Human Amnion Cells Research Articles

Aquaporin 3 Expression in Human Fetal Membranes and its Up-regulation by Cyclic Adenosine Monophosphate in Amnion Epithelial Cell Culture

The cell membrane water channel protein aquaporins (AQPs) may be important in regulating the intramembranous (IM) pathway of amniotic fluid (AF) resorption. The objective of the present study was to determine whether aquaporin 3 (AQP3) is expressed in human fetal membranes and to further determine if AQP3 expression in primary human amnion cell culture is regulated by second-messenger cyclic adenosine monophosphate (cAMP). AQP3 expression in human fetal membranes of normal term pregnancy was studied by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). To determine the effect of cAMP on AQP3 expression, primary human amnion cell cultures were treated in either heat-inactivated medium alone (control), or heat-inactivated medium containing: (1) SP-cAMP, a membrane-permeable and phosphodiesterase resistant cAMP agonist, or (2) forskolin, an adenylate cyclase stimulator. Total RNA was isolated and multiplex real-time RT-PCR employed for relative quantitation of AQP3 expression. We detected AQP3 expression in placenta, chorion, and amnion using RT-PCR. Using IHC, we identified AQP3 protein expression in placenta syncytiotrophoblasts and cytotrophoblasts, chorion cytotrophoblasts, and amnion epithelia. In primary amnion epithelial cell culture, AQP3 mRNA significantly increased at 2 hours following forskolin or SP-cAMP, remained elevated at 10 hours following forskolin, and returned to baseline levels by 20 hours following treatment. This study provides evidence of AQP3 expression in human fetal membranes and demonstrates that AQP3 expression in primary human amnion cell culture is up-regulated by second-messenger cAMP. As AQP3 is permeable to water, urea, and glycerol, modulation of its expression in fetal membranes may contribute to AF homeostasis.

Manufacture of a Cell-free Amnion Matrix Scaffold that Supports Amnion Cell Outgrowth In Vitro

We manufactured a cell-free extracellular matrix scaffolds in order to obtain a support material for amnion cell outgrowth, eventually being used for repair of prematurely ruptured fetal membrane. Human preterm or term amnion tissue was separated into its collagenous extracellular matrix and cell components. The acellular scaffold was explored for its capacity to support regrowth of isolated human amnion epithelial or mesenchymal cells in vitro. The outgrowth of amnion cells on and in the scaffold was investigated by scanning and transmission electron microscopy, and confocal laser scanning microscopy. Cell-free amnion matrix scaffolds demonstrated a porous collagen fiber network similar as in native amnion. Inoculation of acellular amnion scaffolds with human amnion cells revealed that its property to support amnion cell outgrowth was retained. Amnion epithelial and mesenchymal cells were found to grow into dense layers on the surface of the scaffold within 3–4 days and 7–8 days, respectively, and to some extent, invaded the scaffold during the culture period. Manufactured acellular amnion matrix retains structural and functional properties required for cell outgrowth in vitro. It may become useful to repair prematurely ruptured fetal membranes.

The role of CCAAT/enhancer-binding protein β in the transcriptional regulation of COX-2 in human amnion

Human labour is associated with increased prostaglandin synthesis within the uterus by the action of the inducible type-2 cyclo-oxygenase enzyme (COX-2). A major source of prostaglandin is the fetal membranes, in particular the amnion, in which expression of COX-2 increases in late pregnancy and with labour. The COX-2 gene promoter contains several putative transcription factor binding sites including those for NF-kappaB, AP-1 and C/EBP and therefore has the features of a rapid response gene. We have previously shown that, in amnion, the NF-kappaB DNA-binding sites in the COX-2 promoter are essential for gene expression and that there is an increase in NF-kappaB activity in amnion with the onset of labour. In this study, we demonstrate that in primary human amnion cells, CCAAT/enhancer-binding protein beta (C/EBPbeta) DNA-binding sites are crucial for the function of the COX-2 gene promoter. Three potential C/EBPbeta DNA-binding sites were identified within the COX-2 promoter which were shown to bind to C/EBPbeta but not to C/EBPalpha, C/EBPdelta, CREB (cAMP responsive element modulator) or CREM. Luciferase reporter constructs with site-directed mutagenesis of the three C/EBPbeta sites in the COX-2 promoter showed reduced expression of luciferase in transient transfection studies. However, comparison of C/EBPbeta protein levels and their DNA-binding activity from cells obtained before and after labour showed no significant differences. This suggests that although C/EBPbeta plays an essential constitutive role in the expression of COX-2, C/EBPbeta may not be directly involved in its regulation in association with human labour.

