Ο αιματο-εγκεφαλικός φραγμός (ΑΕΦ) παίζει έναν καθοριστικό ρόλο στην ομοιόσταση του Κεντρικού Νευρικού Συστήματος. Οι στενοσύνδεσμοι (TJ) των ενδοθηλιακών κυττάρων των εγκεφαλικών τριχοειδών συνεισφέρουν σημαντικά στη λειτουργία του ΑΕΦ περιορίζοντας την παρακυτταρική διάχυση. Η οκλουδίνη, μια διαμεμβρανική πρωτεΐνη, αποτελεί μείζον συστατικό των TJ και παίζει σημαντικό ρόλο στη ρύθμιση της λειτουργίας τους. Το οξειδωτικό στρες αποτελεί κοινό χαρακτηριστικό πολλών νευροεκφυλιστικών και νευροφλεγμονωδών παθήσεων και η δυσλειτουργία των TJ με τη συνοδό διαταραχή του ΑΕΦ, συνδέονται με αυτό. Το αυξημένο οξειδωτικό φορτίο στα πλαίσια της αποφρακτικής χολόστασης έχει αποδειχθεί σε διάφορα όργανα αρουραίων μεταξύ των οποίων και ο εγκέφαλος. Στην παρούσα μελέτη χρησιμοποιήθηκε ένα πειραματικό μοντέλο αρουραίων με απολίνωση του χοληδόχου πόρου (BDL), για να εξεταστεί η επίδραση της χολόστασης στην εντόπιση της οκλουδίνης στο ενδοθήλιο εγκεφαλικών τριχοειδών με τη χρήση ηλεκτρονικού μικροσκοπίου. Αρσενικοί αρουραίοι Wistar χωρίστηκαν τυχαίως σε δύο ομάδες. Ομάδα Ι ψευδώς χειρουργημένοι αρουραίοι και ομάδα ΙΙ αρουραίοι που υπεβλήθησαν σε απολίνωση χοληδόχου πόρου (BDL) και οι δύο την ίδια ημέρα 0. Την 10η μετεγχειρητική ημέρα, όλα τα επιζήσαντα ζώα θυσιάστηκαν δια αποκεφαλισμού. Μετά από κατάλληλη προετοιμασία για σήμανση με ανοσοχρυσό της οκλουδίνης, δείγματα από το μετωπιαίο λοβό, το μεσεγκέφαλο και την παρεγκεφαλίδα από κάθε ομάδα παρατηρήθηκαν με ηλεκτρονικό μικροσκόπιο για διαφορές στην εντόπιση της οκλουδίνης σε σχέση με τη διενδοθηλιακή σχισμή. Τα αποτελέσματα ανέδειξαν μετακίνηση της οκλουδίνης μακριά από την περιοχή των στενοσυνδέσμων του τριχοειδικού ενδοθηλίου. Σημαντική αύξηση της απόστασης της οκλουδίνης από τη διενδοθηλιακή σχισμή παρατηρήθηκε στο μεσεγκέφαλο και στην παρεγκεφαλίδα και όχι στο μετωπιαίο λοβό, σε σχέση με την ομάδα ελέγχου (control). Τα συγκεκριμένα αποτελέσματα υπαινίσσονται μια εκλεκτική ως προς την περιοχή αποδιοργάνωση της οκλουδίνης σε απάντηση στην ηπατική δυσλειτουργία και ένα σημάδι δυσλειτουργίας των TJ με λογικό συνεπακόλουθο τη δυσλειτουργία και του ΑΕΦ. Προκαταρκτικά δεδομένα της χρήσης αντιοξειδωτικών παραγόντων, όπως η αλλοπουρινόλη, αφήνουν να διαφανεί ένας προστατευτικός ρόλος όσον αφορά τη μετατόπιση της οκλουδίνης σε BDL αρουραίους. Εν συντομία, η παρούσα πειραματική μελέτη παρουσιάζει την επίδραση του οξειδωτικού στρες, στην εντόπιση της πρωτεΐνης των στενοσυνδέσμων οκλουδίνη στο ενδοθήλιο των εγκεφαλικών τριχοειδών αγγείων σε αρουραίους με απολίνωση χοληδόχου πόρου. Για τη μελέτη χρησιμοποιήθηκαν τεχνικές ανοσοσήμανσης συνδυαζόμενες με ηλεκτρονική μικροσκοπία και παρουσιάστηκαν δεδομένα εκλεκτικής ως προς την περιοχή εγκεφαλικής δυσλειτουργίας στην ηπατική πάθηση με δυνητική συσχέτιση με τις κλινικές εκδηλώσεις της ηπατικής εγκεφαλοπάθειας. / The blood–brain barrier (BBB) plays a critical role in central nervous system homeostasis. Interendothelial tight junction (TJ) protein complexes of the brain microvasculature have a major contribution to the BBB function by limiting paracellular diffusion Occludin, a transmembrane protein, is a major component of the TJ which plays a significant role in its regulation. Oxidative stress is a major underlying cause of neurodegenerative and neuroinflammatory disorders while TJ dysfunction leading to BBB disruption is associated with it. The development of increased oxidative stress in the context of obstructive cholestasis has been proven in various rats' organs including the brain. The present study used a rat model with bile duct ligation, to examine the effect of cholestasis, to the localization of occludin in brain capillary endothelium by means of electronic microscopy. Male Wistar rats were randomly divided into Group I, rats subjected to sham operation, and Group II, rats subjected to bile duct ligation (BDL) on day 0 and on post-operative day 10 all surviving animals were sacrificed by instant decapitation. After specific treatment for immunogold labeling of occludin, samples from frontal cortex, midbrain and cerebellum from each group were observed for differences in occludin location in relation to the interendothelial cleft. The results demonstrated a dislocation of occludin away from the tight junction sites of brain endothelial cells. A significant increase of the occludin-interendothelial cleft distance was demonstrated in the midbrain and the cerebellum samples but not in the frontal cortex, compared to the control group samples. These findings imply a brain region selective derangement of occludin in response to liver disease and a sign of TJ impairment and thus, a speculated BBB dysfunction. Preliminary data of use of antioxidant agents, as allopurinol, imply a protective effect concerning the dislocation of occludin in BDL rats. In brief, this experimental study demonstrates the effect of oxidative stress, in the location of TJ protein occludin in brain capillary endothelium of BDL rats. The study used immunolabeling techniques combined with electron microscopy and presented data of region-specific brain abnormalities in liver disease with potential correlation with clinical manifestations of hepatic encephalopathy.