title: Development and pharmacological validation of novel methods of B cell activation in rat whole blood.
abstract: BACKGROUND
  Whole blood functional assays are pharmacologically relevant in the drug discovery process to evaluate potency in a relevant biological matrix, to support establishment of PK/PD relationships and to aid in human dose predictions. However development of B cell activation assays by BCR ligation in rat whole blood has not been previously described. The aim of the present study was to develop novel methods of B cell activation in rat whole blood.
  
  
  METHODS
  B cell activation in rat whole blood was evaluated by measuring CD86 up-regulation via flow cytometry. Rat B cells in whole blood were stimulated with dextran-coupled anti-IgD or a combination of anti-IgD and TLR9 agonist. BTK, SYK, and PI3Kδ inhibitors were added to rat whole blood prior to activation with dextran-coupled anti-IgD or anti-IgD and TLR9 agonist combination for pharmacological validation of the assay.
  
  
  RESULTS
  Both methods of stimulation in rat whole blood evoked robust B cell activation in a uni-modal fashion. Highly selective inhibitors of BTK, SYK, and PI3Kδ dose-dependently attenuated B cell activity evoked by both dextran-coupled anti-IgD and combined anti-IgD and TLR9 agonist. Compound potencies and rank order determined by the two assays were comparable.
  
  
  CONCLUSIONS
  Two novel methods were developed to stimulate B cells in rat whole blood, that have the potential to be used to support drug discovery efforts in the therapeutic targeting of B cells. Furthermore, we pharmacologically validated these whole blood assays using highly selective inhibitors of BTK, SYK, and PI3Kδ, signaling kinases which are downstream of the B cell receptor.
authors.affiliation: Department of Immunology, Oncology & IMR, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA. Electronic address: john.shin@merck.com.
authors.forename: John
authors.initials: J
authors.lastname: Shin
authors.affiliation: Department of Genetics & Pharmacogenomics, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA.
authors.forename: Marianne
authors.initials: M
authors.lastname: Spatz
authors.affiliation: Department of Immunology, Oncology & IMR, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA.
authors.forename: Philip
authors.initials: P
authors.lastname: Brandish
authors.affiliation: Department of Genetics & Pharmacogenomics, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA.
authors.forename: Francois
authors.initials: F
authors.lastname: Gervais
authors.affiliation: Department of In Vivo Pharmacology, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang-Hoover
authors.affiliation: Department of In Vivo Pharmacology, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA.
authors.forename: Erica
authors.initials: E
authors.lastname: Leccese
authors.affiliation: Department of Immunology, Oncology & IMR, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Hicks
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D001769:Blood / Q000187:drug effects* / Q000276:immunology; D004305:Dose-Response Relationship, Drug; D005260:Female; D008213:Lymphocyte Activation / Q000187:drug effects*; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats; D011917:Rats, Inbred Lew; D013329:Structure-Activity Relationship
chemical_list: D047428:Protein Kinase Inhibitors
keywords: B cell; BTK; Dextran-coupled anti-IgD; Methods; PI3Kδ; Rat; SYK; Synergy; TLR9; Whole blood