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Background Platelet (PLT) transfusion was the most practical way to increase patients’ PLT counts before invasive hepatic procedures such as radiofrequency ablation (RFA) for liver cancer. A novel drug that raises the PLT count by acting on the thrombopoietin receptor has recently become available. Methods Lusutrombopag 3mg was administered daily for 7 days to patients who underwent RFA for liver tumors with low PLT counts (− 1). We collected demographic data concerning the patients’ liver function and PLT counts. Results Lusutrombopag was administered to 91 patients, with a median age of 71 years (range 51–86). Forty-two patients had hepatitis C, 12 had hepatitis B, 21 had alcoholic liver disease, 11 had nonalcoholic steatohepatitis, and five had other diseases. The median Child-Pugh score was 7 (range 5–11). Thirty-seven patients had stage I tumors, 41 had Stage II, 12 had stage III, and one had stage IV. PLT count was elevated from 4.4x104 ± 1.4x104 to 8.6x104 ± 2.5x104 PLT µL− 1. Lusutrombopag administration prevented PLT transfusions in 84/91 patients (92%). No patient had bleeding complications after RFA. One had portal thrombosis after lusutrombopag administration. Patients who achieved PLT counts of > 50,000 PLT µL− 1 had higher PLT counts before lusutrombopag administration. The degree of splenomegaly did not affect the rate of PLT count elevation. There was no specific adverse effect by administrating lusutrombopag for patients with PLT counts of around 50,000 µL− 1 but > 50,000 µL− 1. Conclusions Lusutrombopag administration before RFA was safe and effective for hepatocellular carcinoma patients with low PLT counts.

A 78-year-old woman presenting with severe acute liver failure was admitted to our hospital. On screening for the etiology of acute liver failure, it was diagnosed as being due to idiopathic hypereosinophilic syndrome (eosinophil count reported as 4766/μL; 33.8% of the white blood cells). Her medical history included marked eosinophilia, as observed six months prior to this admission. Corticosteroid therapy was initiated. During the clinical course, duodenal perforation occurred but was managed promptly by appropriate surgery. A liver biopsy, following the initiation of corticosteroid therapy, revealed degenerating hepatic cells with mild eosinophilic infiltration. With corticosteroid therapy, the liver function improved.

Hydraulic flip is a common phenomenon that occurs in a short injector nozzle with a contraction flow which results in a separation flow without a wall reattachment. This phenomenon usually occurs in a gasoline direct injection (GDI) injector because the nozzle’s length-to-diameter ratio is low with high injection pressure. The efforts to investigate the hydraulic flip in a fuel injector nozzle have been paid in several studies. However, the investigation on the phenomenon in an actual GDI injector has not been well clarified yet since it is complicated to be quantitatively investigated due to its microscale and the flow location inside a solid non-transparent material. Besides, the hydraulic flip also affects the near-nozzle spray characteristics, but the previous studies are insufficient to explain the whole mechanism and characteristic despite their importance on the engine combustion quality. In this study, the hydraulic flip characteristic is investigated by varying the hydraulic flip thickness. The nozzle with a thinner hydraulic flip resulted from the application of hydro-grinding machining on the nozzle inlet so it forms a nozzle with a round inlet edge and less flow contraction. The characterization of internal flow and its correlation with the injected spray was done by investigating the effect of hydraulic flip thickness. The hydraulic flip phenomenon and the injected spray were captured by a high-speed visualization using a synchrotron X-ray to obtain a phase-contrast image. As a result, the hydraulic flip phenomenon was observed clearly in the tested actual GDI injector nozzles. The hydraulic flip thickness difference and how it affects the spray characteristics, i.e., velocity distribution, flow coefficients, turbulence intensity, spray dispersion, and SMD, were clarified. The results show that the thinner hydraulic flip leads to a higher discharge coefficient, lower turbulence intensity which results in slightly worse atomization, and a slightly narrower spray angle with more uniform spray velocity distribution.

The flash-boiling spray has become a timely topic of seeking the high mixture quality and clean combustion of the gasoline-direction-injection (GDI) engines. The flash-boiling spray, which has the enlarged individual spray cone angles and enhanced plume-plume interactions, are likely to favor the fuel-film formation, otherwise known as the nozzle tip wetting. Recently, the nozzle tip wetting has caused widespread concern because it is considered to be an essential source of particle emissions in GDI engines. In this study, an experimental investigation was proposed to clarify the possible influence of flash-boiling spray on nozzle tip wetting by taking advantage of the X-ray phase-contrast imaging (XPCI) technique. Due to the high brilliance, high energy, and low emittance of X-ray, the fuel flow inside the metal nozzle and spray characteristics at the nozzle exit was observed simultaneously, and results were discussed quantitatively to understand the underlying mechanism. It is found that the post-injected fuel caused by the needle bouncing is one primary source that results in tip wetting at non-flash-boiling conditions. Rising fuel temperature at non-flash-boiling conditions would not significantly affect spray dynamics and tip wetting. At flash-boiling conditions, tip wetting rises quickly during fuel injection, and a maximum appears under transitional flash-boiling conditions. This phenomenon can be understood by the enhanced droplet collision against the nozzle wall and boosted droplet evaporation under flash-boiling.

