Francisella tularensis est une bactérie intracellulaire à Gram négatif et l'un des agents les plus infectieux connu à l'heure actuelle, en particulier par voie respiratoire. L'inhalation d'une dizaine de bactéries suffit à provoquer une maladie mortelle : la tularémie pulmonaire. Sa facilité de dissémination par aérosols, ainsi que le caractère létal de cette pathologie, ont contribué à considérer F. tularensis comme une arme biologique potentielle. La tularémie pulmonaire est une infection aigue qui s'accompagne d'une réponse immunitaire inadaptée. F. tularensis infecte en premier lieu les cellules phagocytaires, notamment les macrophages. Alors que ces derniers sont des acteurs majeurs de la défense contre les agents infectieux, de nombreux mécanismes d'échappement permettent à F. tularensis d'éviter ou de résister aux réponses de l'hôte et ainsi de se multiplier et de disséminer dans l'organisme. Ainsi, après un retard initial dans la mise en place de la réponse immunitaire, la présence d'un grand nombre de bactéries et de signaux de danger libérés par les cellules infectées, conduisent au déclenchement d'une réponse inflammatoire excessive. Celle-ci se caractérise par une tempête cytokinique provoquant un recrutement massif de cellules immunitaires, en particulier de neutrophiles, dans les tissus infectés. Les dommages tissulaires associés à cette réponse inflammatoire sont en grande partie responsable de la mortalité associée aux infections pulmonaires par F. tularensis. La tularémie est actuellement traitée par antibiothérapie. Malheureusement, l'absence de symptômes spécifiques de cette maladie rend le diagnostic difficile et, par conséquent, retarde la prescription du traitement adapté. Or, l'efficacité des antibiotiques est considérablement réduite par cette administration tardive. De nouvelles stratégies thérapeutiques sont donc nécessaires pour remplacer ou compléter l'antibiothérapie. Dans ce contexte, nous avons cherché à déterminer si la modulation de la réponse inflammatoire excessive induite par F. tularensis pouvait être bénéfique pour l'hôte infecté et ainsi être utilisée comme thérapie accessoire. L'objectif de mon travail de thèse était d'évaluer les propriétés anti-inflammatoires et le potentiel bénéfice thérapeutique du mannodendrimère 3T (M3T), un composé synthétisé par l'équipe de J. Nigou, dans un modèle murin d'infection pulmonaire par F. tularensis. Le M3T, conçu pour mimer les propriétés anti-inflammatoires d'un glycolipide de la paroi de Mycobacterium tuberculosis, a précédemment montré un effet inhibiteur sur la production de cytokines pro-inflammatoires et le recrutement de neutrophiles dans un modèle murin d'inflammation pulmonaire aigue induite par le LPS. La souche F. novicida, provoquant chez la souris une pathologie similaire à une infection pulmonaire par F. tularensis, a été utilisée comme souche de substitution dans ces travaux. In vitro, nous avons montré que le M3T inhibe la production de cytokines pro-inflammatoires induite par F. novicida dans des macrophages et cellules dendritiques humaines. D'un point de vue mécanistique, l'ensemble des données suggère que le M3T inhibe la réponse inflammatoire induite par F. novicida via le récepteur TLR2, en activant une voie de signalisation dépendante du récepteur DC-SIGN. In vivo, le M3T a été administré par injection intraveineuse 6 h post-infection, puis quotidiennement pendant 3 jours, en combinaison avec un traitement antibiotique sous-optimal. / Francisella tularensis is an intracellular Gram negative bacterium and the causative agent of tularemia. It is one of the most infectious agents known to date. Infection by the respiratory route leads to the deadly pulmonary form of tularemia. For these reasons, F. tularensis has been considered for years as a potential biological weapon. Pulmonary tularemia is characterized by an acute infection and a defect in immune responses. Particularly, the innate immune system plays a central role in F. tularensis infection and pathology. Macrophages, key cells of the innate immune system, are the main target for F. tularensis. This bacterium has evolved many strategies to escape host defenses that allow it to replicate within the cells and then disseminate into the whole organism. At this systemic stage, bacteria, along with alarm signals from infected cells, are recognized by innate immune receptors, triggering an inappropriate inflammatory response. The latter is characterized by a cytokine storm leading to a massive recruitment of immune cells, particularly neutrophils, in infected tissue. Tissue damages caused by this inflammation are a major cause of mortality associated with F. tularensis infections. Today, the treatment of tularemia is based on antibiotherapy. However, no specific symptoms can be assigned to pulmonary tularemia making its diagnosis difficult. This delays the administration of an appropriate antibiotiotherapy whose efficacy is therefore decreased. Thus, new therapeutic strategies are needed to replace or complement antibiotics. In this context, we investigated whether