Two artificial intelligence models underperform on examinations in a veterinary curriculum.

OBJECTIVE: Advancements in artificial intelligence (AI) and large language models have rapidly generated new possibilities for education and knowledge dissemination in various domains. Currently, our understanding of the knowledge of these models, such as ChatGPT, in the medical and veterinary sciences is in its nascent stage. Educators are faced with an urgent need to better understand these models in order to unleash student potential, promote responsible use, and align AI models with educational goals and learning objectives. The objectives of this study were to evaluate the knowledge level and consistency of responses of 2 platforms of ChatGPT, namely GPT-3.5 and GPT-4.0. SAMPLE: A total of 495 multiple-choice and true/false questions from 15 courses used in the assessment of third-year veterinary students at a single veterinary institution were included in this study. METHODS: The questions were manually entered 3 times into each platform, and answers were recorded. These answers were then compared against those provided by the faculty members coordinating the courses. RESULTS: GPT-3.5 achieved an overall performance score of 55%, whereas GPT-4.0 had a significantly (P < .05) greater performance score of 77%. Importantly, the performance scores of both platforms were significantly (P < .05) below that of the veterinary students (86%). CLINICAL RELEVANCE: Findings of this study suggested that veterinary educators and veterinary students retrieving information from these AI-based platforms should do so with caution.

A Randomized, Self-Controlled Case Series Evaluating Core Osteostixis of Osseous Cyst-Like Lesions of the Navicular Bone to Improve Lameness in Horses with Podotrochlear Syndrome.

Introduction: Podotrochlear syndrome is a common cause of lameness in Quarter Horses involving both soft tissue and bony structures within the heel region. Current surgical treatment of podotrochlear syndrome addresses pathological changes affecting the soft tissue structures of the navicular region but does not address either edema or cyst-like lesions of the navicular bone. Objective: The objective of this randomized, self-controlled case series was to determine whether core osteostixis improved lameness in Quarter Horses with podotrochlear syndrome characterized by bilateral magnetic resonance imaging (MRI) findings of osseous cyst-like lesions of the navicular bone. Methods: Seven Quarter Horses that had not responded to standard medical management were included. Each horse had an affected forefoot randomly assigned to surgical treatment with navicular bursoscopy and core osteostixis; the contralateral limb was assigned to navicular bursoscopy only. Video recordings were used to assign lameness scores and make comparisons of each limb at baseline and 24 weeks post-operatively by an observer blinded to the surgical treatment. A second MRI was performed 24 weeks after surgery to reevaluate navicular bone edema, osseous cyst-like lesions of the navicular bone, and tears of the deep digital flexor tendon (DDFT). Results: Reduction of lameness score from baseline was significantly (P = 0.0254) greater for the limbs treated with core osteostixis than limbs treated with bursoscopy. New DDFT tears were noted in 3 of 7 limbs treated with core osteostixis and in 1 of 7 bursoscopy limbs. Conclusion: Results of this study suggest that core osteostixis of the navicular bone combined with navicular bursoscopy can improve lameness in horses with osseous cyst-like lesions. Further evaluation of this technique is warranted.

Perspectives in Professional Education: Reassessing courses required for admission to colleges of veterinary medicine in North America and the Caribbean to decrease stress among first-year students.

OBJECTIVE To identify courses in which first-year veterinary students struggled academically and to survey veterinarians as to their opinions on existing prerequisite courses and proposed alternatives. DESIGN Electronic surveys. SAMPLE Associate deans for academic affairs at colleges of veterinary medicine and practicing veterinarians in North America and the Caribbean. PROCEDURES Surveys were sent to associate deans of academic affairs seeking information on courses in which first-year veterinary students most commonly struggled academically. The 6 courses most commonly listed as prerequisites for admission to veterinary college were identified, and practitioners were asked to rank the relative importance of those courses for preparing students for veterinary college and to rank the importance of 7 potential alternative courses. RESULTS Data were obtained from 21 associate deans and 771 practicing veterinarians. First-year veterinary students most commonly struggled academically in anatomy, physiology, and histology courses, but these courses were rarely included as prerequisites for admission. Practicing veterinarians agreed that anatomy and physiology should be considered as possible alternatives to 1 or more current prerequisite courses, such as organic chemistry and physics. CONCLUSIONS AND CLINICAL RELEVANCE First-year veterinary students commonly encountered academic difficulties in anatomy, physiology, and histology. Because few surveyed veterinary colleges include these courses as prerequisites for admission, many students were exposed to this material for the first time as veterinary students, potentially adding to their academic difficulties and causing stress and anxiety. To help address this situation, veterinary colleges might consider replacing 1 or more current prerequisite courses (eg, organic chemistry and physics) with anatomy, physiology, and histology.

