Yu Nakagome

Transient elastography for measurement of liver stiffness measurement can detect early significant hepatic fibrosis in Japanese patients with viral and nonviral liver diseases

BackgroundMany studies have reported the efficiency of transient elastography, a noninvasive, reproducible, and reliable method for predicting liver fibrosis, in patients with chronic hepatitis C (CHC) and B (CHB), but there are few reports about nonviral chronic liver disease (CLD) such as primary biliary cirrhosis (PBC), nonalcoholic steatohepatitis (NAFLD), and autoimmune hepatitis (AIH). We therefore compared the efficiency of transient elastography between CHC and nonviral CLD.MethodsWe assessed the accuracy of liver stiffness measurement (LSM) using Fibroscan, and compared these values with those of hyaluronic acid, type 4 collagen, platelet count, prothrombin index, and AST/platelet ratio index (APRI) as indices for the diagnosis of liver fibrosis in 114 patients with a variety of chronic liver diseases: CHC (n = 51), CHB (n = 11), NAFLD (n = 17), PBC (n = 20), and AIH (n = 15). The histology was assessed according to the METAVIR score by two pathologists.ResultsThe number of fibrosis stage (F0/1/2/3/4) with CHC was 9/15/12/6/10, and that with nonviral CLD was 10/21/11/4/6, respectively. The ability, assessed by area under receiver operating characteristic (AUROC) curve, to predict liver fibrosis F ≥ 2 for LSM, HA, type 4 collagen, platelet count, prothrombin index, and APRI, was 0.92, 0.81, 0.87, 0.85, 0.85, and 0.92 in CHC patients, respectively; and 0.88, 0.72, 0.81, 0.67, 0.81, and 0.77 in nonviral CLD patients, respectively.ConclusionsIn patients with nonviral CLD, LSM was most helpful in predicting significant fibrosis (F ≥ 2). Transient elastography is a reliable method for predicting significant liver fibrosis, not only in CHC patients but also in nonviral CLD patients.

Analysis of the full-length genome of hepatitis B virus in the serum and cerebrospinal fluid of a patient with acute hepatitis B and transverse myelitis

Although many extrahepatic manifestations have been described in patients with acute or chronic hepatitis B, there are few reports about neurological disorders. We describe a 55-year-old man who contracted acute hepatitis B virus (HBV) infection and transverse myelitis. His neurological findings were gradually reduced along with the recovery from hepatitis. The cerebrospinal fluid (CSF) was revealed to be positive for HBsAg and HBV DNA. Full-length sequences of HBV in his serum and CSF were determined, and it was revealed that these two isolates had mutations at nucleotide (nt) 1762/1764 in the core promoter region and nt 1896 in the precore region. They were identical to each other except for two ambiguous codes at nt 2020 and 2631 in the CSF isolate. After cloning of the amplicons, substitutions at nt 2020 and 2631 were found in 6 (38%) of the 16 CSF clones. One clone of the 6 CSF clones had an additional substitution at nt 2119. These substitutions were not found in 16 serum clones. The presence of HBV clones unique to CSF suggests that HBV was a possible causative agent of the myelitis.

A case of severe recurrent hepatitis with common variable immunodeficiency

Severe hepatitis with an indistinct etiology manifested in a 16‐year‐old boy who had no particular history. The histological features of the liver and clinical course of the patient were similar to those of patients with autoimmune hepatitis characterized by interface hepatitis and severe lobular inflammation of the liver and recurrent exacerbations of hepatitis. We administered intravenous glycyrrhizin preparation daily or three times a week combined with the oral administration of ursodeoxycholic acid daily throughout the term after the initial onset of disease for the control of disease activity. The normalization of the concentration of alanine aminotransferase in serum was achieved in response to the therapy during the course. The serum concentration of immunoglobulins of the patient gradually decreased from the onset of the disease to an unacceptable level without globulin preparation during the following period of 17 months. Immunological tests revealed impairment of immunoglobulin production bythe B cell population of the patient, which led to the diagnosis of the patient as common variable immunodeficiency (CVID). The patient, with improved liver histology after 27 months from the onset of disease, benefited from the current combination therapy without severe infection through the avoidance of overimmunosuppression. CVID is defined as a heterogeneous syndrome characterized by various degrees of hypogammaglobulinemia without any specific predisposing causes, frequently associated with autoimmunity. Diagnostic criteria and therapeutic options of persistent hepatitis with CVID are to be established, as discussed in the current report.

