Molecular Mechanisms of Pathogenesis of Acute and Chronic Liver Diseases

急慢性肝病发病的分子机制

基本信息

批准号：
8745452
负责人：
Patrizia Farci
金额：
$ 120.33万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/8745452
关键词：
Accounting Acute Acute Hepatitis Acute Liver Failure Affect Animals Antibodies Antibody Formation Area Autoimmune Diseases B-Lymphocytes Basic Science Biological Markers CCL2 gene Capsid Cell Line Cessation of life Chemokine (C-C Motif) Ligand 4 Chronic Chronic Hepatitis C Chronic viral hepatitis Cirrhosis Clinical Clinical Research Collaborations Complement Complex Data Deposition Disease Disease Progression Drops Early Diagnosis Early treatment Employee Strikes Epitopes Etiology Evolution Failure Fibrosis Gene Expression Gene Expression Profiling Genes Goals Hepatic Hepatic Stellate Cell Hepatitis B Hepatitis B Core Antigen Hepatitis B Virus Hepatitis C Hepatitis C virus Hepatitis Viruses Hepatocarcinogenesis Hepatocyte Human Immune response In Vitro Infectious Agent Interferon Type II Intravenous Lasers Lead Liver Liver Cirrhosis Liver Fibrosis Liver Regeneration Malignant - descriptor Malignant Neoplasms Maps Mediating MicroRNAs Modeling Molecular Molecular Profiling National Institute of Arthritis and Musculoskeletal and Skin Diseases Outcome PTEN gene Pan Genus Pathogenesis Pathway interactions Patients Pattern Primary biliary cirrhosis Primary carcinoma of the liver cells Process Progressive Disease RNA Reaction Reporting Risk Factors Role Sampling Serum Severities Severity of illness Signal Pathway Site Specimen Stable Disease Staging Stem cells Syndrome T-Lymphocyte Tissues Transfusion Tretinoin United States Variant Viral Viral hepatitis Viremia Virus Woman Wound Healing bile duct burden of illness chemokine chronic liver disease fibrogenesis hepatoma cell in vivo intrahepatic liver biopsy liver injury novel programs response stem tumor tumorigenesis

