scholarly journals Preservation of Lymphocyte Immunophenotype and Proliferative Responses in Cryopreserved Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus Type 1-Infected Donors: Implications for Multicenter Clinical Trials

2000 ◽  
Vol 7 (3) ◽  
pp. 352-359 ◽  
Author(s):  
Keith A. Reimann ◽  
Miriam Chernoff ◽  
Cynthia L. Wilkening ◽  
Christine E. Nickerson ◽  
Alan L. Landay
Keyword(s):  
Clinical Trials ◽  
Mononuclear Cells ◽  
Lymphocyte Subsets ◽  
In Vitro Assays ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Multicenter Clinical Trials ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infection results in impaired immune function that can be measured by changes in immunophenotypically defined lymphocyte subsets and other in vitro functional assays. These in vitro assays may also serve as early indicators of efficacy when new therapeutic strategies for HIV-1 infection are being evaluated. However, the use of in vitro assays of immune function in multicenter clinical trials has been hindered by their need to be performed on fresh specimens. We assessed the feasibility of using cryopreserved peripheral blood mononuclear cells (PBMC) for lymphocyte immunophenotyping and for lymphocyte proliferation at nine laboratories. In HIV-1-infected patients with moderate CD4+ lymphocyte loss, the procedures of density gradient isolation, cryopreservation, and thawing of PBMC resulted in significant loss of CD19+ B cells but no measurable loss of total T cells or CD4+ or CD8+ T cells. No significant changes were seen in CD28− CD95+lymphocytes after cell isolation and cryopreservation. However, small decreases in HLA-DR+ CD38+ lymphocytes and of CD45RA+ CD62L+ were observed within both the CD4+ and CD8+ subsets. Fewer than 10% of those specimens that showed positive PBMC proliferative responses to mitogens or microbial antigens lost their responsiveness after cryopreservation. These results support the feasibility of cryopreserving PBMC for immunophenotyping and functional testing in multicenter AIDS clinical trials. However, small changes in selected lymphocyte subsets that may occur after PBMC isolation and cryopreservation will need to be assessed and considered in the design of each clinical trial.

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 Drug Combination of AZT and ddl: Synergism of Action and Prevention of Appearance of AZT-Resistance

1994 ◽  
Vol 5 (1) ◽  
pp. 51-55 ◽  
Author(s):  
G. Antonelli ◽  
F. Dianzani ◽  
D. Bellarosa ◽  
O. Turriziani ◽  
E. Riva ◽  
...  
Keyword(s):  
In Vitro Selection ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Resistant Strains ◽  
Azt Resistance ◽  
Hiv 1

Both 3′-azido-3′-deoxythymidine (AZT) and 2′,3′-dideoxynosine (ddl) strongly inhibit the replication of human immunodeficiency virus type 1 (HIV-1). Here, it is shown that combination of AZT and ddl at concentrations that are readily achievable in vivo synergistically inhibit HIV-1 replication in C8166 cells and peripheral blood mononuclear cells. The synergism is significant even when the effect of AZT and ddl alone was negligible. Our findings show that AZT-resistance is less likely to occur when a combination of AZT and ddl is used. Particularly, generation of AZT-resistant strains by in vitro selection is prevented, or delayed, by the combination of AZT plus ddl. Taken together these observations provide a rationale for combination of AZT and ddl in the therapy of AIDS patients.

scholarly journals Inhibition of Human Immunodeficiency Virus Type 1 Activity In Vitro by a New Self-Stabilized Oligonucleotide with Guanosine-Thymidine Quadruplex Motifs

2002 ◽  
Vol 76 (6) ◽  
pp. 3015-3022 ◽  
Author(s):  
Jun-ichiro Suzuki ◽  
Naoko Miyano-Kurosaki ◽  
Tomoyuki Kuwasaki ◽  
Hiroaki Takeuchi ◽  
Gota Kawai ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Mononuclear Cells ◽  
Core Antigen ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT An oligonucleotide with a dimeric hairpin guanosine quadruplex (basket type structure) (dG3T4G3-s), containing phosphorothioate groups, was able to inhibit human immunodeficiency virus type 1 (HIV-1)-induced syncytium formation and virus production (as measured by p24 core antigen expression) in peripheral blood mononuclear cells. This oligonucleotide lacks primary sequence homology with the complementary (antisense) sequences to the HIV-1 genome. Furthermore, this oligonucleotide may have increased nuclease resistance. The activity of this oligonucleotide was increased when the phosphodiester backbone was replaced with a phosphorothioate backbone. In vivo results showed that dG3T4G3-s was capable of blocking the interaction between gp120 and CD4. We also found that dG3T4G3-s specifically inhibits the entry of T-cell line-tropic HIV-1 into cells. This compound is a viable candidate for evaluation as a therapeutic agent against HIV-1 in humans.

