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The gut–liver axis is defined by dietary and environmental communication between the gut, microbiome and the liver with its redox and immune systems, the overactivation of which can lead to hepatic injury. We used media preconditioning to mimic some aspects of the enterohepatic circulation by treating the human Caco-2 intestinal epithelial cell line with 5, 10 and 20 mM paracetamol (N-acetyl-para-aminophenol; APAP) for 24 h, after which cell culture supernatants were transferred to differentiated human hepatic HepaRG cells for a further 24 h. Cell viability was assessed by mitochondrial function and ATP production, while membrane integrity was monitored by cellular-based impedance. Metabolism by Caco-2 cells was determined by liquid chromatography with tandem mass spectrometry. Caco-2 cell viability was not affected by APAP, while cell membrane integrity and tight junctions were maintained and became tighter with increasing APAP concentrations, suggesting a reduction in the permeability of the intestinal epithelium. During 24 h incubation, Caco-2 cells metabolised 64–68% of APAP, leaving 32–36% of intact starting compound to be transferred to HepaRG cells. When cultured with Caco-2-preconditioned medium, HepaRG cells also showed no loss of cell viability or membrane integrity, completely in contrast to direct treatment with APAP, which resulted in a rapid loss of cell viability and membrane integrity and, ultimately, cell death. Thus, the pre-metabolism of APAP could mitigate previously observed hepatotoxicity to hepatic tight junctions caused by direct exposure to APAP. These observations could have important implications for the direct exposure of hepatic parenchyma to APAP, administered via the intravenous route.

Background Chronic liver disease (CLD) is a growing cause of morbidity and mortality worldwide, particularly in low to middle-income countries with high disease burden and limited treatment availability. Coffee consumption has been linked with lower rates of CLD, but little is known about the effects of different coffee types, which vary in chemical composition. This study aimed to investigate associations of coffee consumption, including decaffeinated, instant and ground coffee, with chronic liver disease outcomes. Methods A total of 494,585 UK Biobank participants with known coffee consumption and electronic linkage to hospital, death and cancer records were included in this study. Cox regression was used to estimate hazard ratios (HR) of incident CLD, incident CLD or steatosis, incident hepatocellular carcinoma (HCC) and death from CLD according to coffee consumption of any type as well as for decaffeinated, instant and ground coffee individually. Results Among 384,818 coffee drinkers and 109,767 non-coffee drinkers, there were 3600 cases of CLD, 5439 cases of CLD or steatosis, 184 cases of HCC and 301 deaths from CLD during a median follow-up of 10.7 years. Compared to non-coffee drinkers, coffee drinkers had lower adjusted HRs of CLD (HR 0.79, 95% CI 0.72–0.86), CLD or steatosis (HR 0.80, 95% CI 0.75–0.86), death from CLD (HR 0.51, 95% CI 0.39–0.67) and HCC (HR 0.80, 95% CI 0.54–1.19). The associations for decaffeinated, instant and ground coffee individually were similar to all types combined. Conclusion The finding that all types of coffee are protective against CLD is significant given the increasing incidence of CLD worldwide and the potential of coffee as an intervention to prevent CLD onset or progression.

Background: Type 2 diabetes is associated with increased risk of progression to cirrhosis and hepatocellular carcinoma (HCC) in people with chronic liver diseases, particularly non-alcoholic fatty liver disease (NAFLD). However, the absolute risk of progression is low so it is crucial to accurately identify patients who would benefit most from hepatology referral and intensified management. Current risk-stratification tools are sub-optimal and perform worse in people with diabetes.Aims: To determine whether the addition of complementary biomarker(s) to current NAFLD risk-stratification tools in people with Type 2 diabetes could improve the identification of people who are at increased risk of developing incident cirrhosis or HCC.Methods: The Edinburgh Type 2 diabetes Study (ET2DS) is a cohort study of men and women with Type 2 diabetes (n=1066, age 60-75 at baseline). Cases of cirrhosis and HCC were identified over 11-years of follow-up. Biomarkers were measured at baseline and year one and association with incident disease assessed using logistic regression.Results: Of existing risk-stratification scores tested, the Fibrosis-4 (FIB-4) index and the AST:platelet ratio index (APRI) performed best in this cohort. Addition of hyaluronic acid (cut-point ≥50μg/L) to FIB-4 (cut-point ≥1.3) maintained a false negative rate ≤25% and reduced the number of people incorrectly identified as ‘high-risk’ for incident disease by ~50%.Conclusions: The addition of hyaluronic acid to FIB-4 reduced the proportion of people inappropriately identified as ‘high-risk’ for development of cirrhosis/HCC in a community population of otherwise asymptomatic people with Type 2 diabetes. These findings require validation in independent cohorts.

Introduction The number of deaths and prevalent cases of cirrhosis are increasing worldwide, but there are no licensed antifibrotic or pro-regenerative medicines and liver transplantation is a limited resource. Cirrhosis is characterized by extreme liver fibrosis, organ dysfunction and complications related to portal hypertension. Advances in our understanding of liver fibrosis progression and regression following successful etiological therapy betray vulnerabilities in common and disease-specific mechanisms that could be targeted pharmacologically. Area Covered This review summarizes the cellular and molecular pathogenesis of cirrhosis as a preface to discussion of the current drug development landscape. The dominant indication for global pharma R&D pipelines is cirrhosis related to non-alcoholic steatohepatitis (NASH). We searched Clinicaltrials.gov, GlobalData, Pharmaprojects and PubMed for pertinent information on emerging synthetic drugs for cirrhosis, with a focus on compounds listed in phase 2 and phase 3 trials. Expert Opinion Although cirrhosis can regress following successful etiological treatment, there are no specific antifibrotic or pro-regenerative drugs approved for this condition. Obstacles to drug development in cirrhosis include intrinsic biological factors, a heterogeneous patient population and lack of acceptable surrogate endpoints. Nevertheless, several synthetic drugs are being evaluated in clinical trials and the NASH field is rapidly embracing a drug combination approach.

Current approaches to stage chronic liver diseases have limited utility to predict liver cancer risk. Here, we employed single nucleus RNA sequencing (snRNA-seq) to characterize the cellular microenvironment of healthy and pre-malignant livers using two distinct mouse models. Downstream analyses unraveled a previously uncharacterized disease-associated hepatocyte transcriptional state (daHep). These cells were absent in healthy livers but increasingly prevalent as chronic liver disease progressed. Copy number variation (CNV) analysis of microdissected tissue demonstrated that daHep enriched regions are riddled with structural variants, suggesting these cells represent a pre-malignant intermediary. Integrated analysis of three recent human snRNA-seq datasets confirmed the presence of a similar phenotype in human chronic liver disease and further supported its enhanced mutational burden. Importantly, we show that high daHep levels precede carcinogenesis and predict a higher risk of hepatocellular carcinoma development. These findings may change the way chronic liver disease patients are staged, surveilled and risk-stratified.

