Septic shock is characterized by cardiovascular and vasomotor failure that is induced by an uncontrolled cascade of inflammatory mediators such as TNFα, IL-1β and IL-6. In dogs, systemic bacterial infections, haemorrhage, trauma, gastric dilatation/volvulus and pancreatitis are the major causes of septic shock. While endotoxin is a recognized effector molecule that can initiate an inflammatory cascade, it has been reported that preconditioning with endotoxin can downregulate inflammatory cytokine responses to subsequent endotoxin challenge. This study reports the effect of endotoxin preconditioning on anti-TNFα activity present in plasma from canine donors.
Materials and methods
Plasma from preconditioned (Caniplas®) and normal dogs (FFP) was provided blind to the study by a commercial supplier (Plasvacc Pty Ltd). In vitro anti-TNFα activity in canine donor plasma was determined by a L929 murine cell TNFα inhibition bioassay using recombinant murine TNFα. In vivo effects were tested by a rat subcutaneous skin pouch model. Rats were pretreated for 3 days with either Caniplas®, FFP, saline (2 ml/day, s.c.) or carprofen (5 mg/kg, s.c.) and inflammation was induced by injecting monosodium urate crystals into the pouch (5 mg/ml in 5 ml saline). Fluid was taken from pouches at specified intervals for cell count, TNFα and IL-6 analysis. Data analysis: normalized data were fitted to a four-parameter logistic curve. The fitted midpoints were compared statistically for datasets using an F-test and calculated fitted hill slopes.
In the rat skin pouch model, both Caniplas® and FFP reduced TNFα levels and Caniplas® was a more potent antagonist. The heightened anti-TNFα activity of Caniplas® compared with FFP was confirmed in the in vitro cell bioassay (Figure 1). Neither Caniplas® nor FFP reduced inflammatory cell infiltration or the levels of IL-6.
Figure 1. In vitro TNFα dose-response to canine sera.
While we need to confirm the mechanism, we report that preconditioning with endotoxin does illicit specific anti-TNFα activity and that this observation has been confirmed in both in vitro testing and in vivo animal models. It is plausible that preconditioning animals with endotoxin induces an increase in the concentration of soluble TNFα receptors I and II in donor plasma, and that this is the probable source of TNFα antagonism. This report suggests that preconditioned plasma may be a beneficial treatment where inflammation causes increased expression of TNFα.