Angiogenesis, the formation of new capillaries from preexisting vessels, plays an essential role in revascularization of the myocardium following myocardial infarction (MI). Interleukin-1β (IL-1β), a proinflammatory cytokine increased in the heart following MI, is shown to be essential for angiogenesis in the invasiveness of tumor cells, the progression of arthritic conditions and endometriosis, and the promotion of wound healing. Here we studied the steps of angiogenesis in response to IL-1β in cardiac microvascular endothelial cells (CMECs) and aortic tissue. Cell cycle progression analysis using flow cytometry indicated a G0/G1 phase cell cycle arrest in IL-1β-stimulated cells. IL-1β significantly reduced levels of fibrillar actin in the cytoskeleton, a pre-requisite for tube formation, as indicated by phalloidin-FITC staining. Wound healing assays demonstrated IL-1β prevents cell-to-cell contact formation. On the other hand, vascular endothelial growth factor-D (VEGF-D) initiated restoration of the cell monolayer. IL-1β significantly inhibited in vitro tube formation as analyzed by three-dimensional collagen matrix assay. Aortic ring assay demonstrated that IL-1β inhibits basal and VEGF-D-stimulated microvessel sprouting from aortic rings. The data presented here are novel and of significant interest, providing evidence that IL-1β impedes the process of angiogenesis in myocardial endothelial cells.

Celiac disease is an autoimmune disease which occurs in susceptible individuals after ingestion of food containing gluten. Gluten and its monomeric fraction gliadin induce inflammatory damage of the small intestine by activating the immune cells that react strongly to gluten peptides. Gluten peptides have the ability to activate cells of adaptive as well as innate immune system. This work is focused on the production of interleukin (IL)-1 in antigen presenting cells stimulated with peptic gliadin digest. We found that monocytes and peripheral blood mononuclear cells (PBMC) isolated from blood of celiac patients secrete significantly more IL-1α and IL-1β than cells of healthy donors after stimulation with gliadin digest. The gliadin-induced IL-1β expression is controlled by a signaling cascade that includes MAPK kinase family molecules and transcription factor NF-κB. Moreover, we found that the adaptor proteins MyD88 and TRIF as well as Toll-like receptor (TLR) 2 and 4 play a role in the signaling cascade underlying gliadin-induced IL-1β expression by using murine bone marrow derived dendritic cells (BMDC). The precursor form of IL-1β in gliadin- stimulated PBMC and murine BMDC is maturated by caspase-1. In celiac PBMC the gliadin- induced maturation and secretion of IL-1β depends on the potassium...

La goutte est une maladie inflammatoire particulièrement prévalente causée par la formation de dépôts articulaires et péri-articulaires de cristaux d’urate monosodique (UMS ou MSU) et dépendante de la cytokine interleukine-1β (IL-1β). En 2006, l’inflammasome NLRP3 a été montré comme nécessaire pour la maturation de l’IL-1β, in vitro, en réponse aux cristaux de MSU. Néanmoins, sa nécessité in vivo est un sujet de controverse. Mon travail de thèse a porté sur la caractérisation d’un modèle murin d’inflammation aiguë uratique et l’analyse de la contribution des inflammasomes dans cette pathologie. J’ai d’abord montré que notre modèle par injection sous-cutanée de cristaux de MSU donne lieu une forte inflammation des tissus mous comme cela est souvent observé lors des crises de goutte chez l’Homme. L’emploi de souris invalidées génétiquement et d’inhibitions pharmacologiques m’a permis de décrire son indépendance vis-à-vis de plusieurs composants des inflammasomes et confirme le rôle majeur de l’IL-1β. De manière intéressante, j’ai ensuite montré qu’il est possible de réduire fortement l’inflammation dans ce modèle par un traitement topique à base d’imiquimod (crème ALDARA®), un ligand synthétique de TLR7. Des expériences réalisées in vivo et in vitro m’ont permis de relier l’effet de l’imiquimod à une baisse importante de l’Il1b au niveau transcriptionel, via une signalisation faisant probablement intervenir les interférons de type I et possiblement le facteur RUNX3. Mes données montrent donc que la production d’IL-1β, dans ce modèle, est visiblement indépendante de NLRP3 mais peut être fortement abaissée par l’application topique d’imiquimod. L’imiquimod pourrait ainsi représenter une piste thérapeutique attractive. / Gout is a prevalent inflammatory disease caused by the deposition of monosodium urate crystals (MSU) in articular/periarticular areas, which strongly depends on interleukine-1β (IL-1β). In 2006, the NLRP3 inflammasome has been shown to perform IL-1β maturation in vitro after MSU crystal exposure. However, its in vivo dependence is still matter of controversy. In my thesis project, I focused on the characterization of a murine acute uratic inflammation and analysed the contribution of inflammasome components. I first showed that the subcutaneous injection of MSU crystals in mice generate a strong soft tissue inflammation as observed in human gouty crises. Then, by using genetically-modified mouse lines and pharmacological inhibitions, I demonstrated that this model is inflammasome-independent, while still requiring IL-1β secretion. Interestingly, I observed that the topical application of imiquimod (ALDARA® cream), which is a synthetic TLR7 ligand, strongly dampens inflammation. In vivo and in vitro experiments further demonstrated that this effect is linked to reduced Il1b gene expression, which linkely involves type I interferon signaling and eventually the transcription factor RUNX3. Altogether, my results show that IL-1β production is NLRP3-independent in this mouse model but can be strongly decreased by topical application of imiquimod. Therefore, imiquimod might be an attractive therapeutic option for gouty patients.