Moderate to severe hepatic steatosis leads to overestimation of liver stiffness measurement in chronic hepatitis B patients without significant fibrosis.

BACKGROUND: Liver stiffness measurement (LSM) by transient elastography is a noninvasive method for the diagnosis of hepatic fibrosis. The impact of hepatic steatosis on LSM remains to be explored. AIM: To determine whether LSM is affected by hepatic steatosis in patients with chronic hepatitis B (CHB). METHODS: Consecutive patients with biopsy-proven CHB were prospectively enrolled. Hepatic steatosis was classified by pathology as none (S0, <5%), mild (S1, 5%-33%), and moderate-severe (S2-3, >33%), and quantitatively by controlled attenuation parameter (CAP) as CAP S0 (≤247 dB/m), CAP S1 (248-267 dB/m) and CAP S2-3 (≥268 dB/m). Liver fibrosis was assessed by METAVIR classification and noninvasively by LSM. RESULTS: The prevalence of non-alcoholic fatty liver disease (n = 223) in CHB patients (n = 593) was 37.6%. Forty-eight belonged to S2-3 and 127 belonged to CAP S2-3. In patients without significant fibrosis (F0-1), the median LSM (kPa) was 7.4 in S2-3 and 7.1 in CAP S2-3, which was significantly higher than that in S0/S1 (P = 0.005) and CAP S0/S1 (P = 0.003). No significant difference was found in significant fibrosis (F2-4). For LSM identifying significant fibrosis (F2-4), the negative predictive value was higher in CHB patients with CAP ≥ 268 compared to those with CAP < 268 (0.81 vs 0.73); the positive predictive value was lower in CAP ≥ 268 than its counterpart (0.65 vs 0.76). CONCLUSIONS: Moderate-severe steatosis increased the LSM value in CHB patients without significant fibrosis. A CAP ≥ 268 did not affect LSM for ruling out, but it slightly affected LSM for ruling in significant fibrosis. TRIAL REGISTRATION: ChiCTR-DDT-13003983.

Serum folic acid levels are associated with the presence and severity of liver steatosis in Chinese adults.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is a common and strong risk factor for cardiovascular disease and hepatocellular carcinoma. The rapid acceleration of the increase in NAFLD prevalence has exceeded the trends observed for obesity, and has been driven by multiple factors. The aim of this study was to investigate the correlation between the serum levels of folic acid, the endogenous source of methyl groups for DNA methylation, and NAFLD in Chinese adults. METHODS: The correlations between the serum folic acid levels and NAFLD were investigated in two independent cohorts of 70 subjects who underwent a liver biopsy and 130 subjects with varying liver fat contents, as measured using proton magnetic resonance spectroscopy (1H-MRS). Independent correlations between serum folic acid levels and liver steatosis grades were detected using a multivariate ordinal regression analysis. The diagnostic performances of serum folic acid levels alone and in combination with existing NAFLD prediction scores were compared with those of traditional NAFLD prediction parameters using receiver operating characteristic (ROC) curve analyses. RESULTS: Serum folic acid concentrations were inversely correlated with liver histological steatosis grades (ρ = -0.371, P < 0.001) and the 1H-MRS-measured liver fat content (r = -0.199, P = 0.038). According to the multivariate ordinal regression analysis, serum folic acid levels were inversely correlated with liver steatosis grades (OR 0.739 [0.594-0.918], P = 0.006) independent of age, gender, BMI, components of metabolic syndrome and the serum TC, LDL-c and HOMA-IR levels. The AUROC of serum folic acid for the diagnosis of NAFLD was 0.75 (0.65-0.83), and the addition of serum folic acid to NAFLD prediction scores significantly improved the diagnostic prediction of NAFLD (AUROC = 0.88 [0.81-0.94]). CONCLUSION: Low serum folic acid levels were identified as an independent risk factor for NAFLD in the Chinese population. The addition of the serum folic acid levels to the current existing NAFLD prediction scores significantly improved the prediction of NAFLD.

CSH guidelines for the diagnosis and treatment of drug-induced liver injury.

