Activation and transcriptional profile of monocytes and CD8+ T cells are altered in checkpoint inhibitor-related hepatitis.

BACKGROUND & AIMS: Checkpoint inhibitor-related hepatitis (CPI-Hep) is an emerging clinical challenge. We aimed to gain insights into the immunopathology of CPI-Hep by comprehensively characterising myeloid and lymphoid subsets. METHODS: CPI-treated patients with or without related hepatitis (CPI-Hep; n = 22 and CPI-noHep; n = 7) were recruited. Phenotypic and transcriptional profiling of peripheral immune subsets was performed and compared with 19 healthy controls (HCs). In vitro monocyte-derived macrophages (MoMFs) were assessed for activation and cytokine production. CD163, CCR2, CD68, CD3, CD8 and granzyme B expression was assessed using immunohistochemistry/immunofluorescence (n = 4). RESULTS: A significant total monocyte depletion was observed in CPI-Hep compared with HCs (p = 0.04), along with a proportionate increase in the classical monocyte population (p = 0.0002) and significant upregulation of CCR2, CD163 and downregulation of CCR7. Soluble CD163 levels were significantly elevated in CPI-Hep compared with HCs (p <0.0001). In vitro MoMFs from CPI-Hep showed enhanced production of pro-inflammatory cytokines. CD8+ T cells demonstrated increased perforin, granzyme B, ICOS and HLA-DR expression in CPI-Hep. Transcriptional profiling indicated the presence of activated monocyte and enhanced effector CD8+ T cell populations in CPI-Hep. Immunohistochemistry demonstrated co-localisation of CD8+/granzyme B+ T cells with CD68+CCR2+/CD68+CD163+ macrophages in CPI-Hep liver tissue. CONCLUSIONS: CPI-Hep is associated with activation of peripheral monocytes and an enhanced cytotoxic, effector CD8+ T cell phenotype. These changes were reflected by liver inflammation composed of CD163+/CCR2+ macrophages and CD8+ T cells. LAY SUMMARY: Some patients who receive immunotherapy for cancer develop liver inflammation, which requires cessation of cancer treatment. Herein, we describe ways in which the white blood cells of patients who develop liver inflammation differ from those of patients who receive the same immunotherapy but do not experience liver-related side effects. Targeting some of the pathways we identify may help to prevent or manage this side effect and facilitate cancer treatment.

Thrombin and factor Xa link the coagulation system with liver fibrosis.

BACKGROUND: Thrombin activates hepatic stellate cells via protease-activated receptor-1. The role of Factor Xa (FXa) in hepatic fibrosis has not been elucidated. We aimed to evaluate the impact of FXa and thrombin in vitro on stellate cells and their respective inhibition in vivo using a rodent model of hepatic fibrosis. METHODS: HSC-LX2 cells were incubated with FXa and/or thrombin in cell culture, stained for αSMA and relative gene expression and gel contraction calculated. C57BL/6 J mice were administered thioacetamide (TAA) for 8 weeks with Rivaroxaban (n = 15) or Dabigatran (n = 15). Control animals received TAA alone (n = 15). Fibrosis was scored and quantified using digital image analysis and hepatic tissue hydroxyproline estimated. RESULTS: Stellate cells treated with FXa and thrombin demonstrated upregulation of procollagen, TGF-beta, αSMA and significant cell contraction (43.48%+/- 4.12) compared to culturing with FXa or thrombin alone (26.90%+/- 8.90, p = 0.02; 13.1%+/- 9.84, p < 0.001). Mean fibrosis score, percentage area of fibrosis and hepatic hydroxyproline content (2.46 vs 4.08, p = 0.008; 2.02% vs 3.76%, p = 0.012; 276.0 vs 651.3, p = 0.0001) were significantly reduced in mice treated with the FXa inhibitor compared to control mice. FXa inhibition was significantly more effective than thrombin inhibition in reducing percentage area of fibrosis and hepatic hydroxyproline content (2.02% vs 3.70%,p = 0.031; 276.0 vs 413.1,p = 0.001). CONCLUSIONS: FXa promotes stellate cell contractility and activation. Early inhibition of coagulation using a FXa inhibitor significantly reduces TAA induced murine liver fibrosis and may be a viable treatment for liver fibrosis in patients.

