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.
Love CC.Sperm quality assays have increased in number in the last 10 years. Most of these assays are flow cytometry based in application and are modified from assays that have been developed to measure somatic cell function. The goal of any sperm quality assay should be to advance the clinicians/researchers understanding of sperm cell function and the relationship to fertility. While these assays appear to measure somatic cell-like functions in sperm there tends to be little understanding how the results of these assays relate to fertility.
Gabner S, Ertl R, Velde K, Renner M, Jenner F, Egerbacher M, Hlavaty J.A combination of tissue engineering methods employing mesenchymal stem cells (MSCs) together with gene transfer takes advantage of innovative strategies and highlights a new approach for targeting osteoarthritis (OA) and other cartilage defects. Furthermore, the development of systems allowing tunable transgene expression as regulated by natural disease-induced substances is highly desirable. Bone marrow-derived equine MSCs were transduced with a lentiviral vector expressing interleukin-1 receptor antagonist (IL-1Ra) gene under the control of an inducible nuclear factor-kappa B-responsive prom...
Demyda-Peyrás S, Bottrel M, Acha D, Ortiz I, Hidalgo M, Carrasco JJ, Gómez-Arrones V, Gósalvez J, Dorado J.The aim of this study was to evaluate the effect of different cooling rates on post-thaw quality of cryopreserved donkey spermatozoa. Eighteen ejaculates from six adult Andalusian donkeys (three ejaculates per donkey) were collected using an artificial vagina. Pooled semen samples (two ejaculates per pool) were divided into three aliquots, and frozen in Gent freezing extender using three different cryopreservation protocols (P): P1 (conventional slow freezing, as control): semen pre-cooled in an Equitainer for 2 h and frozen in liquid nitrogen (LN) vapour; P2 (controlled pre-freeze cooling r...
Fortuna PRJ, Bielefeldt-Ohmann H, Ovchinnikov DA, Wolvetang EJ, Whitworth DJ.Horses are susceptible to a number of neurotropic viruses, including West Nile virus (WNV), which is a pathogen of global significance in both horses and humans. However, there are no in vitro models with which to study infectious neuropathic diseases in the horse. In an effort to redress this, we have generated neurons from equine induced pluripotent stem cells (equiPSCs) that express a range of cortical neuron-specific markers, in addition to the membrane-bound ligand ephrin B3, which plays an important role in axon guidance as well as functioning as the receptor through which henipaviruses,...
Jiménez-Trejo F, Coronado-Mares I, Boeta M, González-Santoyo I, Vigueras-Villaseñor R, Arriaga-Canon C, Herrera LA, Tapia-Rodríguez M.Assisted reproductive techniques have been used on several domestic animals to preserve desirable traits in strains of high genetic and commercial value; however in equines its efficiency rate is relatively low. To increase the conception ratio in stallions, some research groups have used pharmacological treatments which promote sperm hyperactivation in order to increase male's fertility rates. In this way, our previous work suggests that serotonin (5-HT) could be a good pharmacological candidate that facilitates conception rate in domestic horses. 5-HT is a neurohormone involved in several re...
Salgado RM, Brom-de-Luna JG, Resende HL, Canesin HS, Hinrichs K.The aim of this study was to evaluate the differential effects of conventional and Piezo-driven ICSI on blastocyst development, and on sperm component remodeling and oocyte activation, in an equine model. Methods: In vitro-matured equine oocytes underwent conventional (Conv) or Piezo ICSI, the latter utilizing fluorocarbon ballast. Blastocyst development was compared between treatments to validate the model. Then, oocytes were fixed at 0, 6, or 18 h after injection, and stained for the sperm tail, acrosome, oocyte cortical granules, and chromatin. These parameters were compared between inject...
Galvão A, Wolodko K, Rebordão MR, Skarzynski D, Ferreira-Dias G.In the present report we describe the involvement of transforming growth factor B1 (TGF) in functional regression and structural luteolysis in the mare. Firstly, TGF and its receptors activin-like kinase (ALK) 5 and TGF receptor 2 were identified in corpus luteum (CL) steroidogenic, endothelial and fibroblast-like cells. Also, TGF and ALK5 protein expression were shown to be increased in Mid-, and Late-CL (p < 0.05). Subsequently, using an in vitro model with Mid-CL cells, we studied the role of TGF on secretory activity and cell viability. Cell treatment with TGF decreased progesterone...
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...