Assessing Academic Self-Efficacy, Knowledge, and Attitudes in Undergraduate Physiology Students.

Academic self-efficacy affects the success of students in the sciences. Our goals were to develop an instrument to assess the self-efficacy and attitudes toward science of students in an undergraduate physiology course. We hypothesized 1) that our instrument would demonstrate that students taking this course would exhibit greater self-efficacy and more positive attitudes toward science than students in a non-science undergraduate course, and 2) that the physiology students' self-efficacy and attitudes would improve after completing the course. A 25-question survey instrument was developed with items investigating demographic information, self-efficacy, content knowledge, confidence, and attitudes regarding science. Students in either an undergraduate physiology course (Group P) or a history course (Group H) completed the survey. Forty-eight students in Group P completed both PRE- and POST-class surveys, while 50 students in Group H completed the pre-class survey. The academic self-efficacy of Group P as assessed by the PRE-survey was significantly higher than Group H (p=0.0003). Interestingly, there was no significant difference between groups in content knowledge in the PRE-survey. The self-efficacy of Group P was significantly higher as assessed by the POST-survey, when compared to the PRE-survey (p<0.0001) coincident with an improvement (p<0.001) in content knowledge for Group P in the POST-survey. This study established a survey instrument with utility in assessing self-efficacy, attitudes, and content knowledge. Our approach has applicability to studies designed to determine the impact of instructional variables on academic self-efficacy, attitudes, and confidence of students in the sciences.

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.

Serial viscoelastic and traditional coagulation testing in horses with gastrointestinal disease.

OBJECTIVE: Objectives of this study were to compare the ability of serial thromboelastography, Sonoclot, and traditional coagulation panels to detect coagulopathies associated with disease category, systemic inflammatory response syndrome (SIRS), complications, and nonsurvival in horses with gastrointestinal disease. DESIGN: Prospective clinical evaluation. SETTING: University referral hospital. ANIMALS: One hundred twenty-one horses admitted as emergencies for gastrointestinal disease and 28 healthy adult horses. INTERVENTION: Blood samples were collected ≤4 times from emergency horses (admission and if surviving and hospitalized on days 2-4) and once from healthy horses. Thromboelastography (with and without tissue factor activation), Sonoclot, and a traditional coagulation panel were performed on each sample. MEASUREMENTS AND MAIN RESULTS: Emergency horses were grouped based on disease category (ie, nonstrangulating medical, nonstrangulating surgical, strangulating, and inflammatory), survival to discharge, SIRS at admission, requirement for exploratory celiotomy, ileus, diarrhea, fever, thrombophlebitis, and laminitis. Changes over time were evaluated individually and compared between disease groups. Horses with gastrointestinal disease had dynamic changes in coagulation and fibrinolysis during the first 4 days of hospitalization that were correlated with disease category, SIRS, complications, and fatality. The multivariate logistic regression model for nonsurvival included activated partial thromboplastin time on day 2 and LY30 on day 3 (overall model significance P < 0.0001). The odds of nonsurvival were 23.75 times higher if activated partial thromboplastin time was >85.6 s on day 2 and 9.38 times higher if LY30 was >1% on day 3. CONCLUSIONS: Horses with gastrointestinal disease have activation of coagulation and fibrinolysis. Magnitude of change in these parameters is small and predictive value moderate, making application of these tests to direct therapy in clinical patients difficult. Effect of specific treatments (eg, surgery) on these tests and coagulation has not been determined. Further studies are required to determine if these tests could be used to help monitor response to treatment in individual animals or specific disease states.

Treatment with aspirin or clopidogrel does not affect equine platelet expression of P selectin or platelet-neutrophil aggregates.

Inflammation-induced P-selectin (CD62P) expression on platelets and endothelial cells facilitates interactions among platelets and polymorphonuclear leukocytes (PMN), and can also promote coagulation. The effects of clopidogrel and aspirin (ASA) on equine platelet CD62P expression were investigated. Six horses were treated in a cross-over design with clopidogrel (2mg/kg PO q 24) or ASA (5mg/kg PO q 24h) for 5 days. Platelets collected at 24, 72, 96, 120, and 168 h after the initiation of therapy were stimulated using 0.1 µg/mL thrombin, followed by flow cytometric analysis using anti-CD41/61 and anti-equine CD62P antibodies. Platelet-PMN aggregates were also enumerated. Baseline CD62P positive platelet numbers were not different between groups (mean ± SD): 4254 ± 1785 (clopidogrel) and 3600 ± 1780 (ASA, P=0. 435). Although expression tended to decrease, there were no significant changes in CD62P+platelets after treatment with either drug (clopidogrel P=0.139, ASA P=0.161). There was also no difference in platelet-PMN aggregates during or after treatment with ASA (P=0.513) or clopidogrel (P=0.543). Due to small numbers of horses, this study may have been underpowered to detect a true decrease in expression, and differences between therapies may have been more pronounced if this study had evaluated horses with systemic inflammation.

