scholarly journals Determination of ddATP Levels in Human Immunodeficiency Virus-Infected Patients Treated with Dideoxyinosine

2004 ◽  
Vol 48 (2) ◽  
pp. 589-595 ◽  
Author(s):  
Cecile Le Saint ◽  
Raphael Terreux ◽  
Daniele Duval ◽  
Jacques Durant ◽  
Helene Ettesse ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Highly Active Antiretroviral Therapy ◽  
Mononuclear Cells ◽  
Exchange Chromatography ◽  
Therapeutic Monitoring ◽  
Target Cells ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus

ABSTRACT Clinical failures of the highly active antiretroviral therapy could result from inefficient intracellular concentrations of antiviral drugs. The determination of drug contents in target cells of each patient would be useful in clinical investigations and trials. The purpose of this work was to quantify the intracellular concentration of ddATP, the active metabolite of dideoxyinosine (ddI), in peripheral blood mononuclear cells (PBMCs) of human immunodeficiency virus (HIV)-infected patients treated with ddI. We have raised antibodies against ddA-citrate, a stable isostere of ddATP selected on the basis of its structural and electronic analogies with ddATP. The anti-ddA-citrate antibodies recognized ddATP and ddA with nanomolar affinities and cross-reacted neither with any of the nucleotide reverse transcriptase inhibitors used in HIV therapy nor with their phosphorylated metabolites. The three phosphorylated metabolites of ddI (ddAMP, ddADP, and ddATP) were purified by anion exchange chromatography and the amount of each metabolite was determined by radioimmunoassay with or without prior phosphatase treatment. The intracellular levels of the three ddI metabolites were measured both in an in vitro model and in PBMCs of HIV-infected patients under ddI treatment. The possibility to measure intracellular levels of ddATP from small blood samples of HIV-infected patients treated with ddI could be exploited to develop individual therapeutic monitoring.

scholarly journals Simultaneous Quantitation of Intracellular Zidovudine and Lamivudine Triphosphates in Human Immunodeficiency Virus-Infected Individuals

2000 ◽  
Vol 44 (11) ◽  
pp. 3097-3100 ◽  
Author(s):  
Jose F. Rodriguez ◽  
Jorge L. Rodriguez ◽  
Jorge Santana ◽  
Hermes García ◽  
Osvaldo Rosario
Keyword(s):  
Human Immunodeficiency Virus ◽  
Highly Active Antiretroviral Therapy ◽  
Mononuclear Cells ◽  
Solid Phase ◽  
Parent Compound ◽  
Hiv Positive ◽  
Peripheral Blood Mononuclear ◽  
Limit Of Quantitation ◽  
Immunodeficiency Virus

ABSTRACT Highly active antiretroviral therapy (HAART) is the standard treatment for infection with human immunodeficiency virus (HIV). The most common HAART regimen consists of the combination of at least one protease inhibitor (PI) with two nucleoside reverse transcriptase inhibitors (NRTIs). Contrary to PIs, NRTIs require intracellular activation from the parent compound of their triphosphate moiety to suppress HIV replication. Simultaneous intracellular determination of two NRTI triphosphates is difficult to accomplish due to their relatively small concentrations in peripheral blood mononuclear cells (PBMCs), requiring large amounts of blood from HIV-positive patients. Recently, we described a method to determine intracellular zidovudine triphosphate (ZDV-TP) concentrations in HIV-infected patients by using solid-phase extraction and tandem mass spectrometry. The limit of quantitation (LOQ) for ZDV-TP was 0.10 pmol, and the method was successfully used for the determination of ZDV-TP in HIV-positive patients. In this study, we enhanced the aforementioned method by the simultaneous quantitation of ZDV-TP and lamivudine triphosphate (3TC-TP) in PBMCs from HIV-infected patients. The LOQ for 3TC-TP was 4.0 pmol, with an interassay coefficient of variation and an accuracy of 7 and 12%, respectively. This method was successfully applied to the simultaneous in vivo determination of the ZDV-TP and 3TC-TP pharmacokinetic profiles from HIV-infected patients receiving HAART.

scholarly journals MDL 74,968, a new acyclonucleotide analog: activity against human immunodeficiency virus in vitro and in the hu-PBL-SCID.beige mouse model of infection.

