Les études d’association pan-génomiques ont révélé plusieurs variants génétiques associés à des traits complexes. Les mesures érythrocytaires ont souvent fait l’objet de ce genre d’études, étant mesurées de façon routinière et précise. Comprendre comment les variations génétiques influencent ces phénotypes est primordial étant donné leur importance comme marqueurs cliniques et leur influence sur la sévérité de plusieurs maladies. En particulier, des niveaux élevés d’hémoglobine fœtal chez les patients atteints d’anémie falciforme est associé à une réduction des complications et une augmentation de l’espérance de vie. Néanmoins, la majorité des variants génétiques identifiés par ces études tombent à l’intérieur de régions génétiques non-codantes, augmentant la difficulté d’identifier des gènes causaux. L’objectif premier de ce projet est l’identification et la caractérisation de gènes influençant les traits complexes, et tout particulièrement les traits sanguins. Pour y arriver, j’ai tout d’abord développé une méthode permettant d’identifier et de tester l’effet de gènes knockouts sur les traits anthropométriques. Malgré un échantillon de grande taille, cette approche n’a révélé aucune association. Ensuite, j’ai caractérisé le méthylome et le transcriptome d’érythroblastes différentiés à partir de cellules souches hématopoïétiques et identifié plusieurs gènes potentiellement impliqués dans les programmes érythroïdes fœtaux et adultes. Par ailleurs, j’ai identifié plusieurs micro-ARNs montrant des motifs d’expression spécifiques entre les stages fœtaux et adultes et qui sont enrichis pour des cibles exprimées de façon opposée. Finalement, j’ai identifié plusieurs variants génétiques associés à l’expression de gènes dans les érythroblastes (eQTL). Cette étude a permis d’identifier des variants associés à l’expression du gène ATP2B4, qui encode le principal transporteur de calcium des érythrocytes. Ces variants, qui sont également associés à des traits sanguins et à la susceptibilité à la malaria, tombent dans un élément d’ADN spécifique aux cellules érythroïdes. La délétion de cet élément par le système CRISPR/Cas9 induit une forte diminution de l’expression du gène et une augmentation des niveaux de calcium intracellulaires. En conclusion, des échantillons de génotypages exhaustifs seront nécessaires pour étudier l’effet de gènes knockouts sur les traits complexes. Les érythroblastes montrent de grandes différences au niveau de leur méthylome et transcriptome entre les différents stages développementaux. Ces différences influencent potentiellement la régulation de l’hémoglobine fœtale et impliquent de nombreux micro-ARNs et régions régulatrices non-codantes. Finalement, l’exemple d’ATP2B4 montre qu’intégrer des études épigénomiques, transcriptomiques et des expériences d’édition de génome est une approche puissante pour caractériser des variants génétiques non-codants. Par ailleurs, ces résultats impliquent ATP2B4 dans l’hydratation des érythroblastes, qui est associé à la susceptibilité à la malaria et la sévérité de l’anémie falciforme. Cibler ATP2B4 de façon thérapeutique pourrait avoir un impact majeur sur ces maladies qui affectent des millions d’individus à travers le monde. / Genome-wide association studies (GWAS) have revealed several genetic variants associated with complex phenotypes. This is the case for red blood cell (RBC) traits, which are particularly amenable to GWAS as they are routinely and accurately measured. Understanding RBC trait variation is important given their significance as clinical markers and modifiers of disease severity. Notably, increased fetal hemoglobin (HbF) production in sickle cell disease (SCD) patients is associated with a higher life expectancy and decreased morbidity. Nonetheless, most variants identified through GWAS fall in non-coding regions of the human genome, increasing the difficulty of identifying causal links. The main goal of this project was to identify and characterize genes influencing complex traits, and in particular RBC phenotypes. First, I developed an approach to identify and test potential gene knockouts affecting anthropometric traits in a large sample from the general population, which did not yield significant associations. Then, I characterized the DNA methylome and transcriptome of erythroblasts differentiated ex vivo from hematopoietic progenitor stem cells (HPSC), and identified several genes potentially implicated in fetal and adult-stage erythroid programs. I also identified microRNAs (miRNA) that show specific developmental expression patterns and that are enriched in inversely expressed targets. Finally, I mapped expression quantitative trait loci (eQTL) in erythroblasts, and identify erythroid-specific eQTLs for ATP2B4, the main calcium ATPase of RBCs. These genetic variants are associated with RBC traits and malaria susceptibly, and overlap an erythroid-specific enhancer of ATP2B4. Deletion of this regulatory element using CRISPR/Cas9 experiments in human erythroid cells minimized ATP2B4 expression and increased intracellular calcium levels. In conclusion, large and comprehensive genotyping datasets will be necessary to test the role of rare gene knockouts on complex phenotypes. The transcriptomes and DNA methylomes of erythroblasts show substantial differences correlating with their developmental stages and that may be implicated in HbF production. These results also suggest a strong implication of erythroid enhancers and miRNAs in developmental stage specificity. Finally, characterizing the erythroid-specific enhancer of ATP2B4 suggest that integrating epigenomic, transcriptomic and gene editing experiments can be a powerful approach to characterize non-coding genetic variants. These results implicate ATP2B4 in erythroid cell hydration, which is associated with malaria susceptibility and SCD severity, suggesting that therapies targeting this gene could impact diseases affecting millions of individuals worldwide.

