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note
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/ mice were also treated with a monoclonal antibody to CD40 (anti-CD40) (100 μg/day, Pharmingen, San Diego, CA) for 7 days after immunization, and C57BL/6 mice were treated with monoclonal antibodies to B7.1 and B7.2 (100 μg/day; Pharmingen, San Diego, CA) every 3 days after immunization for the duration of the experiment.
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51 Cr release was calculated as follows: [(cpm of sample - cpm of spontaneous release)/(cpm of maximal release - cpm of spontaneous release)] × 100. Spontaneous release was determined by culturing target cells in medium, and maximal release was established by culturing target cells in a 1% solution of SDS. The percentage of specific lysis is expressed as a function of different effector-to-target ratios (6:1, 12:1, 25:1, and 50:1).
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/ mice treated with anti-CD40 and C57BL/6 mice treated with anti-B7.1 and anti-B7.2 were harvested 10 days after infusion of H5.010CMVlacZ virus, and the frozen sections were analyzed for germinal center formation. Frozen sections were fixed with methanol for 10 min at -2C°C, air-dried, and rehydrated with phosphate-buffered saline (PBS). After blocking with 10% goat serum for 20 min. sections were incubated with peanut agglutinin conjugated to fluorescein isothiocyanate (FITC) (Sigma; 20 μg/ml) for 1 hour at room temperature. Sections were washed and mounted with Citiflour (Canterbury, UK). Four spleen sections from two mice (2) were stained for the presence of germinal centers (GCs) and analyzed. Data are represented as the number of GCs per section (mean ± SD).
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note
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Antiviral antibodies were evaluated with an ELISA assay in which microtiter plates were coated with 200 ng of viral antigen in 10 μl of PBS per well overnight at 4°C, washed three times in PBS, and blocked with 1% bovine serum albumin (BSA) in PBS for 1 hour at 37°C. Serum samples diluted 1:200 were added to antigen-coated plates and incubated for 4 hours at 37°C. Plates were washed three times with 0.05% Tween-20 in PBS and incubated with biotin-labeled goat antibody to mouse IgG1, IgG2a, or IgM (Pharmingen, San Diego, CA) at 1:1000 dilution overnight at 4°C. Plates were washed as above and anti-biotin phosphatase (Sigma) was added to each well at 1:30,000 dilution for 1 hour at 37°C. Wells were washed again as above and p-nitrophenyl phosphate substrate was added.
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We thank the Vector Core and Clinical Pathology and Animal Service Units of the Institute for Human Gene Therapy for technical help. Supported by the National Institute of Diabetes and Digestive and Kidney Diseases of NIH, the Cystic Fibrosis Foundation, and Genovo, Inc., a company that J.M.W. founded and holds equity in.
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