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.
Phillips T, Ferraz I, Bell S, Clegg PD, Carter SD, Mobasheri A.Glucose serves as the major energy substrate for articular chondrocytes and as the main precursor for the synthesis of extracellular matrix glycosaminoglycans in cartilage. Chondrocytes have been shown to express several glucose transporter (GLUT) isoforms including GLUT1 and GLUT3. The aim of this investigation was to determine the effects of endocrine and cytokine factors on the capacity of equine articular chondrocytes for transporting 2-deoxy-d-[2,6-3H] glucose and on the expression levels of GLUT1 and GLUT3. Chondrocytes maintained in monolayer culture were stimulated for 24 h with TNF-al...
Henderson KM, Eayrs K.To develop a means of determining pregnancy status in horses based on measuring serum oestrone sulphate (OS) concentrations using a rapid lateral flow immunoassay, and to determine the assay's effectiveness using a visual end-point. Methods: Serum samples from mares >100 days post-mating (n=701) were assayed using a nitrocellulose membrane-based lateral flow immunoassay device. The device was developed using membrane-bound 1,3,5 (10)-estratrien-3-ol-17-one conjugated to bovine serum albumin as the capture antigen, and an OS-detection monoclonal antibody coupled to colloidal gold as the visi...
Kankofer M, Kolm G, Aurich J, Aurich C.Sperm cell membranes are susceptible to peroxidative damage by an excess of reactive oxygen species (ROS). Antioxidative defence systems consisting of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) physiologically control the balance between ROS production and neutralization. In the present study the hypothesis was tested that lipid peroxidation occurs during storage of semen at 5 degrees C and that semen extender has positive effects on the antioxidative potential of equine semen. The aim of the study was to determine the activity of GSH-Px, SOD and CAT and the...
Bhuyan AK, Rao DK, Prabhu NP.Proteins meet with the stipulations of Levinthal. Two test tube variants of ferrocytochrome c (ferrocyt c) whose thermodynamic stabilities are vastly different refold to the same global minimum under a given final native condition, and they do so quickly at rates that do not reflect a strong dependence on the thermodynamic driving force. The transition-state ensemble is more unfolded-like, and the folding barrier offered is energetically sizable. The experiments involve neutral- (pH 7) and alkaline ferrocyt c pH (12.7), whose aqueous stabilities are 18 (+/-0.3) and 3 (+/-0.5) kcal mol(-)(1), r...
Lankveld DP, Bull S, Van Dijk P, Fink-Gremmels J, Hellebrekers LJ.Ketamine is widely used in equine anaesthesia. Beside its anaesthetic and analgesic properties, ketamine possesses a cytokine-modulating activity. However, to date, no data are available regarding the inhibitory effect of ketamine on the cytokine response in horses. In horses, cytokines such as tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) play a pivotal role in the pathogenesis of equine endotoxaemia following gastrointestinal disorders. Hence, the objective of this study was to assess the influence of ketamine on LPS-induced TNF-alpha and IL-6 formation in an equine macro...
Fierheller EE, Wilson DG.To determine failure strength, stiffness, and failure mode of 4 suture materials in equine linea alba. Methods: Randomized complete block design. Methods: Linea albas collected from 12 adult horses (mean weight, 475 kg; mean age, 10 years). Methods: The ventral abdominal fascia, including the linea alba, was collected and bisected along the linea alba into right and left halves. Each half was divided into four, 5-cm sections extending from the umbilicus cranially, and randomly assigned to 1 of 4 suture materials: 2 polydioxanone (2 PD), 3 polyglactin 910 (3 PG), 6 polyglactin 910 (6 PG), and 7...
Flaminio MJ, Antczak DF.At days 36-38 of gestation, the equine invasive trophoblast cells migrate into the endometrium of the pregnant mare to form the endometrial cups. During their migration, they become surrounded by maternal CD4+ and CD8+ T lymphocytes, and stimulate a cytotoxic antibody response to the paternal major histocompatibility complex class I antigens that they express. Nevertheless, endometrial cup cells remain viable at the site of uterine invasion up to days 80-100 of gestation, suggesting the participation of immunomodulatory mechanisms to the maternal cellular immune response. To determine the effe...
Albrecht RA, Kim SK, O'Callaghan DJ.The equine herpesvirus 1 (EHV-1) EICP27 protein cooperates with either the immediate-early (IE) or the EICP0 protein to synergistically trans-activate viral promoters. GST-pulldown and co-immunoprecipitation assays revealed that the EICP27 protein's cooperation with the IE or the EICP0 protein involves its physical interaction with these viral proteins. In the case of the IE-EICP27 protein interaction, IE residues 424 to 826 and EICP27 residues 41 to 206 harbor the interactive domains. Electrophoretic mobility shift assays (EMSA) suggested that the EICP27 protein is not a sequence-specific DNA...
