The corticosteroid laminitis story: 2. Science of if, when and how.
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Animals↗
Biomedical subjects
S R Bailey
Publications and source records attributed to S R Bailey.
Platelets in equine recurrent airway obstruction.
Platelets contribute to the pathogenesis of human allergic airway disease. The aim of this study was to compare platelet activating factor (PAF)-induced platelet aggregation and thromboxane (Tx) production, plasma Tx and 5-hydroxytryptamine (5-HT) in ponies with recurrent airway obstruction (RAO), an hypersensitivity to inhaled antigens, and normal ponies, before and after antigen exposure. Plasma 5-HT was significantly higher in ponies with RAO but was not further increased by antigen challenge. There was no difference between PAF-induced platelet aggregation or Tx production, or in plasma Tx before or after challenge. These data suggest there may be a difference between platelet 5-HT uptake in RAO and normal ponies but do not provide evidence of platelet activation following antigen exposure.
Animals↗
Pharmacological characterization of alpha1-adrenoceptors in equine digital veins.
Alpha-adrenoceptors mediate contractile responses in equine digital veins (EDVs) and arteries. Vascular smooth muscle alpha(1)-adrenoceptor subtypes have been implicated in a number of conditions, such as acute equine laminitis, and are therapeutic targets for the treatment of this condition. Digital veins, rather than arteries, were investigated in the present study because they have been specifically implicated in the pathophysiology of acute laminitis. The order of potency of a series of alpha(1)-adrenoceptor-selective agonists and antagonists was determined in isolated rings of EDVs under conditions of isometric tension. A61603 was the most potent agonist, with a higher potency (76-fold greater) than phenylephrine (PHE), suggesting the presence of the alpha(1A)-adrenoceptor subtype. Prazosin (30 nm) caused competitive inhibition of the responses to A61603 and PHE, with pK(b) values of 8.05 +/- 0.28 and 8.20 +/- 0.27, respectively. In addition, the alpha(1A)-adrenoceptor antagonist, WB4101 (10 nm), also caused competitive inhibition of the responses to the two agonists, with pK(b) values of 8.37 +/- 0.16 and 8.54 +/- 0.23, respectively, confirming the presence of the alpha(1A)-adrenoceptor subtype in EDVs. The selective alpha(1D)-adrenoceptor antagonist, BMY7378 (100 nm) did not cause a significant change in the response to the agonists, giving lower pK(b) values (6.97 +/- 0.27 and 6.88 +/- 0.17 vs. A61603 and PHE, respectively). Chloroethylclonidine dihydrochloride (45 microm, 30 min), used to produce selective inactivation of alpha(1B)-adrenoceptors, caused noncompetitive inhibition of the response to PHE, but was without effect on the response to A61603. These findings indicate that EDVs possess at least two different alpha(1)-adrenoceptor populations, which are predominantly of the alpha(1A) and alpha(1B) subtypes. These data may assist in the development of more selective antagonists for therapeutic use in horses.
Adrenergic alpha-1 Receptor Agonists↗
Assessment of endothelium-dependent vasodilation in equine digital resistance vessels.
Haemodynamic disturbances leading to ischaemia and reperfusion injury of the digit are thought to be involved in the pathophysiology of acute equine laminitis. Identification of physiological regulators of blood flow through the equine digit is important in identifying factors, which may predispose animals to laminitis. A method was developed to assess endothelium-dependent responses of the isolated Krebs-perfused equine digit by co-administration of 5-hydroxytryptamine (5-HT) with vasodilator agents, carbachol (CCh), bradykinin (BK) and substance P (SP). Bolus co-administration of CCh (0.02-2 micromol), BK and SP (0.02-0.2 nmol), caused inhibition of the 5-HT pressor response by 50-60%. The vasodilator responses were abolished by the detergent, CHAPS, indicating endothelium dependency; whereas vasoconstrictor responses to 5-HT were potentiated. CCh-induced relaxation was significantly reduced by the nitric oxide synthase inhibitor L-NAME (79.7 +/- 3.4% inhibition), whereas a large proportion of BK and SP-induced relaxation remained (34.1 +/- 6.3% and 33.6 +/- 5.3% inhibition). L-NAME potentiated vasoconstrictor responses to 5-HT. In conclusion, this study demonstrates that endothelium-derived NO modulates the response to vasoconstrictors such as 5-HT and is likely to be an important regulator of blood flow in the digital resistance vascular bed. Other factor(s) released by the endothelium are also important in regulating blood flow, whose identity remains to be established.