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A disfunção mitocondrial tem sido associada a várias doenças, incluindo a colestase hepática, caracterizada pela ativação de células de Kupffer e células fibrogênicas, as quais produzem matriz extracelular excessiva. O acúmulo de sais biliares tóxicos no parênquima hepático leva à lesão crônica com dano mitocondrial, redução da síntese de ATP, aumento de espécies reativas de oxigênio (ROS) e apoptose, resultando em comprometimento da função hepática. Trabalhos anteriores do nosso grupo mostraram o efeito positivo do transplante de células mononucleares da medula óssea (CMMO) na resolução da fibrose hepática e recuperação da função hepática em ratos com colestase, induzida pela ligadura de ducto biliar (LDB). Assim, o presente estudo teve como objetivo analisar a bioenergética mitocondrial após o transplante de CMMO no fígado de ratos com fibrose induzida por ligadura de ducto bilar (LDB). Ratos Wistar machos foram divididos em quatro grupos: animais normais, animais com fibrose hepática após 14 e 21 dias de LDB (F14d e F21d, respectivamente), e animais que após 14 dias de LDB receberam 1x107 CMMO, e foram eutanasiados sete dias após. Nossos dados demonstraram aumento do conteúdo de colágeno tipo I no grupo F21d em relação ao grupo normal, indicativo de fibrose, e sua diminuição após o transplante de CMMO. A análise da fisiologia mitocondrial do fígado mostrou diminuição significativa da taxa respiratória máxima estimulada por ADP (estado 3) nos grupos F14d e F21d, indicando redução da capacidade de oxidação de carboidratos e ácidos graxos. Além disso, a razão do controle respiratório (RCR), indicativa de acoplamento da fosforilação oxidativa com a produção de ATP, apresentou-se significativamente diminuída nos grupos F14d e F21d, sugerindo desacoplamento mitocondrial. No entanto, o transplante de CMMO aumentou significativamente nestes grupos tanto a capacidade oxidativa quanto o acoplamento mitocondrial a níveis semelhantes aos do grupo normal. Estes resultados foram confirmados por análise de western blotting, que mostrou aumento significativo no conteúdo de UCP2 e diminuição do conteúdo de PGC-1α no grupo F21d, com consequente restauração após o transplante de CMMO. Além disso, os resultados mostraram um aumento significativo do conteúdo de 4-HNE no grupo F14d com redução após o transplante CMMO. Assim, podemos concluir que o transplante de CMMO tem um efeito positivo sobre a bioenergética mitocondrial de fígados de ratos com colestase, com aumento da capacidade oxidativa e redução do estresse oxidativo, o que, por sua vez, contribui para a recuperação da função hepática. / Mitochondrial dysfunction has been associated with several diseases, including liver cholestatis, which is characterized by activation of Kupffer cells and fibrogenic cells that produce excessive extracellular matrix. Toxic bile salt accumulation in liver parenchyma leads to chronic injury with mitochondrial damage, ATP synthesis reduction, ROS increase and apoptosis, resulting in liver function impairment. Our previous works showed the positive effect of bone marrow mononuclear cells (BMMNC) transplantation on liver fibrosis resolution and hepatic function recover in cholestatic rats. This study aimed to analyze mitochondrial bioenergetics in rats with hepatic fibrosis induced by bile duct ligation (BDL) after BMMNC transplantation. Wistar male rats were divided into four groups: normal animals, animals with cholestasis after 14 and 21 days BDL (F14d and F21d, respectively), and animals with cholestasis after 14 days of BDL that received 1x107 BMMNC, via jugular vein, and were euthanasia after 7 days. The livers were analyzed by high-resolution respirometry and western blotting. Our data demonstrated increased collagen type I content in livers of F21d group compared to normal group, indicative of fibrosis, and its decrease after BMMNC transplantation. Liver mitochondrial physiology analysis showed that F14d and F21d groups have significantly reduced maximum ADP-stimulated respiratory rates (State 3), indicating reduced carbohydrates and fatty acids oxidation capacity. In addition, respiratory control ratio (RCR), indicative of oxidative phosphorilation coupling to ATP production, was significantly decreased in F14d and F21d groups, suggesting mitochondrial uncoupling. However, BMMNC transplantation significantly increased both State 3 respiration and RCR to levels similar to those of normal group, recovering hepatic mitochondrial function. These results were confirmed by WB analysis, which showed that F21d group had a significantly increase in liver mitochondrial uncoupling protein content, UCP2, and reduced PGC-1α content, a mitochondrial biogenesis co-factor. However, after BMMNC transplantation both proteins returned to levels similar to normal group. In addition, F14d group had a significantly increase in 4-HNE content compared to normal group, indicative of oxidative stress, but after BMMNC transplantation 4-HNE content significantly reduced, compared to normal group, suggesting oxidative stress reduction. Therefore, BMMNC transplantation had a positive effect on hepatic mitochondrial bioenergetics of cholestatic rats, increasing oxidative capacity and reducing oxidative stress, which, in turn, contribute to liver function recover.