Active Liver Disease Research Articles

A Randomised Controlled Trial on the Effectiveness and Adherence of Modified Alternate-day Calorie Restriction in Improving Activity of Non-Alcoholic Fatty Liver Disease

Currently, there is no effective therapy for non-alcoholic fatty liver disease (NAFLD), and although calorie restriction is recommended in guidelines, but adherence is an issue. The current study aimed to determine the effectiveness of eight weeks intermittent fasting (IF) strategy in the control of NAFLD activity and the adherence rate of such strategy. This was a randomized controlled trial with modified alternate-day calorie restriction (MACR), a form of IF, as the active intervention and usual habitual diet as control. The outcome measures included changes in body mass index (BMI), blood lipids (cholesterol, LDL, HDL and triglyceride), fasting blood sugar (FBS), liver enzymes (ALT and AST), and ultrasound measurements of liver steatosis and 2-dimensional shear wave elastography (SWE). Per-protocol (PP) analysis was performed with comparison within (post vs. pre-intervention) and between (MACR vs. control) groups and P < 0.05 as significant. Of 115 individuals with NAFLD, 43 satisfied the study entry criteria, and 33 were randomised to MACR and 10 to control group, and after 8 weeks, 30 from MACR and 9 from control group completed PP. Significant reduction in weight and BMI (P = 0.001 and 0.02 respectively) was observed in MACR vs. control. Likewise, ALT was reduced with MACR but not control (P = 0.02). No reductions in lipid parameters and FBS were found in between-group analyses (all P > 0.22). Both liver steatosis and fibrosis (SWE) scores were significantly reduced in MACR vs. controls (both P < 0.01). Adherence level for MACR remained between 75-83% throughout the study. As a conclusion, eight weeks of IF (MACR) strategy appears more effective than usual habitual diet in the control of NAFLD activity and with good adherence rate.

Agomelatine Drug Utilisation Study in Selected European Countries: A Multinational, Observational Study to Assess Effectiveness of Risk-Minimisation Measures

Hepatotoxic reactions are an important identified risk listed in the agomelatine risk management plan. This post-authorisation safety study evaluated the effectiveness of additional risk-minimisation measures (aRMMs) for agomelatine. The objective of this study was to evaluate, among physicians prescribing agomelatine and their patients, liver function monitoring adherence, compliance with contraindications and patients' reasons for non-compliance with liver monitoring. A non-interventional cohort study was conducted among adults initiating agomelatine in routine clinical practice in Denmark, France, Germany and Spain through a retrospective medical record abstraction (MRA) before and after implementation of aRMMs and a cross-sectional patient survey. Fifty-four sites contributed data on 437 and 404 patients in the before- and after-RMM periods, and 237 patients completed the survey. No patient had cirrhosis in either study period; 98.2% of patients in the before- and 98.0% in the after-RMM period had no active liver disease reported at initiation or during treatment. Compliance to contraindicated medications was > 99% in both periods. The adherence to the liver-monitoring regimen was similar in both periods (15.1% before RMM and 16.3% after RMM). In the after-RMM period, 25.2% of patients had a liver test before or at treatment initiation; 61.5% had a liver test during treatment. Among patients surveyed who did not have a blood test before treatment initiation or during treatment, the most frequently cited reason was a test ordered but not yet performed. The overall adherence to liver-monitoring recommendations remained weakly influenced by aRMMs. However, patients treated with agomelatine are in compliance with relevant contraindications.

Characterization of Cellular Sources and Circulating Levels of Extracellular Vesicles in a Dietary Murine Model of Nonalcoholic Steatohepatitis.

Circulating extracellular vesicles (EVs) are a novel and emerging biomarker for nonalcoholic steatohepatitis (NASH). It has been demonstrated that total circulating EVs and hepatocyte‐derived EVs are elevated in male mice with diet‐induced NASH. How hepatocyte‐derived EVs change over time and other cellular sources of EVs in NASH have not been determined. Our objective was to define the quantitative evolution of hepatocyte‐derived, macrophage‐derived, neutrophil‐derived, and platelet‐derived EVs in male and female mice with dietary NASH. Fluorescently labeled antibodies and a nanoscale flow cytometer were used to detect plasma levels of EVs. Asialoglycoprotein receptor 1 (ASGR1) and cytochrome P450 family 2 subfamily E member 1 (CYP2E1) are markers of hepatocyte‐derived EVs; galectin 3 is a marker of macrophage‐derived EVs; common epitope on lymphocyte antigen 6 complex, locus G/C1 (Ly‐6G and Ly‐6C) is a marker of neutrophil‐derived EVs; and clusters of differentiation 61 (CD61) is a marker of platelet‐derived EVs. Nonalcoholic fatty liver disease activity score (NAS) was calculated using hematoxylin and eosin‐stained liver sections, and magnetic resonance imaging (MRI) was used for measurement of the fat fraction and elastography. Hepatocyte‐derived EVs increased in both male and female mice at 12 and 10 weeks of feeding, respectively, and remained elevated at 24 weeks in both male and female mice and at 48 weeks in male mice and 36 weeks in female mice. Macrophage‐ and neutrophil‐derived EVs were significantly elevated at 24 weeks of dietary feeding concomitant with the histologic presence of inflammatory foci in the liver. In fat‐, fructose‐, and cholesterol‐ (FFC) fed male mice, platelet‐derived EVs were elevated at 12, 24, and 48 weeks, whereas in female mice, platelet derived EVs were significantly elevated at 24 weeks. Hepatocyte‐, macrophage‐ and neutrophil‐derived EVs correlated well with the histologic NAS. Conclusion: Circulating cell‐type‐specific EVs may be a novel biomarker for NASH diagnosis and longitudinal follow up.