Anticoagulation in chronic liver disease.

In this Grand Round presentation, the case of a man with decompensated liver disease is described. He subsequently developed a fatal pulmonary embolism, which may not have occurred if he had been prescribed prophylactic anticoagulation to prevent venous thromboembolic disease. The burden of thrombotic disease in those with chronic liver disease is discussed, before a more detailed analysis of the current evidence, safety data, and clinical dilemmas regarding the use of anticoagulation in patients with chronic liver disease. Finally, the future directions within this field are explored.

Validation of the Baveno VI criteria to identify low risk cirrhotic patients not requiring endoscopic surveillance for varices.

BACKGROUND & AIMS: The Baveno VI guidelines propose that cirrhotic patients with a liver stiffness measurement (LSM) <20kPa and a platelet count >150,000/µl can avoid screening endoscopy as their combination is highly specific for excluding clinically significant varices. The aim of the study was to validate these criteria. METHODS: Transient elastography data was collected from two institutions from 2006-2015. Inclusion criteria were a LSM ⩾10kPa and an upper gastrointestinal endoscopy within 12months, with a diagnosis of compensated chronic liver disease. Exclusion criteria were porto-mesenteric-splenic vein thrombosis and non-cirrhotic portal hypertension. Varices were graded as low risk (grade <2) or high risk (grade ⩾2). RESULTS: The study included 310 patients (169 (55%) hepatitis C, and 275 (89%) Child-Pugh A). Varices were present in 23% cases, with 5% prevalence of high risk varices. Overall 102/310 (33%) met the Baveno VI criteria. Within this group 11% had varices and 2% had high risk varices, representing 2/15 (13%) of all high risk varices. The Baveno VI criteria gave a sensitivity 0.87, specificity 0.34, positive predictive value 0.06, negative predictive value 0.98, positive likelihood ratio 1.31 and negative likelihood ratio 0.39. The AUROC for LSM and platelet count combined was 0.746. CONCLUSIONS: The Baveno VI criteria performed well correctly identifying 98% of patients who could safely avoid endoscopy. LAY SUMMARY: This study examines the effectives of a recent set of guidelines published by the Baveno VI conference, which states that patients with chronic liver disease and a low liver stiffness (<20kPa) and high platelet count (>150) are at low risk of having varices and do not need a screening endoscopy. Varices are a complication of cirrhosis, confer a risk of serious bleeding, and can be diagnosed and treated by endoscopy. Our study reviewed the clinical records of patients who have had liver stiffness scans and endoscopy over a 9-year period at two hospitals. The results show that only about 2% of patients who meet the Baveno VI criteria will be miss-classified as not having varices.

Severe cholestatic jaundice after a single administration of ajmaline; a case report and review of the literature.

BACKGROUND: Ajmaline is a pharmaceutical agent now administered globally for a variety of indications, particularly investigation of suspected Brugada syndrome. There have been previous reports suggesting that repetitive use of this agent may cause severe liver injury, but little evidence exists demonstrating the same effect after only a single administration. CASE PRESENTATION: A 33-year-old man of Libyan origin with no significant past medical history underwent an ajmaline provocation test for investigation of suspected Brugada syndrome. Three weeks later, he presented with painless cholestatic jaundice which peaked in severity at eleven weeks after the test. Blood tests confirmed no evidence of autoimmune or viral liver disease, whilst imaging confirmed the absence of biliary tract obstruction. A liver biopsy demonstrated centrilobular cholestasis and focal rosetting of hepatocytes, consistent with a cholestatic drug reaction. Over the course of the next few months, he began to improve clinically and biochemically, with complete resolution by one year post-exposure. CONCLUSION: Whilst ajmaline-related hepatotoxicity was well-recognised in the era in which the drug was administered as a regular medication, clinicians should be aware that ajmaline may induce severe cholestatic jaundice even after a single dose administration.