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-08-30T20:39:29Z No. of bitstreams: 1 Tâmara Nogueira Caracterização da atividade antinociceptiva do extrato metanólico de adiantum latifolium Lam. em modelos experimentais de dor.pdf: 916333 bytes, checksum: bd09f406974cde64dc9bb0917d1d11b6 (MD5) / Made available in DSpace on 2012-08-30T20:39:29Z (GMT). No. of bitstreams: 1 Tâmara Nogueira Caracterização da atividade antinociceptiva do extrato metanólico de adiantum latifolium Lam. em modelos experimentais de dor.pdf: 916333 bytes, checksum: bd09f406974cde64dc9bb0917d1d11b6 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / Adiantum, um dos gêneros mais amplamente distribuídos da família Pteridaceae é empregado na medicina popular mundialmente. Neste trabalho, nós investigamos as propriedades antinociceptivas do extrato metanólico de Adiantum latifolium (EMA) em modelos animais de dor inflamatória. As propriedades farmacológicas de EMA foram avaliadas nos testes de contorção, formalina, retirada de cauda e nos modelos de edema de pata induzido por carragenina e edema de orelha induzido pelo ácido aracdônico. A toxicidade aguda de EMA, assim como seu efeito sobre o desempenho motor dos camundongos no teste de rota rod, foram investigados. Além disso, o perfil químico de EMA foi avaliado por cromatografia. A administração oral (100-400 mg/Kg) ou intraperitoneal (1-100 mg/Kg) de EMA produziu uma inibição dose-dependente do número de contorções abdominais induzidas pelo ácido acético em camundongos. Do mesmo modo, o tratamento com EMA (100 mg/Kg/IP) inibiu a hipernocicepção induzida pela formalina tanto na fase inicial quanto na fase tardia. Em contraste, EMA não alterou o limiar de resposta a estímulo térmico no teste de retirada de cauda, indicando ausência de ação central. Confirmando sua atividade antiinflamatória, EMA (100 e 200 mg/Kg/IP) inibiu eventos importantes relacionados à resposta inflamatória induzida pela carragenina ou ácido aracdônico: edema local e aumento nos níveis de interleucina-1β tecidual. Camundongos tratados com EMA (200 mg/Kg) não mostraram alteração no desempenho motor no teste de rota rod, ou sinais de toxicidade (1000 mg/Kg) durante um período de 14 dias. A análise fitoquímica preliminar indicou a presença de terpenos, esteróides, flavonóides e ácidos fenólicos, os quais podem ser responsáveis pelos efeitos antinociceptivo e/ou antiinflamatório de EMA. Os extratos metanólicos de diferentes partes da planta apresentaram atividade antinociceptiva de igual magnitude, sugerindo que o princípio ativo de EMA se distribui por toda a planta. Quando as frações do extrato foram avaliadas, a butanólica e de acetato de etila apresentaram maior eficácia, sendo consideradas as frações mais ativas. Nossos resultados demonstram que Adiantum latifolium apresenta consistente atividade antinociceptiva e antiinflamatória em diferentes modelos experimentais, possivelmente pela inibição da produção e/ou liberação de IL-1β, constituindo bom candidato para o desenvolvimento farmacológico. Palavras- / Adiantum, one of the most widely distributed genera of the Pteridaceae family, is employed in folk medicine worldwide. In the present study, we investigated the antinociceptive effects of the methanolic extract of Adiantum latifolium (MEA) in animal models of inflammatory pain. The pharmacological properties of MEA were evaluated by using writhing, formalin and tail flick tests, carrageenan-induced paw oedema and arachidonic acid-induced ear oedema models. Mice motor performance was evaluated in the rota-rod test and the acute toxicity evaluated over 14 days. In the next experiments series, the active part of Adiantum latifolium, as well as the active fraction of MEA, was evaluated. A phytochemical screening for classes of constituents of MEA was carried out by thin layer chromatography (TLC). Oral (100-400 mg/kg) or intraperitoneal (1-100 mg/kg) administration of MEA produced a dose-related inhibition of acetic acid-induced writhing in mice. Furthermore, treatment with MEA (100 mg/kg/IP) inhibited both the early and late phases of formalin induced hypernociception. In contrast, MEA (100 mg/kg/IP) did not prevent the thermal nociception in the tail flick test. In addition, MEA (100 and 200 mg/kg/IP) inhibited important events related to the inflammatory response induced by carrageenan or arachidonic acid: namely local oedema and increase in tissue interleukin-1β levels. MEA (200 mg/kg/IP) treated mice did not show any motor performance alterations. Over the study duration of 14 days, there were no deaths or toxic signs recorded in the group of mice given 1000 mg/kg of MEA. Phytochemical analysis indicated the presence of terpenes, steroids, flavonoids and phenolic acids, which may be responsible for the MEA antinociceptive and/or antiinflammatory effects. Methanolic extracts from different parts of Adiantum latifolium showed equivalent antinociceptive activity, suggesting that the active principle of EMA is homogeny distributed through the plant. Buthanolic and ethyl acetate fractions were the more active fractions of MEA. Our results demonstrate that Adiantum latifolium presents significant antinociceptive and antiinflammatory activities in different experimental models, possibly through an inhibition of IL-1β production and constitute good candidate for pharmacologic development.

