RESUMEN

The impact of hepatitis C virus (HCV) RNA levels on immune status in chronically HCV mono-infected when compared to HIV/HCV co-infected on antiretroviral therapy (ART) remains poorly understood. A total of 78 African American subjects HCV viraemic/naïve to HCV treatment (33 HCV genotype 1 mono-infected, 45 ART-treated HIV/HCV genotype 1 co-infected) were studied. Clinical and liver enzyme measurements were performed. Whole blood was analysed for immune subset changes by flow cytometry. Peripheral blood mononuclear cells (PBMC) were used for same-day constitutive and in vitro Interferon (IFN)-α-induced signal transducer and activator of transcription (STAT) phosphorylation, K562 target cell lysis and K562 target cell recognition-mediated IFN-γ production. Statistical analysis was performed using R (2.5.1) or JMP Pro 11. While both groups did not differ in the level of liver enzymes, HIV/HCV had higher T-cell activation/exhaustion, and constitutive STAT-1 phosphorylation compared to HCV. In contrast, CD4+ FoxP3+ CD25+ frequency, IFN-αR expression on NK cells, as well as constitutive and IFN-α-induced direct cytotoxicity were lower in HIV/HCV. Linear regression models further supported these results. Finally, increase in HCV viral load and CD4+ T-cell count had an opposite effect between the two groups on NK cell activity and T-cell activation, respectively. HCV viral load in ART-treated HIV/HCV co-infection was associated with greater immune activation/exhaustion and NK dysfunction than HCV viral load alone in HCV mono-infection. The more pronounced immune modulation noted in ART-treated HIV-co-infected/untreated HCV viraemic subjects may impact HCV disease progression and/or response to immunotherapy.

Asunto(s)

Coinfección , Infecciones por VIH , Hepacivirus/inmunología , Hepatitis C/inmunología , Hepatitis C/virología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Viremia , Terapia Antirretroviral Altamente Activa , Antivirales/farmacología , Antivirales/uso terapéutico , Biomarcadores , Recuento de Linfocito CD4 , Citocinas/biosíntesis , Citotoxicidad Inmunológica , Quimioterapia Combinada , Femenino , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Hepatitis C/metabolismo , Humanos , Inmunofenotipificación , Células Asesinas Naturales/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/efectos de los fármacos , Carga Viral

RESUMEN

OBJECTIVES: Tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) is approved for pre-exposure prophylaxis (PrEP) against HIV infection. Adherence is critical for the success of PrEP, but current adherence measurements are inadequate for real-time adherence monitoring. We developed and validated a urine assay to measure tenofovir (TFV) to objectively monitor adherence to PrEP. METHODS: We developed a urine assay using high-performance liquid chromatography coupled to tandem mass spectrometry with high sensitivity/specificity for TFV that allowed us to determine TFV concentrations in log10 categories between 0 and 10 000 ng/mL. We validated the assay in three cohorts: (1) HIV-positive subjects with undetectable viral loads on a TDF/FTC-based regimen, (2) healthy HIV-negative subjects who received a single dose of TDF/FTC, and (3) HIV-negative subjects receiving daily TDF/FTC as PrEP for 24 weeks. RESULTS: The urine assay detected TFV with greater sensitivity than plasma-based measures and with a window of measurements within 7 days of the last TDF/FTC dose. Based on the urine log-linear clearance after the last dose and its concordance with all detectable plasma levels, a urine TFV concentration > 1000 ng/mL was identified as highly predictive of the presence of TFV in plasma at > 10 ng/mL. The urine assay was able to distinguish high and low adherence patterns within the last 48 h (> 1000 ng/mL versus 10-1000 ng/mL), as well as nonadherence (< 10 ng/mL) extended over at least 1 week prior to measurement. CONCLUSIONS: We provide proof of concept that a semiquantitative urine assay measuring levels of TFV could be further developed into a point-of-care test and be a useful tool to monitor adherence to PrEP.

