Curcumin and resveratrol act by different ways on NADPH oxidase activity and reactive oxygen species produced by equine neutrophils
Derochette, Sandrine ; Franck, Thierry ; Mouithys-Mickalad, Ange et al
in Chemico-Biological Interactions (2013), 206
In neutrophils (PMNs), superoxide anion (O2●-), the first reactive oxygen species (ROS) produced to kill pathogenic agents, is generated by NADPH oxidase, an enzymatic complex formed by the translocation ... [more ▼]
In neutrophils (PMNs), superoxide anion (O2●-), the first reactive oxygen species (ROS) produced to kill pathogenic agents, is generated by NADPH oxidase, an enzymatic complex formed by the translocation of cytosolic subunits to the membrane flavocytochrome b558. In horses, excessive activation of PMNs is often associated with deadly pathologies and the modulation of their ROS production by acting on NADPH oxidase is a prime target to manage inflammation. We developed a cell-free assay to measure the activity of equine NADPH oxidase assembled in vitro, in order to test the effects of natural or synthetic compounds on the enzyme activity or assembly. The cell-free assay was validated with diphenyleneiodonium chloride and Gp91ds-tat, two inhibitors largely described for human NADPH oxidase. The anti-oxidant effects of curcumin and resveratrol at final concentration ranging from 10-4 to 10-6 M were studied on whole cells by chemiluminescence (CL) and by cell-free assay, in which the molecule was added before or after the enzyme assembly. The CL assay demonstrated that curcumin efficiently inhibited the O2●- production and easily entered into PMNs or interacted with their membrane. Cell-free assay showed that curcumin acted on the reconstitution of NADPH oxidase even at 10-5 M, while resveratrol appeared to be an O2●- scavenger rather than an inhibitor of NADPH oxidase activity, since it acted from outside the cell in CL and after the complex assembly in cell-free assay. By acting directly on NADPH oxidase, curcumin should be a good candidate for the treatment of acute or inflammatory diseases involving an excessive ROS production. [less ▲]Detailed reference viewed: 44 (9 ULg)
Effect of myeloperoxidase and anoxia/reoxygenation on mitochondrial respiratory function of cultured primary equine skeletal myoblasts.
Ceusters, Justine ; Mouithys-Mickalad, Ange ; Franck, Thierry et al
in Mitochondrion (2013), 13(5),
Horses are particularly sensitive to excessive inflammatory reaction where myeloperoxidase, a marker of inflammation, may contribute to mitochondrial dysfunctions. This study investigated the interaction ... [more ▼]
Horses are particularly sensitive to excessive inflammatory reaction where myeloperoxidase, a marker of inflammation, may contribute to mitochondrial dysfunctions. This study investigated the interaction between myeloperoxidase and cultured primary equine skeletal myoblasts, particularly its effect on mitochondrial respiration combined or not with anoxia followed by reoxygenation (AR). We showed that active myeloperoxidase entered into the cells, interacted with mitochondria and decreased routine and maximal respirations. When combined with AR, myeloperoxidase caused a further decrease of these respiratory parameters while the leak increased. Our results indicate that myeloperoxidase amplifies the mitochondrial damages initiated by AR phenomenon and alters the mitochondrial function. [less ▲]Detailed reference viewed: 34 (14 ULg)
Sevoflurane inhibits equine myeloperoxidase release and activity in vitro.
