Hsuan-Shu Lee

Aflatoxin exposure and risk of hepatocellular carcinoma in Taiwan

To investigate the carcinogenic effect of environmental aflatoxin exposure, 56 cases of hepatocellular carcinoma (HCC) diagnosed between 1991 and 1995 were identified and individually matched by age, sex, residence and date of recruitment to 220 healthy controls from the same large cohort in Taiwan. Blood samples were analyzed for hepatitis B and C viral markers and for aflatoxin‐albumin adducts; urine was tested for aflatoxin metabolites. We obtained information about socio‐demographic characteristics, habitual alcohol drinking, cigarette smoking and diet in a structured interview. Hepatitis B virus surface antigen (HBsAg) carriers had a significantly increased risk for HCC. After adjustment for HBsAg serostatus, the matched odds ratio (ORm) was significantly elevated for subjects with high levels of urinary aflatoxin metabolites. When stratified into tertiles, a dose‐response relationship with HCC was observed. The ORm for detectable aflatoxin‐albumin adducts was not significant after adjustment for HBsAg serostatus. HBsAg‐seropositive subjects with high aflatoxin exposure had a higher risk than subjects with high aflatoxin exposure only or HBsAg seropositivity only. In male HBsAg‐seropositive subjects, adjusted ORs were 2.8 (95% confidence interval [Cl] = 0.9–9.1) for detectable compared with non‐detectable aflatoxin‐albumin adducts and 5.5 (Cl = 1.3–23.4) for high compared with low urinary aflatoxin metabolite levels. Our results suggest that environmental aflatoxin exposure may enhance the hepatic carcinogenic potential of hepatitis B virus. A large‐scale study will be needed to evaluate the effect of aflatoxin exposure on HBsAg non‐carriers.

Estimation of Seroprevalence of Hepatitis B Virus and Hepatitis C Virus in Taiwan from a Large-scale Survey of Free Hepatitis Screening Participants

BACKGROUND/PURPOSE Taiwan is a hyperendemic area of liver diseases. Hepatitis B virus (HBV) and hepatitis C virus (HCV) are the two major etiologies of liver diseases in Taiwan. This study investigated the seroprevalence of HBV and HCV in Taiwan. METHODS Since 1996, a series of outreach community-based screening programs for liver diseases have been available to the general population aged > or = 18 years. Blood samples were obtained from the subjects and sent for hepatitis B surface antigen (HBsAg) and antibody to HCV (anti-HCV) tests. RESULTS The prevalence of HBsAg(+) was 17.3% (27,210/157,720), while the prevalence of anti-HCV(+) was 4.4% (6904/157,720). Geographic variation in HBV and HCV seroprevalence was found, with the highest anti-HCV positive rate in Miaoli County, Chiayi County, Chiayi City, and Yunlin County, and the highest HBsAg positive rate in Keelung City and Yilan City. The HBsAg positive rate progressively decreased after the age of 50 years, while the anti-HCV positive rate progressively increased after the age of 20 years. The estimated total number of HBsAg carriers in the general population > 20 years old is 3,067,307, while the estimated number of anti-HCV positive patients is 423,283. CONCLUSION This study estimated a 17.3% seroprevalence of HBV and a 4.4% seroprevalence of HCV in Taiwan. Significant geographic variations in the seroprevalence of HBV and HCV were found. These data suggest the importance of modifying programs for the prevention and treatment of chronic viral hepatitis in Taiwan to reflect its varying prevalence and epidemiology.

Hepatitis C and B viruses in hepatitis B Surface antigen-negative hepatocellular carcinoma

The relative role of hepatitis C virus and hepatitis B virus in hepatitis B surface antigen-negative hepatocellular carcinoma was evaluated by polymerase chain reaction in 31 patients from Taiwan. Twenty-one were positive for antibody to hepatitis C virus (group 1) and 10 were negative (group 2). Of the group 1 patients, hepatitis C viral RNA was detected in the serum by polymerase chain reaction in 16 and in the liver tissue in 17, whereas hepatitis B viral DNA was found in the liver tissue in only 4, and none were found in the serum. In group 2 patients, hepatitis C viral RNA was detected in the serum of 1 and in the liver tissue of another. In contrast, hepatitis B viral DNA was found in the serum of 4 patients and in the liver tissues of 5. It was concluded that hepatitis C virus plays an important role in hepatocarcinogenesis in hepatitis B surface antigen-negative patients in Taiwan, especially in those who had antibody to hepatitis C virus; in those without antibody to hepatitis C virus, hepatitis B virus might still be associated with the development of hepatocellular carcinoma in a significant proportion of such patients.

