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The aim of the study was to assess the effects of tamoxifen and cyclophosphamide on the selected cell-mediated immunity parameters in dogs. The study included 18 dogs aged 5-10 years. The experimental group consisted of 12 animals with neoplastic lesions classified as the first or second staging group (according to the WHO TMN classification). This group was divided into two subgroups: I - six dogs receiving oral tamoxifen, and II - six dogs with cyclophosphamide administered orally. The control group included six healthy dogs. The blood was sampled from the saphenous access vein two times at 14-d intervals before the drug administration, three times every 7 d during administration, and two times every 14 d after completion of the therapy. The basic blood tests were carried out and the subpopulations of TCD4+ and TCD8+ lymphocytes, and phagocytic activity of granulocytes and monocytes were determined using flow cytometry. It was found that tamoxifen induced a marked increase in WBC and neutrophil counts, increased phagocytic activity of monocytes, and changed the CD4+:CD8+ ratio (in favour of cytotoxic lymphocyte subpopulation). These findings indicated the stimulation of cell-mediated immunity mechanisms. Cyclophosphamide caused a substantial decrease in the overall leukocyte pool and reduced the percentage of cells activated for phagocytosis, both neutrophils and monocytes even after completion of its administration, which proves its immunosuppressive effects.
Retinoic acid and transforming growth factor-ß (TGF-ß) affect differentiation, pro­liferation and carcinogenesis of epithelial cells. The effect of both compounds on the proliferation of cells of the hormone sensitive human breast cancer cell line (ER+) MCF-7 was assessed in the presence of estradiol and tamoxifen. The assay was based on [ 3H] thymidine incorporation and the proliferative activity of PCNA- and Ki 67-positive cells. The apoptotic index and expression of the Bcl-2 and p53 antigens in MCF-7 cells were also determined. Exogenous TGF-ß1 added to the cell culture showed antiproliferative activity within the concentration range of 0.003-30 ng/ml. Irrespective of TGF-ß 1 concentrations, a marked reduction in the stimulatory action of estradiol (10-9 and 10-8 M) was observed whereas in combination with tamoxifen (10 -7 and 10 -6 M) only 30 ng/ml TGF-ß 1 caused a statistically significant reduction to aproximately 30% of the proliferative cells. In further experiments we examined the effect of exposure of breast cancer cells to retinoids in combination with TGF-ß 1. The incorporation of [3H]thymidine into MCF-7 cells was inhibited to 52 ± 19% (con­trol = 100%) by 3 ng/ml TGF-ß1, and this dose was used throughout. It was found that addition of TGF-ß1 and isotretinoin to the culture did not decrease proliferation, while TGF-ß1 and tretinoin at low concentrations (3 x 10 -8 and 3 x 10-7 M) reduced the percentage of proliferating cells by aproximately 30% (67±8% and 67±5%, P < 0.05 compared to values in the tretinoin group). Both retinoids also led to a sta­tistically significant decrease in the stimulatory effect of 10-9 M estradiol, attenu­ated by TGF-ß1. In addition, the retinoids in combination with TGF-ß1 and tamoxifen (10–6 M) caused a further reduction in the percentage of proliferating cells. Immunocytochemical analysis showed that all the examined compounds gave a statistically significant reduction in the percentage of cells with a positive reaction to PCNA and Ki 67 antigen. TGF-ß1, isotretinoin and tretinoin added to the culture resulted in the lowest percentage of PCNA positive cells. However, the lowest fraction of Ki 67 positive cells was observed after addition of isotretinoin. The obtained results also confirm the fact that the well-known regulatory proteins Bcl-2 and p53 play an important role in the regulation of apoptosis in the MCF-7 cell line, with lowered Bcl-2 expression accompanying easier apoptotic induction. The majority of the examined compounds act via the p53 pathway although some bypass this important proapoptotic factor.
The study was conducted on 12 male dogs (5 to 10 years of age). The animals were divided into 2 groups: the experimental one (6 tamoxifen-treated dogs) and the control one (6 clinically healthy dogs). Besides, a clinical examination evaluating the overall state of health and sexual impulse, the examination of testosterone level in the blood serum was performed as well as the examination of semen (spermatozoa concentration, percentage of dead spermatozoa, evaluation of spermatozoa morphology, and evaluation of spermatozoa cell membrane activity - HOS test). It was found that tamoxifen had a negative influence on the function of the reproductive system in the dogs and caused, already in the first week of the treatment, the worsening of most of the evaluated characteristics of the semen and later, after the second week of the treatment, it produced aspermia with the complete loss of male fertility. The loss of fertility turned out to be periodical and lasted for about 60 d. After that time, the dogs regained the ability to produce ejaculate, though of much weakened quality, but later it was gradually improving.
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