Relationship between the isoniazid-resistant mutation katGS315T and the prevalence of MDR-/XDR-TB in Osaka, Japan.

OBJECTIVE: To determine the prevalence of katGS315T mutations in isoniazid (INH) resistant Mycobacterium tuberculosis and to elucidate the association of katGS315T mutations with the prevalence of multidrug-resistant tuberculosis (MDR-TB). DESIGN: From 2001 to 2004, 1655 isolates from all newly registered patients who visited the Osaka Prefectural Medical Centre for Respiratory and Allergic Diseases were tested for drug susceptibility. Genotyping was performed using insertion sequence (IS) 6110-restriction fragment length polymorphism (RFLP) in 1629 of 1655 (98.4%) cases. All 145 isolates of INH-resistant M. tuberculosis, including MDR strains, were tested to detect the katGS315T mutation. RESULTS: Five hundred and sixty isolates (34.4%) shared an RFLP pattern. Of the 145 INH-resistant isolates, 18/48 (37.5%) isolates belonging to the RFLP cluster had katGS315T and 23/97 (23.7%) did not have the mutation. Of the 66 MDR-TB cases, 18/29 (62.1%) isolates belonging to the RFLP cluster had katGS315T and 11/37 (29.7%) did not have the mutation. Of the 29 extensively drug-resistant (XDR) TB cases, 17/21 (80.9%) isolates belonging to the RFLP cluster had katGS315T and 3/8 (37.5%) did not have the mutation. CONCLUSION: The clustering rate by IS6110-RFLP was very high among MDR-/XDR-TB isolates with katGS315T. Our study indicates a strong correlation between the katGS315T mutation and the transmission dynamics of MDR-TB, and especially XDR-TB.

Frequency of tuberculin-reactive T-lymphocytes in pleural fluid and blood from patients with tuberculous pleurisy.

A limiting dilution assay was used to determine the frequency of T-lymphocytes reactive to purified protein derivative of tuberculin (PPD). Pleural fluid from patients with tuberculous pleurisy showed higher frequencies of PPD-reactive T-lymphocytes than peripheral blood from the same patients or tuberculin-positive healthy control subjects. The mean frequencies were 1/2,204 T cells in pleural fluid from tuberculous pleurisy, 1/14,970 T cells in peripheral blood from the same patients, and 1/13,130 T cells in peripheral blood from healthy controls. The concentration of tuberculin-reactive lymphocytes in tuberculous pleural fluid could represent selective accumulation or in situ expansion of this population of cell.

Increased circulating activated T-cells in lung cancer.

T-cell activation (Tac) antigens, which are closely associated with the receptors for interleukin 2 (IL 2) and expressed on activated human T-lymphocytes, are found on a small percentage of normal peripheral T-cells. Elevated levels of Tac antigen-positive (Tac+) cells were observed in a high proportion of patients with untreated primary lung cancer assessed by using monoclonal anti-Tac antibody. The mean percentage of Tac+ cells in peripheral blood lymphocytes was 13.1 +/- 6.4 percent in patients with primary lung cancer (n = 67), as compared with 4.3 +/- 1.9 percent in normal controls (n = 30) (p less than 0.001). No significant differences were observed among the cell types of lung cancer examined (adenocarcinoma and squamous and small cell carcinoma). The stages of the disease also showed no significant differences in the development of Tac+ cells. Our results suggest that T-cell-mediated active immune mechanisms against malignant cancer cells are operative in patients with lung cancer, resulting in an increase in activated T-cells in the peripheral blood, although it remains to be elucidated whether these activated T-cells exert a favorable or unfavorable effect on their host.

An analysis of in vitro T cell responsiveness in nontuberculous mycobacterial infection.

In vitro cell-mediated immunity was examined in patients infected with nontuberculous mycobacteria, Mycobacterium avium-intracellulare complex in Japan. Peripheral blood lymphocytes of patients, as compared with those of tuberculous patients or tuberculin-positive healthy donors, showed depressed in vitro blastogenic responses to purified protein derivative of tuberculin (PPD), not only to PPDs of Mycobacterium tuberculosis but also to PPD-B and PPD-Y of M intracellulare and M kansasii, respectively. Nonspecific lymphocyte blastogenic responses to concanavalin A, phytohemagglutinin and pokeweed mitogen were normal. Analysis of defective in vitro PPD-induced lymphocyte blastogenic responses in these patients revealed that PPD-induced interleukin 2 (IL-2) production was impaired whereas PPD-induced IL-2 responsiveness was normally developed after PPD stimulation. Therefore, addition of exogenous recombinant human IL 2 substantially recovered the in vitro depressed PPD-induced blastogenic responses in these patients with nontuberculous mycobacterial infection.

