The rate limiting step of phagocytosis is the binding of collagen to specific receptors, which include β1 integrins and the discoidin domain receptor 1 (DDR1). While these two receptors may interact, the functional nature of these interactions is not defined. We examined the effects of DDR1 over-expression on β1 integrin function and determined that DDR1 over-expression enhanced cell attachment through β1 integrins. These data are consistent with data showing that DDR1 over-expression enhanced cell-surface, but not total, β1 integrin expression and activation. As shown by experiments with endoglycosidase H, DDR1 over-expression increased glycosylation of the β1 integrin subunit. Collectively these data indicate that DDR1 enhances β1 integrin interactions with fibrillar collagen, possibly by affecting the processing and trafficking of β1 integrins to the cell surface. Our data provide insight into the mechanisms by which fibrotic conditions such as cyclosporine A-induced gingival overgrowth are regulated.

Long interspersed nuclear element-1 (L1) is a genetic element that mobilizes throughout the mammalian genome via retrotransposition and damages host DNA via mutational insertions, chromosomal rearrangements, and reprogramming of gene expression. The cellular mechanisms responsible for aberrant L1 expression during cancer pathogenesis are unclear. Previously, we have shown that L1 reactivation in several human cell lines is dependent upon the activation of aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor member of the PAS superfamily of proteins. We also showed that ectopic expression of L1 reprograms the HepG2 genome leading to epithelial-to-mesenchymal transition (EMT). Here we present evidence that reactivation of L1 and modulation of EMT in HepG2 cells by the AhR ligand benzo(a)pyrene (BaP) is effected through the canonical TGF-β1 signaling pathway. BaP increased TGF-β1 mRNA, SMAD2 phosphorylation and decreased expression of E-Cadherin. The functional relevance of these interactions and the involvement of TGFBR1/ALK5 and SMAD2/3 were confirmed by siRNA interference. Furthermore, expression of L1-encoded ORF1p was positively correlated with the activation of TGF-β1 signaling in human hepatocarcinoma samples at various stages of malignant progression. These results indicate that ligand-mediated AhR activation regulates L1 via canonical TGF-β1 signaling and raise important questions about the molecular etiology of human hepatocarcinomas.

Hintergrund: Die interstitielle Fibrose spielt bei der Verschlechterung der Nierenfunktion mit dem Endstadium der Urämie eine große Rolle. In diesem Geschehen kommt der Interaktion der im Krankheitsfall vermehrt filtrierten Proteine mit der proximalen Tubuluszelle, dem Ort der Proteinrückresorption, eine entscheidende Bedeutung zu. Das Ziel der vorliegenden Arbeit war es, anhand eines Zellkulturmodells den Einfluss eines vermehrten Albuminangebots auf die Kollagenhomöostase kultivierter proximaler Tubuluszellen zu untersuchen, dabei involvierte Signaltransduktionswege aufzuzeigen und die zentrale Rolle der Albuminendozytose im Rahmen des Geschehens zu beurteilen. Methoden: Für unsere Untersuchungen verwendeten wir überwiegend die kultivierten proximalen Tubuluszellen des Opossums, welche – wie auch die LLC-PK1 Zellen - die für die Proteinendozytose notwendigen Komponenten - nämlich die Rezeptoren Megalin und Cubilin sowie den Natrium-Protonen-Austauscher 3 – besitzen und somit eine hohe Endozytoserate aufweisen. Diese wurden mit Albuminkonzentrationen - wie man sie bei erhöhter Proteinfiltration unter pathophysiologischen Bedingungen findet - inkubiert. Für Vergleichsstudien zogen wir Zelllinien mit niedriger endozytotischer Aktivität (MDCK-, IHKE-1-, NHE3-defiziente-OK-Zellen) heran. Die Kollagenhomöostase wurde mittels Kollagenase-sensitiven Prolininkorporationsassay, Kollagen-ELISA und Kollagen-Western Blot, die Aktivität der Matrixmetalloproteinasen mittels Zymographie und Gelatinaseassay erfasst. Die Aktivierung von Signaltransduktionswegen wurde mittels eines SEAP-Reporter Gen Assays untersucht. Ergebnisse: Albuminexposition führte bei den Zelllinien mit hoher endozytotischer Aktivität (OK- und LLC-PK1- Zellen) zu einer vermehrten Sekretion von Kollagen Typ I, III und IV. Bei den Zelllinien mit niedriger endozytotischer Aktivität (MDCK-, IHKE-1-, NHE3-defiziente-OK-Zellen) kam es nach Albuminexposition zu einem Rückgang der Kollagensekretion. Im