Incidence of development of hepatocellular carcinoma in Japanese patients infected with hepatitis B virus is equivalent between genotype B and C in long term.

Hepatitis B virus (HBV) genotypes B (HBV/B) and C (HBV/C) are the most prevalent genotypes among Japanese patients with hepatitis. Reportedly, HBV/C infection has been associated with more severe disease progression, manifesting as developing cirrhosis and hepatocellular carcinoma (HCC), than HBV/B infection. However, no long-term studies have examined the development of HCC in HBV/B-infected patients in Japan. The aims of our study were to compare the incidence of HCC in HBV/B- or HBV/C-infected patients. A total of 241 patients were followed up among 295 hepatitis B surface antigen (HBsAg)-positive carriers. Genotypes of HBV were A in 1% (4/295), B in 61% (179/295), C in 37% (110/295) and D in 1% (2/295) patients, and 96% of HBV/B were infected with subgenotype Bj. The mean age at HCC diagnosis was significantly higher in HBV/B than in HBV/C (67.0 ± 10.0 vs 57.7 ± 8.0 years, P < 0.001). The value of FIB-4 index was significantly higher in HBV/B than in HBV/C (P < 0.01). The rate of HCC was higher in HBV/C than in HBV/B, and a significant difference was observed until the 20-year observation period (P = 0.048). However, thereafter, HCC associated with HBV/B increased, and no significant difference was observed between HBV/B and HBV/C. HCC development was consistently observed even in HBV/B infection, especially among elderly patients with advanced fibrosis compared with HBV/C. HBV/B-infected patients developed HCC later in life, and in the long term, we found no differences in incidence of HCC development rates between these two genotypes.

Perinatal Nutritional Reprogramming of the Epigenome Promotes Subsequent Development of Nonalcoholic Steatohepatitis.

With the epidemic of obesity, nonalcoholic fatty liver disease (NAFLD) has become the most common pediatric liver disease. The influence of a perinatal obesity-inducing diet (OID) on the development and progression of NAFLD in offspring is important but incompletely studied. Hence, we fed breeding pairs of C57BL/6J mice during gestation and lactation (perinatally) either chow or an OID rich in fat, fructose, and cholesterol (FFC). The offspring were weaned to either chow or an FFC diet, generating four groups: perinatal (p)Chow-Chow, pChow-FFC, pFFC-Chow, and pFFC-FFC. Mice were sacrificed at 10 weeks of age. We examined the whole-liver transcriptome by RNA sequencing (RNA-seq) and whole-liver genome methylation by reduced representation bisulfite sequencing (RRBS). Our results indicated that the pFFC-FFC mice had a significant increase in hepatic steatosis, injury, inflammation, and fibrosis, as assessed histologically and biochemically. We identified 189 genes that were differentially expressed and methylated in the pFFC-FFC mice versus the pChow-FFC mice. Gene set enrichment analysis identified hepatic fibrosis/hepatic stellate cell activation as the top canonical pathway, suggesting that the differential DNA methylation events in the mice exposed to the FFC diet perinatally were associated with a profibrogenic transcriptome. To verify that this finding was consistent with perinatal nutritional reprogramming of the methylome, we exposed pFFC-Chow mice to an FFC diet in adulthood. These mice developed significant hepatic steatosis, injury, inflammation, and more importantly fibrosis when compared to the appropriate controls. Conclusion: Perinatal exposure to an OID primes the immature liver for an accentuated fibrosing nonalcoholic steatohepatitis (NASH) phenotype, likely through nutritional reprogramming of the offspring methylome. These data have potential clinical implications for monitoring children of obese mothers and risk stratification of children with NAFLD.

Antibodies against adenovirus fiber and penton base proteins inhibit adenovirus vector-mediated transduction in the liver following systemic administration.

