The study of cells in horses encompasses the examination of various cell types and their functions within the equine body. Cells are the basic structural and functional units of life, and in horses, they contribute to numerous physiological processes, including growth, repair, and immune responses. Different cell types, such as red blood cells, white blood cells, and muscle cells, each perform specific roles that are vital for maintaining the health and homeostasis of the horse. This topic includes research on cellular mechanisms, cellular responses to disease or injury, and the application of cellular biology in equine medicine. This page compiles peer-reviewed research studies and scholarly articles that explore the structure, function, and significance of cells in equine biology and health.
Moyo NA, Marchi E, Steinbach F.Dendritic cells (DC) are the main immune mediators inducing primary immune responses. DC generated from monocytes (MoDC) are a model system to study the biology of DC in vitro, as they represent inflammatory DC in vivo. Previous studies on the generation of MoDC in horses indicated that there was no distinct difference between immature and mature DC and that the expression profile was distinctly different from humans, where CD206 is expressed on immature MoDC whereas CD83 is expressed on mature MoDC. Here we describe the kinetics of equine MoDC differentiation and activation, analysing both ph...
Asami K.Horse erythrocytes, murine lymphoblasts (L5178Y) and lipid vesicles that were treated with dipicrylamine (DPA) as a lipophilic ion were studied by dielectric spectroscopy over a frequency range of 10 Hz to 10 MHz. The DPA-treated cells and lipid vesicles showed low-frequency (LF) dielectric dispersion around 1-10 kHz in addition to β-dispersion due to the Maxwell-Wagner effect. The LF dispersion corresponds to that found in previous electrorotation (ROT) studies on DPA-treated cells, being due to the translocation of mobile ions in the plasma membranes. Analysis of the LF dispersion based on ...
Vendrig JC, Coffeng LE, Fink-Gremmels J.Increasing evidence suggests that reactions to lipopolysaccharide (LPS), particularly in the gut, can be partly or completely mitigated by colostrum- and milk-derived oligosaccharides. Confirmation of this hypothesis could lead to the development of new therapeutic concepts. Objective: To demonstrate the influence of equine colostral carbohydrates on the inflammatory response in an in vitro model with equine peripheral blood mononuclear cells (PBMCs). Methods: Carbohydrates were extracted from mare colostrum, and then evaluated for their influence on LPS-induced inflammatory responses in PBMCs...
Sato K, Wakai T, Seita Y, Takizawa A, Fissore RA, Ito J, Kashiwazaki N.A sperm-specific phospholipase C (PLC), PLCzeta (PLCζ), is thought to underlie the initiation of calcium ([Ca(2+) ]i ) oscillations that induce egg activation in mammals. In large domestic species, only bovine, porcine and recently equine PLCζ have been cloned, and the physiological functions of these molecules have not been fully characterized. Here, we evaluated the physiological functions of equine PLCζ (ePLCζ) in mouse oocytes. ePLCζ was cloned from testis using RT-PCR. The expression of ePLCζ messenger RNA was confirmed in testis but not in other tissues. Microinjection of ePLCζ co...
Raabe O, Shell K, Fietz D, Freitag C, Ohrndorf A, Christ HJ, Wenisch S, Arnhold S.Mesenchymal stem cells have become extremely interesting for regenerative medicine and tissue engineering in the horse. Stem cell therapy has been proven to be a powerful and successful instrument, in particular for the healing of tendon lesions. We pre-differentiated equine adipose-tissue-derived stem cells (ASCs) in a collagen I gel scaffold by applying tensile strain, growth differentiation factors (GDFs) and various oxygen tensions in order to determine the optimal conditions for in vitro differentiation toward the tenogenic lineage. We compared the influence of 3% versus 21% oxygen tensio...
Nugraheni AD, Nagao S, Yanagisawa S, Ogura T, Hirota S.We have previously shown that methionine-heme iron coordination is perturbed in domain-swapped dimeric horse cytochrome c. To gain insight into the effect of methionine dissociation in dimeric cytochrome c, we investigated its interaction with cyanide ion. We found that the Soret and Q bands of oxidized dimeric cytochrome c at 406.5 and 529 nm redshift to 413 and 536 nm, respectively, on addition of 1 mM cyanide ion. The binding constant of dimeric cytochrome c and cyanide ion was obtained as 2.5 × 10(4) M(-1). The Fe-CN and C-N stretching (ν (Fe-CN) and ν (CN)) resonance Raman ba...
Giraldo CE, López C, Álvarez ME, Samudio IJ, Prades M, Carmona JU.There is no information on the effects of the breed, gender and age on the cellular content and growth factor (GF) release from equine pure-platelet rich plasma (P-PRP) and pure-platelet rich gel (P-PRG). The objectives of this study were: 1) to compare the cellular composition of P-PRP with whole blood and platelet poor plasma (PPP); 2) to compare the concentration of transforming GF beta 1 (TGF-β1) and platelet derived GF isoform BB (PDGF-BB) between P-PRP treated with non-ionic detergent (P-PRP+NID), P-PRG (activated with calcium gluconate -CG-), PPP+NID, PPP gel (PPG), and plasma and; 3) ...