Human preterm amnion cells cultured in 3-dimensional collagen I and fibrin matrices for tissue engineering purposes

In this study, human preterm amnion cells were investigated in 3-dimensional (3D) cell-matrix culture systems in an attempt to design therapeutic strategies for preterm premature rupture of the membranes. Three-dimensional collagen I and fibrin cell-containing biomatrices were created to mimic the architecture of native amnion. Amnion mesenchymal cells were embedded in 3D matrices, and epithelial cells were placed on top of these matrices. Cell viability and morphology were visualized by DiI-ac-LDL, F-actin, and nuclear staining. Proteolytic activity of matrix metalloproteinases (MMPs) was investigated using gelatine zymography. Preterm amnion epithelial and mesenchymal cells cultured in collagen I and fibrin matrices assume cell morphologies similar to those observed in vivo. Mesenchymal cells were capable of remodelling collagen I, as seen by extensive volume contraction, by 40% at day 1 and 80% at day 5. Matrix contraction was independent of the presence of epithelial cells, and could not be inhibited by GM6001 and/or aprotinin. No contraction was observed in fibrin matrices over 8 days. The migratory response of mesenchymal cells cultured in 3D fibrin matrices supplemented with fibronectin was associated with specific activated MMP-9. Three-dimensional fibrin matrices might be useful in amnion cell tissue engineering, including cell-matrix transplantation.

Secretion of Bioactive Interleukin-6 and Tumor Necrosis Factor-α Proteins from Primary Cultured Human Fetal Membrane Chorion Cells Infected with Influenza Virus

Influenza virus infection during pregnancy is implicated in one of the causes of premature delivery, abortion and stillbirth. Pro-inflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha produced by fetal membranes, are postulated to facilitate premature delivery. We investigated the secretion of IL-6 and TNF-alpha from primary cultured human fetal membrane chorion and amnion cells infected with influenza virus at protein and bioactivity levels in order to understand the pathology of premature delivery during influenza virus infection. Concentrations of IL-6 and TNF-alpha proteins were significantly increased in culture supernatants of chorion cells by influenza virus infection. Culture supernatants of the virus-infected chorion cells stimulated the proliferation of IL-6-sensitive 7-TD-1 cells and induced the cytolysis of TNF-alpha-sensitive L929 cells, both activities of which were inhibited by the addition of respective antibody, whereas no such phenomena were observed in amnion cells. The results demonstrated that only chorion cells secreted significant amounts of bioactive IL-6 and TNF-alpha proteins responding to influenza virus infection. The present study suggests a possibility that the secretion of bioactive IL-6 and TNF-alpha proteins from fetal membrane chorion cells is implicated in the pathogenesis of premature delivery during influenza virus infection.

Study of human amnion cells outgrowth in three-dimensional Collagen I and Fibrin matrices for the treatment of PPROM

