Tri-antennary tri-sialylated mono-fucosylated glycan of alpha-1 antitrypsin as a non-invasive biomarker for non-alcoholic steatohepatitis: a novel glycobiomarker for non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a progressive form of non-alcoholic fatty liver disease (NAFLD) that may lead to liver cirrhosis or hepatocellular carcinoma. Here, we examined the diagnostic utility of tri-antennary tri-sialylated mono-fucosylated glycan of alpha-1 antitrypsin (AAT-A3F), a non-invasive glycobiomarker identified in a previous study of NASH diagnosis. This study included 131 biopsy-proven Japanese patients with NAFLD. We evaluated the utility of AAT-A3F in NASH diagnosis, and conducted genetic analysis to analyse the mechanism of AAT-A3F elevation in NASH. Serum AAT-A3F concentrations were significantly higher in NASH patients than in NAFL patients, and in patients with fibrosis, lobular inflammation, and ballooning. Hepatic FUT6 gene expression was significantly higher in NASH than in NAFL. IL-6 expression levels were significantly higher in NASH than in NAFL and showed a positive correlation with FUT6 expression levels. The serum-AAT-A3F levels strongly correlated with hepatic FUT6 expression levels. AAT-A3F levels increased with fibrosis, pathological inflammation, and ballooning in patients with NAFLD and may be useful for non-invasive diagnosis of NASH from the early stages of fibrosis.

POGLUT1, the putative effector gene driven by rs2293370 in primary biliary cholangitis susceptibility locus chromosome 3q13.33.

Primary biliary cholangitis (PBC) is a chronic and cholestatic autoimmune liver disease caused by the destruction of intrahepatic small bile ducts. Our previous genome-wide association study (GWAS) identified six susceptibility loci for PBC. Here, in order to further elucidate the genetic architecture of PBC, a GWAS was performed on an additional independent sample set, then a genome-wide meta-analysis with our previous GWAS was performed based on a whole-genome single nucleotide polymorphism (SNP) imputation analysis of a total of 4,045 Japanese individuals (2,060 cases and 1,985 healthy controls). A susceptibility locus on chromosome 3q13.33 (including ARHGAP31, TMEM39A, POGLUT1, TIMMDC1, and CD80) was previously identified both in the European and Chinese populations and was replicated in the Japanese population (OR = 0.7241, P = 3.5 × 10-9). Subsequent in silico and in vitro functional analyses identified rs2293370, previously reported as the top-hit SNP in this locus in the European population, as the primary functional SNP. Moreover, e-QTL analysis indicated that the effector gene of rs2293370 was Protein O-Glucosyltransferase 1 (POGLUT1) (P = 3.4 × 10-8). This is the first study to demonstrate that POGLUT1 and not CD80 is the effector gene regulated by the primary functional SNP rs2293370, and that increased expression of POGLUT1 might be involved in the pathogenesis of PBC.

Surgically Diagnosed Primary Hepatic Angiosarcoma.

Primary hepatic angiosarcoma is a rare tumor originating from endothelial cells in the liver and accounts for approximately 1% of all hepatic malignant tumors. It is difficult to diagnose due to the lack of specific symptoms or tumor markers. No effective treatment exists, but complete surgical resection may achieve a good outcome. Since most primary hepatic angiosarcomas are already at an advanced stage at diagnosis, few reports describe tumors smaller than 2 cm. We report a case of surgery for a 1.7-cm sized primary hepatic angiosarcoma. Further studies are required to improve the preoperative diagnosis of primary hepatic angiosarcoma.

Investigation of the utility of various diagnostic guidelines for Wilson's disease.

Wilson's disease is one of many potential differential diagnoses in patients with unknown liver injury or liver cirrhosis. Many patients are diagnosed in early childhood, but rare cases may not be revealed until adulthood, and of these, most present with liver cirrhosis. While some patients can be diagnosed by the examination of serum ceruloplasmin and urinary copper levels, there are patients in whom ATP7B genetic testing and hepatic copper content measurement are indicated. Diagnostic guidelines for Wilson's disease were proposed by the American Association for the Study of Liver Diseases in 2008, the European Association for the Study of the Liver in 2012, and the Japan Society of Hepatology along with affiliated societies in 2015. Even in patients in whom making a diagnosis was problematic, we were able to establish confirmation of Wilson's disease in patients with unknown adulthood liver dysfunction using these established guidelines. In this paper, we present the cases of patients with Wilson's disease treated by the administration of copper chelating agents in our department using diagnostic criteria and further provide flow charts of each guideline. We also report the utility of this approach.

