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1.
Clin Microbiol Infect ; 20(4): O230-8, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24205913

RESUMO

New diagnostics and vaccines for tuberculosis (TB) are urgently needed, but require an understanding of the requirements for protection from/susceptibility to TB. Previous studies have used unbiased approaches to determine gene signatures in single-site populations. The present study utilized a targeted approach, reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA), to validate these genes in a multisite study. We analysed ex vivo whole blood RNA from a total of 523 participants across four sub-Saharan countries (Ethiopia, Malawi, South Africa, and The Gambia) with differences in TB and human immunodeficiency virus (HIV) status. We found a number of genes that were expressed at significantly lower levels in participants with active disease than in those with latent TB infection (LTBI), with restoration following successful TB treatment. The most consistent classifier of active disease was FCGR1A (high-affinity IgG Fc receptor 1 (CD64)), which was the only marker expressed at significantly higher levels in participants with active TB than in those with LTBI before treatment regardless of HIV status or genetic background. This is the first study to identify a biomarker for TB that is not affected by HIV status or geo-genetic differences. These data provide valuable clues for understanding TB pathogenesis, and also provide a proof-of-concept for the use of RT-MLPA in rapid and inexpensive validation of unbiased gene expression findings.


Assuntos
Biomarcadores/sangue , Expressão Gênica , Receptores de IgG/sangue , Tuberculose/diagnóstico , Adolescente , Adulto , África Subsaariana , Sangue , Etnicidade , Feminino , Perfilação da Expressão Gênica , Infecções por HIV/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
2.
Int J Tuberc Lung Dis ; 17(11): 1448-51, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24125449

RESUMO

SETTING: Patients with smear-positive, newly diagnosed pulmonary tuberculosis (TB) presenting to the out-patient TB clinic in Kampala, Uganda. OBJECTIVE: To compare colony-forming unit (cfu) counting and time to positive (TTP) in Mycobacteria Growth Indicator Tube (MGIT) culture as measures of early bactericidal activity (EBA). DESIGN: Patients were enrolled in an EBA feasibility study of standard TB chemotherapy. Sixteen-hour overnight sputum collections were obtained before and on days 2, 4, 7, 10, 12 and 14 of treatment for quantitative culture on selective Middlebrook 7H11 agar media and TTP in the MGIT liquid culture system. RESULTS: Log cfu and TTP were correlated over all time points (r(s) = -0.71, P < 0.001). Within-subject (day to day) variation as a percentage of total variation was very similar between the two measures: 25.7% for cfu and 25% for TTP. Mean EBA 0-14, 0-2 and 2-14 measured by TTP were similar to those previously reported. CONCLUSION: TTP measured by an automated, standardized, commercially available culture system correlates with cfu determinations. EBA measured by TTP provides similar information to cfu counting, and is reproducible across sites and in different patient populations. These findings support replacing cfu counting with TTP as the primary measurement in EBA studies.


Assuntos
Antituberculosos/uso terapêutico , Contagem de Colônia Microbiana , Monitoramento de Medicamentos/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Pulmonar/tratamento farmacológico , Adulto , Automação Laboratorial , Quimioterapia Combinada , Etambutol/uso terapêutico , Estudos de Viabilidade , Feminino , Humanos , Isoniazida/uso terapêutico , Masculino , Mycobacterium tuberculosis/crescimento & desenvolvimento , Valor Preditivo dos Testes , Estudos Prospectivos , Pirazinamida/uso terapêutico , Rifampina/uso terapêutico , Escarro/microbiologia , Fatores de Tempo , Resultado do Tratamento , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologia , Uganda , Adulto Jovem
3.
Int J Tuberc Lung Dis ; 17(3): 361-7, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23407224

RESUMO

OBJECTIVE: To determine the proportion of recurrent tuberculosis (TB) due to relapse with the patient's initial strain or reinfection with a new strain of Mycobacterium tuberculosis 1-2 years after anti-tuberculosis treatment in Uganda, a sub-Saharan TB-endemic country. DESIGN: Records of patients with culture-confirmed TB who completed treatment at an urban Ugandan clinic were reviewed. Restriction fragment length polymorphism (RFLP) patterns were used to determine relapse or reinfection. Associations between human immunodeficiency virus (HIV) positivity and type of TB recurrence were determined. RESULTS: Of 1701 patients cured of their initial TB episode with a median follow-up of 1.24 years, 171 (10%) had TB recurrence (8.4 per 100 person-years). Rate and risk factors for recurrence were similar to other studies from sub-Saharan Africa. Insertion sequence (IS) 6110-based RFLP of paired isolates from 98 recurrences identified 80 relapses and 18 reinfections. Relapses among HIV-positive and -negative patients were respectively 79% and 85% of recurrences. CONCLUSIONS: Relapse was more common and presented earlier than reinfection in both HIV-positive and -negative TB patients 1-2 years after completing treatment. These findings impact both the choice of retreatment drug regimen, as relapsing patients are at higher risk for acquired drug resistance, and clinical trials of new TB regimens with relapse as clinical endpoint.


