Les tiques sont des arthropodes ectoparasites obligatoires qui se nourrissent sur une grande variété de vertébrés sur une large partie du globe. Au cours de leur repas, les tiques sécrètent dans leur salive de nombreux facteurs leur permettant de contourner bon nombre des défenses de l’hôte. Bien que la littérature rapporte beaucoup d’informations au sujet des effets du repas de la tique sur l’hôte, la nature des facteurs actifs exprimés par les glandes salivaires de la tique est peu connue. Au cours d’anciens travaux au sein du laboratoire, le crible de deux banques d’ADN complémentaires - issues de la rétro-transcription des ARN messagers synthétisés par les glandes salivaires de la tique Ixodes ricinus – a permis l’identification de 27 protéines dont l'expression est spécifiquement induite ou régulée positivement pendant le repas sanguin de la tique I. ricinus. Parmi ces protéines, la protéine Seq24, induite au cours du repas sanguin, présente la capacité de moduler les immunités innée et acquise de l’hôte. En conséquence, la protéine Seq24 a été nommée Iris pour « Ixodes ricinus Immunosuppressor ». Au cours de la présente étude, notre but fût de caractériser le rôle d’Iris et de déterminer son importance dans le repas sanguin de la tique I. ricinus.<p>La protéine Iris appartient à la famille des inhibiteurs de sérine protéases et présente une homologie significative avec l’inhibiteur d’élastase de leucocytes. Une analyse in silico a confirmé qu’Iris présentait la structure des serpines, et notamment le RCL (Reactive Center Loop), boucle responsable de l’activité anti-protéasique. Comme attendu (sur base de l’analyse in silico), Iris inhibe de manière spécifique l’activité de plusieurs sérine protéases, et en particulier l’élastase de leucocyte. Ces tests effectués, nous avons essayé de comprendre quel(s) pouvai(en)t être le(s) rôle(s) d’Iris dans l’accomplissement du repas sanguin de la tique, c’est à dire dans la lutte contre les différents systèmes de défenses de l’hôte.<p>Tout d’abord, des tests ont démontré la capacité d’Iris à inhiber les mécanismes de l’hémostase. Des tests sur du plasma et du sang complet ont montré qu’Iris allonge le temps de fibrinolyse, la voie intrinsèque de la coagulation et l’adhésion plaquettaire. L’utilisation de mutants a également démontré que si les deux premières activités sont dépendantes du RCL, et donc d’un mode de fonctionnement anti-protéolytique, l’adhésion plaquettaire est indépendante de ce système. Ce résultat met en évidence l’existence d’autres sites actifs, isolés par analyse in silico, nommés Receptor Binding Domain (RBD).<p>Un travail antérieur du laboratoire avait permis d’indiquer la capacité de la protéine recombinante Iris semi-purifiée à inhiber la production de TNF-a, d’IL-6, et d’IL-8 (cytokines pro-inflammatoires) ainsi que l’IFN-g par des PBMCs (Peripherical Blood Mononuclear Cells) humaines. Ces résultats ont été confirmés avec de la protéine purifiée. Des analyses complémentaires ont démontré qu’un mutant d’Iris - dépourvu d’activité anti-protéasique - conserve l’activité pro-inflammatoire. Là encore, ce mécanisme semble impliquer un ou plusieurs RBD. L’utilisation d’anticorps dirigés contre ces zones a permis de déterminer le domaine d’interaction (aa :105-120) impliqué dans cette fonction. D’autre part, une analyse par FACS a permis de démontrer qu’Iris interagit uniquement avec les cellules d’origine monocytaire.<p>Enfin, nous avons également analysé l’importance d’Iris au cours du repas sanguin de la tique par une approche vaccinale. Les résultats observés indiquent que 30 % des tiques nourries sur des lapins immunisés par la protéine rIris ne survivent pas au repas. / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

For the successful food intake by organisms that feed on blood is essentials presence of antihaemostatic molecules such as vasodilators, anticoagulant molecules and apyrases., Although members of family Diplozoidae (Heteronchoinea) are blood-feeding parasites on the gills of the fish, these molecules, that could disrupt host hemostasis, have not yet been identified. Thus, the aim of this study was to find molecules with potential anticoagulant activity in homogenates of whole worm bodies and excretory/secretory products of the members of family Diplozoidae. Furthermore perform bioinformatics analysis of sequences obtained from transcriptom project of Eudiplozoon nipponicum (Heteronchoinea: Diplozoidae) and selected proteins (protein domain) then expressed in a recombinant form. We tested inhibitory activity in excretory-secretory products and homogenates of members family Diplozoidae towards coagulation factors IIa and Xa and their specific fluorogenic with 4 negative and 1 positive results. From the results of two transcriptome analysis we discovered three protein families of potential anticoagulants - annexins, serpins and Kunitz-domain proteins. For further analyses we focused on the Kunitz protein family. These proteins contain one or more structurally related active domains which are able to...

