Improved detection of hepatocyte proliferation using antibody to the pre-replication complex: an association with hepatic fibrosis and viral replication in chronic hepatitis C virus infection

Summary. To test the hypothesis that hepatitis C virus (HCV) might induce hepatocyte proliferation directly, thereby predisposing HCV carriers to cirrhosis and hepatocellular carcinoma, we have used a new method to identify proliferating hepatocytes, employing a novel monoclonal antibody to minichromosome maintenance (Mcm) proteins, essential components of the pre-replication complex. Antibody to Ki-67, a conventional marker of cell division, was also studied. Eighty-seven patients with chronic HCV infection and a broad spectrum of histological change were studied. Proliferation was observed rarely in hepatocytes from normal liver from healthy controls (always less than 0.01%). However, proliferating hepatocytes were detected in all HCV-infected patients and the proportion of hepatocytes expressing Mcm-2 (3–40%) always exceeded that expressing Ki-67 (1–14%) and correlated positively with increasing stage of fibrosis ( P  = 0.0001) and viral replication ( P  = 0.0004). There were weaker but significant associations between the proportion of hepatocytes expressing Mcm-2 and inflammatory indices including interface hepatitis, portal tract inflammation, lobular inflammation and steatosis. There was no association between the proportion of hepatocytes expressing Mcm-2 and age, gender or past alcohol consumption, but there was a weak association with current consumption of alcohol ( P  = 0.0067). The proportion of Ki-67 hepatocytes did not correlate with any clinical, laboratory or histological parameter. Mcm-2 was also detected in bile duct cells, sinusoidal lining cells and infiltrating lymphocytes, but at low frequency. These data indicate first, that Mcm-2 is a more sensitive marker of hepatocyte proliferation than Ki-67, second that many hepatocytes in chronic HCV infection have entered the cell cycle and third, suggest that interference with the hepatocyte cell cycle might be an alternative approach to therapy.     

799例重型肝炎患者的临床病原学与实验室分析

目的探讨乙型重型肝炎患者乙型肝炎病毒(HBV)DNA定量、e抗原表达与病死率的相关性,为重型肝炎临床治疗提供参考。方法统计我院2000-2004年各型重型肝炎的发病率,进一步应用荧光定量多聚酶链反应方法检测乙型重型肝炎患者血清HBV DNA,应用微粒子方法检测乙型肝炎e抗原表达情况,并分析其与病死率及抗病毒治疗的临床疗效间的关系。结果(1)重型肝炎中乙型肝炎占83．50％,慢性重型肝炎中乙型肝炎占96．77％;(2)5年间慢性乙型重型肝炎患者HBV DNA定量大于1×10,拷贝／ml组,总病死率为53．25%,小于1×105拷贝／ml组,病死率为34．50％,差异有统计学意义(P<0．01);e抗原表达对病死率无影响;(3)2004年,慢性乙型重型肝炎患者HBV DNA定量大于1×105拷贝／ml病例加用拉米夫定抗病毒治疗,病死率由2000年的54．64%下降至2004年的30．38%,差异有统计学意义(P<0．01)。结论重型肝炎以慢性乙型重型肝炎为主,病毒载量高是高病死率关键因素之一,抗病毒治疗可以降低患者的病死率。     

Hepatocyte growth factor gene transfer with naked plasmid DNA ameliorates dimethylnitrosamine-induced liver fibrosis in rats

Aim: Hepatocyte growth factor (HGF) has various biological properties, including antifibrogenic activity. In the present study, we tested the efficacy of HGF gene therapy using naked plasmid DNA in dimethylnitrosamine (DMN)-induced liver fibrosis in a rat model. Methods: Naked plasmid DNA encoding human HGF was injected once, together with a hypertonic solution, into the hepatic artery after DMN treatment on three consecutive days per week for 3 weeks. Naked plasmid DNA encoding beta-galactosidase was injected similarly in the DMN-treated control rats. DMN treatment was continued once weekly after gene transfer for additional 3 weeks. Results: The human HGF protein expression was detected in livers transfected with human HGF naked plasmid DNA, gradually decreasing by day 21. The expression of the endogenous rat HGF protein was also upregulated after human HGF gene transfer. Phosphorylation of c-Met, a HGF receptor, was detected only in livers transfected with human HGF plasmid DNA. Fibrosis was attenuated significantly in livers transfected with the human HGF plasmid. Attenuation wasaccompanied by decreased expression of alpha-smooth muscle actin. Increased portal vein pressure after treatment with DMN was suppressed significantly by HGF gene transfer. The upregulated hepatic protein expression of transforming growth factor-beta (TGF-beta) in response to DMN was markedly attenuated by HGF gene transfer accompanied by the increased protein expression for matrix metalloproteinases (MMP)-3 and -13. Conclusion: The hepatic arterial injection of human naked plasmid HGF DNA was effective in suppressing liver fibrosis induced in rats by DMN. The mechanisms by which HGF expression attenuated liver fibrosis may include the suppression of hepatic TGF-beta expression and the induction of MMP expression.     

