Effect of fetal bovine serum and heat-inactivated fetal bovine serum on microbial cell wall-induced expression of procoagulant activity by equine and canine mononuclear cells in vitro

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 1 stage 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 LPS-binding protein. Increasing concentrations of HIFBS significantly increased LPS- and zymosan-induced 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 LPS and 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.     

Effects of the second-generation synthetic lipid A analogue E5564 on responses to endotoxin in [corrected] equine whole blood and monocytes

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)-alpha production; and mRNA expression of TNF-alpha, interleukin (IL)-1beta, 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-alpha, and cell lysates were assayed to determine PCA. Expression of mRNA for TNF-alpha, IL-1beta, 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-alpha production in a concentration-dependent manner. Furthermore, E5564 significantly inhibited LPS-induced mRNA expression of TNF-alpha, IL-1beta, 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.     

Evaluation of the opsonic capacity of core lipopolysaccharide antiserum of equine origin against smooth Escherichia coli 0111:B4, using macrophage chemiluminescence

A study was performed to determine whether equine antiserum to core lipopolysaccharide (LPS) would enhance phagocytosis of smooth gram-negative (GN) organisms by equine macrophages. Five healthy adult horses (group A) were immunized with a bacterin prepared from the J-5 mutant of Escherichia coli 0111:B4 and Salmonella minnesota R595 to produce antibodies to core LPS. Five horses (group B) served as nonimmunized controls and were given physiologic saline solution instead of the rough mutant bacterin. Serum antibody titers to core LPS and to smooth E coli 0111:B4 were determined by indirect ELISA. Four serum pools were prepared: pool 1 = sera from horses in group B prior to immunization; pool 2 = sera from horses in group A prior to immunization (preimmune serum); pool 3 = sera from horses in group B, 7 days after the last saline injection; pool 4 = sera from horses in group A, 7 days after the last immunization (core LPS antiserum). The serum pools, either unheated or heated 30 minutes at 56 C, in 3 dilutions (1/50, 1/100, 1/500) were used to opsonize smooth E coli 0111:B4 in an assay of equine peritoneal macrophage chemiluminescence (CL). Peritoneal fluid was collected from clinically normal horses and the macrophages were purified by adherence to borosilicate glass scintillation vials. Each serum type and dilution was added to triplicate vials containing 10(7) colony-forming units of E coli 0111:B4. Luminol-dependent CL was measured with a liquid scintillation counter in the out-of-coincidence mode. Each serum dilution was tested in duplicate vials without bacteria to asses serum-induced nonspecific CL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimization of conditions for in vitro production of radical oxygen species and expression of tissue factor by canine mononuclear cells and granulocytes for use in high-throughput assays

The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3h in PBG (PBS containing 0.5% BSA and 5mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01microg/ml or greater, and by zymosan concentrations of 0.1microg/ml or greater. ROS production in response to incubation with PMA was significantly increased starting at 10(-7)M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10(-7)M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3h in RPMI with LPS (0.1microg/ml), zymosan (10 microg/ml), PGN (10 microg/ml), and PMA (10(-7)M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.     

Ethyl pyruvate decreases proinflammatory gene expression in lipopolysaccharide-stimulated equine monocytes

Monocytes are among the initial cells that interact with circulating LPS. Binding of LPS to monocyte surface receptors triggers an intracellular signaling cascade and results in the production of proinflammatory cytokines. Ethyl pyruvate, a stable derivative of pyruvate, has been effective in mitigating LPS induced alterations in isolated human monocytes. We hypothesized that ethyl pyruvate would suppress proinflammatory gene expression in LPS-stimulated equine monocytes without affecting cell viability. Equine monocytes were isolated from whole blood using a sediment-gradient centrifugation protocol and enriched to 76% purity by adhesion to tissue culture dishes. Isolated monocytes were incubated with 0, 1, 5, 10 and 50 mM ethyl pyruvate. Cell viability, production of caspase 3/7, and caspase-3 gene expression were determined. In a separate experiment, monocytes were stimulated with LPS (0.1 ng/ml for 1h) followed by incubation with 0, 1, 5, or 10 mM ethyl pyruvate for 1 h. Proinflammatory gene expression was determined by real-time PCR. Ethyl pyruvate at 50 mM adversely affected monocyte viability. Ethyl pyruvate at 10mM or less had no significant effect on monocyte viability, and did not increase activity of caspase 3/7 nor caspase-3 gene expression. Incubation with LPS alone induced a significant upregulation in proinflammatory gene expression. Subsequent treatment of monocytes with ethyl pyruvate significantly reduced IL-8 expression in LPS stimulated monocytes at 5 mM, and IL-8, TNF-α and COX-2 at 10 mM. No beneficial effect on expression of IL-1β or IL-6 was detected. Overall, 10 mM ethyl pyruvate did not adversely affect monocyte viability and suppressed LPS-induced proinflammatory gene expression. Ethyl pyruvate may be a beneficial anti-inflammatory therapy in equine endotoxemia.     

