Integration of Immunochemical, Genomic & Transcriptomic Analyses of Human Skin

描述（由申请人提供）：伴有严重慢性疼痛的各种周围神经病是最常见和最令人衰弱的人类疾病。现有的治疗方法几乎没有长期的好处，而且往往有严重的副作用。越来越多的证据表明，皮肤及其神经支配的结构和化学中的病理可能是疼痛性周围神经病变的贡献者，并且是潜在治疗的高度优先目标。此外，皮肤及其神经支配是药物副作用的常见部位。因此，人类皮肤的穿刺活检越来越多地被用作诊断周围神经病变和评估潜在治疗剂的功效和风险的工具。大多数分析都局限于少数生物标志物，而不是挖掘这些有价值的活检的全部诊断潜力，例如仅使用抗PGP9.5来评估表皮中严格的感觉末梢密度。该I期STTR提交材料提出通过整合活检结构和化学（特别是神经支配）的多重免疫标记分析与内在皮肤细胞成分（例如表皮、毛囊、血管、乳头状真皮）的基因组学和转录组学的激光捕获显微切割评估，极大地扩展皮肤活检的实用性。Integrated Tissue Dynamics LLC（Intidyn）的创始人在使用广泛的多免疫标记来评估各种实验动物模型和人类神经病变条件下皮肤神经支配的形态学和生物化学方面具有数十年的记录经验。这种专业知识已经记录了不同类型的皮肤神经支配之间的疼痛相关病理，不仅在表皮中，而且与毛囊和脉管系统相关。重要的是，他们最近的免疫化学研究结果表明，表皮中的角质形成细胞在皮肤感觉中起直接作用，并且表皮化学中的病理可能导致慢性疼痛。Intidyn最近成立，以增加基础设施，以满足公共和私营部门对这一专业知识日益增长的需求。认识到内在皮肤病理学对诊断和治疗慢性疼痛的重要性，这项1期STTR提案旨在将我们的免疫化学专业知识与基因组和转录组学检测相结合。正常人和带状疱疹后神经痛患者的活检将通过使用免疫荧光和基因组和转录组阵列的交替切片进行比较。 公共卫生相关性：各种各样的周围神经病，如糖尿病和带状疱疹相关的神经病，是最常见的和衰弱的人类痛苦，由于严重的，顽固性疼痛的发生。现有的治疗方法几乎没有长期的好处，而且往往有严重的副作用。本一期STTR提案中要求的资金将主要研究者在外周神经疾病方面的广泛研究经验转化为更有效疗法的大规模药物开发。最近，一些证据表明皮肤是神经性疼痛的潜在贡献者，皮肤神经支配和皮肤免疫化学都发生了显著变化。在这里，资金被要求进一步表征的病理机制存在于皮肤PHN患者通过整合蛋白质免疫化学结果与基因组和转录组数据，每个从相同的打孔活检获得。这些结果将极大地有助于开发针对皮肤的新型有效的治疗干预措施。

DESCRIPTION (provided by applicant): Wide varieties of peripheral neuropathies accompanied by severe chronic pain are among the most common and debilitating human afflictions. Existing treatments have little long term benefits and often have severe side effects. Increasing evidence indicates that pathologies in the structure and chemistry of the skin and its innervation are likely contributors to painful peripheral neuropathies and are a high priority target for potential therapeutics. Moreover, the skin and its innervation are frequent sites of drug side effects. Consequently, punch biopsies of human skin are increasingly being used as a tool for diagnosing peripheral neuropathies and for assessing the efficacy and risk of potential therapeutics. Instead of tapping the full diagnostic potential of these valuable biopsies, most analyses have been limited to few biomarkers such as the sole use of anti-PGP9.5 to assess the density of sensory endings strictly in the epidermis. This phase I STTR submission proposes to vastly expand the utility of skin biopsies by integrating multi-immunolabeling analyses of biopsy structure and chemistry (particularly the innervation) with laser-capture microdissection assessments of the genomics and transcriptomics of intrinsic skin cellular components (e.g. epidermis, hair follicles, blood vessels, papillary dermis). The founders of Integrated Tissue Dynamics LLC (Intidyn) have decades of documented experience in using extensive multimmunolabeling to assess the morphology and biochemistry of cutaneous innervation in a variety of experimental animal models and in human neuropathic conditions. This expertise has documented pain-related pathologies among different types of cutaneous innervation, not only in the epidermis, but also in association with hair follicles and vasculature. Importantly, their recent immunochemical findings have shown that keratinocytes in the epidermis play a direct role in skin sensation, and that pathologies in the epidermal chemistry likely contribute to chronic pain. Intidyn was recently founded to increase the infrastructure to the growing demand for this expertise from the public and private sector. Recognizing the importance of the intrinsic skin pathology to the diagnosis and treatment chronic pain, this Phase 1 STTR proposal is designed to integrate our immunochemical expertise with genomic and transciptomic assays. Biopsies from normal humans and patients with postherpetic neuralgia, will be compared by using alternating sections for immunofluorescence and genomic and transcriptomic arrays. PUBLIC HEALTH RELEVANCE: A wide variety of peripheral neuropathies, such as diabetic and shingles-related neuropathies, are among the most common and debilitating human afflictions due to the occurrence of severe, intractable pain. Existing treatments have little long term benefits and often have severe side effects. The funding requested in this Phase I STTR proposal is to translate the Principal Investigators' extensive research experience on peripheral nerve diseases to large scale pharmaceutical development of more effective therapeutics. Recently, several lines of evidence have implicated the skin as a potential contributor to neuropathic pain with significant alterations occurring in both the cutaneous innervation and the immunochemistry of the skin. Here, funding is requested to further characterize the pathologic mechanisms present in skin from patients with PHN by integrating protein immunochemistry results with genomic and transcriptomic data each obtained from the same punch biopsy. The results will greatly aid the development of novel and efficacious therapeutic interventions directed at the skin.