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Topic:Cell Culture
Cell culture in horses involves the in vitro cultivation of equine cells under controlled conditions. This technique is employed to study various cellular processes, including growth, differentiation, and response to external stimuli, in an isolated environment. Equine cell cultures can be derived from various tissues, such as skin, muscle, or bone, and are used in a range of research applications, including genetic studies, drug testing, and disease modeling. These cultures provide a valuable platform for understanding cellular mechanisms and developing therapeutic strategies. This page compiles peer-reviewed research studies and scholarly articles that explore the methodologies, applications, and findings related to cell culture in equine research.
Generation and activity of equine osteoclasts in vitro: effects of the bisphosphonate pamidronate (APD). Equine osteoclast-like cells (OCLs) were generated from the bone marrow (BM) of two ponies and one horse in the presence of RANKL, the receptor activator of NF kappa B ligand and macrophage colony-stimulating factor (M-CSF). The phenotype of these cells was confirmed by demonstration of characteristics typical of osteoclasts (OCs) including: the expression of tartrate-resistant acid phosphatase (TRAP), the vitronectin receptor (VNR) and the calcitonin receptor (CTR), the demonstration of responsiveness to calcitonin (CT) and the ability to form resorption lacunae on ivory slices and calcium ph...
Effect of follicle size on in vitro production of steroids and insulin-like growth factor (IGF)-I, IGF-II, and the IGF-binding proteins by equine ovarian granulosa cells. Little is known regarding the hormonal regulation of granulosa cell steroidogenesis and the ovarian insulin-like growth factor (IGF) system in the mare. The objectives of this study were to determine, first, if estradiol, insulin, and/or FSH affect steroid production by equine granulosa cells (experiment 1) and, second, if the components of the IGF system are produced by equine granulosa cells in culture as well as whether estradiol, insulin, and/or FSH affects IGF and/or IGF-binding protein (IGFBP) production by equine granulosa cells (experiment 2). Granulosa cells from small (6-15 mm), medi...
Effects of carprofen (R and S enantiomers and racemate) on the production of IL-1, IL-6 and TNF-alpha by equine chondrocytes and synoviocytes. Chondrocytes and synoviocytes harvested from the joints of healthy horses were maintained in tissue culture. Production of the cytokines interleukin-1 (IL-1), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) in response to lipopolysaccharide (LPS), and the effects of addition of carprofen (racemate and R and S enantiomers) were determined. Lipopolysaccharide failed to stimulate TNF-alpha activity in both cell types but concentrations of IL-1 and IL-6 were both increased in a concentration and time-related manner. Both carprofen enantiomers and the racemic mixture attenuated th...
In vitro development of horse oocytes reconstructed with the nuclei of fetal and adult cells. This study investigated the basic conditions required for the production of horse embryos by the transfer of the nuclei of fetal and adult fibroblast cells to enucleated oocytes. Cumulus-oocyte complexes were recovered from abattoir ovaries and matured in vitro in groups of 20-30 for 28-30 h in tissue culture medium 199 containing 20% v:v fetal bovine serum in coculture with equine oviduct epithelial cells. Fetal fibroblast cells (FFC) were derived from a 32-day-old Thoroughbred x Pony fetus, and adult skin fibroblast cells (SFC) were obtained from subdermal biopsies recovered from a 4-yr-old ...
Suppression of meiosis by inhibitors of m-phase proteins in horse oocytes with low meiotic competence. Germinal vesicle (GV)-stage horse oocytes with diffuse chromatin are meiotically incompetent and degenerate in culture, whereas horse oocytes having condensed chromatin within the GV are meiotically competent. Degeneration of incompetent oocytes in culture may be related to premature GV breakdown, which could possibly be prevented by inhibition of m-phase protein activity. We examined the effects of 6-dimethylaminopurine (6-DMAP), butyrolactone and roscovitine on GV-stage horse oocytes. Culture in the presence of 2 mM 6-DMAP for 24 h suppressed meiosis (2% MI or MII compared with 38% for untre...
Simultaneous flow cytometric analysis of phagocytosis and oxidative burst activity in equine leukocytes. This paper describes a method for simultaneously measuring phagocytosis and oxidative burst activity in equine peripheral blood leukocytes by flow cytometry. Opsonized propidium iodide-labelled Staphylococcus aureus (PI-Sa) was used to measure the uptake of bacteria by equine phacocytes and the oxidative burst activity by oxidation of dihydrorhodamine 123. The requirements to achieve optimal activity of phagocytosis and oxidative burst are described. The advantage of the simultaneous technique is that it provides both independent and comparative values for phagocytosis and the oxidative burst,...