scholarly journals Integration of Human Immunodeficiency Virus Type 1 in Untreated Infection Occurs Preferentially within Genes

2006 ◽  
Vol 80 (15) ◽  
pp. 7765-7768 ◽  
Author(s):  
Hongbing Liu ◽  
Eugene C. Dow ◽  
Reetakshi Arora ◽  
Jason T. Kimata ◽  
Lara M. Bull ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Integration Sites ◽  
Hiv 1

ABSTRACT Previous analyses of human immunodeficiency virus type 1 (HIV-1) integration sites generated in infections in vitro or in patients in whom viral replication was repressed by antiviral therapy have demonstrated a preference for integration within protein-coding genes. We analyzed integration sites in peripheral blood mononuclear cells (PBMCs), spleen, lymph node, and cerebral cortex from patients with untreated HIV-1 infections. The great majority of integration sites in each tissue were within genes. Statistical analyses of the frequencies of integration in genes in PBMCs and lymph tissue demonstrated a strong preference for integration within genes. Although the sample size for brain tissue was too small to demonstrate a clear statistical preference for integration in genes, four of the five integration sites identified in brain were within genes. Taken together, our data indicate that HIV-1 preferentially integrates within genes during untreated infection.

scholarly journals Mechanism of Action and In Vitro Activity of 1′,3′-Dioxolanylpurine Nucleoside Analogues against Sensitive and Drug-Resistant Human Immunodeficiency Virus Type 1 Variants

1999 ◽  
Vol 43 (10) ◽  
pp. 2376-2382 ◽  
Author(s):  
Zhengxian Gu ◽  
Mark A. Wainberg ◽  
Nghe Nguyen-Ba ◽  
Lucille L’Heureux ◽  
Jean-Marc de Muys ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mononuclear Cells ◽  
Nucleoside Analogues ◽  
Drug Resistant ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells ◽  
Anti Hiv ◽  
Hiv 1

ABSTRACT (−)-β-d-1′,3′-Dioxolane guanosine (DXG) and 2,6-diaminopurine (DAPD) dioxolanyl nucleoside analogues have been reported to be potent inhibitors of human immunodeficiency virus type 1 (HIV-1). We have recently conducted experiments to more fully characterize their in vitro anti-HIV-1 profiles. Antiviral assays performed in cell culture systems determined that DXG had 50% effective concentrations of 0.046 and 0.085 μM when evaluated against HIV-1IIIB in cord blood mononuclear cells and MT-2 cells, respectively. These values indicate that DXG is approximately equipotent to 2′,3′-dideoxy-3′-thiacytidine (3TC) but 5- to 10-fold less potent than 3′-azido-2′,3′-dideoxythymidine (AZT) in the two cell systems tested. At the same time, DAPD was approximately 5- to 20-fold less active than DXG in the anti-HIV-1 assays. When recombinant or clinical variants of HIV-1 were used to assess the efficacy of the purine nucleoside analogues against drug-resistant HIV-1, it was observed that AZT-resistant virus remained sensitive to DXG and DAPD. Virus harboring a mutation(s) which conferred decreased sensitivity to 3TC, 2′,3′-dideoxyinosine, and 2′,3′-dideoxycytidine, such as a 65R, 74V, or 184V mutation in the viral reverse transcriptase (RT), exhibited a two- to fivefold-decreased susceptibility to DXG or DAPD. When nonnucleoside RT inhibitor-resistant and protease inhibitor-resistant viruses were tested, no change in virus sensitivity to DXG or DAPD was observed. In vitro drug combination assays indicated that DXG had synergistic antiviral effects when used in combination with AZT, 3TC, or nevirapine. In cellular toxicity analyses, DXG and DAPD had 50% cytotoxic concentrations of greater than 500 μM when tested in peripheral blood mononuclear cells and a variety of human tumor and normal cell lines. The triphosphate form of DXG competed with the natural nucleotide substrates and acted as a chain terminator of the nascent DNA. These data suggest that DXG triphosphate may be the active intracellular metabolite, consistent with the mechanism by which other nucleoside analogues inhibit HIV-1 replication. Our results suggest that the use of DXG and DAPD as therapeutic agents for HIV-1 infection should be explored.

scholarly journals Human immunodeficiency virus types 1 and 2 have different replication kinetics in human primary macrophage culture

