Data were collected using a standard protocol chart containing the following information: identification, clinical complaints, physical examination

and results of laboratory tests. Exclusion criteria were the following: other pathologies and infections, treatment with hormones or immunosuppressants, alcoholism, pregnancy and amenorrhea. All procedures were approved by the Ethical Committee of Hospital Universitário Edgard Santos – UFBA, BA. Age-matched normal volunteers (NV) living in the same endemic area (n = 32; 17 men and 15 women) served as controls for the study. NV had no history of cutaneous lesions characteristic of leishmaniasis and tested negative for the intradermal delayed-type hypersensitivity test (DTH) to Proteases inhibitor the Leishmania antigen. When patients were compared with NV we evaluated only the patients age-matched with the controls (n = 32; 17 men and 15 women). These 32 patients did not show any difference in clinical and immunological markers when compared to other patients of the study. For analyses of correlations of find more hormones with cytokines we used all patients. Clinical evaluation for correlations with hormone or cytokine levels was performed using three parameters: lesion size, time of disease and dose of antimoniate needed to achieve clinical

cure. Lesion size was the measurement of the largest diameter of the largest lesion in cm, time of disease was recorded based on patient information and the dose of antimoniate required was based on the number of treatment cycles received by the patient. Heparinized pheromone peripheral blood was collected between 8 a.m. and 11 a.m., transported on ice to the laboratory and the plasma was stored at −20 °C for measurements of hormone levels. PBMCs were isolated from heparinized venous blood by passage over a Ficoll Hypaque gradient (Sigma–Aldrich). PBMCs were washed three times and resuspended at a concentration of 5 × 106 cells/mL in RPMI

1640 medium (Gibco, NY) supplemented with 2 mM l-glutamine, penicillin (100 U/mL), streptomycin (100 μg/mL) (Gibco, NY) and 10% heat inactivated human AB serum (Sigma–Aldrich). Cells were plated in 24-well tissue culture plates (Costar, Corning Incorporated, NY) at a concentration of 5 × 106 cells/mL and incubated at 37 °C at 5% CO2. Stimulation was performed by adding 10 μg/mL of SLA (soluble Leishmania antigen). The SLA was prepared as described by Carvalho et al. (1985). Briefly, stationary-phase promastigotes of L. amazonensis (MHOMBR86BA-125) were ultrasonicated and centrifuged at 20,000g for 2 h. The supernatant was used at a final concentration of 10 μg/mL. PBMC culture supernatants were harvested at 24, 48 and 96 h after in vitro stimulation and maintained at −20 °C until use.