Dissertationen zum Thema „Hepatocyt“

Resveratrol is reported to possess chemotherapeutic properties in several cancers. In this study, we wanted to investigate the molecular mechanisms of resveratrol-induced cell cycle arrest and apoptosis as well as the impact of resveratrol on NAMPT and SIRT1 protein function and asked whether there are differences in hepatocarcinoma cells (HepG2, Hep3B cells) and non-cancerous primary human hepatocytes. We found a lower basal NAMPT mRNA and protein expression in hepatocarcinoma cells compared to primary hepatocytes. In contrast, SIRT1 was significantly higher expressed in hepatocarcinoma cells than in primary hepatocytes. Resveratrol induced cell cycle arrest in the S- and G2/M- phase and apoptosis was mediated by activation of p53 and caspase-3 in HepG2 cells. In contrast to primary hepatocytes, resveratrol treated HepG2 cells showed a reduction of NAMPT enzymatic activity and increased p53 acetylation (K382). Resveratrol induced NAMPT release from HepG2 cells which was associated with increased NAMPT mRNA expression. This effect was absent in primary hepatocytes where resveratrol was shown to function as NAMPT and SIRT1 activator. SIRT1 inhibition by EX527 resembled resveratrol effects on HepG2 cells. Furthermore, a SIRT1 overexpression significantly decreased both p53 hyperacetylation and resveratrol-induced NAMPT release as well as S-phase arrest in HepG2 cells. We could show that NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and primary hepatocytes and that resveratrol did not act as a SIRT1 activator in hepatocarcinoma cells.

Background Hedgehog signaling plays an important role in embryonic development, organogenesis and cancer. In the adult liver, Hedgehog signaling in non-parenchymal cells has been found to play a role in certain disease states such as fibrosis and cirrhosis. However, whether the Hedgehog pathway is active in mature healthy hepatocytes and is of significance to liver function are controversial. Findings Two types of mice with distinct conditional hepatic deletion of the Smoothened gene, an essential co-receptor protein of the Hedgehog pathway, were generated for investigating the role of Hedgehog signaling in mature hepatocytes. The knockout animals (KO) were inconspicuous and healthy with no changes in serum transaminases, but showed a slower weight gain. The liver was smaller, but presented a normal architecture and cellular composition. By quantitative RT-PCR the downregulation of the expression of Indian hedgehog (Ihh) and the Gli3 transcription factor could be demonstrated in healthy mature hepatocytes from these mice, whereas Patched1 was upregulated. Strong alterations in gene expression were also observed for the IGF axis. While expression of Igf1 was downregulated, that of Igfbp1 was upregulated in the livers of both genders. Corresponding changes in the serum levels of both proteins could be detected by ELISA. By activating and inhibiting the transcriptional output of Hedgehog signaling in cultured hepatocytes through siRNAs against Ptch1 and Gli3, respectively, in combination with a ChIP assay evidence was collected indicating that Igf1 expression is directly dependent on the activator function of Gli3. In contrast, the mRNA level of Igfbp1 appears to be controlled through the repressor function of Gli3, while that of Igfbp2 and Igfbp3 did not change. Interestingly, body weight of the transgenic mice correlated well with IGF-I levels in both genders and also with IGFBP-1 levels in females, whereas it did not correlate with serum growth hormone levels. Conclusions Our results demonstrate for the first time that Hedgehog signaling is active in healthy mature mouse hepatocytes and that it has considerable importance for IGF-I homeostasis in the circulation. These findings may have various implications for mouse physiology including the regulation of body weight and size, glucose homeostasis and reproductive capacity.

Primary human hepatocytes are a scarce resource with variable function, which diminishes with time in culture. As a consequence their use in tissue modelling and therapy is restricted. Human embryonic stem cells (hESCs) could provide a stable source of human tissue due to their properties of self-renewal and their ability to give rise to all three germ layers. hESCs have the potential to provide an unlimited supply of hepatic endoderm (HE) which could offer efficient tools for drug discovery, disease modelling and therapeutic applications. In order to create a suitable environment to enhance HE formation, hESC culture needed to be standardised. As such, a media trail was carried out to define serum free media capable of maintaining hESC in a pluripotent undifferentiated state. We also ensured hESC cultured in the various media could be directly differentiated to HE in a reproducible and efficient manner. The project then focused on the effect of post-translational modifications (PTMs), specifically SUMOylation, in hepatocyte differentiation and its subsequent manipulation to enhance HE viability. SUMOylation is a PTM known to modify a large number of proteins that play a role in various cellular processes including: cell cycle regulation, gene transcription, differentiation and cellular localisation. We hypothesised that SUMO modification may not only regulate hESC self renewal, but also maybe required for efficient hESC differentiation. We therefore interrogated the role of SUMOylation in hESC differentiation to hepatic endoderm (HE). hESC were differentiated and the cellular lysates were analysed by Western blotting for key proteins which modulate the conjugation and de conjugation of SUMO. We demonstrate that peak levels of SUMOylation were detectable in hESC populations and during cellular differentiation to definitive endoderm (DE), day 5. Following commitment to DE we observed a decrease in the level of SUMO modified proteins during cellular specialisation to a hepatic fate, corresponding with an increase in SENP 1, a SUMO deconjugation enzyme. We also detected reduced levels of hepatocyte nuclear factor 4 α (HNF4α), a critical regulator of hepatic status and metabolic function, as SUMOylation decreased. As a result, we investigated if HNF4α was SUMOylated and if this process was involved in modulating HNF4α’s critical role in HE. HNF4α is an important transcription factor involved in liver organogenesis during development and is a key regulator for efficient adult liver metabolic functions. We observed a decreasing pattern of HNF4α expression at day 17 of our differentiation protocol in conjunction with a decrease in SUMO modified proteins. In order to further investigate and validate a role of SUMOylation on HNF4α stability Immunoprecipitation (IP) was employed. HNF4α protein was pulled down and probed for SUMO 2. Results show an increase in the levels of SUMO2 modification as the levels of HNF4α decrease. Through deletion and mutation analysis we demonstrated that SUMO modification of HNF4α was restricted to the C-terminus on lysine 365. Protein degradation via the proteasome was responsible for the decrease in HNF4α, demonstrated by the use of a proteasome 26S inhibitor MG132. Additionally, a group at the University of Dundee has shown that polySUMOylation of promyelocytic leukaemia protein (PML) leads to its subsequent ubiquitination via RNF4, an ubiquitin E3 ligase, driving its degradation. Using an in vitro ubiquitination assay, we show that polySUMOylated HNF4α is preferentially ubiquitinated in the presence of RNF4. Overall polySUMOylation of HNF4α may reduce its stability by driving its degradation, hence regulating protein activity. In conclusion, polySUMOylation of HNF4α is associated with its stability. HNF4α is subsequently important for HE differentiation both driving the formation of the hepatocytes and in maintaining a mature phenotype, in agreement with a number of different laboratories. Creating the ideal environment for sustaining mature functional hepatocytes, primary and those derived from hESCs and iPSCs, is essential for further use in applications such as drug screening, disease modelling and extracorporeal devices.

