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PL
Zbadano adhezję ludzkich granulocytów i limfocytów T do komórek śródbłonka naczyniowego linii HMEC-1 stymulowanych za pomocą endotoksyny (LPS) i enterotoksyny (BFT) B. fragilis. Stwierdzono przyleganie granulocytów oraz limfocytów T spoczynkowych i aktywowanych PMA do komórek endotelialnych po stymulacji endotoksyną i enterotoksyną B. fragilis. Aktywność obu toksyn B. fragilis w procesie stymulacji adhezji leukocytów do śródbłonka naczyniowego jest mniejsza od aktywności LPS E. coli O55:B5.
EN
The aim of presented study was to estimates the number of human granulocytes and T lymphocytes adhering to 1 mm² of vascular endothelial cell culture stimulated by Bacteroides fragilis endotoxins (LPS) and enterotoxin (BFT). HMEC-1 cells were activated with bacterial preparations at the concentration of 10 (µg/ml for 4 and 24 hours. Granulocytes and T lymphocytes were isolated from peripheral blood of healthy blood donors. The adhesion tests of granulocytes and adhesion tests of resting and activated with PMA (at the concentration of 10 ng/ml) T lymphocytes to the non-stimulated and stimulated by B fragilis compounds (LPS and BFT) vascular endothelium were performed. The number of viable leukocytes, which adhered to the endothelium, was determined using inverted microscope (magnification 200 x). The results were presented as the number of viable cells adhering to 1 mm² of the endothelial cell culture. The results of experiments indicate that granulocytes and T lymphocytes (resting and after activation with PMA even in greater number) adhere to the endothelial cells stimulated by B. fragilis endotoxins and enterotoxin. B. fragilis toxins are weaker stimulants of human leukocyte adhesion to the HMEC-1 cells than E. coli O55:B5 LPS. B. fragilis LPS and BFT preparations stimulate endothelial cells to the adhesion of granulocytes in similar manner, whereas the activation of vascular endothelium to the adhesion of T lymphocytes is differentiated.
PL
Badano wpływ klindamycyny na stymulację powstawania cząsteczek adhezyjnych: ICAM-1, VCAM-1 i E-selektyny na komórkach śródbłonka naczyniowego linii HMEC-1 przez lipopolisacharydy izolowane z jednego nieenterotoksynotwórczego (NTBF) i trzech enterotoksynotwórczych (ETBF) szczepów Bacteroides fragilis oraz enterotoksynę (fragilizynę) wytworzoną przez szczep Bacteroides fragilis ATCC 43858. Stwierdzono, że klindamycyna wpływa na powstawanie cząsteczek adhezyjnych na komórkach linii spoczynkowej. Komórki stymulowane preparatami LPS Bacteroides fragilis wykazują obniżenie aktywności ICAM-1 po zadziałaniu klindamycyną. Klindamycyna podwyższa nieznacznie ICAM-1 w reakcjach po stymulacji preparatami fragilizyny. Klindamycyna podwyższa ekspresję VCAM-1 i E selektyny na komórkach linii HMEC-1 stymulowanej LPS i enterotoksyną Bacteroides fragilis.
EN
The influence of clindamycin on expression of B. fragilis endotoxins (LPS) and enterotoxin stimulated cell adhesion molecules: ICAM-1, VCAM-1 and E-selectin on HMEC-1 (human microvascular endothelial cell line) was tested. Lipopolysaccharides from four Bacteroides fragilis strains: one nonenterotoxigenic (NTBF) and three enterotoxigenic (ETBF) were extracted by hot phenol-water method and purified. B fragilis enterotoxin was prepared according to the method described by van Tassel et al. (1992). All bacterial preparations were used for stimulation at concentration 10 µg/ml. Clindamycin was used in concentration of 2 µg/ml. The influence of antimicrobial agent on the endotoxins and enterotoxin stimulation and expression of adhesion molecules was tested by ELISA, using monoclonal mouse anti-human antibodies (Genzyme, USA). Peroxidase-conjugated rabbit anti-mouse immunoglobulins (DAKO A/S Denmark) and OPD (Sigma USA) were used. The coloured reaction product was measured by reading the absorbance at 492 nm in SPECTRA II reader (SLT, Austria), It was observed that clindamycin influenced the expression of cell adhesion molecules on resting cell line. HMEC-1 cells stimulated with Bacteroides fragilis LPS preparations have suppressive effect on ICAM-1 expression. ICAM-1 expression was augmented when stimulated with Tox 1 and Tox 2 preparations. Clindamycin augmented the VCAM-1 expression in tests with all bacterial preparations. All used bacterial preparations of Bacteroides fragilis LPS and enterotoxin enhanced the expression of E-selectin with exception of LPS of NTBF strain.