PPROM occurs in approximately 1% of all pregnancies and 30–40% of preterm deliveries. No satisfactory treatment of PPROM is currently available. Here we studied the human amnion epithelial and mesenchymal cells cultured in three-dimensional (3D) collagen I and fibrin matrices with the objective to develop an implant for application in PPROM. To mimic the architecture of native amnion, amnion mesenchymal cells were embedded in 3D matrices and epithelial cells were placed on top of these matrices. Cell viability, morphology and proteolytic activity of matrix metalloproteinases(MMPs) were investigated. Epithelial and mesenchymal cells in modified 3D fibrin cell-matrix systems remained vital and acquired their natural morphologies. In addition, in fibrin matrices supplemented with fibronectin, mesenchymal cells activated proteolytic programs of tissue repair, demonstrated by the activation of MMP-9. We also studied the growth of amnion cells in collagen I matrix. One limitation of this approach is contraction of collagen I matrices. In vivo, such a size reduction would result in failure of the implant followed by clinical consequences. In contrast to the collagen I matrix, no decrease in the size of fibrin cell-matrices was observed. To conclude 3D fibrin matrices supplied with amnion cells might be useful in treatment of PPROM.

Engraftment Potential of Human Amnion and Chorion Cells Derived from Term Placenta

Fetal membranes are tissues of particular interest for several reasons, including their role in preventing rejection of the fetus and their early embryologic origin. which may entail progenitor potential. The immunologic reactivity and the transplantation potential of amnion and chorion cells, however, remain to be elucidated. Amnion and chorion cells were isolated from human term placenta and characterized by immunohistochemistry, flow cytometric analysis, and expression profile of relevant genes. The immunomodulatory characteristics of these cells were studied in allogeneic and xenogeneic mixed lymphocyte reactions and their engraftment potential analyzed by transplantation into neonatal swine and rats. Posttransplant chimerism was determined by polymerase chain reaction analysis with probes specific for human DNA. Phenotypic and gene expression studies indicated mesenchymal stem cell-like profiles in both amnion and chorion cells that were positive for neuronal, pulmonary, adhesion, and migration markers. In addition, cells isolated both from amnion and chorion did not induce allogeneic nor xenogeneic lymphocyte proliferation responses and were able to actively suppress lymphocyte responsiveness. Transplantation in neonatal swine and rats resulted in human microchimerism in various organs and tissues. Human amnion and chorion cells from term placenta can successfully engraft neonatal swine and rats. These results may be explained by the peculiar immunologic characteristics and mesenchymal stem cell-like phenotype of these cells. These findings suggest that amnion and chorion cells may represent an advantageous source of progenitor cells with potential applications in a variety of cell therapy and transplantation procedures.

Expressional pattern of known and predicted signaling proteins in seven human cell lines

Although a variety of signaling systems and signaling proteins have been described, cell specific expression of these structures has not yet been systematically studied. Human amnion, bronchial epithelial, fibroblast, glial, kidney, lymphocyte and mesothelial cells were subjected to two-dimensional-gel electrophoresis followed by analysis of protein spots by MALDI-TOF and subsequent identification by specific software. A series of well-documented signaling proteins showed cell specific expressional patterns. Five hypothetical proteins—hypothetical 37.5 kDa protein, similar to calsyntenin 1, hypothetical armadillo repeat/plakoglobulin ARM-repeat profile containing protein, 11 days embryo cDNA clone 2700084k13, hypothetical protein flj22171—so far predicted from their nucleic acid sequence only, were identified, complementing already reported signaling cascades. An analytical tool for the concomitant determination of a large series of signaling structures by an antibody independent protein-chemical method is provided.

Induction of suppressor of cytokine signaling-3 by herpes simplex virus type 1 contributes to inhibition of the interferon signaling pathway.

We showed previously that herpes simplex virus type 1 (HSV-1) suppresses the interferon (IFN) signaling pathway during the early infection stage in the human amnion cell line FL. HSV-1 inhibits the IFN-induced phosphorylation of Janus kinases (JAK) in infected FL cells. In the present study, we showed that the suppressor of cytokine signaling-3 (SOCS3), a host negative regulator of the JAK/STAT pathway, is rapidly induced in FL cells after HSV-1 infection. Maximal levels of SOCS3 protein were detected at around 1 to 2 h after infection. This is consistent with the occurrence of HSV-1-mediated inhibition of IFN-induced JAK phosphorylation. The HSV-1 wild-type strain VR3 induced SOCS3 more efficiently than did mutants that are defective in UL41 or UL13 and that are hyperresponsive to IFN. Induction of the IRF-7 protein and transcriptional activation of IFN-alpha4, which occur in a JAK/STAT pathway-dependent manner, were poorly induced by VR3 but efficiently induced by the mutant viruses. In contrast, phosphorylation of IRF-3 and transcriptional activation of IFN-beta, which are JAK/STAT pathway-independent process, were equally well induced by the wild-type strain and the mutants. In conclusion, the SOCS3 protein appears to be mainly responsible for the suppression of IFN signaling and IFN production that occurs during HSV-1 infection.