Animals↗
Reactive oxygen species from smooth muscle mitochondria initiate cold-induced constriction of cutaneous arteries.
Cold constricts cutaneous blood vessels by selectively increasing the activity of smooth muscle alpha2-adrenoceptors (alpha2-ARs). In mouse tail arteries, alpha2-AR constriction is mediated by alpha2A-ARs at 37 degrees C, whereas the cold-induced augmentation in alpha2-AR activity is mediated entirely by alpha2C-ARs. Cold causes translocation of alpha2C-ARs from the trans-Golgi to the plasma membrane, mediated by cold-induced activation of RhoA and Rho kinase. The present experiments analyzed the mechanisms underlying these responses. Mouse tail arteries were studied in a pressure myograph. Cooling the arteries (28 degrees C) caused a rapid increase in reactive oxygen species (ROS) in smooth muscle cells, determined by confocal microscopy of arteries loaded with the ROS-sensitive probes, dichlorodihydrofluorescein or reduced Mitotracker Red. The inhibitor of mitochondrial complex I rotenone (10 micromol/l), the antioxidant N-acetylcysteine (NAC; 20 mmol/l), or the cell-permeable mimic of superoxide dismutase MnTMPyP (50 micromol/l) did not affect vasoconstriction to alpha2-AR stimulation (UK-14304) at 37 degrees C but dramatically inhibited the response at 28 degrees C. Indeed, these ROS inhibitors abolished the cold-induced increase in alpha2-AR constrictor activity. NAC (20 mmol/l) or MnTMPyP (50 micromol/l) also abolished the cold-induced activation of RhoA in human cultured vascular smooth muscle cells and the cold-induced mobilization of alpha2C-ARs to the cell surface in human embryonic kidney 293 cells transfected with the receptor. The combined results suggest that cold-induced constriction is mediated by redox signaling in smooth muscle cells, initiated by mitochondrial generation of ROS, which stimulate RhoA/Rho kinase signaling and the subsequent mobilization of alpha2C-ARs to the cell surface. Altered activity of ROS may contribute to cold-induced vasospasm occurring in Raynaud's phenomenon.
Acetylcysteine↗
Rho kinase mediates cold-induced constriction of cutaneous arteries: role of alpha2C-adrenoceptor translocation.
Cold-induced vasoconstriction in cutaneous blood vessels is mediated in part by increased activity of vascular smooth muscle alpha2-adrenoceptors (VSM alpha2-ARs). In mouse cutaneous arteries, alpha2C-ARs are normally silent at 37 degrees C but mediate cold-induced augmentation of alpha2-AR responsiveness. In transfected HEK293 cells, this functional rescue is mediated by cold-induced translocation of alpha2C-ARs from the Golgi to the plasma membrane. Experiments were performed to determine the role of Rho/Rho kinase signaling in this process. Inhibition of Rho kinase (fasudil, Y27632 or H-1152) did not affect constriction of isolated mouse tail arteries to the alpha2-AR agonist UK 14 304 at 37 degrees C but dramatically reduced the augmented responses to the agonist at 28 degrees C. After Rho kinase inhibition, cooling no longer increased constriction evoked by alpha2-AR stimulation. Cooling (to 28 degrees C) activated Rho in VSM cells and increased the calcium sensitivity of constriction in alpha toxin-permeabilized arteries. Stimulation of alpha2-ARs in VSM cells had no effect on Rho activity or calcium sensitivity at 37 degrees C or 28 degrees C. In HEK293 cells transfected with alpha2C-ARs, cooling (to 28 degrees C) stimulated the translocation of alpha2C-ARs to the plasma membrane and this effect was prevented by inhibition of Rho kinase, using fasudil or RNA interference. Consistent with inhibition of the spatial rescue of alpha2C-ARs, fasudil inhibited alpha2-AR-mediated mobilization of calcium in tail arteries at 28 degrees C but not 37 degrees C. Therefore, cold-induced activation of Rho/Rho kinase can mediate cold-induced constriction in cutaneous arteries by enabling translocation of alpha2C-ARs to the plasma membrane and by increasing the calcium sensitivity of the contractile process.