Synthetic biology is a fairly recent field that aims to engineer novel functions in biological systems. In a broad sense synthetic biology encompasses the development of tools that makes the engineering of biology easier. In this thesis I develop a collection of standard DNA parts (Biobricks) that consists of a tool to build custom eukaryotic plasmids. This is not just intended for biology researchers in the field of synthetic biology, but also for more general use. Besides the development of molecular biology tools that facilitate the engineering of biology, synthetic biology researchers have implemented devices that are electronics-like in behavior and have demonstrated the potential of the field for the production of biofuels, pharmaceutics and biosensors. Here I present a sensor of iron regulatory protein activity, based on Biobricks. To demonstrate its use I apply it to the study of a novel reconstituted two cell-type co-culture (BNL CL.2 and RAW 264.7), surrogate for hepatocyte-macrophage communication
La biología sintética es un campo recientemente desarrollado con el objectivo de implementar nuevas funciones en sistemas biológicos. De forma global, la biología sintética incluye el desarrollo de herramientas para facilitar la ingeniería de sistemas biológicos. En diversas publicaciones, investigadores en el campo de la biología sintética han implementado dispositivos que funcionan de forma similar a circuitos electrónicos y han demonstrado el potencial del campo para la producción de biocarburantes, farmaceuticos y biosensores. Para la presente tesis he creado una colección de plasmidos estandarizados (Biobricks) que pueden ser de interés para biólogos fuera del campo da la biología sintética. Además, utilizando estos Biobricks, he creado un sensor de la actividad de las proteínas reguladas por el hierro. Para demonstrar su aplicación, he utilizado el sensor para estudiar un nuevo sistema de co-cultura de dos tipos celulares (BNL CL.2 y RAW 264.7), substituto para la comunicación entre hepatocitos y macrófagos

The liver is the major detoxifying organ, clearing the blood from drugs and other xenobiotics. The extent of hepatic clearance (CL) determines drug exposure and hence, the efficacy and toxicity associated with exposure. Drug-drug interactions (DDIs) that alter the hepatic CL may cause more or less severe outcomes, such as adverse drug reactions. Accurate predictions of drug CL and DDI risk from in vitro data are therefore crucial in drug development. Liver CL depends on several factors including the activities of transporters involved in the hepatic uptake and efflux. The work in this thesis aimed at developing new in vitro and in silico methods to predict hepatic transporter-mediated CL and DDIs in vivo. Particular emphasis was placed on interactions involving the hepatic uptake transporters OATP1B1, OATP1B3, and OATP2B1. These transporters regulate the plasma concentration-time profiles of many drugs including statins. Inhibition of OATP-mediated transport by 225 structurally diverse drugs was investigated in vitro. Several novel inhibitors were identified. The data was used to develop in silico models that could predict OATP inhibitors from molecular structure. Models were developed for static and dynamic predictions of in vivo transporter-mediated drug CL and DDIs. These models rely on a combination of in vitro studies of transport function and mass spectrometry-based quantification of protein expression in the in vitro models and liver tissue. By providing estimations of transporter contributions to the overall hepatic uptake/efflux, the method is expected to improve predictions of transporter-mediated DDIs. Furthermore, proteins of importance for hepatic CL were quantified in liver tissue and isolated hepatocytes. The isolation of hepatocytes from liver tissue was found to be associated with oxidative stress and degradation of transporters and other proteins expressed in the plasma membrane. This has implications for the use of primary hepatocytes as an in vitro model of the liver. Nevertheless, by taking the altered transporter abundance into account using the method developed herein, transport function in hepatocyte experiments can be scaled to the in vivo situation. The concept of protein expression-dependent in vitro-in vivo extrapolations was illustrated using atorvastatin and pitavastatin as model drugs.

Human anaphylatoxin C3a had previously been shown to increase glycogenolysis in perfused rat liver and prostanoid formation in rat liver macrophages. Surprisingly, human C5a, which in other systems elicited stronger responses than C3a, did not increase glycogenolysis in perfused rat liver. Species incompatibilities within the experimental system had been supposed to be the reason. The current study supports this hypothesis: (1) In rat liver macrophages that had been maintained in primary culture for 72 h recombinant rat anaphylatoxin C5a in concentrations between 0.1 and 10 pg/ml increased the formation of thromboxane A₂, prostaglandin D₂, E₂ and F₂α6- to 12-fold over basal within 10 min. In contrast, human anaphylatoxin C5a did not increase prostanoid formation in rat Kupffer cells. (2) The increase in prostanoid formation by recombinant rat C5a was specific. It was inhibited by a neutralizing monoclonal antibody. (3) In co-cultures of rat hepatocytes and rat Kupffer cells but not in hepatocyte mono-cultures recombinant rat C5a increased glycogen phosphorylase activity 3-fold over basal. This effect was inhibited by incubation of the co-cultures with 500 μM acetylsalicyclic acid. Thus, C5a generated either locally in the liver or systemically e.g. in the course of sepsis, may increase hepatic glycogenolysis by a prostanoid-mediated intercellular communication between Kupffer cells and hepatocytes.

