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Reprod Biol Endocrinol
2011 Jan 21;9:49. doi: 10.1186/1477-7827-9-49.
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In-vitro effects of the antimicrobial peptide Ala8,13,18-magainin II amide on isolated human first trimester villous trophoblast cells.
Sengupta J
,
Khan MA
,
Huppertz B
,
Ghosh D
.
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Research on antimicrobial cationic peptides (AMPs) has gained pace toward using their potential to replace conventional antibiotics. These peptides preferentially interact with negatively charged membrane lipids typically seen in bacteria and thereby lead to membrane perturbations and membrane dysfunction. However, one possible disadvantage of AMP drugs is their potential for toxicity, especially to those cells which display externalization of negatively charged moieties to the outer leaflet of the plasma membrane during the process of syncytialization. Human placental villous trophoblast is one such cell type. Indeed, intra-vaginal administration of an antimicrobial cationic peptide Ala8,13,18-magainin II amide (AMA) which is a synthetic analogue of magainin 2 derived from Xenopus frog has been observed to result in inhibition of pregnancy establishment in monkeys. However, only little is known about the cellular behavior of early placental cytotrophoblasts (CTB) in the presence of cationic antimicrobial peptides. It is believed that suitable cell culture approaches using AMA as a representative alpha-helical AMP may yield tangible knowledge in this regard. Immunocytochemical (ICC) analyses using confocal microscopy (n = 6 for each treatment sub-group) and Western blot (WB) method (n = 5 for each treatment sub-group) of CTB differentiation based on synthesis of beta-hCG and hPL, and apoptosis based on apoptosis-associated cytokeratin 18 neo-epitope (CK18f) were performed for CTB isolated from human first trimester placental villi and grown in serum-free primary culture for 24 h, 48 h and 96 h on rat-tail collagen with and without AMA (1000 ng/ml). Moreover, secretion of beta-hCG and hPL into conditioned media from isolated CTB grown in vitro for 24 h, 48 h and 96 h (n = 6/each sub-group) with and without AMA was examined using enzyme immunoassays. Furthermore, TUNEL assay, and cell viability based on LDH leakage into medium (n = 6/each sub-group) were assessed to examine the phenomenon of cell death with time and administration of AMA. CTB in serum-free primary culture showed increased (P < 0.05) level of synthesis and secretion of beta-hCG and hPL with time, and higher (P < 0.05) level of cellular cytokeratin 18 neo-epitope and number of TUNEL-positive cells, and LDH activity in conditioned medium at 96 h of culture. Exposure of CTB to AMA resulted in lower (P < 0.05) level of synthesis and secretion of beta-hCG and hPL, as well as, an increase (P < 0.05) of cellular cytokeratin 18 neo-epitope and number of TUNEL-positive cells, and LDH activity in conditioned medium at 96 h as compared to the control treatment. Administration of AMA resulted in attenuation of differentiation, enhancement in apoptosis and loss of viability in early placental villi trophoblast cells in primary culture. Thus, it appears that administration of alpha-helical AMP may adversely affect the process of placentation and pregnancy outcome.
Figure 1. Immunocytochemical characterization of isolated villous trophoblast cells. Isolated cells were allowed to attach to the collagen biomatrix and immunostained for cytokeratin 7 (CK7; A; green) and βhCG (B; green) and vitronectin receptor (CD51; C; green). Nuclei are counterstained with DAPI (blue). Bar = 40 μm.
Figure 2. Representative microphotography of isolated villous trophoblast cells grown on collagen biomatrix. Isolated cells were allowed to attach to the collagen biomatrix and grow for 24 h (A, D), 48 h (B, E) and 96 h (C, F) with 1000 ng/ml AMA (D, E, F) and without AMA (A, B, C) in serum-free culture medium. Bar = 20 μm.
Figure 3. Effect of time and magainin on immunopositive levels of βhCG, hPL and cytokeratin 18 neo-epitope (CK18f). Isolated villous trophoblast cells grown on collagen biomatrix for 24 h (A, D, G, J, M, P), 48 h (B, E, H, K, N, Q) and 96 h (C, F, I, L, O, R) show immunopositive staining for βhCG (A-F; green), hPL (G-L; red), and cytokeratin 18 neo-epitope (CK18f; M-R; green) treated with AMA (D-F, J-L, P-R) and without AMA(A-C, G-I, M-O). The replacement of primary antibody with non-immune mouse IgG (S), non-immune rabbit and goat IgGs (T, U), as well as, pre-neutralization of primary antibodies for βhCG (V) and (hPL) (W) with specific target antigens show no immunopositive staining. Nuclei are counterstained with DAPI (blue). Bars = 20 μm.
Figure 4. Representative Western blot analysis of immunopositive levels of βhCG, hPL and cytokeratin 18 neo-epitope (CK18f) in cytotrophoblast cells grown in primary culture. Cell lysates of isolated villous trophoblast cells grown on collagen biomatrix for 24 h, 48 h and 96 h with or without AMA (1000 ng/ml) were electrophoretically separated and subjected to immunoblot analysis using β-actin as the internal control.
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