Little CB, Flannery CR, Hughes CE, Goodship A, Caterson B.To determine whether the focal susceptibility to cartilage degeneration in joints is related to a differential response to cytokine stimulation. Methods: Compare aggrecan and collagen catabolism in in-vitro models of cartilage degradation induced by retinoic acid (RA), interleukin-1 (IL-1), tumor necrosis factor alpha (TNF) and IL-1 plus oncostatin M (OSM). Glycosaminoglycan (GAG) and hydroxyproline (HyPro) quantification and Western immunoblot analyses of aggrecan and collagen degradation products were undertaken in explant cultures of normal cartilage from regions of equine joints with a kno...
Doyle AJ, Stewart AA, Constable PD, Eurell JA, Freeman DE, Griffon DJ.To determine effects of sodium hyaluronate (HA) on corticosteroid-induced cartilage matrix catabolism in equine articular cartilage explants. Methods: 30 articular cartilage explants from fetlock joints of 5 adult horses without joint disease. Methods: Articular cartilage explants were treated with control medium or medium containing methylprednisolone acetate (MPA; 0.05, 0.5, or 5.0 mg/mL), HA (0.1, 1.0, or 1.5 mg/mL), or both. Proteoglycan (PG) synthesis was measured by incorporation of sulfur 35-labeled sodium sulphate into PGs, and PG degradation was measured by release of radiolabeled PGs...
Legardinier S, Duonor-Cérutti M, Devauchelle G, Combarnous Y, Cahoreau C.Equine luteinizing hormone (eLH) and chorionic gonadotropin (eCG) are composed of identical alpha and beta polypeptide chains, but eCG subunits are much more heavily glycosylated and sialylated. Consequently, eCG exhibits a much longer half-life than eLH in blood. Recombinant eLH/CG, expressed in Sf9 and Mimic insect cells, were compared with one another and to the natural hormones eCG and eLH. Mimic cells are stably-transformed Sf9 cells, expressing five mammalian genes encoding glycosyltransferases involved in the synthesis of complex N-carbohydrate chains. Recombinant eLH/CG expressed in Mi...
Benton HP, Esquivel AV, Rice AD, Giri SN.Pirfenidone is under investigation as an anti-inflammatory and anti-fibrotic agent in several organs including lung. Since important features of arthritic conditions include inflammation and long-term damage to articular cartilage, we have investigated whether PD can suppress chondrocyte responses to bacterial lipopolysaccharide (LPS) and interleukin 1 (IL-1); modulators that induce a cascade of inflammatory responses that lead to articular joint tissue damage. PD (0 - 5microM) showed no effect on cell number or viability when incubated with high density primary equine chondrocyte cultures for...
Hornyák A, Dénes B, Szeredi L, Dencsö L, Rusvai M.Two monoclonal antibodies against the Bucyrus strain of equine arteritis virus (EAV) were produced, and according to immunoperoxidase reaction following Western blot of electrophoresed EAV structural proteins, they recognized the nucleocapsid (N) protein antigen (14-kDa protein). Besides reacting with the blotted polypeptide, the antibodies of the two clones (designated 1H1 and 4G6) selected from 576 have shown high affinity and specificity to intracellular virus antigen as well. Both antibodies reacted with the representatives of the different subtypes of equine arteritis virus providing a su...
Gibson VA, Stover SM, Gibeling JC, Hazelwood SJ, Martin RB.We hypothesized that recently formed, incompletely mineralized, and thus, relatively deformable osteons in the equine third metacarpus enhance in vitro load-controlled fatigue life in two ways. Macroscopically, there is a compliance effect, because reduced tissue elastic modulus diminishes the stress required to reach a given strain. Microscopically, there is a cement line effect, in which new osteons and their cement lines more effectively serve as barriers to crack propagation. We studied 18 4 x 10 x 100 mm beams from the medial, lateral, and dorsal cortices of metacarpal bones from 6 thorou...
Gutmann S, Zawatzky R, Müller M.Interferon-gamma (IFN-gamma) is a key cytokine in cell-mediated immunity. To measure IFN-gamma production of equine lymphocytes (eqIFN-gamma), we developed a quantitative ELISA. Monoclonal antibodies (mAb) were produced against bacterially derived eqIFN-gamma. The mAbs recognised recombinant and lymphocyte-derived eqIFN-gamma in ELISA, Western blotting, as well as flow cytometric and microscopic analysis. In contrast to bacterially derived material, mammalian and insect cell-derived eqIFN-gamma was biologically active but could be neutralised by one of the monoclonal antibodies. Unexpectedly, ...