A raised level of soluble CD8 in bronchoalveolar lavage fluid in summer-type hypersensitivity pneumonitis in Japan.

We used ELISA to measure soluble CD8 (sCD8) in the bronchoalveolar lavage fluid (BALF) and serum of patients with summer-type hypersensitivity pneumonitis (HP). The sCD8 levels in BALF were significantly higher in the patients with summer-type HP, surpassing those found in sarcoidosis and the other pulmonary diseases studied; however, the sCD8 levels in the serum of patients with summer-type HP did not differ from the levels of the healthy controls. The numbers of CD8+ T cells were increased in the BALF of the patients with summer-type HP, and there was a correlation between the sCD8 levels and the concentrations of CD8+ T cells. Gel filtration and polyacrylamide gel electrophoresis of the fluid revealed that the anti-CD8 monoclonal antibody-reactive components in the BALF of patients with pneumonitis corresponded to a protein with a molecular weight of between 52 and 54 kDa. Soluble CD8-rich fraction purified from the BALF of patients with summer-type HP augmented in vitro lymphocytes' proliferative responses stimulated with Cryptococcus neoformans, one of the causative agents for summer-type HP. Our result suggests that soluble CD8 in the BALF may play an important role in the pathogenesis of summer-type HP.

Increased production of interleukin-10 by human blood monocytes stimulated with Mycobacterium avium-intracellulare complex.

Macrophages produce various cytokines in response to mycobacteria, including interleukin 10 (IL-10) and tumor necrosis factor alpha (TNF-alpha). IL-10 has been shown to down-regulate numerous macrophage functions, including microbicidal activity against intracellular bacteria and parasites. IL-10 also inhibits interferon-gamma (IFN-gamma) production and antigen-specific proliferation of Th1 cells mediating immunologic resistance to mycobacterial infection. In contrast, TNF-alpha activates macrophages and may augment their mycobacterial activity. In this study, peripheral blood mononuclear cells (PBMC) or blood monocytes obtained from healthy tuberculin reactors were stimulated in vitro with heat-killed Mycobacterium tuberculosis or heat-killed M. avium-intracellulare complex (MAC) to produce IL-10 and TNF-alpha. We studied a total of 26 clinical isolates of M. tuberculosis and 28 isolates of MAC. MAC-stimulated PBMC and monocytes released significantly larger amounts of IL-10 than those cells stimulated with M. tuberculosis. However, there was no difference in induction of TNF-alpha production between MAC and M. tuberculosis. When TNF-activity was neutralized by the addition of anti-TNF-alpha mAb in culture, MAC still induced more IL-10 secretion than did M. tuberculosis. These findings suggest that increased production of IL-10 by MAC-stimulated monocytes may play a role in the intractable disease caused by these organisms.

[Immunological aspects of Mycobacterium avium complex infection].

Tuberculin anergy is common in patients with Mycobacterium avium complex (MAC) infection. We examined in vitro cell-mediated immunity in these patients with MAC infection. Peripheral blood lymphocytes of patients, as compared with those of tuberculous patients or tuberculin-positive healthy donors, showed depressed in vitro blastogenic responses to purified protein derivative of tuberculin (PPD), not only to PPDs of Mycobacterium tuberculosis but also to PPD-B and PPD-Y of M. intracellulare and M. kansasii, respectively. Analysis of defective in vitro PPD-induced lymphocyte blastogenic responses in these patients revealed that PPD-induced interleukin 2 (IL-2) production was impaired whereas PPD-induced IL-2 responsiveness was normally developed after PPD stimulation. In the second half of this report, study was carried out to examine the mechanism of depressed T cell activity in these patients. Heat-killed MAC organisms and their lipid component impaired the capacity of peripheral blood lymphocytes to proliferate in vitro in response to concanavalin A (Con A), PPD, and to a lesser degree, phytohemagglutinin (PHA) stimulation. Inhibition by MAC was not contingent upon prior exposure of the donor to MAC or other mycobacteria and occurred with lymphocytes from tuberculin-negative as well as -positive subjects. The suppression was not due to the toxicity of MAC. Adherent cell depletion and cell mixing experiments with T cells indicated that monocytes and not T cells were a major contributor to the immunosuppression observed. Treatment of monocytes with MAC or MAC-derived lipid resulted in significant decreases in CD11b, a member of the LFA-1 and Leu M3 (CD14) molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

[Immunology of tuberculosis and cytokines].

