Clinical Research of Oral Connective Tissue Program
口腔结缔组织项目临床研究
基本信息
- 批准号:8939440
- 负责人:
- 金额:$ 19.53万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:AffectBiopsyCHS proteinCHS1 geneCandidate Disease GeneCaringCase StudyCell Culture TechniquesCell membraneCellsClinicalClinical ResearchCollaborationsConnective TissueCulture MediaDentalDevelopmentDigestionDiseaseEndocrinologistEscherichia coliEvaluationExhibitsFibroblastsFreezingGene ExpressionGene ProteinsGenesGoalsHemorrhageImmuneImmune responseImmunologicsIndividualInfectionInflammationInflammatory ResponseInterleukin-1Interleukin-6LeadLinkMaintenanceMetabolismMolecular AnalysisMolecular ProfilingMusMutationNational Human Genome Research InstituteNational Institute of Dental and Craniofacial ResearchNatural HistoryNatural regenerationNeurologicOculocutaneous albinism immunodeficiencyOralPTGS2 genePathologyPatientsPeriodontal DiseasesPhenotypeProductionProteinsProtocols documentationRegulator GenesResearchResearch PersonnelRoleRoot ResorptionSalivaSamplingSignal PathwaySkinStaining methodStainsTissue SampleTissuesToll-Like Receptor PathwayToll-like receptorsTooth root structureUnited States National Institutes of HealthWestern Blottingbasechediak-higashi syndromecraniofacial complexcytokinegenome wide association studyimmunogenicmineralizationmutantprogramsprotein expressionrepairedresponsetherapeutic targettissue processingtrafficking
项目摘要
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)是一种罕见的常染色体隐性疾病(溶酶体运输调节剂基因(LYST/CHS1)的突变),其特征在于,其特征是部分眼皮白细胞主义(OCA),免疫缺陷,轻度出血趋势,轻度出血趋势和神经系统的问题。此外,被认为与免疫利益相关的情况,几例病例报告表明严重的牙周疾病。我们假设与对照成纤维细胞相比,从CHS患者获得的原发性皮肤成纤维细胞将表现出增加的细胞因子和免疫调节因子的表达以及对免疫原性挑战的过敏反应。我们在NHGRI的合作者提供了细胞,Interone博士先前是从皮肤活检的酶消化中获得的,并冷冻直至使用。 PCR阵列被用来介绍健康人的成纤维细胞中炎症相关基因的表达,作为对照细胞和CHS患者,有或没有大肠杆菌LPS(10NG/mL)(持续3 h)(n = 3)。在通过PCR-Array评估的84个基因中,基线15上调(包括IL-1β,IL-6和COX2),与对照细胞相比,CHS细胞的变化超过2倍,下调了6个下调(包括TLR-2和-4)。随着LPS挑战,CHS细胞只有84个免疫反应基因中的9.5%显着影响,而对照细胞中受影响的基因的33.3%。此外,对照细胞比CHS细胞(30倍变化)提出的反应更强大(4,000倍)。通过蛋白质印迹评估的蛋白质表达表明,在基线,CHS与对照细胞中相关的TLR -2和-4相关的TLR -2和-4显着降低。虽然对照细胞中的胞质TLR-2蛋白水平明显低于CHS细胞,但胞质TLR-4蛋白在与对照细胞中的CHS细胞中在同一水平上表达,这表明TLR-4向CHS细胞膜膜的出口不高,并且可能受到溶解质量质量调节液的失常的影响,并且可能受到质量症状的效率。此外,通过LPS处理,CHS细胞与对照细胞上TLR的强大升高相比,由于TLR的表达不变,导致了钝化的反应。 Toll样受体途径认为对LPS挑战的反应很高,并且可以诱导细胞因子(例如IL-6)作为启动免疫反应的机制。因此,与对照细胞的培养基相比,TLR的下调基因表达以及TLR蛋白在CHS细胞膜上的表达较少,在基线和LSP治疗的CHS细胞培养基中可能导致显着的IL-6蛋白水平,从而提供了一种机制,从而提供了一种机制,其中CHS患者更易于感染。此外,蛋白质印迹和免疫荧光染色表明,在CHS的细胞膜上TLR-2和TLR-4降低并与RAB11A解离。这项研究的结果表明,TLR-2和TLR-4的运输有缺陷有助于CHS皮肤成纤维细胞对免疫学挑战的不敏反应,从而为CHS中的临床相互作用提供了潜在的治疗靶标。尽管LYST的功能仍未完全理解,但这些发现支持调节指导炎症反应的关键因素的重要作用。
B.矿化疾病:与NIDCR临床研究人员和内分泌学家迈克尔·柯林斯(Michael Collins)合作,我们一直在检查他的护理中的个体,以矿化组织代谢的疾病来改变DOC复合物的组织/细胞的改变。迄今为止,我们已经从一个个体和当前正在处理组织进行评估的组织和样品。
C.特发性牙根吸收:在对BSP KO小鼠的研究期间,我们确定了特发性牙根呼吸表型。基于这一发现,我们正在从具有特发根吸收(和适当对照)的个体中获得唾液样本,以进行基因组广泛的关联分析。我们已经确定了与该疾病相关的候选基因。正在进行的研究集中于确定这些基因是否与BSP信号通路相关。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Martha Somerman其他文献
Martha Somerman的其他文献
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{{ truncateString('Martha Somerman', 18)}}的其他基金
Clinical Research of Oral Connective Tissue Program
口腔结缔组织项目临床研究
- 批准号:
10244803 - 财政年份:
- 资助金额:
$ 19.53万 - 项目类别:
Clinical Research of Oral Connective Tissue Program
口腔结缔组织项目临床研究
- 批准号:
10006390 - 财政年份:
- 资助金额:
$ 19.53万 - 项目类别:
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