Global hemostatic status in patients with acute-on-chronic liver failure and septics without underlying liver disease

Background Even the sickest patients with chronic liver disease (CLD), such as those with acute-on-chronic liver failure (ACLF) remain in hemostatic balance due to a concomitant decline in pro- and antihemostatic factors. Objectives We aimed to study whether the hemostatic status in ACLF is merely an exaggeration from the status in patients with compensated and acutely decompensated cirrhosis, or whether sepsis-associated hemostatic changes contribute. Methods We performed extensive hemostatic profiling in 31 adult patients with ACLF, 20 patients with sepsis without underlying CLD, and 40 healthy controls. Results We found similarly elevated plasma levels of the platelet adhesive protein von Willebrand factor (VWF) and decreased levels of the VWF-regulating protease ADAMTS13 in both groups compared to healthy controls. In vivo markers of activation of coagulation (thrombin-antithrombin III, D-dimer) were similarly elevated in both groups compared to controls, but ex vivo thrombin-generating capacity was similar between patients and controls, despite a much more profound international normalized ratio elevation in ACLF. Plasma fibrinogen levels were much higher in septics, which was accompanied by a decreased ex vivo clot permeability and an increase in ex vivo resistance to clot lysis. All hemostatic parameters were remarkably stable over the first 10 days after admission. Conclusions We have found hemostatic changes in ACLF to partially overlap with that of patients with sepsis, and evidence of preserved hemostatic capacity in both patient groups. The notable difference was a profound hyperfibrinogenemia, associated with a thrombogenic clot structure and a marked ex vivo resistance to fibrinolysis in patients with sepsis

Exaggerated inflammatory response to bacterial products in decompensated cirrhotic patients is orchestrated by interferons IL-6 and IL-8

Cirrhosis-associated immune dysfunction (CAID) contributes to disease progression and organ failure development. We interrogated immune system function in nonseptic compensated and decompensated cirrhotic patients using the TruCulture whole blood stimulation system, a novel technique that allows a more accurate representation than traditional methods, such as peripheral blood mononuclear cell culture, of the immune response in vivo. Thirty cirrhotics (21 decompensated and 9 compensated) and seven healthy controls (HCs) were recruited. Whole blood was drawn directly into three TruCulture tubes [unstimulated to preloaded with heat-killed Escherichia coli 0111:B4 (HKEB) or lipopolysaccharide (LPS)] and incubated in dry heat blocks at 37°C for 24 h. Cytokine analysis of the supernatant was performed by multiplex assay. Cirrhotic patients exhibited a robust proinflammatory response to HKEB compared with HCs, with increased production of interferon-γ-induced protein 10 (IP-10) and IFN-λ1, and to LPS, with increased production of IFN-λ1. Decompensated patients demonstrated an augmented immune response compared with compensated patients, orchestrated by an increase in type I, II, and III interferons, and higher levels of IL-1β, IL-6, and IL-8 post-LPS stimulation. IL-1β, TNF-α, and IP-10 post-HKEB stimulation and IP-10 post-LPS stimulation negatively correlated with biochemical markers of liver disease severity and liver disease severity scores. Cirrhotic patients exposed to bacterial products exhibit an exaggerated inflammatory response orchestrated by IFNs, IL-6, and IL-8. Poststimulation levels of a number of proinflammatory cytokines negatively correlate with markers of liver disease severity raising the possibility that the switch to an immunodeficient phenotype in CAID may commence earlier in the course of advanced liver disease. NEW &amp; NOTEWORTHY Decompensated cirrhotic patients, compared with compensated patients, exhibit a greater exaggerated inflammatory response to bacterial products orchestrated by interferons, IL-6, and IL-8. Postbacterial product stimulation levels of a number of pro-inflammatory cytokines negatively correlate with liver disease severity biomarkers and liver disease severity scores raising the possibility that the switch to an immunodeficient phenotype in cirrhosis-associated immune dysfunction may commence earlier in the course of advanced liver disease.</p

Human Precision-Cut Liver Slices:A Potential Platform to Study Alcohol-Related Liver Disease