马疫锥虫动基体DNA诱导抗dsDNA抗体产生条件与致病性的研究

目的研究不同免疫途径对马疫锥虫动基体DNA(kDNA)诱导正常小鼠产生抗双链DNA(dsDNA)抗体及其致病性的影响。方法将马疫锥虫kDNA与不完全弗氏佐剂乳化混合,通过皮下、腹腔、肌肉及静脉等途径注射入正常BALB/C小鼠。8周后,检测小鼠血清抗dsDNA抗体亚型及滴度、血尿素氮(BUN)、血肌酐(Scr)、补体C3、24h尿蛋白浓度、肾小球免疫复合物沉积强度、肾组织病变活动指数等,并分析肾组织病理学特征。结果上述各组小鼠产生IgG型抗dsDNA抗体量及肾损害程度依次为:皮下组>腹腔组>肌肉组>静脉组(P<0.05);IgG型抗dsDNA抗体与肾组织病变活动指数呈正相关。结论不同免疫途径对马疫锥虫kDNA诱导抗dsDNA抗体产生有明显不同影响,其导致的狼疮样肾脏损害与该抗体亚型有关。     

尘螨主要变应原DNA疫苗对其提取液诱导小鼠肺部变应性炎症的免疫保护作用

目的　探讨尘螨主要变应原DNA疫苗对尘螨提取液诱导的小鼠肺部变应性炎症的免疫治疗作用。方法　分别构建表达Derp1和Derp 2的真核表达质粒pCMV Derp 1和pDerp 2 IRES Derp1。 30只BALB/c小鼠随机分为正常组 (6只 )、对照组 (12只 )、单质粒组 (6只 )和混合组 (6只 )。后三组分别用空白质粒、pDerp 2 IRES Derp 1或混合的pDerp 2 IRES Derp 1和pCMV Derp 1免疫 ,4周后用尘螨提取液致敏、激发 ,观察肺部炎症、计分、肺泡灌洗液 (BALF)细胞总数和分类计数 ;酶联免疫吸附法 (ELISA)检测BALF中白细胞介素 4 (IL 4 )和γ干扰素 (IFN γ)水平。结果　细胞总数和嗜酸细胞(EOS)计数 :混合组 [(6± 4 )× 10 5/ml、0 0 5± 0 0 4 ]和对照组 [(2 1± 13)× 10 5/L、1 80± 1 39]比较差异有显著性 (P <0 0 5 ) ;IL 4水平 :混合组 [(16 8± 2 33)g/L]与对照组 [(5 38± 2 5 6 )g/L]比较差异有显著性 (P<0 0 5 ) ,各组间IFN γ比较差异无显著性 (P >0 0 5 )。结论　尘螨主要变应原的DNA疫苗可有效抑制尘螨提取液诱导的肺部变应性炎症 ,能表达两个主要变应原的DNA疫苗其抑制变应性炎症效果优于只表达一种变应原的疫苗。     

不同类型慢性乙型肝炎血清HBV DNA基因含量与临床的关系

探讨不同类型慢性乙型肝炎 (慢乙肝 )患者病毒复制水平与肝损害程度的关系。 180例慢乙肝患者血清HBVDNA基因含量采用荧光定量PCR方法检测。血清HBeAg阳性组HBVDNA含量 (10 6 3 5± 1 84)显著高于HBeAg阴性组 (10 4 73± 1 88) (P <0 0 1) ,不同类型慢乙肝组血清HBVDNA含量相比较无显著性差异 (P >0 0 5 )。研究证实 ,血清HBeAg的存在影响HBVDNA的水平变化 ,不同类型慢性乙型肝炎肝损害程度与血清HBVDNA基因含量无明显关系     

Germ-line mutations, DNA damage, and global hypermethylation in mice exposed to particulate air pollution in an urban/industrial location

Particulate air pollution is widespread, yet we have little understanding of the long-term health implications associated with exposure. We investigated DNA damage, mutation, and methylation in gametes of male mice exposed to particulate air pollution in an industrial/urban environment. C57BL/CBA mice were exposed in situ to ambient air near two integrated steel mills and a major highway, alongside control mice breathing high-efficiency air particulate (HEPA) filtered ambient air. PCR analysis of an expanded simple tandem repeat (ESTR) locus revealed a 1.6-fold increase in sperm mutation frequency in mice exposed to ambient air for 10 wks, followed by a 6-wk break, compared with HEPA-filtered air, indicating that mutations were induced in spermatogonial stem cells. DNA collected after 3 or 10 wks of exposure did not exhibit increased mutation frequency. Bulky DNA adducts were below the detection threshold in testes samples, suggesting that DNA reactive chemicals do not reach the germ line and cause ESTR mutation. In contrast, DNA strand breaks were elevated at 3 and 10 wks, possibly resulting from oxidative stress arising from exposure to particles and associated airborne pollutants. Sperm DNA was hypermethylated in mice breathing ambient relative to HEPA-filtered air and this change persisted following removal from the environmental exposure. Increased germ-line DNA mutation frequencies may cause population-level changes in genetic composition and disease. Changes in methylation can have widespread repercussions for chromatin structure, gene expression and genome stability. Potential health effects warrant extensive further investigation.     

The use of the hydrodynamic HBV animal model to study HBV biology and anti-viral therapy.

A simple reproducible and versatile small animal model for hepatitis B virus (HBV) infection is still unavailable. We have generated a simple transient liver-targeted transgenic mouse. Hydrodynamics tail vein injection of a head-to-tail dimer of adw HBV genome (pHBVadwHTD) into immunocompetent mice generated HBsAg and HBeAg expression in both serum and hepatocytes, followed by seroconversion. The injection of pHBVadwHTD into SCID mice generated prolonged HBsAg and HBeAg antigenemia and HBV viremia. Our results demonstrate that hydrodynamic injection of naked DNA could support the generation of HBV particles. We used this model for the assessment of anti-viral agents. Administration of our human monoclonal antibodies, HBV-Ab17(XTL) and HBV-Ab19(XTL), as well as Lamivudine (3TC) treatment suppressed HBV viremia. The model presented herein supports long and stable expression of HBV and will enable determination of various biological questions related to HBV life cycle, mutants and could enhance the development of anti-viral reagents.