The difference between the number of patients needing transplantation for chronic liver disease and the number of organ donors is growing, drawing attention to the urgent requirement for novel therapies. Chronic liver injury is commonly caused by viral hepatitis, alcohol consumption, obesity and metabolic disorders. Prolonged liver injury leads to fibrosis, hepatic scarring and eventually cirrhosis. This project is based on previous studies demonstrating the therapeutic effects of bone marrow-derived macrophages (BMDM) in a murine model of liver fibrosis. BMDM facilitated fibrosis regression and improved liver regeneration. Pro-resolution macrophages exhibited increased expression of MMPs, growth factors and phagocytosis-related genes. However, macrophages derived from bone marrow are inherently heterogeneous and difficult to genetically manipulate. To overcome this limitation, our laboratory has established a protocol whereby pure populations of macrophages can be produced in significant numbers from murine embryonic stem cells (ESC) in vitro, providing an essentially limitless source of macrophages. The first goal of this project was to compare macrophages derived from ESCs (ESDM) with classical BMDM. ESDM displayed characteristic macrophage morphology, could be activated and responded to different cytokines in vitro, and were functionally phagocytic. However, they displayed some differences in their gene expression profile, and were found to be less phagocytic than BMDM. We then assessed whether ESDM could be used in the treatment of a murine model of hepatic injury induced by carbon tetrachloride administration. ESDM therapy helped in the regression of liver fibrosis, down-regulated the number of fibrogenic myofibroblasts, and activated liver progenitor cells. However, a higher number of ESDM compared to BMDMs were required to exert that effect. To assess whether ESDM may be similar to yolk sac derived tissue-resident macrophages, rather than monocyte-derived, we compared their behaviour in a Kupffer cell repopulation assay. Macrophages were depleted using liposomal clodronate treatment then animals were transplanted with either ESDM or BMDM. We demonstrated that ESDM were more efficient than BMDM at repopulating the Kupffer cell compartment and reversing the effects of liposomal clodronate treatment in mice. It is well known that macrophages are very difficult to genetically modify. So our strategy was to genetically modify ESC and then differentiate them to macrophages that carry the modification. By genetically modifying ESCs, we attempted to produce pro-fibrolytic ESDM that over-express MMP12 which is a member of the matrix metalloproteinase family of genes that mainly degrades elastin, an extracellular matrix component. We initially employed a Tet-On 3G expression system to create an ESC line where MMP12 could be expressed in an inducible manner in differentiated macrophages. However, although this inducible strategy functioned in undifferentiated ESCs we could not induce the expression of MMP12 in differentiated macrophages. In an attempt to overcome possible gene-silencing issues, we designed and constructed an expression strategy such that Mmp12 was expressed specifically in macrophages. The ESC line was built such that Mmp12 expression would be driven by the promoter of macrophage colony stimulating factor-1 receptor gene (Csf-1r or c-fms). Using the CRISPR/Cas9 strategy, we successfully targeted the Mmp12 cDNA to the Csf-1r locus but ESDM that were differentiated from targeted ESC lines did not express Mmp12. Thus, despite having adopted two independent strategies, we have failed to generate genetically modified macrophages. As a first step to translate the therapeutic effects of macrophages into the clinical setting, we optimized a feeder- and serum-free protocol to efficiently generate macrophages from human induced pluripotent stem cells.

