In vitro research involving horses refers to the study of equine cells, tissues, or biological molecules outside their normal biological context, typically in controlled laboratory environments. This research approach allows scientists to investigate cellular processes, molecular interactions, and the effects of various treatments without the ethical and logistical complexities of in vivo studies. In vitro studies contribute to understanding equine physiology, pathology, and pharmacology by providing insights into cellular responses to pathogens, drugs, and other stimuli. This page compiles peer-reviewed research studies and scholarly articles that explore various in vitro methodologies and their applications in equine science, including cell culture techniques, molecular assays, and drug efficacy testing.
Antoni KP, Natalia L, Maciej S, Leszek N, Małgorzata SS, Robert M, Maciej G, Ewa PO.Adipose tissue functions as an endocrine organ; however, excessive lipid accumulation can lead to obesity and metabolic disorders, such as Equine Metabolic Syndrome (EMS), characterized by insulin resistance, fat deposition, and increased inflammation. Despite the growing prevalence of obesity in horses, knowledge of equine adipocytes and their metabolic functions remains limited. The main objective of the study was to develop and optimize a method for isolating equine adipocytes and to characterize their metabolic activity. Using slaughterhouse-derived horse visceral adipose tissue, we develo...
Castanheira CIGD, Anderson JR, Clarke EJ, Hackl M, James V, Clegg PD, Peffers MJ.This study describes a novel technique to analyze the extracellular vesicle (EV)-derived microRNA (miRNA) crosstalk between equine chondrocytes and synoviocytes. Donor cells (chondrocytes, n = 8; synoviocytes, n = 9) were labelled with 5-ethynyl uridine (5-EU); EVs were isolated from culture media and incubated with recipient cells (chondrocytes [n = 5] were incubated with synoviocyte-derived EVs, and synoviocytes [n = 4] were incubated with chondrocyte-derived EVs). Total RNA was extracted from recipient cells; the 5-EU-labelled RNA was recovered and sequenced. Differential expression analysi...
Laroche N, Grimm P, Julliand S, Sorci G.Sainfoin (Onobrychis viciifolia), a polyphenol-rich plant, has shown promising anti-parasitic properties in ruminants, but results in horses are fewer and inconsistent. The mechanisms of action involved are not fully understood and different factors may influence its anti-parasitic properties. Recently, it has been shown that the effect of sainfoin depends on the horse's diet. Indeed, the inclusion of dehydrated sainfoin pellets in a high starch diet limited the rate of increase in strongyle egg shedding over a short period of time (21 days). The objective of this study was to evaluate, in viv...
Sultan T, Chaudhary AK, Mehta JS, Mehra R, Manuja A, Talluri TR, Bhattacharya TK.Sperm cryopreservation is one of the most important procedures in the development of biotechnologies for assisted reproduction. Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Different substances and compounds can be added to semen extenders to improve sperm quality. Objective: To investigate the effect of supplementing semen extender with zinc nanoparticles (ZnONPs) and gold nanoparticles (AuNPs) on cooled and frozen-thawed spermatozoa of Marwari stallion. Methods: A total of 20 ejaculates from four Marwari ...
Cuervo-Arango J, Sala-Ayala L, Márquez-Moya A, Martínez-Boví R.The objective of this study was to determine the effect of two aspiration pressures (75 vs. 150 mmHg), the follicle flushing method (injection pump controlled by a foot pedal vs. a plastic syringe) and the twisting of the OPU needle on oocyte recovery and in vitro embryo production. OPU data from a total of 104 warmblood sport mares belonging to a commercial OPU-ICSI program were collected as part of a prospective study split into three experiments. Each mare was used only once for OPU. In Experiment 1, the mares' follicles were aspirated using either a high aspiration pressure (flow rate of 1...
Felix MR, Dobbie T, Woodward E, Linardi R, Okada C, Santos R, Hinrichs K.We recently reported successful equine IVF using fresh semen pre-incubated for a prolonged period (22 h) before co-culture with oocytes. In this study, we evaluated the feasibility of equine IVF with frozen-thawed sperm and evaluated capacitation-related changes in these sperm over the pre-incubation period. Sperm selected via a commercial sperm separation device (SSD) yielded significantly higher fertilization than did sperm selected by swim-up or by colloid centrifugation. Using the SSD method, fertilization rates with sperm pre-incubated for 15 min, 3 h, 6 h, and 9 h were 7.1, 22.2, 38...
Arnade H, Ivie IE, Gordon J, Peroni JF.To evaluate the feasibility of short-term 3-D tissue culture in maintaining the structure and function of equine synovial explants and demonstrate that day 4 explant characteristics were not significantly different from day 0. We hypothesized that a 4-day culture period in 3-D explant culture would not significantly disrupt synovial tissue health and function compared to baseline measurements. Unassigned: Synovial explants (n = 24/horse) from healthy carpal joints of 5 horses were cultured in 12-well plates using tissue-stabilizing agar rings. Explants were evaluated in triplicate or quadrupli...
