Lysophosphatidylcholines modulate immunoregulatory checkpoints in peripheral monocytes and are associated with mortality in people with acute liver failure.

BACKGROUND & AIMS: Acute liver failure (ALF) is a life-threatening disease characterised by high-grade inflammation and immunoparesis, which is associated with a high incidence of death from sepsis. Herein, we aimed to describe the metabolic dysregulation in ALF and determine whether systemic immune responses are modulated via the lysophosphatidylcholine (LPC)-autotaxin (ATX)-lysophosphatidylcholinic acid (LPA) pathway. METHODS: Ninety-six individuals with ALF, 104 with cirrhosis, 31 with sepsis and 71 healthy controls (HCs) were recruited. Pathways of interest were identified by multivariate statistical analysis of proton nuclear magnetic resonance spectroscopy and untargeted ultraperformance liquid chromatography-mass spectrometry-based lipidomics. A targeted metabolomics panel was used for validation. Peripheral blood mononuclear cells were cultured with LPA 16:0, 18:0, 18:1, and their immune checkpoint surface expression was assessed by flow cytometry. Transcript-level expression of the LPA receptor (LPAR) in monocytes was investigated and the effect of LPAR antagonism was also examined in vitro. RESULTS: LPC 16:0 was highly discriminant between ALF and HC. There was an increase in ATX and LPA in individuals with ALF compared to HCs and those with sepsis. LPCs 16:0, 18:0 and 18:1 were reduced in individuals with ALF and were associated with a poor prognosis. Treatment of monocytes with LPA 16:0 increased their PD-L1 expression and reduced CD155, CD163, MerTK levels, without affecting immune checkpoints on T and NK/CD56+T cells. LPAR1 and 3 antagonism in culture reversed the effect of LPA on monocyte expression of MerTK and CD163. MerTK and CD163, but not LPAR genes, were differentially expressed and upregulated in monocytes from individuals with ALF compared to controls. CONCLUSION: Reduced LPC levels are biomarkers of poor prognosis in individuals with ALF. The LPC-ATX-LPA axis appears to modulate innate immune response in ALF via LPAR1 and LPAR3. Further investigations are required to identify novel therapeutic agents targeting these receptors. IMPACT AND IMPLICATIONS: We identified a metabolic signature of acute liver failure (ALF) and investigated the immunometabolic role of the lysophosphatidylcholine-autotaxin-lysophosphatidylcholinic acid pathway, with the aim of finding a mechanistic explanation for monocyte behaviour and identifying possible therapeutic targets (to modulate the systemic immune response in ALF). At present, no selective immune-based therapies exist. We were able to modulate the phenotype of monocytes in vitro and aim to extend these findings to murine models of ALF as a next step. Future therapies may be based on metabolic modulation; thus, the role of specific lipids in this pathway require elucidation and the relative merits of autotaxin inhibition, lysophosphatidylcholinic acid receptor blockade or lipid-based therapies need to be determined. Our findings begin to bridge this knowledge gap and the methods used herein could be useful in identifying therapeutic targets as part of an experimental medicine approach.

Von Willebrand factor is an independent predictor of short-term mortality in acutely ill patients with cirrhosis.

BACKGROUND AND AIMS: Levels of von Willebrand factor (VWF) are elevated in patients with cirrhosis, and correlate well with disease severity. In patients with decompensated cirrhosis (DC), plasma VWF is associated with mortality. The value of VWF in predicting short-term mortality risk in patients with acute-on-chronic liver failure (ACLF) is, however, unclear. METHODS: We included patients with DC (n = 111) and ACLF (n = 105). We measured VWF levels and correlated these with other laboratory parameters and prediction models for mortality. Also, we assessed the predictive value of VWF in the prediction of 90- and 30-day mortality in patients with DC and ACLF, respectively, and compared this to the predictive value of clinically used prediction models. Finally, we determined the optimal cut-off value for VWF in patients with ACLF. RESULTS: Sixteen of 111 (14%) patients with DC and 35 of 105 (33%) with ACLF died within 90 and 30 days, respectively. VWF was associated with mortality and correlated closely with other prediction models. In patients with ACLF, VWF levels had a discrimination for 30-day mortality comparable with these models and accurately identified ACLF patients with high 30-day mortality risk. CONCLUSIONS: Levels of VWF associate closely with risk of mortality in patients with DC and ACLF, and may have predictive utility as a laboratory marker of prognosis. Further research is warranted to assess the additional value of VWF in the prediction of mortality and associated complications in chronic liver failure syndromes.