Early gastric cancer, especially cancer confined to the mucosa (stage T1a), is known to have a high cure rate with rare recurrence. We herein report the case of a 40-year-old female who initially presented with biliary tract dilatation, pancreatic duct dilatation and intestinal wall thickening 3 years after curative resection of pT1aN0 stage gastric cancer. The intestinal resection specimen revealed tumor cells spreading through the subserosa to the submucosa sparing mucosal membrane, which made exploratory laparotomy the only approach to confirm the diagnosis. It is always important to be aware of malignancy recurrence and clinicians should not hesitate to choose exploratory laparotomy to avoid any delay in the diagnosis and treatment.

Premixed charge compression ignition technology for diesel engines has been attracting increasing interest in recent years because of its ability to reduce nitrogen oxide emissions. At the same time, homogeneous charge compression ignition technology for gasoline engines also has been expected for high thermal efficiency and reduction in nitrogen oxide emissions. However, both methods have common challenges, such as limited part-load operation and ignition timing control. Additionally, under very low load conditions, thermal efficiency decreases due to the difficulty of providing steady combustion. In this study, a dual-fuel method has been applied to control the ignition timing and combustion duration. As a high ignitability fuel, diesel fuel was supplied to the direct injector, and as an inhibitor, gasoline was supplied to the port injector. Under very low load conditions, negative valve overlap and multiple fuel injection were applied. Diesel fuel injected during the negative valve overlap duration oxidised partially in the high-temperature environment with oxygen-contained exhaust gas of the previous cycle. The oxidation reaction of fuel during the main compression stroke was inhibited by the products from these reactions that occurred during negative valve overlap. Thus, it was possible to control the timing of the heat release immediately after top dead centre. As a consequence, high controllability of combustion and high thermal efficiency at low loads were achieved through independent control of the ratio of diesel fuel injected at negative valve overlap and the fractions of the two fuels.

AIM There have been no established predictors of the outcome on sorafenib therapy for hepatocellular carcinoma (HCC) patients. We aimed to establish a new prognostic model suitable for sorafenib in HCC. METHODS Among 465 HCC patients treated with sorafenib in 14 hospitals, we formed a training cohort with 270 patients at seven hospitals located in West Japan and a validation cohort with 167 patients at seven hospitals located in East Japan. In the training cohort, we examined the relationship between overall survival (OS) and pretreatment clinical factors, and structured a new prognostic model. We verified this model in the validation cohort and compared with four existing staging models. RESULTS Multivariate analysis demonstrated distant metastases, portal invasion, intrahepatic tumor burden of more than 50%, serum α-fetoprotein of 150 ng/dL or more, des-γ-carboxyprothrombin of 1200 mAU/mL or more, albumin of 3.5 g/dL or less and total bilirubin of more than 1.0 mg/dL were significant independent adverse prognostic factors. We calculated a Japan Red Cross (JRC) score with these factors and classified three groups: low-, intermediate- or high-risk. Their median OS were well stratified (18.0, 8.8 and 3.7 months, respectively, P

Background & Aims There have been no established predictive factors of responders to sorafenib in patients with unresectable hepatocellular carcinoma (HCC). This study aimed to investigate the factors predicting a good response to sorafenib in Japanese patients with HCC. Methods A total of 465 patients with unresectable HCC in the Japanese Red Cross Liver Study Group were treated with sorafenib between January 2008 and August 2013, and 316 patients with sufficient clinical data were analysed. To determine the factors predicting a good response, the relationships between radiological response and the following clinicopathological factors were analysed: age, gender, performance status, liver function, tumour status and decrease in serum alpha-foetoprotein (AFP) level after 1 month. Results This study included 259 males and 57 females with a median age of 70 years (range, 37–90 years), of which 191 (60.4%) were classified as Barcelona Clinic Liver Cancer stage C, and 271 (85.8%) had Child-Pugh class A liver function. The median overall survival time was 307 days and progression-free survival time was 109 days. According to the modified Response Evaluation Criteria In Solid Tumours, four patients achieved a complete response, 51 achieved a partial response, 136 had stable disease and 125 had progressive disease. Multivariate analysis identified female gender (P = 0.003) and decreased serum AFP level after 1 month (P = 0.042) as independent predictors of a complete or partial response. Conclusion Our results suggest female gender and a decrease in serum AFP level are independent predictors of good response to sorafenib.

