published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 274-307).

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 67-76).

Thesis (PhD--Bioengineering)--University of Auckland, 2010. / "Supervised by Dr. AP M.H. Tawhai and Dr E.J. Crampin." " A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Bioengineering." "Auckland Bioengineering Institute." Includes bibliographical references (p. 204-255).

Pulmonary tuberculosis (TB) remains one of the most infectious causes of death worldwide. Mycobacterium tuberculosis (M.tb), the causative agent of TB is transmitted via infectious aerosols, and in the majority of cases the bacteria is effectively controlled, by the host, resulting in a chronic latent infection. Currently, the only available vaccine is the Bacillus Calmette-Guérin (BCG), which despite being successful in preventing childhood disseminated forms of TB, has failed to control the adult pulmonary TB epidemic. One of the major contributing factors in the failure of the BCG is that although antigen-specific T cells are present at the time of M.tb infection, the recruitment of such T cells into the site of infection is significantly delayed. This delay, while reduced compared to non-vaccinated hosts, allows the bacteria to replicate unchecked within the lung and establish a “foothold” prior to the arrival of protective T cells and subsequent immune control. Thus, novel initiatives seek to close this “immunological gap” through increasing the level of protective T cell responses within the airway mucosa immediately following M.tb infection. We therefore investigated the impact of deliberate modulation of T cell geography following BCG vaccination on the outcome of pulmonary M.tb infection. In addition, a number of environmental factors are also thought to affect the site of M.tb infection: the respiratory mucosa. However, little is currently known about the effects of environmental exposure to allergens and other substances such as cigarette smoke on the outcome of pulmonary TB. Throughout this thesis we have investigated the mechanisms of immune protection and failure of protection against pulmonary M.tb infection within the respiratory mucosa. / Thesis / Doctor of Philosophy (Medical Science)

Streptococcus pneumoniae (pneumococo) é um patógeno causador de infecções respiratórias que causa a morte de 1 milhão de crianças anualmente no mundo todo. Neste trabalho a propriedade adjuvante da vacina celular pertussis (wP) em combinação com o antígeno PspA5 de S. pneumoniae foi avaliada em modelos animais. Combinações de PspA5 com wP, wPlow (vacina celular pertussis com baixa quantidade de LPS) ou com a vacina DTPlow foram inoculadas em camundongos por via nasal ou subcutânea. A resposta protetora induzida pela imunização nasal com PspA5-wP e PspA5-wPlow ao desafio letal com pneumococo foi caracterizada pelo aumento de anticorpos anti-PspA5 sistêmicos e na mucosa respiratória e infiltração de linfócitos T CD4+, T CD8+ e B no pulmão dos animais. A inoculação subcutânea de PspA5-DTPlow também protegeu contra desafio letal com pneumococo e a resposta induzida foi caracterizada pelo aumento de IgG anti-PspA5 sistêmicos e na mucosa respiratória. Não houve prejuízo às respostas contra difteria, tétano ou pertussis quando PspA5 foi combinado à DTPlow. / Streptococcus pneumoniae (pneumococcus) is a pathogen that causes respiratory infections leading to the death of 1 million children per year, worldwide. Here, the adjuvant property of the whole cell pertussis vaccine (wP) in combination with the PspA5 antigen from S. pneumoniae was evaluated in animal models. Combinations of PspA5 with wP, wPlow (whole cell pertussis vaccine with low amount of LPS) or with the DTPlow vaccine were inoculated in mice by nasal or subcutaneous routes. The protective response induced by nasal immunization with PspA5-wP and PspA5-wPlow against the lethal challenge with pneumococci was characterized by increased systemic and mucosal anti-PspA5 antibodies and influx of TCD4+, TCD8+ and B cells in the lungs of mice. The subcutaneous inoculation of PspA5-DTPlow also protected against the lethal challenge with pneumococci and the response was characterized by increased levels of systemic and mucosal anti-PspA5 IgG. There was no prejudice to the responses against diphtheria, tetanus or pertussis when PspA5 was combined with DTPlow.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 5 uppsatser.

