Protocol to study monocyte transmigration across primary human liver endothelial cells under physiological shear flow conditions in vitro.

Modeling immune cell recruitment by liver endothelial cells in vitro is important to better understand the pathology of chronic inflammatory liver diseases and cancers. Here, we present a protocol for the study of monocyte transmigration across activated primary human liver endothelial cells, under physiological flow conditions. We describe primary endothelial cell isolation from human liver tissues and monocyte isolation from human blood.

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.

Postexercise muscle glycogen synthesis with glucose, galactose, and combined galactose-glucose ingestion.

Ingested galactose can enhance postexercise liver glycogen repletion when combined with glucose but effects on muscle glycogen synthesis are unknown. In this double-blind randomized study participants [7 men and 2 women; V̇o: 51.1 (8.

The role of neutrophils in alcohol-related hepatitis.

Alcohol-related liver disease is a major cause of liver disease-associated mortality, with inpatient care being a major contributor to its clinical and economic burden. Alcohol-related hepatitis (AH) is an acute inflammatory form of alcohol-related liver disease. Severe AH is associated with high short-term mortality, with infection being a common cause of death.

Tracer-Based Metabolic Analysis by NMR in Intact Perfused Human Liver Tissue.

Human metabolic liver disease is dramatically increasing globally and presents an urgent clinical unmet need. Rodent models of non-alcoholic fatty liver disease (NAFLD) are available, but they fail to fully recreate the metabolic and cellular features of human disease. Thus, it is imperative to understand the metabolic interplay in human cells in the context of disease.

Decellularization of the Human Liver to Generate Native Extracellular Matrix for Use in Automated Functional Assays with Stellate Cells.

With the incidence of liver disease on the rise globally, increasing numbers of patients are presenting with advanced hepatic fibrosis and significant mortality risk. The demand far outstrips possible transplantation capacities, and thus there is an intense drive to develop new pharmacological therapies that stall or reverse liver scarring. Recent late-stage failures of lead compounds have highlighted the challenges of resolving fibrosis, which has developed and stabilized over many years and varies in nature and composition from individual to individual.

Liver Disease and Cell Therapy: Advances Made and Remaining Challenges.

The limited availability of organs for liver transplantation, the ultimate curative treatment for end stage liver disease, has resulted in a growing and unmet need for alternative therapies. Mesenchymal stromal cells (MSCs) with their broad ranging anti-inflammatory and immunomodulatory properties have therefore emerged as a promising therapeutic agent in treating inflammatory liver disease. Significant strides have been made in exploring their biological activity.

TGFβ priming enhances CXCR3-mediated mesenchymal stromal cell engraftment to the liver and enhances anti-inflammatory efficacy.

The immunomodulatory characteristics of mesenchymal stromal cells (MSC) confers them with potential therapeutic value in the treatment of inflammatory/immune-mediated conditions. Previous studies have reported only modest beneficial effects in murine models of liver injury. In our study we explored the role of MSC priming to enhance their effectiveness.

The Contribution of Liver Sinusoidal Endothelial Cells to Clearance of Therapeutic Antibody.

Many chronic inflammatory diseases are treated by administration of "biological" therapies in terms of fully human and humanized monoclonal antibodies or Fc fusion proteins. These tools have widespread efficacy and are favored because they generally exhibit high specificity for target with a low toxicity. However, the design of clinically applicable humanized antibodies is complicated by the need to circumvent normal antibody clearance mechanisms to maintain therapeutic dosing, whilst avoiding development of off target antibody dependent cellular toxicity.

Ketohexokinase inhibition improves NASH by reducing fructose-induced steatosis and fibrogenesis.

Background & Aims: Increasing evidence highlights dietary fructose as a major driver of non-alcoholic fatty liver disease (NAFLD) pathogenesis, the majority of which is cleared on first pass through the hepatic circulation by enzymatic phosphorylation to fructose-1-phosphate via the ketohexokinase (KHK) enzyme. Without a current approved therapy, disease management emphasises lifestyle interventions, but few patients adhere to such strategies. New targeted therapies are urgently required.

Inhibition of vascular adhesion protein-1 modifies hepatic steatosis and .

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.

Alcoholic hepatitis and metabolic disturbance in female mice: a more tractable model than animals.

Alcoholic hepatitis (AH) is the dramatic acute presentation of alcoholic liver disease, with a 15% mortality rate within 28 days in severe cases. Research into AH has been hampered by the lack of effective and reproducible murine models that can be operated under different regulatory frameworks internationally. The liquid Lieber-deCarli (LdC) diet has been used as a means of delivery of alcohol but without any additional insult, and is associated with relatively mild liver injury.