MINGUET, Grégory ; de la Rebière de Pouyade, Geoffroy ; Franck, Thierry et al
in Veterinary Anaesthesia & Analgesia (2013), 40
Objective To investigate the effects of the volatile anaesthetic sevoflurane on the release of total and active myeloperoxidase (MPO) by non-stimulated and stimulated polymorphonuclear neutrophils (PMNs ... [more ▼]
Objective To investigate the effects of the volatile anaesthetic sevoflurane on the release of total and active myeloperoxidase (MPO) by non-stimulated and stimulated polymorphonuclear neutrophils (PMNs) in whole blood from healthy horses. Study design In vitro experimental study. Animals Adult healthy horses. Methods Samples of whole venous blood were collected and incubated in air or in air plus 2.3% or 4.6% sevoflurane for 1 hour. PMNs were stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP), with a combination of cytochalasin B (CB) and fMLP or with phorbol myristate acetate (PMA). Total and active MPO contents released by PMNs in blood were measured by enzyme-linked immunosorbent assay (ELISA) and specific immunological extraction followed by enzymatic detection (SIEFED) respectively. Additional experiments were performed to assess the effect of sevoflurane on the peroxidase and chlorination cycles of purified equine MPO using Amplex Red and 3'-(p-aminophenyl) fluorescein as fluorogenic substrates respectively. Results As compared with air alone, 1 hour exposure of whole blood to 4.6% sevoflurane in air significantly inhibited the release of total and active MPO by unstimulated and both fMLP- and CB + fMLP-stimulated PMNs but not by PMA-stimulated PMNs. Although 2.3% sevoflurane had no effect on total MPO release by unstimulated and stimulated PMNs, it significantly reduced the release of active MPO by unstimulated and fMLP-stimulated PMNs. Additionally, sevoflurane reversibly inhibited the activity of MPO, especially the peroxidase cycle of the enzyme. Conclusions and clinical relevance Although our experimental study was not designed to assess the effects of sevoflurane in vivo, this inhibition of MPO release and activity may have relevance for anaesthetized horses and deserves further studies to examine the clinical importance of these findings. [less ▲]Detailed reference viewed: 30 (11 ULg)
Equine myeloperoxidase: A novel biomarker in synovial fluid for the diagnosis of infection.
; ; et al
in Equine Veterinary Journal (2013), 45(3),
REASONS FOR PERFORMING STUDY: Equine joint infection is a life-threatening disorder, and confirmation of the diagnosis can be difficult. Synovial fluid biomarkers may assist the discrimination between ... [more ▼]
REASONS FOR PERFORMING STUDY: Equine joint infection is a life-threatening disorder, and confirmation of the diagnosis can be difficult. Synovial fluid biomarkers may assist the discrimination between infectious and noninfectious joint disease. OBJECTIVES: This study investigates whether the immunological detection of total and enzymatically active myeloperoxidase (MPO) assists the diagnosis of joint infection in horses. METHODS: The following 4 sample groups were included: healthy; osteochondritis dissecans (OCD); traumatic synovitis; and culture-confirmed infected joints. Synovial fluid was analysed for total MPO by a horse-specific sandwich enzyme-linked immunosorbent assay (ELISA) and for active MPO using the specific immunological extraction followed by enzymatic detection (SIEFED) technique. Western blot analysis was performed to confirm the antibody specificity. RESULTS: Synovial fluid from infected joints contained significantly more total and active MPO than samples from healthy joints, joints affected by OCD and joints with traumatic synovitis. Cut-off values were set at 5000 and 350 ng/ml for total and active MPO, respectively, with fair sensitivity, specificity, positive and negative predictive values and likelihood ratios for infection. Correlation coefficients were reported between the total as well as the active MPO levels and the routine synovial fluid parameters, i.e. the white blood cell count, the neutrophil count and the total protein level. No correlation was observed between MPO and either the age of the horse or the joint affected. Western blotting confirmed the antibody specificity for equine MPO. CONCLUSIONS AND POTENTIAL RELEVANCE: Synovial fluid MPO was identified as a very promising biomarker to augment the discrimination of infectious vs. noninfectious joint disease in horses. Both ELISA and SIEFED techniques can be used for its specific and rapid detection. The analysis of synovial fluid MPO can be used as a complementary test to aid in the discrimination between infectious and noninfectious joint disease, especially when the white blood cell counts and the total protein level are inconclusive. [less ▲]Detailed reference viewed: 14 (2 ULg)
Differentiation between stoichiometric and anticatalytic antioxidant properties of benzoic acid analogues: A structure/redox potential relationship study.