reducing the excessive inflammation induced by F. tularensis could be beneficial for the host and be considered as an adjunctive host- directed therapy. The aim of my work was to evaluate the anti-inflammatory properties and the therapeutic potential of mannodendrimer 3T (M3T), a synthetic coumpond designed in the team, in a mouse model of pulmonary infection by F. tularensis. M3T was previously designed to mimic the anti-inflammatory traits of a specific glycolipid from Mycobacterium tuberculosis. It was previously shown to inhibit the production of pro-inflammatory cytokines and neutrophils recruitment in a mouse model of LPS-induced pulmonary inflammation. Here, we used F. novicida as a surrogate for F. tularensis since it induces an identical inflammatory pathology. In vitro, M3T was found to inhibit the production of pro-inflammatory cytokines in human macrophages and dendritic cells infected by F. novicida. M3T modulates inflammatory response triggered by F. novicida via TLR2 most likely by the activation of a DC-SIGN-dependant pathway. In vivo, M3T was administered 6 h post-infection and then, daily for 3 days, by intraveinous injection and combined with a suboptimal antibiotic. This combination increases the survival rate of mice infected with F. novicida as compared to mice treated with antibiotic alone. M3T treatment has no impact on bacterial burden but seems to reduce tissue damages as observed by histological analyses of lungs, liver and spleen of infected mice. Altogether, our data demonstrate that M3T administration provides a therapeutic benefit in a mouse model of pulmonary infection by F. novicida. On a more general perspective our results suggest that targeting inflammation can be considered as an adjunctive treatment in acute pulmonary infections.

Pulmonary infection caused by nontypeable Haemophilus influenzae (NTHi) is a significant cause of morbidity and mortality in both industrialised and developing countries. Previous work from this group resulted in the development of a respiratory model in rodents which has precipitated studies into the pathogenesis of infection by NTHi and investigation of the humoral and cellular mechanisms by which the bacteria are cleared from the lung. Comparison of mucosally immunised with non-immunised animals has demonstrated that not only are bacteria cleared more rapidly from the lungs, but there is a more rapid response and resolution of inflammatory factors in the mucosally immunised animals following challenge with NTHi. This inflammatory response is partially regulated by the ability of the mucosally immunised animals to rapidly produce, then control the production of tumour necrosis factor (TNF)-a. The TNF-a is produced by both macrophages and type I pneumocytes in the alveoli and also by the endothelial cells lining the blood vessels in the lungs. Immunocytochemical studies have identified cellular subsets accumulating in the lung at various time points following infection. Marked differences in cellular infiltration into the lung tissue were noted between immunised and non-immunised animals after challenge with NTHi. Immunised animals demonstrated an early influx of macrophages, CD8+ T cells and Y8+ T cells, followed by enhanced expression of the MHC-II marker, cellular infiltration by polymorphonuclear leukocytes and finally an increased number of both B cells and CD4+ T cells. In contrast, non-immunised animals did not demonstrate any proliferation nor extravasation of lymphocytes or increased expression of MHC-II before total bacterial clearance had occurred. Polymorphonuclear leukocyte infiltration occurred in the non-immunised animals, however at a later time than that seen in immunised animals. Challenging rodents to establish persistent infection highlighted the inappropriately aggressive white blood cell response to an initial challenge when bacteria may be masked by other substances, followed by the inability to amplify the polymorphonuclear leukocyte response on repeated challenge with NTHi. This hyporesponsiveness in the macrophage population, shown by lack of detectable TNF-a production, concomitant with low numbers of NTHi resulted in a continuously high number of macrophages in the alveoli and the possibility of increased damage to the lung tissue. The requirement for cell surface TNF-a and CD8+ T cells to enhance the clearance of NTHi from the lungs further strengthens previous in vitro and in vivo findings of the possible significance of cellular invasion as a mechanism of pathogenicity for NTHi. This thesis has contributed to the understanding of both the immune response to and the pathogenicity mechanisms of pulmonary infection with NTHi. Kinetic studies identifying cellular responses and cytokine levels have emphasised the ability of mucosal immunisation to increase the rate of immune response and resolution of inflammation to NTHi infection in the lung. Observations demonstrating a requirement for macrophages and CD8+ T cells in mechanisms associated with enhancing NTHi clearance from the lung will lead to further investigations.

