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.

Introduction Ammonia is a pathogenic factor implicated in the progression of metabolic-associated steatotic liver disease (MASLD). The contribution of the glutaminase 1 (GLS) isoform, an enzyme converting glutamine to glutamate and ammonia, to hepatic ammonia build-up and the mechanisms underlying its upregulation in metabolic-associated steatohepatitis (MASH) remain elusive. Methods Multiplex transcriptomics and targeted metabolomics analysis of liver biopsies in dietary mouse models representing the whole spectra of MASLD were carried out to characterize the relevance of hepatic GLS during disease pathological progression. In addition, the acute effect of liver-specific GLS inhibition in hepatic ammonia content was evaluated in cultured hepatocytes and in in vivo mouse models of diet-induced MASLD. Finally, the regulatory mechanisms of hepatic GLS overexpression related to the lipopolysaccharide (LPS)/Toll-like receptor 4 (TLR4) axis were explored in the context of MASH. Results In mouse models of diet-induced MASLD, we found that augmented liver GLS expression is closely associated with the build-up of hepatic ammonia as the disease progresses from steatosis to steatohepatitis. Importantly, the acute silencing/pharmacological inhibition of GLS diminishes the ammonia burden in cultured primary mouse hepatocytes undergoing dedifferentiation, in steatotic hepatocytes, and in a mouse model of diet-induced steatohepatitis, irrespective of changes in ureagenesis and gut permeability. Under these conditions, GLS upregulation in the liver correlates positively with the hepatic expression of TLR4 that recognizes LPS. In agreement, the pharmacological inhibition of TLR4 reduces GLS and hepatic ammonia content in LPS-stimulated mouse hepatocytes and hyperammonemia animal models of endotoxemia. Conclusions Overall, our results suggest that the LPS/TLR4 axis regulates hepatic GLS expression promoting liver ammonia build-up as steatotic liver disease progresses to steatohepatitis.

BACKGROUND & AIMS:Neuropsychological and psychophysical tests are recommended to assess the risk of overt hepatic encephalopathy (OHE), but their accuracy is limited. Hyperammonaemia is central in the pathogenesis of OHE, but its predictive utility is unknown. In this study, we aimed to determine the role of neuropsychological or psychophysical tests and ammonia, and to develop a model (AMMON-OHE) to stratify the risk of subsequent OHE development in outpatients with cirrhosis. METHODS:This observational, prospective study included 426 outpatients without previous OHE from three liver units followed for a median of 2.5 years. Psychometric hepatic encephalopathy score (PHES) <-4 or critical flicker frequency (CFF) <39 was considered abnormal. Ammonia was normalized to upper limit of normal (AMM-ULN) at the respective reference laboratory. Multivariable frailty competing risk and random survival forest analyses were performed to predict future OHE and to develop the AMMON-OHE model. External validation was carried out using 267 and 381 patients from two independent units. RESULTS:Significant differences were found in time-to-OHE (log-rank p <0.001) according to PHES or CFF and ammonia, with the highest risk in patients with abnormal PHES plus high AMM-ULN (hazard ratio 4.4; 95% CI 2.4-8.1; p <0.001 compared with normal PHES and AMM-ULN). On multivariable analysis, AMM-ULN but not PHES or CFF was an independent predictor of the development of OHE (hazard ratio 1.4; 95% CI 1.1-1.9; p = 0.015). The AMMON-OHE model (sex, diabetes, albumin, creatinine and AMM-ULN) showed a C-index of 0.844 and 0.728 for the prediction of a first episode of OHE in two external validation cohorts. CONCLUSIONS:In this study, we developed and validated the AMMON-OHE model, comprising readily available clinical and biochemical variables that can be used to identify outpatients at the highest risk of developing a first episode of OHE. IMPACT AND IMPLICATIONS:In this study, we aimed to develop a model to predict which patients with cirrhosis are at risk of developing overt hepatic encephalopathy (OHE). Using data from three units and including 426 outpatients with cirrhosis, we developed the AMMON-OHE model - comprising sex, diabetes, albumin, creatinine and ammonia levels - which demonstrated good predictive ability. The AMMON-OHE model performs better than PHES and CFF to predict the first episode of OHE in outpatients with cirrhosis. This model was validated in 267 and 381 patients from two independent liver units. The AMMON-OHE model is available online for clinical use.

