Immunomodulatory effects of arginine supplementation in colitis and colon cancer

描述(申请人提供)：在亲本R01中，我们正在研究L-精氨酸(L-精氨酸)在小鼠结肠炎模型中的体外和体内实验。自从这笔赠款于2008年9月15日开始以来，我们已经取得了实质性的进展。特别是，在目标2下，我们发现L-精氨酸转运体阳离子氨基酸转运体(CAT)2的敲除显著加剧了小鼠结肠炎，并且在目标3下，我们发现CAT2缺失显著增加了AOM-DSS结肠炎相关癌症模型中的肿瘤数量和大小。在最初提出的研究之外，我们研究了14名正常人和22名溃疡性结肠炎(UC)患者的L精氨酸的利用度。我们发现重度结肠炎患者血清L-精氨酸水平高于中度结肠炎患者和正常对照组。血清L-精氨酸水平与梅奥疾病活动指数(DAI)密切相关。在血清L-精氨酸水平升高的同时，L-鸟氨酸(L-鸟氨酸)和L-赖氨酸(L-赖氨酸)也增加，这两种氨基酸与L-精氨酸竞争被转运蛋白CAT1和CAT2摄取，因此净影响是精氨酸利用率指数没有增加，计算为[L-精氨酸]/[L-奥恩+L-赖氨酸]。作为对亲本R01的补充，我们建议检验以下假设：UC中存在L-精氨酸利用率的功能性缺陷，并且膳食摄入L-精氨酸可能是L-精氨酸利用率和疾病活动性的决定因素。由于L-精氨酸可能代表UC疾病严重程度的生物标志物，我们还将测定DAI并评估与血清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：测定溃疡性结肠炎患者及正常对照组的L-精氨酸利用率，并与疾病活动性进行相关性分析。临床参数、DAI、胃镜评分；组织病理学：C.)血清L-精氨酸、L-奥恩、L-赖斯、AAI和细胞因子；结肠组织L-精氨酸、L-奥恩和L-赖斯；AAI；和细胞因子；组织CAT1和CAT2水平；组织摄取L-精氨酸。我们将比较正常对照组、全结肠炎患者和左侧结肠炎患者。在后者中，我们将比较受累和未受累的粘膜。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。确定膳食中L-精氨酸的摄入量是否与UC的临床状态呈负相关；探讨L-精氨酸摄入量与血清和/或组织中L-精氨酸水平的相关性。这些研究将为辅助治疗IBD的临床问题和营养问题提供新的见解。根据复苏法案的目标，我们将额外雇用两名人员，利用核心设施，并从美国公司购买物资。本项目是对NCCAM关于ARRA竞争性修订指南的响应，该指南的标题为“研究个体或人群的可靠性和结果指标的可变性，这些研究对评估特定的CAM干预措施很重要”，因为建议的研究对于理解未来膳食干预/L-精氨酸补充剂研究所需的生物学和方法是必要的。 与公共卫生相关：超过100万美国人患有炎症性肠病，约一半的患者患有溃疡性结肠炎(UC)，导致许多人寻求各种饮食和替代药物。除了我们在R01AT004821项下研究氨基酸L-精氨酸(L-精氨酸)在动物模型中的重要性的资助研究之外，我们在人类溃疡性结肠炎中有令人兴奋的新数据表明，严重UC患者的血清中存在L-精氨酸的蓄积，这表明L-精氨酸在本病中的利用可能存在缺陷。在这项竞争性修订申请中，我们建议使用ARRA下的补充资金在人类受试者中进行额外的研究，以确定UC是否存在与疾病活动和程度相关的L-Arg功能缺陷，以及L-Arg的饮食水平是否与结肠组织对L-Arg的利用率和UC疾病活动的减少相关。

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.