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Aim: To assess the utility of the perioperative use of direct oral anticoagulants for patients with hepatocellular carcinoma (HCC) with cancer-associated thrombosis., Case Report: An 83-year-old woman was admitted with a solitary HCC (10-cm diameter), as well as with multiple sites of venous thromboembolism and macroscopic portal vein tumor thrombosis. She had appropriate liver function without viral hepatitis, triple-positive tumor markers, and secondary polycythemia. Edoxaban at 30 mg was initiated 10 days before surgery to remove HCC. Complete remission of the pulmonary embolism and stability of the deep vein thrombosis and massive superior mesenteric vein thrombosis were recognized preoperatively. An extended left hepatectomy was successfully performed. To avoid hemorrhage complications, we used intravenous administration of nafamostat mesylate for 2 days, thereafter we restarted edoxaban. Superior mesenteric vein thrombosis resolved 5 months after surgery., Conclusion: Perioperative oral administration of edoxaban was useful in multidisciplinary treatment for a patient with advanced HCC with cancer-associated thrombosis., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: An ectopic hepatocellular carcinoma (EHCC) arises from the ectopic liver which is defined as a hepatic organ or tissue not connected to surrounding tissues. EHCC is a rare disease and it is difficult to diagnose preoperatively. Furthermore, the clinical features are not fully elucidated., Case Summary: A retroperitoneal tumor (6 cm) was located at the dorsal side of the pancreas head on abdominal ultrasonography in an 81-year old woman positive for hepatitis C virus antibody. Contrast enhanced-computed tomography and gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid-enhanced magnetic resonance imaging showed viable HCC patterns with early enhancement and delayed washout. The tumor markers - serum alpha-fetoprotein and alpha-fetoprotein-L3% - were increased to 30.1 ng/mL and 83.1%, respectively. Protein induced by vitamin K absence or antagonist-II was within normal levels (17 mAU/mL). Positron emission tomography-computed tomography showed strong accumulation into the tumor (Standardized Uptake Value max: 13.8), and the tumor cytology following endoscopic ultrasound-guided fine needle aspiration showed poorly differentiated carcinoma. Tumor extirpation was performed, and operative findings showed that the retroperitoneal tumor was disconnected from the pancreas and the liver. Swollen lymph nodes near the tumor were histologically normal. On histological examination, the tumor was finally diagnosed as EHCC with Arginase-1 positive expression., Conclusion: We report our experience of a rare EHCC which was difficult to diagnose, and we present a review of the literature., Competing Interests: Conflict-of-interest statement: The authors declare that they have no conflicts of interest., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.)

Glucokinase is expressed principally in pancreatic β-cells and hepatocytes, and catalyzes the phosphorylation of glucose to glucose-6-phosphate, a rate-limiting step of glycolysis. To better understand the roles of hepatic glucokinase, we generated Gck knockout mice by ablating liver-specific exon 1b. The knockout mice exhibited impaired glucose tolerance, decreased hepatic glycogen content, and reduced Pklr and Fas gene expression in the liver, indicating that hepatic glucokinase plays important roles in glucose metabolism. It has also been reported that hepatic glucokinase regulates the expression of thermogenesis-related genes in brown adipose tissue (BAT) and insulin secretion in response to glucose. However, the liver-specific Gck knockout mice displayed neither altered expression of thermogenesis-related genes in BAT nor impaired insulin secretion by β-cells under a normal chow diet. These results suggest that chronic suppression of hepatic glucokinase has a small influence on intertissue (liver-to-BAT as well as liver-to-β-cell) metabolic communication., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Hepatocellular carcinoma (HCC), the fifth most common cancer type and the third highest cause of cancer death worldwide, develops in different types of liver injuries, and is mostly associated with cirrhosis. However, non-alcoholic fatty liver disease often causes HCC with less fibrosis, and the number of patients with this disease is rapidly increasing. The high mortality rate and the pathological complexity of liver diseases and HCC require blood biomarkers that accurately reflect the state of liver damage and presence of HCC., Methods and Findings: Here we demonstrate that a circulating protein, apoptosis inhibitor of macrophage (AIM) may meet this requirement. A large-scale analysis of healthy individuals across a wide age range revealed a mean blood AIM of 4.99 ± 1.8 µg/ml in men and 6.06 ± 2.1 µg/ml in women. AIM levels were significantly augmented in the younger generation (20s-40s), particularly in women. Interestingly, AIM levels were markedly higher in patients with advanced liver damage, regardless of disease type, and correlated significantly with multiple parameters representing liver function. In mice, AIM levels increased in response to carbon tetrachloride, confirming that the high AIM observed in humans is the result of liver damage. In addition, carbon tetrachloride caused comparable states of liver damage in AIM-deficient and wild-type mice, indicating no influence of AIM levels on liver injury progression. Intriguingly, certain combinations of AIM indexes normalized to liver marker score significantly distinguished HCC patients from non-HCC patients and thus could be applicable for HCC diagnosis., Conclusion: AIM potently reveals both liver damage and HCC. Thus, our results may provide the basis for novel diagnostic strategies for this widespread and fatal disease.

