Objective - evaluate horses with acute airway obstruction using three treatment regimens: tapering doses of dexamethasone (DEX), environmental modification (ENV), and a combination of both treatments (DEX + ENV) by analyzing clinical parameters, pulmonary function testing, bronchoalveolar lavage fluid (BALF) cytology and BALF cell expression of the cytokines IFN-? and IL-4 Animals - 6 horses with recurrent airway obstruction (RAO) Procedures - Clinical examination, pulmonary function test, and collection of BALF prior to treatment and during 22 day treatment period Hypothesis - Alterations in clinical parameters, pulmonary function and airway inflammation in acute equine RAO will return to remission values by treating with DEX, ENV or DEX + ENV Results - All horses demonstrated clinical disease, reduced pulmonary dynamic compliance (Cdyn) and an increased maximum change in pleural pressures (?Pplmax) when in a challenge environment. All treatments improved clinical parameters, ?Pplmax and Cdyn. BALF cytology during an RAO crisis demonstrated neutrophilic inflammation. ENV or DEX + ENV resulted in a significant decrease in airway neutrophilia that was maintained throughout the treatment period. In contrast, treatment with DEX caused a reduction in airway neutrophilia initially followed by a rebound neutrophilia as the period between administrations of dexamethasone (0.05mg/kg) was increased to 72 hours. The rebound neutrophilia was not accompanied by equivalent deterioration in clinical parameters or pulmonary function. Conclusions - Environmental modification is important in the management of RAO horses. Treatment of clinical RAO with a decreasing dosage protocol of corticosteroids in the absence of environmental modification results in the persistence of airway inflammation without recrudescence of clinical disease. / Master of Science

Background – Feeding horses round bale hay (RBH) has been associated with airway inflammation. The purpose of this study was to determine if horses fed RBH for a 6-week period demonstrated more evidence of airway inflammation than horses fed square bale hay (SBH) of comparable quality. Hypothesis - The respiratory health of horses fed RBH will not differ from horses fed SBH of comparable quality. Animals – Two feeding groups of 15 healthy horses (mixed ages, breeds) from the University riding program. Methods – This was a prospective study performed during fall of 2009. At the beginning and end of a 6- week feeding trial, horses were examined (physical, upper airway endoscopic) and samples (tracheal aspirate (TA), bronchoalveolar lavage (BAL)) collected for cytology and/or bacterial/fungal culture. Hay was analyzed for nutritional value and bacterial/fungal content. Results – Horses fed RBH demonstrated an increase in pharyngeal lymphoid hyperplasia (p=0.0143) and percentage neutrophils (p=0.0078) in the TA samples post-feeding as compared to pre-feeding values. Nutritional analysis of hay and measurements of bacterial/fungal load did not differ over time and/or between hay types. Conclusions and clinical importance – The identification of airway inflammation in the horses fed RBH indicates that factors associated with the manner in which the hay is fed and consumed contribute to the development of subclinical airway inflammation. RBH affords horses continuous daily exposure to hay and as horses bury their muzzles in the bale, exposure to particulate matter is likely increased. These factors may partially explain the response in horses fed RBH. Further studies are required to confirm these predictions. / Master of Science

Os modelos experimentais têm sido utilizados para o estudo dos mecanismos fisiopatológicos envolvidos no desenvolvimento da Doença Pulmonar Obstrutiva Crônica (DPOC). O modelo que melhor mimetiza a doença em humanos é o que utiliza a exposição à fumaça de cigarro. No entanto, a utilização deste modelo experimental requer um longo tempo de exposição (6 meses) e a lesão do parênquima obtida é considerada leve. O desequilíbrio protease/anti-protease é considerado um importante mecanismo fisiopatológico envolvido no desenvolvimento da DPOC. Desta forma, neste estudo propomos o desenvolvimento de um modelo experimental no qual associamos a instilação de elastase previamente ao início da exposição ao fumo na tentativa de obter um maior grau de lesão tecidual em um menor espaço de tempo. Para tanto, camundongos C57Bl/6 foram divididos em quatro grupos: Controle, Elastase, Fumo, Fumo/Elastase 1 dose e Fumo/Elastase 2 doses e analisados após dois meses de exposição. Os animais do grupo Fumo/Elastase 1 dose e 2 doses foram submetidos à instilação