Efficacy and safety of telaprevir, a new protease inhibitor, for difficult-to-treat patients with genotype 1 chronic hepatitis C.

The aims of this phase III study were to assess the efficacy and safety of telaprevir in combination with peginterferon alfa-2b (PEG-IFN) and ribavirin (RBV) for difficult-to-treat patients who had not achieved sustained virological response (SVR) to prior regimens in Japan. The subjects were 109 relapsers (median age of 57.0 years) and 32 nonresponders (median age of 57.5 years) with hepatitis C virus genotype 1. Patients received telaprevir (750 mg every 8 h) for 12 weeks and PEG-IFN/RBV for 24 weeks. The SVR rates for relapsers and nonresponders were 88.1% (96/109) and 34.4% (11/32), respectively. Specified dose modifications of RBV that differed from that for the standard of care were introduced to alleviate anaemia. RBV dose reductions were used for 139 of the 141 patients. The SVR rates for relapsers did not depend on RBV dose reduction for 20-100% of the planned dose (SVR rates 87.5-100%, P < 0.05). Skin disorders were observed in 82.3% (116/141). Most of the skin disorders were controllable by anti-histamine and/or steroid ointments. The ratios of discontinuation of telaprevir only or of all the study drugs because of adverse events were 21.3% (30/141) and 16.3% (23/141), respectively. A frequent adverse event leading to discontinuation was anaemia. Telaprevir in combination with PEG-IFN/RBV led to a high SVR rate for relapsers and may offer a potential new therapy for nonresponders even with a shorter treatment period.

Frequent silencing of RUNX3 in esophageal squamous cell carcinomas is associated with radioresistance and poor prognosis.

Radiotherapy is an effective treatment for some esophageal cancers, but the molecular mechanisms of radiosensitivity remain unknown. RUNX3, a novel tumor suppressor of gastric cancer, functions in transforming growth factor (TGF)-beta-dependent apoptosis. We obtained paired samples from 62 patients with advanced esophageal cancers diagnosed initially as T3 or T4 with image diagnosis; one sample was obtained from a biopsy before presurgical radiotherapy, and the other was resected in surgical specimens after radiotherapy. RUNX3 was repressed in 67.7% cases of the pretreatment biopsy samples and 96.7% cases of the irradiated, resected samples. The nuclear expression of RUNX3 was associated with radiosensitivity and a better prognosis than cytoplasmic or no RUNX3 expression (P<0.003); cytoplasmic RUNX3 expression was strictly associated with radioresistance. RUNX3 was downregulated and its promoter was hypermethylated in all radioresistant esophageal cancer cell lines examined. Stable transfection of esophageal cancer cells with RUNX3 slightly inhibited cell proliferation in vitro, enhanced the antiproliferative and apoptotic effects of TGF-beta and increased radiosensitivity in conjunction with Bim induction. In contrast, transfection of RUNX3-expressing cells with a RUNX3 antisense construct or a Bim-specific small interfering RNA induced radioresistance. Treatment with 5-aza-2'-deoxycytidine restored RUNX3 expression, increased radiosensitivity and induced Bim in both control and radioresistant cells. These results suggest that RUNX3 silencing promotes radioresistance in esophageal cancers. Examination of RUNX3 expression in pretreatment specimens may predict radiosensitivity, and induction of RUNX3 expression may increase tumor radiosensitivity.

Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues.

MicroRNAs (miRNAs) are a non-coding family of genes involved in post-transcriptional gene regulation. These transcripts are associated with cell proliferation, cell differentiation, cell death and carcinogenesis. We analysed the miRNA expression profiles in 25 pairs of hepatocellular carcinoma (HCC) and adjacent non-tumorous tissue (NT) and nine additional chronic hepatitis (CH) specimens using a human miRNA microarray. Targets and references samples were co-hybridized to a microarray containing whole human mature and precursor miRNA sequences. Whereas three miRNAs exhibited higher expression in the HCC samples than that in the NT samples, five miRNAs demonstrated lower expression in the HCC samples than in the NT samples (P<0.0001). Classification of samples as HCC or NT by using support vector machine algorithms based on these data provided an overall prediction accuracy of 97.8% (45/46). In addition, the expression levels of four miRNAs were inversely correlated with the degree of HCC differentiation (P<0.01). A comparison of CH and liver cirrhosis samples revealed significantly different pattern of miRNA expression (P<0.01). There were no differences, however, between hepatitis B-positive and hepatitis C-positive samples. This information may help clarify the molecular mechanisms involved in the progression of liver disease, potentially serving as a diagnostic tool of HCC.

