Murine experimental leprosy: evaluation of immune response by analysis of peritoneal lavage cells and footpad histopathology

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, IL­2, IL­4, IL­10, IL­12, IFN­Î³ and TNF levels. Serum levels of anti­PGL­I 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−, IL­2, IL­4, IL­10 and IFN­Î³ production in the course of infection in nude mice; however, in BALB/c, O2− and IL­12 production was higher at 5 months and NO, IFN­Î³ and TNF production was higher at 8 months when there was a decrease in the number of bacilli. The level of anti­PGL­I 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(AU).

Experimental reproduction of the Jorge Lobos disease in BALB/c mice inoculated with Lacazia loboi obtained from a previously infected mouse

Long-term maintenance of Lacazia loboi in the laboratory has not been reported. We report here the use BALB/c mice to maintain the Lacazia loboi for extended period of time. Eight to ten week-old mice were inoculated intradermally in both hind footpads with a fungal suspension from a macerated footpad obtained from an original mouse previous infected with the fungi and sacrificed 8 months after inoculation. The inoculated animals were sacrificed at different time intervals, footpads were excised, the right one was submitted to histopathological examination and the left one was macerated in sterile saline for fungal count and viability index determination. The inoculated animals presented the histopathological picture identical to the mice previously inoculated with material from human lesion. Granulomatous infiltrates with predominance of macrophages and giant cells were observed. The granulomas evolved progressively as observed in the different times of sacrifice. After 7 months of inoculation, macroscopic lesions were observed, and the number of fungi obtained from macerated footpads was higher than the number of inoculated fungi. The pattern of lesion development was similar to what was observed in animals infected with a fungal suspension obtained from a human lesion. Considering the histopathological findings, the clinical manifestations, and the finding of a higher number of fungi obtained than the inoculated into footpads of each mice, we believe the BALB/c mice strain is as an excellent way to amintain L. loboi in laboratory. Moreover, even after serial passages of the funfi, the granulomatous lesions are reproduced consistently in laboratory conditions.

Experimental mycobacterium leprae infection in BALB/c Mice: effect of BCG administration on TNF-a production and granuloma development

In the present study, the experimental model of Mycobacterium leprae infection in the foot pads of BALB/c mice was used to investigate the effects of BCG administration on tumor necrosis factor-alpha (TNF-alpha) production and granuloma development. It was observed that mice intravenously infected with BCG 7 months after M. leprae inoculation into the foot pads presented a more effective mycobacteria clearance, revealed by a significant reduction of BCG-colony forming units in the spleen and by the reduction of acid-fast bacilli (AFB) in the foot pads. BCG infection at the peak of M. leprae infection also modulated the granulomatous response to M. leprae by converting mononuclear granulomas into an epithelioid-cell granuloma. Furthermore, lower TNF-alpha serum levels were detected in M. leprae-infected mice when compared to mice infected with M. leprae + BCG. An analysis of the TNF-alpha gene expression in the spleen by semiquantitative reverse transcription-polymerase chain reactions (RT-PCR) demonstrated that co-infection with BCG induced an earlier expression of TNF-alpha mRNA than in M. leprae-infected mice. The numbers of TNF-alpha-positive cells and apoptotic cells were also enhanced in epithelioid versus non-epithelioid granulomas. As a whole, the data suggest that co-infection of M. leprae-infected mice with BCG modulates TNF-alpha synthesis which, in turn, leads to induction of protective epithelioid granuloma formation in the foot pads and subsequent mycobacterial clearance. Macrophage differentiation into epithelioid cells, in association with the enhancement of TNF-alpha production at the granuloma site, may represent a triggering signal that induced apoptosis in these cells, leading to mycobacterial elimination. Moreover, the rate of apoptosis in epithelioid granulomas may well be related to the extent of immunopathologically mediated tissue damage.

The potent bone-resorbing mediator of Actinobacillus actinomycetemcomitans is homologous to the molecular chaperone GroEL.

