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See detailAre COX-2 Inhibitors Active on Intracellular Oxidative Processes? A Study on In Vitro and Cellular Models
Mouithys-Mickalad, Ange ULg; Deby-Dupont, Ginette; Deby, Carol et al

Book published by Nova Science Publishers, Inc. (2006)

In the last years, there has been an increasing interest of using cyclooxygenase-2 (COX-2) inhibitors to treat the inflammatory pain and chronic inflammatory diseases such as osteoarthritis and rheumatoid ... [more ▼]

In the last years, there has been an increasing interest of using cyclooxygenase-2 (COX-2) inhibitors to treat the inflammatory pain and chronic inflammatory diseases such as osteoarthritis and rheumatoid arthritis. The beneficial effects were to avoid the secondary adverse effects such as bleeding and gastric irritation, generally observed with aspirin and conventional NSAIDs. COX-1 is constitutively expressed in most tissues and involved in the regulation of normal homeostatic functions, while COX-2 is not detected in most tissues but induced by inflammatory stimuli. These outcomes motivated the commercial development of selective COX-2 inhibitors. Recent data suggested that the COX-2 enzyme can be expressed within atherosclerotic lesions and could play a crucial role in various types of cancers, by the way of its activity on the ROS production, gene transcription and prostaglandin (PGE2) production. Consequently, the COX-2 enzyme has become a real target for the study of various classes of compounds and specially the possible additional properties of COX-2 inhibitors. We and other groups have already investigated the pro or antioxidant profile of conventional NSAIDs and some COX-2 inhibitors. With the recent withdrawal of two compounds of the coxib’s family (rofecoxib and celecoxib), for adverse cardiovascular events, concerns regarding the safety of all COX-2 inhibitors have been raised. To answer to these concerns, different approaches were developed by studying on in vitro models, the potential inhibiting-or-stimulating activities on oxidative phenomena of new drugs with already recognized therapeutic effects. Preliminary data obtained with COX-2 inhibitors showed a moderate inhibiting effect on the intracellular oxidant processes and others a stimulating activity. New hypotheses for the treatment of inflammation are now suggested for compounds like nimesulide and its analogous, which are selective towards COX-2 with little activity on COX-1. Here, we reported the in vitro effects of some Cox-2 inhibitors, in comparison with traditional drugs (ibuprofen, diclofenac and aceclofenac) by using two cellular models: a human lung type II alveolar cell line (A549) and a human promonocyte cell line (THP-1). The direct interactions between the drugs and ROS were also investigated in cell-free systems. [less ▲]

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See detailMethod and kit for the measurement of neutrophil cell activation
Serteyn, Didier ULg; Deby-Dupont, Ginette; Franck, Thierry ULg et al

Patent (2006)

The present invention is related to methods and kits (or devices) for the measurement of equine myeloperoxidase (MPO), a specific enzyme of equine neutrophils, either in total [first method], or ... [more ▼]

The present invention is related to methods and kits (or devices) for the measurement of equine myeloperoxidase (MPO), a specific enzyme of equine neutrophils, either in total [first method], or specifically in its active form [second method]. Said methods and kits (or devices), used independently or in combination, find improved applications in the veterinary field and can be adapted for application in human health care. The concept of the second method is applicable to any other enzyme. [less ▲]

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See detailA specific method for measurement of equine active myeloperoxidase in biological samples and in in vitro tests
Franck, Thierry ULg; Kohnen, Stephan ULg; Deby-Dupont, Ginette et al

in Journal of Veterinary Diagnostic Investigation (2006), 18(4), 326-334

An original method called SIEFED (specific immunological extraction followed by enzymatic detection) was developed for the specific detection of the activity of equine myeloperoxidase (MPO). The method ... [more ▼]

An original method called SIEFED (specific immunological extraction followed by enzymatic detection) was developed for the specific detection of the activity of equine myeloperoxidase (MPO). The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by washing (to eliminate proteins and interfering molecules) and measurement of MPO activity using a detection system containing a fluorogenic substrate, hydrogen peroxide, and nitrite as reaction enhancer. The SIEFED technique was applied to study active MPO in horse biological fluids and the effects of 2 polyphenolic molecules, curcumin and resveratrol, on MPO activity. The detection limit of the SIEFED was 0.23 mU/ml. The SIEFED exhibited good precision with intra-assay and interassay coefficients of variation below 10% and 20%, respectively, for MPO activities ranging from 0.25 to 6.4 mU/ml. The activity of MPO was generally higher than 1 mU/ml in the fluids collected from horses with inflammatory diseases. Curcumin and resveratrol exerted a dose-dependent inhibition on MPO activity and, as they were removed before the enzymatic detection of MPO, the results suggest a direct drug-nzyme interaction or an enzyme structure modification by the drug. The SIEFED is a new tool that would be useful for specific detection of active MPO in complex media and for selection of MPO activity modulators. [less ▲]

