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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.
Intracellular proteins of feline immunodeficiency virus and their antigenic relationship with equine infectious anaemia virus proteins. Feline immunodeficiency virus (FIV) grown in cat lymphocyte and thymocyte cultures was labelled with L-[35S]methionine or [3H]glucosamine and virus-coded proteins were identified using immunoprecipitation. Polypeptides with apparent Mr values of 15K, 24K, 43K, 50K, 120K and 160K were detected. An additional polypeptide of 10K was detected by Western blot analysis. The two highest Mr species sometimes appeared as one band, of which only the 120K polypeptide was glycosylated. In the presence of tunicamycin gp120 was no longer detectable and a non-glycosylated precursor of 75K was found instead. ...
Superoxide production by stimulated equine polymorphonuclear leukocytes–inhibition by anti-inflammatory drugs. Polymorphonuclear leukocytes (PMNLs) were isolated from an inflammatory exudate induced in the intercarpal joints of horses by an administration of carrageenin. Their superoxide production at rest and following stimulation with either serum-treated zymosan (STZ) or phorbol myristate acetate (PMA) was measured by cytochrome-c reduction. Stimulation of the cells increased the cytochrome-c reduction 10-15 times that of resting cells. The maxima were 20 nmol of reduced cytochrome-c per 10(6) cells per ml at 120 min (STZ) and 35 nmol of reduced cytochrome-c per 10(6) cells per ml at 60 min (PMA). T...
Pharmacokinetics of tinidazole in the horse. Serum tinidazole concentrations were monitored in five clinically healthy adult horses after intravenous (i.v.) and oral administration of the drug (15 mg/kg and 25 mg/kg, respectively). After i.v. administration, the mean residence time was 7.0 h, the elimination half-life 5.2 h and the body clearance rate 1.6 ml/min/kg. The distribution volume was found to be 660 ml/kg. After oral administration, the mean residence time was 8.5 h, the absorption half-life 1.1 h and the bioavailability essentially 100%. In view of the in-vitro sensitivities of various anaerobic bacteria, a dosage of 10-15 mg/...
Calcium-ionophore-induced formation of platelet-activating factor and leukotrienes by horse eosinophils: a comparative study. Horse eosinophils preincubated with 3H-labelled acetate and stimulated with the Ca2+ ionophores ionomycin or A23187 form a radioactive compound, which we have shown to be 1-O-alkyl-2-[3H]acetyl-sn-glycero-3-phosphocholine (platelet-activating factor). We could detect no 1-O-acyl-2-[3H]acetyl-glycero-3-phosphocholine in the radioactive fraction. The formation of platelet-activating factor was strongly correlated to the generation of leukotriene C4, the main arachidonate metabolite in horse eosinophils, suggesting that platelet-activating factor and leukotriene C4 have a common precursor pool (1...
The characterisation of equine interleukin-1. Equine interleukin-1 has been produced from peripheral blood monocytes by stimulation with E. coli lipopolysaccharide. Sephacryl S200 gel filtration revealed a molecular weight of 17-18 kD. Chromatofocusing of the 17-18 kD peak identified four active fractions. Two major peaks were detected at pH 6.7 and pH 7, with smaller peaks at pH 6.3 and pH 5.9. The pI 7 molecule is probably the equine form of IL-1 beta.
Equine growth hormone. Detection of immunoreactive sequences using poly- and monoclonal antibodies. The immunochemical behavior of several fragments of equine growth hormone (eGH) was examined using competitive binding assays with antibodies (Abs) to eGH obtained from different sources. Antigenicity was detected within the sequences 5-72 and 73-123 by rabbit Abs to eGH and by three mouse monoclonal antibodies (MAbs) produced by using bovine growth hormone as immunogen, but showing heteroclitic properties towards eGH. The polyclonal Abs to eGH also recognized as immunoreactive two smaller peptides corresponding to the amino acid residues 52-72 and 110-123. By contrast, the heteroclitic Abs to...
Three-dimensional structures of maturable and abortive capsids of equine herpesvirus 1 from cryoelectron microscopy. Cryoelectron microscopy and three-dimensional computer reconstruction techniques have been used to compare the structures of two types of DNA-free capsids of equine herpesvirus 1 at a resolution of 4.5 nm. "Light" capsids are abortive, whereas "intermediate" capsids are related to maturable intracellular precursors. Their T = 16 icosahedral outer shells, approximately 125 nm in diameter, are indistinguishable and may be described in terms of three layers of density, totalling 15 nm in thickness. The outermost layer consists of protruding portions of both the hexon and the penton capsomers, ris...
