Targeted transcriptomic analysis of synovial tissues from horses with septic arthritis treated with immune-activated mesenchymal stromal cells reveals induction of T-cell response pathways.

OBJECTIVE: To investigate mechanistically the reported beneficial effects of immune-activated mesenchymal stromal cell (MSC) therapy to treat equine septic arthritis, leveraging Nanostring technology. ANIMALS: 8 Quarter Horses with induced tibiotarsal Staphylococcus aureus septic arthritis treated IA with either Toll-like receptor-3 agonist polyinosinic:polycytidylic acid-activated MSCs + vancomycin antimicrobials (TLR-MSC-VAN; n = 4) or antimicrobials (VAN; 4). METHODS: Synovial tissues were collected and fixed in neutral-buffered 10% formalin, and formalin-fixed paraffin-embedded synovial and osteochondral tissues were sequenced using a custom-designed 200-gene equine Nanostring nCounter immune panel to directly quantify expression of key immune and cartilage-related genes. Immunohistochemistry to detect CD3+ T cells was performed on synovial tissues to further quantify T-cell infiltration in TLR-MSC-VAN- versus VAN-treated joints. RESULTS: Comparison of synovial transcriptomes between groups revealed moderate changes in differential gene expression, with upregulated expression of 9 genes and downregulated expression of 17 genes with fold change ≥ 2 or ≤ -2 and a significant false discovery rate-adjusted P value of ≤ .05. The most upregulated genes in TLR-MSC-VAN-treated horses included those related to T-lymphocyte recruitment and function, while pathways related to innate immune activation and inflammation were significantly downregulated. Immunohistochemistry and quantitation of CD3+ T-cell infiltrates revealed a numerically greater infiltrate in synovial tissues of TLR-MSC-VAN-treated horses, which did not reach statistical significance in this small sample set (P = .20). CLINICAL RELEVANCE: Targeted transcriptomic analyses using an equine Nanostring immune and cartilage health panel provided new mechanistic insights into how innate and adaptive immune cells within synovial tissues respond to TLR-activated MSC treatment when used to treat septic arthritis.

Erythrocyte removal from bone marrow aspirate concentrate improves efficacy as intra-articular cellular therapy in a rodent osteoarthritis model.

Background: Despite the high prevalence of osteoarthritis (OA), there remains a need for additional therapeutic options. Cellular therapies with minimally manipulated cells such as bone marrow aspirate concentrates (BMAC) are increasingly popular in the U.S. but clear-cut evidence of efficacy has not been established. In theory, BMAC injections provide a source of stromal cells to stimulate healing in OA and ligamentous injuries; however, BMAC injections are also often associated with inflammation, short-term pain, and mobility impairment. Given that blood is known to trigger inflammation in joints, we hypothesized that removing erythrocytes [red blood cells (RBCs)] from BMAC preparations prior to intra-articular injection would improve efficacy for OA treatment. Methods: To test this hypothesis, BMAC was collected from the bone marrow of mice. Three treatment groups were pursued: (I) untreated; (II) BMAC; or (III) BMAC depleted of RBCs by lysis. Product was injected into the femorotibial joint of mice 7 days after OA had been induced by destabilization of the medial meniscus (DMM). To assess the impact of treatment on joint function, individual cage monitoring (ANY-mazeTM) and Digigait treadmill-based analyses were performed over 4 weeks. At study completion, joint histopathology was assessed and immune transcriptomes within joint tissues were compared using a species-specific NanoString panel. Results: Significant improvements in activity, gait parameters, and histology scores were seen in animals receiving RBC-depleted BMAC compared to untreated mice; animals treated with non-depleted BMAC did not demonstrate this same extent of consistent significant improvement. Transcriptomic analysis of joint tissues revealed significant upregulation of key anti-inflammatory genes, including interleukin-1 receptor antagonist (IRAP), in mice treated with RBC-depleted BMAC compared to animals treated with non-RBC depleted BMAC. Conclusions: These findings indicate that RBC depletion in BMAC prior to intra-articular injection improves treatment efficacy and reduces joint inflammation compared to BMAC.

Antimicrobial Properties of Equine Stromal Cells and Platelets and Future Directions.

Increasing antimicrobial resistance in veterinary practice has driven the investigation of novel therapeutic strategies including regenerative and biologic therapies to treat bacterial infection. Integration of biological approaches such as platelet lysate and mesenchymal stromal cell (MSC) therapy may represent adjunctive treatment strategies for bacterial infections that minimize systemic side effects and local tissue toxicity associated with traditional antibiotics and that are not subject to antibiotic resistance. In this review, we will discuss mechanisms by which biological therapies exert antimicrobial effects, as well as potential applications and challenges in clinical implementation in equine practice.

Hoof Unevenness in Juvenile Quarter Horses During First 6 Months of Training.