Gugole PM, Zannoni A, Forni M, Iacono E, Zambelli F, Merlo B.This study investigates the effects of overnight holding and naloxone (Nx) supplementation on the vitrification outcomes of equine immature oocytes. Oocytes were divided into six experimental groups based on treatment combinations: fresh (F) and held (H) control oocytes, oocytes vitrified with or without Nx (10 M) (VIT and VIT-Nx), oocytes vitrified after overnight holding with or without Nx (10 M) (H-VIT and H-VIT-Nx). They were assessed for survival, meiotic competence, intracellular oxidative stress, mitochondrial activity and distribution, apoptosis, and apoptotic gene expression. At sur...
Yu Y, Wang J, Ruan L, Chen L, Khan MZ, You A, Wang C, Li L, Ren H, Wang T, Liu W.Equid alphaherpesvirus type 8 (EHV-8) is a contagious pathogen that causes reproductive disorders, respiratory diseases, and viral encephalitis in equids, resulting in significant economic losses for the global horse and donkey industries. Currently, there are no approved antiviral drugs or vaccines available for EHV-8 control. In this study, we investigated the antiviral efficacy of celastrol against EHV-8 both in vitro and in vivo. Our results demonstrated that celastrol significantly inhibited EHV-8 infection in Rabbit kidney (RK-13) and equine dermal cells (NBL-6) in a dose-dependent manne...
Adler DMT, Nielsen AT, Olsen C, Verwilghen D, Damborg P, Olsen RH.To investigate the in vitro efficacy of antibiotics (amikacin, ceftiofur, and gentamicin) in combination with local anesthetics (LAs; bupivacaine hydrochloride, lidocaine hydrochloride, and mepivacaine hydrochloride), a combination commonly performed for IA injectate and regional limb perfusion (RLP) in horses. Unassigned: 17 equine clinical isolates were tested by the checkerboard method for their minimum inhibitory concentration (MIC) against a combination of concentrations of LAs and antibiotics from August 2020 through December 2023. Unassigned: For the majority of combinations, the antibi...
Gaspari G, Lange-Consiglio A, Cremonesi F, Desantis S.Extracellular vesicles (EVs) are important mediators of cell-cell communication thanks to their ability to transfer their bioactive cargo, thus regulating a variety of physiological contexts. EVs derived from amniotic mesenchymal/stromal cells (eAMC-EVs) are internalized by equine endometrial cells (eECs) with positive effects on regenerative medicine treatments. As the cellular uptake of EVs is influenced by the glycan profile of both EVs and target cells, this study is focused on the role of surface glycans in the uptake of eAMC-EVs by recipient eECs. Equine ECs were obtained by enzymatic di...
Fedorka CE, Scoggin KE, Coleman SJ, Hatzel JN, Burleson MD, Troedsson MHT.Alterations during the early stages of embryo development have been associated with long-term effects on the fetus, neonate, and adult, but this has not been investigated in horses. In recent years, intracytoplasmic sperm injection (ICSI) has gained in commercial popularity in the equine population. Research suggests an association between ICSI-produced embryos and placental malformations, but there exists little understanding of the physiology involved. Therefore, we aim to produce a complete transcriptomic analysis of chorioallantois and provide potential pathways that may be impacted follow...
Byambaragchaa M, Park SH, Park MH, Kang MH, Min KS.Equine chorionic gonadotropin (eCG) hormone, comprising highly glycosylated α- and β-subunits, elicits responses similar to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in non-equid species. This study aimed to establish a mass production of recombinant eCG (rec-eCG) using CHO DG44 cells. Single-chain rec-eCG β/α was expressed in CHO DG44 cells. FSH- and LH-like activities were evaluated in CHO-K1 and HEK 293 cells expressing the equine LH/CG receptor (eLH/CGR), rat LH/CGR (rLH/CGR), and rFSHR. pERK1/2 activation and β-arrestin 2 recruitment were assessed in PathHunter ...
Barton CK, Nelson BB, Winther TM, Maker TC, Goodrich LR.To develop and optimize a tenoscopic technique for resection of the manica flexoria utilizing a radiofrequency probe. Methods: Experimental study. Methods: Eighteen cadaver limbs and two experimental horses. Methods: Tenoscopic-guided resection of the manica flexoria using radiofrequency energy proceeded sequentially by transection of the medial, lateral and proximal borders. The technique was performed in ex vivo limbs and then in vivo to replicate clinical scenarios. Time to complete resection of the manica flexoria (mean ± SD) and complications was recorded. Limbs were dissected and sc...
