The DC populations were CD123+, CD11c+CD16+, CD11c+BDCA1+, CD11c+BDCA3+ DC.

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

Monocyte-derived DCs are added at a final concentration of 5x105 cells/ml into 96-well plates.

Serial diluition of yeast cells and preparations are added to the MoDCs.

Intensity values are adjusted by subtracting surrounding background from spots. The median of spot intensities is corrected for background. To eliminate signals that are most prone to estimation error, any spot is excluded from analysis if both the Cy3 and Cy5 mean fluorescence signals are within two standard deviations of the mean background signals for that spot. This procedure avoids artificially inflated measurements of expression due to low signals. Additional to eliminating flagged spots, spots are also visually inspected and flawed ones discarded from the analysis.

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.

AND

The yeast oligonucleotide array is constructed using the S. cerevisiae Genome Oligo Set ™ (Operon Technologies, CA, USA) composed of 6240 optimized oligonucleotides (70mers) each representing one yeast gene.

The polarization is assessed by flow cytofluorimetry and cytokine production.

The cells are collected and the pellets are resuspended in 3.6 ml AE Buffer (AE=50mM NaAcetate pH 5.2, 10mM EDTA) and then transferred in 15 ml tubes wit 200 µl 10% SDS (w/v). 2 ml of preheated acid phenol (pH 4.3) are added and the falcon is mixed by vortex.

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.]

Cells are cultured in RPMI 1640 medium (GIBCO BRL) supplemented with 2 mM L-glutamine (Sigma), 1% (vol/vol) non-essential amino acids, 100 mM sodium pyruvate, 50 U/ml of penicillin and 50 mg/ml of streptomycin (Gibco BRL) containing 10% (vol/vol) FCS (Hyclone).

2 x 200 x 10e6 monocytes are transferred into culture bags by sterile welding and cultured for 5 days at a density of 2.5-5 x 10e6 cells/mL in serum free medium (CellGro, Cell Genix) in the presence of 100 ng/mL GM-CSF (Leukine, Berlex) and 20 ng/mL IL-4 (Cell Genix). After 2 days one volume fresh medium containing 2x concentrated cytokines is added.

A portion of pulsed and unpulsed mature dendritic cells will be frozen and used to monitor immunological responses elicited by the DC vaccine and transferred T cells during the clinical trial.

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

Pulsing of autologous DCs with apoptotic autologous ovarian carcinoma cells.

Day 1: seed cells

The samples are centrifugated at 5000 rpm in an tabletop centrifuge.

After the spin (10’ at 3,000 rpm), the supernatants are precipitated in RNase-free centrifuge tube by adding 1/10 volume 3 M NaAcetate (pH 5.3) and 2.5 volumes of cold ETOH 100% overnight at -20°C. Centrifugation (30’ at 12000 rpm).

Day 3: change of medium