A atresia das vias biliares é afecção hepática da infância. A etiologia é desconhecida, e o diagnóstico baseia-se na biópsia hepática, cujo achado é a proliferação ductular. A ligadura do ducto biliar comum em ratos é modelo utilizado para estudo das doenças colestáticas. A proposta do trabalho foi estudar, em modelo experimental de obstrução biliar, as alterações histológicas hepáticas em ratos jovens e compará-las com o animal adulto. Avaliou-se a semiquantificação da proliferação ductular e inflamação pelo HE; quantificação da fibrose portal e periportal pelo picrosírius; semiquantificação da expressão de desmina e a-actina de músculo liso pelas células estreladas e miofibroblastos. Apesar das respostas de proliferação ductular e inflamação mais lentas no rato jovem, a fibrose e a expressão de desmina foram mais intensas neste grupo / Biliary atresia is an hepatic disease of infancy. Etiology is unknown, and diagnosis is made by liver biopsy, with ductular proliferation being the main histological feature. Bile duct ligation in rats is an useful experimental model of biliary obstruction. The aim of this study of extra-hepatic cholestasis was analyse hepatic histological alterations in young rats compared to adult animals. The responses were studied by semiquantification of ductular proliferation and inflammatory infiltrated by HE stain; quantification of portal and periportal fibrosis with the sirius-red stain; semiquantification of the expression of desmin and a-smooth muscle actin by the hepatic stellated cells and myofibroblasts. In young animals, despite the very slow response of ductular proliferation and inflammation observed with HE, there were significantly more fibrosis and expression of desmin than in adult group

Cadre conceptuel et problématique : La cholestase hépatique est une pathologie à large éventail d’étiologies, affectant fréquemment les nouveau-nés. Un diagnostic approprié est essentiel pour la prise en charge clinique, le choix des traitements et l’amélioration du pronostic. Alors que les prématurés ont un risque accru de développement de la cholestase, les algorithmes de traitement ne proposent qu’une prise en charge unique, quel que soit l’âge gestationnel (AG) du patient. Objectif : Déterminer si le profil clinique, la prise en charge et le pronostic de la cholestase néonatale diffèrent selon l’AG. Méthodologie : Une étude de cohorte rétrospective de nouveau-nés atteints de cholestase et admis en néonatologie au CHU Sainte Justine entre janvier 2014 et décembre 2017 a été menée. La cholestase était définie par au moins deux valeurs consécutives de bilirubine conjuguée ≥ 34 μmol/L. La cohorte a été stratifiée en deux groupes d’AG : les extrêmes et les grands prématurés (< 32 semaines AG) et les prématurés modérés ou tardifs et naissances à terme (≥ 32 semaines AG). Résultats : 125 nouveau-nés sur 3 277 ont développé une cholestase. L’incidence globale était de 4% ; cette incidence était 5 fois plus élevée chez les nouveau-nés < 32 semaines d’AG comparativement aux ≥ 32 semaines d’AG. La cholestase était associée à une nutrition parentérale chez 91% des patients avec AG < 32 semaines et seulement 40% des patients avec AG ≥ 32 semaines (p < 0,01). Alors que l'acide ursodésoxycholique était plus prescrit aux nouveau-nés ≥ 32 semaines AG, les émulsions lipidiques à base d'huile de poisson étaient plus administrées aux nouveau-nés < 32 semaines AG, parmi les patients recevant une nutrition parentérale. Conclusion : La cholestase néonatale est associée à deux profils cliniques différents, basés sur l'AG. Nous recommandons que les tests diagnostics et la prise en charge clinique de la cholestase soient adaptés à l'AG. Une nouvelle approche pour l'évaluation d'un nourrisson atteint d'hyperbilirubinémie conjuguée est proposée. / Background and Aims: Cholestasis is a frequent neonatal disease that has a wide range of etiologies. Appropriate diagnosis is essential to clinical management, treatment choices and improvement of outcomes. Most references discuss neonatal cholestasis as one entity. The goal of this study was to determine if the clinical profile, management and outcome of cholestasis differ according to gestational age (GA). Methods: Medical records of infants with cholestasis in the division of neonatology at CHU Sainte Justine, between January 2014 and December 2017, were retrospectively reviewed. Cholestasis was defined as two or more consecutive conjugated bilirubin values ≥ 34μmol/L. The cohort was stratified into two groups: extremely to very preterm (< 32 weeks GA) and moderate to late preterm and term (≥ 32 weeks GA). Results: 125 of 3,277 patients developed cholestasis. Overall incidence of cholestasis was 4%. Incidence was 5 times higher in neonates < 32 weeks GA compared to neonates ≥ 32 weeks GA. Cholestasis was associated with parenteral nutrition in 91% of patients with GA < 32 weeks and 40% of patients with GA ≥ 32 weeks (p < 0.01). While ursodiol treatment was prescribed more to cholestatic neonates ≥ 32 weeks GA, fish oil lipid was administered more to neonates < 32 weeks GA, among patients receiving parenteral nutrition. Conclusions: Neonatal cholestasis was associated with two different clinical profiles based on GA. We recommend diagnostic tests and clinical management of neonatal cholestasis be adapted to GA. A GA-based approach to the evaluation of an infant with conjugated hyperbilirubinemia is proposed.

