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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.
Morphogenesis of Berne virus (proposed family Toroviridae). In equine dermis cells infected with Berne virus particles were first detected 10 h after infection. Virions were encountered in all parts of the Golgi system and, infrequently, in the rough endoplasmic reticulum. A unique form of budding of preassembled rigid tubular nucleocapsids was demonstrated. Masses of tubular nucleocapsids of a lesser diameter and electron density were prominent in the cytoplasm and the nucleus of infected cells. Within the Golgi system and cytoplasmic cisternae virions appeared as straight or slightly curved rods. Extremely long, aberrant virions (250 nm) were occasio...
Innate immunity during Equid herpesvirus 1 (EHV-1) infection. Intrinsic phagocytosis and killing of C. albicans by equine monocytes and polymorphonuclear leucocytes (PMN) was examined during Equid Herpesvirus 1 (EHV-1) (subtypes 1 or 2) and Adenovirus infections. Monocyte function increased during EHV-1 subtype 2 and Adenovirus infection. Conversely, there was an impairment of monocyte ingestion during EHV-1 subtype 1 infection which was ascribed to virus replication in peripheral blood mononuclear cells. PMN phagocytosis was not decreased in any of the infections studied. The raised levels of haemolytic complement in animals which subsequently developed...
Pathophysiology of equine postoperative ileus: effect of adrenergic blockade, parasympathetic stimulation and metoclopramide in an experimental model. An experimental model of postoperative ileus was developed in ponies using trauma to, and exposure of, a length of small intestine which gave rise to a reproducible and reversible set of changes in gut activity. This was assessed by recordings of electrical and mechanical activity and by propulsion of spheres from stomach to anus. Activity was depressed, especially in the stomach and colon, and transit was slowed. All drugs given increased electromechanical activity but propranolol was the least effective and did not alter the delayed transit of spheres. Yohimbine was more effective and the ad...
The trans-cis isomerization of trans-4′-(2-hydroxy-3,5-dibromo-benzylamino)cyclohexanol in vivo and in vitro in different species. Isomerization of trans-4'-(2-hydroxy-3,5-dibromo-benzylamino)cyclohexanol (HDBC) in vivo has been investigated in horse, cow, dog, rat and man. Following oral administration of 4'-trans-HDBC to the horse, a very efficient first-pass trans-cis isomerization was observed. In the urine of the horse and cow, 40% and 29% respectively of the conjugated alcohols consisted of the 4'-cis isomer. Isomerization in rat and dog took place only to a small extent, and in man no 4'-cis isomer was detected. Oxidation of HDBC to the corresponding ketone, at pH 9.0, was highest with horse- and rat-liver 10 000 g...
Effects of urea and guanidine hydrochloride on the activity and dynamical structure of equine liver alcohol dehydrogenase. The inactivation of equine liver alcohol dehydrogenase by guanidine hydrochloride and urea has been studied by monitoring the intrinsic tryptophan fluorescence and phosphorescence emission. The use of triplet-state lifetimes to probe the flexibility of protein structure at the site of tryptophan-314 reveals a distinct behavior between the two denaturants. At predenaturational concentrations, the loss of enzyme activity in guanidine hydrochloride is associated with a loosening of intramolecular interactions resulting in a greater fluidity of the interior region of the macromolecule. In contrast...
Generation of packaging-defective DNA molecules of equine adenovirus. Equine adenovirus (EAd) DNA prepared from infected bovine kidney (MDBK) cells contained additional sequences of about 100 to 700 bp at the left-hand end of the genome. These aberrant viral genomes were produced even after the first passage of the wild type EAd in MDBK cells and their relative amounts did not change significantly during serial passage. The left terminal fragments of two defective viral DNAs were cloned into the plasmid vector pBR322 and the nucleotide sequences of their terminal regions were analyzed. The data indicate that one viral DNA contained a duplication of the inverted ...
Equine endothelial cells in vitro. Certain in vitro culture conditions were determined for equine endothelial cells obtained from the aorta and pulmonary arteries. Cells were enzymatically isolated from the vessel lumen, using clostridial collagenase (2.5 mg/ml of Hanks's balanced salt solution) incubated at 37 C for 30 minutes. Cells were cultured in alpha minimum essential medium supplemented with plasma-derived and nonplasma-derived bovine fetal sera, endothelial cell-growth supplement, heparin, and antibiotics. Smooth muscle cell growth was not inhibited with nonplasma-derived animal sera, plasma-derived equine serum, or he...
