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 H2 O2 , 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 H2 O2, 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.

Can anti-PGL-1 and anti-NDO-LID-1 antibody titers be used to predict the risk of reactions in leprosy patients?

Leprosy patients may present reactional episodes classified as type I or reversal reaction and type II or erythema nodosum leprosum. Early diagnosis of these reactions is hampered by lack of diagnostic tests. This study aimed at evaluating anti-Mycobacterium leprae antibody levels in reactional and nonreactional leprosy patients at the time of diagnosis. Clinical data and serum samples of 224 patients diagnosed between 2009 and 2010 were collected in the municipality of Rondonópolis-MTBR. Quantification of anti-phenolic glycolipid-1 (PGL-1) IgM antibodies of M. leprae was obtained by the enzyme-linked immunosorbent assay method and anti-natural octyl disacharide-leprosy IDRI diagnostic (NDO-LID-1) IgM/IgG semiquantitative rapid test. We obtained low serological levels of anti-PGL-1 and anti-NDO-LID-1 for tuberculoid (T) (1.56% and 15.62%) and borderline tuberculoid (BT) patients (7.95% and 26.13%), medium levels in the borderline-borderline (BB) (47.91% and 68.75%), and high levels in lepromatous (LL) (93.33% and 100%) and borderline-lepromatous (BL) (88.0% and 100%). When comparing the reactional groups (RI and RII) with without reaction (WR) group at the time of diagnosis, we observed a statistically significant difference between the groups; patients with RII presented higher serological response: 66.66% anti-PGL-1 and 91.66% anti-NDO-LID-1. In respect to patients who developed a reaction after the initial diagnosis, they also showed significant positivity for both anti-PGL-1 and anti-NDO-LID-1 in comparison to the patients who stayed without reaction in the study period (P<0.0001). These results allow us to conclude that serological tests may contribute to an early diagnosis of RII and that the anti-NDO-LID-1 test was demonstrated to be a better indicator.

Synthesis and evaluation of novel dapsone-thalidomide hybrids for the treatment of type 2 leprosy reactions.

We synthesized a series of novel dapsone-thalidomide hybrids (3a-i) by molecular hybridization and evaluated their potential for the treatment of type 2 leprosy reactions. All of the compounds had analgesic properties. Compounds 3c and 3h were the most active antinociceptive compounds and reduced acetic acid-induced abdominal constrictions by 49.8% and 39.1%, respectively. The hybrid compounds also reduced tumor necrosis factor-α levels in lipopolysaccharide-stimulated L929 cells. Compound 3i was the most active compound; at concentrations of 15.62 and 125 µM, compound 3i decreased tumor necrosis factor-α levels by 86.33% and 87.80%, respectively. In nude mice infected with Mycobacterium leprae in vivo, compound 3i did not reduce the number of bacilli compared with controls. Compound 3i did not have mutagenic effects in Salmonella typhimurium strains TA100 and TA102, with or without metabolic activation (S9 mixture). Our results indicate that compound 3i is a novel lead compound for the treatment of type 2 leprosy reactions.

Molecular drug susceptibility testing and genotyping of Mycobacterium leprae strains from South America.

Possible drug resistance in Mycobacterium leprae strains from Venezuela and three other South American countries was surveyed by molecular methods. None of the 230 strains from new leprosy cases exhibited drug resistance-associated mutations. However, two of the three strains from relapsed cases contained dapsone resistance mutations, and one strain also harbored a rifampin resistance mutation. Single nucleotide polymorphism analysis of these strains revealed five subtypes: 3I (73.8%), 4P (11.6%), 1D (6.9%), 4N (6%), and 4O (1.7%).

In vitro and in situ activation of the complement system by the fungus Lacazia loboi.

Since there are no studies evaluating the participation of the complement system (CS) in Jorge Lobo's disease and its activity on the fungus Lacazia loboi, we carried out the present investigation. Fungal cells with a viability index of 48% were obtained from the footpads of BALB/c mice and incubated with a pool of inactivated serum from patients with the mycosis or with sterile saline for 30 min at 37 masculineC. Next, the tubes were incubated for 2 h with a pool of noninactivated AB+ serum, inactivated serum, serum diluted in EGTA-MgCl2, and serum diluted in EDTA. The viability of L. loboi was evaluated and the fungal suspension was cytocentrifuged. The slides were submitted to immunofluorescence staining using human anti-C3 antibody. The results revealed that 98% of the fungi activated the CS by the alternative pathway and no significant difference in L. loboi viability was observed after CS activation. In parallel, frozen histological sections from 11 patients were analyzed regarding the presence of C3 and IgG by immunofluorescence staining. C3 and IgG deposits were observed in the fungal wall of 100% and 91% of the lesions evaluated, respectively. The results suggest that the CS and immunoglobulins may contribute to the defense mechanisms of the host against L. loboi.