AN IMMUNOLOGICAL METHOD TO COMBINE THE MEASUREMENT OF ACTIVE AND TOTAL HUMAN MYELOPEROXIDASE ON THE SAME SAMPLE FROM A COMPLEX MEDIUM
Franck, Thierry ; MINGUET, Grégory ; et al
Conference (2013, September 12)
Active neutrophil myeloperoxidase (MPO) is a powerful producer of oxidant molecules in acute or chronic inflammation, and it is essential to measure its activity in biological samples. We combined two ... [more ▼]
Active neutrophil myeloperoxidase (MPO) is a powerful producer of oxidant molecules in acute or chronic inflammation, and it is essential to measure its activity in biological samples. We combined two immunological techniques, the SIEFED (specific immunologic extraction followed by enzymatic detection) and an ELISA, to measure the active and total contents of human MPO on the same sample and with the same calibration curve, to define an accurate ratio between the active and total enzyme. After the extraction of MPO from aqueous or biological samples by immobilized anti-MPO antibodies followed by a washing to eliminate unbound material, the active and the total contents of the enzyme were sequentially measured without interferences (patent: EP2017351-B1, 2010). Compared to a classical sandwich ELISA, there is one additional step corresponding to the in situ measurement of MPO activity, but this step does not affect the following measurement of the total MPO content. After validation, the combined technique was applied to a whole blood model of in vitro stimulation with phorbol-myristate-acetate (PMA), cytochalasin B/N-formyl-methionyl-leucyl-phenyl-alanine (CB/fMLP) or lipopolysaccharide/ tumor necrosis factor-alpha (LPS/TNF-α) (n=9). The active/total MPO ratio in whole blood reached 0.267±0.126 for non-stimulated condition and was significantly (p<0.05) higher for PMA (0.360±0.106), CB/fMLP (0.380±0.113) and LPS/TNF-α (0.432±0.124) stimulated conditions. These different ratios highlight the real oxidant potential of MPO, which depends on the stimulating conditions, witness of what could happen in pathological situations with diagnostic purpose. The combined SIEFED/ELISA method also appeared as a powerful tool to screen potential inhibitors that could interact directly with the enzyme, either on its active site or on another key position. [less ▲]Detailed reference viewed: 15 (5 ULg)
An in vitro whole blood model to test the effects of different stimuli conditions on the release of myeloperoxidase and elastase by equine neutrophils.
Ceusters, Justine ; Serteyn, Didier ; MINGUET, Grégory et al
in Veterinary Immunology and Immunopathology (2012), 150(3-4), 221-7
Horses are particularly sensitive and exposed to excessive inflammatory responses evolving toward an important stimulation of polymorphonuclear neutrophils (PMNs). The aim of this work was to stimulate ... [more ▼]
Horses are particularly sensitive and exposed to excessive inflammatory responses evolving toward an important stimulation of polymorphonuclear neutrophils (PMNs). The aim of this work was to stimulate equine neutrophils in whole blood and to evaluate their response by measuring the release of total and active myeloperoxidase (MPO) and total elastase, considered as markers of neutrophil stimulation and degranulation. Because of the critical importance of the concomitant presence of LPS and TNF-alpha in equine pathological situations, we combined these two natural mediators to stimulate PMN and compared the response with those obtained after the PMN stimulation with each mediator used alone and well-known artificial stimulation systems such as 12-phorbol 13-myristate acetate (PMA) and the combination of cytochalasin B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP). All the activation systems, PMA, CB/fMLP, TNF-alpha, LPS and LPS/TNF-alpha, induced a significant release of total MPO in whole blood but only the combinations CB/fMLP and LPS/TNF-alpha significantly favored the release of active MPO. Regarding the total elastase, we did not observe a significant release in all the stimulated conditions except with PMA. It appears clearly that the choice of the neutrophil stimulation model is fundamental for the selection of potentially active pharmacological agents, especially on MPO activity. [less ▲]Detailed reference viewed: 31 (17 ULg)
Sevoflurane inhibits equine myeloperoxidase release and activity in vitro.
MINGUET, Grégory ; de la Rebière de Pouyade, Geoffroy ; Franck, Thierry et al
in Veterinary Anaesthesia & Analgesia (2012)
Objective To investigate the effects of the volatile anaesthetic sevoflurane on the release of total and active myeloperoxidase (MPO) by non-stimulated and stimulated polymorphonuclear neutrophils (PMNs ... [more ▼]
Objective To investigate the effects of the volatile anaesthetic sevoflurane on the release of total and active myeloperoxidase (MPO) by non-stimulated and stimulated polymorphonuclear neutrophils (PMNs) in whole blood from healthy horses. Study design In vitro experimental study. Animals Adult healthy horses. Methods Samples of whole venous blood were collected and incubated in air or in air plus 2.3% or 4.6% sevoflurane for 1 hour. PMNs were stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP), with a combination of cytochalasin B (CB) and fMLP or with phorbol myristate acetate (PMA). Total and active MPO contents released by PMNs in blood were measured by enzyme-linked immunosorbent assay (ELISA) and specific immunological extraction followed by enzymatic detection (SIEFED) respectively. Additional experiments were performed to assess the effect of sevoflurane on the peroxidase and chlorination cycles of purified equine MPO using Amplex Red and 3'-(p-aminophenyl) fluorescein as fluorogenic substrates respectively. Results As compared with air alone, 1 hour exposure of whole blood to 4.6% sevoflurane in air significantly inhibited the release of total and active MPO by unstimulated and both fMLP- and CB + fMLP-stimulated PMNs but not by PMA-stimulated PMNs. Although 2.3% sevoflurane had no effect on total MPO release by unstimulated and stimulated PMNs, it significantly reduced the release of active MPO by unstimulated and fMLP-stimulated PMNs. Additionally, sevoflurane reversibly inhibited the activity of MPO, especially the peroxidase cycle of the enzyme. Conclusions and clinical relevance Although our experimental study was not designed to assess the effects of sevoflurane in vivo, this inhibition of MPO release and activity may have relevance for anaesthetized horses and deserves further studies to examine the clinical importance of these findings. [less ▲]Detailed reference viewed: 21 (8 ULg)
Inhibitory effect of sevoflurane on myeloperoxidase release by equine neutrophils
Minguet, Grégory ; de la Rebière de Pouyade, Geoffroy ; Franck, Thierry et al
(2009, March)Detailed reference viewed: 45 (12 ULg)
Preconditioning and protection against ischaemia-reperfusion in non-cardiac organs: a place for volatile anaesthetics?
Minguet, Grégory ; Joris, Jean ; Lamy, Maurice
in European Journal of Anaesthesiology (2007), 24(9), 733-745
There is an increasing body of evidence that volatile anaesthetics protect myocardium against ischaemic insult by a mechanism termed 'anaesthetic preconditioning'. Anaesthetic preconditioning and ... [more ▼]
There is an increasing body of evidence that volatile anaesthetics protect myocardium against ischaemic insult by a mechanism termed 'anaesthetic preconditioning'. Anaesthetic preconditioning and ischaemic preconditioning share several common mechanisms of action. Since ischaemic preconditioning has been demonstrated in organs other than the heart, anaesthetic preconditioning might also apply in these organs and have significant clinical applications in surgical procedures carrying a high risk of ischaemia-reperfusion injury. After a brief review on myocardial preconditioning, experimental and clinical data on preconditioning in non-cardiac tissues will be presented. Potential benefits of anaesthetic preconditioning during non-cardiac surgery will be addressed. [less ▲]Detailed reference viewed: 32 (0 ULg)