Mitochondrial dysfunction in HAART: Point of care tests

描述（由申请人提供）：HAART是HIV感染的有效治疗方法，但可能导致严重的代谢并发症，包括乳酸中毒、脂肪营养不良、周围神经病变和心肌病。有相当多的证据表明，代谢并发症是HAART的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)诊断工具来监测由此产生的线粒体功能障碍。解决这两种需求可以为指导治疗提供理论依据和手段，以便在严重的临床并发症发生之前对HAART进行修改。为此，我们建议建立一套简单的基于试纸的免疫测定方法，将首先用于研究治疗对能量代谢关键线粒体酶的影响，然后发展为护理点诊断。测量的酶构成氧化磷酸化系统（OXPHOS）负责提供几乎所有的细胞ATP。这些酶复合物不仅对线粒体功能至关重要，而且由于mtDNA的耗尽，它们在HAART中也受到影响。我们已经证明，OXPHOS复合物的消耗可以作为mtDNA消耗的替代品，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的水平。最后，与直接测量mtDNA损耗的实时PCR不同，试纸法简单、快速、廉价且具有高通量。我们的原型复合物I特异性试纸可以在含有少至0.1ug线粒体或少至1x105溶解全细胞（成纤维细胞或外周血单个核细胞）的样品中检测复合物I。原型量尺还可以检测mtdna缺失的影响，即mtdna缺失的人类成纤维细胞缺乏复合体i。我们将研究2种不同的方法来用量尺量化蛋白质水平，1种不需要仪器，只使用廉价的自我报告一次性量尺，第二种结合廉价的量尺读取器来提供更大的决策。测试将优化灵敏度、准确性、可靠性和动态范围，使用培养的人类成纤维细胞和来自临床相关组织（如pbmc和皮下脂肪组织）的对照样本。

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.