ABSTRACT
Abstract Mycobacterium tuberculosis (MTB) adopts a special survival strategy to overcome the killing mechanism(s) of host immune system. Amongst the many known factors, small heat shock protein 16.3 (sHSP16.3) of MTB encoded by gene hspX has been reported to be critical for the survival of MTB. In the present study, the effect of recombinant murine interferon-gamma (rmIFN-γ) and recombinant murine interleukin-10 (rmIL-10) on the expression of gene hspX of MTB in murine macrophage RAW264.7 has been investigated. By real-time RT-PCR, it was observed that three increasing concentrations (5, 25 and 50 ng/ml) of rmIFN-γ significantly up-regulated the expression of hspX whereas similar concentrations of rmIL-10 (5, 25 and 50 ng/ml) significantly down-regulated the hspX expression. This effect was not only dependent on the concentration of the stimulus but this was time-dependent as well. A contrasting pattern of hspX expression was observed against combinations of two different concentrations of rmIFN-γ and rmIL-10. The study results suggest that rIL-10 mediated down-regulation of hspX expression, in the presence of low concentration of rIFN-γ, could be used as an important strategy to decrease the dormancy of MTB in its host and thus making MTB susceptible to the standard anti-mycobacterial therapy used for treating tuberculosis. However, as these are only preliminary results in the murine cell line model, this hypothesis needs to be first validated in human cell lines and subsequently in animal models mimicking the latent infection using clinical isolates of MTB before considering the development of modified regimens for humans.
Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial/physiology , Interferon-gamma/pharmacology , Interleukin-10/pharmacology , Macrophages/microbiology , Mycobacterium tuberculosis/genetics , Antigens, Bacterial/metabolism , Time Factors , Bacterial Proteins/genetics , Recombinant Proteins/pharmacology , Down-Regulation/drug effects , Dose-Response Relationship, Drug , Antigens, Bacterial/geneticsABSTRACT
The rotavirus nonstructural glycoprotein, NSP4, has been identified as the first viral enterotoxin capable of inducing diarrhea. To investigate the biological function of NSP4 in the inflammatory process, a cDNA from human rotavirus (Wa strain) RNA segment 10 was amplified by RT-PCR, cloned into TA vector, and subsequently subcloned into pET23b expression plasmid. The expression of NSP4 protein was determined by SDS-PAGE and Western blotting, then, the protein was purified by affinity chromatography on Ni-NTA-agarose column. The inflammatory effects of NSP4, namely, production of nitric oxide (NO), pro-inflammatory cytokines (IL-1β, IL-6, IL-10, and TNF-α), and prostaglandin E2 (PGE₂), was evaluated using NSP4-stimulated RAW 264.7 murine macrophages and compared with those observed after stimulation with lipopolysaccharide (LPS). The levels of IL-1β, IL-6, and TNF-α were significantly increased, and those of NO and PGE₂ also increased in NSP4-stimulated RAW 264.7 cells. These findings indicate that NSP4 plays an important role in the inflammatory response observed during rotavirus infection.
Subject(s)
Humans , Blotting, Western , Chromatography, Affinity , Clone Cells , Cytokines , Diarrhea , Dinoprostone , DNA, Complementary , Electrophoresis, Polyacrylamide Gel , Enterotoxins , Glycoproteins , Inflammation , Interleukin-10 , Interleukin-6 , Macrophages , Nitric Oxide , Plasmids , RNA , Rotavirus Infections , RotavirusABSTRACT
Baerobic, spore forming, and rod-shaped bacterium. Anthrax spores are introduced into macrophage by phagocytosis and multiply after germination. The anthrax spores infected in macrophage produce lethal toxin eventually caused cell death. In this study, we analyzed apoptosis and cytokine TNF-alpha and IL-12 secretion after the infection of spores of B. anthracis Sterne in the murine macrophage RAW264.7 cells and in the primary human macrophages. In murine macrophage RAW264.7 cells infected by spore of B. anthracis Sterne, the cells were markedly changed in secretion of TNF-alpha (482~6,213 pg/ml) by lethal toxin, and induced apoptosis. In case of RAW264.7 cells infected by formalin-inactivated spores of B. anthracis, the cells were not able to produce lethal toxin, which released lower level concentration of TNF-alpha (7.7~97.2 pg/ml), and rarely induced apoptosis. When primary human macrophage cells infected with spores of B. anthracis Sterne, they secreted TNF-alpha (5~16 pg/ml), and induced apoptosis about 1% of total cells. We presented that inducing apoptosis by spores of B. anthracis Sterne capable of expressing lethal toxin is related with the secretion of TNF-alpha in murine macrophage RAW264.7 cells. These studies revealed that human and murine macrophages has affected differently by anthrax lethal toxin produced by spores of B. anthracis Sterne.
Subject(s)
Humans , Anthrax , Apoptosis , Bacillus anthracis , Bacillus , Cell Death , Germination , Interleukin-12 , Macrophages , Phagocytosis , Spores , Tumor Necrosis Factor-alphaABSTRACT
No abstract available.