[Novel type of antimicrobial mechanism in host macrophages against mycobacterial infections].

Macrophages (M(phi)s) play a central role as anti-microbial effector cells in the expression of host resistance to mycobacterial infections. With respect to antimicrobial effector molecules of host M(phi) against mycobacterial pathogens, recent studies suggest the possibility that the reactive nitrogen intermediates (RNI)--and reactive oxygen intermediates-independent antimycobacterial mechanism(s) may be crucial for the antimycobacterial function of host M(phi). In this context, we previously found that free fatty acids (FFAs) such as arachidonic acid (AA) and linolenic acid exhibited potent antimicrobial activity against mycobacterial organisms, including Mycobacterium tuberculosis (MTB) and Mycobacterium avium complex (MAC). In addition, FFAs in combination with RNI played critical roles in manifestation of the activity of M(phi) against mycobacterial organisms. Moreover, our recent studies have shown the following findings. First, anti-MTB activity of IFN-gamma-activated M(phi)s was specifically blocked by arachidonyl trifluoromethylketone (aTFMK), an inhibitor of cytosolic phospholipase A2 (cPLA2). Second, ATP potentiated the anti-MAC bactericidal activity of M(phi)s cultivated in the presence of clarithromycin and rifamycin. This effect of ATP was closely related to intracellular Ca2+ mobilization and was specifically blocked by aTFMK. Third, intramacrophage translocation of membranous AA molecules to MAC-containing phagosomes was also specifically blocked by aTFMK. In the confocal microscopic observation of MAC-infected M(phi)s, ATP enhanced the intracellular translocation of cPLA2 into MAC-containing phagosomes. These findings suggest that FFAs (especially AA) produced by the enzymatic action of cPLA2 play important roles as antimycobacterial effectors in the expression of M(phi) antimicrobial activity against mycobacterial pathogens.

Limited ATP generation in cells of mycobacterium leprae thai-53 strain in enriched kirchner liquid medium containing adenosine

The ATP generation in cells of Mycobacterium leprae Thai-53 strain takes place in vitro when the cells are cultivated in Kirchner liquid medium, pH 7.0, enriched with egg-yolk solution, pyruvate, transferrin, and adenosine at 30 degrees C. Among the supplements, adenosine was key and critical for the ATP generation. The optimal concentration of adenosine was 50 micrograms/ml of the medium. ATP generation, however, was limited; the rates of increase in ATP content extracted from the cells were approximately two- to threefold compared to that of the starting samples, and the increase reached a maximum at 4 or 6 weeks after incubation. No significant ATP generation in M. leprae cells was demonstrated in medium at pH 6.2 or pH 6.6, in the original Kirchner medium with or without adenosine, or when cultured at 37 degrees C, or when containing an antileprosy drug. No detectable increase in the number of M. leprae cells was observed with the increase in intracellular ATP content and DNA replication. No effect was seen with renewal of the cultured medium by freshly prepared medium at 6 weeks' cultivation on the progressive ATP generation in M. leprae.