Pre-existing anti-adenovirus (Ad) neutralizing antibodies (AdNAbs) are a major barrier in clinical gene therapy using Ad vectors and oncolytic Ads; however, it has not been fully elucidated which Ad capsid protein-specific antibodies are involved in AdNAb-mediated inhibition of Ad infection in vivo. In this study, mice possessing antibodies specific for each Ad capsid protein were prepared by intramuscular electroporation of each Ad capsid protein-expressing plasmid. Ad vector-mediated hepatic transduction was efficiently inhibited by more than 100-fold in mice immunized with a fiber protein-expressing plasmid or a penton base-expressing plasmid. An Ad vector pre-coated with FX before administration mediated more than 100-fold lower transduction efficiencies in the liver of warfarinized mice immunized with a fiber protein-expressing plasmid or a penton base-expressing plasmid, compared with those in the liver of warfarinized non-immunized mice. These data suggest that anti-fiber protein and anti-penton base antibodies bind to an Ad vector even though FX has already bound to the hexon, and inhibit Ad vector-mediated transduction. This study provides important clues for the development of a novel Ad vector that can circumvent inhibition with AdNAbs.

Mixed-lineage kinase 3 pharmacological inhibition attenuates murine nonalcoholic steatohepatitis.

With the increase in obesity worldwide, its associated comorbidities, including nonalcoholic steatohepatitis (NASH), have become a public health problem that still lacks effective therapy. We have previously reported that mixed-lineage kinase 3-deficient (MLK3-deficient) mice are protected against diet-induced NASH. Given the critical need to identify new therapeutic agents, we sought to examine whether the small-molecule MLK3 inhibitor URMC099 would be effective in reversing diet-induced murine NASH. C57BL/6J mice were fed either a diet high in saturated fat, fructose, and cholesterol (FFC), or a chow diet for 24 weeks. Mice were treated with either URMC099 (10 mg/kg) twice daily by intraperitoneal injection or its vehicle during the last 2 weeks of the feeding study. FFC-fed mice receiving URMC099 had similar body weight, caloric intake, homeostatic model assessment of insulin resistance, metabolic phenotype, and hepatic steatosis compared with vehicle-treated mice. Furthermore, FFC-fed mice treated with URMC099 had less hepatic macrophage infiltration, activation, and proinflammatory polarization, as well as less liver injury and fibrosis when compared with vehicle-treated mice. In conclusion, URMC099 is well tolerated in mice without obvious toxicities and appears to be efficacious in reversing diet-induced NASH. Hence, URMC099 may serve as a therapeutic agent in human NASH.

Mixed Lineage Kinase 3 Mediates the Induction of CXCL10 by a STAT1-Dependent Mechanism During Hepatocyte Lipotoxicity.

Saturated fatty acids (SFA) and their toxic metabolites contribute to hepatocyte lipotoxicity in nonalcoholic steatohepatitis (NASH). We previously reported that hepatocytes, under lipotoxic stress, express the potent macrophage chemotactic ligand C-X-C motif chemokine 10 (CXCL10), and release CXCL10-enriched extracellular vesicles (EV) by a mixed lineage kinase (MLK) 3-dependent mechanism. In the current study, we sought to examine the signaling pathway responsible for CXCL10 induction during hepatocyte lipotoxicity. Here, we demonstrate a role for signal transducer and activator of transcription (STAT) 1 in regulating CXCL10 expression. Huh7 and HepG2 cells were treated with lysophosphatidylcholine (LPC), the toxic metabolite of the SFA palmitate. In LPC-treated hepatocytes, CXCL10 induction is mediated by a mitogen activated protein kinase (MAPK) signaling cascade consisting of a relay kinase module of MLK3, MKK3/6, and p38. P38 in turn induces STAT1 Ser727 phosphorylation and CXCL10 upregulation in hepatocytes, which is reduced by genetic or pharmacological inhibition of this MAPK signaling cascade. The binding and activity of STAT1 at the CXCL10 gene promoter were identified by chromatin immunoprecipitation and luciferase gene expression assays. Promoter activation was attenuated by MLK3/STAT1 inhibition or by deletion of the consensus STAT1 binding sites within the CXCL10 promoter. In lipotoxic hepatocytes, MLK3 activates a MAPK signaling cascade, resulting in the activating phosphorylation of STAT1, and CXCL10 transcriptional upregulation. Hence, this kinase relay module and/or STAT1 inhibition may serve as a therapeutic target to reduce CXCL10 release, thereby attenuating NASH pathogenesis. J. Cell. Biochem. 118: 3249-3259, 2017. © 2017 Wiley Periodicals, Inc.

