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Der human pathogene Erreger Streptococcus pneumoniae besiedelt symptomlos den Nasenrachenraum des Menschen, löst aber auch Infektionen wie Otitis Media und invasive Erkrankungen wie Pneumonie und Meningitis aus. Einen essentiellen Schritt für den Infektionsprozess stellt die Anheftung von S. pneumoniae an die Epithel- und Endothelzellen des Wirtes dar. Im Infektionsverlauf ist auch das nachfolgende Eindringen der pathogenen Mikroorganismen in das humane Gewebe von wichtiger Bedeutung. An dieser Interaktion zwischen Erreger und Wirtszellen sind sowohl bakterielle Virulenzfaktoren als auch Komponenten des Wirtes beteiligt. Der pneumococcal adherence and virulence factor A (PavA) von S. pneumoniae ist ein Fibronektin-bindendes Oberflächenprotein und ist essentiell für die Virulenz in einem Sepsis- und einem Pneumonie-Mausinfektionsmodell (Holmes et al., 2001; Lau et al., 2001). In der vorliegenden Arbeit konnte PavA zusätzlich in einem experimentellen Maus-Meningitis-Modell als Virulenzfaktor identifiziert werden. In in vitro Infektionsstudien zeigten pavA-defiziente Pneumokokkenmutanten eine signifikant verringerte Adhärenz an und Invasion in alveoläre Epithelzellen A549 von Typ II Pneumozyten, in Larynxkarzinomzellen HEp-2, in humane Hirnendothelzellen HBMEC und in humane Nabelschnurendothelzellen HUVEC. Diese Zelllinien repräsentieren modellhaft typische Gewebezellen, mit denen S. pneumoniae während des Infektionsprozesses in Kontakt treten kann. Die signifikante Reduktion der Adhärenz der pavA-Mutante ist auf die Mutagenese des pavA-Gens zurückzuführen, da die Komplementierung mit einem aktiven pavA-Gen die Adhärenz an die humanen Zellen wiederherstellte. In Inhibitionsstudien blockierte anti-PavA-Antiserum die bakterielle Bindung an immobilisiertes Fibronektin, die über den C-terminalen Bereich des PavA-Proteins vermittelt wird. Im Gegensatz dazu wurde die Adhärenz von S. pneumoniae an die humanen Wirtszellen in Inhibitionsstudien mit anti-PavA-Antiserum oder rekombinantem PavA-Protein nicht blockiert. Zusammenfassend ist PavA ein wichtiger Virulenzfaktor für die Infektion und das Überleben der Erreger in vivo und spielt gleichzeitig auch eine Rolle während der Adhärenz von Pneumokokken an die humanen Wirtszellen in vitro. Allerdings beeinflusst PavA den Anheftungsprozess nicht direkt als Adhäsin, sondern scheint die Funktion anderer, wichtiger, bisher unbekannter Virulenzfaktoren von S. pneumoniae zu regulieren. Im zweiten Abschnitt der Arbeit wurde die Interaktion von S. pneumoniae mit dem Glykoprotein Fibronektin und deren Bedeutung für die Kolonisierung und den Infektionsmechanismus in vitro untersucht. S. pneumoniae bindet direkt an immobilisiertes Fibronektin (van der Flier et al., 1995). In der vorliegenden Arbeit konnte gezeigt werden, dass Pneumokokken Fibronektin sowohl wirtsunabhängig aus dem Plasma rekrutieren als auch reines lösliches Fibronektin binden können. Dabei binden Pneumokokken sowohl die multimere, zelluläre Form des Fibronektins als auch das lösliche, dimere Plasma-Fibronektin. Die Zugabe von Fibronektin verstärkt die Adhärenz von S. pneumoniae an die humanen Nasopharynxepithelzellen Detroit 562, die Larynxzellen HEp-2, die Bronchialepithelzellen A549 und die Hirnendothelzellen HBMEC. Die Fibronektin-vermittelte Anheftung von Pneumokokken an die Nasopharynxzellen Detroit 562 und die Hirnendothelzellen HBMEC erfolgt wahrscheinlich über die Heparin-Bindungsdomäne, da die bakterielle Adhärenz durch die Zugabe von Heparin inhibiert wird. Weitere Inhibitionsstudien zeigten, dass weder die Präinkubation der Erreger mit anti-PavA-Antiserum noch die Zugabe von rekombinantem PavA-Protein die Fibronektin-vermittelte Adhärenz an die Wirtszellen blockierte. Die Interaktion von S. pneumoniae über das Brückenmolekül Fibronektin mit den humanen Wirtszellen findet damit nicht über das Fibronektin-bindende Oberflächenprotein PavA statt. Fibronektin vermittelt nicht nur die Adhärenz von S. pneumoniae an die Wirtszellen, sondern auch deren Internalisierung. In Inhibitionsstudien mit spezifischen Inhibitoren des Aktin-Zytoskeletts und der Mikrotubuli konnte gezeigt werden, dass die Dynamik des Zytoskeletts eine essentielle Rolle für die Fibronektin-vermittelte Internalisierung der Pneumokokken in die Wirtszellen spielt. Außerdem wurde durch die Zugabe von pharmakologischen Inhibitoren der Tyrosin Kinasen, der Familie der Src-Kinasen und der Phosphatidylinositol (PI-3)-Kinase die Fibronektin-vermittelte bakterielle Invasion in humane Zellen signifikant blockiert. Die Invasion von S. pneumoniae in Mausfibroblasten, die defizient für die fokale Adhäsionskinase (FAK) waren, war im Vergleich zu der bakteriellen Invasion in die Wildtyp-Fibroblasten reduziert. Diese Ergebnisse