[Antiviral mechanism of ribavirin against RNA virus].
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Animals↗
Biomedical subjects
Nobuyuki Enomoto
Publications and source records attributed to Nobuyuki Enomoto.
[Virus factors determining the outcome of IFN treatment for chronic hepatitis C].
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Antiviral Agents↗
[Analysis of replicating hepatitis C virus quasispecies in hepatocellular carcinoma tissues].
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Carcinoma, Hepatocellular↗
Hepatitis C virus nonstructural protein 5A inhibits tumor necrosis factor-alpha-mediated apoptosis in Huh7 cells.
To analyze the influence of hepatitis C virus nonstructural protein 5A (NS5A) on apoptosis, we established Huh7 cells that stably express NS5A, and induced apoptosis using tumor necrosis factor (TNF)-alpha. The viability of control Huh7 cells was reduced to 40%, compared with untreated cells, after TNF-alpha treatment, whereas that of Huh7-NS5A cells was reduced only to 80%. DNA fragmentation also decreased to <50% in Huh7-NS5A compared with control cells. Nuclear factor-kappaB activation was the same in both cell types, whereas caspase-8, -9, and -3 activity was decreased in Huh7-NS5A cells, compared with control cells, which indicates that the inhibition of caspase-8 activation is responsible for the antiapoptotic effect of the NS5A protein. The coexpression of NS5A did not inhibit apoptosis induced by caspase-8 or Fas-associating death domain protein expression. These findings suggest that the NS5A protein inhibits the apoptotic effect of TNF-alpha upstream of caspase-8 in the apoptosis cascade.
Apoptosis↗
Sequence elements correlating with circulating viral load in genotype 1b hepatitis C virus infection.
The correlation between hepatitis C virus (HCV) genomic sequences and circulating HCV RNA levels was assessed to investigate the genetic elements affecting viral load. The interferon sensitivity-determining region (ISDR) sequence and the serum viral load were strongly correlated in 226 patients examined. Analysis of the entire HCV genome from six patients (three with a high and the others with a low viral load) with similar ISDR sequences identified several candidate residues associated with viral load. The amino acid (aa) sequences of these candidate residues and flanking regions in 67 additional patients revealed that only the residue at aa 962 varied significantly between the HCV patients with low and high serum loads (P=0.042). At this position, alanine was observed more frequently in the patients with a high viral load. In conclusion, our results strongly suggest that serum HCV RNA loads are inversely correlated with amino acid substitutions in the ISDR, and aa 962 was identified as a possible second determinant of serum HCV RNA load.
Amino Acid Sequence↗
Prevention of ethanol-induced liver injury in rats by an agonist of peroxisome proliferator-activated receptor-gamma, pioglitazone.
Agonists of peroxisome proliferator-activated receptor (PPAR)-gamma have been shown to reduce tumor necrosis factor-alpha (TNF-alpha)-induced insulin resistance. On the other hand, sensitization of Kupffer cells to lipopolysaccharide (LPS) and their production of TNF-alpha are critical for progression of alcoholic liver injury. This study was intended to determine whether pioglitazone, a PPAR-gamma agonist, could prevent alcohol-induced liver injury. Rats were given ethanol (5 g/kg b.wt.) and pioglitazone (500 microg/kg) once every 24 h intragastrically. Ethanol for 8 weeks caused pronounced steatosis, necrosis, and inflammation in the liver. These pathological parameters were diminished greatly by pioglitazone. Kupffer cells were sensitized to LPS after ethanol for 4 weeks as evidenced by aggravation of liver pathology induced by LPS (5 mg/kg) and enhancement of LPS (100 ng/ml)-induced intracellular Ca2+ concentration elevation in Kupffer cells. The parameters were diminished by treatment with pioglitazone. LPS-induced TNF-alpha production by Kupffer cells from the 4-week ethanol group was 3 to 4 times higher than control. This increase was blunted by 70% with pioglitazone. Gut permeability was 10-fold higher in the 4-week ethanol group, and pioglitazone treatment did not change the value. Inclusion of TNF-alpha in culture media of Kupffer cells enhanced CD14 expression, LPS-induced intracellular Ca2+ concentration response, and production of TNF-alpha. These results indicate that pioglitazone prevents alcoholic liver injury through abrogation of Kupffer cell sensitization to LPS.
Animals↗
Dietary glycine prevents chemical-induced experimental colitis in the rat.