The role of hoof morphology is increasingly recognized as a factor associated with lameness incidence in performance horses. The primary objective was to evaluate effect of training initiation on hoof unevenness in Quarter Horses (n = 42; 29 2-year-olds, 13 3-year-olds) over 6-months (m) in training (m0, m2, m4, and m6). Horses were objectively assessed for lameness (inertial sensor system) and photographs and radiographs of feet were taken. Hoof measurements were taken (palmar/plantar angles, frog base width/length, toe length/angle, heel length/angle, heel/foot width, wall height/angle), and analyzed with regards to laterality. Front and hindfoot pairs were determined even if toe angles were within 1.5°. Statistical analyses were performed (Fisher's exact test, mixed-model linear regression, P < .05). There were no differences in distal phalanx palmar/plantar angle between lame/nonlame forelimbs (P = .54) or hindlimbs (P = .20). Unevenness between front feet was seen in toe angle m6 (P < .001), heel length m6 (P = .01) and heel angle over time (P = .006). Unevenness between hind feet was seen at m6 in toe angle (P < .001), heel length (P = .009) and heel angle (P = .02). Lameness incidence did not differ between even and uneven footed horses in forelimbs (P = .64) or hindlimbs (P = .09). In uneven feet, there was no difference in lameness between high versus low foot in forelimbs (P = .34) or hindlimbs (P = .29). Limitations include lack of control group not entering training, lack of consistency in timing data collection to previous trimming, and small sample size. In summary, differences in foot measurements and laterality were noted over time following training initiation in juvenile Western performance horses.

Distinct differences in immunological properties of equine orthobiologics revealed by functional and transcriptomic analysis using an activated macrophage readout system.

Introduction: Multiple biological therapies for orthopedic injuries are marketed to veterinarians, despite a lack of rigorous comparative biological activity data to guide informed decisions in selecting a most effective compound. Therefore, the goal of this study was to use relevant bioassay systems to directly compare the anti-inflammatory and immunomodulatory activity of three commonly used orthobiological therapies (OTs): mesenchymal stromal cells (MSC), autologous conditioned serum (ACS), and platelet rich plasma (PRP). Methods: Equine monocyte-derived macrophages were used as the readout system to compare therapies, including cytokine production and transcriptomic responses. Macrophages were stimulated with IL-1ß and treated 24 h with OTs, washed and cultured an additional 24 h to generate supernatants. Secreted cytokines were measured by multiplex immunoassay and ELISA. To assess global transcriptomic responses to treatments, RNA was extracted from macrophages and subjected to full RNA sequencing, using an Illumina-based platform. Data analysis included comparison of differentially expressed genes and pathway analysis in treated vs. untreated macrophages. Results: All treatments reduced production of IL-1ß by macrophages. Secretion of IL-10 was highest in MSC-CM treated macrophages, while PRP lysate and ACS resulted in greater downregulation of IL-6 and IP-10. Transcriptomic analysis revealed that ACS triggered multiple inflammatory response pathways in macrophages based on GSEA, while MSC generated significant downregulation of inflammatory pathways, and PRP lysate induced a mixed immune response profile. Key downregulated genes in MSC-treated cultures included type 1 and type 2 interferon response, TNF-α and IL-6. PRP lysate cultures demonstrated downregulation of inflammation-related genes IL-1RA, SLAMF9, ENSECAG00000022247 but concurrent upregulation of TNF-α, IL-2 signaling, and Myc targets. ACS induced upregulation of inflammatory IL-2 signaling, TNFα and KRAS signaling and hypoxia, but downregulation of MTOR signaling and type 1 interferon signaling. Discussion: These findings, representing the first comprehensive look at immune response pathways for popular equine OTs, reveal distinct differences between therapies. These studies address a critical gap in our understanding of the relative immunomodulatory properties of regenerative therapies commonly used in equine practice to treat musculoskeletal disease and will serve as a platform from which further in vivo comparisons may build.

Successful surgical removal of a pheochromocytoma in a mare via trans-costal approach.

BACKGROUND: Pheochromocytomas have been previously reported in horses, but successful antemortem diagnosis and surgical removal without recurrence of clinical signs have not been described. OBJECTIVE: To report the clinical presentation, diagnostic evaluation, surgical technique, anaesthetic management and post-operative care of a mare diagnosed with pheochromocytoma. STUDY DESIGN: Clinical case report. METHODS: An 18-year-old Quarter Horse mare presented for recurrent episodes of colic, profuse sweating, muscle fasciculations and agitation over a 2-month period. Clinical, clinicopathologic and ultrasonographic (transcutaneous, transrectal) abnormalities were consistent with a unilateral left-sided adrenal mass. Surgical removal of the mass was performed via a trans-costal approach with removal of the 18th rib and retraction of the left kidney to improve exposure. Associated vasculature was ligated, and the adrenal mass was removed and submitted for histopathology and immunohistochemistry. RESULTS: A trans-costal surgical approach provided excellent visualisation of the adrenal mass and allowed for identification and ligation of associated vessels. Total surgical and anaesthesia time were 86 and 114 min, respectively. Several intraoperative (hypertension, tachycardia) and post-operative (colic with tachycardia, tachypnea, large colon pelvic flexure impaction and nasogastric reflux) complications were encountered and managed successfully. Immunohistochemistry demonstrated positive labelling for synaptophysin and chromogranin A, confirming diagnosis of pheochromocytoma. The mare had recovered well at 6-week recheck post-operatively and returned to training at 6 months post-operatively. No further clinical signs consistent with pheochromocytoma have been observed following removal. CONCLUSIONS: The trans-costal approach allowed for surgical removal of a pheochromocytoma in a mare. Surgical removal of adrenal masses in horses may be associated with complications yet was successfully performed without subsequent recurrence of clinical signs associated with tumour presence and return to athletic use in this mare.

