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- Author or Editor: David J. Hurley x
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Abstract
Objective—To evaluate effects of colostral cells on the ability of neonatal leukocytes to respond in a mixed leukocyte response (MLR) as a means of evaluating specific immune responsiveness.
Animals—10 Holstein calves, their respective dams, and 10 unrelated adult Holstein cows.
Procedure—Soon after birth, their calves were fed maternal whole colostrum or colostrum after cells were removed by centrifugation. Responses for leukocytes obtained from calves during the first 5 weeks after birth, their dams, and unrelated cows were measured by use of 1-way MLR as an indicator of immune development. An internal control treatment, proliferation of lymphocytes stimulated with Staphylococcus enterotoxin B (SEB), was also measured.
Results—Transfer of colostral leukocytes had a significant effect on the MLR and SEB-induced response in calves. Calves receiving whole colostrum had enhanced responses to maternal and unrelated leukocytes 24 hours after ingestion of colostrum. These responses decreased quickly, indicating direct modulation of the neonatal immune response. Calves receiving whole colostrum effectively stimulated the MLR by 24 hours after ingestion of colostrum. In contrast, calves receiving acellular colostrum did not effectively stimulate the MLR until 2 to 3 weeks after birth.
Conclusions and Clinical Relevance—Ingestion of maternal colostral leukocytes immediately after birth stimulates development of the neonatal immune system. These maternal leukocytes enhance development of antigen-presenting capacity as indicated by their ability to stimulate the MLR and SEB response. The influence of ingested maternal cells on neonatal immunity was also indicated by a reduction in reactivity of neonatal cells to maternal alloantigens. (Am J Vet Res 2005;66:1854–1860)
Abstract
Objective—To evaluate effects of black walnut extract (BWE) on equine mononuclear cells and determine whether BWE has direct proinflammatory effects.
Sample—Mononuclear cells separated from blood samples from 8 horses.
Procedures—Aqueous BWE was prepared and processed to eliminate contamination with particulates and microbes. A Limulus amoebocyte lysate assay was used to detect lipopolysaccharide (LPS) contamination in the BWE. Mononuclear cells were incubated in minimal essential medium with or without the addition of 0.6% to 10% (vol/vol) BWE. These mononuclear cells were assessed for viability, activities of caspases 3 and 7, nitric oxide production, procoagulant activity, and tumor necrosis factor-α production. The effect of LPS on cellular responses induced by BWE was assessed by coincubation with 13 U of polymyxin B/mL; mononuclear cells incubated with LPS were used as a reference.
Results—BWE did not cause loss of cell membrane integrity in mononuclear cells but did induce a dose-dependent increase in activities of caspases 3 and 7. Neither BWE nor LPS significantly induced production of nitric oxide. Both BWE and LPS induced comparable amounts of procoagulant activity and tumor necrosis factor-α production; coincubation with polymyxin B reduced the activity for BWE and LPS by 50% and approximately 100%, respectively.
Conclusions and Clinical Relevance—Addition of BWE induced inflammatory activation of equine mononuclear cells, a portion of which was independent of the effects of LPS. Furthermore, BWE and LPS may work in concert to induce systemic inflammatory responses that contribute to the development of acute laminitis in horses.
Abstract
Objective—To evaluate the effects of topical antifungal drugs and delivery vehicles on the morphology and proliferation rate of cultured equine keratocytes.
Study Population—16 corneas obtained from 8 apparently ophthalmologically normal horses < 0.5 hours after euthanasia for reasons unrelated to the study.
Procedures—Primary cultures of equine keratocytes were obtained from corneal stroma and were exposed to several concentrations of 3 commonly used, topically applied antifungals: natamycin, itraconazole, and miconazole. In addition, effects of drug delivery vehicles DMSO, benzalkonium chloride, and carboxymethylcellulose and a combination vehicle composed of polyethylene glycol, methylparaben, and propylparaben were also evaluated. Morphological changes and cellular proliferation were assessed 24, 48, and 72 hours after application.
