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Topic:In Vitro Research
In vitro research involving horses refers to the study of equine cells, tissues, or biological molecules outside their normal biological context, typically in controlled laboratory environments. This research approach allows scientists to investigate cellular processes, molecular interactions, and the effects of various treatments without the ethical and logistical complexities of in vivo studies. In vitro studies contribute to understanding equine physiology, pathology, and pharmacology by providing insights into cellular responses to pathogens, drugs, and other stimuli. This page compiles peer-reviewed research studies and scholarly articles that explore various in vitro methodologies and their applications in equine science, including cell culture techniques, molecular assays, and drug efficacy testing.
Development of neutralizing antibody in horses infected with Ehrlichia risticii. The role of the humoral immune response in ehrlichial infection is unknown. Development of neutralizing antibodies during a course of Ehrlichia risticii infection in a pony was examined in vitro by determining the inhibition of E. risticii infection of P388D1 cells in the presence of the sera. The pony experimentally infected with E. risticii developed significant neutralizing activity in the sera by 15 days postinfection when parasitemia started to decline. Neutralizing activity continued to rise after recovery from the disease up to 34 days postinfection at which time the experiment was term...
Neutrophil chemotaxis in the horse is not mediated by a complex of equine neutrophil elastase and equine alpha-1-proteinase inhibitor. Studies have demonstrated that as a result of proteolytic inactivation or complex formation (with neutrophil elastase), human alpha-1-proteinase inhibitor (API) becomes a potent chemoattractant for human neutrophils. The present study aimed to investigate the in vitro chemotactic response of equine neutrophils to an equivalent complex of equine API and neutrophil elastase. No evidence of neutrophil migration was observed towards purified complex derived from equine neutrophil elastase and the Spi 1 isoform of equine API, or to crude mixtures of porcine pancreatic elastase and unseparated equin...
Effect of polysulfated glycosaminoglycan on osteoarthritic equine articular cartilage in explant culture. Middle carpal cartilage explants from 4 horses with mild osteoarthritis involving that joint were maintained in tissue culture to test the effects of a polysulfated glycosaminoglycan (PSGAG) on proteoglycan synthesis and degradation. Cultures were exposed to 0.025 or 25 mg of PSGAG/ml for 48 hours, after which the medium was replaced with medium containing similar doses of PSGAG and 35S. Subsequently, the sulfated proteoglycan content of the medium and extracts of the explants was measured. Gel filtration chromatography was used to estimate the size and to purify the principal, large proteogly...
Development to blastocysts of one- to two-cell equine embryos after coculture with uterine tubal epithelial cells. Development of 1- to 2-cell in vivo fertilized equine embryos cultured with or without uterine tubal epithelial cells (UTEC) was studied. One- to 2-cell embryos (n = 26) were collected surgically from the uterine tubes of pony mares 1 day after ovulation. Four- to 8-cell embryos (n = 9) were collected 2 days after ovulation. Presumptive zygotes and 2-cell embryos were cultured with (n = 17) or without (n = 9) UTEC, and all 4- to 8-cell embryos were cocultured with UTEC as positive controls. Uterine tubal epithelial cells were used as cell suspensions within 2 weeks after initiation of cultures...
In-vitro susceptibility to antimicrobial drugs of bacterial isolates from horses in The Netherlands. Minimum inhibitory concentrations (MICs) of 30 antimicrobial agents (including the hitherto unreported antimicrobial agents doxycycline, minocycline, vanomycin, 3 quinolones and 3 combinations of antimicrobial agents) for isolates of Salmonella spp. (20), Escherichia coli (17), Klebsiella spp. (8), Proteus spp. (7), Pseudomonas aeruginosa (7), Actinobacillus equuli (5), Rhodococcus equi (4), Streptococcus zooepidemicus (23), Streptococcus equisimilis (6), Streptococcus equi (4), coagulase-positive Staphylococcus spp. (20) and Taylorella equigenitalis (19) were determined using the agar dilutio...
Molecular cloning and expression of an intracellular serpin: an elastase inhibitor from horse leucocytes. Horse blood leucocytes contain an elastase inhibitor (HLEI) belonging to the serpin family. Poly(A)+RNA isolated from these cells was used to construct a cDNA library in lambda gt10, which was first screened with a synthetic degenerate oligonucleotide probe corresponding to the amino acid sequence of the reactive centre of the inhibitor. Three clones were obtained covering the entire coding region of the protein. Sequencing of these clones showed identity with the amino acid sequence obtained from Edman degradation of the elastase inhibitor. The coding sequence of the HLEI cDNA was cloned into...