zeigen die Beteiligung der Src-Kinasen, der PI-3-Kinase und der FAK an der Signaltransduktion, die für die Fibronektin-vermittelte Invasion von S. pneumoniae in eukaryotische Zellen notwendig ist. / The human pathogen Streptococcus pneumoniae causes infections such as otitis media, pneumonia and meningitis. Pneumococci colonize asymptomatically the human nasopharynx. But they can migrate also to the lungs and after breaching the lung barrier spread throughout the human body and induce severe invasive diseases. Adhesion of S. pneumoniae to epithelial and endothelial host cells is an essential first step for the establishment of colonization and invasive infections. Moreover subsequent bacterial invasion into the human tissue is important for developing disease as well. Both, bacterial virulence factors and host components participate in the interaction of pneumococci and human cells. The pneumococcal adherence and virulence factor A (PavA) of S. pneumoniae is a fibronectin binding surface protein and a crucial virulence determinant in a sepsis and in a pneumonia mouse model of infection (Holmes et al., 2001; Lau et al., 2001). Within this study, PavA was identified as an important factor causing disease and promoting survival in an experimental mouse meningitis model. PavA-deficient mutants of S. pneumoniae showed significantly reduced adherence and invasion into the alveolar epithelial cell type A549 of type II pneumocytes, larynx carcinoma epithelial cells HEp-2, human brain derived endothelial cells HBMEC and human umbilical vein derived endothelial cells HUVEC. These cell lines represent typical cells of the human tissue that are involved in the progression of pneumococcal infections. The adhesion level of a pavA-knockout mutant was restored by re-introducing the entire pavA gene, indicating that the observed effect was due to pavA-deficiency. In inhibition studies the addition of anti-PavA antisera inhibited pneumococcal attachment to immobilized fibronectin. Binding of fibronectin was mediated by the C-terminal part of the PavA protein. In contrast, bacterial adherence and invasion into host cells was not influenced by addition of anti-PavA antisera or recombinant PavA protein. In conclusion, PavA plays a pivotal role in pneumococcal adherence to host cells in vitro and is an important virulence factor for the progression of pneumococcal disease and survival in vivo. PavA is not directly involved in the pneumococcal adhesion process by acting as an adhesin. The results indicated that PavA affects colonization and pneumococcal survival by modulating the function of important but yet unknown virulence determinants of S. pneumoniae. In the second part of this study the interaction of S. pneumoniae with the host glycoprotein fibronectin and its role in colonization in vitro was investigated. S. pneumoniae binds directly to immobilized fibronectin (van der Flier et al., 1995). In this study is shown that pneumococci recruite fibronectin from plasma in a species-unspecific manner and bind pure soluble fibronectin. S. pneumoniae interacts with the multimeric cellular form of fibronectin as well as with the soluble dimeric plasma fibronectin. Host-cell bound fibronectin significantly enhances pneumococcal adherence to the human nasopharyngeal epithelial cell line Detroit 562, to larynx cells HEp-2, to alveolar cells A549 and to brain endothelial cells HBMEC. Fibronectin-mediated pneumococcal adherence to the nasopharyngeal cells Detroit 562 and to the brain endothelial cells HBMEC is probably mediated by the heparin binding site due to the fact that bacterial adherence is inhibited by the addition of heparin. Further inhibition studies demonstrated that neither pneumococcal preincubation with anti-PavA antisera nor addition of recombinant PavA protein inhibited fibronectin-mediated adherence. These results indicate that fibronectin-mediated adherence was not due to binding of PavA to fibronectin. Fibronectin plays a key role in pneumococcal adherence to host cells and, in addition, promotes pneumococcal internalization. Inhibition studies with specific inhibitors of the actin cytoskeleton an the microtubulis showed an essential impact of the cytoskeleton dynamic on the fibronectin-mediated pneumococcal invasion into host cells. In addition, presence of pharmacological inhibitors of tyrosine kinases, Src-kinases and Phosphatidylinositol (PI-3)-kinase significantly blocked fibronectin-mediated bacterial invasion in human cells. Pneumococcal invasion into fibroblasts deficient for the focal adhesion kinase (FAK) was reduced compared to wildtype fibroblasts. These results indicate the involvement of Src-kinases, PI-3-kinase and FAK in signal transduction leading to fibronectin-mediated internalization of S. pneumoniae in eukaryotic cells.