RESUMEN
A higher human immunodeficiency virus 1 (HIV-1) viral load at pleural sites infected with Mycobacterium tuberculosis (MTB) than in peripheral blood has been documented. However, the cellular source of productive HIV infection in HIV-1/MTB-coinfected pleural fluid mononuclear cells (PFMCs) remains unclear. In this study, we observed significant quantities of HIV-1 p24(+) lymphocytes in PFMCs, but not in peripheral blood mononuclear cells (PBMCs). HIV-1 p24(+) lymphocytes were mostly enriched in DN T cells. Intracellular CD4 expression was detectable in HIV-1 p24(+) DN T cells. HIV-1 p24(+) DN T cells showed lower surface expression of human leukocyte antigen (HLA)-ABC and tetherin than did HIV-1 p24(+) CD4 T cells. Upon in vitro infection of PFMC CD4 T cells from TB mono-infected subjects, Nef- and/or Vpu-deleted HIV mutants showed lower generation of HIV-1 p24(+) DN T cells than the wild-type virus. These data indicate that productively HIV-1-infected DN T cells, generated through down-modulation of surface CD4, likely by HIV-1 Nef and Vpu, are the predominant source of HIV-1 at pleural sites of HIV/MTB coinfection.
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Coinfección/inmunología , Infecciones por VIH/inmunología , VIH-1/fisiología , Mycobacterium tuberculosis/fisiología , Pleura/inmunología , Linfocitos T/inmunología , Tuberculosis/inmunología , Adulto , Coinfección/microbiología , Coinfección/virología , Femenino , Infecciones por VIH/microbiología , Infecciones por VIH/virología , Humanos , Masculino , Tuberculosis/microbiología , Adulto JovenRESUMEN
The specificities and sensitivities of the Bactec mycobacterial growth indicator tube (MGIT) system for the recovery of Mycobacterium tuberculosis from pleural fluid are not statistically different than those of the Myco/F lytic liquid culture system. The time to positivity is shorter in the MGIT system (12.7 versus 20.7 days, respectively; P=0.007). The Myco/F lytic culture system may be an alternative to the MGIT system for diagnosing pleural tuberculosis.
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Técnicas Bacteriológicas/instrumentación , Técnicas Bacteriológicas/métodos , Mycobacterium tuberculosis/aislamiento & purificación , Derrame Pleural/microbiología , Tuberculosis Pulmonar/diagnóstico , Humanos , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Systemic immune activation is a strong predictor of progression of human immunodeficiency virus type 1 (HIV-1) disease and a prominent feature of infection with Mycobacterium tuberculosis. OBJECTIVE: To understand the role of systemic immune activation and microbial translocation in HIV/tuberculosis dually infected patients over the full spectrum of HIV-1 immunodeficiency, we studied circulating sCD14 and lipopolysaccharide (LPS) and their relationship to HIV-1 activity. METHODS: Two cohorts of HIV/tuberculosis subjects defined by CD4 T-cell count at time of diagnosis of tuberculosis were studied: those with low (<350/µL) and those with high (≥ 350/µL) CD4 T-cell count. Circulating soluble CD14 (sCD14) and LPS were assessed. RESULTS: Levels of sCD14 were higher in HIV/tuberculosis with high (≥ 350/µL) as compared to low CD4 T-cell count (P < .001). Whereas sCD14 levels remained elevated in HIV/tuberculosis subjects with lower CD4 T-cell counts despite treatment of tuberculosis, in HIV/tuberculosis patients with higher CD4 T-cell count (≥ 350/µL), levels declined regardless of whether highly active antiretroviral therapy (HAART) was included with the anti-tuberculosis regimen. Circulating LPS levels in HIV/tuberculosis patients with CD4 T-cell count ≥ 350/µL were unaffected by treatment of tuberculosis with or without HAART. CONCLUSION: During HIV/tuberculosis, systemic immune activation is dissociated from microbial translocation. Changes in circulating sCD14 and LPS are dependent on CD4 T-cell count.
