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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
81

Untersuchungen zur Zellproliferation von Maus-Lungen-Fibroblasten am FACS-Flow-Zytometer unter dem Einfluss von Kulturüberständen bestrahlter Fibroblasten / FACS-flow-studies on cell-proliferations of mouse-lung-fibroblasts under the influence of culture-supernatants of irradiated fibroblasts

Wenemoser, Alexander January 2011 (has links) (PDF)
Experimentelle FACS-Flow-Analysen im Kontext einer radiogenen Lungenfibrose zur Veränderung der Zellproliferation von Maus-Lungen-Fibroblasten unter dem Einfluss von Kulturüberständen bestrahlter Fibroblasten. Additiv einzelne Versuche mit Antikörperzugabe gegen TGF-beta zur Evaluation eines hemmenden Effektes auf eine postulierte Arretierung der Fibroblasten in der G1-Phase der Zellteilung durch die Zytokine der Kulturüberstände bestrahlter Maus-Lungen-Fibroblasten. / FACS-Flow-Analyses on changes of cell-proliferations of mouse-lung-fibroblasts under the influence of culture-supernatants of irradiated fibroblasts in the context of a radiogenic lung fibrosis. Additionally some studies on a postulated inhibitory effect of an tgf-beta-antibody on the g1-phase cell-cycle-arrest of mitosis through cytokines in the supernatants of the irradiated fibroblasts.
82

Wachstums- und Sekretionsverhalten humaner fetaler Lungenfibroblasten nach Applikation von Gamma-Strahlung in vitro / Growth and secretion behavior of human fetal lung fibroblasts after application of gamma-radiation in vitro

