Neutrophil adhesion molecules in term and premature infants: normal or enhanced leucocyte integrins but defective L-selectin expression and shedding.

The functional deficits of neonatal neutrophils are well documented and are thought to contribute to the increased susceptibility of newborn infants to infection. We measured the adhesion molecules L-selectin, CD11a/CD18 and CD11b/CD18 on neutrophils from the cord blood of term (n = 22) and premature (n = 32) infants using a whole blood method with flow cytometry and quantitative bead standards to enumerate cell surface receptors. We also assayed plasma for the shed form of L-selectin (sL-selectin). Our results suggested that L-selectin expression on term infant neutrophils is lower than that on adult neutrophils (unstimulated and stimulated, both P < 0.001), but that stimulated premature infant cell express higher L-selectin than term infants (P < 0.05); it is possible that this deficiency is caused by physiological changes occurring around the normal time of parturition. We observed reduced sL-selectin in term infants (P < 0.001) compared with adults, and even lower concentrations in premature infants (P < 0.001). The sL-selectin concentrations in plasma may be a reflection of granulopoiesis, which may be reduced in premature infants. Our results showed increased resting neonatal neutrophil expression of CD11b/CD18 compared with adults, and the absence of any neonatal deficit of the ability to up-regulate CD11b/CD18 expression on stimulation. These findings are contrary to previous reports. Further studies suggested that the isolation procedures used in previous reports reduces the capability of the cells to respond to a formyl methionine leucine phenylalanine (fMLP) stimulus. This effect is more marked in neonatal neutrophils, suggesting that the previously reported deficiency is in fact due to the isolation techniques used rather than the cells' innate ability to up-regulate CD11b/CD18 expression. The results of our study lead us to propose that the adhesive function of neonatal neutrophils may be less defective than previously thought.     

人的可溶性L-selectin(sL-selectin)在杆状病毒载体系统中的表达与纯化

目的利用昆虫细胞杆状病毒系统表达人的重组可溶性L-型选择蛋白配体凝集素（human recombinant soluble L-selectin：sL-selectin），探索在真核细胞中高效表达人的重组可溶性L-型选择蛋白配体凝集素的新途径。方法将已经重组好的杆状病毒感染昆虫细胞，表达sL-选凝素于细胞培养上清，利用末端连接的ZZ-结构域（蛋白A来源的首尾相连的二聚化Z结构域）与IgG-琼脂糖-6的结合而分离纯化，通过SDS-PAGE鉴定分子量的大小，并用特异性抗体验证，另外通过重组的sL-选凝素与SMMC7721的黏附观察其活性。结果sL-选凝素可在昆虫细胞中表达，产物的分子量约为46000，与人肝癌细胞SMMC7721的结合率可达70％。结论sL-选凝素可通过杆状病毒载体在昆虫细胞中有效表达，分离纯化后依然保持生理活性。     

Soluble L-selectin and neutrophil derived oxidative stress after pacing induced myocardial ischemia in chronic stable coronary artery disease

We studied the effect of atrial pacing induced myocardial ischemia on levels of soluble L-selectin (sL-selectin) and generation of neutrophil derived reactive oxygen species (ROS) in 10 patients with coronary artery disease (CAD) and stable angina and in six individuals without CAD. Myocardial ischemia was measured metabolically by lactate sampling from the coronary sinus (CS) and arterial blood at each pacing step. Before each pacing step, at peak pacing and shortly after cessation, plasma concentrations of sL-selectin and generation of ROS using the chemiluminescence method were measured in CS and femoral artery blood. Baseline sL-selectin levels in CS samples were significantly lower in the CAD compared to the control group (547 +/- 80 vs 836 +/- 82 ng/mL, P = 0.03). At peak pacing, nine of ten patients with CAD developed myocardial ischemia (lactate extraction ratio at rest 28% +/- 7%, at peak pacing -16% +/- 6%). In these patients, luminol-enhanced chemiluminescence (CL, 0.88 +/- 0.45 vs 1.9 +/- 0.9 cpm x 10(5), P = 0.09) and levels of sL-selectin (547 +/- 80 vs 764 +/- 86 ng/mL, P = 0.03) from naive neutrophils increased significantly in CS blood suggesting a potent in vivo activation of neutrophils. In control patients, incremental pacing caused neither myocardial ischemia nor a significant change of chemiluminescence or of sL-selectin levels. In conclusion, myocardial ischemia induced by pacing tachycardia is able to activate neutrophils in patients with chronic stable coronary artery disease leading to increased generation of ROS and shedding of L-selectin into the coronary circulation.     