Kollagen-Western Blot zeigte sich nach Inkubation mit Albumin eine Zunahme des zellulären Kollagens. Mittels Zymographie und Gelatinaseassay konnte eine Albumin-induzierte Abnahme der MMP-Aktivität nachgewiesen werden. Inkubation der OK-Zellen mit Albumin führte im SEAP-Reporter-Gen-Assay zu einer Aktivierung des NF-kappaB-, AP-1- und CRE-Singalweges. Hemmung der NF-kappaB-, PKC- und PKA- Aktivierung hatte eine teilweise Reduktion der Albumin-inudzierten Kollagensekretion zur Folge. Eine Hemmung der Rezeptor-vermittelten Endozytose mittels des NHE3-Inhibitors EIPA verminderte sowohl die Kollagensekretion als auch die Aktivierung der untersuchten Signaltransduktionswege. Diskussion: Unsere Daten zeigen, dass ein Mehrangebot an Albumin die Kollagenhomöostase der proximalen Tubuluszellen aufgrund einer vermehrten Kollagensynthese und eines verminderten Kollagenabbaus stört. Dabei scheinen vor allem Kollagen Typ I und III zur tubulointerstitiellen Fibrose beizutragen. Albuminexposition aktiviert PKC, PKA, NF-kappaB, AP1 und CRE. Für PKC, PKA und NF-kappaB konnte eine direkte Beteiligung an der Albumin-induzierten Kollagensekretion nachgewiesen werden. Albumin muss mittels Rezeptor-vermittelter Endozytose in die proximale Tubuluszelle aufgenommen werden, um die beobachteten Effekte zu vermitteln. Die alleinige Anwesenheit von Protein im proximalen Tubulus reicht dafür nicht aus. Es ist anzunehmen, dass die gestörte Matrixhomöostase zu einer Progression der interstitiellen Fibrose und somit zum Fortschreiten der Niereninsuffizienz führt. Albuminurie ist also nicht nur ein Marker, sondern ein Motor der Nierenfibrose. / Background: Interstitial fibrosis is of major importance for the deterioration of renal function, leading to uremia. Interaction of filtered proteins with proximal tubular cells is crucial for the onset and development of tubulointerstitial damage. In the present study we investigated the effects of albuminuria on collagen homeostasis and signaling pathways of the proximal tubular cells. Methods: Therefore we exposed cultured cells derived from the proximal tubule (OK-, LLC-PK1-cells) with high endocytic activity by expressing the endocytic machinery typical for the proximal tubule (megalin, cubilin, NHE3) to albumin concentrations in a range expected during enhanced protein filtration. For comparative studies we used renal epithelial cells (MDCK-, IHKE1-, NHE3-deficient-OK-cells) with low endocytic activity. Collagen homeostasis was assessed by collagenase-sensitive proline incorporation assay, collagen ELISA and collagen Western blot; matrix metalloproteinase activity was assessed by zymography and gelatinase assay. Signaling pathways were monitored by SEAP reporter gene asssay. Results: Albumin exposure led to an increase of secreted collagen type I, III and IV in cells with high endocytic activity. In cells with low endocytic activity albumin exposure inhibited collagen secretion. Collagen Western blot analysis showed an albumin-induced increase of cellular collagen. MMP activity was significantly decreased by albumin exposure shown by zymography and gelatinase assay. Furthermore, albumin exposure led to activation of the NF-kappaB-, AP1- and CRE-pathways detected by SEAP reporter gene assay. Inhibition of NF-kappaB, PKC and PKA partially reversed the effects of albumin-induced collagen secretion. Inhibition of receptor-mediated albumin endocytosis by NHE3-inhibitor EIPA reduced collagen secretion and activation of the above investigated pathways. Discussion: The data show that protein exposure, in a concentration range expected during enhanced protein filtration, disturbs collagen homeostasis of proximal tubular cells due to increased collagen synthesis and decreased collagen degradation. Especially collagen type I and III may contribute to tubulointerstitial fibrosis. The signaling pathways activated by albumin exposure include PKC, PKA, NF-kappaB, AP1 and CRE; stimulation of PKC, PKA and NF-kappaB directly contribute to collagen secretion. There has to be efficient receptor-mediated protein endocytosis in order to stimulate collagen secretion and signaling pathways; the mere presence of protein in the proximale tubule is not sufficient. The disturbed matrix homeostasis probably supports the progression of interstitial fibrosis, which is of importance for the development of renal insufficiency. Therefore albuminuria is not only a marker, but also a motor or renal fibrosis.