SUMMARYCurrent approaches to stage chronic liver diseases have limited utility to directly predict liver cancer risk. Here, we employed single nucleus RNA sequencing (snRNA-seq) to characterize the cellular microenvironment of healthy and chronically injured pre-malignant livers using two distinct mouse models. Analysis of 40,748 hepatic nuclei unraveled a previously uncharacterized disease-associated hepatocyte transcriptional state (daHep). These cells were absent in healthy livers, but were increasingly prevalent as chronic liver disease progressed towards hepatocarcinogenesis. Gene expression deconvolution of 1,439 human liver transcriptomes from publicly available datasets revealed that daHep frequencies highly correlate with current histopathological liver disease staging systems. Importantly, we show that high daHep levels precede carcinogenesis in mice and humans and predict a higher risk of hepatocellular carcinoma (HCC) development. This novel transcriptional signature with diagnostic and, more importantly, prognostic significance has the potential to change the way chronic liver disease patients are staged, surveilled and risk-stratified.

IntroductionLiver cirrhosis is a growing global healthcare challenge. Cirrhosis is characterised by severe liver fibrosis, organ dysfunction and complications related to portal hypertension. There are no licensed antifibrotic or proregenerative medicines and liver transplantation is a scarce resource. Hepatic macrophages can promote both liver fibrogenesis and fibrosis regression. The safety and feasibility of peripheral infusion of ex vivo matured autologous monocyte-derived macrophages in patients with compensated cirrhosis has been demonstrated.Methods and analysisThe efficacy of autologous macrophage therapy, compared with standard medical care, will be investigated in a cohort of adult patients with compensated cirrhosis in a multicentre, open-label, parallel-group, phase 2, randomised controlled trial. The primary outcome is the change in Model for End-Stage Liver Disease score at 90 days. The trial will provide the first high-quality examination of the efficacy of autologous macrophage therapy in improving liver function, non-invasive fibrosis markers and other clinical outcomes in patients with compensated cirrhosis.Ethics and disseminationThe trial will be conducted according to the ethical principles of the Declaration of Helsinki 2013 and has been approved by Scotland A Research Ethics Committee (reference 15/SS/0121), National Health Service Lothian Research and Development department and the Medicine and Health Care Regulatory Agency-UK. Final results will be presented in peer-reviewed journals and at relevant conferences.Trial registration numbersISRCTN10368050 and EudraCT; reference 2015-000963-15

Biopsy remains the gold‐standard measure for staging liver disease, both to inform prognosis and to assess the response to a given treatment. Semiquantitative scores such as the Ishak fibrosis score are used for evaluation. These scores are utilised in clinical trials, with the US Food and Drug Administration mandating particular scores as inclusion criteria for participants and using the change in score as evidence of treatment efficacy. There is an urgent need for improved, quantitative assessment of liver biopsies to detect small incremental changes in liver architecture over the course of a clinical trial. Artificial intelligence (AI) methods have been proposed as a way to increase the amount of information extracted from a biopsy and to potentially remove bias introduced by manual scoring. We have trained and evaluated an AI tool for measuring the amount of scarring in sections of picrosirius red‐stained liver. The AI methodology was compared with both manual scoring and widely available colour space thresholding. Four sequential sections from each case were stained on two separate occasions by two independent clinical laboratories using routine protocols to study the effect of inter‐ and intra‐laboratory staining variation on these tools. Finally, we compared these methods to second harmonic generation (SHG) imaging, a stain‐free quantitative measure of collagen. Although AI methods provided a modest improvement over simpler computer‐assisted measures, staining variation both within and between laboratories had a dramatic effect on quantitation, with manual assignment of scar proportion being the most consistent. Manual assessment also most strongly correlated with collagen measured by SHG. In conclusion, results suggest that computational measures of liver scarring from stained sections are compromised by inter‐ and intra‐laboratory staining. Stain‐free quantitative measurement using SHG avoids staining‐related variation and may prove more accurate in detecting small changes in scarring that may occur in therapeutic trials.

BACKGROUND & AIMS Nonalcoholic fatty liver disease (NAFLD) is increasing in prevalence worldwide. NAFLD is associated with excess risk of all-cause mortality, and its progression to nonalcoholic steatohepatitis (NASH) and fibrosis accounts for a growing proportion of cirrhosis and hepatocellular cancer and thus is a leading cause of liver transplant worldwide. Noninvasive precise methods to identify patients with NASH, and NASH with significant disease activity and fibrosis when the disease is still modifiable, are crucial. The aim of this study was to examine the clinical utility of corrected T1 (cT1) vs magnetic resonance imaging (MRI) liver fat for identification of NASH participants with nonalcoholic fatty liver disease activity score ≥4 and fibrosis (F) ≥2 (high-risk NASH). METHODS Data from 5 clinical studies (n = 543) with participants suspected of NAFLD were pooled or used for individual participant data meta-analysis. The diagnostic accuracy of the MRI biomarkers to stratify NASH patients was determined using the area under the receiver operating characteristic curve (AUROC). RESULTS A stepwise increase in cT1 and MRI liver fat with increased NAFLD severity was shown, and cT1 was significantly higher in NASH participants with fibrosis grade ≥2 (high-risk NASH). The diagnostic accuracy (AUROC) of cT1 to identify patients with NASH was 0.78 (95% CI, 0.74-0.82), for liver fat was 0.78 (95% CI, 0.73-0.82), and when combined with MRI liver fat was 0.82 (95% CI, 0.78-0.85). The diagnostic accuracy of cT1 to identify patients with high-risk NASH was good (AUROC, 0.78; 95% CI, 0.74-0.82), was superior to MRI liver fat (AUROC, 0.69; 95% CI, 0.64-0.74), and was not substantially improved by combining it with MRI liver fat (AUC, 0.79; 95% CI, 0.75-0.83). The meta-analysis showed similar performance to the pooled analysis for these biomarkers. CONCLUSIONS This study shows that quantitative MRI-derived biomarkers cT1 and liver fat are suitable for identifying patients with NASH, and cT1 is a better noninvasive technology than liver fat to identify NASH patients at greatest risk of disease progression. MRI cT1 and liver fat therefore have important clinical utility to help guide the appropriate use of interventions in NAFLD and NASH clinical care pathways.

