Myeloperoxidase activity decreases in equine semen freezing extenders

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 ▲]

An in vitro whole blood model to test the effects of different stimuli conditions on the release of myeloperoxidase and elastase by equine neutrophils.

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 ▲]

Association of breeding conditions with prevalence of osteochondrosis in foals

in Veterinary Record : Journal of the British Veterinary Association (2012)

Osteochondrosis (OC) is the most common developmental orthopaedic disease in horses and represents a major problem to the horse industry. The complete mechanism of this multifactorial disease is not yet ... [more ▼]

Osteochondrosis (OC) is the most common developmental orthopaedic disease in horses and represents a major problem to the horse industry. The complete mechanism of this multifactorial disease is not yet elucidated, but it is accepted that OC lesions are the result of intrinsic genetic and external factors. The aim of the present work was to evaluate the relationship between breeding management and OC. Breeding conditions were recorded, and a radiological examination was performed in 223 foals. Feeding practice and housing management were analysed in a multivariate model to determine risk factors for OC in three periods: gestation, birth to weaning and weaning to one-year-old. The major breakthrough of this study is the significant relationship between OC development and (1) the maternal nutrition during gestation and (2) the type of housing of the foals during their first year. It appears that mares fed with concentrates during gestation are more likely to produce foals that are subsequently affected by OC compared with other mares (P<0.05). Foals housed exclusively at pasture until one year of age are significantly less affected than foals exclusively housed in box or, alternatively, in box and at pasture (P<0.05). These results underline the role of the energy metabolism and the level of exercise in the aetiologic process of the disease, and help to develop preventive strategies during the crucial period of gestation to one year of age of the foal. [less ▲]

Surgical treatment of open joint injuries: a retrospective study of 22 horses.

Conference (2012, October 19)

Aims: To evaluate the prognosis of horses treated surgically for open joint injuries. Methods: The medical records of horses admitted for surgical treatment of a wound in communication with a joint were ... [more ▼]

Aims: To evaluate the prognosis of horses treated surgically for open joint injuries. Methods: The medical records of horses admitted for surgical treatment of a wound in communication with a joint were reviewed. A telephone questionnaire was used for the long term evaluation. Fisher’s exact tests were used for the statistical analyses. Results: Twenty two horses were included in the study with the following distribution of lesions: 6 carpi, 6 fetlocks, 4 tibiotarsal joints, 2 proximal and 3 distal interphalangeal joints, 1 elbow. The duration of the injury before referral ranged from 3 hours to 10 days. Surgical treatment consisted of 1-3 joint lavages. Of the 22 horses, 4 were euthanatized during hospitalization and 18 were discharged. After discharge, 3 horses died due to colic, 2 were lost and 13 were still alive. The survival was not influenced by the duration of the wound. All horses with cutaneous defects less than 5 cm and all horses affected in the lower limb (below the level of the canon) were discharged. Having an affected joint proximal to the canon was significantly associated to the need of multiple surgeries (OR: 17.5; p= 0.024). Conclusions: Even if the prognosis remains guarded for open joint injuries, a long delay between injury and treatment should not be systematically associated with a bad prognosis for survival. Open joint injuries of the lower limb were associated with survival. They required less often multiple articular lavages than open joint injuries of the upper limb, warranting thus a better prognosis. [less ▲]

Les kératomes: lésions et traitements chez 19 chevaux

Conference (2012, October 12)

Introduction: Les kératomes, masses hyperplasiques de kératine, croissent entre la phalange distale (P3) et la paroi du sabot et provoquent des boiteries chez le cheval. La littérature comporte de ... [more ▼]

Introduction: Les kératomes, masses hyperplasiques de kératine, croissent entre la phalange distale (P3) et la paroi du sabot et provoquent des boiteries chez le cheval. La littérature comporte de nombreux rapports de cas isolés mais peu de séries cliniques. Matériel et méthodes: Les dossiers cliniques des cas de kératomes présentés depuis 2004 à la faculté de médecine vétérinaire de Liège ont été revus. Résultats: Vingt-trois kératomes ont été diagnostiqués sur 19 chevaux boiteux. Un cheval présentait 2 kératomes sur le même pied et 3 chevaux présentaient 2 kératomes sur des pieds différents. Sur les 22 pieds atteints, les lésions observées étaient: un abcès, souvent récidivant (18 cas), une déformation de la muraille (6 cas), une déviation de la ligne blanche (6 cas) et une seime (3 cas). Un kératome était situé sous la sole et 22 sous la muraille. La durée des symptômes variait de 2 semaines à 15 ans (plus d'un an sur 12 pieds). Sur les radiographies, une lyse par compression de P3 était visible en regard de 20 kératomes. Le cheval présentant 2 kératomes sur le même pied a été euthanasié. Trois kératomes ont été traités de manière conservative. Dix-huit kératomes (chez 16 chevaux) ont été excisés chirurgicalement dont 16 par avulsion complète de muraille, 1 par avulsion partielle de muraille et 1 par curetage d'une portion de sole. Aucune complication postopératoire n'a été observée dans 7/18 cas (39%). Les complications rencontrées étaient: une granulation excessive (10 cas), une douleur importante pendant plusieurs jours (5 cas), une légère infection (4 cas), un enfoncement de P3 en regard de la zone avulsée (2 cas) et une récidive (2 cas, dont 1 a été réopéré avec succès ultérieurement). Un cheval est encore en convalescence au moment de cette étude. Le suivi à long terme d’un cheval a été perdu. Des 14 chevaux repris dans l'évaluation postopératoire, 1 a été euthanasié pour une autre raison que le kératome, 3 chevaux ont gardé une boiterie résiduelle au trot et 10 chevaux ont récupéré leur niveau d'activité. Discussion: La littérature décrit les kératomes comme rares1. Or, 21% des chevaux de cette étude (4 chevaux sur 19) présentaient plusieurs kératomes. De plus, dans 54 % des cas, les symptômes duraient depuis plus d’un an. Dès lors, il apparait que les kératomes sont sous diagnostiqués ou le sont souvent tardivement. La chronicité et l’étendue des kératomes de cette étude n’a que très rarement permis leur résection par avulsion partielle de muraille, technique de choix dont les complications postopératoires sont moindres². Un diagnostic précoce par inspection minutieuse des sabots, particulièrement en cas d’abcès de pied récidivant permettrait une excision par une technique moins invasive, qui diminuerait la convalescence et les complications postopératoires. Bibliographie: 1. Sundberg, J.P. et al. Neoplasms of Equidae. Journal of American veterinary medical association, 1977, 170: 150-152. 2. Boys Smith SJ. et al. Complete and partial hoof wall resection for keratoma removal: postoperative complications and final outcome in 26 horses (1994-2004). Equine Veterinary Journal, 2006, 38 (2): 127-133. [less ▲]

Sevoflurane inhibits equine myeloperoxidase release and activity in vitro.

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 ▲]

Simultaneous 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.

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 ▲]

Relationships between young stallions's temperament and their behavioral reactions during standardized veterinary examinations.

in Journal of Veterinary Behavior: Clinical Applications and Research (2012), 7(5), 311-321

Possible intracellular effect of the new water-soluble form of curcumin (NDS27) on the oxidant response of stimulated neutrophils

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 ▲]

Systemisch-entzuendliche Reaktion beim Hochleistungssportpferd

Book published by Leipziger Blaue Hefte (2012)