intranasal de elastase pancreática de porco (0,33UI) e expostos a fumaça de cigarro por dois meses. O grupo controle recebeu o mesmo tratamento com solução fisiológica (NaCl 0.9%). A exposição ao fumo foi feita por 30min, 2 vezes/dia, 5 dias da semana. Após dois meses, os animais foram sacrificados e observamos aumento de LM no grupo Fumo/Elastase 1 dose e 2 doses comparado aos grupos Controle e Fumo; aumento de células positivas para MAC-2 no parênquima (Fumo/Elastase 2 doses) e vias aéreas (Fumo/Elastase 1 dose e 2 doses), MMP-12 no parênquima pulmonar (Fumo/Elastase 2 doses), GP91 no parênquima (Fumo/Elastase 1 dose e 2 doses) e vias aéreas (Fumo e Fumo/Elastase 1 dose) e aumento de proporção de fibras elásticas no parênquima pulmonar do grupo Fumo/Elastase 1 dose e do grupo Fumo, caracterizando presença de enfisema pulmonar. A instilação de elastase pancreática de porco juntamente com a exposição à fumaça de cigarro aumentou a susceptibilidade ao desenvolvimento do enfisema / Experimental models have been used to study the pathophysiological mechanisms involved in the development of COPD. Cigarette Smoke exposure (CS) is considered the best model to mimetize the disease in humans. However, the CS requires a long exposure time (6 months) and the parenchymal destruction obtained is considered mild. The protease / anti - protease imbalance is considered an important pathophysiological mechanism involved in the development of COPD. Thus, in this study we propose the development of an experimental model in which we associate instillation of elastase before the start of exposure to smoke, trying to increase the parenchymal destruction degree in a shorter time. For that, C57BL / 6 mice were divided into four groups: Control, Elastase, Smoke and Smoke/Elastase 1 dose and Smoke/Elastase 2 doses and analyzed in two months after the CS exposition. The Smoke/Elastase 1 dose and 2 doses animals group received an intranasal instillation of porcine pancreatic elastase (0.33 IU) and exposed to cigarette smoke for two months. The control group received the same treatment with saline (NaCl 0.9 %). Animals were exposed to CS for 30min, 2 times / day, 5 days a week. After two months, we observed increased mean linear intercept (LM) and positive cells for MAC-2, MMP-12 and GP91 in the airways and lung parenchyma and increase of elastic fibers in the lung parenchyma characterizing the presence of pulmonary emphysema. The instillation of porcine pancreatic elastase along the exposure to cigarette smoke increased susceptibility to the development of emphysema

Chronic obstructive pulmonary disease (COPD) is characterized by a non-fully reversible airflow limitation and a chronic inflammatory response accompanied by the development of emphysema. The β2-adrenoceptor agonists and anticholinergic agents are widely used in patients with COPD due to their bronchodilator properties. Today, many studies in vitro and in vivo in experimental animal models have shown that these bronchodilators also exert anti-inflammatory effects, but their protective roles against lung inflammation and the development of emphysema in patients with COPD remain to be determined. The imbalance between the activity of matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs) is considered to play a key role in the pathogenesis of COPD, especially in the development of pulmonary emphysema. The modulation of inflammatory responses and emphysema via the inhibition of the MMPs activity induced by the use of synthetic inhibitors of MMPs suggests that MMPs may be therapeutic targets for COPD patients. However, very few studies have demonstrated the regulation exerted by β2-adrenoceptor agonists and anticholinergic agents on the activity of MMPs. The combination of β2-adrenoceptor agonists with anticholinergic agents has been found to exert an additive and even synergistic bronchodilator effect, but nothing is known about their combination on the inflammatory pathogenesis and the development of emphysema. Thus, a better knowledge of the activities of β2-adrenoceptor agonists and anticholinergic agents on controlling pulmonary inflammation and emphysema in COPD could provide a new therapeutic approach in this area. The first goal of the present work was to investigate the effects of formoterol, a β2-adrenoceptor agonist and/or ipratropium bromide, an anticholinergic agent, on acute pulmonary inflammation induced by cadmium inhalation in rats. In addition, we examined whether the expected anti-inflammatory effects of formoterol and/or of ipratropium bromide were associated with a modulation of the gelatinase A (MMP-2), gelatinase B (MMP-9) and macrophage