References of "Deby, Carol"
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See detailThe unexpected hidden face of the Cephalosporin Antibiotic Ceftazidime: From biological to chemical and physical activities against oxidant species produced by phagocytes
Mouithys-Mickalad, Ange ULg; Deby-Dupont, Ginette; Mathy-Hartert, Marianne ULg et al

Conference (2008, October 03)

Background: Over several decades the use of antibiotics to treat infectious and bacterial diseases has been the main challenge. Ceftazidime (CAZ), belonging to the cephalosporin’s family, is known as ... [more ▼]

Background: Over several decades the use of antibiotics to treat infectious and bacterial diseases has been the main challenge. Ceftazidime (CAZ), belonging to the cephalosporin’s family, is known as empiric treatment for severe sepsis. Beside its antibiotic effect, CAZ has been shown to have other properties, making it unique. This study is aimed to investigate unexpected antioxidant properties of CAZ with special emphasis on the mechanism of action. Methods: Four in vitro and ex-vivo experimental models were designed 1) Assay of proteinases inhibitory activity of α2-macroglobulin (α2M) in the presence of trypsine (1.4µg) with or without 10-3 M CAZ, in 0.1 M Tris-HCl at pH 8.1. 2) Assessment of MPO-induced toxicity on endothelial cells or oxidant activities of stimulated phagocytes (PMNs) using 160 µM ferricytochrome C. 3) Effect of CAZ on two models of anoxia/reoxygenation from adherent and suspension alveolar cells (A549, 106 to 1010 cells/ml) using oxymetry coupled to EPR-spin trapping technique. 4) Cell-free systems to investigate: lipoperoxidation of linoleate induced by γ-irradiation, Fe2+/ascorbate system or ferryl species; Quenching of hydroxyl radical (·OH) and singlet oxygen (1O2)-scavenging activity from Mallet’s (H2O2/NaOCl) and Fenton’ reactions. Results: Ex-vivo assays show efficient protective effect of CAZ on plasmatic antiproteinases against oxidative stress; a dose-dependent inhibitory capacity on endothelial and A549 cells against MPO toxicity or excessive production of ROS during anoxia/reoxygenation. On cell-free systems, CAZ had unique 1O2–scavenging activity; and less effect on ferryl species. CAZ exerts its antioxidant effect by chelating activity. Conclusions: Overall results indicate that: 1) CAZ protects endothelial cells against MPO toxicity but also A549 cells towards the effect of anoxia/reoxygenation; 2) CAZ protects α2M and 3) acts as efficient inhibitor of lipid peroxidation of linoleate and hydroxyl radical and as singlet oxygen-scavengers. Antioxidant properties of CAZ might be relevant in clinical situations where excessive activation of leukocytes is known and in anoxia/reoxygenation model cause an increased production of ROS. [less ▲]

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See detailDiselenide Derivative, Potential Successor of Ebselen with High Antioxidant Activity: Assessment on in vitro Models
Mareque-Faez, Juan; Deby, Carol; Lamy, Maurice ULg et al

in Resource-Full Chemistry (2008, May 24)

Oxidative stress plays a key role in several pathophysiological events, including the attack of DNA, cell membrane damage and signaling pathways disruption. The harmful effect of oxidant stress has been ... [more ▼]

Oxidative stress plays a key role in several pathophysiological events, including the attack of DNA, cell membrane damage and signaling pathways disruption. The harmful effect of oxidant stress has been attributed to a high production of reactive oxygen species (ROS) followed by a depletion of antioxidant enzymes, glutathione peroxidase (GPx), a mammalian selenoenzyme which functions as a catalytic antioxidant, has been described to protect various organisms against oxidative stress. In the past two decades, the design of small weight molecules such as ebselen (PZ 51, 2-phenyl-1,2-benzoisoselenazol-3(2H)-one), has renewed the interest of synthetic analogues able to mimic the GPx activity. From four in vitro in vitro models, we previously showed that ebselen and some of its analogues (compounds 1, 2, 3 and 4) not only behaved at various degrees as GPx-like mimics but also as antioxidants especially for diselenide derivative (3). The present study deals with the antioxidant effect of diselenide derivative (3) versus ebselen on cellular model and the enzyme myeloperoxidase (MPO) activity using the in vitro systems. Derivative (3) has been chosen because of its interesting antioxidant profile in cell-free systems. [less ▲]

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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 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 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 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 detailEffects of glucocorticoids on the respiratory burst of Chlamydia-primed THP-1 cells.
Mouithys-Mickalad, Ange ULg; Deby, Ginette ULg; Mathy, Marianne ULg et al

in Biochemical and Biophysical Research Communications (2004), 318(4), 941-8

We previously observed that the respiratory burst of human monocytes (THP-1 cell line) triggered by phorbol myristate acetate was strongly enhanced by a priming of the cells by Chlamydia pneumoniae ... [more ▼]