Interleukin-8 production via protease-activated receptor 2 in human esophageal epithelial cells.

Interaction between proteases and protease-activated receptor (PAR) 2 has been proposed to mediate inflammatory and immune response in the gastrointestinal tract. Recently, increase in interleukin (IL)-8 in the esophageal mucosa has been associated with the pathogenesis of esophagitis induced by reflux of gastric acids, bile acids or trypsin. The aims of the present study were to determine PAR2 expression in normal human esophageal epithelial cells (HEEC) and to evaluate the mediation of IL-8 production by trypsin-PAR2 interaction in HEEC. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis revealed that PAR2 mRNA and protein were constitutively expressed in HEEC without upregulation by the stimulation with tumor necrosis factor alpha or trypsin. IL-8 was produced in a dose-dependent fashion when cells were stimulated with a PAR2 agonist such as trypsin or SLIGKV-amide. Blocking antibody to PAR2, camostat mesilate (a trypsin inhibitor), p-38 mitogen-activated protein kinase (MAPK) inhibitors or ERK1/2 inhibitors reduced IL-8 production from trypsin-stimulated HEEC. Mutation of the NFkappaB-, AP-1- and NF-IL-6-binding site on the IL-8 gene promoter abrogated the induction of luciferase activities stimulated with trypsin by 100, 80 and 50%, respectively. These results indicate that PAR2 activation in HEEC by trypsin induces NFkappaB- and AP-1-dependent IL-8 production in association with activation of p38 MAPK and ERK1/2, suggesting that esophageal inflammation may be induced by PAR2 activation via reflux of trypsin.

Outer dense fiber 2 is a widespread centrosome scaffold component preferentially associated with mother centrioles: its identification from isolated centrosomes.

Because centrosomes were enriched in the bile canaliculi fraction from the chicken liver through their association with apical membranes, we developed a procedure for isolation of centrosomes from this fraction. With the use of the centrosomes, we generated centrosome-specific monoclonal antibodies. Three of the monoclonal antibodies recognized an antigen of ~90 kDa. Cloning of its cDNA identified this antigen as a chicken homologue of outer dense fiber 2 protein (Odf2), which was initially identified as a sperm outer dense fiber-specific component. Exogenously expressed and endogenous Odf2 were shown to be concentrated at the centrosomes in a microtubule-independent manner in various types of cells at both light and electron microscopic levels. Odf2 exhibited a cell cycle-dependent pattern of localization and was preferentially associated with the mother centrioles in G0/G1-phase. Toward G1/S-phase before centrosome duplication, it became detectable in both mother and daughter centrioles. In the isolated bile canaliculi and centrosomes, Odf2, in contrast to other centrosomal components, was highly resistant to KI extraction. These findings indicate that Odf2 is a widespread KI-insoluble scaffold component of the centrosome matrix, which may be involved in the maturation event of daughter centrioles.

Identification of human cancer-related genes by naturally occurring Hepatitis B Virus DNA tagging.

Proviral tagging has been used in animals as a powerful tool for cancer genetics. We show that a similar approach is possible in patients with hepatocellular carcinoma (HCC) infected by Hepatitis B Virus (HBV), a human pararetrovirus which may act by insertional mutagenesis. In this work, the HBV genome is used as a probe to identify cancer-related genes. By using HBV-Alu-PCR, we obtained 21 HBV/cellular DNA junctions from 18 different patients. In six of 21, we found the HBV DNA integrated into a cellular gene: (1) Sarco/Endoplasmic Reticulum Calcium ATPase1 Gene; (2) Thyroid Hormone Receptor Associated Protein 150 alpha Gene; (3) Human Telomerase Reverse Transcriptase Gene; (4) Minichromosome Maintenance Protein (MCM)-Related Gene; (5) FR7, a new gene expressed in human liver and cancer tissues; and (6) Nuclear Matrix Protein p84 Gene. Seven junctions contained unique cellular sequences. In the remaining eight, the HBV DNA was next to repetitive sequences, five of them of LINE1 type. The cellular genes targeted by HBV are key regulators of cell proliferation and viability. Our results show that studies on HBV-related HCCs allow to identify cellular genes involved in cancer. We therefore propose this approach as a valuable tool for functional cancer genomic studies in humans.