Wruck, Robert January 2011 (has links) (PDF)
Der wesentliche Dosis limitierende Faktor einer Strahlentherapie thorakaler Malignome ist die Strahlenempfindlichkeit des Lungenparenchymes, da sich mit einer Häufigkeit von 25-75 % aller Patienten ein Strahlenschaden des Lungengewebes entwickeln kann. Die Inzidenz einer Lungenfibrose nach 6- 12 Monaten liegt bei 15-30%. Die Kombination zytostatischer Medikamente mit ionisierender Strahlung kann die Ansprechraten verbessern, kann andererseits die Inzidenz einer Pneumonitis erhöhen. Die konkreten Mechanismen, die zu einer Pneumonitis und einer strahleninduzierten Fibrose führen, sind bislang noch nicht vollständig bekannt. Es wird vermutet, daß die ortsständigen Zellen der Lunge eine aktivere Rolle in der Pathogenese als bisher angenommen, einnehmen. Tiermodelle der Strahlenschädiung der Lunge zeigten ein sehr frühe Expression von TGF-ß-mRNA and fibronectin-mRNA nach Bestrahlung. TGF-ß und Fibronectin sind in der BALF und Serum von an thorakalen Malignomen erkrankten, strahlentherapeutisch behandelten Patienten erhöht. Neben Makrophagen und Typ II Pneumocyten als zelluläre Quellen der genannten Cytokine, sind Fibroblasten in der Lage beide Agentien in erheblichem Umfang zu synthetisieren. Ziele Um die aktive Rolle von Fibroblasten in der Pathogenese der strahleninduzierten Lungenfibrose in Abwesenheit von Entzündungszellen zu untersuchen, bestrahlten wir Lungenfibroblasten in vitro und beobachteten folgende Parameter. 1. Zellwachstum 2. Synthese von Fibronectin 3. Synthese von Kollagen ( Procollagen-I-Peptid) 4. Synthese von TGF-ß1 Methoden Humane fetale Lungenfibroblasten (MRC-5 ,ICN Biochemicals Eschwege ,Deutschland) wurden in DME Medium kultiviert unter Zugabe von 10% FCS plus L-Glutamine, Penicillin G , Amphotericin B und Gentamycin; Luftfeuchtigkeit 100% , Temperatur 37°, CO2 5%, Medienwechsel erfolgten zweimal wöchentlich und 24 Stunden vor den Messungen. 24h nach der Aussaat der Zellen erfolgte die Strahlenapplikation (CO 60; 4.5, 7.5, 10.5 Gy ). Messungen erfolgten an den Tagen 3,6,9,12,15 nach Bestrahlung. Hierfür wurden folgende Materialien verwandt. Fibronectin (ELISA), Takara TGF beta (ELISA), DPC Biermann Procollagen-I-Peptide (ELISA), Takara LDH ( kinetischer Assay), Sigma Cell counts (Zählkammer) Alle Messungen wurden zweimal unternommen. Ergebnisse: 1. Das Zellwachstum wurde dosisabhängig gehemmt. 2. Beginnend am 3 Tag stieg die Syntheserate des Fibronectin dosisabhängig. 3. Ähnliche Beobachtungen wurde bzgl der Procollagen-I-Peptid Synthese beobachtet. 4. TGF-ß Spiegel fanden sich nach Bestrahlung ab Tag 6 bis zum 4-fachen über dem Ausgangswert erhöht und kehrten ziwschen den Tagen 9 und 15 auf das Ausgangsniveau zurück. 5. Eine Erhöhung des LDH wurde nicht beobachtet. Dies zeigte, dass eine Zytolyse kein wesentlichen Einfluß hatte. Disskusion: Bei Bestrahlung humaner fetaler Lungenfibroblasten wird das Zellwachstum dosisabhängig limitiert. Dies wurde nicht durch einen strahlenbedingt erhöhten Zelltod hervorgerufen , da das bestimmte LDH ( ein Marker der Zytolyse) in den Zellkulturüberständen nicht erhöht war. Wir vermuten, das durch Bestrahlung eine Differenzierung von Progenitor Fibroblasten zu postmitotischen Fibrocyten erfolgte, wie auch bereits von anderen Arbeitsgruppen berichtet. TGF-ß fand sich nach Bestrahlung in den Zellkulturüberständen deutlich erhöht. Es wird angenommen , daß TGF-ß eine Schlüsselrolle in der Pathogenese fibrosierender Erkrankungen der Lunge, der Leber, der Niere spielt und ebenso in die Enstehung der durch ionisierende Bestrahlung hervorgerufene Lungenfibrose eingebunden ist. Unsere Experimente haben gezeigt , daß Fibroblasten in der Lage sind große Mengen TGF-ß and Fibronectin - sogar in Abwesenheit von Entzündungszellen- zu erzeugen und sich vermutlich autokrin stimulieren können. Dieser Mechanismus wird als wichtiger Co-Faktor in der Pathobiologie verschiedener zur Fibrose führender Lungenerkrankungen angenommen. Schlussfolgerung Fibroblasten produzieren erhöhte Mengen TGF-ß und Fibronectin nach Applikation ionisierender Strahlung. Sie könnten in der Pathogenese der Strahlenschädigung der Lunge eine aktivere Rolle spielen als bisher angenommen. / Introduction The major dosis limiting factor of radiation therapy of thoracic malignomas is the lung which may develop radiation injury with a frequency of 25-70% of patients .The incidence of lung fibrosis after 6-12 months ist 15-30 %. Combination of cytostatic drugs with ionizid radiation can improve response rates, but may result in a higher incidence of pneumonitis. The exact mechanisms leading to pneumonitis and radiation induced fibrosis of the lung are yet unknown.The structural cells of the lung are of the lung are probably involved in the pathogenesis in a more active way than thougt until now. Animal models of radiation injury of the lung showed a very early expression of TGF-beta -mRNA and fibronectin-mRNA after irradiation. TGF-ß and Fibronectin were elevated in BALF and in serum. Macrophages and type-II-pneumocytes are thought to be the cellular source, but fibroblasts also are capable to synthesize both agents in large amounts. Aims In order to investigate the active role of fibroblasts in the pathogenesis of radiation fibrosis we irradiated human lung fibroblasts in vitro. We focused on following points: 1. cell growth 2. synthesis of fibronectin 3. synthesis of collagen (procollagen-I-peptid) 4. synthesis of TGF-beta-1 Methods Human fetal lung fibroblasts (MRC-5 ,ICN Biochemicals Eschwege ,Germany) cultured in DME-medium plus 10 % FCS plus L-glutamine, penicillin G, amphotericin B and gentamycine; air humidity 100 %, temp. 37°C, CO2 5%; change of medium twice weekly and 24 hr. before measurements. 24hrs. after seeding, application of ionizing radiation (CO 60; 4.5, 7.5, 10.5 Gy ). Measurements on day 3,6,9,12,15 after irradiation: Fibronectin (ELISA), Takara TGF beta (ELISA), DPC Biermann Procollagen-I-Peptide (ELISA), Takara LDH ( kinetic assay), Sigma Cell counts (counting chamber) All measurements have been done twice. Results 1. cell growth was inhibited in a dose dependent manner. 2. Beginning at day 3 cell related synthesis of fibronectin was increased depending on the dose of irradiation. 3. Similar observations were made in synthesis of procollagen-I-peptide. 4. TGF-beta levels were increased four fold after irradiation beginning on day 6 and returned to basal values between day 9 and 15 (the cells treated with 10.5 Gy were an exception. Here we found a furthermore higher secretion rate ). 5. No elevation of LDH was noticed, showing that cytolysis was not important in these effects. Discussion Irradiation of fetal human lung fibroblasts inihibited cell growth in a dose depend manner. This was not due to cell death initiated by ionizing rays, because LDH ( marker of cytolysis) was not elevated in culture supernatants. We assume that irradiation induces differentiation of progenitor-fibroblasts to promitotic fibrocytes as reported by other groups. TGF-beta was considerably elevated in culture supernatants after irradiation. TGF-beta is assumed to play a key role in fibrosing disease of lung, liver and kidney and may be involved in radiation induced lung fibrosis as well. Our experiments show, that fibroblasts are able to produce high amounts of TGF-beta and fibronectins - even if inflammatory cells are absent- and may stimulate themselves in an autocrine manner.This mechanism is thought to be an important co-factor in the pathobiology of different fibrosing disorders of the lung and may be important in radiation injury of the lung as well. Conclusion Fibroblasts produce increased amounts of TGF-beta and fibronectin after irradiation. They may play a more active role in the pathogenesis of radiation injury than thought up to now.
83