Franck, Thierry ; Mouithys-Mickalad, Ange ; Robert, Thierry et al
in Chemico-biological interactions (2013), 206(2), 194-203
We investigated the antioxidant activities of some phenolic acid derivatives on a cell free system and on cellular and enzymatic models involved in inflammation. The stoichiometric antioxidant activities ... [more ▼]
We investigated the antioxidant activities of some phenolic acid derivatives on a cell free system and on cellular and enzymatic models involved in inflammation. The stoichiometric antioxidant activities of phenolic acid derivatives were studied by measuring their capacity to scavenge the radical cation 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS+) and reactive oxygen species (ROS) produced by stimulated neutrophils. The anticatalytic antioxidant capacity of the molecules was evaluated on the activity of myeloperoxidase (MPO), an oxidant enzyme present in and released by the primary granules of neutrophils. The ROS produced by PMA-stimulated neutrophils were measured by lucigenin-enhanced chemiluminescence (CL) and the potential interaction of the molecules with MPO was investigated without interferences due to medium by Specific Immuno-Extraction Followed by Enzyme Detection (SIEFED). The antioxidant activities of the phenolic compounds were correlated to their redox potentials measured by differential pulse voltammetry (DPV), and discussed in relation to their molecular structure. The ability of the phenolic molecules to scavenge ABTS radicals and ROS derived from neutrophils was inversely correlated to their increased redox potential. The number of hydroxyl groups (three) and their position (catechol) were essential for their efficacy as stoichiometric antioxidants or scavengers. On MPO activity, the inhibitory capacity of the molecules was not really correlated with their redox potential. Likewise, for the inhibition of MPO activity the number of OH groups and mainly the elongation of the carboxylic group were essential, probably by facilitating the interaction with the active site or the structure of the enzyme. The redox potential measurement, combined with ABTS and CL techniques, seems to be a good technique to select stoichiometric antioxidants but not anticatalytic ones, as seen for MPO, what rather involves a direct interaction with the enzyme. [less ▲]Detailed reference viewed: 23 (4 ULg)
Effects of diazoxide, benzothiadiazine and benzopyrane derivatives on mitochondrial proton and electron leaks of cardiomyocytes (H9C2 cell line).
Mouithys-Mickalad, Ange ; Ceusters, Justine ; et al
Background: Mitochondria are double membrane- organelles that play a central role in cellular metabolism, calcium homeostasis and redox signaling. They have been also considered as main producers of ... [more ▼]
Background: Mitochondria are double membrane- organelles that play a central role in cellular metabolism, calcium homeostasis and redox signaling. They have been also considered as main producers of adenosine triphosphate (ATP) and reactive oxygen species (ROS). In many cancer cells those organelles become dysfunctional leading to a shift of energy metabolism from oxidative phosphorylation to active glycolysis and an increase of ROS generation. According to Warberg’ theory, cancer damage might occur at the mitochondrial level, affecting tiny structures within each cell implicated in the energy production through ATP. New insight is that mitochondria might be a good therapeutic target for metabolic syndromes, ischemia/reperfusion injury and organs transplantation. Therefore, search for novel molecules able to keep mitochondria functional are of relevant interest. Methodology: Cardiomyocytes (H9C2 cells) were from ATCC (USA) and grown till confluence. The basal cellular respiratory rate, proton and electron leaks as well as ATP production were measured with the High Resolution Oxygraphy (Oroboros, Austria). All compounds: diazoxide (DIAZ), diazoxide –related analogs (1: BPDZ-259, 2: BPDZ-444), and benzopyran derivatives (3: BPDZ-490, 4: BPDZ-711) were tested at final concentration of 10-5 M, except when specified and compared to control samples (cells with or without DMSO). Results and conclusion: The basal respiratory rate of H9C2 cells (5x106/mL) was changed depending on the chemical structure of the tested compounds: e.g. compound 3 strongly enhanced the routine respiration, while 4 displayed a marked lowering effect. In contrast, the addition of similar concentration of benzothiadiazin derivatives (1, 2) had no effect on routine respiration but also on the other respiratory parameters such as oligomycin-induced leak and ATP production. Similar profile was obtained with the reference molecule: diazoxide. Overall, our findings indicate that both diazoxide-like analogues (1 and 2) and diazoxide were without significant effect on basal respiration, ATP production, even on maximal respiration. Interestingly, two derivatives show opposite effects: compound 3 behaves as a uncoupling agent and the other one (4) exhibits a real lowering effect on respiration but that was reversible. The latter effect might be of interest if this kind of molecules could be used for further use as an agent for organ conservation during transplantation. Our results also demonstrate that diazoxide, a well-known Mito-KATP opener, did not exert its effect beside of clinical situation like ischemia/reperfusion injury. [less ▲]Detailed reference viewed: 51 (3 ULg)
Systemic Active and Total Myeloperoxidase Levels in Coronary Artery Disease.