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Traslocación Bacteriana , Infecciones por VIH/inmunología , VIH-1/inmunología , Mycobacterium tuberculosis/fisiología , Tuberculosis Pulmonar/inmunología , Adulto , Terapia Antirretroviral Altamente Activa , Antituberculosos/uso terapéutico , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Estudios de Casos y Controles , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Tracto Gastrointestinal/microbiología , Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Humanos , Receptores de Lipopolisacáridos/sangre , Lipopolisacáridos/sangre , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/inmunología , Tuberculosis Pulmonar/complicaciones , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/microbiología , Uganda , Carga ViralRESUMEN
Our recent microarray analysis of infected human alveolar macrophages (AMs) found serine protease inhibitor 9 (PI-9) to be the most prominently expressed of a cluster of apoptosis-associated genes induced by virulent Mycobacterium tuberculosis. In the current study, we show that induction of PI-9 occurs within hours of infection with M. tuberculosis H37Rv and is maintained through 7 days of infection in both AMs and blood monocytes. Inhibition of PI-9 by small inhibitory RNA decreased M. tuberculosis-induced expression of the antiapoptotic molecule Bcl-2 and resulted in a corresponding increase in production of caspase 3, a terminal effector molecule of apoptosis. Further, PI-9 small inhibitory RNA mediated a significant reduction in the subsequent survival of M. tuberculosis within AMs. Thus PI-9 induction within human mononuclear phagocytes by virulent M. tuberculosis serves to protect these primary targets of infection from elimination by apoptosis and thereby promotes intracellular survival of the organism.
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Apoptosis , Macrófagos Alveolares/metabolismo , Mycobacterium tuberculosis/patogenicidad , Serpinas/metabolismo , Caspasa 3/metabolismo , Células Cultivadas , Humanos , Macrófagos Alveolares/microbiología , Monocitos/metabolismo , Monocitos/microbiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Interferente Pequeño/metabolismoAsunto(s)
Coinfección , Mycobacterium tuberculosis , Recuento de Linfocito CD4 , Infecciones por VIH , Humanos , TuberculosisRESUMEN
BACKGROUND: Tuberculosis (TB) is the most frequent co-infection in HIV-infected individuals still presenting diagnostic difficulties particularly in developing countries. Recently an assay based on IFN-gamma response to M. tuberculosis RD1 peptides selected by computational analysis was developed whose presence is detected during active TB disease. Objective of this study was to investigate the response to selected RD1 peptides in HIV-1-infected subjects with or without active TB in a country endemic for TB and to evaluate the change of this response over time. METHODS: 30 HIV-infected individuals were prospectively enrolled, 20 with active TB and 10 without. Among those with TB, 12 were followed over time. IFN-gamma response to selected RD1 peptides was evaluated by enzyme-linked immunospot (ELISPOT) assay. As control, response to RD1 proteins was included. Results were correlated with immune, microbiological and virological data. RESULTS: Among patients with active TB, 2/20 were excluded from the analysis, one due to cell artifacts and the other to unresponsiveness to M. tuberculosis antigens. Among those analyzable, response to selected RD1 peptides evaluated as spot-forming cells was significantly higher in subjects with active TB compared to those without (p = 0.02). Among the 12 TB patients studied over time a significant decrease (p =< 0.007) of IFN-gamma response was found at completion of therapy when all the sputum cultures for M. tuberculosis were negative. A ratio of RD1 peptides ELISPOT counts over CD4+ T-cell counts greater than 0.21 yielded 100% sensitivity and 80% specificity for active TB. Conversely, response to RD1 intact proteins was not statistically different between subjects with or without TB at the time of recruitment; however a ratio of RD1 proteins ELISPOT counts over CD4+ T-cell counts greater than 0.22 yielded 89% sensitivity and 70% specificity for active TB. CONCLUSION: In this pilot study the response to selected RD1 peptides is associated with TB disease in HIV-infected individuals in a high TB endemic country. This response decreases after successful therapy. The potential of the novel approach of relating ELISPOT spot-forming cell number and CD4+ T-cell count may improve the possibility of diagnosing active TB and deserves further evaluation.