Proteomic analysis of extracellular matrix from the hepatic stellate cell line LX-2 identifies CYR61 and Wnt-5a as novel constituents of fibrotic liver.

Activation of hepatic stellate cells (HSCs) and subsequent uncontrolled accumulation of altered extracellular matrix (ECM) underpin liver fibrosis, a wound healing response to chronic injury, which can lead to organ failure and death. We sought to catalogue the components of fibrotic liver ECM to obtain insights into disease etiology and aid identification of new biomarkers. Cell-derived ECM was isolated from the HSC line LX-2, an in vitro model of liver fibrosis, and compared to ECM from human foreskin fibroblasts (HFFs) as a control. Mass spectrometry analyses of cell-derived ECMs identified, with ≥99% confidence, 61 structural ECM or secreted proteins (48 and 31 proteins for LX-2 and HFF, respectively). Gene ontology enrichment analysis confirmed the enrichment of ECM proteins, and hierarchical clustering coupled with protein-protein interaction network analysis revealed a subset of proteins enriched to fibrotic ECM, highlighting the existence of cell type-specific ECM niches. Thirty-six proteins were enriched to LX-2 ECM as compared to HFF ECM, of which Wnt-5a and CYR61 were validated by immunohistochemistry in human and murine fibrotic liver tissue. Future studies will determine if these and other components may play a role in the etiology of hepatic fibrosis, serve as novel disease biomarkers, or open up new avenues for drug discovery.

EUS-guided FNA for diagnosis of solid pancreatic neoplasms: a meta-analysis.

BACKGROUND: Preoperative diagnosis of solid pancreatic lesions remains challenging despite advancement in imaging technologies. EUS has the benefit of being a minimally invasive, well-tolerated procedure, although results are operator-dependent. The addition of FNA (EUS-guided FNA) provides samples for cytopathologic analysis, a major advantage over other imaging techniques. OBJECTIVE: To determine the diagnostic accuracy of EUS-FNA for pancreatic cancer. DESIGN: This is a meta-analysis of published studies assessing the diagnostic capability of EUS-FNA. Relevant studies were identified via MEDLINE and were included if they used a reference standard of definitive surgical histology or clinical follow-up of at least 6 months. MAIN OUTCOME MEASUREMENTS: Data from selected studies were analyzed by using test accuracy meta-analysis software, providing a pooled value for sensitivity, specificity, diagnostic odds ratio, and summary receiver operating characteristic curve. Cytology results were classified as inadequate, benign, atypical, suspicious, or malignant. Predefined subgroup analysis was performed. RESULTS: Thirty-three studies published between 1997 and 2009 were included, with a total number of 4984 patients. The pooled sensitivity for malignant cytology was 85% (95% confidence interval [CI], 84-86), and pooled specificity was 98% (95% CI, 0.97-0.99). If atypical and suspicious cytology results were included to determine true neoplasms, the sensitivity increased to 91% (95% CI, 90-92); however, the specificity was reduced to 94% (95% CI, 93-96). The diagnostic accuracy of EUS-FNA was enhanced in prospective, multicenter studies. LIMITATION: Publication bias was not a significant determinant of pooled accuracy. CONCLUSION: This meta-analysis demonstrates that EUS-FNA is a highly accurate diagnostic test for solid neoplasms of the pancreas and should be considered when algorithms for investigating solid pancreatic lesions are being planned.

Pembrolizumab-Induced Pancreatic Exocrine Insufficiency Complicated by Severe Hepatic Steatosis.

Anti-programmed death receptor-1 (anti-PD-1) monoclonal antibodies (mAbs) are used to treat an increasing range of cancers. However, the distinct toxicity profile of immune-related adverse events (irAEs) is a frequent drawback of their clinical application. Among the more common irAEs are hepatitis and colitis, which are diagnosed and graded in patients based on elevated serum liver enzyme levels and increased stool frequency, respectively, and both of which often require treatment with high-dose corticosteroids. Herein, we describe the case of a patient who developed severe transaminase elevation and diarrhoea due to an unusual irAE, which was successfully treated without corticosteroids.

