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In the advanced stages of mycobacterial infections, host immune systems tend to change from a Th1-type to Th2-type immune response, resulting in the abrogation of Th1 cell- and macrophage-mediated antimicrobial host protective immunity. Notably, this type of immune conversion is occasionally associated with the generation of. certain types of suppressor macrophage populations. During the course of infections due to pathogenic mycobacteria, the generation of macrophages which possess strong suppressor activity against host T- and B-cell functions is frequently encountered. This review describes the immunological properties of M1- and M2-type macrophages generated in hosts with certain microbial infections including mycobacteriosis and those generated in tumor-bearing animals. Particularly, this paper highlights the immunological and molecular biological characteristics of M1 and M2 macrophage populations, which are induced by mycobacterial infections

Worldwide, tuberculosis remains the most frequent and important infectious disease to cause morbidity and death. However, the development of new drugs for the treatment and prophylaxis of TB has been slow. Therefore, novel types of antituberculous drugs, which act on the unique drug targets in Mycobacterium tuberculosis, particularly the drug targets related to the establishment of mycobacterial dormancy in host's macrophages, are urgently needed. In this context, it should be noted that current antituberculous drugs mostly target the metabolic reactions and proteins which are essential for the growth of M. tuberculosis in extracellular milieus. It may also be promising to develop another type of drug that exerts an inhibitory action against bacterial virulence factors which cross talk and interfere with signaling pathways of M. tuberculosis-infected host immunocompetent cells such as lymphocytes, macrophages and NK cells, thereby changing the intracellular milieus favorable to intramacrophage survival and growth of infected bacilli. In this review article, I will describe recent approaches to identify and establish novel potential drug targets in M. tuberculosis, especially those related to mycobacterial virulence, dormancy, and cross-talk with cellular signaling pathways.

Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death. One-third of the world's population is infected with Mycobacterium tuberculosis (MTB), the etiologic agent of TB. The World Health Organization estimates that about eight to ten million new TB cases occur annually worldwide and the incidence of TB is currently increasing. In this context, TB is in the top three, with malaria and HIV being the leading causes of death from a single infectious agent, and approximately two million deaths are attributable to TB annually. In particular, pulmonary TB, the most common form of TB, is a highly contagious and life-threatening infection. Moreover, enhanced susceptibility to TB in HIV-infected populations is another serious health problem throughout the world. In addition, multidrug-resistant TB (MDR-TB) has been increasing in incidence in many areas, not only in developing countries but industrialized countries as well, during the past decade. These situations, particularly the global resurgence of TB and the rapid emergence of MDR-TB, underscore the importance of the development of new antituberculous drugs and new protocols for efficacious clinical control of TB patients using ordinary antimycobacterial drugs. Concerning the development of new antituberculous drugs, the following points are of particular importance. (1) Development of drugs which display lasting antimycobacterial activity in vivo is desirable, since they can be administered with long intervals and consequently facilitate directly observed therapy and enhance patient compliance. (2) Development of novel antituberculosis compounds to combat MDR-TB is urgently needed. (3) The eradication of slowly metabolizing and, if possible, dormant populations of MTB organisms that cause relapse, using new classes of anti-TB drugs is very promising for prevention of TB incidence, because it will markedly reduce the incidence of active TB from persons who are latently infected with MTB. Unfortunately, no new drugs except rifabutin and rifapentine has been marketed for TB in the US and other countries during the 40 years after release of rifampicin. There are a number of constraints that have deterred companies from investing in new anti-TB drugs. The research is expensive, slow and difficult, and requires specialized facilities for handling MTB. There are few animal models that closely mimic the human TB disease. Development time of any anti-TB drug will be long. In fact, clinical trials will require the minimum six-month therapy, with a follow-up period of one year or more. In addition, it is hard to demonstrate obvious benefit of a new anti-TB agents over pre-existing drugs, since clinical trials involve multidrug combination therapy using highly effective ordinary anti-TB drugs. Finaly, there is the perceived lack of commercial return to companies engaged in the development of new anti-TB drugs, because over 95% of TB cases worldwide are in developing countries. In this symposium, we reviewed the following areas. 1. Critical new information on the entire genome of MTB recently obtained and increasing knowledge of various mycobacterial virulence genes are greatly promoting the identification of genes that code for new drug targets. In this context, Dr. Namba reviewed the status of new types of compounds which are being developed as anti-TB drug. He also discussed the development of new antimycobacterial drugs according to new and potential pharmacological targets and the best clinical development plans for new-TB drugs in relation to corporate strategy. 2. Using such findings for mycobacterial genomes, bioinformatics/genomics/proteomics-based drug design and drug development using quantitative structure-activity relationships may be possible in the near future. In this context, Dr. Suwa and Dr. Suzuki reviewed the usefulness of chemical genomics in searching novel drug targets for development of new antituberculous drugs. The authors reviewed (1) the history and present status of chemical genomics that is defined as the systemic search for a selective small molecular modulator for each function of all gene products, (2) recent studies of the authors on profiles of the interactions between various kinds of human proteins and small molecule modulators using the new technology devised by Reverse Proteomics Research Institute, and (3) future prospects of the development of new antituberculous drugs based on chemical genomics. 3. It appears also promising to develop new types of drug administration systems using drug vehicles, which enable efficacious drug delivery to their target in vivo. Dr. Izumikawa, Dr. Ohno and Dr. Kohno reviewed the usefulness of liposome- and polymer-based technologies, which enable efficacious delivery of encapsulated drugs at required doses for prolonged periods of time with only a single shot without toxicity, and also enable highly targeted delivery of drugs to their target in vivo. They indicated that the applications of drug delivery system using conventional anti-mycobacterial agents are challenging to improve the compliance of treatment and better clinical outcome. 4. Immunoadjunctive therapy appears to be promising in improving outcome of clinical control of refractory mycobacterial infections, including MDR-TB and M. avium complex infection. Dr. Shimizu, Dr. Sato and Dr. Tomioka reviewed the present status of immunotherapy of mycobacterial infections in combination with antimycobacterial drugs. They indicated that the development of new classes of immunomodulators other than cytokines (IL-2, IFN-gamma, GM-CSF, IL-12, etc.) particularly those with no severe side-effects, are urgently needed. Their review dealed with some promising immunoadjunctive agents, especially ATP and its analogues, which potentiate macrophage antimycobacterial activity via purinergic P2 receptors. The aim of this symposium is to address the future prospects of the development of new drugs and drug regimens for anti-TB chemotherapy. There are a number of difficulties in drug-design for the development of new drug formulations with increased potential for antimycobacterial effects, excellent pharmacokinetics, and tolerability. It should be emphasized that the most urgent goal of chemotherapy of TB and MAC infections, especially that associated with HIV infection, is to develop highly active, low-cost drugs which can be used not only in industrialized countries but also in developing countries, since the incidences of AIDS-associated intractable TB and MAC infections are rapidly increasing in the latter. We strongly wish a great advance of fundametal and practical studies in developing such kinds of new anti-TB drugs in the near future. 1. Prospects for non-clinical or clinical development of new antituberculous drugs in relation to corporate strategy: Kenji NAMBA (New Product Research Laboratories I, Daiichi Pharmaceutical Co., Ltd.) Tuberculosis (TB) remains one of the deadliest threats to public health. No new anti-TB drugs have been brought into the clinic in the past 40 years. Current non-clinical works with progressed technology and Global Alliance for TB Drug Development, a non-profit organization established in 2000, accelerate research and development of faster-acting anti-TB compounds. We reviewed the status of new types of compounds which are being developed as anti-TB drug, such as diarylquinoline (TMC 207), nitroimidazole (PA-824 and OPC-67683), and moxifloxacin (MFLX). We also discussed the best clinical development plans for new-TB drugs in relation to corporate strategy. 2. Exploring novel drug targets through the chemical genomics approach and its possible application to the development of anti-tuberculosis drugs: Yorimasa SUWA (Reverse Proteomics Research Institute Co., Ltd.), Yohji SUZUKI (Teijin Ltd.) Recently, chemical genomics approach has been focused as an emerging technology for the drug discovery. In advance to a very large scale national project in US started last year, Reverse Proteomics Research Institute Co., Ltd. (REPRORI) has developed the core technologies for chemical genomics. Here we describe the outline of chemical genomics study, especially that of REPRORI, and discuss about its possible application to the development of anti-tuberculosis drugs. 3. Anti-mycobacterial agents and drug delivery: Koichi IZUMIKAWA, Hideaki OHNO, Shigeru KOHNO (Second Department of Internal Medicine, Nagasaki University School of Medicine) Mycobacterium infection is a major clinical concern in whole world. Since the newly developed anti-mycobacterial agents are few and still unavailable in clinical settings, the applications of drug delivery system using conventional anti-mycobacterial agents are challenging to improve the compliance of treatment and better efficacy. The efficacy of anti-mycobacterial agents modified by liposome or polymer based technology have been investigated and reported using various animal models. Drug delivery system increased and prolonged the drug concentrations at the blood and targeted organs and the duration of sustained drug release, respectively. These effects lead to decrease in the frequency of drug administrations dramatically and better efficacy rates. The studies, however, were performed only in animal models, the further investigations and evaluations in human are required for practical use. 4. Adjunctive immunotherapy of mycobacterial infections: Toshiaki SHIMIZU, Katsumasa SATO, Haruaki TOMIOKA (Department of Microbiology and Immunology, Shimane University School of Medicine) There is an urgent need to develop new antimicrobials and protocols for the administration of drugs that are potently efficacious against intractable mycobacterial infections. Unfortunately, development of the new drugs for solving this problem is not progressing. (ABSTRACT TRUNCATED)

