Clinical Research of Oral Connective Tissue Program

口腔结缔组织项目临床研究

• 批准号: 8939440
• 负责人: Martha Somerman
• 金额: $ 19.53万
• 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/8939440
• 关键词: Affect; Biopsy; CHS protein; CHS1 gene; Candidate Disease 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; Immunologics; Individual; Infection; Inflammation; Inflammatory Response; Interleukin-1; Interleukin-6; Lead; Link; Maintenance; Metabolism; Molecular Analysis; Molecular Profiling; Mus; Mutation; National Human Genome Research Institute; 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; Signal Pathway; Skin; Staining method; Stains; 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; therapeutic target; tissue processing; trafficking

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 our collaborator at NHGRI, 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. Furthermore, Western blot and immunofluorescent staining revealed that TLR-2 and TLR-4 were diminished on cell membranes of CHS and dissociated from Rab11a. Results from this study indicate defective trafficking of TLR-2 and TLR-4 contributes to the hyposensitive response of CHS skin fibroblasts to immunologic challenge, providing a potential therapeutic target for clinical interaction in CHS. 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 tissue 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. We have identified candidate genes associated with this disease. Ongoing studies are focused on determine if these genes are linked with BSP signaling pathways.

A. Chediak Higashi 综合征 (CHS)，一种罕见的常染色体隐性遗传疾病（溶酶体运输调节基因突变 (LYST/CHS1)），其特征为部分眼皮肤白化病 (OCA)、免疫缺陷、轻度出血倾向和不同的神经系统问题。此外，一些病例报告表明存在严重的牙周病，这被认为与免疫状况有关。我们假设，与对照成纤维细胞相比，从中枢性低通气综合症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表达增加，以及对免疫原性攻击的过敏反应。细胞由我们在 NHGRI 的合作者 Introne 博士提供，之前通过酶消化皮肤活检获得并冷冻直至使用。 PCR 阵列用于分析健康人成纤维细胞（作为对照细胞）和用或不用大肠杆菌 LPS（10ng/mL，3 小时）培养的中枢性低通气综合症患者的成纤维细胞中炎症相关基因的表达（n=3）。在通过 PCR 阵列评估的 84 个基因中，基线时有 15 个基因上调（包括 IL-1β、IL-6 和 COX2），6 个基因下调（包括 TLR-2 和 -4），表达量增加了 2 倍以上与对照细胞相比，CHS 细胞发生变化。在 LPS 攻击下，CHS 细胞的 84 个免疫反应基因中只有 9.5% 受到显着影响，而对照细胞中受影响的基因有 33.3%。此外，对照细胞表现出比 CHS 细胞（30 倍变化）更强烈的反应（4,000 倍变化）。 Western blot 评估的蛋白质表达表明，在基线时，CHS 细胞中细胞膜相关的 TLR-2 和 -4 显着低于对照细胞；虽然对照细胞中胞质 TLR-2 蛋白水平显着低于 CHS 细胞，但 CHS 细胞中胞质 TLR-4 蛋白的表达水平与对照细胞相同，表明 TLR-4 向 CHS 细胞膜的输出效率不高并可能受到突变体 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，与对照细胞上 TLR 的大幅升高相比，由于 TLR 表达未改变，CHS 细胞表现出迟钝的反应。 Toll 样受体途径被认为对 LPS 攻击高度敏感，并且可以诱导 IL-6 等细胞因子的产生，作为启动免疫反应的机制。因此，与对照培养基相比，TLR 基因表达下调以及 CHS 细胞膜上 TLR 蛋白表达减少可能导致 CHS 细胞培养基中基线和 LSP 处理时的 IL-6 蛋白水平显着降低细胞，提供了一种机制，使中枢性低通气综合症患者更容易受到感染。此外，Western blot和免疫荧光染色显示CHS细胞膜上的TLR-2和TLR-4减少并与Rab11a解离。这项研究的结果表明，TLR-2 和 TLR-4 的运输缺陷导致中枢性低通气综合症皮肤成纤维细胞对免疫挑战的低敏感性反应，为中枢性低通气综合症的临床相互作用提供了潜在的治疗靶点。尽管 LYST 的功能仍未完全了解，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：我们与 NIDCR 临床研究员和内分泌学家 Michael Collins 合作，一直在检查他治疗下患有矿化组织代谢障碍的个体 DOC 复合体组织/细胞的变化。迄今为止，我们已从一名个体身上获取了组织和样本，目前正在处理组织以进行评估。 C.特发性牙根吸收：在我们对 Bsp KO 小鼠的研究中，我们鉴定了特发性牙根再吸收表型。基于这一发现，我们正在从患有特发性牙根吸收的个体（和适当的对照）获取唾液样本，以进行全基因组关联分析。我们已经确定了与这种疾病相关的候选基因。正在进行的研究重点是确定这些基因是否与 BSP 信号通路相关。