Non-Alcoholic Fatty Liver Disease and Vascular Disease.

Non-Alcoholic Fatty Liver Disease (NAFLD) represents an increasing cause of liver disease worldwide. However, notably, the primary cause of morbidity and mortality in patients with NAFLD is cardiovascular disease (CVD), with fibrosis stage being the strongest disease-specific predictor. It is globally projected that NAFLD will become increasingly prevalent, especially among children and younger adults. As such, even within the next few years, NAFLD will contribute considerably to the overall CVD burden. In this review, we discuss the role of NAFLD as an emerging risk factor for CVD. In particular, this article aims to provide an overview of pathological drivers of vascular damage in patients with NAFLD. Moreover, the impact of NAFLD on the development, severity and the progression of subclinical and clinical CVD will be discussed. Finally, the review illustrates current and potential future perspectives to screen for CVD in this high-risk population.

Liver function tests and metabolic-associated fatty liver disease: Changes in upper normal limits, does it really matter?

BACKGROUND: Metabolic-associated fatty liver disease (MAFLD) is the commonest cause of abnormal liver function tests (LFTs). Current upper normal of limit (UNL) of LFTs was derived from a "healthy" population, where undiagnosed MAFLD and viral hepatitis might be suspected. AIM: To evaluated potential implications of changes in UNL of alanine aminotransferase (ALT) in MAFLD. METHODS: We retrospectively assessed consecutive first referrals with a diagnosis of MAFLD from 2010 to 2017. The conventional UNL of ALT was 45 IU/L for men and 34 IU/L for women, while a low UNL of ALT was 30 IU/L for men and 19 IU/L for women. The UNL of aspartate aminotransferase (AST) was 40 IU/L. RESULTS: Total 436 patients were enrolled; of these, 288 underwent liver biopsy. Setting a lower UNL reduced the percentage of those with significant disease despite normal ALT; specifically, patients with advanced fibrosis (F ≥ F3) or definite "metabolic-associated steato-hepatitis (MASH)" (NAS ≥ 5) within normal ALT decreased from 10% to 1% and from 28% to 4% respectively. However, the proportion of those with elevated ALT and no evidence of advanced fibrosis or "definite MASH" increased from 39% to 47% and from 3% to 19%. Overall, LFTs performed poorly in distinguishing "definite MASH" from simple steatosis (receiver operating characteristic areas under the curves 0.59 for ALT and 0.55 for AST). CONCLUSION: Liver function tests might both under- and overestimate MASH-related liver disease. Reducing the UNL might not be beneficial and imply an increase in healthcare burden. Risk stratification in MAFLD should rely on a combination of risk factors, not on LFTs alone.

Leucocyte ratios are biomarkers of mortality in patients with acute decompensation of cirrhosis and acute-on-chronic liver failure.

BACKGROUND: In patients with cirrhosis, progression to acute decompensation (AD) and acute-on-chronic liver failure (ACLF) has been associated with poor prognosis. Differential leucocyte ratios might predict mortality in systemic inflammatory conditions. AIM: To evaluate differential leucocyte ratios as prognostic biomarkers in patients with cirrhosis. METHODS: Patients with AD and ACLF were recruited from four centres in three countries. Peripheral blood differential leucocytes were measured (three centres using flow cytometry) on hospital admission and at 48 hours. Ratios were correlated to model for end-stage liver disease (MELD), chronic liver failure-sequential organ failure (CLIF-SOFA), suspected/culture-positive bacterial infection and survival. RESULTS: Nine hundred twenty-six patients (562 (61%) male, median age 55 (25-94) years) were studied. Overall, 350 (37%) did not survive to hospital discharge. Neutrophil-lymphocyte ratio (NLR) and monocyte-lymphocyte ratio (MLR) were elevated in patients with AD and ACLF who died during their hospital stay. On multivariate analysis NLR retained statistical significance independently of CLIF-SOFA or MELD. NLR >30 was associated with an 80% 90-day mortality in patients with ACLF but not AD. On sensitivity analysis for subgroups (alcohol-related liver disease and suspected sepsis), NLR and MLR retained statistically robust accuracy for the prediction of mortality. Significant predictive accuracy was only observed in centres using flow cytometry. CONCLUSION: Leucocyte ratios are simple and robust biomarkers of outcome in ACLF, which are comparable to CLIF-SOFA score but dependent on leucocyte quantification method. NLR and MLR may be used as screening tools for mortality prediction in patients with acutely deteriorating cirrhosis.

