ISSN:0269-9370    

出版商:OVID    

年代:2000

数据来源: OVID

ISSN:0269-9370    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

ObjectiveThe Nef protein has a major influence on disease pathogenesis in HIV-infected individuals. The objective of the present study was to examine the effects of Nef on T lymphocyte activation and associated signalling events.DesignA recombinant vaccinia expression system was used to express Nef in a human T cell line. Stimulation of these cells with anti-CD28 antibody, and either phorbol 12-myristate 13-acetate (PMA) or anti-CD3, activates signal transduction pathways and results in IL-2 production and IL-2 receptor α-chain (CD25) expression. Cellular responses were examined in cells expressing either Nef or an irrelevant control protein.MethodsActivation of signalling was assessed by immunoblot analysis, or by in-vitro phosphatidylinositol 3-kinase (PI3K) assays. IL-2 production was measured by enzyme-linked immunosorbent assay, and CD25 cell surface expression was examined using flow cytometry.ResultsInfection of cells with recombinant vaccinia expressing HIV-nefresulted in a marked increase in the production of IL-2 when cells were activated. The enhanced IL-2 response was accompanied by an increase in the level of PI3K activity. IL-2 production remained sensitive to inhibition with the PI3K competitive inhibitor Ly294002, and to the fungal macrolide, rapamycin. In contrast, CD25 expression was not affected, and there were no measurable changes to nuclear factor κB (NFκB) activation pathways.ConclusionEnhanced IL-2 production in stimulated T cells expressing HIV-Nef is associated with increased activation of PI3K-dependent signalling pathways. The results support a model in which Nef affects HIV disease progression by distorting T cell responses.

ISSN:0269-9370    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

ObjectiveTo compare the architecture and HIV-1 RNA and Gag p24 protein expression in lymph nodes (LN) excised from individuals during chronic highly active antiretroviral therapy (HAART) with LN removed from the same patient after plasma virus rebound following the interruption of HAART.Materials and methodsSix HIV-1-infected patients on HAART, with CD4 cell counts greater than 350 cells/μl, and plasma HIV-1 RNA less than 50 copies/ml, underwent inguinal LN excision upon discontinuation of HAART, and again after rebound of plasma virus. Lymph nodes were evaluated by immunohistochemical staining for Gag p24 antigen and Ki67, in-situ hybridization for HIV-1 RNA and H3-histone, and transmission electron microscopy (TEM).ResultsLN at baseline were quiescent to mildly hyperplastic and generally contained more primary than secondary follicles. Only one LN had detectable follicular dendritic cell (FDC)-associated p24 antigen, none had HIV RNA. Few mononuclear cells (MNC) expressed RNA or p24 antigen. Plasma virus at the second biopsy ranged from 329 to 3.2 × 106copies/ml. CD4 cell count decline ranged from 5 to 51% during drug hiatus, and was greatest in patients with highest viral rebound. Four of six of the second LN were more hyperplastic than the initial LN, two showed paracortical hyperplasia. MNC expression of HIV RNA in the second LN paralleled the level of plasma viremia. Increased Ki67 and H3-histone signal occurred in the second LN.ConclusionQuiescent LN from individuals on HAART rapidly become hyperplastic and activated within 1–2 months after treatment interruption. As in acute HIV infection, virus expression by LN MNC parallels the rebound in plasma viremia and fall in CD4 cell count.

ISSN:0269-9370    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

ObjectiveTo describe the short-term changes in CD4 lymphocyte counts after the interruption of antiretroviral HIV therapy in order to increase the understanding of CD4 lymphocyte dynamics, and so that appropriate monitoring strategies can be designed.MethodsWe studied 35 HIV-infected patients with late-stage disease who had therapy interruptions leading to high viral load levels, median greater than 750 000 RNA log10copies/ml, and in whom two CD4 cell counts (median 28 days apart) were available before beginning a salvage regimen.ResultsOverall, there was a substantial decline in CD4 cell counts from a median of 125 to 83 cells/mm3in the average 28 day period, with median proportionate and absolute losses of 26% and 24 cells/mm3per month, respectively (P< 0.008). This tended to be greater in individuals studied sooner after interrupting therapy (P= 0.03) and in those with CD4 cell counts above the pre-therapy baseline (P= 0.06). There was a strong negative correlation between the proportionate increase in viral load and the absolute change in CD4 cell count (−0.66,P= 0.0002).ConclusionPatients with relatively advanced HIV infection interrupting antiretroviral therapy after failing a protease inhibitor-containing regimen require frequent monitoring because CD4 cell counts appear to fall quite rapidly, at least in the first few weeks after interruption

ISSN:0269-9370    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

ObjectiveTo investigate the relationship between CC chemokine receptor 5 (CCR5) genotype, viral load and co-receptor usage of maternal HIV-1 isolates in perinatal HIV-1 transmission.Patients and methodsA total of 181 mothers and infants were studied at the time of delivery. Wild-type (wt) and Δ32 CCR5 alleles were determined by means of polymerase chain reaction (PCR). The viral load in maternal plasma samples was determined by a quantitative reverse transcriptase–PCR assay; co-receptor usage of maternal isolates was determined by viral infection in cells stably expressing CCR5 or CXC chemokine receptor 4 (CXCR4) co-receptors.ResultsHIV-1 transmission rates in wt/wt and wt/Δ32 mothers (14.7 versus 15.8%), and in wt/wt and wt/Δ32 infants (14.6 versus 14.3%) were similar. Mothers transmitting infection to wt/Δ32 infants had significantly higher HIV-1-RNA levels than those who transmitted infection to wt/wt infants (5.4 versus 4.1 log10copies/ml,P= 0.03). In wt/wt children there was a positive relationship between transmission rate and maternal viral load over the entire range of HIV-1 values, whereas in wt/Δ32 children transmission occurred only at viral loads greater than 4.0 log10copies/ml. Logistic regression analysis confirmed that the relationship between viral load and transmission varied according to the child's CCR5 genotype (P= 0.035; adjusted for zidovudine prophylaxis and mode of delivery,P= 0.090). Moreover, the majority of wt/wt transmitting mothers had R5-type isolates, whereas none of the wt/Δ32 mothers with an R5-type virus transmitted HIV-1 to their wt/Δ32 infants.ConclusionTaken together, these findings suggest that CCR5 Δ32 heterozygosity exerts a protective effect against perinatal transmission in children exposed to a low maternal viral burden of an R5-type isolate.