ObjectiveGenome-wide association studies (GWAS) have identified several risk loci for gallstone disease. As with most polygenic traits, it is likely many genetic determinants are undiscovered. The aim of this study was to identify novel genetic variants that represent new targets for gallstone research and treatment.DesignWe performed a GWAS of 28,627 gallstone cases and 348,373 controls in the UK Biobank and a GWA meta-analysis (43,639 cases and 506,798 controls) with the FinnGen cohort. We assessed pathway enrichment using gene-based then gene-set analysis and tissue expression of implicated genes in Genotype-Tissue Expression project data. We constructed a polygenic risk score (PRS) and evaluated phenotypic traits associated with the score.ResultsSeventy-five risk loci were identified (P−8) of which forty-six were novel. Pathway enrichment revealed associations with lipid homeostasis, glucuronidation, phospholipid metabolism and gastrointestinal motility. ANO1 and TMEM147, both in novel loci, are strongly expressed in the gallbladder and gastrointestinal tract. Both regulate gastrointestinal motility. The gallstone risk allele rs7599-A leads to suppression of hepatic TMEM147 expression suggesting the protein protects against gallstone formation. Individuals in the highest decile of the PRS demonstrated a 6-fold increased risk of gallstones compared to the lowest risk category. The PRS was strongly associated with increased body mass index, serum liver enzyme and C-reactive protein concentrations and decreased lipoprotein cholesterol concentrations.ConclusionThis GWAS demonstrates the polygenic nature of gallstone risk and identifies 46 novel susceptibility loci. For the first time, we implicate genes influencing gastrointestinal motility in the pathogenesis of gallstones.Summary BoxWhat is already known on this subject?Genome-wide association studies (GWAS) have identified 29 genetic variants within independent loci which increase the risk of gallstone disease.Most of these variants lie within or near to genes that regulate lipid or bile acid metabolism. Gallstones are known to have a significant genetic component with 25-50% of gallstone disease due to genetic risk factors.Much of this risk is not accounted for by the known gallstone-susceptibility loci.What are the new findings?We performed a GWAS in the UK Biobank (28,627 gallstone cases, 348,373 controls) and a GWA meta-analysis (43,639 cases and 506,798 controls) with the FinnGen cohort.We identified a total of 75 gallstone-susceptibility loci with 46 of these being new and the remaining 29 being those already identified.We annotated the variants based on their position within or near to genes and assessed pathway enrichment through gene-set analysis.We identify two novel gallstone-susceptibility loci in which the lead variants lie within genes governing gastrointestinal motility which highly expressed in gallbladder (ANO1 and TMEM147).We demonstrate further loci involved in primary cilia function.We report significant association of a polygenic risk score with gallstones using independent subsets of the study population.How might it impact on clinical practice in the foreseeable future?The genes and pathways identified represent novel targets for development of medication targeting primary or secondary prevention of gallstones. This may be of particular benefit to those unable to undergo cholecystectomy.The individual variants or polygenic risk score identified in this GWAS could form the basis for identification of individuals with high risk of gallstones to support screening or treatment of gallstone disease. This screening may be of particular benefit in populations with elevated risk of gallstones such as haemolytic disease or bariatric surgery.

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Genome-wide association studies (GWAS) have identified several risk loci for nonalcoholic fatty liver disease (NAFLD). Previous studies have largely relied on small sample sizes and have assessed quantitative traits. We performed a case-control GWAS in the UK Biobank using recorded diagnosis of NAFLD based on diagnostic codes recommended in recent consensus guidelines. We performed a GWAS of 4,761 cases of NAFLD and 373,227 healthy controls without evidence of NAFLD. Sensitivity analyses were performed excluding other co-existing hepatic pathology, adjusting for body mass index (BMI) and adjusting for alcohol intake. A total of 9,723,654 variants were assessed by logistic regression adjusted for age, sex, genetic principal components, and genotyping batch. We performed a GWAS meta-analysis using available summary association statistics. Six risk loci were identified (P < 5*10-8 ) (apolipoprotein E [APOE], patatin-like phospholipase domain containing 3 [PNPLA3, transmembrane 6 superfamily member 2 [TM6SF2], glucokinase regulator [GCKR], mitochondrial amidoxime reducing component 1 [MARC1], and tribbles pseudokinase 1 [TRIB1]). All loci retained significance in sensitivity analyses without co-existent hepatic pathology and after adjustment for BMI. PNPLA3 and TM6SF2 remained significant after adjustment for alcohol (alcohol intake was known in only 158,388 individuals), with others demonstrating consistent direction and magnitude of effect. All six loci were significant on meta-analysis. Rs429358 (P = 2.17*10-11 ) is a missense variant within the APOE gene determining ϵ4 versus ϵ2/ϵ3 alleles. The ϵ4 allele of APOE offered protection against NAFLD (odds ratio for heterozygotes 0.84 [95% confidence interval 0.78-0.90] and homozygotes 0.64 [0.50-0.79]). Conclusion: This GWAS replicates six known NAFLD-susceptibility loci and confirms that the ϵ4 allele of APOE is associated with protection against NAFLD. The results are consistent with published GWAS using histological and radiological measures of NAFLD, confirming that NAFLD identified through diagnostic codes from consensus guidelines is a valid alternative to more invasive and costly approaches.

Liver cirrhosis is a major cause of death worldwide and is characterized by extensive fibrosis. There are currently no effective antifibrotic therapies available. To obtain a better understanding of the cellular and molecular mechanisms involved in disease pathogenesis and enable the discovery of therapeutic targets, here we profile the transcriptomes of more than 100,000 single human cells, yielding molecular definitions for non-parenchymal cell types that are found in healthy and cirrhotic human liver. We identify a scar-associated TREM2+CD9+ subpopulation of macrophages, which expands in liver fibrosis, differentiates from circulating monocytes and is pro-fibrogenic. We also define ACKR1+ and PLVAP+ endothelial cells that expand in cirrhosis, are topographically restricted to the fibrotic niche and enhance the transmigration of leucocytes. Multi-lineage modelling of ligand and receptor interactions between the scar-associated macrophages, endothelial cells and PDGFRα+ collagen-producing mesenchymal cells reveals intra-scar activity of several pro-fibrogenic pathways including TNFRSF12A, PDGFR and NOTCH signalling. Our work dissects unanticipated aspects of the cellular and molecular basis of human organ fibrosis at a single-cell level, and provides a conceptual framework for the discovery of rational therapeutic targets in liver cirrhosis. Single-cell RNA sequencing is used to characterize and compare the functional diversity of cells from liver biopsies of human scarred and normal liver, and identifies markers for scar-associated macrophages and endothelial cells.