In-hospital mortality is associated with inflammatory response in NAFLD patients admitted for COVID-19.

BACKGROUND & AIMS: Although metabolic risk factors are associated with more severe COVID-19, there is little evidence on outcomes in patients with non-alcoholic fatty liver disease (NAFLD). We here describe the clinical characteristics and outcomes of NAFLD patients in a cohort hospitalised for COVID-19. METHODS: This study included all consecutive patients admitted for COVID-19 between February and April 2020 at Imperial College Healthcare NHS Trust, with either imaging of the liver available dated within one year from the admission or a known diagnosis of NAFLD. Clinical data and early weaning score (EWS) were recorded. NAFLD diagnosis was based on imaging or past medical history and patients were stratified for Fibrosis-4 (FIB-4) index. Clinical endpoints were admission to intensive care unit (ICU)and in-hospital mortality. RESULTS: 561 patients were admitted. Overall, 193 patients were included in the study. Fifty nine patients (30%) died, 9 (5%) were still in hospital, and 125 (65%) were discharged. The NAFLD cohort (n = 61) was significantly younger (60 vs 70.5 years, p = 0.046) at presentation compared to the non-NAFLD (n = 132). NAFLD diagnosis was not associated with adverse outcomes. However, the NAFLD group had higher C reactive protein (CRP) (107 vs 91.2 mg/L, p = 0.05) compared to non-NAFLD(n = 132). Among NAFLD patients, male gender (p = 0.01), ferritin (p = 0.003) and EWS (p = 0.047) were associated with in-hospital mortality, while the presence of intermediate/high risk FIB-4 or liver cirrhosis was not. CONCLUSION: The presence of NAFLD per se was not associated with worse outcomes in patients hospitalised for COVID-19. Though NAFLD patients were younger on admission, disease stage was not associated with clinical outcomes. Yet, mortality was associated with gender and a pronounced inflammatory response in the NAFLD group.

Current and future pharmacological therapies for managing cirrhosis and its complications.

Due to the restrictions of liver transplantation, complication-guided pharmacological therapy has become the mainstay of long-term management of cirrhosis. This article aims to provide a complete overview of pharmacotherapy options that may be commenced in the outpatient setting which are available for managing cirrhosis and its complications, together with discussion of current controversies and potential future directions. PubMed/Medline/Cochrane Library were electronically searched up to December 2018 to identify studies evaluating safety, efficacy and therapeutic mechanisms of pharmacological agents in cirrhotic adults and animal models of cirrhosis. Non-selective beta-blockers effectively reduce variceal re-bleeding risk in cirrhotic patients with moderate/large varices, but appear ineffective for primary prevention of variceal development and may compromise renal function and haemodynamic stability in advanced decompensation. Recent observational studies suggest protective, haemodynamically-independent effects of beta-blockers relating to reduced bacterial translocation. The gut-selective antibiotic rifaximin is effective for secondary prophylaxis of hepatic encephalopathy; recent small trials also indicate its potential superiority to norfloxacin for secondary prevention of spontaneous bacterial peritonitis. Diuretics remain the mainstay of uncomplicated ascites treatment, and early trials suggest alpha-adrenergic receptor agonists may improve diuretic response in refractory ascites. Vaptans have not demonstrated clinical effectiveness in treating refractory ascites and may cause detrimental complications. Despite initial hepatotoxicity concerns, safety of statin administration has been demonstrated in compensated cirrhosis. Furthermore, statins are suggested to have protective effects upon fibrosis progression, decompensation and mortality. Evidence as to whether proton pump inhibitors cause gut-liver-brain axis dysfunction is conflicting. Emerging evidence indicates that anticoagulation therapy reduces incidence and increases recanalisation rates of non-malignant portal vein thrombosis, and may impede hepatic fibrogenesis and decompensation. Pharmacotherapy for cirrhosis should be implemented in accordance with up-to-date guidelines and in conjunction with aetiology management, nutritional optimisation and patient education.

