Prognosis and incidence of immunological and oncological complications after direct-acting antiviral therapy for chronic hepatitis C.

Background and study aims: The long-term comprehensive prognosis of chronic hepatitis C after direct-acting antiviral (DAA) therapy is unclear. This study aimed to investigate the prognosis and incidence of immunological and oncological complications after DAA therapy. Patients and methods: The study included a total of 1461 patients who received DAA therapy in our university hospital and affiliated hospitals between September 3, 2014 and September 30, 2018. Results: The incidence rates of total malignancies in overall or female patients after DAA therapy were significantly greater than expected in the corresponding general population. The same was true for lung malignancies. Predictive risk factors associated with the occurrence and recurrence of hepatic malignancies after DAA therapy in patients with sustained virological response were cirrhosis and insulin use, protein induced by vitamin K absence or antagonist-II level, and albumin-bilirubin score, respectively. Eight (0.5%) patients were diagnosed with autoimmune diseases after starting DAA therapy. Importantly, the attending physician considered a possible causal relationship between DAA therapy and these autoimmune diseases in five cases (four rheumatoid arthritis and one membranoproliferative glomerulonephritis). The 5-year overall survival rate was 91.6%. The most frequent primary cause of death was malignancy in 41 (60.2%) patients, including 25 with hepatic malignancies. Lung and colorectal cancers were the next most common. Conclusions: Given that the incidence of total and lung cancers might increase and DAA-related autoimmune diseases might emerge after DAA therapy, we should be alert for the development of these diseases as well as hepatic malignancies.

Tolvaptan reduces the required amount of albumin infusion in patients with decompensated cirrhosis with uncontrolled ascites : a multicenter retrospective propensity score-matched cohort study.

Background: The aim of this retrospective study was to determine whether tolvaptan treatment reduces the amount of albumin administered, volume of ascites removed, and frequency of paracentesis procedures in patients with decompensated cirrhosis with uncontrolled ascites with conventional diuretics. Patients and methods: The control (C) group included patients treated with conventional diuretics. The tolvaptan (T) group included patients treated with both tolvaptan and conventional diuretics. Both groups were matched according to baseline parameters. The amount of albumin administered, volume of ascites removed, and frequency of paracentesis within 30 days of onset of uncontrolled ascites were compared between the two groups. Results: After matching, 74 patients (C=37, T=37) were included. Baseline parameters (C vs. T group) were as follows: age, 69.5 ± 9.3 vs. 70.4 ± 11.0 years (p = 0.702) ; males, 24 (64.9%) vs. 25 (67.6%) (p = 0.999) ; patients with hepatocellular carcinoma, 17 (45.9%) vs. 18 (48.6%) (p = 0.999) ; serum albumin levels at treatment initiation, 2.76 ± 0.48 vs. 2.73 ± 0.49 g/dL (p = 0.773), and serum creatinine levels at treatment initiation, 1.18 ± 1.23 vs. 1.09 ± 0.48 g/dL (p = 0.679). In the C vs. T groups, respectively, mean amount of albumin administered was 51.0 ± 31.4 vs. 33.4 ± 29.8 g/month (p = 0.016) ; mean volume of ascites removed was 2,905 ± 4,921 vs. 1,824 ± 3,185 mL/month (p = 0.266) ; and mean frequency of paracentesis was 0.92 ± 1.46 vs. 0.89 ± 1.45 procedures (p = 0.937). Conclusions: Tolvaptan reduced the use of albumin infusion in patients with decompensated cirrhosis and was effective and acceptable for uncontrolled ascites.

A change in the timing for starting systemic therapies for hepatocellular carcinoma: the comparison of sorafenib and lenvatinib as the first-line treatment.

