Kazuhiko Sugahara

NAD(P)H oxidase plays a crucial role in PDGF-induced proliferation of hepatic stellate cells.

The proliferation of hepatic stellate cells (HSCs) is a critical step in hepatic fibrogenesis. Platelet‐derived growth factor (PDGF) is the most potent mitogen for HSCs. We investigated the role of nonphagocytic NAD(P)H oxidase–derived reactive oxygen species (ROS) in PDGF‐induced HSC proliferation. The human HSC line, LI‐90 cells, murine primary‐cultured HSCs, and PDGF‐BB were used in this study. We examined the mechanism of PDGF‐BB‐induced HSC proliferation in relation to the role of a ROS scavenger and diphenylene iodonium, an inhibitor of NAD(P)H oxidase. We also measured ROS production with the aid of chemiluminescence. We showed that PDGF‐BB induced proliferation of HSCs through the intracellular production of ROS. We also demonstrated that HSCs expressed key components of nonphagocytic NAD(P)H oxidase (p22phox, gp91phox, p47phox, and p67phox) at both the messenger RNA and protein levels. Diphenylene iodonium suppressed PDGF‐BB–induced ROS production and HSC proliferation. Coincubation of H2O2 and PDGF‐BB restored the proliferation of HSCs that was inhibited by diphenylene iodonium pretreatment. Phosphorylation of the mitogen‐activated protein kinase (MAPK) family constitutes a signal transduction pathway of cell proliferation. Our data demonstrate that NAD(P)H oxidase–derived ROS induce HSC proliferation mainly through the phosphorylation of p38 MAPK. Moreover, an in vivo hepatic fibrosis model also supported the critical role of NAD(P)H oxidase in the activation and proliferation of HSCs. In conclusion, NAD(P)H oxidase is expressed in HSCs and produces ROS via activation of NAD(P)H oxidase in response to PDGF‐BB. ROS further induce HSC proliferation through the phosphorylation of p38 MAPK. (HEPATOLOGY 2005;41:1272–1281.)

Differentiation of bone marrow cells into cells that express liver-specific genes in vitro: implication of the Notch signals in differentiation

Bone marrow (BM) stem cells have been shown to differentiate into liver cells. It remains difficult to sort and culture BM stem cells, and the gene expression of liver-specific proteins in these cells has not been fully investigated. We used a negative selective magnetic cell separation system to obtain stem cell-enriched BM cells. The cells obtained were cultured with hepatocytes or with hepatocyte growth factor (HGF), and the differentiation of BM cells into cells expressing liver-specific genes, hepatocyte nuclear factor (HNF) 1alpha, cytokeratin (CK) 8, alpha-fetoprotein (AFP), and albumin was investigated by the reverse transcription-polymerase chain reaction. We also investigated the gene expressions of Notch receptor-1 (Notch-1) and its ligand Jagged-1 in BM cell differentiation. Sorted BM cells showed positive for Sca-1 (Ataxin-1) by immunofluorescence staining. Fluorescence activated cell sorter analysis showed that 32.6% of sorted BM cells had a high level of expression of the hematopoietic stem cell marker CD90 (Thy-1). When cultured with hepatocytes, these cells expressed the liver-specific genes HNF1alpha and CK8 on culture day 3, AFP and albumin on culture day 7. When cultured with HGF (20ng/ml), the cells expressed HNF1alpha on day 3 and CK8 on day 7. Gene expressions of Notch-1 and Jagged-1 were detected in cultured BM cells on day 3. These results suggest that the negative selective magnetic cell separation system is useful for the rapid preparation of stem cell-enriched BM cells, and that the Notch signaling pathway plays a role in BM cell differentiation into a hepatocyte lineage in vitro.

