IMMUNE RESPONSE TO B BURGDORFERI PROTEINS IN LYME ARTHRITIS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 在美国，一小部分感染伯氏疏螺旋体（Bb）的人会发展成抗生素治疗无效的慢性关节炎。这种情况可以持续数月至数年，尽管明显根除螺旋体。抗生素治疗难治性莱姆关节炎与MHC II类等位基因HLA-DR*0401和HLA-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蛋白衍生肽由疾病易感性MHC II类等位基因呈递。我们已经建立了HLA-DR*0401和 *0101纯合细胞系从莱姆关节炎患者作为抗原呈递细胞在细胞培养。将这些细胞与重组Bb蛋白或Bb全细胞超声处理物一起孵育。使用抗HLA-DR抗体免疫纯化HLA-DR-肽缀合物。将肽酸洗脱，通过离心超滤分离，并通过C18固相萃取纯化。通过LC-MS/MS分析，随后进行数据库检索，鉴定纯化的肽。 作为我们方法的测试，我们已经从单独在培养基中孵育的HLA-DR*0401细胞中纯化了肽。LC-MS/MS分析鉴定了许多自身衍生的肽以及一些血清衍生的肽。这些结果表明，我们的方法工作良好，我们已经着手研究Bb蛋白。我们有2例关节炎但无莱姆病的对照组和2例莱姆病患者的滑膜组织。使用MS数据库鉴定抗原呈递肽，并将四名患者的结果相互比较，以鉴定可能是莱姆病患者和关节炎患者特征的表位，并确定是否存在等位基因特异性肽。严格的标准是为了确保任务的高度可靠性。发现一些肽具有翻译后修饰，并且正在合成适当修饰的肽用于免疫学测试。这些结果最近发表在Molecular and Cellular Proteomics上。目前的研究正在评估患者细胞对初步研究中确定的候选抗原的反应;正在分析更多的患者样本。

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.