Aim: The aim of this retrospective multicenter study was to evaluate the differences in the timing for starting systemic therapies as the first-line treatment for hepatocellular carcinoma (HCC). Methods: A total of 375 patients with HCC treated with sorafenib from May 2009 to March 2018 and 56 patients treated with lenvatinib from March 2018 to November 2018 at our affiliated hospitals were included in this study. Results: The median ages of the sorafenib and lenvatinib groups were 71.0 (interquartile range [IQR]: 64.0-77.0) and 73.5 (IQR: 68.0 -80.0) years old, and 300 (80.0%) and 42 (75.0%) patients were men, respectively. The Barcelona Clinic Liver Cancer stage was early, intermediate and advanced in 39 patients (10.4%), 133 patients (35.5%) and 203 patients (54.1%) in the sorafenib group and 1 patient (1.8%), 17 patients (30.4%) and 38 patients (67.9%) in the lenvatinib group, respectively. In the analysis of intermediate HCC, patients who satisfied the criteria of TACE failure/refractoriness (P=0.017), those with ALBI grade 1 (P=0.040), and those with a serum AFP level < 200 ng/ml (P=0.027) were found more frequently in the lenvatinib group than in the sorafenib group, with statistical significance. The objective response rate (ORR) of lenvatinib was 34.8% in the overall patients and 46.7% in the intermediate-stage HCC patients, which was significantly higher than sorafenib (P=0.001, P=0.017). Conclusions: The emergence of lenvatinib has encouraged physicians to start systemic chemotherapy earlier in intermediatestage HCC patients.

Glutathione content is correlated with the sensitivity of lines of PC12 cells to cisplatin without a corresponding change in the accumulation of platinum.

A study of the involvement of glutathione (GSH) in cellular resistance to cisplatin was performed using methylmercury-resistant sublines (PC12/TM series) of the PC12 line of rat pheochromocytoma cells. The seven clonal sublines of PC12 cells (PC12/TM, PC12/TM2, PC12/TM5, PC12/TM11, PC12/TM15, PC12/TM23, PC12/TM26) used in the study had intracellular levels of GSH that ranged from 8.7-39.9 nmol/mg protein. The intracellular level of GSH was significantly correlated (p < 0.01, r = 0.87) with the sensitivity to cisplatin of PC12 cells and the seven sublines. Among the seven sublines, PC12/TM cells contained the highest concentration of GSH and were the most resistant to cisplatin. Treatment of PC12/TM cells with L-buthionine-SR-sulfoximine, which reduced the level of GSH to that in the parental PC12 cells, significantly reduced the resistance of the cells to cisplatin. The amount of platinum accumulated by resistant PC12/TM cells after treatment with cisplatin was higher than that by sensitive PC12 cells. These results suggest that the intracellular level of GSH might be directly involved in the resistance to cisplatin of these cell lines. However, a high intracellular concentration of GSH does not appear to contribute to a decrease in the accumulation of cisplatin in these cells.

Two nuclear proteins, Cin5 and Ydr259c, confer resistance to cisplatin in Saccharomyces cerevisiae.

In an attempt to identify genes that can confer resistance to cisplatin, we introduced a yeast genomic library into Saccharomyces cerevisiae and selected for transformants that grew in the presence of a normally toxic concentration of cisplatin. Plasmids were rescued from the transformants and were analyzed for the presence of individual open reading frames that conferred resistance to cisplatin. We isolated two genes, CIN5 and YDR259c, that increased resistance to cisplatin when overexpressed in Saccharomyces cerevisiae. These genes encoded two proteins, Cin5 and Ydr259c, that were homologous to yAP-1, a basic leucine zipper transcriptional factor that is known to mediate cellular resistance to various toxic agents. The two proteins exhibited stronger homology to each other (33.2% identity, 49.2% similarity) than to all other gene products in S. cerevisiae. Overexpression of each of these proteins also conferred resistance to two DNA-alkylating agents, methylmethanesulfonate and mitomycin C. An experiment with fusion proteins with green fluorescent protein revealed that Cin5 and Ydr259c were localized constitutively in the nuclei of yeast cells. Our results suggest that Cin5 and Ydr259c might be involved in pleiotropic drug-resistance and might protect yeast against the toxicity of cisplatin and other alkylating agents via a single mechanism. These two nuclear proteins might act as transcriptional factors, regulating the expression of certain genes that confer resistance to DNA-alkylating agents.

