Kazuaki Tabu

Glycoprotein Nonmetastatic Melanoma B (Gpnmb)-Positive Macrophages Contribute to the Balance between Fibrosis and Fibrolysis during the Repair of Acute Liver Injury in Mice.

Background and aims Glycoprotein nonmetastatic melanoma B (Gpnmb), a transmembrane glycoprotein that is expressed in macrophages, negatively regulates inflammation. We have reported that Gpnmb is strongly expressed in the livers of rats fed a choline-deficient, L-amino acid-defined (CDAA) diet. However, the role of macrophage-expressed Gpnmb in liver injury is still unknown. This study aimed to clarify the characteristics of infiltrating macrophages that express Gpnmb, and the involvement of Gpnmb in the repair process in response to liver injury. Methods C57BL/6J, DBA/2J [DBA] and DBA/2J-Gpnmb+ [DBA-g+] mice were treated with a single intraperitoneal injection of carbon tetrachloride (CCl4) at a dose of 1.0 mL/kg body weight. Mice were sacrificed at predetermined time points, followed by measurement of serum alanine aminotransferase (ALT) levels and histological examination. Expression of Gpnmb, pro-/anti-inflammatory cytokines, and profibrotic/antifibrotic factors were examined by quantitative RT-PCR and/or Western blotting. Immunohistochemistry, fluorescent immunostaining and flow cytometry were used to determine the expression of Gpnmb, CD68, CD11b and α-SMA, phagocytic activity, and the presence of apoptotic bodies. We used quantitative RT-PCR and ELISA to examine TGF-β and MMP-13 expression and the concentrations and supernatants of isolated infiltrating hepatic macrophages transfected with siGpnmb. Results In C57BL/6J mice, serum ALT levels increased at two days after CCl4 injection and decreased at four days. Gpnmb expression in the liver was stimulated four days after CCl4 injection. Histological examination and flow cytometry showed that Gpnmb-positive cells were almost positive for CD68-positive macrophages, contained engulfed apoptotic bodies and exhibited enhanced phagocytic activity. Isolated infiltrating hepatic macrophages transfected with siGpnmb showed high MMP-13 secretion. There was no significant difference in the magnitude of CCl4-induced liver injury between DBA-g+ and DBA mice. However, hepatic MMP-13 expression, as well as α-SMA expression and collagen production, increased significantly in DBA-g+ compared with DBA mice. Conclusions Gpnmb-positive macrophages infiltrate the liver during the recovery phase of CCl4–induced acute liver injury and contribute to the balance between fibrosis and fibrolysis in the repair process following acute liver injury.

Expression of glycoprotein nonmetastatic melanoma protein B in macrophages infiltrating injured mucosa is associated with the severity of experimental colitis in mice

Glycoprotein nonmetastatic melanoma protein B (Gpnmb) is a transmembrane glycoprotein, which negatively regulates the inflammatory responses of macrophages. However, the role of Gpnmb in intestinal macrophages remains to be fully elucidated. The present study aimed to investigate the expression of Gpnmb and its effects on colonic mucosal injuries associated with dextran sulfate sodium (DSS)‑induced colitis in BALB/c mice, DBA/2J (D2) mice lacking Gpnmb and Gpnmb‑transgenic DBA/2J mice (D2‑gpnmb+). The colonic expression of Gpnmb increased with the severity of DSS‑induced colitis in BALB/c mice, and macrophages infiltrating the inflamed mucosa were found to express Gpnmb. The D2 mice lacking Gpnmb exhibited more severe DSS‑induced colitis, which was accompanied by higher levels of pro‑inflammatory cytokines, including interleukin (IL)‑1β and IL‑6, compared with the D2‑gpnmb+ mice. Following lipopolysaccharide stimulation, macrophages from the D2 mice expressed higher levels of pro‑inflammatory cytokines and lower levels of IL‑10, compared with the D2‑gpnmb+mice. In addition, in the RAW264.7 murine macrophage cell line, knockdown of Gpnmb by small interfering RNA was associated with increased production of pro‑inflammatory cytokines, which were potentially mediated by the extracellular signal‑regulated kinase (ERK) and p38 signaling pathways. The results of the present study indicated that macrophages infiltrating injured mucosa express Gpnmb, and that Gpnmb‑positive macrophages may ameliorate inflammation in the intestinal mucosa by decreasing pro‑inflammatory cytokine production via the ERK and p38 signaling pathways.