metalloelastase (MMP-12) activity. Compared with the data observed in rats exposed to a single dose of cadmium, the pre-administration of formoterol or ipratropium bromide inhibited the cadmium-induced increase in airway resistance. Formoterol significantly reduced the total cell, neutrophil and macrophage counts in bronchoalveolar lavage fluid (BALF), whereas, ipratropium bromide only reduced the neutrophil number. Both bronchodilators administrated alone attenuated significantly the lung lesions associated with parenchyma inflammatory cell influx and congestion observed in cadmium-group. The increased MMP-9 activity was significantly attenuated. A reduction of pulmonary edema was also detected by measuring the lung wet-to-dry weight ratio. However, no additive or synergistic effect was obtained when formoterol was administrated in combination with ipratropium bromide. In conclusion, formoterol and ipratropium bromide partially protect the lungs against inflammation by reducing the neutrophilic infiltration. This protective effect may be related to the reduction of MMP-9 activity which plays an important role in the acute inflammation. Up to now, the impact of a long-term administration of bronchodilators aiming to control the chronic inflammation and the development of emphysema in experimental animal models and in patients with COPD has been poorly investigated. In this context, it was rational to investigate whether the protective role of formoterol and ipratropium bromide identified in acute conditions persists in a rat model of subacute neutrophilic pulmonary inflammation with an enlargement of airspaces. In the second part of this study, we also intended to determine whether these anti-inflammatory effects are related to the modulation of imbalance between MMPs and TIMPs. Though ipratropium induced no effect on the subacute pulmonary inflammation and the airspace enlargement induced by repeated cadmium inhalations during 5 weeks in rats, formoterol elicited marked anti-inflammatory effects on the increase of total cell and neutrophil counts as well as the activity of MMP-9 mainly expressed in alveolar macrophages and epithelial cells. This drug also prevented the inflammatory infiltration in alveoli and in interstitial tissue and significantly inhibited the airspace enlargement as demonstrated by the significant decrease in the mean linear intercept (Lm). The combination of both bronchodilators at inefficient concentrations induced synergistic inhibitory effects on the total cell and neutrophil counts and on the cadmium-induced increased Lm associated with a reduction of MMP-9 activity in BALF. These data suggest that formoterol alone or combined with ipratropium could protect lungs against subacute pulmonary inflammation and the airspace enlargement by inhibiting neutrophilic infiltration via the reduction of MMP-9 activity. To the best of our knowledge, this is the first report which reveals the anti-inflammatory effects of β2-adrenoceptor agonists and anticholinergic agents in an animal model which mimics the main features of COPD. The data obtained in this work contribute to identify new therapeutic targets in COPD for drugs currently used in clinical practice./ La broncho-pneumopathie chronique obstructive (BPCO) est caractérisée essentiellement par une limitation du débit aérien qui n'est pas entièrement réversible et une inflammation chronique pulmonaire accompagnée dun développement demphysème. Les agonistes β2-adrénergiques et les anticholinergiques sont largement utilisés chez les patients atteints de BPCO en raison de leurs propriétés bronchodilatatrices. Aujourdhui, de nombreuses études expérimentales in vitro et in vivo, utilisant des modèles animaux, ont montré que ces bronchodilatateurs exerçaient également des effets anti-inflammatoires. Leur rôle protecteur contre linflammation pulmonaire et le développement d'emphysème chez des patients souffrant de BPCO reste à déterminer. Le déséquilibre entre les métalloprotéinases de la matrice (MMPs) et leurs inhibiteurs tissulaires (TIMPs) est considéré comme un mécanisme clé dans lévolution de la maladie et surtout dans le développement d'emphysème pulmonaire. La modulation des réactions inflammatoires et de lemphysème obtenue grâce à la réduction de lactivité des MMPs induite par des inhibiteurs synthétiques suggère que les MMPs pourraient être des cibles thérapeutiques importantes dans le traitement de la BPCO. Mais jusquà ce jour, très peu détudes ont été consacrées à la régulation de lactivité des MMPs par les agonistes β2-adrénergiques et les anticholinergiques. Lassociation dun agoniste β2-adrénergique avec un