Sandbaumhüter FA, Thormann W.Glucuronidation catalyzed by uridine-5'-diphospho-glucuronosyl-transferases (UGTs) is the most important reaction in phase II metabolism of drugs and other compounds. O-glucuronidation is more common than N-glucuronidation. The anesthetic, analgesic and antidepressive drug ketamine is metabolized in phase I by cytochrome P450 enzymes to norketamine, hydroxynorketamine (HNK) diastereomers and dehydronorketamine (DHNK). Equine urine samples collected two hours after ketamine injection were treated with β-glucuronidase and analyzed with three enantioselective capillary electrophoresis assays. Co...
Sandbaumhüter FA, Vimercati S, Thormann W, Mevissen M.The anesthetic ketamine is often combined with analgesics and benzodiazepines in equine medicine. Therefore, drug-drug interactions are possible. Enzyme kinetics for ketamine N-demethylation were determined using equine CYP3A94, CYP3A95 and CYP3A97, and the effect of medetomidine, diazepam and methadone on the ketamine metabolism was studied in vitro. Ketamine was incubated with the CYPs or equine liver microsomes (ELM) alone or in presence of medetomidine, diazepam and/or methadone for different times. Norketamine levels were determined using enantioselective capillary electrophoresis (CE) wi...
Byambaragchaa M, Lee SY, Kim DJ, Kang MH, Min KS.Previous studies showed that recombinant equine chorionic gonadotropin (rec-eCGβ/α) exhibits both follicle-stimulating hormone (FSH) and luteinizing hormone (LH)-like activities in rat LHR- and FSHR-expressing cells. In this study, we analyzed signal transduction by eelFSHR and eelLHR upon stimulation with rec-eCGβ/α and native eCG. The cyclic adenosine monophosphate (cAMP) stimulation in CHO-K1 cells expressing eelLHR was determined upon exposure to different doses (0-1,450 ng/mL) of rec-eCGβ/α and native eCG. The EC values of rec-eCGβ/α and native eCG were 172.4 and 786.6 ng/mL, resp...
Rassoli A, Fatouraee N, Guidoin R.The benefit of bioprosthetic aortic valve over mechanical valve replacements is the release of thromboembolism and digression of long-term anticoagulation treatment. The function of bioprostheses and their efficiency is known to depend on the mechanical properties of the leaflet tissue. So it is necessary to select a suitable tissue for the bioprosthesis. The purpose of the present study is to clarify the viscoelastic behavior of bovine, equine, and porcine pericardium. In this study, pericardiums were compared mechanically from the viscoelastic aspect. After fixation of the tissues in glutara...
Anderson SL, Townsend HGG, Singh B.OBJECTIVE To evaluate the effect of lipopolysaccharide (LPS) on apoptosis of equine neutrophils in vitro. SAMPLE Venous blood samples from 40 adult horses. PROCEDURES Neutrophils were isolated from blood samples and cultured with or without LPS from Escherichia coli O55:B5 for 12 or 24 hours. Neutrophil apoptosis was assessed by use of cytologic examination, annexin V and propidium iodide staining quantified with flow cytometry, coincubation with inducers of intrinsic and extrinsic apoptosis or a toll-like receptor (TLR) 4 inhibitor, and measurement of caspase-3, -8, and -9 activities. RESULTS...
Vanderstock JM, Lecours MP, Lavoie-Lamoureux A, Gottschalk M, Segura M, Lavoie JP, Jean D.OBJECTIVE To evaluate in vitro phagocytosis and bactericidal activity of circulating blood neutrophils in horses with severe equine asthma and control horses and to determine whether circulating blood neutrophils in horses with severe equine asthma have an increase in expression of the proinflammatory cytokine tumor necrosis factor (TNF)-α and the chemokine interleukin (IL)-8 and a decrease in expression of the anti-inflammatory cytokine IL-10 in response to bacteria. ANIMALS 6 horses with severe equine asthma and 6 control horses. PROCEDURES Circulating blood neutrophils were isolated from h...
Wang XF, Liu Q, Wang YH, Wang S, Chen J, Lin YZ, Ma J, Zhou JH, Wang X.The equine infectious anemia virus (EIAV) attenuated vaccine was developed by long-term passaging of a field-isolated virulent strain in cross-species hosts, followed by successive cultivation in cells To explore the molecular mechanism underlying the evolution of the EIAV attenuated vaccine, a systematic study focusing on long-terminal-repeat (LTR) variation in numerous virus strains ranging from virulent EIAV to attenuated EIAV was performed over time both and Two hypervariable regions were identified within the U3 region in the enhancer region (EHR) and the negative regulatory element (N...
