such as L. (L.) amazonensis and L. (V.) braziliensis, which are responsible for the opposite ADCL and MCL clinical–immunological forms in the ACL spectrum, respectively, Afatinib manufacturer are scarce and reinforce the importance of studying the parasite species in triggering an efficient cellular

immune response. Thus, the main objective of this study was to evaluate the dynamics of dDCs (CD11c+), LCs (CD207+), CD4+, and CD8+ cells in the dermal site of L. (L.) amazonensis and L. (V.) braziliensis BALB/c mice infection and their relationship with the development of Th1 and Th2 immune responses. Eight-week-old BALB/c mice obtained from the Animal Facility of the São Paulo University, Medical School, Brazil, were maintained in our laboratory during the experiments according to the guidelines of the institutional rules regarding the welfare of experimental animals and with the approval of the Animal Ethics Committee of São Paulo University (protocol number 0589/08). L. (L.) amazonensis (MHOM/BR/1973/M2269) and L. (V.) braziliensis (MHOM/BR/1995/M15280) parasites were isolated from patients with ADCL and MCL,

respectively, being both from Pará state, north of Brazil. The parasites were identified using monoclonal antibodies (14) and isoenzyme electrophoretic profiles (15) at the Leishmaniasis laboratory of Evandro Chagas Institute SCH727965 datasheet (Belém, Pará state, Brazil). L. (L.) amazonensis has been maintained in BALB/c mice footpad, isolated and grown in RPMI-1640 medium (Gibco, Invitrogen, Camarillo, CA, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 10 μg/mL gentamicin, and 1000 U/mL penicillin at 25°C. L. (V.) braziliensis has been

maintained in hamster footpad, isolated and grown in Schneider′s Drosophila medium (Sigma, St. Louis, MO, USA), supplemented with 10% heat-inactivated FBS, 10 μg/mL gentamicin and 100 U/mL penicillin at 25°C. On the 6th day of culture, promastigote forms from the stationary phase of culture growth were centrifuged (1620 g, for 10 min) using phosphate-buffered saline solution (PBS), pH 7·4, and were used for mice infection. BALB/c mice were infected subcutaneously into the hind footpad with 106 promastigote forms from stationary phase either with L. (L.) amazonensis or with L. (V.) braziliensis from a low in vitro passage (≤6 passages) in 50 μL PBS. The control Racecadotril groups were inoculated only with PBS. The hind footpad swelling was weekly evaluated till the 8th weeks PI. The parasite load in the skin lesion was determined using the quantitative limiting-dilution assay as previously described (16). Briefly, the infected footpads were aseptically excised at the 4th and 8th weeks PI and were homogenized in Schneider’s medium. The cellular suspension was subjected to 12 serial dilutions with four replicate wells. The number of viable parasites was determined from the highest dilution that promastigotes could be grown after 10 days of incubation at 25°C.