Wear particles and ions from cobalt-chromium-molybdenum (CoCrMo)-based implants have been shown to cause adverse immune responses, including periprosthetic osteolysis leading to aseptic loosening, the main cause of implant failure. Previous studies have shown that these wear and corrosion products can lead to the release of inflammatory cytokines, including interleukin-1β (IL-1β), suggesting the involvement of the NLRP3 inflammasome. However, the mechanisms leading to IL-1β release have not been fully elucidated. The primary objectives of this thesis were to determine if, in murine macrophages, IL-1β release induced by micrometre-size CoCrMo particles and nanometre-size chromium oxide (Cr2O3) particles is: 1. Caspase-1-dependent; 2. Reduction-oxidation (redox)-dependent; and 3. NLRP3 inflammasome-dependent. Additionally, the effects of metal ions (Co2+, Cr3+, and Ni2+) on NLRP3 inflammasome activation and the effects of matrix metalloproteinase (MMP) inhibition on IL-1β release induced by CoCrMo particles were analyzed. Results showed that IL-1β release induced by CoCrMo particles was partly caspase-1-, redox-, and MMP-dependent, but NLRP3 inflammasome-independent. On the other hand, IL-1β release induced by Cr2O3 particles appeared to be NLRP3 inflammasome-dependent. Finally, IL-1β release induced by Cr3+, but not Co2+, appeared to be NLRP3 inflammasome-dependent, while Ni2+-induced IL-1β release appeared to be only partially NLRP3 inflammasome-dependent, suggesting that other pathways may also be involved. These findings, which provide additional insights into the mechanisms leading to IL-1β release induced by wear particles and ions from CoCrMo-based implants, may help the future development of therapeutic treatments to modulate wear product-induced inflammation and increase implant longevity.

Bakgrund: Användningen av nanopartiklar ökar varje dag men mycket är oklart i vilkenutsträckning nanopartiklar har hälsopåverkan hos människor. I studier har det visats attnanopartiklar under 100 nm kan passera cellmembranet och under 40 nm kan passeranuklearmembran. I dagsläget används guldnanopartiklar (GNP) inom medicin för visualisering, diagnostisering och läkemedelsbärare. En av biverkningar som GNP kan ha är inflammation. Det finns flera studier som försökt redogöra GNP:s effekter samt hur de uppkommer men det är intehelt klarlagt. Vidare är det inte helt tydligt vilka egenskaper hos GNP som står för de olikaeffekterna. Syfte: Syftet med studien är att undersöka rollen som GNP:s storlekar och former har i NLRfamily pyrin domain containing 3 (NLRP3) inflammasomens interleukin-1β-frisättning hoshuman leukemia monocytic cell line (THP1-celler). Metod: Studien är en experimentell studie där THP1-celler differentieras och exponeras för GNPi olika storlekar, former och koncentrationer. LUMIT immunoassay används sedan för att erhållade olika interleukin-1β-koncentrationerna (IL-1β). Resultat: I datan som erhölls efter att experimenten utförts observerades det att GNP 30 nm i 40μg/mL gav störst inflammasominduktion med en IL-1β-koncentration på 645 ±388 pg/mL.Nästhögst IL-1β-koncentration gav GNP 100 nm i 40 μg/mL på 592 ±270 pg/mL. Slutsats: Nakna GNP kan inducera inflammasomaktivering och på så sättIL-1β-koncentrationshöjningar. De “gold urchins” som undersökts visade sig ha svagarepåverkan på cellerna jämfört med de sfäriska GNP.

No description available.