PL
Przeprowadzono testy adhezji ludzkich limfocytów T i granulocytów do komórek śródbłonka naczyniowego linii HMEC-1 stymulowanych przy użyciu polisacharydu otoczkowego (CPS), lipopolisacharydów (LPS) oraz komponentów wielocukrowych (PS) i lipidowych (lipid A) LPS. Stwierdzono, że limfocyty T spoczynkowe i aktywowane PMA oraz granulocyty przylegają do komórek endotelialnych stymulowanych za pomocą antygenów powierzchniowych B. thetaiotaomicron. Aktywność w tym procesie preparatów LPS i CPS B. thetaioitaomicron jest mniejsza od aktywności LPS E. coli 055:B5.
EN
The aim of this study was to assay the degree of human T lymphocyte and granulocyte adhesion to the vascular endothelial cells stimulated by Bacteroides thetaiotaomicron lipopolysaccharides, components of LPS and capsular polysaccharide. HMEC-1 cells were activated with bacterial preparations in concentration 10 ug/ml for 4 and 24 hours. T lymphocytes and granulocytes were isolated from peripheral blood of healthy blood donors. Thereafter, the adhesion tests of granulocytes and adhesion tests of non-activated and activated with PMA ( in concentration 10 ng/ml) T lymphocytes to the resting and stimulated vascular endothelium were performed. The number of viable cells, which adhered to the endothelium, was determined using inverted microscope (magnification 200x). The results were presented as the number of viable cells adhering to 1 mm2 of the endothelial cell culture. The obtained results indicate that granulocytes and T lymphocytes (resting and activated with PMA) adhere to the endothelial cells stimulated by B. thetaiotaomicron cell-surface antigens. B. thetaiotaomicron lipopolysaccharides and capsular polysaccharide are weaker stimulants of human leukocyte adhesion to the HMEC-1 cells than E. coli 055:B5 LPS.
PL
W przedstawionej pracy badano wpływ metronidazolu na stymulację ekspresji cząsteczek adhezyjnych: ICAM-1, VCAM-1 i E-selektyny na komórkach śródbłonka naczyniowego linii HMEC-1 przez lipopolisacharydy izolowane z jednego nieenterotoksynotwórczego (NTBF) i trzech enterotoksynotwórczych (ETBF) szczepów Bacteroides fragilis oraz enterotoksynę (fragilizynę) wytworzoną przez szczep B. fragilis ATCC 43858. Stwierdzono, że metronidazol wpływa na ekspresję cząsteczek adhezyjnych na komórkach linii HMEC-1. Komórki śródbłonka stymulowane przy pomocy preparatów LPS i enterotoksyny szczepów B. fragilis wykazują nieznacznie wyższą ekspresję ICAM-1 niż te, na które podziałano preparatami LPS i metronidazolem. Metronidazol podwyższa ekspresję VCAM-1 na komórkach linii HMEC-1 stymulowanej przez LPS i enterotoksynę B. fragilis. Wpływ metronidazolu i preparatów bakteryjnych na indukcję selektyny E jest zróżnicowany.
EN
The influence of metronidazole on the level of expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin on the surface of vascular endothelial cells activated with B. fragilis endotoxins and enterotoxin was examined. Three enterotoxigenic (ETBF) strains and one nonenterotoxigenic (NTBF) strain were used for lipopolysaccharide extraction. Enterotoxin was prepared from the culture supernatant of the reference B. fragilis ATCC 43858 strain. Expression of adhesion molecules on vascular endothelial cells (HMEC-1 cell line) was determined after their stimulation with bacterial compounds at the concentration of 10 µg/ml in the presence of metronidazole at the concentration of 4 µg/ml. Endothelial cells were activated for 4 hours (E-selectin expression) and for 24 hours (ICAM-1 and VCAM-1 expression). Adhesion molecules were detected in immunoenzymatic test (ELISA) with mouse, monoclonal antibodies against human ICAM-1, VCAM-1 and E-selectin. The results of experiments suggest, that metronidazole enhances the expression of examined adhesion molecules on endothelial cells. This antimicrobial agent causes some changes in the expression of endothelial ICAM-1, VCAM-1 and E-selectin stimulated by B. fragilis endotoxins and enterotoxin.