No description available.

A respiração e a deglutição são vitais para o homem. Enquanto a primeira diz respeito a um ato primitivo da vida, a hematose, a segunda trata da manutenção da vida, oferecendo a energia necessária, nutrindo e hidratando, perpassando pelo prazer alimentar, ato tão importante na sociedade contemporânea. A relação funcional entre essas funções ainda não foi totalmente elucidada, porém é crescente o interesse e o número de estudos sobre esta temática. Considerando que a deglutição eficiente tem como pressuposto a capacidade de proteger via aérea inferior, a alteração de deglutição primária ou secundária a um dano pulmonar pode trazer repercussões severas para a integridade do sistema respiratório. O objetivo desse estudo é analisar o processo sinérgico da deglutição em portadores de bronquiectasia, a fim de verificar se há alteração na fisiologia da deglutição e caracterizá-la, assim como, identificar se há correspondência entre alteração da função pulmonar e alteração da deglutição.Para tal foram selecionados randomicamente 30 pacientes na faixa etária de 18 a 65 anos, atendidos no ambulatório de bronquiectasia do HUPE. Destes, 26 indivíduos responderam a um questionário dirigido sobre hábitos alimentares e possíveis dificuldades de alimentação; foram submetidos à avaliação clínica da deglutição; 22 fizeram espirometria no setor de prova de função pulmonar no HUPE e 17 avaliação videofluoroscópica da deglutição. Dos 26 indivíduos estudados 10 eram homens e 16 mulheres, com média de idade de 46,3 anos. Na avaliação clínica da deglutição observaram-se alterações estruturais e funcionais em todos os indivíduos estudados. As principais alterações estruturais detectadas referem-se e a dinâmica laríngea; enquanto na avaliação funcional detectou-se alterações referentes à ejeção oral, dinâmica hiolaríngea, trânsito faríngeo e presença de deglutições múltiplas. Com relação à espirometria 06 indivíduos apresentaram distúrbio obstrutivo leve; 04 distúrbio obstrutivo moderado e 09 distúrbio obstrutivo acentuado. A videofluoroscopia da deglutição corroborou os achados da avaliação clínica da deglutição e evidenciou episódios de penetração e aspiração laríngea Pode-se concluir que: (1) a avaliação clínica da deglutição associada à avaliação videofluoroscópica são métodos eficientes para a análise do processo sinérgico da deglutição; (2) identificou-se alteração do processo sinérgico da deglutição, nos indivíduos avaliados; (3) a ausculta cervical isoladamente, não demonstrou ser um método eficiente para predizer aspiração e/ou penetração laringotraqueal; (4) houve correspondência entre os resultados da avaliação clínica funcional da deglutição e videofluoroscópica, exceto quanto a presença de penetração e/ou aspiração e quanto a capacidade de avaliar a ejeção oral; (5) não foi possível identificar se há correspondência entre alteração da função pulmonar e processo sinérgico da deglutição. / Breathing, as well as swallowing, is vital to men.While the first accounts for a primitive aspect of life, the transference of gases through the blood-air barrier, the second is about the maintenance of life, supplying the necessary energy, nourishing and hydrating the organism and then touching the subject of feeding pleasure, such an important act in contemporary society. The functional relation between these functions has not been completely elucidated; however the interest is increasing and the number of studies about this theme. Considering that the efficient swallowing has the capacity of protecting the low airway as purpose, the alteration of primary or secondary swallowing to a pulmonary damage may bring severe repercussions to the integrity of the respiratory system. The aim of the study is to analyze the swallowing in patients carrying bronchiectasis, in order to verify whether there is an alteration in the swallowing physiology and characterize it, as well as to identify whether there is a correspondence between the alteration of the pulmonary function and swallowing disorders. 