Influence of the Antiseptic Agents Polyhexanide and Octenidine on FL Cells and on Healing of Experimental Superficial Aseptic Wounds in Piglets

The main target of the combination of octenidine with phenoxyethanol (Octenisept^®) is the antisepsis of acute wounds, whereas polyhexanide combined with polyethylene glycol in Ringer solution (Lavasept^®) is the agent of choice for antisepsis of chronic wounds and burns. Because comparative data for both agents on the effects on wound healing are lacking, we investigated the influence of preparations based on polyhexanide and octenidine versus placebo (Ringer solution) in experimental superficial aseptic skin wounds (n = 108) of 20 mm diameter, using a double-blind, randomised, stratified, controlled, parallel-group design in piglets. Computerised planimetry and histopathological methods were used for the assessment of wound healing. Histologically, no significant differences could be verified at any time between the 3 groups. However, in the early phase (day 9 after wounding), the octenidine-based product retarded wound contraction to a significantly greater extent than placebo and polyhexanide, whereas in the later phase (days 18 and 28), polyhexanide promoted contraction significantly more than did placebo and octenidine. The consequence is complete wound closure after 22.9 days using polyhexanide, in comparison to the placebo after 24.1 days (p < 0.05) and octenidine after 28.3 days (no statistical difference to placebo). This may be explained by the better tolerance of polyhexanide in vitro, which was demonstrated with dose and time dependence in cytotoxicity tests on human amnion cells.

Hypothetical proteins with putative enzyme activity in human amnion, lymphocyte, bronchial epithelial and kidney cell lines

A myriad of predicted proteins have been described based upon nucleic acid sequences but the existence of these structures has not been confirmed at the protein level. The aim of the study was therefore to show expression of hypothetical proteins in several cell lines and to provide the analytical basis for their identification and characterisation. We used two-dimensional gel electrophoresis with in-gel digestion of high protein spots and subsequent MALDI-TOF analysis of cell lysates from human amnion, lymphocyte, bronchial epithelial and kidney cell lines. A p I range from 3 to 10 was selected and second dimension was run using 9–16% gradient gels. A series of structures that have not been described before at the protein level were identified in several cell lines and were assigned to major enzyme systems including proteolysis (proteases, peptidases, ubiquitin), intermediary metabolism and oxidoreductases. We conclude that the proteomic approach used serves as a suitable tool to verify the existence of predicted/hypothetical proteins. The herein identified enzymes may contribute to several pathways/cascades in the human organism. Furthermore, analytical data given are of major relevance as p Is, a prerequisite to find proteins in a map, cannot be predicted from nucleic acid sequences.

Evidence for the existence of hypothetical proteins in human bronchial epithelial, fibroblast, amnion, lymphocyte, mesothelial and kidney cell lines.

The human genome maybe limited to about 30000 genes whereas the human proteome may be represented by a rough estimate of one million proteins. A legion of proteins have been described and information about these structures are readily available in data banks. There remains, however, a large series of unknown or hypothetical proteins (HPs). Many of them have been predicted from nucleic acid sequences only and are therefore named predicted or HPs. Carrying out "protein hunting" by generating large maps of human cell lines, we aimed to find and identify HPs and provide an analytical tool thereof. Cell lysates from human bronchial epithelial, fibroblast, amnion, lymphocyte, mesothelial and kidney cell lines were prepared and proteins run on two-dimensional gel-electrophoresis (2DE) with in-gel digestion and mass spectrometrical analysis using the MALDI-TOF principle.16 HPs were found in these cell lines and some show cell-specific expressional patterns. HPs belong to several protein classes including structural, signaling, transcriptional/translational, chaperone-related and others. We furthermore provide analytical data i.e. pIs that were often different from predicted values in data banks.A list of HPs has been shown to really exist in several human cell lines thus contributing to knowledge on protein machineries and cascades. Observed and predicted pI values are given representing an analytical tool along with unambiguous identification of protein spots by mass spectrometry independent of antibody availability and specificity thus complementing established methods.