Asunto(s)

Antivirales/administración & dosificación , Emtricitabina/administración & dosificación , Infecciones por VIH/prevención & control , Tenofovir/administración & dosificación , Tenofovir/orina , Adulto , Antivirales/uso terapéutico , Cromatografía Liquida , Esquema de Medicación , Emtricitabina/uso terapéutico , Femenino , Humanos , Masculino , Cumplimiento de la Medicación , Persona de Mediana Edad , Proyectos Piloto , Profilaxis Pre-Exposición , Espectrometría de Masas en Tándem , Tenofovir/uso terapéutico , Adulto Joven

RESUMEN

Sex workers practicing in high HIV endemic areas have been extensively targeted to test anti-HIV prophylactic strategies. We hypothesize that in women with high levels of genital exposure to semen changes in cervico-vaginal mucosal and/or systemic immune activation will contribute to a decreased susceptibility to HIV-1 infection. To address this question, we assessed sexual activity and immune activation status (in peripheral blood), as well as cellular infiltrates and gene expression in ectocervical mucosa biopsies in female sex workers (FSWs; n=50), as compared with control women (CG; n=32). FSWs had low-to-absent HIV-1-specific immune responses with significantly lower CD38 expression on circulating CD4(+) or CD8(+) T-cells (both: P<0.001) together with lower cervical gene expression of genes associated with leukocyte homing and chemotaxis. FSWs also had increased levels of interferon-ɛ (IFNɛ) gene and protein expression in the cervical epithelium together with reduced expression of genes associated with HIV-1 integration and replication. A correlative relationship between semen exposure and elevated type-1 IFN expression in FSWs was also established. Overall, our data suggest that long-term condomless sex work can result in multiple changes within the cervico-vaginal compartment that would contribute to sustaining a lower susceptibility for HIV-1 infection in the absence of HIV-specific responses.

Asunto(s)

Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/fisiología , Infecciones por VIH/inmunología , VIH-1/fisiología , Interferones/metabolismo , Membrana Mucosa/inmunología , Trabajadores Sexuales , Adulto , Cuello del Útero/patología , Susceptibilidad a Enfermedades , Femenino , Regulación Viral de la Expresión Génica , Humanos , Tolerancia Inmunológica , Interferón Tipo I/metabolismo , Interferones/genética , Activación de Linfocitos/genética , Membrana Mucosa/virología , Semen/inmunología , Conducta Sexual , Integración Viral/genética , Replicación Viral/genética

RESUMEN

The description of highly exposed individuals who remain seronegative (HESN) despite repeated exposure to human immunodeficiency virus (HIV)-1 has heightened interest in identifying potential mechanisms of HIV-1 resistance. HIV-specific humoral and T cell-mediated responses have been identified routinely in HESN subjects, although it remains unknown if these responses are a definitive cause of protection or merely a marker for exposure. Approximately half of HESN lack any detectible HIV-specific adaptive immune responses, suggesting that other mechanisms of protection from HIV-1 infection also probably exist. In support of the innate immune response as a mechanism of resistance, increased natural killer (NK) cell activity has been correlated with protection from infection in several high-risk cohorts of HESN subjects, including intravenous drug users, HIV-1 discordant couples and perinatally exposed infants. Inheritance of protective NK KIR3DL1(high) and KIR3DS1 receptor alleles have also been observed to be over-represented in a high-risk cohort of HESN intravenous drug users and HESN partners of HIV-1-infected subjects. Other intrinsic mechanisms of innate immune protection correlated with resistance in HESN subjects include heightened dendritic cell responses and increased secretion of anti-viral factors such as ß-chemokines, small anti-viral factors and defensins. This review will highlight the most current evidence in HESN subjects supporting the role of epithelial microenvironment and the innate immune system in sustaining resistance against HIV-1 infection. We will argue that as a front-line defence the innate immune response determines the threshold of infectivity that HIV-1 must overcome to establish a productive infection.

Asunto(s)

Seronegatividad para VIH/inmunología , VIH-1/inmunología , Inmunidad Innata , Inmunidad Mucosa/inmunología , Adulto , Animales , Quimiocinas/fisiología , Defensinas/fisiología , Células Dendríticas/inmunología , Susceptibilidad a Enfermedades/inmunología , Femenino , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/fisiología , Humanos , Inmunoglobulina A/inmunología , Recién Nacido , Transmisión Vertical de Enfermedad Infecciosa , Células Asesinas Naturales/inmunología , Macaca mulatta , Compartición de Agujas , Exposición Profesional , Embarazo , Asunción de Riesgos , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Replicación Viral

RESUMEN

Assessment of circulating CD4 count change over time in HIV-infected subjects on antiretroviral therapy (ART) is a central component of disease monitoring. The increasing number of HIV-infected subjects starting therapy and the limited capacity to support CD4 count testing within resource-limited settings have fueled interest in identifying correlates of CD4 count change such as total lymphocyte count, among others. The application of modeling techniques will be essential to this endeavor due to the typically non-linear CD4 trajectory over time and the multiple input variables necessary for capturing CD4 variability. We propose a prediction based classification approach that involves first stage modeling and subsequent classification based on clinically meaningful thresholds. This approach draws on existing analytical methods described in the receiver operating characteristic curve literature while presenting an extension for handling a continuous outcome. Application of this method to an independent test sample results in greater than 98% positive predictive value for CD4 count change. The prediction algorithm is derived based on a cohort of n = 270 HIV-1 infected individuals from the Royal Free Hospital, London who were followed for up to three years from initiation of ART. A test sample comprised of n = 72 individuals from Philadelphia and followed for a similar length of time is used for validation. Results suggest that this approach may be a useful tool for prioritizing limited laboratory resources for CD4 testing after subjects start antiretroviral therapy.

