[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to explore the potential modulation of equine neutrophil oxidative burst by a series of classical NSAIDs which was subsequently monitored by the luminol or lucigenin-enhanced chemiluminescence (CL) technique. A significant dose-dependent inhibition of the luminol CL was observed with the majority of investigated drugs. This inhibition was very significant for phenylbutazone and Indomethacin; while for aspirin, a higher concentration is required. The action of Ketoprofen was significant during the first 5 min and only when the concentration was above 1 mM. Indomethacin and acetylsalicylic acid result in an inhibition dose-dependent of luminol CL. On the other hand, the phenylbutazone showed an inhibiting effect when used either luminol or lucigenin though luminol is slightly better. When the ketoprofen is considered, an inhibiting effect of luminal CL was observed but less significant than the other NSAIDs investigated. The flunixin meglumine enhances strongly the CL.
Veterinary Research Communications 03/2012; 36(1):29-33. DOI:10.1007/s11259-011-9507-5 · 1.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neutrophil myeloperoxidase (MPO) and elastase can be released in severe inflammatory diseases and cause tissue injuries. Equine enzymes have already been individually purified from large blood quantities. We describe the isolation of both enzymes from a same limited blood volume. Both MPO and elastase were extracted by crushing PMN isolated by centrifugation on a percoll-gradient from a 460 ml blood collection. MPO and elastase were separated by an ionic exchange chromatography phase and further purified by gel filtration chromatography on Superdex 200 and 75, respectively. Enzymes were identified in the collected fractions by specific enzymatic assays. The final purity was verified by electrophoresis. Specific activity was improved to 19.92 and 34.3× for elastase (final yield: 340 μg) and MPO (final yield: 130 μg), respectively, during the procedure. Results show the possibility of isolating both enzymes from the same blood sample with a sufficient yield and purity for future studies on their implication and interaction during inflammatory diseases.
Research in Veterinary Science 12/2009; 87(3):358-363. DOI:10.1016/j.rvsc.2009.04.002 · 1.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The previous experiments have shown that some phenothiazines have antioxidant and anti-inflammatory properties in vitro. In this study the inhibition of the production of reactive oxygen species (ROS) by neutrophils was studied in two groups of horses, which received a dose of 0.1 mg/kg of either acepromazine or promethazine intravenously. Blood samples were collected before (T0) and 0.5, 1, 3 and 5 h after drug administration. The chemiluminescence (CML) response of neutrophils was measured ex vivo in the presence of luminol for a period of 10 min and the maximum CML value (peak value) recorded. There was a significant inhibition of the ROS production in the acepromazine treated group (49% inhibition) at 5 h after administration and in the promethazine group (24% inhibition) at 3 h after administration (P < 0.05 vs. T0). These findings are of therapeutic relevance in the use of phenothiazines in equine patients with inflammatory diseases where neutrophil activation and ROS production are implicated.
Journal of Veterinary Pharmacology and Therapeutics 12/2009; 32(6):541-7. DOI:10.1111/j.1365-2885.2009.01077.x · 1.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The SIEFED ("Specific Immunological Extraction Followed by Enzymatic Detection") method already developed for the specific detection of the activity of equine myeloperoxidase (MPO) was adapted for the specific measurement of active human MPO in biological fluids or tissue extracts. The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by a washing (to eliminate the extraction medium and the biological fluid with their possible interfering molecules) and the measurement of the activity of MPO with a detection system containing a fluorogenic substrate, H(2)O(2) and nitrite ions as reaction enhancer. The SIEFED was applied to study active MPO in human biological fluids (plasma, bronchoalveolar lavage fluid and supernatant from carotids extracts). The SIEFED for human MPO has a sensitivity limit of 0.080 mU/mL and showed good precision with intra- and inter-assay coefficients of variation below 10 and 20% respectively within a broad range of MPO activities establish from 0.156 to 473 mU/mL. The SIEFED for human MPO will be useful for the specific detection of active MPO in complex fluids and can be complementary to an ELISA to determine an active/total MPO ratio in healthy volunteers and patients especially in case of chronic or acute inflammatory diseases.
