Positive [genomic DNA of L. chagasi (MHOM/BR/1972/BH46)] and negative (without DNA) controls were included in each test. Amplified fragments were analyzed MLN2238 by electrophoresis on 8% polyacrylamide gel and ethidium bromide-stained for the PCR product identification. The parasitological investigation was performed until 885 days after L. chagasi challenge. Statistical

analyses were performed using Prism 5.0 software package (Prism Software, Irvine, CA, USA). Normality of the data was demonstrated using a Kolmogorov-Smirnoff test. Paired t-tests were used to evaluate differences in mean values of cytokines levels, considering the comparative analysis of T0 and T3 (Fig. 1) or T90 (Fig. 2) or T885 (Fig. 3), in each group evaluated. Unpaired t-tests were used to evaluate differences in mean of values of TGF-β (Table 1). Analysis of variance

(ANOVA) test followed by Tukey’s multiple comparisons were used in the evaluation between the different treatment groups for cytokines (Fig. 1, Fig. 2 and Fig. 3) and nitric oxide (Fig. 4) analysis. Differences were considered significant when P values were <0.05. To determine the impact of LBSap vaccination on the immune response, we evaluated the cytokine profile (TNF-α, IL-12, IFN-γ, IL-4, and IL-10) in the supernatant of PBMC stimulated with VSA (Fig. 1A) or SLcA (Fig. 1B). In this context, we performed a comparative analysis between T0 and T3, in addition to the comparisons between experimental groups at each time point. In the comparison between T0 and T3, the Sap group showed increased levels (P < 0.05) Ponatinib research buy of TNF-α and IFN-γ production at T3 with VSA stimulation. Additionally, the LB group presented higher levels (P < 0.05) of IL-10 in

VSA-stimulated PBMCs heptaminol at T3, as compared to T0. In contrast, in SLcA-stimulated cultures, the LB group displayed lower levels of TNF-α at T3 as compared to T0 in SLcA-stimulated cultures (P < 0.05). Interestingly, the LBSap vaccine induced higher levels of both IL-12 and IFN-γ at T3 in VSA-stimulated PBMCs. Similarly, in the presence of SLcA, increased levels (P < 0.05) of IFN-γ were observed in the LBSap group at T3. The comparison between the experimental groups, in different time points, revealed increased levels (P < 0.05) of IFN-γ in VSA-stimulated cultures from the LB group, as compared to C group in T3. Interestingly, higher (P < 0.05) levels of this cytokine were observed in the VSA-stimulated culture of LBSap group when compared to C and Sap groups, at T3. Similarly, in SLcA-stimulated cultures, LBSap group displayed increased (P < 0.05) levels of IFN-γ in relation to C, Sap and LB groups at T3. In addition, at T3, LBSap group showed increased (P < 0.05) levels of IL-12 in relation to C and Sap groups, in addition to reduced (P < 0.