The effect of cortisol on the secretion of ACTH by anterior pituitary cells of the horse in culture. Perifused equine anterior pituitary cells were used to investigate the effect of cortisol on the ACTH response to pulses of corticotrophin-releasing hormone (CRH; 0.01 nmol/l) and arginine vasopressin (AVP; 100 nmol/l), given for 5 min every 30 min for 690 min and ACTH measured in 5-min fractions. At the fourth pulse of secretagogue (0 min), a constant perifusion with cortisol began (0 nmol/l (control), 100, 200, 500, 5000 and 50,000 nmol/l) and continued until the ninth pulse (150 min). For each pulse of secretagogue, the amount of ACTH (pmol) secreted in response to each pulse (ACTH response...
Isolation of an inhibitor of tumor necrosis factor-alpha-mediated cytotoxicity liberated from chemotaxin-stimulated equine white blood cell populations. Objectives of this investigation were to extract and isolate protein fractions inhibitory to the cytotoxic properties of tumor necrosis factor-alpha (TNF-alpha). In this context, mixed populations of WBC were harvested from equine blood and were stimulated with a combination of a synthetic chemotactic peptide and a calcium ionophore. Several methods were subsequently applied for the initial preparation of cell-free crude protein extracts, including fractional precipitation with gradient concentrations of ammonium sulfate and preparative-scale isoelectric focusing. In addition, protein fraction...
In vitro concentrative accumulation of D-xylose by jejunum from horses and rabbits. Accumulation of D-xylose by jejunal mucosa from healthy horses and rabbits was studied in vitro. When tissue sheets were incubated with 1 mM D-xylose for 60 minutes, mucosa from horses and rabbits accumulated D-xylose against a concentration gradient. There was no accumulation when equine specimens were incubated with 5 mM D-xylose. By comparison, equine jejunum accumulated D-glucose against a concentration gradient when incubated in 5 mM D-glucose. In equine and rabbit jejunum, 13.3 +/- 7.0% and 36 +/- 11.0%, respectively, of accumulated D-xylose was phosphorylated when sheets were incubated ...
Different in vitro metabolism of 7 alpha-methyl-19-nortestosterone by human and equine aromatases. The ability of human and equine placental microsomes to aromatize 7 alpha-methyl-19-nortestosterone (MNT) was studied. Kinetic analysis indicates that MNT shares the androgen-binding site of human and equine placental microsomal aromatases. Human placental microsomal estrogen synthetase had about a 2.5-fold higher relative affinity for MNT than the equine placental enzyme (KiMNT/Km androstenedione of 32 versus 87). However, MNT was not metabolized by human placental microsomes, whereas it was very actively metabolized by equine placental microsomes. Further studies using purified equine cytoch...
Developmental regulation of insulin like growth factor II expression in the horse. The expression of the insulin like growth factor (IGF) II gene has been examined in the developing equine fetus. It was found that IGF II transcripts were present in abundant quantities in third trimester embryonic and extraembryonic tissues as for example the placenta. The expression of the IGF II gene was high in the fetal liver where two prominent transcripts--4.6 and 4.1--kB were produced. However, these transcripts could not be traced in the adult liver. Instead we found two different transcripts with the sizes of 4.0 and 2.9 kB in the adult liver. These findings taken together with the d...
Factors affecting ACTH release from perifused equine anterior pituitary cells. The multifactorial control of ACTH is well established. We wished to establish and characterize an in-vitro perifusion system, using equine anterior pituitary cells and physiological concentrations of secretagogues, to investigate factors which affect the dynamics of ACTH secretion. Anterior pituitary tissue was divided for dispersion into cells with collagenase, trypsin or dispase, or by mechanical dispersion. After dispersal followed by 18-h incubation, cells were perifused and the ACTH response to 10-min pulses of arginine vasopressin (AVP; 100 nmol/l), corticotrophin-releasing hormone (CRH...
The cDNA sequence of horse transferrin. The cDNA sequence of horse transferrin was determined by sequencing clones isolated from a horse liver cDNA library and clones obtained by PCR. The 2305 bp horse transferrin cDNA sequence included part of the 5' untranslated region and extended to the poly(A) tail. It had 80% sequence identity with the human transferrin cDNA, and encoded a protein of 706 residues, including a signal sequence of 19 amino acids. The horse transferrin sequence had the duplicated structure and conserved iron binding and cysteine residues which are characteristic of the transferrin family.
In vitro effects of alpha 2-adrenergic receptor stimulation on cholinergic contractions of equine distal airways. In horses with noninduced, reversible airway obstruction (heaves), pulmonary function is improved after sedation with the alpha 2-adrenergic agonist xylazine. The mechanism of this effect is undetermined. Because the predominant excitatory innervation of equine airways is cholinergic, the influence of alpha 2-adrenergic receptor stimulation on the response of isolated distal airways to cholinergic stimulation was determined. Distal bronchial segments from 22 healthy horses were suspended in isolated organ baths where their mechanical responses to various stimuli could be studied. Each tissue w...