Hepatitis C virus NS3/4A protease inhibits complement activation by cleaving complement component 4.

Background It has been hypothesized that persistent hepatitis C virus (HCV) infection is mediated in part by viral proteins that abrogate the host immune response, including the complement system, but the precise mechanisms are not well understood. We investigated whether HCV proteins are involved in the fragmentation of complement component 4 (C4), composed of subunits C4α, C4β, and C4γ, and the role of HCV proteins in complement activation. Methods Human C4 was incubated with HCV nonstructural (NS) 3/4A protease, core, or NS5. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then subjected to peptide sequencing. The activity of the classical complement pathway was examined using an erythrocyte hemolysis assay. The cleavage pattern of C4 in NS3/4A-expressing and HCV-infected cells, respectively, was also examined. Results HCV NS3/4A protease cleaved C4γ in a concentration-dependent manner, but viral core and NS5 did not. A specific inhibitor of NS3/4A protease reduced C4γ cleavage. NS3/4A protease–mediated cleavage of C4 inhibited classical pathway activation, which was abrogated by a NS3/4A protease inhibitor. In addition, co-transfection of cells with C4 and wild-type NS3/4A, but not a catalytic-site mutant of NS3/4A, produced cleaved C4γ fragments. Such C4 processing, with a concomitant reduction in levels of full-length C4γ, was also observed in HCV-infected cells expressing C4. Conclusions C4 is a novel cellular substrate of the HCV NS3/4A protease. Understanding disturbances in the complement system mediated by NS3/4A protease may provide new insights into the mechanisms underlying persistent HCV infection.

Human neutrophil peptide-1 promotes alcohol-induced hepatic fibrosis and hepatocyte apoptosis

Background and aims Neutrophil infiltration of the liver is a typical feature of alcoholic liver injury. Human neutrophil peptide (HNP)-1 is an antimicrobial peptide secreted by neutrophils. The aim of this study was to determine if HNP-1 affects ethanol-induced liver injury and to examine the mechanism of liver injury induced by HNP-1. Methods Transgenic (TG) mice expressing HNP-1 under the control of a β-actin-based promoter were established. Ethanol was orally administered to HNP-1 TG or wild-type C57BL/6N (WT) mice. SK-Hep1 hepatocellular carcinoma cells were used to investigate the effect of HNP-1 on hepatocytes in vitro. Results After 24 weeks of ethanol intake, hepatic fibrosis and hepatocyte apoptosis were significantly more severe in TG mice than in WT mice. Levels of CD14, TLR4, and IL-6 in liver tissues were higher in TG mice than in WT mice. Apoptosis was accompanied by higher protein levels of caspase-3, caspase-8, and cleaved PARP in liver tissue. In addition, phosphorylated ASK1, ASK1, phosphorylated JNK, JNK1, JNK2, Bax, Bak and Bim were all more abundant in TG mice than in WT mice. In contrast, the level of anti-apoptotic Bcl2 in the liver was significantly lower in TG mice than in WT mice. Analysis of microRNAs in liver tissue showed that miR-34a-5p expression was significantly higher in TG mice than in WT mice. Furthermore, in the presence of ethanol, HNP-1 increased the apoptosis with the decreased level of Bcl2 in a concentration-dependent manner in vitro. Conclusions HNP-1 secreted by neutrophils may exacerbate alcohol-induced hepatic fibrosis and hepatocyte apoptosis with a decrease in Bcl2 expression and an increase in miR-34a-5p expression.

Utility of percutaneous radiofrequency ablation alone or combined with transarterial chemoembolization for early hepatocellular carcinoma

