25 Adenovirus was diluted in phosphate-buffered saline to a final volume of Bicalutamide clinical trial 10 ml and slowly infused to the anesthetized animal via a peripheral vein at 1.4 �� 1012 viral particles per dose. Immunomodulation therapy. Immunosuppressants were administered as described in Figures 1a and 2a2a. Two-drug regimens consisted of (i) 20 mg Rituximab/kg/dose intravenously (i.v.) (Mabthera, ROCHE, Basel, Switzerland) at days ?9, ?6, ?3 and immediately before adenovirus injections; and (ii) Tacrolimus (FK506; Astellas Pharma, Madrid, Spain) at a dose of 0.25 mg/kg administered orally from day ?2 and daily to the end of the study (Figure 1a). The five-drug regime included (i) Rituximab (20 mg/kg/dose i.v.
) at days ?9, ?6, ?3, immediately before adenovirus injections and weekly after the viral administration, (ii) two doses of 3 mg/kg of rabbit ATG (Genzyme Polyclonals, Marcy l’Etoile, France) at days ?2 and ?1 before the adenovirus injection. (iii) Methyl-prednisolone (Solu-moderin, Pfizer SA, Spain) was applied intramuscularly 10 minutes before the ATG infusion at a dose of 100 mg on day ?2 and 50 mg on day ?1. (iv) MMF (CellCept; Roche Pharma, Madrid, Spain) at a dose of 25�C30 mg/kg/day, and (v) FK506 0.25 mg/kg/day. MMF and FK506 were orally given from day ?2 daily during the indicated periods (Figure 2a). PET analysis. Transgene expression in the liver parenchyma was visualized and quantified by PET13 1 week before and 48 hours after the AdCMVHS1-tk administration (��PET analyses of HSV1-tk expression�� in Supplementary Materials and Methods). Neutralizing antibody assays49 to AdCMVHSV1-tk.
Serial dilutions of macaque serum starting from 1/25 were mixed with 1 �� 105 plaque-forming units of Ad5CMV-luc encoding firefly luciferase (a similar recombinant adenovirus also based on serotype 5 backbone) and incubated at 37 ��C for 1 hour then the mixture was added to PCL-PRF5 cells (1 �� 104 cells/well) in a 96-well plate. Forty-eight hours later, cells were washed in phosphate-buffered saline and d-luciferin substrate (Xenogen, Alameda, CA) was added at a final concentration of 150 ��g/ml and placed in a light-tight chamber. The intensity of light emission from individualized wells was detected using the IVIS cooled charge-coupled device camera (Xenogen) and Living Image 2.20 software package (Xenogen). Sera were scored as positive if the light intensity was 50% when compared to negative control sera.
A curve was adjusted to extrapolate the serum dilution for 50% inhibition (IC50). Fluorescence-activated cell-sorting analysis of the lymphocyte populations and cytokine serum concentrations. The percentage of B, CD4, and CD8 lymphocytes in peripheral blood was determined by flow cytometry as detailed in ��FACS analysis�� in Supplementary Cilengitide Materials and Methods. Serum concentrations of cytokines were measured with a BD Cytometric Bead Array (Inflammatory Cytokine Kit, Ref.: 551811) analyzed in a FACSCalibur (Brussels, Belgium).