The C57BL/6:Slc23a13(-/-);Gpd2(-/-) double-knockout (a.k.a., citrin/mitochondrial glycerol 3-phosphate dehydrogenase double knockout or Ctrn/mGPD-KO) mouse displays phenotypic attributes of both neonatal intrahepatic cholestasis (NICCD) and adult-onset type II citrullinemia (CTLN2), making it a suitable model of human citrin deficiency. In the present study, we show that when mature Ctrn/mGPD-KO mice are switched from a standard chow diet (CE-2) to a purified maintenance diet (AIN-93M), this resulted in a significant loss of body weight as a result of reduced food intake compared to littermate mGPD-KO mice. However, supplementation of the purified maintenance diet with additional protein (from 14% to 22%; and concomitant reduction or corn starch), or with specific supplementation with alanine, sodium glutamate, sodium pyruvate or medium-chain triglycerides (MCT), led to increased food intake and body weight gain near or back to that on chow diet. No such effect was observed when supplementing the diet with other sources of fat that contain long-chain fatty acids. Furthermore, when these supplements were added to a sucrose solution administered enterally to the mice, which has been shown previously to lead to elevated blood ammonia as well as altered hepatic metabolite levels in Ctrn/mGPP-KO mice, this led to metabolic correction. The elevated hepatic glycerol 3-phosphate and citrulline levels after sucrose administration were suppressed by the administration of sodium pyruvate, alanine, sodium glutamate and MCT, although the effect of MCT was relatively small. Low hepatic citrate and increased lysine levels were only found to be corrected by sodium pyruvate, while alanine and sodium glutamate both corrected hepatic glutamate and aspartate levels. Overall, these results suggest that dietary factors including increased protein content, supplementation of specific amino acids like alanine and sodium glutamate, as well as sodium pyruvate and MCT all show beneficial effects on citrin deficiency by increasing the carbohydrate tolerance of Ctrn/mGPD-KO mice, as observed through increased food intake and maintenance of body weight., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Chronic liver inflammation is a critical component of hepatocarcinogenesis. Indeed, inflammatory mediators are believed to promote liver cancer by upholding compensatory proliferation of hepatocytes in response to tissue damage. However, inflammation can also mediate the depletion of malignant cells, but the difference between tumor-suppressive and tumor-promoting inflammation is not defined at the molecular level. Here, we analyzed the role of the major inflammatory mediator IFN-γ in chemical hepatocarcinogenesis of transgenic mice that overexpress IFN-γ in the liver; these mice manifest severe chronic inflammatory liver damage and lasting compensatory regeneration. We found that chronic exposure to IFN-γ suppressed chemical hepatocarcinogenesis, despite overt liver injury. Indeed, IFN-γ-transgenic mice had significantly fewer and significantly less advanced malignant lesions than nontransgenic mice. This tumor-suppressive effect of IFN-γ seemed to be mediated in part by its known immune activating function, indicated by infiltration of IFN-γ-transgenic livers with CD8 T cells, natural killer T cells, and natural killer cells. However, IFN-γ seemed to prevent carcinogenesis also by activating the cell-intrinsic p53 tumor suppressor pathway. Indeed, exposure to IFN-γ in vivo or in vitro was associated with accumulation of p53 in hepatocytes and the sensitization of hepatocytes to apoptosis induced by genotoxic stress. The IFN-γ-induced increase in apoptosis of hepatocytes seemed to be p53 dependent. Thus, chronic inflammation dominated by IFN-γ may prevent hepatocarcinogenesis, despite continued inflammatory liver injury and regeneration. Therefore, the carcinogenic potential of inflammation seems to be determined by type and composition of its mediators and manipulating the type of chronic inflammation may serve the prevention of cancer.