BACKGROUND & AIMS: In this study, the effect of dietary glycine on experimental colitis induced by 2,4,6-trinitrobenzene sulphonic acid (TNBS) and dextran sulfate sodium (DSS) in the rat was evaluated. METHODS: Male Wistar rats were fed a diet containing 5% glycine or casein as controls starting 3 days before experiments, and were given a single intracolonic injection of TNBS (50 mg/rat, dissolved in 50% ethanol). Similarly, some rats were given 3% DSS orally in drinking water for 5 days to induce colitis as a second model. The severity of colitis was evaluated pathologically, and tissue myeloperoxidase (MPO) activity was measured. Further, mRNA and protein levels for interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, cytokine-induced neutrophil chemoattractant (CINC), and macrophage inflammatory protein (MIP)-2 were detected by reverse-transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: A diet containing glycine ameliorated diarrhea and body weight loss caused by TNBS, and improved both macroscopic and histologic scores of colitis significantly. TNBS-induced increases in MPO activities in the colonic tissue were blunted significantly in glycine-fed animals. Further, dietary glycine largely prevented increases in IL-1beta and TNF-alpha in the colon 2 days after TNBS, and TNBS induction of CINC and MIP-2 in the colonic tissue also was abrogated by glycine. Importantly, the protective effect of glycine was significant even when TNBS colitis was once established. Moreover, dietary glycine also was preventive in a second, DSS-induced colitis model. CONCLUSIONS: Dietary glycine prevents chemical-induced colitis by inhibiting induction of inflammatory cytokines and chemokines. It is postulated that glycine may be useful for the treatment of inflammatory bowel diseases as an immunomodulating nutrient.
Animals↗
Amino acid substitutions in PKR-eIF2 phosphorylation homology domain (PePHD) of hepatitis C virus E2 protein in genotype 2a/2b and 1b in Japan and interferon efficacy.
Recently, the envelope protein 2 (E2) of hepatitis C virus (HCV) was reported to interact with double stranded RNA-dependent protein kinase (PKR) through an element homologous to the phosphorylation site of PKR and its target, eukaryotic translation initiation factor (eIF) 2alpha (PKR-eIF2alpha phosphorylation homology domain: PePHD). Inhibition of the kinase activity of PKR by this interaction was postulated as a mechanism for the resistance to interferon (IFN) therapy. The aim of this study was to clarify whether the variation of PePHD amino acid sequences affects IFN efficacy in Japanese population. Amino acid sequences of PePHD (aa. 659-670 in genotype1b, aa. 663-674 in genotype 2a and 2b) were determined in randomly selected 112 patients with chronic hepatitis C (genotype 1b; 83 patients, 2a; 14 patients, 2b; 15 patients) prior to IFN monotheray. In 21 out of the 23 genotype 1b sustained responders (SR) (91%) and 55 out of the 60 non-SR (92%), PePHD sequences were identical to that of the HCV-1b consensus sequence. Only two SR showed one amino acid substitution in PePHD, and five non-SR showed amino acid substitutions in PePHD. Among 14 genotype 2a patients, only two SR had one amino acid substitution comparing to the consensus HCV-2a sequence. Likely, only one SR had an amino acid substitution in PePHD among 15 genotype 2b patients. In conclusion, our study revealed no clinical relationship between the amino acid sequence of PePHD and the outcome of IFN therapy. PePHD polymorphism was not suggested to play a role in predicting IFN efficacy.
Journal Article↗
Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs.
Small interfering RNAs (siRNAs) efficiently inhibit gene expression by RNA interference. Here, we report efficient inhibition, by both synthetic and vector-derived siRNAs, of hepatitis C virus (HCV) replication, as well as viral protein synthesis, using an HCV replicon system. The siRNAs were designed to target the 5' untranslated region (5' UTR) of the HCV genome, which has an internal ribosomal entry site for the translation of the entire viral polyprotein. Moreover, the 5' UTR is the most conserved region in the HCV genome, making it an ideal target for siRNAs. Importantly, we have identified an effective site in the 5' UTR at which approximately 80% suppression of HCV replication was achieved with concentrations of siRNA as low as 2.5 nM. Furthermore, DNA-based vectors expressing siRNA against HCV were also effective, which might allow the efficient delivery of RNAi into hepatocytes in vivo using viral vectors. Our results support the feasibility of using siRNA-based gene therapy to inhibit HCV replication, which may prove to be valuable in the treatment of hepatitis C.
