R. D’Elia and K. J. Else Parasite Immunology Figure 3 Bone marrow dendritic cells (BMDC) stimulated with isolate E/S show increased levels of CD40 compared to unstimulated levels. Bone marrow was isolated and pooled from 3–5 naïve C57BL/6 mice and cultured into BMDCs. The BMDCs were incubated for 48 h, in vitro with media alone – unstimulated (U), 50 μg/mL E isolate E/S (E), 50 μg/mL J isolate E/S (J), 50 μg/mL S isolate E/S (S) or 100 ng Lipopolysaccharide (LPS). The cells were analysed for cell speciﬁc markers CD11c, CD11b, co-stimulatory markers CD80, CD86, CD40 and antigen recognition marker MHCII. A representative scatter plot is shown (a) with analysis performed on cells negative for 7AAD-R1 (b) and double positive for CD11c and CD11b (c). Levels of CD80, CD86, CD40 and MHCII were then measured on these cells with representative histograms showing CD40 expression on unstimulated cells (unﬁlled histogram) compared to its relevant isotype control (ﬁlled histogram) (d), LPS treated (unﬁlled histogram) compared to isotype control (ﬁlled histogram) (e) and LPS CD40 proﬁle (dark line) compared to unstimulated CD40 expression proﬁle (grey line) (f ). Geomeans for unstimulated, E, J or S isolate antigens and LPS stimulated BMDC and isotype controls were calculated. A fold change ratio for Geomean anti-cell surface marker antibody/Geomean Isotype control antibody were plotted for CD80, CD86, CD40 and MHCII expression on CD11c+CD11b+7AAD− cells (e). Experiments were performed four times and data shown are a representative experiment. differences in Th subset responses and thus outcome of infection (5–7). We demonstrate three novel ﬁndings: Firstly BMMΦ cultured with S isolate E/S secrete signiﬁ- cantly higher levels of IL-10 and IL-6 compared to BMMΦ stimulated with the E or J isolate E/S. Secondly this pheno- menon is not restricted to macrophages with BMDC also secreting elevated levels of IL-10 and IL-6 when stimulated with S isolate E/S compared to stimulation with the other two isolates. And ﬁnally, we identify no consistent differences in cell surface expression of co-stimulatory markers and MHC class II on BMMΦ and BMDC after exposure to E, 128 J or S isolate E/S, although BMDCs consistently express increased levels of CD40 after culture with all three types of E/S compared to unstimulated samples. Helminths have often been termed ‘masters of regulation’ (29), and this ability to regulate has been hypothesized to be due to parasite-derived E/S proteins. Several studies have focused on the immunomodulatory effects of E/S from helminths (19,30) but of particular interest has been a molecule secreted by A. viteae, ES-62, which has possible therapeutic potential in autoimmune diseases such as Rheumatoid Arthritis (17). BMDCs exposed to ES-62 are capable of inducing a Th2 © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Parasite Immunology, 31, 123–131