The cellular densities were expressed by cells per square millimetre. Draining lymph nodes were collected aseptically, macerated and cultured in RPMI-1640 medium (Gibco), supplemented with 10% heat-inactivated FBS, 10 μg/mL gentamicin and 1000 U/mL penicillin in 96-well plates containing 106 cells/mL under stimulation with 5 μg/well of L. (L.) amazonensis, L. (V.) braziliensis antigens (specific antigen) (17) or Concanavalin A (ConA) for 48 h at 37°C and 5% CO2. Cells from control group, noninfected mice, were stimulated
with the same antigens or ConA. The quantification of IL-4, IL-10 and IFN-γ in the supernatant of draining lymph nodes cells culture selleck kinase inhibitor was carried out by capture ELISA using commercial kits (BD Bioscience). The differences between BALB/c mice
groups p38 protein kinase were analysed by nonparametric Mann–Whitney test using Bioestat 5.0 (software developed by the University of Para, Belém, Para, Brazil) and P values <0·05 were considered significant. L. (L.) amazonensis induced a progressive growth of skin lesions in BALB/c mice since the 3rd weeks PI. Significant differences were observed from the 3rd to 8th weeks PI when compared with the control group as well as with the BALB/c mice infected with L. (V.) braziliensis (P < 0·05), which showed a small swelling in the skin lesion between the 6th and 7th weeks PI, with regression to control level at the 8th week (Figure 1a). At 4th and 8th weeks, the parasite load, in the skin lesions of mice infected with L. (L.) Mannose-binding protein-associated serine protease amazonensis, was higher (P < 0·05) than that of animals infected with L. (V.) braziliensis. At 4th week, the number of parasites recovered from L. (L.) amazonensis lesions per mg of tissue was 3·05 × 107 promastigotes, while in L. (V.) braziliensis lesions was 3·44 × 103 promastigotes. At 8th week, the parasite load in the hind footpad was 1·37 × 109 promastigotes and 53 promastigotes, respectively. Regarding the evolution of parasite load in both infections, no difference (P > 0·05) was observed in the L. (V.) braziliensis group, but in the L. (L.) amazonensis group, there was
a significant (P < 0·05) increase in parasites at the inoculation site with the evolution of infection (Figure 1b). The skin lesion of BALB/c mice infected with L. (L.) amazonensis showed, at 4th week, a mixed and moderate cellular inflammatory infiltrate characterized by the presence of polymorphonuclear and mainly mononuclear cells with moderate parasitism, and focal areas of necrosis in a few cases (Figure 1C-I). At 8th weeks PI, these lesions in the chronic phase of infection showed an intense and diffuse cellular inflammatory process, with a predominance of vacuolated macrophages heavily parasitized, few polymorphonuclear cells, but with necrotic areas more evident (Figure 1C-IV). On the other hand, the skin lesion of BALB/c mice infected with L. (V.