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The present study aimed to establish a novel isolation strategy for circulating tumor cells (CTCs) using a microcavity array (MCA) system and to evaluate the clinical significance of CTCs in hepatocellular carcinoma (HCC). We examined recovery rates of HCC cell lines spiked into whole blood in MCA assay. Circulating tumor cells were isolated from peripheral blood samples (3 mL) of 7 healthy donors (HD), 14 patients with liver cirrhosis (LC), and 31 patients with HCC using the MCA system. Additionally, we investigated the mRNA expression of liver‐specific genes in isolated CTCs using qPCR. The recovery rates were 65.1% (HepG2), 76.7% (HuH7), and 99.0% (PLC/PRF/5). In HD and patients with LC and HCC, the CTC positivity rate (CTCs ≥10) and average CTC number were as follows: HD 0% and 0.1, LC 14.3% and 5.3, HCC 54.8% and 47.6, respectively. The CTC positivity rate in HCC was significantly higher than that in LC (p, This study describes the detection of circulating tumor cells (CTCs) in the peripheral blood of hepatocellular carcinoma (HCC) patients using a newly developed microcavity array. Our results show that a large number of CTCs was detected in metastatic HCC patients, and mRNA expression of AFP, glypican‐3, EpCAM and albumin was detected in detected CTCs. Identification of positive CTCs or ALB mRNA expression can help predict poor prognosis of HCC patients.

Aim A complete cure for chronic hepatitis B virus (HBV) infection requires elimination of covalently closed circular DNA; however, this remains to be clinically achieved. Interferon (IFN)-γ, a type II IFN, is produced by intrahepatic cytotoxic T lymphocytes and has non-cytolytic antiviral potential. However, the mechanism by which IFN-γ regulates HBV infection has not been fully elucidated. Thus, we developed an in vitro HBV infection assay system and analyzed the molecular signature of HBV regulation by IFN-γ. Methods The in vitro HBV infection assay system was established in primary human hepatocytes infected with HBV derived from the plasmid containing 1.3-mer HBV genome, and treated with IFN-γ. The antiviral effects and signaling pathways of IFN-γ were examined using microarray, and assessed by siRNA knockdown experiments of the related genes. Results IFN-γ treatment suppressed both HBV propagation and transcription as efficiently as IFN-α. Microarray analysis showed that IFN-γ stimulation induced the activation of both IFN-γ and IFN-α signaling, regulating HBV covalently closed circular DNA. HBV production was decreased by IFN-γ through Janus kinase/signal transducer and activator of transcription signaling and interferon-stimulated genes, such as 2'-5'-oligoadenylate synthase 2 and apolipoprotein B mRNA editing enzyme catalytic subunit 3G. Conclusions IFN-γ can suppress HBV propagation and transcription in hepatocytes by activating specific intracellular signaling pathways in hepatocytes, and suggests the future application of these particular signaling pathways or genes for the complete elimination of HBV.

OBJECTIVE To evaluate the long-term outcomes of complete common bile duct (CBD) stone removal and biliary stenting in elderly patients (≥85 years) with CBD stones. METHODS We retrospectively examined 65 patients who underwent complete CBD stone removal (the duct clearance group) and 40 patients who underwent biliary stenting (the biliary stenting group) between July 2006 and March 2016. To reduce selection bias, we also conducted a propensity score matching analysis and generated 30 pairs of patients. Cholangitis recurrence-free survival and overall survival were compared between the two groups and independent prognostic factors of survival were identified by univariate and multivariate analyses. RESULTS Cholangitis recurrence-free survival was significantly better in the duct clearance group than in the biliary stenting group (P < 0.001). Their overall survival did not significantly differ after propensity score matching (P = 0.388). In all cohorts, univariate analysis demonstrated that poor performance status and biliary stenting were factors of poor prognosis, and in multivariate analysis only performance status remained associated with poor prognosis for survival. Similarly, in the propensity score-matched cohort, only performance status independently predicted poorer survival (hazard ratio [HR] 2.726, 95% confidence interval [CI] 1.105-6.675, P = 0.029). The choice of endoscopic treatment was not a significant factor associated with prognosis (HR 1.354, 95% CI 0.678-2.701, P = 0.391). CONCLUSIONS Biliary stenting was similar to complete stone removal in terms of prognosis for long-term survival. Biliary stenting for CBD stones could be an effective therapeutic tool in high-risk elderly patients.

