Thierry Franck

Effect of non-sperm cells removal with single layer colloidal centrifugation on myeloperoxidase concentration in post-thaw equine semen

Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after lysis. Post-thaw semen contains MPO and its concentration is associated with decreased sperm motility. Recently, MPO concentration in post-thaw semen was shown to be associated with the presence of non-sperm cells (NSC). The objective of this study was to evaluate the effect of a single-layer colloidal centrifugation before cryopreservation on NSC and MPO concentrations in equine semen. The experimental design consisted of freezing semen with or without previous centrifugation through two concentrations of single-layer colloid media. Non-sperm cells and MPO concentrations were assessed in pellet and upper layer at each step of the procedure and MPO was detected in cells by immunocytochemistry. Single-layer colloid centrifugation decreased NSC and MPO concentrations in post-thaw semen. The MPO concentration was correlated with concentration of NSC in the upper layer of the supernatant. In post-thaw semen, with or without previous single-layer colloid centrifugation, MPO concentration was correlated with concentration of NSC. Overall, neutrophils were rarely observed and NSC were mainly epithelial cells or cellular debris, as demonstrated by MPO immunocytochemistry. At all steps of the semen processing and cryopreservation, MPO immunostaining was clearly identified only on NSC. In conclusion, our study shows that NSC present in fresh semen release MPO during freezing.

Antioxidant potentiality of three herbal teas consumed in Bandundu rural areas of Congo.

Abstract The aim of this study was to evaluate and compare the cellular antioxidant activities of Lantana montevidensis, Lippia multiflora, and Ocimum gratissimum leaves often consumed as herbal teas in a rural area of Bandundu severely affected by konzo, which is related to oxidative damage. Consequently, dietary supplements with proven antioxidant potentialities could be of real interest to promote in this area. Phytochemical screening by TLC and HPLC-DAD of extracts revealed the presence of verbascoside as a major phenolic compound. Verbascoside in L. montevidensis and O. gratissimum is reported here for the first time. All extracts displayed high ABTS and DPPH radical-scavenging activities at the concentration range of 1–40 μg mL−1 according to order: L. multiflora > O. gratissimum > L. montevidensis. L. multiflora showed the best cellular antioxidant activity using DCFH-DA on HL-60 monocytes assay at 1–20 μg mL−1. These herbal teas may be used as nutraceuticals for their potent antioxidant activity.

Development and evaluation in vitro and in vivo of injectable hydrolipidic gels with sustained-release properties for the management of articular pathologies such as osteoarthritis.

This study aimed to evaluate glycerol monooleate (GMO) as a carrier to develop viscoelastic and injectable sustained-release drug delivery systems. The potential pro- and antioxidant activity of the developed hydrolipidic gels were evaluated by measuring the production of ROS by polymorphonuclear leukocytes (PMNs). In addition, the biocompatibility and effectiveness of two selected gel candidates were evaluated in vivo by evaluating the benefit of a single intraarticular injection of these new treatments in a model of osteoarthritis in rabbits. The in vitro study demonstrated that the carrier F1 did not have a pro-oxidative effect and even protected PMNs against natural auto-activation, regardless of the incorporation of either clonidine chlorhydrate or betamethasone dipropionate. The in vivo study demonstrated that F1 and F1-BDP induced a loss of cartilage quality in comparison to the control and reference groups but that the lesions of cartilage observed were generally mild, with not much full-depth erosion. Moreover, no exacerbating inflammation was observed when considering the synovial membranes and the PGE2 and CRP levels. These results seemed to demonstrate that the sustained-release formulation based on GMO could be well-tolerated after intraarticular injection. Moreover, it could have the potential to prevent inflammatory conditions while sustaining drug activity locally over weeks.

Substitution of egg yolk by a cyclodextrin-cholesterol complex allows a reduction of the glycerol concentration into the freezing medium of equine sperm

The aim of this work was to completely replace the egg yolk a classical diluent for freezing equine semen by a cyclodextrin-cholesterol complex. At the same time, the reduction in the glycerol content used for cryopreservation and the incubation time between sperm and the freezing media were evaluated. Horse ejaculates were frozen with four different freezing extenders: a frozen reference medium (IF) containing egg yolk and 2.5% glycerol and media without egg yolk but supplemented with 1.5xa0mg 2-hydroxypropyl-beta-cyclodextrin cholesterol (HPβCD-C) complex and containing either 1% (G1), 2% (G2) or 3% glycerol (G3). Three incubation times (90, 120 and 180xa0min) at 4xa0°C between the fresh semen and the different media were tested before freezing. Viability and motility analyses were performed with computer assisted semen analysis (CASA). Results showed that the freezing media containing the HPβCD-C complex with 1%, 2% and 3% glycerol significantly improve the 3 inxa0vitro parameters of post thawing semen quality (viability, progressive and total mobilities) compared to IF. The best improvement of the parameters was obtained with G1 medium and the longest contact time. The substitution of egg yolk by HPβCD-C complex allows the decrease of protein charge of the medium while favouring the cholesterol supply to membrane spermatozoa offering it a better resistance to osmotic imbalance and a better tolerance to the glycerol toxicity. Our results highlight that the egg yolk of an extender for the freezing of horse semen can be completely substituted by HPβCD-C complex.

