Feasibility of hemoperfusion using extracorporeal therapy in the horse.

Objective: Develop, implement, and monitor for adverse effects of, a novel hemoperfusion therapy in adult horses. Methods: A prospective, observational feasibility study using three healthy adult horses from the North Carolina State University teaching herd. Health status was determined by physical exam, complete blood count, coagulation panel, and serum biochemistry. Each horse was instrumented with a 14 Fr × 25 cm double-lumen temporary hemodialysis catheter and underwent a 240 min polymer-based hemoperfusion session. Horses were administered unfractionated heparin to maintain anti-coagulation during the session. Given the novelty of this therapy in horses, each horse was treated as a learning opportunity that informed an iterative process of protocol development and modification. Measurements and main results: Our long-term goal is to investigate potential clinical applications of hemoperfusion in horses, including cytokine reduction in horses with severe SIRS/sepsis. Horses were monitored for changes in clinical exam, biochemistry and hematology parameters. Additionally, cytokines were quantified to determine whether extracorporeal hemadsorption therapy alone caused an inflammatory response. Our results show that hemoperfusion therapy was associated with decreased platelet counts and serum albumin concentration. There was no significant change in plasma cytokine concentrations with hemoperfusion therapy. In one horse, the cytokine concentrations decreased, as previously reported with hemoperfusion therapy in humans. Hypothesis: We hypothesized that hemoperfusion therapy could be performed in healthy adult horses without significant adverse effects. Conclusion: Polymer-based hemoperfusion is a feasible extracorporeal therapy (ECT) modality for adult horses. Additional studies are needed to further establish clinical protocols, as well as establish efficacy of polymer-based hemoperfusion for treatment of various conditions in horses, including intoxications, immune-mediated conditions, and sepsis.

Opinions regarding equine veterinarian attire and attributes: results of a horse owner survey.

OBJECTIVE: To investigate potential equine clients' perceptions of equine veterinarians based on attire. SAMPLE: 763 horse owners/lessees. METHODS: Participants were invited to complete a survey shared mainly via equestrian social media pages between August and October 2022.1-3 Survey participants were shown pictures of a male veterinarian and a female veterinarian in 7 outfits ranging from casual to business attire and were asked to score the veterinarian on 7 traits: easygoing attitude, friendliness, compassion, trustworthiness, professionalism, competence, and cost of services. The survey asked which of the traits were most valued in an equine veterinarian, as well as whether various aspects of appearance including tattoos, piercings, and hair dyed a nonorganic color were acceptable for equine veterinarians. RESULTS: Of the 2,655 individuals who opened the survey, 763 responses were included. Respondents were predominantly female (743/763 [97.4%]) from rural areas (493/763 [64.6%]). Only 37.1% (283/763) of respondents agreed that what a veterinarian wears influences their confidence in them. The highest-ranked traits in an equine veterinarian were knowledge/competency (mean ± SD, 1.46 ± 0.98), followed by trustworthiness (2.34 ± 1.08) and compassion (3.50 ± 1.20), with coveralls and scrubs being the preferred attire clients associated with these attributes (with the exception of compassion, for which polo shirt/jeans was the preferred attire). T-shirt/jeans was consistently ranked lowest by respondents in association with these attributes, except in the area of compassion, where polo shirt/black pants was ranked lowest. CLINICAL RELEVANCE: Our findings suggested the attire and appearance of equine veterinarians can impact client perceptions, with veterinarians wearing scrubs and coveralls associated with higher competency and trustworthiness.

Hemadsorption extracorporeal therapy removes cytokines ex vivo in horses.

OBJECTIVE: Plasma cytokine adsorption has shown benefit as an adjunctive therapy in human sepsis but has yet to be investigated in horses. We hypothesized that ex vivo filtration of equine plasma with a novel cytokine adsorption device would significantly reduce concentrations of lipopolysaccharide-stimulated cytokines. We also hypothesized that the device would adsorb medications commonly used to treat sepsis. ANIMALS: 8 horses owned by North Carolina State University. METHODS: Four liters of heparinized whole blood was collected from healthy adult horses (n = 8) and stimulated with lipopolysaccharide (100 ng/mL) for 6 hours (37 °C.) from June 4, 2023, to December 15, 2023. Plasma was filtered through a cytokine adsorption device or sham circuit. Samples were collected at 11 time points for multiplex cytokine analysis. Chemistry analysis was performed before and after filtration. To investigate the impact of the device on medication concentrations, equine plasma containing potassium penicillin, gentamicin, and flunixin meglumine was filtered through the cytokine adsorption device or sham for 6 hours. Drug concentrations before and after filtration were determined by ultra-high-performance liquid chromatography. Prefiltration versus postfiltration sample concentrations were analyzed by Student paired t test using GraphPad Prism 9.0 (P < .05). RESULTS: Filtration of lipopolysaccharide-stimulated equine plasma (n = 8) for 6 hours resulted in significant mean reductions in the cytokines IL-10, IL-5, IL-8, tumor necrosis factor-α (TNF-α), and IL-1ß, as well as albumin. Drug concentrations of potassium penicillin, gentamicin, and flunixin meglumine were also significantly reduced by filtration. CLINICAL RELEVANCE: This work provides proof of concept for further investigation of extracorporeal cytokine adsorption as a potential adjunct treatment for equine sepsis.