Binding of stallion spermatozoa to the equine zona pellucida after coculture with oviductal epithelial cells. The objective of this study was to determine whether coculture of stallion spermatozoa and mare oviductal (uterine tubal) epithelial cells induced sperm cell capacitation in vitro. Capacitation as determined by zona binding and chlortetracycline staining of the sperm cells was compared for stallion spermatozoa: (1) incubated with medium alone (negative control), (2) treated with calcium ionophore A23187 (positive control) or (3) cultured with mare oviductal epithelial cells (OEC) for 4 h. Chlortetracycline staining patterns of sperm cells bound to the zonae were used to group spermatozoa as un...
Focal exocytosis by eosinophils–compound exocytosis and cumulative fusion. We have investigated the granule fusion events during exocytosis in horse eosinophils by time-resolved patch-clamp capacitance measurements. Stimulation with intracellular GTP gamma S leads to a stepwise capacitance increase by 4.0 +/- 0.9 pF. At GTP gamma S concentrations < 20 microM the step size distribution is in agreement with the granule size distribution in resting cells. Above 80 microM the number of steps is reduced and very large steps occur. The total capacitance increase, however, is unaffected. These results show that at high GTP gamma S concentrations granule--granule fusion o...
Characterization of equine infectious anemia virus dUTPase: growth properties of a dUTPase-deficient mutant. The putative dUTPase domain was deleted from the polymerase (pol) gene of equine infectious anemia virus (EIAV) to produce a recombinant delta DUpol Escherichia coli expression cassette and a delta DU proviral clone. Expression of the recombinant delta DUpol polyprotein yielded a properly processed and enzymatically active reverse transcriptase, as determined by immunoblot analysis and DNA polymerase activity gels. Transfection of delta DU provirus into feline (FEA) cells resulted in production of virus that replicated to wild-type levels in both FEA cells and fetal equine kidney cells. In con...
Competitive inhibition of lipolytic enzymes. IX. A comparative study on the inhibition of pancreatic phospholipases A2 from different sources by (R)-2-acylamino phospholipid analogues. The inhibitory power (Z) of a number of (R)-1-alkyl-2-acylamino phospholipid analogues was determined for three mammalian phospholipases A2 from pig, ox and horse pancreas. All three enzymes display a clear preference for anionic (phosphoglycol) inhibitors over the zwitterionic (phosphocholine) derivatives; this effect is most pronounced for the bovine enzyme. Upon variation of the 1-alkyl chain length, the bovine and equine phospholipases, like the porcine enzyme in previous studies, show an optimum in Z for a six-carbon alkyl group. The introduction of a double bond in the 2-acylamino group ...
Studies on the substrate specificity of the proteinase of equine infectious anemia virus using oligopeptide substrates. The proteinase of the equine infectious anemia virus (EIAV), a lentivirus closely related to human immunodeficiency virus (HIV), was purified from concentrated virus. The specificity of the enzyme was characterized using oligopeptides representing naturally occurring cleavage sites in the Gag and Gag-Pol polyproteins. The length of the substrate binding pocket was found to be 1-2 residues longer than that of HIV proteinases. Although the EIAV and HIV proteinases cleaved most of the peptides at the same bond, some were hydrolyzed by only the EIAV enzyme. Oligopeptides representing cleavage site...
A novel response of anion transporter in equine erythrocytes to a fluorescent substrate, N-(2-aminoethyl sulfonate)-7-nitrobenz-2-oxa-3-diazole (NBD-taurine). This report describes a unique response of the anion transporter in equine erythrocytes to the fluorescent substrate N-(2-aminoethyl sulfonate)-7-nitrobenz-2-oxa-3-diazole (NBD-taurine). Equine erythrocytes showed fluxes of NBD-taurine both inward and outward at rates considerably slower than those in human cells. These fluxes were completely abolished by a typical anion transport inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulfonate. Furthermore, NBD-taurine competitively inhibited the uptake of phosphate in equine red cells with an inhibition constant of phosphate that was slightly higher ...
Topography of equine chorionic gonadotropin epitopes relative to the luteinizing hormone and follicle-stimulating hormone receptor interaction sites. In order to localize the epitopes of equine chorionic gonadotropin (eCG) involved in interaction with luteinizing hormone (LH) and follicle-stimulating hormone (FSH) receptors, we used 14 monoclonal anti-eCG antibodies (mAbs). Different effects of these mAbs on the bioactivities of eCG were observed in in vitro bioassays, but the effects of each mAb on the two bioactivities were similar for all but four mAbs. All mAbs were found to inhibit the binding of eCG to LH receptors except 3A3 mAb, in radioreceptor assay. Six mAbs, which were strong inhibitors of eCG binding to LH receptors and of both...