Santos VH, Pfeifer JPH, de Souza JB, Milani BHG, de Oliveira RA, Assis MG, Deffune E, Moroz A, Alves ALG.Mesenchymal stem cells derived from the synovial membrane (MSCSM) have a greater potential for joint regeneration, besides the capacity for chondrogenic differentiation, since they are a source closer to the chondrocytes. This study aimed to cultivate and evaluate viability and differentiation of MSC encapsulated in a three-dimensional alginate hydrogel (HA) scaffold. Samples of the synovial membrane of the metatarsophalangeal joint of 4 horses were collected by astroscopic surgery. These were subjected to enzymatic digestion, isolated mesenchymal cells, cultured in monolayers and encapsulated...
The ultra long-acting β -adrenoceptor agonist olodaterol plus the ultra long-acting muscarinic antagonist tiotropium bromide are known to relax equine airways. In human bronchi combining these drugs elicits a positive interaction, thus we aimed to characterize this information further in equine isolated airways stimulated by electrical field stimulation (EFS) and using the Concentration-Reduction Index (CRI) and Combination Index (CI) equations. The drugs were administered alone and together by reproducing ex vivo the concentration-ratio delivered by the currently available fixed-dose combina...
Varela E, Rey J, Plaza E, Muñoz de Propios P, Ortiz-Rodríguez JM, Álvarez M, Anel-López L, Anel L, De Paz P, Gil MC, Morrell JM....Contaminating bacteria present in stallion ejaculates may compromise sperm quality during storage. Different procedures have been used to reduce the load of microorganisms in semen and avoid bacterial growth during storage. The aims of this study were: 1) to evaluate different techniques to eliminate bacteria in semen 2) to study the relationship between total microflora load (TML) and ROS production; and 3) to determine if TML affects the functionality of cool-stored sperm. Ejaculates from 11 stallions were split and processed in 3 ways: A. extended semen; B. conventional centrifuged semen, a...
Lange-Consiglio A, Lazzari B, Perrini C, Pizzi F, Stella A, Cremonesi F, Capra E.Cell-derived microvesicles (MVs) are a recently discovered mechanism of cell-to-cell communication. Our previous data show that MVs secreted by equine amniotic mesenchymal-derived cells (AMCs) are involved in downregulation of proinflammatory genes in lipopolysaccharide-stressed equine tendon and endometrial cells. The aim of the present study was to evaluate whether AMC-MVs contain selected microRNAs (miRNAs) involved in inflammation. Two pools of cells, derived from 3 amniotic membranes each, and their respective MVs were collected. Small RNAs were extracted and deep sequenced, followed by m...
Naskou MC, Sumner SM, Chocallo A, Kemelmakher H, Thoresen M, Copland I, Galipeau J, Peroni JF.Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) poo...
Castro Martins M, Peffers MJ, Lee K, Rubio-Martinez LM.Intra-articular administration of stanozolol has shown promising results by improving the clinical management of lameness associated with naturally-occurring osteoarthritis (OA) in horses, and by decreasing osteophyte formation and subchondral bone reaction in sheep following surgically induced OA. However, there is limited evidence on the anti-inflammatory and modulatory properties of stanozolol on articular tissues. The objective of the current study was to evaluate the effects of stanozolol on chondrocyte viability and gene expression in normal equine chondrocytes and an inflammatory in vit...
Zayed M, Adair S, Ursini T, Schumacher J, Misk N, Dhar M.Osteoarthritis (OA), the most common form of joint disease affecting humans and horses, is characterized by the advance and decline of cartilage and loss of function of the affected joint. The progression of OA is steadily accompanied with biochemical events, which interfere with the cytokines and proteolytic enzymes responsible for progress of the disease. Recently, regenerative therapies have been used with an assumption that mesenchymal stem cells (MSCs) possess the potential to prevent the advancement of cartilage damage and potentially regenerate the injured tissue with an ultimate goal o...
Suliman Y, Becker F, Wimmers K.Poor fertility of breeding stallions is a recognised problem in the equine industry. The aim of the present study was to detect molecular pathways using two groups of stallions that differed in pregnancy rates as well as in the proportion of normal and motile spermatozoa. RNA was isolated from spermatozoa of each stallion and microarray data were analysed to obtain a list of genes for which transcript abundance differed between the groups (P ≤0.05, fold change ≥1.2). In all, there were 437 differentially expressed (DE) genes between the two groups (P ≤ 0.05, fold change ≥1.2). Next, th...
Khan FA, Chenier TS, Foster RA, Hewson J, Scholtz EL.Emerging research suggests that the nitric oxide system may play a role in persistent breeding-induced endometritis (PBIE) in the mare. Differences in uterine nitric oxide (NO) levels between mares susceptible or resistant to PBIE and a dose-dependent inhibitory effect of NO on uterine contractility have been demonstrated. The objectives of this study were to investigate the difference in total nitric oxide synthase (NOS) activity of the endometrium between susceptible and resistant mares and the effect of a specific inducible nitric oxide synthase (iNOS) inhibitor on the endometrial NOS activ...