One of the unique features characterizing human tuberculosis (TB) is its pathogenesis. The pathogenesis of TB involves cell-mediated immune responses against Mycobacterium tuberculosis. Concisely, macrophages activated by various soluble mediators or cytokines released through the cellular interactions after infection with M. tuberculosis play a pivotal role in the pathogenesis of human TB. In fact, very complex cellular interactions are going on within the host after infection with or endogenous reactivation of M. tuberculosis. Cells communicate by cell-cell contact and by the release of mediators which may originate locally, called cytokines. In TB infection, macrophages can be activated by two ways; directly with mycobacterial organisms or lipid fractions of their cell walls at the earlier phase of infection, and indirectly with cytokines produced by CD4+ T cells specifically activated by mycobacterial peptide antigens at the later phase of infection. The various clinical features of TB are the summarized outcome of cell to cell interactions mediated by diverse cytokines produced by various immune cells which are initially triggered by M. tuberculosis infection. CD4+ T cells can be classified into two subsets according to the patterns of cytokines they produce; Th1 cells give rise to cell-mediated immunity and are characterized by the production of IL-2 and IFN-gamma, whereas Th2 cells are more efficient in mediating antibody production and secrete IL-4, IL-5, IL-6 and IL-10. Th2 cells can control Th1 cells and vice versa. Th2 cells therefore inhibit the production of cytokines by Th1 cells by releasing IL-4 and IL-10. Infection with mycobacteria stimulates macrophage IL-12 production which appears to act directly on naive CD4+ T cells to induce Th1 development and initiation of cell-mediated immunity. IL-12 is a critical component in the development of cell-mediated immunity. In addition, IL-12 also activates NK cells and gamma/delta T cells, both of which secrete various macrophage-activating factors to kill M. tuberculosis. One of the structural characteristics of M. tuberculosis is the cell wall rich in lipid components. Of importance among various biological activities of the cell wall lipids is the stimulation of mononuclear phagocytes to produce a certain number of cytokines or monokines including IL-12 and IL-10, both of which play important roles in regulation of immune responses in mycobacterial infection and in pathogenesis of TB.(ABSTRACT TRUNCATED AT 400 WORDS)

[Immunotherapy for MDR-TB (multi-drug resistant tuberculosis)--its feasibility].

MDR-TB is known to be man-made-disease. Inappropriate treatment of tuberculosis is responsible for the development of MDR-TB. MDR-TB is often accompanied with the immunosuppression of the host. Given that we are unable to develop another potent anti-TB drug in near future, immunotherapy directed at combating immunosuppression and enhancing the host's own immune response is an attractive approach to supplement conventional chemotherapy for MDR-TB. Patients with AIDS and patients with abnormalities of macrophage function have frequent problems with TB. This is suggesting that the host defenses involved in protection against mycobacteria include T-cell and monocyte/macrophage functions. That is cell-mediated immunity. Diverse cytokines are known to play an important role in anti-TB cell-mediated immunity, including IL-2, IL-12, IL-18 and IFN-gamma. Various animal experiments are indicating that administration of these cytokine (s) did recover the suppressed immunity and rescued the host from death by tuberculous infection. However, we have to keep it in mind that the results obtained from animal model of mycobacterial infection on the study of pathogenesis and immune responses in TB is not always applicable to the understanding of human TB. Clinical trial of inhalation therapy with IFN-gamma showed some improvement for drug-resistant TB. Cytokine treatment, however, often gave some deleterious side effects such as high fever, malaise, general edema and even the death of the host. Clinical trials with M. vaccae have been extensively conducted by UK group. The mechanisms underlying its possible therapeutic action remain to be clarified, but when administered at an appropriate dose, it has been shown to elicit a strong Th1 immune response. From the practical view point of immunotherapy for TB, surrogate markers of disease eradication and protective immunity are urgently required. Such markers would facilitate clinical trials by providing early evidence that test compounds or vaccines are effective. Even during the era when no potent chemotherapeutic agents were available, one third of the patients with TB survived the disease and enjoyed the entire lives. Then the question is what determines the alternative: survival or death following development of drug resistant TB. Is it host immune responsiveness or virulence of the microbe, or both? Clearly much more work seems required before we are able to find some definite means to conquer MDR-TB in human.

[Clinical immunology of tuberculosis].