Près de la moitié des patients atteints de sclérodermie systémique (ScS) présentent des troubles de la pigmentation cutanée. La mélanogenèse est sous la dépendance, outre les facteurs épidermiques, de facteurs dermiques produits par les fibroblastes et les cellules endothéliales. Parmi ces facteurs se trouve NOV (CCN3) (protéine appartenant à la même famille que CTGF (CCN2), protéine pro-fibrotique augmentée dans la ScS) qui aurait un rôle anti-fibrotique. Nous souhaitons ainsi disséquer le lien entre la composante fibrotique dermique et la pigmentation épidermique. Pour cela, des biopsies cutanées ont été réalisées chez 21 patients ScS, en zone lésionnelle (scléreuse avec ou sans trouble de pigmentation) et non lésionnelle pour 12 d’entre eux. Ces biopsies ont été séparées en 3 fragments qui ont été soit fixés pour analyses histologiques et immunohistochimiques, soit congelés à -80°C pour analyse protéomique ou transcriptomique, soit dissociés pour isoler les cellules cutanées afin de les analyser en protéomique ou transcriptomique. Des témoins de peau et des cellules saines sont utilisées en contrôle. Nous avons dégagé deux types de troubles pigmentaires : une hyperpigmentation s’apparentant à un photo-vieillissement, et une dépigmentation péri-folliculaire apparaissant de manière précoce. Les niveaux d’expression de CCN2 et CCN3 varient chez les patients en fonction des données cliniques, aussi bien au niveau protéique qu’au niveau immunohistochimique. S’il existe des différences en fonction de l’atteinte pigmentaire, il semble que dans les fibroblastes sclérodermiques, la balance CCN2/CCN3 soit déséquilibrée tant au niveau de la zone lésionnelle que cliniquement non lésionnelle, suggérant que CCN2 et 3 pourraient être dérégulés intrinsèquement ou à des stades précoces. Ce travail pourrait aboutir à l’identification d’un phénotype précoce basé sur l’atteinte pigmentaire, et au développement d’une thérapie basée sur la rééquilibration du ratio CCN2/CCN3. / Systemic Sclerosis (SSc) is a rare but potentially deadly connective tissue disease. Its pathophysiology remains partly unknown but combines auto-immunity, small and large vessels involvement and fibrosis of the connective tissue, affecting all organs. Skin features are considered as diagnostic and prognosis markers and include for some patients the presence of pigmentary disorders. In this work, we looked into pigmentary disorders in SSc and their relationship with the pathophysiology of the disease. First, we analyzed the presence of pigmentary disorders among a local cohort of 239 patients as well as their association with systemic involvement in the disease. We have found that diffuse hyperpigmentation was associated with an increased risk of vascular involvement in SSc, particularly digital ulcers. Then, we investigated the molecular basis behind this association. Proteins of the CCN (CYR61 / CTGF / NOV) family are multimodular proteins secreted in the extra-cellular matrix, where they take part in numerous biological processes, such as cell proliferation, adhesion, collagen secretion. Within this family, CCN3 (also called NOV) is a promising candidate, being implicated both in angiogenesis and epidermal homeostasis. We studied CCN3 expression in the skin of SSc patients presenting or not pigmentary disorders, as compared to healthy controls. We found that CCN3 expression was particularly decreased in the dermal vessels in situ, as well as in endothelial cells in vitro. CCN3 inhibition in endothelial cells resulted in altered angiogenesis in vitro, through a decrease in cell migration. We also studied CCN3 expression in SSc epidermis. SSc patients presenting hyperpigmentation exhibited decreased CCN3 in their melanocytes as well as increased CCN3 in their keratinocytes, compared to patients without pigmentary disorders. Overall, CCN3 represents a promising therapeutic lead for SSc patients with vascular involvement, which could bespotted early thanks to the presence of hyperpigmentation.

Orientador: Márjorie de Assis Golim / Resumo: O vírus da hepatite C (VHC) é responsável por causar hepatite C nas formas aguda ou crônica, sendo a fibrose hepática uma possível consequência da evolução da lesão. Na avaliação da fibrose o método considerado padrão ouro é a biópsia hepática. Com a necessidade de se desenvolver metodologias alternativas à biópsia hepática, escores utilizando biomarcadores séricos têm sido validados, de modo que possam servir para o acompanhamento de indivíduos infectados pelo vírus da hepatite C. Realizou-se estudo retrospectivo, incluindo 94 pacientes portadores crônicos do VHC, genótipo 1, pré-tratamento (naive) ou retratados. Os pacientes foram separados em grupos conforme os resultados da classificação METAVIR dos graus de fibrose (F0 à F4), seja por biópsia hepática ou por elastografia hepática por quantificação de ponto. Além disso, os pacientes foram classificados em grupos de fibrose leve G1 (F1-F2) e fibrose avançada G2 (F3-F4). Metodologias não-invasivas como FIB-4 e APRI foram comparadas ao método denominado FibMaster desenvolvido neste estudo através de análises multivariadas e aprendizado de máquina para elaborar um modelo preditivo de fibrose hepática baseado em variáveis sanguíneas possivelmente associadas ao dano hepático. Os parâmetros estatisticamente mais significantes foram alfa-fetoproteína (AFP), apresentando AUROC de 0.890 para a classificação de fibrose F3-F4 e 0.772 para classificação dos grupos G1-G2, ureia com AUROC de 0.723 para fibrose F2-F3, FIB-4 (AUROC de 0.8... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Hepatitis C virus (HCV) is responsible for causing both acute hepatitis C and chronic form, where the last is commonly associated with liver fibrosis. The evaluation of fibrosis is currently performed mainly through liver biopsy, which is the methodology considered as gold standard in the classification of fibrosis stages. With the necessity of developing new alternative methodologies to hepatic biopsy, scores using serum biomarkers have been validated, thus they can be used to monitor infected individuals with the hepatitis C virus. A retrospective study was carried out including 94 chronic HCV, genotype 1, pre-treatment (naive) or retreated patients. Patients were separated into groups according to the METAVIR classification of degrees of fibrosis (F0 to F4), either by hepatic biopsy or point shear wave-elastography exam. Moreover, patients were classified into groups of mild fibrosis G1 (F1-F2) and advanced fibrosis G2 (F3- F4). Non-invasive methodologies such as FIB-4 and APRI were compared to the method named FibMaster, which was proposed in this study through multivariate analyzes and machine learning, in order to elaborate a predictive model for hepatic fibrosis based on blood biomarkers possibly associated with hepatic injury. The most statistically significant parameters were alpha-fetoprotein (AFP), presenting AUROC of 0.890 for fibrosis classification F3-F4 and 0.772 for the classification of groups G1- G2, urea with an AUROC of 0.723 for fibrosis F2-F3, FIB-4 (AUR... (Complete abstract click electronic access below) / Mestre

Idiopathic pulmonary fibrosis (IPF) is an irreversible disease of unknown etiology that involves progressive scarring of the lung tissue, leading to respiratory failure and death.1 IPF is thought to develop from repetitive lung injury and aberrant wound healing that leads to the generation of fibrous tissue rather than restoration of normal tissue.2 It has been suggested in mice that vascular leak after lung injury contributes to the development of lung fibrosis.2,3 Gadofosveset is an intravascular enhancing, gadolinium-based contrast agent used with magnetic resonance imaging (MRI) to assess a variety of biological processes in vivo because it can reversibly bind to albumin.13-14 Gadofosveset has been used to assess endothelial permeability and function, as it diffuses through the vessel walls via leaky neovessels and damaged endothelium.15 Our research group has developed a new method to assess disease activity in IPF patients using gadofosveset-enhanced lung MRI. In unpublished work, we have demonstrated that this technique can be used to generate an albumin extravasation index (AEI), and we have found that this is significantly and diffusely increased in the lung of patients with idiopathic pulmonary fibrosis compared to healthy controls.16 The AEI is a measure of the change in signal intensity post-contrast minus pre-contrast in predefined regions of interest (ROIs) in the lung parenchyma divided by post- minus pre-contrast signal intensity in the ROI in the aorta. In this study, we compared the AEI in patients with IPF to healthy control (HC) subjects and evaluated the correlation between the AEI and high-resolution computed tomography (HRCT) and pulmonary function testing (PFT). We found that IPF subjects had increased AEI values compared with HC subjects. While not statistically significant, AEI was more strongly correlated with fibrosis (interstitial abnormalities) than ground-glass (alveolar abnormalities) on HRCT. Furthermore, there was a possible correlation between AEI and change in percent predicted forced expiratory volume in the first second (FEV1), forced vital capacity (FVC), and diffusion capacity of carbon monoxide adjusted for hemoglobin (DLCO) [Hb]. Our results demonstrate that AEI calculations from gadofosveset-enhanced lung MRI are a surrogate measure of vascular leak and can potentially serve as an alternative method for predicting the clinical course and severity of IPF through its correlation with fibrosis on HRCT and pulmonary function.

