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See detailSimultaneous measurement of protein-bound 3-chlorotyrosine and homocitrulline by LC-MS/MS after hydrolysis assisted by microwave: application to the study of myeloperoxidase activity during hemodialysis.
Delporte, Cedric; Franck, Thierry ULg; Noyon, Caroline et al

in Talanta (2012), 99

A high degree of uremia is common in patients with end-stage renal disease and has been linked to the development of chronic inflammation and cardiovascular diseases. In conditions where transplantation ... [more ▼]

A high degree of uremia is common in patients with end-stage renal disease and has been linked to the development of chronic inflammation and cardiovascular diseases. In conditions where transplantation is not possible, uremia can be reduced by hemodialysis although the repeated interventions have been implicated in loss of renal function, partially as a result of chronic inflammation and/or oxidative stress processes. In this context, it has been suggested that myeloperoxidase (MPO) can contribute to the oxidative stress during hemodialysis and to the cardiovascular risk. Protein damages due to MPO activity have never been assessed during hemodialysis although two of its reaction products, 3-chlorotyrosine and homocitrulline, are of interest. Indeed, the first one is a specific product of MPO activity and the formation of the second one could be catalyzed by MPO. In order to analyze these products in plasma proteins, a total hydrolysis method followed by liquid chromatography mass spectrometry analysis was developed. Different conditions of hydrolysis were tested and the optimized procedure was assessed for complete hydrolysis and artifactual chlorination. Finally, the method was used for analyzing 3-chlorotyrosine and homocitrulline in plasma proteins during a hemodialysis session in fifteen patients and data were related to measurements of MPO concentration and activity. Both increases in MPO activity and protein-bound 3-chlorotyrosine were observed, highlighting the involvement of MPO in oxidative stress during hemodialysis and further demonstrating the link between hemodialysis and cardiovascular diseases. [less ▲]

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See detailDoes the new water-soluble form of curcumin(NDS27) inhibit the oxidant response of stimulated neutrophils and HL-60 cells?
Derochette, Sandrine ULg; Franck, Thierry ULg; Deby-Dupont, Ginette et al

Poster (2012, September 07)

Neutrophils (PMNs) are involved in host defense against infections through the production of reactive oxygen species (ROS) and the release of an oxidant enzyme, myeloperoxidase (MPO), to kill pathologic ... [more ▼]

Neutrophils (PMNs) are involved in host defense against infections through the production of reactive oxygen species (ROS) and the release of an oxidant enzyme, myeloperoxidase (MPO), to kill pathologic agents. But, an excessive stimulation of PMNs is associated with development of inflammatory diseases. Neutrophils are prime targets to control inflammatory events and the therapeutic use of polyphenols is proposed to lower oxidative stress. The aim of this work was to study antioxidant effect of NDS27, a water-soluble form of curcumin, on stimulated equine PMNs and human promyelocytic leukemia cells (HL-60) which are less differenciated. 2',7'-Dichlorofluorescin diacetate and lucigenin were used to measure ROS production by activated HL-60 cells or PMNs. NDS27 (10-6 to 10-4 M) was pre-incubated with cells and eliminated before their activation to study its intracellular effects on ROS production. The effect of NDS27 on MPO activity released by the cells was determined by SIEFED. Likewise, the ability of NDS27 to enter into the cells was checked by HPLC on the cellular extracts.NDS27 significantly and dose-dependently inhibited the ROS production in both cell types without affecting their viability. Its intracellular effect showed higher efficiency for PMNs while its interaction with HL-60 cells remained better. The activity of MPO released by PMNs and HL-60 cells was decreased by NDS27 with a more efficient effect for PMNs. Our findings suggest that the greater efficiency of NDS27 in mature PMNs is not due to a better membrane permeability or a better interaction between membrane and NDS27, but rather to an inhibitory effect on the ROS production by the more mature cells, probably by targeting the enzymes implied in respiratory burst like MPO and NADPH oxidase. The modulatory effect of NDS27 towards oxidant activity of cells involved in immune and inflammatory response opens therapeutic perspectives to control pathologies with excessive inflammatory reactions. [less ▲]

