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.
Mund SJK, Corbett C, MacPhee DJ, Campbell J, Honaramooz A, Wobeser B, Barber SM.Horses often sustain cutaneous wounds and healing can be prolonged and difficult to treat. Compared to body wounds, limb wounds heal slower and are more likely to develop exuberant granulation tissue. Differences in healing rates and exuberant granulation tissue formation is attributed to abnormal cytokine profiles. CXCL8 and its receptor CXCR2 are involved in acute inflammation whereas CXCL10 and its receptor CXCR3 are involved in inflammation resolution. β- arrestin-2 regulates inflammation through internalization of G-protein coupled receptors (GPCRs) including CXCR2 and CXCR3. Gene expres...
Singh UK, Patel R.In vitro refolding of denatured protein and the influence of the alkyl chain on the refolding of a protein were tested using long chain imidazolium chloride salts, 1-methyl-3-octylimidazolium chloride [Cmim][Cl], and 1-decyl-3-methylimidazolium chloride [Cmim][Cl]. The horse heart cytochrome c (h-cyt c) was denatured by urea and guanidinium hydrochloride (GdnHCl), as well as by base-induced denaturation at pH 13, to provide a broad overview of the overall refolding behavior. The variation in the alkyl chain of the ionic liquids (ILs) showed a profound effect on the refolding of denatured h-cyt...
Ghasemi S, Sardari K, Mirshokraei P, Hassanpour H.Application of synthetic matrix metalloproteinases (MMPs) inhibitors, such as doxycycline is one of the possible therapeutic options for osteoarthritis. However, little is known about the protective mechanism of doxycycline in equine models on MMPs inhibitors as well as on serum amyloid A (SAA) gene expression. This study investigated the effects of doxycycline on mRNA expression of MMP-1, MMP-2, MMP-9, MMP-13, and SAA of equine fibroblast-like synoviocytes (FLSs). The FLSs were established from synovial fluids of clinically normal metacarpophalangeal joints of 6 skeletally mature horses. The ...
Hollis AR, Starkey MP.MicroRNAs are small noncoding RNAs that play a pivotal role in diverse cellular processes through post-transcriptional regulation of gene expression. The dysregulation of specific microRNAs is associated with disease development and progression. In this review, we summarise how microRNAs modulate gene expression, and explain microRNA nomenclature. We discuss the potential applications of microRNAs in equine disease diagnosis and treatment, in the context of the sum of current knowledge about microRNA expression in normal and diseased equine tissues.
Ortved KF, Begum L, Stefanovski D, Nixon AJ.Following joint trauma, a posttraumatic inflammatory cascade drives degeneration of the joint. We aimed to assess whether transduction of chondrocytes with AAV5 overexpressing the immunomodulatory cytokine IL-10 would have protective effects in pellet cultures stimulated with IL-1β. Chondrocytes were isolated from 3 healthy horses and were transduced with AAV5-IL-10 at a dose of 1 x 105vg/cell. Chondrocyte pellets were formed by centrifugation and were stimulated with IL-1β starting 48 hours following transduction. After 2, 6 and 14 days in culture, supernatants were collected for cytokine a...
Baird A, Lindsay T, Everett A, Iyemere V, Paterson YZ, McClellan A, Henson FMD, Guest DJ.Bone fractures occur in horses following traumatic and non-traumatic (bone overloading) events. They can be difficult to treat due to the need for the horse to bear weight on all legs during the healing period. Regenerative medicine to improve fracture union and recovery could significantly improve horse welfare. Equine induced pluripotent stem cells (iPSCs) have previously been derived. Here we show that equine iPSCs cultured for 21 days in osteogenic induction media on an OsteoAssay surface upregulate the expression of osteoblast associated genes and proteins, including , , , , and We al...
