Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 4 uppsatser.

Este trabalho realizou importantes avanços na elucidação do potencial das toxinas termo-lábeis do tipo II (LT-IIs) como adjuvantes por via intradérmica e transcutânea. Os dados gerados indicam que LT-IIb e LT-IIc nativas atuam como potentes adjuvantes vacinais por via intradérmica induzindo respostas imunológicas antígeno específicas, como medido pela produção de anticorpos sistêmicos (IgG) e ativação de linfócitos T CD8+ citotóxicos. Soma-se ao potencial adjuvante demonstrado para as LT-IIs por essa via a baixa reatogenicidade das moléculas, com menores níveis de edema e reduzida migração leucocitária para o sítio de inoculação em comparação a LT-I. Os resultados indicam também que o efeito adjuvante das LTs aplicadas por via transcutânea pode estar relacionado à capacidade de ligação ao gangliosídeo GM1 já que a toxina LT-IIb, incapaz de interagir com este receptor, não apresenta efeito adjuvante por essa via. O conjunto de dados apresentados abre perspectivas para o emprego das LT-IIs nativas como adjuvantes parenterais em vacinas para animais e humanos. / This work has made significant advances in the understanding of the potential of type II heat-labile toxins (LT-IIs) as vaccine adjuvants by intradermal and transcutaneous route. The generated data indicate that native forms of LT-IIb and LT-IIc act as potent vaccine adjuvants when intradermally injected inducing antigen-specific immune responses, as measured by the generation of systemic serum antibody (IgG) and activation of cytotoxic CD8+ T lymphocytes. Besides the adjuvant effects, LT-IIs show reduced side effects, measured by the lesser edema formation and reduced leukocytes migration to the site of injection, in comparison to LT-I. The results also indicate that the adjuvant activity of LTs applied transcutaneously may be related to the the ganglioside GM1 binding property since the LT-IIb toxin, unable to interact with this receptor, has no adjuvant effect by this route. The presented data set opens prospects for the employment of native LT-IIs as parenteral adjuvants in vaccines for animals and humans.

Les systèmes toxine-antitoxine (TA) bactériens ont été découverts il y a une vingtaine d’année sur les plasmides à bas nombre de copie. Ils sont composés de deux gènes organisés en opéron, l’un codant pour une toxine stable et l’autre pour une antitoxine instable capable de neutraliser l’effet de la toxine. Les systèmes TA sont fortement représentés au sein de l’ensemble des génomes bactériens. Ils se localisent aussi bien sur des éléments génétiques mobiles (plasmides, phages, transposons,…) que dans les chromosomes, ce qui suggère que le transfert horizontal de gènes participe à leur dissémination. Le système TA ccd du plasmide F d’Escherichia coli (ccdF) est composé de l’antitoxine CcdA et de la toxine CcdB. Le système ccdF contribue à la stabilité du plasmide F en tuant les bactéries-filles n’ayant pas reçu de copies plasmidiques lors de la division bactérienne (tuerie post-ségrégationelle).<p>Au cours de ce travail, nous avons caractérisé un homologue du système toxine-antitoxine ccd du plasmide F (ccdF) qui se situe dans le chromosome de la souche pathogène E. coli O157:H7 EDL933 entre les gènes folA et apaH (ccdO157). Les systèmes ccdF et ccdO157 coexistent naturellement dans les souches d’E. coli O157:H7, le système ccdF se trouvant sur le plasmide pO157 qui dérive du plasmide F. Nos résultats montrent que l’antitoxine plasmidique CcdAF neutralise l’effet de la toxine chromosomique CcdBO157, tandis que l’antitoxine chromosomique CcdAO157 ne contrecarre pas la toxicité de la toxine plasmidique CcdBF. Nous avons également montré que le système ccdF cause une tuerie post-ségrégationelle, lorsqu’il est cloné dans un plasmide instable, dans une souche possédant le système chromosomique ccdO157. Le système ccdF est donc fonctionnel en présence de son homologue chromosomique. <p>Le système ccdO157 est absent du chromosome de la souche de laboratoire E. coli K-12 MG1655, où une région intergénique de 77 pb sépare les gènes folA et apaH. Celle-ci contient une séquence cible pour la transposition. Nous avons étudié la distribution du système ccdO157 au sein de 523 souches d’E. coli représentatives de l’ensemble des sérogroupes décrits. Nos résultats montrent que le système ccdO157 est présent au sein de souches appartenant à 47 sérogroupes différents. Nos résultats mettent en évidence la diversité de la région intergénique folA-apaH d’E. coli. Celle-ci peut contenir gènes codant pour des protéines présentant de l’homologie avec des protéines d’espèce bactériennes éloignées d’E. coli ou d’organismes eucaryotes, ainsi qu’un élément génétique mobile, l’IS621, ce qui montre que le système ccdO157 a intégré le chromosome d’E. coli via le transfert horizontal de gènes.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

