Cynomolgus monkeys were treated weekly with 0, 2, or 20 mg/kg BR3-Fc for 18 weeks (127 days) and allowed to recover until week 41 (day 287). West Grove, PA), at a density of 2 105 cells per well. Soluble recombinant mouse BAFF was added at a concentration of 5 g/ml for 5 days. Tritiated-thymidine (1 Ci) (Perkin Elmer, Boston, MA) was added during the last 6 hours of culture, and cells were harvested onto UNIFILTER plates (Perkin Elmer) and counted. For survival assays, purified Dihydroeponemycin B cells were Dihydroeponemycin cultured with 1 g/ml soluble recombinant CD40 Ligand (R & D Systems, Minneapolis, MN) and interleukin (IL)-2 (2 ng/ml) (R & D Systems) for 4 days at a density of 2 105 cells per well in six-well plates. Cells were then washed and cultured with soluble recombinant mouse FLAG-tagged BAFF (produced at Genentech using the murine BAFF extracellular domain name cloned into a pCMV-FLAG vector; Sigma, St. Louis, MO), IL-2 (2 ng/ml) (R & D Systems), and indicated blocking reagents (50 g/ml) in six-well plates at a density of 1 1 105 cells per well. Cell viability was assayed at the indicated time points using a Coulter Viacell (Beckman Coulter, Fullerton, CA). Animals This study was conducted at Shin Nippon Biomedical Laboratories USA, Ltd. (SNBL USA, Everett, WA), according to their standard operating procedures and in compliance with applicable regulations concerning the use of laboratory animals. Three- to five-year-old male and female na?ve cynomolgus monkeys (weight range, 2.26 to 3.26 kg for the females and 2.64 to 4.50 kg for the males) were used in the study. All animals were acclimated to the study room for 28 days before the initiation of dosing. Cynomolgus Monkey Study Design Nineteen male and 19 Dihydroeponemycin female cynomolgus monkeys were each given a slow intravenous bolus injection of BR3-Fc at either 2 or 20 mg/kg or the BR3-Fc vehicle control once weekly for 13 (interim necropsy) or 18 weeks (see Table 1 for a detailed summary of the study design). The interim necropsy was performed on four animals (two males and two females) from the control and 20-mg/kg group at week 13; the main necropsy was performed on six animals (three males and three females) from each treatment group at week 18; whereas the recovery necropsy was performed on six animals (three males and three females) from only the control and 20-mg/kg group at week 41. All animals in the 2-mg/kg group were necropsied at week 18. Table 1 Study Design 0.05). IHC For dual-label IHC on paraffin-embedded sections, 4-m sections of spleen and lymph node were deparaffinized and then treated with Target Retrieval answer (Dakocytomation, Carpinteria, CA) heated to 99C in Dihydroeponemycin a boiling water bath. Primary antibodies used in this study were mouse anti-human CD3 (clone SP34-2, used at 5 g/ml; BD/Pharmingen, San Diego, CA), mouse anti-human CD20 (clone L26, used at 1 g/ml; Dakocytomation), and mouse anti-human easy muscle -actin (clone 1A4, used at 0.1 g/ml; Dakocytomation). Isotype control antibodies were mouse IgG1 and mouse IgG2a (BD/Pharmingen). Sections were stained with the first primary antibody, then incubated with biotinylated horse anti-mouse IgG (Vector, Burlingame, CA), and finally incubated with avidin-biotin peroxidase complex (ABC-HRP Elite; Vector). The first primary antibody was detected with metal-enhanced diaminobenzidine (Pierce Chemical, St. Louis, MO). Slides were then subjected to a second round of antigen retrieval, which served to denature and remove the first primary antibody complex. Slides were re-blocked for endogenous biotin and nonspecific protein interactions before incubation with mouse anti-human Dihydroeponemycin CD20. Slides were then incubated with biotinylated horse anti-mouse IgG followed Mouse monoclonal antibody to DsbA. Disulphide oxidoreductase (DsbA) is the major oxidase responsible for generation of disulfidebonds in proteins of E. coli envelope. It is a member of the thioredoxin superfamily. DsbAintroduces disulfide bonds directly into substrate proteins by donating the disulfide bond in itsactive site Cys30-Pro31-His32-Cys33 to a pair of cysteines in substrate proteins. DsbA isreoxidized by dsbB. It is required for pilus biogenesis by streptavidin alkaline phosphatase (Vector). Chromogenic detection of CD20 was performed using Alkaline Phosphatase Substrate kit III (Vector), producing a blue reaction product. Slides were washed, dehydrated in an alcohol series into a limonene-based clearing agent (Grasp Clear), and coverslipped using VectaMount (Vector). For dual-label immunofluorescence, frozen sections of cynomolgus spleen were cut at 5 m. Frozen sections were blocked with 10% normal donkey serum and then incubated with either rabbit anti-human IgD (Dakocytomation) used at 10 g/ml or a mixture of rabbit anti-IgD and mouse anti-smooth muscle actin (clone 1A4; Dako) used at 5 g/ml for 1.
Posted in PDGFR.