1996 ◽  
Vol 40 (5) ◽  
pp. 1072-1077 ◽  
Author(s):  
C G Bridges ◽  
D L Taylor ◽  
P S Ahmed ◽  
T M Brennan ◽  
J M Hornsperger ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Hiv Infection ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Placebo Control ◽  
Intravenous Route ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Orally Bioavailable

The novel acyclonucleotide derivative of guanine, 9-[2-methylidene-3-(phosphonomethoxy)propyl] guanine (MDL 74,968), had antiviral activity comparable to those of 9-(2-phosphonomethoxyethyl) adenine (PMEA) and 2',3'-dideoxyinosine against laboratory strains of both human immunodeficiency virus (HIV) types 1 and 2 cultured in MT-4 cells and several clinical HIV isolates cultured in human peripheral blood mononuclear cells (PBMCs). MDL 74,968 was at least fourfold less toxic than PMEA to MT-4 cells or PBMCs, thereby producing a more favorable in vitro selectivity index for the former compound. Studies of acute toxicity in CD-1 mice showed that MDL 74,968 was not toxic at doses of 1,600 mg/kg of body weight via the intraperitoneal route or at doses of 500 mg/kg via the intravenous route. Furthermore, no adverse effects of MDL 74,968 were apparent when mice were treated at doses of 200 mg/kg twice daily for 5 days. Treatment by continuous subcutaneous infusion of MDL 74,968 or PMEA at the daily dose of 20 mg/kg in the hu-PBL-SCID.beige murine model of HIV infection significantly reduced the severity of infection compared with that in placebo-treated controls. Quantitation of virus recovery by endpoint titration of spleen cells in coculture with mitogen-activated PBMCs demonstrated that MDL 74,968 as well as PMEA significantly reduced the amount of virus (P < 0.02). Moreover, by using DNA extracted from spleens, the mean HIV:HLA PCR product ratio, which takes into account individual variation in immune system reconstitution, were 0.50 and 0.40 for MDL 74,968 and PMEA treatments, respectively, whereas animals receiving the placebo control had significantly higher levels of HIV proviral DNA (mean 0.78; P < 0.02). Taken together, these promising findings suggest that an orally bioavailable prodrug of MDL 74,968 should be developed for the treatment of HIV infection.

scholarly journals Response to Superantigen Stimulation in Peripheral Blood Mononuclear Cells from Children Perinatally Infected with Human Immunodeficiency Virus and Receiving Highly Active Antiretroviral Therapy

2004 ◽  
Vol 11 (5) ◽  
pp. 957-962 ◽  
Author(s):  
Thomas W. McCloskey ◽  
Viraga Haridas ◽  
Lucy Pontrelli ◽  
Savita Pahwa
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Antiretroviral Therapy ◽  
Highly Active Antiretroviral Therapy ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Highly Active ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells ◽  
Ifn Γ

ABSTRACT Our understanding of the pathogenesis of perinatal human immunodeficiency virus (HIV) infection is still evolving. We sought to characterize the response to the bacterial superantigen Staphylococcus enterotoxin B (SEB) of lymphocytes from HIV-infected children receiving treatment with highly active antiretroviral therapy (HAART). Using the flow cytometric methodology, we quantified apoptosis, proliferation, cytokine production, and activation antigen upregulation in CD4 and CD8 T lymphocytes following in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with SEB. The levels of proliferation, CD4 interleukin-2 (IL-2) production, CD8 gamma interferon (IFN-γ) production, and upregulation of CD69 expression by cells from HIV-infected children were indistinguishable from those by cells from controls. However, stimulation with SEB dramatically decreased the ratio of resting apoptotic cells to cycling apoptotic cells in the controls but not in the patients. In addition, unstimulated spontaneous apoptosis of CD4 T cells remained greater in the patients than in the controls. The percentages of IL-2-positive CD8 T cells and IFN-γ-positive CD4 T cells following SEB stimulation were significantly lower in the patients than in the controls. Our multiparameter approach was able to demonstrate differences in lymphocyte superantigen responsiveness in HIV-infected children receiving HAART in comparison to that in uninfected controls, notably, an apoptotic versus a proliferative response to stimulation.

scholarly journals Simian Immunodeficiency Virus SIVagm Efficiently Utilizes Non-CCR5 Entry Pathways in African Green Monkey Lymphocytes: Potential Role for GPR15 and CXCR6 as Viral Coreceptors

2015 ◽  
Vol 90 (5) ◽  
pp. 2316-2331 ◽  
Author(s):  
Nadeene E. Riddick ◽  
Fan Wu ◽  
Kenta Matsuda ◽  
Sonya Whitted ◽  
Ilnour Ourmanov ◽  
...  
Keyword(s):  
T Cells ◽  
Mononuclear Cells ◽  
Rhesus Macaques ◽  
Target Cells ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Sooty Mangabeys ◽  
Natural Hosts