Monocytes and macrophages are mediator cells of cartilage and bone erosion in the synovia of rheumatoid arthritis (RA) patients due to secretion of the inflammatory cytokine Interleukin-1β (IL-1β). Calcium, phosphate and fetuin are liberated from the affected bone matrix, and the formation of calciproteinparticles (CPPs) is likely. IL-1β production in monocytes in vitro is stimulated by high concentrations of extracellular calcium. Additionally, the rise of extracellular calcium concentrations leads to increased macropinocytosis in mononuclear phagocytes. Flow cytometry analyses in this study show that peripheral blood monocytes from patients with RA perform more calcium stimulated macropinocytosis of the fluorescent dye calcein than monocytes from healthy donors. Stimulation of monocytes with calcium and preformed CPPs leads to more IL-1β production, quantified using ELISA, by monocytes from RA patients. Experiments with macrophages show similar results. Furthermore calcium-stimulated macropinocytosis and IL-1β secretion are significantly positively correlated. However, there was no connection of in vitro findings and the severity of RA in patients.:Abbildungsverzeichnis IV Tabellenverzeichnis VI Abkürzungsverzeichnis VII 1 Einleitung 1 1.1 Rheumatoide Arthritis 1 1.1.1 Epidemiologie und Klinik der rheumatoiden Arthritis 1 1.1.2 Ätiopathogenese der rheumatoiden Arthritis 1 1.2 Monozyten und Makrophagen 3 1.2.1 Inflammasomaktivierung und Interleukin-1β-Sekretion in Monozyten und Makrophagen 4 1.2.2 Makropinozytose in Monozyten und Makrophagen 6 1.2.3 Beitrag der Monozyten und Makrophagen zur rheumatoiden Arthritis 7 1.3 Kalzium – lokale Dysregulation trotz systemischer Regulation 9 1.3.1 Entstehung von Kalziumproteinpartikeln 10 1.3.2 Kurzportrait des G-Protein-gekoppelten Kalziumrezeptors CaSR 11 2 Fragestellungen 13 3 Forschungsdesign, Material und Methoden 15 3.1 Forschungsdesign 15 3.2 Materialien 15 3.2.1 Laborgeräte 16 3.2.2 Verbrauchsmaterialien 17 3.2.3 Materialien und Chemikalien 17 3.2.4 Medien, Lösungen und Puffer 19 3.2.5 Stimulanzien und Inhibitoren 20 3.2.6 Fluoreszenzfarbstoffe 20 3.2.7 Software 20 3.3 Methoden 21 3.3.1 Separation von PBMCs mittels Ficolldichtegradientenzentrifugation 21 3.3.2 Separation von Monozyten mittels negativer Magnetseparation 22 3.3.3 Differenzierung von Monozyten zu Makrophagen in Zellkulturbeuteln 23 3.3.4 Makropinozytose von Monozyten und Makrophagen in der Durchflusszytometrie 23 3.3.4.1 Makropinozytose von Calcein in Monozyten 24 3.3.4.2 Makropinozytose fluoreszenzgefärbter Kalziumproteinpartikel in Monozyten und Makrophagen 25 3.3.4.3 Inhibition der Makropinozytose in Monozyten 26 3.3.4.4 Auswertung der am Durchflusszytometer generierten Rohdaten mit FlowJo 26 3.3.5 Makropinozytose von Monozyten in der Fluoreszenzmikroskopie 28 3.3.6 Bestimmung der Interleukin-1β-Produktion von Monozyten und Makrophagen mittels ELISA 29 3.3.7 Erhebung des DAS28 33 3.3.8 Bestimmung von Laborparametern 33 3.4 Statistische Auswertung 33 4 Ergebnisse 35 4.1 Charakterisierung der Kohorten 35 4.2 Vorversuche zur Auswahl eines geeigneten Fluoreszenzfarbstoffes für die Detektion der Makropinozytose 37 4.3 Stimulation von Monozyten mit Kalzium zur Makropinozytose und Interleukin-1β-Produktion 39 4.3.1 Kalziumstimulierte Calceinaufnahme von Monozyten 39 4.3.2 Kalziumstimulierte Interleukin-1β-Produktion von Monozyten 44 4.4 Stimulation von Monozyten mit Kalzium zur Makropinozytose und Interleukin-1β-Produktion unter Zugabe von Kalziumproteinpartikeln 47 4.4.1 Kalziumstimulierte Aufnahme fluoreszierender Kalziumproteinpartikel 47 4.4.2 Kalziumstimulierte Interleukin-1β-Produktion in Monozyten unter Zugabe von Kalziumproteinpartikeln 51 4.5 Stimulation von Makrophagen mit Kalzium zur Makropinozytose und Interleukin-1β-Produktion 53 4.5.1 Kalziumstimulierte Makropinozytose von fluoreszierenden Kalziumproteinpartikeln in Makrophagen 53 4.5.2 Kalziumstimulierte Interleukin-1β-Produktion mit und ohne Zugabe von Kalziumproteinpartikeln in Makrophagen 54 4.5.3. Visualisierung von Monozyten und Makrophagen nach 16 Stunden Inkubation 57 4.6 Korrelation zwischen kalziumstimulierter Makropinozytose und Interleukin-1β-Sekretion 59 5 Diskussion 61 5.1 Kalziumstimulierte Makropinozytoseaktivität von Monozyten und Makrophagen 61 5.2 Kalziumstimulierte Interleukin-1β-Sekretion von Monozyten und Makrophagen 64 5.2.1 Auswirkung der Phosphatkonzentration im Zellkulturmedium auf die kalziumstimulierte Interleukin-1β-Sekretion von Monozyten und Makrophagen 65 5.2.2 Kalziumstimulierte Interleukin-1β-Sekretion von Monozyten und Makrophagen von RA-Patienten und Kontrollprobanden 66 5.3 Zusammenhang von kalziumstimulierter Makropinozytose und Interleukin-1β-Sekretion in Monozyten und Makrophagen von RA-Patienten und Kontrollprobanden 70 5.4 Ausblick und offene Fragen 71 6 Zusammenfassung der Arbeit 73 8 Erklärung über die eigenständige Abfassung der Arbeit 88 9 Danksagung 89