Glucocorticoids induce cytosolic phospholipase A2 and prostaglandin H synthase type 2 but not microsomal prostaglandin E synthase (PGES) and cytosolic PGES expression in cultured primary human amnion cells.

This study examines the regulation of major enzymes in prostaglandin E(2) (PGE(2)) synthesis by glucocorticoids in separate cultures of human amnion epithelial and fibroblast cells at term. Cytosolic phospholipase A(2) (cPLA(2)), cytosolic PGES (cPGES), and microsomal PGES (mPGES) mRNA were expressed at similar levels in both cell types, whereas a greater prostaglandin H synthase type 2 (PGHS-2) mRNA expression was observed in amnion fibroblasts than in epithelial cells. Amnion fibroblasts produced 50-fold more PGE(2) per cell than epithelial cells. Dexamethasone (0.01-1 microM) increased PGE(2) production in amnion fibroblasts in a concentration-dependent manner but did not affect PGE(2) production in amnion epithelial cells. Both mRNA and protein expression of cPLA(2) and PGHS-2 but not cPGES and mPGES were increased in a dose-dependent manner by dexamethasone (0.01-1 microM) in amnion fibroblasts. Induction of cPLA(2) and PGHS-2 mRNA by dexamethasone was blocked by RU486. Dexamethasone did not affect PGHS-2, cPGES, and mPGES mRNA expression in amnion epithelial cells. In conclusion, amnion fibroblasts express a higher level of PGHS-2 mRNA and produced more PGE(2) per cell than amnion epithelial cells at term of human pregnancy. Glucocorticoids increase PGE(2) production only in the amnion fibroblasts mainly through induction of cPLA(2) and PGHS-2 expression.

Sphingosine 1-Phosphate in Amniotic Fluid Modulates Cyclooxygenase-2 Expression in Human Amnion-derived WISH Cells

The metabolism of arachidonic acid, in particular the generation of prostaglandins (PGs), has been proposed to play a key role in the regulation of labor. Moreover, several extracellular proteins have been reported to modulate PG synthesis in amnion cells. In this study, we found that lipid components dissolved in the amniotic fluid modulate PG synthesis in WISH human amnion cells and identified one of these components as a sphingosine 1-phosphate (S1P). WISH cells express several S1P receptors including S1P1, S1P2, and S1P3. When WISH cells were stimulated with S1P, PGE2 synthesis increased in a concentration-dependent manner, showing maximal activity at around 100 nM. S1P treatment also caused the up-regulation of cyclooxygenase-2 (COX-2) mRNA and protein, which was apparent within 3-12 h of stimulation. In terms of the intracellular signaling pathway of S1P-induced WISH cell activation, we found that S1P stimulated two kinds of MAPK, ERK, and p38 kinase. We examined the roles of these two MAPKs in S1P-induced COX-2 expression. S1P-induced COX-2 expression was blocked completely by PD-98059 but not by SB-203580, suggesting that ERK has a critical role in the process. Transfection of S1P1 or S1P3 but not of S1P2 antisense oligonucleotide inhibited S1P-induced COX-2 expression and PGE2 production in WISH cells, indicating the involvements of S1P1 and S1P3 in the processes. This study demonstrates the physiological role of S1P in amniotic fluid and its effect on the modulation of COX-2 expression and PGs synthesis in WISH cells.