Characteristics of rat bone marrow cells differentiated into a liver cell lineage and dynamics of the transplanted cells in the injured liver

BackgroundBone marrow cells (BMCs) have been shown to differentiate into a liver cell lineage, but little is known about their dynamics following transplantation. BMCs were cultured to investigate the expression of liver-specific genes in vitro and transplanted into in vivo liver-injury models to elucidate their dynamics in the liver.MethodsThe mRNA expression of various liver-specific genes in BMCs cocultured with hepatocytes was analyzed using reverse transcription-polymerase chain reaction. BMCs from transgenic rats expressing green fiuorescent protein were transplanted into the spleen of rat liver-injury models induced with 2-acetylaminofiuorene (2-AAF) or carbon tetrachloride (CCl4). BMCs were also transplanted directly into livers treated with CCl4 to determine which route is better for transplantation.ResultsBMCs differentiated into a liver cell lineage in vitro and expressed mRNAs consistent with mature hepatocytes, including albumin. The transplanted BMCs were found in the liver in the CCl4-induced injury model, but not in the 2-AAF-induced model. The hepatocyte growth factor and fibroblast growth factor mRNA levels in the liver were significantly higher in the CCl4-induced model than in the 2-AAF-induced model. Migration of BMCs to the liver was more effective following injection into the liver, rather than into the spleen.ConclusionsCultured BMCs differentiated into a liver cell lineage are a potential source for cell transplantation. Transplantation is successful in the severely injured liver with a high level of expression of mRNAs for growth factors. Injection of BMCs directly into the liver is the preferred route of administration.

The sites for fatty acylation, phosphorylation and intermolecular disulphide bond formation of influenza C virus CM2 protein

The sites for fatty acylation, disulphide bond formation and phosphorylation of influenza C virus CM2 were investigated by site-specific mutagenesis. Cysteine 65 in the cytoplasmic tail was identified as the site for palmitoylation. Removal of one or more of three cysteine residues in the ectodomain showed that all of cysteines 1, 6 and 20 can participate in the formation of disulphide-linked dimers and/or tetramers, although cysteine 20 may play the most important role in tetramer formation. Furthermore, it was found that serine 78, located within the recognition motifs for mammary gland casein kinase and casein kinase I, is the predominant site for phosphorylation, although serine 103 is phosphorylated to a minor extent by proline-dependent protein kinase. The effects of acylation and phosphorylation on the formation of disulphide-linked oligomers were also studied. The results showed that, while palmitoylation has no role in oligomer formation, phosphorylation accelerates tetramer formation without influencing dimer formation. CM2 mutants defective in acylation, phosphorylation or disulphide bond formation were all transported to the cell surface, suggesting that none of these modifications is required for proper oligomerization. When proteins solubilized in detergent were analysed on sucrose gradients, however, the mutant lacking cysteines 1, 6 and 20 sedimented as monomers, raising the possibility that disulphide bond formation, although not essential for proper oligomerization, may stabilize the CM2 multimer. This was supported by the results of chemical cross-linking analysis, which showed that the triple-cysteine mutant can form multimers.

Highly sensitive hepatitis B surface antigen detection by measuring stable nitroxide radical formation with ESR spectroscopy.

In areas where hepatitis B virus (HBV) is prevalent, HBV carriers negative for hepatitis B surface antigen (HbsAg) by enzyme-linked immunosorbent assay (ELISA) have been reported. Moreover, even after screening donor blood for HbsAg and hepatitis B core antibody (HBcAb), post-transfusion hepatitis B continues to occur, though with a decreasing frequency. Therefore, screening tests far more sensitive for detecting HBsAg than those currently available are needed. We developed a highly sensitive method for HBsAg detection. It is based on the recognition of peroxidase activity through measuring the formation of stable nitroxide radical with electron spin resonance (ESR) spectroscopy in the presence of hydrogen peroxide, p-acetamidophenol (p-AP), and 4-hydrazonomethyl-1-hydroxy-2,2,5,5,-tetramethyl-3-imidazoline-3-o xide (HHTIO). A cut-off value was established by testing of 186 healthy adults and 50 HBsAg-positive individuals. The signal to noise (S/N) ratio of less than 1.488 obtained by ESR spectroscopy was considered to be negative and more than 2.181, positive. The p-AP/HHTIO method was found to be 10 times more sensitive than the standard ELISA and reproducibility was excellent. Additional investigations were made on the HBsAg levels in the serum from 26 healthy subjects, in whom cut-off index levels on ELISA were negative but relatively high (range: 0.6 to 1.0); and on 15 patients with non B non C hepatitis. Three of 26 cases and 3 of 15 with non B non C hepatitis were judged to be HBsAg positive. Of these, 5 were found to be positive for HBV DNA by polymerase chain reaction (PCR). It was shown in this study that the p-AP/HHTIO method is practical and useful in screening HBV carriers because of the sensitivity in HBsAg detection, which is comparable to PCR analysis.

