DNA gyrase could be a crucial regulatory factor for growth and survival of Mycobacterium leprae.

Leprosy, an important infectious disease in humans caused by Mycobacterium leprae (Mle), remains endemic in many countries. Notably, the pathogen cannot be cultured in vitro, except in mouse footpads in vivo. The molecular basis of these characteristics and the mechanisms remain unknown. Consequently, analysis of Mle growth and survival is urgently needed to develop novel therapies against leprosy, including rapid, simple, and specific methods to detect infection. Here, we demonstrated the functional role and contribution of Mle-DNA gyrase, which regulates DNA topology, DNA replication, and chromosome segregation to promote bacterial growth and survival, in Mle growth and survival in vitro and in vivo. The optimum temperature for Mle-DNA gyrase activity was 30 °C. When the DNA gyrB-gyrA genes in Mycobacterium smegmatis were replaced with the Mle gyrase genes by allelic exchange, the recombinants could not grow at 37 °C. Moreover, using radiorespirometry analysis for viability of Mle bacilli, we found that Mle growth was more vigorous at 25-30 °C than at 37 °C, but was inhibited above 40 °C. These results propose that DNA gyrase is a crucial factor for Mle growth and survival and its sensitivity to temperature may be exploited in heat-based treatment of leprosy.

An in vitro model of Mycobacterium leprae induced granuloma formation.

BACKGROUND: Leprosy is a contagious and chronic systemic granulomatous disease caused by Mycobacterium leprae. In the pathogenesis of leprosy, granulomas play a key role, however, the mechanisms of the formation and maintenance of M. leprae granulomas are still not clearly understood. METHODS: To better understand the molecular physiology of M. leprae granulomas and the interaction between the bacilli and human host cells, we developed an in vitro model of human granulomas, which mimicked the in vivo granulomas of leprosy. Macrophages were differentiated from human monocytes, and infected with M. leprae, and then cultured with autologous human peripheral blood mononuclear cells (PBMCs). RESULTS: Robust granuloma-like aggregates were obtained only when the M. leprae infected macrophages were co-cultured with PBMCs. Histological examination showed M. leprae within the cytoplasmic center of the multinucleated giant cells, and these bacilli were metabolically active. Macrophages of both M1 and M2 types co-existed in the granuloma like aggregates. There was a strong relationship between the formation of granulomas and changes in the expression levels of cell surface antigens on macrophages, cytokine production and the macrophage polarization. The viability of M. leprae isolated from granulomas indicated that the formation of host cell aggregates benefited the host, but the bacilli also remained metabolically active. CONCLUSIONS: A simple in vitro model of human M. leprae granulomas was established using human monocyte-derived macrophages and PBMCs. This system may be useful to unravel the mechanisms of disease progression, and subsequently develop methods to control leprosy.

A lipopeptide facilitate induction of Mycobacterium leprae killing in host cells.

Little is known of the direct microbicidal activity of T cells in leprosy, so a lipopeptide consisting of the N-terminal 13 amino acids lipopeptide (LipoK) of a 33-kD lipoprotein of Mycobacterium leprae, was synthesized. LipoK activated M. leprae infected human dendritic cells (DCs) to induce the production of IL-12. These activated DCs stimulated autologous CD4+ or CD8+ T cells towards type 1 immune response by inducing interferon-gamma secretion. T cell proliferation was also evident from the CFSE labeling of target CD4+ or CD8+ T cells. The direct microbicidal activity of T cells in the control of M. leprae multiplication is not well understood. The present study showed significant production of granulysin, granzyme B and perforin from these activated CD4+ and CD8+ T cells when stimulated with LipoK activated, M. leprae infected DCs. Assessment of the viability of M. leprae in DCs indicated LipoK mediated T cell-dependent killing of M. leprae. Remarkably, granulysin as well as granzyme B could directly kill M. leprae in vitro. Our results provide evidence that LipoK could facilitate M. leprae killing through the production of effector molecules granulysin and granzyme B in T cells.

Apoptosis-inducing activity of clofazimine in macrophages.

Clofazimine is a riminophenazine compound which has been used for the treatment of leprosy since the 1960s. Although the drug is effective in the management of leprosy reactions because of its anti-inflammatory activity, the mechanism leading to the cessation of inflammation is not well understood. In the present study, it was shown that clofazimine exhibits apoptosis-inducing activity in macrophages. When human monocyte-derived macrophages were cultured in vitro in the presence of clofazimine, the cells exhibited a marked decrease in metabolic activity and showed shrinkage in cell size, indicating cell death. Nuclear condensation and fragmentation were also observed by Giemsa and Hoechst 33248 stains. The endonuclease inhibitor ZnCl(2) inhibited the clofazimine-induced cell death. Significant enhancement of caspase-3 activity was observed in clofazimine-treated macrophages and THP-1 cells. Collectively, these results suggest the apoptosis-inducing activity of clofazimine in macrophages, which may also be responsible for the antibacterial properties of clofazimine.