MiR-139-5p is associated with inflammatory regulation through c-FOS suppression, and contributes to the progression of primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease characterized pathologically by destruction of intrahepatic bile ducts. PBC is largely classified into three subtypes based on clinical course: (i) gradually progressive, (ii) portal hypertension, and (iii) hepatic failure. Previous studies have indicated that serum levels of the pro-inflammatory cytokine TNF-α, is elevated in PBC patients with fibrosis. Although the severity of cholangitis might also be related to the PBC subtype, its etiology has been unclear. Several studies have shown that microRNAs (miRNAs) demonstrate specific expression patterns in various diseases. In the present study, we evaluated miRNA expression patterns among the PBC subtypes using comprehensive deep sequencing. We also carried out histologic examination by laser capture microdissection and investigated how the identified miRNAs were involved in PBC clinical progression using the miRNA transfection method. On average, ~11 million 32-mer short RNA reads per sample were obtained, and we found that the expression levels of 97 miRNAs differed significantly among the four groups. Heat mapping demonstrated that the miRNA profiles from hepatic failure and portal hypertension type were clustered differently from those of the gradually progressive type and controls. Furthermore, we focused on miR-139-5p, which has an adequate number of total short reads. Quantitative reverse transcription PCR showed that miR-139-5p was significantly downregulated in clinically advanced PBC. Also, examination of liver tissues demonstrated that the expression of lymphocyte-derived miR-139-5p was significantly higher in hepatocytes. In vitro, the level of TNF-α was significantly elevated in supernatant of cells with upregulation of miR-139-5p. Furthermore, c-FOS gene transcription was repressed. Thus, we have demonstrated a novel inflammation-regulatory mechanism involving TNF-α and c-FOS transcription through miR-139-5p in the NF-κB signaling pathway. We conclude that the specific miRNA miR-139-5p might be involved in the pathogenesis of PBC, especially during clinical progression.

Dicer functions as an antiviral system against human adenoviruses via cleavage of adenovirus-encoded noncoding RNA.

In various organisms, including nematodes and plants, RNA interference (RNAi) is a defense system against virus infection; however, it is unclear whether RNAi functions as an antivirus system in mammalian cells. Rather, a number of DNA viruses, including herpesviruses, utilize post-transcriptional silencing systems for their survival. Here we show that Dicer efficiently suppresses the replication of adenovirus (Ad) via cleavage of Ad-encoding small RNAs (VA-RNAs), which efficiently promote Ad replication via the inhibition of eIF2α phosphorylation, to viral microRNAs (mivaRNAs). The Dicer knockdown significantly increases the copy numbers of VA-RNAs, leading to the efficient inhibition of eIF2α phosphorylation and the subsequent promotion of Ad replication. Conversely, overexpression of Dicer significantly inhibits Ad replication. Transfection with mivaRNA does not affect eIF2α phosphorylation or Ad replication. These results indicate that Dicer-mediated processing of VA-RNAs leads to loss of activity of VA-RNAs for enhancement of Ad replication and that Dicer functions as a defence system against Ad in mammalian cells.

CXCL10-Mediates Macrophage, but not Other Innate Immune Cells-Associated Inflammation in Murine Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is an inflammatory lipotoxic disorder, but how inflammatory cells are recruited and activated within the liver is still unclear. We previously reported that lipotoxic hepatocytes release CXCL10-enriched extracellular vesicles, which are potently chemotactic for cells of the innate immune system. In the present study, we sought to determine the innate immune cell involved in the inflammatory response in murine NASH and the extent to which inhibition of the chemotactic ligand CXCL10 and its cognate receptor CXCR3 could attenuate liver inflammation, injury and fibrosis. C57BL/6J CXCL10(-/-), CXCR3(-/-) and wild type (WT) mice were fed chow or high saturated fat, fructose, and cholesterol (FFC) diet. FFC-fed CXCL10(-/-) and WT mice displayed similar weight gain, metabolic profile, insulin resistance, and hepatic steatosis. In contrast, compared to the WT mice, FFC-fed CXCL10(-/-) mice had significantly attenuated liver inflammation, injury and fibrosis. Genetic deletion of CXCL10 reduced FFC-induced proinflammatory hepatic macrophage infiltration, while natural killer cells, natural killer T cells, neutrophils and dendritic cells hepatic infiltration were not significantly affected. Our results suggest that CXCL10(-/-) mice are protected against diet-induced NASH, in an obesity-independent manner. Macrophage-associated inflammation appears to be the key player in the CXCL10-mediated sterile inflammatory response in murine NASH.