Abstract Cardiovascular diseases are the leading cause of death worldwide, and coronary artery disease accounts for the majority of them. The treatment of choice for complex coronary artery disease is coronary artery bypass grafting. However, as surgery in general, cardiac surgery is associated with an increased risk of perioperative bleeding and utilization of blood products. The present study aimed to investigate the impact of preoperative anemia, perioperative bleeding and retained blood syndrome as well as blood transfusion on the outcomes after isolated coronary surgery. The severity of perioperative bleeding was assessed mainly using the E-CABG and UDPB stratification criteria. Our analyses showed that severe bleeding is associated with a significantly increased risk of stroke. Furthermore, severe bleeding increased the risk of several adverse events even in low-risk patients. Retained blood syndrome was observed to be a common complication after coronary surgery and was associated with an increased risk of postoperative complications. Preoperative anemia seems to have no significant impact on patient early and late survival. Instead, the frequent exposure to blood products may be the determinant of poorer survival observed among anemic patients. Perioperative blood loss and exposure to allogeneic blood has been shown to increase adverse events. Therefore, prevention of bleeding and measures to optimize patient blood management could improve patient outcomes after cardiac surgery. / Tiivistelmä Sydän ja verisuonitaudit ovat maailmanlaajuisesti yleisin kuoleman aiheuttaja, joista sepelvaltimotaudilla on suurin vaikutus. Sepelvaltimoiden ohitusleikkaus on käypä hoito vakavassa sepelvaltimotaudissa. Kuten kirurgiassa yleisestikin, erityisesti sydänkirurgia on yhdistetty suurentuneeseen verenevuodon ja verituotteiden saannin riskiin. Tutkimukseni tavoitteena oli selvittää preoperatiivisen anemian, perioperatiivisen verenvuodon, verituotteiden annon, sekä leikkausalueelle jääneen veren itsenäisiä vaikutuksia potilaiden lopputulemiin sepelvaltimoiden ohitusleikkauksen jälkeen. Verituotteiden ja perioperatiivisen verenvuodon määrää arvioitiin pääsääntöisesti käyttäen E-CABG ja UDPB verenvuotoluokituksia. Tuloksenamme oli, että vakava verenvuoto lisää merkitsevästi aivoinfarktin riskiä. Lisäksi vakava perioperatiivinen verenvuoto on yhteydessä useisiin komplikaatioihin myös matalan leikkausriskin potilailla. Leikkausalueelle jääneen veren huomattiin olevan yleinen ongelma sepelvaltimoiden ohitusleikkauksen jälkeen, minkä lisäksi se lisäsi riskiä useille haitta-tapahtumille. Preoperatiivisella anemialla ei ollut tilastollisesti merkitsevää vaikutusta potilaiden lyhyen ja pitkän aikavälin ennusteisiin. Sen sijaan, aneemisille potilaille annetut verensiirrot saattaisivat aiheuttaa näillä potilailla huomatun alentuneen elinajan ennusteen. Perioperatiivisen verenvuodon ja altistumisen verituotteille on osoitettu lisäävän haittatapahtumia. Siispä verenvuodon vähentäminen ja verituotteiden säästäminen voisi parantaa potilaiden ennustetta sydänkirurgiassa.

L’arille de Momordica cochinchinensis (gac), un fruit de la famille des Cucurbitacées, est la source végétale la plus riche en lycopène et β-carotène. Ces deux composés ont, respectivement, un rôle de puissant antioxydant et de provitamine A, intéressant les compléments santé. Tout d'abord, un procédé d’extraction fractionnée douce a été développé pour extraire ces caroténoïdes naturels en gardant leur qualité originale. Puis, le lycopène et le β-carotène extraits ont été caractérisés et analysés. Au moins 95 % des extraits sont composés de l’isomère all-trans. Ils ne sont pas dégradés pendant le traitement thermique représentant les procédés de formulation. Leur stéréo-mutation thermique a été évaluée. Le lycopène est plus antioxydant et donc plus rapidement isomérisé que le β-carotène à haute température. L’isomérisation augmente leur activité antioxydante, qui a été évalué par test chimique TEAC et sur l’hémolyse des cellules sanguines (KRL) in vitro. Les deux caroténoïdes de l’arille de gac sont beaucoup plus antioxydants que le Trolox contre l’hémolyse. En comparant avec d’autres sources de β-carotène, les caroténoïdes extraits de gac dans ces conditions douces restent antioxydants même à des concentrations plus élevées contrairement à ceux extraits dans des conditions classiques qui deviennent prooxydants. Ces résultats permettent de discuter la bioactivité des caroténoïdes d'après leur qualité et de leur origine, c’est à dire leur source et leur procédé d’extraction. D’un point de vue applicatif, outre le procédé de fractionnement qui est industrialisable, le traitement thermique appliqué permet de contrôler la fonctionnalité des produits riches en caroténoïdes. / The aril of Momordica cochinchinensis (gac), plant from the Cucurbitaceae family, is the richest source of lycopene and β-carotene, which are a strong antioxidant and a pro-vitamin A, respectively, interesting for