Analysis of drug-resistant strains of Mycobacterium leprae in an endemic area of Vietnam.

BACKGROUND: Multidrug therapy has effectively reduced the number of leprosy cases in the world. However, the rate of reduction has decelerated over the years, giving early detection of Mycobacterium leprae and epidemiological study of relapse renewed relevance in attempts to eliminate the disease. METHODS: A molecular epidemiological survey for drug-resistant M. leprae was conducted in the central and highland regions of Vietnam. A total of 423 samples taken from patients, including 83 patients with new cases, 321 patients receiving treatment, and 19 patients with relapse, were studied for detection of M. leprae with mutations relating to drug resistance by sequencing the drug resistance determining region of the folP1, rpoB, and gyrA genes, which are responsible for dapsone, rifampicin, and ofloxacin resistance, respectively. RESULTS: Nineteen mutations were found in the folP1 gene samples, and no mutations relating to drug resistance were found in either the rpoB or gyrA genes. Samples from patients with relapse showed folP1 mutation rates as high as 57%, and the mutation rates in samples from new and recent cases were <10%. Patients with relapse who had histories of treatment with dapsone monotherapy showed high mutation rates (78%), compared with patients with relapse who had previously only received multidrug therapy (33%). CONCLUSIONS: Our study indicated high rates of dapsone resistance in patients with relapse, compared with patients with new and recent cases of leprosy. Moreover, it was observed that many of the patients with relapse who had dapsone-resistant mutations had histories of treatment with dapsone monotherapy.

[Forefront of vaccine development: tuberculosis and leprosy].

The role of vaccines to tuberculosis and leprosy is to induce a cellular immunity, and as a result to induce the differentiation of memory CD8+ cytotoxic T cells. 'Help' from CD4+ T cells is important for the differentiation of naive CD8+ T cells to effector and memory CD8+ cytotoxic T cells. However, how CD4+ T cell 'help' is involved in the steps instructing T helper (Th) polarization is not yet clear. Peptide-25, a major Th epitope of Ag85B from Mycobacterium tuberculosis, preferentially induced development of Th1 cells. In contrast, altered peptide ligands (APL) that have a substitution of glycine for alanine at position 248 of Peptide-25 induced solely Th2 development. To elucidate the role of Th polarization on the 'Help' function of CD4+ T cells, we established an in vitro culture system using OVA specific CD8+ T cells, Peptide-25 specific CD4+ T cells and splenic dendritic cells (DCs). The DCs that were pre-cultured with Peptide-25 specific CD4+ T cells together with OVA and Peptide-25 induced the proliferation and granzyme B production of OVA specific CD8+ T cells. On the other hand, the DCs that were pre-cultured with Peptide-25 specific CD4+ T cells together with OVA and APL induced only proliferation of OVA specific CD8+ T cells. These results suggest that Th1 immune response induced by Peptide-25 plays an important role in the induction of functional activation of CD8+ cytotoxic T cells.

Temperature dependency for survival of Mycobacterium leprae in macrophages.

Hansen's disease is caused by an infection with an intracellular pathogen, Mycobacterium leprae, which mainly inhabits macrophages and Schwann cells. However, little is known about the survival or growth mechanisms of the bacilli in mouse and human macrophages. In the present study, by using radiorespirometry analysis for the evaluation of the viability of M. leprae, we observed that in vitro incubation of M. leprae-infected macrophages at 35 degrees C was more growth permissive than at 37 degrees C, and supplementation with the immunosuppressive cytokine IL-10 supported the survival of the bacilli in the macrophages for 3 weeks, whereas viability of the bacilli was gradually lost if cultured without IL-10. In human macrophages, M. leprae retained its viability when cultured at 35 degrees C for at least 4 weeks without IL-10. However, the viability of M. leprae was almost lost within 2 weeks if cultured at 37 degrees C. These data suggest that temperature is a crucial factor for the survival of M. leprae in host cells.

Detection of serum antibodies to M. leprae major membrane protein-II in leprosy patients from Indonesia.

