MEASUREMENT OF SERUM ALBUMIN BY PROTEIN ELECTROPHORESIS AND BROMOCRESOL GREEN METHODS IN CANINE AND EQUINE PATIENTSRamery, Eve ; Bureau, Fabrice ![]() in Veterinary Clinical Pathology (in press) Background: Bromocresol green method (BCG) is routinely used in veterinary laboratories to measure albumin. A good agreement between BCG and electrophoresis (SPE) in healthy domestic animals is reported ... [more ▼] Background: Bromocresol green method (BCG) is routinely used in veterinary laboratories to measure albumin. A good agreement between BCG and electrophoresis (SPE) in healthy domestic animals is reported. However, in human medicine, at low albumin and high globulin concentrations, BCG also binds to α- and β-globulins fractions. Surprisingly, there is no recent literature evaluating the BCG method in diseased domestic animals. Objective: The purpose of the present study was therefore to compare the serum albumin values obtained by SPE and BCG in canine and equine patients. Methods: Albumin measured by BCG and SPE was compared in the serum from 30 canine and 30 equine patients. Results: Good correlation was found (Spearman´s rho rs = 0.91 in dogs and 0.84 in horses). However, Altman and Bland analysis of results showed a positive bias of BCG with SPE (3.41 g/l in horses and 0.41 g/l in dogs) that increased when only hypoalbuminemic patients were considered (4.17 g/l in horses and 1.31 g/l in dogs). Conclusion: Results confirm that, despite good correlation, BCG method tends to overestimate albumin, especially in hypoalbuminemic patients. This is particularly evident in horses. [less ▲] Detailed reference viewed: 35 (2 ULg) Successful reduction of cholelithiasis in a holstein cow; Frisee, Vincent ; Ramery, Eve et alin Cattle Practice (2012), 20(1), 93-98 A pregnant 3.5 year-old high-productive Holstein cow in the third month of lactation showed colic signs, marked anorexia, and stopped milk production. Clinical examination revealed tachypnea, pyrexia and ... [more ▼] A pregnant 3.5 year-old high-productive Holstein cow in the third month of lactation showed colic signs, marked anorexia, and stopped milk production. Clinical examination revealed tachypnea, pyrexia and the mucous membranes were all congested. Signs of abdominal discomfort were observed, the abdomen was tense and painful; digestive activity was poor. Blood investigations revealed inflammation, cholestasis, and leucocytosis with marked neutrophilia. Trans-abdominal ultrasonography revealed decreased digestive transit and enlarged gall-bladder. Hepatic or biliary involvement was suspected. A right-flank exploratory laparotomy revealed a gallbladder filled with firm and mobile 0.5 to 1cm diameter fluctuant masses. Digital palpation allowed cholelith extraction through the cystic duct into the duodenum. After the surgery, the cow progressively regained a normal appetite and pain signs decreased. Blood samples analysed 10 days after the surgery still showed inflammation but to a lesser extent. Hepatic enzymes were markedly increased, indicating hepatic injury. Three months after the surgery, the cow was healthy and was inseminated. This case report suggests that cholelithiasis may be diagnosed and successfully treated in early stage of the disease in cattle, though this condition is rare in this species. This is the second report of manual cholelith extraction in a cow. [less ▲] Detailed reference viewed: 20 (4 ULg) EVALUATION OF THE CYTOTOXICITY OF ORGANIC DUST COMPONENTS ON THP1 MONOCYTES-DERIVED-MACROPHAGES USING HIGH CONTENT ANALYSISRamery, Eve ; in Environmental Toxicology (2012) Organic dust contains pathogen-associated molecular patterns (PAMPs) which can induce significant airway diseases following chronic exposure. Mononuclear phagocytes are key protecting cells of the ... [more ▼] Organic dust contains pathogen-associated molecular patterns (PAMPs) which can induce significant airway diseases following chronic exposure. Mononuclear phagocytes are key protecting cells of the respiratory tract. Several studies have investigated the effects of PAMPs and mainly endotoxins, on cytokine production. However the sub-lethal cytotoxicity of organic dust components on macrophages has not been tested yet. The novel technology of high content analysis (HCA) is already used to assess subclinical drug-induced toxicity. It combines the capabilities of flow cytometry, intracellular fluorescence probes, and image analysis and enables rapid multiple analyses in large numbers of