Tanaka T, Otoguro T, Yamashita A, Kasai H, Fukuhara T, Matsuura Y, Moriishi K.The 5' untranslated region (UTR) of hepatitis C virus (HCV), which is composed of four domains (I, II, III, and IV) and a pseudoknot, is essential for translation and viral replication. Equine nonprimate hepacivirus (EHcV) harbors a 5' UTR consisting of a large 5'-terminal domain (I); three additional domains (I', II, and III), which are homologous to domains I, II, and III, respectively, of HCV; and a pseudoknot, in the order listed. In this study, we investigated the roles of the EHcV 5' UTR in translation and viral replication. The internal ribosome entry site (IRES) activity of the EHcV 5'...
Tallmadge RL, Žygelytė E, Van de Walle GR, Kristie TM, Felippe MJB.Equine herpesvirus type 1 (EHV-1) is a ubiquitous and highly contagious pathogen that causes a range of disease severities with outbreaks of notable economic impact. Given the limitations in immune protection of current vaccines and the limited effectiveness of antiviral drugs on EHV-1 infections , improved treatment measures are needed to control disease. The use of drugs that alter the epigenetic state of herpes simplex virus genome has been shown to limit viral primary infection and reactivation both and . Therefore, we tested the hypothesis that maintaining a repressive epigenetic state o...
Duan W, Chen C, Haque M, Hayes D, Lopez MJ.Use of bioscaffolds to direct osteogenic differentiation of adult multipotent stromal cells (MSCs) without exogenous proteins is a contemporary approach to bone regeneration. Identification of in vivo osteogenic contributions of exogenous MSCs on bioscaffolds after long-term implantation is vital to understanding cell persistence and effect duration. This study was designed to quantify in vivo equine MSC osteogenesis on synthetic polymer scaffolds with distinct mineral combinations 9 weeks after implantation in a murine model. Cryopreserved, passage (P)1, equine bone marrow-derived MSCs (BMSC)...
Morgan RE, Clegg PD, Hunt JA, Innes JF, Tew SR.The joint synovium consists of a heterogeneous cell population, chiefly comprised of macrophages, and fibroblast-like synoviocytes (FLS). An inter-species co-culture model was developed to examine interactions between these cells. Equine FLS and the canine macrophage line DH82 were differentially labeled using fluorescent markers and results from direct co-culture compared with those from both indirect co-culture, and conditioned media experiments. The transcript expression of IL-1β, IL-6, ADAMTS4, and ADAMTS5 in each cell type were determined using species-specific qPCR assays. Lipopolysacch...
Ceriotti S, Consiglio AL, Casati L, Cremonesi F, Sibilia V, Ferrucci F.Increasing evidence suggests a role for ghrelin in the control of articular inflammatory diseases like osteoarthritis (OA). In the present study we examined the ability of ghrelin to counteract LPS-induced necrosis and apoptosis of chondrocytes and the involvement of GH secretagogue receptor (GHS-R)1a in the protective action of ghrelin. The effects of ghrelin (10-10 mol/L) on equine primary cultured chondrocytes viability and necrosis in basal conditions and under LPS treatment (100 ng/ml) were detected by using both acridine orange/propidium iodide staining and annexin-5/propidium iodide...
Merlo B, Teti G, Mazzotti E, Ingrà L, Salvatore V, Buzzi M, Cerqueni G, Dicarlo M, Lanci A, Castagnetti C, Iacono E.Wharton's jelly (WJ) is an important source of mesenchymal stem cells (MSCs) both in human and other animals. The aim of this study was to compare human and equine WJMSCs. Human and equine WJMSCs were isolated and cultured using the same protocols and culture media. Cells were characterized by analysing morphology, growth rate, migration and adhesion capability, immunophenotype, differentiation potential and ultrastructure. Results showed that human and equine WJMSCs have similar ultrastructural details connected with intense synthetic and metabolic activity, but differ in growth, migration, a...
Call GB, Wolfe MW.Activation of the luteinizing hormone beta (LHbeta) promoter by gonadotropin-releasing hormone (GnRH) via the transcription factor early growth response protein-1 (Egr1) has been well characterized. To determine the mechanisms affecting Egr1 regulation of LHbeta, we analyzed five different species of LHbeta promoters (equine, mouse, rat, bovine and human). Electrophoretic mobility shift assays (EMSAs) identified multiple transcription factors binding to the Egr regions on the LHbeta promoter. Species-specific differences existed in the binding affinity for Sp1, Sp3, steroidogenic factor-1 (SF-...