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See detailPlasma concentrations of myeloperoxidase in endurance and 3-day event horses after a competition
Art, Tatiana ULg; Franck, Thierry ULg; Gangl, M. et al

in Equine Veterinary Journal. Supplement (2006), 36

REASON FOR PERFORMING STUDY: In man, exercise of any type has been shown to induce neutrophil degranulation and respiratory burst activity, as well as an increase in plasma myeloperoxidase (MPO), a ... [more ▼]

REASON FOR PERFORMING STUDY: In man, exercise of any type has been shown to induce neutrophil degranulation and respiratory burst activity, as well as an increase in plasma myeloperoxidase (MPO), a specific enzyme of neutrophil azurophilic granules with a strong oxidative activity. Until now, it is not known whether this is the same in horses. OBJECTIVES: To study whether degranulation of blood neutrophils may be induced by exercise by measuring the total concentration of MPO and assess the possible influence of type of competition on this exercise-induced adjustment. METHODS: Blood was sampled before, and 30 min after, the course, in 9 ponies performing the Phase D of a national 3-day event championship (CIC*), and in 7 endurance horses participating at the European endurance championship 2005. White blood cells and granulocytes, total plasma proteins, creatine phosphokinase (CK), and total MPO contents were determined from blood samples. In addition, blood was taken from all ponies and 4 of the endurance horses 5 min after completion of the course to give some idea of the intensity of exercise. RESULTS: The mean blood lactate was 15.8 +/- 5.8 mmol/l after the CIC* and 2.7 +/- 0.2 mmol/l after the 160 km course. Performing both competitions induced a significant increase in CK and MPO. After the endurance course, the number of granulocytes significantly increased. Whilst there was no significant correlation between the measurements in CIC* ponies, MPO was significantly correlated with granulocyte count (r2 = 0.776) and CK (r2 = 0.586) in endurance horses. CONCLUSIONS: Intense exercise induces an activation of blood granulocytes, with degranulation of neutrophils and release of MPO. The plasmatic MPO concentration after endurance was higher than the values reported in some inflammatory pathological conditions. POTENTIAL RELEVANCE: This phenomenon may partly contribute to the occurrence of an exercise-induced oxidative stress and to the alteration of muscular membrane permeability. Further studies should be conducted to assess the possible relationship between MPO concentration and markers of oxidative stress in performance horses [less ▲]

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See detailMyeloperoxidase concentration in bronchoalveolar lavage fluid from healthy horses and those with recurrent airway obstruction
Art, Tatiana ULg; Franck, Thierry ULg; Lekeux, Pierre ULg et al

in Canadian Journal of Veterinary Research = Revue Canadienne de Recherche Vétérinaire (2006), 70(4), 291-296

The aim of this work was to measure the myeloperoxidase (MPO) concentration in bronchoalveolar lavage (BAL) fluid collected from horses with recurrent airway obstruction (RAO), both in crisis and in ... [more ▼]

The aim of this work was to measure the myeloperoxidase (MPO) concentration in bronchoalveolar lavage (BAL) fluid collected from horses with recurrent airway obstruction (RAO), both in crisis and in remission, as well as from healthy horses. Seven horses with RAO were exposed to moldy hay until the maximum change in pleural pressure was greater than 1.5 kPa. At that point, BAL was performed, and the total cell counts and percentages in the fluid were immediately determined. To measure the MPO concentration in BAL-fluid supernatant, we used a specific enzyme-linked immunosorbent assay with polyclonal antibodies against equine MPO. The tests were repeated on the horses with RAO after they had spent 2 mo on pasture. Six healthy horses serving as controls underwent the same tests. The absolute and relative neutrophil counts and the MPO concentration in the BAL fluid were significantly greater in the horses with an RAO crisis than in the control horses. After 2 mo on pasture, the horses that had been in RAO crisis were clinically normal, and their neutrophil counts and MPO levels in BAL fluid had significantly decreased; during remission their neutrophil counts were not significantly different from those in the healthy horses, but their MPO concentration remained significantly higher. This study showed that determining the MPO concentration in a horse's BAL fluid is technically possible and that during remission from RAO the concentration remains higher than normal. Thus, MPO may be a marker of neutrophil presence and activation in the lower airways. [less ▲]