ISSN:0269-9370    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

BackgroundResistance against protease inhibitors (PI) can either be analysed genotypically or phenotypically. However, the interpretation of genotypic data is difficult, particularly for PI, because of the unknown contributions of several mutations to resistance and cross-resistance.ObjectiveDevelopment of an algorithm to predict PI phenotype from genotypic data.MethodsRecombinant viruses containing patient-derived protease genes were analysed for sensitivity to indinavir, saquinavir, ritonavir and nelfinavir. Drug resistance-associated mutations were determined by direct sequencing. geno- and phenotypic data were compared for 119 samples from 97 HIV-1 infected patients.ResultsSamples with one or two mutations in the gene for the protease were phenotypically sensitive in 74.3%, whereas 83.6% of samples with five or more mutations were resistant against all PI tested. Some mutations (36I, 63P, 71V/T, 77I) were frequent both in sensitive and resistant samples, whereas others (24I, 30N, 46I/L, 48V, 54V, 82A/F/T/S, 84V, 90M) were predominantly present in resistant samples. Therefore, the presence or absence of a single drug resistance-associated mutation predicted phenotypic PI resistance with high sensitivity (96.5–100%) but low specificity (13.3–57.4%). A more specific algorithm was obtained by taking into account the total number of drug resistance-associated mutations in the gene for the protease and restricting these to certain key positions for the PI. The algorithm was subsequently validated by analysis of 72 independent samples.ConclusionWith an optimized algorithm, phenotypic PI resistance can be predicted by viral genotype with good sensitivity (89.1–93.0%) and specificity (82.6–93.3%). The reliability and relevance of this algorithm should be further evaluated in clinical practice.

ISSN:0269-9370    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Background :Previous studies of the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on HIV-1 replication in macrophages have had inconsistent results, variously reporting no effect, augmentation or inhibition of viral replication.Objective :To investigate the regulation of HIV-1 in monocyte-derived macrophages (MDM) by GM-CSFin vitro.Methods :The role of GM-CSF on HIV-1 replication was assessed as supernatant and intracellular p24 antigen concentrations and by HIV-1 DNA and mRNA production under different culture conditions. Expression of CD4 and CCR5 receptors was examined. The effect of GM-CSF with an E21R mutation, which binds only to the α-chain of GM-CSF receptor, was used as an additional control.Results :GM-CSF consistently suppressed HIV-1 replication in human MDMin vitro, as assessed by supernatant and intracellular p24 antigen concentrations and HIV-1gagmRNA expression. The inhibitory effect of GM-CSF on HIV-1 replication was observed regardless of HIV-1 strain, source of GM-CSF, stage of MDM maturation or timing of GM-CSF exposure in relation to HIV-1 infection. The effect was dose dependent and reversed by addition of a neutralizing monoclonal antibody (4D4). Flow cytometric analysis of surface expression of CD4 and CCR5 indicates that GM-CSF does not affect HIV-1 entry into MDM. Analysis of intracellular HIV-1 DNA and mRNA suggests that HIV-1 replication is inhibited at or before transcription. E21R GM-CSF had no effect on HIV-1 replication in MDM.Conclusions :GM-CSF regulates HIV-1 replication in MDM, inhibiting HIV-1 replication through binding to the β-chain of the GM-CSF receptor.

ISSN:0269-9370    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

ObjectiveTo evaluate the efficacy of a genetic vaccination protocol based on minimalistic, immunogenic defined gene expression (MIDGE) vectors coding for domains of the feline immunodeficiency virus (FIV)envgene and feline IL-12.MethodsThree groups of four cats each were immunized three times within 6 weeks by the ballistic transfer of gold particles coated with MIDGE vectors. Group 1 received non-coated gold beads, groups 2 and 3 MIDGE vectors expressing FIV surface plus part of the transmembrane protein. In addition, group 3 received feline IL-12 DNA. All cats were challenged by intraperitoneal injection of 25 TCID50of infectious FIV Z2. The following criteria were monitored: clinical signs, antibodies to transmembrane protein, antibodies to whole FIV, haematological parameters and kinetics of CD4 and CD8 cells, FIV proviral load (determined by quantitative polymerase chain reaction; PCR) and cytotoxic T lymphocyte (CTL) activity (in selected cats).ResultsNone of the cats developed a detectable antibody response during immunizations. Four weeks after challenge exposure, all cats in group 1 (control) and group 2 (FIV surface-transmembrane protein) had seroconverted and showed a high proviral load until week 19 (end of experiment). In contrast, only one of four cats in group 3 (surface-transmembrane protein and IL-12) showed antibodies; it was provirus positive at reduced virus load. Short-lived CTL activity was found in two cats in group 3.ConclusionGenetic vaccination using a MIDGE-based construct for the expression of the surface-transmembrane protein domain of FIVenvand feline IL-12 DNA led to protection against homologous virus challenge in three out of four vaccinated cats.

ISSN:0269-9370    

出版商:OVID    

年代:2000

数据来源: OVID

ISSN:0269-9370    

出版商:OVID    

年代:2000

数据来源: OVID