Johnson NL, Galuppo LD, Stover SM, Taylor KT.To compare screw insertion variables and pullout mechanical properties between AO 6.5-mm cancellous and 7.3-mm cannulated bone screws in foal femoral bone. Methods: A paired, in vitro mechanical study. Methods: Seven pairs of femora from immature (1-7 months) foals. Methods: The 6.5 cancellous and 7.3-mm cannulated screws were inserted at standardized proximal and distal metaphyseal, and mid-diaphyseal locations. Insertion torque, force, and time to drill, tap (6.5-mm cancellous), guide wire insertion (7.3-mm cannulated), and screw insertion were measured. Screw pullout properties (yield and f...
Beutelspacher SC, Ardjomand N, Tan PH, Patton GS, Larkin DF, George AJ, McClure MO.In this study we compare the ability of self-inactivating Human Immunodeficiency Virus 1 (HIV-1) and Equine Infectious Anaemia Virus (EIAV)-based vectors to mediate gene transfer to rabbit and human corneas and to a murine corneal endothelial cell line. Both vectors were pseudotyped with vesicular stomatitis virus-G (VSV-G) envelope and contained marker transgenes under the control of an internal CMV promoter. For specificity of action, the heterologous promoter in the EIAV-vector was exchanged for an inducible E-Selectin promoter, previously shown to regulate gene-expression in a plasmid syst...
Degueurce C, Chateau H, Denoix JM.Sacroiliac joint (SIJ) disease is associated with poor hindlimb action, lameness and poor performance in horses. However, little is known about the biomechanics of this low-motion joint. Objective: To determine in vitro the capacities of movement of the SIJ in the sagittal plane, and to test the effect of a sacrosciatic and sacrotuberal desmotomy on its stabilisation. Methods: Six anatomical specimens underwent cycles of flexion-extension of the lumbosacral joint (LSJ) before and after desmotomy. Kinematic triads were linked rigidly to the sacrum, spinous process of vertebra L5 and iliac wing....
Hobbs SJ, Mather J, Rolph C, Bower JA, Matuszewski B.Strains during stance on the hoof wall surface have been measured by a number of authors in vitro and in vivo. Histological structure and mechanical properties vary through the wall thickness (radially); radial strain measurements may therefore aid the understanding of mechanical function of the capsule and adjacent tissues. Objective: To develop instrumentation capable of measuring internal hoof strain, and to carry out a preliminary comparison of normal and laminitic hooves. Methods: Six forelimbs from 4 horses, including 2 with laminitis from the same horse, were tested using an Instron tes...
Tomlinson JE, Blikslager AT.Recent studies have shown that flunixin prevented recovery of equine jejunum post ischaemia. However, the use of a purported cyclooxygenase (COX)-2 preferential inhibitor, etodolac, also prevented recovery. These findings may have implications for the use of nonsteroidal anti-inflammatory drugs in colic patients. Objective: To compare the effects of deracoxib, a highly selective canine COX-2 inhibitor, with flunixin on in vitro recovery of ischaemic-injured equine jejunum. Methods: Six horses underwent 2 h jejunal ischaemia, after which mucosa was mounted in Ussing chambers and recovered for 2...
Sanchez LC, Merritt AM.Most current models of visceral sensitivity testing in the horse have required visceral cannulation. Colorectal distention (CRD) is a widely used, noninvasive method for testing in other species and could be adapted for use in horses. Objective: To develop a protocol of controlled CRD in the conscious horse and to evaluate the effect of i.v. xylazine or intrarectal lidocaine on CRD threshold and rectal compliance. Methods: Eight horses were used for baseline studies (3 trials each) and 6 horses to evaluate treatments (4 trials, 2 per treatment). A 45 cm diameter polyvinyl balloon attached to p...
Klinc P, Kosec M, Majdic G.The success rate of artificial insemination following the freezing of stallion semen is limited; therefore, improving the stallion semen quality after the freezing and thawing process is a necessary objective. Objective: To investigate the influence of glass bead column separation on the freezability of stallion semen. Objective: Glass beads in a column separator remove damaged and dead spermatozoa in the ejaculate during centrifugation. Methods: In total, 50 ejaculates from 6 Lipizzaner stallions were studied. Each ejaculate was divided into 2 parts, one half processed following standard proc...
Gamboa S, Ramalho-Santos J.Proteins implicated in the "SNARE hypothesis" for membrane fusion have been characterized in the acrosome of several mammalian species, and a functional role for these proteins during the acrosome reaction has been proposed. We have investigated the presence of SNAREs in equine sperm, using semen samples obtained from stallions with varying fertility. Immunocytochemical analysis revealed that members of different SNARE families can be detected on the acrosome of equine sperm, notably in the acrosomal cap and equatorial segment. These proteins include the t-SNARE syntaxin, the v-SNARE synaptobr...