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.

Evaluation of the ability of aqueous black walnut extracts to induce the production of reactive oxygen species.

OBJECTIVE: To assess the in vitro capability of aqueous black walnut extracts (BWEs) to generate reactive oxygen species in water-based media ranging in makeup from a simple buffer solution to a complex solution containing serum. SAMPLE: 3 BWEs. PROCEDURES: Production of reactive oxygen species by BWEs prepared in water or N-hexane was tested in PBS solution, PBS solution containing 0.5% bovine serum albumin and 5mM glucose (PBG), and RPMI-1640 medium (RPMI) containing 10% fetal bovine serum or 10% donor horse serum. Reactive oxygen species production was measured as conversion of nonfluorescent dihydrorhodamine 123 by reactive oxygen species to its fluorescent product, rhodamine-123. Hydrogen peroxide was used as a standard for reactive oxygen species activity. RESULTS: BWEs prepared in water generated reactive oxygen species in a dose-dependent manner over a 4-hour period, with peak activity detected when the BWEs were added as 10% (vol/vol) of the RPMI. The BWE prepared in N-hexane generated maximal reactive oxygen species activity after incubation for 3 to 4 hours when added at concentrations ranging from 0.3% to 0.5% (vol/vol) of the RPMI. The BWE prepared in water generated the highest fluorescent signal in PBS solution, whereas the BWE prepared in N-hexane generated the highest fluorescent signal in PBG. CONCLUSIONS AND CLINICAL RELEVANCE: The BWEs prepared in water generated a dose-dependent induction of fluorescence in all the water-based solutions tested. These findings indicated that the BWEs, which are used to induce laminitis in horses, generate reactive oxygen species.

Evaluation of the in vitro effects of aqueous black walnut extract on equine mononuclear cells.

OBJECTIVE: To evaluate effects of black walnut extract (BWE) on equine mononuclear cells and determine whether BWE has direct proinflammatory effects. SAMPLE: Mononuclear cells separated from blood samples from 8 horses. PROCEDURES: Aqueous BWE was prepared and processed to eliminate contamination with particulates and microbes. A Limulus amoebocyte lysate assay was used to detect lipopolysaccharide (LPS) contamination in the BWE. Mononuclear cells were incubated in minimal essential medium with or without the addition of 0.6% to 10% (vol/vol) BWE. These mononuclear cells were assessed for viability, activities of caspases 3 and 7, nitric oxide production, procoagulant activity, and tumor necrosis factor-α production. The effect of LPS on cellular responses induced by BWE was assessed by coincubation with 13 U of polymyxin B/mL; mononuclear cells incubated with LPS were used as a reference. RESULTS: BWE did not cause loss of cell membrane integrity in mononuclear cells but did induce a dose-dependent increase in activities of caspases 3 and 7. Neither BWE nor LPS significantly induced production of nitric oxide. Both BWE and LPS induced comparable amounts of procoagulant activity and tumor necrosis factor-α production; coincubation with polymyxin B reduced the activity for BWE and LPS by 50% and approximately 100%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Addition of BWE induced inflammatory activation of equine mononuclear cells, a portion of which was independent of the effects of LPS. Furthermore, BWE and LPS may work in concert to induce systemic inflammatory responses that contribute to the development of acute laminitis in horses.

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.

Teaching veterinary obstetrics using three-dimensional animation technology.

In this three-year study, test scores for students taught veterinary obstetrics in a classroom setting with either traditional media (photographs, text, and two-dimensional graphical presentations) were compared with those for students taught by incorporating three-dimensional (3D) media (linear animations and interactive QuickTime Virtual Reality models) into the classroom lectures. Incorporation of the 3D animations and interactive models significantly increased students' scores on essay questions designed to assess their comprehension of the subject matter. This approach to education may help to better prepare students for dealing with obstetrical cases during their final clinical year and after graduation.

Differential induction of Toll-like receptor gene expression in equine monocytes activated by Toll-like receptor ligands or TNF-α.