Ultrasound screening and risk factors for death from hepatocellular carcinoma in a high risk group in Taiwan.

Although previous studies have demonstrated the ability of ultrasonography (US) screening to detect small asymptomatic hepatocellular carcinoma (HCC), the efficacy of US screening in reducing deaths from HCC still remained unresolved. A 2‐stage screening program was designed to identify a high risk group in 7 townships in Taiwan by 6 markers (of risk for HCC) and repeated US screening was further applied to those with at least 1 positive result for the 6 markers, with a range of 3‐ to 6‐month inter‐screening intervals to those with liver cirrhosis or other chronic liver diseases and an annual screening regime for the remaining subjects with normal findings according to US. The 4,843 subjects in this cohort were followed up for an average of 7 years. We compared 4,385 attenders with 458 non‐attenders, in conjunction with baseline assessment for self‐selection bias. In addition, we assessed baseline variables with respect to their effects on risk of incidence of and mortality from HCC and on risk of incidence of liver cirrhosis. The difference in mortality between attenders and non‐attenders was then re‐estimated adjusting for significant predictors of cirrhosis, HCC incidence and HCC death as a further guard against baseline differences between attenders and non‐attenders in risk profiles. Results of US screening for this high risk group found the mortality was lower by 24% (95% CI: −52 to 62%) in the attenders compared to the non‐attenders. After adjustment for sensitivity, the mean sojourn time (MST) were 1.57 (95% CI: 0.94–4.68) for subjects with liver cirrhosis and 2.66 (95% CI: 1.68–6.37) years for non‐cirrhotic patient. Significant increases in risk of HCC incidence were associated with increasing age, male gender, hepatitis B surface antigen positive (HbsAg), hepatitis C antibody positive (Anti‐HCV), high levels of alanine transaminase (ALT) and alpha‐fetoprotein (AFP) and a family history of HCC. Significantly increased risks of liver cirrhosis were associated with predictors of cirrhosis were increasing age, HbsAg, high levels of ALT and of AFP. Significant or borderline significant increases in risk of HCC death were associated with increasing age, male gender, HbsAg, high levels of AST and AFP. Adjusted for the significant variables, the mortality was lower by 41% (95% CI: −20 to 71%, p = 0.1446) in the attenders compared to the non‐attenders. The present study provides suggestive evidence on the efficacy of US screening in a selective high risk group in an endemic area of hepatitis B. A randomized controlled trial would yield definitive evidence. Within the protocol of such a trial, a shorter interscreening interval for patients with liver cirrhosis is suggested.

Evaluation of articular cartilage repair using biodegradable nanofibrous scaffolds in a swine model: a pilot study

The aim of this study was to evaluate a cell‐seeded nanofibrous scaffold for cartilage repair in vivo. We used a biodegradable poly(ε‐caprolactone) (PCL) nanofibrous scaffold seeded with allogeneic chondrocytes or xenogeneic human mesenchymal stem cells (MSCs), or acellular PCL scaffolds, with no implant as a control to repair iatrogenic, 7 mm full‐thickness cartilage defects in a swine model. Six months after implantation, MSC‐seeded constructs showed the most complete repair in the defects compared to other groups. Macroscopically, the MSC‐seeded constructs regenerated hyaline cartilage‐like tissue and restored a smooth cartilage surface, while the chondrocyte‐seeded constructs produced mostly fibrocartilage‐like tissue with a discontinuous superficial cartilage contour. Incomplete repair containing fibrocartilage or fibrous tissue was found in the acellular constructs and the no‐implant control group. Quantitative histological evaluation showed overall higher scores for the chondrocyte‐ and MSC‐seeded constructs than the acellular construct and the no‐implant groups. Mechanical testing showed the highest equilibrium compressive stress of 1.5 MPa in the regenerated cartilage produced by the MSC‐seeded constructs, compared to 1.2 MPa in the chondrocyte‐seeded constructs, 1.0 MPa in the acellular constructs and 0.2 MPa in the no‐implant group. No evidence of immune reaction to the allogeneically‐ and xenogeneically‐derived regenerated cartilage was observed, possibly related to the immunosuppressive activities of MSCs, suggesting the feasibility of allogeneic or xenogeneic transplantation of MSCs for cell‐based therapy. Taken together, our results showed that biodegradable nanofibrous scaffolds seeded with MSCs effectively repair cartilage defects in vivo, and that the current approach is promising for cartilage repair. Copyright