Os mecanismos responsáveis pela fibrose hepática na infância são pouco conhecidos. Crianças com atresia das vias biliares, quando submetidas a portoenterostomia a Kasai com sucesso, se tornam anictéricas mas mesmo assim desenvolvem cirrose a longo prazo. Da mesma forma, a ocorrência de estenoses biliares segmentares intra-hepáticas no pós-operatório de transplante hepático podem levar ao desenvolvimento de cirrose em todo o órgão. Tais fatos sugerem que mecanismos endócrinos ou parácrinos estejam envolvidos na fibrogênese hepática. Para elucidar este processo o modelo de ligadura seletiva das vias biliares em ratos jovens foi desenvolvido em nosso laboratório. Usando este modelo, identificamos mudanças na expressão do gene da alfa-actina de músculo liso, tanto no parênquima hepático obstruído como no parênquima hepático adjacente à obstrução. No entanto, o perfil de expressão gênica da desmina, uma proteína presente em níveis elevados durante a ativação das células estreladas hepáticas e o TGF-beta1, principal citocina pró-fibrogênica, não demostraram diferenças significantes quando analisados pelo método do RT-PCR semiquantitativo. Assim, os mecanismos moleculares envolvidos na modulação da fibrogênese hepática nesse modelo experimental não estão totalmente compreendidos. A metodologia do qRT-PCR (PCR em tempo real), têm sido previamente descrita como um método mais preciso e sensível, possibilitando a detecção do aumento do número de cópias do gene à medida que ocorre a amplificação, enquanto que o método do RT-PCR semiquantitativo a análise dos transcritos só é realizada após a etapa da amplificação. O objetivo deste estudo foi avaliar as alterações moleculares no modelo experimental de ligadura seletiva das vias biliares e comparar os resultados obtidos entre os métodos do RT-PCR semiquantitativo e do qRT-PCR em tempo real. Foi realizada a ligadura seletiva do ducto biliar em ratos Wistar com 21 dias de vida, os grupos foram separados de acordo com o momento da morte: 7 ou 60 dias após a cirurgia. A expressão da desmina, alfa actina de músculo liso e TGF-beta1 foi avaliada no tecido do parênquima hepático com obstrução biliar (ducto ligado - DL) e no parênquima hepático adjacente à obstrução biliar (ducto não ligado - DNL) usando o RT-PCR semiquantitativo e o qRT-PCR em tempo real. A metodologia do qRT-PCR em tempo real permitiu identificar mudanças no perfil de expressão gênica que não foram demonstrados pelo método semiquantitativo. O parênquima DL mostrou reação fibrogênica mais intensa, com aumento na expressão da alfa actina de músculo liso e TGF-beta1 após 7 dias. O parênquima DNL apresentou resposta fibrogênica tardia, com aumento da expressão da desmina 7 dias e 60 dias após a cirurgia, além de aumento da alfa-actina de músculo liso 60 dias após a cirurgia. O qRT-PCR em tempo real apresentou maior sensibilidade para identificar mudanças no perfil de expressão gênica em relação ao método convencional do RT-PCR semiquantitativo. Nossos resultados ajudam a esclarecer a dinâmica das alterações moleculares envolvidas na modulação da fibrogênese hepática no modelo experimental de ligadura seletiva do ducto biliar e pode ser diretamente aplicado para o estudo de estenoses biliares intra-hepáticas e atresia das vias biliares / The mechanisms responsible for liver fibrosis in childhood are poorly understood. Children suffering from biliary atresia, when submitted to the successful Kasai portoentostomy, become anicteric, but nonetheless develop cirrhosis in the long term. Similarly, the occurrence of intrahepatic biliary stenosis in the postoperative period of liver transplantation may lead to cirrhosis of the whole organ. Such facts suggest that endocrine or paracrine mechanisms are involved in