L'utilisation de virus de bactéries ou bactériophages pourrait être un complément efficace à l’antibiothérapie. Mon travail a porté sur la caractérisation de bactériophages dirigés contre l’espèce Pseudomonas aeruginosa, pathogène opportuniste responsable d'infections des voies respiratoires des patients atteints de mucoviscidose.J'ai tout d'abord déterminé la sensibilité des souches mucoviscidosiques au Pyophage (un cocktail de phages thérapeutiques Géorgien) et identifié six phages lytiques de quatre genres différents. Environ 15% des souches sont résistantes au Pyophage. Ensuite, en utilisant les souches cliniques multi-résistantes aux phages comme bactérie d’enrichissement, 32 phages ont été obtenus à partir des eaux usées de France et Côte d’Ivoire. Tous les phages analysés sont caudés et distribués au sein de dix genres parmi lesquels six exclusivement lytiques. J'ai identifié des souches bactériennes qui demeurent insensibles à tous les phages. J'ai montré que le système CRISPRs-Cas n'est pas associé à la résistance des souches aux phages lytiques. / The use of viruses of bacteria commonly called bacteriophages could constitute an efficient complement to antibiotics. During my PhD, I have characterized phages infecting the opportunistic pathogen Pseudomonas. aeruginosa, responsible for lung infections in cystic fribrosis patients. Firstly, I investigated the efficiency of Pyophage (a cocktail of phages therapeutic Georgian) on clinical P. aeruginosa strains and recovered six lytic phages from four different genus. The Pyophage appears to be unactive on approximately 15% of clinical strains. Secondly, and using multi-phages resistant strains as enrichment bacteria, 32 phages were isolated from waste water of France and Côte d’Ivoire. All phages are tailed and distributed within ten different genus including six exclusively lytic. I identified bacterial strains which remain insensitive to all phages. I also demonstrated that the CRISPRs-cas system plays no role in the resistance of strains to lytic phages.

Ataxia telangiectasia (AT), caused by mutations in the gene encoding ataxia telangiectasia mutated kinase (ATM), is a rare autosomal recessive disorder. AT individuals exhibit neuronal degeneration and are predisposed to cancer. Carriers of this disorder are predisposed to cancer and ischemic heart disease. Heart disease, mostly due to myocardial infarction (MI), is a leading cause of death in the US. Following MI, release of catecholamines in the heart stimulates β- adrenergic receptors (β-AR). Our lab has shown that β-AR stimulation increases ATM expression in the heart and myocytes, and ATM plays an important role in β-AR-stimulated myocardial remodeling with effects on function, fibrosis and apoptosis. Using wild-type (WT) and ATM heterozygous knockout (hKO) mice, this study investigated the role of ATM in the inflammatory, proliferative and maturation phases of infarct healing post-MI. During the inflammatory phase, 1 and 3 days post-MI, a deficiency of ATM resulted in decreased left ventricular dilation as measured by echocardiography. It decreased the number of neutrophils and macrophages in the heart 1 day post-MI. Myocardial fibrosis, expression of alpha-smooth muscle actin (α-sma) and apoptosis were higher in the infarct region of ATM deficient hearts. Akt activation (anti-apoptotic) was lower, while Bax expression (pro-apoptotic) was higher in the infarct region of ATM deficient hearts. During the proliferative phase, 7 days post-MI, ATM deficiency attenuated cardiac dysfunction as measured by echocardiography. ATM deficient hearts exhibited increased fibrosis and expression of α-sma in the infarct region with increased myocyte apoptosis in the border area. During the maturation phase, 14 and 28 days post-MI, ATM deficiency resulted in exaggerated cardiac function. It associated with increased fibrosis, expression of α-sma and decreased cardiac cell apoptosis in the infarct region 28 days post-MI. Myocyte hypertrophy was greater in the non-infarct region during ATM deficiency. ATM deficiency decreased expression of p16 (marker of cell senescence) and activation of proapoptotic protein, GSK-3β. Thus, ATM modulates the remodeling processes of the heart including function, fibrosis, apoptosis and hypertrophy post-MI. ATM (1) delays the inflammatory response post-MI, (2) decreases dilative remodeling during inflammatory and proliferative phases and (3) exaggerates dysfunction during the maturation phase.

The purpose of this review was to identify the history of and advances in cystic fibrosis (CF). New treatment plans, medication developments, and a historical perspective of airway clearance therapy (ACT) will be presented. The importance of treatment compliance and time management in the care of cystic fibrosis patients will also be discussed. Furthermore, the development of cystic fibrosis clinics and the pivotal role they play in the treatment of the disease will be addressed. Lastly, a brief discussion concerning the need for and process of lung transplantation will be reported.