Background and aims: We aimed to compare clinical outcomes and safety after sorafenib therapy between patients with Barcelona Clinic Liver Cancer (BCLC) stage B or C hepatocellular carcinoma (HCC) aged ≥75 years (aged group, n=179) and those with BCLC stage B or C HCC aged 0.999). Objective response rates were 15.1% (27/179) in the aged group and 14.3% (40/279) in the control group (P=0.892). Treatment related SAEs of grade 3 or more were observed in 51 patients (28.5%) in the aged group and in 69 patients (24.7%) in the control group (P=0.385). In the propensity score matched cohort (132 pairs), no significant difference in the two groups was observed in terms of OS (P=0.898) and PFS (P=0.407). Conclusion: In BCLC stage B or C HCC patients treated with sorafenib, life expectancy, disease progression, treatment efficacy and SAEs are unaffected by age over 75 years.

In this study, we suggest the effective enlargement method of (0001) 4H-SiC bulk crystals grown by the sublimation method using long length growth technique (LLG). This method could achieve low thermal strain and rapid enlargement growth comparing with conventional c-axis growth technique. We also demonstrated high quality enlargement growth from 2inch to  4inch of (0001) 4H-SiC by LLG.

A compact-sized in-vehicle Rankine cycle that recovers exhaust heat with high efficiency and fuel economy has been developed. The system integrates a capacity-type compact expansion machine that uses high-pressure and high-temperature steam and a generator. The key component that determines the performance of the expansion machine is the distribution steam valve. The requirements for the valve to satisfy the performance are low leakage and low friction. However, it has been difficult to satisfy the requirements with the existing mechanisms and materials due to problems such as corrosion and lubricity. A valve has been designed that satisfies the requirements by letting water be retained on the surface of sintered carbon to improve wear resistance and, at the same time, by letting the valve surface dither during steam distribution to maintain the water. This is the report of the configuration and performance of the distribution steam valve for a Rankine cycle expansion machine.

In this study, we report defect analysis in 4H-SiC crystals of high nitrogen doping grown by sublimation method, and we discuss key points for defect restraint. The growth was performed in two kinds of growth directions; c-axis and a-axis. In the c-axis grown crystal with carrier density greater than 1×10-19cm-3, defect propagation was confirmed in the vertical direction for a growth direction affected by the doping by x-ray topography. This phenomenon was not observed in the a-axis grown crystals. In sublimation growth, the quantity of impurities tends to increase as growth rate decreases. Therefore, in the c-axis growth of doped 4H-SiC bulk crystals, we have to be careful so that dopant does not increase too much without intention in grown layers with lower growth rate, for example at the beginning and end of the growth.

We report SiC wafer polishing study to achieve high throughput with extremely flat, smooth and damageless surface. The polishing consists of three process, wafer grinding, lapping and chemical mechanical polishing (CMP), which are completed in shortest about 200 minutes in total for 2 inch wafer. Specimens of 4H- and 6H-SiC were provided from slicing single crystal as wafers oriented (0001) with 0 or 8 degrees offset angle toward to . By the first grinding using a diamond whetstone wheel, we realized flat surface on the wafers with small TTV error of 1 μm in 15 minutes. After second process of lapping, the wafers were finished by CMP using colloidal silica slurry. AFM observation showed not only scratch-free surface but also atomic steps on the wafers after CMP. Rms marks extremely flat value of 0.08 nm in 10 μm square area.

BACKGROUND We evaluated the association between variations in hepatitis C virus (HCV) core protein and hepatitis severity in patients with chronic HCV infection who achieved remission without viral eradication and had a biochemical response to interferon (IFN) therapy, to evaluate the effect of HCV core sequence in the absence of the influence of host factors. METHODS. Using serum from 10 patients with a biochemical response and 10 patients with no response, we measured serum levels of interleukin (IL)-1 beta , IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFN- gamma , and tumor necrosis factor- alpha before and after IFN therapy. Expression vectors with the core region were transfected into Huh7 cells, and cytokine induction was evaluated by reporter assay. RESULTS In biochemical responders, only IL-8 levels decreased after IFN therapy (P=.04). Changes in the C-terminal hydrophobic region were observed more frequently in biochemical responders. Activation of the IL-8 promoter by HCV core protein was significantly decreased in biochemical responders after IFN therapy (P=.04). When 69 C-terminal amino acids from before IFN therapy were replaced with those from after IFN therapy in 3 biochemical responders, their ability to transactivate IL-8 decreased. CONCLUSIONS Differences in amino acids in the HCV core protein correlates with hepatitis activity through the modulation of IL-8 induction in HCV-infected patients.

Vitamin K is a cofactor for gamma-glutamyl carboxylase, an enzyme that is important for blood coagulation. Recent studies have shown that vitamin K has other roles, in addition to post-transcriptional modification, such as bone metabolism and antitumoral actions; these findings have indicated that there might be unknown intracellular binding proteins that are specific for vitamin K. In this study, vitamin K-binding proteins were characterized by pull-down experiment using a chemically synthesized biotynylated vitamin K followed by mass spectrometric identification of the pull-downed components. The results indicated that 17beta hydroxy steroid dehydrogenase 4, apolipoportein E, and 40S ribosomal proteins S7 and S13 might be the candidates of the vitamin K-binding proteins. Subsequent experiments showed that vitamin K2 binds 17beta hydroxysteroid dehydrogenase 4 and decreases the intracellular estradiol:estrone ratio, which resulted in the inhibition of the amount of estrogen receptor alpha-binding to its target DNA. These results suggest a possible novel role for vitamin K in modulating estrogen function.

