建立NGAL诊断阈值有利于临床判断和发现急性肾功能损伤

为探讨中性粒细胞相关载脂蛋白(neutrophil gelatinase-associated lipocalin, NGAL)检测在肝胆疾病患者继发急性肾功能损伤(acute kidney injury, AKI)中的诊断性能,本研究回顾性收集了476例肝胆疾病患者与225例健康受试者作为实验组,根据血清中性粒细胞相关载脂蛋白(NGAL)、血清肌酐(serum creatinine,s Cr)、尿素(Urea)、半胱氨酸蛋白酶抑制剂C (cystatin C, CysC)、估算肾小球滤过率(estimating glomerular filtration rate, eGFR)水平和尿量,分为急性肾功能损伤(AKI)组和高风险(high-risk, HR)组、低风险(low-risk,LR)组和对照(health control, HC)组,进而建立上述血清指标在急性肾功能损伤(AKI)诊断性能最大时的判断界值;随后,选取145例肝胆疾病患者作为验证组,以评估各观察指标对肝胆疾病继发急性肾功能损伤(AKI)的诊断性能。结果表明:本实验组中各亚组间所观察指标之血清水平均有统计学差异(p<0.05)。中性粒细胞相关载脂蛋白(NGAL)诊断性能最大时的判断界值为205.2μg/L。验证组患者各指标阳性检出率,在低风险(LR)亚组中中性粒细胞相关载脂蛋白(NGAL)均高于其它指标(p<0.05),但在高风险(HR)亚组中中性粒细胞相关载脂蛋白(NGAL)仅高于血清肌酐(sCr)(p<0.05),而与半胱氨酸蛋白酶抑制剂C (CysC)和估算肾小球滤过率(eGFR)无统计学差异(p>0.05);再经分层风险分析各指标预测高风险(HR)亚组患者急性肾功能损伤(AKI)发生的能力,中性粒细胞相关载脂蛋白(NGAL)的优势比(OR=21.0 (2.3, 192.8)),是半胱氨酸蛋白酶抑制剂C(CysC)或估算肾小球滤过率(eGFR)(OR (95%CI)=3.3 (0.7, 15.3))的6.4倍。本研究初步结论表明,肝胆疾病患者诊断急性肾功能损伤(AKI)时应考虑中性粒细胞相关载脂蛋白(NGAL)肝脏合成代谢的作用。因此实验室应建立适宜的中性粒细胞相关载脂蛋白(NGAL)诊断阈值以有利于临床准确判断及早期发现急性肾功能损伤(AKI)。     

先天性心脏病患儿术后急性肾损伤的影响因素及尿NGAL、KIM-1的诊断价值分析

摘要 目的：探讨影响先天性心脏病患儿术后急性肾损伤（AKI）的影响因素及尿中性粒细胞明胶酶相关脂质运载蛋白（NGAL）、肾损伤分子1（KIM-1）的诊断价值。方法：选择2018年1月至2019年12月我院心胸外科收治的60例先天性心脏病术后并发AKI患儿（AKI组）和同期收治的172例先天性心脏病术后未发生AKI患儿（NAKI组）作为研究对象。收集患儿临床基线资料,检测尿NGAL、KIM-1水平,采用Logistic回归分析先天性心脏病患儿术后发生AKI的影响因素,受试者工作特征曲线（ROC）分析尿NGAL、KIM-1诊断先天性心脏病患儿术后发生AKI的价值。结果：AKI组年龄、体重低于NAKI组（P＜0.05）,手术时间、心肺转流（CPB）时间、主动脉阻断（ACT）时间、机械通气时间、重症监护室（ICU）住院时间长于NAKI组（P＜0.05）,术后平均动脉压（MAP）、尿素氮（BUN）、血肌酐（Scr）、NGAL、KIM-1高于NAKI组（P＜0.05）。Logistic回归分析结果显示低龄、低体重、CPB时间长、高NGAL、KIM-1水平是先天性心脏病患儿术后发生AKI的危险因素（P＜0.05）。ROC分析显示尿NGAL、KIM-1诊断先天性心脏病患儿术后发生AKI的灵敏度分别为81.67%,83.33%,特异度分别为84.30%,87.79%。结论：低龄、低体重、CPB时间长、高NGAL、KIM-1水平是先天性心脏病患儿术后发生AKI的危险因素,尿NGAL、KIM-1诊断先天性心脏病术后AKI具有较高价值。     