anticholinergique donne lieu à une amplification des effets bronchodilatateurs, mais il nest pas certain que leur combinaison débouche sur des effets additifs ou synergiques sur le plan dun meilleur contrôle de la réaction inflammatoire. Ainsi, une meilleure connaissance des activités des agonistes β2-adrénergiques et des anticholinergiques visant au contrôle de l'inflammation pulmonaire et de l'emphysème dans la BPCO pourrait fournir une nouvelle approche thérapeutique dans ce domaine. Lobjectif de la première partie de ce travail était donc d'étudier les effets du formotérol, un agoniste β2-adrénergique et / ou du bromure d'ipratropium, un anticholinergique, sur l'inflammation pulmonaire aiguë provoquée par linhalation de cadmium chez le rat. En outre, nous voulions aussi vérifier si ces effets anti-inflammatoires étaient associés à une modulation de lactivité de la gélatinase A (MMP-2), de la gélatinase B (MMP-9) et de la métallo-élastase du macrophage (MMP-12). Par rapport aux effets observés chez des rats exposés à une dose de cadmium, ladministration préventive de formotérol ou de bromure dipratropium a atténué laugmentation de la résistance des voies aériennes. Le formotérol a induit une diminution significative du nombre de cellules totales, des neutrophiles et des macrophages dans le liquide de lavage broncho-alvéolaire (BALF). Par contre, le bromure dipratropium na entraîné quune diminution du nombre de neutrophiles. Les lésions pulmonaires caractérisées par de la congestion et une réaction inflammatoire du parenchyme ont été significativement inhibées par ces deux bronchodilatateurs administrés séparément. Lélévation remarquable de lactivité de MMP-9 dans le BALF a été significativement atténuée par le prétraitement au formotérol ou au bromure dipratropium. Il en est de même pour ldème pulmonaire évalué par le biais du rapport entre le poids humide et le poids sec du parenchyme. Lorsque les deux principes actifs ont été combinés et administrés préventivement à laction du cadmium, aucun effet synergique ou additif na été constaté. En conclusion, le formotérol et le bromure dipratropium préviennent partiellement linflammation pulmonaire aiguë en réduisant linfiltration neutrophilique du parenchyme pulmonaire faisant suite à une exposition aiguë au cadmium. Cet effet protecteur pourrait être lié à une réduction de lactivité de MMP-9 qui joue un rôle pro-inflammatoire important dans linflammation aiguë. Jusquici, les effets potentiels des bronchodilatateurs contre linflammation chronique et lévolution de lemphysème pulmonaire chez des animaux et chez les patients atteints de BPCO restent mal connus. Il nous restait donc à vérifier si les effets protecteurs du formotérol et du bromure dipratropium révélés par nos premières études au cours desquelles les rats ont été exposés de manière aiguë au cadmium persistent dans un modèle dinflammation pulmonaire subaiguë accompagnée dun élargissement des espaces aériens. Nous voulions également déterminer si ces effets étaient liés à la modulation du déséquilibre entre les MMP-2/9/12 et les TIMP-1/2. Bien que le bromure dipratropium nait aucun effet sur linflammation subaiguë pulmonaire et lélargissement des espaces aériens induits par des inhalations répétées de cadmium chez le rat, le prétraitement par du formotérol a, quant à lui, inhibé significativement laugmentation du nombre de cellules totales et des neutrophiles ainsi que de lactivité de MMP-9 exprimée principalement dans les macrophages et les cellules épithéliales alvéolaires. En outre, une atténuation importante des lésions pulmonaires caractérisées par un élargissement des espaces aériens les plus distaux et une infiltration de cellules inflammatoires dans les alvéoles et le tissu ont été observées. La combinaison des deux bronchodilatateurs, à des concentrations pourtant inefficaces, a provoqué un effet synergique sur la plupart des paramètres étudiés, en particulier sur linfiltration par les neutrophiles et lactivité de MMP-9 dans le BALF. Ce travail suggère que le formotérol, seul ou combiné avec le bromure dipratropium, pourrait protéger partiellement les poumons contre linflammation pulmonaire et lélargissement des espaces aériens en réduisant l'infiltration neutrophilique éventuellement via l'inhibition de lactivité de MMP-9. A notre connaissance, il sagit du premier rapport montrant les effets anti-inflammatoires des agonistes β2-adrénergiques et des anticholinergiques dans un modèle animal mimant les principales caractéristiques physiopathologiques de la BPCO. Les données obtenues dans ce travail pourraient contribuer à identifier de nouvelles cibles thérapeutiques pour cette maladie.