Assuntos
Antituberculosos/uso terapêutico , Doenças Endêmicas , Mycobacterium tuberculosis/patogenicidade , Rifampina/uso terapêutico , Tuberculose Pulmonar/tratamento farmacológico , Saúde da População Urbana , Adulto , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Coinfecção , Feminino , Genótipo , Infecções por HIV/epidemiologia , Humanos , Incidência , Estimativa de Kaplan-Meier , Masculino , Análise Multivariada , Mycobacterium tuberculosis/genética , Fenótipo , Polimorfismo de Fragmento de Restrição , Modelos de Riscos Proporcionais , Recidiva , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Escarro/microbiologia , Fatores de Tempo , Resultado do Tratamento , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/epidemiologia , Tuberculose Pulmonar/microbiologia , Uganda/epidemiologia
4.
J Infect Dis ; 204(6): 884-92, 2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21849285

RESUMO

BACKGROUND: Optimal treatment of human immunodeficiency virus (HIV)-associated tuberculosis in patients with high CD4⁺ T-cell counts is unknown. Suppression of viral replication during therapy for tuberculosis may block effects of immune activation on T cells and slow HIV disease progression. METHODS: We conducted a randomized trial in 214 HIV-infected patients with active tuberculosis and CD4⁺ T-cell counts of ≥ 350 cells/µL to determine whether 6 months of antiretroviral therapy given during tuberculosis treatment would improve clinical outcomes. Subjects were randomized to receive 6 months of abacavir-lamivudine-zidovudine concurrent with tuberculosis therapy or delayed antiretroviral therapy. Endpoints were CD4⁺ T-cell counts of < 250 cells/µL, AIDS, or death. RESULTS: Intervention and comparison arms had similar median CD4⁺ counts (517 and 534 cells/µL, respectively) and HIV RNA levels (4.6 and 4.7 log10 copies/µL, respectively). Viral suppression was achieved in 86% of patients allocated to intervention. Seventeen subjects (15.6%) in the intervention arm developed study outcome compared to 25 subjects (22.8%) in the comparison arm (P = .17). Grade 3 or 4 adverse events were less frequent in the intervention arm. By 2 months, 90% of subjects in both arms were culture-negative for tuberculosis. CONCLUSIONS: Short-term antiretroviral therapy during tuberculosis treatment in patients with CD4⁺T-cell counts of >350 cells/µL was safe and associated with clinical benefits.


Assuntos
Fármacos Anti-HIV/administração & dosagem , Terapia Antirretroviral de Alta Atividade/métodos , Antituberculosos/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/tratamento farmacológico , Tuberculose Pulmonar/tratamento farmacológico , Adolescente , Adulto , Fármacos Anti-HIV/efeitos adversos , Terapia Antirretroviral de Alta Atividade/efeitos adversos , Antituberculosos/efeitos adversos , Contagem de Linfócito CD4 , Didesoxinucleosídeos/administração & dosagem , Feminino , Infecções por HIV/complicações , Infecções por HIV/mortalidade , Humanos , Lamivudina/administração & dosagem , Masculino , Pessoa de Meia-Idade , Análise de Sobrevida , Resultado do Tratamento , Tuberculose Pulmonar/complicações , Tuberculose Pulmonar/mortalidade , Uganda , Adulto Jovem , Zidovudina/administração & dosagem
5.
Tuberculosis (Edinb) ; 91(3): 257-9, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21353641

RESUMO

Testing new drugs is critical to improving the treatment of tuberculosis. Quantitative cultures of Mycobacterium tuberculosis on solid media have been used in Phase 1 and 2 trials, but are time and resource intensive. Time to detection (TTD) of growth of M. tuberculosis in automated liquid culture systems is an alternative. TTD has been shown to correlate with CFU in quantitative cultures, and is faster and simpler to perform. We compared TTD in the BACTEC 460 liquid culture system with CFU in a clinical trial that included 110 subjects. Comparing all sputum cultures collected between baseline and 2 months we found a strong negative correlation between log(10) CFU and TTD (rho = -0.91). In addition, when TTD at baseline was compared with 1 and 2 month sputum culture positivity, subjects whose cultures were negative after 1 and 2 months had a significantly longer median baseline TTD compared with subjects whose cultures were positive at 1 and 2 months (5 vs. 3 days and 3 vs. 2 days, respectively). TTD compares closely with CFU and represents a faster, simpler alternative to quantitative cultures.