Alcohol-related liver disease (ALD) encompasses a range of pathological conditions that are complex to study at the clinical and preclinical levels. Despite the global burden of ALD, there is a lack of effective treatments, and mortality is high. One of the reasons for the unsuccessful development of novel therapies is that experimental studies are hindered by the challenge of recapitulating this multifactorial disorder in vitro, including the contributions of hepatotoxicity, impaired lipid metabolism, fibrosis and inflammatory cytokine storm, which are critical drivers in the pathogenesis of ALD in patients and primary targets for drug development. Here, we present the unique characteristics of the culture of human precision-cut liver slices (PCLS) to replicate key disease processes in ALD. PCLS were prepared from human liver specimens and treated with ethanol alone or in combination with fatty acids and lipopolysaccharide (FA + LPS) for up to 5 days to induce hepatotoxic, inflammatory and fibrotic events associated with ALD. Alcohol insult induced hepatocyte death which was more pronounced with the addition of FA + LPS. This mixture showed a significant increase in the cytokines conventionally associated with the prototypical inflammatory response observed in severe ALD, and interestingly, alcohol alone exhibited a different effect. Profibrogenic activation was also observed in the slices and investigated in the context of slice preparation. These results support the versatility of this organotypic model to study different pathways involved in alcohol-induced liver damage and ALD progression and highlight the applicability of the PCLS for drug discovery, confirming their relevance as a bridge between preclinical and clinical studies.</p

Beta-blockers or Placebo for Primary Prophylaxis (BOPPP) of oesophageal varices:study protocol for a randomised controlled trial

BACKGROUND: Liver disease is within the top five causes of premature death in adults. Deaths caused by complications of cirrhosis continue to rise, whilst deaths related to other non-liver disease areas are declining. Portal hypertension is the primary sequelae of cirrhosis and is associated with the development of variceal haemorrhage, ascites, hepatic encephalopathy and infection, collectively termed hepatic decompensation, which leads to hospitalisation and mortality. It remains uncertain whether administering a non-selective beta-blocker (NSBB), specifically carvedilol, at an earlier stage, i.e. when oesophageal varices are small, can prevent VH and reduce all-cause decompensation (ACD).METHODS/DESIGN: The BOPPP trial is a pragmatic, multicentre, placebo-controlled, triple-blinded, randomised controlled trial (RCT) in England, Scotland, Wales and Northern Ireland. Patients aged 18 years or older with cirrhosis and small oesophageal varices that have never bled will be recruited, subject to exclusion criteria. The trial aims to enrol 740 patients across 55 hospitals in the UK. Patients are allocated randomly on a 1:1 ratio to receive either carvedilol 6.25 mg (a NSBB) or a matched placebo, once or twice daily, for 36 months, to attain adequate power to determine the effectiveness of carvedilol in preventing or reducing ACD. The primary outcome is the time to first decompensating event. It is a composite primary outcome made up of variceal haemorrhage (VH, new or worsening ascites, new or worsening hepatic encephalopathy (HE), spontaneous bacterial peritonitis (SBP), hepatorenal syndrome, an increase in Child-Pugh grade by 1 grade or MELD score by 5 points, and liver-related mortality. Secondary outcomes include progression to medium or large oesophageal varices, development of gastric, duodenal, or ectopic varices, participant quality of life, healthcare costs and transplant-free survival.DISCUSSION: The BOPPP trial aims to investigate the clinical and cost-effectiveness of carvedilol in patients with cirrhosis and small oesophageal varices to determine whether this non-selective beta-blocker can prevent or reduce hepatic decompensation. There is clinical equipoise on whether intervening in cirrhosis, at an earlier stage of portal hypertension, with NSBB therapy is beneficial. Should the trial yield a positive result, we anticipate that the administration and use of carvedilol will become widespread with pathways developed to standardise the administration of the medication in primary care.ETHICS AND DISSEMINATION: The trial has been approved by the National Health Service (NHS) Research Ethics Committee (REC) (reference number: 19/YH/0015). The results of the trial will be submitted for publication in a peer-reviewed scientific journal. Participants will be informed of the results via the BOPPP website ( www.boppp-trial.org ) and partners in the British Liver Trust (BLT) organisation.TRIAL REGISTRATION: EUDRACT reference number: 2018-002509-78. ISRCTN reference number: ISRCTN10324656. Registered on April 24 2019.</p

Using a theory-informed approach to explore patient and staff perspectives on factors that influence clinical trial recruitment for patients with cirrhosis and small oesophageal varices.

