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Background: The roll out of HPV immunization programs across the United States was hindered by controversy. We tracked the debate in the United States through MySpace, then the most popular social networking site, in order to better understand the public's reaction to the vaccine., Methods: We searched MySpace for all blog discourse related to HPV immunization. We analyzed each blog according to the overall portrayal of HPV immunization, identified the characteristics of the bloggers, and developed a content analysis to categorize the types of supporting arguments made., Results: 303 blogs met our inclusion criteria. 157 (52%) of the blogs were classified as positive, 129 (43%) as negative, and 17 (6%) were ambivalent toward HPV immunization. Positive blogs generally argued that HPV infection was effective and there were no reasonable alternatives to immunizing. Negative blogs focused on the risks of immunizing and relied heavily on vaccine-critical publications to support their viewpoint. Of the blogs where gender could be identified, 75 (25%) were posted by men and 214 (71%) by women. 60% of blogs posted by men were explicitly critical about HPV immunization versus 36% of women's blogs. Male bloggers also had larger networks of friends., Conclusions: We describe a novel and promising approach to the surveillance of public opinions and attitudes toward immunization. In our analysis, men were far more likely to hold negative views about HPV immunization than women and disseminate negative messages through larger social networks. Blog analysis is a useful tool for Public health officials to profile vaccine criticism and to design appropriate educational information tailored to respond to alternative media/alternative information actively disseminated via social media tools. Public health officials should examine mechanisms by which to leverage this media to better communicate their message through existing networks and to engage in on-going dialogue with the public., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

In the early 1980's concerns about the safety of the whole cell pertussis vaccine in the United States resulted in declining vaccination rates and the withdrawal of multiple vaccine providers from the market. While the possibility of inflammation and febrile reactions to the vaccine were acknowledged by public health authorities, parents also claimed the vaccine was associated with sudden infant death syndrome and encephalopathy. Epidemiological studies examining this question, however, consistently failed to identify an association. We argue that these reactions may have occurred in metabolically vulnerable children, specifically those with defects in fatty acid oxidation. In these children the combination of anorexia and fever that could be caused by the vaccine may have resulted in hypoglycemic episodes and possibly death. We believe that this association was not detected because these conditions were not recognized at the time and because these conditions are uncommon. Nevertheless, at a population level, enough events could have occurred to cause concern amongst parents.

Background: Increasingly, governments, health care agencies, companies, and private groups have chosen Second Life as part of their Web 2.0 communication strategies. Second Life offers unique design features for disseminating health information, training health professionals, and enabling patient education for both academic and commercial health behavior research., Objectives: This study aimed to survey and categorize the range of health-related activities on Second Life; to examine the design attributes of the most innovative and popular sites; and to assess the potential utility of Second Life for the dissemination of health information and for health behavior change., Methods: We used three separate search strategies to identify health-related sites on Second Life. The first used the application's search engine, entering both generic and select illness-specific keywords, to seek out sites. The second identified sites through a comprehensive review of print, blog, and media sources discussing health activities on Second Life. We then visited each site and used a snowball method to identify other health sites until we reached saturation (no new health sites were identified). The content, user experience, and chief purpose of each site were tabulated as well as basic site information, including user traffic data and site size., Results: We found a wide range of health-related activities on Second Life, and a diverse group of users, including organizations, groups, and individuals. For many users, Second Life activities are a part of their Web 2.0 communication strategy. The most common type of health-related site in our sample (n = 68) were those whose principle aim was patient education or to increase awareness about health issues. The second most common type of site were support sites, followed by training sites, and marketing sites. Finally, a few sites were purpose-built to conduct research in SL or to recruit participants for real-life research., Conclusions: Studies show that behaviors from virtual worlds can translate to the real world. Our survey suggests that users are engaged in a range of health-related activities in Second Life which are potentially impacting real-life behaviors. Further research evaluating the impact of health-related activities on Second Life is warranted.

Immunization is a public health area in which the intergovernmental challenges of formulating a national policy are evident. It is also an area in which harmonization of policy across Canada is particularly critical. The National Immunization Strategy was a F/P/T initiative designed to achieve this policy goal. The combination of national guidelines and flexible federal funding via a trust has, to date, been effective in improving equality of access to vaccines in provincial/territorial programmes with limited intergovernmental discord. The long-term success of the initiative will, however, largely depend on ongoing federal financial support and provincial/territorial views on national guidelines. This approach to immunization is a model that would lend itself well to other public health areas in which there is large variability in provincial/territorial programmes, where uniformity of programmes is particularly important and where there is a reluctance or inability of the federal government to legislatively mandate the harmonization of programmes.

Placental ATP-binding cassette (ABC) transporters limit fetal exposure to xenobiotics by regulating transplacental passage into the fetal circulation; their expression and function in fetal membranes, however, has not been studied. In the present study the expression, localisation and function of ABC transporters in human amnion was examined to explore their potential role in modulating amniotic fluid drug disposition in pregnancy. Single-assay oligo-microarrays were used to profile amnion gene expression, and drug transporters expressed at significant levels were identified and selected for further studies. The expression of ABCG2/breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRP) 1 (ABCC1), 2 (ABCC2) and 5 (ABCC5) was detected on the arrays, and verified by RT-PCR and immunoblotting. On confocal microscopy of fetal membrane cryosections, MRP1 and MRP5 were immunolocalised to both apical and basolateral surfaces of the amniotic epithelium, while MRP2 was expressed at low levels only in the apical membrane. BCRP in contrast showed cytoplasmic staining throughout the amniotic epithelium. In addition to the amnion, MRP1 and BCRP immunostaining was observed in the chorion and the decidua. Cell accumulation studies using selective MRP and BCRP inhibitors showed the transporters to be functionally active in amnion epithelial monolayer cultures. In contrast, transwell transport studies using intact amnion membranes did not show significant vectorial transport. These findings identify the amnion as a novel site of ABC drug transporter expression. Functional studies indicate that they may act primarily to prevent cellular xenobiotic accumulation, rather than to confer fetal protection through reduced accumulation in amniotic fluid.

Trophoblast cells undergo loss of plasma membrane lipid asymmetry during cell fusion without further progression to terminal phases of apoptosis. The nature of the anti-apoptotic mechanisms providing cell survival during this process is unknown. Using a BeWo cell model, we explored the role of the xenobiotic/lipid transporter ABCG2 in promoting cell survival during forskolin-induced differentiation. Suppression of ABCG2 expression by siRNA led to a marked increase in phosphatidylserine externalisation followed by accumulation of ceramides and increased apoptosis. Expression of markers of syncytial formation (beta-hCG and HERV-W) was decreased by ABCG2 silencing, although fusion was unaffected. These findings suggest that ABCG2 protects cells during the period of transient membrane instability associated with cell differentiation and fusion, highlighting a novel, previously unrecognised role of ABCG2 as a survival factor during the formation of the placental syncytium.

Bovine nuclear transfer pregnancies are characterized by a high incidence of placental abnormalities, notably, increased placentome size and deficiencies in trophoblast cell function and establishment of placental vasculature. Alterations in gene expression during placental growth and development may contribute to the appearance of large placentomes in pregnancies derived from nuclear transfer. The placenta synthesizes a number of cytokines and growth factors, including the transforming growth factor-betas (TGF-betas) that are involved in the establishment, maintenance and/or regulation of pregnancy. All forms of TGF-beta and their receptors are present at the fetal-maternal interface of the bovine placentome, where they are thought to play an important role in regulating growth, differentiation, and function of the placenta. Using real-time RT-PCR, we have examined the expression of TGF-beta1, TGF-beta2, TGF-beta3 and the receptors TGF-betaRI and TGF-betaRII in placentomes of artificially inseminated (AI) and nuclear transfer (NT)-derived bovine pregnancies at days 50, 100 and 150 of gestation. TGF-beta1, TGF-beta2 and TGF-beta3 mRNA expression increased by 2.0-2.8-fold, while TGF-betaRI and TGF-betaRII mRNA expression decreased by 1.7-2.0-fold in NT placentomes compared to AI controls at all gestational ages examined. These findings indicate that NT placentomes may be resistant to the growth suppressive effects of TGF-betas and could contribute to the placental proliferative abnormalities observed in NT-derived placentas. Alternatively, deficiencies in placentation may provide a mechanism whereby TGF-betas are dysregulated in NT pregnancies.