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Infecciones por VIH/complicaciones , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis Pulmonar/diagnóstico , Adulto , Antígenos Bacterianos/aislamiento & purificación , Proteínas Bacterianas/inmunología , Recuento de Linfocito CD4 , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Femenino , Estudios de Seguimiento , Humanos , Interferón gamma/inmunología , Masculino , Proyectos Piloto , Juego de Reactivos para Diagnóstico , Tuberculosis Pulmonar/complicaciones , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/terapia , Uganda , Carga ViralRESUMEN
HIV-1 replication remains elevated in dually infected HIV-1/TB subjects at completion of antituberculosis therapy. A viral immunocapture assay was used to examine the cellular origin of HIV-1 within plasma from HIV-1/TB subjects at time of diagnosis of pulmonary TB, at end of TB treatment, and 6 months after completion of treatment. Asymptomatic HIV-1-infected subjects without TB (HIV-1/C) served as controls. Both activated immature macrophage (CD36(+)) and CD4 T cell (CD26(+)) compartments contributed to viral load. Changes in the activation status of either cellular compartment paralleled their contribution to viral load. Levels of HIV-1 originating from activated (HLA-DR(+)) cells and from CD36(+) and CD26(+) mononuclear cells resolved to levels observed in HIV-1/C by the end of treatment. HIV-1 isolated by anti-CD3 immunocapture from HIV-1/TB patients remained significantly higher than from HIV-1/C patients at the end of TB treatment and at 12 months follow-up. Therefore, viral production by lymphocytes extends well beyond the completion of TB treatment.
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Infecciones por VIH/virología , VIH-1/fisiología , Tuberculosis Pulmonar/tratamiento farmacológico , Adolescente , Adulto , Complejo CD3/inmunología , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Comorbilidad , Femenino , Citometría de Flujo , Estudios de Seguimiento , Infecciones por VIH/complicaciones , Infecciones por VIH/inmunología , Antígenos HLA-DR/análisis , Humanos , Inmunohistoquímica , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/virología , Masculino , Estudios Prospectivos , ARN Viral/sangre , Radiografía , Factores de Tiempo , Resultado del Tratamiento , Tuberculosis Pulmonar/complicaciones , Tuberculosis Pulmonar/diagnóstico , Tuberculosis Pulmonar/diagnóstico por imagen , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiología , Carga ViralRESUMEN
Chronic inflammation associated with tumor necrosis factor (TNF)-alpha and reactive oxygen species (ROS) is the hallmark of tuberculosis. Mycobacterium tuberculosis (MTB) directly stimulates human monocytes to secrete TNF-alpha. We show the augmented expression of TNF-alpha mRNA in MTB-infected monocytes by cellular activation and ROS was suppressed by allicin in a dose-dependent manner. Also, allicin enhanced the glutathione peroxidase activity, which correlated inversely with the downregulation of ROS and TNF-alpha in MTB-infected monocytes. Hence, allicin may prove to be a valuable natural antioxidant in combating tuberculosis.
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Monocitos/efectos de los fármacos , Mycobacterium tuberculosis/fisiología , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Ácidos Sulfínicos/farmacología , Factor de Necrosis Tumoral alfa/genética , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Disulfuros , Relación Dosis-Respuesta a Droga , Glutatión Peroxidasa/sangre , Humanos , Monocitos/enzimología , Monocitos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: Infection by MTB or exposure to MTB constituents is associated with intense microbial stimulation of the immune system, through both antigenic and TLR components, and induction of a milieu that is rich in pro-inflammatory/anti-inflammatory cytokines. Here, we addressed the basis of induced regulatory T-cell (iT-reg) expansion in response to MTB stimulation, in the absence of prior T cell antigen responsiveness. METHODS: PBMC from HIV-1 un-infected TST negative and TST positive control subjects were stimulated by virulent MTB H37Rv lysate (L), a French press preparation of MTB that includes all bacterial components. Phenotype of MTB H37RvL induced iT-reg was assessed using immunostaining and flow cytometry. Functional capacity of iT-reg was assessed using 3H-Thymidine incorporation and IFNγ production of non-adherent T cells (NAC) in the presence or absence of iT-reg in corresponding culture supernatants in response to TCR stimulation. Realtime PCR was used to assess IDO and FoxP3 mRNA expression. RESULTS: The capacity of MTB H37RvL to induce CD4+CD25hi+ Foxp3+ T-cells in PBMC from TST negative subjects was robust (p<0.001), and in fact comparable to induction of iT-reg in PBMC from TST positive subjects. MTB-induced CD4+CD25hi+ T-reg were TGFß positive (p<0.05). Further, MTB H37RvL induced CD4+CD25hi+ Foxp3+ iT-reg suppressed 3H-Thymidine incorporation and IFNγ production of non-adherent T cells (NAC) in response to TCR stimulation. MTB H37RvL induction of iT-reg was significantly stronger (p<0.01) than that by TLR-2, TLR-4, TLR-9 ligands, or combination of all TLR ligands. MTB H37RvL inducted indoleamine 2,3-dideoxygenase (IDO) mRNA expression in monocytes (p<0.001), and co-culture with the IDO inhibitor, D-1MT, decreased frequencies of T-reg (p<0.05). Inhibition of TGFß by siRNA reduced Foxp3 mRNA expression in CD4 T cells (p<0.05). CONCLUSION: Therefore, MTB and its components expand functional iT-reg in human mononuclear cells from MTB non-sensitized subjects. Also, MTB-induced iT-reg expansion depends on mononuclear phagocyte expression of both TGFß and IDO.