ABSTRACTAfrican green monkeys (AGM) are natural hosts of simian immunodeficiency virus (SIV), and infection in these animals is generally nonpathogenic, whereas infection of nonnatural hosts, such as rhesus macaques (RM), is commonly pathogenic. CCR5 has been described as the primary entry coreceptor for SIVin vivo, while human-derived CXCR6 and GPR15 also appear to be usedin vitro. However, sooty mangabeys that are genetically deficient in CCR5 due to an out-of-frame deletion are infectible with SIVsmm, indicating that SIVsmm can use alternative coreceptorsin vivo. In this study, we examined the CCR5 dependence of SIV strains derived from vervet AGM (SIVagmVer) and the ability of AGM-derived GPR15 and CXCR6 to serve as potential entry coreceptors. We found that SIVagmVer replicated efficiently in AGM and RM peripheral blood mononuclear cells (PBMC) in the presence of the CCR5 antagonist maraviroc, despite the fact that maraviroc was capable of blocking the CCR5-tropic strains SIVmac239, SIVsmE543-3, and simian-human immunodeficiency virus SHIV-AD8 in RM PBMC. We also found that AGM CXCR6 and AGM GPR15, to a lesser extent, supported entry of pseudotype viruses bearing SIVagm envelopes, including SIVagm transmitted/founder envelopes. Lastly, we found that CCR5, GPR15, and CXCR6 mRNAs were detected in AGM and RM memory CD4+T cells. These results suggest that GPR15 and CXCR6 are expressed on AGM CD4+T cells and are potential alternative coreceptors for SIVagm usein vivo. These data suggest that the use of non-CCR5 entry pathways may be a common feature of SIV replication in natural host species, with the potential to contribute to nonpathogenicity in these animals.IMPORTANCEAfrican green monkeys (AGM) are natural hosts of SIV, and infection in these animals generally does not cause AIDS, whereas SIV-infected rhesus macaques (RM) typically develop AIDS. Although it has been reported that SIV generally uses CD4 and CCR5 to enter target cellsin vivo, other molecules, such as GPR15 and CXCR6, also function as SIV coreceptorsin vitro. In this study, we investigated whether SIV from vervet AGM can use non-CCR5 entry pathways, as has been observed in sooty mangabeys. We found that SIVagmVer efficiently replicated in AGM and RM peripheral blood mononuclear cells in the presence of the CCR5 antagonist maraviroc, suggesting that non-CCR5 entry pathways can support SIVagm entry. We found that AGM-derived GPR15 and CXCR6 support SIVagmVer entryin vitroand may serve as entry coreceptors for SIVagmin vivo, since their mRNAs were detected in AGM memory CD4+T cells, the preferred target cells of SIV.

scholarly journals Design and Profiling of GS-9148, a Novel Nucleotide Analog Active against Nucleoside-Resistant Variants of Human Immunodeficiency Virus Type 1, and Its Orally Bioavailable Phosphonoamidate Prodrug, GS-9131

2007 ◽  
Vol 52 (2) ◽  
pp. 655-665 ◽  
Author(s):  
Tomas Cihlar ◽  
Adrian S. Ray ◽  
Constantine G. Boojamra ◽  
Lijun Zhang ◽  
Hon Hui ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Mononuclear Cells ◽  
Biological Properties ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT GS-9148 [(5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl)phosphonic acid] is a novel ribose-modified human immunodeficiency virus type 1 (HIV-1) nucleotide reverse transcriptase (RT) inhibitor (NRTI) selected from a series of nucleoside phosphonate analogs for its favorable in vitro biological properties including (i) a low potential for mitochondrial toxicity, (ii) a minimal cytotoxicity in renal proximal tubule cells and other cell types, (iii) synergy in combination with other antiretrovirals, and (iv) a unique resistance profile against multiple NRTI-resistant HIV-1 strains. Notably, antiviral resistance analysis indicated that neither the K65R, L74V, or M184V RT mutation nor their combinations had any effect on the antiretroviral activity of GS-9148. Viruses carrying four or more thymidine analog mutations showed a substantially smaller change in GS-9148 activity relative to that observed with most marketed NRTIs. GS-9131, an ethylalaninyl phosphonoamidate prodrug designed to maximize the intracellular delivery of GS-9148, is a potent inhibitor of multiple subtypes of HIV-1 clinical isolates, with a mean 50% effective concentration of 37 nM. Inside cells, GS-9131 is readily hydrolyzed to GS-9148, which is further phosphorylated to its active diphosphate metabolite (A. S. Ray, J. E. Vela, C. G. Boojamra, L. Zhang, H. Hui, C. Callebaut, K. Stray, K.-Y. Lin, Y. Gao, R. L. Mackman, and T. Cihlar, Antimicrob. Agents Chemother. 52:648-654, 2008). GS-9148 diphosphate acts as a competitive inhibitor of RT with respect to dATP (Ki = 0.8 μM) and exhibits low inhibitory potency against host polymerases including DNA polymerase γ. Oral administration of GS-9131 to beagle dogs at a dose of 3 mg/kg of body weight resulted in high and persistent levels of GS-9148 diphosphate in peripheral blood mononuclear cells (with a maximum intracellular concentration of >9 μM and a half-life of >24 h). This favorable preclinical profile makes GS-9131 an attractive clinical development candidate for the treatment of patients infected with NRTI-resistant HIV.