Intra-amniotic secretion and abundance of epithelial cell-derived neutrophil-activating peptide (ENA)-78, a potent chemoattractant and activator of neutrophils, was studied in the context of term and preterm parturition. Staining of ENA-78 immunoperoxidase was localized predominantly to chorionic trophoblasts and amniotic epithelium in term and preterm gestational membranes, with weaker and less consistent staining in decidual cells. The abundance of ENA-78 in membrane tissue homogenates was significantly increased ( approximately 4-fold) with term labor in amnion (n = 15), and with preterm labor ( approximately 30-fold) in amnion and choriodecidua (n = 31). In amnion tissue homogenate extracts, ENA-78 levels were positively correlated with the degree of leukocyte infiltration (r2 = 0.481). In amniotic fluids, median ENA-78 levels from pregnancies with preterm labor without intra-amniotic infection were significantly lower (P < 0.01 by ANOVA) than those from pregnancies with preterm deliveries with infection; levels in samples derived from term pregnancies were similar before and after labor. Production of ENA-78 by amnion monolayers was stimulated in a concentration-dependent fashion by both interleukin-1beta and tumor necrosis factor alpha. Production of ENA-78 by choriodecidual explants was increased modestly after 2-4 h of exposure to lipopolysaccharide (5 microg/ml). An immunoreactive doublet ( approximately 8 kDa) was detected in choriodecidual explant-conditioned media by immunoblotting. We conclude that ENA-78, derived from the gestational membranes, is present in increased abundance in the amniotic cavity in response to intrauterine infection and, hence, may play a role in the mechanism of infection-driven preterm birth and rupture of membranes secondary to leukocyte recruitment and activation.

The elaboration of cytokines, chemokines and immunomodulatory proteins in the placenta and gestational membranes has been extensively investigated in the context of both normal and abnormal pregnancy and delivery. Patterns of expression of cytokines in the foetal membranes and decidua suggest that inflammatory activation occurs modestly with term labour, but much more robustly in preterm delivery, particularly in the presence of intrauterine infection. Enhanced chemokine expression, particularly evident in deliveries with an infected amniotic cavity, is presumably responsible for recruiting infiltrating leukocytes into the membranes thereby amplifying the inflammatory process and hastening membrane rupture and delivery. Anti-inflammatory cytokines suppress inflammatory reactions in the placenta, but under some circumstances may act in a pro-inflammatory fashion in the membranes. Intracellular signalling by cytokines is modulated by proteins such as SOCS (Silencer Of Cytokine Signalling)-1, -2 and -3. Changes in the abundance of these proteins occur with term labour, implicating them as modulators of cytokine actions around the time of parturition. Prostaglandins, released by the membranes in response to stretch and the actions of pro-inflammatory cytokines, act not only upon the myometrium and cervix, but may also exert paracrine/autocrine effects on cell viability and matrix protein integrity. The localization and regulation of prostanoid isomerases, responsible for converting PGH(2) (derived from prostaglandin H synthase-1 and -2) to bioactive prostanoids, are being studied in these tissues, particularly in the context of cytokine interactions. Although the gestational tissues are known to be sources of PGD(2), PGJ(2) and its derivatives, the regulation of production of these prostaglandins has yet to be studied in any detail and their actions, which may include apoptosis and suppression of inflammation, remain poorly defined. A more complete understanding of these aspects of cytokine-prostaglandin interactions in pregnancy and parturition will, no doubt, unfold as current studies come to fruition.

Elevated activin A and inhibin A levels have been associated with pre-eclampsia, a pregnancy-related disorder associated with placental hypoxaemia. We investigated the effect of in vitro hypoxia on the production of inhibin A, activin A and its binding protein follistatin in term villous placental explants (n=4-7) and trophoblast monolayer cultures (n=4). Explants and trophoblasts were incubated for 24-72 h under either normoxic (21 per cent O(2)) or hypoxic (2 per cent O(2)) conditions. Production of activin A, inhibin A, and follistatin was determined by specific ELISA. After 48 h of hypoxia, villous explants exhibited a significant reduction in activin A production rates to 53.2 +/- 8.9 per cent (mean +/- SEM, P<0.05) of normoxic controls which was sustained after 72 h in culture (46.8 +/- 5.9 per cent), whereas production by trophoblast monolayers was not affected by hypoxia. Follistatin production was decreased to 53.7 +/- 9.2 per cent of control (P<0.05) after 48 h of hypoxia. Inhibin A production remained unaltered in both culture systems. Our data demonstrate for the first time that hypoxia lowers term placental activin A and follistatin production in vitro. These findings do not support the notion that elevated circulating activin A levels in pre-eclampsia originate from the placenta as a result of placental hypoxia. Other as yet unknown maternal/placental factors may contribute to elevated activin A production in women with severe pre-eclampsia., (Copyright 2002 Elsevier Science Ltd.)

Summary In an earlier, companion, review, we concluded that cytokines produced by the placenta and associated membranes are likely to be involved in control of the processes of implantation and placental development (Bowen et al., 2002). In this review, we discuss evidence that cytokines continue to be part of a paracrine/autocrine regulatory network in the placenta and membranes throughout the mid and late stages of gestation. Cytokines are involved in regulation of placental growth during these later stages of pregnancy and also function to protect the fetus from pathological organisms. The evidence, while not entirely consistent, suggests that production of certain cytokines within the extraplacental membranes is altered during normal term parturition, whereas in the villous placenta evidence of labour-associated changes is much more equivocal. Roles for cytokines have been postulated in many facets of parturition, including expulsion of the fetus by uterine contractions, membrane rupture, and dilation of the cervix. Imbalances and disruptions to the cytokine milieu have been implicated in a number of diseases of pregnancy involving abnormalities of both placental growth/establishment and initiation of parturition. Cytokine secretion induced by intrauterine infection is associated with increased occurrence or severity of some neonatal diseases. This wealth of data supports the view that cytokines are an integral part of a functional regulatory/communication network operating within the placental-maternal unit during normal gestation., (Copyright 2002 Elsevier Science Ltd.)

Inflammatory processes are implicated in preterm labour (PTL). To identify potential novel markers for PTL, we have used commercial cDNA arrays to generate profiles of differential expression of inflammation-associated genes in gestational membranes with term and PTL. RNA for cDNA probe synthesis was isolated from reflected human amnion and choriodecidua membranes delivered following Caesarean section at term before the onset of labour (TNL, n = 4), spontaneous labour at term (TSL, n = 4), and PTL with and without chorioamnionitis (PTL(+INF) and PTL(-INF) respectively, n = 4 each). Profiles were displayed relative to TNL and statistical comparisons of TSL versus TNL and PTL(+INF) versus PTL(-INF) were performed. Elevated expression of chemokines macrophage inflammatory protein 1beta(MIP-1beta) and pulmonary and activation-regulated chemokine (PARC) was observed in PTL(+INF) compared to PTL(-INF) amnion and choriodecidua respectively (P = 0.03). Likewise, the cytokines oncostatin-M and pre-B cell enhancing factor (PBEF) were more highly expressed in PTL(+INF) compared with PTL(-INF) and in TSL compared with TNL respectively (P = 0.03). Conversely, inhibin A, tissue inhibitors of matrix metalloproteinase (TIMP)-3 and TIMP-4 were all significantly elevated in PTL(-INF) compared with PTL(+INF) (P = 0.03). Furthermore, differential expression patterns of classes of genes, grouped according to function (e.g. chemokines), were noted. The cDNA array approach holds promise for identification of new candidate markers or combinations thereof for prediction or diagnosis of PTL, as well as for increasing our understanding of the particular aetiologies involved.

