBMCs. Coculture of HIV-infected PBMCs with HMVEC-C increased MMP-1 (110-fold) and MMP-2 (307-fold) but not active MMP-7 and -9. The combination of NE, HIV infection, and coculture increased MMP-1 (126-fold) and MMP-2 (467-fold), and active MMP-7 (65-fold) and MMP-9 (75-fold). MMP-3 or-13 was not detected in any of the treatment groups and TIMP-1 and -2 appeared inversely proportional to the observed levels of MMPs. These results suggest that HIV infection, NE, and leukocyte endothelial interactions demonstrate separate and overlapping cooperative effects on the regulation of expression of TIMPs and MMPs associated with AIDS-related cardiomyopathies.

Cell-to-Cell Contact Results in a Selective Translocation of Maternal Human Immunodeficiency Virus Type 1 Quasispecies across a Trophoblastic Barrier by both Transcytosis and Infection

Mother-to-child transmission can occur in utero, mainly intrapartum and postpartum in case of breastfeeding. In utero transmission is highly restricted and results in selection of viral variant from the mother to the child. We have developed an in vitro system that mimics the interaction between viruses, infected cells present in maternal blood, and the trophoblast, the first barrier protecting the fetus. Trophoblastic BeWo cells were grown as a tight polarized monolayer in a two-chamber system. Cell-free virions applied to the apical pole neither crossed the barrier nor productively infected BeWo cells. In contrast, apical contact with human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells (PBMCs) resulted in transcytosis of infectious virus across the trophoblastic monolayer and in productive infection correlating with the fusion of HIV-infected PBMCs with trophoblasts. We showed that viral variants are selected during these two steps and that in one case of in utero transmission, the predominant maternal viral variant characterized after transcytosis was phylogenetically indistinguishable from the predominant child's virus. Hence, the first steps of transmission of HIV-1 in utero appear to involve the interaction between HIV type 1-infected cells and the trophoblastic layer, resulting in the passage of infectious HIV by transcytosis and by fusion/infection, both leading to a selection of virus quasispecies.

BMS-232632, a highly potent human immunodeficiency virus protease inhibitor that can be used in combination with other available antiretroviral agents.

BMS-232632 is an azapeptide human immunodeficiency virus type 1 (HIV-1) protease (Prt) inhibitor that exhibits potent anti-HIV activity with a 50% effective concentration (EC(50)) of 2.6 to 5.3 nM and an EC(90) of 9 to 15 nM in cell culture. Proof-of-principle studies indicate that BMS-232632 blocks the cleavage of viral precursor proteins in HIV-infected cells, proving that it functions as an HIV Prt inhibitor. Comparative studies showed that BMS-232632 is generally more potent than the five currently approved HIV-1 Prt inhibitors. Furthermore, BMS-232632 is highly selective for HIV-1 Prt and exhibits cytotoxicity only at concentrations 6,500- to 23, 000-fold higher than that required for anti-HIV activity. To assess the potential of this inhibitor when used in combination with other antiretrovirals, BMS-232632 was evaluated for anti-HIV activity in two-drug combination studies. Combinations of BMS-232632 with either stavudine, didanosine, lamivudine, zidovudine, nelfinavir, indinavir, ritonavir, saquinavir, or amprenavir in HIV-infected peripheral blood mononuclear cells yielded additive to moderately synergistic antiviral effects. Importantly, combinations of drug pairs did not result in antagonistic anti-HIV activity or enhanced cytotoxic effects at the highest concentrations used for antiviral evaluation. Our results suggest that BMS-232632 may be an effective HIV-1 inhibitor that may be utilized in a variety of different drug combinations.