Engineered Lipids for Intracellular Reactive Oxygen Species Scavenging in Steatotic Hepatocytes.

Intracellular reactive oxygen species (ROS) in steatotic cells pose a problem due to their potential to cause oxidative stress and cellular damage. Delivering engineered phospholipids to intracellular lipid droplets in steatotic hepatic cells, using the cell's inherent intracellular lipid transport mechanisms are investigated. Initially, it is shown that tail-labeled fluorescent lipids assembled into liposomes are able to be transported to intracellular lipid droplets in steatotic HepG2 cells and HHL-5 cells. Further, an antioxidant, an EUK salen-manganese derivative, which has superoxide dismutase-like and catalase-like activity, is covalently conjugated to the tail of a phospholipid and formulated as liposomes for administration. Steatotic HepG2 cells and HHL-5 cells incubated with these antioxidant liposomes have lower intracellular ROS levels compared to untreated controls and non-covalently formulated antioxidants. This first proof-of-concept study illustrates an alternative strategy to equip native organelles in mammalian cells with engineered enzyme activity.

Cardiac dysfunction in patients with cirrhosis and acute decompensation.

The prevalence of cirrhotic cardiomyopathy (CCM) has been reported as high as 60%-70% in patients with liver cirrhosis and is associated with various negative outcomes. There has been a growing understanding of CCM over recent years. Indeed, the development of imaging techniques has enabled new diagnostic criteria to be proposed by the Cirrhotic Cardiomyopathy Consortium. However, important unanswered questions remain over pathophysiological mechanisms, optimal diagnostic modalities and potential treatment options. While there has been an increasing volume of literature evaluating CCM, there is a lack of clarity on its implications in acute decompensation, acute-on-chronic liver failure and following interventions such as transjugular intrahepatic portosystemic shunt insertion and liver transplantation. This review aims to summarise the literature in these challenging domains and suggest where future research should focus. We conclude that systemic inflammation and structural myocardial changes are likely to be crucial in the pathophysiology of the disease, but the relative contribution of different components remains elusive. Furthermore, future studies need to use standardised diagnostic criteria for CCM as well as incorporate newer imaging techniques assessing both myocardial structure and function. Finally, while specific treatments are currently lacking, therapeutics targeting systemic inflammation, microbial dysbiosis and bacterial translocation are promising targets and warrant further research.

Clinical, experimental and pathophysiological effects of Yaq-001: a non-absorbable, gut-restricted adsorbent in models and patients with cirrhosis.

OBJECTIVE: Targeting bacterial translocation in cirrhosis is limited to antibiotics with risk of antimicrobial resistance. This study explored the therapeutic potential of a non-absorbable, gut-restricted, engineered carbon bead adsorbent, Yaq-001 in models of cirrhosis and acute-on-chronic liver failure (ACLF) and, its safety and tolerability in a clinical trial in cirrhosis. DESIGN: Performance of Yaq-001 was evaluated in vitro. Two-rat models of cirrhosis and ACLF, (4 weeks, bile duct ligation with or without lipopolysaccharide), receiving Yaq-001 for 2 weeks; and two-mouse models of cirrhosis (6-week and 12-week carbon tetrachloride (CCl4)) receiving Yaq-001 for 6 weeks were studied. Organ and immune function, gut permeability, transcriptomics, microbiome composition and metabolomics were analysed. The effect of faecal water on gut permeability from animal models was evaluated on intestinal organoids. A multicentre, double-blind, randomised, placebo-controlled clinical trial in 28 patients with cirrhosis, administered 4 gr/day Yaq-001 for 3 months was performed. RESULTS: Yaq-001 exhibited rapid adsorption kinetics for endotoxin. In vivo, Yaq-001 reduced liver injury, progression of fibrosis, portal hypertension, renal dysfunction and mortality of ACLF animals significantly. Significant impact on severity of endotoxaemia, hyperammonaemia, liver cell death, systemic inflammation and organ transcriptomics with variable modulation of inflammation, cell death and senescence in the liver, kidneys, brain and colon was observed. Yaq-001 reduced gut permeability in the organoids and impacted positively on the microbiome composition and metabolism. Yaq-001 regulated as a device met its primary endpoint of safety and tolerability in the clinical trial. CONCLUSIONS: This study provides strong preclinical rationale and safety in patients with cirrhosis to allow clinical translation. TRIAL REGISTRATION NUMBER: NCT03202498.

