Gene therapy targeting for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by a hypoxia-inducible enhancer linked to a human alpha-fetoprotein promoter.

We previously reported that the retroviral vector expressing the herpes simplex virus-thymidine kinase gene under the control of 0.3-kb human alpha-fetoprotein (AFP) gene promoter (AF0.3) provided the cytotoxicity to ganciclovir (GCV) in high-AFP-producing human hepatoma cells but not in low-AFP-producing cells. Therefore, specific enhancement of AFP promoter activity is likely to be required to induce enough cytotoxicity in low-AFP-producing hepatoma cells. In this study, we constructed a hybrid promoter, [HRE]AF, in which a 0.4-kb fragment of human vascular endothelial growth factor 5'-flanking sequences containing hypoxia-responsive element (HRE) was fused to AF0.3 promoter. By means of the reporter gene transfection assay, hypoxia-inducible transcriptions that were mediated by [HRE]AF promoter were detected in low- and non-AFP-producing human hepatoma cells, but not in nonhepatoma cells. When the herpes simplex virus-thymidine kinase gene controlled by [HRE]AF promoter was transduced into hepatoma and nonhepatoma cells by a retroviral vector, the exposure to 1% O2 induced GCV cytotoxicity specifically in the hepatoma cells. Moreover, in nude mice bearing solid tumor xenografts, only the tumors consisting of the virus-infected hepatoma cells gradually disappeared by GCV administration. These results indicate that the hypoxia-inducible enhancer of the human vascular endothelial growth factor gene, which is directly linked to human AFP promoter, involves selective and enhanced tumoricidal activity in gene therapy for hepatocellular carcinoma.

Transduction of antisense cyclin D1 using two-step gene transfer inhibits the growth of rat hepatoma cells.

Cyclin D1, one of the G(1) cyclins, is frequently overexpressed in several types of carcinomas and is thought to play an important role in tumorigenesis and tumor progression including hepatocellular carcinoma. We constructed a retrovirus vector-carrying rat cyclin D1 cDNA in the reverse orientation, resulting in expression of antisense (AS) cyclin D1 mRNA. For efficient transduction of this recombinant retrovirus, two-step gene transfer was performed. The rat hepatoma cell line (dRLh84) was infected with this recombinant retrovirus after preinfection with adenovirus expressing the retrovirus receptor. In the rat hepatoma cells, AS cyclin D1 mRNA was expressed, inducing a decrease in the expression of endogenous cyclin D1 mRNA and an inhibition of cell growth. Moreover, two-step gene transfer of AS cyclin D1 into s.c. hepatoma xenografts resulted in inhibition of tumor growth and prolonged animal survival. In the virus-infected tumor xenografts, expression of cyclin D1 was immunohistochemically inhibited, and apoptosis of hepatoma cells was detected. These findings suggest that transduction of AS cyclin D1 is useful as an adjunct to standard treatments for hepatocellular carcinoma.

Liver cell adenoma in a young man with elevated serum PIVKA-II level.

A 21-year-old man was referred to our hospital because of a liver mass lesion detected by abdominal ultrasonography. He had received no hormonal treatment. Physical examinations revealed no abnormalities, and laboratory data, including hepatic function test results, were within normal ranges, with the exception of elevated levels of serum protein induced by vitamin K absence or antagonist (PIVKA)-II (2.2 AU/ml). Abdominal ultrasonography revealed a hyperechoic mass lesion measuring 10 x 10 cm, with hypoechoic areas located in the right posterior segment of the liver. A low-density area and a hypervascular area were detected in the right posterior segment of the liver by computed tomography and celiac angiography, respectively. As hepatocellular carcinoma could not be completely excluded, the tumor was resected. The tissue consisted of sheets of tumor cells with eosinophilic cytoplasm and round nuclei showing a thin trabecular pattern, and these histological findings indicated liver cell adenoma. After resection of the tumor, serum PIVKA-II returned to the normal level.

A case of chronic hepatitis C developing insulin-dependent diabetes mellitus associated with various autoantibodies during interferon therapy.