Results—At the highest concentrations tested, all antifungals caused marked cellular morphological changes and inhibited proliferation. At low concentrations, natamycin and miconazole induced rounding, shrinking, and detaching of the cells with inhibition of cellular proliferation. Natamycin caused the most severe cellular changes. Itraconazole, at the low concentrations, caused minimal morphological changes and had a minimal effect on proliferation. All vehicles tested had significantly less effects on cellular morphology and proliferation when compared with the antifungals, except for the combination vehicle, which caused severe morphological changes and inhibited proliferation, even at low concentrations. The DMSO had minimal effects on cellular morphology and proliferation, even at high concentrations.
Conclusions and Clinical Relevance—Itraconazole had significantly less cytotoxic effects on equine keratocytes in culture than did natamycin or miconazole. Natamycin had severe cytotoxic effects in vitro.
Abstract
Objective—To determine the effect of fetal bovine serum (FBS) and heat-inactivated FBS (HI-FBS) on lipopolysaccharide (LPS)- and zymosan-induced procoagulant activity of equine and canine mononuclear cells.
Sample Population—Mononuclear cells from 18 horses and 3 dogs.
Procedures—Cells were incubated with various concentrations of FBS, HI-FBS, LPS, zymosan, polymyxin B, and anti–LPS-binding protein monoclonal antibody or combinations of these constituents. A 1stage recalcification assay was used to determine procoagulant activity.
Results—Addition of FBS to media significantly increased procoagulant activity; equine and canine cells were stimulated by 1% and 10% FBS, respectively. Coincubation of cells with FBS and polymyxin B did not reduce this effect, suggesting that the response was not attributable to LPS contamination. Addition of HI-FBS to media did not stimulate procoagulant activity of equine or canine cells, and the sensitivity of the equine cells to LPS was significantly increased by HI-FBS. This increased LPS sensitivity was reduced 40% with monoclonal antibody directed against human recombinant LPSbinding protein. Increasing concentrations of HIFBS significantly increased LPS- and zymosaninduced procoagulant activity of canine cells.
Conclusion and Clinical Relevance—Procoagulant activity production in equine and canine mononuclear cells was significantly increased by addition of FBS, whereas heat inactivation of FBS eliminated this effect. Heat inactivation did not eliminate the function of serum proteins involved in enhancement of LPSand zymosan-induced procoagulant activity. Results suggest that HI-FBS can be used as a source of serum proteins that increase the sensitivity of mononuclear cells to bacterial and yeast cell wall components.
Abstract
Objective—To evaluate the effect of the duration of cold Ischemia on the renin-angiotensin system during renal transplantation In cats and to define the potential Influence of vasoactive factors in renal tissue following cold ischemic storage versus warm ischemic storage
Animals—10 purpose-bred 6-month-old sexually Intact female cats
Procedures—10 cats underwent renal autotransplantation after 30 minutes (n = 5) or 3 hours (5) of simple, ex vivo cold storage of renal autographs. Following autograft reperfusion, direct hemodynamic variables were measured with a telemetric Implant and samples were collected for plasma renin concentration. Activation of vascular-related genes (renin, endothelin, and angiotensin converting enzyme) relative to 2-hour simple cold or warm ischemia was also evaluated.
Results—No significant difference between groups was detected In any of the hemodynamic variables or postreperfusion plasma renin concentrations measured in this study relative to the duration of cold ischemic storage. There was also no difference between warm- and cold-stored kidneys in the expression of vascular-related genes
Conclusions and Clinical Relevance—Prolonged renal Ischemia for clinically relevant durations does not appear to predispose clinically normal cats to altered hemodynamics or high plasma renin concentrations following graft reperfusion. Activation of vasoactive genes does not appear to be Influenced by type of Ischemia over 2 hours. (Am J Vet Res 2010;71:1220-1227)
Abstract
Objective—To evaluate the use of a commercially available 5-carboxyfluorescein–based, intramolecularly quenched, fluorescence resonance energy transfer (FRET) peptide substrate of renin for measurement of plasma renin concentration in cats.
