Clinical Research of Oral Connective Tissue Program

口腔结缔组织项目临床研究

• 批准号: 8750652
• 负责人: Martha Somerman
• 金额: $ 18.81万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8750652
• 关键词: Affect; Biopsy; CHS protein; CHS1 gene; Caring; Case Study; Cell Culture Techniques; Cell membrane; Cells; Clinical; Clinical Research; Collaborations; Connective Tissue; Culture Media; Dental; Development; Digestion; Disease; Endocrinologist; Escherichia coli; Evaluation; Exhibits; Fibroblasts; Freezing; Gene Expression; Gene Proteins; Genes; Goals; Hemorrhage; Immune; Immune response; Individual; Infection; Inflammation; Inflammatory Response; Interleukin-1; Interleukin-6; Lead; Maintenance; Metabolism; Molecular Analysis; Molecular Profiling; Mus; Mutation; National Institute of Dental and Craniofacial Research; Natural History; Natural regeneration; Neurologic; Oculocutaneous albinism immunodeficiency; Oral; PTGS2 gene; Pathology; Patients; Periodontal Diseases; Phenotype; Production; Proteins; Protocols documentation; Regulator Genes; Research; Research Personnel; Role; Root Resorption; Saliva; Sampling; Skin; Tissue Sample; Tissues; Toll-Like Receptor Pathway; Toll-like receptors; Tooth root structure; United States National Institutes of Health; Western Blotting; base; chediak-higashi syndrome; craniofacial complex; cytokine; genome wide association study; immunogenic; mineralization; mutant; programs; protein expression; repaired; response; tissue processing

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.

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/CHS 1）突变），特征为部分眼皮肤白化病（OCA）、免疫缺陷、轻度出血倾向和各种神经系统问题。此外，被认为是与免疫组成的情况下，一些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过敏反应。 细胞由Introne博士提供，之前从皮肤活检的酶消化中获得，并冷冻直至使用。采用PCR芯片检测正常人成纤维细胞和CHS患者成纤维细胞中炎症相关基因的表达，并与大肠杆菌LPS（10 ng/mL，培养3 h）进行比较。在通过PCR阵列评估的84个基因中，在基线时，CHS细胞中有15个上调（包括IL-1、IL-6和COX 2），6个下调（包括TLR-2和TLR-4），与对照细胞相比，变化超过2倍。在LPS攻击下，CHS细胞中84个免疫应答基因中只有9.5%受到显著影响，而对照细胞中受影响的基因为33.3%。此外，对照细胞比CHS细胞（30倍变化）表现出更稳健的反应（4，000倍变化）。蛋白质印迹分析显示，在基线时，CHS细胞膜相关TLR-2和TLR-4显著低于对照细胞;而对照细胞中胞质TLR-2蛋白水平显著低于CHS细胞，CHS细胞中胞质TLR-4蛋白表达水平与对照细胞相同，这表明TLR-4向CHS细胞膜的输出是无效的，并且可能受到由突变体LYST编码的溶酶体运输调节剂的功能障碍的影响。此外，与对照细胞上TLR的强烈升高相比，在LPS处理下，CHS细胞由于TLR的表达不变而呈现钝化反应。Toll样受体途径被认为对LPS攻击高度响应，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，与对照细胞的培养基相比，下调的TLR基因表达以及CHS细胞膜上TLR蛋白的较少表达可能导致CHS细胞培养基中在基线和LSP处理时的IL-6蛋白水平显著更低，提供了CHS患者更容易感染的机制。虽然LYST的功能仍不完全清楚，但这些发现支持在调节指导炎症反应的关键因素中发挥重要作用。 B。矿化障碍：在与NIDCR临床研究员和内分泌学家Michael柯林斯的合作中，我们一直在检查在他的护理下患有矿化组织代谢障碍的个体，以检查DOC复合体的组织/细胞的改变。 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价。 C.特发性牙根吸收：在我们对Bsp KO小鼠的研究中，我们鉴定了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。