samples. In the present study, HCA was used to investigate the cytotoxicity of the 3 major PAMPs contained in organic dust, ie. endotoxin (LPS), peptidoglycan (PGN) and β-glucans (zymosan) on THP-1 monocyte-derived macrophages. LPS was used at concentrations of 0.005, 0.01, 0.02, 0.05, 0.1 and 1 μg/ml; PGN and zymosan were used at concentrations of 1, 5, 10, 50, 100 and 500 μg/ml. Cells were exposed to PAMPs for 24 h. In addition, the oxidative burst and the phagocytic capabilities of the cells were tested. An overlap between PGN intrinsic fluorescence and red/far-red fluorescent dyes occurred, rendering the evaluation of some parameters impossible for PGN. LPS induced sub-lethal cytotoxicity at the lowest dose (from 50 ng/ml). However, the greatest cytotoxic changes occurred with zymosan. In addition, zymosan, but not LPS, induced phagosome maturation and oxidative burst. Given the fact that β-glucans can be up to 100-fold more concentrated in organic dust than LPS, these results suggest that β-glucans could play a major role in macrophage impairment following heavy dust exposure and will merit further investigation in the near future. [less ▲] Detailed reference viewed: 9 (3 ULg) Assessing fitness in endurance horsesFraipont, Audrey ; ; Ramery, Eve et alin Canadian Veterinary Journal = Revue Vétérinaire Canadienne (2012), 53 A field test and a standardized treadmill test were used to assess fitness in endurance horses. These tests discriminated horses of different race levels: horses participating in races of 120km and more ... [more ▼] A field test and a standardized treadmill test were used to assess fitness in endurance horses. These tests discriminated horses of different race levels: horses participating in races of 120km and more showed higher values of VLA4 (velocity at which blood lactate reached 4 mmol/L) and V200 (velocities at which heart rates reached 200 beats per min) than horses of lower race levels. [less ▲] Detailed reference viewed: 33 (12 ULg) Bacterial cholangiohepatitis in a dogRamery, Eve ; ; et alin Canadian Veterinary Journal = Revue Vétérinaire Canadienne (2012), 53(4), 423-425 A 9-year-old female Yorkshire terrier was presented for vomiting and diarrhoea. Blood chemistry tests revealed hepatic dysfunction, cholestasis and inflammation. Liver ultrasonography and liver biopsy ... [more ▼] A 9-year-old female Yorkshire terrier was presented for vomiting and diarrhoea. Blood chemistry tests revealed hepatic dysfunction, cholestasis and inflammation. Liver ultrasonography and liver biopsy were consistent with cholangiohepatitis. Fine needle aspiration of the gall-bladder revealed the presence of bacteria later identified as Clostridium spp. The cholangiohepatitis diagnosed was successfully treated. [less ▲] Detailed reference viewed: 29 (1 ULg) Exercise and epinephrine affect equine leukocytes distribution and gene expression in a distinct but closely related patternRamery, Eve ; ; Art, Tatiana et alin Veterinary Clinical Pathology (2011, December), 40(4), 583-584 Background: In horses-athletes, exercise has been shown to be an important regulator of immune cells and their functions, and catecholamines to be essential components of the physiological response to ... [more ▼] Background: In horses-athletes, exercise has been shown to be an important regulator of immune cells and their functions, and catecholamines to be essential components of the physiological response to exercise. Objectives: The purpose of the present study was to compare immunological reactions and molecular events during exercise and epinephrine infusion. Methods: Horses were submitted to exercise or epinephrine infusion for 10 min. Blood samples were collected at T0, T5, Tl0, T20 and T40 min for biochemistry and haematology at T0 and T20 min for RNA extraction and microarray analysis. Results: In both protocols, packed-cell-volume and neutrophils peaked at T5 and T10, and returned to baseline at T40. Lymphocytes increased at T5 with exercise and at T20 with epinephrine and then remained high until the end of the protocols. Between T0 and T20, 55 probes, coding for 25 genes, and 19 probes, coding for 11 genes, were differentially regulated with epinephrine and exercise respectively (P<0.001). Ten of the genes over-expressed with epinephrine were related to inflammatory response and leukocytes trafficking and activation. Among the genes with the highest significant fold changes following exercise were FOS, DUSP-1 and CXCL1. FOS was over-expressed