Project 2-Three-dimensional modeling of EBV-HPV interactions leading to anogenital cell dysplasia

摘要 感染高危亚型的人乳头瘤病毒(HPV)占所有感染的99%以上 美国的宫颈癌病例。我们与Hagensee博士实验室合作的最新数据显示 宫颈标本中EB病毒和HPV的联合检测增加了并发的风险 宫颈发育不良4-6倍。此外，来自新奥尔良的艾滋病毒阳性妇女(n=531)患有可检测到的宫颈 HPV和EBV并发鳞状上皮内病变的风险(69%)高于 仅有28%的女性仅检测到HPV(P&lt；0.001；OR5.57，95%CI2.19-14.4)。我们假设EB病毒 是HPV相关宫颈癌进展的一个共同因素。我们将直接讨论这一假设 使用创新的三维(3D)上皮细胞联合感染模型。首先，使用一个特色化的3D 终末分化角质形成细胞模型，我们将确定是否合并HPV和EBV感染 与单独使用HPV相比，会对复层多层上皮的正常分化产生不利影响。vbl.使用 有针对性的分子方法，我们将剖析EBV的癌蛋白LMP-1对细胞的增强能力 在高危HPV类型背景下的转变。重要的是，拟议的成果措施还将 在低风险HPV6的背景下解决上皮转化问题，因为HPV6在 女性感染EB病毒的可能性更大，因为EBV的增强能力在其他低风险类型中可能更大。 平行实验将在原代和/或HPV永生化的人宫颈外上皮细胞中进行。 利用Cobre团队所有成员的专业知识，这里使用的创新3D上皮模型将 促进对合并感染EBV是否会增加恶性程度的有力和直接的调查 转型。具体地说，这些模型允许可视化分层的上皮结构和 侵袭下间质层，量化基底细胞增殖，以及一些结果不是 可以用传统的2D(单层)模型来实现。同样是3D模型独一无二的，我们有能力 生产和利用传染性HPV，从而允许与活病毒共同感染，而不是模拟HPV 通过逆转录病毒转导E6/E7癌基因感染。艾莉森·奎尔博士和奥古斯托博士的指导二人组 Ochoa，结合Michelle Ozbun博士和Hagensee博士提供的咨询，将提供极好的 科布雷导师克里斯·麦高文博士的科学领导力。与科布雷的目标一致 在这一机制下，拟议的初级调查员培训导师的综合记录将提供 Chris有一个丰富的环境，有利于将他的发现转化为独立的R01资金。

ABSTRACT Infections with high-risk sub-types of Human Papilloma Virus (HPV) are responsible for more than 99% of all cervical carcinoma cases in the US. Our recent data in collaboration with Dr. Hagensee laboratory has shown that co-detection of Epstein-Barr virus (EBV) with HPV in cervical samples increases the risk of concurrent cervical dysplasia by 4-6 fold. In addition, HIV+ women from New Orleans (n=531) with detectable cervical HPV and EBV are at higher risk (69%) for concurrent squamous intraepithelial lesions (SIL) as compared to only 28% of women with only detectable HPV (p<0.001; OR 5.57, 95%CI 2.19-14.4). We hypothesize that EBV acts as a co-factor for progression of HPV-related cervical cancers. We will address this hypothesis directly using innovative 3-dimensional (3D) epithelial models of co-infection. First, using a well-characterized 3D model of terminally differentiated keratinocytes, we will determine whether co-infection with HPV and EBV adversely affects normal differentiation of the stratified multi-layer epithelium compared to HPV alone. Using targeted molecular methods, we will dissect the augmenting capacity of EBV's oncoprotein, LMP-1, for cellular transformation in the context of high-risk HPV types. Importantly, the proposed outcome measures will also address epithelial transformation in the context of the low-risk HPV6 because HPV6 is found more frequently in women shedding EBV, and since EBV's augmenting capacity might be greater in otherwise low-risk types. Parallel experiments will be performed in primary and/or HPV-immortalized human ectocervical epithelial cells. Utilizing expertise from all members of the COBRE team, the innovative 3D epithelial models utilized herein will facilitate a powerful and direct investigation of whether co-infection with EBV enhances malignant transformation. Specifically, these models allow for visualization of the stratified epithelial architecture and invasion of the underlying stromal layer, quantification of basal cell proliferation, and several outcomes not achievable with conventional 2D (monolayer) models. Also unique to the 3D model, we have the ability to produce and utilize infectious HPV thus allowing for co-infection with live viruses rather than modeling HPV infection by retroviral transduction of E6/E7 oncogenes. The mentoring duo of Drs. Alison Quayle and Augusto Ochoa, combined with consulting provided by Drs. Michelle Ozbun and Hagensee, will provide excellent scientific leadership to proposed COBRE mentee Dr. Chris McGowin. In line with the goals of the COBRE mechanism, the combined track record of the proposed mentors for training of junior investigators will provide Chris a rich environment conducive for translating his findings into independent R01 funding.