Beaumont RE, Smith EJ, David C, Paterson YZ, Faull E, Guest DJ.Tendon injuries are a common problem in humans and horses. There is a high re-injury rate in both species due to the poor regeneration of adult tendon and the resulting formation of scar tissue. In contrast, fetal tendon injuries undergo scarless regeneration, but the mechanisms which underpin this are poorly defined. It is also unclear if tendon cells derived from embryonic stem cells (ESCs) would aid tendon regeneration. In this study we determined the responses of adult, fetal and ESC-derived equine tenocytes to a range of cytokines, chemokines and growth factors that are upregulated follow...
Ma Z, Li Y, Zhao Z, Song Q, Wang Q, Lu S, Wang J.In this study, we purified and identified antioxidant peptides from equine plasma protein hydrolysates and assessed their protective effects against HO-induced oxidative stress in Caco-2 cells. Four antioxidant peptides were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in equine plasma protein hydrolysate, namely: GTMVGC (567.69 Da), FGMTST (662.88 Da), VGYHSHF (847.01 Da) and ALSPFFKE (939.18 Da). Among them, ALSPFFKE showed the strongest antidigestive properties after modelled digestion studies. Moreover, ALSPFFKE enhanced intracellular superoxide dismutase (SO...
Fakheri A, Esmaeilnejad B, Akbari H, Molaei R.Strongyle nematodes pose a major challenge in veterinary parasitology, causing significant economic losses in livestock due to resistance to conventional treatments. Current anthelmintics, like Ivermectin, often encounter resistance issues. This study aims to address these gaps by synthesizing Carbon Quantum Dots (CQDs) and Copper-Doped CQDs (Cu@CQDs) using glucose extract, and evaluating their nematicidal properties against strongyles in vitro. We assessed the nematicidal effects of CQDs and Cu@CQDs through larval feeding inhibition of first-stage larvae (L1), egg hatch inhibition (EHI), and ...
Stafford LS, Plummer CE, Smith WC, Gibson DJ, Sharma J, Vicuna V, Diakite S, Larkin J.Recurrent uveitis (RU), an autoimmune disease, is a leading cause of ocular detriment in humans and horses. Equine and human RU share many similarities including spontaneous disease and aberrant cytokine signaling. Reduced levels of SOCS1, a critical regulator of cytokine signaling, is associated with several autoimmune diseases. Topical administration of SOCS1-KIR, a peptide mimic of SOCS1, was previously correlated to reduced ocular pathologies within ERU patients. Unassigned: To further assess the translational potential of a SOCS1 mimetic to treat RU, we assessed peptide-mediated modulatio...
Hidalgo M.Sperm vitrification is an alternative freezing method, which includes high cooling rates and non-permeable cryoprotectants agents. The first attempt in equids was using the spheres technique by directly dropping small volumes of the sperm into liquid nitrogen. Later, vitrification was developed using 0.25 mL straws with outer covers, which resulted in similar progressive motility when compared to conventional freezing in donkeys (44.3 ± 15.0 % vs. 44.7 ± 18.2 %) or even higher in horses (48.2 ± 2.3 % vs. 37.3 ± 2.2 %). Subsequently, the vitrification of larger volumes of sperm in 0.5 mL st...
Königová A, Babják M, Kuzmina TA, Burcáková Ľ, Syrota Y, Várady M.The study aimed to conduct a survey on the occurrence of benzimidazole (BZ) resistance in strongyles by in vitro egg hatch test (EHT) and larval development test (LDT) and to identify the effective indicators of early resistance detection on horse farms with associated risk factors analysis appraisal. In total, 203 horses from 8 farms underwent the fecal sampling of which 77 horses were selected for in vitro testing. Simultaneously, 18 horses were chosen to analyse the results of in vitro tests compared to the in vivo fecal egg count reduction test (FECRT). The EHT indicated the presence of re...
Bernabe MCM, Fonseca BB, Silva MVC, Pedrosa IE, Silva MB, Sommerfeld S, de Sousa ALP, de Leva Resende BC, Sousa ACP, Dos Santos Freitas A....Endometritis is one of the main reproductive disorders in mares and due to the increasing prevalence of antibiotic resistance, the use of probiotics in the prevention and treatment of endometritis in mares has gained interest, given their potential to restore and maintain a healthy uterine microbiota. Therefore, the aim of this study was to evaluate the antimicrobial properties of total metabolites of Lactobacillus acidophilus (LA) and Lactiplantibacillus plantarum (LP) against common equine endometrial pathogenic bacteria in vitro (Acinetobacter baumannii, Escherichia coli (1), Escherichia co...
Ana A, Agnieszka S, Marta CP, Pawel K, Dariusz S, Graça FD, Szóstek-Mioduchowska A.Equine endometrosis is a major cause of infertility in mares and is characterized by degenerative, functional and fibrotic changes in the endometrium with increased collagen (COL) deposition. Transforming growth factor (TGF)-β1 is one of the major pro-fibrotic factors involved in the excessive deposition of extracellular matrix (ECM) components in the equine endometrium. It has been demonstrated that ovarian steroids, specifically 17β-estradiol (E2) and progesterone (P4), not only regulate the cyclicity of the estrous cycle, but also have been implicated as anti- or pro-fibrotic factors. Thi...
