IMMUNE RESPONSE TO B BURGDORFERI PROTEINS IN LYME ARTHRITIS

莱姆关节炎中对布氏 B 蛋白的免疫反应

基本信息

批准号：
8365544
负责人：
ALLEN C STEERE
金额：
$ 2.92万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-06-01 至 2012-08-09
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8365544
关键词：
Acids Alleles Antibiotic Therapy Antibodies Antigen-Presenting Cells Antigens Arthritis Biology Borrelia burgdorferi Cell Culture Techniques Cell Line Cells Characteristics Chronic Databases Disease Progression Disease susceptibility Epitopes Funding Goals Grant HLA-DR Antigens Immune response Immunologic Tests Incubated Investigation Lyme Arthritis Lyme Disease MHC Class II Genes Mass Spectrum Analysis Medicine Methods Molecular National Center for Research Resources Order Spirochaetales Patients Peptides Persons Phase Post-Translational Protein Processing Principal Investigator Proteins Proteomics Publishing Recombinants Refractory Research Research Infrastructure Resources Sampling Serum Solid Source Testing Tissues Ultrafiltration United States United States National Institutes of Health Ursidae Family Work cost response

项目摘要

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.

该子项目是利用资源的众多研究子项目之一由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持并且子项目的主要研究者可能是由其他来源提供的，包括其他 NIH 来源。子项目可能列出的总成本代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。在美国，一小部分感染伯氏疏螺旋体 (Bb) 的人会患上抗生素治疗难治的慢性关节炎。尽管螺旋体已明显根除，但这种情况仍可能持续数月至数年。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疾病易感性 MHC II 类等位基因呈递哪些 Bb 蛋白衍生肽。我们已经从莱姆关节炎患者建立了 HLA-DR*0401 和 *0101 纯合细胞系，用作细胞培养中的抗原呈递细胞。这些细胞与重组 Bb 蛋白或 Bb 全细胞超声处理一起孵育。使用抗 HLA-DR 抗体对 HLA-DR-肽缀合物进行免疫纯化。肽经酸洗脱、离心超滤分离、C18固相萃取纯化。通过 LC-MS/MS 分析和数据库搜索来鉴定纯化的肽。作为对我们方法的测试，我们从仅在培养基中孵育的 HLA-DR*0401 细胞中纯化了肽。 LC-MS/MS 分析鉴定出许多自体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运行良好，我们已经开始研究 Bb 蛋白。我们分析了 2 名患有关节炎但未患莱姆病的对照者和 2 名莱姆病患者的滑膜组织。使用 MS 数据库鉴定抗原呈递的肽，并将四位患者的结果相互比较，以确定莱姆病患者和关节炎患者可能特有的表位，并确定是否存在等位基因特异性肽。实施严格的标准以确保作业的高度可靠性。一些肽被发现带有翻译后修饰，并且正在合成适当修饰的肽用于免疫学测试。研究结果最近发表在《分子和细胞蛋白质组学》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；正在分析更多患者样本。