30 patients were randomly selected aged between 18 and 62 years old, attended at the bronchiectasis clinic of HUPE. 26 out of those individuals were evaluated. They answered a directed quiz about alimentary habits and possible alimentary difficulties; they were submitted to clinical evaluation of swallowing, 22 were orientated to spirometric evaluation in the sector of pulmonary function test at HUPE and 17 videofluorographic examination of swallowing. From the 26 individuals 10 were male and 16 were female, average age of 26 years old. As for the clinical evaluation of swallowing (structural and functional), all individuals presented some kind of laryngeal alteration in the structural evaluation. In the functional evaluation was noted that the main alterations referred to oral ejection, elevation and anterior movement of the hyoid and larynx, pharyngeal transit and multiple swallowing were also present. In relation to spirometry 06 individuals presented mild obstructive disorder 04 presented moderate obstructive disorders and 09 presented severe obstructive disorders. The videofluorograph examination confirmed the findings of the swallowing clinical evaluation and made clear an episode of larynx penetration of liquids. The present study showed as preliminary results: (1) that the clinical evaluation of swallowing associated with videofluorographic Examination are efficient methods to the analysis of the swallowing process; (2) Alteration in the synergic process of swallowing was identified in the evaluated individuals; (3) Cervical auscutating isolatedly did not prove to be an efficient method to predict larynx-tracheal aspiration and/or penetration; (4) There was a correspondence between the results of clinical evaluation of the swallowing videofluorographic, except for the presence of penetration and/or aspiration and for the capacity of evaluating oral ejection; (5) Was not possible to identify if there is a correlation between alteration of the pulmonary function and swallowing disorders.

BACKGROUND: Older individuals suffer from higher rates of pulmonary infections than younger individuals. In addition, older individuals have increased morbidity and mortality due to pulmonary infections when compared to younger individuals. The physiological and immunological reasons for these aforementioned differences are not clear. Recently, non-invasive markers of the lung's physiologic and immunologic status have been recognized. This study employs one of these non-invasive markers, exhaled nitric oxide, in an attempt to determine how the airways may change with age, predisposing older individuals to pulmonary diseases and poorer outcomes as compared to younger individuals. METHODS: Exhaled nitric oxide measurements were obtained from a group of 25 older subjects (61 to 79 years old, median 72 years old) and a group of 23 younger subjects (21 to 30 years old, median 24 years old) that were non-smokers with no history of pulmonary disease, no recent respiratory infections, and no history of environmental allergies. A focused history and physical exam along with spirometry were used to confirm the normal pulmonary status of each subject. Exhaled nitric oxide was measured following the American Thoracic Society recommendations using the Sievers Nitric Oxide Analyzer 280i. The exhaled nitric oxide values for the old and young groups were compared using the Wilcoxon Rank Sum test. RESULTS: For the older subjects, the median exhaled NO concentration was 36.9 ppb. For the younger subjects, the median exhaled NO concentration was 18.7 ppb. These exhaled NO concentrations are significantly different (p = 0.0011). CONCLUSIONS: The exhaled NO concentrations are significantly higher in older individuals than in younger individuals. The reasons for this difference along with the significance are unclear and further studies will be necessary to further evaluate these issues.

