Possible roles of proinflammatory and chemoattractive cytokines produced by human fetal membrane cells in the pathology of adverse pregnancy outcomes associated with influenza virus infection.

Pregnant women are at an increased risk of influenza-associated adverse outcomes, such as premature delivery, based on data from the latest pandemic with a novel influenza A (H1N1) virus in 2009-2010. It has been suggested that the transplacental transmission of influenza viruses is rarely detected in humans. A series of our study has demonstrated that influenza virus infection induced apoptosis in primary cultured human fetal membrane chorion cells, from which a factor with monocyte differentiation-inducing (MDI) activity was secreted.

Lactate dehydrogenase leakage as a marker for apoptotic cell degradation induced by influenza virus infection in human fetal membrane cells.

Objective: In order to elucidate the implication of apoptosis in lactate dehydrogenase (LDH) leakage from influenza virus-infected cells, the effects of a general caspase inhibitor, N-t-Boc-Asp(OMe)-fluoromethylketone (Boc-D-fmk), on LDH leakage, apoptosis induction and virus proliferation were examined.

Methods: Cultured human fetal membrane chorion and amnion cells were incubated with or without Boc-D-fmk after influenza virus infection. LDH leakage was estimated by measuring LDH activities in the culture supernatants and cell lysates.

Expression of stathmin, a microtubule regulatory protein, is associated with the migration and differentiation of cultured early trophoblasts.

Background: The microtubule-destabilizing protein stathmin is expressed by the villous cytotrophoblasts and invasive extravillous trophoblasts (EVTs) in the first-trimester human placenta. Here, we evaluated the significance of stathmin expression in terms of the functions of trophoblasts.

Methods: We employed two choriocarcinoma cell lines (BeWo and JEG-3), an EVT cell line (HTR-8/SVneo) and isolated first-trimester trophoblast cells.

Imbalance between ROS production and elimination results in apoptosis induction in primary smooth chorion trophoblast cells prepared from human fetal membrane tissues.

We have previously demonstrated that induction of apoptosis was observed in the smooth chorion trophoblast cells of human fetal membranes prepared at term, and that apoptosis progressed rapidly during in vitro incubation of the tissues. Furthermore, we identified the contribution of ROS production system (e.g.

Effects of mitogen-activated protein kinase inhibitors on tumor necrosis factor-alpha gene expression and apoptosis induction in cultured human fetal membrane chorion cells infected with influenza virus.

Objectives: We investigated the involvement of p38 mitogen-activated protein (MAP) kinase in tumor necrosis factor (TNF)-alpha gene expression, apoptosis induction and virus replication in cultured human fetal membrane chorion cells infected with influenza virus.

Methods: Influenza virus-infected chorion cells were incubated in the absence or presence of inhibitors of p38 MAP kinase, SB203580 and SB202190. TNF-alpha mRNA and hemagglutinin viral RNA (HA vRNA) were amplified with reverse transcriptase-polymerase chain reaction techniques.

Characterization of monocyte differentiation-inducing (MDI) factors derived from human fetal membrane chorion cells undergoing apoptosis after influenza virus infection.

Influenza virus infection during pregnancy has been implicated as one of cause of premature delivery, abortion and stillbirth. We have reported that cultured human fetal membrane chorion cells undergoing apoptosis by influenza virus infection secrete unidentified heat-stable monocyte differentiation-inducing (MDI) factors. In this study, cellular, biological and immunochemical characteristics of MDI factors were investigated using human monocytic leukemia THP-1 cells by nitroblue tetrazolium reduction and cell adhesion assays.

Contribution of inducible nitric oxide synthase and cyclooxygenase-2 to apoptosis induction in smooth chorion trophoblast cells of human fetal membrane tissues.

We examined the contribution of apoptosis- and oxidative stress-associated genes to apoptosis induction in trophoblast cells of human fetal membrane tissues undergoing apoptosis during in vitro incubation. RT-PCR analyses demonstrated an increased level of HO-1, Mn-SOD, Cox-2, iNOS, TNFalpha, TNFR1, IL-1beta, IL-6, Bax, Bak, and Bad gene expression, while Bcl-2 mRNA expression level decreased. Western blot analyses demonstrated an increase in iNOS, Cox-2, and HO-1 protein levels; a decrease in pro-caspase-3 and 9, proform-PARP, and Apaf-1 protein levels; a leakage of cytochrome c from the mitochondria.

Inhibition of influenza-virus-induced apoptosis in chorion cells of human fetal membranes by nordihydroguaiaretic Acid.

Objectives: It has been postulated that the pathogenesis of influenza virus infection involves not only the virus-proliferation-mediated apoptotic cell death in infected cells, but also a direct reactive oxygen species (ROS)-induced cellular injury in the infected organs. We examined effects of an antioxidant, nordihydroguaiaretic acid (NDGA), on apoptosis induction and viral proliferation. Subsequently, the results were compared with those of pyrrolidine dithiocarbamate (PDTC), another antioxidant.

