RESUMO
BACKGROUND: Virulent Mycobacterium leprae interfere with host defense mechanisms such as cytokine activation and apoptosis. The mitochondrial pathway of apoptosis is regulated by the Bcl-2 family of proteins. Expression of Fas ligand and apoptotic proteins is found in leprosy lesions and M. leprae has been shown to activate pro-apoptotic Bcl-2 genes, Bak and Bax. However, the mechanism by which M. leprae modulates apoptosis is as yet unclear. We investigated expression of apoptotic genes in THP-1 monocytes in response to infection by M. leprae and non-pathogenic M. bovis BCG. RESULTS: M. leprae did not induce apoptosis in THP-1 cells, while BCG induced a significant loss of cell viability by 18 h post-infection at both (multiplicity of infection) MOI-10 and 20, with an increase by 48 h. BCG-induced cell death was accompanied by characteristic apoptotic DNA laddering in cells. Non-viable BCG had a limited effect on host cell death suggesting that BCG-induced apoptosis was a function of mycobacterial viability. M. leprae also activated lower levels of TNF-alpha secretion and TNF-alpha mRNA expression than BCG. Mycobacterium-induced activation of apoptotic gene expression was determined over a time course of infection. M. leprae reduced Bad and Bak mRNA expression by 18 h post-stimulation, with a further decrease at 48 h. Outcome of cell viability is determined by the ratio between pro- and anti-apoptotic proteins present in the cell. M. leprae infection resulted in downregulation of gene expression ratios, Bad/Bcl-2 mRNA by 39% and Bak/Bcl-2 mRNA by 23%. In contrast, live BCG increased Bad/Bcl-2 mRNA (29 %) but had a negligible effect on Bak/Bcl-2 mRNA. Heat killed BCG induced only a negligible (1-4 %) change in mRNA expression of either Bak/Bcl-2 or Bad/Bcl-2. Additionally, M. leprae upregulated the expression of anti-apoptotic gene Mcl-1 while, BCG downregulated Mcl-1 mRNA. CONCLUSION: This study proposes an association between mycobacterium-induced apoptosis in THP-1 cells and the regulation of Bcl-2 family of proteins. M. leprae restricts apoptosis in THP-1 cells by downregulation of Bad and Bak and upregulation of Mcl-1 mRNA expression.
Assuntos
Apoptose/fisiologia , Mycobacterium leprae/fisiologia , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína de Morte Celular Associada a bcl/genética , Apoptose/genética , Linhagem Celular , Sobrevivência Celular/genética , Regulação para Baixo/genética , Eletroforese em Gel de Ágar/métodos , Regulação da Expressão Gênica/genética , Humanos , Microscopia de Fluorescência/métodos , Mycobacterium bovis/patogenicidade , Mycobacterium bovis/fisiologia , Mycobacterium leprae/patogenicidade , Proteína de Sequência 1 de Leucemia de Células Mieloides , Fatores de Tempo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/genética , VirulênciaRESUMO
Se ha desarrollado un ensayo colorimétrico de hibridación en microplacas para simplificar la detección de Mycobacterium leprae en muestras clínicas. La técnica detecta los productos amplificados por un ensayo muy sensible RT-PCR con diana sobre una secuencia especie-específica del rRNA 16S bacteriano. El test detectó hasta 10 bacilos aislados de los nódulos linfáticos de ratones desnudos infectados o biopsias cutáneas humanas. La sensibilidad para el diagnóstico en nuestras clínicas se evaluó en 58 biopsias cutáneas de 58 pacientes de lepra sin tratar. El ensayo detectó amplificados RT-PCR de M. leprae en el 100 por ciento de las biopsias de pacientes con lepra multibacilar y el 80 por ciento de biopsias de pacientes paucibacilares, con una sensibilidad total de 91.3 por ciento. El test resultó ser muy específico ya que no se detectaron amplificaciones en las biopsias de pacientes normales o afectados de otras enfermedades distintas a la lepra. La variante colorimétrica es más rápido, sensible y simplifica la detección de los amplificados RT-PCR comparado con el análisis por Southern blot. Puede ser útil para el diagnóstico de los casos difíciles de lepra y como el RNA se degrada rápidamente después de la inactivación celular siendo útil para la evaluación de la respuesta al tratamiento y distinción de recidivas de reacción (AU)
Assuntos
Adulto , Feminino , Masculino , Pessoa de Meia-Idade , Humanos , Colorimetria/métodos , Mycobacterium leprae/isolamento & purificação , Mycobacterium leprae/patogenicidade , Biópsia/métodos , Sensibilidade e Especificidade , RNA Complementar , Ribonucleases/isolamento & purificação , Ribonucleases , Eletroforese em Gel de Ágar/métodos , Southern Blotting/métodos , Reação em Cadeia da Polimerase/métodos , Colorimetria/classificação , Colorimetria/tendências , Meios de Cultura/isolamento & purificação , Rifampina , OfloxacinoRESUMO
The ligand "Sepharose-IDA-Cu(II)" was entrapped into an agarose gel used for affinity electrophoresis. The binding of three closely related proteins, namely alpha-chymotrypsinogen A, alpha-chymotrypsin, and alpha-chymotrypsin inactivated with diisopropyl fluorophosphate (DIFP) to the affinity gel, was investigated. When the protein having affinity for the ligand was run in the presence of small amounts of the ligand, the retention of the protein by the ligand caused "tailing" of the sample. This pattern was changed in the presence of increasing amounts of the ligand, leading to a "rocket" shape due to the stronger binding of the protein to the chelated metal ligand entrapped in the gel. The degree of retardation in the gel with the ligand is an expression of the affinity between the protein and the ligand. The migration distance of alpha-chymotrypsin and alpha-chymotrypsin treated with DIFP at a given concentration of the ligand is linearly related to the protein amount deposited on the gel. The dissociation constant for the tested proteins were calculated from the Bøg-Hansen-Takeo plot. The difference in the affinity strength of these structurally related proteins towards the ligand suggests the involvement of the surface topography of histidine residues on their binding to the ligand.