The PBMCs were placed in a humidified incubator overnight with 5% CO2 atmosphere at 37°C. The yields and phenotypes of the 10 effector cells post-thaw were: total yields: 90–99%; CD3+ cells: 53–79%, CD3−CD56+ cells: 9–31%. The long-term, lymphoblastoid cell cultures (MS1533, MS1847, MS1874, MS1946), originating from the PBMCs of MS patients in different disease states, were cultured as described previously [8, selleckchem 9]. In brief, the cells were grown at 0·5 × 106 cells/ml of RPMI-1640 supplemented with 10% inactivated HS. Cells were split three times a week and supplemented with fresh medium. Twenty-four h before use the cells were transferred to AIM-V serum-free medium (Gibco,

Naerum, Denmark) containing 0·03% w/v glutamine, 10 mM HEPES and 0·1 Mio IU/l penicillin. Polyclonal antibodies against Env and Gag from HERV-H/F and Env from HERV-W were raised in New Zealand white rabbits. The antibodies U0126 supplier were raised against 16-mer peptide epitopes localized at equivalent positions in open reading frames (ORFs) of the respective endogenous retroviruses. Both the peptides and the anti-sera were prepared by Sigma Genosys (Haverhill, UK). The polyclonal anti-sera were: anti-HERV-H/F Gag [the peptide translated

from the long putative gag ORF of the HERV-Fc1 sequence (aa380-395) (GenBank AL354685)] in a region with very high similarity to the gag sequences of known HERV-H copies with complete Env ORFs: HERV-H env62/H19, HERV-H env60 and HERV-H env59 [10], anti-HERV-H Env (1–3) and anti-HERV-W

Env (1–3) (these peptides were derived from equivalent positions in the Env ORFs of HERV-H env62/H19 (Env H1TM: aa489–505; Env H3SU: aa 370–386 (10) and syncytin 1 (Env W1TM: aa415–431, Env W3SU: aa301–317) [11], respectively. All peptide sequences fulfil the criteria of immunogenicity, and are localized at equivalent positions in the HERV-H and HERV-W Envs, while having highly dissimilar amino acid sequences. Preimmune sera were collected from all rabbits before immunization. Rabbits were immunized with the peptides, boosted three times, and after the final boost peripheral blood was collected for subsequent measuring of anti-peptide antibodies. mafosfamide The specificity and cross-reactivity of the anti-HERV anti-sera were analysed by enzyme-linked immunosorbent assay (ELISA) and time-resolved immunofluorimetic assay (TRIFMA) assays. The anti-sera were at least 1000 times more reactive towards their relevant peptide antigens than towards non-relevant peptides (data not shown). The polyclonal anti-HERV antibodies were prepared for ADCC by thawing, dilution × 10 in AIM-V medium (Gibco), supplemented as described above, heat-inactivation for 30 min at 56°C and refreezing at −20°C. Immediately before use each diluted serum sample was thawed and added to the prepared target cells.