Cultivation and characterisation of primary and subcultured equine keratinocytes. We describe the establishment and characterisation of equine keratinocyte cultures with maintenance of a high proliferative capacity up to the second passage. Improved attachment and growth were obtained by seeding primary cells on equine feeder layers. Subcultured keratinocytes showed optimal growth when seeded on collagen type I. The proliferation rate of cells on this substrate exceeded that seen for cells seeded on equine feeder layers. By immunohistochemistry, epithelial origin and state of differentiation of the equine keratinocytes were determined. They expressed keratin and desmoplakin...
Echinococcus granulosus: regulation of leukocyte growth by living protoscoleces from horses, sheep, and cattle. To determine whether living hydatid tissue can, like hydatid fluid, regulate leukocyte growth, T-cell, B-cell, and macrophage lines were cocultured with protoscoleces of Echinococcus granulosus and their growth was compared with that of control cultures by thymidine uptake estimates and chemiluminescent assays of cell number. Protoscoleces supported mitosis of IL-1-deprived D10 T cells, but did not increase D10 count. The action of protoscoleces was affected by the species and organ of their origin and the length of time in culture. Unusually marked mitotic reaction, unaffected by parasite age...
Surface immunolocalisation of HPr in the equine pathogen Streptococcus equi. We have investigated the surface localisation of the phosphotransferase system protein HPr in the equine pathogen Streptococcus equi subsp. equi using immunogold localisation and transmission electron microscopy. Like the LppC acid phosphatase lipoprotein, a reference surface antigen, the S. equi HPR could be clearly detected on the surfaces of intact cells. This study is consistent with previous reports that some streptococcal HPr is cell surface associated and suggests that the extracytoplasmic mobilisation and transfer of phosphate groups by streptococci warrant further investigation.
Cell death during natural and induced luteal regression in mares. In mares, little information is available on the type of cell death that occurs during natural and induced luteal regression. Corpora lutea were collected from mares in the early luteal phase, days 3-4 (n = 4); mid-luteal phase, day 10 (n = 5); early regression, day 14 (n = 4); late regression, day 17 (n = 4); and 12 and 36 h (n = 3 per group) after PGF2alpha administration on day 10. Histological and ultrastructural sections were examined and TUNEL was used to detect DNA fragmentation. In early luteal regression, there were more pyknotic luteal cells and extracellular round dense bodies compa...
Equine infectious anemia virus and the ubiquitin-proteasome system. Some retroviruses contain monoubiquitinated Gag and do not bud efficiently from cells treated with proteasome inhibitors, suggesting an interaction between the ubiquitin-proteasome system and retrovirus assembly. We examined equine infectious anemia virus (EIAV) particles and found that approximately 2% of the p9(Gag) proteins are monoubiquitinated, demonstrating that this Gag protein interacts with an ubiquitinating activity. Different types of proteasome inhibitors were used to determine if proteasome inactivation affects EIAV release from chronically infected cells. Pulse-chase immunoprecip...
Phenotypic expression of equine articular chondrocytes grown in three-dimensional cultures supplemented with supraphysiologic concentrations of insulin-like growth factor-1. To assess the effects of supraphysiologic concentrations of insulin-like growth factor-1 (IGF-1) on morphologic and phenotypic responses of chondrocytes. Methods: Articular cartilage obtained from 2 young horses. Methods: Chondrocytes were suspended in fibrin cultures and supplemented with 25, 12.5, or 0 mg of IGF-1/ml of fibrin. Chondrocyte morphology and phenotypic expression were assessed histologically, using H&E and Alcian blue stains, immunoreaction to collagen type I and II, and in situ hybridization. Proteoglycan content, synthesis, and monomer size were analyzed. The DNA content w...
Molecular analysis of Neorickettsia risticii in adult aquatic insects in Pennsylvania, in horses infected by ingestion of insects, and isolated in cell culture. Upon ingestion of adult aquatic insects, horses developed clinical signs of Potomac horse fever, and Neorickettsia risticii was isolated from the blood. 16S rRNA and 51-kDa antigen gene sequences from blood, isolates, and caddis flies fed to the horses were identical, proving oral transmission of N. risticii from caddis flies to horses.
Cultures of equine respiratory epithelial cells and organ explants as tools for the study of equine influenza virus infection. Equine nasal turbinate epithelial cells and tracheal rafts were maintained with sustained viability in culture. Both types of culture supported productive replication of equine influenza virus (equine-2, subtype H3N8) and cell death occurred through apoptosis following viral infection. Thus, primary respiratory epithelial cell and organ cultures of equine origin may be valuable as alternatives to the intact animal for studying the virus-host interaction of equine respiratory viruses including influenza.
