17-ß-Estradiol counteracts the effects of high frequency electromagnetic fields on trophoblastic connexins and integrins.

We investigated the effect of high-frequency electromagnetic fields (HF-EMFs) and 17-ß-estradiol on connexins (Cxs), integrins (Ints), and estrogen receptor (ER) expression, as well as on ultrastructure of trophoblast-derived HTR-8/SVneo cells. HF-EMF, 17-ß-estradiol, and their combination induced an increase of Cx40 and Cx43 mRNA expression. HF-EMF decreased Int alpha1 and ß 1 mRNA levels but enhanced Int alpha5 mRNA expression. All the Ints mRNA expressions were increased by 17-ß-estradiol and exposure to both stimuli. ER-ß mRNA was reduced by HF-EMF but augmented by 17-ß-estradiol alone or with HF-EMF. ER-ß immunofluorescence showed a cytoplasmic localization in sham and HF-EMF exposed cells which became nuclear after treatment with hormone or both stimuli. Electron microscopy evidenced a loss of cellular contact in exposed cells which appeared counteracted by 17-ß-estradiol. We demonstrate that 17-ß-estradiol modulates Cxs and Ints as well as ER-ß expression induced by HF-EMF, suggesting an influence of both stimuli on trophoblast differentiation and migration.

Circadian variation of cell proliferation in HTR-8/SVneo cell line.

Circadian clock controls several physiological processes such as cell proliferation. Extravillous trophoblast proliferation is a tightly regulated function playing a fundamental role in maternal vessel remodeling. We recently demonstrated that clock genes Per2 and Dec1 as well as the clock-controlled genes Dbp and Vegf are rhythmically expressed in human extravillous trophoblast-derived HTR-8/SVneo cells. Analyzing the time course of HTR-8/SVneo cell proliferation, a circadian variation in cell number was found. Moreover, we showed a rhythmic expression of mRNAs for Wee1 and stathmin, two genes involved in cell cycle progression. We suggest that circadian clockwork may orchestrate the functionality of the several factors involved in the control of human trophoblast functions that are fundamental for a successfully pregnancy outcome.

Estrogen metabolites in the release of inflammatory mediators from human amnion-derived cells.

AIMS: Human amnion-derived cells have been used as in vitro models to test the release of inflammatory mediators, such as arachidonic acid (AA) and prostaglandin E(2) (PGE(2)). We compared estrogen metabolites for their ability to induce AA release, to influence PGE(2) production and to interact toward intracellular estrogen receptors (ERs). MAIN METHODS: Metabolite effects on AA and PGE(2) release were examined by radiolabelled substrate incorporation and by colorimetric enzyme immunoassays, respectively. [(3)H]17-ß-estradiol binding displacements were performed on Ro-20-1724 treated whole cells. KEY FINDINGS: In WISH cells, estrone, 2-hydroxy-estrone and estriol induced a rapid dose dependent release of AA that was not inhibited by cycloheximide. Estrone and 2-hydroxy-estrone showed biphasic dose-response curves of PGE(2), whereas estriol and 16-α-hydroxy-estrone increased PGE(2) levels at high concentrations. 2-methoxy-estrone, 4-hydroxy-estradiol and 4-hydroxy-estrone did not significantly affect PGE(2) release. 2-methoxy-estradiol and 2-hydroxy-estradiol decreased the PGE(2) release. Effects of metabolites on PGE(2) were inhibited by cycloheximide and by the ER antagonist tamoxifen. In AV3 cells PGE(2) production was poorly detectable. On Ro-20-1724 treated WISH cells the K(i) of 17-ß-estradiol was 29.2 ± 5.4 nM. Estrone, 2-methoxy-estrone and 2-methoxy-estradiol showed similar affinity values. The hydroxyl substituent at position 2, 4 and 16 decreased or markedly increased the affinity for estradiol or estrone derivatives, respectively. SIGNIFICANCE: The estrogen metabolites induced nongenomic effects on AA release from WISH cells. The influence on PGE(2) release was detectable only on WISH cells. These effects appeared genomic and mediated by intracellular ERs, whose properties seemed strongly dependent on intracellular cAMP levels.

