Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A medula óssea adulta possui duas populações de células-tronco importantes no tratamento de diversas doenças hepáticas: células-tronco hematopoiéticas (CTHs) e células-tronco mesenquimais. A regeneração do fígado após a hepatectomia é um processo complexo que requer a proliferação de todas as células hepáticas. Fatores de crescimento, citocinas e componentes da matriz extracelular são elementos-chave nesse processo. As lamininas são uma família de proteínas de matriz extracelular, com funções adesivas e quimiotáticas pelo recrutamento de integrinas e outros receptores de superfície celular. No fígado normal, a laminina é expressa nas veias porta e centrolobular. O objetivo desse estudo foi investigar a expressão de laminina durante a regeneração hepática induzida por hepatectomia parcial e após o transplante de células mononucleares de medula óssea. As células mononucleares de medula óssea foram obtidas dos fêmures e tíbias de ratos, isoladas, marcadas com DAPI e injetadas pela veia porta em ratos recém-hepatectomizados. Os fígados foram coletados 15 minutos, 1 dia e 3 dias após a hepatectomia e o transplante de células de medula óssea e congelados. Os cortes foram imunomarcados com anticorpos primários anti-CD34 e anti-laminina de rato e observados em microscópio confocal de varredura a laser. Os resultados mostraram que 15 minutos após a hepatectomia parcial, as células-tronco hematopoiéticas CD34+ transplantadas foram encontradas em contato com a laminina localizada nas veias porta e centrolobular, indicando que a laminina poderia participar na adesão inicial das células-tronco a esses vasos logo após o seu transplante. Além disso, 1 e 3 dias após a hepatectomia, as células mononucleares de medula óssea transplantadas foram observadas nos sinusóides hepáticos expressando laminina. Esses resultados sugerem que a laminina pode ser um componente da matriz extracelular importante para a adesão e enxerto de células de medula óssea no fígado após uma lesão. Nós também analisamos a expressão de osteopontina (OPN) em células de medula óssea e CTHs. Os resultados por microscopia confocal demonstraram que a maioria das células mononucleares de medula óssea recém-isoladas expressa quantidades variáveis de OPN. Além disso, algumas CTHs CD34+ também expressam OPN. Após 1 e 4 dias de cultura, observamos uma diminuição de células expressando CD34, e um aumento na expressão de OPN pelas células mononucleares de medula óssea. / The adult bone marrow retains two populations of stem cells with emerging importance for the treatment of diverse liver diseases: hematopoietic stem cells (HSCs) and mesenchymal stem cells. Liver regeneration after partial hepatectomy is a complex process that requires the proliferation of all hepatic cells. Growth factors, cytokines and extracellular matrix molecules are key elements in this process. Laminins are a family of heterotrimeric extracellular matrix proteins with adhesive and chemotactic functions, through recruitment of integrins and other cell surface receptors. In the normal liver, laminin is expressed in portal and centrolobular veins. The aim of this study was to investigate laminin expression during liver regeneration induced by partial hepatectomy and after bone marrow mononuclear cells transplantation. Rat bone marrow mononuclear cells were obtained from tibias and femurs, isolated, stained with DAPI and injected in immediately hepatectomyzed rats via portal vein. Livers were collected 15 minutes, 1 day and 3 days after hepatectomy and bone marrow cells transplantation and frozen. Liver sections were immunolabeled with mouse anti-rat CD34 and rabbit anti-rat laminin primary antibodies and observed under a Laser Scanning Confocal Microscope. Results showed that 15 minutes after partial hepatectomy, transplanted CD34+ HSCs were found in contact with laminin, which was localized principally in portal and centrolobular veins of rat livers, indicating that laminin could participate in stem cell initial attachment to these vessels soon after their transplantation. Furthermore, 1 and 3 days after hepatectomy, transplanted bone marrow mononuclear cells were found in the hepatic sinusoids expressing laminin. These results suggest that laminin could be an important extracellular matrix component for bone marrow cell adhesion and grafting in the injured liver. We also analyzed osteopontin (OPN) expression in bone marrow mononuclear cells and HSCs. Results by confocal microscopy showed that the most freshly isolated bone marrow mononuclear cells express variable amounts of OPN. Furthermore, some CD34+ HSCs also expressed OPN. After 1 and 4 days in culture, we observed a decrease in CD34+ cells, and an increase in OPN expression in bone marrow mononuclear cells.