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See detailMyeloperoxidase as an indicator of endometritis in the mare: preliminary results
Parrilla Hernandez, Sonia ULg; Franck, Thierry ULg; Ponthier, Jérôme ULg et al

in Reproduction in Domestic Animals (2012, August), 47(suppl 5), 65

Diagnosis of endometritis in the mare is routinely based on the presence of polymorphonuclear cells (PMNs) on endometrial smears. Studies show a relation between PMNs and myeloperoxidase (MPO), an enzyme ... [more ▼]

Diagnosis of endometritis in the mare is routinely based on the presence of polymorphonuclear cells (PMNs) on endometrial smears. Studies show a relation between PMNs and myeloperoxidase (MPO), an enzyme released by PMNs during degranulation or after cell lysis, in many fluids and tissues. The aims of this study were to assess the presence and concentration of MPO in the mare’s uterus, and to investigate its relation with PMNs. Thirty-six cycles from 28 mares (ages ranging from 6 to 22 years) were used. Endometrial cytological samples were obtained with a small volume uterine flush and either a uterine cotton swab or a cytobrush, when a follicle >35 mm was observed by ultrasound. The smears were stained with Diff-QuickÒ and one or more PMNs per field (400·) was diagnosed as endometritis. The supernatant of the flushes was used to measure MPO concentration with a specific equine MPO ELISA assay. Our results showed the presence of MPO in the equine uterus during oestrus (mean = 2839 + 2785). MPO concentrations were signifi- cantly (p < 0.05) higher in samples with positive cytological results. Occasionally, some samples with negative cytological results showed high MPO concentration, but the opposite was never observed. Clinical signs of endometritis are not always present, or they may be delayed. An early diagnostic improves the success of treatment. Our results show that high quantities of MPO in endometrial samples indicate the presence of PMNs. Further studies are needed to determine if MPO concentration could be routinely used as a tool of early detection of endometritis. [less ▲]

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See detailPossible intracellular effect of the new water-soluble form of curcumin (NDS27) on the oxidant response of stimulated neutrophils
Derochette, Sandrine ULg; Mouithys-Mickalad, Ange ULg; Deby-Dupont, Ginette et al

Poster (2012, April 18)

Neutrophils (PMNs) are involved in host defense against infections through the production of reactive oxygen species (ROS) to kill pathologic agents. But, an excessive ROS production, called “oxidative ... [more ▼]

Neutrophils (PMNs) are involved in host defense against infections through the production of reactive oxygen species (ROS) to kill pathologic agents. But, an excessive ROS production, called “oxidative stress” is associated with tissue damages and development of chronic or acute inflammatory diseases. PMNs are prime therapeutic targets to control inflammatory events associated to ROS production. Nowadays, there is a growing interest for the use of polyphenolic molecules to modulate the inflammatory response. The aim of this work was to study the antioxidant effect of NDS27 (1), a new highly water-soluble form of the polyphenolic molecule curcumin, on in vitro stimulated equine PMNs. NDS27 (10-6 to 10-4 M) was pre-incubated with cells and eliminated before their activation. The ability of NDS27 to enter into the cells was checked by HPLC from the cellular extracts. The intracellular ROS production by phorbol myristate acetate (PMA) stimulated PMNs was measured by fluorescence using 2’,7’-dichlorofluorescin diacetate. Lucigenin dependent chemiluminescence was used to measure extracellular ROS production. Additionally, the effect of NDS27 was tested on the activity of myeloperoxidase (MPO), a hemic enzyme contributing to the oxidant response of neutrophils. The activity of the released MPO by cytochalazine B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated PMNs was measured by SIEFED (“Specific Immunologic Extraction Followed by Enzymatic Detection”) (2). The HPLC results showed that NDS27 enters into PMNs and interacts with their membrane. NDS27 significantly and dose-dependently inhibited the ROS production in neutrophils without affecting their viability. Likewise, the activity of MPO released by PMNs was lowered by NDS27. Overall, our findings demonstrate that the membrane of neutrophils is permeable to NDS27 or interacts with the drug, suggesting that its inhibitory effect on ROS production is mainly associated to an intracellular effect probably by acting on the enzymes implied in respiratory burst like NADPH oxidase and MPO. The modulatory effect of NDS27 towards the oxidant activity of cells involved in immune and inflammatory response open therapeutic perspectives to control equine or human pathologies with excessive inflammatory reactions. 1. Neven et al. 2011, Patent Application Publication: US2011/0257126 A1 2. Serteyn et al. 2005, European Patent Specification : EP1711817 B1 [less ▲]

