Dermal-Epidermal Junction Disruptors: Mechanistic Insights

真皮-表皮连接破坏者：机制见解

• 批准号: 10359790
• 负责人: Blase Christopher Billack
• 金额: $ 12.3万
• 依托单位: ST. JOHN'S UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-15 至 2023-07-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10359790
• 关键词: 3-Dimensional; Achievement; Acute; Adverse effects; Affect; Antidotes; Antineoplastic Agents; Apoptosis; Autophagocytosis; Biogenesis; Biopsy Specimen; Bulla; CASP3 gene; CASP9 gene; Cell Culture Techniques; Cell Proliferation; Cells; Chemical Warfare Agents; Chemicals; Chemotherapy-Oncologic Procedure; Cicatrix; Complex; Cutaneous; DNA; DNA Damage; DNA Double Strand Break; DNA Probes; Data; Dermal; Dermis; Dose; Down-Regulation; Doxorubicin; EIF4EBP1 gene; Ear; Edema; Electrolytes; Epidermis; Exhibits; Extravasation; FRAP1 gene; Flap Endonucleases; Foundations; Future; Gamma-H2AX; Gelatinase B; Goals; Histologic; Histones; Homeostasis; Human; Hyperplasia; Ifosfamide; Immunofluorescence Immunologic; Immunohistochemistry; In Vitro; Inflammation; Inflammatory; Interleukin-1 beta; Interleukin-6; Intravenous; Knowledge; Lipid Peroxidation; Lipids; Liposomal Doxorubicin; Liquid substance; Literature; Mechlorethamine; Mediating; Medical; Metabolism; Metalloproteases; MicroRNAs; Microscopic; Minor; Mitochondria; Mitochondrial DNA; Modeling; Mus; Mustard Gas; Names; Nuclear; Order Coleoptera; Organoselenium Compounds; Paclitaxel; Pain; Patients; Pharmaceutical Preparations; Phosphorylation; Process; Protein Biosynthesis; Proteins; Punch Biopsy; RTH-1 Nuclease; Raptors; Reaction; Reporting; Research; Research Proposals; Role; Scaffolding Protein; Signal Pathway; Signaling Molecule; Site; Skin; Skin Tissue; Skin injury; Snake Venoms; Supportive care; System; TNF gene; Testing; Thick; Time; Tissue Sample; Tissues; Toxic Environmental Substances; Toxic effect; Up-Regulation; Variant; Vesicants; Work; World War I; base; chemotherapy; cytokine; design; drug development; ebselen; endonuclease; in vitro Model; in vivo; in vivo Model; infection risk; inflammatory marker; innovation; insight; lewisite; member; novel; prototype; recruit; repair enzyme; repaired; response; skin barrier; skin morphogenesis; three dimensional cell culture; wound healing

Project Summary/Abstract A diverse array of chemicals including certain chemical warfare agents (e.g., sulfur mustard), certain intravenous chemotherapy drugs (e.g., mechlorethamine, MEC; paclitaxel, liposomal doxorubicin, ifosfamide, bendamustine), and certain environmental toxins (e.g., viper snake venom metalloproteinases, blister beetle cantharidin) cause acute and severe cutaneous toxicity including dermal-epidermal junction (DEJ) disruption and blistering (vesication). The cutaneous mechanisms involved in chemically-induced DEJ disruption by these types of disruptors remain obscure and current treatments aimed at reducing DEJ disruption remain grossly inadequate. The primary aim of the present proposal, Specific Aim 1, is to investigate novel mechanisms involved in cutaneous responses to DEJ disruptors. Recently, dysregulation of the mammalian target of rapamycin complex 1 (mTORC1) has been implicated in cutaneous injury; with the observation that mouse skin deficient in epidermal expression of Raptor, a component of mTORC1, exhibits epidermal detachments consistent with vesication. Based on this important information, here we will test the hypothesis that DEJ disruption by MEC, a prototype DEJ disruptor, involves both time- and dose- dependent decreases in the epidermal expression of Raptor and phosphorylated forms of S6K and 4E-BP-1. To this end, we will utilize two relevant and complementary skin models: (a) the mouse ear vesicant model (MEVM; in vivo model) and (b) a human skin full thickness 3D culture (in vitro model). Increasing doses of the prototype DEJ disruptor MEC will be applied to mouse ear skin or human skin and punch biopsy samples will be evaluated for cutaneous responses at 0.5, 1, 2, 4, 8, 12 and 24 h after exposure. Dermatotoxicity caused by MEC will be investigated both histologically (by microscopic analyses of edema/ hyperplasia/ inflammation/ vesication) and mechanistically (through the use of immunohistochemical analyses of tissue expression of Raptor and phosphorylated forms of S6K and 4E-BP1 in the epidermis). This highly innovative approach will allow us to decipher the relationship between tissue blistering by DEJ disruptors and components of the mTORC1 signaling pathway. In addition to Specific Aim 1, there are two sub-aims associated with this proposal (Sub-Aim 1 and Sub-Aim 2, respectively). In Sub- Aim 1, the cutaneous expression of several key inflammatory markers (TNFα, IL-1β, iNOS, IL-6, MMP-9) and mIR-132, a microRNA abundant in skin and involved in epidermal cell proliferation and wound healing, will be evaluated in all tissue samples (mouse and human) using qPCR and immunohistochemistry (IHC). In Sub-Aim 2, the time- and dose-dependent effects of MEC on DNA damage sensing and repair responses of the skin will be investigated. DNA damage by MEC will be assessed in all tissue samples (mouse and human) by using immunofluorescence for the presence of DNA damage foci containing phosphorylated γH2AX (the minor H2 histone variant X), and the repair enzymes FEN1 (flap endonuclease 1) and APE1 (apurinic/apyrimidinic endonuclease 1. Mitochondrial DNA (mtDNA) damage by MEC will be investigated by qPCR. All in all, the work proposed here will establish an important foundation of cutaneous responses to MEC and will aid in deciphering novel targets for future drug development designed to eliminate DEJ disruption by other drugs/chemicals.