Rifaximin-α reduces gut-derived inflammation and mucin degradation in cirrhosis and encephalopathy: RIFSYS randomised controlled trial.

BACKGROUND & AIMS: Rifaximin-α is efficacious for the prevention of recurrent hepatic encephalopathy (HE), but its mechanism of action remains unclear. We postulated that rifaximin-α reduces gut microbiota-derived endotoxemia and systemic inflammation, a known driver of HE. METHODS: In a placebo-controlled, double-blind, mechanistic study, 38 patients with cirrhosis and HE were randomised 1:1 to receive either rifaximin-α (550 mg BID) or placebo for 90 days. PRIMARY OUTCOME: 50% reduction in neutrophil oxidative burst (OB) at 30 days. SECONDARY OUTCOMES: changes in psychometric hepatic encephalopathy score (PHES) and neurocognitive functioning, shotgun metagenomic sequencing of saliva and faeces, plasma and faecal metabolic profiling, whole blood bacterial DNA quantification, neutrophil toll-like receptor (TLR)-2/4/9 expression and plasma/faecal cytokine analysis. RESULTS: Patients were well-matched: median MELD (11 rifaximin-α vs. 10 placebo). Rifaximin-α did not lead to a 50% reduction in spontaneous neutrophil OB at 30 days compared to baseline (p = 0.48). However, HE grade normalised (p = 0.014) and PHES improved (p = 0.009) after 30 days on rifaximin-α. Rifaximin-α reduced circulating neutrophil TLR-4 expression on day 30 (p = 0.021) and plasma tumour necrosis factor-α (TNF-α) (p <0.001). Rifaximin-α suppressed oralisation of the gut, reducing levels of mucin-degrading sialidase-rich species, Streptococcus spp, Veillonella atypica and parvula, Akkermansia and Hungatella. Rifaximin-α promoted a TNF-α- and interleukin-17E-enriched intestinal microenvironment, augmenting antibacterial responses to invading pathobionts and promoting gut barrier repair. Those on rifaximin-α were less likely to develop infection (odds ratio 0.21; 95% CI 0.05-0.96). CONCLUSION: Rifaximin-α led to resolution of overt and covert HE, reduced the likelihood of infection, reduced oralisation of the gut and attenuated systemic inflammation. Rifaximin-α plays a role in gut barrier repair, which could be the mechanism by which it ameliorates bacterial translocation and systemic endotoxemia in cirrhosis. CLINICAL TRIAL NUMBER: ClinicalTrials.gov NCT02019784. LAY SUMMARY: In this clinical trial, we examined the underlying mechanism of action of an antibiotic called rifaximin-α which has been shown to be an effective treatment for a complication of chronic liver disease which effects the brain (termed encephalopathy). We show that rifaximin-α suppresses gut bacteria that translocate from the mouth to the intestine and cause the intestinal wall to become leaky by breaking down the protective mucus barrier. This suppression resolves encephalopathy and reduces inflammation in the blood, preventing the development of infection.

Dysregulation of the Lysophosphatidylcholine/Autotaxin/Lysophosphatidic Acid Axis in Acute-on-Chronic Liver Failure Is Associated With Mortality and Systemic Inflammation by Lysophosphatidic Acid-Dependent Monocyte Activation.