Direct Oral Anticoagulants in Patients With Atrial Fibrillation and Liver Disease

BackgroundAdvanced liver disease is known to increase the risk for bleeding and affects the hepatic clearance and metabolism of drugs. Subjects with active liver disease were excluded from pivotal clinical trials of direct oral anticoagulants (DOACs), so the evidence regarding the efficacy and safety of DOACs in patients with liver disease is lacking. ObjectivesThe aim of this study was to compare DOACs with warfarin in patients with nonvalvular atrial fibrillation and liver disease. MethodsUsing the Korean National Health Insurance Service database, subjects with atrial fibrillation and active liver disease treated with oral anticoagulation were included (12,778 with warfarin and 24,575 with DOACs), and analyzed ischemic stroke, intracranial hemorrhage, gastrointestinal bleeding, major bleeding, all-cause death, and the composite outcome. Propensity score weighting was used to balance covariates between the 2 groups. ResultsDOACs were associated with lower risks for ischemic stroke (hazard ratio [HR]: 0.548; 95% confidence interval [CI]: 0.485 to 0.618), intracranial hemorrhage (HR: 0.479; 95% CI 0.394 to 0.581), gastrointestinal bleeding (HR: 0.819; 95% CI: 0.619 to 0.949), major bleeding (HR: 0.650; 95% CI: 0.575 to 0.736), all-cause death (HR: 0.698; 95% CI: 0.636 to 0.765), and the composite outcome (HR: 0.610; 95% CI: 0.567 to 0.656) than warfarin. Among the total study population, 13% of patients (n = 4,942) were identified as having significant active liver disease. A consistent benefit was observed in patients with significant active liver disease (HR for the composite outcome: 0.691; 95% CI: 0.577 to 0.827). ConclusionsIn this large Asian population with atrial fibrillation and liver disease, DOACs showed better effectiveness and safety than warfarin, which was consistent in those with significant active liver disease.

Apical sodium-dependent bile acid transporter inhibition with volixibat improves metabolic aspects and components of non-alcoholic steatohepatitis in Ldlr-/-.Leiden mice.

Interruption of bile acid recirculation through inhibition of the apical sodium-dependent bile acid transporter (ASBT) is a promising strategy to alleviate hepatic cholesterol accumulation in non-alcoholic steatohepatitis (NASH), and improve the metabolic aspects of the disease. Potential disease-attenuating effects of the ASBT inhibitor volixibat (5, 15, and 30 mg/kg) were investigated in high-fat diet (HFD)-fed Ldlr-/-.Leiden mice over 24 weeks. Plasma and fecal bile acid levels, plasma insulin, lipids, and liver enzymes were monitored. Final analyses included liver histology, intrahepatic lipids, mesenteric white adipose tissue mass, and liver gene profiling. Consistent with its mechanism of action, volixibat significantly increased the total amount of bile acid in feces. At the highest dose, volixibat significantly attenuated the HFD-induced increase in hepatocyte hypertrophy, hepatic triglyceride and cholesteryl ester levels, and mesenteric white adipose tissue deposition. Non-alcoholic fatty liver disease activity score (NAS) was significantly lower in volixibat-treated mice than in the HFD controls. Gene profiling showed that volixibat reversed the inhibitory effect of the HFD on metabolic master regulators, including peroxisome proliferator-activated receptor-γ coactivator-1β, insulin receptor, and sterol regulatory element-binding transcription factor 2. Volixibat may have beneficial effects on physiological and metabolic aspects of NASH pathophysiology.