Transient DNA damage induced by high-frequency electromagnetic fields (GSM 1.8 GHz) in the human trophoblast HTR-8/SVneo cell line evaluated with the alkaline comet assay.

One of the most controversial issue regarding high-frequency electromagnetic fields (HF-EMF) is their putative capacity to affect DNA integrity. This is of particular concern due to the increasing use of HF-EMF in communication technologies, including mobile phones. Although epidemiological studies report no detrimental effects on human health, the possible disturbance generated by HF-EMF on cell physiology remains controversial. In addition, the question remains as to whether cells are able to compensate their potential effects. We have previously reported that a 1-h exposure to amplitude-modulated 1.8 GHz sinusoidal waves (GSM-217 Hz, SAR=2 W/kg) largely used in mobile telephony did not cause increased levels of primary DNA damage in human trophoblast HTR-8/SVneo cells. Nevertheless, further investigations on trophoblast cell responses after exposure to GSM signals of different types and durations were considered of interest. In the present work, HTR-8/SVneo cells were exposed for 4, 16 or 24h to 1.8 GHz continuous wave (CW) and different GSM signals, namely GSM-217 Hz and GSM-Talk (intermittent exposure: 5 min field on, 10 min field off). The alkaline comet assay was used to evaluate primary DNA damages and/or strand breaks due to uncompleted repair processes in HF-EMF exposed samples. The amplitude-modulated signals GSM-217 Hz and GSM-Talk induced a significant increase in comet parameters in trophoblast cells after 16 and 24h of exposure, while the un-modulated CW was ineffective. However, alterations were rapidly recovered and the DNA integrity of HF-EMF exposed cells was similar to that of sham-exposed cells within 2h of recovery in the absence irradiation. Our data suggest that HF-EMF with a carrier frequency and modulation scheme typical of the GSM signal may affect the DNA integrity.

Adenylyl cyclases as innovative therapeutic goals.

Pharmacological modulation of intracellular cyclic AMP (cAMP) signalling could provide new therapeutic and experimental tools. Although drugs interfering with this pathway have traditionally targeted membrane receptors, the effector enzyme adenylyl cyclase (AC), which functions as a signalling catalyst, also presents an interesting target. Thus, development of isoform-selective stimulator and/or inhibitor compounds for AC could lead to organ-specific pharmacotherapeutics for treating heart failure, cancer and neurodegenerative diseases. In this review, the potential of AC as the object of drug therapy is discussed.

Effect of high-frequency electromagnetic fields on trophoblastic connexins.

Connexins (Cx) are membrane proteins able to influence trophoblast functions. Here we investigated the effect of high-frequency electromagnetic fields (HF-EMF) on Cx expression and localization in extravillous trophoblast cell line HTR-8/SVneo. We also analysed cell ultrastructural changes induced by HF-EMF exposure. Samples were exposed to pulse-modulated 1817 MHz sinusoidal waves (GSM-217 Hz; 1h: SAR of 2 W/kg). Cx mRNA expression was assessed through semi-quantitative RT-PCR, protein expression by Western blotting, protein localization by indirect immunofluorescence, cell ultrastructure using electron microscopy. HF-EMF exposure significantly and selectively increased Cx40 and Cx43, without altering protein expression. Nevertheless, Cx40 and Cx43 lost their punctuate fluorescence within the cell membrane, becoming diffuse after HF-EMF exposure. Electron microscopy evidenced a sharp decrease in intercellular gap junction-like structures. This study is the first to indicate that exposure of extravillous trophoblast to GSM-217 Hz signals can modify Cx gene expression, Cx protein localization and cellular ultrastructure.