OBJECTIVE: The success of pharmacological randomised controlled trials (RCTs) depends on the recruitment of the required number of participants. Recruitment to RCTs for patients with cirrhosis and small oesophageal varices raises specific additional challenges. The objectives of the study were 1) to explore patient perspectives on factors that influence RCT recruitment, 2) to understand factors that influence the success of recruitment from a staff perspective, and 3) to identify opportunities for tailored interventions to improve trial recruitment in this context. METHODS: The qualitative study was embedded in a multi-centre blinded RCT (BOPPP trial) and was conducted alongside site opening. Semi-structured interviews were conducted with patients who enrolled to participate in the trial (n = 13), patients who declined to take part (n = 5), and staff who were responsible for recruiting participants to the trial (n = 18). An open approach to data collection and analysis was adopted and the Theoretical Domains Framework (TDF) was used to provide a theoretical lens through which to view influences on behaviour. Data was analysed using thematic analysis. RESULTS: The findings consist of 5 overarching themes that outline trial recruitment influences at the patient, staff, team, organisational and trial levels: i) patient risks and benefits ii) staff attitudes, knowledge and capacity, iii) team-based approach, iv) organisational context and v) Trial collective. Patient-generated themes map onto thirteen of the fourteen TDF domains and staff-generated themes map onto all TDF domains. The overarching themes are not mutually exclusive; with evidence of direct interactions between patient and staff-level themes that influence recruitment behaviours. CONCLUSIONS: This study uses a theory-informed approach to gain new insights into improving clinical trial recruitment for patients with cirrhosis and small oesophageal varices. Although people with cirrhosis often display decreased healthcare-seeking behaviours, we found that patients used research to empower themselves to improve their health. Pragmatic trials involving unpredictable populations require staff expertise in building trust, and a deep knowledge of the patient group and their vulnerabilities. RCT recruitment is also more successful when research visits align with what staff identified as the natural rhythm of care. TRIAL REGISTRATION: ISRCTN10324656; https://clinicaltrials.gov/

Human immunocompetent model of neuroendocrine liver metastases recapitulates patient-specific tumour microenvironment

Neuroendocrine liver metastases (LM-NEN) develop in a considerable proportion of patients with gastroenteropancreatic neuroendocrine neoplasms. There is a paucity of experimental models that accurately recapitulate this complex metastatic human liver microenvironment precluding scientific and clinical advancements. Here, we describe the development of a novel personalised immunocompetent precision cut tumour slice (PCTS) model for LM-NEN using resected human liver tissue. The histological assessment throughout the culture demonstrated that slices maintain viability for at least 7 days and retain the cellular heterogeneity of the original tumour. Essential clinical features, such as patient-specific histoarchitecture, tumour grade, neuroendocrine differentiation and metabolic capacity, are preserved in the slices. The PCTS also replicate the tumor-specific immunological profile as shown by the innate and adaptive immunity markers analysis. Furthermore, the study of soluble immune checkpoint receptors in the culture supernatants proves that these immunomodulators are actively produced by LM-NEN and suggests that this process is epithelium-dependent. This model can be employed to investigate these pathways and provides a powerful platform for mechanistic, immunological and pre-clinical studies

Patient-derived precision cut tissue slices from primary liver cancer as a potential platform for preclinical drug testing

BackgroundThe exploitation of anti-tumour immunity, harnessed through immunomodulatory therapies, has fundamentally changed the treatment of primary liver cancer (PLC). However, this has posed significant challenges in preclinical research. Novel immunologically relevant models for PLC are urgently required to improve the translation from bench to bedside and back, explore and predict effective combinatorial therapies, aid novel drug discovery and develop personalised treatment modalities. MethodsWe used human precision-cut tissue slices (PCTS) derived from resected tumours to create a patient-specific immunocompetent disease model that captures the multifaceted and intricate heterogeneity of the tumour and the tumour microenvironment. Tissue architecture, tumour viability and treatment response to single agent and combination therapies were assessed longitudinally over 8 days of ex vivo culture by histological analysis, detection of proliferation/cell death markers, ATP content via HPLC. Immune cell infiltrate was assessed using PCR and immunofluorescence. Checkpoint receptor expression was quantified via Quantigene RNA assay. FindingsAfter optimising the culture conditions, PCTS maintained the original tissue architecture, including tumour morphology, stroma and tumour-infiltrated leukocytes. Moreover, PCTS retained the tumour-specific immunophenotype over time, suggesting the utility of PCTS to investigate immunotherapeutic drug efficacy and identify non-responsiveness. InterpretationHere we have characterised the PCTS model and demonstrated its effectiveness as a robust preclinical tool that will significantly support the development of successful (immuno)therapeutic strategies for PLC. FundingFoundation for Liver Research, London.</p

Patient-derived precision cut tissue slices from primary liver cancer as a potential platform for preclinical drug testing

