CARTILAGE AUTOCATABOLISM AND INFECTIOUS ARTHRITIS

葡萄球菌感染性关节炎在非常年幼的儿童、慢性类风湿性关节炎患者和免疫功能低下患者中仍然是一个严重的临床问题。葡萄球菌性关节感染的后果是残余的关节破坏，尽管适当的抗生素治疗。葡萄球菌破坏关节的病因涉及多个参数，包括对细菌的炎症反应、细菌表达毒力因子、宿主免疫系统状态和细菌粘附因子。我们的研究表明，在体外，金黄色葡萄球菌对软骨代谢的直接作用加速了细胞外基质的破坏。金黄色葡萄球菌对软骨的直接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分解代谢。葡萄球菌因子需要活的软骨细胞，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通过兔膝关节模型的软骨降解试验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尚不清楚。这项建议的目的是解决有关这一强有力的破坏性调解人的性质的根本问题。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纯化葡萄球菌因子至均匀性，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。特异性目标1将包括a部分，重点描述葡萄球菌因子的初级蛋白序列，B部分将重点描述蛋白质结构的碳水化合物修饰。本文的假设是葡萄球菌因子独特的生物学特性来源于其独特的分子结构。分子的结构使其能够诱导软骨破坏，抵抗热，蛋白水解攻击和糖酵解裂解。随着葡萄球菌耐药菌株数量的增加，这项工作的重要性对儿科骨科、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研究对皮肤病学具有重要意义。涉及的实验室技术将包括超滤，凝集素亲和层析，FPLC蛋白质层析，二维SDS-PAGE，微测序和使用质量分光光度法的碳水化合物分析。

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.