Prospective validation of FibroTest in comparison with liver stiffness for predicting liver fibrosis in Asian subjects with chronic hepatitis B

Background and Aims

Diagnostic values of FibroTest (FT) for hepatic fibrosis have rarely been assessed in Asian chronic hepatitis B (CHB) patients. We aimed to validate its diagnostic performances in comparison with liver stiffness (LS).

Methods

From 2008 to 2010, 194 CHB patients who underwent liver biopsies along with FT and transient elastography were prospectively enrolled. Fibrosis stage was assessed according to the Batts and Ludwig system.

Results

To predict significant fibrosis (F≥2), advanced fibrosis (F≥3), and cirrhosis (F = 4), areas under receiver operating characteristic curves (AUROCs) of FT were 0.903, 0.907, and 0.866, comparable to those of LS (0.873, 0.897, and 0.910, respectively). Optimized cutoffs of FT to maximize sum of sensitivity and specificity were 0.32, 0.52, and 0.68 for F≥2, F≥3, and F = 4, while those of LS were 8.8, 10.2, and 14.1 kPa, respectively. According to FT and LS cutoffs, 123 (63.4%) and 124 (63.9%) patients were correctly classified consistent with histological fibrosis (F1, F2, F3, and F4), respectively. Overall concordance between each fibrosis stage estimated by FT and LS was observed in 111 patients, where 88 were correctly classified with histological results. A combination formula adding LS to FT (LS+FT) showed similar AUROC levels (0.885, 0.905, and 0.915), while another multiplying LS by FT (LS×FT) showed the best AUROCs (0.941, 0.931, and 0.929 for F≥2, F≥3, and F4, respectively).

Conclusions

FT provides good fibrosis prediction, with comparable outcomes to LS in Asian CHB patients. FT substantially reduces need for liver biopsy, especially when used in combination with LS.     

The role of serum biomarkers in predicting fibrosis progression in pediatric and adult hepatitis C virus chronic infection

Background/Aims

Liver biopsy represents the gold standard for damage evaluation, but noninvasive serum markers that mirror liver fibrosis progression are actual goals both in adults and especially in children. The aim was to determine specific serum markers that correlate with liver fibrosis progression during chronic HCV infection.

Methods

Liver biopsies and concomitant serum samples from 22 pediatric and 22 adult HCV patients were analyzed. Histological parameters were evaluated. On serum TGF-ß1, tissue inhibitor of matrix metalloprotein inhibitor-1 (TIMP-1), hyaluronic acid (HA) and aminoterminal peptide of procollagen type III (PIIINP) were tested.

Results

Significant fibrosis (F≥2) and advanced fibrosis (F≥3) represented 64% and 20%, respectively in children; while 54% F≥2 and 23% F≥3 in adults. Hyaluronic acid (p = 0.011) and PIIINP (p = 0.016) were related to worse fibrosis stages only in adults, along with TIMP-1 (p = 0.039) just in children; but TGF-ß1 was associated with mild fibrosis (p = 0.022) in adults. The AUROC of TIMP-1 in children to discriminate advanced fibrosis was 0.800 (95%IC 0.598–0.932). In adults, the best AUROCs were that of HA, PIIINP and TGF-ß1 [0.929 (IC95% 0.736–0.994), 0.894 (IC95% 0.689–0.984) and 0.835 (IC95% 0.617–0.957)], respectively. In children, according to the cut off (165.7 ng/mL) value for TIMP-1, biopsies could have been avoided in 72% (18/25). Considering the cut off for HA (109.7 ng/mL), PIIINP (9.1 µg/L), and TGF-ß1 (10,848.3 pg/mL), biopsies could have been avoided in 87% (19/22) of adult patients by using HA and 73% (16/22) using PIIINP or TGF-ß1.

Conclusions

In adults given the diagnostic accuracy of HA, PIIINP, TGF-ß1, their combination may provide a potential useful tool to assess liver fibrosis. This first pediatric study suggests that TIMP-1 is clinically useful for predicting liver fibrosis in HCV patients.     