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is characterized by systemic inflammation, monocyte dysfunction, and susceptibility to infection. Lysophosphatidylcholines (LPCs) are immune-active lipids whose metabolic regulation and effect on monocyte function in ACLF is open for study. APPROACHES & RESULTS: Three hundred forty-two subjects were recruited and characterized for blood lipid, cytokines, phospholipase (PLA), and autotaxin (ATX) concentration. Peripheral blood mononuclear cells and CD14+ monocytes were cultured with LPC, or its autotaxin (ATX)-derived product, lysophosphatidic acid (LPA), with or without lipopolysaccharide stimulation and assessed for surface marker phenotype, cytokines production, ATX and LPA-receptor expression, and phagocytosis. Hepatic ATX expression was determined by immunohistochemistry. Healthy volunteers and patients with sepsis or acute liver failure served as controls. ACLF serum was depleted in LPCs with up-regulated LPA levels. Patients who died had lower LPC levels than survivors (area under the receiver operating characteristic curve, 0.94; P < 0.001). Patients with high-grade ACLF had the lowest LPC concentrations and these rose over the first 3 days of admission. ATX concentrations were higher in patients with AD and ACLF and correlated with Model for End-Stage Liver Disease, Consortium on Chronic Liver Failure-Sequential Organ Failure Assessment, and LPC/LPA concentrations. Reduction in LPC correlated with higher monocyte Mer-tyrosine-kinase (MerTK) and CD163 expression. Plasma ATX concentrations rose dynamically during ACLF evolution, correlating with IL-6 and TNF-α, and were associated with increased hepatocyte ATX expression. ACLF patients had lower human leukocyte antigen-DR isotype and higher CD163/MerTK monocyte expression than controls; both CD163/MerTK expression levels were reduced in ACLF ex vivo following LPA, but not LPC, treatment. LPA induced up-regulation of proinflammatory cytokines by CD14+ cells without increasing phagocytic capacity. CONCLUSIONS: ATX up-regulation in ACLF promotes LPA production from LPC. LPA suppresses MerTK/CD163 expression and increases monocyte proinflammatory cytokine production. This metabolic pathway could be investigated to therapeutically reprogram monocytes in ACLF.

Fibrin clot quality in acutely ill cirrhosis patients: Relation with outcome and improvement with coagulation factor concentrates.

BACKGROUND & AIMS: Patients with liver disease may acquire substantial changes in their hemostatic system, which are most pronounced in patients who are critically ill. Changes in the quality of the fibrin clot in critically ill patients have not been studied in detail. Here we assessed markers of fibrin clot quality and effects of coagulation factor concentrates in patients with acutely decompensated (AD) cirrhosis and acute on chronic liver failure (ACLF). METHODS: We measured plasma levels of fibrinogen, factor XIII, prothrombin and performed thrombin generation assays in 52 AD patients, 58 ACLF patients and 40 controls. In addition, we examined the effects of coagulation factor concentrates on functional assays of fibrin quality. RESULTS: We found increased thrombin generating capacity in both AD and ACLF in comparison with healthy controls. Plasma levels of prothrombin, fibrinogen, and factor XIII were lower in patients compared to controls, appeared lower in ACLF compared to AD patients, and were related to clinical outcomes. Fibrinogen concentrate, but not factor XIII or prothrombin complex concentrate, improved clot quality in vitro. Prothrombin complex concentrate increased the resistance of the clot to break down. CONCLUSIONS: We have demonstrated elevated thrombin generation but decreased plasma levels of prothrombin, fibrinogen and FXIII in acutely ill patients with cirrhosis. In addition, we showed that fibrinogen concentrate and PCCs, but not factor XIII concentrate, improve clot properties in patient plasma. Whether there is true clinical benefit from coagulation factor concentrates in prevention or treatment of bleeding requires further study. LAY SUMMARY: Patients with liver diseases are at risk of bleeding, but mechanisms involved in this bleeding risk are incompletely understood. We studied components that determine the stability of the blood clot and found that concentrations of certain proteins involved in clot stability are present in low levels in acutely ill patients with liver disease. We furthermore demonstrated that some clinically available drugs improve the stability of blood clots from these patients in a test tube.

Mixed Fibrinolytic Phenotypes in Decompensated Cirrhosis and Acute-on-Chronic Liver Failure with Hypofibrinolysis in Those With Complications and Poor Survival.