health-complements. First, a process of soft extraction-fractionation was developed for extracting effectively the natural carotenoids from gac without loss of their original quality. Then, the lycopene and β-carotene extracted from gac were analyzed and characterized. At least 95% of the extracts were composed of the all-trans isomer. They were not degraded during the heat-treatment mimicking formulation processing. Their thermal stereo-mutation was evaluated. Lycopene is more antioxidant, it is thus isomerized more rapidly than β-carotene at high temperature. The isomerization of carotenoids increases their antioxidant activity that was evaluated by the chemical test TEAC and through the hemolysis of red blood cells (KRL) in vitro. The lycopene and β-carotene from gac are notably more antioxidant than Trolox. By comparing with other sources of β-carotene, carotenoids extracted from gac in these soft conditions keep their antioxidant properties, even at high concentration, contrasting with extracts obtained in classical conditions that become prooxidant. From these results, the bioactivity of carotenoids is discussed from their quality and their origin that is their source and extraction process. For application, in addition to the fractionation process which is easily transferable to the industry scale, the heat-treatment used in this study is interesting for controlling products rich in functional carotenoids

Made available in DSpace on 2016-01-26T18:55:35Z (GMT). No. of bitstreams: 1 deyvid.pdf: 251965 bytes, checksum: 4ba035cb4ab8b1227779ef5ba9b57cb3 (MD5) Previous issue date: 2012-09-26 / The white tea in a healthy drink, but this tea can interfere in some growth factors involved in the reproduction and in the metabolism. So, the aim of this work was to verify the effect of the white tea consumption in the metabolism, number of corpora lutea and in the weight of superovulated rats. There were utilized two groups of rats: control (n=30) that received water and the group that received only white tea to drink (n=30). The experiment lasts three months and the liquid and ration consumption were verified. In the end of each month, 10 rats of each group were superovulated with 150 UI/Kg of eCG, and 150UI/Kg of hCG 48 hours later. After 48 hours of the superovulation, the rats were weighted, sacrificed and the ovaries were weighted and the corpora lutea counted, besides the blood of animals were collected for hemogram and serum biochemistry. Não houve diferença no número de corpos lúteos e peso dos ovários entre os grupos. The statistical analysis was ANOVA test followed of Tukey analysis, differences were considered when P<0.05. The group treated with white tea showed higher ingestion of ration (39.63 &#61617; 3.91 g) than control group (37.19 &#61617; 4.00 g; P<0.05). However there wasn t any difference in the animals weight. Moreover, the treated group presented lower cholesterol concentration (32.02 &#61617; 9.62 mg/dL) than control group (76.70 &#61617; 10.41 mg/dL; P<0.05). No one statistical difference was observed in the number of corpora lutea, neither in the ovaries weight between the groups. The conclusion that white tea inhibis the excessive weight gain and decreases the cholesterol concentration; furthermore the white tea does not interfere with number of corpora lutea neither in the ovaries weight of superovulated rats. / O chá branco é uma bebida saudável, porém este chá pode interferir em vários fatores de crescimento envolvidos na reprodução e no metabolismo. Portanto, este trabalho teve como objetivo verificar o efeito do consumo prolongado de chá branco no metabolismo, no número de corpos lúteos e no peso dos ovários de ratas superovuladas. Foram utilizados dois grupos de ratas: controle (n=30) que recebeu água e o grupo que recebeu apenas chá branco para beber (n=30). O experimento durou 3 meses, o consumo de líquido e de ração foi verificado. Ao final de cada mês, 10 ratas de cada grupo eram superovuladas, com 150 UI/Kg de eCG e mais 150UI/Kg de hCG depois de 48 horas. Após 48 horas da superovulação, as ratas foram pesadas, sacrificadas, os ovários foram pesados e os corpos lúteos contados. O sangue dos animais foi colhido para hemograma e bioquímica sérica. A análise estatística foi de ANOVA seguida do teste de Tukey, foram consideradas diferenças estatísticas quando P<0,05. O grupo tratado com chá branco apresentou maior ingestão de ração (39,63 &#61617; 3,91 g) em relação ao controle (37,19 &#61617; 4,00 g; P<0,05), porém não houve diferença no peso dos animais, o grupo tratado apresentou menor concentração de colesterol (32,02 &#61617; 9,62 mg/dL) que o grupo controle (76,70 &#61617; 10,41 mg/dL; P<0,05). Não houve diferença no número de corpos lúteos e peso dos ovários entre os grupos. Conclui-se que o consumo de chá branco evita o ganho excessivo de peso dos animais, diminui a concentração sérica de colesterol e não interfere no número de corpos lúteos e no peso dos ovários de ratas superovuladas.

