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1.
Molecules ; 26(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34361751

RESUMO

Species of Mycobacteriaceae cause disease in animals and humans, including tuberculosis and leprosy. Individuals infected with organisms in the Mycobacterium tuberculosis complex (MTBC) or non-tuberculous mycobacteria (NTM) may present identical symptoms, however the treatment for each can be different. Although the NTM infection is considered less vital due to the chronicity of the disease and the infrequency of occurrence in healthy populations, diagnosis and differentiation among Mycobacterium species currently require culture isolation, which can take several weeks. The use of volatile organic compounds (VOCs) is a promising approach for species identification and in recent years has shown promise for use in the rapid analysis of both in vitro cultures as well as ex vivo diagnosis using breath or sputum. The aim of this contribution is to analyze VOCs in the culture headspace of seven different species of mycobacteria and to define the volatilome profiles that are discriminant for each species. For the pre-concentration of VOCs, solid-phase micro-extraction (SPME) was employed and samples were subsequently analyzed using gas chromatography-quadrupole mass spectrometry (GC-qMS). A machine learning approach was applied for the selection of the 13 discriminatory features, which might represent clinically translatable bacterial biomarkers.


Assuntos
Metaboloma , Mycobacterium abscessus/química , Complexo Mycobacterium avium/química , Mycobacterium avium/química , Mycobacterium bovis/química , Mycobacterium/química , Compostos Orgânicos Voláteis/isolamento & purificação , Biomarcadores/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Aprendizado de Máquina/estatística & dados numéricos , Mycobacterium/metabolismo , Mycobacterium abscessus/metabolismo , Mycobacterium avium/metabolismo , Complexo Mycobacterium avium/metabolismo , Mycobacterium bovis/metabolismo , Análise de Componente Principal , Microextração em Fase Sólida , Compostos Orgânicos Voláteis/classificação , Compostos Orgânicos Voláteis/metabolismo
2.
Vaccine ; 38(48): 7629-7637, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33071000

RESUMO

This work demonstrates the presence of immune regulatory cells in the cervical lymph nodes draining Bacillus Calmette-Guérin (BCG) vaccinated site on the dorsum of the ear in guinea pigs. It is shown that whole cervical lymph node cells did not proliferate in vitro in the presence of soluble mycobacterial antigens (PPD or leprosin) despite being responsive to whole mycobacteria. Besides, T cells from these lymph nodes separated as a non-adherent fraction on a nylon wool column, proliferated to PPD in the presence of autologous antigen presenting cells. Interestingly, addition of as low as 20% nylon wool adherent cells to these, sharply decreased the proliferation by 83%. Looking into what cells in the adherent fraction suppressed the proliferation, it was found that neither the T cell nor the macrophage enriched cell fractions of this population individually showed suppressive effect, indicating that their co-presence was necessary for the suppression. Since BCG induced granulomas resolve much faster than granulomas induced by other mycobacteria such as Mycobacterium leprae the present experimental findings add to the existing evidence that intradermal BCG vaccination influences subsequent immune responses in the host and may further stress upon its beneficial role seen in Covid-19 patients.


Assuntos
Antígenos de Bactérias/farmacologia , Vacina BCG/farmacologia , Granuloma/imunologia , Linfonodos/imunologia , Linfócitos T/imunologia , Tuberculina/farmacologia , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/microbiologia , COVID-19 , Adesão Celular , Proliferação de Células , Infecções por Coronavirus/prevenção & controle , Orelha , Feminino , Granuloma/microbiologia , Cobaias , Humanos , Injeções Intradérmicas , Linfonodos/microbiologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Mycobacterium bovis/imunologia , Mycobacterium leprae/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Remissão Espontânea , Linfócitos T/classificação , Linfócitos T/efeitos dos fármacos , Linfócitos T/microbiologia
3.
Diagn Cytopathol ; 48(4): 371-375, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31858747