项目摘要

Chronic viral hepatitis and its long-term sequelae, cirrhosis and hepatocellular carcinoma (HCC) contribute to a very large burden of disease both in the United States and worldwide. The Hepatic Pathogenesis Section (HPS) has developed an extensive program involving basic and clinical research to study the pathogenesis of acute and chronic liver diseases in humans with a major focus on viral hepatitis. 1. Molecular mechanisms of pathogenesis of acute liver failure (ALF) and liver regeneration. ALF is a dramatic clinical syndrome characterized by sudden loss of hepatic function leading to multiorgan failure. By gene expression profiling we found that HBV-associated ALF, unlike classic acute hepatitis B where liver damage is predominantly T-cell mediated, is characterized by an overwhelming intrahepatic B-cell gene signature associated with a T-cell independent, intrahepatic B-cell response with massive production of antibodies in germline configuration, accompanied by complement deposition. In collaboration with Dr. Stevens from the LSB, NIAMS, the target epitope recognized by these antibodies has been characterized as a novel conformational site on the capsid of HBV, suggesting that antibodies directed to particular HBcAg epitopes may be involved in the pathogenesis of ALF. ALF has also provided a model to study the complex process of liver regeneration in humans. We found that HBV-associated ALF is associated with a strong hepatic stem/progenitor cell (HSPC) gene signature, hepatic stellate cell activation and fibrogenesis, along with an overriding tumorigenesis gene signature, which was previously never reported in liver regeneration. Both HSPC markers and fibrogenesis positively correlated with the extent of histopathological severity, likely reflecting the wound-healing process. 2. Determinants of disease progression, fibrogenesis, and viral evolution in chronic hepatitis C. Chronic hepatitis C may follow a mild and stable disease course or progress rapidly to cirrhosis and liver-related death. The mechanisms underlying the different rates of disease progression are unknown. Using prospectively collected samples from cases of transfusion-associated hepatitis C, we identified outcome-specific features that predict long-term disease severity. Slowly progressing disease correlated with an early ALT peak and antibody seroconversion, transient control of viremia, and significant induction of IFN-gamma and MIP-1beta, all indicative of an effective, albeit insufficient, adaptive immune response. By contrast, rapidly progressive disease correlated with persistent and significant elevations of ALT and the profibrogenic chemokine MCP-1 (CCL2) greater viral diversity and divergence, and a higher rate of synonymous substitution. These studies led to the identification of profibrogenic and proinflammatory chemokines that predict rapid progression of hepatitis C to cirrhosis, providing a new model of HCV-disease pathogenesis and opening new perspectives for early diagnosis and treatment. 3. Host and Viral Factors in the Pathogenesis of Hepatocellular Carcinoma (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. Although the major etiologic agents and risk factors for HCC are well defined, the molecular mechanisms of hepatocarcinogenesis remain unclear. Our aim is to investigate the pathogenesis of HCC by studying the host and the virus. Due to the complexity of HCC, our initial approach was an extensive mapping of gene expression profiles of the entire liver containing HCC associated with HBV, HCV and HDV in order to identify molecular signatures specific for each hepatitis virus as well as early biomarkers of progression to HCC. Strikingly, we detected a distinct molecular signature specific for each hepatitis virus, which was confirmed by profiling of laser-microdissected hepatocytes. This different molecular signature was also reflected by the different major canonical pathways. Altogether, these data emphasize the different molecular features of HCC associated with different hepatitis viruses, suggesting that each virus may induce distinct disease mechanisms. A. Limited replication of hepatitis viruses within HCC tissue. We quantified the levels of HBV, HCV and HDV replication in different areas of the liver within and outside the tumor, in non-HCC cirrhotic livers, and in serum samples from the same patients. Strikingly, we found a significant reduction in the levels of HCV RNA within the tumor compared to the non-tumoral tissue, whereas no differences were seen in multiple liver specimens obtained from non-HCC cirrhosis. HDV RNA levels were also lower in the tumor compared to the surrounding sites. By contrast, the levels of HCV RNA and HDV RNA in serum of HCC patients did not differ from those with non-HCC cirrhosis. Collectively, our data indicate that HBV, HCV and HDV do not grow well in malignant hepatocytes in vivo, in line with the inability or limited efficiency of hepatitis viruses to grow in hepatoma cell lines in vitro, suggesting that malignant hepatocytes express restriction factors that negatively affect viral replication. B. HCV quasispecies within the tumor and the surrounding non-tumoral tissue. To investigate whether the reduced HCV replication in the tumor reflected the presence of viral variants selected by malignant hepatocytes, we performed an extensive HCV quasispecies analysis from the E1/E2 region (inclusive of HVR1) from different liver areas and serum. Cirrhotic patients showed lack of HCV compartmentalization, whereas a different quasispecies distribution was seen in HCC. Cases with 1-log drop in HCV RNA had a pattern similar to that seen in cirrhosis, while those with the greatest drop showed a shift in quasispecies distribution, consistent with selection of viral variants by malignant hepatocytes. C. Identification of microRNA specifically expressed in HCV-associated HCC. Perturbations of miRNAs have been correlated with various diseases and in particular with cancer. Although several studies have investigated the association of miRNAs with HCC, the data are not univocal. We identified 18 miRNAs expressed in HCV-associated HCC, some of which were never previously reported in association with HCC. The HCC-specific miRNAs are connected through a regulatory network pivoting on p53, PTEN and all-trans retinoic acid signaling pathway, which may be directly involved in the pathogenesis of HCC. These findings suggest that HCC-specific miRNAs form a molecular network directly involved in the pathogenesis of HCC. Because of the lower HCV RNA levels found in the tumor, we are also investigating the potential role of HCC-specific miRNAs in modulating HCV replication. 4. Search for new hepatotropic agents: attempt to transmit a putative infectious agent from a patient with primary biliary cirrhosis (PBC). PBC is a chronic progressive disease of the liver bile ducts of unknown etiology that primarily affects women and may lead to cirrhosis and liver-related death. Although it is generally thought to be an autoimmune disease, an infectious agent has not been excluded. The aim of this study was to attempt to transmit a putative infectious agent from PBC to chimpanzees through intravenous inoculation of serum from an Italian patient with early stage PBC (Stage 1) to two chimpanzees, and a second passage to a third animal. Serial liver biopsies have revealed progressive liver damage with increasing degree of bile duct damage and ductular reaction and, importantly, fibrosis. This is a particularly striking finding as liver fibrosis is not commonly seen in chimpanzees, even in those chronically infected with hepatitis viruses for more than 10 years.