Efficient detection of human circulating tumor cells without significant production of false-positive cells by a novel conditionally replicating adenovirus.

Circulating tumor cells (CTCs) are promising biomarkers in several cancers, and thus methods and apparatuses for their detection and quantification in the blood have been actively pursued. A novel CTC detection system using a green fluorescence protein (GFP)-expressing conditionally replicating adenovirus (Ad) (rAd-GFP) was recently developed; however, there is concern about the production of false-positive cells (GFP-positive normal blood cells) when using rAd-GFP, particularly at high titers. In addition, CTCs lacking or expressing low levels of coxsackievirus-adenovirus receptor (CAR) cannot be detected by rAd-GFP, because rAd-GFP is constructed based on Ad serotype 5, which recognizes CAR. In order to suppress the production of false-positive cells, sequences perfectly complementary to blood cell-specific microRNA, miR-142-3p, were incorporated into the 3'-untranslated region of the E1B and GFP genes. In addition, the fiber protein was replaced with that of Ad serotype 35, which recognizes human CD46, creating rAdF35-142T-GFP. rAdF35-142T-GFP efficiently labeled not only CAR-positive tumor cells but also CAR-negative tumor cells with GFP. The numbers of false-positive cells were dramatically lower for rAdF35-142T-GFP than for rAd-GFP. CTCs in the blood of cancer patients were detected by rAdF35-142T-GFP with a large reduction in false-positive cells.

Mixed lineage kinase 3 mediates release of C-X-C motif ligand 10-bearing chemotactic extracellular vesicles from lipotoxic hepatocytes.

UNLABELLED: Mixed lineage kinase 3 (MLK3) deficiency reduces macrophage-associated inflammation in a murine model of nonalcoholic steatohepatitis (NASH). However, the mechanistic links between MLK3 activation in hepatocytes and macrophage-driven inflammation in NASH are uncharted. Herein, we report that MLK3 mediates the release of (C-X-C motif) ligand 10 (CXCL10)-laden extracellular vesicles (EVs) from lipotoxic hepatocytes, which induce macrophage chemotaxis. Primary mouse hepatocytes (PMHs) and Huh7 cells were treated with palmitate or lysophosphatidylcholine (LPC). Released EVs were isolated by differential ultracentrifugation. LPC treatment of PMH or Huh7 cells induced release of EVs, which was prevented by either genetic or pharmacological inhibition of MLK3. Mass spectrometry identified the potent chemokine, CXCL10, in the EVs, which was markedly enriched in EVs isolated from LPC-treated hepatocytes versus untreated cells. Green fluorescent protein (GFP)-tagged CXCL10 was present in vesicular structures and colocalized with the red fluorescent protein (RFP)-tagged EV marker, CD63, after LPC treatment of cotransfected Huh-7 cells. Either genetic deletion or pharmacological inhibition of MLK3 prevented CXCL10 enrichment in EVs. Treatment of mouse bone-marrow-derived macrophages with lipotoxic hepatocyte-derived EVs induced macrophage chemotaxis, an effect blocked by incubation with CXCL10-neutralizing antisera. MLK3-deficient mice fed a NASH-inducing diet had reduced concentrations of total plasma EVs and CXCL10 containing EVs compared to wild-type mice. CONCLUSIONS: During hepatocyte lipotoxicity, activated MLK3 induces the release of CXCL10-bearing vesicles from hepatocytes, which are chemotactic for macrophages.