Isolation and characterization of three forms of luteinizing hormone from the pituitary gland of the horse. Three isoforms of equine luteinizing hormone (eLH-A, eLH-B and eLH-C) have been isolated from horse pituitary glands. Separation was achieved on the basis of charge heterogeneity by ion-exchange chromatography. These charge differences were apparent after final purification, as determined by electrophoretic mobility on polyacrylamide disc gels (RF = 0.14, 0.19 and 0.26 for eLH-A, -B and -C, respectively). Apparent size differences were also noted between the isohormones by gel filtration on Sephadex G-100. Ve/Vo ratios for eLH-A, -B and -C were 1.72, 1.54 and 1.47, respectively. All 3 isoforms...
In vitro interference between equine herpesvirus types 1 and 2. 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 ...
Direct demonstration of intrinsic follicle-stimulating hormone receptor-binding activity in acid-treated equine luteinizing hormone. After dissociating equine gonadotropins as a function of time at pH 3, we examined them by radioligand assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis under nondissociating conditions (low, 0.1% SDS). Equine follicle-stimulating hormone (FSH) rapidly lost its receptor-binding activity, and low SDS-polyacrylamide gels demonstrated dissociation into subunits. Maximum dissociation occurred after 20-30 min of pH 3 incubation. Equine luteinizing hormone (LH), however, retained most biologic activity and was largely intact after 72 h of pH 3 incubation. Dose-response curves of ac...
Two-dimensional 1H NMR studies of cytochrome c: assignment of the N-terminal helix. The 1H resonances of 11 sequential amino acids in the N-terminal helix of horse ferrocytochrome c were studied by two-dimensional nuclear magnetic resonance techniques. All the main-chain protons from Lys-5 through Ala-15 and many of the side-chain protons were assigned. J-Correlated spectroscopy (COSY) was used to distinguish protons on neighboring bonds and to recognize amino acid types. Nuclear Overhauser effect spectroscopy (NOESY) was used to define spatially contiguous protons and to determine amino acid sequence neighbors. The relayed coherence experiment (relay COSY) was used to resolv...
Impaired estrogen production by Leydig cells of the naturally retained testis in unilaterally cryptorchid boars and stallions. Estrogen production in vitro was compared for Leydig cells from cryptorchid and scrotal testes in boars and stallions. Animals with natural and experimental cryptorchidism were used. Purified Leydig cells were prepared from testes of mature animals by collagenase treatment and Percoll density gradients. After incubation for 3 hours (1 X 10(6) cells), estrone sulfate and estrone in the media were measured by direct radioimmunoassay. Androstenedione and testosterone in media extracts also were determined. Cells from the abdominal testis of unilateral cryptorchid boars and stallions showed impair...
Molecular pathogenesis of equine coital exanthema: temperature-sensitive function(s) in cells infected with equine herpesviruses. Preliminary experiments have revealed that several laboratory and wild-type strains of the equine herpesvirus (EHV) triad were temperature-sensitive for growth when assayed at 39 degrees C. The efficiencies of plating (EOP) observed were 10(-2) for both EHV 1 and 2, and 1 X 10(-6) for EHV 3. The EOPs were determined by plaque assays which compared titrations at 34 degrees C and 39 degrees C on equine fetal dermal fibroblast cells. Growth yield experiments, assayed at 34 degrees C, reflected those EOP's, but did not indicate any difference in yields when infected cultures were incubated at 34 d...
A study on the role of evolutionarily invariant leucine 32 of cytochrome c. To investigate the role of evolutionarily invariant leucine 32 of horse cytochrome c, analogs of residues 28-38, (28-38), each containing a substituted amino acid at positions 32 or 35 were synthesized using Merrifield's method. Position 35 is leucine in horse cytochrome c but replaced by nonpolar amino acids in some species. The ability of the analogs to bind to the two-fragment complex of ferri- or ferro heme fragment (1-25)H and apofragment (39-104) was measured using gel filtration and equilibrium dialysis. Replacement of leucine 32 with isoleucine, for example, increased the dissociation ...
