Differential effects of calcineurin inhibitors, tacrolimus and cyclosporin a, on interferon-induced antiviral protein in human hepatocyte cells.

The premise of our study is that selective inhibition of interferon (IFN) by calcineurin inhibitors contribute to the increased severity of hepatitis C virus (HCV) posttransplantation. Therefore, we examined the influence of calcineurin inhibitors in the human hepatocyte cell line on IFN-alpha-induced phosphorylation of Janus kinase (Jak) and signal transducers and activators of transcription (STAT), nuclear translocation of IFN-stimulated gene factor 3 (ISGF-3), IFN-stimulated regulatory element (ISRE)-contained promoter activity, and the expressions of antiviral proteins. Tacrolimus (Tac), but not cyclosporin A (CyA), had an inhibitory effect on IFN-alpha-induced double-stranded ribonucleic acid (RNA)-dependent protein kinase (PKR) in a dose-dependent manner. STAT-1 also acted in a similar fashion to PKR. IFN-alpha combined with Tac attenuated the ISRE-containing promoter gene activity as compared with IFN-alpha alone. In contrast, its expression in pretreated CyA was slightly attenuated. In pretreated Tac, but not CyA, the levels of IFN-alpha-induced tyrosine phosphorylated STAT-1 and -2 were clearly lower than those induced by IFN-alpha alone. Tac and CyA did not decrease the IFN-alpha-induced JAK-1 phosphorylation. The nuclear translocation rate of tyrosine phosphorylated STAT-1 was inhibited by pretreatment of both Tac and CyA by western blotting and immunohistochemistry. In an HCV replicon system, pretreated Tac diminished the replication inhibitory effect of IFN-alpha. In this study, we show that calcineurin inhibitors, especially Tac, are the negative regulators of IFN signaling in the hepatocyte; the greatest cause of such inhibition is the phosphorylation disturbance of STAT-1, next to inhibition of the nuclear translocation of STAT-1. In conclusion, disturbance of tyrosine phosphorylation of STAT-1 resulted in diminished ISRE-containing promoter activity and a decline in antiviral protein expression. Moreover, the replication of HCV was activated. This phenomenon is detrimental to IFN therapy after liver transplantation, and the selection of calcineurin inhibitors may warrant further discussion depending on the transplant situation.

Mental stress induces sustained elevation of blood pressure and lipid peroxidation in postmenopausal women.

Mental stress is thought to underlie cardiovascular events, but there is information on oxidative stress induced by mental stress in association with cardiovascular responses in women. Using a sensitive assay for plasma 4-hydroxy-2-nonenal (HNE), as a marker for oxidative stress, we addressed the relation between pressor responses and oxidative stress induced by mental or physical stress in premenopausal and postmenopausal women. Healthy subjects (7 postmenopausal and 8 premenopausal women, in early and late follicular phases) were subjected to mental and physical stress evoked by a Color Word Test (CWT) and isometric handgrip, respectively. The CWT induced a rapid elevation of diastolic blood pressure (DBP), at a higher level in the postmenopausal than in the premenopausal women (p<0.01), and this higher DBP was sustained during the CWT and recovery (p<0.01). The CWT induced a significant elevation in plasma noradrenaline in premenopausal women in the early follicular phase and in postmenopausal women (p<0.05). Plasma nitric oxide metabolites were higher in postmenopausal than in the premenopausal women in the late follicular phase (p<0.05), but did not change during exposure to the two types of stress in either group. Plasma HNE was increased during recovery from the CWT, but not the handgrip, in postmenopausal women (2.4 times, p<0.05). There was a significant difference in the time course of the CWT-induced HNE response between the postmenopausal and premenopausal women (p<0.05). These findings suggest that mental, but not physical, stress causes sustained diastolic blood pressure elevation in postmenopausal women, accompanied by heightened oxidative stress.

Survivin downregulation by siRNA sensitizes human hepatoma cells to TRAIL-induced apoptosis.