PD-L1 + MDSCs are increased in HCC patients and induced by soluble factor in the tumor microenvironment

Myeloid-derived suppressor cells (MDSCs) could have important roles in immune regulation, and MDSCs can be induced in patients with various malignant tumors. The immune-suppressive functions of MDSCs in hepatocellular carcinoma (HCC) patients have not been clarified. Therefore, we tried to analyze the biological significance of MDSCs in HCC patients. We quantified PD-L1+MDSCs of HCC patients in various conditions by using multi-color flow cytometry analysis. PBMCs from HCC patients contained significantly higher percentages of PD-L1+MDSCs in comparison to those from healthy subjects (p < 0.001). The percentages of PD-L1+MDSCs were reduced by curative treatment for HCC (p < 0.05), and the percentages of PD-L1+MDSCs before treatment were inversely correlated with disease-free survival time. After we cocultivated PBMCs and several liver cancer cell lines in a transwell coculture system, the percentages of PD-L1+MDSCs were significantly increased compared with control (p < 0.05). The expression of M-CSF and VEGFA was higher in the cell lines that strongly induced PD-L1+MDSCs. Peripheral blood from HCC patients had significantly higher percentages of PD-L1+MDSCs in comparison to those of healthy subjects, and the percentages of PD-L1+MDSCs were reduced by HCC treatment, suggesting that we might use PD-L1+MDSCs as a new biomarker of HCC.

A case of HIV co-infected with hepatitis B virus precore/core deletion mutant treated by entecavir.

We report a case of a HIV and hepatitis B virus (HBV)‐co‐infected patient to whom entecavir (ETV) was administered initially before the notification regarding the potential mutagenesis effect on HIV against the nucleoside analog. Since initial evaluations indicated the advanced stage of chronic hepatitis B and preserved numbers of peripheral CD4+ lymphocytes without the manifestation of immunodeficiency, priority was given to the management of HBV. We started HBV therapy with ETV at a dose of 0.5 mg daily without using any HIV drugs. The viral loads of both HBV and HIV‐1 decreased gradually during the 5 months following the initial administration of ETV. HBV was well controlled by the gradual replacement of ETV with highly‐active antiretroviral therapy against HIV with a regimen including atazanavir, emtricitabine, and tenofovir. HBV was genotyped as A2 with the quasispecies pool consisting of the −1G precore/core deletion mutant strain.

Shifting hepatitis B virus genotypes of acute hepatitis B patients in northeast Japan

It has been reported that acute hepatitis B (AHB) patients with genotype A HBV (HBV/A) have been increasing since the 1990s in metropolitan areas in Japan. However, little is known about the trends of HBV genotypes in AHB patients in northeast Japan where genotype B HBV (HBV/B) prevails more than in other areas. In this study, we aimed to clarify the changes in the HBV genotypes and clinical characteristics of AHB patients in this area. HBV genotypes were determined by direct sequencing (n = 125) or enzyme immunoassay (n = 9) using serum samples from AHB patients including fulminant hepatitis in 1987–2014. Among 134 patients, 26 (19%), 33 (25%), and 75 (56%) patients were infected with HBV of genotypes A, B, and C, respectively. HBV/A emerged from 2001 and the percentage was increased gradually up to 48% in 2010–2014, whereas HBV/B was reduced from 40% in 1987–1994 to 10% in 2010–2014. Phylogenetic analysis showed that three major subgenotype A2 strains had come into this area serially. The levels of HBV DNA and prothrombin time were higher in HBV/A patients than other genotypes. This study could not show significant difference in the HBsAg‐positive period among genotypes nor the effect of nucleoside analogues to shorten the HBsAg‐positive period. A higher level of initial HBV DNA was associated with late disappearance of HBsAg. In conclusion, the percentage of HBV/A in AHB patients has been increasing in northeast Japan since 2001, which is later than metropolitan areas, whereas that of HBV/B is decreasing. J. Med. Virol. 88:69–78, 2016.