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A disfunção mitocondrial tem sido associada a várias doenças, incluindo a colestase hepática, caracterizada pela ativação de células de Kupffer e células fibrogênicas, as quais produzem matriz extracelular excessiva. O acúmulo de sais biliares tóxicos no parênquima hepático leva à lesão crônica com dano mitocondrial, redução da síntese de ATP, aumento de espécies reativas de oxigênio (ROS) e apoptose, resultando em comprometimento da função hepática. Trabalhos anteriores do nosso grupo mostraram o efeito positivo do transplante de células mononucleares da medula óssea (CMMO) na resolução da fibrose hepática e recuperação da função hepática em ratos com colestase, induzida pela ligadura de ducto biliar (LDB). Assim, o presente estudo teve como objetivo analisar a bioenergética mitocondrial após o transplante de CMMO no fígado de ratos com fibrose induzida por ligadura de ducto bilar (LDB). Ratos Wistar machos foram divididos em quatro grupos: animais normais, animais com fibrose hepática após 14 e 21 dias de LDB (F14d e F21d, respectivamente), e animais que após 14 dias de LDB receberam 1x107 CMMO, e foram eutanasiados sete dias após. Nossos dados demonstraram aumento do conteúdo de colágeno tipo I no grupo F21d em relação ao grupo normal, indicativo de fibrose, e sua diminuição após o transplante de CMMO. A análise da fisiologia mitocondrial do fígado mostrou diminuição significativa da taxa respiratória máxima estimulada por ADP (estado 3) nos grupos F14d e F21d, indicando redução da capacidade de oxidação de carboidratos e ácidos graxos. Além disso, a razão do controle respiratório (RCR), indicativa de acoplamento da fosforilação oxidativa com a produção de ATP, apresentou-se significativamente diminuída nos grupos F14d e F21d, sugerindo desacoplamento mitocondrial. No entanto, o transplante de CMMO aumentou significativamente nestes grupos tanto a capacidade oxidativa quanto o acoplamento mitocondrial a níveis semelhantes aos do grupo normal. Estes resultados foram confirmados por análise de western blotting, que mostrou aumento significativo no conteúdo de UCP2 e diminuição do conteúdo de PGC-1α no grupo F21d, com consequente restauração após o transplante de CMMO. Além disso, os resultados mostraram um aumento significativo do conteúdo de 4-HNE no grupo F14d com redução após o transplante CMMO. Assim, podemos concluir que o transplante de CMMO tem um efeito positivo sobre a bioenergética mitocondrial de fígados de ratos com colestase, com aumento da capacidade oxidativa e redução do estresse oxidativo, o que, por sua vez, contribui para a recuperação da função hepática. / Mitochondrial dysfunction has been associated with several diseases, including liver cholestatis, which is characterized by activation of Kupffer cells and fibrogenic cells that produce excessive extracellular matrix. Toxic bile salt accumulation in liver parenchyma leads to chronic injury with mitochondrial damage, ATP synthesis reduction, ROS increase and apoptosis, resulting in liver function impairment. Our previous works showed the positive effect of bone marrow mononuclear cells (BMMNC) transplantation on liver fibrosis resolution and hepatic function recover in cholestatic rats. This study aimed to analyze mitochondrial bioenergetics in rats with hepatic fibrosis induced by bile duct ligation (BDL) after BMMNC transplantation. Wistar male rats were divided into four groups: normal animals, animals with cholestasis after 14 and 21 days BDL (F14d and F21d, respectively), and animals with cholestasis after 14 days of BDL that received 1x107 BMMNC, via jugular vein, and were euthanasia after 7 days. The livers were analyzed by high-resolution respirometry and western blotting. Our data demonstrated increased collagen type I content in livers of F21d group compared to normal group, indicative of fibrosis, and its decrease after BMMNC transplantation. Liver mitochondrial physiology analysis showed that F14d and F21d groups have significantly reduced maximum ADP-stimulated respiratory rates (State 3), indicating reduced carbohydrates and fatty acids oxidation capacity. In addition, respiratory control ratio (RCR), indicative of oxidative phosphorilation coupling to ATP production, was significantly decreased in F14d and F21d groups, suggesting mitochondrial uncoupling. However, BMMNC transplantation significantly increased both State 3 respiration and RCR to levels similar to those of normal group, recovering hepatic mitochondrial function. These results were confirmed by WB analysis, which showed that F21d group had a significantly increase in liver mitochondrial uncoupling protein content, UCP2, and reduced PGC-1α content, a mitochondrial biogenesis co-factor. However, after BMMNC transplantation both proteins returned to levels similar to normal group. In addition, F14d group had a significantly increase in 4-HNE content compared to normal group, indicative of oxidative stress, but after BMMNC transplantation 4-HNE content significantly reduced, compared to normal group, suggesting oxidative stress reduction. Therefore, BMMNC transplantation had a positive effect on hepatic mitochondrial bioenergetics of cholestatic rats, increasing oxidative capacity and reducing oxidative stress, which, in turn, contribute to liver function recover.

