Studies on cellular engraftment and hepatocytic differentiation in liver injury and repair

BACKGROUND: In recent years, considerable attention has been directed towards the use of cellular therapies in the management of acute and chronic liver failure. The majority of clinical studies have utilised hepatocyte infusions, although there is limited understanding of the factors which limit the engraftment of cells in the face of liver injury. The use of stem cells as sources of hepatocytes which can contribute to liver repair has been examined in rodent models, but the extrapolation of these findings to human stem cells is unclear.AIM: In this thesis the factors which regulate the adhesion and survival of hepatocytes in the face of acute liver injury environment are examined. In addition, factors which regulate the differentiation of human stem cells towards hepatocytes are examined in vitro and in vivo.MATERIALS AND METHODS: Human hepatoblastoma (HepG2) cells were used as a model of human hepatocytes to study the effect of serum from patients with acute liver failure. Various laboratory assays were used to determine effects on adhesion, cell necrosis/apoptosis, integrin expression (flow cytometry) and integrin activation. Human cord blood was used as a source of human stem cells for both in vitro experiments and the in vivo work with the NOD-SCID miceRESULTS: Paracetamol-induced liver injury results in the marked up regulation of collagen on hepatic sinusoids. Adhesion of HepG2 cells to Collagen after exposure to fulminant serum was reduced within only a few hours. Apoptosis occurred approximately 24-48 hours after incubation and is associated with caspase3 activation. Furthermore, fulminant serum reduces the adhesive capabilities of HepG2 cells by a rapid down-regulation of their Pi-integrin activity. Loss of cellular adhesion and subsequent apoptosis can be reversed by treatment ofHepG2 cells with the stimulatory mAb TS2/16. Human cord blood could not be directed in vitro down the hepatocytic lineage under any of the different combinations of cytokines/matrix. In vivo however infused human cord blood cells are capable of engrafting into NOD-SCID mouse liver and differentiating down the hepatocytic lineage without fusion to host hepatocytesCONCLUSION: In this thesis, mechanisms regulating the engraftment and survival of HepG2 cells during exposure to fulminant serum were identified. Human stem cells were also demonstrated in vivo (but not in vitro) to differentiate into hepatocytes within the NOD-SCID mouse liver with no evidence of cellular fusion

Mesenchymal stromal cell therapy in liver disease: opportunities and lessons to be learnt?

End-stage liver disease is responsible for 30,000 deaths per year in the United States alone, and it is continuing to increase every year. With liver transplantation the only curative treatment currently available, new therapies are in great demand. Mesenchymal stem cells (MSC) offer an opportunity to both treat liver inflammatory damage, as well as reverse some of the changes that occur following chronic liver injury. With the ability to regulate both the innate and adaptive immune system, as well as both inhibit and promote apoptosis of effector inflammatory cells, there are numerous therapeutic opportunities for MSC in acute and chronic liver disease. This article critically appraises the potential therapeutic roles of MSC in liver disease, as well as the barriers to their adoption into clinical practice

Care standards for non-alcoholic fatty liver disease in the United Kingdom 2016: a cross-sectional survey

Objective Guidelines for the assessment of non-alcoholic fatty liver disease (NAFLD) have been published in 2016 by National Institute for Health and Care Excellence and European Associations for the study of the Liver–European Association for the study of Diabetes–European Association for the study of Obesity. Prior to publication of these guidelines, we performed a cross-sectional survey of gastroenterologists and hepatologists regarding NAFLD diagnosis and management. Design An online survey was circulated to members of British Association for the Study of the Liver and British Society of Gastroenterology between February 2016 and May 2016. Results 175 gastroenterologists/hepatologists responded, 116 completing the survey, representing 84 UK centres. 22% had local NAFLD guidelines. 45% received >300 referrals per year from primary care for investigation of abnormal liver function tests (LFTs). Clinical assessment tended to be performed in secondary rather than primary care including body mass index (82% vs 26%) and non-invasive liver screen (86% vs 32%) and ultrasound (81% vs 37%).Widely used tools for non-invasive fibrosis risk stratification were aspartate transaminase (AST)/alanine transaminase (ALT) ratio (53%), Fibroscan (50%) and NAFLD fibrosis score (41%). 78% considered liver biopsy in selected cases. 50% recommended 10% weight loss target as first-line treatment. Delivery of lifestyle interventions was mostly handed back to primary care (56%). A minority have direct access to community weight management services (22%).Follow-up was favoured by F3/4 fibrosis (72.9%), and high-risk non-invasive fibrosis tests (51%). Discharge was favoured by simple steatosis at biopsy (30%), and low-risk non-invasive scores (25%). Conclusions The survey highlights areas for improvement of service provision for NAFLD assessment including improved recognition of non-alcoholic steatohepatitis in people with type 2 diabetes, streamlining abnormal LFT referral pathways, defining non-invasive liver fibrosis assessment tools, use of liver biopsy, managing metabolic syndrome features and improved access to lifestyle interventions