Drug-induced liver injury (DILI) is an important clinical problem, which has received more attention in recent decades. It can be induced by small chemical molecules, biological agents, traditional Chinese medicines (TCM), natural medicines (NM), health products (HP), and dietary supplements (DS). Idiosyncratic DILI is far more common than intrinsic DILI clinically and can be classified into hepatocellular injury, cholestatic injury, hepatocellular-cholestatic mixed injury, and vascular injury based on the types of injured target cells. The CSH guidelines summarized the epidemiology, pathogenesis, pathology, and clinical manifestation and gives 16 evidence-based recommendations on diagnosis, differential diagnosis, treatment, and prevention of DILI.

Histological outcome for chronic hepatitis B patients treated with entecavir vs lamivudine-based therapy.

AIM: To compare the histological outcome of chronic hepatitis B (CHB) patients treated with entecavir (ETV) or lamivudine (LAM)-based therapy. METHODS: We conducted a retrospective analysis of data from 42 CHB patients with advanced fibrosis (baseline Ishak score ≥ 2) or cirrhosis who were treated with ETV or LAM-based therapy in Beilun People's Hospital, Ningbo between January 2005 and May 2012. The patients enrolled were more than 16 years of age and underwent a minimum of 12 mo of antiviral therapy. We collected data on the baseline characteristics of each patient and obtained paired liver biopsies pre- and post-treatment. The Knodell scoring system and Ishak fibrosis scores were used to evaluate each example. An improvement or worsening of necroinflammation was defined as ≥ 2-point change in the Knodell inflammatory score. The progression or regression of fibrosis was defined as ≥ 1-point change in the Ishak fibrosis score. The continuous variables were compared using t-test or Mann-Whitney test, and the binary variables were compared using χ(2) test or Fisher's exact test. The results of paired liver biopsies were compared with a Wilcoxon signed rank test. RESULTS: Nineteen patients were treated with ETV and 23 patients were treated with LAM therapy for a mean duration of 39 and 42 mo, respectively. After long-term antiviral treatment, 94.74% (18/19) of the patients in the ETV arm and 95.65% (22/23) in the LAM arm achieved an HBV DNA level less than 1000 IU/mL. The majority of the patients (94.74% in the ETV arm and 73.91% in the LAM arm) had normalized ALT levels. The median Knodell necroinflammatory score decreased from 11 to 0 in the patients receiving ETV, and the median Knodell score decreased from 9 to 3 in the patients receiving LAM (P = 0.0002 and < 0.0001, respectively). The median Ishak fibrosis score showed a 1-point reduction in ETV-treated patients and a 2-point reduction in LAM-treated patients (P = 0.0019 and 0.0205, respectively). The patients receiving ETV showed a more significant improvement in necroinflammation than the LAM-treated patients (P = 0.0003). However, there was no significant difference in fibrotic improvement between the two arms. Furthermore, two patients in each arm achieved a fibrosis score of 0 post-treatment, which indicates a full reversion of fibrosis after antiviral therapy. CONCLUSION: CHB patients with advanced fibrosis or cirrhosis benefit from antiviral treatment. ETV is superior to LAM therapy in improving necroinflammatory but not fibrotic outcome.

Inherent lipid metabolic dysfunction in glycogen storage disease IIIa.

We studied two patients from a nonconsanguineous family with life-long abnormal liver function, hepatomegaly and abnormal fatty acid profiles. Abnormal liver function, hypoglycemia and muscle weakness are observed in various genetic diseases, including medium-chain acyl-CoA dehydrogenase (MCAD) deficiency and glycogen storage diseases. The proband showed increased free fatty acids, mainly C8 and C10, resembling fatty acid oxidation disorder. However, no mutation was found in ACADM and ACADL gene. Sequencing of theamylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase (AGL) gene showed that both patients were compound heterozygotes for c.118C > T (p.Gln40X) and c.753_756 del CAGA (p.Asp251Glufsx29), whereas their parents were each heterozygous for one of these mutations. The AGL protein was undetectable in EBV-B cells from the two patients. Transcriptome analysis demonstrated a significant different pattern of gene expression in both of patients' cells, including genes involving in the PPAR signaling pathway, fatty acid biosynthesis, lipid synthesis and visceral fat deposition and metabolic syndrome. This unique gene expression pattern is probably due to the absence of AGL, which potentially accounts for the observed clinical phenotypes of hyperlipidemia and hepatocyte steatosis in glycogen storage disease type IIIa.

CD25 is a novel marker of hepatic bile canaliculus.