Serum markers of hepatocyte death and apoptosis are non invasive biomarkers of severe fibrosis in patients with alcoholic liver disease

Background

Quantification of hepatotocyte death is useful to evaluate the progression of alcoholic liver diseases. Our aims were to quantify and correlate the circulating levels of Cytokeratin 18 (CK18) and its caspases-generated fragment to disease severity in heavy alcoholics.

Methodology/Principal Findings

CK18 and CK18-fragment were evaluated in the serum of 143 heavy alcoholics. Serum levels of markers of hepatocyte death (CK18), apoptosis (CK18 fragment) and necrosis (CK18 -CK18 fragment) increased in patients with severe fibrosis compared to patients with mild fibrosis. These markers strongly correlated with Mallory-Denk bodies, hepatocyte ballooning, fibrosis and with hepatic TNFα and TGFβ assessed in the liver of 24 patients. Elevated levels of serum hepatocyte death and apoptotic markers were independent risk factors in predicting severe fibrosis in a model combining alkaline phosphatase, bilirubin, prothrombin index, hyaluronate, hepatocyte death and apoptotic markers. The level of markers of hepatocyte death and apoptosis had an area under the receiving operator curve that predicted severe fibrosis of 0.84 and 0.76, respectively.

Conclusion/Significance

Death of hepatocytes can be easily evaluated with serum markers and correlated with severe fibrosis in heavy alcohol drinkers. These biomarkers could be useful to rapidly evaluate liver injuries and the efficacy of therapies.     

Osteopontin is required for the early onset of high fat diet-induced insulin resistance in mice

Background

Insulin resistance is manifested in muscle, adipose tissue, and liver and is associated with adipose tissue inflammation. The cellular components and mechanisms that regulate the onset of diet-induced insulin resistance are not clearly defined.

Methodology and Principal Findings

We initially observed osteopontin (OPN) mRNA over-expression in adipose tissue of obese, insulin resistant humans and rats which was normalized by thiazolidinedione (TZD) treatment in both species. OPN regulates inflammation and is implicated in pathogenic maladies resulting from chronic obesity. Thus, we tested the hypothesis that OPN is involved in the early development of insulin resistance using a 2–4 week high fat diet (HFD) model. OPN KO mice fed HFD for 2 weeks were completely protected from the severe skeletal muscle, liver and adipose tissue insulin resistance that developed in wild type (WT) controls, as determined by hyperinsulinemic euglycemic clamp and acute insulin-stimulation studies. Although two-week HFD did not alter body weight or plasma free fatty acids and cytokines in either strain, HFD-induced hyperleptinemia, increased adipose tissue inflammation (macrophages and cytokines), and adipocyte hypertrophy were significant in WT mice and blunted or absent in OPN KO mice. Adipose tissue OPN protein isoform expression was significantly altered in 2- and 4-week HFD-fed WT mice but total OPN protein was unchanged. OPN KO bone marrow stromal cells were more osteogenic and less adipogenic than WT cells in vitro. Interestingly, the two differentiation pathways were inversely affected by HFD in WT cells in vitro.

Conclusions

The OPN KO phenotypes we report reflect protection from insulin resistance that is associated with changes in adipocyte biology and adipose tissue inflammatory status. OPN is a key component in the development of HFD-induced insulin resistance.     

Serum Osteopontin Predicts Degree of Hepatic Fibrosis and Serves as a Biomarker in Patients with Hepatitis C Virus Infection

Background & Aims

Osteopontin (OPN) is a matricellular protein that upregulates during pathogenesis of hepatic fibrosis. The present study was aimed to evaluate whether serum OPN could be used as a biomarker to assess the degree of hepatic fibrosis in patients with hepatitis C virus (HCV) infection.

Methods

Needle biopsy was performed on HCV patients and scored as zero fibrosis (F0), mild fibrosis (F1), moderate fibrosis (F2), severe fibrosis (F3) and liver cirrhosis (F4) based on Masson’s trichrome and α-smooth muscle actin (α-SMA) staining. Serum OPN levels were measured using ELISA and correlated with the degree of fibrosis. Furthermore, the OPN values were correlated and evaluated with platelets count, serum hyaluronic acid (HA), and collagen type IV and subjected to receiver operating characteristic (ROC) curve analysis.