We report a case of chronic hepatitis C presenting insulin-dependent diabetes mellitus (IDDM) associated with various autoantibodies including possible anti-insulin receptor antibody (AIRA) during interferon (IFN) therapy. A 57-year-old man having chronic hepatitis C virus (HCV) infection with chronic thyroiditis received IFN therapy. The thyroid function was well-controlled by administration of thyroid hormone, although thyroid autoantibodies were positive. At 15 weeks after starting IFN (reaching 530 million units of total dose), marked thirst happened, with increased fasting plasma glucose level (488 mg/dl) and decreased daily urinary C peptide immunoreactivity level (less than 4.2 microg/day). IDDM occurred with anti-nuclear antibody (ANA), anti-DNA antibody and possible AIRA, and thyroid autoantibodies titers increased, but without pancreatic islet cell antibody and anti-glutamic acid decarboxylase antibody. Administration of IFN was stopped and insulin treatment was started, but plasma glucose level was not controlled well. AIRA became negative 2 months later, however, insulin antibody (IA) was positive when tested after 18 months. Serum HCV RNA has been negative, and a normal level of serum transaminase has been observed since IFN therapy. It is likely that IFN therapy induced the immunological disturbance and resulted in occurrence of various autoantibodies and IDDM in the patient.

A case of acute hepatitis A with marked hemophagocytosis in bone marrow.

A previously well 18-year-old female was referred to our hospital because of abnormalities of blood biochemistry and slight jaundice. Because serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were elevated more than 6000 IU/L, the patient was suspected to have acute viral hepatitis. The platelet count on admission was 9.7x10(4)/µl, which was decreased from the initial value of 21x10(4)/µl for 3 days. The coagulation tests revealed marked elevation of D-dimmer, fibrinogen degradation products and thrombin-antithrombin III complex suggesting increase in fibrinolysis. Serum levels of high density lipoprotein cholesterol and ferritin were markedly decreased and increased, respectively. The bone marrow smears revealed proliferation of mature histiocytes ingesting platelets and erythrocytes, these pathological findings were consistent with those of hemophagocytic syndrome (HPS). In addition, anti-hepatitis A IgM antibody in the serum and hepatitis A virus (HAV) RNA in the stool were positive. Therefore, the patient was diagnosed as having acute hepatitis A with probable HPS. Since a fulminant clinical course was suspected, glucocorticoid pulse therapy was started immediately 7 days after onset and a favorable clinical outcome resulted.

[A case of systemic lupus erythematosus showing abnormal lipoproteins due to accompanied drug induced hepatitis].

We describe here a 28-years-male with AIHA and SLE who had lipid and lipoprotein abnormalities during cholestasis induced by PGE1 administration. High free cholesterol level, 792 mg/dl was found in his serum, and markedly elevated, phospholipid level 1,614 mg/dl. But, LCAT activity was within normal range in this case. An agarose gel electrophoresis of lipoproteins showed abnormal bands which were located in slow alpha 2, pre beta and slow beta, and between beta and origin point. Moreover, it was detected formation of Lp-X from serum of the patient. Serum levels of apoprotein B, C-II, C-III, and E were higher, while apoprotein A-I, A-II were very lower than reference value. From these results, it was suspected that the patient might occur transient abnormal lipid metabolism according to the drug induced hepatic injury.

Evidence-based clinical practice guidelines for nonalcoholic fatty liver disease/nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is currently the most common cause of chronic liver disease in industrialized countries worldwide, and has become a serious public health issue not only in Western countries but also in many Asian countries including Japan. Within the wide spectrum of NAFLD, nonalcoholic steatohepatitis (NASH) is a progressive form of disease, which often develops into liver cirrhosis and increases the risk of hepatocellular carcinoma. In turn, a large proportion of NAFLD/NASH is the liver manifestation of metabolic syndrome, suggesting that NAFLD/NASH plays a key role in the pathogenesis of systemic atherosclerotic diseases. Currently, a definite diagnosis of NASH requires liver biopsy, though various noninvasive measures are under development. The mainstays of prevention and treatment of NAFLD/NASH include dietary restriction and exercise; however, pharmacological approaches are often necessary. Currently, vitamin E and thiazolidinedione derivatives are the most evidence-based therapeutic options, although the clinical evidence for long-term efficacy and safety is limited. This practice guideline for NAFLD/NASH, established by the Japanese Society of Gastroenterology in cooperation with The Japan Society of Hepatology, covers lines of clinical evidence reported internationally in the period starting from 1983 to January 2012, and each clinical question was evaluated using the GRADE system. Based on the primary release of the full version in Japanese, this English summary provides the core essentials of this clinical practice guideline comprising the definition, diagnosis, and current therapeutic recommendations for NAFLD/NASH in Japan.(AU)

Usefulness of a new immuno-radiometric assay to detect hepatitis C core antigen in a community-based population.