Einleitung: Osteoarthrose (OA) ist eine der häufigsten muskuloskelettalen Erkrankungen beim Hund und wird definiert durch einen fortschreitenden Verlust der Knorpelschicht auf den Gelenksflächen. Therapeutika erster Wahl, wie nichtsteroidale Antiphlogistika, wirken rein palliativ und haben insbesondere bei der Langzeitgabe schädliche Nebenwirkungen. Bei der Suche nach Alternativen rückten Ergänzungsfuttermittel in den Focus der Forschung. Dabei zeigten mehreren Studien, dass eine Fütterung von Fischöl die klinischen Symptome von OA bei Hunden verbessert. Der dahinterliegende Wirkmechanismus ist jedoch beim Hund nicht erforscht. Ziele der Untersuchungen: Ziel dieser Studie war demnach, den genauen Wirkmechanismus von mehrfach ungesättigten Fettsäuren (PUFA) auf ausgewählte Schlüsselfaktoren der Knorpeldegeneration in einem Modellsystem der caninen OA zu untersuchen. Material und Methoden: Wir etablierten ein Protokoll zur Isolierung von caninen Chondrozyten, kultivierten die Zellen erfolgreich bis zur Konfluenz und kryokonservierten sie für die weiteren Versuche. Um die Ansprechbarkeit der Zellen auf verschiedene Zytokine zu untersuchen, wurden die Zellen über 24 h mit 10 ng/ml Interleukin‑1β (IL‑1β) und mit 1 bis 10 ng/ml Transforming Growth Faktor‑β (TGF‑β) stimuliert und die Genexpression von Matrixproteinasen, induzierbarer Stickstoffmonoxid Synthase (iNOS) und Cyclooxygenase‑2 (COX‑2) sowie die Produktion von Stickstoffmonoxid (NO) und Prostaglandin E (PGE) gemessen (4 Spender, N=3). Auf Basis des etablierten Models wurden in einer zweiten Versuchsreihe die Zellen über 6 Tage mit n-3 Eicosapentaensäure (EPA), n-3 Docosahexaensäure (DHA) oder n-6 Arachidonsäure (AA) kultiviert und anschließend über 48 h mit IL‑1β stimuliert. Die Veränderung der IL‑induzierte Genexpression von Matrixproteinasen, iNOS und COX‑2 sowie die Produktion von PGE und NO unter Einfluss der Fettsäuren wurde analysiert (6 Spender, N=4). Darüber hinaus wurde die Fettsäureaufnahme und der Fettsäuremetabolismus via Gaschromatographie überprüft. Um die Wirkungen der Fettsäuren und Stimulantien zu verifizieren, wurden die Daten einer zweifachen Varianzanalyse (AV) unterzogen. Bei signifikanten Einflüssen der Faktoren folgte ein Post-hoc-Test (Dunnett’s Test für die PUFA bzw. ein Bonferroni Test für die Stimulanzien). Die Signifikanzniveaus für AV- und die Post-hoc Tests wurden auf α = 0,01 eingestellt. Ergebnisse: Unter der Stimulation mit IL‑1β kam es zu einem signifikanten Anstieg von degenerativen und entzündlichen Markern. Das Muster der Marker entsprach dabei dem Muster, welches unter natürlichen Bedingungen in erkrankten Gelenken vorzufinden ist. Somit haben wir ein simples und dennoch repräsentatives Modell für die canine OA etabliert. Im Gegensatz zu IL‑1β reduzierte TGF‑β konzentrationsabhängig signifikant den von IL‑1β induzierten Anstieg der inflammatorischen Marker und stellt sich somit als Gegenspieler zu diesem proinflammatorischen Zytokin dar. Die gegensätzliche Antwort der Zellen auf beide Zytokine bewies, dass die Chondrozyten in unserem Modell Charakteristika von differenzierten Zellen aufweisen. Alle Fettsäuren wurden konzentrationsabhängig in die Zellen aufgenommen und metabolisiert. EPA reduzierte die IL‑induzierte Expression von iNOS und die damit im Zusammenhang stehende Produktion von NO signifikant. AA reduzierte ebenfalls iNOS und NO und außerdem die Expression von Matrixmetalloproteinase‑3 signifikant. Auf der anderen Seite steigerte AA signifikant die Produktion von PGE und die Expression von Aggrecanase ADAMTS‑5. DHA hatte auf keinen der untersuchten Marker einen Effekt. Schlussfolgerungen: Zusammenfassend bestätigen die Ergebnisse dieser Studie die in Fütterungsversuchen dokumentierte positive Wirkung von n-3 PUFAs bei der caninen OA und bieten zudem eine Erklärung für die dahinterliegenden Mechanismen. Hervorzuheben ist, dass in dieser Studie nicht nur EPA, sondern auch AA eine positive Wirkung auf einzelne Marker erzielen. EPA scheint von allen untersuchten Fettsäuren die höchste Effektivität zu haben. Eine geringe Dosis an Fettsäuren über einen längeren Zeitraum führte nicht zu einer Akkumulation der Fettsäuren und hatte daher auf einige untersuchte Marker nur eine geringe Wirkung. Insgesamt lässt sich schlussfolgern, dass eine ausreichende Menge und ausgewogenes Verhältnis von n‑3 und n‑6 PUFA als unterstützende Therapie bei chronischen Gelenkserkrankungen beim Hund von großer Wichtigkeit ist. Besonders ein hoher Gehalt an EPA scheint dabei eine hohe Bedeutung für die Effektivität zu haben.:INHALTSVERZEICHNIS 1 Einleitung 1 2 Literaturübersicht 3 2.1 Physiologie und Anatomie des hyalinen Knorpels 3 2.1.1 Feinstruktur des Gewebes 3 2.1.2 Bedeutung der Chondrozyten 4 2.1.3 Aufbau der Extrazellulären Matrix (ECM) 5 2.2 Osteoarthrose (OA) 6 2.2.1 Pathogenese 6 2.2.1.1 Kausale