La numération plaquettaire est une analyse délicate et le résultat est souvent erroné notamment du fait d’une tendance à l’agrégation in vitro dans certaines espèces animales. Il a ainsi pu être démontré chez le Chat que ce phénomène peut être inhibé par l’association d’un anticoagulant avec des inhibiteurs plaquettaires : le CTAD (Citrate, Théophylline, Adénosine et Dipyridamole). Cette association permet ainsi l’obtention de numérations plaquettaires fiables sans affecter les autres populations sanguines, mais également d’effectuer des analyses d’hémostase et de biochimie. De nouveaux intervalles de référence ont dû être établis pour certaines variables hématologiques avec les analyseurs utilisés en laboratoire et dans les cliniques vétérinaires. Par ailleurs, si les effets antiagrégants du CTAD sont moins nets chez le Chien, il peut également servir d’anticoagulant « universel », permettant de réduire le nombre de prélèvements et d’améliorer ainsi le bien-être des animaux. / The platelet count is a delicate measurement, which may often be erroneous because of the tendency of platelets from some animal species to aggregate in vitro. This study demonstrated that this effect can be inhibited in cats using CTAD (Citrate, Theophylline, Adenosine and Dipyridamole) composed of an anticoagulant and platelet inhibitors. This association provides reliable platelet counts without affecting other blood populations and also allows hemostasis and biochemical analyses. New hematological reference intervals have been established for some variables with analyzers used in clinical pathology laboratories and veterinary clinics. Furthermore, if the antiplatelet clumping effects of CTAD are less marked in canine species, the CTAD can also serve as "universal" anticoagulant, reducing the number of blood samples and thus improving animal welfare.

Hemocoagulation, blood coagulation, is an important indicator of hemostatic balance in the human body. There are many ways how to investigate blood clotting. In practice, next to the tests investigating time of coagulation cascade from view of internal way (APTT – activated partial tromboplastin time) and external way (PT – prothrombin time) is often used determining of fibrinogen concentration by Clauss method. Derived fibrinogen method determined fibrinogen concentration, too, by subtracting form the clotting curve in PT test. The reaction for Clauss method is not necessary here. Derived fibrinogen is not used much in practice. This is the reason, why the thesis related to this project will try to find relationship between concentration of fibrinogen and standard tests APTT and PT. Clinical data will be used for this.

Le choc septique est caractérisé par une intense activation cellulaire marquée par une génération excessive de microparticules (MPs), libérées dans l’espace extracellulaire suite à un remaniement de la membrane plasmique. Les MPs participeraient à la dysfonction cardiovasculaire et à la coagulopathie du choc septique. Nous avons exploré l’intérêt des MPs circulantes comme marqueurs pathogènes, en étudiant l’effet d’acides gras exogènes sur le remodelage de la membrane plasmique et la genèse des MPs, in-vitro dans un modèle de cellules en culture stimulées par une endotoxine et in vivo chez le rat septique. Nous avons ensuite caractérisé l’activation cellulaire du choc septique chez l’homme, en utilisant les MPs circulantes comme témoins de la dysfonction du compartiment vasculaire, puis montré la place des MPs comme cibles thérapeutiques potentielles au cours du choc septique, par leur modulation pharmacologique dans un modèle de choc septique chez le rat.Nous montrons ainsi l’intérêt des MPs comme un nouvel outil dans l’exploration de nouvelles pistes physiopathologiques du choc septique et comme cibles pharmacologiquement modulables à des fins éventuellement thérapeutiques. / Septic shock is characterized by an intense cell activation marked by an excessive generation of microparticles (MPs), released into the extracellular space after plasma membrane remodeling. MPs take part in cardiovascular dysfunction and coagulopathy of septic shock. We investigated the role of circulating MPs as pathogenic markers, by studying the effects of exogenous fatty acids on plasma membrane remodeling and MP generation, in vitro with cultured cells stimulated by an endotoxin and and in vivo in septic rats. We have then characterized the cellular activation of septic shock in humans, using circulating MPs as evidence of vascular dysfunction and shown the place of MPs as potential therapeutic targets, through their pharmacological modulation in a rat model of septic shock.We have therefore shown the interest of MPs as a new tool to explore septic shock pathophysiology and as therapeutic targets than can be pharmacologically modulated.