BACKGROUND: Sero-diagnostic methods are the easiest way of diagnosing an infectious disease in developing countries. In leprosy, phenolic glycolipid-1 (PGL-I) based methods for the detection of leprosy are currently available, but the use of these methods has been hindered due to the inherent problems of sensitivity. We previously showed that antibodies to Major Membrane Protein-II (MMP-II) derived from Mycobacterium leprae could be used to diagnose leprosy in Japan. METHODS: Sera from patients and healthy individuals were collected with informed consent and the anti-MMP-II antibody levels of the sera were measured by enzyme-linked immunosorbent assay. The study was conducted at South Sulawesi and Bali, in Indonesia. The study population included 40 each of multibacillary leprosy and paucibacillary leprosy patients, 30 tuberculosis and 16 patients with typhoid. RESULTS: We evaluated the anti-MMP-II antibody levels in Indonesian individuals. The cut-off value was determined from receiver operator characteristic curve as 0.124 using the O.D. titers for patients with multibacillary leprosy, so that the sensitivity of the test was 97.5% and the specificity taking healthy individuals as controls was 984%. Using the determined cut-off values, 98% of multibacillary (MB) leprosy and 48% of paucibacillary (PB) leprosy patients had positive levels of anti-MMP-II antibodies, 13% of patients with typhoid and 22% of the household contacts of MB leprosy had positive levels of anti-MMP-II antibodies. CONCLUSIONS: Our results suggest that measuring anti-MMP-II antibody levels could facilitate the detection of leprosy in endemic countries.

Serological diagnosis of leprosy in patients in vietnam by enzyme-linked immunosorbent assay with Mycobacterium leprae-derived major membrane protein II.

A serological diagnostic test using phenolic glycolipid-I (PGL-I) developed in the 1980s is commercially available, but the method is still inefficient in detecting all forms of leprosy. Therefore, more-specific and -reliable serological methods have been sought. We have characterized major membrane protein II (MMP-II) as a candidate protein for a new serological antigen. In this study, we evaluated the effectiveness of the enzyme-linked immunosorbent assay (ELISA) using the MMP-II antigen (MMP-II ELISA) for detecting antibodies in leprosy patients and patients' contacts in the mid-region of Vietnam and compared to the results to those for the PGL-I method (PGL-I ELISA). The results showed that 85% of multibacillary patients and 48% of paucibacillary patients were positive by MMP-II ELISA. Comparison between the serological tests showed that positivity rates for leprosy patients were higher with MMP-II ELISA than with PGL-I ELISA. Household contacts (HHCs) showed low positivity rates, but medical staff members showed comparatively high positivity rates, with MMP-II ELISA. Furthermore, monitoring of results for leprosy patients and HHCs showed that MMP-II is a better index marker than PGL-I. Overall, the epidemiological study conducted in Vietnam suggests that serological testing with MMP-II would be beneficial in detecting leprosy.

Evaluation of major membrane protein-II as a tool for serodiagnosis of leprosy.

As serodiagnosis is the easiest way of diagnosing a disease, the utility of Mycobacterium leprae-derived major membrane protein-II (MMP-II), one of the immuno-dominant antigens, in the serodiagnosis of leprosy was examined. The percent positivity by an enzyme-linked immunosorbent assay for anti-MMP-II antibody was 82.4% for multi-bacillary leprosy, and the specificity of the test was 90.1%. For pauci-bacillary leprosy where cell-mediated immunity predominates, 39.0% showed positive results. These percentage values were significantly higher than these values obtained for existing phenolic glycolipid-I based methods, suggesting that MMP-II antibody detection would facilitate the diagnosis of leprosy.

Contribution of GM-CSF on the enhancement of the T cell-stimulating activity of macrophages.

Mycobacterium leprae is an intracellular parasitic organism that multiplies in macrophages (MØ). It inhibits the fusion of mycobacterial phagosome with lysosome and induces interleukin (IL)-10 production from macrophages. However, macrophages are heterogenous in various aspects. We examined macrophages that differentiated from monocytes using either recombinant (r) granulocyte-MØ colony-stimulating factor (GM-CSF) (these MØ are named as GM-MØ) or rMØ colony-stimulating factor (M-CSF) (cells named as M-MØ) in terms of the T cell-stimulating activity. Although both macrophages phagocytosed the mycobacteria equally, GM-MØ infected with M. leprae and subsequently treated with IFN-gamma- and CD40 ligand (L) stimulated T cells to produce interferon-gamma (IFN-gamma), but M-MØ lacked the ability to stimulate T cells. While M-MØ mounted a massive IL-10 production, GM-MØ did not produce the cytokine on infection with M. leprae. M. leprae-infected, IFN-gamma- and CD40L-treated GM-MØ expressed a higher level of HLA-DR and CD86 Ags than those of M-MØ, and expressed one of the dominant antigenic molecules of M. leprae, Major Membrane Protein-II on their surface. These results indicate that GM-CSF, but not M-CSF, contributes to the up-regulation of the T cell-stimulating activity of M. leprae-infected macrophages.

