Exercise and aspirin increase levels of circulating gliadin peptides in patients with wheat-dependent exercise-induced anaphylaxis.

BACKGROUND: Food-dependent exercise-induced anaphylaxis (FDEIA) is an allergic reaction characteristically induced by intense exercise combined with the ingestion of causative food. Recent reports have shown that aspirin intake is a contributing factor in some patients with FDEIA. Wheat is known to be the most frequent causative food, and the IgE-binding epitopes of a major wheat allergen (omega-5 gliadin) in wheat-dependent exercise induced anaphylaxis (WDEIA) have already been clarified. However, the mechanism of eliciting the symptom in WDEIA remains not fully understood. OBJECTIVES: The aim of this study was to examine the relationship of serum gliadin levels and allergic symptoms induced by exercise or aspirin in patients with WDEIA. METHODS: Six patients with a history of recurrent anaphylaxis associated with wheat ingestion were diagnosed as having WDEIA by the provocation test, which included wheat ingestion, exercise, aspirin intake and a combination of these challenges. During the tests, serum levels of gliadins were monitored by gliadin-specific sandwich ELISA. The effects of exercise and aspirin on serum gliadin levels were also investigated in four healthy subjects. RESULTS: Immunoreactive gliadins appeared in the sera of patients during the provocation test with both wheat-exercise and wheat-aspirin challenges in parallel with allergic symptoms. Serum gliadin levels also increased under the two same challenge conditions in the healthy subjects, although they exhibited no allergic symptoms. However, low levels of gliadin were detected in the sera of both patients and healthy subjects when challenged with wheat alone. CONCLUSION: We demonstrated for the first time that blood gliadin levels correlate with clinical symptoms induced by exercise and aspirin in patients with WDEIA. These findings suggest that exercise and aspirin facilitate allergen absorption from the gastrointestinal tract.

[Invasion and intracellular growth of Mycobacterium tuberculosis and Mycobacterium avium complex adapted to intramacrophagic environment within macrophages and type II alveolar epithelial cells].

Profiles of the invasion and intracellular growth of M. tuberculosis (MTB) and M. avium complex (MAC), which had been adapted to intramacrophagic environment, within Mono Mac 6 human macrophages (MM6-M phi s) and A-549 human type II alveolar epithelial cells (A-549 cells) were studied. In this study, we used the organisms grown in MM6-M phi s (intracellularly-adapted: I-type) and those passaged in 7H9 liquid medium (extracellularly-adapted: E-type). First, I-type MTB was less efficient than E-type MTB in invading into MM6-M phi s, while I-type MTB invasion into A-549 cells was greater than of E-type MTB. On the other hand, I-type MAC was more efficient than E-type MAC in entering both into MM6-M phi s and A-549 cells. Second, the ability of MTB and MAC to replicate within MM6-M phi s was increased by intracellular passage of these organisms through MM6-M phi s. In contrast, the ability of these organisms to grow within A-549 cells was decreased to some extent by intramacrophagic passage. These findings suggest that growth within M phi s changes the efficiency of MTB and MAC in invading and replicating in M phi s and type II alveolar epithelial cells.

Comparative roles of free fatty acids with reactive nitrogen intermediates and reactive oxygen intermediates in expression of the anti-microbial activity of macrophages against Mycobacterium tuberculosis.

We assessed the role of free fatty acids (FFA) in the expression of the activity of macrophages against Mycobacterium tuberculosis in relation to the roles of two major anti-microbial effectors, reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI). Intracellular growth of M. tuberculosis residing inside macrophages was accelerated by treatments of macrophages with either quinacrine (phospholipase A2 (PLA2) inhibitor), arachidonyl trifuloromethylketone (type IV cytosolic PLA2 inhibitor), NG-monomethyl-L-arginine (nitric oxide synthase inhibitor), and superoxide dismutase plus catalase (ROI scavengers). In addition, M. tuberculosis-infected macrophages produced and/or secreted these effectors sequentially in the order ROI (0-3 h), FFA (0-48 h), and RNI (3 to at least 72 h). Notably, membranous FFA (arachidonic acid) of macrophages translocated to M. tuberculosis residing in the phagosomes of macrophages in phagocytic ability- and PLA2-dependent fashions during cultivation after M. tuberculosis infection. FFA, RNI and H2O2-mediated halogenation system (H2O2-halogenation system) displayed strong activity against M. tuberculosis in cell-free systems, while ROI alone exerted no such effects. Combinations of 'FFA + RNI' and 'RNI + H2O2-halogenation system' exhibited synergistic and additive effects against M. tuberculosis, respectively, while 'FFA + H2O2-halogenation system' had an antagonistic effect. Moreover, a sequential attack of FFA followed by RNI exerted synergistic activity against M. tuberculosis. Since M. tuberculosis-infected macrophages showed simultaneous production of RNI with FFA secretion for relatively long periods (approx. 45 h) and prolonged RNI production was seen thereafter, RNI in combination with FFA appear to play critical roles in the manifestation of the activity of macrophages against M. tuberculosis.

