The effect of canine hyperimmune plasma on inflammation in a rat pouch model

The effect of canine hyperimmune plasma on inflammation in a rat pouch model

Inflammation is a series of complex interactions among soluble mediators and cells that can occur in any tissue in response to insults, such as trauma and infection. The process normally progresses to recovery, however, if the biological insult and repair are not properly phased inflammation can lead to chronic tissue damage. Inflammation is significantly orchestrated by pro-inflammatory cytokines such as tumor necrosis factor-α (TNFα), interleukin 1 beta (IL-1ß) and interleukin 6 (IL-6) which serve as a means of systemic communication among the cellular components. Despite the application of significant resources devoted to exploring the therapeutic potential of pro-inflammatory cytokine antagonists, outcomes have generally been of limited value, particularly in control of acute systemic inflammatory events. Whilst the use of TNFα antagonists has been approved for the management of rheumatoid arthritis when conventional therapies have failed, their experimental use in the control of syndromes such as sepsis has been less rewarding. The main aims of this study were: to characterise the active TNFα antagonists present in canine hyperimmune frozen plasma (HFP), investigate the efficacy of HFP in an in vivo model of inflammation; to develop a mathematical model which described the inter-relationship between varying concentrations of TNFα and its primary antagonist, soluble TNFα receptor 1 (sTNFR1). Finally, using comparative genomic analysis to study and identify the role of genetic variance present in inflammation related genes.

In part 1 of the study, the nature of a TNFα antagonist present in HFP was characterised by resolving plasma proteins from canine HFP and fresh frozen plasma (FFP) donors by Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (PAGE) and by an in house developed immunofluorescent dot blot assay. Western Blot analysis was used to characterise the target protein. The analysis revealed that HFP contained significantly greater levels of sTNFR1 than were present in FFP. These findings suggested that HFP has an increased capacity (compared to FFP) to moderate the effects of TNFα. In summary, it was postulated that administration of HFP may down grade the pro-inflammatory effects of TNFα and may have a therapeutic role in the control of systemic inflammatory diseases mediated by this cytokine.

The identification of elevated levels of sTNFR1 in HFP prompted the undertaking of a second investigation, which explored the efficacy of HFP to down regulate levels of TNFα in an animal model of inflammation. TNFα is a powerful pro-inflammatory cytokine with a broad spectrum of activity and plays a central role as an acute endogenous mediator of sepsis and in particular endotoxic shock. The over or inappropriate production of TNFα is also recognised to play a role in the development of a variety of chronic inflammatory diseases. In this part of the study, results from the characterisation experiments were tested in an in vivo model of inflammation. The effects of subcutaneous administration of HFP, native fresh frozen plasma (FFP), Carprofen and normal saline were studied in a rat air pouch model of inflammation in which either Escherichia coli derived lipopolysaccharide (LPS) or monosodium urate crystals (MSU) were used as inflammatory agonists. The effect of each treatment was monitored at 1, 6, 12, 24 and 48 hours by irrigating the pouch cavity with saline and analysing the fluid for: TNFα and sTNFR1 levels and leukocyte concentration. Key outcomes were that at 6 hours post LPS and or MSU antagonism, rats treated with HFP showed significantly reduced levels of TNFα concentration compared to FFP, Carprofen and saline treatments (p < 0.05). At the same time point, no leukocytes were detected in HFP treated animals compared to FFP, Carprofen or saline treatment groups. In summary, HPF treatment of rats revealed a significant reduction of TNFα levels in rat air pouch exudates.

In an attempt to explore, the effect that varying concentrations of sTNFR1on TNFα levels and leukocytes infiltration, a semi-quantitative immunofluorescent dot blot assay was used to determine sTNFR1 concentration in pouch fluid following instillation of LPS and MSU. The results suggested that at 6 hours post HFP treatment, levels of sTNFR1 were significantly higher than for FFP, Carprofen and saline treatments (p < 0.05). A linear regression equation derived from a standard curve using recombinant murine sTNFR1 was used to determine the actual concentration of sTNFR1. Importantly, the result also showed significant inverse correlations of r = -0.73, p < 0.0001 (LPS) and r = -0.84, p < 0.0001 (MSU) between the levels of sTNFR1 concentration and TNFα response in each rat pouch fluid. Similarly, significant inverse correlations of r = -0.82, p < 0.0001 (LPS) and r = -0.78, p < 0.0001 (MSU) were observed between the levels of sTNFR1 concentration and level of leukocyte infiltration. These results reveal a possible correlation between sTNFR1 concentration and inflammatory markers such as TNFα concentration and leukocyte infiltration. In summary, the strength of these correlations suggests that sTNFR1 is bound to sTNFα in an approximate ratio of 1:1. That HFP administration increases the levels of sTNFR1 concentration in the pouch and reduces TNFα concentration and leukocyte infiltration. Consequently, it is possible that subsequent inflammatory reactions and pathology could be attenuated by the infusion of HFP.

