Molecular Mechanisms of Pathogenesis of Acute and Chronic Liver Diseases

急慢性肝病发病的分子机制

• 批准号: 8157000
• 负责人: Patrizia Farci
• 金额: $ 55.13万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8157000
• 关键词: Acute; Chronic; Liver diseases; Molecular; Pathogenesis

Chronic viral hepatitis and its long-term sequelae, cirrhosis and HCC, represent a major global health problem. Considerable progress has been made in the control and treatment of chronic viral hepatitis, but the success is still limited, and further progress will depend on a more thorough knowledge of the molecular mechanisms of pathogenesis. The chimpanzee model has been fundamental for the discovery of the etiologic agents and for understanding the diseases caused by the hepatitis viruses, but it is not a suitable model for fulminant hepatitis and liver fibrogenesis. To investigate the pathogenesis of liver diseases in humans, we started a collaboration with the Liver Transplanation Center and the Liver Unit of the University of Cagliari, Italy, which is an invaluable source of clinical samples. This collection of specimens serves as the basis for the translational research that I have initiated at the NIH since I joined the LID as a senior investigator in 2007. Our main research strategy is to combine the molecular analysis with the clinical, virologic and histopathologic data. 1. Molecular mechanisms of pathogenesis of acute liver failure (ALF) Hepatitis B virus (HBV)-associated ALF is a dramatic clinical syndrome due to a sudden loss of hepatic cells leading to multiorgan failure. While liver damage in classic acute hepatitis B is T-cell mediated, the pathogenesis of HBV-associated ALF is unknown. Access to liver tissue collected at the time of liver transplantation from two well-defined cases of HBV-associated ALF provided a unique opportunity to establish a molecular definition of this disease (Farci et al. PNAS 2010). By gene expression analysis, we demonstrated that ALF is characterized by an overwhelming B-cell signature centered in the liver with massive accumulation of plasma cells secreting IgG and IgM, accompanied by complement deposition. By phage-display libraries, we discovered that the molecular target of these antibodies is the hepatitis B core antigen (HBcAg); that these antibodies display a restricted variable heavy chain (VH) repertoire and lack somatic mutations; and that unrelated individuals with ALF use an identical predominant VH gene with unmutated variable domain (VH1-3) for both IgG and IgM anti-HBc antibodies, indicating that HBcAg is the target of a germline human VH gene. Thus, in contrast to acute hepatitis B, our data strongly suggest that HBV-associated ALF is mediated by a T cell-independent intrahepatic B-cell response against the core antigen of HBV that is associated with complement-mediated massive hepatocellular damage. 2. Molecular mechanisms of pathogenesis of chronic liver diseases Cirrhosis develops in 30-40% of patients with chronic hepatitis B and C, but up to 80% in those with chronic hepatitis D. It may be a stable disease for decades or it may lead to liver-related death for decompensation or HCC. The biological reasons why some patients die of liver cirrhosis complications and some do not are presently unknown. However, this variable clinical outcome suggests that not all cirrhosis is the same. To investigate these important questions, we started extensive transcriptional studies in patients with end-stage liver cirrhosis of different etiology (HBV, HCV, HCV plus