Animals↗
In vitro and in vivo studies of homocysteine in equine tissues: implications for the pathophysiology of laminitis.
REASONS FOR PERFORMING STUDY: Elevated plasma homocysteine (HCy) concentration is a risk factor for cardiovascular diseases associated with endothelial dysfunction, including the human digital ischaemic disease, Raynaud's phenomenon. HYPOTHESIS: HCy causes dysfunction of equine vascular endothelium and elevated plasma concentrations predispose to laminitis. OBJECTIVES: To determine 1) the concentration of HCy in vitro, which inhibits equine vascular endothelial cell function and 2) any association between risk of laminitis and plasma HCy concentration. METHODS: Endothelial function was studied by measuring endothelium-dependent vasodilatory responses of the equine isolated perfused digit and basal nitric oxide (NO) production by cultured equine digital vein endothelial cells (EDVECs). Total plasma HCy (tHCy) concentrations were measured in samples collected in the winter and spring from normal ponies and ponies predisposed to laminitis. RESULTS: HCy (10 and 100 micromol/l) inhibited endothelial function and, at concentrations above 100 micromol/l, inhibited NO production by EDVECs. Plasma tHCy concentration ranged from 13 to 14.7 micromol/l. There was no effect of season or disease status on the concentration measured. CONCLUSIONS: In vitro, HCy was shown to interfere with endothelial cell function at physiologically relevant concentrations. No evidence was found for an association between risk of laminitis and high plasma concentrations of HCy. POTENTIAL RELEVANCE: Elevated plasma HCy concentrations could adversely affect endothelial cell function and mangement regimens that lead to increases in plasma HCy concentration should be avoided in ponies predisposed to laminitis.
Animals↗
The pathogenesis of acute laminitis: fitting more pieces into the puzzle.
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Acute Disease↗
Endotoxin-induced digital vasoconstriction in horses: associated changes in plasma concentrations of vasoconstrictor mediators.
REASONS FOR PERFORMING STUDY: Lipopolysaccharide (LPS) infusion reduces digital perfusion, but the mediators responsible remain undetermined. OBJECTIVES: To identify vasoconstrictor mediators released following LPS infusion and relate their appearance in plasma to digital blood flow alterations. METHODS: Blood flow in the lateral digital vessels of 6 Thoroughbred horses, following a 30 min infusion of LPS (E. coli 055:B5; 30 ng/kg), was measured using Doppler ultrasonography. Concomitant measurements of hoof wall and coronary band surface temperatures (HWST and CBST) were made. Serial blood samples were collected and plasma LPS, tumour necrosis factor alpha (TNFalpha), 5-HT, thromboxane B2 (TxB2) and endothelin measured. RESULTS: Plasma LPS concentrations reached a maximum of 13.2 pg/ml during the infusion, followed by an increase in plasma TNFalpha concentration. Digital arterial and venous blood flow decreased by 43 and 63%, respectively; HWST and CBST similarly decreased. Systemic blood pressure remained unaltered. Plasma concentrations of TxB2 and 5-HT increased, coinciding with the onset of digital hypoperfusion. Plasma endothelin concentrations remained unchanged. CONCLUSIONS: The temporal relationship between the onset of digital hypoperfusion and increases in plasma 5-HT and TxB2 concentrations is consistent with these platelet-derived mediators being associated with LPS-induced laminitis. POTENTIAL RELEVANCE: These experimental data support the use of anti-platelet therapy in the prevention of laminitis associated with endotoxaemic conditions.
Animals↗
The effects of vasoactive amines found in the equine hindgut on digital blood flow in the normal horse.