Virtually all known cytokines have been demonstrated to be expressed in the placenta and associated fetal and maternal membranes during normal gestation. In addition to playing their traditional roles as modulators of immunological function, cytokines derived from the placenta and extraplacental membranes, together with other locally-derived growth factors, appear to be implicated in various aspects of implantation and placental development. Imbalances in the intrauterine cytokine milieu around the time of implantation and invasion may play a causative role in disorders associated with early pregnancy failure, and are also associated with the abnormal trophoblast development seen in gestational trophoblastic disease. Cytokines thus appear to be an important component of a paracrine/autocrine communication network operating within the feto-maternal interface to ensure the successful establishment of pregnancy., (Copyright 2002 Elsevier Science Ltd.)

Glutathione (GSH) is a major antioxidant system in the mammalian central nervous system (CNS). Abnormalities of GSH metabolism have been associated with many disorders of the CNS, including Parkinson's, Alzheimer's, and Huntingdon's diseases and ischaemic/reperfusion injury. Investigation of GSH levels in the CNS generally relies on biochemical assays from cultures enriched for different cell types. Because glia influence neuronal metabolism, we have studied cultures in which neurons and glia are cocultured. This approach demands fluorescence imaging to differentiate between the different cell types in the culture, permitted by the use of monochlorobimane (MCB), which reacts with GSH to produce a fluorescent product. We have defined the conditions required to ensure steady-state MCB loading and show the specificity of MCB for GSH through a reaction catalysed by glutathione-S-transferase (GST). [GSH] was consistently higher in glia than in neurons, and [GSH] in both cell types decreased with time in culture. Inhibition of GSH synthesis by buthionine sulfoximine (BSO) caused a greater proportional depletion of GSH in glia than in neurons. The depletion of GSH induced by BSO was significantly greater in cells cultured for >10 days. Furthermore, release of GSH from glia and its breakdown by the ectoenzyme gamma-glutamyltranspeptidase (gammaGT) maintains [GSH] in neurons. In older cultures, inhibition of gammaGT by acivicin caused significant depletion of neuronal GSH. After inhibition of GSH synthesis by BSO, inhibition of the glia-neuron trafficking pathway by acivicin caused widespread neuronal death. Such neurotoxicity was independent of the endogenous glutamate and nitric oxide synthase, suggesting that it is not due to secondary excitotoxicity., (Copyright 2001 Wiley-Liss, Inc.)

Apoptosis at the site of rupture has been proposed to play a role in premature rupture of the fetal membranes, a condition associated with increased risk of neonatal sepsis and preterm birth. We investigated the ability of peroxisome proliferator-activated receptor (PPAR)-gamma ligands 15-deoxy-delta12,14PGJ2 (15d-PGJ2), delta12PGJ2, ciglitizone and rosiglitazone to induce apoptosis in the amnion-like WISH cell line. 15d-PGJ2 (10 microM) induced morphological characteristics of apoptosis within 2 h, with biochemical indices (caspase activation and substrate cleavage) following shortly after; maximum cell death (approximately 60%) was observed by 16 h, with an EC50) of approximately 7 microM 15d-PGJ2. Delta12-PGJ2 also induced apoptosis but was less potent and acted at a much slower rate. While ciglitizone also induced apoptosis, rosiglitazone had no effect on cell viability. The mechanism of induction of apoptosis by 15d-PGJ2 and delta12PGJ2, which may be independent of PPAR-gamma activation, requires further elucidation.

Problem: The pro-inflammatory cytokine interleukin (IL)-1beta has been shown to stimulate the production of prostaglandins (PG) in gestational tissues. Increased PG synthesis is considered a key step in the initiation of labor both at term and preterm. In this study. IL-1beta mRNA in the uterus and gestational tissues of mice during mid to late pregnancy was studied to characterize its tissue specific as well as gestational age expression., Method of Study: Gestational tissues (placenta. decidual cap and fetal membranes). uterus, and cervix were collected from pregnant mice during gestation. Total RNA was isolated and probed for the expression of IL-1beta mRNA., Results: There was a significantly increased expression of IL-1beta mRNA in the uterus on day 18 of pregnancy. In the decidual caps, there was increased expression of IL-1beta mRNA on day 14 of pregnancy and a decrease in expression with the onset of labor. In the fetal membranes and placenta, IL-1beta mRNA expression significantly increased on days 14 and 18 of pregnancy. respectively, and then remained elevated for the duration of pregnancy. In the cervix, there was a decrease in expression with labor onset., Conclusions: The increases in IL-1beta mRNA in the fetal membranes and placenta late in pregnancy are consistent with a localized, tissue specific inflammatory activation involved in the initiation of parturition.

These studies were undertaken to evaluate the changes in mRNA expression of prostaglandin H synthase (PGHS)-1 and -2 in murine gestational tissues during the latter half of pregnancy. Gestational tissues (decidual caps, membranes surrounding the fetus, and placentae), uterus, and cervix were collected from pregnant mice at days 12, 14, 16, 18, and 19 (am and pm) of gestation (n = 4), and total RNA was isolated and evaluated for PGHS-1 and PGHS-2 expression by northern blot analysis. Expression was normalized to GAPDH. There were no significant increases in PGHS-2 mRNA expression in any of the tissues studied through gestation. In contrast, expression of PGHS-1 mRNA increased significantly at term in the uterus and fetal membranes. In the placenta, mRNA for PGHS-1 was elevated at day 18 and remained elevated over the remainder of the study. These findings suggest that, in the mouse, increased production of PGs by uterine and intrauterine tissues during pregnancy is associated with up-regulation of PGHS-1 and not PGHS-2.

Prostanoids have been proposed to play a major role in the regulation of uteroplacental blood flow. We examined the effect of hypoxia on the production of prostaglandin E(2)(PGE(2)) thromboxane B(2)(TXB(2)), and prostacyclin (measured as 6-keto-PGF(1alpha)) by human term trophoblast cells and villous placental explants. Explants (n=8) and purified trophoblast cells (n=5) were incubated for 24-72 h under either normoxic (21 per cent O(2)) or hypoxic (2 per cent O(2)) conditions. In trophoblast monolayer cultures, hypoxia attentuated PGE(2)production rates to 52+/-9.4 per cent (mean+/-sem, P< 0.05) but recovered to control rates within 48 h. In villous explants, PGE(2)production was significantly decreased after 48 and 72 h of hypoxia versus the normoxic control, accompanied by increased production of 6-keto-PGF(1alpha)to 173.9+/-26.7 per cent after 48 h. TXB(2)production was increased to 172.3+/-25.9 per cent and 653.2+/-135.7 per cent (P< 0.05) control after 48 and 72 h of hypoxia, respectively. These results were confirmed in villous explants (n=3) cultured in the presence of exogenous 10 microm arachidonic acid. Hypoxia had no significant effect on TXB(2)and 6-keto-PGF(1alpha)in trophoblast cells. In summary, our findings suggest that hypoxia could be responsible for abnormal profiles of prostanoid production commonly observed in women with pre-eclampsia. These results indicate a putative link between hypoxia and compromised placental perfusion., (Copyright 2001 Harcourt Publishers Ltd.)

We evaluated the changes in mRNA expression of cytosolic phospholipase A(2)(cPLA(2)) and 15-hydroxyprostaglandin dehydrogenase (PGDH) in intrauterine and gestational tissues during mid-late murine pregnancy. Tissues (decidual caps, fetal membranes, and placentae, uterus, and cervix) were collected from pregnant mice at days 12, 14, 16, 18, and 19 (am and pm) of gestation. Total RNA was isolated and evaluated for cPLA(2)and PGDH expression by northern blot analysis normalized to GAPDH expression. Expression of mRNA for cPLA(2)increased in the placentae and decidual caps on day 18 and 19 pm, respectively. There was also increased expression for PGDH mRNA in the placenta and fetal membranes at the later stages of pregnancy. The tissue specific differences in expression of cPLA(2)and PGDH suggest that changes in enzymatic regulation of PG production and degradation may be crucial for the initiation of labour., (Copyright 2001 Harcourt Publishers Ltd.)

