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.

MicroRNAs were first discovered as small endogenous RNA molecules and some viruses have been reported to interact with host miRNAs. By investigating miRNA expression in serum derived from HBV-infected patients, we have clarified the relationship between miRNA expression and chronic HBV infection. Additionally, we demonstrate the use of miRNAs as both novel biomarkers and new therapies against HBV. We included the sera of 20 patients with chronic HBV infection, sera of 20 patients with HCV infection and sera of 10 healthy controls in this study. The miRNA libraries were sequenced using a 32-mer single end sequence. The validation study of circulating miRNA in serum was conducted by qRT-PCR. The HBV genomic regions of genotype B and genotype C that were speculated to be targeted by miRNA were constructed using complementary oligonucleotides in the vectors. Reporter assays were performed 48 h after transfection. The expression levels of 21 miRNAs were found to be differentially expressed in the three groups. 10 miRNAs (hsa-miR-100-5p, miR-125b-5p, miR-193b-3p, miR-194-3p, miR-30a-3p, miR-30c-2-3p, miR-3591-5p, miR-4709-3p, miR-574-3p and miR-99a-5p) were found to be upregulated in CH-B by deep sequence analysis. The computer analysis showed that two regions of HBsAg are potential targets of miR-125b-5p and miR-30c-2-3p and that these miRNAs may downregulate the expression of HBV-S. The HBV genotype C segment speculated to be targeted by hsa-miR-125b-5p significantly decreased the expression of the reporter. This study indicated that expression of miR-125b-5p was related to the etiology of chronic hepatitis B infection and regulated the expression of HBsAg.

Increasing evidence supports the important role of cancer stem cells (CSCs). Many reports suggest that epithelial cell adhesion molecule (EpCAM) is a useful marker for cancer stem cells in hepatocellular carcinoma (HCC). To elucidate the mechanisms of cancer stem cells, the development of specific molecular targeted drugs has become very important. In the present study, we examined the EpCAM expression pattern and its characteristic expression in resected HCC. We studied the drug resistance of EpCAM expression cells. EpCAM expression was detected significantly more frequently with hepatitis B virus (HBV) than with other etiologies. In HCC resection patients who had received prior treatment (transcatheter arterial embolization or hepatic arterial infusion chemotherapy), EpCAM was strongly expressed. In particular, very strong expression was observed after hepatic arterial infusion chemotherapy. The PLC/PRF/5 human HCC cell line expressed bimodal EpCAM, and EpCAM-positive cells had CSC cell potency. The EpCAM expression in EpCAM-positive cells increased significantly by treatment with cisplatin. EpCAM-positive cells showed better viability than EpCAM-negative cells when treated with ciplatin. Collectively, our results suggest that cancer stem cells are highly expressed in hepatitis B and have potential anticancer drug resistance.

Objective1,25(OH)2 vitamin D3 can affect immune cells. However, the mechanism responsible for the favorable effects of 1(OH) vitamin D3, which becomes 1,25(OH)2 vitamin D3 in the liver, is not clear. The aim of this study is to analyze the immunological response of 1(OH) vitamin D3 supplementation in CH-C patients.DesignForty-two CH-C patients were treated with 1(OH) vitamin D3/Peg-IFNα/RBV. Forty-two case-matched controls were treated with Peg-IFNα/RBV. The expression of Interferon-stimulated genes (ISGs)-mRNA in the liver biopsy samples and JFH-1 replicating Huh-7 cells were quantified by real-time PCR. Ten kinds of cytokines in the plasma were quantified during treatment by using a suspension beads array. A trans-well co-culture system with peripheral blood mononuclear cells (PBMCs) and Huh-7 cells was used to analyze the effect of 1(OH) vitamin D3. The activities of the Th1 response were compared between subjects treated with 1(OH) vitamin D3/Peg-IFN/RBV and those treated with Peg-IFN/RBV therapy alone.Results1(OH) vitamin D3/Peg-IFN/RBV treatment could induce rapid viral reduction, especially in IL28B T/T polymorphism. Several kinds of cytokines including IP-10 were significantly decreased after 4 weeks of 1(OH) vitamin D3 treatment (p<0.05). Th1 responses in the subjects treated with 1(OH) vitamin D3/Peg-IFN/RBV were significantly higher than those treated with Peg-IFN/RBV at 12 weeks after Peg-IFN/RBV therapy (p<0.05). The expression of ISGs in the patient’s liver biopsy samples was significantly lower than in those treated without 1(OH) vitamin D3 (p<0.05).Conclusion1(OH) vitamin D3 could improve the sensitivity of Peg-IFN/RBV therapy on HCV-infected hepatocytes by reducing the IP-10 production from PBMCs and ISGs expression in the liver.

Aim Hepatitis B virus genotype B (HBV/B) has been reported to have less risk of liver cirrhosis and hepatocellular carcinoma (HCC), but long‐term observation has rarely been reported. We aimed to clarify the characteristics of HBV/B in nucleos(t)ide analog‐treated patients in an area where HBV/B is more prevalent than in other areas of Japan. Methods A total of 498 chronically HBV‐infected patients treated with nucleos(t)ide analog (lamivudine, entecavir, tenofovir disoproxil fumarate, or tenofovir alafenamide fumarate) for >6 months (mean 70.6 months) were included from nine hospitals in northeast Japan. The frequencies of hepatitis B surface antigen loss and HCC occurrence were analyzed. Results Among 427 patients whose genotype could be determined, 34.0% and 64.4% were infected with HBV/B and genotype C (HBV/C), respectively. The age of patients with HBV/B was significantly older than those with HBV/C (57.7 vs. 48.1). The cumulative rate of hepatitis B surface antigen loss was significantly higher in HBV/B than in HBV/C (3.6% vs. 0.7% at 10 years). Among 480 patients without HCC history, HCC occurrence was found in 40 patients (13.4% at 10 years). There was no cumulative rate difference of HCC occurrence among the genotypes, but after propensity score matching for age/sex, it was significantly lower in HBV/B than in HBV/C (5.3% vs. 18.5% at 10 years). Conclusions Although a lower rate of HCC occurrence in HBV/B was shown by an age/sex‐matched analysis than that in HBV/C, patients with HBV/B were significantly older and had a comparative risk of HCC occurrence in nucleos(t)ide analog‐treated patients.