Novel ocular immunotherapy induces tumor regression in an equine model of ocular surface squamous neoplasia.

Ocular surface squamous neoplasia (OSSN) is the major cause of corneal cancer in man and horses worldwide, and the prevalence of OSSN is increasing due to greater UVB exposure globally. Currently, there are no approved treatments for OSSN in either species, and most patients are managed with surgical excision or off-label treatment with locally injected interferon alpha, or topically applied cytotoxic drugs such as mitomycin C. A more broadly effective and readily applied immunotherapy could exert a significant impact on management of OSSN worldwide. We therefore evaluated the effectiveness of a liposomal TLR complex (LTC) immunotherapy, which previously demonstrated strong antiviral activity in multiple animal models following mucosal application, for ocular antitumor activity in a horse spontaneous OSSN model. In vitro studies demonstrated strong activation of interferon responses in horse leukocytes by LTC and suppression of OSSN cell growth and migration. In a trial of 8 horses (9 eyes), treatment with topical or perilesional LTC resulted in an overall tumor response rate of 67%, including durable regression of large OSSN tumors. Repeated treatment with LTC ocular immunotherapy was also very well tolerated clinically. We conclude therefore that ocular immunotherapy with LTC warrants further investigation as a novel approach to management of OSSN in humans.

Liposomal bupivacaine is both safe and effective when administered via local infiltration at surgical site and mesovarium for laparoscopic ovariectomy in mares.

BACKGROUND: Liposomal local anaesthetic solutions may provide extended-duration analgesia postoperatively but have not been assessed following intra-peritoneal local infiltration in any species. OBJECTIVES: To evaluate two doses of 1.33% liposomal bupivacaine (LB) versus 0.75% bupivacaine HCL (BHCl) for analgesia following laparoscopic ovariectomy in mares. STUDY DESIGN: Prospective cohort study. METHODS: Fifteen healthy Quarter Horse mares (age 2-20 years) with normal bilateral ovarian palpation and appearance were enrolled. Horses were restrained in standing stocks and administered an α-2 agonist, butorphanol, and flunixin meglumine, followed by a variable rate infusion of sedation with α-2 agonists. Bilateral paralumbar fossa ovariectomies were performed. Treatment with either 30 ml 0.75% BHCl followed by 20 or 40 ml LB 13.3% (LB20 and LB40) volume expanded with saline to 80 ml total (n = 6/group) or 80 ml BHCl alone (n = 3, BCHL) was infused around incision sites and each mesovarium (LB or BHCl) prior to ovariectomy. Horses were monitored for 72 h by physical examination, algometry, and pain scoring (composite pain scale by Bussieres et al., Horse Grimace Scale). Abdominocentesis with peritoneal fluid analysis was performed at 72 h. RESULTS: Analgesia achieved with all treatment protocols allowed completion of ovariectomy procedures. Pressure algometry scores were lower in BHCl-treated horses versus both LB groups overall. Pain scores were improved with LB-treated horses in a dose-dependent fashion (Horse Grimace Scale scores LB40 < LB20 < BHCL; composite pain scale scores LB40 < BHCL, LB20 < BHCL, BHCL, and LB20 did not differ). Peritoneal fluid total protein was lower in LB40 versus LB20 and BHCL horses. No complications from LB administration were appreciated. MAIN LIMITATIONS: Small patient sample size, lack of follow-up past 72 h or histopathology. CONCLUSIONS: Analgesia duration was extended and pain scores improved postoperatively with LB versus BHCl in a dose-dependent fashion. Further clinical evaluation of extended-duration local anaesthetics in horses for improved postoperative pain management is warranted.

Effect of age at training initiation on hoof morphology and lameness in juvenile American Quarter Horses.

BACKGROUND: Lameness, discipline, training intensity, environmental variability, and shoeing are all factors demonstrated to affect hoof loading and therefore act as adaptive stimuli to alter hoof morphology. OBJECTIVES: To evaluate the effect of age at training initiation on hoof morphology and lameness incidence and determine if specific hoof morphology measurements correlate with lameness in juvenile American Quarter Horses. STUDY DESIGN: Prospective cohort study. METHODS: American Quarter Horses (n = 42; 29 two-year-olds, 13 three-year-olds) entering training were monitored for hoof morphology and lameness over 6 months (months 0, 2, 4, and 6). Hoof measurements (palmar/plantar angles, frog base width/length, toe length/angle, heel length/angle, heel and foot width, wall height/angle) from radiographs and photographs were recorded. Lameness was graded subjectively and objectively (Lameness locator®). Statistical analyses were performed with Fisher's exact test and repeated measures ANOVA with p < 0.05. RESULTS: 25/42 horses developed subclinical lameness (16/42 forelimb, 19/42 hindlimb), with 3-year-olds developing lameness more frequently compared to 2-year-olds overall (p = 0.04; 84.6 vs. 48.3%) and in forelimbs (p = 0.05; 61.5% vs. 27.6%); no difference was noted between 2- versus 3-year-olds in hindlimbs (p = 0.2; 61.5% vs. 37.9%). In lame versus sound forelimbs, 3-year-olds had decreased foot width (p = 0.03; 11.48 cm [CI 10.68-12.28] vs. 12.21 cm [CI 11.99-12.42]), decreased toe length (p = 0.03; 6.02 cm [CI 5.69-6.36] vs. 6.45 cm [CI 6.32-6.58]), shorter lateral wall height (p = 0.03; 4.64 cm [CI 4.31-4.96] vs. 5.11 cm [CI 5.03-5.2]), and shorter medial wall height (p = 0.02; 4.58 cm [CI 4.06-5.10] vs. 5.15 cm [CI 4.99-5.30]). In lame versus sound hindlimbs, horses overall (p = 0.05; 3.74, CI 3.53-3.96 vs. 3.55, CI 3.48-3.61) and 3-year-olds had longer heels p = 0.01; 3.90 cm (CI 3.5-4.3) vs. 3.50 cm (CI 3.39-3.61). MAIN LIMITATIONS: Small sample size, lack of control group not entering training. CONCLUSIONS: Three-year-old American Quarter Horses entering training were more likely to develop forelimb lameness than 2-year-olds. This subclinical lameness was associated with specific hoof morphology characteristics (decreased foot width, toe length, heel length, and lateral/medial wall height; greater toe angle).