Assuntos
Contagem de Colônia Microbiana , Meios de Cultura/farmacologia , Mycobacterium tuberculosis/isolamento & purificação , Escarro/microbiologia , Tuberculose Pulmonar/microbiologia , Adolescente , Adulto , Ensaios Clínicos Fase II como Assunto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Tempo , Tuberculose Pulmonar/epidemiologia , Uganda/epidemiologia , Adulto Jovem
6.
J Clin Microbiol ; 48(1): 46-51, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19923475

RESUMO

mRNA is a marker of cell viability. Quantifying Mycobacterium tuberculosis mRNA in sputum is a promising tool for monitoring response to antituberculosis therapy and evaluating the efficacy of individual drugs. mRNA levels were measured in sputum specimens from patients with tuberculosis (TB) receiving monotherapy in an early bactericidal activity study of fluoroquinolones and in those receiving a standard rifampin-based regimen in an interleukin-2 (IL-2) trial. In the early bactericidal activity study, sputum for quantitative culture and mRNA analysis was collected for 2 days before and daily during 7 days of study drug administration. In the IL-2 trial, sputum was collected for quantitative culture, Bactec 460 liquid culture, and mRNA analysis throughout the intensive treatment phase. RNA was isolated from digested sputum and tested in quantitative reverse transcription-PCR assays for several gene targets. mRNA for the glyoxylate cycle enzyme isocitrate lyase declined at similar rates in patients receiving isoniazid, gatifloxicin, levofloxacin, and moxifloxacin monotherapy. Isocitrate lyase mRNA correlated highly with CFU in sputum prior to therapy and during 7 days of monotherapy in all treatment arms. Isocitrate lyase mRNA was detectable in sputum of culture-positive TB patients receiving a rifampin-based regimen for 1 month. At 2 months, sputum for isocitrate mRNA correlated more closely with growth in liquid culture than did growth on solid culture medium. Data suggest that isocitrate lyase mRNA is a reliable marker of M. tuberculosis viability.


Assuntos
Antituberculosos/uso terapêutico , Monitoramento de Medicamentos/métodos , Mycobacterium tuberculosis/genética , RNA Bacteriano/isolamento & purificação , RNA Mensageiro/isolamento & purificação , Escarro/microbiologia , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/tratamento farmacológico , Contagem de Colônia Microbiana , Humanos , Viabilidade Microbiana , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/isolamento & purificação , RNA Bacteriano/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Estatística como Assunto , Adulto Jovem
7.
Int J Tuberc Lung Dis ; 11(2): 168-74, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17263287

RESUMO

OBJECTIVES: Voluntary counseling and testing (VCT) for the human immunodeficiency virus (HIV) is recommended for persons treated for tuberculosis (TB). Opportunities to diagnose HIV may be missed by limiting HIV testing to only persons diagnosed with TB. Among TB suspects in Uganda, we determined HIV prevalence, risk behaviors, and willingness to refer family for VCT. METHODS: Consenting adult patients presenting for evaluation at a referral TB clinic received same-day VCT. TB diagnosis data were abstracted from clinical records. RESULTS: Among 665 eligible patients, 565 (85%) consented to VCT. Among these, 238 (42%) were HIV-positive. Of the HIV-infected patients, 37% had received a non-TB diagnosis. HIV seroprevalence was higher in patients with a non-TB diagnosis (49%) than those diagnosed with TB (39%) (P = 0.02). Fewer than 6% of HIV-infected patients reported always using condoms with sexual partners. The majority of patients (86%) reported being 'very willing' to refer family members for VCT. CONCLUSIONS: Over 35% of HIV-infected cases in our population would have been undetected if HIV testing was limited to cases with diagnosed TB. The high HIV seroprevalence in both TB and non-TB cases merits HIV testing for all patients evaluated at TB clinics. HIV-infected TB suspects reporting high-risk behavior are at risk for HIV transmission, and should receive risk-reduction counseling.


Assuntos
Infecções por HIV/epidemiologia , Soroprevalência de HIV , Tuberculose Pulmonar/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Comorbidade , Aconselhamento , Feminino , Infecções por HIV/prevenção & controle , Humanos , Masculino , Programas de Rastreamento/métodos , Pessoa de Meia-Idade , Assunção de Riscos , Uganda/epidemiologia
8.
Int J Tuberc Lung Dis ; 10(6): 605-12, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16776446