Safety and efficacy of daclatasvir and asunaprevir in hepatitis C virus-infected patients with renal impairment.

AIM: Hepatitis C virus (HCV) infection is a risk factor for end-stage renal disease, renal graft failure, and hemodialysis patient mortality. However, the efficacy of direct-acting antiviral therapy for HCV-infected patients with renal impairment is unclear. Additionally, the promising NS5B inhibitor sofosbuvir has not been recommended for patients with severe renal impairment. In this prospective, multicenter study, we evaluated the efficacy and safety of daclatasvir and asunaprevir combination therapy, with a focus on patients with renal impairment. METHODS: The study included 322 genotype 1 HCV-infected patients who received daclatasvir and asunaprevir combination therapy. The safety and sustained virological response was examined at 12 weeks after the end of treatment and safety was evaluated according to renal function. RESULTS: Of 322 patients, 5% (16/322) and 2.5% (8/322) had chronic kidney disease stage G3b (estimated glomerular filtration rate [eGFR], 30-44 mL/min/1.73 m2 ) and stage G4/5 (eGFR, 15-29/<15 mL/min/1.73 m2 ), respectively. Baseline presence of the NS5A resistance-associated variant, previous simeprevir treatment, and HCV RNA titers, which were predictors of a sustained viral response, were similar between patients with eGFR <45 mL/min/1.73 m2 and eGFR >45 mL/min/1.73 m2 . Notably, the 12-week sustained viral response rate was comparable in patients with eGFR <45 mL/min/1.73 m2 (100%, 24/24) and those with eGFR >45 mL/min/1.73 m2 (88.9%, 265/298; P = 0.07). Treatment discontinuation rates and adverse events, including alanine aminotransferase elevation, anemia, and renal disorders, were similar between the two groups. CONCLUSION: Daclatasvir and asunaprevir combination therapy for patients with renal dysfunction was highly effective and safe.

Combination of neutrophil-to-lymphocyte ratio and early des-γ-carboxyprothrombin change ratio as a useful predictor of treatment response for hepatic arterial infusion chemotherapy against advanced hepatocellular carcinoma.

AIM: Hepatic arterial infusion chemotherapy (HAIC) is a potent therapeutic option for advanced hepatocellular carcinoma (HCC). However, there are few known predictive factors of treatment response to HAIC. We clarified the most accurate predictive factors early on in treatment. METHODS: Study subjects were 70 patients with advanced HCC who had been treated with HAIC. We assessed the relationships between patient characteristics, change ratios of early tumor markers, tumor response, progression-free survival (PFS), and overall survival. RESULTS: After two courses of HAIC, 1 (1.4%), 16 (22.9%), 30 (42.8%), and 23 (32.9%) of the 70 patients showed complete response, partial response, stable disease, and progressive disease, respectively. Overall survival was related to Child-Turcotte-Pugh score, extrahepatic metastasis, and the des-γ-carboxyprothrombin (DCP) response. Univariate and multivariate analyses identified the neutrophil-to-lymphocyte ratio (NLR) and DCP response as significant determinants of treatment response and PFS. Progression-free survival with a low NLR (<2.87) was significantly longer than with a high NLR (median, 8.4 months vs. 2.8 months, respectively). Progression-free survival was 7.2 months for patients with a responsive DCP (<0.7) and 2.3 months for an unresponsive DCP (≥0.7). Additionally, even with baseline high NLR, patients with responsive DCP achieved better PFS. CONCLUSION: Baseline NLR and early DCP response were significant predictors of treatment response and PFS after HAIC for patients with advanced HCC. The combination of baseline NLR and early DCP response could be accurate and useful predictive factors of response to HAIC and could help optimize treatments for patients with advanced HCC.

Anti-adipogenic and antiviral effects of l-carnitine on hepatitis C virus infection.

