Mitochondrial dysfunction in HAART: Point of care tests

HAART 中的线粒体功能障碍：护理点测试

• 批准号: 6893169
• 负责人: Michael F Marusich
• 金额: $ 10万
• 依托单位: MITOSCIENCES, INC
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-15 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6893169
• 关键词: AIDS therapy; antiAIDS agent; bioenergetics; clinical research; diagnostic tests; drug adverse effect; high throughput technology; human tissue; immunologic assay /test; mitochondrial disease /disorder; oxidative phosphorylation; rapid diagnosis; respiratory enzyme; technology /technique development; tissue /cell culture

DESCRIPTION (provided by applicant): HAART is an effective treatment for HIV infection but can cause serious metabolic complications including lacticacidemia, lipodystrophy, peripheral neuropathies, and cardiomyopathies. There is considerable evidence that the metabolic complications are the result of unintended mitochondrial toxicity of HAART. Because these complications can limit treatment, there are pressing needs: 1) to better understand the molecular basis of this toxicity, and 2) for diagnostic tools to monitor the resulting mitochondrial dysfunction. Addressing both needs could provide the rationale and means to guide therapy so HAART could be modified before serious clinical complications ensue. To this end, we propose to make a set of simple dipstick-based immunoassays that will be used first in research directed at understanding the effects of therapy on key mitochondrial enzymes of energy metabolism, and then developed into point of care diagnostics. The enzymes measured constitute the oxidative phosphorylation system (OXPHOS) responsible for providing almost all cellular ATP. Not only are these enzyme complexes essential for mitochondrial function, but they are known to be affected in HAART as a result of the depletion of mtDNA. We have shown that depletion of OXPHOS complexes can be used as a surrogate for mtDNA depletion, as mtDNA encodes 13 protein subunits that are essential components of 4 of the 5 complexes of the OXPHOS system. Moreover, it is the levels of functional OXPHOS enzymes rather than mtDNA levels that most accurately reflects mitochondrial dysfunction Finally, the dipstick assays, unlike real time PCR used to measure mtDNA depletion directly, are simple quick, inexpensive and capable of high throughput. Our prototype Complex I specific dipsticks can detect Complex I in samples containing as little as 0.1ug of mitochondria or as few as 1x105 solubilized whole cells (fibroblasts or peripheral blood mononuclear cells). The prototype dipsticks can also detect the effects of mtDNA-depletion, i.e., that mtDNA-depleted human fibroblasts lack Complex I. We will examine 2 different ways to quantify protein levels with the dipsticks, 1 instrument-free using only inexpensive self reporting disposable dipsticks, the second incorporating an inexpensive dipstick reader to provide greater Decision. The tests will be optimized for sensitivity, accuracy, reliability and dynamic range, using cultured human fibroblasts and control samples from clinically relevant tissues, such as PBMCs and subcutaneous adipose tissue.

描述（申请人提供）：HAART 是治疗 HIV 感染的有效方法，但可引起严重的代谢并发症，包括乳酸血症、脂肪营养不良、周围神经病和心肌病。有大量证据表明代谢并发症是HAART 意外线粒体毒性的结果。由于这些并发症可能限制治疗，因此迫切需要：1）更好地了解这种毒性的分子基础，2）用于监测由此产生的线粒体功能障碍的诊断工具。满足这两种需求可以提供指导治疗的原理和方法，以便在严重的临床并发症发生之前对 HAART 进行修改。为此，我们建议制作一套简单的基于试纸的免疫测定法，该测定法将首先用于旨在了解治疗对能量代谢关键线粒体酶的影响的研究，然后发展为护理点诊断。测量的酶构成了负责提供几乎所有细胞 ATP 的氧化磷酸化系统 (OXPHOS)。这些酶复合物不仅对线粒体功能至关重要，而且已知它们在 HAART 中会因 mtDNA 耗尽而受到影响。我们已经证明，OXPHOS 复合物的消耗可以用作 mtDNA 消耗的替代物，因为 mtDNA 编码 13 个蛋白质亚基，这些亚基是 OXPHOS 系统 5 个复合物中的 4 个的重要组成部分。此外，最准确反映线粒体功能障碍的是功能性 OXPHOS 酶的水平，而不是 mtDNA 水平。最后，试纸测定与用于直接测量 mtDNA 损耗的实时 PCR 不同，简单快速、廉价且能够实现高通量。我们的原型复合物 I 特异性试纸可以检测含有少至 0.1ug 线粒体或少至 1x105 溶解的全细胞（成纤维细胞或外周血单核细胞）的样品中的复合物 I。原型试纸还可以检测 mtDNA 耗尽的影响，即 mtDNA 耗尽的人成纤维细胞缺乏复合物 I。我们将研究用试纸定量蛋白质水平的 2 种不同方法，1 无需仪器，仅使用廉价的自报告一次性试纸，第二个结合了廉价的量油尺读取器以提供更大的决策。这些测试将使用培养的人类成纤维细胞和来自临床相关组织（例如 PBMC 和皮下脂肪组织）的对照样本，针对灵敏度、准确性、可靠性和动态范围进行优化。