A artroplastia é, em última análise, a opção final para o tratamento da osteoartrose. Com o aumento do número de indicações deste procedimento, a troca (artroplastia total de quadril de revisão – ATQR) dos componentes também passou a ser mais frequente. Os defeitos ósseos ou falhas ósseas são problemas que podem ser encontrados quando se realiza uma ATQR e, eles devem ser reparados, ou seja, o quadril do paciente a receber outra prótese necessita ser reconstruído. Para isto a grande maioria das técnicas empregadas requer o uso de enxerto ósseo e, devido a este motivo este tecido tem se tornado um dos tecidos mais transplantados na atualidade. Porém a demanda para utilização dos enxertos, na maioria provenientes de bancos de tecidos ósseos, tem aumentado, mas o suprimento é insuficiente. Portanto faz-se necessário a busca por novas tecnologias e alternativas aos bancos de tecidos. O enxerto bovino liofilizado (EBL) é uma destas opções, sua produção em livre demanda suas características físicas e químicas semelhantes ao osso humano e sua boa repercussão clínico-radiológica o torna uma alternativa viável. Este estudo tem o objetivo de verificar e quantificar a neoformação óssea com o uso do EBL pelo uso da avaliação histológica. Realizou-se um estudo de casos de Julho de 2000 a Abril de 2013 no Hospital de Clínicas de Porto Alegre (HCPA), onde se incluíram sujeitos que foram submetidos a uma cirurgia prévia cirurgia de ATQR onde foi utilizado o EBL os quais internaram posteriormente para uma segunda cirurgia de ATQR não relacionada a falha do enxerto e sim a falha da prótese. Nesta segunda cirurgia realizou-se a biópsia óssea. Quatorze sujeitos foram analisados, sendo 64,3% do sexo feminino. A média de idade dos pacientes foi 52,36±18,55. Neoformação óssea estava presente em 85,7% dos sujeitos, e constituiu 61,79% da área total de matriz óssea. O diagnóstico de absorção do EBL estava presente em 12 sujeitos. Uma forte correlação de proporção inversa foi constatada pelo teste de Pearson entre a porcentagem de osso neoformado e a porcentagem de EBL na área total de matriz óssea (p=0,001). Nenhuma resposta inflamatória foi encontrada. Concluiu-se que houve neoformação óssea adequada na grande maioria dos casos sendo o EBL uma boa estrutura osteocondutora, podendo ser considerado uma alternativa aos outros enxertos ósseos no tratamento das falhas ósseas. / Arthroplasty is, ultimately, the final option for the treatment of osteoarthritis. With the increasing number of indications of this procedure, the exchange (total hip arthroplasty revision surgery- THARS) also became more frequent. The bone defects are problems that can be encountered when conducting a THARS and this defect must be repaired, in other words, the patient's hip needs to be rebuilt before receive a new prosthesis. Most of techniques used to rebuild requires the use of bone graft, and because of this reason, this tissue has become one of the most transplanted tissues today. However the demand for the use of grafts, mostly from bone tissue banks, has increased, but the supply is insufficient. Therefore it is necessary to search for new technologies and alternatives to tissue banks. The bovine lyophilized xenograft (BLX) is one of these options; the production on free demand, its physical and chemical characteristics similar to human bone and its good clinical and radiological outcomes makes it a viable alternative. The aim of this study was to verify and quantify new bone formation by the histological analysis in subjects who received the BLX. This was a case series from July 2000 to April 2013 realized in the Hospital de Clínicas de Porto Alegre. This study included patients who underwent to a THARS where was used the BLX, which later was admitted to a second THARS surgery, not related to the xenograft failure but a mechanical failure of the implant. In this second surgery was performed the bone biopsy. Fourteen subjects were analyzed, 64.3% were female. The average age of patients was 52.36 ± 18.55 years. New bone formation was present in 85.7% of subjects, and constituted 61.79% of the total bone matrix. The diagnosis of BLX absorption was present in 12 subjects. A strong inverse correlation founded by the Pearson's test was observed between the proportion of new bone and the proportion of BLX (p=0.001). No inflammatory response was found. Was concluded that there was suitable bone formation in the vast majority of the cases, as well as the BLX is a good osteoconductive scaffold and can be considered an alternative to other bone graft in the treatment of bone defects.