PL
Badano wpływ antygenów powierzchniowych: polisacharydu otoczkowego, lipopolisacharydu i komponentów LPS B. thetaiotaomicron na ekspresję molekuł adhezyjnych ICAM-1, VCAM-1 i selektyny E na powierzchni komórek śródbłonka naczyniowego (linia HMEC-1). Cząsteczki adhezyjne oznaczano za pomocą testu immunoenzymatycznego ELISA z przeciwciałami monoklonalnymi dla ludzkich: ICAM-1, VCAM- 1 i selektyny E. Najsilniejszymi stymulatorami cząsteczek adhezyjnych były lipopolisacharydy, a następnie części lipidowe LPS (lipid A). Polisacharyd otoczkowy (CPS) stymulował ekspresję wszystkich molekuł adhezyjnych, ale tylko w największym stężeniu. Nąjsłabszymi stymulatorami ICAM-1, VCAM-1 i selektyny E były części polisacharydowe (PS) LPS. Niektóre preparaty PS nie wykazały aktywności stymulacyjnej.
EN
The aim of this study was to assay the influence of capsular polysaccharide (CPS), lipopolysaccharide (LPS) and components of B. thetaiotaomicron lipopolysaccharide - polysaccharide part (PS) and lipid part (lipid A) on the expression of adhesion molecules associated with inflammation (ICAM-1, VCAM-1, E-selectin) on the surface of vascular endothelial cells. Capsular polysaccharide was isolated by the method of Poxton and Ip (1981). Lipopolysaccharides were extracted using the hot phenol-water method (Westphal and Jann, 1965). Components of LPS were prepared by mild acid hydrolysis of lipopolysaccharide. Experiments with bacterial compounds at concentrations 10, 1, 0.1 and 0.01 (mg/ml) were performed on HMEC-1 cell line (human dermal microvascular endothelial cells). Immunoenzymatic ELISA test with mouse monoclonal antibodies against human: ICAM-1, VCAM-1 and E-selectin was applied to determine adhesion molecules. Resting HMEC-1 and E. coli 055:B5 LPS were used as controls in each experiment. Lipopolysaccharides were the strongest stimulants of endothelial adhesion molecules. Capsular polysaccharide caused the expression of three adhesion molecules, but only at the highest concentration (10 mg/ml). The stimulatory activities of LPS lipid components were much higher than the activities of polysaccharide parts. PS preparations did not reveal the property of adhesion molecule stimulation or their activities were weak. The activity of B. thetaiotaomicron cell-surface antigens in the process of adhesion molecule stimulation on vascular endothelium was lower than the activity of E. coli LPS.
PL
Przygotowano lipopolisacharydy jednego nieenterotoksynotwórczego (NTBF) i trzech enterotoksynotwórczych (ETBF) szczepów B. fragilis oraz enterotoksynę B. fragilis. Badano wpływ polimyksyny B na stymulację ekspresji cząsteczek adhezyjnych: selektyny E, ICAM-1 i VCAM-1 na powierzchni komórek ludzkiego śródbłonka naczyniowego przez przygotowane preparaty bakteryjne. Do oznaczenia cząsteczek adhezyjnych zastosowano test ELISA z przeciwciałami monoklonalnymi. Stwierdzono, że polimyksyna B zmienia poziom ekspresji selektyny E, ICAM-1 i VCAM-1 podczas jednoczesnego działania z lipopolisacharydami lub enterotoksyną B. fragilis.
EN
The aim of this study was to examine the influence of polymyxin B on the level of expression of adhesion molecules E-selectin, ICAM-1, and VCAM-1 on human vascular endothelium activated with B. fragilis endotoxins or enterotoxin. Lipopolysaccharides were extracted by phenol-water method from one nonenterotoxigenic (NTBF) and three enterotoxigenic (ETBF) B. fragilis strains. LPS preparations were purified with nucleolytic enzymes and ultracentrifugation. Enterotoxin (BFT) was prepared from the supernatant of reference B. fragilis ATCC 43858 culture by precipitation with ammonium sulphate. BFT preparations were purified with the application of ion-exchange chromatography and hydrophobic chromatography. Adhesion molecule expression on the surface of human vascular endothelial cells (HMEC-1 cell line) was determined after simultaneous stimulation with bacterial compounds at the concetration of 10 μg/ml and polymyxin B at the concentration of 20 μg/ml. Endothelial cells were activated for 4 hours (E-selectin expression) or for 24 hours (ICAM-1 and VCAM-1 expression). Adhesion molecules were detected in immunoenzymatic test (ELISA) with the use of mouse, monoclonal antibodies against human ICAM-1, VCAM-1, and E-selectin. The results of performed experiments suggest, that polymyxin B changes the level of adhesion molecule expression on human vascular endothelium. This antibiotic causes changes in the expression of endothelial ICAM-1, VCAM-1, and E-selectin during simultaneous stimulationof endothelium with B. fragilis endotoxins or enterotoxin. In the majority of cases the addition of polymyxin B leads to the up-regulation of examined adhesion molecules.
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