The platelet receptor CLEC-2 blocks neutrophil mediated hepatic recovery in acetaminophen induced acute liver failure.

Acetaminophen (APAP) is the main cause of acute liver failure in the West. Specific efficacious therapies for acute liver failure (ALF) are limited and time-dependent. The mechanisms that drive irreversible acute liver failure remain poorly characterized.

Platelet GPIbα is a mediator and potential interventional target for NASH and subsequent liver cancer.

Non-alcoholic fatty liver disease ranges from steatosis to non-alcoholic steatohepatitis (NASH), potentially progressing to cirrhosis and hepatocellular carcinoma (HCC). Here, we show that platelet number, platelet activation and platelet aggregation are increased in NASH but not in steatosis or insulin resistance. Antiplatelet therapy (APT; aspirin/clopidogrel, ticagrelor) but not nonsteroidal anti-inflammatory drug (NSAID) treatment with sulindac prevented NASH and subsequent HCC development.

A framework for tracer-based metabolism in mammalian cells by NMR.

Metabolism changes extensively during the normal proliferation and differentiation of mammalian cells, and in cancer and inflammatory diseases. Since changes in the metabolic network reflect interactions between genetic, epigenetic and environmental changes, it is helpful to study the flow of label from isotopically labelled precursors into other metabolites rather than static metabolite levels. For this Nuclear Magnetic Resonance (NMR) spectroscopy is an attractive technique as it can quantify site-specific label incorporation.

Liver sinusoidal endothelial cells - gatekeepers of hepatic immunity.

Liver sinusoidal endothelial cells (LSECs) line the low shear, sinusoidal capillary channels of the liver and are the most abundant non-parenchymal hepatic cell population. LSECs do not simply form a barrier within the hepatic sinusoids but have vital physiological and immunological functions, including filtration, endocytosis, antigen presentation and leukocyte recruitment. Reflecting these multifunctional properties, LSECs display unique structural and phenotypic features that differentiate them from the capillary endothelium present within other organs.

Changes in human hepatic metabolism in steatosis and cirrhosis.

Aim: To understand the underlying metabolic changes in human liver disease we have applied nuclear magnetic resonance (NMR) metabolomics analysis to human liver tissue.

Methods: We have carried out pilot study using H-NMR to derive metabolomic signatures from human liver from patients with steatosis, nonalcoholic steatohepatitis (NASH) or alcohol-related liver damage (ARLD) to identify species that can predict outcome and discriminate between alcohol and metabolic-induced liver injuries.

Results: Changes in branched chain amino acid homeostasis, tricarboxylic acid cycle and purine biosynthesis intermediates along with betaine were associated with the development of cirrhosis in both ARLD and nonalcoholic fatty liver disease.

In Vitro and Ex Vivo Models to Study T Cell Migration Through the Human Liver Parenchyma.

The liver is the largest internal organ and filters around 3 pints of blood per minute. This continuous flux of blood should not be confused with rapid egress of lymphocytes through the liver; this organ has intricate corridors of specialized sinusoidal spaces, ensuring that immune cells decelerate to shear flow rates, and providing ample opportunities to interact with parenchymal cells. Migration has been intricately linked to T cell function; it is therefore important to study liver T cell biology into context within the liver microenvironment.

Using Ex Vivo Liver Organ Cultures to Measure Lymphocyte Trafficking.

Lymphocyte recruitment to different organs, and even alternate anatomical regions within the same organ, is differentially regulated. Key combinations of adhesion molecules and chemokines govern compartmentalization, and these can change depending upon the nature and duration of tissue injury. We are interested in understanding lymphocyte recruitment to the liver during human disease, and thus need models of the liver inflammatory milieu that are as representative as possible.

Introduction to Lymphocyte Trafficking in Disease.

Lymphocytes are the key effector cells of the adaptive immune response but are also implicated in the development of chronic inflammatory diseases such as type 2 diabetes, cardiovascular disease, inflammatory bowel disease, and allergy. In order to understand the mechanisms responsible for the global increase in such debilitating conditions it is vital to appreciate the complexity of immune cell trafficking into tissue under normal and inflamed conditions. In this chapter we introduce the mechanisms of immune cell recruitment into tissue and highlight how these processes have been targeted by new therapeutic entities based on blocking integrin or chemokine function.