Evidence for circadian rhythms in human trophoblast cell line that persist in hypoxia.

Circadian clock governs daily rhythmicity of a number of physiological processes such as reproductive functions. The existence of circadian clocks in the placenta is not clearly established. In order to investigate whether human placenta may function as circadian oscillator, we utilized HTR-8/SVneo cells derived from human first-trimester trophoblast. In serum-shocked cells we found circadian expressions for the clock genes Per2 and Dec1 as well as for Dbp, a canonical clock-controlled gene. We obtained similar results for Vegf, a circadian output involved in the control of placental vasculogenesis and trophoblast functions. Interestingly, circadian oscillations persisted and even enhanced in cells experimentally rendered hypoxic with CoCl(2). These results could be explained since the hypoxic milieu of the first-trimester placenta is considered the optimal condition for normal placentation. These data collectively support a possible role for the differential rhythmic expression of Vegf, influenced by circadian clock, in the adjustment of placental vascularization and trophoblast functions to the specific requirements of the different gestational ages.

HSP70 expression in human trophoblast cells exposed to different 1.8 Ghz mobile phone signals.

The heat-shock proteins (HSPs) are important cellular stress markers and have been proposed as candidates to infer biological effects of high-frequency electromagnetic fields (EMFs). In the current study, HSP70 gene and protein expression were evaluated in cells of the human trophoblast cell line HTR-8/SVneo after prolonged exposure (4 to 24 h) to 1.8 GHz continuous-wave (CW) and different GSM signals (GSM-217Hz and GSM-Talk) to assess the possible effects of time and modulation schemes on cell responses. Inducible HSP70 protein expression was not modified by high-frequency EMFs under any condition tested. The inducible HSP70A, HSP70B and the constitutive HSC70 transcripts did not change in cells exposed to high-frequency EMFs with the different modulation schemes. Instead, levels of the inducible HSP70C transcript were significantly enhanced after 24 h exposure to GSM-217Hz signals and reduced after 4 and 16 h exposure to GSM-Talk signals. As in other cell systems, in HTR-8/SVneo cells the response to high-frequency EMFs was detected at the mRNA level after exposure to amplitude-modulated GSM signals. The present results suggest that the expression analysis for multiple transcripts, though encoding the same or similar protein products, can be highly informative and may account for subtle changes not detected at the protein level.

An RPE cell line as a useful in vitro model for studying retinoic acid receptor beta: expression and affinity.

Retinoids mediate their biological effect by interacting with specific nuclear receptors. Of the several known RAR (retinoic acid receptor) subtypes, RAR-beta is of particular interest, since its expression is silenced in many cancers and it is believed to be a tumour suppressor. Specific ligands of RAR-beta can potentially be used in anti-cancer therapy. In the present study, we have investigated the feasibility of using HRPE cells (human retinal pigment epithelial cells) as an experimental model for characterizing RAR-beta-ligand interaction. RT-PCR (reverse transcription-PCR) and Western blot analyses show that HRPE cells specifically express only RAR-beta and none of the other receptor subtypes. In addition, we show that the expression of RAR-beta increases with increasing passage number of the cells. Interestingly, the increase in RAR-beta expression is not associated with telomere shortening, a typical biomarker of cellular senescence. In the present study, we also describe a protocol for characterizing RAR-beta-ligand interactions using nuclear extract from late passage HRPE cells as a source of endogenous RAR-beta. Using [(3)H]CD367 as the ligand, RAR-beta in HRPE cells showed an affinity of 9.6 +/- 0.6 nM and a B(max) of 780 +/- 14 fmol/mg of protein. We have confirmed the feasibility of using this assay to detect the interaction of ligands with RAR-beta by investigating the ability of certain flavonoids to inhibit the binding of [(3)H]CD367 to nuclear extracts from HRPE cells. The inhibition constant of the flavonoids for RAR-beta was between approx. 1-30 microM, showing that the flavonoids interact with RAR-beta with low affinity.