[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.

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

WQ-3034 is a newly synthesized acidic fluoroquinolone. We assessed its in vitro activity against Mycobacterium tuberculosis and 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 (MIC(50)s, and MIC(90)s, respectively) for the test quinolones for rifampin (RMP)-susceptible M. tuberculosis strains were in the order SPFX < LVFX 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-Mphis. 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.

Comparative in vitro antimicrobial activities of the newly synthesized quinolone HSR-903, sitafloxacin (DU-6859a), gatifloxacin (AM-1155), and levofloxacin against Mycobacterium tuberculosis and Mycobacterium avium complex.

We compared the in vitro antimycobacterial activity of a new fluoroquinolone, HSR-903, with strong activity against gram-positive cocci with those of levofloxacin (LVFX), sitafloxacin (STFX), and gatifloxacin (GFLX). The MICs of the quinolones for Mycobacterium tuberculosis and Mycobacterium avium complex were in the order STFX approximately GFLX < LVFX <== HSR-903 and STFX <== GFLX <== HSR-903 <== LVFX, respectively. HSR-903 effectively eliminated intramacrophagial M. tuberculosis, as did LVFX, and exhibited bacteriostatic effects against M. tuberculosis replicating in type II alveolar cells.

[Internalization and replication of Mycobacterium tuberculosis and M. avium complex within type II alveolar epithelial cell line].

Profiles of internalization and replication of Mycobacterium tuberculosis (MTB) or M. avium complex (MAC) within A-549 human type II alveolar epithelial cell line (A-549 cells) were studied and the results were compared with the mode of internalizing and proliferative behaviors of the organisms within murine peritoneal macrophages (M phi s) and various M phi-like cell lines (murine J774A. 1, human THP-1, and human MONO-MAC-6). First, MTB and MAC internalized not only in peritoneal M phi s and M phi-like cell lines but also in A-549 cells. Secondly, MTB and MAC replicated within A-549 cells and these organisms displayed much more vigorous intracellular multiplication in A-549 cells than in murine peritoneal M phi s and J774A. 1 M phi-like cell line. Human M phi-like cell lines (THP-1, MONO-MAC-6) allowed the growth of MTB and MAC equally or occasionally more rapidly, as compared to the case of A-549 cells.

[Effects of the Chinese traditional medicines "mao-bushi-saishin-to" and "yokuinin" on the antimycobacterial activity of murine macrophages against Mycobacterium avium complex infection].

We previously examined the effects of two Chinese traditional medicines "Mao-Bushi-Saishin-To" (MBST) and "Yokuinin", on the therapeutic efficacies of a benzoxazinorifamycin, KRM-1648, against Mycobacterium avium complex (MAC) infection induced in mice. MBST but not Yokuinin potentiated the therapeutic activity of KRM-1648 against MAC infection. In the present study, we examined the effects of these traditional medicines on some M phi cell functions. First, MBST significantly potentiated M phi anti-MAC antimicrobial activity, while Yokuinin did so to a much lesser extent. Secondly, MBST and Yokuinin each strongly inhibited production of nitric oxide (NO) in MAC-infected M phi s. Thirdly, treatment of M phi s with MBST or Yokuinin caused reductions in the accumulation of IL-10 in culture fluids by MAC-infected M phi s during the first 2-days cultivation. On the other hand, in the separate experiment, treatment of M phi s with these drugs caused no significant change in the accumulation of TGF-beta by MAC-infected M phi s at day 7. These findings suggest that these Chinese traditional medicines, particularly MBST, potentiate M phi anti-MAC antimicrobial activity, however, NO do not appear to be crucial effectors in the anti-MAC activity of MBST- or Yokuinin-treated M phi s. Moreover, MBST- and Yokuinin-mediated down-regulation of the production of IL-10 in MAC-infected M phi s may be related to their potentiating effects on M phi anti-MAC activity.