Physical and functional characterization of transcriptional control elements in the equine infectious anemia virus promoter. Equine infectious anemia virus (EIAV) is a lentivirus that causes a chronic disease of horses characterized by cyclic episodes of fever, anemia, and viremia. Although the genome and promoter of EIAV are much less complex than those of its relatives the primate immunodeficiency viruses, the cellular proteins that activate and regulate transcription of EIAV have not yet been identified. In this report, we show by electrophoretic mobility shift assays and DNase I footprinting that the EIAV promoter contains multiple binding sites for ubiquitous, cell type-specific, and inducible cellular proteins...
Culture confirmation of the carrier status of Babesia caballi-infected horses. Culture of horse blood for Babesia caballi identified four carrier horses among nine previously infected horses. Three of the carriers had no detectable parasitemias on stained blood smears, and sera from two carrier horses were complement fixation test negative. Three cultures were continuously cultivated. Cryopreserved fourth-passage B. caballi was successfully reestablished in vitro. Blood from a 10th horse previously subinoculated with blood from a suspected carrier was cultured, with negative results.
Column separation of motile sperm from stallion semen. Subfertility in stallions is common, and methodologies are needed to increase the fertility in these animals. In other species, removal of the dead sperm from semen increases the quality and fertility of semen. With horse semen we evaluated 48 combinations of column separation techniques using micro-spin chromatography columns. The greatest improvement in motility was observed with glass wool, whereas glass beads exhibited the greatest recovery of motile sperm. Although centrifugation time did not influence recovery rate or percent motility, a column length of 2 cm was superior for recovery of...
ADP induces desensitisation of equine platelet aggregation responses: studies using ADP beta S, a stable analogue of ADP. Pre-incubation of equine platelets in platelet-rich plasma with adenosine 5'-diphosphate (ADP) induced a reduction in aggregation responsiveness to subsequent additions of ADP. The desensitisation was shown to be homologous since the responsiveness to platelet-activating factor, thrombin, collagen, 5-hydroxytryptamine or ionomycin remained unchanged. Adenosine 5'-(beta-thio)-diphosphate (ADP beta S), a non-hydrolysable analogue of ADP, was shown to act as an agonist inducing aggregation by interaction with the ADP receptor. ADP beta S was then used in the desensitisation studies in which resid...
ACh release from horse airway cholinergic nerves: effects of stimulation intensity and muscle preload. This study was conducted to determine the effects of stimulation parameters and muscle preload on acetylcholine (ACh) release induced by electrical field stimulation (EFS) of horse airway cholinergic nerves. Trachealis strip bundles were prepared and suspended in 2-ml tissue baths. The tissues were stimulated three to five times for 30 min each. Increasing frequency (0.5-16 Hz) and voltage (5-20 V) increased ACh release; increasing pulse duration (0.5-3 ms) had only a minor effect. Alterations in muscle preload (2-20 g) had no effect on ACh release. ACh release was fairly constant for up to fi...
Structural features of the trans-activation response RNA element of equine infectious anemia virus. A 25-nucleotide RNA with the sequence of the trans-activation response (TAR) element of equine infectious anemia virus (EIAV) was analyzed by biochemical methods and by one- and two-dimensional NMR spectroscopy. NMR, nuclease probing, and polyacrylamide gel migration rates show that the RNA consists of an A-helical stem capped by two non-Watson-Crick U-G base pairs and a compact four-nucleotide loop. The loop is stabilized by base stacking, with loop nucleotides C12 and C15 stacked upon U11 and G16, respectively. Near the 5' end of the molecule, the stem contains a bulge at nucleotide C2, most...
Free radical oxidation products in plasma and synovial fluid of horses with synovial inflammation. Free radical oxidation products, namely conjugated dienes, ultraviolet fluorescence (excitation 325 nm, emission 395 nm) and visible fluorescence (excitation 360 nm, emission 460 nm) were measured in equine synovial fluid exposed to free radicals in vitro and in the plasma and synovial fluids of horses with synovial effusions. The synovial effusions were induced by intra-articularly administered carrageenin (0.3 ml, 1%), which rarely resulted in clinical lameness. The free radicals were generated in vitro by mixtures of iron and ethylene diamine tetra acetate (Fe/EDTA) or mixtures of hypoxanth...
Comparative study of the stability of the folding intermediates of the calcium-binding lysozymes. Unfolding profiles of two calcium-binding lysozymes, equine milk lysozyme and pigeon egg-white lysozyme, were obtained by circular dichroism and proton NMR measurements. Equine lysozyme unfolds through a stable molten globule intermediate. The molten globule of equine lysozyme was characterized as more ordered than that of bovine alpha-lactalbumin. On the other hand, pigeon lysozyme unfolds by a two-state mechanism and the intermediate could not be observed in guanidine or thermal unfolding, the same as with conventional non-calcium-binding lysozymes. Thus, from the point of view of the unfold...
