Is it the systemic inflammatory response syndrome or endotoxemia in horses with colic?

Some veterinarians describe particularly sick horses or neonatal foals as being endotoxemic, whereas others refer to the same animals as having the systemic inflammatory response syndrome. This article reviews the basis for the use of each of these terms in equine practice, and highlights the mechanisms underlying the response of the horse's innate immune system to key structural components of the microorganisms that initiate these conditions, including how some of those responses differ from other species. Current approaches used to treat horses with these conditions are summarized, and caution advised on extrapolating findings from other species to the horse.

Disparities in TLR5 expression and responsiveness to flagellin in equine neutrophils and mononuclear phagocytes.

As sentinel cells of the innate immune system, neutrophils and mononuclear phagocytes use specific TLRs to recognize the conserved molecular patterns that characterize microbes. This study was performed to compare the responses of equine neutrophils and mononuclear phagocytes to LPS and flagellin, components of bacteria that are recognized by TLR4 and TLR5, respectively. Neutrophils and mononuclear phagocytes isolated from healthy horses were incubated in vitro with LPS, flagellin, or pronase-inactivated flagellin in the presence or absence of polymyxin B. Production of reactive oxygen species and expression of mRNA for proinflammatory cytokines were used as readouts for activation of neutrophils; production of TNF-α was used for the mononuclear cells. Western blot analysis and flow cytometry were used to detect TLR5 protein in both cell types. Although the neutrophils responded to both LPS and flagellin by producing reactive oxygen species and expressing mRNA for proinflammatory cytokines, flagellin had no stimulatory effect on monocytes or macrophages. Although both neutrophils and monocytes expressed mRNA for TLR5, it appeared to be translated into protein only by the neutrophils. Incubation with neither LPS nor IFN-γ altered TLR5 expression by the monocytes. These findings indicate that flagellin has disparate effects on neutrophils and mononuclear phagocytes isolated from horses, a species that is exquisitely sensitive to the TLR4 ligand, LPS, and that equine mononuclear phagocytes, unlike corresponding cells of other mammalian species, lack surface expression of TLR5 and do not respond to flagellin.

Characterization of cytokines associated with Th17 cells in the eyes of horses with recurrent uveitis.

OBJECTIVE: Equine recurrent uveitis (ERU) is a spontaneous disease that is the most common cause of blindness in horses, affecting up to 15% of the horse population. Th17 cells are a major cell population driving the pathogenesis in several mouse models of autoimmune inflammation, including experimental autoimmune uveitis. The purpose of this study is to investigate the role a Th17 cell-mediated response plays in the pathogenesis of ERU. PROCEDURE: Banked, Davidson's-fixed equine globes histopathologically diagnosed with ERU (n = 7) were compared immunohistochemically with healthy control globes (n = 7). Immunohistochemical staining was performed using a pan-Leptospira antibody and antibodies against IL-6, IL-17, and IL-23. Additionally, immunostaining was performed for T-cell (CD3) and B-cell (CD79α) markers. Specificity of immunoreactivity was confirmed by western blot analysis. RESULTS: Immunohistochemical staining was positive for IL-6, IL-17, and IL-23 within the cytoplasm of nonpigmented ciliary epithelial cells and mononuclear inflammatory cells infiltrating the iris, and ciliary body of ERU horses (n = 7) but negative in controls (n = 7). ERU-affected eyes were CD3 positive (n = 7) and CD79α negative (n = 7). Staining for Leptospira was negative in all ERU and control globes. CONCLUSIONS: Strong immunoreactivity for IL-6, IL-17, and IL-23, in conjunction with the fact that T lymphocytes are the predominating inflammatory cells present in ERU, suggests that IL-17-secreting helper T-cells play a role in the pathogenesis of ERU. These findings suggest that horses with ERU may serve as a naturally occurring animal model for autoimmune uveitis.

Effects of low-dose hydrocortisone therapy on immune function in neonatal horses.

Low-dose hydrocortisone (LDHC) therapy modulates inflammatory responses in adults and improves outcomes in some septic adults and neonates, but its immunologic effects have not been evaluated in neonates. The objective of this study was to evaluate effects of LDHC therapy on ex vivo immune function in neonatal horses (foals). We hypothesized that LDHC treatment would dampen proinflammatory responses without impairing neutrophil function. Hydrocortisone (1.3 mg/kg/d i.v.) was administered to foals in a tapering 3.5 d course. Peripheral blood leukocytes were collected from foals before, during, and after hydrocortisone treatment. A separate group of age-matched untreated foals served as controls. Endotoxin-induced peripheral blood mononuclear cell gene expression of inflammatory cytokines was measured by real-time quantitative RT-PCR. Neutrophils were incubated with labeled, killed Staphylococcus aureus or Escherichia coli for assessment of phagocytosis, and with phorbol myristate acetate, zymosan, or endotoxin for measurement of reactive oxygen species (ROS) production. Neutrophil phagocytosis and ROS production were similar in both groups. Foals receiving hydrocortisone had significantly decreased endotoxin-induced expression of TNF-α, IL-6, IL-8, and IL-1ß. These data suggest that this LDHC treatment regimen ameliorates endotoxin-induced proinflammatory cytokine expression in neonatal foals without impairing innate immune responses needed to combat bacterial infection.