Cappelli K, Porceddu A, Verini-Supplizi A, Capomaccio S, Marchis FD, Falcinelli M, Gaiti A, Silvestrelli M.The identification of differentially expressed genes is a fundamental prerequisite for understanding the molecular regulation of most physiological and pathological processes. Among the procedures employed to compare mRNA populations, those that are gel-based appear to hold great promise and are considered excellent tools for studying gene expression in species, such as the equine one, for which little genomic information is available. In the present study, we evaluated two techniques for studying mRNA profiles in horse tissue, one referred to the cDNA-amplified fragment length polymorphism (A...
Hinrichs K, Choi YH, Love LB, Varner DD, Love CC, Walckenaer BE.We evaluated the relationship of initial chromatin configuration to time of oocyte recovery and to nuclear maturation after culture in horse oocytes having compact (Cp) and expanded (Ex) cumuli. In addition, we evaluated the effect of oocyte type, time of recovery, and duration of culture on blastocyst development after intracytoplasmic sperm injection. In oocytes collected within 1 h of slaughter, fibrillar and intermediate chromatin configurations were more prevalent in Cp than in Ex oocytes (68% and 12%, respectively). In Cp oocytes collected after a 5- to 9-h delay, the proportions in the ...
Hosaka Y, Sakamoto Y, Kirisawa R, Watanabe T, Ueda H, Takehana K, Yamaguchi M.Although tumor necrosis factor (TNF) alpha is an important key factor in degeneration of equine superficial digital flexor tendon (SDFT), the dynamism of TNF receptors and associated factors on tendinocytes has not been elucidated. To reveal signaling events mediated by TNF-receptors (TNF-Rs) in tendinocytes, we focused on four signaling factors, TNF-R1, TNF-R2, TNF-R-associated factor 2 (TRAF2) and nuclear factor-kappa B (NF-kappaB), and investigated the distribution and production of these factors. Cultured tendinocytes were obtained from SDFTs of thoroughbred horses. The tendinocytes were t...
Amann RP.Apparent fertility of a male or group of females is considered frequently by veterinarians or animal scientists. Unfortunately, concepts of experimental design and statistics impacting validity and interpretation of values for average pregnancy rate frequently are ignored. The magnitude of this problem was documented by examination of published papers; 51 of 67 (76%) were considered flawed for one or more reasons. The discussion considers why: (a) conclusions from most published fertility studies reporting no significant difference due to treatment(s) are suspect, because too few males and/or ...
Breathnach CC, Soboll G, Suresh M, Lunn DP.A commercial bovine IFN-gamma-specific monoclonal antibody was used to measure antigen-specific IFN-gamma production by equine lymphocytes. Paired PBMC samples were collected from six ponies prior to and 10 days after challenge infection with equine herpesvirus-1 (EHV-1). Each sample was stimulated in vitro with EHV-1, virus-free medium, or PMA and ionomycin, and labelled with monoclonal antibodies specific for various equine lymphocyte subset markers. Following fixation, intracellular IFN-gamma was detected using a FITC-conjugated bovine IFN-gamma-specific monoclonal antibody. In vitro restim...
Albrizio M, Guaricci AC, Maritato F, Sciorsci RL, Mari G, Calamita G, Lacalandra GM, Aiudi GG, Minoia R, Dell'Aquila ME, Minoia P.The development of fertilizing ability in sperm cells is associated with changes in the plasma membrane. However, to date the exact nature of sequentially activated primary receptors and channels and the signal transduction pathways derived from these remains elusive. We analyzed the expression and localization of the mu-opioid receptor in equine spermatozoa. A transcript corresponding to the third extracellular loop that selectively binds mu agonists was amplified, sequenced and compared with the known sequences in humans, rats and cattle. The amplification product showed a high degree of nuc...
Haupt JL, Frisbie DD, McIlwraith CW, Robbins PD, Ghivizzani S, Evans CH, Nixon AJ.This study evaluated the potential of gene induced synoviocyte expression of a combination of insulin-like growth factor-I (AdIGF-I) and interleukin-1 receptor antagonist protein (AdIL-1Ra) to control articular cartilage degradation in vitro. Cartilage explants and synovial membrane were harvested from young mature horses. Synovial monolayers were established and either (1) maintained as untransduced controls; (2) transduced with AdIGF-I at 200 MOI in 500 microl serum-free medium; (3) transduced with AdIL-1Ra at 100 MOI; or (4) transduced with a combination of AdIGF-I (200 MOI) and AdIL-1Ra (1...
