Antibody Generation of ROS and Macular Degeneration

描述(申请人提供)：年龄相关性黄斑变性(AMD)是发展中国家盲目注册的主要原因，但其发病机制仍知之甚少。然而，越来越多的证据支持免疫系统和氧化应激在疾病进展中的作用，这些作用源于超氧阴离子、过氧化氢和单线态氧等活性氧物种(ROS)的产生和破坏的失衡。也有明确的证据表明，通过吸收紫外线和可见光，储存在视网膜色素上皮(RPE)细胞中的脂褐素中的生色团被光化学敏化，导致ROS的产生和老化眼睛的损害。我们最近已经证明，所有的抗体分子，无论是物种还是抗原决定簇，在化学或光化学来源的单线态氧存在下，都有一种内在的能力来产生一系列有效的氧化剂。抗体催化的水氧化途径(ACWOP)的最终产物是过氧化氢，因此，这一途径通过拦截光化学产生的单线态氧在AMD中发挥作用的可能性是真实的，并将导致整体氧化应激的增加。我们打算通过以下三个具体目标来研究这一假说：1.具体目标1，从细胞毒性、细胞凋亡和热休克蛋白(HSP)的表达方面，确定ACWOP产生的氧化剂是否对传代的人RPE细胞或原代培养的人RPE细胞造成损伤。2.特殊目的#2确定A2E负载的RPE细胞能否通过光照射激活ACWOP，以及这种激活是否增强了这些传代和原代培养的人RPE细胞的光化学损伤。3.确定ACWOP在全血、血浆和血清中激活时是否会影响RPE细胞的存活

DESCRIPTION (provided by applicant): Age-related macular degeneration (AMD) is the leading cause of blind registration in the developing world and yet its pathogenesis remains poorly understood. However, there is increasing evidence to support a role for the immune system and oxidative stress, arising from an imbalance in the production and destruction of reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide and singlet oxygen, in disease progression. There is also clear evidence that photochemical sensitization of chromophores stored within lipofuscin in the retinal pigment epithelial (RPE) cells via absorption of both ultraviolet and visible light, leads to ROS generation and damage in the ageing eye. We have recently shown that all antibody molecules, regardless of species or antigenic determinant, have an intrinsic ability to generate a cascade of potent oxidants when presented with either a chemical or photochemical source of singlet dioxygen. The end product of the antibody-catalyzed water-oxidation pathway (ACWOP) is hydrogen peroxide and thus the implication for this pathway to play a role in AMD via interception of photochemically generated singlet oxygen is a real possibility and would lead to an increase in overall oxidative stress. We intend to investigate this hypothesis with the three following specific aims: 1. Specific Aim #1 To determine whether oxidants generated by the ACWOP, are damaging to passaged human RPE cells or primary cultured human RPE cells, in terms of cytotoxicity, apoptosis and expression of heat shock protein (Hsp) 27. 2. Specific Aim #2 To determine whether the ACWOP can be activated by photoirradiation of A2E-loaded RPE cells and whether such activation enhances photochemical damage to these passaged and primary cultures of human RPE cells. 3. Specific Aim #3 To determine whether the ACWOP when activated in whole blood, plasma and serum can affect RPE cell survival