红细胞分布宽度、中性粒细胞与淋巴细胞比值与晚期非小细胞肺癌患者临床病理特征及预后的关系研究

目的:探讨红细胞分布宽度(RDW)、中性粒细胞与淋巴细胞比值(NLR)与晚期非小细胞肺癌(NSCLC)患者临床病理特征及预后的关系。方法:选择2017年5月至2019年5月我院收治的106例确诊为晚期NSCLC患者(NSCLC组)和门诊接诊的102例体检正常者(对照组)作为研究对象。检测RDW、NLR,分析RDW、NLR与NSCLC患者临床病理特征以及预后的关系。结果:NSCLC组RDW、NLR高于对照组(P<0.05),年龄≥60岁、淋巴结转移NSCLC患者RDW高于年龄<60岁、无淋巴结转移NSCLC患者(P<0.05),TNM分期为Ⅳ期、淋巴结转移NSCLC患者NLR高于TNM分期为Ⅲ期、无淋巴结转移患者(P<0.05)。Kaplan-Meier生存曲线分析结果显示高RDW组、高NLR组NSCLC患者生存率低于低RDW组、低NLR组(P<0.05)。单因素COX回归分析显示分化程度、TNM分期、淋巴结转移、RDW、NLR与NSCLC预后有关(P<0.05),多因素COX回归分析显示淋巴结转移、RDW、NLR与NSCLC患者预后相关(P<0.05)。结论:晚期NSCLC患者RDW、NLR较高,RDW与年龄、淋巴结转移有关,NLR与TNM分期和淋巴结转移有关,高水平RDW、NLR预示着NSCLC预后不良,可作为预后评估的辅助指标。     

Salivary extracellular heat shock protein 70 (eHSP70) levels increase after 59 min of intense exercise and correlate with resting salivary secretory immunoglobulin A (SIgA) levels at rest

This study aimed to identify the response of a salivary stress protein, extracellular heat shock protein (eHSP70), to intense exercise and to investigate the relationship between salivary eHSP70 and salivary immunoglobulin A (SIgA) levels in response to exercise. Sixteen healthy sedentary young males (means ± SD 23.8 ± 1.5 years, 172.2 ± 6.4 cm, 68.3 ± 7.4 kg) performed 59 min of cycling exercise at 75 % VO2_max. Saliva and whole blood samples were collected before (Pre), immediately after (Post), and at 1, 2, 3, and 4 h after completion of the exercise (1, 2, 3, and 4 h). The salivary eHSP70 and SIgA levels were measured by enzyme-linked imunosorbent assay (ELISA), and the secretion rates were computed by multiplying the concentration by the saliva flow rate. White blood cells were analyzed using an automated cell counter with a direct-current detection system. The salivary eHSP70 secretion rates were 1.11 ± 0.86, 1.51 ± 1.47, 1.57 ± 1.32, 2.21 ± 2.04, 3.36 ± 2.72, and 6.89 ± 4.02 ng · min⁻¹ at Pre, Post, and 1, 2, 3, and 4 h, respectively. The salivary eHSP70 secretion rate was significantly higher at 4 h than that at Pre, Post, 1, and 3 h (p < 0.05). The SIgA secretion rates were 26.9 ± 12.6, 20.3 ± 10.4, 19.6 ± 11.0, 21.8 ± 12.8, 21.5 ± 11.9, and 21.9 ± 11.7 μg · min⁻¹ at Pre, Post, 1, 2, 3, and 4 h, respectively. The salivary SIgA secretion rate was significantly lower between 1 and 4 h than that at Pre (p < 0.05). There was a positive correlation between salivary eHSP70 and SIgA in both concentration and secretion rates before exercise (p < 0.05). The absolute number of white blood cells significantly increased after exercise, with a maximum at 2 h (p < 0.05). The neutrophil/lymphocyte ratio was significantly increased from 1 to 4 h when compared with that in the Pre samples (p < 0.05). The present study revealed that salivary eHSP70 significantly increased at 4 h after the 59 min of intense exercise in sedentary male subjects. Exercise stress can induce elevated salivary eHSP70 level and upregulate oral immune function partially.     