Finlay M, Yuan Z, Morgan IM, Campo MS, Nasir L.Bovine papillomavirus type 1 infects not only cattle but also equids and is a causative factor in the pathogenesis of commonly occurring equine sarcoid tumours. Whilst treatment of sarcoids is notoriously difficult, cisplatin has been shown to be one of the most effective treatment strategies for sarcoids. In this study we show that in equine fibroblasts, BPV-1 sensitises cells to cisplatin-induced and UVB-induced apoptosis, a known cofactor for papillomavirus associated disease, however BPV-1 transformed fibroblasts show increased clonogenic survival, which may potentially limit the therapeut...
Wach-Gygax L, Burger D, Malama E, Bollwein H, Fleisch A, Jeannerat E, Thomas S, Schuler G, Janett F.In this study annual fluctuations of DNA fragmentation and quality of cold-stored equine sperm were evaluated. Ejaculates were collected weekly during one year from 15 stallions. Ejaculate volume, sperm concentration and total sperm count were determined and semen was then extended and cold-stored for 48 h. Sperm motility was evaluated by CASA before and after 24 as well as 48 h of cold storage. In addition, the percentages of sperm with intact plasma membrane and acrosome (PMAI %) and with low intracellular Ca2+ level were determined in cold-stored semen (24 h, 48 h). SCSA™ was performe...
Boschwitz JS, Timoney JF.The antiphagocytic property of equine fibrinogen for Streptococcus equi subsp. equi strain CF32 was examined in vitro. The results of bactericidal assays demonstrated that the presence of fibrinogen enhanced the ability of overnight and early log-phase cultures of strain CF32 to resist killing by equine neutrophils by 12-fold and seven-fold, respectively (p > 0.01). In addition, fibrinogen-coated bacteria treated with fibrinogen specific F(ab')2 fragments were 32% more susceptible to killing by equine neutrophils after opsonization in serum (p > 0.05), indicating that specific epitopes o...
Choi YH, Velez IC, Macías-García B, Riera FL, Ballard CS, Hinrichs K.Equine intracytoplasmic sperm injection (ICSI) is being used clinically for foal production, but little information is available on factors affecting the efficiency of this procedure. We examined factors that may influence blastocyst development when ICSI is performed clinically, i.e., on oocytes recovered from live mares by transvaginal ultrasound-guided follicle aspiration (TVA), using sperm from the stallion of the client's choice. In a clinical setting, there may be a delay from the time of TVA to isolation of oocytes from the aspirated fluid. In a preliminary study, oocytes from fluid hel...
Ricci G, Del Boccio G, Pennelli A, Aceto A, Whitehead EP, Federici G.Glutathione transferase (EC 2.5.1.18) from horse erythrocytes has been purified and some molecular and kinetic properties have been investigated. It appears to be a dimeric protein composed of subunits of about 23 kDa, indistinguishable either in sodium dodecyl sulfate or in urea electrophoresis. Amino acid composition, substrate specificities, sensitivity to inhibitors, CD spectra, and immunological studies provide evidence that the horse enzyme is related to the pi class transferases. This enzyme has only two reactive thiol groups/dimer whose integrity appears to be essential for the activit...
Morita H, Shiratori C, Murakami M, Takami H, Kato Y, Endo A, Nakajima F, Takagi M, Akita H, Okada S, Masaoka T.Two strains, KBL13(T) and GBL13, were isolated as one of intestinal lactobacilli from the faecal specimens from different thoroughbreds of the same farm where they were born in Hokkaido, Japan. They were Gram-positive, facultatively anaerobic, catalase-negative, non-spore-forming and non-motile rods. KBL13(T) and GBL13 homofermentatively metabolize glucose, and produce lactate as the sole final product from glucose. The 16S rRNA gene sequence, DNA-DNA hybridization, DNA G+C content and biochemical characterization indicated that these two strains, KBL13(T) and GBL13, belong to the same species...
Zheng YH, Sentsui H, Kono Y, Ikuta K.An attenuated equine infectious anemia virus (EIAV), named V26, was previously obtained after 50 passages of the Japanese virulent strain V70 in primary macrophage culture. To clarify the differences between both viruses, their full-length sequences were determined. There were higher mutations in S2 (6.15% amino acid difference) and LTR (10.7% nucleotide difference). The presumed initiation codon of the S2 gene was absent from the sequence of V26. There was a large insertion within the long-terminal repeat (LTR) U3 hypervariable region of V26. In addition, there were minor mutations in gag (1....