RESUMO

Bacille Calmette-Guerin (BCG) vaccine is administered worldwide to neonates and considered safe. Serious complications like disseminated BCGosis are extremely rare occurrences (<1 per million vaccinations). A 6 months male was brought to paediatric outpatient department with fever and swelling over the dorsum of the left hand for 5 days. On examination, he was febrile and had hepatosplenomegaly. X-ray of the hand showed lytic lesions in the first and second metacarpals. Provisional clinical diagnosis included Langerhans cell histiocytosis, congenital syphilis, and haematological malignancy. Fine Needle Aspiration Cytology (FNAC) was done from the swelling and showed diffuse sheets of histiocytes with both intracellular and extracellular rod-shaped unstained structures along with inflammatory cells. These ghost images stained positive with ZN stain. A cytological diagnosis of atypical mycobacteria vs leprosy was made. Child was revisited and found to have an active BCG scar. Further investigations showed low serum IgM and positive AFB culture. These bacilli were confirmed by GenoType MTBDR plus test as Mycobacterium bovis. Despite Antitubercular therapy, the patient succumbed to death. This case highlights the variable clinical presentation of BCGosis. Its occurrence may unmask any underlying immunodeficiency. If unfamiliar with the above cytological features and in absence of routinely performed special stains, the cytopathologist may miss these notorious organisms and treat such cases like suppurative lesions. To conclude, an early and definitive diagnosis of BCGosis can be established on FNAC which would ensure timely management and better outcome in this highly lethal entity.


Assuntos
Antituberculosos/administração & dosagem , Vacina BCG/efeitos adversos , Mycobacterium bovis , Tuberculose , Citodiagnóstico , Evolução Fatal , Humanos , Lactente , Masculino , Tuberculose/diagnóstico , Tuberculose/tratamento farmacológico
4.
Virulence ; 10(1): 1026-1033, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31782338

RESUMO

In this study, we characterized the role of Rv2617c in the virulence of Mycobacterium tuberculosis. Rv2617c is a protein of unknown function unique to M. tuberculosis complex (MTC) and Mycobacterium leprae. In vitro, this protein interacts with the virulence factor P36 (also named Erp) and KdpF, a protein linked to nitrosative stress. Here, we showed that knockout of the Rv2617c gene in M. tuberculosis CDC1551 reduced the replication of the pathogen in a mouse model of infection and favored the trafficking of mycobacteria to phagolysosomes. We also demonstrated that Rv2617c and P36 are required for resistance to in vitro hydrogen peroxide treatment in M. tuberculosis and Mycobacterium bovis, respectively. These findings indicate Rv2617c and P36 act in concert to prevent bacterial damage upon oxidative stress.


Assuntos
Proteínas de Bactérias/genética , Mycobacterium bovis/genética , Mycobacterium bovis/patogenicidade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Estresse Oxidativo , Fatores de Virulência/genética , Animais , Pulmão/microbiologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Virulência
5.
Front Immunol ; 9: 629, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29670618

RESUMO

Background: Notwithstanding its beneficial immunoprophylactic outcomes regarding leprosy and childhood TB, BCG vaccination may cause adverse events, particularly of the skin. However, this local hyper-immune reactivity cannot be predicted before vaccination, nor is its association with protection against leprosy known. In this study we investigated the occurrence of adverse events after BCG (re)vaccination in contacts of leprosy patients and analyzed whether the concomitant systemic anti-mycobacterial immunity was associated with these skin manifestations. Methods: Within a randomized controlled BCG vaccination trial in Bangladesh, 14,828 contacts of newly diagnosed leprosy patients received BCG vaccination between 2012 and 2017 and were examined for adverse events 8 to 12 weeks post-vaccination. From a selection of vaccinated contacts, venous blood was obtained at follow-up examination and stimulated with Mycobacterium leprae (M. leprae) antigens in overnight whole-blood assays (WBA). M. leprae phenolic glycolipid-I-specific antibodies and 32 cytokines were determined in WBAs of 13 individuals with and 13 individuals without adverse events after vaccination. Results: Out of the 14,828 contacts who received BCG vaccination, 50 (0.34%) presented with adverse events, mainly (80%) consisting of skin ulcers. Based on the presence of BCG scars, 30 of these contacts (60%) had received BCG in this study as a booster vaccination. Similar to the pathological T-cell immunity observed for tuberculoid leprosy patients, contacts with adverse events at the site of BCG vaccination showed elevated IFN-γ levels in response to M. leprae-specific proteins in WBA. However, decreased levels of sCD40L in serum and GRO (CXCL1) in response to M. leprae simultaneously indicated less T-cell regulation in these individuals, potentially causing uncontrolled T-cell immunity damaging the skin. Conclusion: Skin complications after BCG vaccination present surrogate markers for protective immunity against leprosy, but also indicate a higher risk of developing tuberculoid leprosy. Clinical Trial Registration: Netherlands Trial Register: NTR3087.