Background & Aims: Hepatocellular carcinoma (HCC) is one of the most common human malignancies. Its high mortality rate is mainly a result of high intrahepatic recurrence. The novel synthetic retinoid acyclic retinoid (ACR) has been reported to prevent the recurrence of human HCC after surgical resection of primary tumors, but the molecular mechanisms underlying its effects remain to be elucidated. In this study, we clarified the molecular targets of ACR. Methods: The inhibitory effects by ACR on growth were examined. Intracellular signaling induced by ACR was comprehensively studied by a reporter assay. Gene expression changes by ACR were examined using a microarray. From these results, a candidate signaling pathway modulated by ACR was determined and whether antagonizing this pathway reverses the effect was examined. Results: We show that ACR inhibits the growth of HCC cells through the down-regulation of fibroblast growth factor (FGF) receptor 3 expression and FGF-mediated signaling, which in turn suppresses the activity of Rho and serum response factor-mediated transcription. Conversely, overexpression of the active form of FGF receptor 3 or the addition of FGF reverses the ACR-mediated inhibition of growth. In addition, silencing the FGF receptor 3 gene by RNA interference inhibits cell growth. Conclusions: These studies show that ACR is a potent inhibitor of FGF signaling and that selective blocking of the FGFmediated pathway could be a promising therapeutic approach for the management of patients with HCC.

Previous studies indicated that hepatitis C virus core protein influences cellular apoptosis. However, the precise mechanisms of the effects are not fully understood. Therefore, in this study, we examined the mechanisms of the effects on cell apoptosis by core protein, using transiently transfected and magnetically collected core-producing HepG2 cells. First, to elucidate the target site of core protein in the apoptotic pathway, we examined the activation of caspases after anti-Fas antibody stimulation. Core protein inhibited the apoptotic cascade downstream from caspase 8 and upstream from caspase 3. Next, to clarify more direct mechanisms of this effect, mRNA levels of several bcl-2-related genes were examined. An RNase protection assay showed that the mRNA of bcl-xl increased in the core-producing cells. We showed that this increase was mediated by the enhancement of bcl-x promoter activity by core protein through an extracellular-regulated kinase pathway. These results suggest that core protein inhibits apoptosis at the mitochondria level through augmentation of Bcl-x expression, resulting in an inhibition of caspase 3 activation.

There have been no established predictors of the outcome on sorafenib therapy for hepatocellular carcinoma (HCC) patients. We aimed to establish a new prognostic model suitable for sorafenib in HCC.Among 465 HCC patients treated with sorafenib in 14 hospitals, we formed a training cohort with 270 patients at seven hospitals located in West Japan and a validation cohort with 167 patients at seven hospitals located in East Japan. In the training cohort, we examined the relationship between overall survival (OS) and pretreatment clinical factors, and structured a new prognostic model. We verified this model in the validation cohort and compared with four existing staging models.Multivariate analysis demonstrated distant metastases, portal invasion, intrahepatic tumor burden of more than 50%, serum α-fetoprotein of 150 ng/dL or more, des-γ-carboxyprothrombin of 1200 mAU/mL or more, albumin of 3.5 g/dL or less and total bilirubin of more than 1.0 mg/dL were significant independent adverse prognostic factors. We calculated a Japan Red Cross (JRC) score with these factors and classified three groups: low-, intermediate- or high-risk. Their median OS were well stratified (18.0, 8.8 and 3.7 months, respectively, P 0.001) in the training cohort. In the validation cohort, OS were also statistically stratified (23.9, 10.3 and 2.9 months, P 0.001). C-statistics of the JRC score was 0.755, the highest in the five models, indicating its novel predictability.Our proposed JRC score well predicts the prognosis of sorafenib therapy, and would be useful to plan individualized strategies for unresectable HCC.

The aims of the present study were to examine whether unresectable hepatocellular carcinoma (HCC) patients treated with initial dose of sorafenib of 400 mg/day (half-dose group) had comparable treatment efficacy, safety and survival merit as compared with those treated with initial dose of sorafenib of 800 mg/day (standard-dose group) in a multicenter large study. For reducing the bias in patient selection, we compared clinical outcomes of these two groups using propensity score matching analysis. A total of 465 patients were treated with sorafenib at fourteen hospitals in Japanese Red Cross Liver Study Group from 2008 to 2013. After propensity score matching, 139 matched HCC patients were selected for analysis in both groups. We retrospectively compared overall survival (OS), progression-free survival (PFS), best treatment response and sorafenib related serious adverse events (SAEs) in the two groups. There were no relevant differences in terms of OS (median OS intervals: 9.2 months in the standard-dose group and 9.7 months in the half‑dose group, P=0.350), PFS (median PFS intervals: 3.4 months in the standard-dose group and 3.2 months in the half-dose group, P=0.729) and best treatment efficacy (objective response rate: P=0.416; disease control rate: P=0.719). Grade 3 or more SAEs were observed in 37 patients (26.6%) in the standard-dose group and 33 patients (23.7%) in the half-dose group (P=0.580). Furthermore, in all subgroup analyses according to Child-Pugh classification and Barcelona Clinic Liver Cancer stage, there were no significant differences in the two groups. In conclusion, unresectable HCC patients treated with initial half‑dose sorafenib had comparable prognosis compared with those treated with initial standard-dose sorafenib.