Sabban S, Ye H, Helm B.The binding of immunoglobulin E (IgE) to its high-affinity receptor (FcɛRI) is the central protein interaction in IgE-mediated allergic reactions. The cross-linking of the IgE/FcɛRI complex, through cognate allergens, on the surface of mast cells and basophil cells results in mediator release, and thus leads to the symptoms of type I hypersensitivity responses in mammals. To develop a baseline value for subsequent equine anti-allergy drug and vaccine research, the interaction of equine IgE with its high-affinity FcɛRI receptor was investigated following the cloning and expression of equine ...
Zamboulis DE, Senior JM, Clegg PD, Gallagher JA, Carter SD, Milner PI.Purinergic pathways are considered important in pain transmission, and P2X receptors are a key part of this system which has received little attention in the horse. The aim of this study was to identify and characterise the distribution of P2X receptor subtypes in the equine digit and associated vasculature and nervous tissue, including peripheral nerves, dorsal root ganglia and cervical spinal cord, using PCR, Western blot analysis and immunohistochemistry. mRNA signal for most of the tested P2X receptor subunits (P2X1-5, 7) was detected in all sampled equine tissues, whereas P2X6 receptor su...
Wang L, Pawlak EA, Johnson PJ, Belknap JK, Eades S, Stack S, Cousin H, Black SJ.The digital laminae is a two layer tissue that attaches the distal phalanx to the inner hoof wall, thus suspending the horse's axial skeleton in the hoof capsule. This tissue fails at the epidermal:dermal junction in laminitic horses, causing crippling disease. Basal epithelial cells line the laminar epidermal:dermal junction, undergo physiological change in laminitic horses, and lose versican gene expression. Versican gene expression is purportedly under control of the canonical Wnt signaling pathway and is a trigger for mesenchymal-to-epithelial transition; thus, its repression in laminar ep...
Seo MS, Park SB, Kim HS, Kang JG, Chae JS, Kang KS.Recent studies have shown that mesenchymal stem cells (MSCs) are able to differentiate into multi-lineage cells such as adipocytes, chondroblasts, and osteoblasts. Amniotic membrane from whole placenta is a good source of stem cells in humans. This membrane can potentially be used for wound healing and corneal surface reconstruction. Moreover, it can be easily obtained after delivery and is usually discarded as classified waste. In the present study, we successfully isolated and characterized equine amniotic membrane-derived mesenchymal stem cells (eAM-MSCs) that were cultured and maintained i...
Goodrich LR, Phillips JN, McIlwraith CW, Foti SB, Grieger JC, Gray SJ, Samulski RJ.Osteoarthritis (OA) affects over 40 million people annually. We evaluated interleukin-1 receptor antagonist (IL-1ra) gene transfer in an equine model based on IL-1ra protein therapy which inhibits inflammation through blocking IL-1. Using the self-complementary adeno-associated virus (scAAV)IL-1ra equine gene as a starting construct, we optimized the transgene cassette by analyzing promoters (cytomegalovirus (CMV) versus chicken β-actin hybrid (CBh)), coding sequences (optimized versus unoptimized), vector capsid (serotype 2 versus chimeric capsid), and biological activity in vitro. AAV serot...
Link KA, Koenig JB, Silveira A, Plattner BL, Lillie BN.To compare the effect of extracorporeal shock wave therapy (ESWT) on expression of fibroblast growth factor-7 (FGF-7), transforming growth factor-β1 (TGF-β1), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor-A (PDGF), and vascular endothelial growth factor-A (VEGF) in skin with surgically created skin wounds and intact skin in horses. Methods: 14 healthy horses. Methods: 8 horses were treated with ESWT at 6 locations along the neck at 36, 24, 12, 6, 2, or 1 hour prior to collection of full-thickness biopsy specimens from each location; a control specimen was collected fro...
Pereira GR, Lorenzo PL, Carneiro GF, Bilodeau-Goeseels S, Kastelic JP, Esteller-Vico A, Lopez-Bejar M, Liu IK.Immature oocytes synthesize a variety of proteins that include the enzyme glucose-6-phosphate dehydrogenase (G6PDH). Brilliant cresyl blue (BCB) is a vital blue dye that assesses intracellular activity of G6PDH, an indirect measure of oocyte maturation. The objective was to evaluate the BCB test as a criterion to assess developmental competence of equine oocytes and to determine if equine growth hormone (eGH) enhanced in vitro maturation (IVM) of equine oocyte. Cumulus-oocytes complexes (COCs) were recovered by aspirating follicles 0.05). Maturation was not affected significantly by BCB class...