RESUMEN

We demonstrate the application and comparative interpretations of three tree-based algorithms for the analysis of data arising from flow cytometry: classification and regression trees (CARTs), random forests (RFs), and logic regression (LR). Specifically, we consider the question of what best predicts CD4 T-cell recovery in HIV-1 infected persons starting antiretroviral therapy with CD4 count between 200 and 350 cell/muL. A comparison to a more standard contingency table analysis is provided. While contingency table analysis and RFs provide information on the importance of each potential predictor variable, CART and LR offer additional insight into the combinations of variables that together are predictive of the outcome. In all cases considered, baseline CD3-DR-CD56+CD16+ emerges as an important predictor variable, while the tree-based approaches identify additional variables as potentially informative. Application of tree-based methods to our data suggests that a combination of baseline immune activation states, with emphasis on CD8 T-cell activation, may be a better predictor than any single T-cell/innate cell subset analyzed. Taken together, we show that tree-based methods can be successfully applied to flow cytometry data to better inform and discover associations that may not emerge in the context of a univariate analysis.

RESUMEN

BACKGROUND: Natural killer (NK) cells and plasmacytoid and myeloid dendritic cells (DCs) are depleted, and their function impaired, in advanced adult human immunodeficiency virus (HIV)-1 infection. Studies in perinatally infected children are lacking. METHODS: Percentages of NK cells and plasmacytoid and myeloid DCs were evaluated by flow cytometry. Forty children with perinatal HIV-1 infection were compared with 11 age-matched, uninfected children. Plasmacytoid and myeloid DC function was evaluated by activation-induced cytokine secretion. RESULTS: Virally suppressed children had normal levels of circulating plasmacytoid and myeloid DCs and total NK cells but had sustained depletion of a mature (CD3-/161+/56+/16+) NK cell subset and decreased interferon- alpha secretion by plasmacytoid DCs. Despite similar viral loads, percentages of myeloid and plasmacytoid DCs and mature NK cells were significantly lower in viremic children with a history of decreasing CD4+ cell percentages, compared with children with stable CD4+ cell counts. CONCLUSIONS: Children achieve partial reconstitution of myeloid and plasmacytoid DCs and NK cells during viral suppression; irrespective of viral load, a clinical history of decreasing CD4+ cell percentage is associated with greater depletion of these subsets. We hypothesize that the evaluation of selected innate-immunity effector cells may serve as a marker of CD4+ cell loss in pediatric HIV-1 infection.

Asunto(s)

Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida/inmunología , Recuento de Linfocito CD4 , Células Dendríticas/inmunología , Células Asesinas Naturales/inmunología , Síndrome de Inmunodeficiencia Adquirida/sangre , Niño , Citocinas/sangre , Femenino , Citometría de Flujo , VIH-1 , Antígenos HLA-DR/sangre , Humanos , Interferón-alfa/sangre , Subgrupos Linfocitarios/inmunología , Masculino , Valores de Referencia

RESUMEN

CD8 T cells are believed to play a key role in the immune control of human immunodeficiency virus-1 (HIV-1) infection in children as well as in adults. We have used an enhanced EliSpot (AmpliSpot) assay to quantitate CD8 T-cell responses directed to five human leucocyte antigen (HLA)-A2-presented HIV-1 epitopes derived from the key viral antigen Nef. Responses were assayed in one group of 21 children with vertically acquired HIV infection and one group of 19 adult subjects with chronic infection. The paediatric group displayed significantly weaker and more narrowly focused CD8 T-cell responses as compared with the adult subjects. Two epitopes stood out as the most frequently and strongly recognized, suggesting that they should be considered immunodominant in the CD8 T-cell response to HIV-1 Nef. Interestingly, the most frequently and strongly recognized epitope in both adults and children was previously identified in HLA-A2-transgenic mice, demonstrating the usefulness of such mice in finding natural viral epitopes. These findings indicate significant weakness in strength and breadth of the CD8 T-cell response to the target protein Nef in infected children and prompt renewed efforts into the immunology of vertically acquired HIV-1 infection.

