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），以部分皮肤白化病（OCA）、免疫缺陷、轻度出血倾向和各种神经系统问题为特征。此外，一些病例报告显示严重的牙周病，被认为与免疫系统的情况有关。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会表现出细胞因子和免疫调节因子的表达增加，以及对免疫原性挑战的超敏反应。细胞由Introne博士提供，之前从皮肤活组织组织的酶解中获得并冷冻直至使用。采用pcr阵列分析健康人成纤维细胞中炎症相关基因的表达，作为对照细胞，用或不加大肠杆菌LPS （10ng/mL，培养3小时）培养CHS患者（n=3）。在PCR-array评估的84个基因中，在基线时，CHS细胞中有15个基因上调（包括il -1、IL-6和COX2）， 6个基因下调（包括TLR-2和-4），与对照细胞相比变化超过2倍。在LPS刺激下，CHS细胞中84个免疫应答基因中只有9.5%受到显著影响，而对照细胞中有33.3%的基因受到显著影响。此外，对照细胞表现出比CHS细胞（30倍变化）更强的反应（4000倍变化）。Western blot蛋白表达评估显示，在基线时，与对照细胞相比，CHS的细胞膜相关TLR-2和-4显著降低；而在对照细胞中，胞质TLR-2蛋白水平明显低于CHS细胞，而胞质TLR-4蛋白在CHS细胞中的表达水平与对照细胞相同，这表明TLR-4向CHS细胞膜的输出效率不高，可能受到突变体LYST编码的溶酶体运输调节因子功能障碍的影响。此外，在LPS处理下，CHS细胞表现出迟钝的反应，这是由于TLRs的表达不变，而对照组细胞的TLRs则大幅升高。toll样受体途径被认为对LPS具有高度反应性，并且可以诱导细胞因子如IL-6的产生，作为启动免疫应答的机制。因此，TLRs基因表达下调，同时CHS细胞膜上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处理时均明显低于对照细胞培养液，为CHS患者更易感染提供了机制。尽管LYST的功能仍不完全清楚，但这些发现支持在调节炎症反应的关键因素中发挥重要作用。

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.