Animal models of primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is characterized histologically by the presence of chronic non-suppurative destructive cholangitis of the small interlobular bile duct, leading to chronic progressive cholestasis. Most PBC patients are asymptomatic and have a reasonable prognosis, but a few develop esophageal varices or jaundice, rapidly leading to liver failure within a short period. As multiple factors appear to be involved in the onset of PBC, its clinical course may be complicated. Therefore, the use of an animal model would be valuable for clarifying the pathogenesis of PBC. Here, we review recent data of selected PBC models, particularly spontaneous models, xenobiotic immunized models, and infection-triggered models. There are a number of spontaneous models: the NOD.c3c4, dominant-negative TGF-ß receptor II, IL-2Rα-/-, Scurfy, and Ae2a,b-/- mice. These animal models manifest distinct clinical and immunological features similar, but also often different, from those of human PBC. It is clear that a combination of genetic predisposition, environmental factors, and immunological dysfunction contribute to the pathogenesis of PBC. The diverse clinical course and complexity of the immunological mechanisms of PBC cannot be fully recapitulated solely any single animal model. The challenge remains to develop a progressive PBC disease model that exhibits fibrosis, and ultimately hepatic failure.

Epiregulin promotes the emergence and proliferation of adult liver progenitor cells.

We have previously reported that epiregulin is a growth factor that seems to act on liver progenitor cells (LPCs) during liver regeneration. However, the relationship between epiregulin and LPCs has remained unclear. The aim of the present study was to clarify the role of epiregulin during liver regeneration. The serum levels of epiregulin in patients with acute liver failure were examined. A liver injury model was developed using mice fed a diet containing 0.1% 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) to induce LPCs. We then evaluated the expression of epiregulin and LPCs in these mice. The proliferation of epithelial cell adhesion molecule + LPCs cultured with epiregulin was examined in vitro, and finally epiregulin was overexpressed in mouse liver. In patients with acute liver failure, serum epiregulin levels were elevated significantly. In DDC mice, LPCs emerged around the portal area. Epiregulin was also detected around the portal area during the course of DDC-induced liver injury and was partially coexpressed with Thy1. Serum epiregulin levels in DDC mice were also significantly elevated. Recombinant epiregulin augmented the proliferative capacity of the LPCs in a dose-dependent manner. In mice showing overexpression of epiregulin, the expression of PCNA on hepatocytes was increased significantly. Finally, LPCs emerged around the portal area after epiregulin gene delivery. We concluded that epiregulin promotes the proliferation of LPCs and DNA synthesis by hepatocytes and is upregulated in the serum of patients with liver injury. Furthermore, induction of epiregulin leads to the appearance of LPCs. Epiregulin would be a useful biomarker of liver regeneration.

Clinical manifestations of liver injury in patients with anorexia nervosa.

AIM: The number of Japanese patients with anorexia nervosa (AN) is increasing as society changes. Mild liver injury is a complication of AN in around 30% of cases. In some rare instances, patients present with severe liver injury similar to acute liver failure. However, there are numerous uncertainties over the clinical characteristics of this condition. The objective of the present study was to clarify the clinical characteristics of AN complicated by liver injury and to investigate the factors related to hepatic complications. METHODS: Thirty-seven patients hospitalized at our institution with a diagnosis of AN were enrolled as the study subjects. The study used clinical data obtained at the time of hospitalization. The enrolled patients underwent subgroup analysis and were categorized into three groups: (i) normal alanine aminotransferase (ALT), (ii) moderately elevated ALT, and (iii) highly elevated ALT. RESULTS: All of the study subjects were female with a median age of 24 years and presenting with marked weight loss (mean body mass index, 13 kg/m(2) ). Thirteen of the subjects had liver injury. We found that patients in the highly elevated ALT group had a significantly high blood urea nitrogen (BUN)/creatinine ratio, and a low blood sugar level. CONCLUSIONS: Our present findings indicate that AN patients with highly elevated ALT have a severe dehydration. This suggests that dysfunction of hepatic circulation accompanying severe dehydration due to malnutrition may be an important factor in the development of liver injury in AN patients.

Suppression of leaky expression of adenovirus genes by insertion of microRNA-targeted sequences in the replication-incompetent adenovirus vector genome.

