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: 7964599
• 负责人: Patrizia Farci
• 金额: $ 63.77万
• 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/7964599
• 关键词: Accounting; Acute; Acute Liver Failure; Address; Age; Alcohols; Antibodies; Autoantibodies; Autoimmune Diseases; Autoimmune Process; B-Lymphocytes; Biliary; Biochemical; Biological Markers; Biopsy; Blast Cell; Cessation of life; Chronic; Chronic Hepatitis B; Chronic Hepatitis C; Chronic Hepatitis D; Chronic viral hepatitis; Cirrhosis; Clinical; Collaborations; Communities; Complement; Confocal Microscopy; Data; Deposition; Development; Diagnostic; Diffuse; Disease; Disease Progression; Early Diagnosis; Elderly; Employee Strikes; Etiology; Failure; Female; Fibrinolysis; Gender; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Genes; Genetic Predisposition to Disease; Goals; Hepatic; Hepatic Fibrogenesis; Hepatitis; Hepatitis B; Hepatitis B Core Antigen; Hepatitis B Virus; Hepatitis C virus; Hepatitis Viruses; Hepatocarcinogenesis; Heterogeneity; Human; Humoral Immunities; IgG1; Immune response; Immunity; Immunoglobulin G; Immunoglobulin Genes; Immunoglobulin M; Individual; Infection; Infectious Agent; Infiltration; Inflammatory Infiltrate; Injury; Italy; Knowledge; Lead; Libraries; Liver; Liver Cirrhosis; Liver Fibrosis; Liver diseases; Mature B-Lymphocyte; Mediating; Medicine; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Molecular Analysis; Natural History; Natural Immunity; Outcome; Pan Genus; Pathogenesis; Patients; Persons; Phage Display; Pharmaceutical Preparations; Plasma; Plasma Cells; Population; Primary carcinoma of the liver cells; Process; Production; Research; Research Personnel; Resources; Risk Factors; Role; Sampling; Sardinia; Sequence Analysis; Serum; Specimen; Stable Disease; Staging; Staining method; Stains; Syndrome; T-Cell Activation; T-Lymphocyte; Techniques; Transcript; Translational Research; United States National Institutes of Health; Universities; Up-Regulation; Viral; Viral Antigens; Viral hepatitis; age group; base; design; inhibitor/antagonist; interest; intrahepatic; light microscopy; liver biopsy; liver transplantation; male; sample collection; success; treatment strategy

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. However, chimpanzees are 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, (Sardinia, Italy), which is an invaluable resource of clinical samples from well-characterized patients with acute and chronic liver diseases. 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, biochemical, virologic and histopathologic data. 1. Molecular mechanisms of pathogenesis of acute liver failure Few conditions in medicine are more dramatic and challenging than acute liver failure (ALF), a clinical syndrome characterized by a sudden loss of hepatic function leading to multiorgan failure in a person without preexisting liver disease. Viral hepatitis and drug-induced liver injury account for most cases of ALF. Although the pathogenic mechanisms of virally-induced ALF are presently unknown, we demonstrated that in HCV ALF the extent of liver damage correlated with the magnitude of viral replication and with an unusually homogeneous viral population. By contrast, in fulminant hepatitis B, HBV replication is barely detectable or undetectable concomitant with an early and enhanced immune response to several viral antigens. Taking advantage of gene expression analysis and phage-display library, we have provided evidence that in fulminant hepatitis B the liver damage is mediated by B-cell immunity. Liver specimens obtained from two patients with HBV-associated ALF (4 samples per liver) and individual liver specimens from 8 liver donors were processed by microarray. Multivariate permutation analysis identified 1,368 transcripts that were differentially expressed in ALF; 709 were up-regulated and 659 down-regulated. ALF was characterized by an overriding B-cell signature comprising genes related to mature B cells and plasma cells with abundant polyclonal expression of immunoglobulin genes. By contrast, there was a limited T-cell signature and up-regulation of several inhibitors of T-cell activation. Immunohistochemical analysis confirmed the prominent B-cell signature showing diffuse liver infiltration by plasma blasts and plasma cells with strong cytoplasmic staining for IgM and IgG, associated with a significant deposition of complement factors. Analysis of 4 phage display Fab libraries of IgG1 and IgM generated from the liver of the 2 patients with ALF identified HBcAg as the major target of the antibodies produced in