Nogacka AM, García A, de Los Reyes-Gavilán CG, Arboleya S, Gueimonde M.Horses are hindgut fermenters that harbor a complex intestinal microbiota (IM) which provides key enzymes aiding in the breakdown of complex carbohydrates present in their herbivorous diet. Therefore, these animals are deeply dependent on their IM for digestion and nutrition. Consequently, IM imbalances may result in alteration of fermentation patterns with impact on the animal health and the risk of disease. In this context, strategies for assisting the maintenance of a healthy IM in horses are of interest. However, there is limited research concerning the use of probiotics to improve hindgut...
Guidoni K, Chiaradia E, Pepe M, Di Meo A, Tognoloni A, Seccaroni M, Beccati F.Intra-articular corticosteroids, such as triamcinolone acetonide (TA), help reduce pain related to osteoarthritis (OA), but they may impair cartilage metabolism. In contrast, platelet-rich plasma (PRP) therapy, a regenerative therapy, has shown potential to promote healing and regeneration of articular cartilage. This study investigates the effects of combining PRP with TA to treat osteoarthritis in racehorses. The study proposes that PRP injection following TA treatment could reduce side effects and improve treatment outcomes. Firstly, in the in vitro study, chondrocytes were exposed to diffe...
Egli P, Boone L, Huber L, Higgins C, Gaonkar PP, Arrington J, Naskou MC, Peroni J, Gordon J, Lascola KM.Platelet lysate (PL) demonstrates antimicrobial and anti-inflammatory properties offering potential for treatment of bacterial pneumonia in horses. It remains unknown whether nebulization is suitable for PL administration in horses. This pilot study characterized particle size and flow rate of pooled equine PL (single preparation) nebulized using an equine-specific nebulizer (Flexivent®). Unassigned: Protein composition and antimicrobial activity were compared before and after nebulization. Protein composition was evaluated according to growth factor, antimicrobial peptide and cytokine concen...
Samper JC, Hernandez Aviles JC, Ramirez-Agamez LF, Love CC, Gonzalez-Marin C, Fleury P, Dini P, De La Fuente A, Foss R, Campos FL, Ross PJ.The application of sex-sorted semen in horses has historically lagged the bovine industry due to differences in the reproductive physiology and grater variability in equine semen quality. Recent advancements, including SuperGen™ sorters and Ultraplus™ semen processing methods developed by STgenetics® (Navasota, Texas), have significantly improved the efficiency of the sex-sorting process and reduced the sperm damage previously reported. To facilitate the broader use sex-sorted semen in the equine industry, it is essential to address challenges such as shipping cooled semen to a central so...
Elkhenany HA, Linardi RL, Ortved KF.Osteoarthritis (OA) remains a challenging joint disorder necessitating effective anti-inflammatory interventions. In this study, our primary objective was to establish an in vitro protocol that replicates the clinical investigation of anti-inflammatory drugs intended for OA management. Focusing on recombinant IL-10 (r.IL-10) as a potential anti-inflammatory treatment, we designed and implemented two distinct protocols to evaluate the efficacy of r.IL-10 in modulating chondrocyte and synoviocyte inflammation.The experimental design involved sequential stimulation with IL-1β and TNF-α for 24 ...
de Lima AE, Mendes RP, Piñeros DDV, Alibrando BB, de Zoppa ALV, da Silva LCLC, de Freitas SH, Corrêa RR.Sinusitis is common in horses. Sinoscopy is used for visualization of sinus cavities and can be used for diagnostic and therapeutic purposes. In this surgical procedure, a larger chamber is created and communication with other sinus compartments facilitated. To compare the applicability of different surgical approaches and rigid endoscopy systems for sinus inspection and fenestration of the maxillary septal bulla, and to examine the impact of different lens angles on the effectiveness of the procedure. Six chemically preserved cadaveric equine heads were used. Frontal and caudal maxillary port...
Broothaers K, Pascottini OB, Hedia M, Angel-Velez D, De Coster T, Peere S, Polfliet E, Van den Branden E, Govaere J, Van Soom A, Smits K.Previous studies in the horse highlight the potential benefit of prolonged in vitro maturation (IVM) (34 h) compared to short IVM (24 h) with or without prior oocyte holding, but little is known about the optimal IVM duration within this interval. To determine the effect of oocyte holding and duration of IVM ranged between 28 and 34 h on nuclear maturation, cleavage, blastocyst formation, and pregnancy rates, a retrospective study was performed in an equine clinical OPU-ICSI setting. The study included data of 2114 aspirated oocytes from 201 OPU-ICSI sessions. Duration of IVM was divided in...