Pathogenese 6 2.2.1.2 Formale Pathogenese 7 2.2.2 Die Rolle der Zytokine 9 2.2.2.1 Interleukin-1β (IL-1β) 9 2.2.2.2 Transforming Growth Factor-β (TGF-β) 10 2.2.3 Rolle der Matrixproteinasen MMP und ADAMTS 12 2.2.4 Rolle der Entzündungsmediatoren 14 2.2.4.1 Stickstoffmonoxid (NO) 14 2.2.4.2 Prostaglandin E2 (PGE2) 15 2.3 Zellkulturmodelle für OA 16 2.4 Die canine OA 17 2.4.1 Ätiologie und Diagnose 17 2.4.2 Therapieansätze in der caninen OA 18 2.5 Mehrfach ungesättigte Fettsäuren (PUFAs) als Ergänzungsfuttermittel 20 2.5.1 Grundlagen des Fettsäurestoffwechsels 20 2.5.2 Fettsäurestoffwechsel im Knorpelgewebe 21 2.5.3 n-3 PUFAs als Therapieoption bei caniner OA 22 3 Ziel der Arbeit 25 4 Ergebnisse 26 4.1 Publikation 1 26 4.2 Publikation 2 36 5 Diskussion 47 5.1 Isolation und Anzucht von caninen Chondrozyten 47 5.2 IL-stimulierte canine Chondrozyten als Osteoarthrosemodell 51 5.3 TGF‑β als Gegenspieler von IL-1β 52 5.4 Fettsäuremetabolismus der Chondrozyten 54 5.5 Fettsäuren als Therapieoption bei der caninen OA 59 6 Schlussfolgerung 65 7 Zusammenfassung 66 8 Summary 68 9 Literaturverzeichnis 70 10 Danksagung 90 / Introduction: Osteoarthrosis (OA) is one of the most common musculoskeletal diseases in dogs and is defined by the progressive loss of the protective joint cartilage layer. First-choice treatments, like nonsteroidal anti-inflammatory drugs, act palliative only and are known to have harmful side effects especially in long-term usage. The urge to find alternative treatments moved nutraceuticals into the focus of research. In multiple trials, feeding fish oil improved the clinical symptoms of osteoarthritis in dogs. However, the underlying molecular mechanisms have not been investigated in dogs. Aim of the Study: The aim of this study was therefore to further clarify how polyunsaturated fatty acids affect key factors of cartilage degeneration in a cell culture model for canine OA. Materials and methods: We established a protocol for the isolation of canine chondrocytes, cultivated them successfully until confluency and cryoconservated them for further experiments. To examine the response of the cells to different cytokines, cells were stimulated for 24 h with 10 ng/ml Interleukin‑1β (IL‑1β) or 1 to 10 ng/ml Transforming Growth Factor‑β (TGF‑β). The gene expression of matrix proteinases, inducible nitric oxide synthase (iNOS) and cyclooxygenase‑2 (COX‑2), the production of nitric oxide (NO) and prostaglandin E (PGE) were measured (4 donors, N=3). Using the established model, cells in the second trial were incubated with eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) for 6 days and subsequently stimulated with IL‑1β for another 48 h. The change in IL‑induced gene expression of matrix proteinases, iNOS and COX‑2 as well as the production of NO and PGE under the influence of n‑3 EPA, n‑3 DHA and n‑6 AA was analysed (6 donors, N=4). To verify the effects of the fatty acids and the stimulants, data were analysed with a two‑factorial analysis of variance (ANOVA). When there was a significant effect of the factors, a post-hoc test was performed (Dunnett’s test for PUFA, Bonferroni test for stimulants). Significance level for ANOVA- and the post hoc test was set at α = 0.01. Results: Stimulation with IL-1β led to a significant increase of degenerative and inflammatory markers. This pattern of responsiveness was comparable to the events in naturally occurring OA. We therefore have established a simple yet representative model for canine OA. In contrast to IL‑1β, TGF‑β significantly decreased the IL‑induced inflammatory markers in a dose-dependent manner and therefore could counteract IL‑1β. The response of the cells to the cytokines indicates, that in our model, the chondrocytes have characteristics of differentiated cells. All fatty acids were incorporated in a dose-dependent manner and metabolized. EPA decreased the IL-induced gene expression of iNOS and reduced the concomitant production of NO significantly. AA also decreased iNOS and NO significantly and downregulated the gene expression of matrix metalloproteinase‑3 significantly. On the other hand, AA significantly increased the production of PGE and the gene expression of the aggrecanase ADAMTS‑5. DHA had no effect on any of the markers. Conclusions: Taking together, the results of this study add further weight to the beneficial effect of n‑3 PUFAs in canine OA and furthermore provide an explanation for the underlying mechanism. It is of importance, that not only the n‑3 fatty acid EPA but also the n‑6 fatty acid AA could provide beneficial effects on several markers. EPA seems to be the most effective of all fatty acids included in this study. A low concentration of fatty acids over a long period of time did not lead to an accumulation of fatty acids and therefore had a rather small impact on some of the investigated markers. Therefore, an optimal ratio between n‑3 and