2006 ◽  
Vol 87 (2) ◽  
pp. 411-418 ◽  
Author(s):  
David Marchant ◽  
Stuart J. D. Neil ◽  
Áine McKnight
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Steady Rate ◽  
Immunodeficiency Virus ◽  
Load Characteristics ◽  
Kinetics Of ◽  
Hiv 1

This study compares the replication of primary isolates of human immunodeficiency virus type 2 (HIV-2) and type 1 (HIV-1) in monocyte-derived macrophages (MDMs). Eleven HIV-2 and five HIV-1 primary isolates that use CCR5, CXCR4 or both coreceptors to enter cells were included. Regardless of coreceptor preference, 10 of 11 HIV-2 viruses could enter, reverse transcribe and produce fully infectious virus in MDMs with efficiency equal to that in peripheral blood mononuclear cells. However, the kinetics of replication of HIV-2 compared with HIV-1 over time were distinct. HIV-2 had a burst of virus replication 2 days after infection that resolved into an apparent ‘latent state’ at day 3. HIV-1, however, continued to produce infectious virions at a lower, but steady, rate throughout the course of infection. These results may have implications for the lower pathogenesis and viral-load characteristics of HIV-2 infection.

scholarly journals Enhancement of Human Immunodeficiency Virus Type 1 Replication Is Not Intrinsic to All Polyanion-Based Microbicides

2009 ◽  
Vol 53 (8) ◽  
pp. 3565-3568 ◽  
Author(s):  
Secondo Sonza ◽  
Adam Johnson ◽  
David Tyssen ◽  
Tim Spelman ◽  
Gareth R. Lewis ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Sexual Transmission ◽  
Intrinsic Property ◽  
Enhancement Effect ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Polyanion-based microbicides have been developed to prevent the sexual transmission of human immunodeficiency virus (HIV). Recent data suggest that polyanions have the capacity to enhance HIV type 1 (HIV-1) replication at threshold antiviral concentrations. Evaluation of the microbicide candidates SPL7013 and PRO 2000 revealed no specific enhancement of two CCR5 HIV-1 strains in human peripheral blood mononuclear cells compared to enfuvirtide (Fuzeon). The enhancement effect is likely to be a function of the assay conditions and is not an intrinsic property of these polyanions.

Human serum alpha 1 acid glycoprotein reduces uptake, intracellular concentration, and antiviral activity of A-80987, an inhibitor of the human immunodeficiency virus type 1 protease.

1996 ◽  
Vol 40 (6) ◽  
pp. 1491-1497 ◽  
Author(s):  
J A Bilello ◽  
P A Bilello ◽  
K Stellrecht ◽  
J Leonard ◽  
D W Norbeck ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiviral Activity ◽  
Peripheral Blood Mononuclear Cells ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Acid Glycoprotein ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells ◽  
Hiv 1

The therapeutic utility of a human immunodeficiency virus type 1 (HIV-1) protease inhibitor may depend on its intracellular concentration, which is a property of its uptake, metabolism, and/or efflux. Previous studies in our laboratory indicated that the addition of alpha 1 acid glycoprotein (alpha 1 AGP) to the medium markedly increased the amount of the drug required to limit infection in vitro. In this study, physiologically relevant concentrations of alpha 1 AGP and a radiolabeled inhibitor, A-80987, were used to determine both the uptake and activity of the agent in HIV-1-infected human peripheral blood mononuclear cells and cell lines. Both the uptake and efflux of 14C-labeled A-80987 were rapid (t1/2, < 5 min). Uptake of the drug was linearly dependent on the concentration but insensitive to the metabolic inhibitors KF, sodium cyanide, or CCCP (carbonyl cyanide m-chlorophenyl hydrazone). The amount of A-80987 which entered the cells was inversely proportional to the concentration of alpha 1 AGP (r2, 0.99) and directly proportional to the amount of extracellular non-protein-bound drug (r2, 0.99). Most importantly, the antiviral activity of the drug in HIV-1-infected peripheral blood mononuclear cells and MT-2 cells was directly related to the amount of intracellular A-80987. This study demonstrates that A-80987 binds to alpha 1 AGP, resulting in a free fraction below 10%. Cellular uptake of A-80987 is proportionally decreased in the presence of alpha 1 AGP, which results in less-than-expected antiviral activity. Importantly, we demonstrate for the first time that the inhibition of HIV protease is highly correlated with the amount of intracellular inhibitor.

scholarly journals In Vitro Antiviral Activity of the Novel, Tyrosyl-Based Human Immunodeficiency Virus (HIV) Type 1 Protease Inhibitor Brecanavir (GW640385) in Combination with Other Antiretrovirals and against a Panel of Protease Inhibitor-Resistant HIV