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See detailEffect of cross country on plasma levels of myeloperoxidase in saddle ponies
Art, Tatiana ULg; Franck, Thierry ULg; Gangl, M. et al

in Abstracts book of iceep (2006)

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See detailDevelopment of an enzyme-linked immunosorbent assay for specific equine neutrophil myeloperoxidase measurement in blood
Franck, Thierry ULg; Grulke, Sigrid ULg; Deby-Dupont, Ginette et al

in Journal of Veterinary Diagnostic Investigation (2005), 17(5), 412-419

Equine inflammatory disease is accompanied by a neutrophil activation resulting in the release of granulocytic enzyme myeloperoxidase (MPO). To measure MPO in horse plasma as marker of neutrophil ... [more ▼]

Equine inflammatory disease is accompanied by a neutrophil activation resulting in the release of granulocytic enzyme myeloperoxidase (MPO). To measure MPO in horse plasma as marker of neutrophil activation, the authors purified equine neutrophil MPO and developed a specific enzyme immunoassay using 2 specific polyclonal antibodies obtained from rabbit (primary antibody) and guinea pig (secondary antibody). The sandwich complex "primary antibody-MPO-secondary antibody" was detected using a goat anti-guinea pig immunoglobulin antibody conjugated to alkaline phosphatase. The enzyme-linked immunosorbent assay (ELISA) showed good precision and accuracy, with intra- and interassay coefficients of variation below 10% for MPO concentrations ranging from 0.78 to 50 ng/ml. A stable plasma MPO value, unaffected by time elapsed between blood collection and centrifugation, was obtained with plasma from EDTA anticoagulated blood. The mean MPO value measured in 38 healthy horses was 181.80 +/- 64.74 ng/ml. In 20 horses suffering from obstruction of the large or small intestine, MPO concentrations measured at the time of arrival at the intensive care unit were significantly higher than mean normal value, ranging from 477.88 to 2,748.13 ng/ml. Work is in progress to apply this MPO ELISA technique to other biological fluids and other equine diseases. [less ▲]

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See detailResveratrol and curcumin reduce the respiratory burst of Chlamydia-primed THP-1 cells
Deby-Dupont, Ginette; Mouithys-Mickalad, Ange ULg; Serteyn, Didier ULg et al

in Biochemical and Biophysical Research Communications (2005), 333(1), 21-27

The intracellular bacterium Chlamydia pneumoniae is involved in the inflammation process of atherosclerosis. We previously demonstrated that C. pneumonia infected monocytes (THP-1 cells) responded to ... [more ▼]

The intracellular bacterium Chlamydia pneumoniae is involved in the inflammation process of atherosclerosis. We previously demonstrated that C. pneumonia infected monocytes (THP-1 cells) responded to stimulation by an increased respiratory burst linked to an increased NADPH oxidase (NOX) activity. We now tested agents acting on the assembly of the NOX subunits or on protein kinase C, a trigger of NOX activity. Apocynin, resveratrol, rutin, quercetin, curcumin, and tocopherols were tested. The cells were pre-incubated with Chlamydia and the agent for 19 h, and then stimulated with phorbol myristate acetate. The NOX activity was monitored by measuring the hydrogen peroxide production. Resveratrol and curcumin (10(-4)-10(-6) M) were better inhibitors than apocynin. alpha-Tocopherol was inactive, and gamma-tocopherol inhibitor at 10(-4) M only. Quercetin was inactive, and rutin a moderate but significant inhibitor. The inhibition by resveratrol was increased by 10(-6) M rutin or quercetin. Resveratrol and curcumin thus appeared to be interesting for atherosclerosis treatment. [less ▲]

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See detailEarly release of neutrophil markers of activation after direct stenting in patients with unstable angina
Gach, Olivier ULg; Biemar, Christian; Nys, Monique ULg et al

in Coronary Artery Disease (2005), 16(1), 59-65

Objective To assess polymorphonuclear neutrophils activation after stenting in acute coronary syndromes studied by myeloperoxydase, lactoferrin and elastase release in this clinical setting. Methods ... [more ▼]

