Final publication is avilable at http://www.sciencedirect.com/science/article/pii/S0954611115300172 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19577号 / 医博第4084号 / 新制||医||1013(附属図書館) / 32613 / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊達 洋至, 教授 平家 俊男, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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An estimated 45% of all deaths can be attributed to various chronic fibroproliferative diseases. Idiopathic pulmonary fibrosis (IPF) is the most common form of interstitial lung disease which is characterized by progressive decline in lung function. While the pathogenesis of IPF is not fully understood, alternatively activated macrophages (M2) have been implicated as a key contributor to the fibrotic process. The plasticity of macrophages in vivo challenges the ability to specifically target the M2 macrophage phenotype across species. Previous bioinformatic analysis from our lab identified Dectin-1/Clec7a as a unique marker of M2 macrophages in both human and murine model systems. The expression of the transmembrane receptor Dectin-1 has not been elucidated in the context of pulmonary fibrosis. To prevent the progression of fibrosis by targeting alternatively activated macrophages, we investigated the expression of Dectin-1 in IPF and an experimental model of fibrotic lung disease. Our data demonstrated that while protein expression of Dectin-1 was increased in archived lung tissues of patients with IPF, mRNA expression of this receptor was downregulated in the tissues of these IPF patients. Gene expression of Dectin-1 was shown to be increased in monocyte-derived macrophages, further suggesting a circulatory component contributing to lung fibrosis. As expected, we confirmed that Dectin-1 was highly expressed past the injury phase of the bleomycin-model of induced pulmonary fibrosis which aligns with the increased immune infiltrates at this time point. Preliminary work into the time dependency of the resolution phase of the bleomycin-induced model of lung fibrosis was shown. All in all, our data suggests that Dectin-1 may be a useful marker in characterizing and differentiating phenotypes of macrophages implicated in the fibrotic process. Future efforts aim to gain insight into the functional requirement of Dectin-1 in the alternative activation of profibrotic macrophages to identify novel therapeutic targets for fibrotic lung disease. / Thesis / Master of Science (MSc)

La Radiothérapie occupe la deuxième place dans la liste de traitement du cancer le plus important après chirurgie. Le progrès technique récent, comme la radiothérapie avec modulation d'intensité (IMRT) ou la radiothérapie guidée par l'image (IGRT), en combinaison avec de nouveaux médicaments à action spécifique tels que les anticorps monoclonaux, sont une garantie d'augmentation de l'index thérapeutique. Cependant, la radiothérapie peut provoquer un’ altération du processus normal de réparation et d'induire le développement d'un cadre de fibrose dans un sous-ensemble de patients sensibles et dans les survivants à long terme au cancer. La principale caractéristique de la fibrose radio-induit est l'accumulation excessive et anormale de collagène composé principalement des éléments fibrillaire et immatures de la matrice extracellulaire (ECM).Les organes qui peuvent être touchés par ce phénomène sont le foie, la peau, les intestins, les reins et les poumons. D'un point de vue clinique, la fibrose peut être considérée comme une condition irréversible, sans solution. Nous et d'autres ont récemment montré que, outre l'activation de la TGF-β/Smad canonique, d'autres voies sont activées dans les tissus fibreux tels que la cascade de signalisation intracellulaire Rho/ROCK. Fait intéressant, la façon dont Rho/ROCK semble spécifiquement activé dans la fibrose intestinale radio-induite, fournis une justification pour un stratégie anti-fibrotique ciblé. L’ inhibition pharmacologique de Rho avec les statines, en fait, est en mesure de prévenir et même inverser les phénomènes de fibrose post-actinique intestinale.Avec ces prémisses, dans nos études, nous avons montré le rôle des statines (Simvastatine et Pravastatine) et d'un inhibiteur spécifique de ROCK (Y-27632) dans un modèle murin de fibrose pulmonaire obtenue