A new immuno-radiometric assay (IRMA) to detect hepatitis C virus (HCV) core antigen (HCVcAg) has been developed. The aim of the present study was to investigate the sensitivity and specificity of this IRMA to measure HCV antigenemia, based on the detection of HCV RNA as the gold standard, and to assess the utility of the IRMA in a community-based population. Anti-HCV positive residents in a hyperendemic area of HCV infection in Japan were studied. Serum levels of HCVcAg were measured using IRMA, and the presence of HCV RNA was determined by a qualitative reverse transcription-polymerase chain reaction (RT-PCR) assay. The sensitivity and the specificity of the IRMA were 96.4 and 100%, respectively. The sensitivity of the IRMA was similar between serological HCV group I (HCV genotypes 1a and 1b) (97.6%) and group II (HCV genotypes 2a and 2b) (94.0%). There was a strong correlation between serum HCVcAg level and HCV-RNA measured by a quantitative RT-PCR (r = 0.832, P < 0.0001). There also was a very strong correlation of HCVcAg level between IRMA measurements performed on serum and those performed on plasma (r = 0.984, P < 0.0001). In conclusion, this new IRMA is useful for the detection of HCV core antigen in a community-based population.

A case of 11q-syndrome associated with abnormalities of the retinal vessels.

A case of 11q- syndrome associated with abnormalities of the retinal vessels similar to those of familial exudative vitreoretinopathy was followed from 6 months to 3 years of age. The patient was a female infant born at 39 weeks of gestation and weighing 2,360 g without oxygen therapy. A chromosomal analysis showed a 46,XX,del(11)(q23.3). An ocular examination revealed exotropia and a positive kappa angle of the left eye with psychomotor retardation. An abnormal pattern of the retinal vessels and an avascular retina was observed in the periphery of both eyes. Therefore we concluded that the abnormalities of the retinal vessels may be related to the deletion of the long arm of chromosome 11.

Hepatoma-specific gene therapy through retrovirus-mediated and targeted gene transfer using an adenovirus carrying the ecotropic receptor gene.

Tissue-specific transcriptional regulatory sequences have been inserted in retrovirus vectors for therapeutic gene expression in cancer gene therapy. However, the transcriptional activity of these sequences is generally low, and the proviral DNA integration appears to increase the possibility of genomic DNA alteration in nontarget cells. Therefore, retrovirus-mediated targeted gene transduction into human hepatoma cells was evaluated using transient expression of an ecotropic receptor gene, mouse cationic amino acid transporter-1 (MCAT-1). Two recombinant adenoviruses, AxCAMCAT and AxAFMCAT, carrying the MCAT-1 gene under the control of the CAG and human alpha-fetoprotein (AFP) promoter, respectively, were generated. The preinfection with AxCAMCAT allowed highly efficient ecotropic retroviral infection of human cells. In addition, after AxAFMCAT infection, retroviral infection occurred only in AFP-producing hepatoma cells, resulting in selective cytotoxicity induced by the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) system. Transient expression of the MCAT-1 gene under the control of the AFP promoter permits ecotropic retrovirus-mediated gene transduction specifically in AFP-producing human hepatoma cells, resulting in selective induction of the suicide killing effect while the therapeutic gene is driven from ubiquitously expressed promoters.

Additive and inhibitory effects of simultaneous treatment with growth factors on DNA synthesis through MAPK pathway and G1 cyclins in rat hepatocytes.

Several growth factors play an important role in liver regeneration. Once hepatic injury occurs, liver regeneration is stimulated by hepatocyte growth factor (HGF), transforming growth factor (TGF)-alpha, and heparin-binding epidermal growth factor-like growth factor (HB-EGF), whereas TGF-beta1 terminates liver regeneration. In this study, we analyzed the effect of a combination of HGF and epidermal growth factor (EGF) on mitogen-activated protein kinase (MAPK) activity and G1 cyclin expression in primary cultured rat hepatocytes. Treatment with a combination of HGF and EGF, in comparison with that of either HGF or EGF, induced tyrosine phosphorylation of both c-Met and EGF receptor (EGFR) independently and additively stimulated MAPK activity and cyclin D1 expression, resulting in additive stimulation of DNA synthesis. On the other hand, although TGF-beta1 treatment did not affect tyrosine phosphorylation of c-Met and EGFR, MAPK activity, and cyclin D1 expression, which were stimulated by HGF and EGF, DNA synthesis was completely inhibited through a marked decrease in cyclin E expression. These results indicate that potent mitogens, such as HGF, TGF-alpha, and HB-EGF, could induce the additive enhancement of liver regeneration cooperatively through an increase in Ras/MAPK activity followed by cyclin D1 expression, and that TGF-beta1 suppresses the growth factor-induced signals between cyclin D1 and cyclin E, resulting in the inhibition of DNA synthesis.

