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.

慢性病毒肝炎及其长期后遗症肝硬化和HCC代表了一个主要的全球健康问题。在控制和治疗慢性病毒肝炎方面已经取得了长足的进步，但是成功仍然有限，进一步的进步将取决于对发病机理的分子机制的更彻底的了解。黑猩猩模型对于发现病因学和理解由肝炎病毒引起的疾病的基础是基础，但这不是用于暴发性肝炎和肝纤维发生的合适模型。为了研究人类肝病的发病机理，我们与意大利卡利亚里大学的肝移植中心和肝脏单位进行了合作，这是临床样本的宝贵来源。自从我在2007年加入LID以自加入盖子以来，我在NIH上发起的转化研究的基础是我在2007年开始的转化研究的基础。我们的主要研究策略是将分子分析与临床，病毒和组织病理学数据相结合。 1。急性肝衰竭（ALF）发病机理的分子机制 丙型肝炎病毒（HBV）相关的ALF是一种引人注目的临床综合征，因为肝细胞突然丧失导致多器官衰竭。尽管经典急性肝炎中的肝损伤是T细胞介导的，但与HBV相关的ALF的发病机理尚不清楚。从两个定义明确的HBV相关的ALF病例从肝移植时收集的肝组织提供了一个独特的机会来建立该疾病的分子定义（Farci等人PNAS，2010年）。通过基因表达分析，我们证明了ALF的特征是以肝脏中为中心的压倒性B细胞特征，并伴随着补体沉积，其分泌IgG和IgM的血浆细胞大量积累。通过噬菌体播放文库，我们发现这些抗体的分子靶标是丙型肝炎核心抗原（HBCAG）。这些抗体显示出受限的可变链（VH）曲目，并且缺乏体细胞突变。 ALF的无关个体对IgG和IgM抗HBC抗体都使用了具有未突出可变结构域（VH1-3）的主要VH基因（VH1-3），这表明HBCAG是人类VH基因的靶标。因此，与急性肝炎B相反，我们的数据强烈表明与HBV相关的ALF是由与HBV的核心HBV核心抗原相关的肝内B细胞反应介导的，该反应与补体介导的大规模肝细胞损伤有关。 2。慢性肝病发病机理的分子机制 肝硬化在30-40％的慢性乙型肝炎患者中发生，但慢性乙型肝炎的患者多达80％。几十年来，这可能是一种稳定的疾病，或者可能导致与肝脏相关的死亡或HCC的肝脏死亡。某些患者死于肝硬化并发症而有些患者目前尚不清楚的生物学原因。但是，这种可变的临床结果表明并非所有肝硬化都是相同的。为了调查这些重要问题，我们在不同病因（HBV，HCV，HCV加酒精，HDV，酒精和自身免疫性）以及正常肝脏供体的终阶段肝硬化患者中开始了广泛的转录研究。我们研究了74种正常肝脏，不仅是为了建立一个对照组与肝病进行比较，还研究了为什么在HCV感染时年龄较大的年龄（> 40岁）和男性性别较差。有趣的是，我们发现根据年龄和性别差异表达的基因，现在我们正在确定这些特定基因特征是与先天或适应性免疫反应相关，纤维纤维蛋白发生增加或纤维蛋白溶解降低。总体而言，通过微阵列处理了对终末期肝肝硬化的原位肝移植患者的275个肝脏标本。我们的数据显示了每种类型的肝硬化的独特基因特征，即使在生物学上相关的疾病（例如HBV和HDV肝硬化）之间，具有惊人和意外的异质性。与肝硬化相关的基因的彻底表征对于确定哪些基因参与肝硬化向HCC的发展至关重要。 3。HCC的发病机理并寻找生物标志物以早期检测到HCC 已经鉴定出HCC的主要病因学剂，但是肝癌发生的机制仍然未知。特别是，关于围绕肿瘤的非肿瘤组织中存在的变化的信息有限。由于难以研究来自同一患者的顺序肝样品，因此我们决定绘制肿瘤病变以及肝脏未受影响的区域，以查看基因表达的特定变化在附近的组织中是否也存在。与Zamboni博士合作，我们设计了一项协议，其中包括5个来自肿瘤的活检，其中1个来自外围（B）的中心（A）和4（北，南，东，西），肿瘤外12个，在1 cm（c）外4个，4个在3 cm（d），d）和4位。到目前为止，我们已经通过微阵列462个肝脏标本的HCC患者进行了分析。值得注意的是，我们发现了每个肿瘤的分子特征不同，但是更重要的差异表达基因表现出了从非HCC肝硬化到肿瘤的组织到肿瘤组织的独特表达梯度。令人惊讶的是，在与HCV相关的HCC中，我们发现在肿瘤中差异表达的选定基因也从最遥远的部位开始的非肿瘤周围组织中也改变了。 HCC周围非肿瘤组织中基因表达改变的鉴定可能会揭示肝癌发生的多步过程，并为HCC提供新的诊断或预后标记。 4。HCV感染中病毒进化与临床结果之间的相关性 HCV准菜的早期演变被证明可以预测急性肝炎C的结果（Farciet。AlScience 2000），但对于缓慢和快速进步者之间的肝炎进化的模式是否差异很少。选择六名患者代表两种不同的临床结果：3例患有稳定疾病的缓慢症状，3岁且患有迅速进行性疾病，导致感染开始后5至10年内与肝脏相关的死亡。从第一个PCR阳性样品（在感染后的2周内）研究了HCV准特性，长达23年。用新精制的生物信息学工具分析了来自E1和E2基因的1793个序列，包括高变化区域。 我们的研究为慢性HCV感染的早期阶段提供了遗传瓶颈的证据，显示瓶颈后既有菌株的持久性与疾病快速进展之间存在相关性。因此，宿主免疫控制的有效性可能会严重影响疾病进展的速度。 5。寻找新的肝炎剂 尽管尚未排除感染力，但普遍认为PBC是一种自身免疫性疾病。我们已经确定了一名早期PBC患者（第1阶段）。这项研究的目的是试图将传染剂从PBC传播到黑猩猩。我们目前正在跟踪患有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活检，现在我们正在分析数据，以识别与先天免疫或线粒体或其他细胞基因有关的特定基因。如果有传染剂的证据，我们将尝试通过分子技术识别推定的药物。