Ginette Deby

In vitro effects of aceclofenac and its metabolites on the production by chondrocytes of inflammatory mediators.

Abstract. Objectives: To investigate the mechanisms of action underlying the anti-inflammatory action of aceclofenac in vivo, we studied in vitro the effect of aceclofenac and its main metabolite, 4′-hydroxyaceclofenac, in comparison with diclofenac, another metabolite, on cyclooxygenases activity as well as interleukin-1β, -6 and -8, nitric oxide, and prostaglandin E2 production by human osteoarthritic and normal articular chondrocytes. Methods: Enzymatically isolated human chondrocytes were cultured for 72 h in the absence or presence of interleukin-1β (IL-1β) or lipopolysacharride (LPS) and with or without increased amounts (1 to 30 μM) of aceclofenac or metabolites. The production of different cytokines was measured by Enzyme Amplified Sensitivity Immunoassays (EASIA). Prostaglandin E2 was quantified by a specific radioimmunoassay. Nitrite and nitrate concentrations in the culture supernatants were determined by spectrophotometric method based upon the Griess reaction. Cyclooxygenase-2, inducible NO synthase and IL-1β gene expression were quantified by reverse transcription of mRNA followed by real time and quantitative polymerase chain reaction. Finally, cyclooxygenase inhibitory potency of the drugs was also tested in both a cell-free system using purified ovine cyclooxygenase-1 and -2 (COX-1 and COX-2) and at a cellular level using human whole blood assay. Results: We have demonstrated that aceclofenac, 4′-hydroxyaceclofenac and diclofenac significantly decreased interleukin-6 production at concentrations ranged among 1 to 30 μM and fully blocked prostaglandin E2 synthesis by IL-1β- or LPS-stimulated human chondrocytes. Aceclofenac and diclofenac had no effect on interleukin-8 production while 4′-hydroxyaceclofenac slightly decreased this parameter at the highest dose (30 μM). Aceclofenac was without effect on IL-1β- or LPS-stimulated nitric oxide production. At 30 μM, 4′-hydroxyaceclofenac inhibited both IL-1β or LPS-stimulated nitric oxide production while diclofenac inhibited only the LPS-stimulated production. Finally, at 30 μM, the three drugs significantly decreased IL-1β mRNA. In the whole blood test, aceclofenac and 4′-hydroxyaceclofenac weakly inhibited COX-1 with IC50 values superior to 100 μM, but decreased by 50% COX-2 activity at the concentration of 0.77 and 36 μM, respectively. Diclofenac strongly inhibited both COX-1 and COX-2 with IC50 values of 0.6 and 0.04 μM, respectively. On the other hand, aceclofenac and diclofenac weakly inhibited purified ovine cyclooxygenases with IC50 values superior to 100 μM, whereas 4′-hydroxyaceclofenac was without effect. Conclusions: These results suggest that aceclofenac actions are multifactorial and that metabolites could contribute to its anti-inflammatory actions.

Modulating effects of acepromazine on the reactive oxygen species production by stimulated equine neutrophils

OBJECTIVE To investigate the effect of acepromazine (ACP) on reactive oxygen species (ROS) production by stimulated equine neutrophils. STUDY DESIGN Ex vivo biochemical experiments. ANIMALS Isolated neutrophils from healthy untreated horses. METHODS Neutrophils were incubated with ACP at concentrations of 10(-4), 10(-5) or 10(-6) M and then stimulated with phorbol-myristate-acetate (PMA) before measurement of lucigenin-enhanced chemiluminescence (CL). In a second experiment neutrophils were incubated in the presence of α-keto-γ methylthiobutyric acid (KMB) and treated with ACP at concentrations of 10(-4), 10(-5) or 10(-6) M. Subsequent PMA stimulation lead to neutrophilic ROS production and decomposition of KMB to ethylene, which is measured by gas chromatography. Electron paramagnetic resonance-spin trapping (EPR) analysis was performed with PMA-stimulated neutrophils in the presence of ACP (10(-4), 10(-5) or 10(-6) M) directly added to the cell suspension. In the second experiment, the same concentrations of ACP were pre-incubated with neutrophils, then centrifuged to eliminate the excess of ACP and re-suspended in phosphate buffer before stimulation with PMA. In all experiments, the results of ACP-treated and ACP-untreated stimulated neutrophils were compared. RESULTS Overall, results obtained with lucigenin-enhanced CL and KMB oxidation were in agreement with those seen in electron paramagnetic resonance spectroscopy. Acepromazine induced a dose-dependent inhibitory effect on neutrophilic ROS production. Electron paramagnetic resonance also showed, at high ACP concentration, the appearance of a cation radical derived from ACP. In contrast, electron paramagnetic resonance study performed with pre-incubated neutrophils showed an important dose-dependent inhibitory effect of ACP. CONCLUSION The results indicate that ACP can neutralize O.-2 or its by-products during the stimulation of neutrophils. CLINICAL RELEVANCE These findings may have a therapeutic relevance when phenothiazines are used in horses suffering from inflammatory diseases in which neutrophil activation and ROS production are implicated.

