[Guidelines for the treatment of Hansen's disease in Japan (third edition)].

ad hoc committee of Japanese Leprosy Association recommends revised standard treatment protocol of leprosy in Japan, which is a modification of World Health Organization's multidrug therapy (WHO/MDT, 2010). For paucibacillary (PB) leprosy, 6 months treatment by rifampicin and dapsone (MDT/PB) is enough. However, for high bacterial load multibacillary (MB) leprosy, 12 months treatment seems insufficient. Thus, (A) For MB with bacterial index (BI) > 3 before treatment, 2 years treatment by rifampicin, dapsone and clofazimine (MDT/MB) is necessary. When BI becomes negative and active lesion is lost within 2 years, no maintenance therapy is necessary. When BI is still positive, one year of MDT/MB is added (3 years in total), followed by maintenance therapy by dapsone and clofazimine until BI negativity and loss of active lesions. (B) For MB with BI < 3 or fresh MB (less than 6 months after the onset of the disease) with BI > 3, 1 year treatment by MDT/MB is necessary. When BI becomes negative and active lesion is lost within one year, no maintenance therapy is necessary. When BI is still positive or active lesion is remaining, additional therapy with MDT/MB for one more year is recommended. Brief summary of diagnosis, purpose of therapy, character of drugs, and prevention of deformity is also described.

Clofazimine modulates the expression of lipid metabolism proteins in Mycobacterium leprae-infected macrophages.

Mycobacterium leprae (M. leprae) lives and replicates within macrophages in a foamy, lipid-laden phagosome. The lipids provide essential nutrition for the mycobacteria, and M. leprae infection modulates expression of important host proteins related to lipid metabolism. Thus, M. leprae infection increases the expression of adipophilin/adipose differentiation-related protein (ADRP) and decreases hormone-sensitive lipase (HSL), facilitating the accumulation and maintenance of lipid-rich environments suitable for the intracellular survival of M. leprae. HSL levels are not detectable in skin smear specimens taken from leprosy patients, but re-appear shortly after multidrug therapy (MDT). This study examined the effect of MDT components on host lipid metabolism in vitro, and the outcome of rifampicin, dapsone and clofazimine treatment on ADRP and HSL expression in THP-1 cells. Clofazimine attenuated the mRNA and protein levels of ADRP in M. leprae-infected cells, while those of HSL were increased. Rifampicin and dapsone did not show any significant effects on ADRP and HSL expression levels. A transient increase of interferon (IFN)-ß and IFN-γ mRNA was also observed in cells infected with M. leprae and treated with clofazimine. Lipid droplets accumulated by M. leprae-infection were significantly decreased 48 h after clofazimine treatment. Such effects were not evident in cells without M. leprae infection. In clinical samples, ADRP expression was decreased and HSL expression was increased after treatment. These results suggest that clofazimine modulates lipid metabolism in M. leprae-infected macrophages by modulating the expression of ADRP and HSL. It also induces IFN production in M. leprae-infected cells. The resultant decrease in lipid accumulation, increase in lipolysis, and activation of innate immunity may be some of the key actions of clofazimine.

[Structure and anti-M. leprae activity relationships of new quinolones].

Due to the emergence of drug resistant M. leprae, there is a need to look for new drugs for the treatment of leprosy. We evaluated the effectiveness of new quinolones in vitro as well as in vivo. The in vitro and in vivo results suggested that a cyclopropyl group at the 1-position, COOH at the 3-position, OH at the 4-position, NH2 or OH-substitutions at the 5-position, F at the 6-position, 5- and 6-membered rings at the 7-position, halogen (F or Cl) or OCH3 at the 8-position of the quinolone core structure remarkably enhance anti-M. leprae activities of the drug.

[In-vitro and in-vivo activities of moxifloxacin and garenoxacin against Mycobacterium leprae].