The third part of the study was directed at determining if the sensitivity of TNFα to its specific receptor (sTNFR1) could be qualitatively described using a systems biology approach. Using a system-theoretic approach a model was formulated in which TLR4-mediated TNFα signalling pathways were used to analyse the effects of TNFα positive feedback mechanisms in response to the modulation of sTNFR1. Published TNFα/sTNFR1 association and dissociation rate constants were used in the design of the model. The signalling pathway was mapped using a set of non-linear ordinary deferential equations (ODEs) based on the mass action law. The simulation results showed that sTNFR1 is effective in interrupting further amplification of TNFα levels and is integral in controlling threshold levels of TNFα expression. Furthermore, the results show that as the concentration of sTNFR1 is varied from 1 nM, to 5 nM, 10 nM and 15 nM the levels of TNFα concentration decreases from ~22 nM to ~18 nM, ~11 nM and ~7 nM respectively. The results also indicate that as the rate of change in concentration of sTNFR1 increases with time, the rate of change in concentration of TNFα decreases, suggesting that as TNFα becomes more sensitive to increased sTNFR1 as the concentration of TNFα/sTNFR1 complex increase. This suggests that a temporal increase in the concentration of TNFα/sTNFR1 complex is directly related to the sTNFR1 concentration. The results also quantitatively reveal that the rate of change in the concentration of sTNFR1 and TNFα/sTNFR1 complex are mirrored. The modelling also revealed a sigmoidal response that showed that as the sTNFR1 concentration increased, the sigmoidal characteristic became more pronounced. It is then plausible to conclude that TNFα is ultrasensitive to the sTNFR1 ligand, such that it may have the potential to interrupt the ligand binding to membrane bound TNFR1 (mTNFR1), thus, a condition in which TNFα production and sTNF-R1 (inhibition factor) are in equilibrium. Consequently, it appears that the system-theoretic model suggested here is validated by correlations with experimental data. This model may be useful in predicting in a quantitative manner, the effects of variations in the concentration of sTNFR1 and TNFα activity. The significant modulation of TNFα by sTNFR1 suggests that the presence of HFP in biological systems is also of physiological importance and its use may prove efficacious in preventing the deleterious effects of uncontrolled levels of TNFα.

The fourth part rounded out this study by using in-silico computation approach incorporating comparative genomic analytical methods to explore a range of conserved genetic sequences to identify potential variance in inflammation related genes. There is increasing evidence of genetic factors influencing patient outcomes, although the degree and nature of the influence remains to be fully elucidated. The progress of a sepsis event is often subject to an uncontrolled pro-inflammatory cascade mediated by inflammatory mediators such as cascade of endogenous mediators, TNF-α, IL1β and IL-6. Whilst activation of other factors such as AKT-1, NFκβ and less defined environmental variables also appear to influence patient outcomes. The variability of genes encoding endogenous mediators that constitute the inflammatory pathway have been shown to influence patient predisposition to sepsis and prognosis. In this study, we propose to utilize candidate genes that have been characterized in case control studies as being important in the pathogenesis of sepsis-induced organ dysfunction. Specifically, the Toll-like receptor TLR-4 was used to identify putative genes associated in the signalling pathway and was then applied in a comparative genomics analysis to identify gene polymorphisms. To date, we have identified a number of genetic polymorphisms within the highly conserved cording region of human AKT1. This protein is an intracellular serine/threonine kinase involved in regulating cell survival. The analysis is based on characteristic pattern searching comparative genomics, evolutionary conservation and expression studies that were applied in order to find genetic variation amongst different species. Furthermore, we hypothesised that these genetic variations can induce over expression of AKT signalling, which is associated with the onset of oedema and vascular malformations and may lead to sepsis-induced organ dysfunction. It is then possible that this genetic information may be used to identify patients at high risk of developing fulminant sepsis and associated catastrophic multiple organ failure. This may lead to the development of screening procedures that may be able to categorise patient according to risk and consequently improve outcomes because of better formulated therapeutic interventions. The reported genomic information may be used to identify patients with high risk of developing severe sepsis and associated multiple organ dysfunctions.