Development of an HPTLC method for determination of hypoglycin A in aqueous extracts of seedlings and samaras of Acer species.

Hypoglycin A (HGA) is a toxin contained in seeds of the sycamore maple tree (Acer pseudoplatanus). Ingestion of this amino acid causes equine atypical myopathy (AM) in Europe. Another variety, A. negundo, is claimed to be present where AM cases were reported in the US. For unknown reasons, occurrence of this disease has increased. It is important to define environmental key factors that may influence toxicity of samaras from Acer species. In addition, the content of HGA in seedlings needs to be determined since AM outbreaks, during autumn period when the seeds fall but also during spring when seeds are germinating. The present study aims to validate a reliable method using high performance thin layer chromatography for determination and comparison of HGA in samaras and seedlings. The working range of the method was between 20 μg HGA to 408 μg HGA per ml water, corresponding to 12 - 244 mg/kg fresh weight or 40 - 816 mg/kg dry weight, taking into account of an arbitrary average dry matter content of 30%. Instrumental limit of detection and limit of quantification were of 10 μg HGA/ml and 20 μg HGA/ml water, respectively. Instrumental precision was 4% (RSD on 20 repeated measurements) while instrumental accuracy ranged between 86% and 121% of expected value. The HGA recovery of the analytical method estimated from spiked samaras and seedlings samples ranged between 63 and 103%. The method was applied to 9 samples of samaras from Acer pseudoplatanus, A. platanoides and A. campestre and 5 seedlings samples from A. pseudoplatanus. The results confirm detection of HGA in samaras from A. pseudoplatanus and the absence of detection in samaras of other tested species. They also suggest that detected levels of HGA are highly variable. This confirmed the suitability of the method for HGA detection in samaras or seedling.

Comparison of metabolic profiles and bioactivities of the leaves of three edible Congolese Hibiscus species

Abstract Methanolic and dichloromethane extracts from the leaves of Congolese Hibiscus species were characterised by chromatographic and spectroscopic techniques and their in vitro biochemical activities against ROS production were evaluated in cellular models and on an enzyme, myeloperoxidase (MPO), involved in inflammation. Hibiscus acetosella has a chemical fingerprint different from Hibiscus cannabinus and Hibiscus sabdariffa both having similar fingerprints. Major compounds were polyphenols, represented mainly by caffeoyl-hydroxycitric acid for H. acetosella and neochlorogenic acid for the two other species. All extracts displayed high cellular antioxidant activity with IC50 values ranging from 0.5 to 3 μg mL−1 using lucigenin on neutrophils. Dichloromethane extracts showed more efficient effects on extracellular ROS production and MPO activity. Antioxidant and anti-inflammatory activities of caffeoyl-hydroxycitric acid were significantly higher than those of neochlorogenic acid. The bioactivities of Hibiscus species were positively correlated with their phytochemical content and could justify their use as local nutraceutical resources and medicines.

Polyphenols from Silybum marianum inhibit in vitro the oxidant response of equine neutrophils and myeloperoxidase activity.

A recent study showed that silymarin, a standardized extract of S. marianum might be used in the prevention of equine laminitis. We investigated the effects of quercetin and some compounds found in silymarin (silybin, taxifolin and dehydrosilybin) on reactive oxygen species (ROS) production and myeloperoxidase (MPO) release by stimulated equine neutrophils (PMNs) and on MPO activity. All compounds (tested between 100xa0nm and 100xa0μm) inhibited superoxide anion production by stimulated PMNs in a dose-dependent manner. Dehydrosilybin and quercetin inhibited superoxide production and MPO release from 10xa0μm. Classical MPO assay showed quercetin as the most potent inhibitor, followed by taxifolin, dehydrosilybin and silybin. SIEFED MPO assay highlighting the binding of tested compounds to MPO showed that only quercetin and taxifolin maintained an efficient inhibition above 90% at 10xa0μm. Altogether, our results showed a strong inhibition of PMN activation by planar compounds such as quercetin and dehydrosilybin and a strong inhibition of MPO activity by the smallest molecules, quercetin and taxifolin. In conclusion, the compounds from silymarin may be useful for modulating the oxidative response of PMNs, involved in the pathogenesis of laminitis, but further inxa0vivo studies are needed.

