IDENTIFICATION OF TREATMENT-RESISTANT LYME ARTHRITIS AUTOANTIGENS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。抗生素治疗难治性莱姆关节炎的特征是在伯氏疏螺旋体感染明显消除后，患病关节滑膜组织中的B-T细胞聚集体和浆细胞群。据推测，关节中的这种细胞活动是由于自身反应性B细胞的激活和随后产生的自身抗体所致。这项工作的重点是从滑膜组织中鉴定潜在的自身抗原蛋白。体外培养病变关节的显微解剖浆细胞，从细胞上清液中提纯抗体。这些潜在的自身抗体被偶联到琼脂糖珠上，并用于从滑液中亲和纯化抗原蛋白。蛋白质从小球上洗脱下来，用十二烷基硫酸钠-PAGE分离。从凝胶中切下蛋白条带，进行凝胶内胰酶消化。用MALDI-TOF质谱仪对从凝胶内消化中提取的多肽进行蛋白质鉴定。我们已经确定了一种细胞角蛋白，它存在于一个样本中，但不存在于对照空白凝胶切片中。我们目前正在通过蛋白质印迹来确认这一发现，并继续在其他样本中寻找其他蛋白质。

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. Antibiotic treatment-resistant Lyme arthritis is characterized by B-T cell aggregates and plasma cell clusters in synovial tissue from affected joints, after apparent eradication of Borrelia burgdorferi infection. It is suspected that this cellular activity in the joint is a due to activation of self-reactive B cells with subsequent production of autoantibodies. This work focuses on identification of potential self antigenic proteins from synovial tissue. Microdissected plasma cells from affected joints were cultured in vitro and the antibodies were purified from the cell supernates. These potential autoantibodies were conjugated to agarose beads and used to affinity purify antigenic proteins from synovial fluid. Proteins were eluted from the beads and separated by SDS-PAGE. Protein bands were excised from gels and subjected to in-gel trypsin digestion. Peptides extracted from the in-gel digests were analyzed by MALDI-TOF mass spectrometry for protein identification. We have identified a cytokeratin that was present in one sample, but not in control blank gel slices. We are currently in the process of confirming the finding by western blot as well as continuing to search for other proteins in additional samples.