IDENTIFICATION OF IMMUNOGENIC GLYCOLIPIDS FROM BORRELIA BURGDORFERI

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 研究中心，而研究中心不一定是研究者所在的机构。 莱姆病是由蜱传播的螺旋体伯氏疏螺旋体（Bb）引起的。在美国，一小部分莱姆关节炎患者在接受2 - 3个月的口服和/或IV抗生素治疗后，关节炎持续数月甚至数年，称为难治性莱姆关节炎。在这些患者中，尽管B检测结果为阴性，关节炎仍持续存在。在抗生素后时期，关节液和滑膜组织中的伯氏DNA。这项工作的重点是鉴定一种Bb糖脂，它能诱导莱姆病患者的免疫反应，但在螺旋体杀灭后，这种反应可预见地消失了。为了实现这一目标，我们正在提取和分馏Bb脂质，并使用ELISA检测莱姆病患者的血清或未感染供体的对照血清来测试单个组分。正相色谱法用于初始脂质分级分离。这通过（头基）类别分离脂质。ELISA阳性的那些级分通过反相色谱法亚分级分离，该反相色谱法通过其疏水部分分离脂质。再次通过ELISA测试这些亚级分。在血清反应性的指导下，复合脂质提取物被分级分离，因此离线质谱法可用于鉴定脂质结构，包括免疫原性组分的结构。 我们从Bb中提取总脂质，并通过正相色谱法分离它们。我们分析了脂质馏分MALDI-TOF和ESI qoTOF质谱法，以评估其复杂性，并确定完整的脂质随后串联MS结构鉴定。该方法通过完整质量鉴定了几种已知的Bb脂质，其将用于鉴定螺旋体的可能的免疫原性脂质或脂蛋白。我们还进行了血清ELISA测定酰化胆固醇半乳糖苷（ACG）纯化的脂质馏分，免疫原性糖脂的BB。初步分析表明，这种脂质具有免疫原性，我们现在正在开发一种可靠的ELISA，以评估对这种抗原的抗体反应。描述脂质分析结果的手稿已被接受发表在临床免疫学（Epub 2009年4月2日）上。

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. Lyme disease is caused by the tick-transmitted spirochete Borrelia burgdorferi (Bb). In the United States, a small percentage of patients with Lyme arthritis have persistent arthritis for months or even several years after treatment with 2-3 months of oral and/or IV antibotic therapy, termed antibiotic-refractory Lyme arthritis. In these patients, the arthritis persists despite negative results for B. burgdorferi DNA in joint fluid and synovial tissue in the post-antibiotic period. This work is focused on identification of a Bb glycolipid that induces an immune response in patients with Lyme disease, but the response disappears predictably after spirochetal killing. Towards this goal, we are extracting and fractionating Bb lipids, and testing individual fractions using ELISA assays with sera from Lyme disease patients or control sera from uninfected donors. Normal phase chromatography is employed for the initial lipid fractionation. This separates the lipids by (headgroup) class. Those fractions that are ELISA-positive are sub-fractionated by reversed-phase chromatography, which separates lipids by their hydrophobic moieties. These sub-fractions are again be tested by ELISA. Guided by serum reactivity, the complex lipid extract is being fractionated, so that off-line mass spectrometry can be used to identify the lipid structures, including that of the immunogenic component. We have extracted total lipids from Bb and separated them by normal phase chromatography. We have analyzed lipid fractions by MALDI-TOF and ESI qoTOF mass spectrometry to assess their complexity and to identify intact lipids for subsequent tandem MS structural identification. This approach has identified, by intact mass, several known Bb lipids, which will be used to identify possible immunogenic lipids or lipoproteins of the spirochete. We have also performed serum ELISA assays on acylated cholesteryl galactoside (ACG) purified from lipid fractions, an immunogenic glycolipid of Bb. Preliminary analysis shows that this lipid is immunogenic, and we are now working on the development of a reliable ELISA to assess the antibody response to this antigen. A manuscript describing the results from the lipid analyses has been accepted for publication in Clinical Immunology (Epub Apr 2, 2009).