In this paper, we report a new polishing technique regarding the elimination of step bunching on the silicon carbide (SiC) surface. The step bunching generation is often observed as frequent phenomenon on the surface of SiC epilayers grown on low off-angle (0001) SiC wafers and on SiC devices after annealing to activate the dopants. We polished the step bunching surface using a chemical mechanical polishing (CMP) technique reported in a previous study, and we succeeded to improve the morphology with a flat and smooth surface which showed a small Rms value of around 0.1nm. We especially found an excellent polishing effect for the control of leakage current in reverse I-V characteristics of SiC Schottky barrier diodes (SBD).

Parallel processing for the DSMC method (direct simulation Monte Carlo method) using a multiprocessor computer is discussed in this paper. Rarefied gas flows through a finite length circular tube, over a flat plate, and in aclosed cavity were simulated by the DSMC method. The performance of parallel processing with a dynamic domain decomposition technique is evaluated in terms of parallel processing efficiencies and acceleration. An estimation method of parallel processing for evaluation of the DSMC method is proposed.

Dilute gas flows through finite length circular tubes were numerically simulated by the Direct Simulation of Monte Carlo method. The Direct Simulation of Monte Carlo method can simulate both the collisions of molecule-molecule and molecule-solid surfaces. The simulated transmission probabilities of monoatomic gas molecules through the tubes were compared with the free molecular flow theory in order to evaluate the numerical accuracy of the method. The numerical probabilities in the free molecular range coincide with the theory (to an accuracy of ±2%). In the flow range from transition flows to slip flows, the ratios of the conductances through the finite length tubes to the conductances through a zero-length circular slit at the free molecular flow are in accord with the experimental equation (to an accuracy of ±5%).

Background: Double-stranded RNA-activated protein kinase (PKR), an interferon (IFN)-stimulated gene, is activated by binding with double-stranded RNA, a putative replicative intermediate of the hepatitis C virus (HCV). Activated PKR phosphorylates the α subunit of eukaryotic initiation factor-2 to inhibit the translation of viral protein. Aims/methods: We established stable PKR knockdown Huh7 cells using RNA interference and investigated the effect of PKR against HCV replication using a subgenomic replicon that expressed luciferase reporter protein and the JFH1 full-length HCV genome. Results: In stable PKR knockdown cells that harboured a subgenomic replicon, luciferase activity was approximately three times higher than that of control cells, indicating that the subgenomic replicon replicated with a higher efficiency in stable PKR knockdown cells than that in control cells. Furthermore, stable PKR knockdown cells secreted significantly more HCV particles than did control cells after transfection with the full-length HCV genome. The replication of the subgenomic replicon was suppressed by the addition of IFN-α in both cells. Although the extent of suppression was significantly lower in stable PKR knockdown than control cells using a low concentration (2.5–5 U/ml) of IFN-α, even 10 U/ml IFN-α suppressed the replication of subgenomic replicon by >98% in both cells. Conclusions: Double-stranded RNA-activated protein kinase plays an important role in suppressing HCV replication in an innate state, but may not be essential in IFN therapy.

Innate immunity is part of the antiviral response. Interferon (IFN)-beta plays a leading role in this system. To investigate the influence of hepatitis C virus (HCV) on innate immunity, we examined the effect of viral proteins on IFN-beta induction. HepG2 cells were co-transfected with plasmids for seven HCV proteins (core protein, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) and the IFN-beta promoter luciferase. Toll-like receptor (TLR) 3 and Toll/IL-1 receptor domain-containing adapter inducing IFN-beta (TRIF) play key roles in dsRNA-mediated activation of interferon regulatory factor (IRF)-3 and IFN-beta; therefore, the participation of TLR3/TRIF in NS5B-mediated IFN induction was examined. Among seven HCV proteins, only NS5B, a viral RNA-dependent RNA polymerase (RdRp), activated the IFN-beta promoter. However, mutant NS5B without RdRp activity or template/primer association did not activate the IFN-beta promoter. Activation of the IFN-beta promoter by NS5B required the positive regulatory domain III, a binding sequence for IRF-3. Moreover, IRF-3 was phosphorylated by NS5B. Both inhibition of TLR3 expression by small interfering RNA and expression of the dominant negative form of TRIF significantly reduced NS5B-induced activation of IFN-beta. Of the six other HCV proteins, NS4A, NS4B, and NS5A efficiently inhibited this activation. HCV NS5B is a potent activator of the host innate immune system, possibly through TLR3/TRIF and synthesis of dsRNA. Meanwhile, NS4A, NS4B, and NS5A block IFN-beta induction by NS5B, which may contribute toward the persistence of this virus.