GACH, Olivier ; MAGNE, Julien ; Franck, Thierry et al
in Cardiology (2013), 126(Suppl. 2), 1-521
Backgound: Measurement of total Myeloperoxidase (MPO) by ELISA is considered as a marker of neutrophil activation but is not the true indicator of the degree of its activity. In a dynamic pathology such ... [more ▼]
Backgound: Measurement of total Myeloperoxidase (MPO) by ELISA is considered as a marker of neutrophil activation but is not the true indicator of the degree of its activity. In a dynamic pathology such as atherosclerosis, it may be important to measure the real active part of MPO because it represents the true witness of the oxidant potential of the enzyme. Aim: To identify the relation between coronary artery disease identified by coronaro-angiography on measured serum total and active MPO levels and evaluate the correlation between these MPO levels and the presence of clinically defined unstable condition. Methods: Prospective analyse of serum samples of patients before (within 30 min) coronaro-angiography. Total and active MPO concentrations were assessed by sandwich Elisa and SIEFED® method’s respectively. Results: Two hundred and twenty patients were included in this study (age: 66.1±10.7 years, 67% of male). Among these, 62% presented significant coronary artery disease (stenosis more than 60% at least in one épicardial coronary artery). Twenty four patients (11%) presented unstable coronary syndrome. Mean active and total MPO in the general population were 50.1±63.5 and 147.6±223.3 ng.mL-1 respectively. In comparison, mean active MPO was 47.1±47.9 ng.mL-1 in stable patients and 75.1±135.2 ng.mL-1 in unstable patients (p=0.04). Mean total MPO was 146.3±224.7 ng.mL-1 in stable patients and 158.2±215.8 ng.mL-1 in the unstable’s one (p=0.8). There was a significant correlation between active MPO levels and instability (r=0.14, p=0.04) not present for total MPO levels (r=0.016, p=0.8). Conclusion: We observed a correlation between active MPO and clinical instability while there was no correlation with total MPO. Our preliminary results suggest that this marker could be a powerful indicator of instability which could possess an important prognostic impact. This hypothesis requires an evaluation in wider population and during a prolonged follow-up. [less ▲]Detailed reference viewed: 34 (2 ULg)
An in vitro whole blood model to test the effects of different stimuli conditions on the release of myeloperoxidase and elastase by equine neutrophils.