Investigation Of Transforming Growth Factor-Alpha And Its Potential Role In Promoting Ovarian Follicular Dominance

Lundberg, Allie Lynn 01 January 2019 (has links)
Intraovarian growth factors play a vital role in influencing the fate of ovarian follicles. They affect proliferation versus apoptosis of granulosa cells (GCs), and can influence whether small antral follicles continue their growth or undergo atresia. Transforming Growth Factor-alpha (TGFα), an oocyte-derived growth factor, is thought to regulate granulosa cell function, yet has been largely overshadowed by current interest in TGF-beta superfamily members, such as bone morphogenetic proteins (BMPs) and anti-Mullerian hormone (AMH). In the current study, effects of TGFα on bovine GC proliferation, intracellular signaling and cytokine-induced apoptosis were evaluated. Briefly, all small antral follicles (3-5mm) from bovine ovaries were aspirated and the cells were initially plated in T25 flasks containing DMEM/F12 medium, 10% FBS, and antibiotic-antimycotic, and incubated at 37 degrees Celsius in 5% CO2 for 3-4 days. Once confluent, the cells were sub-cultured to 96-, 12- or 6-well plates in serum-free conditions (insulin 100 ng/mL; transferrin 55 ng/mL; sodium selenite 6.7 pg/mL). 24-hour treatment of bovine GCs with TGFα (10 and 100ng/mL) stimulated cell proliferation compared to control (p<0.05; n = 7 ovary pairs). Cell proliferation was accompanied by a concomitant increase in mitogen-activated protein kinase (MAPK) signaling within 2 hours of treatment, as measured by phosphorylated ERK1/2 expression (p<0.05, n = 3 ovary pairs). These effects were entirely negated, however, by the MAPK inhibitor, U0126 (10uM, p<0.05). Additionally, pre-exposure of the bovine GCs to TGFα (100ng/mL) failed to prevent Fas Ligand (100ng/mL)-induced apoptosis, as determined by caspase 3/7 activity (P<0.05, n = 7 ovary pairs). Collectively, the results indicate TGFα stimulates proliferation of bovine GCs from small antral follicles via a MAPK/ERK-mediated mechanism, but this action alone fails to prevent apoptosis, suggesting TGFα may be incapable of promoting the persistence of follicles during the process of follicular selection and deviation.
84

Bambi – Charakterisierung eines inhibitorischen BMP-Pseudorezeptors / Bambi - characterization of an inhibitoric BMP pseudoreceptor

