To assess the importance of ROS production in DC killing ability, DPI (10 microM) is added 30 minutes before stimulation.

Yeasts/spores are biotinylated using 10 mg/ml sulfo-NHS-LC-biotin (Sigma-Aldrich) in 50 mM NaHCO3 pH 8.5 for 2 hours at 4°C. The remaining reactive biotin molecules are inactivated by incubation in 100 mM Tris-HCl pH 8.0 for 40 minutes at 4°C. DCs are then treated with biotinylated spores/yeasts. After 1 hour, cells are permeabilized and labeled with aHLA-DR-FITC. After zymolyase treatment, intracellular yeasts/spores are detected using APC-labeled streptavidin and analyzed by flow cytometry.

Blue formazan particles are dissolved using 2M KOH and DMSO.

DCs are stimulated with spores or yeasts, washed, supplemented with 0.1 mg/ml of NBT.

DCs are treated with zymolyase.

DCs are stimulated over 6 hours.

Serotype A C. albicans strain SC5314 is cultured overnight in Saboraud medium at 28°C and then shifted to RPMI medium for 18 hours at 37°C to allow hyphal growth. The culture is treated as for yeast cells. After microscopical inspection of the purity of hyphal culture, this is treated as for yeast cells. [It should be noted that in all the experiments performed in this study live microrganisms were used.]

In order to test homogeneous yeast populations, pure spore cultures are obtained by using this strain whose sporulation efficiency is 100%. To prepare spores, cells are grown on YPD plates, replicated on SPOIV medium (2% potassium acetate, 0.25% yeast extract, 0,1% glucose) and sporulation is assessed by optical microscopy. Zymolyase (2 mg/ml) is used to digest ascum and liberate spores. [We initially performed experiments to evaluate zymoliase sensitivity of asci for this specific strain and we assessed that 15 minutes incubation was the minimum time needed ...

Saccharomyces cerevisiae strain SK1 (MATa/alpha HO gal2 cupS can1R BIO, Kane SM and Roth J. 1974 Bacteriol. 118: 8-14) is cultured in complete medium (YPD, 2% yeast extract, 1% peptone, 2% glucose) for 18 hours, then collected, washed twice with sterile water and resuspended at 108 cells/ml. S. cerevisiae strains BY4741 (genotype, Mata his3delta1 leu2delta0 met15delta0 ura3delta0) and BY4741 och1 (Mata his3delta1 leu2delta0 met15delta0 ura3delta0 OCH1::kanMX4) are cultured in complete medium till exponentially phase and treated as before.

PBMC are stimulated in limiting dilution conditions with a pool of overlapping peptides (15 amino acids) covering the protein.

The DC with blocking receptors are exposed to fungi.

Advanced melanoma patients are vaccinated with the DC vaccine.

This step is repeated several times to do several stimulations.

Patients with advanced prostate carcinoma are vaccinated with the DC vaccine.

Vaccination with a vaccine composed by autologous DCs pulsed with apoptotic autologous ovarian carcinoma cells. Apoptosis is induced by UV.

Pulsing of autologous DCs with apoptotic autologous ovarian carcinoma cells.

Autologous DC are pulsed with the apoptotic allogenic prostate carcinoma cell line LNCap to produce the vaccine for patients with advanced prostate carcinoma..

Leukapheresis products from cancer patients is used as a starting material for monocyte enrichment by elutriation technology with ELUTRA® (Gambro).

The primary objective of this trial is to determine toxicity and feasibility of treatment of patients with advanced melanoma with DC vaccine and adoptive transfer.

The patients will be vaccined with the DC vaccine that consists of monocyte derived DC loaded with tumor lysate generated from the excised lesion.