Ceusters, Justine ; Serteyn, Didier ; MINGUET, Grégory et al
in Veterinary Immunology and Immunopathology (2012), 150(3-4), 221-7
Horses are particularly sensitive and exposed to excessive inflammatory responses evolving toward an important stimulation of polymorphonuclear neutrophils (PMNs). The aim of this work was to stimulate ... [more ▼]
Horses are particularly sensitive and exposed to excessive inflammatory responses evolving toward an important stimulation of polymorphonuclear neutrophils (PMNs). The aim of this work was to stimulate equine neutrophils in whole blood and to evaluate their response by measuring the release of total and active myeloperoxidase (MPO) and total elastase, considered as markers of neutrophil stimulation and degranulation. Because of the critical importance of the concomitant presence of LPS and TNF-alpha in equine pathological situations, we combined these two natural mediators to stimulate PMN and compared the response with those obtained after the PMN stimulation with each mediator used alone and well-known artificial stimulation systems such as 12-phorbol 13-myristate acetate (PMA) and the combination of cytochalasin B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP). All the activation systems, PMA, CB/fMLP, TNF-alpha, LPS and LPS/TNF-alpha, induced a significant release of total MPO in whole blood but only the combinations CB/fMLP and LPS/TNF-alpha significantly favored the release of active MPO. Regarding the total elastase, we did not observe a significant release in all the stimulated conditions except with PMA. It appears clearly that the choice of the neutrophil stimulation model is fundamental for the selection of potentially active pharmacological agents, especially on MPO activity. [less ▲]Detailed reference viewed: 36 (18 ULg)
INFLUENCE OF MYELOPEROXIDASE ACTIVITY ON EQUINE POST-THAW SEMEN QUALITY
Ponthier, Jérôme ; Franck, Thierry ; Parrilla Hernandez, Sonia et al
in Edeas, Marvin (Ed.) Proceedings of the 2nd ISANH World congress on Fertility and Antioxidants (2012, December 06)
This study confirms that active MPO is associated with cellular fraction of the ejaculate, as previously suggested for total MPO concentration in thawed semen (2). However, active MPO concentrations were ... [more ▼]
This study confirms that active MPO is associated with cellular fraction of the ejaculate, as previously suggested for total MPO concentration in thawed semen (2). However, active MPO concentrations were dramatically lower than total MPO concentrations observed in equine semen (3), which could be explained by presence of inactive MPO subunits in semen. MPO activity in sperm-rich pellet can be used as a predictive marker of post-thaw semen quality. Moreover, methods to inhibit MPO should be investigated in semen. [less ▲]Detailed reference viewed: 40 (9 ULg)
EFFECT OF MYELOPEROXIDASE ON MITOCHONDRIAL RESPIRATORY FUNCTION OF PERMEABILIZED PRIMARY EQUINE SKELETAL MYOBLASTS IN CULTURE.
Ceusters, Justine ; Mouithys-Mickalad, Ange ; Franck, Thierry et al
Poster (2012, November)Detailed reference viewed: 34 (8 ULg)
Effect of 120 minutes of high pressure-volume and low pressure-volume mechanical ventilation on plasmatic markers of systemic inflammation in horses during general anaesthesia
; Cerri, Simona ; Gougnard, Alexandra et al
Poster (2012, October 19)Detailed reference viewed: 19 (4 ULg)
Simultaneous measurement of protein-bound 3-chlorotyrosine and homocitrulline by LC-MS/MS after hydrolysis assisted by microwave: application to the study of myeloperoxidase activity during hemodialysis.
; Franck, Thierry ; et al
in Talanta (2012), 99
A high degree of uremia is common in patients with end-stage renal disease and has been linked to the development of chronic inflammation and cardiovascular diseases. In conditions where transplantation ... [more ▼]
A high degree of uremia is common in patients with end-stage renal disease and has been linked to the development of chronic inflammation and cardiovascular diseases. In conditions where transplantation is not possible, uremia can be reduced by hemodialysis although the repeated interventions have been implicated in loss of renal function, partially as a result of chronic inflammation and/or oxidative stress processes. In this context, it has been suggested that myeloperoxidase (MPO) can contribute to the oxidative stress during hemodialysis and to the cardiovascular risk. Protein damages due to MPO activity have never been assessed during hemodialysis although two of its reaction products, 3-chlorotyrosine and homocitrulline, are of interest. Indeed, the first one is a specific product of MPO activity and the formation of the second one could be catalyzed by MPO. In order to analyze these products in plasma proteins, a total hydrolysis method followed by liquid chromatography mass spectrometry analysis was developed. Different conditions of hydrolysis were tested and the optimized procedure was assessed for complete hydrolysis and artifactual chlorination. Finally, the method was used for analyzing 3-chlorotyrosine and homocitrulline in plasma proteins during a hemodialysis session in fifteen patients and data were related to measurements of MPO concentration and activity. Both increases in MPO activity and protein-bound 3-chlorotyrosine were observed, highlighting the involvement of MPO in oxidative stress during hemodialysis and further demonstrating the link between hemodialysis and cardiovascular diseases. [less ▲]Detailed reference viewed: 31 (0 ULg)
Effect of high pressure-volume and low pressure-volume mechanical ventilation on plasmatic levels of IL-6, TNF-α,
; Cerri, Simona ; Gougnard, Alexandra et al
Poster (2012, September 12)Detailed reference viewed: 21 (3 ULg)
Does the new water-soluble form of curcumin(NDS27) inhibit the oxidant response of stimulated neutrophils and HL-60 cells?