5' Untranslated Regions↗
Protective effect of thalidomide on endotoxin-induced liver injury.
BACKGROUND: Activation of Kupffer cells by lipopolysaccharide (LPS) plays a pivotal role in the onset of pathophysiological events that occur during endotoxemia, and intracellular calcium concentration ([Ca2+]i) is involved in LPS-stimulated cytokine production. Tumor necrosis factor (TNF)-alpha is produced exclusively by the monocyte-macrophage lineage, which is mostly made up of Kupffer cells, and thalidomide has been shown to reduce TNF-alpha production from macrophages. However, there is increasing evidence that TNF-alpha may play a role in the initiation or progression of multiple organ failure syndrome. Therefore, the purpose of this work was to determine whether thalidomide could prevent LPS-induced liver injury. METHODS: Rats were given a single oral dose of thalidomide (5 mg/kg). To assess the sensitization of Kupffer cells, LPS (5 or 10 mg/kg) was administered intravenously, and mortality, liver histology, and transaminases were evaluated 24 hr later. Kupffer cells were isolated 2 hr after thalidomide treatment. After the addition of LPS, [Ca2+]i was measured by using a microspectrofluorometer with the fluorescent indicator fura-2, and TNF-alpha was measured by enzyme-linked immunosorbent assay. RESULTS: LPS caused focal necrosis with neutrophil infiltration in the liver. Moreover, LPS dramatically increased transaminases. These pathologic parameters and increases of serum transaminases were diminished markedly by thalidomide. In isolated Kupffer cells, LPS-induced increases in [Ca2+]i and TNF-alpha production were suppressed by treatment with thalidomide. To further explore the mechanism by which thalidomide directly abrogated Kupffer cell sensitivity to LPS, we determined the effect of thalidomide (5 microM) in vitro on LPS-induced [Ca2+]i response and TNF-alpha production. With the addition of thalidomide (5 microM) in vitro to the culture media for 2 hr before LPS, these parameters were suppressed. CONCLUSIONS: Thalidomide prevents LPS-induced liver injury via mechanisms dependent on the suppression of TNF-alpha production from Kupffer cells.
Animals↗
Dalteparin sodium prevents liver injury due to lipopolysaccharide in rat through suppression of tumor necrosis factor-alpha production by Kupffer cells.
BACKGROUND: Sensitization of Kupffer cells (KC) to lipopolysaccharide (LPS) and overproduction of tumor necrosis factor (TNF)-alpha play important roles in the pathogenesis of alcoholic liver damage and sepsis-associated organ injury. Therefore, suppression of TNF-alpha should prove useful for treatment of LPS-induced liver injury. Recently, heparin has been reported to diminish TNF-alpha production from macrophages in response to LPS. Dalteparin sodium (DS) is a low-molecular-weight heparin with a mean molecular weight of 5,000. DS elicits an antithrombotic effect through a mechanism depending on anti-factor Xa activity but not on the antithrombin activity. DS is thus suitable for treatment of disseminated intravascular coagulation because it has a much smaller prohemorrhagic property. In this study, we evaluated whether DS could prevent LPS-induced liver injury. METHODS: Female Wistar rats were administered DS (50 IU/kg intraperitoneally) followed by challenge with LPS (5 mg/kg intravenously) 2 hr later. Livers and sera were collected 24 hr later. KC from rats were isolated and cultured in RPMI 1640 supplemented with 10% fetal bovine serum. After the addition of LPS (10 microg/ml) to the culture media, intracellular Ca2+ was measured by using a fluorescent indicator, fura-2. RESULTS: LPS (5 mg/kg intravenously) caused focal necrosis and neutrophil infiltration in the control liver. The histological changes and increased alanine aminotransferase levels caused by LPS injection were diminished by treatment with DS. LPS increased intracellular Ca2+ of KC in control rats from the basal level (26 +/- 6 nmol/liter) to 280 +/- 18 nmol/liter. This increase was blunted by DS (126 +/- 28 nmol/liter). The DS treatment decreased the LPS-induced TNF-alpha production by KC from 911 +/- 78 pg/ml to 309 +/- 45 pg/ml (p < 0.05). CONCLUSIONS: These results indicate that DS reduces the LPS-induced liver injury through suppression of TNF-alpha production.
Animals↗
Chronic hepatitis delta virus infection with genotype IIb variant is correlated with progressive liver disease.
