乳铁蛋白-DNA复合物检测评价甲型H1N1重症流感患者血浆中性粒细胞胞外诱捕网水平
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摘要
目的建立乳铁蛋白-DNA复合物的相对定量检测方法,评价甲型H1N1重型流感及其他病原体感染者血浆样本中性粒细胞胞外诱捕网(NETs)的含量。方法将DNA含量明确的血浆样本进行梯度稀释作为标准品,应用酶联免疫吸附法(ELISA)检测血浆中乳铁蛋白-DNA复合物含量。采用乳铁蛋白-DNA ELISA方法比较19例甲型H1N1重症流感患者与19例献血者(对照组)血浆乳铁蛋白-DNA复合物含量差异。结果甲型H1N1重型重症流感患者血浆中乳铁蛋白-DNA复合物水平与游离DNA(cf DNA)和组蛋白-DNA复合物水平均呈正相关(r=0.6763、P <0.0001,r=0.7560、P <0.0001)。甲型H1N1重症流感患者血浆中乳铁蛋白-DNA复合物含量为[2.082(1.169,5.021)μg/ml],显著高于对照组[0.233(0.170,0.376)μg/ml],差异有统计学意义(P <0.0001),且乳铁蛋白-DNA复合物含量与甲型H1N1重症流感患者的急性生理与慢性健康评分(APACHEⅡ评分)呈正相关(r=0.7379、P=0.0005)。结论血浆乳铁蛋白-DNA复合物含量可用于评价甲型H1N1重症流感患者NETs水平。
Objective To establish a relative quantitative assay for lactoferrin-DNA complex, and to assess the levels of neutrophil extracellular traps(NETs)in plasma samples from patients with severe influenza A(H1N1) and other pathogen infectious diseases. Methods Plasma samples with clear DNA content were serially diluted as a standard, and the level of lactoferrin-DNA complex in plasma was determined by Enzymelinked immunosorbent assay(ELISA). The lactoferrin-DNA ELISA method was used to compare the difference of plasma lactoferrin-DNA complex between 19 patients with severe influenza A(H1N1) and 19 blood donors(control group). Results Level of plasma lactoferrin-DNA complex of patients in severe influenza A(H1N1)was positively correlated with that of cfDNA(cell-free DNA) and histone-DNA complex, respectively(r =0.6763, P < 0.0001; r = 0.756, P < 0.0001). The plasma lactoferrin-DNA complex content in patients with severe influenza A(H1N1) was 2.082(1.169, 5.021) μg/ml, which was significantly higher than that of the controls [0.233(0.170, 0.376) μg/ml, P < 0.0001], and positively correlated with Acute Physiological and Chronic Health Evaluation Ⅱ score(APACHE Ⅱ score)(r = 0.7379, P = 0.0005). Conclusion Plasma lactoferrin-DNA complex level could be used to assess NETs level in patients with severe influenza A(H1N1).
Objective To establish a relative quantitative assay for lactoferrin-DNA complex, and to assess the levels of neutrophil extracellular traps(NETs)in plasma samples from patients with severe influenza A(H1N1) and other pathogen infectious diseases. Methods Plasma samples with clear DNA content were serially diluted as a standard, and the level of lactoferrin-DNA complex in plasma was determined by Enzymelinked immunosorbent assay(ELISA). The lactoferrin-DNA ELISA method was used to compare the difference of plasma lactoferrin-DNA complex between 19 patients with severe influenza A(H1N1) and 19 blood donors(control group). Results Level of plasma lactoferrin-DNA complex of patients in severe influenza A(H1N1)was positively correlated with that of cfDNA(cell-free DNA) and histone-DNA complex, respectively(r =0.6763, P < 0.0001; r = 0.756, P < 0.0001). The plasma lactoferrin-DNA complex content in patients with severe influenza A(H1N1) was 2.082(1.169, 5.021) μg/ml, which was significantly higher than that of the controls [0.233(0.170, 0.376) μg/ml, P < 0.0001], and positively correlated with Acute Physiological and Chronic Health Evaluation Ⅱ score(APACHE Ⅱ score)(r = 0.7379, P = 0.0005). Conclusion Plasma lactoferrin-DNA complex level could be used to assess NETs level in patients with severe influenza A(H1N1).
引文
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[24]Yoo DG,Floyd M,Winn M,et al.NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes[J].Immunol Lett,2014,160(2):186-194.
[25]Knaus WA,Draper EA,Wagner DP,et al.APACHEⅡ:a severity of disease classification system[J].Crit Care Med,1985,13(10):818-829.