Human gastric signet ring carcinoma (KATO-III) cell apoptosis induced by Vitex agnus-castus fruit extract through intracellular oxidative stress.

We have previously reported that an ethanol extract of the dried ripe fruit of Vitex agnus-castus (Vitex) displays cytotoxic activity against certain kinds of human cancer cell line resulting in the induction of apoptosis. In this paper, we investigate the molecular mechanism of apoptosis induced by Vitex using a human gastric signet ring carcinoma cell line, KATO-III. DNA fragmentation was observed in Vitex-treated KATO-III cells in a time- and dose-dependent manner.

Induction of pro-inflammatory cytokine gene expression and apoptosis in human chorion cells of fetal membranes by influenza virus infection: possible implications for maintenance and interruption of pregnancy during infection.

Human fetal membranes are composed of amnion, chorion and decidua tissues, which play a critical role in defense barriers as well as maintenance of pregnancy and parturition. Pro-inflammatory cytokines, such as interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha, produced by the tissues are postulated to facilitate parturition. Influenza virus infection is one of causes of pregnancy-associated complications, such as premature delivery, abortion and stillbirth.

Apoptosis induced by influenza virus-hemagglutinin stimulation may be related to fluctuation of cellular oxidative condition.

Primary cultivated amnion epitherial cells prepared from amnion tissue of human fetal membrane (Amnion-cells) were stimulated with influenza virus-hemagglutinin (IV-HA), fractionated from a commercialized IV-HA vaccine by DEAE Sephacel column chromatography. From 72-96 h after stimulation, chromosomal DNA fragmentation and the appearance of in situ TUNEL stained-positive cells were revealed. Amnion-cell DNA fragmentation was inhibited in the presence of glycophorin A or C purified from the human erythrocyte membrane fraction, but not inhibited with free N-acetyl-neuraminic acid.

Effect of antioxidants on apoptosis induced by influenza virus infection: inhibition of viral gene replication and transcription with pyrrolidine dithiocarbamate.

Influenza virus (IV) infection induced apoptotic DNA fragmentation and the moderate overproduction of reactive oxygen species (ROS) in primary cultured chorion cells prepared from human fetal membranes, and IV particles were released from the infected cells. The antioxidant pyrrolidine dithiocarbamate (PDTC) inhibited the induced DNA fragmentation, ROS overproduction and IV particle release. Although Trolox inhibited ROS overproduction, it did not inhibit DNA fragmentation or IV production.

Semi-quantitative RT-PCR-based assay, improved by Southern hybridization technique, for polarity-specific influenza virus RNAs in cultured cells.

Complementary (c) DNAs against viral (v) RNA of negative polarity and complementary and/or messenger (c/m) RNA of positive polarity for influenza virus hemagglutinin (HA) were synthesized from total cellular RNA extracted from influenza virus- and mock-infected cells using polarity-specific primers, respectively. HA vRNA and c/mRNA were amplified readily by polymerase chain reaction (PCR) from influenza virus-infected cells during a virus productive period; however, non-specific PCR product was prone to amplification from mock-infected cells and cells at once after virus infection. Southern blots of the PCR products were hybridized with biotinylated DNA probe, which enabled the generation of specific signals to HA vRNA and c/mRNA.

Differentiation of monocytes to macrophages induced by influenza virus-infected apoptotic cells.

The effect of the culture supernatant of influenza virus (IV)-infected apoptotic and non-apoptotic cells on the differentiation of monocytes to macrophages was investigated. IV infection induced apoptotic DNA fragmentation in cultured chorion cells but not in amnion cells prepared from human foetal membrane tissue. To examine the differentiation of monocytes to macrophages, an adhesion assay was employed using the human monocytic leukaemia THP-1 cell line.

Differential mRNA expression of inflammatory cytokines in cultured human fetal membrane cells responding to influenza virus infection.

We examined the expression of mRNAs for inflammatory cytokines and Fas in cultured human fetal membrane cells responding to influenza virus (IV) infection using the reverse transcriptase-polymerase chain reaction (RT-PCR). Primary cultured chorion and amnion cells prepared from human fetal membranes were infected with IV. Chorion cells expressed significant amounts of interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF)-alpha, interferon (IFN)-beta, IFN-gamma and granulocyte macrophage colony-stimulating factor (GM-CSF) mRNAs and small amounts of Fas mRNA in response to IV infection.

Apoptosis in cultured human fetal membrane cells infected with influenza virus.

We investigated the induction of apoptosis in cultured human fetal membrane cells infected with influenza virus type A. We found that influenza virus yield in supernatants of primary cultured chorion and amnion cells prepared from human fetal membranes increased 6 h after infection. Chromosomal DNA was fragmented into oligonucleosomes at 48 h after influenza virus infection in chorion cells but not in mock-infected chorion cells, mock-infected amnion cells or influenza virus-infected amnion cells.