Streptococcus pneumoniae (pneumococo) é um patógeno causador de infecções respiratórias que causa a morte de 1 milhão de crianças anualmente no mundo todo. Neste trabalho a propriedade adjuvante da vacina celular pertussis (wP) em combinação com o antígeno PspA5 de S. pneumoniae foi avaliada em modelos animais. Combinações de PspA5 com wP, wPlow (vacina celular pertussis com baixa quantidade de LPS) ou com a vacina DTPlow foram inoculadas em camundongos por via nasal ou subcutânea. A resposta protetora induzida pela imunização nasal com PspA5-wP e PspA5-wPlow ao desafio letal com pneumococo foi caracterizada pelo aumento de anticorpos anti-PspA5 sistêmicos e na mucosa respiratória e infiltração de linfócitos T CD4+, T CD8+ e B no pulmão dos animais. A inoculação subcutânea de PspA5-DTPlow também protegeu contra desafio letal com pneumococo e a resposta induzida foi caracterizada pelo aumento de IgG anti-PspA5 sistêmicos e na mucosa respiratória. Não houve prejuízo às respostas contra difteria, tétano ou pertussis quando PspA5 foi combinado à DTPlow. / Streptococcus pneumoniae (pneumococcus) is a pathogen that causes respiratory infections leading to the death of 1 million children per year, worldwide. Here, the adjuvant property of the whole cell pertussis vaccine (wP) in combination with the PspA5 antigen from S. pneumoniae was evaluated in animal models. Combinations of PspA5 with wP, wPlow (whole cell pertussis vaccine with low amount of LPS) or with the DTPlow vaccine were inoculated in mice by nasal or subcutaneous routes. The protective response induced by nasal immunization with PspA5-wP and PspA5-wPlow against the lethal challenge with pneumococci was characterized by increased systemic and mucosal anti-PspA5 antibodies and influx of TCD4+, TCD8+ and B cells in the lungs of mice. The subcutaneous inoculation of PspA5-DTPlow also protected against the lethal challenge with pneumococci and the response was characterized by increased levels of systemic and mucosal anti-PspA5 IgG. There was no prejudice to the responses against diphtheria, tetanus or pertussis when PspA5 was combined with DTPlow.

