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: 8336223
• 负责人: Patrizia Farci
• 金额: $ 87.54万
• 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/8336223
• 关键词: ATM Signaling Pathway; Accounting; Acute; Acute Hepatitis; Acute Hepatitis C; Acute Liver Failure; Affect; Age; Alcohols; Antibodies; Antigens; Autoimmune Process; B-Lymphocytes; Basic Science; Binding; Bioinformatics; Biological Markers; Cessation of life; Chronic; Chronic Hepatitis B; Chronic Hepatitis C; Chronic Hepatitis D; Chronic viral hepatitis; Cirrhosis; Clinical; Clinical Medicine; Collaborations; Complement; Complementarity Determining Regions; Control Groups; Cryoelectron Microscopy; Data; Deposition; Development; Disease Progression; Early Diagnosis; Effectiveness; Employee Strikes; Epitopes; Etiology; Evolution; Family; Fibrinolysis; Gender; Gene Expression; Gene Expression Profiling; Genes; Genetic; Goals; Hepatic; Hepatitis; Hepatitis B; Hepatitis B Core Antigen; Hepatitis B Virus; Hepatitis C; Hepatitis C virus; Hepatitis Viruses; Hepatocarcinogenesis; Hepatocellular Damage; Heterogeneity; Human; Immune response; Immunoglobulin G; Immunoglobulin M; Immunologics; Infection; Italy; Knowledge; Lasers; Lead; Libraries; Liver; Liver Cirrhosis; Liver diseases; Maps; Mediating; Methionine Metabolism Pathway; Microdissection; Molecular; Molecular Models; Molecular Profiling; Molecular Target; National Institute of Arthritis and Musculoskeletal and Skin Diseases; Outcome; Pathogenesis; Pathway Analysis; Pathway interactions; Patients; Pattern; Phage Display; Phase; Plasma Cells; Primary carcinoma of the liver cells; Progressive Disease; RNA Polymerase II; RNA Viruses; Research; Risk Factors; Sampling; Science; Source; Specimen; Stable Disease; Staging; T-Lymphocyte; Time; Tissues; Universities; Viral; Virus; anti-IgM; disease natural history; global health; image reconstruction; intrahepatic; male; molecular modeling; novel marker; programs; ras Oncogene; response; tool; tumor

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 further progress will depend on a more thorough knowledge of the molecular mechanisms of pathogenesis. The Hepatic Pathogenesis Section (HPS) has developed a comprehensive research program to study the pathogenesis of acute and chronic liver diseases in humans in collaboration with the Liver Transplanation Center and the Liver Unit of the University of Cagliari, Italy, which is an invaluable source of clinical samples. Our main research strategy is to combine basic research with clinical medicine. 1. Molecular mechanisms of pathogenesis of acute liver failure (ALF) While the liver damage in classic acute hepatitis B is T-cell mediated, the pathogenesis of HBV-associated ALF is unknown. 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, and 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 are in germline configuration; and that unrelated ALF patients use an identical unmutated VH gene (VH1-3) for both IgG and IgM anti-HBc antibodies. Thus, 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 (Farci et al. PNAS 2010). In collaboration with Dr. Steven from the LSBR, NIAMS, we have characterized the epitope of these antibodies using cryo-electron microscopy, image reconstruction, and molecular modeling. The antibodies associated to ALF bind to a region in which epitopes have not been detected before. Thus, our data are consistent with the hypothesis that antibodies directed to a particular epitope of HBcAg may be involved in the pathogenesis of HBV-associated ALF. 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 rapidly lead to liver-related death for decompensation or HCC. This variable clinical outcome suggests that not all cirrhosis is the same. To investigate these questions, we started extensive transcriptional studies in patients with end-stage liver cirrhosis of different etiology (HBV, HCV, HDV, alcohol, and autoimmune), as well as normal liver donors as a control group. 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, which may have implications for pathogenesis, treatment and natural history of these diseases. Interestingly, among liver donors, 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 since an older age (>40) at the time of infection and the male gender are associated with a worse outcome. 3. Pathogenesis of HCC and search for biomarkers for the early detection of HCC Hepatitis viruses (HBV, HCV and HDV) account for about 80% of HCC, and cirrhosis is the single most important risk factor. However, the molecular mechanisms of hepatocarcinogenesis are still unclear. Our major goal is to investigate the molecular mechanisms of hepatocarcinogenesis by studying simultaneously both the host and the virus. Because of the difficulty in studying sequential liver samples from the same patient progressing toward HCC, we investigated by microarray the molecular heterogeneity within and outside the tumor by mapping the entire liver containing HCC (up to 21 specimens for each patient). We have so far analyzed by microarray 462 liver specimens from patients with HCC of different etiology. Selected samples from the tumor and the periphery were also analyzed after laser microdissection. Remarkably, we found a high proportion of differentially expressed genes that were confirmed using both whole and microdissected tissue, confirming the robustness of our data. Interestingly, each tumor showed a distinct molecular signature with pathways specific for each tumor. An enrichment of genes related to the Ras oncogene family was documented in HBV-associated HCC and confirmed by Pathway Analysis (IPA9), whereas ATM signaling and methionine metabolism were the top-scored network functions in HCV-associated HCC. There are currently no data on the molecular pathogenesis of cirrhosis and HCC associated with HDV, a unique defective RNA virus that uses the host RNA polymerase II for replication. Our data provide the first evidence that genetic instability is the most important feature of HDV-associated HCC. We are now comparing the gene expression profiling of cirrhosis without HCC versus cirrhosis containing the tumor to see whether genes associated with liver cirrhosis are involved in the progression of cirrhosis toward HCC. The results of these studies may pave the way for the identification of new markers to predict the development of HCC, which are badly needed for the early diagnosis of 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.