Jørgensen E, Lazzarini G, Pirone A, Jacobsen S, Miragliotta V.Information on microscopic anatomy of equine skin is sparse. In horses, limb wounds often become chronic and/or non-healing whereas body wounds heal normally. These dissimilarities in healing patterns might be a product of different phenotypic characteristics of body and limb skin. The objective of this study was to investigate microscopic anatomy, epidermal thickness, keratinocyte proliferation and differentiation as well as the presence of mast cells in normal equine skin of body and limb. Methods: The study involved body and limb skin biopsies from six horses. Histological characteristics o...
Park MJ, Lee J, Byeon JS, Jeong DU, Gu NY, Cho IS, Cha SH.Mesenchymal stem cells (MSCs) are useful candidates for tissue engineering and cell therapy fields. We optimize culture conditions of equine adipose tissue-derived MSCs (eAD-MSCs) for treatment of horse fractures. To investigate enhancing properties of three-dimensional (3D) culture system in eAD-MSCs, we performed various sized spheroid formation and determined changes in gene expression levels to obtain different sized spheroid for cell therapy. eAD-MSCs were successfully isolated from horse tailhead. Using hanging drop method, spheroid formation was generated for three days. Quantitative re...
Löfgren M, Svala E, Lindahl A, Skiöldebrand E, Ekman S.Osteoarthritis is an inflammatory and degenerative joint disease commonly affecting horses. To identify genes of relevance for cartilage pathology in osteoarthritis we studied the time-course effects of interleukin (IL)-1β on equine articular cartilage. Articular cartilage explants from the distal third metacarpal bone were collected postmortem from three horses without evidence of joint disease. The explants were stimulated with IL-1β for 27 days and global gene expression was measured by microarray. Gene expression was compared to that of unstimulated explants at days 3, 9, 15, 21 and 27...
Rotter Sopasakis V, Wickelgren R, Sukonina V, Brantsing C, Svala E, Hansson E, Enerbäck S, Lindahl A, Skiöldebrand E.Chondrocytes are responsible for remodeling and maintaining the structural and functional integrity of the cartilage extracellular matrix. Because of the absence of a vascular supply, chondrocytes survive in a relatively hypoxic environment and thus have limited regenerative capacity during conditions of cellular stress associated with inflammation and matrix degradation, such as osteoarthritis (OA). Glucose is essential to sustain chondrocyte metabolism and is a precursor for key matrix components. In this study, we investigated the importance of glucose as a fuel source for matrix repair dur...
Read JE, Cabrera-Sharp V, Kitscha P, Cartwright JE, King PJ, Fowkes RC, de Mestre AM.Equine chorionic gonadotrophin (eCG) is a placental glycoprotein critical for early equine pregnancy and used therapeutically in a number of species to support reproductive activity. The factors in trophoblast that transcriptionally regulate eCGβ-subunit (), the gene which confers the hormones specificity for the receptor, are not known. The aim of this study was to determine if glial cells missing 1 regulates promoter activity. Here, studies of the proximal promoter identified four binding sites for glial cells missing 1 (GCM1) and western blot analysis confirmed GCM1 was expressed in equi...
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...
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...
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...
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...
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...
Brandão JS, Alvarenga ML, Pfeifer JPH, Dos Santos VH, Fonseca-Alves CE, Rodrigues M, Laufer-Amorim R, Castillo JAL, Alves ALG.The superficial digital flexor tendon (SDFT) is a structure frequently affected by injuries in high-performance athletic horses, and there are limited therapeutic options. Regenerative medicine has evolved significantly in treating different illnesses. However, understanding the cellular behaviour during mesenchymal stem cell (MSC) transplantation in healthy tissues is not fully known yet. To address the inflammatory response induced by allogeneic MSC transplantation, this study evaluated the local inflammatory response after the application of allogeneic adipose tissue-derived mesenchymal ste...
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...
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...
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...
Otto IA, Levato R, Webb WR, Khan IM, Breugem CC, Malda J.Paramount for the generation of auricular structures of clinically-relevant size is the acquisition of a large number of cells maintaining an elastic cartilage phenotype, which is the key in producing a tissue capable of withstanding forces subjected to the auricle. Current regenerative medicine strategies utilize chondrocytes from various locations or mesenchymal stromal cells (MSCs). However, the quality of neo-tissues resulting from these cell types is inadequate due to inefficient chondrogenic differentiation and endochondral ossification, respectively. Recently, a subpopulation of stem/pr...