Asunto(s)

Linfocitos T CD8-positivos/inmunología , Productos del Gen nef/inmunología , Infecciones por VIH/transmisión , Antígeno HLA-A2/inmunología , Transmisión Vertical de Enfermedad Infecciosa , Adulto , Animales , Niño , Epítopos de Linfocito T/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Inmunidad Celular , Productos del Gen nef del Virus de la Inmunodeficiencia Humana

RESUMEN

OBJECTIVE: To investigate the infectivity of T-helper (Th)1 and Th2 cells (derived from ccr5 wild-type and homozygous ccr5 Delta 32) to R5 and X4 HIV-1. DESIGN: It remains unclear whether infection of Th1 and Th2 CD4 cells by R5 and X4 viruses mirrors their co-receptor expression profile as no direct quantitation of coreceptor levels and infection has been performed. In addition, it is unknown whether the lack of CCR5 expression affects the degree of Th1/Th2 polarization. METHODS: Surface expression of CCR5 and CXCR4 was determined by quantitative fluorescence activated cell sorter analysis on in vitro differentiated Th1 and Th2 cells. R5 (Ba-L) and X4 (IIIB) HIV-1 isolates were used for infection studies and the efficiency of viral entry was determined by quantitative real time polymerase chain reaction detection of reverse transcribed proviral DNA. RESULTS: Cell surface density of CCR5 molecules was eight-fold higher in Th1 versus Th2 subsets (P = 0.005) whereas CXCR4 surface density was four-fold higher in Th2 versus Th1 subsets (P = 0.006). Preferential infection and entry of Th1 cells by R5 HIV-1 was not associated with preferential replication, as eventually the R5-virus replicated to a higher level in Th2 cells in spite of lower initial viral infection/entry. By contrast, Th2 cells preferentially supported X4-virus infection and replication. High beta chemokine secretion by Th1 cells was associated with a lower R5 replication rate. CONCLUSIONS: Th1 and Th2 cells differ in their infection efficiency for R5 and X4 HIV-1. ccr5 Delta 32-homozygous individuals maintain the ability for Th1/Th2 polarization, i.e., the expression of CCR5 is not required for Th1/Th2 polarization.

Asunto(s)

VIH-1/patogenicidad , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Células TH1/virología , Células Th2/virología , Línea Celular , Polaridad Celular , Quimiocinas CC/metabolismo , ADN Viral/sangre , VIH-1/clasificación , VIH-1/genética , VIH-1/fisiología , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células TH1/metabolismo , Células Th2/metabolismo , Replicación Viral

RESUMEN

Initially thought to be functionally redundant with IL-4 as a predominant anti-inflammatory factor secreted during type-2 T-cell responses, IL-13 possesses a number of additional properties that distinguish it from IL-4 in addition to having both anti-inflammatory and immune activating properties. This review centers primarily on the role of IL-13 in the regulation of cellular functions of innate immunity and acquired immunity against certain microbial pathogens. First, we discuss IL-13's regulation of innate cell targets and its impact on inflammation, antigen uptake and antigen presentation. Second, we focus on IL-13's involvement in acquired immunity to infectious helminths and protozoa. The role of this cytokine in immune responses is still being determined but evidence to date suggests this molecule has been conserved as an important regulatory factor involved in both early innate and late adaptive responses.

Asunto(s)

Interleucina-13/inmunología , Adaptación Fisiológica , Animales , Formación de Anticuerpos , Quimiocinas/biosíntesis , Citocinas/biosíntesis , Humanos , Inmunidad Celular , Cambio de Clase de Inmunoglobulina , Infecciones/inmunología , Interleucina-4/inmunología , Óxido Nítrico/biosíntesis , Transducción de Señal