The standard tuberculin skin test has been known as the prototype of delayed type hypersensitivity testing which is mediated by T cells and macrophages and plays an important role in the pathogenesis of tuberculosis. Tuberculosis is indeed a chronic infectious disease, but variation in the host immune responses to tubercle bacilli results in the various clinical manifestations of the disease ranging from an immunologically hyperreactive state observed in pleural fluid lymphocytes in tuberculous pleurisy to an almost totally unresponsive state observed in those severely ill with refractory tuberculosis. In tuberculous pleurisy, T cells in pleural fluid respond remarkably in vitro to PPD tuberculin whereas T cells in peripheral blood responded poorly to PPD stimulation. Compartmentalization of PPD-reactive T cells in the pleural fluid and immunosuppression by T cells and/or macrophages in the peripheral blood were responsible for this immunological difference observed between the lymphocytes in pleural fluid and those in peripheral blood of tuberculous pleurisy. In advanced, drug-resistant tuberculosis as well as in nontuberculous mycobacterial infection, the proliferative responses of T cells in vitro to PPD stimulation were impaired. This depressed T cell response was due to depressed interleukin-2 (IL-2) production and not due to depressed IL-2 responsiveness. Therefore, the addition of exogenous IL-2, returned the depressed PPD-induced lymphocyte proliferation in vitro in these patients to the level of the response observed in lymphocytes from patients with newly-diagnosed tuberculosis. Our results suggest that recombinant IL-2 offers a novel approach to the therapy of advanced, drug-resistant tuberculosis and nontuberculous mycobacterial infection. Preliminary clinical trials of immunotherapy with recombinant IL-2 reveals the effectiveness of this therapy and encourages us to extend the trial to a larger scale. Tubercle bacilli have various biological activities. Research on tuberculosis and tubercle bacilli have contributed much to the progress of biochemistry, pathology and immunology. Mycobacterium is a fascinating organism, which now presents another big appeal to those studying immunology: Study of immunological interaction between gamma delta T cells and the highly conserved protein in mycobacteria, HSP, heat shock protein will contribute to the elucidation of the mechanism of immunological surveillance and the mechanism of autoimmune diseases. In addition, it will also contribute to the development of a new mycobacterial vaccine which will give direct, protective immunity against tuberculosis.

[Riddles in human tuberculous infection].

Tuberculosis is indeed an infectious disease caused by Mycobacterium tuberculosis. However, only a small percentage of individuals infected develops overt disease, tuberculosis whereas the infected bacilli persist alive years long within the vast majority of persons infected but remained healthy. There are several riddles or enigmas in the natural history of M. tuberculosis infection in humans. Some of them are as follows: 1. What is the virulence of M. tuberculosis? 2. How does M. tuberculosis persist dormant within the host? 3. What determines the development of disease from remaining healthy after infection with M. tuberculosis? 4. What is the mechanism of "endogenous reactivation" of dormant M. tuberculosis within the host? 5. Can we expect more potent anti-TB vaccine than BCG in near future? Most of these issues cited above remain unsolved. What is urgently needed today to answer correctly to these questions is the production of appropriate animal model of tuberculosis infection which mimics human tuberculosis. Murine TB does not reflect human TB at all. What characterizes the mycobacterial organism is its armour-plated unique cell wall structure which is rich in lipid and carbohydrate. Cord factor or trehalose dimycolate (TDM), the main component of cell wall, has once been regarded as the virulence factor of mycobacteria. Cord factor is responsible for the pathogenesis of TB and cachexia or even death of the patients infected. However, cord factor in itself is not toxic but exerts its detrimental effect to the host through the excessive stimulation of the host's immune system to produce abundant varied cytokines including TNF-alpha. How to evade this embarrassing effect of mycobacterial cell wall component on the host immune system seems very important for the future development of better TB vaccine than the currently used BCG.

[From the immunological aspect--interleukin-10 production by human blood mononuclear cells stimulated with multidrug-resistant Mycobacterium tuberculosis].

Interleukin-10 (IL-10) has been shown to down-regulate a number of different macrophage functions, including microbicidal activity against intracellular bacteria or parasites. In this study, peripheral blood mononuclear cells (PBMC) obtained from a healthy tuberculin-reactor were stimulated in vitro with various strains of multidrug-resistant Mycobacterium tuberculosis (MDRTB) or drug-sensitive M. tuberculosis (DSTB) to produce IL-10. We obtained one mycobacterial strain from each patient, preparing a total of 10 strains of DSTB, 5 strains of MDRTB. PBMC were cultured with LPS or mycobacterial preparations for 1, 3 and 5 days. IL-10 concentration in the culture supernatants was measured by ELISA using IL-10-specific monoclonal antibodies. The mean IL-10 production by PBMC stimulated with MDRTB was greater than that with DSTB, statistically significant at day 3 (MDRTB 171.4 +/- 18.2 pg/ml, DSTB 106.3 +/- 17.2 pg/ml, p < 0.05). Cell separation experiments indicated that cells producing IL-10 when stimulated with M. tuberculosis or LPS were monocytes, not T lymphocytes. Next, we examined PBMC from refractory tuberculosis patient who had continuously excreted MDRTB. PBMC from those patients secreted greater, but statistically not significant, amounts of IL-10 in response to LPS or TB bacilli than those of healthy subjects. Increased production of IL-10 by MDRTB-stimulated PBMC might be responsible for intractable tuberculosis.