We determined the sequence of the hepatitis delta virus (HDV) genome in 40 Japanese patients, most of whom were from the Miyako Islands, Okinawa, Japan. Consensus sequences from 33 HDV full genomes out of a total of 40 patients were determined by directly sequencing four partially overlapping PCR products. Phylogenetic tree analysis classified these 33 complete HDV genomes as HDV genotype I (two patients), genotype IIa (one patient) and genotype IIb (30 patients). Among the 30 genotype IIb patients, there were two clusters of genetic variants. One group consisted of six isolates showing significant homology with genotype IIb, previously reported from Taiwan. The other group consisted of 24 isolates, whose sequences formed a new genetic subgroup (genotype IIb-Miyako; IIb-M). When the genetic structures were compared in detail between IIb and IIb-M, characteristic variations were found in the C-terminal sequence of the large delta antigen-conferring packaging signal as well as the RNA editing site. Determination of subclasses of genotype IIb in a total of 37 patients, including seven HDV patients whose partial HDV sequence was determined, revealed eight patients with IIb and 29 patients with IIb-M. Although there was no significant difference in the clinical background or virological state of hepatitis B virus between these two groups, patients with genotype IIb-M showed greater progression of chronic hepatitis and cirrhosis than those with genotype IIb (P=0.0009). These data indicate the existence of a genetic subgroup of HDV genotype IIb, which is associated with different clinical characteristics and which could be related to genetic variations in functionally important parts of the HDV genome.
Adult↗
Chronic hepatitis B with flare due to co-infection of hepatitis delta virus during lamivudine therapy.
In 1997, a 27-year-old homosexual man contracted acute hepatitis B that developed into chronic hepatitis. Because of repeated flares, administration of lamivudine was started in March 2002. Hepatitis B virus (HBV) DNA immediately decreased, but the serum level of alanine aminotransferase gradually increased. Drug-induced hepatitis due to lamivudine was excluded. It was suspected that the progression of liver damage was caused by hepatitis delta virus (HDV), because the patient was positive for both anti-HDV antibody and HDV RNA. Co-infection of HDV should be considered a possibility if liver injury is not improved by lamivudine therapy.
Adult↗
[Hepatitis C virus infection].
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Animals↗
[Ethanol changes sensitivity of Kupffer cells to endotoxin].
Gut-derived endotoxin plays an important role in alcoholic liver injury. Intestinal sterilization with antibiotics (polymyxin B and neomycin) or inactivation of Kupffer cells with gadolinium chloride can prevent early alcohol-induced liver injury in the Tsukamoto-French model. Although short-term administration of alcohol enhances endotoxin hepatotoxicity, a majority of studies report that short-term ethanol inactivates Kupffer cells. It is therefore paradoxical that Kupffer cells are involved in alcoholic liver injury based on in vivo data with gadolinium chloride and antibiotics, yet ethanol blunts activation of isolated Kupffer cells. Accordingly, this review focuses on understanding this paradox by studying the temporal effect of ethanol in vivo on the response of subsequently isolated Kupffer cells. Mice were given ethanol intragastrically, and LPS was injected later. One hour after ethanol treatment, serum transaminases after LPS were 60% of control, while ethanol increased these parameters about 3-fold 21 hours after ethanol. Pretreatment with antibiotics blocked these effects of ethanol. Two hours after ethanol administration, the LPS-induced increases in intracellular calcium concentration and TNF alpha release by Kupffer cells was diminished by 50% of control, and these parameters were reciprocally enhanced two-fold at 24 hours. Sterilization of the gut with antibiotics blocked both effects of ethanol on intracellular calcium concentration and TNF alpha release. Twenty-four hours after ethanol, CD14 in Kupffer cells was elevated to about five-fold. In Kupffer cells from mice treated with ethanol 1 hour earlier, IRAK expression and activity and NF kappa B were decreased to 50-60% of control. In contrast, in Kupffer cells from mice treated with ethanol 21 hours earlier, LPS-induced TNF alpha production, expression and activity of IRAK were increased 1.5-fold over controls, while NF kappa B activation was elevated 3-fold. Kupffer cells isolated from rodents early after ethanol exhibited tolerance to LPS, whereas sensitization was observed later. In conclusion, acute ethanol alters the expression of endotoxin receptors and intracellular signaling molecules, and causes both tolerance and sensitization of Kupffer cells to endotoxin. It is postulated that tolerance of Kupffer cells contributes to the impairment of innate immune system in alcoholism, while sensitization to endotoxin enhances progression of alcoholic liver injury.