BACKGROUND AND AIMS: Patients with liver disease acquire complex changes in their hemostatic system, which results in a fragile rebalanced status. The status of the fibrinolytic system is controversial, as is the role of fibrinolytic dysfunction in bleeding and thrombosis in patients with cirrhosis. Here, we aimed to determine fibrinolytic status and its relationship with outcome in acutely ill patients with cirrhosis. APPROACH AND RESULTS: We assessed plasma fibrinolytic potential in a large cohort of patients with acutely decompensated cirrhosis (AD, n = 52) or acute-on-chronic liver failure (ACLF, n = 57). Compared with 40 healthy volunteers, median clot lysis times (CLTs) were shorter in patients with AD but comparable to controls in patients with ACLF. However, the variability in CLTs in patients was much larger than in healthy controls, and in both patient groups, a proportion of patients had clearly prolonged or shortened CLTs. The variability in CLTs in patients was not readily explained by variations in plasma levels of key fibrinolytic proteins. However, CLTs were clearly related to clinical characteristics, with longer CLTs in patients with sepsis and patients with any organ failure (as defined by the European Foundation for the Study of Chronic Liver Disease organ failure scores). CLTs were not different between patients that did or did not experience bleeding or a thrombotic event during follow-up. Baseline CLTs were substantially longer in patients that died within 30 days of admission. CONCLUSIONS: Our study demonstrates a mixed fibrinolytic phenotype in acutely ill patients with cirrhosis with baseline hypofibrinolysis associated with sepsis, organ failure, and short-term mortality. These associations may be explained by defective clearance of intraorgan microthrombi that have been proposed to drive organ failure.

CD14+ CD15- HLA-DR- myeloid-derived suppressor cells impair antimicrobial responses in patients with acute-on-chronic liver failure.

OBJECTIVE: Immune paresis in patients with acute-on-chronic liver failure (ACLF) accounts for infection susceptibility and increased mortality. Immunosuppressive mononuclear CD14+HLA-DR- myeloid-derived suppressor cells (M-MDSCs) have recently been identified to quell antimicrobial responses in immune-mediated diseases. We sought to delineate the function and derivation of M-MDSC in patients with ACLF, and explore potential targets to augment antimicrobial responses. DESIGN: Patients with ACLF (n=41) were compared with healthy subjects (n=25) and patients with cirrhosis (n=22) or acute liver failure (n=30). CD14+CD15-CD11b+HLA-DR- cells were identified as per definition of M-MDSC and detailed immunophenotypic analyses were performed. Suppression of T cell activation was assessed by mixed lymphocyte reaction. Assessment of innate immune function included cytokine expression in response to Toll-like receptor (TLR-2, TLR-4 and TLR-9) stimulation and phagocytosis assays using flow cytometry and live cell imaging-based techniques. RESULTS: Circulating CD14+CD15-CD11b+HLA-DR- M-MDSCs were markedly expanded in patients with ACLF (55% of CD14+ cells). M-MDSC displayed immunosuppressive properties, significantly decreasing T cell proliferation (p=0.01), producing less tumour necrosis factor-alpha/interleukin-6 in response to TLR stimulation (all p<0.01), and reduced bacterial uptake of Escherichia coli (p<0.001). Persistently low expression of HLA-DR during disease evolution was linked to secondary infection and 28-day mortality. Recurrent TLR-2 and TLR-4 stimulation expanded M-MDSC in vitro. By contrast, TLR-3 agonism reconstituted HLA-DR expression and innate immune function ex vivo. CONCLUSION: Immunosuppressive CD14+HLA-DR- M-MDSCs are expanded in patients with ACLF. They were depicted by suppressing T cell function, attenuated antimicrobial innate immune responses, linked to secondary infection, disease severity and prognosis. TLR-3 agonism reversed M-MDSC expansion and innate immune function and merits further evaluation as potential immunotherapeutic agent.

MerTK expressing hepatic macrophages promote the resolution of inflammation in acute liver failure.

