Le syndrome de détresse respiratoire aiguë (SDRA) se développe suite à une atteinte pulmonaire lésionnelle, induisant un œdème et une inflammation excessive, généralement suivis d’une réparation atypique menant à la fibrose. Malgré de signifiants progrès dans les traitements, la mortalité reste élevée : ~ 40 %. Mon hypothèse de travail est que l’atténuation de l’œdème ou de la réponse inflammatoire pourrait freiner le développement ou la sévérité de la phase exsudative. Nous avons évalué cette hypothèse à l’aide d’un modèle de phase exsudative du SDRA, i.e. instillation intra-trachéale de bléomycine, chez les souris.  La modulation des fluides alvéolaires est étudiée avec des souris transgénique (Tg) pour le canal ENaC, qui sont sensibles à la formation d’un œdème. Cependant, ces souris Tg ne sont pas plus sensibles au développement de la phase exsudative en condition lésionnelle (bléomycine). Nous avons déterminé par une étude électrophysiologique des cellules épithéliales alvéolaires de type II (AT II) que ce n’est pas lié à une inhibition par la bléomycine de la fonction du canal ENaC.  Le traitement de la réponse inflammatoire associée au SDRA par des glucocorticoïdes est une thérapie potentielle mais controversée. Les glucocorticoïdes dans notre modèle murin ne réduisent pas la sévérité des lésions. Nous avons pu déterminé lors d’expériences in vitro que ce serait dû à une réduction de la capacité de réparation des AT II. En résumé :  La modulation du canal ENaC ne modifie pas le développement de la phase exsudative, suggérant que la régulation de l’œdème n’est pas suffisante pour modifier l’évolution du SDRA.  La modulation de l’inflammation par les glucocorticoïdes est ineffective, possiblement à cause d’une altération de la réparation. Mon étude suggère que le traitement de la phase exsudative du SDRA est complexe. En effet, la régulation de l’œdème ou de l’inflammation de façon isolée ne peut pas modifier l’évolution du SDRA. L'hétérogénéité des sources du SDRA et la redondance des mécanismes cellulaires impliqués dans l’évolution des lésions pulmonaires suggèrent que le traitement nécessitera une approche visant plusieurs cibles mécanistiques afin d’en accélérer la résolution. / Although much has been learned about the mechanisms leading to acute respiratory distress syndrome (ARDS), mortality remains high: ~ 40%. This syndrome is associated with lung injury where alveolar edema and excessive inflammatory response can progress to abnormal epithelial repair and fibrosis. The hypothesis of the work presented in this thesis is that attenuation of edema or of the inflammatory response in the initial stage of the acute lung injury would decrease the severity of injury. I evaluated this hypothesis in an ARDS acute phase, modeled by an intratracheal instillation of bleomycin in mice, using two distinct experimental strategies.  The importance of edema clearance was studied in a transgenic (Tg) ENaC mouse, a mouse known to be sensitive to the formation of edema. However, our results show that these Tg mice were not more susceptible to the development of the ARDS acute phase induced by bleomycin. Furthermore, we have been able to show that bleomycin itself did not interfere with the ENaC channel function of alveolar epithelial cells type II (AT II).  The treatment of the inflammatory response associated with ARDS by glucocorticoid therapy is subject to controversy. In our mouse model, glucocorticoids decrease the level of cytokine in the alveolar milieu but did not decrease the severity of lung injury. Using in vitro experiments, we show that this lack of response could be secondary to the impact of the treatment on the epithelial repair capacity of AT II. In summary:  The ENaC channel expression did not have an impact on the development of the exudative phase, suggesting that the regulation of edema is not sufficient to alter the course of ARDS.  The modulation of inflammation by glucocorticoids was ineffective, possibly because of impaired repair of the epithelium. These results suggest that the control of edema or inflammation separately does not modify the evolution of lung injury. The heterogeneity of the ARDS origins and the redundancy of cellular mechanisms involved in lung injury will require therapy aimed at multiple pathophysiological targets to permit the resolution of lung injury.