A self-inactivating lentiviral vector expressing shRNA specific for STAT3 is now available in the laboratory.

Serotype A C. albicans strain SC5314 was cultured overnight in Saboraud medium at 28°C and then shifted to RPMI medium for 18 hours at 37°C to allow hyphal growth.

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.

Shuttle plasmids into which antigens are inserted are available from a discontinued clinical program where an HSV vector expressing full length tyrosinase, gp100 and MART-1 was used to transduce DC from melanoma patients which were then to be used for vaccination. These could be of use to the project if partners would like to use HSV-based technology for the delivery of antigens to 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.

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.

This is spleen from patients that undergo surgery due to clinical situations that do not affect the immune system (most frequently malformations). We have attempted to identify and characterize CD8T lymphocyte populations in humans. We compared the distribution of phenotypically distinct cell sets using seven color staining in the blood, lymph nodes and spleen. We found that the addition of other markers allow to subdivide N, CM, TEM and TEMRA populations in additional cell types.

The capacity of splenic DC, pulsed in vitro with protein antigens or peptides, to induce immunity was monitored after transfer into syngeneic animals. To improve existing strategies, the amplitude and polarisation of T cell responses was monitored in the presence or absence of regulatory T cells.

In order to test homogeneous yeast populations, pure spore cultures were obtained by using this strain whose sporulation efficiency is 100%. To prepare spores, cells were grown on YPD plates, replicated on SPOIV medium (2% potassium acetate, 0.25% yeast extract, 0,1% glucose). Zymolyase (2 mg/ml) was used to digest ascum and liberate spores. 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 disc...

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.

These stage III/IV melanoma patients underwent surgical removal of a metastatic lesion and lymphodepletion prior to vaccination and adoptive transfer of autologous T cells.

These patients receive DC transfected with RNA encoding MelanA, Mage3 and Survivin +/- E/L-selectin by the i.v. route. 2nd phase of the DERMA-ER-DC 06 trial.

Cells stained in PE-alpha DC80 in PBS + 2 % FCS for FACS analysis for activation markers.

The D2SC1/Flt3-DC were stained (Mini67-1KT or Mini26-1KT (PKH67/PKH26 green/red fluorescent cell linker mini kit for general cell membrane labelling) from Sigma) for a process of dead cell uptake.