A role of mast cells for hepatic fibrosis in primary sclerosing cholangitis.

We encountered four patients with overt primary sclerosing cholangitis (PSC) which were histologically classified into stage 2 or 3. We examined the expression of stem cell factor (SCF), a ligand of c-kit, in injured bile ducts by immunohistochemistry, and mast cells were identified by immunohistochemistry using anti-HMCT (human mast cell tryptase) and anti-c-kit antibodies to clarify their relation with portal fibrosis coincident with destroyed bile ducts. SCF was detected in the epithelia of most bile ducts in PSC, and many HMCT- and c-kit-positive mast cells were found in portal tracts. Image analysis showed more significant numbers of c-kit-positive mast cells per area of portal tract in PSC than in chronic hepatitis C, and they might increase from stage 2 to 3. c-Kit-positive cells infiltrated into the portal tracts with SCF-positive destroyed bile ducts, and c-kit mast cells should be investigated in detail to make a role for portal fibrosis in PSC.

Tyrosine kinase-dependent modulation by interferon-alpha of the ATP-sensitive K+ current in rabbit ventricular myocytes.

We examined the effects of interferon-alpha on the ATP-sensitive K+ current (IK,ATP) in rabbit ventricular cells using the patch-clamp technique. IK,ATP was induced by NaCN. Whole-cell experiments indicated that interferon-alpha (5 x 10(2) - 2.4 x 10(4) U/ml) inhibited IK,ATP in a concentration-dependent manner (60.7+/-7.5% with 2.4 x 10(4) U/ml). In cell-attached configuration, interferon-alpha (2.4 x 10(4) U/ml) applied to the external solution also inhibited the activity of the single ATP-sensitive K+ (KATP) channel by 56.0+/-5.8% without affecting the single channel conductance. The inhibitory effect of IK,ATP by interferon-alpha was blocked by genistein and herbimycin A, tyrosine kinase inhibitors, but was not affected by N-(2-metylpiperazyl)-5-isoquinolinesulfoamide (H-7), an inhibitor of protein kinase C and cAMP-dependent protein kinase. These findings suggest that interferon-alpha inhibits the cardiac KATP channel through the activation of tyrosine kinase. The tyrosine kinase-mediated inhibition of IK,ATP by cytokines may aggravate cell damage during myocardial ischemia.

Vitamin K concentrations in the plasma and liver of surgical patients.

Vitamin K deficiency has been reported in patients who were treated with antibiotics and placed on poor diets after surgery. High-performance liquid chromatography (HPLC) was used to study the influence of dietary intake on vitamin K concentrations in surgical patients (n = 22). Plasma phylloquinone decreased rapidly from 1.19 +/- 0.16 to 0.47 +/- 0.12 nmol/L (means +/- SEM, n = 11) on a low-phylloquinone diet and from 1.16 +/- 0.12 to 0.36 +/- 0.07 nmol/L (n = 11) by postoperative fasting. A small amount of phylloquinone and a large amount of menaquinone were found in liver tissue. Phylloquinone concentration was 28.0 +/- 4.3 pmol/g liver (wet weight) on the standard diet (n = 7) whereas it was 6.8 +/- 1.1 pmol/g on the low-phylloquinone diet after 3 d (n = 8). Because phylloquinone is rapidly depleted by fasting, it may be difficult to prevent vitamin K deficiency by dietary phylloquinone alone during long-term fasting after surgery.

Highly efficient suicide gene expression in hepatocellular carcinoma cells by epstein-barr virus-based plasmid vectors combined with polyamidoamine dendrimer.

