Baldauf C, Adam D
Klinikum Innenstadt der LMU, Abteilung für Antimikrobielle Therapie und Infektionsimmunologie im Dr. von Haunerschen Kinderspital, Lindwurmstr. 4, D-80337 München, Germany.
Eur J Med Res. 2000 Oct 30;5(10):455-9.
The aim of this study was to investigate the influence of fluconazole, an antimycotic on phagocytosis, oxidative burst and killing activity of phagocytes in human whole blood with Candida albicans as a test strain using a flow cytometric method.
Candida albicans was stained with Calcein AM, a greenfluorescent dye from Bioprobes (Molecular Probes, Inc., P.O. Box 22010, Eugene, OR 97402-0469, USA). To measure phagocytosis and burst activity diluted monoclonal antibody (CD-13-R-PE, Molecular Probes, Inc., P.O. Box 22010, Eugene, OR 97402-0469, USA) attaching at the surface of granulocytes and monocytes was added as well as Dihydroethidium solution (Molecular Probes, Inc., P.O. Box 22010, Eugene, OR 97402-0469, USA) which changes into red fluorescent Ethidium by oxidation when killing activity takes place. With Ethidium-Homodimer-1-solution (Molecular Probes, Inc., P.O. Box 22010, Eugene, OR 97402-0469, USA) killing activity can be observed. Three different tests, one incubating the Candida for 1- 4 hrs in advance, another incubating whole blood for 1 h, and the third incubating neither yeast nor blood, and a combined main test were carried out. Measurement of phagocytosis, burst- and killing activtiy was performed with a flow cytometric method (Coulter Company, type: Epics-Profile II).
Three different concentrations of fluconazole (5, 20 and 100 microg/ml) show neither decreasing nor increasing influence on phagocytosis and burst activity, irrespective of whether yeasts or phagocytes had been incubated with fluconazole in advance or not. Also after incubating the drug with phagocytes for 1 h, neither an increase nor a decrease of killing activity was observed. A significant increase was, however, found with increasing incubation time of yeasts and fluconazole. - The minimum concentration of fluconazole, just enough to show a significant increase of the killing rate was 1 microg/ml after 3hrs of incubation. No further significant increase was detected when the concentration exceeded 5 microg/ml.
1 h incubation of human phagocytes with fluconazole does not have any significant influence on cellular activities. After advanced incubation of Candida a corresponding increase of the intracellular killing rate in phagocytes occurs, probably due to changes of the cytomorphology of yeasts.
本研究旨在采用流式细胞术，以白色念珠菌为测试菌株，研究抗真菌药氟康唑对人全血中吞噬细胞的吞噬作用、氧化爆发及杀伤活性的影响。
用来自Bioprobes（美国俄勒冈州尤金市邮编97402 - 0469，Molecular Probes公司，邮政信箱22010）的绿色荧光染料钙黄绿素乙酰甲酯对白色念珠菌进行染色。为测定吞噬作用和爆发活性，加入稀释的单克隆抗体（CD - 13 - R - PE，来自美国俄勒冈州尤金市邮编97402 - 0469，Molecular Probes公司，邮政信箱22010），其附着于粒细胞和单核细胞表面，以及二氢乙锭溶液（来自美国俄勒冈州尤金市邮编97402 - 0469，Molecular Probes公司，邮政信箱22010），当发生杀伤活性时，该溶液通过氧化转变为红色荧光的乙锭。使用乙锭同源二聚体 - 1溶液（来自美国俄勒冈州尤金市邮编97402 - 0469，Molecular Probes公司，邮政信箱22010）可观察杀伤活性。进行了三种不同的试验，一种是预先将白色念珠菌孵育1 - 4小时，另一种是将全血孵育1小时，第三种既不孵育酵母也不孵育血液，以及一个联合的主要试验。采用流式细胞术（库尔特公司，型号：Epics - Profile II）测量吞噬作用、爆发和杀伤活性。
三种不同浓度的氟康唑（5、20和100微克/毫升）对吞噬作用和爆发活性均未显示出降低或增加的影响，无论酵母或吞噬细胞是否预先用氟康唑孵育。在将药物与吞噬细胞孵育1小时后，也未观察到杀伤活性的增加或降低。然而，随着酵母与氟康唑孵育时间的增加，发现有显著增加。 - 孵育3小时后，刚好足以显示杀伤率显著增加的氟康唑最低浓度为1微克/毫升。当浓度超过5微克/毫升时，未检测到进一步的显著增加。
人吞噬细胞与氟康唑孵育1小时对细胞活性没有任何显著影响。白色念珠菌预先孵育后，吞噬细胞内的杀伤率相应增加，可能是由于酵母细胞形态的改变。
