Activation of equine neutrophils by phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine induces a different response in reactive oxygen species production and release of active myeloperoxidase.

Neutrophil (PMN) contribution to the acute inflammatory processes may lead to an excessive generation of reactive oxygen metabolites species (ROS) and secretion of granule enzymes. We compared the effects of either phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) in combination with a pre-treatment by cytochalasin B (CB) on the production of ROS and the release of total and active myeloperoxidase (MPO) by isolated equine PMNs. The ROS production was assessed by lucigenin dependent chemiluminescence (CL) and ethylene release by alpha-keto-gamma-methylthiobutyric acid (KMB) oxidation.

Direct stimulation of the oxidative activity of isolated equine neutrophils by TNF-alpha and IL-1beta.

The capacity of the two cytokines TNF-alpha and IL-1beta to directly stimulate the oxidative activity of polymorphonuclear neutrophils remains debated. The purpose of this study was to verify if a direct stimulation of equine neutrophils by TNF-alpha and IL-1beta was possible. Equine neutrophils were isolated from blood by discontinuous density gradient centrifugation.

Resveratrol inhibits the activity of equine neutrophil myeloperoxidase by a direct interaction with the enzyme.

Resveratrol is a polyphenolic antioxidant present in beverage and food known for its multiple protective effects. We report the inhibitory effects of resveratrol on equine myeloperoxidase (MPO), a hemic peroxidase present in the granules of the neutrophils involved in the inflammatory response. Resveratrol inhibited the production of reactive oxygen species (ROS) by stimulated equine neutrophils by acting as a direct scavenger of the ROS released by the cells but did not modify the degranulation of the stimulated neutrophils as the amounts of released MPO were unchanged.

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

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.

Development of an enzyme-linked immunosorbent assay for specific equine neutrophil myeloperoxidase measurement in blood.

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.

Resveratrol and curcumin reduce the respiratory burst of Chlamydia-primed THP-1 cells.

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.

Catalytic activation of copper (II) salts on the reaction of peroxynitrite with propofol in alkaline medium.

We report here on the role of copper (II) salts on the acceleration of peroxynitrite (ONOO-) decomposition and ONOO- reaction with the anaesthetic agent propofol (2,6-diisopropylphenol) in alkaline medium. We observed a strong acceleration of the ONOO- decomposition in alkaline medium in the presence of copper (I and II) salts. After 18 h of ONOO- reaction with propofol, we observed nitrosated, nitrated, and oxidized (quinone and diphenylquinone) derivatives of propofol, but in the presence of Cu(II) (20% molar vs ONOO-), the yields of quinone and nitrosopropofol strongly increased.

Early release of neutrophil markers of activation after direct stenting in patients with unstable angina.

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).

Viability of equine articular chondrocytes in alginate beads exposed to different oxygen tensions.

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.

Hyperhydricity of Prunus avium shoots cultured on gelrite: a controlled stress response.

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).

In vitro evaluation of glutathione peroxidase (GPx)-like activity and antioxidant properties of some Ebselen analogues.

Four analogues of Ebselen were synthesized and their glutathione peroxidase activity and antioxidant property evaluated and compared to Ebselen. Among the studied compounds, only diselenide [3] exhibited both glutathione peroxidase activity and radical-scavenging capability. Compounds [3] and [4] showed a strong inhibitory effect (53% and 43%, respectively) on the lipid peroxidation of linoleic acid compared to Ebselen and selenide derivatives ([1] and [2]) which were less active (28%, 26% and 18% inhibition, respectively).

Effects of glucocorticoids on the respiratory burst of Chlamydia-primed THP-1 cells.

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.

Oxidative stress, KLF6 and transforming growth factor-beta up-regulation differentiate non-alcoholic steatohepatitis progressing to fibrosis from uncomplicated steatosis in rats.

Background/aims: Pathogenesis of non-alcoholic steatohepatitis (NASH) remains poorly understood. Cytochrome P450 2E1 (CYP 2E1), cytokines, oxidative stress and activation of hepatic stellate cells seem to play a role in this process. The aim was to determine the potential implication of these factors in the progression from uncomplicated steatosis to steatohepatitis with progressive fibrosis.

Investigation of the reaction of peroxynitrite with propofol at acid pH: predominant production of oxidized, nitrated, and halogenated derivatives.

We reported here the reaction, in acidic conditions, of peroxynitrite (ONOO(-)) with the anaesthetic agent propofol (2,6-diisopropylphenol, PPF). The most interesting finding is that peroxynitrite is able to nitrate and to oxidize propofol leading to 4-nitropropofol, quinone, and diphenylquinone as the major products. More surprisingly, we also found that peroxynitrite is capable of halogenating propofol in the presence of halide ions, suggesting the formation of nitrosyl chloride (NOCl) or nitryl chloride (NO(2)Cl) from the reaction of peroxynitrite with chloride ions.

Equine trypsin: purification and development of a radio-immunoassay.

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 > or = 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.

