Identification of hepatitis B virus aetiologic antigens,HBx and Pre‐S2, in diffuse large B‐cell lymphoma

Diffuse large B‐cell lymphoma (DLBCL) has been reported to have a significant association with the hepatitis B virus (HBV) infection. However, there has been no experimental evidence to determine whether the components of the hepatitis B virus are expressed in lymphoid cells. In this study, we used immunohistochemical methods to explore whether the antigens of hepatitis B virus are expressed in DLBCL lymphoma cells in HBsAg‐positive DLBCL patients (HBsAg + DLBCL). HBx antigen was detected in 48.9% of HBsAg + DLBCL patients, and the expression rate of the Pre‐S2 antigen was 57.2%. HBx expression was significantly associated with high‐level expression of c‐Myc, while the Pre‐S2 antigen was not. In this study, we demonstrated that HBx antigen and Pre‐S2 antigen could be detected in lymphoma cells, and HBx expression was related to c‐Myc expression. Our findings provide a strong basis for further study of the HBV‐infected DLBCL and molecular mechanism underlying the lymphomagenesis.     

2009H1N1流感病毒对A549细胞和BEAS-2B细胞作用的初步研究

目的 探求2009H1N1流感病毒对A549细胞和BEAS-2B细胞作用,为研究2009H1N1流感病毒的致病机理提供线索。方法 不同来源(死亡、重症、普通病例分离)的2009H1N1流感病毒和季节性H1N1流感病毒分别感染A549和BEAS-2B细胞12、24、48、72 h后用流式细胞术检测细胞凋亡和细胞周期。结果 感染A549细胞12和24 h,普通病例分离的2009H1N1流感病毒组的细胞凋亡率最高(P<0.05),重症组的细胞凋亡率最低(P<0.05);48 h和72 h,死亡组细胞凋亡率最高(P<0.05)。感染BEAS-2B细胞12 h,重症组细胞凋亡率最高(P<0.05);48 h,死亡组和重症组细胞凋亡率高(P<0.05);72 h,死亡组和普通组细胞凋亡率高(P<0.05)。4株病毒主要将A549细胞阻滞在S期,将BEAS-2B细胞阻滞在G0/G1期。结论 在细胞凋亡和细胞周期的细胞学观察水平上2009H1N1流感病毒和季节性H1N1流感病毒之间存在差异,不同来源的2009H1N1流感病毒之间也存在差异。     

基于树突状细胞表型及T淋巴细胞功能对HBV相关慢加急性肝衰竭患者中医虚实属性实质的分析

目的归纳HBV相关慢加急性肝衰竭(HBV-ACLF)的中医虚实属性,分析其与树突状细胞(DC)表型及T淋巴细胞亚群的关系。方法收集2012年3月~(-1)1月于湖南中医药大学第一附属医院就诊的30例HBV-ACLF患者的基本资料、分期、中医证候等,归纳其中医虚实属性,分为实证组和虚证组;另选同期健康研究生志愿者10例作为对照组。应用流式细胞仪检测外周血CD3+、CD4+、CD8+T淋巴细胞百分比、CD4+CD25highCD127low表达率,以及外周血单个核细胞体外分离诱导培养的DC表型CD1α、HLA-DR、CD80、CD83、CD86表达率。计量资料符合正态分布的组间比较采用t检验,非正态分布的组间比较采用Wilcoxon秩和检验;计数资料组间比较用χ~2检验。结果与健康对照组相比,HBV-ACLF患者外周血CD3+、CD4+、CD8+T淋巴细胞百分比、CD4+与CD8+细胞比值及DC表型CD1α、HLA-DR、CD80、CD83、CD86的表达率均显著降低,CD4+CD25highCD127low表达率显著增高,差异均有统计学意义(P值均0.01)。HBV-ACLF患者中,与实证组比较,虚证组外周血CD3+、CD4+T淋巴细胞百分比、CD4+与CD8+细胞比值及CD4+CD25highCD127low表达率均显著降低,差异均有统计学意义(P值均0.05);与虚证组比较,实证组患者DC表型CD1α、CD83、CD86分子表达率均显著增高,差异均有统计学意义(P值均0.05)。结论 HBV-ACLF虚证组、实证组患者均存在T淋巴细胞亚群功能下降及DC活化功能不足,其在虚证组更为显著;DC表型及T淋巴细胞亚群等免疫指标可以作为HBV-ACLF患者中医虚实属性的客观参考指标。     

Patients with HBV‐related acute‐on‐chronic liver failure have increased concentrations of extracellular histones aggravating cellular damage and systemic inflammation

