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See detailConcentration, Activity and Biochemical Characterization of Myeloperoxidase in Fresh and Post-thaw equine semen and their Implication on Freezability
Ponthier, Jérôme ULg; Franck, Thierry ULg; Parrilla Hernandez, Sonia ULg et al

in Reproduction in Domestic Animals (in press)

Myeloperoxidase (MPO) is a pro-oxidant enzyme associated with decreased motility in thawed equine semen. This study aimed to describe MPO concentration, activity and subunits in raw and thawed semen and ... [more ▼]

Myeloperoxidase (MPO) is a pro-oxidant enzyme associated with decreased motility in thawed equine semen. This study aimed to describe MPO concentration, activity and subunits in raw and thawed semen and to correlate these data with motilities in raw and thawed semen. Semen samples from five stallions were collected four times. Motilities were assessed in raw and thawed semen. MPO assays were performed in raw seminal plasma, raw sperm-rich pellet and thawed semen. Total and active MPO concentrations were, respectively, assayed by enzyme-linked immunosorbent assay and specific immunological extraction followed by enzymatic detection. MPO subunits present in semen were characterized by Western 3 blot. Purified active MPO was added in PBS and freezing extender to control its activity during freezing procedure. Differences between medians were determined using Kruskal– Wallis test, and correlations were determined using Spearman’s test for nonparametric data. Active MPO concentration was low in seminal plasma and thawed semen, but high in pellet (p = 0.0058), as the opposite relation was observed for total MPO concentration (p < 0.0001). In seminal plasma and post- thaw semen, inactive 86-kDa MPO precursor was mainly observed. Purified MPO activity was decreased in the extender (p = 0.0286). MPO activity in pellet was highly correlated with thawed progressive motility (r = 􏰑0.5576, p = 0.0086). Inac- tive MPO precursor and unknown low molecular weight inactive MPO precursor subunits explain low MPO activity in semen. Major MPO activity was observed in pellet, and post- thaw loss of activity is partially explained by MPO inactiva- tion in extender. Thawed semen motility was negatively correlated with MPO activity in pellet, becoming a potential freezability predictor. [less ▲]

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See detailThe challenge of understanding myopathies in horses using permeabilized muscle cells
Votion, Dominique ULg; Mouithys-Mickalad, Ange ULg; Ceusters, Justine ULg et al

in In proceedings 9th Conference on Mitochondrial Physiology (2013, September)

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See detailMyeloperoxidase activity decreases in equine semen freezing extenders
Ponthier, Jérôme ULg; Franck, Thierry ULg; Niesten, Ariane ULg et al

in Amir, Arav (Ed.) Proceedings of the 3rd Cryo Congress (2013, March 23)

Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils, and associated with decreased post-thaw motility of equine semen. This study aimed to compare MPO activity in pure ... [more ▼]

Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils, and associated with decreased post-thaw motility of equine semen. This study aimed to compare MPO activity in pure equine freezing extender, raw and post-thaw semen. Active MPO Concentration (AMC) was measured with Specific Immunologic Extraction Followed by Enzymatic Detection in 20 ejaculates. Raw semen intra cellular AMC was determined in the supernatant after membrane lysis, each pellet containing 100x106 spermatozoa. AMC was also assayed in supernatants of semen frozen following a conventional method using INRA FreezeTM (IMV, France). Effect of freezing procedure on AMC was tested by experimentally adding 500ng of purified active MPO (Calbiochem, Germany) in 4 samples either with 5ml of PBS or INRA FreezeTM before assay. AMC was higher in sperm-rich pellet (0.306ng/ml) than in post-thaw semen (0.002ng/ml) (p=0.058). After experimental MPO addition, no activity variation was observed during the freezing procedure (after dilution, 1, 2 hours of cooling and post-thawing) within the same medium. Purified MPO activity was decreased in INRA FreezeTM when compared to PBS at all timings of sampling (p=0.0286). When all samples were pooled, remaining activity in INRA FreezeTM was 23.93±13.13%. MPO fixation on large proteins contained in the extender experimentally reduces AMC, as previously observed in plasma. However, AMC decrease observed during semen freezing is more important than after experimental addition. That could be explained by a MPO interaction with seminal plasma, a partial MPO release or a MPO inactivation during equine semen freezing. [less ▲]

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See detailLa microbiopsie musculaire : un nouvel outil pour le suivi sportif et la détection précoce des dysfonctions musculaires
Votion, Dominique ULg; Fraipont, Audrey ULg; Robert, Céline et al

in 36ème Journée de la Recherche Equine (2010)

The objective of this study was to confirm the practical value of high-resolution respirometry (HRR) applied to biopsies to determine, in horses, the level of training, their athletic ability and for the ... [more ▼]

The objective of this study was to confirm the practical value of high-resolution respirometry (HRR) applied to biopsies to determine, in horses, the level of training, their athletic ability and for the early detection of muscular dysfunction. Materials and methods – The muscle mitochondrial respiration was determined by HRR in 20 endurance horses and 10 trotters sampled at the triceps brachii at different stages of their training. Results – Training increases mitochondrial respiration, in addition, the best performers had the highest rate of respiration. A trotter had abnormally low levels of muscle mitochondrial respiration for its level of training. This horse has presented several episodes of rhabdomyolysis during its racing season. Discussion – The biopsy is easily achievable by the attending veterinarian. This study shows that the athletic ability of horses is closely linked to respiratory muscle function, and suggests the value of HRR for performance prediction. In addition, the RHR has the ability to demonstrate mitochondrial dysfunction potentially responsible for exercise-induced rhabdomyolysis. [less ▲]