The ornithine decarboxylase antizyme inhibitor (AZI) was discovered as a protein that binds to the regulatory protein antizyme and inhibits the ability of antizyme to interact with the enzyme ornithine decarboxylase (ODC). Several studies showed that the AZI protein is important for cell growth in vitro. However, the function of this gene in vivo remained unclear. In our study, we analyzed the transcriptional profiles of livers on the 19th day of pregnancy of Azin1 knock-out mice and wild-type mice using the Agilent oligonucleotide array. Compared to the wild-type mice, in the liver of Azin1 knock-out mice 1812 upregulated genes (fold change > or = 2) and 1466 downregulated genes (fold change < or = 0.5) were showed in the microarray data. Altered genes were then assigned to functional categories and mapped to signaling pathways. These genes have functions such as regulation of the metabolism, transcription and translation, polyamine biosynthesis, embryonic morphogenesis, regulation of cell cycle and proliferation signal transduction cascades, immune response and apoptosis. Real-time PCR was used to confirm the differential expression of some selected genes. Overall, our study provides novel understanding of the biological functions of AZI in vivo.

Human transthyretin (TTR) has about 110 variants, more than 90 of which are associated with human amyloidosis. Several groups have generated transgenic mice that carry various mutant TTR genes. However, formation of mouse/human TTR heterotetramers has been shown to be inhibitory to dissociation and subsequent amyloid formation. To avoid the effect of mouse Ttr and produce humanized mice carrying different TTR variants at high efficiency, we first produced a null allele in the mouse transthyretin locus using targeting vector that contained a neomycin resistance gene flanked by lox71 and loxP. Then, through Cre-mediated recombination, we created a replacement allele that carried either a human normal (Val30) or mutant (Met30) TTR cDNA. This replacement resulted in a humanized TTR mouse with similar tissue-specific profile of human TTR as that of the endogenous mouse Ttr gene. The expression levels of human TTR mRNA and protein in the liver of homozygous human TTR (Val30/Val30) mice were about twice those of heterozygous mouse/human TTR (+/Val30) mice. However, the serum human TTR levels in the Val30/Val30 mice were much less than those in the +/Val30 mice. This contradictory expression was due to unstable Val30 tetramers caused by low binding affinity to mouse retinol binding protein.