alcohol, HDV, alcohol, and autoimmune), as well in normal liver donors. We studied 74 normal livers not only to build a control group for comparison with liver disease but also to study why an older age (>40) at the time of HCV infection and the male gender are associated with a worse outcome. Interestingly, we found genes differentially expressed according to age and gender, and we are now determining whether these specific gene signatures are associated with innate or adaptive immune responses, increased fibrinogenesis, or decreased fibrinolysis. Overall, 275 liver specimens from patients who underwent orthotopic liver transplantation for end-stage liver cirrhosis were processed by microarray. Our data show a distinct gene signature for each type of cirrhosis, with a striking and unexpected heterogeneity even between biologically related conditions such as HBV and HDV cirrhosis. A thorough characterization of genes associated with liver cirrhosis will be critical to identify which genes are involved in the progression of cirrhosis toward HCC. 3. Pathogenesis of HCC and search for biomarkers for the early detection of HCC The major etiologic agents of HCC have been identified but the mechanisms of hepatocarcinogenesis are still unknown. In particular, there is limited information on the alterations present in the nontumor tissue that surrounds the tumor. Because of the difficulty in studying sequential liver samples from the same patient progressing toward HCC, we have decided to map the tumor lesion as well as the unaffected areas of the liver to see whether specific alterations in gene expression are also present in the neighboring tissue. In collaboration with Dr. Zamboni, we have designed a protocol that includes 5 biopsies from the tumor, 1 from the center (A) and 4 (North, South, East, West) from the periphery (B), and 12 outside the tumor, 4 at 1 cm (C), 4 at 3 cm (D) and 4 at the edge of the liver (E). We have so far analyzed by microarray 462 liver specimens from patients with HCC of different etiology. Remarkably, we found a different molecular signature for each tumor, but even more important differentially expressed genes showed a distinct gradient of expression from non-HCC cirrhosis to tumor-surrounding tissues to tumor tissues. Strikingly, in HCV-related HCC, we found that selected genes differentially expressed in the tumor were also altered in nontumoral surrounding tissues starting from the most distant sites. The identification of gene expression alterations in nontumoral liver tissue surrounding HCC may shed light on the multistep process of hepatocarcinogenesis and provide new diagnostic or prognostic markers for HCC. 4. Correlation between viral evolution and clinical outcome in HCV infection The early evolution of the HCV quasispecies was shown to predict the outcome of acute hepatitis C (Farci et. al Science 2000), but little is known on whether the pattern of viral evolution in progressing hepatitis differs between slow and rapid progressors. Six patients were selected to represent two different clinical outcomes: 3 were slow progressors with a stable disease for > 20 yrs and 3 had a rapidly progressive disease leading to liver-related death within 5 to 10 years from the onset of the infection. HCV quasispecies was studied from the first PCR-positive sample, within 2 weeks of infection, for up to 23 years. A total of 1793 sequences from the E1 and E2 genes, including the hypervariable region 1, were analyzed with newly refined bioinformatic tools. Our study provides evidence for a genetic bottleneck during the early phase of chronic HCV infection, showing a correlation between persistence of pre-existing strains after the bottleneck and rapid disease progression. Thus, the effectiveness of the host immunologic control may critically affect the pace of disease progression. 