N-钙黏蛋白在烟曲霉黏附及侵袭内皮细胞中的作用

目的 探讨N-钙黏蛋白在内皮细胞黏附吞噬烟曲霉过程中的作用.方法 观察提取人脐静脉内皮细胞蛋白与烟曲霉的结合过程,了解N-钙黏蛋白是否可与烟曲霉结合,建立内皮细胞黏附及吞噬烟曲霉的体外模型,通过单克隆抗体阻断上皮细胞膜受体N-钙黏蛋白,再次观察脐静脉内皮细胞黏附及吞噬烟曲霉情况.结果 脐静脉内皮细胞膜蛋白N-钙黏蛋白可与烟曲霉结合,抗体阻断N-钙黏蛋白后,脐静脉内皮细胞黏附和吞噬烟曲霉能力明显下降.结论 N-钙黏蛋白是脐静脉内皮细胞黏附吞噬烟曲霉孢子的相关受体.     

Infection of vascular endothelial cells with herpes simplex virus enhances tissue factor activity and reduces thrombomodulin expression.

Latent infection of vascular cells with herpes-viruses may play a pathogenic role in the development of human atherosclerosis. In a previous study, we found that cultured human umbilical vein endothelial cells (HUVECs) infected with herpes simplex virus 1 (HSV-1) became procoagulant, exemplified both by their enhanced assembly of the prothrombinase complex and by their inability to reduce adhesion of platelets. We now report two further procoagulant consequences of endothelial HSV infection: loss of surface thrombomodulin (TM) activity and induction of synthesis of tissue factor. Within 4 hr of infection of HUVECs, TM activity measured by thrombin-dependent protein C activation declined 21 +/- 3% (P less than 0.05) and by 18 hr, 48 +/- 5% (P less than 0.001). Similar significant TM decrements accompanied infection of bovine aortic endothelial cells. Identical TM loss was induced with HSV-2 infection but not with adenovirus infection. Decreased surface expression of TM antigen (measured by the specific binding of a polyclonal antibody to bovine TM) closely paralleled the loss of TM activity. As examined by Northern blotting, these losses apparently reflected rapid onset (within 4 hr of HSV infection) loss of mRNA for TM. In contrast, HSV infection induced a viral-dose-dependent increase in synthesis of tissue factor protein, adding to the procoagulant state. The results indicate that loss of endothelial protein-synthetic capacity is not a universal effect of HSV infection. We suggest that the procoagulant state induced by reduction in TM activity and amplified tissue factor activity accompanying HSV infection of endothelium could contribute to deposition of thrombi on atherosclerotic plaques and to the "coagulant-necrosis" state that characterizes HSV-infected mucocutaneous lesions.     

Human natural killer cells exhibit direct activity against Aspergillus fumigatus hyphae, but not against resting conidia

Because natural killer (NK) cells kill tumor cells and combat infections, there is growing interest in adoptively transferring NK cells to hematopoietic stem cell recipients. Unfortunately, in humans, the activity of NK cells against Aspergillus species, the major cause of invasive fungal infection in stem cell recipients, are poorly characterized. Our results show that unstimulated and interleukin-2 prestimulated human NK cells kill Aspergillus fumigatus hyphae but do not affect resting conidia. Killing is also induced by the supernatant of prestimulated NK cells and human perforin. The high levels of interferon-γ and granulocyte macrophage colony-stimulating factor produced by prestimulated NK cells are significantly reduced by Aspergillus, indicating an immunosuppressive effect of the fungus. Whereas Aspergillus hyphae activate NK cells, resting, and germinating, conidia and conidia of ΔrodA mutants lacking the hydrophobic surface layer do not. Our results suggest that adoptively transferred human NK cells may be a potential antifungal tool in the transplantation context.     