Hepatic stellate cells are believed to play a key role in the development of liver fibrosis. Several studies have reported that bone marrow cells can give rise to hepatic stellate cells. We hypothesized that hepatic stellate cells are derived from hematopoietic stem cells. To test this hypothesis, we generated chimeric mice by transplantation of clonal populations of cells derived from single enhanced green fluorescent protein (EGFP)-marked Lin(-)Sca-1(+)c-kit(+)CD34(-) cells and examined the histology of liver tissues obtained from the chimeric mice with carbon tetrachloride (CCl(4))-induced injury. After 12 weeks of CCl(4) treatment, we detected EGFP(+) cells in the liver, and some cells contained intracytoplasmic lipid droplets. Immunofluorescence analysis demonstrated that 50% to 60% of the EGFP(+) cells were negative for CD45 and positive for vimentin, glial fibrillary acidic protein, ADAMTS13, and alpha-smooth muscle actin. Moreover, EGFP(+) cells isolated from the liver synthesized collagen I in culture. These phenotypes were consistent with those of hepatic stellate cells. The hematopoietic stem cell-derived hepatic stellate cells seen in male-to-male transplants revealed only one Y chromosome. Our findings suggest that hematopoietic stem cells contribute to the generation of hepatic stellate cells after liver injury and that the process does not involve cell fusion.

Acceleration of amyloid deposition by administration of amyloid fibrils and transmissibility of disease have been reported in several types of amyloidoses. Families with a variant transthyretin (TTR V30M)-associated familial amyloidotic polyneuropathy (FAP) exhibit genetic anticipation, with TTR V30M-amyloid depositing at an earlier age in successive generations. The molecular bases of anticipation in FAP have remained to be determined. We asked if administration of TTR-amyloid fibrils (ATTR) extracted from the heart of an FAP TTR V30M patient would accelerate ATTR deposition in transgenic mice expressing the human mutant ttr gene responsible for FAP TTR V30M and indeed the administration did accelerate deposition of apolipoprotein A-II-amyloid fibrils (AApoAII), and not A TTR. Our experiments present, for the first time, evidence that the degree of inducibility of ATTR is low relative to AApoAII and we suggest that administration of ATTR may not explain the genetic anticipation which occurs in FAP.

Cholesteryl ester transfer protein (CETP) is one of the key proteins in reverse cholesterol transport (RCT). The role of CETP in atherosclerosis remains controversial. In this study we investigated the associations between polymorphisms of CETP (mutations in intron 14 and exon 15, and Taq1B), hepatic lipase (C-514T), lipoprotein lipase ( PvuII and HindIII), and ATP-binding cassette transporter 1 (R219K) loci and longevity in 256 centenarians and 190 healthy younger controls. Although heterozygous CETP deficiency and the B2 allele of the Taq1B polymorphism was consistently associated with higher HDL-C concentrations both in centenarians and controls, the allelic frequencies of those polymorphisms did not differ between the two groups. The allelic frequencies of other gene polymorphisms in RCT were not different between the two groups. Centenarians with lipoprotein lipase P(-/-) genotype had significantly higher HDL-C concentration than those with P(-/+) or with P(+/+), in contrast, there was no such a relationship among controls. In stepwise multiple regression analysis, serum albumin, CETP deficiency and lipoprotein lipase PvuII genotype were independently associated with HDL-C in centenarians. Sex, CETP deficiency, and the Taq1B genotype were also independently associated with HDL-C; however, lipoprotein lipase PvuII genotype had no significant effect on their HDL-C in controls. In conclusion, we observed that CETP deficiency and other gene polymorphisms in RCT have no impact on longevity for Japanese centenarians.

Antigen presenting cells, especially the antigen presenting dendritic cells (DC) in the tissue, regulate the magnitude of antigen-specific immune response. A role of impaired and narrowly focused specific immune response has been implicated in the pathogenesis of chronic hepatitis due to hepatitis B virus and hepatitis C virus. In order to clarify this role, we studied liver DC from interferon gamma (IFN-gamma) transgenic mouse (TgM), an animal model of chronic hepatitis. These mice had high serum levels of alanine transaminase and histological evidence of chronic hepatitis. Transgene negative offspring (littermate control) with normal serum transaminase levels and without any evidence of hepatitis were used as controls. The stimulatory capacity of the liver DC from IFN-gamma TgM in allogenic mixed leukocyte reaction was significantly lower than that of the liver DC from control mouse. The endocytosis capacity was significantly lower in liver DC from IFN-gamma TgM than in that from the control mouse. Most importantly, liver DC from IFN-gamma TgM were unable to induce antigen-specific proliferation. The impaired function of liver DC from these mice may be attributable to increased production or induction of suppressor cytokines such as interleukin-10 and nitric oxide. Defective capacity of liver DC from mouse with chronic hepatitis (IFN-gamma TgM) may be related to impaired magnitude of specific immune response in the liver.