Made available in DSpace on 2016-07-18T17:53:10Z (GMT). No. of bitstreams: 1 deyvid.pdf: 251965 bytes, checksum: 4ba035cb4ab8b1227779ef5ba9b57cb3 (MD5) Previous issue date: 2012-09-26 / The white tea in a healthy drink, but this tea can interfere in some growth factors involved in the reproduction and in the metabolism. So, the aim of this work was to verify the effect of the white tea consumption in the metabolism, number of corpora lutea and in the weight of superovulated rats. There were utilized two groups of rats: control (n=30) that received water and the group that received only white tea to drink (n=30). The experiment lasts three months and the liquid and ration consumption were verified. In the end of each month, 10 rats of each group were superovulated with 150 UI/Kg of eCG, and 150UI/Kg of hCG 48 hours later. After 48 hours of the superovulation, the rats were weighted, sacrificed and the ovaries were weighted and the corpora lutea counted, besides the blood of animals were collected for hemogram and serum biochemistry. Não houve diferença no número de corpos lúteos e peso dos ovários entre os grupos. The statistical analysis was ANOVA test followed of Tukey analysis, differences were considered when P<0.05. The group treated with white tea showed higher ingestion of ration (39.63 &#61617; 3.91 g) than control group (37.19 &#61617; 4.00 g; P<0.05). However there wasn t any difference in the animals weight. Moreover, the treated group presented lower cholesterol concentration (32.02 &#61617; 9.62 mg/dL) than control group (76.70 &#61617; 10.41 mg/dL; P<0.05). No one statistical difference was observed in the number of corpora lutea, neither in the ovaries weight between the groups. The conclusion that white tea inhibis the excessive weight gain and decreases the cholesterol concentration; furthermore the white tea does not interfere with number of corpora lutea neither in the ovaries weight of superovulated rats. / O chá branco é uma bebida saudável, porém este chá pode interferir em vários fatores de crescimento envolvidos na reprodução e no metabolismo. Portanto, este trabalho teve como objetivo verificar o efeito do consumo prolongado de chá branco no metabolismo, no número de corpos lúteos e no peso dos ovários de ratas superovuladas. Foram utilizados dois grupos de ratas: controle (n=30) que recebeu água e o grupo que recebeu apenas chá branco para beber (n=30). O experimento durou 3 meses, o consumo de líquido e de ração foi verificado. Ao final de cada mês, 10 ratas de cada grupo eram superovuladas, com 150 UI/Kg de eCG e mais 150UI/Kg de hCG depois de 48 horas. Após 48 horas da superovulação, as ratas foram pesadas, sacrificadas, os ovários foram pesados e os corpos lúteos contados. O sangue dos animais foi colhido para hemograma e bioquímica sérica. A análise estatística foi de ANOVA seguida do teste de Tukey, foram consideradas diferenças estatísticas quando P<0,05. O grupo tratado com chá branco apresentou maior ingestão de ração (39,63 &#61617; 3,91 g) em relação ao controle (37,19 &#61617; 4,00 g; P<0,05), porém não houve diferença no peso dos animais, o grupo tratado apresentou menor concentração de colesterol (32,02 &#61617; 9,62 mg/dL) que o grupo controle (76,70 &#61617; 10,41 mg/dL; P<0,05). Não houve diferença no número de corpos lúteos e peso dos ovários entre os grupos. Conclui-se que o consumo de chá branco evita o ganho excessivo de peso dos animais, diminui a concentração sérica de colesterol e não interfere no número de corpos lúteos e no peso dos ovários de ratas superovuladas.

Einleitung Zytokine bewirken maßgeblich die Kommunikation und Koordination der zellulären und humoralen Effektorsysteme der angeborenen und erworbenen Immunität. Die Immunzellen stellen selbst die Hauptproduzenten der Zytokine dar. Pro- und anti-inflammatorische Zytokine nehmen nicht nur innerhalb der Zellkommunikation ablaufender Immun- und Entzündungsreaktionen eine Schlüsselrolle ein, sondern sind somit ebenso am Ablauf von Pathogenesen zahlreicher Erkrankungen beim Pferd beteiligt. Trotz verschiedener Studien anhand unterschiedlicher Modelle existiert keine einheitliche Datenlage zu validierten, vergleichbaren in vivo-nahen Zellkultursystemen, die es erlauben die Kinetiken equiner Zytokine als Grundlage zur weiterführenden Erforschung von Zytokinwechselwirkungen sowie zur Testung potentieller Arzneimittel abzubilden. Aktuell werden insbesondere Glukokortikoide weiterhin aufgrund ihrer anti-inflammatorischen und immunmodulatorischen Eigenschaften häufig, aber in Bezug auf Zytokine unspezifisch zur Therapie equiner Erkrankungen eingesetzt. Ziele der Untersuchung Das Ziel der vorliegenden Studie bestand darin, eine einfache, schnelle, günstige und reproduzierbare Methodik zur ex vivo-Messung von Zytokinen (Tumornekrosefaktor-alpha [TNF-α], Interleukin-1 Rezeptor-Antagonist [IL-1Ra] und Interferon gamma [IFN-γ]) und deren zeit- und konzentrationsabhängige Freisetzung in der equinen Vollblutzellkultur zu entwickeln. Anhand dessen sollte weiterführend der Effekt der Glukokortikoide Dexamethason (DEX) und Hydrocortison (HC) auf die Produktion von TNF-α, IL-1Ra und IFN-γ im equinen Vollblut untersucht werden. Zurzeit sind Zytokine, ihre Freisetzung sowie das Eintreten ihrer vermittelten Effekte Gegenstand der gegenwärtigen Forschung, mit dem Ziel spezifische Wechselwirkungen aufzuzeigen und somit zielgerichtete Therapeutika etablieren zu können. Insbesondere Pferde, die eine Anfälligkeit gegenüber mit Sepsis einhergehenden Erkrankungen oder für equines Asthma aufweisen, könnten davon profitieren. Material und Methoden Hierfür wurde Pferdevollblut, in den Verdünnungen zu 10%, 20% und 50% eingesetzt und mit Lipopolysaccharid (LPS), einer Kombination aus Phytohämagglutinin, Concanavalin A, Pokeweed-Mitogen und LPS (PCPwL) oder equinem rekombinantem TNF-a (erTNF-α) zur Zytokinfreisetzung stimuliert. Zur Erstellung der Zytokinkinetiken wurden Zellkulturüberstände zu verschiedenen Zeitpunkten gesammelt und die Konzentrationen von TNF-α, IL-1Ra und IFN-γ mit spezifischen enzyme-linked immunosorbent assays (ELISA) analysiert. In weiterführenden Versuchen wurden in der etablierten equinen Vollblutzellkultur DEX und HC in Konzentrationen von 10-12 - 10-5 M eingesetzt, um die LPS- oder PCPwL- induzierte Zytokinfreisetzung zu modulieren. Statistische Analysen erfolgten über die Berechnungen der Mittelwerte mit den dazugehörigen Standardfehlern. Signifikanzen wurden über ein- und zweifaktorielle ANOVA bestimmt. Ergebnisse Die durchgeführten Untersuchungen ergaben, dass die optimale Detektion der Zytokine in equinen Vollblutzellkulturen mit einem Blutanteil von 20% durchgeführt werden kann. TNF-α, IL-1Ra und IFN-γ wurden zeitabhängig freigesetzt und zeigten unterschiedliche Freisetzungskinetiken. Die PCPwL- induzierte TNF-α- und IL-1Ra-Freisetzung stiegen jeweils kontinuierlich über 24 - 48 Stunden an. In ähnlicher Weise erreichte die LPS- stimulierte TNF-α-Konzentration ein Maximum zu Zeitpunkten zwischen 8 - 12 Stunden und begann daraufhin abzufallen, wohingegen die Konzentration von IL-1Ra 24 Stunden später gipfelte und vielmehr fortgeführt über 48 Stunden hinaus akkumulierte. Equines rekombinantes TNF-α konnte ebenso die IL-1Ra-Freisetzung induzieren. Die PCPwL-induzierte IFN-γ-Freisetzung begann zeitversetzt und verlief kontinuierlich ansteigend über 48 - 72 Stunden. In weiterführenden konzentrationsabhängigen Untersuchungen konnte anhand der equinen Vollblutzellkultur eine stärkere Suppression der LPS-induzierten TNF-α- und IL-1Ra-Produktion sowie der PCPwL-induzierten IFN-γ-Produktion durch DEX als durch HC nachgewiesen werden. DEX hemmte die Zytokinfreisetzung mit einer mittleren inhibitorischen Konzentration (IC50) von 0,09 μM (TNF-α), 0,453 μM (IL-1Ra) und 0,001 μM (IFN-γ), während HC IC50 Werte von 1,45 μM (TNF-α), 2,96 μM (IL-1Ra) und 0,09 μM (IFN-γ) aufwies. Schlussfolgerungen Schlussfolgernd kann zusammengefasst werden, dass sich das Model der equinen Vollblutzellkultur hervorragend eignet, um nach erfolgreicher Mitogenstimulation den zeitabhängigen Freisetzungsverlauf von Zytokinen evaluieren zu können. Somit bietet das Model der equinen Vollblutzellkultur durch die Vorteile einer einfachen, günstigen Durchführung im in vivo-nahen, physiologischen Milieu, die Möglichkeit den Zytokinstatus gesunder sowie kranker Pferde zu beurteilen und stellt seinen Nutzen und die Verlässlichkeit unter Beweis potentielle Arzneimittel und immunologische Zusammenhänge des Pferdes untersuchen zu können.:INHALTSVERZEICHNIS I ABBILDUNGSVERZEICHNIS III TABELLENVERZEICHNIS III ABKÜRZUNGSVERZEICHNIS IV 1 EINLEITUNG 1 2 LITERATURÜBERSICHT 3 2.1 Allgemeine wissenschaftliche Hintergründe 3 2.1.1 Das Blut und das Immunsystem des Pferdes 3 2.1.1.1 Zusammensetzung des equinen Blutes 3 2.1.1.2 Allgemeiner Aufbau des Immunsystems 4 2.1.2 Zytokine und Entzündungsreaktionen - Mediation der Immunantwort durch Zytokine und Chemokine 14 2.1.2.1 Pro-inflammatorische Zytokine: Tumornekrosefaktor-α und Interferon-γ 17 2.1.2.2 Anti-inflammatorische Zytokine: Interleukin-1 Rezeptor-Antagonist 19 2.2 Therapeutische Beeinflussung der Zytokin- und Mediator-Freisetzung 21 2.2.1 Inhibition der Zytokinfreisetzung durch Glukokortikoide 21 2.2.2 Inhibition der Zytokinfreisetzung durch weitere Pharmaka und Substanzen 23 2.2.2.1 NSAID 23 2.2.2.2 Small molecules und Anti-Zytokinantikörper 24 2.3 Equine Zellkulturmodelle zur Zytokindetektion 25 2.3.1 Stimulation der Zytokinfreisetzung 26 2.3.2 Vollblutzellkultursysteme und Systeme mit isolierten Zellen 27 2.4 Fragestellung der