Briefly, calf-thymus DNA was passed through a 045?m nitrocellulose Millex syringe filter (Millipore Corporation, Bedford, MA, USA) to remove any ssDNA fragments. tested in experimental HIV 19 and as a tumour vaccine 20, while MF59 is included in seasonal influenza and influenza A (H1N1) vaccine preparations 21C22. However, actually the last generation of adjuvants (SQU adjuvants), like the early one 23, specifically elicited anti-nuclear antibody in non-autoimmune susceptible Balb/c mice 24 and pathogenic anti-phospholipid antibodies in C57/B6 mice 25. Indeed, DW14800 Balb/c mice receiving adjuvants developed either an autoimmune glomerulonephritis 26 or autoantibody to ribonuclear protein/Smith (RNP/Sm) and to single-stranded DNA (ssDNA) 23, the specificity profile/pattern of these autoantibodies being dependent upon the type of adjuvant used 27. Recently, a clinical syndrome referred to as autoimmune/autoinflammatory syndrome induced by adjuvant (ASIA), encompassing four different clinical entities, namely siliconosis 28C29, the gulf war syndrome 30, macrophagic myofasciitis syndrome 31 and post-vaccination phenomena 29C34, has been described in humans. The symptoms are DW14800 overlapping and have been attributed to the probable effect of adjuvants (reviewed in 35C38). All the above findings raised concerns as to whether adjuvants alone could be harmful in our animal model of SLE, by acting as a sort of autoimmune disease activator, and so reversing the potential beneficial effect of a CD20-mimotope peptide vaccine. To identify the least harmful adjuvant, as no definite data are yet available on the effects of adjuvants on human-like SLE-prone mice BWF1, we evaluated the effects of IFA, CFA, ALU and SQU on the disease course in this mouse strain. Materials and methods Reagents and antibodies Keyhole limpet haemocyanin (KLH) was purchased from Calbiochem (San Diego, CA, USA). IFA and CFA were purchased from Thermo Fisher Scientific Inc. (Rockford, IL, USA). SQU adjuvant (AddaVax) and ALU adjuvant (Alhydrogel) were purchased from Invivogen (San Diego, CA, USA). Double-stranded calf thymus DNA was purchased from Sigma (St Louis, MO, USA). RNP/Sm antigen was purchased from Arotec Diagnostics Limited (Wellington, New Zealand). Phycoerythrin-cyanin 51 (PE-Cy5)-conjugated anti-mouse CD3 antibody, phycoerythrin (PE)-conjugated anti-mouse CD8 antibody, and fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 and CD19 antibodies were purchased from BD Pharmingen (Franklin Lakes, NJ, USA). Mice and immunization strategy Nine-week-old BWF1 female mice were purchased from Harlan Laboratories (San Pietro al Natisone, Italy). Mice were kept at the University of Bari with a 12-h light/dark cycle and experiments were performed in accordance with institutional and internationally approved guidelines on animal research. Animal study protocols were approved by the University of Bari Medical School ethical review Committee. BWF1 mice were treated with adjuvants that are commercially available for human use (Table?1), namely IFA, SQU and ALU, which were mixed with KLH before injection. Immunogen (200?l of adjuvant emulsion/injection) was prepared by mixing 100?l of phosphate-buffered saline (PBS) containing 100?g KLH with an equal volume of adjuvant. Four groups of mice (four mice per group) were primed at the 9th week (when Rabbit Polyclonal to IP3R1 (phospho-Ser1764) no proteinuria was detected in any of the mice) with IFA, CFA, SQU or ALU adjuvant. Boosters were given fortnightly (up to the 15th week) until proteinuria 100?mg/dl was detected in at least one mouse in each group. Two additional groups of mice treated with PBS-only (adjuvant untreated group; UNT) and with CFA, served as the negative and positive 39 control groups. Groups with only one mouse left were excluded from further evaluations. Table 1 Adjuvants most commonly employed in vaccine for human use B, hepatitis A/B, human papilloma computer virus, anthraxIncomplete Freund’s adjuvantMineral oil-in-water emulsionInfluenza, experimental HIV, tumourSqualene-based (MF59?)Organic oil-in-water emulsionSeasonal influenza (Fluad?), pandemic influenza (Focetria?), pre-pandemic influenza (Aflunov?) Open in a separate window Blood was collected every 2 DW14800 weeks by an orbital technique from the ophthalmic venous plexus. An aliquot of blood was mixed rapidly with 900?l of PBS containing 18?mg K3-ethylenediamine tetraacetic acid (EDTA) and another aliquot was used for serum recovery. Sera were stored at ?80C until used, while EDTA blood was used immediately for white blood cell (WBC) counts and cytofluorimetric assays. Mice were.