Development and validation of the AMMON-OHE model to predict risk of overt hepatic encephalopathy occurrence in outpatients with cirrhosisJournal of HepatologyVol. 79Issue 4PreviewNeuropsychological and psychophysical tests are recommended to assess the risk of overt hepatic encephalopathy (OHE), but their accuracy is limited. Hyperammonaemia is central in the pathogenesis of OHE, but its predictive utility is unknown. In this study, we aimed to determine the role of neuropsychological or psychophysical tests and ammonia, and to develop a model (AMMON-OHE) to stratify the risk of subsequent OHE development in outpatients with cirrhosis. Full-Text PDF It has come to our attention that the description of Fig. 4 in the published version of our manuscript could be confusing. The current title of the figure is “Prediction for the risk of future OHE using the AMMON-OHE random forest model”, but the y-axis of the figure is described as survival instead of risk. Both terms are complementary as risk could be defined as one minus the survival at a given point, but this could cause some confusion. Thus, the figure should be described as “Kaplan-Meier curve for the probability of survival to OHE using the AMMON-OHE random forest model”. In addition, the colors of the figure have been changed according to the Journal format and therefore the previous description of “Ribbons have been drawn by predicting OHE probability at one year with the AMMON-OHE model in all patients of the training cohort and using 0% (lower red), 33% (upper red/lower blue), 66% (higher blue/lower green) and 100% (upper green) percentiles” has to be changed to “Ribbons have been drawn by predicting OHE probability at one year with the AMMON-OHE model in all patients of the training cohort and using 0% (lower light blue), 33% (upper light blue/lower dark blue), 66% (upper dark blue/lower grey) and 100% (upper grey) percentiles”. Therefore, the legend to the new Fig. 4 should be as follows: Fig. 4. Kaplan-Meier curve for the probability of survival to OHE using the AMMON-OHE random forest model. The figure shows the probability of development of OHE in three hypothetical patients with high-risk (woman with diabetes mellitus, creatinine of 3.6 mg/dl, albumin of 2.7 g/dl and AMM-ULN of 2), medium-risk (man without diabetes, creatinine of 1.5 mg/dl, albumin of 3.2 g/dl and AMM-ULN of 1.1) and low-risk (man without diabetes, creatinine of 0.7 mg/dl, albumin of 4.5 g/dl and AMM-ULN of 0.5). Ribbons have been drawn by predicting OHE probability at 1 year with the AMMON-OHE model in all patients of the training cohort and using 0% (lower light blue), 33% (upper light blue/lower dark blue), 66% (upper dark blue/lower grey) and 100% (upper grey) percentiles.

Mechanistically, the symptomatology and disease progression of non-alcoholic fatty liver disease (NAFLD) remain poorly understood, which makes therapeutic progress difficult. In this review, we focus on the potential importance of decreased urea cycle activity as a pathogenic mechanism. Urea synthesis is an exclusive hepatic function and is the body's only on-demand and definitive pathway to remove toxic ammonia. The compromised urea cycle activity in NAFLD is likely caused by epigenetic damage to urea cycle enzyme genes and increased hepatocyte senescence. When the urea cycle is dysfunctional, ammonia accumulates in liver tissue and blood, as has been demonstrated in both animal models and patients with NAFLD. The problem may be augmented by parallel changes in the glutamine/glutamate system. In the liver, the accumulation of ammonia leads to inflammation, stellate cell activation and fibrogenesis, which is partially reversible. This may be an important mechanism for the transition of bland steatosis to steatohepatitis and further to cirrhosis and hepatocellular carcinoma. Systemic hyperammonaemia has widespread negative effects on other organs. Best known are the cerebral consequences that manifest as cognitive disturbances, which are prevalent in patients with NAFLD. Furthermore, high ammonia levels induce a negative muscle protein balance leading to sarcopenia, compromised immune function and increased risk of liver cancer. There is currently no rational way to reverse reduced urea cycle activity but there are promising animal and human reports of ammonia-lowering strategies correcting several of the mentioned untoward aspects of NAFLD. In conclusion, the ability of ammonia-lowering strategies to control the symptoms and prevent the progression of NAFLD should be explored in clinical trials.