A artroplastia é, em última análise, a opção final para o tratamento da osteoartrose. Com o aumento do número de indicações deste procedimento, a troca (artroplastia total de quadril de revisão – ATQR) dos componentes também passou a ser mais frequente. Os defeitos ósseos ou falhas ósseas são problemas que podem ser encontrados quando se realiza uma ATQR e, eles devem ser reparados, ou seja, o quadril do paciente a receber outra prótese necessita ser reconstruído. Para isto a grande maioria das técnicas empregadas requer o uso de enxerto ósseo e, devido a este motivo este tecido tem se tornado um dos tecidos mais transplantados na atualidade. Porém a demanda para utilização dos enxertos, na maioria provenientes de bancos de tecidos ósseos, tem aumentado, mas o suprimento é insuficiente. Portanto faz-se necessário a busca por novas tecnologias e alternativas aos bancos de tecidos. O enxerto bovino liofilizado (EBL) é uma destas opções, sua produção em livre demanda suas características físicas e químicas semelhantes ao osso humano e sua boa repercussão clínico-radiológica o torna uma alternativa viável. Este estudo tem o objetivo de verificar e quantificar a neoformação óssea com o uso do EBL pelo uso da avaliação histológica. Realizou-se um estudo de casos de Julho de 2000 a Abril de 2013 no Hospital de Clínicas de Porto Alegre (HCPA), onde se incluíram sujeitos que foram submetidos a uma cirurgia prévia cirurgia de ATQR onde foi utilizado o EBL os quais internaram posteriormente para uma segunda cirurgia de ATQR não relacionada a falha do enxerto e sim a falha da prótese. Nesta segunda cirurgia realizou-se a biópsia óssea. Quatorze sujeitos foram analisados, sendo 64,3% do sexo feminino. A média de idade dos pacientes foi 52,36±18,55. Neoformação óssea estava presente em 85,7% dos sujeitos, e constituiu 61,79% da área total de matriz óssea. O diagnóstico de absorção do EBL estava presente em 12 sujeitos. Uma forte correlação de proporção inversa foi constatada pelo teste de Pearson entre a porcentagem de osso neoformado e a porcentagem de EBL na área total de matriz óssea (p=0,001). Nenhuma resposta inflamatória foi encontrada. Concluiu-se que houve neoformação óssea adequada na grande maioria dos casos sendo o EBL uma boa estrutura osteocondutora, podendo ser considerado uma alternativa aos outros enxertos ósseos no tratamento das falhas ósseas. / Arthroplasty is, ultimately, the final option for the treatment of osteoarthritis. With the increasing number of indications of this procedure, the exchange (total hip arthroplasty revision surgery- THARS) also became more frequent. The bone defects are problems that can be encountered when conducting a THARS and this defect must be repaired, in other words, the patient's hip needs to be rebuilt before receive a new prosthesis. Most of techniques used to rebuild requires the use of bone graft, and because of this reason, this tissue has become one of the most transplanted tissues today. However the demand for the use of grafts, mostly from bone tissue banks, has increased, but the supply is insufficient. Therefore it is necessary to search for new technologies and alternatives to tissue banks. The bovine lyophilized xenograft (BLX) is one of these options; the production on free demand, its physical and chemical characteristics similar to human bone and its good clinical and radiological outcomes makes it a viable alternative. The aim of this study was to verify and quantify new bone formation by the histological analysis in subjects who received the BLX. This was a case series from July 2000 to April 2013 realized in the Hospital de Clínicas de Porto Alegre. This study included patients who underwent to a THARS where was used the BLX, which later was admitted to a second THARS surgery, not related to the xenograft failure but a mechanical failure of the implant. In this second surgery was performed the bone biopsy. Fourteen subjects were analyzed, 64.3% were female. The average age of patients was 52.36 ± 18.55 years. New bone formation was present in 85.7% of subjects, and constituted 61.79% of the total bone matrix. The diagnosis of BLX absorption was present in 12 subjects. A strong inverse correlation founded by the Pearson's test was observed between the proportion of new bone and the proportion of BLX (p=0.001). No inflammatory response was found. Was concluded that there was suitable bone formation in the vast majority of the cases, as well as the BLX is a good osteoconductive scaffold and can be considered an alternative to other bone graft in the treatment of bone defects.