AlcoChange: A digital therapeutic for patients with alcohol-related liver disease.

Background & Aims: Maintenance of abstinence in alcohol-related liver disease (ARLD) is a major unmet therapeutic need. Digital therapeutics can deliver ongoing behavioural therapy, in real-time, for chronic conditions. The aim of this project was to develop and clinically test AlcoChange, a novel digital therapeutic for ARLD. Methods: AlcoChange was developed using validated behaviour change techniques and a digital alcohol breathalyser. This was an open-label, single-centre study. Patients with ARLD, ongoing alcohol use (within 1 month) and possession of a suitable smartphone were eligible. Patients were recruited from inpatient and outpatient settings, and received AlcoChange therapy for 3 months. The primary outcome was reduction in alcohol use from baseline to 3 months, measured by timeline follow-back. Secondary outcomes included: (i) compliance with the AlcoChange app, (ii) alcohol-related and all-cause hospital re-admissions up to 1 year, (iii) qualitative analysis to determine factors associated with compliance. Results: Sixty-five patients were recruited, of whom 41 completed the study per protocol. Patients compliant with the intervention (>60 logins over 3 months) had a significant reduction in alcohol use from baseline compared to non-compliant patients (median [IQR]: -100% [100% to -55.1%] vs. -57.1% [-95.3% to +32.13%], p = 0.029). The proportion attaining abstinence at 3 months was higher in the compliant group (57.1% vs. 22.2%, p = 0.025). The compliant group had a significantly decreased risk of subsequent alcohol-related re-admission up to 12 months (p = 0.008). Qualitative analysis demonstrated that receiving in-app feedback and the presence of a health-related 'sentinel event' were predictors of compliance with the intervention. Conclusions: Use of the novel digital therapeutic, AlcoChange, was associated with a significant reduction in alcohol use and an increase in the proportion of patients with ARLD attaining abstinence. Definitive randomised trials are warranted for this intervention. Impact and implications: Alcohol-related liver disease (ARLD) is an increasing health problem worldwide. The main cause of death and disability in ARLD is ongoing alcohol consumption, but few patients receive medications or talking therapy to maintain abstinence. This study demonstrated that a digital therapeutic, linked to a smartphone, may help reduce alcohol consumption and alcohol-related hospital admissions in these patients. If validated in larger, randomised, trials, digital therapeutics may have a role in the primary and secondary prevention of complicatons from ARLD.

Cognitive dysfunction in early experimental metabolic dysfunction-associated steatotic liver disease is associated with systemic inflammation and neuroinflammation.