Results

Serum OPN levels were remarkably increased from F0 through F4 in a progressive manner and the differences were significant (P < 0.001) between each group. The data were highly correlated with the degree of hepatic fibrosis. The ROC curve analysis depicted that serum OPN is an independent risk factor and an excellent biomarker and a prognostic index in HCV patients.

Conclusions

The results of the present study indicate that serum OPN levels reflect the degree of hepatic fibrosis and could be used as a biomarker to assess the stage of fibrosis in HCV patients which would help to reduce the number of liver biopsies. Furthermore, serum OPN serves as a prognostic index towards the progression of hepatic fibrosis to cirrhosis and hepatocellular carcinoma.     

Prospective Validation of ELF Test in Comparison with Fibroscan and FibroTest to Predict Liver Fibrosis in Asian Subjects with Chronic Hepatitis B

Background and Aims

Liver stiffness measurement (LSM) and FibroTest (FT) are frequently used as non-invasive alternatives for fibrosis staging to liver biopsy. However, to date, diagnostic performances of Enhanced Liver Fibrosis (ELF) test, which consists of hyaluronic acid, aminoterminal propeptide of procollagen type-III, and tissue inhibitor of matrix metalloproteinases-1, have not been compared to those of LSM and FT in Asian chronic hepatitis B (CHB) patients.

Methods

Between June 2010 and November 2011, we prospectively enrolled 170 CHB patients who underwent liver biopsies along with LSM, FT, and ELF. The Batts system was used to assess fibrosis stages.

Results

Areas under receiver operating characteristic curves (AUROCs) to predict significant fibrosis (F≥2), advanced fibrosis (F≥3), and cirrhosis (F = 4) were 0.901, 0.860, and 0.862 for ELF, respectively; 0.937, 0.956, and 0.963 for LSM; and 0.896, 0.921, and 0.881 for FT. AUROCs to predict F≥2 were similar between each other, whereas LSM and FT had better AUROCs than ELF for predicting F≥3 (both p<0.05), and LSM predicted F4 more accurately than ELF (p<0.05). Optimized cutoffs of ELF to maximize sum of sensitivity and specificity were 8.5, 9.4, and 10.1 for F≥2, F≥3, and F = 4, respectively. Using suggested ELF, LSM and FT cutoffs to diagnose F1, F2, F3, and F4, 91 (53.5%), 117 (68.8%), and 110 (64.7%) patients, respectively, were correctly classified according to histological results.

Conclusions

ELF demonstrated considerable diagnostic value in fibrosis staging in Asian CHB patients, especially in predicting F≥2. However, LSM consistently provided better performance for predicting F≥3 and F4.     

Performance of Real-Time Elastography for the Staging of Hepatic Fibrosis: A Meta-Analysis

Background

With the rapid development of real-time elastography (RTE), a variety of measuring methods have been developed for the assessment of hepatic fibrosis. We evaluated the overall performance of four methods based on RTE by performing meta-analysis of published literature.

Methods

Online journal databases and a manual search from April 2000 to April 2014 were used. Studies from different databases that meet inclusion criteria were enrolled. The statistical analysis was performed using a random-effects model and fixed-effects model for the overall effectiveness of RTE. The area under the receiver operating characteristic curve (AUROC) was calculated for various means. Fagan plot analysis was used to estimate the clinical utility of RTE, and the heterogeneity of the studies was explored with meta-regression analysis.

Results

Thirteen studies from published articles were enrolled and analyzed. The combined AUROC of the liver fibrosis index (LFI) for the evaluation of significant fibrosis (F≥2), advanced fibrosis (F≥3), and cirrhosis (F = 4) were 0.79, 0.94, and 0.85, respectively. The AUROC of the elasticity index (EI) ranged from 0.75 to 0.92 for F≥2 and 0.66 to 0.85 for F = 4. The overall AUROC of the elastic ratio of the liver for the intrahepatic venous vessels were 0.94, 0.93, and 0.96, respectively. The AUROC of the elastic ratio of the liver for the intercostal muscle in diagnosing advanced fibrosis and cirrhosis were 0.96 and 0.92, respectively. There was significant heterogeneity in the diagnostic odds ratio (DOR) for F≥2 of LFI mainly due to etiology (p<0.01).

