Tatsuya Akaki

Effector molecules in expression of the antimicrobial activity of macrophages against Mycobacterium avium complex: roles of reactive nitrogen intermediates, reactive oxygen intermediates, and free fatty acids.

We studied microbicidal activities of reactive nitrogen intermediates (RNI), free fatty acids (FFA), and reactive oxygen intermediates (ROI) against Mycobacterium aviumcomplex (MAC) and the mode of macrophage (mΦ) production of these effectors. (1) Intracellular growth of MAC in murine peritoneal mΦs was accelerated by scavengers for ROI or RNI and inhibitors of nitric oxide synthase or phospholipase A2, indicating roles of ROI, RNI, and FFA in mΦ anti‐MAC functions. (2) Acidified NaNO2‐derived RNI, FFA (linolenic and arachidonic acids), and the H2O2‐mediated halogenation system exhibited a significant anti‐MAC bactericidal activity. The combination of RNI with FFA showed a synergistic effect However, the H2O2‐halogenation system in combination with either RNI or FFA showed an antagonism. When Listeria monocytogenes(Lm) was used as a target organism, the combinations of RNI + FFA and RNI + H2O2‐halogenation gave a synergistic effect, whereas FFA + H2O2‐halogenation showed an antagonism in exerting bactericidal activity. In addition, when ROI generated by the xanthine oxidase‐acetaldehyde system was combined with RNI, anti‐Lm but not anti‐MAC activity was potentiated. (3) ROI production by murine peritoneal mΦs was observed immediately after contact with MAC organisms (MAC stimulation) and ceased within 2 h. FFA release was seen 1‐24 h after MAC stimulation. RNI production was initiated from 3 h and increased during the first 36 h and continued at least for 4 days. These findings suggest that RNI and FFA rather than ROI are important effectors of anti‐MAC functions of mΦs, and the collaborating action of RNI with FFA temporarily participates in mΦ‐mediated killing of MAC in the relatively early phase after MAC stimulation. J. Leukoc. Biol. 62: 795–804; 1997.

Comparative antimicrobial activities of the newly synthesized quinolone WQ-3034, levofloxacin, sparfloxacin, and ciprofloxacin against Mycobacterium tuberculosis and Mycobacterium avium complex.

ABSTRACT WQ-3034 is a newly synthesized acidic fluoroquinolone. We assessed its in vitro activity against Mycobacterium tuberculosisand M. avium complex using levofloxacin (LVFX), ciprofloxacin (CPFX), sparfloxacin (SPFX), and KRM-1648 (KRM) as reference drugs. The MICs of these agents were determined by the agar dilution method with 7H11 medium. The MICs at which 50 and 90% of the test strains were inhibited (MIC50s, and MIC90s, respectively) for the test quinolones for rifampin (RMP)-susceptible M. tuberculosis strains were in the order SPFX < LVFX ≦ WQ-3034 ≦ CPFX, while those for RMP-resistant M. tuberculosis strains were in the order SPFX ≦ WQ-3034 ≦ LVFX < CPFX. The MICs of KRM for RMP-susceptible M. tuberculosis were much lower than those of the test quinolones, while the MIC90 of KRM for RMP-resistant M. tuberculosis strains was higher than those of the quinolones. The MIC50s and MIC90s of the test drugs for M. avium were in the order KRM < SPFX < CPFX ≦ WQ-3034 ≦ LVFX, while those forM. intracellulare were in the order KRM < SPFX < WQ-3034 ≒ LVFX ≦ CPFX. Next, we compared the antimicrobial activities of the test drugs against M. tuberculosisorganisms residing in cells of the Mono Mac 6 macrophage (Mφ)-like cell line (MM6-Mφs) and of the A-549 type II alveolar cell line (A-549 cells). When drugs were added at the concentration that achieves the maximum concentration in blood, progressive killing or inhibition of the M. tuberculosis organisms residing in MM6-Mφs and A-549 cells was observed in the order KRM > SPFX ≧ LVFX > WQ-3034 > CPFX. The efficacies of all quinolones against intracellular M. tuberculosis organisms were significantly lower in A-549 cells than in MM6-Mφs. WQ-3034 at the MIC caused more marked growth inhibition of intramacrophage M. tuberculosis than did LVFX. These findings indicate that the in vitro anti-M. tuberculosis activity of WQ-3034 is greater than that of CPFX and is comparable to that of LVFX.

Antimicrobial activities of levofloxacin, clarithromycin, and KRM-1648 against Mycobacterium tuberculosis and Mycobacterium avium complex replicating within Mono Mac 6 human macrophage and A-549 type II alveolar cell lines

The antimicrobial activities of levofloxacin, clarithromycin and KRM-1648 against Mycobacterium tuberculosis (MTB) and Mycobacterium avium complex (MAC) residing in Mono Mac 6 human macrophage-like cells (MM6-Mphis) and A-549 human type II alveolar epithelial cells (A-549 cells) were studied. We measured the antimicrobial activity of test drugs in terms of effects on the behaviour of intracellular organisms during a 7-day cultivation of MTB- or MAC-infected cells in the medium containing the drugs at Cmax doses. Microbicidal action of levofloxacin against intracellular MTB within A-549 cells was markedly less than its activity against the same organisms in MM6-Mphis. The same effect was also noted for the action of KRM-1648 against MAC organisms but this did not occur with clarithromycin. The MIC of KRM-1648 for MAC multiplying within A-549 cells was 32 times larger than that for MAC residing in MM6-Mphis. These findings indicate that MTB and MAC organisms replicating in the type II lung epithelial cells resist the action of certain antimycobacterial agents such as quinolones and rifamycin derivatives but not when the organisms are contained in macrophages. It appears that the antimicrobial action of certain drugs against intracellular mycobacteria is differentially manifested depending on the types of host cells, i.e. professional phagocytes (MM6-Mphis) or non-professional phagocytes (A-549cells), in which the organisms are contained.

Combined Effects of ATP on the Therapeutic Efficacy of Antimicrobial Drug Regimens against Mycobacterium avium Complex Infection in Mice and Roles of Cytosolic Phospholipase A2-Dependent Mechanisms in the ATP-Mediated Potentiation of Antimycobacterial Host Resistance

ATP, which serves as a mediator of intramacrophage signaling pathways through purinoceptors, is known to potentiate macrophage antimycobacterial activity. In this study we examined the effects of ATP in potentiating host resistance to Mycobacterium avium complex (MAC) infection in mice undergoing treatment with a drug regimen using clarithromycin and rifamycin and obtained the following findings. First, the administration of ATP in combination with the clarithromycin and rifamycin regimen accelerated bacterial elimination in MAC-infected mice without causing changes in the histopathological features or the mRNA expression of pro- or anti-inflammatory cytokines from those in the mice not given ATP. Second, ATP potentiated the anti-MAC bactericidal activity of macrophages cultivated in the presence of clarithromycin and rifamycin. This effect of ATP was closely related to intracellular Ca2+ mobilization and was specifically blocked by a cytosolic phospholipase A2 (cPLA2) inhibitor, arachidonyl trifluoromethylketone. Third, intramacrophage translocation of membranous arachidonic acid molecules to MAC-containing phagosomes was also specifically blocked by arachidonyl trifluoromethylketone. In the confocal microscopic observation of MAC-infected macrophages, ATP enhanced the intracellular translocation of cPLA2 into MAC-containing phagosomes. These findings suggest that ATP increases the host anti-MAC resistance by potentiating the antimycobacterial activity of host macrophages and that the cPLA2-dependent generation of arachidonic acid from the phagosomal membrane is essential for such a phenomenon.