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Science Johannesburg, 2012 / This experiment was designed to evaluate whether an allogeneic fibrin-fibronectin protein concentrate (AFFP) can not only prevent ankylosis and root resorption of autotransplanted roots during healing but contribute to regenerate a periodontal attachment as well. In two adult male baboons (Papio ursinus), four horizontal alveoli, 2 to 3 mm deep, were prepared bilaterally in the buccal alveolar and basal bone adjacent to the first and second mandibular molars to receive the roots of the adjacent two molars. Following hemisection, the first and second mandibular molars were extracted, the coronal two-thirds of the roots were planed to remove the remnants of the periodontal ligament and cementum and a notch was placed at the junction between the planed and non-planed surfaces. The planed surfaces were demineralised with citric acid at pH 1 for 3 min. Before transplantation, the crowns were resected and the experimental roots and alveoli were coated with the AFFP prepared from pooled fresh-frozen baboon plasma. The animals were killed 55 days after the transplantations. Histometrical evaluation was performed on serial sections cut in a bucco-lingual direction parallel to the long axis of the transplanted roots. An analysis of variance, in relation to the extent of ankylosis and root resorption, revealed minimal differences between the treatments of experimental and control roots both in the planed and non-planed sections. In this primate autotransplantation model, the treatment with AFFP did not prevent ankylosis and root resorption and did not result in the establishment of a new periodontal attachment.

Bioengineered skin substitutes have been developed to treat burn and non-healing wounds; however limitations still hinder their clinical success rates. Optimizing these current design strategies requires an understanding of how biochemical and topographical features of the native tissue modulate keratinocyte processes involved in tissue functionality. In this thesis, a novel bioengineered skin substitute was developed that contains a microfabricated basal lamina analog that recapitulates the native microenvironment found at the dermal-epidermal junction (DEJ). In native skin, this microenvironment consists of both biochemical and topographical cues which play critical roles in maintaining tissue architecture and overall homeostasis with the external environment. Therefore, we hypothesize that microfabricated basal lamina analogs with extracellular matrix cues and three-dimensional features that mimics the cellular microenvironment of the DEJ will promote enhanced epithelialization and increase epidermal stem cell clustering on the surface of bioengineered skin substitutes. We determined that the extracellular matrix protein fibronectin (FN) found in the cellular microenvironment of the DEJ enhanced keratinocyte attachment, proliferation, and epithelialization of a collagen based basal lamina analog. It was also found that the collagen material used to create the basal lamina analog as well as the FN conjugation strategy to this material significantly influenced the bioactivity of FN and its ability to modulate keratinocyte functions through integrin based mechanism. To investigate spatial tissue organization and the role it plays in the cellular microenvironment of the DEJ on epithelialization and epidermal stem cell localization, we used photolithography coupled with materials processing techniques to create microfabricated basal lamina analogs. It was determined that epidermal thicknesses found in narrow channels of microfabricated basal lamina analogs (50 µm and 100 µm widths with 200 µm depths) were similar to cultures on de-epithelialized acellular dermis and native foreskin tissues after 7 days of in vitro culture. We also determined that the microfabricated basal lamina analogs created an epidermal stem cell niche that promoted epidermal stem cell clustering in the channels which is critical for longevity of the tissue. Overall, we developed a platform technology that was specifically used to produce a highly functional bioengineered skin substitute with regenerative capacity that mimics native skin. We anticipate through the use of this technology, we can further improve bioengineered skin substitutes by incorporating epidermal structures of native skin including hair follicles and sweat glands as well as improve overall cosmetic appearance. Additionally, this novel bioengineered skin substitute can serve as a model system to further our understanding of pathological conditions and diseases of the skin as well as facilitate robust preclinical screenings of epidermal responses to new therapeutic agents as well as to cosmetic and chemical products.

