In a process of total RNA extraction from S. cerevisia, yeast cells were collected and the pellets were resuspended in 3.6 ml AE buffer (AE=50mM NaAcetate pH 5.2, 10mM EDTA).

These T cells were isolated during elutriation of a leukapheresis product (patients with stage III or stage IV melanoma).

These T cells were obtained from isolation during elutriation and were used for subsequent adoptive transfer.

DCs were pulsed with allogenic melanoma peptides (SK-Mel 24, HLA-A1 and HLA-A2 positive) plus KLH as immunological tracer and subsequently used to produce a vaccine for melanoma.

Zymolyase was used to treat DC, which were used in a study of microrganism survival following uptake by DCs.

These are strains of Saccharomyces cerevisiae that were cultured and collected in different conditions: - different growth phase (exponential and stationary phase) - different growth media (standard and promoting pseudohyphal growth) - different cell form (spheroplast, spore and whole cell)

In a spore culture, zymolyase (2 mg/ml) was 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 to damage the ascus, without touching the spore cell wall. Zymoliase was inactivated by heat (65°C for 2 minutes) and washed away carefully together with the remainings of the empty ascus, by resuspending twice the spore culture in 500 µl of distilled water centrifuging and discarding the supernatant; th...

In a process of total RNA extraction from S. cerevisia, the cells were collected and the pellet were resuspend in 3.6 ml AE Buffer.

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

Vectors were engineered to express distinct chemokines at the local skin site of mice. In particular, attention has been focussed on the recruitment of distinct subsets of DC to skin.

Melanoma patients were vaccinated with 4 peptides (MAGE-A3, gp100, NA17 and tyrosinase) presented by HLA-A2 and monitored. We measured responses to these peptides injected with Montanide, but did not know whether the Montanide adjuvant was important for these T cell responses. We therefore injected the same peptides without adjuvant, and no response was observed. We conclude that Montanide had a clear adjuvant effect for the CD8 T cell responses measured against these four peptides.

These constructs were prepared for a per-clinical model of breast cancer.

Yeast cells in different conditions of culture (along with R848, Curdland, yeast RNA and LPS) were used to induce DC activation.

Yeast RNA (along with R848, Curdland, LPS and yeast cells in different conditions of culture) was used to induce DC activation.

Untouched naive CD4+ T cells are co-coltured with fungi -pulsed DCs to prime naive T cells.

These T cells are to be purified using MACS cell separation to contain only CD8+ T cells. The latter are then co-cultured with electroporated DC, stimulated and become evaluated on their cytokine secretion, cytotoxicity and % of antigen specific T cells.

TNF alpha was used for DC maturation with the final aim to produce a DC vaccine.

TLR ligands (simultaneous activation of TLR4 and TLR8 signaling cascades) were used as stimuli for MDDC and resulted in a marked inhibition of the secretion of the proinflammatory chemokine CCL2 with respect to stimulation through a single TLR.

TNF-alpha is used during maturation of the DCs.

The immature monocyte-derived dendritic cells where transduced with high doses of lentiviral vectors to monitor activation of the immature DC.