Conclusion

The elastic ratio of the liver for the intrahepatic vein has excellent precision in differentiating each stage of hepatic fibrosis and is recommend to be applied to the clinic.     

Micro-RNA profiling in human serum reveals compartment-specific roles of miR-571 and miR-652 in liver cirrhosis

Background and Aims

Micro-RNAs (miRNAs) have recently emerged as crucial modulators of molecular processes involved in chronic liver diseases. The few miRNAs with previously proposed roles in liver cirrhosis were identified in screening approaches on liver parenchyma, mostly in rodent models. Therefore, in the present study we performed a systematic screening approach in order to identify miRNAs with altered levels in the serum of patients with chronic liver disease and liver cirrhosis.

Methods

We performed a systematic, array-based miRNA expression analysis on serum samples from patients with liver cirrhosis. In functional experiments we evaluated the relationship between alterations of miRNA serum levels and their role in distinct cellular compartments involved in hepatic cirrhosis.

Results

The array analysis and the subsequent confirmation by qPCR in a larger patient cohort identified significant alterations in serum levels of miR-513-3p, miR-571 and miR-652, three previously uncharacterized miRNAs, in patients with alcoholic or hepatitis C induced liver cirrhosis. Of these, miR-571 serum levels closely correlated with disease stages, thus revealing potential as a novel biomarker for hepatic cirrhosis. Further analysis revealed that up-regulation of miR-571 in serum reflected a concordant regulation in cirrhotic liver tissue. In isolated primary human liver cells, miR-571 was up-regulated in human hepatocytes and hepatic stellate cells in response to the pro-fibrogenic cytokine TGF-β. In contrast, alterations in serum levels of miR-652 were stage-independent, reflecting a concordant down-regulation of this miRNA in circulating monocytes of patients with liver cirrhosis, which was inducible by proinflammatory stimuli like bacterial lipopolysaccharide.

Conclusion

Alterations of miR571 and miR-652 serum levels in patients with chronic liver disease reflect their putative roles in the mediation of fibrogenic and inflammatory processes in distinct cellular compartments involved in the pathogenesis of liver cirrhosis.     

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.     

Hedgehog signaling antagonist promotes regression of both liver fibrosis and hepatocellular carcinoma in a murine model of primary liver cancer

Objective

Chronic fibrosing liver injury is a major risk factor for hepatocarcinogenesis in humans. Mice with targeted deletion of Mdr2 (the murine ortholog of MDR3) develop chronic fibrosing liver injury. Hepatocellular carcinoma (HCC) emerges spontaneously in such mice by 50–60 weeks of age, providing a model of fibrosis-associated hepatocarcinogenesis. We used Mdr2^−/− mice to investigate the hypothesis that activation of the hedgehog (Hh) signaling pathway promotes development of both liver fibrosis and HCC.

Methods

Hepatic injury and fibrosis, Hh pathway activation, and liver progenitor populations were compared in Mdr2^−/− mice and age-matched wild type controls. A dose finding experiment with the Hh signaling antagonist GDC-0449 was performed to optimize Hh pathway inhibition. Mice were then treated with GDC-0449 or vehicle for 9 days, and effects on liver fibrosis and tumor burden were assessed by immunohistochemistry, qRT-PCR, Western blot, and magnetic resonance imaging.

Results

Unlike controls, Mdr2^−/− mice consistently expressed Hh ligands and progressively accumulated Hh-responsive liver myofibroblasts and progenitors with age. Treatment of aged Mdr2-deficient mice with GDC-0449 significantly inhibited hepatic Hh activity, decreased liver myofibroblasts and progenitors, reduced liver fibrosis, promoted regression of intra-hepatic HCCs, and decreased the number of metastatic HCC without increasing mortality.

Conclusions

Hh pathway activation promotes liver fibrosis and hepatocarcinogenesis, and inhibiting Hh signaling safely reverses both processes even when fibrosis and HCC are advanced.     

Interleukin-8 is activated in patients with chronic liver diseases and associated with hepatic macrophage accumulation in human liver fibrosis

Background

Interleukin-8 (IL-8, CXCL8) is a potent chemoattractant for neutrophils and contributes to acute liver inflammation. Much less is known about IL-8 in chronic liver diseases (CLD), but elevated levels were reported from alcoholic and hepatitis C-related CLD. We investigated the regulation of IL-8, its receptors CXCR1 and CXCR2 and possible IL-8 responding cells in CLD patients.

