Detection of anti-TNFalpha activity in canine hyperimmune serum using a TNFalpha inhibition assay.
BACKGROUND
Increased serum tumor necrosis factor-alpha (TNFalpha) activity has been associated with onset of serious inflammatory diseases in dogs. Development of treatment with TNFalpha-antagonists has been limited by the unavailability of suitable reagents and potency assays for TNFalpha.


OBJECTIVE
The objectives of this study were to optimize a cell-based assay to measure anti-TNFalpha activity in serum and plasma from hyperimmune (vaccinated with an Escherichia coli J5 bacterin) and unvaccinated canine donors; to use the assay to determine whether hyperimmune serum inhibits TNFalpha activity in vivo; and to determine whether soluble TNF receptor-1 (sTNFR1, a naturally occurring TNFalpha antagonist) contributes to anti-TNFalpha activity.


METHODS
Commercial plasma and serum from hyperimmune-frozen plasma (HFP) donors and unvaccinated fresh-frozen plasma (FFP) donors were used in the study. An L929-cell TNFalpha-inhibition assay (LTIA) was optimized to measure anti-TNFalpha activity. Using a rat subcutaneous pouch model of inflammation, the effects of HFP, FFP, a synthetic TNFalpha antagonist (Etanercept), and carprofen on TNFalpha activity were compared in vivo. Immunofluorescence was used to measure soluble sTNFR1 concentration.


RESULTS
Using the optimized LTIA, HFP serum but not FFP serum decreased canine TNFalpha activity (P<.01). HFP plasma and Etanercept (but not FFP plasma or carprofen) significantly decreased TNFalpha activity in pouch exudates (P<.05). A significantly higher concentration of sTNFR1 was found in HFP than FFP serum.


CONCLUSIONS
Using the LTIA, anti-TNFalpha activity is readily measured in canine serum and inflammatory exudates. sTNFR1 appears to contribute to anti-TNFalpha activity in HFP serum. These results suggest HFP should be investigated further as a potential immunotherapeutic agent for controlling canine diseases in which TNFalpha is implicated.
Kotiw
Michael
M
Department of Biological and Physical Sciences, Centre for Systems Biology, University of Southern Queensland, Toowoomba, Qld, Australia. kotiw@usq.edu.au
Morgan
Michael
M
Taylor
Stephen M
SM
Shiels
Ian A
IA
D000818:Animals; D001428:Bacterial Vaccines / Q000276:immunology; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D004285:Dogs; D004926:Escherichia coli / Q000276:immunology; D000068800:Etanercept; D015200:Fluoroimmunoassay / Q000662:veterinary; D007106:Immune Sera / Q000032:analysis*; D007074:Immunoglobulin G / Q000494:pharmacology; D008297:Male; D051379:Mice; D051381:Rats; D017207:Rats, Sprague-Dawley; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis* / Q000037:antagonists & inhibitors*
D001428:Bacterial Vaccines; D007106:Immune Sera; D007074:Immunoglobulin G; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D000068800:Etanercept