Effects of ischemia and the cyclooxygenase inhibitor flunixin on in vitro passage of lipopolysaccharide across equine jejunum

OBJECTIVE: To determine whether ischemia and flunixin affect in vitro lipopolysaccharide (LPS) absorption in samples of the jejunum of horses. ANIMALS: 12 horses. PROCEDURE: Horses were anesthetized, a midline celiotomy was performed, and the jejunum was located. Two 30-cm sections of jejunum (60 cm apart) were selected. One segment was designated as control tissue; ischemia was induced in the other segment for 120 minutes. Horses were then euthanatized. Mucosa from each jejunal segment was mounted on Ussing chambers and treated with or without flunixin. Tissues from 6 horses were used to assess permeability to radiolabeled LPS; mucosal samples from the remaining 6 horses were incubated with fluorescent-labeled LPS (FITC-LPS) and examined histologically. Production of tumor necrosis factor-alpha (TNF-alpha) and production of LPS-binding protein (LBP) were assessed as indicators of mucosal response to LPS. RESULTS: Ischemia significantly increased mucosal permeability to LPS, but by 180 minutes, the mucosa was not more permeable than control tissue. Flunixin treatment adversely affected intestinal barrier function throughout the experiment but did not result in increased mucosal permeability to LPS. Compared with control tissues, LBP production was increased by ischemia and reduced by exposure to LPS. In ischemic tissue, FITC-LPS entered the lamina propria but TNF-alpha was produced on the mucosal side only, indicating little response to the absorbed LPS. CONCLUSIONS AND CLINICAL RELEVANCE: Ischemia increased LPS passage across equine jejunal mucosa. Flunixin delayed mucosal recovery but did not exacerbate LPS absorption. Evaluation of the clinical importance of flunixin-associated delayed mucosal recovery requires further in vivo investigation.     

Rapid infusion of a phospholipid emulsion attenuates the effects of endotoxaemia in horses

REASONS FOR PERFORMING STUDY: Endotoxaemia currently is associated with a poor prognosis in horses. The results of recent trials in other species indicate that phospholipid emulsions reduce the deleterious effects of endotoxin (LPS). However, in a previous study in horses, a 2 h infusion of emulsion caused an unacceptable degree of haemolysis. HYPOTHESIS: Rapid administration of a lower total dose of emulsion would reduce the effects of LPS and induce less haemolysis; the emulsion would reduce inflammatory effects of LPS in vitro. METHODS: Twelve healthy horses received an i.v. infusion either of saline or a phospholipid emulsion (100 mg/kg), followed immediately by E. coli 055:B5 LPS (30 ng/kg). Clinical parameters, haematological profiles, serum tumour necrosis factor (TNF) activity, serum lipid profiles, urine analyses and severity of haemolysis were monitored before and at selected times after LPS. Monocytes were also incubated in vitro with LPS in the presence or absence of emulsion, after which TNF and tissue factor activities were determined. RESULTS: Clinical signs of endotoxaemia were reduced in horses receiving the emulsion, including clinical score, heart rate, rectal temperature, serum TNF activity, and the characteristic leucopenic response to LPS, when compared to horses not receiving the emulsion. Three horses receiving the emulsion had none, 2 had mild and one had moderate haemolysis. There were no differences in urinalysis results and creatinine concentrations, either within the groups over time or between the groups. Serum concentrations of phosphatidylcholine, bile acids and triglycerides peaked immediately after the infusion; there were no significant changes in concentrations of nonesterified fatty acids or cholesterol. Incubation of equine monocytes with emulsion prevented LPS-induced TNF and tissue factor activities. CONCLUSIONS: Rapid administration of emulsion significantly reduced inflammatory effects of LPS in vivo and caused a clinically insignificant degree of haemolysis. The results of the in vitro studies indicate that emulsion prevents not only LPS-induced synthesis of cytokines, but also expression of membrane-associated mediators (i.e. tissue factor). POTENTIAL RELEVANCE: Rapid i.v. administration of emulsions containing phospholipids that bind endotoxin may provide a clinically useful method of treating endotoxaemia in horses.     