Hepatitis C virus (HCV) has been reported to hijack fatty acid metabolism in infected hepatocytes, taking advantage of lipid droplets for virus assembly. In this study, we analyzed the anti-HCV activity of l-carnitine, a substance involved in the transport of fatty acids into mitochondria. JFH-1 or HCV replicon-transfected Huh7.5.1 cells were treated with or without l-carnitine to examine its anti-HCV effects. The effects of l-carnitine on HCV entry, HCV-induced adipogenesis and lipid droplet formation, and HCV-induced oxidative stress were examined. Treatment of JFH-1-infected cells with l-carnitine inhibited HCV propagation in a concentration-dependent manner. In contrast, l-carnitine had no anti-HCV activity in the HCV replicon system, which is lacking viral assembly. In addition, l-carnitine did not affect HCV entry. However, l-carnitine treatment decreased intracellular lipid droplets, which are crucial for HCV assembly in JFH-1-infected cells. The expression level of CPT-1 was decreased in JFH-1-infected cells, and l-carnitine treatment restored this expression. HCV-infected cells exhibited increased production of reactive oxygen species and glutathione oxidation. l-carnitine decreased oxidative stress induced by JFH-1-infection, as shown by glutathione/glutathione disulfide assays and MitoSOX staining. l-carnitine exhibited anti-HCV activity, possibly by inhibiting HCV assembly and through its anti-adipogenic activity in HCV-infected cells. Moreover, l-carnitine has antioxidant properties in HCV-infected hepatocytes. Overall, these results indicated that l-carnitine may be an effective adjunctive agent in antiviral therapies to treat chronic hepatitis C. J. Med. Virol. 89:857-866, 2017. © 2016 Wiley Periodicals, Inc.

Hepatitis B virus X protein impairs α-interferon signaling via up-regulation of suppressor of cytokine signaling 3 and protein phosphatase 2A.

Hepatitis B Virus (HBV) causes liver cirrhosis and hepatocellular carcinoma. Standard therapy includes treatment with interferon (IFN); however, its efficacy is limited. HBV has been reported to impair IFN signaling; however, the mechanism is unclear. Here, the relationship between HBV X protein (HBx) and IFN signaling was investigated by establishing HepG2 cells, stably expressing HBx (HepG2/HBx) via retrovirus-mediated gene transfer. Subsequently, IFN negative-regulator expression and its mechanism were studied. HepG2/HBx cells showed reduced expression of IFN-stimulated genes and expressed higher levels of suppressor of cytokine signaling 3 (SOCS3) and protein phosphatase 2A (PP2A) suppressor compared with control cells. Knockdown of SOCS3 and PP2A restored IFN sensitivity. Moreover, HepG2/HBx cells showed higher phosphorylation levels of signal transducers and activators of transcription 3 and endoplasmic reticulum stress, which are inducers of SOCS3 and PP2A, respectively. Additionally, HBx-knockdown restored IFN sensitivity in HepG2.2.15.7 cells. It was also confirmed that SOCS3 and PP2A expression levels were up-regulated in the liver of patients with HBV infection. The results of this study demonstrated that HBx impairs IFN signaling via increased expression of SOCS3 and PP2A, a novel mechanistic insight, providing a potential therapeutic target to enhance the efficiency of IFN therapy. J. Med. Virol. 89:267-275, 2017. © 2016 Wiley Periodicals, Inc.

Prevalence and characteristics of naturally occurring sofosbuvir resistance-associated variants in patients with hepatitis C virus genotype 1b infection.

AIM: Sofosbuvir (SOF), a nucleotide analog pro-drug, targets hepatitis C virus (HCV) NS5B polymerase and shows potential for treating HCV infection, given its high efficacy and good barrier to resistance. However, in addition to the rare resistant-associated variant (RAV) of non-structural protein NS5B S282T, several new potential RAVs of SOF have been reported, especially related to HCV genotype 1b. However, the prevalence and characteristics of these RAVs have not been clarified. METHODS: We analyzed the prevalence of variants in the NS3/NS5A/NS5B regions in 96 patients treated with simeprevir (SMV) combination therapy, and the prevalence of RAVs in patients showing treatment failure was determined by direct- or deep-sequencing methods. Associations between these potential RAVs and clinical factors were also analyzed. RESULTS: Prevalence of NS5B RAV C316N was high (46.9%, 45/96), whereas that of NS5B L159F was relatively low (1.04%, 1/96); however, deep sequencing showed that 30.0% of patients with C316N also had NS5B RAV L159F. Additionally, there was no significant relationship between the existence of potential NS5B and NS5A or NS3 RAVs. However, the presence of NS5B C316N was significantly associated with an HCV core amino acid 91 substitution. No significant difference was detected between each RAV and sustained virological response in simeprevir combination therapy. CONCLUSION: We provide clear evidence of the high prevalence of two potential naturally occurring NS5B RAVs (C316N and L159F) in Japan. It may be important to pay particular attention to these new potential RAVs, especially when using SOF-based therapy in patients with RAVs due to previous direct-acting antiviral therapy failure.

Efficacy and safety of daclatasvir and asunaprevir combination therapy in chronic hemodialysis patients with chronic hepatitis C.

