Effect of dialyzable leukocyte extract, sodium butyrate, and valproic acid in the development of anergy in murine leprosy.

Background: Murine leprosy is a chronic granulomatous disease caused by Mycobacterium lepraemurium (MLM) in mice and rats. The disease evolves with the development of cellular anergy that impedes the production of interferon gamma (IFNγ), tumor necrosis factor-alpha (TNFα), and nitric oxide (NO) required to kill the microorganism. In this study we investigated whether histone deacetylase inhibitors (HDACi) (valproic acid and sodium butyrate [NaB]) and the immunomodulator transfer factor in dialyzable leukocyte extracts (DLE) can prevent anergy in murine leprosy. Methods: Five groups of six Balb/c mice were intraperitoneally inoculated with 2 × 107 MLM. Thirty-days post inoculation, treatment was started; one group received no treatment, one was treated with rifampicin-clofazimine (R-C), one with sodium valproate (VPA), one with NaB, and one with DLE. The animals were monitored for the evidence of disease for 96 days. After euthanasia, their spleens were removed and processed for histologic, bacteriologic, and cytokine studies. Results: R-C completely controlled the ongoing disease. DLE and NaB significantly reduced the development of lesions, including granuloma size and the number of bacilli; VPA was less effective. DLE, NaB, and VPA reverted the anergic condition in diverse grades and allowed the expression of IFNγ, TNFα, and inducible NO synthase, also in diverse grades. Conclusion: Anergy in leprosy and murine leprosy allows disease progression. In this study, anergy was prevented, in significant degree, by DLE (an immunomodulator) and NaB (HDACi). VPA was less effective. These results suggest potential beneficial effects of DLE and NaB in the ancillary treatment of leprosy.

Late leprosy reaction presenting as erythema multiforme-like erythema nodosum leprosum with underlying rifampicin resistance and its potential implications.

Erythema multiforme (EM)-like erythema nodosum leprosum (ENL) is a rare atypical presentation, and its late appearance after the completion of multidrug therapy (MDT) is unusual. We describe the case of a lepromatous leprosy patient who after the completion of MDT presented to us with late EM-like ENL and was found to be resistant to rifampicin. We discuss the implications of this finding and the potential role of resistant bacilli in causing reactions with atypical presentations.

Mycobacterium lepraemurium uses TLR-6 and MR, but not lipid rafts or DC-sign, to gain access into mouse macrophages.

OBJECTIVE/BACKGROUND: Mycobacterium lepraemurium (MLM), the etiologic agent of murine leprosy, is an intracellular parasite of macrophages; the mechanism used by this bacterium to enter macrophages is not known. The fate of the MLM phagosome inside macrophages is also unknown. This study was conducted to investigate how MLM enters macrophages and to define the maturation process of MLM phagosome inside macrophages. MATERIALS AND METHODS: Peritoneal macrophages were incubated in the presence of mannan-bovine serum albumin (BSA), and antibodies to known macrophage receptors, including, anti-FcγRIII/RII (anti-CD16/32), anti-CD35 (anti-CR1), anti-TLR2, anti-TLR4, anti-TLR6, anti-CD14, and anti-dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). Then, macrophages were challenged with Iris Fuchsia-stained MLM, at a multiplicity of infection of 50:1. The blocking effect of the antibodies (and mannan-BSA) used was analyzed using direct microscopy and flow cytometry. The maturation process of MLM phagosomes was visualized by their interaction with antibodies to Rab5, Rab7, proton ATPase, and cathepsin D, by confocal microscopy. RESULTS: Only mannan-BSA and anti-TLR6 antibody significantly blocked the entry of MLM into macrophages. None of the other antibodies, including that for DC-SIGN, meaningfully inhibited the endocytic process. We also found that MLM is a fusiogenic mycobacterium. This was deduced from the orderly association of MLM phagosomes with Rab5, Rab7, Proton ATPase, and lysosomes (cathepsin D). CONCLUSION: Fusion of MLM phagosomes with lysosomes seems to be a necessary event for the intracellular multiplication of MLM; similar to Mycobacterium leprae, this microorganism hardly grows on artificial, synthetic, bacteriologic media.

The use of the microplate alamar blue assay (MABA) to assess the susceptibility of Mycobacterium lepraemurium to anti-leprosy and other drugs.