Production of biologically active equine interleukin 12 through expression of p35, p40 and single chain IL-12 in mammalian and baculovirus expression systems. Interleukin-12 (IL-12) is a key cytokine in the development of cell-mediated immune responses. Bioactive IL-12 is a heterodimeric cytokine composed of disulphide linked p35 and p40 subunits. The aim of this study was to verify biologically activity of the products expressed from equine interleukin-12 (IL-12) p35 and p40 cDNAs and to establish whether equine IL-12 could be expressed as a p35/p40 fusion polypeptide, as has been reported for IL-12a of several mammalian species. We report production of equine IL-12 through expression of p35 and p40 subunits in mammalian and insect cells and of a p...
Investigation of mRNA expression of tumor necrosis factor-alpha, interleukin-1beta, and cyclooxygenase-2 in cultured equine digital artery smooth muscle cells after exposure to endotoxin. To determine messenger RNA expression of cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-alpha, and interleukin- (IL)-1beta from cultured equine smooth muscle cells (SMC). Methods: Segments of palmar digital artery harvested from 6 clinically normal adult horses. Methods: Explants were collected from the tunica media of arteries for primary culture of SMC. Equine mononuclear cells were used as control cells. Subcultured vascular SMC and control cells were exposed to lipopolysaccharide (20 microg/ml and 100 ng/ml, respectively). Northern blot analysis with equine-specific probes for COX-2, ...
Cryopreservation of equine sperm: optimal cooling rates in the presence and absence of cryoprotective agents determined using differential scanning calorimetry. Optimization of equine sperm cryopreservation protocols requires an understanding of the water permeability characteristics and volumetric shrinkage response during freezing. A cell-shape-independent differential scanning calorimeter (DSC) technique was used to measure the volumetric shrinkage during freezing of equine sperm suspensions at cooling rates of 5 degrees C/min and 20 degrees C/min in the presence and absence of cryoprotective agents (CPAs), i.e., in the Kenney extender and in the lactose-EDTA extender, respectively. The equine sperm was modeled as a cylinder of length 36.5 microm a...
Caspase activation in equine influenza virus induced apoptotic cell death. Equine influenza virus (EIV) is the leading cause of acute respiratory infection in horses worldwide. In recent years, the precise mechanism by which influenza infection kills host cells is being re-evaluated. In this report, we examined whether caspases, a group of intracellular proteases, are activated following EIV infection and contribute to EIV-mediated cell death. Western blotting analysis indicated that a nuclear target of caspase-3, poly(ADP-ribose) polymerase (PARP) was proteolytically cleaved in EIV-infected MDCK cells, but not in mock-infected cells. In comparison with caspase-3 spe...
Capacitation and the acrosome reaction in equine sperm. During sexual reproduction, the sperm and oocyte must fuse before the production of a diploid zygote can proceed. In mammals such as equids, fusion depends critically on complex changes in the plasma membrane of the sperm and, not surprisingly, this membrane differs markedly from that of somatic cells. After leaving the testes, sperm cease to synthesize plasma membrane lipids or proteins, and vesicle-mediated transport stops. When the sperm reaches the female reproductive tract, it is activated by so-called capacitation factors that initiate a delicate reorientation and modification of molecul...
Construction of chimeric arteriviruses reveals that the ectodomain of the major glycoprotein is not the main determinant of equine arteritis virus tropism in cell culture. The recent development of arterivirus full-length cDNA clones makes possible the construction of chimeric arteriviruses for fundamental and applied studies. Using an equine arteritis virus (EAV) infectious cDNA clone, we have engineered chimeras in which the ectodomains of the two major envelope proteins, the glycoprotein GP(5) and the membrane protein M, were replaced by sequences from envelope proteins of related and unrelated RNA viruses. Using immunofluorescence microscopy, we monitored the transport of the hybrid GP(5) and M proteins to the Golgi complex, which depends on their heterodime...
Immortalization of equine trophoblast cell lines of chorionic girdle cell lineage by simian virus-40 large T antigen. Immortalized cell lines have many potential experimental applications including the analysis of molecular mechanisms underlying cell-specific gene expression. We have utilized a recombinant retrovirus encoding the simian virus-40 (SV-40) large T antigen to construct several immortalized cell lines of equine chorionic girdle cell lineage - the progenitor cells that differentiate into the equine chorionic gonadotropin (eCG) producing endometrial cups. Morphologically, the immortalized cell lines appear similar to normal chorionic girdle cells. Derivation of the immortalized cell lines from a cho...
Identification and characterisation of beta-adrenoceptors on intact equine peripheral blood lymphocytes with the radioligand (-)-[125I]-iodocyanopindolol. In this study, beta-adrenoceptors of intact equine lymphocytes were identified and subclassified by (-)-[125I]-iodocyanopindolol (ICYP) binding. ICYP binding to intact equine lymphocytes was rapid, saturable (maximal number of binding sites 320 +/- 20 ICYP binding sites/cell, n = 12) and of high affinity (KD value for ICYP 14.4 +/- 1.7 pmol/l, n = 12). Binding was stereospecific as shown by the 10 times greater potency of (-)-propranolol to inhibit binding than its (+)-isomer. Beta-adrenoceptor agonists inhibited ICYP binding with an order of potency: (-)-isoprenaline >(-)-adrenaline >(-...
