DEVELOPMENT OF BRIDGE LOOP RESONATORS FOR IN VIVO L BAND STUDIES

开发了一种新的基于EPR的方法来获得选择性 关于特定细胞内隔室中PO2的信息 (噬菌体)。该方法利用选择性地合并 氧敏探针4-(三甲铵) 2，2，6，6，-四甲基哌啶-D17-1-氧基碘(D-CAT1) 酵母多糖刺激的巨噬细胞吞噬小体。因为这个信号 由中性氮氧化物产生 4-氧代-2，2，6，6，-(15N)-四甲基哌啶-D16-1-氧基(15N-PDT) 与D-CAT1的信号不重叠，可以监控这些信号 同时。脂多糖(LPS)是一种来自于 革兰氏阴性细菌的外膜，与 感染性休克患者的高发病率和高死亡率。我们的 先前的研究表明，内毒素可以影响线粒体 在各种细胞类型中的氧气消耗和改变氧气 实验性感染性休克中器官的利用。它也是 提示内毒素可以增强与以下相关的呼吸爆发 巨噬细胞的吞噬作用。将D-CAT1加入到小鼠的细胞中 酵母多糖刺激巨噬细胞系RAW-264.7 诱导吞噬作用。清洗后，探头仍留在 在没有刺激吞噬作用的情况下，探针不能 结合在一起，可以通过洗涤去除。将15N-PDT添加到 相同的样本提供有效的胞外氧气 浓度(因为只有不到5%的PDT信号来自内部 细胞)。吞噬体内D-CAT1和15N-PDT的细胞 用内毒素处理，并测量氧浓度 两者计算后的吞噬体内和细胞外部位 在纯净的空气和氮气中探测。结果表明，内毒素可降低 吞噬体内的氧气浓度几乎降低了一半。 此外，EPR自旋捕获实验(使用DMPO捕获-OH 和-哦自由基)表明，内毒素刺激持续的呼吸 在这些巨噬细胞中破裂，高于单独酵母多糖诱导的巨噬细胞。 这些结果表明，内毒素可以影响巨噬细胞的吞噬功能， 在某些情况下，吞噬小体内的低氧浓度 可能会限制巨噬细胞的杀微生物活性 感染。

A new EPR-based method has been developed to obtain selective information on pO2 in a specified intracellular compartment (phagosome). The method utilises the selective incorporation of the oxygen-sensitive probe 4-(Trimethylammonium) 2,2,6,6,-tetramethylpiperidine-D17-1-Oxyl iodide (D-CAT1) into phagosomes of macrophages stimulated with zymosan. Since the signal arising from the neutral nitroxide 4-oxo-2,2,6,6,-(15N)-tetramethylpiperidine-D16-1-Oxyl (15N-PDT) does not overlap with that from the D-CAT1, these signals can be monitored simultaneously. Lipopolysaccharide (LPS) is the endotoxin from the outer membrane of Gram-negative bacteria that is associated with the high morbidity and mortality in patients with septic shock. Our previous studies have shown that LPS can influence mitochondrial oxygen consumption in a variety of cell types and alter the oxygen utilisation of organs in experimental septic shock. It is also suggested that LPS can augment the respiratory burst associated with phagocytosis in macrophages. D-CAT1 was added to cells of the murine macrophage cell lines RAW 264.7 followed by zymosan stimulation to induce phagocytosis. After washing, the probe remained in the phagosome; without stimulation of phagocytosis the probe was not incorporated and could be removed by washing. 15N-PDT was added to the same samples to give the effective extracellular oxygen concentration (as less than 5% of the PDT signal arises from inside the cells). Cells with intraphagosomal D-CAT1 and 15N-PDT were then treated with LPS and the oxygen concentrations measured for intraphagosomal and extracellular sites after callibration of the two probes in pure air and nitrogen. The results show that LPS reduces intraphagosomal oxygen concentrations by almost one half. Furthermore, EPR spin-trapping experiments (using DMPO to trap - OH and -OOH radicals) showed that LPS stimulates a sustained respiratory burst in these macrophages above that induced by a zymosan alone. These results suggest that LPS can influence macrophage phagocytosis, and in certain cases the low oxygen concentration within phagosomes can potentially limit macrophage microbicidal funtion during infections.