BACKGROUND & AIMS:Acute-on-chronic liver failure (ACLF) is characterised by high short-term mortality, systemic inflammation, and failure of hepatic regeneration. Its treatment is a major unmet medical need. This study was conducted to explore whether combining TAK-242, a Toll-like receptor-4 (TLR4) antagonist, with granulocyte-colony stimulating factor (G-CSF), could reduce inflammation whilst enhancing liver regeneration.METHODS:Two mouse models of ACLF were investigated. Chronic liver injury was induced by carbon tetrachloride; lipopolysaccharide (LPS) or galactosamine (GalN) were then administered as extrahepatic or hepatic insults, respectively. G-CSF and/or TAK-242 were administered daily. Treatment durations were 24 hours and 5 days in the LPS model and 48 hours in the GalN model.RESULTS:In a mouse model of LPS-induced ACLF, treatment with G-CSF was associated with significant mortality (66% after 48 hours vs. 0% without G-CSF). Addition of TAK-242 to G-CSF abrogated mortality (0%) and significantly reduced liver cell death, macrophage infiltration and inflammation. In the GalN model, both G-CSF and TAK-242, when used individually, reduced liver injury but their combination was significantly more effective. G-CSF treatment, with or without TAK-242, was associated with activation of the pro-regenerative and anti-apoptotic STAT3 pathway. LPS-driven ACLF was characterised by p21 overexpression, which is indicative of hepatic senescence and inhibition of hepatocyte regeneration. While TAK-242 treatment mitigated the effect on senescence, G-CSF, when co-administered with TAK-242, resulted in a significant increase in markers of hepatocyte regeneration.CONCLUSION:The combination of TAK-242 and G-CSF inhibits inflammation, promotes hepatic regeneration and prevents mortality in models of ACLF; thus, this combination could be a potential treatment option for ACLF.LAY SUMMARY:Acute-on-chronic liver failure is associated with severe liver inflammation and poor short-term survival. Therefore, effective treatments are urgently needed. Herein, we have shown, using mouse models, that the combination of granulocyte-colony stimulating factor (which can promote liver regeneration) and TAK-242 (which inhibits a receptor that plays a key role in inflammation) could be effective for the treatment of acute-on-chronic liver failure.

Principal component analysis of all disease states Conclusion:The results of this first untargeted blood metabolomics study that specifically investigated the changes in metabolic pathways involved in HRS suggests the presence of mitochondrial dysfunction, which improves following treatment response with terlipressin and albumin.Furthermore, findings may help to uncover novel biomarkers that could be useful in differentiating HRS from ATN, as well as in predicting treatment response.

BACKGROUND & AIMS:Hyperammonaemia is central in the pathogenesis of hepatic encephalopathy. It also has pleiotropic deleterious effects on several organ systems, such as immune function, sarcopenia, energy metabolism and portal hypertension. This study was performed to test the hypothesis that severity of hyperammonaemia is a risk factor for liver-related complications in clinically stable outpatients with cirrhosis.METHODS:We studied 754 clinically stable outpatients with cirrhosis from 3 independent liver units. Baseline ammonia levels were corrected to the upper limit of normal (AMM-ULN) for the reference laboratory. The primary endpoint was hospitalisation with liver-related complications (a composite endpoint of bacterial infection, variceal bleeding, overt hepatic encephalopathy, or new onset or worsening of ascites). Multivariable competing risk frailty analyses using fast unified random forests were performed to predict complications and mortality. External validation was carried out using prospective data from 130 patients with cirrhosis in an independent tertiary liver centre.RESULTS:Overall, 260 (35%) patients were hospitalised with liver-related complications. On multivariable analysis, AMM-ULN was an independent predictor of both liver-related complications (hazard ratio 2.13; 95% CI 1.89-2.40; p <0.001) and mortality (hazard ratio 1.45; 95% CI 1.20-1.76; p <0.001). The AUROC of AMM-ULN was 77.9% for 1-year liver-related complications, which is higher than traditional severity scores. Statistical differences in survival were found between high and low levels of AMM-ULN both for complications and mortality (p <0.001) using 1.4 as the optimal cut-off from the training set. AMM-ULN remained a key variable for the prediction of complications within the random forests model in the derivation cohort and upon external validation.CONCLUSION:Ammonia is an independent predictor of hospitalisation with liver-related complications and mortality in clinically stable outpatients with cirrhosis and performs better than traditional prognostic scores in predicting complications.LAY SUMMARY:We conducted a prospective cohort study evaluating the association of blood ammonia levels with the risk of adverse outcomes in 754 patients with stable cirrhosis across 3 independent liver units. We found that ammonia is a key determinant that helps to predict which patients will be hospitalised, develop liver-related complications and die; this was confirmed in an independent cohort of patients.

