Low levels of microRNA-21 in neutrophil-derived exosomes may contribute to airway smooth muscle hyperproliferation in horses with severe asthma.

OBJECTIVE: Neutrophilic inflammation is associated with the degree of airway obstruction in severe equine asthma (SEA), but the contribution of these leukocytes to bronchial remodeling remains ill defined. Neutrophils could cause structural alterations of the airways by the release of exosomes, a type of cell-derived nanoparticles that can modify the biology of local and distant cells. Neutrophil-derived exosomes have been shown to increase airway smooth muscle (ASM) cell proliferation in humans and horses. Therefore, this study aimed to identify neutrophil exosomal microRNAs (miRs) implicated in the regulation of ASM biology in SEA. ANIMALS: 6 horses with SEA and 6 healthy controls. METHODS: The expression of selected miRs in exosomes from peripheral neutrophils was studied by quantitative PCR. The effects of miR-21 transfection in ASM cells were evaluated by gene expression analysis and proliferation studies. RESULTS: The miR-21 was downregulated in neutrophil exosomes from SEA horses, and it attenuated the proliferation of ASM cells stimulated with lipopolysaccharide. CLINICAL RELEVANCE: The lower level of miR-21 in neutrophil-derived exosomes could contribute to ASM hyperproliferation, which could, in turn, promote the thickening of the bronchial wall in SEA.

Iron-related markers of inflammation in horses with colic.

The aim of the study was to compare and correlate levels of ferritin, transferrin, iron and APPs in healthy horses and those surgically treated for strangulating colic. On admission, measurements of inflammatory markers related to iron and total protein, fibrinogen, albumin, haptoglobin and ceruloplasmin were made. The study comprised 22 horses, divided into a control group (CG) of healthy horses (n = 10) and horses with surgically treated acute abdomen (n = 12), obstruction group (OG). The OG was subdivided according to the affected intestinal segment (small vs. large) and according to outcome (survivors vs. non survivors). The OG had higher haptoglobin (34.8±14.2 mg/dL vs 20.8±7.21 mg/dL) and transferrin (487±161 mg/dL vs 369±71.4 mg/dL) values and lower iron (96.9±65 µg/dL vs 218±105 µg/dL) values than the CG. The OG horses with large intestine obstruction had lower values of transferrin (374.6±130 mg/dL) than horses with small intestinal obstruction (598.6±98.9 mg/dL). There was no difference in outcome between horses with large and small intestinal obstruction. Ferritin levels were moderately correlated with total protein (r = 0.594; P = 0.042) and albumin (r = 0.584; P = 0.046) in OG. In the multivariate exploratory analysis, fibrinogen levels were higher in animals that did not survive. In conclusion, haptoglobin, transferrin and iron were useful inflammatory markers for colic in horses. The correlation of ferritin with other APPs shows a possible role of ferritin as an APP in horses. Fibrinogen levels are higher in horses with greater risk of death from strangulating obstructions.

Genome-wide association study suggests genetic candidate loci of insulin dysregulation in Finnhorses.

Equine metabolic syndrome (EMS) is a common welfare problem in horses worldwide. It is characterized by insulin dysregulation (ID), predisposition to laminitis and often obesity. EMS is multifactorial by nature, with both the environment and genetics contributing to the phenotype. Environmental factors, such as feeding and exercise, can be controlled, thus forming the basis for treatment and prevention. Genetic factors, by contrast, are less well-known and not easily controllable. The aim of this study was to identify potential genetic loci influencing ID/EMS in Finnhorses. A single-breed (Finnhorse) case-control genome-wide association study (GWAS) of ID was conducted with controls that included age-appropriate non-ID horses. ID status was determined with an oral sugar test (OST) for fasted horses. Seventy-one Finnhorses participated (n = 34 ID, n = 37 control). DNA samples (hair roots) were genotyped for 65 157 single-nucleotide polymorphisms (SNPs) with the Illumina Equine SNP70 BeadChip, and these data were analysed for association and FST outliers with genomic tools. P-values that exceeded the suggestive threshold (P = 1.00 ×10-5) were found in SNP BIEC2_383954 (P = 3.45 ×10-6) in chromosome 17 and SNP BIEC2_312374 (P = 1.89 ×10-5) in chromosome 15. Hierarchical and Bayesian FST outlier tests also detected these SNPs. Potential candidate genes associated with the ID close to SNP BIEC2_383954, with functions in carbohydrate metabolism, were Arginine and Glutamate Rich 1 (ARGLU1) and Ephrin-B2 (EFNB2).