Methodology

Serum IL-8 levels were measured in CLD patients (n = 200) and healthy controls (n = 141). Intrahepatic IL-8, CXCR1 and CXCR2 gene expression was quantified from liver samples (n = 41), alongside immunohistochemical neutrophil (MPO) and macrophage (CD68) stainings. CXCR1 and CXCR2 expression was analyzed on purified monocytes from patients (n = 111) and controls (n = 31). In vitro analyses explored IL-8 secretion by different leukocyte subsets.

Principal Findings

IL-8 serum levels were significantly increased in CLD patients, especially in end-stage cirrhosis. Interestingly, patients with cholestatic diseases exhibited highest IL-8 serum concentrations. IL-8 correlated with liver function, inflammatory cytokines and non-invasive fibrosis markers. Intrahepatically, IL-8 and CXCR1 expression were strongly up-regulated. However, intrahepatic IL-8 could only be associated to neutrophil infiltration in patients with primary biliary cirrhosis (PBC). In non-cholestatic cirrhosis, increased IL-8 and CXCR1 levels were associated with hepatic macrophage accumulation. In line, CXCR1, but not CXCR2 or CXCR3, expression was increased on circulating monocytes from cirrhotic patients. Moreover, monocyte-derived macrophages from CLD patients, especially the non-classical CD16⁺ subtype, displayed enhanced IL-8 secretion in vitro.

Conclusions

IL-8 is strongly activated in CLD, thus likely contributing to hepatic inflammation. Our study suggests a novel role of IL-8 for recruitment and activation of hepatic macrophages via CXCR1 in human liver cirrhosis.     

Angiopoietin-2 Serum Levels Improve Noninvasive Fibrosis Staging in Chronic Hepatitis C: A Fibrogenic-Angiogenic Link

Aims

Accurate liver fibrosis staging is crucial for the management of chronic hepatitis C (CHC). The invasiveness and cost burden of liver biopsy have driven the search for new noninvasive biomarkers of fibrosis. Based on the link between serum angiopoietin-1 and 2 levels and CHC progression, we aimed to determine the value of these angiogenic factors as noninvasive biomarkers of liver fibrosis.

Methods

Serum levels of angiopoietin-1 and -2 were measured by ELISA in 108 CHC patients who underwent pretreatment liver biopsy. The correlation between angiopoietins and clinical and demographic variables with liver fibrosis was analyzed by univariate regression. Significant factors were then subjected to multivariate analysis, from which we constructed a novel noninvasive liver fibrosis index (AngioScore), whose performance was validated in an independent series of 71 CHC patients. The accuracy of this model was compared with other documented fibrosis algorithms by De Long test.

Results

Angiopoietins correlated significantly with hepatic fibrosis; however, only angiopoietin-2 was retained in the final model, which also included age, platelets, AST, INR, and GGT. The model was validated and behaved considerably better than other fibrosis indices in discriminating all, significant, moderate and severe liver fibrosis (0.886, 0.920, 0.923). Using clinically relevant cutoffs, we classified CHC patients by discarding significant fibrosis and diagnosing moderate and severe fibrosis with greater accuracy, sensitivity, and specificity.

Conclusions

Our novel noninvasive liver fibrosis model, based on serum angiopoietin-2 levels, outperforms other indices and should help substantially in managing CHC and monitoring long-term follow-up prognosis.     

E3 ubiquitin ligase synoviolin is involved in liver fibrogenesis

Background and Aim

Chronic hepatic damage leads to liver fibrosis, which is characterized by the accumulation of collagen-rich extracellular matrix. However, the mechanism by which E3 ubiquitin ligase is involved in collagen synthesis in liver fibrosis is incompletely understood. This study aimed to explore the involvement of the E3 ubiquitin ligase synoviolin (Syno) in liver fibrosis.

