HEPATITIS C VIRUS IN ETIOLOGY OF WA RHEUMATOID FACTORS

我们已经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交叉二型（XId）阳性单克隆类风湿因子（WA mRF）的关联，以及这些冷球蛋白中HCV和极低密度脂蛋白（VLDL）的选择性浓度。我们还证实LDL受体介导HCV和黄病毒科其他成员的内吞作用，并且HCV感染患者的载脂蛋白E ε 2等位基因使发生MC的风险增加了三倍。该提案的广泛、长期目标是研究WA mRF在MC-II中是如何产生的，以及它们如何影响慢性HCV感染。两种假设将被验证：1)在与HCV感染相关的MC-II患者中，WA mRF是由于HCV和VLDL复合物对B细胞的慢性刺激而产生的。根据这一假设，最初会产生WA+ RF- IgM，类风湿因子活性是慢性HCV感染时CDR3点突变的结果。将确定：a) WA+ RF- IgM是否对HCV VLDL具有抗体活性，b) WA mRF与同一抗原具有交叉反应性，c)产生WA mRF- IgM的细胞是产生WA mRF+ IgM的细胞的前体。HCV感染合并混合低温球蛋白血症患者的肝活检中，将检测WA+ RF-和WA+ RF+ B细胞的存在，并将结果与来自相同肝活检和配对外周血的WA序列的DNA和mRNA分析进行比较。2) LDL受体内吞作用是HCV感染肝细胞的主要途径。WA抗体的主要生理作用是阻断LDL受体对HCV VLDL复合物的内吞作用。含载脂蛋白E2的HCV-VLDL复合物通过LDL受体的滞吞作用是发生MC-II的载脂蛋白E2危险因素的潜在机制。流式细胞术、原位杂交和定量PCR技术将用于研究HCV-脂蛋白复合物的内吞作用，以确定a)不同载脂蛋白E表型和不同HCV基因型的HCV- vldl的内吞率；b) WA mRF和WA+ RF-IgM对内吞率的影响。此外，还将确定脂蛋白浓度、载脂蛋白E表型和HCV基因型对HCV在低温球蛋白血症和非低温球蛋白血症HCV感染个体中脂蛋白分布的影响，以及MC-II中HCV对VLDL的选择性浓度的影响。这些建议的研究可能为MC-II的病因、HCV感染的机制以及天然抗体系统在HCV免疫反应中的作用提供见解，并可能导致更好的治疗、早期发现和预防疾病。

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.