Aim/methods: A series of 10 cases of chest wall tuberculosis (man/woman=7/3, average age 62 +/- 17 years old) during past seven years were reviewed., Results: Acid-fast bacillus was detected from an abscess in 60% by smear, 30% by culture, and 75% by polymerase chain reaction (PCR). It was characteristic that enhanced CT of abscess revealed a low density mass with peripheral enhancement, 'rim enhancement findings', in all cases. CT also showed ipsilateral pleural thickening in all cases, suggesting lymphogenous pathogenesis of chest wall lesions from tuberculous pleurisy. As for the treatment, antituberculosis chemotherapies were done in all cases. In addition, open drainage was done in 8 cases and curettage of abscess was performed in 5 cases. None of these ten cases had relapsed during the follow-up periods for 12 to 77 months., Conclusions: Chest wall tuberculosis is still important as a disorder of a chest wall mass requiring differential diagnosis. Contrasting CT is thought to be useful for the diagnosis. It should be emphasized that 50% of the cases had good outcome without curettage.

Unlabelled: End-stage renal failure patients on chronic dialysis are high risk groups of tuberculosis due to attenuated cellular immunity. Patients receiving haemodialysis stay prolonged time inside the health-care facilities, thereby increased risk of tuberculosis transmission if a patient has active disease. So management of active pulmonary tuberculosis undergoing haemodialysis is important, however, the number of hospitals which are capable of taking care of such patients is estimated to be few in Japan., Methods: From August 1994 through July 2002, 1059 active pulmonary tuberculosis patients (mean age; 57 +/- 19, male/female = 773/286) were admitted to Nishi-Kobe Medical Center, a 500-bed teaching hospital. Out of them, patients undergoing haemodialysis were retrospectively studied to describe the clinical characteristics of such cases. Then we conducted a questionnaire survey regarding the management of active pulmonary tuberculosis patients undergoing haemodialysis for 86 self-governing bodies in Japan., Results: (1) Clinical characteristics of active pulmonary tuberculosis undergoing haemodialysis. We encountered 14 cases (mean age; 65 +/- 11, male/female = 7/7) of pulmonary tuberculosis undergoing haemodialysis during 8 years. In addition to pulmonary involvement, 3 pleural, one knee joint and one lymph node involvement was detected. Primary renal disease included diabetic nephropathy (n = 3), chronic glomerulonephritis (n = 3), congenital anomaly (n = 1), and unknown (n = 7). Nine cases were referred to our hospital from health-care facilities located out of city or prefecture. In five cases it took more than three months from the onset or detection of abnormal chest X-ray findings to the admission to our hospital. Five cases developed pulmonary tuberculosis within the first year after the initiation of dialysis. None of the patients had a past history of tuberculosis. Cavitary lesion on chest X-ray was observed in only one case. Triple antituberculosis therapy was used in 9 patients, and 4 antituberculosis drugs were used in 5 patients. Antituberculosis therapy was successfully done in all cases except two patients who died of apoplexy and cerebral infarction. (2) The nation-wide questionnaire survey. Of the 86 self-governing bodies we mailed, 66 self-governing bodies replied. Of them, 31% reported that they have experienced difficulties in the management of active pulmonary tuberculosis patients undergoing haemodialysis, and 25% reported the lack of health-care facilities to take care of such cases in their territory. They have referred such patients to hospitals located in the nearby prefectures or they have recommended antituberculosis therapy visiting a local haemodialysis facility., Conclusion: There are sometimes difficulties to manage active pulmonary tuberculosis patients undergoing haemodialysis in Japan. Health-care facilities to take care of such patients should be arranged and the formation of the network is necessarily.

There has not yet been systematic studies which attempt to elucidate detailed profiles of the interaction between antimicrobial drugs and macrophage microbicidal mechanisms. We examined the effects of antisense oligo DNAs (AsDNAs) against oxyR and ahpC on the susceptibility of Mycobacterium avium complex (MAC) to the H2O2-halogenation system and combined antimycobacterial drugs [clarithromycin (CAM) + rifampicin (RFP)], both separately and in combination. It was found that AsDNA treatment of MAC did not affect the susceptibility of the organisms to any of the antimicrobial systems tested. Since the present AsDNAs did not efficiently reduce the expression of AhpC mRNA, attempts to increase bacterial uptake of AsDNAs are necessary to achieve significant increase in the drug susceptibility of MAC organisms due to AsDNA treatment.

Studied were made on the profiles of the therapeutic efficacy of KRM-1648 (KRM) against Mycobacterium avium complex (MAC) infection, which was induced in mice at different challenge doses, in reducing bacterial growth in the visceral organs and altering the profiles of cytokine mRNA expression at the sites of infection. First, bacterial growth in the lungs of mice infected with either high or low challenge doses of MAC, was reduced due to KRM treatment. This effect was noted even in the early phase of infection (week 4) in mice, that were given a high-dose infection. Second, marked therapeutic efficacy of KRM was observed in mice, that were given low-dose MAC infection, in terms of the reduction in bacterial loads in the spleen. However, in mice given a high-dose bacterial challenge, KRM did not exhibit such an efficacy. Third, the expression of both proinflammatory cytokines (TNF-alpha, IFN-gamma) and anti-inflammatory cytokines (IL-10, TGF-beta) in mRNA levels were increased at 4 weeks after infection. Notably, all of the cytokines tested for the mRNA expression levels were higher in mice given a low-dose MAC infection as compared to those in mice given a high-dose infection. KRM treatment increased the mRNA levels of these cytokines at week 4, while TGF-beta mRNA expression at week 8 was conversely decreased by KRM treatment. These findings suggest that the profiles of the therapeutic efficacy of KRM vary in mice given low- or high-dose MAC infection.

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.

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.

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.

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.

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.

