The discovery that proprotein convertase subtilisin/kexin type 9 (PCSK9) mediates degradation of low-density lipoprotein receptors (LDLR) indicates a critical role in LDL metabolism. PCSK9 is a secreted protein that binds to the epidermal growth factor-like (EGF)-A domain of LDLR and directs the receptor for degradation in lysosomes by an unknown mechanism. A gain-of-function mutation, D374Y, increases binding to LDLR EGF-A >10-fold and is associated with a severe form of hypercholesterolemia in humans. Similar to previous studies, data obtained in my project has established that PCSK9 was capable of promoting robust LDLR degradation in liver-derived cell lines; however, minimal effects on LDLR levels were detected in several lines of fibroblast cells despite normal LDLR-dependent cellular uptake of PCSK9. Importantly, a PCSK9 degradation assay showed that 125I-labeled wild-type PCSK9 was internalized and degraded equally in both hepatic and fibroblast cells, indicating dissociation of wild-type PCSK9 from recycling LDLRs in fibroblasts. Moreover, PCSK9 recycling assays confirmed that no recycling of wild-type PCSK9 to the cell surface could be detected in fibroblast cells. In contrast, more than 60% of internalized PCSK9-D374Y recycled to the cell surface in these cells, and thus had reduced ability to direct the LDLR for lysosomal degradation despite persistent binding. Co-localization studies indicated that PCSK9-D374Y trafficked to both lysosomes and recycling compartments in fibroblast cells, whereas wild-type PCSK9 exclusively trafficked to lysosomes. We conclude that two factors diminish PCSK9 activity in fibroblast cells: i) an increased dissociation from the LDLR in early endosomal compartments, and ii) a decreased ability of bound PCSK9 to direct the LDLR to lysosomes for degradation. Finally, an LDLR variant that binds to PCSK9 in a Ca2+-independent manner could partially restore wild-type PCSK9 activity, but not PCSK9-D374Y activity, in fibroblast cells.

Le carcinome hépatocellulaire (CHC) est un cancer très fréquent - au 5ème rang de l’échelon mondial - dont l’incidence ne cesse d’augmenter. Lié aux maladies chroniques du foie (hépatite C, syndrome métabolique et, le plus souvent, cirrhose), il représente désormais un véritable problème de santé publique. C’est la nature du foie sous-jacent qui détermine les modalités de sa prise en charge. Lorsque le foie ne présente pas de maladie chronique (foie sain), on se trouve généralement devant une tumeur déjà évoluée; dans ce cas on a recours essentiellement à la résection hépatique. Lorsque le foie présente une maladie chronique (foie pathologique), qu’il s’agisse de fibrose, cirrhose ou hépatite, c’est le stade tumoral au moment du diagnostic qui oriente le choix du traitement ; à part la transplantation, limitée dans ses indications, les options thérapeutiques comportent la résection hépatique, la destruction par voie sous-cutanée (radio fréquence) et un traitement par voie artérielle (chimioembolisation). Cependant, ces traitements à visée curative, ne peuvent être envisagés actuellement que dans 30% des cas. Notre travail porte uniquement sur la résection hépatique. Partout disponible, cette intervention chirurgicale représente en effet le traitement de référence dans la prise en charge du CHC. Nous nous appuyons sur une vaste enquête nationale qui, développée sur une période de 15 ans – de 1990 à 2005 – à partir de 23 centres de chirurgie, à porté sur plus de 2590 dossiers de patients. Grâce à ces données de base, particulièrement précieuses par leur nombre et leur précisions, nous avons tenté de donner une image panoramique des pratiques (indications, techniques opératoires) et des résultats (survie, récidive, morbidité, mortalité) de la résection hépatique pour CHC en France. Il s’agit de la plus grande étude multicentrique chirurgicale menée sur le CHC en France à ce jour. On a classé 102 paramètres dans 6 groupes de données ont été colligés pour chaque malade inclus dans l’étude: Terrain, Bilan préopératoire, Chirurgie, Anatomopathologie, Morbidité et traitements adjuvants et Evolution. Au total, cette enquête a permis de recueillir une somme considérable de données dont l’analyse multivariée avait pour l’objectif d’aboutir à des critères prédictifs de mortalité opératoire et de survie après résection sur foie sain et pathologique. Cette analyse a confirmé le développent et la qualité de la chirurgie hépatique en France. Aussi, l’analyse a montré que la résection hépatique est un traitement efficace du CHC sur foie sain et pathologique. Ces résultats et leur implication pour l’approche multidisciplinaire