To induce maturation, DCs were incubated with 10 µg/ml LPS (Sigma-Aldrich, Saint Louis, MO, USA) for 48 h. To analyse the effect of parasites on DC maturation, LPS- or IFN-γ- (10 ng/ml; BD Pharmingen) check details or IFN-γ/LPS-stimulated cells were incubated in the presence of Lm clones for 48 h. Cytospins were prepared using a cytocentrifuge

set at 100 g for 5 min. DCs were then May–Grünwald–Giemsa-stained and the percentage of infected cells and the number of intracellular parasites were determined by light microscopic analysis, after counting 100 cells per slide. Cytokines (IL-12p70, TNF-α and IL-10) were detected on cell-free 48 h culture supernatants using commercially available ELISA kits (BD optEIA; BD Biosciences). Recombinant cytokines were used to obtain standard curves to calculate cytokine concentration in the supernatants. Results are expressed as mean ± standard error of the mean (s.e.m.) of at least six independent experiments. Statistical significance

between treated and control cultures was analysed by Mann–Whitney U-test. P-values of P < 0·05 were considered find more statistically significant. To analyse the effect of virulence on the capacity of Leishmania parasites to enter and multiply within human DC we used two Lm clones differing by their virulence, which was established in BALB/c mice and two other Lm clones, HVΔlmpdi and LVΔlmpdi, generated from HV and LV, respectively, and invalidated for the lmpdi gene. We showed that HV promastigotes were internalized by DCs of all (n = 10) tested individuals with an infection rate (IR) and a parasite burden (PB) that increased significantly during the 3-day period (mean IR and mean PB ± s.e.m. were 42·3% ± 7·83 and 6·7 ± 0·99 at 24 h; 50·1% ± 7·64 and 12·4 ± 2·15 at 48 h; 66·3% ± 7·06 and 22·5 ± 7·29 at 72 h , respectively ) (Figs 1 and 2a,e). Interestingly, LV promastigotes failed to enter DCs from five

of 10 individuals (Fig. 1). In the other five donors, IR and PB were significantly lower than those observed in HV-infected DCs (5·9% ± 2·63 and 1·46 ± 0·6 at 24 h; 9·3% ± 4·43 and 2·9 ± 1·29 at 48 h; 11·7% ± 5·4 and 4·5 ± 2·27 at 72 h) Buspirone HCl (Fig. 2a,e). Differences observed in IR and PB between HV and LV were highly significant (P ≤ 0·0003 for IR and P ≤ 0·002 for PB during the 3-day culture). PB was significantly higher in HVΔlmpdi-infected DCs compared with LVΔlmpdi-infected DCs (P ≤ 0·01). For IR, a significant decrease was observed in LVΔlmpdi-infected DCs only at 72 h (P = 0·008) (Fig. 2b,f). Interestingly, IR and PB were lower in HVΔlmpdi-infected DCs when compared with HV-infected DCs. This result was significant for IR at 72 h (P = 0·03) and PB at 48 h and 72 h (P ≤ 0·01) (Fig. 2c,g).