Objective: To compare the cervicovaginal cytokines IL-1beta, IL-6 and IL-8 with fetal fibronectin (fFN) and cervical dilatation in the prediction of preterm delivery., Study Design: Cervicovaginal cytokine concentration and fFN status were measured in 104 women with symptoms of preterm labour and intact membranes between 24(0) and 33(6) weeks and related to delivery within 2 and 7 days., Results: A group of 18% had cervical dilatation > or = 1cm and 18% were positive for fFN. Preterm delivery within 2 and 7 days occurred in 5 and 12%, respectively. Only IL-6 demonstrated any ability to predict delivery within 2 and 7 days (area under the ROC curve = 0.63 and 0.75, respectively). Using 35pg/ml (75th centile) as a cut-off, IL-6 had a sensitivity and specificity of 60 and 77% for predicting delivery within 2 days, and 62 and 80% for predicting delivery within 7 days. This is similar to the performance of cervical dilatation or fFN status., Conclusions: Measurement of cervicovaginal cytokines has limited ability to predict imminent delivery apart from cervicovaginal IL-6 concentrations, which, in this population, is equivalent to that of fFN status and cervical dilatation > or = 1cm.

1. Exposure of hippocampal neurones to glutamate at toxic levels is associated with a profound collapse of mitochondrial potential and deregulation of calcium homeostasis. We have explored the contributions of reactive oxygen species (ROS) to these events, considered to represent the first steps in the progression to cell death. 2. Digital imaging techniques were used to monitor changes in cytosolic Ca2+ concentration ([Ca2+]c; fura-2FF) and mitochondrial potential (Deltapsim; rhodamine 123); rates of ROS generation were assessed using hydroethidium (HEt); and membrane currents were measured with the whole-cell configuration of the patch clamp technique. 3. Inhibitors of lipid peroxidation (trolox plus ascorbate) and scavengers of superoxide or hydrogen peroxide (manganese(III) tetrakis(4-benzoic acid) porphyrin (MnTBAP) and TEMPO plus catalase), had only minimal impact on the mitochondrial depolarisation and the sustained increase in [Ca2+]c during and following a 10 min exposure to glutamate. 4. The antioxidants completely suppressed ROS generated by xanthine with xanthine oxidase. No significant increase in ROS production was detected with HEt during a 10 min glutamate exposure. 5. A combination of antioxidants (TEMPO, catalase, trolox and ascorbate) delayed but did not prevent the glutamate-induced mitochondrial depolarisation and the secondary [Ca2+]c rise. However, this was attributable to a transient inhibition of the NMDA current by the antioxidants. 6. Despite their inability to attenuate the glutamate-induced collapse of Deltapsim and destabilisation of [Ca2+]c homeostasis, the antioxidants conferred significant protection in assays of cell viability at 24 h after a 10 min excitotoxic challenge. The data obtained suggest that antioxidants exert their protective effect against glutamate-induced neuronal death through steps downstream of a sustained increase in [Ca2+]c associated with the collapse of Deltapsi(m).

We sought to determine whether the gestational age of the pregnant mouse had any relationship with its lipopolysaccharide (LPS) responsiveness. Murine decidual caps from days 13, 15 and 17 of gestation (term is day 20) were dissected out, placed in inserts and equilibrated in media overnight. The following day, media were removed, replaced with fresh media (+/-LPS at 10 microg/mL). After LPS stimulation (24 h), prostaglandin (PG)E2 production by decidual caps from days 13 and 15 increased by 80-fold and 5-fold, respectively. PGF2alpha, 6-keto-PGF1a and TxB2 production also increased. Day 17 decidual caps were unaffected by LPS, pregnant mice inoculated i.p. with LPS (50 microg) at day 13 of gestation induced 100% delivery within 24 h. However, mice treated at days 15 and 17 had an equal occurrence of premature delivery or fetal resorption. This change in LPS responsiveness may indicate changes in the fetal-maternal immune system in late pregnancy.

The fluorescent compound 4-methylumbelliferone (4MU) can be used to detect uridine diphosphate glucuronosyl transferase activity by observing the fall in fluorescence as the compound is converted to 4-methylumbelliferone glucuronide. A microplate assay has been developed that has improved sensitivity and is faster and cheaper than the historical extraction method. Activity is detectable with approximately 10% of the protein required in the extraction method. Absence of extraction and cleanup procedures and the ability to observe reaction rate directly are also of great advantage to the researcher. Michaelis-Menten kinetic data from one healthy female human liver is presented. The extraction method yielded a mean V(max) of 19.9 nmol/min/mg of protein and a mean K(m) of 652.5 microM on 1 day [n = 6, coefficients of variation (CV) 15 and 24%, respectively]. For the microplate method on 1 day, the mean V(max) was 36.21 +/- 1.3 nmol/min/mg of protein (CV = 3.7%), significantly (P <.0001) higher than for the extraction method. The mean K(m), 175. 4 +/- 24.2 microM (CV = 14.5%), was significantly lower (P <.0001) than observed in the extraction method. The assay was performed in replicates of six over 6 days; average intra- and interassay coefficients of variation were 9 and 22% for V(max) and 8 and 35% for K(m), respectively, for the microplate method. The microplate method has also detected activity in the placental trophoblast-derived cell lines JEG-3, JAr, and BeWo (5.5, 4.1, and 2. 6 nmol/min/mg of protein, respectively, at 200 microM 4MU concentration), indicating that placental cells may be capable of glucuronidating 4MU.

Objective: The aim of this study was to determine prospectively whether serum concentrations of corticotropin-releasing hormone, corticotropin-releasing hormone-binding protein, and activin A (1) predict preterm birth within 10 days of hospital admission or at <37 weeks' gestation among women with symptoms of preterm labor and (2) are affected by glucocorticoid therapy., Study Design: Serum concentrations of corticotropin-releasing hormone and activin A were measured in 94 women with symptoms of preterm labor between 24 and 34 weeks' gestation, and delivery outcomes were monitored. Corticotropin-releasing hormone-binding protein concentrations were measured in 71 of these women. In a subgroup of 15 women the serum analytes were assayed in conjunction with estriol before and 12 to 24 hours after administration of dexamethasone., Results: Forty-six percent (6/13) of the women who were delivered within 10 days of hospital admission had a raised serum corticotropin-releasing hormone level, but the predictive relationship was not significant (chi(2) = 1.7; P =.2). Among the 31 women (including the 6 previously mentioned) who were delivered at <37 weeks' gestation, 39% (12/31) had a raised corticotropin-releasing hormone level. Although a raised corticotropin-releasing hormone concentration was positively associated with delivery at <37 weeks' gestation (chi(2) = 9; P =.003), the predictive diagnostic value was poor, with sensitivity, specificity, and positive and negative predictive values of 39%, 90%, 67%, and 75%, respectively. The serum concentrations of corticotropin-releasing hormone-binding protein and activin A were unrelated to gestational age at delivery. Dexamethasone markedly lowered the serum estriol level (P <.001) but had no effect on concentrations of corticotropinreleasing hormone, corticotropin-releasing hormone-binding protein, and activin A., Conclusion: Serum concentrations of corticotropin-releasing hormone, corticotropin-releasing hormone-binding protein, and activin A are not clinically useful for the prediction of preterm delivery among women with symptoms of preterm labor and are not affected by administration of glucocorticoids.

