title: Methods: implementation of in vitro and ex vivo phagocytosis and respiratory burst function assessments in safety testing.
abstract: Functional innate immune assessments, including phagocytosis and respiratory burst, are at the forefront of immunotoxicology evaluation in pre-clinical animal species. Although in the clinic and in academic science, phagocytosis, and respiratory burst assessments have been reported for over two decades, the implementation of phagocytosis and respiratory burst analyses in toxicology safety programs is just recently gaining publicity. Discussed herein are general methods, both microtiter plate-based and flow cytometric-based, for assessing phagocytosis and respiratory burst in pre-clinical species including mouse, rat, dog, and monkey. This methods-centric discussion includes a review of technologies and descriptions of method applications, with examples of results from analyses testing reported inhibitors (rottlerin, wortmannin, and SB203580) of phagocytosis and respiratory burst. Justification of implementation, strategic experimental design planning, and feasibility aspects of evaluating test article effects on phagocytosis and respiratory burst function are described within the context of a case study. The case study involves investigation of the effects of a small molecule p38 kinase inhibitor, BMS-582949, on phagocytosis and respiratory burst functions in rat and monkey neutrophils and monocytes in vitro, as well as ex vivo in these innate immune cells from monkeys administered BMS-582949 during a 1-week repeat dose investigative study. The results of the in vitro and ex vivo assessments demonstrated that BMS-582949 inhibited phagocytosis and respiratory burst. These findings correlated with incidences of opportunistic infections observed in rat and monkey toxicity studies.
authors.affiliation: Department of Immunotoxicology, Drug Safety Evaluation, Bristol-Myers Squibb, New Brunswick, NJ, USA. wendy.freebern@bms.com
authors.forename: Wendy J
authors.initials: WJ
authors.lastname: Freebern
authors.forename: Tammy J
authors.initials: TJ
authors.lastname: Bigwarfe
authors.forename: Karen D
authors.initials: KD
authors.lastname: Price
authors.forename: Helen G
authors.initials: HG
authors.lastname: Haggerty
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001578:Benzopyrans / Q000494:pharmacology; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004285:Dogs; D004353:Drug Evaluation, Preclinical; D000882:Haplorhini; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007113:Immunity, Innate / Q000187:drug effects; D007159:Immunologic Tests; D051379:Mice; D009000:Monocytes / Q000187:drug effects; D009504:Neutrophils / Q000187:drug effects; D010587:Phagocytosis / Q000187:drug effects*; D011725:Pyridines / Q000494:pharmacology; D011758:Pyrroles / Q000008:administration & dosage / Q000009:adverse effects*; D051381:Rats; D016897:Respiratory Burst / Q000187:drug effects*; D014227:Triazines / Q000008:administration & dosage / Q000009:adverse effects*; D000077191:Wortmannin
chemical_list: C552704:4-(5-(cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-N-propylpyrrolo(1,2-f)(1,2,4)triazine-6-carboxamide; D000098:Acetophenones; D000730:Androstadienes; D001578:Benzopyrans; D007093:Imidazoles; D011725:Pyridines; D011758:Pyrroles; D014227:Triazines; C085746:rottlerin; C093642:SB 203580; D000077191:Wortmannin