Diamond R D, Clark R A, Haudenschild C C
J Clin Invest. 1980 Nov;66(5):908-17. doi: 10.1172/JCI109958.
In previous studies, we noted that Candida hyphae and pseudohyphae could be damaged and probably killed by neutrophils, primarily by oxygen-dependent nonphagocytic mechanisms. In extending these studies, amount of damage to hyphae again was measured by inhibition of [(14)C]cytosine uptake. Neutrophils from only one of four patients with chronic granulomatous disease damaged hyphae at all, and neutrophils from this single patient damaged hyphae far less efficiently than simultaneously tested neutrophils from normal control subjects. Neutrophils from neither of two subjects with hereditary myeloperoxidase deficiency damaged the hyphae. This confirmed the importance of oxidative mechanisms in general and myeloperoxidase-mediated systems in particular in damaging Candida hyphae. Several potentially fungicidal oxidative intermediates are produced by metabolic pathways of normal neutrophils, but their relative toxicity for Candida hyphae was previously unknown. To help determine this, cell-free in vitro systems were used to generate these potentially microbicidal products. Myeloperoxidase with hydrogen peroxide, iodide, and chloride resulted in 91.2% damage to hyphal inocula in 11 experiments. There was less damage when either chloride or iodide was omitted, and no damage when myeloperoxidase was omitted or inactivated by heating. Azide, cyanide, and catalase (but not heated catalase) inhibited the damage. Systems for generation of hydrogen peroxide could replace reagent hydrogen peroxide in the myeloperoxidase system. These included glucose oxidase, in the presence of glucose, and xanthine oxidase, in the presence of either hypoxanthine or acetaldehyde. In the presence of myeloperoxidase and a halide, the toxicity of the xanthine oxidase system was not inhibited by superoxide dismutase and, under some conditions, was marginally increased by this enzyme. This suggested that superoxide radical did not damage hyphae directly but served primarily as an intermediate in the production of hydrogen peroxide. The possible damage to hyphae by singlet oxygen was examined using photoactivation of rose bengal. This dye damaged hyphae in the presence of light and oxygen. The effect was almost completely inhibited by putative quenchers of singlet oxygen: histidine, tryptophan, and 1,4-diazobicyclo[2.2.2]octane. These agents also inhibited damage to hyphae by myeloperoxidase, halide, and either hydrogen peroxide or a peroxide source (xanthine oxidase plus acetaldehyde). Myeloperoxidase-mediated damage to hyphae was also inhibited by dimethyl sulfoxide, an antioxidant and scavenger of the hydroxyl radical. These data support the involvement of oxidative mechanisms and the myeloperoxidase-H(2)O(2)-halide system, in particular in damaging hyphae in vitro and perhaps in vivo as well.
在先前的研究中，我们注意到念珠菌的菌丝和假菌丝可能会被中性粒细胞破坏甚至杀死，主要是通过氧依赖的非吞噬机制。在拓展这些研究时，对菌丝的损伤程度再次通过抑制[（14）C]胞嘧啶摄取来测定。在四名慢性肉芽肿病患者中，只有一名患者的中性粒细胞能够损伤菌丝，而且该患者的中性粒细胞损伤菌丝的效率远低于同时检测的正常对照受试者的中性粒细胞。两名遗传性髓过氧化物酶缺乏症患者的中性粒细胞均未损伤菌丝。这证实了氧化机制总体上的重要性，尤其是髓过氧化物酶介导的系统在损伤念珠菌菌丝方面的重要性。正常中性粒细胞的代谢途径会产生几种潜在的杀真菌氧化中间体，但它们对念珠菌菌丝的相对毒性此前尚不清楚。为了帮助确定这一点，使用无细胞体外系统来生成这些潜在的杀微生物产物。在11次实验中，髓过氧化物酶与过氧化氢、碘化物和氯化物一起导致菌丝接种物91.2%的损伤。省略氯化物或碘化物时损伤较少，省略髓过氧化物酶或加热使其失活时则无损伤。叠氮化物、氰化物和过氧化氢酶（但不是加热的过氧化氢酶）抑制了这种损伤。过氧化氢生成系统可以替代髓过氧化物酶系统中的试剂过氧化氢。这些系统包括葡萄糖存在下的葡萄糖氧化酶，以及次黄嘌呤或乙醛存在下的黄嘌呤氧化酶。在存在髓过氧化物酶和卤化物的情况下，黄嘌呤氧化酶系统的毒性不受超氧化物歧化酶的抑制，并且在某些条件下，该酶会使其略有增加。这表明超氧阴离子自由基不会直接损伤菌丝，而是主要作为过氧化氢生成过程中的中间体。使用孟加拉玫瑰红的光活化来检测单线态氧对菌丝可能的损伤。这种染料在光和氧存在的情况下会损伤菌丝。这种效应几乎完全被假定的单线态氧猝灭剂抑制：组氨酸、色氨酸和1,4 - 二氮杂双环[2.2.2]辛烷。这些试剂也抑制髓过氧化物酶、卤化物以及过氧化氢或过氧化物源（黄嘌呤氧化酶加乙醛）对菌丝的损伤。髓过氧化物酶介导的对菌丝的损伤也被二甲基亚砜抑制，二甲基亚砜是一种抗氧化剂和羟基自由基清除剂。这些数据支持氧化机制以及髓过氧化物酶 - H₂O₂ - 卤化物系统的参与，特别是在体外损伤菌丝，或许在体内也是如此。