Survivin, an anti-apoptotic protein, is abundantly expressed in a variety of cancer cells, including hepatoma cells, resulting in the resistance of these cells to various apoptotic stimuli. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known to induce cancer cell-specific apoptosis, but hepatoma cells are resistant to TRAIL-induced apoptosis. In the present study, we have examined whether the downregulation of survivin by short interfering RNA (siRNA) promotes spontaneous or TRAIL-induced apoptosis in Huh-7 human hepatoma cells. Survivin siRNA transfection downregulated the expression of survivin in Huh-7 cells and reduced cell viability by 20% through inducing spontaneous apoptosis. TRAIL (1 to 2 ng/ml) only slightly induced apoptosis in Huh-7 cells; however, survivin siRNA transfection apparently enhanced TRAIL-induced apoptosis. These results suggest that the level of survivin is linked to the susceptibility of Huh-7 cells to TRAIL. It is possible that survivin downregulation by siRNA combined with TRAIL administration may provide a new therapeutic strategy against hepatoma.

Different mechanisms for anti-tumor effects of low- and high-dose cyclophosphamide.

It is known that, besides its direct cytotoxic effect as an alkylating chemotherapeutic agent, cyclophosphamide also has immuno-modulatory effects, such as depletion of CD4+CD25+ regulatory T cells. However, its optimal concentration has not yet been fully elucidated. Therefore, we first compared the effects of different doses of cyclophosphamide on T cell subsets including CD4+CD25+ T cells in mice. Cyclophosphamide (20 mg/kg) decreased the numbers of splenocytes, CD4+ and CD8+ T cells by approximately 50%, while a decline in CD4+CD25+ T cell number was more profound, leading to the remarkably lower ratios of CD4+CD25+ T cells to CD4+ T cells. In contrast, 200 mg/kg cyclophosphamide severely decreased the numbers of all the T cell subsets by > 90% although the decreased ratios of CD4+CD25+ T cells to CD4+ T cells were still observed. Next, low-dose cyclophosphamide significantly inhibited in vivo growth of murine hepatoma MH129 tumor in immuno-competent but not immuno-deficient mice. This anti-tumor effect was abolished by CD4+CD25+ T cell repletion. In contrast, high-dose cyclophosphamide exhibited similar anti-tumor effects in both mice. In addition, contrary to antibody-mediated CD4+CD25+ T cell depletion, administration of low-dose cyclophosphamide after tumor inoculation was more efficacious than the prior administration. Our data show that low-dose cyclophosphamide selectively depletes CD4+CD25+ T cells, leading to enhanced anti-tumor effects against pre-existing tumors, while the anti-tumor effect of high-dose cyclophosphamide is solely attributed to its direct cytotoxicity. These findings appear to be highly crucial in a clinical setting of combined chemotherapy and immunotherapy for cancer treatment.

Vaccination with dendritic cells pulsed with apoptotic cells elicits effective antitumor immunity in murine hepatoma models.

Dendritic cell (DC)-based vaccine is a developing strategy to treat cancer including hepatoma. We evaluated the antitumor efficacy of vaccination with DCs pulsed with apoptotic cells, as compared to vaccination with DCs pulsed with cell lysates, in murine hepatoma models. Murine hepatoma cells, Hepa1-6, MH134 and BNL1ME.A.7R.1, and their syngeneic mice, C57BL/6, C3H/HeN and BALB/c, respectively, were used in the study. Protective and therapeutic antitumor effects of vaccination with bone marrow-derived DCs pulsed with irradiation or sulindac-induced apoptotic cells or cell lysates were analyzed. Immature DCs efficiently phagocytosed apoptotic cells and increased expression of CD86, a cell surface maturation marker. Vaccination with apoptotic cell-pulsed, but not cell lysate-pulsed, DCs promoted significant protective immunity against parental hepatoma in vivo. Spleen cells from mice vaccinated with apoptotic cell-pulsed DCs showed higher cytolytic activity and contained higher number of IFN-gamma producing cells against parental hepatoma cells than those from mice vaccinated with cell lysate-pulsed DCs in vitro. Polyriboinosinic polyribocytidylic acid [poly (I:C)], double strand RNA, further enhanced CD86 expression and the therapeutic efficacy of vaccination with DCs pulsed with apoptotic cells for pre-established hepatoma. These results suggest that vaccination with DCs pulsed with apoptotic cells and treated with poly (I:C) appears to be a promising approach as a new therapeutic means for hepatoma.