Wooldridge AA, Eades SC, Hosgood GL, Moore RM.To characterize the in vitro effects of oxytocin, acepromazine, xylazine, butorphanol, detomidine, dantrolene, isoproterenol, and terbutaline on skeletal and smooth muscle from the equine esophagus. Methods: 14 adult horses without digestive tract disease. Methods: Circular and longitudinal strips from the skeletal and smooth muscle of the esophagus were suspended in tissue baths, connected to force-displacement transducers interfaced with a physiograph, and electrical field stimulation was applied. Cumulative concentration-response curves were generated for oxytocin, acepromazine, xylazine, d...
Araujo-Viel MS, Juliano L, Prado ES.The synthetic tridecapeptide Gly-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg was used as a model substrate for horse urinary and porcine pancreatic kallikreins. The Met-Lys bond is hydrolyzed selectively by both enzymes. Oxidation of the methionine residue to sulfoxide made the peptide resistant to both kallikreins. Substitution of either the methionine or lysine residues by norleucine led to peptides in which the Nle-Lys or the Met-Nle bonds, respectively, were susceptible to the urinary kallikrein. The esterolytic and Met-Lys bond-splitting activities of both enzymes were inhibited simil...
Haerdi-Landerer MC, Habermacher J, Wenger B, Suter MM, Steiner A.The search for an effective treatment for septic arthritis is ongoing. Current therapies are expensive since they require repeated joint lavage and long term antibiotic treatment. Local application of antimicrobial drugs is advantageous because high concentrations can be attained at the infection site, although repeated injections increase the risk of superinfection of the joint. Thus, slow release formulations, which have the advantage of local treatment yet single application of the drug, are appealing. Antibiotics used in slow release formulations are selected for tissue compatibility, an a...
Satoh K, Yasuda I, Nagai F, Ushiyama K, Akiyama K, Kano I.In the folk-medicine, several kinds of crude drugs are used as diuretics. Twenty three kinds of diuretic drugs were chosen, and examined for their effects on the horse kidney (Na+ + K+)-adenosine triphosphatase (ATPase), which is an intrinsic enzyme of the plasma membrane and responsible for the active transport of Na+ and K+ across the membrane. Twenty one out of twenty three kinds of ethanol extracts of diuretic drugs inhibited the kidney (Na+ + K+)-ATPase activity. The intensity of the inhibition of these drugs was compared by estimating the amounts of their ethanol extracts which inhibited...
Jablonka-Shariff A, Roser JF, Bousfield GR, Wolfe MW, Sibley LE, Colgin M, Boime I.To study structure-activity relationships and the role of equine gonadotropins in the normal and pathophysiology of equine reproduction, the availability of purified hormones is essential. Previous expression studies in transfected CHO cells showed inefficient assembly of the human and bovine alpha and beta subunits, resulting in low levels of recombinant LH. The ability to express a single chain bearing genetically linked alpha and beta subunits bypasses this rate-limiting assembly step. A chimera was constructed by overlap PCR in which the carboxy terminal end of the eLHbeta subunit was gene...
Roach J, Schnobrich M, Ellerbrock R, Feijo L, Bradecamp E, Alvarenga MA, Kline K, Canisso I.This study compares two methods for seminal plasma removal by evaluating sperm recovery rates, and motility and morphology of cooled-stored semen. Ejaculates were divided into three groups: control, filtration and cushioned centrifugation. Semen was extended to 25 million sperm/ml using a skim-milk-based extender and stored at 5°C for all groups. Sperm motility (total motility (%TM) and progressive motility (%PM)) was determined at 0, 24, 48 and 72 hours by a computer-assisted sperm analyser. Sperm morphology was assessed using differential interference microscopy. Overall, %TM of the cen...
Turowska A, Pajak B, Godlewski MM, Dzieciatkowski T, Chmielewska A, Tucholska A, Banbura M.Viruses can reorganize the cytoskeleton and restructure the host cell transport machinery. During infection viruses use different cellular cues and signals to enlist the cytoskeleton for their mission. However, each virus specifically affects the cytoskeleton structure. Thus, the aim of our study was to investigate the cytoskeletal changes in homologous equine dermal (ED) and heterologous Vero cell lines infected with either equine herpesvirus 1 (EHV-1) strain Rac-H or Jan-E. We found that Rac-H strain disrupted actin fibers and reduced F-actin level in ED cells, whereas the virus did not infl...
Suthar A, Maji C, Gopalkrishnan A, Raval SH, Kumar R, Kumar S.Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis (EP). Currently, imidocarb dipropionate (ID) is the only available drug for treating the clinical form of EP. Serious side effects and incomplete clearance of infection is a major drawback of ID. Heat-shock proteins (Hsp) play a vital role in the life cycle of these haemoprotozoans by preventing alteration in protein conformation. These Hsp are activated during transmission of EP sporozoites from the tick vector (poikilotherm) to the natural host (homeotherm) and facilitate parasite survival. In the present stu...