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A medula óssea adulta possui duas populações de células-tronco importantes no tratamento de diversas doenças hepáticas: células-tronco hematopoiéticas (CTHs) e células-tronco mesenquimais. A regeneração do fígado após a hepatectomia é um processo complexo que requer a proliferação de todas as células hepáticas. Fatores de crescimento, citocinas e componentes da matriz extracelular são elementos-chave nesse processo. As lamininas são uma família de proteínas de matriz extracelular, com funções adesivas e quimiotáticas pelo recrutamento de integrinas e outros receptores de superfície celular. No fígado normal, a laminina é expressa nas veias porta e centrolobular. O objetivo desse estudo foi investigar a expressão de laminina durante a regeneração hepática induzida por hepatectomia parcial e após o transplante de células mononucleares de medula óssea. As células mononucleares de medula óssea foram obtidas dos fêmures e tíbias de ratos, isoladas, marcadas com DAPI e injetadas pela veia porta em ratos recém-hepatectomizados. Os fígados foram coletados 15 minutos, 1 dia e 3 dias após a hepatectomia e o transplante de células de medula óssea e congelados. Os cortes foram imunomarcados com anticorpos primários anti-CD34 e anti-laminina de rato e observados em microscópio confocal de varredura a laser. Os resultados mostraram que 15 minutos após a hepatectomia parcial, as células-tronco hematopoiéticas CD34+ transplantadas foram encontradas em contato com a laminina localizada nas veias porta e centrolobular, indicando que a laminina poderia participar na adesão inicial das células-tronco a esses vasos logo após o seu transplante. Além disso, 1 e 3 dias após a hepatectomia, as células mononucleares de medula óssea transplantadas foram observadas nos sinusóides hepáticos expressando laminina. Esses resultados sugerem que a laminina pode ser um componente da matriz extracelular importante para a adesão e enxerto de células de medula óssea no fígado após uma lesão. Nós também analisamos a expressão de osteopontina (OPN) em células de medula óssea e CTHs. Os resultados por microscopia confocal demonstraram que a maioria das células mononucleares de medula óssea recém-isoladas expressa quantidades variáveis de OPN. Além disso, algumas CTHs CD34+ também expressam OPN. Após 1 e 4 dias de cultura, observamos uma diminuição de células expressando CD34, e um aumento na expressão de OPN pelas células mononucleares de medula óssea. / The adult bone marrow retains two populations of stem cells with emerging importance for the treatment of diverse liver diseases: hematopoietic stem cells (HSCs) and mesenchymal stem cells. Liver regeneration after partial hepatectomy is a complex process that requires the proliferation of all hepatic cells. Growth factors, cytokines and extracellular matrix molecules are key elements in this process. Laminins are a family of heterotrimeric extracellular matrix proteins with adhesive and chemotactic functions, through recruitment of integrins and other cell surface receptors. In the normal liver, laminin is expressed in portal and centrolobular veins. The aim of this study was to investigate laminin expression during liver regeneration induced by partial hepatectomy and after bone marrow mononuclear cells transplantation. Rat bone marrow mononuclear cells were obtained from tibias and femurs, isolated, stained with DAPI and injected in immediately hepatectomyzed rats via portal vein. Livers were collected 15 minutes, 1 day and 3 days after hepatectomy and bone marrow cells transplantation and frozen. Liver sections were immunolabeled with mouse anti-rat CD34 and rabbit anti-rat laminin primary antibodies and observed under a Laser Scanning Confocal Microscope. Results showed that 15 minutes after partial hepatectomy, transplanted CD34+ HSCs were found in contact with laminin, which was localized principally in portal and centrolobular veins of rat livers, indicating that laminin could participate in stem cell initial attachment to these vessels soon after their transplantation. Furthermore, 1 and 3 days after hepatectomy, transplanted bone marrow mononuclear cells were found in the hepatic sinusoids expressing laminin. These results suggest that laminin could be an important extracellular matrix component for bone marrow cell adhesion and grafting in the injured liver. We also analyzed osteopontin (OPN) expression in bone marrow mononuclear cells and HSCs. Results by confocal microscopy showed that the most freshly isolated bone marrow mononuclear cells express variable amounts of OPN. Furthermore, some CD34+ HSCs also expressed OPN. After 1 and 4 days in culture, we observed a decrease in CD34+ cells, and an increase in OPN expression in bone marrow mononuclear cells.

The biological half-life of several pro-inflammatory mediators involved in the pathogenesis of rheumatoid arthritis (RA) is controlled by molecules exhibiting dipeptidyl peptidase-IV (DPP-IV)-like enzymatic activity (Dipeptidyl peptidase-IV activity and/or structure homologues- DASH). The aim of this thesis was to identify the molecular source of the DPP-IV-like enzymatic activity in the peripheral blood and synovial fluid in patients with rheumatoid arthritis as compared to control patients with osteoarthritis (OA), and to evaluate the association of DPP-IV with the disease activity. We found that the main source of the DPP-IV-like enzyme activity in the plasma and in the synovial fluid in patients with RA is the canonical DPP-IV. DPP-IV-like enzymatic activity and canonical DPP-IV were also detected on the cell surface of blood and synovial fluid mononuclear cells. Significantly lower DPP-IV-like enzymatic activity and DPP-IV expression in the synovial fluid mononuclear cells was found in RA as opposed to OA patients. In the synovial fluid of RA patients there was also a negative correlation between the concentration of the pro-inflammatory DPP-IV substrate SDF (stromal cell-derived factor-1 and the proportion of the DPP-IV+ T cells. The blood plasma DPP-IV-like enzymatic activity and...