5. Search for new hepatitis agents PBC is generally thought to be an autoimmune disease though an infectious agent has not been excluded. We have identified a Sardinian patient with early PBC (stage 1). The aim of this study was to attempt to transmit an infectious agent from PBC to chimpanzees. We are currently following a chimpanzee inoculated with serum from the patient with PBC (week 104 post-inoculation). We have examined all weekly liver biopsies by microarray and we are now analyzing the data to identify specific genes related to innate immunity or mitochondria or other cellular genes. If there is evidence of an infectious agent, we will attempt to identify the putative agent by molecular techniques.

慢性病毒性肝炎及其长期后遗症、肝硬化和肝癌是一个主要的全球健康问题。慢性病毒性肝炎的控制和治疗已经取得了相当大的进展，但成功仍然有限，进一步的进展将取决于对发病机制分子机制的更深入了解。黑猩猩模型对于发现病因和了解肝炎病毒引起的疾病至关重要，但它并不是暴发性肝炎和肝纤维化的合适模型。为了研究人类肝脏疾病的发病机制，我们开始与意大利卡利亚里大学的肝脏移植中心和肝脏科合作，该中心是临床样本的宝贵来源。自从我 2007 年作为高级研究员加入 LID 以来，这些标本集合是我在 NIH 发起的转化研究的基础。我们的主要研究策略是将分子分析与临床、病毒学和组织病理学数据相结合。 1. 急性肝衰竭（ALF）发病的分子机制 乙型肝炎病毒 (HBV) 相关的 ALF 是一种由于肝细胞突然丢失导致多器官衰竭而引起的严重临床综合征。虽然经典急性乙型肝炎的肝损伤是 T 细胞介导的，但 HBV 相关 ALF 的发病机制尚不清楚。对两例明确的 HBV 相关 ALF 病例进行肝移植时收集的肝组织的获取，为建立该疾病的分子定义提供了独特的机会（Farci 等人，PNAS 2010）。通过基因表达分析，我们证明 ALF 的特点是以肝脏为中心的压倒性 B 细胞特征，分泌 IgG 和 IgM 的浆细胞大量积累，并伴有补体沉积。通过噬菌体展示文库，我们发现这些抗体的分子靶点是乙型肝炎核心抗原（HBcAg）；这些抗体显示出受限的可变重链（VH）库并且缺乏体细胞突变；患有 ALF 的无关个体对 IgG 和 IgM 抗 HBc 抗体使用具有未突变可变域 (VH1-3) 的相同显性 VH 基因，表明 HBcAg 是种系人类 VH 基因的靶标。因此，与急性乙型肝炎相反，我们的数据强烈表明，HBV 相关的 ALF 是由 T 细胞独立的肝内 B 细胞针对 HBV 核心抗原的反应介导的，而该反应与补体介导的大规模肝细胞损伤有关。 2. 慢性肝病发病的分子机制 30-40% 的慢性乙型肝炎和丙型肝炎患者会出现肝硬化，但慢性丁型肝炎患者的比例高达 80%。这种疾病可能会稳定数十年，也可能因失代偿或 HCC 导致与肝脏相关的死亡。目前尚不清楚一些患者死于肝硬化并发症而另一些患者则没有的生物学原因。然而，这种可变的临床结果表明并非所有肝硬化都是相同的。为了研究这些重要问题，我们开始对不同病因的终末期肝硬化患者（HBV、HCV、HCV 加酒精、HDV、酒精和自身免疫）以及正常肝脏捐献者进行广泛的转录研究。我们研究了 74 个正常肝脏，不仅是为了建立一个对照组与肝脏疾病进行比较，也是为了研究为什么 HCV 感染时年龄较大（> 40 岁）和男性与较差的结果相关。有趣的是，我们发现基因根据年龄和性别而有差异表达，我们现在正在确定这些特定的基因特征是否与先天性或适应性免疫反应、纤维蛋白生成增加或纤维蛋白溶解减少有关。总体而言，对来自因终末期肝硬化接受原位肝移植的患者的 275 份肝脏标本进行了微阵列处理。我们的数据显示每种类型的肝硬化都有独特的基因特征，即使在 HBV 和 HDV 肝硬化等生物学相关疾病之间也存在惊人且意想不到的异质性。全面表征与肝硬化相关的基因对于确定哪些基因参与肝硬化向 HCC 的进展至关重要。 3. HCC发病机制及寻找早期发现HCC的生物标志物 HCC 的主要病因已被确定，但肝癌发生的机制仍不清楚。特别是，关于肿瘤周围非肿瘤组织中存在的改变的信息有限。由于研究同一患者进展为 HCC 的连续肝脏样本存在困难，我们决定绘制肿瘤病变以及肝脏未受影响区域的图谱，以了解邻近组织中是否也存在基因表达的特定改变。我们与 Zamboni 博士合作设计了一个方案，其中包括来自肿瘤的 5 个活检，其中 1 个来自中心 (A)，4 个来自外围 (B)（北、南、东、西），以及肿瘤外部的 12 个活检，4 个位于 1 厘米处 (C)，4 个位于 3 厘米处 (D)，4 个位于肝脏边缘 (E)。迄今为止，我们已通过微阵列分析了来自不同病因的 HCC 患者的 462 份肝脏标本。值得注意的是，我们发现每个肿瘤都有不同的分子特征，但更重要的差异表达基因显示出从非 HCC 肝硬化到肿瘤周围组织再到肿瘤组织的明显表达梯度。引人注目的是，在 HCV 相关的 HCC 中，我们发现肿瘤中差异表达的选定基因也在非肿瘤周围组织中从最远的部位开始发生改变。 HCC 周围非肿瘤肝组织中基因表达变化的鉴定可能有助于揭示肝癌发生的多步骤过程，并为 HCC 提供新的诊断或预后标志物。 4. HCV感染中病毒进化与临床结果的相关性 HCV 准种的早期进化被证明可以预测急性丙型肝炎的结果（Farci et. al Science 2000），但对于慢速进展型肝炎和快速进展型肝炎病毒进展模式是否不同，我们知之甚少。选择 6 名患者代表两种不同的临床结果：3 名患者病情稳定，病情稳定 > 20 年；3 名患者病情快速进展，在感染后 5 至 10 年内导致与肝脏相关的死亡。在感染后 2 周内，从第一个 PCR 阳性样本开始对 HCV 准种进行了长达 23 年的研究。使用新改进的生物信息学工具分析了来自 E1 和 E2 基因的总共 1793 个序列，包括高变区 1。 我们的研究为慢性丙型肝炎病毒感染早期阶段的遗传瓶颈提供了证据，显示了瓶颈后先前存在的菌株的持续存在与疾病快速进展之间的相关性。因此，宿主免疫控制的有效性可能严重影响疾病进展的速度。 5. 寻找新的肝炎药物 PBC 通常被认为是一种自身免疫性疾病，但也不排除感染因素。我们发现一名撒丁岛患者患有早期 PBC（1 期）。本研究的目的是尝试将传染源从 PBC 传播给黑猩猩。我们目前正在追踪一只接种了 PBC 患者血清的黑猩猩（接种后第 104 周）。我们已经通过微阵列检查了所有每周的肝脏活检，我们现在正在分析数据以识别与先天免疫或线粒体或其他细胞基因相关的特定基因。如果有证据表明存在传染性病原体，我们将尝试通过分子技术识别假定的病原体。