OBJECTIVE: Acute liver failure (ALF) is characterised by overwhelming hepatocyte death and liver inflammation with massive infiltration of myeloid cells in necrotic areas. The mechanisms underlying resolution of acute hepatic inflammation are largely unknown. Here, we aimed to investigate the impact of Mer tyrosine kinase (MerTK) during ALF and also examine how the microenvironmental mediator, secretory leucocyte protease inhibitor (SLPI), governs this response. DESIGN: Flow cytometry, immunohistochemistry, confocal imaging and gene expression analyses determined the phenotype, functional/transcriptomic profile and tissue topography of MerTK+ monocytes/macrophages in ALF, healthy and disease controls. The temporal evolution of macrophage MerTK expression and its impact on resolution was examined in APAP-induced acute liver injury using wild-type (WT) and Mer-deficient (Mer-/-) mice. SLPI effects on hepatic myeloid cells were determined in vitro and in vivo using APAP-treated WT mice. RESULTS: We demonstrate a significant expansion of resolution-like MerTK+HLA-DRhigh cells in circulatory and tissue compartments of patients with ALF. Compared with WT mice which show an increase of MerTK+MHCIIhigh macrophages during the resolution phase in ALF, APAP-treated Mer-/- mice exhibit persistent liver injury and inflammation, characterised by a decreased proportion of resident Kupffer cells and increased number of neutrophils. Both in vitro and in APAP-treated mice, SLPI reprogrammes myeloid cells towards resolution responses through induction of a MerTK+HLA-DRhigh phenotype which promotes neutrophil apoptosis and their subsequent clearance. CONCLUSIONS: We identify a hepatoprotective, MerTK+, macrophage phenotype that evolves during the resolution phase following ALF and represents a novel immunotherapeutic target to promote resolution responses following acute liver injury.

In vitro efficacy of pro- and anticoagulant strategies in compensated and acutely ill patients with cirrhosis.

BACKGROUND & AIMS: A simultaneous decline in pro- and anticoagulant drivers in patients with liver diseases results in a "rebalanced" haemostatic system, even in acutely ill patients. Nevertheless, both bleeding and thrombotic events are common. Here, we explored efficacy of pro- and antihaemostatic strategies in compensated and acutely ill cirrhotics which may be unpredictable given the profound haemostatic changes. METHODS: We tested the effects in vitro of the addition of clinically relevant doses of commonly used pro- and antihaemostatic strategies in plasma from healthy individuals (n = 30) and patients with compensated (n = 18) and acutely decompensated cirrhosis (n = 18), and acute-on-chronic liver failure (n = 10). We used thrombin generation tests and fibrin clot permeability assays to assess potency of various approaches. RESULTS: Fresh frozen plasma and recombinant factor VIIa modestly increased thrombin generation (10%-20%). Prothrombin complex concentrate increased thrombin generation two-fold in controls and 2-4-fold in patients. Clot permeability decreased after addition of fibrinogen concentrate by 51% in controls and by 50%-60% in patients. Low molecular weight heparin decreased thrombin generation by 18% in controls and by 23%-54% in patients. Similarly, dabigatran decreased thrombin generation by 33% in controls and by 47%-100% in patients. In contrast, rivaroxaban decreased thrombin generation by 55% in controls, but only by 11%-38% in patients. CONCLUSIONS: These in vitro data suggest little prohaemostatic effect of fresh frozen plasma and recombinant factor VIIa in acutely ill cirrhotics, whereas prothrombin complex concentrate and fibrinogen concentrate clearly improved haemostasis. Furthermore, our data suggest the requirement for dose adjustments of commonly used anticoagulants in these patients.

Therapeutic plasma exchange as a novel treatment for severe intrahepatic cholestasis of pregnancy: Case series and mechanism of action.

INTRODUCTION: Intrahepatic cholestasis of pregnancy is characterised by pruritus and elevated serum bile acids. The pruritus can be severe, and pharmacological options achieve inconsistent symptomatic improvement. Raised bile acids are linearly associated with adverse fetal outcomes, with existing management of limited benefit. We hypothesised that therapeutic plasma exchange removes pruritogens and lowers total bile acid concentrations, and improves symptoms and biochemical abnormalities in severe cases that have not responded to other treatments. METHODS: Four women with severe pruritus and hypercholanemia were managed with therapeutic plasma exchange. Serial blood biochemistry and visual analogue scores of itch severity were obtained. Blood and waste plasma samples were collected before and after exchange; individual bile acids and sulfated progesterone metabolites were measured with HPLC-MS, autotaxin activity and cytokine profiles with enzymatic methods. Results were analysed using segmental linear regression to describe longitudinal trends, and ratio t tests. RESULTS: Total bile acids and visual analogue itch scores demonstrated trends to transiently improve following plasma exchange, with temporary symptomatic benefit reported. Individual bile acids (excluding the drug ursodeoxycholic acid), and the sulfated metabolites of progesterone reduced following exchange (P = .03 and P = .04, respectively), whilst analysis of waste plasma demonstrated removal of autotaxin and cytokines. CONCLUSIONS: Therapeutic plasma exchange can lower potentially harmful bile acids and improve itch, likely secondary to the demonstrated removal of pruritogens. However, the limited current experience and potential complications, along with minimal sustained symptomatic benefit, restrict its current use to women with the most severe disease for whom other treatment options have been exhausted.