Background & AimsIn cirrhosis, astrocytic swelling is believed to be the principal mechanism of ammonia neurotoxicity leading to hepatic encephalopathy (HE). The role of neuronal dysfunction in HE is not clear. We aimed to explore the impact of hyperammonaemia on mitochondrial function in primary co-cultures of neurons and astrocytes and in acute brain slices of cirrhotic rats using live cell imaging.MethodsTo primary cocultures of astrocytes and neurons, low concentrations (1 and 5 μM) of NH4Cl were applied. In rats with bile duct ligation (BDL)-induced cirrhosis, a model known to induce hyperammonaemia and minimal HE, acute brain slices were studied. One group of BDL rats was treated twice daily with the ammonia scavenger ornithine phenylacetate (OP; 0.3 g/kg). Fluorescence measurements of changes in mitochondrial membrane potential (Δψm), cytosolic and mitochondrial reactive oxygen species (ROS) production, lipid peroxidation (LP) rates, and cell viability were performed using confocal microscopy.ResultsNeuronal cultures treated with NH4Cl exhibited mitochondrial dysfunction, ROS overproduction, and reduced cell viability (27.8 ± 2.3% and 41.5 ± 3.7%, respectively) compared with untreated cultures (15.7 ± 1.0%, both p <0.0001). BDL led to increased cerebral LP (p = 0.0003) and cytosolic ROS generation (p <0.0001), which was restored by OP (both p <0.0001). Mitochondrial function was severely compromised in BDL, resulting in hyperpolarisation of Δψm with consequent overconsumption of adenosine triphosphate and augmentation of mitochondrial ROS production. Administration of OP restored Δψm. In BDL animals, neuronal loss was observed in hippocampal areas, which was partially prevented by OP.ConclusionsOur results elucidate that low-grade hyperammonaemia in cirrhosis can severely impact on brain mitochondrial function. Profound neuronal injury was observed in hyperammonaemic conditions, which was partially reversible by OP. This points towards a novel mechanism of HE development.Lay summaryThe impact of hyperammonaemia, a common finding in patients with liver cirrhosis, on brain mitochondrial function was investigated in this study. The results show that ammonia in concentrations commonly seen in patients induces severe mitochondrial dysfunction, overproduction of damaging oxygen molecules, and profound injury and death of neurons in rat brain cells. These findings point towards a novel mechanism of ammonia-induced brain injury in liver failure and potential novel therapeutic targets.

Background and Aims Acute-on-chronic liver failure (ACLF) is characterised by lack of regeneration. Granulocyte colony stimulating factor (G-CSF) carries pro-regenerative properties and has been shown to be of benefit in ACLF. However, the large trial of G-CSF (GRAFT study) in patients with ACLF showed no benefit and in certain groups mortality tended to be higher. This study was performed to define the mechanisms underlying the negative effect of G-CSF and determine whether its beneficial effect could be harnessed using a toll-like receptor 4 (TLR4) antagonist. Method Two mouse models of ACLF were used: CCL4 (0.5mg/ml,6w) to induce chronic liver injury followed by LPS i.p. (Klebsiella, 4mg/kg) (n=4–10) or Galactosamine (GalN) i.p. (1000mg/kg) as a second hit (n=8). 1h after, G-CSF 250µg/kg s.c. and/or TLR4-inhibitor TAK-242 10mg/kg i.p. were injected and continued every 24h. The treatment duration was 24h and 5d in the LPS model and 48h in the GalN model. Samples were stored and analysed for liver injury, inflammation, senescence and regeneration. Results 6w CCL4 led to bridging fibrosis, TLR4 up-regulation and infiltration of G-CSFr expressing cells. LPS increased ALT levels, cell death (TUNEL+), enhanced hepatic infiltration of neutrophils (Ly6G+), macrophages (F4/80+) and TNFa. G-CSF increased the 48h mortality from 0% to 66%, aggravated liver inflammation with macrophage and NK cell (CD45+,CD49b+,CD3-,CD19-) infiltration and IL6 expression. G-CSF+TAK-242 reduced the mortality to 0%, abrogated the liver injury (TUNEL) and liver inflammation (macrophages, neutrophils, TNFa, IL6) significantly. In the second model, GalN also induced a significant liver injury. Treatment with G-CSF+TAK-242 was significantly more effective than the individual therapies. G-CSF+TAK-242 was associated with increased liver regeneration evidenced by increased tissue expression of pSTAT3 and BCL2. CCL4+LPS induced a p53 and p16-dependent cell cycle arrest and lack of proliferation (CyclinA) in hepatocytes. G-CSF+TAK-242 mitigated senescence and significantly increased the rate of CyclinA expressing hepatocytes (figure) suggesting enhanced liver regeneration. Conclusion The present study shows that G-CSF is deleterious in LPS-associated ACLF through further activation of inflammatory pathways and immune cell infiltration. TLR4 inhibition with TAK-242 prevented G-CSF driven tissue injury and induced liver regeneration showing evidence of synergy between the two molecules thereby providing a novel therapeutic strategy for ACLF patients.

