PATHOGENESIS OF IDIOPATHIC LIVER DISEASE

特发性肝病的发病机制

• 批准号: 3080865
• 负责人: T. Jake Liang
• 金额: $ 8.74万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-05-01 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3080865
• 关键词: DNA virus; Hepadnaviridae; Hepatovirus; acute disease /disorder; chronic disease /disorder; endonuclease; genetic manipulation; genome; hepatitis B; hepatitis B antigens; hepatitis B virus group; hepatitis vaccine; human subject; laboratory mouse; molecular pathology; monoclonal antibody; nucleic acid sequence; nucleocapsid; polymerase chain reaction; recombinant DNA; restriction fragment length polymorphism; serum; surface antigens; virus genetics

Hepatitis B virus is the most common cause of acute and chronic liver disease in the world. We have recently detected by monoclonal antibodies and recombinant DNA techniques, hepatitis B virus related antigens and genome inpatients with and without conventional serologic markers that include hepatitis B surface antigen, antibodies to hepatitis B surface antigen (anti-HBs), hepatitis B core antigen (anti-HBc) and hepatitis B e antigen (anti-HBe). These agents have been shown to be present in serum and are infectious since they will produce a long incubation hepatitis infection in chimpanzees. We wish to investigate these patients further and characterize such hepatitis viral agents in more detail at he molecular and antigenic level and to evaluate their significance in the pathogenesis of liver disease of unknown etiology. We plan to do the following: 1) Employ the polymerase chain reaction (PCR) to detect and amplify HBV related DNA sequences in serum and liver. For this procedure, we will first capture on a solid-phase support, HBV and related genome with different high affinity monoclonal anti-HBs antibodies that recognize all known subtypes of HBV and thus bind to different alpha domain epitopes. Next, we will amplify defined regions in the captured HBV genomes such as those conserved in all known hepadnaviruses (pre-core and core region) as well as those sequences in the more variable pre-S and S gene domains. Since the PCR developed in our laboratory in combination with monoclonal anti-HBs monoclonal antibodies will amplify and thus detect DNA sequences at a level of less than 10 viral particles per assay, we have the capability to clone amplify sequences directly through primary liners at the restriction enzyme site. 2) We have recently developed a new method of measuring heterogeneity in the HBV genome by restriction endonuclease fragment analysis. This approach will allow us to rapidly assess variations within the S region following amplification by the antibody capture-PCR technique. 3) We wish to determine nucleotide sequence variability of the surface antigen region which may be the molecular basis for different antigenic composition or immunologic reactivity. 4) Comparisons will be made to serologic findings that employ a newly developed monoclonal based second generation immunoradiometric assay (M2- IRMA). This assay detects as low as 10-15 pg/il of HBsAg associated epitopes. 4) Selected patient populations will be studied for the presence and significance of low level HBV related infection. In this regard, we will assess patients with acute and chronic liver disease and HBV serologic markers of previous infection (anti-HBc and/or anti-HBs or both). In addition, patients with acute and chronic liver disease without any HBV markers, including those classified as viral hepatitis of unknown etiology and idiopathic liver disease,k will be studied. Finally, we will evaluate HBV vaccine non-responders for the presence of low level HBV infection. Based on the preliminary data obtained thus far, we are optimistic that new information will be obtained on the characteristics of HBV and related agents. Low level HBV related genome will be identified and characterized at the molecular level. We believe that these agents may play a previously unrecognized role in the pathogenesis of acute and chronic liver disease of uncertain etiology.

B型肝炎病毒是最常见的急性和慢性肝病的原因 世界上的疾病。 我们最近通过单克隆抗体检测到 和重组DNA技术，B肝炎病毒相关抗原， 有和没有常规血清学标志物的基因组住院患者， 包括B型肝炎表面抗原、针对B型肝炎表面抗体 抗原（抗-HBs）、B型肝炎核心抗原（抗-HBc）和B e型肝炎 抗-HBe抗原。 这些药物已被证明存在于血清中 并且具有传染性，因为它们会产生长期潜伏的肝炎 黑猩猩的感染。 我们希望进一步调查这些患者 并在分子水平上更详细地表征这些肝炎病毒因子 和抗原水平，并评估其在发病机制中的意义 病因不明的肝病 我们计划做以下工作：1） 应用聚合酶链反应（PCR）检测和扩增HBV 血清和肝脏中的相关DNA序列。 在这个过程中，我们将 首次在固相支持物上捕获HBV和相关基因组， 不同的高亲和力单克隆抗-HBs抗体， 已知的HBV亚型，因此结合不同的α结构域表位。 接下来，我们将扩增捕获的HBV基因组中的定义区域，例如 在所有已知嗜肝DNA病毒中保守的那些（前核心区和核心区）， 以及在更易变的前S和S基因结构域中的那些序列。 自本实验室发展PCR技术与单克隆抗体结合以来， 抗-HBs单克隆抗体将扩增并因此检测DNA序列 在每次检测少于10个病毒颗粒的水平下，我们得到了 直接通过初级衬管克隆扩增序列的能力， 限制性内切酶位点 2)我们最近发明了一种新方法 通过限制性内切酶测定HBV基因组的异质性 片段分析 这种方法将使我们能够快速评估 抗体扩增后S区内的变异 捕获PCR技术。 3)我们希望确定核苷酸序列 表面抗原区域的可变性可能是 不同的抗原组成或免疫反应性。 四、 将与采用新的 开发了基于单克隆的第二代免疫放射分析（M2- IRMA）。 该检测试剂盒可检测低至10-15 pg/il的HBsAg相关 表位 4)将对选定的患者人群进行研究， 低水平HBV相关感染的意义。 在这方面我们 将评估急性和慢性肝病患者和HBV血清学 既往感染的标志物（抗-HBc和/或抗-HBs或两者）。 在 此外，无任何HBV的急慢性肝病患者 标志物，包括那些被归类为病因不明的病毒性肝炎的标志物 和特发性肝病，k将被研究。 最后，我们将评估 对于存在低水平HBV感染的HBV疫苗无应答者。 根据迄今为止获得的初步数据，我们乐观地认为， 将获得有关HBV特征和相关信息 剂. 将鉴定和表征低水平HBV相关基因组 在分子水平上。 我们认为这些特工可能在之前 在急性和慢性肝病发病机制中的作用尚未得到认识， 病因不明。