RESUMEN
Systemic immune activation is critical to the pathogenesis of HIV-1 disease, and is accentuated in HIV/TB co-infected patients. The contribution of immune activation at sites of HIV/TB co-infection to viral activity, CD4 T cell count, and productive HIV-1 infection remain unclear. In this study, we measured markers of immune activation both in pleural fluid and plasma, and in T cells in pleural fluid mononuclear cell (PFMC) and peripheral blood mononuclear cell (PBMC) in HIV/TB co-infected subjects. The relationship between soluble and T cell activation markers with viral load in pleural fluid and blood CD4 T cell count were assessed. The T cell phenotype and activation status of HIV-1 p24 + T cells in PFMC and PBMC from HIV/TB patients were determined. We found that T cell and macrophage-specific and non-specific soluble markers of immune activation, sCD27, sCD163, IL1Ra, and sCD14, were higher in pleural fluid as compared to plasma from HIV/TB co-infected subjects, and higher as compared to pleural fluid from TB mono-infected subjects. Intestinal fatty acid-binding protein, a marker of intestinal tract damage, in plasma from HIV/TB co-infected patients was not different than that in HIV+ subjects. Expression of HLADR and CD38 double positive (HLADR/CD38) on CD4 T cells, and CD69+ on CD8 T cells correlated with pleural fluid viral load, and inversely with blood CD4 T cell count. Higher expression of HLADR/CD38 and CCR5 on CD4 T cells, and HLADR/CD38 and CD69 on CD8 T cells in PFMC were limited to effector memory populations. HIV-1 p24+ CD8 negative (includes CD4 + and double negative T cells) effector memory T cells in PFMC had higher expression of HLADR/CD38, Ki67, and CCR5 compared to HIV-1 p24- CD8 negative PFMC. Cumulatively, these data indicate that sites of HIV/TB co-infection are the source of intense immune activation.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Coinfección/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis Pulmonar/inmunología , Adulto , Anciano , Antígenos CD/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/patología , Femenino , Infecciones por VIH/microbiología , Infecciones por VIH/patología , Antígenos HLA-DR/inmunología , Humanos , Memoria Inmunológica , Masculino , Persona de Mediana Edad , Tuberculosis Pulmonar/patología , Tuberculosis Pulmonar/virologíaRESUMEN
BACKGROUND: CD4 T-cells expressing Foxp3 are expanded systemically during active tuberculosis (TB) regardless of HIV-1 co-infection. Foxp3+ CD4 T cells are targets of HIV-1 infection. However, expansion of HIV-1 infected Foxp3+ CD4 T cells at sites of HIV/TB co-infection, and whether they contribute to promotion of HIV-1 viral activity is not known. METHODS: Pleural fluid mononuclear cells (PFMC) from HIV/TB co-infected patients with pleural TB were characterized by immune-staining and FACS analysis for surface markers CD4, CD127, CCR5, CXCR4, HLA-DR and intracellular expression of Foxp3, HIVp24, IFN-γ and Bcl-2. Whole PFMC and bead separated CD4+CD25+CD127- T cells were assessed for HIV-1 LTR strong stop (SS) DNA by real-time PCR, which represents viral DNA post cell entry and initiation of reverse transcription. RESULTS: High numbers of HIV-1 p24 positive Foxp3+ and Foxp3+CD127- CD4 T cells were identified in PFMC from HIV/TB co-infected subjects. CD4+Foxp3+CD127- T cells displayed high expression of the cellular activation marker, HLA-DR. Further, expression of the HIV-1 co-receptors, CCR5 and CXCR4, were higher on CD4+Foxp3+T cells compared to CD4+Foxp3- T cells. Purified CD4+CD25+CD127- T cells isolated from PFMC of HIV/TB co-infected patients, were over 90% CD4+Foxp3+T cells, and exhibited higher HIV-1 SS DNA as compared to whole PFMC, and as compared to CD4+CD25+CD127- T cells from an HIV-infected subject with pleural mesothelioma. HIV-1 p24+ Foxp3+ CD4+T cells from HIV/TB patients higher in Bcl-2 expression as compared to both HIV-1 p24+ Foxp3- CD4 T cells, and Foxp3+ CD4+T cells without HIV-p24 expression. CONCLUSION: Foxp3+ CD4 T cells in PFMC from HIV/TB co-infected subjects are predisposed to productive HIV-1 infection and have survival advantage as compared to Foxp3 negative CD4 T cells.