REASONS FOR PERFORMING STUDY: Disturbances of digital blood flow are thought to be fundamental to the pathophysiology of acute laminitis. However, factors linking the initiating events in the equine hindgut with these disturbances in the foot remain to be determined. HYPOTHESIS: Amine compounds, formed by bacteria in the equine hindgut, have digital vasoconstrictor effects in vivo. METHODS: Tryptamine (1.6 microg/kg/min) and phenylethylamine (2.13 microg/kg/min) were infused i.v. into standing nonsedated horses. Digital blood flow was measured by Doppler ultrasound and foot surface temperature was monitored. Plasma 5-hydroxytryptamine (5-HT) concentrations were measured by HPLC. RESULTS: Tryptamine and phenylethylamine infusions had no effect on systemic arterial blood pressure or heart rate, but caused significant decreases in digital arterial blood flow (mean +/- s.e. 29.2 +/- 8.5 and 18.4 +/- 6.8%, respectively). Both amines also caused decreases in dorsal hoof wall temperature (0.6 +/- 0.1 and 0.5 +/- 0.1 degrees C for tryptamine and phenylethylamine, respectively) and concomitant increases in plasma 5-HT concentration. CONCLUSIONS: Tryptamine and phenylethylamine caused reduction of digital blood flow, effects which may have been mediated, in part, via displacement of 5-HT from platelets. POTENTIAL RELEVANCE: Amine compounds occurring in the equine hindgut, if released into the circulation following carbohydrate overload, could contribute to selective digital vasoconstriction. Further work in ponies and horses, with naturally occurring laminitis, is necessary to determine whether amines represent a therapeutic target in this disease.
Acute Disease↗
Cloning, expression and biological activity of equine interleukin (IL)-5.
The cytokine, interleukin (IL)-5 stimulates eosinophil differentiation, activation and survival and can prime these cells, increasing the response to other mediators. In view of its many effects on eosinophils, IL-5 has been implicated in the pathogenesis of allergic disease in man. Here we report the cloning of equine IL-5 and expression of the recombinant protein by transfection of Chinese hamster ovary (CHO) cells. The cloned cDNA sequence consisted of 405 nucleotides and encoded a protein of 135 amino acids. There is >85% identity with feline, bovine, ovine, canine, and human IL-5 sequences at the nucleotide and protein level. Supernatants containing equine IL-5 were also examined for biological activity. CHO supernatant containing equine recombinant (eqr) IL-5, like the human ortholog (hrIL-5), induced concentration dependent equine eosinophil adherence to autologous serum-coated plastic (9.7+/-1.5% with a 1:100 dilution of eqrIL-5 and 9.1+/-1.6% adherence with 1 nM hrIL-5; n = 4). The eqr protein also caused concentration dependent superoxide production (11.9+/-2.4 nmol (reduced cytochrome (cyt) C)/10(6) cells at a 1:50 dilution, n = 4). In contrast, hrIL-5 only caused significant superoxide production when diluted in conditioned CHO medium, an effect that was inhibited by the anti-human mAb, TRFK5 (4.4+/-0.3 versus 0.3+/-0.4 nmol/10(6) cells for 0.5 nM hrIL-5 in the presence of the isotype matched IgG1 control (10 microM) and TRFK5 (10 microM), respectively). TRFK5 also significantly inhibited hrIL-5 induced adherence at concentrations of 0.3 microg/ml and above but had no significant inhibitory effect on either superoxide or adherence caused by eqrIL-5. These results demonstrate that equine IL-5 expressed by CHO cells stimulates equine eosinophils, suggesting that this cytokine could play a role in eosinophil recruitment and activation in equine allergic disease. The anti-human and murine moAb TRFK5 does not appear to recognise the equine protein.
Animals↗
Health status and participation in a range of activities in an Australian community.