Animals↗
A double-strand decoy DNA oligomer for NF-kappaB inhibits TNFalpha-induced ICAM-1 expression in sinusoidal endothelial cells.
Altered gene expression of liver sinusoidal endothelial cells (SECs) is associated with impaired immune response. Here we report that the decoy technique effectively suppresses TNFalpha-induced ICAM-1 expression in SEC. An NF-kappaB decoy (NF-kappaB31: 5(')-TGGGGACTTTCCAGTTTCTGGAAAGTCCCCA-3), which contains a consensus sequence for NF-kappaB, was complexed to PLL-g-HA [hyaluronate-grafted poly(L-lysine) copolymer] that permits transfer of exogenous DNA selectively to the SEC. The PLL-g-HA/NF-kappaB31 complex was added to the culture media of LSE cells, a human SEC-derived cell line. Then, cells were stimulated with TNFalpha (5ng/mL). PLL-g-HA/NF-kappaB31, but not control oligodeoxynucleotides having a reverse or scrambled sequence, inhibited the intranuclear localization of NF-kappaB induced by TNFalpha, with almost complete inhibition at 2.5microg/mL as DNA. NF-kappaB31 attenuated the increase in ICAM-1 mRNA as well as protein levels in LSE cells. The decoy technique in combination with PLL-g-HA may provide a novel strategy for manipulation of SEC functions.
Active Transport, Cell Nucleus↗
Pioglitazone prevents early-phase hepatic fibrogenesis caused by carbon tetrachloride.
Here we investigated the effect of pioglitazone, a peroxisome proliferator-activated receptor (PPAR)-gamma ligand, on early-phase hepatic fibrogenesis in vivo caused by acute carbon tetrachloride (CCl(4)) administration in the rat. Pioglitazone (1 mg/kg BW) prevented pericentral fibrosis and induction of alpha-smooth muscle actin (SMA) 72 h after CCl(4) administration (1 ml/kg BW). CCl(4) induction of alpha1(I)procollagen mRNA in the liver was blunted by pioglitazone to the levels almost 2/3 of CCl(4) alone. Pioglitazone also prevented CCl(4)-induced hepatic inflammation and necrosis, as well as increases in serum tumor necrosis factor-alpha levels. Further, pioglitazone inhibited the induction of alphaSMA and type I collagen in primary cultured hepatic stellate cells in a dose-dependent manner. In conclusion, pioglitazone inhibits both hepatic inflammation and activation of hepatic stellate cells, thereby ameliorating early-phase fibrogenesis in the liver following acute CCl(4).
Actins↗
Senescence marker protein-30 knockout mouse liver is highly susceptible to tumor necrosis factor-alpha- and Fas-mediated apoptosis.
Senescence marker protein-30 (SMP30) is a calcium-binding protein that decreases in an androgen-independent manner with aging. To elucidate the physiological role of this protein, we introduced a null mutation of the SMP30 gene into the germ line of mice. Despite the complete lack of SMP30 (SMP30-/-), these mutant mice were indistinguishable from their wild-type (SMP30+/+) littermates in terms of development and fertilization capability. We then investigated the tissue susceptibility for apoptosis induced by cytokine using primary cultured hepatocytes, because SMP30 could rescue cells from cell death caused by calcium influx, using a calcium ionophore as previously described. SMP30-/- hepatocytes were found to be more susceptible to apoptosis induced by tumor necrosis factor-alpha (TNF-alpha) plus actinomycin D (ActD) than SMP30+/+ hepatocytes. In addition, the TNF-alpha/ActD-induced caspase-8 activity in SMP30-/- hepatocytes was twofold greater than that in SMP30+/+ hepatocytes. In contrast, no significant difference was observed in the TNF-alpha/ActD-induced nuclear factor-kappa B activation of SMP30+/+ versus SMP30-/- hepatocytes, indicating that SMP30 is not related to TNF-alpha/ActD-induced nuclear factor-kappa B activation itself. Moreover, deletion of the SMP30 gene enhanced liver injury after treatment in vivo with anti-Fas antibody and the SMP30+/- mice showed intermediate susceptibility to Fas-induced apoptosis. Collectively, these results demonstrate that SMP30 acts to protect cells from apoptosis.
Actins↗