Ramos-Vara JA, Frank CB, DuSold D, Miller MA.The immunoreactivity of PNL2, Melan A, and protein gene product (PGP) 9.5 was compared with that of S100 protein in 50 formalin-fixed, paraffin-embedded equine melanocytic neoplasms. PNL2, PGP 9.5, and S100 protein were detected in all 50 neoplasms; none expressed Melan A. PNL2 was not expressed in 62 nonmelanocytic tumors (equine sarcoids, schwannomas, carcinomas, sarcomas, endocrine tumors, sex-cord stromal tumors, germ cell tumors, and leukocytic tumors) or in normal tissues other than epidermis. In summary, antibody PNL2 is a sensitive marker of equine melanocytic neoplasms and is more spe...
Nürnberger S, Meyer C, Ponomarev I, Barnewitz D, Resinger C, Klepal W, Albrecht C, Marlovits S.Treatment of cartilage defects poses challenging problems in human and veterinary medicine, especially in horses. This study examines the suitability of applying scaffold materials similar to those used for human cartilage regeneration on equine chondrocytes. Chondrocytes gained from biopsies of the talocrural joint of three horses were propagated in 2D culture and grown on two different scaffold materials, hyaluronan (HYAFF®) and collagen (BioGide®), and evaluated by light and electron microscopy. The equine chondrocytes developed well in both types of materials. They were vital and physiol...
Almeida J, Conley AJ, Ball BA.Cryptorchidism affects 2-8% of male horses and the affected testis undergoes a disruption of normal spermatogenesis. The underlying molecular changes are poorly understood in the cryptorchid equine testis. Objective: Compare the expression of anti-Müllerian hormone (AMH), anti-Müllerian hormone receptor (AMHR2), androgen receptor (AR), cyclin kinase inhibitor (CDKN1B), connexin 43 (Cx43), 3β hydroxysteroid dehydrogenase/Δ(5) -Δ(4) - isomerase (3βHSD), P450c17 hydroxylase/lyase (P450c17) and cytochrome P450 aromatase (P450arom) in the undescended testis of cryptorchid stallions with that ...
Fernandez NJ, Hecker KG, Gilroy CV, Warren AL, Léguillette R.Reliable enumeration of mast cells and eosinophils in equine bronchoalveolar lavage (BAL) fluid is important because small increases in the percentages of these cells support the clinical diagnosis of inflammatory airway disease (IAD). Increases in BAL neutrophils also occur with IAD but are not specific due to overlap between IAD and recurrent airway obstruction (RAO). Objective: The objectives of this study were to evaluate the reliability of a standard 400-cell leukocyte differential count and an alternate method evaluating 5 microscopic fields at 500× magnification in equine BAL fluid cyt...
McCandless EE, Rai SK, Mwangi D, Sly L, Franz LC.Hydrocortisone is widely accepted as an anti-inflammatory agent and there are many products available containing hydrocortisone as an active ingredient. Surprisingly, there is little data available specifically on the immunological effects of hydrocortisone in large animals. Glucocorticoids are well-characterized for their ability to repress inflammation via a wide variety of mechanisms including suppression of cytokine production. In this study the effects of hydrocortisone on IFN-γ production by equine, bovine, and ovine PBMCs were assessed using flow cytometric or ELISpot analysis. Hydroco...
Lee CM, Kisiday JD, McIlwraith CW, Grodzinsky AJ, Frisbie DD.To develop an in vitro model of cartilage injury in full-thickness equine cartilage specimens that can be used to simulate in vivo disease and evaluate treatment efficacy. Methods: 15 full-thickness cartilage explants from the trochlear ridges of the distal aspect of the femur from each of 6 adult horses that had died from reasons unrelated to the musculoskeletal system. Methods: To simulate injury, cartilage explants were subjected to single-impact uniaxial compression to 50%, 60%, 70%, or 80% strain at a rate of 100% strain/s. Other explants were left uninjured (control specimens). All speci...
Zaniboni A, Merlo B, Zannoni A, Bernardini C, Lavitrano M, Forni M, Mari G, Bacci ML.Sperm mediated gene transfer (SMGT) has been reported to be a powerful tool for producing transgenic livestock with applications in biomedicine and agriculture. To date, two studies have reported the production of transgenic equine embryo with, however, low efficiency in blastocyst production and transgene expression. The aim of the present study was to develop a method which allowed the efficient production of transgene-expressing embryos of the equine species through SMGT. To overcome problems due to in vitro fertilization (IVF) in horses, the ICSI procedure was associated with SMGT. The upt...
Ceusters JD, Mouithys-Mickalad AA, Franck TJ, Derochette S, Vanderplasschen A, Deby-Dupont GP, Serteyn DA.Horses are particularly sensitive to excessive inflammatory reaction where myeloperoxidase, a marker of inflammation, may contribute to mitochondrial dysfunctions. This study investigated the interaction between myeloperoxidase and cultured primary equine skeletal myoblasts, particularly its effect on mitochondrial respiration combined or not with anoxia followed by reoxygenation (AR). We showed that active myeloperoxidase entered into the cells, interacted with mitochondria and decreased routine and maximal respirations. When combined with AR, myeloperoxidase caused a further decrease of thes...
