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...

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.

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.

BruCells has been actively involved in the discussion of the EMEA guideline on Cell Based Medicinal Products (CBMP). Catherine De Greef was present at the DC-THERA Cluster 4 Meeting in Bamberg (July 2007) where among other topics these guidelines were discussed and where the basis for a written statement has been worked out. During the finalisation of the statement, BruCells has mainly put forward the notion that the guidelines are not suitable to CBMP for the authorization of investigational trials, but are applicable in the procedure of obtaining market approval.

A standard operating procedure for the identification of preclinical tests for cellular vaccines and release criteria for DC vaccines has been generated in our lab.

Draft templates for IMP have been generated.

Draft templates for CTP have been generated.

We possess an SOP for the assessment of Th subsets via flow cytometry.

Array construction: The yeast oligonucleotide array was constructed using the S. cerevisiae Genome Oligo Set ™ (Operon Technologies, CA, USA) composed of 6240 optimized oligonucleotides (70mers) each representing one yeast gene. Probe preparation and hybridization: We used the indirect labeling method. Briefly, the reactive amine derivative of dUTP, 5-(3- aminoallyl)-2?-deoxyuridine 5?-triphosphate (Sigma) was incorporated into cDNA using the Superscript II reverse transcriptase (Invitrogen) and oligo dT (Invitrogen) and random examers (Roche). After synthesi...

1 Saccharomyces cerevisiae cell stimuli Saccharomyces cerevisiae strain SK1 (MATa/alpha HO gal2 cupS can1R BIO, Kane SM and Roth J. 1974 Bacteriol. 118: 8-14) was cultured in complete medium (YPD, 2% yeast extract, 1% peptone, 2% glucose) for 18 hours, then collected, washed twice with sterile water and resuspended at 108 cells/ml. S. cerevisiae strains BY4741 (genotype, Mata his3delta1 leu2delta0 met15delta0 ura3delta0) and BY4741 och1 (Mata his3delta1 leu2delta0 met15delta0 ura3delta0 OCH1::kanMX4) were cultured in complete medium till exponentially phase and t...

Staining of D2SC1/Flt3-DC and staining of the cells to be taken up by Mini67-1KT or Mini26-1KT (PKH67/PKH26 green/red fluorescent cell linker mini kit for general cell membrane labelling) from Sigma Staining of GM-DC/Raw with anti-CD11c antibodies Coculture of DCs and cells to be taken up (1(-2)x105 + 1(-2)x105 cells in 12-well) for 4 h to 24 h at 37°C Take supernatant and freeze at -80°C for analysis of DC-stimulation Trypsinize cells have and fix in 4 % PFA (or formalin) FACS analysis

We have efficiently transfected DC with RNA encoding a functional protein (E/L-Selectin) which allows entry of DC into LN from HEV. RNA transfected human DC could be frozen and thawed without loosing their functionality.

Protocol for the documentation and the analysis of the interaction between RIG-I, a viral sensing protein, and NS1, an inhibitor of interferon production that is encoded by influenza A virus. This protocol has been made available.

For quantitative proteomic studies, our group recently described the SILAC (Stable Isotope Labelling by Amino Acids in Cell Culture) approach. In this method, independent populations of cells are grown in medium containing different non-radioactive isotope forms of essential amino acids (e.g. arginine and lysine). Because the cells cannot synthesize these amino acids, after some generations all proteins are labelled and therefore can be distinguished and quantified by the mass spectrometer.

The cells were collected and the pellet were resuspended in 3.6 ml AE Buffer (AE=50mM NaAcetate pH 5.2, 10mM EDTA) and then transferred in 15 ml tubes wit 200 µl 10% SDS (w/v). 2 ml of preheated acid phenol (pH 4.3) were added and the falcon were mixed by vortex. After 10’ of incubation in 65 °C water bath, the samples were incubated on ice and then centrifugated at 5000 rpm in an tabletop centrifuge. The supernatants were transferred to a new tube and added 2 ml of acid phenol to repeat the extraction. After the centrifugation (10’ at 5000 rpm) the supernata...

The company has now successfully developed a technique for the large-scale generation of DC in serum-free medium under GMP conditions, and are willing to share their know-how with Network Partners.

We have worked out SOPs for the cytometric assessment of Th-cell immunity based on antigen-induced CD154 expression, a technology developed by us in the frame of previous DC-THERA activities. Now the entire antigen-specific Th-cells specific for defined antigens can be monitored simultaneously with respect to quantity and quality. Tests can either be performed on standard 4 colour cytometers or on 12 colour high-end cytometric analysers. Tests that can be either performed to analyse fixed or isolate live antigen-specific Th-cells have been adapted also to be used ...

Argos successfully developed a manual DC manufacturing method with excellent reproducibility in terms of quality, yield, and viability of DCs. The process developed has the following unique advantages: - The manufacturing process has been optimized for producing DC from non-frozen day-old pheresis material which allows centralized manufacturing. The pheresis product is sent in a specially designed shipper to the manufacturing facility. - The final vaccine product is formulated for direct injection (i.e., no clinical site participation required except for bedside thaw and i.d. injection)

We are progressing steadily towards the delivery of a standardized method for the immuno-monitoring of DC vaccination trials using a finite number of defined tumor antigens. Our starting point was the method that we had developed previously to estimate blood frequencies of CD8 T lymphocytes against one defined antigenic peptide. It was based on an in vitro restimulation of blood lymphocytes with the peptide and growth factors over two weeks followed by detection with the appropriate HLA class I tetramer. In order to estimate frequencies of responding cells, these ...

Different strains of Saccharomyces cerevisiae were cultured and collected in different conditions: * different growth phase (exponential and stationary phase) * different growth media (standard and promoting pseudohyphal growth) * different cell form (spheroplast, spore and whole cell) Monocyte-derived DCs were added at a final concentration of 5x105 cells/ml into 96-well plates. Serial diluition of yeast cells and preparations were added to the MoDCs. Cytokine accumulation was evaluated in the supernatants at 24h by ELISA, according to a standard protocol and ...