慢性病毒肝炎及其长期后遗症，肝硬化和肝细胞癌（HCC）在美国和全球范围内造成了很大的疾病负担。肝发病机理部分（HPS）开发了一项广泛的计划，涉及基础和临床研究，以研究人类急性和慢性肝病的发病机理，主要关注病毒性肝炎。 1。急性肝衰竭（ALF）和肝脏再生的发病机理的分子机制。 ALF是一种戏剧性的临床综合征，其特征是肝功能突然丧失导致多器官衰竭。通过基因表达分析，我们发现与经典的急性肝炎相关的ALF与经典的急性肝炎B不同，其中肝损伤主要是介导的T细胞，其特征是具有压倒性的肝内B细胞基因签名，与T-cell独立的，独立的eply eplatic eplatic eplatic B-cell响应相关的抗本质中的抗体构造中，由抗原构造的抗体构造，由伴随着抗生素的补充。与Niams的LSB的Stevens博士合作，这些抗体认可的靶表位置已被描述为HBV帽上的一种新型构象位点，这表明可能参与ALF的病原体，可能涉及针对特定HBCAG表位的抗体。 ALF还提供了一个模型来研究人类肝脏再生的复杂过程。我们发现与HBV相关的ALF与强肝茎/祖细胞（HSPC）基因特征，肝星状细胞活化和纤维发生有关，以及过压力肿瘤发生基因信号，以前从未在肝脏再生中报道过。 HSPC标记和纤维发生都与组织病理严重程度的程度正相关，这可能反映了伤口治疗过程。 2。慢性丙型肝炎的疾病进展，纤维发生和病毒进化的决定因素。慢性丙型肝炎可能遵循温和稳定的疾病病程，或者迅速发展为肝硬化和与肝脏相关的死亡。疾病进展率不同的机制尚不清楚。使用前瞻性收集的来自输血相关的丙型肝炎病例的样品，我们确定了预测长期疾病严重程度的结果特异性特征。缓慢进展的疾病与早期的ALT峰和抗体血清转化，病毒血症的瞬时控制以及IFN-GAMMA和MIP-1BETA的显着诱导，都表明有效的，尽管不足，适应性免疫反应。相比之下，快速进行性疾病与ALT的持续和显着升高以及纤维化趋化因子MCP-1（CCL2）相关，更大的病毒多样性和差异以及更高的同义替代率。这些研究导致了鉴定能够预测丙型肝炎向肝硬化的快速发展的纤维化和促炎性趋化因子，从而提供了HCV-疾病发病机理的新模型，并为早期诊断和治疗提供了新的观点。 3.肝细胞癌（HCC）发病机理中的宿主和病毒因子，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。尽管HCC的主要病因和危险因素是很好的定义，但肝癌发生的分子机制尚不清楚。我们的目的是通过研究宿主和病毒来研究HCC的发病机理。由于HCC的复杂性，我们的初始方法是对包含与HBV，HCV和HDV相关的整个肝脏的基因表达谱图进行了广泛的映射，以鉴定针对每种肝炎病毒的分子特征以及早期进展到HCC的分子特征。引人注目的是，我们检测到针对每种肝炎病毒的独特分子特征，通过对激光 - 微分子释放的肝细胞的分析证实了这一点。不同的主要规范途径也反映了这种不同的分子特征。总的来说，这些数据强调了与不同肝炎病毒相关的HCC的不同分子特征，这表明每种病毒可能诱导不同的疾病机制。 A. HCC组织中肝炎病毒的复制有限。我们量化了肿瘤内外不同区域，非HCC肝硬化肝脏以及同一患者的血清样品中的HBV，HCV和HDV复制水平。令人惊讶的是，我们发现与非肿瘤组织相比，肿瘤内HCV RNA水平的水平显着降低，而在非HCC cirrhosis获得的多个肝脏样本中没有发现差异。与周围部位相比，肿瘤中的HDV RNA水平也较低。相比之下，HCC患者血清中HCV RNA和HDV RNA的水平与非HCC肝硬化的HCV RNA和HDV RNA水平没有差异。总的来说，我们的数据表明，HBV，HCV和HDV在体内的恶性肝细胞中不能很好地生长，这与肝炎病毒在体外的无能或有限的肝炎病毒效率一致，这表明恶性肝细胞表达了对病毒重复产生负面影响的恶性肝细胞。 B.肿瘤和周围非肿瘤组织内的HCV准蛋白酶。为了研究肿瘤中HCV复制的减少是否反映了由恶性肝细胞选择的病毒变异，我们从不同肝脏区域和血清进行了E1/E2区域（包括HVR1）进行了广泛的HCV准准分析分析。肝硬化患者表现出缺乏HCV分室化，而在HCC中则观察到不同的准特性分布。 HCV RNA中1-log下降的病例的模式与肝硬化相似，而下降最大的情况则显示出准特性分布的变化，这与恶性肝细胞选择病毒变异剂一致。 C.在HCV相关HCC中特异性表达的microRNA的鉴定。 miRNA的扰动已与各种疾病，尤其是癌症相关。尽管有几项研究调查了miRNA与HCC的关联，但数据并非单次。我们确定了在HCV相关的HCC中表达的18个miRNA，其中一些以前从未与HCC相关。 HCC特异性的miRNA通过p53，PTEN和全反式视黄酸信号通路的调节网络连接，这可能直接参与HCC的发病机理。这些发现表明，HCC特异性miRNA形成了直接参与HCC发病机理的分子网络。由于肿瘤中发现的HCV RNA水平较低，因此我们还研究了HCC特异性miRNA在调节HCV复制中的潜在作用。 4。搜索新的肝硬化剂：尝试从原发性胆汁性肝硬化患者（PBC）中传播推定的感染剂。 PBC是未知病因的肝胆管的慢性进行性疾病，主要影响妇女，并可能导致肝硬化和肝脏相关死亡。尽管通常认为它是一种自身免疫性疾病，但尚未排除感染剂。这项研究的目的是试图通过静脉接种早期PBC（1阶段）（第1阶段）的血清静脉接种血清将推定的感染剂从PBC传播到黑猩猩，并将其第二次传递给第三只动物。连续肝活检表明，胆管损伤程度的增加，促进性肝脏损伤和螺丝剂反应，重要的是纤维化。这是一个特别引人注目的发现，因为在黑猩猩中通常看不到肝纤维化，即使在长期感染乙型肝病毒的人已超过10年中。