Pathogenesis of Parenteral Viral Hepatitis

我们正在研究病毒性肝炎的发病机制，以及这些重要病原体毒力和减毒的分子基础。在与斯克里普斯研究所Frank Chisari博士的合作研究中，我们在黑猩猩身上研究了宿主清除乙肝病毒感染的机制，以及这些机制与临床疾病的关系。我们证明，主要由肝脏中的干扰素-伽玛干扰素介导的非细胞溶解机制可以消除肝细胞中用于合成乙肝病毒的模板--共价闭合环状HBVDNA的清除。残留的含有乙肝病毒抗原的肝细胞的清除是由细胞溶解的CD8阳性T细胞介导的较晚的事件，并且与感染的肝炎阶段有关。通过对黑猩猩在乙肝感染过程中上调和下调的肝内信使RNA的微阵列分析，进一步研究了与急性乙肝相关的病毒诱导和免疫反应相关基因的谱。令人惊讶的是，我们没有检测到对感染的先天免疫反应的证据，这表明乙肝病毒可以颠覆宿主免疫反应，但我们确实在感染的清除阶段检测到了强烈的适应性免疫反应；这与上面描述的抑制病毒复制和清除感染细胞相关。有趣的是，在对黑猩猩慢性乙肝病毒感染的其他研究中，我们发现了一种先天免疫反应，这种免疫反应在急性、自限性的乙肝病毒感染中缺失。这发生在肝脏受损的时候，这表明死亡和濒临死亡的肝细胞释放的病毒和细胞产物可能会触发其他固有的宿主防御，如TLR-3。然而，上调的先天免疫反应很弱，不足以影响病毒复制。 丙型肝炎病毒的遗传异质性被认为在其致病性中起着重要作用。我们以前曾通过确定从输血后感染的患者身上恢复的丙型肝炎病毒分离株的遗传异质性来检验这种关系，以便研究感染的早期阶段，以及从接受干扰素治疗的患者那里恢复的丙型肝炎病毒分离株的遗传异质性，以便研究慢性感染后期的变化。在感染的最初几周内，观察到病毒克隆序列的动态变化的独特模式，这些变化与感染的结果相关。同样，干扰素治疗过程中序列的动态变化模式可以预测结果。这些发现可能有助于在疗程早期预测干扰素治疗的结果。 虽然从这些对患者的纵向研究中已经获得了大量的信息，但很难研究这些系统的发病机制。黑猩猩是除人类外唯一易感染丙型肝炎病毒的动物，为研究宿主和病毒在丙型肝炎发病机制中的相互作用提供了实验模型。与Frank Chisari的合作研究表明，就像在乙肝病毒感染中一样，在丙型肝炎病毒感染中，细胞免疫反应在非溶细胞性下调病毒复制和清除残留感染细胞方面发挥着重要作用。这两种机制是相继和重叠的，前者似乎是由干扰素γ介导的，后者是由CD8阳性细胞介导的，可能是通过其促炎活性而由干扰素伽马介导的。这些研究还表明，1型干扰素(干扰素α/β)激活的抗病毒蛋白在病毒感染时表达，但丙型肝炎病毒对这种先天免疫反应的抗病毒活性具有抵抗力。对病毒性肝炎的宿主免疫反应以及肝炎病毒如何试图绕过这些反应的微阵列研究，正在产生关于这些疾病发病机制的重要信息，这些研究正在扩展到其他肝炎病毒，以描绘这些因素在单一宿主--黑猩猩--中的比较发病机制，这是唯一对所有人类肝炎病毒敏感的非人类宿主。 目前，我们还在通过微阵列分析研究HDV感染的发病机制，并已确定了对此类感染的先天和获得性免疫反应。对HDV感染的免疫反应与我们在丙型肝炎病毒感染期间观察到的类似：既有很强的先天免疫反应，也有很强的获得性免疫反应。先天免疫反应足以下调重叠感染HDV的慢性感染乙肝病毒的黑猩猩体内的乙肝病毒复制。因此，尽管乙肝病毒不能触发先天免疫反应，但当由另一种病毒提供时，它对这种反应高度敏感。

We are studying the pathogenesis of viral hepatitis and the molecular basis for virulence and attenuation of these important pathogens. In collaborative studies with Dr. Frank Chisari (Scripps Institute) we have studied in chimpanzees the mechanism by which the host clears a hepatitis B virus infection and the relationship of these mechanisms to clinical disease. We demonstrated that the clearance of the template for HBV synthesis, covalently closed circular HBV DNA, is eliminated from hepatocytes by non-cytolytic mechanisms mediated principally by interferon gamma in the liver. Elimination of residual hepatocytes containing HBV antigens is a later event that is mediated by cytolytic CD8 positive T cells and is temporally related to the hepatitis phase of the infection. The spectrum of virus-induced and immune response-related genes involved in acute hepatitis B were further studied by microarray analysis of intrahepatic messenger RNAs up-regulated and down-regulated during the course of hepatitis B infections in chimpanzees. Surprisingly, we could not detect evidence of an innate immune response to infection, suggesting that HBV can subvert the host immune response, but we did detect a strong adaptive immune response during the clearance phase of infection; this correlated with the inhibition of viral replication and removal of infected cells described above. Interestingly, in additional studies of chronic HBV infection in chimpanzees, we found an innate immune response that was missing in acute, self-limiting infections of HBV. This occurred at a time of liver damage, suggesting that viral and cellular products released from dead and dying hepatocytes could trigger other innate host defenses, such as