Ultrasound-guided cutting biopsy for the diagnosis of hepatocellular carcinoma — a study based on 420 patients

BACKGROUND/AIMS To evaluate ultrasound-guided cutting biopsy for hepatocellular carcinoma, we report findings from 10 years of experience. METHODS We performed 455 ultrasound-guided cutting biopsies of hepatic tumors in 420 patients with hepatocellular carcinoma from 1981 to 1990. RESULTS Liver tissues were adequately sampled for a histological diagnosis of hepatocellular carcinoma in the initial biopsy in 391 sessions. The remaining 64 were proved to have hepatocellular carcinoma after subsequent studies. Ultrasound-guided biopsy changed the initial diagnosis in 9 of the 420 patients: three had been diagnosed with liver abscess, and six with metastatic liver tumors. Complications of the biopsy were rare: the tumor had spread to the chest wall in nine, and internal bleeding was noted in five patients. There was no mortality and no other sequelae. CONCLUSIONS Ultrasound-guided biopsy of hepatic tumors is important in the diagnosis of liver cancer, but this technique should be applied only when the image diagnosis and results of fine needle biopsy are equivocal to minimize possible complications. For patients with small HCCs, who are candidates for surgical resection of hepatocellular carcinoma or liver transplantation, it should not be considered as a first-step invasive procedure.

Multipotential mesenchymal stem cells from femoral bone marrow near the site of osteonecrosis.

Stem cell‐based therapies for degenerative disorders and injuries are promising in the new era. Multipotential mesenchymal stem cells (MSCs) from bone marrow (BM) are on the leading edge because they are easy to expand in culture while maintaining their multilineage potential. In vitro assessment of the chondrogenic and osteogenic potentials of cultured MSCs has been established, and the BM used in those experiments was exclusively from healthy donors via iliac crest aspiration. It is unknown whether human marrow obtained from femurs also contains these multipotential MSCs. We collected marrow from proximal femurs of two patients undergoing total hip replacement surgery for femoral head osteonecrosis and isolated and culture expanded MSCs to about 20 population doublings. These cells were homogeneously positive for β1‐integrin. When pelleted into aggregates and cultured in a medium containing transforming growth factor‐β3 for 14 days, the cells began to express mRNA for aggrecan and collagen type II and to deposit immunoreactive collagen type II and sulfated proteoglycans in the matrix, hallmarks of chondrogenic differentiation. These MSCs could also be differentiated into osteocytic lineage in vitro, as shown by increased expression of alkaline phosphatase activity and deposition of mineral content onto culture plates. These results indicate that femoral BM obtained during hip surgeries also contained multipotential MSCs. These data imply that direct replacement therapy using MSCs from in situ marrow may be possible in the future and that an MSC bank may be established by using marrow from this approach, bypassing the necessity for iliac marrow aspiration from healthy donors.

Dynamic contrast-enhanced magnetic resonance imaging with Gd-EOB-DTPA for the evaluation of liver fibrosis in chronic hepatitis patients

AbstractObjectivesTo develop a non-invasive MRI method for evaluation of liver fibrosis, with histological analysis as the reference standard.MethodsThe study protocol was approved by the Institutional Review Board for Human Studies of our hospital, and written informed consent was obtained from all subjects. Seventy-nine subjects who received dynamic contrast-enhanced MRI (DCE-MRI) with Gd-EOB-DTPA were divided into three subgroups according to Metavir score: no fibrosis (n = 30), mild fibrosis (n = 34), and advanced fibrosis (n = 15). The DCE-MRI parameters were measured using two models: (1) dual-input single-compartment model for arterial blood flow (Fa), portal venous blood flow, total liver blood flow, arterial fraction (ART), distribution volume, and mean transit time; and (2) curve analysis model for Peak, Slope, and AUC. Statistical analysis was performed with Student’s t-test and the nonparametric Kruskal-Wallis test.ResultsSlope and AUC were two best perfusion parameters to predict the severity of liver fibrosis (>F2 vs. ≦F2). Four significantly different variables were found between non-fibrotic versus mild-fibrotic subgroups: Fa, ART, Slope, and AUC; the best predictor for mild fibrosis was Fa (AUROC:0.701).ConclusionsDCE-MRI with Gd-EOB-DTPA is a noninvasive imaging, by which multiple perfusion parameters can be measured to evaluate the severity of liver fibrosis. Key Points • Dynamic Gd-EOB-DTPA contrast-enhanced-MRI can help evaluate the severity of liver fibrosis.• Slope and AUC were two best perfusion parameters to predict severity.• Absolute arterial blood flow was the best predictor for mild fibrosis.