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.

Dilated cardiomyopathy (DCM) represents an important subgroup of patients suffering from heart failure. The disease is supposed to be associated with autoimmune mechanisms in about one third of the cases. In the latter patients functionally active conformational autoantibodies directed against the second extracellular loop of the β1-adrenergic receptor (AR, β1ECII-aabs) have been detected. Such antibodies chronically stimulate the β1-AR thereby inducing the adrenergic signaling cascade in cardiomyocytes, which, in the long run, contributes to heart failure progression. We analyzed the production of cAMP after aab-mediated β1-AR activation in vitro using a fluorescence resonance energy transfer (FRET) assay. This assay is based on HEK293 cells stably expressing human β1-AR as well as the cAMP-sensor Epac1-camps. The assay showed a concentration-dependent increase in intracellular cAMP upon stimulation with the full agonist (-) isoproterenol. This response was comparable to results obtained in isolated adult murine cardiomyocytes and was partially blockable by a selective β1-AR antagonist. In the same assay poly- and monoclonal anti-β1ECII-abs (induced in different animals) could activate the adrenergic signaling cascade, whereas isotypic control abs had no effect on intracellular cAMP levels. Using the same method, we were able to detect functionally activating aabs in the serum of heart failure patients with ischemic and hypertensive heart disease as well as patients with DCM, but not in sera of healthy control subjects. In patients with DCM we observed an inverse correlation between the stimulatory potential of anti-β1-aabs and left ventricular pump function. To adopt this assay for the detection of functionally activating anti-β1ECII-aabs in clinical routine we attempted to establish an automated large-scale approach. Neither flow cytometry nor FRET detection with a fluorescence plate reader provided an acceptable signal-to-noise ratio. It was possible to detect (-) isoproterenol in a concentration-dependent manner using two different FRET multiwell microscopes. However, due to focus problems large-scale detection of activating anti-β1ECII-abs could not be implemented. Neutralization of anti-β1-aabs with the corresponding epitope-mimicking peptides is a possible therapeutic approach to treat aab-associated autoimmune DCM. Using our FRET assay we could demonstrate a reduction in the stimulatory potential of anti-β1ECII-abs after in vitro incubation with β1ECII-mimicking peptides. Cyclic (and to a lesser extent linear) peptides in 40-fold molar excess acted as efficient ab-scavengers in vitro. Intravenously injected cyclic peptides in a rat model of DCM also neutralized functionally active anti-β1ECII-abs efficiently in vivo. For a detailed analysis of the receptor-epitope targeted by anti-β1ECII-abs we used sequentially alanine-mutated β1ECII-mimicking cyclic peptides. Our data revealed that the disulfide bridge between the cysteine residues C209 and C215 of the human β1-AR appears essential for the formation of the ab-epitope. Substitution of further amino acids relevant for ab-binding in the cyclic scavenger peptide by alanine reduced its affinity to the ab and the receptor-activating potential was blocked less efficiently. In contrast, the non-mutant cyclic peptide almost completely blocked ab-induced receptor activation. Using this ala-scan approach we were able to identify a “NDPK”-epitope as essential for ab binding to the β1ECII. In summary, neutralization of conformational activating anti-β1ECII-(a)abs by cyclic peptides is a plausible therapeutic concept in heart failure that should be further exploited based on the here presented data. / Dilatative Kardiomyopathie (DCM) stellt eine wichtige Subgruppe von Patienten mit Herzinsuffizienz dar und ist vermutlich in etwa einem Drittel der Fälle mit einem Autoimmunmechanismus assoziiert. In