Infection by hepatitis C virus (HCV) usually results into chronic hepatitis that can ultimately lead to cirrhosis and hepatocellular carcinoma. Type 1 interferons (IFN-α/β) constitute the primary cellular defense against viral infection including HCV. IFN binding to their receptors activates associated Jak1 and Tyk2 kinases, which ultimately leads to phosphorylation and assembly of a signal transducer and activator of transcription protein (STAT)1-STAT2-interferon regulatory factor (IRF)9 trimetric complex called interferon-stimulated gene factor 3 that translocates into the nucleus and binds to the interferon- stimulated response elements (ISRE), leading to transcriptional induction of several antiviral genes, including double-stranded RNA-activated protein kinase (PKR), 2′,5′- oligoadenylate synthetase (OAS), and myxovirus resistance protein A (MxA). Understanding the mechanisms of how the virus evades this cellular innate defense and establishes a chronic infection is the key for the development of better therapeutics against HCV infection. Here, we demonstrate that p53 could have a crucial role in the cellular innate defense against HCV. We observed significantly higher levels of HCV RNA replication and viral protein expression in the Huh7 cells when their p53 expressions were knocked down. Moreover, IFN treatment was less effective in inhibiting the HCV RNA replication in the p53–knocked-down (p53kd) Huh7 cells. In fact, the activation of the ISRE and the induction of ISGs were significantly attenuated in the p53kd Huh7 cells and p53 was found to directly interact with IRF9. Conclusion: These observations underscore the potential contributions of the tumor suppressor p53 in cellular antiviral immunity against HCV with possible therapeutic implications. (HEPATOLOGY 2008.)

Hepatitis C virus (HCV) infection is a major risk factor for developing hepatocellular carcinoma (HCC). The host genetic factors that are involved in the development of HCC in patients with HCV infection remain to be investigated. To search for single nucleotide polymorphisms (SNPs) in HCC susceptibility genes, 393 SNPs in 171 candidate genes were examined in 188 Japanese patients with chronic HCV infection, including 77 patients with HCC. HCC-related SNPs were then examined in another 188 patients (including 93 patients with HCC) with chronic HCV infection. Haplotype analyses of HCC-related genes were performed in a total of 376 patients. Of the 393 SNPs, 31 SNPs in 29 genes were significantly associated with HCC based on an initial screening (P < .05). Of these 31 SNPs, 3 SNPs of 3 genes (SCYB14, GFRA1, and CRHR2) were significantly associated with HCC in a secondary screening. Haplotype analyses of these 3 genes identified 2 haplotype blocks associated with HCC. In conclusion, these SNPs and haplotypes located in the SCBY14, CRHR2, and GFRA1 genes will be used as markers to identify a subgroup of Japanese patients with chronic HCV infection who are at high risk of developing HCC.

The persistent nature of hepatitis C virus (HCV) infection suggests that HCV encodes proteins that enable it to overcome host antiviral responses. Toll-like receptor 3 (TLR3)-mediated signaling, which recognizes the double-stranded RNA that is produced during viral replication and induces type I interferons, including interferon beta (IFN-beta), is crucial to the host defense against viruses. Recent studies suggest that a TIR domain-containing adaptor protein, TRIF, and two protein kinases, TANK-binding kinase-1 (TBK1) and IkappaB kinase-epsilon (IKKepsilon), play essential roles in TLR3-mediated IFN-beta production through the activation of the transcriptional factor interferon regulatory factor 3 (IRF-3). We report that the HCV NS3 protein interacts directly with TBK1, and that this binding results in the inhibition of the association between TBK1 and IRF-3, which leads to the inhibition of IRF-3 activation. In conclusion, these results suggest the mechanisms of the inhibition of the innate immune responses of HCV infection by NS3 protein.

Hepatocellular carcinoma (HCC) is a common human malignancy. Its high mortality rate is mainly a result of high intrahepatic recurrence and portal venous invasion (PVI). We previously reported that the development of PVI is related to levels of des-gamma-carboxy prothrombin (DCP), a serum protein that increases at a notably higher rate in patients with HCC. Because DCP is produced by a vitamin K shortage, we examined the biological effects of extrinsic supplementation of vitamin K(2) in HCC cells in vitro and in vivo. Consequently, vitamin K(2) inhibits the growth and invasion of HCC cells through the activation of protein kinase A, which modulates the activities of several transcriptional factors and inhibits the small GTPase Rho, independent of suppression of DCP. In addition, administration of vitamin K(2) to nude mice inoculated with liver tumor cells reduced both tumor growth and body weight loss. In conclusion, similar to an acyclic retinoid--which was previously reported to prevent the recurrence of HCC--vitamin K(2), another lipid-soluble vitamin, may be a promising therapeutic means for the management of HCC.