CsrA is a global regulator and the main player of the carbon storage regulator (Csr) network, a well- conserved regulatory network in the bacterial world. CsrA is involved in regulation of many physiological processes, including pathways of central carbon metabolism, biofilm formation, motility and virulence in pathogenic species. CsrA acts at the post-transcriptional level by binding specific sequences on its target mRNAs, leading to mRNA destabilization or stabilization. The majority of studies were analyzing a csrA mutant of E. coli K-12 encoding a truncated form of the CsrA protein, retaining residual activity.<p>This work aims at further characterize the roles of CsrA by deleting the entire csrA gene in a recently isolated strain, the uropathogenic E. coli CFT073 strain. Deletion of csrA leads to a marked growth defect, indicating that this gene, although not essential, is primordial for growth. We performed experimental evolution of csrA deletion mutants. Compensatory mutants totally outcompete the original csrA deletion mutant after six days of culture, indicating that the applied selective pressures are strong. The ÄcsrA and three ÄcsrA evolved mutants were extensively analyzed by combining molecular techniques such as genetics, microscopy and use of fluorescent reporters, and global approaches, including comparative proteomics and whole genome sequencing.<p>Our data indicate that csrA deletion strongly affects central metabolism and energy status, constituting an endogenous metabolic stress that, in turn, induces specific stress responses. This work illustrates the interconnection of multiple regulation networks for responding to specific conditions and demonstrates the flexibility of metabolic network to compensate for genetic perturbations in E. coli.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Introdução: O tratamento da Rinossinusite Crônica (RSC) tem sofrido poucos avanços nas últimas décadas. Uma das barreiras na aquisição de novas terapias é a falta de conhecimento pleno sobre sua fisiopatogenia. A carência de avanço decorre principalmente da complexa e provável multifatorialidade da RSC, associada à inexistência de um bom modelo animal que possa mimetizar os fenômenos biológicos que ocorrem em humanos. A maioria dos modelos animais de RSC descrita na literatura mimetiza uma infecção aguda ou promove bloqueio das vias de drenagem que, na maioria das vezes, não corresponde aos mecanismos encontrados nas RSC em humanos. Por outro lado, diversas evidências indicam que as bactérias exercem importante papel na fisiopatogenia da RSC, possivelmente pela presença de biofilmes ou indução de inflamação crônica promovida por endo e exotoxinas. Objetivo: Neste estudo avaliou-se a viabilidade de um modelo experimental de RSC em coelhos, utilizando-se a exposição crônica de toxinas bacterianas em animais previamente sensibilizados à ovalbumina (OVA), analisando seus efeitos histopatológicos sobre a mucosa nasossinusal. Material e Métodos: Após indução de sensibilização com injeção subcutânea de OVA 2,5% e 0,4% de hidróxido de alumínio por duas semanas, os coelhos foram submetidos à implantação de cateter de longa duração em seio maxilar direito. Após, foram submetidos à irrigação nasossinusal com OVA 2,5% três vezes por semana, por duas semanas, e em seguida, irrigação de soluções contendo diferentes toxinas bacterianas (enterotoxina estaflocócica B (SEB) 1 ?g/mL, lipopolissacáride (LPS) 100 ng/mL e ácido lipotecóico (LTA) 100 ng/mL) por quatro semanas. Os animais foram sacrificados 24 horas após a última irrigação e a mucosa do seio maxilar direito (teste) e esquerdo (controle interno) foi coletada para avaliação histopatológica. Resultados: A exposição nasossinusal ao SEB causou espessamento epitelial, infiltração celular, eosinofilia e neutrofilia tecidual, além de redução do epitélio ciliado. A exposição ao LPS causou espessamento epitelial e subepitelial, infiltração celular, eosinofilia epitelial e subepitelial e aumento da fibrose subepitelial. O LTA causou espessamento epitelial e subepitelial, infiltração celular e eosinofílica subepitelial e aumento da fibrose subepitelial. Conclusão: A exposição crônica de toxinas bacterianas na mucosa nasossinusal promoveu alterações histológicas, como espessamento da mucosa e infiltração celular, semelhantes às encontradas em pacientes com RSC. O presente estudo demonstrou que este é um modelo animal viável de