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See detailEffect of intensive exercise on plasmatic neutrophil elastase level in eventing and endurance horses
Lejeune, Jean-Philippe ULg; Sandersen, Charlotte ULg; Votion, Dominique ULg et al

in Equine Veterinary Journal. Supplement (2010), 48

Reasons for performing the study – Intensive exercise induces a systemic inflammatory response characterized by an increase of blood neutrophil count and myeloperoxidase (MPO) release. Neutrophil elastase ... [more ▼]

Reasons for performing the study – Intensive exercise induces a systemic inflammatory response characterized by an increase of blood neutrophil count and myeloperoxidase (MPO) release. Neutrophil elastase (NE) could also contribute to tissues lesions by their proteinase activities. Objective – To compare plasmatic NE concentrations before and after different forms of intensive exercise. Materials and Methods – EDTA blood samples were taken from 51 eventing horses (EvH) and 32 endurance horses (EndH) were sampled before the race (T0). Blood sampling was performed 2 h (T1) after completing either phase D of a one or two star eventing competition (n=51) or a 120 or 160 km endurance race (n=32). Plasmatic NE and MPO were measured by a specific equine ELISA. Neutrophil counts and creatine kinase (CK) levels were also measured. A Wilcoxon test for paired samples was used to compare mean values of neutrophils, CK, MPO and NE at T0 and T1 in EvH and in EndH. Correlations were calculated between all the 4 parameters in EvH and EndH. Results – At T0, mean NE levels were 14.43 ± 3.63 ng/ml for EvH and 11.7 ± 2.11 ng/ml for EndH. The competition induced a significant increase of NE levels in (58.57 ± 24.06 ng/mL) EvH and (95.74 ± 22.70 ng/mL) EndH (p < 0,05). NE was significantly (p < 0,0001) correlated to MPO in EvH (r = 0.293) and EndH (0.594) and to CK (r = 0.297) in EndH (p<0.0001). Neutrophils, CK and MPO were significantly increased between T0 and T1 in both types of horses. Conclusions – Significant increase of NE was observed after intense exercise with a significant correlation between NE and MPO. The huge variability in MPO and ELT, indicates, that not all horses show the same intensity of systemic inflammatory response. [less ▲]

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See detailAlterations in mitochondrial respiratory function in response to endurance training and endurance racing
Votion, Dominique ULg; Fraipont, Audrey ULg; Goachet, Anne-Gaëlle et al

in Equine Veterinary Journal. Supplement (2010), 42(38), 268-274

Objectives: To determine effects of training and racing on muscle oxidative phosphorylation (OXPHOS) and electron transport system (ETS) capacities in horses with high-resolution respirometry (HRR).

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See detailAMADEOX method : combination of various techniques to distinguish stoichiometric and anticatalytic activities of antioxidant molecules or natural extracts
Franck, Thierry ULg; Mouithys-Mickalad, Ange ULg; Kohnen, Stephan et al

in International Society of Antioxidants in Nutrition & Health, 5th International Conference on Polyphenols Applications, Bridging Bioefficacy to Innovations & Applications, October 29th-30th 2009, Malta (2009, October 29)

We developed an original method called AMADEOX (for « Action Modulatrice de l'Activité Des Enzymes Oxydantes ») able to distinguish stoichiometric, metabolic and anticatalytic activities of antioxidant ... [more ▼]

We developed an original method called AMADEOX (for « Action Modulatrice de l'Activité Des Enzymes Oxydantes ») able to distinguish stoichiometric, metabolic and anticatalytic activities of antioxidant molecules or natural extracts on stimulated neutrophils and purified myeloperoxidase (MPO), a key enzyme released by neutrophil and acting as one of the major ROS generating systems. [less ▲]

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See detailA new easy method for specific measurement of active myeloperoxidase in human biological fluids and tissue extracts
Franck, Thierry ULg; Kohnen, Stephan; Zouaoui Boudjeltia, Karim et al

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 ▲]

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See detailActivation 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.
Franck, Thierry ULg; Kohnen, Stéphane; de la Rebière de Pouyade, Geoffroy ULg et al

in Veterinary Immunology and Immunopathology (2009)

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 ... [more ▼]

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. In the supernatant of activated PMNs, total equine MPO was measured by ELISA and active MPO by the SIEFED (Specific Immunologic Extraction Followed by Enzymatic Detection) technique that allows for the study of the interaction of a compound directly with the enzyme. The stimulation of PMNs with CB-fMLP only modestly increased the release of MPO, but more than 70% of released MPO was active. PMA stimulation markedly increased the production of ROS and release of MPO, but more than 95% of released MPO was inactive. When PMNs were pre-incubated with superoxide dismutase (SOD) prior to PMA activation, the lucigenin enhanced CL, which is linked to the superoxide anion (O(2)(-)) production, was much more decreased than KMB oxidation, linked to the hydroxyl-like radical production. The addition of SOD prior to the activation of PMNs by PMA also limited the loss of the activity of released MPO. These results confirm the key role of O(2)(-) generation in the ROS cascade in PMN and reveal its critical role on MPO inactivation. [less ▲]

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