MARCKS Inhibition Alters Bovine Neutrophil Responses to Salmonella Typhimurium.

Neutrophils are innate immune cells that respond quickly to sites of bacterial infection and play an essential role in host defense. Interestingly, some bacterial pathogens benefit from exuberant neutrophil inflammation. Salmonella is one such pathogen that can utilize the toxic mediators released by neutrophils to colonize the intestine and cause enterocolitis. Because neutrophils can aid gut colonization during Salmonella infection, neutrophils represent a potential host-directed therapeutic target. Myristoylated alanine-rich C-kinase substrate (MARCKS) is an actin-binding protein that plays an essential role in many neutrophil effector responses. We hypothesized that inhibition of MARCKS protein would alter bovine neutrophil responses to Salmonella Typhimurium (STm) ex vivo. We used a MARCKS inhibitor peptide to investigate the role of MARCKS in neutrophil responses to STm. This study demonstrates that MARCKS inhibition attenuated STm-induced neutrophil adhesion and chemotaxis. Interestingly, MARCKS inhibition also enhanced neutrophil phagocytosis and respiratory burst in response to STm. This is the first report describing the role of MARCKS protein in neutrophil antibacterial responses.

A myristoylated alanine-rich C-kinase substrate (MARCKS) inhibitor peptide attenuates neutrophil outside-in ß2-integrin activation and signaling.

MARCKS is an actin and PIP2-binding protein that plays an essential role in neutrophil migration and adhesion; however, the molecular details regarding MARCKS function in these processes remains unclear. Neutrophil adhesion and migration also require the cell surface receptors ß2-integrins. We hypothesized that MARCKS inhibition would alter neutrophil ß2-integrin activation and signaling. We utilized a MARCKS-targeting peptide to inhibit MARCKS in inside-out and outside-in ß2-integrin activation in neutrophils. MANS-mediated MARCKS inhibition had no significant effect on inside-out ß2-integrin activation. MANS treatment significantly attenuated ICAM-1/Mn2+-stimulated static adhesion, cell spreading and ß2-integrin clustering, suggesting a role for MARCKS function in outside-in ß2-integrin activation. Additional work is needed to better understand the molecular mechanisms of MARCKS role in outside-in ß2-integrin activation and signaling.

Targeting Neutrophil ß2-Integrins: A Review of Relevant Resources, Tools, and Methods.

Neutrophils are important innate immune cells that respond during inflammation and infection. These migratory cells utilize ß2-integrin cell surface receptors to move out of the vasculature into inflamed tissues and to perform various anti-inflammatory responses. Although critical for fighting off infection, neutrophil responses can also become dysregulated and contribute to disease pathophysiology. In order to limit neutrophil-mediated damage, investigators have focused on ß2-integrins as potential therapeutic targets, but so far these strategies have failed in clinical trials. As the field continues to move forward, a better understanding of ß2-integrin function and signaling will aid the design of future therapeutics. Here, we provide a detailed review of resources, tools, experimental methods, and in vivo models that have been and will continue to be utilized to investigate the vitally important cell surface receptors, neutrophil ß2-integrins.

Asthma: The Use of Animal Models and Their Translational Utility.

Asthma is characterized by chronic lower airway inflammation that results in airway remodeling, which can lead to a permanent decrease in lung function. The pathophysiology driving the development of asthma is complex and heterogenous. Animal models have been and continue to be essential for the discovery of molecular pathways driving the pathophysiology of asthma and novel therapeutic approaches. Animal models of asthma may be induced or naturally occurring. Species used to study asthma include mouse, rat, guinea pig, cat, dog, sheep, horse, and nonhuman primate. Some of the aspects to consider when evaluating any of these asthma models are cost, labor, reagent availability, regulatory burden, relevance to natural disease in humans, type of lower airway inflammation, biological samples available for testing, and ultimately whether the model can answer the research question(s). This review aims to discuss the animal models most available for asthma investigation, with an emphasis on describing the inciting antigen/allergen, inflammatory response induced, and its translation to human asthma.