Cytokine release in healthy donors and patients with chronic granulomatous disease upon stimulation with Aspergillus fumigatus

The release of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and IL-10 upon stimulation with non-viable conidia and hyphal fragments from Aspergillus fumigatus was investigated in an ex vivo whole-blood model. In healthy volunteers, high numbers of conidia (between 10(6) and 3 x 10(8)/ml) induced a moderate release of TNF-alpha and IL-6. Hyphal fragments (2.5 x 10(5)/ml) were more potent in stimulating the release of these pro-inflammatory cytokines. Although some IL-10 release was observed upon stimulation with either conidia or hyphal fragments, it was not significantly different from that in unstimulated controls. In comparison, in whole blood obtained from 4 patients with chronic granulomatous disease (CGD), a high release of pro-inflammatory cytokines together with a significantly higher IL-10 release than in the healthy controls was seen after stimulation with A. fumigatus. In conclusion, A. fumigatus can trigger the release of pro-inflammatory cytokines in a human whole-blood system, which is likely to be central to the activation of antifungal defence mechanisms. In contrast, A. fumigatus stimulates a higher release of anti-inflammatory cytokines in CGD patients, which may suggest that a dysregulation between pro- and anti-inflammatory cytokines contributes to the increased susceptibility to invasive aspergillosis in this patient group.     

烟曲霉刺激后内皮细胞膜受体C型植物血凝素-1及Toll样受体的表达

目的 研究内皮细胞接触烟曲霉后表面受体树突状细胞相关性C型植物血凝素-1(Dectin-1)及Toll样受体2(TLR2)的表达变化,探讨其在免疫过程中的作用.方法 烟曲霉与人脐静脉内皮细胞共孵育,分不同时间(1、2、4及6 h)点收集细胞,流式细胞仪检测Dectin-1受体及TLR2表达的变化;提取细胞总蛋白,应用Western blot法检测细胞受体蛋白表达水平的变化;应用间接免疫荧光染色法观察各受体在细胞内外的分布情况.结果 (1)静息状态下,内皮细胞膜表面可见Dectin-1及TLR2表达,其平均荧光强度值分别为45和13.经烟曲霉刺激后,TLR2表达逐渐下降至18,Dectin-1表达先升高后下降,接触2 h后平均荧光强度值升至35,6 h后又降至18;(2)Western blot 法检测结果显示,内皮细胞经烟曲霉刺激后2 h Dectin-1蛋白表达最明显,4 h次之,6 h最低;而TLR2在各时间点均无明显变化;(3)共聚焦显微镜下可见部分烟曲霉出芽孢子和菌丝进入内皮细胞,细胞膜、孢子和菌丝表面均可见 Dectin-1表达;TLR2仅在细胞表面表达.结论 烟曲霉感染时,内皮细胞可表达Dectin-1及TLR2,这对识别是否为烟曲霉感染有重要价值.

Abstract：

Objective To observe the changes of TLR-2, Dectin-1 expression on endothelial cells,and to explore their role in the immune response after contact with Aspergillus fumigatus. Methods Aspergillus fumigatus and human umbilical vein endothelial cells were co-incubated. Cells were collected respectively after incubation for 0 h, 1 h, 2 h, 4 h and 6 h. TLR2 and Dectin-1 receptor expressions were detected by flow cytometry, and their protein was measured by Western blot. The distribution of the receptors in the cells were observed by immunofluorescence. Result TLR2 and Dectin-1 were expressed on the endothelial cell surface in quiescent condition. The mean fluorescence intensity of TLR2 on endothelial cells decreased from 45 to 13 stimulation by Aspergillus fumigatus, but the mean fluorescence intensity of Dectin-1increased from 13 to 35 in the first 2 hours and then decreased. By Western blot, the electrophoresis strip of Dectin-1 was most bright in 2 hours after contact with the fungus, and then decreased 4 and 6 hours. TLR2did not change significantly. Dectin-1 with fluorescent labeling was seen in spores and hyphae as well as in the cell membrane under confocal microscope. TLR2 was detected only on cell surface. Conclusion TLR2and Dectin-1 were expressed by endothelial cells, and may be useful in the identification of Aspergillus fumigatus.     

Serine proteinases with gelatinolytic activity in an Aspergillus fumigatus allergenic extract.