In vitro bactericidal efficacy of equine polymorphonuclear leukocytes against Corynebacterium equi. Polymorphonuclear leukocytes from adult horses were separated from whole blood, using a 2-step Percoll gradient, and were tested for bactericidal function against Corynebacterium equi. Staphylococcus aureus, an organism against which equine neutrophils have proved efficacy, was a positive control. The percentage of uptake after a 15-minute preincubation of the neutrophils and bacteria in the presence of normal horse serum was also calculated. The results indicated that equine neutrophils effectively phagocytosed and killed C equi and S aureus. The percentage of uptake for S aureus (95% +/- 3%)...
Assessment of spermatozoal function using dual fluorescent staining and flow cytometric analyses. Spermatozoa from bulls, boars, dogs, horses, mice, and men were examined using a fluorogenic stain consisting of the membrane-permeant substrate carboxyfluorescin diacetate (CFDA) and the relatively membrane-impermeant nuclear stain propidium iodide (PI). Three distinct populations of spermatozoa were discernible in samples from each species upon microscopic examination. Individual spermatozoa, presumed to be viable because of their motility, retained products of the fluorescein chromophore throughout the cell. A second population of spermatozoa in which the nuclei stained red with PI retained...
Cytolytic activity of liposomes containing stearylamine. In order to develop the cytotoxic liposome, the cytolytic effect of polycationic liposome was examined. Upon incubation of the stearylamine-containing liposome (stearylamine-liposome) with rabbit erythrocyte, a significant extent of hemolysis was observed. Hemolytic activity of the liposome depends on the amount of stearylamine in the liposome membrane. The plots of the initial rate of hemolysis versus the concentration of stearylamine-liposome showed a sigmoidal curve, suggesting that stearylamine-liposomes act cooperatively on the erythrocyte membrane. Hemolytic activity of stearylamine-lipo...
Joint report of the Third International Workshop on Lymphocyte Alloantigens of the Horse, Kennett Square, Pennsylvania, 25-27 April 1984. The Third International Workshop on Lymphocyte Alloantigens of the Horse was held on 25-27 April 1984 in Kennett Square, Pennsylvania. Twelve laboratories from five countries participated. The principal purpose of this Workshop was to determine the phenotypic and gene frequencies of the 10 equine lymphocyte antigens (ELA) and a non-ELA lymphocyte antigen, ELY-2.1, in several breeds of horse. A total of 86 alloantisera characterized in previous workshops were tested against lymphocytes from 1179 horses. In addition, several experimental antisera were also tested against the same panel of lympho...
Increased acidophilia of eosinophil granules after EDTA treatment. The acidophilic reaction of eosinophil leucocyte granules from human, pig and horse blood smears was investigated by using May-Grünwald-Giemsa staining after previous treatment with EDTA and sodium citrate solutions. The same peak at 530 nm, but absorption values considerably higher than those of controls, were found in eosinophil granules after application of chelating agents, indicating that removal of metal cations could unmask basic groups in these structures.
Opiate-like and adrenocorticotrophin-like materials in equine pancreas. Equine pancreatic acetone powder was extracted with an acetone-water-HCl mixture. An acid acetone powder resulted by adding a copious volume of acetone to the extract. The powder was subjected to salt fractionation, gel filtration and chromatography on CM-cellulose. Steroidogenic activity, ACTH-like immunoreactivity and opiate receptor binding activity were distributed among the CM-cellulose chromatographic fractions derived from material unretarded as well as from material retarded on Sephadex G-25. The data indicates a separation of steroidogenic and opiate receptor binding activities, and t...
Single-radial-immunodiffusion potency tests of inactivated influenza vaccines for use in man and animals. Single-radial-immunodiffusion (SRD) provides a sensitive and reproducible in vitro assay for haemagglutinin (HA) concentration in inactivated influenza vaccines. The use of SRD for human influenza vaccine standardization and application for equine and avian influenza vaccines is discussed. In clinical trials, vaccine HA concentration measured by SRD has been shown to be directly related to antibody responses and to protection against challenge. The use of SRD may considerably reduce the usage of animals for potency testing of veterinary influenza vaccines.
Electromyographic, myomechanical, and intraluminal pressure changes associated with acute extraluminal obstruction of the jejunum in conscious ponies. Bipolar electrodes, strain gauge force transducers, intraluminal pressure recording catheters, and extraluminal intestinal obstructors were surgically implanted in 4 ponies to record myoelectrical and mechanical activity of the distal portion of the jejunum and ileum. After determining normal intestinal activity and pressures, the distal portion of the jejunum was obstructed with an extraluminal obstructor. Myoelectrical and mechanical activity recorded from jejunal segments proximal to the obstruction increased significantly (P less than 0.01), whereas activity distal to the obstruction remai...