Progress in drug delivery to the central nervous system by the prodrug approach.

This review describes specific strategies for targeting to the central nervous system (CNS). Systemically administered drugs can reach the brain by crossing one of two physiological barriers resistant to free diffusion of most molecules from blood to CNS: the endothelial blood-brain barrier or the epithelial blood-cerebrospinal fluid barrier. These tissues constitute both transport and enzymatic barriers. The most common strategy for designing effective prodrugs relies on the increase of parent drug lipophilicity. However, increasing lipophilicity without a concomitant increase in rate and selectivity of prodrug bioconversion in the brain will result in failure. In these regards, consideration of the enzymes present in brain tissue and in the barriers is essential for a successful approach. Nasal administration of lipophilic prodrugs can be a promising alternative non-invasive route to improve brain targeting of the parent drugs due to fast absorption and rapid onset of drug action. The carrier-mediated absorption of drugs and prodrugs across epithelial and endothelial barriers is emerging as another novel trend in biotherapeutics. Several specific transporters have been identified in boundary tissues between blood and CNS compartments. Some of them are involved in the active supply of nutrients and have been used to explore prodrug approaches with improved brain delivery. The feasibility of CNS uptake of appropriately designed prodrugs via these transporters is described in detail.

Adenylyl cyclase/cAMP system involvement in the antiangiogenic effect of somatostatin in the retina. Results from transgenic mice.

Neoangiogenesis is a response to retinal hypoxia that is inhibited by somatostatin (SRIF) through its subtype 2 receptor (sst2). Using a mouse model of hypoxia-induced retinopathy, we investigated whether inhibition of adenylyl cyclase (AC) is involved in SRIF anti-angiogenic actions. Hypoxia increased AC responsiveness in wild type (WT) retinas and in retinas lacking sst2, but not in sst2-overexpressing retinas. Hypoxia also altered AC isoform expression with different patterns depending on sst2 expression level. The AC VII isoform mRNA and protein resulted the most affected. Indeed, in hypoxia AC VII expression was enhanced in WT retinas and it was further increased in sst2-lacking retinas, whereas in sst2 overexpressing retinas the increase of AC VII was lower than in WT retinas. These data suggest an involvement of AC/cAMP in mediating both hypoxia-evoked retinal neoangiogenesis and SRIF protective actions. The AC VII isoform is a candidate to a main role in these mechanisms.

Evaluation of HSP70 expression and DNA damage in cells of a human trophoblast cell line exposed to 1.8 GHz amplitude-modulated radiofrequency fields.

The aim of this study was to determine whether high-frequency electromagnetic fields (EMFs) could induce cellular effects. The human trophoblast cell line HTR-8/SVneo was used as a model to evaluate the expression of proteins (HSP70 and HSC70) and genes (HSP70A, B, C and HSC70) of the HSP70 family and the primary DNA damage response after nonthermal exposure to pulse-modulated 1817 MHz sinusoidal waves (GSM-217 Hz; 1 h; SAR of 2 W/kg). HSP70 expression was significantly enhanced by heat, which was applied as the prototypical stimulus. The HSP70A, B and C transcripts were differentially expressed under basal conditions, and they were all significantly induced above basal levels by thermal stress. Conversely, HSC70 protein and gene expression was not influenced by heat. Exposing HTR-8/SVneo cells to high-frequency EMFs did not change either HSP70 or HSC70 protein or gene expression. A significant increase in DNA strand breaks was caused by exposure to H(2)O(2), which was used as a positive stimulus; however, no effect was observed after exposure of cells to high-frequency EMFs. Overall, no evidence was found that a 1-h exposure to GSM-217 Hz induced a HSP70-mediated stress response or primary DNA damage in HTR-8/SVneo cells. Nevertheless, further investigations on trophoblast cell responses after exposure to GSM signals of different types and durations are needed.

