anti-Mtb Activity Research Articles

AntiMtb activity of triterpenoid-rich fractions from Spondias mombin L.

Spondias mombin L. used in traditional medicine because of its antimicrobial properties was found to contain cardiac glycosides, flavonoids, tannins, and anthraquinones in the stem bark. Bioassaydirected fractionation of the methanol extract of S. mombin was carried out with VLC and HPLC. Isolates were evaluated for antimtb activity which led to the isolation of a series of potential molecules. A semi pure triterpenoid that demonstrated potency of 92.8% inhibition against Mycobaterium tuberculosis (Mtb) at a concentration of 64
g/ml was further purified by HPLC.

抗酸菌に対する新しいタイプのマクロファージ殺菌メカニズム

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.

Changes in antibacterial activity of murine peritoneal macrophages against Mycobacterium tuberculosis after prolonged in vitro precultivation

We examined profiles of intramacrophagial growth of M. tuberculosis (MTB) when mouse peritoneal macrophages (M phi s) were infected with the organisms at day 0 or day 7 after in vitro precultivation, and obtained the following results. First, the growth rate of the virulent MTB H37Rv strain as well as attenuated H37Ra strain was slower in M phi s which had been precultured for 7 days (M phi s [day 7]) than in freshly prepared M phi s without precultivation (M phi s [day 0]). The doubling time of MTB H37Rv was 2.2 and 2.9 days in M phi s [day 0] and M phi s [day 7], respectively, and that of MTB H37Ra was 2.9 and 3.6 days in M phi s [day 0] and M phi s [day 7], respectively. Second, MTB-mediated cytotoxicity in terms of the LDH release from infected M phi s was less marked in M phi s [day 7] than in M phi s [day 0], when they were infected with MTB of either the H37Rv or H37Ra strain. MTB H37Ra strain exhibited much weaker cytotoxic effects on host M phi s than did H37Rv strain. Third, when M phi s [day 7] were infected with MTB of either the H37Rv or H37Ra strain, they showed markedly lowered levels of reactive oxygen intermediate (ROI) production than did M phi s [day 0]. In contrast, the reactive nitrogen intermediate (RNI) producing ability of M phi s in response to MTB infection was not so markedly reduced in M phi s [day 7] from that of M phi s [day 0]. As mentioned above, the M phi s [day 7] did not permit accelerated growth of infected MTB, compared to the MTB growth in the M phi s [day 0]. It thus appears that ROI played a trivial role in the antimicrobial activity against MTB of murine peritoneal M phi s which had been precultured for long periods. Although it is regarded that RNI played more critical roles in M phi anti-MTB activity than did ROI, the present results also suggest that other kinds of antimicrobial effectors are required in M phi antimicrobial activity against MTB organisms, particularly in the case of M phi s after prolonged in vitro cultivation.

Induction of In Vitro Human Macrophage Anti-Mycobacterium tuberculosis Activity: Requirement for IFN-γ and Primed Lymphocytes

Abstract Mycobacterium tuberculosis (Mtb) is the world’s leading infectious cause of mortality. Despite the overwhelming data supporting the critical role of cellular immunity, little is known of the early microbial and immune cell interactions and whether human macrophages can be activated to express anti-Mtb activity. We report the reconstitution of an in vitro system whereby human macrophages express anti-Mtb activity only in coculture with PBL and with IFN-γ. Omission of IFN-γ in the cocultures or Mtb lysate/IFN-γ-primed lymphocytes was associated with high growth of Mtb, high IL-10 and IL-12 p40, nearly undetectable IL-12 p70 levels, and the highest percentages of CD4 and CD8 T cells. In contrast, IFN-γ treatment of cocultures containing Mtb lysate/IFN-γ-primed PBL reduced bacilli count by ∼2.5 log, decreased the production of IL-10 by 5.7-fold, increased IL-12 p70 by ∼50-fold, and reduced the percentages of CD4 and CD8 T cells. Activation of anti-Mtb activity was time and dose dependent. At 2000 U/ml of IFN-γ, bactericidal activity was achieved (10-fold reduction from initial inoculum). Anti-Mtb activity against several strains of M. tuberculosis (H37Ra and H37Rv, and C, a clinical isolate) was observed and was associated with expression of inducible nitric oxide synthase. These data suggest that induction of human macrophage anti-Mtb activity required dual signaling from PBL and IFN-γ. Thus, the development of an in vitro human system may greatly facilitate studies to delineate immune cells, cytokines, and effector functions/genes critical in controlling Mtb. Defining the mechanisms may also provide novel treatment strategies for tuberculosis.