Testicular expression of heat SHOCK proteins 60, 70, and 90 in cryptorchid horses.

Heat shock proteins are the most evolutionarily conserved protein families induced by stressors including hyperthermia. In the context of pathologies of the male reproductive tract, cryptorchidism is the most common genital defect that compromises the reproductive potential of the male because it induces an increase in intratesticular temperature. In equine species, cryptorchidism affects almost 9 % of newborns and few studies have been carried out on the molecular aspects of the retained testis. In this study, the expression pattern of HSP60, 70, and 90 in abdominal and inguinal testes, in their contralateral descended normally testes, and in testes of normal horses were investigated by Western blot and immunohistochemistry. The histomorphological investigation of retained and scrotal testes was also investigated. The seminiferous epithelium of the retained testes showed a vacuolized appearance and displayed a completely blocked spermatogenesis for lacking meiotic and spermiogenetic cells. On the contrary, the contralateral scrotal testes did not show morphological damage and the seminiferous epithelium displayed all phases of the spermatogenetic cycle as in the normal testes. The morphology of Leydig cells was not affected by the cryptorchid state. Western blot and immunohistochemistry evidenced that equine testis (both scrotal and retained) expresses the three investigated HSPs. More in detail, the Western blot evidenced that HSP70 is the more expressed chaperone and that together with HSP90 it is highly expressed in the retained gonad (P < 0.05). The immunohistochemistry revealed the presence of the three HSPs in the spermatogonia of normal and cryptorchid testes. Spermatogonia of retained testes showed the lowest expression of HSP60 and the highest expression of HSP90. Spermatocytes, spermatids of scrotal testes, and the Sertoli cells of retained and scrotal testes did not display HSP60 whereas expressed HSP70 and HSP90. These two proteins were also localized in the nucleus of the premeiotic cells. The Leydig cells displayed the three HSPs with the higher immunostaining of HSP70 and 90 in the cryptorchid testes. The results indicate that the heat stress condition occurring in the cryptorchid testis influences the expression of HSPs.

Use of Biologics and Stem Cells in the Treatment of Other Inflammatory Diseases in the Horse.

Mesenchymal stem cells (MSCs) are powerful immunomodulatory cells that act via multiple mechanisms to coordinate, inhibit, and control the cells of the immune system. MSCs act as rescuers for various damaged or degenerated cells of the body via (1) cytokines, growth factors, and signaling molecules; (2) extracellular vesicle (exosome) signaling; and (3) direct donation of mitochondria. Several studies evaluating the efficacy of MSCs have used MSCs grown using xenogeneic media, which may reduce or eliminate efficacy. Although more research is needed to optimize the anti-inflammatory potential of MSCs, there is ample evidence that MSC therapeutics are worthy of further development.

Equine Platelet-Rich Plasma.

Platelet-rich plasma (PRP) is an orthobiologic therapy composed of platelets, leukocytes, red blood cells, and plasma proteins. PRP has been used for 20 years, but progress determining efficacy has been slow. The definitions and classification of PRP are reviewed, and the use of PRP for tendon, ligament, and joint disease is discussed with a focus on findings of basic science and clinical studies, platelet activation, concurrent administration of nonsteroidal anti-inflammatory drugs, and treatment complications. Finally, the advantages of platelet lysates and freeze-dried platelets are discussed. The promising results of a PRP lysate optimized for antibiofilm and antimicrobial properties are introduced.

Equine Bone Marrow Aspirate Concentrate.