avec une approche pharmacologique (Bléomycine - BLM) . Par la suite, nous avons développé un modèle de fibrose pulmonaire induite par l'irradiation complet du thorax et évalué la réponse à l'administration de la Pravastatine. La confirmation de la participation de la voie Rho/ROCK/CTGF dans la fibrose pulmonaire a été montré par immunohistochimie: le traitement à la Pravastatine normalise l'expression de trois marqueurs: RhoB, TGFβ-RII et CTGF.Après, dans deux modèles de fibrose radio-induite (intestinal et pulmonaire), nous avons analysé, grâce à l’immunohistochimie, les mécanismes sous-jacents l'action antifibrotique de la Pravastatine via l’axe MMP2-TIMP2. Très intéressant, quand la pravastatine a été administré à titre préventif ou curatif, nous avons trouvé un impact différent sur la fibrolyse.Enfin, in vitro, nous avons étudié par zymographie l'expression des gélatinases (MMP2 et MMP9) dans des cultures primaires des fibroblastes pulmonaire murins exposées à différentes doses de rayonnement et de Pravastatine. Le métalloprotéases semble être à son tour impliquée dans les mécanismes pro-fibrolytiques induits par les statines.Dans notre modèle animal de fibrose pulmonaire, la Pravastatine est capable d'inverser le processus fibrotique et les métalloprotéases semblent être impliqués à leur tour, in vivo et in vitro, dans les mécanismes pro-fibrolyse induits par le médicament.La multiplicité des acteurs impliqués dans la physiopathologie de lésions fibrotiques explique pourquoi la mise en place d'une stratégie thérapeutique efficace est si complexe. La recherche dans les processus mécaniques de dommages aux tissus normaux ont ouvert la voie à de nouvelles approches thérapeutiques. Ces nouvelles cibles comprennent la réduction de l’inflammation, de l'activation vasculaire et de la thrombose, ainsi que la découverte de nouvelles cibles moléculaires. Il existe une variété de modèles précliniques et des stratégies efficaces, mais de nombreux efforts doivent être déployés pour atteindre l'objectif difficile de protéger les tissus normaux des effets secondaires de la radiothérapie. / Radiotherapy is the second most important treatment modality after surgery in the treatment of cancer. Recent technical advancements, such as intensity-modulated radiation therapy (IMRT) or image-guided radiation therapy (IGRT), combined with new targeted drugs have significant promise for therapeutic outcome. However radiation treatment could result in disabling normal tissue injury and in the development of progressive fibrosis in a subset of sensitive patients and in long-term cancer survivors. The main feature of tissue fibrosis is excessive accumulation of abnormal and cross-linked collagen mainly composed of fibrillar and immature extracellular matrix (ECM) components.The organs that can be affected by this phenomenon are liver, skin, intestine, kidneys and lungs. From a clinical point of view, fibrosis can be seen as an irreversible condition, without solution. We and others recently showed that beside the activation of the canonical TGF-β/Smad pathway, other intracellular signaling cascades including the Rho/ROCK pathway are switched on in fibrotic tissues. Interestingly, the Rho/ROCK pathway seems differentially activated in radiation-induced intestinal fibrosis, thereby providing a rationale for a specific, targeted anti-fibrotic strategy. Pharmacological inhibition of Rho using statins indeed prevent and even reverse intestinal radiation fibrosis.In our studies, we showed the role of Statin (Pravastatin e Simvastatin) and a specific inhibitor ROCK inhibitors (Y-27632) in a mice model of pulmonary induced-fibrosis obtained by a pharmacological approach (Bleomycin – BLM). Indeed, we developed a model of lung fibrosis by complete irradiation of chest and tested Pravastatin action. Confirmation of the involvement of Rho/ROCK/CTGF pathway in lung fibrosis are shown by immunohistochemistry: Pravastatin-treament normalized the expression of three markers: RhoB, TGF-RII and CTGF.Then, in models of radiation induced gut and lung fibrosis, we analysed, from a immunohistological point of view, the underlying mechanisms of the antifibrotic action of Pravastatin via MMP2-TIMP2 axis. Interestingly we found a different impact on fibrolysis when Pravastatin was