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Thesis (M. Tech.) -- Central University of Technology, Free State, 2007 / Cerebrovascular disease and coronary heart disease (CHD) are of the most important causes of morbidity and mortality amongst South Africans. The risk factor prevalence for stroke and CHD becomes altered by changes in lifestyle, including diet. In general it is suggested that lifestyle management should be the first choice when having to treat patients with increased cardiovascular risk. The prudent low-fat, high-fibre diet is regarded as an apparently healthy diet. It is suspected that this diet is effective for the control of known coronary risk factors as well as raised clotting factors. Research studies have shown the addition of dietary fibre to the diet as a promising therapeutic agent for the limited control of known coronary risk factors. The physiological effects of dietary fibre in humans are significantly influenced by the degree to which fibre is fermented in the colon. Fermentation results in the production of short-chain fatty acids (SCFAs); acetate, propionate and butyrate. The aim of this study was to examine the possible effects of different combinations of short-chain fatty acids on some metabolic risk markers. In this study a group of westernised African male volunteers was recruited and randomly assigned to three groups. Group one received a placebo. Group two received a supplement containing 50% acetate and 50% propionate. Group three received a SCFA supplement in the ratio of 70% acetate, 15% propionate and 15% butyrate. Supplementation was sustained for a period of six weeks. Blood samples were drawn during the different visits. At baseline the study group represented a group of black African men without any apparent metabolic or physical abnormalities. All measured variables fell within the normal range. In the placebo group, there was a statistically significant decrease in plasma fibrinogen levels from baseline to the end of supplementation. In the acetatepropionate supplement study group a statistically significant decrease in factor VIII (from 91.1 ± 11.2 to 90.9 ± 8.3%, respectively), and ATIII (from 114.3 ± 13.1 to 108.34 ± 9.5%), as well as a statistically significant decrease in low-density lipoprotein cholesterol (LDL-C) from 3.10 ± 0.79 to 2.64 ± 0.73 mmol/L. The significant increase in %HDL-C from 26.3 ± 6.5 to 30.2 ± 9.3% should also be noted. Both triglycerides (8%) and plasma fibrinogen (2%) showed a statistically significant increase. However, these changes are of no clinical significance. For the high-acetate supplement study group (with the addition of butyrate), a statistically significant decrease in factor VII (from 102.5 ± 13.7 to 101.1 ± 6.4%), VIII (from 92.6 ± 12.8 to 87.6 ± 6.0%), ATIII (from 109.2 ± 16.0 to 103.0 ± 9.9%) as well as fibrin monomer concentration (from 13.9 ± 2.2 to 12.1 ± 3.6 mg/L), were measured. Fibrin network compaction increased significantly from 14.2 ± 4.6 to 13.7 ± 4.0%. Other changes include a statistically significant increase in the serum-TC of 4.2%. From the results it is evident that the acetate-propionate supplement, with exclusion of butyrate, has a beneficial effect on metabolic parameters when compared to a highacetate- propionate supplement. The results do provide evidence of a possible therapeutic application for the propionate-acetate containing supplement. The specific mechanism should, however, still be investigated. It can be concluded from this study that acetate, propionate and butyrate each have different effects on human metabolism. It is evident that the use of a mixture of acetate and propionate may have a beneficial effect on patients at risk of developing CVD. Further studies that investigate the optimum ratio of these two products may lead to the development of a naturally derived therapeutic product for the prevention or treatment of CVD in black African men, as well as the population at large.