Legacy and emerging per- and polyfluoroalkyl substances suppress the neutrophil respiratory burst.

Per- and polyfluoroalkyl substances (PFASs) are used in a multitude of processes and products, including nonstick coatings, food wrappers, and fire-fighting foams. These chemicals are environmentally-persistent, ubiquitous, and can be detected in the serum of 98% of Americans. Despite evidence that PFASs alter adaptive immunity, few studies have investigated their effects on innate immunity. The report here presents results of studies that investigated the impact of nine environmentally-relevant PFASs [e.g. perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid potassium salt (PFOS-K), perfluorononanoic acid (PFNA), perfluorohexanoic acid (PFHxA), perfluorohexane sulfonic acid (PFHxS), perfluorobutane sulfonic acid (PFBS), ammonium perfluoro(2-methyl-3-oxahexanoate) (GenX), 7H-perfluoro-4-methyl-3,6-dioxa-octane sulfonic acid (Nafion byproduct 2), and perfluoromethoxyacetic acid sodium salt (PFMOAA-Na)] on one component of the innate immune response, the neutrophil respiratory burst. The respiratory burst is a key innate immune process by which microbicidal reactive oxygen species (ROS) are rapidly induced by neutrophils in response to pathogens; defects in the respiratory burst can increase susceptibility to infection. The study here utilized larval zebrafish, a human neutrophil-like cell line, and primary human neutrophils to ascertain whether PFAS exposure inhibits ROS production in the respiratory burst. It was observed that exposure to PFHxA and GenX suppresses the respiratory burst in zebrafish larvae and a human neutrophil-like cell line. GenX also suppressed the respiratory burst in primary human neutrophils. This report is the first to demonstrate that these PFASs suppress neutrophil function and support the utility of employing zebrafish larvae and a human cell line as screening tools to identify chemicals that may suppress human immune function.

Withaferin A Inhibits Neutrophil Adhesion, Migration, and Respiratory Burst and Promotes Timely Neutrophil Apoptosis.

Neutrophils play a major role in many equine conditions, including equine asthma, laminitis, and intestinal ischemia and reperfusion injury, and therefore represent an attractive target for innovative therapeutic approaches. Novel strategies for reducing neutrophilic inflammation include modulation of neutrophil functions and lifespan. Withaferin A (WFA) is a phytochemical with well-established in vitro and in vivo anti-inflammatory properties, but its direct effects on neutrophils are largely unknown. We hypothesized that WFA would inhibit adhesion, migration, and respiratory burst by equine neutrophils and promote timely apoptosis of primed equine neutrophils. Consistent with this hypothesis, our data show that WFA causes a significant, concentration-dependent inhibition of equine neutrophil adhesion, migration, and respiratory burst in response to diverse stimuli. Further, WFA treatment increased apoptosis of equine neutrophils exposed to GM-CSF for 24 h. This pro-apoptotic effect of WFA was not observed in unprimed neutrophils, nor at the 2-h time point relevant to our functional neutrophil experiments. Our data demonstrate that WFA may reduce neutrophil-mediated inflammation through multiple mechanisms, including suppression of inflammatory responses and promotion of apoptosis. Additional research is needed to elucidate the molecular mechanisms for these effects and evaluate the potential clinical use of WFA in veterinary and human patients.

Investigation of plasma cell-free DNA as a potential biomarker in horses.

Plasma cell-free DNA (cfDNA) is a biomarker of ischemia, systemic inflammation, and mortality in humans with gastrointestinal disease. Cell-free DNA has not been investigated as a biomarker for equine colic, to our knowledge. We hypothesized that cfDNA could be measured accurately in neat equine plasma using a benchtop fluorometer and that plasma cfDNA would be elevated in emergency patients compared to healthy horses. Plasma was obtained from blood collected in Roche DNA stabilizing tubes. We used the Qubit 4 fluorometer and 1× dsDNA HS assay kit to measure cfDNA concentration in neat patient plasma and following DNA extraction of plasma with a commercial kit. Assay precision and linearity of dilution were satisfactory for neat plasma cfDNA, but DNA spike and recovery results were variable. Further, cfDNA concentrations in paired neat plasma and extracted-plasma samples (n = 66) were not correlated. Median extracted-plasma cfDNA was higher in emergency patients (n = 50) and a subgroup of colic patients (n = 36), compared to healthy horses (n = 19). Our results with extracted-plasma samples provide proof of concept for further investigation of plasma cfDNA as a biomarker in horses.