administered preventively or curatively.Finally, in vitro, we investigate by zymography the expression of Gelatinases (MMP2 and MMP9) in primary lung fibroblasts cultures exposure at the different radiation and Pravastatin doses. Metalloproteases would appear to be in turn involved in pro-fibrolytic mechanisms induced by statin.The multiplicity of actors involved in the pathogenesis of fibrotic lesions explains why the definition of an effective therapeutic strategy is so complex.Researches in mechanistic processes of normal tissue damage paved the way for new therapeutic approaches. These new targets include reduction of vascular activation, inflammation and thrombosis and new molecular targets definition. Effective strategies are multiple on preclinical models, but numerous efforts have to be made to achieve the complicated goal of protection of normal tissues from the side effects of radiation therapy. / La radioterapia è la seconda modalità di trattamento più importante dopo chirurgia neltrattamento delle neoplasie. I recenti progressi tecnici, come la terapia ad intensità modulata(IMRT) o l’image-guided radioterapia (IGRT), in combinazione con nuovi farmaci ad azionemirata come gli anticorpi monoclonali, costituiscono ulteriore garanzia di incrementodell’indice terapeutico. Tuttavia il trattamento radiante può causare un’alterazione delnormale processo di riparazione e indurre lo sviluppo di un quadro di fibrosi in unsottogruppo di pazienti sensibili e nei lungo-sopravviventi al cancro. La caratteristicacardinale della fibrosi radioindotta è l’eccessivo ed anomalo accumulo di collagene compostoprincipalmente di componenti fibrillari e immature della matrice extracellulare (ECM).Gli organi che possono essere interessati da questo fenomeno sono fegato, pelle,intestino, reni e polmoni. Da un punto di vista clinico, la fibrosi può essere vista come unacondizione irreversibile, senza soluzione. Noi ed altri recentemente abbiamo mostrato cheaccanto alla attivazione della via canonica TGF-β/Smad, altre vie vengono attivate nei tessutifibrotici come la cascata di segnalazione intracellulare della via Rho/ROCK. Interessantenotare che la via Rho/ROCK sembra specificatamente attivata nella radiazione indotta fibrosiintestinale, fornendo così una spiegazione razionale per una specifica, mirata strategia antifibrotica.L'inibizione farmacologica di Rho con le statine infatti è in grado di prevenire eaddirittura invertire i fenomeni di fibrosi intestinale post-attinica.Grazie a queste premesse, nei nostri studi, abbiamo mostrato il ruolo delle statine(Pravastatina e Simvastatina) e di uno specifico inibitore di ROCK (Y-27632) in un modellomurino di fibrosi polmonare indotta ottenuto con un approccio farmacologico (bleomicina -BLM). In seguito, abbiamo sviluppato un modello di fibrosi polmonare indottadall’irradiazione completa del torace e valutata la risposta alla somministrazione dellaPravastatina. In questo modello ed in un modello di fibrosi intestinale indotto da radiazioni,abbiamo analizzato, da un punto di vista immunoistologico, i meccanismi sottostanti l'azione9antifibrotica della pravastatina e il ruolo delle metalloproteasi (MMP2 e TIMP2). Infine, invitro, abbiamo indagato, mediante zimografia, l'espressione delle gelatinasi (MMP2 e MMP9)in culture primarie di fibroblasti polmonari murini esposti a differenti dosi di radiazione epravastatina.Nel nostro modello animale di fibrosi polmonare, la Pravastatina è in grado di renderereversibile il processo fibrotico e le metalloproteasi parrebbero essere a loro volta coinvolte,in vivo and in vitro, nei meccanismi pro-fibrolitici indotti dal farmaco.La molteplicità di attori coinvolti nella patogenesi delle lesioni fibrotiche spiegaperché la definizione di una strategia terapeutica efficace è così complessa. Ricerche neiprocessi meccanicistici di danno ai tessuti normali hanno aperto la strada a nuovi approcciterapeutici. Questi nuovi obiettivi comprendono la riduzione dell’ attivazione vascolare,dell'infiammazione e della trombosi, oltre alla definizione di nuovi target molecolari. Esistonomolteplici ed efficaci strategie su modelli preclinici, ma numerosi sforzi devono essere fattiper raggiungere il complicato obiettivo di proteggere i tessuti normali dagli effetti collateralidella radioterapia.