Cytoskeletal Regulation of CD44 Membrane Organization and Interactions with E-selectin

Interactions of CD44 on neutrophils with E-selectin on activated endothelial cells mediate rolling under flow, a prerequisite for neutrophil arrest and migration into perivascular tissues. How CD44 functions as a rolling ligand despite its weak affinity for E-selectin is unknown. We examined the nanometer scale organization of CD44 on intact cells. CD44 on leukocytes and transfected K562 cells was cross-linked within a 1.14-nm spacer. Depolymerizing actin with latrunculin B reduced cross-linking. Fluorescence resonance energy transfer (FRET) revealed tight co-clustering between CD44 fused to yellow fluorescent protein (YFP) and CD44 fused to cyan fluorescent protein on K562 cells. Latrunculin B reduced FRET-reported co-clustering. Number and brightness analysis confirmed actin-dependent CD44-YFP clusters on living cells. CD44 lacking binding sites for ankyrin and for ezrin/radixin/moesin (ERM) proteins on its cytoplasmic domain (ΔANKΔERM) did not cluster. Unexpectedly, CD44 lacking only the ankyrin-binding site (ΔANK) formed larger but looser clusters. Fluorescence recovery after photobleaching demonstrated increased CD44 mobility by latrunculin B treatment or by deleting the cytoplasmic domain. ΔANKΔERM mobility increased only modestly, suggesting that the cytoplasmic domain engages the cytoskeleton by an additional mechanism. Ex vivo differentiated CD44-deficient neutrophils expressing exogenous CD44 rolled on E-selectin and activated Src kinases after binding anti-CD44 antibody. In contrast, differentiated neutrophils expressing ΔANK had impaired rolling and kinase activation. These data demonstrate that spectrin and actin networks regulate CD44 clustering and suggest that ankyrin enhances CD44-mediated neutrophil rolling and signaling.     

Siglec-E Promotes β2-Integrin-dependent NADPH Oxidase Activation to Suppress Neutrophil Recruitment to the Lung

Siglec-E is a sialic acid-binding Ig-like lectin expressed on murine myeloid cells. It has recently been shown to function as a negative regulator of β2-integrin-dependent neutrophil recruitment to the lung following exposure to lipopolysaccharide (LPS). Here, we demonstrate that siglec-E promoted neutrophil production of reactive oxygen species (ROS) following CD11b β2-integrin ligation with fibrinogen in a sialic acid-dependent manner, but it had no effect on ROS triggered by a variety of other stimulants. Siglec-E promotion of ROS was likely mediated via Akt activation, because siglec-E-deficient neutrophils plated on fibrinogen exhibited reduced phosphorylation of Akt, and the Akt inhibitor, MK2206, blocked fibrinogen-induced ROS. In vivo imaging showed that siglec-E also promoted ROS in acutely inflamed lungs following exposure of mice to LPS. Importantly, siglec-E-promoted ROS were required for its inhibitory function, as the NADPH oxidase inhibitor, apocynin, reversed the siglec-E-mediated suppression of neutrophil recruitment and blocked neutrophil ROS production in vitro. Taken together, these results demonstrate that siglec-E functions as an inhibitory receptor of neutrophils via positive regulation of NADPH oxidase activation and ROS production. Our findings have implications for the inhibitory role of siglec-9 on human neutrophils in sepsis and acute lung injury.     