Background & AimsGenome-wide association studies (GWAS) have identified several risk loci for non-alcoholic fatty liver disease (NAFLD). Previous studies have largely relied on small sample sizes and have assessed quantitative traits. We performed a case-control GWAS in the UK Biobank using recorded diagnosis of NAFLD based on diagnostic codes recommended in recent consensus guidelines.Approach & ResultsWe performed a GWAS of 4,761 cases of NAFLD and 373,227 healthy controls without evidence of NAFLD. Sensitivity analyses were performed excluding other co-existing hepatic pathology, adjusting for BMI and adjusting for alcohol intake. 9,723,654 variants were assessed by logistic regression adjusted for age, sex, genetic principal components and genotyping batch. We performed a GWA meta-analysis using available summary association statistics from two previously published case-control GWAS of NAFLD. Six risk loci were identified (PConclusionsThis GWAS demonstrates that the ∈4 allele of APOE is strongly associated with protection against NAFLD.

Background & Aims The development of COVID-19 vaccines has progressed with encouraging safety and efficacy data. Concerns have been raised about SARS-CoV-2 vaccine responses in the large population of patients with non-alcoholic fatty liver disease (NAFLD). The study aimed to explore the safety and immunogenicity of COVID-19 vaccination in NAFLD. Methods This multicenter study included patients with NAFLD without a history of SARS-CoV-2 infection. All patients were vaccinated with 2 doses of inactivated vaccine against SARS-CoV-2. The primary safety outcome was the incidence of adverse reactions within 7 days after each injection and overall incidence of adverse reactions within 28 days, and the primary immunogenicity outcome was neutralizing antibody response at least 14 days after the whole-course vaccination. Results A total of 381 patients with pre-existing NAFLD were included from 11 designated centers in China. The median age was 39.0 years (IQR 33.0–48.0 years) and 179 (47.0%) were male. The median BMI was 26.1 kg/m2 (IQR 23.8–28.1 kg/m2). The number of adverse reactions within 7 days after each injection and adverse reactions within 28 days totaled 95 (24.9%) and 112 (29.4%), respectively. The most common adverse reactions were injection site pain in 70 (18.4%), followed by muscle pain in 21 (5.5%), and headache in 20 (5.2%). All adverse reactions were mild and self-limiting, and no grade 3 adverse reactions were recorded. Notably, neutralizing antibodies against SARS-CoV-2 were detected in 364 (95.5%) patients with NAFLD. The median neutralizing antibody titer was 32 (IQR 8-64), and the neutralizing antibody titers were maintained. Conclusions The inactivated COVID-19 vaccine appears to be safe with good immunogenicity in patients with NAFLD. Lay summary The development of vaccines against coronavirus disease 2019 (COVID-19) has progressed rapidly, with encouraging safety and efficacy data. This study now shows that the inactivated COVID-19 vaccine appears to be safe with good immunogenicity in the large population of patients with non-alcoholic fatty liver disease.

It is not uncommon for clinicians to encounter varying degrees of hepatic steatosis in patients undergoing resection for colorectal liver metastases (CRLM). Magnetic resonance imaging is currently the preferred investigation for identification and pre-operative planning of these patients. An objective assessment of liver quality and degree of steatosis is paramount for planning a safe resection, which is seldom provided by routine MRI sequences. We studied two patients who underwent an additional pre-operative multiparametric MRI scan (LiverMultiScanTM) as a part of an observational clinical trial (HepaT1ca, NCT03213314) to assess the quality of liver. Outcome was assessed in the form of post-hepatectomy liver failure. Both patients (Patient 1 and 2) had comparable pre-operative characteristics. Both patients were planned for an extended right hepatectomy with an estimated future liver remnant of approximately 30%. Conventional preoperative contrast MRI showed mild liver steatosis in both patients. Patient one developed post-hepatectomy liver failure leading to prolonged hospital stay compared to patient two who had uneventful post-operative course. Retrospective evaluation of multiparametric MRI scan revealed findings consistent with fibro-inflammatory disease and steatosis (cT1 829 ms, PDFF 14%) for patient 1 whereas patient two had normal parameters (cT1 735 ms, PDFF 2.4%). These findings corresponded with the resection specimen histology. Multiparametric MRI can objectively evaluate future liver health and volume which may help refine surgical decision-making and improve patient outcomes

Microcirculatory dysfunction is associated with organ failure, poor response to vasoactive drugs and increased mortality in cirrhosis, but monitoring techniques are not established. We hypothesized that the chorioretinal structures of the eye could be visualized as a non-invasive proxy of the systemic microvasculature in cirrhosis and would correlate with renal dysfunction. Optical Coherence Tomography (OCT) was performed to image the retina in n = 55 cirrhosis patients being assessed for liver transplantation. OCT parameters were compared with established cohorts of age- and sex-matched healthy volunteers (HV) and patients with chronic kidney disease (CKD). Retinal thickness, macular volume and choroidal thickness were significantly reduced relative to HV and comparable to CKD patients (macular volume: HV vs. cirrhosis mean difference 0.44 mm3 (95% CI 0.26–0.61), p ≤ 0.0001). Reduced retinal thickness and macular volume correlated with renal dysfunction in cirrhosis (macular volume vs. MDRD-6 eGFR r = 0.40, p = 0.006). Retinal changes had resolved substantially 6 weeks following transplantation. There was an inverse association between choroidal thickness and circulating markers of endothelial dysfunction (endothelin-1 r = −0.49, p ≤ 0.001; von Willebrand factor r = −0.32, p ≤ 0.05). Retinal OCT may represent a non-invasive window to the microcirculation in cirrhosis and a dynamic measure of renal and endothelial dysfunction. Validation in different cirrhosis populations is now required.