Oladunni FS, Sarkar S, Reedy S, Balasuriya UBR, Horohov DW, Chambers TM.Equine herpesvirus-1 (EHV-1) infection is an important and highly prevalent disease in equine populations worldwide. Previously we have demonstrated that a neuropathogenic strain of EHV-1, T953, suppresses the host cell's antiviral type-I interferon (IFN) response in vitro. Whether or not this is unique to EHV-1 strains possessing the neuropathogenic genotype has been undetermined. Here, we examined whether there is any direct relationship between neuropathogenic genotype and the induced IFN-β response in equine endothelial cells (EECs) infected with 10 different strains of EHV-1. The extent ...
Smith AD, Panickar KS, Urban JF, Dawson HD.Vitamins and minerals (micronutrients) play an important role in regulating and shaping an immune response. Deficiencies generally result in inadequate or dysregulated cellular activity and cytokine expression, thereby affecting the immune response. Decreased levels of natural killer, granulocyte, and phagocytic cell activity and T and B cell proliferation and trafficking are associated with inadequate levels of micronutrients, as well as increased susceptibility to various adverse health conditions, including inflammatory disorders, infection, and altered vaccine efficacy. In addition, most s...
DeBoer ML, Martinson KM, Pampusch MS, Hansen AM, Wells SM, Ward C, Hathaway M.Leucine has been shown to stimulate the mammalian/mechanistic target of rapamycin (mTOR) signaling pathway which plays numerous key regulatory roles in cell growth, survival, and metabolism including protein synthesis in a number of species. However, previous work with equine satellite cells has suggested distinct species differences in regards to physiological effects and the magnitude of responses to growth factors and regulators. Because there is limited research available regarding the role of leucine in regulating equine skeletal muscle protein synthesis, the objective of this study was t...
Olivera R, Moro LN, Jordan R, Pallarols N, Guglielminetti A, Luzzani C, Miriuka SG, Vichera G.Cell plasticity is crucial in cloning to allow an efficient nuclear reprogramming and healthy offspring. Hence, cells with high plasticity, such as multipotent mesenchymal stem cells (MSCs), may be a promising alternative for horse cloning. In this study, we evaluated the use of bone marrow-MSCs (BM-MSCs) as nuclear donors in horse cloning, and we compared the in vitro and in vivo embryo development with respect to fibroblasts. Methods: Zona-free nuclear transfer was performed using BM-MSCs (MSC group, n=3432) or adult fibroblasts (AF group, n=4527). Embryos produced by artificial insemination...
Suagee JK, Corl BA, Crisman MV, Wearn JG, McCutcheon LJ, Geor RJ.The lipogenic capacities of equine liver and adipose tissue explants were investigated in vitro. Preference for glucose or acetate as the primary carbon source for de novo fatty acid synthesis was determined using (14)C labeled substrates. Additional aims included determining the relative contribution of NADPH generating pathways to reducing equivalent generation and comparing the lipogenic activity of two adipose depots, mesenteric and subcutaneous harvested from the crest region of the neck. Mesenteric adipose tissue had greater lipogenic activity than subcutaneous adipose tissue, and liver ...
Santos EM, Paula JF, Motta PM, Heinemann MB, Leite RC, Haddad JP, Del Puerto HL, Reis JK.We compared three different protocols for DNA extraction from horse peripheral blood mononuclear cells (PBMC) and lung fragments, determining average final DNA concentration, purity, percentage of PCR amplification using beta-actin, and cost. Thirty-four samples from PBMC, and 33 samples from lung fragments were submitted to DNA extraction by three different protocols. Protocol A consisted of a phenol-chloroform and isoamylic alcohol extraction, Protocol B used alkaline extraction with NaOH, and Protocol C used the DNAzol((R)) reagent kit. Protocol A was the best option for DNA extraction from...