Ek N.The Pr protein, which is one of the major equine acidic prealbumins and which consists of a large number of phenotypes, has been studied with regard to its chemical identity. Serum samples of known Pr phenotype which had been treated with varying amounts of bovine trypsin were subjected to starch gel electrophoresis at pH 4.8. When a certain amount of trypsin was used, the Pr protein was markedly affected, whereas the other acidic prealbumins retained their normal electrophoreitic pattern. Extracts from three different regions of the acidic prealbumin field were tested by the casein precipitat...
Von Dollen KA, Jones M, Beachler T, Harris TL, Papich MG, Lyle SK, Bailey CS.The use of antimicrobials for the management of equine uterine disease is commonplace, with antibiotic selection generally based on empirical evidence or in vitro sensitivity results. However, the potential disconnect between these laboratory results and clinical efficacy in the mare raises concern for antibiotic failure and subsequent development of resistant organisms. In this work, we attempt to bridge this gap by using an ex vivo model of the equine postpartum uterus to quantitatively evaluate the antimicrobial activity of two commonly used antibiotic treatments in the mare (ceftiofur an...
Ferrari G, Pang LY, De Moliner F, Vendrell M, Reardon RJM, Higgins AJ, Chopra S, Argyle DJ.Bleomycin is a chemotherapy agent that, when administered systemically, can cause severe pulmonary toxicity. Bleosome is a novel formulation of bleomycin encapsulated in ultra-deformable (UD) liposomes that may be applicable as a topical chemotherapy for diseases such as non-melanoma skin cancer. To date, the ability of Bleosome to effectively penetrate through the skin has not been evaluated. In this study, we investigated the ability of Bleosome to penetrate through ex vivo skin explants from dogs and horses. We visualized the penetration of UD liposomes through the skin by transmission elec...
Balena V, Pradhan SS, Bera BC, Anand T, Sansanwal R, Khetmalis R, Madhwal A, Bernela M, Supriya K, Pavulraj S, Tripathi BN, Virmani N.Equid alphaherpesvirus 1 (EHV-1) infection causes significant health problems in equines. The EHV-1 infection leads to abortion storm in mares, respiratory disease and myeloencephalopathy. Despite the wide use of vaccines, the outbreaks of EHV-1 infections keep occurring globally, suggesting the need for the development of improved vaccines. Gene deletion attenuated mutant viruses could be a good candidate for the development of modified live vaccines. Here, we report the generation of mutant EHV-1 by deleting virulence (glycoprotein E & internal repeat 6; IR6) and immune evasive (pUL43 &a...
Meng Q, Li S, Liu L, Xu J, Liu Y, Zhang Y, Zhang X, Shao Y.Equine Infectious Anemia Virus (EIAV), like other lentiviruses, has a transmembrane glycoprotein with an unusually long cytoplasmic tail (CT). Viral envelope (Env) proteins having CT truncations just downstream the putative membrane-spanning domain (PMSD) are assumed to exist among all wild-type budded virions, and also in some cell-adapted strains. To determine whether CT-truncated Env proteins can cause particularly deleterious effects on the Env expressing cells and/or their neighboring cells, plasmids encoding codon-optimized env gene including full-length (pE863) or CT-truncated (pE686* a...
Umbilical cord matrix mesenchymal stem cells (UCM-MSCs) present a wide range of potential therapeutical applications. The extracellular calcium-sensing receptor (CaSR) regulates physiological and pathological processes. We investigated, in a large animal model, the involvement of CaSR in triggering osteogenic and neurogenic differentiation of two size-sieved UCM-MSC lines, by using AMG641, a novel potent research calcimimetic acting as CaSR agonist. Results: Large (>8 µm in diameter) and small (<8 µm) equine UCM-MSC lines were cultured in medium with high calcium (Ca2+) concentration (...
Colbath AC, Kisiday JD, Phillips JN, Goodrich LR.Both bone marrow-derived mesenchymal stem cells (BMDMSCs) and extracorporeal shockwave (ESW) have shown promise for enhancing fracture repair. If exposure of BMDMSCs to ESW enhances osteogenic differentiation, these therapies may be combined in vivo or used as a method for preconditioning BMDMSCs. The objective of this study was to determine the effect of ESW on the osteogenic ability of equine BMDMSCs. We hypothesized that ESW would promote osteogenesis evidenced by increased gene expression, alkaline phosphatase (ALPL) expression, slide morphologic score, and protein expression. BMDMSCs were...