The present study was aimed at devising an efficient nonviral strategy for suicide gene therapy of hepatocellular carcinoma (HCC). To improve the efficiency of DNA delivery and expression, we applied Epstein-Barr virus (EBV)-based plasmid vectors instead of conventional plasmid vectors and combined them with cationic liposome (EBV/lipoplex) or polyamidoamine dendrimer (PAAD) (EBV/polyplex). When the beta-galactosidase gene was transferred to HuH7, PLC/PRF/5, or HLE cells, < or =50-fold higher beta-galactosidase activities were demonstrated in the cells transfected with EBV vector compared with those transfected with conventional plasmid vectors. PAAD-mediated transfection of HCC with pSES.Tk (an EBV-based vector carrying the herpes simplex virus-1 thymidine kinase gene) resulted in a marked reduction in viable cell number by the addition of ganciclovir (GCV). The HCC cells transfected with pSES.Tk/PAAD showed 100- to 1000-fold higher susceptibilities to GCV than those transfected with pS.Tk (a conventional plasmid vector carrying herpes simplex virus-1 thymidine kinase gene)/PAAD. The pSES.Tk-transfected HCC cells were effectively killed by day 9 in culture with a clinically feasible concentration of GCV (25 microM), whereas the pS.Tk-transfected cells survived the culture. These results demonstrate highly efficient suicide gene transfer into various HCC cells by EBV-based plasmid vectors in vitro, suggesting the possible application of this nonviral vector system to gene therapy of HCC.

Targeted killing of carcinoembryonic antigen (CEA)-producing cholangiocarcinoma cells by polyamidoamine dendrimer-mediated transfer of an Epstein-Barr virus (EBV)-based plasmid vector carrying the CEA promoter.

The present study reports a novel nonviral method to efficiently and specifically target carcinoembryonic antigen (CEA)-producing cholangiocarcinoma (CC) cells in vitro. Epstein-Barr virus (EBV)-based and conventional plasmid vectors were constructed that possess the beta-galactosidase (beta-gal) or herpes simplex virus-1 (HSV-1) thymidine kinase (Tk) genes as well as tandem repeats of the human genomic sequence -82 to -42 bp from the transcriptional start site of the CEA gene. The plasmids were transfected by means of polyamidoamine dendrimer into CEA-positive (HuCC-T1) or -negative cell lines. Transfection of the conventional plasmid vector with the CEA promoter and beta-gal gene resulted in a very low or undetectable level of marker gene expression even in the CEA-positive cell line. Transferring the HSV-1 Tk gene by conventional plasmid did not affect the susceptibility of HuCC-T1 cells to ganciclovir. In marked contrast, strong beta-gal expression was specifically obtained in HuCC-T1 cells by transfecting the EBV-based plasmid in which the CEA promoter and a ubiquitous promoter (SRalpha) are employed to drive the EBV-encoded nuclear antigen 1 (EBNA1) and beta-gal genes, respectively (pTES.beta). Furthermore, CEA-positive but not -negative tumor cells were rendered highly susceptible to ganciclovir when transfected with the EBV-based vector that carries the CEA promoter-EBNA1 and SRalpha-HSV-1 Tk genes (pTES.Tk). These results strongly suggest that the EBV-based plasmid vector/cationic polymer system (EBV/polyplex) equipped with the CEA promoter provides an efficient nonviral method for the targeted gene therapy of CEA-producing malignancies.

The effect of rebamipide on Helicobacter pylori extract-mediated changes of gene expression in gastric epithelial cells.

BACKGROUND: Recent studies have shown that Helicobacter pylori affects intracellular signal transduction in host cells, leading to the activation of transcriptional factors and the induction of pro-inflammatory cytokines. On the other hand, rebamipide, an anti-gastritis and anti-ulcer agent, could scavenge reactive oxygen species and reduce interleukin-8 (IL-8) expression in gastric epithelial cells induced by H. pylori-stimulation through the attenuated activation of nuclear factor-kappaB (NF-kappaB). AIMS: In this study, we investigated the effects of rebamipide on gene expression in H. pylori-stimulated epithelial cells using DNA chip. METHODS: H. pylori water extract (HPE) was prepared from NCTC11637, the type strain of H. pylori. Total RNA was extracted from MKN45 cells, a human gastric cancer cell line, following HPE-stimulation with and without rebamipide for 3 h, and differences in gene expression profiles were observed using GeneChip and Human 6800 probe array. RESULTS: The GeneChip analysis demonstrated that 132 up-regulated genes and 873 down-regulated genes, such as growth factors, chemokines and transcription factors, were detected in MKN45 cells 3 h after stimulation of H. pylori. Among them, several genes, including bFGF, RANTES and MIP-2beta, were previously unknown to be expressed in H. pylori-stimulated human gastric cells. Rebamipide reduced expression of 119 genes encoding cytokines, growth factors and their receptors and transcription factors. CONCLUSIONS: These findings suggest that rebamipide could inhibit inflammatory reactions and tumour progression by modifying H. pylori infection-induced gene expression in gastric epithelial cells.