Background Yaq-001 is a non-absorbable, synthetic carbon with high adsorptive capacity for bacterial products including lipopolysaccharide (LPS) and pro-inflammatory cytokines. Studies in cirrhotic rats showed reduced endotoxemia, improvements in end-organ dysfunction and reduced sensitisation to LPS. The aims of this study were to evaluate the safety and tolerability of Yaq-001 in decompensated cirrhotic (DC) patients and characterise immunological and metabolic effects. Methods This EU-funded H2020 study (CARBALIVE:634579) recruited patients with diuretic-responsive cirrhotic ascites. 28-patients from 8-EU centres were randomised to receive 4 g of Yaq-001 (Y) or placebo (P) for 12-weeks. Safety assessments were performed at baseline, weeks 1, 4, 8 and 12. Endotoxin activity assay (EAA, data used for analysis: CV<20%), organ function, nutrition, markers of systemic and gut inflammation, gut permeability, urinary 1HMRS, and next generation sequencing of stool were evaluated at baseline, W4 and W12. Data were summarized by group and visit as means (SD) with select efficacy parameters modeled using MMRM. Results 14-patients were randomised to each group (Y: Age: 57.8±7 yrs, M: 71%, MELD: 12.4±0.9; P: Age: 57.3±10, M: 75%, MELD: 13.9±1). Safety: Yaq-001 was well tolerated with no SAEs, stable MELD score, decompensation events and nutritional status. Compliance: 93%(Y) 67%(P). Endotoxemia: Trends to reduction in EAA ratio (Y:-2.4% vs P:29.6%) and constitutive whole blood ROS (Y:-37.3% vs P:+34.3%) were observed in the active group (figure 1). Systemic inflammation: Reductions in WCC, CRP, IL6 and CXCL10 in the active group were observed compared with placebo (Y(%):-8.8,-4.3,-3.6,-16.7; P(%)+0.4,+23.9,+13.8,+31.8) (figure 1). Gut inflammation/permeability: Improvements in faecal cytokines in Yaq-001 patients trended towards published values for healthy control. Reduction in plasma D-lactate was observed in Yaq-001 compared to placebo suggestive of an improvement in gut barrier integrity (figure 1). Metabolism: Yaq-001 reduced stool ammonia with a trend to increased urinary hippurate. Microbiome: No change in bacterial diversity was observed. Conclusion Yaq-001 was safe and well tolerated. The data suggest proof of mechanism that Yaq-001, modulates systemic endotoxemia and inflammation by impacting gut inflammation and its permeability.

Background & Aims: Failure to control oesophago-gastric variceal bleeding (OGVB) and acute-on-chronic liver failure (ACLF) are both important prognostic factors in cirrhosis. The aims of this study were to determine whether ACLF and its severity define the risk of death in OGVB and whether insertion of rescue transjugular intrahepatic shunt (TIPS) improves survival in patients with failure to control OGVB and ACLF. Methods: Data on 174 consecutive eligible patients, with failure to control OGVB between 2005 and 2015, were collected from a prospectively maintained intensive care unit registry. Rescue TIPS was defined as technically successful TIPS within 72 hours of presentation with failure to control OGVB. Cox-proportional hazards regression analyses were applied to explore the impact of ACLF and TIPS on survival in patients with failure to control OGVB. Results: Patients with ACLF (n = 119) were significantly older, had organ failures and higher white cell count than patients with acute decompensation (AD, n = 55). Mortality at 42-days and 1-year was significantly higher in patients with ACLF (47.9% and 61.3%) than in those with AD (9.1% and 12.7%, p <0.001), whereas there was no difference in the number of endoscopies and transfusion requirements between these groups. TIPS was inserted in 78 patients (AD 21 [38.2%]; ACLF 57 [47.8%]; p = 0.41). In ACLF, rescue TIPS insertion was an independent favourable prognostic factor for 42-day mortality. In contrast, rescue TIPS did not impact on the outcome of patients with AD. Conclusions: This study shows that in patients with failure to control OGVB, the presence and severity of ACLF determines the risk of 42-day and 1-year mortality. Rescue TIPS is associated with improved survival in patients with ACLF. Lay summary: Variceal bleeding that is not controlled by initial endoscopy is associated with high risk of death. The results of this study showed that in the occurrence of failure of the liver and other organs defines the risk of death. In these patients, insertion of a shunt inside the liver to drain the portal vein improves survival. (C) 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.