2017 ACVIM Forum Research Abstract Program

The objective of our current project was to evaluate the concentration-dependent effects of MPA on canine lymphocyte function utilizing a CD4 cell activation assay, and B and T cell lymphocyte proliferation assays. Peripheral blood mononuclear cells (PBMCs) were isolated from six healthy hound dogs using Histopaque Ã’ density gradient centrifugation. Once isolated, the PBMCs were plated, incubated in escalating concentrations of MPA (1.25, 2.5, 5, 10, 20, 40, 80, 160 , and 320 ng/mL), vehicle (DMSO) control, and media only control, and then stimulated with either pokeweed mitogen (PWM) or concanavalin A (ConA). The CD4 cell activation assay was modeled on a commercially available human assay of activated CD4 positive T cell ATP synthesis that is marketed as a non-specific test of the efficacy of immunosuppressive agents. Briefly, CD4 positive cells were magnetically sorted from PBMCs following activation with ConA, cells were lysed to release intracellular ATP, a luminescence reagent (luciferin/luciferase) activated by ATP was added, and resultant bioluminescence was measured using a luminometer. B and T cell proliferation were evaluated by measuring incorporation of tritiated thymidine via a scintillation beta-counter following activation with PWM and ConA, respectively.