INTRODUCTION: The aim of the research was to determine the relationship between levels of participation in a community and self-assessed health status of people in a rural and regional setting. METHOD: A cross-sectional design, using a mailed, self-administered questionnaire was used. Questionnaires were mailed to a random sample of people aged 18 years and over who were registered on the electoral roll of a regional city and rural area, the Barwon and Otway regions of Victoria, Australia. The sample consisted of 1752 participants: 990 females (57%), 739 males (42%) and 23 sex undisclosed (1%). The range of participants was 18-98 years, and the mean age was 50.53 years (SD = 17.19). RESULTS: Self-assessed physical and mental health were measured using the SF-12 scale. Participants with low incomes, and those with low self-assessed physical and mental health scores, were significantly more likely than other participants to agree with one or more of the social isolation items, indicating that they experienced some social isolation. Low levels of participation in social, sports, leisure or support activities were associated with low self-assessed physical and mental health. Disengagement with the local community was associated with low levels of self-assessed mental health. While younger people were more likely than older people to participate in social, sports, leisure or support activities, they were less involved as members of their community. Females were more likely than males to have been involved in five or more sports, leisure or support activities. Participation in civic activities was associated with high income. Levels of participation in the four different types of activities were combined (social activities, sport, leisure or support activities, community and group activities, and civic activities). Participants classified as low participators were more likely to be older participants, to have a low income and to have low scores for both physical and mental health. CONCLUSION: An association was found between health and community participation in a range of activities, and between health and engagement with the community in this rural and regional population. These findings are consistent with those reported from similar research with a metropolitan population sample. The current research suggests that the groups of people of most concern in terms of low participation rates, are people who have low incomes, people aged over 65 years, people who may be defined as possessing poor physical health and people who may be defined as possessing poor mental health. The relationship between age, community participation and health is complex and needs further exploration because it is not known whether poor health reduces community participation or whether reduced community participation results in poor health. However, current research suggests that developing and implementing strategies to promote people's engagement with and involvement in their local community is one important way of promoting the health of the community as a whole.
Journal Article↗
Identification and quantification of amines in the equine caecum.
Acute laminitis has been associated with the release of compounds, as yet unidentified, produced by hindgut fermentation which affect blood flow to the digit. The objectives of this study were to identify amine compounds in equine caecal and colonic contents, some of which are known to have vasoactive properties. In addition, the concentrations of amines in caecal contents of horses fed either grass or hay diets were compared. Fifteen amines were identified in equine hindgut contents in concentrations greater than 1 microM. The caecal concentrations of phenylethylamine, isoamylamine, cadaverine, diaminoheptane and spermidine were significantly higher in horses on spring/summer grass compared with those on winter grass or hay. These data show that many amines are present in the equine hindgut, some of which may have the potential to cause peripheral vasoconstriction if released into the circulation from the gastrointestinal tract.
Amines↗
Identification of equine cecal bacteria producing amines in an in vitro model of carbohydrate overload.
Acute laminitis has been associated with the overgrowth of gram-positive bacteria within the equine hindgut, causing the release of factor(s) leading to ischemia-reperfusion of the digits. The products of fermentation which trigger acute laminitis are, as yet, unknown; however, vasoactive amines are possible candidates. The objectives of this study were to use an in vitro model of carbohydrate overload to study the change in populations of cecal streptococci and lactobacilli and to establish whether certain species of these bacteria were capable of producing vasoactive amines from amino acids. Cecal contents from 10 horses were divided into aliquots and incubated anaerobically with either corn starch or inulin (fructan; both at 1 g/100 ml). Samples were taken at 6-h intervals over a 24-h period for enumeration of streptococci, lactobacilli, and gram-negative anaerobes by a dilution method onto standard selective growth media. The effects of the antibiotic virginiamycin (1 mg/100 ml) and calcium hydrogen phosphate (CaHPO(4); 0.3 g/100 ml) were also examined. Fermentation of excess carbohydrate was associated with increases in numbers of streptococci and lactobacilli (2- to 3.5-log unit increases; inhibited by virginiamycin) but numbers of gram-negative anaerobes were not significantly affected. A screening agar technique followed by 16S rRNA gene sequence analysis enabled the identification of 26 different bacterial strains capable of producing one or more vasoactive amines. These included members of the species Streptococcus bovis and five different Lactobacillus spp. These data suggest that certain bacteria, whose overgrowth is associated with carbohydrate fermentation, are capable of producing vasoactive amines which may play a role in the pathogenesis of acute laminitis.
Acute Disease↗
Uptake of 5-hydroxytryptamine by equine digital vein endothelial cells: inhibition by amines found in the equine caecum.