Secretory leukocyte protease inhibitor (SLPI), a serine protease inhibitor abundantly found in mucous secretions of lung, is thought to serve as an important protective component in the secretory fluids at sites of degenerative and inflammatory diseases. In this study, we examined the effects of SLPI on the production of a proinflammatory cytokine, TNF-alpha, and immunosupressive cytokines, IL-10 and transforming growth factor-beta (TGF-beta) by macrophages (M phi s), in response to lipopolysaccharide (LPS)-mediated stimulation and M. avium complex (MAC) infection, using recombinant half-sized SLPI (1/2 SLPI) consisting of C-terminal domain (Arg55-Ala107) of intact SLPI. In addition, effects of SLPI on the production of nitric oxide radicals (NO), important antimicrobial effectors of M phi s against micro-organisms, by these M phi s were studied. First, when the number of TNF-alpha producing cells in the LPS-stimulated M phi population was counted by the ELISPOT assay, more than half of the M phi s acquired TNF-alpha secreting ability at 24 hr after LPS stimulation. On the other hand, MAC-infected M phi s produced detectable amounts of TNF-alpha into culture fluid during the first 24 hr. In both cases, 1/2 SLPI did not affect the LPS- or MAC-induced TNF-alpha production by M phi s. Second, when the production of IL-10 and TGF-beta by M phi s was determined by measuring the amounts of these cytokines accumulated in M phi culture fluids by ELISA, the following was observed. M phi IL-10 production was rapidly increased in the early phase of cultivation after LPS stimulation or MAC infection, peaking on day 1 and thereafter declining to normal level by day 14. Half-sized SLPI did not affect IL-10 production of LPS-stimulated M phi s, while it caused slight enhancement of IL-10 production by MAC-infected M phi s. M phi TGF-beta production was initiated in the middle phase (day 7) of M phi cultivation and continued until day 14. Notably, 1/2 SLPI markedly potentiated the TGF-beta producing ability of the LPS-stimulated M phi s. Moreover, 1/2 SLPI caused moderate increase in the TGF-beta production by MAC-infected M phi s. Third, significantly potentiated NO production was observed in M phi s during the first 2 days after LPS stimulation and MAC infection. Half-sized SLPI did not affect the NO production by LPS-stimulated or MAC-infected M phi s. These findings indicate that SLPI up-regulates the production of some immunoregulatory cytokines including IL-10 and TGF-beta, particularly the latter, by M phi s in response to LPS stimulation or MAC infection, thereby suggesting the possibility that SLPI may exhibit antiinflammatory effects in vivo, especially patients with bacterial infections including MAC diseases, through the modulation of M phi expression of some immunosuppressive cytokines.

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.

Profiles of expression of the antimicrobial activities of LVFX, KRM-1648 (KRM), and CAM against M. tuberculosis (MTB) and M. avium complex (MAC) residing in MONO-MAC-6 human macrophage like cells (MM6-M phi s) and A-549 human type II alveolar pneumocyte cells (A-549 cells) were determined. First, the antimicrobial activities of LVFX, KRM, and CAM against intracellular organisms of MTB Kurono and MAC N-444 strains were examined under conditions in which infected MM6-M phi s and A-549 cells were cultured for up to 7 days or longer in medium containing the antimicrobials at their Cmaxs in the blood, achievable after oral administration of clinical dosages of these drugs. The antimicrobial effects of LVFX and KRM against respectively MTB and MAC within A-549 cells were significantly less than the activities they displayed against the same organisms residing in MM6-M phi s. Notably, it was also found that KRM had a markedly larger MIC (0.25 microgram/ml) for MAC N-444 within A-549 cells than its MIC (0.008 microgram/ml) for the same strain residing in MM6-M phi s. Thus, the profiles of LVFX- and KRM-mediated killing or inhibition of intracellular MTB or MAC organisms in A-549 cells were markedly different from those observed for the organisms residing in MM6-M phi s. Second, invasive and multiplicative phenotypes of MTB and MAC organisms, which had been adapted to either an extracellular or intracellular environment (designated as E- and I-type organisms, respectively), were studied. In the case of MTB, I-type organisms (retrieved from infected MM6-M phi s after bacterial growth within the M phi s during 5-day cultivation) were less efficient than E-type organisms (prepared by cultivating the organisms in 7H9 medium) in entering MM6-M phi s, whereas I-type organisms were more efficient than E-type organisms in invading A-549 cells. On the other hand, in the case of MAC, infectivity of I-type organisms not only in MM6-M phi s but also in A-549 cells was larger than that of E-type organisms. Next, while I-type organisms of MTB and MAC displayed more vigorous replication within MM6-M phi s than E-type organisms, the growth rate of E-type organisms within A-549 cells was more rapid than that of I-type organisms residing in A-549 cells. These findings indicate that there are significant differences between E- and I-type organisms of MTB or MAC in ability to invade and multiply within M phi s (professional phagocytes) and alveolar epithelial cells (nonprofessional phagocytic cells).

A tuberculosis epidemic occurred among 4 relatives who live in the neighborhood of the index case. A thirty-three year old female was admitted to a hospital in July 1994 with high fever and cervical lymphoadenopathy. Culture examination of her sputum was positive for acid-fast bacilli and her chest X-ray showed diffuse small nodules. During the following sixteen months, five new patients with pulmonary tuberculosis were found among the relatives who lived in the neighborhood of the index case. The contact examination was first limited in her own family members, however, after detection of the second case, the examination was extended to other relatives living nearby, and another four patients were found. The results of PPD skin test of ten contact children showed strongly positive reaction, and chemoprophylaxis was indicated. Contacts examination is very important especially for patients with highly infectious tuberculosis.

In this study, we investigated 45 foreign patients who had been diagnosed as having tuberculosis in Chugoku-Shikoku area during the past 12 years. Regarding regional characteristics, in Hiroshima prefecture an epidemic of tuberculosis was experienced among patients coming from South America, but antituberculous therapy was performed for 87% of the patients because of the high coverage of the health insurance scheme. But in Okayama prefecture, most of the patients were female and came from Asian countries, such as, the Philippines. Antituberculous therapy was not performed for nine patients because of no coverage of the health insurance scheme. In the other prefectures, only a few cases of tuberculosis were experienced, but in Yamaguchi prefecture two of three foreign patients were multidrug-resistant tuberculosis.

We examined the effects of such drugs having anti-inflammatory activity as half-sized secretory leukocyte protease inhibitor (1/2 SLPI) and Chinese traditional medicines, Yokuinin (YOK) and Mao-Bushi-Saishin-To (MBST), on therapeutic efficacies of benzoxazinorifamycin KRM-1648 against Mycobacterium avium complex (MAC) infection induced in mice, since it is possible that these agents inhibit the increase in tissue levels of immunosuppressive cytokines due to MAC infection. First, Zymosan A-induced murine peritoneal macrophages treated with either 1/2 SLPI, YOK or MBST at 37 degrees C for 2 days were infected with M. avium N-444 and further cultivated in the medium with or without addition of 1/2 SLPI, YOK or MBST at 37 degrees C for up to 7 days. Treatment of macrophages with these drugs caused some decrease in the intracellular growth of the organisms. Secondly, we evaluated effects of 1/2 SLPI, YOK and MBST on the therapeutic efficacy of benzoxazinorifamycin KRM-1648 against M. avium infection induced in mice. When MAC-infected mice were given KRM-1648 (20 mg/kg) alone, or in combination with 1/2 SLPI (100 mg/kg), YOK (50 mg/kg), or MBST (50 mg/kg), by gavage, except for 1/2 SLPI which was given via i.p. route, once a week, from day 1 for up to 8 weeks after infection, these drugs did not affect the expression of therapeutic activity of KRM-1648. When MAC-infected mice were given KRM-1648 alone (once a week), or in combination with YOK (five times per week) or MBST (five times per week), MBST increased the expression of therapeutic activity of KRM-1648. These findings indicate that suppression of inflammatory reactions using MBST is capable to improve the therapeutic efficacy of KRM-1648 in MAC infection. Moreover, these results also mean that combined use of these drugs in MAC patients receiving KRM-1648 therapy may not cause any disadvantages to the therapeutic efficacy of KRM-1648.