Two phase kinetics of the inflammatory response from hepatocyte-peripheral blood mononuclear cell interactions

Active liver diseases are characterized by an infiltration of inflammatory immune cells, which interact locally with hepatocytes. Co-cultures between non- and -activated human peripheral blood mononuclear cells (PBMCs) and human hepatoma HepaRG cells were used to determine the role of these cell interactions in the inflammatory response. At the early stage, PBMC-HepaRG cell interactions increased mRNA expression and/or secretion of IL-6, IL-8, CCL-20 and MCP-1, in part through direct cell contact and the induction was higher in PHA-activated conditions. The pro-inflammatory cytokines IL-17 and/or TNFα contributed to the increase of IL-6 and IL-8 secretion. HepaRG cells modulated T cell polarization by increasing Th1 cell transcription factor expression and by reducing CD3+ CD4+ IL-17+ cell frequency when PBMCs were activated with PHA. At a later stage, the presence of HepaRG cells inhibited PHA-induced HLA-DR expression on PBMCs, and PBMC proliferation. In contrast, the presence of skin fibroblasts had no effect of PBMC proliferation induced by PHA. After a first pro-inflammatory phase, PBMC-HepaRG cell interactions may down-regulate the immune response. The PBMC-hepatocyte interactions can thus participate first to the initiation of hepatitis and later to the maintenance of immune tolerance in liver, possibly contributing to chronicity.

PB2292 ROLE OF SMOKING ON HEMATOLOGICAL PARAMETERS AND FERRITIN LEVELS

Background:Cigarette smoking is associated with an increased risk of cardiovascular diseases, including coronary artery disease, peripheral vascular disease, ischemic heart disease, atherosclerosis, myocardial infarction and stroke. Our aim was to define possible effects of smoking on hematological parameters as well as ferritin and transferrin saturation.Aims:In this study, we evaluated the association between hematological parameters and smoking status. Our aim was to define the possible effects of smoking on hematological parameters as well as ferritin and transferrin saturation.Methods:This study has been performed in a retrospective manner. One hundred and forty nine patients who admitted to Hacettepe University Hospitals between September 2018 and November 2018 were evaluated. Inclusion criterion in this study were ≥ being older than 18 years of age, having no chronic diseases such as diabetes mellitus, ischemic heart disease, peripheral vascular disease, active liver and kidney disease, chronic pancreatitis, gastrointestinal disease, inflammatory bowel diseases, hypertension, any infectious or debilitating illness. In addition, patients who were on medications such as antibiotics, steroids, thiazide diuretics, aspirin, non‐steroidal anti‐inflammatory drugs, immunomodulatory drugs and drugs that may interfere with ferritin and CBC parameters were excluded from the study.Results:There were 95(63.7%) healthy non‐smokers and 54 (36.3%) healthy smokers included in this study. The median age was 32 (18‐61) years for all groups. There were 47 (31.5%) males and 102 (68.5%) females in this study. Hemoglobin (p&lt;0.001), HCT (p&lt;0.001) and MCV (p = 0.002) were statistically significantly higher in smokers group than in non‐smokers group. Leukocyte (p&lt;0.001), neutrophil (p = 0.001) and lymphocyte (p = 0.04) counts were statistically significantly higher in smokers group compared to non‐smokers group.Summary/Conclusion:In conclusion, our study showed that cigarette smoking has severe adverse effects on hematological parameters such as hemoglobin, leucocytes, lymphocytes, MCV and HCT. These alterations might be associated with a greater risk of developing secondary polycythemia, atherosclerosis, chronic obstructive pulmonary disease and cardiovascular diseases.image

136-OR: Efficacy of Cenicriviroc (CVC) in Adults with NASH-Induced Liver Fibrosis