Oxygen consumption and electron spin resonance studies of free radical production by alveolar cells exposed to anoxia: inhibiting effects of the antibiotic ceftazidime.

By EPR spectroscopy, we investigated free radical production by cultured human alveolar cells subjected to anoxia/re-oxygenation (A/R), and tested the effects of ceftazidime, an antibiotic previously demonstrated to possess antioxidant properties. Two A/R models were performed on type II pneumocytes (A549 cell line), either on cells attached to culture dishes (monolayer A/R model; 3.5 h of anoxia, 30 min of re-oxygenation) or after cell detachment (suspension A/R model; 1 h of anoxia, 10 min of re-oxygenation).

Plasma trypsin level in horses suffering from acute intestinal obstruction.

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.

Oxidation of tetrahydrobiopterin by peroxynitrite or oxoferryl species occurs by a radical pathway.

The molecular mechanisms of tetrahydrobiopterin (BH4) oxidation by peroxynitrite (ONOO-) was studied using ultra-weak chemiluminescence, electron paramagnetic resonance (EPR) and UV-visible diode-array spectrophotometry, and compared to BH4 oxidation by oxoferryl species produced by the myoglobin/hydrogen peroxide (Mb/H2O2) system. The oxidation of BH4 by ONOO- produced a weak chemiluminescence, which was altered by addition of 50 mM of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN). EPR spin trapping demonstrated that the reaction occurred at least in part by a radical pathway.

Oxidative processes in human promonocytic cells (THP-1) after differentiation into macrophages by incubation with Chlamydia pneumoniae extracts.

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.

In vitro study of the antioxidant properties of non steroidal anti-inflammatory drugs by chemiluminescence and electron spin resonance (ESR).

Objectives: To determine the antioxidant activities of nonsteroidal anti-inflammatory drugs (NSAIDS), we examined by chemiluminescence (CL) and electron spin resonance (ESR) their scavenging properties towards lipid peroxides, hypochlorous acid and peroxynitrite.

Methods: The antioxidant properties of nimesulide (NIM), 4-hydroxynimesulide (4-HONIM), aceclofenac (ACLO), 4-hydroxyaceclofenac (4-HOA-CLO), diclofenac (DICLO) and indomethacin (INDO) were tested on four different reactive oxygen species (ROS) generating systems: (I) phorbol-myristate acetate (PMA)-activated neutrophils, (II) Fe2+/ascorbate-induced lipid peroxidation, (III) HOCl-induced light emission, (IV) the kinetics of ONOO- decomposition followed by spectrophotometry. ROS production was monitored by luminol-enhanced CL or by ESR using two different spin traps.

In vitro study of the antioxidant properties of nimesulide and 4-OH nimesulide: effects on HRP- and luminol-dependent chemiluminescence produced by human chondrocytes.

Objectives: Reactive oxygen species (ROS) are now recognized to play an important role in the pathogenesis of rheumatic diseases and constitute an interesting therapeutic target for drugs. This in vitro study was designed to evaluate the antioxidant properties of nimesulide (NIM), a nonsteroidal antiinflammatory drug of the sulfonanilide class, and its main metabolite 4-OH nimesulide (4-OHNIM).

Methods: The scavenging effects of NIM and 4-OH NIM on hydroxyl radical ((.

Cytotoxicity of stimulated equine neutrophils on equine endothelial cells in culture.

We studied the interactions of isolated equine neutrophils with endothelial cells in culture, mimicking a situation of acute inflammation. Our main purpose was to demonstrate that the supernatant of activated neutrophils was sufficient to damage endothelial cells. Equine endothelial cells (from carotid arteries) were covered either with increased numbers of equine neutrophils stimulated by phorbol myristate acetate, or with the supernatant collected after an in vitro stimulation of the neutrophils.

High concentrations of histamine stimulate equine polymorphonuclear neutrophils to produce reactive oxygen species.

Objective And Design: Because high concentrations of histamine are locally released in inflammation, we investigated the effects of supraphysiological doses of histamine on the production of reactive oxygen species (ROS) by neutrophils.

Materials And Methods: Isolated equine neutrophils were activated by 10(-4) to 5 x 10(-3) M histamine. The production of ROS and free radicals was estimated by luminol-enhanced chemiluminescence (CL) and electron spin resonance (ESR) with spin trapping technique.

Plasma myeloperoxidase level and polymorphonuclear leukocyte activation in horses suffering from large intestinal obstruction requiring surgery: preliminary results.

Myeloperoxidase (MPO) is a specific enzyme of neutrophil azurophilic granules with a strong oxidative activity. Thanks to a radioimmunoassay of equine myeloperoxidase, the authors have observed a significantly higher plasma level of MPO in horses operated for strangulation obstruction of the large intestine (n = 6) than in horses suffering from a non-strangulating displacement of the large intestine (n = 9). For the 2 groups, 3 phases were distinguished: reception (P1), intensive care (P2) and terminal phase (P3).