Clinical Research of Oral Connective Tissue Program

A.Chediak Higashi综合征(CHS)，一种罕见的常染色体隐性遗传病(溶酶体运输调节基因(Lyst/CHS1)突变)，特征是部分眼皮肤白化病(OCA)、免疫缺陷、轻度出血倾向和各种神经系统问题。此外，被认为与免疫组成情况有关的几个病例报告表明，严重的牙周病。我们假设，与对照成纤维细胞相比，从CHS患者获得的原代皮肤成纤维细胞将表现出细胞因子和免疫调节因子的表达增加，以及对免疫原性攻击的超敏反应。细胞由Dr.Introne提供，之前从皮肤活组织切片的酶消化中获得，并冷冻直到使用。用聚合酶链式反应芯片检测正常对照细胞和慢性阻塞性肺疾病患者成纤维细胞中炎症相关基因的表达。在用聚合酶链式反应阵列评估的84个基因中，15个基因在基线水平上调(包括IL-1、IL-6和COX2)，6个基因下调(包括TLR-2和-4)，CHS细胞与对照细胞相比变化超过2倍。在LPS刺激下，CHS细胞在84个免疫应答基因中只有9.5%受到显著影响，而对照组细胞中受影响基因的比例为33.3%。此外，对照细胞表现出比CHS细胞(30倍变化)更强健的反应(4000倍变化)。Western印迹分析结果表明，CHS细胞膜相关TLR-2和TLR-4蛋白表达水平显著低于对照组，而CHS细胞胞浆TLR-2蛋白水平显著低于CHS细胞，而CHS细胞胞浆TLR-4蛋白表达水平与CHS细胞相同，提示TLR-4向CHS细胞膜的输出效率低下，可能受Lyst突变体编码的溶酶体转运调节因子功能障碍的影响。此外，在脂多糖处理下，CHS细胞由于TLRs的表达不变而表现出钝化的反应，而与对照细胞TLRs的强劲升高相比。Toll样受体通路被认为对内毒素攻击具有高度的反应性，可以诱导细胞因子如IL-6的产生，作为启动免疫反应的机制。因此，TLRs基因表达下调和CHS细胞膜TLR蛋白表达减少可能导致CHS细胞培养液中IL-6蛋白水平在基线和LSP处理后显著低于对照细胞培养液，从而为CHS患者更容易感染提供了机制。尽管Lyst的功能仍不完全清楚，但这些发现支持了其在调节引导炎症反应的关键因素方面的重要作用。 B.矿化障碍：与NIDCR临床研究员和内分泌学家Michael Collins合作，我们一直在检查他照顾下的矿化组织代谢障碍患者DOC复合体的组织/细胞的变化。到目前为止，我们已经从一个人身上获得了组织和样本，目前正在处理组织进行评估。 C.特发性牙根吸收：在我们对BSP KO小鼠的研究中，我们发现了一种特发性牙根吸收表型。基于这一发现，我们正在从特发性牙根吸收患者(和适当的对照)中获取唾液样本，用于全基因组关联分析。

A. Chediak Higashi syndrome (CHS), a rare autosomal recessive disease (mutations in the lysosomal trafficking regulator gene (LYST/CHS1)) characterized by partial oculocutaneous albinism (OCA), immunodeficiency, mild bleeding tendency, and varying neurologic problems. Furthermore, considered to be related to the immune-comprised situation, several case reports indicate severe periodontal disease. We hypothesized that primary skin fibroblasts obtained from CHS patients would exhibit increased expression of cytokines and immune regulatory factors, as well as a hypersensitive response to immunogenic challenge, compared to control fibroblasts. Cells were provided by Dr. Introne, previously obtained from enzymatic digestion of skin biopsies and frozen until use. PCR-arrays were used to profile the expression of inflammation-related genes in fibroblasts of healthy people as control cells and CHS patients cultured with or without Escherichia coli LPS (10ng/mL for 3 h) (n=3). Of the 84 genes evaluated by PCR-array, at baseline 15 were up-regulated (include IL-1β, IL-6, and COX2) and 6 down-regulated (include TLR-2 and -4) with more than 2-fold change in CHS cells compared to control cells. With LPS challenge, CHS cells had only 9.5% of the 84 immune responsive genes significantly affected compared to 33.3% of the genes affected in control cells. Furthermore, control cells presented a more robust response (4,000-fold change) than CHS cells (30-fold change). Protein expression evaluated by Western blot shows that at baseline, cell membrane associated TLR-2 and -4 were significantly lower in CHS versus control cells; while cytosolic TLR-2 protein level was significantly lower in control cells than CHS cells, cytosolic TLR-4 protein was expressed at the same level in CHS cells as in control cells, suggesting that exportation of TLR-4 to CHS cell membrane was not efficient and may be affected by malfunction of lysosomal trafficking regulator encoded by mutant LYST. Furthermore, with LPS treatment, CHS cells presented a blunted response due to unchanged expression of TLRs compared to robust elevation of TLRs on control cells. The Toll-like receptor pathway is consider highly responsive to LPS challenge and can induce production of cytokines such as IL-6 as a mechanism to initiate immune responses. Thus, down-regulated gene expression of TLRs together with less expression of TLR protein on CHS cell membrane may lead to the significant less IL-6 protein levels in CHS cell culture medium both at baseline and with LSP treatment compared to the culture medium of control cells, providing a mechanism in which CHS patients are more susceptible to infection. Although the function of LYST remains incompletely understood, these findings support an important role in modulating key factors directing inflammatory responses. B. Disorders of mineralization: In collaboration with Michael Collins, a NIDCR clinical researcher and endocrinologist, we have been examining individuals under his care with disorders of mineralized tissues metabolism for alterations in tissues/cells of the DOC complex. To date we have obtained tissues and samples from one individual and currently processing tissues for evaluation. C. Idiopathic tooth root resorption: During our research on the Bsp KO mice we identified an idiopathic tooth root resportption phenotype. Based on this finding we are obtaining saliva samples from individuals with idiopathic root resorption (and appropriate controls) for genome wide association analysis.