Protocols were developed to measure human immune responses to melanoma antigens to be measured using ELISpot and tetramers. The antigens in question were also inserted into adenovirus, lentivirus and vaccinia vectors for re-stimulation studies.

After 6 hour of stimulation, DCs were collected, washed 3 times with PBS, treated with zymolyase, washed twice and cells, lysated with a hypotonic solution (KCl 0.05%), were plated on YPD. Survival of yeast cells, spores or hyphae after uptake was reported as percentage of colony forming units after 3 days relative to the total number of cells growing in the absence of DCs exposure. To assess the importance of ROS production in DC killing ability, DPI (10 microM) was added 30 minutes before stimulation and survival of microorganisms was assessed using the same met...

Leukapheresis products from cancer patients was used as a starting material for monocyte enrichment by elutriation technology with ELUTRA® (Gambro). The monocytes were cultured for five days to immature DC (imDC´s) and subsequently transfected with different constructs encoding for the oncogene Her-2/neu and as control PSA using the technology of Amaxa biosystems or an adeno virus Her-2 full length construct. Levels of Her-2 expression was highest following transfection with 3 different truncated Transmembrane-extracellular constructs, in particular a Her-2 ra...

We have developed a technology to obtain from melanoma metastases melanin-free total tumor RNA, suitable for in vitro amplification and DC transfection.

The optimization for the mass spectrometry analysis methods to be used in the generation of a initial proteomic map of mouse DCs (WP2) and stimuli-related phosphorylation cascades (WP5) was carried out by Lyris M. F. de Godoy (Post-doc) and Jesper V. Olsen (PhD student). Recently, our lab has been developing new techniques in quantitative proteomics and has also acquired new instrumentation to assess these types of questions in depth. This, of course, involves a lot of optimization and, consequently, large amounts of sample. However, to obtain high numbers of dend...

To generate a proteomic map of DCs (WP2) which includes quantitative information that can be used further to study signalling cascades (WP5) it is also necessary to optimize the SILAC labelling of DCs. This work was performed by Christian A. Luber (PhD student). First, we have set up a system for SILAC labelling of bone marrow derived murine dendritic cells (BM-DC). The principal method for generating BM-DC with GM-CSF was adapted from Lutz et al. (J Immunol Methods 1999, 223: 77-92). After some modifications of the protocol the FACS analysis of DC, which were gen...

These procedures have led to an improvement in processing of small RNA samples. Studies of 3’ IVT microarrays expression profiling are obtainable starting from 2 ng of total RNA.

Pathway analysis performed with Eu.Gene (Cavalieri et al., 2005), following the algorithm developed by Beltrame et al. (2009).

This is a Pilot Study (Phase I/II) testing the immunologic activity and safety of AGS-004, an autologous HIV immunotherapeutic, in HIV-infected adults on HAART. Study Design: Patients that are successfully treated with highly active anti-retroviral therapy (HAART) with no measurable viral load AND have a cryopreserved infectious plasma sample that was drawn immediately prior to initiation of HAART will be administered 4 monthly doses of the Arcelis product.

A Phase II Study Testing the Activity and Safety in successfully ART-Treated Subjects Infected with HIV in Combination with ART Followed by ART Interruption. Study Design: The study design is identical to the Phase I study except that after the fourth dose patients will begin a drug treatment interruption period while receiving additional immunizations during that period. This provides an opportunity to assess the impact of the Arcelis therapy on viral load. Current status: Argos has received regulatory approval for this study and is actively accruing patients.

We are preparing a protocol for a phase I clinical trial in patients with advanced (stage III or IV) melanoma. Patients will receive a DC vaccination regimen followed by 3 adoptive transfers of autologous in vitro expanded T cells. This novel approach combines in vivo priming by the DC vaccine with passive immunotherapy by lymphocyte infusion. Patients will undergo surgical removal of a metastatic lesion and leukapheresis at the beginning of the trial to provide sources for tumor antigens as well as monocytes and T cells, which will be isolated from the leukaphe...

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

Untouched naive CD4+ T cells are co-coltured with fungi -pulsed DCs and then the polarization is assessed by flow cytofluorimetry and cytokine production.

Production of lentiviruses: Producing lentivirus in 293 FT Cells: Day 1: 4 x 106 293 FT in 10 ml DMEM (+10 % FCS (Tet free), +P/S) in a 10cm plate Day 2: transfection Day 3: change of medium Day 4: harvesting

A protocol for obtaining highly pure integral membrane proteins has been set up in the last two months in the lab of Paola Castagnoli in collaboration with the lab of Juri Rappsilber at IFOM.

This methodology incorporates a unique sample preparation procedure based on 96 well plate complex sample trypsinization, a nano-LC-based peptide separation coupled to MSMS analysis sequencing. This procedure led us to the identification of 600 phagosomal proteins from murine DC phagosomes.

We established protocols for the efficient in vitro transfection of primary dendritic cells based on viral and non-viral (nucleofection) methods (A. Mantei). These have been applied to modify DC surface molecules (Notch ligands) in the murine system (S. Vaddakadathou) or deliver antigens in both murine and human DC into either the MHC I or MHC II presentation pathways (A. Sattler, M. Dziubainau). For the latter we used a MHC II presentation pathway targeting vector originally published by Wang et al. 1999. Proof of principle studies for antigen delivery have been ...

Protocols were developed to refold in vitro CD1 molecules, which were used for the generation of CD1 tetramers. The ability to generate CD1d tetramers has provided us with the opportunity of comparing a broad panel of CD1d binding compounds for their ability to stimulate iNKT cells.

A retrotranscriptional reaction is performed on RNA extracted from T cells or DCs. Transcripts for interested genes are quantified by real-time quantitative PCR on an Perkin-Elmer Applied Biosystems with predesigned TaqMan Gene Expression Assays according to the manufacturer’s instructions.