INTRODUCTION/CONTEXTE: Les boiterie, discipline, intensité d'entraînement, variabilité environnementale et ferrage ont tous été établis comme facteurs affectant le port de poids au niveau du sabot. Ils contribuent aux stimuli adaptatifs qui peuvent altérer la morphologie du sabot. OBJECTIFS: Évaluer l'effet de l'âge en début d'entraînement sur la morphologie du sabot, l'incidence de boiterie et déterminer si des mesures spécifiques de morphologie du sabot pourraient être corrélées avec une boiterie chez les chevaux Quarter Horse Américains. TYPE D'ÉTUDE: Étude de cohorte prospective. MÉTHODES: Des Quarter Horse Américains (n = 42; 29 2 ans, 13 3 ans) en début d'entraînement ont été suivi pour la présence de boiterie et la conformation de leur sabot sur une période de 6 mois (mois 0, 2, 4, 6). Des mesures de sabot (angles palmaires/plantaires, largeur/longueur de la base de la fourchette, longueur/angle de la pince, longueur/angle des talons, largeur du pied et des talons, hauteur/angle de la muraille) à partir de radiographies et de photographies ont été recueillies. Les boiteries ont été gradées subjectivement et objectivement (Lameness locator®). Des analyses statistiques ont été effectuées avec la méthode exacte de Fisher et ANOVA pour mesures répétées avec un p < 0.05. RÉSULTATS: 25/42 chevaux ont développé une boiterie sous-clinique (16/42 membre antérieur, 19/42 membre postérieur). Les chevaux âgés de 3 ans ont développé une boiterie de façon plus fréquente comparativement aux 2 ans (p = 0.04; 84.6 vs. 48.3%) et aux membres antérieurs (p = 0.05; 61.5% vs. 27.6%); il n'y avait pas de différence au niveau des membres postérieurs entre les 2 et 3 ans. En comparant les chevaux boiteux des antérieurs avec ceux qui ne boitaient pas, les 3 ans avaient une largeur de sabot diminuée (p = 0.03; 11.48 cm [IC 10.68-12.28] vs. 12.21 cm [IC 11.99-12.42]), une longueur de pince plus courte (p = 0.03; 6.02 cm [IC 5.69-6.36] vs. 6.45 cm [IC 6.32-6.58]), une hauteur de muraille latérale plus courte (p = 0.032; 4.64 cm [IC 4.31-4.96] vs. 5.11 cm [IC 5.03-5.2]) et une hauteur de muraille médiale plus courte également (p = 0.024; 4.58 cm [IC 4.06-5.10] vs. 5.15 cm [IC 4.99-5.30]). En comparant les chevaux boiteux des postérieurs avec ceux qui ne boitaient pas, chevaux dans l'ensemble (p = 0.05; 3.74, CI 3.53-3.96 vs. 3.55, CI 3.48-3.61) et les 3 ans avaient des talons plus long (p = 0.01; 3.90 cm [IC 3.5-4.3] vs. 3.50 cm [IC 3.39-3.61]). LIMITES PRINCIPALES: Petite taille d'échantillon, aucun groupe contrôle n'ayant pas commencé l'entraînement. CONCLUSIONS: Les Quarter Horse Américains âgés de 3 ans qui débutent l'entraînement sont plus à risque de développer une boiterie des antérieurs comparativement aux chevaux de 2 ans. Une boiterie sous-clinique était associée à des caractéristiques morphologiques spécifiques au sabot (Largeur du sabot, longueur de la pince, longueur des talons et hauteur des murailles latérales et médiales toutes diminuées; angle de la pince augmenté).

Intravenous injection of adipose-derived mesenchymal stromal cells benefits gait and inflammation in a spontaneous osteoarthritis model.