Although murine leprosy is no longer a common illness, our understanding of the biology of this disease is incomplete. One particular example of this concerns the etiologic agent Mycobacterium lepraemurium (MLM). MLM is a fastidious microorganism that is difficult to grow in axenic media; in a way, this has hampered attempts to thoroughly study its physiological and metabolic characteristics. MLM is an obligate intracellular bacillus that invades macrophages and replicates profusely with a generation time that oscillates between 0.5 and 11 days. In the present study, we have successfully maintained MLM alive for more than 12 days in vitro, providing us with an opportunity to study its susceptibility to several anti-leprosy agents and other drugs. To achieve this, we used a fluorescence reduction assay of alamar blue (a resazurin) in a microplate format (microplate-alamar-blue-assay; MABA), which is a highly sensitive, practical, and inexpensive method for assaying cell viability. We found that MLM was highly susceptible to clofazimine and rifampicin and was less susceptible to streptomycin, thiacetazone, kanamycin, dapsone, and ethionamide, in that order. MLM was not susceptible to four plant triterpenoids (oleanolic acid, neolignan-c, sitosterol, and ursolic acid) for which bactericidal activity has been reported in M. tuberculosis. Because the MABA has high sensitivity, it can be used to monitor the activity of microorganisms that are difficult to cultivate (such as M. lepraemurium), in response to various drugs, thus offering a method to complement the study of murine leprosy, about which many questions remain unanswered.

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.

Propagation of Mycobacterium lepraemurium on supplemented minimal medium and its experimental pathogenesis.

The splenic tissue of a mouse experimentally infected with M. lepraemurium (Hawaiian strain, M-65) and developing 'rat leprosy', yielded a pure culture of an acid - fast bacterium having all the characteristics of M. lepraemurium on mineral salt minimal medium supplemented with simple sources of C and N, e.g., NH4 -salts, liquid paraffin, urea, gelatin etc. This could be maintained, by serial passages in vitro with good growth. Its indefinite propagation with tissue - free washed, small inoculum on complex media including Ogawa medium was difficult, and its serial sub-culture was practically impossible. The in vitro isolate from supplemented minimal medium could produce pathological lesions in mice typical of rat leprosy.

Palmitate oxidation by in vivo and in vitro grown Mycobacterium lepraemurium.

Oxidation of palmitate by Mycobacterium lepraemurium isolated from C3H mice lepromata (in vivo) and also grown on Ogawa egg-yolk medium (in vitro) was investigated. Palmitate was found to be oxidized, after a lag period of about 8 h, by both the in vivo and in vitro grown bacilli. Cell-free extracts prepared from in vivo and in vitro grown cells catalysed an active oxidation of palmitate after a lag period of 3-4 h. The amount of ATP increased, with the increase in time during oxidation of palmitate by the cell-free extracts. The generation of ATP was strongly inhibited by the inhibitors rotenone, antimycin A and cyanide as well as by the uncouplers 2,4-dinitrophenol and 2,6-dibromophenol. These results indicated that oxidation of palmitate by the in vivo and in vitro grown M. lepraemurium is mediated through the respiratory chain using oxygen as the terminal electron acceptor.

[Energy production in Mycobacterium lepraemurium cultured in vitro]. / Production d'énergie chez Mycobacterium lepraemurium cultivé in vitro.

Cell-free extracts prepared from in vitro cultured Mycobacterium lepraemurium catalysed phosphorylation coupled to the oxidation of NADH and succinate, yielding P/O ratios of 0.52 and 0.34, respectively. No ATP synthesis occurred during oxidation of ascorbate. Oxidative phosphorylation was uncoupled by dinitrophenol and dibromophenol. Oxidation of NADH and coupled phosphorylation was markedly inhibited by rotenone, whereas this inhibitor had no effect on succinate oxidation and associated ATP synthesis. Oxidative phosphorylations and coupled oxidations of NADH and succinate were strongly inhibited by antimycin A and cyanide.

Modulation of Mycobacterium lepraemurium growth in murine macrophages: beneficial effect of tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor.

Mycobacterium lepraemurium grew progressively in monolayers of Proteose Peptone-elicited macrophages from C57BL/6 mice. Treatment of macrophage monolayers with gamma interferon led to an enhancement of growth of M. lepraemurium in macrophages. Treatment with tumor necrosis factor alpha or granulocyte-macrophage colony-stimulating factor led to restriction of mycobacterial growth in macrophages.