Assuntos
Hanseníase/imunologia , Mycobacterium bovis/imunologia , Mycobacterium leprae/fisiologia , Úlcera Cutânea/imunologia , Pele/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Anticorpos Antibacterianos/sangue , Bangladesh , Ligante de CD40/sangue , Quimiocina CXCL1/sangue , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Interferon gama/metabolismo , Hanseníase/complicações , Ativação Linfocitária , Masculino , Úlcera Cutânea/etiologia , Vacinação/efeitos adversos , Adulto Jovem
7.
PLoS Negl Trop Dis ; 10(8): e0004881, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27479467

RESUMO

Mycobacterium leprae is the causative agent of leprosy and also known to possess unique features such as inability to proliferate in vitro. Among the cellular components of M. leprae, various glycolipids present on the cell envelope are well characterized and some of them are identified to be pathogenic factors responsible for intracellular survival in host cells, while other intracellular metabolites, assumed to be associated with basic physiological feature, remain largely unknown. In the present study, to elucidate the comprehensive profile of intracellular metabolites, we performed the capillary electrophoresis-mass spectrometry (CE-MS) analysis on M. leprae and compared to that of M. bovis BCG. Interestingly, comparison of these two profiles showed that, in M. leprae, amino acids and their derivatives are significantly accumulated, but most of intermediates related to central carbon metabolism markedly decreased, implying that M. leprae possess unique metabolic features. The present study is the first report demonstrating the unique profiles of M. leprae metabolites and these insights might contribute to understanding undefined metabolism of M. leprae as well as pathogenic characteristics related to the manifestation of the disease.


Assuntos
Aminoácidos/metabolismo , Citoplasma/metabolismo , Hanseníase/microbiologia , Mycobacterium leprae/metabolismo , Animais , Antígenos de Bactérias/metabolismo , Células Cultivadas , Eletroforese Capilar , Glicolipídeos/metabolismo , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mycobacterium bovis/metabolismo
8.
J Infect Dis ; 214(2): 311-20, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27190175

RESUMO

Cytosolic detection of nucleic acids elicits a type I interferon (IFN) response and plays a critical role in host defense against intracellular pathogens. Herein, a global gene expression profile of Mycobacterium leprae-infected primary human Schwann cells identified the genes differentially expressed in the type I IFN pathway. Among them, the gene encoding 2'-5' oligoadenylate synthetase-like (OASL) underwent the greatest upregulation and was also shown to be upregulated in M. leprae-infected human macrophage cell lineages, primary monocytes, and skin lesion specimens from patients with a disseminated form of leprosy. OASL knock down was associated with decreased viability of M. leprae that was concomitant with upregulation of either antimicrobial peptide expression or autophagy levels. Downregulation of MCP-1/CCL2 release was also observed during OASL knock down. M. leprae-mediated OASL expression was dependent on cytosolic DNA sensing mediated by stimulator of IFN genes signaling. The addition of M. leprae DNA enhanced nonpathogenic Mycobacterium bovis bacillus Calmette-Guerin intracellular survival, downregulated antimicrobial peptide expression, and increased MCP-1/CCL2 secretion. Thus, our data uncover a promycobacterial role for OASL during M. leprae infection that directs the host immune response toward a niche that permits survival of the pathogen.


Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Interações Hospedeiro-Patógeno , Proteínas de Membrana/metabolismo , Viabilidade Microbiana , Mycobacterium leprae/fisiologia , Células de Schwann/microbiologia , Células Cultivadas , Células Epiteliais/microbiologia , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Hanseníase/microbiologia , Hanseníase/patologia , Macrófagos/microbiologia , Mycobacterium bovis/fisiologia
9.
J Bacteriol ; 198(15): 2020-8, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27185825