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.

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.

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.

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.

Growth inhibition of the intracellular mycobacteria such as Mycobacterium tuberculosis, M. bovis, M. kansasii, M. avium, M. intracellulare, M. fortuitum, and M. chelonae subsp. abscessus by interferon-gamma (IFN-gamma)- or tumor necrosis factor-alpha (TNF-alpha)-treated murine peritoneal macrophages elicited by proteose peptone was studied in vitro. Macrophages were infected with slowly growing mycobacteria and the extracellular mycobacteria were washed out. Then, macrophages were treated with IFN-gamma or TNF-alpha at a concentration of 10 to 1000 U/ml for 2 days. In another experiment, macrophages were pretreated with these cytokines for 1 day then infected with rapidly growing mycobacteria as before. Macrophages were cultured with or without IFN-gamma or TNF-alpha for additional day. Mycobacterial growth was assessed by determination of colony-forming units on 7H11 agar plates after destruction of the macrophages. Stimulation of macrophages with IFN-gamma reduced the growth of mycobacteria. However, except for M. tuberculosis and M. bovis, growth was not inhibited by macrophages treated with TNF-alpha. IFN-gamma seems to be an important cytokine for the activation of mycobactericidal mechanisms in murine macrophages. Stimulation with IFN-gamma or TNF-alpha and subsequent phagocytosis of M. tuberculosis or M. intracellulare increased O2- production, which was assayed by the method of cytochrome C reduction by murine peritoneal macrophages. Phorbol myristate acetate-triggered-O2- production was also elevated by the cytokine pretreatment of the macrophages, suggesting that mycobacterial growth inhibition did not parallel the production of reactive oxygen intermediates in TNF alpha-activated murine peritoneal macrophages. These data suggest that bactericidal mechanisms of murine macrophages against nontuberculous mycobacteria may not depend on reactive oxygen intermediates.

The interaction of LFA-1 on T lymphocytes with ICAM-1 on antigen presenting cells (APCs) is critical in determining conjugate formation between the APCs and T cells as well as activation of T cells. Recently, it was found that stimulation of THP-1 cells, a human monocyte M phi cell line, with Mycobacterium tuberculosis or its lipoarabinomannan, elicited the increase in the ICAM-1 expression. In addition, in cases of lepromatous leprosy patients with a serious defect in the M. leprae antigen-specific cellular immunity, keratinocytes in the leprosy lesions were lacking in the ICAM-1 expression. Therefore, ICAM-1 seems to participate in the host response to mycobacterial infections. Here, we studied the mode of the expression of ICAM-1 molecules on murine peritoneal M phis in response to stimulation with M. avium complex (MAC). In addition, the regulatory effect of some cytokines including TNF-alpha, IL-10, and transforming growth factor-beta (TGF-beta) on the ICAM-1 expression was studied. Monolayer cultures of peptone-starch induced murine peritoneal M phis were cultured in RPMI-1640 medium in the presence of MAC with or without test agents. At intervals, the M phis were stained with anti-ICAM-1 antibody (Ab) and then treated with alkaline phosphatase (Ap)-conjugated anti-1g Ab. After color development with NBT-BCIP substrate, percentage of the blue-stained (ICAM-1 positive) M phis was determined microscopically. The concentrations of TNF-alpha, IL-10, and TGF-beta in the M phi culture fluid was measured by ELISA using capture Ab, biotin-labelled capping Ab, and Ap-conjugated streptavidin. When M phis infected with MAC organisms were cultured in RPMI medium containing 10% fetal bovine serum or in serum-free GPI medium, a significant increase in their ICAM-1 expression was observed, reaching the peak at days 1 to 3, thereafter rapidly decreased and returned to the normal level by day 14. Further addition of TNF-alpha caused no significant change in the mode of the MAC-induced expression of ICAM-1. A transient increase in the IL-10 production of MAC-infected M phis was observed during the first 3-days cultivation, as in the case of TNF-alpha. On the other hand, TGF-beta production of the Mø population was initiated from day 3, and therafter increased gradually until day 14. ICAM-1 expression of the MAC-infected M phis was not influenced by the addition of IL-10, while anti-IL-10 Ab retarded the decline of ICAM-1 expression at day 7. On the other hand, the addition of TCF-beta attenuated the MAC infection-induced increase in the ICAM-1 expression significantly. In addition, anti-TGF-beta Ab significantly delayed the reduction of the ICAM-1 expression of MAC-infected M phis during days 3 to 7. These results indicate that, in the MAC-infected M phis. TGF-beta rather than IL-10 play important roles as a mediators for the late-phase down-regulation of ICAM-1 expression which had been transiently elevated by the action of TNF-alpha in the early phase of the Mø cultivation.

In vitro antimicrobial activities of the benzoxazinorifamycin derivative KRM-1648 (KRM) against 50 strains of Mycobacterium tuberculosis isolated from patients with mainly intractable pulmonary tuberculosis were studied. MIC90 values of KRM, rifabutin (RBT) and rifampicin (RFP) for RFP-sensitive strains (27 strains; defined as those with MICRFP values of < 1.56 micrograms/ml) were 0.013, 0.1 and 0.4 micrograms/ml, respectively, when determined by the agar dilution method using 7H11 medium. MIC90 values of KRM, RBT, and RFP for RFP-resistant strains (23 strains; defined as those having MICRFP values of > or = 1.56 micrograms/ml) were 100, 12.5 and > 100 micrograms/ml, respectively. MICs of KRM against 50 clinical isolates of M. tuberculosis distributed over a much lower range than those of RFP. KRM showed more potent antimicrobial activity than RBT against the organisms with low MIC values (< or = 1.56 micrograms/ml), while it was not so active as RBT against the organisms with high MIC values (> or = 3.13 micrograms/ml). Cross-resistance between KRM and RFP or RBT was observed for M. tuberculosis.

The BBL MGIT Mycobacteria Growth Indicator Tube is a novel broth based culture system for the detection of mycobacteria from clinical specimens. The tubes consist of a fluorescent indicator embedded in silicone on the bottom of a 16x100 mm round-bottom tube, filled with 4ml of an enriched BBL Middlebrook 7H9 broth base, with 0.25% glycerol. Actively growing mycobacteria consume the oxygen dissolved in the medium and fluorescence will occur when the tubes are observed with a 365nm transilluminator. The purpose of this study is to evaluate comparatively MGIT with 1% Ogawa egg medium by using two hundred and forty-five clinical specimens. The samples were digested, decontaminated and concentrated for culture using N-acetyl-L-cysteine-sodium hydroxide method. Fifty-nine of 245 (24%) clinical samples were culture positive for mycobacteria (43 M. tuberculosis complex, 12 M. avium complex and 4 other species) by one or both test systems. The MGIT detected 4 isolates of M. tuberculosis complex and 6 isolates of M. avium complex not recovered by the Ogawa egg medium, respectively. The mean time of detection of M. tuberculosis complex was 13 days (range: 2-26 days) and 19 days (range: 8-31 days) for MGIT and Ogawa egg medium, respectively, and that of M. avium complex was 5 days (range: 2-8 days) and 16 days (range: 6-22 days) for the MGIT and Ogawa egg medium, respectively. Overall, the MGIT is a sensitive culture system for the detection of mycobacteria from clinical specimens, is easy to use and may be applicable to clinical laboratories.