diesen Patienten konnten funktionell aktive konformationelle Autoantikörper gegen die zweite extrazelluläre Schleife des β1-adrenergen Rezeptors (Anti-β1ECII-AAK) nachgewiesen werden. Diese AK stimulieren chronisch den β1-AR und induzieren dadurch die adrenerge Signaltransduktionskaskade in Kardiomyozyten, was, auf lange Sicht, zur Verschlimmerung der Herzinsuffizienz beiträgt. Mittels eines Fluoreszenz-Resonanz Energie Transfer (FRET) Assays analysierten wir die Bildung von cAMP nach aab-vermittelter β1-AR Aktivierung in vitro. Dieser Assay verwendet HEK293 Zellen, die den humanen β1-AR sowie den cAMP-Sensor Epac1-camps stabil exprimieren. Der Assay zeigte einen konzentrationsabhängigen Anstieg von cAMP bei Stimulation mit dem vollen Agonisten (-) Isoproterenol. Diese Antwort war mit den Ergebnissen an isolierten adulten murinen Kardiomyozyten vergleichbar und konnte durch einen selektiven β1-AR Antagonist blockiert werden. In verschiedenen Tieren induzierte poly- und monoklonale Anti-β1¬ECII-abs konnten die adrenerge Signalkaskade in dem gleichen FRET Assay aktivieren, wogegen isotypische Kontroll-AK keine intrazellulären cAMP Änderungen herbeiführten. Mit dem gleichen Ansatz konnten wir funktionell aktivierende AAK im Serum von Herzinsuffizienzpatienten mit ischämischer und hypertensiver Herzerkrankung sowie bei Patienten mit dilatativer Kardiomyopathie nachweisen, jedoch nicht im Serum von gesunden Kontrollpersonen. Bei Patienten mit dilatativer Kardiomyopathie beobachteten wir eine inverse Korrelation zwischen dem stimulierenden Potential der Anti-β1-AAKs und der linksventrikulären Pumpfunktion. Um diesen Assay zur Detektion funktionell aktivierender Anti-β1ECII-aabs in der klinischen Routinediagnostik einzusetzen, versuchten wir ein automatisiertes Hochdurchsatzverfahren zu etablieren. Weder die Durchflusszytometrie noch die FRET Detektion mittels eines Fluoreszenz Plattenlesegeräts wiesen einen akzeptabelen Signal-zu-Rausch-Quotienten auf. Es gelang mit zwei verschiedenen FRET-Multiwell-Mikroskopsystemen die Aktivierung durch (-) isoproterenol konzentrationsabhängig zu detektieren, allerdings war es aufgrund von Fokusproblemen nicht möglich aktivierende Anti- β1¬ECII-AAKs im Hochdurchsatzverfahren zu detektieren. Einen möglichen Therapieansatz zur Behandlung der Anti-β1-AAK-assoziierten autoimmunen DCM stellt die Neutralisierung der AAKs mittels korrespondierender Epitop-imitierender Peptide dar. Wir konnten im FRET Assay eine Reduktion des aktivierenden Effekts von Anti-β1¬ECII-AAKs nach in vitro Inkubation mit β1¬ECII-imitierenden Peptiden nachweisen. Hierbei erwiesen sich zyklische (in geringerem Maß als lineare) Peptide in 40-fachem molarem Überschuss als effektive Antikörper-„Fänger“ in vitro. Eine intravenöse Gabe der Zyklopeptide in einem Rattenmodell der DCM neutralisierte funktionell aktivierende Anti-β1¬ECII-abs ebenfalls effizient in vivo. Zur genaueren Analyse des von Anti-β1¬ECII-AAK gebundenen Rezeptor-Epitops setzten wir sequentiell Alanin-mutierte β1ECII-imitierende Zyklopeptide ein. Unsere Daten zeigten, dass die Disulfidbrücke zwischen den Cysteinresten C209 und C215 des humanen β1¬AR essentiell für die Ausbildung des AAK-Epitops zu sein scheinen. Ein Austausch weiterer AK-bindungsrelevanter Aminosäuren im zyklischen Fängerpeptid durch Alanin verminderte dessen Avidität zum AK und inhibierte dessen Rezeptor-aktivierendes Potential weniger effizient. Im Gegensatz dazu verhinderte das nicht-mutierte Zyklopeptid nahezu vollständig die AK-vermittelte Rezeptoraktivierung. Durch diesen Ala-Scan Ansatz konnten wir ein „NDPK“-Epitop identifizieren, das für die AK-Bindung an β1¬ECII essentiell zu sein scheint. Zusammenfassend ist festzuhalten, dass die Neutralisation von konformationellen aktivierenden Anti-β1¬ECII-(A)AKs durch Zyklopeptide ein viel versprechendes Therapiekonzept bei Herzinsuffizienz darstellt, das im Hinblick auf die hier vorgestellten Daten weiter ausgebaut werden sollte.