Bianchera A, Borghetti P, Ravanetti F, Bertocchi L, De Angelis E, Bettini R.Hyaluronic acid (HA) is a key component of synovial fluid as it plays a crucial role in joint physiology. Its biological activity is influenced by molecular weight, local concentration, and persistence in joints. High-molecular-weight HA has a consolidated history of clinical use, whereas little is known about the metabolic effect of low-molecular-weight hyaluronate on cartilage differentiation. This study explores the potential of HA-based nanoparticles (NPs) on chondrocytes differentiation in vitro. Starting from 25 kDa and 250 kDa sodium hyaluronate solutions, two types of NPs were prepared...
Hondalus MK, Mosser DM.Rhodococcus equi is a facultative intracellular bacterium of macrophages that can cause serious pneumonia in both young horses and immunocompromised people. Essential to understanding rhodococcus pathogenesis is a quantitative documentation of the intracellular events that follow macrophage phagocytosis of the organism. By using a bacterial immunofluorescence staining assay, we verified the intracellular survival and replicative potential of R. equi in both murine peritoneal macrophages and equine alveolar macrophages in vitro. Following an initial lag period of 6 to 12 h, the intracellular nu...
Vidal MA, Kilroy GE, Johnson JR, Lopez MJ, Moore RM, Gimble JM.To characterize equine bone marrow (BM)-derived mesenchymal stem cell (MSC) growth characteristics and frequency as well as their adipogenic and osteogenic differentiation potential. Methods: In vitro experimental study. Methods: Foals (n=3, age range, 17-51 days) and young horses (n=5, age range, 9 months to 5 years). Methods: Equine MSCs were harvested and isolated from sternal BM aspirates and grown up to passage 10 to determine cell-doubling (CD) characteristics. Limit dilution assays were performed on primary and passaged MSCs to determine the frequency of colony-forming units with a fibr...
Toupadakis CA, Wong A, Genetos DC, Cheung WK, Borjesson DL, Ferraro GL, Galuppo LD, Leach JK, Owens SD, Yellowley CE.To determine the optimal osteogenic source of equine mesenchymal stem cells (eMSCs) and optimize collection of and expansion conditions for those cells. Methods: 10 adult Quarter Horses and 8 newborn Thoroughbred foals. Methods: eMSCs were isolated from bone marrow (BM), adipose tissue, and umbilical cord blood and tissue, and the osteogenic potential of each type was assessed. Effects of anatomic site, aspiration volume, and serum type on eMSC yield from BM were investigated. Results: BM-eMSCs had the highest overall expression of the osteogenic genes Cbfa1, Osx, and Omd and staining for ALP ...
Whittle BJ, Moncada S, Vane JR.The activity of prostacyclin (PGI2), PGE1 or PGD2 as inhibitors of platelet aggregation in plasma from human, dog, rabbit, rat, sheep and horse was investigated. Prostacyclin was the most potent inhibitor in all species. PGD2 was a weak inhibitor in dog, rabbit and rat plasma whereas PGE1 and prostacyclin were highly active. Theophylline or dipyridamole potentiated the inhibition of human platelet aggregation by prostacyclin, PGE1 or PGD2. Compound N-0164 abolished the inhibition by PGD2 of human platelet aggregation but did not inhibit the effects of PGE1 or prostacyclin. The results suggest ...
Barberini DJ, Freitas NP, Magnoni MS, Maia L, Listoni AJ, Heckler MC, Sudano MJ, Golim MA, da Cruz Landim-Alvarenga F, Amorim RM.Studies with mesenchymal stem cells (MSCs) are increasing due to their immunomodulatory, anti-inflammatory and tissue regenerative properties. However, there is still no agreement about the best source of equine MSCs for a bank for allogeneic therapy. The aim of this study was to evaluate the cell culture and immunophenotypic characteristics and differentiation potential of equine MSCs from bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and umbilical cord (UC-MSCs) under identical in vitro conditions, to compare these sources for research or an allogeneic therapy cell bank. Methods: The BM-MS...
Vidal MA, Robinson SO, Lopez MJ, Paulsen DB, Borkhsenious O, Johnson JR, Moore RM, Gimble JM.To compare the chondrogenic potential of adult equine mesenchymal stem cells derived from bone marrow (MSCs) or adipose tissue (ASCs). Methods: In vitro experimental study. Methods: Adult Thoroughbred horses (n=11). Methods: BM (5 horses; mean [+/-SD] age, 4+/-1.4 years) or adipose tissue (6 horses; mean age, 3.5+/-1.1 years) samples were obtained. Cryopreserved MSCs and ASCs were used for pellet cultures in stromal medium (C) or induced into chondrogenesis+/-transforming growth factor-3 (TGFbeta(3)) and bone morphogenic factor-6 (BMP-6). Pellets harvested after 3, 7, 14, and 21 days were exam...
