PATHOGENESIS OF IDIOPATHIC LIVER DISEASE

乙肝病毒是引起急性和慢性肝脏的最常见原因 世界上最大的疾病。我们最近用单抗检测到了 和重组DNA技术，乙肝病毒相关抗原和 有和没有常规血清学标志物的基因组住院患者 包括乙肝表面抗原、乙肝表面抗体 抗原(抗-HBs)、乙肝核心抗原(抗-HBc)和乙肝e 抗原(抗-HBe)。这些药物已被证明存在于血清中。 并具有传染性，因为它们会产生长期潜伏期的肝炎 黑猩猩的感染。我们希望对这些患者进行进一步的调查 以及更详细地描述这些肝炎病毒病原体的分子 和抗原水平，并评价其在发病机制中的意义 不明原因的肝病。我们计划做以下工作：1) 应用聚合酶链式反应(PCR)检测和扩增乙肝病毒 血清和肝脏中的相关DNA序列。对于这个过程，我们将 首次在固相载体上捕获乙肝病毒和相关基因组 识别ALL的不同高亲和力抗-HBs单抗 已知的乙肝病毒亚型，从而与不同的阿尔法区表位结合。 接下来，我们将扩增捕获的乙肝病毒基因组中的特定区域，如 在所有已知的庚型核糖核酸病毒(前核心区和核心区)中保守的那些基因 以及S和S基因前更易变的区域中的那些序列。 由于本实验室将聚合酶链式反应与单抗相结合， 抗HBs的单抗将被放大，从而检测DNA序列 在每次检测少于10个病毒颗粒的水平上，我们有 直接通过初级线路克隆扩增序列的能力 限制性内切酶的位置。2.我们最近开发了一种新方法 用限制性内切酶测定乙肝病毒基因组的异质性 片断分析。这种方法将使我们能够快速评估 抗体扩增后S区域内的变异 捕获-聚合酶链式反应技术。3)我们希望确定核苷酸序列 表面抗原区的可变性可能是其分子基础 针对不同的抗原成分或免疫反应性。4) 将与新发现的血清学结果进行比较 建立了基于单抗的第二代免疫放射分析(M2- Irma)。该检测方法可检测到低至10-15pg/il的相关乙肝表面抗原。 表位。4)将对选定的患者群体进行研究 以及低水平乙肝病毒相关感染的意义。在这方面，我们 将评估急慢性肝病患者和乙肝病毒血清学 既往感染的标志物(抗-HBc和/或抗-HBs)。在……里面 此外，无任何乙肝病毒的急慢性肝病患者 标记物，包括归类为原因不明的病毒性肝炎的标记物 和特发性肝病，k将进行研究。最后，我们将评估 乙肝疫苗无应答者存在低水平的乙肝病毒感染。 根据迄今获得的初步数据，我们乐观地认为，新的 将获得有关乙肝病毒的特征和相关信息 探员们。低水平的乙肝病毒相关基因组将被识别和表征 在分子水平上。我们认为，这些经纪人可能扮演了以前的角色 在急、慢性肝病发病机制中的未知作用 不明原因。

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.