the liver. In conclusion, the prominent B-cell gene signature with intrahepatic Ig production primarily directed against HBcAg, associated with complement deposition, suggests a pivotal role of the humoral immunity in the pathogenesis of HBV-associated ALF. 2. Molecular mechanisms of pathogenesis of chronic liver diseases The proportion of patients who develop cirrhosis is 30-40% among patients with chronic hepatitis B and C, and up to 80% in chronic hepatitis D. We have long been interested in understanding the mechanisms underlying the different outcomes observed in patients with chronic viral hepatitis. Presently, it is not possible to predict which individuals will sustain more dire outcomes, although age at infection has been shown to have a significant impact on the pace of disease progression in chronic hepatitis C, which is more rapid in the elderly. Likewise, male gender is associated with a worst outcome. To investigate the role of age and gender in liver fibrosis progression, we have completed an extensive study of gene expression analysis in normal livers obtained from individuals in different age groups. We are now determining whether specific gene signatures associated with innate or adaptive immune response, increased fibrinogenesis, or decreased fibrinolysis are modulated according to age or if there are significant differerences in gene expression between males and females. The results of this study may help to understand the influence of age and gender in the natural history of chronic hepatitis C, as well as in other forms of chronic liver diseases. Cirrhosis may be a stable disease for decades in some patients, whereas in others it may lead to liver-related death for decompensation or hepatocellular carcinoma (HCC). The reasons why some patients eventually die of complications of liver cirrhosis and some dont are presently unknown. To address these important questions, we have designed and completed a study in which a large number of liver specimens rigorously selected to represent clean cases of cirrhosis of different etiologies (HBV, HBV+HDV, HCV, HCV+alcohol, alcohol, autoimmune) were analyzed by microarray. We have found that each subtype of cirrhosis is characterized by a specific gene expression signature, with a striking and unexpected heterogeneity even between biologically related conditions such as HBV vs. HBV+HDV cirrhosis. Gene expression profiling will be integrated by virological studies using confocal microscopy and sequencing analysis. 3. Pathogenesis of HCC and search for biomarkers for the early detection of HCC. The clinical heterogeneity of HCC and the lack of good diagnostic markers and treatment strategies have made this disease one of the most important challenges for the scientific community. The presence of cirrhosis of any etiology is the strongest risk factor for the development of HCC. Although the major etiologic agents of HCC have been identified, the mechanisms of hepatocarcinogenesis are unclear and genetic predisposition has rarely been described. In collaboration with Dr. Zamboni, from the Liver Transplantation Center in Cagliari, Italy, we have designed a study in which multiple biopsies obtained from a single liver have been studied. We have collected more than 500 liver specimens from patients with HCC and about 200 have already been subjected to microarray analysis.The study is currently in progress. 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. 4. Search for new hepatitis agents. The cause of primary biliary (PBC)cirrhosis is unknown,but because of the presence of autoantibodies against mitochondrial protein in 90% of the patients, PBC is generally thought to be an autoimmune disease. However, an infectious agent has not been excluded. We have identified a Sardinian patient with early PBC (stage 1). The aim of this study is to attempt to transmit an infectious agent from PBC to chimpanzees. We are currently following a chimpanzee that was inoculated with serum from the patient with PBC (week 77 post-inoculation). Interestingly, we have observed ultrastructural alterations of the mitochondria by EM, as well as portal inflammatory infiltrates by light microscopy. We are planning to examine all the weekly liver biopsies by microarray to see whether there are changes in genes related to innate immunity, which may reflect the nonspecific, but universal, immune response elicited by an infectious agent, genes related to mithocondria or other cellular genes. If there is evidence of an infectious agent, we will attempt to to identify the putative agent by molecular techniques.