Metallothionein mediates gene expression of 3.1 mRNA (PTZ17) related to epileptic seizure.

Genes differentially expressed in association with disruption of the metallothionein gene were screened using two hepatic stellate cell lines isolated and established from the livers of normal 129/Sv (IMS/N cells) and transgenic mice deficient in the genes for metallothionein-I and -II (IMS/MT (-) cells). We found one cDNA (tentatively named NM31) that was expressed only in IMS/IN cells. Transfecting IMS/MT (-) cells with the genes for both metallothionein-I and -II resulted in NM31 expression. These results suggest that metallothionein is essential for NM31 gene expression. The nucleotide sequence of NM31 (294 bp) was identical to the 3' region of 3.1 mRNA (PTZ 17), which is abundant in the embryonic mouse brain and is related to chemically induced seizures. The present study indicates that metallothionein mediates the expression of specific genes. This is a novel explanation for some of the functions of metallothionein.

Activation of the interferon-inducible 2'-5'-oligoadenylate synthetase gene by hepatitis C virus core protein.

The effects of hepatitis C virus (HCV) proteins on several signal transduction pathways in human nonneoplastic hepatocyte PH5CH8 cells were investigated using expression vectors encoding HCV proteins derived from HCV-infected human nonneoplastic cultured T-lymphocyte and hepatocyte cells (MT-2C and PH5CH7), which could support HCV replication. The amino acid sequences of HCV proteins obtained from HCV-infected human cells were identical or very close to the consensus sequences of the proteins derived from the original inoculum used for HCV infection. During the course of the study, we found that HCV core protein specifically activated the 40/46-kDa 2'-5'-oligoadenylate synthetase (2'-5'-OAS) gene promoter in a dose-dependent manner in different human hepatocyte cell lines (PH5CH8, HepG2, and PLC/PRF/5). We also found that the activation by core protein was further enhanced in the cells treated with alpha interferon. The expression of E1 or E2 envelope protein or nonstructural NS5A protein did not activate the 2'-5'-OAS gene promoter. We demonstrated that the activation by core protein in the hepatocyte cells was suppressed by antisense RNA complementary to core-encoding RNA. Deletion mutant analysis of core protein and deletion analysis of the 2'-5'-OAS gene promoter have been performed. Finally, we demonstrated that the activation of the 2'-5'-OAS gene occurred at the transcriptional level and furthermore demonstrated that the endogenous 2'-5'-OAS gene was also activated by core protein. This is the first report to show that a viral protein activated the 2'-5'-OAS gene.

Hepatitis C virus NS5B RNA replicase specifically binds ribosomes.

Hepatitis C virus (HCV) non-structural protein 5B (NS5B) is an RNA replicase. We expressed full-length NS5B (591 amino acid residues) in Escherichia coli as a fusion protein with maltose binding protein (MBP-NS5B). MBP-NS5B was recovered in the soluble fraction after centrifugation at 40,000 x g and affinity-purified with amylose resin. The purified MBP-NS5B had a high-level of poly (A), oligo (U)-dependent UMP incorporation with a Km of 2 microM for UTP. Surprisingly, the enzymatically active MBP-NS5B was sedimented by ultracentrifugation at 160,000 x g. The pellet contained 16S and 23S ribosomal RNAs, suggesting that ribosomes were associated with MBP-NS5B. Ribosomes and MBP-NS5B were subsequently co-purified on amylose resin. Deletion study revealed that either the N-terminal (amino acid residues 1-107) or the C-terminal (amino acid residues 498-591) region of NS5B were sufficient for this association with ribosomes. We further found that NS5B also bound with human ribosomes. Our results implicate a novel mechanism of coupling between replication and translation of the viral genome in the life cycle of HCV.

HeLa cell transformants overproducing mouse metallothionein show in vivo resistance to cis-platinum in nude mice.