Coleman M, Kuskie K, Liu M, Chaffin K, Libal M, Giguère S, Bernstein L, Cohen N.The objective of this study was to determine the in vitro antimicrobial activity of gallium maltolate (GaM) against Rhodococcus equi. A total of 98 virulent bacterial isolates from equine clinical cases were examined, of which 19 isolates were known to be resistant to macrolides and rifampin. Isolates were cultured with various concentrations of GaM and minimal inhibitory concentration (MIC) values were determined after 24 and 48 h. Both the MIC(50) and the MIC(90) after 24h of growth were 558 ng/mL (8 μM) and after 48 h of growth were 2230 ng/mL (32 μM). There were no apparent differences b...
Radcliffe PA, Sion CJ, Wilkes FJ, Custard EJ, Beard GL, Kingsman SM, Mitrophanous KA.Effective gene therapy for haemophilia A necessitates a vector system that is not subject to a pre-existing immune response, has adequate coding capacity, gives long-term expression and preferably can target non-dividing cells. Vector systems based on lentiviruses such as equine infectious anaemia virus (EIAV) fulfil these criteria for the delivery of factor VIII (FVIII). We have found that B domain-deleted (BDD) FVIII protein inhibits functional viral particle production when co-expressed with the EIAV vector system. Although particle numbers (as measured by reverse transcriptase activity) ar...
Tözsér J, Friedman D, Weber IT, Bláha I, Oroszlan S.The proteinase of the equine infectious anemia virus (EIAV), a lentivirus closely related to human immunodeficiency virus (HIV), was purified from concentrated virus. The specificity of the enzyme was characterized using oligopeptides representing naturally occurring cleavage sites in the Gag and Gag-Pol polyproteins. The length of the substrate binding pocket was found to be 1-2 residues longer than that of HIV proteinases. Although the EIAV and HIV proteinases cleaved most of the peptides at the same bond, some were hydrolyzed by only the EIAV enzyme. Oligopeptides representing cleavage site...
Hisey E, Ross PJ, Meyers SA.OCT4 is a core transcription factor involved in pluripotency maintenance in the early mammalian embryo. The POU5F1 gene that encodes the OCT4 protein is highly conserved across species, suggesting conserved function. However, studies in several species including mice, cattle, and pigs, suggest that there are differences in where and when OCT4 is expressed. Specifically, in the horse, several studies have shown that exposure to the uterine environment may be necessary to induce OCT4 expression restriction to the inner cell mass (ICM) of the developing embryo, suggesting that there may be equine...
Ascenzi P, di Masi A, Gullotta F, Mattu M, Ciaccio C, Coletta M.Cyanide binds to ferric heme-proteins with a very high affinity, reflecting the very low dissociation rate constant (k(off)). Since no techniques are available to estimate k(off), we report herewith a method to determine k(off) based on the irreversible reductive nitrosylation reaction to trap ferric myoglobin (Mb(III)). The k(off) value for cyanide dissociation from ferric cyanide horse heart myoglobin (Mb(III)-cyanide) was determined at pH 9.2 and 20.0 degrees C. Mixing Mb(III)-cyanide and NO solutions brings about absorption spectral changes reflecting the disappearance of Mb(III)-cyanide w...
Bundgaard L, Stensballe A, Elbæk KJ, Berg LC.Stem cells have great potential for tissue regeneration, but before stem cell populations can be used in the clinic, it is crucial that the stem cells have been definitely characterized by a set of specific markers. Although there have been attempts to identify a set of immunophenotypic markers to characterize equine mesenchymal stromal cells (MSCs), immunophenotyping of equine MSCs is still challenging due to the limited availability of suitable antibodies of high quality and consistent performance across different laboratories. The aim of this study was to evaluate a strategy for mapping the...
Messick JB, Rikihisa Y.The effects of equine antiserum, immunoglobulin G (IgG) specific for Ehrlichia risticii, and its Fab fragment on E. risticii binding to, internalization into, and proliferation in P388D1 cells were studied by immunofluorescence flow cytometry. Anti-E. risticii equine serum or IgG inhibited E. risticii at a stage beyond binding and internalization. In contrast, monovalent anti-E. risticii equine Fab fragments inhibited E. risticii binding and internalization into P388D1 cells. In the presence of control equine serum, IgG, or its Fab fragment, E. risticii cells were bound, were internalized and ...