Thompson JM, Whitmore AC, Konopka JL, Collier ML, Richmond EM, Davis NL, Staats HF, Johnston RE.Vaccination represents the most effective control measure in the fight against infectious diseases. Local mucosal immune responses are critical for protection from, and resolution of, infection by numerous mucosal pathogens. Antigen processing across mucosal surfaces is the natural route by which mucosal immunity is generated, as peripheral antigen delivery typically fails to induce mucosal immune responses. However, we demonstrate in this article that mucosal immune responses are evident at multiple mucosal surfaces after parenteral delivery of Venezuelan equine encephalitis virus replicon pa...
Baker TS, Newcomb WW, Booy FP, Brown JC, Steven AC.Cryoelectron microscopy and three-dimensional computer reconstruction techniques have been used to compare the structures of two types of DNA-free capsids of equine herpesvirus 1 at a resolution of 4.5 nm. "Light" capsids are abortive, whereas "intermediate" capsids are related to maturable intracellular precursors. Their T = 16 icosahedral outer shells, approximately 125 nm in diameter, are indistinguishable and may be described in terms of three layers of density, totalling 15 nm in thickness. The outermost layer consists of protruding portions of both the hexon and the penton capsomers, ris...
Gaspar D, Spanoudes K, Holladay C, Pandit A, Zeugolis D.The last decade has seen significant developments in cell therapies, based on permanently differentiated, reprogrammed or engineered stem cells, for tendon injuries and degenerative conditions. In vitro studies assess the influence of biophysical, biochemical and biological signals on tenogenic phenotype maintenance and/or differentiation towards tenogenic lineage. However, the ideal culture environment has yet to be identified due to the lack of standardised experimental setup and readout system. Bone marrow mesenchymal stem cells and tenocytes/dermal fibroblasts appear to be the cell populat...
Guest DJ, Smith MR, Allen WR.Autologous mesenchymal progenitor cells (MPCs) purified from bone marrow aspirates are being used in the treatment of superficial digital flexor tendon (SDFT) injuries in the horse with promising results. In this study the fate of autologous and allogeneic MPCs following injection into the SDFT was monitored by stable transfection of MPCs with green fluorescent protein (GFP). Small lesions were created manually in one forelimb SDFT of 2 horses and injected with autologous MPCs, allogeneic MPCs or bone marrow supernatant alone. Post mortem examinations performed after 10 or 34 days revealed GFP...
Wagner B, Freer H.The detection and quantification of equine cytokines has been hampered by the lack of antibodies for many years. With the development of antibody pairs for equine cytokines during the past years, the quantification of these essential regulators of the immune response became possible. After being successfully tested by enzyme-linked immunosorbent assays (ELISA), three of these anti-cytokine reagents were used here to establish the first cytokine multiplex assay for equine IL-4, IL-10 and IFN-alpha. A fluorescent bead-based system was used as matrix for this assay that allows the simultaneous de...
Patterson-Kane JC, Becker DL, Rich T.The equine superficial digital flexor tendon (SDFT) is a frequently injured structure that is functionally and clinically equivalent to the human Achilles tendon (AT). Both act as critical energy-storage systems during high-speed locomotion and can accumulate exercise- and age-related microdamage that predisposes to rupture during normal activity. Significant advances in understanding of the biology and pathology of exercise-induced tendon injury have occurred through comparative studies of equine digital tendons with varying functions and injury susceptibilities. Due to the limitations of in-...
Yu L, Wei Y, Sun HX, Mahdi AK, Pinzon Arteaga CA, Sakurai M, Schmitz DA, Zheng C, Ballard ED, Li J, Tanaka N, Kohara A, Okamura D, Mutto AA, Gu Y....Dynamic pluripotent stem cell (PSC) states are in vitro adaptations of pluripotency continuum in vivo. Previous studies have generated a number of PSCs with distinct properties. To date, however, no known PSCs have demonstrated dual competency for chimera formation and direct responsiveness to primordial germ cell (PGC) specification, a unique functional feature of formative pluripotency. Here, by modulating fibroblast growth factor (FGF), transforming growth factor β (TGF-β), and WNT pathways, we derived PSCs from mice, horses, and humans (designated as XPSCs) that are permissive for dire...
Aguilar PV, Weaver SC, Basler CF.Eastern equine encephalitis virus (EEEV) causes sporadic but often severe cases of human and equine neurological disease in North America. To determine how EEEV may evade innate immune responses, we screened individual EEEV proteins for the ability to rescue the growth of a Newcastle disease virus expressing green fluorescent protein (NDV-GFP) from the antiviral effects of interferon (IFN). Only expression of the EEEV capsid facilitated NDV-GFP replication. Inhibition of the antiviral effects of IFN by the capsid appears to occur through a general inhibition of cellular gene expression. For ex...