OBJECTIVE: Although many antigens have been investigated, the method for the bile canaliculus staining using optical microscopy needs to be improved. The aim of the present study was to assess the expression pattern of a candidate marker, CD25, in normal and diseased liver tissue. METHODS: Immunohistochemistry, immunofluorescence, and immune electron microscopy assays were performed with 41 liver sections and 2 different anti-CD25 monoclonal antibodies. A polyclonal antibody against carcinoembryonic antigen (CEA) was also used to stain bile canaliculus as a control. CD25 expression levels in normal and diseased liver tissue were also determined. RESULTS: CD25 was predominantly localized at the bile canaliculus of adult and infantile liver, evidenced by both immunohistochemistry and immunofluorescence assays. The electron microscopy assay showed that there were obvious amorphous electron-dense deposits at the bile canaliculus. In contrast, the CEA-positive area included bile canaliculus as well as basolateral aspects of hepatocytes. CD25 expression levels did not differ significantly among different disease states. CONCLUSION: This study provides the first evidence that CD25 is a novel marker of bile canaliculus. Characteristics of CD25 expression may shed light on immunohistochemistry and immunofluorescence analysis of bile canaliculus in both basic and clinical hepatic investigations.

Clinical and pathological characteristics of Alagille syndrome in Chinese children.

BACKGROUND: Alagille syndrome (AS) is regarded as the most common cause of chronic cholestasis in childhood associated with specific phenotypic features in western countries. This study was undertaken to investigate the significance of AS in Chinese children with chronic cholestasis and to describe its clinical and histological features. METHODS: From October 2004 to January 2007, 157 children who presented with conjugated jaundice from less than 3 months of age were admitted to a tertiary hospital in Shanghai. Investigations of the heart, spine, eyes and kidneys were conducted in 13 children who experienced prolonged cholestasis beyond 1 year of age after exclusion of biliary atresia and familial progressive intrahepatic cholestasis type 1 or 2. In patients with interlobular bile duct paucity, AS was diagnosed if 3 or more of the following 5 major features were present: cardiac murmur, posterior embryotoxon, butterfly-like vertebrae, renal abnormalities and characteristic faces. In patients without interlobular bile duct paucity or who did not receive liver biopsy, 4 or more features were required for the diagnosis. RESULTS: Of the 13 children, 6 were diagnosed with AS at ages ranging from 1 year and 7 months to 3 years and 11 months. Jaundice was noticed in early infancy and then pruritus developed in all the 6 patients, of whom 5 presented with acholic stool and 4 had been misdiagnosed as having presumed biliary atresia by hepatobiliary scintigraphy or laparoscopic cholangiography. Biochemical examinations demonstrated increased concentration of total bile acid and hyperlipidemia. Interlobular bile duct paucity was demonstrated histologically in 5 patients who received liver biopsy. Vertebral abnormalities, heart murmur, characteristic faces and failure to thrive were found in all the 6 patients. Two patients had evidence of renal involvement. Micropenis, empty scrotum, and gall stone were seen in 1 patient. CONCLUSION: AS is also an important cause of prolonged cholestasis in Chinese children. It is difficult to differentiate AS from biliary atresia. Liver biopsy and spine X-ray may be helpful in the early detection of AS.

Hepatitis B virus mutations associated with in situ expression of hepatitis B core antigen, viral load and prognosis in chronic hepatitis B patients.

In this retrospective study, we investigated the prevalence and significance of mutations in part of the hepatitis B virus (HBV) x gene, and tried to clarify their relationship with clinicopathological or histopathological characteristics and prognosis in patients with chronic hepatitis B (CHB). A total of 83 consecutive CHB patients (1986-1994) were chosen for the present study. Sequence analysis was performed using polymerase chain reaction (PCR) and the direct sequencing method. The histological activity index was described using Scheuer scores. Two-step immunohistochemical staining showed the expression of viral antigens in situ. Tissue HBV DNA levels were determined by fluorescence quantitative real-time PCR. For the prognostic study, all the patients were followed up using clinical and laboratory data. Mutation at nt1726-1730 correlated significantly with decreased expression of HBcAg in situ (P = 0.006) and with lower HBV DNA levels in the liver (P = 0.004). In particular, the CTGAC mutation showed the strongest decrease of the viral load (P = 0.007). By contrast, nt1762/1764 mutation correlated with increased HBcAg (P = 0.005) and higher HBV DNA levels (P = 0.006). The mutants with the wild-type of nt1726-1730 or nt1762/1764 mutation were more prevalent in hepatocellular carcinoma (HCC) patients than in CHB patients. Although the mutations did not correlate with cirrhosis, the frequency of nt1762/1764 mutation in patients with hepatocarcinogenesis was significantly higher than in those without hepatocarcinogenesis (P = 0.011). Mutations at nt1726-1730 and nt1762/1764 are associated with in situ expression of HBcAg and viral load. Higher HBV DNA levels in the liver may be associated with hepatocarcinogenesis. Mutation at nt1762/1764 remarkably increases the risk of hepatocarcinogenesis.