Evaluation of hyaluronidase activity in equine and bovine sera and equine synovial fluid samples by use of enzyme zymography

OBJECTIVE: To investigate the activities of hyaluronidases in equine sera and synovial fluid samples and sera from fetal and adult bovids and evaluate the extent to which the degradation of hyaluronan is influenced by chondrocytes. SAMPLE POPULATION: Commercial and noncommercial samples of equine (n = 6) and bovine (6) sera and 16 synovial fluid samples from horses. PROCEDURE: Hyaluronidase activities in sera and synovial fluid samples were assessed via enzyme zymography (performed at pH 4, 5, 6, or 7). Chondrocytes were isolated from equine cartilage and cultured with or without hyaluronan (1 mg/mL); the degradation of hyaluronan was assessed via agarose gel electrophoresis. RRESULTS: Hyaluronidase activity was detected in equine sera and synovial fluid samples at pH 4, but not at pH 7, and in bovine sera at both pH values. In all samples at pH 4, a major band of activity (molecular weight, approx 60 kd) and some additional higher molecular weight bands were detected; high- and low-molecular-weight activities were detected in bovine sera at pH 7 Hyaluronan in tissue culture medium with or without fetal calf serum was degraded in the presence, but not the absence, of equine chondrocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Hyaluronidase activity was detected in equine sera and synovial fluid at pH 4 and in bovine sera at pH 4 and 7. Primary chondrocytes in monolayer culture can degrade exogenous hyaluronan. Modulating native hyaluronidase activity may offer a new approach to improve the quantity and quality of hyaluronan in articular joints.     

Endotoxin induction of nitric oxide synthase and cyclooxygenase-2 in equine alveolar macrophages

OBJECTIVE: To determine the amount of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzymes induced in vitro in equine alveolar macrophages in response to lipopolysaccharide (LPS). Sample Population-Alveolar macrophages obtained from 12 horses. PROCEDURE: Alveolar macrophages were collected by bronchoalveolar lavage from 12 horses and incubated for 6 hours with LPS (0.001 to 10 microg/ml) or vehicle. Total RNA was extracted and purified. After first-strand cDNA synthesis, mRNA induction was measured, using a polymerase chain reaction (PCR) technique for COX-2, iNOS, and glyceraldehyde 3-phosphate dehydrogenase. In a second study, cells were incubated with LPS or vehicle for 24 hours. Culture medium was assayed for COX-2 and iNOS activity by determining prostaglandin E2 (PGE2) and total nitrite concentrations, respectively. RESULTS: Lipopolysaccharide induces COX-2 and iNOS mRNA in equine alveolar macrophages. Sequencing revealed that PCR products for COX-2 and iNOS had a high degree of nucleotide homology with the human sequences (91% COX-2, 93% iNOS). Production of mRNA for COX-2 and iNOS was accompanied by induction of enzyme activity. Comparing PCR fragment production, expression of mRNA for iNOS appeared to be less than that for COX-2. Induction of COX-2, but not iNOS, was LPS-concentration dependent. Conclusion-Lipopolysaccharide induces COX-2 and iNOS in equine macrophages. CLINICAL RELEVANCE: The induction of iNOS and COX-2 by LPS in equine macrophages suggests these enzymes may be important in the pathophysiology of sepsis. Pharmacologic modulation of iNOS and COX-2 activity may represent a novel therapeutic target in the management of endotoxemia in horses.     

An investigation of the role of soluble CD14 in hospitalized,sick horses

The objectives of this study were to (1) evaluate the effects of equine soluble CD14 (sCD14) and monoclonal antibodies (mAb) to equine CD14 on lipopolysaccharide-induced tumor necrosis factor α (TNF-α) secretion from equine peripheral blood mononuclear cells (PBMC); and to (2) determine serum concentrations of sCD14 in a population of horses with gastrointestinal diseases or other illnesses likely to result in endotoxemia. Equine PBMC isolated from 10 healthy horses were incubated with Escherichia coli LPS plus CD14 mAb or sCD14 and assayed for TNF-α activity. Pre-incubation with CD14 mAb did not inhibit LPS-induced TNF-α production, whereas use of sCD14 inhibited LPS-induced TNF-α production in a concentration-dependent manner. Additionally, blood samples from 55 ill and 23 healthy horses were used to determine serum concentrations of sCD14. Concentrations of sCD14 were positively correlated to respiratory rate, duration of clinical signs and band neutrophil count. Although serum sCD14 was significantly increased in the ill horses compared to healthy horses, sCD14 did not correlate with outcome. Results of this study indicate that release of sCD14 is increased in ill horses and that TNF-α production by PBMC is decreased when cells are treated with sCD14.     