Percutaneous radiofrequency ablation (RFA) combined with transarterial chemoembolization (TACE) is an effective, standard therapy against small hepatocellular carcinoma (HCC). However, there is debate regarding the effectiveness of RFA combined with TACE (RFA/TACE) compared with RFA alone. These two approaches were compared for the treatment of early HCC. The present study examined 83 HCC tumors in 83 patients treated with RFA between April 2007 and August 2014 at three medical institutions. All HCCs were single hypervascular tumors, with a median diameter of 16 mm (range, 6-30 mm). The overall survival (OS) rate of all patients (n=83) was 97.5% at 1 year, 82.8% at 3 years and 48.6% at 5 years, and the local recurrence rate of all patients was 14.3% at 1 year, 32.3% at 3 years and 36.5% at 5 years. The tumor-free survival (TFS) rate of all patients was 95.1% at 1 year, 56.3% at 3 years and 23.4% at 5 years. Compared with RFA alone, RFA/TACE significantly improved OS (P<0.001), intrahepatic distant recurrence (IDR; P=0.038) and TFS (P=0.010). A univariate analysis of prognostic indicators revealed that age <70 years (P=0.008), aspartate transaminase <40 IU/l (P=0.003), alanine aminotransferase <40 IU/l (P=0.006) and platelet count >10×104/µl (P=0.05) were associated with a high survival rate. Multivariate analysis identified RFA/TACE [hazard ratio (HR), 0.108; P=0.001] as an independent prognostic indicator. RFA/TACE was identified as the only independent indicator of IDR (HR: 0.467; P=0.042) and TFS (HR: 0.452; P=0.012). RFA/TACE improved OS rate, IDR and TFS compared with RFA alone. The data suggested that RFA/TACE should be considered for the treatment of single hypervascular HCC.

The co-existence of NS5A and NS5B resistance-associated substitutions is associated with virologic failure in Hepatitis C Virus genotype 1 patients treated with sofosbuvir and ledipasvir

Objective The present study aimed to reveal the factors associated with virologic failure in sofosbuvir and ledipasvir (SOF/LDV)-treated patients, and identify baseline NS5A or NS5B resistance-associated substitutions (RASs). Methods Four hundred ninety-three patients with Hepatitis C Virus (HCV) genotype 1b infection were treated with SOF/LDV; 31 had a history of interferon (IFN)-free treatment with daclatasvir and asunaprevir. The effect of baseline RASs on the response to SOF/LDV therapy was analyzed. Results Overall, a sustained virologic response at 12 weeks (SVR12) was achieved in 476 patients (96.6%). The SVR12 rates in the patients with IFN-free treatment-naïve and retreatment were 97.6% and 80.6%, respectively. HCV elimination was not achieved in 17 patients, 11 (including 5 with IFN-free retreatment) of whom had virologic failure. Eight patients had coexisting NS5A RASs of Q24, L28 and/or R30, L31, or Y93 and one patient had coexisting NS5A RASs of P32L and A92K. Interestingly, 10 and 8 patients had NS5B A218S and C316N RAS respectively. According to a multivariate analysis, coexisting NS5A RASs, NS5A P32 RAS, NS5B A218 and/or C316 RASs, and γ-glutamyltranspeptidase were associated with virologic failure. In the naïve patients, all patients without NS5B A218 and/or C316 RAS achieved an SVR12. Notably, the SVR12 rates of patients with coexisting NS5A and NS5B RASs were significantly lower (83.3%). Conclusions Although SOF/LDV therapy resulted in a high SVR12 rate, coexisting NS5A and NS5B RASs were associated with virologic failure. These results might indicate that the coexisting baseline RASs influence the therapeutic effects of SOF/LDV.

Image support system in transarterial chemoembolization contributes to reduction of the quantity of radiation exposure and contrast media

We confirmed the clinical utility of a three‐dimensional navigation system during transarterial chemoembolization.

Sustained virologic response after eight weeks of treatment with sofosbuvir, ledipasvir, and ribavirin in a decompensated cirrhotic patient with hepatitis C virus genotype 1b: Therapy for decompensated cirrhosis