<p>Uvod: Sepsa je jedan od vodećih uzroka smrtnosti u jedinicama intenzivnog lečenja i van njih uprkos implementaciji novih dijagnostičkih i terapijskih protokola &scaron;irom sveta. Multiorganska disfunkcija (MODS), koja predstavlja najtežu formu nepovoljnog toka sepse, je u osnovi svojih patofiziolo&scaron;kih de&scaron;avanja obeležena promenama, koje se de&scaron;avaju na nivou kapilara, pre svega u endotelu. Poremećaji koagulacije koji se javljaju kao posledica ovih promena u endotelu su prepoznati kao jedan od dijagnostičkih kriterijuma prema najnovijim smernicama za dijagnostiku i lečenje sepse, međutim njihov značaj u predviđanju toka i ishoda ovog oboljenja jo&scaron; uvek nije precizno definisan. Cilj istraživanja: Odrediti koncentraciju markera endotelne aktivacije, aktivacije koagulacije, aktivnost prirodnih inhibitora koagulacije i funkcionalnost fibrinolize kod obolelih od sepse u odnosu na njihove vrednosti u zdravoj populaciji. Ispitati mogućnost upotrebe markera endotelne disfunkcije i pokazatelja poremećaja mehanizma hemostaze za postavljanje dijagnoze sepse i predikciju pojave komplikacija. Ispitati mogućnost upotrebe markera endotelne disfunkcije i pokazatelja poremećaja mehanizma hemostaze za procenu ishoda kod obolelih od sepse. Materijal i metode: Istraživanje je sprovedeno analitičkom metodom u formi studije preseka, a obuhvatilo je pacijente lečene na Odeljenju anestezije i reanimacije Urgentnog centra Kliničkog centra Vojvodine i na Klinici za infektivne bolesti Kliničkog centra Vojvodine, u Novom Sadu. Istraživanje je sprovođeno tokom 2012. i 2013. godine u trajanju od dve godine. U studiju je bilo uključeno 180 ispitanika od kojih je 150 imalo postavljenu dijagnozu sepse,a 30 ispitanika su činili kontrolnu grupu su klinički i biohemijski zdravih ispitanika, dobrovoljni davaoci krvi. Ispitanici su kategorisani u četiri grupe u odnosu na kliničko stanje i laboratorijske nalaze unutar prvih 24 časa od prijema: bolesnici sa sepsom, te&scaron;kom sepsom, septičkim &scaron;okom i multiorganskom disfunkcijom na prijemu. Nakon kategorizacije ispitanika, izračunati su APACHE II i SOFA numerički pokazatelji procene težine bolesti ispitanika. U roku 24 časa od trenutka postavljanja dijagnoze sepse, iz uzoraka krvi ispitanika, izvr&scaron;ene su predviđene laboratorijske analize u cilju praćenja endotelne aktivacije, aktivacije koagulacije i inhibicije antikoagulantnih mehanizama. U toku 48 časova od prijema, bolesnici koji nisu imali MODS na prijemu su intenzivno praćeni u cilju evidentiranja razvoja multiorganske disfunkcije, dok su bolesnici koji su imali MODS praćeni radi evidentiranja perzistiranja ili eventualne rezolucije MODS-a. Zdravstveno stanje bolesnika je praćeno tokom 28 dana od trenutka uključivanja u studiju i nakon tog perioda je evidentiran ishod lečenja u smislu preživljavanja ili smrtnog ishoda. Statistička analiza je izvr&scaron;ena pomoću statističkog paketa IBM SPSS 20 Statistics. Podaci su predstavljeni tabelarno i grafički, a statistička značajnost određivana je na nivou p&lt; 0,05. Rezultati: Vrednosti biolo&scaron;kih markera endotelne aktivacije i aktivacije koagulacije su statistički značajno povi&scaron;ene kod obolelih od sepse u odnosu na njihove vrednosti u zdravoj populaciji, dok su vrednosti prirodnih inhibitora koagulacije statistički značajno snižene kod obolelih od sepse u odnosu na njihove vrednosti u zdravoj populaciji. Vrednosti APTT-a, PT-a, D-dimera, fibrinogena, prirodnih inhibitora koagulacije i markera endotelne aktivacije (endokan i vWF antigena i aktivnosti) imaju značajan i veoma visok dijagnostički potencijal. Vrednosti biomarkera endotelne disfunkcije i pokazatelja poremećaja hemostaznog mehanizma su značajni prediktori komplikacija kod bolesnika sa sepsom. APTT, PT, D-dimer, broj trombocita, vrednosti priorodnih inhibitora koagulacije, trombomodulina, endokana i ETP-a su jednako validni u inicijalnoj proceni toka kliničke slike sepse kao i prediktivni APACHE II i SOFA skorovi. Koncentracija trombomodulina, D-dimera, ETP-a i PC su dobri prediktori nastanka MODS-a u prvih 48 časova u toku sepse. Endokan, PT, APTT, koncentracija fibrinogena, prirodnih inhibitora koagulacije i vrednosti ETP-a su značajni u predikciji mortaliteta kod bolesnika sa sepsom. Zaključci: Ukoliko bi pokazatelji aktivacije endotela i mehanizma hemostaze bili inkorporirani u određeni sistem skorovanja u cilju procene težine bolesti u smislu ishoda kod bolesnika sa sepsom, to bi moglo doneti doprinos boljoj klasifikaciji bolesnika, te primeni pravovremene i adekvatne terapije u cilju postizanja pozitivnog ishoda kod bolesnika sa sepsom. Prilikom interpretacije pokazatelja inflamacije i koagulacije neophodno je steći uvid u celokupnu sliku pro-i antikoagulantnih de&scaron;avanja koja se odvijaju tokom sepse, odnosno adekvatno proceniti pravac toka disbalansa mehanizma hemostaze da bi se eventualnim terapijskim merama mogao postići pozitivan učinak.