Development and Formative Evaluation of a Low-Fidelity Equine Castration Model for Veterinary Education.

Entrustable Professional Activities (EPAs) are units of activity that early-stage professionals perform in the workplace that necessitate simultaneous integration of multiple competencies. EPA #6 requires students to perform a common surgical procedure on a stable patient, including pre-operative and post-operative management. Castration is one of the most common surgeries performed by equine primary care practitioners and is considered an "entry-level competency" for veterinary graduates entering equine private practice, however, to our knowledge there are no equine castration models available for veterinary student education. Therefore, we developed an inexpensive, low-fidelity model of equine field castration and evaluated it using a mixed-methods approach. Two different groups of students, with or without model experience, completed surveys before and after live horse castration. Students who used the model also completed model specific surveys. Videos of the students completing the model were evaluated by at least two different equine veterinary faculty using a 15-point rubric, and inter-rater reliability of the rubric was determined. After completing the model, students reflected on strengths and weaknesses of their performance. From our student survey results, we determined that student attitudes toward the model were mostly positive. Interestingly, there were several student attitudes toward the model that became significantly more favorable after live horse castration. Prior to live horse castration, there was no significant difference in confidence in model vs. no-model groups. Following live horse castration, students who used the model had higher confidence in procedure preparation and hand-ties than students who did not use the model, but they had lower scores for confidence during patient recovery. When reflecting on model castration, students most commonly cited preparation and surgical description as strengths, and ligature placement and hand-ties as weaknesses. Experts provided several suggestions to improve the model, including incorporation of emasculators and the need for better model stabilization. Our findings suggest that both students and veterinary educators feel that this low-fidelity model has educational value. Rubric performance metrics were favorable, but additional steps are needed to improve grading consistency among educators. Future research will determine whether student performance on the model is predictive of competence score during live-horse castration.

Differential gene expression and Ingenuity Pathway Analysis of bronchoalveolar lavage cells from horses with mild/moderate neutrophilic or mastocytic inflammation on BAL cytology.

Mild to moderate equine asthma syndrome (mEAS) affects horses of all ages and breeds. To date, the etiology and pathophysiology of mEAS are active areas of research, and it remains incompletely understood whether mEAS horses with different immune cell 'signatures' on BAL cytology represent different phenotypes, distinct pathobiological mechanisms (endotypes), varied environmental conditions, disease severity, genetic predispositions, or all of the above. In this descriptive study, we compared gene expression data from BAL cells isolated from horses with normal BALF cytology (n = 5), to those isolated from horses with mild/moderate neutrophilic inflammation (n = 5), or mild/moderate mastocytic inflammation (n = 5). BAL cell protein lysates were analyzed for cytokine/chemokine levels using Multiplex Bead Immunoassay, and for select proteins using immunoblot. The transcriptome, determined by RNA-seq and analyzed with DEseq2, contained 20, 63, and 102 significantly differentially expressed genes in horses with normal vs. neutrophilic, normal vs. mastocytic, and neutrophilic vs. mastocytic BALF cytology, respectively. Pathway analyses revealed that BAL-isolated cells from horses with neutrophilic vs. normal cytology showed enrichment in inflammation pathways, and horses with mastocytic vs. normal cytology showed enrichment in pathways involved in fibrosis and allergic reaction. BAL cells from horses with mastocytic mEAS, compared to neutrophilic mEAS, showed enrichment in pathways involved in alteration of tissue structures. Cytokine analysis determined that IL-1ß was significantly different in the lysates from horses with neutrophilic inflammation compared to those with normal or mastocytic BAL cytology. Immunoblot revealed significant difference in the relative level of MMP2 in horses with neutrophilic vs. mastocytic mEAS. Upregulation of mRNA transcripts involved in the IL-1 family cytokine signaling axis (IL1a, IL1b, and IL1R2) in neutrophilic mEAS, as well as KIT mRNA in mastocytic mEAS, are novel, potentially clinically relevant, findings of this study. These findings further inform our understanding of inflammatory cell subtypes in mEAS.

The Role of Neutrophils in the Pathophysiology of Asthma in Humans and Horses.

Asthma is a common and debilitating chronic airway disease that affects people and horses of all ages worldwide. While asthma in humans most commonly involves an excessive type 2 immune response and eosinophilic inflammation, neutrophils have also been recognized as key players in the pathophysiology of asthma, including in the severe asthma phenotype where neutrophilic inflammation predominates. Severe equine asthma syndrome (sEAS) features prominent neutrophilic inflammation and has been increasingly used as a naturally occurring animal model for the study of human neutrophilic asthma. This comparative review examines the recent literature in order to explore the role of neutrophil inflammatory functions in the pathophysiology and immunology of asthma in humans and horses.