RESUMO

OBJECTIVE: To evaluate the early bactericidal activity (EBA) of the new fluoroquinolones levofloxacin, gatifloxacin and moxifloxacin in patients with pulmonary tuberculosis (PTB). DESIGN: Randomized, open-label trial. Forty adults with newly diagnosed smear-positive PTB (10 per arm) were assigned to receive isoniazid (INH) 300 mg, levofloxacin 1000 mg, gatifloxacin 400 mg, or moxifloxacin 400 mg daily for 7 days. Sputum for quantitative culture was collected for 2 days before and daily during 7 days of monotherapy. Bactericidal activity was estimated by measuring the decline in bacilli during the first 2 days (EBA 0-2) and last 5 days of monotherapy (extended EBA, EBA 2-7). Laboratory staff were blinded to treatment assignment. RESULTS: The EBA 0-2 of INH (0.67 log10 cfu/ml/day) was greater than that of moxifloxacin and gatifloxacin (0.33 and 0.35 log10 cfu/ml/day, respectively), but not of levofloxacin 1000 mg daily (0.45 log10 cfu/ml/day) (P = 0.14). Bactericidal activity between days 2 and 7 was similar for all three fluoroquinolones. In a pooled comparison, the EBA 2-7 of the fluoroquinolones was greater than for INH. CONCLUSION: Moxifloxacin, gatifloxacin, and high-dose levofloxacin have excellent EBA, only slightly less than for INH, and greater extended EBA. These drugs warrant further study in the treatment of drug-susceptible TB.


Assuntos
Antibacterianos/uso terapêutico , Anti-Infecciosos/uso terapêutico , Compostos Aza/uso terapêutico , Fluoroquinolonas/uso terapêutico , Levofloxacino , Ofloxacino/uso terapêutico , Quinolinas/uso terapêutico , Tuberculose Pulmonar/tratamento farmacológico , Adulto , Feminino , Gatifloxacina , Humanos , Masculino , Pessoa de Meia-Idade , Moxifloxacina , Método Simples-Cego
9.
Clin Exp Immunol ; 144(1): 25-34, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16542361

RESUMO

Active tuberculosis (TB) is associated with prolonged suppression of Mycobacterium tuberculosis (MTB)-specific immune responses, but mechanisms involved are understood incompletely. We investigated a potential role for CD4+CD25+ regulatory T cells in depressed anti-MTB immunity by evaluating serially CD4 cell phenotype and interferon (IFN)-gamma production by mononuclear cells from patients with TB. At diagnosis, frequencies of CD4+CD25+ T cells were increased in blood from TB patients compared to healthy purified protein derivative (PPD)-positive controls (with a history of prior TB exposure), and remained elevated at completion of therapy (6 months). By contrast, expression of another activation marker, CD69, by CD4 T cells was increased at diagnosis, but declined rapidly to control levels with treatment. Among CD4+CD25+ T cells from TB patients at diagnosis those expressing high levels of CD25, probably representing regulatory T cells, were increased 2.9-fold when compared to control subjects, while MTB-stimulated IFN-gamma levels in whole blood supernatants were depressed. A role for CD4+CD25+ T cells in depressed IFN-gamma production during TB was substantiated in depletion experiments, where CD25+-depleted CD4 T cells produced increased amounts of IFN-gamma upon MTB stimulation compared to unseparated T cells. At follow-up, IFN-gamma production improved most significantly in blood from TB patients with high baseline frequencies of CD4+CD25+ T cells (more than threefold higher than controls for both total and CD25hi+ CD4 T cells), who also had a significant drop in frequencies of both total and 'regulatory' CD4+CD25+ T cells in response to treatment. Expansion of CD4+CD25+ regulatory T cells during active TB may play a role in depressed T cell IFN-gamma production.


Assuntos
Receptores de Interleucina-2/imunologia , Linfócitos T Reguladores/imunologia , Tuberculose Pulmonar/imunologia , Adulto , Antígenos de Bactérias/imunologia , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Humanos , Interferon gama/imunologia , Interleucina-10/imunologia , Lectinas Tipo C , Pulmão/imunologia , Masculino , Monócitos/imunologia , Mycobacterium tuberculosis/imunologia , Fenótipo , Fator de Crescimento Transformador beta/imunologia , Tuberculina/imunologia , Tuberculose Pulmonar/microbiologia , Fator de Necrose Tumoral alfa/imunologia
10.
Tuberculosis (Edinb) ; 83(1-3): 98-106, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12758197