The soil-borne filamentous fungus Trichoderma virens is a biocontrol agent with a well known ability to produce antibiotics, parasitize pathogenic fungi and induce systemic resistance in plants. Here we report the identification, purification and characterization of an elicitor secreted by T. virens; a small protein designated Sm1 (small protein 1). Confrontation and disk assays demonstrated that Sm1 lacks toxic activity against plants and microbes. Native, purified Sm1 triggers production of reactive oxygen species in rice (Oryza sativa) and cotton (Gossypium hirsutum), and induces the expression of defense related genes both locally and systemically in cotton. Gene expression analysis revealed that SM1 is expressed throughout fungal development and is transcriptionally regulated by nutrient conditions and the presence of a host plant. When T. virens was co-cultured with cotton in an axenic hydroponic system, SM1 expression and secretion of the protein was significantly higher than when the fungus was grown alone. These results indicate that Sm1 is involved in plant-Trichoderma recognition and the induction of resistance by activation of plant defense mechanisms. Following the cloning of SM1, strains disrupted in or over-expressing SM1 were generated. Targeted gene disruption revealed that SM1 was not involved in fungal development. Expression of defense related genes in cotton and maize (Zea mays) was induced locally and systemically following colonization by T. virens in the hydroponic system. Low levels of expression of cotton or maize defense genes were found when seedlings were grown with a T. virens strain disrupted in SM1, ssupporting the Sm1-elicitor hypothesis. Additionally, unique proteins in T.virens-cotton/maize interaction were identified. Thus, the induction of defense responses in two agriculturally important crops appears to be microbially mediated. Functional analysis of a cell wall degrading enzyme, beta-1,6-glucananse (Tv-bgn3) from T. virens, demonstrated involvement of this enzyme indirectly in mycoparasitic activity of T. virens. Protein extracts from the strain disrupted in TV-BGN3 displayed reduced capability to inhibit growth of Pythium ultimum as compared to the wild-type. Additionally, protein extracts from the strains co-expressed with TV-BGN2 (beta-1,3-glucananse) from T. virens showed a significantly increased capability to inhibit growth of P. ultimum and Rhizoctonia solani hyphae.

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.

Articular cartilage functions to maintain joint mobility. The loss of healthy, functional articular cartilage due to osteoarthritis or injury can severely compromise quality of life. To address this issue, cartilage tissue engineering approaches are currently in development. Bone marrow-derived mesenchymal progenitor cells (MPCs) hold much promise as an alternative cell source for cartilage tissue engineering. While previous studies have established that MPCs from humans and multiple other species undergo in vitro chondrogenic differentiation, additional research is needed to define conditions that will enhance MPC differentiation, increase matrix production by differentiating cultures, and support development of functional tissue-engineered cartilage constructs. Mechanical loading may be an important factor regulating chondrogenic differentiation of MPCs and cartilage matrix formation by chondrogenic MPCs. This thesis work evaluated the influence of oscillatory unconfined compressive mechanical loading on in vitro MPC chondrogenic activity and biosynthesis within hydrogel suspension. Loading was conducted using MPCs cultured in media supplements supporting chondrogenic differentiation. Possible interactions between the number of days in chondrogenic media preceding loading initiation and the ability of the MPC culture to respond to mechanical stimulation were explored in two different loading studies. The first loading study investigated the effects of 3 hour periods of daily oscillatory mechanical stimulation on subsequent chondrogenic activity, where chondrogenic activity represented an assessment of cartilage matrix production by differentiating MPCs. This study found that oscillatory compression of MPCs initiated during the first seven days of culture did not enhance chondrogenic activity above the level supported by media supplements alone. The second loading study evaluated changes in biosynthesis during a single 20 hour period of oscillatory mechanical stimulation to assess mechanoresponsiveness of the MPC cultures. This study found that MPCs modulated proteoglycan and protein synthesis in a culture time-dependent and frequency-dependent manner upon application of oscillatory compression. Together the two loading studies provide an assessment of dynamic compressive mechanical loading influences on MPC cultures undergoing chondrogenic differentiation. The information gained through in vitro studies of differentiating MPC cultures will increase basic knowledge about progenitor cells and may also prove valuable in guiding the future development of cartilage tissue engineering approaches.