项目总结/摘要 各种各样的化学品，包括某些化学战剂（例如，硫芥），某些静脉注射 化疗药物（例如，氮芥，MEC;紫杉醇，脂质体阿霉素，异环磷酰胺，苯达莫司汀），和 某些环境毒素（例如，蝰蛇蛇毒金属蛋白酶，斑蝥素）引起急性和严重的 皮肤毒性，包括真皮-表皮连接（DEJ）破坏和起泡（水疱）。皮肤 这些类型的干扰物在化学诱导DEJ干扰中涉及的机制仍然是模糊的和当前的 旨在减少DEJ破坏的治疗仍然严重不足。本提案的主要目的是， 目的1，是研究皮肤对DEJ干扰物反应的新机制。最近， 哺乳动物雷帕霉素靶蛋白复合物1（mTORC 1）与皮肤损伤有关， 缺乏Raptor（mTORC 1组分）表皮表达小鼠皮肤表现出表皮脱落， 与水疱形成一致基于这一重要信息，我们将在这里检验以下假设： MEC是DEJ破坏剂的原型，涉及Raptor表皮表达的时间和剂量依赖性降低 以及S6 K和4 E-BP-1的磷酸化形式。为此，我们将使用两个相关且互补的皮肤模型： (a)小鼠耳部水疱模型（MEVM;体内模型）和（B）人皮肤全厚度3D培养物（体外模型）。 将增加剂量的原型DEJ破坏剂MEC应用于小鼠耳皮肤或人类皮肤，并进行穿刺活检 在暴露后0.5、1、2、4、8、12和24 h评价样品的皮肤反应。皮肤毒性由 将对MEC进行组织学研究（通过水肿/增生/炎症的显微镜分析）， 囊泡化）和机制（通过使用Raptor和 表皮中S6 K和4 E-BP 1的磷酸化形式）。这种高度创新的方法将使我们能够破译 DEJ破坏剂引起的组织起泡与mTORC 1信号通路组分之间的关系。此外 除了具体目标1之外，还有两个次级目标与本提案相关（分别为次级目标1和次级目标2）。在子- 目的：1、检测皮肤炎症标志物TNFα、IL-1β、iNOS、IL-6、MMP-9和mIR-132的表达， 一种在皮肤中丰富并参与表皮细胞增殖和伤口愈合的微小RNA，将在所有组织中进行评估。 使用qPCR和免疫组织化学（IHC）对样品（小鼠和人）进行检测。在子目标2中，时间和剂量依赖性 将研究MEC对皮肤的DNA损伤感测和修复反应的影响。MEC的DNA损伤将 在所有组织样本（小鼠和人）中通过使用免疫荧光法评估DNA损伤灶的存在 含有磷酸化γ H2 AX（次要H2组蛋白变体X）和修复酶FEN 1（瓣状核酸内切酶1） 和APE 1（脱嘌呤/脱嘧啶核酸内切酶1。MEC对线粒体DNA（mtDNA）的损伤将通过以下方法进行研究： qPCR。总而言之，本文提出的工作将为MEC的皮肤反应奠定重要基础， 帮助破译未来药物开发的新目标，旨在消除其他药物/化学品对DEJ的破坏。

项目成果

Selected N-Terpenyl Organoselenium Compounds Possess Antimycotic Activity In Vitro and in a Mouse Model of Vulvovaginal Candidiasis.

选定的N-替苯基有细胞烯化合物在体外和小鼠外阴阴道念珠菌病模型中具有抗菌毛活性。

• DOI: 10.3390/molecules28217377
• 发表时间: 2023-10-31
• 期刊: Molecules (Basel, Switzerland)
• 作者: Liang X;Pacuła-Miszewska AJ;Obieziurska-Fabisiak M;Vartak R;Mao G;Patel K;Fedosova NU;Ścianowski J;Billack B
• 通讯作者: Billack B

Combination of ebselen and hydrocortisone substantially reduces nitrogen mustard-induced cutaneous injury.

• DOI: 10.1016/j.crtox.2021.10.002
• 发表时间: 2021
• 期刊: Current research in toxicology
• 影响因子: 3.3
• 作者: Tumu HCR;Cuffari BJ;Billack B
• 通讯作者: Billack B