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Orientador: José Roberto Fioretto / Resumo: A Síndrome do Desconforto Respiratório Agudo (SDRA) cursa com alta morbi-mortalidade apesar dos avanços no entendimento de sua fisiopatologia e tratamento. A terapia ventilatória baseia-se na proteção pulmonar, sendo a ventilação oscilatória de alta frequência (VOAF) uma opção de método protetor. A posição prona (PP) é terapia adjuvante que possibilita homogeneização da distribuição do volume corrente (VC) e promove recrutamento alveolar. O objetivo do estudo foi investigar o efeito da posição prona associada à VOAF e ventilação mecânica convencional (VMC) protetora sobre a oxigenação, inflamação, dano oxidativo e histologia pulmonares, comparando-a à posição supina em ambos os modos ventilatórios. Foram instrumentados 75 coelhos com traqueostomia e acessos vasculares. A lesão pulmonar aguda (LPA) foi induzida por lavagem traqueal de salina aquecida (30mL/Kg, 38°C). Os animais foram então aleatorizados em cinco grupos (n=15): 1) GC (Controle): animais sadios em VMC protetora basal; 2) GVMS: animais com LPA em VMC protetora e posição supina; 3) GVMP: animais com LPA em VMC protetora e posição prona; 4) GVAFS: animais com LPA em VOAF e posição supina; 5) GVAFP: animais com LPA em VOAF e posição prona. Após, foram submetidos a quatro horas de VMC protetora (modo pressão regulada-volume controlado, PEEP 10 cmH2O, VC 6mL/kg, Ti 0,5s, FR 40 rpm e FiO2 1) ou VOAF (MAP 15 mmHg, FR 10Hz, amplitude 22 e FiO2 1). O nível de significância foi de 5%. Após a indução, os grupos apresentaram... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Acute Respiratory Distress Syndrome (ARDS) presents with high morbidity and mortality despite advances in the understanding of its pathophysiology and treatment. Ventilatory therapy is based on the intention of injuring less, with high frequency oscillatory ventilation (HFOV) being a protective method option. Prone position (PP) is an adjuvant therapy that enables homogenization of volume tidal (VT) distribution and promotes alveolar recruitment. The aim of this study was to investigate the effects of prone position associated with HFOV and protective conventional mechanical ventilation (CMV) on oxygenation and lung inflammation, oxidative damage and histology, comparing it with the supine position in both ventilatory modes. Seventy five rabbits were submitted to tracheostomy and vascular accesses. ALI was induced by tracheal infusion of heated saline (30mL/kg, 38° C). The subjects were then ramdomized in five groups (n=15): 1) CG (Control): healthy animals in basal protective CMV; 2) MVSG: animals with ALI in protective CMV and supine position; 3) MVPG animals with ALI in protective CMV and prone position; 4) HFSG: animals with ALI in HFOV and supine position; 5) HFPG: animals with ALI in HFOV and prone position. After that, they were submitted to four hours of protective VMC (PRV mode, PEEP 10 cmH2O, VC 6ml/kg, Ti 0,5s, FR=40 rpm and FiO2 1) or HFOV (MAP 15 mmHg, FR 10 Hz, amplitude 22 and FiO2 1). The level of significance was 5%. After induction, the groups presented simi... (Complete abstract click electronic access below) / Doutor

Submitted by Rafaelle Batistella Pires (rafaelle.pires@gmail.com) on 2018-03-07T15:41:30Z No. of bitstreams: 1 Tese Rafaelle Batistella Pires.pdf: 2978722 bytes, checksum: f79b8076fd69934911d1a338cd131aa3 (MD5) / Approved for entry into archive by Luciana Pizzani null (luciana@btu.unesp.br) on 2018-03-08T20:07:06Z (GMT) No. of bitstreams: 1 pires_rb_dr_bot.pdf: 2978722 bytes, checksum: f79b8076fd69934911d1a338cd131aa3 (MD5) / Made available in DSpace on 2018-03-08T20:07:06Z (GMT). No. of bitstreams: 1 pires_rb_dr_bot.pdf: 2978722 bytes, checksum: f79b8076fd69934911d1a338cd131aa3 (MD5) Previous issue date: 2018-02-20 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A Síndrome do Desconforto Respiratório Agudo (SDRA) cursa com alta morbi-mortalidade apesar dos avanços no entendimento de sua fisiopatologia e tratamento. A terapia ventilatória baseia-se na proteção pulmonar, sendo