Dissertation 30 3 PUBLIKATIONEN 31 3.1 Freisetzungskinetik von TNF-α und IL-1Ra im equinen Vollblut 32 3.2 Modulation der Freisetzung von TNF-α, IL-1Ra und IFN-γ in der equinen Vollblutzellkultur durch Glukokortikoide 40 4 DISKUSSION 45 4.1 Zytokinfreisetzung im equinen Vollblut 46 4.2 Der Einfluss von Glukokortikoiden auf die Zytokinfreisetzung 52 4.3 Schlussfolgerungen 56 4.4 Ausblick 56 5 ZUSAMMENFASSUNG 58 6 SUMMARY 60 7 LITERATURVERZEICHNIS 62 8 ANHANG 72 8.1 Freisetzungskinetik von IFN-γ 72 8.2 Konzentration von TNF-α, IL-1Ra und IFN-γ in der equinen Vollblutzellkultur 72 9 DANKSAGUNG 74 / Introduction The communication and coordination between the cellular and humoral effector compartments of the innate and adaptive immunity were mainly accomplished by cytokines. Immunocompetent cells themselves represent the main source for cytokines. Pro- and anti-inflammatory cytokines not only play a pivotal role within the cell signaling of expiring immune- and inflammatory reactions but also take part in the pathogenesis of several equine diseases. Despite various studies based on different experimental setups no uniform availability of data about validated, comparable in vivo cell culture systems exists which enables the description of the kinetically time course of cytokines as foundation of further investigations of cytokine interactions as well as the testing of potential drugs. These days especially glucocorticoids are still frequently used for treatment of equine diseases because of their anti-inflammatory and immunomodulatory, but with respect to cytokines unspecific properties. Objectives of the investigations The aim of the study was to develop an easy, quick, cheap and reproducible ex vivo method for measuring cytokines (tumor necrosis factor alpha [TNF-α], interleukin-1 receptor antagonist [IL-1Ra] and interferon gamma [IFN-γ]) and their time- and concentration-dependent release in the equine whole blood cell culture. Whereby the impact of the glucocorticoids dexamethasone (DEX) and hydrocortisone (HC) on production of TNF-α, IL-1Ra and IFN-γ should be investigated subsequently. Currently, cytokines, their release and eventuation of their mediated effects are objects of actual research with the aim to reveal specific interactions and thus be able to establish purposeful therapeutic agents. This could be beneficial especially for horses which display a susceptibility to septic diseases or equine asthma. Material and Methods Therefore horse whole blood diluted to 10%, 20% and 50% was stimulated with lipopolysaccharide (LPS), a combination of phytohemagglutinin, concanavalin A, pokeweed mitogen and LPS (PCPwL) or equine recombinant TNF-α (erTNF-α). To generate cytokine kinetics TNF-α, IL-1Ra and IFN-γ were analyzed in culture supernatants, which were collected at different time points using specific enzyme-linked immunosorbent assays (ELISA). In further investigations within the equine whole blood cell culture DEX and HC were applied with concentrations between 10-12 and 10-5 M to modulate LPS- or PCPwL-induced cytokine release. Statistics were performed by calculation of means with associated standard errors. Statistical significances were assessed by one- and two-way analysis of variance. Results The evaluations revealed that cytokines could be detected optimal in whole blood cell cultures with 20% blood volume. TNF-α, IL-1Ra and IFN-γ were released time-dependently and differing kinetics were displayed. PCPwL-induced TNF-α and IL-1Ra release was enhanced continuously over 24 - 48 hours, respectively. Similarly, LPS-stimulated TNF-α was at maximum at time points between 8 - 12 hours and started to decrease thereafter, whereas IL-1Ra peaked 24 hours later and rather continued to accumulate beyond 48 hours. ErTNF-α could induce also the IL-1Ra release. PCPwL- induced IFN-γ release started time displaced and showed a continuously enhanced course over 48 - 72 hours. In subsequent investigations within equine whole blood cell culture, LPS-induced TNF-α and IL-1Ra as well as PCPwL-induced IFN-γ production were more potently suppressed concentration-dependently by DEX than by HC. DEX inhibited cytokine release with the inhibition concentration (IC50) 0.09 μM (TNF-α), 0.453 μM (IL-1Ra) and 0.001 μM (IFN-γ), whereas HC with IC50 values of 1.45 μM (TNF-α), 2.96 μM (IL-1Ra) and 0.09 μM (IFN-γ). Conclusion In conclusion our results could suggest the eminent suitability of equine whole blood cell culture to assess the release of a variety of cytokines following successful mitogen stimulation. Therefore the model of the equine whole blood cell culture provides, because of its advantages including simple and cheap performance in an in vivo close physiological ambient, the opportunity to evaluate the cytokine status of healthy and