Mycobacterium tuberculosis-induced neutrophil ectosomes decrease macrophage activation.

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2014-11-12T14:04:23Z No. of bitstreams: 1 Duarte TA Mycobacterium....pdf: 1042368 bytes, checksum: 72a82354af873a14f7ec429585669248 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2014-11-12T14:04:32Z (GMT) No. of bitstreams: 1 Duarte TA Mycobacterium....pdf: 1042368 bytes, checksum: 72a82354af873a14f7ec429585669248 (MD5)Made available in DSpace on 2014-11-12T14:22:42Z (GMT). No. of bitstreams: 1 Duarte TA Mycobacterium....pdf: 1042368 bytes, checksum: 72a82354af873a14f7ec429585669248 (MD5) Previous issue date: 2012Federal University of Bahia. Health Science Institute. Salvador, BA, Brasil / Federal University of Rio de Janeiro. Clementino Fraga Filho University Hospital, Department of Internal Medicine. Multidisciplinary Research Laboratory. Rio de Janeiro, RJ, BrasilUniversity College London. Gower Street, London, UK / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFederal University of Rio de Janeiro. Clementino Fraga Filho University Hospital, Department of Internal Medicine. Multidisciplinary Research Laboratory. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFederal University of Rio de Janeiro. Clementino Fraga Filho University Hospital, Department of Internal Medicine. Multidisciplinary Research Laboratory. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFederal University of Rio de Janeiro. Clementino Fraga Filho University Hospital, Department of Internal Medicine. Multidisciplinary Research Laboratory. Rio de Janeiro, RJ, BrasilBACKGROUND: The existence of ectosome-like microvesicles released by neutrophils was proposed a few decades ago. Other studies revealed that the innate immune response during mycobacterial infection is accompanied by an intense migration of neutrophils to the site of infection, which may be important during the acute phase of tuberculosis. We found that the ectosomes derived from infected neutrophils are biologically active and can influence the survival of Mycobacterium tuberculosis within macrophages. METHODS: Mycobacteria were cultured on supplemented Middlebrook-7H9 broth. All strains were grown to the exponential phase and quantitated by serial dilution. Human neutrophils and macrophages were infected with mycobacteria. Ectosomes from neutrophils were isolated post-infection and characterized by transmission electron microscopy and flow cytometry. To determine whether these microvesicles influenced mycobactericidal activity, mycobacteria-infected macrophages were treated with isolated ectosomes. RESULTS: Ectosomes were released from neutrophils infected with mycobacteria. These ectosomes were derived from neutrophil plasma membrane and a small proportion stained with PKH26. These microvesicles, when incubated with infected macrophages, influenced antimycobacterial activity. CONCLUSIONS: This is the first study to demonstrate that ectosomes that are shed from infected neutrophils influence mycobactericidal activity in macrophages in vitro, suggesting that these microvesicles have biological significance. Nevertheless, major gaps in our knowledge of microvesicle biology remain

Genotyping did not evidence any contribution of Mycobacterium bovis to human tuberculosis in Brazil