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。肝发病机理科（HPS）已开发了一项全面的研究计划，以研究人类与肝脏移植中心和意大利Cagliari大学的肝脏肝脏疾病的发病机理，这是意大利Cagliari大学的肝脏单位，这是临床样本的宝贵来源。我们的主要研究策略是将基础研究与临床医学相结合。 1。急性肝衰竭（ALF）发病机理的分子机制 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。通过基因表达分析，我们证明了ALF的特征是以压倒性的B细胞签名为中心，其肝细胞分泌IgG和IgM，以及补体沉积。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）。这些抗体显示有限的可变重链（VH）曲目，并处于种系配置；并且无关的ALF患者对IgG和IgM抗HBC抗体均使用相同的未经未经形的VH基因（VH1-3）。因此，我们的数据强烈表明，与HBV相关的ALF是由与HBV核心抗原的T细胞独立肝内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（Farci等人，PNAS 2010）。 NIAMS与LSBR的Steven博士合作，我们使用冷冻电子显微镜，图像重建和分子建模来表征这些抗体的表位。与ALF相关的抗体结合了以前未检测到表位的区域。因此，我们的数据与以下假设一致：针对HBCAG特定表位的抗体可能与HBV相关ALF的发病机理有关。 2。慢性肝病发病机理的分子机制 肝硬化在30-40％的慢性丙型肝炎患者中发生，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，或者可能会迅速导致与肝脏相关的死亡或HCC的肝脏相关死亡。这种可变的临床结果表明，并非所有肝硬化都是相同的。为了调查这些问题，我们开始对不同病因（HBV，HCV，HDV，酒精和自身免疫性）以及正常肝脏供体作为对照组的终阶段肝肝硬化患者进行广泛的转录研究。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，这可能对这些疾病的发病机理，治疗和自然病史有影响。有趣的是，在肝脏供体中，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的基因特征是否与先天或适应性免疫反应有关，纤​​维纤维纤维纤维化的增加或自感染和男性时年龄较大的年龄（> 40）的纤维蛋白溶解降低与男性性别较差的情况相关。 3。HCC的发病机理并寻找生物标志物以早期检测到HCC 肝炎病毒（HBV，HCV和HDV）约占HCC的80％，肝硬化是最重要的危险因素。但是，肝癌发生的分子机制尚不清楚。 我们的主要目标是通过同时研究宿主和病毒来研究肝癌发生的分子机制。由于难以研究来自同一患者的顺序肝样品，因此我们通过微阵列通过映射包含HCC的整个肝脏（每位患者的21个标本）来研究肿瘤内部和外部的分子异质性。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。激光显微解剖后，还分析了来自肿瘤和周围的样品。值得注意的是，我们发现使用整个和微分化组织证实了大量差异表达的基因，从而证实了我们数据的鲁棒性。有趣的是，每个肿瘤显示出一个明显的分子特征，并具有针对每个肿瘤的途径。在HBV相关的HCC中记录了与RAS癌基因家族相关的基因的富集，并通过途径分析（IPA9）证实，而ATM信号和蛋氨酸代谢是HCV相关HCC中最高的网络功能。目前尚无有关肝硬化和HCC分子发病机制的数据，HDV是一种独特的缺陷RNA病毒，它使用宿主RNA聚合酶II进行复制。我们的数据提供了第一个证据，表明遗传不稳定性是HDV相关HCC的最重要特征。现在，我们正在比较肝硬化的基因表达分析而没有HCC与含有肿瘤的肝硬化，以查看与肝硬化相关的基因是否参与肝硬化向HCC的发展。这些研究的结果可能为识别新标记的预测HCC的发展铺平了道路，而HCC的早期诊断非常需要。 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。