Tappenbeck K, Hoppe S, Hopster K, Kietzmann M, Feige K, Huber K.Postoperative ileus (POI) is a severe complication following small intestinal surgery in horses. It was hypothesised that prokinetic effects of lidocaine, the most commonly chosen drug for treatment of POI, resulted from drug integration into smooth muscle (SM) cell membranes, thereby modulating cell membrane properties. This would probably depend on the structural and lipophilic characteristics of lidocaine. To assess the influence of molecular structure and lipophilicity on prokinetic effects in vitro, the current study compared the effects of lidocaine with four structure-related drugs, nam...
Haag KT, Magalhães-Padilha DM, Fonseca GR, Wischral A, Gastal MO, King SS, Jones KL, Figueiredo JR, Gastal EL.A Biopsy Pick-Up (BPU) method was tested to determine the feasibility of retrieving preantral follicles from mare ovaries in vivo. A total of 33 ovarian biopsy procedures were performed on 18 mares during the breeding season. Mares were 5 to 21 years old and biopsies were performed during the estrous and/or diestrous phase, as confirmed by transrectal ultrasonography. Follicles were mechanically isolated using a tissue chopper, counted, and classified as normal or abnormal and primordial or primary. Viability of isolated follicles was determined by Trypan Blue dye. A total of 256 biopsy attemp...
Caron JP, Gandy JC, Schmidt M, Hauptman JG, Sordillo LM.To compare the effects of triamcinolone acetonide (TA) and methylprednisolone acetate (MPA) on expression of selected chondrocyte genes in recombinant equine interleukin-1β (reIL-1β) stimulated articular cartilage explants. Methods: In vitro experiment. Methods: Horses (n = 6). Methods: Articular cartilage explants from 2- to 3- year-old horses were exposed to reIL-1β in the presence and absence of TA and MPA at 10(-7) and 10(-6) M. Resting levels of mRNA of anabolic and catabolic genes of chondrocyte origin were quantified using qPCR after 6- and 12-hour incubations. Genes of interest incl...
Adams AA, Horohov DW.The weaning process of foals involves a period of considerable stress which likely contributes to an increased risk of infectious disease in these young horses. Mechanisms responsible for this heightened risk of infection remain unknown, although likely due to compromised cell-mediated immunity. Parapoxvirus ovis (PPVO), an immmunomodulator, has been shown to limit the severity of infectious disease outbreaks among horses and has been shown to enhance CMI responses. Thus, an objective of this study was to investigate the effect of PPVO therapy on cell-mediated immune (CMI) responses of abruptl...
Antczak DF, de Mestre AM, Wilsher S, Allen WR.A remarkable feature of equine pregnancy is the development of the invasive trophoblast of the chorionic girdle and its formation of the gonadotrophin-secreting endometrial cup cells in early gestation. The details of this process have been revealed only slowly over the past century, since the first description of the endometrial cups in 1912. This centennial presents an opportunity to review the characteristics of the cells and molecules involved in this early, critical phase of placentation in the mare. The invasiveness of the chorionic girdle trophoblast appears to represent an atavistic at...
Di Stefano DA, Andreasi Bassi M, Cinci L, Pieri L, Ammirabile G.While deproteinized bovine bone and bovine membranes have been well studied and can yield good results when used to treat bone defects and peri-implant dehiscences, enzymatically deantigenated equine bone and equine membranes have emerged as possible alternative biomaterials. The objective of this study was the clinical and histological assessment of such materials: equine bone granules, an equine collagen membrane and an equine pericardium membrane. Methods: Enzymatically deantigenated equine bone and an equine collagen membrane were used to restore a bone defect caused by the removal of a bo...
Hansen S, Sun L, Baptiste KE, Fjeldborg J, Horohov DW.Diseases of the lower airways represent some of the most common conditions affecting horses of all ages, but the type and severity tends to follow the horses' age. The age-related dysregulation of pro-inflammatory cytokines may, in part contribute to the development of the diseases. Therefore, we hypothesize that the expression of pro-inflammatory cytokines increases with age. Peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage (BAL) cells from clinically healthy horses of different ages were used for the investigation. The cells were stimulated and the production of IFN-γ an...
Artiushin S, Timoney JF, Fettinger M, Fallon L, Rathgeber R.Enterocolitis caused by Clostridium difficile (C. difficile) is a serious, sometimes fatal, disease of neonatal foals and older horses. Toxins A and B (TcdA and B) produced by C. difficile are important virulence factors. Immunisation of mares with receptor binding domains of toxins may prevent or reduce the severity of C. difficile colitis in foals. Objective: To determine whether antibodies generated in the pregnant mare to the binding regions of TcdA and B will neutralise TcdA and B toxicity. Methods: Sequences encoding the binding domains of each toxin were isolated by PCR amplification fr...