Leaky expression of adenovirus (Ad) genes occurs following transduction with a conventional replication-incompetent Ad vector, leading to an induction of cellular immunity against Ad proteins and Ad protein-induced toxicity, especially in the late phase following administration. To suppress the leaky expression of Ad genes, we developed novel Ad vectors by incorporating four tandem copies of sequences with perfect complementarity to miR-122a or miR-142-3p into the 3'-untranslated region (UTR) of the E2A, E4, or pIX gene, which were mainly expressed from the Ad vector genome after transduction. These Ad vectors easily grew to high titers comparable to those of a conventional Ad vector in conventional 293 cells. The leaky expression of these Ad genes in mouse organs was significantly suppressed by 2- to 100-fold, compared with a conventional Ad vector, by insertion of the miRNA-targeted sequences. Notably, the Ad vector carrying the miR-122a-targeted sequences into the 3'-UTR of the E4 gene expressed higher and longer-term transgene expression and more than 20-fold lower levels of all the Ad early and late genes examined in the liver than a conventional Ad vector. miR-122a-mediated suppression of the E4 gene expression in the liver significantly reduced the hepatotoxicity which an Ad vector causes via both adaptive and non-adaptive immune responses.

Possible autoimmune hepatitis induced after chronic active Epstein-Barr virus infection.

Chronic active Epstein-Barr virus infection (CAEBV) can be manifested in a variety of systemic conditions, including interstitial pneumonia, malignant lymphoma, and coronary aneurysm. Sometimes it may be associated with hepatic failure, although the mechanism underlying CAEBV-related hepatotoxicity remains unclear. We encountered a case of autoimmune hepatitis (AIH) associated with CAEBV. A 61-year-old male was referred to our hospital because of abnormal liver enzyme levels after initial diagnosis of CAEBV had been made by laboratory tests and liver biopsy. On admission, positivity for anti-nuclear antibody was evident, and examination of the liver biopsy specimen showed findings compatible with AIH. Steroid administration was initiated, and the liver function parameters subsequently improved. Although phenotypic changes in liver biopsy specimens are rare in this condition, the present case could provide clues to the possible pathogenesis of AIH.

Dynamics of serum metabolites in patients with chronic hepatitis C receiving pegylated interferon plus ribavirin: a metabolomics analysis.

OBJECTIVES: Serum samples from patients with chronic hepatitis C were subjected to metabolomics analysis to clarify the pretreatment characteristics of their metabolites and also changes in specific metabolites resulting from antiviral therapy with pegylated interferon plus ribavirin (PegIFN/RBV). MATERIALS/METHODS: The serum levels of low-molecular-weight metabolites in the twenty patients before and 24weeks after completion of PegIFN/RBV therapy were analyzed using capillary electrophoresis and liquid chromatography-mass spectrometry. RESULTS: Ten patients showed a non-virological response (NVR) and 10 achieved a sustained virological response (SVR) with eradication of viremia. The pretreatment levels of tryptophan were significantly higher in the patients of SVR than in those of NVR (p=0.010). The area under the curve (AUC) value of tryptophan calculated from the receiver operating characteristic (ROC) curve for discriminating SVR from NVR was 0.84 (95% confidential interval, 0.66-1.02, p=0.010). The ROC curve of multiple logistic regression model incorporating the pretreatment levels of tryptophan and γ-glutamate-arginine showed that the AUC value was highly significant (AUC=0.92, 95% confidential interval, 0.79-1.05, p=0.002). Twenty four weeks after completion of treatment, the levels of γ-glutamyl dipeptides, glutamic acid, 5-oxoproline, glucosamine and methionine sulfoxide were decreased, whereas those of 5-methoxy-3-indoleacetate, glutamine, kynurenine and lysine were increased significantly (p<0.05) in both the NVR and SVR patients. CONCLUSIONS: The pretreatment serum levels of certain metabolites including tryptophan are associated with the response to PegIFN/RBV therapy. PegIFN/RBV therapy can ameliorate the oxidative stress responsible for glutathione metabolism.

Impaired mitochondrial ß-oxidation in patients with chronic hepatitis C: relation with viral load and insulin resistance.