BACKGROUND: HCV infection in chronic hemodialysis patients is high, has a poor prognosis and high risk of renal graft failure, and requires nosocomial infection control measures. However, options of anti-HCV therapy in such patients are limited and unsatisfactory. In this study, we report effectiveness and safety of HCV-NS5A-inhibitor daclatasvir (DCV) and protease-inhibitor asunaprevir (ASV) combination therapy for hemodialysis patients with HCV infection. METHODS: This study was registered at the UMIN Clinical Trials Registry as UMIN000016355. Thirty-four dialysis patients were treated with DCV/ASV combination therapy between January 2015 and November 2015. Of those, 21 patients who were followed more than 12 weeks after treatment ended were included. We evaluated the 12-week sustained virologic response (SVR12) and adverse events during treatment. RESULTS: Of the 21 patients, four had compensated liver cirrhosis and three had resistance-associated variant of NS5A (NS5A RAVs)-Y93H at baseline. Overall, total of 95.5 % (20/21) of the patients achieved SVR12. Of note, all patients with cirrhosis or NS5A RAVs achieved SVR12. One relapser patient at 4 weeks post-treatment had NS3 D168E RAVs at baseline. A total of 20 patients (95.5 %) completed the 24-week therapy. One patient discontinued treatment at week 12 due to ALT elevations and achieved SVR12. CONCLUSIONS: DAV and ASV combination therapy for chronic hemodialysis patients with HCV infection was highly effective and well tolerated, even in elderly patients and patients with liver cirrhosis and NS5A-RAVs.

A pivotal role of Krüppel-like factor 5 in regulation of cancer stem-like cells in hepatocellular carcinoma.

In hepatocellular carcinoma (HCC), there exists a highly tumorigenic subset of cells defined by high expression of CD44 and CD133 that has been reported to contain cancer stem-like cells (CSCs). Krüppel-like factor 5 (KLF5) regulates many factors involved in cell cycle, migration, inflammation, angiogenesis and stemness and has cancer-promoting effects in some cancers. While some reports have indicated that KLF5 may have important roles in regulation of CSCs, what role, if any, KLF5 plays in regulation of CSCs in HCC remains to be elucidated. Flow cytometric analysis of CD44 and CD133 in HCC cell lines revealed subpopulations of CD44(High)/CD133(High) and CD44(Low)/CD133(Low) cells. We subsequently sorted these subpopulations and identified KLF5 as a gene that is significantly upregulated in CD44(High)/CD44(High) cells via RNA sequencing using next generation sequencing technology. Moreover, KLF5 overexpression enriched the CD44(High)/CD133(High) subpopulation and, consistent with the up-regulation of CD44(High)/CD133(High) cells, KLF5 overexpressing cells were more resistant to anti-cancer drugs and displayed enhanced colony-formation capacity. By contrast, knock-down of KLF5 by siRNA diminished the CD44(High)/CD133(High) subpopulation. When KLF5 was acetylated by TGF-ß1, the KLF5-mediated CD44(High)/CD133(High) subpopulation enrichment was abrogated. Oppositely, ectopic expression of an acetylation-deficient KLF5 mutant further increased CD44(High)/CD133(High) subpopulations as compared to cell expressing wild-type KLF5. These findings provide novel mechanistic insight into a pivotal role for KLF5 in the regulation of CSCs in HCC.

Intratumoral artery on contrast-enhanced computed tomography imaging: differentiating intrahepatic cholangiocarcinoma from poorly differentiated hepatocellular carcinoma.

AIM: Differentiating intrahepatic cholangiocarcinoma (ICC) from poorly differentiated hepatocellular carcinoma (p-HCC) is often difficult, but it is important for providing appropriate treatments. The purpose of this study was to examine the features differentiating ICC from p-HCC on contrast-enhanced dynamic-computed tomography (CT). METHODS: This study examined 42 patients with pathologically confirmed ICC (n = 19) or p-HCC (n = 23) for which contrast-enhanced dynamic CT data were available. CT images were analyzed for enhancement patterns during the arterial phase, washout pattern, delayed enhancement, satellite nodules, capsular retraction, lesion shape, and presence of an intratumoral hepatic artery, intratumoral hepatic vein, intratumoral portal vein, and bile duct dilation around the tumor, portal vein tumor thrombus, lobar atrophy, or lymphadenopathy. RESULTS: Univariate analysis revealed the presence of rim enhancement (p = 0.037), lobulated shape (p = 0.004), intratumoral artery (p < 0.001), and bile duct dilation (p = 0.006) as parameters significantly favoring ICC, while a washout pattern significantly favored p-HCC (p < 0.001). Multivariate analysis revealed intratumoral artery as a significant, independent variable predictive of ICC (p = 0.037), and 15 ICCs (78.9%) showed this feature. Washout pattern was a significant, independent variable favoring p-HCC (p = 0.049), with 15 p-HCCs (65.2%) showing this feature. CONCLUSION: The presence of an intratumoral artery in the arterial phase on contrast-enhanced dynamic CT was a predictable finding for ICC, and the presence of a washout pattern was a predictable finding for p-HCC, differentiating between ICC and p-HCC.