The interferon-gamma (IFN-gamma) transgenic mouse expresses the exogenous IFN-gamma gene in the liver and develops chronic hepatitis. For the present experiment, four IFN-gamma transgene (+) mice of 48 weeks of age and 16 IFN-gamma transgene (+) mice of 8 weeks of age were used. The four IFN-gamma transgene (+) mice of 48 weeks of age showed significantly elevated plasma alanine aminotransferase (ALT) and expressed the inducible form of nitric oxide synthase (iNOS) mRNA in the liver. Of the 16 IFN-gamma transgene (+) mice of 8 weeks of age, iNOS mRNA was expressed in the livers of three. These three mice exhibited higher plasma ALT levels than the other mice of 8 weeks of age. The present results suggest that iNOS mRNA expression in the liver might be correlated with the progression of hepatitis.

Interferon-gamma (IFN-gamma) transgenic mice expressed the IFN-gamma gene in the liver and developed chronic hepatitis. The expression of cyclooxygenase (Cox)-1 and -2 mRNA were studied in the livers of these mice. A faint band of Cox-1 mRNA was observed in the livers of both normal and transgenic mice. Slight expression of Cox-2 mRNA was observed in the normal mouse liver, and this mRNA expression was induced in the transgenic mouse liver. The present results showed the induction of Cox-2 mRNA expression in the livers of transgenic mice with chronic hepatitis.

Seroconversion from HBeAg to alphaHBe of persons chronically infected by HBV is usually associated with a transient exacerbation of liver disease and subsequent normalization of liver histology. It is speculated that these clinico-pathological features may be due to the activation of cytodestructive mechanisms by alphaHBe antibodies. The aim of the present study was to investigate the pathogenic potential of alphaHBe antibodies in a transgenic mouse model. Therefore, alphaHBe autoantibodies were elicited in double-transgenic mice expressing high amounts of HBeAg and interferon-gamma in the liver. Interferon-gamma has reviously been shown to play an important role in the development of hepatic necroinflammation associated with hepadnaviral infection, probably via tumor-necrosis-factor-alpha secreted by activated macrophages. We found no evidence that alphaHBe antibodies have the potential to destroy HBeAg-secreting hepatocytes even if the cells were predisposed to injury due to high-level interferon-gamma expression. We conclude that seroconversion from HBeAg to alphaHBe of persons chronically infected with HBV seems to be an immunological epiphenomenon without pathogenic significance.

There are few reports regarding the effects of benzene and ethanol being administered simultaneously. In our experiments with 4 groups (controls, ethanol, benzene and ethanol plus benzene) Wistar male rats were treated with ethanol (20%) for 5 weeks, and then exposed to benzene (0.26 g/kg) for 5 days per week for 3 weeks. We also investigated the effects of benzene on the body weight, organ weight, peripheral hematology and hepatic drug metabolizing enzymes in the ethanol administrated rats. The liver weight increased significantly, but spleen weight decreased significantly in the benzene exposed group. Hematological examination showed a decrease of leukocyte in the two groups of benzene and ethanol plus benzene in comparison with the controls, but an effect promoted by ethanol was not found. Serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) values were not significantly different in the exposure groups when compared with the controls. The contents of microsomal cytochrome P450 in all the exposed groups showed a tendency to increase, but they were not significantly different in comparison with the controls. On the other hand, hepatic glutathione S-transferase activity in the exposed groups increased significantly.