Methods

The expression and localization of synoviolin in the liver were analyzed in CCl₄-induced hepatic injury models and human cirrhosis tissues. The degree of liver fibrosis and the number of activated hepatic stellate cells (HSCs) was compared between wild type (wt) and Syno^+/− mice in the chronic hepatic injury model. We compared the ratio of apoptosis in activated HSCs between wt and Syno^+/− mice. We also analyzed the effect of synoviolin on collagen synthesis in the cell line from HSCs (LX-2) using siRNA-synoviolin and a mutant synoviolin in which E3 ligase activity was abolished. Furthermore, we compared collagen synthesis between wt and Syno^−/− mice embryonic fibroblasts (MEF) using quantitative RT-PCR, western blotting, and collagen assay; then, we immunohistochemically analyzed the localization of collagen in Syno^−/− MEF cells.

Results

In the hepatic injury model as well as in cirrhosis, synoviolin was upregulated in the activated HSCs, while Syno^+/− mice developed significantly less liver fibrosis than in wt mice. The number of activated HSCs was decreased in Syno^+/− mice, and some of these cells showed apoptosis. Furthermore, collagen expression in LX-2 cells was upregulated by synoviolin overexpression, while synoviolin knockdown led to reduced collagen expression. Moreover, in Syno^−/− MEF cells, the amounts of intracellular and secreted mature collagen were significantly decreased, and procollagen was abnormally accumulated in the endoplasmic reticulum.

Conclusion

Our findings demonstrate the importance of the E3 ubiquitin ligase synoviolin in liver fibrosis.     

Osteopontin Deletion Prevents the Development of Obesity and Hepatic Steatosis via Impaired Adipose Tissue Matrix Remodeling and Reduced Inflammation and Fibrosis in Adipose Tissue and Liver in Mice

Osteopontin (OPN) is a multifunctional extracellular matrix (ECM) protein involved in multiple physiological processes. OPN expression is dramatically increased in visceral adipose tissue in obesity and the lack of OPN protects against the development of insulin resistance and inflammation in mice. We sought to unravel the potential mechanisms involved in the beneficial effects of the absence of OPN. We analyzed the effect of the lack of OPN in the development of obesity and hepatic steatosis induced by a high-fat diet (HFD) using OPN-KO mice. OPN expression was upregulated in epididymal white adipose tissue (EWAT) and liver in wild type (WT) mice with HFD. OPN-KO mice had higher insulin sensitivity, lower body weight and fat mass with reduced adipose tissue ECM remodeling and reduced adipocyte size than WT mice under a HFD. Reduced MMP2 and MMP9 activity was involved in the decreased ECM remodeling. Crown-like structure number in EWAT as well as F4/80-positive cells and Emr1 expression in EWAT and liver increased with HFD, while OPN-deficiency blunted the increase. Moreover, our data show for the first time that OPN-KO under a HFD mice display reduced fibrosis in adipose tissue and liver, as well as reduced oxidative stress in adipose tissue. Gene expression of collagens Col1a1, Col6a1 and Col6a3 in EWAT and liver, as well as the profibrotic cytokine Tgfb1 in EWAT were increased with HFD, while OPN-deficiency prevented this increase. OPN deficiency prevented hepatic steatosis via reduction in the expression of molecules involved in the onset of fat accumulation such as Pparg, Srebf1, Fasn, Mogat1, Dgat2 and Cidec. Furthermore, OPN-KO mice exhibited higher body temperature and improved BAT function. The present data reveal novel mechanisms of OPN in the development of obesity, pointing out the inhibition of OPN as a promising target for the treatment of obesity and fatty liver.     

Clinical Benefit of Liver Stiffness Measurement at 3 Months after Kasai Hepatoportoenterostomy to Predict the Liver Related Events in Biliary Atresia

Background

The progression of hepatic fibrosis may result in decompensated hepatic failure with cirrhosis, liver related events (LRE) such as ascites, variceal bleeding, and death after successful and timely Kasai hepatoportoenterostomy (HPE) in biliary atresia. The aim of this study is to suggest clinical benefit of the liver stiffness measurement (LSM) using transient elastography at 3 months after the Kasai operation to predict LRE.

Methods

Between January 2007 and December 2011, 69 eligible biliary atresia patients who underwent Kasai HPE and performed transient elastography before and 3 months after HPE were included. The occurrences of LRE were analyzed for all patients. All patients were divided into 2 groups (with and without LRE) for comparison. Multivariate analysis was used to detect the independent risk factors of LRE. The area under the receiver operation characteristics curve (AUROC) was used to establish the LSM optimal cutoff value of 3 months after Kasai operation in predicting LRE.