Bone marrow concentrate is generated by centrifugation of bone marrow aspirate. It contains mesenchymal stromal cells, anabolic chemokines/cytokines, and supraphysiological concentrations of interleukin-1 receptor antagonist protein (IL-1RA). It is an effective treatment for osteoarthritis or desmitis, or as an adjunct in surgery to enhance bone or cartilage repair.

Characteristic Inflammatory Biomarkers in an Equine Model of Persistent Synovitis Induced By the Intra-Articular Administration of Monoiodoacetic Acid.

Persistent synovitis damages the articular cartilage in horses. To evaluate the effectiveness of treatment for synovitis using a model induced by intra-articular administration of monoiodoacetic acid (MIA), it is necessary to identify inflammatory biomarkers characteristic of the MIA model. Synovitis was induced by administering MIA into the unilateral antebrachiocarpal joints of five horses, and saline was injected into the contralateral joints as a control on day 0. Clinical and ultrasonographic examinations and synovial fluid collection were performed on days 0, 1, 2, 7, 14, 21, 28, and 35. Leukocyte, lactate dehydrogenase (LDH), tumor necrosis factor-α (TNF-α), interleukin-1 receptor antagonist (IL-1Ra), interleukin-6 (IL-6), and transforming growth factor-ß1 (TGF-ß1) concentrations in the synovial fluid were measured. Synovium was obtained after euthanasia on day 42 and histologically examined before quantification of the gene expression of inflammatory biomarkers by real-time PCR. Acute inflammatory symptoms persisted for approximately 2 weeks before returning to control levels. However, some indicators of chronic inflammation remained elevated until day 35. On day 42, synovitis continued histologically, with osteoclasts. The expressions of matrix metalloproteinase 13 (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), receptor activator of nuclear factor kappa-Β ligand (RANKL), and collagen type I α2 chain (Col1a2) were significantly higher in the MIA model than in the control. In the MIA model, representative inflammatory biomarkers in the chronic inflammatory stage were persistently expressed in both synovial fluid and tissue, suggesting that they may be useful for the assessment of the anti-inflammatory effect of drugs.

Serum nerve growth factor in horses with osteoarthritis-associated lameness.

BACKGROUND: Nerve growth factor (NGF) is a neurotrophin that is increased in osteoarthritic joints of horses. In humans, NGF has been associated with pain, and both synovial and serum NGF concentrations are increased in osteoarthritic patients. Studies in humans also have shown that serum NGF concentration can increase with stress. Serum NGF concentration should be evaluated in horses with osteoarthritis-associated lameness. OBJECTIVES: Quantify and compare serum NGF concentration in horses with osteoarthritis-associated lameness and sound horses. Additionally, the impact of short-term stress on serum NGF concentration was investigated. ANIMALS: Lame horses with radiographic evidence of osteoarthritis (n = 20), lame horses without radiographic changes in the affected joint (n = 20) and sound horses (n = 20). In addition, horses with acute fractures (n = 9) were sampled. To determine the effect of stress, serum from horses subjected to a stressful event (transportation, n = 5; stress confirmed by increased serum cortisol concentration) was analyzed. METHODS: Cross-sectional clinical study (lame, sound, and fracture cohorts) and experimental longitudinal study (stress cohort). Serum NGF concentration was determined using a quantitative sandwich ELISA. RESULTS: Serum NGF concentration was increased in lame horses with radiographic evidence of osteoarthritis (P < .0001; median, 238 pg/mL; interquartile range [IQR], 63-945 pg/mL) and in lame horses without radiographic evidence of osteoarthritis in the painful joint (P < .05; median, 31 pg/mL; IQR, 31-95 pg/mL) compared with sound horses (median, 31 pg/mL; IQR, 31-46 pg/mL). Serum NGF concentration did not increase with short-term stress and was low in horses with fracture-associated pain. CONCLUSIONS AND CLINICAL IMPORTANCE: Serum NGF concentration was high in the cohort with advanced osteoarthritis and should be investigated as a marker for osteoarthritis-associated pain.