RSC. Mais estudos serão necessários para elucidar se os mecanismos patogênicos deste modelo são semelhantes aos observados em humanos. / Background: The treatment of chronic rhinosinusitis (CRS) has had little evolvement in the last decades. One of the barriers to the development of new therapies is the lack of knowledge about CRS pathophysiology. The complexity and multifactoriality of this disease, together with the inexistence of a proper animal model of CRS, are probably the causes for the few advances in CRS therapy. Most of the animal models of CRS resemble acute infection or promote sinonasal obstructions, which are not a very common etiologies in CRS patients. However, there has been a lot of evidence that bacteria play an important role in the pathophysiology of CRS, probably due to the presence of biofilms, or the chronic inflammation induced by endo e exotoxins. Objective: This study aims to evaluate the viability of an experimental model of CRS in rabbits through the use of bacterial toxins in previously sensitized animals with ovalbumin, analyzing its histopathological effects onto the sinonasal mucosa. Materials and Methods: After inducing ovalbumin (OVA) sensitization by intradermic injection of OVA 2,5% and 0,4% aluminum hydroxide for 2 weeks, rabbits underwent maxillary sinus instillation of OVA 2,5% three times a week for 2 weeks followed by sinus lavage with either one bacterial toxin (Staphylococcus aureus enterotoxin B (SEB) 1 ?g/mL, lipopolysaccharide (LPS) 100 ng/mL, lipoteichoic acid (LTA), 100 ng/mL) for 4 weeks. Rabbits were euthanised 24 hours after the last sinus lavage and the mucosa of right maxillary sinus (tested side) and left side (control) were collected for histopathological evaluation. Results: The sinonasal exposure to SEB resulted in epithelial thickening, inflammatory cells infiltration (tissue eosinophilia and neutrophilia) and reduction of ciliated cells. The exposure to LPS resulted in epithelial and subepithelial thickening, inflammatory cells infiltration, epithelial and subepithelial eosinophilia and increased subepithelial fibrosis. The exposure to LTA resulted in epithelial and subepithelial thickening, subepithelial inflammatory cells infiltration and eosinophilia and increased subepithelial fibrosis. Conclusion: This study reported the effects of bacterial toxins on the the sinonasal mucosa of ovalbumin-sensitized rabbits, demonstrating similar changes that are observed in CRS patients. Our results show that this is a viable animal model of CRS. Further studies are need to elucidate whether the pathomechanisms in this model are similar to what are observed in humans.

Indiana University-Purdue University Indianapolis (IUPUI) / The patients with Cystic fibrosis (CF), an inherent genetic disorder, suffer from chronic bacterial infection in the lung. In CF, modification of epithelial cells leads to alteration of the lung environment, such as inhibition of ciliary bacterial clearance and accumulation of thickened mucus in the airways. Exploiting these conditions, opportunistic pathogens like Pseudomonas aeruginosa cause lifelong persistent infection in the CF lung by forming into antibiotic-resistant aggregated communities called biofilms. Airway infections as well as inflammation are the two major presentations of CF lung disease. P. aeruginosa strains isolated from CF lungs often contain mutations in the mucA gene, and this mutation results in higher level expression of bacterial polysaccharides and toxic lipoproteins. In a previous work, we have found a putative lipoprotein gene (PA4326) which is overexpressed in antibiotic-induced biofilm formed on cultured CF-derived airway cells. In the current work, we speculated that this particular putative lipoprotein affects cellular cytotoxicity and immune-stimulation in the epithelial cells. We found that mutation of this gene (ΔPA4326) results in reduced airway cell killing without affecting other common virulence factors.Moreover, we observed that this gene was able to stimulate secretion of the proinflammatory cytokine IL-8 from host cells. Interestingly, we also found that ΔPA4326 mutant strains produced less pyocyanin exotoxin compared to the wild type. Furthermore, our results suggest that PA4326 regulates expression of the pyocyanin biosynthesis gene phzM, leading to the reduced pyocyanin phenotype. Overall, these findings implicate PA4326 as a virulence factor in Pseudomonas aeruginosa. In the future, understating the molecular interplay between the epithelial cells and putative lipoproteins like PA4326 may lead to development of novel anti-inflammatory therapies that would lessen the suffering of CF patients.

