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In this study, we compared the anti-leishmanial activity of three crotalic venoms (Crotalus durissus terrificus-Cdt, Crotalus durissus cascavella-Cdca, and Crotalus durissus collilineatus-Cdcol). Different concentrations of each venom incubated with Leishmania (Leishmania) amazonensis promastigotes were used. Cdt venom exhibited a higher anti-leishmanial activity (Inhibitory concentration-IC50-value of 4.70+/-1.72 microg/ml) in comparison with that of Cdca venom (IC50 value of 9.41+/-1.21 microg/ml), while Cdcol venom increased parasite numbers in 50% at a concentration of 44.30+/-2.18 microg/ml. In addition, this venom showed a low anti-leishmanial activity in higher concentrations (IC50 value of 281.00+/-9.50 microg/ml). The main fractions of Cdca venom were isolated and assayed under similar conditions used for assessing crude venom. The most active fractions were gyroxin and crotamine that had IC50 values of 3.80+/-0.52 microg/ml and 19.95+/-4.21 microg/ml, respectively. Convulxin also inhibited parasite growth rate, although this effect was not dose-dependent. Crotoxin was the least effective fraction with an IC50 value of 99.80+/-2.21 microg/ml. None of the protein fractions presented cytotoxic effects against J774 cells in culture. In vivo assays using BALB/c mice revealed that crotoxin and crotamine were the main toxic fractions. In conclusion, C. durissus cascavella venom has three main fractions with anti-leishmanial activity. These results open new possibilities to find proteins that might be used as possible agents against cutaneous leishmaniasis.

In Honduras, Leishmania (L.) infantum chagasi causes both visceral leishmaniasis (LV) and nonulcerated or atypical cutaneous leishmaniasis (NUCL). NUCL is characterized by mononuclear inflammatory infiltration of the dermis, composed mainly of lymphocytes followed by macrophages with discrete parasitism. Considering that little is known about the pathogenesis of NUCL, the aim of this study was to evaluate the regulatory response in situ in skin lesions of patients affected by NUCL. Biopsies (n = 20) from human cutaneous nonulcerative lesions were collected and processed by usual histological techniques. The in situ regulatory immune response was evaluated by immunohistochemistry using antihuman CD4, FoxP3, IL-10, and TGF-β antibodies. CD4+, FoxP3+, TGF-β+, and IL-10+ cells were observed in the dermis with inflammatory infiltration in all studied cases and at higher densities compared to the normal skin controls. A positive and strong correlation was observed between CD4+ and FoxP3+ cells, and a positive and moderate correlation was observed between FoxP3+ and TGF-β+ but not with IL-10+ cells. The data suggest that T regulatory FoxP3+ cells and the regulatory cytokines, especially TGF-β, play an important role in the immunopathogenesis of NUCL, modulating a cellular immune response in the skin, avoiding tissue damage, and leading to low tissue parasitic persistence.

In this study, the effect of phospholipase A2 (PLA2) derived from Crotalus durissus collilineatus was evaluated in vitro and in vivo on experimental cutaneous leishmaniasis. The promastigote and amastigote forms treated with PLA2 presented increased growth rate. In vivo studies showed that PLA2-treated Leishmania (Leishmania) amazonensis promastigotes increased the size of lesions in BALB/c mice, and histopathological analysis showed numerous necrotic regions presenting a higher density of polymorphonuclear, mononuclear, and amastigote cells. Additionally, infected macrophages treated with PLA2 were able to generate prostaglandin E2 (PGE2). Cytokine quantification showed that the supernatant from infected macrophages presented moderate and high amounts of IL-2 and IL-10, respectively. However, in PLA2-treated infected macrophages, suppression of IL-2 levels occurred, but not of IL-10 levels. Observation also revealed that both the supernatant and lysate of L. (L.) amazonensis promastigotes exhibited PLA2 activity, which, in the presence of dexamethasone, showed no reduction in their activities; while glucocorticoid maintained the ability of promastigote forms to infect macrophages, which presented values similar to controls. In conclusion, the results indicate that PLA2 may be a progression factor for cutaneous leishmaniasis, since the PLA2 effect suppressed IL-2 levels and generated PGE2, an inflammatory lipid mediator.

OBJECTIVE This study investigated the serological status of dogs living in a visceral leishmaniasis-endemic area and its correlation with the parasitological condition of the animals.METHODS Canine humoral response was evaluated using the sera of 134 dogs by enzyme-linked immunosorbent assay and immunohistochemistry to detect parasites in the skin, lymph node, and spleen of the animals. The specific antibodies investigated were IgG, IgG1, IgG2, and IgE.RESULTS According to the parasitological, laboratory, and clinical findings, the dogs were placed into one of four groups: asymptomatic with (AP+, n = 21) or without (AP-, n = 36) Leishmania tissue parasitism and symptomatic with (SP+, n = 52) or without (SP-, n = 25) parasitism. Higher IgG and IgE levels were positively correlated with the infection condition and parasite load, but not with the clinical status. In all groups, total IgG was the predominant antibody, which occurred at the expense of IgG2 instead of IgG1. Most of the infected dogs tested positive for IgG (SP+, 98.1%; AP+, 95.2%), whereas this was not observed with IgE (SP+, 80.8%; AP+, 71.2%). The most relevant finding was the high positivity of the uninfected dogs for Leishmania-specific IgG (SP-, 60.0%; AP-, 44.4%), IgE (SP-, 44.0%; AP-, 27.8%), IgG1 (SP-, 28.0%; AP-, 22.2%), and IgG2 antibodies (SP-, 56.0%; AP-, 41.7%).CONCLUSIONS The serological status of dogs, as determined by any class or subclass of antibodies, did not accurately distinguish dogs infected with L. (L.) infantum chagasi from uninfected animals. The inaccuracy of the serological result may impair not only the diagnosis, but also epidemiological investigations and strategies for visceral leishmaniasis control. This complex serological scenario occurring in a visceral leishmaniasis-endemic area highlights the challenges associated with canine diagnosis and points out the difficulties experienced by veterinary clinicians and coordinators of control programs.

BackgroundThere are only a few studies reporting the role of nitric oxide metabolites for controlling macrophage intracellular parasitism, and these are controversial. Therefore, the present study aimed to evaluate the expression of inducible nitric oxide synthase (iNOS) in the lymph nodes and spleen of dogs affected by visceral leishmaniasis through immunohistochemistry and to determine its correlation with tissue parasite burden and serum interferon (IFN)-γ levels. Twenty-eight dogs were selected and assigned to one of two groups, symptomatic (n = 18) and asymptomatic (n = 10), according to clinical status and laboratory evaluation. A negative control group (n = 6) from a non-endemic region for visceral leishmaniasis was included as well.ResultsParasite density (amastigotes/mm2) was similar between clinical groups in the lymph nodes (P = 0.2401) and spleen (P = 0.8869). The density of iNOS+ cells was higher in infected dogs compared to controls (P < 0.05), without a significant difference in lymph node (P = 0.3257) and spleen (P = 0.5940) densities between symptomatic and asymptomatic dogs. A positive correlation was found between the number of iNOS+ cells in lymph nodes and interferon-γ levels (r = 0.3776; P = 0.0303), and there was a negative correlation between parasites and iNOS+ cell densities both in lymph nodes (r = −0.5341; P = 0.0034) and spleen (r = −0.4669; P = 0.0329).ConclusionThe negative correlation observed between tissue parasitism and the expression of iNOS may be a reflection of NO acting on the control of parasites.