Radiation Therapy Is a Reasonable Option for Improving the Prognosis in Hepatocellular Carcinoma.

Radiation therapy (RT) may be suitable for treating patients with hepatocellular carcinoma (HCC) who are difficult to treat with any other option. However, it remains unclear whether RT extends survival in these patients. Among the 957 HCC patients treated at Tohoku University Hospital from January 2007 to December 2013, only 49 patients received RT. We therefore retrospectively analyzed the outcomes of these patients; they were divided into three groups based on the reasons for choosing RT: 27 patients at Stage IV A (67.1 ± 1.6 years, 50.5 ± 2.1 Gy), 9 patients with alternative therapy (72.2 ± 2.4 years, 58.9 ± 1.1 Gy), and 13 patients who received RT after transarterial chemoembolization (TACE) (75.6 ± 2.1 years, 56.5 ± 1.5 Gy). RT was employed to ensure the local control of the lesion. The patients at Stage IV A were treated with radical RT (n = 16) or with palliative RT (n = 11). In radical RT group, the response rate was 37.5% and the complete response rate was 25%. The survival rate was 12.5 ± 2.6 months after radical RT. This is considered relatively good for Stage IV A. The disease-free survival rate was 13.0 ± 2.8 months after RT. This excellent disease-free survival indicates that RT is an alternative to other treatments. In the TACE group, patients who received the RT had the significantly long disease-free survival rate than only-TACE (18.0 ± 3.8 months vs. 11.2 ± 0.58 months). We propose that RT is effective and safe for HCC.

Bioinformatic approach for cholangiocyte pathophysiology

The heterogeneous functions of cholangiocytes regulate the physiology of the biliary epithelium regarding secretory, proliferative and apoptotic activities. However, due to their technical difficulties for isolation and culture, the pathophysiology of cholangiocytes was poorly understood. Recently, cutting‐edge technology, such as the microarray, has given the opportunity for investigating cholangiocytes. We have found the distinct expression profiles of two murine cholangiocytes lines, termed small and large, revealed by microarray analysis. The features of the two cholangiocyte cell lines, categorized partly according to gene ontology, indicate the specific physiological role of each cell line. Namely, large cholangiocytes are characterized as “transport” and “immune/inflammatory responses”. In contrast to large, small cholangiocytes are associated with properties of limited physiological functional ability and proliferating/migratingpotential with specific molecules like Eph receptors, comparable to mesenchymal cells. Further analysis using other modern technology, such as proteomics, will provide more information to understand the pathophysiology of cholangiocytes. These novel methods enable us to investigate the key molecules and their mutual relationship. Although the evaluation of some important biological regulatory processes like protein modification requires methodologies other than microarray, microarray technology is anticipated to grow with the development of data‐analysis theory for the comprehension of cellular cross‐talk in the liver.

Su1000 Hepatoma With HBV Replication Could Attract CX3CR1+ NKG2Dlow NK Cells and CX3CR1+PD-1+ CD8 T Cells

