Modeling Novel Treatments for Dry Eye

描述（由申请人提供）：我们的多学科和多机构申请从我们发现的新的人促分泌有丝分裂原“lacritin”的角度解决了干眼综合征。泪蛋白在泪腺和睑板腺以及角膜和结膜中限制性表达，在那里它似乎能够保护泪腺-角膜轴的上皮细胞免于炎症相关的细胞死亡。干眼症患者泪液中促炎细胞因子TNF α水平升高与眼表上皮细胞损伤相关事实上，向培养的人角膜上皮细胞中加入TNF α通过半胱天冬酶-8和-3促进死亡。最近，我们观察到，半胱天冬酶的激活和死亡是完全防止列入10 nM的lacritin。这一观察结果已得到加强的催泪蛋白缺失分析，揭示了催泪蛋白的C-末端内的细胞保护性网站。Lacritin利用独特的细胞靶向机制：乙酰肝素酶解除细胞表面多配体蛋白聚糖-1的N-末端胞外域内的lacritin结合位点。然后结合的催乳素可能被呈递给GPCR。由于乙酰肝素酶具有较低的最适pH值，因此可能的是，lacritin对假设的突然低pH值“危险信号”具有保护作用，该“危险信号”被认为是某些个体中原发性干燥综合征干眼症开始的基础。在兔临床前研究中，局部应用催泪素可促进泪液流量增加至少4小时，且无毒性-甚至连续治疗30天。在细胞培养中，催泪蛋白刺激泪腺泡细胞分泌泪液-催泪蛋白也是从相同的细胞分泌的。它还促进角膜上皮MUC 16和lacritin的表达。由于催泪蛋白是一种天然泪液蛋白，这表明上游和下游自分泌刺激机制延长催泪蛋白的细胞保护和促分泌作用。泪液蛋白可能作为生物活性复合物起作用。这些活动可以利用重组蛋白工程。我们的工作假设是，催泪素是天然保护对干眼症炎症。我们的近期目标是优化催乳素的细胞保护活性并了解其作用机制。我们的第一个目标是设计最小和最具细胞保护性的催乳素。我们的第二个目标是找出其细胞保护活性的生物学途径，以寻找治疗协同作用或禁忌症。我们的第三个目的是在干眼动物模型中临床前测试局部应用或遗传诱导的催泪蛋白。

DESCRIPTION (provided by applicant): Our multi-disciplinary and multi-institutional application addresses dry eye syndromes from the perspective of the new human prosecretory mitogen 'lacritin' discovered by us. Lacritin is restrictively expressed in lacrimal and meibomian glands and in the cornea and conjunctiva, where it appears to be capable of protecting epithelia of the lacrimal-corneal axis against inflammation-associated cell death. Increased levels of proinflammatory cytokine TNFa in tears of dry eye patients is associated with damage to ocular surface epithelia. Indeed, adding TNFa to cultured human corneal epithelial cells promotes death via caspases-8 and -3. Recently we observed that caspase activation and death is completely prevented by inclusion of 10 nM lacritin. This observation has been reinforced by lacritin deletion analysis that reveals a cytoprotective site within lacritin's C-terminus. Lacritin's utilizes a unique cell targeting mechanism: heparanase unblocks a lacritin binding site within the N-terminal ectodomain of cell surface syndecan-1. Bound lacritin is then likely presented to a GPCR. Since heparanase has a lower pH optimum, it is possible that lacritin is protective against the hypothetical sudden low pH 'danger signal' thought to underlie the initiation of primary Sjogren's syndrome dry eye in some individuals. In rabbit preclinical studies, topical application of lacritin promotes increased tear flow for at least 4 hr without toxicity - even over 30 days of continuous treatment. In cell culture, lacritin stimulates tear secretion by lacrimal acinar cells - the same cells from which it is secreted. It also promotes corneal epithelial MUC16 and lacritin expression. Since lacritin is a natural tear protein, this suggests mechanisms of upstream and downstream autocrine stimulation that prolong lacritin's cytoprotective and prosecretory effects. Tear proteins likely function as bioactive complexes. These activities can be harnessed by recombinant protein engineering. Our working hypothesis is that lacritin is naturally protective against dry eye inflammation. Our immediate goal is to optimize lacritin's cytoprotective activity and understand its mechanism of action. Our first aim is to engineer the smallest and most cytoprotective form of lacritin. Our second aim is to work out biological pathways that underlie its cytoprotective activity in a search for treatment synergies or counterindications. Our third aim is to preclinically test topically applied or genetically induced lacritin in animal models of dry eye.