OBJECTIVE To validate primer sets for use in reverse transcription quantitative PCR assays to measure gene expression of cytosolic phospholipase A2 (cPLA2) and microsomal prostaglandin E2 synthase 1 (mPGES1) in equine mononuclear cells and determine the effects of firocoxib, a selective cyclooxygenase 2 (COX-2) inhibitor, on COX-2, cPLA2, and mPGES1 gene expression following incubation of mononuclear cells with lipopolysaccharide (LPS).
ANIMALS 8 healthy adult horses.
PROCEDURES Peripheral blood mononuclear cells were isolated by density gradient centrifugation and incubated at 37°C with medium alone, firocoxib (100 ng/mL), LPS (1 ng/mL or 1 μg/mL), or combinations of firocoxib and both LPS concentrations. After 4 hours, supernatants were collected and tested for prostaglandin E2 (PGE2) concentration with an enzyme inhibition assay, and gene expression in cell lysates was measured with PCR assays.
RESULTS Primer pairs for cPLA2 and mPGES1 yielded single products on dissociation curve analyses, with mean assay efficiencies of 102% and 100%, respectively. Incubation with firocoxib and LPS significantly decreased PGE2 supernatant concentrations and significantly reduced COX-2 and mPGES1 gene expression, compared with values following incubation with LPS alone.
CONCLUSIONS AND CLINICAL RELEVANCE Primer sets for mPGES1 and cPLA2 gene expression in equine mononuclear cells were successfully validated. Firocoxib significantly decreased LPS-induced COX-2 and mPGES1 expression, suggesting that it may be useful in the control of diseases in which expression of these genes is upregulated.
Objective—To determine the effect of fetal bovine serum (FBS) and heat-inactivated FBS (HI-FBS) on lipopolysaccharide (LPS)- and zymosan-induced procoagulant activity of equine and canine mononuclear cells.
Sample Population—Mononuclear cells from 18 horses and 3 dogs.
Procedures—Cells were incubated with various concentrations of FBS, HI-FBS, LPS, zymosan, polymyxin B, and anti–LPS-binding protein monoclonal antibody or combinations of these constituents. A 1stage recalcification assay was used to determine procoagulant activity.
Results—Addition of FBS to media significantly increased procoagulant activity; equine and canine cells were stimulated by 1% and 10% FBS, respectively. Coincubation of cells with FBS and polymyxin B did not reduce this effect, suggesting that the response was not attributable to LPS contamination. Addition of HI-FBS to media did not stimulate procoagulant activity of equine or canine cells, and the sensitivity of the equine cells to LPS was significantly increased by HI-FBS. This increased LPS sensitivity was reduced 40% with monoclonal antibody directed against human recombinant LPSbinding protein. Increasing concentrations of HIFBS significantly increased LPS- and zymosaninduced procoagulant activity of canine cells.
Conclusion and Clinical Relevance—Procoagulant activity production in equine and canine mononuclear cells was significantly increased by addition of FBS, whereas heat inactivation of FBS eliminated this effect. Heat inactivation did not eliminate the function of serum proteins involved in enhancement of LPSand zymosan-induced procoagulant activity. Results suggest that HI-FBS can be used as a source of serum proteins that increase the sensitivity of mononuclear cells to bacterial and yeast cell wall components.
Objective—To evaluate the effect of the duration of cold Ischemia on the renin-angiotensin system during renal transplantation In cats and to define the potential Influence of vasoactive factors in renal tissue following cold ischemic storage versus warm ischemic storage
Procedures—10 cats underwent renal autotransplantation after 30 minutes (n = 5) or 3 hours (5) of simple, ex vivo cold storage of renal autographs. Following autograft reperfusion, direct hemodynamic variables were measured with a telemetric Implant and samples were collected for plasma renin concentration. Activation of vascular-related genes (renin, endothelin, and angiotensin converting enzyme) relative to 2-hour simple cold or warm ischemia was also evaluated.
Results—No significant difference between groups was detected In any of the hemodynamic variables or postreperfusion plasma renin concentrations measured in this study relative to the duration of cold ischemic storage. There was also no difference between warm- and cold-stored kidneys in the expression of vascular-related genes
Conclusions and Clinical Relevance—Prolonged renal Ischemia for clinically relevant durations does not appear to predispose clinically normal cats to altered hemodynamics or high plasma renin concentrations following graft reperfusion. Activation of vasoactive genes does not appear to be Influenced by type of Ischemia over 2 hours. (Am J Vet Res 2010;71:1220-1227)
Objective—To determine concentrations of 2 acute-phase
proteins (serum amyloid A [SAA] and
lipopolysaccharide-binding protein [LBP]) in serum
samples obtained from horses with colic and identify
relationships among these acute-phase proteins and
Animals—765 horses with naturally developing gastrointestinal
tract diseases characterized by colic (ie,
clinical signs indicative of abdominal pain) and 79
healthy control horses; all horses were examined at 2
university teaching hospitals.