Background: The exploitation of anti-tumour immunity, harnessed through immunomodulatory therapies, has fundamentally changed the treatment of primary liver cancer (PLC). However, this has posed significant challenges in preclinical research. Novel immunologically relevant models for PLC are urgently required to improve the translation from bench to bedside and back, explore and predict effective combinatorial therapies, aid novel drug discovery and develop personalised treatment modalities. / Methods: We used human precision-cut tissue slices (PCTS) derived from resected tumours to create a patient-specific immunocompetent disease model that captures the multifaceted and intricate heterogeneity of the tumour and the tumour microenvironment. Tissue architecture, tumour viability and treatment response to single agent and combination therapies were assessed longitudinally over 8 days of ex vivo culture by histological analysis, detection of proliferation/cell death markers, ATP content via HPLC. Immune cell infiltrate was assessed using PCR and immunofluorescence. Checkpoint receptor expression was quantified via Quantigene RNA assay. / Findings: After optimising the culture conditions, PCTS maintained the original tissue architecture, including tumour morphology, stroma and tumour-infiltrated leukocytes. Moreover, PCTS retained the tumour-specific immunophenotype over time, suggesting the utility of PCTS to investigate immunotherapeutic drug efficacy and identify non-responsiveness. / Interpretation: Here we have characterised the PCTS model and demonstrated its effectiveness as a robust preclinical tool that will significantly support the development of successful (immuno)therapeutic strategies for PLC. / Funding: Foundation for Liver Research, London

Evidence for alcohol-mediated hemolysis and erythrophagocytosis

Alcohol-related liver disease (ALD) is the most common liver disease worldwide; however, its underlying molecular mechanisms remain poorly understood. Here, we identify ethanol-mediated hemolysis and erythrophagocytosis as major contributors to ALD pathogenesis using both in vitro and in vivo models, as well as surrogate markers such as heme oxygenase-1 (HO-1) and CD163, a scavenger receptor for hemoglobin-haptoglobin complexes. A key initial observation was the direct optical evidence of serum hemolysis in heavy drinkers, which diminished after one week of alcohol withdrawal. In parallel, soluble CD163 (sCD163) levels declined during alcohol detoxification correlating with liver damage and fibrosis stages. Moreover, red blood cells (RBCs) from heavy drinkers exhibited increased fragility under hemolytic stress. In ethanol-fed mice, we also observed serum hemolysis. Erythrophagocytosis in liver tissue was visualized by co-localization of CD163 and hemoglobin autofluorescence. In vitro studies confirmed that ethanol – at concentrations transiently present in the upper gastrointestinal tract during alcohol ingestion – directly induces hemolysis and primes RBCs for erythrophagocytosis through eryptosis, marked by externalization of phosphatidylserine. Both heme, released during hemolysis, and bilirubin, its degradation product, further amplified erythrophagocytosis at clinically relevant concentrations, suggesting a self-perpetuating cycle. The antioxidant N-acetylcysteine efficiently blocked ethanol-induced RBC priming for erythrophagocytosis. In conclusion, alcohol triggers a cascade of hemolysis, eryptosis, and erythrophagocytosis that may contribute to the pathogenesis of alcoholic hepatitis and end-stage ALD. sCD163 could serve as a noninvasive marker of hemolysis-associated macrophage activation. This mechanism opens new avenues for antioxidant-based therapies and may help to explain typical iron abnormalities, including ferroptosis, and hyperbilirubinemia in ALD

Comprehensive analysis of extracellular matrix remodelling via cyclophilin inhibition in human models of alcohol-related liver fibrosis

Background and Purpose: Chronic liver disease and hepatic fibrosis constitute a threat to global health. Clinical translation of preclinical research has been limited, highlighting an urgent need for novel treatments. Cyclophilin inhibitors have shown beneficial effects in liver disease; however, the underlying mechanism of action and the effect across different aetiologies remain elusive. Here, we investigate the impact of a pan-cyclophilin inhibitor (rencofilstat, RCF) in human models of fibrosis and alcohol-related liver disease. Experimental Approach: RCF was tested in human precision-cut liver slices (PCLS) and primary human hepatic stellate cells (HSCs). Fibrosis and cell activation were assessed using transcriptomic and protein analysis. A comprehensive characterisation of changes in extracellular matrix (ECM) biochemical and structural composition was performed in PCLS and HSC-derived matrix using proteomics, imaging and bioinformatic tools to study ECM alignment. PCLS stiffness upon treatment was assessed by atomic force microscopy. Key Results: Transcriptomic and proteomic analyses of PCLS revealed a dramatic impact of RCF on ECM organisation and remodelling. Biochemical composition and fibre alignment analysis of the ECM obtained from HSCs showed a reduction in the amount of ECM core proteins and associated enzymes by RCF, reshaping the architecture of matrix fibres without affecting the HSC activation. The disordered matrix detected in RCF-treated HSC cultures reflected a less-stiff ECM, which was confirmed in the PCLS. Conclusions and Implications: This work provides evidence for a novel mechanism linking cyclophilins and ECM remodelling in advanced 3D models of liver disease, with potential applications in therapeutic development.</p