Background: Nonalcoholic steatohepatitis (NASH) is considered the hepatic manifestation of type 2 diabetes and can progress to advanced fibrosis (AF), associated with significant increase in morbidity and mortality. CVC is an oral C-C chemokine receptor 2/5 antagonist being evaluated to treat liver fibrosis in adults with NASH. This study describes antifibrotic effects seen in the Phase 2b CENTAUR study overall and those with AF (F3) specifically. Methods: Adults with confirmed NASH, nonalcoholic fatty liver disease activity score ≥4, and liver fibrosis Stages F1-3 (NASH CRN) received CVC 150 mg daily (Arm A) or placebo (PBO; Arm C) for 2 years; Arm B received PBO in Year (Y)1 and CVC in Y2. Liver biopsies were assessed blind at baseline (BL), Y1, and Y2 by a central pathologist. Improvement in fibrosis and serum inflammatory markers were assessed in subjects with evaluable biopsies at BL, Y1, and Y2 (modified intent-to-treat [mITT] population). Results: Of 289 adults randomized (mean age 54 years, mean BMI 33.9 kg/m2, 52.0% diabetes), 28.6% (36/126) vs. 19.0% (24/126) achieved ≥1-stage fibrosis improvement at Y1 in CVC vs. PBO arms (mITT) overall. Of 97 AF subjects at BL (mean age 57.8 years, mean BMI 34.1 kg/m2, 69.0% diabetes), 38.3% (18/47) vs. 28.0% (14/50) achieved this endpoint in CVC vs. PBO arms (mITT). Overall, 60.0% (18/30) in Arm A vs. 30.0% (3/10) in Arm C maintained benefit at Y2 (mITT). In AF subjects, 85.7% (12/14) in Arm A vs. 60.0% (3/5) in Arm C maintained benefit at Y2 (mITT). Greater reductions in hs-CRP, fibrinogen, IL-6, IL-8, and IL-1β were observed with CVC. In AF subjects, these changes were more pronounced. Overall and with AF, adverse events were comparable for CVC and PBO; no deaths occurred. Conclusion: CVC was well tolerated and provided antifibrotic benefit in adults with NASH and fibrosis, especially those with AF. Most subjects achieving ≥1-stage fibrosis improvement at Y1 maintained benefit at Y2, with reductions in inflammatory biomarkers, supporting the ongoing Phase 3 study of CVC in NASH. Disclosure N. Alkhouri: Speaker's Bureau; Self; Gilead Sciences, Inc., Intercept Pharmaceuticals, Inc. Other Relationship; Self; Allergan, GENFIT, Gilead Sciences, Inc., Intercept Pharmaceuticals, Inc., Madrigal. R.W. Charlton: Employee; Self; Allergan. E.B. Martins: Employee; Self; Allergan. Stock/Shareholder; Self; Allergan. H. Landgren: Employee; Self; Allergan. A.D. Coviello: Consultant; Self; Novo Nordisk Inc. Research Support; Self; Allergan, Bristol-Myers Squibb Company, GENFIT. Speaker's Bureau; Self; Novo Nordisk Inc. M.F. Abdelmalek: Advisory Panel; Self; Allergan, Bristol-Myers Squibb Company, Madrigal Pharmaceuticals, NGM Biopharmaceuticals, Prometic Life Sciences Inc. Consultant; Self; TaiwanJ Pharmaceuticals Co., Ltd. Research Support; Self; Akcea Therapeutics, Allergan, Boehringer Ingelheim Pharmaceuticals, Inc., Bristol-Myers Squibb Company, Conatus Pharmaceuticals Inc., Galectin Therapeutics Inc., GENFIT, Gilead Sciences, Inc., Intercept Pharmaceuticals, Inc., Madrigal Pharmaceuticals, NGM Biopharmaceuticals, Novartis Pharmaceuticals Corporation, Prometheus, Shire, TaiwanJ Pharmaceuticals Co., Ltd. Speaker's Bureau; Self; Alexion Pharmaceuticals, Inc.

N-ACETYLCYSTEINE AND/OR URSODEOXYCHOLIC ACID ASSOCIATED WITH METFORMIN IN NON-ALCOHOLIC STEATOHEPATITIS: AN OPEN-LABEL MULTICENTER RANDOMIZED CONTROLLED TRIAL.

Nowadays, pharmacological treatment of non-alcoholic fatty liver disease (NAFLD) is still limited and it is based on the treatment of conditions associated comorbities. Oxidative stress and insulin resistance are the mechanisms that seem to be mostly involved in its pathogenesis. To evaluate the efficacy of N-acetylcysteine (NAC) in combination with metformin (MTF) and/or ursodeoxycholic acid (UDCA) for treatment of non-alcoholic steatohepatitis (NASH). Open-label multicenter randomized trial was conducted for 48 weeks. It included patients with biopsy-proven NASH. The patients were randomized into three groups: NAC (1.2 g) + UDCA (15 mg/kg) + MTF (850-1500 mg/day) (n=26); UDCA (20 mg/kg) + MTF (850-1500 mg/day) (n=13); NAC (1.2g) + MTF (850-1500 mg/day) (n=14) for 48 weeks. Clinical, laboratory and the second liver biopsies were performed after 48 weeks. A total of 53 patients were evaluated; 17 (32.1%) were males; median age ±54 (IQR=15, 21-71) years. In the baseline, no difference was seen between groups according clinical and histological parameters. The groups differed only in cholesterol, LDL and triglycerides. No significant differences in biochemical and histologic parameters were found between these the three groups after 48 weeks of treatment. In the intragroup analysis (intention-to-treat) comparing histological and biochemical features, there were significant improvements in the steatosis degree (P=0.014), ballooning (0.027) and, consequently, in the NAFLD Activity Score (NAS) (P=0.005), and in the ALT levels at the end of the treatment only in the NAC + MTF group. No significant evidence of modification in the liver fibrosis could be observed in any of the groups. This multicenter study suggests that the association of NAC + MTF could reduce the liver disease activity in patients with NASH. These data stimulate further controlled studies with this therapy for these patients.

Role of Vitamin E for Nonalcoholic Steatohepatitis in Patients With Type 2 Diabetes: A Randomized Controlled Trial.

While vitamin E has shown to improve nonalcoholic steatohepatitis (NASH) in patients without diabetes, information on patients with type 2 diabetes mellitus (T2DM) is lacking. The aim of this study was to determine whether vitamin E, alone or combined with pioglitazone, improves histology in patients with T2DM and NASH. This was a proof-of-concept, randomized, double-blind, placebo-controlled trial conducted from 2010 to 2016. Patients with T2DM and biopsy-proven NASH (n = 105) were randomized to vitamin E 400 IU b.i.d., vitamin E 400 IU b.i.d. plus pioglitazone 45 mg/day, or placebo. Eighty-six patients completed the 18-month study. The primary end point was a two-point reduction in the nonalcoholic fatty liver disease activity score from two different parameters, without worsening of fibrosis. Secondary outcomes were resolution of NASH without worsening of fibrosis, individual histological scores, and metabolic parameters. More patients on combination therapy achieved the primary outcome versus placebo (54% vs. 19%, P = 0.003) but not with vitamin E alone (31% vs. 19%, P = 0.26). Both groups showed improvements in resolution of NASH compared with placebo (combination group: 43% vs. 12%, P = 0.005; vitamin E alone: 33% vs. 12%, P = 0.04). While steatosis assessed by histology improved with combination therapy (P < 0.001) and vitamin E alone (P = 0.018), inflammation (P = 0.018) and ballooning (P = 0.022) only improved with combination therapy. No improvement in fibrosis was observed in any group. In this proof-of-concept study, combination therapy was better than placebo in improving liver histology in patients with NASH and T2DM. Vitamin E alone did not significantly change the primary histological outcome.

Tropifexor-Mediated Abrogation of Steatohepatitis and Fibrosis Is Associated With the Antioxidative Gene Expression Profile in Rodents.

Farnesoid X receptor (FXR) agonism is emerging as an important potential therapeutic mechanism of action for multiple chronic liver diseases. The bile acid‐derived FXR agonist obeticholic acid (OCA) has shown promise in a phase 2 study in patients with nonalcoholic steatohepatitis (NASH). Here, we report efficacy of the novel nonbile acid FXR agonist tropifexor (LJN452) in two distinct preclinical models of NASH. The efficacy of tropifexor at <1 mg/kg doses was superior to that of OCA at 25 mg/kg in the liver in both NASH models. In a chemical and dietary model of NASH (Stelic animal model [STAM]), tropifexor reversed established fibrosis and reduced the nonalcoholic fatty liver disease activity score and hepatic triglycerides. In an insulin‐resistant obese NASH model (amylin liver NASH model [AMLN]), tropifexor markedly reduced steatohepatitis, fibrosis, and profibrogenic gene expression. Transcriptome analysis of livers from AMLN mice revealed 461 differentially expressed genes following tropifexor treatment that included a combination of signatures associated with reduction of oxidative stress, fibrogenesis, and inflammation. Conclusion: Based on preclinical validation in animal models, tropifexor is a promising investigational therapy that is currently under phase 2 development for NASH.

Effects of Super Nutritional Hepatic Copper Accumulation on Hepatocyte Health and Oxidative Stress in Dairy Cows.

Concerns regarding excessive hepatic copper concentrations in dairy cows have increased. The objective of this study was to determine the association of hepatic copper concentrations with evidence of liver disease. Blood and liver samples were collected at the time of slaughter in cull dairy cows (n=100). Liver samples were analyzed for copper using inductively coupled plasma mass spectrometry and crude fat using liquid-liquid extraction and gravimetry. Serum samples were analyzed for glutamate dehydrogenase, γ-glutamyltransferase, sorbitol dehydrogenase, aspartate aminotransferase activities, and bile acid concentrations. Liver samples were examined histologically for inflammation, fibrosis, and rhodanine staining. Animals were stratified by hepatic copper concentration and samples in the highest and lowest quintiles (Q5 and Q1) were evaluated for oxidative stress. Systemic indices of oxidative stress included serum reactive oxygen and nitrogen species (RONS) and total antioxidant potential (AOP). Tissue-level oxidative stress was assessed by immunohistochemistry using 4-hydroxynonenal (4HNE) and 3-nitrotyrosine (3NIT) stains to score the relative abundance and distribution of oxidized lipid and protein products, respectively. Mean hepatic copper concentration was 496.83 μg/g and median 469.72 μg/g and ranged from 70.56 to 1264.27 μg/g dry tissue. No association was found between hepatic copper concentrations and clinicopathological or histological evidence of hepatic damage or dysfunction. There was a significant increase in the amount of IHC staining of 4HNE and 3NIT in Q5 compared with Q1. Moreover, the IHC staining mirrored the distribution of the copper-specific stain rhodanine. These results demonstrate that cows with elevated hepatic copper concentrations had no evidence of active liver disease but had increased hepatic oxidative stress.

Prevalence and Impact of Hepatitis E Virus Infection Among Persons With Chronic Hepatitis B Living in the US and Canada.

Patients with chronic hepatitis B virus (HBV) may experience spontaneous biochemical flares of liver disease activity. This study aimed to determine (i) the prevalence of prior and possible acute hepatitis E virus (HEV) infection among persons with chronic HBV and (ii) whether HEV infection is associated with liver disease flares among persons with chronic HBV. Serum from a random sample of 600 adults in the Hepatitis B Research Network Cohort Study was tested for HEV RNA and anti-HEV IgM and IgG. Logistic regression models were used to estimate crude and adjusted odds ratios of anti-HEV prevalence for participant characteristics. Anti-HEV IgG and IgM seroprevalence was 28.5% and 1.7%, respectively. No participants had detectable HEV RNA. Of the 10 anti-HEV IgM+ participants, only 1 had elevated serum ALT at seroconversion. The odds of anti-HEV seropositivity (IgG+ or IgM+) were higher in older participants, males, Asians, less educated people, and those born outside the United States and Canada. Acute HEV infection is a rare cause of serum ALT flares among persons with chronic HBV. The high seroprevalence of anti-HEV IgG among the chronic HBV patients is strongly associated with various demographic factors in this largely Asian American cohort.

Hepatic Knockdown of RECK Increases NASH Susceptibility

IntroductionNonalcoholic steatohepatitis (NASH) is characterized by hepatic steatosis, hepatocellular inflammation and fibrosis. Reversion‐inducing‐cysteine‐rich protein with Kazal motifs (RECK) exerts anti‐fibrotic effects in the heart, although its role in NASH is unknown. We have previously shown that western diet (WD)‐induced NASH and fibrosis were associated with RECK downregulation in the liver. Here, we examined whether RECK knockdown, specifically in liver, increases NASH susceptibility.MethodsHomozygous RECK floxed mice on a C57/BL6J background were injected with either an AAV8‐TBG‐Cre (1×1012) (RECK‐KD) for liver‐specific knockdown of RECK or an AAV8‐TBG‐GFP control (RECK‐CON). Following AAV injections, mice (n = 5–8) were fed either a control diet (CD; 10% fat) or WD (45% fat, 17% sucrose) for 8 weeks. An additional set of mice injected with AAV8‐TBG‐Cre or AAV8‐TBG‐GFP were fed WD for 30 weeks (n = 8).ResultsAfter 8 weeks, WD feeding significantly increased body weight and body fat percentage vs CD (p≤0.05). There were no differences in animal characteristics between RECK‐KD and RECK‐CON in either CD or WD groups. Nonalcoholic fatty liver disease activity score (NAS; combination of steatosis, ballooning, and inflammation) was significantly elevated in WD vs CD fed mice (p&lt;0.05), with further exacerbation with RECK‐KD for NAS and inflammation scores (main effect for genotype, p&lt;0.05). Furthermore, RECK‐KD mice had a ~3‐fold increase in hepatic fibrosis compared to RECK‐CON (main effect for genotype, p≤0.05). Thirty weeks of WD feeding significantly elevated NAS (p&lt;0.01) and fibrosis scores (P&lt;0.01) compared with 8 weeks of WD feeding. In addition, RECK knockdown exacerbated NAS (p&lt;0.05), inflammation score (p=0.001), and fibrosis (p=0.06) compared to RECK‐CON mice following 30 weeks of WD feeding. Further investigations revealed that RECK‐KD (vs. RECK‐CON) expressed significantly higher IKK‐β gene expression (p&lt;0.05), along with trends for increases in hepatic inflammatory markers NF‐κB, TGF‐β, and JUN at 30 weeks. Expression of matrix metalloproteases‐2 and −14 also tended to be elevated in RECK‐KD mice (vs RECK‐CON) at 30 weeks.ConclusionHere we demonstrate for the first time that liver‐specific RECK knockdown exacerbates western diet induced NASH. Further investigation is warranted to determine the role and precise mechanisms by which RECK regulates hepatocellular inflammation and NASH progression.Support or Funding InformationVA Merit Grant I01BX003271‐01 (R.S.R.), VA Merit Grant I01BX004220‐01 (B.C.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Can we stop nucleoside analogues before HBsAg loss?

Most of the current guidelines and the existing data suggest that long-term therapy with nucleos(t)ide analogue(s) [NA(s)] may be stopped in carefully selected chronic hepatitis B patients who remain HBsAg positive. In particular, NA(s) may be discontinued in such patients without pre-existing cirrhosis who achieved long-term on-therapy virological remission (>12months of HBeAg seroconversion and HBV DNA undetectability for initially HBeAg-positive cases; ≥3years of HBV DNA undetectability for HBeAg-negative cases) and are expected to remain under close follow-up after NA(s) discontinuation. The majority of patients will develop post-NA(s) virological relapses and a proportion of them will have biochemical relapses and occasionally flares, but prompt retreatment can reintroduce remission. No reliable predictor(s) of post-NA(s) relapses have been identified so far. HBsAg loss develops in a progressively increasing proportion of chronic hepatitis B patients who discontinue NA(s) with HBsAg loss rates being higher in Caucasian patients with HBeAg-negative chronic hepatitis B. Follow-up at least every 3months for the first year seems to be appropriate for all chronic hepatitis B patients who discontinue NA(s), while HBeAg-negative patients need to be followed more closely (monthly) during the first 3months. Predefined criteria for retreatment are quite important, and the best candidates for retreatment are probably the patients with persistent (≥3months) liver disease activity and those with severe flares.