[Therapeutic effects of benzoxazinorifamycin KRM-1648 administered alone or in combination with glycyrrhizin against Mycobacterium avium complex infection in mice].

We previously examined the effects of a Chinese medicine "Mao-Bushi-Saishin-To" (MBST) which has anti-inflammatory activity on the therapeutic efficacies of a benzoxazinorifamycin, KRM-1648 (KRM), against, Mycobacterium avium complex (MAC) infection induced in mice. MBST potentiated the therapeutic activity of KRM against MAC infection. In the present study, we examined the effects of another anti-inflammatory drug Glycyrrhizin, which is effective for chronic hepatitis, on the therapeutic efficacy of KRM against MAC infection induced in mice. First, KRM significantly inhibited the bacterial growth in the lungs and spleen of MAC-infected mice. Glycyrrhizin exhibited no therapeutic activity against MAC infection and did not affect the expression of the therapeutic efficacy of KRM. Secondly, treatment of murine peritoneal macrophages (M phi s) with Glycyrrhizin caused no significant changes in the M phi anti-MAC activity.

[MICs and MBCs of levofloxacin, clarithromycin, and KRM-1648 for Mycobacterium tuberculosis and M. avium complex residing in MONO-MAC-6 human macrophage-like cell and A-549 human type II alveolar epithelial cell lines].

In this study, we determined the MICs and MBCs of levofloxacin (LVFX), clarithromycin (CAM), and KRM-1648 (KRM) for Mycobacterium tuberculosis (MTB) strain Kurono and M. avium complex (MAC) strain N-444 residing in MONO-MAC-6 human macrophage-like cells (MM6-M phi s) and A-549 human type II alveolar epithelial cells (A-549 cells). First, the MICs of LVFX for MTB replicating in MM6-M phi s (1 microgram/ml) and A-549 cells (2 micrograms/ml) were 4 to 8 times higher than its MICs for extracellular MTB growing in 7HSF medium. In contrast, the MICs of CAM for intracellular MTB residing in both the cells (2-4 micrograms/ml) were 4 to 8 times less than its MICs for extracellular MTB organisms. On the other hand, the MICs of KRM for extracellular MTB were nearly the same as its MICs for intracellular MTB residing in both types of the cells. Secondly, the MICs of LVFX and CAM for extracellular MAC were not significantly different from their MICs for intracellular MAC residing in MM6-M phi s and A-549 cells. The MIC of KRM for MAC residing in A-549 cells was 0.25 microgram/ml, and this value was 32 times higher than its MIC for MAC residing in MM6-M phi s (0.008 microgram/ml). Thirdly, the MBCs of test drugs for intracellular MTB and MAC residing in both types of the cells were somewhat longer than their MBCs for extracellular organisms. These findings indicate that, in the case of pulmonary infections with MTB or MAC, the therapeutic efficacy of a given drug, especially KRM, is more or less influenced by the bacterial location in the host lung tissues where the mycobacterial pathogens survive and multiply, i.e., either alveolar M phi s, type II alveolar epithelial cells, or surrounding environment.

Effects of the Chinese traditional medicine mao-bushi-saishin-to on therapeutic efficacy of a new benzoxazinorifamycin, KRM-1648, against Mycobacterium avium infection in mice.