hepatic fibrogenesis. To elucidate this process, the selective bile duct ligation model in young rats was developed in our laboratory. Using this model, we identified changes in the expression of smooth muscle alpha-actin both in the obstructed parenchyma and the hepatic parenchyma adjacent to the obstruction. However, the expression profiles of desmin, a protein present at high levels during activation of hepatic stellate cells and TGF-beta1, the main pro-fibrogenic cytokine, were unchanged when analyzed with semiquantitative RT-PCR. Thus, the molecular mechanisms involved in the modulation of hepatic fibrogenesis in this experimental model are not fully understood. The methodology of qRT-PCR (real time PCR) has previously been described as a more precise and sensitive method, allowing the detection of increased copy number of the gene while amplification occurs, whereas by semiquantitative RT-PCR analysis transcripts is only perfomed after the amplification step. This study aimed to evaluate the molecular changes in experimental model of selective bile duct ligation and compare the results between semiquantitative RT-PCR and real-time qRT-PCR methods. Selective biliary duct ligation was performed on Wistar rats with 21 days of life, the groups were separated according to the moment of death: 7 or 60 days after surgery. The expression of desmin, alpha-actin smooth muscle and TGF-beta1 was examined in tissue from hepatic parenchyma with biliary obstruction (duct ligation - DL) and in the adjacent hepatic parenchyma (duct non-ligated - DNL) using semiquantitative RT-PCR and real-time qRT-PCR. The methodology of the real-time qRT-PCR allowed to identify changes in gene expression profile that were not shown by semiquantitative method. The DL parenchyma showed a more severe fibrogenic reaction, with increased alpha-actin smooth muscle and TGF-beta1 expression after 7 days. The DNL parenchyma presented a later fibrotic response, with increased desmin expression 7 and 60 days after surgery, besides of increased alpha-actin smooth muscle 60 days after surgery. Real-time qRT-PCR was more sensitive to identify changes in gene expression profile comparated to the semiquantitative method. Our results help to clarify the dynamic of molecular changes involved in the modulation of hepatic fibrogenesis in an experimental model of selective bile duct ligation and can be directly applied to the study of intrahepatic biliary stenosis and biliary atresia

Os mecanismos responsáveis pela fibrose hepática na infância são pouco conhecidos. Crianças com atresia das vias biliares, quando submetidas a portoenterostomia a Kasai com sucesso, se tornam anictéricas mas mesmo assim desenvolvem cirrose a longo prazo. Da mesma forma, a ocorrência de estenoses biliares segmentares intra-hepáticas no pós-operatório de transplante hepático podem levar ao desenvolvimento de cirrose em todo o órgão. Tais fatos sugerem que mecanismos endócrinos ou parácrinos estejam envolvidos na fibrogênese hepática. Para elucidar este processo o modelo de ligadura seletiva das vias biliares em ratos jovens foi desenvolvido em nosso laboratório. Usando este modelo, identificamos mudanças na expressão do gene da alfa-actina de músculo liso, tanto no parênquima hepático obstruído como no parênquima hepático adjacente à obstrução. No entanto, o perfil de expressão gênica da desmina, uma proteína presente em níveis elevados durante a ativação das células estreladas hepáticas e o TGF-beta1, principal citocina pró-fibrogênica, não demostraram diferenças significantes quando analisados pelo método do RT-PCR semiquantitativo. Assim, os mecanismos moleculares envolvidos na modulação da fibrogênese hepática nesse modelo experimental não estão totalmente compreendidos. A