Os modelos experimentais têm sido utilizados para o estudo dos mecanismos fisiopatológicos envolvidos no desenvolvimento da Doença Pulmonar Obstrutiva Crônica (DPOC). O modelo que melhor mimetiza a doença em humanos é o que utiliza a exposição à fumaça de cigarro. No entanto, a utilização deste modelo experimental requer um longo tempo de exposição (6 meses) e a lesão do parênquima obtida é considerada leve. O desequilíbrio protease/anti-protease é considerado um importante mecanismo fisiopatológico envolvido no desenvolvimento da DPOC. Desta forma, neste estudo propomos o desenvolvimento de um modelo experimental no qual associamos a instilação de elastase previamente ao início da exposição ao fumo na tentativa de obter um maior grau de lesão tecidual em um menor espaço de tempo. Para tanto, camundongos C57Bl/6 foram divididos em quatro grupos: Controle, Elastase, Fumo, Fumo/Elastase 1 dose e Fumo/Elastase 2 doses e analisados após dois meses de exposição. Os animais do grupo Fumo/Elastase 1 dose e 2 doses foram submetidos à instilação intranasal de elastase pancreática de porco (0,33UI) e expostos a fumaça de cigarro por dois meses. O grupo controle recebeu o mesmo tratamento com solução fisiológica (NaCl 0.9%). A exposição ao fumo foi feita por 30min, 2 vezes/dia, 5 dias da semana. Após dois meses, os animais foram sacrificados e observamos aumento de LM no grupo Fumo/Elastase 1 dose e 2 doses comparado aos grupos Controle e Fumo; aumento de células positivas para MAC-2 no parênquima (Fumo/Elastase 2 doses) e vias aéreas (Fumo/Elastase 1 dose e 2 doses), MMP-12 no parênquima pulmonar (Fumo/Elastase 2 doses), GP91 no parênquima (Fumo/Elastase 1 dose e 2 doses) e vias aéreas (Fumo e Fumo/Elastase 1 dose) e aumento de proporção de fibras elásticas no parênquima pulmonar do grupo Fumo/Elastase 1 dose e do grupo Fumo, caracterizando presença de enfisema pulmonar. A instilação de elastase pancreática de porco juntamente com a exposição à fumaça de cigarro aumentou a susceptibilidade ao desenvolvimento do enfisema / Experimental models have been used to study the pathophysiological mechanisms involved in the development of COPD. Cigarette Smoke exposure (CS) is considered the best model to mimetize the disease in humans. However, the CS requires a long exposure time (6 months) and the parenchymal destruction obtained is considered mild. The protease / anti - protease imbalance is considered an important pathophysiological mechanism involved in the development of COPD. Thus, in this study we propose the development of an experimental model in which we associate instillation of elastase before the start of exposure to smoke, trying to increase the parenchymal destruction degree in a shorter time. For that, C57BL / 6 mice were divided into four groups: Control, Elastase, Smoke and Smoke/Elastase 1 dose and Smoke/Elastase 2 doses and analyzed in two months after the CS exposition. The Smoke/Elastase 1 dose and 2 doses animals group received an intranasal instillation of porcine pancreatic elastase (0.33 IU) and exposed to cigarette smoke for two months. The control group received the same treatment with saline (NaCl 0.9 %). Animals were exposed to CS for 30min, 2 times / day, 5 days a week. After two months, we observed increased mean linear intercept (LM) and positive cells for MAC-2, MMP-12 and GP91 in the airways and lung parenchyma and increase of elastic fibers in the lung parenchyma characterizing the presence of pulmonary emphysema. The instillation of porcine pancreatic elastase along the exposure to cigarette smoke increased susceptibility to the development of emphysema