Expression of procoagulant activity by equine lung macrophages: stimulation by blood lymphocytes

Increases in procoagulant activities (PCA) in equine lung macrophages were induced by non-adherent blood lymphocytes which were prestimulated with phytohaemagglutinin for 48 to 72 hours or by supernatants harvested from prestimulated blood lymphocyte cultures. However, prestimulated lymphocyte suspensions themselves expressed PCA which was most probably derived from contaminating monocytes. Because non-adherent cells from lymphocyte suspensions may have attached to adherent macrophages, cells within lymphocyte suspensions might have contributed to the PCAs expressed by lymphocyte-stimulated lung macrophages. Stimulation of lung macrophages for 24 hours by supernatants of phytohaemagglutinin-prestimulated blood lymphocytes induced a significantly greater PCA increase than stimulation by phytohaemagglutin alone. Thus, cytokines from lymphocyte cultures might have triggered or enhanced PCA induction. Direct stimulation of lung cell preparations with phytohaemagglutinin for 48 hours resulted in a progressive increase of PCA in only two of five specimens tested. The failure to induce PCA in three specimens could be due to the absence of sufficient numbers of T cells within the adherent lung cell preparations. In conclusion, PCA response of equine lung macrophages might be lymphocyte-stimulated in which case PCA might be a useful tool for monitoring the processes of cell-mediated immunity in horses.     

Rapid and specific serodiagnosis of western equine encephalitis virus infection in horses

Paired sera from 28 nonvaccinated horses with serologically confirmed western equine encephalitis (WEE) virus infections were evaluated for immunoglobulin (Ig)M and IgG directed against WEE virus, by use of enzyme immunoassay. Twenty-one of the horses developed greater than or equal to 4-fold increases or decreases in serum IgM titers in paired serum samples, confirming the diagnosis of WEE in these horses. Of the remaining 7 horses, 1 had stable IgM titers, 1 had a 2-fold increase in IgM titer between paired sera, 2 had 2-fold decreases in IgM titer, and for 3 horses adequate volumes were not available for both sera of the pair. Twenty-nine of 56 blood samples collected from these 28 horses had been collected within the first 3 days after clinical disease was recognized; all 28 horses and 48 of 53 available serum samples had IgM antibody to WEE virus. Immunoglobulin M also was detected in sera of 27 of 45 other nonvaccinated horses that had illnesses clinically compatible with WEE. Sera with IgM did not have cross-reacting IgM against eastern equine encephalitis virus. Therefore, the sensitivity, specificity, and lack of persistence of IgM was useful in the rapid diagnosis of WEE virus infections in horses.     

Adenosine A2A receptor agonists inhibit lipopolysaccharide-induced production of tumor necrosis factor-alpha by equine monocytes

Adenosine is an endogenous nucleoside that regulates many physiological processes by activating one or more adenosine receptor subtypes, namely A1, A2A, A2B and A3. The results of previous studies indicate that adenosine analogues inhibit lipopolysaccharide (LPS)-induced production of reactive oxygen species (ROS) by equine neutrophils primarily through activation of A2A receptors. Because peripheral blood monocytes produce cytokines that are responsible for many of the deleterious effects of LPS, the current study was performed to evaluate the effects of an array of novel adenosine receptor agonists on LPS-induced production of tumor necrosis factor-alpha (TNF-alpha), and to assess the selectively of these agonists for equine adenosine A2A over the A1 receptor. Radioligand binding studies performed with equine tissues expressing adenosine A1 and A2A receptor subtypes yielded a rank order of affinity for the equine A2A receptor of ATL307>ATL309 approximately ATL310 approximately ATL313>ATL202 approximately ATL361 approximately ATL376>ATL372>CGS21680>NECA. Co-incubation of equine peripheral blood monocytes with LPS and these agonists resulted in inhibition of TNF-alpha production with a rank order of potency that strongly correlated with their binding affinities for equine adenosine A2A receptors. Results of experiments performed with one of the adenosine receptor agonists (ATL313) and selective adenosine receptor antagonists confirmed that inhibition of LPS-induced production of TNF-alpha occurred via stimulation of A2A receptors. Although incubation of monocytes with IB-MECA, a compound purported to act as an adenosine A3 receptor agonist, reduced LPS-induced TNF-alpha production, this effect of IB-MECA was inhibited by the A2A selective antagonist ZM241385 but not by the A3 receptor antagonist MRS1220. These results indicate that the adenosine receptor subtype responsible for regulation of LPS-induced cytokine production by equine monocytes is the A2A receptor. To address the signal transduction mechanism responsible for the anti-inflammatory effects of ATL313 in equine monocytes, production of cAMP was compared in the presence and absence of either the adenosine A2A receptor antagonist ZM241385 or the adenosine A2B receptor antagonist MRS1706. In the absence of the antagonists, ATL313 increased production of cAMP; ZM241385 inhibited this effect of ATL313, whereas MRS1706 did not. Furthermore, incubation of monocytes with either the stable analogue of cAMP, dibutyryl cAMP, or forskolin, an activator of adenylyl cyclase, also inhibited LPS-induced production of TNF-alpha production by equine monocytes. Collectively, the results of the current study indicate that adenosine analogues inhibit LPS-induced production of TNF-alpha by equine monocytes primarily via activation of adenosine A2A receptors and do so in a cAMP-dependent manner. The results of this study indicate that stable adenosine analogues that are selective for adenosine A2A receptors may be suitable for development as anti-inflammatory drugs in horses.     