Thomas LM, Huntington PJ, Mead LJ, Wingate DL, Rogerson BA, Lew AM.The use of the bacterial expression vector, pGex, to produce an abundant, soluble fusion protein of gp45 from equine infectious anaemia virus is described. Purification of the recombinant protein was achieved by one step affinity chromatography on immobilized glutathione using competitive elution so no harsh conditions were required. This provides a readily available antigen that is defined, plentiful and cheap. Yields of 3.5 mg of purified soluble protein/litre of bacterial culture were obtained. This antigen was found to be suitable for ELISA. Background reactivity to either the glutathione-...
Giese A, Jude R, Kuiper H, Raudsepp T, Piumi F, Schambony A, Guérin G, Chowdhary BP, Distl O, Töpfer-Petersen E, Leeb T.The cysteine-rich secretory protein (CRISP) family consists of three members called acidic epididymal glycoprotein 1 (AEG1), AEG2, and testis-specific protein 1 (TPX1), which share 16 conserved cysteine residues at their C-termini. The CRISP proteins are primarily expressed in different sections of the male genital tract and are thought to mediate cell-cell interactions of male germ cells with other cells during sperm maturation or during fertilization. Therefore, their genes are of interest as candidate genes for inherited male fertility dysfunctions and as putative quantitative trait loci fo...
Wilson CR, Sauer JM, Carlson GP, Wallin R, Ward MP, Hooser SB.A comparative study of vitamin K(1) 2,3-epoxide reductase (VKOR) activity in vitro was conducted across species. The apparent kinetic constants K(m app), V(max), and Cl(int app) were determined in bovine, canine, equine, human, murine, ovine, porcine, and rat hepatic microsomes. In addition to these enzyme kinetic constants, the IC(50) of warfarin for VKOR was determined in human, murine, porcine, and rat hepatic microsomes. Interspecies differences were observed when comparing the K(m app) (range, 2.41-6.46 microM), V(max) (range, 19.5-85.7 nmol/mg/min), and Cl(int app) (range, 8.2-18.4 ml/mg...
Saad B, Corradin G, Bosshard HR.The antigenic determinants for two monoclonal antibodies directed against horse apo-cytochrome c, a protein of disordered structure, as judged by spectroscopic and hydrodynamic criteria, have been studied by a combination of methods: antigen competition in solution by radio immunoassay and enzyme-linked immunoassay, and differential acetylation of free and antibody-bound antigen. In the latter method the accessibility of lysine residues of the antigen in the antigen-antibody complex is compared to the accessibility in the free antigen. The two antibodies against the heme-free protein do not re...
Kumar S, Kanwar S, Bansal V, Sehgal R.Diphtheria toxin has the capacity to block protein synthesis in cultured mammalian cells, and thus causing cell death. This capacity of diphtheria toxin was utilized for in-vitro neutralization test to determine antibody titer, using Vero cells, which have been found to be susceptible to diphtheria toxin. In the present study, a Vero cell assay was standardized and validated for potency estimation of diphtheria antitoxin serum (DATS). The results obtained by Vero cell assay were compared with in-vivo biological assay. High degree of correlation (+0.98) was found between in-vivo biological assa...
Groot MT, Merkx M, Koper MT.The redox properties of horse and yeast cytochrome c electrostatically immobilized on carboxylic acid-terminated self-assembled monolayers (SAMs) have been determined over a broad pH range (pH 3.5-8) in the absence and presence of nitric oxide. Below pH 6, both proteins exhibit comparable midpoint potentials, coverages, and electron-transfer rate constants, which suggests that they are adsorbed on the SAM in a similar fashion. Above pH 6, a sharp decrease in electron-transfer rate constants is observed for immobilized yeast cytochrome c, which is indicative of a change in the electron tunnelin...
Huang Z, Depoortere I, De Clercq P, Peeters T.Motilin is involved in the regulation of the fasting motility pattern in man and in dog, but may have a different role in other species. Immunoreactive motilin has been demonstrated in several species, but the sequence is mostly unknown. The aim of this study was to isolate and sequence the cDNA encoding the motilin precursor from several mammalian species and from chicken. Total RNA was isolated from the duodenal mucosa of the chicken, dog, cow and horse. In each case single stranded cDNA was synthesized. Motilin cDNA fragments were amplified by PCR, ligated into a plasmid and cloned. Clones ...
Goehring LS, Brandes K, Ashton LV, Wittenburg LA, Olea-Popelka FJ, Lunn DP, Soboll Hussey G.Equine herpesvirus-associated myeloencephalopathy is the result of endothelial cell infection of the spinal cord vasculature with equine herpesvirus-1 (EHV-1) during cell-associated viraemia. Endothelial cell infection requires contact between infected peripheral blood mononuclear and endothelial cells. Inflammation generated during viraemia likely upregulates adhesion molecule expression on both cell types increasing contact and facilitating endothelial cell infection. Objective: Evaluating the role of anti-inflammatory drugs in decreasing endothelial cell infection with EHV-1. Methods: In vi...