Problem: Clinically useful tests for the prediction and diagnosis of preterm labor and delivery remain to be established. We have hypothesized that soluble intercellular adhesion molecule-1 (sICAM-1) in the cervicovaginal fluid of women with preterm labor may be a useful diagnostic tool., Method of Study: The cervicovaginal fluid of 103 women between 24(0) and 33(6) weeks gestation with preterm contractions and intact membranes was assayed for sICAM-1., Results: Elevated sICAM-1 concentrations predicted short intervals to delivery (area under receiver operator characteristic (ROC) curves, 0.70-0.72 for delivery within 3, 7 and 10 days), with high specificity. Characteristics for delivery within 3 days at a 3 ng/mL threshold for a positive test were sensitivity 33.3%, specificity 98.9%, and positive and negative predictive values of 75.0% and 93.9%, respectively. Predictive ability was independent of and complementary to that of fetal fibronectin (fFN)., Conclusions: Measurement of sICAM-1 in cervicovaginal fluid has potential as a predictor of preterm delivery in women with symptoms of preterm labor, particularly in conjunction with fFN testing.

Problem: To determine whether leptin exhibits cytokine-like properties in gestational tissues in light of its homologies with the class I family of cytokines., Method of Study: WISH and JEG3 cells, and amnion and choriodecidua explants, were treated inflammatory modulators (interleukin-1beta [IL-1beta], tumor necrosis factor-alpha [TNF-alpha] and bacterial lipopolysaccharide [LPS]) and leptin production was measured by immunoassay. Other agents known to regulate adipocyte leptin production were also tested for comparative purposes. In addition, WISH cells, JAR cells and placental explants were treated with leptin to assess its effects on production of IL-8, IL-6 and prostaglandin E2 (PGE2)., Results: Leptin production by all cells and tissues studied was unaffected by treatment with IL-1beta (2.5 ng/mL), TNF-alpha (25 ng/mL) and LPS (2.5 microg/mL). Dexamethasone stimulated leptin production over two-fold by WISH and JEG3 cells, whereas insulin also stimulated a two-fold increase in leptin production in JEG3 cells. IL-6 production by JAR cells and placental explants was stimulated (two- to three-fold) by leptin (300 ng/mL). PGE2 production was unaffected., Conclusions: Leptin derived from gestational tissues is unlikely to play a role in inflammatory reactions within the placenta, but may regulate placental cytokine production. The physiological significance of amnion-derived leptin remains to be established.

RNA was extracted from human gestational membranes and villous placental tissue following spontaneous delivery (n = 15) or elective caesarean section (n = 15) at term. The samples were subjected to Northern analysis, using a 2 kb cDNA probe for peroxisome proliferator activated receptor (PPAR)-gamma. The mRNA was detectable in all choriodecidual and villous placental samples, irrespective of mode of delivery, but was only rarely detectable in the amnion. The JEG3 choriocarcinoma cell line also expressed PPARgamma. In order to evaluate PPAR mediated transcriptional activation in JEG3 cells, the cells were transfected with pTK-PPREx3-luc, a PPAR response element (PPRE) containing luciferase reporter construct. Subsequent treatment with 10 microm 15-deoxy-delta(12,14)prostaglandin J(2)(15dPGJ(2)) resulted in an eight-fold stimulation of luciferase production relative to controls transfected with the same construct lacking the PPRE. This stimulation was concentration-dependent. These results suggest roles for PPARgamma and its ligand in lipid, steroid and inflammatory mediator homeostasis and in remodelling of gestational tissues., (Copyright 2000 Harcourt Publishers Ltd.)

There is a growing appreciation of the importance of activin as a modulator of immune function. The aim of the present study was to determine whether activin A exerts any effects on cytokine and prostaglandin (PG) production by the tissues of pregnancy. Explant cultures were established for amnion, choriodecidual and placental tissues derived from pregnancies delivered at term by Caesarean section (n=5 placentae). Explants were treated with activin A (0.5, 5 and 50 ng/ml) in serum-free Ham's F12/DME media for 24 h (n=3-4 replicates). Production rates of interleukin (IL)-1beta, IL-6, IL-8, tumour necrosis factor-alpha (TNF-alpha) and PGE(2)were determined using immunoassay. Differences between treatment groups were analysed by ANOVA followed by Dunnett's test;P< 0.05 was considered to be significant. Amnion IL-6 production exhibited biphasic responses to activin A: at 5 ng/ml activin A, IL-6 production was significantly stimulated (to 246+/-74.6 per cent of control (mean+/-sem), while at 50 ng/ml it was significantly inhibited (to 46+/-7.4 per cent of control). IL-8 and PGE(2)production by amnion showed significant responses to activin A that were similar to those of IL-6. No significant effects of activin A were observed on choriodecidual and placental IL-6, IL-8 and PGE(2)production. However, TNF-alpha production was significantly inhibited by 50 ng/ml activin A in both choriodecidual and placental explants (to 43+/-9.7 per cent and 51+/-6.7 per cent of control, respectively). Placental IL-1beta production was not altered by treatment with activin A at any concentration. These findings support the concept of activin as an immune modulator in tissues of pregnancy., (Copyright 2000 Harcourt Publishers Ltd.)

To evaluate the association between intercellular adhesion molecule-1 (ICAM-1) in the amnion and preterm labour and delivery, we have assessed ICAM-1 mRNA abundance by Northern analysis and protein levels by enzyme-linked immunosorbent assay (ELISA), in samples of this tissue after term and preterm delivery. The median ICAM-1 mRNA expression following preterm delivery (PTD, n=30) was 24 times greater (P< 0.05) than following elective caesarean section prior to labour at term (CST, n=14). ICAM-1 expression following vaginal delivery after spontaneous labour at term (SLT, n=11) was seven times greater than in the CST group (P< 0.05). The concentration of ICAM-1 protein in the PTD samples (n=31) was four-fold greater than (P< 0.05) in CST (n=14). It was also three-fold greater than in the SLT (n=15) samples (P< 0.05). The results were substantially the same when a preterm spontaneous labour group (PTL) (n=26), exclusive of deliveries complicated by pre-eclampsia (n=1) or intrauterine growth restriction (n=3), was compared to the CST and SLT groups. The ICAM-1 mRNA expression did not differ significantly (P=0.93) between PTL with (n=12) or without (n=14) indicators of intrauterine infection. The results were similar when ICAM-1 protein concentrations were compared (P=0.43) between these two groups. These findings indicate that ICAM-1 is expressed by the human amnion and that this expression is elevated with preterm labour and delivery., (Copyright 2000 Harcourt Publishers Ltd.)

The effects of pro-inflammatory cytokines on 15-hydroxyprostaglandin dehydrogenase (PGDH) expression and prostaglandin (PG) metabolizing activity were investigated in placental tissue. Treatment of trophoblast cells in primary culture with interleukin-1beta (IL-1beta) or tumour necrosis factor-alpha (TNF-alpha) resulted in decreased prostaglandin metabolizing activity. This was assessed both by determining the ratio of production of prostaglandins to their metabolites, and by Northern blot analysis of PGDH mRNA abundance. Dexamethasone had similar inhibitory effect. We hypothesize that proinflammatory cytokines act to enhance prostaglandin actions through coordinated effects on activities of enzymes of both prostaglandin biosynthesis and metabolism, perhaps in concert with effects on receptor function.

Increased production of prostaglandins and cytokines by amnion, particularly prostaglandin (PG) E2, interleukin (IL)-6 and IL-8, is thought to be an important event in infection-associated preterm labour. We characterized the amnion-derived AV3 cell line to determine its appropriateness as a model for investigation of the regulation of amnion cytokine and PG production. Amnion-derived AV3 cells were treated with tumour necrosis factor-alpha (TNF-alpha, interleukin-1beta (IL-1beta), epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) and IL-6, IL-8 and prostaglandin production was determined by immunoassay. Production of IL-6 and IL-8 rose dramatically with all treatments. PGE2, but not PGF2alpha or 6-keto-PGF1alpha, biosynthesis was also increased in a concentration-dependent manner with all treatments. A rapid increase in PGHS-2 (but not PGHS-1) mRNA expression was observed in response to TNF-alpha and IL-1beta. We conclude that the AV3 cell line inflammatory response profile is similar to those observed in primary amnion and other amnion-derived cell lines, and is an appropriate model for human amnion.