Differential DNA Methylation Associated with Hepatitis B Virus Infection in Hepatocellular Carcinoma

Gene inactivation through DNA hypermethylation plays a pivotal role in carcinogenesis. This study aimed to profile aberrant DNA methylation in different stages of liver disease, namely noncirrhosis, cirrhosis and hepatocellular carcinoma (HCC), and also to clarify the influence of hepatitis B virus (HBV) infection on the aberrant DNA methylation in HCCs. Promoter methylation in p14ARF, p16INK4a, O6‐methylguanine‐DNA methyltransferase (MGMT), glutathione S‐transferase pi (GSTP1) and E‐cadherin (E‐Cad) genes of 58 HCCs paired with adjacent nontumorous tissues was assayed by methylation‐specific PCR. HBV infection was determined using a hepatitis B virus surface antigen (HBsAg) serological assay. The frequency of p16INK4a promoter methylation increased from noncirrhotic, cirrhotic, to HCC tissues (noncirrhotic vs. HCC, p < 0.001), while that of GSTP1 promoter methylation increased in cirrhotic tissues compared to noncirrhotic ones (p = 0.029). The frequency of GSTP1 promoter hypermethylation is significantly higher in HCC than in nontumorous tissues (p = 0.022) from HBsAg‐positive patients, but not the HBsAg‐negative controls (p = 0.289). While the frequency of E‐Cad promoter hypermethylation remained high in both nontumorous tissues and HCCs from HBsAg‐positive patients (p = 0.438), it was lower in HCCs than in nontumorous tissues from HBsAg‐negative patients (p = 0.002). In contrast, the frequency of p16INK4a, MGMT and p14ARF promoter hypermethylation in HCCs was unrelated to HBsAg status. In conclusion, aberrant DNA methylation may begin at different stages of liver disease in a gene‐dependent manner. Moreover, HBV infection may enhance or maintain GSTP1 and E‐Cad promoter methylation and thereby affect hepatocarcinogenesis.

Green tea extract supplement reduces D-galactosamine-induced acute liver injury by inhibition of apoptotic and proinflammatory signaling.

Oxidative stress and inflammation contributed to the propagation of acute liver injury (ALI). The present study was undertaken to determine whether D-galactosamine (D-GalN) induces ALI via the mitochondrial apoptosis- and proinflammatory cytokine-signaling pathways, and possible mechanism(s) by which green tea (GT) extract modulates the apoptotic and proinflammatory signaling in rat. D-GalN induced hepatic hypoxia/hypoperfusion and triggered reactive oxygen species (ROS) production from affected hepatocytes, infiltrated leukocytes, and activated Kupffer cells. D-GalN evoked cytosolic Bax and mitochondrial cytochrome C translocation and activated proinflammatory nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1) translocation, contributing to the increase of intercellular adhesion molecule-1 expression, terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL)-positive hepatocytes, multiple plasma cytokines and chemokines release, and alanine aminotransferase (ALT) activity. An altered biliary secretion profile of several acute phase proteins directly indicates oxidative stress affecting intracellular trafficking in the hepatocyte. GT pretreatment attenuated ROS production, mitochondrial apoptosis- and proinflammatory cytokine-signaling pathway, plasma ALT and cytokines levels, biliary acute phase proteins secretion and hepatic pathology by the enhancement of anti-apoptotic mechanisms. In conclusion, D-GalN induced ALI via hypoxia/hypoperfusion-enhanced mitochondrial apoptosis- and proinflammatory cytokine-signaling pathway, contributing to oxidative stress and inflammation in the liver. GT can counteract the D-GalN-induced ALI via the attenuation of apoptotic and proinflammatory signaling by the upregulation of anti-apoptotic mechanism.