The role of IL-18 in the modulation of matrix metalloproteinases and migration of human natural killer (NK) cells.

In this study, we examined whether interleukin-18 (IL-18) affects natural killer (NK) cells' migration and matrix metalloproteinases (MMPs) production. We demonstrated that chemotaxis of human NK cells through basement membrane-like Matrigel was augmented by IL-18. As well, IL-18 stimulation induces the production of activated forms of matrix metalloproteinase-2 (MMP-2) as well as the production of pro-MMP-2 from NK cells. We also demonstrated that MT1-MMP expression on human NK cells, which is a major activator of MMP-2, was induced by IL-18 stimulation coordinated with MMP-2 activation. These data suggest that the MT1-MMP/MMP-2 system participates in the degradation of basement membrane components and thus contributes to NK cell migration.

Diverse efficacy of vaccination therapy using the alpha-fetoprotein gene against mouse hepatocellular carcinoma.

Antitumor vaccination therapy approaches using naked plasmid DNA or recombinant viruses encoding tumor-associated antigens are currently in development. In the present study, we examined the therapeutic efficacy of vaccination using the mouse alpha-fetoprotein (AFP) gene in mouse hepatocellular carcinoma (HCC) cells. C57L/J or C3H/HeN mice were primed with an injection of naked plasmid DNA expressing mouse AFP followed by a booster of replication-defective adenovirus expressing mouse AFP (plasmid-AFP prime/adenovirus-AFP booster vaccination). The mice were then challenged with high AFP-producing Hepa1-6 cells or low AFP-producing MH134 cells, respectively, and the tumor growth rate was monitored. Plasmid-AFP prime/adenovirus-AFP booster vaccination promoted protective immunity against Hepa1-6 cells, and significantly increased the number of interferon-gamma-producing splenic cells in C57L/J mice. In addition, this vaccination protocol repressed the growth of pre-established Hepa1-6 tumors in C57L/J mice. However, plasmid-AFP prime/adenovirus-AFP booster vaccination did not induce protective immunity against MH134 cells in C3H/HeN mice. These results suggest that vaccination with the AFP gene is a promising strategy to treat HCC, but its outcome may be affected by the level of AFP expression in HCC or by the immunological response of the host.

Analysis of anti-HBs levels in healthcare workers over 10 years following booster vaccination for hepatitis B virus.

In this study, we analyzed anti-HBs levels in 104 Japanese healthcare workers who received three booster HBV surface antigen (HBsAg) vaccines because 80 became anti-HBs-negative at a mean of 2.4 years after the primary vaccination and 24 did not respond to primary vaccination. Of the re-vaccinees, 96% achieved a level of 10 mIU/ml or more of anti-HBs (i.e. seroprotected), 1 month after booster vaccination. Although anti-HBs levels of re-vaccinees decreased as rapidly as those of primary immunized vaccinees, at 10 years post-booster, 64% of re-vaccinees maintained anti-HBs levels at 10 mIU/ml or higher. Our results suggest that the additional three-dose protocol of booster HBsAg vaccination is beneficial in maintaining a seroprotective level of anti-HBs until new immunogenic vaccination protocols are established.

