GENETICS VARIATION IN 5LO PRODUCT PRODUCTION

哮喘是一种异质性综合征，有多种潜在的途径导致气道阻塞，这是它的标志。在已确定的哮喘气道阻塞介质中有白三烯。白三烯是多种酶催化作用的产物，其中包括5-脂氧合酶（5-LO）。考虑到哮喘效应机制的异质性，以及半胱氨酸白三烯作为哮喘效应分子家族之一的重要性，了解白三烯合成调节中个体差异的因素对基础和临床科学都有应用价值。该提案解决了5-LO产物生产调控的潜在位点之一，即由于5-LO核心启动子中存在基因突变而进行的调控。我们发现了一系列突变，其中在5-LO核心启动子中增加或删除Sp1/Egr-1结合基序（即- gggggg -）；这些突变改变了报告基因结构中的基因转录。本文回顾了我们的初步数据，表明携带核心启动子突变形式的哮喘患者对5-LO抑制的有益治疗反应减弱。从这一观察中，我们间接地断言，5-LO抑制的较低治疗反应反映了5-LO核心启动子突变形式纯合的患者合成5-LO产物的减少。拟议研究的目的是基于对一项大型临床试验结果的分析，确定这一论断在个别患者的5-LO产物形成水平上是否正确。为了验证这一假设，我们将追求两个具体目标。在第一个目标中，我们将确定具有相同严重程度的特应性哮喘的患者队列，这些患者携带野生型或突变的5-LO核心启动子。这些受试者将用于确定从外周血中分离的基础和A23 187挑战的中性粒细胞和嗜酸性粒细胞或通过支气管肺泡灌洗分离的肺泡巨噬细胞中定量产生LTE4，半胱氨酸白三烯和脂毒素。还将通过在吸入全肺过敏原刺激前后测量尿LTE4，在每个基因典型定义的哮喘患者队列中研究5-LO产品的生产。除了全肺过敏原激射外，患者还将进行肺段过敏原激射，并比较来自激射段的5-LO产品与对照段的恢复情况。如果这个假设是正确的，那么与核心启动子野生型的患者相比，携带核心启动子突变型的患者应该减少5-LO产物的产生。此外，在过敏原攻击（包括全肺和部分肺）后，核心启动子突变型患者的5-LO产物产量应该低于核心启动子野生型患者。

Asthma is a heterogenous syndrome with multiple potential pathways leading to the airway obstruction which is its hallmark. Among the substances which have an established role as mediators of the airway obstruction in asthma are the leukotrienes. The leukotrienes are the products of the catalytic action of a number of enzymes among which is 5-Lipoxygenase (5-LO). Given the perceived heterogeneity of the effector mechanisms in asthma and the established importance of the cysteinyl leukotrienes as one of the families of effector molecules in asthma, an understanding of the factors responsible for variations among individuals in the regulation of leukotriene synthesis has applications to both basic and clinical science. This proposal addresses one of the potential sites of regulation of 5-LO product production, i.e., regulation due to the presence of genetic mutations in the 5-LO core promoter. We have discovered a series of mutations in which there is the addition of or the deletion of Sp1/Egr-1 binding motifs (i.e.,- GGGCGG-) in the 5-LO core promoter; these mutations modify gene transcription in reporter gene constructs. Our preliminary data, reviewed herein, indicate that patients with asthma harboring the mutant forms of the core promoter have a diminished salutary therapeutic response to 5-LO inhibition. From this observation we have indirectly asserted that the lesser therapeutic response to 5- LO inhibition reflects decreased synthesis of 5-LO products by patients who are homozygous for mutant forms of the 5-LO core promoter. The goal of the proposed research is to determine if this assertion, based on analysis of the results of a large clinical trial, is true at the level of 5-LO product formation in individual patients. To test this hypothesis we will pursue two specific aims. In the first aim we will identify cohorts of patients, with atopic asthma of equivalent severity, who harbor either the wild-type or the mutant 5-LO core promoter. These subjects will be used to ascertain the quantitative production of LTE4, cysteinyl leukotrienes and lipoxins from basal and A23 187 challenged neutrophils and eosinophils isolated from the peripheral blood, or from alveolar macrophages isolated by bronchoalveolar lavage. 5-LO product production will also be studied in each of the genotypically defined cohorts of asthma patients by measurement of urinary LTE4 before and after inhaled whole lung allergen stimulation. In addition to whole lung allergen challenge, patients will undergo pulmonary segmental allergen challenge and the recovery of 5-LO products from the challenged versus the control segment compared. If the hypothesis is correct, then patients harboring the mutant forms of the core promoter should have diminished 5-LO product production when compared to patients with the wild type form of the core promoter. Furthermore, after allergen challenge (both whole lung and segmental), the yield of 5-LO products should be less in patients with the mutant forms of the core promoter than in patients with the wild type form of the core promoter.