What can be revealed by extending the sensitivity of HBsAg detection to below the present limit

BACKGROUND/AIMS We investigated what can be revealed by extending the sensitivity of HBsAg detection to below the present limit. METHODS We examined the sensitivity of this immunoassay in comparison with real-time PCR detection of HBV DNA using serially diluted sera from HBV carriers. Low HBsAg was measured in 210 healthy volunteers and 368 patients with non-B chronic liver diseases who were negative for HBsAg by a standard EIA method. RESULTS The radical immunoassay was able to detect HBsAg at a concentration of 0.025 ng/ml. Low HBsAg was positive in 6 of 210 normal volunteers (2.86%), 5 of 65 non-B, non-C cirrhosis patients (7.69%), 6 of 62 non-B, non-C hepatocellular carcinoma patients (9.68%: p=0.04 vs. volunteers), 12 of 134 chronic hepatitis C patients (8.96%: p<0.02 vs. volunteers), and 11 of 107 hepatocellular carcinoma patients complicated by chronic hepatitis C (10.28%: p<0.008 vs. volunteers). Although no HBV DNA was positive in healthy volunteers, 9 patients with non-B chronic liver diseases were positive for HBV DNA by real-time PCR analysis. CONCLUSIONS Increasing the sensitivity of HBsAg detection to below the present limit has revealed that infection with HBV, including occult HBV, is far more endemic than suspected previously.

Serum levels of stem cell factor and thrombopoietin are markedly decreased in fulminant hepatic failure patients with a poor prognosis.

Background and Aim:  Hematopoietic growth factors including stem cell factor (SCF), thrombopoietin (TPO) and granulocyte colony stimulating factor (G‐CSF) have a potential role in inducing bone marrow hematopoietic stem cells to move into the circulation, and the association of these factors with liver regeneration has received a lot of attention recently. The aim of this study was to determine the serum levels of such factors in patients with acute liver injury.

Risk of Hepatocellular Carcinoma and Secondary Structure of Hepatitis C Virus (HCV) NS3 Protein Amino-Terminus, in Patients Infected with HCV Subtype 1b

We conducted a retrospective study of 65 patients with chronic hepatitis C, to determine whether the secondary structure of the amino-terminal 120 residues of the hepatitis C virus (HCV) NS3 protein is associated with an increased risk of development of hepatocellular carcinoma (HCC). The cumulative incidence of HCC was highest among patients infected with group B HCV-1b, wherein the risk of HCC significantly increased compared with that among patients infected with group A (hazard ratio, 4.95 [95% CI, 1.43-17.11]) after adjustment for age and histological stage. This HCV-1b grouping may be a useful marker for detecting the risk of development of HCC.

Successful percutaneous radiofrequency ablation of adrenal metastasis from hepatocellular carcinoma.