Toll-like receptors (TLRs) function as sentinels for the innate immune system, detecting microbial ligands during infection and inflammation. Previous studies indicate that activation of these receptors on equine monocytes leads to discrete pro- and anti-inflammatory responses that are mediated through the induction of specific cytokine genes. However, less is known regarding the regulation of TLR gene expression in these cells. Therefore, we investigated the effects of ligands recognized by TLR2, 3 or 4 upon TLR2, 3 and 4 gene expression by equine monocytes. We determined that incubation of monocytes with TLR2 and 4 ligands, which signal through the intracellular adaptor protein MyD88, induces expression of the TLR2 and 4 genes, but not the TLR3 gene. Conversely, incubation with a TLR3 ligand, which recruits the TRIF adaptor protein, selectively induces expression of the TLR3 gene, but not TLR2 or 4 genes. Furthermore, incubation of these cells with TNF-α, the pro-inflammatory cytokine that is a hallmark of TLR activation, does not affect expression of the three TLR genes. These findings suggest that exposure of equine monocytes to microbial ligands but not to endogenous inflammatory mediators may initiate responses that alter the horse's sensitivity to other microbial components during infections.

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.

Evaluation of the possible role of prostaglandin F(2 alpha) in laminitis induced in horses by nasogastric administration of black walnut heartwood extract.

OBJECTIVE: To provide insights into the role of prostaglandin F(2 alpha) (PGF(2 alpha)) in the developmental stages of laminitis induced in horses by ingestion of black walnut heartwood extract (BWHE). SAMPLE POPULATION: 10 adult mixed-breed horses. PROCEDURES: Horses were separated into 2 groups and were euthanatized at 12 hours after placebo (water) administration (control horses) or after BWHE administration and development of Obel grade 1 laminitis. Blood samples were obtained to determine plasma PGF(2 alpha) concentrations hourly for the first 4 hours and subsequently every 2 hours after substance administration. Laminar arteries and veins were isolated, and responses to increasing concentrations of PGF(2 alpha) were measured before and after preincubation of blood vessels with prostanoid and thromboxane receptor antagonists SQ 29,548, SC-19220, and AH 6809. RESULTS: Plasma PGF(2 alpha) concentrations increased in horses given BWHE; the WBC count decreased concurrently. In control horses, PGF(2 alpha) was a potent contractile agonist for laminar veins but not for laminar arteries. In horses given BWHE, PGF(2 alpha) was similarly selective for laminar veins; however, the magnitude of PGF(2 alpha)-induced venoconstriction was less than that in control horses. After preincubation with SQ 29,548, laminar veins from control horses responded to PGF(2 alpha) with a small degree of dilation, whereas laminar veins from horses given BWHE did not. CONCLUSIONS AND CLINICAL RELEVANCE: PGF(2 alpha) may play a role in the inflammatory and vascular dysfunction associated with the prodromal stages of laminitis. Prostanoids such as PGF(2 alpha) may be viable targets for the prevention of acute laminitis in horses.

Lipopolysaccharide and TNF-alpha modify adenosine A(2A) receptor expression and function in equine monocytes.

Stimulation of adenosine A(2A) receptors results in anti-inflammatory effects in a variety of cell types. Lipopolysaccharide (LPS) and pro-inflammatory cytokines, such as TNF-alpha and IL-1, have been reported to up-regulate the expression of adenosine A(2A) receptors and thereby enhance the functional activity of adenosine A(2A) receptors in human and murine monocyte/macrophage cell lines and in monocytes/macrophages isolated from those species. In this study, we investigated the effects of LPS and TNF-alpha on the expression and functional activity of adenosine A(2A) receptors in isolated equine peripheral blood monocytes. The results of this study indicate that LPS and TNF-alpha up-regulate the transcription of adenosine A(2A) receptors for up to 24h; the response to LPS was of greater magnitude than the response to TNF-alpha. In this study, incubation with LPS, but not with TNF-alpha, resulted in down-regulation of adenosine A(3) receptor mRNA expression. Furthermore, incubation of these cells with LPS significantly increases the surface density of adenosine A(2A) receptors, and incubation with low concentrations of either LPS or TNF-alpha significantly increases the potency of the adenosine A(2A) receptor agonist, ATL313, to inhibit LPS-induced production of TNF-alpha. These findings suggest that the increased expression of adenosine A(2A) receptors and the enhanced functional potency of adenosine A(2A) receptor agonists after exposure to pro-inflammatory substances such as LPS or TNF-alpha may render adenosine A(2A) receptor agonists particularly important in the treatment of the systemic inflammatory response syndrome that occurs secondary to endotoxemia and bacterial infections in adult horses and neonatal foals.

Equine neutrophil elastase in plasma, laminar tissue, and skin of horses administered black walnut heartwood extract.