Acute Phase Protein Response in Native and Imported Horses After Routine Combination Vaccination Protocol.

Fibrinogen and serum amyloid A (SAA) are commonly measured equine acute phase proteins. Limited data exist on SAA and fibrinogen responses to combination vaccination protocols in horses. A prospective cohort study evaluating SAA, fibrinogen, and rectal temperature following a standard combination vaccination. Blood for measurement of SAA and serum fibrinogen and rectal temperatures were obtained before (0 hour) and after vaccination (24, 48, 72, 96, 168 hours). After vaccination, SAA and fibrinogen increased in all horses. Imports had elevated SAA from 24-168 hours, whereas native horses returned to baseline by 168 hours. Compared to native horses, SAA was significantly higher in imports (coefficient 24-168 hours 358, 95%CI: 46-671 mg/L; P = .03). Fibrinogen increased significantly from 24 to 168 hours postvaccination, but groups did not differ (coefficient -16; 95%CI: -69 to 37 mg/dL; P = .5). Absolute rectal temperatures were significantly higher in imports throughout, including 0 hour (median 37.8; IQR 37.7-38.0 vs. 37.3; 37.1-37.3; P = .002). At 24 hours postvaccination when temperatures significantly increased above baseline in both groups, there was a small but significant difference in the percent change relative to baseline (coefficient 1.9; 95%CI 0.8%-2.9%; P = .002). A standard combination vaccination protocol elicited an acute phase response in all horses. Compared to native previously vaccinated horses, imports had a stronger SAA response. The observed response is worthy of consideration when examining recently vaccinated imported horses.

Effect of platelet lysate on Schwann-like cell differentiation of equine bone marrow-derived mesenchymal stem cells.

Currently, treatment for peripheral nerve injuries in horses primarily relies upon physical therapy and anti-inflammatory drugs. In humans, various treatments using mesenchymal stem cells (MSCs) are being attempted. Therefore, in this study, Schwann-like cell differentiation cultures of equine MSCs were prepared using fetal bovine serum (FBS) and equine platelet lysate (ePL). ePL increased the platelet count to 1 × 106/µl, the optimal concentration for culture. In both groups, an elongated morphology at both ends, characteristic of Schwann cells, was observed under the microscope. Real-time PCR analysis of the expression levels of neuronal markers showed that the ePL group tended to express higher levels of Nestin, Musashi1, and Pax3 than the FBS group. p75 was expressed at low levels in both groups. Immunostaining results showed localization of Nestin in both groups of differentiated cells, but the positive cell rate was significantly higher in the ePL group than in the FBS group. Overall, the ePL gro showed good results for Schwann-like cell differentiation, which may be useful for future use in the treatment of equine motor neuron disease. This knowledge could be applied translationaly in the treatment of amyotrophic lateral sclerosis in humans.Overall, the ePL group showed good results for Schwann-like cell differentiation, which may be useful for future use in the treatment of equine motor neuron disease and in the treatment of amyotrophic lateral sclerosis in humans.

Equine metabolic syndrome: Role of the enteroinsular axis in the insulin response to oral carbohydrate.

Equine insulin dysregulation (ID) comprises amplified insulin responses to oral carbohydrates or insulin resistance, or both, which leads to sustained or periodic hyperinsulinaemia. Hyperinsulinaemia is important in horses because of its clear association with laminitis risk, and the gravity of this common sequela justifies the need for a better understanding of insulin and glucose homoeostasis in this species. Post-prandial hyperinsulinaemia is the more commonly identified component of ID and is diagnosed using tests that include an assessment of the gastrointestinal tract (GIT). There are several factors present in the GIT that either directly, or indirectly, enhance insulin secretion from the endocrine pancreas, and these factors are collectively referred to as the enteroinsular axis (EIA). A role for key components of the EIA, such as the incretin peptides glucagon-like peptide-1 and 2, in the pathophysiology of ID has been investigated in horses. By comparison, the function (and even existence) of many EIA peptides of potential importance, such as glicentin and oxyntomodulin, remains unexplored. The incretins that have been examined all increase insulin responses to oral carbohydrate through one or more mechanisms. This review presents what is known about the EIA in horses, and discusses how it might contribute to ID, then compares this to current understanding derived from the extensive studies undertaken in other species. Future directions for research are discussed and knowledge gaps that should be prioritised are suggested.