A respiração e a deglutição são vitais para o homem. Enquanto a primeira diz respeito a um ato primitivo da vida, a hematose, a segunda trata da manutenção da vida, oferecendo a energia necessária, nutrindo e hidratando, perpassando pelo prazer alimentar, ato tão importante na sociedade contemporânea. A relação funcional entre essas funções ainda não foi totalmente elucidada, porém é crescente o interesse e o número de estudos sobre esta temática. Considerando que a deglutição eficiente tem como pressuposto a capacidade de proteger via aérea inferior, a alteração de deglutição primária ou secundária a um dano pulmonar pode trazer repercussões severas para a integridade do sistema respiratório. O objetivo desse estudo é analisar o processo sinérgico da deglutição em portadores de bronquiectasia, a fim de verificar se há alteração na fisiologia da deglutição e caracterizá-la, assim como, identificar se há correspondência entre alteração da função pulmonar e alteração da deglutição.Para tal foram selecionados randomicamente 30 pacientes na faixa etária de 18 a 65 anos, atendidos no ambulatório de bronquiectasia do HUPE. Destes, 26 indivíduos responderam a um questionário dirigido sobre hábitos alimentares e possíveis dificuldades de alimentação; foram submetidos à avaliação clínica da deglutição; 22 fizeram espirometria no setor de prova de função pulmonar no HUPE e 17 avaliação videofluoroscópica da deglutição. Dos 26 indivíduos estudados 10 eram homens e 16 mulheres, com média de idade de 46,3 anos. Na avaliação clínica da deglutição observaram-se alterações estruturais e funcionais em todos os indivíduos estudados. As principais alterações estruturais detectadas referem-se e a dinâmica laríngea; enquanto na avaliação funcional detectou-se alterações referentes à ejeção oral, dinâmica hiolaríngea, trânsito faríngeo e presença de deglutições múltiplas. Com relação à espirometria 06 indivíduos apresentaram distúrbio obstrutivo leve; 04 distúrbio obstrutivo moderado e 09 distúrbio obstrutivo acentuado. A videofluoroscopia da deglutição corroborou os achados da avaliação clínica da deglutição e evidenciou episódios de penetração e aspiração laríngea Pode-se concluir que: (1) a avaliação clínica da deglutição associada à avaliação videofluoroscópica são métodos eficientes para a análise do processo sinérgico da deglutição; (2) identificou-se alteração do processo sinérgico da deglutição, nos indivíduos avaliados; (3) a ausculta cervical isoladamente, não demonstrou ser um método eficiente para predizer aspiração e/ou penetração laringotraqueal; (4) houve correspondência entre os resultados da avaliação clínica funcional da deglutição e videofluoroscópica, exceto quanto a presença de penetração e/ou aspiração e quanto a capacidade de avaliar a ejeção oral; (5) não foi possível identificar se há correspondência entre alteração da função pulmonar e processo sinérgico da deglutição. / Breathing, as well as swallowing, is vital to men.While the first accounts for a primitive aspect of life, the transference of gases through the blood-air barrier, the second is about the maintenance of life, supplying the necessary energy, nourishing and hydrating the organism and then touching the subject of feeding pleasure, such an important act in contemporary society. The functional relation between these functions has not been completely elucidated; however the interest is increasing and the number of studies about this theme. Considering that the efficient swallowing has the capacity of protecting the low airway as purpose, the alteration of primary or secondary swallowing to a pulmonary damage may bring severe repercussions to the integrity of the respiratory system. The aim of the study is to analyze the swallowing in patients carrying bronchiectasis, in order to verify whether there is an alteration in the swallowing physiology and characterize it, as well as to identify whether there is a correspondence between the alteration of the pulmonary function and swallowing disorders. 30 patients were randomly selected aged between 18 and 62 years old, attended at the bronchiectasis clinic of HUPE. 26 out of those individuals were evaluated. They answered a directed quiz about alimentary habits and possible alimentary difficulties; they were submitted to clinical evaluation of swallowing, 22 were orientated to spirometric evaluation in the sector of pulmonary function test at HUPE and 17 videofluorographic examination of swallowing. From the 26 individuals 10 were male and 16 were female, average age of 26 years old. As for the clinical evaluation of swallowing (structural and functional), all individuals presented some kind of laryngeal alteration in the structural evaluation. In the functional evaluation was noted that the main alterations referred to oral ejection, elevation and anterior movement of the hyoid and larynx, pharyngeal transit and multiple swallowing were also present. In relation to spirometry 06 individuals presented mild obstructive disorder 04 presented moderate obstructive disorders and 09 presented severe obstructive disorders. The videofluorograph examination confirmed the findings of the swallowing clinical evaluation and made clear an episode of larynx penetration of liquids. The present study showed as preliminary results: (1) that the clinical evaluation of swallowing associated with videofluorographic Examination are efficient methods to the analysis of the swallowing process; (2) Alteration in the synergic process of swallowing was identified in the evaluated individuals; (3) Cervical auscutating isolatedly did not prove to be an efficient method to predict larynx-tracheal aspiration and/or penetration; (4) There was a correspondence between the results of clinical evaluation of the swallowing videofluorographic, except for the presence of penetration and/or aspiration and for the capacity of evaluating oral ejection; (5) Was not possible to identify if there is a correlation between alteration of the pulmonary function and swallowing disorders.

