Immunostaining of PD-1/PD-Ls in liver tissues of patients with hepatitis and hepatocellular carcinoma

AIM: To investigate the expression of programmed death (PD)-1,PD ligand 1 (PD-L1) and PD-L2 in liver tissues in the context of chronic hepatitis and hepatocellular carcinoma (HCC).METHODS: Liver biopsies and HCC specimens from patients were collected and histologically examined.The expression of PD-1,PD-L1,and PD-L2 in biopsy specimens of chronic hepatitis and HCC specimens was evaluated by immunohistochemical staining.The association between the expression level of PD-1,PD-L1,and PD-L2 and clinical and pathological variables was analyzed statistically.RESULTS: Expression of PD-1 was found in liverinfiltrating lymphocytes.In contrast,PD-L1 and PD-L2 were expressed in non-parenchyma liver cells and tumor cells.The expression of PD-L1 was significantly correlated with hepatitis B virus infection (1.42 ± 1.165 vs 0.50 ± 0.756,P = 0.047) and with the stage of HCC (7.50 ± 2.121 vs 1.75 ± 1.500 vs 3.00 ± 0.001,P = 0.018).PD-1 and PD-Ls were significantly up-regulated in HCC specimens (1.40 ± 1.536 vs 5.71 ± 4.051,P = 0.000;1.05 ± 1.099 vs 4.29 ± 3.885,P = 0.004;1.80 ± 1.473 vs 3.81 ± 3.400,P = 0.020).CONCLUSION: PD-L1 may contribute to negative regulation of the immune response in chronic hepatitis B.PD-1 and PD-Ls may play a role in immune evasion of tumors.

Animal models for the study of hepatitis B virus infection

Even with an effective vaccine, an estimated 240 million people are chronically infected with hepatitis B virus （HBV） worldwide. Current antiviral therapies, including interferon and nucleot（s）ide analogues, rarely cure chronic hepatitis B. Animal models are very crucial for understanding the pathogenesis of chronic hepatitis B and developing new therapeutic drugs or strategies. HBV can only infect humans and chimpanzees, with the use of chimpanzees in HBV research strongly restricted. Thus, most advances in HBV research have been gained using mouse models with HBV replication or infection or models with HBV-related hepadnaviral infection. This review summarizes the animal models currently available for the study of HBV infection.

Inhibition of hepatitis B virus replication by APOBEC3G in vitro and in vivo

AIM： To investigate the effect of APOBEC3G mediated antiviral activity against hepatitis B virus （HBV） in cell cultures and replication competent HBV vector-based mouse model. METHODS： The mammalian hepatoma cells Huh7 and HepG2 were cotransfected with various amounts of CMV-driven expression vector encoding APOBEC3G and replication competent 1.3 fold over-length HBV. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA. The expression of HBcAg in transfected cells was detected by western blot. HBV DNA and RNA from intracellular core particles were examined by Northern and Southern blot analyses. To assess activity of the APOBEC3G in vivo, an HBV vector-based model was used in which APOBEC3G and the HBV vector were co-delivered via high-volume tail vein injection. Levels of HBsAg and HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by ELISA and quantitative PCR analysis respectively. RESULTS： There was a dose dependent decrease in the levels of intracellular core-associated HBV DNA and extracellular production of HBsAg and HBeAg. The levels of intracellular core-associated viral RNA also decreased, but the expression of HBcAg in transfected cells showed almost no change. Consistent with in vitro results, levels of HBsAg in the sera of mice were dramatically decreased. More than 1.5 log10 decrease in levels of serum HBV DNA and liver HBV RNA were observed in the APOBEC3G-treated groups compared with the control groups.CONCLUSION： These findings indicate that APOBEC3G could suppress HBV replication and antigen expression both in vivo and in vitro, promising an advance in treatment of HBV infection.