Introdução: O diagnóstico histológico das estenoses biliares é fundamental na definição da terapêutica a ser empregada, devido à heterogeneidade dos resultados dos estudos comparando o uso do escovado citológico e da biópsia transpapilar na colangiopancreatografia retrógada endoscópica (CPRE) com a punção aspirativa ecoguiada com agulha fina (ECO-PAAF) no diagnóstico histológico da estenose biliar maligna, e o fato de não existirem revisões sistemáticas e metanálises comparando esses métodos, este estudo propõe comparar esses dois métodos no diagnóstico histológico da estenose biliar maligna, através de revisão sistemática e metanálise da literatura. Métodos: Utilizando as bases de dados eletrônicas Medline, Embase, Cochrane, LILACS, CINAHL, e Scopus foram pesquisados estudos datados anteriormente a novembro de 2014. De um total de 1009 estudos publicados, foram selecionados três estudos prospectivos comparando ECO-PAAF e CPRE no diagnóstico histológico da estenose biliar maligna e cinco estudos transversais comparando ECO-PAAF com o mesmo padrão-ouro dos outros três estudos comparativos. Todos os pacientes foram submetidos ao mesmo padrão-ouro. Foram calculadas as variáveis do estudo (prevalência, sensibilidade, especificidade, valores preditivos positivos e negativos e acurácia) e realizada a metanálise utilizando os softwares Rev Man 5 e Meta-DiSc 1.4. Resultados: Um total de 294 pacientes foi incluído na análise. A probabilidade pré-teste para estenose biliar maligna foi de 76,66%. As sensibilidades médias da CPRE e da ECO-PAAF para o diagnóstico histológico da estenose biliar maligna foram de 49% e 76,5%, respectivamente; especificidades foram de 96,33% e 100%, respectivamente. As probabilidades pós-teste também foram determinadas: valores preditivos positivos de 98,33% e 100%, respectivamente, e valores preditivos negativos de 34% e 58,87%. As acurácias foram 60,66% e 82,25%, respectivamente. Conclusão: A ECO-PAAF é superior a CPRE com escovado citológico e/ou biópsia transpapilar no diagnóstico histológico da estenose biliar maligna. No entanto, um teste de ECO-PAAF ou CPRE com amostra histológica negativa não pode excluir a estenose biliar maligna, pois ambos os testes apresentam baixo valor preditivo negativo / Background and Aims: Due the heterogeneity of the results of studies comparing the use of ERCP-based brush cytology and forceps biopsy and EUS-guided fine-needle aspiration for the diagnosis of malignant biliary stricture, and the fact that there are no systematic reviews and meta-analysis comparing these methods, in this review, we will compare ERCP against EUS-FNA for tissue diagnosis of malignant biliary stricture. Design: A systematic review of comparative studies (prospective or retrospective) was conducted analyzing EUS and ERCP for tissue diagnosis of malignant biliary stricture. Methods: The databases Medline, EMBASE, Cochrane, LILACS, CINAHL, and Scopus were searched for studies dated previous to November 2014. We identified three prospective studies comparing EUS-FNA and ERCP for the diagnosis of malignant biliary stricture and five cross sectional studies comparing EUS-FNA with the same gold standard of the other three studies. All patients were submitted to the same gold standard method. We calculated study variables (prevalence, sensitivity, specificity, positive and negative predictive values, and accuracy) and performed a meta-analysis using the Rev Man 5 and Meta-DiSc 1.4 softwares. Results: A total of 294 patients were included in the analysis. The pretest probability for malignant biliary stricture was 76.66%. The mean sensitivities of ERCP and EUS-FNA for tissue diagnosis of malignant biliary stricture were 49% and 76.5%, respectively; specificities were 96.33% and 100%, respectively. The post-test probabilities, positive predictive value (98.33% and 100%, respectively) and negative predictive value (34% and 58.87%, respectively) were determined. The accuracies were 60.66% and 82.25%, respectively. Conclusion: EUS- FNA is superior to ERCP with brush cytology and forceps biopsy for diagnosing malignant biliary strictures. However, a negative EUS-FNA or ERCP test may not exclude malignant biliary stricture because both have low negative post-test probabilities

La cholestase intrahépatique représente environ 40% des lésions hépatiques induites par les médicaments et se caractérise par une accumulation intracellulaire des acides biliaires (AB). Les mécanismes impliqués sont encore mal connus et sa prédiction reste difficile. Le but de ce travail était de caractériser dans la cholestase d’origine médicamenteuse et de développer des méthodes de screening pour sa prédiction précoce, en utilisant la lignée humaine hépatique HepaRG et les hépatocytes humains. Tout d’abord, nous avons démontré que la motilité des canalicules biliaires (CB) est indispensable à la clairance des AB et requiert une alternance de phosphorylation/déphosphorylation de la chaine légère de la myosine (MLC), contrôlé par la voie Rho-kinase/Myosin Light Chain Kinase (ROCK/MLCK). Nous avons ensuite montré pour la première fois que les médicaments cholestatiques altèrent la voie ROCK/MLCK/MLC et la dynamique