Results

LSM value, aminotransferase, albumin, bilirubin, and PT-INR significantly differed among the two groups. Multivariate analysis demonstrated LSM value as the most powerful independent factor of the development of LRE. The cut-off value of 19.9 kPa was calculated to be optimal for predicting LRE development with total sensitivity and specificity of 1.804. AUROC resulted in 0.943, with sensitivity of 85.3% and specificity of 95.2%.

Conclusions

The LSM value of 3 months after Kasai HPE can be a useful predictor of LRE development.     

Serum Hepatic Enzyme Activity and Alcohol Drinking Status in Relation to the Prevalence of Metabolic Syndrome in the General Japanese Population

Background

Studies on the combined associations of elevated serum hepatic enzyme activity and alcohol drinking with metabolic syndrome are rare. Our objectives were to evaluate the associations of elevated serum hepatic enzyme activity with the prevalence of metabolic syndrome in the general Japanese population and whether alcohol drinking had a modifying effect on these associations.

Methods

We conducted a cross-sectional study with 1,027 men and 1,152 women throughout Japan during 2002–2010. Biochemical factors including alanine aminotransferase (ALT) and gamma-glutamyl transferase (GGT) were determined in overnight fasting blood, and a survey on lifestyle was conducted by questionnaire. Serum ALT and GGT levels were divided into tertiles in men and women, and their associations with the prevalence of metabolic syndrome were evaluated by logistic regressions.

Results

Elevated serum ALT and GGT, even within the reference range, were independently associated with increased metabolic syndrome prevalence and were associated with most of its components in both sexes, except for the association between GGT and low high-density lipoprotein (HDL) cholesterol in men. Stratified analyses by alcohol drinking status revealed that within the same tertile category of serum ALT and GGT, subjects classified as alcohol abstainers showed higher adjusted odds ratios for metabolic syndrome prevalence than those classified as regular alcohol drinkers in both sexes. The interaction effects of serum GGT with alcohol drinking status on metabolic syndrome prevalence were significant in both sexes.

Conclusions

These results suggest that elevated serum ALT and GGT, even within the reference range, are independently associated with increased metabolic syndrome prevalence, especially in alcohol abstainers, in Japanese men and women.     

Cytosolic Malic Enzyme 1 (ME1) Mediates High Fat Diet-Induced Adiposity,Endocrine Profile,and Gastrointestinal Tract Proliferation-Associated Biomarkers in Male Mice

Background

Obesity and associated hormonal disturbances are risk factors for colon cancer. Cytosolic Malic Enzyme (ME1) generates NADPH used for lipogenesis in gastrointestinal (GI), liver and adipose tissues. We have reported that inclusion of soy protein isolate (SPI) in the diet lowered body fat content and colon tumor incidence of rats fed AIN-93G diet, while others have demonstrated SPI inhibition of rat hepatic ME1 expression. The present study examined the individual and combined effects of dietary SPI and absence of ME1 on: 1) serum concentrations of hormones implicated in colon cancer development, 2) expression of lipogenic and proliferation-associated genes in the mouse colon and small intestine, and 3) liver and adipose expression of lipogenic and adipocytokine genes that may contribute to colon cancer predisposition.

Methods

Weanling wild type (WT) and ME1 null (MOD-1) male mice were fed high-fat (HF), iso-caloric diets containing either casein (CAS) or SPI as sole protein source for 5 wks. Somatic growth, serum hormone and glucose levels, liver and adipose tissue weights, GI tissue parameters, and gene expression were evaluated.

Results

The MOD-1 genotype and SPI-HF diet resulted in decreases in: body and retroperitoneal fat weights, serum insulin, serum leptin, leptin/adiponectin ratio, adipocyte size, colon mTOR and cyclin D1 mRNA abundance, and jejunum FASN mRNA abundance, when compared to WT mice fed CAS-HF. Regardless of diet, MOD-1 mice had reductions in liver weight, liver steatosis, and colon crypt depth, and increases in adipose tissue expression of IRS1 and IRS2, compared to WT mice. SPI-HF diet reduced ME1 gene expression only in retroperitoneal fat.