Interferon-alpha sensitizes human hepatoma cells to TRAIL-induced apoptosis through DR5 upregulation and NF-kappa B inactivation.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces apoptosis in a variety of cancer cells with little or no effect on normal cells. Human hepatoma cells, however, are resistant to TRAIL-induced apoptosis. Since interferon-alpha (IFN-alpha) is capable of enhancing TNF-alpha-induced apoptosis in certain cancer cells, we evaluated the effect of IFN-alpha on TRAIL-induced apoptosis of human hepatoma cells. IFN-alpha pretreatment enhanced TRAIL-induced apoptosis of HuH-7 and Hep3B cells, in which IFN-alpha upregulated the expression of DR5, a death receptor of TRAIL, and downregulated the expression of survivin, which has an antiapoptotic function. In contrast, IFN-alpha did not enhance TRAIL-induced apoptosis of HepG2 cells, in which expression of DR5 and survivin was not affected by IFN-alpha. On the other hand, TRAIL activated NF-kappa B composed of RelA-p50 heterodimer, a key transcription factor regulating cell survival, in HuH-7 and HepG2 cells. However, IFN-alpha pretreatment repressed the TRAIL-mediated activation of NF-kappaB and decreased its transcriptional activity in HuH-7 but not in HepG2 cells. Moreover, IFN-alpha pretreatment clearly augmented TRAIL-mediated caspase-8 activation in HuH-7 cells. Our results suggest that IFN-alpha could sensitize certain human hepatoma cells to TRAIL-induced apoptosis by stimulating its death signaling and by repressing the survival function in these cells.

Immuno-gene therapy with adenoviruses expressing fms-like tyrosine kinase 3 ligand and CD40 ligand for mouse hepatoma cells in vivo.

Introduction of genes encoding immuno-stimulatory cytokines into cancer cells is known to enhance antitumor immunity. CD40 ligand (CD40L, CD154) and fms-like tyrosine kinase 3 ligand (Flt3L) are recently identified cytokines, which have been demonstrated to stimulate antitumor immunity in several cancer models. However little is known about antitumor activity of Ftl3L and CD40L against hepatocellular carcinoma (HCC). In the present study, we constructed replication-defective adenoviruses expressing Flt3L and CD40L and examined their therapeutic efficacy on mouse HCC, MH134 cells. Subcutaneous injection of MH134 cells genetically engineered to express Flt3L and/or CD40L developed tumors in all the syngeneic immunocompetent mice, but tumor growth was significantly delayed as compared to control mice. Partial inhibition of this antitumor effect in athymic nude mice suggests that both innate and adaptive immunity appear to play a role. It was shown by immunodepletion of NK cells with anti-asialo-GM1 antibody that the effector cells involved in innate immunity are NK cells. In a therapeutic setting, however, injection of adenovirus expressing Flt3L or CD40L into pre-established MH134 tumors exhibited no efficacy. These data demonstrate that Flt3L and CD40L induce significant, but only weak, antitumor immunity against MH134 cells presumably through both innate and adaptive immunity. Our results suggest that immuno-gene therapy with Flt3L and CD40L may need adjuvant modalities to achieve strong immune response.

Thyroid cancer immuno-therapy with retroviral and adenoviral vectors expressing granulocyte macrophage colony stimulating factor and interleukin-12 in a rat model.

BACKGROUND: Introduction of genes encoding immuno-stimulatory cytokine(s) into cancer cells is well known to enhance anti-tumour immunity. AIM: The present studies were designed to evaluate the therapeutic efficacy of retroviral- and adenoviral-mediated delivery of IL-12 and/or granulocyte macrophage colony-stimulating factor (GM-CSF) genes for thyroid cancer in an immuno-competent rat model. METHODS: A rat thyroid cancer cell line FRTL-Tc syngeneic to Fisher rat was used. RESULTS: Expression of these exogenous cytokines did not affect in vitro cell growth. Subcutaneous injection of FRTL-Tc cells retrovirally transduced with IL-12 or GM-CSF genes formed significantly smaller tumours than that of the parental cells, but had little effect on growth of distant tumours, suggesting no vaccine effect. Similarly, injection of the cells infected with adenovirus expressing IL-12 or GM-CSF (AdIL-12 or AdGM-CSF) almost completely abolished tumourigenicity and injection of AdGM-CSF into pre-established tumours significantly inhibited growth of the tumours injected; neither, however, showed a systemic vaccine affect. On the other hand, injection of AdIL-12 into the pre-established tumours significantly inhibited growth of not only the tumours injected but also distant tumours, indicating induction of systemic anti-tumour immunity. Serum IL-12 was detectable only in this approach. There was neither a synergistic or additive effect of these two cytokines. CONCLUSIONS: Our data demonstrate in a rat thyroid cancer model that only injection of AdIL-12 into the pre-established tumours elicited systemic anti-tumour immunity, but injection of AdGM-CSF or injection of the cells expressing IL-12 or GM-CSF elicited only local effect, indicating that in situdelivery of IL-12 gene with adenovirus appears most efficacious but may still require adjuvant modalities to enhance the anti-tumour effect.

