Development of the Cellular Biomarker for Diabetic Foot Ulcers

描述（由申请人提供）：本申请涉及广泛挑战领域04，“临床研究”和两个特定挑战主题：03-DK-101，“发现NIDDK感兴趣的疾病的疾病风险、进展或治疗反应的生物标志物；”和04- dk -102，“改进的临床诊断技术，详细的临床表型，临床疾病分期和NIDDK感兴趣的疾病的活动。”该项目的目标是确定与糖尿病足溃疡（DFUs）愈合结果相关的客观、定量的生物分子参数。慢性不愈合dfu是一个广泛而严重的临床问题，具有高发病率、致残率和死亡率，包括下肢截肢的高风险。根据目前的治疗标准，约有一半的dfu未能及时或完全治愈。DFU迅速和完全愈合的主要障碍之一是无法预测哪些伤口不太可能对标准治疗方案产生反应，需要其他干预措施。慢性伤口边缘含有表达愈合抑制剂的组织，对伤口愈合刺激没有反应。该项目的挑战是开发一种可行的定量方法来区分具有愈合能力的组织和不具有愈合能力的组织，从而确定非愈合表型。本项目旨在寻找能够客观定量识别dfu中未愈合组织的生物标志物。两种蛋白标记物——¿-catenin的核定位和c-myc的诱导表达——与其他慢性伤口的愈合损伤有关，但尚未在DFUs中进行研究。因此，本项目的目的是确定伤口边缘¿-catenin和/或c-myc的表达是否与DFU伤口闭合的客观定量指标相关。连续50例接受目前慢性DFUs标准治疗的患者将在初始治疗（第0周）和4周后进行小伤口组织活检。第4周的伤口愈合程度将作为伤口愈合的替代结果，并将通过组织病理学证实。目的1是确定-catenin在细胞核中的定位是否与不愈合相关。-catenin定位将通过免疫组织化学定量。主要结局指标将是第4周伤口愈合程度与核¿-catenin染色之间的相关性。目的2是确定c-myc蛋白表达是否与不愈合相关。免疫组织化学定量检测C-myc的表达。主要结局指标将是伤口愈合程度与核-连环蛋白染色之间的相关性。次要结局指标将是伤口愈合与这两种标志物的表达之间的相关性。该项目非常适合美国国立卫生研究院挑战计划的目标和选定的挑战主题，因为它通过确定预测愈合结果的客观细胞生物标志物来推进伤口愈合科学，并提供改进的定量技术，可用于监测治疗效果和临床结果。该项目很有可能在两年内实现重要的里程碑，因为它得到了一个成熟且富有成效的合作者团队的支持，该团队已经获得了机构审查委员会（IRB）对DFU活检和分析的批准，并且所有方法和方案都在先前的出版物中得到了验证和充分描述。这笔资金将创造全职和兼职工作，并支持学生进行临床和转化研究。这项研究计划的长期目标是减少截肢的数量，改善数百万患有慢性无法愈合的皮肤溃疡的人的愈合结果。该项目的成功完成将导致鉴定可靠的生物标记物，可用于识别未愈合的伤口组织，并预测哪些糖尿病足溃疡在没有及时和积极干预的情况下无法愈合。

DESCRIPTION (provided by applicant): This application addresses Broad Challenge Area 04, "Clinical Research" and two specific Challenge Topics: 03-DK-101, "Discovery of biomarkers for disease risk, progression or response to therapy in diseases of interest of NIDDK;" and 04-DK-102, "Improved techniques for clinical diagnosis, detailed clinical phenotyping, and clinical disease staging and activity for conditions of interest to NIDDK." The goal of this project is to identify objective, quantitative bio-molecular parameters that correlate with healing outcomes for diabetic foot ulcers (DFUs). Chronic non-healing DFUs are a widespread and serious clinical problem with high rates of morbidity, disability and mortality, including a high risk for lower limb amputations. About half of all DFUs fail to heal promptly or completely in response to the current standard of care. One of the major obstacles to prompt and complete DFU healing is the inability to predict which wounds are not likely to respond to standard treatment protocols and will require alternative interventions. The edges of chronic wounds contain tissue that expresses inhibitors of healing and does not respond to wound-healing stimuli. The challenge of this project is to develop a feasible quantitative method of distinguishing between tissue that has the capacity to heal and tissue that does not, thus identifying non-healing phenotype. This project proposes to identify biomarkers that can objectively and quantifiably identify non-healing tissue in DFUs. Two protein markers -- nuclear localization of ¿-catenin and induced expression of c-myc -- have been associated with healing impairment in other chronic wounds but have not been studied in DFUs. Therefore, the objective of this project is to determine whether expression of ¿-catenin and/or c-myc at the wound edge is correlated with objective quantitative measures of DFU wound closure. Fifty consecutive patients receiving the current standard of care for chronic DFUs will provide small wound tissue biopsies at the initial treatment (week 0) and 4 weeks later. The extent of wound closure at 4 weeks will be used as a surrogate outcome for wound healing and will be confirmed by histopathology. Aim 1 is to determine whether ¿-catenin localization in the nucleus is correlated with non-healing. ¿-catenin localization will be quantified by immunohistochemistry. The primary outcome measure will be the correlation between the extent of wound closure at week 4 and nuclear ¿-catenin staining. Aim 2 is to determine whether c-myc protein expression is correlated with non-healing. c-myc expression will be quantified by immunohistochemistry. The primary outcome measure will be the correlation between extent of wound closure and nuclear ¿-catenin staining. The secondary outcome measure will be the correlation between wound closure and expression of both markers together. This project is ideally suited to the goals of the NIH Challenge Initiative and the selected Challenge Topics because it advances the science of wound healing by identifying objective cellular biomarkers that predict healing outcomes and provides improved quantitative techniques that can be used to monitor treatment efficacy and clinical outcomes. This project has a high likelihood of achieving important milestones within two years because it is supported by an established and productive team of collaborators that has existing Institutional Review Board (IRB) approvals for DFU biopsy and analysis and has all of the methods and protocols validated and fully described in prior publications. Funds from this grant will create full-time and part- time jobs and support students to clinical and translational research. The long-term goal of this research program is to reduce the number of amputations and improve healing outcomes for millions of individuals suffering from the consequences of chronic non-healing skin ulcers. Successful completion of this project will result in identification of reliable biological markers that can be used to identify non-healing wound tissue and predict which diabetic foot ulcers are incapable of healing without timely and aggressive intervention.