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

Differentiation of monocytes into dendritic cells is promoted, along with granulocyte-macrophage colony-stimulating factor, by addition of recombinant IL-4 (1000U/ml, R&D Systems).

A long-standing collaboration with Centre for Ecology & Hydrology (CEH, UK) has led to the identification of an activity in salivary gland extracts of the tick Rhipicephalus appendiculatus that profoundly modulates the responses of human monocyte-DC towards extrinsic stimuli. Recombinant protein has been generated from the gene for this molecule (Japanin) which had been cloned. This material inhibits the up-regulation of costimulatory molecules in response to certain TLR agonists, and some cytokines but not others, and modulates the function of the DC in ...

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.

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

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.

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.

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 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.

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.

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.