in both conditions. Among genes differentially regulated, Ingenuity software identified 3 overlapping functional networks with epinephrine and 1 functional network with exercise that overlapped the 3 epinephrine networks. Conclusions: Circulating leukocytes distribution and gene expression are rapidly affected following exercise and epinephrine infusion. Distinct but closely-related sets of gene activities were shown to be altered with exercise and epinephrine, with lesser changes in the immunological pattern with physical stress than epinephrine alone, arguing for compensatory mechanisms in exercise. [less ▲] Detailed reference viewed: 48 (9 ULg) EVALUATION OF ORGANIC DUST COMPONENTS CYTOTOXICITY ON THP1 MONOCYTES-DERIVED-MACROPHAGES USING HIGH CONTENT ANALYSISRamery, Eve ; in Veterinary Clinical Pathology (2011, December), 40(4), 583 Background: Organic dust contains pathogen associated molecular patterns (PAMPs) which can induce, following chronic exposure, significant airway diseases. Mononuclear phagocytes are key protecting cells ... [more ▼] Background: Organic dust contains pathogen associated molecular patterns (PAMPs) which can induce, following chronic exposure, significant airway diseases. Mononuclear phagocytes are key protecting cells of the respiratory tract. Several studies have investigated the effects of PAMPs, and mainly endotoxins, on cytokine production. However the sub-lethal cytotoxicity of organic dust components on macrophages has not been tested yet. The novel technology of high content analysis (HCA) is already used to assess subclinical drug-induced toxicity. It combines the capabilities of flow cytometry, intracellular fluorescence probes, and image analysis and enables to perform rapid multiple analysis in large numbers of samples. Objectives: The purpose of the present study was, by using HCA, to investigate the cytotoxicity of the 3 major PAMPs contained in organic dust, ie. endotoxin (LPS), peptidoglycan (PGN) and β-glucans (zymosan) on THP-1 monocyte-derived macrophages. Methods: LPS was used at concentrations of 0.005, 0.01, 0.02, 0.05, 0.1 and 1 μg/ml; PGN and zymosan were used at concentrations of 1, 5, 10, 50, 100 and 500 μg/ml. Cells were exposed to PAMPs for 24 hours. In addition, the oxidative burst and the phagocytic capabilities of the cells were tested. Results: An overlap between PGN intrinsic fluorescence and red/far-red fluorescent dyes occurred, rendering the evaluation of some parameters impossible for PGN. LPS induced sub-lethal cytotoxicity at the lowest dose (from 10 ng/ml). However, the most spectacular changes occurred with zymosan. In addition, zymosan, but not LPS, induced phagosome maturation and oxidative burst. Conclusions: Given the fact that β-glucans can be up to 100 fold more concentrated in organic dust than LPS, these results suggest that β-glucans could play a major role in macrophages impairment following heavy dust exposure and will deserve further investigation in the near future. [less ▲] Detailed reference viewed: 23 (6 ULg) Kyste hématique valvulaire chez une vache HolsteinLamain, Guillaume ; Ramery, Eve ; Guyot, Hugues ![]() in Point Vétérinaire (2011), 320 Une vache laitière haute productrice est présentée pour une fièvre récidivante. L’animal présente des symptômes cardiaques et digestifs. Un phénomène inflammatoire et une atteinte cardiaque sont mis en ... [more ▼] Une vache laitière haute productrice est présentée pour une fièvre récidivante. L’animal présente des symptômes cardiaques et digestifs. Un phénomène inflammatoire et une atteinte cardiaque sont mis en évidence. Une endocardite bactérienne est suspectée. L’autopsie de la vache révèle l’existence d’un kyste hématique sur la valve tricuspide et une péritonite localisée ayant pour point de départ un abcès périruménal. [less ▲] Detailed reference viewed: 27 (0 ULg) Successful reduction of cholelithiasis in a Holstein cowLamain, Guillaume ; Frisee, Vincent ; Ramery, Eve et alPoster (2011, September) Detailed reference viewed: 20 (5 ULg) Accuracy of inflammation field tests in cattle practiceGuyot, Hugues ; Lamain, Guillaume ; Detilleux, Johann et alin Société Belge Francophone de Buiatrie & Vlaamse vereniging voor Buiatrie (Ed.) Proceedings of the 6th European Congress of Bovine Health Management (2011, September) Detailed reference viewed: 24 (7 ULg) Computed tomography diagnosis of pituitary abscess in a dairy cowGuyot, Hugues ; Michiels, Marie-Pierre ; Dahmani, Abdelkadir et alPoster (2011, September) Detailed reference viewed: 44 (21 ULg) Outils diagnostiques des maladies infectieuses utilisables en fermeGuyot, Hugues ; Ramery, Eve ![]() in Point Vétérinaire (2011), N° Spécial: Maladies Infectieuses des Ruminants: Actualités(314), 78-84 La médecine vétérinaire se développe et s’équipe d’outils de terrain précis pour diagnostiquer la présence d’infections voire même d’en déterminer l’agent étiologique. Des tests de terrain ont été conçus ... [more ▼] La médecine vétérinaire se développe et s’équipe d’outils de terrain précis pour diagnostiquer la présence d’infections voire même d’en déterminer l’agent étiologique. Des tests de terrain ont été conçus pour pouvoir affiner les diagnostics au chevet du patient. Pour savoir si la maladie d’un bovin examiné a une origine infectieuse, on peu recourir à des tests mesurant l’inflammation, des tests indiquant la présence de cellules inflammatoires, des tests mettant en évidence des agents infectieux au sein de prélèvements biologiques et des tests mesurant les conséquences métaboliques du passage d’un agent infectieux. Par ailleurs, des méthodes de diagnostic de terrain ont été développées pour rechercher de manière spécifique des agents infectieux dans les selles (diarrhées), le lait (mammites), l’urine (infections du système urinaire), le sang (parasites sanguins), et enfin dans les poumons (broncho-pneumonies). Par ailleurs, il est possible de prévoir une morbidité et mortalité accrues chez les jeunes veaux en testant le transfert passif de l’immunité colostrale. La recherche d’un diagnostic étiologique en médecine vétérinaire des ruminants conduit à l’établissement de thérapeutiques ciblées. Il ne faut toutefois pas oublier que les laboratoires d’analyse restent les centres de référence pour confirmer la plupart des étiologies des maladies infectieuses chez les ruminants et autres espèces. [less ▲] Detailed reference viewed: 63 (15 ULg) Effect of a combined iodine and selenium supplementation on I and Se status of cows and their calvesGuyot, Hugues ; ; Ramery, Eve et alin Journal of Trace Elements in Medicine and Biology (2011), 25(2), 118-124 Iodine (I) and selenium (Se) deficiencies are commonly reported in cattle, however, there are also studies regarding a very high iodine supply. The aim of the study was to determine the long-term effect ... [more ▼] Iodine (I) and selenium (Se) deficiencies are commonly reported in cattle, however, there are also studies regarding a very high iodine supply. The aim of the study was to determine the long-term effect of I and Se supplementation on non-pregnant cows, pregnant cows and their calves. The hypothalamus pituitary axis was investigated (TSH, T4, T3 assays) during a TRH challenge on non-pregnant cows. Twenty-four cows, half of them pregnant, were assigned into 2 diet-groups, one group with a low I (0.45 ppm) and Se (0.15 ppm) diet (LISe), the other with a high I (5.45 ppm) and Se (0.45 ppm) diet (HISe), for a period of 120 days. Nutritional (plasma iodide, urinary I, plasma Se, I content in colostrum and foetal fluids) and functional (thyrotropin, thyroid hormones, glutathione-peroxidase activity in erythrocytes) markers of I and Se status were assayed in dams at regular intervals for 120 days and in their calves at birth. A TRH challenge was performed on 8 non-pregnant cows at day 110 of the trial. At the end of the study, I and Se nutritional markers were higher in dams in the HISe group, compared to the LISe group, except for plasma Se. At birth, I nutritional markers in calves in the HISe group were higher compared to the LISe group. Reactivity of the pituitary–thyroid-axis was not influenced by I and Se supplementation. I and Se supplementation is efficient in improving newborn status. [less ▲] Detailed reference viewed: 51 (13 ULg) Le microdamier d’expression : un outil performant dans la compréhension de la réponse; Art, Tatiana ; Pirottin, Dimitri et alin Annales de Médecine Vétérinaire (2011), 154 Malgré de multiples études, les interrelations entre l’exercice, le stress et la réponse immune sont peu définies. Or, la compréhension de ces interrelations pourrait jouer un rôle très important dans ... [more ▼] Malgré de multiples études, les interrelations entre l’exercice, le stress et la réponse immune sont peu définies. Or, la compréhension de ces interrelations pourrait jouer un rôle très important dans l’amélioration de la santé et des résultats sportifs des athlètes. En effet, l’exercice peut être reconnu comme un stress. Il