[Chronic hepatitis B virus infection and the methylation status of p16INK4A promoter].

OBJECTIVE: To explore the relationship among HBV-associated histopathological indexes, x gene mutations and the methylation status of p16INK4A promoter in liver with chronic hepatitis B virus infection, in order to illustrate their role in p16INK4A hypermethylation and HCC progression. METHODS: Twenty-three cases of surgically resected HBV-associated hepatocellular carcinoma and twenty-five fine needle aspiration biopsy cases of chronic hepatitis B were chosen for this study. The methylation status of the p16INK4A promoter in tumors, their corresponding peritumoral samples and chronic hepatitis B cases was determined by methylation-specific polymerase chain reaction (MSP). EnVision two-step immunohistochemical staining showed the expression of viral antigens in situ. Tissue HBV DNA levels were determined by real-time fluorescence quantitative PCR. Polymerase chain reaction and the direct sequencing method was used for mutation analysis of HBV x gene. RESULTS: In peritumoral samples (P = 0. 025) and chronic hepatitis B cases (P = 0.029), the expression of HBx protein in methylated groups was all significantly higher than that in unmethylated groups of p16INK4A gene. But in tumors, there was no such significant difference. Other HBV antigens including HBsAg and HBcAg, tissue HBV DNA levels and point mutations of HBV x gene did not show a relationship with the methylation status of p16INK4A gene. CONCLUSION: The data suggest that p16INK4A hypermethylation correlated closely with higher HBx expression in precancerous lesions. HBx may play an important role in the early stage of HBV-associated hepatocarcinogenesis via induction of hepermethylation of p16INK4A promoter.

Association of p16INK4A hypermethylation with hepatitis B virus X protein expression in the early stage of HBV-associated hepatocarcinogenesis.

The aim of the present study was to explore the relationship between methylation status of the p16(INK4A) promoter and some HBV-related factors, and the role of these factors in p16(INK4A) hypermethylation and hepatocellular carcinoma (HCC) progression. Twenty-three cases of surgically resected HBV-associated HCC and 25 fine-needle aspiration biopsy cases of chronic hepatitis B (CHB) were studied. The methylation status of the p16(INK4A) promoter was determined by methylation-specific polymerase chain reaction (PCR). Two-step immunohistochemical staining showed the expression of viral antigens in situ. Tissue HBV-DNA levels were determined by fluorescence quantitative real-time PCR. PCR and the direct sequencing method were used for mutation analysis. In peritumoral tissues (P = 0.025) and CHB samples (P = 0.029), the expression of hepatitis B virus X protein (HBx) was higher in methylated groups of p16(INK4A) promoter than in unmethylated groups. Other HBV factors including hepatitis B surface antigen and hepatitis B core antigen, tissue HBV-DNA levels and HBV x gene mutations had no relation to the methylation status of p16(INK4A) promoter. The data indicate that p16(INK4A) promoter hypermethylation correlated closely with higher HBx expression in the precancerous lesions, suggesting that HBx may play an important role in the early stage of HBV-associated hepatocarcinogenesis via induction of hypermethylation of p16(INK4A) promoter.

Roles of EGFR-Stat3 signal pathway in carcinogenesis of experimental hepatoma in rats.

