CELLULAR AND MOLECULAR BIOLOGY OF CHOLESTEATOMA

描述（由申请人提供）：耳源性中耳炎是一种上皮囊肿样结构，扩张到邻近的骨骼中，导致慢性感染和听力丧失。本研究旨在探讨铜绿假单胞菌（PA）感染中耳瘤的致病机制和致病力。这些细菌在中耳瘤内形成生物膜，导致对抗生素和宿主防御无效的慢性感染。感染性中耳瘤的病理生理学是复杂的，涉及细菌因素、下层上皮和邻近骨之间的相互作用。在特定目标I中，我们将使用实验性中耳瘤的沙鼠模型来评估从人类中耳瘤中分离的耳病原性PA分离株的发病机制。我们选择了具有代表性的菌株OPPA 8进行测序和分析。定点缺失突变的评估将提供深入了解必要的基因为中耳炎的发展和增强的侵略性。我们还计划通过使用关键毒力基因（galU，algC，algD，lasI，lasR，fliC，exsA）的靶向缺失来评估沙鼠模型中的一些已知毒力因子。在特定目标II中，我们的目标是阐明PA LPS在宿主损伤和反应中的机制，特别是失调的角质形成细胞增殖和骨吸收。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，研究脂多糖在体外角质形成细胞增殖和分化中的作用。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来进一步研究PA LPS诱导体外增殖的机制。通过比较来自PA LPS刺激的源自TLR 2-/-、TLR 4-/-和MyD 88-/-敲除小鼠的角质形成细胞的细胞因子谱，我们希望描绘该过程发生的机制。此外，我们将使用小鼠局部骨吸收模型评估PA LPS诱导体内骨吸收的机制。我们实验室先前的研究表明PA LPS通过TLR 4，MyD 88依赖性途径在体外诱导骨吸收，但初步的体内数据也表明TLR 2的作用。了解PA在中耳炎中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，从而预防或改善与这种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.