Lab on a chipApril 26, 2020
Volume 20, Issue 9 1621-1627 doi: 10.1039/d0lc00304b
Sun F, Ganguli A, Nguyen J, Brisbin R, Shanmugam K, Hirschberg DL, Wheeler MB, Bashir R, Nash DM, Cunningham BT.Rapid, sensitive and specific detection and reporting of infectious pathogens is important for patient management and epidemic surveillance. We demonstrated a point-of-care system integrated with a smartphone for detecting live virus from nasal swab media, using a panel of equine respiratory infectious diseases as a model system for corresponding human diseases such as COVID-19. Specific nucleic acid sequences of five pathogens were amplified by loop-mediated isothermal amplification on a microfluidic chip and detected at the end of reactions by the smartphone. Pathogen-spiked horse nasal swab...
Harr KE, Flatland B, Nabity M, Freeman KP.As all laboratory equipment ages and contains components that may degrade with time, initial and periodically scheduled performance assessment is required to verify accurate and precise results over the life of the instrument. As veterinary patients may present to general practitioners and then to referral hospitals (both of which may each perform in-clinic laboratory analyses using different instruments), and given that general practitioners may send samples to reference laboratories, there is a need for comparability of results across instruments and methods. Allowable total error (TEa ) is ...
Shin EK, Perryman LE, Meek K.The equine SCID defect is more severe than its murine counterpart in that SCID foals are incapable of forming either coding or signal joints, whereas SCID mice manifest normal signal joint formation. To determine the basis of this difference and whether DNA-dependent kinase, catalytic subunit (DNA-PK(CS)), is involved in signal joint formation, equine DNA-PK(CS) transcripts were cloned and sequenced from normal and SCID cell lines. In the mutant allele, a frame-shift mutation truncates the protein N terminal of the domain with homology to the phosphatidylinositol 3-kinase family resulting in c...
Kubickova S, Cernohorska H, Musilova P, Rubes J.Laser microbeam microdissection and laser pressure catapulting procedure were used for the construction of chromosome-specific painting probes, arm-specific probes and probes for chromosomal subfragments. We report on a method for generation of fluorescence in-situ hybridization probes from laser dissected chromosomes of farm animals. So far, using the described method, a set of chromosome-specific painting probes has been obtained for all porcine chromosomes, 17 chromosomes of cattle and selected equine chromosomes. It is concluded that the laser technology appears to be a useful and powerful...
Radcliffe CH, Flaminio MJ, Fortier LA.Mesenchymal progenitor cells (MPCs) are often characterized using surface markers after expansion and treatment in culture. There are no studies directly comparing gene and protein markers in undifferentiated samples during the very early phases of culture. The goal of this study was to evaluate temporal gene and protein expression changes during establishment of equine MPC cultures. Bone marrow aspirate was obtained from 35 horses and processed by density gradient centrifugation. In freshly isolated bone marrow, mononuclear cells had variable expression of CD44, CD11a/CD18, CD90, and CD45RB c...
Lafont R, Dinan L.Ecdysteroids are widely used as inducers for gene-switch systems based on insect ecdysteroid receptors and genes of interest placed under the control of ecdysteroid-response elements. We review here these systems, which are currently mainly used in vitro with cultured cells in order to analyse the role of a wide array of genes, but which are expected to represent the basis for future gene therapy strategies. Such developments raise several questions, which are addressed in detail. First, the metabolic fate of ecdysteroids in mammals, including humans, is only poorly known, and the rapid catabo...
Cassano JM, Schnabel LV, Goodale MB, Fortier LA.Inflammatory licensed mesenchymal stem cells (MSCs) have the ability to promote functional tissue repair. This study specifically sought to understand how the recipient tissue environment reciprocally affects MSC function. Inflammatory polarized macrophages, modeling an injured tissue environment, were exposed to licensed MSCs, and the resultant effects of MSC immunomodulation and functionality of the MSC secretome on chondrocyte homeostasis were studied. Inflammatory licensed MSCs were generated through priming with either IFN-γ or polyinosinic:polycytidylic acid (poly I:C). Macrophages were...
Marędziak M, Marycz K, Lewandowski D, Siudzińska A, Śmieszek A.The aim of this work study was to evaluate the cytophysiological activity of equine adipose-derived stem cells (ASCs) cultured under conditions of static magnetic field. Investigated cells were exposed to a static magnetic field (MF) with the intensity of 0.5 T. In order to investigate the effects of magnetic field on stem cell signaling, the localization and density and content of microvesicles (MVs) as well as morphology, ultrastructure, and proliferation rate of equine ASCs were evaluated. Results showed that potential of equine adipose-derived mesenchymal stem cells was accelerated when ma...