Huang W, Yaraghi NA, Yang W, Velazquez-Olivera A, Li Z, Ritchie RO, Kisailus D, Stover SM, McKittrick J.The equine hoof has been considered as an efficient energy absorption layer that protects the skeletal elements from impact when galloping. In the present study, the hierarchical structure of a fresh equine hoof wall and the energy absorption mechanisms are investigated. Tubules are found embedded in the intertubular matrix forming the hoof wall at the microscale. Both tubules and intertubular areas consist of keratin cells, in which keratin crystalline intermediate filaments (IFs) and amorphous keratin fill the cytoskeletons. Cell sizes, shapes and IF fractions are different between tubular a...
Rockey JH.Eight antigenically unique immunoglobulins have been identified in purified equine anti-p-azophenyl-beta-lactoside (Lac) antibody isolated from a single horse. The Fc fragments of the gammaGa-, gammaGb-, gammaGc-, and -gammaA-globulins have been shown to possess unique antigenic determinants. Common gammaG- and gammaA-Fc fragment antigenic determinants, which were absent from the 10Sgamma(1)- and gammaM-globulins, have also been observed. All antibody populations share two antigenically distinct light (B, L) chain variants. The association of anti-Lac antibody with the hapten p-(p-dimethylamin...
Galiazzo G, Tagliavia C, Giancola F, Rinnovati R, Sadeghinezhad J, Bombardi C, Grandis A, Pietra M, Chiocchetti R.Colic is a common digestive disorder in horses and one of the most urgent problems in equine medicine. A growing body of literature has indicated that the activation of cannabinoid receptors could exert beneficial effects on gastrointestinal inflammation and visceral hypersensitivity. The localisation of cannabinoid and cannabinoid-related receptors in the intestine of the horse has not yet been investigated. The purpose of this study was to immunohistochemically localise the cellular distribution of canonical and putative cannabinoid receptors in the ileum of healthy horses. Distal ileum spec...
Nielsen SB, Wilhelm K, Vad B, Schleucher J, Morozova-Roche LA, Otzen D.The normal function of equine lysozyme (EL) is the hydrolysis of peptidoglycan residues of bacterial cell walls. EL is closely related to alpha-lactalbumins with respect to sequence and structure and further possesses the calcium binding site of alpha-lactalbumins. Recently, EL multimeric complexes with oleic acids (ELOAs) were shown to possess tinctorial and morphological properties, similar to amyloidal aggregates, and to be cytotoxic. ELOA's interactions with phospholipid membranes appear to be central to its biological action, similar to human alpha-lactalbumin made lethal to tumor cells. ...
Litzke LE, Wagner E, Baumgaertner W, Hetzel U, Josimović-Alasević O, Libera J.Damaged adult articular cartilage has very limited capacity to heal. Autologous chondrocyte transplantation (ACT) has been used clinically and studied in experimental animals in an attempt to provide biologically based cartilage regeneration. This study evaluated cartilage repair following ACT in a large animal model over a period of 2 years. Articular cartilage defects (10 mm in diameter, full-thickness) were created in the minor load-bearing area on the lateral talus of tibiotarsal joints of eight adult horses. In each animal, the right joint was repaired using autologous chondrocytes inject...
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...
Cullier A, Cassé F, Manivong S, Contentin R, Legendre F, Garcia Ac A, Sirois P, Roullin G, Banquy X, Moldovan F, Bertoni L, Audigié F, Galéra P....Osteoarthritis (OA) is a degenerative and heterogeneous disease that affects all types of joint structures. Current clinical treatments are only symptomatic and do not manage the degenerative process in animals or humans. One of the new orthobiological treatment strategies being developed to treat OA is the use of drug delivery systems (DDS) to release bioactive molecules over a long period of time directly into the joint to limit inflammation, control pain, and reduce cartilage degradation. Two vasoactive peptides, endothelin-1 and bradykinin, play important roles in OA pathogenesis. In this ...