The soil-borne filamentous fungus Trichoderma virens is a biocontrol agent with a well known ability to produce antibiotics, parasitize pathogenic fungi and induce systemic resistance in plants. Here we report the identification, purification and characterization of an elicitor secreted by T. virens; a small protein designated Sm1 (small protein 1). Confrontation and disk assays demonstrated that Sm1 lacks toxic activity against plants and microbes. Native, purified Sm1 triggers production of reactive oxygen species in rice (Oryza sativa) and cotton (Gossypium hirsutum), and induces the expression of defense related genes both locally and systemically in cotton. Gene expression analysis revealed that SM1 is expressed throughout fungal development and is transcriptionally regulated by nutrient conditions and the presence of a host plant. When T. virens was co-cultured with cotton in an axenic hydroponic system, SM1 expression and secretion of the protein was significantly higher than when the fungus was grown alone. These results indicate that Sm1 is involved in plant-Trichoderma recognition and the induction of resistance by activation of plant defense mechanisms. Following the cloning of SM1, strains disrupted in or over-expressing SM1 were generated. Targeted gene disruption revealed that SM1 was not involved in fungal development. Expression of defense related genes in cotton and maize (Zea mays) was induced locally and systemically following colonization by T. virens in the hydroponic system. Low levels of expression of cotton or maize defense genes were found when seedlings were grown with a T. virens strain disrupted in SM1, ssupporting the Sm1-elicitor hypothesis. Additionally, unique proteins in T.virens-cotton/maize interaction were identified. Thus, the induction of defense responses in two agriculturally important crops appears to be microbially mediated. Functional analysis of a cell wall degrading enzyme, beta-1,6-glucananse (Tv-bgn3) from T. virens, demonstrated involvement of this enzyme indirectly in mycoparasitic activity of T. virens. Protein extracts from the strain disrupted in TV-BGN3 displayed reduced capability to inhibit growth of Pythium ultimum as compared to the wild-type. Additionally, protein extracts from the strains co-expressed with TV-BGN2 (beta-1,3-glucananse) from T. virens showed a significantly increased capability to inhibit growth of P. ultimum and Rhizoctonia solani hyphae.

Cellular adhesion plays a crucial role in the biological function of cells, allowing them to communicate and signal, as well as physically anchor, by enabling them to adhere to either other cells or the extra cellular matrix (ECM). This process is regulated by several factors including intrinsic bond kinetics, internal cellular signaling, environment, force exerted on the bond, and force history of the bond. Concerning the force and force history dependence, the observation of catch bonds in integrin binding has asked as more questions than it has answered. To explore the force and force history dependence this process, each bond was loaded to a peak force before relaxing to a much lower force that was held for the duration of the measurement. Two different integrins were studied, both of which have in previous works exhibited a catch bond. Furthermore, the effects of different metal ion conditions and an allosteric antagonist were also studied to elucidate the conformational effects on force priming of integrin. What was observed was that I domain, or αA domain, possessing integrin, whether tested against its more active or less active binding state, changed very little in terms of off rate once the priming force was applied. However in the I domain, or αA domain, lacking integrin, the observed off rate changed as well. It seems that force priming is capable of causing integrin to bind in a stronger manner regardless of the other conditions used to either activate or inhibit binding. However the way in which the binding is strengthened depends on the receptors structure.