Replication of clinical hepatitis B virus isolate and its application for selecting antiviral agents for chronic hepatitis B patients

AIM: To establish a cell model harboring replicative clinical hepatitis B virus (HBV) isolates and evaluate its application in individualized selection of anti-HBV agents for chronic hepatitis B (CHB) patients. METHODS: The full-length HBV genomic DNA from 8 CHB patients was amplified by polymerase chain reaction (PCR). All the patients were treated with lamivudine for at least seven months and finally became resistant to lamivudine. The amplified HBV DNA fragments were inserted into pHY106 vectors by Sap Ⅰ?digestion. The recombinant plasmids containing 1.1 copies of HBV genome were transiently transfected into Huh7 cell line, and the levels of HBsAg, HBeAg and intercellular HBV replicative intermediates were determined by ELISA and Southern blot analysis, respectively, with or without lamivudine and adefovir treatment. The antiviral treatment with adefovir was administered to the patients and analyzed in parallel. RESULTS: A total of 25 independent HBV isolateswere obtained from the sera of 8 patients, each patient had at least two isolates. One isolate from each individual was selected and subcloned into pHY106 vector, including 5 isolates with YVDD mutation and 3 isolates with YIDD mutation. All recombinant plasmids harboring HBV isolates were transfected into Huh7 cells. The results indicated that HBV genome carried in HBV replicons of clinical HBV isolates could effectively replicate and express in Huh7 cells. Adefovir, but not lamivudine, inhibited HBV replication both in vitro and in vivo, and in vitro inhibition was dose-dependent. CONCLUSION: The novel method described herein enables individualized selection of anti-HBV agents in clinic and is useful in future studies of antiviral therapy for CHB.

Expression of PTEN,PPM1A and P-Smad2 in hepatocellular carcinomas and adjacent liver tissues

AIM： To investigate the expressions of PTEN, PPMIA and P-Smad2 in hepatocellular carcinoma （HCC） and their significance.METHODS： The expressions of PTEN, PPMIA and P-Smad2 in 31 HCC tissues, 25 adjacent liver tissues and 13 non-tumor liver tissues were detected by using Envision immunohistochemical technique. RESULTS： The positive expression （64.52%） and staining intensity （4.19 ± 3.31） of PTEN in the cytoplasm of HCC were significantly lower and weaker than those in the adjacent or non-tumor liver tissues （97.37%, 7.88 ± 0.93; 100%, 7.77 ± 0.93, respectively） （P 〈 0.05）, and its staining intensity in the cytoplasm of HCC, which belongs to Edmondson pathologic grades Ⅱ-Ⅲ and above, was also lower than that of grade I and I-Ⅱ. Furthermore, its location in the nucleus or cytoplasm of liver cells was negatively correlated with the progression of liver disease （r = -0.339, P = 0.002）; most of PPMIA might be only expressed in the nucleus of adjacent liver tissues, non-HCC tissues or Edmondson grade I and I - Ⅱ HCC, but it was mainly expressed in the cytoplasm of HCC with Edmondson grade ≥ Ⅱ, weakly or negatively expressed in the nucleus （P 〈 0.05）, and its location was negatively correlated with the progression of liver disease （r = -0.45, P = 0.0000）. P-Smad2, which was mostly located in the nucleus and cytoplasm of grade I and I -Ⅱ HCC, surrounding or non-tumor liver tissues, was only in the nucleus of HCC with Edmondson grade Ⅱ and above （P 〈 0.001）, and its location was positively correlated with the disease progression （r = 0.224, P = 0.016）. Spearman correlation analysis revealed that P-Smad2 was significantly negatively correlated with PTEN and PPMIA （r = -0.748, P = 0.000; r = -0.366, P = 0.001, respectively）; and PTEN and PPMIA were positively correlated with HCC carcinogenesis （r = 0.428, P = 0.000）.CONCLUSION： The aberrant location of expression and staining intensity of PTEN, PPMIA and P-Smad2 in HCC and their relationship might have an impact on the pathogenesis of HCC.