Kottmair, Mathias January 2014 (has links) (PDF)
Die TGF-β-Proteinfamilie umfasst eine Vielzahl von zumeist homodimeren sezernierten Liganden in höheren Tieren, die viele Vorgänge und Entwicklungen im Embryo wie im adulten Lebewesen über absolute oder graduelle Einflussnahme steuern. Die Signalweiterleitung ins Zytoplasma und den Nukleus erfolgt über promisk paarig rekrutierte Typ-I- und Typ-II-Rezeptoren, ehe vorwiegend rezeptorabhängig verschiedene SMAD-Proteine von Typ-I-Kinasen der Rezeptoren aktiviert werden, in den Kern translozieren und die Transkription induzieren. Zu jedem Zeitpunkt dieser Signalweiterleitung kann mittels verschiedener endogener Inhibitoren regulatorisch eingegriffen werden. Dem bisher einzig bekannten membranständigen Pseudorezeptor Bambi (BMP and Activine membrane bound inhibitor) wurde in vorangegangenen Arbeiten inhibitorisches Potential gegenüber dem BMP- und Activin-vermittelten Signalweg über Bindung an distinkte ligandenadressierte Rezeptoren zugeschrieben, wobei die genaue Wirkweise bislang noch vollkommen unklar war. In der vorliegenden Arbeit wurde initial ein Homologiemodell der extrazellulären Domäne von hBambi anhand der gelösten Kristallstruktur der extrazellulären Domäne von BR1A im gebundenen Zustand (PDB-ID: 1REW) erstellt. Anhand dieses Modells wurde eine Arbeitshypothese entwickelt und es gelang in der Folge, biologisch aktives rekombinantes Protein zum einen aus transfizierten Insektenzellen sowie aus der Renaturierung aus bakteriellen Einschlusskörpern in hinreichender Menge herzustellen und chromatographisch aufzureinigen. Nach einer vergleichenden Qualitätskontrolle beider Exprimate wurden mittels CD-Spektroskopie und analytischer Gelfiltration der Anteil der Sekundärstrukturelemente sowie der Oligomerisierungsgrad erfolgreich bestimmt. In SPR-Bindestudien wurde der Beweis erbracht, dass hBambi-ECD Affinität zu annähernd allen getesteten Liganden der BMP-/GDF-Gruppe, die den SMAD-1/-5-/-8-Signalweg aktivieren, zeigt. Bekannte Typ-I- und Typ-II-Bindungsmutanten von BMP-2 wurden ebenfalls von hBambi-ECD quasi wildtypisch gebunden. Verschiedene Rezeptorektodomänen sowie ActivinA wurden, wie bisher in der Literatur fälschlich angenommen wurde, hingegen nicht gebunden. Die propagierte Homooligomerisierung von Bambi wird überdies nicht über die extrazelluläre Domäne vermittelt. Eingesetzt in Stimulationsversuche mit BMP-responsiven Zellen wurde eine konzentrationsabhängige inhibierende Wirkung von freier hBambi-ECD auf die BMP-2-vermittelte Signalweiterleitung mit unterschiedlichen Nachweismethoden ermittelt, welche die Ergebnisse aus den SPR-Versuchen erfolgreich bestätigten. In einem weiteren Teil dieser Arbeit wurden verschiedene chimäre Konstrukte aus für Bambi- und BR1A-Domänen kodierenden Sequenzen kloniert, in HEK Ad293-Zellen zusammen mit BMP- und Activin-responsiven Reportergenkonstrukten transient transfiziert und Stimulationsversuche mit BMP-2 und ActivinA durchgeführt. Wildtypisches Bambi zeigte hierbei ein ambivalentes Verhalten in Bezug auf die Regulation des BMP-2-Signals: geringe Mengen wirken agonistisch, höhere Mengen antagonistisch auf die Ausbildung des Reporters. Im Fall von ActivinA zeigte sich hingegen kein antagonistischer Einfluss von Bambi. In den Experimenten mit chimären Varianten erfolgte durch die erhaltenen Daten die Eingrenzung der Bindestelle von hBambi-ECD an BMP-2 auf den Bereich der Typ-I-Bindestelle. Ein direkter Einfluss der intrazellulären Domäne auf den BMP-2-Signalweg wurde ausgeschlossen. Weiterhin konnte gerade in Versuchen mit einem Antikörper gegen BR1A-ECD eine weitere Eigenheit der Bindung von Bambi an den Liganden offenbart werden: so bildet das Konstrukt aus hBambi-ECD und der intrazellulären BR1A-Domäne mit zugehöriger GS-Box und Typ-I-Kinase einen korrekt in den signalaktiven heterohexameren Komplex rekrutierten funktionellen Typ-I-Rezeptor. Mit den in dieser Arbeit erzielten Ergebnissen, nämlich der gelungenen Erstellung eines Herstellungsprotokolls der ECD, deren erfolgreich identifizierten Bindepartnern sowie der Charakterisierung der Bindung an BMP-2 ist der Grundstein für die Strukturaufklärung von hBambi-ECD gelegt, welche weitere Klarheit in die Funktionalität dieses Modulators der BMP-/GDF-vermittelten Signalweiterleitung bringen wird. Ebenso sind erste das Verständnis der ICD aufklärende Ergebnisse erzielt worden, die das Fundament für weitere Experimente und darauf folgende Kenntnisgewinne darstellen werden. / The protein family of TGF-β-proteins consists of a huge number of predominantly homodimeric secreted ligands in higher animals, regulating many processes and developmental procedures in embryonic and adult forms via absolute or gradual influence. Signal transduction to cytoplasm and nucleus occurs by the aid of promiscuous, pairwisely recruited type-I- and type-II-receptors, usually activating a variety of receptor-dependent nucleus-permissive SMAD-proteins by an intracellular kinase-phosphorylation-cascade. After translocation they induce transcription of bre-promoted genes. Every signal transduction step may be interfered by endogenous inhibitors. So far the known membrane-bound pseudoreceptor Bambi (BMP and activine membrane-bound inhibitor) is hypothesized to have inhibitory potential against BMP- and activin-mediated signaling pathway via binding of distinct ligand-dependent receptors. However, its proper function still remains unclear. At the beginning of this work a homology model of the extracellular domain of hBambi was built based on a solved 3D-structure of BR1A-ECD bound to its ligand (PDB-ID: 1REW). Upon this model a work hypothesis was issued and biologically active recombinant protein was obtained successfully, both from transfected insect cells and from refolding ex bacterial inclusion bodies in sufficient amount and purified by chromatography, respectively. After comparative quality control of both samples the degree of oligomerisation and secondary structure composition was analyzed via CD spectroscopy and analytical gelfiltration runs. Binding affinity towards nearly all ligands of BMP-/GDF-group assigned to SMAD-1/-5-/-8-signaling was shown for hBambi-ECD by SPR experiments. Known BMP-2-mutants lacking affinity for type-I- or type-II-receptors exhibited wildtype-like binding to hBambi-ECD, respectively. Contrary to the current opinion, neither various ectodomains of receptors nor ActivinA did show any specific binding. Moreover, proposed homooligomerisation of Bambi is not mediated via ECD. Introduction of hBambi-ECD into stimulation experiments on several BMP-responsive cells with differing detection modes yielded in concentration-dependent inhibition of BMP-2-signalling, confirming the results of SPR-attempts well. In a further part of this work various chimeric constructs consisting of Bambi- and BR1A-coding sequence were cloned and effectively co-transfected into HEK Ad293 cells together with plasmids coding for BMP- and Activin-dependent reporters. Stimulation experiments with BMP-2 and ActivinA provided insights into Bambi participation within cellular processes. Full length Bambi exhibited ambivalent behaviour with respect to BMP-2-signaling: small amounts have an agonistic effect, while increasing levels revert it into an antagonistic one with respect to reporter formation. Regarding ActivinA no antagonistic effect was observed. Assays with chimeric constructs led to a containment for Bambi-epitope to the type-I-binding-site of BMP-2. Direct impact of Bambi-ICD on BMP-2-signaling could be blackballed. Furthermore, attempts with an antibody against BR1A-epitope revealed another feature of Bambi-binding to the ligand: a construct of Bambi-ECD fused to intracellular BR1A-domain including kinase and GS-Box forms a functional type-I-receptor correctly orientated and incorporated into heterohexameric signaling-complex. Upon gained results within this work, namely the successful establishment of a purification protocol for the extracellular domain of hBambi, the identification of its binding partners as well as the characterization of a prominent binding partner the basis for successful structure determination of hBambi-ECD aiming to unravel the function of this modulator of BMP-/GDF-signaling is laid. Likewise, first knowledge of the ICD was acquired that will be the basis for further experiments leading to continuative scientific findings.
85