Tomlinson JE, Wagner B, Felippe MJB, Van de Walle GR.Immune phenotyping of equine peripheral blood mononuclear cells (PBMC) is commonly described by single or double marker labeling, which limits complex phenotypic descriptions and subpopulation identification. Our objective was to develop a new multispectral flow cytometry protocol to identify and sort equine lymphocyte subpopulations using commercially available, pre-conjugated monoclonal antibodies to cell surface markers. Two clones of anti-equine CD8 (CVS8 and CVS21) were compared in combination with CD3. Clone CVS21 bound non-T CD3 cells in addition to CD8 T cells. Further analysis using c...
Lee KH, Park C, Kim J, Moon C, Ahn M, Shin T.The morphological characteristics and glycoconjugate composition of the vomeronasal organ (VNO) of the horse was investigated using histological, immunohistochemical, and lectin histochemical methods. The VNO is bilaterally located at the base of the nasal septum, has a tubular structure surrounded by cartilage, and consists of sensory and non-sensory epithelia. Immunohistochemical examination showed that the vomeronasal sensory epithelium (VSE) consisted of receptor cells positive for both olfactory marker protein (OMP) and protein gene product 9.5 (PGP 9.5), supporting cells, and basal cells...
Winter RL, Tian Y, Caldwell FJ, Seeto WJ, Koehler JW, Pascoe DA, Fan S, Gaillard P, Lipke EA, Wooldridge AA.Endothelial colony forming cells (ECFCs) may be useful therapeutically in conditions with poor blood supply, such as distal limb wounds in the horse. Encapsulation of ECFCs into injectable hydrogel microspheres may ensure cell survival and cell localization to improve neovascularization and healing. Autologous ECFCs were isolated from 6 horses, labeled with quantum nanodots (QD), and a subset were encapsulated in poly(ethylene) glycol fibrinogen microspheres (PEG-Fb MS). Full-thickness dermal wounds were created on each distal limb and injected with empty PEG-Fb MS, serum, ECFCs, or ECFCs enca...
Gargala G, Le Goff L, Ballet JJ, Favennec L, Stachulski AV, Rossignol JF.Sarcocystis neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis (EPM). The aim of this work was to document inhibitory activities of nitazoxanide (NTZ, [2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide]) and new thiazolides/thiadiazolides on S. neurona in vitro development, and investigate their structure-activity relationships. S. neurona was grown in bovine turbinate cell cultures. At concentrations varying from 1.0 to 5.0mg/L, nitazoxanide and 21 of 32 second generation thiazolide/thiadiazolide agents exerted a > or =95% maximum inhibition on S. neuro...
Lange-Consiglio A, Romele P, Magatti M, Silini A, Idda A, Martino NA, Cremonesi F, Parolini O.Equine amniotic mesenchymal stromal cells (AMSCs) and their conditioned medium (CM) were evaluated for their ability to inhibit in vitro proliferation of peripheral blood mononuclear cells (PBMCs) with and without priming. Additionally, AMSC immunogenicity was assessed by expression of MHCI and MHCII and their ability to counteract the in vitro inflammatory process. Horse PBMC proliferation was induced with phytohemagglutinin. AMSC priming was performed with 10 ng/ml of TNF-α, 100 ng/ml of IFN-γ, and a combination of 5 ng/ml of TNF-α and 50 ng/ml of IFN-γ. The CM generated from na...
van der Haegen A, Griot-Wenk M, Welle M, Busato A, von Tscharner C, Zurbriggen A, Marti E.The aim of the present study was to investigate, with immunohistochemistry and in situ hybridisation, if immunoglobulin-E (IgE) and mast cells are involved in the pathogenesis of insect bite hypersensitivity (IBH), an allergic dermatitis of horses. In tissue sections fixed in paraformaldehyde (PFA) for <24 h, significantly more IgE protein-bearing cells were found in the dermis and epidermis of acute and chronic IBH lesions than in skin biopsies from healthy horses (medians = 466, 236 and 110 cells/mm2, respectively; P < or = 0.01). More IgE-mRNA positive (+) cells were observed in the dermis ...
Magnuson NS, Perryman LE, Wyatt CR, Mason PH, Talmadge JE.Peripheral blood mononuclear cells from foals with hereditary severe combined immunodeficiency (SCID) have morphologic characteristics of large granular lymphocytes (LGL). Attempts to demonstrate cytotoxic activity were without success unless the LGL were incubated with 100 U of human recombinant interleukin 2 (rIL 2)/ml for 24 hr. With rIL 2 incubation, low effector to target ratios (10:1) consistently yielded high levels of cytotoxic activity (30 to 50%) in a standard 4-hr 51Cr-release assay using YAC-1 lymphoma or K562 erythroleukemia cell lines as targets. Monoclonal antibody EqT12 reacted...