Receptor-independent augmentation of adenovirus-mediated gene transfer with chitosan in vitro.

Recombinant adenovirus is one of the most widely used viral vectors for gene delivery. This study was designed to evaluate the ability of chitosan, a cationic, linear polysaccharide composed of beta(1,4) linked glucosamine partly containing N-acetyl-glucosamine, to enhance the in vitro infectivity of adenovirus to mammalian cells. Wild type and a fiber-mutant replication-defective recombinant adenoviruses expressing beta-galactosidase were used. In the latter, an RGD peptide, the binding site for alpha(v)beta3 and alpha(v)beta5 integrin, was introduced in the fiber knob enabling adenovirus receptor-independent viral infection. Enhanced effect of chitosan on the infectivity of both adenoviruses was observed in Chinese hamster ovary cells that do not express the receptor for adenovirus with beta-galactosidase activity assay and x-gal staining. These data indicate the receptor-independent mechanism(s) for this enhancement effect. In addition, we found that pH of the culture medium, and molecular mass and concentration of chitosan are also critical factors. Thus, the highest effect was obtained with 0.1-1 microg/ml of chitosan with molecular mass of 19K and 40K in the culture medium of pH 6.4; on the other hand, the effect was negligible with the higher chitosan concentrations (10 microg/ml or more), lower or higher molecular mass (11K and 110K) of chitosan, or at pH of 7.4. Studies using several cell lines with variable levels of adenoviral infectivity revealed that this enhanced effect is evident in the cells with poor infectivity to adenovirus. Since chitosan is biocompatible and inexpensive, these data indicate that chitosan may be a potential candidate for a non-viral vector to safely increase adenoviral infectivity to mammalian cells, particularly those with poor susceptibility to adenoviral infection.

p48 Overexpression enhances interferon-mediated expression and activity of double-stranded RNA-dependent protein kinase in human hepatoma cells.

BACKGROUND/AIMS: Double-stranded RNA-dependent protein kinase (PKR) is a key factor involved in interferon (IFN)-induced antiviral actions. Since p48, together with signal transducers and activators of transcription 1 and 2 (STAT1 and STAT2), is an indispensable mediator in IFN-alpha signaling pathways, we investigated the effect of p48 gene transduction on PKR expression and its activity in HuH-7 human hepatoma cells. METHODS: HuH-7 cells were infected or transfected with p48 gene expression adenoviral vector or plasmid vector, respectively, and incubated with or without IFN-alpha, then PKR expression and phosphorylation of alpha-subunit of eukaryotic protein synthesis initiation factor-2 (eIF2alpha) in the cells were examined. In addition, PKR activity inhibiting protein translation was determined by the decrease of chloramphenicol acetyltransferase (CAT) gene translation or alpha-fetoprotein secretion. RESULTS: p48 overexpression itself could not stimulate PKR expression. However, p48 overexpression in combination with interferon-alpha treatment caused a marked increase in PKR expression and augmented the phosphorylation of eIF2alpha, by which the transfected CAT gene translation, as well as the endogenous alpha-fetoprotein synthesis, was blocked without affecting their mRNA levels. CONCLUSIONS: These results suggest that p48 gene transduction may provide a strategy to enhance the IFN-mediated PKR expression and its activity in hepatocytes.

Association between nonalcoholic fatty liver, markers of obesity, and serum leptin level in young adults.