Rodríguez-Roldán V, García-Heredia JM, Navarro JA, Hervás M, De la Cerda B, Molina-Heredia FP, De la Rosa MA.Two synthetic genes coding for human and Arabidopsis cytochrome c, respectively, have been designed and constructed, and the recombinant proteins have been over-expressed in Escherichia coli cells. Thus a comparative analysis of the two heme proteins, including horse cytochrome c as a reference, has been performed. In addition to their physico-chemical properties, the redox behavior of the three proteins has been analyzed by following the kinetics of both their reduction by flavin semiquinones (lumiflavin, riboflavin, and FMN) and oxidation by cytochrome c oxidase. The resulting data indicate ...
Siemieniuch MJ, Szóstek AZ, Gajos K, Kozdrowski R, Nowak M, Okuda K.Mares that fail to conceive or lose their embryos, without showing typical signs of clinical endometritis, should be suspected of subclinical endometritis (SE). In this study, the question was addressed: does SE fully activate selected mechanisms of innate immunity in mares? For this aim, expression of mRNAs for Toll-like Receptor 2 and 4 (TLR 2/4), interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor α (TNF) was examined in control mares versus either mares suffering from chronic endometritis (ChE) or subacute suppurative endometritis (SSE). The concentrations of IL-1β, ...
Hinrichs K, Schmidt AL, Friedman PP, Selgrath JP, Martin MG.The chromatin configuration of resting horse oocytes and the time course of in vitro oocyte maturation was characterized using a fluorescent, DNA-specific label. Oocytes were classified as having either compact (CP) or expanded (EX) cumuli at the time of collection. Centrifugation of oocytes was effective in allowing visualization of the germinal vesicle. Two main chromatin configurations were found in oocytes known to have a germinal vesicle: condensed chromatin (CC), in which the chromatin formed a dense mass surrounding the nucleolus; and fluorescing nucleus (FN), in which the entire nucleu...
Rode K, Sieme H, Richterich P, Brehm R.The formation of the blood-testis barrier (BTB) is defined as occurring with the first appearance of spermatocytes at around puberty and is vital for normal spermatogenesis. This barrier between two adjacent Sertoli cells (SCs) consists of a cell junctional protein complex, which includes tight junctions (TJs), adherens junctions, and gap junctions. In many mammalian species, BTB composition has already been investigated, whereas little is known about the equine BTB. In the present study, immunohistochemistry and qualitative Western Blot analysis were used to assess the expression and distribu...
van Berlo MF, Rottier PJ, Spaan WJ, Horzinek MC.Intracellular virus-specific proteins induced by equine arteritis virus (EAV) have been compared with in vitro translation products of virion and intracellular EAV RNAs. In infected BHK-21 cells, the two major virion proteins (C and E1) and polypeptides with mol. wt. of 60,000 (p60), 42,000 (p42) and 30,000 (p30) were found. There were no indications that the viral proteins were processed from a larger precursor as shown by pulse-chase, amino acid analogue and protease inhibitor experiments. The six polyadenylated RNAs that occur in EAV-infected cells were isolated and translated in an mRNA-de...
Koch DW, Berglund AK, Messenger KM, Gilbertie JM, Ellis IM, Schnabel LV.Tendon injury in the horse carries a high morbidity and monetary burden. Despite appropriate therapy, reinjury is estimated to occur in 50-65% of cases. Although intralesional mesenchymal stem cell (MSC) therapy has improved tissue architecture and reinjury rates, the mechanisms by which they promote repair are still being investigated. Additionally, reevaluating our application of MSCs in tendon injury is necessary given recent evidence that suggests MSCs exposed to inflammation (deemed MSC licensing) have an enhanced reparative effect. However, applying MSC therapy in this context is limited...
Ketchum CC, Larsen CD, McNeil A, Meyer-Ficca ML, Meyer RG.Chromatin remodeling during spermatogenesis culminates in the exchange of nucleosomes for transition proteins and protamines as an important part of spermatid development to give rise to healthy sperm. Comparative immunofluorescence analyses of equine and murine testis histological sections were used to characterize nucleoprotein exchange in the stallion. Histone H4 hyperacetylation is considered a key event of histone removal during the nucleoprotein transition to a protamine-based sperm chromatin structure. In the stallion, but not the mouse, H4 was already highly acetylated in lysine residu...