Procedure—Serum concentrations of SAA and LBP
were determined by immunoturbidometric and dotblot
Results—SAA and LBP concentrations were determined
for 718 and 765 horses with colic, respectively.
Concentrations of SAA were significantly higher in
nonsurvivors than in survivors, and horses with
enteritis or colitis and conditions characterized by
chronic inflammation (eg, abdominal abscesses, peritonitis,
or rectal tears) had SAA concentrations significantly
greater than those for horses with other conditions.
Serum concentrations of LBP did not correlate
with outcome, disease process, or portion of the
gastrointestinal tract affected.
Conclusions and Clinical Relevance—Circulating
concentrations of SAA were significantly higher at
admission in horses with colic attributable to conditions
having a primary inflammatory cause (eg, enteritis,
colitis, peritonitis, or abdominal abscesses) and
were higher in horses that failed to survive the
episode of colic, compared with concentrations in
horses that survived. Serum concentrations of LBP
did not correlate with survival. Analysis of these findings
suggests that evaluation of SAA concentrations
may be of use in identifying horses with colic attributable
to diseases that have inflammation as a primary
component of pathogenesis. (Am J Vet Res
Objective—To assess the anti-inflammatory effects of an adenosine analogue on lipopolysaccharide (LPS)-stimulated equine neutrophils.
Sample Population—Neutrophils obtained from 10 healthy horses.
Procedures—An adenosine analogue (5′-N-ethylcarboxamidoadenosine [NECA]) was tested for its ability to inhibit production of reactive oxygen species (ROS) in LPS-stimulated equine neutrophils. Selective adenosine receptor antagonists were used to identify the receptor subtype responsible for effects. To assess the mechanism of action of NECA, cAMP concentrations were measured, and effects of dibutyryl cAMP (a stable analogue of cAMP) and rolipram (a type 4 phosphodiesterase inhibitor) were investigated.
Results—NECA elicited concentration-dependent inhibition of ROS production that was inhibited by ZM241385, a selective adenosine A2A receptor antagonist; this effect of NECA was not affected by the adenosine A2B receptor antagonist MRS1706. Also, ZM241385 blocked NECA-induced increases in cAMP concentrations, whereas MRS1706 did not alter this effect of NECA. Rolipram potentiated NECA-induced inhibition of ROS production, and dibutyryl cAMP also inhibited ROS production.
Conclusions and Clinical Relevance—Activation of adenosine A2A receptors inhibited ROS production by LPS-stimulated equine neutrophils in a cAMP-dependent manner. These results suggest that stable adenosine A2A receptor agonists may be developed as suitable anti-inflammatory drugs in horses.
Objective—To evaluate anti-inflammatory effects of several novel adenosine receptor agonists and to determine their specificity for various adenosine receptor subtypes on neutrophils, cells heterologously expressing equine adenosine receptors, or equine brain membranes.
Sample Population—Neutrophils isolated from 8 healthy horses.
Procedures—Radioligand binding experiments were performed to compare binding affinities of adenosine receptor agonists to equine adenosine A1, A2A, and A3 receptor subtypes. Effects of these agonists on endotoxin-induced production of reactive oxygen species (ROS) by equine neutrophils and roles of specific adenosine receptor subtypes and cAMP production in mediating these effects were determined.
Results—Radioligand binding experiments yielded a ranked order of affinity for the brain equine A2A receptor on the basis of 50% inhibitory concentrations (IC50) of the agonists as follows: ATL307 (IC50 = 1.9nM) and ATL313 > ATL309 and ATL310 > ATL202 > 2-([p-2- carboxyethyl] phenylethylamino)-5′-N-ethylcarboxyamidoadenosine > 5′-N-ethylcarboxamidoadenosine. Furthermore, ATL313 had approximately 100-fold greater selectivity for A2A over A1 and A3 receptors. In functional assays with equine neutrophils, the compounds inhibited endotoxin-induced ROS production and stimulated production of cAMP with the same ranked order of potency. Results of experiments performed with selective adenosine receptor antagonists indicated that functional effects of ATL313 were via stimulation of A2A receptors.
Conclusions and Clinical Relevance—Results indicated that activation of A2A receptors exerted anti-inflammatory effects on equine neutrophils and that stable, highly selective adenosine A2A receptor agonists may be developed for use in management of horses and other domestic animals with septic and nonseptic inflammatory diseases.