The presence of proteolytic activity in Aspergillus fumigatus (Af) extract was analyzed in polyacrylamide gelatine containing gels at different pHs. The gelatinolytic pattern showed three major activities (180, 70, and 34 kDa bands) over a broad pH range (pH 4-8). All bands showed maximum activity at pH 8, and the activity declined at lower pH, showing no activity at pH 2. The susceptibility of the proteolytic activity to protease inhibitors confirmed that the fungal extract contained serine proteinases, and one of them, the 34 kDa band, showed a chymotrypsin-like proteinase. The immunochemical fungal extract properties were studied by western blot, and the samples were run in the presence or absence of denaturing and reducing (DTT) conditions. Two protein bands located at approximately 180 and 84 kDa showed immunoreactivity with allergenic patients' anti-Af serum, when the samples were run under denaturing and reducing conditions. In the native form an additional third immunoreactive band of about 70 kDa was found. There were no immunoreactive bands toward IgE about 34 kDa. Periodate treatment caused the decrease or disappearance of the immunoreactivity in the bands, demonstrating that epitopes recognized by serum IgE specific to Af are partially composed by carbohydrates (180 and 70 kDa bands) while others (84 kDa) are carbohydrates. Although further investigations are needed, these preliminary results suggest alkaline serine proteinases from Aspergillus fumigatus as one of the factors responsible for the fungal allergenicity.     

Neutrophil-activating activity and platelet-activating factor synthesis in cytokine-stimulated endothelial cells: reduced activity in growth-arrested cells

The reactivity of endothelial cells (ECs) to proinflammatory cytokines is critically important for the pathogenesis of vascular diseases. Here, we studied functional alterations of human ECs during culture under a confluent condition; i.e., the alterations of neutrophil-activating activity, platelet-activating factor (PAF) synthesis, and granulocyte-macrophage colony-stimulating factor (GM-CSF) production in cytokine-stimulated ECs. Human umbilical vein-derived ECs exhibited the increased activity in neutrophil activation, PAF synthesis, and GM-CSF production when stimulated by proinflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha). The activity of cytokine-stimulated ECs to stimulate superoxide release in human neutrophils and to produce PAF declined markedly in parallel as ECs became growth-arrested during culture under a confluent condition. By contrast, GM-CSF production induced by cytokine stimulation was modestly increased, and up-regulation of intercellular adhesion molecule-1 (ICAM-1) and activation of mitogen-activated protein kinases were not altered. The neutrophil-activating activity of cytokine-stimulated ECs was dependent on PAF synthesis and GM-CSF production from ECs. These findings indicate that the reduced neutrophil-activating activity in growth-arrested ECs may be, at least in part, ascribed to down-regulation of PAF synthesis.     

Interactions of Chlamydia pneumoniae with human endothelial cells

In order to fulfill the "biological plausibility" criterion of a role for infection with Chlamydia pneumoniae in the pathogenesis of human atherosclerosis, detailed studies on the interaction of this organism with the cell types involved are necessary. This article summarizes the current knowledge on the interaction of C. pneumoniae with human endothelial cells. In vitro, C. pneumoniae can infect human endothelial cells and induce the expression of many molecules that are important mediators of atherogenesis including cytokines, adhesion molecules, chemokines, and molecules with procoagulant activity.     

Synthesis of thromboplastin (tissue factor) by endothelial cells

Molecular genetics, biochemistry, and cell biology of thromboplastin are briefly reviewed with special emphasis on its biosynthesis by endothelial cells.     

In vitro stimulation of monocyte tissue factor activity by autologous platelets

Washed human platelets were found to enhance phytohemagglutinin (PHA)-stimulated tissue factor (TF) synthesis when incubated with autologous mononuclear cell cultures. Furthermore, platelets increased TF synthesis even when no other stimulator was present during the incubation. Experiments utilizing similar cultures derived from blood of patients with Glanzmann's disease, von Willebrand's disease, and platelet storage pool disease indicated that platelets with each of these genetic defects possessed the capacity to enhance the synthesis of this initiator of coagulation by unstimulated cells as did normal platelets. The degree of platelet enhancement varied between individuals, but for any given donor, the extent of the effect depended on the concentration of platelets used. The effect was demonstrable at platelet/monocyte ratios ranging from a low of approximately 15 to the highest ratio used, about 300. For comparison the ratio of these two cellular elements in normal human blood is estimated to be approximately 1,000. These findings may reflect a relationship between these two cell types that can occur in vivo.     