[Show abstract][Hide abstract] ABSTRACT: Neutrophil (PMN) contribution to the acute inflammatory processes may lead to an excessive generation of reactive oxygen metabolites species (ROS) and secretion of granule enzymes. We compared the effects of either phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) in combination with a pre-treatment by cytochalasin B (CB) on the production of ROS and the release of total and active myeloperoxidase (MPO) by isolated equine PMNs. The ROS production was assessed by lucigenin dependent chemiluminescence (CL) and ethylene release by alpha-keto-gamma-methylthiobutyric acid (KMB) oxidation. In the supernatant of activated PMNs, total equine MPO was measured by ELISA and active MPO by the SIEFED (Specific Immunologic Extraction Followed by Enzymatic Detection) technique that allows for the study of the interaction of a compound directly with the enzyme. The stimulation of PMNs with CB-fMLP only modestly increased the release of MPO, but more than 70% of released MPO was active. PMA stimulation markedly increased the production of ROS and release of MPO, but more than 95% of released MPO was inactive. When PMNs were pre-incubated with superoxide dismutase (SOD) prior to PMA activation, the lucigenin enhanced CL, which is linked to the superoxide anion (O2-) production, was much more decreased than KMB oxidation, linked to the hydroxyl-like radical production. The addition of SOD prior to the activation of PMNs by PMA also limited the loss of the activity of released MPO. These results confirm the key role of O2- generation in the ROS cascade in PMN and reveal its critical role on MPO inactivation.
[Show abstract][Hide abstract] ABSTRACT: Collagens are major constituents of connective tissues in the animal kingdom. During aging and inflammatory-related diseases, the collagen network undergoes oxidation that leads to structural and biochemical alterations within the collagen molecule. Collagen oxidation appears to be a key determinant of aging and a critical physiopathologic mechanism of numerous diseases. Further, the detection of oxidized-collagen peptides seems to be a promising approach for the diagnosis and the prognosis of inflammatory diseases. This chapter reviews the structural and biochemical changes to collagen induced by reactive oxygen and nitrogen species and discusses recent data on the use of collagen-derived biomarkers for measuring oxidative damage.
Advances in clinical chemistry 01/2009; 49:31-55. · 2.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As heparins are sometimes used to prevent equine laminitis, the interactions between equine neutrophil myeloperoxidase (MPO), unfractionated (UFH) and fractionated low molecular weight (LMWH) heparins and digital endothelium have been investigated. The effects of the heparins on purified equine MPO activity were tested by immunocapture followed by enzymatic detection. Endothelium–MPO interactions were assessed by measuring total and active MPO uptake by arterial and venous digital endothelial cells in culture with or without the addition of heparins. A dose-dependent MPO inhibition by UFH and LMWH was seen, with the greatest reduction in MPO activity noted with the highest concentration of LMWH. The MPO capture was greater in arterial cells, but heparins better inhibited MPO capture in venous cells. The activity of cell-bound MPO was almost completely suppressed by the heparins, and no differences were observed between UFH and LMWH. The results confirm the anti-inflammatory properties of heparins and allow a better understanding of the potential role of MPO in laminitis.
The Veterinary Journal 10/2008; 178(1):62-69. DOI:10.1016/j.tvjl.2007.08.033 · 1.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Beside matrix metalloproteinases, reactive oxygen species (ROS) are the main biochemical factors of cartilage degradation. To prevent ROS toxicity, chondrocytes possess a well-coordinated enzymatic antioxidant system formed principally by superoxide dismutases (SODs), catalase (CAT) and glutathione peroxidase (GPX). This work was designed to assess the effects of interleukin (IL)-1beta and IL-6 on the enzymatic activity and gene expression of SODs, CAT and GPX in bovine chondrocytes.
Bovine chondrocytes were cultured in monolayer for 4-96 h in the absence or in the presence of IL-1beta (0.018-1.8ng/ml) or IL-6 (10-100 ng/ml). To study signal transduction pathway, inhibitors of mitogen-activated protein kinases (MAPK) (PD98059, SB203580 and SP600125) (5-20 microM) and nuclear factor (NF)-kappaB inhibitors [BAY11-7082 (1-10 microM) and MG132 (0.1-10 microM)] were used. SODs, CAT and GPX enzymatic activities were evaluated in cellular extract by using colorimetric enzymatic assays. Mn SODs, Cu/Zn SOD, extracellular SOD (EC SOD), CAT and GPX gene expressions were quantified by real-time and quantitative polymerase chain reaction (PCR).