La démonstration du rôle de la CDT (« Cytolethal Distending Toxin ») de Helicobacterhepaticus dans le développement de l’hépatocarcinome murin fait de cette toxine un candidatpertinent dans l'activation de processus pro-cancéreux. Comme la toxine CagA de Helicobacterpylori, la sous-unité active CdtB de la CDT pourrait être une oncoprotéine. Nous avons étudié lerôle de la CdtB des Hélicobacters dans l’inflammation et la carcinogenèse digestive via unestratégie lentivirale d’expression constitutive ou conditionnelle de la CdtB ou de son mutant pourl’activité DNase. Nous avons réalisé une étude du transcriptome et montré que la CdtB deH. hepaticus induisait une réponse inflammatoire en surexprimant des cytokines, chimiokines,peptides antimicrobiens et en activant la voie du NF-κB des cellules épithéliales. La CdtB réguleégalement l’expression et la localisation nucléaire du facteur de transcription et oncogène MafB.Ces résultats ont été confirmés pour la CdtB de Helicobacter pullorum. Des expériencesd'infection des cellules avec des souches sauvages et mutées pour la CDT (deH. hepaticus & H. pullorum) ont permis de valider les résultats obtenus et de les attribuer à laCdtB et notamment à son activité DNase. Nous avons aussi développé un nouveau modèle dexénogreffes de cellules épithéliales inductibles pour l’expression de la CdtB de H. hepaticus.Dans ce modèle, la CdtB, en plus de ses effets déjà connus, retarde la croissance tumorale,induit l’apoptose, la sénescence et la surexpression du marqueur nucléaire de prolifération,Ki-67, suggérant la survie cellulaire. L’ensemble de ces résultats fournit de nouveaux argumentsen faveur du potentiel oncogénique de la CDT. / The demonstration of the role of the Cytolethal Distending Toxin (CDT) of Helicobacter hepaticusin the development of hepatocarcinoma in mice, makes this toxin a relevant candidate in theactivation of precancerous processes. As in the case of the CagA toxin of Helicobacter pylori, theCdtB active subunit of CDT could be an oncoprotein. We studied the role of Helicobacter CdtB ininflammation and digestive carcinogenesis using a lentiviral strategy for constitutive or conditionalexpression of the CdtB subunit or its corresponding DNase mutant. We conducted a study of thetranscriptome and showed that CdtB induced an inflammatory response by overexpressingcytokines, chemokines, antimicrobial peptides and activating the NF-kB pathway in epithelialcells. The CdtB also regulated the expression and nuclear localization of the transcription factorand oncogene MafB. These results were confirmed for the CdtB of Helicobacter pullorum.Infection of cells with wild type strains and the corresponding CDT-mutant strains (of H. hepaticus& H. pullorum) were used to validate the results and to attribute the effects to the CdtB and, inparticular, to its DNase activity. We also developed a novel epithelial cell xenograft model toevaluate the inducible expression of H. hepaticus CdtB. In this model, the CdtB, in addition to itspreviously well-known effects, delayed tumor growth, induced apoptosis, senescence and theoverexpression of nuclear proliferation marker, Ki-67, suggesting cell survival. All of these resultsprovide new arguments in favor of the oncogenic potential of the CDT.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23062号 / 医博第4689号 / 新制||医||1048(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 妹尾 浩, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