RESUMEN

The RNA genome of human immunodeficiency virus type 1 (HIV-1) is converted into DNA after infection in order to integrate into the host cell DNA. However, a large number of these reverse-transcribed genomes remain unintegrated in the nucleus of infected cells. Currently, there are no data available about the intranuclear distribution pattern of unintegrated HIV-1 DNA in relation to nuclear structures as observed on the single-cell level. In the present study, we investigated the intranuclear fate of unintegrated viral DNA in cell lines expressing CD4 and coreceptors (HOS-CD4.CCR5 and U373-MAGI-CXCR4(CEM)) infected with HIV-1 (strain 89.6). We used a novel approach to distinguish in situ unintegrated from integrated viral DNA by performing fluorescent in situ hybridization on cells in which stress-induced chromosome condensation had been induced, a procedure that contracts chromosomes independent of the cell cycle. Cells infected for 15 h accumulated large amounts of HIV-1 DNA which was located between the condensed chromosome strands, allowing the identification of this viral DNA as unintegrated. In contrast, in HeLa/LAV, a cell line carrying integrated HIV-1 genomes, the great majority of viral DNA colocalized with the cellular DNA. We show that unintegrated HIV-1 DNA does not evenly distribute within the host cell nucleus but tends to aggregate into clusters containing many copies of the viral genomes. The formation of these DNA clusters was independent of viral DNA replication and thus appeared to result solely from multiple infections. The DNA aggregates remained in the nuclei of infected cells for at least 25 h after the infection was stopped. The emergence of transcription sites, which most likely denote sites of the integrated provirus, lagged clearly behind the accumulation of viral DNA. These transcription foci could not be linked to unintegrated DNA molecules, suggesting that this DNA type is unable to transcribe, at least at levels comparable to those of integrated DNA. Neither unintegrated HIV-1 DNA nor transcription foci nor integrated DNA was observed to associate with nuclear domain 10 (ND10), a nuclear structure known to represent the site where several DNA viruses replicate and transcribe. Also, HIV-1 does not modify ND10 at early or late times of infection. There was no specific association of HIV-1 transcripts with splicing factor SC35 domains, in contrast to what has been reported for a number of both cellular and viral genes. Surprisingly, unintegrated HIV-1 DNA was found to accumulate within or in close association with SC35 domains, demonstrating a specific distribution of the viral DNA within the host cell nucleus. Taken together, our results demonstrate that unintegrated proviral HIV-1 DNA does not randomly localize within infected cells but preferentially aggregates in the nucleus within SC35 domains.

Asunto(s)

ADN Viral/fisiología , Infecciones por VIH/virología , VIH-1/fisiología , Transporte Biológico , Núcleo Celular/genética , Núcleo Celular/virología , Infecciones por VIH/genética , Células HeLa , Humanos , Integración Viral

RESUMEN

Endotoxin tolerance, the transient, secondary down-regulation of a subset of endotoxin-driven responses after exposure to bacterial products, is thought to be an adaptive response providing protection from pathological hyperactivation of the innate immune system during bacterial infection. However, although protecting from the development of sepsis, endotoxin tolerance also can lead to fatal blunting of immunological responses to subsequent infections in survivors of septic shock. Despite considerable experimental effort aimed at characterizing the molecular mechanisms responsible for a variety of endotoxin tolerance-related phenomena, no consensus has been achieved yet. IL-12 is a macrophage- and dendritic cell (DC)-derived cytokine that plays a key role in pathological responses to endotoxin as well as in the induction of protective responses to pathogens. It recently has been shown that IL-12 production is suppressed in endotoxin tolerance, providing a likely partial mechanism for the increased risk of secondary infections in sepsis survivors. We examined the development of IL-12 suppression during endotoxin tolerance in mice. Decreased IL-12 production in vivo is clearly multifactorial, involving both loss of CD11c(high) DCs as well as alterations in the responsiveness of macrophages and remaining splenic DCs. We find no demonstrable mechanistic role for B or T lymphocytes, the soluble mediators IL-10, TNF-alpha, IFN-alphabeta, or nitric oxide, or the NF-kappaB family members p50, p52, or RelB.

Asunto(s)

Células Dendríticas/inmunología , Tolerancia Inmunológica , Interleucina-12/antagonistas & inhibidores , Interleucina-12/biosíntesis , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/inmunología , Macrófagos Peritoneales/inmunología , Animales , Apoptosis/inmunología , Linfocitos B/inmunología , Células Dendríticas/citología , Células Dendríticas/metabolismo , Relación Dosis-Respuesta Inmunológica , Regulación hacia Abajo/inmunología , Femenino , Tolerancia Inmunológica/genética , Inmunización Secundaria , Inyecciones Intraperitoneales , Integrina alfaXbeta2/biosíntesis , Interferón Tipo I/deficiencia , Interferón Tipo I/genética , Interleucina-10/deficiencia , Interleucina-10/genética , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , FN-kappa B/deficiencia , FN-kappa B/genética , Subunidad p50 de NF-kappa B , Óxido Nítrico/deficiencia , Óxido Nítrico/genética , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Bazo/citología , Bazo/inmunología , Linfocitos T/inmunología , Factor de Transcripción ReIB , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/deficiencia , Factor de Necrosis Tumoral alfa/genética

RESUMEN

Although therapy-mediated suppression of HIV-1 is effective and results in a degree of immune reconstitution, the complications resulting from life-long treatment emphasize the need for alternatives. This review discusses the use of structured interruptions in antiviral therapy to induce drug-free periods of immune-mediated control of HIV-1. Such an approach has the ultimate objective of harnessing anti-viral immune responses, reducing drug exposure (toxicity and cost) and potentially extending the clinical benefits of a suppressive treatment regimen.