Dahm AM, de Bruin A, Linat A, von Tscharner C, Wyder M, Suter MM.We describe the establishment and characterisation of equine keratinocyte cultures with maintenance of a high proliferative capacity up to the second passage. Improved attachment and growth were obtained by seeding primary cells on equine feeder layers. Subcultured keratinocytes showed optimal growth when seeded on collagen type I. The proliferation rate of cells on this substrate exceeded that seen for cells seeded on equine feeder layers. By immunohistochemistry, epithelial origin and state of differentiation of the equine keratinocytes were determined. They expressed keratin and desmoplakin...
Tozaki T, Ohnuma A, Nakamura K, Hano K, Takasu M, Takahashi Y, Tamura N, Sato F, Shimizu K, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Hamilton NA....The creation of genetically modified horses is prohibited in horse racing as it falls under the banner of gene doping. In this study, we developed a test to detect gene editing based on amplicon sequencing using next-generation sequencing (NGS). We designed 1012 amplicons to target 52 genes (481 exons) and 147 single-nucleotide variants (SNVs). NGS analyses showed that 97.7% of the targeted exons were sequenced to sufficient coverage (depth > 50) for calling variants. The targets of artificial editing were defined as homozygous alternative (HomoALT) and compound heterozygous alternative (ALT1/...
Hanel RM, Crawford PC, Hernandez J, Benson NA, Levy JK.To determine whether passive transfer of IgG in neonatal kittens affects plasma opsonic capacity and neutrophil phagocytic and oxidative burst responses to bacteria in vitro. Methods: 22 kittens from 6 specific pathogen-free queens. Methods: Kittens were randomized at birth into the following treatment groups: colostrum-fed, colostrum-deprived, or colostrum-deprived supplemented with feline or equine IgG. Blood samples were collected at intervals from birth to 56 days of age. Plasma IgG concentrations were determined by radial immunodiffusion assay. Neutrophil function was assessed by a flow c...
Michler JK, Hillmann A, Savkovic V, Mülling CKW.The easily accessible niche represented by skin and its appendages may serve as a promising source to complement modern regenerative medicine for horses. In humans and in animal models for human medicine, the hair follicle and its stem cell niches are well characterized. Since literature in this field of equine research is scarce, we sought to analyze cells of the dermal stem cell niche of the equine hair follicle morphologically and for a subset of markers useful for cell characterization via immunolabeling. We cultured equine forelock skin explants to obtain cultures with cells migrating fro...
Gil MC, Ferrusola CO, Anel-López L, Ortiz-Rodriguez JM, Alvarez M, de Paz P, Anel L, Peña FJ.Spermatozoa undergo apoptotic changes during the cryopreservation process. These changes, recently termed spermptosis, resemble the cryopreservation induced delayed onset of cell death observed after thawing of somatic cells. Due to its importance in cryobiology, methods to easily identify spermptotic cells are warranted. In this study, a well-validated method for identification of spermatozoa with caspase 3 activity was compared with use of the combination of Hoechst 33342 (H-42) and ethidium homodimer (Eth-1). Live, dead and apoptotic spermatozoa assessed with each method were compared using...
Hosaka Y, Ozoe S, Kirisawa R, Ueda H, Takehana K, Yamaguchi M.The aim of this study was to clarify whether matrix metalloproteinases (MMP-2 and -9: gelatinases) and pro-inflammatory cytokines [tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta] are induced by heat in tendon tissue in vitro and to test the hypothesis that heat exposure causes tendinocytes to synthesize pro-inflammatory cytokines and that synthesis of these cytokines, in turn, leads to up-regulation of synthesis of gelatinases. Isolated tendinocytes from equine superficial digital flexor tendons were cultured and all experiments were performed on cells passaged 3 or 4 times. In t...
Dowling BA, Dart AJ, Hodgson DR, Rose RJ, Walsh WR.To evaluate the effect of recombinant equine growth hormone (rEGH) on the in vitro biomechanical properties of normal adult equine superficial digital flexor tendon (SDFT). Methods: Completely randomized design. Methods: Nine Standardbred horses, 6 to 9 years of age with ultrasonographically normal forelimb SDFT. Methods: Six horses were administered intramuscular (IM) rEGH at 10 microg/kg/day for 1 week, and then 20 microg/kg/day for another 5 weeks; 3 horses (control subjects) were administered an equivalent daily volume of sterile water IM. Horses were killed at the end of the 6-week treatm...