Comparison of specificity of human and horse leucocyte proteinases with synthetic peptide substrates. Highly purified horse leucocyte proteinases 1, 2A and 2B hydrolyze synthetic substrates which are decomposed also by human leucocyte elastase but they are unable to hydrolyze typical substrates of cathepsin G. Thus in distinction to other mammalian species horse leucocytes are devoid of cathepsin G and contain only elastases.
Characteristics of cells derived from the girdle region of the pre-implantation blastocyst of the donkey. The establishment of a monolayer culture of cells derived from the girdle region of a 34-day-old donkey conceptus is described. These cells have had over 100 repeated passages in culture. Low levels of pregnant mares' serum gonadotrophin (PMSG, eCG) could be detected in the cells by indirect immunofluorescence using some monoclonal anti-eCG antibodies, but the cells did not secrete eCG as measured by radioimmunoassay or inhibition of haemagglutination. There was marked nuclear polymorphism with binucleate and occasional multinucleate cells. The cells were strongly reactive with wheatgerm agglu...
Spontaneous expression of an endogenous retrovirus by the equine sarcoid-derived MC-1 cell line. A retrovirus is spontaneously released into the culture medium of the equine sarcoid-derived MC-1 cell line. The MC-1 virus did not exhibit in vitro transforming activity or replication when tested on equine fibroblasts or a variety of other mammalian cell cultures. Complementary DNA, synthesized using detergent-activated MC-1 virus RNA-dependent DNA polymerase, detected homologous sequences in the DNA of an established equine dermal cell line and in the DNA of primary equine dermal fibroblasts. Iododeoxyuridine or azacytidine induced a replication-deficient endogenous retrovirus in the normal...
The interaction between some serine proteinases and horse leucocyte inhibitor. Horse blood leucocyte cytosol exhibits a broad inhibitory activity against serine proteinases. The purified inhibitor was exposed to investigated enzymes (trypsin, chymotrypsin, elastases and serine proteinase from S. aureus) for variable time and the products were analyzed by gradient polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molar ratio I:E, association rate constants k on and inhibition constants Ki for the enzymes and inhibitor were determined. The examined elastases form stable, stoichiometric complexes with the inhibitor (Ki less than 10(-10) M), ...
Analysis of pharmaceutical dosage forms for oxfendazole: II. Simultaneous liquid chromatographic determination of oxfendazole and trichlorfon in equine paste. A reverse phase liquid chromatographic procedure is described for the simultaneous determination of oxfendazole [2-(methoxycarbonylamino)-5-phenylsulfinylbenzimidazole] and trichlorfon [(2,2,2-trichloro-1-hydroxyethyl)phosphonic acid dimethyl ester] in equine paste. The sample is extracted by sonication in methanol. Insoluble excipients are removed by centrifugation and an aliquot plus internal standard are diluted with dilution solvent (water-acetonitrile-phosphoric acid, 80 + 20 + 1). The samples are filtered and injected onto a Partisil-5 ODS-3 column with acetonitrile-0.01 M phosphate buff...
Comparison of neutrophil elastases and of neutrophil protease inhibitors in the horse and man. Neutral neutrophil protease, elastase activities, and cytosol protease inhibitors of these enzymes of horses and man were compared. Human neutrophils had 5 times the elastase activity of equine neutrophils, and neutral protease activity was approximately 50% greater in human neutrophils than that in equine neutrophils. Cytosol inhibitors for elastase and neutral proteases were not found in human neutrophils, whereas large amounts were found in equine neutrophils. Using fibrinogen-agarose electrophoresis, 4 cytosol inhibitors of different enzyme specificities were detected. These cytosol inhibi...
Identification of 3 beta-hydroxy-5,7-androstadien-17-one as a secretory product of the fetal horse gonad in vivo and in vitro. Isolation of 3 beta-hydroxy-5,7-androstadien-17-one, as a major component of steroids extracted from vein blood of the fetal gonads of the horse, supports the proposed role for the compound as a precursor for equilin formation in the placenta of the mare. The 5,7-diene was extracted from blood collected from gonadal veins of fetal ovaries and testes in situ, and from a fetal testis connected to an artery in the neck region of the mare. Perfusion of fetal gonads in the laboratory was carried out to allow longer periods of collection. In addition, isolated cell preparations from a fetal testis w...