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See detailSystemisch-entzuendliche Reaktion beim Hochleistungssportpferd
Sandersen, Charlotte ULg; Lejeune, Jean-Philippe ULg; Votion, Dominique ULg et al

Book published by Leipziger Blaue Hefte (2012)

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See detailMyeloperoxidase in Equine Semen: Concentration and Localization during Freezing Processing
Ponthier, Jérôme ULg; Desvals, Maud; Franck, Thierry ULg et al

in Journal of Equine Veterinary Science (2012), (32), 32-37

Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutro- phils during degranulation or after lysis. Post-thaw semen contains MPO, and concen- tration of this enzyme is associated ... [more ▼]

Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutro- phils during degranulation or after lysis. Post-thaw semen contains MPO, and concen- tration of this enzyme is associated with decreased motility. The aim of this study was to determine kinetics of MPO concentration during freezing, its origin, and its impact on frozenethawed semen. Forty ejaculates were used. Semen was frozen using the classical freezing procedure. MPO concentrations were assayed in fresh semen, after centrifuga- tion, and after cooling down to 4 C. Post-thaw MPO assay results and spermogram characteristics were determined. MPO immunocytochemistry was performed on 4 different ejaculates at each step of freezing procedure. MPO concentration increased after cooling down to 4 C and thawing compared with fresh semen. As temperature decreased, MPO was higher or tended to be higher in post-thaw poor quality samples. Nonsperm cells showed various degrees of MPO immunostaining and were observed as epithelial cells with nuclear pyknosis and keratinization. MPO immunostaining increased in medium and decreased in nonsperm cells during freezing. Our study shows that MPO concentration in equine semen increases when temperature decreases. We hypothesize that nonsperm cells present in fresh semen could release MPO. [less ▲]

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See detailAssessment of reactive oxygen species production in cultured equine skeletal myoblasts in response to conditions of anoxia followed by reoxygenation with or without exposure to peroxidases.
Ceusters, Justine ULg; Mouithys-Mickalad, Ange ULg; de la Rebière de Pouyade, Geoffroy ULg et al

in American Journal of Veterinary Research (2012), 73(3), 426-434

Objective—To culture equine myoblasts from muscle microbiopsy specimens, examine myoblast production of reactive oxygen species (ROS) in conditions of anoxia followed by reoxygenation, and assess the ... [more ▼]

Objective—To culture equine myoblasts from muscle microbiopsy specimens, examine myoblast production of reactive oxygen species (ROS) in conditions of anoxia followed by reoxygenation, and assess the effects of horseradish peroxidase (HRP) and myeloperoxidase (MPO) on ROS production. Animals—5 healthy horses (5 to 15 years old). Procedures—Equine skeletal myoblast cultures were derived from 1 or 2 microbiopsy specimens obtained from a triceps brachii muscle of each horse. Cultured myoblasts were exposed to conditions of anoxia followed by reoxygenation or to conditions of normoxia (control cells). Cell production of ROS in the presence or absence of HRP or MPO was assessed by use of a gas chromatography method, after which cells were treated with a 3,3′-diaminobenzidine chromogen solution to detect peroxidase binding. Results—Equine skeletal myoblasts were successfully cultured from microbiopsy specimens. In response to anoxia and reoxygenation, ROS production of myoblasts increased by 71%, compared with that of control cells. When experiments were performed in the presence of HRP or MPO, ROS production in myoblasts exposed to anoxia and reoxygenation was increased by 228% and 183%, respectively, compared with findings for control cells. Chromogen reaction revealed a close adherence of peroxidases to cells, even after several washes. Conclusions and Clinical Relevance—Results indicated that equine skeletal myoblast cultures can be generated from muscle microbiopsy specimens. Anoxia-reoxygenation– treated myoblasts produced ROS, and production was enhanced in the presence of peroxidases. This experimental model could be used to study the damaging effect of exercise on muscles in athletic horses. [less ▲]

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See detailRelationship between exercise-induced systemic inflammatory like reaction and racing performance in endurance horses
Serteyn, Didier ULg; Caudron, Isabelle ULg; Lejeune, Jean-Philippe ULg et al

in Comparative Exercise Physiology (2012), 8(3/4), 213218

This study showed that systemic inflammatory like reaction is not clearly related to performance but also to horse-related factors such as intinsic capacity or training.