L’apparition de fibrose pariétale dans la maladie de Crohn est responsable de sténoses et fistules, principales indications chirurgicales. Le but de notre travail était d’évaluer différentes séquences IRM pour différencier l’inflammation de la fibrose à partir d’un modèle original de colite radio-induite. Nous avons inclus un groupe « contrôle » de 10 rats, un groupe « inflammation » avec inflammation et fibrose sous-muqueuse de 24 rats et un groupe « mixte » avec inflammation et fibrose transmurale de 39 rats. Nous avons montré que ce modèle était reproductible et mis au point sur une IRM petit animal 7 T des séquences dont les résultats étaient homogènes, montrant la qualité à la fois des séquences et du modèle. Les signes IRM ont été comparés à l’analyse histologique des pièces (inflammation et fibrose). Les séquences utilisées (pondération T2 et T1, diffusion, transfert d’aimantation et perfusion sans injection) montraient des différences significatives entre les rats des groupes inflammation et mixte. Les séquences ayant la meilleure performance diagnostique pour différencier les deux groupes étaient l’intensité en pondération T2, la diffusion et le transfert d’aimantation. Les combinaisons de signes IRM ayant les meilleures AUC étaient celles comprenant la perfusion, à 0.95 pour la meilleure. Une analyse multivariée de la corrélation entre les signes IRM et les scores histologiques d’inflammation et de fibrose a montré de plus que la perfusion était le seul paramètre lié à la fibrose. Les perspectives sont maintenant de tester ces séquences pour évaluer des traitements anti-fibrosants en cours de développement et de les transposer à des patients pour mieux adapter la prise en charge thérapeutique. / Crohn's disease transmural bowel wall inflammation can lead to fibrosis causing luminal narrowing and stricture formation which are the main indications to surgery. The aim of our study was to evaluates MR sequences to distinguish inflammation from fibrosis using an original model of radiation-induced colitis. We included a “control” group of 10 rats, an “inflammation” group of 24 rats with inflammation and submucosal fibrosis and a “mixed” group of 39 rats with inflammation and transmural fibrosis. We showed that this model was very reproducible and developed sequences on a 7 T MR which results were homogeneous, showing both the quality of the sequences and of the model. MR data were compared with the histological analysis (inflammation and fibrosis) of the resection pieces. MR sequences (T2 and T1 weighted, diffusion weighted, magnetization transfer and FAIR perfusion) showed significant differences between irradiated and control rats and between inflammation and mixed groups. Sequences with the best AUC to differentiate the two groups were T2-weighted intensity, diffusion and magnetization transfer. The combinations of MR signs with the best AUC were those including perfusion, at 0.95 for the best. A multivariate analysis of the correlations between MR imaging and pathologic inflammation and fibrosis scores showed that perfusion was the only parameter related to fibrosis. Prospects are now to test these sequences to evaluate antifibrotic treatments currently under development and to transpose these sequences to patients to evaluate intestinal fibrosis and improve patients’ management.