Identification and functional characterization of TGF-β inducible, immunosuppressive miRNAs in human CD8+ T cells / Identifizierung und funktionelle Charakterisierung von TGF-β induzierbaren, immunsuppressiven miRNAs in humanen CD8+ T Zellen

Premachandran Nair, Anoop Chandran January 2014 (has links) (PDF)
While TGF-β is able to regulate miRNA expression in numerous cell types, TGF-β-dependent changes in the miRNA profile of CD8+ T cells had not been studied before. Considering that TGF-β suppresses CD8+ T cell effector functions in numerous ways, we wondered whether induction of immune-regulatory miRNAs could add to the known transcriptional effects of TGF-β on immune effector molecules. In this study, we used miRNA arrays, deep sequencing and qRT-PCR to identify miRNAs that are modulated by TGF-β in human CD8+ T cells. Having found that the TGF-β-dependent downregulation of NKG2D surface expression in NK cells and CD8+ T cells does not go along with a corresponding reduction in mRNA levels, this pathway appeared to be a possible target of TGF-β-inducible miRNAs. However, this hypothesis could not be confirmed by miRNA reporter assays. Instead, we observed that DAP10 transcription is suppressed by TGF-β which in turn negatively affects NKG2D surface expression. In spite of promising preliminary experiments, technical difficulties associated with the transfection of primary NK cells and NK cell lines unfortunately precluded the final proof of this hypothesis. Instead, we focused on the TGF-β-induced changes in the miRNome of CD8+ T cells and confirmed the induction of the miR-23a cluster members, namely miR-23a, miR-27a and miR-24 by three different techniques. Searching for potential targets of these miRNAs which could contribute to the immunosuppressive action of TGF-β in T cells, we identified and confirmed a previously unknown regulation of IFN-γ mRNA by miR-27a and miR-24. Newly generated miRNA reporter constructs further revealed that LAMP1 mRNA is a target of miR-23a. Upon modulation of the miR-23a cluster in CD8+ T cells by the respective miRNA antagomirs and mimics, significant changes in IFN-γ expression confirmed the functional relevance of our findings. Effects on CD107a/LAMP1 expression were, in contrast, rather minimal. Still, overexpression of the miR-23a cluster attenuated the cytotoxic activity of antigen-specific CD8+ T cells. Taken together, these functional data reveal that the miR-23a cluster not only is induced by TGF-β, but also exerts a suppressive effect on CD8+ T-cell effector functions, even in the absence of TGF-β signaling. / Obwohl bekannt war, dass TGF- die miRNA Expression in zahlreichen Zelltypen moduliert, waren TGF- abhängige Veränderung des miRNA Profils in CD8+ T Zellen noch nicht untersucht worden. Da TGF-β die Effektorfunktionen von CD8+ T Zellen aber in vielfältiger Weise inhibiert, fragten wir uns, ob die transkriptionellen Effekte, die TGF-β bekanntermaßen auf Immuneffektormoleküle ausübt, noch durch die Induktion immunregulatorischer miRNAs ergänzt werden. Daher nutzten wir miRNA Arrays, Genomsequenzierungstechniken und Echtzeit-PCR um miRNAs zu identifizieren, welche in humane CD8+ T Zellen von TGF- moduliert werden. Die Beobachtung, dass die TGF--abhängige Herunterregulation der NKG2D Oberflächenexpression in Natürlichen Killerzellen und CD8+ T Zellen nicht mit einer entsprechenden Verringerung der mRNA Menge einhergeht, ließ zudem vermuten, dass dieser Signalweg über miRNAs reguliert werden könnte. Nach verschiedenen miRNA Reporterassays musste diese Hpothese jedoch verworfen werden. Stattdessen zeigte sich, dass TGF- die Transkription von DAP10 inhibiert was wiederum die Oberflächenexpression von NKG2D limitieren sollte. Trotz viel versprechender initialer Experimente scheiterte der letzgültige Beweis dieser Hypothese aber an der ungenügenden Transfizierbarkeit von primären NK Zellen sowie von NK Zelllinien. Daher konzentrierten wir uns im Weiteren auf die durch TGF- induzierten Veränderungen im miRNom von CD8+ T Zellen und konnten mit drei verschiedenen Techniken die Induktion des miR-23a Clusters (mit den einzelnen miRNAs miR-23a, miR-27a und miR-24) bestätigen. Auf der Suche nach potentiellen immunregulatorisch relevanten Zielgenen dieser miRNAs konnten wir erstmals eine Regulation von IFN- durch miR-27a und miR-24 nachweisen. Zu diesem Zweck generierte miRNA Reporterkonstrukte zeigten zudem, dass LAMP1 durch miR-23a reguliert wird. Nach Modulation des miR-23a Clusters durch die entsprechenden miRNA Antagomir und Surrogat-Konstrukte konnten wir auch in CD8+ T Zellen signifikante Veränderungen der IFN-γ Expression nachweisen und somit die funktionelle Relevanz unserer Befunde bestätigen. Die Effekte auf die Expression von CD107a/LAMP1 waren hingegen nur minimal. Trotzdem führte die Überexpression des miR-23a Clusters zu einer Verringerung der zytotoxischen Aktivität von antigenspezifischen CD8+ T Zellen. Zusammen genommen belegen diese funktionellen Untersuchungen, dass das miRNA-23a Cluster, welches durch TGF-β induziert wird, zur Hemung der Effektorfunktionen in CD8+ T Zellen beiträgt, und zwar sowohl in Gegenwart als auch in Abwesenheit von TGF-β.
86