[Background] We previously reported that soluble factors produced from hepatocyte could contribute to the immune pathogenesis of HBV-related inflammation and progression of HCC. (JID2010, JG2012). It has been reported that the progression of fibrosis and HCC is different among the patients infected with various HBV genotypes and HCV. [Aim] The aim of this study is to analyze the effects of chemokines derived from HBV (genotype A, B and C) and HCV -infected hepatocytes and hepatoma cells for the various kinds of immune cells in vitro and in vivo. [Methods] Plasmid Construction: At least 5 clones of each fragment were sequenced and consensus sequence was identified and used as template for 1.24-fold the HBV genome of different genotypes (2 strains each for A, B and C). Cells: Huh7 and HepG2 cells were used for the experiment of transfection. cDNA array: cDNA array for 84chemokine-related genes were carried out. Flow-cytometry analysis: The expression of NKG2D and NKG2A on the NK cells, PD1 on CD8+ cells and MHC class I-related molecule A (MICA) on the HCC cells were analyzed by FACS canto-II. Migration Assay: various kinds of human primary lymphocyte were negatively isolated by MACS beads and used for the migration assay. NOG Mouse model: NOG mice were used for HBV-expressed HCC-transplanted-mouse model. Human primary lymphoid cells were introduced into this mouse. Immunohistochemistry: The liver samples obtained from 20 HBV related HCC patients, HCV-related HCC patients and non HBV/HCV-related HCC patients were stained with CX3CL1, CX3CR1, CD3 and CD56 antibodies. [Results] CCBP2, IL18, CMTM3, CMTM4, CX3CL1, CXCL12, TYMP, GPR81, LTB4R, SDF2 and VHL were well expressed (threshold cycle value range<28) in HBV-replicated-Huh7 cells and significantly up-regulated in comparison to mock transfected Huh7 cells (p<0.01). Among these chemokines-related genes, the expression level of CX3CL1 mRNA was significantly different among genotype A, B and C (relative expression: 1.41, 2.04 and 3.31) (p<0.05). The migration activity of CD16+CD56dimNK cells, CD4+ cells, CD8+ cells and CD14+ Monocyte but not CD19+ B cells or Tregs cells were significantly different among genotype A, B and C in vitro (p<0.05). Moreover, the neutralization of CX3CL1 could cancel these differences of migration activities. Significantly lower expression of NKG2D on CX3CR1+NK cells were observed in primary human peripheral blood mononuclear cells obtained from HBV-related HCC patients and the Tumor-infiltrating lymphocytes in HCC with HBV expression in NOG mouse model in comparison to those is non-HBV related HCC. (p<0.05). [Conclusion] The differential expre s s ion l eve l s o f CX3CL1 and MICA and the d i f f e r en t i a l a t t r ac t i on of NKG2DlowCX3CR1+NK cells and PD-1+ CX3CR1+ CD8 T cells might be involved in HBV persistent infection and hepatocellular carcinoma by affecting immune cells.

Sa1694 MicroRNA-133b Plays an Onco-Suppressive Role in Hepatocellular Carcinoma

INTRODUCTION. Colon cancer (CC) remains the third leading cause of worldwide cancerrelated death in men and women. Currently, available prognostic and/or predictive markers for colon cancer lack specificity and sensitivity. Developing new biomarker for early detection, accurate diagnosis and therapeutic treatment for CC is of great importance in improving the clinical outcome of the disease. Cathepsin B (CB), a lysosomal cysteine protease, is expressed constitutively in lysosomes, however its expression and localization change in cancer. High levels of expression of CB at both gene and protein levels have been observed in different types of cancer. AIM. The aim of this study was to analyze the CB expression in different stages of CC progression and to evaluate its clinical relevance. MATERIALS & METHODS. We examined for first time, using quantitative real time PCR, the expression of CB in 185 colonic tissue specimens from 130 patients; 50 were pairs of cancerous-normal tissues, 17 were cancerous tissues and 63 were adenomas for 5 of which normal paired mucosa were also available. RESULTS. We proved that CB was up regulated in the cancer specimens in comparison to their normal pairedmucosa (p<0.001), as well as in the adenomas in comparison to normal tissue (p<0.001). CB expression was found to be associated with histological grade (p=0.037). Cox proportional hazard regression model using univariate and multivariate analysis revealed that high status CB expression is a significant factor for disease-free survival(DFS) (p=0.037 and 0.0038, respectively) and overall survival (OS)(p= 0.003 and p=0.0037, respectively) of patients. Receiver-operating characteristic (ROC) analysis of our results showed that CB has discriminatory value between CC and adenomas tissues (area under the curve [AUC]=0.711). Kaplan-Meier survival curves demonstrated that CB expression of low status is significantly associated with longer DFS (p=0.023) as well as OS (p=0.002). CONCLUSION. Present results suggest that CB gene expression may represent a useful marker of unfavorable prognosis for CC patients with discriminatory power between CC and adenoma patients.