REASONS FOR PERFORMING STUDY: 5-hydroxytryptamine (5-HT; serotonin) is a potent vasoconstrictor of equine digital blood vessels and has been implicated in the pathogenesis of acute laminitis. OBJECTIVES: The aims of this study were firstly to examine whether cells of the digital blood vessel wall exhibited an active uptake mechanism for 5-HT and to characterise its efficiency; and secondly, to study the potential inhibitory effect on this process of other amines, produced in the equine caecum. METHODS: Confluent monolayers of equine digital vein endothelial cells (EDVEC) and equine digital vein smooth muscle cells (EDVSMC) were incubated with [3H]5-HT (0.1-250 micromol/l) and the total and active uptake calculated. Equine pulmonary vein endothelial cells (EPVEC) were used as a positive control. RESULTS: Both EDVEC and EDVSMC showed uptake of [3H]5-HT by nonfaci litated diffusion; however, only EDVEC showed evidence of saturable facilitated uptake mechanism, with a Km of 41.6 +/- 9.3 micromol/l, which was significantly higher than that of EPVEC (9.9 +/- 2.1 micromol/l). All 6 caecally-derived amines examined (tyramine, spermine, isoamylamine, tryptamine, phenylethylamine and isobutylamine) inhibited the total uptake of [3H]5-HT in a concentration-dependent manner, tyramine having the lowest IC50 (3.7 x 10(-6) mol/l). CONCLUSIONS: These data suggest that facilitated uptake into the endothelium could play a role in moderating the vasoconstrictor response to 5-HT in the equine digital circulation. POTENTIAL CLINICAL RELEVANCE: The vasoconstrictor action of 5-HT could be potentiated by gut-derived amines, providing a feasible link between GI disturbances and the pathophysiology of laminits.
Amines↗
Production of amines in equine cecal contents in an in vitro model of carbohydrate overload.
Acute laminitis can be induced experimentally in horses by the administration of carbohydrate, resulting in fermentation within the cecum and ischemia-reperfusion of the digits. The products of fermentation that trigger acute laminitis are as yet unknown; however, compounds such as amines might play a role due to their potential vasoactive properties. The objectives of this study were to quantify the amines present in equine cecal contents and to use a model of carbohydrate overload in vitro to test the hypothesis that carbohydrate fermentation is associated with increased amine production. Cecal contents from each horse were divided into aliquots and incubated anaerobically with either cornstarch or inulin (a form of fructan carbohydrate; both 1 g/100 mL). The pH was measured and samples were taken at the same time for amine measurement by HPLC at 2-h intervals over a 24-h period. In a second set of experiments, the effects of the antibiotic virginiamycin (1 mg/100 mL), calcium (CaPO4; 0.3 g/100 mL), and plant steroidal saponin (Yucca schidigera extract; 0.1 g/100 mL) were examined on pH and amine concentrations in cecal contents incubated with starch or inulin. Both starch and inulin caused significant time-dependent falls in pH, from 6.7 +/- 0.1 at 0 h to 5.2 +/- 0.1 (starch) and 5.0 +/- 0.1 (inulin) at 24 h. Fermentation of carbohydrate was also associated with increased production of phenylethylamine and isoamylamine (two- to threefold increases) as well as putrescine and cadaverine (1.5- to twofold increases). Virginiamycin inhibited the fall in pH and increases in production of phenylethylamine and isoamylamine, while calcium phosphate moderated the changes in pH only. Yucca schidigera extract was without effect. These data show that fermentation of carbohydrate by equine cecal microbiota may lead to increased production of amines.
Acute Disease↗
Assessment of neurobehavioral function with computerized tests in a population of hispanic adolescents working in agriculture.
In recent years there has been heightened concern over the potential of occupational or environmental exposures to affect neurological function in children and adolescents. The current study was designed to develop computerized tests to effectively assess neurobehavioral function in Hispanic adolescents working in agriculture and to evaluate those tests in Hispanic youths working in agriculture and in a non-agricultural group. After exclusions, 96 adolescents currently working in agriculture (AG) and 51 adolescents currently non-migratory and not working in agriculture (Non-AG) were tested. Neurobehavioral tests were selected from the computerized Behavioral Assessment and Research System. AG test performance was significantly below Non-AG performance on the cognitive tests. However, educational and cultural differences between the AG and Non-AG groups may explain this difference. Repeat testing of the AG group revealed substantially improved performance, further supporting educational or cultural differences as an explanation for the group differences. Together, these results expose the limitations in case-control or cross-sectional designs for testing migrant worker populations in the United States. Longitudinal or cross-sectional designs with repeat testing offer more promise and may be essential for drawing accurate conclusions in migrant worker groups where there are no truly equivalent comparison or control groups.
Adolescent↗