Background & Aims: Cognitive dysfunction is an increasingly recognised manifestation of metabolic dysfunction-associated steatotic liver disease (MASLD), but the mechanistic link remains unclear. The aim of this study was to investigate the hypothesis that experimental MASLD leads to cognitive dysfunction via systemic inflammation and neuroinflammation. Methods: Twenty male Sprague Dawley rats were randomised to a high-fat high-cholesterol (HFHC) diet to induce MASLD, or a standard diet (n = 10/group), for 16 weeks. Assessments included: MASLD severity (histology), neurobehaviour, inflammation (liver, plasma and cerebrospinal fluid), brain microglia and astrocyte activation, and synaptic density. Results: The HFHC diet induced MASLD with extensive steatosis and lobular inflammation without fibrosis. Several plasma cytokines were elevated (CXCL1, IL-6, IL-17, MIP-1α, MCP-1, IL-10; all p <0.05) and correlated with increases in hepatic chemokine gene expression. Cerebrospinal fluid concentrations of CXCL1 were elevated (p = 0.04). In the prefrontal brain cortex, we observed a 19% increase in microglial activation confirmed by Iba1 immunohistochemistry (p = 0.03) and 3H-PK11195 autoradiography (p <0.01). In parallel, synaptic density was reduced to 92%, assessed by 3H-UCB-J autoradiography (p <0.01). MASLD animals exhibited impaired memory to previously encountered objects in the novel object recognition test (p = 0.047) and showed depression-like behaviour evidenced by increased immobility time (p <0.01) and reduced swimming time (p = 0.03) in the forced swim test. Conclusions: Experimental non-fibrotic MASLD, as a model to reflect the early stage of human disease, results in cognitive impairment and depression-like behaviour. This is associated with an inflammatory phenotype not only in the liver but also in the plasma and brain, which together with diminished synaptic density, provides a pathophysiological link between liver disease and cognitive dysfunction in MASLD. Impact and implications: Cognitive dysfunction is an increasingly recognised comorbidity in patients with metabolic dysfunction-associated steatotic liver disease (MASLD), yet the underlying mechanisms remain unclear. This study provides evidence of impaired memory and depression-like symptoms in early experimental MASLD and indicates that hepatic inflammation may drive a systemic inflammatory response, resulting in neuroinflammation and reduced brain synaptic density. The evidence of impaired memory in MASLD and establishing its underlying pathophysiological link provides insights that could guide the development of potential new treatments for this increasingly common condition in people of working age. The study also emphasises the need to develop better tools for clinical cognitive testing, which will enable physicians to assess and manage brain dysfunction early in MASLD.

Antioxidant Microgels Support Peroxide-Challenged Hepatic Cells.

Access to therapeutic strategies that counter cellular stress induced by reactive oxygen species (ROS) is an important, long-standing challenge. Here, the assembly of antioxidant artificial cells is based on alginate hydrogels equipped with non-native catalysts, namely platinum nanoparticles and an EUK compound. These artificial cells are able to preserve the viability and lower the intracellular ROS levels of challenged hepatic cells by removing peroxides from the extracellular environment. Conceptually, this strategy illustrates the potential use of artificial cells with a synthetic catalyst toward long-term support of hepatic cells and potentially other mammalian cells.

Assessment of bilirubin levels in patients with cirrhosis via forehead, sclera and lower eyelid smartphone images.

One of the key biomarkers evaluating liver disease progression is an elevated bilirubin level. Here we apply smartphone imaging to non-invasive assessment of bilirubin in patients with cirrhosis. Image data was processed using two different approaches to remove variation introduced by ambient conditions and different imaging devices-a per-image calibration using a color chart in each image, and a two-step process using pairs of flash/ no-flash images to account for ambient light in combination with a one-time calibration. For the first time, results from the forehead, sclera (white of the eye) and lower eyelid were compared. The correlation coefficients between the total serum bilirubin and the predicted bilirubin via the forehead, sclera and lower eyelid were 0.79, 0.89 and 0.86 (all with p<0.001, n = 66), respectively. Given the simpler image capture for the sclera, the recommended imaging site for this patient cohort is the sclera.

Mechanisms and treatment approaches for ACLF.

Acute-on-chronic liver failure (ACLF) is a life-threatening syndrome characterized by decompensation of cirrhosis, severe systemic inflammation and organ failures. ACLF is frequently triggered by intra- and/or extrahepatic insults, such as bacterial infections, alcohol-related hepatitis or flares of hepatic viruses. The imbalance between systemic inflammation and immune tolerance causes organ failures through the following mechanisms: (i) direct damage of immune cells/mediators; (ii) worsening of circulatory dysfunction resulting in organ hypoperfusion and (iii) metabolic alterations with prioritization of energetic substrates for inflammation and peripheral organ 'energetic crisis'. Currently, the management of ACLF includes the support of organ failures, the identification and treatment of precipitating factors and expedited assessment for liver transplantation (LT). Early LT should be considered in patients with ACLF grade 3, who are unlikely to recover with the available treatments and have a mortality rate > 70% at 28 days. However, the selection of transplant candidates and their prioritization on the LT waiting list need standardization. Future challenges in the ACLF field include a better understanding of pathophysiological mechanisms leading to inflammation and organ failures, the development of specific treatments for the disease and personalized treatment approaches. Herein, we reviewed the current knowledge and future perspectives on mechanisms and treatment of ACLF.