Osteoarthritis (OA) is a leading cause of morbidity among aging populations, yet symptom and/or disease-modification remains elusive. Adipose-derived mesenchymal stromal cells (adMSCs) have demonstrated immunomodulatory and anti-inflammatory properties that may alleviate clinical signs and interrupt disease onset and progression. Indeed, multiple manuscripts have evaluated intra-articular administration of adMSCs as a therapeutic; however, comparatively few evaluations of systemic delivery methods have been published. Therefore, the aim of this study was to evaluate the short-term impact of intravenous (IV) delivery of allogeneic adMSCs in an established model of spontaneous OA, the Hartley guinea pig. Animals with moderate OA received once weekly injections of 2 × 106 adMSCs or vehicle control for 4 weeks in peripheral veins; harvest occurred 2 weeks after the final injection. Systemic administration of adMSCs resulted in no adverse effects and was efficacious in reducing clinical signs of OA (as assessed by computer-aided gait analysis) compared to control injected animals. Further, there were significant decreases in key inflammatory mediators (including monocyte chemoattractant protein-1, tumor necrosis factor, and prostaglandin E2 ) both systemically (liver, kidney, and serum) and locally in the knee (joint tissues and synovial fluid) in animals treated with IV adMSCs relative to controls (as per enzyme-linked immunosorbent assay and/or immunohistochemistry, dictated by tissue sample). Thus, systemic administration of adMSCs by IV injection significantly improved gait parameters and reduced both systemic and intra-articular inflammatory mediators in animals with OA. These findings demonstrate the potential utility of alternative delivery approaches for cellular therapy of OA, particularly for patients with multiple affected joints.

Preliminary evaluation of safety and migration of immune activated mesenchymal stromal cells administered by subconjunctival injection for equine recurrent uveitis.

Introduction: Equine recurrent uveitis (ERU), an immune mediated disease characterized by repeated episodes of intra-ocular inflammation, affects 25% of horses in the USA and is the most common cause of glaucoma, cataracts, and blindness. Mesenchymal stromal cells (MSCs) have immunomodulatory properties, which are upregulated by preconditioning with toll-like receptor agonists. The objective was to evaluate safety and migration of TLR-3 agonist polyinosinic, polycytidylic acid (pIC)-activated MSCs injected subconjunctivally in healthy horses prior to clinical application in horses with ERU. We hypothesized that activated allogeneic MSCs injected subconjunctivally would not induce ocular or systemic inflammation and would remain in the conjunctiva for >14 days. Methods: Bulbar subconjunctiva of two horses was injected with 10 × 106 pIC-activated (10 µg/mL, 2 h) GFP-labeled MSCs from one donor three times at two-week intervals. Vehicle (saline) control was injected in the contralateral conjunctiva. Horses received physical and ophthalmic exams [slit lamp biomicroscopy, rebound tonometry, fundic examination, and semiquantitative preclinical ocular toxicology scoring (SPOTS)] every 1-3 days. Systemic inflammation was assessed via CBC, fibrinogen, and serum amyloid A (SAA). Horses were euthanized 14 days following final injection. Full necropsy and histopathology were performed to examine ocular tissues and 36 systemic organs for MSC presence via IVIS Spectrum. Anti-GFP immunohistochemistry was performed on ocular tissues. Results: No change in physical examinations was noted. Bloodwork revealed fibrinogen 100-300 mg/dL (ref 100-400) and SAA 0-25 µg/mL (ref 0-20). Ocular effects of the subjconjucntival injection were similar between MSC and control eyes on SPOTS grading system, with conjunctival hypermia, chemosis and ocular discharge noted bilaterally, which improved without intervention within 14 days. All other ocular parameters were unaffected throughout the study. Necropsy and histopathology revealed no evidence of systemic inflammation. Ocular histopathology was similar between MSC and control eyes. Fluorescent imaging analysis did not locate MSCs. Immunohistochemistry did not identify intact MSCs in the conjunctiva, but GFP-labeled cellular components were present in conjunctival phagocytic cells. Discussion: Allogeneic pIC-activated conjunctival MSC injections were well tolerated. GFP-labeled tracking identified MSC components phagocytosed by immune cells subconjunctivally. This preliminary safety and tracking information is critical towards advancing immune conditioned cellular therapies to clinical trials in horses.

TLR-activated mesenchymal stromal cell therapy and antibiotics to treat multi-drug resistant Staphylococcal septic arthritis in an equine model.

Background: Rapid development of antibiotic resistance necessitates advancement of novel therapeutic strategies to treat infection. Mesenchymal stromal cells (MSC) possess antimicrobial and immunomodulatory properties, mediated through antimicrobial peptide secretion and recruitment of innate immune cells including neutrophils and monocytes. TLR-3 activation of human, canine and equine MSC has been shown to enhance bacterial killing and clearance in vitro, in rodent Staphylococcal biofilm infection models and dogs with spontaneous multi-drug-resistant infections. The objective of this study was to determine if intra-articular (IA) TLR-3-activated MSC with antibiotics improved clinical parameters and reduced bacterial counts and inflammatory cytokine concentrations in synovial fluid (SF) of horses with induced septic arthritis. Methods: Eight horses were inoculated in one tarsocrural joint with multidrug-resistant Staphylococcus aureus (S. aureus). Bone marrow-derived MSC from three unrelated donors were activated with TLR-3 agonist polyinosinic, polycytidylic acid (pIC). Recipient horses received MSC plus vancomycin (TLR-MSC-VAN), or vancomycin (VAN) alone, on days 1, 4, 7 post-inoculation and systemic gentamicin. Pain scores, quantitative bacterial counts (SF, synovium), SF analyses, complete blood counts, cytokine concentrations (SF, plasma), imaging changes (MRI, ultrasound, radiographs), macroscopic joint scores and histologic changes were assessed. Results were reported as mean ± SEM. Results: Pain scores (d7, P=0.01, 15.2±0.2 vs. 17.9±0.5), ultrasound (d7, P=0.03, 9.0±0.6 vs. 11.8±0.5), quantitative bacterial counts (SF d7, P=0.02, 0±0 vs. 3.4±0.4; synovium P=0.003, 0.4±0.4 vs. 162.7±18.4), systemic neutrophil (d4, P=0.03, 4.6±0.6 vs. 7.8±0.6) and serum amyloid A (SAA) (d4, P=0.01, 1,106.0±659.0 vs. 2,858.8±141.3; d7, P=0.02, 761.8±746.2 vs. 2,357.3±304.3), and SF lactate (d7, P<0.0001, 5.4±0.2 vs. 15.0±0.3), SAA (endterm, P=0.01, 0.0 vs. 2,094.0±601.6), IL-6 (P=0.03, 313.0±119.2 vs. 1,328.2±208.9), and IL-18 (P=0.02, 11.1±0.5 vs. 13.3±3.8) were improved in TLR-MSC-VAN vs. VAN horses. Study limitations include the small horse sample size, short study duration, and lack of additional control groups. Conclusions: Combined TLR-activated MSC with antibiotic therapy may be a promising approach to manage joint infections with drug resistant bacteria.