Le développement d’une nouvelle sonde dite « activity-based probe » pour réaliser la détection de formes actives de protéases appartenant à la famille des protéases à zinc de la matrice (MMP) a été réalisé dans ce travail, en partant d’un inhibiteur phosphinique puissant des MMP dans lequel a été introduit un groupement photoactivable de type diazérine. Ce composé se révèle un inhibiteur puissant de plusieurs MMP avec des affinités nanomolaires. Ce composé incubé avec différentes MMP est par ailleurs capables de modifier de façon covalente un grand nombre de MMP au niveau de leur site actif, avec des rendements de modification variant de plus de 50% à 11%, selon la nature des MMP. En ayant choisi comme moyen de détection la radioactivité, nous démontrons qu’avec cette nouvelle sonde qu’il est possible de détecter des formes actives de MMP avec des sensibilités de l’ordre de la femtomole dans des systèmes modèles de protéomes complexes. Appliquée à l’analyse de lavages broncho alvéolaires de souris traitées par voie pulmonaire avec des nanoparticules pour induire une réponse inflammatoire, cette nouvelle sonde permet de mettre en évidence la présence de formes actives du domaine catalytique de la MMP-12, une métalloprotéase à zinc exprimée par les macrophages, mais pas dans les animaux contrôles. En revanche l’analyse de carotides humaines de patients souffrant d’athérosclérose ne nous pas conduit avec cette sonde à la détection de formes actives de MMP. Malgré ce résultat, il est à noter que la détection de forme active de MMP dans un fluide pathologique est une première dans ce domaine. Cette sonde étant validée pour sa capacité à détecter des formes actives de MMP, elle permettra dans l’avenir de tester d’autres fluides pathologiques d’origine humaine ou bien des extraits de tissu comme des tumeurs pour lesquels les MMP pourraient être des marqueurs de ces pathologies. / A new activity-based probe able to covalently modify the active site of proteases belonging to the matrix metalloprotease family (MMPs) has been developed in this thesis project. The probe was shown to behave as potent inhibitor of several MMPs, with nanomolar Ki values. This probe was also able to modify specifically only the free active site of MMPs, with particular high yields of cross-linking varying from 50 % to 11 %, depending of the MMPs tested. Using radioactivity as means of detection, this probe was able to detect active form of MMPs with a threshold of 1 femtomole. Applied to the study of bronchoalvelolar fluids (BAL) from mice exposed to nanoparticles by a lung aspiration protocol, this probe revealed the presence of the catalytic domain of MMP-12 under its active form, but not in control animals. When used to detect active form of MMPs from extracts obtained from human arteries of patient suffering from atherosclerosis, the probe was not able to detect such MMP active forms. Despite this negative result, the detection of active form of MMP in pathological fluid like BAL has never been reported before this work. Having validated this novel MMP activity-based probe, it will be possible to use it now for detecting MMPs from other pathological fluids or tissues extracts in which MMPs can be good markers of the pathology.