Primary human hepatocytes are a scare resource with limited lifespan and variable function which diminishes with time in culture. As a consequence, their use in tissue modelling and therapy is restricted. Human embryonic stem cells (hESC) could provide a stable source of human tissue due to their self-renewal properties and their ability to give rise to all the cell types of the human body. Therefore, hESC have the potential to provide an unlimited supply of hepatocytes. To date, the use of hESCs-derived somatic cells is limited due to the undefined, variable and xeno-containing microenvironment that influences the cell performance and life span, limiting scale-up and downstream application. Therefore, the development of highly defined cell based systems is required if the true potential of stem cell derived hepatocytes is to be realised. In order to replace the use of animal derived culture substrates to differentiate and maintain hESCs-derived hepatocytes, an interdisciplinary approach was employed to define synthetic materials, which maintain hepatocyte-like cell phenotype in culture. A simple polyurethane, PU134, was identified which improved hepatocyte performance and stability when compared to biological matrices. Moreover, the synthetic polymer was amenable to scale up and demonstrated batch-to-batch consistency. I subsequently used the synthetic polymer surface to probe the underlying biology, identifying key modulators of hepatocyte-like cell phenotype. This resulted in the identification of a novel genetic signature, MMP13, CTNND2 and THBS2, which was associated with stable hepatocyte performance. Importantly, those findings could be translated to two hESC lines derived at GMP. In conclusion, hepatocyte differentiation of pluripotent stem cells requires a defined microenvironment. The novel gene signature identified in this study represents an example of how to deliver stable hESCs-derived hepatocytes.

The primary objective of this study was to identify, develop and characterise a novel bioactive surface capable of binding hepatocytes and enabling the retention of hepatocyte-specific cell function during in-vitro culture. The materials were designed to exploit a unique characteristic of hepatocyte biology, with β-galactose moieties displayed to allow cellular adhesion via the specific asialoglycoprotein receptors (ASGP-R) found on hepatocytes. Hydrogels were created by modifying a commercially available block co-polymer of polyethylene glycol (PEG) and acrylamide, (PEGA) with galactose moieties contained within lactobionic acid (LA), producing a unique bioactive sugar-based gel. A control sugar, D-glucuronic acid (GA), was used as a non-ASGP-R binding control. Monomers used were mono- and bis-acryloamido PEG (Mw=1900 gmol-1), and dimethylacrylamide. The pendant PEGA amine groups were used as ligands to bind to the sugars. The resultant gels were characterised using Fourier Transform Infrared Spectroscopy (FT-IR), protein adsorption, Fmoc-Phe and dansyl chloride labelling. The biocompatibility of the gel surfaces was evaluated using a hepatocyte cell line and the degree of attachment, proliferation, and morphology was characterised using light microscopy, live/dead assays, DNA assays, immunochemical staining, flow cytometry and reverse-transcription polymerase chain reaction (RT-PCR).FT-IR analysis of LA revealed a distinctive band at approximately 1740cm-1 corresponding to carbonyl stretching (C=O) of carboxylic acid. This unique peak disappeared as the galactose moieties within the LA were incorporated into the PEGA gel. A similar trend was also observed with the control GA sugar within the PEGA gel, confirming that the sugars had been integrated into the material. Protein adsorption assays confirmed the non-fouling nature of PEGA. Cell culture experiments showed that hepatocytes attached preferentially to the sugar surfaces, with few cells seen on the PEGA surfaces. It was observed that cells on the PEGA with LA surface were more metabolically active, than the controls and proliferated to a monolayer by day 7 in culture. Immunocytochemical staining of the cells for actin, vinculin and phosphorylated focal adhesion kinase illustrated differences in cell morphology between cells grown on different surfaces. It was determined that the sugar PEGA surfaces maintained some characteristics of hepatocyte functionality e.g. urea synthesis over the course of 7 days. To improve the reproducibility of the surfaces generated, a preliminary investigation of two-dimensional PEG monolayer surfaces as a well defined platform for surface reactions was conducted. These were chemically functionalised in a stepwise manner with the sugars. The number of coupling steps and the choice of solvent were shown to affect the efficiency of the reaction. Further more, the need for careful sample preparation was highlighted as contamination could potentially inhibit the interpretation of the surface chemistry.The overall conclusion of this work is that saccharides within non-fouling surfaces composed of thin layers of PEG-acrylamide hydrogels are able to support hepatocyte attachment and the retention of cell type specific functions in culture. However, this preliminary work has shown that much further research is necessary to elucidate the role that the surface chemistry plays in the attachment of hepatocytes.