des CB. En utilisant la famille des antibiotiques résistant à la pénicillinase, dont fait partie la flucloxacilline, responsable de nombreux cas de cholestase, nous avons observé que la dérégulation de ROCK pouvait se faire par activation de HSP27, associée aux voies de signalisation PKC/P38 et PI3K/AKT. Enfin, nous avons montré une capacité variable des médicaments cholestatiques à moduler les profils des AB. En effet, les médicaments cholestatiques majeurs induisent une accumulation préférentielle des AB hydrophobes toxiques, in vitro, dans les premières 24h, qui résulte d’une inhibition de leur amidation. Au total, l’ensemble du travail a permis de progresser dans la compréhension des mécanismes impliqués dans la cholestase d’origine médicamenteuse et de mettre en évidence de nouveaux biomarqueurs utiles pour sa prédiction. / Intrahepatic cholestasis represents around 40% of drug-induced liver injuries and is characterized by intracellular accumulation of bile acids (BA); mechanisms involved and its accurate prediction remains challenging. The aim of the current work was to characterize the mechanisms involved in drug-induced cholestasis and to develop screening methods for its early prediction, using human differentiated HepaRG and primary human hepatocytes. First, we demonstrated that bile canaliculi (BC) motility is essential for BA clearance and requires alternating phosphorylation/dephosphorylation of myosin light chain (MLC) that is controlled by the Rho-kinase/Myosin Light Chain Kinase (ROCK/MLCK) signaling pathway. Then, we showed, for the first time that cholestatic drugs could alter the ROCK/MLCK/MLC pathway and BC dynamics. Using the penicillinase-resistant antibiotics family, including flucloxacillin that is responsible for many cases of cholestasis, we found that deregulation of ROCK could be modulated by HSP27, associated with PKC/P38 and PI3K/AKT signaling pathways. Finally, we evidenced variable potency of cholestatic drugs to modulate BA profiles. Indeed, the well-known cholestatic drugs induced a preferential accumulation of unconjugated toxic hydrophobic BA in vitro within the first 24h that resulted from inhibition of their amidation. Altogether, these data bring new information on the understanding of the mechanisms involved in drug-induced cholestasis and highlight new morphological and molecular predictive biomarkers of drug-induced cholestasis.

La cholestase est l'une des manifestations les plus courantes des lésions induitespar des médicaments. Dans 40% des cas elle n’est pas prévisible; une meilleure prédictibilité représente donc un défi majeur. Tout d'abord, nous avons démontré que les cellules hépatiques humaines HepaRG différenciées sont un modèle approprié pour étudier la cholestase induite par les médicaments en comparant la localisation et l’activité des transporteurs d'influx et d'efflux avec les hépatocytes humains primaires. Tous les transporteurs d'efflux et d’influx testés ont été correctement localisés au niveau des membranes canaliculaire (BSEP, MRP2, MDR1 et MDR3) et basolatéral (NTCP, MRP3) et sont fonctionnels. En outre, ces cellules expriment également les enzymes qui métabolisent les acides biliaires (ABs) et ont la capacité de les synthétiser et de les conjuguer avec la taurine, la glycine et le sulfate, à un taux similaire à celui des hépatocytes primaires. Des changements ont été observés sur la répartition des ABs totaux après traitements de cellules HepaRG par un médicament cholestatique, la cyclosporine A (CsA), de manière concentration- dépendante. L'inhibition de l'efflux et de l'influx de taurocholate a été observée après 15 min et 1 h respectivement. Ces premiers effets ont été associés à la dérégulation de la voie des cPKC et l'induction d’un stress du réticulum endoplasmique puis d’un stress oxydant. Nous avons également montré pour la première fois une accumulation intracellulaire d’ABs endogènes avec un médicament cholestatique in vitro. En outre, notre travail apporte des preuves que la motilité des canalicules biliaires (BC) est indispensable à la clairance des ABs. La voie ROCK et le complexe actomyosine sont fortement impliqués. Nous avons fourni la première démonstration que la voie ROCK et les dynamiques des BC sont des cibles majeures des composés cholestatiques. Nos données devraient contribuer à l'élaboration de méthodes de screening pour la prédiction précoce des effets secondaires induits par les médicaments cholestatiques. / Cholestasis is one of the most common manifestations of drug-induced liver injury (DILI). Since up to now it is unpredictable in 40% of all cases its accurate prediction represents a major challenge. First, we validated that differentiated HepaRG human liver cells are a suitable in vitro model to study drug-induced cholestasis, by comparing localization of influx and efflux transporters and their functional activity in these cells and primary human hepatocytes. All tested influx and efflux transporters were correctly localized to canalicular (BSEP, MRP2, MDR1, and MDR3) or basolateral (NTCP, MRP3) membrane domains and were functional. In addition, the HepaRG cell line also exhibits bile acids (BAs) metabolizing