A 68‐year‐old Japanese man with decompensated cirrhosis due to hepatitis C virus (HCV) genotype 1b infection was treated with sofosbuvir (SOF; 400 mg/day), ledipasvir (LDV; 90 mg/day), and ribavirin (RBV; 400 mg/day). Before treatment, his Child–Pugh and Model for End‐Stage Liver Disease (MELD) scores were 10 (class C) and 13 points, respectively. Although RBV was initially given at two‐thirds the normal dose due to anemia, his hemoglobin level gradually declined, and RBV was reduced to 200 mg daily on day 11, and 200 mg every other day on day 14. His alanine aminotransferase level gradually decreased during combination therapy; and HCV‐RNA was undetectable on day 28. He complained of fatigue from day 49, and RBV was ceased. On day 56, he asked to discontinue treatment because of strong fatigue and insomnia. As hepatic encephalopathy occurred just after the cessation of direct‐acting antivirals, diuretics were discontinued, and treatment with synthetic disaccharides and intractable antibiotics were given, after which his consciousness returned to normal. Ascites gradually disappeared, and a sustained virologic response (SVR) was achieved. At 1.5 years after treatment, his Child–Pugh and MELD scores had improved to 6 (class A) and 10 points, respectively. Although he did not experience hepatic encephalopathy during the observation period, his blood ammonia concentration persistently increased. We reported a case of decompensated cirrhosis in a patient who achieved SVR with SOF/LDV plus RBV for 8 weeks. Although his liver function improved after treatment, careful long‐term observation is required for complications of liver cirrhosis, even after HCV elimination.

Early sorafenib induction after transarterial chemoembolization for unresectable hepatocellular carcinoma: Can sorafenib after TACE improve loco‑regional control?

This prospective study aimed to estimate the efficacy of sorafenib therapy after transarterial chemoembolization (TACE) for unresectable hepatocellular carcinoma (HCC). Between July 2011 and March 2013, 17 patients were enrolled, 11 of whom received sorafenib therapy. Patients who previously received TACE for HCC and whose disease progressed within a six-month period were given 400-800 mg sorafenib orally, once or twice daily, within the 3 weeks after a second TACE (sorafenib after TACE group). The response to treatment, time to progression (TTP), overall survival (OS), and adverse events (AEs) were recorded. Of the 113 patients who underwent initial TACE for unresectable HCC between January 1995 and January 2013, 23 patients were selected who were treated with TACE alone, and for whom the interval between the second and third TACE treatments was <6 months (TACE alone group). The interval (TTP) was calculated between the third and fourth TACE treatments, then TTP was compared among the three groups: Sorafenib after TACE for > or <4 months; and TACE alone. During a median follow-up period of 34.4 months (range, 5.9-51.7 months) in both groups receiving sorafenib after TACE, sorafenib prolonged TTP (3.9 months) and OS (34.4 months). It was demonstrated that sorafenib use for >4 months prolonged TTP (5.7 months) significantly compared with use for <4 months (3.0 months) (P=0.002). The OS of patients given sorafenib for >4 months (35.9 months) was longer than that of patients who received the drug for <4 months (17.2 months), but this difference was not significant. In the TACE alone group, the median TTP between the third and fourth TACE treatments was 4.3 months. TTP decreased among the groups in the following order: Sorafenib for >4 months, TACE alone, and sorafenib for <4 months. There were three AEs of grade 3 in the present study. Two patients demonstrated a decrease in liver reserve function following sorafenib treatment, but improved immediately after sorafenib administration was stopped. Sorafenib induction early after TACE for unresectable HCC was generally well tolerated and significantly improved TTP. Further studies are required to confirm the safety and efficacy of this combination therapy.

Horizontal transmission of de novo hepatitis B between spouses: A case report.

We report a female patient with acute hepatitis B due to horizontal transmission of hepatitis B virus from her husband, who suffered from de novo hepatitis B. A 48‐year‐old man underwent peripheral blood stem cell transplantation (PBSCT) for adult T‐cell leukemia/lymphoma. Nine months after the initial treatment, he was referred to our hospital because of jaundice. Laboratory data showed elevated serum aminotransferase levels and hepatitis B surface antigen (HBsAg) positivity. We diagnosed de novo hepatitis B because a pre‐PBSCT serum sample was negative for HBsAg and positive for anti‐hepatitis B core antibody (HBcAb). His liver function improved with entecavir therapy. Two months after his diagnosis of hepatitis B, his 31‐year‐old wife was admitted with fever and appetite loss. She was diagnosed with acute hepatitis B because of increased serum aminotransferase levels and HBsAg and immunoglobulin M HBcAb positivity. Sequencing of HBV DNA in the serum obtained from both patients showed 99.9% homology. Therefore, we diagnosed her acute hepatitis B as due to horizontal transmission of de novo hepatitis B from her husband. HBV derived from de novo hepatitis B should be considered a potential source of infection, although intrafamilial transmission of de novo hepatitis B is rare.