Randomized, controlled clinical trial of the DIALIVE liver dialysis device versus standard of care in patients with acute-on- chronic liver failure.

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is characterized by severe systemic inflammation, multi-organ failure and high mortality rates. Its treatment is an urgent unmet need. DIALIVE is a novel liver dialysis device that aims to exchange dysfunctional albumin and remove damage- and pathogen-associated molecular patterns. This first-in-man randomized-controlled trial was performed with the primary aim of assessing the safety of DIALIVE in patients with ACLF, with secondary aims of evaluating its clinical effects, device performance and effect on pathophysiologically relevant biomarkers. METHODS: Thirty-two patients with alcohol-related ACLF were included. Patients were treated with DIALIVE for up to 5 days and end points were assessed at Day 10. Safety was assessed in all patients (n = 32). The secondary aims were assessed in a pre-specified subgroup that had at least three treatment sessions with DIALIVE (n = 30). RESULTS: There were no significant differences in 28-day mortality or occurrence of serious adverse events between the groups. Significant reduction in the severity of endotoxemia and improvement in albumin function was observed in the DIALIVE group, which translated into a significant reduction in the CLIF-C (Chronic Liver Failure consortium) organ failure (p = 0.018) and CLIF-C ACLF scores (p = 0.042) at Day 10. Time to resolution of ACLF was significantly faster in DIALIVE group (p = 0.036). Biomarkers of systemic inflammation such as IL-8 (p = 0.006), cell death [cytokeratin-18: M30 (p = 0.005) and M65 (p = 0.029)], endothelial function [asymmetric dimethylarginine (p = 0.002)] and, ligands for Toll-like receptor 4 (p = 0.030) and inflammasome (p = 0.002) improved significantly in the DIALIVE group. CONCLUSIONS: These data indicate that DIALIVE appears to be safe and impacts positively on prognostic scores and pathophysiologically relevant biomarkers in patients with ACLF. Larger, adequately powered studies are warranted to further confirm its safety and efficacy. IMPACT AND IMPLICATIONS: This is the first-in-man clinical trial which tested DIALIVE, a novel liver dialysis device for the treatment of cirrhosis and acute-on-chronic liver failure, a condition associated with severe inflammation, organ failures and a high risk of death. The study met the primary endpoint, confirming the safety of the DIALIVE system. Additionally, DIALIVE reduced inflammation and improved clinical parameters. However, it did not reduce mortality in this small study and further larger clinical trials are required to re-confirm its safety and to evaluate efficacy. CLINICAL TRIAL NUMBER: NCT03065699.

Negative impact of the pandemic on hospital admissions, morbidity and early mortality for acute cirrhosis decompensation.

