Each experimental group included five animals unless otherwise stated. Control mice (mock infection) received 100 μL of RPMI-1640. Metacestode vesicles were obtained by an in vitro system as described elsewhere (16). Vesicles were maintained in RPMI-1640 alone for 48 h. Subsequently, the supernatant containing Everolimus datasheet the excreted and secreted compounds (E/S) was collected, concentrated to 500 μg protein per mL and stored in aliquots at −80°C until use. The vesicular fluid (VF), containing 950 μg protein per mL, was aspirated with a needle (0·4 × 19 mm) mounted on a syringe, from individual
metacestode vesicles (cysts). VF antigen was stored in aliquots at −80°C until use. Peritoneal exudate cells from naive and infected mice, sacrificed after 6 weeks at the early stage and 12–16 weeks at the late stage of infection, were collected by peritoneal rinsing with 10 mL RPMI-1640. Cells were subsequently washed twice with HBSS and resuspended in RPMI-1640. Pe-DCs and CD4+ pe-T cells were enriched from the peritoneal
cell suspension of each group of naive and AE-infected mice after incubation of cells in 5 mL RPMI-1640 + 20% FCS in a Petri dish for 2h at 37°C, with an atmosphere containing 5% CO2. Nonadherent cells separated from macrophage-enriched adherent cells were subsequently divided into two parts; EPZ015666 concentration the first part was used for the positive selection of pe-DCs using the mouse CD11c easySep kit (STEMCELL Technologies SA, Grenoble, France). The second part was employed for the selection of CD4+ pe-T from cells using
the mouse CD4+ T-cell enrichment easySep kit (StemCell). With both kits, the selected cells were retained from the original cell population using a magnetic cell separation (MACS) system according to manufacturer’s instructions. Highly enriched (>90% purity) pe-DCs as well as CD4+ pe-T cells were washed and suspended in complete RPMI-1640. To quantify the amount of peritoneal DCs following the intraperitoneal secondary infection with E. multilocularis metacestodes, peritoneal cells from naive and AE-infected mice were prepared in HBSS, washed and resuspended in staining buffer (PBS, 0·05% NaN3 and 0·5% BSA). Aliquots of 106 cells per 50 μL per well were incubated each with 1 μg of anti-CD16/CD32 for 20 min in the dark, to block nonspecific binding of antibodies to the FcγIII and FcγII receptors, and subsequently incubated for 30 min with 1μg of phycoerythrin (PE)-labelled anti-CD11c antibody. To analyse whether the expression of adhesive and co-stimulatory molecules on DCs of AE-infected mice was modified, these cells were isolated from the peritoneal cavity of AE-infected mice taken at the early and late stages of infection and from mock-infected naive mice (as control) separately, all cell preparations were resuspended in staining buffer.