RESUMO

A hallmark of M. tuberculosis infection is the ability of most (90-95%) healthy adults to control infection through acquired immunity, in which antigen specific T cells and macrophages arrest growth of M. tuberculosis bacilli and maintain control over persistent bacilli. In addition to CD4+ T cells, other T cell subsets such as, gammadelta, CD8+ and CD1-restricted T cells have roles in the immune response to M. tuberculosis. A diverse T cell response allows the host to recognize a wider range of mycobacterial antigens presented by different families of antigen-presenting molecules, and thus greater ability to detect the pathogen. Macrophages are key antigen presenting cells for T cells, and M. tuberculosis survives and persists in this central immune cell. This is likely an important factor in generating this T cell diversity. Furthermore, the slow growth and chronic nature of M. tuberculosis infection results in prolonged exposure to antigens, and hence further T cell sensitization. The effector mechanisms used by T cells to control M. tuberculosis are poorly understood. To survive in macrophages, M. tuberculosis has evolved mechanisms to block immune responses. These include modulation of phagosomes, neutralization of macrophage effector molecules, stimulating the secretion of inhibitory cytokines, and interfering with processing of antigens for T cells. The relative importance of these blocking mechanisms likely depends on the stage of M. tuberculosis infection: primary infection, persistence, reactivation or active tuberculosis. The balance of the host-pathogen interaction in M. tuberculosis infection is determined by the interaction of T cells and infected macrophages. The outcome of this interaction results either in control of M. tuberculosis infection or active disease. A better understanding of this interaction will result in improved approaches to treatment and prevention of tuberculosis.


Assuntos
Apresentação de Antígeno , Mycobacterium tuberculosis/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Humanos , Imunidade Celular , Receptores de Antígenos de Linfócitos T gama-delta/análise
11.
Infect Immun ; 70(9): 5322-7, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12183593

RESUMO

Although neutrophils have been identified as sources of inflammatory cytokines and chemokines, little is known about their immunologic function during mycobacterial infection in the lungs. In this study, we examined the growth of Mycobacterium bovis BCG in the lungs under experimental conditions that altered neutrophil recruitment to the lungs. Depletion and recruitment of neutrophils was associated with respective increases and decreases in M. bovis BCG growth. Thus, neutrophils may enhance mycobacteriocidal immunity in the lungs.


Assuntos
Pulmão/imunologia , Mycobacterium bovis/imunologia , Mycobacterium bovis/patogenicidade , Neutrófilos/imunologia , Tuberculose Pulmonar/imunologia , Animais , Quimiocina CXCL2 , Quimiocinas/administração & dosagem , Feminino , Interferon gama/biossíntese , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium bovis/crescimento & desenvolvimento , Linfócitos T/imunologia , Tuberculose Pulmonar/microbiologia
12.
J Exp Med ; 194(10): 1421-32, 2001 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-11714749

RESUMO

Mycobacterium tuberculosis (MTB) inhibits phagosomal maturation to promote its survival inside macrophages. Control of MTB infection requires CD4 T cell responses and major histocompatibility complex (MHC) class II (MHC-II) processing of MTB antigens (Ags). To investigate phagosomal processing of MTB Ags, phagosomes containing heat-killed (HK) or live MTB were purified from interferon-gamma (IFN-gamma)-activated macrophages by differential centrifugation and Percoll density gradient subcellular fractionation. Flow organellometry and Western blot analysis showed that MTB phagosomes acquired lysosome-associated membrane protein-1 (LAMP-1), MHC-II, and H2-DM. T hybridoma cells were used to detect MTB Ag 85B(241-256)-I-A(b) complexes in isolated phagosomes and other subcellular fractions. These complexes appeared initially (within 20 min) in phagosomes and subsequently (>20 min) on the plasma membrane, but never within late endocytic compartments. Macrophages processed HK MTB more rapidly and efficiently than live MTB; phagosomes containing live MTB expressed fewer Ag 85B(241-256)-I-A(b) complexes than phagosomes containing HK MTB. This is the first study of bacterial Ag processing to directly show that peptide-MHC-II complexes are formed within phagosomes and not after export of bacterial Ags from phagosomes to endocytic Ag processing compartments. Live MTB can alter phagosome maturation and decrease MHC-II Ag processing, providing a mechanism for MTB to evade immune surveillance and enhance its survival within the host.


Assuntos
Aciltransferases , Apresentação de Antígeno , Antígenos de Bactérias , Proteínas de Bactérias/metabolismo , Antígenos de Histocompatibilidade Classe II/análise , Antígenos de Histocompatibilidade Classe II/metabolismo , Mycobacterium tuberculosis/imunologia , Fagossomos/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/análise , Western Blotting , Centrifugação com Gradiente de Concentração , Proteínas de Membrana Lisossomal , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
13.
J Immunol ; 167(5): 2734-42, 2001 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-11509617