High-Speed Quantitative UPLC-MS Analysis of Multiple Amines in Human Plasma and Serum via Precolumn Derivatization with 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate: Application to Acetaminophen-Induced Liver Failure.

A targeted reversed-phase gradient UPLC-MS/MS assay has been developed for the quantification /monitoring of 66 amino acids and amino-containing compounds in human plasma and serum using precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQTag Ultra). Derivatization of the target amines required minimal sample preparation and resulted in analytes with excellent chromatographic and mass spectrometric detection properties. The resulting method, which requires only 10 µL of sample, provides the reproducible and robust separation of 66 analytes in 7.5 min, including baseline resolution of isomers such as leucine and isoleucine. The assay has been validated for the quantification of 33 amino compounds (predominantly amino acids) over a concentration range from 2 to 20 and 800 µM. Intra- and interday accuracy of between 0.05 and 15.6 and 0.78-13.7% and precision between 0.91 and 16.9% and 2.12-15.9% were obtained. A further 33 biogenic amines can be monitored in samples for relative changes in concentration rather than quantification. Application of the assay to samples derived from healthy controls and patients suffering from acetaminophen (APAP, paracetamol)-induced acute liver failure (ALF) showed significant differences in the amounts of aromatic and branched chain amino acids between the groups as well as a number of other analytes, including the novel observation of increased concentrations of sarcosine in ALF patients. The properties of the developed assay, including short analysis time, make it suitable for high-throughput targeted UPLC-ESI-MS/MS metabonomic analysis in clinical and epidemiological environments.

High-volume plasma exchange in patients with acute liver failure: An open randomised controlled trial.

BACKGROUND & AIMS: Acute liver failure (ALF) often results in cardiovascular instability, renal failure, brain oedema and death either due to irreversible shock, cerebral herniation or development of multiple organ failure. High-volume plasma exchange (HVP), defined as exchange of 8-12 or 15% of ideal body weight with fresh frozen plasma in case series improves systemic, cerebral and splanchnic parameters. METHODS: In this prospective, randomised, controlled, multicentre trial we randomly assigned 182 patients with ALF to receive either standard medical therapy (SMT; 90 patients) or SMT plus HVP for three days (92 patients). The baseline characteristics of the groups were similar. The primary endpoint was liver transplantation-free survival during hospital stay. Secondary-endpoints included survival after liver transplantation with or without HVP with intention-to-treat analysis. A proof-of-principle study evaluating the effect of HVP on the immune cell function was also undertaken. RESULTS: For the entire patient population, overall hospital survival was 58.7% for patients treated with HVP vs. 47.8% for the control group (hazard ratio (HR), with stratification for liver transplantation: 0.56; 95% confidence interval (CI), 0.36-0.86; p=0.0083). HVP prior to transplantation did not improve survival compared with patients who received SMT alone (CI 0.37 to 3.98; p=0.75). The incidence of severe adverse events was similar in the two groups. Systemic inflammatory response syndrome (SIRS) and sequential organ failure assessment (SOFA) scores fell in the treated group compared to control group, over the study period (p<0.001). CONCLUSIONS: Treatment with HVP improves outcome in patients with ALF by increasing liver transplant-free survival. This is attributable to attenuation of innate immune activation and amelioration of multi-organ dysfunction.

Patients with acute-on-chronic liver failure have increased numbers of regulatory immune cells expressing the receptor tyrosine kinase MERTK.