急性脑梗死患者外周血sE-选择素?sL-选择素水平变化及其临床意义

目的:探讨急性脑梗死患者外周血sE-选择素、sL-选择素表达水平的变化、临床意义.方法:对30例急性脑梗死患者采用酶联免疫吸附技术(EUsA)定量测定发病<72 h、7天时外周血血清sE-选择素、sL-选择素的表达水平,并与20例健康者作对照.结果:发病<72 h、7天时外周血sE-选择素的表达水平分别为(10.49±8.70)ng/ml、(5.74±4.40)ng/ml,均显著高于对照组(均P<0.01);且与发病<72 h相比,发病7天时下降明显,其差异有显著性(P<0.01).发病<72 h、7天时外周血sL-选择素的表达水平分别为(4.29±2.22)ng/ml、(4.27±2.11)ng/ml,均显著低于对照组(均P<0.01);7天与<72 h相比其表达水平差异无显著性(P0.05).结论:急性脑梗死时患者外周血sE-选择素表达上调而sL-选择素表达下调;它们可能参与了急性脑梗死的病理过程,对临床治疗有参考价值.     

Correlation between serum level of soluble L-selectin and leukocyte count in chronic myeloid and lymphocytic leukemia and during bone marrow transplantation

Abstract: L-selectin is a glycoprotein which is one of three members in a family of cell adhesion molecules called selectins. L-selectin is present in distinct forms on both neutrophil granulocytes and lymphocytes, and it appears to play an important role in the early stages of leukocyte-endothelial cell interaction. Activation of leukocytes leads to shedding of the extracellular part of L-selectin which thus forms a soluble adhesion molecule, sL-selectin, which retains functional capacity and can be detected in serum. In the present study we have developed a specific, sensitive sandwich ELISA to measure the serum level of sL-selectin in patients with hematological and infectious disorders. Three patients with acute myeloid leukemia in remission and 1 patient with chronic myeloid leukemia in chronic phase were followed during bone marrow transplantation and the level of sL-selectin was found to correlate closely to the leukocyte counts with no detectable sL-selectin during periods of severe leukopenia. In 11 patients with chronic phase chronic myeloid leukemia and 13 patients with chronic lymphocytic leukemia the sL-selectin level was also found to correlate closely to the leukocyte count (R = 0.98; p = 0.001 and R = 0.83; p = 0.004 respectively). One CML patient with a leukocytosis of 385 times 10⁹/1 was found to have an sL-selectin concentration 625 times above normal. Ten patients with acute pneumonia were evaluated at diagnosis and at the time of follow-up 4–8 weeks later. In all patients the initial sL-selectin level was higher than at follow-up. However, no close correlation between sL-selectin and leukocyte count or CRP (C-reactive protein) at the time of diagnosis was found. In summary, we have found that the sL-selectin level in human serum closely correlates to the leukocyte count in both CML and CLL and during bone marrow transplantation. –     

Serum levels of sL-selectin and tumour necrosis factor-α in children with type 1 diabetes mellitus

Abstract Type 1 diabetes mellitus (DM) is a result of inflammation and destruction of α-cells in the pancreatic islet cells. The aim of this study is to evaluate the associations of diabetes with soluble L-selectin (sL-selectin) and tumour necrosis factor-α (TNF-α) in children with type 1 DM; and also to evaluate the associations of these parameters with the disease period, glycaemic control state and puberty stage. Serum sL-selectin and TNF-α levels were measured in 44 children with type 1 DM and 44 healthy children. Neither the patients nor the control group showed significant difference between the levels of sL-selectin and TNF-α (sequence mean 12.17±1.62 ng/ml vs. 12.62±1.56 ng/ml and 7.27±3.1 pg/ml vs. 7.88±2.7 pg/ml). There was no statistically significant difference between children with duration of diabetes longer than 5 years and children with duration of diabetes shorter than 1 year. There was also no statistically significant difference between poor glycaemic control and good–acceptable glycaemic control patients. The present results indicate that sL-selectin and TNF-α serum levels are not increased and cannot be used as prognostic predictors in type 1 DM; and also sL-selectin and TNF-α do not change with the disease period, glycaemic control state and puberty stage.     