Abstract Pulmonary fibrosis, lung cancer and chronic obstructive pulmonary disease (COPD) are severe diseases and common death causes worldwide. Due to the lack of an effective therapy, the investigation of cell biological mechanisms behind these diseases is essential. An activation of stromal cells, including myofibroblasts, is a main feature found in the pathogenesis of lung diseases. Myofibroblasts express alpha-smooth muscle actin (α-SMA), have specific ultrastructure, produce extracellular matrix proteins and possess contractile capacity. Detailed structure and function of myofibroblasts and their roles in healthy and diseased lung are not yet wholly understood. The investigation of the myofibroblasts may further offer novel tools for the acquisition of proper diagnosis, prognosis and medical treatment. The study aimed to characterize the ultrastructural, functional and disease-specific features of stromal cells, particularly myofibroblasts, in interstitial and malignant lung diseases. The functional properties evaluated here were differentiation, invasive and contractile properties. The study material included in vitro stromal cells cultured from bronchoalveolar lavage (BAL) fluids. The appearance and location of myofibroblasts in different lung compartments of non-smokers and the COPD-patients were examined in vivo. The cells were investigated by light and electron microscopy. The α-SMA expression was analysed by gene or protein assays. The study demonstrated that stromal cells could be cultured from diagnostic BAL fluid samples and lung tissues. Cultured cells were a mixture of fibroblasts and myofibroblasts. A small proportion of cells exhibited progenitor-like features. Myofibroblasts revealed differential features in electron microscopy and invasive or contractile assays. When studying tissues from healthy and COPD lungs, myofibroblasts were located both in alveoli and airways. In alveoli myofibroblasts localized in widened alveolar tips which were newly described structures and locations of myofibroblasts in healthy and diseased lung. The amount of myofibroblasts in large airways, but not in peripheral lung, was increased in COPD. We concluded that myofibroblasts have several locations in normal and COPD lung, which suggests a function both in pulmonary regeneration and the pathogenesis of COPD. Smoking altered the phenotype of myofibroblasts regardless of its origin. / Tiivistelmä Keuhkofibroosi, keuhkosyöpä ja keuhkoahtaumatauti (COPD) ovat kansallisesti ja maailmanlaajuisesti yleisiä ja kuolemaan johtavia sairauksia. Taudinmääritys ja hoito ovat vaativia, eikä kaikille potilaille ole parantavaa hoitoa. Keuhkosairauksien kaikkia solubiologisia mekanismeja ei vielä tunneta, mikä on yksi syy lääkekehityksen ongelmiin. Interstitiaaleissa ja pahanlaatuisissa keuhkosairauksissa esiintyy paljon aktiivisia sidekudossoluja, kuten muuntuneita fibroblasteja eli myofibroblasteja. Ne tunnistetaan hienorakenteesta, jota voidaan tutkia elektronimikroskoopilla. Myofibroblastit ilmentävät myös solun sisäistä sileän lihaksen alfa-aktiinia (α-SMA), tuottavat sidekudoksen proteiineja ja kykenevät supistumaan. Myofibroblastien hienorakenteen ja toiminnan selvittäminen voi antaa lisätietoa keuhkosairauksien syntymekanismeista, jolloin diagnostiikkaa, ennustetta sekä hoitoja voidaan arvioida paremmin. Väitöskirjassa selvitettiin myofibroblastien hienorakennetta ja toimintaa eri keuhkosairauksissa. Tutkitut toiminnalliset ominaisuudet olivat erilaistumispotentiaali, invasiivisuus ja supistumiskyky. Sairauksien kliinistä käyttäytymistä ja potilaiden tupakointitottumuksia tarkasteltiin suhteessa solubiologiatason havaintoihin. Tutkimusmateriaali kerättiin taudinmäärityksen yhteydessä interstitiaalisia keuhkosairauksia, keuhkoahtaumatautia tai keuhkosyöpää sairastavilta potilailta. Tulosten mukaan bronkoalveolaarihuuhtelunesteestä (BAL) ja keuhkokudospaloista voidaan soluviljelymenetelmin kasvattaa ja ylläpitää solulinjoja. Viljellyt solut muodostivat sekasolupopulaatiota, joissa esiintyi pääosin fibroblasteja ja vaihteleva osuus myofibroblasteja. Pieni osa soluista ilmensi kantasoluille tyypillisiä piirteitä. Myofibroblastien tyyppipiirteet ja toiminnalliset ominaisuudet vaihtelivat taudeittain. Kudoksessa myofibroblasteja ilmentyi sekä keuhkorakkuloissa että ilmateissä. Keuhkorakkulatasolla myofibroblastit sijoittuivat irrallisten alveoliseinämien laajentuneisiin päihin, joita ei ole aiemmin tutkittu tieteellisessä kirjallisuudessa myofibroblastien yhteydessä. Keuhkoahtaumatauti ja tupakointi vähensivät näiden rakenteiden määrää perifeerisessä keuhkossa, kun taas suurissa ilmateissä keuhkoahtaumatauti lisäsi myofibroblasteja. Päättelimme, että myofibroblastit edistävät keuhkoahtaumataudin syntyä isoissa ilmateissä, mutta saattavat osallistua keuhkojen korjaukseen keuhkorakkuloissa ja pienissä ilmateissä.