CELLULAR AND MOLECULAR BIOLOGY OF CHOLESTEATOMA

描述（由申请人提供）：耳部胆脂瘤是上皮囊肿样结构，可扩张至邻近骨，导致慢性感染和听力丧失。本研究旨在探讨铜绿假单胞菌（Pseudomonas aeruginosa， PA）感染的胆脂瘤发病机制和毒力中细菌-宿主相互作用的机制。这些细菌在胆脂瘤内形成生物膜，导致慢性感染，对抗生素和宿主防御不起作用。感染胆脂瘤的病理生理是复杂的，涉及细菌因素、下层上皮和邻近骨之间的相互作用。在Specific Aim I中，我们将使用沙鼠实验性胆脂瘤模型来评估从人胆脂瘤中分离的耳致病性PA分离物的发病机制。我们选择了一株具有代表性的菌株OPPA8进行测序和分析。位点定向缺失突变的评估将提供洞察胆脂瘤发展和增强侵袭性所必需的基因。我们还计划通过靶向删除关键毒力基因（galU, algC, algD, lasI, lasR, fliC, exsA）来评估沙鼠模型中许多已知的毒力因子。在Specific Aim II中，我们的目标是阐明PA LPS在宿主损伤和反应中的机制，特别是角化细胞增殖和骨吸收的失调。我们将使用永生化细胞系、原代人和原代小鼠角质形成细胞，在体外研究脂多糖对角质形成细胞增殖和分化的影响。我们将通过评估参与PA LPS介导的增殖和分化的下游效应物，进一步研究PA LPS诱导体外增殖的机制。通过比较PA LPS刺激的TLR2-/-、TLR4-/-和MyD88-/-敲除小鼠角质形成细胞的细胞因子谱，我们希望描述这一过程发生的机制。此外，我们将利用小鼠局部骨吸收模型来评估PA LPS诱导骨吸收的体内机制。我们实验室之前的研究表明，PA LPS通过TLR4、MyD88依赖途径诱导骨吸收，但初步的体内数据也表明TLR2也有作用。了解胆脂瘤中PA的致病性和毒力可能会导致药物治疗或治疗方式的新靶点，从而预防或改善与该疾病相关的听力损失。

DESCRIPTION (provided by applicant): Aural cholesteatomas are epithelial cyst-like structures that expand into adjacent bone, resulting in chronic infection and hearing loss. The overall goal of this proposal is to investigate the mechanisms of bacterial-host interactions in the pathogenesis and virulence of cholesteatomas infected with Pseudomonas aeruginosa (PA). These bacteria form biofilms within the cholesteatoma resulting in chronic infection which is recalcitrant to antibiotics and host defenses. The pathophysiology of infected cholesteatomas is complex involving interactions among bacterial factors, the underlying epithelium and adjacent bone. In Specific Aim I, we will use a gerbil model of experimental cholesteatoma to evaluate the pathogenesis of otopathogenic PA isolates isolated from human cholesteatomas. We have chosen a representative strain, OPPA8 for sequencing and analysis. Evaluation of site directed deletion mutants will provide insight into the genes necessary for cholesteatoma development and enhanced aggressiveness. We also plan to evaluate a number of known virulence factors in the gerbil model by using targeted deletions of key virulence genes (galU, algC, algD, lasI, lasR, fliC, exsA). In Specific Aim II, our goal is to elucidate the mechanisms of PA LPS in host injury and response, specifically deregulated keratinocyte proliferation and bone resorption. We will examine the effect of lipopolysaccharide in the proliferation and differentiation of keratinocytes in vitro using an immortalized cell line, primary human and primary mouse keratinocytes. We will further examine the mechanism of PA LPS induced proliferation in vitro by evaluating downstream effectors involved in PA LPS mediated proliferation and differentiation. By comparing cytokine profiles from PA LPS stimulated keratinocytes derived from TLR2-/-, TLR4-/- and MyD88-/- knockout mice we hope to delineate the mechanism by which this process occurs. Additionally, we will evaluate the mechanisms of PA LPS induced bone resorption in vivo using a murine model of localized bone resorption. Previous studies in our laboratory demonstrated PA LPS induces bone resorption in vitro through a TLR4, MyD88 dependent pathway but preliminary in vivo data also suggests a role for TLR2. An understanding of the pathogenicity and virulence of PA in cholesteatoma may lead to new targets of drug therapy or treatment modalities that will prevent or ameliorate the hearing loss associated with this disease.