2007 ◽  
Vol 51 (9) ◽  
pp. 3147-3154 ◽  
Author(s):  
Richard Hazen ◽  
Robert Harvey ◽  
Robert Ferris ◽  
Charles Craig ◽  
Phillip Yates ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiviral Activity ◽  
Protease Inhibitor ◽  
Reverse Transcriptase ◽  
In Vitro Assays ◽  
Immunodeficiency Virus ◽  
Anti Hiv ◽  
Hiv 1

ABSTRACT Brecanavir, a novel tyrosyl-based arylsulfonamide, high-affinity, human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI), has been evaluated for anti-HIV activity in several in vitro assays. Preclinical assessment of brecanavir indicated that this compound potently inhibited HIV-1 in cell culture assays with 50% effective concentrations (EC50s) of 0.2 to 0.53 nM and was equally active against HIV strains utilizing either the CXCR4 or CCR5 coreceptor, as was found with other PIs. The presence of up to 40% human serum decreased the anti-HIV-1 activity of brecanavir by 5.2-fold, but under these conditions the compound retained single-digit nanomolar EC50s. When brecanavir was tested in combination with nucleoside reverse transcriptase inhibitors, the antiviral activity of brecanavir was synergistic with the effects of stavudine and additive to the effects of zidovudine, tenofovir, dideoxycytidine, didanosine, adefovir, abacavir, lamivudine, and emtricitabine. Brecanavir was synergistic with the nonnucleoside reverse transcriptase inhibitor nevirapine or delavirdine and was additive to the effects of efavirenz. In combination with other PIs, brecanavir was additive to the activities of indinavir, lopinavir, nelfinavir, ritonavir, amprenavir, saquinavir, and atazanavir. Clinical HIV isolates from PI-experienced patients were evaluated for sensitivity to brecanavir and other PIs in a recombinant virus assay. Brecanavir had a <5-fold increase in EC50s against 80% of patient isolates tested and had a greater mean in vitro potency than amprenavir, indinavir, lopinavir, atazanavir, tipranavir, and darunavir. Brecanavir is by a substantial margin the most potent and broadly active antiviral agent among the PIs tested in vitro.

scholarly journals Multifunctional Human Immunodeficiency Virus (HIV) Gag-Specific CD8+ T-Cell Responses in Rectal Mucosa and Peripheral Blood Mononuclear Cells during Chronic HIV Type 1 Infection

2007 ◽  
Vol 81 (11) ◽  
pp. 5460-5471 ◽  
Author(s):  
J. William Critchfield ◽  
Donna Lemongello ◽  
Digna H. Walker ◽  
Juan C. Garcia ◽  
David M. Asmuth ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mononuclear Cells ◽  
Rectal Mucosa ◽  
Peripheral Blood Mononuclear ◽  
Tnf Α ◽  
Immunodeficiency Virus ◽  
Ifn Γ ◽  
Hiv 1

ABSTRACT The intestinal tract is a lymphocyte-rich site that undergoes severe depletion of memory CD4+ T cells within days of simian immunodeficiency virus or human immunodeficiency virus type 1 (HIV-1) infection. An ensuing influx of virus-specific CD8+ T cells, which persist throughout the chronic phase of infection, has also been documented in the gastrointestinal tract. However, little is known of the functionality of these effector cells or their relationship to the disease course. In this study, we measured CD8+ T-cell responses to HIV-1 peptides in paired rectal and blood samples from chronically infected patients. In both blood and rectum, there was an immunodominant CD8+ T-cell response to HIV Gag compared to Pol and Env (P < 0.01). In contrast, cytomegalovirus pp65 peptides elicited gamma interferon (IFN-γ) secretion strongly in peripheral blood mononuclear cells (PBMC) but weakly in rectal CD8+ T cells (P = 0.015). Upon stimulation with HIV peptides, CD8+ T cells from both sites were capable of mounting complex responses including degranulation (CD107 expression) and IFN-γ and tumor necrosis factor alpha (TNF-α) production. In rectal tissue, CD107 release was frequently coupled with production of IFN-γ or TNF-α. In patients not on antiretroviral therapy, the magnitude of Gag-specific responses, as a percentage of CD8+ T cells, was greater in the rectal mucosa than in PBMC (P = 0.054); however, the breakdown of responding cells into specific functional categories was similar in both sites. These findings demonstrate that rectal CD8+ T cells are capable of robust and varied HIV-1-specific responses and therefore likely play an active role in eliminating infected cells during chronic infection.

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