Significant differences ( 0.05) were detected in Compact disc4 T-cell responses between your 10 and 50 g DNA dosages of OxMan-PLL on times 2 and 3 from the proliferation assay. Open in another window Figure 4 Proliferative responses in C57BL/6 mice immunized with different DNA vaccine formulations. tumours. Both reduced and oxidized mannan delivery was more advanced than DNA alone or DNA-poly-L-lysine. These studies show the potential of oxidized and decreased mannan for efficient Posaconazole receptor-mediated gene delivery efficacy of OxMan and RedMan as receptor-mediated gene transfer ligands. Using a poly cationic linker, PLL, to link OxMan and RedMan to DNA, we exhibited successful immune responses (CD8, CD4 and antibody responses) leading to tumour protection in mice. Materials and methods Preparation of oxidized and reduced mannan-PLLTo prepare oxidized mannan (OxMan), 14 mg of mannan (from values of 0, 0.25, 0.5, 0.75 and 1) in the same final NaCl concentration was added in a stepwise manner (10 ml per addition) for 1 hr. The conjugates, OxMan-PLL-DNA and RedMan-PLL-DNA, were incubated at RT for 30 min before use. The degree of complexation between OxMan-PLL and DNA under different conditions, i.e. different concentrations and values, was determined from the extent of DNA retardation in 0.6% (w/v) agarose gel electrophoresis run for 1 hr at 100 mV. The amount of conjugate in micrograms refers to the amount of plasmid DNA unless otherwise stated. Cytotoxicity of carriersJ774 cells (1 105) in a volume of 150 ml of medium were seeded into each well of a 96-well microtitre plate. A volume of 50 ml of OxMan, Redman, OxMan-PLL, RedMan-PLL and PLL at various concentrations was incubated with the cells for 16 hr at 37 and subsequently 1 Posaconazole Ci of thymidine was added and the mixture was incubated for a further 6 hr. Cells were harvested and [3H]thymidine uptake was measured using a -scintillation counter (Top Count Gamma Counter; Packard, Meriden, CT). Mice and immunizationsFemale C57BL/6 mice aged 6C10 weeks were used in all experiments. Mice were immunized twice intradermally (i.d.) into the base of the tail with 100 l of the following: DNA 10 g, DNA 50 g, DNA-PLL 10 g, DNA-PLL 50 g, RedMan-PLL-DNA 10 g, RedMan-PLL-DNA 50 g, OxMan-PLL-DNA 10 g or OxMan-PLL-DNA 50 g. Spleens were removed 10C14 days after the last immunization and immune responses were assessed. All studies were reviewed and approved by the Austin Health animal ethics committee. AntigensFor stimulation, the following antigens were used: chicken egg OVA (Sigma, Steinheim, Germany), OVA CD4 epitope ISQAVHAAHAEINEAGR (OVA323-339) and OVA CD8 epitope SIINFEKL. All peptides were synthesized by Mimotopes (VIC, Clayton, Australia) and were ?95% real by mass spectrometry and high-performance liquid chromatography (HPLC). Proliferation assaySplenocytes (2 105) in 100 l of complete medium [RPMI supplemented with 10% fetal calf serum (FCS), penicillin/streptomycin and glutamine] were seeded into 96-well round-bottom plates and cultured in triplicate with 10 g/ml OVA, OVA CD8 epitope, OVA CD4 epitope or medium alone (unfavorable control) and incubated at 37 and 5% CO2. Concanavalin A (Con A) at a concentration of 1 1 g/ml was used as an internal positive control. Proliferation was assessed on days 2C5 by adding 1 Ci of [3H]thymidine per well to Posaconazole one plate per time-point. Cells were incubated for a further 6 hr before harvesting onto glass fibre filters. [3H] uptake was measured using a -scintillation counter (Top Count Gamma Counter). Enzyme-linked immunosorbent spot-forming cell assay (ELISPOT)Ninety-six-well IL8 plates (MAIP S4510; Millipore, Moisheim, France) Posaconazole were pre-wet with 50 l of 70% ethanol, washed six occasions with 200 l of sterile PBS and coated with 70 l of 5 g/ml anti-interferon (IFN)- coating antibody, AN18 (Mabtech, Stockholm, Sweden), overnight at 4. Plates were washed six occasions with sterile PBS and blocked with 200 l of complete medium for 2 hr at 37. Spleen cells (5 105) in 100 l of complete medium were incubated with 10 g/ml OVA, 10 g/ml OVA CD8 epitope, 10 g/ml OVA CD4 epitope or medium alone for 18 hr at 37. Con A at a concentration of 1 1 g/ml was used as an internal positive control. Triplicate wells were set up for each condition..