This thesis describes the establishment of a primary cell culture system to investigate IFN-γ-induced hepatocyte apoptosis. We hypothesised that the hepatocyte response to IFN signalling is context-dependent, influenced by both external stimuli, such as growth factors, and the internal state of the cell. This was tested using a dual approach; firstly to assess the importance of known genes, IRF-l, p53 and p21 on apoptosis in this system and the contribution of known apoptotic inducers, such as CD95 and c-myc, to IFN-γ signalling. Secondly, to assess the feasibility of a functional gene trap assay in primary cells to identify unknown effectors of IFNγ-induced apoptosis. The results show that IFNΔ induces primary hepatocyte apoptosis in the context of serum- deprivation, an effect requiring IRF-1 but not p53. A deficiency of p21 potentiated apoptosis. Rather than generating a single outcome, the cellular response to IFNγ is modulated by external factors. IFNγ-induced apoptosis was inhibited by specific cytokines, while IFNγ potentiated primary hepatocyte apoptosis induced by death receptor signalling and DNA damage, in an IRF-1-dependent manner. Further, thyroid hormone potentiated IFNγ-induced apoptosis via MAPK- mediated phosphorylation of STAT1 at serine 727. This occurred via an extra-nuclear, that is non-genomic, signalling pathway. For the first time in primary hepatocytes it is demonstrated that IFNγ-mediated apoptotic signalling required the cell surface interaction of CD95 and its ligand and that IFNγ induces soluble CD95 ligand release from hepatocyte monolayers. The characteristics of IFNγ-mediated apoptosis identified in this research led to a hypothesis that c-myc contributes to IFNγ-induced apoptosis.

Specifically we set out to identify the upstream caspases that were activated by IFNγ and the role of the mitochondria in apoptosis. We selected an in vitro primary murine hepatocytes system to investigate this pathway. Despite experimental difficulties and questions about the relevance of animal research to human disease, this system was chosen as it provided a practical model of cell injury. We demonstrate that apoptosis occurs in a significant proportion of the population 72 hrs after administration of IFNγ and that this apoptosis is subject to modulation. Serum was shown to have an effect on the kinetics of apoptosis. An interaction of the IFNγ and Fas apoptosis pathways is demonstrated with the use of the Jo2 anti-Fas antibody. Using caspase inhibitors and western blot analysis it was demonstrated that both caspase 8 and caspase 9 are crucially involved in hepatocytes apoptosis. Following 48 hours of IFNγ treatment evidence of Bid cleavage is seen. Following 72 hours of IFNγ treatment cleavage and activation of caspase 9 is seen. These results demonstrate for the first time that in IFNγ treated hepatocytes caspase 8 is activated followed by caspase 9. We hypothesised that a type II membrane death receptor pathway was operating. This hypothesis was tested with the FasL blocking antibody MFL3. IFNγ-induced apoptosis was successfully blocked with this agent. Our results lead us to conclude that IFNγ acts through the Fas pathway to achieve hepatocytes apoptosis. This finding suggests manipulation of components in the Fas pathway may be a beneficial treatment strategy in hepatitis. To investigate the involvement of the mitochondria in the pathway we administered the drug CsA to IFNγ treated cells. CsA was effective in reducing the percentage of cells showing apoptotic morphology at 72 hours. Studies of the mitochondrial membrane potential revealed that a large proportion of the population (~80%) undergo mitochondrial depolarisation. Membrane depolarisation was found to occur despite the presence of apoptosis inhibitors.  Both CsA and the caspase 8 inhibitor z-IETD-fmk, were unable to prevent mitochondrial depolarisation indicating that it is not dependent on the mitochondrial permeability transition pore or the presence of tBid.

Hepatocyte transplantation is an attractive method for the treatment of metabolic liver disease and acute liver failure. The clinical application of this method has been hampered by a large initial loss of transplanted cells. This thesis has identified and characterized an instant blood-mediated inflammatory reaction (IBMIR), which is a thromboinflammatory response from the innate immunity that may partly explain the observed loss of cells. In vitro perifusion experiments were performed and established that hepatocytes in contact with blood activate the complement and coagulation systems and induce clot formation in conjunction with the recruitment of neutrophils.  Within an hour, the hepatocytes were surrounded by platelets and entrapped in a clot infiltrated by neutrophils. Furthermore, hepatocytes expressed tissue factor (TF), and the reactions were shown to be initiated through the TF pathway. Monitoring of hepatocyte transplantation in vivo revealed activation of the same parameters as were noted in vitro. For the first time, von Willebrand factor (vWF) was identified on the hepatocyte surface, being demonstrated by flow cytometry and confocal microscopy. mRNA for vWF was also confirmed in hepatocytes. Complex formation between platelets and hepatocytes was also identified. Addition of antibodies targeting the binding site for vWF on the platelets reduced the complex formation. Two different strategies, systemic and local intervention, were applied to diminish the thromboinflammation elicited from the hepatocytes in contact with ABO-matched blood. Systemic inhibition with LMW-DS, in a clinically applicable dose, was found to be superior in controlling the IBMIR in vitro when compared to heparin. Cryopreserved hepatocytes elicited the IBMIR to the same extent as did fresh hepatocytes, and the IBMIR was equally well controlled with LMW-DS in both cryopreserved and fresh cells. Hepatocytes were coated with two layers of immobilized heparin in an attempt to protect the cells from the IBMIR. In vitro perifusion experiments showed heparinized hepatocytes triggered a significantly lower degree of IBMIR.

The transdifferentiation of pancreatic acinar cells towards hepatocytes is an event that occurs in vitro and in vivo in rodents. The B-13 cell line is a model for studying this phenomenon in vitro; it readily transdifferentiates into hepatocyte-like cells in response to glucocorticoids such as dexamethasone (DEX). The transdifferentiation event is dependent on a transient suppression of Wnt signalling followed by induction of Serine/threonine-protein kinase 1(SGK1) via interactions with the glucocorticoid receptor. This thesis has aimed to further explore pancreatic to hepatic transdifferentiation, using the B-13 cell as a model and also investigated the phenomenon in human cells. As hepatic stellate cells are involved in liver regeneration and may support the progenitor niche in liver, coculture experiments were conducted to assess their effects on B-13 transdifferentiation. Transdifferentiation was enhanced in cocultures and found to be dependent on cell-cell interaction that resulted in further suppression of the Wnt signalling pathway by myofibroblasts. B-13 transdifferentiation was shown to be able to take place in vivo for the first time; cells were found to engraft only into the liver and pancreas of NOD/SCID mice. Interestingly, only cells within the liver environment showed expression of hepatocyte-specific genes. B-13 cells were also cultured in 3D bioreactor devices where they transdifferentiated into functional hepatocyte-like cells with gene expression at levels comparable to primary rat hepatocytes. Elucidating the mechanisms involved during B-13 transdifferentiation will support the isolation of an equivalent human pancreatic cell. Studies with a human cell line and primary exocrine cells demonstrated that glucocorticoids also induce hepatocyte-gene expression, and thus the generation/isolation of a human equivalent to the B-13 is a realistic goal.