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See detailAntioxidant and anti-inflammatory activities of Ribes nigrum extracts.
Tabart, Jessica ULg; Franck, Thierry ULg; Kevers, Claire ULg et al

in Food Chemistry (2012), 131(4), 1116-1122

Blackcurrant berries contain high amounts of flavonoids with various health benefits as anti-inflammatory properties attributed to their antioxidant potential. Leaves and buds actually used to produce ... [more ▼]

Blackcurrant berries contain high amounts of flavonoids with various health benefits as anti-inflammatory properties attributed to their antioxidant potential. Leaves and buds actually used to produce food supplement could also exhibit such interesting properties. <br />In the literature, various methods are often used and valid indicators of the antioxidant potential of dietary substances. However these assays do not provide evidence that antioxidants have in vivo or ex vivo activity when consumed. To obtain biologically relevant information, the antioxidant activities of the extracts were evaluated on cellular models implicating the measurement of blood haemolysis, the Cellular Antioxidant Activity on endothelial cells and the anti-inflammatory activities on isolated equine stimulated neutrophils and purified myeloperoxidase. <br />These tests generally showed that the blackcurrant leaf extract have the highest antioxidant and <br />anti-inflammatory (inhibition of MPO activity and ROS production on activated neutrophils) capacities correlated to the highest total phenolics content. [less ▲]

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See detailAntioxidant and anti-inflammatory like properties of benzoic acid analogs on the oxidant response of neutrophils: structure/redox potential relationship study.
Franck, Thierry ULg; Mouithys-Mickalad, Ange ULg; Robert, Thierry ULg et al

in Free Radical Biology & Medicine (2012), 53(supplement 1),

We investigated the antioxidant capacity of phenolic acid derivatives by measuring their capacity to prevent ABTS oxidation and evaluating their anti-inflammatory like-properties on the oxidant response ... [more ▼]

We investigated the antioxidant capacity of phenolic acid derivatives by measuring their capacity to prevent ABTS oxidation and evaluating their anti-inflammatory like-properties on the oxidant response of neutrophils, especially on superoxide anion production and the activity of myeloperoxidase (MPO), an oxidant enzyme present and released by the primary granules of neutrophils. The superoxide anion production by PMA-stimulated neutrophils was measured by lucigenin-enhanced chimiluminescence (CL) and the activity of MPO by SIEFED to study the potential interaction of a molecule with the enzyme without interferences due to medium. The antioxidant and anti-inflammatory activities of the phenolic compounds were correlated to their redox potentials measured by voltammetry method, and discussed in relation to their molecular structure. The ability of the phenolic molecules to decrease ABTS oxidation and CL production was inversely correlated to their redox potential increasing as follows: propyl gallate > gallic acid > caffeic acid > 3,4-dihydroxybenzoic acid > ferulic acid > syringic acid > 2,6-dihydroxybenzoic acid > salicylic acid > benzoic acid. The number of hydroxyl groups (3) and their position (catechol) were essential for the efficacy of the molecules as stoichiometric antioxidants or scavengers. On MPO activity, the inhibitory capacity of the molecules was not really correlated with their redox potential and increased as follows: gallic acid > caffeic acid > 2,6-dihydroxybenzoic acid > propyl gallate > ferulic acid = syringic acid > 3,4-dihydroxybenzoic acid = salicylic acid > benzoic acid. The number of OH groups and the elongation of the carboxyl group were essential for the inhibition of MPO activity, probably by facilitating the interaction with the MPO active site or structure. The redox potential measurement seems to be a good technique to select stoichiometric antioxidants, but not anti-catalytic ones. [less ▲]