Gene Therapy for Mice Sarcoma with Oncolytic Herpes Simplex Virus-1 Lacking the Apoptosis-inhibiting Gene, icp34.5

A mutant herpes simplex virus 1, mtHSV, was constructed by inserting the E. coli beta-galactosidase gene into the loci of icp34.5, the apoptosis-inhibiting gene of HSV. The mtHSV replicated in and lysed U251 (human glioma cells), EJ (human bladder cells), and S-180 (mice sarcoma cells), but not Wish (human amnion cells) cells. With its intact tk (thymidine kinase) gene, mtHSV exhibited susceptibility to acyclovir (ACV), which provided an approach to control viral replication. An in vivo test with mtHSV was conducted in immune-competent mice bearing sarcoma S-180 tumors, which were treated with a single intratumoral injection of mtHSV or PBS. Tumor dimensions then were measured at serial time points, and the tumor volumes were calculated. Sarcoma growth was significantly inhibited with prolonged time and reduced tumor volume. There was microscopic evidence of necrosis of tumors in treated mice, whereas no damage was found in other organs. Immunohistochemical staining revealed that virus replication was exclusively confined to the treated tumor cells. HSV-1 DNA was detected in tumors, but not in the other organs by a polymerase chain reaction analysis. From these experiments, we concluded that mtHSV should be a safe and promising oncolytic agent for cancer treatment.

Regulation of human oxytocin receptor (OTR) in primary human amnion cells

Oxytocin-stimulated PGE2 release is enhanced by the up-regulation of oxytocin receptors at the end of pregnancy. We have shown previously that OTR is upregulated by IL-1β and synergistically upregulated by C/EBPβ and NF-κB transcription factors in human myocytes. The present work was directed toward understanding the regulation of OTR in human amnion at term. OTR mRNA before and after labour was measured using real-time RT-PCR. The expression of OTR in labour (+) primary amnion cells is 16-fold higher than in labour (−) amnion cells (P < 0.001). Treatment with IL-1β resulted in a significant upregulation of OTR which peaked with a 17-fold induction after 6 hours (P < 0.001). IL-1β caused a 16-fold increase in OTR mRNA levels in labour (–) cells, bringing the expression level up to that found in labour (+) cells. Amnion cells were transfected with an OTR promoter reporter vector. Promoter activity was increased fivefold by co-transfection with C/EBPβ or NF-κB p65 (P < 0.05). Co-transfection of C/EBPβ and NF-κB p65 together increased OTR promoter activity 50-fold (P < 0.05).

Nuclear factor kappa B activation and regulation of cyclooxygenase type-2 expression in human amnion mesenchymal cells by interleukin-1beta.

Interleukin-1beta (IL-1beta) has been shown in numerous studies to increase prostaglandin (PG) output by up-regulating the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in PG synthesis. In this study, we investigated the possible role of the nuclear factor kappa B (NFkappaB) in IL-1beta signaling, leading to the expression of COX-2 in human amnion cell culture. Fetal amnion was obtained following vaginal delivery and digested with collagenase, and the subepithelial (mesenchymal) cells were isolated. Cultures were characterized with antisera to keratin (epithelial cells) and vimentin (mesenchymal cells). Confluent cells were stimulated with human recombinant IL-1beta, and activation of NFkappaB was assessed by measuring changes in the inhibitory protein IkappaB (total IkappaB and phosphorylated IkappaB) using Western blot analysis as well as by nuclear binding of NFkappaB using an electrophoretic mobility shift assay. COX-2 protein levels were determined by Western blot analysis. After 5 min of stimulation with IL-1beta, phosphorylated IkappaB began to appear, 90% of which was degraded within 15 min. This was temporally associated with decreased total IkappaB and increased nuclear NFkappaB DNA-binding activity. In the IL-1beta-treated group, COX-2 protein began to increase after 6 h; this response was time-dependent, with a significant increase until 24 h after IL-1beta stimulation. When NFkappaB translocation was blocked by using SN50 (a cell-permeable inhibitory peptide of NFkappaB translocation), the synthesis of COX-2 protein was inhibited. These results suggest that NFkappaB is involved in the IL-1beta-induced COX-2 expression in the mesenchymal cells of human amnion.