Plasmid pSV2MT-I encoding mouse metallothionein-I (MT-I) designed to be expressed under the control of an SV40 promoter was introduced into human HeLa S3 cells. Several transformants (HeLa/MTH) carrying multi-copies of mouse MT-I cDNA in their genomes were isolated. These transformants produced 4 to 20-fold larger amounts of MT than their parent cells. The MT levels in HeLa/MTH were well correlated with the extent of resistance to cadmium, but not with that to cis-platinum (cis-DDP) in vitro. To study the role of MT in resistance to cis-DDP in vivo, nude mice were inoculated subcutaneously with two independent HeLa/MTH clones. MT levels in these tumors were about 3-fold higher than those in the parental cells. The growth of tumors derived from either HeLa/MTH clone was not inhibited in the presence of 15 micromol/kg of cis-DDP, which completely inhibited the growth of tumors derived from the parental HeLa cells. These data strongly suggest that the elevated level of MT confers resistance to cis-DDP in vivo but not in vitro. Thus, the results of this study indicate that in vitro determinations of the influence of MT on cis-DDP resistance may underestimate its importance in in vivo situations.

GFAT as a target molecule of methylmercury toxicity in Saccharomyces cerevisiae.

Using a genomic library constructed from Saccharomyces cerevisiae, we have identified a gene GFA1 that confers resistance to methylmercury toxicity. GFA1 encodes L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT) and catalyzes synthesis of glucosamine-6-phosphate. Transformed yeast cells expressing GFA1 demonstrated resistance to methylmercury but no resistance to p-chloromercuribenzoate, a GFAT inhibitor. The cytotoxicity of methylmercury was inhibited by loading excess glucosamine 6-phosphate into yeast. Considering that GFAT is an essential cellular enzyme, our findings suggest that GFAT is the major target molecule of methylmercury in yeasts. This report is the first to identify the target molecule of methylmercury toxicity in eukaryotic cells.

Characterization of antiviral activity of lactoferrin against hepatitis C virus infection in human cultured cells.

We recently found that bovine lactoferrin (bLF), a milk glycoprotein belonging to the iron transporter family, prevented hepatitis C virus (HCV) infection in human hepatocyte PH5CH8 cells, that are susceptible to HCV infection, and demonstrated that the anti-HCV activity of bLF was due to the interaction of bLF and HCV. In this study we further characterized the anti-HCV activity of bLF and the mechanism by which bLF prevents HCV infection. We found that bLF inhibited viral entry to the cells by interacting directly with HCV immediately after mixing of bLF and HCV inoculum. The anti-HCV activity of bLF was lost by heating at 65 degrees C, and other milk proteins (mucin, beta-lactoglobulin and casein) did not prevent HCV infection, indicating that bLF prevented HCV infection in a rather specific manner. Furthermore, we found that bovine lactoferricin, a basic N-terminal loop of bLF that is an important region for antibacterial activity, did not exhibit any anti-HCV activity, suggesting that some other region is involved in anti-HCV activity. We confirmed that prevention of HCV infection by bLF was a general phenomenon, because bLF inhibited HCV infection with all five inocula examined, and bLF inhibited HCV infection in human MT-2C T-cells, that were susceptible to HCV infection. In addition, infection with hepatitis G virus, which is distantly related to HCV, was prevented also by bLF. In conclusion, lactoferrin is a natural glycoprotein which effectively protects against HCV infection in hepatocytes and lymphocytes by neutralizing the virus.

[A case of acute respiratory distress syndrome induced by fulminant influenza A (H3 N2) pneumonia].

We report a rare case of acute respiratory distress syndrome (ARDS) induced by Influenza A (H3 N2) without secondary microbiological infection. A 69-year-old woman was admitted to our hospital because of cough and severe dyspnea. We diagnosed ARDS, because of the severe respiratory failure resistant to high-dose oxygen, the diffuse bilateral infiltrates without cardiomegaly on chest radiography, and the normal pulmonary artery wedge pressure. This patient was treated with high doses of methylprednisolone, antibiotics, globulins, urinastatin, neutrophilic elastase inhibitor, nitric oxide inhalation, and extracorporeal membrane oxygenation, but died on the thirteenth hospital day. Our final diagnosis was ARDS induced by fulminant influenza (A/Hong Kong/68 (H3 N2)) virus pneumonia, because the antibody titers of H3 N2 influenza of paired sera showed a 128-fold increase.