Mn SOD and GPX activities were dose and time-dependently increased by IL-1beta. In parallel, IL-1beta markedly enhanced Mn SOD and GPX gene expressions, but decreased Cu/Zn SOD, EC SOD and CAT gene expressions. Induction of SOD enzymatic activity and Mn SOD mRNA expression were inhibited by NF-kappaB inhibitors but not by MAPK inhibitors. IL-6 effects were similar but weaker than those of IL-1beta.
In conclusion, IL-1beta, and to a lesser extend IL-6, dysregulates enzymatic antioxidant defenses in chondrocyte. These changes could lead to a transient accumulation of H(2)O(2) in mitochondria, and consequently to mitochondria damage. These changes contribute to explain the mitochondrial dysfunction observed in osteoarthritis chondrocytes.
Osteoarthritis and Cartilage 08/2008; 16(7):756-63. DOI:10.1016/j.joca.2007.10.009 · 4.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The capacity of the two cytokines TNF-alpha and IL-1beta to directly stimulate the oxidative activity of polymorphonuclear neutrophils remains debated. The purpose of this study was to verify if a direct stimulation of equine neutrophils by TNF-alpha and IL-1beta was possible. Equine neutrophils were isolated from blood by discontinuous density gradient centrifugation. The cell viability after isolation was >98%. The neutrophils were used at 1.25 x 10(6) cells by assay, immediately after isolation. The oxidative activity of neutrophils was measured by luminol- or lucigenin-enhanced chemiluminescence (CL), and the CL was recorded for 60 min. TNF-alpha and IL-1beta were used at concentrations ranging from 0.001 to 100 ng (0.0017-167 ng ml(-1)) for 1.25 x 10(6) neutrophils, and added to the cells just before the CL measurement. Both cytokines highly stimulated the lucigenin-enhanced CL of equine neutrophils in a dose-dependent manner. TNF-alpha was already active at 0.001 ng and IL-1beta at 0.01 ng. The CL response obtained with TNF-alpha was maximal after 5 min and more pronounced with luminol than with lucigenin. With IL-1beta, the luminol-enhanced CL response of neutrophils was short-lived and inversely proportional to the cytokine concentration: the CL response returned to baseline after 12 min, and became even lower than the baseline value for 10 and 100 ng IL-1beta. As luminol (but not lucigenin) enters the cell, we hypothesized that a rapid intracellular consumption of the luminol molecules occurred, explaining the rapid and intense CL response. The choice of the CL enhancer used in previous CL studies of neutrophils stimulation by cytokines could perhaps explain that controversial results were reported. In conclusion, we demonstrated a direct activation of the oxidative activity of equine neutrophils by TNF-alpha and IL-1beta, which was dose-dependent and obtained with very low doses equivalent to the plasma concentrations measured for both cytokines in equine septic shock. TNF-alpha and IL-1beta can thus aggravate neutrophils oxidative activity during septic shock in horses.
[Show abstract][Hide abstract] ABSTRACT: Several breeds of draft horses suffer from degenerative digital osteoarthropathy, resulting in a reduced active lifespan. A group of 30 Ardenner horses was followed, in standardized conditions, from 15 to 28 months of age to detect the early manifestations of the disease. The severity of the disease was assessed according to a personal grading system including clinical and radiographic items. Coll 2-1, a peptide of the helical region of type II collagen, and its nitrated form (Coll 2-1 NO(2)) were assayed in blood plasma collected at 452 +/- 18 days, 504 +/- 20 days, 558 +/- 18 days, 613 +/- 19 days, 675 +/- 19 days, 752 +/- 21 days and 852 +/- 19 days of age. At the end of the follow-up period, 53.3% of Ardenner horses were affected by a degenerative digital osteoarthropathy. A significant effect (p<0.05) of time, sex and pathology was observed for Coll 2-1 NO(2). Variations of Coll 2-1 were not significant except for the time effect. The elevation of Coll 2-1 NO(2) in the pathological group could indicate an inflammatory process during the growth of the affected horses, as nitration of tyrosine is mediated through reactive oxygen/nitrogen species and/or myeloperoxidase activity. Coll 2-1 NO(2) appears to be an interesting early marker of cartilage degradation and oxidation in degenerative osteoarthropathy.