The Effects of Non-Surgical Interventions on Osteoarthritis-Like Changes in the Mouse Knee

Anemaet, Wendy K 31 March 2008 (has links)
Osteoarthritis (OA) is a debilitating condition affecting over 21 million persons in the United States. This number is expected to rise in the coming decades. Treatment approaches for OA focus on symptom modifying measures (i.e., pain relief) as disease modifying interventions do not currently exist. However, some of the interventions used to alleviate the symptoms of OA are also thought to have disease-modifying benefits. Two such non-surgical interventions for OA are intra-articular hyaluronan (HA) injections and physical exercise. In order to effectively study their effects in human OA, animal models that are amenable for studying intervention outcomes are needed. The research focused on developing and characterizing a progressive non-surgical model of knee OA in adult mice. This model was used to firstly, examine the capacity of intra-articular HA injections to prevent knee joint degeneration, and secondly to examine the capacity of moderate exercise to prevent onset and progression of joint degeneration. Intra-articular injections of TGF--β1 into murine knees produce synovial hyperplasia, osteophyte formation, and fibrotic changes on cartilage surfaces and joint capsules. However, additional exposure of the joints to high intensity treadmill running (biomechanical overuse) results in more widespread and focal OA-like cartilage erosions of both the tibial and femoral surfaces, similar to that described for the pathological appearance of late human knee OA. Taken together, these data support that synovitis and soft-tissue activation in early OA joints may precede and/or accelerate the process cartilage degeneration characteristic of progressive and late stage osteoarthritis. Intra-articular injections of high molecular weight HA one day following TGF--β1 injections resulted in decreased synovial hyperplasia, minimized osteophyte formation, and significantly decreased severity of cartilage lesions. A four week, alternate day, low intensity aerobic treadmill running program prior to TGF--β1 injections and overuse also resulted in decreased severity of cartilage lesions.
87

The Role of Endoglin in the Resolution of Inflammation

Peter, Madonna 26 November 2012 (has links)
Endoglin, a co-receptor of the TGF-β superfamily, is predominantly expressed in endothelial cells and in some myeloid cells and implicated as a potential modulator of immune responses. We previously demonstrated that Endoglin heterozygous (Eng+/-) mice subjected to the dextran sulfate sodium colitis model developed persistent inflammation and epithelial ulceration, while Eng+/+ mice recovered following the acute phase of disease. Our aim was to assess potential alterations in distribution and number of immune cells, expression of inflammatory mediators and mechanisms of oxidative burst in Eng+/- mice. While the number of overall T, B and myeloid cells was unaltered between the genotypes, changes in neutrophil regulating cytokines and angiogenesis mediating factors were observed in Eng+/- mice. In addition, downregulation of phagocyte oxidative burst enzymes point to potential defects in microbial clearance in Eng+/- mice. These findings suggest a role for endoglin in regulating immune and vascular functions during inflammation.
88