In vivo regulation of plasma [H+] in ponies during acute changes in PCO2. The major objective of this study was to test the hypothesis that in ponies the change in plasma [H+] resulting from a change in PCO2 (delta H+/delta PCO2) is less under acute in vivo conditions than under in vitro conditions. Elevation of inspired CO2 and lowering of inspired O2 (causing hyperventilation) were used to respectively increase and decrease arterial PCO2 (Paco2) by 5-8 Torr from normal. Arterial and mixed venous blood were simultaneously sampled in 12 ponies during eucapnia and 5-60 min after Paco2 had changed. In vitro data were obtained by equilibrating blood in a tonometer at f...
Effect of palosein (superoxide dismutase) and catalase upon oxygen derived free radical induced degradation of equine synovial fluid. The effect of oxygen derived free radicals (ODFR) upon the specific viscosity of equine synovial fluid was studied. ODFR were generated either by a mixture of ferrous iron and EDTA (Fe/EDTA) or by a mixture of hypoxanthine and xanthine oxidase (HX/XO). Incubation of the synovial fluid with both free radical generating systems decreased its specific viscosity. When the synovial fluid was incubated with Fe/EDTA the specific viscosity of the synovial fluid was reduced rapidly. By 2 mins, it was 53 +/- 3 per cent of the original specific viscosity and by 30 mins it was reduced to 39 +/- 5 per cent...
An improved method for the study of equine haptoglobin heterogeneity. Equine serum haptoglobin was separated by polyacrylamide gel isoelectric focusing and visualized by protein staining or Western blotting. Conventional protein staining revealed up to three bands in the pI range 4.17 to 4.44. The blotting technique, however, showed an anodal group of 8 to 10 bands with a pI range of 4.11 to 4.52 and a cathodal group of 4 to 6 bands with a range of 4.55 to 5.14. The blotting method revealed that equine haptoglobin migrates outside the prealbumin area, in contrast to previous reports.
Allergens of horse epithelium. I. Physicochemical and immunochemical characterization of five different horse epithelium raw materials used for allergen extract preparation. We investigated five horse epithelial allergen extracts prepared from different qualities of raw material by several biochemical and immunochemical methods. Horse serum albumin and horse serum were used to identify serum-related antigens. We found high similarities as well as marked differences between the extracts. There were strong differences in the protein contents, the protein patterns obtained by isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the total allergenic activities obtained by radioallergosorbent test inhibition assays and the amounts and num...
An attempt to determine the tissue origin of equine serum alkaline phosphatase by isoelectric focusing. The main purpose of this study was to ascertain whether isoelectric point determination of alkaline phosphatase (AP) using an isoelectric focusing technique on agarose gels could define the isoenzymes present in healthy equine serum. The isoelectric points of AP extracted from nine tissues ranged from pH 3.5 to 7.5 with all tissues having multiple bands. There was considerable similarity in band pattern among tissues, with only pancreatic and colostral AP having substantially different isoelectric points from the others. Sera contained thirteen bands with isoelectric points ranging from pH 3.5...
Equus przewalskii plasma protease inhibitor (Pi) system. A detailed biochemical characterization of four of the five previously described alleles of the plasma protease inhibitor (Pi) system of Equus przewalskii was performed using both one- and two-dimensional electrophoretic techniques. The proteins have been characterized in terms of isoelectric point, relative molecular mass, inhibitory activity to bovine trypsin and chymotrypsin, immunochemical cross-reactivity, terminal sialic acid content and enzyme:inhibitor complex formation and the oxidation sensitivity of this interaction. Using these functional criteria, only three loci (Spi 1, 2 and 3) ...
In vitro isolation of a neutralization escape mutant of equine infectious anemia virus (EIAV). A neutralization escape mutant (A/1 E) of equine infectious anemia virus was isolated after 13 passages in cell culture in the presence of serum containing antibodies to type- and group-specific determinants of EIAV envelope glycoproteins. Loss of neutralization by the selecting serum correlated with loss of two epitopes in the major envelope glycoprotein gp90 of A/1 E which were present in a parallel variant isolated from a persistently infected pony.
Effects of indomethacin on neural and myogenic components in equine airway smooth muscle. Equine airway smooth muscle is innervated by vagal efferents and, in addition, displays spontaneous mechanical activity. The preparation thus appears to contain at least two discrete excitable components, the cholinergic neural elements and the smooth muscle membrane. Indomethacin (INDO), a cyclooxygenase (CO) inhibitor, exerts a considerable potentiation of function in this preparation. The latter may be effected indirectly, through loss of the inhibitory effect of endogenous prostaglandin E2 (PGE2) on neural acetylcholine release and through direct effects on smooth muscle of the generally a...