Derochette, Sandrine ; Franck, Thierry ; et al
Poster (2012, September 07)
Neutrophils (PMNs) are involved in host defense against infections through the production of reactive oxygen species (ROS) and the release of an oxidant enzyme, myeloperoxidase (MPO), to kill pathologic ... [more ▼]
Neutrophils (PMNs) are involved in host defense against infections through the production of reactive oxygen species (ROS) and the release of an oxidant enzyme, myeloperoxidase (MPO), to kill pathologic agents. But, an excessive stimulation of PMNs is associated with development of inflammatory diseases. Neutrophils are prime targets to control inflammatory events and the therapeutic use of polyphenols is proposed to lower oxidative stress. The aim of this work was to study antioxidant effect of NDS27, a water-soluble form of curcumin, on stimulated equine PMNs and human promyelocytic leukemia cells (HL-60) which are less differenciated. 2',7'-Dichlorofluorescin diacetate and lucigenin were used to measure ROS production by activated HL-60 cells or PMNs. NDS27 (10-6 to 10-4 M) was pre-incubated with cells and eliminated before their activation to study its intracellular effects on ROS production. The effect of NDS27 on MPO activity released by the cells was determined by SIEFED. Likewise, the ability of NDS27 to enter into the cells was checked by HPLC on the cellular extracts.NDS27 significantly and dose-dependently inhibited the ROS production in both cell types without affecting their viability. Its intracellular effect showed higher efficiency for PMNs while its interaction with HL-60 cells remained better. The activity of MPO released by PMNs and HL-60 cells was decreased by NDS27 with a more efficient effect for PMNs. Our findings suggest that the greater efficiency of NDS27 in mature PMNs is not due to a better membrane permeability or a better interaction between membrane and NDS27, but rather to an inhibitory effect on the ROS production by the more mature cells, probably by targeting the enzymes implied in respiratory burst like MPO and NADPH oxidase. The modulatory effect of NDS27 towards oxidant activity of cells involved in immune and inflammatory response opens therapeutic perspectives to control pathologies with excessive inflammatory reactions. [less ▲]Detailed reference viewed: 40 (5 ULg)
Myeloperoxidase as an indicator of endometritis in the mare: preliminary results
Parrilla Hernandez, Sonia ; Franck, Thierry ; Ponthier, Jérôme et al
in Reproduction in Domestic Animals (2012, August), 47(suppl 5), 65
Diagnosis of endometritis in the mare is routinely based on the presence of polymorphonuclear cells (PMNs) on endometrial smears. Studies show a relation between PMNs and myeloperoxidase (MPO), an enzyme ... [more ▼]
Diagnosis of endometritis in the mare is routinely based on the presence of polymorphonuclear cells (PMNs) on endometrial smears. Studies show a relation between PMNs and myeloperoxidase (MPO), an enzyme released by PMNs during degranulation or after cell lysis, in many fluids and tissues. The aims of this study were to assess the presence and concentration of MPO in the mare’s uterus, and to investigate its relation with PMNs. Thirty-six cycles from 28 mares (ages ranging from 6 to 22 years) were used. Endometrial cytological samples were obtained with a small volume uterine flush and either a uterine cotton swab or a cytobrush, when a follicle >35 mm was observed by ultrasound. The smears were stained with Diff-QuickÒ and one or more PMNs per field (400·) was diagnosed as endometritis. The supernatant of the flushes was used to measure MPO concentration with a specific equine MPO ELISA assay. Our results showed the presence of MPO in the equine uterus during oestrus (mean = 2839 + 2785). MPO concentrations were signifi- cantly (p < 0.05) higher in samples with positive cytological results. Occasionally, some samples with negative cytological results showed high MPO concentration, but the opposite was never observed. Clinical signs of endometritis are not always present, or they may be delayed. An early diagnostic improves the success of treatment. Our results show that high quantities of MPO in endometrial samples indicate the presence of PMNs. Further studies are needed to determine if MPO concentration could be routinely used as a tool of early detection of endometritis. [less ▲]Detailed reference viewed: 105 (15 ULg)