OBJECTIVES: The aim of the present study was to clarify the risk factors for nonalcoholic fatty liver in young adults. METHODS: One thousand two hundred two students, aged 18-21 yr, received matriculation health examinations, including measurements of body mass index and percent body fat and determination of serum levels of ALT at Nagasaki University in 1998. One hundred twenty-nine were found to have borderline or elevated levels of serum ALT, and 105 of the 129 students (75 men and 30 women) were subjected to further analysis for the presence of fatty liver using ultrasonography, by which both the degree of steatosis and the abdominal wall fat index (AFI) corresponding to the ratio of visceral to s.c. adipose tissue (V/S ratio) were evaluated, in addition to determination of the serum level of leptin. RESULTS: Of 105 students, 74 (70%) had fatty liver. The incidence of moderately to severely fatty liver was significantly higher in men than in women. In parameters related to obesity, the close correlation between body mass index and percent body fat was observed in both sexes. The serum level of leptin correlated well with percent body fat and AFI (V/S ratio) in women, whereas it did not correlate with AFI (V/S ratio) in men. Multiple logistic regression analysis indicated that AFI (V/S ratio) was the only independent risk factor for fatty liver in both sexes. CONCLUSIONS: These results suggest that visceral fat distribution is a key risk factor for nonalcoholic fatty liver in young adults.

Involvement of IL-1beta and IL-10 in IFN-alpha-mediated antiviral gene induction in human hepatoma cells.

Crosstalk between interferons (IFNs) and several cytokines is likely to play an important role in viral clearance in chronic hepatitides B and C. We investigated the influence of this phenomenon on IFN-inducible antiviral gene expression in HuH-7 human hepatoma cells. HuH-7 cells were treated with IFN-alpha in the absence or presence of interleukin-1beta (IL-1beta) or IL-10 and the expression of antiviral genes such as 2(')5(')-oligoadenylate synthetase (2(')5(')-OAS) and double-stranded RNA-dependent protein kinase (PKR), as well as activation of signal transducer and activator of transcription 1 (STAT1), a key step for relaying the IFN-alpha signals, was analyzed by Northern blotting, Western blotting, and the reporter gene transfection assay. IL-1beta potentiated IFN-alpha-induced 2(')5(')-OAS and PKR gene expression, similar to expression of the transfected reporter genes containing the IFN-stimulated regulatory elements, while IL-10 suppressed IFN-alpha-stimulated gene expression. With regard to IFN-alpha signaling, IL-1beta enhanced both tyrosine and serine phosphorylation of STAT1 through p38 mitogen-activated protein kinase activation. In contrast, IL-10 inhibited IFN-alpha-mediated tyrosine phosphorylation of STAT1 by induction of a Janus kinase inhibitor, JAB. IL-1beta and IL-10 interact with IFN-alpha to up- and down-regulate antiviral gene expression, respectively, by modulating STAT1 activation induced by IFN-alpha.

Transactivation of human alpha-fetoprotein gene by X-gene product of hepatitis B virus in human hepatoma cells.

The X-gene product of hepatitis B virus (HBX) modulates a variety of viral and cellular genes relevant to hepatocarcinogenesis, where alpha-fetoprotein (AFP) is produced by hepatoma cells. In the present study, the possible mechanism by which HBX regulates AFP expression was investigated using three human hepatoma cells, HepG2, HuH-7 and Hep3B, which are known to contain the wild-type, the mutant-type and the deletion of p53, respectively. Transfection with the HBX expression vector stimulated the co-transfected AFP reporter gene expression in HepG2 cells and HuH-7 cells, but not in Hep3B cells. Transfection with the p53 expression vector repressed the AFP reporter gene expression in all three hepatoma cells, while overexpression of HBX counteracted the p53-induced repression. In addition, a G-->A substitution at nucleotide -119 in the AFP promoter sequence abrogated the stimulatory effect of HBX on the AFP promoter in HepG2 cells. These results suggest that HBX interacts with p53 to up-regulate AFP gene transcription probably by restoration of the p53-mediated repression of the AFP promotor activity.