RESUMO

Cytotoxic effector phenotype and function of MHC-restricted Mycobacterium tuberculosis (MTB)-reactive CD4(+) and CD8(+) T lymphocytes were analyzed from healthy tuberculin skin test-positive persons. After stimulation in vitro with MTB, both CD4(+) and CD8(+) T cells up-regulated mRNA expression for granzyme A and B, granulysin, perforin, and CD95L (Fas ligand). mRNA levels for these molecules were greater for resting CD8(+) than CD4(+) T cells. After MTB stimulation, mRNA levels were similar for both T cell subsets. Increased perforin and granulysin protein expression was confirmed in both in CD4(+) and CD8(+) T cells by flow cytometry. Both T cell subsets lysed MTB-infected monocytes. Biochemical inhibition of the granule exocytosis pathway in CD4(+) and CD8(+) T cells decreased cytolytic function by >90% in both T cell subsets. Ab blockade of the CD95-CD95L interaction decreased cytolytic function for both T cell populations by 25%. CD4(+) and CD8(+) T cells inhibited growth of intracellular MTB in autologous monocytes by 74% and 84%, respectively. However, inhibition of perforin activity, the CD95-CD95L interaction, or both CTL mechanisms did not affect CD4(+) and CD8(+) T cell mediated restriction of MTB growth. Thus, perforin and CD95-CD95L were not involved in CD4(+) and CD8(+) T cell mediated restriction of MTB growth.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/microbiologia , Mycobacterium tuberculosis/imunologia , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/metabolismo , Atividade Bactericida do Sangue/imunologia , Citotoxicidade Imunológica , Proteína Ligante Fas , Granzimas , Humanos , Técnicas In Vitro , Complexo Principal de Histocompatibilidade , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Perforina , Proteínas Citotóxicas Formadoras de Poros , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Regulação para Cima , Receptor fas/genética , Receptor fas/imunologia
14.
J Immunol ; 167(2): 910-8, 2001 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-11441098

RESUMO

Mycobacterium tuberculosis (MTB) induces vigorous immune responses, yet persists inside macrophages, evading host immunity. MTB bacilli or lysate was found to inhibit macrophage expression of class II MHC (MHC-II) molecules and MHC-II Ag processing. This report characterizes and identifies a specific component of MTB that mediates these inhibitory effects. The inhibitor was extracted from MTB lysate with Triton X-114, isolated by gel electroelution, and identified with Abs to be MTB 19-kDa lipoprotein. Electroelution- or immunoaffinity-purified MTB 19-kDa lipoprotein inhibited MHC-II expression and processing of both soluble Ags and Ag 85B from intact MTB bacilli. Inhibition of MHC-II Ag processing by either MTB bacilli or purified MTB 19-kDa lipoprotein was dependent on Toll-like receptor (TLR) 2 and independent of TLR 4. Synthetic analogs of lipopeptides from Treponema pallidum also inhibited Ag processing. Despite the ability of MTB 19-kDa lipoprotein to activate microbicidal and innate immune functions early in infection, TLR 2-dependent inhibition of MHC-II expression and Ag processing by MTB 19-kDa lipoprotein during later phases of macrophage infection may prevent presentation of MTB Ags and decrease recognition by T cells. This mechanism may allow intracellular MTB to evade immune surveillance and maintain chronic infection.


Assuntos
Aciltransferases , Apresentação de Antígeno/imunologia , Antígenos de Bactérias , Proteínas de Bactérias/farmacologia , Proteínas de Drosophila , Antígenos de Histocompatibilidade Classe II/biossíntese , Imunossupressores/farmacologia , Lipoproteínas/farmacologia , Macrófagos/imunologia , Glicoproteínas de Membrana/fisiologia , Mycobacterium tuberculosis/imunologia , Receptores de Superfície Celular/fisiologia , Animais , Apresentação de Antígeno/genética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Detergentes , Epitopos/metabolismo , Feminino , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Octoxinol , Polietilenoglicóis , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-Like
15.
J Immunol ; 167(3): 1542-9, 2001 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-11466375

RESUMO

Certain microbial substances, e.g., LPS, can activate neutrophils or prime them to enhance their response to other activating agents, e.g., fMLP. We investigated the role of the Mycobacterium tuberculosis (MTB) 19-kDa lipoprotein in activation of human neutrophils. MTB 19-kDa lipoprotein initiated phenotypic changes characteristic of neutrophil activation, including down-regulation of CD62 ligand (L-selectin) and up-regulation of CD35 (CR1) and CD11b/CD18 (CR3, Mac-1). In addition, exposure of neutrophils to MTB 19-kDa lipoprotein enhanced the subsequent oxidative burst in response to fMLP as assessed by oxidation of dihydrorhodamine 123 (determined by flow cytometry). LPS also produced these effects with similar kinetics, but an oligodeoxynucleotide containing a CpG motif failed to induce any priming or activation response. Although the effects of LPS required the presence of serum, neutrophil activation by MTB 19-kDa lipoprotein occurred independently of serum factors, suggesting the involvement of different receptors and signaling mechanisms for LPS and MTB 19-kDa lipoprotein. Thus, MTB 19-kDa lipoprotein serves as a pathogen-associated molecular pattern that promotes neutrophil priming and activation.