BACKGROUND The incidence of cirrhosis and hepatocellular carcinoma (HCC) is increased in Type 2 diabetes, primarily secondary to non-alcoholic fatty liver disease (NAFLD). European guidelines recommend screening for NAFLD in Type 2 diabetes. American guidelines, while not advocating a screening protocol, suggest using non-invasive markers of fibrosis for risk-stratification and guiding onward referral. AIMS To test the ability of individual fibrosis scores and the European screening algorithm to predict 11-year incident cirrhosis/HCC in an asymptomatic community cohort of older people with Type 2 diabetes. METHODS The Edinburgh Type 2 Diabetes Study investigated men and women with Type 2 diabetes (n = 1066, aged 60-75 at baseline). Liver markers were measured at baseline and year 1; steatosis and fibrosis markers were calculated according to independently published calculations. During 11 years of follow-up, cases of cirrhosis and HCC were identified. RESULTS Forty-three out of 1059 participants with no baseline cirrhosis/HCC developed incident disease. All scores were significantly associated with incident liver disease by odds ratio (P < .05). The ability of the risk-stratification tools to accurately identify those who developed incident cirrhosis/HCC was poor with low-positive predictive values (5-46%) and high false-negative and -positive rates (up to 60% and 77%) respectively. When fibrosis risk scores were used in conjunction with the European algorithm, they performed modestly better than when applied in isolation. CONCLUSIONS In a cohort with a moderately low incidence of cirrhosis/HCC, existing risk scores did not reliably identify participants at high risk. Better prediction models for cirrhosis/HCC in people with Type 2 diabetes are required.

IntroductionLate stage clinical trials in non-alcoholic steatohepatitis (NASH) are currently required by the FDA to use liver biopsy as a primary endpoint. The well-reported limitations with biopsy, such as associated risks and sampling error, coupled with patient preference, are driving investigation into non-invasive alternatives. MRI-derived biomarkers proton density fat fraction (PDFF) and iron-corrected T1 mapping (cT1) are gaining traction as emerging alternatives to biopsy for NASH. Our aim was to explore the correlations between cT1 and PDFF (from LiverMultiScan®), with the histological components on the NAFLD-NASH spectrum in a large cohort of cross-sectional data, in order to calibrate the measurement to histology, and to infer what might constitute a clinically meaningful change when related to the FDA’s criteria.Materials and MethodsIn a retrospective analysis of data combined from three previously published observational NASH studies, in which adult participants who underwent liver biopsy on suspicion of NAFLD or NASH and had an MRI scan measuring cT1 and PDFF (LiverMultiScan®, Perspectum Ltd, UK), associations between imaging biomarkers and histology were tested using Spearman’s rank correlation coefficient (rs), and further exploration of the relationships between the imaging variables and histology were performed using linear regression.ResultsN = 264 patients with mean age of 54 (SD:9.9), 39% female, and 69% with BMI ≥ 30kg.m−2 were included in the analysis. cT1 and PDFF both correlated with all features of the NAFLD activity score (NAS). cT1 was also positively correlated with Kleiner-Brunt fibrosis. Partial correlations, adjusting for steatosis, revealed cT1 correlated with inflammation and fibrosis, whereas PDFF did not, and both were still associated with the NAS, but correlation was weaker with PDFF than cT1. An estimated difference of 88 ms in cT1, or 21% relative difference in PDFF was related to a two-point difference in overall NAS.ConclusionThe correlations between cT1 and PDFF with the histopathological hallmarks of NASH demonstrate the potential utility of both cT1 and PDFF as non-invasive biomarkers to detect a pharmacodynamic change in NASH, with cT1 showing superiority for detecting changes in inflammation and fibrosis, rather than liver fat alone.

Objectives COVID-19 has rapidly become a major health emergency worldwide. Patients with IBD are at increased risk of infection, especially when they have active disease and are taking immunosuppressive therapy. The characteristics and outcomes of COVID-19 in patients with IBD remain unclear. Design This Italian prospective observational cohort study enrolled consecutive patients with an established IBD diagnosis and confirmed COVID-19. Data regarding age, sex, IBD (type, treatments and clinical activity), other comorbidities (Charlson Comorbidity Index (CCI)), signs and symptoms of COVID-19 and therapies were compared with COVID-19 outcomes (pneumonia, hospitalisation, respiratory therapy and death). Results Between 11 and 29 March 2020, 79 patients with IBD with COVID-19 were enrolled at 24 IBD referral units. Thirty-six patients had COVID-19-related pneumonia (46%), 22 (28%) were hospitalised, 7 (9%) required non-mechanical ventilation, 9 (11%) required continuous positive airway pressure therapy, 2 (3%) had endotracheal intubation and 6 (8%) died. Four patients (6%) were diagnosed with COVID-19 while they were being hospitalised for a severe flare of IBD. Age over 65 years (p=0.03), UC diagnosis (p=0.03), IBD activity (p=0.003) and a CCI score >1 (p=0.04) were significantly associated with COVID-19 pneumonia, whereas concomitant IBD treatments were not. Age over 65 years (p=0.002), active IBD (p=0.02) and higher CCI score were significantly associated with COVID-19-related death. Conclusions Active IBD, old age and comorbidities were associated with a negative COVID-19 outcome, whereas IBD treatments were not. Preventing acute IBD flares may avoid fatal COVID-19 in patients with IBD. Further research is needed.

SummaryBackgroundPatients with pre-existing cirrhosis are considered at increased risk of severe coronavirus disease 2019 (COVID-19) but the clinical course in these patients has not yet been reported. This study aimed to provide a detailed report of the clinical characteristics and outcomes among COVID-19 patients with pre-existing cirrhosis.MethodsIn this retrospective, multicenter cohort study, we consecutively included all adult inpatients with laboratory-confirmed COVID-19 and pre-existing cirrhosis that had been discharged or had died by 24 March 2020 from 16 designated hospitals in China.Demographic, clinical, laboratory and radiographic findings on admission, treatment, complications during hospitalization and clinical outcomes were collected and compared between survivors and non-survivors.FindingsTwenty-one patients were included consecutively in this study, of whom 16 were cured and 5 died in hospital. Seventeen patients had compensated cirrhosis and hepatitis B virus infection was the most common etiology. Lymphocyte and platelet counts were lower, and direct bilirubin levels were higher in patients who died than those who survived (p= 0·040, 0·032, and 0·006, respectively). Acute respiratory distress syndrome and secondary infection were both the most frequently observed complications. Only one patient developed acute on chronic liver failure. Of the 5 non-survivors, all patients developed acute respiratory distress syndrome and 2 patients progressed to multiple organ dysfunction syndrome.InterpretationLower lymphocyte and platelet counts, and higher direct bilirubin level might represent poor prognostic indicators in SARS-CoV-2-infected patients with pre-existing cirrhosis.