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See detailPolyphenol Content and Modulatory Activities of Some Tropical Dietary Plant Extracts on the Oxidant Activities of Neutrophils and Myeloperoxidase
Tsumbu, César Ndele ULg; Deby-Dupont, Ginette; Tits, Monique ULg et al

in International Journal of Molecular Sciences (2012), 13(1), 628-650

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See detailAntioxidant and Antiradical Activities of Manihot esculenta Crantz (Euphorbiaceae) Leaves and Other Selected Tropical Green Vegetables Investigated on Lipoperoxidation and Phorbol-12-myristate-13-acetate (PMA) Activated Monocytes
Tsumbu, César Ndele ULg; Deby-Dupont, Ginette; Tits, Monique ULg et al

in Nutrients (2011), 3(9), 818-838

Abelmoschus esculentus (Malvaceae), Hibiscus acetosella (Malvaceae), Manihot esculenta Crantz (Euphorbiaceae) and Pteridium aquilinum (Dennstaedtiaceae) leaves are currently consumed as vegetables by ... [more ▼]

Abelmoschus esculentus (Malvaceae), Hibiscus acetosella (Malvaceae), Manihot esculenta Crantz (Euphorbiaceae) and Pteridium aquilinum (Dennstaedtiaceae) leaves are currently consumed as vegetables by migrants from sub-Saharan Africa living in Western Europe and by the people in the origin countries, where these plants are also used in the folk medicine. Manihot leaves are also eaten in Latin America and some Asian countries. This work investigated the capacity of aqueous extracts prepared from those vegetables to inhibit the peroxidation of a linoleic acid emulsion. Short chain, volatile C-compounds as markers of advanced lipid peroxidation were measured by gas chromatography by following the ethylene production. The generation of lipid hydroperoxides, was monitored by spectroscopy using N-N′-dimethyl-p-phenylene-diamine (DMPD). The formation of intermediate peroxyl, and other free radicals, at the initiation of the lipid peroxidation was investigated by electron spin resonance, using α-(4-pyridyl-1-oxide)-N-tert-butylnitrone as spin trap agent. The ability of the extracts to decrease the cellular production of reactive oxygen species (ROS) in “inflammation like” conditions was studied by fluorescence technique using 2′,7′-dichlorofluorescine-diacetate as fluorogenic probe, in a cell model of human monocytes (HL-60 cells) activated with phorbol ester. Overall the extracts displayed efficient concentration-dependent inhibitory effects. Their total polyphenol and flavonoid content was determined by classic colorimetric methods. An HPLC-UV/DAD analysis has clearly identified the presence of some polyphenolic compounds, which explains at least partially the inhibitions observed in our models. The role of these plants in the folk medicine by sub-Saharan peoples as well as in the prevention of oxidative stress and ROS related diseases requires further consideration. [less ▲]

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See detailSpecific Immuno Extraction Followed by Enzymatic Detection (SIEFED), a new tool to measure active myeloperoxidase in complex media and to screen potential inhibitors of this enzyme.
Serteyn, Didier ULg; Mouithys-Mickalad, Ange ULg; Deby-Dupont, Ginette et al

in The 7th International Human Peroxidase Meeting, 22-25 May 2011, Brussels (2011, May 25)

A method called SIEFED (“Specific Immuno Extraction Followed by Enzymatic Detection”) was developed for the specific measurement of myeloperoxidase (MPO) activity without interference of the sample medium ... [more ▼]

A method called SIEFED (“Specific Immuno Extraction Followed by Enzymatic Detection”) was developed for the specific measurement of myeloperoxidase (MPO) activity without interference of the sample medium. In this method, MPO is first extracted out of aqueous or biological samples by its capture by immobilized anti-MPO antibodies. A washing step then allows to eliminate the solution or biological fluid with interfering materials (proteins, lipids, reducing or oxidizing molecules, ...) and in a last step the activity of MPO bound to its antibodies is measured by using a sensitive detection system containing a fluorogenic substrate, hydrogen peroxide and nitrite as reaction enhancer. SIEFED is a method of choice to measure easily, quantitatively, and specifically the active part of MPO present in biological samples or complex media. Results obtained with this technique applied to biological samples emphasize the importance to distinguish the total MPO concentration of a sample obtained by ELISA from the active part of MPO, which is the real witness of the oxidant potential of the enzyme. SIEFED is also a powerful tool to study compounds or natural extracts that could have an inhibitory effect on the activity of MPO. Since the potential inhibitor is first incubated with the enzyme solution, and further eliminated by washing after the immunocapture of MPO, the tested compound or extract cannot interfere with the chromogenic substrate used to measure the activity of MPO or with products derived from the enzyme activity. Thus, an inhibitory effect could only be attributed to a direct interaction of the tested compound with the enzyme, either on the active site or another key position of the structure. In conclusion, the SIEFED technique opens new perspectives to study pathologies in which the release of active MPO is relevant and to select interesting compounds or extracts able to modulate the MPO activity. [less ▲]