Assessment of systemic inflammation by time-trends of blood granulocyte count and plasma myeloperoxidase and elastase concentrations following colic surgery in horses.

OBJECTIVEnTo determine changes in blood granulocyte counts and in plasma myeloperoxidase (MPO) and elastase (ELT) concentrations in surgical colic cases, and to determine the relationship between these changes and the surgical procedure performed, occurrence of postoperative ileus, and final outcome.nnnDESIGNnProspective clinical study conducted over a 12-month period.nnnSETTINGnUniversity teaching hospital.nnnANIMALSnFifty-three horses undergoing emergency laparotomy and surviving at least 12 hours postoperatively.nnnINTERVENTIONSnBlood samples were taken before surgery, during surgery, at the recovery from anesthesia, and then serially until the 150th hour after the first blood sampling. Granulocyte counts were performed by an automated cell hematology analyzer. Specific ELISAs were performed for the MPO and ELT measurements. Mixed models were used to compare the time-trends of the 3 parameters.nnnMEASUREMENTS AND MAIN RESULTSnTaking all horses together, the time-trends of MPO and ELT were not significantly different from each other, but they were significantly different from the granulocyte time-trend. The type of surgical procedure did not influence the time-trends of the 3 parameters. Significant changes in the granulocyte time-trends were associated with postoperative ileus and outcome. Significant changes in the MPO time-trends were associated with outcome. The ELT time-trends were not influenced by ileus or outcome.nnnCONCLUSIONSnGranulocyte counts and MPO change over time and are related to the severity of the inflammatory reaction in surgical colic cases. These time-trends may allow evaluation of treatment efficacy in an effort to modulate excessive granulocyte activation and degranulation.

Effects of isoflurane and sevoflurane on the neutrophil myeloperoxidase system of horses

Volatile anaesthestics have shown to modulate the oxidative response of polymorphonuclear neutrophils (PMNs). We investigated the effects of isoflurane and sevoflurane on the degranulation of total and active myeloperoxidase (MPO) from horse PMNs and their direct interaction with MPO activity. Whole blood from horse was incubated in 1 and 2 minimal alveolar concentrations (MAC) of isoflurane or sevoflurane for 1h and PMNs were stimulated with cytochalasin B (CB) plus N-formyl-méthionyl-leucyl-phenylalanine (fMLP). After stimulation, the plasma was collected to measure total and active MPO by enzyme-linked immunosorbent assay (ELISA) and specific immunological extraction followed by enzymatic detection (SIEFED) respectively. The effects of 1 and 2 MAC of isoflurane and sevoflurane on the peroxidase and chlorination activity of pure MPO were assessed by fluorescence using Amplex red and 3-(p-aminophenyl) fluorescein (APF) respectively and in parallel with a SIEFED assay to estimate the potential interaction of the anaesthetics with the enzyme. Although isoflurane and sevoflurane had inconsistent effects on total MPO release, both volatile agents reduced active MPO release and showed a direct inhibition on the peroxidase and the chlorination activity of the enzyme. A persistent interaction between MPO and anaesthetics was evidenced with isoflurane but not with sevoflurane.

High concentrations of Myeloperoxidase in the equine uterus as an indicator of endometritis

Intraluminal fluid and excessive abnormal hyperedema are regularly used for the diagnosis of endometritis in the mare, which is routinely confirmed by the presence of neutrophils on endometrial smears. Studies show a relation between neutrophils and myeloperoxidase (MPO), an enzyme contained in and released by neutrophils during degranulation or after cell lysis. This enzyme has been found in many fluids and tissues, and associated with different inflammatory pathologies in the horse. The aims of this study were to assess the presence and concentration of MPO in the equine uterus, and to investigate its relation with neutrophils, and other clinical signs of endometritis. Mares (n = 51) were evaluated for the presence of intraluminal fluid and excessive endometrial edema before breeding, and a small volume lavage and cytology samples were obtained. From 69 cycles, supernatant of the uterine flushes was analyzed with a specific equine MPO ELISA assay to measure MPO concentration. Cytology samples were used for the diagnosis of endometritis. Myeloperoxidase was present in the uterus of all estrus mares in highly variable concentrations. Myeloperoxidase concentrations were significantly (P < 0.05) higher in samples with positive cytologies and in the presence of intraluminal fluid. Occasionally, some samples with negative cytologies showed high MPO concentration, but the opposite was never observed. Cycles presenting hyperedema werent associated with high concentration of MPO, intraluminal fluid, or positive cytology, making it a poor diagnostic tool of endometritis.