The Chinese traditional medicine mao-bushi-saishin-to (MBST), which has anti-inflammatory effects and has been used to treat the common cold and nasal allergy in Japan, was examined for its effects on the therapeutic activity of a new benzoxazinorifamycin, KRM-1648 (KRM), against Mycobacterium avium complex (MAC) infection in mice. In addition, we examined the effects of MBST on the anti-MAC activity of murine peritoneal macrophages (M phi s). First, MBST significantly increased the anti-MAC therapeutic activity of KRM when given to mice in combination with KRM, although MBST alone did not exhibit such effects. Second, MBST treatment of M phi s significantly enhanced the KRM-mediated killing of MAC bacteria residing in M phi s, although MBST alone did not potentiate the M phi anti-MAC activity. MBST-treated M phi s showed decreased levels of reactive nitrogen intermediate (RNI) release, suggesting that RNIs are not decisive in the expression of the anti-MAC activity of such M phi populations. MBST partially blocked the interleukin-10 (IL-10) production of MAC-infected M phi s without affecting their transforming growth factor beta (TGF-beta)-producing activity. Reverse transcription-PCR analysis of the lung tissues of MAC-infected mice at weeks 4 and 8 after infection revealed a marked increase in the levels of tumor necrosis factor alpha, gamma interferon (IFN-gamma), IL-10, and TGF-beta mRNAs. KRM treatment of infected mice tended to decrease the levels of the test cytokine mRNAs, except that it increased TGF-beta mRNA expression at week 4. MBST treatment did not affect the levels of any cytokine mRNAs at week 8, while it down-regulated cytokine mRNA expression at week 4. At week 8, treatment of mice with a combination of KRM and MBST caused a marked decrease in the levels of the test cytokines mRNAs, especially IL-10 and IFN-gamma mRNAs, although such effects were obscure at week 4. These findings suggest that down-regulation of the expression of IL-10 and TGF-beta is related to the combined therapeutic effects of KRM and MBST against MAC infection.

Differential potentiation of anti-mycobacterial activity and reactive nitrogen intermediate-producing ability of murine peritoneal macrophages activated by interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha).

The anti-mycobacterial activities of IFN-gamma and TNF-alpha-treated murine peritoneal macrophages were determined. Resident macrophages pretreated with IFN-gamma or TNF-alpha for 2 days were infected with test organisms and subsequently cultured for up to 7 days. First, the early-phase growth of Mycobacterium tuberculosis (days 0-3) was strongly suppressed in IFN-gamma-treated macrophages, and progressive bacterial elimination was subsequently observed. Although TNF-alpha treatment of macrophages did not affect the early phase growth of organisms, bacterial killing was observed in the later phase of cultivation. Second, although IFN-gamma-treated macrophages killed M. avium during the first 3 days of culture, regrowth of the intracellular organisms was subsequently observed. TNF-alpha treatment of macrophages did not influence the mode of intracellular growth of M. avium. Third, IFN-gamma but not TNF-alpha enhanced production of reactive nitrogen intermediates (RNI) by macrophages infected with M. tuberculosis or M. avium, whereas both cytokines increased macrophage release of reactive oxygen intermediates (ROI). The present findings therefore show that IFN-gamma and TNF-alpha potentiated the anti-mycobacterial activity of murine peritoneal macrophages in different fashions. They also suggest that RNI played more important roles than did ROI in the expression of macrophage anti-mycobacterial, particularly anti-M. avium, activity.

[Usefulness of MycoDot test for the detection of anti-mycobacterial antibodies as an aid in the diagnosis of experimental Mycobacterium tuberculosis and Mycobacterium avium complex infections in mice].

The MycoDot is a new diagnostic kit for tuberculosis which was devised by DynaGen Inc., USA. The MycoDot test is based on the detection of anti-mycobacterial antibodies in the serum samples of patients by employing plastic combs coated with lipoarabinomannan (LAM) antigen which is a highly immunogenic lipopolysaccharide presenting in the cell wall of all species of mycobacteria. It has been reported that healthy infected and BCG-vaccinated individuals do not react to the MycoDot test, while a positive reaction occurs in patients with active tuberculosis or atypical mycobacteriosis with good sensitivity and specificity. In this study, we evaluated the efficacy of MycoDot test for the detection of anti-LAM antibodies in sera of mice infected with Mycobacterium tuberculosis or M. avium complex (MAC). By using the MycoDot test, anti-LAM antibodies were positive in 2 out of 4 mice infected with M. tuberculosis 2 weeks before, while all of M. intracellulare-infected mice were negative at the same phase of infection. On the other hand, anti-mycobacterial (MB) antibodies were detected in the serum samples of mice infected with M. intracellulare as well as M. tuberculosis by home-made ELISA testing using whole cells of test mycobacteria as antigen. In the next experiment, mice were infected with M. avium. All the serum samples of mice obtained at 13 weeks after infection were negative for anti-LAM antibodies in MycoDot test, whereas they reacted positively to anti-MB antibodies in ELISA test. These results indicate that the MycoDot test is capable of detecting M. tuberculosis infection but not MAC infection induced in mice.

