IMMUNE RESPONSE TO B BURGDORFERI PROTEINS IN LYME ARTHRITIS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 在美国，感染伯氏疏螺旋体(BB)的一小部分人会患上对抗生素治疗无效的慢性关节炎。这种情况可以持续几个月到几年，尽管螺旋体显然已经根除。抗生素治疗难治性莱姆关节炎与MHC II类等位基因人类白细胞抗原DR*0401和人类白细胞抗原DR*0101相关，这表明宿主对BB的反应在疾病进展中很重要。该项目的目标是确定哪些BB蛋白衍生的多肽是由疾病易感性MHC II类等位基因呈现的。我们已经建立了来自莱姆关节炎患者的人类白细胞抗原-DR*0401和*0101纯合子细胞系，用于在细胞培养中作为抗原提呈细胞。这些细胞与重组BB蛋白或BB全细胞超声酸盐孵育。使用抗人类白细胞抗原DR抗体免疫纯化人类白细胞抗原DR多肽结合物。多肽经酸洗脱，离心超滤分离，C18固相萃取纯化。通过LC-MS/MS分析和数据库检索对纯化的多肽进行鉴定。作为对我们方法的测试，我们从单独培养的人类白细胞抗原-DR*0401细胞中提纯了多肽。LC-MS/MS分析鉴定了大量的自体源多肽和一些血清来源的多肽。这些结果表明，我们的方法工作良好，我们已经开始研究BB蛋白。我们从2名患有关节炎但没有莱姆病的对照组和2名莱姆病患者身上提取了滑膜组织。利用MS数据库对抗原呈递的多肽进行鉴定，并将四名患者的结果相互比较，以确定可能是莱姆病患者和关节炎患者特有的表位，并确定是否存在等位基因特异性多肽。实施了严格的标准，以确保任务的高度可靠性。一些多肽被发现具有翻译后修饰，适当修饰的多肽正在合成中用于免疫学测试。这些结果最近发表在《分子和细胞蛋白质组学》杂志上。目前的研究正在评估患者细胞对初步调查中确定的候选抗原的反应；进一步的患者样本正在分析中。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. In the United States, a small percentage of persons infected with the spirochete Borrelia burgdorferi (Bb) develop a chronic arthritis that is refractory to antibiotic treatment. This condition can last for months to years despite apparent eradication of the spirochete. Antibiotic treatment refractory Lyme arthritis is associated with the MHC class II alleles HLA-DR*0401 and HLA-DR*0101, which suggests that the host response to Bb is important in disease progression. The goal of this project is to determine which Bb protein-derived peptides are presented by the disease susceptibility MHC class II alleles. We have established HLA-DR*0401 and *0101 homozygous cell lines from Lyme arthritis patients for use as antigen-presenting cells in cell culture. These cells are incubated with recombinant Bb proteins or Bb whole cell sonicates. HLA-DR-peptide conjugates are immunopurified using anti-HLA-DR antibodies. Peptides are acid eluted, separated by centrifugal ultrafiltration, and purified by C18 solid-phase exraction. Purified peptides are identified by LC-MS/MS analysis followed by database searching. As a test of our methods, we have purified peptides from HLA-DR*0401 cells incubated in media alone. LC-MS/MS analysis identified numerous self-derived peptides as well as some serum-derived peptides. These results indicate that our methods are working well and we have proceeded to study Bb proteins. We have anakyzed synovial tissue from 2 controls who have arthritis but not Lyme disease and from 2 Lyme disease patients. The antigen-presented peptides were identified using MS databases and the results for the four patients were compared to one another to identify epitopes that may be characteristic for the Lyme patients and for the arthritis patients, and to determine whether there are allele-specific peptides. Rigorous criteria were imposed to assure high reliability in the assignments. Some peptides were found to bear post-translational modifications and the appropriately modified peptides are being synthesized for immunological testing. Ther esults were recently published in Molecular and Cellular Proteomics. Current studies are evaluating the response of patient cells to candidate antigens identified in the initial investigation; further patient samples are being analyzed.