Association of AIM, a novel apoptosis inhibitory factor, with hepatitis via supporting macrophage survival and enhancing phagocytotic function of macrophages.

A hallmark of many inflammatory diseases is the destruction of tissue cells by infiltrating hematopoietic cells including lymphocytes, neutrophils, and macrophages. The regulation of apoptosis of both target tissue cells and the infiltrating cells is one of the key events that defines the initiation and the progression of inflammation. However, the precise picture of the apoptosis regulation of the cells at the inflammatory sites is still unclear. We recently isolated a novel apoptosis inhibitory factor, termed AIM, which is secreted exclusively by tissue macrophages. In this report, we present unique characteristics of AIM associated with liver inflammation (hepatitis), identified by introducing an experimental hepatitis in both AIM-transgenic mice, which overexpress AIM in the body, and normal mice. First, endogenous AIM expression in macrophages is rapidly increased in response to inflammatory stimuli. Second, AIM appears to inhibit the death of macrophages in the inflammatory regions, judging by the remarkably increased number of macrophages observed in the liver from transgenic mice. In addition, we show that AIM also enhances the phagocytosis by macrophages, which emphasizes the multifunctional character of AIM. All these findings strongly provoke an idea that AIM may play an important role in hepatitis pathogenesis in a sequential manner; first AIM expression is up-regulated by inflammatory stimuli, and then in an autocrine fashion, AIM supports the survival of infiltrating macrophages as well as enhances phagocytosis by macrophages, which may result in an efficient clearance of dead cells and infectious or toxic reagents.

Retinal complications during interferon therapy for chronic hepatitis C.

OBJECTIVES: Various side effects of interferon (IFN) therapy have been reported. In this study, we examined retinal change during IFN therapy. METHODS: We performed ophthalmological examinations before, during, and after therapy on 63 patients with chronic hepatitis C who were receiving either natural IFA-alpha or recombinant IFN-alpha 2a or 2b. RESULTS: No retinal lesion was detected before IFN therapy, but, during therapy, retinal abnormality or retinopathy developed in 36 (57.1%) of 63 patients, including retinal hemorrhage in 25 patients and cotton-wool spots in 28 patients. They were noted early in the course of IFN therapy, within the first 4 wk in 67% (24/36) and within 8 wk in 86% (31/36). The incidence was not influenced by the type of IFN but was higher among diabetic (11/12, 92%, p < 0.05) or hypertensive patients (4/5, 80%, not significant) than among patients without either diabetes or hypertension (24/49, 49%). There was no relation between the incidence of retinopathy and the level of ALT activity or white blood cell or platelet counts. However, retinopathy occurred in most patients receiving IFN therapy after white blood cell count and platelet count reached a nadir. The levels of LDL-cholesterol and the atherosclerotic index in patients with retinopathy were slightly higher than those in the patients without retinopathy. CONCLUSIONS: These results suggest that retinopathy often occurs in patients with chronic hepatitis C who are receiving IFN and that we should closely monitor patients for retinal complications during IFN therapy.

Fatal propionic acidemia in mice lacking propionyl-CoA carboxylase and its rescue by postnatal, liver-specific supplementation via a transgene.

Propionic acidemia (PA) is an inborn error of metabolism caused by the genetic deficiency of propionyl-CoA carboxylase (PCC). By disrupting the alpha-subunit gene of PCC, we created a mouse model of PA (PCCA(-/-)), which died in 24-36 h after birth due to accelerated ketoacidosis. A postnatal, liver-specific PCC expression via a transgene in a far lower level than that in wild-type liver, allowed PCCA(-/-) mice to survive the newborn and early infant periods, preventing a lethal fit of ketoacidosis (SAP(+)PCCA(-/-) mice). Interestingly, SAP(+)PCCA(-/-) mice, in which the transgene expression increased after the late infant period, continued to grow normally while mice harboring a persistent low level of PCC died in the late infant period due to severe ketoacidosis, clearly suggesting the requirement of increased PCC supplementation in proportion to the animal growth. Based on these results, we propose a two-step strategy to achieve an efficient PA prevention in human patients: a partial PCC supplementation in the liver during the newborn and early infant periods, followed by a larger amount of supplementation in the late infant period.