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HIV and Mycobacterium tuberculosis not only co-circulate throughout the developing world but each has contributed to prevalence and mortality caused by the other. Several reports have described how HIV-1 increases the incidence of new M. tuberculosis infections, exacerbates the severity of tuberculosis (TB), and re-activates latent M. tuberculosis. However, the converse relationship is more difficult to understand considering TB can emerge in asymptomatic individuals and as an opportunistic infection during AIDS. Development of TB in HIV infected individuals with higher CD4 cell counts (> 200/mm3) appears to increase the rate of disease progression and mortality. Higher viral loads, increased HIV-1 diversity, and changes in cytokine/chemokine levels in HIV-infected individuals with TB appear to be related to a localized immune stimulation. Specifically, increased levels of TNF alpha and MCP-1, induced by TB, may activate HIV replication in lymphocytes, monocytes, and macrophages that are resident or have migrated to M. tuberculosis infected organs (e.g. pleura or lung). The HIV-1 found in blood following this TB-mediated burst in load and diversity appear to be phylogenetically-related to HIV-1 clones that have evolved independently in the lung or pleural compartments, now infected by M. tuberculosis.
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Infecciones por VIH/complicaciones , Infecciones por VIH/fisiopatología , VIH-1/fisiología , Mycobacterium tuberculosis/patogenicidad , Tuberculosis Pulmonar/complicaciones , Infecciones Oportunistas Relacionadas con el SIDA/microbiología , Progresión de la Enfermedad , Variación Genética , Infecciones por VIH/virología , VIH-1/genética , VIH-1/patogenicidad , Humanos , Replicación ViralRESUMEN
Sites of HIV/TB coinfection are characterized by increased HIV-1 replication and a TH1 profile. However, expression of HIV-1 restriction factors, such as APOBEC3G (A3G) in situ, is unknown. Using an RT-profiler focused on genes related to HIV-1 expansion, we examined pleural fluid mononuclear cells (PFMCs) from patients with HIV/TB coinfection in comparison to HIV-uninfected patients with TB disease. Significant expression of interferon (IFN)-γ and restriction factors A3G and A3F and TRIM5α in PFMCs was found. Genes correlating significantly with the expression of IFN-γ included A3G and A3F. However, pleural fluid HIV-1 viral load and HIV-1 gag/pol mRNA in PFMCs did not correlate with A3G activity.
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Líquidos Corporales/citología , Citidina Desaminasa/biosíntesis , Infecciones por VIH/inmunología , Interferón gamma/biosíntesis , Leucocitos Mononucleares/inmunología , Derrame Pleural , Tuberculosis Pulmonar/inmunología , Desaminasa APOBEC-3G , Adulto , Líquidos Corporales/virología , Perfilación de la Expresión Génica , Infecciones por VIH/complicaciones , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Humanos , Masculino , Tuberculosis Pulmonar/complicaciones , Carga ViralRESUMEN
CD4+ T cell counts of HIV-infected individuals with pulmonary TB (PTB) are higher than with other opportunistic infections suggesting that progression to PTB is not merely due to T cell depletion but also dysfunction. There are limited data examining T cell functional signatures in human HIV-TB co-infection particularly in PTB which accounts for about 80% of active TB disease overall. We examined a cohort of HIV-infected anti-retroviral naïve individuals in Kampala, Uganda, a TB endemic area using multiparametric flow cytometry analysis to determine IFN-γ, IL-2, IL-17, and TNF-α production in CD4+ memory T cell subsets. The cytokine frequency and polyfunctionality profile of Mycobacterium tuberculosis (MTB)-specific CD4+ T cells in HIV-infected persons with latent TB infection (LTBI) or PTB is comparable. This similarity suggests that LTBI may represent a smoldering state of persistent MTB replication rather than dormant infection. This may be a contributory mechanism to the significantly increased risk of progression to PTB in this population.