Moxifloxacin(MFLX) and garenoxacin(GRNX), new synthetic antibacterial agents, were assessed for in vitro anti-M. leprae activities. The anti-bacterial activities of these two drugs were compared to those of sparfloxacin (SPFX), gatifloxacin(GFLX), levofloxacin(LVFX) and rifampicin (RFP). The anti-M leprae activity obtained by Buddemeyer system was stronger in order of RFP, MFLX, SPFX, GFLX and GRNX and LVFX. The anti-M. leprae activity of MFLX or GRNX was also examined by the nude mouse footpad method. MFLX completely inhibited the growth of M. leprae inoculated into nude mouse footpads, when given orally at a daily dose of 10 mg/kg , while GRNX completely inhibited at a daily dose of 60 mg/kg. Both in vitro and in vivo tests indicated that MFLX was equal or superior to SPFX, but GRNX was equipotent to LVFX, in terms of anti-M. leprae activities.

[Guideline for the treatment of Hansen's disease in Japan (Second edition)].

ad hoc committee of Japanese Leprosy Association recommends revised standard treatment protocol of leprosy in Japan, which is a modification of World Health Organization's multidrug therapy (WHO/MDT, 1997). For paucibacillary (PB) leprosy, 6 months treatment by rifampicin and dapsone (MDT/PB) is enough. However, for high bacterial load multibacillary (MB) leprosy, 12 months treatment seems insufficient. Thus, (A) For MB with bacterial index (BI) > or = 3 before treatment, 2 years treatment by rifampicin, dapsone and clofazimine (MDT/MB) is necessary. When BI become negative and active lesion is lost within 2 years, no maintenance therapy is necessary. When BI is still positive, one year of MDT/MB is added (3 years in total), followed by maintenance therapy by dapsone and clofazimine until BI negativity and loss of active lesions. (B) For MB with BI < 3 or fresh MB (less than 6 months after the onset of the disease) with BI > or = 3, 1 year treatment by MDT/MB is necessary. When BI become negative and active lesion is lost within one year, no maintenance therapy is necessary. When BI is still positive or active lesion is remaining, additional therapy with MDT/MB for one more year is recommended. This is a simplification of first version in 2000. Brief summary of diagnosis, purpose of therapy, character of drugs, and prevention of deformity is also described.

[Basic evaluation for new antimicrobial susceptibility testing of Mycobacterium leprae by bioluminescence assay (ATP method)].

Antimicrobial susceptibility testing of Mycobacterium leprae by non-radioactive bioluminescence assay was developed. Optimization of the assay conditions such as temperature and time for ATP extraction, bacteria dose, preparation of bacteria suspension and pH of culture medium was carried out using M. leprae Thai 53 strain. Samples of bacterial suspension of M. leprae were first treated with filamentous cell treatment reagent at room temperature for 30 minutes and ATP was extracted from the leprosy bacilli by heating at 60 degrees for five minutes. Luciferin luciferase was added to the extract after cooling to room temperature followed by measurement of relative light units (RLU) of each sample using a luminometer. The concentrations of the drugs used for the evaluation of antimicrobial activities of rifampin (RFP), clofazimine (CLF), ofloxacin (OFLX) and clarithromycin (CAM) were 0.125, 0.50, 2.0 and 8.0 microg/ml respectively. Middlebrook 7H9 broth medium was used (pH6.6) as the basal medium and the bacilli were cultivated at 32 C for 0-14 days. ATP was extracted from 0.1 ml of culture suspension and inhibition of the luminescent activity was calculated. The results were compared to that obtained by radio-active CO2 detection system, Buddemeyer method which is commonly used for measuring anti-M. leprae activity. There was a good correlation between the results obtained by ATP method on the tenth day of culture and the results obtained by Buddemeyer method on the seventh day of culture. ATP method may be useful for the determination of drug susceptibility ofM. leprae.

[In vitro and in vivo activities of newly synthesized fluoroquinolones WQ-3345 and WQ-3402 against Mycobacterium leprae].