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...
Lepage SIM, Lee OJ, Koch TG.Mesenchymal stromal cells (MSCs) are the most common cell population studied for therapeutic use in veterinary medicine. MSCs obtained from neonatal sources such as umbilical cord tissue (CT-MSCs) or cord blood (CB-MSCs) are appealing due to the non-invasive nature of procurement and the time allowed for characterization of cells before use. However, it remains unclear as to whether CB- or CT-MSCs have equivalent progenitor and non-progenitor functions. CB-MSCs have been shown to have superior chondrogenic potential to MSCs from other sources, whereas their immunomodulatory capacity does not s...
Sayasith K, Sirois J, Lussier JG.The luteinizing hormone preovulatory surge stimulates several signal pathways essential for ovulation, and the regulator of G-protein signaling protein-2 (RGS2) is thought to be involved in this process. The objectives of this study were to characterize the regulation of RGS2 transcripts in equine and bovine follicles prior to ovulation and to determine its transcriptional control in bovine granulosa cells. To assess the regulation of equine RGS2 prior to ovulation, RT-PCR was performed using total RNA extracted from equine follicles collected at various times after human chorionic gonadotropi...
Futas J, Horin P.Natural killer (NK) cells have important functions in immunity. NK recognition in mammals can be mediated through killer cell immunoglobulin-like receptors (KIR) and/or killer cell lectin-like Ly49 receptors. Genes encoding highly variable NK cell receptors (NKR) represent rapidly evolving genomic regions. No single conservative model of NKR genes was observed in mammals. Single-copy low polymorphic NKR genes present in one mammalian species may expand into highly polymorphic multigene families in other species. In contrast to other non-rodent mammals, multiple Ly49-like genes appear to exist ...
Seeto WJ, Tian Y, Winter RL, Caldwell FJ, Wooldridge AA, Lipke EA.A common challenge in cell therapy is the inability to routinely maintain survival and localization of injected therapeutic cells. Delivering cells by direct injection increases the flexibility of clinical applications, but may cause low cell viability and retention rates due to the high shear forces in the needle and mechanical wash out. In this study, we encapsulated endothelial colony forming cells (ECFCs) in poly(ethylene glycol)-fibrinogen (PF) hydrogel microspheres using a custom-built microfluidic device; this system supports rapid encapsulation of high cell concentrations (10 million c...
Go YY, Li Y, Chen Z, Han M, Yoo D, Fang Y, Balasuriya UB.The objective of this study was to investigate the effect of equine arteritis virus (EAV) on type I interferon (IFN) production. Equine endothelial cells (EECs) were infected with the virulent Bucyrus strain (VBS) of EAV and expression of IFN-β was measured at mRNA and protein levels by quantitative real-time RT-PCR and IFN bioassay using vesicular stomatitis virus expressing the green fluorescence protein (VSV-GFP), respectively. Quantitative RT-PCR results showed that IFN-β mRNA levels in EECs infected with EAV VBS were not increased compared to those in mock-infected cells. Consistent wit...
Noiman S, Yaniv A, Sherman L, Tronick SR, Gazit A.The pattern of expression of the equine infectious anemia virus (EIAV) genome in a persistently infected canine cell line was determined. Five EIAV-specific transcripts (8.2, 5.0, 4.0, 2, and 1.8 kilobases [kb]) were detected by using subgenomic restriction enzyme fragments of EIAV DNA and EIAV-specific oligonucleotides as probes. The 8.2-kb mRNA could be shown to represent viral genomic RNA, whereas the smaller transcripts were generated by splicing events. Evidence was obtained that indicated that each subgenomic RNA species shared a common 5'-splice donor. The 5.0-kb mRNA was found to be ex...