Palatability, glycemic, and insulinemic responses to various carbohydrate formulations: Alternatives for the diagnosis of insulin dysregulation in horses?

BACKGROUND: Oral glycemic challenge (GC) tests are recommended for diagnosis of insulin dysregulation (ID). Various protocols are used, but all have limitations in terms of palatability, ease of use, variable composition, geographic availability, or some combination of these. HYPOTHESIS/OBJECTIVE: To evaluate newly developed formulations with defined carbohydrate composition for use as oral GCs. ANIMALS: Thirty-four horses and ponies in various metabolic states. METHODS: Our objectives were carried out in 2 separate cross-over experiments. First, the palatability and acceptance of various GCs (2 syrups, 1 granulate) offered for free intake were compared to glucose mixed in a chaff-based diet. Subsequently, syrups were administered by syringe and compared to an oral glucose test using naso-gastric tubing (tube OGT) to investigate the glycemic and insulinemic responses. Second, these variables were compared in the best performing GC-formulations (granulate further optimized to pelleted formulation and 1 syrup) and a tube OGT. All GCs were administered with equivalent amounts of 0.5 g glycemic carbohydrates per kg body weight. RESULTS: Only the GC pellets were consumed completely by all horses (consumption time 5 ± 2 min). When administered by syringe, the GC syrup also was well accepted. The insulin concentrations at 120 min correlated significantly between tube OGT and GC pellets (r = .717; P < .001) or GC syrup (r = .913; P < .001). The new GC syrup and GC pellets discriminate between healthy and ID horses. CONCLUSIONS AND CLINICAL SIGNIFICANCE: The GC pellets (DysChEq)™ and GC syrup can be used as palatable and well-accepted oral GC tests for assessment of ID in horses.

Visual Assessment of Adiposity in Elite Hunter Ponies.

Obesity is a critical problem in the equine industry, with ponies being at particularly high risk of developing obesity-related conditions such as metabolic syndrome and laminitis. The purpose of this study was to estimate the level of adiposity in an elite level of competitive show ponies, and to determine if adiposity was related to performance based on their model score (judged on conformation and appearance). Two researchers visually evaluated the body condition score (BCS) and cresty neck score (CNS) of 377 ponies at a national hunter competition. Thirty five percent of the ponies in the competition were considered obese, with BCS scores of 7 or higher. Mean BCS of the ponies was 6.7 ± 0.6 with a range of 5.25 - 8.25 and mean CNS was 2.8 ± 0.6, with a range of 1.75 - 4.5. Medium ponies had higher BCS (P<0.0001) and higher CNS (P=0.015) than the large ponies. There was a tendency for a relationship between body condition score and the model score in all ponies (r = 0.08; P = 0.059), and this was stronger within the large ponies (r =0.20; P <0.01). These findings show that elite competition ponies are dangerously overweight and that adiposity may influence performance in a judged event. This is of grave concern to the horse industry and needs to be addressed.

Proteomic Differences Between the Ovulatory and Anovulatory Sides of the Mare's Follicular and Oviduct Fluid.

The follicular fluid and oviduct fluid play major roles in oocyte maturation, sperm activation, and fertilization. To better understand the physiological environments for equine oocyte maturation and fertilization, here we conducted the proteome analysis and comparison on follicular fluids and oviduct fluids from the ovulatory side and the anovulatory side. The results showed that there is no significant difference between two side oviduct fluids, but a total of 71 differential abundance proteins (DAPs) were identified between two side follicular fluids, of which 9 are up-regulated and 62 are down-regulated in ovulatory side follicle fluid versus anovulatory side follicle fluid. As we expected, the function classification and enrichment results indicate that up- and down-regulated proteins are largely related to oocyte meiosis, maturation and ovulation. Noticeably, among 9 up-regulated DAPs in ovulatory side follicle fluid, as the DAP with the greatest fold change, PLA2G1B may be a newly discovered component that influences the efficacy of horse IVM/IVF. The current findings add to our knowledge of the in vivo conditions and regulation of equine reproduction, as well as the regulatory mechanism underpinning alternative ovulation.