Comparative Review of Asthma in Farmers and Horses.

PURPOSE OF REVIEW: Farmers are routinely exposed to organic dusts and aeroallergens that can have adverse respiratory health effects including asthma. Horses are farm-reared large animals with similar exposures and can develop equine asthma syndrome (EAS). This review aims to compare the etiology, pathophysiology, and immunology of asthma in horses compared to farmers and highlights the horse as a potential translational animal model for organic dust-induced asthma in humans. RECENT FINDINGS: Severe EAS shares many clinical and pathological features with various phenotypes of human asthma including allergic, non-allergic, late onset, and severe asthma. EAS disease features include variable airflow obstruction, cough, airway hyperresponsiveness, airway inflammation/remodeling, neutrophilic infiltrates, excess mucus production, and chronic innate immune activation. Severe EAS is a naturally occurring and biologically relevant, translational animal disease model that could contribute to a more thorough understanding of the environmental and immunologic factors contributing to organic dust-induced asthma in humans.

A Suspected Case of Rocky Mountain Spotted Fever in an Adult Horse in the Southeastern United States.

A 20-year-old Paint gelding was evaluated for fever of unknown origin. History and clinical signs were consistent with potential tick-borne disease. Samples were collected and submitted for tick-borne disease panel, herpes virus, complete blood count, and serum biochemistry. Based on physical examination findings and vaccination history, the gelding was treated for suspected tick-borne disease with oxytetracycline (8 mg/kg intravenously BID) for 5 days, followed by doxycycline (10 mg/kg PO BID) for an additional 5 days. Although titers to Borrelia burgdorferi, Anaplasma phagocytophilum, and Neorickettsia risticii on Days 4 and 8 were negative, the Rickettsia rickettsii titer went from 1:1,600 on Day 4 to 1:800 on Day 8, 1:100 on Day 21, and was seronegative by Day 38. Although complete blood polymerase chain reaction for Rickettsia rickettsii was negative, the clinical and serologic features of this case are extremely consistent with clinical cases of Rocky Mountain Spotted Fever (RMSF) described in both dogs and humans. Therefore, we submit this case report to document suspected clinical infection of an adult horse in the southeastern United States with Rickettsia rickettsii, the causative agent of RMSF. Other relevant differentials (i.e., Rickettsia parkeri, Theileria equi, and Babesia caballi) are also discussed.

A Comparative Review of Equine SIRS, Sepsis, and Neutrophils.

The most recent definition of sepsis in human medicine can be summarized as organ dysfunction caused by a dysregulated host response to infection. In equine medicine, although no consensus definition is available, sepsis is commonly described as a dysregulated host systemic inflammatory response to infection. Defense against host infection is the primary role of innate immune cells known as neutrophils. Neutrophils also contribute to host injury during sepsis, making them important potential targets for sepsis prevention, diagnosis, and treatment. This review will present both historical and updated perspectives on the systemic inflammatory response (SIRS) and sepsis; it will also discuss the impact of sepsis on neutrophils, and the impact of neutrophils during sepsis. Future identification of clinically relevant sepsis diagnosis and therapy depends on a more thorough understanding of disease pathogenesis across species. To gain this understanding, there is a critical need for research that utilizes a clearly defined, and consistently applied, classification system for patients diagnosed with, and at risk of developing, sepsis.

Analysis of Final Year Veterinary Students' Telephone Communication Skills at a Veterinary Teaching Hospital.

Client communication is a core clinical skill that is taught as part of the required curriculum at many veterinary colleges. Although much client communication occurs face-to-face, telephone communication is used to provide patient updates, relay results of diagnostic tests, and check on discharged patients. This research explored fourth year veterinary medical students' telephone communication skills. We recorded and analyzed the transcripts of 25 calls students made to clients of three different services in the Veterinary Teaching Hospital. Additionally, we explored the perspectives of veterinary educators by distributing a survey to university faculty and house officers (n = 57). Results indicate that students excelled at identifying the patient and purpose of the call and incorporating professional language and clear explanations. They require development in providing structure and incorporating core communication skills. Compared with our survey results, the student findings are at odds with clinicians' expectations of students' communication abilities. We conclude that additional training is required to familiarize students with expectations regarding telephone communication, including reviewing the case thoroughly, preparing to answer questions and provide explanations, following organizational protocol, and incorporating open ended questions, reflective listening, and empathy. This data will inform design, and help to measure the impact, of telephone communication education and training that will be incorporated into the existing veterinary communication curriculum.