Immune Activated Cellular Therapy for Drug Resistant Infections: Rationale, Mechanisms, and Implications for Veterinary Medicine.

Antimicrobial resistance and biofilm formation both present challenges to treatment of bacterial infections with conventional antibiotic therapy and serve as the impetus for development of improved therapeutic approaches. Mesenchymal stromal cell (MSC) therapy exerts an antimicrobial effect as demonstrated in multiple acute bacterial infection models. This effect can be enhanced by pre-conditioning the MSC with Toll or Nod-like receptor stimulation, termed activated cellular therapy (ACT). The purpose of this review is to summarize the current literature on mechanisms of antimicrobial activity of MSC with emphasis on enhanced effects through receptor agonism, and data supporting use of ACT in treatment of bacterial infections in veterinary species including dogs, cats, and horses with implications for further treatment applications. This review will advance the field's understanding of the use of activated antimicrobial cellular therapy to treat infection, including mechanisms of action and potential therapeutic applications.

Evaluation of factors associated with surgical site infection in equine proximal interphalangeal joint arthrodesis: 54 cases (2010-2019).

BACKGROUND: The frequency of surgical site infection (SSI) following orthopaedic implant placement in horses has been reported but not compared with respect to specific antibiotic protocols administered. OBJECTIVES: To determine factors associated with SSI in horses undergoing proximal interphalangeal joint (PIPJ) arthrodesis including perioperative antibiotic protocols. METHODS: Records were evaluated (2010-2019), and horses undergoing PIPJ arthrodesis were identified. Patient signalment, supervising surgeon, reason for surgery, limb, implants placed, anaesthetic time, duration casting/coaptation postoperatively, antibiotic regimen and incidence/onset SSI were recorded. Bayesian and frequentist logistic regressions were used to estimate the contribution of covariates to infection occurrence. RESULTS: Fifty-four PIPJ arthrodeses were performed. SSI occurred in 2/54 (3.7%) on day 15,30. Arthrodesis was performed most commonly for osteoarthritis (33/54, 61.1%), fracture (11/54, 20.4%), and subluxation (5/54, 9.3%). Perioperative systemic antibiotics were administered 1-3 days (15/54, 27.8%) or > 3 days (39/54, 72.2%). Antibiotic protocols included cefazolin/gentamicin (20/54, 37%), cefazolin/gentamicin/doxycycline (14/54, 25.9%) and potassium penicillin/gentamicin (10/54, 18.5%). Regional limb perfusion was performed preoperatively 31/54 (57.4%) and postoperatively 7/54 (13%). Survival to dismissal was 98.1% (53/54 horses) with one horse euthanized due to support limb laminitis. No association was identified between antibiotic selection or duration (1-3 vs. > 3 days), pre-operative regional antibiotic perfusion, intraoperative antibiotic lavage or anaesthetic time (< or > 3 h) and SSI; however, modelling was complicated by quasi-complete or complete separation of the data. Bayesian analysis (but not frequentist analysis) indicated an association between post-operative regional antibiotic perfusion and SSI. Limitations include the retrospective nature of data collection and the low rate of infection overall. CONCLUSIONS: The prevalence of SSI in this population was lower than that in previous reports of equine orthopaedic internal fixation. There was no difference in SSI rate in cases administered systemic antibiotics for 1-3 days or >3 days, or for those horses that did or did not receive preoperative regional antibiotic perfusion.

Liposomal bupivacaine provides longer duration analgesia than bupivacaine hydrochloride in an adjustable sole-pressure model of equine lameness.