INTRODUÇÃO: O transplante de pulmão é parte fundamental no tratamento das doenças terminais do pulmão, constituindo uma modalidade terapêutica eficaz para pacientes com doença pulmonar incapacitante, progressiva e em estágio final. No entanto, as drogas imunossupressoras usadas para evitar a rejeição do enxerto podem causar efeitos colaterais em diversos tecidos. O sistema mucociliar, presente nas vias aéreas, é um dos principais mecanismos de defesa do trato respiratório e pode ser alterado por ação das drogas imunossupressoras. Desta forma, o objetivo deste estudo foi avaliar o sistema mucociliar traqueobrônquico de ratos submetidos a dois esquemas de terapia tríplice imunossupressora. MÉTODOS: Foram utilizados 90 ratos machos Wistar distribuídos em 3 grupos conforme o tratamento: controle (C) = solução salina; terapia 1 (TI) = tacrolimus + micofenolato de mofetil + prednisona; terapia 2 (TII) = ciclosporina + azatioprina + prednisona. Após o período de tratamento (7, 15 ou 30 dias), os animais foram sacrificados e realizadas as seguintes medidas: transportabilidade do muco (TM), frequência de batimento ciliar (FBC), quantificação de muco neutro e ácido, velocidade de transporte mucociliar (VTMC), e contagem total e diferencial de células no lavado broncoalveolar (LBA). RESULTADOS: A TM não foi afetada pelas terapias em nenhum dos tempos estudados. Ambas as terapias causaram significativa redução da FBC dos animais tratados por 7 e 15 dias. A produção de muco neutro foi menor nos animais tratados com a TI por 7, 15 e 30 dias. Porém, com a TII, essa redução ocorreu apenas aos 7 dias. Por outro lado, a quantidade de muco ácido foi significativamente maior em todos os animais tratados com as duas terapias. Todos os animais tratados com as terapias imunossupressoras apresentaram redução da VTMC nos três tempos. Houve aumento do número total de células e de macrófagos e neutrófilos no grupo TI em 7 dias. CONCLUSÕES: Ambas as terapias imunossupressoras foram prejudiciais ao transporte mucociliar das vias aéreas de ratos, tanto pela redução da FBC e da VTMC, quanto pela maior produção de muco ácido e menor produção de muco neutro. A TI foi mais prejudicial ao sistema mucociliar em comparação à TII / INTRODUCTION: Lung transplantation is an essential part in the treatment of terminal lung diseases, providing an effective therapeutic modality for patients with disabling, progressive and final stage lung disease. However, the immunosuppressant drugs used to prevent graft rejection may cause side effects in several tissues. The mucociliary system, present in the airways, is a major defense mechanism of the respiratory tract and can be changed by action of immunosuppressive drugs. Thus, the aim of this study was to evaluate the tracheobronchial mucociliary system of rats submitted to two triple immunosuppressive therapy regimens. METHODS: We used 90 male Wistar rats divided into 3 groups according to treatment: control (C) = saline solution; therapy 1 (TI) = tacrolimus + mycophenolate mofetil + prednisone therapy; therapy 2 (TII) = cyclosporine + azathioprine + prednisone. After the period of treatment (7, 15, or 30 days), the animals were sacrificed and the following measures taken: mucus transportability (MT), ciliary beating frequency (CBF), quantification of neutral and acid mucus, mucociliary transport velocity (MCTV), and total and differential counting of cells in bronchoalveolar lavage (BAL). RESULTS: MT was not affected by treatments in any of the periods studied. Both therapies have caused significant reduction of CBF of animals treated for 7 and 15 days. The neutral mucus production was lower in animals treated with TI for 7, 15 and 30 days. But with TII, this reduction occurred only at 7 days. Moreover, the amount of acid mucus was significantly higher in all animals treated with both therapies. All animals treated with immunosuppressive therapies had reduced MCTV at the three times. There was an increase of total cells and macrophages and neutrophils in the TI group in 7 days. CONCLUSIONS: Both immunosuppressive therapies were harmful to the mucociliary clearance of the airways of rats, either by reducing the CBF and MCTV, as by the increased production of acid mucus and decreased production of neutral mucus. TI was more harmful to the mucociliary system in comparison to TII