Objective To assess polymorphonuclear neutrophils activation after stenting in acute coronary syndromes studied by myeloperoxydase, lactoferrin and elastase release in this clinical setting. Methods Myeloperoxydase, lactoferrin, elastase, C-reactive protein and cytokines serum levels were assessed in 20 patients undergoing catheterization for unstable angina. Serial sampling starting before arteriography and continued up to 24 h was carried out in 15 patients undergoing direct stenting (group A) and in five patients assessed by coronary angiography only (group B). Results Myeloperoxydase, lactoferrin and elastase levels remained unchanged following catheterization, whereas a significant increase in myeloperoxydase (P=0.0009) and lactoferrin (P=0.004) was observed after stenting. No change in levels of tumour necrosis factor alpha, interleukin (IL)-8 and IL-12 was found in group B after catheterization at the different sampling times, although IL-8 and IL-12 levels increased transiently following stenting. IL-6 values increased in both groups. Baseline values of C-reactive protein were similar in each group. A progressive increase in C-reactive protein was noted in both groups and appeared to be larger following stenting (group A: P=0.0002; group B: P=0.01). Conclusions In patients with unstable angina, stenting is associated by immediate neutrophil activation followed by release of inflammatory cytokines (IL-6, IL-8, IL-12) and C-reactive protein elevation. This study points out a potential role of myeloperoxydase as a trigger for inflammatory reaction in patients with unstable coronary syndromes undergoing percutaneous coronary intervention. (C) 2005 Lippincott Williams WillZins. [less ▲]

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See detailSYNOVIOCYTES BUT NOT ARTICULAR CHONDROCYTES RELEASE FREE RADICALS AFTER REPETITIVE CYCLES OF ANOXIA/REOXYGENATION: AN ELECTRON SPIN RESONANCE STUDY.
Schneider, Nicole ULg; Lejeune, Jean-Philippe ULg; Mouithys-Mickalad, Ange ULg et al

Poster (2005)

Objective : We investigated if short recurrent periods of anoxia/reoxygenation (A/R), mimicking the in vivo conditions of hypoxia, could stimulate the production of free radicals by synoviocytes and ... [more ▼]

Objective : We investigated if short recurrent periods of anoxia/reoxygenation (A/R), mimicking the in vivo conditions of hypoxia, could stimulate the production of free radicals by synoviocytes and chondrocytes. Material and Methods : Synoviocytes (immortalised rabbit line HIG-82 and equine synoviocytes isolated from synovial membranes of the stifle joint) and chondrocytes (isolated from the same equine joint) were cultured for 48 hours. The respiration rate of the cells (107 cells/assay) was studied by oxymetry and the free radical production monitored by electron spin resonance (ESR) in the presence of the spin trap (-[-4-pyridyl 1-oxide]-N-tert butyl nitrone)/ethanol mixture (2%v/v). The A/R consisted of three periods of 20 minutes anoxia, each anoxia period being followed by re-oxygenation. Oxygen consumption by the cells was measured before starting the anoxia/reoxygenation cycles and after each reoxygenation. At the end of the last A/R period, the cells were transferred into the ESR flat cell and in the cavity, and the free radical formation was monitored. Results : The equine chondrocytes showed a low respiration rate, weakly affected by A/R and no production of free radicals (n=3). Synoviocytes showed a higher respiration rate (at least 20 times higher) which was affected by recurrent A/R (decrease of the slope of oxygen consumption), and a free radical production as evidenced by the appearance of the 4-POBN/EtOH adducts (n=6). Conclusions :These observations suggest that synoviocytes, but not chondrocytes, responded to repeated anoxia-reoxygenation conditions by the production of free radicals. Synoviocytes could thus be responsible for the production of free radicals in the joints, what could be an important factor in the onset of osteoarthritis. [less ▲]

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See detailHyperhydricity of Prunus avium shoots cultured on gelrite: a controlled stress response
Franck, Thierry ULg; Kevers, Claire ULg; Gaspar, Thomas ULg et al

in Plant Physiology & Biochemistry (2004), 42(6), 519-527

Hyperhydricity is a physiological disorder frequently affecting shoots vegetatively propagated in vitro. Hyperhydric shoots are characterised by a translucent aspect due to a chlorophyll deficiency, a not ... [more ▼]

