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Biomarker and Cytokine Measurements in Dogs with Endotoxemia

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Title: Biomarker and Cytokine Measurements in Dogs with Endotoxemia
Author: Floras, Alexandra Nicole Karrys
Department: Department of Clinical Studies
Program: Veterinary Science
Advisor: Holowaychuk, Marie K
Abstract: Rapid identification of sepsis enables prompt intervention and improved patient survival. Procalcitonin (PCT) is a biomarker used to diagnose sepsis in people; assays for its measurement have not been validated in dogs. In dogs, serum N-terminal pro-C-natriuretic peptide (NT-proCNP) concentration, at admission, differentiates naturally occurring sepsis from non-septic inflammation; however, its concentrations during the course of sepsis are unknown. This study investigated the validity of a commercially available enzyme-linked immunosorbent assay (ELISA) for the measurement of canine PCT, and determined kinetics of serum NT-proCNP and select cytokine concentrations in dogs with endotoxemia, a model of canine sepsis. Serum samples from three dogs with sepsis and one healthy dog were examined. The PCT ELISA’s ability to detect recombinant and native canine PCT was investigated, coefficients of variability were calculated, and mass spectrometry of the standard solution was performed. Subsequently, 8 healthy adult Beagles were randomized to receive an intravenous bolus of lipopolysaccharide (LPS, 5 μg/kg) or saline placebo in a randomized crossover study. Serum was collected, and NT-proCNP, and 13 cytokines and chemokines were measured at 0, 1, 2, 4 and 24 hours. The PCT ELISA generated inconsistent results. Intra- and inter-assay variability was 18.9-77.4%, and 56.1-79.5%, respectively. Mass spectrometry of the PCT ELISA standard solution did not confirm presence of PCT. Serum NT-proCNP concentrations did not differ significantly between LPS- and placebo-treated dogs at any time. When comparing serum cytokine concentrations, LPS-treated dogs had higher interleukin (IL)-6 IL-10, tumor necrosis factor-α and KC-like at 1, 2, and 4 hours; higher C-C motif chemokine ligand-2 at 1, 2, 4 and 24 hours; and higher IL-8 and C-X-C motif chemokine ligand-10 at 4 hours compared to placebo-treated dogs (p < 0.05). There were no significant differences in serum granulocyte macrophage-colony stimulating factor, interferon-γ, IL-2, IL-7, IL-15 or IL-18 concentration between LPS- and placebo-treated dogs. These results do not support use of this commercial ELISA for detection of PCT in dogs. Serum NT-proCNP concentration did not change in response to LPS administration; however, certain serum cytokine and chemokine concentrations warrant further investigation of using cytokine profiles for the detection of sepsis in dogs.
Date: 2014-07
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