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Linfocitos T CD4-Positivos/inmunología , Coinfección , Infecciones por VIH/inmunología , Tuberculosis Latente/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis Pulmonar/inmunología , Adulto , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/microbiología , Linfocitos T CD4-Positivos/virología , Citocinas/sangre , Citocinas/inmunología , Femenino , Citometría de Flujo , Infecciones por VIH/sangre , Infecciones por VIH/diagnóstico , Infecciones por VIH/virología , Interacciones Huésped-Patógeno , Humanos , Memoria Inmunológica , Tuberculosis Latente/sangre , Tuberculosis Latente/diagnóstico , Tuberculosis Latente/virología , Masculino , Mycobacterium tuberculosis/crecimiento & desarrollo , Tuberculosis Pulmonar/sangre , Tuberculosis Pulmonar/diagnóstico , Tuberculosis Pulmonar/microbiología , UgandaRESUMEN
Transforming growth factor beta 1 (TGF-beta1) has been implicated in the pathogenesis of a number of diseases including infection with intracellular pathogens such as Mycobacterium avium complex (MAC). In this study, we developed an ELISPOT assay for measurement of active TGF-beta1 produced by peripheral blood mononuclear cells (PBMC) from healthy individuals in response to LPS or MAC. The frequency of TGF-beta1 producing cells was significantly (p<0.04) higher in response to LPS (10 microg/ml) as compared to unstimulated cells (n=4). Moreover, the frequency of TGF-beta1 producing cells was threefold higher in monocyte (MN)-enriched cell population than those in PBMC indicating that the source of TGF-beta1 producing cells in PBMC was MN. In addition, the frequency of TGF-beta1 producing cells in response to MAC (10:1, cfu:MN) was significantly higher (p<0.03) than unstimulated cells. However, the frequency of TGF-beta1 producing cells in response to MAC (10:1) was eight to ninefold lower than that by LPS (10 microg/ml). Moreover, there was a correlation between the level of total TGF-beta1 in 24-h culture supernatants and the number of TGF-beta1 producing cells upon MAC stimulation. TGF-beta1 ELISPOT-assay may be a sensitive and a powerful tool for detection of TGF-beta1 producing cells, and may be helpful in elucidation of the nature of TGF-beta1 production at sites of diseases.
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Inmunoensayo/métodos , Monocitos/inmunología , Monocitos/microbiología , Mycobacterium avium , Factor de Crecimiento Transformador beta/análisis , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Humanos , Sensibilidad y Especificidad , Factor de Crecimiento Transformador beta1 , Tuberculosis/sangre , Tuberculosis/inmunologíaRESUMEN
Circulating free HIV-1 viral protein R (Vpr) is found in up to one third of subjects with HIV-1 infection. Free Vpr presumably shares some of the immunopathogenic effects of cell-associated Vpr. Here we assessed Vpr in plasma and pleural fluid from HIV/tuberculosis (TB) dually infected subjects with pleural TB and from plasma of patients with pulmonary HIV/TB. Vpr was assessed by western blot analysis. In plasma from HIV/TB subjects with pulmonary TB free Vpr could be detected in 47%. Only one subject, among 26 tested, with HIV monoinfection showed plasma Vpr activity. The majority (87.5%) of patients with pleural HIV/TB demonstrated free Vpr reactivity in their plasma. However, no Vpr activity was found in autologous pleural fluid samples from pleural HIV/TB patients. Standard (s) Vpr reactivity was reduced markedly by the addition of sVpr to pleural fluid from HIV-uninfected subjects. A high incidence of plasma Vpr reactivity in HIV/TB patients implies heightened processing and release of this HIV-1 accessory protein during HIV/TB coinfection. The contribution of free Vpr to HIV-1 immunopathogenesis during HIV/TB needs to be studied.