We previously observed that the respiratory burst of human monocytes (THP-1 cell line) triggered by phorbol myristate acetate was strongly enhanced by a priming of the cells by Chlamydia pneumoniae [Biochem. Biophys. Res. Commun. 287 (2001) 781]. We describe here the modifications of the responses of Chlamydia-primed THP-1 cells to hydrocortisone (HCT) and methylprednisolone (MPL). HCT and MPL inhibited the production of the cytokines TNFalpha and IL-8. But HCT, which inhibited the respiratory burst in LPS-primed monocytes, paradoxically stimulated the phenomenon in Chlamydia-primed cells; MPL exerted no significant effect. Both glucocorticoids did not significantly modify the triggering effect of Chlamydia on NF-kappaB binding activity. On the expression of p22(phox), a protein subunit of the NADPH oxidase, HCT had an increasing and MPL a decreasing effect. Glucocorticoids thus had unexpected effects on the inflammatory response of Chlamydia-primed monocytes. [less ▲]

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See detailEquine trypsin: purification and development of a radio-immunoassay
Grulke, Sigrid ULg; Deby-Dupont, G.; Gangl, M. et al

in Veterinary Research (2003), 34(3, May-Jun), 317-330

Shock is accompanied by generalised splanchnic hypoperfusion, and splanchnic organs like the pancreas can be damaged, as shown in animal experimental models and in humans, by the presence of high plasma ... [more ▼]

Shock is accompanied by generalised splanchnic hypoperfusion, and splanchnic organs like the pancreas can be damaged, as shown in animal experimental models and in humans, by the presence of high plasma concentrations of trypsin and other pancreatic enzymes. In order to design a radioimmunoassay technique (RIA) for the measurement of equine trypsin-like immunoreactivity (TLI) in biological fluids, trypsin was purified (with purity greater than or equal to 96 %) from the equine pancreas by extraction in an acid medium, ammonium sulfate precipitations, gel filtration chromatography and, after activation of trypsinogen into trypsin, affinity chromatography. Gel polyacrylamide electrophoresis showed a monomeric enzyme with a molecular weight of 27 kDa. The purified equine trypsin served for the immunisation of rabbits in order to obtain a specific antiserum, and the labelled antigen was prepared by iodination of equine trypsin with I-125. The RIA was based on the binding of the antigen to the antibody followed by the separation of the antigen-antibody complex by immunoprecipitation in the presence of sheep anti-rabbit gammaglobulins and the assay of the radioactivity in the precipitate. The RIA showed good sensitivity, specificity, precision, accuracy and reproducibility. The reference mean value of TLI in the plasma of healthy horses (n = 20) was 30.01 +/- 6.84 ng/mL (upper confidence limit 50.52 ng/mL; p < 0.01). Three horses with non strangulating intestinal obstruction without shock showed TLI values within normal limits whereas 5 of 7 horses with strangulation obstruction showed TLI levels above the upper confidence limit. Further studies using the RIA and the enzymatic assay should be performed in order to confirm the role of the pancreas in equine intestinal obstruction. [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 detailHow hyperhydric shoots try to survive
Franck, Thierry ULg; Kevers, Claire ULg; Gaspar, Thomas ULg et al

in Free Radical Research (2003), 37(Suppl. 1), 74-74

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See detailPlasma trypsin level in horses suffering from acute intestinal obstruction
Grulke, Sigrid ULg; Gangl, M.; Deby, Ginette ULg et al

in Veterinary Journal (2002), 163(3), 283-291

Gastrointestinal disorders in horses leading to endotoxic shock could have further consequences on other splanchnic organs such as the pancreas, as can be seen in humans suffering from septic shock. In ... [more ▼]

Gastrointestinal disorders in horses leading to endotoxic shock could have further consequences on other splanchnic organs such as the pancreas, as can be seen in humans suffering from septic shock. In this study, the range of enzymatically active trypsin (EAT) in healthy horses was established and is similar to the range observed in healthy humans. EAT values were determined in horses with acute abdominal crises on admission as well as during anaesthesia and in the postoperative phase. A significant increase in plasma EAT was found in 59% of the horses with surgical colic when compared to our established reference range. Significantly higher values were found in severe shock cases. When separated in groups according to the duration of colic before referral, significantly higher EAT values were observed in the non-survivor group compared to the survivor group of colics of short duration. EAT plasma values increased significantly during the postoperative phase, and were significantly higher in small intestine obstructions than in large bowel disorders. In human medicine, hypovolaemic or septic shock patients show an increase in pancreatic proteases. Splanchnic hypoperfusion during shock could lead to pancreatic damage resulting in trypsin liberation into the peritoneal space and an increase in plasma levels. Trypsin is able to activate inflammatory cascades and leucocytes and could play a role in multiple organ failure. Further studies are needed to evaluate the implications of changes in plasma trypsin in the disease process of equine acute abdomen and to demonstrate possible pancreatic damage. [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 detailAn Electron Spin Resonance (Esr) Study on the Mechanism of Ascorbyl Radical Production by Metal-Binding Proteins
Mouithys-Mickalad, Ange ULg; Deby, Carol; Dupont, Ginette ULg et al

in Biometals (1998), 11(2), 81-8

The mechanism of ascorbate oxidation by metal-binding proteins (ceruloplasmin, albumin and transferrin) was investigated in vitro and in isolated plasma by the measurement of the ascorbyl free radicals ... [more ▼]