Sample Population—Plasma samples obtained during a previous study of renal autograft ischemia-reperfusion injury in 10 cats and samples of fetal bovine serum containing recombinant human renin (rh-renin).
Procedures—Experiments involving samples of fetal bovine serum containing rh-renin were conducted to identify a suitable control vehicle, optimal substrate concentration, and appropriate duration of incubation. With the use of the identified assay conditions, a standard curve was constructed to allow conversion of relative fluorescent units into values of renin concentration (ng/mL). Subsequently, plasma samples obtained from cats before and after renal autograft ischemia-reperfusion injury were assayed to determine endogenous renin concentration.
Results—Under conditions of a 1:50 substrate dilution and 4-hour incubation period, the assay detected small amounts of rh-renin in fetal bovine serum. A linear relationship (R 2 = 0.996) between the relative fluorescent units generated and exogenous rh-renin concentration was evident. The assay detected renin in plasma samples obtained from cats after renal autograft ischemia-reperfusion, and renin concentrations on days 1 and 2 after transplant differed significantly.
Conclusions and Clinical Relevance—The study data indicated that the assay involving the FRET peptide substrate of renin is potentially a rapid and specific method for measurement of plasma renin concentration in cats.
Abstract
Objective—To assess the in vitro capability of aqueous black walnut extracts (BWEs) to generate reactive oxygen species in water-based media ranging in makeup from a simple buffer solution to a complex solution containing serum.
Sample—3 BWEs.
Procedures—Production of reactive oxygen species by BWEs prepared in water or N-hexane was tested in PBS solution, PBS solution containing 0.5% bovine serum albumin and 5mM glucose (PBG), and RPMI-1640 medium (RPMI) containing 10% fetal bovine serum or 10% donor horse serum. Reactive oxygen species production was measured as conversion of nonfluorescent dihydrorhodamine 123 by reactive oxygen species to its fluorescent product, rhodamine-123. Hydrogen peroxide was used as a standard for reactive oxygen species activity.
Results—BWEs prepared in water generated reactive oxygen species in a dose-dependent manner over a 4-hour period, with peak activity detected when the BWEs were added as 10% (vol/vol) of the RPMI. The BWE prepared in N-hexane generated maximal reactive oxygen species activity after incubation for 3 to 4 hours when added at concentrations ranging from 0.3% to 0.5% (vol/vol) of the RPMI. The BWE prepared in water generated the highest fluorescent signal in PBS solution, whereas the BWE prepared in N-hexane generated the highest fluorescent signal in PBG.
Conclusions and Clinical Relevance—The BWEs prepared in water generated a dose-dependent induction of fluorescence in all the water-based solutions tested. These findings indicated that the BWEs, which are used to induce laminitis in horses, generate reactive oxygen species.
Abstract
Objective—To determine within a cat shelter effects of dietary lysine supplementation on nasal and ocular disease and detection of nucleic acids of Chlamydophila felis, feline calicivirus (FCV), and feline herpesvirus (FHV-1).
Animals—261 adult cats.
Procedures—Cats were fed a diet containing 1.7% (basal diet; control cats) or 5.7% (supplemented diet; treated cats) lysine for 4 weeks. Plasma concentrations of lysine and arginine were assessed at the beginning (baseline) and end of the study. Three times a week, cats were assigned a clinical score based on evidence of nasal and ocular disease. Conjunctival and oropharyngeal swab specimens were tested for FHV-1, FCV, and C felis nucleic acids once a week.
