Immunomodulatory effects of arginine supplementation in colitis and colon cancer

描述（由申请人提供）：在母体R01中，我们正在研究l -精氨酸（L-Arg）在结肠炎小鼠模型中的体外和体内作用。自2008年9月15日开始拨款以来，我们已经取得了实质性进展。特别是，在Aim 2下，我们发现敲除L-Arg转运蛋白阳离子氨基酸转运蛋白（CAT）2显著加重小鼠结肠炎，在Aim 3下，我们发现在AOM-DSS结肠炎相关癌症模型中，CAT2缺失显著增加肿瘤数量和大小。在最初提出的研究之外，我们研究了14名正常受试者和22名溃疡性结肠炎（全结肠）患者的l -精氨酸可用性。我们发现严重结肠炎患者血清l -精氨酸水平高于中度结肠炎或正常对照组。血清L-Arg水平与Mayo疾病活动指数（DAI）有很强的相关性。在血清l -精氨酸水平升高的同时，氨基酸l -鸟氨酸（L-Orn）和l -赖氨酸（L-Lys）也增加了，它们与l -精氨酸竞争，被转运体CAT1和CAT2吸收到细胞中，因此净效应是精氨酸可利用指数（AAI）没有增加，计算为[L-Arg]/[L-Orn + L-Lys]。作为对我们的亲本R01的补充，我们提出验证UC中存在L-Arg可用性功能缺陷的假设，并且饮食中L-Arg的摄入量可能是L-Arg可用性和疾病活性的决定因素。由于l -精氨酸可能代表UC疾病严重程度的生物标志物，我们还将确定DAI并评估其与血清l -精氨酸的相关性。我们建议以前瞻性的方式实现两个具体目标：确定UC患者和正常对照中l -精氨酸的可用性，并通过评估与疾病活动的相关性：临床参数、DAI、内镜评分；b .)组织组织病理学;c .)血清l -精氨酸、L-Orn、l -赖氨酸；AAI公司;细胞因子;d .)结肠组织L-Arg、L-Orn和L-Lys；AAI公司;细胞因子;大肠)组织CAT1、CAT2水平；和f)组织l -精氨酸摄取。我们将比较正常对照、全结肠炎患者和左侧结肠炎患者。对于后者，我们将比较受累粘膜和未受累粘膜。2)。使用专门的软件评估UC患者和对照组饮食中l -精氨酸的摄入量：a)确定饮食中l -精氨酸的摄入量是否与UC临床状况呈负相关；和b)确定l -精氨酸摄入量是否与血清和/或组织中l -精氨酸水平相关。这些研究将为IBD辅助治疗和营养问题的临床问题提供新的见解。根据《复苏法案》的目标，我们将额外雇用两名工作人员，使用核心设施，并从美国公司购买物资。该项目是对NCCAM ARRA竞争性修订指南的响应，该指南的标题是“对评估特定CAM干预措施重要的个体或群体结果测量的可靠性和可变性的研究”，因为所提出的研究对于了解未来饮食干预/L-Arg补充研究所需的生物学和方法是必要的。

DESCRIPTION (provided by applicant): In the Parent R01, we are studying L-arginine (L-Arg) in vitro and in vivo in mouse models of colitis. Since the start of this grant on 9/15/08, we have made substantial progress. In particular, under Aim 2 we have found that knockout of the L-Arg transporter cationic amino acid transporter (CAT)2 significantly exacerbates murine colitis and under Aim 3 we have found that CAT2 deletion dramatically increases tumor number and size in the AOM-DSS colitis-associated cancer model. In studies beyond those originally proposed, we have studied L-Arg availability in 14 normal human subjects and 22 patients with ulcerative colitis (UC) involving the entire colon (pancolitis). We have found increased serum L-Arg levels in severe colitis vs. moderate colitis or normal controls. There was a strong correlation of serum L-Arg level with the Mayo Disease Activity Index (DAI). In parallel to the increase in serum L-Arg levels, there were also increases in the amino acids L-ornithine (L-Orn) and L-lysine (L-Lys), which compete with L-Arg for uptake into cells by the transporters CAT1 and CAT2, so the net effect was no increase in the arginine availability index (AAI), calculated as the [L-Arg]/[L-Orn + L-Lys]. As a supplement to our Parent R01, we propose to test the hypotheses that there is a functional deficiency of L-Arg availability in UC, and that dietary consumption of L-Arg may be a determinant of L-Arg availability and disease activity. Because L-Arg may represent a biomarker for UC disease severity, we will also determine the DAI and assess for correlation with serum L-Arg. We propose to undertake two specific aims in a prospective manner: 1.) To determine L-Arg availability in UC patients and normal controls, and correlate with disease activity by assessing: A.) Clinical parameters, DAI, and endoscopy score; B.) Tissue histopathology; C.) Serum L-Arg, L-Orn, L-Lys; AAI; and cytokines; D.) Colonic tissue L-Arg, L-Orn, and L-Lys; AAI; and cytokines; E.) Tissue CAT1 and CAT2 levels; and F.) Tissue L-Arg uptake. We will compare normal control subjects, patients with pancolitis, and patients with left-sided colitis. In those with the latter, we will compare involved and uninvolved mucosa. 2.) To assess L-Arg intake in the diet of UC patients and control subjects, using specialized software: A.) To determine if L-Arg intake in the diet correlates inversely with UC clinical status; and B.) To determine if L-Arg intake correlates with serum and/or tissue levels of L-Arg. These studies will provide new insights into the clinical problem of adjunctive treatments for IBD and nutritional issues. Consistent with the goals of the Recovery Act, we will hire two additional personnel, utilize core facilities, and purchase supplies from US companies. This project is responsive to the NCCAM Guidelines for ARRA Competitive Revisions under "studies examining individual or population reliability and variability of outcome measures important to evaluation of specific CAM interventions" because the studies proposed are necessary to understand the biology and methods needed for a future dietary intervention/L-Arg supplementation study. PUBLIC HEALTH RELEVANCE: Over one million Americans have inflammatory bowel disease and about half of those affected have ulcerative colitis (UC), leading many to seek a variety of diets and alternative medicines. In addition to our funded studies under R01 AT004821 studying the importance of the amino acid L-arginine (L-Arg) in animal models, we have exciting new data in humans with ulcerative colitis indicating that there is accumulation of L-Arg in the serum of patients with severe UC, which suggests that there may be a defect in utilization of L-Arg in this disease. In this competitive revision application we propose to use supplemental funds under ARRA to conduct additional studies in human subjects to establish whether there is a functional deficiency of L-Arg in UC that correlates with disease activity and extent, and if dietary levels of L-Arg correlate with L-Arg availability to colonic tissues and reduction of UC disease activity.