We studied hsBAFF activity in in vitro mouse splenic B cells. hsBAFF effects on intracellular free Ca 2+ concentration ([Ca 2+ ] i ) were assayed, using a laser scanning confocal microscope with fluorescent probe, Fluo-3/AM. We showed that treatment of B cells with 0.5-5 μ g/ml hsBAFF resulted in significantly higher [Ca 2+ ] i levels in a dose-dependent fashion at 12 and 24 h, respectively (p<0.05 or p<0.01 vs. control). Furthermore, we noticed that 2.5 μ g/ml hsBAFF-treated cells were significantly resistant to decrease of cellular viability induced by thapsigargin (Tg), an endoplasmic reticulum (ER) Ca 2+ -ATPase inhibitor (p<0.05 hsBAFF plus Tg group vs. Tg group). Thus hsBAFF may promote B cell survival by direct upregulation of [Ca 2+ ] i physiological homeostasis contri buting to prevention of [Ca 2+ ] i dysfunction. Using immunocytochemistry and Western blot analysis, we found that the activation of ERK1/2 due to hsBAFF was triggered by a [Ca 2+ ] i -dependent pathway, leading to elevation of B cell proliferation. This is supported by the findings that intracellular Ca 2+ chelator BAPTA/AM attenuated phosphorylated ERK1/2 expression and cell proliferation in hsBAFF-stimulated B cells. hsBAFF-stimulated B cell proliferation was obviously reduced by mitogen extracellular kinase 1/2 (MEK1/2, upstream of ERK1/2) inhibitor U0126. Taken together, the main finding of this study is that hsBAFF elicits higher but homeostatic [Ca 2+ ] i levels, which regulates ERK1/2 activity and cell proliferation in in vitro B cells., J. Q. Liang, W. Zhang, L. Wen, W. Gao, S. Q. Zhang, L. Chen., and Obsahuje bibliografii
Tagging cells of experimental organisms with genetic markers is commonly used in biomedical research. Insertion of artificial gene constructs can be highly beneficial for research as long as this tagging is functionally neutral and does not alter the tissue function. The transgenic UBC-GFP mouse has been recently found to be questionable in this respect, due to a latent stem cell defect compromising its lymphopoiesis and significantly influencing the results of competitive transplantation assays. In this study, we show that the stem cell defect present in UBC-GFP mice negatively affects T-lymphopoiesis significantly more than B-lymphopoiesis. The production of granulocytes is not negatively affected. The defect in T-lymphopoiesis causes a low total number of white blood cells in the peripheral blood of UBC-GFP mice which, together with the lower lymphoid/myeloid ratio in nucleated blood cells, is the only abnormal phenotype in untreated UBCGFP mice to have been found to date. The defective lymphopoiesis in UBC-GFP mice can be repaired by transplantation of congenic wild-type bone marrow cells, which then compensate for the insufficient production of T cells. Interestingly, the wild-type branch of haematopoiesis in chimaeric UBC-GFP/wild-type mice was more active in lymphopoiesis, and particularly towards production of T cells, compared to the lymphopoiesis in normal wild-type donors.