Prediction of nonalcoholic fatty liver disease (NAFLD) activity score (NAS) with multiparametric hepatic magnetic resonance imaging and elastography.

To investigate the use of MR elastography (MRE)-derived mechanical properties (shear stiffness (|G*|) and loss modulus (G″)) and MRI-derived fat fraction (FF) to predict the nonalcoholic fatty liver disease (NAFLD) activity score (NAS) in a NAFLD mouse model. Eighty-nine male mice were studied, including 64 training and 25 independent testing animals. An MRI/MRE exam and histologic evaluation were performed. Pairwise, nonparametric comparisons and multivariate analyses were used to evaluate the relationships between the three imaging parameters (FF, |G*|, and G″) and histologic features. A virtual NAS score (vNAS) was generated by combining three imaging parameters with an ordinal logistic model (OLM) and a generalized linear model (GLM). The prediction accuracy was evaluated by ROC analyses. The combination of FF, |G*|, and G″ predicted NAS > 1 with excellent accuracy in both training and testing sets (AUROC > 0.84). OLM and GLM predictive models misclassified 3/54 and 6/54 mice in the training, and 1/25 and 1/25 in the testing cohort respectively, in distinguishing between "not-NASH" and "definite-NASH." "Borderline-NASH" prediction was poorer in the training set, and no borderline-NASH mice were available in the testing set. This preliminary study shows that multiparametric MRI/MRE can be used to accurately predict the NAS score in a NAFLD animal model, representing a promising alternative to liver biopsy for assessing NASH severity and treatment response. • MRE-derived liver stiffness and loss modulus and MRI-assessed fat fraction can be used to predict NAFLD activity score (NAS) in our preclinical mouse model (AUROC > 0.84 for all NAS levels greater than 1). • The overall agreement between the histological-determined NASH diagnosis and the imaging-predicted NASH diagnosis is 80-92%. • The multiparametric hepatic MRI/MRE has great potential for noninvasively assessing liver disease severity and treatment efficacy.

The Role of Three-Dimensional Magnetic Resonance Elastography in the Diagnosis of Nonalcoholic Steatohepatitis in Obese Patients Undergoing Bariatric Surgery.

The lack of reliable, noninvasive methods to diagnose early nonalcoholic steatohepatitis (NASH) is a major unmet need. We aimed to determine the diagnostic accuracy of three-dimensional magnetic resonance elastography (3D-MRE), with shear stiffness measured at 60 Hz, damping ratio at 40 Hz, and magnetic resonance imaging proton density fat fraction (MRI-PDFF) in the detection of NASH in individuals undergoing bariatric surgery. Obese adults at risk for NASH were enrolled between 2015 and 2017 (prospective cohort, n=88) and 2010 and 2013 (retrospective cohort, n=87). The imaging protocol consisted of multifrequency 3D-MRE (mf3D-MRE) with shear waves delivered at different frequencies to explore parameters that best correlated with histologic NASH, and MRI-PDFF to estimate steatosis. The prospective cohort was used to establish the optimal mf3D-MRE technical parameters for NASH detection. The two cohorts were then combined to derive predictive models of NASH and disease activity by nonalcoholic fatty liver disease activity score (NAS) using the three imaging parameters that correlated with NASH. A total of 175 patients (median age 45, 81% women, and 81 [46%] with histologic NASH) were used for model derivation. From the complex shear modulus output generated by mf3D-MRE, the damping ratio at 40 Hz and shear stiffness at 60 Hz best correlated with NASH. The fat fraction obtained from MRI-PDFF correlated with steatosis (P<0.05 for all). These three parameters were fit into a logistic regression model that predicted NASH with cross-validated area under the receiver operating characteristic curve (AUROC)=0.73, sensitivity=0.67, specificity=0.80, positive predictive value=0.73 and negative predictive value=0.74, and disease activity by NAS with cross-validated AUROC=0.82. Conclusion: The mf3D-MRE allows identification of imaging parameters that predict early NASH and disease activity. This imaging biomarker represents a promising alternative to liver biopsy for NASH diagnosis and monitoring. The results provide motivation for further studies in nonbariatric cohorts.

Improvements in Histologic Features and Diagnosis Associated With Improvement in Fibrosis in Nonalcoholic Steatohepatitis: Results From the Nonalcoholic Steatohepatitis Clinical Research Network Treatment Trials.