metodologia do qRT-PCR (PCR em tempo real), têm sido previamente descrita como um método mais preciso e sensível, possibilitando a detecção do aumento do número de cópias do gene à medida que ocorre a amplificação, enquanto que o método do RT-PCR semiquantitativo a análise dos transcritos só é realizada após a etapa da amplificação. O objetivo deste estudo foi avaliar as alterações moleculares no modelo experimental de ligadura seletiva das vias biliares e comparar os resultados obtidos entre os métodos do RT-PCR semiquantitativo e do qRT-PCR em tempo real. Foi realizada a ligadura seletiva do ducto biliar em ratos Wistar com 21 dias de vida, os grupos foram separados de acordo com o momento da morte: 7 ou 60 dias após a cirurgia. A expressão da desmina, alfa actina de músculo liso e TGF-beta1 foi avaliada no tecido do parênquima hepático com obstrução biliar (ducto ligado - DL) e no parênquima hepático adjacente à obstrução biliar (ducto não ligado - DNL) usando o RT-PCR semiquantitativo e o qRT-PCR em tempo real. A metodologia do qRT-PCR em tempo real permitiu identificar mudanças no perfil de expressão gênica que não foram demonstrados pelo método semiquantitativo. O parênquima DL mostrou reação fibrogênica mais intensa, com aumento na expressão da alfa actina de músculo liso e TGF-beta1 após 7 dias. O parênquima DNL apresentou resposta fibrogênica tardia, com aumento da expressão da desmina 7 dias e 60 dias após a cirurgia, além de aumento da alfa-actina de músculo liso 60 dias após a cirurgia. O qRT-PCR em tempo real apresentou maior sensibilidade para identificar mudanças no perfil de expressão gênica em relação ao método convencional do RT-PCR semiquantitativo. Nossos resultados ajudam a esclarecer a dinâmica das alterações moleculares envolvidas na modulação da fibrogênese hepática no modelo experimental de ligadura seletiva do ducto biliar e pode ser diretamente aplicado para o estudo de estenoses biliares intra-hepáticas e atresia das vias biliares / The mechanisms responsible for liver fibrosis in childhood are poorly understood. Children suffering from biliary atresia, when submitted to the successful Kasai portoentostomy, become anicteric, but nonetheless develop cirrhosis in the long term. Similarly, the occurrence of intrahepatic biliary stenosis in the postoperative period of liver transplantation may lead to cirrhosis of the whole organ. Such facts suggest that endocrine or paracrine mechanisms are involved in hepatic fibrogenesis. To elucidate this process, the selective bile duct ligation model in young rats was developed in our laboratory. Using this model, we identified changes in the expression of smooth muscle alpha-actin both in the obstructed parenchyma and the hepatic parenchyma adjacent to the obstruction. However, the expression profiles of desmin, a protein present at high levels during activation of hepatic stellate cells and TGF-beta1, the main pro-fibrogenic cytokine, were unchanged when analyzed with semiquantitative RT-PCR. Thus, the molecular mechanisms involved in the modulation of hepatic fibrogenesis in this experimental model are not fully understood. The methodology of qRT-PCR (real time PCR) has previously been described as a more precise and sensitive method, allowing the detection of increased copy number of the gene while amplification occurs, whereas by semiquantitative RT-PCR analysis transcripts is only perfomed after the amplification step. This study aimed to evaluate the molecular changes in experimental model of selective bile duct ligation and compare the results between semiquantitative RT-PCR and real-time qRT-PCR methods. Selective biliary duct ligation was performed on Wistar rats with 21 days of life, the groups were separated according