Plasma syndecan-1 concentration as a biomarker for endothelial glycocalyx degradation in septic adult horses.

BACKGROUND: Limited information is available regarding endothelial glycocalyx degradation during sepsis in horses. Plasma syndecan-1 concentrations are increased in consequence of sepsis in other species and have been useful for prognostication. OBJECTIVES: To determine whether plasma syndecan-1 levels are increased in adult horses affected with sepsis. STUDY DESIGN: Retrospective cohort study. METHODS: Adult horses were assigned to one of three groups based on results of physical and laboratory examinations, clinical diagnosis, and results of previously described SIRS classification: Group 1 horses included healthy, nonseptic horses; Group 2 included horses in which clinical illness was identified but that were not considered to be septic; Group 3 included horses with a clinical diagnosis of sepsis. Plasma syndecan-1 concentration was determined in blood obtained at admission into the hospital for each horse, using an equine specific ELISA. Data were analysed using ANOVA and linear regression (p ≤ 0.05). RESULTS: One hundred and ninety-one horses were included and divided into three groups. Scores for SIRS were highest for Group 3 horses and lowest in Groups 1 and 2. Plasma syndecan-1 concentrations in Group 3 horses (50.73 ± 84.24 µg/ml; n = 42) were greater than those for Group 1 (15.69 ± 11.28 µg/ml; n = 66) and Group 2 (16.88 ± 15.30 µg/ml; n = 83). There was no difference regarding syndecan concentrations between Groups 1 and 2. MAIN LIMITATIONS: Retrospective study design, solitary time point of measurement for each patient, and lack of a widely accepted consensus regarding definitive diagnosis of sepsis in adult horses. CONCLUSIONS: Circulating plasma levels of syndecan-1, a biochemical marker of endothelial glycocalyx damage, are increased in septic adult horses.

Conditioned serum in vitro treatment of chondrocyte pellets and osteoarthritic explants.

BACKGROUND: Autologous conditioned serum (ACS) is used to treat osteoarthritis in horses, although its effects are not fully investigated. OBJECTIVES: To investigate the effects of equine serum and conditioned serum on chondrocytes stimulated with interleukin (IL)-1ß and cartilage explants with mild osteoarthritis. STUDY DESIGN: In vitro experimental study. METHODS: The effect of three different serum preparations (unincubated control [PS], serum incubated 24 h [PS24h] and serum incubated 24 h in ACS containers [PCS]) pooled from lame horses were tested in two in vitro models. IL-1ß and IL-1 receptor antagonist (IL-1Ra) concentrations were measured in all sera. In model 1, chondrocyte pellet cultures were stimulated with IL-1ß prior to treatment with the serum preparations for 2 and 48 h. Microarray, polymerase chain reaction, and matrix metallopeptidase-13 analyses were performed. In model 2, cartilage explants from horses with structural osteoarthritis were treated with PS or PCS on days 0, 6 and 12, or left untreated, and evaluated at day 24 using the OARSI grading scale for histological evaluation of articular cartilage. RESULTS: The IL-1Ra concentration in PS24h and PCS was significantly higher than in PS. In model 1, inflammation- and cartilage matrix degradation-related genes were upregulated after 48 h in all treatment groups versus untreated controls. Cartilage matrix molecules, aggrecan and collagens, were downregulated in PS24h- and PCS-treated pellets versus untreated controls. Growth factor signalling genes were upregulated-FGF7 in all treatment groups, BMP2 in PS24h-, and INHBA in PCS-treated-compared with untreated controls. In model 2, the OARSI score at day 24 was not significantly different between treatment groups. MAIN LIMITATIONS: Results from in vitro models cannot be directly translated to in vivo situations. CONCLUSIONS: In vitro treatment with conditioned serum did not alleviate IL-1ß-induced responses in chondrocyte pellets or lead to morphological improvement in osteoarthritic cartilage explants.