The Role of Endoglin in the Resolution of Inflammation

Peter, Madonna 26 November 2012 (has links)
Endoglin, a co-receptor of the TGF-β superfamily, is predominantly expressed in endothelial cells and in some myeloid cells and implicated as a potential modulator of immune responses. We previously demonstrated that Endoglin heterozygous (Eng+/-) mice subjected to the dextran sulfate sodium colitis model developed persistent inflammation and epithelial ulceration, while Eng+/+ mice recovered following the acute phase of disease. Our aim was to assess potential alterations in distribution and number of immune cells, expression of inflammatory mediators and mechanisms of oxidative burst in Eng+/- mice. While the number of overall T, B and myeloid cells was unaltered between the genotypes, changes in neutrophil regulating cytokines and angiogenesis mediating factors were observed in Eng+/- mice. In addition, downregulation of phagocyte oxidative burst enzymes point to potential defects in microbial clearance in Eng+/- mice. These findings suggest a role for endoglin in regulating immune and vascular functions during inflammation.
89

Use of autologous platelet concentrates for the treatment of musculoskeletal injuries in the horse

Carmona Ramírez, Jorge Uriel 18 May 2006 (has links)
Las plaquetas (PLTs) son protagonistas en la reparación de las heridas, ya que contienen factores de crecimiento (GFs), los cuales producen quimiotaxis, proliferación y diferenciación celular, neovascularización y deposición de matriz extracelular (ECM). Se ha propuesto la utilización de concentrados de plaquetas (PCs) autólogos para acelerar la reparación de las heridas y estimular la capacidad de regeneración de los tejidos lesionados. En los capitulos III y IV de esta tesis se presentan dos estudios clínicos sobre el efecto de un concentrado plaquetario (PC) autólogo en caballos con patología músculo-esquelética grave. Este PC fue preparado por medio de una nueva técnica de doble centrifugación en tubo. Una caracterización celular y molecular de este PC se presenta en el cápitulo V de ésta tesis. Se evaluó el efecto de la inyección intra-articular del PC en 7 caballos con enfermedad articular grave clasificada así según el grado de cojera (DL) y la efusión sinovial (JE). El PC produjo una mejoría estadísticamente significativa del DL y JE (p<0.05). La mejoría más notable fue observada a los dos meses de finalizado el tratamiento y permaneció hasta 8 meses después (ver cápitulo III). En el capitulo IV se evaluó el efecto del PC en 7 caballos con lesiones tendinosas (tendonitis del tendón flexor digital superficial (SDFT) y ligamentosas (desmitis del ligamento suspensorio (DSL)). Todos los caballos mejoraron significativamente su DL y la respuesta a la prueba de flexión (p<0.05). Los registros ultrasonográficos mejoraron en los caballos con tendinopatías, pero no cambiaron en los que tenían desmopatías. Dos caballos con tendinitis retornaron con exito a su nivel de competición sin recidivar. Un caballo con tendinosis crónica recidivó. El resto de pacientes con desmopatías volvieron a su nivel de competición pre-lesión. Se obtuvieron un promedio de 250 ± 71.8 x 106 plaquetas, 8.68 ± 3.78 leucocitos x 106 y 12515 ± 2443 pg de TGF- ?1/ml de PC. No se observaron signos clínicos adversos asociados con el tratamiento. En el cápitulo V se realizó un análisis celular y molecular del PC usado clínicamente (PC¬C), sangre entera y 4 fracciones de PCs adicionales (PC-A, PC-B y PC-D), los cuales son obtenidos durante la elaboración del PC-C. El objetivo fue evaluar el método de obtención de este PC, en el que son necesarios dos periodos de centrifugación. Todas las muestras fueron analizadas mediante citometría de flujo y determinación de los niveles de TGF- ?1. Las concentraciones de plaquetas para PC-A, PC-B, PC-C y PC-D fueron un 45%, 44%, 71% y 21%, respectivamente más altas en comparación con cada PC y la sangre entera. Las concentraciones de TGF- ?1 para PC-A, PC-B, PC-C y PC-D fueron un 38%, 44%, 44% and 37%, respectivamente más altas en comparación con cada PC y la sangre entera. Se concluyó que el método empleado para concentrar plaquetas es valido para producir PCs con niveles potencialmente terapéuticos de TGF-?1. Los resultados obtenidos en esta tesis abren un nuevo y prometedor campo de investigación para conocer los efectos clínicos y moleculares de los PCs en caballos con enfermedad crónica músculo-esquelética. Los resultados que se puedan obtener en caballos serán de gran valor para conocer el uso potencial de los PCs autólogos en seres humanos con similares patologías. / Platelets (PLTs) play a central role in wound healing, since they contain growth factors (GFs), which produce chemotaxis, cellular proliferation and differentiation, neovascularization, and extra¬cellular matrix (ECM) deposition. The use of autologous platelet concentrates (PCs) has been proposed to accelerate wound repair and to stimulate the regenerative capacity of injured tissues. Two clinical studies about the effect of an autologous platelet concentrate (PC) in horses with severe musculoskeletal pathology are presented in chapters III and IV of this thesis. The PC was prepared by a novel double centrifugation tube method. A cellular and molecular characterization of this PC is presented in chapter V of this thesis.The effect of the intraarticular injection of this PC in 7 horses with severe joint disease was evaluated on the basis on degree of lameness (DL) and joint effusion (JE). When PCs were injected into the joint a statistically significant improvement in both DL and JE (p<0.05) were observed. The most marked improvement was maximun 2 months after the last injection and persisted up to 8 months later (see chapter III). The clinical effect of PC injection in 7 horses with soft tissue musculoskeletal injuries namely: SDFT tendinopathy and desmopathy of the susensory ligament (DSL) was also evaluated (see the chapter IV). All the horses presented a clinical and a statistical (p<0.05) decrease of the DL and the response to flexion test. Ultrasonic appearance improved in the horses with SDFT lesions, but remained the same in the horses with DSL. Two horses with acute SDFT tendinopathy returned successfully to competition level without reinjury. One horse with chronic SDFT tendinopathy relapsed. The rest of the horses with DSL returned successfully to competition level without reinjury. A mean of 250 ± 71.8 x 106 platelets, 8.68 ± 3.78 leucocytes x 106, and 12515 ± 2443 pg TGF-?1 were obtained per ml of the PC. No adverse reactions resulted from this treatment. A cellular and a molecular study of the PC clinically used in this thesis (PC-C),of whole blood and of three additional PCs (PC-A, PC-B, and PC-D) obtained during the PC-C preparation were performed (see the chapter V) to compare the single and the double centrifugation tube methods for concentrating equine platelets. Whole blood and the 4 PCs were analyzed using flow cytometry for cellular quantification and determination of TGF- ?1 in all the samples. Platelet concentrations for PC-A, PC-B, PC-C and PC-D were 45%, 44%, 71% and 21% higher, respectively, compared to the same values for citrated whole blood samples. TGF- ?1 concentrations for PC-A, PC-B, PC-C and PC-D were 38%, 44%, 44% and 37% higher, respectively, compared to citrated whole blood sample values. In conclusion, the single and double centrifugation tube methods are reliable methods for concentrating equine platelets and obtaining potentially therapeutic TGF- ?1 levels. The results obtained in this thesis open a new encouraging research field on clinical and molecular effects of PCs in equine chronic musculoskeletal pathology. The future potential results obtained in horses can be of key value to determine the potenetial use of autologous PCs in human beings with chronic musculoskeletal pathology.
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Investigation on the Pathological Role of Hepatoma-Derived Growth Factor in Hepatic Fibrogenesis