Bakterielle Toxine stellen eine wirkungsvolle und effektive Alternative zur Therapie von Tumorerkrankungen dar. Das vom Clostridium perfringens Typ A produzierte Clostridium perfringens enterotoxin (CPE) gehört zu der Gruppe der porenbildenden Toxine und weist eine rezeptorspezifische zytotoxische Wirkung auf, welche über die Membranrezeptoren Cldn3 und Cldn4 entfaltet wird. Diese liegen vor allem in Epithelialkarzinomen wie dem Brust-, Prostata-, oder Kolon-, sowie dem Pankreaskarzinom (PK) stark hochreguliert vor. Ziel dieser Arbeit war die Anwendung des neuen selektiven und effizienten „Onkoleaking“ Suizid-Gentherapie Konzepts für die Behandlung von Cldn3 / 4 überexprimierender PK unter Verwendung eines nicht-viralen translations-optimierten CPE exprimierenden Vektors (optCPE). Weiterhin sollte in dieser Arbeit der genaue molekulare Mechanismus der CPE-vermittelten Zytotoxizität in vitro und auch in vivo analysiert werden. Für die in vitro Analysen wurden verschiedene humane PK Zelllinien, Patienten abgeleitete Xenotransplantate (PDX) und deren abgeleiteten Zellen bezüglich ihrer Cldn3 / 4 Expression und Sensitivität sowohl gegenüber rekombinantem CPE (rekCPE) als auch nach optCPE Gentransfer untersucht. Es konnte eine positive Korrelation zwischen der Effizienz CPE vermittelter Zytotoxizität und der Höhe der Cldn3 / 4 Überexpression gezeigt werden. Des Weiteren wurde die Verfügbarkeit und Zugänglichkeit der CPE Rezeptoren für die Toxinbindung als kritischer Faktor für die durch Porenbildung induzierte Zytotoxizität beschrieben. Auch eine detaillierte Analyse verschiedener apoptotischer und nekrotischer Signalwege und deren Schlüsselmoleküle waren vom besonderen Interesse. Von noch größerer Wichtigkeit war jedoch die Anwendbarkeit und der Nachweis der antitumoralen Wirksamkeit der optCPE-basierten Suizid-Gentherapie mit Hilfe des intratumoralen Jet-Injektion Gentransfers in verschiedenen Luziferase-exprimierenden CDX und PDX Modellen des PK. Alle in vivo Studien zeigten eine selektive optCPE vermittelte Verminderung der Tumorvitalität in Verbindung mit Nekrose, die in fast allen Fällen mit einer Reduktion des Tumorvolumens einher ging. Die tierexperimentellen Studien belegen damit die Effektivität der CPE-basierten Gentherapie im Pankreaskarzinom. Mit diesen neu gewonnenen Erkenntnissen zum „Onkoleaking“ Konzept der CPE Suizid-Gentherapie und deren Wirkungsmechanismen sind Kombinationen mit konventionellen Therapien möglich. / Bacterial toxins have evolved to an effective therapeutic option for cancer therapy and numerous studies demonstrated their antitumoral potential. The Clostridium perfringens enterotoxin (CPE), produced by the anaerobic Clostridium perfringes bacteria, is a pore-forming (oncoleaking) toxin, which binds to its receptors claudin-3 and -4 (Cldn3 / 4) and exerts a selective, receptor-dependent cytotoxicity. The transmembrane tight junction proteins Cldn3 and Cldn4 are known CPE receptors and are highly upregulated in several human epithelial cancers such as breast, colon, ovarian and pancreatic cancer. This study aimed at the evaluation of the potential of oncoleaking gene therapy using a non-viral translation optimized CPE vector (optCPE) as a new suicide approach for the treatment of Cldn3  /  4 overexpressing pancreatic cancer (PC) in vitro and in vivo. We demonstrated the successful in vitro use of optCPE gene transfer in a panel of human PC cells and more importantly patient derived PC xenograft (PDX) derived cells. We showed significant reduction of cell viability in all Cldn3 / 4 overexpressing PC cells after optCPE transfection. Furthermore a positive correlation between CPE cytotoxicity and level of claudin expression was shown. We revealed accessibility of CPE receptors for toxin binding as determining for optCPE mediated cytotoxicity. Since investigation of optCPE induced cell death mechanism was of particular interest, detailed analyses of apoptotic and necrotic key players were performed. By this, caspase dependent- and independent apoptosis and necrosis activation after gene transfer was demonstrated, which was dependent on amount of expressed optCPE and accessibility of Cldn. More importantly, this study demonstrated the applicability and antitumoral efficacy of optCPE gene therapy by the non-viral intratumoral jet-injection gene transfer in vivo in different luciferase-expressing CDX and PDX pancreatic cancer models. The animal experiments demonstrated the selective CPE mediated tumor growth inhibition, associated with reduced tumor viability and effective induction of tumor necrosis. This further corroborated the advantages of this novel oncoleaking strategy. With this gain of knowledge about our new oncoleaking concept of suicidal gene therapy and its mechanism of action, novel combinations with conventional therapies are possible to further improve therapeutic efficacy and to overcome resistance in pancreas carcinoma.

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).