Non-alcoholic steatohepatitis (NASH) is major health burden lacking effective pharmacological therapies. Clinical trials enrol patients with histologically-defined NAFLD (non-alcoholic fatty liver disease) activity score (NAS) ≥ 4 and Kleiner-Brunt fibrosis stage (F) ≥ 2; however, screen failure rates are often high following biopsy. This study evaluated a non-invasive MRI biomarker, iron-corrected T1 mapping (cT1), as a diagnostic pre-screening biomarker for NASH. In a retrospective analysis of 86 biopsy confirmed NAFLD patients we explored the potential of blood and imaging biomarkers, both in isolation and in combination, to discriminate those who have NAS ≥ 4 and F ≥ 2 from those without. Stepwise logistic regression was performed to select the optimal combination of biomarkers, diagnostic accuracy was determined using area under the receiver operator curve and model validated confirmed with and fivefold cross-validation. Results showed that levels of cT1, AST, GGT and fasting glucose were all good predictors of NAS ≥ 4 and F ≥ 2, and the model identified the combination of cT1-AST-fasting glucose (cTAG) as far superior to any individual biomarker (AUC 0.90 [0.84–0.97]). This highlights the potential utility of the composite cTAG score for screening patients prior to biopsy to identify those suitable for NASH clinical trial enrolment.

Background In preclinical models, recombinant human relaxin-2 (serelaxin) had anti-fibrotic effects and ameliorated portal hypertension (PH). A small exploratory study in patients with cirrhosis also suggested that serelaxin could reduce portal pressure. Methods In a phase 2, double-blind, randomised controlled study conducted in a single centre (Royal Infirmary of Edinburgh, UK), male and female adult participants with cirrhosis and clinically significant PH (CSPH; hepatic venous pressure gradient (HVPG) > 10 mmHg) were enrolled. Participants were allocated to serelaxin or placebo in a 3:1 ratio. The placebo was matched to serelaxin on appearance and administration protocol to create and maintain blinding. The primary endpoint was the change from baseline in fasting HVPG after 2 h of peripheral i.v. serelaxin infusion (80 μg/kg/day for 60 min followed by 30 μg/kg/day for at least 60 min). Secondary endpoints included the change from baseline in hepatic blood flow and systemic haemodynamics (cardiac index, systemic vascular resistance index and aortic pulse wave velocity). Short-term safety and tolerability of serelaxin were assessed. Results A total of 17 participants were screened, 15 were randomised and 11 completed the study (n = 9 serelaxin, n = 2 placebo). Reasons for withdrawal were baseline HVPG n = 2) and technical failure (n = 2). The trial ended early due to manufacturer discontinuation of the study drug. The median age was 56 (range 43–69) years and 73% of participants were male. Alcohol was the commonest cirrhosis aetiology (n = 10). Participants had a median Model for End-Stage Liver Disease score of 10 (range 6–14). The mean baseline HVPG was 16.3 (range 10.3–21.7) mmHg. Individual responses were variable, but overall there was no statistically significant change in HVPG after 2 h of i.v. serelaxin (arithmetic mean of difference ± SD was 0.4 ± 3.5 mmHg (95% CI –2.3, 3.1; p = 0.76)). There were also no substantial changes from baseline in hepatic or systemic haemodynamics. We recorded 12 adverse events in 7 participants treated with serelaxin; none were significant, and most were unrelated to the investigational medicinal product. There were no serious adverse events. Conclusion In a small randomised, phase 2, proof-of-concept study in patients with cirrhosis and CSPH, serelaxin infusion was safe and well-tolerated but had a neutral effect on HVPG. Trial registration ClinicalTrials.gov, NCT02669875. Registered on 1 February 2016.

The risk of poor post-operative outcome and the benefits of surgical resection as a curative therapy require careful assessment by the clinical care team for patients with primary and secondary liver cancer. Advances in surgical techniques have improved patient outcomes but identifying which individual patients are at greatest risk of poor post-operative liver performance remains a challenge. Here we report results from a multicentre observational clinical trial (ClinicalTrials.gov NCT03213314) which aimed to inform personalised pre-operative risk assessment in liver cancer surgery by evaluating liver health using quantitative multiparametric magnetic resonance imaging (MRI). We combined estimation of future liver remnant (FLR) volume with corrected T1 (cT1) of the liver parenchyma as a representation of liver health in 143 patients prior to treatment. Patients with an elevated preoperative liver cT1, indicative of fibroinflammation, had a longer post-operative hospital stay compared to those with a cT1 within the normal range (6.5 vs 5 days; p = 0.0053). A composite score combining FLR and cT1 predicted poor liver performance in the 5 days immediately following surgery (AUROC = 0.78). Furthermore, this composite score correlated with the regenerative performance of the liver in the 3 months following resection. This study highlights the utility of quantitative MRI for identifying patients at increased risk of poor post-operative liver performance and a longer stay in hospital. This approach has the potential to inform the assessment of individualised patient risk as part of the clinical decision-making process for liver cancer surgery.

Background Accurate assessment of liver health prior to undertaking resectional liver surgery or chemoembolisation for primary and secondary cancers is essential for patient safety and optimal outcomes. LiverMultiScan™, an MRI-based technology, non-invasively quantifies hepatic fibroinflammatory disease, steatosis and iron content. We hypothesise that LiverMultiScan™can quantify liver health prior to surgery and inform the risk assessment for patients considering liver surgery or chemoembolization and seek to evaluate this technology in an operational environment. Methods/Design HepaT1ca is an observational cohort study in two tertiary-referral liver surgery centres in the United Kingdom. The primary outcome is correlation between the pre-operative liver health assessment score (Hepatica score - calculated by weighting future remnant liver volume by liver inflammation and fibrosis (LIF) score) and the post-operative liver function composite integer-based risk (Hyder-Pawlik) score. With ethical approval and fully-informed consent, individuals considering liver surgery for primary or secondary cancer will undergo clinical assessment, blood sampling, and LiverMultiScan™multiparametric MRI before and after surgical liver resection or TACE. In nested cohorts of individuals undergoing chemotherapy prior to surgery, or those undergoing portal vein embolization (PVE) as an adjunct to surgery, an additional testing session prior to commencement of treatment will occur. Tissue will be examined histologically and by immunohistochemistry. Pre-operative liver health assessment scores and the post-operative risk scores will be correlated to define the ability of LiverMultiScan™to predict the risk of post-operative morbidity and mortality. Because technology performance in this setting is unknown, a pragmatic sample size will be used. For the primary outcome, n = 200 for the main cohort will allow detection of a minimum correlation coefficient of 0.2 with 5% significance and power of 80%. Discussion This study will refine the technology and clinical application of multiparametric MRI (including LiverMultiScan™), to quantify pre-existing liver health and predict post-intervention outcomes following liver resection. If successful, this study will advance the technology and support the use of multiparametric MRI as part of an enhanced pre-operative assessment to improve patient safety and to personalise operative risk assessment of liver surgery/non-surgical intervention. Trial registration This study is registered on ClinicalTrials.gov Identifier: NCT03213314. Electronic supplementary material The online version of this article (10.1186/s12885-018-4737-3) contains supplementary material, which is available to authorized users.