The interferon-gamma (IFN-gamma) transgenic mouse develops chronic hepatitis. In the present study, lymphoid cell infiltration and expression of the Fas system were studied in the transgenic mouse liver. Morphological analysis showed the infiltration of lymphoid cells at the portal area and necro-inflammation in the lobules. Furthermore, the infiltrating lymphoid cells were found to be present in the areas of piecemeal necrosis and the intralobular region. The polymerase chain reaction revealed induction of Fas antigen and Fas ligand mRNA expressions. The chronic hepatitis in this mouse reflects the profiles of human chronic hepatitis.

Carcinogen-resistant inbred DRH rats developed from the Donryu strain showed a remarkably low incidence of liver tumors when they were fed diets containing hepatocarcinogens such as 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). In this work, we examined various characteristics of male DRH and Donryu rats during 3'-Me-DAB administration for 8 weeks. 32P-Postlabeling analysis showed that essentially similar levels of DNA-adducts were generated by the metabolites of 3'-Me-DAB in the livers of these two strains of rats at several time points. However, both GADD45 (growth arrest and DNA damage-inducible) and O6-methylguanine methyltransferase (putatively DNA damage-inducible) mRNA levels were increased significantly in Donryu rat livers, but were increased to a lesser extent in DRH rats. [3H]Thymidine incorporation into hepatic DNA began to increase around 10 to 20 days after the start of 3'-Me-DAB administration in Donryu rats probably due to DNA repair, while no significant change occurred in DRH rats under the same conditions. Furthermore, inductions of heme oxygenase (due to degradation of heme-proteins) and hepatocyte growth factor (HGF; cell death and regeneration of hepatocytes) mRNAs were greater in Donryu rat livers than those of DRH, suggesting that the former were more sensitive to cytotoxic effects of 3'-Me-DAB than the latter. Another remarkable difference observed between these two strains was the significant induction of cytochrome P-450 2E1 mRNA in Donryu rat livers; this may contribute to the generation of reactive oxygen intermediates. Finally, increases of glutathione S-transferase (P-form) and gamma-glutamyltranspeptidase mRNAs as marker enzymes of preneoplastic changes of hepatocytes were clearly seen only in Donryu rat livers at 6 to 8 weeks after the start of 3'-Me-DAB administration. These results indicate that the different susceptibility to hepatocarcinogenesis between these two strains of rats may arise from events other than the DNA adduct formation.

N-Acetylglucosaminyltransferase III (GnT-III) produces "bisecting-GlcNAc" and regulates the branching of N-glycans. GnT-III activity is elevated during hepatocarcinogenesis, which is in contrast to the undetectable level found in normal hepatocytes. To determine the biological significance of GnT-III in hepatocytes, transgenic mice that specifically express GnT-III in the liver were established and characterized. The transgenic hepatocytes had a swollen oval-like morphology, with many lipid droplets. Apolipoprotein B, which contained increased level of bisecting-GlcNAc accumulated in the transgenic hepatocytes. In the transgenic serum, triglycerides, the beta- and pre-beta-lipoprotein fractions, and apolipoprotein B100 were significantly decreased, compared with levels in nontransgenic serum. These abnormal phenotypes were more prominent in the mice with more copies of the transgene and a resulting high GnT-III activity. We demonstrate that aberrant glycosylation, as the direct result of the formation of bisecting-GlcNAc, disrupts the function of apolipoprotein B, leading to the generation of fatty liver. This observation suggests a novel mechanism for the pathogenesis of fatty liver.