OBJECTIVES: Models for the development of new treatment options in vestibular schwannoma (VS) treatment are lacking. The purpose of this study is to establish a quantifiable human VS xenograft model in mice. STUDY DESIGN AND METHODS: Both rat malignant schwannoma cells (KE-F11 and RT4) and human malignant schwannoma (HMS-97) cells were implanted near the sciatic nerve in the thigh of severe combined immunodeficiency (SCID) mice. Additionally, human benign VS specimens were implanted in another set of SCID mice. Three-dimensional tumor volumes were calculated from magnetic resonance images over the next 6 months. RESULTS: Mice implanted with malignant schwannoma cells developed visible tumors within 2 weeks. Imaging using a 4.7-tesla magnetic resonance imaging and immunohistopathologic examination identified solid tumors in all KE-F11 and HMS-97 xenografts, whereas RT4 xenografts consistently developed cystic schwannomas. VS xenografts demonstrated variability in their growth rates similar to human VS. The majority of VS xenografts did not grow but persisted throughout the study, whereas two of 15 xenografts grew significantly. Histopathologic examination and immunohistochemistry confirmed that VS xenografts retained their original microscopic and immunohistochemical characteristics after prolonged implantation. CONCLUSIONS: This study describes the first animal model for cystic schwannomas. Also, we demonstrate the use of high-field magnetic resonance imaging to quantify VS xenograft growth over time. The VS xenografts represent a model complimentary to Nf2 transgenic and knockout mice for translational VS research.

Final publication is available at http://www.bloodjournal.org/ / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19614号 / 医博第4121号 / 新制||医||1015(附属図書館) / 32650 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 山田 亮, 教授 三森 経世 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

A major hurdle for exploring RNA interference (RNAi) in a therapeutic setting is still the issue of in vivo delivery of small RNA molecules (siRNAs). The chemical modification of polyethylenimines (PEIs) offers a particularly attractive avenue towards the development of more efficient non-viral delivery systems. Here, we explore tyrosine-modified polyethylenimines with low or very low molecular weight (P2Y, P5Y, P10Y) for siRNA delivery. In comparison to their respective parent PEI, they reveal considerably increased knockdown efficacies and very low cytotoxicity upon tyrosine modification, as determined in different reporter and wildtype cell lines. The delivery of siRNAs targeting the anti-apoptotic oncogene survivin or the serine/threonine-protein kinase PLK1 (polo-like kinase 1; PLK-1) oncogene reveals strong inhibitory effects in vitro. In a therapeutic in vivo setting, profound anti-tumor effects in a prostate carcinoma xenograft mouse model are observed upon systemic application of complexes for survivin or PLK1 knockdown, in the absence of in vivo toxicity. We thus demonstrate the tyrosine-modification of (very) low molecular weight PEIs for generating effcient nanocarriers for siRNA delivery in vitro and in vivo, present data on their physicochemical and biological properties, and show their efficacy as siRNA therapeutic in vivo, in the absence of adverse effects.

No description available.

No description available.

No / AQ4N (banoxantrone) is a prodrug that, under hypoxic conditions, is enzymatically converted to a cytotoxic DNA-binding agent, AQ4. Incorporation of AQ4N into conventional chemoradiation protocols therefore targets both oxygenated and hypoxic regions of tumors, and potentially will increase the effectiveness of therapy. This current pharmacodynamic and efficacy study was designed to quantify tumor exposure to AQ4 following treatment with AQ4N, and to relate exposure to outcome of treatment. A single dose of 60 mg/kg AQ4N enhanced the response of RT112 (bladder) and Calu-6 (lung) xenografts to treatment with cisplatin and radiation therapy. AQ4N was also given to separate cohorts of tumor-bearing mice 24 hours before tumor excision for subsequent analysis of metabolite levels. AQ4 was detected by high performance liquid chromatography/mass spectrometry in all treated samples of RT112 and Calu-6 tumors at mean concentrations of 0.23 and 1.07 microg/g, respectively. These concentrations are comparable with those shown to be cytotoxic in vitro. AQ4-related nuclear fluorescence was observed in all treated tumors by confocal microscopy, which correlated with the high performance liquid chromatography/mass spectrometry data. The presence of the hypoxic marker Glut-1 was shown by immunohistochemistry in both Calu-6 tumors and RT112 tumors, and colocalization of AQ4 fluorescence and Glut-1 staining strongly suggested that AQ4N was activated in these putatively hypoxic areas. This is the first demonstration that AQ4N will increase the efficacy of chemoradiotherapy in preclinical models; the intratumoral levels of AQ4 found in this study are comparable with tumor AQ4 levels found in a recent phase I clinical study, which suggests that these levels could be potentially therapeutic.