Hyperhydricity is a physiological disorder frequently affecting shoots vegetatively propagated in vitro. Hyperhydric shoots are characterised by a translucent aspect due to a chlorophyll deficiency, a not very developed cell wall and a high water content. Hyperhydricity of Prunus avium shoots was expressed in vitro in one multiplication cycle by replacing the gelling agent agar (normal shoots: NS) by gelrite (hyperhydric shoots: HS). P. avium shoots evolving towards the hyperhydric state produced higher amounts of ethylene, polyamines (PAs) and proline, which are substances considered as stress markers. A higher activity of glutathione peroxidase (GPX; EC 1.11.1.9), involved in organic hydroperoxide elimination, suggested an increased production of these compounds in HS. The unchanged free fatty acid composition indicated no HS membrane damages compared to NS. The ploidy level of HS nuclei was not affected, but the bigger size and the lower percentage of nuclei during the S phase suggested a slowing down of the cell cycle. The results argued for a stress response of the HS, but no signs of oxidative damages of lipid membrane and nucleus were observed. The discussion points out paradoxical results in a classical analysis of stress and suggests an alternative way of defense mechanisms in HS, involving homeostatic regulation and controlled degradation processes to maintain integrity and vital functions of the cell. (C) 2004 Elsevier SAS. All rights reserved. [less ▲]

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See detailViability of equine articular chondrocytes in alginate beads exposed to different oxygen tensions.
Schneider, Nicole ULg; Lejeune, Jean-Philippe ULg; Deby, Carol et al

in Veterinary Journal (2004), 168

Ischaemia and reperfusion are suspected to alter chondrocyte metabolism. Here, we studied the effects of three oxygen (O2) tensions on the viability of equine articular chondrocytes isolated from the ... [more ▼]

Ischaemia and reperfusion are suspected to alter chondrocyte metabolism. Here, we studied the effects of three oxygen (O2) tensions on the viability of equine articular chondrocytes isolated from the cartilage of the distal interphalangeal joint of horses. Chondrocytes were cultured in alginate beads under 1%, 5% or 21% gas phase O2 concentration for 14 days, cellular growth kinetics were measured (n=6), and the cells were observed by light microscopy after staining for necrotic and apoptotic cell detection. For information about the metabolic status, the intracellular adenosine triphosphate (ATP) content was measured. The number of chondrocytes remained stable for the first eight days, then decreased especially at 1% and 21% O2. At 21% O2, normal cells decreased and necrotic cells increased at the end of the 14 day-period. No significant variations were found at 5% O2 except for a decrease in necrotic cells at day 14. Most apoptotic cells were found at 1% O2 from days 5 to 11, and normal cells decreased during the same period. But an unexpected increase in normal cells and decrease in apoptotic cells were observed at day 14. The intracellular ATP content remained stable. It was concluded that, in a three-dimensional culture model of equine articular chondrocytes, O2 tension affected the viability of the cells after an 11-day period, with the most important effects observed at 21% and 1% O2 conditions. [less ▲]

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See detailInfluence of oxygen tension on nitric oxide and prostaglandin E2 production by bovine chondrocytes
Burton, Sandrine; Mathy, Marianne ULg; Deby-Dupont, Ginette et al

in Osteoarthritis and Cartilage (2003, October), 11(Suppl.1), 58

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See detailBronchoalveolar lavage fluids of ventilated patients with acute lung injury activate NF-kappa B in alveolar epithelial cell line: role of reactive oxygen/nitrogen species and cytokines
Nys, Monique ULg; Deby-Dupont, Ginette; Habraken, Yvette ULg et al

in Nitric Oxide (2003), 9(1), 33-43

In human alveolar epithelial cell line, we investigated the binding activity of NF-kappaB induced by the bronchoalveolar lavage fluids (BALs) from ventilated patients with acute lung injury (ALI), in ... [more ▼]

In human alveolar epithelial cell line, we investigated the binding activity of NF-kappaB induced by the bronchoalveolar lavage fluids (BALs) from ventilated patients with acute lung injury (ALI), in correlation with the concentrations of inflammatory cytokines, RNOS, and the severity of the ALI. In BALs obtained in 67 patients (16 bronchopneumonia, 14 infected ARDS, 20 ARDS, and 17 ALI patients without bronchopneumonia and no ARDS), we measured endotoxin, IL-1beta, IL-8, and nitrated proteins (NTP), the activity of mycloperoxidase, and the capacity to activate the NF-kappaB in alveolar A549 cells by electrophoretic mobility shift and supershift assays. The neutrophil counts and mean IL-1beta, IL-8, myeloperoxidase, and NTP values were increased in bronchopneumonia and infected ARDS groups compared to ARDS and ALI without bronchopneumonia and no ARDS groups (P < 0.001). The number of neutrophils was correlated to those of IL-1beta, IL-8, myeloperoxidase, NTP, and endotoxin in all groups (P < 0.0001). NF-kappaB activity was induced in alveolar like cells by BALs in all groups, was higher in bronchopneumonia and infected ARDS groups (P < 0.02), and was correlated to IL-1beta (P = 0.0002), IL-8 (P = 0.02), NTP (P = 0.014), myeloperoxidase (P = 0.016), and neutrophil counts (P = 0.003). BALs of bronchopneumonia and infected ARDS patients had increased inflammatory mediators (compared to ARDS and ALI without bronchopneumonia and no ARDS patients) that correlated to neutrophil counts and to the NF-kappaB-binding activity. These mediators and NF-kappaB activation may induce an amplification of inflammatory phenomena. By in vitro studies, we confirmed that NO-derived species (10(-6) to 10(-5) M peroxynitrite and 10(-5) M nitrites) and myeloperoxidase (at concentration equivalent to that found in BALs) can participate in the NF-kappaB activation. (C) 2003 Elsevier Inc. All rights reserved. [less ▲]