Laminitis is a local manifestation of a systemic inflammatory response that is characterized by neutrophil activation and movement of neutrophils into the laminar tissues. Given the evidence for the involvement of neutrophils in the development of laminitis, we measured concentrations of neutrophil elastase, a serine protease released from the azurophilic granules of neutrophils, in plasma, skin and laminar tissues obtained from control horses and horses given black walnut heartwood extract (BWHE) to induce laminitis. Healthy horses (5-15 years old) were randomly assigned to 4 groups: 3 experimental groups given BWHE via nasogastric tube, and a control group given an equal volume of water. The experimental groups consisted of horses euthanized 1.5h (n=5), 3h (n=6) or 12h (n=10) after BWHE administration. Control horses (n=7) were euthanized 12h after intragastric administration of water. Plasma samples were collected in all horses of the control and 12h BWHE groups at 0, 1, 2, 3, 4, 6, 8, 10, and 12h after treatment, and laminar tissue and skin from the middle region of the neck were harvested at the time of euthanasia in all 1.5 and 3h BWHE horses, in 6 of the 12h BWHE horses and in 5 of the control horses. Plasma and tissue concentrations of neutrophil elastase were determined using an equine specific ELISA, and statistical significance was set at p<0.05. Plasma concentrations of neutrophil elastase in the BWHE group were significantly higher at 6 and 8h compared to the control group and at 8 and 10h compared to time 0. Concentrations of neutrophil elastase in skin and laminar tissue were significantly higher in the 3 and 12h BWHE groups compared to the control group. Concentrations of neutrophil elastase were significantly higher in the skin than in the lamina in the 12h BWHE horses. The administration of BWHE thus results in significant increases in the concentration of neutrophil elastase in the circulation, skin and laminar tissue. These results confirm a role for neutrophils in the developmental phase of laminitis, and the systemic nature of the inflammatory process. Furthermore, neutrophil elastase may play a key role in the disintegration of the hoof basal membrane and be a target for the development of new treatments for laminitis.

Innate immune responses of primary murine macrophage-lineage cells and RAW 264.7 cells to ligands of Toll-like receptors 2, 3, and 4.

Although studies have been performed to characterize responses of macrophages from individual anatomical sites (e.g., alveolar macrophages) or of murine-derived macrophage cell lines to microbial ligands, few studies compare these cell types in terms of phenotype and function. We directly compared the expression of cell surface markers and functional responses of primary cultures of three commonly used cells of monocyte-macrophage lineage (splenic macrophages, bone marrow-derived macrophages, and bone marrow-derived dendritic cells) with those of the murine-leukemic monocyte-macrophage cell line, RAW 264.7. We hypothesized that RAW 264.7 cells and primary bone marrow-derived macrophages would be similar in phenotype and would respond similarly to microbial ligands that bind to either Toll-like receptors 2, 3, and 4. Results indicate that RAW 264.7 cells most closely mimic bone marrow-derived macrophages in terms of cell surface receptors and response to microbial ligands that initiate cellular activation via Toll-like receptors 3 and 4. However, caution must be applied when extrapolating findings obtained with RAW 264.7 cells to those of other primary macrophage-lineage cells, primarily because phenotype and function of the former cells may change with continuous culture.

Differential modulation of lipopolysaccharide-induced expression of inflammatory genes in equine monocytes through activation of adenosine A2A receptors.

Adenosine is an endogenous nucleoside that has potent receptor-mediated immunomodulatory effects on macrophage/monocyte function. In this study, we determined the effects of an adenosine A(2A) receptor agonist, ATL313, on the expression of mRNAs for four pro-inflammatory mediators, IL-1beta, IL-8, COX-2, and TNF-alpha, and the mRNA and protein for the anti-inflammatory cytokine, IL-10 in equine monocytes incubated with lipopolysaccharide (LPS). The results indicate that ATL313 significantly reduces LPS-induced expression of COX-2 and TNF-alpha, enhances the expression of IL-10 and IL-8, but does not alter the expression of IL-1beta. These effects of ATL313 were reversed by co-incubation with the selective adenosine A(2A) antagonist ZM241385, and were mimicked by the cAMP analogue dibutyryl cAMP. These differential effects of adenosine A(2A) receptor activation were in contrast to those obtained using the P38 MAPK inhibitor, SB203580, which nearly abolished all LPS-induced changes in mRNA expression as well as the production of TNF-alpha protein. These findings, which indicate that adenosine A(2A) receptor activation modulates the transcription of several, but not all, pro-inflammatory mediators and exerts a synergistic effect on the induction of at least one anti-inflammatory cytokine, suggest that selective adenosine A(2A) agonists may reduce the early pro-inflammatory effects of endotoxemia in horses.