Evaluation of lipopolysaccharide-induced activation of equine neutrophils

OBJECTIVE: To evaluate lipopolysaccharide (LPS)-induced activation of equine neutrophils in blood. SAMPLE POPULATION: Blood samples from 5 healthy adult Thoroughbreds. PROCEDURES: Neutrophil integrin (CD11/CD18) expression, size variation, degranulation, and deformability were measured with and without incubation with LPS. Time and concentration studies were done. The mechanism of endotoxin-induced neutrophil activation was investigated by inactivating complement or preincubating neutrophils with inhibitors of tumor necrosis factor-alpha (TNF-alpha) synthesis, prostaglandin-leukotriene synthesis, or platelet-activating factor. RESULTS: Incubation of equine neutrophils with LPS increased cell surface expression of CD11/CD18, decreased neutrophil deformability, increased and decreased neutrophil size, and induced neutrophil degranulation. The LPS-induced neutrophil activation was attenuated by addition of inhibitors of TNF-alpha and prostaglandin-leukotriene synthesis. CONCLUSIONS AND CLINICAL RELEVANCE: Equine neutrophils are readily activated in vitro by LPS, resulting in increased expression of integrin adhesion molecules, decreased deformability, variation in neutrophil size, and degranulation. The tests used to detect activated neutrophils in this study may be useful in detecting in vivo neutrophil activation in horses with sepsis and endotoxemia.     

Stability of sorbitol dehydrogenase activity in bovine and equine sera

Serum sorbitol dehydrogenase (SDH) activities in 10 cows and nine horses were measured using an automated clinical analyzer. The serum samples were divided into aliquots that were stored at room temperature (21 degrees C), refrigerated (0-5 degrees C), or frozen (-30 degrees C). The stability of the SDH activity was monitored at various intervals. SDH activity in bovine sera remained stable for at least 5 hours at room temperature, 24 hours refrigerated, and 72 hours frozen without any significant (p < 0.05) differences from the initial serum values. In equine sera, SDH activity remained stable for at least 5 hours at room temperature and 48 hours frozen. The activity of the refrigerated equine sera was stable for at least 5 hours but less than 24 hours. An evaluation of fresh bovine serum and heparinized plasma samples indicated that there was no significant difference (p < 0.05) between the two sampling methods and that either may be employed for automated measurement of SDH activity following the established protocol. Sample type comparison indicated that there was a small but statistically significant (p < 0.05) difference between the results obtained comparing fresh serum and heparinized plasma samples for the horse. A reference range for Holstein cows was established using sera from 71 clinically healthy cattle (mean -/+ 2 SD = 32 -/+ 26 U/L).     

Generation and characterization of monoclonal antibodies to equine CD16

The low-affinity Fc receptor CD16 plays a central role in the inflammatory and innate immune responses of many species, but has not yet been investigated in the horse. Using the predicted extracellular region of equine CD16 expressed as a recombinant fusion protein with equine IL-4 (rIL-4/CD16), we generated a panel of mouse monoclonal antibodies (mAbs) that recognize equine CD16. Nine mAbs were chosen for characterization based upon recognition of CD16, but not IL-4, in ELISA. All nine mAbs recognized full-length, cell-surface CD16 expressed as a GFP fusion protein by CHO cells, but not the closely related Fc receptor CD32 expressed in the same system. In flow cytometric analysis with equine peripheral leukocytes, the mAbs labeled cells in the granulocyte, monocyte, and lymphocyte populations in a pattern consistent with other species. Monocytes that were strongly labeled with CD16 mAb 9G5 were also positive for the LPS receptor CD14. Cytospins made with peripheral leukocytes were immunohistochemically labeled and showed mAb recognition of primarily mononuclear cells. ELISA revealed that the nine mAbs can be grouped into three patterns of epitope recognition. These new antibodies will serve as useful tools in the investigation of equine immune responses and inflammatory processes.