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See detailEFFECTS OF O2 TENSION AND GLUCOSE CONCENTRATION ON THE CELLULAR RESPIRATION OF EQUINE ARTICULAR CHONDROCYTES IN CULTURE.
Schneider, Nicole ULg; Lejeune, Jean-Philippe ULg; Duyckaerts, Claire ULg et al

Poster (2003)

In vivo, articular chondrocytes are exposed to 5 to 10% O2. Chondrocytes are also suspected to produce reactive oxygen species when submitted to anoxia/re-oxygenation. We were interested to study the ... [more ▼]

In vivo, articular chondrocytes are exposed to 5 to 10% O2. Chondrocytes are also suspected to produce reactive oxygen species when submitted to anoxia/re-oxygenation. We were interested to study the effects of O2 and glucose variations on cellular respiration, glucose consumption and lactate production. Equine articular chondrocytes were cultured in suspension for 2 days under 5 or 21 % O2 in the gaseous phase, and with 0, 1.0 or 4.5 g/L glucose. The viable cells were then counted and the respiration rate (O2 consumption) of 10.106 cells was monitored by oxymetry for 2 hours; after oxymetry, glucose and lactate were measured in the medium (enzymatic assays). After 2 days, the cell viability was the best at 5% O2 and 1g/L glucose; it decreased at 4.5 g/L glucose and was the worst at 0g/L glucose, for the two O2 tensions (n=3). There was no obvious difference of the respiration rate between cells cultured at 5 and 21% O2, but respiration of chondrocytes was surprisingly low. When cells were submitted to 20 min anoxia at 0% O2, the O2 consumption was doubled at re-oxygenation for cells previously cultured at 21% O2. Glucose and lactate values found in the medium after oxymetry: lactate release in medium was similar (36.23 and 34.57 mg/L respectively) for cells cultured with 1g glucose and 5 or 21% O2 conditions; lactate values were low (2.03 and 8,63 mg/L respectively) for 4.5 g glucose and 5 or 21% O2. Glucose uptake was not different whatever the culture conditions. These results indicate a low cellular respiration with a lactate production linked to the glucose concentrationin the medium, and raise the question of the capacity of chondrocytes to produce ROS in vivo starting from the mitochondrial chain. [less ▲]

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See detailEffects of O2 tension and glucose concentration on the cellular respiration of equine articular chondrocytes in culture.
Schneider, Nicole ULg; Lejeune, Jean-Philippe ULg; Mouithys-Mickalad, Ange ULg et al

Poster (2003)

In vivo, articular chondrocytes are exposed to 5 to 10% O21,2. Chondrocytes are also suspected to produce reactive oxygen species when submitted to anoxia/re-oxygenation3,4. We were interested to study ... [more ▼]

In vivo, articular chondrocytes are exposed to 5 to 10% O21,2. Chondrocytes are also suspected to produce reactive oxygen species when submitted to anoxia/re-oxygenation3,4. We were interested to study the effects of O2 and glucose variations on cellular respiration, glucose consumption and lactate production. Equine articular chondrocytes were cultured in suspension for 2 days under 5 or 21 % O2 in the gaseous phase, and with 0, 1.0 or 4.5 g/L glucose. The viable cells were then counted and the respiration rate (O2 consumption) of 10.106 cells was monitored by oxymetry for 2 hours; after oxymetry, glucose and lactate were measured in the medium (enzymatic assays). After 2 days, the cell viability was the best at 5% O2 and 1g/L glucose; it decreased at 4.5 g/L glucose and was the worst at 0g/L glucose, for the two O2 tensions (n=3). There was no obvious difference of the respiration rate between cells cultured at 5 and 21% O2, but respiration of chondrocytes was surprisingly low. When cells were submitted to 20 min anoxia at 0% O2, the O2 consumption was doubled at re-oxygenation for cells previously cultured at 21% O2. Glucose and lactate values found in the medium after oxymetry : lactate release in medium was similar (36.23 and 34.57 mg/L respectively) for cells cultured with 1g glucose and 5 or 21% O2 conditions; lactate values were low (2.03 and 8,63 mg/L respectively) for 4.5 g glucose and 5 or 21% O2. Glucose uptake was not different whatever the culture conditions. These results indicate a low cellular respiration with a lactate production linked to the glucose concentration in the medium, and raise the question of the capacity of chondrocytes to produce ROS in vivo starting from the mitochondrial chain. [less ▲]