Pathology of acute hepatitis A in humans. Comparison with acute hepatitis B.

Little is known about the pathologic characteristics of viral hepatitis A in humans. The authors compared the histologic features in liver biopsy specimens taken within 30 days of the onset of illness from 15 patients with hepatitis A and 14 patients with acute hepatitis B. In both hepatitis A and B, liver cell damage and necrosis were diffusely located, with accentuation in the centrilobular and midzonal areas in which ballooning degeneration and variation in cytoplasmic staining quality were observed frequently. One case of epidemic hepatitis A showed prominent periportal liver cell necrosis with inconspicuous centrilobular liver cell alterations. Kupffer cell mobilization was mild in hepatitis A, but more striking in hepatitis B. The portal inflammation was more pronounced and rich in plasma cells in hepatitis A than in hepatitis B. In summary, there were no major differences in the pathologic features of acute hepatitis A and B as sampled within 30 days of the onset of illness.

Hepatic damage influences the decay of nitroxide radicals in mice--an in vivo ESR study.

To determine the role of the liver in the elimination of free radicals from the body, the clearance rate (K) of nitroxide radicals (Tempol) at the hepatic domain was compared with that at the pelvic domain of live mice, using L-band ESR spectroscopy. The reduction of Tempol in biopsy specimens (liver tissue and femoral muscle) and blood obtained from Tempol-treated mice was also monitored using X-band ESR spectroscopy. Results indicated that the reduction of nitroxide radicals was delayed in both the liver and peripheral tissues when the liver was damaged. The decrease in both blood supply and reductants in the damaged liver might be involved in delaying the reduction in the whole body, because the liver can reduce the radicals supplied via the blood from the peripheral tissues, and the reductants such as reduced, glutathione in the peripheral tissues are supplied from the liver.

Islet-cell hyperplasia causing hyperinsulinemic hypoglycemia in an adult.

We report a patient, a 23-year-old man, who had clinical and laboratory findings suggestive of insulinoma. Although imaging studies did not reveal any tumors in the pancreas, distal pancreatectomy was performed because the possibility of small insulinoma could not be completely excluded. Grossly, the surgically removed pancreas did not reveal any tumors. Microscopically, the pancreas exhibited islet cell hyperplasia and nesidioblastosis. To our knowledge, this is the first authentic reported case of islet-cell hyperplasia occurring in a Japanese adult.

Hemodynamic effects of combined treatment with somatostatin analogue (SMS 201-995) and low-dose isosorbide dinitrate on portal hypertension in conscious cirrhotic rats.

The authors investigated whether combined treatment with the somatostatin analogue, SMS 201-995, and low-dose isosorbide dinitrate enhanced the hemodynamic effects of the individual agents on rats with thioacetamide-induced cirrhosis. Four groups of cirrhotic rats received SMS 201-995 (0.1 microgram.min-1.kg-1), isosorbide dinitrate (10 micrograms.min-1.kg-1), both agents, or placebo, respectively. Hemodynamics were measured serially in conscious rats, using a radioactive microsphere method. SMS 201-995 reduced portal venous inflow 21 +/- 4% and portal pressure 17 +/- 3%. Isosorbide dinitrate decreased portal venous inflow 20 +/- 4%, by inducing splanchnic vasoconstriction mediated by low pressure baroreflexes, and this agent also decreased portal pressure, by 14 +/- 2%. Portal venous resistance rose 7.6 +/- 3% with isosorbide dinitrate alone, but decreased 18 +/- 4% with combination therapy. This effect may have been induced by the pronounced vasodilatory effect of isosorbide dinitrate on the venous vasculature, since the reflex splanchnic vasoconstriction that occurs with low-dose isosorbide dinitrate disappears when this agent is combined with SMS 201-995. The decrease in portal pressure was more marked (22 +/- 4%) and changes in systemic hemodynamics were milder with the combined treatment. It was concluded that combination therapy with SMS 201-995 and low-dose isosorbide dinitrate may be beneficial for portal hypertension in liver cirrhosis.