HISTORIAL: Suero autólogo acondicionado (ACS) es usado para tartar osteoartritis en caballos, aunque sus efectos no han sido completamente investigados. OBJETIVOS: Investigar los efectos de suero equino y suero acondicionado en condrocitos estimulados con interleukina (IL)-1ß y explantes de cartílago con osteoartritis leve. DISEÑO DEL ESTUDIO: Estudio experimental in vitro. MÉTODOS: El efecto de tres preparaciones séricas diferentes (control no incubado (PS), suero incubado 24 h (PS24h), y suero incubado 24 h en frascos ACS (PCS)) combinados y obtenidos de caballos cojos fueron probados en dos modelos in vitro. Las concentraciones de IL-1ß y de receptor antagonista de IL-1 (IL-1Ra) fueron medidas en todos los sueros. En el modelo 1, los cultivos de pellets de condrocitos fueron estimulados con IL-1ß antes de ser tratados con las preparaciones séricas durante 2 y 48 h. Se realizaron análisis de micromatrices, reacciones de polimerasa en cadena y de matriz de metalopeptidasa-13. En el modelo 2, explantaciones de cartílago proveniente de caballos con osteoartritis estructural fueron tratados con PS o PCS en los días 0, 6 y 12, o dejados sin tartar, y evaluados al día 24 usando la escala de graduación OARSI para evaluación histológica de cartílago articular. RESULTADOS: La concentración de IL-1Ra en PS24h y PCS fue significativamente mayor que en PS. En el modelo 1, los genes relacionados a la inflamación y a la degradación de la matriz cartilaginosa estaban aumentados después de 48 h en todos los grupos tratados en comparación a los controles no tratados. Las moléculas de matriz cartilaginosa, agrecanos y colágenos estaban disminuidos en los pellets PS24h y PCS versus los controles no tratados. Los genes de señales de factores de crecimiento FGF7 estaban aumentados en todos los grupos tratados, BMP2 en PS24h y INHBA in PCS en comparación con los controles no tratados. En el modelo 2, la escala OARSI al día 24 no fue significativamente distinta entre los grupos de tratamientos. LIMITACIONES PRINCIPALES: Los resultados de modelos in vitro no pueden ser directamente aplicados a situaciones in vivo. CONCLUSIONES: El tratamiento in vitro con suero acondicionado no alivió las respuestas inducidas por IL-1ß en pellets de condrocitos o llevo a mejoramiento morfológico en explantes de cartílago con osteoartritis.

Residual effects of intra-articular betamethasone and triamcinolone acetonide in an equine acute synovitis model.

BACKGROUND: Intra-articular (IA) corticosteroids are regularly used in equine athletes for the control of joint inflammation. OBJECTIVES: The goal of this study was to use an acute synovitis inflammation model to determine the residual effects of IA betamethasone and triamcinolone acetonide on various inflammatory parameters and lameness. STUDY DESIGN: Crossover randomised trial. METHODS: Five mixed-breed, 2-year-old horses were randomly allocated to an IA treatment of the radiocarpal joint with 9 mg of either betamethasone or triamcinolone acetonide. Two weeks following treatment, horses were injected with 1 µg of lipopolysaccharide (LPS) diluted in 1 ml of saline. Following LPS injection, horses were crossed-over and both sets of injections were repeated after a washout period. Blood samples were collected at multiple time points for mRNA analysis, as well as serum amyloid A (SAA) and cortisol determination. At each time point, lameness was also subjectively scored. Additional injections with saline-only or LPS-only (twice) were conducted as negative and positive controls, respectively. Two-way repeated measures analysis of variance was used to analyse all data. RESULTS: Corticosteroid-only treatments result in significant mRNA expression differences, as well as significant and prolonged cortisol suppression. Following LPS injection, there was a residual treatment effect with triamcinolone evidenced by a significant treatment effect on IL-6 and PTGS1 (cyclooxygenase-1), lameness, SAA and cortisol concentrations, while only IL-6 expression was affected by betamethasone. MAIN LIMITATIONS: The acute synovitis model used here results in significant inflammation and is not representative of the low-grade inflammation seen with typical joint disease and residual anti-inflammatory effects may be more profound in naturally occurring joint disease. CONCLUSIONS: Current regulatory guidelines may be insufficient if the concern is residual anti-inflammatory effects. Additionally, intra-articular corticosteroid administration is not without risk, as evidenced by a significant suppression of serum cortisol concentration and, as such, the benefits of their administration should be weighed against those risks.

