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lijunqing525金虫 (正式写手)
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英译汉 麻烦帮翻译下
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Due to the complex interplay between platelets and monocytes, both alone and together, during immune pathogenesis of HIV infection, it is essential to devise a better methodology to detect PMCs. With this goal in mind, a method to quantitate PMCs in whole blood was standardized and was subsequently employed to assess the percentages of circulating PMCs in blood collected from persons infected with HIV. Although several previous reports have enumerated PMCs using flow cytometry in regard to various disease conditions, the method-ologies used thus far have failed to consider spontaneous platelet activation and the resulting consequence on de novo PMC forma-tion (Gkaliagkousi et al., 2009; Joseph et al., 2001; Ogura et al., 2001; Passacquale et al., 2011; Sevush et al., 1998). Thus, in the efforts to formulate an optimized method for detection of PMCs, fixing the blood samples immediately following collection was given prime importance, as it helped to conserve PMCs and avoid experimen-tal artifacts. Demonstrated herein, is a fix–wash–lyse–wash–stain method to fluorescently label blood samples, which when used along with a progressive gating strategy and FMO (fluorescence minus one) controls, allows for efficient enumeration of PMCs. The additional wash step performed after sample fixation was neces-sary to avoid the toxicity caused by excessive exposure to PFA. One significant concern in adopting this method was that the fixing of samples prior to staining might alter the antibody binding capacity to some extent; however this was deemed a reasonable trade-off given the advantages of this method as outlined above. Nonetheless, in vitro whole blood treatments with LPS and colla-gen were performed, to serve as monocyte and platelet activating reagents, respectively, in order to ensure that it is possible to cap-ture the changes in PMC percentages induced by these treatments using the method of detection described here. Results indicated that platelet–monocyte interaction increased significantly upon platelet activation as compared to monocyte activation. These results corroborate an earlier report by Rinder et al. who showed that platelet–leukocyte complexes increased upon platelet acti-vation and that this interaction was dependent on monocyte activation only to a very limited extent (Rinder et al., 1991). Upon validation of the procedure, persons with or without HIV infection (without any incidence of cardiovascular disease at least one year prior to enrollment in the study) were enrolled to assess whether HIV infection had any effect on platelet–monocyte inter-actions. All of the individuals enrolled in the study were on ART and had low viral load with CD4+ T cell count above 500 cells/mm 3(data not shown). The results show that despite the viral suppres-sion induced by successful ART, the number of circulating PMCs was significantly higher in the inflammatory monocyte subset (i.e. CD16+ monocytes) of these individuals as compared to healthy indi-viduals without HIV infection, and correlated positively with the extent of platelet activation. The PMC percentages in the classi-cal CD16− monocyte subset were also higher in samples obtained from persons infected with HIV, although the effects were not sta-tistically different from controls. Consistently, our data (not shown) and previous reports (Pulliam et al., 1997; Thieblemont et al., 1995) demonstrate a significant increase in CD16+ monocyte percentages in samples obtained from persons infected with HIV. This study defines a flow cytometric method to quantify PMCs in clinical specimens. The findings suggest that persons infected with HIV have increased levels of platelet–monocyte complexes, particularly within the inflammatory monocyte population despite successful ART. Furthermore, this data suggests that either platelets have an increased preference to associate with CD16+ mono-cytes, or that alternatively, the platelet–monocyte cross-talk causes maturation of classical monocytes to the CD16+ phenotype; thus warranting further investigation into whether this interaction enhances the monocyte capacity to cross the blood brain barrier, as well as the role of these monocytes in HIV associated neuro-cognitive impairment. Acknowledgements |
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xzx8066
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爱与雨下: 金币+10 2012-04-10 16:43:22
lijunqing525: 金币+100, 翻译EPI+1, ★有帮助 2012-04-11 22:37:21
爱与雨下: 金币+10 2012-04-10 16:43:22
lijunqing525: 金币+100, 翻译EPI+1, ★有帮助 2012-04-11 22:37:21
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在人类免疫缺陷病毒（HIV）感染的免疫发病机制中，血小板和单核细胞相互作用，无论是单独作用还是共同作用，都非常复杂，因此很有必要建立一个能更好地检测外周血单核细胞（PMC）的方案。怀着这个目标，我们将一个定量检测全血中PMCs的方法进行标准化，随后应用它来评定HIV感染者血液标本中循环PMCs百分比。 尽管以前有几个关于应用流式细胞仪计数不同疾病状态PMCs的研究报道(Gkaliagkousi et al., 2009; Joseph et al., 2001; Ogura et al., 2001; Passacquale et al., 2011; Sevush et al., 1998)，然而他们应用的方法不能解决自发性血小板激活导致的初始PMC形成这个问题。要构想出最佳的检测PMCs的方法，血液标本采集后迅速固定极其重要，因为这样有助于保护PMCs，而且可避免实验操作导致的人为破坏。 这里介绍的是一种“固定-冲洗-细胞溶解-冲洗-染色”方法检测荧光标记血液标本。当与渐进门控技术和荧光减一控制技术一起应用时，它可以高效地计数PMCs。样本固定后冲洗这一步骤对于避免暴露多聚甲醛（PFA）过多导致的毒性是很有必要的。采用这种方法时一个非常重要的顾虑就是染色之前固定样本在某种程度上可能会改变抗体的结合能力，然而相对于这种方法的优势来说，这可以认为是一个合理的折衷。虽然如此，体外应用脂多糖（LPS）和胶原蛋白（作为单核细胞和血小板的激活剂）分别处理全血标本，应用这里描述的方法确保能捕捉到这些激活剂诱导的PMC百分比的变化。 结果显示，与激活单核细胞相比，激活血小板可引起血小板-单核细胞相互作用明显增加。Rinder等曾研究发现激活血小板明显增加血小板-白细胞复合体，这种相互作用很少依赖单核细胞激活(Rinder et al., 1991)。本研究结果与这是一致的。 为了评估这种方法的有效性，本研究纳入HIV感染和非HIV感染个体（所有个体纳入研究前，至少1年内没有任何心血管疾病史）评价HIV感染是否对血小板-单核细胞相互作用有影响。本研究纳入的所有个体均接受抗逆转录病毒治疗（ART），处于低病毒载量且CD4+ T细胞含量在500个细胞/mm3时期（数据未显示）。 结果显示，尽管有效的ART会导致病毒抑制，与无HIV感染的健康个体相比，这些个体循环PMCs中炎性单核细胞亚群（如CD16+单核细胞）数量明显增加，并且与血小板激活程度呈正相关。HIV感染者样本中经典单核细胞（CD16-）亚群百分比有所增高，然而与对照组相比无统计学差异。一致地，我们的结果（未显示）和以前研究（Pulliam et al., 1997; Thieblemont et al., 1995）结果表明HIV感染者样本中CD16+单核细胞百分比明显增加。 这个研究精确地描述了一个量化计数临床样本中PMCs的流式方法。 即使是在有效的ART情况下，这些结果显示HIV感染者血小板-单核细胞复合体水平明显增加，尤其是炎性单核细胞亚群。此外，这些结果还显示血小板优先与CD16+单核细胞增加相关，或者CD16+单核细胞优先与血小板增加相关；而且血小板-单核细胞交互作用促使经典单核细胞转变为成熟单核细胞（CD16+）。进而，这可确保我们进一步研究这种相互作用是否会增加单核细胞穿透血脑屏障的能力，以及这些单核细胞在HIV感染相关神经认知损害中作用。致谢！ |
2楼2012-04-10 16:15:59