en cancérologie contribueront à améliorer les connaissances et la prise en charge du CHC. Finalement, à coté de la transplantation hépatique, limitée par ses indications restreintes et la pénurie de greffons, la résection du CHC occupe une place importante qui doit continuer de croître du fait de ses bons résultats et de l’augmentation constante de l’incidence du CHC / Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, and the third most common cause of cancer-related death. It is a major health problem worldwide, which represents the most prevalent primary liver cancer and constitutes the third most frequent cause of cancer-related deaths. The major risk factor for HCC is cirrhosis. All types of cirrhosis predispose to HCC, but the incidence is particularly high in persistent infection with hepatitis B (HBV) and hepatitis C (HCV) and in alcoholic liver disease. The clinical presentation and management of HCC depends on whether the liver is cirrhotic and whether there is underlying viral hepatitis. Therapeutic options fall into four main categories (1) surgical interventions, including tumor resection and liver transplantation, (2) percutaneous interventions, including ethanol injection and radiofrequency thermal ablation, (3) transarterial interventions, including embolisation and chemoembolisation and (4) drugs as well as gene and immune therapies. Potentially curative therapies are tumor resection, liver transplantation, and percutaneous interventions that can result in complete responses and improved survival in a high proportion of patients. Liver resection offers the greatest impact on survival when patients do not meet transplantation criteria and this is considered as the optimal treatment for HCC. The objective of this thesis, based on a retrospective survey, was to give an overview on conditions of realization and the results of the resection of HCC in France, in the period from 1990-2005. All the French centers of excellence in the hepatobiliary surgery were contacted and most of them accepted to participate. All the contacted units were essentially localized in University Clinical Centers, all of them experts in hepatic surgery and most of them were centers for liver transplantation. More than 2590 cases with hepatic resection were collected in this study. The file consisted of 102 questions and contained following headings: demographic data, underlined liver pathology, circumstances of diagnosis, imaging, evaluation of underlined liver pathology: biological, morphological, histological, preparation for resection: neoadjuvant treatment of the tumor, portal embolization, surgical intervention: approach, clamping, vascular control, nature and the extent of the exeresis, anatomic or non-anatomic features, histopathological analysis of the removed tissues, results: mortality, morbidity, recurrence, survival, lost from analysis. In total, this survey enabled us to collect a considerable sum of data in order to give a more precise overview on predictive criteria of per operative mortality and survival, as well as recurrence rates, after the resection of normal and pathological livers. It confirmed the development and the quality of the hepatic surgery in France

O fígado é um órgão importante para se analisar como o animal reage a agressões químicas. Um dos métodos utlizado para estudar a regeneração hepática é a hepatectomia parcial (HP). Assim, nosso objetivo foi verificar a influência da hepatectomia de 30% e de 70% sobre a taxa de proliferação das células hepática medida pela incorporação de BrdU na região da incisão e no resto do fígado e a proliferação de ductos biliares e de células epiteliais pré-ductulares biliares (CEPDBs) com o uso de uma combinação de 2 anticorpos específicos para citoqueratinas humanas, AE1/AE3. O pico de proliferação nos animais que sofreram HP de 30% do parênquima hepático deu-se após 1 dia da cirurgia enquanto que os animais que sofreram a HP de 70% o pico ocorreu no terceiro dia após a cirurgia.O índice de proliferação foi semelhante para a região próxima ou distante do corte. Para ambas as HP não houve diferença na regeneração do tecido retirado próximo ou distante da área de incisão. Nas regiões distantes da HP havia maior número de ductos havendo o pico do número de ductos coincidindo com o pico de proliferação celular de ambas as HP. Depois da HP o número de CEPDBs cresce consideravelmente. Os pico de proliferação também ocorreram no primeiro e terceiro dia para a HP de 30% e de 70% respectivamente, sendo mais altos na região distante do corte. Assim, durante a regeneração do fígado de C. gariepinus ocorre hiperplasia