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See detailOxidant activity of rabbit synoviocytes (HIG-82) demonstrated by oxymetry and ethylene production.
Schneider, Nicole ULg; Lejeune, Jean-Philippe ULg; Mouithys-Mickalad, Ange ULg et al

Poster (2003)

We are interested in a possible role of synoviocytes in the ROS production implicated in osteoarthritis, therefore we studied the response of a rabbit synoviocyte cell line (HIG-82) to variable oxygen ... [more ▼]

We are interested in a possible role of synoviocytes in the ROS production implicated in osteoarthritis, therefore we studied the response of a rabbit synoviocyte cell line (HIG-82) to variable oxygen tensions and the oxidant activity of these cells in response to stimuli. Synoviocytes were cultured at 5 and 21 % O2, their O2 consumption (cellular respiration, monitored with Clark electrode) was measured at 21% O2 and after anoxia, before and after stimulation with phorbol myristate acetate (PMA), and their oxidant response to PMA stimulation was quantified by measuring ethylene (gas chromatography) released when the substrate, alpha-keto-gamma-methylbutyric acid, is oxidised by the ROS produced by the cells. Cell growth was faster at 21 % O2 than at 5% O2, and microscopic observation revealed 2 cell populations: a few small round cells in suspension and many adherent cells. By oxymetry, we observed that a 106 synoviocytes suspension in 2 ml completely consumed O2 within 15 min, that anoxia (7 min) slightly slowed the respiration rate down and that PMA stimulation increased O2 consumption (150 % increase). The oxidant activity (ethylene production) of the cells was stimulated by PMA in a dose-dependent manner (10-9 to 10-7M) but the cell response was highly variable (from 150 to 1500 % increase) and was largely reduced by diphenyliodonium, an inhibitor of NADPH-oxidase and NO-synthase. The capacity to produce free radical species was confirmed for the small round cells by detection of an electron paramagnetic resonance (EPR) signal after stimulation. These results thus demonstrate a sensibility to O2 and an oxidant activity of synoviocytes at least related to ROS production by NADPH-oxidase activity. [less ▲]

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See detailOxidative Processes in Human Promonocytic Cells (Thp-1) after Differentiation into Macrophages by Incubation with Chlamydia Pneumoniae Extracts
Mouithys-Mickalad, Ange ULg; Deby-Dupont, Ginette; Nys, Monique ULg et al

in Biochemical and Biophysical Research Communications (2001), 287(3), 781-8

Human monocytes differentiated into macrophages by Chlamydia pneumoniae were able to oxidize blood lipoproteins, as discovered by Kalayoglu et al. (1998). Using a model of human promonocytic cells (THP-1 ... [more ▼]

Human monocytes differentiated into macrophages by Chlamydia pneumoniae were able to oxidize blood lipoproteins, as discovered by Kalayoglu et al. (1998). Using a model of human promonocytic cells (THP-1), the cells were differentiated into macrophages by preincubation with C. pneumoniae extract, and further stimulated by phorbol myristate acetate. In these conditions, the differentiated cells oxidized a thiol compound and released superoxide anion as demonstrated respectively by gas liquid chromatography and electron spin resonance. The thiol oxidation and superoxide anion release were inhibited by diphenyliodonium, a NADPH oxidase and NOsynthase inhibitor, proving that the respiratory burst and the NOsynthase were involved in the oxidation processes occurring in the differentiated THP-1. The role of H(2)O(2) (derived from superoxide anion) was indicated by the enhancing effect of a peroxidase on the thiol oxidation. The presence of alpha-tocopherol in the surrounding medium strongly diminished the oxidation of the thiol target. [less ▲]

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See detailPotential Antioxidant properties of Aceclofenac and its Metabolites: Investigation on an in vitro model
Mouithys-Mickalad, Ange ULg; Mathy, Marianne ULg; Deby, Carol et al