Organisation of the cytoskeleton during in vitro maturation of horse oocytes. Meiotic maturation of mammalian oocytes is a complex process during which microfilaments and microtubules provide the framework for chromosomal reorganisation and cell division. The aim of this study was to use fluorescence and confocal laser scanning microscopy to examine changes in the distribution of these important cytoskeletal elements and their relationship to chromatin configuration during the maturation of horse oocytes in vitro. Oocytes were cultured in M199 supplemented with pFSH and eLH and, at 0, 12, 24, and 36 hr after the onset of culture, they were fixed for immunocytochemistry ...
Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor-beta1 in monolayer and insulin-like growth factor-I in a three-dimensional matrix. This study evaluated chondrogenesis of mesenchymal progenitor stem cells (MSCs) cultured initially under pre-confluent monolayer conditions exposed to transforming growth factor-beta1 (TGF-beta1), and subsequently in three-dimensional cultures containing insulin-like growth factor I (IGF-I). Bone marrow aspirates and chondrocytes were obtained from horses and cultured in monolayer with 0 or 5 ng of TGF-beta 1 per ml of medium for 6 days. TGF-beta 1 treated and untreated cultures were distributed to three-dimensional fibrin disks containing 0 or 100 ng of IGF-I per ml of medium to establish fou...
Developmental capacity of equine oocytes matured and cultured in equine trophoblast-conditioned media. The objective was to compare culture media for in vitro maturation of equine oocytes and for in vitro culture of zygotes produced from IVF of partially zona-removed oocytes. Cumulus-oocyte complexes from slaughterhouse-derived ovaries were washed in m-Dulbecco's PBS and cultured in TCM-199, F10-DMEM or c-F10-DMEM (50% F10-DMEM + 50% F10-DMEM conditioned medium from culture of an equine trophoblast monolayer for 3 or 4 days). All media included FSH, LH, E2, and 10% FCS. After 28 to 30 h maturation, cumulus expansion was scored from 0 (no expansion) to 4 (fully expanded). Oocytes with a 1st pola...
Inflammatory mediators induce endothelium-dependent adherence of equine eosinophils to cultured endothelial cells. Accumulation of equine eosinophils at sites of parasite infestation or allergic inflammation depends upon their adherence to vascular endothelial cells and subsequent migration through the endothelium and extracellular matrix. This study has examined whether cytokines, which cause endothelial cell-dependent eosinophil adherence in other species, and histamine and substance P, which increase adherence of equine eosinophils to protein coated plastic, induce equine eosinophil adherence to cultured equine digital vein endothelial cell (EDVEC) monolayers. The EDVEC monolayers were stimulated with r...
The effects of pyrantel tartrate on Sarcocystis neurona merozoite viability. Sarcocystis neurona is the etiologic agent of equine protozoal myeloencephalitis, a neurologic disease of horses. The present study was designed to test the hypothesis that pyrantel tartrate can kill S. neurona merozoites growing in equine dermal cell culture. Sarcocystis neurona merozoites were exposed to a range of concentrations of pyrantel tartrate or sodium tartrate ranging from 0.001 to 0.01 M. Merozoites were then placed onto equine dermal cell cultures and incubated for 2 weeks to check for viability. At 1 and 2 weeks after inoculation, plaque counts were compared between treatments an...
Activation of cumulus-free equine oocytes: effect of maturation medium, calcium ionophore concentration and duration of cycloheximide exposure. Two different culture media (TCM-199 and follicular fluid), two activation treatments (10 and 50 micromol calcium ionophore l(-1)) and three culture periods with cycloheximide were evaluated to find effective culture conditions for activation of cumulus-free equine oocytes. Oocytes were collected by scraping the follicle walls of ovaries obtained from an abattoir. Oocytes with expanded cumuli were matured at 38.2 degrees C in a humidified atmosphere of 5% CO(2) in air, in either TCM-199 with 10% fetal bovine serum (FBS) and 5 microU FSH ml(-1), or in 100% follicular fluid derived from a preovu...
DH82 cells: a macrophage cell line for the replication and study of equine infectious anemia virus. In vivo, tissue macrophages have been implicated as an important cell for the replication of equine infectious anemia virus (EIAV). Laboratory investigations of EIAV/macrophage interactions, however, have been hampered by the laborious blood monocyte isolation procedures. In addition, adherent equine macrophage cultures generally have poor long-term viability and are resistant to transfection. This report describes an adherent canine macrophage-like cell line, DH82, that supports the replication of EIAV. This cell line was easily transfectable and supported EIAV Tat transactivation of the LTR....