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Infecciones Oportunistas Relacionadas con el SIDA/sangre , Infecciones por VIH/sangre , Tuberculosis Pleural/sangre , Tuberculosis Pulmonar/sangre , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/sangre , Adulto , Anciano , Recuento de Linfocito CD4 , Coinfección/sangre , Femenino , Infecciones por VIH/complicaciones , Infecciones por VIH/virología , VIH-1 , Humanos , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/aislamiento & purificación , Derrame Pleural/virología , Tuberculosis Pleural/complicaciones , Tuberculosis Pleural/microbiología , Tuberculosis Pulmonar/complicaciones , Tuberculosis Pulmonar/microbiología , Adulto JovenRESUMEN
OBJECTIVE AND DESIGN: Predisposition to opportunistic infections by Mycobacterium tuberculosis (MTB) is a concomitant of HIV-1 infection and occurrence of tuberculosis is independent of circulating CD4(+) T-cell count in HIV-1-infected patients. Infection of mononuclear phagocytes from healthy individuals by virulent MTB is associated with expression of the antiapoptotic molecule protease inhibitor 9 (PI-9), and PI-9 contributes to successful parasitism of macrophages by MTB. Here we studied the contribution of PI-9 to successful MTB infection of monocytes from HIV-1-infected patients. METHODS: Blood monocytes obtained from HAART-treated HIV-1-infected patients (HIV+) and healthy controls were assessed for support of MTB H37Rv growth by assessment of MTB 16S ribosomal (r)RNA in cell lysates on day 1 and day 7 by real-time reverse transcription-PCR. PI-9 expression in monocyte cell lysates was assessed by ELISA and by reverse transcription-PCR. Inhibition of intracellular PI-9 was achieved by siRNA to PI-9 and compared to control constructs. RESULTS: Monocytes from HIV-infected patients supported higher MTB growth [MTB 16S rRNA (d7/d1)] as compared with monocytes from healthy controls. Both PI-9 protein and mRNA were significantly higher in monocytes from HIV-infected patients as compared with healthy controls. PI-9 protein levels prior to MTB infection correlated with MTB replication on day 7, and with plasma soluble CD14 levels. Silencing of PI-9 by transfection of monocytes from HIV-1-infected patients with PI-9-specific siRNA prior to infection improved intracellular containment of MTB. CONCLUSION: Increased intracellular PI-9 activity in mononuclear phagocytes from HIV-infected patients contributes to successful intracellular infection by virulent MTB.
Asunto(s)
Infecciones por VIH/inmunología , Interacciones Huésped-Patógeno , Leucocitos Mononucleares/microbiología , Viabilidad Microbiana , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/fisiología , Serpinas/metabolismo , Adulto , Anciano , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , ARN Ribosómico 16S/análisis , ARN Ribosómico 16S/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: The contribution of HIV-infection to periodontal disease (PD) is poorly understood. We proposed that immunological markers would be associated with improved clinical measures of PD. METHODS: We performed a longitudinal cohort study of HIV-infected adults who had started highly active antiretroviral therapy (HAART) <2 years. PD was characterized clinically as the percent of teeth with ≥ 1 site with periodontal probing depth (PPD) ≥ 5.0mm, recession (REC) >0mm, clinical attachment level (CAL) ≥ 4.0mm, and bleeding on probing (BOP) at ≥ 4 sites/tooth and microbiologically as specific periodontopathogen concentration. Linear mixed-effects models were used to assess the associations between immune function and PD. RESULTS: Forty (40) subjects with median 2.7 months on HAART and median nadir CD4+ T-cell count of 212 cells/µl completed a median 3 visits. Over 24 months, CD4+ T-cell count increased by a mean 173 cells/µl (p<0.001) and HIV RNA decreased by 0.5 log10 copies/ml (p<0.001); concurrently, PPD, CAL and BOP decreased by a mean 11.7%, 12.1%, and 14.7% respectively (all p<0.001). Lower nadir CD4+ T-cell count was associated with worse baseline REC (-6.72%; p=0.04) and CAL (9.06%; p<0.001). Further, lower nadir CD4+ T-cell count was associated with a greater relative longitudinal improvement in PPD in subjects with higher baseline levels of Porphyromonas gingivalis (p=0.027), and BOP in subjects with higher baseline levels of Porphyromonas gingivalis or Treponema denticola (p=0.001 and p=0.006 respectively). Longitudinal changes from baseline in CD4+ T-cell count and level of HIV RNA were not independently associated with longitudinal changes in any clinical markers of PD. CONCLUSION: Degree of immunosuppression was associated with baseline gingival recession. After HAART initiation, measures of active PD improved most in those with lower nadir CD4+ T-cell counts and higher baseline levels of specific periodontopathogens. Nadir CD4+ T-cell count differentially influences periodontal disease both before and after HAART in HIV-infected adults.