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和理解由肝炎病毒引起的疾病至关重要。但是，黑猩猩不是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，我们与肝脏移植中心和Cagliari大学（意大利萨迪尼亚）的肝脏移植中心和肝脏部门进行了合作，这是来自急性和慢性肝脏疾病的临床样本的宝贵临床样本资源。自从我在2007年加入LID以来，这是我在NIH上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，生化，病毒学和组织病理学数据相结合。 1。急性肝衰竭发病机理的分子机制 与急性肝衰竭（ALF）相比，医学中很少有疾病更具戏剧性和具有挑战性，这是一种临床综合征，其特征是肝功能突然丧失，导致人的多机器人失败而没有肝病。大多数ALF病毒肝炎和药物诱导的肝损伤。尽管目前尚不清楚病毒诱导的ALF的致病机制，但我们证明，在HCV ALF中，肝损伤的程度与病毒复制的大小以及异常均匀的病毒种群相关。相比之下，在暴发性乙型肝炎B中，HBV复制几乎无法检测到或无法检测到的早期和增强对几种病毒抗原的免疫反应。 利用基因表达分析和噬菌体播放文库，我们提供了证据，表明在暴发性丙型肝炎中，肝损伤是由B细胞免疫介导的。通过微阵列处理从两名患有HBV相关的ALF患者（每个肝脏4个样本）和来自8个肝脏供体的单个肝样本的患者获得的肝标本。多元置换分析确定了1,368份在ALF中差异表达的转录本。 709被上调，659下调。 ALF的特征是由与成熟的B细胞和血浆细胞相关的基因和具有丰富多克隆表达的免疫球蛋白基因基因的基因。相比之下，T细胞的T细胞特征有限，并且对T细胞激活的几种抑制剂的上调。免疫组织化学分析证实了突出的B细胞特征，表明血浆爆炸和血浆细胞具有较强的IgM和IgG的血浆细胞，与补体因子的显着沉积有关。分析来自2例ALF患者的肝脏产生的IgG1和IgM的Fab库，将HBCAG鉴定为肝脏产生的抗体的主要靶标。总之，主要针对HBCAG的肝内IG产生的突出的B细胞基因信号与补体沉积有关，这表明体液免疫在HBV相关的ALF的发病机理中具有关键作用。 2。慢性肝病发病机理的分子机制 在慢性乙型肝炎和C的患者中，发生肝硬化的患者比例为30-40％，在慢性丙型肝炎中高达80％。我们长期以来一直有兴趣了解慢性病毒性肝炎患者观察到的不同结果的基础机制。目前，尽管事实证明，感染时代的年龄对慢性乙型肝炎的疾病进展速度产生重大影响，但不可能预测哪些人会维持更多的可怕结果，这在老年人中更快。同样，男性性别与最糟糕的结果有关。为了研究年龄和性别在肝纤维化进展中的作用，我们已经完成了从不同年龄段的个体获得的正常肝脏中基因表达分析的广泛研究。现在，我们正在确定与先天或适应性免疫反应相关的特定基因特征，纤维纤维蛋白发生的增加或纤维蛋白溶解的减少是根据年龄调节的，还是男性和女性之间的基因表达存在显着差异。这项研究的结果可能有助于了解年龄和性别在慢性丙型肝炎的自然史以及其他形式的慢性肝疾病中的影响。 在某些患者中，肝硬化可能是数十年来的稳定疾病，而在另一些患者中，肝硬化可能导致与肝脏相关的死亡或肝细胞癌（HCC）。某些患者最终死于肝肝硬化的并发症而有些患者目前未知的原因。为了解决这些重要问题，我们设计并完成了一项研究，其中严格选择了大量肝脏标本，以代表不同病因的清洁病例（HBV，HBV，HBV+HDV，HCV，HCV+HCV+酒精，酒精，酒精，自免疫）。我们发现，肝硬化的每种亚型的特征都具有特定的基因表达特征，即使在生物学上相关的条件（例如HBV vs. HBV+HDV肝硬化）之间，具有惊人和意外的异质性。基因表达分析将通过使用共聚焦显微镜和测序分析的病毒学研究整合。 3。HCC的发病机理并寻找生物标志物以早期检测HCC。 HCC的临床异质性以及缺乏良好的诊断标记和治疗策略使该疾病成为科学界最重要的挑战之一。任何病因的肝硬化的存在是HCC发展的最强危险因素。尽管已经鉴定出HCC的主要病因学剂，但肝癌发生的机制尚不清楚，很少描述遗传易感性。与意大利卡利亚里肝移植中心的Zamboni博士合作，我们设计了一项研究，研究了从单个肝脏获得的多次活检。我们已经从HCC患者那里收集了500多个肝脏标本，并且已经进行了微阵列分析。该研究目前正在进行中。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。 4。寻找新的肝炎剂。原发性胆道（PBC）肝硬化的原因尚不清楚，但是由于90％的患者中存在针对线粒体蛋白的自身抗体，PBC通常被认为是一种自身免疫性疾病。但是，尚未排除感染力。我们已经确定了一名早期PBC患者（第1阶段）。这项研究的目的是尝试将传染剂从PBC传播到黑猩猩。我们目前正在关注一种与PBC患者血清接种血清的黑猩猩（接种后第77周）。 有趣的是，我们已经观察到EM对线粒体的超微结构改变以及光学显微镜的门户炎性浸润。我们计划通过微阵列检查所有每周的肝活检，以查看与先天免疫相关的基因发生变化，这可能反映了与Mithocondia或其他细胞基因有关的非特异性但普遍的免疫反应。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。