enzymes and has the capacity to synthesize BAs and to further amidate these BAs with taurine and glycine as well as sulfate, at a rate similar to that of primary hepatocytes. Concentration- dependent changes were observed in total BAs disposition after treatment of HepaRG cells by the cholestatic drug cyclosporine A (CsA). Inhibition of efflux and uptake of taurocholate was evidenced as early as 15 min and 1 h respectively. These early effects were associated with deregulation of cPKC pathway and induction of endoplasmic reticulum stress that preceded generation of oxidative stress. We also showed for the first time intracellular accumulation of endogenous BAs by a cholestatic drug in vitro. In addition, our work brings evidences that motility of bile canaliculi (BC) is essential for BAs clearance where ROCK pathway and actomyosin complex are highly implicated. We provided the first demonstration that ROCK pathway and BC dynamics are major targets of cholestatic compounds. Our data should help in the development of screening methods for early prediction of drug-induced cholestatic side effects.

La cholestase intrahépatique est une manifestation fréquente des lésions hépatiques induites par les médicaments; Cependant, les mécanismes impliqués sont peu connus. Nous avons cherché à étudier les mécanismes de la cholestase induite par les médicaments afin d’améliorer sa détection précoce en utilisant les cellules HepaRG humaines. Tout d'abord, nous avons prouvé que les canalicules biliaires (BC) subissaient des contractions spontanées, essentielles pour l'efflux d’acides biliaires et nécessitaient des séries d’alternance dans la phosphorylation/déphosphorylation de la chaîne légère de myosine (MLC2). La courte exposition à des composés cholestatiques a révélé que la modulation des BC était associée à des perturbations de la voie de signalisation ROCK/MLCK. Afin de confirmer notre étude, 12 médicaments cholestatiques et six non cholestatiques ont été analysés et nous avons démontré que tous les médicaments cholestatiques classés sur la base de résultats cliniques provoquaient des perturbations dans la dynamique des BC (dilatation ou constriction), et altéraient la voie de signalisation ROCK/MLCK, tandis que les composés non cholestatiques n'avaient pas d'effet. Nous avons également prouvé que ces changements étaient plus spécifiques que la mesure de l'inhibition de l’efflux comme marqueurs prédictifs non cliniques de la cholestase induite par les médicaments. Afin de confirmer et d’étendre ces conclusions, nous avons analysé les mécanismes impliqués dans les effets cytotoxiques et cholestatiques induits par 4 médicaments de la famille des antagonistes des récepteurs de l'endothéline: deux ayant un lien avec des cas cliniques d'hépatotoxicité (sitaxentan) et/ou cholestase (bosentan), et deux n’ayant pas été impliqués dans l’élévation de transaminases hépatiques ou de bilirubine (ambrisentan et macitentan). Les résultats montrent que le macitentan récemment commercialisé et ayant une structure chimique similaire à celle du bosentan, était capable de causer, comme ce dernier, des altérations in vitro des BC. En revanche, l'ambrisentan n’était pas hépatotoxique et le sitaxentan qui a été retiré du marché pour des cas d’hépatotoxicité, n’affectait pas la dynamique canaliculaire. / Intrahepatic cholestasis represents a frequent manifestation of drug-induced liver injuries; however, the mechanisms involved in such injuries are poorly understood. We aimed to investigate mechanisms underlying drug-induced cholestasis and improve its early detection using human HepaRG cells. First, we proved that bile canaliculi (BC) underwent spontaneous contractions, which are essential for bile acid efflux and required alternations in myosin light chain (MLC2) phosphorylation/dephosphorylation. A short exposure to cholestatic compounds revealed that BC dynamics was altered and associated with impairment of the ROCK/MLCK pathway. Then, in order to confirm our study, 12 cholestatic drugs and six noncholestatic drugs were analyzed and we demonstrated that all cholestatic drugs classified on the basis of reported clinical findings caused disturbances of both BC dynamics (dilatation or constriction), and alteration of the ROCK/MLCK signaling pathway, whereas noncholestatic compounds had no effect. We also proved that these changes were more specific than efflux inhibition measurements alone as predictive nonclinical markers of drug-induced cholestasis. To confirm and extend these conclusions, we analyzed the mechanisms involved in cytotoxic and cholestatic effects induced by the 4 main drugs from the endothelin receptor antagonists family: two related to clinical cases of hepatotoxicity (sitaxentan) and/or cholestasis (bosentan), and two that have not been reported to cause elevation of liver transaminases or bilirubin (ambrisentan and macitentan). The results showed that like bosentan, the structurally similar recently marketed drug, macitentan, could cause in vitro major BC alterations. By contrast, ambrisentan appeared as a safe drug and sitaxentan that has been withdrawn from the market for hepatotoxic cases, did not impair BC dynamics.