Asunto(s)

Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Fármacos Anti-VIH/administración & dosificación , VIH-1/efectos de los fármacos , Síndrome de Inmunodeficiencia Adquirida/inmunología , Enfermedad Aguda , Enfermedad Crónica , Esquema de Medicación , VIH-1/inmunología , Humanos , Inmunosupresores/administración & dosificación , Factores de Riesgo

RESUMEN

The HIV-1 vpr gene encodes a 14-kDa virion-packaged protein that has been implicated in viral pathogenesis. Vpr exhibits profound effects on human primary cells influencing proliferation, differentiation, apoptosis, and cytokine production, in part through NF-kappaB-mediated transcription. NF-kappaB, a potent transcription factor, activates many proinflammatory cytokines/chemokines upon infection. Here, we analyzed the effect of extracellular Vpr as well as the virion-associated Vpr on beta chemokines (MIP-1alpha, MIP-1beta, and RANTES) production in human macrophages and primary lymphocytes (PBLs). Macrophages and PBLs exposed to HIV-1 vpr+ viruses or to recombinant Vpr protein produced significantly less beta chemokines compared with cells infected with HIV-1 vpr-viruses or irrelevant control protein (Gag)-exposed cells. These results suggest that a Vpr-mediated increase in virus replication could be in part through down-regulation of chemokine production.

Asunto(s)

Quimiocinas CC/biosíntesis , Productos del Gen vpr/fisiología , VIH-1/genética , Linfocitos/metabolismo , Macrófagos/metabolismo , Quimiocinas CC/genética , Regulación Viral de la Expresión Génica/fisiología , Productos del Gen vpr/genética , Productos del Gen vpr/farmacología , Genes prv , VIH-1/fisiología , Células HeLa , Humanos , Linfocitos/virología , Macrófagos/virología , Proteínas Recombinantes/farmacología , Virión/genética , Virión/metabolismo , Replicación Viral/fisiología , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana

RESUMEN

HIV-1 infection elicits a broad range of host responses, many of which interfere with the regulatory pathways of gene expression of interleukin-12 (IL-12), a heterodimeric cytokine essential for cell-mediated immunity against microbial infection. The inhibition of IL-12 production by accessory cells after HIV-1 infection has been identified as a potential factor responsible for impaired innate and Th1 cell-mediated responses observed in AIDS patients. The mechanism by which HIV-1 infection suppresses IL-12 gene expression is largely uncharacterized. Here we review all pathways identified that could potentially mediate HIV-induced impairment of IL-12 gene expression, such as IL-10, transforming growth factor beta, interferon-alpha/beta, tumor necrosis factor alpha, Fc receptors, complement regulatory proteins, and receptors. Also discussed is the decreased CD40 ligand induction in CD4 T cells during HIV infection, which may have a strong impact on T cell-dependent IL-12 production that is critical for the establishment and maintenance of a Th1 response.

Asunto(s)

Infecciones por VIH/metabolismo , VIH-1 , Interleucina-12/biosíntesis , Animales , Regulación de la Expresión Génica/fisiología , Infecciones por VIH/inmunología , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Interleucina-12/genética , Interleucina-12/inmunología

RESUMEN

Immunologic and virologic outcomes of treatment interruption were compared for 5 chronically human immunodeficiency virus (HIV)-infected persons who have maintained antiretroviral therapy-mediated virus suppression, as compared with 5 untreated controls. After a median interruption of 55 days of therapy accompanied by rebound of virus, reinitiated therapy in 4 of 5 subjects resulted in suppression of 98.86% of plasma virus load by 21-33 days and no significant decrease in CD4 T cell percentage from baseline. Increased T helper responses against HIV-1 p24 antigen (P=. 014) and interferon-gamma-secreting CD8 T cell responses against HIV-1 Env (P=.004) were present during interruption of therapy and after reinitiation of treatment. The remaining subject whose treatment was interrupted did not resume treatment and continued to have a low virus load (<1080 HIV-1 RNA copies/mL) and persistent antiviral cell-mediated responses. In summary, cellular immunity against autologous HIV-1 has the potential to be acutely augmented in association with temporary treatment interruption in chronically infected persons.