induit des modifications de l’équilibre homéostatique qui peuvent à leur tour altérer la réponse immunitaire de l’hôte et donc sa susceptibilité aux maladies. L’adrénaline est la molécule essentielle de tout processus de stress. La technologie des microdamiers, outil majeur d’investigation transcriptomique, permet l’étude de l’expression génique de l’ensemble du génome. Son utilisation devrait donc permettre de mieux caractériser et définir les interrelations entre l’exercice, le stress et la réponse immune. Cette revue recense les interrelations connues entre la réponse immune à un stress adrénergique d’une part et la réponse immune à l’exercice d’autre part. Elle considère en outre la contribution potentielle des microdamiers à une meilleure compréhension des effets d’un stress, et plus particulièrement celui lié à l’exercice, sur l’immunité. [less ▲] Detailed reference viewed: 39 (11 ULg) Sub-clinical diseases underlying poor performance in endurance horses: diagnostic methods and predictive testsFraipont, Audrey ; ; Ramery, Eve et alPoster (2011) Detailed reference viewed: 32 (7 ULg) Subclinical diseases underlying poor performance in endurance horses: diagnostic methods and predictive tests.Fraipont, Audrey ; ; Ramery, Eve et alin Veterinary Record : Journal of the British Veterinary Association (2011), 169(6), 154 Thirty-eight endurance horses underwent clinical and ancillary examinations, including haematological and biochemical evaluation, standardised exercise tests both on a treadmill and in the field, Doppler ... [more ▼] Thirty-eight endurance horses underwent clinical and ancillary examinations, including haematological and biochemical evaluation, standardised exercise tests both on a treadmill and in the field, Doppler echocardiography, impulse oscillometry, video endoscopy and collection of respiratory fluids. All of the examined poorly performing horses were affected by subclinical diseases, and most of them had multiple concomitant disorders. On the contrary, the well-performing horses were free of any subclinical disease. The most frequently diagnosed diseases were respiratory disorders, followed by musculoskeletal and cardiac problems. Poor performers exhibited lower speeds at blood lactate concentration of 4 mmol/l (VLA4) and at heart rates of 160 (V160) and 200 bpm (V200) on the treadmill and in the field, as well as slower recovery of heart rate. [less ▲] Detailed reference viewed: 36 (3 ULg) Emergence of bovine ehrlichiosis in Belgian cattle herdsGuyot, Hugues ; Ramery, Eve ; et alin Ticks and Tick-borne Diseases (2011), 2 Bovine ehrlichiosis is a tick-borne rickettsial disease caused by Anaplasma phagocytophilum. The disease can also be transmitted to humans. Outbreaks in cattle have been described in many European ... [more ▼] Bovine ehrlichiosis is a tick-borne rickettsial disease caused by Anaplasma phagocytophilum. The disease can also be transmitted to humans. Outbreaks in cattle have been described in many European countries. In Belgium, infections caused by Anaplasma phagocytophilum have been reported in humans and dogs; however this paper details the first report of ehrlichiosis in cattle herds in Belgium. The first case described was in a dairy herd located in east Belgium. Clinical signs included hyperthermia, polypnea and swelling of the limbs. The other case was diagnosed in a second, mixed purpose, herd in west Belgium. Within the second herd all of the affected animals came from the same pasture. All animals in that pasture showed recurrent hyperthermia and some also showed signs of mastitis and late-term abortions. Blood smears and serology revealed the presence of Anaplasma phagocytophilum in the majority of animals with pyrexia. Furthermore, the presence of leptospirosis, Neospora caninum and Q fever antibodies was tested by serological analysis but all results were negative. Paired serology for Adenovirus, BHV-4, BHV-1, BVD, PI3 and RSV-B did not show any significant seroconversion. Milk samples from cows affected by mastitis revealed minor pathogens. Fecal testing for the presence of Dictyocaulus viviparus in the first herd was negative. Recurrent pyrexia in pastured cattle is a non-specific sign, and can be related to several different pathogens. Bovine ehrlichiosis is transmitted by the tick species Ixodes ricinus which is known to be present throughout Belgium. Belgian practitioners should include ehrlichiosis in their differential diagnosis when confronted with pastured cattle