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Thirty 15-month-old ewes were randomly allocated to three groups of 10 sheep each. One group was given a single infection of 60,000 T. colubriformis 3rd stage larvae (L3). The second group 2 was infected orally with 20,000, 40,000 and 60,000 L3 T. colubriformis at 21 day intervals and 21 days after the third dose the sheep were treated with Oxfendazole. Seven days later Groups 1 and 2 sheep were challenged with 60,000 L3 T. colubriformis. The third group was left as uninfected controls. All sheep were bled at weekly intervals and the blastogenic responses to Con A, PHA, PWM and LPS of peripheral blood lymphocytes were evaluated using tritiated thymidine incorporation into DNA. When lymphocytes were cultured with Con A, PHA, PWM or LPS, a progressive decrease of blastogenic activity was observed up to 35 days after a single infection of sheep. In multiple infected sheep, the blastogenic responses to the mitogens were generally depressed from the second infection or shortly thereafter with a return to control levels observed only with LPS. Non-mitogen stimulated lymphocytes from single and multiple infected sheep showed significantly increased blastogenic activity on 35 day after each infection. The data indicate that this type of infection with T. colubriformis may have led to activation of peripheral lymphocytes, coinciding with diminished T-cell responsiveness to the mitogens.
The main aim of this study was to compare the reaction of quiescent and proliferating, i.e. phytohemagglutinin (PHA)-stimulated, human peripheral blood mononuclear cells (PBMCs) to γ-radiation, and analyse changes of proteins related to repair of DNA damage and apoptosis, such as γH2A.X, p53, p53 phosphorylation at serines-15 and -392, and p21 and their dose dependence. Freshly isolated PBMCs in peripheral blood are predominantly quiescent, in G0phase, and with very low amounts of proteins p53 and p21. Using confocal microscopy we detected dose dependent (0.5–5 Gy) induction of foci containing γH2A.X (1 h after γ-ray exposure), which are formed around radiation-induced double strand breaks of DNA. Apoptosis was detected from 24 h after irradiation by the dose of 4 Gy onwards by Annexin V binding and lamin B cleavage. Seventy two hours after irradiation 70% of CD3+lymphocytes were A+. Neither increase in p53 nor its phosphorylation on serine-392 after irradiation was detected in these cells. However, massive increase in p21 (cyclin-dependent kinase inhibitor 1A) was detected after irradiation, which can be responsible for late occurrence of apoptosis in these quiescent cells. PHA-stimulation itself (72 h) caused an increase in early apoptosis (A+PI–) in comparison to non-stimulated PBMCs (38% A+resp. 13.4%). After PHA-stimulation also the amount of γH2A.X, p53, and p21 increased, but no phosphorylation of p53 on serine-392 or -15 was detected. Reaction to γ-radiation was different in PHA-stimulated lymphocytes: the p53 pathway was activated and p53 was phosphorylated on serines-15 and -392 4 h after irradiation by the dose of 4 Gy. Phosphorylation of p53 at serine-15 increased in a dose-dependent manner in the studied dose range 0.2–7.5 Gy. Also the amount of p21 increased after irradiation. Seventy two hours after irradiation of PHA-stimulated CD3+T lymphocytes by the dose of 4 Gy 65% of cells were A+.
Immunosenescence is viewed as a remodeling process with the exhaustion of naïve T cells and filling up of the immunological space with memory cells. In this study some phenotypic changes of CD8+ human cells during in vivo ageing were compared with those observed in long term cultures of lymphocytes derived from cord blood or from peripheral blood from donors of different age. Both in vivo and in vitro a significant decrease of the fraction of CD8+CD28+ cells was observed. Comparing the proportions of other T cell subpopulations (the CD4/CD8+ ratio, CD56, CD57, CD27) made it possible to conclude that replicative senescence in vitro partially reflects in vivo ageing.
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