Secretion of prostaglandins and progesterone by cells from corpora lutea of mares. Corpora lutea (CL) were collected from mares during early (Day 4-5), mid- (Day 8-9), and late (Day 12-13) dioestrus. Dispersed cell suspensions were obtained by enzymic digestion of tissue. Two distinct luteal cell populations (large and small) were observed. The proportion of small luteal cells significantly increased as age of CL advanced. Cells (2 x 10(6)) from CL which were incubated for 24 h secreted prostaglandin (PG) F, PGE-2 and 6-keto-PGF-1 alpha (the stable metabolite of prostacyclin). Higher concentrations of all PGs were produced by cells from CL at early dioestrus than from those ...
Effects of age and prosthesis material on in vitro cartilage retention of laryngoplasty prostheses in horses. Cartilage retention strengths of laryngoplasty prostheses were compared in larynges of 2-, 3-, and 4-year-old horses, using doubled polyester and expanded polytetrafluoroethylene prostheses. Bilateral laryngoplasties were performed on each of 15 (seven 2-year-old, two 3-year-old, and six 4-year-old) larynges, which were collected at an abbatoir. Prostheses were secured to a mechanical testing machine, and tension causing arytenoid cartilage abduction was applied, until total failure of the cartilage or prosthesis resulted. Tension caused cricoid cartilage failure in 1 specimen, and muscular pr...
Transbilayer movement of phosphatidylserine in nonhuman erythrocytes: evidence that the aminophospholipid transporter is a ubiquitous membrane protein. A 31-32-kDa integral membrane protein has been previously identified in erythrocytes as the protein most likely to be responsible for the transbilayer movement of phosphatidylserine (PS) [Connor & Schroit (1988) Biochemistry 27, 848-851]. Using similar techniques, we have identified analogous proteins of identical molecular weights in bovine, equine, ovine, porcine, canine, caprine, and rhesus red blood cells. Similar to human red blood cells, all of the mammalian cells were able to specifically transport an exogenously supplied fluorescent PS analogue from their outer-to-inner membrane le...
Biotransformation of 5(10)-estrene-3 alpha,17 beta-diol by equine testicular preparations in vitro. The research investigates the biosynthesis of a particular isomer called 5(10)-estrene-3 alpha, 17 beta-diol in stallion testes and how it affects the formation of 19-nor steroids and oestrogens. Summary of […]
Effect of calcium-channel blockers and salbutamol on the isolated mare uterus–interaction with the calcium agonist Bay K 8644. The effects of nifedipine, verapamil and diltiazem were investigated in the isolated mare uterus in comparison with salbutamol. All the calcium-channel blockers and salbutamol inhibited the spontaneous, KC1- and electrically induced contractions; nifedipine and salbutamol were the most potent compounds. The calcium agonist Bay K 8644 (10(-8)-10(-6) mol/l) competitively antagonized the inhibitory effect of nifedipine (pA2 value = 8.54 +/- 0.06), whereas it was only slightly or totally ineffective against verapamil, diltiazem and salbutamol. These results indicate that calcium-channel blockers a...
Antagonism in isolated equine digital vessels of contraction induced by epinephrine in the presence of hydrocortisone and an aqueous extract of black walnut (Juglans nigra). Prazosin, isoxsuprine, and nifedipine were screened for ability to reverse contraction of isolated equine digital vascular strips produced by epinephrine (Epi) in the presence of hydrocortisone (Hc) and an aqueous extract of black walnut (Juglans nigra) (BW). Two arteries and two veins from each of three horses for each drug (n = 9) were maintained in isolated tissue baths in Krebs' bicarbonate buffer with 95% oxygen at 37 degrees C. Six-point Epi concentration-response (C-R) curves were obtained for each vessel in the presence of Hc, BW, and the appropriate vehicle. This was repeated for each...
In vitro transport of L-alanine by equine cecal mucosa. When sheets of mucosa from the cecum of clinically normal horses were incubated in vitro with radiolabeled L-alanine, they could accumulate this amino acid against an apparent concentration gradient after 60 to 150 minutes of incubation. The active transport system for L-alanine was on the serosal surface of the mucosal sheet only. L-Alanine accumulation at 60 minutes was partly inhibited by 20 mM glycine (P less than 0.01), 0.5 mM ouabain (P less than 0.05), and Na deprivation (P less than 0.02). Anoxia for 60 minutes increased L-alanine accumulation, but had adverse effects on cell structure...