Although various antimicrobial drugs show appreciable bactericidal activity in the early phase (2 to 4 weeks after infection) of Mycobacterium avium complex (MAC) infections in mice, no drug, as far as we know, can continue to exert the growth inhibiting activity against the bacteria at the site of infection in the progressed stage. Here, we studied the mechanisms of the bacterial regrowth which usually starts around 2-4 weeks after infection. First, the changes in the level of TNF-alpha, IFN-gamma, IL-6 and IL-10 in the lungs and spleen during the course of MAC infections was examined. Tissue levels of TNF-alpha and IL-10 increased around weeks 2 to 4, then rapidly decreased thereafter, and returned to the normal levels by week 8, while levels of IFN-gamma and IL-6 remained very low throughout the observation period. In this experiment, the bacterial CFUs rapidly decreased during the first 2 weeks of the treatment with a rifamycin derivative, KRM-1648, and thereafter the regrowth of the organisms was observed even in mice treated continuously with KRM-1648, although the rate of bacterial growth in the treated mice was much lower than in untreated control mice. Second, effect of either anti-TGF-beta or anti-IL-10 antibody on intracellular growth of MAC in human monocytes cultured in vitro in the medium with or without addition of TNF-alpha or IFN-gamma were examined. Anti-TGF-beta and anti-IL-10 antibodies potently reduced the bacterial growth in monocytes. Effects of TNF-alpha and IFN-gamma in reducing the bacterial growth was potentiated by the addition of either anti-TGF-beta or anti-IL-10 antibody. Third, anti-IL-10 antibody augmented to some extent the chemotherapeutic efficacy of KRM-1648 against MAC infection, when the drug was given to mice at weeks 2 and 4 after infection. From these results, it is suggested that IL-10 derived from MAC-infected macrophages in response to stimulation with some bacterial components, such as lipoarabinomannan, might downregulate the antimicrobial function of host macrophages against MAC.

JTP3309, a newly synthesized lipid A-subunit analogue, was examined for in vitro and in vivo antimycobacterial activities. Firstly, the effect of JTP3309 on the in vitro antimycobacterial activity of murine peritoneal macrophages (M phi s) was studied. When resident peritoneal M phi s from CBA mice which had phagocytosed Mycobacterium avium were treated with JTP3309 once from 0 hr to 24 hr after the initiation of M phi cultivation or three times from 0 hr to 24 hr, 48 hr to 72 hr and 96 hr to 120 hr of culture period, there was no increase in the anti-M. avium activity of the treated M phi s. On the other hand, the bacterial growth was slightly inhibited in peritoneal M phi s from mice which had been injected with JTP3309, in comparison with that seen in resident M phi s. Secondly, protective and therapeutic efficacies of JTP3309 against M. avium and M. fortuitum infections in mice were studied, based on the suppression of the bacterial growth in the visceral organs including the lungs, kidneys and spleen. BALB/c mice infected i.v. with M. avium were given JTP3309 i.v. at the doses of 10 and 20 micrograms/mouse according to the following protocols; protocol A, two injections 4 and 1 day(s) before the infection; protocol B, once daily at days 1, 3, 5, 12, 19, 26, 33, 40, 47 and 54 after the infection; protocol C, once daily at days 1, 8, 15, 22, 29, 36, 43 and 50 after the infection. Only protocol A could reduce the number of CFU recovered from the spleen of infected mice.(ABSTRACT TRUNCATED AT 250 WORDS)

We evaluated the in vitro antimicrobial activity of T-3761 and ofloxacin against representative pathogenic mycobacteria by the agar dilution method, using 7H11 agar medium. T-3761 showed appreciable antimicrobial activity against Mycobacterium tuberculosis (MIC90: 3.13 micrograms/ml), M. kansasii (MIC90: 6.25 micrograms/ml) and M. fortuitum (MIC90: 3.13 micrograms/ml), whereas the agent was not active against M. marinum (MIC90: 25 micrograms/ml), M. scrofulaceum (MIC90: 50 micrograms/ml), M. avium (MIC90: > 100 micrograms/ml), M. intracellulare (MIC90: > 100 micrograms/ml), M. chelonae subsp. abscessus (MIC90: > 100 micrograms/ml) and M. chelonae subsp. chelonae (MIC90: 50 micrograms/ml). The in vitro antimicrobial activity of T-3761 against M. fortuitum was a little more potent than that of ofloxacin, whereas the activity of T-3761 against the other mycobacteria was slightly inferior to that of ofloxacin. The antimycobacterial activity of T-3761 against M. tuberculosis H37Rv (MIC in vitro: T-3761 = 3.13 micrograms/ml, OFLX = 0.78 micrograms/ml) phagocytosed in murine peritoneal macrophages was less active than that of ofloxacin, when the macrophages were cultured in RPMI-1640 medium containing 1 microgram/ml or 10 micrograms/ml of these agents for up to 5 days. The activity of 10 micrograms/ml of T-3761 was equivalent to that of 1 microgram/ml of ofloxacin.

Because of the recent AIDS endemic, there is a worldwide increase in intractable mycobacterial infections including extrapulmonary tuberculosis due to multidrug-resistant M. tuberculosis and disseminated M. avium complex (MAC) infections. Therefore, development of new anti-tuberculous drugs having an excellent antimycobacterial activity and protocols for clinical use of presently available antimicrobials are urgently desired. In this study, we performed in vitro and in vivo experiments to assess the chemotherapeutic efficacy of a newly synthesized benzoxazinorifamycin derivative, KRM-1648 (KRM), against experimental infections due to MAC and M. tuberculosis. In addition, we attempted to improve the therapeutic efficacy of the KRM against MAC infections by changing its administration protocols and timing or by combined use with other antimicrobials, including clarithromycin (CAM), clofazimine (CFZ), sparfloxacin (SPFX), streptomycin (SM), and ethambutol (EB). Furthermore, we examined therapeutic efficacy of KRM against rifampicin (RFP)-resistant M. tuberculosis in details. KRM showing much more potent in vitro activity against the MAC organisms compared to rifabutin (RBT) and RFP, also exerted markedly greater therapeutic efficacy against the MAC infections induced in mice or rabbits in terms of reducing the incidence and the extent of gross pulmonary lesions and the bacterial loads in the lungs and spleens. However, in the case of mouse experimental infections, regrowth of the organisms was initiated after week 4 to 6 even in the animals given KRM. Since KRM-resistant organisms could not be isolated from infected mice given continuous KRM administrations, some unknown mechanisms other than the acquisition of drug resistance by infected organisms may be important for the establishment of the regrowth of MAC organisms.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to establish an animal model for disseminated M. avium complex (MAC) infections frequently encountered in AIDS patients, we studied growth of M. intracellulare in visceral organs (lungs, livers, spleens, kidneys), in blood, and in footpads of mice with defined immunodeficiencies, such as SCID mice with T and B cell-defect, BALB/c athymic nude mice with matured T cell-defect, and beige mice with NK cell-defect. In addition, Sprague-Dawley rats with acquired immunodeficiency induced by cyclosporine-treatment were also examined. The following results were obtained. 1) SCID mice: First, SCID mice were infected sc with 6.1 x 10(6) CFU of M. intracellulare N-260 (virulent SmT colonial variant) into the hind footpad. The organisms grew in the footpad remarkably during the 12 weeks after infection in SCID mice, where the growth rate was much greater than that in CB-17 strain mice with the same genotype as SCID mice and in BALB/c mice with Bcgs genotype (CB-17 and BALB/c mice are MAC-susceptible). Furthermore, in SCID mice, bacteremia and dissemination of organisms to the visceral organs were observed but not in the two control strains of mice. Second, SCID mice were infected i.v. with 4.8 x 10(6) CFU. The bacterial loads in the viscera of SCID mice after infection were larger than those of CB-17 mice except for livers. However, the incidence and the degree of gross lung lesions were much less in SCID mice compared to CB-17 mice, presumably due to the defect in T cell-mediated immune reactions in SCID mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Therapeutic efficacy of KRM-1648 was studied in BALB/c mice infected with Mycobacterium intracellulare. Mice were infected intravenously with 1.0 x 10(7) CFU of the organisms/mouse and then given 0.2 mg or 0.4 mg of KRM-1648 emulsified in 2.5% gum arabic-0.2% Tween 80 by gavage, once daily, 6 days per week, from day 1 to the death of mice. The therapeutic efficacy of the drug was evaluated on the basis of survival times, incidence and degree of gross lung lesions and bacterial loads in the lungs and spleen. The lung lesions were not observed in any experimental groups at 4 weeks after the infection. At 8 weeks after the infection, the lung lesions were observed in all control mice, but 2 of 5 mice treated with 0.2 mg of KRM and 4 of 5 mice treated with 0.4 mg of KRM did not show any lung lesions, and the degree of the lesions was much milder in KRM-1648-treated mice than in the control mice. All mice of treated and untreated groups died, and median survival times were 141 days for the control mice, 216 days for mice treated with 0.2 mg of KRM-1648 and 220 days for mice treated with 0.4 mg of KRM-1648. At the death, lung lesions were observed in all mice. The CFUs of M. intracellulare in the lungs and spleen in mice treated with KRM-1648 were fewer than those in the control mice at 4 and 8 weeks after the infection.(ABSTRACT TRUNCATED AT 250 WORDS)