Transdifferentiation induces chromatin reconstructions and epigenetic changes that affect gene expression spectum and cause cell remodeling in general. Direct conversion of mature somatic cell line into another mature cell type occures during the transdifferentiation thereby differences betweeen individual germ layers are eliminated. The aim of the master thesis is transdifferentation of mesenchymal cells - mouse embryonic fibroblast into endodermal cells - hepatocytes in vitro, using combination of transcripion factors Hnf4α and Foxa1. Detection of fibroblasts transformation has been initiated immediately after retroviral transduction and final generation of induced hepatocyte culture was confirmed by morphological and function analysis. The population of mouse induced hepatocytes served as a possible model for human liver disease in case of a pacient whose liver proteins could not be detected immunohistochemically. Genome editing of induced hepatocytes was realized by CRISPR/Cas9 technology which is based on cooperation of guideRNA and Cas9 nuclease followed in addition to generation of DNA-specific double strand breaks. These specific breaks in the Tight junction protein 2 gene were repaired via homologous recombination that induced a missense mutation with amino acid changes in the target...

La cholestase intrahépatique de la grossesse (CIG) est la maladie du foie la plus répandue au cours de la grossesse. Elle est caractérisée par un prurit et est associée à une augmentation de la concentration des acides biliaires dans le sang, ce qui peut mener à un risque accru de conséquences périnatales indésirables, y compris un accouchement prématuré spontané et une augmentation des risques de mort de l’enfant à l’accouchement, entre autres. Le traitement médical de cette maladie repose actuellement sur l’acide ursodésoxycholique (UDCA) qui diminue le prurit et les anomalies biochimiques maternelles dans certains cas. Actuellement, le diagnostic de la CIG est posé suite à un test de quantification des acides biliaires sériques totaux par une méthode enzymatique. Nous émettons l'hypothèse que certains profils d’acides biliaires permettraient d’évaluer le risque de complications chez les femmes atteintes de CIG. En analysant les profilages, il pourrait être possible de déterminer la ou les espèces responsables de ces complications et ainsi déterminer des sous-groupes de patientes plus à risque de complications ou qui répondraient mieux au traitement. De plus, nous pensons que le traitement à l’UDCA, étant lui-même un acide biliaire, pourrait interférer lors de la quantification des acides biliaires totaux sériques, particulièrement dans les cas les plus problématiques de CIG où de fortes doses de ce composé sont administrées. Si c’était le cas, cela ferait en sorte que les valeurs de référence pourraient être modifiées en fonction du traitement administré. Le projet de recherche présenté vise au développement d’une méthode de quantification des acides biliaires sériques par la chromatographie liquide couplée à un spectromètre de masse en tandem (LC-MS/MS), qui permettrait un profilage des acides biliaires sériques chez les femmes enceintes atteintes de la CIG et qui permettrait également d’évaluer l’effet du traitement à l’UDCA sur ce profilage. Une méthode de quantification des acides biliaires par chromatographie liquide couplée à un spectromètre de masse en tandem a été développée et validée. Les surnageants obtenus par précipitation de protéines avec le méthanol ont été injectés sur le LC-MS/MS. La séparation est réalisée par chromatographie en phase inverse sur une colonne C18 de type interactions hydrophobes. Les transitions ioniques sur le spectromètre de masse ont été déterminées pour toutes les espèces d’acides biliaires au préalable et l’acide cholique deutéré, l’acide chénodésoxycholique deutéré ainsi que l’acide désoxycholique deutéré ont été utilisés comme standards internes. Quinze acides biliaires, y compris les acides biliaires conjugués et libres, ont été séparés et quantifiés par LC–MS/MS en utilisant l’ionisation par électro nébulisation (ESI) en mode ion négatif. La quantification a été réalisée en mode de surveillance de réactions multiples (MRM) avec des méthodes de courbes d'étalonnage externes. Les coefficients de corrélation des courbes standards pour tous les acides biliaires étaient supérieurs à 0,9966. La méthode développée a démontré une précision acceptable, avec une imprécision intra analyse inférieure à 3,2% pour toutes les espèces d’acide biliaire étudiées (pour des échantillons à 0,8 et 5 μg/mL) et une imprécision inter analyse inférieure à 15%. Une suppression d’ion moyenne de 8,2% a été observée, qui a été jugée acceptable. Une bonne corrélation a été obtenue entre la méthode LC-MS/MS et une méthode enzymatique (r=0,964). En conclusion, une méthode fonctionnelle, efficace et rapide a été développée pour quantifier les acides biliaires sériques individuels et différents profils d’acides biliaires représentant une large gamme de concentrations ont été comparés. La comparaison des profilages d’acides biliaires suggère que les acides biliaires principaux responsables de l’augmentation de la concentration des acides biliaires totaux dans le sang pour des échantillons à une concentration de plus de 10 μmol/L sont l’acide cholique glyco-conjugué (GCA), l’acide cholique tauro-conjugué (TCA) ainsi que l’acide ursodésoxycholique glyco- conjugué (GUDCA). Cette nouvelle méthode validée, et les données préliminaires sur les profils d’acides biliaires dans les échantillons cliniques, permettront de lancer des analyses cliniques prospectives pour évaluer l’effet du traitement par l’UDCA sur les concentrations totales d’acides