Henson FM, Davenport C, Butler L, Moran I, Shingleton WD, Jeffcott LB, Schofield PN.The effects of insulin and insulin-like growth factors (IGFs) I and II on fetal and foal chondrocytes were investigated in vitro. Chondrocytes from the lateral trochlear ridge of the distal femur were obtained from 2 fetuses (280 and 320 days gestation) and one 4-day-old foal and cultured. Membrane proteins consistent with type 1 and type 2 IGF receptors were demonstrated by radioligand cross linking and equilibrium binding analysis. It was demonstrated that both IGF-I and IGF-II acted as mitogens for isolated equine chondrocytes when present as the sole mitogenic factor in monolayer culture. ...
Pickles K, Pirie RS, Rhind S, Dixon PM, McGorum BC.Bronchoalveolar lavage fluid (BALF) samples are often subject to time delays, possibly with temperature fluctuations, between collection and processing. The aim of this study was to evaluate the effects of time, temperature and 2 different fixatives on equine BALF cytology, in order to develop guidelines for optimal equine BALF storage conditions. Total nucleated cell count (TCC), differential cell counts (DCC), absolute cell counts (ACC), cell viability, cell morphology and bacterial growth of BALF samples stored at 4, 18 (+/- addition of formalin- or alcohol-based fixatives) and 38 degrees C...
Goudet G, Belin F, Bézard J, Gérard N.In the equine species, a large proportion of oocytes fail to complete meiosis during in-vitro culture. The biochemical and molecular basis of this failure is unknown. The meiotic cell cycle is controlled in part by the maturation-promoting factor (MPF) and the mitogen-activated protein kinase (MAPK). In this study, we evaluated the oocyte competence for in-vitro maturation and the expression of MPF components (p34cdc2 and cyclin B) and MAPK after in-vitro culture. The maturation rate was influenced by the culture medium and the physiological stage of the mare at the time of oocyte recovery. We...
Claessen C, Favoreel H, Ma G, Osterrieder N, De Schauwer C, Piepers S, Van de Walle GR.Equid herpesvirus 1 (EHV1) is an ubiquitous alphaherpesvirus that can cause respiratory disease, abortion and central nervous disorders. EHV1 is known to infect a variety of different cell types in vitro, but its tropism for cultured primary equine mesenchymal stem cells (MSC) has never been explored. We report that equine MSC were highly permissive for EHV1 and supported lytic replication of the virus in vitro. Interestingly, we observed that an infection of MSC with EHV1 resulted in a consistent downregulation of cell surface molecules CD29 (β1-integrin), CD105 (endoglin), major histocompat...
Caron JP, Gandy JC, Schmidt M, Hauptman JG, Sordillo LM.To compare the effects of triamcinolone acetonide (TA) and methylprednisolone acetate (MPA) on expression of selected chondrocyte genes in recombinant equine interleukin-1β (reIL-1β) stimulated articular cartilage explants. Methods: In vitro experiment. Methods: Horses (n = 6). Methods: Articular cartilage explants from 2- to 3- year-old horses were exposed to reIL-1β in the presence and absence of TA and MPA at 10(-7) and 10(-6) M. Resting levels of mRNA of anabolic and catabolic genes of chondrocyte origin were quantified using qPCR after 6- and 12-hour incubations. Genes of interest incl...
Anderson SL, Singh B.Horses are susceptible to a multitude of inflammatory conditions that are characterized by a strong neutrophilic response. Here, we review basic equine neutrophil biology and explore the role of neutrophils in inflammatory conditions with emphasis on intestinal ischemia and reperfusion injury and lung inflammation. In addition, unique aspects of equine neutrophil biology have been highlighted. Neutrophils comprise the highest proportion of circulating white blood cells in equine blood. The concentration of circulating equine neutrophils is a primary indicator of systemic inflammation. Addition...
Yates AC, Stewart AA, Byron CR, Pondenis HC, Kaufmann KM, Constable PD.To determine the effects of sodium hyaluronate (HA) in combination with methylprednisolone acetate (MPA) on interleukin-1 (IL-1)-induced inflammation in equine articular cartilage pellets. Methods: Chondrocytes collected from 7 horses euthanatized for problems unrelated to the musculoskeletal system. Methods: Chondrocyte pellets were treated with medium (negative control); medium containing IL-1 (positive control); or medium containing IL-1 with MPA only (0.05 or 0.5 mg/mL), HA only (0.2 or 2 mg/mL), or MPA (0.05 or 0.5 mg/mL) and HA (0.2 or 2 mg/mL) in combination. Proteoglycan (PG) synthesis...