1. Glutamate neurotoxicity has been attributed to cellular Ca2+ overload. As mitochondrial depolarisation may represent a pivotal step in the progression to cell death, we have used digital imaging techniques to examine the relationship between cytosolic Ca2+ concentration ([Ca2+]c) and mitochondrial potential (DeltaPsim) during glutamate toxicity, and to define the mechanisms underlying mitochondrial dysfunction. 2. In cells of > 11 days in vitro (DIV), exposure to 50 mM potassium or 100 microM glutamate had different consequences for DeltaPsim. KCl caused a small transient loss of DeltaPsim but in response to glutamate there was a profound loss of DeltaPsim. In cells of 7-10 DIV, glutamate caused only a modest and reversible drop in DeltaPsim. 3. Using fura-2 to measure [Ca2+]c, responses to KCl and glutamate did not appear significantly different. However, use of the low affinity indicator fura-2FF revealed a difference in the [Ca2+]c responses to KCl and glutamate, which clearly correlated with the loss of DeltaPsim. Neurons exhibiting a profound mitochondrial depolarisation also showed a large secondary increase in the fura-2FF ratio. 4. The glutamate-induced loss of DeltaPsim was dependent on Ca2+ influx. However, inhibition of nitric oxide synthase (NOS) by L-NAME significantly attenuated the loss of DeltaPsim. Furthermore, photolysis of caged NO at levels that had no effect alone promoted a profound mitochondrial depolarisation when combined with high [Ca2+]c, either in response to KCl or to glutamate in cultures at 7-10 DIV. 5. In cells that showed only modest mitochondrial responses to glutamate, induction of a mitochondrial depolarisation by the addition of NO was followed by a secondary rise in [Ca2+]c. These data suggest that [Ca2+]c and nitric oxide act synergistically to cause mitochondrial dysfunction and impaired [Ca2+]c homeostasis during glutamate toxicity.

Apoptosis has been described in placental (trophoblast) tissues during both normal and abnormal pregnancies. We have studied the effects of the cyclopentenone prostaglandins (PGs) on trophoblast cell death using JEG3 choriocarcinoma cells. PGJ(2), Delta(12)PGJ(2), and 15-deoxy-Delta(12,14)-PGJ(2) (15dPGJ(2)) (10 microM) significantly reduced mitochondrial activity (MTT assay) over 16 h by 17.4 +/- 4.7%, 28 +/- 9.3%, and 62.5 +/- 2.8%, respectively (mean +/- sem), while PGA(2) and PGD(2) had no effect. The synthetic PPAR-gamma ligand ciglitizone (12.5 microM) had a potency similar to 15dPGJ(2) (69 +/- 3% reduction). Morphological examination of cultures treated with PGJ(2) and its derivatives revealed the presence of numerous cells with dense, pyknotic nuclei, a hallmark of apoptosis. FACS analysis revealed an abundance (approximately 40%) of apoptotic cells after 16-h treatment with 15dPGJ(2) (10 microM). The caspase inhibitor ZVAD-fmk (5 microM) significantly diminished the apoptotic effects of Delta(12)PGJ(2) and 15dPGJ(2). JEG3 cells expressed PPAR-gamma mRNA by Northern analysis. These novel findings imply a role for PPAR-gamma ligands in various processes associated with pregnancy and parturition., (Copyright 1999 Academic Press.)

1. The objective of this study was to clarify the relationships between loss of mitochondrial potential and the perturbation of neuronal Ca2+ homeostasis induced by a toxic glutamate challenge. Digital fluorescence imaging techniques were employed to monitor simultaneously changes in cytoplasmic Ca2+ concentration ([Ca2+]i) and mitochondrial potential (DeltaPsim) in individual hippocampal neurones in culture coloaded with fura-2 AM or fura-2FF AM and rhodamine 123 (Rh 123). 2. In most cells (96 %) at 6-7 days in vitro (DIV) and in a small proportion of cells (29 %) at 11-17 DIV the [Ca2+]i increase induced by exposure to 100 microM glutamate for 10 min was associated with a small mitochondrial depolarisation, followed by mitochondrial repolarisation, and a degree of recovery of [Ca2+]i following glutamate washout. In the majority of neurones at 11-17 DIV (71 %), exposure to glutamate for 10 min induced a profound mono- or biphasic mitochondrial depolarisation, which was clearly correlated with a sustained [Ca2+]i plateau despite the removal of glutamate. 3. Addition of glutamate receptor antagonists (15 microM MK-801 plus 75 microM 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX)) to the washout solution did not affect the post-glutamate [Ca2+]i plateau in neurones exhibiting a profound mitochondrial depolarisation but greatly improved [Ca2+]i recovery in those neurones undergoing only a small mitochondrial depolarisation, suggesting that the release of endogenous glutamate delays [Ca2+]i recovery in the postglutamate period. 4. Cyclosporin A (500 nM) or N-methyl Val-4-cyclosporin A (200 nM) delayed or even prevented the development of the second phase of mitochondrial depolarisation in cells at 11-17 DIV and increased the proportion of neurones exhibiting a small monophasic mitochondrial depolarisation and [Ca2+]i recovery upon glutamate removal. 5. We have thus described a striking correlation between mitochondrial depolarisation and the failure of cells to restore [Ca2+]i following a toxic glutamate challenge. These data suggest that mitochondrial dysfunction plays a major role in the deregulation of [Ca2+]i associated with glutamate toxicity.

Parturition is associated with changes in the production of inflammatory mediators by gestational tissues. An explant system was established to study the change in response of human amnion to various regulating factors during labour. Disks of tissue (6 mm) were excised from amnion membranes obtained either at term by Caesarian section before labour (n = 5-6) or after spontaneous vaginal delivery (n = 3-7). After 24 h equilibration in media, the tissues were treated with interleukin 1 beta (10 ng ml-1), tumour necrosis factor alpha (100 ng ml-1), lipopolysaccharide (5 micrograms ml-1) and dexamethasone (1 mumol l-1) or an appropriate vehicle control for 24 h (n = 3 wells per treatment). Media were harvested and interleukin 10, interleukin 6 and prostaglandin E2 concentrations were determined by immunoassay. In tissues taken both before and after the onset of labour, basal interleukin 10 production by amnion explants was near to the limit of detection. Basal production rates of PGE2 by amnion explants were significantly higher (P < 0.0012; Mann-Whitney U test) in tissues taken during labour than in tissues taken before the onset of labour, while interleukin 6 production was not significantly altered by labour. Production rates of interleukin 6 and prostaglandin E2 were significantly increased by interleukin 1 beta, tumour necrosis factor alpha and lipopolysaccharide in explants from tissues taken during and before labour, while the responsiveness of interleukin 10 production to these treatments was inconsistent. Dexamethasone had no effect on interleukin 6 production by amnion explants, but significantly inhibited prostaglandin E2 production, although this inhibition was approximately 30% lower in tissues obtained after the onset of labour. These results support the presence of inflammatory positive feedback cycles, coincident with a deficiency of an anti-inflammatory factor within gestational tissue, which may be involved in the progression or maintenance of labour.

Preterm labor is frequently associated with ascending intrauterine infection, accompanied by leukocytes infiltration and enhanced local production of cytokines and other inflammatory mediators. The resulting amplification of the inflammatory response, and of prostanoid production in particular, is postulated to be a principal mechanism of infection-driven preterm labor. In this review the effects of pro- and anti-inflammatory cytokines are discussed with respect to the expression of enzymes involved in three key steps of prostanoid biosynthesis and metabolism: liberation of arachidonic acid (AA), conversion of AA to bioactive prostanoids, and prostanoid catabolism. We suggest that by exerting coordinate actions on all three key steps, through multiple molecular mechanisms, inflammatory cytokines acutely up-regulate prostanoid production in intrauterine tissues.