The relationship between loss expression of DPC4/Smad4 gene and carcinogenesis of pancreatobiliary carcinoma

Objective: To clarify the relationship between loss of DPCA gene expression and pathogenesis of pancreato- biliary carcinoma. Methods: 75 slides of normal duct (20), hyperplasia (15), dysplasia (15), invasive carcinoma (25) from patients with pancreatic diseases including pancreatic carcinoma (25 patients), chronic pancreatitis (6), pancreas injury (2) and 71 slides of common bile duct (CBD) carcinoma (38), gallbladder carcinoma (18), hilar bile duct (HBD) carcinoma (15) from patients with primary biliary tract carcinoma were analyzed for the expression of DPC4 protein by im- munohistochemical staining. Results: All specimens from 20 cases of normal duct and 15 cases of hyperplasia showed marked expres- sion of DPC4 protein. The frequency of loss expres- sion of the DPC4 gene was 33 % in dysplasia, and 48% in invasive carcinoma. There was a significant statistical difference between byperplasia and dyspla- sia (P<0.01) and in dysplasia vs invasive carcinoma (P<0.05). The frequency of loss expression of the DPC4 gene was 47.3% in CBD carcinoma, 11% in gallbladder carcinoma, and 13% in HBD carcinoma. The frequency of loss expression of the DPCA gene was significantly different in CBD carcinoma vs gall- bladder carcinoma and HBD carcinoma (P<0.01). Conclusions: Inactivation of the DPC4 gene occurs late in the neoplastic progression of pancreatic carci- noma. The frequency of DPC4 gene alternation was different in various locations of biliary tract carcino- ma. In CBD carcinoma, this frequency is similar to that in pancreatic carcinoma, indicating their similar molecular alternations.

N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication

AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo. METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA.The expression of hepatitis B virus core antigen (HBcAg) in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively. RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells, and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly, the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls. CONCLUSION: Our findings provide probably the first evidence showing that APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain could suppress HBV replication in vitro and in vivo.

Construction and Characterization of a Hepatitis B Virus Replicon

建立复制细胞肝炎 B 病毒(HBV ) 当模特儿并且在抗病毒的药评估决定它的应用程序，我们构造了表情包含了 HBV 染色体的 1.3 个拷贝，并且在 Huh7 房间在短暂 transfection 以后测量了病毒的复制的水平的 plasmid。我们然后观察了抗病毒的药管理的效果。HBV (ayw ) 基因碎片的 1.3 褶层被 PCR 和限制 endonuclease 消化克隆进 pCR2.1。recombinant plasmid 是进 Huh7 房间， HBsAg， HBeAg 和 HBV 的短暂 transfected 在 Huh7 房间的上层清液的 DNA 被 ELISA 和即时 PCR 分别地测量；细胞内部的 HBV replicative 中介和细胞内部的 HBV 抄本被南部的污点和北污点分别地检测。adefovir 的抗病毒的效果，新奇 anti-HBV 核苷酸类似物，在这个细胞的模型系统被评估。结果显示 HBV replicon 的 recombinant plasmid 成功地被构造；在 plasmid pHBV1.3 带的 HBV 染色体能高效地复制并且被表示在哈 7 个房间， adefovir 能在这个细胞的模型，和抑制禁止 HBV 复制是剂量依赖者。结论是 HBV replicon，能在 hepatoma 房间高效地开始病毒的复制，可以是在 HBV 复制和抗病毒的药的学习的一个有用工具。关键词肝炎 B 病毒 - 传染 replicon - 表示向量 CLC 数字 R373 基础条款：国家自然科学基础(No.30271170， No.30170889 ) 。

Association of CX3CL1 and CX3CR1 Expression with Liver Fibrosis in a Mouse Model of Schistosomiasis