compensatória por proliferação de hepatócitos, de ductos biliares e de CEPDBs. A hepatectomia de 30% gerou uma resposta regenerativa intensa e é menos traumática para o animal, a região distante do corte reage mais intensamente que a região próxima do corte no que se refere aos ductos biliares e as CEPDBs. / The liver is an important organ in studies aimed to the verification of animals reactions to chemical injuries. The partial hepatectomy (PH) is one of the methods that are usually employed in hepatic regeneration experiments. Our objectives in this work were to verify the influence of 30% and 70% PH on the hepatic cells proliferation index as it is measured by BrdU nuclear uptake in two regions: close to the hepatic surgery region and in a farther region. The biliary ducts and bile preductular epithelial cells (BPDECs) proliferation was quantified with a combination of two antibodies against the human cytokeratins AE1/AE3. The proliferation index peak in 30% hepatectomy animals occurred one day after the surgery, but in 70% hepatectomy animals the peak was only observed after three days from the surgery. Differences between the proliferation index of the close and the far region were not observed and likewise histological differences were absent. In the regions far from the PH site more biliary ducts were counted than in closer regions, but the peaks were coincident in both regions with the hepatocytes proliferation index. It was also observed that the quantity of BPDECs increased. The peaks occurred in the first and third days for the 30% and 70% PH respectively, and both peaks were higher in the region far from the PH site. We may conclude that C. gariepinus liver regeneration after 30% and 70% PH occurs by means of compensatory hyperplasia by hepatocytes, biliary ducts and BPDECs proliferation. The 30% HP stimulated a strong regenerative response and is less traumatic to the animals. The regions far from the PH site reacts more intensely that the close region concerning the stimulation of biliary ducts and BPDECs proliferation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22696号 / 医博第4640号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 妹尾 浩, 教授 木村 剛 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

BACKGROUND: After proteolysis, the majority of released amino acids from dietary protein are transported to the liver for gluconeogenesis or to peripheral tissues where they are used for protein synthesis and eventually catabolized, producing ammonia as a byproduct. High ammonia levels in the brain are a major contributor to the decreased neural function that occurs in several pathological conditions such as hepatic encephalopathy when liver urea cycle function is compromised. Therefore, it is important to gain a deeper understanding of human ammonia metabolism. The objective of this study was to predict changes in blood ammonia levels resulting from alterations in dietary protein intake, from liver disease, or from partial loss of urea cycle function. METHODS: A simple mathematical model was created using MATLAB SimBiology and data from published studies. Simulations were performed and results analyzed to determine steady state changes in ammonia levels resulting from varying dietary protein intake and varying liver enzyme activity levels to simulate liver disease. As a toxicity reference, viability was measured in SH-SY5Y neuroblastoma cells following differentiation and ammonium chloride treatment. RESULTS: Results from control simulations yielded steady state blood ammonia levels within normal physiological limits. Increasing dietary protein intake by 72% resulted in a 59% increase in blood ammonia levels. Simulations of liver cirrhosis increased blood ammonia levels by 41 to 130% depending upon the level of dietary protein intake. Simulations of heterozygous individuals carrying a loss of function allele of the urea cycle carbamoyl phosphate synthetase I (CPS1) gene resulted in more than a tripling of blood ammonia levels (from roughly 18 to 60 μM depending on dietary protein intake). The viability of differentiated SH-SY5Y cells was decreased by 14% by the addition of a slightly higher amount of ammonium chloride (90 μM). CONCLUSIONS: Data from the model suggest decreasing protein consumption may be one simple strategy to decrease blood ammonia levels and minimize the risk of developing hepatic encephalopathy for many liver disease patients. In addition, the model suggests subjects who are known carriers of disease-causing CPS1 alleles may benefit from monitoring blood ammonia levels and limiting the level of protein intake if ammonia levels are high.