to the moment of death: 7 or 60 days after surgery. The expression of desmin, alpha-actin smooth muscle and TGF-beta1 was examined in tissue from hepatic parenchyma with biliary obstruction (duct ligation - DL) and in the adjacent hepatic parenchyma (duct non-ligated - DNL) using semiquantitative RT-PCR and real-time qRT-PCR. The methodology of the real-time qRT-PCR allowed to identify changes in gene expression profile that were not shown by semiquantitative method. The DL parenchyma showed a more severe fibrogenic reaction, with increased alpha-actin smooth muscle and TGF-beta1 expression after 7 days. The DNL parenchyma presented a later fibrotic response, with increased desmin expression 7 and 60 days after surgery, besides of increased alpha-actin smooth muscle 60 days after surgery. Real-time qRT-PCR was more sensitive to identify changes in gene expression profile comparated to the semiquantitative method. Our results help to clarify the dynamic of molecular changes involved in the modulation of hepatic fibrogenesis in an experimental model of selective bile duct ligation and can be directly applied to the study of intrahepatic biliary stenosis and biliary atresia

Exposure to toxic bile acids (BA) and retinoic acids (RA) is implicated in toxicities related to excessive oxidative stress. This thesis examined roles and mechanisms of the oxidative stress-responsive nuclear factor (erythroid 2-like) factor 2 (Nrf2) in adaptive cell defense against BA and RA toxicities. Using liver cells and mouse models, many antioxidant proteins known to be Nrf2 target genes, particularly the rate-limiting enzyme for glutathione (GSH) biosynthesis, i.e., glutamate-cysteine ligase subunits (GCLM/GCLC), were induced by BA [lithocholic acid (LCA)] or RA (all-trans, 9-cis and 13-cis) treatment. Evidence for increased Nrf2 transactivation by LCA and all-trans-RA was exemplified in HepG2 by: (1) reduced constitutive and inducible expression of GCLM/GCLC upon Nrf2 silencing via small-interfering RNA; (2) increased inducible expression of GCLM/GCLC genes by Nrf2 overexpression, but overexpression of dominant-negative Nrf2 decreased it; (3) increased nuclear accumulation of Nrf2 as signature event of receptor activation; (4) enhanced Nrf2-dependent antioxidant-response-element (ARE) reporter activity as indicative of increased Nrf2 transactivation; and (5) increased Nrf2 occupancy to AREs of GCLM and GCLC. Additionally, in BA-treated HepG2 cells, we observed concomitant increases of many ATP-binding cassette (ABC) transporters (MRPs 1-5, MDR1 and BCRP) in parallel with increased cellular efflux. Nrf2 silencing in HepG2 cells decreased constitutive and inducible expression of MRP2, MRP3 and ABCG2. However, Nrf2-silenced mouse hepatoma cells, Hepa1c1c7, and Nrf2-/- mice had decreased constitutive and/or inducible expression of Mrps 1-4, suggesting species differences in Nrf2-dependent regulation of hepatic ABC transporters. Protection by Nrf2 against BA and RA toxicities was confirmed by observations that Nrf2 silencing increased cell susceptibility to BA- and RA-induced cell death. Moreover, Nrf2-/- mice suffered more severe liver injury than the wildtype. Increased GSH and efflux activity following increased GCLM/GCLC and ABC transporters, respectively, can mitigate LCA toxicity. Activation of MEK1-ERK1/2 MAPK was shown to primarily mediate Nrf2 transactivation and LCA-induced expression of antioxidant proteins and Nrf2-dependent and -independent ABC transporters. In conclusion, Nrf2 activation by BA and RA led to coordinated induction of antioxidant and ABC proteins, thereby counteracting resultant oxidative cytotoxicity. The potential of targeting Nrf2 in management of BA and RA toxicities merits further investigation.