Rationale & Objective: Chronic kidney disease (CKD) is a leading cause of morbidity and mortality worldwide, with limited strategies for prevention and treatment. Coffee is a complex mixture of chemicals, and consumption has been associated with mostly beneficial health outcomes. This work aimed to determine the impact of coffee consumption on kidney function. Study Design: Genome-wide association study (GWAS) and Mendelian randomization. Setting & Participants: UK Biobank baseline data were used for a coffee consumption GWAS and included 227,666 participants. CKDGen Consortium data were used for kidney outcomes and included 133,814 participants (12,385 cases of CKD) of mostly European ancestry across various countries. Exposure: Coffee consumption. Outcomes: Estimated glomerular filtration rate (eGFR), CKD GFR categories 3 to 5 (G3-G5; eGFR < 60 mL/min/1.73 m2), and albuminuria. Analytical Approach: GWAS to identify single-nucleotide polymorphisms (SNPs) associated with coffee consumption in UK Biobank and use of those SNPs in Mendelian randomization analyses of coffee consumption and kidney outcomes in CKDGen. Results: 2,126 SNPs were associated with coffee consumption (P < 5 × 10−8), 25 of which were independent and available in CKDGen. Drinking an extra cup of coffee per day conferred a protective effect against CKD G3-G5 (OR, 0.84; 95% CI, 0.72-0.98; P = 0.03) and albuminuria (OR, 0.81; 95% CI, 0.67-0.97; P = 0.02). An extra cup was also associated with higher eGFR (β = 0.022; P = 1.6 × 10−6) after removal of 3 SNPs responsible for significant heterogeneity (Cochran Q P = 3.5 × 10−15). Limitations: Assays used to measure creatinine and albumin varied between studies that contributed data and a sex-specific definition was used for albuminuria rather than KDIGO guideline recommendations. Conclusions: This study provides evidence of a beneficial effect of coffee on kidney function. Given widespread coffee consumption and limited interventions to prevent CKD incidence and progression, this could have significant implications for global public health in view of the increasing burden of CKD worldwide.

Background & aims Analysis of volatile organic compounds (VOCs) in exhaled breath, ‘volatomics’, provides opportunities for non-invasive biomarker discovery and novel mechanistic insights into a variety of diseases. The purpose of this pilot study was to compare breath VOCs in an initial cohort of patients with non-alcoholic fatty liver disease (NAFLD) and healthy controls. Methods Breath samples were collected from 15 participants with Child-Pugh class A NAFLD cirrhosis, 14 with non-cirrhotic NAFLD, and 14 healthy volunteers. Exhaled breath samples were collected using an established methodology and VOC profiles were analysed by gas chromatography-mass spectrometry. The levels of 19 VOCs previously associated with cirrhosis were assessed. Peaks of the VOCs were confirmed and integrated using Xcalibur® software, normalised to an internal standard. Receiver-operating characteristic (ROC) curves were used to determine the diagnostic accuracy of the candidate VOCs. Results Terpinene, dimethyl sulfide, and D-limonene provided the highest predictive accuracy to discriminate between study groups. Combining dimethyl sulfide with D-limonene led to even better discrimination of patients with NAFLD cirrhosis from healthy volunteers (AUROC 0.98; 95% CI 0.93–1.00; p, Graphical abstract, Highlights • Metabolic dysfunction in liver disease is reflected in the biocomposition of exhaled breath. • Specific volatile organic compounds can be measured in breath samples (volatomics) and have diagnostic potential in chronic liver disease. • Levels of alfa-terpinene, dimethyl sulfide, and D-limonene in exhaled breath can be used to stratify patients with non-alcoholic fatty liver disease.

LiverMultiScan is an emerging diagnostic tool using multiparametric MRI to quantify liver disease. In a two-centre prospective validation study, 161 consecutive adult patients who had clinically-indicated liver biopsies underwent contemporaneous non-contrast multiparametric MRI at 3.0 tesla (proton density fat fraction (PDFF), T1 and T2* mapping), transient elastography (TE) and Enhanced Liver Fibrosis (ELF) test. Non-invasive liver tests were correlated with gold standard histothological measures. Reproducibility of LiverMultiScan was investigated in 22 healthy volunteers. Iron-corrected T1 (cT1), TE, and ELF demonstrated a positive correlation with hepatic collagen proportionate area (all p MultiScan showed excellent test/re-test reliability (coefficients of variation ranging from 1.4% to 2.8% for cT1). Overall failure rates for LiverMultiScan, ELF and TE were 4.3%, 1.9% and 15%, respectively. LiverMultiScan is an emerging point-of-care diagnostic tool that is comparable with the established non-invasive tests for assessment of liver fibrosis, whilst at the same time offering a superior technical success rate and contemporaneous measurement of liver steatosis and iron accumulation.

Background: Validated diagnostic tools that are accurate, cost effective and acceptable to patients are required for disease stratification and monitoring in NAFLD. Aims: We investigated the performance and cost of multiparametric MRI alongside existing biomarkers in the assessment of NAFLD. Methods: Adult patients undergoing standard of care liver biopsy for NAFLD were prospectively recruited at two UK liver centres and underwent multiparametric MRI, blood sampling and transient elastography withing 2 weeks of liver biopsy. Non-invasive markers were compared to histology as the gold standard. Results: Data were obtained in 50 patients and 6 healthy volunteers. Corrected T1 (cT1) correlated with NAFLD activity score (Rho=0.514, p

Targeting of the peptide hormone relaxin to injured mouse liver, via a nanoparticle/gene therapy approach, switches pro-fibrotic hepatic macrophages to a restorative phenotype that orchestrates tissue repair.