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See detailEffect of benzoic acid analogs on myeloperoxidase activity measured by a new technique to study their direct interaction with the enzyme.
Franck, Thierry ULg; Mazloum, Ali; Mouithys-Mickalad, Ange ULg et al

Poster (2011, May)

Myeloperoxidase (MPO) plays a key role in inflammatory response and constitutes a target for new drug development. The effects of some benzoic acid analogs were studied on the specific activity of human ... [more ▼]

Myeloperoxidase (MPO) plays a key role in inflammatory response and constitutes a target for new drug development. The effects of some benzoic acid analogs were studied on the specific activity of human MPO measured by SIEFED (“Specific Immunologic Extraction Followed by Enzymatic Detection”), an original method that consists of incubation of the compound with MPO, followed by capture of the enzyme by specific antibodies, washing (elimination of the compounds) and enzymatic detection of the immunocaptured enzyme. The compounds tested at 10-4, 10-5 and 10-6 M were studied in terms of structure activity relationship. Gallic acid (3,4,5-trihydroxybenzoic acid) with 3 hydroxyl groups had an important dose dependent inhibitory effect on MPO activity. Other molecules with less or without hydroxyl groups [3,4-dihydroxybenzoic acid, 2-hydroxybenzoic acid (salicylic acid) and benzoic acid] had rather an activator effect at 10-5 and 10-6 M. 2,4,6-Trihydroxybenzoic acid, with two hydroxyl groups adjacent to the carboxyl group, had a less efficient inhibitory effect. Caffeic acid (3,4-dihydroxycinnamic acid) with a propenoic acid group presented a dose dependent inhibitory effect on MPO activity contrary to its analog 3,4-dihydroxybenzoic acid. The esterification of the carboxyl group of gallic acid to obtain propyl gallate induced an activation of MPO at 10-5 and 10-6 M. Finally, the substitution of one or two hydroxyl groups by methoxyl ones (ferulic and syringic acids) decreased the efficiency of the molecules on the enzyme inhibition. The SIEFED technique appears as an innovative pharmacological tool to study the direct interaction of compounds with MPO. Number and position of hydroxyl groups and the extension of the carboxyl group of benzoic acid play a crucial role in the inhibition of MPO activity probably by facilitating the interaction with the active site or another elements of the enzyme structure. [less ▲]

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See detailModulating effects of acepromazine on the reactive oxygen species production by stimulated equine neutrophils
Sandersen, Charlotte ULg; Mouithys-Mickalad, Ange ULg; de la Rebière de Pouyade, Geoffroy ULg et al

in Veterinary Anaesthesia & Analgesia (2011), 38

To investigate the effect of acepromazine (ACP) on reactive oxygen species (ROS) production by stimulated equine neutrophils.

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See detailFlow cytometric detection of myeloperoxidase in horse neutrophils: a novel technique in equine diagnostic research.
Wauters, Jella; Franck, Thierry ULg; Pille, Frederik et al

in Veterinary Immunology and Immunopathology (2011), 144(3-4), 417-22

Myeloperoxidase (MPO) is a protein of interest due to its involvement in equine pathologies. Until now, results in equine diagnostic research were achieved through extracellular MPO detection. However ... [more ▼]

Myeloperoxidase (MPO) is a protein of interest due to its involvement in equine pathologies. Until now, results in equine diagnostic research were achieved through extracellular MPO detection. However, studying the cellular MPO content in neutrophils has revealed important insights in human diseases. This study aimed to develop a technique for the specific detection of MPO on the single cell level defining a flow cytometric protocol for the detection of both equine surface-bound and cellular MPO. Both indirect and direct labeling techniques are described which include the comparison of two secondary antibodies and two linking-fluorochromes, respectively. [less ▲]

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