RESUMO

UNLABELLED: Phthiocerol dimycocerosates (PDIM) are a group of cell surface-associated apolar lipids of Mycobacterium tuberculosis and closely related mycobacteria, such as Mycobacterium bovis and Mycobacterium leprae A characteristic methoxy group of these lipids is generated from the methylation of a hydroxyl group of the direct precursors, the phthiotriols. The precursors arise from the reduction of phthiodiolones, the keto intermediates, by a ketoreductase. The putative phthiodiolone ketoreductase (PKR) is encoded by Rv2951c in M. tuberculosis and BCG_2972c in M. bovis BCG, and these open reading frames (ORFs) encode identical amino acid sequences. We investigated the cofactor requirement of the BCG_2972c protein. A comparative analysis based on the crystallographic structures of similar enzymes identified structural elements for binding of coenzyme F420 and hydrophobic phthiodiolones in PKR. Coenzyme F420 is a deazaflavin coenzyme that serves several key functions in pathogenic and nonpathogenic mycobacteria. We found that an M. bovis BCG mutant lacking F420-dependent glucose-6-phosphate dehydrogenase (Fgd), which generates F420H2 (glucose-6-phosphate + F420 → 6-phosphogluconate + F420H2), was devoid of phthiocerols and accumulated phthiodiolones. When the mutant was provided with F420H2, a broken-cell slurry of the mutant converted accumulated phthiodiolones to phthiocerols; F420H2 was generated in situ from F420 and glucose-6-phosphate by the action of Fgd. Thus, the reaction mixture was competent in reducing phthiodiolones to phthiotriols (phthiodiolones + F420H2 → phthiotriols + F420), which were then methylated to phthiocerols. These results established the mycobacterial phthiodiolone ketoreductase as an F420H2-dependent enzyme (fPKR). A phylogenetic analysis of close homologs of fPKR revealed potential F420-dependent lipid-modifying enzymes in a broad range of mycobacteria. IMPORTANCE: Mycobacterium tuberculosis is the causative agent of tuberculosis, and phthiocerol dimycocerosates (PDIM) protect this pathogen from the early innate immune response of an infected host. Thus, the PDIM synthesis system is a potential target for the development of effective treatments for tuberculosis. The current study shows that a PDIM synthesis enzyme is dependent on the coenzyme F420 F420 is universally present in mycobacteria and absent in humans. This finding expands the number of experimentally validated F420-dependent enzymes in M. tuberculosis to six, each of which helps the pathogen to evade killing by the host immune system, and one of which activates an antituberculosis drug, PA-824. This work also has relevance to leprosy, since similar waxy lipids are found in Mycobacterium leprae.


Assuntos
Proteínas de Bactérias/metabolismo , Desidrogenases de Carboidrato/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Lipídeos/biossíntese , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Desidrogenases de Carboidrato/genética , Mycobacterium bovis/enzimologia , Mycobacterium tuberculosis/enzimologia , Filogenia
10.
Int J Tuberc Lung Dis ; 20(6): 848-52, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27155192

RESUMO

BACKGROUND: The nasopharynx is a known gateway for some mycobacterial species such as Mycobacterium bovis and M. leprae. M. tuberculosis can cross lymphoepithelial barriers in vitro, but its ability to colonise the nasopharyngeal mucosa in vivo has not been established. OBJECTIVE: To determine if M. tuberculosis can be transiently detected in nasopharyngeal mucosa of tuberculosis (TB) contacts as a preliminary step in the development of tuberculous infection. DESIGN: Exploratory study conducted among asymptomatic household contacts of pulmonary TB cases. A chest X-ray, QuantiFERON(®) TB-Gold or tuberculin skin test and a bilateral nasopharyngeal swab for Xpert(®) MTB/RIF and mycobacterial culture were performed at baseline and repeated 8-12 weeks later. RESULTS: Eighty-nine contacts were enrolled a median of 9 days after the diagnosis of the index case. At baseline, 29.9% were positive for latent tuberculous infection and one subject (1.1%) had a positive Xpert in the nasopharyngeal swab with a normal chest X-ray, negative QuantiFERON and negative induced sputum. After 12 weeks' follow-up, this subject developed a new cough and upper lobe infiltrates and M. tuberculosis grew in sputum. No other cases of active TB were detected at follow-up. CONCLUSION: The detection of M. tuberculosis DNA in the nasopharyngeal mucosa of contacts is an infrequent event that in this instance preceded the development of pulmonary TB. Its pathogenic role requires further investigation.


Assuntos
DNA Bacteriano/isolamento & purificação , Tuberculose Latente/diagnóstico , Membrana Mucosa/microbiologia , Mycobacterium tuberculosis/isolamento & purificação , Nasofaringe/microbiologia , Tuberculose Pulmonar/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Estudos Transversais , Feminino , Seguimentos , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mycobacterium bovis/isolamento & purificação , Escarro/microbiologia , Teste Tuberculínico , Adulto Jovem
11.
Int Immunol ; 28(9): 435-41, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-26921215

