HEPATITIS C VIRUS IN ETIOLOGY OF WA RHEUMATOID FACTORS

我们建立了丙型肝炎病毒(HCV)感染与II型冷球蛋白血症(MC-II)和WA交叉型(XID)阳性的单抗类风湿因子(WA MRF)的关系，以及这些冷球蛋白中丙型肝炎病毒(HCV)和极低密度脂蛋白(VLDL)的选择性浓度。我们还证实，低密度脂蛋白受体介导了丙型肝炎病毒和黄病毒科其他成员的内吞作用，并且丙型肝炎病毒感染患者的载脂蛋白E epsilon2等位基因使发生MC的风险增加了三倍。这项建议的广泛、长期目标是调查MC-II如何产生WA MRF，以及它们如何影响慢性丙型肝炎病毒感染。将检验两个假设：1)在与丙型肝炎病毒感染相关的MC-II患者中，Wa MRF是由于丙型肝炎病毒和极低密度脂蛋白复合体对B细胞的慢性刺激而产生的。根据这一假设，最初产生了WA+RF-IgM，类风湿因子活性是慢性丙型肝炎病毒感染的CDR3点突变的结果。将确定：a)WA+RF-IgM是否具有抗丙型肝炎病毒极低密度脂蛋白的抗体活性，b)WA MRF与同一抗原发生交叉反应，c)产生WA MRF-IgM的细胞是产生WA MRF+IgM的前体细胞。丙型肝炎病毒感染的混合性低温球蛋白血症患者的肝活检组织中的淋巴聚集物将被检测是否存在WA+RF-和WA+RF+B细胞，并将结果与来自相同肝活检组织和配对外周血的WA序列的DNA和mRNA分析进行比较。2)低密度脂蛋白受体内吞作用是肝细胞感染丙型肝炎病毒的主要途径。WA抗体的主要生理作用是阻断低密度脂蛋白受体对丙型肝炎病毒极低密度脂蛋白复合体的内吞作用。通过低密度脂蛋白受体抑制含载脂蛋白E2的丙型肝炎病毒-极低密度脂蛋白复合体的内吞作用是导致载脂蛋白E2发生MC-II的危险因素的机制。采用流式细胞术、原位杂交和定量聚合酶链式反应等方法研究丙型肝炎病毒-脂蛋白复合体的内吞作用，以确定a)不同载脂蛋白E表型和不同丙型肝炎病毒基因型的丙型肝炎病毒极低密度脂蛋白的内吞速率和b)WA MRF和WA+RF-IgM对内吞速率的影响。此外，还将确定脂蛋白浓度、载脂蛋白E表型和丙型肝炎病毒基因型别对丙型肝炎病毒感染者和无冷球蛋白血症患者中丙型肝炎病毒在脂蛋白中分布的作用，以及对MC-II中丙型肝炎病毒对极低密度脂蛋白的选择性浓度的影响。这些研究可能会对MC-II的病原学、丙型肝炎病毒感染的机制以及天然抗体系统在丙型肝炎免疫应答中的作用提供见解，并可能导致更好的治疗、早期发现和预防。

We have established the association of hepatitis C virus (HCV) infection with type II cryoglobulinemia (MC-II) and with the WA crossidiotype (XId) positive monoclonal rheumatoid factors (WA mRF), and the selective concentration of HCV and very low density lipoprotein (VLDL) in these cryoglobulins. We have also established that the LDL receptor mediates endocytosis of HCV and other members of the Flaviviridae family and that the apolipoprotein E epsilon2 allele in HCV infected patients increases the risk of developing MC three-fold. The broad, long-term objective of this proposal is to investigate how WA mRF are produced in MC-II and how they may affect chronic HCV infection. Two hypotheses will be tested: 1) In patients with MC-II associated with HCV infection, the WA mRF is produced as a result of chronic stimulation of B cells by complexes of HCV and VLDL. It is postulated from this hypothesis that initially a WA+ RF- IgM is produced and that rheumatoid factor activity arises as a result of a point mutation in the CDR3 with chronic HCV infection. It will be determined whether: a) WA+ RF- IgM has antibody activity to HCV VLDL, b) WA mRFs ve cross reactivity with the same antigen, and c) cells producing WA mRF- IgM are the precursors of those producing WA mRF+ IgM. Lymphoid aggregates in liver biopsies from HCV infected patients with mixed cryglobulinemia will be examined for the presence of WA+ RF- and WA+ RF+ B cells and the results compared to DNA and mRNA analysis for WA sequences from the same liver biopsies and paired peripheral bloods. 2) LDL receptor endocytosis is a major route of HCV infection of hepatocytes. The main physiologic role of WA antibodies is to block endocytosis of HCV VLDL complexes by the LDL receptors. The retarded endocytosis of HCV-VLDL complexes containing apolipoprotein E2 via the LDL receptor is the mechanism underlying the apo E2 risk factor for developing MC-II. Flow cytometry, in situ hybridization, and quantitative PCR assays will be used to study the endocytosis of HCV-lipoprotein complexes to determine a) the rate of endocytosis of HCV-VLDL of various apo E phenotypes and various HCV genotypes and b) the effect of WA mRF and WA+ RF-IgM on the rates of endocytosis. In addition, the role of lipoprotein concentration, apo E phenotypes and HCV genotypes on the distribution of HCV among lipoproteins in HCV infected individuals with and without cryoglobulinemia and on the selective concentration on VLDL with HCV in MC-II will be determined. The proposed studies may provide insights into the etiology of MC-II, the mechanism of HCV infection, and the role of natural antibody systems in the immune response to HCV, and may lead to better therapy, early detection and prophylaxis of the disease.