Clinical significance of serum levels of soluble intercellular adhesion molecule-1 and soluble L-selectin in malignant melanoma

Although several molecules have been evaluated as tumor markers of malignant melanoma (MM) progression, there are few available markers sensitive enough to detect recurrence or metastasis. The objective of the present study was to determine clinical significance of serum soluble adhesion molecules in the monitoring of progression in patients with MM. Serum levels of soluble L-selectin (sL-selectin), sE-selectin, sP-selectin, and soluble intercellular adhesion molecule-1 (sICAM-1) were determined by ELISA in 57 MM patients. In a retrospective longitudinal study, nine serum samples from two MM patients were analyzed during a follow-up period of 4.0 and 4.3 years, respectively. Serum sICAM-1 levels in MM patients were significantly higher than those in healthy controls and tended to be elevated as the disease stage progressed. In contrast, serum sL-selectin levels were significantly lower in MM patients compared to healthy controls and tended to decrease as the disease stage progressed. There was no significant difference in serum sE-selectin and sP-selectin levels between MM patients and normal control. In a longitudinal study, increased sICAM-1 and decreased sL-selectin levels were generally associated with the progression of MM. These results suggest that monitoring both sICAM-1 and sL-selectin is available to evaluate the progression of MM.     

Serial changes in neutrophil-endothelial activation markers during the course of sepsis associated with disseminated intravascular coagulation

INTRODUCTION: For systematic elucidation of serial changes in neutrophil-endothelial activation markers as well as to investigate the correlationship among the inflammation markers, disseminated intravascular coagulation (DIC), and multiple organ dysfunction syndrome (MODS) in patients with sepsis, we made this prospective study. MATERIALS AND METHODS: Forty-five patients with sepsis, severe sepsis, and septic shock were subdivided into two groups, 27 with DIC and 18 without DIC. Eight normal healthy volunteers served as control subjects. Serial levels of soluble L-, P-, and E-selectins, intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), thrombomodulin (sTM), and neutrophil elastase were measured within 12 h after the diagnosis of sepsis (day 0) and on days 1-4 after the diagnosis. The numbers of systemic inflammatory response syndrome (SIRS) criteria that patients met and the DIC score were determined simultaneously. RESULTS: Acute Physiology and Chronic Health Evaluation (APACHE) II score was identical between the two groups. In the DIC patients, higher DIC scores, lower platelet counts, and more maximum numbers of SIRS criteria being met were observed compared with the non-DIC patients. The incidence of MODS and the number of the dysfunctioning organs were higher in the patients with DIC than those without DIC, and the DIC patients had poor outcome. Soluble L-selectin (sL-selectin) levels in both groups tended to be lower than those in the control subjects. All other parameters both in the two groups were continuously higher than those in the control subjects during study period. The levels of soluble E-selectin (sE-selectin), sICAM-1, sVCAM-1, neutrophil elastase, and sTM were more elevated in the DIC patients than those in the non-DIC patients. There were no differences in the sP-selectin levels between the two groups; however, more increased sP-selectin levels per platelet were found in the DIC patients compared with the non-DIC patients. Maximum DIC scores in the DIC group positively correlated with the peak levels of neutrophil elastase and sTM and the number of the dysfunctioning organs. CONCLUSIONS: We found close relations among the neutrophil-endothelial cell interactions, DIC, and MODS in patients with sepsis, severe sepsis, and septic shock. The results indirectly confirm the concept that DIC can produce organ dysfunction and that DIC reflects an inflammatory disorder of the microvasculature.