This thesis has looked at various aspects of polyploidisation in the mouse liver to obtain more information about the function of polyploidisation: The effect of the non-genotoxic carcinogen, sodium phenobarbitone (PB) on ploidy and proliferation was determined over 21 days in p53 wild type (+/+), p53 +/- and p53 null (-/-) mice. PB induced a significant increase in the proportion of 8n nuclei and this increase occurred independently of p53. Whether polyploidisation occurs as a protective mechanism or acceleration of ageing in this case is unknown. However, the results confirm that the p53 +/- mouse model, often used in short-term bioassays, may not be suitable for the identification of non-genotoxic carcinogens. The function of polyploidy with respect to increasing cell size, receptor expression and susceptibility to apoptosis was also studied. The first accurate measure of volume of isolated hepatocytes differing in ploidy and nuclearity was carried out using confocal image analysis. The increase in volume associated with increasing DNA content was found to be proportional to intercellular adhesion molecule-1 (ICAM-1) surface expression, measured by flow cytometry. Apoptosis induced by interferon-gamma (IFN-γ) in culture did not occur preferentially in polyploid hepatocytes. This would suggest that the susceptibility of polyploid cells to apoptosis depends on the circumstances. Analysis of gene expression in the different hepatocyte populations also provides information regarding the function of polyploid cells. Evaluation of the effect of fluorescent activated cell sorting (FACS) and Hoechst 33342 on RNA from sorted hepatocytes found that the quality was not affected by the procedure and the RNA was suitable for subsequent gene expression analysis. Initial experiments using microarray and real-time PCR identified several genes that were induced or repressed in cells containing greater amounts of DNA. Further biological studies will hopefully determine whether these genes are important to the function or maintenance of polyploidy in hepatocytes.

This thesis is an investigation into effect of elevated levels of hepatocyte growth factor (HGF) on immune responses. High levels of HGF are found in cancer such as in MPM patient serum and pleural fluid. However, little is known about the consequence of high HGF levels on the development and function of dendritic cells and contribution of these effects to tumour immune evasion. A pre-treatment in vitro model system was applied to study the effects of high HGF concentrations on the development of dendritic cells (DC) from monocytes. The effects of HGF on the phenotype of dendritic cell, functional characteristics, including migration, phagocytosis and T cell stimulation were analysed. Using this model system I discovered a previously un-reported immature DC-like phenotype, caused by the pre-treatment of monocytes for 24 h prior to induction of iDC development by GM-CSF/IL-4: Delayed differentiation of DC alone generates a Th2 bias, which is further enhanced by the presence of HGF. HGF pre-treated DCs express both monocyte marker CD 14 and DC marker CD209 (DC-SIGN). They are able to take up antigen by phagocytosis. However, they produce increased levels of IL-10 and express elevated levels Programmed Death 1 (PD-1) ligand, PD-L1. HGF-pre-treated DC also display impaired ability to stimulate allo-T cell proliferation and antigen-specific IFN-y production. HGF pre-treated DC induces increased IL-10 production by T cells. Blocking IL-10 with a neutralizing antibody restores normal DC differentiation, partially reduced PD-L1 levels and restored T cell stimulatory capacity of DC. The physiological relevance of these findings was demonstrated by similar effects on DC developed in the presence of mesothelioma pleural fluid, in a HGF dependent manner. This thesis demonstrates that HGF is an immunosuppressive factor that can contribute to tumour-induced regulation of DC function and T cell responses.