INTRODUCTION: The global pandemic has diverted resources away from management of chronic diseases, including cirrhosis. While there is increasing knowledge on COVID-19 infection in liver cirrhosis, little is described on the impact of the pandemic on decompensated cirrhosis admissions and outcomes, which was the aim of this study. METHODS: A single-centre, retrospective study, evaluated decompensated cirrhosis admissions to a tertiary London hepatology and transplantation centre, from October 2018 to February 2021. Patients were included if they had an admission with cirrhosis decompensation defined as new-onset jaundice or ascites, infection, encephalopathy, portal hypertensive bleeding or renal dysfunction. RESULTS: The average number of admissions stayed constant between the pre-COVID-19 (October 2018-February 2020) and COVID-19 periods (March 2020-February 2021). Patients transferred in from secondary centres had consistently higher severity scores during the COVID-19 period (UK Model for End-Stage Liver Disease 58 vs 54; p=0.007, Model for End-Stage Liver Disease-Sodium 22 vs 18; p=0.006, EF-CLIF Acute Decompensation (AD) score 55.0 vs 51.0; p=0.055). Of those admitted to the intensive care without acute-on-chronic liver failure, there was a significant increase in AD scores during the COVID-19 period (58 vs 48, p=0.009). In addition, there was a trend towards increased hospital readmission rates during the COVID-19 period (29.5% vs 21.5%, p=0.067). When censored at 30 days, early mortality postdischarge was significantly higher during the COVID-19 period (p<0.001) with a median time to death of 35 days compared with 62 days pre-COVID-19. DISCUSSION: This study provides a unique perspective on the impact that the global pandemic had on decompensated cirrhosis admissions. The findings of increased early mortality and readmissions, and higher AD scores on ICU admission, highlight the need to maintain resourcing for high-level hepatology care and follow-up, in spite of other disease pressures.

Evaluation of CirrhoCare® - a digital health solution for home management of individuals with cirrhosis.

BACKGROUND & AIMS: Individuals with cirrhosis discharged from hospital following acute decompensation are at high risk of new complications. This study aimed to assess the feasibility and potential clinical benefits of remote management of individuals with acutely decompensated cirrhosis using CirrhoCare®. METHODS: Individuals with cirrhosis with acute decompensation were followed up with CirrhoCare® and compared with contemporaneous matched controls, managed with standard follow-up. Commercially available monitoring devices were linked to the smartphone CirrhoCare® app, for daily recording of heart rate, blood pressure, weight, % body water, cognitive function (CyberLiver Animal Recognition Test [CL-ART] app), self-reported well-being, and intake of food, fluid, and alcohol. The app had 2-way patient-physician communication. Independent external adjudicators assessed the appropriateness of CirrhoCare®-based decisions. RESULTS: Twenty individuals with cirrhosis were recruited to CirrhoCare® (mean age 59 ± 10 years, 14 male, alcohol-related cirrhosis [80%], mean model for end-stage liver disease-sodium [MELD-Na] score 16.1 ± 4.2) and were not statistically different to 20 contemporaneous controls. Follow-up was 10.1 ± 2.4 weeks. Fifteen individuals showed good engagement (≥4 readings/week), 2 moderate (2-3/week), and 3 poor (<2/week). In a usability questionnaire, the median score was ≥9 for all questions. Five CirrhoCare®-managed individuals had 8 readmissions over a median of 5 (IQR 3.5-11) days, and none required hospitalisation for >14 days. Sixteen other CirrhoCare®-guided patient contacts were made, leading to clinical interventions that prevented further progression. Appropriateness was confirmed by adjudicators. Controls had 13 readmissions in 8 individuals, lasting a median of 7 (IQR 3-15) days with 4 admissions of >14 days. They had 6 unplanned paracenteses compared with 1 in the CirrhoCare® group. CONCLUSIONS: This study demonstrates that CirrhoCare® is feasible for community management of individuals with decompensated cirrhosis with good engagement and clinically relevant alerts to new decompensating events. CirrhoCare®-managed individuals have fewer and shorter readmissions justifying larger controlled clinical trials. IMPACT AND IMPLICATIONS: As the burden of cirrhosis grows worldwide, increasing demands are being placed on limited healthcare resources, necessitating the adoption of more sustainable care models that allow for at-home patient management. The CirrhoCare® management system was developed to fill this care gap, deploying a novel combination of hardware, apps, and algorithms, to monitor and intervene in individuals at risk of new decompensation. This study highlights the possibility of reducing hospital readmissions for cirrhosis by optimising specialist community care, reducing the need for interventions such as paracentesis, while providing a more sustainable care pathway that is acceptable to patients. However, given the pilot and non-randomised nature of this study, the outcomes require further validation in a larger randomised controlled trial, to assess both clinical effectiveness and cost-effectiveness. Moreover, the data generated will also facilitate data modelling and further research to refine the CirrhoCare® algorithms to increase their detection sensitivity and utility.

