Objective—To use in vitro assays to evaluate the effects of a novel immunosuppressive agent, FTY720, on biological functions (migration, phagocytosis, and production of reactive-oxygen species [ROS]) of feline peripheral neutrophils and determine the cytotoxic effects of FTY720 on feline peripheral neutrophils.
Sample Population—Peripheral neutrophils obtained from 8 healthy cats.
Procedure—Peripheral neutrophils were isolated from blood samples obtained from the 8 cats and exposed to the phosphorylated form of FTY720 (FTY720-P). A fluorescence-based in vitro evaluation of migration was performed. Phagocytosis of microbes and production of ROS were evaluated by use of a 2-color flow cytometry system. Samples of whole blood obtained from the cats were incubated with various concentrations of FTY720-P, fluorescein-labeled Staphylococcus aureus, and dihydroethidium. Cytotoxic effects were evaluated by use of propidium iodide staining.
Results—Addition of FTY720-P caused a slight non-significant decrease in phagocytosis and production of ROS by feline peripheral neutrophils. Migration activity of feline peripheral neutrophils was significantly increased by the addition of FTY720-P. Addition of FTY720-P at concentrations considered for clinical use did not increase the death rate of feline peripheral neutrophils.
Conclusions and Clinical Relevance—FTY720 does not inhibit critical functions of feline peripheral neutrophils in vitro.
Objective—To determine whether the active metabolite of leflunomide, A77 1726 (A77), inhibits replication of feline herpesvirus-1 (FHV-1) in cell culture.
Study Population—Crandell Rees feline kidney (CRFK) cell cultures.
Procedures—Cell cultures were inoculated with FHV-1 and treated simultaneously with concentrations of A77 ranging from 0 to 200μM. The antiviral effect of A77 was determined by use of conventional plaque reduction assays. The effect of A77 on viral load was determined via real-time PCR analysis, and transmission electron microscopy was used to evaluate the effect of A77 on viral morphology. To determine whether the antiviral effect was attributable to alterations in CRFK cell viability and number, CRFK cells were treated with various concentrations of A77 and stained with Annexin V and propidium iodide to assess apoptosis and a mitochondrial function assay was used to determine cell viability.
Results—Concentrations of A77 ≥ 20μM were associated with substantial reduction in plaque number and viral load. Concentrations ≥ 100μM were associated with complete suppression of plaque formation. At low concentrations of A77, clusters of intracytoplasmic virus particles that appeared to lack tegument and an external membrane were detected. Treatment of uninfected CRFK cell monolayers with A77 was associated with reduction in mitochondrial function with minimal evidence of apoptosis.
Conclusions and Clinical Relevance—Leflunomide may be an alternative to current calcineurin-based immunosuppressive protocols used in feline organ transplantation because of its antiherpesviral activity.