Immunopathological mechanisms of schistosomiasis,a debilitating parasitic disease,are still unclear.In this study,we investigated the involvement of CX3C chemokine ligand 1(CX3CL1)and its sole receptor CX3CR1 in the development of liver fibrosis in schistosomiasis.The animal model of schistosomiasis was established by infection of C57BL/6 mice with Schistosoma japonicum cercariae;mice injected with carbon tetrachloride(CCl4)were used as positive control of liver injury.After 4 and 8 weeks,the degree of liver lesions was assessed by hematoxylin and eosin staining,serum levels of hyaluronic acid(HA)were analyzed by a chemiluminescence immunoassay,liver fibrosis was evaluated by immunohistochemistry analysis ofα-smooth muscle actin(α-SMA)expression,and CX3CL1 and CX3CR1 expression in the liver was measured by immunohistochemistry and real-time PCR.The results showed that at 8 weeks after Schistosoma infection,serum HA levels were increased andα-SMA-expressing cells appeared in the liver,indicating fibrogenesis.CX3CL1-and CX3CR1-positive cells were observed in the outer layer of granulomas formed around Schistosoma eggs in liver tissues,which was consistent with the significant upregulation of hepatic CX3CL1 and CX3CR1 mRNA expression at 4 and 8 weeks post-infection.Furthermore,correlation analysis revealed positive association between CX3CL1 and CX3CR1 expression and serum HA levels at 8 weeks post-infection,indicating a link between fibrogenesis and the CX3CL1/CX3CR1 axis in schistosomiasis.In conclusion,our data suggest the involvement of CX3CL1 and CX3CR1 in the progression of liver fibrosis caused by Schistosoma infection.

Silencing of UBP43 by shRNA Enhances the Antiviral Activity of Interferon against Hepatitis B Virus

Previous studies have shown that expression of the interferon-sensitive gene （ISG）15 protease UBP43 is increased in the liver biopsy specimens of patients who do not respond to interferon （IFN）-α therapy. We hypothesized that UBP43 might hinder the ability of IFN to inhibit HBV replication. In this study, we investigated whether vector-based siRNA promoted by HI （psiUBP43） could enhance IFN inhibiting HBV replication in cell culture. UBP43 was specifically silenced using shRNA. In HepG2.2.15 cells, the HBeAg and HBV DNA levels were significantly reduced by IFN after transfection of shRNA, imphicated that vector-based siRNA promoted by H1 （psiUBP43） could enhance IFN inhibiting HBV replication in cell culture. These data suggest that UBP43 modulates the anti-HBV type I IFN response, and is a possible therapeutic target for the treatment of HBV infection.

Preparation of Pd/c-Al_(2)O_(3)/nickel foam monolithic catalyst and its performance for selective hydrogenation in a rotating packed bed reactor

Selective hydrogenation plays an important role in chemical industries,yet its selectivity is usually limited by the mass transfer.In this work,the enhanced hydrogenation selectivity was achieved in a rotating packed bed(RPB)reactor with excellent mass transfer efficiency.Aiming to be used under the centrifugal filed,a monolithic catalyst Pd/c-Al_(2)O_(3)/nickel foam suiting for the shape and size of the rotor of RPB reactor was prepared by the electrophoretic deposition method.The mechanical strength of the catalyst can meet the requirement of high centrifugal force in the RPB.The hydrogenation selectivity in the RPB reactor using the 3-methyl-1-pentyn-3-ol hydrogenation system was 3–8 times higher than that in a stirred tank reactor under similar conditions.This work proves the feasibility of intensifying the selectivity of hydrogenation process in the RPB reactor.

An Evaluation of Metronidazole Degradation in a Plasma-Assisted Rotating Disk Reactor Coupled with TiO2 in Aqueous Solution

Pollution involving pharmaceutical components in bodies of water is an increasingly serious environmental issue.Plasma discharge for the degradation of antibiotics is an emerging technology that may be relevant toward addressing this issue.In this work,a plasma-assisted rotating disk reactor(plasma-RDR)and a photocatalyst—namely,titanium dioxide(TiO_(2))—were coupled for the treatment of metronidazole(MNZ).Discharge uniformity was improved by the use of a rotating electrode in the plasma-RDR,which contributed to the utilization of ultraviolet(UV)light radiation in the presence of TiO_(2).The experimental results showed that the degradation efficiency of MNZ and the concentration of generated hydroxyl radicals respectively increased by 41%and 2.954 mg∙L^(-1) as the rotational speed increased from 0 to 500 r∙min^(-1).The synergistic effect of plasma-RDR plus TiO_(2) on the generation of hydroxyl radicals was evaluated.Major intermediate products were identified using three-dimensional(3D)excitation emission fluorescence matrices(EEFMs)and liquid chromatography-mass spectrometry(LC-MS),and a possible degradation pathway is proposed herein.This plasma-catalytic process has bright prospects in the field of antibiotics degradation.