Radiotherapy is the second most important treatment modality after surgery in the treatment of cancer. Recent technical advancements, such as intensity-modulated radiation therapy (IMRT) or image-guided radiation therapy (IGRT), combined with new targeted drugs have significant promise for therapeutic outcome. However radiation treatment could result in disabling normal tissue injury and in the development of progressive fibrosis in a subset of sensitive patients and in long-term cancer survivors. The main feature of tissue fibrosis is excessive accumulation of abnormal and cross-linked collagen mainly composed of fibrillar and immature extracellular matrix (ECM) components.The organs that can be affected by this phenomenon are liver, skin, intestine, kidneys and lungs. From a clinical point of view, fibrosis can be seen as an irreversible condition, without solution. We and others recently showed that beside the activation of the canonical TGF-β/Smad pathway, other intracellular signaling cascades including the Rho/ROCK pathway are switched on in fibrotic tissues. Interestingly, the Rho/ROCK pathway seems differentially activated in radiation-induced intestinal fibrosis, thereby providing a rationale for a specific, targeted anti-fibrotic strategy. Pharmacological inhibition of Rho using statins indeed prevent and even reverse intestinal radiation fibrosis.In our studies, we showed the role of Statin (Pravastatin e Simvastatin) and a specific inhibitor ROCK inhibitors (Y-27632) in a mice model of pulmonary induced-fibrosis obtained by a pharmacological approach (Bleomycin - BLM). Indeed, we developed a model of lung fibrosis by complete irradiation of chest and tested Pravastatin action. Confirmation of the involvement of Rho/ROCK/CTGF pathway in lung fibrosis are shown by immunohistochemistry: Pravastatin-treament normalized the expression of three markers: RhoB, TGF-RII and CTGF.Then, in models of radiation induced gut and lung fibrosis, we analysed, from a immunohistological point of view, the underlying mechanisms of the antifibrotic action of Pravastatin via MMP2-TIMP2 axis. Interestingly we found a different impact on fibrolysis when Pravastatin was administered preventively or curatively.Finally, in vitro, we investigate by zymography the expression of Gelatinases (MMP2 and MMP9) in primary lung fibroblasts cultures exposure at the different radiation and Pravastatin doses. Metalloproteases would appear to be in turn involved in pro-fibrolytic mechanisms induced by statin.The multiplicity of actors involved in the pathogenesis of fibrotic lesions explains why the definition of an effective therapeutic strategy is so complex.Researches in mechanistic processes of normal tissue damage paved the way for new therapeutic approaches. These new targets include reduction of vascular activation, inflammation and thrombosis and new molecular targets definition. Effective strategies are multiple on preclinical models, but numerous efforts have to be made to achieve the complicated goal of protection of normal tissues from the side effects of radiation therapy.

BACKGROUND: Cystic fibrosis (CF) patients grow poorly and tend to be malnourished. They frequently suffer from lung infections necessitating the repeated use of antibiotics. AIM: This study was conducted to determine whether supplementation with a probiotic Lactobacillus reuteri (L. reuteri) could reduce the incidence and duration of lung infections, and whether this would impact on their anthropometric data. The secondary purpose was to compare the nutritional status of the CF patients attending CF clinics in Kwazulu-Natal (KZN) with CF patients attending CF clinics in Cape Town (CT). METHODS: Twenty three CF patients 6-31 years of age from 2 CF clinics in Kwazulu-Natal started the study although only 16 patients completed it. The study was a randomized, double blind, placebo controlled crossover trial with six months on placebo and six months on probiotic. Weight, height, mid arm circumference (MAC), triceps skin fold thickness (TSF), forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) were measured, sputum collected and a symptom diary completed over the 12 month period. Anthropometric data of CF patients attending CF clinics in CT was obtained from the publication by Westwood & Saitowitz (1999). RESULTS: Compliance with taking the L. reuteri was poor. Most took only 50% of the required daily dose. Probiotic supplementation showed a slight (non significant) trend to improve FEV1 and FVC, while no significant difference could be seen in the number and duration of the lung infections. Sputum analysis showed a non significant trend towards the probiotic reducing the number of bacteria in the sputum. There was a significant reduction of symptoms for fever, running nose, sore throat and ear ache while on placebo. There was a significant increase in weight gained off probiotic compared to the probiotic period. The changes in height, weight for age (WFA) percentiles, height for age (HFA) percentiles, WFA and HFA Z-scores, percentage expected weight for age and percentage expected height for age all showed no difference whether on or off probiotic. Over half the CF children in the KZN clinics were underweight for their actual height compared to one third in the CT clinics with a higher number of subjects below the 5th percentile for MAC and TSF readings compared to CT. CONCLUSION: Due to a small sample size and poor compliance no firm conclusions could be drawn. However a slight (non significant) improvement could be seen in favour of the probiotic for FEV1, FVC, and sputum analysis. Although all other findings were not significantly different it would be of benefit to carry out further investigation with improved compliance with the probiotic to see if the parameters set out above could be improved. The KZN and CT CF groups were comparable and the nutritional status of CF patients on KZN was well below that of the CT CF clinics and further monitoring would need to be carried out.