Asunto(s)

Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/virología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , VIH-1 , Adulto , Fármacos Anti-VIH/uso terapéutico , Esquema de Medicación , Femenino , Humanos , Inmunidad Celular , Interferón gamma/metabolismo , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Carga Viral

RESUMEN

We show that IL-13 in the presence of TNF-alpha effected an equal or greater antiviral activity against a dual-tropic HIV-1 (R5X4) in macrophages. A temporary or continued exposure of macrophages to both cytokines significantly decreased the infection and replication of R5X4 HIV-1(89.6) (median, 128-fold, n = 9, p = 0.024) in macrophages as compared to untreated controls when analyzed over six decreasing multiplicities of infection. A quantitative flow cytometric assay revealed that IL-13 induced a significant (approximately 50 %) reduction in the number of CD4 and CC chemokine receptor 5 (CCR5) antibody binding sites while completely abrogating surface expression of CXC chemokine receptor 4 (CXCR4). In the presence of IL-13 and TNF-alpha, expression of CCR5 was completely abrogated while the expression of CD4 and CXCR4 remained significantly reduced as compared to untreated controls. A reduction in CD4 and HIV-1 coreceptors was associated with a decrease in reverse-transcribed viral DNA at 24 h post-infection. Quantification of viral gene expression using amphotropic MLV Env pseudotyped luciferase reporter viruses suggested that IL-13 inhibited HIV-1 gene expression within 24 h by up to 90 % in the presence or absence of TNF-alpha. In conclusion, our data suggest that IL-13 is a powerful counter-regulatory agent against TNF-alpha-induced HIV-1 expression while also acting with TNF-alpha in inhibiting de novo infection of macrophages.

Asunto(s)

Antígenos CD4/inmunología , VIH-1/fisiología , Interleucina-13/inmunología , Macrófagos/inmunología , Macrófagos/virología , Receptores CCR5/inmunología , Receptores CXCR4/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Células Cultivadas , Sinergismo Farmacológico , Regulación Viral de la Expresión Génica/efectos de los fármacos , Regulación Viral de la Expresión Génica/inmunología , Humanos , Interleucina-13/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Replicación Viral/efectos de los fármacos , Replicación Viral/inmunología

RESUMEN

Human immunodeficiency virus type 1 (HIV-1) is known for its ability to infect immune cells, including T-cells and macrophages. The 96-amino acid Vpr, a virion-associated protein, is essential for viral replication in monocytes/macrophages and increases viral replication in primary and established T-cell lines. The Vpr protein regulates a number of host cellular events, including proliferation, differentiation, apoptosis, cytokine production, and NF-kappaB-mediated transcription. Most of these functions have been analyzed using either endogenous Vpr protein or cells transfected with a Vpr expression plasmid. We developed a lentiviral vector complemented with a Vpr expression plasmid that results in viral particles packaged with Vpr protein. To facilitate identification of the target cells infected with the particles containing Vpr, we fused green fluorescent protein (GFP) with the Vpr open reading frame and analyzed the biology of this novel particle. Vpr itself is expressed as a 14-kDa protein; however, in vitro translation of the pVpr-GFP plasmid resulted in the expression of 39-kDa fusion protein. The fusion molecule exhibited the same activity in arresting the cell cycle in G2 as does the wildtype Vpr molecule. Subcellular localization of Vpr and Vpr-GFP by immunofluoresence in human and murine cell lines indicated that Vpr by itself or with the reporter GFP showed a perinuclear staining pattern. Replication kinetics showed no significant difference between Vpr-GFP and native complemented pseudovirus replication in a single-round infectivity assay. A flow cytometry analysis of peripheral blood lymphocytes and macrophages infected with Vpr-GFP-packaged virions and selected by GFP showed 56.7% infectivity for lymphocytes and 84.6% infectivity for macrophages. Additional analysis of CD24 (HSA)-positive cells showed infection of CD4+ cells, macrophages, and, importantly, dendritic cells. This system will allow us to identify specific cell populations including antigen-presenting cells, and allow quantitative analysis of the precise effect of Vpr on both target and bystander cells in vitro as well as in vivo.