L'émergence rapide de bactéries résistantes et l’absence de nouveaux traitements efficaces ont conduit à réutiliser d’anciens antibiotiques. Le chloramphenicol (CHL) et le thiamphenicol (THA) ont ainsi été proposés pour traiter les infections respiratoires multirésistantes. Leur administration directe dans les poumons sous forme d’aérosols thérapeutiques devrait augmenter leur efficacité et minimiser l’exposition systémique responsable d’effets secondaires, en particulier lors de traitements prolongés. Ce travail de thèse a eu pour objectifs de réaliser des étudies biopharmaceutiques et de développer des formulations d’aérosols pour la voie pulmonaire. La perméabilité membranaire du CHL et du THA a été évaluée sur le modèle d’épithélium bronchique Calu-3 et leur pharmacocinétique a été réalisée chez le rat après administrations intratrachéale et intraveineuse. La perméabilité membranaire in vitro du CHL s’est révélée élevée, et intermédiaire pour le THA. Les deux antibiotiques sont substrats de transporteurs membranaires d’efflux. Les études pharmacocinétiques, cohérents avec les études in vitro, ont montré un impact nul de la voie d’administration dans cas du CHL et modéré dans le cas du THA. Par conséquent, pour prolonger l’exposition pulmonaire à ces antibiotiques, des formulations à libération prolongées basées sur des nanoparticules ont été incluses dans des poudres sèches de microsphères pour inhalation. Ces poudres se caractérisent par une teneur optimale, des propriétés aérodynamiques satisfaisantes et un profil de libération prolongée, et sont donc prometteuses pour l’administration pulmonaire de CHL ou de THA sous la forme d’aérosols. / The rapid emergence of resistant bacteria and the lack of new efficient treatments lead to re-use old forgotten, but still effective, antimicrobials. In particular, chloramphenicol (CHL) and thiamphenicol (THA) have been proposed to treat multidrug-resistant pulmonary bacterial infections. Their direct administration into the lungs as therapeutic aerosols should increase their efficiency and minimize whole body exposure responsible for adverse effects, particularly in the case of prolonged treatments. The purpose of these PhD. works was to perform biopharmaceutical studies and to develop an effective aerosol formulation for lung delivery. The membrane permeability of CHL and THA was evaluated in vitro in the Calu-3 bronchial epithelial cell model and pharmacokinetic (PK) studies were carried out in rats after intratracheal and intravenous administration. In vitro membrane permeability of CHL was high, but intermediate for THA. Both compounds were shown to be substrates of membrane efflux transporters. In agreement with these findings, the PK studies showed that the administration route had no impact in the case of CHL and a moderate one in the case of THA. Therefore, in order to prolong lung exposure to CHL and THA, nanoparticle-based formulations with sustained release properties were formulated using the palmitate ester prodrugs of CHL and THA. To ease administration, nanoparticles were included in microsphere-based dry powder for inhalation. These powders showed an optimal content, satisfactory aerodynamic properties and sustained drug release, which make them promising formulations for lung delivery of CHL and THA as aerosols.