A novel smartphone scleral-image based tool for assessing jaundice in decompensated cirrhosis patients.

BACKGROUND AND AIM: Serum bilirubin is an established marker of liver disease. Reliable tools for non-invasive assessment of jaundice in cirrhosis patients, at risk of clinical decompensation, are highly desirable. While smartphone-based imaging has been described in neonatal jaundice, it has not been investigated in advanced cirrhosis patients. METHODS: We included 46 hospitalized patients with acute cirrhosis decompensation and jaundice. Scleral images using an Android smartphone were taken to derive "Scleral Color Values (SCV)," which were matched with same day serum bilirubin measurements. In 29 patients, repeat SCV and bilirubin measurements were performed over time. We analyzed the relationship of SCV and its dynamics with serum bilirubin, clinical scores, and patient outcomes. RESULTS: Of 46 patients, 26 (57%) had alcoholic hepatitis as the decompensation precipitant. Seven patients died during admission; a further 12 following hospital discharge. SCV had an excellent linear correlation with serum bilirubin (rho = 0.90, P < 0.001); changes in SCV and serum bilirubin across different time points, were also closely associated (rho = 0.77, P < 0.001). SCV correlated significantly with CLIF Consortium Acute Decompensation score (rho = 0.38, P < 0.001) and grade of Acute-on-Chronic Liver Failure (rho = 0.42, P = 0.039). SCV was higher in patients who died, however, not significantly (86.1 [IQR 83.0-89.7] vs 82.3 [IQR 78.5-83.3], P = 0.22). The associations of SCV with clinical parameters mirrored those of serum bilirubin. CONCLUSION: Smartphone-based assessment of jaundice shows excellent concordance with serum bilirubin and is associated with clinical parameters in acute cirrhosis decompensation. This approach offers promise for remote assessment of cirrhosis patients at-risk of decompensation, post hospital discharge.

The role of brain inflammation and abnormal brain oxygen homeostasis in the development of hepatic encephalopathy.

Hepatic encephalopathy (HE) is a frequent complication of chronic liver disease (CLD) and has a complex pathogenesis. Several preclinical and clinical studies have reported the presence of both peripheral and brain inflammation in CLD and their potential impact in the development of HE. Altered brain vascular density and tone, as well as compromised cerebral and systemic blood flow contributing to the development of brain hypoxia, have also been reported in animal models of HE, while a decrease in cerebral metabolic rate of oxygen and cerebral blood flow has consistently been observed in patients with HE. Whilst significant strides in our understanding have been made over the years, evaluating all these mechanistic elements in vivo and showing causal association with development of HE, have been limited through the practical constraints of experimentation. Nonetheless, improvements in non-invasive assessments of different neurophysiological parameters, coupled with techniques to assess changes in inflammatory and metabolic pathways, will help provide more granular insights on these mechanisms. In this special issue we discuss some of the emerging evidence supporting the hypothesis that brain inflammation and abnormal oxygen homeostasis occur interdependently during CLD and comprise important contributors to the development of HE. This review aims at furnishing evidence for further research in brain inflammation and oxygen homeostasis as additional therapeutic targets and potentially diagnostic markers for HE.

Imaging features for the prediction of clinical endpoints in chronic liver disease: a scoping review protocol.