[Comparison of the newly developed MYCOACID system with mycobacteria growth indicator tube (MGIT) and newly developed 2% Ogawa medium (S) for recovery of mycobacteria in clinical specimens].

The detection rate of mycobacteria from patients' specimens and the time required to get positive culture were compared among newly developed MYCOACID SYSTEM, MGIT, Ogawa K medium and 2% Ogawa medium (S). A total of 249 sputum samples taken from patients were used as the study subjects and 124 kinds of mycobacteria were isolated. For 135 cases clinically diagnosed as pulmonary tuberculosis, the detection rate was 44.4% for MYCOACID, 47.4% for MGIT and 38.5% for Ogawa K medium, showing that there are no significant differences in the detection rate between MYCOACID and MGIT, and MYCOACID and Ogawa K medium but the differences was significant between MGIT and Ogawa K medium (p = 0.02). The mean days needed for detection of Mycobacterium tuberculosis complex was 12.3 days for MYCOACID, 13.4 days for MGIT, and 26.8 days for Ogawa K medium, indicating significant differences in the time to get positive culture between Ogawa K medium and either of both liquid media (p < 0.001). Furthermore, 2% Ogawa medium (S) was used only for the detection of mycobacteria among previously untreated tuberculosis and there were no significant differences in the detection rate between 2% Ogawa medium (S) and either of both liquid media. The time to get positive culture for 2% Ogawa medium (S) was 18.2 days, which was longer than that for either of liquid media, MYCOACID and MGIT, but it was significantly shorter (7.9 days) than that for Ogawa K medium (p = 0.003). These results demonstrate that the liquid culture systems both MYCOACID and MGIT were very useful for the detection of mycobacteria compared with Ogawa K medium.

[Analysis of Mycobacterium tuberculosis-derived substance which induces interleukin-12 production from macrophages].

Protection of hosts against tuberculosis depends on expression of cellular immunity. To express cellular immunity, interleukin 12 (IL-12) has been shown to play an important role. Although Mycobacterium tuberculosis is known to induce IL-12 from macrophages (M phi s), the mechanism for the induction is still unclear. To understand the mechanisms of IL-12 induction from M phi s by M. tuberculosis, the IL -12-inducing ability of substances derived from M. tuberculosis was investigated in vitro. Production of IL-12 in culture medium of M phi s was measured by ELISA system using specific antibodies. Live M. tuberculosis H37Rv induced slightly higher IL-12 production than live M. tuberculosis H37Ra upon stimulation of human or mouse alveolar macrophages (hAM phi s or mAM phi s). Heat-killed M. tuberculosis failed to induce IL-12 production of alveolar macrophages (AM phi). The responses of hAM phi s and mAM phi s to M. tuberculosis were remarkably different. mAM phi s produced five times larger amount of IL-12, compared with that from hAM phi s. Human peripheral blood mononuclear cells (PBMC) obtained by the density gradient centrifugation were also used for induction of IL-12 production. Although production levels of IL-12 from PBMC stimulated with M. tuberculosis were below the detectable level, addition of interferon-gamma (IFN-gamma) or neutralizing antibody against IL-10 augmented the production of IL-12 from PBMC, suggesting that IFN-gamma and IL-10 regulate the production of IL-12 from M phi positively and negatively, respectively. To characterize the physicochemical properties of IL-12-inducing molecules, M. tuberculosis H37Rv was disrupted by pressing with 1,000 bar and centrifuged and separated into cytosol and cell wall fraction. The culture filtrate was also examined on IL-12-inducing activity. Among the three subjects examined, cytosol was found to induce the highest production of IL-12 from mAM phi s 1 day after the stimulation. Addition of IFN-gamma to the cytosol fraction markedly increased the production of IL-12 from mAM phi s. The molecular weight of IL-12-inducing substance was shown to be more than 30kDa by fractionating with molecular filters. Treatment of 30kDa-fraction with IL-12-inducing activity by proteinase K completely abolished the activity. Furthermore, approximately 90% of IL-12-inducing activity of 30kDa-fraction was lost by proteinase K treatment even in the presence of IFN-gamma. These results indicate that the major component of IL-12-inducing activity is a protein. The identification of this IL-12-inducing active substance may provide a new therapeutic tool for tuberculosis.