利用合成六肽对急性破坏性肺炎患儿中性粒细胞两个亚群的实验性体外表型重编程

I. Nesterova, V. N. Chapurina, G. Chudilova, V. Tarakanov
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引用次数: 0

摘要

中性粒细胞的效应功能障碍通常与抗菌免疫防御中失调过程的发生有关。急性破坏性肺炎是一种严重的脓性炎症性疾病,与中性粒细胞效应机制的功能失调和负转化细胞亚群的出现有关。因此,寻找新的实验方法,旨在通过各种免疫物质重新定位急性破坏性肺炎儿童中性粒细胞不同亚群的负性表型改变是非常重要的。本研究的目的是评估人工合成六肽(精氨酸- α -天冬氨酸-赖氨酸-缬氨酸-酪氨酸-精氨酸)对非典型急性破坏性肺炎患儿中性粒细胞主要亚群(CD16+CD64-CD32+CD11b+)和次要亚群(CD16+CD64+CD32+CD11b+)的含量和表型的调节作用。我们检测了10例急性破坏性肺炎患儿的20份外周血样本和20例2-4岁健康儿童的40份血液样本。根据膜受体的表达密度,按照MFI标准,将中性粒细胞分为2个亚群进行免疫分型。用Hexapeptide (10-6 g/L;37℃,60分钟)。在急性破坏性肺炎患儿中,与有条件健康儿童相比,中性粒细胞亚群的阴性转化存在以下变异:主要亚群的比例显著下降,即从98.0%(96.9-98.7)%降至55.8% (35.3-74.8)%,MFI显示CD16和CD11b密度表达降低,次要中性粒细胞亚群的比例显著增加。从1.3(0.4-1.6)%增加到52.6 (41.8-54.9)%,CD11b受体表达增加,CD64表达减少。六肽对急性破坏性肺炎儿童中性粒细胞的免疫调节作用已在封闭的体外系统中得到证实,在没有显著定量变化的情况下,两种细胞亚群的表型重塑均呈阳性。因此,在用六肽治疗后,我们发现激活受体CD16、CD11b在主要亚群中的表达显著增加,而在次要亚群中的表达显著降低至健康儿童的典型水平。同时,六肽不影响健康儿童中性粒细胞亚群的研究,除了在小亚群中CD64表达增加。获得的数据可用于未来开发靶向免疫治疗的新方法,旨在纠正儿童急性破坏性肺炎中性粒细胞亚群的表型。
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Experimental in vitro phenotype reprogramming of two subsets of neutrophilic granulocytes in children with acute destructive pneumonia by means of a synthetic hexapeptide
Effector dysfunctions of neutrophil granulocytes are often associated with the occurrence of dysregulatory processes in the antibacterial immune defense. Acute destructive pneumonia is a severe purulent-inflammatory disease associated with discordant functions of effector mechanisms of neutrophil granulocytes and emergence of negatively transformed cell subsets. Therefore, the search for new experimental approaches aimed at re-orientation of negatively altered phenotype of distinct subsets of neutrophilic granulocytes in the children with acute destructive pneumonia by means of various immunotropic substances is quite relevant. The aim of the study was to evaluate the modulating effects of synthetic hexapeptide (Arginyl-alpha-Aspartyl-Lysyl-Valyl-Tyrosyl-Arginine) on the contents and phenotype of 2 functionally significant subsets of major (CD16+CD64-CD32+CD11b+) and minor (CD16+CD64+CD32+CD11b+) subpopulations of neutrophils in a closed in vitro experimental system sampled in the children with atypical acute destructive pneumonia. We have examined twenty peripheral blood samples from 10 children with acute destructive pneumonia, and 40 blood samples of 20 healthy children 2-4 years old. Immunophenotyping of neutrophil granulocytes classified in 2 subsets was performed on the basis of expression density of membrane receptors, according to MFI criteria. Phenotypic features of neutrophil granulocyte subsets were evaluated in the in vitro system before and after incubation of peripheral blood with Hexapeptide (10-6 g/L; 37 C, 60 min). In children with acute destructive pneumonia, compared with conditionally healthy children, the following variants of negative transformation of the neutrophil subsets were established: a significant decrease in the ratios of the major subset, i.e., from 98.0 (96.9-98.7) % o 55.8 (35.3-74.8) %, with a decreased CD16 and CD11b density expression according to MFI, and a significantly increase ratio of the minor neutrophil subset: from 1.3 (0.4-1.6) % to 52.6 (41.8-54.9) %, with increased expression of CD11b receptor, and a decrease in CD64 expression. Immunomodulatory effects of Hexapeptide upon neutrophil granulocytes of children with acute destructive pneumonia have been demonstrated in the closed in vitro system showing positive phenotype remodeling of both cell subsets in the absence of significant quantitative changes. Thus, upon treatment with the hexapeptide, we have found a significantly increased expression of activation receptors CD16, CD11b in the major subset, and a significant decrease in their expression for the minor subset to the levels typical to healthy children. At the same time, hexapeptide did not affect the studied subsets of neutrophils from healthy children, except of increased CD64 expression in the minor subset. The obtained data can be used in future to develop new approaches to the targeted immunotherapy aimed at correcting the phenotype of neutrophil granulocyte subsets in acute destructive pneumonia in children.
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