Serum granulysin levels as a predictor of serious telaprevir-induced dermatological reactions.

AIM: Telaprevir-based therapy for chronic hepatitis C patients is effective; however, the high prevalence of dermatological reactions is an outstanding issue. The mechanism and characteristics of such adverse reactions are unclear; moreover, predictive factors remain unknown. Granulysin was recently reported to be upregulated in the blisters of patients with Stevens-Johnson syndrome (SJS). Therefore, we investigated the risk factors for severe telaprevir-induced dermatological reactions as well as the association between serum granulysin levels and the severity of such reactions. METHODS: A total of 89 patients who received telaprevir-based therapy and had complete clinical information were analyzed. We analyzed the associations between dermatological reactions and clinical factors. Next, we investigated the time-dependent changes in serum granulysin levels in five and 14 patients with grade 3 and non-grade 3 dermatological reactions, respectively. RESULTS: Of the 89 patients, 57 patients had dermatological reactions, including nine patients with grade 3. Univariate analysis revealed that grade 3 dermatological reactions were significantly associated with male sex. Moreover, serum granulysin levels were significantly associated with the severity of dermatological reactions. Three patients with grade 3 dermatological reaction had severe systemic manifestations including SJS, drug-induced hypersensitivity syndrome, and systemic lymphoid swelling and high-grade fever; all were hospitalized. Importantly, among the three patients, two patients' serum granulysin levels exceeded 8 ng/mL at onset and symptoms deteriorated within 6 days. CONCLUSION: Male patients are at high risk for severe telaprevir-induced dermatological reactions. Moreover, serum granulysin levels are significantly associated with the severity of dermatological reactions and may be a predictive factor in patients treated with telaprevir-based therapy.

Hepatosplenic gamma-delta T-cell lymphoma associated with Epstein-Barr virus.

Hepatosplenic gamma-delta T-cell lymphoma (HSTCL) is a rare, aggressive subset of peripheral T-cell lymphoma. It has been reported that Epstein-Barr virus (EBV) infection can cause HSTCL; however, such cases are extremely rare, with only a few cases having been reported to date. We herein report an autopsy case of HSTCL associated with EBV infection. The presence of EBV infection was confirmed in serum EBV DNA and on in-situ hybridization, and cytotoxic molecules, such as granzyme B, perforin and T-cell intracytoplasmic antigen (TIA)-1, were all positive in lymphoma cells. These findings indicate that stimulation of persistent EBV infection may have caused HSTCL in this patient.

Efficacy of therapy for advanced hepatocellular carcinoma: intra-arterial 5-fluorouracil and subcutaneous interferon with image-guided radiation.

BACKGROUND AND AIM: To evaluate the efficacy of intra-arterial 5-fluorouracil (5-FU) and subcutaneous interferon (IFN) combined with image-guided radiation therapy (IGRT) in advanced hepatocellular carcinoma (HCC) with portal vein tumor thrombosis (PVTT). METHODS: Twenty HCC patients with PVTT were treated with 5-FU and IFN combined with image-guided radiation therapy (IGRT) (IGRT group), and as controls, 20 patients with PVTT were treated with 5-FU and IFN alone (non-IGRT group). Overall survival (OS) time, response rates, time to progression (TTP) and safety were compared across groups. RESULTS: Complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD) of PVTT were 5%, 55%, 40% and 0% in the IGRT group and 0%, 30%, 35% and 35%, in the non-IGRT group, respectively. CR, PR, SD, and PD of the whole tumor were 0%, 35%, 45% and 20% in the IGRT group and 0%, 30%, 35% and 35%, in the non-IGRT group, respectively. Overall median survival was significantly longer in the IGRT group (12.0 months 95% confidence interval [CI], 9.3-17.6 months) than in the non-IGRT group (9.1 months [95% CI, 5.5-11.1 months]) (P = 0.041). TTP was significantly longer in the IGRT group (6.9 months [95% CI, 5.6-10.2 months]) than in the non-IGRT group (4.0 months [95% CI, 3.3-6.4 months]) (P = 0.034). CONCLUSIONS: The response rates, median OS time and TTP in patients with advanced HCC with PVTT who received this novel combination therapy of intra-arterial 5-FU and subcutaneous IFN with IGRT are encouraging, and this combination therapy warrants further investigation.