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Adverse pregnancies outcomes present a clinical dilemma in Perinatal medicine. This is partly due to lack of accuracy of current biomarkers to predict high-risk pregnancies at an earlier stage. The placental alkaline phosphatase (PLAP) carrying extracellular vesicles (EVs), and their cargo have been reported as a source of biomarkers for placental health and an indication of pre-eclampsia progression. We postulate that PLAP is not only placental but also expressed in other fetal organs, suggesting that PLAP + ve EVs are not only a functional indicator of placental function alone. We evaluated PLAP in the placenta, fetal membranes, maternal decidua, myometrial cells, and the EVs derived from them. Various bioanalytical techniques were used to detect PLAP expressions in the cells and EVs. The EVs were characterized by size/quantity, PLAP as a cargo, and canonical EV protein markers. PLAP expression was determined in the chorion trophoblast cells (CTCs) of the fetal membranes and the placental trophoblasts; however, it was absent in the amnion layer of the fetal membranes and the maternal uterine cells used in this study. Using multiple experimental approaches, we further verified the cellular sources of PLAP and confirmed that the EVs from the chorion and placental trophoblasts contain PLAP. Our studies suggest that PLAP is not truly placental. Instead, cells of trophoblast lineage in both fetal membranes and the placenta express PLAP in cells and their EVs. Although PLAP + ve EVs for biomarkers are not exclusively placental, they still represent real-time fetal-specific tissues conditions during pregnancy. • PLAP carrying EVs cannot be considered as placental origin. • The cells of trophoblast lineage in both fetal membranes and the placenta express PLAP in cells and their EVs. • Maternal upper and lower uterine cells do not express PLAP and the EVs are negative for PLAP. [ABSTRACT FROM AUTHOR]

Introduction: To develop protocols for isolation and culture of human chorionic mesenchymal and trophoblast cells and test their differential responsiveness to oxidative stress.Methods: Chorion trophoblast cells (CTC) and chorion mesenchymal cells (CMC) were isolated from term fetal membranes by modifying current protocols. Their purity and characteristics were tested using bright field microscopy and after staining for cytokeratin (CK)-7 and vimentin. Cigarette smoke extract (CSE) was used to stimulate cells, and we determined reactive oxygen species (ROS) production using 2'7'-dichlorodihydro-fluorescein assay, stress signaler p38MAPK activation (Western blot) and senescence by flow cytometry. Co-treatment with antioxidant N-acetyl cystine (NAC) either alone or in combination with SB203580 (p38MAPK inhibitor) was used to test oxidative stress (OS)- and p38MAPK-mediated effects.Results: The isolation and cell culture protocol used in this study yielded 92% pure CTC and 100% pure CMC. CSE treatment significantly induced ROS production, P-p38MAPK activation, and senescence in both cell types compared to controls. Cotreatment with NAC reduced ROS production and p38MAPK activation, and co-treatment with both NAC and SB203580 reduced senescence. ROS response in CMC was higher than CTC; however, senescence of CTC was 10-fold higher than CMC.Conclusions: We introduce approaches for proper isolation and culture of CTC and CMC without any influence or overgrowth of one specific type cell that can confound results. Using this approach, we determined differential effects of CTC and CMC to OS condition seen at term labor. Both CTC and CMC undergo p38MAPK-mediated senescence; however, the rate of senescence is higher in CTC. [ABSTRACT FROM AUTHOR]

Introduction: Throughout gestation, amnion membranes undergo mechanical and or physiological stretch, scratch, or stress which is withstood by repairing and remodeling processes to protect the growing fetus. At term, increased oxidative stress (OS) activates p38MAPK, induces senescence, and inflammation contributing to membrane dysfunction to promote labor. However, the signaling initiated by stretch and scratch is still unclear. This study compares the induction of p38MAPK mediated senescence by stretch, scratch, and stress in human amnion epithelial cells (AECs).Methods: Primary AECs from term, not-in-labor, fetal membranes were cultured using the following conditions (N = 3); 1) CellFlex chambers with or without 20% biaxial stretch, 2) 8-well coverslips with or without scratch, and 3) cells exposed to cigarette smoke extract (CSE) inducing OS. p38MAPK (Western blot or immunocytochemistry), senescence activation, and inflammation (matrix metalloproteinases 9 [MMP9] activity-ELISA) were determined in cells exposed to various conditions. T-test and One-Way ANOVA was used to assess significance.Results: Biological membrane extension, mimicked by 20% biaxial stretch of AEC, maintained an epithelial morphology and activated P-p38MAPK (P = 0.02) compared to the non-stretch controls, but did not induce senescence or MMP9 activation. AEC scratches were healed within 40-hrs, which included proliferation, migration, and cellular transitions aided by p38MAPK activation but not senescence. CSE induced OS increased p38MAPK (P = 0.018) activation, senescence (P = 0.019), and MMP9 (P = 0.02).Conclusion: Physiologic stretch and scratch experienced during gestation can cause p38MAPK activation without causing senescence or inflammation. This may be indicative of p38MAPK's role in tissue remodeling during pregnancy. Overwhelming OS, experienced at term, results in P-p38MAPK mediated senescence and inflammation to disrupt membrane remodeling. [ABSTRACT FROM AUTHOR]

Summary Currently there are no approved vaccines or specific therapies to prevent or treat Zika virus (ZIKV) infection. We interrogated a library of FDA-approved drugs for their ability to block infection of human HuH-7 cells by a newly isolated ZIKV strain (ZIKV MEX_I_7). More than 20 out of 774 tested compounds decreased ZIKV infection in our in vitro screening assay. Selected compounds were further validated for inhibition of ZIKV infection in human cervical, placental, and neural stem cell lines, as well as primary human amnion cells. Established anti-flaviviral drugs (e.g., bortezomib and mycophenolic acid) and others that had no previously known antiviral activity (e.g., daptomycin) were identified as inhibitors of ZIKV infection. Several drugs reduced ZIKV infection across multiple cell types. This study identifies drugs that could be tested in clinical studies of ZIKV infection and provides a resource of small molecules to study ZIKV pathogenesis. [ABSTRACT FROM AUTHOR]