Possible intracellular effect of the new water-soluble form of curcumin (NDS27) on the oxidant response of stimulated neutrophils
Derochette, Sandrine ; Mouithys-Mickalad, Ange ; et al
Poster (2012, April 18)
Neutrophils (PMNs) are involved in host defense against infections through the production of reactive oxygen species (ROS) to kill pathologic agents. But, an excessive ROS production, called “oxidative ... [more ▼]
Neutrophils (PMNs) are involved in host defense against infections through the production of reactive oxygen species (ROS) to kill pathologic agents. But, an excessive ROS production, called “oxidative stress” is associated with tissue damages and development of chronic or acute inflammatory diseases. PMNs are prime therapeutic targets to control inflammatory events associated to ROS production. Nowadays, there is a growing interest for the use of polyphenolic molecules to modulate the inflammatory response. The aim of this work was to study the antioxidant effect of NDS27 (1), a new highly water-soluble form of the polyphenolic molecule curcumin, on in vitro stimulated equine PMNs. NDS27 (10-6 to 10-4 M) was pre-incubated with cells and eliminated before their activation. The ability of NDS27 to enter into the cells was checked by HPLC from the cellular extracts. The intracellular ROS production by phorbol myristate acetate (PMA) stimulated PMNs was measured by fluorescence using 2’,7’-dichlorofluorescin diacetate. Lucigenin dependent chemiluminescence was used to measure extracellular ROS production. Additionally, the effect of NDS27 was tested on the activity of myeloperoxidase (MPO), a hemic enzyme contributing to the oxidant response of neutrophils. The activity of the released MPO by cytochalazine B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated PMNs was measured by SIEFED (“Specific Immunologic Extraction Followed by Enzymatic Detection”) (2). The HPLC results showed that NDS27 enters into PMNs and interacts with their membrane. NDS27 significantly and dose-dependently inhibited the ROS production in neutrophils without affecting their viability. Likewise, the activity of MPO released by PMNs was lowered by NDS27. Overall, our findings demonstrate that the membrane of neutrophils is permeable to NDS27 or interacts with the drug, suggesting that its inhibitory effect on ROS production is mainly associated to an intracellular effect probably by acting on the enzymes implied in respiratory burst like NADPH oxidase and MPO. The modulatory effect of NDS27 towards the oxidant activity of cells involved in immune and inflammatory response open therapeutic perspectives to control equine or human pathologies with excessive inflammatory reactions. 1. Neven et al. 2011, Patent Application Publication: US2011/0257126 A1 2. Serteyn et al. 2005, European Patent Specification : EP1711817 B1 [less ▲]Detailed reference viewed: 49 (4 ULg)
Systemisch-entzuendliche Reaktion beim Hochleistungssportpferd
Sandersen, Charlotte ; Lejeune, Jean-Philippe ; Votion, Dominique et al
Book published by Leipziger Blaue Hefte (2012)Detailed reference viewed: 32 (7 ULg)
Myeloperoxidase in Equine Semen: Concentration and Localization during Freezing Processing
Ponthier, Jérôme ; ; Franck, Thierry et al
in Journal of Equine Veterinary Science (2012), (32), 32-37
Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutro- phils during degranulation or after lysis. Post-thaw semen contains MPO, and concen- tration of this enzyme is associated ... [more ▼]
Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutro- phils during degranulation or after lysis. Post-thaw semen contains MPO, and concen- tration of this enzyme is associated with decreased motility. The aim of this study was to determine kinetics of MPO concentration during freezing, its origin, and its impact on frozenethawed semen. Forty ejaculates were used. Semen was frozen using the classical freezing procedure. MPO concentrations were assayed in fresh semen, after centrifuga- tion, and after cooling down to 4 C. Post-thaw MPO assay results and spermogram characteristics were determined. MPO immunocytochemistry was performed on 4 different ejaculates at each step of freezing procedure. MPO concentration increased after cooling down to 4 C and thawing compared with fresh semen. As temperature decreased, MPO was higher or tended to be higher in post-thaw poor quality samples. Nonsperm cells showed various degrees of MPO immunostaining and were observed as epithelial cells with nuclear pyknosis and keratinization. MPO immunostaining increased in medium and decreased in nonsperm cells during freezing. Our study shows that MPO concentration in equine semen increases when temperature decreases. We hypothesize that nonsperm cells present in fresh semen could release MPO. [less ▲]Detailed reference viewed: 62 (16 ULg)
Assessment of reactive oxygen species production in cultured equine skeletal myoblasts in response to conditions of anoxia followed by reoxygenation with or without exposure to peroxidases.
Ceusters, Justine ; Mouithys-Mickalad, Ange ; de la Rebière de Pouyade, Geoffroy et al
in American Journal of Veterinary Research (2012), 73(3), 426-434
Objective—To culture equine myoblasts from muscle microbiopsy specimens, examine myoblast production of reactive oxygen species (ROS) in conditions of anoxia followed by reoxygenation, and assess the ... [more ▼]
Objective—To culture equine myoblasts from muscle microbiopsy specimens, examine myoblast production of reactive oxygen species (ROS) in conditions of anoxia followed by reoxygenation, and assess the effects of horseradish peroxidase (HRP) and myeloperoxidase (MPO) on ROS production. Animals—5 healthy horses (5 to 15 years old). Procedures—Equine skeletal myoblast cultures were derived from 1 or 2 microbiopsy specimens obtained from a triceps brachii muscle of each horse. Cultured myoblasts were exposed to conditions of anoxia followed by reoxygenation or to conditions of normoxia (control cells). Cell production of ROS in the presence or absence of HRP or MPO was assessed by use of a gas chromatography method, after which cells were treated with a 3,3′-diaminobenzidine chromogen solution to detect peroxidase binding. Results—Equine skeletal myoblasts were successfully cultured from microbiopsy specimens. In response to anoxia and reoxygenation, ROS production of myoblasts increased by 71%, compared with that of control cells. When experiments were performed in the presence of HRP or MPO, ROS production in myoblasts exposed to anoxia and reoxygenation was increased by 228% and 183%, respectively, compared with findings for control cells. Chromogen reaction revealed a close adherence of peroxidases to cells, even after several washes. Conclusions and Clinical Relevance—Results indicated that equine skeletal myoblast cultures can be generated from muscle microbiopsy specimens. Anoxia-reoxygenation– treated myoblasts produced ROS, and production was enhanced in the presence of peroxidases. This experimental model could be used to study the damaging effect of exercise on muscles in athletic horses. [less ▲]Detailed reference viewed: 61 (28 ULg)
Relationship between exercise-induced systemic inflammatory like reaction and racing performance in endurance horses
Serteyn, Didier ; Caudron, Isabelle ; Lejeune, Jean-Philippe et al
in Comparative Exercise Physiology (2012), 8(3/4), 213218
This study showed that systemic inflammatory like reaction is not clearly related to performance but also to horse-related factors such as intinsic capacity or training.Detailed reference viewed: 30 (10 ULg)