Objective—To evaluate proinflammatory effects of the second-generation synthetic lipid A analogue E5564 on equine whole blood and isolated monocytes and to determine the ability of E5564 to prevent LPS (lipopolysaccharide)-induced procoagulant activity (PCA); tumor necrosis factor (TNF)-α production; and mRNA expression of TNF-α, interleukin (IL)-1β, IL-6, and IL-10 by equine monocytes.
Sample Population—Venous blood samples obtained from 19 healthy horses.
Procedures—Whole blood and monocytes were incubated with Escherichia coli O111:B4 LPS, E5564, or E5564 plus E coli O111:B4 LPS. Whole blood and cell supernatants were assayed for TNF-α, and cell lysates were assayed to determine PCA. Expression of mRNA for TNF-α, IL-1β, IL-6, and IL-10 by monocytes was determined by use of real-time quantitative PCR assay.
Results—Minimal proinflammatory effects were detected in whole blood and monocytes. In addition, E5564 inhibited LPS-induced PCA and TNF-α production in a concentration-dependent manner. Furthermore, E5564 significantly inhibited LPS-induced mRNA expression of TNF-α, IL-1β, and IL-10 and decreased LPS-induced expression of IL-6.
Conclusions and Clinical Relevance—The second-generation synthetic lipid A analogue E5564 lacked agonist activity in equine whole blood and monocytes and was a potent antagonist of enteric LPS. Therefore, E5564 appeared to be the first lipid A analogue that has potential as an effective therapeutic agent in horses with endotoxemia.
Objective—To investigate whether expression of inflammation-associated genes in leukocytes from horses with gastrointestinal tract (GIT) diseases correlated with the type of disease and outcome.
Animals—10 healthy horses and 50 horses with GIT disease.
Procedures—A blood sample was collected from each healthy horse or horse with GIT disease (during admission to the hospital). Leukocytes were isolated, diluted to a standard concentration, and frozen until RNA extraction. Expression of 14 genes associated with inflammation was quantified by use of a real-time quantitative reverse transcription PCR assay. Results were grouped by GIT disease type and disease outcome for comparison.
Results—Horses with GIT disease had colic of unknown etiology (n = 8 horses), GIT inflammation or strangulation (19), or nonstrangulating GIT obstruction (23). Among the 45 horses receiving treatment, 38 were discharged from the hospital, and 7 died or were euthanized. Compared with healthy horses, horses with colic of unknown etiology had similar gene expression. Significant differences in expression of the interleukin-8, leukocyte-selectin molecule, matrix metalloproteinase-9, platelet-selectin molecule, mitochondrial superoxide dismutase, Toll-like receptor 4, and tumor necrosis factor-A genes were detected between healthy horses and horses with GIT disease. Significant differences in expression of the interleukin-1 receptor antagonist, interleukin-8, leukocyte-selectin molecule, matrix metalloproteinase-9, platelet-selectin molecule, mitochondrial superoxide dismutase, Toll-like receptor 4, and tumor necrosis factor-A genes were detected among healthy horses and horses grouped by disease outcome.
Conclusions and Clinical Relevance—Inflammatory gene expression in leukocytes of horses with GIT disease appeared to be related to disease pathogenesis and prognosis.
Objective—To investigate the effect of ex vivo exposure to lipopolysaccharide (LPS) on the expression of inflammatory genes in leukocytes from horses with gastrointestinal (Gl) disease and determine whether the pattern or magnitude of the response to LPS correlated with the type of disease and outcome.
Animals—49 horses with Gl disease and 10 healthy horses
Procedures—Leukocytes were isolated from blood samples and submitted to 3 protocols: immediate freezing, freezing after 4-hour incubation in medium, and freezing after 4-hour incubation in medium containing LPS. Expression of 14 genes associated with inflammation was assessed via PCR assay. Results were compared by disease type and outcome
Results—Horses with Gl disease had colic of unknown etiology (n = 8), Gl inflammation or strangulation (18), or nonstrangulating Gl obstruction (23). Among the 44 horses receiving treatment, 38 were discharged from the hospital and 6 died or were euthanized. Incubation of leukocytes in medium alone changed the expression of several genes. Incubation with LPS resulted in increased expression of interleukin-10 and monocyte chemotactic protein-3 in leukocytes from healthy and sick horses. Leukocytes from horses with nonstrangulating obstruction and horses that survived had less pronounced LPS-induced increases in interleukin-10 expression than did cells from healthy horses. The opposite was evident for monocyte chemotactic protein-3.
Conclusions and Clinical Relevance—No evidence existed for a reduced response of leukocytes from horses with gastrointestinal disease to ex vivo exposure to LPS. Leukocyte expression of inflammatory genes after ex vivo incubation with LPS appeared to be related to pathogenesis and prognosis. (Am J Vet Res 2010;71:1162—1169)