[Nontuberculous mycobacteriosis; the present status and in the future. Mechanisms of host resistance to Mycobacterium avium complex and Mycobacterium tuberculosis infection].

In order to know profiles of the antimicrobial action of some microbicidal effector molecules against Mycobacterium avium complex (MAC) and M. tuberculosis (MTB), profiles of collaborating effects among reactive nitrogen intermediates (RNI), free fatty acids (FFA), and reactive oxygen intermediates (ROI) were studied, RNI and FFA exerted synergistic effects in killing MAC and MTB, while the combination of ROI (H2O2-mediated halogenation system) with FFA conversely caused antagonism. The combination of RNI with ROI displayed additive effects in killing MTB, whereas the same combination showed antagonistic effects against MAC. Murine peritoneal macrophages (M phi s) produced and/or released these three antimicrobial effectors in the order ROI, FFA, and RNI in response to cellular stimulation induced by their contact with MAC or MTB organisms. These findings indicate that the collaborating effect of RNI with FFA is crucial for M phi-mediated intracellular killing of MAC and MTB. Secondly, we examined the modes of bacterial growth of MAC and MTB in murine peritoneal M phi s and A-549 type II alveolar epithelial cell line. The growth rate of these organisms was much larger in A-549 cells than in M phi s. In addition, the growth rate of high-virulence MAC (N-260 strain) was significantly larger than that of low-virulence MAC (N-444 strain), when they were residing in M phi or A-549 cells. Although a high virulence MTB (strain Kurono) also showed much more rapid growth in M phi s than did low-virulence MTB (strain H37Ra), such a phenomenon was not observed for their intracellular growth in A-549 cells. MTB exhibited strong cytotoxic effects against M phi s but not against A-549 cells when resided in these cells. On the other hand, MAC organisms did not cause cytotoxicity even in M phi s. Although MAC and MTB infections caused significant increase in RNI production by M phi s but not by A-549 cells, there was no significant relationship between the degree of M phi RNI production by a given mycobacterial organism and its virulence. These findings indicate some important roles of type II alveolar epithelial cells as a target cell for primary invasion and transient growth of mycobacterial organisms in the host lungs.

Antimicrobial activities of benzoxazinorifamycin KRM-1648, clarithromycin and levofloxacin against intracellular Mycobacterium avium complex phagocytosed by murine peritoneal macrophages.

The in-vitro activities of KRM-1648, a new benzoxazinorifamycin, clarithromycin and levofloxacin against clinical isolates of Mycobacterium avium complex (MAC) were measured using various methods of assay and compared with their in-vivo therapeutic activities against MAC infection in mice. The MICs varied according to drug in the order KRM-1648 << clarithromycin < levofloxacin. However, KRM-1648 and clarithromycin but not levofloxacin had similar therapeutic outcomes in MAC-infected mice. KRM-1648 and clarithromycin given at clinical dosages caused 1 to 2 log unit reductions in bacterial loads in the lungs of host mice. The values of Cmax (lung)/MBC were more closely related to the therapeutic efficacy of these drugs in mice than were MICs and MBCs alone. Potent microbicidal activity was observed with KRM-1648 and clarithromycin but not with levofloxacin against extracellularly growing MAC (EG-MAC) in a liquid medium. These two agents caused more than 3 log unit killing of MAC during a 5 day incubation, when added at concentrations equivalent to Cmax (lung). The anti-EG-MAC bactericidal activity of these drugs was greater than their efficacy in mice in vivo. KRM-1648 and clarithromycin but not levofloxacin caused respectively 2 and 0.5 log unit killing of intracellularly growing MAC (IG-MAC) in murine peritoneal macrophages. The profiles of bacterial killing effects of these agents against IG-MAC accurately reflected their therapeutic effects in mice, although the in-vivo activity of KRM-1648 was still overestimated using even this parameter.

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 avium complex (MAC) and the mode of macrophage (mphi) production of these effectors. (1) Intracellular growth of MAC in murine peritoneal mphis 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 mphi 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 mphis 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 mphis, and the collaborating action of RNI with FFA temporarily participates in mphi-mediated killing of MAC in the relatively early phase after MAC stimulation.

Effector molecules of the host defence mechanism against Mycobacterium avium complex: the evidence showing that reactive oxygen intermediates, reactive nitrogen intermediates, and free fatty acids each alone are not decisive in expression of macrophage antimicrobial activity against the parasites.