Asunto(s)

Productos del Gen vpr/genética , VIH-1/fisiología , Productos del Gen vpr/biosíntesis , Técnicas de Transferencia de Gen , Genes Reporteros , Proteínas Fluorescentes Verdes , VIH-1/genética , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Recombinantes de Fusión/biosíntesis , Rabdomiosarcoma , Células Tumorales Cultivadas , Virión/genética , Virión/fisiología , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana

RESUMEN

IL-12 is a pivotal cytokine that links the innate and adaptive immune responses. TNF-alpha also plays a key role in orchestrating inflammation and immunity. The reciprocal influence of these two inflammatory mediators on each other may have significant impact on the cytokine balance that shapes the type and extent of immune responses. To investigate the relationship between TNF-alpha and IL-12 production, we analyzed the effects of exposure of human monocyte-derived macrophages to TNF-alpha on LPS- or Staphylococcus aureus-induced IL-12 production in the presence or absence of IFN-gamma. TNF-alpha is a potent inhibitor of IL-12 p40 and p70 secretion from human macrophages induced by LPS or S. aureus. IL-10 is not responsible for the TNF-alpha-mediated inhibition of IL-12. TNF-alpha selectively inhibits IL-12 p40 steady-state mRNA, but not those of IL-12 p35, IL-1alpha, IL-1beta, or IL-6. Nuclear run-on analysis identified this specific inhibitory effect at the transcriptional level for IL-12 p40 without down-regulation of the IL-12 p35 gene. The major transcriptional factors identified to be involved in the regulation of IL-12 p40 gene expression by LPS and IFN-gamma, i.e., c-Rel, NF-kappaB p50 and p65, IFN regulatory factor-1, and ets-2, were not affected by TNF-alpha when examined by nuclear translocation and DNA binding. These data demonstrate a selective negative regulation on IL-12 by TNF-alpha, identifying a direct negative feedback mechanism for inflammation-induced suppression of IL-12 gene expression.

Asunto(s)

Inmunosupresores/farmacología , Interleucina-12/antagonistas & inhibidores , Interleucina-12/biosíntesis , Macrófagos/inmunología , Macrófagos/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Transporte Biológico/inmunología , Núcleo Celular/inmunología , Núcleo Celular/metabolismo , ADN/metabolismo , Regulación de la Expresión Génica/inmunología , Humanos , Interferón gamma/fisiología , Interleucina-10/fisiología , Interleucina-12/genética , Interleucina-12/metabolismo , Cinética , Monocitos/inmunología , Monocitos/metabolismo , FN-kappa B/inmunología , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/inmunología , Unión Proteica/inmunología , Transducción de Señal/inmunología , Staphylococcus aureus/inmunología , Transcripción Genética/inmunología , Activación Transcripcional/inmunología

RESUMEN

The increase in the incidence of AIDS-related tuberculosis over the last decades has fueled the dissemination of multiple drug resistance tuberculosis (including resistant strains to INH and rifampin). This has now been recognized in a variety of settings including hospitals, prisons and shelters. We have identified a nosocomial epidemic at the Muñiz Hospital in the city of Buenos Aires, Argentina. This has evolved as one of the largest institutional outbreaks yet to be recognized. The purpose of this paper is to characterize the evolution of this outbreak which at the end of 1997 had involved in excess of 500 cases. Among the 3,322 patients discharged at the Muñiz Hospital during the years 1996-1997 with the diagnosis of tuberculosis, 440 (13.24%) were discharged with the diagnosis of multiple drug resistance tuberculosis. The immediate mortality (during the ensuing four months following the bacteriological diagnosis) was of 91.3% of cases in 1995 and decreased progressively to 65.9% in 1996 and 55.9% in 1997. The bacteriological confirmation of the diagnosis was made after the patients death in a decreasing number of cases, going from 72.5% of the cases in 1995 to 28.3% of the cases in 1997. Despite the significant progress achieved with regard to the diagnosis and treatment of multiple drug resistance tuberculosis, the measures undertaken to decrease the spread of the cases have had limited success. This is chiefly attributable to the inability to isolate cases. This has continued to promote nosocomial spread of multiple drug resistance tuberculosis in our environment.

Asunto(s)

Infecciones Oportunistas Relacionadas con el SIDA/epidemiología , Infección Hospitalaria/epidemiología , Brotes de Enfermedades , Tuberculosis Resistente a Múltiples Medicamentos/epidemiología , Adulto , Antibióticos Antituberculosos/uso terapéutico , Antituberculosos/uso terapéutico , Argentina/epidemiología , Estudios de Cohortes , Femenino , Humanos , Isoniazida/uso terapéutico , Masculino , Rifampin/uso terapéutico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico