Evaluation of Fucosylated Haptoglobin and Mac-2 Binding Protein as Serum Biomarkers to Estimate Liver Fibrosis in Patients with Chronic Hepatitis C

Fucosylated haptoglobin (Fuc-Hpt) and Mac-2 binding protein (Mac-2 bp) are identified as cancer biomarkers, based on the results from a glyco-proteomic analysis. Recently, we reported that these glyco-biomarkers were associated with liver fibrosis and/or ballooning hepatocytes in patients with nonalcoholic fatty liver disease (NAFLD). We evaluated the ability of these glycoproteins to estimate liver fibrosis in 317 patients with chronic hepatitis C. We measured the serum Fuc-Hpt and Mac-2 bp levels using a lectin-antibody ELISA and ELISA, respectively. The serum levels of both Fuc-Hpt and Mac-2 bp increased with the progression of liver fibrosis. The multivariate analysis revealed that Mac-2 bp was an independent factor associated with moderate liver fibrosis (F ≥ 2). In contrast, Fuc-Hpt was an independent factor associated with advanced liver fibrosis (F ≥ 3). In terms of evaluating liver fibrosis, the serum levels of these glycomarkers were correlated with well-known liver fibrosis indexes, such as the aspartate aminotransferase to platelet ratio index (APRI) and Fibrosis-4 (FIB4) index. An assay that combined the APRI or FIB4 index and the Fuc-Hpt or Mac-2 bp levels increased the AUC value for diagnosing hepatic fibrosis. Interestingly, the cumulative incidence of hepatocellular carcinoma (HCC) was significantly higher in the patients with elevated serum levels of Fuc-Hpt and Mac-2 bp. In conclusion, both Fuc-Hpt and Mac-2 bp could be useful glyco-biomarkers of liver fibrosis and predictors of HCC in patients with chronic hepatitis C.     

Glucose-Induced Glucagon-Like Peptide 1 Secretion Is Deficient in Patients with Non-Alcoholic Fatty Liver Disease

Background & Aims

The incretins glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are gastrointestinal peptide hormones regulating postprandial insulin release from pancreatic β-cells. GLP-1 agonism is a treatment strategy in Type 2 diabetes and is evaluated in Non-alcoholic fatty liver disease (NAFLD). However, the role of incretins in its pathophysiology is insufficiently understood. Studies in mice suggest improvement of hepatic steatosis by GLP-1 agonism. We determined the secretion of incretins after oral glucose administration in non-diabetic NAFLD patients.

Methods

N = 52 patients (n = 16 NAFLD and n = 36 Non-alcoholic steatohepatitis (NASH) patients) and n = 50 matched healthy controls were included. Standardized oral glucose tolerance test was performed. Glucose, insulin, glucagon, GLP-1 and GIP plasma levels were measured sequentially for 120 minutes after glucose administration.

Results

Glucose induced GLP-1 secretion was significantly decreased in patients compared to controls (p<0.001). In contrast, GIP secretion was unchanged. There was no difference in GLP-1 and GIP secretion between NAFLD and NASH subgroups. All patients were insulin resistant, however HOMA2-IR was highest in the NASH subgroup. Fasting and glucose-induced insulin secretion was higher in NAFLD and NASH compared to controls, while the glucose lowering effect was diminished. Concomitantly, fasting glucagon secretion was significantly elevated in NAFLD and NASH.

Conclusions

Glucose-induced GLP-1 secretion is deficient in patients with NAFLD and NASH. GIP secretion is contrarily preserved. Insulin resistance, with hyperinsulinemia and hyperglucagonemia, is present in all patients, and is more severe in NASH compared to NAFLD. These pathophysiologic findings endorse the current evaluation of GLP-1 agonism for the treatment of NAFLD.     

Association between Serum Atypical Fibroblast Growth Factors 21 and 19 and Pediatric Nonalcoholic Fatty Liver Disease

Atypical fibroblast growth factors (FGF) 21 and 19 play a central role in energy metabolism through the mediation of Klotho coreceptor. Contradictory findings are available about the association of FGF21 and FGF19 with nonalcoholic fatty liver disease (NAFLD) in humans. We investigated the association of serum FGF21, FGF19 and liver Klotho coreceptor with non-alcoholic steatohepatitis (NASH) and fibrosis in children with NAFLD. Serum FGF21 and FGF19 were measured in 84 children with biopsy-proven NAFLD and 23 controls (CTRL). The hepatic expression of Klotho coreceptor was measured in 7 CTRL, 9 patients with NASH (NASH+) and 11 patients without NASH (NASH−). FGF21 and FGF19 showed a tendency to decrease from CTRL (median FGF21 = 196 pg/mL; median FGF19 = 201 pg/mL) to NASH− (FGF21 = 89 pg/mL; FGF19 = 81 pg/mL) to NASH+ patients (FGF21 = 54 pg/mL; FGF19 = 41 pg/mL) (p<0.001 for all comparisons) and were inversely associated with the probability of NASH and fibrosis in children with NAFLD. The hepatic expression of Klotho coreceptor was inversely associated with NASH (R² = 0.87, p<0.0001) and directly associated with serum FGF21 (R² = 0.57, p<0.0001) and FGF19 (R² = 0.67, p<0.0001). In conclusion, serum FGF19 and FGF21 and hepatic Klotho expression are inversely associated with hepatic damage in children with NAFLD and these findings may have important implications for understanding the mechanisms of NAFLD progression.     

Limited Utility of Plasma M30 in Discriminating Non-Alcoholic Steatohepatitis from Steatosis – A Comparison with Routine Biochemical Markers

Introduction

The utility of Cytokeratin-18 fragment, namely CK18Asp396 (M30), for the diagnosis of non-alcoholic steatohepatitis (NASH) is currently uncertain. We aimed to provide further data in this area among multi-ethnic Asian subjects with NAFLD.

Materials and Methods

The accuracy of M30 for detecting NASH was compared with serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma glutamyl transpeptidase (GGT) levels in consecutive adult subjects with biopsy-proven non-alcoholic fatty liver disease (NAFLD).

Results

Data for 93 NAFLD subjects (mean age 51.0±11.1 years old and 51.6% males) and 20 healthy controls (mean age 50.2±16.4 years old and 33.3% males) were analyzed. There were 39 NASH subjects (41.9%) and 54 non-NASH subjects (58.1%) among the NAFLD subjects. Plasma M30 (349 U/L vs. 162 U/L), and serum ALT (70 IU/L vs. 26 IU/L), AST (41 IU/L vs. 20 IU/L) and GGT (75 IU/L vs. 33 IU/L) were significantly higher in NAFLD subjects than in healthy controls. Serum ALT (86 IU/L vs. 61 IU/L), AST (58 IU/L vs. 34 IU/L) and GGT (97 IU/L vs. 56 IU/L) were significantly higher in NASH subjects compared to non-NASH subjects, but no significant difference was observed with plasma M30 (435 U/L vs. 331 U/L). The accuracy of plasma M30, and serum ALT, AST and GGT was good for predicting NAFLD (AUROC 0.91, 0.95, 0.87 and 0.85, respectively) but less so for NASH (AUROC 0.59, 0.64, 0.75 and 0.68, respectively). Serum ALT and AST, but not plasma M30 showed a significant trend with increasing grades of ballooning and lobular inflammation.

Conclusion

The utility of M30 in the detection of NASH in clinical practice appears limited, in comparison to routine biochemical markers.     

Cytokeratin 18, Alanine Aminotransferase,Platelets and Triglycerides Predict the Presence of Nonalcoholic Steatohepatitis

Background

Nonalcoholic fatty liver disease (NAFLD) is one of the critical public health problems in China. The full spectrum of the disease ranges from simple steatosis and nonalcoholic steatohepatitis (NASH) to cirrhosis and hepatocellular carcinoma(HCC). The infiltration of inflammatory cells characterizes NASH. This characteristic contributes to the progression of hepatitis, fibrosis, cirrhosis, and HCC. Therefore, distinguishing NASH from NAFLD is crucial.

Objective and Methods

Ninety-five patients with NAFLD, 44 with NASH, and 51 with non-NASH were included in the study to develop a new scoring system for differentiating NASH from NAFLD. Data on clinical and biological characteristics, as well as blood information, were obtained. Cytokeratin-18 (CK-18) fragments levels were measured using an enzyme-linked immunosorbant assay.

Results

Several indexes show significant differences between the two groups, which include body mass index (BMI), waist-on-hip ratio (WHR), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GT), platelets, uric acid (UA), hs-C-reactive protein (hs-CRP), triglycerides (TG), albumin (ALB), and CK-18 fragments (all P < 0.05). The CK-18 fragment levels showed a significant positive correlation with steatosis severity, ballooning, lobular inflammation, and fibrosis stage (all P < 0.05). Therefore, a new model that combines ALT, platelets, CK-18 fragments, and TG was established by logistic regression among NAFLD patients. The AUROC curve in predicting NASH was 0.920 (95% CI: 0.866 - 0.974, cutoff value = 0.361, sensitivity = 89%, specificity = 86%, positive predictive value = 89%, negative predictive value = 89%).

Conclusion

The novel scoring system may be considered as a useful model in predicting the presence of NASH in NAFLD patients.     

Pharmacological Inhibition of the Chemokine CXCL16 Diminishes Liver Macrophage Infiltration and Steatohepatitis in Chronic Hepatic Injury

Non-alcoholic fatty liver disease (NAFLD) is a major cause of morbidity and mortality in developed countries, resulting in steatohepatitis (NASH), fibrosis and eventually cirrhosis. Modulating inflammatory mediators such as chemokines may represent a novel therapeutic strategy for NAFLD. We recently demonstrated that the chemokine receptor CXCR6 promotes hepatic NKT cell accumulation, thereby controlling inflammation in experimental NAFLD. In this study, we first investigated human biopsies (n = 20), confirming that accumulation of inflammatory cells such as macrophages is a hallmark of progressive NAFLD. Moreover, CXCR6 gene expression correlated with the inflammatory activity (ALT levels) in human NAFLD. We then tested the hypothesis that pharmacological inhibition of CXCL16 might hold therapeutic potential in NAFLD, using mouse models of acute carbon tetrachloride (CCl₄)- and chronic methionine-choline-deficient (MCD) diet-induced hepatic injury. Neutralizing CXCL16 by i.p. injection of anti-CXCL16 antibody inhibited the early intrahepatic NKT cell accumulation upon acute toxic injury in vivo. Weekly therapeutic anti-CXCL16 administrations during the last 3 weeks of 6 weeks MCD diet significantly decreased the infiltration of inflammatory macrophages into the liver and intrahepatic levels of inflammatory cytokines like TNF or MCP-1. Importantly, anti-CXCL16 treatment significantly reduced fatty liver degeneration upon MCD diet, as assessed by hepatic triglyceride levels, histological steatosis scoring and quantification of lipid droplets. Moreover, injured hepatocytes up-regulated CXCL16 expression, indicating that scavenging functions of CXCL16 might be additionally involved in the pathogenesis of NAFLD. Targeting CXCL16 might therefore represent a promising novel therapeutic approach for liver inflammation and steatohepatitis.     

LDL-Migration Index (LDL-MI), an Indicator of Small Dense Low-Density Lipoprotein (sdLDL), Is Higher in Non-Alcoholic Steatohepatitis than in Non-Alcoholic Fatty Liver: A Multicenter Cross-Sectional Study

Background

Non-alcoholic fatty liver disease (NAFLD) is associated with increased risks of atherosclerotic diseases, including cardiovascular disease. However, the difference in risk between patients with non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) has not yet been determined. Accumulating evidence has shown that high amounts of small dense low-density lipoprotein (sdLDL) are closely associated with atherosclerotic diseases. This study investigated differences in risk factors for atherosclerotic diseases, especially LDL-migration index (LDL-MI), an indicator of sdLDL, between patients with NAFL and NASH.

Methods

LDL-MI was analyzed in a primary cohort of 156 patients with NAFLD, including 53 with NAFL and 103 with NASH, and a validation cohort of 69 patients with NAFLD, including 25 with NAFL and 44 with NASH.

Results

In the primary cohort, NASH was associated with elevated LDL-MI (p = 0.039). Multiple regression analysis showed that NASH and the non-use of lipid lowering medications were independently correlated with higher LDL-MI in all patients with NAFLD. Among patients not on lipid lowering medications, those with NASH had significantly higher LDL-MI than those with NAFL (p = 0.001). These findings were confirmed in a validation cohort, in that LDL-MI was significantly higher in patients with NASH than with NAFL (p = 0.043).

Conclusion

This study is the first to show that LDL-MI, an indicator of sdLDL, was higher in patients with NASH than with NAFL, suggesting that the risk of atherosclerotic diseases may be higher in NASH than NAFL. Patients with NASH should be followed closely, especially for the progression of liver pathology and atherosclerotic diseases.

Trial Registration

UMIN000009614     

Genetic Polymorphism of Cytochrome P450 4F2, Vitamin E Level and Histological Response in Adults and Children with Nonalcoholic Fatty Liver Disease Who Participated in PIVENS and TONIC Clinical Trials

Vitamin E improved liver histology in children and adults with NAFLD who participated in TONIC and PIVENS clinical trials, but with significant inter-individual variability in its efficacy. Cytochrome P450 4F2 (CYP4F2) is the major enzyme metabolizing Vit E, with two common genetic variants (V433M, rs2108622 and W12G, rs3093105) found to alter its activity. We investigated the relationship between CYP4F2 genotypes, α-tocopherol levels and histological improvement in these two trials. V433M and W12G variants were genotyped in TONIC (n = 155) and PIVENS (n = 213) DNA samples. The relationships between CYP4F2 genotypes, plasma α-tocopherol levels at baseline and weeks 48 (w48) and 96 (w96) and histological end points (overall improvement in liver histology and resolution of NASH) were investigated. As a result, the V433M genotype was significantly associated with baseline plasma α-tocopherol in the TONIC trial (p = 0.004), but not in PIVENS. Among those receiving Vit E treatment, CYP4F2 V433M genotype was associated with significantly decreased plasma α-tocopherol levels at w48 (p = 0.003 for PIVENS and p = 0.026 for TONIC) but not at w96. The w96 α-tocopherol level was significantly associated with resolution of NASH (p = 0.006) and overall histology improvement (p = 0.021)in the PIVENS, but not in the TONIC trial. There was no significant association between CYP4F2 genotypes and histological end points in either trial. Our study suggested the a moderate role of CYP4F2 polymorphisms in affecting the pharmacokinetics of Vit E as a therapeutic agent. In addition, there may be age-dependent relationship between CYP4F2 genetic variability and Vit E pharmacokinetics in NAFLD.     

Risk of Obstructive Sleep Apnea with Daytime Sleepiness Is Associated with Liver Damage in Non-Morbidly Obese Patients with Nonalcoholic Fatty Liver Disease

Background

A high prevalence of obstructive sleep apnea syndrome (OSAS) has been reported in severely obese patients with nonalcoholic fatty liver disease (NAFLD), but few studies have evaluated OSAS in non-morbidly obese NAFLD patients.

Aims

To determine the prevalence of risk for OSAS with or without daytime sleepiness in non-morbidly obese patients with NAFLD and evaluate the association with the severity of liver damage.

Methods

We considered 159 consecutive patients with histological NAFLD and body mass index (BMI) <35 Kg/m², and 80 controls without ultrasonographic steatosis matched for age, sex, and BMI. OSAS risk was determined by positivity for Berlin questionnaire (BQ), and daytime sleepiness by the Sleepness Epworth Scale (ESS). Liver damage was evaluated according to the NAFLD activity score.

Results

In NAFLD patients, BQ alone was positive in 39 (25%), ESS in 8 (5%), and both in 13 (8%, OSAS with sleepines); p = ns vs. controls without steatosis. In NAFLD patients at risk for OSAS with (but not in those without) sleepiness, we observed a higher prevalence of nonalcoholic steatohepatitis (NASH; 11/13, 85% vs. 72/146, 49%; p = 0.018), and of clinically significant fibrosis (stage>1; 9/13, 69% vs. 39/146, 27%; p = 0.003). At multivariate logistic regression analysis, OSAS with sleepiness was strongly associated with NASH and fibrosis>1 independently of known clinical risk factors such as age, gender, BMI, diabetes, and ALT levels (OR 7.1, 95% c.i. 1.7–51, p = 0.005 and OR 14.0, 95% c.i. 3.5–70, p = 0.0002, respectively).

Conclusions

A proportion of NAFLD patients without severe obesity is at risk for OSAS with daytime sleepiness, which is associated with the severity of liver damage independently of body mass and other cofactors.     

Inhibition of MAP4K4 signaling initiates metabolic reprogramming to protect hepatocytes from lipotoxic damage

The primary hepatic consequence of obesity is non-alcoholic fatty liver disease (NAFLD), affecting about 25% of the global adult population. Non-alcoholic steatohepatitis (NASH) is a severe form of NAFLD characterized by liver lipid accumulation, inflammation, and hepatocyte ballooning, with a different degree of hepatic fibrosis. In the light of rapidly increasing prevalence of NAFLD and NASH, there is an urgent need for improved understanding of the molecular pathogenesis of these diseases. The aim of this study was to decipher the possible role of STE20-type kinase MAP4K4 in the regulation of hepatocellular lipotoxicity and susceptibility to NAFLD. We found that MAP4K4 mRNA expression in human liver biopsies was positively correlated with key hallmarks of NAFLD (i.e., liver steatosis, lobular inflammation, hepatocellular ballooning, and fibrosis). We also found that the silencing of MAP4K4 suppressed lipid deposition in human hepatocytes by stimulating β-oxidation and triacylglycerol secretion, while attenuating fatty acid influx and lipid synthesis. Furthermore, downregulation of MAP4K4 markedly reduced the glycolysis rate and lowered incidences of oxidative/endoplasmic reticulum stress. In parallel, we observed suppressed JNK and ERK and increased AKT phosphorylation in MAP4K4-deficient hepatocytes. Together, these results provide the first experimental evidence supporting the potential involvement of STE20-type kinase MAP4K4 as a component of the hepatocellular lipotoxic milieu promoting NAFLD susceptibility.     

Imaging Mass Spectrometry Reveals Alterations in N-Linked Glycosylation That Are Associated With Histopathological Changes in Nonalcoholic Steatohepatitis in Mouse and Human

Nonalcoholic steatohepatitis (NASH) is the progressive form of nonalcoholic fatty liver disease (NAFLD) and is characterized by inflammation, hepatocyte injury, and fibrosis. Further, NASH is a risk factor for cirrhosis and hepatocellular carcinoma. Previous research demonstrated that serum N-glycan profiles can be altered in NASH patients. Here, we hypothesized that these N-glycan modifications may be associated with specific liver damage in NAFLD and NASH. To investigate the N-glycome profile in tissue, imaging mass spectrometry was used for a qualitative and quantitative in situ N-linked glycan analysis of mouse and human NAFLD/NASH tissue. A murine model was used to induce NAFLD and NASH through ad libitum feeding with either a high-fat diet or a Western diet, respectively. Mice fed a high-fat diet or Western diet developed inflammation, steatosis, and fibrosis, consistent with NAFLD/NASH phenotypes. Induction of NAFLD/NASH for 18 months using high caloric diets resulted in increased expression of mannose, complex/fucosylated, and hybrid N-glycan structures compared to control mouse livers. To validate the animal results, liver biopsy specimens from 51 human NAFLD/NASH patients representing the full range of NASH Clinical Research Network fibrosis stages were analyzed. Importantly, the same glycan alterations observed in mouse models were observed in human NASH biopsies and correlated with the degree of fibrosis. In addition, spatial glycan alterations were localized specifically to histopathological changes in tissue like fibrotic and fatty areas. We demonstrate that the use of standard staining’s combined with imaging mass spectrometry provide a full profile of the origin of N-glycan modifications within the tissue. These results indicate that the spatial distribution of abundances of released N-glycans correlate with regions of tissue steatosis associated with NAFLD/NASH.     

Non-Invasive Separation of Alcoholic and Non-Alcoholic Liver Disease with Predictive Modeling

Background & Objective

Currently, a major clinical challenge is to distinguish between chronic liver disease caused by metabolic syndrome (non-alcoholic fatty liver disease, NAFLD) from that caused by long term or excessive alcohol consumption (ALD). The etiology of severe liver disease affects treatment options and priorities for liver transplantation and organ allocation. Thus we compared physiologically similar NAFLD and ALD patients to detect biochemical differences for improved separation of these mechanistically overlapping etiologies.

Methods

In a cohort of 31 NAFLD patients with BMI below 30 and a cohort of ALD patient with (ALDC n = 51) or without cirrhosis (ALDNC n = 51) serum transaminases, cell death markers and (adipo-)cytokines were assessed. Groups were compared with One-way ANOVA and Tukey''s correction. Predictive models were built by machine learning techniques.

Results

NAFLD, ALDNC or ALDC patients did not differ in demographic parameters. The ratio of alanine aminotransferase/aspartate aminotransferase - common serum parameters for liver damage - was significantly higher in the NAFLD group compared to both ALD groups (each p<0.0001). Adiponectin and tumor necrosis factor(TNF)-alpha were significantly lower in NAFLD than in ALDNC (p<0.05) or ALDC patients (p<0.0001). Significantly higher serum concentrations of cell death markers, hyaluronic acid, adiponectin, and TNF-alpha (each p<0.0001) were found in ALDC compared to ALDNC. Using machine learning techniques we were able to discern NAFLD and ALDNC (up to an AUC of 0.9118±0.0056) or ALDC and ALDNC (up to an AUC of 0.9846±0.0018), respectively.

Conclusions

Machine learning techniques relying on ALT/AST ratio, adipokines and cytokines distinguish NAFLD and ALD. In addition, severity of ALD may be non-invasively diagnosed via serum cytokine concentrations.     

Complement Alternative Pathway Activation in Human Nonalcoholic Steatohepatitis

The innate immune system plays a major role in the pathogenesis of nonalcoholic steatohepatitis (NASH). Recently we reported complement activation in human NASH. However, it remained unclear whether the alternative pathway of complement, which amplifies C3 activation and which is frequently associated with pathological complement activation leading to disease, was involved. Here, alternative pathway components were investigated in liver biopsies of obese subjects with healthy livers (n = 10) or with NASH (n = 12) using quantitative PCR, Western blotting, and immunofluorescence staining. Properdin accumulated in areas where neutrophils surrounded steatotic hepatocytes, and colocalized with the C3 activation product C3c. C3 activation status as expressed by the C3c/native C3 ratio was 2.6-fold higher (p<0.01) in subjects with NASH despite reduced native C3 concentrations (0.94±0.12 vs. 0.57±0.09; p<0.01). Hepatic properdin levels positively correlated with levels of C3c (r_s = 0.69; p<0.05) and C3c/C3 activation ratio (r_s = 0.59; p<0.05). C3c, C3 activation status (C3c/C3 ratio) and properdin levels increased with higher lobular inflammation scores as determined according to the Kleiner classification (C3c: p<0.01, C3c/C3 ratio: p<0.05, properdin: p<0.05). Hepatic mRNA expression of factor B and factor D did not differ between subjects with healthy livers and subjects with NASH (factor B: 1.00±0.19 vs. 0.71±0.07, p = 0.26; factor D: 1.00±0.21 vs. 0.66±0.14, p = 0.29;). Hepatic mRNA and protein levels of Decay Accelerating Factor tended to be increased in subjects with NASH (mRNA: 1.00±0.14 vs. 2.37±0.72; p = 0.22; protein: 0.51±0.11 vs. 1.97±0.67; p = 0.28). In contrast, factor H mRNA was downregulated in patients with NASH (1.00±0.09 vs. 0.71±0.06; p<0.05) and a similar trend was observed with hepatic protein levels (1.12±0.16 vs. 0.78±0.07; p = 0.08). Collectively, these data suggest a role for alternative pathway activation in driving hepatic inflammation in NASH. Therefore, alternative pathway factors may be considered attractive targets for treating NASH by inhibiting complement activation.     

Soluble CD14 Levels Reflect Liver Inflammation in Patients with Nonalcoholic Steatohepatitis

Background & Aims

Liver inflammation is a risk factor for the progression of nonalcoholic fatty liver disease (NAFLD). However, the diagnosis of liver inflammation is very difficult and invasive liver biopsy is still the only method to reliably detect liver inflammation. We previously reported that overexpression of CD14 in Kupffer cells may trigger the progression to nonalcoholic steatohepatitis (NASH) via liver inflammation following hyper-reactivity to low-dose lipopolysaccharide. Therefore, the aim of this study was to investigate the relationship between soluble type of CD14 (sCD14) and histological features in patients with NAFLD.