The biological half-life of several pro-inflammatory mediators involved in the pathogenesis of rheumatoid arthritis (RA) is controlled by molecules exhibiting dipeptidyl peptidase-IV (DPP-IV)-like enzymatic activity (Dipeptidyl peptidase-IV activity and/or structure homologues- DASH). The aim of this thesis was to identify the molecular source of the DPP-IV-like enzymatic activity in the peripheral blood and synovial fluid in patients with rheumatoid arthritis as compared to control patients with osteoarthritis (OA), and to evaluate the association of DPP-IV with the disease activity. We found that the main source of the DPP-IV-like enzyme activity in the plasma and in the synovial fluid in patients with RA is the canonical DPP-IV. DPP-IV-like enzymatic activity and canonical DPP-IV were also detected on the cell surface of blood and synovial fluid mononuclear cells. Significantly lower DPP-IV-like enzymatic activity and DPP-IV expression in the synovial fluid mononuclear cells was found in RA as opposed to OA patients. In the synovial fluid of RA patients there was also a negative correlation between the concentration of the pro-inflammatory DPP-IV substrate SDF (stromal cell-derived factor-1 and the proportion of the DPP-IV+ T cells. The blood plasma DPP-IV-like enzymatic activity and...

Qualitativ unterschiedliche genomische und nichtgenomische Mechanismen vermitteln die starken anti-inflammatorischen und immunmdulatorischen Eigenschaften der Glucocorticoide (GC). Der genomisch vermittelte Mechanismus ist bereits gut untersucht und dokumentiert, während der nichtgenomisch vermittelte Mechanismen noch einen Gegenstand vielseitiger Untersuchungen darstellt. Wir haben uns daher die Frage gestellt, ob Beclometason und Clobetasol besonders geeignet für die topische Applikation sind, weil sie sich in ihrem Wirkungsspektrum von systemisch zu applizierenden GC wie Dexamethason unterscheiden. Wir verglichen dazu die Effekte auf den Sauerstoffverbrauch mittels der Clark-Elektrode (nichtspezifisch nichtgenomischer Mechanismus), auf die IL-6-Synthese mittels ELISA (genomischer Mechanismus) und auf die Apoptose mittels Durchflusszytometrie (nichtgenomischer und genomischer Mechanismus) in ruhenden und stimulierten humanen PBMC. Dabei zeigten Beclometason und Clobetasol in sehr niedrigen Konzentrationen (10-10, 10-8 M) einen stärkeren Effekt auf den Sauerstoffverbrauch, waren aber in hohen Konzentrationen (10-5, 10-4 M) weniger potent im Vergleich zu Dexamethason. Auch hinsichtlich ihrer genomischen Potenz waren die topischen GC in einer Konzentration von 10-10 M und 10-8 M effektiver als Dexamethason, in höheren Konzentrationen unterschieden sie sich aber nicht. Alle drei GC induzierten Apoptose konzentrationsabhängig und unterschieden sich nicht in Konzentrationen zwischen 10-8 M und 10-5 M. In einer Konzentration von 10-4 M war die Induktion von Apoptose durch die topischen GC in PBMC und Jurkat-T-Zellen aber signifikant stärker im Vergleich zu Dexamethason. Diese Ergebnisse zeigen, dass sich topische und systemische GC in ihrer genomischen und nichtgenomischen Potenz signifikant unterscheiden. Es ist daher davon auszugehen, dass nichtgenomische Effekte eine deutlichere klinische Relevanz besitzen als bisher angenommen. / Several different genomic and non-genomic mechanisms mediate the important anti-inflammatory and immunomodulatory effects of glucocorticoids (GCs). The genomic effects are the most important while the clinical relevance of non-genomic actions is still a matter of debate. We therefore investigated whether beclomethasone and clobetasol are particularly suitable for topical application because they differ in their spectrum of activity from systemically administered GCs such as dexamethasone. We compared effects on oxygen consumption as measured with a Clark electrode (nonspecific non-genomic glucocorticoid effects), on interleukin-6 synthesis by means of ELISA (genomic effects) and on apoptosis using flow cytometry (non-genomic and genomic effects) in quiescent and mitogen-stimulated PBMCs. Beclomethasone and clobetasol had stronger effects on the oxygen consumption of quiescent and stimulated cells at lower concentrations (10-10, 10-8 M) but were less potent at higher concentrations (10-5, 10-4 M) in comparison with dexamethasone. Also in terms of genomic potency, topical GCs were more effective than dexamethasone at 10-10 M and 10-8 M but gave similar results at higher concentrations. The ability of all three GCs to induce apoptosis was found to be concentration-dependent and similar at concentrations between 10-8 and 10-5 M but, compared with 10-4 M dexamethasone, 10-4 M beclomethasone or clobetasol was significantly more effective at inducing apoptosis in both PBMCs and Jurkat T cells. These results show that systemic and topical GCs differ significantly in their ability to induce genomic and non-genomic effects. This suggests that non-genomic effects are more therapeutically relevant in certain clinical conditions than currently assumed.