Control of human trophoblast function.

The trophoblast, i.e. the peripheral part of the human conceptus, exerts a crucial role in implantation and placentation. Both processes properly occur as a consequence of an intimate dialogue between fetal and maternal tissues, fulfilled by membrane ligands and receptors, as well as by hormone and local factor release. During blastocyst implantation, generation of distinct trophoblast cell types begins, namely the villous and the extravillous trophoblast, the former of which is devoted to fetal-maternal exchanges and the latter binds the placental body to the uterine wall. Physiological placentation is characterized by the invasion of the uterine spiral arteries by extravillous trophoblast cells arising from anchoring villi. Due to this invasion, the arterial structure is replaced by amorphous fibrinoid material and endovascular trophoblastic cells. This transformation establishes a low-resistance, high-capacity perfusion system from the radial arteries to the intervillous space, in which the villous tree is embedded. The physiology of pregnancy depends upon the orderly progress of structural and functional changes of villous and extravillous trophoblast, whereas a derangement of such processes can lead to different types of complications of varying degrees of gravity, including possible pregnancy loss and maternal life-threatening diseases. In this review we describe the mechanisms which regulate trophoblast differentiation, proliferation, migration and invasiveness, and the alterations in these mechanisms which lead to pathological conditions. Furthermore, based on the growing evidence that proper inflammatory changes and oxidative balance are needed for successful gestation, we explain the mechanisms by which agents able to influence such processes may be useful in the prevention and treatment of pregnancy disorders.

Circadian clocks regulate adenylyl cyclase activity rhythms in human RPE cells.

Genes and components of the circadian clock may represent relevant drug targets for diseases involving circadian dysfunctions. By exploiting an established cell line derived from human retinal pigment epithelium (HRPE), the cell constituting the blood-retinal barrier that is essential to maintain the visual functions of the sensorineural retina, we showed serum-shock induction of rhythmic changes in forskolin-evoked adenylyl cyclase (AC) activity. In the presence of Ca2+ and protein kinase A, the forskolin-induced AC activity is significantly, but not completely inhibited, suggesting the involvement of both Ca2+-sensitive and Ca2+-insensitive AC isoforms in the regulation of circadian rhythmicity in these cells. Semi-quantitative RT-PCR showed circadian profile in the expression of three AC isoforms, the Ca2+-inhibitable AC5 and AC6 and the Ca2+-insensitive AC7, and the clock genes hPer1 and hPer2. Our results demonstrate for the first time circadian rhythmicity in a human cell line, identifying the isoforms involved in the circadian profile of AC activity and showing a rhythmicity of the clock gene mRNA expression in these cells. Therefore, the results reported here provide evidence for an intertwine between AC/[Ca2+]i signalling pathways and Per genes in the HRPE circadian clockwork.

Evidence for the presence of N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptor ligands in human amniotic fluid and fMLP receptor modulation by physiological labour.

OBJECTIVES: The presence of amniotic binding sites for N-formyl-methionyl-leucyl-phenylalanine (fMLP), an inflammatory peptide, and its ability to induce prostaglandin E2 synthesis in the human amnion prompted us to investigate for: (1) the presence of fMLP receptor ligands (fMLPRL) in the amniotic fluid; (2) expression of the fMLP receptor in amniotic tissue; (3) the effect of amniotic fMLPRL on neutrophil cyclic AMP (cAMP) level and calcium concentration ([Ca2+]i) during physiological pregnancy and labour. METHODS: Binding assays were performed on neutrophils to determine the presence of fMLRL in the amniotic fluid at the 17th week of pregnancy, as well as at term, before and after the onset of labour. The expression of fMLP receptor mRNA was evaluated by RT-PCR, the cAMP level by a radiochemical assay, and the calcium concentration by Fura-2 AM fluorescence measurement. RESULTS: fMLPRLs were detectable in amniotic fluid throughout pregnancy, and their levels did not vary during gestation. Labour significantly increased both the amniotic fMLPRL level and the expression of fMLP receptor in amnion tissue. The increased amniotic fMLPRL concentration noted during labour significantly increased neutrophil cAMP level and [Ca2+]i. CONCLUSIONS: These findings demonstrate for the first time the presence of fMLP receptor ligands in amniotic fluid, and indicate a modulation of the fMLP system by the events of physiological labour.