BACKGROUND & AIMS: Characteristics of decompensated cirrhosis and acute-on-chronic liver failure (ACLF) include susceptibility to infection, immuneparesis, and monocyte dysfunction. MER receptor tyrosine kinase (MERTK) is expressed by monocytes and macrophages and contributes to down-regulation of innate immune responses. We investigated whether MERTK expression is altered on monocytes from patients with liver failure. METHODS: We analyzed blood and liver samples collected from patients admitted to the liver intensive therapy unit at King's College Hospital in London from December 2012 through July 2014. Patients had either ACLF (n = 41), acute decompensation of cirrhosis without ACLF (n = 9), cirrhosis without decompensation (n = 17), or acute liver failure (n = 23). We also analyzed samples from healthy individuals (controls, n = 29). We used flow cytometry to determine the level of innate immune function, and associated the findings with disease severity. We developed an assay to measure recruitment and migration of immune cells from the tissue parenchyma. Immunohistochemistry and confocal microscopy were used to determine levels of MERTK in bone marrow, liver, and lymph node tissues. We performed immunophenotype analyses and measured the production of tumor necrosis factor and interleukin 6 and intracellular killing of Escherichia coli by monocytes and peritoneal macrophages incubated with lipopolysaccharide, with or without an inhibitor of MERTK (UNC569). RESULTS: The number of monocytes and macrophages that expressed MERTK was greatly increased in the circulation, livers, and lymph nodes of patients with ACLF, compared with patients with stable cirrhosis and controls. MERTK expression (mean fluorescence intensity) correlated with the severity of hepatic and extrahepatic disease and systemic inflammatory responses. Based on immunophenotype, migration, and functional analyses, MERTK-expressing monocytes migrate across the endothelia to localize into tissue sites and regional lymph nodes. Expression of MERTK reduced the response of cultured monocytes to lipopolysaccharide; the addition of UNC569 restored production of inflammatory cytokines in response to lipopolysaccharide. CONCLUSIONS: Patients with ACLF have increased numbers of immunoregulatory monocytes and macrophages that express MERTK and suppress the innate immune response to microbes. The number of these cells correlates with disease severity and the inflammatory response. MERTK inhibitors restore production of inflammatory cytokines by immune cells from patients with ACLF, and might be developed to increase the innate immune response in these patients.

Clinical science workshop: targeting the gut-liver-brain axis.

A clinical science workshop was held at the ISHEN meeting in London on Friday 11th September 2014 with the aim of thrashing out how we might translate what we know about the central role of the gut-liver-brain axis into targets which we can use in the treatment of hepatic encephalopathy (HE). This review summarises the integral role that inter-organ ammonia metabolism plays in the pathogenesis of HE with specific discussion of the roles that the small and large intestine, liver, brain, kidney and muscle assume in ammonia and glutamine metabolism. Most recently, the salivary and gut microbiome have been shown to underpin the pathophysiological changes which culminate in HE and patients with advanced cirrhosis present with enteric dysbiosis with small bowel bacterial overgrowth and translocation of bacteria and their products across a leaky gut epithelial barrier. Resident macrophages within the liver are able to sense bacterial degradation products initiating a pro-inflammatory response within the hepatic parenchyma and release of cytokines such as tumour necrosis factor alpha (TNF-α) and interleukin-8 into the systemic circulation. The endotoxemia and systemic inflammatory response that are generated predispose both to the development of infection as well as the manifestation of covert and overt HE. Co-morbidities such as diabetes and insulin resistance, which commonly accompany cirrhosis, may promote slow gut transit, promote bacterial overgrowth and increase glutaminase activity and may need to be acknowledged in HE risk stratification assessments and therapeutic regimens. Therapies are discussed which target ammonia production, utilisation or excretion at an individual organ level, or which reduce systemic inflammation and endotoxemia which are known to exacerbate the cerebral effects of ammonia in HE. The ideal therapeutic strategy would be to use an agent that can reduce hyperammonemia and reduce systemic inflammation or perhaps to adopt a combination of therapies that can address both.

Bile acid profiling and quantification in biofluids using ultra-performance liquid chromatography tandem mass spectrometry.