biliaires sériques et sur les profils d’acides biliaires individuels chez les patientes atteintes de la CIG. / Intrahepatic cholestasis of pregnancy (ICP) is the most common liver disease during pregnancy. It is characterized by pruritus and is associated with an increased concentration of bile acids in blood, which may lead to an increased risk of perinatal consequences, including spontaneous preterm delivery and an increased risk of death at birth, among others. The medical treatment of this disease currently relies on ursodeoxycholic acid (UDCA) which reduces pruritus and maternal biochemical abnormalities in some cases. Currently, the diagnosis of ICP is made using an enzymatic assay to measure total serum bile acids. We hypothesize that profiling of the individual bile acids would make it possible to assess the risk of complications in women with ICP. By analyzing the bile acid profiles, it could be possible to determine which specie(s) is responsible for these complications and thus to distinguish subgroups of patients at higher risk of complications or who would respond better to treatment. In addition, we believe that UDCA treatment, being a bile acid itself, could interfere with the quantification of total serum bile acids, particularly in the most problematic cases of CIG where high doses of this compound are administered. If this was the case, it would mean that the reference values would need to be changed depending on the administered treatment. The research project aims to develop and validate a method for quantifying bile acids in serum by liquid chromatography coupled to a tandem mass spectrometer (LC-MS/MS), which would allow profiling of serum bile acids in affected women and which would also make it possible later to evaluate the effects of UDCA treatment on this profiling. A method for the quantification of bile acids by liquid chromatography coupled to tandem mass spectrometry has been developed and validated. The supernatants obtained by precipitation of proteins with methanol were injected onto the LC-MS/MS. The separation was carried out using reversed-phase chromatography on a C18 hydrophobic interactions type column. Ionic transitions on the mass spectrometer were determined for all bile acids species beforehand and deuterated cholic acid, deuterated chenodeoxycholic acid and deuterated deoxycholic acid were used as internal standards. Fifteen bile acids, including conjugated and free bile acids, were separated and quantified by LC–MS/MS using electrospray ionization (ESI) in negative ion mode. Quantification was performed in multiple reaction monitoring (MRM) mode with external calibration curve methods. Correlation coefficients for standard curves for all bile acids were greater than 0.9966. The method developed showed acceptable precision, with intra-assay imprecision of less than 3.2% for all the bile acid species studied (for samples at 0.8 and 5 μg/mL) and inter-assay imprecision under 15%. An average ion suppression of 8.2% was observed, which was judged acceptable. Finally, a good correlation was obtained between the LC-MS/MS method and an enzymatic method (r = 0.964). In conclusion, a functional, efficient and rapid method was developed to quantify the individual serum bile acids and different bile acids profiles representing a wide range of concentrations were compared. The comparison of the bile acid profiles suggests that the main bile acids responsible for the increase in total bile acids concentration in blood for samples at a concentration of more than 10 μmol/L are glycocholic acid (GCA), taurocholic acid (TCA), glycoursodeoxycholic acid (GUDCA). This new validated method, and the preliminary data on bile acid profiles in clinical samples, will allow us to initiate prospective clinical analyses to assess the effect of UDCA treatment on total bile acid concentrations and profiles in patients with intrahepatic cholestasis of pregnancy.

The differentiation, in an infant with cholestasis, between extrahepatic biliary atresia (EHBA) and the neonatal hepatitis syndrome (NHS) is important in that laparotomy is always indicated in EHBA but is undesirable in NHS. This differentiation is particularly difficult in those infants with complete cholestasis. Hepatobiliary scintigraphy is a commonly used investigation in infants with obstructive jaundice. The scintigraphic demonstration of excretion into the gut excludes extrahepatic obstruction, however, absence of excretion may be due to EHBA, severe cholestasis with patent extrahepatic bile ducts or poor uptake of the agent, and is therefore not diagnostic. This study has examined the quantitative measurement of the hepatic uptake of p-butyl IDA and Sn colloid, and an estimation of liver shape, in a group of patients with complete cholestasis in whom conventional scan interpretation, based on excretion into the-gut, would not be useful. The scans were recorded as dynamic studies and the resultant time-activity curves were subjected to curve fitting to calculate a rate constant for uptake of radiopharmaceutical. Liver shape was determined from the anterior static image of the colloid scan. The results show a significant difference between the EHBA and the NHS patients in the rate of uptake of p-butyl IDA, in the ratio of the rate of uptake of p-butyl IDA/the rate of uptake of colloid and in the measurements used to express liver shape. Using this method of scan interpretation, a diagnostic accuracy of 85% was achieved in this study of patients who clinically, and on scan, had no evidence of bile flow. Hepatic scintigraphy is therefore a useful investigation in the diagnostic work-up of infants presenting with obstructive jaundice even when bile flow is completely absent.