Pancreatic cellular injury after cardiopulmonary bypass

The pancreas is both an endocrine and exocrine gland, but it is its exocrine portion which is more easily described as the target of cellular alterations in shock and other pathological situations [1]. This exocrine pancreatic injury is often characterized by an increase in the blood levels of pancreatic enzymes such as the P-type amylase, the lipase and the trypsinogen, precursor of the proteolytic enzyme trypsin.

Oxygen Therapy in Intensive Care Patients: A Vital Poison?

Hypoxemia is a frequent phenomenon in patients with acute lung injury (ALI) from various origins related or unrelated to pulmonary diseases. One of the most effective therapies is the administration of supplemental oxygen to reverse hypoxemia, using increasing concentrations until an inspired fraction of oxygen (FiO2) of 100% is reached. This therapy is often effective in reversing hypoxemia and restoring the partial pressure of oxygen (PaO2) levels to near normal values. It is a relatively low-cost therapy that can be administered using several modalities, and titrated to exact concentrations. However, it must be kept in mind that oxygen is a drug with indications, contraindications, dosage ranges and the potential for toxicity. If administration of oxygen is vital to hypoxic patients to restore efficient breathing and maintain organ viability, its potential toxicity must be considered for long and aggressive use, particularly in inflamed and damaged lungs, and at high values of FiO2.

Clinical significance of active myeloperoxidase in carotid atherosclerotic plaques.

Assessment of vulnerable carotid artery plaques is a majorunresolved issue. Patients presenting unstable angina have evidenceof unstable carotid artery plaque [1] suggesting systemic vascularvulnerability [2].Neutrophil myeloperoxidase (MPO) is a powerful producer ofoxidant molecule [3]. MPO level is higher in patients with coronaryartery disease and in patients undergoing percutaneous coronaryintervention for unstable coronary syndrome compared to stablepatients [4,5], carrying predictive value of adverse outcome [6].MPO levels measured by immunological methods (ELISA or RIA)quantify the total concentration of the enzyme without providing itsenzymatic activity [7]. MPO activity is currently measured byspectrophotometry or fluorimetry [8,9]. However, these methodsare not applicable to complex biological samples such as humantissue. We recently developed a new technique, the SIEFED (SpecificImmunological Extraction Followed by Enzymatic Detection) tomeasure the activity of the enzyme in biological fluids and developedit for equine MPO and thereafter for human use [10,11]. The aims ofthis study were to assess the presence of the active form of MPO inhuman carotid atherosclerotic lesions, to compare active MPO withtotal MPO, and to assess the potential significance of total and/oractive MPO by comparing their levels in symptomatic and asymp-tomtic patients.We prospectively collected 90 carotid artery plaque specimensobtained by surgical thrombo-endarteriectomy according to theNASCET criteria [12].Carotid atherosclerotic plaques were surgically collected andrinsed with 0.9% NaCl solution before freezing at −20 °C. Methodol-ogy and validation of SIEFED technique as well as ELISA method havebeen previously described [7,10,11]. The activity of the total MPOexpressed in ng/mL was reported to the total protein concentration(mg/mL) measured in the supernatant of carotid extracts using the

Biochemical Mediators in Acute Respiratory Distress Syndrome (ARDS) after Burning Injury

Seventeen burned patients, 5 of whom developed the adult respiratory distress syndrome (ARDS), were investigated. Occurrence of ARDS was correlated with the severity of burn in ARDS patients (UBS: 82±27) and non ARDS patients (UBS: 36 ± 18) respectively (p < 0.005) and with inhalation injury.