In vivo Models of Defensin Activity

描述（由申请人提供）： 昆虫、低等脊椎动物和哺乳动物揭示了一种以前未被认识的 动物宿主防御的组成部分。抗菌肽是基因编码的 对多种微生物有活性的抗生素。防御素是 哺乳动物中这类肽的主要家族。我们小组和其他人的研究 已经发现防御素肽由哺乳动物粘膜表达， 上皮细胞，使它们有能力参与当地宿主 防御虽然肠上皮是一个持续接触的表面， 由于管腔内容物充满微生物，感染是不常见的。我们 基本假设是，在人类中，上皮防御素HD5和HD6 有助于肠粘膜的抗微生物防御。在这份赠款中， 建议，我们将测试上皮抗菌剂的生物学功能， 通过在小鼠中转基因表达HD5和HD6肽在体内表达HD5和HD6肽。 我们认为这些人防御素的转基因表达可以为小鼠提供 具有增强的抵抗细菌挑战的能力。根据初步 我们建立的转基因小鼠的研究，这里描述的实验将 更清楚地了解人类抗菌素的贡献 肽对宿主先天防御的作用 为了验证我们的假设，目标1将分析 转基因小鼠中人HD5表达。我们将描述 HD5转基因小鼠，与对照野生型小鼠相比，抵抗肠 感染鼠伤寒沙门氏菌（目标1A），平行实验将 扩展到其他肠道病原体（目标1B）。我们将研究 转基因HD5在小鼠肠道常驻菌群上的表达（目的 1C）。转基因表达所贡献的抗微生物活性将是 体外定量，包括分离的隐窝分析（Aim 1D）。HD6 基因和肽与HD5共享很少的序列同一性，但它们表达 在Paneth细胞中。在目标2中，我们将产生HD6转基因小鼠（Aim 2A）使用重组HD6开发用于免疫测定的抗体（Aim 2B），和 在基因和蛋白水平上表征HD6的转基因表达 (Aim 2C）。然后，我们将表征转基因HD6表达对 对肠道细菌定植和感染的抗性（Aim 2D） 目标1中开发的方法。最后，通过血统杂交， 建立HD5/HD6复合转基因小鼠，以确定这两种肽是否具有 体内协同活性（Aim 2E）。拟议的调查和其他 先天免疫的研究，可能会提供新的治疗方法， 防治传染病的目标和方法。

DESCRIPTION (provided by applicant): The discovery of antimicrobial peptides in insects, lower vertebrates and mammals has unveiled a previously unrecognized component of animal host defense. Antimicrobial peptides are gene-encoded antibiotics with activity against many classes of microbes. Defensins are the predominant family of such peptides in mammals. Studies by our group and others have discovered that defensin peptides are expressed by mammalian mucosal epithelial cells, providing them with the capacity to participate in local host defense. Although the intestinal epithelium is a surface in continual contact with luminal contents variably laden with microbes, infection is uncommon. Our underlying hypothesis is that in humans, the epithelial defensins HD5 and HD6 contribute to antimicrobial defense of the enteric mucosa. In this grant proposal, we will test biological functions of epithelial antimicrobial peptides in vivo through transgenic expression of HD5 and HD6 peptides in mice. We propose that transgenic expression of these human defensins may provide mice with an enhanced capacity to resist bacterial challenges. Based on preliminary studies of our established transgenic mice, the experiments described here will establish a clearer understanding of the contributions of human antimicrobial peptides to innate host defense. To test our hypotheses, Aim 1 will assay the immunological consequences of human HD5 expression in transgenic mice. We will characterize the ability of HD5 transgenic mice, compared to control wild-type mice, to resist enteric infection by Salmonella typhimurium (Aim 1A), and parallel experiments will extend to other enteric pathogens (Aim 1B). We will examine the impact of transgenic HD5 expression on resident microflora of the mouse intestine (Aim 1C). The antimicrobial activity contributed by transgenic expression will be quantitated in vitro, including analysis of isolated crypts (Aim 1D). The HD6 gene and peptide share little sequence identity to HD5, yet they are expressed together in Paneth cells. In Aim 2, we will generate HD6 transgenic mice (Aim 2A) use recombinant HD6 to develop an antibody for immunoassays (Aim 2B), and characterize the transgenic expression of HD6 at the gene and protein level (Aim 2C). We will then characterize the effects of transgenic HD6 expression on resistance to enteric bacterial colonization and infection (Aim 2D) using the approaches developed in Aim 1. Finally, through lineage interbreeding we will create HD5/HD6 compound transgenic mice to determine if these two peptides have synergistic activities in vivo (Aim 2E). The proposed investigations, and other studies of innate immunity, may provide insights yielding novel therapeutic targets and approaches to combat infectious disease.