Made available in DSpace on 2014-06-11T19:31:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-01Bitstream added on 2014-06-13T21:02:10Z : No. of bitstreams: 1 nakajima_v_dr_botfm.pdf: 679137 bytes, checksum: a82e171acc95c833c38592445769661d (MD5) / Universidade Estadual Paulista (UNESP) / Objetivo: Estudar a expressão da Galectina-3 e a distribuição das proteínas de matriz, laminina, fibronectina e colágeno IV, em 30 amostras teciduais de carcinoma de nasofaringe (CNF) e correlacionar com as características clínicopatológicas, agressividade tumoral e sobrevida dos indivíduos. Forma de estudo: clínico retrospectivo. Casuística e Material: Foram estudadas por método imunohistoquímico 30 amostras teciduais de 26 pacientes com diagnóstico de Carcinoma de Nasofaringe (CNF), cujos blocos parafinados estavam arquivados no Departamento de Patologia da Faculdade de Medicina de Botucatu, UNESP. As lâminas foram revisadas e reclassificadas de acordo com a classificação de 2005 da WHO. As proteínas de matriz e a galectina-3 foram analisadas por método imunohistoquímico. Resultado: A análise mostrou que a media etária foi de 48anos, com o pico de prevalência entre 60 a 69 anos, e predominância do sexo masculino de 2:1. O Carcinoma Escamoso Não Ceratinizante Indiferenciado (CENCI) foi mais comuns em 23 amostras (76,7%), o Carcinoma Escamoso Não Ceratinizante Diferenciado (CENCD) em 4 amostras (13.3%) e Carcinoma Escamoso Ceratinizante (CEC) em 3 amostras (10,0%). A expressão da laminina que normalmente é restrita à parede dos vasos e na lâmina própria, estava muito aumentada na matriz das células neoplásicas em 23 amostras (76,7%); a fibronectina foi positiva em 13 amostras (43%) e a galectina-3 foi positiva em 21 amostras (70%). Tivemos correlação positiva da laminina, fibronectina e galectina-3 em 7 amostras (23,3%) e entre laminina e galectina-3 em 11amostras (36,6%). Tivemos um caso de CEC na recidiva de um CENCD. Conclusão: A expressão positiva da Galectina-3 e da laminina não apresentaram significância estatística quanto a agressividade tumoral e a sobrevida dos pacientes. A presença da fibronectina parece reduzir a chance de recidiva do tumor e também sobrevida maior ao contrário da laminina / Objectives: Analyze the expression of galectin-3 and distribution of matrix proteins, laminin, fibronectin and collagen IV, in 30 paraffinated samples of nasopharynx carcinoma(NFC); and correlate the to clinicopathological characteristics, as tumor aggressiveness and survival of individuals. Study design: retrospective clinical study. Methods: The analyses were performed by immunohistochemistry in 30 tissue samples of 26 patients diagnosed with NPC were archived in the Pathology Department of Botucatu of Medical School, São Paulo State University, UNESP, Brazil. The slides were reviewed and reclassified according to the WHO classification of 2005. The matrix proteins and galectin-3 analyses were performed by immunohistochemistry. Results: The analysis showed that the patients mean age was 48 years-old, with the age peak prevalence was from 60 to 69 years-old, with male predominance of 2:1. The undifferentiated non-keratinized squamous cell carcinoma (UNKSCC) was predominant in 23 samples (79%), differentiated non-keratinized squamous cell carcinoma (DNKCC) was found in 4 samples (13.3%) and keratinized squamous cell carcinoma (KCC) was found in 3 samples (10%). The laminin expression, which is normally restricted to the vessel walls and lamina propria, was increased in the neoplasic cell matrix in 23 samples (76.7%). Fibronectin was positive in 23 samples (43%). Galectin-3 was observed in 21 samples (70%). Seven cases showed positive correlation between all three proteins. Eleven cases presented positive correlation of laminin and galectin-3 immunoexpression. There was one case of KCC in recurrence of DNKCC. Conclusions: The statistical analysis was not conclusive, maybe because of the sample size. Nevertheless, fibronectin presence seems to reduce the chance of tumor recurrence and also, increase the survival rate, differently from laminin and galectin-3

Thesis (doctoral)--Technische Hochschule, Aachen, 2000.