To the Editor: Recently, radiofrequency ablation (RFA) has been widely used as therapy for hepatocellular carcinoma (HCC), because it is safe and only minimally invasive to the human body.1,2 However, the therapeutic efficacy of RFA for extrahepatic metastasis of HCC is still unclear. The adrenal gland is one of the organs often affected by hematogenous metastasis of HCC. As a recent report has indicated that percutaneous RFA is a safe and well-tolerated procedure for the treatment of unresectable adrenocortical carcinoma,3 it appears that RFA could also be applicable for the treatment of HCC metastasis to the adrenal gland. So far, surgical procedures have been employed for the removal of adrenal metastasis from HCC, but the optimal treatment regimen is still inconclusive. Here, we describe the use of ultrasonography (US)-guided percutaneous RFA for the treatment of a patient with adrenal metastasis of HCC. We found that this procedure allowed complete ablation of the metastasis without any severe adverse events. The patient was a 69-year-old woman suffering from Child’s class A, compensated cirrhosis due to hepatitis C. Computed tomography (CT) scan showed that an HCC, 20 mm in diameter, in the right lobe of the liver, had metastasized to the right adrenal gland and right portal vein, resulting in a 20-mm adrenal tumor mass and tumor emboli, respectively. The HCC was treated by arterial infusion of the chemotherapeutic agents, fluorouracil and cisplatin, according to the planned regimen, via a subcutaneously implanted injection port. Six months after the start of chemotherapy, CT imaging showed that both the primary HCC in the liver and the tumor emboli were reduced markedly, to an undetectable level, and the serum alpha-fetoprotein (AFP) level had a decreased from 29 740 ng/ml before therapy to 6280 ng/ml after. However, the metastatic lesion in the adrenal gland was found to have increased in diameter, to 40 mm (Fig. 1a). Histological examination of a biopsy sample of the adrenal tumor, taken using a 21-gauge needle, showed the features of well-differentiated HCC. Immunohistochemical analysis of the tumor cells revealed positive staining for AFP. After local anesthesia was carried out with intradermal and subcutaneous lidocaine (1%), percutaneous transhepatic thermoablation of the metastatic tumor was performed twice, under US guidance, using a cool-tip needle radiofrequency system (Radionics, Burlington, MA, USA), comprising a 17-gauge, 3-cm active-tip RF electrode and radiofrequency generator, with an allowable output power of 120 W for 12 min. The first RFA was performed on the upper side of the tumor, and the second RFA was performed on the lower side of the tumor. There were no procedure-related complications. Both the plasma cortisol and catecholamine levels were measured 1 week before and after RFA. The plasma cortisol levels before and after RFA were 13.6 and 10.8 mg/dl, respectively. The plasma catecholamine levels before and after RFA were 16.0 and 10.0 pg/ml (adrenaline), 188.0 and 94.0 pg/ml (noradrenaline), and 8.0 and 5.0pg/ml (dopamine), respectively. All levels of adrenal hormones were within the normal ranges. CT scan 6 months after the RFA showed that the adrenal metastatic HCC had been completely ablated and devascularized, with loss of contrast enhancement (Fig. 1b). The serum AFP level returned to the normal range, below 10 ng/ml, 4 months after the RFA. To our knowledge, this is the first report of the successful treatment of adrenal metastasis of HCC using percutaneous RFA. This procedure was safe and well-tolerated and allowed complete ablation of the localized adrenal metastasis in this patient. Recently, it has been reported that unintended injury to normal adrenal tissue during RFA of adrenal tumors can lead to hypertensive crisis, a potentially catastrophic complication.4 Therefore, we must pay sufficient attention to serious adverse

Mucosal sulfhydryl compounds evaluation by in vivo electron spin resonance spectroscopy in mice with experimental colitis.

Background: Sulfhydryl (SH) compounds are essential in maintaining mucosal integrity in the gastrointestinal tract. A decrease in colonic mucosal SH compounds affects the redox status of the mucosa, resulting in vulnerability to further attacks. Therefore, there is a strong need for in vivo evaluation of SH compounds in the colonic mucosa. Aims: The aim of the current study was to establish a method of evaluating levels of SH compounds in the colonic mucosa of live animals before and after induction of colitis. Methods: Murine experimental colitis was induced by instillation of trinitrobenzene sulphonic acid (TNBS) dissolved in 50% ethanol into the colon via the anus. For evaluation of mucosal SH compounds in the colon, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL), a stable nitroxide radical, was instilled into the colonic lumen of live mice and the spin clearance rate was measured by L-band electron spin resonance (ESR) spectroscopy. Results: Morphological study showed that mucosal damage was severe one or two days after TNBS instillation. The colonic mucosa started to regenerate at four days, and looked normal at seven days, after induction of colitis. The spin clearance rate of carbamoyl-PROXYL decreased significantly at 0.5, 1, 2, and 4 days after induction of colitis compared with mice before TNBS instillation. Surprisingly, although the colonic mucosa looked normal seven days after TNBS administration, the spin clearance rate still remained significantly slow. The spin clearance rate returned to normal 14 days after induction of colitis. The change in in vivo spin clearance rate was consistent with the time dependent change in mucosal reduced glutathione, a major component of SH compounds. Conclusion: The spin clearance rate obtained by L-band ESR spectroscopy in combination with carbamoyl-PROXYL can give an estimate of the level of colonic mucosal SH compounds in live animals and is useful for evaluating the mucosal defence system against oxidative stress.