BACKGROUND: Hepatic steatosis is often seen in patients with chronic hepatitis C (CH-C). It is still unclear whether these patients have an impaired mitochondrial ß-oxidation. In this study we assessed mitochondrial ß-oxidation in CH-C patients by investigating ketogenesis during fasting. METHODS: This study consisted of thirty patients with CH-C. Serum levels of insulin and hepatitis C virus (HCV) core protein were measured by chemiluminescence enzyme immunoassay. The subjects were then fasted, and venous blood samples were drawn 12 h and 15 h after the start of fasting. The levels of blood ketone bodies were measured by an enzymatic cycling method. The rate of change in total ketone body concentration was compared with that in eight healthy volunteers. RESULTS: The rate of change in total ketone body concentration between 12 h and 15 h after the start of fasting was significantly lower in CH-C patients than in healthy volunteers (129.9% (8.5-577.3%) vs. 321.6% (139.6-405.4%); P <0.01). The rate of change in total ketone body concentration in patients with a serum level of HCV core protein of 10000 fmol/L or higher was significantly lower than in patients with a level of less than 10000 fmol/L (54.8% (8.5-304.3%) vs. 153.6% (17.1-577.3%); P <0.05). The rate of change in total ketone body concentration in patients with a homeostasis model assessment of insulin resistance (HOMA-IR) of 2.5 or higher was significantly lower than in patients with a HOMA-IR of less than 2.5 (56.7% (8.5-186.7%) vs. 156.4% (33.3-577.3%); P <0.01). CONCLUSIONS: These results suggest that mitochondrial ß-oxidation is impaired, possibly due to HCV infection in patients with CH-C.

Bone marrow cell-based regenerative therapy for liver cirrhosis.

Bone marrow cells are capable of differentiation into liver cells. Therefore, transplantation of bone marrow cells has considerable potential as a future therapy for regeneration of damaged liver tissue. Autologous bone marrow infusion therapy has been applied to patients with liver cirrhosis, and improvement of liver function parameters has been demonstrated. In this review, we summarize clinical trials of regenerative therapy using bone marrow cells for advanced liver diseases including cirrhosis, as well as topics pertaining to basic in vitro or in vivo approaches in order to outline the essentials of this novel treatment modality.

Further reduction in adenovirus vector-mediated liver transduction without largely affecting transgene expression in target organ by exploiting microrna-mediated regulation and the Cre-loxP recombination system.

In order to detarget undesirable transduction in the liver by an adenovirus (Ad) vector, we previously demonstrated that insertion of sequences perfectly complementary to liver-specific miR-122a into the 3'-untranslated region (UTR) of transgene specifically reduced the transgene expression in the liver by approximately 100-fold; however, a certain level of residual transgene expression was still found in the liver. In order to further suppress the hepatic transduction, we developed a two-Ad vector system that uses the microRNA (miRNA)-regulated transgene expression system and the Cre-loxP recombination system, i.e., insertion of miR-122a target sequences and loxP sites into the transgene expression cassette and coadministration of a Cre recombinase-expressing Ad vector. In addition, to maintain as much as possible the transgene expression in the spleen, which is the target organ of this study, spleen-specific miR-142-3p target sequences were inserted into the 3'-UTR of the Cre recombinase gene to suppress Cre recombinase expression in the spleen. The spleen is an attractive target for immunotherapy because the spleen plays important roles in the immune system. Coadministration of Ad vector possessing CMV promoter-driven Cre recombinase expression cassette with miR-142-3p target sequences resulted in a further 24-fold reduction in the hepatic transgene expression by the Ad vector containing miR-122a target sequences and loxP sites, compared with coadministration of control Ad vector. On the other hand, there was no significant reduction of transgene expression in the spleen.

Intramuscular DNA immunization with in vivo electroporation induces antigen-specific cellular and humoral immune responses in both systemic and gut-mucosal compartments.

Mucosal delivery of antigens induces antigen-specific immune responses in both systemic and mucosal compartments, and is an attractive approach for preventing initial infection with mucosal pathogens. It has been shown that the intramuscular (i.m.) immunization of plasmid DNA by in vivo electroporation (DNA e.p.) induces both cellular and humoral immune responses in the airway-mucosal compartment as well as in the systemic compartment, implying there is a mechanism that bridges between the systemic and mucosal immune responses. An important question is whether the i.m. DNA e.p.-immunization alone can induce antigen-specific immune responses in the gut-mucosal compartment. Here, we investigated the induction of antigen-specific CD8(+) T cells and antibodies in both systemic and gut-mucosal compartments following i.m. DNA e.p.-immunization to mice. Surprisingly, the i.m. DNA e.p.-immunization induced the antigen-specific CD8(+) T cells and antigen-specific antibodies in the gut-mucosal as well as the systemic compartment. These results suggest that the i.m. DNA e.p.-immunization should be considered as an effective vaccine strategy for the prevention of gut-mucosal infectious diseases.