Cocaine unbalances endothelial tissue factor and tissue factor pathway inhibitor expression

Cocaine consumption can lead to myocardial infarction. Tissue factor (TF) has been implicated in acute coronary syndromes, and the balance of TF and tissue factor pathway inhibitor (TFPI) determines initiation of thrombus formation. This study was designed to investigate the effect of cocaine on endothelial TF and TFPI expression. Cocaine (10(-8)-10(-5) mol/l) increased thrombin-induced TF expression by 24% at 10(-7) mol/l (P < 0.001) without affecting basal TF expression. In contrast, cocaine reduced endothelial TFPI expression by 47% at 10(-7) mol/l (P < 0.01). Moreover, thrombin impaired endothelial TFPI expression, and cocaine (10(-8) mol/l) further reduced TFPI expression by 33% as compared to thrombin (P < 0.02). These effects occur at cocaine concentrations usually present in plasma of consumers. Given the importance of TF in the pathogenesis of acute coronary syndromes, TF induction in conjunction with TFPI suppression may be relevant for the increased frequency of myocardial infarction observed in cocaine consumers.     

MicroRNA-223 inhibits tissue factor expression in vascular endothelial cells

Objective: Atherosclerosis is a chronic inflammatory process, in which vascular endothelial cells (ECs) become dysfunctional owing to the effects of chemical substances, such as inflammatory factor and growth factors. Tissue factor (TF) expression is induced by the above chemical substances in activated ECs. TF initiates thrombosis on disrupted atherosclerotic plaques which plays an essential role during the onset of acute coronary syndromes (ACS). Increasing evidences suggest the important role of microRNAs as epigenetic regulators of atherosclerotic disease. The aim of our study is to identify if microRNA-223 (miR-223) targets TF in ECs. Methods and results: Bioinformatic analysis showed that TF is a target candidate of miR-223. Western blotting analysis revealed that tumor necrosis factor α (TNF-α) increased TF expression in aorta of C57BL/6J mice and cultured ECs (EA.hy926 cells and HUVEC) after 4 h treatment. In TNF-α treated ECs, TF mRNA was also increased measured by real-time PCR. Real-time PCR results showed that miR-223 levels were downregulated in TNF-α-treated aorta of C57BL/6J mice and cultured ECs. Transfection of ECs with miR-223 mimic or miR-223 inhibitor modified TF expression both in mRNA and protein levels. Luciferase assays confirmed that miR-223 suppressed TF expression by binding to the sequence of TF 3′-untranslated regions (3′UTR). TF procoagulant activity was inhibited by overexpressing miR-223 with or without TNF-α stimulation. Conclusions: MiR-223-mediated suppression of TF expression provides a novel molecular mechanism for the regulation of coagulation cascade, and suggests a clue against thrombogenesis during the process of atherosclerotic plaque rupture.     

A20基因抑制内皮细胞组织因子表达的研究

目的　探讨外源性A2 0基因在内皮细胞获得表达及对脂多糖诱导的内皮细胞组织因子表达的影响。方法　DOTAP脂质体介导的pCAGGSEHA2 0和pMAMneo基因转染 ,经G4 18筛选阳性单克隆 ,再用免疫荧光组织化学检测A2 0基因的表达 ,将转染A2 0基因的内皮细胞与未转染的内皮细胞分别加脂多糖 ,经原位杂交及免疫荧光组织化学检测组织因子基因的转录和表达 ,采用裂解内皮细胞一步复钙凝块时间测定法测定组织因子促凝活性。结果　成功构建了pCAGGSEHA2 0真核表达重组体 ,A2 0基因在经G4 18筛选后的内皮细胞中得到有效表达 ,能抑制 70 %组织因子的表达及凝块的形成。结论　A2 0基因能够明显抑制脂多糖诱导的内皮细胞组织因子的表达 ,在心血管血栓形成的预防和治疗中有重要作用     