CD8+T cells from patients with cirrhosis display a phenotype that may contribute to cirrhosis-associated immune dysfunction.

BACKGROUND: Cirrhosis-associated immune dysfunction (CAID) contributes to high sepsis risk in patients with chronic liver disease. Various innate and; to a lesser extent; adaptive immune dysfunctions have been described as contributors to CAID leading to immune-paresis and impaired anti-microbial response in cirrhosis. In this study, we examined the phenotype of CD8+T cells in chronic liver disease with the aim to evaluate changes that might contribute to impaired immune responses. METHODS: Sixty patients with cirrhosis were prospectively recruited for this study. CD8+T cells from peripheral blood, ascites and liver explants were characterized using flow cytometry and immunohistochemistry, respectively. The transcriptional signature of flow-sorted HLA-DR+CD8+T cells was performed using Nanostring™ technology. HLA-DR+CD8+T cells interactions with PBMCs and myeloid cells were tested in vitro. FINDINGS: Peripheral CD8+T cells from cirrhotic patients displayed an altered phenotype characterized by high HLA-DR and TIM-3 surface expression associated with concomitant infections and disease severity, respectively. Paired peritoneal CD8+T cells expressed more pronounced levels of HLA-DR and PD-1 compared to peripheral CD8+T cells. HLA-DR+CD8+T cells were enriched in cirrhotic livers compared to controls. TIM-3, CTLA-4 and PD-1 levels were highly expressed on HLA-DR+CD8+T cells and co-expression of HLA-DR and PD1 was higher in patients with poor disease outcomes. Genes involved in cytokines production and intracellular signalling pathways were strongly down-regulated in HLA-DR+CD8+T cells. In comparison to their HLA-DR- counterparts, HLA-DR+CD8+T cells promoted less proliferation of PBMCs and induced phenotypic and functional dysfunctions in monocytes and neutrophils in vitro. INTERPRETATION: In patients with cirrhosis, CD8+T cells display a phenotypic, functional and transcriptional profile which may contribute to CAID. FUND: This work was supported by Medical Research Council, the Rosetrees Charitable Trust, Robert Tournut 2016 grant (Sociéte Nationale Française de GastroEntérologie), Gilead® sciences, and NIHR Imperial Biomedical Research Centre.

Alcoholic ketoacidosis with multiple complications: a case report.

Alcoholic ketoacidosis is a poorly diagnosed medical emergency usually identified in chronic alcohol misusers following an abrupt cessation or reduction of alcohol consumption. A high index of suspicion should be maintained by acute physicians as response to treatment is rapid with complete resolution of metabolic derangements. Complications are usually the result of not instituting the correct treatment or not addressing associated conditions. We describe a case of alcoholic ketoacidosis with multiple complications at presentation.

Gastrointestinal Motility Disorders and Their Clinical Implications in Cirrhosis.

Gastrointestinal motility is impaired in a substantial proportion of patients with cirrhosis. Cirrhosis-related autonomic neuropathy, increased nitric oxide production, and gut hormonal changes have been implicated. Oesophageal dysmotility has been associated with increased frequency of abnormal gastro-oesophageal reflux. Impaired gastric emptying and accommodation may result in early satiety and may have an impact on the nutritional status of these patients. Small intestinal dysmotility might be implicated in small intestinal bacterial overgrowth and increased bacterial translocation. The latter has been implicated in the pathophysiology of hepatic encephalopathy and spontaneous bacterial peritonitis. Enhanced colonic motility is usually associated with the use of lactulose. Pharmacological interventions aiming to alter gastrointestinal motility in cirrhosis could potentially have a beneficial effect reducing the risk of hepatic decompensation and improving prognosis.