Developing advanced therapeutics through the study of naturally occurring immune-mediated ocular disease in domestic animals.

This review, which is part of the "Currents in One Health" series, describes the importance of the study of immune-mediated ocular disease in the development of innovative therapeutics, such as cell and gene therapy for the eye. Recent examples of cell and gene therapy studies from the author's laboratory are reviewed to emphasize the importance of One Health initiatives in developing innovative therapies for ocular diseases. Spontaneous immune-mediated corneal disease is common in horses, cats, dogs, and humans. Autologous bone marrow-derived mesenchymal stem cells (BM-MSCs) injected subconjunctivally resulted in the resolution of naturally occurring immune-mediated keratitis (IMMK) without adverse effects. These results support that autologous subconjunctival BM-MSC therapy may be a viable treatment alternative for IMMK. Furthermore, the use of subconjunctival MSCs may be an effective method to treat ocular surface immune-mediated diseases in humans and other species, including herpetic stromal keratitis and immunologic dry eye disease. Furthermore, the use of adeno-associated viral (AAV) vectors to deliver the immunosuppressive transgene cDNA of equine interleukin 10 (eqIL-10) or human leukocyte antigen G injected intravitreally was shown to be safe and inhibited the development of uveitis in the experimental autoimmune uveitis rat model. Efficacy and safety studies of ocular gene therapy in models will pave the way for clinical trials in animals with naturally occurring immune-mediated diseases, such as a therapeutic clinical trial for AAV-eqIL-10 in horses with equine recurrent uveitis.

Equine insulin dysregulation causes tissue specific alterations of proinflammatory cytokines and acute phase proteins in a NF-kB independent manner.

Similar to human diabetes, equine metabolic syndrome (EMS) causes insulin dysregulation leading to debilitating sequela including laminitis. The pathophysiological mechanisms underlying EMS and laminitis are not well known. Therefore, using an insulin-resistant equine model, we hypothesized that insulin dysregulation induces an increased expression of inflammatory proteins in a tissue specific manner. Two groups of horses (n = -5/group) were categorized as insulin-resistant (IR) or insulin-sensitive (IS), using a frequently sampled intra-venous glucose tolerance test. Biopsies from skeletal muscle, and visceral and subcutaneous adipose tissues were collected in both groups. Protein expression was quantified via Western blotting in order to investigate HSP90, α 2 macroglobulin (A2M), Fibrinogen α, ß, γ isoforms as well as cytokines, including interleukin-1ß (IL-1ß) and interleukin-6 (IL-6), in muscle and adipose tissues. Protein expression of HSP90, A2M and IL1-ß was significantly greater in visceral adipose tissue of IR horses compared to IS horses. Fibrinogen (α and γ) expression was only significantly increased in subcutaneous adipose tissue of IR group compared to IS group. In contrast, no statistically significant difference in protein expression of proinflammatory cytokines and acute phase proteins was reported in skeletal muscle of IR vs. IS horses. Relative protein expression of total and phospho-NFκB protein expression was not statistically significantly changed in adipose tissues of IR horses compared to IS horses. In conclusion, proinflammatory cytokines and acute phase proteins were upregulated in adipose tissue, but not in skeletal muscle, through an NF-kB independent pathway. Insights from this study could reveal novel biomarkers and potential therapeutic targets for EMS.