TLR-3. However, the up-regulated innate immune responses were weak and insufficient to affect virus replication. The genetic heterogeneity of hepatitis C virus is believed to play an important role in its pathogenicity. We have previously examined this relationship by determining the genetic heterogeneity of HCV isolates that were recovered from patients who were infected following transfusion in order to study the early phase of infection and from patients undergoing interferon therapy in order to study changes during the later phase of chronic infection. Distinctive patterns of dynamic change in the sequence of viral clones during the first several weeks of infection were observed and these correlated with the outcome of infection. Similarly, the pattern of dynamic changes in sequence during interferon therapy was predictive of the outcome. These findings may be useful in predicting the outcome of therapy with interferon early in the course of treatment. Although considerable information has been gained from these longitudinal studies of patients, it is difficult to study the mechanisms of pathogenesis in such systems. Chimpanzees, which are the only animals other than man that are susceptible to infection with HCV, provide an experimental model for studying the interactions of the host and the virus in the pathogenesis of hepatitis C. Collaborative studies with Frank Chisari have demonstrated that, as in hepatitis B virus infections, in hepatitis C virus infections the cellular immune response plays an important role in noncytolytic down-regulation of viral replication and cytolytic removal of residual infected cells. These two mechanisms are sequential and overlapping and the former appears to be mediated by interferon gamma and the latter by CD8 positive cells and, perhaps by interferon gamma through its proinflammatory activity. These studies have also revealed that type 1 interferon (interferon alpha/beta)-activated antiviral proteins are expressed in response to the viral infections, but that HCV is resistant to the antiviral activity of this innate immune response. Microarray studies of the host immune responses to viral hepatitis and how the hepatitis viruses attempt to circumvent the responses, are yielding important information on pathogenesis of these diseases, and the studies are being extended to the other hepatitis viruses in order to delineate the comparative pathogenesis of these agents in a single host, the chimpanzee, which is the only non-human host that is susceptible to all human hepatitis viruses. Currently, we are also studying the pathogenesis of HDV infections by microarray analysis and have identified both innate and adaptive immune responses to such infections. Immune responses to HDV infection were similar to those we observed during HCV infection: both a strong innate and a strong adaptive immune response. The innate immune response was sufficient to down-regulate HBV replication in HBV chronically infected chimpanzees that were superinfected with HDV. Thus, HBV, although unable to trigger an innate immune response, is highly sensitive to such a response when provided by another virus.