There is siRNA available in the lab.

For transient transfection of synthetic siRNA, commercially available siRNA Smart Pool from Dharmacon targeted against the STAT3 transcription factor have been successfully transfected in human MDDCs (Lipofectamine 2000 as transfection reagent). The efficiency of silencing, as detected by western blot or FACS analysis of the protein was of 50-80% (depending on the donor), and peaked at 72 h post-transfection. Transfection efficiency was reproducibly higher than 80% and did not induce any type I IFN production.

100 mM sodium pyruvate were added to the monocyte cell culture.

Src kinases were found to be required for the initiation of some maturation characteristics of human monocytes derived dendritic cells upon stimulation with several toll like receptor (TLR) agonists but not of others, since their inhibition was able to dissociate cytokines and chemokines production from the induction of surface maturation markers.

These inhibitors were used to test the role of Src kinases in DCs during synergic engagement of TLR8 with either TLR4 or TLR3.

We have identified some genes differentially expressed in different stimulation conditions. In particular, the most relevant gene upregulated in presence of stimuli typically inducing Th1 responses (LPS, CpG, Poly I:C) but not in presence of the Th2 stimulus, Pam3Cys, was Stat-1.

It was evaluated whether STAT-1 was phosphorylated in DC following activation with TLR-dependent Th1 and Th2 stimuli and we found that only in presence of TLR stimuli able to induce Th1 responses STAT-1 was activated.

50 mg/ml of streptomycin (Gibco BRL) containing 10% (vol/vol) FCS (Hyclone) were added to the monocyte cell culture.

10 mg/ml sulfo-NHS-LC-biotin (Sigma-Aldrich) in 50 mM NaHCO3 pH 8.5 were used to biotinylate yeasts / spores for 2 hours at 4°C. The remaining reactive biotin molecules were inactivated by incubation in 100 mM Tris-HCl pH 8.0 for 40 minutes at 4°C. DCs were then treated with biotinylated spores/yeasts.

The TcR-Tg clones are specific for the 0T-1-OVA antigen. In the individual T cells, we studied the expression of twenty different genes either mediating effector functions, or coding for different receptors involved in T cell differentiation and memory generation.

These TcR-Tg clones are specific for the P14-GP33 antigen. In the individual T cells, we studied the expression of twenty different genes either mediating effector functions, or coding for different receptors involved in T cell differentiation and memory generation.

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.

Used for stimulation in the evaluation of the role of Src kinases in DCs during synergic engagement of TLR8 with either TLR4 or TLR3.

Used for stimulation in the evaluation of the role of Src kinases in DCs during synergic engagement of TLR8 with either TLR4 or TLR3.

Used for stimulation in the evaluation of the role of Src kinases in DCs during synergic engagement of TLR8 with either TLR4 or TLR3.

These conjugates were tested in vivo by either direct vaccination or ex vivo DC loading prior to vaccination. Both approaches show similar effectiveness in CTL priming.

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

TNF-alpha is used during maturation of the DCs.

Commercially available siRNA Smart Pool from Dharmacon targeted against the STAT3 transcription factor have been successfully transfected in human MDDCs.

We have shown recently that the T cell stimulatory capacity of DCs pulsed with tumorantigen-derived peptides can be considerably increased by activating the DCs through electroporation with CD40L, CD70 and constitutively active TLR4 encoding mRNA (TriMix DCs). This vaccine will also be tested in the multicentre trial.