A study was performed on the therapeutic efficacy of KRM-1648 combined with LC9018 in beige mice infected with Mycobacterium intracellulare. Each mouse was intravenously infected with 3.9 x 10(6) CFU, and then given 0.2 mg of KRM-1648 emulsified in 2.5% gum arabic-0.2% Tween 80 by gavage once a day, six times per week, beginning 24 h after infection for up to eight weeks. A sc injection of 0.1 mg LC9018 in saline was given once or twice per week, beginning two weeks before infection to the end of experiment. An evaluation of the therapeutic efficacy of these drugs against the infection was performed on the basis of incidence and degree of gross lung lesions and bacterial loads in the lungs and spleen. Lung lesions were not observed in all experimental groups at four weeks after infection. However, at eight weeks after infection, although lung lesions were observed in all experimental groups, the degree of lesions was much milder in those mice receiving KRM-1648 than in the control mice. The degree of lesions in the mice receiving LC9018 was much the same as that in the control mice. Moreover, the degree of lung lesions in mice treated with KRM-1648 alone. The CFUs of organisms in the lungs and spleen of the mice treated with KRM-1648 were considerably lower than those of the control mice (P < 0.05), but the CFUs in the mice treated with KRM-1648 and LC9018 were nearly the same as those mice treated with KRM-1648.

Functional changes in T lymphocytes and macrophages (M phi s) in host mice during the course of Mycobacterium intracellulare infection were studied. In both strains of mice, BALB/c or C57BL/6 (susceptible to M. avium complex) and CBA/JN or C3H/He (resistant to M. avium complex), the smooth, opaque and dome-shaped colonial (SmD) variants of M. intracellulare were easily eliminated from the sites after week 2 of infection. In contrast, the smooth, transparent and irregularly shaped colonial (SmT) variants showed steady growth in the former strains of mice and persisted for long time even in the latter strains of mice. No difference was found between persistence of the organisms in euthymic (+/+) and athymic (nu/nu) BALB/c mice during the first 4 weeks after infection. Thereafter, more rapid growth was seen in the spleens and lungs of nu/nu mice. Thus, matured T cells may be important for the prevention of the progression of M. intracellulare infection to the terminal state. Next, the profiles of generation and characteristics of splenic M phi s which suppress the Con A mitogenic response of splenic T cells in host CBA/JN or BALB/c mice during the course of M. intracellulare infection were investigated. In M. intracellulare--infected mice, reduction in some cellular functions of host splenic T cells, such as the Con A mitogenic response and mixed leucocyte reaction, were seen around 2 weeks after infection, and this was accompanied by appearance of immunosuppressive M phi s in spleen cells (SPCs).(ABSTRACT TRUNCATED AT 250 WORDS)

Recent AIDS endemic causes the worldwide increase in intractable mycobacterial infections including extrapulmonary tuberculosis due to drug resistant organisms and disseminated Mycobacterium avium complex infections. Therefore, new antituberculous (antimycobacterial) drugs and development of regimens and protocols for clinical treatment of such mycobacterial infections are urgently needed. Here, I described the present situations of new antituberculous agents, in particular new rifamycin derivatives including rifabutin and benzoxazinorifamycin (KRM-1648), new macrolides such as clarithromycin and azithromycin, and new quinolones including sparfloxacin, ofloxacin, ciprofloxacin, fleroxacin, AM-1155 and so on. Their in vitro antimycobacterial activities, therapeutic efficacy against experimental infections induced in animals especially in mice, and clinical trials using these drugs are summarized by referring to recent studies by worldwide investigators including us. Moreover, this paper dealt with some of recent attempts for chemotherapy of mycobacterial infections employing the drug delivery system using liposomal microvesicles as a carrier of drugs. Although these new drugs and development in new regimens appreciably potentiated the efficacy of controlling mycobacterial infections, in particular M. avium complex infections, it remains very difficult to achieve a complete elimination of the organisms from the sites of infection, even if provided multi-drug regimens using new drugs having excellent antimycobacterial activity and sufficient dosages. Therefore, it seems important to make an effort to elucidate the mechanisms of induction of immuno-unresponsiveness in hosts in progressed state of mycobacterial infection, as well as to develop new drugs possessing more potent antimycobacterial activity.

Mice were infected intravenously with M. intracellulare (5.2 x 10(6) CFU/mouse) and then were given 0.4 mg of KRM-1648 emulsified in 2.5% gum arabic-0.2% Tween 80 by gavage, once daily 1, 3 or 6 times per week, from 24h after infection to the end of experiment (week 8). Evaluation of the therapeutic efficacy of the drug against the infection was done on the basis of incidence and degree of gross lung lesions, organ weight (square root of organ (mg)/body (g) x 10), and bacterial loads in the lungs and spleen. The lung lesions were not observed in all experimental groups at 4 weeks after infection. At 8 weeks after infection, the lung lesions observed in all control and solute (2.5% gum arabic-0.2% Tween 80) control mice, whereas 3 of the 5 mice given KRM-1648, 3 times per week and all of 5 mice given KRM-1648, 6 times per week showed no lung lesions. Although lung lesions were observed in all mice given KRM-1648 only once per week, the degree of the lesions was much more milder in KRM-1648-treated mice than in solute control mice. The spleen weight of solute control mice and KRM-treated mice differed from each other 4 and 8 weeks after infection, especially in mice administered 6 times per week. The CFUs of organisms in the lungs and spleen were lower in mice treated with the agent than in mice given solute at 4 and 8 weeks after infection, in orders of administration of 6, 3 and 1 times per week.