Ríos DL, López C, Álvarez ME, Samudio IJ, Carmona JU.Platelet-rich plasma (PRP) preparations are a common treatment in osteoarthritis (OA) and inflammatory synovitis. However, there is ambiguity regarding the ideal concentration of leukocytes and platelets in these preparations necessary to induce an adequate anti-inflammatory and anabolic response in joint tissues, such as the synovial membrane. This research aimed to study, in normal synovial membrane explants (SME) challenged with lipopolysaccharide (LPS), the temporal effects (at 48 and 96h) of leukocyte- and platelet-rich gel (L-PRG) and pure platelet-rich gel (P-PRG) supernatants on the pr...
Baumann U, Bode W, Huber R, Travis J, Potempa J.The crystal structure of active-site cleaved equine leucocyte elastase inhibitor, a member of the serpin superfamily, has been solved and refined to a crystallographic R-factor of 17.6% at 1.95 A resolution. Despite being an intracellular inhibitor with rather low sequence homology of 30% to human alpha 1-antichymotrypsin and alpha 1-proteinase inhibitor, the three-dimensional structures are very similar, with deviations only at the sites of insertions and few mobile secondary structure elements. The better resolution in comparison with the structures of other cleaved serpins allows a more pre...
Elashry MI, Baulig N, Wagner AS, Klymiuk MC, Kruppke B, Hanke T, Wenisch S, Arnhold S.Combination of mesenchymal stem cells (MSCs) and biomaterials is a rapidly growing approach in regenerative medicine particularly for chronic degenerative disorders including osteoarthritis and osteoporosis. The present study examined the effect of biomaterial scaffolds on equine adipose-derived MSC morphology, viability, adherence, migration, and osteogenic differentiation. MSCs were cultivated in conjunction with collagen CultiSpher-S Microcarrier (MC), nanocomposite xerogels B30 and combined B30 with strontium (B30Str) biomaterials in osteogenic differentiation medium either under static or...
Boeer U, Buettner FF, Klingenberg M, Antonopoulos GC, Meyer H, Haverich A, Wilhelmi M.The limited biocompatibility of decellularized scaffolds is an ongoing challenge in tissue engineering. Here, we demonstrate the residual immunogenicity of an extensively decellularized equine carotid artery (dEAC(intens)) and identify the involved immunogenic components. EAC were submitted to an elaborated intensified decellularization protocol with SDS/sodium desoxycholate for 72 h using increased processing volumes (dEAC(intens)), and compared to dEAC(ord) prepared by an ordinary protocol (40 h, normal volumes). Matrix integrity was checked via correlative volumetric visualization which rev...
Azab W, Tsujimura K, Maeda K, Kobayashi K, Mohamed YM, Kato K, Matsumura T, Akashi H.Heparan sulfate moieties of cell surface proteoglycans serve as receptors for several herpesviruses. For herpes simplex virus 1, pseudorabies virus and equine herpesvirus 1, glycoprotein C (gC) homologues have been shown to mediate the binding to cell surface heparan sulfate. However, the role of gC in equine herpesvirus 4 (EHV-4) infection has not yet been analyzed. Using pull-down assay, we first determined that EHV-4 gC as well as gB are heparin-binding glycoproteins. To study the role of gC in EHV-4 infection, we constructed a gC-deletion mutant, WA79DeltagC, where the kanamycin resistant ...
Olivera R, Moro LN, Jordan R, Pallarols N, Guglielminetti A, Luzzani C, Miriuka SG, Vichera G.Cell plasticity is crucial in cloning to allow an efficient nuclear reprogramming and healthy offspring. Hence, cells with high plasticity, such as multipotent mesenchymal stem cells (MSCs), may be a promising alternative for horse cloning. In this study, we evaluated the use of bone marrow-MSCs (BM-MSCs) as nuclear donors in horse cloning, and we compared the in vitro and in vivo embryo development with respect to fibroblasts. Methods: Zona-free nuclear transfer was performed using BM-MSCs (MSC group, n=3432) or adult fibroblasts (AF group, n=4527). Embryos produced by artificial insemination...