Assuntos
Proteínas de Bactérias/farmacologia , Lipoproteínas/farmacologia , Mycobacterium tuberculosis/imunologia , Ativação de Neutrófilo/imunologia , Antígenos CD18/biossíntese , Membrana Celular/imunologia , Membrana Celular/metabolismo , Meios de Cultura Livres de Soro , Relação Dose-Resposta Imunológica , Regulação para Baixo/imunologia , Humanos , Selectina L/biossíntese , Lipopolissacarídeos/farmacologia , Antígeno de Macrófago 1/biossíntese , Mycobacterium tuberculosis/patogenicidade , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/metabolismo , Receptores de Complemento 3b/biossíntese , Explosão Respiratória/imunologia , Regulação para Cima/imunologia
16.
Infect Immun ; 69(3): 1755-65, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11179353

RESUMO

Despite the continued importance of tuberculosis as a world-wide threat to public health, little is known about the mechanisms used by human lymphocytes to contain and kill the intracellular pathogen Mycobacterium tuberculosis. We previously described an in vitro model of infection of human monocytes (MN) with virulent M. tuberculosis strain H37Rv in which the ability of peripheral blood lymphocytes to limit intracellular growth of the organism could be measured. In the current study, we determined that lymphocyte-mediated killing of intracellular M. tuberculosis occurs within the first 24 h of coculture with infected MN. Natural killer (NK) cells isolated from both purified protein derivative (PPD)-positive and PPD-negative subjects were capable of mediating this early killing of intracellular H37Rv. NK cell-mediated killing of intracellular M. tuberculosis was not associated with the production of gamma interferon. Transferred supernatants of cocultured NK cells and M. tuberculosis-infected MN could not mediate the killing of intracellular M. tuberculosis, and Transwell studies indicated that direct cell-to-cell contact was required for NK cells to mediate the killing of the organism. Killing was not dependent upon exocytosis of NK cell cytotoxic granules. NK cells induced apoptosis of mycobacterium-infected MN, but neither killing of intracellular M. tuberculosis by NK cells nor NK cell-induced apoptosis of infected MN was inhibited by blocking the interaction of FasL and Fas. Thus, human NK cells may mediate killing of intracellular M. tuberculosis via alternative apoptotic pathways.


Assuntos
Apoptose , Grânulos Citoplasmáticos/metabolismo , Células Matadoras Naturais/imunologia , Mycobacterium tuberculosis/imunologia , Adulto , Comunicação Celular , Técnicas de Cocultura , Proteína Ligante Fas , Humanos , Interferon gama/metabolismo , Células Matadoras Naturais/microbiologia , Linfócitos/imunologia , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Mycobacterium bovis/imunologia , Receptor fas/metabolismo
17.
J Clin Microbiol ; 39(1): 298-303, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11136787

RESUMO

To characterize changes in serum cytokine levels in human immunodeficiency virus type 1 (HIV-1)-infected persons with Mycobacterium avium complex (MAC) bacteremia, the levels of IL-1alpha (interleukin-1alpha), IL-6, IL-10, tumor necrosis factor alpha (TNF-alpha), soluble type II TNF receptor (sTNF-RII), and transforming growth factor beta (TGF-beta) in serum were measured in two cohorts of HIV-1-infected persons with MAC bacteremia. The first cohort was part of a MAC prophylaxis study. Patients with bacteremia were matched with controls without bacteremia. Elevated IL-6, IL-10, TNF-alpha, sTNF-RII, and TGF-beta levels were noted at baseline for all subjects, a result consistent with advanced HIV-1 disease. IL-1alpha was not detected. No differences in cytokine levels in serum were noted at baseline and at the time of bacteremia between patients with MAC and controls. In the second cohort, subjects had serum samples collected at the time of MAC bacteremia and thereafter while on macrolide therapy. Serum samples at time of bacteremia were collected from HIV-1-infected persons at a time when neither highly active antiretroviral therapy (HAART) nor MAC prophylaxis was used routinely. MAC treatment resulted in decreased levels of IL-6 and TNF-alpha in serum, which were evident for IL-6 by 4 to 6 weeks and for TNF-alpha by 8 to 16 weeks. Thus, antibiotic treatment for MAC results in decreased levels of IL-6 and TNF-alpha in serum in HIV-1-infected persons who are not on HAART.


