Mycobacterium leprae and Mycobacterium tuberculosis antimicrobial resistance has been followed with great concern during the last years, while the need for new drugs able to control leprosy and ...tuberculosis, mainly due to extensively drug-resistant tuberculosis (XDR-TB), is pressing. Our group recently showed that M. leprae is able to induce lipid body biogenesis and cholesterol accumulation in macrophages and Schwann cells, facilitating its viability and replication. Considering these previous results, we investigated the efficacies of two statins on the intracellular viability of mycobacteria within the macrophage, as well as the effect of atorvastatin on M. leprae infections in BALB/c mice. We observed that intracellular mycobacteria viability decreased markedly after incubation with both statins, but atorvastatin showed the best inhibitory effect when combined with rifampin. Using Shepard's model, we observed with atorvastatin an efficacy in controlling M. leprae and inflammatory infiltrate in the BALB/c footpad, in a serum cholesterol level-dependent way. We conclude that statins contribute to macrophage-bactericidal activity against Mycobacterium bovis, M. leprae, and M. tuberculosis. It is likely that the association of statins with the actual multidrug therapy effectively reduces mycobacterial viability and tissue lesion in leprosy and tuberculosis patients, although epidemiological studies are still needed for confirmation.
Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for ...growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1(nu)). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.
Summary
This study evaluated the immune response of nude and BALB/c mice inoculated in the footpads (FP) with Mycobacterium leprae after 3, 5 and 8 months. At each timepoint peritoneal cells, ...peripheral blood, FP and popliteal lymph nodes (PLN) were collected. Peritoneal cell cultures were performed to measure the H2O2, O2−, NO, IL‐2, IL‐4, IL‐10, IL‐12, IFN‐γ and TNF levels. Serum levels of anti‐PGL‐I antibodies were also quantified. The results showed that the infection was progressive in nude mice with bacterial multiplication, development of macroscopic lesions in the FP and presence of bacilli in the PLN at 8 months. In BALB/c mice, the infection reached a plateau of bacillary multiplication at 5 months and regressed at 8 months. Histopathological analysis of FP revealed a mononuclear inflammatory infiltrate with a large number of neutrophils at 5 months, with a higher number in nude mice. At 8 months, the number of neutrophils decreased and the infiltrate was predominantly mononuclear in both mouse strains. There was no H2O2, O2−, IL‐2, IL‐4, IL‐10 and IFN‐γ production in the course of infection in nude mice; however, in BALB/c, O2− and IL‐12 production was higher at 5 months and NO, IFN‐γ and TNF production was higher at 8 months when there was a decrease in the number of bacilli. The level of anti‐PGL‐I antibodies was higher in BALB/c mice. Thus, nude and BALB/c mice can be used as experimental models for the study of various aspects of leprosy.
Introdução: a hanseníase é uma doença in fecciosa crônica causada pelo Mycobacterium leprae (M. leprae), um parasita intracelular obrigatório. Assim, a resistência do hospedeiro a esse patógeno ...depende da imunidade celular. O uso de modelos experimentais tem permitido o estudo da hanseníase do ponto de vista imunológico, microbiológico e terapêutico, entretanto, as diferenças na progressão da infecção entre os modelos mais empregados (camundongos imunocompetentes, BALB/c, e camundongos congenitamente atímicos, nude) são pouco estudadas. Objetivo: comparar a evolução da infecção pelo M. leprae em camundongos BALB/c e nude quanto à multiplicação bacilar e avaliação do perfil inflamatório sistêmico pela quantificação sérica de citocinas e óxido nítrico (NO). Métodos: os camundongos foram inoculados com M. leprae nos coxins plantares e avaliados aos 3, 5 e 8 meses após a infecção. Resultados: camundongos nude apresentaram multiplicação bacilar progressiva nos coxins plantares. Em camundongos BALB/c, o número de bacilos foi maior aos 5 meses. Em relação à quantificação de citocinas, nos camundongos BALB/c houve aumento de IL-2 e IL-17A e diminuição de IL-6 e NO aos 8 meses de inoculação. Nos camundongos nude, verificou-se o aumento do TNF aos 8 meses de inoculação e manutenção dos níveis de NO. Conclusão: os resultados encontrados sugerem que em camundongos BALB/c ocorre a ativação de uma resposta imune capaz de controlar a multiplicação do M. leprae, em contrapartida em camundongos nude a infecção é progressiva a despeito de altos níveis de TNF.