Asunto(s)
Recuento de Linfocito CD4 , Infecciones por VIH/complicaciones , Infecciones por VIH/inmunología , Enfermedades Periodontales/complicaciones , Enfermedades Periodontales/inmunología , Adulto , Terapia Antirretroviral Altamente Activa , Femenino , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Factores de Tiempo , Carga ViralRESUMEN
At sites of Mycobacterium tuberculosis (MTB) infection, HIV-1 replication is increased during tuberculosis (TB). Here we investigated the role of positive transcription elongation factor (P-TEFb), comprised of CycT1 and CDK9, as the cellular cofactor of HIV-1 Tat protein in transcriptional activation of HIV-1 in mononuclear cells from HIV-1-infected patients with pleural TB. Expression of CycT1 in response to MTB was assessed in mononuclear cells from pleural fluid (PFMC) and blood (PBMC) from HIV/TB patients with pleural TB, and in blood monocytes (MN) from singly infected HIV-1-seropositive subjects. We then examined whether the CDK9 inhibitor, Indirubin 3'-monoxime (IM), was effective in inhibition of MTB-induced HIV-1 mRNA expression. We found higher expression of CycT1 mRNA in PFMCs as compared to PBMCs from HIV/TB-coinfected subjects. MTB induced the expression of CycT1 and HIV-1 gag/pol mRNA in both PFMCs from HIV/TB subjects and MN from HIV-1-infected subjects. CycT1 protein was also induced by MTB stimulation in PFMCs from HIV/TB patients, and both MN and in vitro-derived macrophages. Inhibition of CDK9 by IM in both PFMCs from HIV/TB and MN from HIV-1-infected subjects in response to MTB led to inhibition of HIV-1 mRNA expression. These data imply that IM may be useful as an adjunctive therapy in control of HIV-1 replication in HIV/TB dually infected subjects.
Asunto(s)
Infecciones por VIH/metabolismo , VIH-1/fisiología , Indoles/farmacocinética , Mycobacterium tuberculosis/efectos de los fármacos , Oximas/farmacocinética , Factor B de Elongación Transcripcional Positiva/metabolismo , Tuberculosis/metabolismo , Adulto , Western Blotting , Coinfección , Ciclina T/efectos de los fármacos , Femenino , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , VIH-1/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/metabolismo , Factor B de Elongación Transcripcional Positiva/efectos de los fármacos , Factor B de Elongación Transcripcional Positiva/genética , Activación Transcripcional/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Tuberculosis/genética , Uganda/epidemiología , Replicación Viral/efectos de los fármacosRESUMEN
The determinants of HIV-associated cardiovascular disease (CVD) are not well understood. Periodontal disease (PD) has been linked to CVD but this connection has not been examined in HIV infection. We followed a cohort of HIV-infected adults to ascertain whether PD was associated with carotid artery intima media thickness (IMT) and brachial artery flow-mediated dilation (FMD). We performed a longitudinal observational study of HIV-infected adults on HAART for <2 years with no known heart disease. PD was characterized clinically and microbiologically. Cardiovascular disease was assessed by IMT/FMD. Linear mixed models assessed cross-sectional and longitudinal associations between PD and FMD/IMT. Forty three HIV(+) adults completed a median of 24 (6-44) months on the study. Defining delta to be the change in a variable between baseline and a follow-up time, longitudinally, on average and after adjusting for change in time, CVD-specific and HIV-specific potential confounding covariates, a 1-log(10) increase in delta Porphyromonas gingivalis was associated with a 0.013 mm increase in delta IMT (95% CI: 0.0006-0.0262; p=0.04). After adjusting for the same potential confounding covariates, a 10% increase in delta gingival recession was associated with a 2.3% increase in delta FMD (95% CI: 0.4-4.2; p=0.03). In a cohort of HIV-infected adults, an increase in subgingival Porphyromonas gingivalis, a known periodontal pathogen, was significantly associated with longitudinal increases in IMT, while increased gingival recession, which herein may represent PD resolution, was significantly associated with longitudinal improvement in FMD. In the context of HIV infection, PD may contribute to CVD risk. Intervention studies treating PD may help clarify this association.