a ventilação oscilatória de alta frequência (VOAF) uma opção de método protetor. A posição prona (PP) é terapia adjuvante que possibilita homogeneização da distribuição do volume corrente (VC) e promove recrutamento alveolar. O objetivo do estudo foi investigar o efeito da posição prona associada à VOAF e ventilação mecânica convencional (VMC) protetora sobre a oxigenação, inflamação, dano oxidativo e histologia pulmonares, comparando-a à posição supina em ambos os modos ventilatórios. Foram instrumentados 75 coelhos com traqueostomia e acessos vasculares. A lesão pulmonar aguda (LPA) foi induzida por lavagem traqueal de salina aquecida (30mL/Kg, 38°C). Os animais foram então aleatorizados em cinco grupos (n=15): 1) GC (Controle): animais sadios em VMC protetora basal; 2) GVMS: animais com LPA em VMC protetora e posição supina; 3) GVMP: animais com LPA em VMC protetora e posição prona; 4) GVAFS: animais com LPA em VOAF e posição supina; 5) GVAFP: animais com LPA em VOAF e posição prona. Após, foram submetidos a quatro horas de VMC protetora (modo pressão regulada-volume controlado, PEEP 10 cmH2O, VC 6mL/kg, Ti 0,5s, FR 40 rpm e FiO2 1) ou VOAF (MAP 15 mmHg, FR 10Hz, amplitude 22 e FiO2 1). O nível de significância foi de 5%. Após a indução, os grupos apresentaram comportamentos semelhantes, com diminuição da relação PaO2/FiO2 e da complacência pulmonar, e aumento do índice de oxigenação (IO) e da pressão média de via aérea (p > 0,05). Ao final do experimento, houve aumento da PaO2/FiO2 nos grupos VOAF comparado aos grupos em VMC (p < 0,05). Houve queda do IO para os grupos em VOAF comparados ao GVMS (p < 0,05), porém o GVMP não diferiu deles (p > 0,05). Não houve diferença estatística quanto à contagem de células polimorfonucleares no lavado broncoalveolar (BAL) nos grupos com LPA. Não houve diferença estatística entre os grupos com lesão para a medida de TNF-alfa no plasma e para sua expressão gênica em tecido pulmonar. Entretanto, a medida de TNF-alfa no lavado broncoalveolar (BAL) e no tecido pulmonar no grupo GVMP foi menor, assemelhando-se ao controle (p > 0,05). Não houve diferença no dano oxidativo avaliado no tecido pulmonar entre os grupos (p > 0,05) e, também, na comparação entre regiões ventral e dorsal dos pulmões. O escore de lesão histológica foi menor nos grupos em VOAF, efeito potencializado no grupo em prona quando comparado aos grupos em VMC (GC = GVAFP < GVMS = GVMP), sem diferença na regionalização pulmonar. Concluimos que, em modelo de LPA por lavagem alveolar com salina aquecida em coelhos: a VOAF melhora a oxigenação quando comparados à VMC; na VMC, a PP atenua a lesão inflamatória avaliada pela medida de TNF-alfa no BAL e tecido pulmonar; os modos ventilatórios e as posições não modificam o grau de estresse oxidativo quando avaliados pelo método de malondialdeído; a VOAF melhora o escore histopatológico de lesão pulmonar, independemente da posição, mas a associação de VOAF e PP atenua a lesão histopatológica quando comparada com a VMC protetora, seja em posição prona ou supina. / Acute Respiratory Distress Syndrome (ARDS) presents with high morbidity and mortality despite advances in the understanding of its pathophysiology and treatment. Ventilatory therapy is based on the intention of injuring less, with high frequency oscillatory ventilation (HFOV) being a protective method option. Prone position (PP) is an adjuvant therapy that enables homogenization of volume tidal (VT) distribution and promotes alveolar recruitment. The aim of this study was to investigate the effects of prone position associated with HFOV and protective conventional mechanical ventilation (CMV) on oxygenation and lung inflammation, oxidative damage and histology, comparing it with the supine position in both ventilatory modes. Seventy five rabbits were submitted to tracheostomy and vascular accesses. ALI was induced by tracheal infusion of heated saline (30mL/kg, 38° C). The subjects were then ramdomized in five groups (n=15): 1) CG (Control): healthy animals in basal protective CMV; 2) MVSG: animals with ALI in protective CMV and supine position; 3) MVPG animals with ALI in protective CMV and prone position; 4) HFSG: animals with ALI in HFOV and supine position; 5) HFPG: animals with ALI in HFOV and prone position. After that, they were submitted to four hours of protective VMC (PRV mode, PEEP 10 cmH2O, VC 6ml/kg, Ti 0,5s, FR=40 rpm and FiO2 1) or HFOV (MAP 15 mmHg, FR 10 Hz, amplitude 22 and FiO2 1). The level of significance was 5%. After induction, the groups presented similar behaviors, with a decrease in the PaO2/FiO2 ratio and lung compliance, and an increase in oxygenation index (OI) and mean airway pressure (p > 0.05). At the end of experimental time, PaO2/FiO2 increased in the HFOV groups compared to the CMV groups (p < 0.05). There was a decrease in OI for HFOV groups compared to MVSG (p < 0.05), but MVPG did not differ from them (p > 0.05). There was no statistically significant difference in polymorphonuclear cell counts in bronchoalveolar lavage (BAL) in the groups with ALI. There was no difference between ALI groups regarding the TNF-alfa dosage in plasma and its gene expression in lung tissue. However, TNF-alpha measurement in BAL and in lung tissue was smaller, resembling control (p > 0.05). There was no difference in the oxidative damage assessed in the lung tissue between the groups (p > 0.05), nor between the lung regions. The histological damage score was lower in the HFOV groups, potentiated effect in the prone group when compared to the CMV groups (CG = HFPG < MVSG = MVPG), no difference in pulmonary regionalization. We conclude that, in the model of ALI induced by alveolar lavage with heated saline in rabbits: HFOV improves oxygenation if compared to CMV; PP in CMV attenuates lung inflammation, evaluated by TNF-alfa dosage in BAL and in lung tissue; ventilatory modes and positions don’t modify the oxidative stress whan evaluated by malondialdehyde method; HFOV improves histopathological lung lesion score, regardless of position, but HFOV and prone position association attenuates histopathological injury compared to protective CMV, either in the prone or supine positions. / FAPESP: 2010/06242-8

A isquemia/reperfusão intestinal (I/R-i) se associa ao desenvolvimento de inflamação pulmonar aguda, que pode ser modulada por mediadores inflamatórios presentes na linfa. Avaliamos os efeitos da I/R-i sob a inflamação pulmonar e a participação do sistema linfático. Wistar machos foram submetidos a 45 min de isquemia intestinal e 24, 72 ou 120 h de reperfusão. Outro grupo teve o ducto linfático bloqueado antes da isquemia. Os resultados revelaram maior inflamação pulmonar nos animais reperfundidos por 120 h em relação aos outros períodos de reperfusão estudados. Os animais apresentaram aumento de MPO e permeabilidade. Aumento de VEGF e de IL-1<font face=\"Symbol\">&#946 e diminuição de IL-10 no explante pulmonar. Diminuição de vWf e aumento de integrina <font face=\"Symbol\">&#9461, PECAM-1 e colágeno I e IV no endotélio pulmonar. Os dados indicam que mecanismos temporais modulam a resposta inflamatória decorrente da I/R-i. Mediadores na linfa e na circulação participam do desencadeamento/manutenção da inflamação pulmonar alterando a integridade do endotélio e ativando o pulmão que libera mediadores adicionais. / Intestinal ischemia/reperfusion (i-I/R) is associated with the development of acute lung inflammation, which can be modulated by inflammatory mediators present in the lymph. We evaluated the effects of i-I/R in lung inflammation and the involvement of the lymphatic system. Wistar rats were subjected to 45 min of intestinal ischemia and 24, 72 or 120 h of reperfusion. Another group had the lymphatic duct blocked before ischemia. The results revealed greater lung inflammation in animals reperfused for 120 h in comparison to other periods studied. These animals showed increased MPO and permeability. Increased VEGF and IL-1<font face=\"Symbol\">&#946 and decreased IL-10 in lung explants. Decreased vWf and increased <font face=\"Symbol\">&#9461 integrin, PECAM-1 and collagen I and IV in the pulmonary endothelium. These data indicate that temporal mechanisms modulate the inflammatory response due to i-I/R. Mediators in the lymph and circulation participate in the initiation / maintenance of lung inflammation by altering the integrity of the endothelium and activating the lung which release additional mediators.