Mitochondria Regulate Neutrophil Activation by Generating ATP for Autocrine Purinergic Signaling

Polymorphonuclear neutrophils (PMNs) form the first line of defense against invading microorganisms. We have shown previously that ATP release and autocrine purinergic signaling via P2Y₂ receptors are essential for PMN activation. Here we show that mitochondria provide the ATP that initiates PMN activation. Stimulation of formyl peptide receptors increases the mitochondrial membrane potential (Δψm) and triggers a rapid burst of ATP release from PMNs. This burst of ATP release can be blocked by inhibitors of mitochondrial ATP production and requires an initial formyl peptide receptor-induced Ca²⁺ signal that triggers mitochondrial activation. The burst of ATP release generated by the mitochondria fuels a first phase of purinergic signaling that boosts Ca²⁺ signaling, amplifies mitochondrial ATP production, and initiates functional PMN responses. Cells then switch to glycolytic ATP production, which fuels a second round of purinergic signaling that sustains Ca²⁺ signaling via P2X receptor-mediated Ca²⁺ influx and maintains functional PMN responses such as oxidative burst, degranulation, and phagocytosis.     

Granule swelling and cleavage of mitogen-activated protein kinases in human neutrophils undergoing apoptosis

Extracellular signal-regulated kinase and p38 have been shown to be cleaved in human neutrophils undergoing apoptosis induced by tumor necrosis factor-α and cycloheximide. However, the cleavage products of these molecules were undetected when apoptotic neutrophils were pretreated with phenylmethylsulfonyl fluoride or disrupted by nitrogen cavitation before preparation of cell lysates. The electron microscopy revealed that granules in apoptotic neutrophils were significantly swollen than those in control cells. These findings suggest that granule membrane may become destabilized during neutrophil apoptosis, leading to rapid proteolysis of these molecules by granule-derived serine proteases during preparation of cell lysates with the conventional lysis buffer.     

Increased NaCl-induced interleukin-8 production by human bronchial epithelial cells is enhanced by the DeltaF508/W1282X mutation of the cystic fibrosis transmembrane conductance regulator gene

A satisfactory model describing the airway surface fluid (ASF) in the airways of persons with cystic fibrosis (CF) remains to be established due to theoretical challenges to both the "Hydration Hypothesis" and the "Salt Hypothesis." Irrespective of these models, inhaled hypertonic saline is often used to facilitate clearance of inspissated secretions. Hypertonicity induces interleukin-8 (IL-8) expression, a potent chemokine for neutrophils. The objectives of this study were: (i) to determine the relative contribution of three potential cis-regulatory elements in the regulation of NaCl-induced IL-8 production in BEAS-2B human bronchial epithelial cells, (ii) to compare NaCl-induced IL-8 expression in IB3-1 bronchial epithelial cells, which have the DeltaF508/W1282X mutation of the CF transmembrane conductance regulator (CFTR) gene, with that in C38 cells, which are IB3-1 cells stably transfected with a truncated but functional CFTR gene, and (iii) to compare equal osmolar concentrations of NaCl and D-sorbitol in the induction of IL-8 in all three cell types. In human bronchial epithelial cells, binding sites for NFkappaB, AP-1, and NF-IL6 in the 5'-flanking region of the IL-8 promoter are necessary for optimal NaCl induction of IL-8. Human bronchial epithelial cells with the DeltaF508/W1282X CFTR mutation produce an exaggerated amount of basal and NaCl-induced IL-8.     

Increased FITC fluorescence on LPS stimulated neutrophils cultured in whole blood

Recently, it has been observed that Annexin V labelling of phosphatidylserine (PS) on non-apoptotic cells can vary in different leukocyte populations and with the activation of cells, due to differences in the absolute level of exposed PS. We have also observed changes in the absolute level of Annexin V-FITC intensity, but under conditions where absolute PS expression did not change. In the present study, we have explored the effect of neutrophil cell activation on Annexin V-FITC fluorescence intensity by comparing alternatively labelled matched antibodies against Annexin V. Human venous whole blood was cultured with and without stimulation with lipopolysaccharide (LPS). Apoptosis in the neutrophil and lymphocyte populations was analyzed by flow cytometry and the intensity of FITC labelling was compared to matched fluorochromes conjugated to the same cell surface markers. There was an increase in the intensity of Annexin V-FITC in non-apoptotic neutrophils when stimulated with LPS, which did not correlate with increased apoptosis. Furthermore, CD65-FITC intensity also increased on activated neutrophils. Activated neutrophils exhibited higher amounts of FITC fluorescence that were not associated with changes in extracellular PS expression. This effect appears to be fluorochrome related, likely due to an increase in the pH surrounding activated neutrophils.