Purpose: Genetic polymorphisms of UDP-glucuronosyltransferase 1A7 (UGT1A7), which detoxifies endogenous and environmental carcinogens, have been reported to be associated with hepatocellular carcinoma (HCC) in German populations. On the other hand, we reported that interleukin-1β (IL-1β) gene polymorphisms were associated with hepatitis C virus (HCV)-related HCC. In this study, we evaluated the association of both genes with the risk of HCC in Japanese HCV-infected patients. Experimental Design: Genetic polymorphisms of UGT1A7 and IL-1β were investigated in 280 Japanese patients (122 with HCC and 158 without HCC) with chronic HCV infections, by use of standard PCR-based genotyping techniques. Results: We designated the UGT1A7*1 allele (a haplotype conferring higher activity) as H and the *2, *3, and *4 alleles (haplotypes conferring lower activity) as L. The proportions of UGT1A7 L/L and H/L alleles (genotypes) in patients with HCC (25% and 45%, respectively) were higher than those in patients without HCC (15% and 39%, respectively) with odds ratios of 2.73 (95% confidence interval, 1.40–5.35) and 1.80 (95% confidence interval, 1.05–3.09), respectively, compared with the UGT1A7 H/H alleles. Multivariate analyses revealed that UGT1A7 L/L and IL-1β/−31T/T−511C/C genotypes, the presence of cirrhosis, age >60 years, male sex, and α-fetoprotein >20 μg/ml were associated with the presence of HCC (odds ratios, 2.33, 2.67, 4.20, 3.12, 3.09, and 2.90, respectively). Conclusion: The UGT1A7 polymorphisms together with IL-1β were associated with the presence of HCC in Japanese HCV-infected patients.

The majority of persons with chronic hepatitis C virus (HCV) infection develop liver fibrosis. Transforming growth factor (TGF)-beta 1 plays a pivotal role in the pathogenesis of post-inflammatory liver scarring. To clarify the influence of HCV infection on liver fibrosis, a reporter assay was used to investigate the effect of viral proteins on TGF-beta 1 expression in human hepatoma cells. Of all HCV proteins investigated (core, E1/E2/p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B), only the core protein activated the TGF-beta 1 promoter and upregulated TGF-beta 1 expression measured by an RNase protection assay. Bases -376 to -331 bp in the promoter region of TGF-beta 1 are responsible for upregulation by HCV core protein, and the nuclear protein that binds to this region increased with the stimulation of HCV core protein. Blocking the mitogen-activated protein kinase pathway prevented upregulation of TGF-beta 1 by HCV core protein. The immunological response is supposed to be a major factor to cause the secretion of TGF-beta 1 from non-parenchymal cells, but the results suggest that the HCV core protein expression may upregulate directly TGF-beta 1 transcription in parenchymal cells and suggest a new paradigm for exacerbation of liver fibrosis by HCV infection.

A comprehensive profile of genes expressed at the mRNA level in various human tissues is considered to be important for understanding the molecular mechanisms of the tissue-specific function and the pathogenesis of related diseases. Here, the gene expression profiling in three human digestive tissues, liver, stomach, and pancreas, was catalogued by generating a large number of expressed sequence tags, and clarified how quantitatively the gene expressions are different. After assembling the redundant clones among three tissues, the results showed that only 1.7% among the assembled genes was expressed commonly in the investigated tissues. These results suggest that the significant functional divergences in different tissues must be related to the divergence of the gene expression profiles. Recently, microarray technologies are widely used. Considering the results that different genes express in different tissues, however, it is important to spot the cDNAs derived from the same tissues or cells examined to acquire information efficiently. For the study of digestive diseases, we constructed an in-house microarray by using the cDNA sets derived from the digestive tissues (liver and gastric chip). In addition, because the amount of information acquired by the microarray analyses is huge, the power of bioinformatics for unifying the obtained data is indispensable. Some examples of the strategies for handling the microarray data obtained by our in-house microarrays are shown in this article.

Generally, hepatoma is not a chemosensitive tumor, and the mechanism of resistance to anticancer drugs is not fully elucidated. We aimed to comprehensively evaluate the relationship between chemosensitivity and gene expression profile in human hepatoma cells, by using microarray analysis, and analyze the data by constructing relevance networks. In eight hepatoma cell lines (HLE, HLF, Huh7, Hep3B, PLC/PRF/5, SK-Hep1, Huh6, and HepG2), the baseline expression levels of 2300 genes were measured by cDNA microarray. The concentrations of eight anticancer drugs (nimustine, mitomycin C, cisplatin, carboplatin, doxorubicin, epirubicin, mitoxantrone, and 5-fluorouracil) needed for 50% growth inhibition were examined and used as a measure of chemosensitivity. These data were combined and comprehensive pair-wise correlations between gene expression levels and the 50% growth inhibition values were calculated. Significant correlations with significance were used to construct networks of similarity. Fifty-two relations, including 42 genes, were selected. Among them, nearly 20% were various types of transporters, and most of them negatively correlated with chemosensitivity. Transporter associated with antigen processing 1 was associated with resistance to mitoxantrone, consistent with previous reports. Other transporters were not reported previously to associate with chemosensitivity. Resistance to doxorubicin and its analogue, epirubicin, were positively correlated with topoisomerase II beta expression, whereas it negatively correlated with expression of carboxypeptidases A3 and Z. Response to nimustine was associated with expression of superoxide dismutase 2. Relevance networks identified several negative correlations between gene expression and resistance, which were missed by hierarchical clustering. Our results suggested the necessity of systematically evaluating the transporting systems that may play a major role in resistance in hepatoma. This may provide useful information to modify anticancer drug action in hepatoma.