RESUMO

BACKGROUND: Immunological characterization of mycobacterial peptides may help not only in the preparation of a vaccine for leprosy but also in developing in vitro T-cell assays that could perhaps be used as an in vitro correlate for treatment outcome. The main goal of this study was to evaluate the use of Mycobacterium bovis recombinant 32-kDa protein (r32-kDa) antigen-stimulated T-cell assay as a surrogate marker for treatment outcome and monitor vitamin D receptor (VDR)-mediated anti-microbial responses during multidrug therapy (MDT) in leprosy. METHODS: Newly diagnosed tuberculoid and lepromatous leprosy patients were enrolled and followed up during their course of MDT at 6 and 12 months. IFN-γ, IL-10, IL-17 and IL-23 levels in culture supernatants and expression of VDR, TLR2, LL37 and DEFB in r32-kDa-stimulated PBMCs were measured. Controls comprised household contacts (HHCs) and healthy endemic subjects (HCs). RESULTS: Significant differences were observed in the levels of IFN-γ, IL-17, IL-23, VDR and anti-microbial peptides LL37 and DEFB after treatment and when compared with that of HHCs and HCs, respectively. CONCLUSIONS: These findings suggest that responses to r32-kDa antigen reflect an improved immunological and anti-microbial response in leprosy patients during therapy, thereby indicating its potential use as an immune correlate in the treatment of leprosy patients.


Assuntos
Antígenos de Bactérias/farmacologia , Proteínas de Bactérias/farmacologia , Citocinas/imunologia , Hanseníase/imunologia , Mycobacterium bovis , Linfócitos T/imunologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Peptídeos Catiônicos Antimicrobianos , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Catelicidinas/imunologia , Feminino , Seguimentos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Hanseníase/patologia , Masculino , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Linfócitos T/patologia , Receptor 2 Toll-Like/imunologia
12.
s.l; s.n; 2016. 10 p. tab, graf.
Não convencional em Inglês | SES-SP, HANSEN, SESSP-ILSLPROD, SES-SP, SESSP-ILSLACERVO, SES-SP | ID: biblio-1095379

RESUMO

Cytosolic detection of nucleic acids elicits a type I interferon (IFN) response and plays a critical role in host defense against intracellular pathogens. Herein, a global gene expression profile of Mycobacterium leprae-infected primary human Schwann cells identified the genes differentially expressed in the type I IFN pathway. Among them, the gene encoding 2'-5' oligoadenylate synthetase-like (OASL) underwent the greatest upregulation and was also shown to be upregulated in M. leprae-infected human macrophage cell lineages, primary monocytes, and skin lesion specimens from patients with a disseminated form of leprosy. OASL knock down was associated with decreased viability of M. leprae that was concomitant with upregulation of either antimicrobial peptide expression or autophagy levels. Downregulation of MCP-1/CCL2 release was also observed during OASL knock down. M. leprae-mediated OASL expression was dependent on cytosolic DNA sensing mediated by stimulator of IFN genes signaling. The addition of M. leprae DNA enhanced nonpathogenic Mycobacterium bovis bacillus Calmette-Guerin intracellular survival, downregulated antimicrobial peptide expression, and increased MCP-1/CCL2 secretion. Thus, our data uncover a promycobacterial role for OASL during M. leprae infection that directs the host immune response toward a niche that permits survival of the pathogen.


Assuntos
Humanos , Células de Schwann/microbiologia , Células Cultivadas , Perfilação da Expressão Gênica , Células Epiteliais/microbiologia , Viabilidade Microbiana , Interações Hospedeiro-Patógeno , Técnicas de Silenciamento de Genes , Hanseníase/microbiologia , Hanseníase/patologia , Macrófagos/microbiologia , Proteínas de Membrana/metabolismo , Mycobacterium bovis/fisiologia , Mycobacterium leprae/fisiologia
13.
Artigo em Inglês | MEDLINE | ID: mdl-26577191

