Equine skeletal muscle cells submitted to anoxia/reoxygenation produce reactive oxygen species.

Poster (2009)

A new easy method for specific measurement of active myeloperoxidase in human biological fluids and tissue extracts

in Talanta (2009), 80

The SIEFED (“Specific Immunological Extraction Followed by Enzymatic Detection”) method already developed for the specific detection of the activity of equine myeloperoxidase (MPO) was adapted for the ... [more ▼]

The SIEFED (“Specific Immunological Extraction Followed by Enzymatic Detection”) method already developed for the specific detection of the activity of equine myeloperoxidase (MPO) was adapted for the specific measurement of active human MPO in biological fluids or tissue extracts. [less ▲]

The unexpected hidden face of the Cephalosporin Antibiotic Ceftazidime: From biological to chemical and physical activities against oxidant species produced by phagocytes

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 ▲]

Diselenide Derivative, Potential Successor of Ebselen with High Antioxidant Activity: Assessment on in vitro Models

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 ▲]

Does propofol alter membrane fluidity at clinically relevant concentrations? An ESR spin label study

in Biophysical Chemistry (2007), 129(1), 82-91

General anesthetics have been shown to perturb the membrane properties of excitable tissues. Due to their lipid solubility, anesthetics dissolve in every membrane, penetrate into organelles and interact ... [more ▼]

General anesthetics have been shown to perturb the membrane properties of excitable tissues. Due to their lipid solubility, anesthetics dissolve in every membrane, penetrate into organelles and interact with numerous cellular structures in multiple ways. Several studies indicate that anesthetics alter membrane fluidity and decrease the phase-transition temperature. However, the required concentrations to induce such effects on the properties of membrane lipids are by far higher than clinically relevant concentrations. In the present study, the fluidizing effect of the anesthetic agent propofol (2,6-diisopropyl phenol: PPF), a general anesthetic extensively used in clinical practice, has been investigated on liposome dimyristoyi-L-alpha phosphatidylcholine (DMPC) and cell (erythrocyte, Neuro-2a) membranes using electron spin resonance spectroscopy (ESR) of nitroxide labeled fatty acid probes (5-, 16-doxyl stearic acid). A clear effect of PPF at concentrations higher than the clinically relevant ones was quantified both in liposome and cell membranes, while no evident fluidity effect was measured at the clinical PPF doses. However, absorption spectroscopy of merocyanine 540 (MC540) clearly indicates a PPF fluidizing capacity in liposome membrane even at these clinical concentrations. PPF may locally influence the structure and dynamics of membrane domains, through the formation of small-scale lipid domains, which would explain the lack of ESR information at low PPF concentrations. (c) 2007 Elsevier B.V. All rights reserved. [less ▲]

Plasma concentrations of a type II collagen-derived peptide and its nitrated form in growing ardenner sound horses and in horses suffering from juvenile digital degenerative osteoarthropathy

in Veterinary Research Communications (2007), 31(5), 591-601

Several breeds of draft horses suffer from degenerative digital osteoarthropathy, resulting in a reduced active lifespan. A group of 30 Ardenner horses was followed, in standardized conditions, from 15 to ... [more ▼]

Several breeds of draft horses suffer from degenerative digital osteoarthropathy, resulting in a reduced active lifespan. A group of 30 Ardenner horses was followed, in standardized conditions, from 15 to 28 months of age to detect the early manifestations of the disease. The severity of the disease was assessed according to a personal grading system including clinical and radiographic items. Coll 2-1, a peptide of the helical region of type II collagen, and its nitrated form (Coll 2-1 NO2) were assayed in blood plasma collected at 452 +/- 18 days, 504 +/- 20 days, 558 +/- 18 days, 613 +/- 19 days, 675 +/- 19 days, 752 +/- 21 days and 852 +/- 19 days of age. At the end of the follow-up period, 53.3% of Ardenner horses were affected by a degenerative digital osteoarthropathy. A significant effect (p < 0.05) of time, sex and pathology was observed for Coll 2-1 NO2. Variations of Coll 2-1 were not significant except for the time effect. The elevation of Coll 2-1 NO2 in the pathological group could indicate an inflammatory process during the growth of the affected horses, as nitration of tyrosine is mediated through reactive oxygen/nitrogen species and/or myeloperoxidase activity. Coll 2-1 NO2 appears to be an interesting early marker of cartilage degradation and oxidation in degenerative osteoarthropathy. [less ▲]

Oxygen consumption of equine articular chondrocytes: Influence of applied oxygen tension and glucose concentration during culture.

in Cell Biology International (2007), 31

We investigated the oxygen (O(2)) uptake of equine articular chondrocytes to assess their reactions to anoxia/re-oxygenation. They were cultured under 5% or 21% gas phase O(2) and at glucose ... [more ▼]

We investigated the oxygen (O(2)) uptake of equine articular chondrocytes to assess their reactions to anoxia/re-oxygenation. They were cultured under 5% or 21% gas phase O(2) and at glucose concentrations of 0, 1.0 or 4.5g/L in the culture medium (n=3). Afterwards, the O(2) consumption rate of the chondrocytes was monitored (oxymetry) before and after an anoxia period of 25min. The glucose consumption and lactate release were measured at the end of the re-oxygenation period. The chondrocytes showed a minimal O(2) consumption rate, which was hardly changed by anoxia. Independently from the O(2) tension, glucose uptake by the cells was about 30% of the available culture medium glucose, thus higher for cells at 4.5g/L glucose (n=3). Lactate release was also independent from O(2) tension, but lower for cells at 4.5g/L glucose (n=3). Our observations indicated that O(2) consumption by equine chondrocytes was very low despite a functional mitochondrial respiratory chain, and nearly insensitive to anoxia/re-oxygenation. But the chondrocytes metabolism was modified by an excess of O(2) and glucose. [less ▲]

Le rôle des synoviocytes dans l’articulation diarthrodiale enflammée

in Annales de Médecine Vétérinaire (2007), 151

The degenerative osteoarthritis is a common source of disease in the horse, causing pain and leading finally to the destruction of the cartilage in the diarthrodial joints. The synovial membrane is a ... [more ▼]

The degenerative osteoarthritis is a common source of disease in the horse, causing pain and leading finally to the destruction of the cartilage in the diarthrodial joints. The synovial membrane is a constitutive element of the joint, but often neglected due to its less obvious alterations, despite its crucial role. This review of literature considers the embryology, the histology of synoviocytes in vivo and in vitro, gives information about the production of inflammatory mediators, degrading enzymes, free oxygen radicals, about the composition of the synovial fluid and the interaction with chondrocytes. The number of studies on equine synoviocytes in osteoarthritis is very restricted, therefore we included studies in other species and other types of joint diseases to achieve a more complete image of the complex, interdependent and mutual acting mechanisms. [less ▲]

Are COX-2 Inhibitors Active on Intracellular Oxidative Processes? A Study on In Vitro and Cellular Models

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 ▲]

A specific method for measurement of equine active myeloperoxidase in biological samples and in in vitro tests

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 ▲]