Kevin J. Fagan

The portal inflammatory infiltrate and ductular reaction in human nonalcoholic fatty liver disease

Although nonalcoholic fatty liver disease (NAFLD) is conventionally assessed histologically for lobular features of inflammation, development of portal fibrosis appears to be associated with disease progression. We investigated the composition of the portal inflammatory infiltrate and its relationship to the ductular reaction (DR), a second portal phenomenon implicated in fibrogenesis. The portal inflammatory infiltrate may contribute directly to fibrogenesis as well as influence the fate of the DR hepatic progenitor cells (HPCs), regulating the balance between liver repair and fibrosis. The presence of portal inflammation in NAFLD was strongly correlated with disease severity (fibrosis stage) and the DR. The portal infiltrate was characterized by immunostaining NAFLD liver biopsy sections (n = 33) for broad leukocyte subset markers (CD68, CD3, CD8, CD4, CD20, and neutrophil elastase) and selected inflammatory markers (matrix metalloproteinase 9 and interleukin [IL]‐17). Cells expressing all markers examined were identified throughout the liver lobules and in portal tracts, although portal tracts were more densely populated (P < 0.01), and dominated by CD68+ macrophages and CD8+ lymphocytes, at all stages of disease. An increase in portal macrophages in NAFLD patients with steatosis alone (P < 0.01) was the earliest change detected, even before elevated expression of the proinflammatory cytokines, IL1B and TNF, in patients with early NASH (P < 0.05). Portal and periductal accumulation of all other cell types examined occurred in progressed NASH (all P < 0.05). Conclusion: Knowledge of the complex cellular composition of the portal inflammatory infiltrate and HPC/DR niche in NAFLD will shape future functional studies to elucidate the contribution of portal inflammation to HPC differentiation and NAFLD pathogenesis. (Hepatology 2014;59:1393‐1405)

ELF score ≥9.8 indicates advanced hepatic fibrosis and is influenced by age, steatosis and histological activity

There is increasing need to identify individuals with advanced liver fibrosis, who are at risk of complications such as hepatocellular carcinoma. The commercially available enhanced liver fibrosis (ELF) test provides a non‐invasive assessment of fibrosis severity. This study was designed to determine the diagnostic accuracy of the manufacturers cut‐off value (≥9.8) in identifying advanced fibrosis.

The Enhanced liver fibrosis score is associated with clinical outcomes and disease progression in patients with chronic liver disease

Current tools for risk stratification of chronic liver disease subjects are limited. We aimed to determine whether the serum‐based ELF (Enhanced Liver Fibrosis) test predicted liver‐related clinical outcomes, or progression to advanced liver disease, and to compare the performance of ELF to liver biopsy and non‐invasive algorithms.

Ascites Bacterial Burden and Immune Cell Profile Are Associated with Poor Clinical Outcomes in the Absence of Overt Infection

Bacterial infections, most commonly spontaneous bacterial peritonitis in patients with ascites, occur in one third of admitted patients with cirrhosis, and account for a 4-fold increase in mortality. Bacteria are isolated from less than 40% of ascites infections by culture, necessitating empirical antibiotic treatment, but culture-independent studies suggest bacteria are commonly present, even in the absence of overt infection. Widespread detection of low levels of bacteria in ascites, in the absence of peritonitis, suggests immune impairment may contribute to higher susceptibility to infection in cirrhotic patients. However, little is known about the role of ascites leukocyte composition and function in this context. We determined ascites bacterial composition by quantitative PCR and 16S rRNA gene sequencing in 25 patients with culture-negative, non-neutrocytic ascites, and compared microbiological data with ascites and peripheral blood leukocyte composition and phenotype. Bacterial DNA was detected in ascitic fluid from 23 of 25 patients, with significant positive correlations between bacterial DNA levels and poor 6-month clinical outcomes (death, readmission). Ascites leukocyte composition was variable, but dominated by macrophages or T lymphocytes, with lower numbers of B lymphocytes and natural killer cells. Consistent with the hypothesis that impaired innate immunity contributes to susceptibility to infection, high bacterial DNA burden was associated with reduced major histocompatibility complex class II expression on ascites (but not peripheral blood) monocytes/macrophages. These data indicate an association between the presence of ascites bacterial DNA and early death and readmission in patients with decompensated cirrhosis. They further suggest that impairment of innate immunity contributes to increased bacterial translocation, risk of peritonitis, or both.

BMI but not stage or etiology of nonalcoholic liver disease affects the diagnostic utility of carbohydrate-deficient transferrin.

BACKGROUND A reliable biomarker is required in hepatology clinics for detection and follow-up of heavy alcohol consumption. Carbohydrate-deficient transferrin (CDT) increases with sustained heavy alcohol consumption and is the most specific biomarker of ethanol (EtOH) consumption. Recent introduction of a standardized method for measuring CDT has improved its clinical application. This study was designed to determine whether alcohol-independent factors influence CDT levels in patients with chronic liver disease (CLD). METHODS The relationship between serum %CDT and self-reported history of alcohol consumption was examined in 254 patients referred for evaluation of liver disease. CDT analysis was performed on serum collected at time of liver biopsy. RESULTS CDT levels were not affected by severity or etiology of nonalcoholic liver disease. Thirteen of 254 subjects had a %CDT >1.7, predictive of heavy alcohol intake, 6 of whom did not acknowledge heavy drinking. Twelve of these 13 subjects were suspected heavy drinkers on review of their medical records and clinical results. Conversely, not all acknowledged heavy drinkers had %CDT >1.7. Heavy drinkers with a body mass index (BMI) in the overweight or obese range had significantly lower %CDT than lean heavy drinkers. This persisted even when lean body weight was used as an approximation of the EtOH volume of distribution. CONCLUSIONS An elevated BMI reduces the diagnostic utility of CDT at higher alcohol intake in subjects with CLD using the standardized method. In a hepatology outpatient setting, this assay is likely to be useful to confirm suspicion of heavy drinking in subjects who are not overweight, but cannot reliably identify moderate drinkers or heavy drinkers who are overweight.