Expression of inflammation-associated genes in circulating leukocytes collected from horses with gastrointestinal tract disease.

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.

Expression of genes associated with inflammation induced by ex vivo exposure to lipopolysaccharide in peripheral blood leukocytes from horses with gastrointestinal disease.

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.

Temporal aspects of laminar gene expression during the developmental stages of equine laminitis.

The results of recent studies indicate that inflammatory responses occurring in the early stages of equine laminitis lead to downstream events that eventually result in failure of the bond between the hoof wall and the distal phalanx. In order to gain further insights into the molecular mechanisms involved in the development of laminitis, an equine-specific cDNA microarray consisting of transcripts for more that 3000 genes was used to assess temporal changes in gene expression in laminar tissues at 1.5, 3 and 12 h after administration of either a laminitis-inducing agent (black walnut heartwood extract; BWHE) or an equal volume of water (control). As early as 1.5 h after BWHE administration, pro-inflammatory genes associated with leukocyte activation and emigration, including MCP-3/CCL7, MCP-1/CCL2, IP-10/CXCL10 and ICAM-1 were up-regulated. At both 1.5 and 3h after administration of BWHE, expression of B-cell specific transcripts (e.g., Ig-gamma 3, Ig-gamma 1 and lambda-light chain) were decreased in the laminar tissues. At the onset of Obel grade 1 lameness in horses administered BWHE, other genes involved in inflammatory processes (e.g., serum amyloid A, calgranulin C and NFAT-activation molecule 1), regulation of inflammation (e.g., inter-alpha-trypsin inhibitor, BiP/GRP78 [Ig binding protein], L-plastin, serpin and nexin-1), antioxidant responses (e.g., superoxide dismutase), matrix turnover (e.g., MMP-9 and TIMP-1), and anti-microbial responses (e.g., serotransferrin, beta-defensin-1 and elafin) were up-regulated. These results provide convincing evidence that genes associated with inflammation, activation and extravasation of leukocytes, antimicrobial activities, and destruction of the lamellar basement membrane are induced during the early stages of development of laminitis in response to administration of BWHE.

Differential induction of MyD88- and TRIF-dependent pathways in equine monocytes by Toll-like receptor agonists.

Our understanding of the innate immune response in the horse has been limited by a lack of definitive data concerning cell signaling in response to microbial products. Toll-like receptors (TLRs) recognize conserved molecular motifs of microbes and elicit immune responses through their coupling with intracellular adaptor molecules, particularly MyD88 and TRIF. To provide a more definitive characterization of TLR signaling in the horse, the objectives of this study were to: (1) characterize the responses of equine monocytes to TLR ligands that signal through MyD88, TRIF or both in other species, and (2) determine the profiles of gene expression initiated utilizing these adaptor molecules. Monocytes were used to establish concentration response curves for Escherichia coli lipopolysaccharide (LPS; TLR4 ligand) and N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine x 3 HCl (Pam(3)CSK(4); TLR2 ligand) based on expression of procoagulant activity (PCA) and production of tumor necrosis factor-alpha (TNF-alpha); effects of polyinosine-polycytidylic acid (Poly I:C; TLR3 ligand) were determined by quantifying expression of mRNA for interferon-beta (IFN-ss). Expression of genes associated with the MyD88- (TNF-alpha, IL-1ss, IL-6 and IL-10) and TRIF-dependent pathways (IFN-ss, IP-10, RANTES and TRAF1) were measured at intervals spanning 20 h. LPS and Pam(3)CSK(4) induced significantly higher expression of TNF-alpha, IL-1ss, and IL-10 than did Poly I:C. Poly I:C induced significantly higher expression of IFN-ss, IP-10 and RANTES than did either the TLR2 or TLR4 ligands. High concentrations of E. coli LPS did not significantly increase expression of genes associated with the TRIF-dependent pathway. The results of this study suggest that equine monocytes utilize a common intracellular pathway in response to TLR2 and TLR4 ligands, but a distinct pathway in response to TLR3 ligands.