Assuntos
Infecções Oportunistas Relacionadas com a AIDS/imunologia , Antibacterianos/uso terapêutico , Antígenos CD/sangue , Citocinas/sangue , Infecção por Mycobacterium avium-intracellulare/imunologia , Receptores do Fator de Necrose Tumoral/sangue , Infecções Oportunistas Relacionadas com a AIDS/tratamento farmacológico , Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Fármacos Anti-HIV/uso terapêutico , Bacteriemia/tratamento farmacológico , Bacteriemia/imunologia , Bacteriemia/microbiologia , Estudos de Casos e Controles , Estudos de Coortes , Quimioterapia Combinada , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Humanos , Interleucina-10/sangue , Interleucina-6/sangue , Macrolídeos , Complexo Mycobacterium avium/imunologia , Complexo Mycobacterium avium/isolamento & purificação , Infecção por Mycobacterium avium-intracellulare/tratamento farmacológico , Infecção por Mycobacterium avium-intracellulare/microbiologia , Infecção por Mycobacterium avium-intracellulare/prevenção & controle , Receptores Tipo II do Fator de Necrose Tumoral , Fator de Crescimento Transformador beta/sangue , Fator de Necrose Tumoral alfa/metabolismo
18.
J Infect Dis ; 182(6): 1658-63, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11069237

RESUMO

To characterize the influence of highly active antiretroviral therapy (HAART) on cell-mediated immunity (CMI) to Mycobacterium avium complex (MAC), we measured immune responses to M. avium in human immunodeficiency virus (HIV)-infected individuals before and during HAART, in subjects with a history of disseminated MAC (DMAC), and in HIV-uninfected control subjects. Forty-seven percent of untreated HIV-infected patients and 78% of control subjects exhibited in vitro proliferative responses to M. avium (P=.03). Proliferative responses to M. avium increased after HAART for 3 months and were present in 77% of subjects after 6 months. Mean interferon-gamma production increased from 199 to 1156 pg/mL after HAART (P=.06). Proliferative responses to M. avium occurred in 76% of DMAC subjects receiving HAART. CD4 and CD8 but not gammadelta T cells expanded in response to M. avium. CMI to M. avium reconstitutes rapidly after HAART and appears sustained even with partial viral suppression.


Assuntos
Infecções por HIV/tratamento farmacológico , HIV-1 , Mycobacterium avium/imunologia , Adolescente , Adulto , Idoso , Terapia Antirretroviral de Alta Atividade , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Estudos de Coortes , Infecções por HIV/complicações , Infecções por HIV/imunologia , Humanos , Imunidade Celular , Interferon gama/análise , Ativação Linfocitária , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Tuberculose Miliar/complicações , Tuberculose Miliar/imunologia
19.
Infect Immun ; 68(11): 6505-8, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11035768

RESUMO

Latency-associated peptide of transforming growth factor beta (TGF-beta) (LAP) was used to determine whether in vivo modulation of TGF-beta bioactivity enhanced pulmonary immunity to Mycobacterium bovis BCG infection in C57BL/6 mice. LAP decreased BCG growth in the lung and enhanced antigen-specific T-cell proliferation and gamma interferon mRNA expression. Thus, susceptibility of the lung to primary BCG infection may be partially mediated by the immunosuppressive effects of TGF-beta.


Assuntos
Pulmão/imunologia , Pulmão/microbiologia , Mycobacterium bovis/imunologia , Fator de Crescimento Transformador beta/fisiologia , Tuberculose/imunologia , Animais , Feminino , Interferon gama/biossíntese , Interferon gama/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/análise , Linfócitos T/imunologia
20.
Cell Immunol ; 201(1): 63-74, 2000 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-10805975

RESUMO

Infection of murine bone-marrow-derived macrophages with viable Mycobacterium tuberculosis (MTB) H37Ra inhibited surface expression of MHC class II (MHC-II) molecules and processing of exogenous antigens for presentation to CD4(+) T hybridoma cells. The inhibition was not dependent on bacterial viability, since it was also produced by exposure to dead bacilli and MTB cytosol preparations, suggesting that it was initiated by a constitutively expressed bacterial component. Northern blot analysis demonstrated that MTB bacilli or cytosol decreased MHC-II mRNA, and immunoprecipitation of biosynthetically labeled molecules confirmed that MHC-II protein synthesis was diminished. Exposure to MTB or MTB cytosol also decreased expression of H2-DM, but H2-DM expression was still sufficient to catalyze conversion of MHC-II to SDS-stable dimers, a measure of MHC-II peptide loading. Thus, infection with MTB decreased both MHC-II and H2-DM expression, but diminished MHC-II synthesis provided the major limitation to antigen processing.


Assuntos
Apresentação de Antígeno/imunologia , Células da Medula Óssea/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Animais , Células da Medula Óssea/microbiologia , Antígenos HLA-D/biossíntese , Antígenos de Histocompatibilidade Classe II/biossíntese , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos CBA
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