In this study, we evaluated the roles of reactive oxygen intermediates (ROI), reactive nitrogen intermediates (RNI), and free fatty acids (FFA) as effectors of the macrophage-mediated host defence mechanism against Mycobacterium avium complex (MAC). First, M. avium (three strains) and M. intracellulare (two strains) were treated with the H2O2-Fe2+-mediated halogenation system, acidified NaNO2-derived RNI, or FFA (linolenic acid) in sodium acetate buffer pH 5.5, and then counted for the number of residual colony-forming units (CFU) of organisms. Although these effectors exerted strong bactericidal activity against the MAC, the susceptibility of test organisms markedly varied from strain to strain. There was no significant relationship between the degree of resistance of a given MAC strain to these effectors and its virulence in mice, indicating that ROI, RNI, and FFA each alone are not decisive as the effector components of the host defence mechanism against the MAC. Second, the increase in ROI-producing ability in murine peritoneal macrophages due to tumour necrosis factor-alpha (TNF-alpha) treatment was not accompanied by parallel potentiation of anti-MAC activity of the same macrophage population. This excludes the possibility that ROI play a central role in macrophage-mediated killing and inhibition of MAC organisms. Third, anti-MAC activity of BAM3 macrophage cell line was not significantly attenuated by N(G)-monomethyl-L-arginine (NO synthase-inhibitor causing reduction of RNI production) or by quinacrine (phospholipase A2-inhibitor causing reduction of FFA release), indicating that RNI and FFA each alone do not play crucial roles in the expression of macrophage antimicrobial activity against the MAC. The present findings suggest important roles of collaborating actions of various antimicrobial effectors and/or the participation of other kinds of effectors in macrophage-mediated killing and inhibition of MAC organisms.

[In vivo activities of benzoxazinorifamycin KRM-1648, clarithromycin, and levofloxacin, or combination of KRM-1648 with diclofenac sodium against Mycobacterium avium infection induced in mice].

We evaluated the in vivo therapeutic activities of benzoxazinorifamycin KRM-1648, clarithromycin (CAM) and levofloxacin (LVFX) against Mycobacterium avium infection induced in mice. Mice infected intravenously with M. avium (1.4 x 10(7)) were given KRM-1648 (20 mg/kg), CAM (10 mg/kg), or LVFX (5 mg/kg) alone, or combination of KRM-1648 with diclofenac sodium (1.25 mg/kg) by gavage, once daily, five times per week, from day 1 for up to 8 weeks. The bacterial loads in the lungs and spleens were determined by counting colony forming units of the organisms in the tissue homogenates of the visceral organs using 7H11 agar plates. Both KRM-1648 and CAM caused significant levels of bacteriological response in mice treated with these drugs, while LVFX exerted no appreciable therapeutic effect. The therapeutic efficacies of test antimicrobials were in the order, KRM-1648 > CAM > > LVFX. The combined use of diclofenac sodium with KRM-1648 did not affect the expression of therapeutic activity of KRM-1648. This excludes the possibility that cyclooxygenase-dependent inflammatory reactions may be involved in the establishment of persistent bacterial growth of M. avium organisms at the sites of infection in mice. Furthermore, the present study showed that the parameters of in vitro antimicrobial activities of drugs such as MIC and MBC values are not useful in predicting their therapeutic outcome in M. avium-infected mice.

[Anti-Mycobacterium avium complex activities of KRM-1648, clarithromycin and levofloxacin in 7HSF medium at peak or average blood concentrations after their oral administration of clinical dosages].

We evaluated the anti-Mycobacterium avium complex (MAC) activities of KRM-1648 (KRM), clarithromycin (CAM) and levofloxacin (LVFX) in 7HSF medium at concentrations in human blood after their oral administration of clinical dosages. MAC organisms were inoculated into tubes containing the medium with or without the addition of test drugs at either 1/10 Cmax, Cmax or C0-8h (average concentrations during the first 8 h) in the blood. KRM at Cmax or C0-8h showed strong bactericidal activity against MAC, CAM showed weak or moderate degree of bactericidal activity at Cmax and C0-8h, while LVFX added at Cmax or C0-8h showed only a weak bacteriostatic effect against M. avium, but did not show any effect against M. intracellulare.