Neste estudo, foram realizados três experimentos distintos (1) analisando os efeitos da administração de material particulado em camundongos BALB/c com inflamação pulmonar alérgica crônica induzida por ovalbumina; (2) comparando camundongos AIRmax e AIRmin com inflamação pulmonar alérgica crônica induzida por ovalbumina; (3) comparando camundongos AIRmax e AIRmin que receberam material particulado (resíduo da queima de óleo diesel - ROFA) por via intranasal. Para a indução da inflamação pulmonar alérgica crônica, os camundongos foram sensibilizados com ovalbumina (OVA) através de duas injeções intraperitoneais de alérgeno com o adjuvante hidróxido de alumínio (dias 0 e 14) e quatro inalações de OVA 1% (dias 22, 24, 26 e 28). Os animais que foram expostos ao material particulado, receberam ROFA (60 ?g) nos dias 0, 2, 4 e 6 no experimento do efeito do material particulado ou nos dias dos desafios com OVA no experimento do efeito da administração de material particulado em animais com inflamação pulmonar induzida pela OVA. Os grupos controle foram tratados com solução salina 0,9 % seguindo o mesmo protocolo. Quarenta e oito horas após o último desafio, a responsividade pulmonar foi medida por broncoprovocação àr metacolina através da pletismografia de corpo inteiro, após a qual os animais foram sacrificados e tiveram os pulmões removidos para analise histológica. O estudo realizado com animais BALB/c para análise dos efeitos do material particulado sobre a resposta inflamatória induzida pela ovalbumina mostrou um aumento de responsividade nos animais inflamados e co-expostos ao material particulado, sem efeito sobre a inflamação ou remodelamento epitelial induzidos pela OVA. A exposição ao material particulado por si só levou a uma diminuição da área ocupada por células ciliadas e a um aumento da responsividade pulmonar. O experimento realizado com camundongos das linhagens AIRmax e AIRmin para estudar eventuais diferenças entre a resposta destas linhagens à inflamação pulmonar crônica induzida pela OVA mostrou uma maior susceptibilidade dos animais AIRmin, com maior infiltrado eosinofílico nas vias aéreas e responsividade pulmonar. Por outro lado, o estudo realizado para verificar a ação do material particulado nas linhagens AIRmax e AIRmin mostrou um intenso remodelamento epitelial, com infiltrado macrofágico ao redor das vias aéreas de ambas as linhagens, porém os animais AIRmin apresentaram maior responsividade pulmonar que os AIRmax quando expostos ao material particulado. Assim pudemos concluir que (1) a exposição ao material particulado na presença de inflamação pulmonar alérgica crônica amplifica o remodelamento epitelial e a responsividade pulmonar; o background genético influencia (2) a inflamação eosinofílica e a responsividade pulmonar induzidas pela inflamação alérgica crônica bem como (3) a responsividade pulmonar induzida pela exposição ao material particulado. / In this study, three distinct experiments were performed (1) examining the effects of administration of particulate matter in BALB/c mice with chronic allergic pulmonary inflammation induced by ovalbumin; (2) comparing AIRmax and AIRmin mice with chronic allergic pulmonary inflammation induced by ovalbumin; (3) comparing AIRmax and AIRmin mice receiving particulate matter (residue from the burning of diesel oil - ROFA) intranasally. For the induction of chronic allergic pulmonary inflammation, the mice were sensitized to ovalbumin (OVA) through two intraperitoneal injections of allergen with adjuvant aluminum hydroxide (days 0 and 14) and four inhalations of OVA 1% (days 22, 24, 26 and 28). The animals that were exposed to particulate matter received ROFA (60 ?g) on days 0, 2, 4 and 6 in the study of the effect of the particulate matter or after the challenges with OVA in experiment of the effect of administration of particulate matter in animals with pulmonary inflammation induced by OVA. The control groups were treated with saline 0.9% following the same protocol. Forty eight hours after the last challenge, pulmonary responsiveness was measured through broncoprovocation to methacholine by whole body plethysmography, after which the animals were sacrificed and the lungs were removed for histologic analysis. The study conducted on animals BALB/c for analysis of the effects of particulate matter on the inflammatory response induced by ovalbumin showed an increase of responsiveness in animals with allergic inflammation and exposed to the particulate matter, without effects on inflammation or epithelial remodeling induced by OVA. Exposure to particulate matter led to a reduction in the area occupied by ciliated cells and increased lung responsiveness. The experiment in AIRmax AIRmin mice to study possible differences in the response of these strains to chronic lung inflammation induced by OVA showed greater susceptibility of AIRmin, with more eosinophilic infiltration in airway and greater lung responsiveness. The study to check the action of particulate matter in the lines AIRmax and AIRmin showed an intense epithelial remodeling, with macrophagic infiltrate around the airways of both strains. However, AIRmin mice had higher pulmonary responsiveness than AIRmax when exposed to particulate matter. We conclude that (1) exposure to particulate matter in the presence of chronic allergic pulmonary inflammation amplifies the epithelial remodeling and pulmonary responsiveness; the genetic background influences (2) the pulmonary responsiveness and eosinophilic inflammation induced by chronic allergic inflammation and (3) the pulmonary responsiveness induced by exposure to particulate matter.