Poster (2001, June 22)

Introduction: Recent studies have shown that some steroidal anti-inflammatory drugs (NSAIDs) could exert their actions by multifactorial processes. Among them, the potential antioxidant activity of ... [more ▼]

Introduction: Recent studies have shown that some steroidal anti-inflammatory drugs (NSAIDs) could exert their actions by multifactorial processes. Among them, the potential antioxidant activity of certain NSAIDs towards various reactive oxygen species (ROS) is often suggested and could have pharmacological relevance. Objective: This study was designed to assess the potential antioxidant properties (IC50 values) of aceclofenac and its metabolites (4’OH-aceclofenanc and diclofenac) on three different systems of ROS production, using chemiluminescence (CL) technique with luminal and electron spin resonance (ESR) spin trapping. Material and Methods: Isolated human PMNs (1x106 cells) were activated with 5x10-7 M PMA in the presence of luminal (CL assays) with or without drug addition. For spin trapping experiments, 100 mM DMPO, a radical trapping agent, was added to the reaction milieu containing 6x106 cells/ml. For free-cell experiments, the Fenton’s reagent was used for generation of ·OH and xanthine/xanthine-oxidase system for O2-radicals. The NaOCl-induced CL, amplified by luminal, was used to test the drug effects on HOCl. Results: On the model of PMA-activated PMNs, 4’OH-aceclofenac exhibited the best antioxidant profile (IC50 = 10 µM) while the effect of the parent drug was less pronounced (IC50 = 100 µM). Diclofenac did not inhibit CL response even at the high dose of 1 mM. Quite similar results were obtained on the NaOCl-induced chemiluminescence, where the efficacy of the drug was as follows: 4’HO-ACE (25 µM) > ACE (1 mM) > DICLO (no effect at 1 mM). By ESR technique, 4’HO-ACE also showed an inhibitory effect (501 µM) on the ROS production by PMA-activated PMN as well as on the ·OH production, while ACE (IC50 = 100 µM) was less efficient and DICLO (IC50 = 1 mM) without significant effect. These findings indicate that beside its anti-inflammatory effects, aceclofenac acts as an antioxidant, at least in part, by the way of its metabolite especially 4’HO-ACE. [less ▲]

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See detailKetoprofen and Phenylbutazone Attenuation of PAF-Induced Lung Inflammation in Calves
Van de Weerdt, ML; Coghe, Joost; Uystepruyst, Christophe et al

in Veterinary Journal (1999), 157(1), 39-49

The purposes of this study were: (1) to investigate which arachidonic acid metabolites contributed to platelet-activating factor (PAF) induced pulmonary dysfunction; and (2) to compare the effect of two ... [more ▼]

The purposes of this study were: (1) to investigate which arachidonic acid metabolites contributed to platelet-activating factor (PAF) induced pulmonary dysfunction; and (2) to compare the effect of two non-steroidal anti-inflammatory drugs, phenylbutazone and ketoprofen in a model of PAF-induced reversible lung inflammation in six calves. In placebo and phenylbutazone groups, PAF infusion induced significant dysfunctions in the pattern of breathing, mechanics of breathing and gas exchange. These dysfunctions were prevented by ketoprofen pretreatment, except for the mechanics of breathing which was moderately but significantly altered by the PAF challenge. In all calves, leukotriene (LT) B4 plasma concentrations did not significantly increase above baseline values at any time. Prostaglandin (PG) E2 plasma concentrations showed a minor significant increase in phenylbutazone pretreated calves (55.8 +/- 25.8 pg/mL from 36.7 +/- 16.13 pg/mL). Thromboxane (TX) B2 plasma concentration was significantly increased during PAF challenge in placebo- and phenylbutazone-pretreated groups, but not in ketoprofen-pretreated calves (1580.0 +/- 1370 from 42.7 +/- 10.7 pg/mL; 2340 +/- 477 from 63 +/- 32 pg/mL; and 36.5 +/- 4.12 from 39.3 +/- 12.0 pg/mL, respectively). These data suggest that TXA2 is an important cyclooxygenase metabolite of arachidonic acid produced in response to PAF and that ketoprofen (intramuscular injection, 3 mg/kg) is more effective than phenylbutazone (intramuscular injection, 10 mg/kg) in preventing respiratory dysfunctions induced by the PAF challenge 30 min after drug administration. Ketoprofen did not suppress totally the PAF-induced changes in mechanics of breathing, which suggests that PAF or a secondary release of mediators could have a direct action on airway smooth muscle. [less ▲]

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