Choi YH, Harding HD, Hartman DL, Obermiller AD, Kurosaka S, McLaughlin KJ, Hinrichs K.The reported patterns of trophectodermal expression of POU5F1 protein in blastocysts vary among species, and are possibly related to the differences in placental growth and function. This study investigated the pattern of embryonic POU5F1 expression in the horse, a species with delayed placental formation. Immature equine oocytes expressed POU5F1 protein in the cytoplasm and nucleus. Staining for POU5F1 protein in in vitro-produced (IVP) embryos decreased to day 5 of culture, then the nuclear staining increased to day 7. IVP day-7 to -11 blastocysts showed POU5F1 staining in nuclei throughout ...
Stanley RL, Goodship AE, Edwards B, Firth EC, Patterson-Kane JC.The injury-prone, energy-storing equine superficial digital flexor tendon (SDFT) of the mature performance horse has a limited ability to respond to exercise in contrast with the noninjury-prone, anatomically opposing common digital extensor tendon (CDET). Previous studies have indicated low levels of cellular activity in the mature SDFT, but in foal tendons the tenocytes may still have the ability to adapt positively to increased exercise. Objective: To measure tenocyte densities and types in histological sections from the SDFT and CDET of horses from controlled long-term, short-term and foal...
Choi YH, Love CC, Chung YG, Varner DD, Westhusin ME, Burghardt RC, Hinrichs K.We investigated the use of direct nuclear injection using the Piezo drill and activation by injection of stallion sperm cytosolic extract for production of cloned equine embryos. When metaphase II horse oocytes were injected with either of two dosages of sperm extract and cultured 20 h, similar activation rates (88% vs. 90%) and cleavage rates (49% vs. 46%) were obtained. The successful reconstruction rate of horse oocytes with horse somatic cell donor nuclei after direct injection using the Piezo drill was 82%. Four dosages of sperm extract (containing 59, 176, 293, or 1375 microg/ml protein)...
Hammond SA, Horohov D, Montelaro RC.Naive T cells can be activated both in vivo and in vitro by specialized antigen presenting cells, dendritic cells (DC), with potent antigen-specific, immunostimulatory activity. Indeed, DC can provide an extremely powerful and important immunological tool by which to potentiate the immune response for specific recognition of foreign antigens. Until recently, the direct isolation of DC from PBMC required laborious procedures with extremely poor yields (<0.1%). Methods have been developed for the human, lower primate, and murine model systems to propagate large numbers of DC from PBMC or bone...
Dykgraaf S, Dechant JE, Johns JL, Christopher MM, Bolt DM, Snyder JR.To determine the effect of intrathecal amikacin administration and repeated tenovaginocentesis on the total nucleated cell count (TNCC), total protein (TP) concentration and cytologic characteristics of synovial fluid of the equine digital flexor tendon sheath (DFTS). Methods: Randomized, cross-over experimental design. Methods: Adult horses (n=8). Methods: Synovial fluid was aseptically collected from the DFTS and either 1 mL amikacin sulfate (250 mg/mL) or lactated Ringer's solution (LRS) was injected into the DFTS. Serial synovial fluid samples were obtained at 0, 12, 24, 48, and 72 hours. ...
Grosche A, Morton AJ, Graham AS, Sanchez LC, Blikslager AT, Polyak MM, Freeman DE.Ultrastructural changes in the epithelium can provide information on early changes in barrier properties, repair and inflammation in equine colon after ischaemia and reperfusion (I/R). Objective: To describe the morphology and ultrastructure of the epithelium in equine large colonic mucosa after I/R, and the response of inflammatory cells to injury. Methods: Ischaemia was induced for 1 h followed by 4 h of reperfusion in a 40 cm segment of the pelvic flexure in 6 horses. Mucosal biopsies before and after ischaemia, and after 1, 2 and 4 h of reperfusion were fixed in glutaraldehyde/paraformalde...
Osborne RL, Coggins MK, Walla M, Dawson JH.The heme-containing respiratory protein, myoglobin (Mb), best known for oxygen storage, can exhibit peroxidase-like activity under conditions of oxidative stress. Under such circumstances, the initially formed ferric state can react with H2O2 (or other peroxides) to generate a long-lived ferryl [Fe(IV)=O] Compound II (Cpd II) heme intermediate that is capable of oxidizing a variety of biomolecules. In this study, the ability of Mb Cpd II to catalyze the oxidation of carcinogenic halophenols is demonstrated. Specifically, 2,4,6-trichlorophenol (TCP) is converted to 2,6-dichloro-1,4-benzoquinone...
Nelis H, D'Herde K, Goossens K, Vandenberghe L, Leemans B, Forier K, Smits K, Braeckmans K, Peelman L, Van Soom A.Equine embryos remain for 6 days in the oviduct and thus there is a need for an in vitro model to study embryo-oviductal interactions in the horse, since this subtle way of communication is very difficult to analyse in vivo. Until now, no equine oviduct explant culture model has been characterised both morphologically and functionally. Therefore, we established a culture system for equine oviduct explants that maintained epithelial morphology during 6 days of culture, as revealed by light microscopy and transmission electron microscopy. We demonstrated the presence of highly differentiated, ta...