INTRODUCTION: Chronic liver disease is a growing cause of morbidity and mortality in the UK. Acute presentation with advanced disease is common and prioritisation of resources to those at highest risk at earlier disease stages is essential to improving patient outcomes. Existing prognostic tools are of limited accuracy and to date no imaging-based tools are used in clinical practice, despite multiple anatomical imaging features that worsen with disease severity.In this paper, we outline our scoping review protocol that aims to provide an overview of existing prognostic factors and models that link anatomical imaging features with clinical endpoints in chronic liver disease. This will provide a summary of the number, type and methods used by existing imaging feature-based prognostic studies and indicate if there are sufficient studies to justify future systematic reviews. METHODS AND ANALYSIS: The protocol was developed in accordance with existing scoping review guidelines. Searches of MEDLINE and Embase will be conducted using titles, abstracts and Medical Subject Headings restricted to publications after 1980 to ensure imaging method relevance on OvidSP. Initial screening will be undertaken by two independent reviewers. Full-text data extraction will be undertaken by three pretrained reviewers who have participated in a group data extraction session to ensure reviewer consensus and reduce inter-rater variability. Where needed, data extraction queries will be resolved by reviewer team discussion. Reporting of results will be based on grouping of related factors and their cumulative frequencies. Prognostic anatomical imaging features and clinical endpoints will be reported using descriptive statistics to summarise the number of studies, study characteristics and the statistical methods used. ETHICS AND DISSEMINATION: Ethical approval is not required as this study is based on previously published work. Findings will be disseminated by peer-reviewed publication and/or conference presentations.

Non-alcoholic Fatty Liver Disease: Also a Disease of the Brain? A Systematic Review of the Preclinical Evidence.

Non-alcoholic fatty liver disease (NAFLD) currently affects 25% of the global adult population. Cognitive impairment is a recently recognised comorbidity impeding memory, attention, and concentration, affecting the patients' activities of daily living and reducing their quality of life. This systematic review provides an overview of the evidence for, and potential pathophysiological mechanisms behind brain dysfunction at a neurobiological level, in preclinical NAFLD. We performed a systematic literature search for animal models of NAFLD studying intracerebral conditions using PubMed, Embase and Scopus. We included studies that reported data on neurobiology in rodent and pig models with evidence of steatosis or steatohepatitis assessed by liver histology. 534 unique studies were identified, and 30 studies met the selection criteria, and were included. Findings of neurobiological changes were divided into five key areas: (1) neuroinflammation, (2) neurodegeneration, (3) neurotransmitter alterations, (4) oxidative stress, and (5) changes in proteins and synaptic density. Despite significant heterogeneity in the study designs, all but one study of preclinical NAFLD reported changes in one or more of the above key areas when compared to control animals. In conclusion, this systematic review supports an association between all stages of NAFLD (from simple steatosis to non-alcoholic steatohepatitis (NASH)) and neurobiological changes in preclinical models.

Inhibition of TLR7 and TLR9 Reduces Human Cholangiocarcinoma Cell Proliferation and Tumor Development.

BACKGROUND: Toll-like receptors (TLRs) are key players in innate immunity and modulation of TLR signaling has been demonstrated to profoundly affect proliferation and growth in different types of cancer. However, the role of TLRs in human intrahepatic cholangiocarcinoma (ICC) pathogenesis remains largely unexplored. AIMS: We set out to determine if TLRs play any role in ICCs which could potentially make them useful treatment targets. METHODS: Tissue microarrays containing samples from 9 human ICCs and normal livers were examined immunohistochemically for TLR4, TLR7, and TLR9 expression. Proliferation of human ICC cell line HuCCT1 was measured by MTS assay following treatment with CpG-ODN (TLR9 agonist), imiquimod (TLR7 agonist), chloroquine (TLR7 and TLR9 inhibitor) and IRS-954 (TLR7 and TLR9 antagonist). The in vivo effects of CQ and IRS-954 on tumor development were also examined in a NOD-SCID mouse xenograft model of human ICC. RESULTS: TLR4 was expressed in all normal human bile duct epithelium but absent in the majority (60%) of ICCs. TLR7 and TLR9 were expressed in 80% of human ICCs. However, TLR7 was absent in all cases of normal human bile duct epithelium and only one was TLR9 positive. HuCCT1 cell proliferation in vitro significantly increased following IMQ or CpG-ODN treatment (P < 0.03 and P < 0.002, respectively) but decreased with CQ (P < 0.02). In the mouse xenograft model there was significant reduction in size of tumors from CQ and IRS-954 treated mice compared to untreated controls. CONCLUSION: TLR7 and TLR9 should be further explored for their potential as actionable targets in the treatment of ICC.