Heterozygous mutations and deletions of the gene that encodes the transcription factor hepatocyte nuclear factor 1β (HNF1B) are the commonest known monogenic cause of developmental kidney disease. However, diagnosis remains challenging due to phenotypic variability and frequent absence of a family history. There is also no consensus as to when HNF1B genetic testing should be performed. This thesis includes work looking at the identification of HNF1B-associated disease. An HNF1B score was developed in 2014 to help select appropriate patients for genetic testing. The aim in chapter 2 was to test the clinical utility of this score in a large number of referrals for HNF1B genetic testing to the UK diagnostic testing service for the HNF1B gene. An HNF1B score was assigned for 686 referrals using clinical information available at the time of testing; performance of the score was evaluated by receiver-operating characteristic curve analysis. Although the HNF1B score discriminated between patients with and without a mutation/deletion reasonably well, the negative predictive value of 85% reduces its clinical utility. HNF1B-associated disease is due to an approximate 1.3 Mb deletion of chromosome 17q12 in about 50% of individuals. This deletion includes HNF1B plus 14 additional genes and has been linked to an increased risk of neurodevelopmental disorders, such as autism. The aim in chapter 3 was to compare the neurodevelopmental phenotype of patients with either an HNF1B intragenic mutation or 17q12 deletion to determine whether haploinsufficiency of the HNF1B gene is responsible for this aspect of the phenotype. Brief behavioural screening showed high levels of psychopathology and impact in children with a deletion. 8/20 (40%) patients with a deletion had a clinical diagnosis of a neurodevelopmental disorder compared to 0/18 with a mutation, P=0.004. 17q12 deletions were also associated with more autistic traits. Two independent clinical geneticists were able to predict the presence of a deletion with a sensitivity of 83% and specificity of 79% when assessing facial dysmorphic features as a whole. These results demonstrate that the 17q12 deletion but not HNF1B intragenic mutations are associated with neurodevelopmental disorders; we conclude that the HNF1B gene is not involved in the neurodevelopmental phenotype of these patients. Extra-renal phenotypes frequently occur in HNF1B-associated disease, including diabetes mellitus and pancreatic hypoplasia. Faecal elastase-1 levels have only been reported in a small number of individuals, the majority of which have diabetes. In chapter 4 we measured faecal elastase-1 in patients with an HNF1B mutation or deletion regardless of diabetes status and assessed the degree of symptoms associated with pancreatic exocrine deficiency. We found that faecal elastase-1 deficiency is a common feature of HNF1B-associated renal disease even when diabetes is not present and pancreatic exocrine deficiency may be more symptomatic than previously suggested. Faecal elastase-1 should be measured in all patients with a known HNF1B molecular abnormality complaining of chronic abdominal pain, loose stools or unintentional weight loss. Hypomagnesaemia is a common feature of HNF1B-associated disease and is due to renal magnesium wasting. The aim in chapter 5 was to measure both serum and urine magnesium and calcium levels in individuals with an HNF1B molecular defect and compare to a cohort of patients followed up in a general nephrology clinic in order to assess their potential as biomarkers for HNF1B-associated disease. The results of this pilot study show that using a cut-off for serum magnesium of ≤0.75 mmol/L was 100% sensitive and 87.5% specific for the presence of an HNF1B mutation/deletion. All individuals in the HNF1B cohort had hypermagnesuria with fractional excretion of magnesium >4%; a cut-off of ≥4.1% was 100% sensitive and 71% specific. This suggests serum magnesium levels and fractional excretion of magnesium are highly sensitive biomarkers for HNF1B-associated renal disease; if these results are confirmed in a larger study of patients with congenital anomalies of the kidneys or urinary tract they could be implemented as cheap screening tests for HNF1B genetic testing in routine clinical care.