Tracheobronchial submucosal glands (SMGs) develop from clusters of epithelial progenitor cells basally orientated within the surface airway epithelium called primordial glandular placodes (PGPs). Signal transduction events that coordinate the transitional process from PGPs into fully developed SMGs consisting of intricately branched networks of tubular secretary structures are still poorly understood. Wnt/β-catenin dependent induction of lymphoid enhancing factor-1 (Lef-1) expression in PGP progenitor/stem cells is required for SMG formation and maturation in the airway. In an effort to better understand the regulatory mechanisms that control Lef-1 during airway SMG development, I have studied its transcriptional regulation. I discovered that Sox2 expression is predominantly confined to the surface airway epithelium (SAE) and is repressed as Lef-1 is induced within PGPs. Deletion of Sox2 in polarized primary airway epithelia significantly enhances Lef-1 mRNA expression. Consequently, my hypothesis is that Sox2 functions as a negative regulator of Lef-1 expression in the SAE. I demonstrated that Sox2 modulates the expression of Lef-1 both independent and dependent on Wnt/β-catenin signaling. I discovered that a Sox2-binding site located in the Wnt Responsive Element (WRE) region of the 2.5Kb Lef-1 promoter is required for Sox2-mediated inhibition of β-catenin-dependent Lef-1 promoter transcription. It is important to understand the biology of SMG development because SMGs are the major mucus-producing structures in the proximal airway and are important in regulating the innate immunity of the lung in response to various neural signals. SMG ducts have also been proposed as a potential protective niche for slowly cycling progenitor cells (SCPCs). Hence, aberrant SMG function is thought to aggravate the pathoprogression of lung disease. Cystic fibrosis (CF) is a disease caused by a defect in the gene that encodes a chloride ion channel called cystic fibrosis transmembrane conductance regulator (CFTR). The absence of CFTR in serous cells within SMG ducts contributes to defective airway secretion, which alters the microenvironment within SMGs. I hypothesized that the glandular SCPC niche may be dysfunctional in CF. I reported that the neural peptide, calcitonin gene-related peptide (CGRP) activates CFTR-dependent SMG secretions and that this signaling pathway is hyperactivated in CF human, pig, ferret, and mouse SMGs. CFTR-deficient mice failed to maintain glandular SCPCs following airway injury, suggesting that the glandular SCPC niche may be dysfunctional in CF. CGRP levels increase following airway injury and function as an injury-inducible mitogen that stimulates progenitor cell proliferation. However, components of the receptor for CGRP (RAMP1 and CLR) were expressed in a very small subset of SCPCs, suggesting that CGRP indirectly stimulates SCPC proliferation through paracrine mechanisms. This discovery may have important implications for injury/repair mechanisms in the CF airway.

Cystic fibrosis (CF) is the most common autosomal recessive genetic disease in Caucasian populations. CF affects multiple organ systems including pancreas, liver, intestines, sweat glands, and male reproductive organs, however the leading cause of morbidity and mortality in CF patients is chronic lung disease. CF is caused by a mutant cystic fibrosis transmembrane conductance regulator (CFTR) gene which leads to chloride (Cl-) and bicarbonate (HCO3-) anion dysregulation at the airway surface. Without adequate anion exchange, thick, viscous mucus accumulates at the airway surface allowing bacterial colonization to occur. Complementing CFTR in the appropriate airway cells restores the anion channel activity in CFTR-deficient cells. The ultimate goal for CF gene therapy is to design an integrating vector that would lead to persistent and efficient expression of CFTR in the airways. Performing gene therapy experiments is dependent upon a relevant animal model. The CF pig is a large animal model similar in size, anatomy, and physiology to humans. Importantly, the CF pig recapitulates human lung disease. From the CF pig, we have learned much about CF lung disease and have developed relevant assays to measure anion channel correction. We have learned that loss of CFTR leads to a decreased airway surface ASL pH, bacterial killing ability, and increased mucus viscosity. Standardized assays have been developed to evaluate the change in current by Ussing chambers, ASL pH, bacterial killing in vivo and ASL pH and viscosity on primary airway cultures in vitro. Ultimately, these metrics allow us to make conclusions about the efficiency of CFTR restoration. Viral vectors are promising candidates for CF gene therapy. Viral vectors such as adenovirus (Ad), adeno-associated virus (AAV), and pseudotyped lentiviral vectors such as feline immunodeficiency virus (FIV) or human immunodeficiency virus (HIV) can efficiently transduce airway cells and express CFTR. Ad and AAV have both been tested in CF clinical trials, but CFTR expression was transient, if detected at all. Understanding vector biology and overcoming barriers in the lung have allowed us to improve vector delivery to the airways. However, the next major hurdle was achieving persistent expression. Ad and AAV are both transiently expressing vectors, and vector readministration is implausible due to the presence of neutralizing antibodies that develop against the vector. Creating a hybrid nonviral/viral vector in which the integrating nonviral piggyBac transposon system is delivered by an Ad or AAV vector has allowed us to achieve persistent expression in mice. In a third integrating vector system, lentiviral vectors have historically been challenging to work with due to low titer levels. However, improvement in vector purification methods have allowed us to validate a lentiviral vector as a viable gene therapy option. In total, we have validated three integrating vector systems by restoring CFTR to CF pigs to correct the phenotypic defect.