Kao, Ying-hsien 25 August 2009 (has links)
Liver fibrosis, a major medical problem with significant morbidity and mortality, is considered as a wound-healing response to a variety of chronic stimuli. It is characterized by an excessive deposition of extracellular matrix (ECM) proteins, which disrupts the normal architecture of liver and ultimately leads to pathophysiological damage to liver. Hepatoma-derived growth factor (HDGF), a growth factor originally purified from hepatoma cells, is highly expressed in fetal hepatocytes and hepatoma. It is known to play multifunctional roles in mitogenesis, organogenesis, embryogenesis, and tumorigenesis. Its expression correlates with the proliferating state of hepatocellular carcinoma (HCC) and serves as a prognostic factor. Since liver fibrosis frequently occurs prior to HCC development, the specific aim of this study is to investigate the role of HDGF in the progression of liver fibrosis by using animal models of mice receiving either bile duct ligation surgery or carbon tetrachloride administration. Quantitative real-time PCR and Western blotting analysis showed a significant elevation of HDGF expression in both models. HDGF levels correlated with progression of liver fibrosis in a time-dependent manner as well as paralleled with the expression of other two fibrotic markers, transforming growth factor-b1 (TGF-b1) and pro-collagen type I, in fibrotic livers. Intriguingly, the over-expressed HDGF protein was localized mainly in perivenous hepatocytes of fibrotic livers. Besides, adenovirus-mediated HDGF gene delivery potentiated the production of TGF-b1 and pro-collagen type I, thereby enhancing the intrahepatic collagen matrix deposits as evidenced by Sirius red stain and morphometrical analysis. In cultured hepatocytes, TGF-b1 and HDGF mutually up-regulated their de novo synthesis only when grown on collagen-coated matrix, strongly suggesting that the TGF-b1- and/or HDGF-driven pro-fibrogenic signaling is collagen-dependent and a vicious circle may exist at the initial stage of hepatic fibrogenesis. Moreover, administration with recombinant HDGF stimulated BrdU uptake and synthesis of both a-smooth muscle actin and pro-collagen type I in cultured hepatic stellate cells, implicating that a mode of paracrinal action lies between these two cell types. In conclusion, HDGF plays a pro-fibrogenic role during liver fibrosis and blockade of HDGF pathway may potentially constitute the preventive or therapeutic strategies for chronic liver diseases.

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