RESUMO

Control of bovine tuberculosis (bTB) continues to be a problem world-wide because of difficulties in identifying infected animals at all stages of infection. The use of the IFN-γ release assays (IGRA) as an ancillary test with the tuberculin skin tests has improved the ability to identify infected animals. However, infected animals may still be missed. The objective of the present study was to evaluate a rapid flow-cytometric assay based on intracellular cytokine staining as an alternative to the in vitro IFN-γ release assay (IGRA). Antigen-specific cells producing IFN-γ were identified after a 6h stimulation with PPD-B, PPD-A and ESAT-6/CFP-10. Defined groups of animals naturally infected with Mycobacterium bovis (Mbv), animals infected with non-tuberculous mycobacteria (NTM), and uninfected control animals were analysed to evaluate the sensitivity and specificity of the optimized assay. Both antemortem and postmortem diagnostic tests were carried out to verify the status of infection. We show that IFN-γ is induced in T cells from whole blood samples from cattle infected with Mbv 6h post stimulation with PPD-B, PPD-A and ESAT-6/CFP-10, whereas non-infected animals did not respond. Four colour flow cytometric analysis demonstrated responding cells were CD45R0(+)CD69(+)CD4(+) memory T cells. Also, the response to stimulation with ESAT-6/CFP-10 can be used to distinguish between cattle infected with Mbv and cattle exposed to NTM. Although further studies are needed, the results indicate that detection of intracellular IFN-γ may represent an important alternative approach for improved method of detection of cattle secreting IFN-γ below levels of detection in culture medium.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Citometria de Fluxo/métodos , Testes de Liberação de Interferon-gama/métodos , Interferon gama/sangue , Tuberculose Bovina/diagnóstico , Animais , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Bovinos , Memória Imunológica , Lectinas Tipo C/imunologia , Antígenos Comuns de Leucócito/imunologia , Linfocinas , Mycobacterium bovis/imunologia , Peptídeos/imunologia , Sensibilidade e Especificidade , Teste Tuberculínico
14.
Lepr Rev ; 86(2): 180-5, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26502690

RESUMO

Cutaneous complications of Bacillus Calmette-Guerin (BCG) vaccine, especially in the form of generalised disease, are uncommon and mostly occur in immunocompromised individuals. There is a paucity of data on the cutaneous adverse reactions secondary to BCG immunotherapy in leprosy. We report two unique cases of disseminated cutaneous BCG infection following immunotherapy in patients with lepromatous leprosy. To our knowledge, cutaneous BCG infection presenting as widespread lesions after immunotherapy and confirmed by isolation of Mycobacterium bovis by polymerase chain reaction (PCR) has not been described. A high index of suspicion is required when leprosy patients who receive BCG immunotherapy develop new lesions that cannot be classified as either reaction or relapse, and diagnosis may be confirmed on histopathology and PCR.


Assuntos
Vacina BCG/efeitos adversos , Hanseníase Virchowiana/complicações , Mycobacterium bovis , Tuberculose Cutânea/etiologia , Adulto , Humanos , Masculino , Tuberculose Cutânea/complicações , Tuberculose Cutânea/patologia
15.
Infect Immun ; 82(12): 5317-26, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25287928

RESUMO

Mycobacteria, the etiological agents of tuberculosis and leprosy, have coevolved with mammals for millions of years and have numerous ways of suppressing their host's immune response. It has been suggested that mycobacteria may contain genes that reduce the host's ability to elicit CD8(+) T cell responses. We screened 3,290 mutant Mycobacterium bovis bacillus Calmette Guerin (BCG) strains to identify genes that decrease major histocompatibility complex (MHC) class I presentation of mycobacterium-encoded epitope peptides. Through our analysis, we identified 16 mutant BCG strains that generated increased transgene product-specific CD8(+) T cell responses. The genes disrupted in these mutant strains had disparate predicted functions. Reconstruction of strains via targeted deletion of genes identified in the screen recapitulated the enhanced immunogenicity phenotype of the original mutant strains. When we introduced the simian immunodeficiency virus (SIV) gag gene into several of these novel BCG strains, we observed enhanced SIV Gag-specific CD8(+) T cell responses in vivo. This study demonstrates that mycobacteria carry numerous genes that act to dampen CD8(+) T cell responses and suggests that genetic modification of these genes may generate a novel group of recombinant BCG strains capable of serving as more effective and immunogenic vaccine vectors.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Deleção de Genes , Mycobacterium bovis/genética , Mycobacterium bovis/imunologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Animais , Tolerância Imunológica , Camundongos Endogâmicos C57BL , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia
16.
Infect Immun ; 82(9): 3900-9, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25001602

