IMMUNE RESPONSE TO B BURGDORFERI PROTEINS IN LYME ARTHRITIS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 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。在我们最初的实验中，我们表达了重组Bb外表面蛋白A（OspA），它已标记与13 C6-赖氨酸。选择赖氨酸是因为OspA序列含有大量在整个蛋白质中均匀分布的赖氨酸残基。 当标记的和未标记的OspA-MBP混合在一起，并添加到抗原呈递细胞，处理的肽可以很容易地通过质谱鉴定为间隔正好为6 Da的对。这些对，然后进行自动LCMS数据采集过程中的序列分析。然后，我们进行了患者来源的样品的分析，并分析了来自2名患有关节炎但没有莱姆病的对照和2名莱姆病患者的滑膜组织。正在使用MS数据库鉴定抗原呈递肽，并将四名患者的结果相互比较，以鉴定可能是莱姆关节炎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。正在实施严格的标准，以确保任务的高度可靠性。已经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。一份手稿即将在《分子和细胞蛋白质组学》上发表。姚博士已被邀请在犹他湖城举行的2010年ASMS会议上对结果进行口头介绍。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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. In our initial experiments, we expressed recombinant Bb outer surface protein A (OspA) which had been labeled with 13C6-lysine. Lysine was selected because the OspA sequence contains numerous lysine residues well distributed throughout the protein. When labeled and unlabeled OspA-MBP were mixed together and added to antigen-presenting cells, the processed peptides could be readily identified by mass spectrometry as pairs separated by intervals of exactly 6 Da. These pairs were then subjected to sequence analysis during automated LCMS data acquisition. We then progressed to the analysis of patient-derived samples and have analyzed synovial tissue from 2 controls who have arthritis but not Lyme disease and from 2 Lyme disease patients. The antigen-presented peptides are being identified using MS databases and the results for the four patients are being 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 are being imposed to assure high reliability in the assignments. Some peptides have been found to bear post-translational modifications and the appropriately modified peptides are being synthesized for immunological testing. A manuscript is nearly ready to be submitted for publication in Molecular and Cellular Proteomics. Dr. Yao has been invited to give an oral presentation of the results at the 2010 ASMS meeting in Salt lake City, UT.