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<p dir="ltr">The recent COVID-19 pandemic has emphasized the risk of respiratory infections leading to acute respiratory distress syndrome (ARDS). A significant factor contributing to poor ARDS outcomes is the impairment of lung surfactant due to infiltrating surface-active proteins and phospholipases during lung inflammation. Lung surfactant's vital role in stabilizing alveoli by reducing air-water interfacial tension becomes evident as its dysfunction severely compromises respiratory function. Although lung surfactant (LS) replacement therapy effectively addresses neonatal LS deficiencies, its efficacy in ARDS treatment for adults remains limited. The challenge lies in the chemical similarity between current animal-extracted surfactants and human lung surfactant which are both phospholipid-based. To address this issue, this dissertation outlines a transformative "polymer lung surfactant (PLS)" designed to overcome the limitations of conventional exogenous surfactants in treating ARDS.</p><p dir="ltr">Firstly, a formulation method, referred to as equilibration-nanoprecipitation (ENP), is established which achieves reproducibility, controls sizing, and limits dispersity of the PLS formulation consisting of block copolymer (BCP) kinetically "frozen" micelles/nanoparticles suspended in water. The method uses a two-step approach of 1) equilibrating the BCP nanoparticles in a water/co-solvent mixture and 2) removing co-solvent using dialysis against a large water reservoir. Comparison of ENP with a conventional solvent-exchange technique through experimental and computational analysis yields further insights into ENP's advantages.</p><p dir="ltr">Next, various studies are highlighted which provide fundamental characterizations of the air-water surface behavior and physical properties of BCP nanoparticles in water. The air-water surface properties of block copolymers have been studied extensively when spread as free chains in organic solvent; however, little was previously known about air-water interfacial behavior of water-spread polymer nanoparticles. The studies address such topics as the effect of nanoparticle size, effect of nanoparticle core chemistry, and the effect of temperature on surface-mechanical behavior. Insights into nanoparticle molecular structure at the interface are provided through X-ray reflectivity and grazing incidence X-ray diffraction. The effect of temperature is further characterized by developing novel NMR and Langmuir trough methods to determine the physical state (glassy vs rubbery) of the core domain in the nanoconfined state at temperatures above and below physiologic temperature.</p><p dir="ltr">Lastly, <i>in vivo </i>studies are presented which demonstrate the detailed and promising proof-of-concept results on the efficacy of the PLS technology in mouse models of lung injury. The PLS therapy not only improves biomechanical function of the lung, but it also significantly lowers the extent of lung injury as shown by histological analysis and inflammatory marker measurements. An additional <i>in vivo </i>study is presented which highlights challenges in the delivery of the liquid PLS suspension to the lungs. The <i>in vivo </i>studies ultimately provide solid motivation for continued research into the development of the PLS therapy.</p><p dir="ltr">Given the promising potential of the PLS technology shown in the <i>in vivo</i> studies, the materials characterizations shared in this presentation offer valuable insights into the design of a novel PLS therapy. From these insights, key design parameters such as nanoparticle size characteristics, core chemistry, and core molecular weight can be chosen to produce the most desirable material properties. Overall, this dissertation furthers the progress of PLS therapeutic development and will hopefully ultimately contribute to improved health outcomes in patients suffering from ARDS.</p>

Coronavirus eller SARS-CoV-2 (severe acute respiratory syndrom coronavirus 2) som fick utbrott i december 2019 har väckt en stor uppmärksamhet och påverkat mänskligheten världen runt och inte minst påverkat den socioekonomiska balansen. Viruset som har ursprung i staden Wuhan i Kina sprider sig snabbt mellan människorna genom frekvent rekombination av det genetiska materialet. Redan efter ett år från utbrottet beräknades antal fall till 98 miljoner och dödsfallen till 2 miljoner globalt. Förutom lunginflammation leder infektionen till högre halter av proinflammatoriska markörer som CRP och höga nivåer av cytokiner som IL-6 som i slutändan kan resultera i en cytokinstorm. Därför anses en blockering av IL6 produktionen och/eller blockering av receptorbindningen vara en terapeutisk lösning för att begränsa patogenicitet. Tocilizumab som är den första immunmodulator som introducerades och testades mot covid-19 pneumoni har i flera studier gett upphov till heterogent resultat angående dess effekt på sjukdomen. Syftet med detta litteraturabete är att studera tocilizumabs effekt på covid-19 sjuka patienter med pneumoni och med måttlig till svår sjukdom. Genom sökning på databasen PubMed hittades sex randomiserade kliniska studier som valdes för att undersökas i detta arbete. Studierna jämförde effekten av standardbehandling med och utan tocilizumab. Resultatet blev heterogent men en signifikant förbättring observerades för tocilizumab vad gäller överlevnad, sjukdomsprogression och hälsostatus. Trots att tocilizumabs fördelar överväger dess nackdelar krävs det dock mer forskning kring läkemedlet för att kunna dra bättre samband mellan tocilizumab och dess effekter.