Bile acids are important end products of cholesterol metabolism. While they have been identified as key factors in lipid emulsification and absorption due to their detergent properties, bile acids have also been shown to act as signaling molecules and intermediates between the host and the gut microbiota. To further the investigation of bile acid functions in humans, an advanced platform for high throughput analysis is essential. Herein, we describe the development and application of a 15 min UPLC procedure for the separation of bile acid species from human biofluid samples requiring minimal sample preparation. High resolution time-of-flight mass spectrometry was applied for profiling applications, elucidating rich bile acid profiles in both normal and disease state plasma. In parallel, a second mode of detection was developed utilizing tandem mass spectrometry for sensitive and quantitative targeted analysis of 145 bile acid (BA) species including primary, secondary, and tertiary bile acids. The latter system was validated by testing the linearity (lower limit of quantification, LLOQ, 0.25-10 nM and upper limit of quantification, ULOQ, 2.5-5 µM), precision (≈6.5%), and accuracy (81.2-118.9%) on inter- and intraday analysis achieving good recovery of bile acids (serum/plasma 88% and urine 93%). The ultra performance liquid chromatography-mass spectrometry (UPLC-MS)/MS targeted method was successfully applied to plasma, serum, and urine samples in order to compare the bile acid pool compositional difference between preprandial and postprandial states, demonstrating the utility of such analysis on human biofluids.

Delayed Postoperative Intracerebral Hemorrhage Associated With Oral Multikinase Inhibitor Therapy for Cancer: A Case Report.

Regorafenib is a multikinase inhibitor with anti-vascular endothelial growth factor receptor (VEGF) activity used as an antiangiogenic agent for metastatic colorectal cancer treatment and has been studied as a potential therapeutic agent for several other cancer treatments. Adverse reactions commonly reported with the use of regorafenib and similar oral multikinase inhibitors include hemorrhage, gastrointestinal fistulas, hypertension, and incomplete wound healing. We report a case of a 59-year-old man with metastatic colorectal adenocarcinoma post-colostomy on regorafenib treatment presenting to the emergency department with altered mental status. MRI showed a left frontoparietal mass, which was resected with a left frontal craniotomy. Postoperative MRI showed a resection cavity without significant hemorrhage. He had been prescribed regorafenib preceding his hospitalization, which was continued after admission before surgery and on postoperative day 1. Thirty-two hours after surgery, the patient exhibited sudden right-sided facial droop and right arm weakness. Imaging revealed an acute intraparenchymal hemorrhage within and adjacent to the tumor resection bed, which was managed conservatively. The patient was subsequently discharged to an inpatient rehabilitation facility. The unusual timing of the hemorrhage suggests that the hemorrhage was due to adverse effects of regorafenib. Patients undergoing neurosurgery should have regorafenib discontinued in preparation for surgery. Similar management should be considered for other anti-VEGF medications to avoid serious complications.

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.

Superficial siderosis: comparison of two cases indicates two distinct diagnostic entities. Illustrative cases.

BACKGROUND: Superficial siderosis is the deposition of hemosiderin in the superficial layers of the central nervous system. It has been described in patients with chronic leakage of blood into the cerebrospinal fluid or with amyloid angiopathy, often associated with Alzheimer's disease (AD). OBSERVATIONS: We present two cases of superficial siderosis with vastly different symptomatologies and treatment courses. The patient in case 1 had diffuse superficial siderosis demonstrated on T2-weighted magnetic resonance imaging (MRI), appearing mostly in the inferior cerebellum and extending throughout the neuraxis. He presented with hearing loss, spasticity, gait abnormalities, and urinary incontinence. Ultimately, surgical exploration of the thoracic spinal dura revealed an arteriovenous fistula, which was obliterated. His clinical course stabilized but with persistent deficits. The patient in case 2 had a family history of AD and underwent MRI to evaluate for memory impairment, which demonstrated superficial siderosis of the left occipital lobe. Lumbar puncture demonstrated only traumatic contamination by red blood cells, but tau protein analysis was consistent with the diagnosis of AD. LESSONS: Superficial siderosis is a diagnostic term prompted by findings on MRI that can arise due to two different pathological entities. The diagnosis in case 1 should be termed diffuse superficial siderosis and in case 2 should be termed lobar cortical siderosis.