Hepatitis B virus (HBV) reactivation can be triggered by immunosuppressive chemotherapy. HLA class II molecules may play a role in HBV reactivation. Genetic polymorphism and mRNA expression of HLA class II were examined in patients with latent HBV infection treated with immunosuppressive therapies. Subjects with resolved HBV infection who had undergone treatment with immunosuppressive chemotherapies were retrospectively enrolled (n = 42) and divided into reactivated (n = 9) and non-reactivated groups (n = 33). Patients were genotyped for 17 single nucleotide polymorphisms (SNPs) within HLA class II DPA1, and DPB1, and mRNA expression levels of HLA class II genes were assessed. The frequency of the AA genotype of rs872956, a SNP in HLA-DPB1, was significantly higher in the reactivated group than in the non-reactivated group (55.6% vs 12.1%, P < 0.05). The frequencies of the T allele and non-AA genotypes (AT/TT) of rs3116996 (located in DPB1) were significantly higher in the reactivated group (T allele frequency: 16.7% vs 0.0% [P < 0.01], non-AA genotype frequency: 22.2% vs 0.0% [P < 0.05]). Multivariate logistic regression identified the AA genotype of rs872956 as an independent protective factor against HBV reactivation (odds ratio [OR] = 18.1, 95% confidence interval [CI] = 2.6-126.7, P < 0.01). mRNA expression of HLA-DPB1 was lower in the HBV reactivated group than in the non-reactivated group (median 276.1 ± 165.6/β-actin vs 371.4 ± 407.5/β-actin [P < 0.05]). These results suggest the involvement of HLA class II molecules in HBV reactivation after treatment with immunomodulatory agents.

Recent genome-wide studies have demonstrated that HLA class II gene may play an important role in viral hepatitis. We studied genetic polymorphism and RNA expression of HLA class II genes in HCV-related liver diseases. The study was performed in groups consisting of 24 patients with HCV-related liver disease (12 of persistent normal ALT: PNALT group and 12 of advanced liver disease: ALD group) and 26 patients without HCV infection (control group). In PBMC samples, RNA expression of HLA class II genes (HLA-DPA1, DPB1, DQA1, DQB1, and DRB1) was analyzed by real-time RT-PCR. Furthermore, 22 single nucleotide polymorphisms (SNPs) in HLA class II gene and two SNPs in IL28B gene were genotyped by genetic analyzer (GENECUBE®). In expression analysis, only DPB1 level was significantly different. Mean expression level of DPB1gene in control group was 160.0, PNALT group 233.8, and ALD group 465.0 (P