suffering from recurrent pyrexia. [less ▲] Detailed reference viewed: 52 (16 ULg) Genomic expression evaluation: introduction and perspectivesRamery, Eve ; Bureau, Fabrice ; Guyot, Hugues et alConference (2010, July) In a way, disease could be considered as the result of gene expression deregulation. Knowing what genes are expressed in a particular cell or tissue and how they are regulated allow to better understand ... [more ▼] In a way, disease could be considered as the result of gene expression deregulation. Knowing what genes are expressed in a particular cell or tissue and how they are regulated allow to better understand disease mechanisms. Transcriptomic is the study of the expression of the entire genome. This technical challenge has been rendered possible as different species’ genomes began to be sequenced. Several techniques have rendered efficient this process of measuring simultaneously the expression level of a large number of genes. To date, micro-array technology is probably the best known. However, with the development of more affordable techniques, sequencing/ resequencing could compete with microarrays in the future. All together, these techniques open new ways for early diagnosis and prognosis for the practioner ‘in the field’. [less ▲] Detailed reference viewed: 36 (7 ULg) Pulmonary function, airway cytology and bronchoalveolar lavage fluid drug concentration after aerosol administration of cefquinome to horsesArt, Tatiana ; Ramery, Eve ; Fraipont, Audrey et alin Equine Veterinary Education (2010), 22(9), 473-479 The administration of antibiotics by aerosol to horses suffering from respiratory infections may partially circumvent the limitations of antimicrobial therapy, e.g. large injection volumes, low ... [more ▼] The administration of antibiotics by aerosol to horses suffering from respiratory infections may partially circumvent the limitations of antimicrobial therapy, e.g. large injection volumes, low bioavailability and risk of diarrhea. Only injectable formulations are available currently and usually contain other substances that could irritate the mucosa and induce coughing and bronchospasm. In addition, the quality of the aerosol, particularly in terms of the delivery of antibiotics to the deep parts of the lung, is unknown. Although used under field conditions, cefquinome delivered by aerosol has never been studied in horses. This study examined the safety of cefquinome injectable solution, administered by aerosol at a dose of 225 mg/inhalation to 7 healthy horses, by assessing (1) pulmonary function before and 15 min after a single inhalation, at the first day (Day 1) and the fifth day (Day 5) of a 5 day period treatment; and (2) the inflammatory status of the lung, i.e. percentage neutrophils and myeloperoxidase concentration, based on bronchoalveolar lavage (BAL) at D1 and D5. In addition, cefquinome concentrations were measured in bronchoalveolar lavage fluid after aerosol, intravenous (i.v.) and intramuscular (i.m.) administrations. A single aerosol of cefquinome injectable solution did not induce any immediate nor delayed pulmonary side effects in healthy horses and produced cefquinome concentrations in bronchoalveolar lavage (BAL) within 30 min that were higher than the minimal inhibitory concentration of the main equine respiratory pathogens. These results should stimulate further studies, especially in horses suffering from bronchial hyper-reactivity. Aerosol delivery of antibiotics may well have a role in equine therapeutics. [less ▲] Detailed reference viewed: 53 (19 ULg) Identification d’une nouvelle molécule d’intérêt chez le cheval atteint d’obstruction récurrente des voies respiratoires: La Pentraxine 3Ramery, Eve ![]() Doctoral thesis (2010) L’ORVR ou obstruction récurrente des voies respiratoires (ORVR) est la cause la plus fréquente de maladie pulmonaire chronique chez le cheval adulte. La maladie se caractérise par une hyperréactivité ... [more ▼] L’ORVR ou obstruction récurrente des voies respiratoires (ORVR) est la cause la plus fréquente de maladie pulmonaire chronique chez le cheval adulte. La maladie se caractérise par une hyperréactivité bronchique, une production excessive de mucus et une inflammation neutrophilique pulmonaire qui ont pour effet de réduire la compliance dynamique du poumon et d’augmenter la résistance des voies respiratoires au débit aérien. Alors que la maladie est une entité documentée dans la littérature depuis plus de 200 ans, les mécanismes moléculaires qui