[2]Lefrancais E,Mallavia B,Zhuo H,et al.Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury[J].JCIInsight,2018,3(3):e98178.
[3]Czaikoski PG,Mota JM,Nascimento DC,et al.Neutrophil extracellular traps induce organ damage during experimental and clinical sepsis[J].PLoS One,2016,11(2):e148142.
[4]Sorensen OE,Borregaard N.Neutrophil extracellular traps--the dark side of neutrophils[J].J Clin Invest,2016,126(5):1612-1620.
[5]Zhu L,Liu L,Zhang Y,et al.High level of neutrophil extracellular traps correlates with poor prognosis of severe influenza A infection[J].J Infect Dis,2018,217(3):428-437.
[6]Papayannopoulos V.Neutrophil extracellular traps in immunity and disease[J].Nat Rev Immunol,2018,18(2):134-147.
[7]Hampson P,Dinsdale RJ,Wearn CM,et al.Neutrophil dysfunction,immature granulocytes,and cell-free DNA are early biomarkers of sepsis in burn-injured patients:A prospective observational cohort study[J].Ann Surg,2017,265(6):1241-1249.
[8]Vong L,Sherman PM,Glogauer M.Quantification and visualization of neutrophil extracellular traps(NETs)from murine bone marrow-derived neutrophils[M]//Allen IC.Methods in Molecular Biology:Mouse Models of Innate Immunity.New Jersey:Humana Press,2013,1031:41-50.
[9]van der Strate BW,Beljaars L,Molema G,et al.Antiviral activities of lactoferrin[J].Antiviral Res,2001,52(3):225-239.
[10]Wakabayashi H,Oda H,Yamauchi K,et al.Lactoferrin for prevention of common viral infections[J].J Infect Chemother,2014,20(11):666-671.
[11]Sivanandham R,Brocca-Cofano E,Krampe N,et al.Neutrophil extracellular trap production contributes to pathogenesis in SIV-infected nonhuman primates[J].J Clin Invest,2018,128(11):5178-5183
[12]钟南山,李兰娟,王辰,等.甲型H1N1流感诊疗方案(2009年第3版)[J].中华医学杂志,2009,89(45):559.
[13]Paules C,Subbarao K.Influenza[J].Lancet,2017,390(10095):697-708.
[14]Mantovani A,Cassatella MA,Costantini C,et al.Neutrophils in the activation and regulation of innate and adaptive immunity[J].Nat Rev Immunol,2011,11(8):519-531.
[15]Mayadas TN,Cullere X,Lowell CA.The multifaceted functions of neutrophils[J].Annu Rev Pathol,2014,9:181-218.
[16]Matthay MA,Ware LB,Zimmerman GA.The acute respiratory distress syndrome[J].J Clin Invest,2012,122(8):2731-2740.
[17]Porto BN,Stein RT.Neutrophil Extracellular Traps in Pulmonary Diseases:Too Much of a Good Thing?[J].Front Immunol,2016,7:311.
[18]Narayana MA,Narasaraju T,Rai P,et al.In vivo and in vitro studies on the roles of neutrophil extracellular traps during secondary pneumococcal pneumonia after primary pulmonary influenza infection[J].Front Immunol,2013,4:56.
[19]Narasaraju T,Yang E,Samy RP,et al.Excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis[J].Am J Pathol,2011,179(1):199-210.
[20]Maruchi Y,Tsuda M,Mori H,et al.Plasma myeloperoxidaseconjugated DNA level predicts outcomes and organ dysfunction in patients with septic shock[J].Crit Care,2018,22(1):176.
[21]朱鏐娈,张玥,李国力,等.建立体外诱导和检测中性粒细胞胞外诱捕网的方法[J/CD].中华实验和临床感染病杂志(电子版),2015(3):405-408.
[22]Hamaguchi S,Akeda Y,Yamamoto N,et al.Origin of circulating free DNA in sepsis:analysis of the CLP mouse model[J].Mediat Inflamm,2015,2015:1-9.
[23]Bronkhorst AJ,Aucamp J,Pretorius PJ.Cell-free DNA:Preanalytical variables[J].Clin Chim Acta,2015,450:243-253.
[24]Yoo DG,Floyd M,Winn M,et al.NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes[J].Immunol Lett,2014,160(2):186-194.
[25]Knaus WA,Draper EA,Wagner DP,et al.APACHEⅡ:a severity of disease classification system[J].Crit Care Med,1985,13(10):818-829.