IMMUNE RESPONSE TO B BURGDORFERI PROTEINS IN LYME ARTHRITIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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。这些细胞将与重组BB蛋白或BB全细胞超声波孵育。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，我们已经从单独在培养基中孵育的人类白细胞抗原-DR*0401细胞中纯化了多肽。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。我们表达了13C6-赖氨酸标记的重组BB外表面蛋白A(OspA)。选择碳-13作为标记物是因为它对液相色谱保留时间的影响最小。之所以选择赖氨酸，是因为OspA序列包含大量赖氨酸残基，均匀分布在整个蛋白质中。每个结合的赖氨酸残基正好增加了6个道尔顿的多肽的质量。当标记和未标记的OspA-MBP混合在一起并加入到抗原提呈细胞中时，经处理的多肽可以通过质谱学很容易地以恰好6Da的间隔分隔成对。然后，在自动获取LC MS数据期间，对这些对进行序列分析。我们现在已经进展到对患者来源的样本的分析。

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 will be 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, 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 begun to study Bb proteins. We have expressed recombinant Bb outer surface protein A (OspA) which is labeled with 13C6-lysine. Carbon-13 was chosen as a label since it has a minimal effect on liquid chromatographic retention time. Lysine was selected because the OspA sequence contains numerous lysine residues well distributed throughout the protein. Each incorporated lysine residue adds exactly 6 Daltons to the mass of the peptide. When labeled and unlabeled OspA-MBP are mixed together and added to antigen-presenting cells, the processed peptides can be readily identified by mass spectrometry as pairs separated by intervals of exactly 6 Da. These pairs are then subjected to sequence analysis during automated LCMS data acquisition. We have now progressed to the analysis of patient-derived samples.