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.

该副本是利用资源的众多研究子项目之一由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持而且，副投影的主要研究员可能是其他来源提供的包括其他NIH来源。列出的总费用可能代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。在美国，一小部分感染了螺旋体渗透症伯氏菌（BB）的人会产生一种对抗生素治疗难治性的慢性关节炎。尽管明显消除了螺旋体，但这种情况仍可以持续数月到几年。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有关，这表明宿主对BB的反应在疾病进展中很重要。该项目的目的是确定疾病易感性MHC II类等位基因提出了哪些BB蛋白来源的肽。我们已经建立了HLA-DR *0401和 *0101来自莱姆关节炎患者的纯合细胞系，用于在细胞培养中用作抗原呈现细胞。这些细胞与重组BB蛋白或BB全细胞超声孵育。使用抗HLA-DR抗体对HLA-DR肽结合物进行免疫菌株。肽被酸洗脱，通过离心超滤分离，并通过C18固相灭绝纯化。通过LC-MS/MS分析鉴定纯化的肽，然后进行数据库搜索。作为对我们方法的测试，我们仅在培养基中孵育的HLA-DR*0401细胞中纯化了肽。 LC-MS/MS分析确定了许多自源肽以及一些血清衍生的肽。这些结果表明我们的方法运作良好，我们开始研究BB蛋白。我们的2个患有关节炎但莱姆病和2名莱姆病患者的对照组中有分析性的滑膜组织。使用MS数据库鉴定出抗原呈递的肽，并将四个患者的结果彼此比较，以鉴定可能是莱姆患者和关节炎患者可能特征的表位，并确定是否有特异性特异性肽。施加了严格的标准以确保作业的高可靠性。发现一些肽进行翻译后修饰，并合成适当的修饰肽进行免疫测试。最近发表在分子和细胞蛋白质组学中。当前的研究正在评估患者细胞对初始研究中鉴定的候选抗原的反应。进一步的患者样本正在分析。