Coeliac disease is a chronic enteropathy triggered by gluten. Patients produce antibodies to gliadin and the autoantigen tissue transglutaminase (tTG). These anti-tTG autoantibodies are disease specific and used in diagnosis. The autoantibodies can be detected by immunofluorescence (the endomysial antibody tests) or by ELISA using recombinant tTG. In vivo tTG associates with fibronectin, which may account for the greater sensitivity of the endomysial antibody assay compared to the ELISA. This project had two aims:  to determine whether GST-tagged tTG bound fibronectin and then, using the fibronectin bound tTG, whether a two-tiered ELISA increased anti-tTG binding in coeliac disease patients. First fibronectin was coupled to a solid support and then incubated with tTG. This was then analysed using SDS-PAGE. Secondly, an ELISA with a two-tiered antigen coating was created by coupling tTG to Fn. This mimics the in vivo orientation of the antigen and could theoretically increase anti-tTG binding. Comparative ELISAs were then run to see if anti-tTG binding differed between tTG and fibronectin-coupled tTG antigen coatings. Results showed GST-tagged tTG bound fibronectin. Coupling of tTG to fibronectin gave no improved binding of anti-tTG. On the contrary, most patients tested had decreased anti-tTG binding compared to the normal tTG based ELISA.

Restenosis is the major cause of the failure of reconstruction methods to restore the blood flow in atherosclerotic arteries. Restenosis results from neointima formation and consequent constrictive remodelling. Vascular smooth muscle cell (VSMC) migration from the tunica media toward the intima is crucial in neointima genesis. The prerequisite for VSMC migratory activity is the modulation from the differentiated (contractile) to the de-differentiated (noncontractile) phenotype. VSMC phenotype change is associated with the altered expression of integrins. alpha8beta1 integrin is upregulated in cell types with contractile properties, including myofibroblasts and mesangial kidney cells. It is one of the integrins that is intensely expressed in mature VSMCs. alpha8beta1 integrin expression during vascular injury and its role in VSMC function have not been studied so far. / In this work, a rat model of carotid angioplasty was used to mimic vascular injury in humans. alpha8beta1 integrin was downregulated in the tunica media concomitantly with loss of the contractile phenotype. In vitro study revealed that it is a differentiation marker of VSMCs. To test the functional significance of the association between alpha8 integrin and the VSMC phenotype, short interference RNA was deployed to silence the alpha8 integrin gene. alpha8 integrin gene silencing heightened VSMC migratory activity as well as modulation of the VSMC phenotype in favour of the noncontractile state. In addition, alpha8 integrin overexpression induced re-differentiation of VSMCs and attenuated their migratory activity. It is, therefore, suggested that alpha8 integrin overexpression after vascular injury might control VSMC migration and neointima formation. On the other hand, alpha8 integrin gene silencing led to a reduced growth rate, which indicated a dichotomy between VSMC migration and proliferation. / In the later stages of neointima formation, constrictive remodeling plays a major role in late lumen loss. Our data demonstrated that alpha8 integrin is upregulated in the neointima during constrictive remodeling with concomitant luminal narrowing. The importance of this finding was highlighted by results showing that alpha8 integrin was required for the VSMC contractile phenotype evoked by transforming growth factor-beta (TFG-beta) and TFG-beta-induced myofibroblastic differentiation of Rat1 fibroblasts. Thus, it appears that alpha8 integrin expression blockade might reduce contractile remodeling and late lumen loss. Although the mechanism of alpha8 integrin signaling is not yet clear, our findings demonstrate that the alpha8 integrin-induced contractile phenotype is blocked by RhoA inhibitors. Furthermore, alpha8 integrin and RhoA are co-immunoprecipitated, and alpha8 integrin gene silencing reduces RhoA activity. Hence, it is postulated that alpha8-RhoA signaling might be closely intertwined. / Altogether, these studies indicate that alpha8 integrin is a contractile marker of VSMCs and a negative regulator of VSMC migration. Therefore, forced alpha8 integrin expression may be applied to reduce neointima formation. However, alpha8 integrin upregulation during constrictive remodeling concomitant with late lumen loss suggest that it could be involved in lumen narrowing. It seems likely that in therapeutic strategies to reduce restenosis the timeline of interference might be very important. Therefore, alpha8 integrin gene silencing in the later stages of neointima formation might be beneficial.