The role and modulation of the oxidative balance in pregnancy.

Oxidative processes exert a fundamental regulatory function during pregnancy. It depends on the influence of oxygen, nitric oxide, reactive oxygen species and reactive nitrogen species metabolic pathways upon the vascular changes in the maternal organism, as well as on the regulation of uterine and cervical tone throughout gestation and delivery. These functions are strictly linked with the mediators of the inflammatory pathway. At the beginning of pregnancy, when a certain grade of inflammatory change is necessary to the trophoblast invasion of maternal tissue, the activation of the process by nitric oxide and reactive nitrogen species is welcome. Indeed, these products modulate the metalloproteinases, which are responsible for the remodelling of uterine extracellular matrix. At this stage estrogens are involved as well in the regulation of the delicate balance of pro-oxidant and anti-oxidant effects. Furthermore, reactive oxygen and nitrogen species appear to play an important role both in normal and pathologic embryogenesis. During advanced pregnancy, a derangement of the oxidative balance can lead to the improper activation of inflammatory changes, thus triggering premature labour as well as other complications, such as foetal growth restriction and preeclampsia. Although a number of pro- and anti-oxidant agents are available to influence the above-mentioned processes, there is no way to adequately measure the oxidative needs in single cases, in order to modulate the oxidative balance in clinical practice. Pharmacological research should be addressed to the development of new drugs, as well as to selective methods of delivery to the gestational tissues.

Ascorbic and 6-Br-ascorbic acid conjugates as a tool to increase the therapeutic effects of potentially central active drugs.

Ascorbic acid (AA) or 6-Br-ascorbate (BrAA) conjugation has been investigated as a tool to improve brain drug delivery by the Vitamin C transporter SVCT2. To this aim, the effects of AA- or BrAA-conjugation on drug affinity and uptake have been assessed in vitro, by using human retinal pigment epithelium (HRPE) cells, and compared in vivo on mice. Nipecotic, kynurenic and diclofenamic acids were chosen as model drugs. Kinetic and inhibition experiments referred to [(14)C]AA uptake into HRPE cells showed that nipecotic and kynurenic acids became able to interact with SVCT2, as competitive inhibitors, only when conjugated to AA or BrAA. Surprisingly, diclofenamic acid itself appeared able to interact with SVCT2, with an affinity that was significantly increased or decreased by AA or BrAA conjugation, respectively. HPLC analysis, performed on HRPE cells, confirmed the SVCT2 mediated transport for the BrAA-conjugate of nipecotic acid, whereas kynurenic acids conjugates although interacting with the transporter did not enter the cells. In accordance, only the nipecotic acid conjugates showed anticonvulsant activity after systemic injection in mice.

Design, synthesis and in vitro evaluation on HRPE cells of ascorbic and 6-bromoascorbic acid conjugates with neuroactive molecules.