Taylor SE, Vaughan-Thomas A, Clements DN, Pinchbeck G, Macrory LC, Smith RK, Clegg PD.There is a paucity of data regarding molecular markers that identify the phenotype of the tendon cell. This study aims to quantify gene expression markers that distinguish between tendon fibroblasts and other mesenchymal cells which may be used to investigate tenogenesis. Methods: Expression levels for 12 genes representative of musculoskeletal tissues, including the proposed tendon progenitor marker scleraxis, relative to validated reference genes, were evaluated in matched samples of equine tendon (harvested from the superficial digital flexor tendon), cartilage and bone using quantitative P...
Rushlow K, Olsen K, Stiegler G, Payne SL, Montelaro RC, Issel CJ.The nucleotide sequence of the envelope (env) gene region of equine infectious anemia virus (EIAV), a member of the lentivirus subfamily of retroviruses, has been determined from a clone of integrated proviral DNA for which the gag and pol sequences have been reported previously. The env gene is 859 codons in length and the sequence reported here is consistent with the published biochemical properties of EIAV glycoproteins. The env gene region of EIAV shares considerable structural similarities but negligible sequence homologies with the env genes of other members of the lentivirus subfamily, ...
Carrade Holt DD, Wood JA, Granick JL, Walker NJ, Clark KC, Borjesson DL.Mesenchymal stem cells (MSCs) are used in both human clinical trials and veterinary medicine for the treatment of inflammatory and immune-mediated diseases. MSCs modulate inflammation by decreasing the cells and products of the inflammatory response. Stimulated equine MSCs from bone marrow (BM), adipose tissue (AT), cord blood (CB), and umbilical cord tissue (CT) inhibit lymphocyte proliferation and decrease inflammatory cytokine production. We hypothesized that equine MSCs inhibit T cell proliferation through secreted mediators and that MSCs from different tissue sources decrease T cell proli...
Abrahams M.The mechanical behaviour of horse and human tendon, as characterised by the stress-strain curve, has been examined with respect to load-strain cycling and strain rate. It was found that the tendon stress-strain curve for successive cycles was reporducible provided that strain on the specimen did not exceed 2·0–4·0%. If this strain level was exceeded, a permanent deformation occurred. This phenomenon was verified by histological studies on strained tendon which showed that some of the collagen fibres did not return to their original orientation. Variation in the rate of strain was found to ...
Iacono E, Brunori L, Pirrone A, Pagliaro PP, Ricci F, Tazzari PL, Merlo B.Mesenchymal stem cells (MSCs) have been derived from multiple sources of the horse including umbilical cord blood (UCB) and amnion. This work aimed to identify and characterize stem cells from equine amniotic fluid (AF), CB and Wharton's Jelly (WJ). Samples were obtained from 13 mares at labour. AF and CB cells were isolated by centrifugation, while WJ was prepared by incubating with an enzymatic solution for 2 h. All cell lines were cultured in DMEM/TCM199 plus fetal bovine serum. Fibroblast-like cells were observed in 7/10 (70%) AF, 6/8 (75%) CB and 8/12 (66.7%) WJ samples. Statistically ...
Klymiuk MC, Balz N, Elashry MI, Heimann M, Wenisch S, Arnhold S.Mesenchymal stem cells are used for different therapeutic approaches, e.g. for osteoarthritis, lesions of the tendon as well as for bone defects. Current research on the mechanism of stem cells on the repair of damaged tissue suggest an important role of a cell-to-cell communication through secreted extracellular vesicles, mainly represented by exosomes. To enhance the scarce knowledge on the functional role of exosomes we compared as a first step different techniques to isolate and identify exosomes from the supernatant of equine adipose derived mesenchymal stem cells for further characteriza...
Hidaka C, Goodrich LR, Chen CT, Warren RF, Crystal RG, Nixon AJ.Cartilage has a limited capacity to heal. Although chondrocyte transplantation is a useful therapeutic strategy, the repair process can be lengthy. Previously we have shown that over expression of bone morphogenetic protein-7 (BMP-7) in chondrocytes by adenovirus-mediated gene transfer leads to increased matrix synthesis and cartilage-like tissue formation in vitro. In this context we hypothesized that implantation of genetically modified chondrocytes expressing BMP-7 would accelerate the formation of hyaline-like repair tissue in an equine model of cartilage defect repair. Methods: Chondrocyt...
Dakin SG, Werling D, Hibbert A, Abayasekara DR, Young NJ, Smith RK, Dudhia J.Macrophages (Mφ) orchestrate inflammatory and reparatory processes in injured connective tissues but their role during different phases of tendon healing is not known. We investigated the contribution of different Mφ subsets in an equine model of naturally occurring tendon injury. Post mortem tissues were harvested from normal (uninjured), sub-acute (3-6 weeks post injury) and chronically injured (>3 months post injury) superficial digital flexor tendons. To determine if inflammation was present in injured tendons, Mφ sub-populations were quantified based on surface antigen expression of...