</p> / <p>Introduction: Sepsis is one of the main causes of death in intensive care units and other hospital wards in spite of implementation of new sepsis treatment guidelines in everyday hospital practice worldwide. Changes that occur in the microvasculature, affecting primarily endothelial cell, are the basis of the pathophysiology of multiorgan dysfunction (MODS) in sepsis. Coagulation abnormalities which occur as a consequence of endothelial changes are recognized as diagnostic criteria for sepsis, but significance of these changes in the outcome prognosis and prediction of the course of sepsis is still not accurately defined. Aims: Evaluation of hemostasis related parameters and endothelial activation biomarkers values in patients with sepsis and healthy volunteers. Determination whether the levels of hemostasis-related parameters and biomarkers of endothelial activation have diagnostic significance and are they associated with MODS development and persistence in the first 48 hours of hospitalization and 28-day mortality in patients with sepsis. Material and methods: This is cross-sectional study conducted in 2012 and 2013 in the Department of Anesthesia and Reanimation at the Emergency Center of the Clinical Center of Vojvodina and in the Clinic of Infectious Disease at the Clinical Center of Vojvodina. 150 patients who fulfilled criteria for diagnosis of sepsis were included in the study. Patients were divided into 4 groups: sepsis, severe sepsis, septic shock and MODS. 30 healthy volunteers, blood donors were the control group. After the categorization of patients, during the first 24 hours of hospitalization, predictive APACHE II and SOFA scores were calculated. Hemostasis related parameters and endothelial activation biomarkers concentrations were determined within the first 24 hours of the onset of the disease. To assess the development of complication of the disease, patients were monitored for 48 hours for MODS development and persistence or resolution and for 28 days from the onset of sepsis for outcome assessment. Data were analyzed using SPSS 20.0 software and are presented in tables and graphs, statistical significance was set at p&lt; 0,05. Results: Biomarkers of endothelial and coagulation activation are significantly higher in patients with sepsis in comparison to their values in healthy volunteers, while concentrations of natural anticoagulants are significantly lower in patients with sepsis than in healthy volunteers. APTT, PT, D-dimer, fibrinogen, natural anticoagulants and biomarkers od endothelial activation (endocan and vWF antigen and activity) have diagnostic significance in patients with sepsis. Hemostasis related parameters and endothelial activation biomarkers are good prognostic factors for complication development in patients with sepsis. APTT, PT, D-dimer, platelet count, natural anticoagulants, thrombomodulin, endocan and ETP are equally valuable in early prediction of sepsis development as APACHE II and SOFA scores. Thrombomodulin, D-dimer, ETP and PC are good predictors of MODS development during the first 48 hours from sepsis onset. Endocan, PT, APTT, fibrinogen concentration, values of natural anticoagulants and ETP values are significant in 28-day mortality prediction in patients with sepsis. Conclusion: A combination of markers of endothelial dysfunction with widely used ICU scores and organ failure assessment could contribute to an early recognition of complication development and consequent death in patients with sepsis. It is necessary to obtain the full insight in pro-and anticoagulant dynamic evaluation while interpreting coagulation and inflammation processes in sepsis development, in order to accurately lead early resuscitation therapy.</p>