la sous-tendent restent incomplètement élucidés à ce jour. De précédentes études ont montré l'implication de variations d'expression génique, pour la plupart grâce à l'utilisation combinée de la transcription inverse et de la réaction en chaîne par polymérase (RT-PCR). Cette technique bien connue ne permet l'étude que d'un nombre limité de gènes en simultané. Les microdamiers apparaissent comme la technique de choix pour l'étude de l'expression génique à large échelle. A l’heure où cette étude a débuté, il n’existait pas de microdamier d’expression spécifiquement dédié au cheval sur le marché. Un microdamier hétérologue humain (Human Genome U133 Plus 2.0 GeneChip, Affymetrix®) a donc été utilisé pour tenter d’identifier de nouveaux gènes d’intérêt pour comprendre la pathogénie de l’ORVR chez le cheval. L'utilisation du microdamier a permis de détecter 46 gènes dont l’expression variait d'un facteur supérieur à 4 entre chevaux sains et atteints. Parmi les gènes mis en avant par le microdamier, trois gènes (CYBB, MARCKS et PTX3) présentaient un intérêt particulier au regard de la littérature et leur variation d’expression a été confirmée par RT-PCR quantitative (RT-qPCR). La pentraxine 3 (PTX3) notamment appartient à la composante humorale de la réponse immune innée. Elle agit comme un ancêtre fonctionnel des anticorps. En effet, elle reconnaît les microbes, active le complément et facilite la reconnaissance de certains pathogènes spécifiques, dont Aspergillus fumigatus (A. fumigatus), par les phagocytes. La suite du travail s’est donc focalisée sur PTX3. L’ADN complémentaire (ADNc) codant pour PTX3 et l’ADN codant pour le second exon de PTX3 ont été clonés et séquencés. L’ADN codant pour le second exon de PTX3 équin présentait une délétion de 120 paires de bases par rapport à la séquence humaine. Par ailleurs, un épissage alternatif du second exon de PTX3 a été mis en évidence, résultant en deux formes de la protéine, "épissée" (32 kilodaltons) et "complète" (42 kilodaltons). Afin d'étudier les effets de l’exposition à des poussières de foin sur l'expression de PTX3 dans les voies respiratoires, six chevaux sains et six chevaux atteints d’ORVR ont été soumis pendant dix jours à un challenge environnemental aux poussières de foin. Les chevaux ont été étudiés via des tests cliniques, fonctionnels et biochimiques. Les lavages broncho-alvéolaires (LBA) ont été récoltés lors de l'initiation du protocole et après les dix jours d'exposition aux poussières de foin. Par ailleurs, des sections bronchiques ont été réalisées chez deux chevaux atteints d'ORVR en crise et euthanasiés pour raisons éthiques et deux chevaux indemnes d'affections respiratoires provenant de l'abattoir. PTX3 était détectée dans les macrophages du LBA après exposition aux poussières de foin et dans les cellules épithéliales bronchiques. PTX3 était surexprimée dans les macrophages du LBA chez tous les chevaux après dix jours d'exposition aux poussières de foin. Cependant, le niveau de PTX3 était plus élevé dans le surnageant des cellules provenant des chevaux atteints d'ORVR que dans celui provenant des cellules des chevaux sains. Par ailleurs, il semble que PTX3 soit surexprimée dans les cellules épithéliales bronchiques chez le cheval atteint d'ORVR. Cette piste est intéressante sur le plan thérapeutique puisque l'activation de l'expression de PTX3 dans les cellules épithéliales bronchiques est sous le contrôle du facteur c-Jun N-terminal kinase (JNK), un facteur différent de celui qui contrôle l'expression de PTX3 dans les leucocytes, à savoir le facteur nucléaire kappa B (NF-κB). L'effet de différents stimuli sur la production et l’excrétion de PTX3 par les cellules du LBA a été étudié ex-vivo après mise en culture et stimulation à la poussière de foin, au lipopolysaccharide (LPS), à A. fumigatus et avec une solution saline tamponnée pour le phosphate (PBS), utilisée comme contrôle. La présence de PTX3 a été mesurée par Western Blot six heures et vingt-quatre heures après les différentes stimulations dans les cellules et dans les surnageants respectivement. PTX3 était détectée dans les cellules et dans les surnageants mais aucune différence significative d'expression n’a pu être mise en évidence que ce soit entre les différents stimuli, entre les différents temps ou avec le contrôle. Ces résultats suggèrent que la poussière de foin est un inducteur indirect de PTX3. [less ▲] Detailed reference viewed: 38 (5 ULg) |
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