We have conducted a phase I long peptide vaccination trial with p53 peptides in Montanide adjuvant in patients with colorectal cancer or ovarium cancer.

The effect of environmental factors on development of high affinity CTL was investigated using a system whereby OT-1 cells were primed in vitro by engineered APC. After a 20 h priming phase, CTL were transferred to recipient mice that were either naïve, or had been injected with activated dendritic cells one day earlier, thus creating a reactive lymph node.

DMSO was used to dissolve blue formazan particles.

2M KOH were used to dissolve blue formazan particles.

DCs were exposed to cytochalasin D (10 microg/ml, TebuBio) for 30 minutes at 4°C.

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.

In a spore culture, zymolyase (2 mg/ml) was used to digest ascum and liberate spores. We initially performed experiments to evaluate zymoliase sensitivity of asci for this specific strain and we assessed that 15 minutes incubation was the minimum time needed to damage the ascus, without touching the spore cell wall. 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 discarding the supernatant; th...

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...

S. cerevisiae strain BY4741 och1 (Mata his3delta1 leu2delta0 met15delta0 ura3delta0 OCH1::kanMX4) was cultured in complete medium till exponentially phase. The cellswere biotinylated, labeled and - after treatment of DCs - detected using APC-labeled streptavidin and analyzed by flow cytometry.

S. cerevisiae strains BY4741 (genotype, Mata his3delta1 leu2delta0 met15delta0 ura3delta0) was cultured in complete medium till exponentially phase. The cells were biotinylated, labeled and - after treatment of DCs - detected using APC-labeled streptavidin and analyzed by flow cytometry.

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. The cells were biotinylated, labeled and - after treatment of DCs - detected using APC-labeled streptavidin and analyzed by flow cytometry.

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...

There is siRNA available in the lab.

There are lentiviral vectors available in the lab.

Tissue biopsies of the last vaccination site and of the VIN lesion prior to the first vaccination and 3 months after the last vaccination were analysed for the presence of HPV16-specific T cells. The IFN?-ELISPOT analysis and proliferation with its accompanying cytokine production revealed a strong and broad vaccine-induced T cell response. The strength of the immune response (defined as breath and magnitude of the response) was significantly higher in the patients with a complete remission (CR) than in the patients with no changes (NC) in the lesion size. Ph...

Tissue biopsies of the last vaccination site and of the VIN lesion prior to the first vaccination and 3 months after the last vaccination were analysed for the presence of HPV16-specific T cells. The IFN?-ELISPOT analysis and proliferation with its accompanying cytokine production revealed a strong and broad vaccine-induced T cell response. The strength of the immune response (defined as breath and magnitude of the response) was significantly higher in the patients with a complete remission (CR) than in the patients with no changes (NC) in the lesion size. Ph...

Tissue biopsies of the last vaccination site and of the VIN lesion prior to the first vaccination and 3 months after the last vaccination were analysed for the presence of HPV16-specific T cells. The IFN?-ELISPOT analysis and proliferation with its accompanying cytokine production revealed a strong and broad vaccine-induced T cell response. The strength of the immune response (defined as breath and magnitude of the response) was significantly higher in the patients with a complete remission (CR) than in the patients with no changes (NC) in the lesion size. ...

Tissue biopsies of the last vaccination site and of the VIN lesion prior to the first vaccination and 3 months after the last vaccination were analysed for the presence of HPV16-specific T cells. The IFN?-ELISPOT analysis and proliferation with its accompanying cytokine production revealed a strong and broad vaccine-induced T cell response. The strength of the immune response (defined as breath and magnitude of the response) was significantly higher in the patients with a complete remission (CR) than in the patients with no changes (NC) in the lesion size. Ph...

We evaluated the capacity of autologous DC and HEK293 cells transfected with relevant HLA alleles to function as T cell targets in Elispot assays upon transfection with tumor antigen encoding plasmids. Due to strong background from the autologous DC we decided that HEK293 transfected with one HLA-allele at a time plus simultaneously transfected with up to 5 tumor antiges would be optimal to screen for antigen specificity in the patients T cells. However, during a second T cell culture to expand large numbers of T cells for this screening procedure a drastic ex...

We evaluated the capacity of autologous DC and HEK293 cells transfected with relevant HLA alleles to function as T cell targets in Elispot assays upon transfection with tumor antigen encoding plasmids. Due to strong background from the autologous DC we decided that HEK293 transfected with one HLA-allele at a time plus simultaneously transfected with up to 5 tumor antiges would be optimal to screen for antigen specificity in the patients T cells. However, during a second T cell culture to expand large numbers of T cells for this screening procedure a drastic ex...