diseased horses. Furthermore it could give the proof of its benefit and reliability to evaluate potential equine drugs and immunological coherences of the horse.:INHALTSVERZEICHNIS I ABBILDUNGSVERZEICHNIS III TABELLENVERZEICHNIS III ABKÜRZUNGSVERZEICHNIS IV 1 EINLEITUNG 1 2 LITERATURÜBERSICHT 3 2.1 Allgemeine wissenschaftliche Hintergründe 3 2.1.1 Das Blut und das Immunsystem des Pferdes 3 2.1.1.1 Zusammensetzung des equinen Blutes 3 2.1.1.2 Allgemeiner Aufbau des Immunsystems 4 2.1.2 Zytokine und Entzündungsreaktionen - Mediation der Immunantwort durch Zytokine und Chemokine 14 2.1.2.1 Pro-inflammatorische Zytokine: Tumornekrosefaktor-α und Interferon-γ 17 2.1.2.2 Anti-inflammatorische Zytokine: Interleukin-1 Rezeptor-Antagonist 19 2.2 Therapeutische Beeinflussung der Zytokin- und Mediator-Freisetzung 21 2.2.1 Inhibition der Zytokinfreisetzung durch Glukokortikoide 21 2.2.2 Inhibition der Zytokinfreisetzung durch weitere Pharmaka und Substanzen 23 2.2.2.1 NSAID 23 2.2.2.2 Small molecules und Anti-Zytokinantikörper 24 2.3 Equine Zellkulturmodelle zur Zytokindetektion 25 2.3.1 Stimulation der Zytokinfreisetzung 26 2.3.2 Vollblutzellkultursysteme und Systeme mit isolierten Zellen 27 2.4 Fragestellung der Dissertation 30 3 PUBLIKATIONEN 31 3.1 Freisetzungskinetik von TNF-α und IL-1Ra im equinen Vollblut 32 3.2 Modulation der Freisetzung von TNF-α, IL-1Ra und IFN-γ in der equinen Vollblutzellkultur durch Glukokortikoide 40 4 DISKUSSION 45 4.1 Zytokinfreisetzung im equinen Vollblut 46 4.2 Der Einfluss von Glukokortikoiden auf die Zytokinfreisetzung 52 4.3 Schlussfolgerungen 56 4.4 Ausblick 56 5 ZUSAMMENFASSUNG 58 6 SUMMARY 60 7 LITERATURVERZEICHNIS 62 8 ANHANG 72 8.1 Freisetzungskinetik von IFN-γ 72 8.2 Konzentration von TNF-α, IL-1Ra und IFN-γ in der equinen Vollblutzellkultur 72 9 DANKSAGUNG 74

IntroduktionDe humana blodgrupperna består av A, B, AB samt O. Antigen kan detekteras av alloantikroppar, att en reaktion sinsemellan kan ske är ett krav för att en molekyl ska kunna kallas antigen. Antigen och antikroppar är vitala för analys inom transfusionsmedicin och möjliggör transfusioner av blod samtidigt som man förhindrar allvarliga transfusionsreaktioner. För att effektivisera manuella serologiska analyser används saltlösning med låg jonstyrka. Syftet med arbetet var att verifiera lösningen MLB 2 (Bio-Rad Medical Diagnostics GmbH, Dreieich, Tyskland). Denna lösning ska förstärka reaktioner i antikroppsidentifiering, blodgruppskontroll med antikropps-screening samt förenlighetsprövning, men även för fenotypning av erytrocyter gällande antigenen s, Fya, Fyb, Kpa, Kpb samt Lua. MetodTotalt fenotypades 61 prover varav 31 var positiva och 30 var negativa för sökt fenotyp. Av de 31 positiva var 17 fenotyper av känt heterozygot anlag. Vid varje analystillfälle utfördes kontroller som bestod av heterozygot positiva samt negativa celler från Örebropanelen för vardera fenotyp. För antikroppsidentifiering ämnade två antikroppar, anti-E samt anti-Lea, att analyseras från två prover där de tidigare identifierats. Beträffande blodgruppskontroll med antikroppsscreening samt förenlighetsprövning analyserades tre prover från patienter som varit i behov av transfusioner och som tidigare påvisat antikroppar. Resultat och slutsatsSamtliga provers resultat överensstämde fullständigt vid fenotypning. För antikroppsidentifiering bekräftas tidigare påvisad anti-E för ett av proven, men för det andra provet erhölls inte resultat fullständigt överensstämmande med patientens anti- Lea. För blodgruppskontroller med antikropps-screen samt förenlighetsprövning överensstämde samtliga resultat. Effekten av förstärkningslösningen MLB 2 bedöms som god. Tydligt blir dock att gelteknik, som idag används som golden standard, är överlägsen de äldre rörteknikerna. / BackgroundHuman blood groups are defined as A, B, AB and O. Antigens are detectable by alloantibodies and a reaction between them is necessary in order to denominate as such. Antigens and antibodies respectively are essential within immunohematology to enable transfusion therapy while evading severe transfusion reactions. To improve the efficiency of manual serological test, low ionic strength saline is used. The purpose of this study was to verify MLB 2 (Bio-Rad Medical Diagnostics GmbH, Dreieich, Germany) for use as low ionic saline solution (LISS) in indirect agglutination technique (IAT) tube-tests for antibody identification, blood group verification with antibody-screen and compatibility test, also for phenotyping antigens s, Fya, Fyb, Kpa, Kpb and Lua. MethodsA total of 61 samples, of which 31 were previously known positive and 31 negative were tested. Of the 31 positive, 17 were heterozygous. At every assay, positive heterozygous and negative cells for each of the phenotype was applied as