A significant unmet need exists for accurate, reproducible, noninvasive diagnostic tools to assess and monitor portal hypertension (PHT). We report the first use of quantitative MRI markers for the haemodynamic assessment of nonselective beta-blockers (NSBB) in PHT. In a randomized parallel feasibility study in 22 adult patients with PHT and a clinical indication for NSBB, we acquired haemodynamic data at baseline and after 4 weeks of NSBB (propranolol or carvedilol) using phase-contrast MR angiography (PC-MRA) in selected intra-abdominal vessels. T1 mapping of liver and spleen was undertaken to assess changes in tissue composition. Target NSBB dose was achieved in 82%. There was a substantial reduction from baseline in mean average flow in the superior abdominal aorta after 4 weeks of NSBB therapy (4.49±0.98versus3.82±0.86 L/min,P=0.03) but there were no statistically significant differences in flow in any other vessels, even in patients with >25% decrease in heart rate (47% of patients). Mean percentage change in liver and spleen T1 following NSBB was small and highly variable. In conclusion, PC-MRA was able to detect reduction in cardiac output by NSBB but did not detect significant changes in visceral blood flow or T1. This trial was registered with the ISRCTN registry (ISRCTN98001632).

Therapies to reduce liver fibrosis and stimulate organ regeneration are urgently needed. We conducted a first-in-human, phase 1 dose-escalation trial of autologous macrophage therapy in nine adults with cirrhosis and a Model for End-Stage Liver Disease (MELD) score of 10–16 (ISRCTN 10368050). Groups of three participants received a single peripheral infusion of 107, 108 or up to 109 cells. Leukapheresis and macrophage infusion were well tolerated with no transfusion reactions, dose-limiting toxicities or macrophage activation syndrome. All participants were alive and transplant-free at one year, with only one clinical event recorded, the occurrence of minimal ascites. The primary outcomes of safety and feasibility were met. This study informs and provides a rationale for efficacy studies in cirrhosis and other fibrotic diseases.

Background: Liver cirrhosis is a large burden on global health, causing over one million deaths per year. Observational studies have reported an inverse association between coffee and cirrhosis. Aims: To perform a systematic review and meta-analysis to characterise the relationship between coffee consumption and cirrhosis. Methods: We searched for studies published until July 2015 that reported odds ratios, relative risks (RR) or hazard ratios for cirrhosis stratified by coffee consumption. We calculated RRs of cirrhosis for an increase in daily coffee consumption of two cups for each study and overall. We performed analyses by study design, type of cirrhosis and mortality. We assessed the risk of bias in each study and the overall quality of evidence for the effect of coffee on cirrhosis. Results: We identified five cohort studies and four case–control studies involving 1990 cases and 432 133 participants. We observed a dose–response in most studies and overall. The pooled RR of cirrhosis for a daily increase in coffee consumption of two cups was 0.56 (95% CI 0.44–0.68; I2 83.3%). The RR pooled from cohort studies for a daily increase of two cups was 0.58 (95% CI 0.41–0.76; I2 91.1%) and from case–control studies it was 0.52 (95% CI 0.40–0.63; I2 0.0%). The pooled RR of alcoholic cirrhosis for a daily increase of two cups was 0.62 (95% CI 0.51–0.73; I2 0%) and of death from cirrhosis it was 0.55 (95% CI 0.35–0.74; I2 90.3%). Conclusion: This meta-analysis suggests that increasing coffee consumption may substantially reduce the risk of cirrhosis.

Objectives: To evaluate the existing evidence for associations between coffee consumption and multiple health outcomes.Design: Umbrella review of the evidence across meta-analyses of observational and interventional studies of coffee consumption and any health outcome.Data: sources PubMed, Embase, CINAHL, Cochrane Database of Systematic Reviews, and screening of references.Eligibility criteria for selecting studies: Meta-analyses of both observational and interventional studies that examined the associations between coffee consumption and any health outcome in any adult population in all countries and all settings. Studies of genetic polymorphisms for coffee metabolism were excluded.Results: The umbrella review identified 201 meta-analyses of observational research with 67 unique health outcomes and 17 meta-analyses of interventional research with nine unique outcomes. Coffee consumption was more often associated with benefit than harm for a range of health outcomes across exposures including high versus low, any versus none, and one extra cup a day. There was evidence of a non-linear association between consumption and some outcomes, with summary estimates indicating largest relative risk reduction at intakes of three to four cups a day versus none, including all cause mortality (relative risk 0.83, 95% confidence interval 0.83 to 0.88), cardiovascular mortality (0.81, 0.72 to 0.90), and cardiovascular disease (0.85, 0.80 to 0.90). High versus low consumption was associated with an 18% lower risk of incident cancer (0.82, 0.74 to 0.89). Consumption was also associated with a lower risk of several specific cancers and neurological, metabolic, and liver conditions. Harmful associations were largely nullified by adequate adjustment for smoking, except in pregnancy, where high versus low/no consumption was associated with low birth weight (odds ratio 1.31, 95% confidence interval 1.03 to 1.67), preterm birth in the first (1.22, 1.00 to 1.49) and second (1.12, 1.02 to 1.22) trimester, and pregnancy loss (1.46, 1.06 to 1.99). There was also an association between coffee drinking and risk of fracture in women but not in men.Conclusion: Coffee consumption seems generally safe within usual levels of intake, with summary estimates indicating largest risk reduction for various health outcomes at three to four cups a day, and more likely to benefit health than harm. Robust randomised controlled trials are needed to understand whether the observed associations are causal. Importantly, outside of pregnancy, existing evidence suggests that coffee could be tested as an intervention without significant risk of causing harm. Women at increased risk of fracture should possibly be excluded.

The peptide hormone human relaxin-2 (H2-RLX) has emerged as a potential therapy for cardiovascular and fibrotic diseases, but its short in vivo half-life is an obstacle to long-term administration. The discovery of ML290 demonstrated that it is possible to identify small molecule agonists of the cognate G-protein coupled receptor for H2-RLX (relaxin family peptide receptor-1 (RXFP1)). In our efforts to generate a new medicine for liver fibrosis, we sought to identify improved small molecule functional mimetics of H2-RLX with selective, full agonist or positive allosteric modulator activity against RXFP1. First, we confirmed expression of RXFP1 in human diseased liver. We developed a robust cellular cAMP reporter assay of RXFP1 signaling in HEK293 cells transiently expressing RXFP1. A high-throughput screen did not identify further specific agonists or positive allosteric modulators of RXFP1, affirming the low druggability of this receptor. As an alternative approach, we generated novel ML290 analogues and tested their activity in the HEK293-RXFP1 cAMP assay and the human hepatic cell line LX-2. Differences in activity of compounds on cAMP activation compared with changes in expression of fibrotic markers indicate the need to better understand cell- and tissue-specific signaling mechanisms and their disease-relevant phenotypes in order to enable drug discovery.