“…After, TMB (tetramethylbenzidine) substrate solution (BD Pharmingen, San Diego, CA, USA) was used for assay revelation, which was read on a microplate Venous blood was collected in two different time periods: (1) at the moment of patient's admission, just after clinical evaluation and before any treatment regimen and (2) at the end of specific treatment for mucocutaneous leishmaniasis (40-day period of Glucantime ® regimen). In both periods, peripheral blood mononuclear cells (PBMC) were separated using Ficoll-Paque TM Plus gradient (GE Health Care, Uppsala, Sweden) and cultured in RPMI 1640 (GIBCO, Grand Island, NY, USA) in 8 different culture conditions: medium alone, 2 ug/mL Phytohaemagglutinin (Sigma, St. Louis, MO, USA), 3:1 T cruzi (Y strain) or 3:1 L. braziliensis (Lb2904 strain) live parasites, as well as 5 ug/mL T. cruzi and 5 ug/mL L. braziliensis soluble proteins, in a 5% CO 2 atmosphere at 37 °C for 24 h, as described elsewhere [11,12]. The supernatants were used for cytokine quantification, IL-6, IL-10, TNF-α and IL-12, by ELISA assay as previously described [11,13].…”