Canine Whole Blood Research Articles

Comparison of TNFα responses induced by Toll-like receptor ligands and probiotic Enterococcus faecium in whole blood and peripheral blood mononuclear cells of healthy dogs

The assessment of in vitro responses of blood-derived cells has traditionally been performed with peripheral blood mononuclear cells (PBMCs). However, stimulation of whole blood (WB) has advantages: ease of experimental setup, avoidance of blood cell manipulation and lower assay cost and time. WB stimulation is widely used in human research, but only infrequently in small animals. The aim of this study was to compare the response generated in canine WB and PBMCs with Toll-like receptor ligands and probiotic bacteria using TNFα as measured endpoint. WB and PBMCs were derived from a total of 15 healthy dogs. Stimulations were performed with LPS (1ngml−1), Pam3CSK4 (100ngml−1), flagellin (1μgml−1) and Enterococcus faecium (EF; 1×107cfuml−1). In 4 of the dogs, PBMC numbers were matched to the numbers of PBMCs found in WB. TNFα was detected in supernatants via ELISA. TNFα production from WB was generally higher than from PBMCs (repeated measures ANOVA p<0.0128). PBMCs produced TNFα inconsistently for all stimulants apart from EF. There was no correlation between results of WB or PBMC stimulation, similar to studies that found that humanWB cytokine production correlates with stimulating monocytes, but not PBMCs. In conclusion, WB stimulation should be considered a valid alternative to PBMC stimulation in the canine system.

Comparative structure–activity relationship studies of 1-(5-methylsulfonylpyrid-2-yl)-5-alkyl and (hetero)aryl triazoles and pyrazoles in canine COX inhibition

Structure-activity relationship (SAR) studies of novel 5-alkyl and 5-aryl/heteroaryl substituted 1,2,4-triazoles are described. The in vitro activity is compared to the pyrazole class of compounds with analogous side chains to delineate the contribution of the triazole ring nitrogen in binding to the active site. Both series are quite potent and selective in the canine whole blood (CWB) COX-2 assay, suggesting the increased binding contribution of the hydrophobic side chains.

5-Heteroatom-substituted pyrazoles as canine COX-2 inhibitors: Part 2. Structure–activity relationship studies of 5-alkylethers and 5-thioethers

Structure–activity relationship (SAR) studies of novel 2-[3-trifluoromethyl-5-alkyl(thio)ether pyrazo-1-yl]-5-methanesulfonyl pyridine derivatives for canine COX enzymes are described. The 4-cyano-5-alkyl ethers were found to have excellent potency and selectivity, whereas the 5-thioethers were potent but less selective than the ether analogs in a canine whole blood (CWB) COX-2 assay.

Use of a time-resolved immunofluorometric assay for determination of canine C-reactive protein concentrations in whole blood

To develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for measurement of C-reactive protein (CRP) in canine whole blood. 12 healthy dogs and 35 dogs with inflammatory processes. CRP was isolated from acute-phase serum by affinity chromatography and used as a standard for calibration. Analytic and functional limit of detection and intra-assay and interassay precision were calculated. Accuracy was evaluated by recovery assays and by comparison with results of a commercial ELISA. Correlation between CRP concentrations in whole blood and corresponding plasma fractions was tested by use of TR-IFMA. Stability of blood samples at 4 degrees C was assessed during a 1-month period, and effects of anticoagulants were evaluated. Measurements of CRP in blood samples from 12 healthy dogs were compared with those of 35 dogs with inflammatory diseases. Analytic and functional limits of detection were 0.53 and 3.26 microg/mL, respectively. Intra-assay and interassay coefficients of variation varied between 2.1% to 8.9% and 8.0% to 12.3%, respectively. Mean recoveries of added CRP were 104% and 114%. Measurements of CRP by use of TR-IFMA and ELISA were highly correlated (R2 = 0.97). Measurements of CRP in whole blood and in corresponding plasma fractions by use of TR-IFMA were also highly correlated (R2 = 0.97). Neither storage nor use of anticoagulants disturbed measurement of CRP concentrations in whole blood. Concentrations of CRP in whole blood of dogs with inflammation were significantly higher than in healthy dogs. Determination of CRP concentrations in whole blood may provide a diagnostic test for inflammation in dogs.