CARTILAGE AUTOCATABOLISM AND INFECTIOUS ARTHRITIS

软骨自动代谢和感染性关节炎

• 批准号: 2902540
• 负责人: ROBERT Lane SMITH
• 金额: $ 15.82万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-15 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2902540
• 关键词: SDS polyacrylamide gel electrophoresis; Staphylococcus aureus; Staphylococcus infection; affinity chromatography; animal tissue; antigen antibody reaction; bacterial antigens; biotransformation; blood toxicology; carbohydrate structure; cartilage metabolism; chondrocytes; chronic disease /disorder; extracellular matrix; high performance liquid chromatography; mass spectrometry; medical complication; protein purification; protein sequence; rheumatoid arthritis; tissue /cell culture

Staphylococcal septic arthritis remains a serious clinical problem in very young children, in individuals with chronic rheumatoid arthritis and in immunocompromised patients. The consequence of Staphylococcal joint infection is residual joint destruction often in spite of appropriate antibiotic therapy. The etiology of Staphylococcal joint destruction involves multiple parameters that include the inflammatory response to the bacteria, bacterial expression of virulence factors, the state of the host immune system and bacterial adherence factors. Our studies demonstrate that direct effects of Staphylococcus aureus on cartilage metabolism accelerate extracellular matrix destruction in vitro. The direct effects of S. aureus on cartilage occur through the release of a soluble protein factor (Staph Factor) that triggers chondrocyte-mediated autocatabolism. Staph Factor requires living chondrocytes and induces the expression of matrix metalloproteinases, interleukin-6 and prostaglandin E2 while selectively inhibiting the synthesis of cartilage matrix macromolecules. Staph Factor has been partially purified and the range of its biological activity has been confirmed in vitro and in vivo using cartilage degradation assays and proteoglycan loss in a rabbit knee model. However, the precise moleculer structure of Staph Factor remains unknown. The purpose of this proposal is to address the fundamental question regarding the nature of this potent destructive mediator. Preliminary data suggest that the molecule does not fall into the peptidoglycan or endotoxin superantigen paradigms. The Specific Aim of this proposal is to purify the Staph Factor to homogeneity so that the molecular mechanisms by which Staphylococcus aureus induces cartilage destruction may be further understood. Specific Aim 1 will consist of a Part A that will focus on the characterization of the primary protein sequence of the Staph Factor and a Part B that will focus on the characterization of the carbohydrate modifications of the protein structure. The hypothesis being addressed is that the unique biological properties of Staph Factor derive from the unique molecular structure. The structure of the molecule renders its capacity to induce cartilage destruction resistant to heat, proteolytic attack and glycolytic cleavage. With the advent of greater numbers of antibiotic resistant strains of Staphylococcal organisms, the importance of this work is critical to the fields of pediatric orthopaedics, rheumatology and adult orthopaedics. The activity of Staph Factor on dermal endothelial cells makes it important to dermatology. The laboratory techniques involved will include ultrafiltration, lectin affinity chromatography, FPLC protein chromatography, two-dimensional SDS-PAGE, microsequencing and carbohydrate analysis using mass- spectrophotometry.

葡萄球菌脓毒性关节炎仍然是幼儿、慢性类风湿性关节炎患者和免疫功能低下患者的严重临床问题。 葡萄球菌关节感染的后果是残余关节破坏往往尽管适当的抗生素治疗。葡萄球菌关节破坏的病因学涉及多个参数，包括对细菌的炎症反应、细菌毒力因子的表达、宿主免疫系统的状态和细菌粘附因子。 我们的研究表明，金黄色葡萄球菌对软骨代谢的直接影响加速了体外细胞外基质的破坏。 S.金黄色葡萄球菌对软骨的作用是通过释放一种可溶性蛋白因子（葡萄球菌因子）来触发软骨细胞介导的自体催化剂。葡萄球菌因子需要活的软骨细胞并诱导基质金属蛋白酶、白细胞介素-6和前列腺素E2的表达，同时选择性地抑制软骨基质大分子的合成。 葡萄球菌因子已被部分纯化，其生物活性范围已在体外和体内使用兔膝关节模型中的软骨降解测定和蛋白聚糖损失得到证实。 然而，葡萄球菌因子的精确分子结构仍然未知。 这一建议的目的是解决关于这个强大的破坏性调解人的性质的基本问题。初步数据表明，该分子不属于肽聚糖或内毒素超抗原范式。 本提案的具体目的是将葡萄球菌因子纯化至均一，以便进一步了解金黄色葡萄球菌诱导软骨破坏的分子机制。具体目标1将由A部分和B部分组成，A部分侧重于葡萄球菌因子一级蛋白质序列的表征，B部分侧重于蛋白质结构的碳水化合物修饰的表征。 正在解决的假设是，葡萄球菌因子的独特生物学特性来自独特的分子结构。 该分子的结构使得其诱导软骨破坏的能力对热、蛋白水解攻击和糖酵解裂解具有抗性。 随着越来越多的耐抗生素葡萄球菌菌株的出现，这项工作的重要性对于儿科骨科、风湿病学和成人骨科领域至关重要。葡萄球菌因子对真皮内皮细胞的活性使其在皮肤病学中具有重要意义。 所涉及的实验室技术将包括超滤、凝集素亲和层析、FPLC蛋白层析、二维SDS-PAGE、微测序和使用质谱法的碳水化合物分析。