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Four B. fragilis strains were studied nonenterotoxigenic (NTBF) and three enterotoxigenic (ETBF). Endotoxin and enterotoxin which are released into the culture medium during the growth of strains were detected. Cultures in BHI broth were incubated for 48 hours at 37ºC. After 4, 8, 16, 24 and 48 hours of cultivation, samples of bacterial culture were colleted and the optical density was measured. Then the samples were centrifuged, supernatans were filtered through 0.45 um filters and concentrated three times with 5000 D ultrafilters. Prepared samples were kept frozen at 71ºC until use. The presence of endotoxin in samples was revealed by means of immunoelectroprecipitation (IEP) and immunoenzymatic test (dot-ELISA). The assays were performed with antibacterial rabbit immune sera. The activity of enterotoxin was detected on a human colon adenocarcinoma cell line HT 29/C1. The results of the study indicate that endotoxin is released spontaneously by nonenterotoxigenic IPL E 323 strain into the culture medium at the early stages of cultivation. The presence of endotoxin is not demonstrated by means of immunoelectroprecipitation in culture filtrated of ETBF strains. Trace amounts or endotoxin are revealed with dot-ELISA. The activity of enterotoxin is detected after 16 hours of incubation of ETBF strains.
The aim of this study was the evaluation of LAL test with chromogenic substrate usefulness for the quantitative detection of B. fragilis endotoxin and the determination of the amount of endotoxin in culture filtrates of the strains of this species. Also, the trial was undertaken to determine the influence of clindamycin un endotoxin release from B. fragilis rods to the culture medium. Four B. fragilis strains were examined: one nonenterutoxigenic (NTBF) and three enterotoxigenic (ETBF). The growth of cultures was determined and endotoxin liberated to the culture medium during growth of strains was detected. BHI broth and BHI broth with addition of sub inhibitory doses (sub-MIC) of clindamycin were applied. Bacterial cultures were incubated for 48 hours at 37°C. Samples of bacterial cultures were collected after 4, 8, 16, 24 and 48 hours of cultivation, and the optical density was measured. Then the samples were centrifuged, supenatants were filtered through 0.45 µm filters and concentrated three times with 5000 D ultrafilters. Prepared samples were kept frozen at -70°C until used. The amount of endotoxin in samples was determined using quantitative LAL test with chromogenic substrate S- 2423. The results of the experiments indicate that LAL, test is the useful method for determination of B. fragilis endotoxin concentration. This endotoxin activates the enzymatic system present in Limulus polyphemus amcbocyte lysate. Endotoxin is shed spontaneously by B. fragilis rods to the culture medium during growth. Clindamycin at subinhibitory concentrations (sub-MIC) inhibits the growth of cultures of examined strains. The antibiotic caused increase in endotoxin amount in culture medium.
Bacterial endotoxins as contamination of biopreparations have been estimated by chromogenie LAL test. Study on some compounds (aluminium hydroxide, formaldehyde and mertiolate) being components of vaccines showed no effect on the result of LAL test. The level of endotoxins in virus vaccines with the limits defined in producers certificate was adequate, the level of endotoxin was also low in virus vaccines of undefined requirements. The concentration of endotoxin in bacterial vaccines was differentiated. Considering the results of our experiments, as well as the fact, that the requirements for endotoxin contamination of bacterial vaccines are not available it seems necessary to establish the limits for these group of biopreparations.
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.
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.
Przedmiot badań stanowiły lipopolisacharydy wyekstrahowane z ośmiu referencyjnych i dwóch klinicznych szczepów grupy B. fragilis (BFG). Biolo#giczną aktywność preparatów LPS oznaczono przy użyciu fotometrycznego testu BET (poprzednio LAL). Aktywność lipopolisacharydów pochodzących z pałeczek rodzaju Bacteroides porównano z aktywnością LPS E. coli O55:B5. Największy aktywnością w reakcji z odczynnikiem LAL odznaczały się lipopolisacharydy tych gatunków pałeczek BFG, które są ważne z klinicznego punktu widzenia - B. fragilis i B. thetaiotaomicron.
The study was carried out on 16 clinically normal stallions of Polish Primitive Horses in the in the reproductive season (April - July). Four stallions were infused with endotoxin (LPS) from Escherichia coli 055:B5 at doses of 0.3 mg/kg b.w. (group ENDO). Twelve stallions were treated with flunixin meglumine (1.1 mg/kg b.w., IV) for 5 min. (group E+FM1, n=4), 30 min. (group E+FM2, n=4), and 60 min. (group E+ FM3, n=4) after the administration of LPS. The study was meant to determine the concentration of testosterone (T), estrone (E1), estradiol-17b (E2b), total protein (TPR), albumin (ALB), zinc (Zn), cholesterol (CHOL), and aspartate aminotransferase (AST) activity in the seminal plasma of stallions. The administration of endotoxin had an influence on the T, E2b, E1 and TPR, ALB, CHOL concentration, as well as on AST activity in seminal plasma (in the period of the 24h-9th weeks after LPS administration). There has been a statistically significant decrease noted noted in the T, to 0.18 ng/ml (ΔT - 21.7%), and an increase in the E1 to 45,3 pg/ml (Δ E2b - 46.1%), E2 β, to 92.9 pg/ml, (Δ E2b - 45.6%), as well as in the: TPR, to 22.3 g/l (Δ TP - 76.9%); ALB, to 1.9 g/l (Δ ALB - 94.0%); CHOL, to 0.52 mmol/l (Δ CHOL - 21.0%), and AST, to 68.1 mU/109pl. (Δ AST-138.4%). The positive effects of treatment with flunixin meglumine were noted on T, E2β, ALB and CHOL concentrations in seminal plasma, especially of stallions group E+FM1.
Lydium KLP (dimer lysozyme) used in veterinary practice as an immunostimulator has different effects of action on humoral immunity of rabbits compared with endotoxin (LPS) activity. E. coli endotoxin (Kroeger O8) given intravenously induced humoral immunity in rabbits after its first injection while the first administration of Lydium KLP hardly stimulated humoral immunity in comparison to the second dose. LPS is commonly regarded as a pirogen while Lydium KLP is not. The results obtained indicate different effects of Lydium KLP and LPS on antibody production and on temperature release.
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