Activated hepatic stellate cells participate in liver fibrosis in a patient with transfusional iron overload.

We describe liver fibrosis caused by iron overload after a long history of blood transfusion in a patient with chronic renal failure. Pertinent laboratory data were: serum (s)-Fe 148 microg/dl; unsaturated iron binding capacity (UIBC) 14 microg/dl; s-ferritin 9350 ng/ml; human leukocyte antigen (HLA) A2, A24, B39, B55, Cw1, Cw7. Computed tomography revealed a high density in the liver, and laparoscopy revealed a brown liver. Liver histology showed bridging fibrosis from portal tracts. A heavy iron deposit was seen in Kupffer cells as well as in hepatocytes surrounded by fibrosis around the portal tracts. Immunocytochemistry revealed alpha-smooth muscle actin in many stellate cells distributed along the fibrotic area, and electron microscopy revealed infiltrating myofibroblastic stellate cells coexisting with collagen fibers around degenerated hepatocytes containing iron deposits. The findings are consistent with the notion that stellate cells play an important role in liver fibrogenesis in both genetic and transfusional iron overload hemochromatosis.

Identification of apoptotic hepatocytes in situ in rat liver after lead nitrate administration.

Apoptosis plays a major role in the regression of mitogen (lead nitrate)-induced hepatic hyperplasia. We compared the in situ end-labeling (ISEL) technique with the conventional detection of apoptotic bodies in this process. In hematoxylin and eosin (H&E) sections, apoptosis is usually recognizable by the presence of apoptotic bodies (apoptosis phase 2). Although the early phase of apoptosis (apoptosis phase 1) can be detected as a prekaryorrhectic appearance in H&E sections, it is difficult to detect and is easily overlooked. On the other hand, ISEL presents intense staining mainly in phase 1 and weak or negative staining in phase 2. Thus, simultaneous investigation by these two methods in two serial sections is the most reliable way to calculate the incidence of apoptosis and gives us precise information on the stages of apoptosis in situ. Since the colorized signals of ISEL are much easier to detect than apoptotic bodies in H&E sections, ISEL is particularly useful for liver tissues, where the incidence of apoptosis is low.

Evaluation of hepatic proliferative activity in chronic liver diseases and hepatocellular carcinomas by proliferating cell nuclear antigen (PCNA) immunohistochemical staining of methanol-fixed tissues.

The proliferative activity of chronic liver diseases and hepatocellular carcinomas (HCCs) was studied by PCNA immunohistochemistry. Human liver tissues were obtained by surgical operation or needle biopsy, and PCNA was detected by immunohistochemistry. PCNA-labelling indices (PCNA-LIs) of methanol-fixed tissues corresponded with the incidence of S-phase cells previously reported, whereas paraformaldehyde-fixed tissues showed extremely high PCNA-LIs in all specimens. Therefore, methanol-fixed tissues were used for evaluation. The PCNA-LIs of the methanol-fixed tissues were: normal liver 0.78 +/- 0.38%, chronic persistent hepatitis 1.06 +/- 0.86%, chronic aggressive hepatitis 2A 1.01 +/- 0.50%, chronic aggressive hepatitis 2B 4.20 +/- 1.79%, inactive cirrhosis 0.81 +/- 0.49%, active cirrhosis 1.96 +/- 0.93%, HCC of Edmondson's type I 4.83 +/- 1.98%, type II 6.65 +/- 1.69%, and type III 38.7 +/- 30.6%. PCNA-positive cells showed little specific distribution; in periportal areas in chronic hepatitis, at the margins of pseudolobules in cirrhosis, and throughout the tumor in HCC. These findings indicated that proliferative activity increased during the progression of chronic hepatitis, but that it decreased at the stage of cirrhosis. In chronic liver diseases, the PCNA-LIs reflected hepatitis activity. HCC showed higher proliferative activity than liver cirrhosis, and the histological grade was correlated with the PCNA-LI.