Monocytes were transferred into culture bags by sterile welding and cultured for 5 days in serum free medium (CellGro, Cell Genix) in the presence of 100 ng/mL GM-CSF (Leukine, Berlex) and 20 ng/mL IL-4 (Cell Genix).

The model antigen OVA was fused to the C1C2 exosome-binding domain of MFG-E8/lactadherin. This construct was used in a study on the capture of antigen-bearing exosomes by DCs and cross-presentation of tumour antigen in exosomes.

Expression vectors for GFP-RIG-I used to analyse the interaction between RIG-I, a viral sensing protein, and NS1, an inhibitor of interferon production that is encoded by influenza A virus have been made available.

In the process of extracting total RNA from yeast cells, the pellets were washed with 70% Ethanol two times.

Total RNA was extracted from yeast cells after buffering, incubation and centrifugation by phenol extraction.

The FC-block was used for incubation of cells (1:50 in PBS + 2 % FCS) for FACS staining of activation markers.

Fabs that bind strongly to L-SIGN, but to a lesser degree or not at all to dendritic cell-specific ICAM-grabbing nonintegrin (DC-SIGN) were isolated and characterized. We believe that the isolated Abs may be useful for selective delivery of Ags to DC-SIGN- or L-SIGN-bearing APCs for the modulation of immune responses and for blocking viral infections.

To assess the importance of ROS production in DC killing ability, 2-(3,4-Dihydroxyphenylimino)-imidazolidine (10 microM) was added to the dendritic cells 30 minutes before stimulation.

DMSO was used to dissolve blue formazan particles.

This vaccine was used for RCC patients who were deficient in T cell IFN-gamma and IL-2 production pre-vaccination.

Constructs for fusion proteins of the DC specific receptor DC-SIGN fused to GFP have been completed and tested.

This DC vaccine was produced from the Leukapheresis product, after elutriation, of a metastatic lesion of stage III/IV melanoma patients.

This DC vaccine was produced from matured DC (matured using TNF alpha, IFN alpha and PGE2) which were subsequently transfected with CD40L-encoding RNA and tumor RNA.

Autologous dendritic cells are loaded with apoptotic leukemic cells from patients with B-CLL to be used as vaccine in patients with chronic lymphocytic leukemia. Preliminary data from a clinical study indicate reduction of circulating tumor cells. No adverse effects have been observed.

Three patients were enrolled in a clinical trial and DC vaccine was produced. The DC vaccine was administered into irradiated tumours in RCC of one patient only due to cancer progression in the other two patients. The patient treated is in complete remission 6 months after treatment.

These constructs were used to transfect immature DCs.

Using curdlan as a specific agonist of dectin-1, we have shown that this C-type lectin couple to Syk kinase leads to activation of ERK, JNK and p38 MAPKs, as well as NF-kappaB.

DC based vaccines expressing the tumor antigens PSA or Her2/neu were produced in a pre-clinical work.

We have prepared several new constructs for the expression of (modified) adhesion receptors in DC.

100 micrograms/ml curdlan (Wako) was used (along with R848, LPS, yeast RNA and yeast cells in different conditions of culture) to induce DC activation.