OBJECTIVE: To compare the efficacy and duration of action for perineural analgesia with liposomal bupivacaine (LB) versus bupivacaine hydrochloride (BHCl) in a sole-pressure induced model of forelimb lameness in horses. ANIMALS: 6 healthy adult research horses. PROCEDURES: In 1 randomly assigned forelimb, grade 3/5 lameness was induced by use of a sole-pressure lameness model. Objective lameness (vector sum [VS]) was determined with an inertial sensor system at 0, 1, 6, and 24 hours after lameness induction to evaluate the model. Mechanical nociceptive thresholds (MNTs) and objective lameness (VS and force platform kinetics) were recorded prior to and at 1, 6, 24, 48, and 72 hours after perineural anesthesia of the palmar nerves at the level of the proximal sesamoid bones with LB or BHCl in random order, with a 1-week washout period between crossover treatments. Data analysis was performed with mixed-model ANOVA. RESULTS: When evaluating the lameness model, there was a decrease in lameness at 24 hours in at least 1 limb of each horse (7/12 limbs); thus, screw length was increased by 1 to 2 mm at each 24-hour interval to maintain lameness. Compared with results at baseline, horses treated with BHCl had significant improvements in median MNT and VS identified at only 1 hour after injection, whereas treatment with LB yielded significantly improved median MNT, VS score, and peak vertical force for up to 24 hours. DISCUSSION: In this experimental model of forelimb lameness, LB provided longer analgesia when compared with BHCl and should be further investigated for treatment of pain in horses.

Outcomes after cervical vertebral interbody fusion using an interbody fusion device and polyaxial pedicle screw and rod construct in 10 horses (2015-2019).

BACKGROUND: Further development of surgical techniques for equine cervical stabilisation is necessary to make the procedure less technically demanding, reduce complications and improve outcomes. OBJECTIVE: To describe clinical outcomes and owner reports in horses undergoing placement of an interbody fusion device and polyaxial pedicle screw and rod construct for cervical vertebral fusion in horses with cervical vertebral compressive myelopathy. STUDY DESIGN: Retrospective case series. METHODS: Data were retrieved from medical records of 10 horses undergoing cervical vertebral fusion (2015-2019). Records were evaluated for signalment, duration of clinical signs, number and location of compression sites, grade of ataxia, duration of hospitalisation and complications. Long-term follow-up was obtained through clinical re-evaluation, postoperative radiographs and owner contact. RESULTS: Breeds were mixed. Median age was 24 (range 12-168) months. There were 2/10 mares, 4/10 geldings and 4/10 stallions. Preoperative grade of ataxia ranged from 1-3/5. Fusion was performed at one (n = 3) or two (n = 7) sites. Two horses were euthanised within the first year. In 6 of 8 horses with ≥1-year follow-up, ataxia improved by 1-3 grades, with an average improvement of 1.25 grades. In four horses, ataxia improved to grade 0-1. In two horses the gait was unaffected, but neck comfort improved. Complications included seroma formation (n = 9), pain (n = 5), fever (n = 4), upper respiratory tract obstruction (n = 2), azotemia (n = 2), screw breakage (n = 2), progression of neurological signs (n = 1), Horner's Syndrome (n = 1), dysphagia (n = 1), hives (n = 1), implant infection (n = 1) and nondisplaced fracture (n = 1). MAIN LIMITATIONS: Small case series, heterogeneous patient population. CONCLUSIONS: This technique resulted in ≥1 grade gait improvement in 6/10 cases operated and 6/8 cases for which ≥1-year follow-up was available, similar to other methods. Fatal complications related to implant placement did not occur. This technique may represent a safer alternative to current techniques of ventral interbody fusion with similar outcomes.

Comparison of equine synovial sepsis rate following intrasynovial injection in ambulatory versus hospital settings.

BACKGROUND: Frequency of synovial sepsis in horses following intrasynovial injection has been reported, but not compared with respect to the environment in which the injection was performed. OBJECTIVES: To describe occurrence of synovial sepsis following intrasynovial injections performed in ambulatory vs hospital settings. STUDY DESIGN: Retrospective cohort study. METHODS: Records from the Colorado State University were evaluated (2014-2018) and horses receiving intrasynovial injections were identified. Patients presenting for septic synovial structures were excluded. Patient signalment, primary supervising service, medications injected, location (field/hospital), whether synovial sepsis resulted, and at what time sepsis was recognised were recorded. Logistic regression was used to estimate the contributions of covariates to the occurrence of synovial sepsis following injection. RESULTS: During the study period, 3866 intrasynovial injections were performed in 1112 horses during 1623 sessions, with 643/1623 sessions performed in the field. The most frequently used medications were hyaluronate (846/1623, 52.1%), triamcinolone acetonide (780 /1623, 48.1%) and amikacin sulfate (684/1623, 42.1%). Four horses developed synovial sepsis (0.2% sessions, 0.1% synovial structures); 3/4 were injected in the field, 2/4 received antibiotics with the injection. The frequency of septic synovitis was 10.4 cases per 10 000 injections, or 1 in 967 injections. All horses recovered following synovial lavage and antibiotic therapy. Performing injections in the field (P = .2) or without antibiotics (P = .7) did not alter the risk of synovial sepsis. MAIN LIMITATIONS: Limitations include the retrospective nature of data collection and low rate of infection overall, which prohibited evaluation of individual medication regimes as factors associated with resultant infection. CONCLUSIONS: The frequency of synovial sepsis in this population of horses was not higher when injections were performed in the field or without concurrent antibiotic administration. These data may help to inform practitioners and clients regarding the relative potential risk of complications following intrasynovial medication in different environmental settings.

Intra-articular administration of antibiotics in horses: Justifications, risks, reconsideration of use and outcomes.