n‑6 and the adequate amount of fatty acids seems to be of high importance when treating chronic degenerative joint disease in dogs. However, a high content of EPA seems to be particularly effective.:INHALTSVERZEICHNIS 1 Einleitung 1 2 Literaturübersicht 3 2.1 Physiologie und Anatomie des hyalinen Knorpels 3 2.1.1 Feinstruktur des Gewebes 3 2.1.2 Bedeutung der Chondrozyten 4 2.1.3 Aufbau der Extrazellulären Matrix (ECM) 5 2.2 Osteoarthrose (OA) 6 2.2.1 Pathogenese 6 2.2.1.1 Kausale Pathogenese 6 2.2.1.2 Formale Pathogenese 7 2.2.2 Die Rolle der Zytokine 9 2.2.2.1 Interleukin-1β (IL-1β) 9 2.2.2.2 Transforming Growth Factor-β (TGF-β) 10 2.2.3 Rolle der Matrixproteinasen MMP und ADAMTS 12 2.2.4 Rolle der Entzündungsmediatoren 14 2.2.4.1 Stickstoffmonoxid (NO) 14 2.2.4.2 Prostaglandin E2 (PGE2) 15 2.3 Zellkulturmodelle für OA 16 2.4 Die canine OA 17 2.4.1 Ätiologie und Diagnose 17 2.4.2 Therapieansätze in der caninen OA 18 2.5 Mehrfach ungesättigte Fettsäuren (PUFAs) als Ergänzungsfuttermittel 20 2.5.1 Grundlagen des Fettsäurestoffwechsels 20 2.5.2 Fettsäurestoffwechsel im Knorpelgewebe 21 2.5.3 n-3 PUFAs als Therapieoption bei caniner OA 22 3 Ziel der Arbeit 25 4 Ergebnisse 26 4.1 Publikation 1 26 4.2 Publikation 2 36 5 Diskussion 47 5.1 Isolation und Anzucht von caninen Chondrozyten 47 5.2 IL-stimulierte canine Chondrozyten als Osteoarthrosemodell 51 5.3 TGF‑β als Gegenspieler von IL-1β 52 5.4 Fettsäuremetabolismus der Chondrozyten 54 5.5 Fettsäuren als Therapieoption bei der caninen OA 59 6 Schlussfolgerung 65 7 Zusammenfassung 66 8 Summary 68 9 Literaturverzeichnis 70 10 Danksagung 90

O endométrio é a mucosa que reveste o útero. A receptividade uterina é definida como um estado em que o endométrio se encontra receptivo à implantação do blastocisto. E, a preparação do endométrio para a implantação não é somente uma questão de estimulação hormonal adequada, depende da interação entre o blastocisto e o endométrio. Esta interação envolve uma complexa sequência de eventos de sinalização e uma variedade de moléculas. As concentrações de interleucina-8 (IL-8) e interleucina-1β (IL-1β) estão correlacionadas com o processo de implantação. Em humanos, a taxa de insucesso desse processo é alta e ocasionada por diversos fatores. A síndrome dos ovários policísticos (SOP) é um distúrbio endócrino-ginecológico que afeta de 6 a 8 % das mulheres em idade reprodutiva, e se caracteriza, principalmente, por anovulação crônica e hiperandrogenismo, estando diretamente relacionada à infertilidade feminina. Apesar da incerteza sobre a causa primária da SOP, há relatos sobre a importância da hiperinsulinemia na sua promoção. O objetivo deste trabalho foi estabelecer um modelo de hiperinsulinemia e hiperandrogenismo em células estromais endometriais in vitro, simulando características de SOP; identificar o melhor gene normalizador para estudos de expressão gênica em amostras das células em cultivo; avaliar o efeito da metformina sobre a proliferação celular e expressão gênica da IL-8 e IL-1β no modelo proposto. O tecido endometrial foi obtido de pacientes submetidas a histerectomia. A cultura primária das células estromais foi padronizada e as células foram divididas em sete grupos de tratamento: estradiol (G1); estradiol e progesterona (G2); estradiol, progesterona e insulina (G3); estradiol, progesterona e diidrotestosterona (G4); estradiol, progesterona e metformina (G5); estradiol, progesterona, insulina e diidrotestosterona (G6); estradiol, progesterona, insulina, diidrotestosterona e metformina (G7). Foi realizada análise de imunocitoquímica para vimentina para confirmação do cultivo com células estromais. Para avaliar a viabilidade e proliferação celular ao longo do tempo foi utilizado o ensaio de MTT em dois tempos diferentes de cultivo. As extrações de RNA foram realizadas e o cDNA obtido das amostras foi utilizado para a amplificação do mRNA de cinco genes candidatos a normalizadores e para avaliar a expressão dos genes da IL-8 e IL-1β através de PCR em tempo real.O estabelecimento da cultura de células estromais foi confirmado através da coloração positiva para a proteína vimentina. As células mantiveram-se viáveis durante todo o período de cultivo, apresentando aumento significativo na proliferação celular no tempo 8 em relação ao tempo 4 em todos os grupos. O grupo G7 (tratado durante 48 horas com metformina) apresentou uma menor taxa de proliferação em relação aos grupos G2, G3 e G6. Para análise de expressão gênica nestas células, o gene que mostrou os melhores parâmetros de estabilidade de expressão no modelo celular proposto foi o gene HPRT1. Observamos uma maior expressão do gene da IL-8 no grupo G5 tratado durante 48 horas em relação ao mesmo grupo tratado durante o período de 24 horas. Verificou-se maior expressão do gene da IL-1β no grupo G5 quando comparado a todos os outros grupos no período de 48 horas de tratamento com