Mouse embryonic stem cells can differentiate into various cell types within cell aggregates called embryoid bodies (EBs). This structure consists of ectodermal, mesodermal, and endodermal tissues, which resemble the embryo of egg-cylinder stage. After 8-10 days in culture, about half of the EBs expand into large cystic structures homologous to visceral yolk sac of postimplantation embryos. To study endoderm differentiation at molecular level, we examined expression of endoderm marker genes during the processes of EB development. alpha-Fetoprotein (AFP) and transthyretin (TTR) transcripts increased at the stage when embryoid bodies began to form yolk-sac-like structures and were expressed strongly thereafter. Expression of hepatocyte nuclear factor (HNF) 4, a variant form of HNF1 (also called HNF1beta), and HNF3beta started before the onset of AFP and TTR expression. HNF1 (also called HNF1alpha) expression began a few days after the onset of the expression of the transcription factors described above. Serum albumin (ALB) transcript was only found in late large cystic EBs. Also, AFP gene expression preceded ALB gene expression. These results suggest that the patterns of endoderm gene expression during EB development reflect the order found during mouse development in vivo, and EB formation may serve as an in vitro system to study the differentiation process.

Cytokine gene expression in the liver of transgenic mouse which specifically expresses interferon-gamma gene (IFN-gamma) was measured using polymerase chain reaction analysis. IFN-gamma mRNA was found to be expressed in the transgenic mouse liver along with TNF-alpha mRNA. Messenger RNAs of CD4 and CD8, maturation markers of T cells, were expressed in both normal and transgenic mouse livers, indicating the presence of resident T cells. The level of CD4 and CD8 expression was slightly elevated in the transgenic mouse case, indicating an increased number of CD4+ and/or CD8+ T cells, but the lack of the activation marker IL-2 mRNA suggested a nonactivated state. TGF-beta 1 mRNA was expressed in both normal and transgenic mouse livers without any increase in the latter, and therefore does not appear to be involved in the development of chronic active hepatitis in this transgenic mouse. Since TNF-alpha is a terminal mediator of liver injury in several animal liver injury models, its high expression, presumably a result of the IFN-gamma which is transcribed from the introduced gene, might be involved in the development of chronic active hepatitis in this transgenic mouse.

The enhancer of the rat ornithine transcarbamylase gene is located 11 kilobases upstream from the transcription start site and has been shown to be hepatoma cell-specific. Using transgenic mice, we showed that this enhancer is capable of activating transcription in a liver-specific manner, inverting the tissue specificity of the homologous promoter that is by itself more active in the small intestine than in the liver. Transient transfection analysis with cultured hepatoma cells indicated that the enhancer activity resides in the approximately 110-base pair region containing four protein-binding sites, two for hepatocyte nuclear factor-4 (HNF-4) and two for CCAAT/enhancer binding protein (C/EBP), both of which are liver-selective transcription factors. Concatemerization of a region containing one HNF-4 and one C/EBP site led to reconstitution of the hepatoma cell-specific enhancer, and intactness of these two sites was strictly required for the enhancer activity. Furthermore, cotransfection experiments showed that both HNF-4 and C/EBP beta are necessary, and neither alone sufficient, for activation of the reconstituted enhancer in nonhepatic cells. Requirement of combinatorial operation of at least two liver-enriched transcription factors for transcriptional activation successfully explains why these liver-selective but not strictly liver-specific factors can confer more restricted liver specificity on transcription of their target genes.

We previously developed a method for introducing foreign genes into liver tissue using liposomes with incorporated hemagglutinating virus of Japan (HVJ, Sendai virus), and found that liver cells transfected with the E. coli beta-galactosidase gene or the gene for hepatitis B virus (HBV) surface protein (HBsAg) expressed these proteins in vivo. Here, we analyzed cellular reactions leading to hepatitis in the liver by expressing the genes of HBV in vivo. Lymphocytes were eluted directly from liver transfected with the HBsAg genes and shown to be cytotoxic only to cells expressing HBsAg in vitro. These lymphocytes were identified as cytotoxic T lymphocytes with the CD4- CD8+ phenotype. Transfer of these lymphocytes to transgenic mice with the whole HBV genome led to elevation of the serum glutamic-pyruvic transaminase (SGPT) level, indicating the induction of hepatitis due to the cytotoxic T lymphocytes in vivo. Similarly, direct transfer of the gene for the HBV secretory core protein (HBeAg) induced expression of HBeAg in hepatocytes and the appearance of antibody against HBeAg in the serum. However, using this system, we found that the lymphocytes infiltrating the transfected liver showed no cytotoxicity specific for HBeAg. These results indicate that expression of HBsAg, one of the components of virions, in animal liver induced hepatitis efficiently through generation of specific cytotoxic T lymphocytes (CTL) without any expression of the other viral components. This in vivo experimental system should be useful for evaluating how expression of a given gene induces cellular reactions and intrinsic functions in the living body.