Therapeutic efficacy of a newly synthesized benzoxazinorifamycin, KRM-1648, administered at the different periods of infection in Mycobacterium intracellulare-infected mice was studied. Mice were infected intravenously with M. intracellulare (9.8 x 10(6) CFU/mouse) and then were given 0.4 mg of KRM-1648 emulsified in 2.5% gum arabic-0.2% Tween 80 by gavage, once daily six times per week, from day 1 to week 4, week 2 to week 6, week 4 to week 8, and week 8 to the end of experiment (week 12). Judgement of the therapeutic efficacy of the drug against the infection was done on the basis of incidence and degree of gross lung lesions, % organ weight (organ weight/body weight x 100), and bacterial loads in the lungs and spleen. The lung lesions were not observed in the control and KRM-treated mice at 4 weeks after infection (KRM treatment: day 1 to week 4). At 6 weeks after infection (KRM treatment: week 2 to week 6), the lung lesions were observed in all control mice, whereas 3 of the 5 mice given KRM-1648 did not show the lesions. At 8 weeks after infection (KRM treatment: week 4 to week 8), the lung lesions were observed in all control and KRM-1648-treated mice, but the degree of the lung lesions was much more slight in mice given KRM-1648 than in control mice. The incidence and the degree of the lung lesions at 12 weeks after infection (KRM treatment: week 8 to week 12) was not different in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

We evaluated the in vitro antimicrobial activity of NM394 and ofloxacin (OFLX) against representative pathogenic mycobacteria by the agar dilution method, using 7H11 agar medium. NM394 showed appreciable antimicrobial activity against Mycobacterium tuberculosis (MIC90 = 0.78 micrograms/ml), M. kansasii (MIC90 = 6.25 micrograms/ml), M. marinum (MIC90 = 3.13 micrograms/ml) and M. fortuitum (MIC90 = 3.13 micrograms/ml), whereas the agent was not active against M. scrofulaceum (MIC90 = > 100 micrograms/ml), M. avium (MIC90 = 50 micrograms/ml), M. intracellulare (MIC90 = > 100 micrograms/ml), M. chelonae subsp. abscessus (MIC90 = > 100 micrograms/ml) and M. chelonae subsp. chelonae (MIC90 = 25 micrograms/ml). The in vitro antimicrobial activity of the agent against M. fortuitum was a little more active than that of OFLX, whereas the activity of NM394 against the other mycobacteria was slightly inferior to that of OFLX. The antimycobacterial activity of NM394 against M. tuberculosis H37Rv (MIC:NM394 = 0.78 micrograms/ml, OFLX = 0.78 micrograms/ml) phagocytosed in murine peritoneal macrophages was less active than that of OFLX, when the macrophages were cultured in RPMI-1640 medium containing 1 microgram/ml or 10 micrograms/ml of these agents for up to 5 days.

Various new quinolones were measured for their in vitro antimicrobial activities against Mycobacterium avium and M. intracellulare by the two-fold dilution methods using two types of media, 7H11 agar medium and 1% Ogawa egg medium. The MIC90s of test quinolones determined on 7H11 agar medium and Ogawa egg medium were as follows. M. avium (20 strains): ofloxacin (OFLX), 50 and 50 micrograms/ml; ciprofloxacin (CPFX), 12.5 and 25 micrograms/ml; sparfloxacin (SPFX), 6.25 and 12.5 micrograms/ml; fleroxacin (FLRX), 50 and 50 micrograms/ml; Y-26611, 100 and 100 micrograms/ml; OPC-17116, 12.5 and 50 micrograms/ml, for 7H11 agar and Ogawa egg medium, respectively. M. intracellulare (20 strains): OFLX, 50 and 50 micrograms/ml; CPFX, 25 and 25 micrograms/ml; SPFX, 12.5 and 12.5 micrograms/ml; FLRX, 50 and 50 micrograms/ml; Y-26611, > 100 and > 100 micrograms/ml; OPC-17116, 12.5 and > 100 micrograms/ml, for 7H11 agar and Ogawa egg medium, respectively. Thus, the MIC values determined by the 7H11 agar medium were comparable to those by the 1% Ogawa egg medium, except for OPC-17116. Moreover, from the above MIC values, it can be regarded that the potencies of in vitro antimicrobial activity of test quinolones against M. avium are in the order of SPFX > CPFX > or = OPC-17116 > or = OFLX > or = FLRX > Y-26611 and those against M. intracellulare are in the order of SPFX > OPC-17116 not equal to CPFX > OFLX = FLRX > Y-26611. There was found the tendency that M. avium has somewhat higher susceptibility to these quinolones as compared to M. intracellulare.

A new macrolide, clarithromycin (CAM), with increased acid-stability and thus having a markedly improved absorption efficiency from gastrointestinal tract, was evaluated for its in vitro antimicrobial activity against various mycobacterial species. CAM had nearly the same level of anti-mycobacterial activity as that of sparfloxacin (SPFX) and slightly higher activity than rifampicin (RFP), except that its anti-M. tuberculosis activity was much lower than those of SPFX and RFP. Anti-M. avium complex activity of CAM (MIC90 values against M. avium and M. intracellulare were 12.5 and 6.25 micrograms/ml, respectively) was in similar level as SPFX and RFP. However, MIC distribution pattern revealed that anti-M. avium activity was in the order of SPFX > CAM > RFP, while anti-M. intracellulare activities of them were almost the same with each other. Moreover, CAM showed bactericidal action against M. intracellulare growing in 7H9 medium. Furthermore, we investigated the therapeutic efficacy of CAM against M. intracellulare infection induced in mice and also determined its combined effect with other antimicrobials including KRM-1648, SPFX and ethambutol (EB). When CAM suspended in 5% gum arabic-saline was given s.c. to mice infected i.v. with M. intracellulare (8 x 10(6) CFU) at 0.2 to 2 mg/mouse/day, once daily six times per week from day 1 for 8 weeks, CAM exhibited a potent therapeutic efficacy, in terms of reduction in the incidence of gross lung lesions and reduction of bacterial loads in the visceral organs (reduction of bacterial CFU by 0.9-3.4 log units in the lung and by 0.4-4.6 log units in the spleen during week 4 to 8, depending on its administration dose).(ABSTRACT TRUNCATED AT 250 WORDS)

In order to know the possibility that gamma delta TCR+ T cells induced by Mycobacterium avium complex (MAC) infections participate in the expression of host resistance and in the occurrence of Behçet disease, we examined the behaviour of them in MAC-infected host mice. In both BALB/c (Bcgs; MAC-susceptible) and CBA/JN (Bcgr; MAC-resistant) strain mice, a transient but appreciable increase in the number of gamma delta TCR+ T cells in the host peritoneal lymphocytes was noted around week 1 to 2 after M. intracellulare infection via ip. route. The degree of induction of gamma delta TCR+ T cells was somewhat higher in CBA/JN mice than in BALB/c mice. Therefore, gamma delta TCR+ T cells are partly responsible for the expression of host resistance against the MAC in the early phase of infection. However, the subsequent decrease in the level of gamma delta TCR+ T cells was observed by week 5. Thus, in the case of chronic state of MAC infection, the size of gamma delta TCR+ T cell-pool seems to be in normal level. This suggests that per cell activity of gamma delta TCR+ T cells rather than mobilizing number of them is important factor in the mechanisms for occurrence of allergic diseases including Behçet disease. Although, the early increase in gamma delta TCR+ T cells of peritoneal cells was also observed during the course of M. fortuitum infection, the degree of induction of gamma delta TCR+ T cells in A/J mice (M. fortuitum-susceptible) was in similar level as that in BALB/c mice (M. fortuitum-resistant).(ABSTRACT TRUNCATED AT 250 WORDS)

Recent advance of technology in isolation and identification methods for mycobacteria, including BACTEC 460 TB System, polymerase chain reaction (PCR), DNA probe test, etc., enabled us to perform rapid and accurate identification of mycobacterial pathogens from various clinical specimens. Our recent study revealed usefulness of BACTEC 460 TB System, on the basis of the high efficiency in detecting the organisms in sputum specimens with much shortened recovery time, as compared to the Ogawa method. Moreover, accurate identification was possible, when the diagnostic system was combined with AccuProbe test. PCR using appropriate primers specific to Mycobacterium, M. tuberculosis complex (MTC), or M. avium complex (MAC) is useful to detect mycobacterial organisms directly in clinical specimens in a rapid manner. By this technique, in particular nested PCR, it is now possible to detect very small number of the organisms, as few as one bacterium, in test specimen, although it is still needed to resolve some difficulties due to contaminating inhibitors in the specimens. In our nested PCR system using Mav17 and Mav18 primers specific for Mycobacterium, especially MAC, we found that detectable degree of DNA amplification was possible even by replacing purified DNA sample, extracted from the organisms by considerably time consuming procedures, with bacterial suspension as a DNA template, by adding about 10(4) of organisms. The three DNA probe testings, Gen-Probe, AccuProbe and SNAP kits, are useful for rapid identification of MAC, MTC, M. kansasii and M. gordonae, and consistent results were obtained by these diagnostic kits. SNAP is able to distinguish the X component of MAC from the other two types of MAC.(ABSTRACT TRUNCATED AT 250 WORDS)