Background: The prognosis of patients with advanced hepatoma is grim. Although chemotherapy is adapted to such patients, the efficacy is low and the outcome cannot be predicted before therapy. In this study, we aimed to identify genes associated with sensitivity to 5-fluorouracil and cisplatin, drugs widely used in treatment, using gene expression profiles. Methods: Gene expression was evaluated in eight human hepatoma cell lines using an in-house cDNA microarray including 2300 known genes. The 50% growth inhibitory concentrations (GI50) of 5-fluorouracil and cisplatin were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and designated as chemosensitivity. Genes with expression ratios associated with GI50 were selected using the permutation test. Results: For 5-fluorouracil and cisplatin, 21 and 40 genes, respectively, were selected. From among the genes associated with 5-fluorouracil and cisplatin, several encoding metabolic enzymes were selected. In addition, several genes involved in the cell cycle and transcription were identified. Conclusions: We identified genes that may be associated with sensitivity to 5-fluorouracil and cisplatin. A list of these genes may be useful to elucidate how these drugs work on human hepatoma.

Hepatitis C virus (HCV) causes a persistent infection, chronic hepatitis, cirrhosis, and hepatocellular carcinoma. To explore the influence of HCV infection on hepatocytes, the effects of HCV proteins on intracellular signal transduction pathways, especially those related to apoptosis, fibrosis, and cell growth, were investigated. The effects of nine HCV proteins (core, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) on the CRE-, SRE-, NFKB-, AP-1-, SRF-, ISRE-, HSE-, GRE-, and p53-associated pathways were investigated by use of a reporter assay. The effects of core protein on apoptosis were examined by DNA laddering and Western blotting after induction of apoptosis by Fas stimulation. The possible mechanisms of anti-apoptotic effects of core protein were investigated by RNase protection assay and a reporter assay. The effects of HCV proteins on TGF beta production were determined by a reporter assay. Among seven HCV proteins investigated, core protein had the strongest influence on intracellular signaling, especially for the SRE-, AP-1-, NFKB-, and p53-associated pathways. Core protein promoted Bc1-xL expression through the mitogen-activated protein kinase (MAPK) pathway and inhibited apoptosis. Among HCV proteins, only core protein caused TGF beta promoter activity through the MAPK pathway. From these results, core protein was considered to regulate cell growth exquisitely in hepatocytes, inhibit apoptosis, and promote liver fibrosis directly without any inflammation or the mediation of any other cells. These functions may reflect the direct action of HCV proteins on the intracellular signal transduction pathways related to chronic hepatitis pathogenesis.

Hepatitis C virus (HCV) infection is a major risk factor for developing hepatocellular carcinoma (HCC), a life-threatening sequel. However, the factors that affect disease progression to HCC have not been thoroughly elucidated. Genetic polymorphisms in proinflammatory cytokines, the interleukin 1 (IL-1) family (IL-1beta and IL-1ra) and tumor necrosis factor-alpha (TNF-alpha), were studied in 274 Japanese patients with chronic HCV infection and 55 healthy individuals using standard polymerase chain reaction-based genotyping techniques. The association between these polymorphisms and disease status was evaluated while controlling for confounding clinical variables. The proportion of patients with HCC in the IL-1beta-31 T/T (55%, odds ratio to C/C was 2.63, P =.009) genotype was higher than in the T/C (44%, odds ratio to C/C was 1.64, P =.149) and C/C genotypes (35%). The IL-1beta-31 and -511 loci were in near complete linkage disequilibrium, and the IL-1beta-511/-31 haplotype C-T was significantly associated with the presence of HCC (odds ratio of 1.51, P =.02). Polymorphisms in the TNF-alpha gene were not associated with disease. A multivariate analysis revealed that the IL-1beta-31 T/T genotype, alpha-fetoprotein20 microg/L, presence of cirrhosis, male sex, and age60 years were associated with the presence of HCC at odds ratios of 3.73 (T/T vs. C/C), 4.12, 4.03, 3.89, and 3.27, respectively. In conclusion, the IL-1beta-31 genotype T/T or the IL-1beta-511/-31 haplotype C-T is associated with the presence of HCC in Japanese patients with chronic HCV infection.