A malária é uma das doenças infecciosas mais importantes a nível mundial, sendo anualmente responsável por mais de 1 milhão de mortes. O agente causador da doença é o parasita intracelular, denominado Plasmodium, que possui um ciclo de vida bastante complexo. A infecção tem início com a inoculação de esporozoítos através da picada de um mosquito fêmea Anopheles, o vector de transmissão da doença. Uma vez na corrente sanguínea, o esporozoíto migra até ao fígado onde infecta a célula hospedeira, o hepatócito. No fígado, o parasita replica-se e desenvolve-se até atingir o seu próximo estado de maturação – o merozoíto. Dada esta complexidade, é natural que o Plasmodium provoque no hospedeiro uma variedade de mecanismos distintos, especialmente ao nível imunitário. Por isso, a resposta imune desenvolvida pelo hospedeiro, contra o parasita, é caracterizada como sendo complexa e específica em relação à espécie e ao estádio do mesmo. De forma a adquirir uma resposta imune protectora contra a malária é necessário que o indivíduo seja infectado consecutivamente durante a vida. Mesmo assim o resultado obtido é apenas uma imunidade parcial contra o parasita. Melhorias significativas têm sido registadas, no que diz respeito à compreensão dos mecanismos de protecção envolvidos na doença, assim como na identificação de novas moléculas que possam ser utilizadas no desenvolvimento de novas vacinas. No entanto, ainda não está disponível uma vacina que seja eficaz conferindo uma protecção total. O uso de esporozoítos irradiados em imunizações induz uma protecção total contra a doença, que é mediada pela activação de linfócitos T CD8+ específicos para antigénios do parasita. O início desta resposta é mediado por células dendríticas, embora a origem dos antigénios intervenientes seja ainda desconhecida. Os esporozoítos irradiados conseguem infectar os hepatócitos. Contudo, não são capazes de progredir para a fase sanguínea da doença. Este desenvolvimento incompleto da fase hepática é uma característica fundamental para que ocorra imunidade. Embora alguns dos mecanismos protectores induzidos pela infecção com esporozoítos irradiados já tenham sido identificados é, ainda, necessário proceder a uma caracterização detalhada dos mesmos. Sendo o fígado um local de extrema importância durante o ciclo de vida do parasita da malária, qualquer descoberta ao nível das interacções que se estabelecem entre o Plasmodium e o hepatócito, terá uma repercussão no melhoramento do processo de indução de uma resposta imune contra a doença. Utilizando um modelo murino, demonstrou-se que os hepatócitos infectados com esporozoítos irradiados entram em apoptose logo após o início da infecção. Durante esta fase as célulasdendríticas são recrutadas para o fígado, local onde fagocitam os corpos apoptóticos provenientes dos hepatóctios que entraram em morte celular. Uma vez que estas células são capazes de apresentar antigénios exógenos e ainda induzir o priming e activação das células T, os resultados por nós obtidos sugerem que os hepatócitos infectados apoptóticos são a fonte de antigénios do parasita utilizada pelas células dendríticas durante a iniciação de uma resposta imune contra a malária. Durante o curso de uma infecção, a morte celular possui um papel fundamental no estabelecimento de uma resposta imune contra um agente patogénico. Os parasitas possuem a capacidade de modular esta resposta através da indução ou inibição da morte da célula hospedeira, de forma a possibilitar o seu desenvolvimento e sobrevivência. Previamente, foi demonstrado que durante a migração dos esporozoítos através dos hepatócitos, as células atravessadas secretam um factor de crescimento específico, o HGF -“hepatocyte growth factor”, que aumenta a susceptibilidade celular à infecção. Esta via de sinalização iniciada pelo HGF através do seu receptor, o MET, provoca uma série de efeitos em diversos tipos de células. Entre eles destaca-se a protecção contra a morte celular programada. Considerando tal facto, estudou-se qual o efeito desta protecção durante a infecção por Plasmodium, tendo como hipótese que a activação da via de sinalização do HGF/MET induziria uma protecção da apoptose nas células infectadas. Os resultados por nós obtidos confirmaram esta teoria. A inibição desta via de sinalização induziu um aumento na quantidade de morte celular observada. Tendo em conta que, usualmente, a activação da sinalização do HGF ocorre segundo o sinal de transdução do PI3K/Akt, testou-se se o bloqueio desta via produzia algum efeito na infecção. De facto, os resultados observados indicam que esta via de sinalização é utilizada durante a infecção quando o HGF/MET é activado. Estas observações demonstram que a inibição da apoptose da célula hospedeira durante a infecção por Plasmodium é necessária para ocorrer doença. Na parte final deste trabalho, procurou-se ainda identificar um gene do parasita responsável pela inibição da morte do hepatócito. Algumas observações preliminares levaram-nos a sugerir que a proteína HSP70 do parasita possa exercer uma função neste processo, daí que sugerimos que no futuro este envolvimento seja mais aprofundado. Assim, os resultados apresentados nesta tese contribuem para um maior esclarecimento e compreensão das interacções que se estabelecem no fígado aquando da infecção por Plasmodium, e para um conhecimento mais alargado da relação entre o parasita e o hepatócito.
Malaria is one of the most predominant infectious diseases worldwide, accounting for more than 1 million deaths annually. The intracellular parasite Plasmodium is the causative agent of malaria which undergoes a complicated life cycle. Infection is initiated by inoculation of sporozoites through mosquito bite, which journey to the liver where they must migrate and invade hepatocytes in order to replicate and mature. Immunity to malaria is complex and is essentially both species and stage specific, thus a wide variety of distinct immune mechanisms are provoked by the parasite in the host. The generation and maintenance of protective immune responses requires repeated infections over the lifetime of an individual and even though only partial immunity is achieved against the disease. Despite the significant advances in understanding mechanisms of protection and identifying new targets for vaccine design, an effective protection against malaria is still not available. However, immunization with irradiated Plasmodium sporozoites induces antigen-specific CD8+ T cells immune response that confers complete protection against malaria. The initiation of this response is mediated by dendritic cells, but the source of parasite antigens intervening in this response remains unknown. Irradiated sporozoites are capable of infecting hepatocytes but do not progress into blood stages forms. Both this incomplete liver development and the hepatic stage itself are indispensable steps for the outcome of a successful malaria protection. Although some protective mechanisms conferred by irradiated sporozoites have been identified, a thorough characterization is still needed. The liver plays a key role in the life cycle of the malaria parasite and therefore insights into Plasmodium-hepatocyte interactions will have a promising effect in improving the process of triggering an immune response against the disease. Using a rodent malaria model, we show that hepatocytes infected with irradiated Plasmodium sporozoites undergo apoptosis shortly after infection. In addition, after infection dendritic cells are recruited to the liver where they phagocytose apoptotic bodies derived from infected hepatocytes. Given that dendritic cells are capable of cross-presenting exogenous antigens and elicit the priming and activation of T cells, our results suggest that the apoptotic Plasmodium infected hepatocytes provide a source of parasite antigens for the initiation of the protective immune response against the disease. Cell death plays a central role in the course of an infection helping establish an immune response against a pathogen. Furthermore, some parasites have the capacity to modulate this response by apoptosis induction or inhibition of the infected host cell, in order to survive and develop within the host.Previously it was shown that wounding of hepatocytes by sporozoite migration induces the secretion of hepatocyte growth factor (HGF) by traversed cells, which renders neighbor epatocytes susceptible to infection. The signaling initiated by HGF through its receptor MET has multifunctional effects on various cell types. Survival signals and protection of host cells is one of these features of HGF/MET signaling. The role of this protection on Plasmodium infected hepatocytes was also a subject of study in this thesis. Therefore, we hypothesize that HGF/MET would induce in infected host cells protection from apoptosis, which in turn would lead to an increased infection. Our data confirms that HGF/MET signaling protects infected cells from apoptosis, since an increase in apoptosis of infected cells was observed when the signaling pathway was inhibited. Given that HGF inhibits cell death primarily through the PI3-kinase/Akt signal transduction pathway, we tested if the infection susceptibility increase was impaired by inhibition of this pathway. In fact, inhibition of PI3-kinase completely abrogates the HGF effect on malaria infection. Taken together, these results implicate that the permissive effect of HGF for susceptibility to malaria infection is, at least in part, mediated by its anti-apoptotic signal. To our knowledge, these results demonstrate for the first time that active host’s cell apoptosis inhibition during infection by Plasmodium is required for a successful infection. Finally, an attempt at identifying a Plasmodium candidate gene responsible for the apoptosis inhibition of the host cell was carried out. Preliminary results evidence a promising role for Plasmodium heat shock protein 70 which broad function should be studied in the future. In summary, data presented in this thesis contributes to a wider understanding of the events that occur in the liver during a malaria infection and expand our knowledge within the interactions established between the malaria parasite and its host.