Veterinary Research Communications 08/2007; 31(5):591-601. DOI:10.1007/s11259-007-3518-2 · 1.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the horse, the inflammation response to various pathologies (intestinal strangulations, laminitis, etc.) involves an excessive stimulation of the polymorphonuclear neutrophils releasing reactive oxygen species (ROS) and myeloperoxidase (MPO). The aim of the present work was to study the effect of natural polyphenols, curcuminoids and tetrahydrocurcuminoids (THC) on isolated stimulated equine neutrophils and on the activity of purified MPO. The ROS production and the release of MPO by activated neutrophils were measured by chemiluminescence and ELISA techniques, respectively. The activity of purified MPO was measured by studying its nitration, chlorination or oxidation capacity and by using an original method called SIEFED allowing the study of drug interaction with the enzyme without interferences of the medium. Curcuminoids and THC had dose-dependent inhibitory effects on ROS production and MPO release by activated neutrophils and on purified MPO activity. We suggest that the higher efficacy of curcuminoids versus THC could be explained, at least partially, by its chemical structure: the conjugated double bounds and the plane structure of curcuminoids made easier the neutralization of the radical species generated by activated neutrophils and the interaction of the drug with the active site of MPO. These inhibitory effects of curcuminoids on the oxidant activity of equine neutrophils and on MPO activity open therapeutic perspectives in equine pathologies with excessive inflammatory reactions.
Physiological research / Academia Scientiarum Bohemoslovaca 08/2007; 57(4):577-87. · 1.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Markers of cartilage breakdown enable studying the degradation of cartilage matrix in equine joint pathologies. This study was designed to determine the levels of Coll2-1, a peptide of the triple helix of type II collagen, and Coll2-1NO(2), its nitrated form in the plasma of healthy horses (controls; n=37) and horses suffering from osteochondrosis (n=34). Clinical and arthroscopic scores were attributed reflecting the severity of lesions and were related to the plasma levels of Coll2-1 and Coll2-1NO(2). The median of Coll2-1 was significantly higher in the control group, whereas the mean of Coll2-1NO(2) showed significant elevation in the pathological group. However, the measurement means of scoring classes did not vary significantly. The markers were able to differentiate the group of horses suffering from osteochondrosis from the group of healthy horses. The elevation of Coll2-1NO(2) in the pathological group indicates an inflammation, mediated through reactive oxygen species and/or increased myeloperoxidase activity.
Research in Veterinary Science 03/2007; 82(1):68-75. DOI:10.1016/j.rvsc.2006.03.008 · 1.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Degenerative osteoarthropathy resulting in a reduced active lifespan was observed in Ardenner horses. In the context of joint biology, insulin-like growth factor I (IGF-I) is a potential candidate to affect the anabolism of cartilage matrix molecules. A group of 30 Ardenner horses reared under standardized conditions from weaning were evaluated periodically from 15 to 28 months of age to detect the early manifestations of the disease. At the end of this period, horses were classified in two pathological groups related to the degree of interphalangeal degenerative osteoarthropathy based on clinical and radiographic evaluations: healthy (46.7%) and pathological (53.3%) horses. Seven sequential blood samples were taken from each horse (during the evaluation period) to study the variation of IGF-I plasma concentration. We tested the variations of the IGF-I plasma concentration during growth, and the effect of sex and of pathological classes. Significant variations were observed during the research period, with a maximum value corresponding to spring and a minimum in autumn. A significant reduction of the IGF-I plasma concentration was also observed in the pathological horses (433.5 +/- 19.5 ng/ml) compared to the healthy horses (493.9 +/- 18.2 ng/ml). An alteration in the level of this growth factor could induce a disregulation of the mechanisms involved in the local control of joint and bone tissue development.
Veterinary Research Communications 03/2007; 31(2):185-95. DOI:10.1007/s11259-006-3385-2 · 1.24 Impact Factor