Dans mon projet de doctorat, j’ai étudié des fonctions primordiales de l’épithélium respiratoire telles que la régulation du transport ionique, la clairance liquidienne et la réparation épithéliale. J’ai particulièrement mis l’emphase sur le rôle des canaux potassiques qui interviennent dans ces trois fonctions de l’épithélium respiratoire. J’ai tout d’abord prouvé que la modulation des canaux potassiques régulait l’activité du promoteur de αENaC, en partie via la voie de signalisation ERK1/2, dans des cellules alvéolaires. Cette régulation entraîne une variation de l’expression génique et protéique du canal ENaC. Physiologiquement, il en résulte une augmentation du phénomène de clairance liquidienne suite à l’activation des canaux K+, tandis que l’inhibition de ces canaux la diminue sévèrement. J’ai aussi pu démontrer que l’absence de canal KvLQT1 entraînait une diminution du courant (ENaC) sensible à l’amiloride, dans les cellules de trachée en culture primaire, isolées de souris KO pour kcnq1. Dans la seconde partie de mon étude, j’ai évalué l’impact de l’hyperglycémie sur la capacité de transport ionique et de réparation de cellules épithéliales bronchiques saines ou Fibrose Kystique. Mes résultats montrent que l’hyperglycémie diminue le transport transépithélial de chlore et le transport basolatéral de potassium. Des études préalables du laboratoire ayant montré que les canaux K+ et Cl- contrôlent les processus de réparation, j’ai donc évalué si ceux-ci étaient modifiés par l’hyperglycémie. Et en effet, l’hyperglycémie ralentit la vitesse de réparation des cellules issues des voies aériennes (CFBE-wt et CFBE-ΔF508). J’ai donc démontré que le transport de potassium intervenait dans des fonctions clés de l’épithélium respiratoire, comme dans la régulation génique de canaux ioniques, le contrôle de la clairance liquidienne alvéolaire, et que l’hyperglycémie diminuait le transport ionique (K+ et Cl-) et la réparation épithéliale. / During my Ph.D. training, I studied 3 important functions of respiratory epithelium : regulation of ion transport, liquid clearance and epithelial repair. I focused on potassium channels, because they control these three respiratory epithelial functions. First, I proved that αENaC promoter activity was regulated following K+ channel modulation, in alveolar cells. This regulation of αENaC promoter which might be through a modification of ERK1/2 phosphorylation, was followed by ENaC mRNA and protein expression regulation. I then showed that activation of KvLQT1 and KATP channels increased alveolar liquid clearance, whereas inhibition of these K+ channels decreased the alveolar clearance. I showed that the absence of KvLQT1 channel inhibited the amiloride-sensitive current (ENaC), in tracheal epithelial cells isolated from KvLQT1-KO mice. In the second part of my Ph.D. project, I studied the impact of hyperglycemia on Cystic Fibrosis (CF) and non-CF epithelial cells. I first observed that K+ and Cl- currents were reduced by hyperglycemia. Because we have previously shown that wound-healing process was dependant on K+ and Cl- channels, I then evaluated the impact of hyperglycemia on wound-healing. As expected, hyperglycemia slowed the repair rate of non-CF (CFBE-wt) and CF (CFBE-ΔF508) cell monolayers.