Y-26611, a newly developed fluoroquinolone having a morpholine moiety at the 7 position was examined for in vitro antimycobacterial activity by the agar dilution method using 7H11 medium. The MIC90 values of Y-26611 were as follows: Mycobacterium tuberculosis (25 strains), 0.4 micrograms/ml; M. kansasii (19 strains), 6.25 micrograms/ml; M. marinum (10 strains), 25 micrograms/ml; M. scrofulaceum (19 strains), 50 micrograms/ml; M. avium (18 strains), 50 micrograms/ml; M. intracellulare (31 strains), greater than 100 micrograms/ml; M. fortuitum (20 strains), 0.4 micrograms/ml; M. chelonae subsp. abscessus (15 strains), greater than 100 micrograms/ml; and M. chelonae subsp. chelonae (20 strains), 100 micrograms/ml. The MICs against M. tuberculosis and M. fortuitum were lower than those of ofloxacin (OFLX), although it had somewhat higher MICs against M. avium complex than OFLX. Antimicrobial activity of Y-26611 against M. tuberculosis phagocytosed in cultured murine peritoneal macrophages were somewhat lower, as compared to that of OFLX. When M. fortuitum-infected (iv) A/J mice were treated with Y-26611 by gavage at doses of 0.5-2 mg/mouse, once daily, six times per week, from day 1 for up to 4 weeks after infection, mice were protected from death and the number of CFU recovered from their visceral organs, such as the lungs, spleen and kidneys were reduced. The therapeutic efficacy of Y-26611 was similar as that of OFLX.

Fourty-five sputum specimens were subjected to isolation for mycobacteria either MB Check system (MB method; F. Hoffmann-La Roche Ltd., Basel, Switzerland) or 3% Ogawa egg medium (Ogawa method). Test sputum was treated with 4 volumes of 4% NaOH for 1-2 min and 0.1 ml of the resulting mixture was inoculated onto 3% Ogawa egg medium. The remaining portion of the mixture was neutralized with IN HCl, diluted with 1/15 M phosphate buffer (PB; pH 6.8), and subsequently centrifuged at 3,000 rpm for 20 min. The sediment was suspended in 1.5 ml of PB and 0.5 ml each was inoculated into MB Check M bottle (20 ml) supplemented with M supplement (1 ml). In MB method, bacterial growth was measured on Middlebrook 7H11 agar medium and Middlebrook 7H11 agar medium containing NAP (p-nitro-alpha-acetylamino-beta-hydroxy-propiophenone). Among 45 sputum specimens, the number of positive specimens for mycobacterial growth in the above two cultivation methods and time required for growth were as follows: 3% Ogawa egg medium; 12 specimens (26.7%) gave positive growth, including 7 of M. tuberculosis complex strains on 14-35 days (average 22 days) and 5 of M. avium complex strains on 14-21 days (average 18 days); MB method; 15 of specimens (33.3%) gave positive growth, including 8 of M. tuberculosis complex strains on 7-21 days (average 15 days), 6 of M. avium complex strains on 7-14 days (average 11 days) and 1 of M. scrofulaceum strain on 28 days. There was no specimen which was positive for mycobacterial growth on 3% Ogawa egg medium but negative on MB medium.

Fourty-five sputum specimens collected at the National Sanatorium Hiroshima Hospital were subjected to cultivation using either BACTEC 460 TB System (BACTEC method; Becton Dickinson Co., Towson, Md., U.S.A.) or 3% Ogawa egg medium. Test sputum was treated with four volumes of 4% NaOH for approximately two minutes, after which 0.1 ml of the treated sputum was immediately inoculated onto 3% Ogawa egg medium. After neutralizing the remaining pretreated sputum with 1N HCl, and diluting with 1/15 M phosphate buffer PB; pH 6.8), it was then centrifuged at 3,000 rpm for 20 min and the sediment was suspended in 1.5 ml of PB. Volumes of 0.5 ml each were inoculated into BACTEC 12B medium (4 ml), containing PANTA for prevention of contamination and POES for promoting the growth of mycobacteria. In the BBCTEC method, bacterial growth was measured in terms of increases in the Growth Index (GI) values which were determined by the amount of 14CO2 released from the 14C-labelled palmitate during cultivation at 37 degrees C (positive growth; GI greater than or equal to 50). Moreover, rho-nitro-alpha-acetylamino-beta-hydroxy-propiophenone (NAP)-sensitivity testing was done by transferring a part of the BACTEC 12B culture showing positive growth to a NAP vial, and thereafter subjected to further cultivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Macrophage chemiluminescence (M phi CL)-triggering activities of Mycobacterium avium complex (MAC) strains belonging to various serovars were examined. When SmT colonial variant (smooth, transparent, irregularly shaped) of M. intracellulare N-260 strain was compared with its SmD variant (smooth, opaque, dome-shaped) for M phi CL-triggering function and resistance to antimicrobial activity of murine resident peritoneal M phi s, the SmT variant showed much lower M phi CL-triggering activity accompanied by its high resistance to M phi microbicidal functions. Thus, difference in the virulence of the two MAC colonial variants seems to be originated from their different activities in M phi-triggering to be stimulated state in terms of O2-dependent antimicrobial functions. When disease-associated MAC strains belonging to serovars 1, 14, 16 (major serovars seen in Japan), 8 (intermediate serovar) and 9 (minor serovar) were challenged to mice, their virulence, in terms of mortality of host animals and growth of the organisms in the lungs, was nearly in the order of serovar 16, 14, 8, 1 and 9. However, there was found no obvious serovar-dependent difference in M phi CL-triggering activity of these MAC strains. Thus, no significant correlation was found between virulence of the MAC strains of various serovars and their triggering activities for M phi active oxygen production, which is important for the O2-dependent microbicidal mechanisms of host M phi s.(ABSTRACT TRUNCATED AT 250 WORDS)

Profiles of generation and characteristic of splenic macrophages (M phi s), which suppress the ConA mitogenic response of splenic T cells, induced in mice during the course of Mycobacterium avium complex infections were investigated. In MAC-infected mice, reduction of ConA mitogenesis of spleen cells (SPCs) was seen after week 2 and the reduced state thereafter durated as long as at least for 8 weeks. Splenic M phi s with a potent suppressive activity against SPC mitogenesis were transiently induced around 2 weeks after infection. Splenic M phi s of BALB/c (MAC-susceptible) mice had similar but somewhat weaker suppressor activity, as compared to those of CBA/JN (MAC-resistant) mice. The suppressor activity of BALB/c M phi s durated much longer than did CBA/JN M phi s. MAC-induced splenic M phi s were markedly elevated in chemiluminescence and this coincided with increase in immunosuppressive activity. These M phi s inhibited the generation of IL-2-reactive T cell populations in response to ConA-signals, without showing any inhibitory effect against IL-2 producing ability of target T cells. Because immunosuppressive M phi s having the same characteristics were also induced in BALB/c athymic nude mice, thereby indicating that mature T cells are not prerequisite for induction of the present suppressor M phi s in MAC-infected host mice. Prostaglandins (PGs), nitric oxide radical (NO.) and free long chain fatty acids are thought to play certain role in the expression of suppressor action of MAC-induced M phi s, because of the following reasons.(ABSTRACT TRUNCATED AT 250 WORDS)