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.

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.

The equine TLR4/MD-2 complex mediates recognition of lipopolysaccharide from Rhodobacter sphaeroides as an agonist.

Lipopolysaccharide (LPS) antagonists inhibit the response of inflammatory cells to LPS, presumably by competitive inhibition, and may be of therapeutic value in the treatment of endotoxemia and sepsis. The inhibitory effects of some LPS antagonists are restricted to certain host species, however, as the same molecules can have significant endotoxic activity in other species. This species-specific recognition appears to be mediated by Toll-like receptor 4 (TLR4) and/or MD-2. We have shown previously that LPS from Rhodobacter sphaeroides ( RsLPS) is an LPS antagonist in human cells but an agonist (or LPS mimetic) in equine cells. In the present study, HEK293 cells were transfected with combinations of human and equine CD14, TLR4 and MD-2, and incubated with either RsLPS or with LPS from Escherichia coli as an endotoxin control. NF-kappaB activation was measured in a dual luciferase assay as an indicator of cellular activation. Our results indicate that E. colic LPS activated NF-kappaB in cells transfected with all combinations of the three receptor proteins, whereas RsLPS activated NF-kappaB only in cells expressing the single combination of equine TLR4 and equine MD-2. We conclude that the TLR4/MD-2 complex is responsible for recognition of RsLPS as an agonist in equine cells.

Effects of stimulation of adenosine A2A receptors on lipopolysaccharide-induced production of reactive oxygen species by equine neutrophils.

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 A(2A) receptor antagonist; this effect of NECA was not affected by the adenosine A(2B) 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 A(2A) receptors inhibited ROS production by LPS-stimulated equine neutrophils in a cAMP-dependent manner. These results suggest that stable adenosine A(2A) receptor agonists may be developed as suitable anti-inflammatory drugs in horses.

Pharmacologic characterization of novel adenosine A2A receptor agonists in equine neutrophils.

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 A(1), A(2A), and A(3) 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 A(2A) receptor on the basis of 50% inhibitory concentrations (IC(50)) of the agonists as follows: ATL307 (IC(50) = 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 A(2A) over A(1) and A(3) 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 A(2A) receptors. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that activation of A(2A) receptors exerted anti-inflammatory effects on equine neutrophils and that stable, highly selective adenosine A(2A) receptor agonists may be developed for use in management of horses and other domestic animals with septic and nonseptic inflammatory diseases.

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.

Dynamic changes in circulating leukocytes during the induction of equine laminitis with black walnut extract.

Administration of black walnut heartwood extract (BWHE) via nasogastric tube induces acute laminitis in horses. However, the processes responsible for the development of laminitis, including laminitis induced with BWHE, remain unclear. The results of recent studies indicate that administration of BWHE initiates an inflammatory response in the laminar tissues and that this response may be due to extravasation of activated leukocytes from the circulation. This study examines the effects of BWHE administration on the dynamics of circulating neutrophils and monocytes, and the capacity of blood leukocytes to produce radical oxygen species (ROS) over the time period from administration of BWHE to the development of lameness consistent with Obel grade I laminitis. Individual horses, free of pre-existing musculoskeletal disease, were administered either 6l of BWHE or an equal volume of water at time 0 (T=0). Blood samples were collected prior to dosing and at 1, 2, 3, 4, 6, 8, 10 and 12h after dosing, or until the onset of Obel grade I laminitis. For each sample, total leukocyte counts were determined followed by collection of buffy coats and removal of erythrocytes by hypotonic lysis. Leukocytes were either fixed for flow cytometric assessment of differential counts or maintained in culture to measure endogenous and phorbol ester-induced production of ROS. At each sample time, the number of cells recovered and the flow cytometric differential counts were compared with corresponding total leukocyte counts determined by the Clinical Pathology laboratory. Horses administered BWHE had a significant reduction in circulating leukocytes at 3-4 h relative to values for horses administered the same volume of water. Horses that developed Obel grade I laminitis had a significant reduction in circulating leukocytes when compared to values for horses administered BWHE that did not become lame. Flow cytometric analysis revealed a consistent decrease in the total number of monocytes obtained from horses that developed laminitis. In these same horses, the endogenous level of ROS production was significantly higher at T=0 than for horses that did not become lame. Furthermore, production of ROS by leukocytes from horses that developed laminitis increased significantly and coincided with the decrease in circulating leukocytes. Collectively, these findings support a role for systemic activation of leukocytes and induction of inflammation by BWHE as a factor in the early pathogenesis of acute laminitis. Because laminitis often develops as a sequel to diseases characterized by systemic inflammatory events, activation and emigration of neutrophils and monocytes may be important factors in the early pathogenesis of laminitis in clinical cases.