Actinobacillus actinomycetemcomitans is a Gram-negative bacterium implicated in the pathology of localized juvenile periodontitis, a condition involving rapid destruction of alveolar bone. We have established that gentle extraction of this bacterium in saline releases a proteinaceous fraction (which we have termed surface-associated material [SAM] which has potent osteolytic activity in the murine calvarial bone resorption assay. Fractionation of the SAM has now revealed that activity is associated with a 62-kD protein. This bone-resorbing activity can be blocked by a monoclonal antibody (raised to the whole bacterium) that is claimed to recognize a protein homologous to the Escherichia coli molecular chaperone GroEL. Purification of this bone-resorbing protein to homogeneity has been achieved by a combination of anion exchange, gel filtration, and ATP-affinity chromatography and the NH2-terminal sequence shows > 95% homology to E. coli GroEL. This GroEL homologue is found in the SAM of A. actinomycetemcomitans but is not found in the osteolytically active SAM from other Gram-negative or Gram-positive bacteria. The GroEL protein from E. coli, but not from Mycobacterium tuberculosis and Mycobacterium leprae, also showed activity in the bone resorption assay. We believe this to be the first observation that a molecular chaperone has the capacity to stimulate the breakdown of connective tissue.

A strategy to improve the efficacy of vaccination against tuberculosis and leprosy.

The pathogens responsible for leprosy, tuberculosis and the leishmaniases can induce different classes of immunity, but protection is provided only by a cell-mediated response. Here, Peter Bretscher proposes a strategy to achieve an immunological imprint that ensures a stable cell-mediated response upon natural infection.

Defective activation of granuloma macrophages from Mycobacterium leprae-infected nude mice.

We have previously demonstrated that granuloma macrophages from the foot pads of Mycobacterium leprae-infected nude mice are functionally normal despite heavy intracellular burdens of bacilli. However, unlike peritoneal macrophages, these macrophages fail to restrict the intracellular growth of Toxoplasma gondii when stimulated with recombinant murine gamma interferon (Mu IFN-gamma) and thus appear defective in their response to macrophage-activating factor(s). In further characterizing this defect we have examined tumoristatic capacity, superoxide radical formation, and expression of la antigens on granuloma macrophages before and after treatment with Mu IFN-gamma. By all three criteria, M. leprae-burdened granuloma macrophages failed to become activated by doses of Mu IFN-gamma that readily activate peritoneal macrophages from M. leprae-infected nude mice or normal Balb/c mice. M. leprae-infected granuloma macrophages produced elevated levels of prostaglandin E2 (PGE2) in vitro, which was suppressed by indomethacin. However, the inhibition of PGE2 production for 48 hr in vitro did not restore normal responses to Mu IFN-gamma.

Production and characterization of a murine monoclonal antibody recognizing a shared mycobacterial polysaccharide.

An IgM monoclonal antibody specific for mycobacterial arabinomannan was produced by the fusion of splenocytes from BALB/c mice immunized with purified arabinomannan with NSI/1 myeloma cells. Specificity was demonstrated by gel-radioimmunoassay, and by inhibition of binding using the purified polysaccharide. The monoclonal antibody recognized the arabinomannans from all 18 species of mycobacteria tested, including Mycobacterium leprae. This antibody expands the number of defined mycobacterial antigens against which monoclonal antibodies have been produced, and has potential application in studies concerning the pathogenesis of mycobacterial disease.

Mitsuda-type lepromin reactions as a measure of host resistance in Mycobacterium lepraemurium infection.

The footpad reaction to autoclaved whole Mycobacterium lepraemurium organisms (MLM lepromin) in high-resistance (C57BL) and low-resistance (BALB/c) mice was studied. Infected C57BL mice gave a prolonged footpad response persisting for 4 weeks after skin testing with high and low doses of lepromin. This was accompanied by mononuclear cell infiltration. Uninfected C57BL mice gave no response. The majority of infected BALB/c mice gave no increase in footpad thickness. However, a high proportion of infected and control BALB/c mice tested with the high dose showed mononuclear cell infiltration which resembled that in C57BL mice. The low dose caused little infiltration in infected or control BALB/c mice. The course of infection in the two strains was different. Dissemination of organisms from the infected footpad was minimal in C57BL mice 5 months after infection. In BALB/c mice, dissemination to the draining lymph node and to some extent to the liver had occurred by 5 months. The draining lymph node of BALB/c mice showed histological evidence of local antibody formation, which uas not found in C57BL mice. On the basis of these findings, it was possible to fit murine leprosy in these two strains into a classification similar to that used for human leprosy.