Enhancement of growth of Mycobacterium lepraemurium in macrophages by gamma interferon.

Gamma interferon, an immune lymphokine that protects mouse macrophages against infection by several parasites, was ineffective against Mycobacterium lepraemurium. On the contrary, it significantly stimulated multiplication of M. lepraemurium in the macrophages. Simultaneous treatment of macrophages with gamma interferon and interleukin-4 or interleukin-2 or a combination of all three did not enhance the macrophage resistance to infection with M. lepraemurium, but instead stimulated growth of M. lepraemurium.

Antimycobacterial activities of two newer ansamycins, R-76-1 and DL 473.

The antimycobacterial activities of two newer ansamycins, isobutylpiperazinylrifamycin SV (R-76-1) and cyclopentylrifamycin SV (DL 473), were compared with those of rifampin (RMP) both in vitro and in vivo. In terms of minimal inhibitory concentrations against a number of cultivable mycobacteria, R-76-1 was about eight times more active in vitro than RMP; whereas DL 473 was only slightly more active than RMP. Therapeutic activities of R-76-1 versus RMP and DL 473 versus RMP were compared, respectively, in the experimental infection of mice with Mycobacterium lepraemurium by different treatment schedules (immediate and delayed) and dosage regimens. R-76-1 appeared to have been three times more effective than RMP; DL 473 was also more effective than RMP in that an equivalent therapeutic effect could be obtained by fewer doses of DL 473 than of RMP, and in that DL 473 exerted a more prolonged activity than RMP. With the kinetic method and a dosage of 0.001% in the diet, R-76-1 demonstrated a bactericidal-type effect against M. leprae whereas RMP did not; with the proportional bactericidal method, R-76-1 possessed about three times the bactericidal activity of RMP against M. leprae. When drugs were administered once in 4 weeks, the RMP dose required to prevent multiplication of M. leprae in the foot pads of half of the mice was in the range of 1.25 to 2.5 mg/kg; whereas that of DL 473 was less than 0.625 mg/kg. With the proportional bactericidal method, even a single dose of 1.25 mg DL 473 per kg was active against M. leprae; whereas the smallest single active dose of RMP was 10 mg/kg. DL 473 in single doses of 5 mg/kg and 10 mg/kg was significantly more effective than RMP in equal doses and, among the intermittent regimens administered four times, once every 4 weeks, no significant differences of bactericidal activity were observed between RMP at 20 mg/kg and DL 473 at 0.625 mg/kg. A preliminary clinical trial of R-76-1 in 20 patients with lepromatous leprosy showed that the compound, administered in a dosage of 150 mg daily, was very effective.

Further studies of the use of cycloheximide for cultivating Mycobacterium lepraemurium in cell culture.

The advantage of using cycloheximide for cultivating Mycobacterium lepraemurium in cell culture was further demonstrated. Continuous multiplication of the bacillus in successive subcultures was obtained in MFP, HEp-2 and Vero cells maintained in culture medium containing 0.1 microgram of cycloheximide per ml. Growth characteristics were comparable to those observed in the cultures of A31 cells previously reported. The procedure was simple and convenient. Comparable results, however, have not been obtained in cultures of other established cell lines, HeLa 229, L, MDCK, and Neuro-2a.

Use of cycloheximide for cultivating Mycobacterium lepraemurium in cell culture.

A serial increase in the number of Mycobacterium lepraemurium with successful subcultures has been obtained in cell culture with cycloheximide treatment. The infected cells seldom floated off the culture vessel. They could be maintained and would support the bacillary multiplication in good condition for ten weeks or more without changing the medium frequently. An overall generation time of the intracellular bacilli up to the tertiary culture for the total period of 35 weeks was 22.1 days.

In vitro sensitivity of Mycobacterium lepraemurium for antimycobacterial drugs.

The sensitivity of Mycobacterium lepraemurium for isoniazide, dapsone, ethionamide, pyrazinamide, rifampicin, p-aminosalicylic acid, ethambutol and various sulfonamides was determined using Ogawa medium. The sensitivities of M. lepraemurium for these drugs are different from those obtained with M. leprae. Our results illustrate that M. lepraemurium is not a model for the drug sensitivities of M. leprae.