Methods

Our cohort consisted of 113 patients with liver biopsy-confirmed NAFLD and 21 age-matched healthy controls. Serum sCD14 levels were measured by an enzyme-linked immunosorbent assay.

Results

Serum sCD14 levels were significantly associated with diagnosis of NASH and the area under the receiver operator characteristic curve (AUROC) to distinguish between not NASH and NASH was 0.802. Moreover, serum sCD14 levels were significantly associated with the disease activity based on NAFLD activity score and hepatic CD14 mRNA expression, which is correlated with membrane CD14 (mCD14) expression, in patients with NAFLD. In multiple regression analysis, the serum sCD14 levels were independently associated with liver inflammation. The AUROC to distinguish between mild and severe liver inflammation in patients with NAFLD was 0.752.

Conclusions

We found that serum sCD14 levels increased significantly with increasing liver inflammation grade in patients with NAFLD, reflecting increased hepatic CD14 expression. Serum sCD14 is a promising tool to predict the worsening of liver inflammation, and may offer a potential biomarker for evaluation of therapeutic effects in NAFLD.     

Prognostic Value of Non-Invasive Fibrosis and Steatosis Tools,Hepatic Venous Pressure Gradient (HVPG) and Histology in Nonalcoholic Steatohepatitis

Background & Aims

Non-invasive diagnostic methods for liver fibrosis predict clinical outcomes in viral hepatitis and nonalcoholic fatty liver disease (NAFLD). We specifically evaluated prognostic value of non-invasive fibrosis methods in nonalcoholic steatohepatitis (NASH) against hepatic venous pressure gradient (HVPG) and liver histology.

Methods

This was a retrospective cohort study of 148 consecutive patients who met the following criteria: transjugular liver biopsy with HVPG measurement; biopsy-proven NASH; absence of decompensation; AST-to-Platelets Ratio Index (APRI), fibrosis-4 (FIB-4), NAFLD fibrosis score, ultrasound, hepatic steatosis index and Xenon-133 scan available within 6 months from biopsy; a minimum follow-up of 1 year. Outcomes were defined by death, liver transplantation, cirrhosis complications. Kaplan–Meier and Cox regression analyses were employed to estimate incidence and predictors of outcomes, respectively. Prognostic value was expressed as area under the curve (AUC).

Results

During a median follow-up of 5 years (interquartile range 3-8), 16.2% developed outcomes, including 7.4% who died or underwent liver transplantation. After adjustment for age, sex, diabetes, the following fibrosis tools predicted outcomes: HVPG >10mmHg (HR=9.60; 95% confidence interval [CI] 3.07-30.12), histologic fibrosis F3-F4 (HR=3.14; 1.41-6.95), APRI >1.5 (HR=5.02; 1.6-15.7), FIB-4 >3.25 (HR=6.33; 1.98-20.2), NAFLD fibrosis score >0.676 (HR=11.9; 3.79-37.4). Prognostic value was as follows: histologic fibrosis stage, AUC=0.85 (95% CI 0.76-0.93); HVPG, AUC=0.81 (0.70-0.91); APRI, AUC=0.89 (0.82-0.96); FIB-4, AUC=0.89 (0.83-0.95); NAFLD fibrosis score, AUC=0.79 (0.69-0.91). Neither histologic steatosis nor non-invasive steatosis methods predicted outcomes (AUC<0.50).

Conclusions

Non-invasive methods for liver fibrosis predict outcomes of patients with NASH. They could be used for serial monitoring, risk stratification and targeted interventions.     

Enhanced HBsAg Synthesis Correlates with Increased Severity of Fibrosis in Chronic Hepatitis B Patients

Background and Aims

Little is known about whether low serum HBsAg levels result from impaired HBsAg synthesis or a reduced number of hepatocytes caused by advanced liver fibrosis. Therefore, we investigated the capacity for HBsAg synthesis in a cross-sectional cohort of treatment-naïve chronic hepatitis B patients.

Methods

Chronic hepatitis B patients (n = 362) were enrolled; liver biopsies were performed and liver histology was scored, and serum HBsAg and HBV DNA levels were investigated. In the enrolled patients, 183 out of 362 have quantitative serum HBsAg levels. Tissue HBsAg was determined by immunohistochemistry.