RESUMO

Leprosy remains a major global health problem and typically occurs in regions in which tuberculosis is endemic. Vaccines are needed that protect against both infections and do so better than the suboptimal Mycobacterium bovis BCG vaccine. Here, we evaluated rBCG30, a vaccine previously demonstrated to induce protection superior to that of BCG against Mycobacterium tuberculosis and Mycobacterium bovis challenge in animal models, for efficacy against Mycobacterium leprae challenge in a murine model of leprosy. rBCG30 overexpresses the M. tuberculosis 30-kDa major secretory protein antigen 85B, which is 85% homologous with the M. leprae homolog (r30ML). Mice were sham immunized or immunized intradermally with BCG or rBCG30 and challenged 2.5 months later by injection of viable M. leprae into each hind footpad. After 7 months, vaccine efficacy was assessed by enumerating the M. leprae bacteria per footpad. Both BCG and rBCG30 induced significant protection against M. leprae challenge. In the one experiment in which a comparison between BCG and rBCG30 was feasible, rBCG30 induced significantly greater protection than did BCG. Immunization of mice with purified M. tuberculosis or M. leprae antigen 85B also induced protection against M. leprae challenge but less so than BCG or rBCG30. Notably, boosting rBCG30 with M. tuberculosis antigen 85B significantly enhanced r30ML-specific immune responses, substantially more so than boosting BCG, and significantly augmented protection against M. leprae challenge. Thus, rBCG30, a vaccine that induces improved protection against M. tuberculosis, induces cross-protection against M. leprae that is comparable or potentially superior to that induced by BCG, and boosting rBCG30 with antigen 85B further enhances immune responses and protective efficacy.


Assuntos
Aciltransferases/imunologia , Antígenos de Bactérias/imunologia , Vacina BCG/imunologia , Proteínas de Bactérias/imunologia , Proteção Cruzada/imunologia , Mycobacterium leprae/imunologia , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Feminino , Imunização/métodos , Hanseníase/imunologia , Hanseníase/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovis/imunologia , Tuberculose/imunologia , Tuberculose/prevenção & controle , Vacinação/métodos
17.
Bioorg Med Chem ; 22(9): 2816-24, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24690527

RESUMO

The flavin-dependent thymidylate synthase X (ThyX), rare in eukaryotes and completely absent in humans, is crucial in the metabolism of thymidine (a DNA precursor) in many microorganisms including several human pathogens. Conserved in mycobacteria, including Mycobacterium leprae, and Mycobacterium tuberculosis, it represents a prospective anti-mycobacterial therapeutic target. In a M. tuberculosis ThyX-enzyme inhibition assay, N-(3-(5-(2'-deoxyuridine-5'-phosphate))prop-2-ynyl)octanamide was reported to be the most potent and selective 5-substituted 2'-deoxyuridine monophosphate analogue. In this study, we masked the two charges at the phosphate moiety of this compound using our ProTide technology in order to increase its lipophilicity and then allow permeation through the complex mycobacterial cell wall. A series of N-(3-(5-(2'-deoxyuridine))prop-2-ynyl)octanamide phosphoroamidates were chemically synthesized and their biological activity as potential anti-tuberculars was evaluated. In addition to mycobacteria, several DNA viruses depend on ThyX for their DNA biosynthesis, thus these prodrugs were also screened for their antiviral properties.


Assuntos
Amidas/química , Antituberculosos/química , Antivirais/química , Desoxiuridina/química , Antituberculosos/síntese química , Antituberculosos/farmacologia , Antivirais/síntese química , Antivirais/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Linhagem Celular , Herpesvirus Humano 3/efeitos dos fármacos , Herpesvirus Humano 3/enzimologia , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/enzimologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Simplexvirus/efeitos dos fármacos , Simplexvirus/enzimologia , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/metabolismo
18.
RBM rev. bras. med ; 70(10)out. 2013.
Artigo em Português | LILACS | ID: lil-704890

RESUMO

Cytokines play a key role in the regulation of the immune response against infectious diseases. In leprosy, the polymorphisms of pro-inflammatory cytokine genes may contribute to host susceptibility to Mycobacterium leprae. The objective of this study was to investigate the association between cytokine gene polymorphisms and BCG protection against leprosy in Brazilian leprosy cases and controls. DNA samples were obtained from 46 patients with leprosy and 83 healthy controls (not leprosy contacts). All genotyping (TNF alpha, IFN gamma, IL-6, IL-10 and TGF beta) measurements were taken using sequence-specific primers (SSP) - PCR. When compared to the healthy controls, no significant associations were observed between the cytokine gene polymorphisms studied and their susceptibility to leprosy. The most frequent genotypes in this population were TNF alpha ?G? allele and G/G genotype at position -308, ?A? allele of IFN gamma at position +874, G allele in the IL-6 at position -174, G allele (codon 25) and T/C-G/G genotype in TGF beta, and ?A? allele in IL-10 at position -1082. For those individuals that had a BCG scar, the TGF beta and IFN gamma genotype polymorphisms did not show difference among leprosy patients compared to healthy controls. Polymorphisms of the cytokine genes studied were not associated with an increased occurrence of leprosy.


Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Citocinas , Hanseníase , Mycobacterium bovis
19.
PLoS One ; 8(6): e64748, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23798993

RESUMO

Herein, we performed microarray experiments in Schwann cells infected with live M. leprae and identified novel differentially expressed genes (DEG) in M. leprae infected cells. Also, we selected candidate genes associated or implicated with leprosy in genetic studies and biological experiments. Forty-seven genes were selected for validation in two independent types of samples by multiplex qPCR. First, an in vitro model using THP-1 cells was infected with live Mycobacterium leprae and M. bovis bacillus Calmette-Guérin (BCG). In a second situation, mRNA obtained from nerve biopsies from patients with leprosy or other peripheral neuropathies was tested. We detected DEGs that discriminate M. bovis BCG from M. leprae infection. Specific signatures of susceptible responses after M. leprae infection when compared to BCG lead to repression of genes, including CCL2, CCL3, IL8 and SOD2. The same 47-gene set was screened in nerve biopsies, which corroborated the down-regulation of CCL2 and CCL3 in leprosy, but also evidenced the down-regulation of genes involved in mitochondrial metabolism, and the up-regulation of genes involved in lipid metabolism and ubiquitination. Finally, a gene expression signature from DEG was identified in patients confirmed of having leprosy. A classification tree was able to ascertain 80% of the cases as leprosy or non-leprous peripheral neuropathy based on the expression of only LDLR and CCL4. A general immune and mitochondrial hypo-responsive state occurs in response to M. leprae infection. Also, the most important genes and pathways have been highlighted providing new tools for early diagnosis and treatment of leprosy.


Assuntos
Quimiocinas/metabolismo , Hanseníase/metabolismo , Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Transcriptoma , Células Cultivadas , Quimiocinas/genética , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno , Humanos , Hanseníase/imunologia , Hanseníase/microbiologia , Masculino , Mitocôndrias/microbiologia , Mycobacterium bovis/imunologia , Mycobacterium leprae/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Nervos Periféricos/metabolismo , Células de Schwann/imunologia , Células de Schwann/metabolismo , Células de Schwann/microbiologia
20.
PLoS Negl Trop Dis ; 7(1): e2015, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23350010

RESUMO

Leprosy is a persistent infectious disease caused by Mycobacterium leprae that still affects over 200,000 new patients annually. The host genetic background is an important risk factor for leprosy susceptibility and the PARK2 gene is a replicated leprosy susceptibility candidate gene. The protein product of PARK2, Parkin, is an E3 ubiquitin ligase that is involved in the development of various forms of Parkinsonism. The human macrophage is both a natural host cell of M. leprae as well as a primary mediator of natural immune defenses, in part by secreting important pro-inflammatory cytokines and chemokines. Here, we report that down-regulation of Parkin in THP-1 macrophages, human monocyte-derived macrophages and human Schwann cells resulted in a consistent and specific decrease in interleukin-6 (IL-6) and monocyte chemoattractant protein 1 (MCP-1/CCL2) production in response to mycobacteria or LPS. Interestingly, production of IL-6 at 6 hours by THP-1 cells stimulated with live M. leprae and M. bovis BCG was dependent on pretreatment with 1,25-dihydroxyvitamin D(3) (VD). Parkin knockdown in VD-treated cells blocked IL-6 induction by mycobacteria. However, IκB-α phosphorylation and levels of IκB-ξ, a nuclear protein required for IL-6 expression, were not affected by Parkin silencing. Phosphorylation of MAPK ERK1/2 and p38 was unaffected by Parkin silencing while JNK activation was promoted but did not explain the altered cytokine production. In a final set of experiments we found that genetic risk factors of leprosy located in the PARK2 promoter region were significantly correlated with M. leprae sonicate triggered CCL2 and IL6 transcript levels in whole blood assays. These results associated genetically controlled changes in the production of MCP-1/CCL2 and IL-6 with known leprosy susceptibility factors.


Assuntos
Quimiocina CCL2/biossíntese , Regulação da Expressão Gênica , Interleucina-6/biossíntese , Macrófagos/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Células Cultivadas , Feminino , Humanos , Lipopolissacarídeos/imunologia , Masculino , Mycobacterium bovis/imunologia , Mycobacterium leprae/imunologia , Células de Schwann/imunologia , Transdução de Sinais
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