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.

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.

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

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.

The LPS was used for stimulating DC. It was shown in mice that both IL-10 as well as IL-12 production is dependent on the signalling adaptor molecules either MyD88 or TRIF, respectively in response to CpG, LPS or PolyIC. Manufacturer: Alexis

There is siRNA available in the lab.

We have chemically coupled the S-epimer of the Pam3CSK4 to long peptides containing a CTL epitope and used this structure to investigate the behaviour of the diastereomer.

We are examining the effects of a schistosome-derived protein on intestinal immune responses, and have shown that oral administration leads to the rapid appearance of cytokines in lymph.

Studies were carried out demonstrating that DC and other cell types can be activated in vitro by transfection with single stranded viral or synthetic RNA containing 5’ phosphates.

It was shown in mice that both IL-10 as well as IL-12 production is dependent on the signalling adaptor molecules either MyD88 or TRIF, respectively in response to CpG, LPS or PolyIC.

It was shown in mice that both IL-10 as well as IL-12 production is dependent on the signalling adaptor molecules either MyD88 or TRIF, respectively in response to CpG, LPS or PolyIC.

This shRNA was cloned into the pLVTHM lentiviral vector (obtained from the repository Addgene). This vector has been already successfully used to generate self-inactivating lentiviral vectors previously tested in DCs for transfection efficiency and absence of effects on their phenotype.

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

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.

This vaccine consists of DC loaded with a single MAA-derived peptide (MAGE-3.A1 or -A2 or Na17.A2). It was tested in a trial in small cohorts of patients (3-9) who were vaccinated with differently composed DC-vaccines.

We showed that, in the absence of additional stimuli, inflammatory signals such as pro-inflammatory stroma-derived cytokines on DC are ineffectual in promoting DC activation and cannot substitute for engagement of innate receptors directly on DC.

We are characterising (in collaboration with Sebastian Amigorena) rat lymph exosomes and will determine if they transport intestinally-delivered scrapie ME7.

This PSA was used as a control in the transfection of immature DC. Transfection of immature DCs was done with different constructs encoding for the oncogene Her-2/neu or an adeno virus Her-2 full length construct.

The protein vaccines were injected combined with a combination of iNKT cell-agonist alpha-GalCer and MPL, a detoxified version of LPS that signals through TLR4. This combination treatment stimulates antigen-specific T and B cell responses that are greater than those elicited with alpha-GalCer or MPL alone.

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