The metabolism of arachidonic acid results in the production of prostaglandins (PGs), which are involved in the initiation of labour at term and preterm. The fetal membranes are a source of pro-inflammatory cytokines which promote increased PG biosynthesis via increased release of arachidonic acid and its conversion to biologically active metabolites such as PGE2 and PGF2alpha. In the amnion, the liberation of arachidonic acid from membrane glycerophospholipid stores can be catalysed by cytosolic phospholipase A2 (cPLA2). In amnion-derived WISH cells, the addition of tumour-necrosis factor alpha (TNF-alpha) (50 ng/ml) provoked a time-dependent increase in the expression of the cPLA2 mRNA which was greatest at 8 and 16 h post-treatment (3.62+/-0.52 and 3.15+/-0.45-fold of control, n=3). The increase in cPLA2 mRNA expression by TNF-alpha was unaffected by the prior addition of interleukin-4 (IL-4) (10 ng/ml), a known inhibitor of prostaglandin endoperoxide H synthase (PGHS)-2 mRNA and protein expression in WISH cells. TNF-alpha also increased the level of immunoreactive cPLA2 protein in a time-dependent manner with the highest levels evident after 8 and 16 h. As with the mRNA, cPLA2 protein levels were unaffected by pre-incubation with IL-4. The inclusion of the cPLA2-specific inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) resulted in a concentration-dependent inhibition of PGE2 biosynthesis in WISH cells treated with TNF-alpha (>95 per cent at 2 microM). We conclude that TNF-alpha increases the abundance of the cPLA2 mRNA and protein in amnion epithelial cells, an effect which plays an important role in amnion PG biosynthesis in the presence of intrauterine infection.

Cytokines and growth factors have been proposed to act as in vivo modulators of amnion prostaglandin production at parturition. To characterize the effects of the 'anti-inflammatory' cytokine interleukin (IL)-4 on amnion prostaglandin production, amnion epithelium-derived WISH cells were treated with IL-4 in the presence/absence of IL-1beta, tumour necrosis factor-alpha (TNF-alpha) or epidermal growth factor (EGF). IL-4 (0.08-10 ng/ml) potently inhibited cytokine-stimulated PGE2 production over 16 h (maximal inhibition approximately 66% at 2.0 ng/ml IL-4). Delaying addition of IL-4 (1 ng/ml) by up to 8 h after IL-1beta addition only slightly attenuated its inhibitory effects, from approximately 65% to approximately 50%. EGF-stimulated PGE2 production was either not inhibited or slightly stimulated by IL-4. Immunoblotting studies revealed that IL-4 (10 ng/ml) significantly suppressed prostaglandin-H synthase-2 (PGHS-2) levels in cells stimulated with IL-1beta and TNF-alpha over 16 h, but had no consistent effects on cytosolic phospholipase A2 (cPLA2) levels under any condition. In the presence of arachidonic acid (10 microM), IL-4 again inhibited cytokine-stimulated, but not EGF-stimulated, PGE2 production. The presence of IL-4 also failed to alter the amount of arachidonic acid released in response to EGF. These findings suggest a role and potential therapeutic application for IL-4 in inhibiting amnion PGHS-2 expression and hence prostaglandin production in infection-driven preterm labour, but not labour in the absence of inflammatory initiators.

Mitochondria are known targets of ischemia-reperfusion injury in adult brain. Although neonates are more resistant to ischemic episodes, the mechanisms accounting for this are not yet fully understood. The aim of this study therefore was to determine whether a difference in post-ischemic mitochondrial function may play a role in the heightened recovery of the neonatal brain following ischemia-reperfusion. We have therefore compared the effects of an in vitro model of ischemia on the enzymes of the mitochondrial respiratory chain in isolated nerve terminals (synaptosomes) from neonatal and adult rats. Ischemia caused a significant, reversible decrease in mitochondrial Complex I activity in both adult and neonatal preparations. In neonatal preparations alone, ischemia also led to a significant decrease in mitochondrial Complexes II-III activity. Following 30 min of reperfusion mitochondrial Complexes II-III and IV exhibited decreased activity in synaptosomes from adult, but not neonatal rats. These data suggest a difference in the susceptibility of adult as compared to neonatal nerve terminal mitochondria to ischemia-reperfusion. These data show for the first time that nerve terminal mitochondria from immature animals remain undamaged following a period of ischemia and reperfusion, in contrast to nerve terminal mitochondria from the adult brain. This adds to the growing body of evidence that mitochondrial function plays a key role in neuronal death following cerebral ischemia reperfusion., (Copyright 1999 Elsevier Science B.V.)

To evaluate the association between intercellular adhesion molecule-1 (ICAM-1) in the choriodecidua and preterm labour and delivery, ICAM-1 mRNA abundance was assessed by northern analysis, and protein levels by ELISA, in samples of this tissue after term and preterm delivery. The median ICAM-1 mRNA expression following preterm delivery (PTD) was 4.8 and 3.8 times (P<0.05), respectively, those following elective Caesarean section prior to labour at term (CST) and following vaginal delivery after spontaneous labour at term (SLT). The concentration of ICAM-1 protein in the PTD samples was 2.2 and 3.0 times (P<0.05) those in CST and SLT samples, respectively. The differences between the term groups were not significant. The results were substantially the same when a preterm spontaneous labour (PTL) subgroup, exclusive of deliveries complicated by pre-eclampsia or intrauterine growth restriction, was compared with the term groups. Choriodecidual ICAM-1 mRNA expression, but not ICAM-1 protein concentration, significantly correlated to the degree of leukocyte infiltration of the PTD gestational membranes. Neither correlated significantly to clinical indications of intrauterine or neonatal infection. These findings indicate that ICAM-1 is expressed by the human choriodecidua and that this expression is elevated with preterm labour and delivery, particularly with increased leukocyte infiltration.

Parturition is associated with increased production of proinflammatory mediators by gestational tissues. Interleukin-10 (IL-10) is an antiinflammatory cytokine produced by human chorion, decidual, and trophoblast tissues. To study the effects of immunomodulators on IL-10, IL-6, and PGE2 production by human choriodecidua before and after labor, an organ explant system was established. Tissue disks (6 mm) were excised from choriodecidual membranes obtained at term by cesarean section before labor (n=6-7) or after spontaneous vaginal delivery (n=7-8). After 24-h equilibration in medium, the tissues were treated with IL-1beta (10 ng/mL), tumor necrosis factor-alpha (100 ng/mL), lipopolysaccharide (5 microg/mL), dexamethasone (1 micromol/ L), or an appropriate vehicle control (n=3 wells/treatment) for 24 h. Media were harvested, and IL-10, IL-6, and PGE2 concentrations were determined by immunoassay. Basal choriodecidual production rates of IL-10 were significantly decreased with labor (P < 0.001), whereas PGE2 and IL-6 production rates increased. The production of all three substances was increased by IL-1beta, tumor necrosis factor-alpha, and lipopolysaccharide, but inhibited by dexamethasone. In contrast to PGE2 and IL-6, there was significantly increased responsiveness of IL-10 production to inflammatory stimuli after labor, but decreased responsiveness to the inhibitory effects of dexamethasone. These data indicate that IL-10 could play a role in modulating or promoting resolution of the inflammatory processes associated with labor at term and with intrauterine infection-associated preterm labor.