Treatment With Simvastatin and Rifaximin Restores the Plasma Metabolomic Profile in Patients With Decompensated Cirrhosis.

Patients with decompensated cirrhosis, particularly those with acute-on-chronic liver failure (ACLF), show profound alterations in plasma metabolomics. The aim of this study was to investigate the effect of treatment with simvastatin and rifaximin on plasma metabolites of patients with decompensated cirrhosis, specifically on compounds characteristic of the ACLF plasma metabolomic profile. Two cohorts of patients were investigated. The first was a descriptive cohort of patients with decompensated cirrhosis (n = 42), with and without ACLF. The second was an intervention cohort from the LIVERHOPE-SAFETY randomized, double-blind, placebo-controlled trial treated with simvastatin 20 mg/day plus rifaximin 1,200 mg/day (n = 12) or matching placebo (n = 13) for 3 months. Plasma samples were analyzed using ultrahigh performance liquid chromatography-tandem mass spectroscopy for plasma metabolomics characterization. ACLF was characterized by intense proteolysis and lipid alterations, specifically in pathways associated with inflammation and mitochondrial dysfunction, such as the tryptophan-kynurenine and carnitine beta-oxidation pathways. An ACLF-specific signature was identified. Treatment with simvastatin and rifaximin was associated with changes in 161 of 985 metabolites in comparison to treatment with placebo. A remarkable reduction in levels of metabolites from the tryptophan-kynurenine and carnitine pathways was found. Notably, 18 of the 32 metabolites of the ACLF signature were affected by the treatment. Conclusion: Treatment with simvastatin and rifaximin modulates some of the pathways that appear to be key in ACLF development. This study unveils some of the mechanisms involved in the effects of treatment with simvastatin and rifaximin in decompensated cirrhosis and sets the stage for the use of metabolomics to investigate new targeted therapies in cirrhosis to prevent ACLF development.

The role of RIPK1 mediated cell death in acute on chronic liver failure.

Acute-on-chronic liver failure (ACLF) is characterized predominantly by non-apoptotic forms of hepatocyte cell death. Necroptosis is a form of programmed lytic cell death in which receptor interacting protein kinase (RIPK) 1, RIPK3 and phosphorylated mixed lineage kinase domain-like (pMLKL) are key components. This study was performed to determine the role of RIPK1 mediated cell death in ACLF. RIPK3 plasma levels and hepatic expression of RIPK1, RIPK3, and pMLKL were measured in healthy volunteers, stable patients with cirrhosis, and in hospitalized cirrhotic patients with acutely decompensated cirrhosis, with and without ACLF (AD). The role of necroptosis in ACLF was studied in two animal models of ACLF using inhibitors of RIPK1, necrostatin-1 (NEC-1) and SML2100 (RIPA56). Plasma RIPK3 levels predicted the risk of 28- and 90-day mortality (AUROC, 0.653 (95%CI 0.530-0.776), 0.696 (95%CI 0.593-0.799)] and also the progression of patients from no ACLF to ACLF [0.744 (95%CI 0.593-0.895)] and the results were validated in a 2nd patient cohort. This pattern was replicated in a rodent model of ACLF that was induced by administration of lipopolysaccharide (LPS) to bile-duct ligated rats and carbon tetrachloride-induced fibrosis mice administered galactosamine (CCL4/GalN). Suppression of caspase-8 activity in ACLF rodent model was observed suggesting a switch from caspase-dependent cell death to necroptosis. NEC-1 treatment prior to administration of LPS significantly reduced the severity of ACLF manifested by reduced liver, kidney, and brain injury mirrored by reduced hepatic and renal cell death. Similar hepato-protective effects were observed with RIPA56 in a murine model of ACLF induced by CCL4/GalN. These data demonstrate for the first time the importance of RIPK1 mediated cell death in human and rodent ACLF. Inhibition of RIPK1 is a potential novel therapeutic approach to prevent progression of susceptible patients from no ACLF to ACLF.