control, from the Örebro panel. For antibody identification, two previously positive samples were used. For blood group verification with antibody-screen and compatibility test, three samples with previous need for transfusion therapy and positive antibody screen were used. Results and conclusionAll samples phenotyped present fully consistent results compared to the previous ones. For antibody identification the results for one of the samples are completely confirmed. The second sample showed inconsistencies to the previous result. For blood group verification with antibody-screen and compatibility test all samples had equivalent results. The effect from MLB 2 in IAT-LISS tube tests are adequate. A distinct observation can be made; gel column technology is superior to the tube tests.

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<p>The pathological hallmarks of sickle cell disease originate from a single mutation of the beta hemoglobin gene resulting in a valine at position 6 instead of the canonical glutamic acid. This small change perpetuates many factors, manifesting into chronic embolic processes in the microvasculature, causing painful vaso-occlusive episodes and eventual organ failure. There have been numerous therapies developed to reduce the mortality of sickle cell ranging from agents to induce production of fetal hemoglobin to chronic blood transfusions. Although each of these options are effective at improving the quality of life for sickle cell patients, they only treat one aspect of the disease and, for some, become ineffective over time. In the hope of producing a better therapy, a better understanding of the pathogenesis of vaso-occlusive episodes is needed. While many models have been offered to account for these vaso-occlusive events, one recently proposed mechanism stems from the elevated tyrosine phosphorylation of the cytoplasmic domain of the major erythrocyte membrane protein, Band 3. Band 3 serves as a hub for many critical proteins in the red cell. It binds ankyrin, which associates the spectrin cortical cytoskeleton to the red cell membrane, deoxygenated hemoglobin, the kinases Wnk1 and OSR1, which regulate cation transport, and a glycolytic enzyme metabolon that regulates the production of ATP and glutathione. When Band 3 is tyrosine phosphorylated, each of these proteins dissociate, causing significant changes to red cell homeostasis. These changes include an accumulation of reactive oxygen species, vesiculation and release of prothrombotic microvesicles, leakage of cell free hemoglobin, and a decrease in cell volume. Normally, Band 3 exists in a predominantly unphosphorylated state, however, in sickle cell disease, Band 3 is abundantly tyrosine phosphorylated. Reduction in the tyrosine phosphorylation of Band 3 has been documented to prevent the release of microvesicles and hemoglobin from sickle cell red blood cells. Because these microvesicles and cell free hemoglobin contribute to the vaso-occlusive episodes in sickle cell patients, inhibiting the mechanism for their release offers a potential therapeutic option. But to accomplish this, the molecular cause for the elevated tyrosine phosphorylation in sickle cell disease must be identified. Since tyrosine phosphorylation is performed by a tyrosine kinase and removed by a tyrosine phosphatase, the elevation in phosphorylation must be due to changes in both of these processes. Unfortunately, the identity and nature of these kinases and phosphatases are poorly understood. In this dissertation, I identified the tyrosine kinases Syk, Lyn, and Src attributed to Band 3</p> <p>15</p> <p>phosphorylation that facilitates the release of microvesicles and hemoglobin in sickle cell red blood cells. Inhibition of Syk or one of the two Src family kinases is sufficient to prevent the destabilization of the red blood cell membrane. These kinases function in a hierarchy, where one of the three Src family kinase, Lyn phosphorylates Syk, activating it, and promoting the phosphorylation of Band 3 at tyrosines 8 and 21. Prevention of either phosphorylation event prevents the release of microvesicles and cell free hemoglobin. I also report the identification of PTP1B as the tyrosine phosphatase responsible for maintaining Band 3 in an unphosphorylated state. Interestingly, in sickle cell disease, this tyrosine phosphatase is proteolytically cleaved, resulting in a reduction in dephosphorylating potential. It has been reported previously that PTP1B is a substrate of the calcium dependent protease, calpain and that calpain inhibitors improve the cell morphology of sickle erythrocytes. Inhibition of this proteolytic process may offer an additional therapeutic option for the treatment of sickle cell disease.</p>

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