A reliable internally controlled RT-nested PCR method for the detection of hepatitis C virus RNA.

We have developed a reliable internally controlled RT-nested PCR method for the detection of hepatitis C virus (HCV) RNA using in vitro synthesized Renilla luciferase (Rluc) RNA as an internal control. Using this method, the 5'-noncoding region of HCV RNA (144 nucleotides) and Rluc RNA (276 nucleotides) were efficiently amplified in a single tube, and the sensitivity and specificity of this method were comparable to standard RT-nested PCR. This method was successfully performed on RNA specimens obtained from in vitro HCV-infected human hepatocyte PH5CH8 cells, which support HCV replication. In addition, we demonstrated that this method was useful for the evaluation of antiviral reagents by confirming the anti-HCV activity of bovine lactoferrin, which we previously found to be a new inhibitor of HCV infection. Therefore, this method may be useful for the studies of not only HCV but also of other viruses.

Modulation of adriamycin toxicity by tissue-specific induction of metallothionein synthesis in mice.

The effect of tissue specific induction of metallothionein (MT) by preadministration of metal compounds on the antitumor activity and adverse effects of adriamycin (ADR) was examined using mice bearing colon 38 adenocarcinoma. Significant increase in MT concentration was observed in the heart and bone marrow but not in the tumor tissue of the mice given bismuth (Bi) compound. Copper (Cu) increased MT in the tumor tissue but did not induce MT either in bone marrow or in the heart, whereas zinc (Zn) increased MT level in the heart and bone marrow as well as in the tumor tissue. ADR exerted cardiotoxicity, indicated by increase in lipid peroxidation in the heart, bone marrow toxicity, indicated by decrease in number of peripheral leukocytes, and antitumor activity, assessed by reduction of tumor weight, in tumor-bearing mice untreated with MT inducing metal compounds. Preadministration of Bi significantly reduced the cardiotoxicity and bone marrow toxicity without compromising the antitumor activity of ADR. Cu pretreatment did not affect the extent of cardiotoxicity and bone marrow toxicity but significantly suppressed the antitumor effect. Pretreatment with Zn markedly reduced not only the adverse side effects but also the antitumor activity. The results described above suggest that ADR toxicity can be attenuated in the tissues in which the MT level was elevated and that the tissue specific induction of MT synthesis may provide a promising regimen for cancer chemotherapy.

Protective effects of triterpene compounds against the cytotoxicity of cadmium in HepG2 cells.

The effects of triterpene compounds on cadmium toxicity were investigated in HepG2 cells. Ten triterpene compounds were examined, namely, betulin, soyasapogenol A, soyasapogenol B, ursolic acid, uvaol, oleanolic acid, friedelin, glycyrrhizin, 18alpha-glycyrrhetinic acid, and 18beta-glycyrrhetinic acid, and betulin, soyasapogenol A, and uvaol were found to reduce the toxicity of CdCl(2). In particular, betulin almost completely abolished the cytotoxicity of CdCl(2) at concentrations as low as 0. 1 microg/ml. The effects of betulin were particularly apparent when added to the culture medium before the addition of CdCl(2). Moreover, when HepG2 cells were incubated with betulin and then incubated in fresh betulin-free medium before the addition of CdCl(2), the toxic effects of cadmium were reduced. Betulin had no significant effect on the intracellular accumulation of cadmium, nor did it bind to cadmium, at least not in a test tube. When HepG2 cells were treated first with cycloheximide or actinomycin D, the subsequent protective effect of betulin against cadmium toxicity was significantly reduced, suggesting that betulin might protect cells against cadmium toxicity by inducing the synthesis of a certain protein or proteins. The synthesis of metallothionein, a protein that is known to reduce the toxicity of heavy metals, was not induced by betulin. However, using the differential display method, we confirmed that betulin promoted the expression of several genes. Our findings suggest that betulin might reduce cadmium toxicity by promoting the synthesis of certain proteins that protect cells against the toxic effects of cadmium.