The monocyte chemotactic proteins (MCPs) form a distinct structurally related subclass of C–C chemokines. MCPs select specific target cells due to binding to a distinct set of chemokine receptors and because of their effects on monocytes, and may participate in the process of granuloma formation during bacterial and/or mycobacterial infections. The aetiology of primary biliary cirrhosis (PBC) is still unclear, although bacterial infection and autoimmune processes have been implicated. In this study, the expression of three of the most potent monocyte chemoattractants, MCP-1, -2, and -3, was examined in patients with PBC and the data were compared with results for other liver diseases including primary sclerosing cholangitis (PSC), chronic viral hepatitis C, hepatic sarcoidosis, and normal liver. MCP-1 was expressed mainly in biliary epithelial cells of all liver specimens, irrespective of the cause of disease. Some mononuclear leukocytes in the portal tract expressed MCP-1 in all the disease groups examined and there were no significant differences in frequency between these groups. In contrast, more than 80% of PBC livers showed MCP-2- and MCP-3-positive mononuclear leukocyte infiltration in portal tracts, particularly around the bile ducts, whereas such cells were far less frequent in the other disease groups or in normal livers. Epithelioid granulomata of PBC patients contained MCP-2- and MCP-3-positive cells at their edge. In double staining experiments, more than 60% of the MCP-positive mononuclear cells co-expressed CD68, suggesting that a proportion of MCP-2- and MCP-3-positive cells are derived from monocytes. These monocytes expressing MCP-2 and MCP-3 may be responsible for the chemotactic activity of more monocytes. Such an expression pattern of MCP-1, -2 and -3 in portal tracts seems to be distinctive for PBC. This pattern underlines the importance of MCP-1, -2, and -3 in the recruitment of monocytes and possibly T lymphocytes into portal tracts, around the injured bile ducts, and into epithelioid granulomata in PBC. The data further implicate bacterial materials derived from bile in the overall pathogenesis of PBC. Copyright © 2000 John Wiley & Sons, Ltd.

Bacterial infection of the biliary tree and bile stasis may be causally related to hepatolithiasis, but which bacterial species are involved and their roles in the pathogenesis of hepatolithiasis have not been ascertained. Recently, the Helicobacter genus was detected in human bile and biliary mucosal samples by molecular techniques, and its association with several biliary diseases has been suggested. The Campylobacter genus, which is closely related to the Helicobacter genus, has also recently been identified as causative of human gastrointestinal diseases. This study attempted to elucidate whether Helicobacter and/or Campylobacter bacteria are present in bile samples and biliary mucosal specimens from hepatolithiasis patients and whether they are involved in the pathogenesis of hepatolithiasis. The 16S rRNA gene of the Helicobacter and of the Campylobacter genus was examined by polymerase chain reaction in DNA samples extracted from bile and/or microdissected biliary epithelium from 69 patients with hepatolithiasis and control patients with choledocholithiasis, cholecystolithiasis, and normal gall bladders. The Helicobacter genus was detected in 1 of 8 (13%) biliary epithelial samples in hepatolithiasis and 1 of 10 (10%) bile samples in choledocholithiasis. The Campylobacter genus was detected in 3 of 14 (21%) bile samples and 5 of 8 (63%) epithelial samples in hepatolithiasis, and in 2 of 15 (13%) bile samples and 1 of 8 (13%) epithelial samples in cholecystolithiasis. The detection rate for Campylobacter in biliary epithelium of hepatolithiasis was significantly higher than in the bile or biliary epithelium of control groups (p

Aims/Background: Apoptosis, including the Fas system, has been implicated in progressive bile duct loss in primary biliary cirrhosis (PBC). In this study, we attempted to analyze Fas ligand (FasL) expressing mononuclear cells infiltrating in the portal tracts of PBC. Methods: We immunohistochemically assessed co-expression of leukocyte markers and FasL on infiltrating mononuclear cells in 18 patients with PBC. Twenty-five patients with chronic hepatitis C (CH-C) were used as controls. Results: In PBC, FasL expressing cells were scattered in the portal tracts, and some were accentuated around the damaged bile ducts. In addition, these cells co-expressed CD68 (71%), a marker of monocytes, but not UCHL-1, CD3 and CD57, markers of activated T cells and natural killer cells. By contrast, in CH-C, the biliocentric pattern of FasL expression was not evident, and about half of FasL expressing cells (42–56%) co-expressed UCHL-1, CD3 or CD57. CD14, a receptor for bacterial products such as lipopolysaccharides, was also detected on a proportion of FasL expressing mononuclear cells around the damaged bile ducts in PBC. Conclusion: The results suggest that in PBC, FasL expressing CD68+ monocytes are at least partly involved in apoptotic bile duct loss mediated by the Fas system, and a surface molecule, CD14, participates in this process.