Carnevale EM.Assisted reproductive techniques that are based on oocyte manipulations have gained acceptance in the equine industry. Methods to collect and handle immature or maturing oocytes have been developed, and systems to ship oocytes now allow for collection in one location and intracytoplasmic sperm injection (ICSI) in another. Subsequently, ICSI-produced embryos can be transferred onsite, shipped to another location, or cryopreserved. Methods for the collection, identification, culture, maturation, and shipment of equine oocytes are reviewed, with an emphasis on procedures from laboratories providi...
Gray WL, Yalamanchili R, Raengsakulrach B, Baumann RP, Staczek J, O'Callaghan DJ.Equine herpesvirus type 1 (EHV-1) preparations enriched in defective interfering particles (DIPs) have previously been demonstrated to mediate the coestablishment of persistent infection and oncogenic transformation in primary hamster embryo fibroblasts. In this study, it was demonstrated that infection of a rabbit kidney (RK) cell line with EHV-1 DIP-enriched preparations also results in the establishment of persistent infection. Viral transcription was characterized in RK cells infected with DIP-enriched stocks and compared to viral transcription in RK cells infected with standard (STD) EHV-...
Gigante A, Bevilacqua C, Cappella M, Manzotti S, Greco F.Articular cartilage defects do not heal. Biodegradable scaffolds have been studied for cartilage engineering in order to implant autologous chondrocytes and help cartilage repair. We tested some new collagen matrices differing in collagen type, origin, structure and methods of extraction and purification, and compared the behavior of human chondrocytes cultured on them. Human chondrocytes were grown for three weeks on four different equine type I collagen matrices, one type I, III porcine collagen matrix and one porcine type II collagen matrix. After 21 days, samples were subjected to histoche...
Berglund AK, Long JM, Robertson JB, Schnabel LV.Allogeneic mesenchymal stem cells (MSCs) are a promising cell therapy for treating numerous diseases, but major histocompatibility complex (MHC)-mismatched MSCs can be rejected by the recipient's immune system. Pre-treating MSCs with transforming growth factor-β2 (TGF-β2) to downregulate surface expression of MHC molecules may enhance the ability of allogeneic MSCs to evade immune responses. We used lymphocyte proliferation assays and ELISAs to analyze the immunomodulatory potential of TGF-β2-treated equine bone marrow-derived MSCs. T cell activation and cytotoxicity assays were then used t...
Nash D, Lane E, Herath S, Sheldon IM.Endometritis after insemination is ubiquitous in the horse and is associated with semen and/or bacteria in the uterus. In up to 40% of horses, inflammation persists causing infertility. An endometrial explant culture was developed to study uterine secretion of prostaglandin F(2alpha) (PGF(2alpha)) in response to physiological and pathological challenge. Methods: Uteri were collected from mares, the endometrium dissected and explants from the uterine body or horn cultured in William's or RPMI medium. The response of explants to oxytocin, semen or bacteria compared to untreated tissue was tested...
Broeckx SY, Maes S, Martinello T, Aerts D, Chiers K, Mariën T, Patruno M, Franco-Obregón A, Spaas JH.Besides the presence of somatic stem cells in hair follicles and dermis, the epidermis also contains a subpopulation of stem cells, reflecting its high regenerative capacity. However, only limited information concerning epidermis-derived epithelial-like stem/progenitor cells (EpSCs) is available to date. Nonetheless, this stem cell type could prove itself useful in skin reconstitution after injury. After harvesting from equine epidermis, the purified cells were characterized as EpSCs by means of positive expression for CD29, CD44, CD49f, CD90, Casein Kinase 2β, p63, and Ki67, low expression f...
Handing KB, Shabalin IG, Szlachta K, Majorek KA, Minor W.Serum albumin (SA) is the main transporter of drugs in mammalian blood plasma. Here, we report the first crystal structure of equine serum albumin (ESA) in complex with antihistamine drug cetirizine at a resolution of 2.1Å. Cetirizine is bound in two sites--a novel drug binding site (CBS1) and the fatty acid binding site 6 (CBS2). Both sites differ from those that have been proposed in multiple reports based on equilibrium dialysis and fluorescence studies for mammalian albumins as cetirizine binding sites. We show that the residues forming the binding pockets in ESA are highly conserved in h...
Treulen F, Aguila L, Arias ME, Jofré I, Felmer R.During cryopreservation procedures, the spermatozoa are exposed to physical and chemical stressors that generate an increase in the intracellular concentration of reactive oxygen species (ROS). If ROS concentrations are too great, this can lead to a state of oxidative stress that are detrimental to sperm quality. The aim of this study was to ascertain the profile the ROS production and assess the effects of post-thaw supplementation of a semen extender with different antioxidant compounds on the quality and function variables of frozen-thawed stallion spermatozoa incubated in vitro. Frozen-tha...