The murine model of Jorge Lobo's disease is characterized by histological alterations similar to those seen in human disease, including a large number of viable fungi. This study evaluated the immune ...response of mice with early and late macroscopic lesions (5 and 13 months post-inoculation p.i., respectively) by the analysis of peritoneal lavage cells and footpad (FP) histology. The FP of mice were inoculated with 1 × 106 fungi (viability index of 41%). At 5 and 13 months p.i., the granuloma mainly consisted of macrophages and multinucleated giant cells, but a larger number of neutrophils was observed at 5 months and lymphocytes at 13 months. The number of fungi in the FP and fungal viability were 1.8 ± 1.1 × 106 fungi/ml and 38.5% at 5 months p.i. and 30.8 ± 11.7 × 106 fungi/ml and 9% at 13 months (P < .05). Higher production of H2O2, O2
−, IL-10, and TNF-α were observed at 13 months (P < .05), but there was no significant difference in the production of NO, IL-2, IL-4, IL-12 and IFN-γ. The results showed significant differences between early and late lesions and support the use of BALB/c mice for evaluation of the different phases of infection.
The aim of this study was to evaluate the effects of the protein-calorie malnutrition in BALB/c isogenic mice infected with Lacazia loboi, employing nutritional and histopathological parameters. Four ...groups were composed: G1: inoculated with restricted diet, G2: not inoculated with restricted diet, G3: inoculated with regular diet, G4: not inoculated with regular diet. Once malnutrition had been imposed, the animals were inoculated intradermally in the footpad and after four months, were sacrificed for the excision of the footpad, liver and spleen. The infection did not exert great influence on the body weight of the mice. The weight of the liver and spleen showed reduction in the undernourished groups when compared to the nourished groups. The macroscopic lesions, viability index and total number of fungi found in the footpads of the infected mice were increased in G3 when compared to G1. Regarding the histopathological analysis of the footpad, a global cellularity increase in the composition of the granuloma was observed in G3 when compared to G1, with large numbers of macrophages and multinucleated giant cells, discrete numbers of lymphocytes were present in G3 and an increase was observed in G1. The results suggest that there is considerable interaction between Jorge Lobo's disease and nutrition.
Summary
There are no studies investigating the role of nutritional status and immunity associated with Jorge Lobo's disease. The objective of this study was to evaluate the effects of protein‐calorie ...malnutrition on the immune response of BALB/c mice inoculated with Lacazia loboi. In this study,the animals were divided into four groups: G1: inoculated with restricted diet, G2: not inoculated with restricted diet, G3: inoculated with regular diet, G4: not inoculated with regular diet. The animals of groups G1 and G2 were submitted to malnutrition for 20 days and once installed the animals were inoculated intradermally into the footpad. After 4 months, they were euthanised for the isolation of peritoneal lavage cells and removal of the footpad. The production of IL‐2, IL‐4, IL‐10, IL‐12, IFN‐γ, TNF‐α, H2O2 and nitric oxide (NO) was evaluated in the peritoneal lavage cells. The footpad was evaluated regarding the size of macroscopic lesions, number of fungi and viability index. The results showed that the infection did not exert great influence on the body weight of the mice and previous malnutrition was an unfavourable factor for viability index, number of fungi, macroscopic lesion size in the footpad and production of H2O2, NO, IL‐12, IL‐10 and IFN‐γ, suggesting that malnutrition significantly altered fungal activity and peritoneal cells. The results suggest considerable interaction between nutrition and immunity in Jorge Lobo's disease.
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