Antibiotics have been injected intra-articularly by equine veterinarians for decades, either prophylactically when other drugs are administered for osteoarthritis or therapeutically to treat septic arthritis. This route of administration has also more recently gained attention in human orthopaedic clinical practice, particularly as an alternative to systemic antibiotic administration to treat infections following prosthetic arthroplasty. While the rationale for injecting antibiotics intra-articularly has been largely focused on achieving high local drug concentrations, there has been relatively little focus on pharmacokinetic parameters of antibiotics administered by this route, or on the potential for local toxicity. The increasing incidence of antibiotic resistance in veterinary and human medicine prompts reconsideration of off-label antibiotic usage and evaluation of evidence-based dosing strategies. The purpose of this review was to summarise the current literature describing intra-articular antibiotic usage, including specific studies where pharmacokinetics, potential safety and toxicity have been evaluated. This review will advance practitioners' understanding of the use of intra-articularly administered antibiotics, including the overall pros and cons of the approach.

Outcomes following single, caudally based bilateral versus unilateral frontonasal sinusotomy for treatment of equine paranasal sinus disease.

BACKGROUND: Bilateral sinus disease is relatively uncommon in horses, accounting for 3%-4.5% of horses with sinonasal disease, but may require bilateral paranasal surgery for complete resolution. Complications and recurrence following bilateral sinusotomy have not been reported or compared to those following unilateral procedures. OBJECTIVE: To describe clinical features and outcomes in horses undergoing standing single, caudally based bilateral frontonasal sinusotomy compared to unilateral frontonasal surgery. METHODS: Records of horses (n = 37) undergoing surgical treatment for sinus disease (five bilateral, 32 unilateral) were retrospectively reviewed (2010-2017) for signalment, presenting complaint, duration of signs preoperatively, diagnostic imaging, treatments administered, duration hospitalization, complications, and owner satisfaction with the procedure. Mann-Whitney testing was used to compare age, duration of hospitalization, and follow-up time in horses undergoing unilateral or bilateral procedures. Fisher's exact testing was used to determine if sex predilection was present for unilateral or bilateral disease. Survival time and time to recurrence were compared by Kaplan-Meier survival curves and log-rank curve comparison testing. Significance was assessed at p < 0.05. RESULTS: Length of signs prior to admission did not differ between horses with unilateral and bilateral disease (p = 0.09), but there was a tendency for horses with bilateral disease to have clinical signs for longer. Age (p = 0.19) and hospitalization duration (p = 0.53) did not differ between horses undergoing unilateral versus bilateral procedures. Recurrence or failure to resolve signs was reported in 11/32 (34%) of unilateral and 0/5 bilateral cases (p = 0.07). CONCLUSIONS: The bilateral single, caudally based sinusotomy approach may be considered to effectively treat bilateral paranasal sinus disease without concern for increased risk of life-threatening complications or longer hospitalization duration than would be typical for unilateral sinusotomy procedures.

Toll-like receptor activation of equine mesenchymal stromal cells to enhance antibacterial activity and immunomodulatory cytokine secretion.

OBJECTIVE: To evaluate effects of Toll-like and nucleotide-binding oligomerization domain (NOD)-like receptor (TLR, NLR) ligand stimulation of equine mesenchymal stromal cells (MSCs) on antibacterial and immunomodulatory properties in vitro. STUDY DESIGN: Controlled laboratory study. SAMPLE POPULATION: Equine bone-marrow-derived MSCs (three horses). METHODS: MSCs were stimulated with TLR (polyinosinic:polycytidylic acid [pIC] and lipopolysaccharide [LPS]) and NLR agonists (γ-d-Glu-mDAP [IE-DAP]) for 2 h, and plated at 1 × 105 cells/well 24 h. MSC-conditioned media (MSC-CM) were collected and assessed for antimicrobial peptide cathelicidin/LL-37 production, bactericidal action against multidrug-resistant planktonic and biofilm Staphylococcus aureus and neutrophil phagocytosis. Bacterial growth was measured by plating bacteria and counting viable colonies, reading culture absorbance, and live-dead staining with confocal microscopy imaging. Following initial comparison of activating stimuli, TLR3-agonist pIC protocols (cell density during activation and plating, culture time, %serum) were further optimized for bactericidal activity and secretion of interleukin-8 (IL-8), monocyte-chemoattractant-protein (MCP-1), and cathelicidin/LL37. RESULTS: MSCs stimulation with pIC (p = .004) and IE-DAP (p = .03) promoted increased bactericidal activity, evidenced by reduced viable planktonic colony counts. PIC stimulation (2 × 106 cells/ml, 2 h, 10 µg/ml) further suppressed biofilm formation (p = .001), enhanced neutrophil bacterial phagocytosis (p = .009), increased MCP-1 secretion (p < .0001), and enhanced cathelicidin/LL-37 production, which was apparent when serum concentration in media was reduced to 1% (p = .01) and 2.5% (p = .05). CONCLUSION: TLR-3 pIC MSCs activation was most effective to enhance antibacterial and cytokine responses, which were affected by serum reduction. CLINICAL SIGNIFICANCE: In vitro TLR-3 activation of equine MSCs tested here may be a strategy to improve antibacterial properties of MSCs to treat antibiotic-resistant infections.