Acute‐on‐chronic liver failure (ACLF) is the most common type of liver failure and associated with grave consequences. Systemic inflammation has been linked to its pathogenesis and outcome, but the identifiable triggers are absent. Recently, extracellular histones, especially H4, have been recognized as important mediators of cell damage in various inflammatory conditions. This study aimed to investigate whether extracellular histones have clinical implications in patients with hepatitis B virus (HBV)‐related ACLF. One hundred and twelve patients with HBV‐related ACLF, 90 patients with chronic hepatitis B, 88 patients with HBV‐related liver cirrhosis and 40 healthy volunteers were entered into this study. Plasma histone H4 levels, cytokine profile and clinical data were obtained. Besides, patient's sera were incubated overnight with human L02 hepatocytes or monocytic U937 cells in the presence or absence of antihistone H4 antibody, and cellular damage and cytokine production were evaluated. We found that plasma histone H4 levels were greatly increased in patients with ACLF as compared with chronic hepatitis B, liver cirrhosis and healthy control subjects and were significantly associated with disease severity, systemic inflammation and outcome. Notably, ACLF patients' sera incubation decreased cultured L02 cell integrity and induced profound cytokine production in the supernatant of U937 cells. Antihistone H4 antibody treatment abrogated these adverse effects, thus confirming a cause‐effect relationship between extracellular histones and organ injury/dysfunction. The data support the hypothesis that the increased extracellular histone levels in ACLF patients may aggravate disease severity by inducing cellular injury and systemic inflammation. Histone‐targeted therapies may have potentially interventional value in clinical practice.     

Schistosome infection aggravates HCV‐related liver disease and induces changes in the regulatory T‐cell phenotype

Schistosome infections are renowned for their ability to induce regulatory networks such as regulatory T cells (Treg) that control immune responses against homologous and heterologous antigens such as allergies. However, in the case of co‐infections with hepatitis C virus (HCV), schistosomes accentuate disease progression and we hypothesized that expanding schistosome‐induced Treg populations change their phenotype and could thereby suppress beneficial anti‐HCV responses. We therefore analysed effector T cells and n/iTreg subsets applying the markers Granzyme B (GrzB) and Helios in Egyptian cohorts of HCV mono‐infected (HCV), schistosome‐co‐infected (Sm/HCV) and infection‐free individuals. Interestingly, viral load and liver transaminases were significantly elevated in Sm/HCV individuals when compared to HCV patients. Moreover, overall Treg frequencies and Helios^posTreg were not elevated in Sm/HCV individuals, but frequencies of GrzB⁺Treg were significantly increased. Simultaneously, GrzB⁺ CD8⁺ T cells were not suppressed in co‐infected individuals. This study demonstrates that in Sm/HCV co‐infected cohorts, liver disease is aggravated with enhanced virus replication and Treg do not expand but rather change their phenotype with GrzB possibly being a more reliable marker than Helios for iTreg. Therefore, curing concurrent schistosome disease could be an important prerequisite for successful HCV treatment as co‐infected individuals respond poorly to interferon therapy.     

SMMC-7721肝癌细胞膜上β_2糖蛋白I可能的受体

研究β2糖蛋白I(β2GPI)与肝细胞相互作用的过程,以进一步探讨β2GPI在乙型肝炎病毒感染肝细胞过程中所发挥的作用。采用L igand b lot技术,从SMMC-7721、HL-60及SGC-7901三个细胞株中,筛选出具有与人β2GPI特异结合蛋白成分的细胞。SMMC-7721细胞在40kD处出现一特异染色带,而HL-60及SGC-7901二种细胞则无此反应。在SMMC-7721细胞中存在有与人β2GPI特异结合的蛋白,这种蛋白可能参与了HBV感染肝细胞的过程。     

CIAPIN1对HepG2细胞增殖和细胞周期的影响

目的 观察调控细胞因子诱导的凋亡抑制分子1（CIAPIN1）基因表达对肝癌细胞株HepG2细胞增殖的影响,并分析其作用机制。方法 分别构建重组慢病毒CIAPIN1表达载体和CIAPIN1沉默载体,感染HepG2细胞,获得稳定高表达和低表达CIAPIN1基因的细胞后,实验分为4组,即CIAPIN1高表达组、CIAPIN1低表达组、无关沉默RNA（siRNA）干扰组和空白对照组,检测各组细胞增殖及CIAPIN1基因和蛋白表达,使用流式细胞仪检测细胞周期,采用Western blotting法检测cyclinD1、CDK4、cyclin E、CDK2水平以及总蛋白中IKKβ、磷酸化IKBα、p65和磷酸化p65水平。结果 CIAPIN1低表达组细胞增殖受到明显抑制,G0/G1期细胞比例为（73.2±2.5）%,明显高于GIAPIN1高表达组【（58.8±2.2）%,P<0.05】、无关siRNA干扰组【（62.4±1.8）%,P<0.05】或空白对照组【（63.2±2.6）%,P<0.05】;CIAPIN1 高表达组cyclinD1、CDK4、CDK2、cyclinE相对表达量明显高于其它各组,差异有统计学意义（P<0.05）;CIAPIN1高表达组磷酸化IKBα、磷酸化P65相对表达量分别为（1.335±0.182）和（0.731±0.106）,明显高于GIAPIN1低表达组【（0.108±0.035）和（0.028±0.010）,P均<0.05】、无关siRNA干扰组【（0.251±0.082）和（0.318±0.058）,P均<0.05】或空白对照组【（0.238±0.067）和（0.322±0.061）,P均<0.05】。结论 CIAPIN1对肝癌细胞增殖有促进作用,可能与其对NF-кB信号通路的激活作用有关。     