Conclusions

Data suggest that the pharmacological targeting of ME1 or the inclusion of soy protein in the diet may provide avenues to reduce obesity and its associated pro-tumorigenic endocrine environment and improve insulin sensitivity, potentially disrupting the obesity-colon cancer connection.     

Non-coding keratin variants associate with liver fibrosis progression in patients with hemochromatosis

Background

Keratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with end-stage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis.

Methods

The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previously-generated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed.

Results

We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. In mice, or ex vivo, the K8 G62C variant did not affect iron-accumulation in response to iron-rich diet or the extent of iron-induced hepatocellular injury.

Conclusion

In patients with hemochromatosis, intronic but not exonic K8/K18 variants associate with liver fibrosis development.     

Performance of Transient Elastography for the Staging of Liver Fibrosis in Patients with Chronic Hepatitis B: A Meta-Analysis

Background

Transient elastography (TE), a non-invasive tool that measures liver stiffness, has been evaluated in meta-analyses for effectiveness in assessing liver fibrosis in European populations with chronic hepatitis C (CHC). However, these data cannot be extrapolated to populations in Asian countries, where chronic hepatitis B (CHB) is more prevalent. In this study, we performed a meta-analysis to assess the overall performance of TE for assessing liver fibrosis in patients with CHB.

Methods

Studies from the literature and international conference abstracts which enrolled only patients with CHB or performed a subgroup analysis of such patients were enrolled. Combined effects were calculated using area under the receiver operating characteristic curves (AUROC) and diagnostic accuracy values of each study.

Result

A total of 18 studies comprising 2,772 patients were analyzed. The mean AUROCs for the diagnosis of significant fibrosis (F2), severe fibrosis (F3), and cirrhosis (F4) were 0.859 (95% confidence interval [CI], 0.857–0.860), 0.887 (95% CI, 0.886–0.887), and 0.929 (95% CI, 0.928–0.929), respectively. The estimated cutoff for F2 was 7.9 (range, 6.1–11.8) kPa, with a sensitivity of 74.3% and specificity of 78.3%. For F3, the cutoff value was determined to be 8.8 (range, 8.1–9.7) kPa, with a sensitivity of 74.0% and specificity of 63.8%. The cutoff value for F4 was 11.7 (range, 7.3–17.5) kPa, with a sensitivity of 84.6% and specificity of 81.5%.

Conclusion

TE can be performed with good diagnostic accuracy for quantifying liver fibrosis in patients with CHB.     

The progression of liver fibrosis is related with overexpression of the miR-199 and 200 families

Background

Chronic hepatitis C (CH) can develop into liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Liver fibrosis and HCC development are strongly correlated, but there is no effective treatment against fibrosis because the critical mechanism of progression of liver fibrosis is not fully understood. microRNAs (miRNAs) are now essential to the molecular mechanisms of several biological processes. In order to clarify how the aberrant expression of miRNAs participates in development of the liver fibrosis, we analyzed the liver fibrosis in mouse liver fibrosis model and human clinical samples.

Methodology

In a CCL₄-induced mouse liver fibrosis model, we compared the miRNA expression profile from CCL₄ and olive oil administrated liver specimens on 4, 6, and 8 weeks. We also measured expression profiles of human miRNAs in the liver biopsy specimens from 105 CH type C patients without a history of anti-viral therapy.

Principle Findings

Eleven mouse miRNAs were significantly elevated in progressed liver fibrosis relative to control. By using a large amount of human material in CH analysis, we determined the miRNA expression pattern according to the grade of liver fibrosis. We detected several human miRNAs whose expression levels were correlated with the degree of progression of liver fibrosis. In both the mouse and human studies, the expression levels of miR-199a, 199a*, 200a, and 200b were positively and significantly correlated to the progressed liver fibrosis. The expression level of fibrosis related genes in hepatic stellate cells (HSC), were significantly increased by overexpression of these miRNAs.

Conclusion

Four miRNAs are tightly related to the grade of liver fibrosis in both human and mouse was shown. This information may uncover the critical mechanism of progression of liver fibrosis. miRNA expression profiling has potential for diagnostic and therapeutic applications.