metformina. Entretanto, o grupo G7, também tratado com metformina, não apresentou diferença estatística em relação ao tempo de tratamento em nenhum dos genes estudados. Esses resultados demonstram que o modelo de hiperinsulinemia e hiperandrogenismo em cultura de células estromais endometriais é viável. Neste modelo, em que foram testados cinco genes em relação à sua estabilidade de expressão, o gene HPRT1 apresentou uma boa estabilidade, ao contrário de outros genes frequentemente utilizados como genes de referência. O tratamento com metformina apresentou um efeito antiproliferativo nas células do grupo hiperinsulinêmico e hiperandrogênico. No período de 48 horas aumentou a expressão do gene da IL-1β no grupo tratado somente com o medicamento. Sugerindo uma ação inibitória da insulina sobre a expressão dos genes da IL-8 e IL-1β no grupo hiperinsulinêmico e hiperandrogênico. Mais estudos são necessários para melhor entendimento do efeito da metformina nos fatores envolvidos durante a implantação. / The endometrium is the mucosa lining the uterus. The uterine receptivity is defined as a condition in which the endometrium is receptive to implantation of the blastocyst. The preparation of the endometrium for implantation is not only a matter of proper hormonal stimulation, it depends on the interaction between the blastocyst and the endometrium. This interaction involves a complex sequence of events and a variety of signaling molecules. The concentrations of interleukin-8 (IL-8) and interleukin-1β (IL-1β) are correlated to the implantation process. In humans, the failure rate of this process is high and caused by several factors. The polycystic ovary syndrome (PCOS) is an endocrine-gynecological disorder that affects from 6 to 8 % of women of reproductive age. It is characterized mainly by chronic anovulation and hyperandrogenism and directly related to female infertility. Despite the uncertainty about the primary cause of PCOS, there are reports about the importance of hyperinsulinemia in promoting it. The aim of this study was to establish a model of hyperinsulinemia and hyperandrogenism in endometrial stromal cells in vitro, simulating features of PCOS; to identify the best housekeeping gene for gene expression studies in the cultured cells; to evaluate the effects of metformin on cell proliferation, as well as IL-8 and IL-1β gene expression in the proposed culture model. The human endometrial tissue was obtained from patients undergoing hysterectomy. The primary culture of stromal cells was standardized and divided into seven treatment groups: estradiol (G1); estradiol and progesterone (G2); estradiol, progesterone and insulin (G3); estradiol, progesterone and dihydrotestosterone (G4); estradiol, progesterone and metformin (G5); estradiol, progesterone, insulin and dihydrotestosterone (G6); estradiol, progesterone, insulin, dihydrotestosterone and metformin (G7). Immunocytochemistry analysis for vimentin were performed. Cell viability and proliferation were evaluated by MTT assay at two days in different times of cultivation. RNA extractions were performed and the cDNA obtained from primary culture was used to amplify five candidates to housekeeping genes mRNA and to evaluate IL-8 and IL1β expression by real time PCR. The stromal culture cell establishment was confirmed by positive staining for vimentin. The cells remained viable throughout the cultivation period, with significant cell proliferation increase at day 8 compared to day 4 in all groups. The G7 group (metformin treated for 48 hours) showed lower proliferation rate than G2, G3 and G6 groups. For gene expression analysis in these cells, the gene showing the best parameters of stability of expression was HPRT1. Increased gene expression of IL-8 was observed in G5 group treated for 48 hours compared to the same group during 24 hours. Similarly, the G5 group showed higher IL-1β gene expression when compared to all other groups treated with metformin for 48 hours. However, the G7 group, also metformin treated, did not show statistically significant difference in treatment time of any studied genes. These results suggest that model of hyperinsulinemia and hyperandrogenism in endometrial stromal cells is viable and provides a good cell sampling to molecular analysis under different experimental conditions. In this model, several genes were tested for expression stability. HPRT1 presented the best values, unlike others classical housekeeping genes. The metformin treatment showed an antiproliferative effect on cells in hyperinsulinemic and hyperandrogenic group and at 48 hours increased IL-1β gene expression in the treated group with the drug alone. It suggests an inhibitory action of insulin on these genes expression in the hyperinsulinemic and hyperandrogenic group. More studies are needed to better understand the effect of metformin on the factors involved during implantation.