Exposure to toxic bile acids (BA) and retinoic acids (RA) is implicated in toxicities related to excessive oxidative stress. This thesis examined roles and mechanisms of the oxidative stress-responsive nuclear factor (erythroid 2-like) factor 2 (Nrf2) in adaptive cell defense against BA and RA toxicities. Using liver cells and mouse models, many antioxidant proteins known to be Nrf2 target genes, particularly the rate-limiting enzyme for glutathione (GSH) biosynthesis, i.e., glutamate-cysteine ligase subunits (GCLM/GCLC), were induced by BA [lithocholic acid (LCA)] or RA (all-trans, 9-cis and 13-cis) treatment. Evidence for increased Nrf2 transactivation by LCA and all-trans-RA was exemplified in HepG2 by: (1) reduced constitutive and inducible expression of GCLM/GCLC upon Nrf2 silencing via small-interfering RNA; (2) increased inducible expression of GCLM/GCLC genes by Nrf2 overexpression, but overexpression of dominant-negative Nrf2 decreased it; (3) increased nuclear accumulation of Nrf2 as signature event of receptor activation; (4) enhanced Nrf2-dependent antioxidant-response-element (ARE) reporter activity as indicative of increased Nrf2 transactivation; and (5) increased Nrf2 occupancy to AREs of GCLM and GCLC. Additionally, in BA-treated HepG2 cells, we observed concomitant increases of many ATP-binding cassette (ABC) transporters (MRPs 1-5, MDR1 and BCRP) in parallel with increased cellular efflux. Nrf2 silencing in HepG2 cells decreased constitutive and inducible expression of MRP2, MRP3 and ABCG2. However, Nrf2-silenced mouse hepatoma cells, Hepa1c1c7, and Nrf2-/- mice had decreased constitutive and/or inducible expression of Mrps 1-4, suggesting species differences in Nrf2-dependent regulation of hepatic ABC transporters. Protection by Nrf2 against BA and RA toxicities was confirmed by observations that Nrf2 silencing increased cell susceptibility to BA- and RA-induced cell death. Moreover, Nrf2-/- mice suffered more severe liver injury than the wildtype. Increased GSH and efflux activity following increased GCLM/GCLC and ABC transporters, respectively, can mitigate LCA toxicity. Activation of MEK1-ERK1/2 MAPK was shown to primarily mediate Nrf2 transactivation and LCA-induced expression of antioxidant proteins and Nrf2-dependent and -independent ABC transporters. In conclusion, Nrf2 activation by BA and RA led to coordinated induction of antioxidant and ABC proteins, thereby counteracting resultant oxidative cytotoxicity. The potential of targeting Nrf2 in management of BA and RA toxicities merits further investigation.

The aim of this thesis was to establish methods for selected PAPP-A (Pregnancy- Associated Plasma Protein A) gene polymorphisms analysis and to study genetic background of PAPP-A and biochemical background of PAPP-A and PlGF (Placental Growth Factor) in relation to risk pregnancy. Secondly, the aim was to establish method for two-dimensional (2D) electrophoresis of amniotic fluid. Methods for analysis of ten PAPP-A gene polymorphisms were established. These polymorphisms, PAPP-A and PlGF levels were studied in together 165 women in third trimester pregnancies complicated with threatening preterm labor (n=98), preeclampsia (n=35), IUGR (Intrauterine Growth Restriction) (n=34) and ICP (Intrahepatic Cholestasis of Pregnancy) (n=15). 114 healthy pregnant women served as controls. The method for 2D electrophoresis of amniotic fluid was established. Preeclamptic patients had significantly higher frequency of TT genotype of Cys327Cys (C/T) PAPP-A gene polymorphism compared to controls. Patients with ICP had increased serum levels of PAPP-A compared to controls, in patients with threatening preterm labor PAPP-A levels were rather decreased. PlGF levels did not differ from control group in patients with ICP and threatening preterm labor. Positive correlation was found between PAPP-A and PlGF in group of...

The aim of this thesis was to establish methods for selected PAPP-A (Pregnancy- Associated Plasma Protein A) gene polymorphisms analysis and to study genetic background of PAPP-A and biochemical background of PAPP-A and PlGF (Placental Growth Factor) in relation to risk pregnancy. Secondly, the aim was to establish method for two-dimensional (2D) electrophoresis of amniotic fluid. Methods for analysis of ten PAPP-A gene polymorphisms were established. These polymorphisms, PAPP-A and PlGF levels were studied in together 165 women in third trimester pregnancies complicated with threatening preterm labor (n=98), preeclampsia (n=35), IUGR (Intrauterine Growth Restriction) (n=34) and ICP (Intrahepatic Cholestasis of Pregnancy) (n=15). 114 healthy pregnant women served as controls. The method for 2D electrophoresis of amniotic fluid was established. Preeclamptic patients had significantly higher frequency of TT genotype of Cys327Cys (C/T) PAPP-A gene polymorphism compared to controls. Patients with ICP had increased serum levels of PAPP-A compared to controls, in patients with threatening preterm labor PAPP-A levels were rather decreased. PlGF levels did not differ from control group in patients with ICP and threatening preterm labor. Positive correlation was found between PAPP-A and PlGF in group of...