scholarly journals Induction of Murine Mucosal CCR5-Reactive Antibodies as an Anti-Human Immunodeficiency Virus Strategy

2005 ◽  
Vol 79 (11) ◽  
pp. 6848-6858 ◽  
Author(s):  
C. Barassi ◽  
E. Soprana ◽  
C. Pastori ◽  
R. Longhi ◽  
E. Buratti ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Flock House Virus ◽  
Peripheral Blood Mononuclear ◽  
Genital Mucosa ◽  
Conformationally Constrained ◽  
Main Site ◽  
Immunodeficiency Virus

ABSTRACT The genital mucosa is the main site of initial human immunodeficiency virus type 1 (HIV-1) contact with its host. In spite of repeated sexual exposure, some individuals remain seronegative, and a small fraction of them produce immunoglobulin G (IgG) and IgA autoantibodies directed against CCR5, which is probably the cause of the CCR5-minus phenotype observed in the peripheral blood mononuclear cells of these subjects. These antibodies recognize the 89-to-102 extracellular loop of CCR5 in its native conformation. The aim of this study was to induce infection-preventing mucosal anti-CCR5 autoantibodies in individuals at high risk of HIV infection. Thus, we generated chimeric immunogens containing the relevant CCR5 peptide in the context of the capsid protein of Flock House virus, a presentation system in which it is possible to engineer conformationally constrained peptide in a highly immunogenic form. Administered in mice via the systemic or mucosal route, the immunogens elicited anti-CCR5 IgG and IgA (in sera and vaginal fluids). Analogous to exposed seronegative individuals, mice producing anti-CCR5 autoantibodies express significantly reduced levels of CCR5 on the surfaces of CD4+ cells from peripheral blood and vaginal washes. In vitro studies have shown that murine IgG and IgA (i) specifically bind human and mouse CD4+ lymphocytes and the CCR5-transfected U87 cell line, (ii) down-regulate CCR5 expression of CD4+ cells from both humans and untreated mice, (iii) inhibit Mip-1β chemotaxis of CD4+ CCR5+ lymphocytes, and (iv) neutralize HIV R5 strains. These data suggest that immune strategies aimed at generating anti-CCR5 antibodies at the level of the genital mucosa might be feasible and represent a strategy to induce mucosal HIV-protective immunity.

Immunophenotypic Analysis of Peripheral Blood Mononuclear Cells Undergoing In Vitro Apoptosis After Isolation From Human Immunodeficiency Virus–Infected Children

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4230-4237 ◽  
Author(s):  
Thomas W. McCloskey ◽  
Saroj Bakshi ◽  
Soe Than ◽  
Parisa Arman ◽  
Savita Pahwa
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Mononuclear Cells ◽  
Apoptotic Cell ◽  
Costimulatory Molecule ◽  
T Cell Subsets ◽  
Live Cells ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus

Abstract Lymphocytes of human immunodeficiency virus (HIV)-infected individuals undergo accelerated apoptosis in vitro, but the subsets of cells affected have not been clearly defined. This study examined the relationship between lymphocyte phenotype and apoptotic cell death in HIV-infected children by flow cytometry. Direct examination of the phenotype of apoptotic lymphocytes was accomplished using a combination of surface antigen labeling performed simultaneously with the Tdt mediated Utp nick end-labeling (TUNEL) assay. In comparison to live cells, apoptotic lymphocytes displayed an overrepresentation of CD45RO and HLA-DR expressing cells, while CD28 and CD95 expressing cells were underrepresented. Lymphocytes expressing CD4, CD8, and CD38 were equally represented in apoptotic and live populations. When percent lymphocyte apoptosis follow- ing culture was examined independently with lymphocyte subsets in fresh blood, apoptosis was negatively correlated with the percentage of CD4 cells, but not with specific CD4 T-cell subsets. Although not correlated with the percentage of total CD8 cells, apoptosis was positively correlated with specific CD8 T-cell subsets expressing CD45RO and CD95 and negatively correlated for CD8 T cells expressing CD45RA. These results provide direct evidence that a population of activated lymphocytes with the memory phenotype lacking the costimulatory molecule CD28 are especially prone to undergo apoptosis. The findings related to CD95 expression in fresh and apoptotic cells implicate Fas-dependent and Fas-independent pathways of apoptosis in HIV disease in children.

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