The purpose of this study is to investigate if the EGFR-Stat3 signal pathway contributes to the carcinogenesis of hepatoma in rats. Hepatoma was induced in rats by 3'Me-DAB as a model. EGFR, TGFalpha, Stat3, p-Stat3 in different stages of carcinogenesis were detected by immunohistochemistry and Western blot. In situ hybridization was applied to investigate the expression of Stat3 mRNA. The expressions of signal molecules were assessed by KS400 Image Analysis system. The data were statistically evaluated. EGFR, TGFalpha, Stat3 were highly expressed in the stages of liver necrosis and repairment. All hepatocellular carcinoma cases revealed elevated expression of EGFR, TGFalpha. Elevation of Stat3 mRNA and protein levels were identified, increase of activation of Stat3 was also observed. In HCC, there was positive correlation between p-Stat3 level and the expression of TGFalpha and PCNA. Increased expression of Bcl-2 (P < 0.05) coincided with elevated level of p-Stat3. Therefore, the EGFR-Stat3 signal pathway was related to the development of hepatoma in rats. TGFalpha-EGFR autocrine ring formation may lead to the activation of Stat3 and in turn, promote proliferation and regulate the transcription of genes regulating cell apoptosis and cell cycle.

[Expression and significance of hTERT mRNA in breast carcinoma and its relation to p53].

OBJECTIVE: This study was designed to investigate the significance of hTERT mRNA in breast carcinogenesis and to explore the diagnostic efficacy, and to study the effect of tumor suppressor gene p53 on the expression of hTERT mRNA. METHODS: The expression of hTERT mRNA was examined by in situ hybridization in 12 cases of normal breast tissue nearby cancer, 7 of simple ductal hyperplasia, 20 of atypical hyperplasia, 18 of ductal carcinoma in situ and 25 with invasive ductal carcinoma. The expression of p53 protein were examined by immunohistochemistry in 43 carcinomas. RESULTS: hTERT was not detected in normal breast tissue nearby cancer and simple ductal hyperplasia. The positive rate of hTERT mRNA in atypical hyperplasia, ductal carcinoma in situ and invasive ductal carcinoma were 25.0%, 83.3% and 88.0%, respectively. The prevalence and intensity of hTERT mRNA expression were much greater in carcinoma than those in simple or atypical hyperplasia and normal breast tissue nearby cancer (P < 0.05). The expression of hTERT was not correlated with tumor size and lymph node metastasis (P > 0.05). The positive correlation between hTERT mRNA and p53 was found in breast carcinoma (r = 0.5540, P < 0.01). CONCLUSION: hTERT mRNA expression is closely related to the malignant transformation of breast tissue. Semi-quantitative detection of hTERT mRNA expression in situ is helpful in differentiated diagnosis of carcinoma in situ and atypical hyperplasia. Inactivation of p53 may play a role in the transcriptive activation of hTERT gene in breast carcinoma.

In situ detection of TGF betas, TGF beta receptor II mRNA and telomerase activity in rat cholangiocarcinogenesis.

AIM: Initial report on the in situ examination of the mRNA expression of transforming growth factor betas (TGFbetas), TGFbeta type II receptor (TbetaRII) and telomerase activity in the experimental rat liver tissue during cholangiocarcinogenesis. METHODS: Rat liver cholangiocarcinogenesis was induced by 3'-methyl 4-dimethylazobenzene (3'Me-DAB). In situ hybridization was used to examine the TGFbetas) and TGFbeta type II receptor (TbetaRII) mRNA, in situ TRAP was used to check the telomerase activity in the tissue samples. RESULTS: There was no TGFbetas, TbetaRII mRNA expression or telomerase activity in the control rat cholangiocytes. The expression of TGFbeta1, TbetaRII was increased in regenerative, hyperplastic, dysplastic cholangiocytes and cholangiocarcinoma (CC) cells. The expression of TGFbeta2 mRNA was observed in only a part of hyperplastic, dysplastic cholangiocytes. TGFbeta3 expression was very weak, only in hyperplastic lesion. There was positive telomerase activity in the regenerative, hyperplastic, dysplastic cholangiocytes, and CC cells. Stroma fibroblasts of these lesions also showed positive TGFbetas, TbetaRII mRNA expression and telomerase activity. CONCLUSION: There were TGFbetas, TbetaRII expression and telomerase activity in hyperplastic, dysplastic cholangiocytes, cholangiocarcinoma cells as well as in stroma fibroblasts during cholangiocarcinogenesis. Their expression or activity is important in cholangiocarcinogenesis andstroma formation.