Overexpression of L-glutamine:D-fructose-6-phosphate amidotransferase provides resistance to methylmercury in Saccharomyces cerevisiae.

To identify novel genes that confer resistance to methylmercury (MeHg), a yeast genomic DNA library was transfected into Saccharomyces cerevisiae. Two functional plasmids were isolated from transfected yeast clones D1 and H5 that exhibited resistance to MeHg. The yeast transfected with plasmid isolated from clone H5 was several-fold more resistant than yeast transfected with plasmid from clone D1. Functional characterization of the genomic DNA fragment obtained from clone H5 determined that the GFA1 gene conferred resistance to MeHg. GFA1 was reported to encode L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT) which catalyzes the synthesis of glucosamine-6-phosphate from glutamine and fructose-6-phosphate. Accumulation of mercury in yeast clone W303B/pGFA1, which contains the transfected GFA1 gene, did not differ from that in control yeast clone W303B/pYES2. The W303B/pGFA1 strain did not show resistance to mercuric chloride, zinc chloride, cadmium chloride or copper chloride, suggesting that the resistance acquired by GFA1 gene transfection might be specific to MeHg. This is the first report of a gene involved in MeHg resistance in eukaryotic cells identified by screening a DNA library.

PF1070A, a novel and potent inducer of the synthesis of metallothionein.

Using mouse Ltk(-) cells (L13-17 cells) that had been transfected with a plasmid in which the lacZ gene had been ligated downstream of 1.4 kbp of the sequence of the promoter of the mouse gene for metallothionein-I (MT-I) as a reporter gene, we examined 268 organic compounds for the ability to activate this promoter. We found that PF1070A, an antibiotic produced by Humicola sp., efficiently activated the MT promoter and caused marked enhancement of beta-galactosidase activity in L13-17 cells. The extent of activation by PF1070A was almost equivalent to that by of zinc ions, the most effective known inducer of the synthesis of MT. PF1070A also caused marked elevation of the levels of the mRNA for MT and of MT itself in L13-17 cells. A similar result was obtained in human HeLa-S3 cells. When PF1070A was added to the culture medium simultaneously with cadmium ion or dexamethasone, the level of expression of the reporter gene was markedly elevated, compared to the level of expression induced by each agent independently. The effect of PF1070A was reduced considerably by deletion of nucleotides at positions -150 and -149 from the site of initiation of transcription in the promoter region of the MT gene and also by deletion of the seven bases located at positions -49 to -43. Since no known cis element was found in these two regions, PF1070A might be a new type of inducer of MT synthesis that promotes expression of the gene for MT via a mechanism completely different from those exploited by other known agents. These results also suggest the presence of a system for control of transcription of the gene for MT that has not previously been recognized. Both cadmium ions and bismuth ions induce the synthesis of MT by acting on the metal response element (MRE). Bismuth ions had no significant effect on the promoter activity that had already reached a maximum level in response to treatment with the optimal concentration of cadmium ion. By contrast, PF1070A further and markedly increased the promoter activity. This result suggests that it is possible to increase the concentration of MT in tissue using PF1070A as an inducer even in cases where the MRE-mediated activation of the MT promoter has already been induced by the accumulation of cadmium, as is the case in a clinical setting. PF1070A may prove to be an excellent inducer of MT synthesis that is effective and clinically applicable. Moreover, use of PF1070A in combination with salts of heavy metals might be useful in controlling expression of a transfected gene that is regulated by the MT promoter since PF1070A can activate the MT promoter to an extent that cannot be achieved with heavy metal ions alone, when PF1070A is used in combination with zinc ions at a concentration of the latter considerably below the toxic level.