These DC were co-electroporated with constitutively active TLRs and TAA encoding mRNA. They were used to compare different maturation methods.

These plasmacytoid pDC that were stimulated with both CpG and the TLR-7 ligand R848 were shown to produce very high levels of IL-12p70 (in the ng/ml range per 5 x 105 cells/ml) and IFN-gamma.

The agonists were used as adjuvants in vivo to induce DC activation and CD4+ T cell and antibody responses.

The immature monocyte-derived dendritic cells where transduced with high doses of lentiviral vectors to monitor activation of the immature DC.

Human DC exposed to TLR ligands and activated iNKT cells in vitro. They had enhanced expression of maturation markers, suggesting that a cooperative action of TLR ligands and iNKT cells on DC function is a generalizable phenomenon across species. These studies highlight the potential for manipulating the interactions between TLR ligands and iNKT cell activation in the design of effective vaccine adjuvants.

In our effort to demonstrate that pathogen-derived signals to DC mediated via TLRs can be modulated by activated iNKT cells, DC isolated from animals treated simultaneously with TLR and iNKT cell ligands were potent stimulators of naive T cells in vitro compared with these DC from animals treated with the ligands individually.

In our effort to demonstrate that pathogen-derived signals to DC mediated via TLRs can be modulated by activated iNKT cells, these DC isolated from animals treated simultaneously with TLR and iNKT cell ligands were potent stimulators of naive T cells in vitro compared with DC from animals treated with the ligands individually.

The vaccine for ovarian carcinoma is composed by autologous DCs pulsed with apoptotic autologous ovarian carcinoma cells. Apoptosis is induced by UV.

These autologous DC were electroporated with mRNA encoding CD40L, CD70, caTLR4 and one tumorantigen (gp100, Tyrosinase, Mage-C2 or Mage-A3) to produce a vaccine for immunotherapy of advanced melanoma patients.

CX3CR1 GFP mice, in which bmDC are green fluorescent (GFP), were analyzed according to their bmDC localization. It was found that the cells were organized in unique clusters, mainly located in the endosteum of the BM. These DC co-localize with B cells and these clusters are occupying architecturally definable niches and are reminiscent to the niches that were found in the cranial BM parenchyma.

Alternatively activated DC (AA-DC) were obtained by culturing immature DC in the presence of maturative agonists (such as, LPS, CD40L or TNF) and calcitriol, IL-10 or prostaglandin E2 for a study of molecular signatures for alternatively activated DC. AA-DC showed a decrease in the production of IL-12 but retained most of the ability to secrete other cytokines, such as TNF and CXCL8. The hallmark of AA-DC was the production of the angiogenic cytokine VEGF in vitro, and in vivo when the cells were implanted into the chicken embryo CAM assay.

These carcinoma cells were used to pulse autologous DCs for vaccine production for ovarian carcinoma patients.

These autologous dendritic cells were used for vaccine production. They were pulsed with apoptotic autologous ovarian carcinoma cells.

Autologous DC were pulsed with apoptotic allogenic prostate carcinoma cell line LNCap to produce the advanced prostate carcinoma vaccine.

The vaccine is composed by autologous DCs pulsed with apoptotic allogenic prostate carcinoma cell line LNCap. It is used for vaccination of patients with advanced prostate carcinoma.

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.

These mice were immunized with OVA and used in a DC staining experiment. LN cells were purified 2 days later, and stained with either 2C44, 2F74, 2E60 or 2X8 mAb. None of these mAb stained DC purified from non immunized mice. However, among the 4 mAb, only 2C44 could stain DC in LACK-immunized mice, but not in OVA-immunized mice.

These mice were immunized with LACK and used in a DC staining experiment. LN cells were purified 2 days later, and stained with either 2C44, 2F74, 2E60 or 2X8 mAb. None of these mAb stained DC purified from non immunized mice. Among the 4 mAb, only 2C44 could stain DC in LACK-immunized mice, but not in OVA-immunized mice.

To address the issue whether infected DC process pathogen-derived antigen and stimulate antigen-specific CD4+ T cells in vivo, we have infected susceptible BALB/c (H2-d) mice with a recombinant Leishmania major parasite expressing a fluorescent tracer. We have directly visualized antigen-presenting cells using a mAb reacting to an antigenic peptide derived from the parasite LACK antigen bound to I-Ad MHC class II molecule. I-Ad/LACK complexes were readily detected at the surface of freshly purified parasite-containing DC which expressed a CD8?- CD11b+ F4/80+ g...

We have worked to strengthen vaccine developments with an improved humanized mouse model. We focus on recombinant vaccines against HIV driven by a powerful poxvirus vaccine system termed MVA BN. To improve our understanding MVA BN has been analyzed thoroughly on the nucleotide level, safety in cell cultures, immune deficient mice as well as in healthy, HIV–infected and other immune deficient humans.