IDENTIFICATION OF TREATMENT-RESISTANT LYME ARTHRITIS AUTOANTIGENS

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。所列机构为 中心，不一定是研究者的机构。 抗生素治疗抗性莱姆关节炎的特征在于在伯氏疏螺旋体感染明显根除后，受影响关节的滑膜组织中的B-T细胞聚集体和浆细胞簇。怀疑关节中的这种细胞活性是由于自身反应性B细胞的激活以及随后自身抗体的产生。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。从受累关节显微切割的浆细胞体外培养，并从细胞上清液中纯化抗体。将这些潜在的自身抗体与琼脂糖珠缀合，并用于从滑液中亲和纯化抗原蛋白。将蛋白质从珠上洗脱并通过SDS-PAGE分离。从凝胶上切下蛋白条带，并进行凝胶内胰蛋白酶消化。通过MALDI-TOF质谱法分析从凝胶内酶中提取的肽用于蛋白质鉴定。我们已经确定了一个细胞角蛋白，这是目前在一个样本，但不是在对照空白凝胶切片。我们已经通过Western blot证实了这一发现，并继续在其他样品中寻找其他蛋白质。描述我们结果的手稿最近已经出版。

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 have confirmed the finding by Western blot and continue to search for other proteins in additional samples. A manuscript describing our results has recently been published.