The mechanism of ascorbate oxidation by metal-binding proteins (ceruloplasmin, albumin and transferrin) was investigated in vitro and in isolated plasma by the measurement of the ascorbyl free radicals (AFR) by electron spin resonance (ESR). In plasma of 13 healthy volunteers, a spontaneous and variable production of AFR was detected, which was increased by a 10(-4) M ascorbate overloading; however, this increase was not correlated to the intensity of the spontaneous AFR signal. The addition of Cu2+ and ceruloplasmin to plasma increased the ESR signal, while the addition of transferrin decreased the signal intensity in a dose-dependent manner. In vitro, we demonstrated that ascorbate was oxidized by human serum albumin and by ceruloplasmin, and that this oxidase-like activity was lost by trypsin or heat treatment of these proteins. These two proteins positively interacted in the oxidation of ascorbate, since addition of crude albumin to a solution of ascorbate and ceruloplasmin increased the intensity of ESR signal in a dose-dependent manner. The treatment of albumin by a metal chelator (DDTC) abolished these positive interactions. The respective roles of copper and iron in ascorbate oxidation were studied and showed a dose-dependent effect of these ions on ascorbate oxidation. The role of iron was confirmed by the inhibiting effect of metal-free transferrin on iron-dependent ascorbate oxidation. Concerted actions between iron carrying albumin and copper carrying ceruloplasmin appear responsible for the production of AFR in vitro and in vivo. [less ▲]

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See detailPropofol reacts with peroxynitrite to form phenoxyl radicals. Demonstration by ESR
Mouithys-Mickalad, Ange ULg; Hans, Pol ULg; Deby-Dupont, Ginette et al

in Biochemical and Biophysical Research Communications (1998), 249(3), 833-837

Peroxynitrite (ONOO-), resulting from the reaction of nitric oxide with superoxide anion, is a powerful oxidant produced in activated macrophages, during ischemia-reperfusion processes as well as in ... [more ▼]

Peroxynitrite (ONOO-), resulting from the reaction of nitric oxide with superoxide anion, is a powerful oxidant produced in activated macrophages, during ischemia-reperfusion processes as well as in neurodegenerative disorders. This study investigated the reaction of the anesthetic agent propofol (PPF) with ONOO-, using electron spin resonance (ESR) and UV-visible spectrometry. Peroxynitrite was synthetized either from acidified hydrogen peroxide and nitrite, or from sodium azide and ozone. The addition of ONOO- to PPF in alkaline solution (pH 12) allowed to detect a, short lifetime, ESR signal corresponding to a phenoxyl radical. This finding was confirmed by a UV-visible study, resulting in the appearance of 427 nm peak and the disappearance of the peak located at 239 nm. The 291 nm peak remained unchanged. The identification of the end-product of the reaction of PPF with ONOO- needs further investigations. [less ▲]

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See detailEquine neutrophil myeloperoxidase in plasma: design of a radio-immunoassay and first results in septic pathologies.
Deby, Ginette ULg; Grulke, Sigrid ULg; Caudron, I. et al

in Veterinary Immunology and Immunopathology (1998), 66(3-4), 257-71

The strangulated intestinal pathologies of horses are accompanied by a local activation of the neutrophils, that can be revealed by measuring the tissular enzymatic activity of the granulocytic enzyme ... [more ▼]

The strangulated intestinal pathologies of horses are accompanied by a local activation of the neutrophils, that can be revealed by measuring the tissular enzymatic activity of the granulocytic enzyme myeloperoxidase (MPO). To estimate the possible spreading of this neutrophil activation to the systemic circulation, we designed a radioimmunoassay (RIA) for equine neutrophil myeloperoxidase (MPO) (EC 1.11.1.7) using a specific rabbit antiserum. MPO was labeled with 1 mCi 125I by a technique of self-labeling in the presence of 10(-4) M hydrogen peroxide. The RIA was performed by incubation of 100 microl diluted antiserum, 100 microl labeled MPO (+/-30,000 cpm) and 100 microl of the reference molecule (unlabeled MPO) solution or the unknown sample, at room temperature for 18 h. The antibody-antigen complexes were isolated by double antibody precipitation. The sensitivity of the RIA was 2 ng/ml. The RIA showed good precision and accuracy with intra- and inter-assay coefficients of variation 6% and 8%, respectively, for MPO concentrations ranging from 2 ng/ml to 60 ng/ml. The best sampling technique for MPO measurement in plasma was to collect blood into EDTA, which allowed us to get a plasmatic value stable with time. The mean MPO value in normal horses was 69.5 +/- 19.4 ng/ml in EDTA anticoagulated plasma (n = 48). The stress of transport and anaesthesia did not modify the mean plasmatic value of MPO. No significant increase of plasma MPO was observed in 17 horses submitted to surgery for pathologies without systemic impact. But, in 25 horses with obstructive intestinal pathologies, persistent abnormal MPO concentrations were measured (until 740 ng/ml). [less ▲]

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