Activities of newly synthesized fluoroquinolnes WQ-3345 and WQ-3402 against M. leprae were measured by using the Buddemeyer method. The % inhibition of the examined drugs for M. leprae was in the order of RFP > WQ-3402 > SPFX > GFLX > WQ-3345 > LVFX. The anti-M. leprae activity of WQ-3402 was found to be strongest in these five fluoroquinolones when examined by this method, and the activity of WQ-3345 was weaker than that of GFLX. The anti-M. leprae activities of WQ-3345 and WQ-3402 were measured by a mouse footpad method using nude mice. The inhibitory effects on the growth of M. leprae inoculated into the footpads were found to be incomplete after orally administered with WQ-3345 or WQ-3402 respectively at dosages of 10 and 20 mg/kg, and the incomplete inhibition was again was again found even at a dosage of 30, 40, or 50 mg/kg im the latter.

[Guideline for the treatment of leprosy by new quinolones].

Ofloxacin(OFLX) is often applied today as a substitution drug of MDT for drug resistance to dapsone, rifampicin or clofazimine. However, OFLX resistance is also becoming a great concern. Low and/or irregular administration are considered to be the major causes of OFLX resistance. OFLX should be used as a combined therapy, and minimal daily dose of 400 mg of OFLX or 200-300 mg of levofloxacin is required. Quinolone resistance should be considered when no improvement of clinical and/or bacterial index is observed after the treatment for 6 months. In such cases, resistance gene detection is necessary.

Assessment of cell mediated immunogenicity of Mycobacterium leprae-derived antigens.

The antigenicity of Mycobacterium leprae (M. leprae)-derived cell membrane fraction was examined using human dendritic cells (DCs). Immature DCs internalized and processed the cell membrane components, and expressed M. leprae-derived antigens (Ags) on their surface. The expression of MHC class II, CD86, and CD83 Ags on DCs and CD40 ligand (L)-associated IL-12 p70 production from DCs were up-regulated by the membrane Ags. Moreover these stimulated DCs induced significantly higher level of interferon-gamma (IFN-gamma) production by autologous CD4(+) and CD8(+) T cells than those pulsed with equivalent doses of live M. leprae or its cytosol fraction. Both subsets of T cells from tuberculoid leprosy patients also produced several fold more IFN-gamma than those from normal individuals. Furthermore, the intracellular perforin production in CD8(+) T cells was up-regulated in an Ag-dose dependent manner. These results suggest that M. leprae membrane Ags might be useful as the vaccinating agents against leprosy.

Assessment of cell mediated immunogenicity of Mycobacterium leprae-derived antigens

The antigenicity of Mycobacterium leprae (M. leprae)-derived cell membrane fraction was examined using human dendritic cells (DCs). Immature DCs internalized and processed the cell membrane components, and expressed M. leprae-derived antigens (Ags) on their surface. The expression of MHC class II, CD86, and CD83 Ags on DCs and CD40 ligand (L)-associated IL-12 p70 production from DCs were up-regulated by the membrane Ags. Moreover these stimulated DCs induced significantly higher level of interferon-gamma (IFN-gamma) production by autologous CD4(+) and CD8(+) T cells than those pulsed with equivalent doses of live M. leprae or its cytosol fraction. Both subsets of T cells from tuberculoid leprosy patients also produced several fold more IFN-gamma than those from normal individuals. Furthermore, the intracellular perforin production in CD8(+) T cells was up-regulated in an Ag-dose dependent manner. These results suggest that M. leprae membrane Ags might be useful as the vaccinating agents against leprosy.

[Clinical cure of leprosy--a criteria in Japan(2002)].

In Japan, a cautious definition of clinical cure of leprosy has been used since 1988. This report presents a new definition of clinical cure for leprosy patients after multi-drug treatment is completed. When the patients complete the standard treatment published in 2000, they are defined as "clinically cured". The doctor in charge should inform the patient of the cure of the disease clearly. On the release from the treatment, it is important to explain necessary cares for protection against injuries and prevention from deformities. The patient should be careful about signs of relapse and reactions.