Preliminary investigations allowed us to anticipate that conjugation of nipecotic acid with L-ascorbate (AA) gave a prodrug endowed with anticonvulsant activity in mice. In view of these results, and in order to get deepen insight into the molecular aspects at the base of the transport mechanism, a second generation of compounds, based on 6-bromo-6-deoxy-L-ascorbic acid (BrAA) as the carrier molecule was designed and synthesized. Effects of the chirality of the transported drug was also investigated on R- and S-nipecotic acid. Interaction and uptake modalities were evaluated in our in vitro model based on human retinal pigment epithelium cells (HRPE), which expresses the membrane L-ascorbic acid (AA) SVCT2 transporters. A remarkable increase on SVCT2 affinity was found going from AA to BrAA conjugates, that is, 11 (Ki=1187+/-78 microM) versus 19 (Ki=193+/-14 microM) and 12 (Ki=39.8+/-3.2 microM) versus 20, (Ki=7.4+/-0.8 microM). Taken together, these data are in agreement with our initial hypothesis on the possibility to achieve better affinities by conjugation with AA analogs, and also consent to hypothesize the presence of accessory interactions that may improve transporters recognition.

Somatostatin coupling to adenylyl cyclase activity in the mouse retina.

The peptide somatostatin-14 (SRIF) acts in the mammalian retina through its distinct receptors (sst(1-5)). Scarce information is available on SRIF function in the retina, including the elucidation of transduction pathways mediating SRIF action. We have investigated SRIF and SRIF receptor modulation of adenylyl cyclase (AC) activity in both wild-type (WT) retinas and sst1 or sst2 knock-out (KO) retinas, which are known to over-express sst2 or sst1 receptors respectively. In WT retinas, application of SRIF compounds does not affect forskolin-stimulated AC activity. In contrast, activation of sst1 or sst2 receptors inhibits AC in the presence of sst2 or sst1 receptor antagonists respectively. Results from sst1 KO retinas demonstrate that either SRIF or the sst2 receptor preferring agonist octreotide, pertussis toxin-dependently inhibit AC activity. In contrast, in sst2 KO retinas, neither SRIF nor CH-275, an sst1 receptor agonist, are found to influence AC activity. As revealed by immunoblotting experiments, in sst1 KO retinas, levels of G(o)alpha proteins are 60% higher than in WT retinas and this increase in G(o)alpha protein levels is concomitant with an increase in sst2A receptor expression. We conclude that interactions between sst1 and sst2 receptors may prevent SRIF effects on AC activity. In addition, we suggest that the density of sst2 receptors and/or G(o)alpha proteins may represent the rate-limiting factor for the sst2 receptor-mediated inhibition of AC.

Inhibition of amniotic interleukin-6 and prostaglandin E2 release by ampicillin.

OBJECTIVE: To test the effect of ampicillin on amniotic interleukin-6 (IL-6) and prostaglandin E2 (PGE2) release. METHODS: In an in vitro study, IL-6 and PGE2 release from amnion-like Wistar Institute Susan Hayflick cells was assayed under basal conditions, as well as after incubation with ampicillin. In an in vivo study, amniotic fluid IL-6 was assayed in a total of 212 patients submitted to genetic amniocentesis during the 17th week of their singleton physiological pregnancy. The study population was subdivided as follows: 92 patients sampled before ampicillin administration, 70 patients sampled 4 hours after administration of 1 g ampicillin, and 50 patients sampled 12 hours after administration of 1 g ampicillin. RESULTS: At doses ranging from 10-7 to 10-4 M, ampicillin decreased IL-6 release from Wistar Institute Susan Hayflick cells. The drug effect was already statistically significant (-30%; P <.05) at the lowest concentration tested (10-7 M), reaching the maximum (-50%) at 10-6 M after 4 hours of incubation. Moreover, ampicillin concentrations ranging from 10-7 to 10-4 M decreased PGE2 release from Wistar Institute Susan Hayflick cells; maximal inhibition was reached at 10-6 M after 4 hours (-40%; P <.05). Finally, IL-6 levels measured in amniotic fluid of patients sampled 4 hours after ampicillin administration proved strongly and significantly reduced when compared with those sampled either before or 12 hours after treatment (P <.001). CONCLUSION: The capacity of ampicillin to directly decrease amniotic IL-6 and PGE2 release should be considered in the management of bacterial and nonbacterial inflammatory complications of pregnancy mediated by the cytokine and prostanoid interaction.