Effect of dietary linolenic acid on endotoxin-induced thromboxane and prostacyclin production by equine peritoneal macrophages. In laboratory animals, the incorporation of alpha linolenic acid or other n-3 series fatty acids into the diet results in marked changes in cell membrane composition as well as arachidonic acid metabolism. The purpose of the present study was to determine whether endotoxin-induced thromboxane A2 (TxA2) and/or prostacyclin (PGI2) production by equine peritoneal macrophages was altered by feeding horses a diet containing 8% linseed oil as a source of alpha linolenic acid for 8 weeks. Peritoneal macrophages were cultured in vitro in the presence of endotoxin (LPS) (0.5-500 ng/ml) or calcium ionop...
A hemolytic assay for the measurement of equine complement. A hemolytic assay was developed for the measurement of functional equine complement activity. The assay utilizes antibody sensitized chicken erythrocytes as the target cell and was specific for classical pathway (antibody dependent) complement activity. The assay was found to be reproducible and more sensitive than previous reports using other species of target cells. Total serum complement (CH50) values were determined for five mares and their foals and followed over a period of 3 months.
Influence of technical parameters on the in vitro motility of equine neutrophils in the presence of streptococcal culture supernatant. To identify the influence of technical factors on the in vitro motility of equine neutrophils towards streptococcus culture supernatant in an under-agarose assay, we studied the changes in eight cell migration parameters. The distances the phagocytes travelled by directed, random and spontaneous migration increased with incubation time, cell concentration and the gelatin and serum contents of the migration plates. The contribution of chemotaxis to the phagocyte migrations, however, decreased simultaneously. The directed and random, though not the spontaneous, migrations of the phagocytes incre...
Histochemical and molecular determination of fiber types in chemically skinned single equine skeletal muscle fibers. Until now, there has been no reliable method for histochemical determination of fiber types of single skinned muscle fibers. The major problem arises from the fact that most histochemical techniques use cross-sections of a large group of fibers and compare a given fiber with those surrounding it. This is not possible with a single skinned fiber which has been separated from a bundle to be used for mechanical analysis. A further problem is that the skinning procedure itself may alter the staining pattern. We have developed a procedure by which multiple cross-sections of single skinned fibers ca...
Comparison of the binding of Ca2+ and Mn2+ to bovine alpha-lactalbumin and equine lysozyme. The enthalpy change of the binding of Ca2+ and Mn2+ to equine lysozyme was measured at 25 degrees C and pH 7.5 by batch microcalorimetry: delta H degrees Ca2+ = -76 +/- 5 kJ mol-1, delta H degrees Mn2+ = -21 +/- 10 kJ mol-1. Binding constants, log KCa2+ = 6.5 +/- 0.2 and log KMn2+ = 4.1 +/- 0.5, were calculated from the calorimetric data. Therefore, delta S degrees Ca2+ = -131 +/- 20 JK-1 mol-1 and delta S degrees Mn2+ = 8 +/- 44 JK-1 mol-1. Removal of Ca2+ induces small but significant changes in the circular dichroism spectrum, indicating the existence of a partially unfolded apo-conformatio...
Difference in content ratio of components among horse serum transferrin variants. Each of five genetic variants of horse serum transferrin (Tf), D, F, H, O, and R, was separated into two bands by polyacrylamide gel isoelectric focusing (PAGIEF). The more acidic band, termed component a, was more abundant than the other one, termed component b, in all variants. Components a and b of TFO variant were immunologically indistinguishable from each other by double immunodiffusion test. Determination of the content ratio of component a to component b in each variant revealed that the variants were classified into two groups: one group (D, F, and H) had a relatively high ratio withi...
In vitro ultrasonographic appearance of the normal and verminous equine aorta, cranial mesenteric artery, and its branches. Ninety-one equine aortic and cranial mesenteric arterial segments were evaluated ultrasonographically in a water bath. On the basis of pathologic evidence of verminous arteritis, arterial segments were classified into 4 categories, and the ultrasonographic characteristics of each group were evaluated. Normal arteries (class 1) were ultrasonographically characterized by a smooth luminal surface layer and uniform wall thickness and echogenicity. Arteries with only histopathologic evidence of verminous arteritis (class 2) were ultrasonographically characterized by a smooth luminal surface layer, ...
Isolation and characterization of equine microvascular endothelial cells in vitro. The use of cultured tissue has not yet become widespread in research involving equine disease, and this may be attributable in part to the scarcity of published reports concerning tissue culture methods for this species. We report here the isolation of equine microvascular endothelium (EMVE) from fresh omental tissue of horses and ponies. Fresh donor tissue was minced, subjected to collagenase digestion, and filtered. Cells were layered on 5% bovine serum albumin for gravity sedimentation, the bottom layer was collected, and the cells were plated onto fibronectin-coated flasks. Medium consiste...