Results—Data were collected from 123, 74, 59, and 47 cats during study weeks 1, 2, 3, and 4, respectively. By study end, plasma lysine concentration in treated cats was greater than that in control cats and had increased from baseline. There was no difference between dietary groups in the proportion of cats developing mild disease. However, more treated cats than control cats developed moderate to severe disease during week 4. During week 2, FHV-1 DNA was detected more commonly in swab specimens from treated versus control cats.
Conclusions and Clinical Relevance—Dietary lysine supplementation in the amount used in our study was not a successful means of controlling infectious upper respiratory disease within a cat shelter. Rather, it led to increases in disease severity and the incidence of detection of FHV-1 DNA in oropharyngeal or conjunctival mucosal swab specimens at certain time points.
Abstract
Objective—To evaluate anti-inflammatory effects of several novel adenosine receptor agonists and to determine their specificity for various adenosine receptor subtypes on neutrophils, cells heterologously expressing equine adenosine receptors, or equine brain membranes.
Sample Population—Neutrophils isolated from 8 healthy horses.
Procedures—Radioligand binding experiments were performed to compare binding affinities of adenosine receptor agonists to equine adenosine A1, A2A, and A3 receptor subtypes. Effects of these agonists on endotoxin-induced production of reactive oxygen species (ROS) by equine neutrophils and roles of specific adenosine receptor subtypes and cAMP production in mediating these effects were determined.
Results—Radioligand binding experiments yielded a ranked order of affinity for the brain equine A2A receptor on the basis of 50% inhibitory concentrations (IC50) of the agonists as follows: ATL307 (IC50 = 1.9nM) and ATL313 > ATL309 and ATL310 > ATL202 > 2-([p-2- carboxyethyl] phenylethylamino)-5′-N-ethylcarboxyamidoadenosine > 5′-N-ethylcarboxamidoadenosine. Furthermore, ATL313 had approximately 100-fold greater selectivity for A2A over A1 and A3 receptors. In functional assays with equine neutrophils, the compounds inhibited endotoxin-induced ROS production and stimulated production of cAMP with the same ranked order of potency. Results of experiments performed with selective adenosine receptor antagonists indicated that functional effects of ATL313 were via stimulation of A2A receptors.
Conclusions and Clinical Relevance—Results indicated that activation of A2A receptors exerted anti-inflammatory effects on equine neutrophils and that stable, highly selective adenosine A2A receptor agonists may be developed for use in management of horses and other domestic animals with septic and nonseptic inflammatory diseases.
Abstract
Objective—To evaluate the effects of a standardized exercise test to exhaustion in horses on leukocyte function ex vivo.
Animals—6 Thoroughbred geldings.
Procedures—Blood samples were obtained from each horse before exercise; at exhaustion (termed failure); and at 2, 6, 24, 48, and 72 hours after exercise to evaluate hematologic changes, rate of leukocyte apoptosis, and leukocyte production of reactive oxygen species (ROS) ex vivo. To assess leukocyte function, leukocyte ROS production in response to stimulation with lipopolysaccharide, peptidoglycan, zymosan, and phorbol myristate acetate was evaluated. Apoptosis was evaluated via assessment of caspase activity in leukocyte lysates.
Results—In response to lipopolysaccharide, production of ROS by leukocytes was significantly increased at 2 hours and remained increased (albeit not significantly) at 6 hours after exercise, compared with the preexercise value. In the absence of any stimulus, leukocyte ROS production was significantly increased at 6 and 24 hours after exercise. In contrast, ROS production in response to phorbol myristate acetate was significantly decreased at 6, 24, and 72 hours after exercise. Leukocyte ROS production induced by zymosan or peptidoglycan was not altered by exercise. Leukocytosis was evident for 24 hours after exercise, and neutrophilia was detected during the first 6 hours. A significant increase in the rate of leukocyte apoptosis was detected at failure and 72 hours after exercise.
Conclusions and Clinical Relevance—Results indicated that strenuous exercise undertaken by horses causes alterations in innate immune system functions, some of which persist for as long as 72 hours after exercise.