Hemostasis and cancer: tumor cells induce the expression of tissue factor-like procoagulant activity on endothelial cells

BACKGROUND AND OBJECTIVES: Clotting activation and thromboembolic manifestations are common features in patients with cancer. The two-way interaction between tumor cells and host cells is of crucial importance in this context. In the present study we investigated the effect of tumor cell-endothelial cell co-culture on the expression of procoagulant activity in the mixed cell populations. DESIGN AND METHODS: Human tumor cell lines (HL60 promyelocytic leukemia and HeLa uterine cervical cancer) and human umbilical vein endothelial cells (HUVEC) were cultured in vitro according to standard procedures. Procoagulant activity was studied in a coagulometer and was found to be tissue factor-like. A calibration curve was obtained with decreasing concentrations of rabbit brain thromboplastin (RBT) and the procoagulant activity of both tumor cells and HUVEC was expressed as RBT U/10(5) cells. RESULTS: Before incubation procoagulant activity (means S.E.) was found to be 0.18 +/- 0.04 U in HUVEC, 9.8 +/- 1.9 U in HL60 cells, 11.9 +/- 2.2 U in HeLa cells, 7.2 +/- 1.4 U in a mixed HL60 cell-HUVEC population (ratio 2:1) and 8.5 +/- 2.0 in a mixed HeLa cell-HUVEC population (ratio 2:1). Incubation at 37 degrees C for up to 4 hours of tumor cells or HUVEC alone did not produce any change in procoagulant activity. In contrast, co-incubation of tumor cells with HUVEC for 4 hours was followed by a significant increase in procoagulant activity of the mixed cell populations. Addition of supernatants from tumor cells, HUVEC or tumor cell-HUVEC co-cultures to HUVEC or tumor cells showed that the tissue factor-like procoagulant activity generated during coincubation was localized on HUVEC. INTERPRETATION AND CONCLUSIONS: Our results show that the close interaction of tumor cells with endothelial cells may induce surface expression of tissue factor in the latter. This effect could represent an additional mechanism of clotting activation in patients with cancer.     

五种抗深部真菌药物对烟曲霉体外抗菌活性的实验研究

目的 测定五种抗深部真菌药物对烟曲霉体外抗菌活性,为临床治疗侵袭性烟曲霉感染提供理论依据.方法 应用美国临床实验室标准化协会(CLSI)的微量液基稀释法 M38-A方案,测定烟曲霉临床株对伊曲康唑(ICZ)、两性霉素B(AmB)、伏立康唑(VRC)、卡泊芬净(CBF)、 氟康唑(FCZ)的敏感性.结果 MIC几何均数VRC为 0.29 μg/ml、CBF为0.45 μg /ml、ICZ为0.52 μg /ml、AmB为0.55 μg /ml、FCZ为62.58 μg /ml.结论 VRC、CBF、ICZ、AmB均有很强的抗烟曲霉作用,FCZ对烟曲霉均耐药.     

Characterization of antigens from Aspergillus fumigatus. II. Fractionation and electrophoretic, immunologic, and biologic activity.

Mycelial extracts of Aspergillus fumigatus grown in completely synthetic medium were fractionated by precipitation with ammonium sulfate, by ion-exchange chromatography, and by gel filtration. The fractions were characterized chemically, by analytic polyacrylamide gel electrophoresis, and by tests for serologic and biologic activity. The distribution of the antigens in the fractions was determined by double diffusion in gel and fused-rocket immunoelectrophoresis. Activity greater than that of the parent mycelial extract serologically, in guinea pig skin tests, and in in vitro lymphocyte transformation was observed with one fraction that was found in the 75 per cent ammonium sulfate precipitable portion and was eluted early by gel filtration. Another ammonium sulfate-precipitable component and one that was not precipitable at 75 per cent ammonium sulfate, but was firmly bound by ion-exchange columns, was found to have activity equal to unfractionated mycelial extract. These fractions were also active by a radioallergosorbent test for IgE. A carbohydrate component comprising almost 40 per cent of mycelial extract was found to be devoid of any immunologic activity.