Submitted by Sandra Infurna ([email protected]) on 2016-10-11T15:30:42Z No. of bitstreams: 1 alexandre_santos_etal_IOC_2011.pdf: 239001 bytes, checksum: 50d5454a5f61743435cbeee1d8103caf (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2016-10-11T15:46:25Z (GMT) No. of bitstreams: 1 alexandre_santos_etal_IOC_2011.pdf: 239001 bytes, checksum: 50d5454a5f61743435cbeee1d8103caf (MD5)Made available in DSpace on 2016-10-11T15:46:25Z (GMT). No. of bitstreams: 1 alexandre_santos_etal_IOC_2011.pdf: 239001 bytes, checksum: 50d5454a5f61743435cbeee1d8103caf (MD5) Previous issue date: 2011Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Doenças Torácicas. Laboratório de Micobacteriologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Doenças Torácicas. Laboratório de Micobacteriologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Ministério da Saúde. Fundação Oswaldo Cruz. Centro de Referência Nacional em Tuberculose Prof. Hélio Fraga. Rio de Janeiro, RJ, Brasil.Clínica de Tuberculose Augusto Guimarães, Centro de Referencia Regional em Tuberculose. Program de Controle de Tuberculose. Campos, RJ, Brasil;.EMBRAPA. Divisão de Saúde Animal. Juiz de Fora, MG, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Doenças Torácicas. Laboratório de Micobacteriologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Faculdade de Medicina. Departamento de Medicina Interna. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Hospital Universitário Clementino Fraga Filho. Laboratório de Pesquisa Multidisciplinar. Rio de Janeiro, RJ, Brasil.The contribution of Mycobacterium bovis to the global burden of tuberculosis (TB) in man is likely to be underestimated due to its dysgonic growth characteristics and because of the absence of pyruvate in most used media is disadvantageous for its primary isolation. In Brazil Mycobacterium culture, identification and susceptibility tests are performed only in TB reference centers, usually for selected cases. Moreover, solid, egg-based, glycerol-containing (without pyruvate supplementation) Löwenstein-Jensen (L-J) or Ogawa media are routinely used, unfavouring M. bovis isolation. To determine the importance of M. bovis as a public health threat in Brazil we investigated 3046 suspected TB patients inoculating their clinical samples onto routine L-J and L-J pyruvate enriched media. A total of 1796 specimens were culture positive for Mycobacterium spp. and 702 TB cases were confirmed. Surprisingly we did not detect one single case of M. bovis in the resulting collection of 1674 isolates recovered from M. bovis favourable medium analyzed by conventional and molecular speciation methods. Also, bacillary DNA present on 454 sputum smears from 223 TB patients were OxyR genotyped and none was recognized as M. bovis. Our data indicate that M. bovis importance on the burden of human TB in Brazil is marginal

Drug susceptibility of Brazilian strains of Mycobacterium bovis using traditional and molecular techniques

Transmission of Mycobacterium bovis from cattle to humans has been reported and can cause tuberculosis (Tb) and a problem in certain risk populations. Therefore, knowledge of resistance of M. bovis towards antibiotics used for therapy of human Tb could help avoiding cure delay and treatment cost increase when dealing with drug resistant organisms. We therefore evaluated the susceptibility of M. bovis isolates towards streptomycin, isoniazide, rifampicin, ethambutol, and ethionamide, the first line antibiotics for human Tb. Therefore, 185 clinical samples from cattle with clinical signs of tuberculosis were processed and submitted to culturing and bacterial isolates to identification and drug susceptibility testing using the proportion method. Among 89 mycobacterial strains, 65 were identified as M. bovis and none were resistant to any of the antibiotics used. Confirmation of present results by future studies, enrolling a large number of isolates and designed to properly represent Brazilian regions, may favor the idea of using isoniazide preventive therapy as part of a Tb control strategy in special situations. Also, nucleic acids from bacterial isolates were submitted to rifoligotyping, a recently described reverse hybridization assay for detection of mutations causing resistance towards rifampicin. Concordance between the conventional and the molecular test was 100%, demonstrating the use of such methodology for rapid evaluation of drug susceptibility in M. bovis

Down-Modulation of Lung Immune Responses by Interleukin-10 and Transforming Growth Factor β (TGF-β) and Analysis of TGF-β Receptors I and II in Active Tuberculosis

Immune factors influencing progression to active tuberculosis (TB) remain poorly defined. In this study, we investigated the expression of immunoregulatory cytokines and receptors by using lung bronchoalveolar lavage cells obtained from patients with pulmonary TB, patients with other lung diseases (OLD patients), and healthy volunteers (VOL) by using reverse transcriptase PCR, a transforming growth factor β (TGF-β) bioactivity assay, and an enzyme immunoassay. TB patients were significantly more likely than OLD patients to coexpress TGF-β receptor I (RI) and RII mRNA, as well as interleukin-10 (IL-10) mRNA (thereby indicating the state of active gene transcription in the alveolar cells at harvest). In contrast, gamma interferon (IFN-γ) and IL-2 mRNA was seen in both TB and OLD patients. Likewise, significantly elevated pulmonary steady-state protein levels of IL-10, IFN-γ, and bioactive TGF-β were found in TB patients versus those in OLD patients and VOL. These data suggest that the combined production of the immunosuppressants IL-10 and TGF-β, as well as coexpression of TGF-β RI and RII (required for cellular response to TGF-β), may act to down-modulate host anti-Mycobacterium tuberculosis immunity and thereby allow uncontrolled bacterial replication and overt disease. Delineating the underlying mechanisms of M. tuberculosis-triggered expression of these immune elements may provide a molecular-level understanding of TB immunopathogenesis