Cystic Fibrosis (CF) is a common life-shortening autosomal recessive disease that affects over 100.000 people worldwide people worldwide. It is caused by mutations in the CF trans-membrane conductance regulator (CFTR) gene, that encodes for a membrane channel localized at the apical surface of epithelial cells where it has a crucial role in the secretion of chloride and bicarbonate. Over 2100 different CFTR mutations have been reported and among the pathogenic once the most common is F508del, located in the nucleotide-binding domain 1 (NBD1). F508del is a three-nucleotide deletion that results in the loss of a phenylalanine at position 508 in the protein and in the consequent CFTR degradation by the ubiquitin-proteasome system. Different attempts to correct F508del-CFTR gene were made using genome editing approaches, however deletions like F508del remain difficult to be repaired. Several studies reported that additional mutations (revertant mutations) in the F508del-CFTR gene can rescue both CFTR folding and activity, suggesting a potential novel strategy to correct F508del. For this reason, the first aim of this work was the identification of novel F508del-CFTR revertants that can rescue CFTR localization and function. We generated a library of mutants introducing random substitutions into the F508del-CFTR gene. Revertant mutations were isolated based on their ability to rescue the presence of CFTR at the plasma membrane (PM) in HEK293T cells and identified by Sanger sequencing. Restoration of CFTR maturation, localization, and function of the identified revertants was evaluated by western blot, flow cytometry analysis and YFP assay, reaching levels similar to the wild type CFTR. Then we used CRISPR-Cas technology to introduce selected revertant mutations, such as I539T, R553Q, G550E, R555K and R1070W, in the endogenous F508del-CFTR gene. Adenine and cytosine base editors (ABE and CBE) allow the insertion of the desired base conversion without the formation of double strand breaks. Efficient editing was evaluated through Sanger sequencing, reaching up to 60% of base conversion. CFTR rescue at the PM in edited cells was analyzed by flow cytometry showing different degrees of recovery compared to the wild type CFTR. In this work, we confirmed that revertant mutations can rescue F508del CFTR localization and function. In addition, we demonstrated that CRISPR-base editors are valid tools to introduce these mutations in the endogenous F508del-CFTR gene, leading to a permanent correction. The proposed strategy could overcome the limits that genome editing strategies faced till now in the correction of F508del, providing a new potential therapeutic approach to treat CF.

Master of Science / Biochemistry and Molecular Biophysics / John M. Tomich / NC-1007 (CK₄-M2GlyR) (PARVGLGITTVLTMTTQSSGSRAKKKK) is a synthetic peptide modeled after the second transmembrane segment of the spinal cord glycine receptor’s α-subunit, and has demonstrates the capacity to oligomerize to form transmembrane channels with Cl[superscript]- permselectivity. While studies into the effects of truncation on both CK[subcript]4 (C-terminal tetra-lysl adducted) and NK[subscript]4 (N-terminal tetra-lysl adducted) led to more control over solution aggregation in the NK[subscript]4 variant, the work presented explore whether C-terminal sequential substitutions with a tryptophan residue could similarly stabilize the aqueous structure in monomeric form or further define the pore registry in such a way as to promote an increase ion permeability. Tryptophan was substituted for amino acids in the 18[superscript]th, 19[superscript]th, 20[superscript]th, and 21[superscript]st positions of the peptide sequence (SSGS, respectively), and changes in aggregation profiles, secondary structure, and channel ion permeability were observed. Synthesized peptides show circular dichroism spectral profiles indicating that the studied tryptophan substitutions did not result in a reduction of the characteristic helicity of the peptide; however, the tryptophan substitution also did little to decrease solution aggregation as demonstrated by comparative studies by reverse-phase high- performance liquid chromatography. All peptides demonstrated channel activity, directly measured by recordings of transepithelial short-circuit current. with profiles that suggest trends in electrostatic interactions and membrane registry relative to substitution position. One peptide in particular, NC-1007 S21W displayed atypical activity, which could not be effectively described by the standard Hill-based model but may be indicative of an ill-defined registry due to the substituted peptide’s proximity to another strongly pore-defining residue. Further studies in the effects of sequence modification to channel-forming peptides will elucidate how sequences may be altered to optimize synthetic peptide solubility, resistance to in-solution aggregation, and ability to form selective and permeable ion channels. The understanding gained from this study will improve our ability to develop peptides that could serve as a therapeutic treatments for a number of endogenous channelopathies.