Increased prostaglandin biosynthesis during intrauterine infection may be a possible mechanism by which preterm labour is initiated. Inflammatory cytokines and growth factors are known to stimulate prostaglandin production through an increase in prostaglandin endoperoxide H synthase (PGHS)-2 synthesis and activity. Interleukin-4 (IL-4), an anti-inflammatory cytokine, can downregulate PGHS-2 expression and inhibit prostaglandin production. Therefore, the aims of the current study were to determine the effects of IL-4 on PGHS-1 and PGHS-2 expression in amion-derived WISH cells treated with inflammatory cytokines and growth factors. In WISH cells, near-maximal production of the PGHS-2 mRNA occurred using 5 ng/ml EGF, 1 ng/ml IL-1beta or 50 ng/ml TNF-alpha. Time-course experiments determined that the PGHS-2 mRNA was induced maximally by these stimuli by 1 h. Pretreatment of WISH cells with IL-4 reduced PGHS-2 mRNA levels at 1 h by 67% in cells treated with EGF, 62% in cells treated with IL-1beta and 54% in cells treated with TNF-alpha. Pretreatment with IL-4 more effectively inhibited PGHS-2 expression than simultaneous addition with EGF or IL-1beta but not TNF-alpha. Immunoblot analysis showed a correlation between inhibition of mRNA levels and levels of PGHS-2 protein, although stimulation of PGHS-2 protein production by EGF was undetectable. Levels of PGHS-1 protein and mRNA remained unchanged in all experiments. Increased production of prostaglandin E2 (PGE2) in response to TNF-alpha and IL-1beta treatment was attenuated by IL-4 pretreatment, by 52% and 72%, respectively. No attenuation of EGF-stimulated PGE2 levels was seen. We conclude that IL-4 inhibits PGHS-2 mRNA and protein production in cytokine-stimulated WISH cells, but does not affect EGF-stimulated PGE2 production, suggesting that EGF can induce prostaglandin biosynthesis by a mechanism other than through increased PGHS-2 expression.

Problem: In hematopoietic cells, interleukin (IL)-13 shares many actions with IL-4. The effects of IL-13 in gestational tissues have yet to be reported, however. We compared the effects of IL-4 and IL-13 on the production of cytokines and prostaglandin E2 (PGE2) in epithelial amnion-derived WISH cells., Method of Study: WISH cells were treated with IL-4 or IL-13 (0.08-10 ng/ml) with/without cotreatment with IL-1 beta (0.2 ng/ml), tumor necrosis factor-alpha (10 ng/ml) or epidermal growth factor (5 ng/ml). The production of IL-6, IL-8, and PGE2 was measured by immunoassay after 16 hr., Results: Both IL-4 and IL-13 inhibited PGE2 production with indistinguishable concentration-response curves, under basal or stimulated conditions. The maximal inhibition of IL-1 beta-stimulated PGE2 production (to 28% +/- 10% of control) was seen at 10 ng/ml of IL-4 or IL-13. Basal IL-6 production was stimulated approximately twofold by IL-4 and IL-13, whereas IL-4 and IL-13 both inhibited cytokine-stimulated (but not basal) IL-8 production by approximately 50%. In the presence of 1 ng/ml of IL-4, IL-13 was unable to further inhibit PGE2 production., Conclusions: The inhibition of PGE2 and IL-8 production by IL-4 in WISH cells is mimicked by IL-13. Both cytokines, probably through binding to a common receptor complex, may share a role in suppressing inflammatory reactions within intrauterine tissues.

Problem: To determine the effects of anti-inflammatory cytokines on the production of inflammatory mediators by placental cells., Method of Study: Cells from term human placentas were isolated and cultured in vitro in the presence of anti-inflammatory cytokines interleukin (IL)-4, IL-10, and transforming growth factor (TGF)-beta. Their effects on the production of IL-8 and prostaglandin E2 (PGE2) were investigated under basal conditions and after stimulation with IL-1 beta and tumor necrosis factor (TNF)-alpha., Results: Both IL-10 and IL-4 inhibited IL-1 beta- and TNF-alpha-induced PGE2 production but had no significant effects on the production of PGE2 under basal conditions. TGF-beta 1 was without effect in stimulated cells, whereas under basal conditions TGF-beta 1 stimulated PGE2 production. Similar trends were seen for IL-8 production, with the exceptions that TGF-beta 1 decreased the TNF-alpha-induced production and IL-4 decreased basal IL-8 production., Conclusions: The anti-inflammatory effects shown by IL-4, IL-10, and (to lesser extent) TGF-beta may play a role in ameliorating the potentially harmful effects of pro-inflammatory mediators in the feto-placental unit.

Activin-betaA subunits are expressed by the human placenta and extraplacental membranes at term and preterm. The regulation of activin-A production by these tissues has not been characterized to date, however. To determine the effects on activin-A production of pro-inflammatory cytokines, amnion, decidual and placental cells were isolated by enzyme dispersion and treated in primary culture with interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha). Activin-A production (determined by ELISA) by amnion, decidual and placental cultures was 1.2 +/-0.27, 31.1+/-9.9, and 50.7+/-28.5 pg/microg protein/16 h, respectively (mean+/-SEM; n=5-7 experiments). Both IL-1beta and TNF-alpha stimulated activin-A production in a concentration-dependent fashion in all cultures; maximal stimulation was achieved at 0.25-1.0 ng/ml IL-1beta and 25-50 ng/ml TNF-alpha, respectively. In amnion, decidual and placental cultures IL-1beta stimulated activin-A production to 747+/-274, 190+/-11, and 254+/-60.2 per cent of controls, while TNF-alpha stimulated production to 312+/-81.5, 194+/-22.5, and 193+/-12.5 per cent, respectively (mean+/-SEM; n=5; P<0.05 by ANOVA). These studies show for the first time that pro-inflammatory cytokines are potent stimulators of activin-A production by intrauterine tissues. This may provide an explanation for the elevated concentrations of activin-A measured in the sera of some women in preterm labour.

Amniotic fluid at term contains high concentrations of interleukin (IL)-6 and IL-8. The source of these cytokines has not been identified, although the fetal membranes (amnion and chorion) are likely contributors. Amnion cytokine production was investigated by using amnion cells isolated by enzymatic digestion (from placentas delivered at term before labor) and cultured in vitro. IL-6 and IL-8 were measured in conditioned media by ELISA. Amnion cells produced detectable amounts of both IL-6 and IL-8 throughout the 7-day culture period. The ratio of IL-8 to IL-6 was approximately 5:1, similar to the ratio found in amniotic fluid. Production of both IL-6 and IL-8 was stimulated in a concentration-dependent fashion by interleukin-1beta (0.1-10 ng/ml), tumor necrosis factor-alpha (1-100 ng/ml), and bacterial lipopolysaccharide (0.1-10 microg/ml), and also by 100 nM phorbol 12-myristate 13-acetate. Epidermal growth factor (1-25 ng/ml) had only minimal effects on amnion cytokine production. Dexamethasone (10 nM) inhibited IL-6/-8 production by approximately 50% throughout the culture period. Production of IL-6/-8 by cultured amniotic fibroblasts, which under basal conditions was much lower than that by epithelial cells, was regulated by all the agents tested in a fashion similar to that of the epithelial cells. These findings suggest that the amnion contributes to the pool of IL-6 and IL-8 in amniotic fluid. We speculate that amnion-derived cytokines might have functions during normal human parturition that are distinct from their conventional roles as inflammatory mediators.

Problem: To determine whether amnion cells produce interleukin (IL)-6 and -8 and thus may contribute to the high concentrations of these cytokines in amniotic fluid at term., Method of Study: Amnion-derived WISH cells were treated in culture with stimuli over 16 hr, and IL-6 and IL-8 concentrations in the conditioned media were measured by enzyme-linked immunosorbent assay or bioassay (IL-6 only)., Results: IL-8 production was approximately 5-fold higher than that of IL-6 under basal and stimulated conditions. Significant (by Dunnett's test after analysis of variance) stimulation of production of both cytokines was achieved by IL-1 beta (> 0.2 ng/ml), TNF alpha (> 10 ng/ml), and the phorbol ester, phorbol 12-myristate 13-acetate (> 2 nM), over a 16-hr culture period. Epidermal growth factor at 10 ng/ml induced a small increase in production of IL-8, but not of IL-6, whereas bacterial lipopolysaccharide had minimal effects on production of either cytokine. Basal and cytokine-stimulated IL-6 and IL-8 production was inhibited by dexamethasone at concentrations equal to or greater than 1 nM., Conclusion: These findings suggest that amnion may be a significant contributor to the IL-6 and IL-8 content of amniotic fluid, and that WISH cells may be a suitable model for the study of cytokine production by amnion epithelial cells.