Results

A positive correlation between serum HBsAg and HBV DNA levels was revealed in HBeAg(+) patients (r = 0.2613, p = 0.0050). In HBeAg(+) patients, serum HBsAg and severity of fibrosis were inversely correlated (p = 0.0094), whereas tissue HBsAg levels correlated positively with the stage of fibrosis (p = 0.0280). After applying the mean aminopyrine breath test as a correction factor, adjusted serum HBsAg showed a strong positive correlation with fibrosis severity in HBeAg(+) patients (r = 0.5655, p<0.0001). The adjusted serum HBsAg values predicted ‘moderate to severe’ fibrosis with nearly perfect performance in both HBeAg(+) patients (area under the curve: 0.994, 95% CI: 0.983–1.000) and HBeAg(−) patients (area under the curve: 1.000, 95% CI: 1.000–1.000).

Conclusions

Although serum HBsAg levels were negatively correlated with fibrosis severity in HBeAg(+) patients, aminopyrine breath test-adjusted serum HBsAg and tissue HBsAg, two indices that are unaffected by the number of residual hepatocytes, were positively correlated with fibrosis severity. Furthermore, adjusted serum HBsAg has an accurate prediction capability.     

血清IL-1RA水平与非酒精性脂肪性肝病的相关性及无创诊断的临床价值

目的:探究血清白介素-1受体拮抗剂(IL-1RA)与非酒精性脂肪性肝病(NAFLD)进展的相关性,以及无创性预测诊断NASH的临床价值。方法:选取确诊NAFLD病例60例,配套肝穿刺活检组织标本及肝穿前一周血清。经B超检查无脂肪肝症状的正常健康人群30例,作为对照。测量身高、体重、腰围等生理指标,计算BMI;检测ALT、AST、AST/ALT、ALP、GGT、TC、TG、HDL、LDL等生化指标;双抗夹心法测定血清中IL-1RA浓度水平。结果:血清IL-1RA与ALT、AST及GGT具有相关性(P0.05),与肝细胞脂肪变性、气球样变、小叶内炎症、汇管区炎症及纤维化程度呈高度相关(P0.01);随NAS评分增高其浓度增大,呈高度正相关(r=0.915,P0.01);NASH患者血清中IL-1RA明显高于Non-NASH患者(t=2.88,P0.01),经ROC曲线分析,曲线下面积AUROC=0.986,利用Youden指数确定最佳敏感性为89.7%,特异性为97.9%,最佳cut-off值为171.8 ng/L。结论:NASH患者血清IL-1RA水平明显升高,血清IL-1RA可作为评价NASH及其严重程度的独立预测因子,可以作为一个无创性诊断指标对NASH进行诊断。     

Hepatic expression patterns of inflammatory and immune response genes associated with obesity and NASH in morbidly obese patients

Background

Obesity modulates inflammation and activation of immune pathways which can lead to liver complications. We aimed at identifying expression patterns of inflammatory and immune response genes specifically associated with obesity and NASH in the liver of morbidly obese patients.

Methodology/Principal Findings

Expression of 222 genes was evaluated by quantitative RT-PCR in the liver of morbidly obese patients with histologically normal liver (n = 6), or with severe steatosis without (n = 6) or with NASH (n = 6), and in lean controls (n = 5). Hepatic expression of 58 out of 222 inflammatory and immune response genes was upregulated in NASH patients. The most notable changes occurred in genes encoding chemokines and chemokine receptors involved in leukocyte recruitment, CD and cytokines involved in the T cell activation towards a Th1 phenotype, and immune semaphorins. This regulation seems to be specific for the liver since visceral adipose tissue expression and serum levels of MCP1, IP10, TNFα and IL6 were not modified. Importantly, 47 other genes were already upregulated in histologically normal liver (e.g. CRP, Toll-like receptor (TLR) pathway). Interestingly, serum palmitate, known to activate the TLR pathway, was increased with steatosis.

Conclusion/Significance

The liver of obese patients without histological abnormalities already displayed a low-grade inflammation and could be more responsive to activators of the TLR pathway. NASH was then characterized by a specific gene signature. These findings help to identify new potential actors of the pathogenesis of NAFLD.