Assessment of alcohol histories obtained from patients with liver disease: opportunities to improve early intervention

Alcohol is an important primary and comorbid cause of liver injury in patients referred for investigation and management of liver disease. Early assessment and documentation of alcohol consumption is therefore essential, and recommended in both general practice and hospital settings.

Multiplex serum protein analysis identifies novel biomarkers of advanced fibrosis in patients with chronic liver disease with the potential to improve diagnostic accuracy of established biomarkers

Background and Aims Non-invasive markers of liver fibrosis are urgently required, especially for use in non-specialist settings. The aim of this study was to identify novel serum biomarkers of advanced fibrosis. Methods We performed an unbiased screen of 120 serum analytes including cytokines, chemokines and proteases in 70 patients (35 without fibrosis, 35 with cirrhosis on biopsy), and selected a panel of 44 candidate biomarkers, which were subsequently measured in a mixed-etiology cohort of 432 patients with known serum HA, PIIINP and TIMP1 (which comprise the validated Enhanced Liver Fibrosis (ELF) test). Multivariate logistic regression modelling was used to generate models for the prediction of advanced or significant fibrosis (METAVIR ≥F3 and ≥F2, respectively); in addition to identifying biomarkers of disease activity and steatohepatitis. Results Seventeen analytes were significantly differentially expressed between patients with no advanced fibrosis and patients with advanced fibrosis, the most significant being hyaluronic acid (HA) and matrix metalloproteinase (MMP) 7 (p = 2.9E-41 and p = 1.0E-26, respectively). The optimal model for the prediction of advanced fibrosis comprised HA, MMP7, MMP1, alphafetoprotein (AFP) and the AST to platelet ratio index (APRI). We demonstrate enhanced diagnostic accuracy (AUROC = 0.938) compared to a model comprising HA, PIIINP and TIMP1 alone (ELF) (AUROC = 0.898, p<0.0001, De Long’s test). Conclusions We have identified novel serum biomarkers of advanced liver fibrosis, which have the potential to enhance the diagnostic accuracy of established biomarkers. Our data suggest MMP7 is a valuable indicator of advanced fibrosis and may play a role in liver fibrogenesis.

Hepatic expression profiling identifies steatosis-independent and steatosis-driven advanced fibrosis genes

Chronic liver disease (CLD) is associated with tissue-destructive fibrosis. Considering that common mechanisms drive fibrosis across etiologies, and that steatosis is an important cofactor for pathology, we performed RNA sequencing on liver biopsies of patients with different fibrosis stages, resulting from infection with hepatitis C virus (HCV) (with or without steatosis) or fatty liver disease. In combination with enhanced liver fibrosis score correlation analysis, we reveal a common set of genes associated with advanced fibrosis, as exemplified by those encoding the transcription factor ETS-homologous factor (EHF) and the extracellular matrix protein versican (VCAN). We identified 17 fibrosis-associated genes as candidate EHF targets and demonstrated that EHF regulates multiple fibrosis-associated genes, including VCAN, in hepatic stellate cells. Serum VCAN levels were also elevated in advanced fibrosis patients. Comparing biopsies from patients with HCV with or without steatosis, we identified a steatosis-enriched gene set associated with advanced fibrosis, validating follistatin-like protein 1 (FSTL1) as an exemplar of this profile. In patients with advanced fibrosis, serum FSTL1 levels were elevated in those with steatosis (versus those without). Liver Fstl1 mRNA levels were also elevated in murine CLD models. We thus reveal a common gene signature for CLD-associated liver fibrosis and potential biomarkers and/or targets for steatosis-associated liver fibrosis.

Response to ELF cut-off points: aetiology is also a relevant factor.

235–41. 5. Trembling PM, Lampertico P, Parkes J, et al. Performance of enhanced liver fibrosis test and comparison with transient elastography in the identification of liver fibrosis in patients with chronic hepatitis b infection. J Viral Hepat 2014; 21: 430–8. 6. Fernandes FF, Dellavance A, Andrade LE, et al. New cutoff points of enhanced liver fibrosis (elf) panel for chronic hepatitis c patients. Hepatology 2014; 60(Suppl): 924(A).

Economic consideration for reducing hospital presentations among patients with decompensated cirrhosis: not all presentations avoided can be considered equal

Individuals with decompensated cirrhosis and ascites requiring paracentesis utilize exceptionally high levels of hospital resources. Consequently, potential modifications to existing models of healthcare to assist patients in the management of their liver disease and reduce the need for hospital encounters have potential to improve patients’ health and reduce demand on acute hospital services. However, there is a paucity of data examining how much healthcare resources could be re-directed to interventions that prevent hospitalizations without net annual budgetary disadvantage (from the hospital’s perspective). The purpose of this study was to probabilistically examine how much healthcare resourcing could be saved per hospital presentation avoided among this clinical population.