[Current advances in leprosy research activities].

Due to the advent of multi-drug therapy (MDT) recommended by the WHO, for the treatment of leprosy, presently, leprosy is regarded as a "curable disease". The number of new cases in Japan is relatively very low, due to which the disease is likely to be neglected, but on scientific grounds, there is a necessity to perform in depth studies. Leprosy caused by M. leprae is still unclear on various aspects including transmission, immunology, nerve damage etc. Here we introduce the recent advances in the field of basic leprosy research.

[Functional changes of macrophages in Hansen's disease].

As an obligate intracellular pathogen, the principal host cells for Mycobacterium leprae are mononuclear phagocytes or macrophages. The macrophage is a primitive cell type being found in both early and advanced life forms, and possesses a variety of functions, such as phagocytosis of invaded bacteria, production of cytokines, antigen presentation and tumor killing. Hansen's disease is a chronic infectious disease characterized by specific host immune responses against M. leprae. In this article the macrophage is focused to dissect its functions in the disease.

Active surveillance of leprosy contacts in country with low prevalence rate.

For advanced control of leprosy in Pakistan where the World Health Organization leprosy elimination goal was achieved in 1996, we conducted surveillance of Mycobacterium leprae-seropositive patients and their contacts and drug resistant strains of M. leprae. We measured anti-PGL-I antibody level in sera from leprosy patients and their contacts for early detection of M. leprae infection. Out of 34 leprosy patients undergoing treatment, 4 lepromatous leprosy patients were antibody positive, and 6.8 to 23.7 percent of occupational or household contacts were seropositive. Furthermore, three cases (1.2%) had a high antibody titer. For surveillance of drug resistant strains of M. leprae, dapsone and rifampin were targeted. Four out of 18 polymerase chain reaction (PCR) positive samples had mutation in folP gene, and among 10 PCR positive samples, one had a mutation in the rpoB gene. These results indicate that serological analysis of patient contacts might be useful to find out high risk individuals, and there are M. leprae strains resistant to chemotherapeutic agents in Pakistan.

IL-10 treatment of macrophages bolsters intracellular survival of Mycobacterium leprae.

In these studies, metabolically active Mycobacterium leprae were maintained for as long as 8 weeks in monolayer cultures of mouse peritoneal macrophages (MPhi). Supplemental IL-10, but not TGF-beta, bolstered, directly or indirectly, M. leprae metabolism in mouse MPhi. In the cell culture system temperature setting is extremely important and 31 to 33 degrees C incubation temperature was more permissive than 37 degrees C. Acid fast staining and transmission electron microscopy (TEM) of intracellular M. leprae revealed visible elongation of bacilli cultured under the above ideal conditions.

Dharmendra antigen but not integral M. leprae is an efficient inducer of immunostimulant cytokine production by human monocytes, and M. leprae lipids inhibit the cytokine production

Killed integral Mycobacterium leprae, Mitsuda antigen, and chloroform-treated M. leprae, Dharmendra antigen (Dh-Ag), have been used for the classification of leprosy patients based on cell-mediated immunity. Heat-killed M. leprae also were used as a component of the Convit vaccine. Human blood monocytes were stimulated with M. leprae or Dh-Ag and their cytokine-inducing ability was compared. Monocytes were cultured in the presence of fresh human serum because of the efficiency of cytokine induction and the phagocytosis of M. leprae have been shown to be optimal in the presence of fresh serum. M. leprae and Dh-Ag were equally phagocytosed by monocytes. Dh-Ag was more potent than M. leprae in the induction of immunostimulatory/proinflammatory cytokines, interleukin-1 (IL-1), IL-6 and tumor necrosis factor (TNF). In contrast, a comparable level of IL-1ra, an immunosuppressive cytokine, was induced by M. leprae and Dh-Ag. The lipids extracted from M. leprae induced none of these cytokines by monocytes. Nevertheless, when monocytes were pretreated with the lipids followed by stimulation with Dh-Ag, productions of IL-1, IL-6 and TNF were all inhibited in a dose-dependent manner. However, the lipids did not inhibit the cytokine production induced by other stimuli including BCG and lipopolysaccharide. Moreover the lipids did not affect the production of IL-1ra. These results suggest that the lipids from M. leprae are responsible for the poor cytokine-inducing ability of M. leprae, thus favoring their infection. These results also suggest that Dh-Ag rather than integral M. leprae may be useful as a vaccine candidate because Dh-Ag is able to induce a large amount of cytokines from monocytes.