Abstract:In vitro studies have shown that amnion-produced growth factors participated in angiogenesis, re-epithelialization, and immunomodulation. The aim of our study was to investigate the growth factors and receptors produced by human amnion tissue and amniotic cells. Human amnions (hAM) were isolated, and amnion circles were dissected for in vitro analysis. Some amnion fragments were digested by the use of different methods to obtain two cell fractions, which were analysed for mesenchymal and epithelial cell markers. Amniotic circles and human amniotic cell fractions were cultured in a protein-free medium. Proteins secreted into the culture medium were analysed with a human growth factor antibody array. Conditioned culture media were added to human umbilical vein epithelial cells (HUVECs) to test for stimulation of migration (scratch test) and proliferation (Ki67 expression). Fraction 1 cells expressed both cytokeratin and mesenchymal cell markers which indicated that it was composed of a mixture of human amnion epithelial cells (hAECs) and mesenchymal stromal cells (hAMSCs). Fraction 2 cells mainly expressed cytokeratin and, therefore, were designed as hAECs. Secretion of proteins by the cultured cells increased with time. The hAM cultures secreted EGF-R, IGF, and IGFBP-2, -3 and -6; Cell Fraction 1 secreted NT-4, whereas Cell Fraction 2 secreted G-CSF, M-CSF, and PDGF. Conditioned media of hAM cultures stimulated HUVECs migration. We have showed for the first time that human amnions and amniotic cells secreted IGFBP-6, MCSF-R, PDGF-AB, FGF-6, IGFBP-4, NT-4, and VEGF-R3. We found that Cell Fraction 1, Cell Fraction 2, and the whole amnion secreted different proteins, possibly due to different proportions of amnion-derived cells and different cell-cell interactions. The hAM cell factors remained functional in vitro and induced intensified migration of HUVECs. The growth factors and receptors found in amnion or amniotic cell media might be used for regenerative medicine.
Amnion is a membrane surrounding the embryo/fetus which determine growth factors and interleukins with angiogenic, immunogenic, and anti-inflammatory properties. The aim of the present study was to investigate the effects of conditioned culture medium from 24-h cultures of human amnion (hAM CCM) on migration and proliferation of human umbilical vein endothelial primary cells (HUVECs), freshly isolated bone marrow mononuclear cells (BM MNCs), and Jurkat leukemia cell line. Amnion membrane was freshly isolated from healthy placenta and its fragments cultured in vitro to produce hAM CCM. Members of the IGFBP protein family made up one third of all assayed proteins present in the hAM medium. The hAM CCM did not affect the proliferation rate of HUVECs or MNCs, but we observed more intensive migration of those cells, and lower expression of CD31 surface antigen on HUVECs as compared to control cultures. In contrast, Jurkat cells did not respond to hAM CCM treatment by proliferation or mobility change. The conditioned medium from 24-h cultures of human amnion is easy to obtain and is a convenient source of various growth and other factors that may be useful in practical medicine.
Small B-cell lymphocytic lymphoma/chronic lymphocytic leukemia, which typically affects elderly people, is a group of conditions that are not clinically uniform. It has been suggested that using the combined activity of the monoclonal antibody anti-CD20 (rituximab) and Listeria monocytogenes toxin listeriolysin O (LLO) for this condition could produce an enhanced treatment effect. Here, we tested the effect of the joint activity of rituximab and LLO, which is a cell membrane toxin, in human leukemia cell lines. The human B-leukemia Raji cell line, which expresses CD20, and the T-cell Jurkat cell line, which does not express CD20, for comparison were used in model tests. Cell cytotoxicity of rituximab or LLO and both applied jointly to the cell lines was compared in the presence of human plasma complement. Optimal cytotoxic effects dependent on rituximab or LLO concentration were tested separately. LD50 values were determined and used for optimal application of a mixture of the two factors. The cytotoxic effect on Raji cells of both rituximab and LLO was more than 2.5 times that of LLO alone and 1.5 times that of rituximab alone. At the highest tested concentrations, a mixture of the tested factors had a non-specific cytotoxic effect on the Jurkat cell line, as well. The rituximab and LLO binding sites appear to be in a similar region of the Raji leukemia cell membrane, suggesting an effective interaction of both factors. The joint interaction of these compounds in cell membrane pore formation suggests an explanation for the more effective cytotoxic activity that their combination was observed in this experiment.
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