A descriptive pilot study of cytokine production following stimulation of ex-vivo whole blood with commercial therapeutic feline hydrolyzed diets in individual healthy immunotolerant cats

Abstract

Background: Hydrolyzed diets are used in companion animals for the diagnosis and treatment of adverse food reaction. Similarly, hydrolyzed formulas are used in human infants with severe inflammatory bowel disease or milk allergy, and these must meet the standard of hypoallergenicity through rigorous testing. Unfortunately, no standards are currently applied to hydrolyzed veterinary therapeutic diets, and data for the immunogenicity of feline diets is also not available. Therefore, the main aim of this pilot study was to determine if ex-vivo whole blood stimulation assays could be used to characterize the cytokine response to hydrolyzed commercial diets in a small number of individual healthy immunotolerant cats. This approach has also been used to investigate cytokine production in response to cow milk protein in humans and currently similar studies do not exist in companion animals. Nine healthy cats previously eating the same basal diet were divided into groups and fed one of three hydrolyzed diets exclusively for 6 weeks. Heparinized whole blood was collected from each cat before and after the feeding trial. Ex-vivo whole blood stimulation assays were performed using crude extracts of the basal diet as a positive control, as this diet contained the same proteins present in the hydrolyzed diet but were intact, saline as a negative control, and each cat's respective hydrolyzed diet. Supernatants were collected and analyzed for tumor necrosis factor-alpha, interleukin-10 (IL-10), and interleukin-4 using enzyme-linked immunosorbant assay. Results: Seven cats produced detectable amounts of the anti-inflammatory cytokine IL-10 upon stimulation with the basal diet. Two cats produced detectable amounts of IL-10 upon stimulation with a hydrolyzed soy-based diet and one cat produced a detectable amount of IL-10 upon stimulation with a hydrolyzed chicken-based diet (>125 pg/mL). Conclusions: Results from this pilot study suggest that in some healthy immunotolerant cats, some hydrolyzed diets may elicit a similar cytokine response compared to their basal diet, which contained the same proteins intact. Therefore, animals may be able to recognize and react to some hydrolyzed forms of tolerated proteins, and may also suggest IL-10 as a target for investigation as a potential marker for dietary tolerance in cats, however further studies would be necessary to corroborate this. Further studies are also needed to determine if this would also be the same in immunologically naïve, sensitized and clinically hypersensitized cats.

A preliminary study assessing cytokine production following ex vivo stimulation of whole blood with diet in dogs with chronic enteropathy

“…Similar to our previous study, a clean pestle and mortar was used to grind ten grams of the relevant food with sterile water and the mixture transferred to a 50 mL sterile tube1 [7]. The sterile tubes containing the mixture were incubated for 2 to 12 h at 4-degrees on a rotator and then centrifuged at 4000 rpm for 12 min at room temperature.…”

“…The bicinchoninic assay4 was used to determine the protein content of the filtrate and a working solution of 500 μg/mL was prepared using sterile phosphate buffered saline5 (PBS). This concentration was chosen based on the results of our previous study [7].…”

“…All samples were analyzed in triplicates for a total of 6 wells for dogs eating one diet type and a total of 9 wells for dogs eating two diet types. Similar to our previous study, all plates were incubated at 37 degrees centigrade in 5% carbon dioxide for 24 h,9 after which time they were then spun at 2000 × g for 4 min and the supernatant collected and stored at − 80 ° C until analyzed [7].…”

“…The author has previously published a study in healthy immunotolerant colony cats using sterile preparations of commercial dry diets to stimulate whole blood ex vivo and measure cytokine production in the supernatant following incubation for 24 h [7]. These stimulation assays used whole blood instead of peripheral blood mononuclear cells (PBMCs) in order to replicate the natural environment of the cells, to prevent changes in phenotype that may occur with isolation of the cells and to minimize the volume of blood needed, as well as the processing time.…”

“…A further study suggested a role for serum IL-10 in the diagnosis of food tolerance in humans [11]. Similarly, the results from the author’s study suggested that increases in IL-10 production in ex vivo whole blood stimulation assays may be used as a marker of tolerance to diet in companion animals [7]. Studies have shown an increase in IL-4 and a decrease in IL-10 concentrations in food-induced GI disorders in humans and mice [12–14].…”