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 p14 ARF , p16 INK4a , O 6 -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 p16 INK4a 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 5 0.029). The frequency of GSTP1 promoter hypermethylation is significantly higher in HCC than in nontumorous tissues (p 5 0.022) from HBsAg-positive patients, but not the HBsAg-negative controls (p 5 0.289). While the frequency of E-Cad promoter hypermethylation remained high in both nontumorous tissues and HCCs from HBsAg-positive patients (p 5 0.438), it was lower in HCCs than in nontumorous tissues from HBsAg-negative patients (p 5 0.002). In contrast, the frequency of p16 INK4a , MGMT and p14 ARF 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. ' 2007 Wiley-Liss, Inc.

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 β β1 1-integrin. When pelleted into aggregates and cultured in a medium containing transforming growth factor-β 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. Stem

Intravital imaging of hepatobiliary excretion is vital for elucidating liver metabolism. In this work, we describe a novel method to observe the intravital dynamics of the uptake, processing, and excretion of an organic anion, 6-carboxyfluorescein diacetate (6-CFDA) in the hepatobiliary system. This is achieved by the use of multiphoton microscopy and an intravital hepatic imaging chamber. The high-quality images show sequential uptake and processing of 6-CFDA from the hepatocytes and the subsequent excretion into bile canaliculi within approximately 50 min. This is a promising technique to study intravital hepatic physiology and metabolism.

Although increases in collagen content (by hydroxyproline) approximately paralleled increases in fibrosis stage (by histology), there was considerable overlap and some discrepancies. Much work remains to be done to clarify the significance of the apparent huge variations in fibrosis within diseased livers. It is advisable for clinicians to interpret staging diagnoses by histological staging with some caution.

Lee HS. In vivo dynamic metabolic imaging of obstructive cholestasis in mice. Am J Physiol Gastrointest Liver Physiol 296: G1091-G1097, 2009. First published February 26, 2009 doi:10.1152/ajpgi.90681.2008.-We tried to image obstructive cholestasis by using a newly developed imaging system to measure the alterations of hepatobiliary function in living mice with their bile ducts ligated. A hepatic imaging window was installed on the upper abdomen soon after the mice underwent ligation of the common bile duct. On the next day, the mice received intravenous injection of rhodamine B isothiocyanate-dextran and carboxyfluorescein diacetate. The later would be transformed into fluorogenic carboxyfluorescein (detected at ϳ500 -550 nm) by hepatocytes and then excreted into bile canaliculi. The images were acquired by multiphoton microscopy. The fluorescence intensities at ϳ500 -550 nm within hepatocytes or sinusoids were measured in time series. In mice with bile duct ligation, bile canaliculi failed to appear during the whole observation period over 100 min following carboxyfluorescein diacetate injection, whereas the fluorescence was retained much longer within sinusoids. Furthermore, the fluorescence intensities in sinusoids were persistently higher than in hepatocytes during the course. Bile duct ligation impedes hepatocytes to excrete carboxyfluorescein into bile canaliculi. The kinetics of fluorescence intensities in hepatocytes and sinusoids indicated there is an active machinery operating backflow of this fluorogenic bile solute from hepatocytes into sinusoids in the liver with obstructive cholestasis. multiphoton microscopy; hepatocyte transporter; fluorescence imaging; common bile duct ligation