Freshly isolated hepatocytes from fed and starved rats were used as a model in the investigation of the mechanisms by which cadmium chloride exerts its toxic effects at the cellular level. Exposure to cadmium chloride resulted in a slight decrease in viability, more pronounced in hepatocytes from starved rats. Morphological changes preceded the increase in membrane permeability. Hepatocytes exhibited a rapid initial uptake of cadmium chloride, followed by a second slower phase. The accumulation of more metal in hepatocytes from starved rats may contribute to their enhanced susceptibility to cadmium chloride. Adverse metabolic effects of cadmium chloride included an increase in the lactate:pyruvate ratio in hepatocytes from fed rats, with a concomitant decrease in the 3-hydroxybutyrate:acetoacetate ratio in hepatocytes from fed and starved rats. Incubation with cadmium chloride resulted in increased glycogenolysis and glycolysis. Decreased rates of gluconeogenesis from lactate and pyruvate reflected the decreased uptake of gluconeogenic precursors. Studies of intracellular lactate concentrations could not resolve whether the decrease in gluconeogenesis was due to an inhibition of lactate transport into the hepatocyte or due to a decrease in its metabolism. Cadmium chloride caused a slight decrease in the basal and pyruvate-stimulated rates of cellular respiration, a marked dose-related decrease with lactate, and no significant effects with succinate. Carbonyl- cyanide-m-chlorophenylhydrazone was less effective in stimulating respiration in hepatocytes incubated with cadmium chloride, this effect being more pronounced with lactate and pyruvate than with succinate. Cadmium chloride had little effect on the uncoupled rates of FADH2 oxidation with succinate suggesting that electron transport from succinate dehydrogenase to cytochrome a/a3 was not impaired. The results from these studies suggest a primary effect of cadmium chloride on mitochondrial function and cellular energy production, resulting in secondary metabolic changes in an attempt to overcome the declining levels of ATP within the cell.

Liver surgery, be it for resection or transplantation, causes injury to the organ. Pre-treatment of the liver to protect it from injury would be of clinical interest as poor liver function following surgery can be life threatening. Such “preconditioning” treatments could be applied to both elective liver surgery and liver transplantation. The aim of this project was to establish the ability of certain pharmacological agents to induce the stress response in human hepatocyte, and to protect them from the type of injury seen in surgical practice.  Curcumin, an extract of the spice turmeric, was found to induce the enzyme heme oxygenase-1. It preconditioned hepatocytes to survive in models mimicking both ischaemia-reperfusion injury and the cold preservation injury which occurs during transplantation. Cyclosporin A was also studied as a putative preconditioning agent. However, although it did have some effect on the stress response, it did not precondition human hepatocytes. As an extension of the work on heme oxygenase-1, the effect of a promoter polymorphism in the human HO-1 gene was studied. This work has demonstrated that curcumin does pharmacologically precondition human hepatocytes in vitro. It has also strengthened the evidence for heme oxygenase-1 as a target for preconditioning strategies.

The electrophysiologic technique (Reuss, L., Proc. Natl. Acad. Sci. USA 82:6014, 1985) was modified to measure changes in steady-state hepatocyte volume during osmotic stress. Hepatocytes in mouse liver slices were loaded with tetramethylammonium ion (TMA$\sp{+}$) during transient exposure of cells to nystatin. Intracellular TMA$\sp{+}$ activity (a$\sp{\rm i}\sb{\rm TMA}$) was measured with TMA$\sp{+}$-sensitive, double-barreled microelectrodes. Loading hepatocytes with TMA$\sp{+}$ did not change their membrane potential (V$\sb{\rm m}$), and under steady-state conditions a$\sp{\rm i}\sb{\rm TMA}$ remained constant over 4 min in single impalements. Hyperosmotic solutions (50, 100, & 150 mM sucrose added to media) and hyposmotic solutions (sucrose in media reduced by 50 & 100 mM) increased and decreased a$\sp{\rm i}\sb{\rm TMA}$, respectively, which suggested transmembrane water movements. The regression coefficient of the ratio of control a$\sp{\rm i}\sb{\rm TMA}$/experimental a$\sp{\rm i}\sb{\rm TMA}$ versus the relative osmolality of media (experimental mOsm/control mOsm) was -0.34 $\pm$ 0.03, p $<$ 0.001, which is less than expected for a perfect osmometer. Corresponding measurements of V$\sb{\rm m}$ showed that its magnitude increased with hyposmolality and decreased with hyperosmolality. When Ba$\sp{2+}$ (2 mM) was present during hyposmotic stress of 0.66 $\times$ 286 mOsm (control), cell water volume increased by a factor of 1.44 $\pm$ 0.02 compared with that of hyposmotic stress alone, which increased cell water volume by a factor of only 1.12 $\pm$ 0.02, p $<$ 0.001. Ba$\sp{2+}$ also decreased the hyperpolarization of V$\sb{\rm m}$ due to hyposmotic stress from a factor of 1.62 $\pm$ 0.04 to 1.24 $\pm$ 0.09, p $<$ 0.01. When verapamil (50 $\mu$M) was present during hyposmotic stress of 0.69 $\times$ 292 mOsm (control), cell water volume increased by a factor of 1.42 $\pm$ 0.02 compared with that of hyposmotic stress alone, which increased cell water volume by a factor of only 1.19 $\pm$ 0.02, p $<$ 0.001. Verapamil also decreased the hyperpolarization of V$\sb{\rm m}$ due to hyposmotic stress from a factor of 1.34 $\pm$ 0.07 to 1.08 $\pm$ 0.08, p $<$ 0.05. Similar results were obtained when exposing hepatocytes to Ca$\sp{2+}$-free medium plus EGTA (5 mM). It was concluded that hepatocytes partially regulate their steady-state volume during hypo- and hyperosmotic stress. However, volume regulation during hyposmotic stress diminished along with hyperpolarization of V$\sb{\rm m}$ in the presence of the K$\sp{+}$-channel blocker, Ba$\sp{2+}$, the Ca$\sp{2+}$-channel blocker, verapamil and the Ca$\sp{2+}$-chelator, EGTA. This indicated that cell calcium and membrane potassium conductance (g$\sb{\rm K}$) were involved in hepatocyte volume regulation mechanism and that variation in V$\sb{\rm m}$ provides an intercurrent, electromotive force for hepatocyte volume regulation.