What is known and objective: Amiodarone, a commonly used class III antiarrhythmic agent notable for a relatively long half-life of up to 6 months and its pronounced adverse effect profile, is used for both acute and chronic management of cardiac arrhythmias. Chronic use of amiodarone has been associated with asymptomatic hepatotoxicity; however, acute toxicity is thought to be uncommon. There are only six reported cases of acute liver failure (ALF) secondary to amiodarone. In all these cases the outcome of death during the same hospitalization resulted. We aimed to report the only case of acute liver failure secondary to amiodarone infusion in the existing literature where the patient survived. Case summary: A 79-year-old woman admitted with atrial flutter was being treated with intravenous (IV) amiodarone when she abruptly developed coagulopathy, altered mental status and liver enzyme derangement. She was diagnosed with acute liver failure (ALF) secondary to an amiodarone adverse drug reaction, with a calculated score of seven on the Naranjo adverse drug reaction probability scale. Amiodarone was immediately withheld, and N-acetylcysteine (NAC) was initiated. Clinical improvement was seen within 48 hours of holding the drug and within 24 hours of initiating NAC. On post-hospital follow-up visit she was reported to have complete recovery. What is new and conclusion: This report emphasizes the importance of monitoring liver enzymes and mental status while a patient is being administered IV amiodarone. N-acetylcysteine administration may have possibly contributed to the early and successful recovery from ALF in our patient. To date, she is the only patient in the existing literature who has been reported to survive ALF secondary to amiodarone administration.

This investigation addressed several aspects of the covalent interaction of metabolites of the insecticide chlordane with cellular macromolecules in vitro. Microsomal preparations from the liver of mice and rats were used and covalent binding to microsomal protein and RNA or to added calf thymus DNA was studied. Pure 14C-labelled cis- and trans-chlordane isomers as well as an isomeric mixture (14C-cis-chlordane plus 14C-trans-chlordane 3:1, w:w) were used as substrates for the in vitro system. Biochemical parameters investigated included inhibition of microsomal mixed-function oxidase and epoxide hydratase plus the induction of these enzymes by pretreatment with chlordane or phenobarbital. The effect of these manipulations on covalent binding of the metabolites to the macromolecules was of interest. Isolation of the protein, RNA and DNA from the in vitro microsomal systems and determination of unextractable radioactivity indicated that the chlordane derived material bound to each of the macromolecules investigated. The only exception was that mouse liver microsomes did not activate trans-chlordane to a form which bound to DNA in measurable amounts under the conditions employed. Microsomal epoxide hydratase and aminopyrine demethylase activity were increased in both the rat and the mouse following chlordane pretreatment. The effect of this induction on the macromolecular interaction of chlordane metabolites was variable for both chlordane and phenobarbital pretreated groups. Generally, for the mouse, induction increased binding to protein and DNA but decreased binding to RNA. In the rat, induction decreased binding to each of the macromolecular species. The effect of enzyme inhibition was variable in both species under the different conditions tested except for the binding of cis-chlordane derived material to DNA in the mouse liver system. In that case inhibition of epoxide hydratase clearly reduced the concentration of material covalently interacting with the DNA to unmeasurable levels. The results indicated little possibility that the primary epoxide metabolite of chlordane, oxychlordane, is involved in the binding. The effects of epoxide hydratase inhibition, however, indicate that some secondary epoxide is involved in the cis-chlordane binding to DNA in the mouse. The possible analogy between the binding behavior of chlordane found in this study and the binding behavior of other well characterized toxic compounds is discussed.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19172号 / 医博第4014号 / 新制||医||1010(附属図書館) / 32164 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 平岡 眞寛, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20794号 / 医博第4294号 / 新制||医||1025(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 浅野 雅秀, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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