Transgenic mice which carry the integrated human hepatitis B virus (HBV) genome and produce HBV particles in the liver were treated with antiviral compounds, and the amount of viral DNA in the liver and serum was monitored by Southern analyses. Mouse alpha interferon (200,000 U per injection, twice daily for 3 days) and a novel nucleoside, oxetanocin G (15 mg/kg of body weight per injection, twice daily for 7 days), inhibited viral DNA synthesis in the liver almost completely. This animal model should prove useful in evaluating anti-HBV agents.

Male Wistar strain rats were exposed to acute stress from broad-band noise [100 dB (A) for 1 h] or cold (4 degrees C for 24 h). Following exposure to noise for 1 h, serum and adrenal gland showed about 1.8 times and 2.5 times the corticosterone levels of the control group, but this enhancement was not observed after exposure to cole. Both stresses increased Zn-thionein significantly in the liver. Furthermore, the two stresses increased the content of Zn in the 30,000 g supernatant fraction of the liver, though serum Zn concentration was not altered. It is believed that thionein is a regulatory protein with a specific function for Zn metabolism in organs. Although it is known that corticosterone affects Zn metabolism in man and animals, there is little investigation of the effect of environmental stresses on Zn metabolism. The present data suggest that Zn metabolism may be important in the investigation of the effects of repeated and long term exposure to environmental stresses involving noise and cold.

Three lines of transgenic mice carrying hepatitis B virus (HBV) DNA were produced. In a pSHB1BB transgenic mouse carrying one copy of BamHI fragment of HBV with a deletion of Bg/II-Bg/II fragment, no RNA transcription was observed. Transgenic mice that had integrated either one or three tandem copies of HBV DNA produced RNA in various tissues except liver. However, neither initiation nor termination of transcription occurred at the correct place. Hepatitis B surface antigen and hepatitis B e antigen were not detectable in these transgenic mice. HBV DNA was methylated at all CCGG sequences, which might have resulted in aberrant RNA production. In two out of five transgenic mice HBV DNA was integrated into the Y chromosome. Although the frequency is unusually high, the mechanism is not known yet.

Summary. Seroconversion from HBeAg to αHBe of persons chronically infected by HBV is usually associated with a transient exacerbation of liver disease and subsequent normalization of liver histology. It is speculated that these clinicopathological features may be due to the activation of cytodestructive mechanisms by αHBe antibodies. The aim of the present study was to investigate the pathogenic potential of αHBe antibodies in a transgenic mouse model. Therefore, αHBe autoantibodies were elicited in double-transgenic mice expressing high amounts of HBeAg and interferon-γ in the liver. Interferon-γ has previously been shown to play an important role in the development of hepatic necroinflammation associated with hepadnaviral infection, probably via tumor-necrosis-factor-α secreted by activated macrophages. We found no evidence that αHBe antibodies have the potential to destroy HBeAg-secreting hepatocytes even if the cells were predisposed to injury due to high-level interferon-γ expression. We conclude that seroconversion from HBeAg to αHBe of persons chronically infected with HBV seems to be an immunological epiphenomenon without pathogenic significance. [ABSTRACT FROM AUTHOR]