Regulation of cyclooxygenase-2 expression by phospholipase D in human amnion-derived WISH cells.

Prostaglandins (PGs) are known to play a key role in the initiation of labor, but the mechanisms regulating their synthesis in amnion are largely unknown. In this study, the regulatory mechanisms for PGE(2) production during phospholipase D (PLD) and p38-dependent activation of WISH cells were investigated. We found that the stimulation of WISH cells with interleukin (IL)-1 beta elicited dose-dependent synthesis of cyclooxygenase-2 (COX-2) mRNA, protein, and their products, PGE(2). Moreover, the treatment of [(3)H]myristate-labeled cells in the presence of 1-butanol caused the dose-dependent formation of [(3)H]phosphatidylbutanol (PBt), a product specific to PLD activity. Pretreating the cells with 1-butanol and Ro 31-8220 inhibited the IL-1 beta-induced COX-2 expression, but 3-butanol did not affect this response. In addition, evidence that PLD was involved in the stimulation of COX-2 expression was provided by the observations that COX-2 expression was stimulated by the dioctanoyl phosphatidic acid (PA) and that the prevention of PA dephosphorylation by 1-propranolol potentiated COX-2 expression by IL-1 beta. Moreover, IL-1 beta stimulation of the cells caused the phosphorylation of p38 and extracellular signal-regulated kinase (ERK), and IL-1 beta-induced COX-2 expression was inhibited by the pretreatment of WISH cells with a p38 inhibitor, in contrast ERK upstream inhibitor had no effect. Furthermore, Ro 31-8220 inhibited IL-1 beta-induced p38 phosphorylation but not ERK phosphorylation. The results of this study indicate that in human amnion cells, IL-1 beta might activate PLD through an upstream protein kinase C to elicit p38 and finally induce COX-2 expression.

Effect of Astragali radix extract on lipopolysaccharide-induced inflammation in human amnion.

The effects of Astragali radix extract on interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha productions, prostaglandin E2 (PGE2) biosynthesis, and leukotriene C4 (LTC4) production from lipopolysaccharide (LPS)-stimulated human amnion cells were investigated. Amnion cells produced detectable amounts of both IL-6 and TNF-alpha under LPS-stimulated conditions. Astragalus extract inhibited the production of IL-6. However, TNF-alpha production was not inhibited by the extract on L929 cytotoxicity assay. Treatment of amnion cells with LPS for up to 24 h resulted in an increase in PGE2 release in a concentration- and time-dependent manner. The extract (150 mg/ml) significantly inhibited the output of PGE2 by amnion cells (p<0.01). The arachidonate lipoxygenase metabolite (LTC4) was increased by LPS treatment of amnion cells. Astragalus extract (30 mg/ml) inhibited LTC4 production by approximately 65% throughout the culture period. These results suggest that Astragali radix extract may have a role in inhibiting bacterial infection-associated preterm labor by suppressing the productions of IL-6, PGE2, and LTC4 by human amnion cells.

Progesterone receptor isoform A inhibits isoform B-mediated transactivation in human amnion

Human amnion cells were transfected with progesterone receptor A and/or B, and the progesterone-dependent reporter construct, mouse mammary tumor virus promoter (MMTV), linked to a luciferase gene. In progesterone receptor B-expressing amnion that had been cultured before the onset of labour, treatment with progesterone resulted in an eightfold increase of the reporter activity, whereas in laboured cells, no such increase was seen. In contrast, progesterone receptor A was a weak activator of transcription in laboured and non-laboured amniocytes. When the isoforms A and B of the progesterone receptor were co-transfected, progesterone receptor A exhibited a marked inhibitory effect on progesterone receptor B-mediated transcription. These results show that progesterone receptors A and B function differentially, and progesterone receptor A is a transdominant repressor of progesterone receptor B-mediated transcription in human term amnion.