Ranz AI, Miguet JG, Anaya C, Venteo A, Cortés E, Vela C, Sanz A.A panel of 32 hybridoma cell lines secreting monoclonal antibodies (MAbs) reactive with African horsesickness virus serotype 4 (AHSV-4) has been developed. Four of the MAbs recognized the major core antigen VP7, twenty recognized the outer capsid protein VP2 and eight reacted with the non-structural protein NS1. With the VP7-specific MAbs a rapid and sensitive double antibody sandwich immunoassay has been developed to detect viral antigen in infected Vero cells and in spleen tissue from AHSV-infected horses. The sensitivity of the assay is 10 ng viral antigen per 100 microliters. The NS1-speci...
Schnabel CL, Babasyan S, Freer H, Larson EM, Wagner B.Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine, that is involved in acute inflammation and is employed as a biomarker of inflammatory diseases in several species for which reliable quantification is available. We aimed to develop suitable tools to quantify TNF-α in equine samples. We generated two new mAbs against equine TNF-α (clones 48 and 292), evaluated their specificity for this cytokine, and confirmed detection of native TNF-α in stimulated equine PBMC. The TNF-α mAbs were paired in a fluorescent bead-based assay for quantification of equine TNF-α. The TNF-α assay had ...
Sun C, Zhang B, Jin J, Montelaro RC.The identification and characterization of a functional cellular receptor for equine infectious anemia virus (EIAV), designated equine lentivirus receptor-1 (ELR1), a member of the tumour necrosis factor receptor protein family, has been reported previously [Zhang, B. et al. (2005). Proc Natl Acad Sci U S A, 102 , 9918-9923]. The finding of a single receptor for EIAV is distinct from feline, simian and human immunodeficiency viruses, which typically utilize two co-receptors for infection, but is similar to avian and murine oncoviruses, which use single receptors. This study sought to determine...
Dam Van P, Desmecht D, Garigliany MM, Bui Tran Anh D, Van Laere AS.Type I/III interferons provide powerful and universal innate intracellular defense mechanisms against viruses. Among the antiviral effectors induced, Mx proteins of some species appear as key components of defense against influenza A viruses. It is expected that such an antiviral protein must display a platform dedicated to the recognition of said viruses. In an attempt to identify such platform in human MxA, an evolution-guided approach capitalizing on the antagonistic arms race between MxA and its viral targets and the genomic signature it left on primate genomes revealed that the surface-ex...
Larson EM, Babasyan S, Wagner B.IL-8 is a potent chemokine that recruits neutrophils and basophils to promote inflammation in many species. IL-8 is produced by many cell types, including monocytes. In this study, we report a novel role for IgE-binding monocytes, a rare peripheral immune cell type, to promote allergic inflammation through IL-8 production in a horse model of natural IgE-mediated allergy. We developed a mAb with confirmed specificity for both recombinant and native equine IL-8 for flow cytometric analysis. Equine IL-8 was produced by CD14/MHC class II/CD16 monocytes, including a subpopulation of IgE-binding mon...
Carter RA, Engiles JB, Megee SO, Senoo M, Galantino-Homer HL.Abnormal epidermal stem cell regulation may contribute to the pathogenesis of equine chronic laminitis. Objective: To analyse the involvement of p63, a regulator of epidermal stem cell proliferative potential, in chronic laminitis. Methods: Epidermal tissues from skin, coronet and lamellae of the dorsal foot were harvested from 5 horses with chronic laminitis and 5 control horses. Tissues were analysed using histopathology, immunofluorescence microscopy and quantitative immunoblotting. Results: Hoof lamellae of laminitic horses had a lower frequency of p63 positive cells than control lamellae,...
Dutta SK, Myrup AC, Thaker SR.Interference between equine herpesvirus types 1 (EHV-1) and 2 (EHV-2) was studied in equine dermis (ED) monolayer cell cultures and equine lymphocyte cultures. Cell cultures were infected with EHV-2, and after a short incubation period, the cultures were superinfected with EHV-1. At various intervals, different measurements of EHV-1 expression in dually infected cultures, compared with those in cultures infected with EHV-1 alone, were studied. In dually infected ED cell cultures, the EHV-1 cytopathic effect, EHV-1 titer, and EHV-1 enzyme-linked immunosorbent assay antigen titer were maximally ...
Zamansky GB, Arundel C, Nagasawa H, Little JB.The growth of two human diploid skin fibroblast cell lines, originally grown in medium supplemented with foetal bovine serum and later adapted to medium supplemented with newborn bovine, bovine calf or horse serum, has been studied. Prolonged generation times increased cell volumes and decreased plating efficiencies were observed in cultures grown in newborn bovine, bovine calf or horse serum. In general, the deleterious effects were most severe as a result of growth in bovine calf or horse serum. In the light of the present findings, we believe investigators should exert great caution in swit...