Tang X, Sayre LM, Tochtrop GP.Cytochrome c is a key mitochondrial respiratory protein that is particularly susceptible to modification during oxidative stress. The nature of this susceptibility is linked to the mitochondrial membrane being rich in esterified linoleic acid, which predisposes this organelle to the formation of lipid peroxidation products such as 4-hydroxy-2-(E)-nonenal (4-HNE). To better understand the nature of cytochrome c modification by 4-HNE, we initiated an in vitro study utilizing a combination of MALDI-TOF mass spectrometry, LC-ESI-MS/MS and isotope labeling to monitor 4-HNE modification of cytochrom...
Jiang CG, Gao X, Ma J, Lin YZ, Wang XF, Zhao LP, Hua YP, Liu D, Zhou JH.Preliminary studies revealed that the gene of the gp45 transmembrane protein (TM) of the attenuated equine infectious anemia virus (EIAV) vaccine strain EIAV(FDDV13) had a high frequency of a premature stop codon at position 261W, which generated a 154-residue truncation at the C-terminus. EIAV(FDDV-TM36), a recombinant virus with the TM truncated at the intracytoplasmic (CT) domain due to the presence of a stop codon, was constructed based on EIAV(FDDV)3-8, which is a proviral derivative of the vaccine. EIAV(FDDV-TM36) had a significantly reduced replication capability compared to EIAV(FDDV)3...
Range F, Mundhenk L, Gruber AD.The equine putative chloride channel protein eCLCA1 is thought to be critically involved in the pathogenesis of recurrent airway obstruction (RAO) via modulation of the hydration of airway mucins. A recent study revealed a strong increase of eCLCA1 messenger ribonucleic acid (mRNA) in the lungs of horses with RAO. In this study, eCLCA1 protein and mRNA expression were quantified in airway goblet cells of 9 horses affected with RAO and 9 control horses by using immunohistochemistry and laser microdissection followed by real-time quantitative reverse transcription polymerase chain reaction, resp...
Pouran B, Arbabi V, Zadpoor AA, Weinans H.The metabolic function of cartilage primarily depends on transport of solutes through diffusion mechanism. In the current study, we use contrast enhanced micro-computed tomography to determine equilibrium concentration of solutes through different cartilage zones and solute flux in the cartilage, using osteochondral plugs from equine femoral condyles. Diffusion experiments were performed with two solutes of different charge and approximately equal molecular weight, namely iodixanol (neutral) and ioxaglate (charge=-1) in order to isolate the effects of solute's charge on diffusion. Furthermore,...
Shibeshi W, Abraham G, Kneuer C, Ellenberger C, Seeger J, Schoon HA, Ungemach FR.Culture of airway epithelial cells is a useful model to investigate physiology of airway epithelia and airway disease mechanisms. In vitro models of airway epithelial cells are established for various species. However, earlier published method for isolation and culture of equine tracheal epithelial cells requires significant improvements. In this report, the development of a procedure for efficient isolation, characterization, culture, and passage of primary equine tracheal epithelial cells are described. Epithelial cells were isolated from adult equine trachea by exposing and stripping the mu...
Murray SJ, Santangelo KS, Bertone AL.To evaluate early cellular influences of bone morphogenetic protein (BMP)12 and BMP2 on equine superficial digital flexor tenocytes (SDFTNs) and equine bone marrow-derived mesenchymal stem cells (BMDMSCs). Methods: 9 adult clinically normal horses. Methods: BMDMSCs and SDFTNs were cultured in monolayer, either untreated or transduced with adenovirus encoding green fluorescent protein, adenovirus encoding BMP12, or adenovirus encoding BMP2. Cytomorphologic, cytochemical, immunocytochemical, and reverse transcriptase-quantitative PCR (RT-qPCR) analyses were performed on days 3 and 6. Genetic pro...
Gondim LSQ, Jesus RF, Ribeiro-Andrade M, Silva JCR, Siqueira DB, Marvulo MFV, Aléssio FM, Mauffrey JF, Julião FS, Savani ESMM, Soares RM, Gondim LFP.Sarcocystis neurona and Neospora spp. are protozoan parasites that induce neurological diseases in horses and other animal species. Opossums (Didelphis albiventris and Didelphis virginiana) are definitive hosts of S. neurona, which is the major cause of equine protozoal myeloencephalitis (EPM). Neospora caninum causes abortion in cattle and infects a wide range of animal species, while N. hughesi is known to induce neurologic disease in equids. The aims of this study were to investigate S. neurona and N. caninum in tissues from opossums in the northeastern Brazil, and to isolate Brazilian stra...