Stepwise application of fibrosis index based on four factors,red cell distribution width–platelet ratio,and aspartate aminotransferase–platelet ratio for compensated hepatitis B fibrosis detection

Background and Aim

Fibrosis index based on four factors (FIB‐4) and aspartate aminotransferase–platelet ratio (APRI) were validated with unsatisfactory efficiency. Routine hematology index red cell distribution width–platelet ratio (RPR) had been tried in liver fibrosis detection. This study tries to evaluate the stepwise application of FIB‐4, RPR, and APRI in detecting chronic hepatitis B (CHB) fibrosis.

Methods

A total of 246 compensated CHB patients who underwent liver biopsies, transient elastography, and routine blood tests including complete blood count were included. Dual cut‐offs were determined to exclude or include cirrhosis diagnosis. Performance of stepwise combining routine biomarkers including RPR, FIB‐4, and APRI were statistically analyzed.

Results

The Metavir F0, F1, F2, F3, and F4 were identified in 2.4%, 22.0%, 32.1%, 24.0%, and 19.5% of the eligible patients, respectively. The area under receiver operating characteristics curves for detecting significant fibrosis and cirrhosis were 0.853 and 0.883 for transient elastography; 0.719 and 0.807 for FIB‐4; 0.638 and 0.791 for RPR; 0.720 and 697 for APRI; and 0.618 and 0.760 for mean platelet volume–platelet ratio, respectively. The proportion of patient determined as cirrhosis or non‐cirrhosis was 65.9% by transient elastography, 36.9% by FIB‐4, 30.5% by RPR, and 19.5% by APRI, respectively. These numbers for determining significant fibrosis were 49.6%, 24.2%, 21.5%, and 23.6% in the same order. Detected by stepwise application of FIB‐4, RPR, and APRI, 41.5% and 52.8% of patients could be determined the state of significant fibrosis and cirrhosis, respectively.

Conclusions

In source‐limited settings without transient elastography, stepwise applying FIB‐4, RPR, and APRI could free nearly half of CHB patients from liver biopsies in detecting significant fibrosis and cirrhosis.     

Effects of oxymatrine on the serum levels of T helper cell 1 and 2 cytokines and the expression of the S gene in hepatitis B virus S gene transgenic mice: a study on the anti-hepatitis B virus mechanism of oxymatrine

BACKGROUND: Oxymatrine has been shown to have a remarkable inhibitory activity to hepatitis B virus (HBV) infection with a hepatitis B virus e antigen (HBeAg) serum conversion rate of approximately 45%. In order to explore the anti-HBV mechanism of oxymatrine, the effects of oxymatrine on serum levels of T helper (h)1 cytokines (interferon (IFN)-gamma and interleukin (IL)-2) and Th2 cytokines (IL-4 and IL-10), and the expression of S gene in HBV S gene transgenic mice were studied. METHODS: Each transgenic mouse was either injected with oxymatrine or saline intraperitoneally once a day for 30 days. Serum levels of IFN-gamma, IL-2, IL-4 and IL-10 were quantitated and compared to the data before the treatment. The expression of HBV S gene in transgenic mice was analyzed at the DNA, mRNA and protein levels. RESULTS: The serum levels of IFN-gamma in transgenic mice before or after oxymatrine treatment were 3.108 +/- 3.172 and 11.059 +/- 6.971 pg/mL, respectively. In contrast, serum levels before and after oxymatrine treatment for IL-4 were 29.045 +/- 13.235 and 13.024 +/- 9.002 pg/mL, respectively (P < 0.001). The serum levels of IL-2 in the control (saline injection) and oxymatrine-treated mice were 1.070 +/- 0.447 and 5.537 +/- 2.887 pg/mL, respectively (P < 0.0001); and that of IL-10 were 97.226 +/- 73.306 and 33.607 +/- 23.154 pg/mL, respectively (P < 0.01). No significant differences were observed in the expression of HBV S gene in the transgenic mice at the DNA, mRNA and protein levels before or after oxymatrine treatment. CONCLUSIONS: The fact that Th1 cytokines are increased while Th2 cytokines are decreased suggests that oxymatrine treatment triggers the change of immune response to hepatitis B infection in transgenic mice, which leads to improved HBV inhibitory activities. The study can help us better understand the mechanisms of the anti-HBV drug, oxymatrine, and how it has potential as an application in clinical chronic hepatitis B treatment.