Hepatocellular injury and inflammation are believed to be the primary drivers of fibrogenesis that ultimately lead to cirrhosis in patients with nonalcoholic steatohepatitis (NASH). This study sought associations between observed improvements in fibrosis with improvement in specific histologic features, nonalcoholic fatty liver disease activity score (NAS) ≥2, diagnostic category, and primary histologically based outcomes of two adult NASH treatment trials. The primary outcome for the study was fibrosis improvement from baseline to end of treatment, defined as a 1-point or more improvement in fibrosis stage. This is a retrospective analysis of biopsy data collected from the NASH Clinical Research Network Pathology Committee of Pioglitazone versus Vitamin E versus Placebo for the Treatment of Nondiabetic Patients with NASH Trial (PIVENS) and Farnesoid X Receptor Ligand Obeticholic Acid in NASH Treatment Trial (FLINT) baseline and final biopsies. Treatment group-adjusted univariable and multivariable logistic regression models related improvement in fibrosis to improvements in other histologic variables, resolution of steatohepatitis, and improvement in the NAS ≥2. In PIVENS 221 subjects had baseline and 96-week biopsies, and in FLINT 200 subjects had baseline and 72-week biopsies. Improvement in fibrosis was found in 38% of PIVENS and 29% of FLINT biopsies; fibrosis improvement was more likely in treated than placebo subjects in both studies. Controlling for treatment group, fibrosis improvement was associated most strongly with resolution of NASH (PIVENS, odds ratio [OR], 3.9; 95% confidence interval [CI] 2.0-7.6; P<0.001; FLINT, OR, 8.0; 95% CI 3.1-20.9; P<0.001), and improved NAS by ≥2 (PIVENS, OR, 2.4; 95% CI 1.3-4.3; P=0.003; FLINT, OR, 4.2; 95% CI 2.1-8.3; P<0.001). Improvement in histologic features associated with improved fibrosis for both studies included steatosis, ballooning, Mallory-Denk bodies, and portal, but not lobular, inflammation. Conclusion: These findings support a strong link between histologic resolution of steatohepatitis with improvement in fibrosis in NASH.

Disease progression of nonalcoholic steatohepatitis in Taiwanese patients: a longitudinal study of paired liver biopsies

Nonalcoholic steatohepatitis (NASH) might progress to fibrosis, cirrhosis, and hepatocellular carcinoma. However, the natural history of NASH has not been fully clarified. This study aimed to investigate the disease progression in NASH patients receiving paired liver biopsies. We also aimed to examine the factors associated with NASH progression. Ten NASH patients who had received liver biopsies during June 2001 and February 2010 were consecutively enrolled. The histopathological changes were examined retrospectively, including nonalcoholic fatty liver disease activity score (NAS) and fibrosis stage. The associated clinical profiles were also analyzed. The median duration between paired biopsies was 20.5 months (range: 12-106 months). According to NAS and fibrosis stage, disease progression, stable disease, and disease regression were observed in seven patients, two patients, and one patient, respectively. Six (60%) patients had increased NAS on second biopsy, and two were lean NASH patients. The only patient with an improvement in NAS had achieved body weight reduction (13.3%) between paired biopsies. None of the 10 patients experienced an improvement in fibrosis. Five (50%) patients showed progression of fibrosis on second biopsy and the annual fibrosis progression rate was 0.32/year. Two of the five patients who showed progression of fibrosis were of the nonobese phenotype, whereas three patients were nondiabetic. NASH is a progressive disease in Taiwanese patients. The disease progression should be further clarified in lean and nondiabetic NASH patients.

Haptoglobin 2 Allele is Associated With Histologic Response to Vitamin E in Subjects With Nonalcoholic Steatohepatitis.

Haptoglobin (Hp) genotype has been linked to oxidative stress and cardiovascular outcomes in response to vitamin E (VitE) among patients with diabetes mellitus. Its effect on histologic response to VitE in nonalcoholic steatohepatitis (NASH) is unknown. Our objective was to determine if Hp genotype associates with response to VitE in patients with NASH. A post hoc analysis of 228 patients receiving VitE or placebo in 2 clinical trials was performed. Regression analysis was used to assess the effect of VitE versus placebo, by Hp genotype (1-1, 2-1, or 2-2), on histologic features and laboratory markers of nonalcoholic fatty liver disease, comparing baseline to end of treatment values. An interaction term was included in the regression models to assess differential treatment effect across Hp genotype. Hp 2-2 patients treated with VitE versus placebo showed significant histologic improvement (51% vs. 20%; OR=4.2; P=0.006), resolution of steatohepatitis (44% vs. 12%; OR=6.2; P=0.009), decrease in nonalcoholic fatty liver disease Activity Score (NAS) (-2.2 vs. -0.6; P=0.001), and decrease in liver enzymes alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and γ-glutamyl transpeptidase. Hp 2-1 patients on VitE versus placebo showed improved resolution of steatohepatitis, NAS and liver enzymes. Hp 1-1 patients showed no significant improvement in histology or liver enzymes. VitE had no effect on fibrosis stage in any group. Regression analysis showed incremental benefit of having Hp 2-2 or 2-1 versus 1-1 for all liver enzyme. Hp 2 allele is associated with greater histologic and biological improvement in NASH with VitE treatment compared with the Hp 1 allele.