Clinical effectiveness of cell therapies in patients with chronic liver disease and acute-on-chronic liver failure: a systematic review protocol

PRISMA-P (Preferred Reporting Items for Systematic review and Meta-Analysis Protocols) 2015 checklist: recommended items to address in a systematic review protocol*. (DOC 82 kb

Circulating myeloid populations have prognostic utility in alcohol-related liver disease

Introduction: Alcohol-related liver disease (ARLD) accounts for over one third of all deaths from liver conditions, and mortality from alcohol-related liver disease has increased nearly five-fold over the last 30 years. Severe alcohol-related hepatitis almost always occurs in patients with a background of chronic liver disease with extensive fibrosis or cirrhosis, can precipitate ‘acute on chronic’ liver failure and has a high short-term mortality. Patients with alcohol-related liver disease have impaired immune responses, and increased susceptibility to infections, thus prompt diagnosis of infection and careful patient management is required. The identification of early and non-invasive diagnostic and prognostic biomarkers in ARLD remains an unresolved challenge. Easily calculated predictors of infection and mortality are required for use in patients who often exhibit variable symptoms and disease severity and may not always present in a specialized gastroenterology unit.Methods: We have used a simple haematological analyser to rapidly measure circulating myeloid cell parameters across the ARLD spectrum.Results and Discussion: We demonstrate for the first time that immature granulocyte (IG) counts correlate with markers of disease severity, and our data suggests that elevated counts are associated with increased short-term mortality and risk of infection. Other myeloid populations such as eosinophils and basophils also show promise. Thus IG count has the potential to serve alongside established markers such as neutrophil: lymphocyte ratio as a simply calculated predictor of mortality and risk of infectious complications in patients with alcohol-related hepatitis. This would allow identification of patients who may require more intensive management

Inhibition of vascular adhesion protein-1 modifies hepatic steatosis in vitro and in vivo

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance and dyslipidaemia and currently is estimated to affect up to a third of all individuals in developed countries. Current standard of care for patients varies according to disease stage, but includes lifestyle interventions common insulin sensitizers, antioxidants and lipid modifiers. However, to date specific therapies have shown little histological or fibrosis stage improvement in large clinical trials, and there is still no licensed therapy for NAFLD. Given the high prevalence, limited treatment options and significant screening costs for the general population, new treatments are urgently required. AIM: To assess the potential for inhibition of the amine oxidase enzyme vascular adhesion protein-1 (VAP-1) to modify hepatic lipid accumulation in NAFLD. METHODS: We have used immunochemical and qPCR analysis to document expression of VAP-1 and key functional proteins and transporters across the NAFLD spectrum. We then utilised hepatocytes in culture and human precision cut liver slices in concert with selective enzyme activity inhibitors to test the effects of activating the semicarbazide-sensitive amine oxidase activity of VAP-1 on hepatic lipid uptake and triglyceride export. A murine model of NAFLD was also used to determine the consequences of VAP-1 knockout and gene expression arrays were used to quantify the effects of VAP-1 activity on key lipid modifying and proinflammatory gene expression. RESULTS: We confirmed that increasing severity of NAFLD and progression to cirrhosis was associated with a significant increase in hepatocellular VAP-1 expression. Hepatocytes in vitro exposed to recombinant VAP-1 and its substrate methylamine showed increased lipid accumulation as determined by quantification of Oil Red O uptake. This was recapitulated using hydrogen peroxide, and lipid accumulation was accompanied by changes in expression of the lipid transporter molecules FABP3, FATP6, insulin receptor subunits and PPARα. Human liver tissue exposed to recombinant VAP-1 or substrates for endo/exogenous VAP-1 produced less triglyceride than untreated tissue and demonstrated an increase in steatosis. This response could be inhibited by using bromoethylamine to inhibit the SSAO activity of VAP-1, and mice deficient in VAP-1/AOC3 also demonstrated reduced steatosis on high fat diet. Exposure of human liver tissue to methylamine to activate VAP-1 resulted in increased expression of FABP2 and 4, FATP3-5, caveolin-1, VLDLR, PPARGC1 and genes associated with the inflammatory response. CONCLUSION: Our data confirm that the elevations in hepatic VAP-1 expression reported in nonalcoholic steatohepatitis can contribute to steatosis, metabolic disturbance and inflammation. This suggests that targeting the semicarbazide sensitive amine oxidase capacity of VAP-1 may represent a useful adjunct to other therapeutic strategies in NAFLD