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.

Nerve growth factor activates kinases that phosphorylate atypical protein kinase C.

Activation of atypical protein kinase C by nerve growth factor (NGF) involves phosphorylation. In order to identify kinases that regulate atypical PKC (aPKC), we surveyed PC12 cell lysates for protein kinases that are activated by NGF and which could phosphorylate aPKC. Employing an in-gel kinase assay where aPKC-zeta was copolymerized within the gel matrix as a substrate, three kinases, pp175, pp87 and pp60, were identified as enzymes that phosphorylated aPKC. Phosphorylation of aPKC by these three kinases coincided with NGF-induced activation of the enzyme. Each kinase possessed a unique subcellular distribution pattern and could be activated by either ceramide or H(2)0(2), second messengers that mimic NGF signaling events. Upstream, pp175 and pp60 lie in a ras pathway, whereas pp87 lies in a pathway dependent upon src. Altogether, these findings reveal that the aPKCs are subject to regulation by a novel group of kinases.

Concentrations of serum amyloid A and lipopolysaccharide-binding protein in horses with colic.

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 clinical data. 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 dot-blot assays, respectively. 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.

Using 3D animations to teach intracellular signal transduction mechanisms: taking the arrows out of cells.

Traditional methods of teaching intracellular biological processes and pathways use figures or flowcharts with the names of molecules linked with arrows. Many veterinary students, presented with such material, simply memorize the names or chemical structures of the molecules and are then likely to forget the material once the examination is completed. To address this problem, the authors designed, created, and field-tested new teaching media that incorporate realistic three-dimensional (3D) animations depicting the dynamic changes that occur in intracellular molecules during cellular activation. Testing found that veterinary students taught using traditional teaching media (e.g., lectures, handouts, textbooks) are proficient in memorizing the names and order of intracellular molecules but unable to appreciate the interactions between these elements or their spatial relationships within cells. In contrast, more than 90% of veterinary students taught using 3D animations not only recall the facts about the intracellular elements but also develop accurate mental images of the interactions among these molecules and their spatial relationships. These findings strongly suggest that the comprehension of complex biological processes by veterinary students can be enhanced by the use of dynamic 3D depictions of these processes in the classroom.

Relationships between intrauterine infusion of N-acetylcysteine, equine endometrial pathology, neutrophil function, post-breeding therapy, and reproductive performance.

Persistent endometritis in the mare is associated with hypersecretion of mucus by endometrial epithelium and migration of neutrophils into the uterine lumen. This study examines the relationships between N-acetylcysteine (NAC), a mucolytic agent with anti-inflammatory properties, and endometrial architecture, serum neutrophil function, post-breeding therapy, and reproductive performance of NAC-treated mares in a clinical setting. In study 1, endometrial biopsies from mares receiving intrauterine saline (fertile-control, n = 6) or 3.3% NAC (fertile-treatment, n = 6; barren-treatment, n = 10) were evaluated by histology and image analysis. In study 2, phagocytic activity of serum-derived neutrophils was measured after adding 0.5% or 3% NAC. In study 3, pregnancy rates of repeat breeders (n = 44) receiving an intrauterine infusion of 3.3% NAC 24-36 hours before mating (group 1) was recorded, as was first cycle of the season pregnancy rates of reproductively normal mares (group 2, n = 85), and mares treated for bacterial endometritis the cycle before mating (group 3, n = 25). Intrauterine NAC did not adversely affect endometrial histology. Extracellular mucus thickness and staining intensity were reduced in fertile-treatment mares (P < 0.03). Neutrophil function was inhibited by 3% NAC solution, but not by 0.5% NAC (P < 0.05). In study 3, for groups 1, 2, and 3, respectively, the first-cycle pregnancy rates were 77%, 74%, and 56%, and early embryonic death rates were 15%, 13%, and 7%. In group 2 mares treated with uterine lavage and oxytocin post-mating, the pregnancy rate was 89% (39/44), whereas in mares treated with uterine lavage and 1 g ceftiofur, it was 60% (24/40). Of the oxytocin-treated mares, 18% (8/44) had ≥ 1 cm of intrauterine fluid or marked uterine edema, whereas 80% (32/40) of the antibiotic-treated mares did. In conclusion, intrauterine infusion of a 3.3% solution of NAC was not irritating and inhibited the oxidative burst of neutrophils. Repeat breeder mares, with evidence of mucus hypersecretion, but no uterine pathogens, when treated with NAC followed by post-mating uterine lavage and oxytocin (and in some cases intrauterine antibiotics), achieved a pregnancy rate of 77%.